CN111500743B - Method for increasing weight of Chinese and cattle loin - Google Patents

Abstract

The invention provides a method for improving Chinese and cattle Ridge weight, wherein the adopted SNP marked locus is 54648801 nucleotide locus on chromosome No. 22 of International cattle reference genome UMD3.1 version, and the basic group of the locus is C 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 Rizhao weight of China and cattle, accelerate the genetic improvement progress of cattle and effectively improve the economic benefit of beef cattle breeding.

Description

Method for increasing weight of Chinese and cattle loin

Technical Field

The invention relates to analysis of Chinese and bovine chromosome gene loci and related traits, in particular to SNP loci related to the weight of Ridge on Chinese and bovine chromosomes and application thereof.

Background

The beef cattle industry is an important component of the animal husbandry in China, the development level of the beef cattle industry is an important sign of the national agricultural development level, and the beef cattle industry has great significance for the construction of Chinese vegetable basket engineering, the increase of the income of farmers, the guarantee of the meat consumption safety of people and the social stability. In the aspect of production efficiency, the average carcass weight of beef cattle in developed countries of beef cattle industry such as America, Canada and the like is more than 340 kg, and the beef cattle are mostly slaughtered at 15-18 months old, while only 154 kg is slaughtered in China, and the slaughtering time is 18-24 months old, so that the production efficiency of the beef cattle in China is far lower than that in developed countries. The Chinese cattle and the Fuzhou cattle are filial generations of the Heniu cattle and the Fuzhou cattle, and the Fuzhou cattle is a contemporary variety of Liaoning province in China, and aims to produce high-grade beef, and the production is positioned to be a high-grade income group. In order to accelerate the molecular breeding process of beef cattle in China, the significant sites influencing the Riji weight traits of China and cattle are identified by adopting whole genome association analysis. Research shows that the weight of the Chinese Ridgeon belongs to the moderate heritability trait, about 0.39, so that the genetic means is feasible for improving the weight of the Chinese Ridgeon and the cattle Ridgeon in resource groups. Ridge 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 Ridge weight. At present, by using a genome-wide association analysis (GWAS) method, more than a plurality of QTLs related to the weight of the internal spine and a large number of Single Nucleotide Polymorphisms (SNPs) are identified on a cattle genome, and SNP with a remarkable effect is added into molecular Marker Assisted Selection (MAS) and Genome Selection (GS), so that the genetic improvement progress of the internal spine weight can be remarkably improved, the internal spine weight of the offspring beef cattle can be improved, the beef yield can be improved, and the market competitiveness of commercial beef production of breeding enterprises can be enhanced.

Disclosure of Invention

The invention aims to provide a method for genetic improvement of cattle. Significant SNP influencing Chinese and cattle Rizhao weight is identified through a GWAS analysis strategy, and is used in molecular marker-assisted selection and genome selection to select a genotype favorable for improving Rizhao weight for seed reservation, so that the gene frequency of dominant alleles is improved generation by generation, the breeding improvement process of cattle can be accelerated, and great economic benefit is brought to beef cattle breeding.

The invention is realized by the following steps:

the method comprises the following steps: determining the sites of the above molecular markers affecting the weight of the cattle Ridge of the cattle in the cattle resource population, and making corresponding selection according to the molecular markers: selecting cattle individuals with CC, CA and AA genotypes at 54648801 th site on UMD3.1 version 22 chromosome of the international cattle reference genome, eliminating cattle individuals with CC genotypes at 54648801 th site, and increasing the frequency of allele A at the site by generations so as to increase the internal ridge weight of the offspring cattle.

The method specifically comprises the following steps:

(1) extracting the genomic DNA of the cattle to be detected;

(2) performing PCR amplification on the genomic DNA of the cattle to be detected by using the primer pair of claim 3 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 according to claim 1 or 2 of the bovine subject to be tested based on the sequencing result.

Compared with the prior art, the invention has the following advantages and effects:

the invention researches and determines the molecular marker related to the weight of the cattle loin, verifies the influence effect of the molecular marker on the weight of the cattle loin, 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 cattle for improving the weight of the loin, thereby improving the weight of the future generation cattle, improving the meat yield and further increasing the market competitiveness of breeding enterprises.

Drawings

FIG. 1 is a Global genome Association analysis (GWAS) Manhattan plot for Ridge weight on chromosome 22 in China and cattle; wherein: the abscissa indicates the chromosome number (bp) of cattle; the ordinate represents the-logP value.

FIG. 2 shows the internal ridge weights of Chinese and bovine species with different genotypes.

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 used in the invention are all from 462 Chinese cattle of Dalian Xuelongte GmbH of Liaoning province, and are Chinese and cattle resource groups established by cattle genetic breeding innovation team of Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences.

