Methods and reagents for determining desired meat quality traits in sheep animals and their use in animal breeding

Abstract

The present disclosure relates generally to methods and test kits for identifying SNPs associated with desired meat quality traits in sheep animals. Specifically, the present disclosure relates to an SNP-based diagnostic test and method for identifying sheep animals having a desired fat melting point (FMP), intramuscular fat (IMF), and omega-3 long chain polyunsaturated fatty acids (n-3LC PUFA) characteristic of Australian white sheep or lambs (e.g., Tattykeel Australian white lambs), and to a method for identifying sheep animals having a desired fat melting point (FMP), intramuscular fat (IMF), and omega-3 long chain polyunsaturated fatty acids (n-3 LC PUFA) characteristic of Australian white sheep or lambs (e.g., Tattykeel Australian white lambs); and the use of these tests and methods in animal breeding planning.

Description

Technical Field

[0001] The present disclosure generally relates to methods and test kits for identifying SNPs associated with desirable meat eating quality traits in sheep animals. Specifically, the present disclosure relates to a SNP-based diagnostic test and method for identifying sheep animals having desirable fat melting point (FMP), intramuscular fat (IMF), and ω-3 long chain polyunsaturated fatty acids (n-3 LC PUFA) characteristic of Australian White sheep or lambs (e.g., Tattykeel Australian White lambs); and the use of these tests and methods in animal breeding programs. Background Art

[0002] Eating quality is the biggest determinant of consumer acceptability and satisfaction with meat products. The eating and nutritional quality of lamb is influenced by intramuscular fat (IMF) content (Thomas et al., (2021). Animal, 15:100-136), fat melting point (FMP), tenderness, juiciness, flavor (Pewan et al., (2020) Antioxidants, 9:11-18), and health-promoting ω-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs), which can optimize retinal, maternal, and child brain function while minimizing risks associated with cardiovascular and chronic diseases (Heck et al., (2021) Curr. Opin. Food Sci. 40:6-12; Pewan et al., (2020) Genes, 11:587).

[0003] In a recent review of the development, calibration, and validation of objective measurement techniques for carcass composition, lean, fat, and edible meat quality traits in the Australian and New Zealand livestock industry, Gardner et al. (2021) Meat Sci. 179:108556 highlighted the inherent difficulties associated with poor measurement of edible meat quality and lean percentage. Attempts to predict IMF (Fowler et al. (2021) Meat Sci. 177:108505; Lambe et al. (2021) Meat Sci. 17:108286; Alvarenga et al. (2021) Meat Sci. 181:108404), intramuscular connective tissue (Andueza et al. (2021) Meat Sci. 179:108537), composition and quality traits (Pat =El et al. (2021) Meat Science, 178:108518), tenderness, extreme pH and IMF content (Dixit et al. (2020) Meat Science, 162:108026020; Dixit et al. (2021) Meat Science, 181:108410; Knight et al. (2019) Meat Science, 155:102–108), characterized by low accuracy, inconsistency and discrepancies between calibration and validation data. Such inaccuracies result in inefficient lambing and an estimated $130 million in value chain waste for the Australian beef industry each year (Gardner et al. (2021) Meat Science, 179:108556).

[0004] However, meat quality data are only available after slaughter, when it is too late to make selection decisions on live animals. Estimated breeding values ​​for carcasses (Knight et al. (2020) Meat Science, 170:108236; Anderson et al. (2016) Meat Science, 116:243-252), visual marbling scores and meat imaging camera marbling systems (Stewart et al. (2021) Meat Science, 181:108369), and fat, lean, and bone composition of lamb carcasses determined by computed tomography based on dual X-ray absorptiometry scanners (Connaughton et al. (2021) Meat Science, 181:108413) are all useful technological advances, but there are still precision issues due to low accuracy, and by the time an informed decision on the genetic merit of meat quality is made, the animal is already dead. In a study of the relationship between estimated breeding values ​​and objective meat quality measurements in sires and sensory scores in Australian lambs, Pannier et al. (2014) Meat Science, 96:1076-1087 confirmed growing concerns that selection for leanness can reduce consumer eating quality and concluded that selection programs need to be carefully monitored to maintain lamb eating quality. In an experimental trial to understand the effects of ram leanness breeding values ​​on carcass composition, meat quality, and nutrient and mineral content in Australian lambs, Knight et al. (2020) Meat Science, 170:1082-36 concluded that in order to avoid deterioration in meat quality, lamb nutritional content, and fresh meat color, Australian sheep producers need to incorporate other aspects of meat quality when selecting rams for increased leanness. To date, the use of conventional laboratory-based fat extraction, 'slipping' and gas chromatography remains the most accurate technique not only for measuring IMF, FMP and n-3 LC-PUFA, but also for predicting consumer acceptance of beef and lamb (Holman et al., (2021) Meat Science, 181:108586). Therefore, there is a need for improved methods for measuring IMF, FMP and n-3 LC-PUFA in livestock species.

[0005] Any discussion of documents, acts, materials, devices, articles of manufacture or the like included in this specification is not to be taken as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present disclosure by virtue of their existence before the priority date of each claim of this application. Summary of the invention

[0006] The present disclosure is based, at least in part, on the inventors' recognition that improved methods are needed for measuring IMF, FMP and n-3 LC-PUFA in sheep species and specifically in lambs bred for meat, in order to aid genetic, breeding and selection programs for meat eating quality. To this end, the inventors combined (1) minimally invasive thoracic lumbar longissimus muscle biopsy sampling of Tattykeel Australian White (TAW) sheep, (2) laboratory-based IMF, FMP and fatty acid analysis, and (3) next generation sequencing (NGS) of single nucleotide polymorphisms (SNPs) within lipid metabolism genes to directly quantify the genetic value of health-promoting n-3 LC-PUFA, IMF and FMP in live lambs.

[0007] Based on this work, the inventors identified several functional SNPs that were strongly associated with n-3 LC-PUFA, IMF and FMP, as well as SNP markers that provided a unique DNA marker signature for TAW sheep genetics. Based on this discovery, the inventors developed a SNP-based test for identifying sheep with desirable edible meat quality traits, including n-3 LC-PUFA, IMF and FMP, for use in animal breeding programs. The SNP-based test can also be used to determine and / or confirm that a sheep / lamb is a 'true to type' TAW sheep / lamb.

[0008] Thus, in one example, the present disclosure provides a method for identifying a sheep animal having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to the general population of animals of the species, the method comprising:

[0009] (a) obtaining SNP genotype data of the sheep animal at one or more single nucleotide polymorphism (SNP) loci, wherein the loci are selected from: (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C 2 SNPs with r values ​​≥ 0.7; (ii) FASN g.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 (iii) SNPs with r values ​​≥ 0.7; and / or (iii) FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7; and

[0010] (b) determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general animal population of the species if the sheep animal has: (i) a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype; (ii) a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4g.62829478A>T, or a genotype in linkage disequilibrium with the AA genotype.

[0011] In some examples, the method comprises:

[0012] (a) obtaining the sheep animal having a gene at the SNP locus SCD g.23881050T>C or a gene in linkage disequilibrium with SCD g.23881050T>C 2 SNP genotype data at SNPs with values ​​≥ 0.7; and

[0013] (b) if the sheep animal has a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general animal population of the species.

[0014] For example, the method comprises:

[0015] (a) obtaining the SNP genotype data of the sheep animal at SCD g.23881050T>C; and

[0016] (b) if the sheep animal has a TT genotype in SCD g.23881050T>C, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general population of animals of the species.

[0017] In some examples, the method comprises:

[0018] (a) obtaining the sheep animal having a gene at the SNP locus FASN g.12323864A>G or a gene in linkage disequilibrium with FASN g.12323864A>G 2 SNP genotype data at SNPs with values ​​≥ 0.7; and

[0019] (b) if the sheep animal has a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype, then determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to the general animal population of the species.

[0020] For example, the method comprises:

[0021] (a) obtaining SNP genotype data of the sheep animal at the SNP locus FASN g.12323864A>G; and

[0022] (b) if the sheep animal has a GG genotype in FASN g.12323864A>G, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general population of animals of the species.

[0023] In some examples, the method comprises:

[0024] (a) obtaining the sheep animal having a SNP locus FABP4g.62829478A>T or a linkage disequilibrium with FABP4g.62829478A>T 2 SNP genotype data at SNPs with values ​​≥ 0.7; and

[0025] (b) if the sheep animal has an AA genotype in FABP4g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general animal population of the species.

[0026] For example, the method comprises:

[0027] (a) obtaining SNP genotype data of the sheep animal at the SNP locus FABP4g.62829478A>T; and

[0028] (b) if the sheep animal has the AA genotype in FABP4g.62829478A>T, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general animal population of the species.

[0029] In some examples, the method comprises:

[0030] (a) obtaining SNP genotype data of the sheep animal at two or three of the following SNP positions: (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C 2 SNPs with r values ​​≥ 0.7; (ii) FASNg.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 (iii) SNPs with r values ​​≥ 0.7; and / or (iii) FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7; and

[0031] (b) If the sheep animal has one or more of the following SNP genotypes, it is determined that the animal has or will have the desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general animal population of the species: (i) a TT genotype in SCD g.23881050T>C or a genotype that is disequilibrium with the TT genotype; (ii) a GG genotype in FASNg.12323864A>G or a genotype that is disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4g.62829478A>T or a genotype that is disequilibrium with the AA genotype.

[0032] In some examples, the sheep animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to the general population of animals of the species if the animal has one of the following SNP genotypes:

[0033] (i) a TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype and a GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype;

[0034] (ii) a TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype and an AA genotype in FABP4 g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype;

[0035] (iii) a GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype and an AA genotype in FABP4 g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype; and

[0036] (vi) TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype, GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype, and AA genotype in FABP4 g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype.

[0037] For example, the sheep animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general population of animals of the species if the animal has one of the following SNP genotypes:

[0038] (i) TT genotype in SCD g.23881050T>C and GG genotype in FASN g.12323864A>G;

[0039] (ii) TT genotype in SCD g.23881050T>C and AA genotype in FABP4g.62829478A>T;

[0040] (iii) GG genotype in FASN g.12323864A>G and AA genotype in FABP4g.62829478A>T;

[0041] (vi) TT genotype in SCD g.23881050T>C, GG genotype in FASN g.12323864A>G, and AA genotype in FABP4 g.62829478A>T.

[0042] The method may further comprise selecting and / or breeding from ovine animals identified as having desirable n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to the general population of animals of the species based on the SNP genotype.

[0043] In one example, the method identifies an ovine animal having a desired n-3 LC-PUFA compared to the general animal population of the species. In one example, the method identifies an ovine animal having a desired IMF compared to the general animal population of the species. In one example, the method identifies an ovine animal having a desired FMP compared to the general animal population of the species.

[0044] As described herein, the inventors have also identified SNP markers that provide a unique DNA marker signature for Tattykeel Australian White (TAW) sheep genetics. These markers can be used in genetic testing to determine and / or confirm that a sheep / lamb is a 'pure bred' TAW sheep / lamb. Thus, the present disclosure also provides a method for determining whether a sheep animal or a part thereof is an Australian White lamb or a part thereof, the method comprising:

[0045] (a) obtaining SNP genotype data of the sheep animal at one or more single nucleotide polymorphism (SNP) loci, wherein the loci are selected from: (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C 2 SNPs with r values ​​≥ 0.7; (ii) FASN g.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 (iii) SNPs with r values ​​≥ 0.7; and / or (iii) FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7; and

[0046] (b) determining that the sheep animal or a part thereof is an Australian White lamb or a part thereof if the sheep animal has: (i) a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype; (ii) a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4g.62829478A>T, or a genotype in linkage disequilibrium with the AA genotype.

[0047] In some examples, the method comprises:

[0048] (a) obtaining the sheep animal having a gene at the SNP locus SCD g.23881050T>C or a gene in linkage disequilibrium with SCD g.23881050T>C 2 SNP genotype data at SNPs with values ​​≥ 0.7; and

[0049] (b) if the sheep animal has a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype, then determining that the animal or the part thereof is an Australian White lamb or the part thereof.

[0050] For example, the method comprises:

[0051] (a) obtaining the SNP genotype data of the sheep animal at SCD g.23881050T>C; and

[0052] (b) if the sheep animal has the TT genotype in SCD g.23881050T>C, determining that the animal or part thereof is an Australian White lamb or part thereof.

[0053] In some examples, the method comprises:

[0054] (a) obtaining the sheep animal having a gene at the SNP locus FASN g.12323864A>G or a gene in linkage disequilibrium with FASN g.12323864A>G 2 SNP genotype data at SNPs with values ​​≥ 0.7; and

[0055] (b) if the sheep animal has a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype, then determining that the animal or the part thereof is an Australian White lamb or the part thereof.

[0056] For example, the method comprises:

[0057] (a) obtaining SNP genotype data of the sheep animal at the SNP locus FASN g.12323864A>G; and

[0058] (b) if the sheep animal has a GG genotype in FASN g.12323864A>G, then determining that the animal or part thereof is an Australian White Lamb or part thereof.

[0059] In some examples, the method comprises:

[0060] (a) obtaining the sheep animal having a SNP locus FABP4g.62829478A>T or a linkage disequilibrium with FABP4g.62829478A>T 2 SNP genotype data at SNPs with values ​​≥ 0.7; and

[0061] (b) if the sheep animal has an AA genotype in FABP4g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general animal population of the species.

[0062] For example, the method comprises:

[0063] (a) obtaining SNP genotype data of the sheep animal at the SNP locus FABP4g.62829478A>T; and

[0064] (b) if the sheep animal has the AA genotype in FABP4g.62829478A>T, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general animal population of the species.

[0065] In some examples, the method comprises:

[0066] (a) obtaining SNP genotype data of the sheep animal at two or three of the following SNP positions: (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C 2 SNPs with r values ​​≥ 0.7; (ii) FASNg.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 (iii) SNPs with r values ​​≥ 0.7; and / or (iii) FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7; and

[0067] (b) determining that the sheep animal or a part thereof is an Australian White lamb or a part thereof if the sheep animal has one or more of the following SNP genotypes: (i) a TT genotype in SCD g.23881050T>C or a genotype that is in linkage disequilibrium with the TT genotype; (ii) a GG genotype in FASN g.12323864A>G or a genotype that is in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4g.62829478A>T or a genotype that is in linkage disequilibrium with the AA genotype.

[0068] In some examples, the sheep animal is determined to be an Australian White Lamb or a part thereof if the sheep animal has one of the following genotypes:

[0069] (i) a TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype and a GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype;

[0070] (ii) a TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype and an AA genotype in FABP4 g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype;

[0071] (iii) a GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype and an AA genotype in FABP4 g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype; and

[0072] (vi) TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype, GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype, and AA genotype in FABP4 g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype.

[0073] For example, if the sheep animal has one of the following genotypes, then the animal is determined to be an Australian White Lamb or a part thereof:

[0074] (i) TT genotype in SCD g.23881050T>C and GG genotype in FASN g.12323864A>G;

[0075] (ii) TT genotype in SCD g.23881050T>C and AA genotype in FABP4g.62829478A>T;

[0076] (iii) GG genotype in FASN g.12323864A>G and AA genotype in FABP4g.62829478A>T;

[0077] (vi) TT genotype in SCD g.23881050T>C, GG genotype in FASN g.12323864A>G, and AA genotype in FABP4 g.62829478A>T.

[0078] The method may further comprise selecting and / or breeding from sheep animals or parts thereof identified as Australian White Lambs based on the SNP genotype.

[0079] In each of the foregoing examples, the method may further comprise generating the SNP genotype data from a nucleic acid sample from the sheep animal. Any method known in the art for generating SNP genotype data from a nucleic acid sample is contemplated. Exemplary methods are described herein.

