Use of rs341165849 in assessing boar sperm quality and / or boar genetic selection

Abstract

The application relates to a molecular genetic marker site related to the abnormality rate of boar sperm, which is rs341165849; the rs341165849 is located at the 6138326th position of chromosome 5 of a pig genome, and corresponds to base T or C; the pig genome is Sscrofa11.1, and the accession number in NCBI is GCF_000003025.6. In a Duroc boar population, the abnormality rate of sperm of a boar with a genotype CC at the molecular genetic marker site is significantly lower than that of boars with a genotype CT or a genotype TT, the boar with the genotype CC at the molecular genetic marker site is selected, the abnormality rate of sperm of the boar population can be significantly reduced, the sperm production performance of the boar population is effectively improved, and the utilization efficiency of the boar is improved.

Description

Technical Field

[0001] This application relates to the field of molecular genetic marker-assisted breeding technology, and in particular to the application of rs341165849 in assessing boar sperm quality and / or in boar genetic selection. Background Technology

[0002] Different pig breeds possess unique genetic resources: Duroc pigs have the advantage of rapid growth, Large White pigs have the advantage of high litter size, and local breeds of pigs excel in meat quality and disease resistance. Crossbreeding between different pig breeds can effectively utilize hybrid vigor, but the realization of this genetic potential depends on a variety of factors, among which semen quality is particularly crucial, and this is also a prerequisite for achieving efficient artificial insemination.

[0003] In recent years, with the development of large-scale and intensive breeding, more and more pig farms are using fresh semen for artificial insemination. Artificial insemination has become a powerful tool in countries with highly intensive pig production. The genes of excellent boars can be rapidly spread to the population through artificial insemination technology, effectively improving the production performance of commercial pigs and playing a huge role in ensuring pork supply.

[0004] The heritability estimates for boar semen quality traits ranged from 0.099 to 0.342, with semen volume and density showing higher heritability (approximately 0.27), while sperm motility showed lower heritability (approximately 0.14), indicating potential for genetic improvement.

[0005] Genome-wide association study (GWAS) is an effective strategy for studying complex quantitative traits and genetic variations. Its core is to study the association between genotype data and target phenotypic traits. In particular, with the continuous development of modern high-throughput sequencing and high-resolution detection technologies, as well as the application of various bioinformatics technologies and statistical genetics methods, the foundation has been laid for the fine localization of causal variations of complex morphology.

[0006] Currently, more genes and loci need to be discovered, and the relationship between genotype and boar sperm quality needs to be precisely located so as to select boars with better semen quality for breeding. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and to provide the application of rs341165849 in evaluating boar sperm quality and / or boar genetic selection.

[0008] The first objective of this invention is to provide a molecular genetic marker locus associated with the sperm abnormality rate in boars.

[0009] A second objective of this invention is to provide the application of rs341165849 in assessing boar sperm quality and / or in boar genetic selection.

[0010] A third objective of this invention is to provide a detection reagent for detecting the genotype at the aforementioned molecular genetic marker sites.

[0011] The fourth objective of this invention is to provide a detection kit for detecting genotypes at the aforementioned molecular genetic marker sites.

[0012] A fifth objective of this invention is to provide the application of the above-described detection reagents and / or the above-described detection kits in the preparation of products for assessing boar sperm quality and / or boar genetic selection.

[0013] The sixth objective of this invention is to provide a method for evaluating boar sperm quality.

[0014] The seventh objective of this invention is to provide a method for the genetic selection and breeding of boars.

[0015] To achieve the above objectives, the present invention is implemented through the following solution: This invention seeks protection for a molecular genetic marker locus associated with sperm abnormality rate in boars, the molecular genetic marker locus being rs341165849; The rs341165849 is located at position 6138326 on chromosome 5 of the pig genome, and the corresponding base is T or C; The pig genome is Sscrofa11.1, and its accession number in NCBI is GCF_000003025.6.

[0016] Preferably, the accession number of chromosome 5 in NCBI is NC_010447.5.

[0017] Preferably, the boar is a Duroc boar.

[0018] This invention collects semen phenotypic data (sperm deformity rate data) of Duroc boars, performs resequencing on individual Duroc boars, and fills the data based on SNP chip formulation data. Then, it uses GWAS analysis to mine key sites of semen phenotypic characteristics of Duroc boars, determines the relationship between the genotype of rs341165849 and the semen phenotypic characteristics of Duroc boars, and selects Duroc boars with lower sperm deformity rates for breeding based on the genotype at rs341165849.

