SNP molecular markers associated with the trait of sows giving birth to deformed piglets and their application

Abstract

The present invention belongs to the technical field of pig molecular markers, and specifically discloses SNP molecular markers related to the trait of sows giving birth to deformed piglets and their applications. Genetic typing is performed using gene chip technology, and genome-wide association analysis is performed based on SNP molecular markers. The analysis screens out four SNP molecular marker sites related to the trait of sows giving birth to deformed piglets. The SNP molecular marker sites are located at NCOA1 Gene fragments and chromosome 9 PRCP and DIAS Gene fragments can be used for marker-assisted selection of sows that produce deformed piglets.

Description

Technical Field

[0001] The invention belongs to the technical field of pig molecular markers, and in particular relates to SNP molecular markers related to the trait of sows giving birth to deformed piglets and applications thereof. Background Art

[0002] my country is the country with the largest scale of pig farming in the world, and its citizens' consumption of pork products ranks first in the world. As the source of pig farming, effective progress in pig breeding can bring huge economic benefits to my country's total pig production and related industries (Liu Wentao and Gu Liwei, 2016). By the end of 2021, according to statistics, the number of sows in my country reached 43.29 million, with an annual birth rate of about 2.3 births, an average litter size of about 13 piglets, and a piglet deformity rate of 2.9%. my country will lose about 37.537 million piglets every year (Wang Minghua, 2022). In recent years, with the development of breeding work, some traits of local pigs in my country have been effectively improved. Using conventional breeding methods, significant progress has been made in improving production traits with medium to high heritability, such as back fat thickness and lean meat percentage. However, there are greater difficulties in improving reproductive traits with low heritability, such as the number of live piglets born per litter, the number of stillbirths, and the number of deformed piglets (Zhang et al., 2019).

[0003] With the development of molecular biology and multi-omics sequencing technology, molecular marker-assisted selection breeding has gradually become the mainstream means of breeding. Many studies have shown that molecular marker-assisted selection has significant advantages in the selection of low heritability traits, greatly improving breeding efficiency (Ding et al, 2021). Domestic and foreign scholars have conducted a large number of studies on molecular markers related to piglet deformity traits and the number of deformed piglets, and have explored potential functional genes. Hao et al. studied three populations of Duroc, Landrace and Large White pigs, and screened out 7 SNPs significantly associated with piglet splayleg syndrome (PSL) through GWAS, and located 4 candidate genes (Hao et al, 2017). Траспов et al. identified 24 SNPs significantly associated with deformity traits such as piglet anal atresia, limb and hoof problems and piglet tremor in the Large White pig population through GWAS, involving 11 functional genes (Траспов et al, 2020). In addition, Yang Jie et al. identified SNPs that were significantly associated with the number of deformed piglets born by sows, making a certain contribution to the work of molecular marker-assisted selection breeding in pigs (Yang Jie et al., 2021). Looking at domestic and foreign research, there are few studies on the identification of molecular markers associated with sows giving birth to deformed piglets.

[0004] The present invention performed whole-genome association analysis using GMAT software and screened out 4 SNP sites that were significantly associated with the trait of sows giving birth to deformed piglets, providing a theoretical basis and application approach for pig molecular marker-assisted selection breeding (Wang et al, 2020). Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art, use gene chip technology for genotyping, conduct genome-wide association analysis with the trait of sows giving birth to deformed piglets, screen out 4 SNPs that are significantly associated with the trait of sows giving birth to deformed piglets, and provide a new SNP molecular marker resource for sow reproductive traits.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The applicant screened SNP molecular markers significantly associated with the trait of sows giving birth to deformed piglets through whole genome association analysis, and obtained the nucleotide sequence of 100bp upstream and downstream of each SNP based on the Ensmble database pig 11.1 version reference genome, as follows:

[0008] The nucleotide sequence containing the SNP1 marker is shown in SEQ ID NO. 1 and 2. The SNP1 marker is located at position 101 of the sequence, corresponding to position 114038560 of chromosome 3 of the pig genome. The corresponding gene is NCOA1, and the polymorphic site is A or C. When the genotype of the site is AC, the sow has a high rate of producing deformed piglets.

[0009] The nucleotide sequence containing the SNP2 marker is shown in SEQ ID NO. 3 and 4. The SNP1 marker is located at the 101st position of the sequence, corresponding to the 17003320th position of chromosome 9 of the pig genome. The corresponding gene is PRCP, and the accession number in the dbSNP database is rs338617139. The polymorphic site is G or A. When the genotype of the site is GA, the sow has a high rate of producing deformed piglets.

[0010] The nucleotide sequence containing the SNP3 marker is shown in SEQ ID NO. 5 and 6. The SNP1 marker is located at the 101st position of the sequence, corresponding to the 17029982nd position of chromosome 9 of the pig genome. The corresponding gene is PRCP, and the accession number in the dbSNP database is rs318571326. The polymorphic site is T or A. When the genotype of the site is TA, the sow has a high rate of producing deformed piglets.

