Analysis of molecular marker combinations and their applications in dairy goat breeds

Abstract

The present invention discloses a molecular marker combination for analyzing dairy goat breeds and an application thereof, relating to the field of biotechnology. The present invention provides 2014 molecular markers capable of analyzing dairy goat breed characteristics, wherein the physical location information of the molecular markers is determined based on genome sequence alignment of the goat reference genome ARS1. A molecular probe combination, a gene chip, and a kit prepared using the 2014 SNP molecular markers can perform genetic evaluation on individual goats, perform individual selection on dairy goat breeds that were difficult to measure in the early stages, shorten the generation interval, accelerate the breeding process, and thus save a large amount of breeding costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to the field of biological detection technology, and more specifically to a SNP site combination of dairy goat breeds and an application thereof. Background Art

[0002] Goats (Capra hircus) are among the earliest domesticated animals (ZEDER, MA, 2006). Taxonomically, they belong to the class Mammalia, order Artiodactyla, suborder Ruminantia, family Bovidae, subfamily Caprinae, and genus Capra. Estimates suggest that the global population of domestic goats exceeds one billion (AMILLS, M, et al., 2017), encompassing over 700 different breeds (FAO, https: / / www.fao.org / home / en). This widespread distribution and large population size are attributed to their natural adaptability to harsh environments, low nutritional requirements, and ease of management (NADERI, S, et al., 2007). They are adaptable to a wide range of environments, from tropical to temperate, arid to humid, cold to hot, and steep to mountainous. Goats, as versatile producers of dairy, meat, and wool in many regions, play a vital role in human survival and economic development (CLUTTON-BROCK, J, 1990; COLLI, L, et al., 2018). The global sheep and goat population approaches 2.2 billion, with approximately one-fifth engaged in dairy production (PULINA, G, et al., 2018). Goat milk products are a crucial nutritional source for the human diet. Furthermore, goat milk production is genetically polymorphic (there is genetic variation between dairy and non-dairy goats). Given the diverse and variable nature of goat breeds, rapidly screening and identifying dairy goats is a pressing issue. Therefore, designing a SNP array suitable for Chinese goat populations that can rapidly and effectively detect milk production is crucial. Summary of the Invention

[0003] In order to meet the current needs of goat breed research and dairy goat breed detection in agricultural production in my country, the present invention provides a molecular probe combination, gene chip, kit and application for analyzing dairy goat breeds. By utilizing the site information provided by the present invention, dairy goat breed screening, breed identification, breed traceability, and goat breeding can be achieved quickly and accurately, which is beneficial to the protection and improvement of germplasm resources, with short time consumption, low cost and broad market benefits.

[0004] In order to achieve the technical purpose of the present invention, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides an application of a 2014 SNP locus combination in analyzing dairy goat breeds. The physical locations of the 2014 SNP locus combination are shown in Table 1:

[0006] Table 1 Location information of 2014 site combinations

[0007]

[0008]

[0009]

[0010]

[0011]

[0012]

[0013] Its physical location was determined based on the ARS1 genome sequence alignment with the goat reference genome.

[0014] A second aspect provides a method for analyzing dairy goat breeds, wherein the genotypes of 2014 SNP sites in the genomic DNA of a test goat are compared with the genotypes of the 2014 SNP sites in the genomic DNA of a control goat;

[0015] Among them, the 2014 SNP sites are the 2014 SNP sites described in Table 1.

[0016] The third aspect provides a molecular probe combination for analyzing dairy goat breeds, which detects the SNP site combination shown in Table 1 in the sample to be tested, and the physical position information of the site combination in Table 1 is determined based on the genome sequence comparison of the goat reference genome ARS1.

[0017] In the fourth aspect, a gene chip for analyzing dairy goat breeds is provided, wherein the gene chip is loaded with the molecular probe combination described in the third aspect.

[0018] A fifth aspect is a kit for analyzing dairy goat breeds, which comprises the molecular probe combination described in the third aspect or the gene chip described in the fourth aspect.

[0019] In a sixth aspect, a method for analyzing dairy goat breeds is provided, wherein the sample to be tested is detected using the molecular probe combination described in the third aspect, the gene chip described in the fourth aspect, or the kit described in the fifth aspect.

