A snp molecular marker related to the number of healthy piglets produced by sows and application thereof

Abstract

The application belongs to the technical field of pig molecular marker, and particularly discloses a SNP molecular marker related to the litter size of sows and application thereof. SLC22A12 A SNP site significantly related to the litter size of Landrace sows is screened out by using second-generation gene sequencing and gene chip technology for genotyping and whole genome association analysis of 732 Landrace sows, the SNP site is located on the upstream of a gene on chromosome 2 of pigs, corresponding to the 7565970th nucleotide on chromosome 2 of the international pig 11.1 version reference genome, and can be used for predicting or assisting breeding of the litter size of sows.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pig molecular markers, and particularly relates to a SNP molecular marker on pig chromosome 2 related to the litter size of sows and application thereof. BACKGROUND

[0002] Increasing the number of marketable pigs and the number of weaned piglets per sow is an important production goal in the industry, and the number of healthy piglets per sow per litter is closely related thereto. For such low-heritability reproductive traits as the number of healthy piglets per sow, the genetic basis is very complex, and the genetic progress using traditional breeding methods is often very slow. In order to better understand the genetic basis of the litter size, it is necessary to screen important SNPs, chromosomal regions and candidate genes. In recent years, molecular biology tools such as marker-assisted selection and genome selection have also become important animal breeding methods (Zhang et al, 2014), and greatly improve the breeding efficiency (Ding et al, 2021), which can identify important SNPs related to traits through whole-genome resequencing, gene chip technology and whole-genome association analysis (GWAS), but there are still very few reliable molecular markers at present.

[0003] The present application carries out whole-genome association analysis on the number of healthy piglets per sow of Large White sows, and screens SNP sites significantly associated with the number of healthy piglets per sow, thereby providing a theoretical basis and application approach for pig molecular marker-assisted selection breeding. SUMMARY

[0004] The present application aims to overcome the defects and deficiencies of the prior art, and screen SNP molecular markers significantly associated with the number of healthy piglets per sow, thereby providing new molecular marker resources for the number of healthy piglets per sow and providing a new application approach for marker-assisted selection breeding of pigs.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] The present application provides a method for screening molecular markers associated with the number of healthy piglets per sow, which comprises the following steps:

[0007] (1) Collecting blood tissues of 732 Large White pigs, extracting DNA and performing quality detection;

[0008] (2) Taking 611 samples for genome resequencing, and 121 samples for gene chip sequencing;

[0009] (3) After filling the chip data, merging with the genome resequencing data, and quality control;

[0010] (4) Estimating the DEBV (de-regression estimated breeding value) of the number of healthy piglets per sow to correct the parity effect and reconstruct the phenotype;

[0011] (5) The application utilizes rMVP software to perform whole genome association analysis based on a FarmCPU model.

[0012] The SNP molecular marker significantly related to the number of healthy piglets of sows is screened through whole genome association analysis, and the nucleotide sequence of 100bp upstream and downstream of each SNP is obtained according to the reference genome (Sus_scrofa.Sscrofa11.1-106) of pig 11.1 version of Ensmble database, the SNP nucleotide sequence is shown as SEQ ID NO. 1 or 2, the 101st base is G or A, and the allele mutation causes the nucleotide polymorphism of the above sequence, the site (chr2:7565970) is located at 219bp upstream of SLC22A12 gene, and the accession number in dbSNP database is rs81359036.

[0013] The application has the beneficial effects that compared with the traditional screening method, the application has the outstanding advantages of precision and rapidness. When the application is applied to the selection breeding of the number of healthy piglets of sows, the number of healthy piglets per pregnancy of sows can be increased, and the production income of pig raising enterprises can be increased. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 : Manhattan plot of the SNP molecular marker screened by the application and significantly related to the number of healthy piglets of Landrace sows.

[0015] Figure 2 : QQ plot of the SNP molecular marker screened by the application and significantly related to the number of healthy piglets of Landrace sows.

[0016] Figure 3 : LD block diagram of the SNP molecular marker screened by the application and significantly related to the number of healthy piglets of Landrace sows and part of SNP of 5' end of SLC22A12 gene. DETAILED DESCRIPTION

[0017] Example 1: Genotyping detection

[0018] (1) Sample collection

[0019] 121 Landrace sows of French line, 338 Landrace sows of plus line and 273 Landrace sows of Dan line were collected from Anhui, Xinjiang and Inner Mongolia respectively.