[0011] The nucleotide sequence containing the SNP4 marker is shown in SEQ ID NOs. 7 and 8. The SNP1 marker is located at position 101 of the sequence, corresponding to position 17083124 of chromosome 9 of the pig genome. The corresponding gene is DDIAS, and the accession number in the dbSNP database is rs340077666. The polymorphic site is A or G. When the genotype of the site is AG, the sow has a high rate of producing deformed piglets.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] Compared with the traditional screening method, the present invention has the outstanding advantages of being simple and fast. Applying it to the selection and breeding of sows with deformed piglets can reduce the total number of deformed piglets, greatly reduce the economic losses of pig farming enterprises, and increase production income; from the overall national perspective, it can increase the effective industrial scale of the pig farming industry, improve the domestic pig breeding level, and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : The Manhattan plot produced in Example 2 visualizes the results of the GWAS analysis. There are 5 SNPs above the threshold line, of which 1 SNP on chromosome 14 is located in the intergenic region and belongs to the non-coding region. The remaining 4 SNPs located in the gene coding region are SNP molecular markers screened by the present invention that are significantly associated with the trait of Large White sows producing deformed piglets.

[0015] Figure 2 : The QQ plot produced in Example 2 is used to further determine the reliability of the GWAS results by comparing the quantiles of the probability distribution of the actual -log(P) value and the expected -log(P) value to compare the two probability distributions. DETAILED DESCRIPTION

[0016] Example 1 Genotyping Detection and Data Processing

[0017] (1) DNA extraction

[0018] ① Collect ear tissues from 1175 large white sows, crush the collected ear tissues and place them in a glass homogenizer, add an equal volume of cell lysis buffer composed of 100mmol / L Tris-saturated phenol, 500mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 20mmol / L sodium chloride (NaCL), 10% sodium dodecyl sulfate (SDS), and 20ug / ml pancreatic RNAse, then add 10ng / mL proteinase K, mix well, and place in a 65℃ constant temperature water bath for 30min;

[0019] ② Slowly shake the centrifuge tube for 15 minutes, centrifuge it at 12000 rpm for 5 minutes, and take the supernatant into another centrifuge tube;

[0020] ③ Add equal volumes of phenol, chloroform, and isoamyl alcohol (volume ratio of 25:24:1), shake and mix, centrifuge at 12000 rpm for 5 min, and take the supernatant into another centrifuge tube;

[0021] ④ Add equal volumes of phenol, chloroform, and isoamyl alcohol (volume ratio of 25:24:1), shake and mix, centrifuge at 12000 rpm for 10 min, and take the supernatant into another centrifuge tube;

[0022] ⑤ Add 2 times the volume of pre-cooled anhydrous ethanol, let it stand until the ethanol evaporates, pick out the DNA precipitate and use ultrapure water to dissolve the DNA;

[0023] ⑥ Use a DNA concentration meter and agarose gel electrophoresis to detect DNA quality.

[0024] (2) Genotyping

[0025] ① Based on the DNA extracted above, genotyping was performed using the 50K SNP whole genome chip developed by Illumina, which contains more than 50,000 SNP molecular marker sites;

[0026] ② Based on the whole genome resequencing data of 152 Large White sows, the original sequencing data was genotyped, and the obtained SNP molecular marker loci were quality controlled using PLINK v1.9 software, and SNPs with a detection rate of less than 90% and a minimum allele frequency of less than 0.05 were eliminated. Finally, 17,985,419 SNPs were used for genotype filling;

[0027] (3) Genotype filling

[0028] Beagle v5.1 software was used to fill in the chip data with the whole genome resequencing data of Large White pigs as the reference group, and 10,197,628 SNP molecular marker sites with a filling accuracy higher than 0.8 were screened for subsequent analysis.

[0029] Example 2 Genome-wide association analysis of SNP molecular markers and the trait of Large White sows producing deformed piglets

[0030] (1) Phenotypic definition

[0031] The birth of deformed piglets by sows was regarded as a case-control trait. According to the original phenotypic records of each sow giving birth to deformed piglets in each litter, sows that gave birth to two or more deformed piglets were taken as cases, and sows that did not give birth to deformed piglets or gave birth to only one deformed piglet were taken as controls. New phenotypes were constructed from case-control for subsequent whole-genome association analysis.

[0032] (2) SNP molecular marker quality control

[0033] According to the reliable SNP molecular marker sites selected after the above genotype filling, PLINK v1.9 software was used to perform quality control on the SNPs of the case phenotype and control phenotype after filling. For the SNP data of the case phenotype, SNPs with a minimum allele frequency of less than 0.01 were removed, and for the SNP data of the control phenotype, SNPs with a minimum allele frequency of less than 0.01 were removed. After merging, completely overlapping SNP molecular markers were obtained. Finally, 1175 samples and 7851901 SNPs were used for genome-wide association analysis.