[0080] The nucleic acid sample can be obtained or prepared from any suitable biological sample (such as tissue, fiber or fluid (for example, blood, blood fraction or semen) or a combination thereof) obtained from a sheep animal. In some instances, the nucleic acid sample is prepared by tissue (for example, muscle) taken from the longissimus dorsi muscle of the animal. In other instances, the nucleic acid sample is prepared by reproductive materials selected from semen, oocytes or embryos of the animal. In other instances, the nucleic acid sample is prepared by fibers obtained from the animal or cells attached thereto (for example, wool fibers or hair follicle cells attached thereto). In other instances, the nucleic acid sample is prepared by blood obtained from the animal (for example, whole blood or blood fractions, such as serum). In each of the foregoing instances, the nucleic acid sample can be DNA. However, in some instances, RNA may need to be prepared by the animal.

[0081] As described herein, any method known in the art for generating SNP genotype data from nucleic acid samples is contemplated. However, in some instances, the SNP genotype data is generated using one or more assays selected from the group consisting of: DNA amplification assay, DNA hybridization assay, DNA sequencing, denaturing high performance liquid chromatography (DHPLC) or a combination thereof. In one example, DNA amplification is employed in the method. For example, SNP alleles can be detected using polynucleotides that allow different alleles of SNP to be distinguished from each other by one or more DNA amplification assays. Exemplary DNA amplification assays for the disclosed method are described herein. However, in one example, the DNA amplification assay is polymerase chain reaction (PCR), such as amplification-retarded mutation system (ARMS) PCR.

[0082] In certain examples, the methods can include detecting SNP alleles by one or more Southern hybridization assays using polynucleotides that allow different alleles of the SNP to be distinguished from one another.

[0083] In other examples, the method may comprise detecting the SNP allele in each ovine animal by sequencing a gene comprising the or each SNP, or a subsequence of the gene.Exemplary DNA sequencing methods contemplated for use in the methods of the present disclosure are described herein.

[0084] The present disclosure also provides a system for determining the occurrence of a nucleotide at one or more sheep SNPs, the SNPs being selected from SCD g.23881050T>C, FASN g.12323864A>G, and FABP4g.62829478A>T. The system may comprise a hybridization medium and / or a solid substrate, the hybridization medium and / or the solid substrate comprising at least two oligonucleotides, each of which is configured to selectively hybridize to a specific allele of a sheep SNP selected from SCD g.23881050T>C, FASN g.12323864A>G, and FABP4g.62829478A>T. For example, where the system is used to determine the occurrence of nucleotides at two or more (e.g., three) sheep SNPs selected from SCDg.23881050T>C, FASN g.12323864A>G, and FABP4g.62829478A>T, the hybridization medium and / or matrix comprises at least two oligonucleotides for each SNP, wherein each oligonucleotide is configured to selectively hybridize to a specific allele of the corresponding sheep SNP. For example, a solid support may be provided to which the oligonucleotides are directly or indirectly attached.

[0085] The present disclosure also provides a test kit comprising a plurality of oligonucleotide primers and / or probes configured to distinguish alleles at one or more SNP loci in sheep animals, wherein the SNP loci are selected from (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C; 2 SNPs with r values ​​≥ 0.7; (ii) FASN g.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 SNPs with r values ​​≥ 0.7 and / or FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7.

[0086] In one example, the test kit may include:

[0087] One or more oligonucleotide primers and / or probes configured to distinguish between allele C and allele T at the SNP locus SCD g.23881050T>C;

[0088] One or more oligonucleotide primers and / or probes configured to distinguish between allele G and allele A at the SNP locus FASN g.12323864A>G; and / or

[0089] One or more oligonucleotide primers and / or probes configured to distinguish between allele A and allele T at the SNP locus FABP4g.62829478A>T.

[0090] In one example, the test kit comprises one or more oligonucleotide primers and / or probes configured to distinguish allele C and allele T at the SNP locus SCD g.23881050T>C. For example, the oligonucleotide primers can be configured to distinguish allele C and allele T at the SNP locus SCDg.23881050T>C by amplification-retarded mutation system polymerase chain reaction (ARMS PCR). In a specific example, the oligonucleotide primers configured to distinguish allele C and allele T at the SNP locus SCD g.23881050T>C by ARMS PCR are:

[0091] SCD g.23881050T>C_forward internal (SEQ ID NO: 1);

[0092] SCD g.23881050T>C_reverse internal (SEQ ID NO: 2);

[0093] SCD g.23881050T>C_positive external (a) (SEQ ID NO:3);

[0094] SCD g.23881050T>C_reverse external (a) (SEQ ID NO:4);

[0095] SCD g.23881050T>C_positive external (b) (SEQ ID NO:5); and

[0096] SCD g.23881050T>C_reverse external (b) (SEQ ID NO:6).

[0097] The test kit may also contain one or more or each of the following:

[0098] at least one DNA polymerase;

[0099] dinucleotide triphosphates (dNTPs);

[0100] magnesium salts; and

[0101] Buffer.

[0102] The test kit can be used in the methods of the present disclosure. For example, the test kit can be used in a method as described herein for identifying or determining an ovine animal having a desired n-3 LC-PUFA, intramuscular fat (IMF), fat melting point (FMP), or any combination thereof compared to a general animal population of the species. Alternatively or additionally, the test kit can be used in a method as described herein for determining whether an ovine animal or a part thereof is an Australian White Lamb or a part thereof.

[0103] The present disclosure also provides a method of producing reproductive or regenerative material from an ovine animal determined to have a desirable n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to a general population of animals of the species, the method comprising:

[0104] (i) performing a method as described herein on one or more animals to thereby identify a sheep animal having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, as compared to said general population of animals of said species;

[0105] (ii) selecting the animal identified in (i) as having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general population of animals of the species; and

[0106] (iii) obtaining reproductive or regenerative material from the selected animal.

[0107] The present disclosure also provides a method of producing reproductive or regenerative material from an ovine animal determined to have a SNP genotype indicative of an AWL genetic trait, the method comprising:

[0108] (i) performing a method as described herein on one or more animals to thereby identify sheep animals having a SNP genotype indicative of an AWL genetic characteristic;

[0109] (ii) selecting an animal identified in (i) as having a SNP genotype indicative of an AWL genetic characteristic; and

[0110] (iii) obtaining reproductive or regenerative material from the selected animal;

[0111] Wherein the SNP genotype indicating the AWL genetic characteristic comprises (i) a TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype; (ii) a GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype. In some instances, the SNP genotype indicating the AWL genetic characteristic comprises one or more of the following: a TT genotype in SCD g.23881050T>C, a GG genotype in FASN g.12323864A>G, or an AA genotype in FABP4g.62829478A>T. In a specific instance, the SNP genotype indicating the AWL genetic characteristic comprises a TT genotype in SCD g.23881050T>C.

[0112] The present disclosure also provides a reproductive or regenerative material from an ovine animal, the reproductive or regenerative material produced according to the method as described herein.

[0113] The present disclosure also provides a method of breeding an ovine animal having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to a general population of animals of the species, the method comprising:

[0114] (i) performing a method as described herein on one or more animals to thereby identify one or more ovine animals having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, as compared to said general population of animals of said species;

[0115] (ii) selecting one or more animals identified in (i) as having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, as compared to the general population of animals of the species;

[0116] (iii) breeding from the selected animal to produce one or more offspring therefrom.

[0117] The present disclosure also provides a sheep animal bred according to the methods described herein.

[0118] The present disclosure also provides a method of selectively breeding Australian White Lamb (AWL), the method comprising:

[0119] (i) performing a method as described herein on one or more animals to thereby identify one or more sheep animals having a SNP genotype indicative of an AWL genetic characteristic;

[0120] (ii) selecting one or more animals identified in (i) as having a SNP genotype indicative of an AWL genetic characteristic;

[0121] (iii) breeding from the selected animal to produce one or more offspring therefrom;

[0122] Wherein the SNP genotype indicating the AWL genetic characteristic comprises: (i) TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype; (ii) GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) AA genotype in FABP4g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype. In some examples, the SNP genotype indicating the AWL genetic characteristic comprises one or more of the following: TT genotype in SCD g.23881050T>C, GG genotype in FASN g.12323864A>G and / or AA genotype in FABP4g.62829478A>T. In a specific example, the SNP genotypes indicative of the AWL genetic characteristic include one or more of the TT genotypes in SCD g.23881050T>C.

[0123] The present disclosure also provides a sheep animal bred according to the methods described herein.

[0124] The present disclosure also provides a method for improving the eating quality of meat of a herd of sheep animals, the method comprising:

[0125] (i) performing a method as described herein on one or more animals in said population to thereby identify one or more ovine animals having a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to said general population of animals of said species;

[0126] (ii) selecting one or more animals identified in (i) as having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, as compared to the general population of animals of the species;

[0127] (iii) obtaining reproductive or regenerative material from the selected animal; and

[0128] (iv) producing one or more offspring or one or more generations of animals from the reproductive or regenerative material.

[0129] In each of the foregoing examples, the reproductive material can be an oocyte, an embryo and / or semen. Similarly, the regenerative material can be an oocyte, an embryo and / or semen, but can further include primordial germ cells (PGCs) that produce oocytes or sperm. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1 Experimental design for selection, breeding and evaluation of n-3 LC-PUFA, IMF and FMP in Tattykeel Australian White sheep.

[0131] Figure 2 FASN fragment 1 PCR products in Tattykeel Australian White (WL) lambs, Polled Dorset (PD) lambs and Texel (TX) lambs.

[0132] Figure 3 FASN fragment 2 PCR products in Tattykeel Australian White (WL) lambs, Polled Dorset (PD) lambs and Texel (TX) lambs.

[0133] Figure 4 FASN fragment 3 PCR products in Tattykeel Australian White (WL) lambs, Polled Dorset (PD) lambs and Texel (TX) lambs.

[0134] Figure 5 .SCD PCR products in Tattykeel Australian White (WL) lambs, Polled Dorset (PD) lambs and Texel (TX) lambs.

[0135] Figure 6 . FABP4 PCR products in Tattykeel Australian White (WL) lambs, Polled Dorset (PD) lambs and Texel (TX) lambs.

[0136] Figure 7 .Correlation between SCD gene SNP loci, IMF, FMP and fatty acids in TAW lambs.

[0137] Figure 8 .Correlation between FASN gene SNP loci, IMF, FMP and fatty acids in TAW lambs.

[0138] Fig. 9 .Correlation between FABP4 gene SNP loci, IMF, FMP and fatty acids in TAW lambs.

[0139] Fig.10 .TAWL, Texel and Dorset (10 uM tetra primers with forward outer / reverse outer primers (a) and 0.5 uL gDNA).

[0140] Fig.11 .TAWL, Texel and Dorset (100uM forward outer primer (a), 10uM forward inner / reverse inner and reverse outer primers (a), 0.5uL gDNA).

[0141] Fig.12 .TAWL, Texel and Dorset (10 uM tetra primers with forward outer / reverse outer primers (b) and 0.5 uL gDNA).

[0142] Fig.13 .TAWL, Wagyu, Angus, Hereford and Brahman (10 uM tetra primers with forward outer / reverse outer primers (b) and 0.5 uL gDNA).

[0143] Explanation of Sequence Listing Symbols

[0144] SEQ ID NO: 1: Primer SCD g.23881050T>C_Forward internal oligonucleotide sequence

[0145] SEQ ID NO: 2: Primer SCD g.23881050T>C__ reverse internal oligonucleotide sequence

[0146] SEQ ID NO: 3: Oligonucleotide sequence of primer SCD g.23881050T>C_forward outer (a)

[0147] SEQ ID NO: 4: Oligonucleotide sequence of primer SCD g.23881050T>C_reverse external (a)

[0148] SEQ ID NO: 5: Oligonucleotide sequence of primer SCD 23881050T>C_forward outer (b)

[0149] SEQ ID NO:6: Oligonucleotide sequence of primer SCD g.23881050T>C_reverse external (b) DETAILED DESCRIPTION

[0150] General Techniques and Selected Definitions

[0151] Unless specifically defined otherwise, all technical and scientific terms used herein should be considered to have the same meaning as commonly understood by one of ordinary skill in the art.

[0152] As used herein, the singular forms "a," "an," and "the" include plural forms of these terms unless the context clearly dictates otherwise.

[0153] The term "and / or", for example, "X and / or Y" should be understood to mean "X and Y" or "X or Y", and should be considered to provide clear support for both meanings or either meaning.

[0154] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps or groups of elements, integers or steps but not the exclusion of any other elements, integers or steps or groups of elements, integers or steps.

[0155] The term "about" is used herein to mean approximately. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the limits above and below the numerical values. In general, the term "about" is used herein to modify a numerical value, such as an amount of time, concentration, temperature, etc., above and below the specified value by ±10%, more preferably ±5%, even more preferably ±1% and still more preferably ±0.1%, to be suitable for performing the disclosed methods.

[0156] Those skilled in the art will appreciate that, in addition to those specifically described, the present disclosure is susceptible to changes and modifications. It should be understood that the present disclosure includes all such changes and modifications. The present disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification, individually or jointly, and any and all combinations of any two or more steps or features in the steps or features. Therefore, each feature of any particular aspect or embodiment of the present disclosure can be applied to any other aspect or embodiment of the present disclosure after making necessary changes.

[0157] The scope of the present disclosure is not to be limited by the specific embodiments described herein, which are intended for purposes of illustration only. As described herein, functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0158] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter shall be considered to cover both one and more (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise.

[0159] Unless otherwise indicated, the present disclosure as described herein can be performed using conventional molecular biology techniques. Such procedures are described, for example, in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 2nd edition (1989), all of Volumes I, II and III; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, in its entirety; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984), IRL Press, Oxford, in its entirety, and in particular Gait, pp. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151; 4. Nucleic Acid Hybridization: A Practical Approach. Acid Hybridization: A Practical Approach (B.D. Hames and S.J. Higgins, eds., 1985) IRL Press, Oxford, full text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, full text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods in Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), full text, Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, RL (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, RB (1963). Journal of the American Chemical Society (J.Am.Chem.Soc.) 85, 2149-2154; Barany, G.12. Wünsch, E., ed., (1974) Synthese von Peptiden in Houben-Weyls Methods of Organic Chemistry (Synthese von Peptiden in Houben-Weyls Methods of Organic Chemistry (Synthese von Peptiden in Houben-Weyls Methods of Organic Chemistry (Müler, E., ed.), Volume 15, 4th Edition, Parts 1 and 2, Thieme, Stuttgart); Bodanszky, M. (1984) Principles of Peptide Synthesis (Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. and Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, 3rd ed. (John R. W. Masters, ed., 2000), ISBN 0199637970, full text. .

[0160] Methods of animal selection

[0161] The present inventors have identified several functional SNPs in lipid metabolism genes that are closely associated with n-3 LC-PUFA, IMF and FMP and provide a unique DNA marker signature for TAW sheep genetics. Based on this discovery, the present inventors have developed a SNP-based test for identifying sheep with desirable edible meat quality traits, including n-3 LC-PUFA, IMF and FMP, for use in animal breeding programs. The SNP-based test can also be used to determine and / or confirm that a sheep / lamb is (or is not) a 'pure breed' TAW sheep / lamb, for example for breeding purposes.

[0162] Thus, in one example, the present disclosure provides a method for identifying a sheep animal having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to the general population of animals of the species, the method comprising:

[0163] (a) obtaining SNP genotype data of the sheep animal at one or more single nucleotide polymorphism (SNP) loci, wherein the loci are selected from: (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C 2 SNPs with r values ​​≥ 0.7; (ii) FASN g.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 (iii) SNPs with r values ​​≥ 0.7; and / or (iii) FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7; and

[0164] (b) determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general animal population of the species if the sheep animal has: (i) a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype; (ii) a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4g.62829478A>T, or a genotype in linkage disequilibrium with the AA genotype.

[0165] As described herein, the methods of the present disclosure can be used to identify sheep animals having desired n-3 LC-PUFA, IMF, FMP, or any combination thereof. It has been shown that consumers prefer meat containing low FMP, moderate IMF, and a fatty acid composition with proportionally more n-3 LC-PUFAs that promote health. Therefore, the methods of the present disclosure can be used to select animals whose meat has a low FMP, moderate IMF, and a fatty acid composition with proportionally more n-3 LC-PUFAs that promote health.

