CN111705137B - SNP (Single nucleotide polymorphism) locus related to live weight of Chinese cattle and cattle before slaughter and application - Google Patents
SNP (Single nucleotide polymorphism) locus related to live weight of Chinese cattle and cattle before slaughter and application Download PDFInfo
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- CN111705137B CN111705137B CN202010197710.3A CN202010197710A CN111705137B CN 111705137 B CN111705137 B CN 111705137B CN 202010197710 A CN202010197710 A CN 202010197710A CN 111705137 B CN111705137 B CN 111705137B
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Abstract
The invention provides SNP loci related to liveweight of Chinese cattle before slaughter on a cattle No. 14 chromosome, wherein the locus of the SNP marker is 25284162 nucleotide loci on the international cattle reference genome UMD3.1 version No. 14 chromosome, and the base of the locus is A or C. By optimizing the dominant allele of the SNP, the invention can increase the frequency of the dominant allele generation by generation, improve the pre-slaughter liveliness 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 pre-slaughter live weight on Chinese and bovine chromosomes and application thereof.
Background
China is a world animal husbandry production big country, and the beef cattle production value is close to 2000 million yuan, accounting for 25 percent of the animal husbandry production value. 10% of cattle all over the world are bred in China, the first few cattle slaughter ranks worldwide, and the beef yield is third worldwide. However, the average carcass weight of the beef cattle does not exceed 154 kg and is only close to one half of the weight of the beef cattle in developed countries in Europe and America, so that the method not only limits the long-term stable development of the beef cattle industry in China, but also is a risk factor of the sustainable development of the beef cattle industry such as international market competitiveness, epidemic diseases and the like. In order to accelerate the molecular breeding process of carcass weight of beef cattle, the significant loci influencing the pre-slaughter live weight of China and cattle are identified by adopting whole genome association analysis. Research shows that the pre-slaughter live weight belongs to the medium heritability character, about 0.33, so that the improvement of the pre-slaughter live weight of Chinese and cattle in resource groups by using a genetic means is feasible. The pre-slaughter live weight is a quantitative trait controlled by a plurality of genes, and a large number of Quantitative Trait Loci (QTLs) influencing the pre-slaughter live weight exist on the cattle genome. At present, a genome-wide association analysis (GWAS) method is used for identifying more than a plurality of QALs related to pre-slaughter live weight and a large number of Single Nucleotide Polymorphism (SNPs) on a cattle genome, and SNP with a remarkable effect is added into molecular Marker Assisted Selection (MAS) and Genome Selection (GS), so that genetic improvement progress of the pre-slaughter live weight can be accelerated, further the pre-slaughter live weight of the offspring beef cattle is improved, and the economic benefit of a breeding enterprise is increased.
Disclosure of Invention
The obvious SNP influencing the live weight of China and cattle before slaughter is identified by a GWAS analysis strategy, and is used for molecular marker-assisted selection and genome selection to select the genotype favorable for improving the live weight before slaughter for seed reservation, 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 beef cattle breeding.
The invention is realized by the following steps:
the first aim is to determine an SNP molecular marker which influences the live weight of Chinese cattle before slaughter.
The molecular marker is positioned on a nucleotide sequence on No. 14 chromosomes of China and cattle, and the SNP locus is the nucleotide mutation of A71-C71 with the marked position of the SEQ ID NO. 1 sequence as position 71; the SNP locus of the molecular marker corresponds to 25264162 th A > C mutation on chromosome 14 of the reference sequence of international bovine genome UMD3.1 version.
The invention also aims to provide a method for screening cattle individuals with high live weight before slaughtering by using the molecular marker, and particularly, the molecular marker on the 14 th chromosome of the cattle is detected, the 161 th nucleotide of the 5' end of the molecular marker is A or C, and the A is eliminated and the C is reserved.
Another object of the present invention is to provide a primer pair for identifying the above molecular marker affecting the pre-slaughter live weight of cattle, the primer pair having the following nucleic acid sequences:
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 identifying the influence on the live weight of the cattle before slaughtering.
The application of the primer pair in the selection of bovine genome is provided.
The primer pair is applied to improving the live weight of the cattle before slaughtering.
It is also an object of the present invention to provide a method for genetic improvement in cattle, the method comprising: determining the sites of the above molecular markers affecting the live weight of the cattle before slaughtering of the cattle in the cattle resource population, and making corresponding selection according to the molecular markers: selecting cattle individuals with AA, AC and CC genotypes at 25264162 th site on chromosome 14 of UMD3.1 version of the international cattle reference genome, eliminating cattle individuals with AA genotypes at 25264162 th site, and increasing the frequency of allele C at the site by generations, thereby increasing the pre-slaughter live 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 pre-slaughter live weight of the cattle, verifies the influence effect of the molecular marker on the pre-slaughter live 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 cattle for improving the pre-slaughter live weight, thereby improving the pre-slaughter live weight of the offspring 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 on chromosome 14 for pre-slaughter live weights in China and cattle; wherein: the abscissa indicates the chromosome number (bp) of cattle; the ordinate represents the-logP value.
FIG. 2 shows the pre-slaughter live weights of Chinese and cattle of 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 from China and 458 cattle of Xuelongte GmbH, Dalian, Liaoning province, and are Chinese and cattle resource groups established by cattle genetic breeding innovation team of Beijing animal husbandry and veterinary institute, national academy of agricultural sciences.