[0034] (3) Genome-wide association analysis of traits of deformed piglets produced by Large White sows

[0035] The experimental pig group used for the whole genome association analysis in this example is Large White pigs, including 1175 sows. According to the information of malformed piglets recorded in the experimental pig group, based on the mixed linear model method, the uvlmm analysis module in the GMAT software was used to perform a whole genome association analysis on SNP molecular markers and the malformed piglet traits of Large White sows. The specific model is as follows:

[0036] y=μ+Wα+xβ+Zu+e

[0037] Among them, y is the phenotype vector; μ is the population mean; W is the fixed effect design matrix; α is the categorical variable of the fixed effect vector; x is the SNP vector; β is the SNP effect; Z is the design matrix; u is the individual effect; and e is the random residual.

[0038] According to the results of GWAS, the P value of each SNP site is calculated as -log(P), and the significant threshold line calculation formula is -log(1 / SNP number). When the -log(P) value of each SNP site is calculated by the present invention to be greater than or equal to 6.889143, it indicates that there is a significant association between the SNP and the trait, and the CMplot package of R language is used to draw the Manhattan plot and QQ plot ( Figure 1 , Figure 2 ).

[0039] Table 1: Candidate SNP loci for the trait of deformed piglets in Large White sows identified based on GWAS

[0040]

[0041] Table 2: Distribution of genotype frequency and malformed piglet rate of candidate SNP loci in Large White sows

[0042]

[0043] Notes on Table 2: The data in brackets shows the frequency of sows of this genotype giving birth to 2 or more piglets with deformities.

[0044] As described in Table 1, four SNP loci significantly associated with the trait of sows producing malformed piglets were screened, and the number of individuals with different genotypes at each SNP locus and the number of individuals with 2 or more malformed piglets of the corresponding genotype were further counted, and the rate of producing malformed piglets of different genotypes was calculated (the number of individuals with 2 or more malformed piglets / the number of individuals with the genotype). As shown in Table 2, for the four SNP loci 3:114038560, 9:17003320, 9:17029982, and 9:17083124, when the genotypes were AC, GA, TA, and AG, respectively, the frequency of sows producing 2 or more malformed piglets was higher than that of individuals with other genotypes.

[0045] Main references:

[0046] [1] Liu Wentao, Gu Liwei. Comparative study on pig farming in China and the United States [J]. China Animal Husbandry Magazine, 2016, 52(06): 3-7.

[0047] [2] Wang Minghua: Grain output hits new high, livestock production grows steadily [EB / OL]. http: / / www.stats.gov.cn / xxgk / jd / sjjd2020 / 202201 / t20220118_1826603.html, 2022–01–18 / 2022–02–23.

[0048] [3] Yang Jie, Wu Zhenfang, Qiu Yibin, Ding Rongrong, Zheng Enqin, Cai Gengyuan, Li Zicong, Hong Linjun, Xu Zheng. A SNP molecular marker on porcine chromosome 13 associated with the number of deformed piglets born by sows and its use [P]. Guangdong Province: CN113637768A, 2021-11-12.

[0049] [4]Ding R,Qiu Y,Zhuang Z,Ruan D,Wu J,Zhou S,Ye J,Cao L,Hong L,Xu Z,Zheng E,Li Z,Wu Z,Yang J.Genome-wide association studies reveals polygenicgenetic architecture of litter traits in Duroc pigs.Theriogenology,2021,173:269-278.[5]Hao X,Plastow G,Zhang C,Xu S,Hu Z,Yang T,Wang K,Yang H,Yin X,LiuS,Wang Z,Wang Z,Zhang S.Genome-wide association study identifies candidategenes for piglet splay leg syndrome in different populations.BMC Genet,2017,18(1):64.

[0050] [6]ТрасповАА,КостюнинаОВ,БелоусАА,КарпушкинаТВ,СвеженцеваНА, ЗиновьеваНА.Whole-genome association studies ofdistribution of developmental abnormalities and other breeding-valuablequalitative traits in offspring of the Russian large-white boars.VavilovskiiZhurnal Genet Selektsii,2020,24(2):185-190.

[0051] [7]Wang D,Tang H,Liu JF,Xu S,Zhang Q,Ning C.Rapid epistatic mixed-model association studies by controlling multiple polygenic effects[J].Bioinformatics,2020,36:4833-4837.

