Application of pig SNP site, primer and kit in pig litter size trait screening

Abstract

The application relates to the technical field of biological breeding, and particularly relates to application of a pig SNP site, a primer and a kit in pig litter quantity trait screening. The application first mines an important molecular marker Ssc.X:101892220C>T capable of affecting the litter quantity trait of a pig, effectively improves the total litter quantity and the live litter quantity of the pig through selection of an individual of the TT type of the Ssc.X:101892220C>T site, and lays a foundation for rapidly and effectively improving the reproduction performance of the pig and improving the economic benefit of the pig.

Description

Technical Field

[0001] The present invention relates to the technical field of biological breeding, and in particular to the application of a pig SNP site, a primer and a kit in screening pig litter traits. Background Art

[0002] With the development of molecular biology-related technologies and the widespread application of computer science, more genetic information has been discovered, and the efficiency of information utilization has greatly improved. Animal breeding has evolved from traditional selection and breeding methods based solely on phenotypic data to genomic selection (GS), which combines genetic information with phenotypic data. Pigs are important food animals both domestically and internationally, and the majority of pork supply comes from hybrid offspring. Therefore, the application of genomic selection in pig hybrid breeding to improve the performance of commercial pigs requires further exploration. As a major pig-raising country with the world's largest pork consumption market, China has initially applied genomic selection to improve pig production performance and the core competitiveness of the pig industry, with significant success in improving purebred pig breeds and improving local pigs.

[0003] The Juema pig is a small, unique, primitive Tibetan pig breed domesticated from wild boar. Juema pigs have a low litter size: one-year-old sows produce one litter per year, while multiparous sows produce two litters per year or three litters every two years, with an average litter size of 3.74 pigs. First-time sows produce 1-3 pigs per litter, while multiparous sows produce 4-7 pigs per litter, with a maximum of 8-10 pigs. As a branch of the Tibetan pig native to Gannan, Gansu Province, Juema pigs have been a popular breed for many years. Traditional breeding methods have proven ineffective, particularly for traits with low heritability and difficult-to-measure traits. Therefore, to meet the current enormous consumer market and demand, there is an urgent need to adopt more efficient and accurate breeding technologies, such as molecular marker-assisted breeding and genomic selection, to promote the rapid and stable development of the livestock farming industry. Summary of the Invention

[0004] To address the above issues, the present invention provides a single-nucleotide polymorphism (SNP) site, primers, and a kit for use in screening for litter size in Juema pigs. This invention, for the first time, identifies a key molecular marker, Ssc.X:101892220C>T, that influences Juema pig litter size, laying the foundation for rapidly and effectively improving the reproductive performance and economic benefits of Juema pigs.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] Application of the Ssc.X:101892220C>T site in screening pig litter traits, wherein the site is located at the C>T mutation point at position 101892220bp of the X chromosome, and the site is the genomic position of Sus Scorfa version 11.1.

[0007] Furthermore, the pig reproductive performance identification includes the total number of piglets born and the number of piglets born alive.

[0008] Furthermore, the pig is a Juema pig.

[0009] Furthermore, the litter size performance of pigs was identified by detecting the genotype of the Ssc.X:101892220C>T site. The litter size performance of individuals with the TT genotype of the Ssc.X:101892220C>T site was better than that of other genotypes.

[0010] Primers for amplifying the Ssc.X:101892220C>T site:

[0011] The upstream sequence is: 5'GGACCATTTCATCGG 3' (shown in SEQ ID NO: 1),

[0012] The downstream sequence is: 5'ATAGCCTAACAAAGAGTG 3' (shown in SEQ ID NO: 2). The amplified sequence is shown in SEQ ID NO: 3.

[0013] A kit for identifying pig litter performance, comprising the above-mentioned primers.

[0014] Application of the primers or the kit in screening pig litter traits.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The present invention screened out a molecular marker Ssc.X:101892220C>T that can significantly affect the litter size of Juema pigs. Through the selection and breeding of TT-type individuals at the Ssc.X:101892220C>T locus, the total number of piglets born was increased from 5.88 to 10.67, and the number of live piglets was increased from 5.58 to 9.33. This molecular marker can be effectively used in the identification of litter size performance and / or breeding of Juema pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Ssc.X:101892220C>T site sequencing peak diagram. DETAILED DESCRIPTION

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0019] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof.

[0020] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0021] Example

[0022] The pig samples, reproductive performance data, and pedigree data described in this example were from Gansu Juema pigs.

[0023] The SNP sites involved in the present invention were obtained from GWAS analysis of the GenoBaits Porcine 50K Pane gene chip sequencing results.

[0024] Statistics of pig GenoBaits Porcine 50K Pane gene chip typing data, SNP chip quality control conditions:

[0025] 1. Sites with minor allele frequency (MAF) less than 0.05 were removed.

[0026] 2. Greater than 20% of sites are removed.

[0027] 3. Sites with a heterozygous ratio greater than 50% were removed.