[0166] The term "fat melting point (FMP)" as used herein refers to the temperature at which the solid and liquid phases of the fat are in equilibrium. The melting point is determined by, among other things, the fatty acid composition, the molecular structure of these fatty acids, and the way these fatty acids 'stack together'. Fatty acids are monocarboxylic acids composed of a fatty chain containing 4 to 22 carbon atoms and a terminal carboxyl group (COOH). Fatty acids can be saturated or unsaturated, branched or unbranched, and may or may not include one or more hydroxyl groups. A low fat melting point is desirable for the eating quality of the meat. The fat melting point (or FMP) of sheep can range from about 25°C to about 50°C, depending on the sheep breed and genetic characteristics. According to the method of the present disclosure, the desired FMP is below 40°C. In one example, the desired FMP is between 28°C and 39°C (e.g., about 28°C, or about 29°C, or about 30°C, or about 31°C, or about 32°C, or about 33°C, or about 34°C, or about 35°C, or about 36°C, or about 37°C, or about 38°C, or about 39°C). In a particular example, the desired FMP is between about 30°C and 36°C on average. For example, the FMP of an animal having a desired FMP selected according to the methods of the present disclosure may be about 32°C to 36°C (e.g., about 34°C) on average. In one example, the method comprises identifying a sheep animal having a desired FMP compared to a general population of animals of the species.

[0167] As used herein, " intramuscular fat (IMF) " refers to the fat between and inside the muscle fibers (cells), and is commonly referred to as "marbling". There is an indirect relationship between the tenderness, juiciness, flavor and outward appearance of IMF and meat. There are many methods that can be used to measure IMF, and each of these methods is considered herein. However, perhaps the most practical and repeatable method for measuring the IMF of living animals is ultrasound. The IMF level of " expectation " as described herein is at least about 2.5% to about 10% (for example, about 2.5%, or about 3.0%, or about 3.5%, or about 4.0%, or about 4.5%, or about 5.0%, or about 5.5%, or about 6.0%, or about 6.5%, or about 7.0%, or about 7.5%, or about 8.0%, or about 8.5%, or about 9.0%, or about 9.5%, or about 10.0%). In some examples, the "desirable" IMF level is about 3.5% to about 8.5%. More preferably, the "desirable" IMF level can be about 4.0% to 6.0%. In a specific example, the desired IMF percentage (average) is about 4.4%. In one example, the method comprises identifying a sheep animal having a desired IMF level compared to the general animal population of the species.

[0168] As described herein, the method can be used to identify animals with desired levels of ω-3 long-chain polyunsaturated fatty acids (n-3LC-PUFA) that promote health. As used herein, the term "n-3LC-PUFA" or "ω-3 long-chain polyunsaturated fatty acids" refers to polyunsaturated fatty acids (PUFAs) in which the final olefinic bond is 3 carbons away from the terminal methyl group (including the terminal methyl group) of the fatty acid. n-3LC-PUFA is a known health-promoting fatty acid that has been shown to optimize retinal, maternal and childhood brain function while minimizing the risks associated with cardiovascular and chronic diseases. Exemplary n-3LC-PUFAs include alpha linolenic acid (C18:3; "ALA"), eicosapentaenoic acid (C20:5; "EPA"), and docosahexaenoic acid (C22:6; "DHA"). Other n-3 LC-PUFAs include, but are not limited to, linoleic acid (C18:2n-6), palmitoleic acid (C16:0), stearic acid (C18:0), trans-9-octadecenoic acid (C18:1n-9), octadecatetraenoic acid (C18:4), eicosatetraenoic acid (C20:4), and docosapentaenoic acid (C22:5). Thus, the term "desirable" in the context of n-3 LC-PUFAs should be understood to mean a desired level or concentration of n-3 LC-PUFAs in animal meat. The desired level or concentration of n-3 LC-PUFAs would be at least about 40.0 mg / 100 g meat, such as about 40.0 mg / 100 g meat to about 150.0 mg / 100 g meat. For example, the desired n-3 LC-PUFA level can be about 40.0 mg / 100 g meat, or about 50.0 mg / 100 g meat, or about 60.0 mg / 100 g meat, or about 70.0 mg / 100 g meat, or about 80.0 mg / 100 g meat, or about 90.0 mg / 100 g meat, or about 100.0 mg / 100 g meat, or about 110.0 mg / 100 g meat, or about 120.0 mg / 100 g meat, or about 130.0 mg / 100 g meat, or about 140.0 mg / 100 g meat, or about 150.0 mg / 100 g meat. In some instances, the desired n-3 LC-PUFA level is about 45.0 mg / 100 g meat to about 120.0 mg / 100 g meat. In some examples, the desired n-3 LC-PUFA level is about 50.0 mg / 100 g meat to about 100.0 mg / 100 g meat. In one example, the desired n-3 LC-PUFA level is about 55.0 mg / 100 g to 60.0 mg / 100 g. In a specific example, the desired n-3 LC-PUFA level is (on average) about 57.9 mg / 100 g meat. In one example, the method comprises identifying a sheep animal having a desired n-3 LC-PUFA level compared to a general animal population of the species.

[0169] The term "general population" as used in the context of sheep animals is understood to mean a plurality of sheep animals which, when considered as a population or group, are heterozygous at one or more of the SNP loci described herein, i.e., one or more SNP loci located at a position selected from the group consisting of: (i) SCD g.23881050T>C or a locus in linkage disequilibrium with SCD g.23881050T>C; 2 SNPs with r values ​​≥ 0.7; (ii) FASN g.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 (iii) SNPs with r values ​​≥ 0.7; and / or (iii) FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with a value of ≥0.7. In some instances, after an animal with a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof is identified according to the methods described herein, the general population may not have undergone selective breeding. In other instances, after an animal with a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof is identified according to the methods described herein, the general population may have undergone selective breeding, but still includes animals that are heterozygous at one or more SNP loci described herein, or animals that are homozygous for alleles at one or more SNP loci described herein that do not confer the desired n-3 LC-PUFA, IMF, and / or FMP. That is, the population includes the genetic diversity at the SNP loci described herein. The general population of sheep animals can include male and / or female animals, and can include animals of any age and any breed. However, in a specific instance, the general population mainly includes sheep, which are meat breeds of sheep or dual-purpose breeds of sheep. The general population can include any number of animals, such as 2 animals or 1000 or more animals.

[0170] The present disclosure also provides a method for determining whether a sheep animal or a part thereof is an Australian White Lamb or a part thereof, the method comprising:

[0171] (a) obtaining SNP genotype data of the sheep animal at one or more single nucleotide polymorphism (SNP) loci, wherein the loci are selected from: (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C 2 SNPs with r values ​​≥ 0.7; (ii) FASN g.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2SNPs with r values ​​≥ 0.7; and / or FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7; and

[0172] (b) determining that the sheep animal or a part thereof is an Australian White lamb or a part thereof if the sheep animal has: (i) a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype; (ii) a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4g.62829478A>T, or a genotype in linkage disequilibrium with the AA genotype.

[0173] As used herein, the term "ovine animal" refers to sheep. Sheep can be male or female, and of any age. Ovine animals can also be sheep of any breed. However, the methods of the present disclosure are particularly useful for meat breeds and dual-purpose breeds. In some instances, the ovine animal using the methods of the present disclosure is a meat sheep breed. Exemplary sheep breeds that tend to be used for meat production include, but are not limited to, Australian White Sheep (also known as Australian White Lamb (AWL) or Australian White Sheep), Poll Dorset, Suffolk, White Suffolk and Dorper. In other instances, the ovine animal using the methods of the present disclosure is a dual-purpose sheep breed (i.e., a sheep breed for meat and fiber). Exemplary dual-purpose breeds include, but are not limited to, Border Leicester, Corriedale, Kupworth, Texel and South African Meat Merino (SAMM).

[0174] The term "Australian White" as used herein in the context of sheep or lamb, i.e. "Australian White sheep", "Australian White Lamb", "AWL" or the like, refers to a breed of meat sheep bred in Australia. The terms "Australian White", "Australian White Sheep", "Australian White Lamb" and "AWL" are used interchangeably herein. The breed was developed through selective breeding of White Dorper, Van Ruy, Polled Dorset and Texel sheep with the aim of producing a large, white sheep suited to Australian conditions with a self-shedding fleece. The breed produces hardy, large-bodied, heavy lambs that reach slaughter weight early. The breed also has a high reproductive capacity and an early maturing, open breeding season, making it a self-replacing herd.

[0175] The term "part thereof" as used in the context of Australian White Lamb refers to a part of an animal identified as an Australian White Lamb. In some instances, in the context of Australian White Lamb, "part thereof" may be a gutted carcass or piece of meat obtained from an animal identified as an Australian White Lamb. In another instance, "part thereof" in the context of Australian White Lamb may be a biological sample, such as a tissue sample, a fiber sample, a blood sample, or a semen sample. Other exemplary biological samples are described and contemplated herein. In other instances, "part thereof" may be reproductive material obtained from an animal identified as an Australian White Lamb. Exemplary reproductive material is described herein, but may include oocytes, embryos, and / or semen.

[0176] To date, assessment of meat quality (including meat eating quality) has typically been performed after slaughter. This can make it difficult to incorporate meat eating quality data into breeding decisions. As discussed herein, the present disclosure is based on the identification of specific SNPs within the lipid metabolism genes SCD, FASN and FABP4, which the inventors have demonstrated to be associated with specific meat eating quality traits (i.e., n-3 LC-PUFA, IMF and / or FMP). Therefore, these SNPs make it possible to predict whether an animal is likely to have the desired n-3 LC-PUFA, IMF and FMP, and thereby make early, informed decisions about the breeding value of live sheep based on meat eating quality traits. It has also been identified that these SNP markers provide a unique DNA marker signature for Tattykeel Australian White (TAW) sheep genetics. These SNP markers can be used in genetic testing to determine and / or confirm that a sheep / lamb is a 'pure bred' Australian White lamb, specifically a TAW sheep / lamb.

[0177] As used herein, the term "single nucleotide polymorphism" or "SNP" refers to a variation ("polymorphism") at a single nucleotide position in a genome. Specifically, SNP is a germline replacement of a single nucleotide at a specific position in a genome, which is present in a sufficiently large part of an organism population (e.g., 1% or more). For example, in a part of a population, there is an 'A' at a specific nucleotide position, and another part of the population has a 'C', 'G' or 'T' at the same specific position in the genome, and the nucleotide position constitutes a SNP. Different SNP variants, such as 'A', 'C', 'G' or 'T' at a specific nucleotide position, are referred to as "alleles". In the case where the organism is a diploid species, such as in the case of sheep, the animal will have two copies of the polynucleotide containing the SNP, and therefore has two SNP alleles. The SNP alleles at a specific SNP position are collectively referred to as SNP "genotypes". However, "genotype" can refer to the genetic information carried by an individual at one or more SNP positions in a genome. In the case where a diploid animal has two copies of the same SNP allele, such as AA, GG, TT, or CC, the animal is referred to as being "homozygous" for the SNP. In contrast, in the case where an animal has non-identical SNP alleles (e.g., AG, AC, AT, CG, CT, or GT) at the SNP position on the corresponding homologous chromosome, the animal is referred to as being "heterozygous" at the particular SNP.

[0178] As described herein, SNPs associated with desired n-3 LC-PUFA, IMF and / or FMP can be selected from: (i) SCD g.23881050T>C; (ii) FASN g.12323864A>G; and / or (iii) FABP4 g.62829478A>T. These SNP positions have been defined by reference to the following coding sequences in the National Center for Biotechnology Information (NCBI) database (Genbank): SCD (NC_040273.1), FASN gene (accession number: NC_040262.1), FABP4 (NC_040260.1). In this regard, the inventors have shown that (i) SCD g.23881050T>C is associated with IMF and n-3 LC-PUFA C22:6n-3 (docosahexaenoic acid) and C22:5n-3 (docosapentaenoic acid); (ii) FASN g.12323864A>G is associated with FMP, long-chain saturated fatty acids C18:3n-3 (alpha linoleic acid), C18:1n-9 (elaidic acid), C18:0 (stearic acid), C16:0 (palmitic acid) and monounsaturated fatty acids, and (iii) FABP4g.62829478A>T is associated with IMF. Therefore, the methods of the present disclosure can include interrogating an animal's genotype at one or more of SCD g.23881050T>C, FASN g.12323864A>G, and FABP4g.62829478A>T. In one example, the TT genotype in SCD g.23881050T>C was associated with desired IMF and n-3 LC-PUFAs compared to TA or TT genotypes in SCD g.23881050T>C. In one example, the GG genotype in FASN g.12323864A>G was associated with desired FMP, long chain saturated fatty acids, and monounsaturated fatty acids compared to GA or AA genotypes in FASN g.12323864A>G. In one example, the AA genotype in FABP4g.62829478A>T was associated with IMF compared to AG or GG genotypes in FABP4g.62829478A>T.

[0179] The inventors have also determined that the above SNP genotypes useful for predicting expected n-3 LC-PUFA, IMF and / or FMP can be used to determine whether a sheep or lamb is a 'pure' Australian White lamb, specifically a 'pure' Tattykeel Australian White (TAW) sheep / lamb.

[0180] In other examples, the methods of the present disclosure may comprise making a selection decision based on one or more SNPs or other polymorphic genetic markers in linkage disequilibrium (LD) with SCD g.23881050T>C, FASNg.12323864A>G, or FABP4g.62829478A>T. This is based on the fact that other SNP or polymorphic gene markers that are very close to SCD g.23881050T>C, FASNg.12323864A>G or FABP4g.62829478A>T will also be associated with the desired n-3 LC-PUFA, IMF and / or FMP, because markers in linkage disequilibrium with SCD g.23881050T>C, FASN g.12323864A>G or FABP4g.62829478A>T will also be in linkage disequilibrium with genes affecting n-3 LC-PUFA, IMF and / or FMP.

[0181] "Linkage disequilibrium" or "LD" as used herein refers to the non-random association of alleles at two or more loci in a particular population. In other words, linkage disequilibrium is the preferential association of a particular allele or gene marker with a particular allele or gene marker at an adjacent chromosomal position, with a frequency higher than the chance expectation of any particular allele frequency in the population. Therefore, linkage disequilibrium can be measured as a deviation from the null hypothesis of linkage equilibrium, in which each allele at one locus is randomly associated with each allele at a second locus in an individual genome population. For example, if locus X has alleles A and B with the same frequency of occurrence, and linkage locus Y has alleles C and D with the same frequency of occurrence, one would expect the frequency of occurrence of combination AC to be 0.25. If AC occurs more frequently, then alleles A and C are in linkage disequilibrium. Linkage disequilibrium may be caused by the natural selection of certain allele combinations, or it may be because the time when the alleles are introduced into the population is too short to reach equilibrium with the linked alleles. For biallelic markers, one of the most commonly used measures of LD is r. 2 , which is the square of the correlation coefficient between two indicator variables, one indicating the presence or absence of a particular allele at a first locus, and the other indicating the presence or absence of a particular allele at a second locus.

[0182] As used herein, the term "locus" or "loci" refers to the site or position of one or more genes or polynucleotide sequences on a chromosome.

[0183] In some instances, SNPs or other polymorphic markers (also collectively referred to as "gene markers") that are in LD with SCD g.23881050T>C, FASN g.12323864A>G, or FABP4g.62829478A>T can be used instead of SCDg.23881050T>C, FASN g.12323864A>G, or FABP4g.62829478A>T, respectively, to predict the likelihood that an animal will have the desired n-3 LC-PUFA, IMF, and / or FMP. Similarly, genetic markers in LD with SCD g.23881050T>C, FASN g.12323864A>G or FABP4g.62829478A>T may be used in place of SCD g.23881050T>C, FASN g.12323864A>G or FABP4g.62829478A>T, respectively, to determine whether a sheep or lamb is a 'pure' Australian White lamb, specifically a 'pure' Tattykeel Australian White (TAW) sheep / lamb.

