SNP (Single Nucleotide Polymorphism) molecular marker related to sheep fecundity and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to sheep fecundity and application of the SNP molecular marker. The SNP molecular marker comprises an SNP1 (Single Nucleotide Polymorphism) and / or an SNP2; the SNP1 is located at a base at the 115747587 site of a chromosome 6 of a sheep reference genome AR-UIRamV2.0 version, and the polymorphism of the SNP1 is T / G; the SNP2 is located at a basic group at the 41419566th site of a chromosome 22 of a sheep reference genome AR-UIRamV2.0 version, and the polymorphism of the SNP2 is A / G. According to the scheme, the SNP molecular marker related to sheep fecundity is provided, sheep with the high fecundity character can be efficiently and accurately identified and screened out, and the marker is high in typing quality, single in copy and high in polymorphism and can be used for sheep fecundity molecular marker assisted breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a SNP molecular marker related to sheep fertility and its application. Background Technology

[0002] Sheep (Ovis aries) are an important economic animal globally, and their reproductive performance directly affects the productivity and economic benefits of animal husbandry. Increasing litter size, shortening lambing intervals, and mitigating reproductive seasonality are core objectives of sheep genetic improvement. With the rapid development of high-throughput sequencing technology and bioinformatics analysis methods, our understanding of the genetic basis controlling sheep reproductive traits has deepened. Identifying reproduction-related genes, quantitative trait loci (QTLs), and other genetic variations, and precisely locating their positions on the reference genome, is crucial for implementing modern breeding strategies such as marker-assisted selection (MAS) and genome-wide selection (GS).

[0003] Although the sheep industry has gradually developed towards large-scale and intensive farming in recent years, there are still significant shortcomings in terms of standardization, industrialization, and economic efficiency. Especially against the backdrop of the rapid development of intensive stall farming, the market demand for high-fertility sheep breeds is increasingly urgent, necessitating genetic improvement to enhance industry efficiency.

[0004] Sheep fertility is mainly influenced by two factors: litter size and estrous cycle. Through long-term natural selection and artificial breeding, a rich variety of local sheep breeds have been developed. Among them, superior breeds such as the Small-tailed Han sheep and the Hu sheep exhibit year-round estrus and high fertility, with lambing rates exceeding 200%. In contrast, while breeds such as Tibetan sheep, Xinjiang local sheep, Yunnan local sheep, and Mongolian sheep possess advantages such as strong resistance and good adaptability, they generally suffer from low fertility due to seasonal estrus and high single-birth rates, severely hindering the improvement of breeding efficiency.

[0005] At present, sheep reproductive phenotype identification is mainly assessed through lambing records. The main disadvantages of this method are: (1) it is affected by environmental factors (such as nutrition and management), which affects the accuracy of phenotypic data; (2) it requires a lot of manpower and material resources, and the breeding cost is high; (3) the breeding cycle is long (1-2 years) and the breeding efficiency is low.

[0006] Genetic studies both domestically and internationally have reported several genes related to sheep reproduction. Among them, BMP15, GDF9, and BMPR1B are members of the TGF-β superfamily, playing indispensable roles in follicle development and ovulation. Mutations in these genes are a major cause of polyfertility in many high-producing sheep breeds (such as the Small-tailed Han sheep and Booroola Merino sheep). The PRLR gene is associated with the prolactin signaling pathway and is believed to play a role in regulating the seasonal reproductive rhythms of sheep. Other genes, such as TMEM154 and CCNB2, although their specific functional mechanisms are still under investigation, have been confirmed by multiple studies to be significantly associated with litter size or ovarian development.