[0020] (2) DNA extraction

[0021] ① Take 200 μL of whole blood, add an equal volume of cell lysis buffer prepared from 100 mmol / L Tris saturated phenol, 500 mmol / L disodium EDTA, 20 mmol / L sodium chloride (NaCL), 10% sodium dodecyl sulfate (SDS), 20 μg / ml of trypsin RNA, add 10 ng / mL of proteinase K, mix well, and place in a 65°C constant temperature water bath for 30 min;

[0022] ② Slowly shake the centrifuge tube for 15 min, place it in a centrifuge at 12000 rpm for 5 min, then take the supernatant into another centrifuge tube;

[0023] ③ Add an equal volume of phenol-chloroform-isoamyl alcohol mixture (25:24:1), shake well, place in a centrifuge at 12000 rpm for 5 min, then take the supernatant into another centrifuge tube;

[0024] ④ Add an equal volume of phenol-chloroform-isoamyl alcohol mixture (25:24:1) again, shake well, place in a centrifuge at 12000 rpm for 10 min, then take the supernatant into another centrifuge tube;

[0025] ⑤ Add 2 volumes of pre-cooled anhydrous ethanol, let it stand until the ethanol evaporates, then pick out the DNA precipitate and dissolve the DNA with ultrapure water;

[0026] ⑥ Use a DNA concentration detector and agarose gel electrophoresis to detect the quality of the DNA.

[0027] (3) Genotyping

[0028] ① According to the extracted DNA, 611 samples were subjected to second-generation whole genome resequencing (average sequencing depth of about 18x) using DNBSEQ-T7 gene sequencer, reference genome (Sus_scrofa.Sscrofa11.1-106) was aligned using BWA software, and SNP calling was performed using GATK software; in addition, 121 samples were subjected to genotyping using 50K SNP whole genome chip, and Beagle v5.4 was used for filling.

[0029] ② After merging the two parts of the genotyping file using BCFtools software, the SNP molecular marker sites obtained were subjected to quality control using PLINK v1.9 software, and SNPs with detection rate less than 90%, minimum allele frequency less than 0.05, Hardy-Weinberg equilibrium P value less than 1 x 10 -6 , and filling accuracy less than 0.8 were removed, and finally 732 samples with a total of 5,782,534 SNPs were obtained for subsequent analysis.

[0030] Example 2 Application of SNP molecular markers in whole genome association analysis of litter size of Large White sows (1) Phenotype statistics and reconstruction

[0031] The present application collects 732 sows per litter data of healthy piglets, part of the sow records multiple data of the data, a total of 1117 data, the specific descriptive statistics results are as follows, see Table 1.

[0032] Table 1: Descriptive statistics of litter size of Large White pig population

[0033]

[0034] As can be seen from Table 1, the coefficient of variation of the litter size phenotype is greater than 20%, indicating that the trait has a large selection space.

[0035] In order to eliminate the influence of genetic information in GWAS analysis, and correct the effect of parity, the present application reconstructs the original litter size phenotype, and uses the de-regression estimated breeding value (DEBVs) weighted by the estimated breeding value (EBVs) as the response variable (Garrick et al, 2009; Ostersen et al, 2011). The present application uses the ssGBLUP method of HiBLUP software to estimate EBVs, and the model is as follows:

[0036] y = Xb + Zu + e

[0037] Where y is the original phenotype vector; b represents the fixed effect and covariate, including birth season, delivery season, production condition, individual number, delivery age and the first three principal components, X is the correlation matrix; u is the random additive effect, Z is the correlation matrix of additive effect and phenotype record; e is the residual vector. The weight coefficient (w i ) of the individual i is calculated as follows:

[0038]

[0039] Where h 2 is the heritability of the trait, is the reliability of the EBV of the i th individual, and c is the proportion of genetic variation that cannot be explained by genetic information, which is set to 0.5 in the present application.

