阅读说明：本技术 一种与猪***数相关的snp位点及其检测方法和应用 (SNP (Single nucleotide polymorphism) locus related to number of pig breasts as well as detection method and application thereof ) 是由 胡晓湘 郭晓莉 王宇哲 杨瑞飞 朱迪 李宁 于 2019-09-03 设计创作，主要内容包括：本发明涉及一种与猪乳头数相关的SNP位点及其检测方法和应用,所述SNP位点位于基因组版本Ensembl Sscrofa 11.1的chr7:97570528,该位点的等位基因为A和G。本发明利用Tn5转座酶建库,对3616头具有重要经济性能记录的杜洛克猪核心群体进行低深度测序,通过填充获得高质量的全基因组重测序数据。通过与猪乳头数性状的GWAS分析,检测到七个显著影响猪乳头数的SNP位点,包括一个新发现的影响最为显著的SNP位点SSC7:97570528。这些SNP位点可作为分子标记应用于猪乳头数优势群体的选育。(The invention relates to a SNP locus related to the number of pig breasts, and a detection method and application thereof, wherein the SNP locus is positioned at chr7:97570528 of a genome version Ensembl Sscrifa 11.1, and alleles of the locus are A and G. According to the invention, a Tn5 transposase is used for establishing a library, 3616 Duroc core populations with important economic performance records are subjected to low-depth sequencing, and high-quality whole genome re-sequencing data are obtained through filling. Through GWAS analysis of pig head number traits, seven SNP loci which significantly affect the pig head number are detected, and a newly found SNP locus which has the most significant effect SSC7:97570528 is included. The SNP loci can be used as molecular markers for breeding pig breast number dominant groups.)

1. A SNP site related to the number of pig breasts, wherein the SNP site is located at chr7:97570528 of the genome version EnsemblScrofa 11.1, and alleles of the site are A and G.

2. The SNP site according to claim 1, wherein the number of pig's breasts with the allele A is higher than that with the allele G at the SNP site.

3. The SNP site according to claim 1 or 2, wherein the SNP site is located at the 101 st base in the nucleotide sequence shown in SEQ ID No. 1.

4. A method for detecting SNP sites related to the number of pig breasts is characterized by comprising the following steps:

(1) establishing a gene library for the sample pig by using Th5 transposase, and performing whole genome re-sequencing;

(2) performing genotyping on the whole genome re-sequencing result of the step (1) by using Basevar, and filling by using STITCH to obtain SNP locus gene data;

(3) performing genome-wide association analysis on the number of the nipples of all sample pigs and the SNP locus gene data obtained in the step (2).

The application of the SNP locus in marker-assisted selective breeding of the porcine papilla number trait is characterized in that the SNP locus is located in chr7:97570528, ch7:97574214, ch7:98073927, ch7:98074438, ch7:98074524, ch7:98074744 and/or ch7:98101170 of the genome version Ensembl Scrofa 11.1.

6. The use according to claim 5, wherein the SNP sites located in the genomic version Ensembl Sscofa 11.1 of ch7:98073927, ch7:98074438, ch7:98074524, ch7:98074744 and ch7:98101170 are in complete linkage disequilibrium, ch7: 97570528 is in high linkage disequilibrium with the above 5 SNP sites, and ch7:97574214 is in low linkage disequilibrium with the above 6 SNP sites.

7. Use according to claim 5, characterized in that it comprises the following steps:

(1) detecting the genotype of the sample pig at the SNP locus;

(2) selecting sample pigs with dominant allelic gene types to breed dominant population of papillary number.

8. The use according to claim 7, characterized in that in the genomic version Ensembl Ssccrofa 11.1, the dominant allele genotype at the SNP site at chr7:97570528 is AA;

the dominant allele genotype of the SNP locus at ch7:97574214 is CC;

the dominant allele type of the SNP locus at ch7:98073927 is AA;

the dominant allele type of the SNP locus at ch7:98074438 is TT;

the dominant allele type of the SNP locus at ch7:98074524 is TT;

the dominant allele genotype of the SNP locus at ch7:98074744 is CC;

the dominant allele type of the SNP site located at ch7:98101170 is GG.