[0184] More specifically, the methods of the present disclosure may include determining that the sheep animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general animal population of the species if the sheep animal has: (i) a gene marker genotype that is in linkage disequilibrium with the TT genotype in SCD g.23881050T>C; (ii) a gene marker genotype that is in linkage disequilibrium with the GG genotype in FASN g.12323864A>G and / or (iii) a gene marker genotype that is in linkage disequilibrium with the AA genotype in FABP4g.62829478A>T. Similarly, genetic marker genotypes in linkage disequilibrium with the TT genotype in SCDg.23881050T>C, genetic marker genotypes in linkage disequilibrium with the GG genotype in FASN g.12323864A>G and / or genetic marker genotypes in linkage disequilibrium with the AA genotype in FABP4g.62829478A>T can be used to determine whether a sheep or lamb is a 'pure bred' Australian White lamb, specifically a 'pure bred' Tattykeel Australian White (TAW) sheep / lambs.

[0185] In each of the foregoing examples, the gene marker or marker genotype is in LD with (i) the TT genotype in SCD g.23881050T>C or SCD g.23881050T>C, (ii) the GG genotype in FASN g.12323864A>G or FASN g.12323864A>G, or (iii) the AA genotype in FABP4g.62829478A>T or FABP4g.62829478A>T. 2 The value will be at least about 0.60 (e.g., about 0.60, or 0.65, or 0.70, or 0.75, or 0.80, or 0.85, or greater). In one example, the r value of a gene marker or marker genotype that is in LD with (i) the TT genotype in SCD g.23881050T>C or SCD g.23881050T>C, (ii) the GG genotype in FASN g.12323864A>G or FASN g.12323864A>G, or (iii) the AA genotype in FABP4g.62829478A>T or FABP4g.62829478A>T. 2 The value is at least about 0.7.

[0186] The SNP data for the method of the present disclosure can be generated by any method known in the art. Exemplary methods are described herein. In some instances, the method of the present disclosure can include generating SNP genotype data from a nucleic acid sample from an ovine animal. Any method known in the art for generating SNP genotype data from a nucleic acid sample is contemplated.

[0187] In the method disclosed herein, the nucleic acid sample to be queried can be prepared by any suitable biological sample obtained from sheep animals. Therefore, in some instances, the method disclosed herein can include first obtaining a biological sample containing nucleic acid from the animal to be tested. For example, the biological sample can be solid tissue, fiber or biological fluid. Suitable biological fluids include but are not limited to whole blood, serum, plasma, cerebrospinal fluid, lymph fluid, semen and various external secretions of the respiratory tract, intestinal tract and urogenital tract, tears, saliva, milk, leukocytes, etc., and biological fluids, such as cell extracts, cell culture supernatants, fixed tissue samples and fixed cell samples. Suitable solid tissue samples include but are not limited to tissues from ears (including cartilage), muscles, hair bulbs or skin, including tissue biopsies or autopsy samples. Suitable fiber samples from which nucleic acid can be obtained include wool. In some instances, wool fibers can be attached with hair follicles or hair bulbs, which can also be the source of nucleic acid.

[0188] In some instances, the nucleic acid sample is prepared from tissue (e.g., muscle) taken from the longissimus dorsi muscle of the animal. In other instances, the nucleic acid sample is prepared from reproductive material selected from semen, oocytes, or embryos of the animal. In other instances, the nucleic acid sample is prepared from blood or blood fractions obtained from the animal. In other instances, the nucleic acid sample is prepared from fibers (e.g., wool fibers and / or hair follicles attached thereto) obtained from the animal.

[0189] As used herein, the term "nucleic acid" or "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs, and derivatives, fragments, and homologs thereof. Nucleic acid molecules can be single-stranded or double-stranded, but are advantageously double-stranded DNA. "DNA" refers to a polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in single-stranded or double-stranded helical form. This term refers only to the primary and secondary structures of the molecule, and is not limited to any specific tertiary form. Therefore, this term includes double-stranded DNA found especially in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. When discussing the structure of a specific double-stranded DNA molecule, the sequence can be described herein according to the conventional convention of only giving the sequence in the 5' to 3' direction along the non-transcribed DNA chain (i.e., the chain with a sequence homologous to mRNA). "Isolated" nucleic acid molecules are nucleic acid molecules separated from other nucleic acid molecules present in the natural source of nucleic acid (e.g., biological sample). In some instances, genomic DNA is preferred because most SNPs within the genome are located in untranslated regions. However, for the avoidance of doubt, and where the context permits, the term "nucleic acid sample" as used herein encompasses genomic DNA, cDNA and mRNA.

[0190] The term "nucleoside" refers to a base linked to a sugar. The base can be adenine (A), guanine (G) (or its substitute, inosine (I)), cytosine (C) or thymine (T) (or its substitute, uracil (U)). The sugar can be ribose (the sugar of natural nucleotides in RNA) or 2-deoxyribose (the sugar of natural nucleotides in DNA). "Nucleotide" refers to a nucleoside linked to a single phosphate group.

[0191] The method of the present disclosure may be included in the step of determining the presence or absence of one or more SNPs as described herein, separating nucleic acids (e.g., DNA) from biological samples. The term "separation (isolation, isolating or isolate)" as used herein in the context of DNA and applied to the methods and assays for preparing DNA from samples described herein, refers to and encompasses any method or approach known in the art, whereby DNA can be obtained, acquired, prepared, purified or separated so that it is suitable for analysis, amplification and / or sequencing provided in the methods and assays disclosed herein. As known and practiced by any skilled technician, various methods for separating or obtaining nucleic acids can be used. Such methods may include methods for separating DNA in various forms and purity states, and do not necessarily involve or require separation of DNA from all cell debris, proteins, etc.

[0192] Although it is contemplated that the SNP data of the animal can be obtained prior to performing the method, in some instances, the method may further comprise generating SNP genotype data from a nucleic acid sample from an ovine animal. Any method known in the art for generating SNP genotype data from a nucleic acid sample is contemplated. Such methods include, but are not limited to, DNA sequencing (including amplicon sequencing), fluorescence spectroscopy, hybridization analysis based on fluorescence resonance energy transfer (or "FRET"), high throughput screening, mass spectrometry, selective nucleic acid hybridization, DNA amplification (including amplification methods based on polymerase chain reaction (PCR)), RFLP analysis, and size chromatography (e.g., capillary or gel chromatography) and denaturing high performance liquid chromatography (DHPLC), all of which are well known to those skilled in the art.

[0193] In some instances, the presence or absence of the SNP genotype described herein can be determined by sequencing the region across the polymorphic locus in the genomic DNA sample. For example, a suitable DNA amplification reaction can be used to amplify the DNA fragment across the SNP position of interest. "Amplification" or "DNA amplification" as used herein refers to the increase in the number of copies of a particular nucleic acid target of interest, wherein the copy is also referred to as an "amplicon" or "amplification product". Therefore, the term "amplification assay" as used in the context of DNA or nucleic acid amplification is understood to refer to a determination configured to increase the number of copies of a particular nucleic acid or DNA target of interest. Exemplary amplification assays are described herein. Illustrative methods include polymerase chain reaction (PCR), long-range PCR, amplification-retarded mutation system PCR (ARMS PCR), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), nucleic acid sequence-based amplification (NASBA), branching amplification method (RAM), strand displacement amplification (SDA), recombinase-polymerase amplification (RPA), multiple strand displacement amplification (MDA), single primer isothermal amplification (SPIA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), exponential amplification reaction (EXPAR), smart-amplification method (SMAP), etc.

[0194] The amplified product or "amplicon" can then be analyzed to detect the presence or absence of a specific SNP (e.g., SCD g.23881050T>C, FASN g.12323864A>G, or FABP4g.62829478A>T). Detection and analysis of SNPs in DNA amplicons or sequences can be performed using any method known in the art. Exemplary methods include UHT Genotyping System (Beckman / Coulter, Fullerton, CA) (Boyce-Jacino and Goelet patents), Mass Array TM System (Sequenom, San Diego, CA) (Storm, N. et al. (2002) Methods in Molecular Biology. 22:241-262.), BeadArray available from Illumina (San Diego, CA) TM SNP genotyping system (Oliphant, A. et al., (June 2002)) (Supplement to Biotechnology) and TaqMan TM(Applied Biosystems, Foster City, CA). Alternatively, the occurrence of a specific nucleotide at a SNP position can be determined using the DNAMassARRAY system (Signal, San Diego, CA). This system combines a proprietary SpectroChips TM , microfluidics, nanodistribution, biochemistry, and MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry).

[0195] In other examples, detection and analysis of SNPs in DNA amplicons or sequences can be performed by DNA sequencing. Any method or platform known in the art for DNA sequencing is considered to be used in the method of the present disclosure. Exemplary sequencing techniques, platforms and techniques include, but are not limited to, capillary electrophoresis, microarrays, systems based on connection, systems based on polymerase, systems based on hybridization, direct or indirect nucleotide identification systems, pyrophosphate sequencing, detection systems based on ions or pH, systems based on electronic signatures, next generation sequencing (NGS), etc.

[0196] As used herein, the term "next generation sequencing (NGS)" refers to various high-throughput sequencing technologies that parallelize the sequencing process and generate thousands or millions of sequence reads at a time. The NGS parallelization of sequencing reactions can generate hundreds of megabases to gigabases of nucleotide sequence reads in a single instrument run. Unlike conventional sequencing technologies (such as Sanger sequencing) that usually report the average genotype of a collection of molecular aggregates, NGS technology usually digitally tabulates the sequences of many single DNA fragments (sequence reads discussed in detail below), so that low-frequency variants can be detected (e.g., variants present at a frequency of less than about 10%, 5% or 1% in a heterogeneous population of nucleic acid molecules). The term "massively parallel" can also be used to refer to the simultaneous generation of sequence information from many different template molecules by NGS. NGS sequencing platforms may include, but are not limited to, the following: massively parallel signature sequencing (Lynx Therapeutics); 454 pyrosequencing technology (454 Life Sciences / Roche Diagnostics); solid phase, reversible dye terminator sequencing (Solexa / Illumina); SOLiD technology (Applied Biosystems); ion semiconductor sequencing (ion Torrent); and DNA nanoball sequencing (Complete Genomics).Descriptions of some NGS platforms can be found in: Shendure et al., "Next-generation DNA sequencing", Nature, 2008, Vol. 26, No. 10, pp. 135-1145; Mardis, "The impact of next-generation sequencing technology on genetics", Trends in Genetics, 2007, Vol. 24, No. 3, pp. 133-141; Su et al., "Next-generation sequencing and its applications in molecular diagnostics", Expert Rev Mol Diagn, 2011, 11(3):333-43; and Zhang et al., "The impact of next-generation sequencing on genomics", J Genet Genomics, 2011, 11(3):333-43. Genomics)》,201,38(3):95-109.

[0197] In other examples, SNP detection and analysis can also be performed by hybridization-based methods, including dynamic allele-specific hybridization (DASH) genotyping, molecular beacons using specifically engineered single-stranded oligonucleotide probes, SNP microarrays, etc. A variety of enzymes including DNA ligase, DNA polymerase, and nuclease are also used to generate high-fidelity SNP genotyping methods. These methods include restriction fragment length polymorphism (RFLP), PCR-based methods such as four-primer amplification retardation mutation system PCR (ARMS-PCR), oligonucleotide ligation assays, etc.

[0198] After performing the methods disclosed herein, sheep animals identified as having desired meat quality traits (including n-3 LC-PUFA, IMF and FMP) can be selected (e.g., from a population) for further animal breeding activities. Similarly, sheep animals identified as 'pure' Australian white sheep / lambs, specifically Tattykeel Australian white lambs (TAWL) can be selected (e.g., from a population) for further animal breeding activities. Methods of animal breeding are described herein, including the production of reproductive material for animal breeding.

[0199] On the other hand, sheep animals identified as having genotypes at SNP positions SCD g.23881050T>C, FASN g.12323864A>G and / or FABP4g.62829478A>T that are not associated with the desired meat quality traits can be selected (e.g., from the population) for removal from the population. Similarly, sheep animals identified as not being 'purebred' Australian white sheep / lambs can be selected (e.g., from the population) for removal from the population. Removing animals from the population can include physically isolating the animals from the population, selling the animals, or culling the animals.

[0200] SNP Detection System

[0201] The present disclosure also provides a system for determining the occurrence of nucleotides at one or more sheep SNPs, the SNPs selected from SCD g.23881050T>C, FASN g.12323864A>G, and FABP4g.62829478A>T. The system may include a hybridization medium and / or a matrix, the hybridization medium and / or the matrix comprising at least two oligonucleotides, each of which is configured to selectively hybridize to a specific allele of a sheep SNP selected from SCD g.23881050T>C, FASN g.12323864A>G, and FABP4g.62829478A>T. For example, where the system is used to determine the occurrence of nucleotides at two or more (e.g., three) sheep SNPs selected from SCD g.23881050T>C, FASNg.12323864A>G, and FABP4g.62829478A>T, the hybridization medium and / or matrix comprises at least two oligonucleotides for each SNP, wherein each oligonucleotide is configured to selectively hybridize to a specific allele of the corresponding sheep SNP. For example, a solid support may be provided to which the oligonucleotides are directly or indirectly attached.

[0202] The oligonucleotides of the system are used to determine the nucleotide occurrence of sheep SNPs selected from SCD g.23881050T>C, FASN g.12323864A>G and FABP4g.62829478A>T, which the inventors have shown to be associated with meat quality traits such as IMF, FMP and n-3LC-PUFA. The nucleotide occurrence of sheep SNPs can be determined by selecting oligonucleotides that bind at or near the genomic position of the corresponding SNP and are designed to selectively hybridize with specific SNP alleles. The system of the present disclosure generally includes a reagent processing mechanism that can be used to apply a reagent (usually a liquid) to a solid phase carrier. The ability of the oligonucleotides of the system to bind to nucleic acids from the genome of the sheep animal may be affected by the occurrence of nucleotides at the SNP position. The system may include a mechanism that can effectively move the solid phase carrier and a detection mechanism. The oligonucleotides of the system can be detectably labeled so that the binding or labeling of the oligonucleotides can be detected (for example, in the method of the present disclosure). Detectable labels of oligonucleotides are well known in the art and are described herein. Specific non-limiting examples of detectable labels include chemiluminescent labels, fluorescent labels, radioactive labels, enzymes, haptens, or even unique oligonucleotide sequences.

[0203] Detection and analysis of SNPs within a DNA amplicon or sequence using the SNP detection system of the present disclosure can be performed using any method known in the art. Exemplary methods include Ovine SNP50 DNA Analysis Kit (Illumina, California, USA), Axiom TM Sheep Genotyping Array (Thermo Fisher Scientific, MA, USA), Sheep 600K-SNP BeadChip (Illumina, CA, USA), and Sheep InfiniumHD 600K SNP BeadChip (Illumina, CA, USA).

[0204] Test Kits

[0205] The present disclosure also provides a test kit that can be used in the method of the present disclosure. That is, the test kit of the present disclosure can be used to interrogate SNPs selected from SCD g.23881050T>C, FASN g.12323864A>G and / or FABP4g.62829478A>T, and thereby determine the genotype of the animal at the corresponding SNP position. This SNP genotype data can then be used to determine whether the sheep animal has or will have the desired FMP, IMF and / or n-3 LC-PUFA, and / or determine whether the sheep animal or its part is a 'pure' Australian white lamb or its part, specifically Tattykeel Australian white lamb (TAWL) or its part.

[0206] In one example, the present disclosure provides a test kit comprising a plurality of oligonucleotide primers and / or probes configured to distinguish alleles at one or more SNP loci in a biological sample taken from an ovine animal, wherein the SNP loci are selected from (i) SCD g.23881050T>C or r in linkage disequilibrium with SCDg.23881050T>C; 2 SNPs with r values ​​≥ 0.7, (ii) FASN g.12323864A>G or in linkage disequilibrium with FASNg.12323864A>G 2 SNPs with r values ​​≥ 0.7 and / or FABP4g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7.

