CN110760592B - SNP (single nucleotide polymorphism) site related to bone weight on chromosome 6 of meat Simmental cattle and application - Google Patents

SNP (single nucleotide polymorphism) site related to bone weight on chromosome 6 of meat Simmental cattle and application Download PDF

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CN110760592B
CN110760592B CN201910930931.4A CN201910930931A CN110760592B CN 110760592 B CN110760592 B CN 110760592B CN 201910930931 A CN201910930931 A CN 201910930931A CN 110760592 B CN110760592 B CN 110760592B
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朱波
李俊雅
徐凌洋
高会江
庄站伟
高雪
张路培
陈燕
蔡文涛
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Inner Mongolia okos animal husbandry Co.,Ltd.
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Abstract

The invention provides an SNP locus related to bone weight on a chromosome 6 of a meat Simmental cattle and application thereof, wherein the locus of the SNP marker is 38576012 th nucleotide locus on a chromosome 6 of UMD3.1 version of the international cattle reference genome, and the base of the locus is G or A. By optimizing the dominant allele of the SNP, the invention can increase the frequency of the dominant allele generation by generation, improve the bone weight of the meat Simmental cattle, accelerate the genetic improvement progress of the cattle and effectively improve the economic benefit of beef cattle breeding.

Description

SNP (single nucleotide polymorphism) site related to bone weight on chromosome 6 of meat Simmental cattle and application
Technical Field
The invention relates to an SNP locus related to bone weight on a chromosome 6 of a meat Simmental cattle and application thereof.
Background
The Simmental beef cattle is taken as a main beef cattle variety in China, and a large amount of beef is provided for consumers in China every year. Therefore, the molecular breeding process of beef cattle in China is accelerated, and more high-quality beef is produced to meet the demand of people on good life quality. The bone weight is the sum of the bone weights of the parts of the body of a beef cattle after slaughtering, and generally, the larger the bone weight is, the larger the body size of the cattle is, and therefore, the amount of beef of the cattle is also large. Bone weight is a quantitative trait controlled by multiple genes, and there are a number of Quantitative Trait Loci (QTLs) on the bovine genome that affect bone weight. At present, more than a plurality of bone weight-related QTLs and a large number of Single Nucleotide Polymorphisms (SNPs) are identified on a bovine genome by using a genome-wide association analysis (GWAS) method, and the SNP with a significant effect is added into molecular Marker Assisted Selection (MAS) and Genome Selection (GS) so as to significantly improve the genetic improvement progress of the bone weight and further improve the bone weight of the offspring beef cattle.
The whole genome association analysis is an analysis method for identifying the relationship between the influence phenotype and the genotype by a statistical analysis strategy based on linkage disequilibrium among SNPs, and plays an important role in identifying molecular markers influencing important economic traits of cattle. The obvious SNP influencing the bone weight of the meat Simmental cattle is identified by a GWAS analysis strategy, and the SNP is used for molecular marker-assisted selection and genome selection to select the genotype favorable for improving the bone weight for reservation, so that the gene frequency of the dominant allele is improved generation by generation, the breeding improvement process of the cattle can be accelerated, and great economic benefit is brought to the breeding of the beef cattle.
Disclosure of Invention
In order to achieve the above objects, the first object of the present invention is to provide a SNP site on chromosome 6 of a bovine animal associated with the bone weight of a meat simmental bovine animal, wherein the nucleotide sequence of the molecular marker is as set forth in SEQ ID NO: 1, wherein M in the sequence is G or A, resulting in differences in bovine bone weight.
The molecular marker is positioned on a nucleotide sequence on a chromosome 6 of a meat Simmental cattle, and the SNP locus of the molecular marker is SEQ ID NO: 1, nucleotide mutation of G206-a206 at position 206 of the sequence notation; the SNP locus of the molecular marker corresponds to the 38576012 th G & gtA mutation on the chromosome 6 of the reference sequence of the international bovine genome UMD3.1 version.
Another object of the present invention is to provide a method for screening high bone weight bovine individuals by using the above molecular marker, specifically, the molecular marker of claim 1 on chromosome 6 of a bovine individual is detected, the 206 th nucleotide of the 5' end of the molecular marker is G or A, and the A is retained in the eliminated G. The cattle is selected from western siemens cattle resource groups for meat in pasture of inner Mongolia Silo Allium management area.
Another object of the present invention is to provide a primer pair for identifying the above molecular marker affecting bovine bone weight, wherein the nucleic acid sequence of the primer pair is as follows:
the sequence of the forward primer is shown as SEQ ID NO: 2 is shown in the specification;
the reverse primer sequence is shown as SEQ ID NO: 3, respectively.
The primer pair is applied to identification of influence on bovine bone weight.
The application of the primer pair in the selection of bovine genome is provided.
The primer pair is applied to the improvement of the weight of the bovine bone.
The invention aims to provide a method for genetic improvement of cattle, which comprises the following steps: determining the sites of the above molecular markers affecting the weight of the bovine bone of the bovine in the bovine resource population, and making corresponding selections according to the molecular markers: selecting cattle individuals with GG, GA and AA genotypes at 38576012 th site on UMD3.1 version 6 chromosome of the international cattle reference genome, and eliminating cattle individuals with GG genotypes at 38576012 th site to increase the frequency of allele A at the site generation by generation, thereby increasing the bone weight of the offspring cattle.
Compared with the prior art, the invention has the following advantages and effects:
the invention researches and determines the molecular marker related to the bone weight of the cattle, verifies the influence effect of the molecular marker on the bone weight, finally establishes an efficient and accurate genome selective breeding technology, and applies the efficient and accurate genome selective breeding technology to genetic improvement of the bone weight of the cattle, thereby improving the bone weight of the cattle of offspring and further increasing the market competitiveness of breeding enterprises.
Drawings
FIG. 1 is a genome-wide association analysis (GWAS) Manhattan plot of meat Simmental cattle on chromosome 6 for bone weight; wherein: the abscissa represents the chromosome number of cattle; the ordinate represents the-logP value.
FIG. 2 shows the bone weight of different genotypes of beef Simmental cattle.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The above object of the present invention is specifically achieved by:
example 1
1. Laboratory animal