In this experiment, 462 Chinese and cattle in the resource population were selected. Chinese and cattle resource groups are expanded every year, and newly increased 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 24-30 months, namely about 11 months in the third year, all China and cattle are slaughtered in batches. 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/T27643 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

Collecting 50ml venous blood from 462 meat of the above resource groups of each individual of Simmental cattle, extracting whole genome DNA by standard phenol-chloroform method, and accurately determining DNA concentration and OD ratio (OD260/280, OD260/230) of each sample by 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. And mixing 6 mu l of the extracted DNA sample to be detected with 2 mu l of Loading Buffer, Loading the mixture into 1% agarose gel, carrying out electrophoresis for 25min 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, 507,812 effective genotype data of the SNPs are obtained.

4. Genome-wide association (GWAS) analysis

In order to eliminate the population stratification effect, the invention adopts a compressed linear mixed model single-point regression analysis and combines with an R language GAPIT software package to carry out GWAS analysis, and the stratification effect is corrected by utilizing the similarity of genomes among individuals in an analysis model. Determining a significance threshold of the degree of association of the SNP with the Ridge weight by using a False Discovery Rate (FDR):

P＝FDR×n/m

FDR was set to 0.01, n is the number of SNPs with P value <0.01, and m is the number of total SNPs.

The GWAS analysis results are shown in fig. 1. As can be seen from fig. 1, there are sites in the meat simmental cow chromosome 22 that significantly affect the weight of the inner ridge, and the most strongly associated SNP is g.97c > a (P ═ 7.24E-6).

5. Association analysis of different genotypes with Ridge weight phenotype

As can be seen from Table 1, the SNP site g.97C > A of the molecular marker is very significantly related to the weight of the inner ridge (P <0.001), which shows that the molecular marker significantly affects the weight of the inner ridge of cattle, and the weight of the inner ridge of the population can be increased by auxiliary selection of the SNP site of cattle, thereby accelerating the breeding process of the trait.

Also, as can be seen from table 1, the AA and CA forms had higher weight of the inner ridge than the CC form, indicating that the CC form cattle individuals were not favorable for selecting the weight of the inner ridge, and therefore, the AA and CA forms were preferentially retained. The weight of the inner spine is an important index for measuring the beef yield, and the improvement of the weight of the inner spine of the cattle is beneficial to improving the beef yield, so that the market competitiveness is improved. Therefore, in the breeding process, CC-type cattle need to be gradually eliminated, AA-type and CA-type cattle are preferentially reserved, and the frequency of the allele A of the locus is increased generation by generation.

TABLE 1 correlation of SNP site g.97C > A of molecular markers with Ridge weight

6. Amplification and sequencing of DNA sequences of interest

(1) Primer design

The DNA sequence of SEQ ID NO:1 on chromosome 22 of cattle was downloaded via Ensembl website (http:// asia. ensemble. org/index. html). And primers were designed using primer premier 5.0, primer design software.

The DNA sequences of the designed primers are shown below:

p001 forward direction: 5'-TGAAGATCAAGGACCCCACG-3' the flow of the air in the air conditioner,

p002 reverses: 5'-GGTTACATACTTAGCCTCAG-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 60 ℃ 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:

TGAAGATCAAGGACCCCACGGGGCGCAACTAGGACCCTCGTAGGCAAATCAACTAATTTTAAAAAGTGATAACCAAGCAAAATGCTTACTTCCTCTM(C>A)CACACCCTTCCAGGAACACTGATTCCAACTGGGACATGGCTCTGCCTTGTACTCCCTTGGGTTACATACTTAGCCTCAG

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.97C > A effect analysis

By the auxiliary selection of the molecular marker, the cattle with CC gene type in the group are eliminated, the internal ridge weight of the group can be obviously improved, the meat yield is improved, and more economic benefits are brought to enterprises.

The invention provides a new molecular marker for bovine molecular marker-assisted selection and genome selection by detecting the 97 th base mutation site in the SEQ ID NO. 1 sequence and primarily applying the correlation analysis between the genotype and the bovine internal spine weight.

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.

Claims (2)

1. A method of increasing the weight of chinese and beef loins, comprising the steps of:

detecting the genotype of 54648801 th nucleotide sites on chromosome of UMD3.1 version 22 of the international reference genome of cattle, and selecting 54648801 th nucleotide sites of AA and CA individuals as cattle.

2. The method of claim 1, wherein the method for detecting the genotype of the 54648801 th nucleotide site on the chromosome 22 of the international bovine reference genome UMD3.1 version of the bovine 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 according to claim 1 of the bovine to be tested based on the sequencing result.

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