A SNP marker for evaluating pig growth traits and its detection method and application

Abstract

The present invention discloses a method for identifying or assisting in the identification of pig growth traits, the method comprising: detecting the genotype of a SNP marker in the genome of the pig to be tested, the SNP marker being the 251st nucleotide of the sequence shown in sequence 1 on chromosome 13 of version 10.2 of the pig reference genome, which is A or G. The present invention provides a SNP molecular marker associated with the age trait of pigs reaching 100 kg in weight and its application. By measuring the growth performance data (age at 100 kg in weight) of 1,173 pigs (Duroc, Landrace and Large White pigs), and performing genotyping on the target pig group, a whole-genome association analysis was performed after quality control. The analysis obtained a SNP site that was significantly associated with the age at which the pig reached 100 kg in weight, and the site was located on chromosome 13 of the pig.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a SNP marker related to pig growth performance, a detection method and an application thereof. Background Art

[0002] Animal husbandry is a key industry supporting my country's agricultural economic growth. Pig farming is a crucial component of this sector. As one of the primary sources of animal protein for Chinese residents, pork has always held a dominant position in the country's meat consumption market. Improving pork production and quality is directly linked to improvements in people's quality of life, the economic benefits of businesses, and the stable development of the pig industry.

[0003] In the pig farming industry, pig growth traits are crucial factors influencing pork production and quality. Days to 100kg (D100) is a key indicator of growth performance. Selecting pigs with a low D100 age helps improve feed utilization, lean meat percentage, and daily weight gain in the pig farming industry, allowing for timely adjustments and control of market timing to maximize economic benefits and optimize pork quality. This is also crucial for preserving and expanding the core breeding population of local pig breeds, increasing farming scale, and clarifying the direction and progress of breed improvement.

[0004] Pig growth traits are quantitative traits controlled by multiple genes. At present, the QTL regions related to the age at which pigs reach 100 kg body weight are distributed on all autosomes except sex chromosomes. Most of the QTL maps for growth traits that have been reported so far have relatively large confidence intervals, making it impossible to accurately locate key genes for growth performance. Single nucleotide polymorphisms (SNPs) mainly include four types: transversion, transition, insertion and deletion. Due to their large number, high density and wide distribution, they are widely used in the evaluation of economic traits in pig breeding. Whole-genome association analysis is currently an important means to explore SNP sites related to complex phenotypic traits, and can be used for accurate molecular breeding marker identification of target traits. At present, second-generation sequencing technology, high-density SNP chips and whole-genome association analysis have been effectively used in whole-genome design breeding and excellent trait evaluation and diagnosis of animals and plants. Therefore, the present invention uses pig SNP typing data and phenotypic data related to its growth traits to conduct genome-wide association analysis, and finds SNP marker sites that are significantly associated with the age at which pigs reach 100 kg body weight, laying the foundation for the optimization and improvement of pig growth traits. Summary of the Invention

[0005] In response to the problems that the confidence intervals of some QTLs related to the age at which pigs reach 100 kg body weight are relatively large, making it difficult to accurately locate key genes and sites, the present invention uses whole-genome association analysis to solve and realize the accurate and rapid detection of excellent breed pigs (the age at which they reach 100 kg body weight is low).

[0006] In view of the shortcomings of the existing technology for molecular breeding of pig growth traits, the purpose of the present invention is to provide SNP markers related to the age at which pigs reach 100 kg body weight for molecular breeding of pig growth traits.

[0007] The present invention provides a method for identifying or assisting in the identification of pig growth traits, comprising: detecting the genotype of a SNP marker ALGA0072703 in the pig genome to be tested, wherein the SNP marker ALGA0072703 is located at the 251st nucleotide of the sequence shown in sequence 1 on chromosome 13 of the 10.2 version of the pig reference genome, and is A or G.

[0008] The growth trait is the age at which the pig reaches 100 kg of body weight, wherein the age at which the pigs with GG genotype and GA genotype reach 100 kg of body weight is greater than the age at which the pigs with AA genotype reach 100 kg of body weight;

[0009] The SNP marker ALGA0072703 of the AA genotype is AA;

[0010] The SNP marker ALGA0072703 of the AG genotype is AG;

[0011] The SNP marker ALGA0072703 of the GG genotype is GG.

[0012] The use of a substance for detecting the polymorphism or genotype of the SNP marker ALGA0072703 in the following A1-A2 should also be within the scope of protection of the present invention; the SNP marker ALGA0072703 is located at nucleotide position 251 of the sequence shown in sequence 1 on chromosome 13 of the 10.2 version of the porcine reference genome, which is A or G:

[0013] A1, application in identifying or assisting in identifying pigs up to 100 kg in weight;

[0014] A2, Application in pig breeding.

[0015] The pig breeding in A2 is to cultivate pigs of small breeds with a body weight of 100 kg per day.