[0028] 4. Removal of non-biallelic sites.

[0029] The SNP quality control results are as follows, and the remaining 74,651 SNP results are used for subsequent sample quality control.

[0030] Table 1 Statistics of tag filtering results

[0031]

[0032] The samples were filtered based on the 74,651 loci after the above filtering. Completeness filtering selected samples with a SNP detection rate exceeding 80%, that is, for a single sample, at least 80 out of 100 markers had a confirmed genotype.

[0033] After GWAS analysis of the total piglet number and live piglet number traits of Juema pig, the P value was less than 1×E using the formula Ps=α / N -5 (-log 10(P-value) = 5) to perform subsequent analysis. The total number of piglets and live piglets traits of the pig were analyzed by GLM model, GLM(Q) model, MLM(K) model and MLM(QK) model, and a plurality of significant SNP sites were found, wherein the site involved in the patent is distributed in SscX chromosome, and the results are as follows in Table 2. -5 ) to perform subsequent analysis. The total number of piglets and live piglets traits of the pig were analyzed by GLM model, GLM(Q) model, MLM(K) model and MLM(QK) model, and a plurality of significant SNP sites were found, wherein the site involved in the patent is distributed in SscX chromosome, and the results are as follows in Table 2.

[0034] Table 2 Whole genome association significant site statistics of total number of piglets and live piglets of the pig (P < 5 x 10 -5 )

[0035]

[0036] 1 Chr. = Chromosome; 2 P-val. = P value;

[0037] From the above Table 2, the association degree of SscX: 101892220 site with the number of piglets and the number of live piglets is less than 1 x 10 -2.5 , and the association is significant.

[0038] Determination of SscX: 101892220 C > T site

[0039] (1) Collect pig ear samples, and extract pig ear tissue DNA by using a DNA extraction kit.

[0040] (2) PCR amplification is performed by using the following primers

[0041] The upstream sequence of the primer is 5'GGACCATTTCATCGG 3', and the downstream sequence is 5'ATAGCCTAACAAAGAGTG 3'.

[0042] (3) PCR amplification is performed by using the above primers with pig ear DNA as a template.

[0043] (4) Sequencing and sequencing results are as follows:

[0044] The PCR amplification product is sequenced. The C > T mutation at the chromosome 101892220 C > T site is shown in Table 3, and the sequencing peak diagram is shown in Figure 1 .

[0045] Table 3 Mutation site position information and mutation type information

[0046]

[0047] The genotype of the pig and the phenotype value of the total number of piglets and live piglets of the pig are analyzed by the least square method.

[0048] 140 Juema pigs were selected to verify the 101892220C>T locus:

[0049] Table 4 below shows the phenotypic values ​​for litter size for these 140 Juema pigs. The parity distribution for the entire main herd was wide, ranging from 1 to 8. The minimum total piglet born was 2, and the maximum was 14. The minimum number of piglets born alive was 0, and the maximum was 11. The average parity was 1.69, the average total piglet born was 5.88, and the average number of piglets born alive was 5.58.

[0050] Table 4 Descriptive statistics of litter characteristics of Juema pigs

[0051]

[0052] The correlation results are shown in Table 5 below:

[0053] Table 5 Association analysis between X_101892220 locus and reproductive traits of Juema Tibetan pigs

[0054]

[0055]

[0056] By selecting and breeding TT-type individuals at the Ssc.X:101892220C>T locus, the total number of piglets born increased from 5.88 to 10.67, and the number of live piglets increased from 5.58 to 9.33. This molecular marker can be effectively used in the identification of piglet performance and / or breeding of Juema pigs.

[0057] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The use of primers for detecting the Ssc.X:101892220C>T site in screening for litter size traits in Juema pigs is characterized by: The litter size traits are the total number of piglets born and the number of piglets born alive. The piglet size performance is identified by detecting the genotype of the Ssc.X:101892220C>T site. The litter size performance of individuals with the TT genotype of the Ssc.X:101892220C>T site is better than that of other genotypes. The site is the genomic position of Sus Scorfa11.1 version.

2. The use according to claim 1, characterized in that The sequences of the primers are: The upstream sequence is: 5'GGACCATTTCATCGG 3', The downstream sequence is: 5'ATAGCCTAACAAAGAGTG 3'.

3. Application of a kit for screening litter traits in Juema pigs, characterized in that: The kit contains the following primers for amplifying the Ssc.X:101892220C>T site: The upstream sequence is: 5'GGACCATTTCATCGG 3', The downstream sequence is: 5′ATAGCCTAACAAAGAGTG 3′; The litter size traits are the total number of piglets born and the number of piglets born alive. The piglet size performance is identified by detecting the genotype of the Ssc.X:101892220C>T site. The litter size performance of individuals with the TT genotype of the Ssc.X:101892220C>T site is better than that of other genotypes. The site is the genomic position of Sus Scorfa11.1 version.

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