[0020] The seventh aspect, the molecular probe combination described in the third aspect, the gene chip described in the fourth aspect, or the kit described in the fifth aspect has any of the following uses:

[0021] (1) Application in goat breed screening;

[0022] (2) Application in goat breed identification;

[0023] (3) Application in goat breed traceability;

[0024] (4) Application in goat breeding;

[0025] (5) Application in germplasm resource conservation;

[0026] (6) Application in germplasm resource improvement.

[0027] Beneficial effects:

[0028] 1. Based on research on the genetic resources of numerous goats at home and abroad, the present invention provides a SNP locus combination consisting of 2014 SNP loci for analyzing dairy goat breeds. The SNP locus combination provided by the present invention not only has good universality both domestically and internationally, but also can quickly evaluate dairy goat breeds that are not visible at the early stage at the genetic level, obtain more accurate breeding assessment information, and control the breeding process. The above locus combination can also be used to screen, identify, and trace goat breeds, providing technical support for the protection and improvement of germplasm resources.

[0029] 2. The probe combination, gene chip, and kit for analyzing dairy goat breeds provided by the present invention also have the characteristics of low throughput, low cost, and easier analysis. They are widely applicable and have broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the identification map of potential selection areas of FST and π ratio dairy goat population;

[0031] Figure 2 It is the Manhattan plot of Tajima's D for dairy goats and non-dairy goats;

[0032] Figure 3 is the Manhattan plot of the ZHP of the dairy goat group and the non-dairy goat group;

[0033] Figure 4 This is a comparison chart of genes selected by different methods;

[0034] Figure 5 This is a result diagram of the significance test of the judgment results of the group threshold analysis in this application. DETAILED DESCRIPTION

[0035] The present invention is further illustrated below with reference to the detailed description of specific embodiments, but these embodiments are merely illustrative and should not be construed as limiting the present invention. If not otherwise specified, the technical means adopted in the embodiments are conventional means well known to those skilled in the art, and can be carried out with reference to the third edition of the original work "Bioinformatics and Functional Genomics" or related books, and the bioinformatics software and products adopted are also commercially available. Various processes and methods not described in detail are conventional methods well known in the art, and the materials used, trade names, and those that are necessary to list their components are all indicated when first occurring, and thereafter, same reagents used, if not otherwise specified, are identical with the content indicated for the first time.

[0036] In addition, it should be noted that the site combinations and applications provided by the present invention were completed by the inventors through arduous creative labor and optimization work.

[0037] The features and advantages described in the site combination section above are also applicable to the molecular probe combination, gene chip, kit and their applications formed based on the site combination, and will not be repeated here.

[0038] The SNP referred to in the present invention refers to single nucleotide polymorphism (SNP), which mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. The variation of the single nucleotide includes variation caused by conversion, transversion, insertion or deletion of a single base.

[0039] It should be noted that the molecular markers referred to in the present invention are all heritable and detectable DNA sequences or proteins, including but not limited to molecular markers based on molecular hybridization, such as RFLP and Minisatellite DNA; molecular markers based on PCR technology, such as RAPD, STS, SSR, and SCAR; DNA markers based on restriction enzyme digestion and PCR technology; molecular markers based on DNA chip technology, such as SNP; analytical marker technology based on EST database development, etc. The molecular markers provided by the present invention can be used for genome mapping, gene localization research, map-based gene cloning, species relationship and systematic classification, etc.

[0040] It should be noted that the probe referred to in the present invention is a nucleic acid sequence (DNA or RNA) with a detection label and a known sequence that is complementary to the target gene, such as a Taqman-MGB probe.

[0041] It should be noted that the kit referred to in the present invention is any box commonly used in the art that contains reagents for detection or experimentation, which can relieve the operator of the tedious process of reagent preparation and optimization. In one embodiment of the present invention, it contains primers for amplifying the site information provided by the present invention, molecular markers or probes or gene chips for detecting the site information provided by the present invention, enzymes and buffers for amplification, and optionally fluorescent markers for detection.

[0042] Example 1 Obtaining a combination of SNP sites for milk performance

[0043] To meet the current demand for microarray locus function detection and functional research in breeding production and to reflect the genetic characteristics of dairy goats worldwide, this study relies on high-depth whole-genome resequencing data from 147 dairy goats and 53 non-dairy goats from around the world. Using the goat ARS1.2 genome as a reference and combining existing research related to goat function, a 5K goat SNP chip targeting goat milk performance was designed. The specific method is as follows.