[0016] The material for detecting the polymorphism or genotype of the SNP marker ALGA0072703 is a SNP chip for detecting the polymorphism or genotype of the SNP marker ALGA0072703.

[0017] The present invention provides a product, comprising the substance for detecting the polymorphism or genotype of the SNP marker ALGA0072703.

[0018] The present invention provides a pig breeding method, which comprises: detecting the genotype of the SNP marker ALGA0072703 in the genome of the pig to be tested, and selecting pigs with homozygous AA genotype for breeding.

[0019] The application of the product or the method in pig breeding should also be within the scope of protection of the present invention.

[0020] The purpose of the breeding is to produce pigs that reach 100 kg body weight per day.

[0021] The pigs mentioned above are Duroc, Landrace and Large White.

[0022] The beneficial effects of the present invention are as follows: the present invention uses pigs as research objects, and through genotyping and whole genome association analysis, obtains a SNP marker ALGA0072703 that is significantly correlated with the age at which pigs reach 100 kg in weight, and the site is located on pig chromosome 13. The present invention provides a SNP molecular marker related to the trait of the age at which pigs reach 100 kg in weight and its application. By measuring the growth performance data (age at 100 kg in weight) of 1,173 pigs (Duroc, Landrace and Large White pigs), and performing genotyping on the target pig group, a whole genome association analysis was performed after quality control. The analysis obtained the SNP site ALGA0072703 that is significantly correlated with the age at which pigs reach 100 kg in weight, and the site is located on pig chromosome 13. By detecting different genotypes of this SNP marker, it is possible to promote the determination and genetic evaluation of my country's breeding pigs, improve the production performance of my country's breeding pig core group, and promote the genetic improvement of breeding pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the association analysis between SNP site ALGA0072703 and pig growth traits, D100: age at 100 kg body weight. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0025] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0026] Implementation Cases:

[0027] 1. Experimental animals: Duroc pigs, Landrace pigs and Large White pigs from Hebei Meishen original pig farm.

[0028] 2 Extraction of pig genomic DNA

[0029] Four ear tissue samples were collected from each of 1,173 pigs, one of which was used for individual DNA extraction;

[0030] Refer to the instructions of the tissue DNA extraction kit of Tiangen Biotechnology Co., Ltd. and perform the extraction in the following order:

[0031] ① First, add 68 mL of anhydrous ethanol to buffer GD and 200 mL of anhydrous ethanol to rinse solution PW, respectively, and mix thoroughly.

[0032] ② Collect approximately 100 mg of tissue sample and place it in a 2 mL EP tube. After completely mincing, add 200 μL of buffer GA and shake until completely suspended.

[0033] ③ Add 20 μL of proteinase K solution, mix well, and digest in a 56°C metal bath overnight until the ear sample tissue is dissolved. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0034] ④ Add 200 μL of buffer GB, mix thoroughly by inversion, and place in a 70°C metal bath for 10 min. The solution should become clear. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0035] ⑤ Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0036] ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, place the adsorption column in a collection tube, and then centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid and place the adsorption column CB3 back into the collection tube.

[0037] ⑦ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0038] ⑧ Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0039] ⑨Repeat step ⑧.

[0040] ⑩ Return the adsorption column CB3 to the collection tube and centrifuge at 12,000 rpm for 2 minutes. Discard the waste liquid. Leave the adsorption column CB3 at room temperature for several minutes to completely dry any remaining rinse solution from the adsorption material.

[0041] Transfer the adsorption column CB3 to a clean centrifuge tube, add 100 μL of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 2 minutes, collect the solution into a centrifuge tube, add the solution obtained by centrifugation to the adsorption column CB3, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 2 minutes, and collect the solution into a centrifuge tube.

[0042] After the mass and concentration were detected by Nanodrop-2000 spectrophotometer, the concentration was uniformly diluted to 50 ng / μL and stored at -20°C for future use.

[0043] 3. SNP typing detection based on Neogen's Neogen_POR80K chip platform

[0044] The SNP-based typing platform software GenCall Version 7.0.0 was used to type data from 1,173 individuals: this process covered quality control of phenotypic and genotypic data, and integration of genotype and phenotype, resulting in the genotype of the ALGA0072703 locus. The SNP marker ALGA0072703 is located at the 251st nucleotide of the sequence shown in sequence 1 on chromosome 13 of the 10.2 version of the porcine reference genome, which is A or G, and is represented by R in the sequence listing.

