CN111500743A - SNP (single nucleotide polymorphism) locus related to Rizhao weight on China and cattle No. 22 chromosome and application technical field - Google Patents
SNP (single nucleotide polymorphism) locus related to Rizhao weight on China and cattle No. 22 chromosome and application technical field Download PDFInfo
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Abstract
The invention provides SNP molecular sites related to Chinese and cattle and Rizhao weight on cattle No. 22 chromosome, wherein the site of the SNP marker is 54648801 nucleotide sites on international cattle reference genome UMD3.1 version No. 22 chromosome, and the base of the site 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
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 livestock industry in China, the development level of the beef cattle industry is an important mark of the national agricultural development level, the beef cattle industry has great significance for vegetable basket engineering construction in China, increase of farmer income, guarantee of meat consumption safety of people and social stability, production efficiency is far lower than that of developed countries in China, China and cattle are crossbreeded with cattle and cattle in Rihou, Rihou cattle are contemporary varieties of Liaoning province in China and aim at producing high-grade beef, production is positioned to be high-grade groups, in order to accelerate the molecular breeding process of beef cattle in China, the significant sites influencing the Ri weight traits of China and cattle are identified by whole genome correlation analysis, the Ri weight belongs to the medium-level force transfer traits and is about 0.39, therefore, improvement of resource groups and the Ri weight of Chinese groups by using genetic means is a significant improvement of the weight of the Ri weight traits of the cattle, the quantitative trait of the Ri weight of the Ri-cattle is improved by a large number of genetic marker (QT) and the genetic marker, the yield of the Wu-related cattle is improved by the GWSe weight marker (QT) and the genetic marker method for improving the weight of the GWSe related genes, the weight of the GWSe-related to the related beef cattle, the related genetic marker of the related cattle, the yield of the cattle, the cattle breeding method has the cattle, the cattle breeding method has the cattle can be improved by the cattle, the cattle can be improved by the weight of the cattle, the cattle can be improved by the cattle, the cattle variety of the cattle, the cattle can be improved by the cattle, the cattle variety of.
Disclosure of Invention
The obvious SNP influencing the Chinese and cattle Rizhao weight 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 Rizhao weight for reserving seeds, so that the gene frequency of the dominant allele is improved generation by generation, the breeding improvement process of cattle can be accelerated, and great economic benefit is brought to the cattle breeding.
The invention is realized by the following steps:
the primary purpose is to provide an SNP molecular marker which influences the immediate weight of China and cattle. The molecular marker is positioned on a nucleotide sequence on a No. 22 chromosome of China and cattle, and the SNP locus of the molecular marker is the nucleotide mutation of C97-A97 with the position 97 marked by a SEQ ID NO. 1 sequence; the SNP locus of the molecular marker corresponds to 54648801 th C > A mutation on the chromosome 22 of the reference sequence of the international bovine genome UMD3.1 version.
The invention also aims to provide a method for screening cattle with high internal spine weight by using the molecular marker, in particular to detect the molecular marker as claimed in claim 1 on cattle chromosome 22, wherein the 97 th single nucleotide at the 5' end of the molecular marker is C or A, and C is eliminated and A is reserved. The cattle is specifically embodied in China and cattle resource groups of Daliang Xuelongte GmbH of Liaoning province.
Another object of the present invention is to provide a primer pair for identifying the above molecular marker affecting the weight of the sirloin, 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 application of the primer pair in identifying the influence on the weight of the Ribes nivalis.
The application of the primer pair in the selection of bovine genome is provided.
The application of the primer pair in improving the weight of the Ribes nigrum.
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 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 of venous blood from each of 462 meat simmental cattle selected from the resource population, extracting whole genome DNA by a standard phenol-chloroform method, accurately measuring the concentration and OD ratio (OD260/280 and OD260/230) of DNA of each sample by a Nanodrop2000/2000C nucleic acid protein detector, detecting qualified DNA samples by a Nanodrop2000/2000C nucleic acid protein detector, diluting the DNA to about 50 ng/mu L according to the detected concentration, mixing 6 mu l of the extracted DNA samples to be detected with 2 mu l of L ° loading Buffer, loading the mixture into 1% agarose gel, performing electrophoresis at 150V for 25min, observing and photographing under an ultraviolet spectrometer and a gel imaging device, and observing the integrity of the DNA.
DNA samples were sent to Neuger Biotechnology (Shanghai) Co., Ltd and bovine whole genome Illumina Bovine HD chip 770K SNP chip (Illumina, USA) was performed according to the company's standard procedures) And (3) carrying out quality control on all 770K chip scanning typing data of the sample by utilizing P L INK v1.90 software, wherein 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 significance level of the Hardy-Weinberg equilibrium 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
A reaction system of 10u L is added with a DNA template of 1u L, double distilled water of 3.4u L, 2 × Tag PCR StanMux with L attached Dye 5u L, and primers P001 and P002 are respectively 0.3ul, wherein the PCR reaction conditions are that after pre-denaturation at 94 ℃ is carried out for 5min, denaturation at 94 ℃ is carried out for 30s, annealing at 60 ℃ is carried out for 30s, extension at 72 ℃ is carried out for 45s, 35 cycles are carried out, and finally extension at 72 ℃ is carried out 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:
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 (9)
1. An SNP molecular marker influencing Chinese and cattle Rizhao weight is characterized in that the locus of the SNP marker is 54648801 th nucleotide locus on chromosome No. 22 of International cattle reference genome UMD3.1 version, and the base of the locus is C or A.
2. The SNP molecular marker according to claim 1, wherein the sequence of the SNP marker
The 97 th base from the 5' end is C or A.
3. A primer set for detecting the SNP marker according to claim 1 or 2, wherein the nucleic acid sequences of the primer set are as follows:
p001 forward direction: 5'-TGAAGATCAAGGACCCCACG-3' the flow of the air in the air conditioner,
p002 reverses: 5'-GGTTACATACTTAGCCTCAG-3' are provided.
4. A kit for detecting the SNP marker according to claim 1 or 2, wherein the kit comprises the primer set according to claim 3.
5. 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.
6. The method of claim 5, 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) 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.
7. The method of claim 5, wherein the bovine population comprises Chinese and bovine and their synthetic lines.
8. Use of the SNP marker according to claim 1 or 2 for increasing the weight of Ribes nivalium.
9. Use of the primer pair according to claim 3 or the kit according to claim 4 for increasing the weight of Ribes nivalis.
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