[0207] As used herein, the term "primer" or similar terms refers to an enzymatically extendable oligonucleotide comprising a defined sequence, which is designed to selectively hybridize with a complementary primer-specific portion of a target (or template) nucleic acid sequence in an antiparallel manner. This is referred to as "annealing" of a primer to its target. Primers, which are generally provided in molar excess relative to their target polynucleotide sequence, initiate template-dependent enzymatic DNA synthesis and amplification of the target sequence. Primers are generally used when polymerase chain reaction (PCR) is performed. Primer oligonucleotide sequences do not need to have 100% complementarity with their template subsequences for primer extension to occur. In this regard, primers with less than 100% complementarity can be sufficient for hybridization and enzymatic (e.g., polymerase) extension, as long as there is enough complementarity to hybridize with the target sequence, and the penultimate base at the 3' end of the primer can be base-paired with the template nucleic acid. Primers are preferably, but not necessarily, synthetic, and are generally about 10 to about 100 nucleotides in length. However, generally primers contain about 15-26 consecutive nucleotides.

[0208] The term "probe" or "probes" refers to an oligonucleotide sequence used to detect identical, similar or complementary nucleic acid sequences by hybridization. Oligonucleotides can have different lengths, but are generally of sufficient length to allow selective hybridization, including selective hybridization to a specific target sequence.

[0209] Oligonucleotide sequences used as primers or probes can be labeled with fluorescent dyes (or fluorophores) to facilitate detection of amplified products. In some instances, amplified products can be detectably labeled during the nucleic acid amplification process. For example, one or more oligonucleotide primers used in the amplification assay can be detectably labeled with "fluorescent markers" or "fluorescent dyes" (also referred to as "fluorophores") so that the fluorescent dyes are incorporated into the amplified products. As used herein, the term "fluorescent marker" includes a signal transduction portion that transmits information through the fluorescence absorption and / or emission characteristics of one or more molecules. Such fluorescent characteristics include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer, etc.

[0210] Exemplary fluorescent dyes that can be conjugated or linked to the oligonucleotide primers include, but are not limited to, fluorescein / Oregon Green, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein, tetramethylrhodamine, Texas Red, dansyl, AlexaFluor 488, BODIPY FL, Lucifer Yellow, and Alexa Fluor 405 / Cascade Blue fluorophores. Fluorescent labels and their attachment to oligonucleotides are described in many reviews, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, 9th ed. (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd ed. (Stockton Press, New York, 1993); Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Wetmur (1991), Critical Reviews in Biochemistry and Molecular Biology, 26:227-259; etc.

[0211] Commercially available fluorescent nucleotide analogs that are easily incorporated into polynucleotides during nucleic acid amplification include, for example, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ, USA), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED TM -5-dUTP、CASCADEBLUE TM -7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREEN TM -5-dUTP、OREGON GREENR TM 488-5-dUTP, TEXAS RED TM -12-dUTP, BODIPYTM 630 / 650-14-dUTP, BODIPYTM 650 / 665-14-dUTP, ALEXA FLUOR TM 488-5-dUTP、ALEXAFLUOR TM 532-5-dUTP、ALEXA FLUOR TM 568-5-dUTP、ALEXA FLUOR TM 594-5-dUTP、ALEXAFLUOR TM 546-14-dUTP, fluorescein-12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED TM -5-UTP, mCherry, CASCADE BLUE TM -7-UTP, BODIPYTM FL-14-UTP, BODIPYTM TR-14-UTP, BODIPYTM TR-14-UTP, RHODAMINE GREEN TM -5-UTP、ALEXA FLUOR TM 488-5-UTP、ALEXA FLUOR TM546-14-UTP (Molecular Probes, Eugene, Oregon). Custom synthesis protocols for nucleotides with other fluorophores are available. Henegariu et al., "Custom Fluorescent-Nucleotide Synthesis as an Alternative Method for Nucleic Acid Labeling," Nature Biotechnol. 18:345-348 (2000).

[0212] Other fluorophores that can be attached post-synthesis include, among others, ALEXA FLUOR TM 350. ALEXA FLUOR TM 532. ALEXA FLUOR TM 546. ALEXA FLUOR TM 568. ALEXA FLUOR TM 594. ALEXA FLUOR TM 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, Lissamine Rhodamine B, Ocean Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, DYLIGHT TM DYES (e.g., DYLIGHT TM 405,DYLIGHT TM 488,DYLIGHT TM 549,DYLIGHT TM 594,DYLIGHT TM 633,DYLIGHT TM 649,DYLIGHT TM 680,DYLIGHT TM 750,DYLIGHT TM800, etc.) (available from Thermo Fisher Scientific, Rockford, Illinois), Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), and Cy2, Cy3.5, Cy5.5, and Cy7 (available from Amersham Biosciences, Piscataway, NJ USA, etc.).

[0213] FRET tandem fluorophores such as PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7; as well as PE-Alexa dyes (610, 647, 680) and APC-Alexa dyes may also be used.

[0214] Metallic silver particles can be coated onto the array surface to enhance the signal of fluorescently labeled oligonucleotide sequences bound to the array. Lakowicz et al. (2003) BioTechniques 34:62.

[0215] Labeling can also be done with quantum dots, as disclosed in the following patents and patent publications: U.S. Pat. Nos. 6,322,901; 6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392; 2002 / 0045045; 2003 / 0017264, etc.

[0216] The amplification product produced in the DNA amplification assay described herein can also be detected by characteristic size, for example, on polyacrylamide or agarose gel stained with ethidium bromide. Alternatively, the amplified target sequence can be detected by a detection probe (as described herein), which is an oligonucleotide labeled with a detectable label, which hybridizes specifically with the target sequence in the nucleic acid under conditions that allow hybridization, thereby allowing detection of the target sequence or amplification product. In an example, at least one labeled detection probe can be used for detecting the allele-specific amplification product by hybridization. Preferably, the detection probe is designed to hybridize with the sequence in the target sequence between the amplification primers, that is, it is an internal detection probe hybridized with the amplification product. The probe can be labeled to promote detection as described herein. The exemplary method of labeled oligonucleotides is described in the context of amplification primers herein, and should be modified mutatis mutandis, applied to each example of the detection probe describing the test kit.

[0217] Primers and probes are considered to be "selectively hybridizable" or "capable of selective hybridization" if they are able to specifically hybridize to a nucleic acid template or variants thereof (e.g., a specific SNP allele), or are able to specifically initiate a polymerase chain reaction under stringent conditions. Typical hybridization and washing conditions are described, for example, in Sambrook et al., supra and Nucleic Acid Hybridization, supra. Washing conditions that allow for reduced stringency of up to about 25-30% base pair mismatches include, for example: 2×SSC, 0.1% SDS, room temperature twice for 30 minutes each; then 2×SSC, 0.1% SDS, 37° C., once for 30 minutes; then 2×SSC room temperature twice for 10 minutes each. The selection of primers for a typical polymerase chain reaction (PCR) under standard conditions is described, for example, in Saiki et al., (1988) Science 239:487-491. Preferably, the primers and probes of the present disclosure are capable of hybridizing under stringent conditions. That is, it is capable of selectively hybridizing to its cognate template under stringent conditions.

[0218] As used herein, the term "capable of hybridizing under stringent conditions" etc. refers to the ability of a primer and / or probe to hybridize or anneal with its homologous template under stringent conditions. Stringent hybridization conditions generally allow a nucleic acid molecule with at least 70% nucleic acid sequence identity to hybridize with a nucleic acid molecule used as a probe in a hybridization reaction. The hybridization of a probe or primer with its homologous template sequence can be carried out under stringent conditions (e.g., high temperature and / or low salt content), which are often unfavorable for the hybridization of different nucleotide sequences. Alternatively, the hybridization of a probe or primer with its homologous template sequence can be carried out under conditions that reduce stringency, such as low temperature and / or high salt content that are often conducive to the hybridization of different nucleotide sequences. Low stringency hybridization conditions can be followed by high stringency conditions or medium stringency conditions to increase the selectivity of primers and / or probes combined with homologous templates. Hybridization conditions may further include reagents, such as but not limited to dimethyl sulfoxide (DMSO) or formamide, to further be unfavorable for the hybridization of different nucleotide sequences. Suitable hybridization protocols may involve, for example, hybridization in 6×SSC (where 1×SSC contains 0.015 M sodium citrate and 0.15 M sodium chloride) at 65° C. in aqueous solution, followed by washing with 1×SSC at 65° C. Formulas for calculating appropriate hybridization and washing conditions to achieve hybridization that allows 30% or less mismatches between two nucleic acid molecules are disclosed, for example, in Meinkoth et al. (1984) Anal. Biochem. 138: 267-284; the contents of which are incorporated herein by reference in their entirety. Protocols for hybridization techniques are well known to those skilled in the art, and standard molecular biology manuals may be referenced to select suitable hybridization protocols without undue experimentation. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, the contents of which are incorporated herein by reference in their entirety.

[0219] Stringent conditions may be those in which the salt concentration is less than about 1.5 M sodium ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts), from about pH 7.0 to about pH 8.3, and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37°C using a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1-2×SSC at 50°C to 55°C. Exemplary moderate stringency conditions include hybridization at 37°C in 40% to 45% formamide, 1 M NaCl, 1% SDS, and washing in 0.5-1×SSC at 55°C to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C.

[0220] In one example, the test kit comprises:

[0221] One or more oligonucleotide primers and / or probes configured to distinguish between allele C and allele T at the SNP locus SCD g.23881050T>C;

[0222] One or more oligonucleotide primers and / or probes configured to distinguish between allele G and allele G at the SNP locus FASN g.12323864A>G; and / or

[0223] One or more oligonucleotide primers and / or probes configured to distinguish between allele A and allele T at the SNP locus FABP4g.62829478A>T.

[0224] In one example, the test kit may include one or more oligonucleotide primers and / or probes configured to distinguish between allele C and allele T at the SNP locus SCDg.23881050T>C. According to this example, the test kit may further include one or more oligonucleotide primers and / or probes configured to distinguish between allele G and allele G at the SNP locus FASN g.12323864A>G. Alternatively or additionally, the test kit may further include one or more oligonucleotide primers and / or probes configured to distinguish between allele A and allele T at the SNP locus FABP4g.62829478A>T. In a specific example, the test kit comprises one or more oligonucleotide primers and / or probes configured to distinguish between allele C and allele T at the SNP locus SCD g.23881050T>C, one or more oligonucleotide primers and / or probes configured to distinguish between allele G and allele G at the SNP locus FASN g.12323864A>G, and one or more oligonucleotide primers and / or probes configured to distinguish between allele A and allele T at the SNP locus FABP4g.62829478A>T.

[0225] In one example, the oligonucleotide primers in the test kit are configured to distinguish allele C and allele T at the SNP locus SCD g.23881050T>C by amplification-retarded mutation system polymerase chain reaction (ARMS PCR). Exemplary oligonucleotide primers configured to distinguish allele C and allele T at the SNP locus SCD g.23881050T>C by ARMS PCR include:

[0226] A primer comprising the sequence shown in SEQ ID NO: 1 or a sequence substantially identical thereto (named “SCDg.23881050T>C_Forward Internal”);

[0227] A primer comprising the sequence shown in SEQ ID NO:2 or a sequence substantially identical thereto (designated as "SCDg.23881050T>C_reverse internal");

[0228] A primer comprising the sequence shown in SEQ ID NO: 3 or a sequence substantially identical thereto (designated as “SCDg.23881050T>C_forward external (a)”);

[0229] A primer comprising the sequence shown in SEQ ID NO:4 or a sequence substantially identical thereto (designated as "SCDg.23881050T>C_reverse external (a)");

[0230] A primer comprising the sequence shown in SEQ ID NO:5 or a sequence substantially identical thereto (designated as "SCDg.23881050T>C_Forward Exterior (b)"); and

[0231] Primers comprising the sequence shown in SEQ ID NO: 6 or a sequence substantially identical thereto (named "SCDg.23881050T>C_reverse outer (b)"). These primers are also collectively referred to as "SCD g.23881050T>C primers".

[0232] In one example, one or more of the SCD g.23881050T>C_Forward Internal, SCD g.23881050T>C_Reverse Internal, SCD g.23881050T>C_Forward External (a), SCD g.23881050T>C_Reverse External (a), SCDg.23881050T>C_Forward External (b) and SCD g.23881050T>C_Reverse External (b) can be at least 85% identical to the primer sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively. In one example, one or more of the SCD g.23881050T>C_Forward Internal, SCD g.23881050T>C_Reverse Internal, SCD g.23881050T>C_Forward External (a), SCD g.23881050T>C_Reverse External (a), SCD g.23881050T>C_Forward External (b) and SCDg.23881050T>C_Reverse External (b) can be at least 90% identical to the primer sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively. In one example, one or more of the SCDg.23881050T>C_Forward Internal, SCDg.23881050T>C_Reverse Internal, SCD g.23881050T>C_Forward External (a), SCD g.23881050T>C_Reverse External (a), SCD g.23881050T>C_Forward External (b) and SCD g.23881050T>C_Reverse External (b) can be at least 95% identical to the primer sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.In one example, one or more of the SCD g.23881050T>C_Forward Internal, SCD g.23881050T>C_Reverse Internal, SCDg.23881050T>C_Forward External(a), SCD g.23881050T>C_Reverse External(a), SCD g.23881050T>C_Forward External(b) and SCD g.23881050T>C_Reverse External(b) can be at least 96% identical to the primer sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQID NO:6, respectively. In one example, one or more of the SCD g.23881050T>C_Forward Internal, SCD g.23881050T>C_Reverse Internal, SCD g.23881050T>C_Forward External (a), SCD g.23881050T>C_Reverse External (a), SCD g.23881050T>C_Forward External (b) and SCD g.23881050T>C_Reverse External (b) can be at least 97% identical to the primer sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively. In one example, one or more of the SCD g.23881050T>C_Forward Internal, SCD g.23881050T>C_Reverse Internal, SCDg.23881050T>C_Forward External(a), SCD g.23881050T>C_Reverse External(a), SCD g.23881050T>C_Forward External(b) and SCD g.23881050T>C_Reverse External(b) can be at least 98% identical to the primer sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQID NO:6, respectively.In one example, one or more of the SCD g.23881050T>C_Forward Internal, SCD g.23881050T>C_Reverse Internal, SCD g.23881050T>C_Forward External (a), SCD g.23881050T>C_Reverse External (a), SCD g.23881050T>C_Forward External (b) and SCD g.23881050T>C_Reverse External (b) can be at least 99% identical to the primer sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively. In one example, one or more of the SCD g.23881050T>C_Forward Internal, SCD g.23881050T>C_Reverse Internal, SCDg.23881050T>C_Forward External(a), SCD g.23881050T>C_Reverse External(a), SCD g.23881050T>C_Forward External(b) and SCD g.23881050T>C_Reverse External(b) can be 100% identical to the primer sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQID NO:6, respectively. In a specific example, SCD g.23881050T>C_forward internal, SCD g.23881050T>C_reverse internal, SCD g.23881050T>C_forward external(a), SCD g.23881050T>C_reverse external(a), SCD g.23881050T>C_forward external(b) and SCD g.23881050T>C_reverse external(b) comprise the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0233] The final concentration of each SCD g.23881050T>C primer in the test kit can vary. In one example, the corresponding primers can each be present at a final concentration in the range of about 0.1 μM to about 2 μM. In one example, each primer is present at the same final concentration. In another example, one or more primers are present in the test kit at different final concentrations (relative to the other primers in the test kit). For example, the outer primer and the inner primer can be provided at a concentration ratio of about 2: 1.

[0234] According to some examples, two or more of the SCD g.23881050T>C primers are packaged together as a mixture. In other examples, the SCD g.23881050T>C primers are packaged individually.

[0235] The test kit may also contain one or more or each of the following:

[0236] at least one DNA polymerase;

[0237] dinucleotide triphosphates (dNTPs);

[0238] magnesium salts; and

[0239] Buffer.