[0007] Some reproductive traits in sheep (such as total number of lambs and conception rate) are complex quantitative traits controlled by multiple minor genes. Quantitative trait locus (QTL) studies have revealed the widespread presence of regions influencing reproductive traits throughout the sheep genome. These QTLs are distributed across multiple chromosomes, including 1, 3, 5, 6, 10, 11, 15, 18, and 20. However, QTL mapping intervals are often large, containing numerous genes, making it difficult to directly pinpoint target genes. Nevertheless, QTL mapping remains an indispensable tool for identifying trait-related genes. Genome-wide association studies (GWAS) utilize high-density SNP markers covering the entire genome to more precisely identify genetic loci associated with target traits at the population level, typically with higher resolution than traditional linkage analysis. GWAS analysis advances QTL mapping from broad chromosomal regions to specific SNP loci and neighboring candidate genes. It not only verifies the roles of known major genes in different populations but also continuously uncovers new candidate genes with relatively small effects, such as FBXW8 and NOS1.

[0008] Therefore, conducting research on the molecular genetic mechanisms of sheep reproductive traits has significant practical implications. By identifying major genes and key variation sites that regulate lambing number and estrous cycle, we can not only provide targets for gene editing breeding but also offer a theoretical basis for marker-assisted selection breeding. This is of great value in promoting efficient and precise breeding in my country's sheep industry. Summary of the Invention

[0009] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a SNP molecular marker related to sheep fertility.

[0010] The present invention also proposes a primer set for detecting the above-mentioned SNP molecular markers.

[0011] The present invention also proposes a reagent kit.

[0012] This invention also proposes the application of the above-mentioned SNP molecular markers, primer sets, and / or kits.

[0013] This invention also proposes a method for identifying or assisting in the identification of sheep fertility.

[0014] This invention also proposes a sheep breeding method.

[0015] According to a first aspect of the invention, a SNP molecular marker associated with sheep fertility is proposed, said SNP molecular marker including SNP1 and / or SNP2;

[0016] The SNP1 is located at position 115747587 on chromosome 6 of the sheep reference genome ARS-UI_Ramb_V2.0, and the polymorphism is T / G. The SNP2 is located at position 41419566 on chromosome 22 of the sheep reference genome ARS-UI_Ramb_V2.0, and its polymorphism is A / G.

[0017] According to a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular markers is provided.

[0018] In some embodiments of the present invention, the primer set includes: (1) A primer set for amplifying SNP1, comprising specific primers with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; and / or, (2) Primer set for amplifying SNP2, containing specific primers with sequences as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0019] In some embodiments of the present invention, the primer set further includes universal primers with sequences as shown in SEQ ID NO.3 and / or SEQ ID NO.6.

[0020] In some embodiments of the present invention, the specific primers are respectively linked to different fluorescent groups.

[0021] In some embodiments of the present invention, the fluorescent group is selected from FAM, JOE, VIC, HEX, ROX, CY3 or CY5.

[0022] According to a third aspect of the present invention, a kit is provided, the kit comprising the above-described primer set.

[0023] According to a fourth aspect of the present invention, the use of the above-described SNP molecular markers, primer sets, or kits in any of the following is proposed: 1) To detect or assist in the detection of sheep fertility; 2) Prepare products for testing or assisting in the testing of sheep fertility; 3) Select and breed sheep with high reproductive capacity; 4) Prepare and breed sheep products with high reproductive capacity; 5) Sheep breeding; 6) Prepare products for sheep breeding.

[0024] In some embodiments of the present invention, sheep with high reproductive capacity are sheep that produce more than one lamb per litter.

[0025] In some embodiments of the present invention, sheep with low reproductive capacity are sheep that give birth to only one lamb per litter.

[0026] According to a fifth aspect of the present invention, a method for identifying or assisting in the identification of sheep fertility using the above-mentioned molecular markers is provided, the method comprising the following steps: S1. Extract genomic DNA from the sheep to be tested; S2. Perform polymorphism detection of the SNP molecular markers on the genomic DNA extracted in step S1, and determine the fertility of the sheep to be tested based on the genotype.