In the experiment, 458 Chinese and cattle in the resource group are 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/A27643 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 each individual of 458 beef simmental cattle selected from the resource groups, the whole genome DNA is extracted by a standard phenol-chloroform method, and the concentration and OD ratio (OD260/280, OD260/230) of the 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, 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 GAPIA 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. And determining the significance threshold of the correlation degree of the SNP and the pre-slaughter live weight trait by adopting a modified Bonferrini method, wherein the genome level significance threshold is 1 divided by the total number of SNP loci, namely the genome significance threshold is 1.97 e-6.
The GWAS analysis results are shown in fig. 1. As can be seen from fig. 1, there are sites in chromosome 14 of the meat simmental cattle that significantly affect pre-slaughter live weight, and the most strongly associated SNP is g.97c > a (P < 0.05).
5. Association analysis of different genotypes with pre-slaughter live-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 pre-slaughter live weight (P <0.001), which shows that the molecular marker significantly affects the pre-slaughter live weight of cattle, and the pre-slaughter live weight of the group can be improved by auxiliary selection of the SNP site of cattle, thereby accelerating the breeding process of the trait.
As is clear from Table 1, the types AC and CC are heavier than those of the type AA before slaughter, indicating that the type AA cattle are unfavorable for screening the before-slaughter live weight, and therefore, the types AC and CC cattle are preferentially kept. The pre-slaughter live weight is an important index for measuring the beef yield, and the improvement of the pre-slaughter live weight of the cattle is beneficial to improving the beef yield, so that the market competitiveness is improved. Therefore, in the breeding process, AA type cattle need to be gradually eliminated, and AC and CC type cattle need to be preferentially reserved so as to increase the frequency of the allele C of the locus generation by generation.
TABLE 1 correlation of SNP site g.97C > A of molecular markers with pre-slaughter live weight
6. Amplification and sequencing of DNA sequences of interest
(1) Primer design
The DNA sequence of SEQ ID NO:1 on chromosome 14 of cattle was downloaded via the Ensembl website (hAAP:// asia. Ensembl. org/index. hAML). 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'-AGAAGGTGGCTGAGAAACGT-3', respectively;
p002 reverses: 5'-GGAACTGAGAAGTGTCACGTG-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:
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.71A > C effect analysis
By the auxiliary selection of the molecular marker, the cattle with AA as the gene type in the group are eliminated, and the pre-slaughter liveness of the group can be obviously improved, so that the meat yield is improved, and more economic benefits are brought to enterprises.
The invention provides a new molecular marker for the molecular marker auxiliary selection and the genome selection of cattle by detecting the 71 th base mutation site in the SEQ ID NO. 1 sequence and primarily applying the correlation analysis between the genotype and the pre-slaughter live weight of cattle.
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 (4)
1. The method for improving the live weight of Chinese cattle before slaughtering is characterized by comprising the following steps:
detecting the genotype of 25264162 nucleotide loci on chromosome No. 14 of UMD3.1 version of international reference genome of cattle, and selecting individuals of CA and CC types of 25264162 nucleotide loci as cattle, wherein the individuals of CA and CC types have high liveness before slaughter.
2. The method of claim 1, wherein the method for detecting the genotype of the 25264162 th nucleotide site on the chromosome 14 of the international reference genome UMD3.1 version of 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 primer pairs 5'-AGAAGGTGGCTGAGAAACGT-3' and 5'-GGAACTGAGAAGTGTCACGTG-3' to obtain a PCR amplification product;
(3) sequencing the PCR amplification product to obtain a sequencing result;
(4) determining the genotype of 25264162 nucleotide sites on chromosome 14 of UMD3.1 version of the international reference genome of the test cow as described in claim 1 based on the sequencing result, wherein individuals with CA and CC types have high pre-mortem liveness.
3. The application of a primer pair for detecting the genotype of 25264162 nucleotide sites on chromosome 14 of UMD3.1 version of international reference genome of cattle in improving the live weight of Chinese cattle before slaughter comprises the following nucleic acid sequences:
p001 forward direction: 5'-AGAAGGTGGCTGAGAAACGT-3', respectively;
p002 reverses: 5'-GGAACTGAGAAGTGTCACGTG-3' are provided.
4. Use of a kit comprising the primer pair of claim 3 for increasing pre-slaughter live weight in china and cattle.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110438238A (en) * | 2019-06-27 | 2019-11-12 | 中国农业科学院北京畜牧兽医研究所 | SNP site relevant to forehoof weight and application on No. 6 chromosomes of meat Simmental |
| CN110468212A (en) * | 2019-06-27 | 2019-11-19 | 中国农业科学院北京畜牧兽医研究所 | SNP site relevant to fatty acid C14:0 content and application on No. 19 chromosomes of meat Simmental |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110438238A (en) * | 2019-06-27 | 2019-11-12 | 中国农业科学院北京畜牧兽医研究所 | SNP site relevant to forehoof weight and application on No. 6 chromosomes of meat Simmental |
| CN110468212A (en) * | 2019-06-27 | 2019-11-19 | 中国农业科学院北京畜牧兽医研究所 | SNP site relevant to fatty acid C14:0 content and application on No. 19 chromosomes of meat Simmental |
Non-Patent Citations (2)
| Title |
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| B. An等.Genome-wide association study reveals candidate genes associated with body measurement traits in Chinese Wagyu beef cattle.《Stichting International Foundation for Animal Genetics》.2019,第50卷(第4期),第386–390页. * |
| Genome-wide association study reveals candidate genes associated with body measurement traits in Chinese Wagyu beef cattle;B. An等;《Stichting International Foundation for Animal Genetics》;20190610;第50卷(第4期);第386页左栏第2段以及右栏第1段，表2 * |
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