[0019] The present invention also claims protection for the use of rs341165849 in assessing boar sperm quality and / or in boar genetic selection, wherein the boar is a Duroc boar.

[0020] Preferably, the boar genetic selection involves selecting boars with low sperm deformity rates for breeding.

[0021] More preferably, the boar genetic selection involves selecting boars with the genotype CC at rs341165849 as boars with low sperm deformity rates for breeding.

[0022] Preferably, the evaluation of boar sperm quality specifically involves: detecting the genotype at rs341165849 of the boar; boars with genotype GG have the best sperm quality, boars with genotype CG have the second best sperm quality, and boars with genotype GG have the worst sperm quality.

[0023] The present invention also claims protection for a detection reagent for detecting genotypes at the aforementioned molecular genetic marker sites.

[0024] The present invention also claims protection for a detection kit for detecting genotypes at the aforementioned molecular genetic marker sites, comprising the aforementioned detection reagents.

[0025] This invention also claims protection for the use of the above-described detection reagents and / or the above-described detection kits in the preparation of products for assessing boar sperm quality and / or boar genetic selection, wherein the boar is a Duroc boar.

[0026] This invention also claims protection for a method for assessing boar sperm quality, the method comprising: detecting the genotype of the boar at the aforementioned molecular genetic marker loci, and assessing the boar sperm quality based on the genotype; Boars with the CC genotype have the best sperm quality, followed by those with the CT genotype, and those with the TT genotype have the worst sperm quality. The boar in question is a Duroc boar.

[0027] Preferably, the detection specifically involves: extracting genomic DNA from a boar, and using the aforementioned detection reagent or kit to determine the genotype of the boar at the aforementioned molecular genetic marker sites.

[0028] This invention also claims protection for a method for genetic selection of boars, the method specifically comprising: Determine the genotype of boars at the molecular genetic marker loci mentioned above, retain boars with the genotype CC for breeding, and increase the frequency of allele C at the molecular genetic marker locus generation by generation, thereby reducing the sperm abnormality rate of offspring boars; The boar in question is a Duroc boar.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention seeks protection for a molecular genetic marker locus associated with sperm abnormality rate in boars, the molecular genetic marker locus being rs341165849; rs341165849 is located at position 6138326 on chromosome 5 of the pig genome, corresponding to the bases T or C; the pig genome is Sscrofa11.1, with NCBI accession number GCF_000003025.6. In Duroc boar populations, boars with the genotype CC at the indicated molecular genetic marker locus have a significantly lower sperm abnormality rate than boars with the genotypes CT or TT. Therefore, selecting boars with the genotype CC at this molecular genetic marker locus can significantly reduce the sperm abnormality rate of the boar population, thereby effectively improving the semen production performance of the boar population, increasing boar utilization efficiency, and facilitating the genetic improvement of boar reproductive performance, thus solving the problem of large-scale culling of production boars due to poor semen quality. Attached Figure Description

[0030] Figure 1 This is a graph showing the results of a genome-wide association analysis performed using sperm phenotype data from Duroc boars in Example 1. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0032] The Duroc pigs used in the embodiments of this invention all come from large-scale farms in Guangxi Zhuang Autonomous Region; all Duroc pigs are scientifically managed, fed according to standards, and are healthy and disease-free; The Duroc boar population included in the genome-wide association analysis consisted of 2,081 Duroc boars. From this population, 400 Duroc boars were randomly selected as the validation population, and the remaining 1,681 Duroc boars were used as the analysis population.

[0033] Example 1: Development of molecular genetic marker loci associated with abnormal sperm morphology in boars I. Experimental Methods (1) Collection of experimental animals and phenotypic data For Duroc boars in the analysis group, semen data from 220,426 collections from 1,681 Duroc boars were collected and analyzed to obtain the sperm deformity rate of each Duroc boar as the semen phenotypic data of each Duroc boar in the analysis group; the sperm deformity rate was obtained through fully automated analysis using the UltiMate™ CASA system (Hamilton Thorne Company, Beverly, Massachusetts, USA).

[0034] (2) Sample collection and sequencing For each Duroc boar in the analysis group, 2g of ear tissue was collected and placed in a 1.5mL sampling tube containing 75% alcohol for later use.

[0035] DNA samples were extracted from ear tissues of Duroc boars using a magnetic bead-based tissue genome extraction kit (catalog number: NMG0611) manufactured by Wuhan Namag Biotechnology Co., Ltd., along with a fully automated DNA extractor. The DNA samples were then subjected to quality control according to general genotyping standards (A260 / A280 value 1.8–2.0; DNA concentration ≥50 ng / μL; clear main band on 1% agarose gel electrophoresis without obvious tailing), resulting in qualified DNA samples from each Duroc boar.