[0052] [8] Zhang Z, Chen Z, Ye S, He Y, Huang S, Yuan X, Chen Z, Zhang H, LiJ. Genome-Wide Association Study for Reproductive Traits in a Duroc PigPopulation. Animals, 2019, 9(10):732. Sequence Listing <110> Lingnan Modern Agricultural Science and Technology Guangdong Provincial Laboratory Huazhong Agricultural University <120> SNP molecular markers associated with the trait of sows giving birth to deformed piglets and their application <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 201 <212> DNA <213> Artificial Sequence <400> 1 attgctctca ttactcagtc aggaagttgg ttaaagatga taatcttaaa ttgtccattg 60 ctctgctcca cacattaatt cctcaatctt attgcatttt agaaatctaa cctaaacaaa 120 ataattat ttaagaatca aagaaactct acatggctgt ctaccatcag tactgagtat 180 tacaagatag tttttttgcc t 201 <210> 2 <211> 201 <212> DNA <213> Artificial Sequence <400> 2 attgctctca ttactcagtc aggaagttgg ttaaagatga taatcttaaa ttgtccattg 60 ctctgctcca cacattaatt cctcaatctt attgcatttt cgaaatctaa cctaaacaaa 120 ataattatat ttaagaatca aagaaactct acatggctgt ctaccatcag tactgagtat 180 tacaagatag tttttttgcc t 201 <210> 3 <211> 201 <212> DNA <213> Artificial Sequence <400> 3 gatcaaaagt tactcaatgc ttccttctca gactggcata gaaatctttg atccactgct 60 tcatatatct gacttccaag gagcgggcta agagcacagt ggtgggatca aaggcagtgt 120 tggctcggag atctagatga tgggctccct ttgggatgac aatcgcaact aaggtgtctg 180 taacattctt ggttacacca c 201 <210> 4 <211> 201 <212> DNA <213> Artificial Sequence <400> 4 gatcaaaagt tactcaatgc ttccttctca gactggcata gaaatctttg atccactgct 60 tcatatatct gacttccaag gagcgggcta agagcacagt agtgggatca aaggcagtgt 120 tggctcggag atctagatga tgggctccct ttgggatgac aatcgcaact aaggtgtctg 180 taacattctt ggttacacca c <210> 5 <211> 201 <212> DNA <213> Artificial Sequence <400> 5 ccgaaccacc tcactgagcc agtcctaatt tcttactcat gcgaactgtg agataacaag tatttattat ggttttaagc tactaaggtt tgaggtaaac tgtcatgtag caatagataa ctaatatagc tacatacaca father ccctacttga ccttcgtgct ttggtctcac 201. cctctgacg gctccccacca <210> 6 <211> 201 <212> DNA <213> Artificial Sequence <400> 6 ccgaaccacc tcactgagcc agtcctaatt tcttactcat gcgaactgtg agataacaag tatttattat ggttttaagc tactaaggtt tgaggtaaac agtcatgtag caatagataa ctaatatagc tacatacaca father ccctacttga ccttcgtgct ttggtctcac 201. cctctgacg gctccccacca <210> 7 <211> 201 <212> DNA <213> Artificial Sequence <400> 7 cagtagctaa tgcctaaaag gatcttactg cattttttcc caccagccaa tgaatatctt 60 tttgccattt gctggagttt accaagtgaa acaccatagt aaaaatactt cagtgagctt 120 tttcttttca gttctcaaac ttaccattgt tatagttttc cagatatact tatttctcca 180 ctagagggca ataaatgctt a 201 <210> 8 <211> 201 <212> DNA <213> Artificial Sequence <400> 8 cagtagctaa tgcctaaaag gatcttactg cattttttcc caccagccaa tgaatatctt 60 tttgccattt gctggagttt accaagtgaa acaccatagt gaaaatactt cagtgagctt 120 tttcttttca gttctcaaac ttaccattgt tatagttttc cagatatact tatttctcca 180 ctagagggca ataaatgctt a 201

Claims

1. The application of SNP markers in the selection of deformed piglets in Large White pigs is characterized by: The SNP markers are the following 4 SNP combinations: The nucleotide sequence containing the SNP1 marker is shown in SEQ ID NOs. 1 and 2, the SNP1 marker is located at position 101 of the sequence, and the polymorphic site is A or C; The nucleotide sequence containing the SNP2 marker is shown in SEQ ID NOs. 3 and 4, the SNP2 marker is located at position 101 of the sequence, and the polymorphic site is G or A; The nucleotide sequence containing the SNP3 marker is shown in SEQ ID NOs. 5 and 6, the SNP3 marker is located at position 101 of the sequence, and the polymorphic site is T or A; and The nucleotide sequence containing the SNP4 marker is shown in SEQ ID NOs. 7 and 8. The SNP4 marker is located at the 101st position of the sequence, and the polymorphic site is A or G.

2. The use according to claim 1, characterized in that: When the genotype of the SNP marker is heterozygous, the sow has a high rate of giving birth to deformed piglets.

Images