[0044] 1. Obtaining the total SNP set

[0045] A total of 5.5 terabytes of resequencing data were collected from 200 goats, including 147 dairy goats from nine breeds and 53 non-dairy goats from five breeds. The goat breed information is shown in Table 2. Sequencing data were downloaded from the NCBI (https: / / www.ncbi.nlm.nih.gov / ), EBI (https: / / www.ebi.ac.uk / ), and NGDC (https: / / ngdc.cncb.ac.cn / ?lang=zh) databases. The average sequencing depth for each sample was 22.99× (range, 8.80–42.64×). Clean data were obtained by filtering the raw data from the whole-genome resequencing of all dairy and non-dairy goats to remove adapter-containing and low-quality reads. Valid sequencing data were aligned to the reference genome using BWA software. Alignment results were deduplicated using SAMTOOLS. Repeat sequences in the BAM files were labeled and removed using GATK. The processed BAM files were then indexed using SAMTOOLS. GATK was used to detect variants in each sample, generate gVCF files, perform variant typing, generate VCF-formatted variant call files, filter out unfiltered variants, and perform further quality control. In one embodiment of the present invention, data quality control was performed using the plink tool, setting the minimum allele frequency to 0.01, the maximum missing rate for genotypes and individuals to 0.1, and allowing non-standard chromosome numbering. The final VCF file for analysis was generated, and a total of 28,651,586 SNP variants were detected.

[0046] Table 2 Goat breed sample information

[0047]

[0048] 2. Screening of candidate genes and their functional regions

[0049] Goat breeds worldwide were grouped according to their significant differences in dairy performance, and all breeds of dairy goats and non-dairy goats were summarized. The dairy goats and non-dairy goats were divided into two groups for selection elimination analysis. The dairy goat breeds (Alpine goats, Ampere goats, Guanzhong dairy goats, Laoshan dairy goats, Nubian goats, Peacock goats, Saanen goats, Toggenburg goats, Tangshan dairy goats) and non-dairy goat breeds (Boots goats, Gesen striped goats, Guishan goats, Longlin goats, Yunshang black goats) were grouped together. ST ,π ratio,H P The intersection of the four methods, Tajima's D, was used to screen out the functional regions related to milk production traits, and then find the genes in this region ( Figure 1-4 ).

[0050] Finally, 37 candidate genes related to milk performance and with well-defined functions were identified, as shown in Table 3. The functional regions corresponding to the candidate genes were then determined using a Perl script.

[0051] Table 3

[0052]

[0053] 3. Obtaining SNP loci combinations for milk production

[0054] The snpEff was used to search the SNP sites corresponding to the functional regions of the candidate genes determined in step 2 in the total SNP set, and a milk performance site combination containing 2014 SNP sites was obtained.

[0055] Example 2: Using the SNP site combination of dairy goats for preparation of primer combination and probe combination

[0056] A person skilled in the art designs primers based on the sequence information of each site in the SNP site combination of dairy goats analyzed provided by the present invention, and performs secondary structure evaluation and Tm value evaluation on the designed primers, and finally obtains primers with good specificity and high sensitivity that can achieve the detection purpose under the same reaction conditions.

[0057] The secondary structure and Tm value evaluation can be performed using any method commonly used in the art, such as using a DNA folding form to evaluate the secondary structure, see (http: / / unafold.rna.albany.edu / ?q=mfold / DNA-Folding-Form) for details, and then using the software RaW-Probe to evaluate the Tm value.

[0058] The above methods are all conventional methods. According to the site information in the milk production SNP site combination provided in this application, it can be obtained without any creative labor. Therefore, the primers obtained according to the SNP site combination provided by the present invention also fall within the scope of protection of the present invention.

[0059] Similarly, the use of the SNP site combination provided by the present invention to prepare probes, such as tanqman probes, also falls within the scope of protection of the present invention.

[0060] Example 3: Combining SNPs for milk performance analysis to prepare gene chips

[0061] The SNP gene chip of the present invention is prepared by fixing the primers or probes obtained in Example 2 on a polymer substrate, such as a nylon membrane, nitrocellulose membrane, plastic, silica gel wafer, micro magnetic beads, etc., using conventional methods, or fixing the probes on a glass plate, or directly synthesizing the primers or probes obtained in Example 2 on a hard surface such as glass. The method of using the SNP gene chip of the present application is the same as the conventional method.

[0062] It should be noted that those skilled in the art can prepare SNP gene chips for detecting dairy goat breeds in any manner, and can also entrust a biological company to prepare them. However, SNP gene chips prepared based on the SNP site combination for milk performance analysis provided in this application all fall within the scope of protection of the present invention.