9. A primer set for amplifying a region containing the SNP site according to claim 1, which is used for amplifying a fragment containing the SNP site from the sequence shown by SEQ ID N0.1.

The application of the SNP locus in identifying the dominant population of the number of pig teats is characterized in that the SNP locus is positioned at chr7:97570528, ch7:97574214, ch7:98073927, ch7:98074438, ch7:98074524, ch7:98074744 and/or ch7:98101170 of the genome version Ensembl Scrofa 11.1.

Technical Field

The invention relates to the field of molecular genetics, in particular to a porcine papilla number-related SNP (single nucleotide polymorphism) locus and a detection method and application thereof, and specifically relates to a newly discovered porcine papilla number-related missense mutation and application thereof.

Background

China is the largest pig producing country and consuming country, the market demand for pork yield and quality is increasing, the pork yield is increased, the pork carcass quality is improved, and the method becomes a work which is continuously explored by breeding scientists for a long time. Early breeding efforts focused primarily on phenotypic selection in swine, and with the continued push in genomic work and the widespread development of genetic markers, molecular selection is becoming a reliable and efficient selection method.

Single Nucleotide Polymorphism (SNP) markers are third-generation molecular markers, and refer to a polymorphism generated by mutation of a single base on a genomic DNA sequence, wherein the mutation comprises single base transversion, conversion, insertion and deletion. The SNP has the advantages of large amount, high frequency, low mutation rate and the like, and is widely applied to genome analysis, biological information automatic detection, genetic research of simple and complex diseases, livestock breeding markers, global ethnic genetics and other researches. The molecular marker assisted selective breeding is to select target characters on a molecular level, is not influenced by environment, and reduces linkage drag through genetic background selection, thereby accelerating the breeding process and accuracy.

Genome-wide association assays (GWAS) are important methods for livestock and poultry economic trait genetic improvement and mechanism analysis. With the development of the second-generation sequencing technology, the whole genome re-sequencing and simplified genome sequencing technology becomes a powerful tool for high-throughput SNP typing. However, the limited marker density limits the use of simplified genomes in fine localization. The whole genome correlation analysis relates to all variation on a genome, but the cost is high, and the large sample sequencing cannot be realized.

Currently, the most commonly used two-step sequencing method is to perform high-depth re-sequencing (10 x) on a small sample size to obtain a high-quality SNP (single nucleotide polymorphism) collection, then perform low-depth sequencing on a large sample size, and infer the genotype of a region which is not completely covered by sequencing through a linkage disequilibrium law. And (3) carrying out GWAS with important traits by filling the obtained whole genome re-sequencing data to obtain SNP (single nucleotide polymorphism) obviously associated with the traits. The low-depth re-sequencing method greatly reduces the cost, and has great advantages in fine positioning compared with simplified genome because of high marker density obtained after filling. Currently, low depth retesting methods are applied in rice (Huang et al, 2010), mouse (Nicod et al, 2016) and human (Rustagi et al, 2017; Liu et al, 2018).

The characters of the number of the pig nipples are related to the reproductive performance of the pig such as lactation force, litter size and the like, so that the research on the number of the pig nipples has important research significance in breeding.

Disclosure of Invention

The invention provides an SNP locus related to the number of pig breasts as well as a detection method and application thereof, which are used for solving the problems in the prior art.

The first purpose of the invention is to provide SNP sites related to the number of pig nipples, wherein the SNP sites are located at chr7:97570528 of genome version Ensembl Sscrifa 11.1, and alleles of the sites are A and G.

Further, the number of pig teats with allele A at the SNP site is higher than that of pig with allele G.

Further, the SNP site is located at the 101 st base in the nucleotide sequence shown in SEQ ID NO. 1.