[0036] For qualified DNA samples from Duroc boars, genotyping was performed using the GeneSeek Genome Profile (GGP) 50K SNP chip (GeneSeek, Lincoln, Nebraska, USA), resulting in SNP chip data for 1681 Duroc boars. This data was then aligned to the pig reference genome (Sus scrofa 11.1, NCBI: GCF_000003025.6). Autosomal SNP loci were retained, while loci with a detection rate below 0.90 or a minor allele frequency (MAF) below 0.01 were removed. The remaining 48,829 autosomal SNP loci were used for subsequent analysis.

[0037] Following existing techniques (Druet T, Macleod IM, Hayes BJ. Toward genomic prediction from whole-genome sequence data: impact of sequencing design on genotype imputation and accuracy of predictions. Heredity (Edinb). 2014 Jan;112(1):39-47. doi: 10.1038 / hdy.2013.13. Epub 2013 Apr 3. PMID: 23549338; PMCID:PMC3860159.), 95 highly representative Duroc pig individuals (including boars and sows, with 66 Duroc boars from the analysis population) were selected from the Duroc pig population based on kinship coefficients. Based on these 95 highly representative Duroc pig individuals, data were analyzed using Illumina Novaseq... 150bp paired-end whole-genome resequencing (average coverage depth of 10×) was performed on the 6000 platform, yielding 3608.06 Gb of raw sequencing data from 95 Duroc pigs, including 3600.28 Gb of effective read sequencing data.

[0038] The effective read sequencing data of 95 Duroc pigs were aligned to the Sus Scrofa 11.1 reference genome (http: / / ftp.ensembl.org / ) using BWA software. After SamTools format conversion and Picard deduplication, SNP detection was performed using the Genome Analysis Toolkit (GATK V4.0), which identified 19,548,256 original SNP loci. Quality control was then performed to obtain 8,429,368 high-quality SNP loci. The quality control steps include: (1) using the GATK Variant Quality Score (VQSR) tool to distinguish between true variants and sequencing noise based on the following parameter thresholds: QualByDepth (QD) < 2.0, RMSMappingQuality (MQ) < 40.0, FisherStrand (FS) > 60.0, StrandOddsRatio (SOR) > 3.0, MappingQualityRankSumTest (MQRankSum) < -12.5, and ReadPosRankSumTest (ReadPosRankSum) < -8.0; (2) then using a detection rate ≥ 90%, a minor allele frequency (MAF) ≥ 0.1, and conforming to Hardy-Weinberg genetic equilibrium (HWE p > 1.0 × 10⁻⁶). -6 High-quality SNP loci were obtained by screening using ) as the standard.

[0039] From 95 highly representative Duroc pig individuals, 87 Duroc pigs with a genotypic identity of over 90% between their SNP chip genotype (obtained using GeneSeek Genome Profile 50K SNP chip) and their paired-end whole-genome resequencing genotype were selected as a reference group for chip data filling. The Duroc pig individuals in the reference group had both SNP chip data and whole-genome resequencing data. The 48,829 autosomal SNP loci retained in the SNP chip data were directly compared with the 8,429,368 high-quality SNP loci obtained from the whole-genome resequencing data, resulting in 32,641 common SNP loci. After removing 770 SNP loci with allele inconsistencies between the SNP chip data and the whole-genome resequencing data, 31,871 SNP loci remained (denoted as reference SNP loci). Based on reference SNP loci from 1681 Duroc boars (derived from SNP microarray data, containing 31871 SNP loci), Beagle software (v5.1) was used to perform sequence-level genotyping of the reference SNP loci from the 1681 Duroc boars using 8429368 high-quality SNP loci, resulting in the filled SNP loci for each Duroc boar (including 8429368 SNP loci). Then, plink (v1.9) software was used to remove SNP loci with a genotyping rate <90%, an SNP detection rate <90%, a minor allele frequency (MAF) <0.01, and significantly deviating from Hardy-Weinberg equilibrium (HWE p < 1 × 10⁻⁶) based on preset parameters. -5 The SNP loci of the 1681 Duroc boars were identified, and then high linkage disequilibrium (LD) SNP loci were removed based on the variance inflation factor. Finally, 6,949,508 SNP loci (denoted as genome-wide association analysis loci) located on 18 autosomes were retained for genome-wide association analysis.