[0240] Technicians will appreciate that the selection of DNA polymerase will depend on the type of DNA amplification assay adopted.Technicians can select appropriate enzyme based on the selection of amplification assay.For example, according to the example in which the test kit is configured for the PCR amplification assay that depends on thermal conditions, a thermostable DNA polymerase (i.e., a DNA polymerase derived from a thermophilic organism) can be included in the test kit.Many different thermostable DNA polymerases are known to those skilled in the art and are considered herein.The DNA polymerase is preferably a thermophilic DNA polymerase with strong strand displacement activity.Non-limiting examples of thermostable DNA polymerases suitable for inclusion in the test kit disclosed herein include naturally occurring A-type DNA polymerases (also referred to as A family DNA polymerases). A-type DNA polymerases are classified based on amino acid sequence homology to Escherichia coli polymerase I (Braithwaite and Ito, (1993) Nuc. Acids. Res. 21:787-802) and include Escherichia coli pol I, Thermus aquaticus DNA pol I (Taq polymerase), Thermus flavus DNA pol I, Streptococcus pneumoniae DNA pol I, Bacillus stearothermophilus pol I, bacteriophage polymerase T5, phage polymerase T7, mitochondrial DNA polymerase pol γ, and additional polymerases discussed below. A-family DNA polymerases are commercially available and include Taq polymerase (New England BioLabs), E. coli pol I (New England BioLabs), E. coli pol I Klenow fragment (New England BioLabs), T7 DNA polymerase (New England BioLabs), and Bacillus stearothermophilus (Bst) DNA polymerase (New England BioLabs).

[0241] According to the different examples that wherein test kit is configured for isothermal PCR amplification assay, the mesophilic enzyme for amplifying nucleic acid under isothermal conditions can be included in the test kit. The selection of mesophilic enzyme will depend on the selection of used isothermal amplification assay. The technician can select appropriate enzyme based on the selection of isothermal amplification assay. For example, when the test kit is configured for LAMP assay, the test kit will include one or more DNA polymerases suitable for LAMP assay. The DNA polymerase is preferably a thermophilic DNA polymerase with strong chain displacement activity. For example, the DNA polymerase can be selected from the group consisting of: Bst DNA polymerase, Bsm DNA polymerase, Gst DNA polymerase, SD DNA polymerase and a combination thereof.

[0242] Those skilled in the art will appreciate that the amount of DNA polymerase included in the test kit will depend on the number of reactions to be performed using the test kit. In this regard, the test kit can be scaled to accommodate any number of reactions. However, in a specific example, the test kit can include about 8 units of DNA polymerase in each reaction. However, those skilled in the art will appreciate that this can vary depending on the efficiency of the polymerase and the specific DNA amplification assay employed.

[0243] As described herein, the reagent mixture will contain dNTPs, which are building blocks for synthesizing new DNA during DNA amplification assays. The dNTPs will contain dATP, dCTP, dGTP, and dTTP. These four dNTPs are preferably present in equimolar amounts to obtain optimal base incorporation during DNA amplification assays. This is typically a final concentration of about 0.2 mM for each dNTP. However, the relative molar amounts and final concentrations can be varied as desired. For example, in the presence of high levels of Mg 2+ In some cases, higher concentrations of dNTPs may be required because Mg 2+ Binds to dNTPs and reduces their availability for incorporation into new DNA strands during amplification. However, the skilled artisan will also appreciate that too high a concentration of dNTPs may also inhibit amplification. For example, a DNA amplification assay performed using a test kit may contain dNTPs at a final concentration of about 0.8 mM to about 2 mM. In some instances, the test kit may be configured so that each assay contains dNTPs within this concentration range.

[0244] The test kit may also contain a magnesium salt. In this regard, the magnesium ion (Mg 2+ ) acts as a cofactor for the activity of DNA polymerase by enabling the incorporation of dNTPs during polymerization. The magnesium ion at the enzyme active site catalyzes the formation of a phosphodiester bond between the 3'-OH of the primer and the phosphate group of the dNTP. 2+The formation of a complex between a primer or probe and a DNA template is promoted by stabilizing the negative charge on its phosphate backbone. The magnesium salt can be provided in any suitable form known to those skilled in the art. For example, the magnesium salt can be magnesium chloride (MgCl2) or magnesium sulfate (MgSO4). In one example, the magnesium salt in the reagent mixture is MgSO4. The concentration of the magnesium salt can be optimized according to the conditions of the isothermal amplification assay, for example by titration. In this regard, too low Mg may increase the activity of the DNA polymerase. 2+ On the other hand, too high a Mg concentration may result in little or no amplification product. 2+ Concentrations of Mg may result in nonspecific amplification products due to increased stability of the primer-template complex and increased replication errors due to dNTP misincorporation. 2+ Typical final concentrations of Mg are in the range of 1-10 mM. Therefore, the test kit can be configured so that each assay contains Mg in this concentration range. 2+ .

[0245] As described herein, the reagent mixture can also include a buffer to ensure a suitable chemical environment for DNA polymerase activity during DNA amplification assays. There are many buffers known in the art and commercially available for nucleic acid amplification assays, and these are also considered herein. The selection of buffer can depend on the selection of the enzyme for amplification (e.g., the selection of DNA polymerase). For example, there is a Bst DNA polymerase (e.g., Bst 2.0 In the case of a Bst DNA polymerase, the reagent mixture may include an amplification buffer suitable for the Bst DNA polymerase (e.g., 10X isothermal amplification buffer (Bst 2.0)).

[0246] In some examples, the test kit can be stored at frozen temperatures (eg, -20°C or lower).

[0247] In other examples, it is contemplated that certain components of the test kit (e.g., primers and / or probes) may be provided in a dried form, such as lyophilized. This may facilitate storage and transportation at ambient temperature. According to examples in which certain reagents in the test kit are lyophilized, these reagents may further comprise a cryoprotectant. Suitable cryoprotectants for lyophilization are known in the art and are contemplated herein. The cryoprotectant may be a sugar selected from the group consisting of sucrose, trehalose, glucose, galactose, maltose, mannitol, lactose, and derivatives thereof. The cryoprotectant may be present in an amount of about 7% w / v to about 8% w / v.

[0248] The test kit may also contain one or more assay positive controls.

[0249] As described above, the test kit components can be packaged together as separate components. The test kit of the present disclosure may further include instructions for use. The instructions for use may provide guidance for using the test kit to determine the genotype of the animal at the corresponding SNP position according to the method of one or more embodiments of the present disclosure. This SNP genotype data can then be used to determine whether the sheep animal has or will have the desired FMP, IMF and / or n-3 LC-PUFA, and / or determine whether the sheep animal or its part is a 'pure bred' Australian white lamb or its part, specifically Tattykeel Australian white lamb (TAWL) or its part.

[0250] Animal breeding and reproductive material

[0251] As described herein, after performing the methods of the present disclosure, sheep animals identified as having desirable meat quality traits (including n-3 LC-PUFA, IMF and FMP) can be selected (e.g., from a population) for animal breeding activities. Similarly, sheep animals identified as 'pure' Australian white sheep / lambs, specifically Tattykeel Australian white lambs (TAWL) can be selected for animal breeding activities.

[0252] Thus, in one example, the present disclosure provides a method of breeding sheep animals, wherein the sheep animals have been identified as having a desired n-3 LC-PUFA, IMF, FMP or any combination thereof according to the methods of the present disclosure. In other examples, the methods of breeding sheep animals of the present disclosure comprise:

[0253] (i) selecting one or more animals having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, as compared to an average population of animals of the species based on the results of the methods described herein, which identify one or more sheep animals having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, as compared to an average population of animals of the species; and

[0254] (ii) breeding from the selected animal to produce one or more offspring therefrom. In some examples, the method of breeding animals comprises the step of performing a method as described herein to identify one or more sheep animals having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to a general population of animals of the species. That is, prior to performing the selection step.

[0255] As described herein, an animal having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof will comprise one or more SNP genotypes selected from the group consisting of: TT genotype in SCD g.23881050T>C, GG genotype in FASN g.12323864A>G, and / or AA genotype in FABP4g.62829478A>T. In this regard, the inventors have shown that (i) the TT genotype in SCD g.23881050T>C is associated with desirable IMF and n-3 LC-PUFAs compared to the TA or TT genotypes in SCD g.23881050T>C, (ii) the GG genotype in FASNg.12323864A>G is associated with desirable FMPs, long chain saturated fatty acids, and monounsaturated fatty acids compared to the GA or AA genotypes in FASN g.12323864A>G, and (iii) the AA genotype in FABP4g.62829478A>T is associated with IMF compared to the AG or GG genotypes in FABP4g.62829478A>T. Thus, animals bred to have desirable n-3 LC-PUFAs, IMFs, FMPs, or any combination thereof, will comprise one or more of the above-described SNP genotypes.

[0256] Also provided is a method of breeding Australian White Lamb (AWL), the method comprising:

[0257] (i) selecting one or more animals identified as having a SNP genotype indicative of an AWL genetic characteristic; and

[0258] (ii) breeding from the selected animal to produce one or more offspring therefrom. In some examples, the method of breeding for AWL further comprises the step of performing a method as described herein on one or more animals to thereby identify one or more sheep animals having a SNP genotype indicative of an AWL genetic characteristic. That is, prior to performing the selection step.

[0259] The SNP genotype indicating the AWL genetic characteristic comprises (i) a TT genotype in SCD g.23881050T>C or a genotype in linkage disequilibrium with the TT genotype; (ii) a GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype. In some examples, the SNP genotype indicating the AWL genetic characteristic comprises one or more of the following: a TT genotype in SCD g.23881050T>C, a GG genotype in FASN g.12323864A>G, or an AA genotype in FABP4g.62829478A>T. In a specific example, the SNP genotype indicating the AWL genetic characteristic comprises a TT genotype in SCD g.23881050T>C.

[0260] According to the foregoing examples, the present disclosure also provides a sheep animal bred according to the method described herein.

[0261] Animal breeding can be carried out by any means known to the skilled person. For example, breeding can be carried out by natural mating of male animals to female animals or using artificial or assisted reproductive technology (ART). Artificial reproductive technology can include artificial insemination, in vitro fertilization (IVF), embryo transfer (ET), multiple ovulation embryo transfer (MOET), young animal in vitro embryo transfer (JIVET) or a combination thereof. In some instances, male animals and female animals (or their corresponding reproductive materials) have been selected using the methods disclosed herein. That is, both male animals and female animals (or their corresponding reproductive materials) are selected based on their desired meat quality traits (including n-3LC-PUFA, IMF and FMP) and / or are 'purebred' Australian white sheep / lambs, specifically Tattykeel Australian white lambs (TAWL). In other examples, only one of the males and females (or their corresponding reproductive material) is selected based on their having desirable meat eating quality traits (including n-3 LC-PUFA, IMF and FMP) and / or being 'pure' Australian White sheep / lambs, specifically Tattykeel Australian White Lambs (TAWL). In the case of the latter example, it may be desirable to introgress one or more SNP genotypes associated with desirable meat eating quality traits (including n-3 LC-PUFA, IMF and FMP) into a population of sheep animals where the SNP genotype associated with the desirable meat eating quality traits is not present or is present at a lower frequency than desired.

[0262] Reproductive or regenerative material may also be obtained from animals identified according to the methods of the present disclosure as having desirable n-3 LC-PUFA, IMF, FMP or any combination thereof, and / or animals identified according to the methods of the present disclosure as 'purebred' Australian White sheep / lambs, specifically Tattykeel Australian White Lambs (TAWL).

[0263] "Reproductive material" as described herein should include oocytes, embryos and / or semen. Similarly, "regenerative material" as used herein can be oocytes, embryos and / or semen, but can further include primordial germ cells (PGCs) that produce oocytes or sperm. Therefore, the present disclosure contemplates the preparation of such reproductive or regenerative materials by animals selected using the methods of the present disclosure for future breeding activities. Reproductive or regenerative materials can be used fresh or frozen and stored.

[0264] Examples

[0265] Example 1: Identification of SNP DNA markers by NGS to select for intramuscular fat, fat melting point ω-3 long-chain polyunsaturated fatty acids and meat eating quality in TattyKeel Australian White (TAW) MARGRA lambs

[0266] The inventors used targeted NGS of functional SNPs in several lipid metabolism genes such as stearoyl-CoA desaturase (SCD), fatty acid binding protein-4 (FABP4) and fatty acid synthase (FASN) to identify unique DNA marker signatures for TAW genetics, breeding and selection programs for meat eating quality. This was performed on muscle tissue obtained from live lambs using a minimally invasive thoracic lumbar longissimus muscle biopsy sampling technique for selection of ω-3 long-chain polyunsaturated fatty acids (n-3LC-PUFA), intramuscular fat (IMF) and fat melting point (FMP) traits.

[0267] Materials and Methods

[0268] Animals and experimental design

[0269] Experimental design for selection, breeding and evaluation of n-3 LC-PUFA, IMF and FMP in Tattykeel Australian White (TAW) sheep Figure 1 Shown in.

[0270] Three composite generations - parent, first (F1) and second (F2) composite lambs - were bred, reared and maintained under the same management at Tattykeel Australian White Stud Farm in Black Springs, Oberon, New South Wales, Australia. The parent composite consisted of 47 rams mated to 500 ewes, and after evaluating their longissimus dorsi muscle biopsies, the minimum thresholds for health-promoting n-3 LCPUFA, IMF and FMP were set at 30 mg / 100 g, 3.0% and 35°C, respectively. The first 10 rams and 200 ewes were selected and mated to produce 150 offspring, whose muscle biopsies were tested in the laboratory for n-3 LC-PUFA, IMF, FMP, and genomic DNA sequencing at 10 months of age, and then completed at a commercial feedlot. Horned Dorset and Texel were used as positive controls, and Rambouillet as negative control in laboratory evaluation of extracted genomic DNA, polymerase chain reaction products, and next-generation sequencing procedures.

[0271] Muscle Biopsy Sampling Procedure

[0272] The biopsy procedure for sampling the longissimus dorsi muscle from the 12th-13th rib was first described in cattle [Malau-Aduli, AEO et al. 1998. Journal of Animal Science (J. Anim. Sci.) 76, 766–773] and modified in sheep [Pewan, SB et al. 2020. Antioxidants. 9, 1118]. This method is also described in more detail in Pewan, SB et al. 2020. Antioxidants. 9, 1118.

[0273] Determine intramuscular fat

[0274] Details of the procedures for laboratory analysis of intramuscular fat are described in more detail in Pewan, SB et al. 2020. Antioxidants. 9, 1118, Holman, B. et al. 2014. Int. J. Vet. Med. Res. Rep. 1-9 and Flakemore, AR et al. 2014. Int. J. Nutr. Food Sci. 3, 203-209.

[0275] Determining the melting point of fat

[0276] Details of laboratory analysis of fat melting point are described in more detail in Pewan, SB et al. 2020. Antioxidants. 9, 1118, Holman, B. et al. 2014. International Veterinary Medicine: Research and Reports 1-9 and Flakemore, AR et al. 2014. International Journal of Food Sciences and Nutrition 3, 203-209.

[0277] Determine fatty acid composition

[0278] Fatty acid composition, including n-3 LC-PUFA analysis of longissimus dorsi muscle biopsy samples, was analyzed by Malau-Aduli, AEO et al. 2016. Anim. Prod. Sci. 56, 2122-2132 based on a modified method in Bligh, EG et al. 1959. Can. J. Biochem. Phys. 37, 911-917, Miller, MR et al. 2006. Lipids 2006, 41, 865-876 and Clayton, E. 2014. Graham Centre Monograph 4; Charles Sturt University: Wagga-Wagga, NSW, Australia using a well-described gas chromatography-mass spectrometry procedure. The details have been described in more detail in Pewan, SB et al. 2020. Antioxidants. 9, 1118.

[0279] Blood collection and genomic DNA extraction

[0280] About 10 mL of blood was collected from Tattykeel Australian White, Hornless Dorset and Texel (positive control) lambs of the same age and under the same management conditions by puncturing the jugular vein into a vacuum blood collection tube containing EDTA. The blood samples were stored at -80 ° C until genomic DNA (gDNA) can be extracted. According to the manufacturer's protocol, gDNA was extracted from 2 mL of blood using NucleoSpin blood kit (Macherey-Nagel GmbH and Co.KG, Neumann-Neander-Str.6-8.52355Duren,Germany, Neumann-Neander-Str.6-8.52355Duren,Germany). The gDNA yield was quantified using NanoDrop ND-1000 spectrophotometer (NanoDrop, Thermo Fisher Scientific Australia Pty Ltd, Scoresby, Victoria, Australia).

