CN110760593B - SNP (single nucleotide polymorphism) site related to spleen weight on chromosome 6 of meat Simmental cattle and application - Google Patents
SNP (single nucleotide polymorphism) site related to spleen weight on chromosome 6 of meat Simmental cattle and application Download PDFInfo
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Abstract
The invention provides SNP sites related to spleen weight on a chromosome 6 of a meat Simmental cattle and application thereof, wherein the site marked by the SNP is 38852378 th nucleotide site on a chromosome 6 of UMD3.1 version 6 of an international cattle reference genome, and the base of the site 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 spleen weight of the Simmental cattle for meat, accelerate the genetic improvement progress of the cattle and effectively improve the economic benefit of beef cattle breeding.
Description
Technical Field
The invention relates to an SNP locus related to spleen weight on a chromosome 6 of a meat Simmental cattle and application thereof.
Background
The meat Simmental cattle is the most popular mating breed of cattle hybridization group in China, has the characteristics of high growth speed and high meat yield, and is often used as a terminal male parent in breeding production. The cattle spleen is commonly used as a traditional Chinese medicine in China, and has the effects of tonifying spleen, stimulating appetite, removing food retention and removing abdominal mass. Currently, breeding of simmental cattle for meat mainly aims at improving growth traits such as meat yield and growth rate, and attention on spleen weight breeding is less. In addition, spleen weight is a quantitative trait controlled by multiple genes, and there are a large number of Quantitative Trait Loci (QTLs) associated with it on the genome. Therefore, it is difficult to rapidly and accurately improve the progress of breeding of a target trait by using a conventional breeding method. The rapid development of molecular marker technology provides possibility for accurate selection of such traits.
In the past, QTL detection has been performed by Candidate gene approach (Candidate gene approach) and QTL Mapping (QTL Mapping). Although the candidate gene method has the advantages of simple method, convenient operation and the like, the candidate gene method only aims at genes with known biological functions; however, the confidence region of candidate position genes identified by the QTL positioning method is also large, which greatly limits the application of the complex character molecular marker in livestock genetic breeding. With the development of high-throughput sequencing technology and the appearance of whole Genome chips, the whole Genome-wide Association Study (GWAS) has significant effect in the genetic analysis of complex traits, and the bottleneck of molecular marker identification of related traits of cattle carcasses is broken.
Disclosure of Invention
In order to achieve the above objects, the first object of the present invention is to provide SNP sites on chromosome 6 of a bovine animal, wherein the SNP sites are related to spleen weight of a meat siemens cattle, and the nucleotide sequence of the molecular marker is as shown in SEQ ID NO: 1, wherein M in the sequence is G or A, resulting in differences in bovine spleen 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 sequence marking the nucleotide mutation of G69-A69 at position 69; the SNP locus of the molecular marker corresponds to the 38852378 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 bovine individuals with high spleen weight by using the above molecular marker, specifically, the molecular marker of claim 1 on bovine chromosome 6 is detected, the 69 th nucleotide at the 5' end of the molecular marker is G or A, and G is retained in knockout A. 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 spleen 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 spleen 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 spleen.
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 spleen weight of the cattle in the cattle resource population, and making corresponding selection according to the molecular markers: selecting cattle individuals with GG, AG and AA genotypes at 38852378 th site on UMD3.1 version 6 chromosome of the international cattle reference genome, and eliminating cattle individuals with AA genotypes at 38852378 th site to increase the frequency of allele G of the site generation by generation, thereby increasing the spleen weight of the offspring cattle.
Compared with the prior art, the invention has the following advantages and effects:
according to the invention, the molecular marker related to the spleen weight of the cattle is researched and determined, the influence effect of the molecular marker on the spleen weight is verified, and finally, an efficient and accurate genome selective breeding technology is established and applied to genetic improvement of the spleen weight of the cattle, so that the spleen weight of the offspring cattle is improved, and further, the market competitiveness of breeding enterprises is increased.