[0040] Based on 6,949,508 SNP loci located on 18 autosomes from 1,681 Duroc boars, the linkage disequilibrium level r among the SNP loci was calculated. 2 ; Linkage disequilibrium level r among SNP loci 2 A correlation coefficient ≥0.2 indicates that the SNP sites are not independent of each other; the linkage disequilibrium level r between SNP sites is... 2 <0.2 indicates that the SNP sites shown are independent of each other (the number of independent segments is 2). Of the 6,949,508 SNP sites shown, based on the linkage disequilibrium level r 2 176,296 independent genomic segments were identified.

[0041] (3) Genome-wide association analysis First, principal component analysis (PCA) was performed on the genome-wide association analysis sites of 1681 Duroc boars using GCTA software to obtain the top 5 principal components.

[0042] Next, the genome-wide association analysis (GWAS) method based on the mixed linear model was used in GCTA software. Based on the sperm phenotypic data (sperm abnormality rate) of 1681 Duroc boars, genome-wide association analysis (GWAS) was performed for each GWAS locus to obtain the p-value of each GWAS locus. The GWAS analysis model is as follows: ; in This is a vector of phenotypic observations, namely, sperm abnormality rate data for Duroc boars; The fixed effects vector includes the field-year-season of semen collection, the semen collection interval (days), the age of semen collection in months, and the top 5 principal components in the principal component analysis. This is a vector of polygenic inheritance effects. Let G be the variance of polymorphic genetic effects, and G be the genomic relation matrix, represented as... , The minor allele frequency (MAF) of each genome-wide association analysis locus. The MAF matrix is... The code represents the genotype of each genome-wide association analysis locus, encoded as 0, 1, or 2, where 0 represents a dominant homozygote, 1 represents a heterozygote, and 2 represents a recessive homozygote, and m represents the number of SNP markers. The SNP effect at a single SNP site; It is a random residual vector. For residual variance, It is the identity matrix; , This is the correlation matrix between fixed effects and polygenic inheritance effects.

[0043] Based on the p-value of the genome-wide association analysis (GWIA) loci obtained from GWIA, a significance threshold of 1 / (independent genomic segment) was used (i.e., 1 / 176296 = 5.67 × 10⁻⁶). -6 ), p value < 5.67 × 10 -6 The site was identified as a significantly associated site.

[0044] II. Experimental Results The results of genome-wide association analysis combining sperm phenotype data from Duroc boars are shown in the figure below. Figure 1 As shown, the results indicate that the SNP locus (numbered rs341165849, allele C / T) located at the 6138326th base pair on chromosome 5 (NC_010447.5) of the pig genome Sscrofa11.1 (NCBI: GCF_000003025.6) showed a significant association with the sperm phenotype data of Duroc boars in genome-wide association analysis.

[0045] Example 2: Validation of the rs341165849 molecular genetic marker locus I. Experimental Methods Based on the SNP locus (numbered rs341165849, hereinafter referred to as the significant association locus) located at the 6138326th base pair on chromosome 5 (NC_010447.5) of the pig genome Sscrofa11.1 (NCBI: GCF_000003025.6) obtained in Example 1, the influence of the significant association locus on the sperm abnormality rate of Duroc boars was analyzed in the analysis population (1681 Duroc boars) and the validation population (400 Duroc boars) using one-way ANOVA, F-test, and multiple comparisons. Specifically, the Duroc boars in the analysis and validation populations were analyzed according to the genotype (CC, CT, and TT) of the significant association locus, and then one-way ANOVA, F-test, and multiple comparisons were used to detect whether the significant association locus was significantly associated with the sperm abnormality rate of Duroc boars.

[0046] II. Experimental Results 1. Validation results in the analyzed population The results of the analysis of sperm abnormality rates at significantly associated loci on chromosome 5 of the population are shown in Table 1.

[0047] Table 1. Results of sperm abnormality rates in individuals with different genotypes at significantly associated loci on chromosome 5 of the population.

[0048] In the table, the superscript letters in the column for sperm abnormality rate are used to indicate significant differences.

[0049] The results showed that among the 1681 Duroc boars in the analyzed population, the sperm abnormality rate differed significantly among different genotypes at the SNP locus (rs341165849) at position 6138326 on chromosome 5 (NC_010447.5) of the porcine genome Sscrofa11.1 (NCBI: GCF_000003025.6), with a p-value of 3.24 × 10⁻⁶ for the F-test. -6 This indicates that the SNP locus is significantly associated with the sperm abnormality rate of Duroc boars. Multiple comparisons of sperm abnormality rates of Duroc boars with three genotypes at this significantly associated locus in the analyzed population showed that the sperm abnormality rate of Duroc boars with genotypes CC or CT at this location was significantly lower than that of Duroc boars with genotype TT at this location, and the sperm abnormality rate of Duroc boars with genotype CC at this location was the lowest.