[0281] Primer design for FASN, FABP4 and SCD

[0282] All primers were designed using the Geneious Prime software program 2020v.2.2 (http: / / www.geneious.com). A targeted candidate gene approach for lipid metabolism genes (FASN, FABP4, and SCD) was utilized. The single coding sequence of each gene of FASN, FABP4, and SCD of sheep breeds stored in the National Center for Biotechnology Information (NCBI) database (Genbank) was used as a reference point. In order to amplify the 18kb FASN gene (accession number: NC_040262.1), the gene sequence was divided into 3 overlapping fragments of 8.5kb each (FASN1, FASN2, and FASN3) using a long-range PCR method, containing approximately 91% of the total gene sequence. For the 4kb FABP4 (NC_040260.1) and 12kb SCD (NC_040273.1) gene fragments, individual primer sets were designed as shown in Table 1. All primers were synthesized at Integrated DNA Technologies Pte. Ltd., Melbourne, Australia.

[0283] Long-range PCR

[0284] Due to the different fragment lengths and DNA compositions, it was necessary to use 3 different long-range PCR methods to amplify the FASN, FABP4 and SCD genes. During optimization, all 3 methods were tested for all 3 genes, but only the best performing combination was utilized.

[0285] FASN amplification

[0286] FASN PCR amplification assays were performed using TakaRa PrimeSTAR GXL Master Mix (TaKaRa Bio Inc., Kusatsu, Shiga, Japan). PCR reaction assays were set up in a total volume of 50 μL containing 10 μL 5× TakaRa PrimeSTAR GXL buffer, 200 μM TaKaRa dNTP mix, 1.25 units of TaKaRa PrimeSTAR GXL DNA polymerase, 0.2 μM of each primer (IDT, Melbourne, Australia), and 100 ng of DNA template. The following conditions were used in SimpliAmp TM PCR was performed in a thermal cycler (Thermo Fisher Scientific, Melbourne, Australia) with a 2-step protocol: initial denaturation at 98°C for 1 min (1 cycle); denaturation at 98°C for 10 s; annealing / extension at 68°C for 10 min, 30 cycles. Figure 2-4 As depicted, PCR success was checked in 0.8% agarose gel electrophoresis.

[0287] FABP4 and SCD amplification

[0288] For the FAPB4 gene, use Platinum TM SuperFi TM II PCR Master Mix (Thermo Fisher Scientific, Australia), while for the SCD gene, Hot Start II High Fidelity PCR Master Mix (Thermo Fisher Scientific, Australia) was used under the same PCR conditions. Amplification reactions were performed in a total volume of 50 μL, containing 25 μL 2× Platinum TM SuperFi TM II PCR Master Mix or Phusion Hot Start II High Fidelity PCR Master Mix (Thermo Fisher Scientific, Australia), 0.5 μM of each primer (IDT, Australia) and 100 ng of DNA template. The following conditions were used in SimpliAmp TMPCR was performed in a thermal cycler (Thermo Fisher Scientific, Australia) with a 3-step protocol: initial denaturation at 98°C for 1 min (1 cycle); denaturation at 98°C for 15 s; annealing at 60°C (FABP4) / 65°C (SCD) for 15 s; extension at 72°C for 9 min; final extension at 72°C for 9 min; and hold at 4°C for 35 cycles. Figure 5 and 6 As depicted, PCR success was checked in 0.8% agarose gel electrophoresis.

[0289] PCR cleanup

[0290] Sera-Mag TM SpeedBeads were prepared according to Faircloth, BC et al. 2014. Illumina Library Prep Protocol. Version 2.1 and used to clean PCR products using a Zephyr NGS workstation (Caliper Lifesciences, Perkin-Elmer) and quantified using the Promega dsDNA Quantifluor system kit (reference number: E2670,00002484139) on an Enspire workstation (Perkin-Elmer). Five different PCR products were combined at approximately 0.4 nM using the Quantifluor dsDNA system (Promega, Madison, WI, USA) to ensure uniform coverage during sequencing. Products were normalized to 2 ng / μL using 10 mM Tris-HCl (pH 8.0). Final dilution to 0.2 ng / μL was performed using 10 mM Tris-HCl (pH 8.0) in preparation for library preparation using Illumina NexteraXT DNA and final accuracy check.

[0291] Library preparation, quantification, normalization, and sequencing

[0292] Libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, CA, USA) according to the manufacturer's protocol, using 5 μL of the recommended input of 0.2 ng / μL gDNA per sample. TMSpeedBeads purification was performed and washed twice with 80% ethanol to select fragments >250 bp and remove unincorporated adapters. The size and concentration of each DNA library fragment was determined using Agilent High Sensitivity D5000 reagents and ScreenTape on a Tape Station 4200 instrument (Agilent Technologies, Santa Clara, CA, USA) according to the Agilent Assay Quick Guide. All individual libraries were quantified using a dsDNA system (Promega, Madison, WI, USA) to give additional concentration estimates. Size and concentration data from the TapeStation and Quantifluor systems were used to normalize each library to 4 nM by diluting with 10 mM Tris-HCl (pH 8.5) prior to pooling. Equal volumes of 5 μL were pooled and sequenced on an Illumina MiSeq desktop sequencer using a 500 cycle MiSeq reagent Nano kit v2, with 10 pM input and 10% PhiX spike-in.

[0293] Bioinformatics and next-generation sequencing data analysis

[0294] Genomic data analysis was performed using the commercial bioinformatics program Geneious Prime software program 2020v.2.24 (http: / / www.geneious.com (accessed on June 12, 2021)) to analyze the sequences. The following reference sequences stored in the NCBI database were used for comparative analysis: NC_040262.1, NC_040260.1, and NC_040273.1 for FASN, FABP4, and SCD genes, respectively. Next-generation sequencing data were retrieved from Dashboard-BaseSpace Sequence Hub (https: / / basespace.illumina.com / dashboard, accessed on July 15, 2021) as paired read data in 2 separate forward and reverse read lists in FASTQ format. Quality control measurements were performed on the retrieved raw reads. The BBDuk trimmer in Geneious Prime 2020v.2.2 with default settings for paired end reads was used to trim the reads and remove adapters. The quality (Q) value of the Phred score was set to 20 to improve the sequencing data and increase the probability of calling true SNPs to 99%. Short reads with a minimum length of 20 bp were discarded to produce clean reads. When calling SNPs using Annotation and Prediction → Find Low / High Coverage, regions with low coverage were excluded. The reads were mapped to the reference in Geneious. The reference sequences were retrieved from the NCBI database (Genbank) of FASN, FABP4, and SCD of sheep breeds. The sensitivity was set on the medium sensitivity / fast and fine-tuning (up to 5 iterations) options, which were selected to improve the results by aligning the reads to each other in addition to the reference sequence. The major allele frequencies in the next generation sequence data based on the observed and expected genotypes were calculated using the Hardy-Weinberg equilibrium principle as described in Graffelman, J. et al. 2017. Hum. Genet. 136, 727–741. Statistical analysis

[0295] All statistical analyses of associations between the detected SNPs of the three genes and meat eating quality traits were performed using R statistical software version 3.6.3 (http: / / www.R-project.org / ). Linkage disequilibrium, as an indicator of non-random association between alleles at different loci, was estimated as the difference between the frequency of gametes carrying a pair of alleles A and B at two loci (pAB) and the product of these allele frequencies (pA and pB), D AB=pAB-pApB, where the allele pair AB is a haplotype, and pAB is the haplotype frequency [Slatkin, M. 2008. "Nature Review: Genetics (Nat. Rev. Genet.)" 9, 477-485]. Major and minor allele frequencies were calculated, and the Hardy-Weinberg equilibrium was tested using the chi-square test. Pearson residual correlation analysis was performed to examine the relationship between genomic variants and meat quality traits (FA, FMP and IMF). The linear mixed model procedure was used to study the differences in FMP, IMF and fatty acid profiles of TAW lambs caused by FABP4, SCD and FASN variants, which were consistent with the fixed effects of allele substitutions of individual SNPs and the random effects of animals (for pedigrees) that explained the compound generation effect. The association of functional allele mutations at the coding regions of the identified FABP4, SCD and FASN loci with FMP, IMF and fatty acids was statistically analyzed. The least squares means were compared using the Tukey-adjusted multiple comparison test. The complete statistical model is:

[0296] Y ij =μ+α i + γ1 FA ij + γ2 SC ij + γ3 SK ij +e ij

[0297] in:

[0298] Y ij = dependent variables (FMP, IMF, FA) of the jth TAW of the i-th composite generation;

[0299] μ = population mean;

[0300] α i = the effect of the ith compound generation;

[0301] FA = genotype FASN (AA, GA, and GG);

[0302] SC = genotype SCD (CC, CT, and TT);

[0303] SK = genotype FABP4 (GG, GA, and AA);

[0304] γ = effect of genotype; and

[0305] e ij = residual error.

[0306] result

[0307] according to Figure 1 The experimental design shown, this study of SCD, FASN and FABP4 lipogenic gene SNPs in muscle biopsy samples of TAW lambs for breeding, selection and evaluation was based on genetically designed primers whose sequences are presented in Table 1 and successful polymerase chain reaction (PCR) products were obtained in Figure 2-6 Presented in.

[0308] Table 1. Primer sequences used for FABP4, FASN and SCD polymerase chain reaction assays # .

[0309]

[0310] # FASN, fatty acid synthase; FABP4, fatty acid binding protein 4; SCD, stearoyl-CoA desaturase; T a , annealing temperature.

[0311] SCD, FASN, and FABP4 gene SNP variants and genotypes

[0312] Using polled Dorset and Texel as positive controls and Rambouillet effect negative controls, eight SCD gene SNP loci with major allele frequencies ranging from 0.53 to 0.93 were identified (g.23880613A>G; g.23881050T>C; g.23883280G>A; g.23885910C>A; g.23887165A>G; g.23888763C>T; g.23889346T>G; g.23890209T>C), as depicted in Table 2. It is evident from Table 2 that in the parental, first and second composite generations, TAW lambs were heterozygous at all three loci (g.23881050T>C, g.23883280G>A, g.23885910C>A), thereby being genetically distinct from the homozygous variants seen in the polled Dorset, Texel and Rambouillet controls.

[0313] Table 2. SCD gene SNPs (major allele frequency) in TAW 1, Polled Dorset (+control) and Rambouillet (-control) lambs.

[0314]

[0315] 1 TAW, Tattykeel Australian White.

[0316] Nine functional SNPs covering 91% of the FASN gene sequence were identified as depicted in Table 3. The genotypes at the nine loci were identical in TAW, indicating a consistent inheritance pattern from the composite TAW parents to the first and second generations, which was distinct from the Rambouillet negative control variety.

[0317] Table 3. FASN gene SNPs (major allele frequency) in TAW 1, Polled Dorset (+control) and Rambouillet (-control) lambs.

[0318]

[0319] 1 TAW, Tattykeel Australian White.

[0320] For the FABP4 gene, three SNP loci were genotyped with major allele frequencies ranging from 0.50 to 0.97 (Table 4).

[0321] Table 4. FABP4 gene SNPs (major allele frequency) in TAW 1, Polled Dorset (+control) and Rambouillet (-control) lambs.

[0322]

[0323] 1 TAW, Tattykeel Australian White.

[0324] Correlation between SCD, FASN and FABP4 gene SNPs, FMP, IMF and fatty acids

[0325] Figure 7 Significant correlations between the detected SCD SNP loci, several fatty acids and other meat quality traits were shown. Among the SCD SNP loci, the highest correlation of 0.98 was observed between g.23888763C>T and g.23881050T>C; between g.23889346T>G and g.23887165A>G. Moderate correlations ranging from 0.37 to 0.47 were observed between health-promoting n-3 LC-PUFAs (EPA, DHA and DPA) and the g.23888763C>T and g.23881050T>C loci. IMF was moderately to highly correlated with n-3 LC-PUFA (0.38-0.66), while FMP was negatively correlated with IMF (-0.66) and DHA (-0.42). Very high correlations of up to 0.99 were evident among the different fatty acids and their sum ( Figure 7 ).

[0326] Figure 8The results show that, among the FASN gene SNPs, there are highly significant correlations between loci, while the correlations between the g.12323864A>G locus and most fatty acids are negative, ranging from -0.3 to -0.34. Negative correlations were also observed between IMF and FMP (-0.66) and DHA (-0.42), while the highest positive correlations were observed between various fatty acids ( Figure 8 ).

[0327] Fig. 9 It was shown that, among the FABP4 gene SNPs, the highest correlation was 0.53 between the loci g.62826965C>G and g.62826961T>C, while a negative correlation of -0.42 was observed between g.62826965C>G and g.62829478A>T. It was also observed that IMF had a continuous positive correlation with n-3 LC-PUFA, with a positive correlation of up to 0.66 with DHA, a positive correlation of up to 0.47 with DPA, and a positive correlation of up to 0.38 with EPA, while the highest positive correlation was between the various fatty acids and their sum ( Fig. 9 ).

[0328] Associations between SCD, FASN and FABP4 SNPs, FMP, IMF and fatty acids

[0329] Table 5 presents the mean, standard deviation and coefficient of variation of meat quality traits and descriptive statistics for the full set of fatty acid breakdown. The mean of FMP was 33.65°C, with a standard deviation of 2.74 and a coefficient of variation of 8.14%, while the mean of IMF was 4.43%, with a standard deviation of 1.31 and a coefficient of variation of 29.58%. Table 5 also shows that the SCDg.23881050T>C SNP was significantly associated with IMF (p<0.0089) and DHA (p<0.0111), while the FABP4g.62829478A>GSNP was only associated with IMF (p<0.0539). FASN g.12323864A>G SNP was associated with FMP (p<0.0544), ALA (p<0.0033), MUFA (p<0.0025), SFA (p<0.0025), C18:2n-6 (p<0.0138), C16:0 (p<0.0039), C18:0 (p<0.0012), and C18:1n-9 (p<0.0023) fatty acids ( Table 5 ).

[0330] Table 5. Association of SNP mutations with FMP, IMF and fatty acids in TAW lambs # .

[0331] Tukey-adjusted multiple comparison tests for significant SNPs, FMP, IMF, and fatty acids

[0332] # *p<0.05, **p<0.01; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; SD, standard deviation; CV, coefficient of variation.

[0333] As depicted in Table 6, the Tukey-adjusted multiple genotype comparison test performed at the SCD g.23881050T>C SNP locus confirmed significant differences, where the homozygous TT genotype had the highest DHA (11.00±2.34 mg / 100g), IMF (5.43±0.516%), and DPA (27.1±3.26 mg / 100g) compared to the CC genotype with the lowest DHA (7.00±2.11 mg / 100g), IMF (3.98±0.312%), and DPA (17.9±6.81 mg / 100g). The heterozygous genotype CT had an intermediate DPA (7.64±2.09 mg / 100g), IMF (4.39±0.287%), and DPA (19.4±6.74 mg / 100g) between the highest and lowest values ​​(Table 6). There were many more significant genotype variations at the FASN g.12323864A>G SNP mutations associated with FMP, ALA, MUFA, SFA, C18:2n-6, C18:1n-9, C18:0 and C16:0, where the homozygous genotype GG had the highest value for all variables, while the AA genotype had the lowest value (Table 6), except for the lowest C18:2n-6 in the heterozygous GA genotype. In contrast, at the FABP4g.62829478A>G SNP locus, only the IMF variation tended to be significant between genotypes (p<0.06).

[0334] Table 6. Tukey-adjusted multiple comparisons of SNP mutations with FMP, IMF and fatty acids in TAW lambs # .

[0335]

[0336]

[0337] **p<0.05, **p<0.01; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; SD, standard deviation; CV, coefficient of variation.

[0338] The data presented here provide insights into the shared genetic control of FMP, IMF content, and health-promoting ω-3 long-chain fatty acid composition traits, aiding the design of breeding strategies to genetically improve meat eating quality traits in TAW lambs while they are still alive. Early decision making using this minimally invasive thoracic lumbar longissimus muscle biopsy sampling technique for direct quantification of genetic merit in live lambs advantageously overcomes the problem of waiting to collect meat quality data after slaughter, when it is too late to make selection decisions about live animals. The identified SNPs of these lipid metabolism genes can also be used for breed-specific identification and marker-assisted selection of TAW sheep for premium meat eating quality. NGS of the FABP4, FASN, and SCD genes also provided data demonstrating their roles in fatty acid metabolism specific to the TAW breed.