The experimental cattle groups used in the invention are all derived from Simmental cattle 1219 for meat in pasture of Wula Gagai management area of Guo of inner Mongolia stannum, and are meat Simmental cattle resource groups established by cattle genetic breeding innovation teams of Beijing animal veterinary institute of Chinese academy of agricultural sciences.
In the experiment, 1219 Simmental cattle for meat are selected from the resource group. The Simmental cattle resource population for the meat is expanded every year, and newly added individuals generally go through 3 stages of birth, fattening and slaughtering. After the calf born in 3-5 months per year is stocked and managed for a period of time, the calf genetic breeding innovation team performs unified birth weight and body size measurement in the same year in 7 months, and simultaneously performs measurement on the basic cow. And uniformly and intensively fattening young cattle of 5-9 months of age in the same year in 10 months, collecting phenotype data of growth and development traits, and simultaneously carrying out genotyping on Illumina Bovine HD chips to obtain genotype data. When the fattening period of all individuals reaches 10-12 months, namely about 11 months in the next year, all meat is slaughtered in batches by Simmental cattle. The slaughtering process is strictly executed according to meat procurement specifications, and slaughtering data, carcass data and meat quality data are strictly measured according to the requirements of GB/G27643 plus 2011 guidelines for measuring carcass traits and meat quality traits after slaughter.
2. Sample collection
Collecting venous blood 50ml of all individuals of the cattle group by using a blood collection tube, and storing the venous blood in a refrigerator at the temperature of 80 ℃ below zero for later use. 3. SNP (single nucleotide polymorphism) judgment of cattle whole genome 770K high-density chip
Venous blood of 50ml is collected from each individual of 1219 meat simmental cattle selected from the above resource groups, whole genome DNA is extracted by standard phenol-chloroform method, and DNA concentration and OD ratio (OD260/280, OD260/230) of each sample are accurately determined by a Nanodrop2000/2000C nucleic acid protein detector. And detecting qualified DNA samples by a NanoDrop2000/2000C nucleic acid protein detector, and diluting the DNA to about 50 ng/. mu.L according to the detected concentration. Then mixing 6 mul of extracted DNA sample to be detected with 2 mul of Loading Buffer, Loading the sample into 1.5% agarose gel, carrying out electrophoresis for 20min under the voltage of 150V, observing and photographing under an ultraviolet spectrophotometer and gel imaging equipment, and observing the integrity of the DNA.
DNA samples were sent to Neuggium Biotechnology (Shanghai) Co., Ltd and genotype determination of cattle Whole genome Illumina Bovine HD chip 770K SNP chip (Illumina, USA) was carried out according to the company standard procedures. Quality control is carried out on all 770K chip scanning typing data of the sample by utilizing PLINK v1.90 software, the rejection rate is lower than 90%, the family Mendelian error rate is higher than 0.1, the minimum allele frequency is lower than 0.05, and the Hardy-Weinberg equilibrium significance level is higher than 10-6Finally, 671,204 effective genotype data of the SNPs are obtained.
4. Genome-wide association (GWAS) analysis
In order to eliminate the population stratification effect, the GWAS analysis is carried out by adopting single-point regression analysis of a linear mixed model and combining with an R language GenABEL software package, and the stratification effect is corrected by utilizing the similarity of genomes among individuals in an analysis model. Determining a significance threshold value of the association degree of the SNP and the bone weight by adopting a Bonferrini method, wherein the genome level significance threshold value is 0.05 divided by the number of effective SNP loci, namely the genome significance level threshold value is 7.45e-8, namely 0.05/671,204 (the number of effective SNPs); the chromosome level significance threshold was 1 divided by the number of effective SNP sites, i.e., the chromosome significance threshold was 1.49e-6, i.e., 1/671,204 (effective SNP number).
The GWAS analysis results are shown in fig. 1. As can be seen from fig. 1, there are sites in chromosome 6 of meat simmental cattle that significantly affect bone weight, and the most strongly associated SNP is g.206g > a (P ═ 7.13E-10).
5. Association analysis of different genotypes with bone remodelling types
As can be seen from Table 1, the SNP site g.206G > A of the molecular marker is extremely obviously related to the bone weight (P < 0.001), which shows that the molecular marker obviously affects the bone weight of cattle, and the bone weight of cattle in the group can be improved by auxiliary selection of the SNP site of the cattle, thereby accelerating the breeding process of target traits.
Also, as is clear from table 1, AA type and GA type are heavier than GG type bones, indicating that GG type cattle are disadvantageous for screening of high bone weight, and therefore, AA and GA type cattle are preferentially retained. Therefore, it is necessary to gradually eliminate GG-type cattle in the breeding process to increase the frequency of allele A at the locus generation by generation.
TABLE 1 correlation of SNP site g.206G > A of molecular marker with bone weight
Figure BSA0000191361020000051
6. Amplification and sequencing of DNA sequences of interest
(1) Primer design
Downloading the sequence of SEQ ID NO on chromosome 6 of cattle through Ensemb1 website (hGGp:// asia. ensembl. org/index. hGml): 1. And primers were designed using primer premier 6.0, primer design software.
The DNA sequences of the designed primers are shown below:
p001 forward direction: 5'-GGACAGTGAGGGAAGGCATC-3' the flow of the air in the air conditioner,
p002 reverses: 5'-GGGCTGCACTTGCTGATACT-3', respectively;
(2) PCR amplification
To a 10uL reaction system, 1uL DNA template, 3.4uL double distilled water, 2 Xtag PCR StanMix with Loading Dye 5uL, and 0.3uL each of primers P001 and P002 were added. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 45s, 35 cycles, and final extension at 72 ℃ for 5 min.
(3) DNA sequencing
DNA sequencing identification: the two reactions of the gene fragment were measured in Beijing Biotechnology technology Ltd. The measured sequence was compared with the NCBI genomic sequence to obtain the mutation of the corresponding SNP site. The sequencing results are shown below:
Figure BSA0000191361020000052
Figure BSA0000191361020000061
note: m marked in the sequence listing is a mutation site and is shown by underlining (the mutation base is shown in parentheses, and is an allelic mutation), and the head and the tail of the sequence are shown in bold as the designed primer sequence position.
7. Molecular marker SNP site g.206G & gtA effect analysis
Through the auxiliary selection of the molecular marker, cattle with GG gene types in the colony are eliminated, the bone weight of the colony can be obviously improved, and more economic benefits are brought to enterprises.
The invention relates to a method for preparing a polypeptide shown in SEQ ID NO: 1 sequence, and preliminarily performing the correlation analysis between the genotype and the bovine bone weight, thereby providing a new molecular marker for bovine molecular marker-assisted selection and genome selection.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Figure RE-ISB0000185261800000011