[0027] In some embodiments of the present invention, when the SNP molecular marker is SNP1, if the genotype obtained by SNP1 detection is GG, the sheep being tested has a low fertility trait; if the genotype obtained by detection is TG or TT, the sheep being tested has a high fertility trait. When the SNP molecular marker is SNP2, if the genotype obtained by SNP2 detection is AA, the sheep being tested has the trait of low fertility; if the genotype obtained by detection is AG or GG, the sheep being tested has the trait of high fertility.

[0028] In some embodiments of the present invention, in step S2, the SNP molecular marker is detected using KASP (competitive allele-specific PCR) technology.

[0029] In some embodiments of the present invention, the composition of the KASP reaction mixture for detecting SNP molecular markers using KASP technology is as follows:

[0030] In some embodiments of the present invention, the amplification program for detecting SNP molecular markers using KASP technology is as follows: 92-95℃ for 13-17 min; 92-95℃ for 18-22 s, 65℃-57℃ for 55-65 s, 8-12 cycles; 92-95℃ for 18-22 s, 55-58℃ for 55-65 s, 30-35 cycles.

[0031] In some embodiments of the present invention, the amplification program for detecting SNP molecular markers using KASP technology is as follows: 94℃ for 15 min; 94℃ for 20 s, 65℃-57℃ for 60 s, 10 cycles; 94℃ for 20 s, 57℃ for 60 s, 33 cycles.

[0032] According to a sixth aspect of the present invention, a sheep breeding method is proposed, comprising the following steps: using the above-mentioned SNP molecular markers to identify or assist in identifying sheep fertility, selecting sheep with high fertility traits for subsequent breeding.

[0033] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention provides an SNP molecular marker related to sheep fertility, which can efficiently and accurately identify and screen sheep with high fertility traits. The marker has high genotyping quality, single copy, high polymorphism (MAF≥0.4 in 611 resequencing data), and high sample detection rate (>99%), and can be used for molecular marker-assisted breeding of sheep fertility.

[0034] This invention provides a KASP-based SNP detection method, enabling rapid, high-throughput, and low-cost identification of sheep fertility genotypes. Based on the Douglas Array Tape platform, this method achieves 90% automation, significantly reducing laboratory manpower and human error. It boasts high throughput and speed, obtaining 122,880 data points in 8 hours, 10 times faster than traditional 96-well plate SNP genotyping methods. The detection reaction requires minimal reagents (only 0.8 μL / reaction), reducing reagent and consumable costs by 70%-90% compared to traditional 96-well plate SNP genotyping methods. Attached Figure Description

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a flowchart of the molecular marker screening and verification experiment in Example 1 of the present invention; Figure 2 This is a graph showing the results of a genome-wide association analysis of sheep fertility in Example 1 of the present invention; Figure 3 This is a typing diagram of the OV900013 molecular marker in Example 1 of the present invention; Figure 4 This is a typing diagram of the OV900014 molecular marker in Example 1 of the present invention. Detailed Implementation

[0036] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0037] Example 1: A SNP molecular marker associated with sheep fertility This embodiment provides a SNP molecular marker related to sheep fertility. The specific screening and verification process is as follows: Figure 1 As shown, the method is as follows: 1. Site screening (1) Analysis of sheep resequencing data By analyzing the genome sequencing data of 611 sheep from different breeds, SNP mining was performed using the sheep reference genome version ARS-UI_Ramb_V2.0 to obtain SNP loci throughout the genome.

[0038] (2) GWAS analysis and site extraction Association analysis was performed by combining whole-genome SNP genotypes with sheep reproductive phenotypic traits.

[0039] The results of the correlation analysis are as follows Figure 2 As shown in Table 1, two highly significant associated loci, OV900013 and OV900014, were located on chromosomes 6 and 22, respectively. Information on these two loci is presented in Table 1. Specifically, molecular marker OV900013 exhibits a T / G variation at position 115747587 on chromosome 6 of the sheep reference genome ARS-UI_Ramb_V2.0, while molecular marker OV900014 exhibits an A / G variation at position 41419566 on chromosome 22 of the sheep reference genome ARS-UI_Ramb_V2.0.