[0040] (2) Whole genome association analysis of litter size of Large White sows

[0041] The experimental pig population used for the genome-wide association analysis in this example was Large White pigs (n=732). A genome-wide association analysis was performed using the FarmCPU model in rMVP software to analyze SNP molecular markers and alternative phenotypes (DEBVs) for the litter size trait in Large White sows. The FarmCPU model iteratively used a fixed-effects model and a random-effects model. The fixed-effects model is shown below:

[0042] y=M t b t +S j d j +e

[0043] Where y is the vector of DEBVs; M t is the genotype matrix of t pseudo-QTNs used as fixed effects; b t It's M t The correlation design matrix of S j is the jth marker to be tested, d j is the corresponding effect; e is the residual effect vector, A random effects model was used to select the most appropriate pseudo-QTN, which was:

[0044] y=u+e

[0045] where y and e are the same as in the fixed-effect model; u is the genetic effect, where K is the relationship matrix defined by the pseudo-QTN.

[0046] The Manhattan plot based on the GWAS analysis results is shown in Figure 1 The Bonferroni correction threshold was used to detect genome-wide significant SNPs, which was defined as 0.05 / N (N is the number of effective independent SNPs inferred using simpleM software, which is 2963141). The QQ plot is shown in Figure 2 .Depend on Figure 1 As can be seen, a total of 6 SNP sites were detected. The present invention only focuses on the SNP site located at the first position on chromosome 2 (the SNP indicated by the arrow in the figure). This SNP site is closely linked to multiple SNP sites in the 5' UTR and exons of the SLC22A12 gene, of which 3 SNP sites can cause non-synonymous mutations in the coding region (see Figure 3 ), therefore, this SNP is highly likely to interact with surrounding SNPs causing nonsynonymous mutations in the structure and function of the SLC22A12 protein, thereby providing a potential SNP marker for the genetic interpretation of healthy piglet number in sows. Basic genetic parameters for this SNP are shown in Table 2.

[0047] Table 2: Information on candidate SNPs for the healthy litter size trait in Large White sows identified based on GWAS

[0048]

[0049] Table 2 shows that the GWAS result log10(1 / P) > 7.772782 is a significant association.

[0050] From Table 2, it can be seen that the screened candidate SNP site (rs81359036) is significantly associated with the number of healthy piglets born by the Large White sow. The present application statistically analyzes part of the information of different genotypes of the SNP site in the population, which is shown in Table 3.

[0051] Table 3: Frequency of different genotypes of the candidate SNP site in the Large White sow and the number of healthy piglets born by the sow

[0052]

[0053] From Table 3, it can be seen that for the above SNP site (rs81359036), the number of healthy piglets born by the individual with the homozygous of the secondary allele (AA genotype) is lower than that of the individuals with the other two genotypes.

[0054] Main references:

[0055] 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. (2021). Genome-wide association studies reveals polygenic genetic architecture of litter traits in Duroc pigs. Theriogenology, 173, 269-278.

[0056] Garrick, D. J., Taylor, J. F., & Fernando, R. L. (2009). Deregressing estimated breeding values and weighting information for genomic regression analyses. Genetics, Selection, Evolution: GSE, 41(1), 55.

[0057] Ostersen, T., Christensen, O. F., Henryon, M., Nielsen, B., Su, G., & Madsen, P. (2011). Deregressed EBV as the response variable yield more reliable genomic predictions than traditional EBV in pure-bred pigs. Genetics, selection, evolution: GSE, 43(1), 38. Zhang, Z., Ober, U., Erbe, M., Zhang, H., Gao, N., He, J., Li, J., & Simianer, H. (2014). Improving the accuracy of whole genome prediction for complex traits using the results of genome wide association studies. PloS one, 9(3), e93017.

Claims

1. Application of SNP molecular markers in the prediction of healthy litter size or in assisting breeding of Large White pigs, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO. 1 or 2, the 101st nucleotide is G or A, and compared with sows with GG or GA genotypes, sows with AA genotype have a lower number of healthy piglets.

2. The use of the kit in predicting healthy piglet number or assisting breeding in Large White pigs is characterized by: The kit contains a reagent for detecting the polymorphic site 7565970 on chromosome 2 of the sow, wherein the polymorphic site is G or A, and the reference genome version is Sus_scrofa.Sscrofa11.1-106. Compared with sows with GG or GA genotypes, sows with AA genotype have a lower number of healthy piglets.

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