Claims (4)

1. A method for increasing the bone weight of a meat simmental cow, comprising the steps of:
detecting the genotype of 38576012 th nucleotide sites on chromosome 6 of UMD3.1 version 6 of the international reference genome of cattle, and selecting the AA and GA type individuals at 38576012 th nucleotide sites as cattle.
2. The method as claimed in claim 1, wherein the method for detecting the genotype of the 38576012 th nucleotide site on the chromosome 6 of the international bovine reference genome UMD3.1 version 6 of the cattle comprises the following steps:
(1) extracting the genomic DNA of the cattle to be detected;
(2) carrying out PCR amplification on the genomic DNA of the cattle to be detected by adopting a primer pair so as to obtain a PCR amplification product;
(3) sequencing the PCR amplification product so as to obtain a sequencing result;
(4) determining the genotype of the SNP marker of the cattle to be detected based on the sequencing result;
the nucleic acid sequence of the primer pair is shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification;
the sequence of the SNP marker is shown as SEQ ID NO: 1, the sequence shown in SEQ ID NO: 1 is G or A at the 206 th base from the 5' end.
3. The application of the SNP marker in increasing the bone weight of the meat Simmental cattle is characterized in that the sequence of the SNP marker is shown as SEQ ID NO: 1, the sequence shown in SEQ ID NO: 1 is G or A at the 206 th base from the 5' end.
4. The application of a primer pair in improving the weight of bones of a meat Simmental cattle is characterized in that the nucleic acid sequence of the primer pair is shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
dbSNP build 148 rs109315366;ucsc;《UCSC》;20160730;全文 *
Effect of single nucleotide polymorphisms on intramuscular fat content in Hungarian Simmental cattle;István Anton et al;《AJAS》;20180930;第31卷(第9期);第1415-1419页 *
西门塔尔牛骨重和酮体重复合策略全基因组关联分析;苗健;《福建农林大学硕士学位论文》;20181231;全文 *

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