The second purpose of the invention is to provide a method for detecting SNP sites related to pig papillary number, which comprises the following steps:

(1) establishing a gene library for the sample pig by using Th5 transposase, and performing whole genome re-sequencing;

(2) performing genotyping on the whole genome re-sequencing result of the step (1) by using Basevar, and filling the STITCH to obtain SNP locus gene data;

(3) and (3) carrying out genome-wide association analysis on the number of the nipples of all sample pigs and the SNP locus gene data segment obtained in the step (2).

Further, Bonferroni correction is carried out on the SNP sites detected by the detection method by adopting independent mark numbers, and the calculation of the independent mark numbers is obtained by using a PLINK index-pair command.

Therefore, the invention obtains 7 SNP loci related to the number of papillae, wherein SSC7:97570528 is a newly found SNP locus, SSC7:98073927, SSC7:98074438, SSC7:98074524, SSC7:98074744 and SSC7:98101170 are in complete linkage disequilibrium and are in high linkage disequilibrium with SSC7:97570528, and SSC7:97574214 is in lower linkage disequilibrium with the other 6 SNP loci. In the population with few papillae, the number of the papillae of the population can be increased by selecting the pigs with the dominant genotype for breeding in the following way:

SSC7:97570528 locus, AA is the dominant genotype. SSC7:97574214 locus, CC is dominant genotype; SSC7:98073927 locus, AA is dominant genotype; SSC7:98074438 locus, TT is dominant genotype; SSC7:98074524 locus, TT is dominant genotype; SSC7:98074744 locus, CC is dominant genotype; SSC7:98101170 locus, GG is dominant genotype.

Furthermore, the SNP loci in linkage disequilibrium indicate that the genotypes of the loci are all related to the characters of the number of the pigs, and show a consistent trend, which means that when the bred pigs have the dominant genotype at the SSC7:97570528 loci, the SNP loci in linkage disequilibrium also show the dominant genotype.

The third purpose of the invention is to provide the application of the seven SNP sites and the detection mode in the marker-assisted selective breeding of the pig nipple number characters.

The SNP locus can be used for identifying pig nipple number dominant groups.

Further, the method comprises the following steps:

(1) detecting the genotype of the sample pig at the SNP locus;

(2) selecting sample pigs with dominant allelic gene types to breed dominant population of papillary number.

Further, the step (1) can be performed by direct sequencing or by amplifying a fragment containing the SNP site and detecting the amplified fragment.

Another object of the present invention is to provide a primer set for amplifying a SNP fragment containing the new discovery of the present invention. The primer pair has the following functions: amplifying a fragment containing the SNP locus from the sequence shown in SEQ ID NO. 1.

The invention provides an SNP locus related to the number of pig breasts and application thereof, and has the following beneficial effects:

according to the invention, a Tn5 transposase is used for establishing a library, 3616 Duroc core populations with important economic performance records are subjected to low-depth sequencing, and high-quality whole genome re-sequencing data are obtained through filling. Through GWAS analysis of pig head number traits, an LD block which obviously influences the pig head number is obtained, the interval is further analyzed, 7 missense mutations are detected, wherein the most obvious SSC7:97570528 locus is a newly found SNP locus, the SNP locus can be applied to breeding of pig head number dominant groups, and the pig head number can be improved by selecting individuals of SSC7:97570528 locus allele AA for breeding. The method can be used for predicting the number of the pig breasts in an early, rapid, low-cost and effective manner by detecting the SNP sites, has a wide application prospect in the aspect of pig breed improvement, and can obtain excellent economic value.

Drawings

Fig. 1 is a manhattan graph of GWAS analysis results of pig teat number traits provided in example 1 of the present invention;

FIG. 2 is a diagram of the fine positioning analysis result of pig breast number traits in candidate QTL regions obtained by GWAS provided by embodiment 1 of the present invention;

FIG. 3 is a diagram of linkage disequilibrium relationship of SNP loci SSC7:97570528, SSC7:97574214, SSC7:98073927, SSC7:98074438, SSC7:98074524, SSC7:98074744 and SSC7:98101170 provided in example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.