[0050] 2. Validation results in the validation group The sperm abnormality rates of individuals with different genotypes at significantly associated loci on chromosome 5 of the validation population are shown in Table 2.

[0051] Table 2. Sperm abnormality rates of different genotypes at significantly associated loci on chromosome 5 in the validation population.

[0052] In the table, the superscript letters in the column for sperm abnormality rate are used to indicate significant differences.

[0053] The results showed that in the validation population (an independent population of 400 Duroc boars), the p-value of the F-test for sperm abnormality rate at the SNP locus (rs341165849) at position 6138326 on chromosome 5 (NC_010447.5) of the pig genome Sscrofa11.1 (NCBI: GCF_000003025.6) among different Duroc boar genotypes was 0.01587, indicating that this SNP locus was also significantly associated with sperm abnormality rate in Duroc boars in the validation population. Furthermore, in the validation population, the sperm abnormality rate of the three genotypes of Duroc boars at this significantly associated locus was extremely significant. Among them, the sperm abnormality rate of Duroc boars with genotype TT at the significantly associated locus was significantly higher than that of Duroc boars with genotype CC, with a difference of 2.73%.

[0054] The results show that at the SNP locus (rs341165849) at position 6138326 of chromosome 5 (NC_010447.5) on Sscrofa11.1 (NCBI: GCF_000003025.6) of the pig genome, Duroc boars with the CC genotype have a significantly lower sperm abnormality rate. Therefore, in the process of genetic breeding, based on the gene sequencing results of this significantly associated locus, Duroc boars with the CC genotype can be selected for breeding to improve the sperm production performance of the Duroc boar population.

[0055] Example 3: A genetic selection method for Duroc boars A genetic selection method for Duroc boars, specifically as follows: Genomic DNA was extracted from Duroc boars, and nucleic acid information at the 6,138,326th base pair site on chromosome 5 of Duroc boars was detected (using Sscrofa11.1 as the reference genome, NCBI: GCF_000003025.6). The genotype of Duroc boars at this site was determined, and Duroc boars with the CC genotype were retained for breeding. The frequency of allele C at this site was increased generation by generation, thereby reducing the sperm abnormality rate of offspring Duroc boars.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of rs341165849 in evaluating boar sperm quality and / or in boar genetic selection, characterized in that, The rs341165849 is located at position 6138326 on chromosome 5 of the pig genome, and the corresponding base is T or C; The pig genome is Sscrofa11.1, and its NCBI accession number is GCF_000003025.6; the boar is a Duroc boar.

2. The application according to claim 1, characterized in that, The boar genetic selection involves selecting boars with low sperm deformity rates for breeding.

3. The application according to claim 2, characterized in that, The boar genetic selection involved selecting boars with the genotype CC at rs341165849 as breeding stock to reduce sperm abnormality rates.

4. A detection reagent for detecting the genotype at rs341165849 as described in claim 1.

5. A detection kit for detecting the genotype at rs341165849 as described in claim 1, characterized in that, It contains the detection reagent as described in claim 4.

6. The use of the detection reagent of claim 4 or the detection kit of claim 5 in the preparation of products for assessing boar sperm quality and / or boar genetic selection, characterized in that, The boar in question is a Duroc boar.

7. A method for assessing boar sperm quality, characterized in that, The method is as follows: detect the genotype of boar at rs341165849 as described in claim 1, and assess boar sperm quality based on the genotype; Boars with the CC genotype have the best sperm quality, followed by those with the CT genotype, and those with the TT genotype have the worst sperm quality. The boar in question is a Duroc boar.

8. The method according to claim 7, characterized in that, The specific detection method involves: extracting genomic DNA from the boar, and using the detection reagent described in claim 4 or the detection kit described in claim 5 to determine the genotype of the boar at rs341165849 as described in claim 1.

9. A method for genetic selection and breeding of boars, characterized in that, The method is specifically as follows: Determine the genotype of boars at rs341165849 as described in claim 1, retain boars with genotype CC for breeding, and increase the frequency of allele C at rs341165849 generation by generation, thereby reducing the sperm abnormality rate of offspring boars. The boar in question is a Duroc boar.

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