[0063] Example 4 Analysis Kit for Dairy Goat Breeds

[0064] The SNP detection kit for milk performance analysis provided in this application includes primers, probes, or gene chips obtained based on the SNP site combination obtained in Example 1. Depending on the type of use, corresponding detection reagents are also included. For example, when the SNP site combination obtained in Example 1 is a TaqMan probe, the kit also includes a buffer, ligase, AceQUniversal U+ Probe Master Mix V2, TaqMan Probe, etc. commonly used in fluorescent quantitative PCR reactions.

[0065] Those skilled in the art can configure different SNP kits for detecting dairy goat breeds according to different usage modes, but the dairy goat breed SNP detection kits configured based on the milk performance SNP site combination provided in this application all fall within the scope of protection of the present invention.

[0066] Example 5 Detection of dairy goat breeds

[0067] Based on the SNP site combination for analyzing dairy goat breeds provided in Example 1 of the present application, the dairy performance of known dairy and non-dairy goats was tested. According to the test results, and in combination with the known dairy and non-dairy goat phenotypes, the accuracy of the test was judged, specifically:

[0068] The peripheral blood of goats was collected by conventional methods, and whole genomic DNA was extracted therefrom to obtain whole genomic DNA samples;

[0069] A gene chip was designed based on the site information in the SNP site combination provided by the present invention using conventional methods, and a whole-genome DNA sample of a goat was tested to obtain the typing results of each site in the goat (i.e., whether each site is homozygous, heterozygous, mutant homozygous, or base deleted). The frequency value of the typing result for each site was calculated and compared with the population threshold. The comparison results showed that the genetic test results were consistent with the phenotype of the corresponding dairy goat breed.

[0070] It should be noted that the population threshold value of the present application is obtained by analyzing different populations of dairy goats and non-dairy goats using the same method as above.

[0071] This application conducted a significance test (independent sample Mann-Whitney U test) on the analysis results of the dairy goat group and the non-dairy goat group. The results are as follows Figure 5 As shown in the figure, it can be seen from the results that P<0.01, the difference is extremely significant, which shows that the results determined by the method of the present invention are accurate and effective.

[0072] Industrial Applications

[0073] Based on the SNP site combination for analyzing dairy goat breeds provided by this application, which consists of only 2014 SNP sites, a SNP probe combination, a gene chip, and a kit for analyzing dairy goat breeds can be prepared by those skilled in the art. The SNP probe combination, gene chip, and kit for analyzing dairy goat breeds can be analyzed at the genomic level, genetic information can be evaluated, breeds can be screened, breeds can be identified, and the breeding process can be controlled. The combination can also be applied to goat breed tracing, goat pedigree reconstruction, germplasm resource protection, and germplasm resource improvement.

[0074] The above description is only a preferred example to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can be subject to various changes and modifications. Without violating the spirit of the present invention, various changes or modifications made by those skilled in the art to the present invention on this basis should also fall within the scope of the present invention.

Claims

1. Application of 2014 SNP loci combination in analyzing whether a goat is a dairy goat breed. The physical locations of the 2014 SNP loci combination are shown in the following table: ； ； ； ； ； ； Its physical location was determined based on genomic sequence alignment with the goat reference genome ARS1.

2. A method for analyzing whether the goat is a dairy goat breed is to compare the 2014 SNP site genotypes of the genomic DNA of the test goat with the 2014 SNP site genotypes of the genomic DNA of the control goat; in, The 2014 SNP sites are the 2014 SNP sites described in claim 1.

3. A molecular probe combination for analyzing whether the sample is a dairy goat breed, wherein the molecular probe combination detects the SNP site combination as described in claim 1 in the sample to be tested.

4. A gene chip for analyzing whether the goat is a dairy goat breed, wherein the gene chip is loaded with the molecular probe combination according to claim 3.

5. A kit for analyzing whether a goat is a dairy goat breed, comprising the molecular probe combination according to claim 3 or the gene chip according to claim 4.

6. A method for analyzing whether the sample is a dairy goat breed, comprising detecting the sample using the molecular probe combination of claim 3, the gene chip of claim 4, or the kit of claim 5.

7. Use of the molecular probe combination according to claim 3, the gene chip according to claim 4, or the kit according to claim 5 in screening for dairy goat breeds.

8. Use of the molecular probe combination according to claim 3, the gene chip according to claim 4, or the kit according to claim 5 in identifying the breed of dairy goats.

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