[0045] 4.2 Genome-wide association analysis

[0046] The analysis software package used for the genome-wide association analysis in this study was the R language package GAPIT (http: / / www.zzlab.net / GAPIT / , developed by Professor Zhiwu Zhang's laboratory at the University of Washington). This package uses the compressed mixed linear model as the statistical model. GAPIT is designed to accurately perform GWAS and genomic prediction on large datasets. Mixed linear models (MLMs) include both fixed and random effects. Including individuals as random effects allows MLMs to incorporate information about the relationships between individuals. This information about relationships is conveyed via the kinship (K) matrix, which serves as the variance-covariance matrix between individuals in the MLM. When the genetic marker-based kinship matrix (K) is used in conjunction with the population structure (often referred to as the "Q" matrix, which can be used to perform principal component analysis or structure analysis), the "Q + K" matrix approach exhibits higher statistical power than approaches using only the "Q" matrix. MLM can be described using Henderson's matrix notation as follows: Y = Xβ + Z u + e, where Y is the value of the observed phenotype; β is the unknown value containing fixed effects, including genetic markers, population structure (Q matrix) and intercept; u is the unknown value of the random additive genetic effect of multiple background QTLs from individuals or lines; X and Z are the known design matrices; and e is the unobserved residual vector.

[0047] 4.3 100kg weight age correction

[0048] Select male and female pigs weighing between 80 and 105 kg and weigh them using an electronic weighing platform. Record the pig's ear ID, weight, and actual age at the time of measurement. Convert the corrected age to 100 kg using the following formula: Corrected age = measured age - [(actual weight - 100) / CF];

[0049] Where: Boar CF value = (actual weight / measured age) × 1.826040;

[0050] Sow CF value = (actual weight / measured age) × 1.714615.

[0051] 5 Results

[0052] PopGene 3.2 was used to calculate the genotype frequency and allele frequency of SNP marker sites, the genotype frequency distribution of different traits, and genome-wide association analysis. The statistical results are shown in Tables 1 and 2:

[0053] Table 1 Allele frequency and genotype frequency of ALGA0072703 locus in this population

[0054]

[0055] Table 2 Genome-wide association analysis of growth traits among individuals with different genotypes at the ALGA0072703 locus

[0056]

[0057] Tables 1 and 2 show the effects of the ALGA0072703 mutation site A / G on pig eye muscle area and age at 100 kg weight in pig populations. As shown above, the ALGA0072703 site is significantly associated with both growth traits (P < 0.01). Among the mutant individuals, AA-type individuals exhibited significantly better growth traits (age at 100 kg weight) than AG-type and GG-type individuals (P < 0.05). This suggests that in pig populations, successive breeding of AA-type individuals with the ALGA0072703 site can gradually reduce the age at 100 kg weight, thereby reducing breeding costs and improving meat production in breeding pigs.

[0058] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims. Sequence Listing <110> Foshan Kunpeng Modern Agriculture Research Institute Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences <120> A SNP marker for evaluating pig growth traits and its detection method and application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 501 <212> DNA <213> Artificial Sequence <400> 1 agttgaagat caggacaaaa atgtattccc tattttacat gattataatt cccctgctaa 60 acccatttat ttacagctta agaaataaag aggttatagg taccttgaga agagttgcaa 120 agaagtaaac acctctcaag ggaaatttca aattcttcct cctttcactc tttgcataaa 180 caggtcccca agtcttgcac attggccaca gttctcccct ttcatcagag caccagaggt 240 catggtcacc ragcactggc tgaaggaacc cagcttcaag caatgtttac ttggatgtgg 300 aaattactat tgcagttgta gttactactt cagttgaaat gactcacctc attcacagtt 360 aattagaaat attgtaattc ctttttatct gaactttagg ctttgaaaat ttgctctctg 420 cagggaactc ctcctcctaa ttttattaat ttttttaaat aactgacagt aactgaggct 480 ggtatataag tgtgagtcat t 501

Claims

1. A method for identifying or assisting in identifying pig growth traits, characterized in that: The method comprises: detecting the genotype of a SNP marker in a pig genome to be tested, wherein the SNP marker is located at the 251st nucleotide of the sequence shown in sequence 1 on chromosome 13 of the 10.2 version of the pig reference genome, and the nucleotide is A or G, and the pig is a Duroc pig, a Landrace pig, or a Large White pig; The growth trait is the age at which the pig reaches 100 kg of body weight, wherein the age at which the pigs with GG genotype and GA genotype reach 100 kg of body weight is greater than the age at which the pigs with AA genotype reach 100 kg of body weight; The AA genotype is the homozygous type with SNP marker A; The AG genotype is a heterozygous type with SNP markers A and G; The GG genotype is a homozygous type with the SNP marker G.

2. A pig breeding method, characterized in that: The method comprises: detecting the genotype of the SNP marker described in claim 1 in the genome of the pig to be tested, selecting pigs with homozygous AA genotype for breeding, wherein the pigs are Duroc pigs, Landrace pigs or Large White pigs, and the pig breeding is to cultivate pigs of a small breed with a body weight of 100 kg per day.

3. Application of the method according to claim 1 in pig breeding, wherein the pig breeding is to cultivate pigs of the smallest age with a body weight of 100 kg.

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