Drawings
FIG. 1 is a GWAS Manhattan map of Western Mentagar beef on chromosome 6 for spleen weight; wherein: the abscissa represents the chromosome number of cattle; the ordinate represents the-logP value.
FIG. 2 shows spleen weights 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 1093 Simmental cattle for meat in pasture of Wula Gai management area of Guo of inner Mongolia stannum, and are meat Simmental cattle resource groups established by cattle genetic breeding innovation team of Beijing animal veterinary institute of Chinese academy of agricultural sciences.
In the experiment, 1093 meat Simmental cattle in the resource group are selected. 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
50ml of venous blood is collected from 1093 meat selected from the resource groups by each individual in Simmental cattle, whole genome DNA is extracted by a standard phenol-chloroform method, and the concentration and OD ratio (OD260/280, OD260/230) of DNA of each sample are accurately measured 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. 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, 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 degree of the re-association of the SNP and the spleen by adopting a Bonferrini method, wherein the genome level significance threshold value is 0.05 divided by the number of effective SNP sites, 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 significantly affecting spleen weight in the meat simmental cattle chromosome 6, and the most strongly associated SNP is g.69g > a (P ═ 4.91E-17).
5. Association analysis of different genotypes with spleen phenotype
As can be seen from Table 1, the SNP site g.69G & gtA of the molecular marker is extremely significantly related to the spleen weight (P & lt, 0.001), which shows that the molecular marker significantly affects the spleen weight of cattle, and the spleen weight of the population can be increased by auxiliary selection of the SNP site of cattle, thereby accelerating the breeding process of the target character.
Also, as is clear from Table 1, GG-type and AG-type cattle are heavier than AA-type spleen, and it is considered that AA-type cattle are disadvantageous in terms of spleen weight screening, and therefore GG-type and AG-type cattle are preferentially retained. Therefore, in the breeding process, AA type cattle need to be gradually eliminated, so as to increase the frequency of the allele G of the locus generation by generation.
TABLE 1 correlation of SNP site g.69G > A of molecular marker with spleen weight
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 Ensembl 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'-ATCTCCAGTTGCCATACCGAG-3' the flow of the air in the air conditioner,
p002 reverses: 5'-ACTAGAACAGTACATCAAGCGGC-3', respectively;
(2) PCR amplification
To a 10uL reaction system, 1uL DNA template, 3.4uL double distilled water, 2 XAGg PCR StanMux 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:
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.69G & gtA effect analysis
By the molecular marker-assisted selection, cattle with AA as a gene type in a group are eliminated, the spleen weight of the group 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, and preliminarily performing association analysis between the genotype and the bovine spleen weight, and 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.
Claims (3)
1. A method for screening the spleen weight of a simmental cattle for meat, which comprises the following steps:
detecting the genotype of 38852378 th nucleotide sites on the chromosome of UMD3.1 version 6 of the international reference genome of cattle, wherein the GG type and the AG type are heavier than the spleen of the AA type, and selecting the individuals of the GG type and the AG type at 38852378 th nucleotide sites as cattle.
2. The method as claimed in claim 1, wherein the method for detecting the genotype of the 38852378 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 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 69 th base from the 5' end;
the nucleic acid sequence of the primer pair is shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
3. The application of the SNP marker in improving spleen weight of meat Simmental cattle is characterized in that the sequence of the SNP marker is as shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 1 is G or A at the 69 th base from the 5' end, wherein GG type and AG type are heavier than AA type spleen.
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| Genome-wide association study identifies loci and candidate genes for internal organ weights in Simmental beef cattle;Bingxing An等;《Physiol Genomics》;20191231;第50卷;第523-531页 * |
| rs110891752;第1页;《Ensembl》;20181031;第1页 * |
| 中国西门塔尔牛肉用群体重要经济性状遗传参数估计;牛红等;《畜牧兽医学报》;20161231;第47卷(第9期);第1817-1823页 * |
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