[0339] Example 2: DNA marker identity test based on single nucleotide polymorphism (SNP)

[0340] Based on the findings in Example 1, the inventors further developed a SNP-based DNA marker identity test as a diagnostic measure for determining purebred Tattykeel Australian White Lambs (TAWL) characterized by high FMP, IMF and n-3 LC-PUFA. The following procedure was performed to create a DNA marker identity test. Genomic DNA (gDNA) extracted from TAWL was diluted to 100 ng / uL. The concentration of gDNA was checked in a Quantifluor dsDNA system (Promega, USA) to ensure accuracy.

[0341] Using Platinum TM SuperFi TM PCR amplification assays were performed using 5 μL of 2X Platinum II PCR Master Mix (Thermo Fisher Scientific, Australia). PCR amplification assays were performed in a total volume of 10 μL containing 5 μL of 2X Platinum TM SuperFi TM IIPCR master mix, 4uL of 10μM each of forward internal / reverse internal and forward external / reverse external four-primer amplification blocking mutation system (four-primer ARMS-PCR: Singapore IDT) and 0.5uL of 100ng gDNA template. The following conditions were used in SimpliAmp TMPCR was performed in a thermocycler (Thermo Fisher Scientific, Melbourne, Australia) with a 3-step protocol: initial denaturation at 98°C for 1 minute (1 cycle); denaturation at 98°C for 15 seconds; annealing at 65°C for 15 seconds; extension at 72°C for 9 minutes; final extension at 72°C for 9 minutes; for 35 cycles and hold at 4°C. Changing the annealing temperature between 50 and 62 did not improve the PCR product. Using 100 uM of the forward outer primer (a) improved the PCR product and facilitated genotyping of different sheep breeds ( Figure 2 ).like Figure 1-4 As depicted, PCR success was checked in 1.5% agarose gel electrophoresis set for 2 hours.

[0342] Table 1: Name and alleles of SNPs, primers and amplicon sizes used for four-primer ARMS-PCR genotyping in Tattykeel Australian White lambs.

[0343]

[0344] Those skilled in the art will appreciate that many changes and / or modifications may be made to the above embodiments without departing from the general broad scope of the present disclosure. Therefore, the present embodiments should be considered in all aspects as illustrative and not restrictive.

Claims

1. A method for identifying an ovine animal having a desired ω-3 long chain polyunsaturated fatty acid (n-3 LC-PUFA), intramuscular fat (IMF), fat melting point (FMP), or any combination thereof, compared to an average population of animals of the species, the method comprising: (a) obtaining SNP genotype data of the sheep animal at one or more single nucleotide polymorphism (SNP) loci, wherein the loci are selected from: (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C 2 SNPs with r values ​​≥ 0.7; and / or (ii) FASN g.12323864A>G or in linkage disequilibrium with FASNg.12323864A>G 2 SNPs with r values ​​≥ 0.7; and / or (iii) FABP4 g.62829478A>T or in linkage disequilibrium with FABP4 g.62829478A>T 2 SNPs with values ​​≥ 0.7; and (b) determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general animal population of the species if the sheep animal has: (i) a TT genotype in SCDg.23881050T>C or a genotype in linkage disequilibrium with the TT genotype; and / or (ii) a GG genotype in FASN g.12323864A>G or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4 g.62829478A>T or a genotype in linkage disequilibrium with the AA genotype.

2. The method according to claim 1, comprising: (a) obtaining the sheep animal having a gene at the SNP locus SCD g.23881050T>C or a gene in linkage disequilibrium with SCDg.23881050T>C 2 SNP genotype data at SNPs with values ​​≥ 0.7; and (b) if the sheep animal has a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general animal population of the species.

3. The method according to claim 2, comprising: (a) obtaining the SNP genotype data of the sheep animal at SCD g.23881050T>C; and (b) if the sheep animal has a TT genotype in SCD g.23881050T>C, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general population of animals of the species.

4. The method according to any one of claims 1 to 3, comprising: (a) obtaining the sheep animal having a SNP locus FASN g.12323864A>G or a linkage disequilibrium with FASNg.12323864A>G 2 SNP genotype data at SNPs with values ​​≥ 0.7; and (b) if the sheep animal has a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype, then determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to the general animal population of the species.

5. The method according to claim 4, comprising: (a) obtaining SNP genotype data of the sheep animal at the SNP locus FASN g.12323864A>G; and (b) if the sheep animal has a GG genotype in FASN g.12323864A>G, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general population of animals of the species.

6. The method according to any one of claims 1 to 5, comprising: (a) obtaining the sheep animal having a SNP locus FABP4 g.62829478A>T or a linkage disequilibrium with FABP4g.62829478A>T 2 SNP genotype data at SNPs with values ​​≥ 0.7; and (b) if the sheep animal has an AA genotype in FABP4 g.62829478A>T, or a genotype in linkage disequilibrium with the AA genotype, determining that the animal has or will have a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general animal population of the species.

7. The method according to claim 6, comprising: (a) obtaining SNP genotype data of the sheep animal at the SNP locus FASN g.12323864A>G; and (b) if the sheep animal has the AA genotype in FABP4 g.62829478A>T, determining that the animal has or will have the desired n-3 LC-PUFA, compared to the general animal population of the species IMF, FMP or any combination thereof.

8. The method of any one of claims 1 to 7, further comprising selecting and / or breeding from sheep animals identified as having desirable n-3 LC-PUFA, IMF, FMP, or any combination thereof compared to the general animal population of the species based on SNP genotype.

9. A method for determining whether a sheep animal or a part thereof is an Australian White Lamb or a part thereof, the method comprising: (a) obtaining SNP genotype data of the sheep animal at one or more single nucleotide polymorphism (SNP) loci, wherein the loci are selected from: (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C 2 SNPs with r values ​​≥ 0.7; (ii) FASN g.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 (iii) SNPs with r values ​​≥ 0.7; and / or (iii) FABP4 g.62829478A>T or in linkage disequilibrium with FABP4g.62829478A>T 2 SNPs with values ​​≥ 0.7; and (b) determining that the sheep animal or a part thereof is an Australian White lamb or a part thereof if the sheep animal has: (i) a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype; (ii) a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype; and / or (iii) an AA genotype in FABP4 g.62829478A>T, or a genotype in linkage disequilibrium with the AA genotype.

10. The method according to claim 9, comprising: (a) obtaining the sheep animal having a gene at the SNP locus SCD g.23881050T>C or a gene in linkage disequilibrium with SCD g.23881050T>C 2 SNP genotype data at SNPs with values ​​≥ 0.7; and (b) if the sheep animal has a TT genotype in SCD g.23881050T>C, or a genotype in linkage disequilibrium with the TT genotype, then determining that the animal or the part thereof is an Australian White lamb or the part thereof.

11. The method according to claim 10, comprising: (a) obtaining the SNP genotype data of the sheep animal at SCD g.23881050T>C; and (b) if the sheep animal has the TT genotype in SCD g.23881050T>C, determining that the animal or part thereof is an Australian White lamb or part thereof.

12. The method according to any one of claims 9 to 11, comprising: (a) obtaining the sheep animal having a gene at the SNP locus FASN g.12323864A>G or a gene in linkage disequilibrium with FASN g.12323864A>G 2 SNP genotype data at SNPs with values ​​≥ 0.7; and (b) if the sheep animal has a GG genotype in FASN g.12323864A>G, or a genotype in linkage disequilibrium with the GG genotype, then determining that the animal or the part thereof is an Australian White lamb or the part thereof.

13. The method according to claim 12, comprising: (a) obtaining SNP genotype data of the sheep animal at the SNP locus FASN g.12323864A>G; and (b) if the sheep animal has a GG genotype in FASN g.12323864A>G, then determining that the animal or part thereof is an Australian White Lamb or part thereof.

14. The method according to any one of claims 9 to 13, comprising: (a) obtaining the sheep animal having a SNP locus FABP4 g.62829478A>T or a linkage disequilibrium with FABP4g.62829478A>T 2 SNP genotype data at SNPs with values ​​≥ 0.7; and (b) if the sheep animal has an AA genotype in FABP4 g.62829478A>T, or a genotype in linkage disequilibrium with the AA genotype, then determining that the animal or the part thereof is an Australian White lamb or the part thereof.

15. The method according to claim 14, comprising: (a) obtaining SNP genotype data of the sheep animal at the SNP locus FASN g.12323864A>G; and (b) if the sheep animal has the AA genotype in FABP4 g.62829478A>T, determining that the animal or the part thereof is an Australian White lamb or the part thereof.

16. The method of any one of claims 9 to 15, further comprising selecting and / or breeding from sheep animals or parts thereof identified as Australian White Lambs based on the SNP genotype.

17. The method of any one of claims 1 to 17, further comprising generating the SNP genotype data from a nucleic acid sample from the ovine animal.

18. The method of claim 17, wherein the nucleic acid sample is prepared from tissue taken from the longissimus dorsi muscle of the animal.

19. The method of claim 17, wherein the nucleic acid sample is prepared from reproductive material of an animal selected from the group consisting of semen, oocytes, or embryos.

20. The method of claim 17, wherein the nucleic acid sample is prepared from blood or a blood fraction.

21. The method of any one of claims 17 to 20, wherein the nucleic acid sample is DNA.

22. The method according to any one of claims 1 to 21, wherein the SNP genotype data is generated using one or more assays selected from the group consisting of: DNA amplification assays, DNA hybridization assays, DNA sequencing, denaturing high performance liquid chromatography (DHPLC), or a combination thereof.

23. The method of claim 22, wherein the SNP alleles are detected by one or more DNA amplification assays using polynucleotides that allow different alleles of the SNP to be distinguished from one another.

24. The method of claim 23, wherein the DNA amplification assay is polymerase chain reaction (PCR).

25. The method of claim 24, wherein the PCR is an amplification-retarded mutation system (ARMS) PCR.

26. The method according to any one of claims 22 to 25, wherein the SNP alleles are detected by one or more Southern hybridization assays using polynucleotides that allow different alleles of the SNP to be distinguished from each other.

27. The method of claim 21, wherein the SNP alleles of each ovine animal are detected by sequencing a subsequence of a gene containing each SNP.

28. A test kit comprising a plurality of oligonucleotide primers and / or probes configured to distinguish alleles at one or more SNP loci in a sheep animal, wherein the SNP loci are selected from (i) SCD g.23881050T>C or r in linkage disequilibrium with SCD g.23881050T>C; 2 SNPs with r values ​​≥ 0.7, (ii) FASN g.12323864A>G or in linkage disequilibrium with FASN g.12323864A>G 2 SNPs with r values ​​≥ 0.7, and / or (iii) FABP4 g.62829478A>T or in linkage disequilibrium with FABP4 g.62829478A>T 2 SNPs with values ​​≥ 0.

7.

29. The test kit according to claim 26, comprising: One or more oligonucleotide primers and / or probes configured to distinguish between allele C and allele T at the SNP locus SCD g.23881050T>C; One or more oligonucleotide primers and / or probes configured to distinguish between allele G and allele G at the SNP locus FASN g.12323864A>G; and / or One or more oligonucleotide primers and / or probes configured to distinguish between allele A and allele G at the SNP locus FASN g.12323864A>G.

30. The test kit according to claim 28 or 29, comprising one or more oligonucleotide primers and / or probes configured to distinguish between allele C and allele T at the SNP locus SCDg.23881050T>C.

31. The test kit according to claim 30, comprising oligonucleotide primers configured to distinguish allele C and allele T at the SNP locus SCD g.23881050T>C by amplification-retarded mutation system polymerase chain reaction (ARMS PCR).

32. The test kit of claim 31, wherein the oligonucleotide primers configured to distinguish between allele C and allele T at the SNP locus SCD g.23881050T>C by ARMS PCR comprise: A primer comprising the sequence shown in SEQ ID NO: 1 or a sequence substantially identical thereto (named "SCD g.23881050T>C_positive interior"); A primer comprising the sequence shown in SEQ ID NO: 2 or a sequence substantially identical thereto (designated as "SCD g.23881050T>C_reverse_internal"); A primer comprising the sequence shown in SEQ ID NO:3 or a sequence substantially identical thereto (designated as "SCD g.23881050T>C_positive external (a)”); A primer comprising the sequence shown in SEQ ID NO:4 or a sequence substantially identical thereto (designated as "SCD g.23881050T>C_reverse external (a)”); A primer comprising the sequence shown in SEQ ID NO:5 or a sequence substantially identical thereto (designated as "SCDg.23881050T>C_forward external (b)"); and A primer comprising the sequence shown in SEQ ID NO:6 or a sequence substantially identical thereto (designated as "SCDg.23881050T>C_reverse external (b)").

33. The test kit according to claim 31 or 32, wherein the oligonucleotide primer configured to distinguish between allele C and allele T at the SNP locus SCD g.23881050T>C by ARMS PCR is: SCD g.23881050T>C_forward internal (SEQ ID NO: 1); SCD g.23881050T>C_reverse internal (SEQ ID NO: 2); SCD g.23881050T>C_positive external (a) (SEQ ID NO:3); SCD g.23881050T>C_reverse external (a) (SEQ ID NO:4); SCD g.23881050T>C_positive external (b) (SEQ ID NO:5); and SCD g.23881050T>C_reverse external (b) (SEQ ID NO:6).

34. A test kit according to any one of claims 28 to 33, comprising one or more or each of the following: at least one DNA polymerase; dinucleotide triphosphates (dNTPs); magnesium salts; and Buffer.

35. The test kit of any one of claims 28 to 34 for use in detecting ovine animals having desirable ω-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), intramuscular fat (IMF), fat melting point (FMP), or any combination thereof, compared to the general population of animals of said species.

36. A test kit according to any one of claims 28 to 35 when used in a method according to any one of claims 1 to 8 or according to any one of claims 17 to 27 when dependent on claim 1.

37. A test kit according to any one of claims 28 to 34 for use in determining whether a sheep animal, or a part thereof, is an Australian White Lamb, or a part thereof.

38. A test kit according to any one of claims 28 to 34 or 37 when used in a method according to any one of claims 9 to 16 or according to any one of claims 17 to 27 when dependent on claim 9.

39. A method of producing reproductive or regenerative material from an ovine animal determined to have a desirable ω-3 long chain polyunsaturated fatty acid (n-3 LC-PUFA), intramuscular fat (IMF), fat melting point (FMP), or any combination thereof, compared to an average population of animals of said species, the method comprising: (i) performing a method according to any one of claims 1 to 8 or any one of claims 17 to 27 on one or more animals to thereby identify a sheep animal having a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general population of animals of the species; (ii) selecting the animal identified in (i) as having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, compared to the general population of animals of the species; and (iii) obtaining reproductive or regenerative material from the selected animal.

40. A reproductive or regenerative material from an ovine animal produced according to the method of claim 39.

41. A method of breeding an ovine animal having a desired ω-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), intramuscular fat (IMF), fat melting point (FMP), or any combination thereof, compared to an average population of animals of said species, the method comprising: (i) performing a method according to any one of claims 1 to 8 or any one of claims 17 to 27 on one or more animals to thereby identify one or more ovine animals having a desired n-3 LC-PUFA, IMF, FMP or any combination thereof compared to the general population of animals of the species; (ii) selecting one or more animals identified in (i) as having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, as compared to the general population of animals of the species; (iii) breeding from the selected animal to produce one or more offspring therefrom.

42. A sheep animal bred according to the method of claim 41.

43. A method for improving the eating quality of meat from a population of sheep animals, the method comprising: (i) performing a method according to any one of claims 1 to 8 or any one of claims 17 to 27 on one or more animals in the population to thereby identify one or more sheep animals having a desired ω-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), intramuscular fat (IMF), fat melting point (FMP), or any combination thereof, compared to the general population of animals of the species; (ii) selecting one or more animals identified in (i) as having a desired n-3 LC-PUFA, IMF, FMP, or any combination thereof, as compared to the general population of animals of the species; (iii) obtaining reproductive or regenerative material from the selected animal; and (iv) producing one or more offspring or one or more generations of animals from the reproductive or regenerative material.

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