[0040] When the genotype obtained by OV900013 is GG, the sheep being tested has the trait of low fertility; if the genotype obtained is TG or TT, the sheep being tested has the trait of high fertility. When the genotype obtained by OV900014 is AA, the sheep being tested has the trait of low fertility; if the genotype obtained is AG or GG, the sheep being tested has the trait of high fertility. Among them, the high fertility trait is: more than 1 lamb per litter, and the low fertility trait is: only 1 lamb per litter.

[0041] Table 1. SNP loci significantly associated with sheep fertility.

[0042] 2. Verification of specific loci (1) Primer design For the SNP loci obtained above, KASP marker primers were designed based on the sheep reference genome ARS-UI_Ramb_V2.0 using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ). Each marker group had three primers, with two specific primers having FAM and HEX fluorescent sequences linked to their 5' ends, respectively. After design, genome-wide copy number analysis was performed on the primer sequences, ultimately yielding two high-quality single-copy KASP marker loci (Table 2). The primers were synthesized by Invitrogen.

[0043] Table 2. Information on the developed KASP markers and primers

[0044] (2) Verification and detection of the markers Ninety-three ewe samples (completely different from the 611 sheep samples of different breeds mentioned above) were collected for testing and verification. All ninety-three ewe samples were of known fertility. The criteria for high fertility was: more than one lamb per litter, and the criteria for low fertility was: only one lamb per litter.

[0045] KASP marker validation and detection were performed using Douglas Scientific's ArrayTape system. The ArrayTape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis.

[0046] PCR amplification system: The PCR amplification system was automatically assembled using NEXAR, and the PCR amplification system is shown in Table 3 below.

[0047] Table 3 PCR amplification system for KASP marker genotyping

[0048] PCR amplification: PCR amplification was performed using SOELLEX under the following conditions: 94℃ for 15 minutes; 94℃ for 20 seconds, 65℃-57℃ (annealing temperature decreased by 0.8℃ per cycle) for 60 seconds, 10 cycles; 94℃ for 20 seconds, 57℃ for 60 seconds, 33 cycles.

[0049] Signal scanning and genotyping: After the PCR reaction, the fluorescence signal of the reaction system was scanned using ARAYA; then, genotyping and data analysis were performed using INTELLICS. In the KASP marker genotyping detection, the genotypes of the samples were divided into three clusters: the X cluster, the Y cluster, and the heterozygous genotype cluster (see...). Figure 3-4 The X cluster indicates that the sample contains a homozygous X allele at this KASP marker locus (marked in red in the upper left corner of the genotyping graph), the Y cluster indicates that the sample contains a homozygous Y allele at this KASP marker locus (marked in blue in the lower right corner of the genotyping graph), and the heterozygous genotype cluster indicates that the sample contains heterozygous X and Y alleles at this KASP marker locus (marked in purple in the genotyping graph).

[0050] Quality validation of KASP marker genotyping for sheep testing: 93 sheep samples with differential fertility were validated using KASP markers OV900013 and OV900014, respectively. The developed KASP marker genotyping map is shown below. Figure 3-4 Verification showed that the two homozygous and heterozygous clusters of each KASP marker were well-differentiated and compact, with single-copy loci and detection rates exceeding 99%, which was consistent with the phenotypic statistical results.

[0051] Based on the genotyping results of KASP markers OV900013 and OV900014 in 93 sheep samples, the fertility of sheep with different genotypes was statistically analyzed. The results are shown in Table 4. It can be seen that the homozygous genotype TT of marker OV900013 was highly fertile (100%), 91.9% of the heterozygous genotype TG was highly fertile, while the homozygous genotype GG was mostly low-fertile (60%). Similarly, the homozygous genotype GG of marker OV900014 was highly fertile (100%), 92.7% of the heterozygous genotype AG was highly fertile, while the homozygous genotype AA was mostly low-fertile (60%). The genotyping results of KASP markers OV900013 and OV900014 are consistent with the actual results and fully meet the requirements for accurate detection of sheep fertility genotypes.

[0052] Table 4. Fertility statistics of sheep with different genotypes

[0053] In actual breeding, two markers, OV900013 and OV900014, can be used for screening in the early stages of ewe growth and development. Individuals with the TT and TG genotypes of marker OV900013, or the GG and AG genotypes of marker OV900014, can be selected efficiently to identify ewes with high fertility (Table 4). If both markers are used for screening simultaneously, except for the relatively low proportion of high-fertility individuals (7.1%) when both markers are homozygous unfavorable genotypes (OV900013-GG and OV900014-AA), the proportion of high-fertility individuals for other genotype combinations is relatively high, with an overall proportion of 93.7% (66.7-100%), which is basically consistent with the known results (Table 5).

[0054] Table 5. Statistical results of the identification of the combination of markers OV900013 and OV900014

[0055] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A SNP molecular marker associated with sheep fertility, characterized in that, The SNP molecular markers include SNP1 and / or SNP2; The SNP1 is located at position 115747587 on chromosome 6 of the sheep reference genome ARS-UI_Ramb_V2.0, and the polymorphism is T / G. The SNP2 is located at position 41419566 on chromosome 22 of the sheep reference genome ARS-UI_Ramb_V2.0, and its polymorphism is A / G.

2. A primer set for amplifying the SNP molecular marker as described in claim 1.

3. The primer set according to claim 2, characterized in that, The primer set includes: (1) A primer set for amplifying SNP1, comprising specific primers with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; and / or, (2) Primer set for amplifying SNP2, containing specific primers with sequences as shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. The primer set according to claim 3, characterized in that, The specific primers are each linked to a different fluorescent group; Preferably, the fluorescent group is selected from FAM, JOE, VIC, HEX, ROX, CY3 or CY5.

5. A reagent kit, characterized in that, The kit comprises the primer set as described in any one of claims 2-4.

6. The use of the SNP molecular marker of claim 1, the primer set of any one of claims 2-4, or the kit of claim 5 in any of the following: 1) To detect or assist in the detection of sheep fertility; 2) Prepare products for testing or assisting in the testing of sheep fertility; 3) Select and breed sheep with high reproductive capacity; 4) Prepare and breed sheep products with high reproductive capacity; 5) Sheep breeding; 6) Prepare products for sheep breeding.

7. A method for identifying or assisting in the identification of sheep fertility using the molecular markers described in claim 1, characterized in that, The method includes the following steps: S1. Extract genomic DNA from the sheep to be tested; S2. Perform polymorphism detection of the SNP molecular markers on the genomic DNA extracted in step S1, and determine the fertility of the sheep to be tested based on the genotype.

8. The method according to claim 7, characterized in that, When the SNP molecular marker is SNP1, if the genotype obtained by SNP1 detection is GG, the sheep being tested has the trait of low fertility; if the genotype obtained by SNP1 detection is TG or TT, the sheep being tested has the trait of high fertility. When the SNP molecular marker is SNP2, if the genotype obtained by SNP2 detection is AA, the sheep being tested has the trait of low fertility; if the genotype obtained by detection is AG or GG, the sheep being tested has the trait of high fertility.

9. The method according to claim 7, characterized in that, In step S2, the SNP molecular markers are detected using KASP technology; Preferably, the composition of the KASP reaction mixture for detecting SNP molecular markers using KASP technology is as follows: Preferably, the amplification program for detecting SNP molecular markers using KASP technology is as follows: 92-95℃ for 13-17 min; 92-95℃ for 18-22 s, 65℃-57℃ for 55-65 s, 8-12 cycles; 92-95℃ for 18-22 s, 55-58℃ for 55-65 s, 30-35 cycles.

10. A sheep breeding method, characterized in that, The method includes the following steps: using the method described in any one of claims 7-9, selecting sheep with high reproductive capacity for subsequent breeding.

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