CN111705136A - SNP (single nucleotide polymorphism) locus related to weight of Chinese and bovine tendrils and application - Google Patents
SNP (single nucleotide polymorphism) locus related to weight of Chinese and bovine tendrils and application Download PDFInfo
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Abstract
The invention provides SNP loci related to the weight of Chinese and bovine tendrils of money on a bovine chromosome 21, wherein the locus of the SNP marker is 63471788 nucleotide loci on the international bovine reference genome UMD3.1 version chromosome 21, and the base of the locus is T 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 weight of the Chinese and cattle tendinous tendon, 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 desmodium tendon on Chinese and bovine chromosomes and application thereof.
Background
The beef cattle industry is an important component of the livestock industry in China, the price of live cattle and beef in China is gradually increased at the present stage, the number of stocked beef cattle is continuously reduced, and the cattle source is extremely tense. From the market supply perspective, the beef demand in China still has a huge gap. From the beef consumption situation, beef still has the rigidity requirement in China. With the improvement of the living standard of people, the beef demand of China has already entered the age of quality and quantity integration, the consumption demand of people on high-grade beef is increasing day by day, and the market share of the domestic high-grade beef is very low due to the higher technical requirements of breeding and feeding of the high-grade beef and production of the high-grade beef. The tendril of Christina loosestrife, the beef front tendon meat, is an important component of beef cattle carcass, has high economic value, and can adopt genome-wide association analysis to identify significant sites influencing the weight of the tendril of China and cattle in order to accelerate the molecular breeding process of the character. Research shows that the weight of the golden belt tendon belongs to the high heritability trait, about 0.42, so that the improvement of the weight of the golden belt tendon of China and cattle in resource groups by using a genetic means is feasible. The weight of the tendrils is a quantitative trait controlled by multiple genes, and a large number of Quantitative Trait Loci (QTLs) affecting the weight of the tendrils are present on the bovine genome. At present, by using a genome-wide association analysis (GWAS) method, more than a plurality of QTLs related to the weight of the bovine tendon and a large number of Single Nucleotide Polymorphisms (SNPs) have been identified on the bovine genome, and adding SNPs having significant effects into molecular Marker Assisted Selection (MAS) and Genomic Selection (GS) can accelerate the genetic improvement progress of the weight of the bovine tendon, thereby improving the weight of the bovine tendon in offspring and increasing the economic benefit of breeding enterprises.
Disclosure of Invention
The obvious SNP which affects the weight of the Chinese and bovine Lysimachia sinensiformis tendon is identified by a GWAS analysis strategy, and the SNP is used for molecular marker-assisted selection and genome selection to select the genotype which is favorable for improving the weight of the Lysimachia sinensiformis tendon 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 benefits are brought to beef cattle breeding.
The invention is realized by the following steps:
the primary aim is to determine a SNP molecular marker affecting the weight of Chinese and bovine Lysimachia tendons. The molecular marker is positioned on No. 21 chromosomes of China and cattle, and the SNP locus of the molecular marker is the nucleotide mutation of T161-C161 of which the sequence mark position is 161 of SEQ ID NO. 1; the SNP locus of the molecular marker corresponds to 63471788 th T > C mutation on the chromosome 21 of the international bovine genome UMD3.1 version reference sequence. The 97 th base of the sequence shown as SEQ ID NO. 1 from the 5' end of the SNP marker is C or A;
the invention also aims to provide a method for screening cattle individuals with high tendinous mass by using the molecular marker, which is specifically characterized in that the molecular marker on the cattle chromosome 21 is detected, the 161 th nucleotide of the 5' end of the molecular marker is T or C, T is eliminated and C is reserved, and the CA and CC type individuals at the 63471788 th nucleotide sites are selected as cattle;
another object of the present invention is to provide a primer pair for identifying the above molecular markers affecting the weight of bovine Lysimachia tendon, wherein the nucleic acid sequences of the primer pair are as follows:
p001 forward direction: 5'-TGGCTCTTCCCTATACTCCAAG-3', respectively;
p002 reverses: 5'-TCCATATGCCTTAGAGCAAC-3' are provided.
The application of the primer pair in identifying the influence on the weight of the bovine Lysimachia tendon.
The application of the primer pair in the selection of bovine genome is provided.
The application of the primer pair in increasing the weight of the bovine Lysimachia tendon.
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 Lysimachia tendon of the cattle in the cattle resource population, and making corresponding selection according to the molecular markers: selecting cattle individuals with TT, TC and CC genotypes at the 63471788 th site on the international cattle reference genome UMD3.1 version 21 chromosome from the cattle resource population, eliminating the cattle individuals with TT genotypes at the 63471788 th site, and increasing the frequency of allele C at the site by generations so as to increase the weight of the bovine tendon of offspring, which 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.
The cattle group comprises Chinese cattle and synthetic lines thereof.
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 bovine Lysimachia christinae Hance, verifies the influence effect of the molecular marker on the weight of the bovine Lysimachia christinae Hance, 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 bovine Lysimachia christinae Hance for improving the weight of the bovine Lysimachia christinae Hance, thereby improving the weight of the bovine Lysimachia christinae Hance and the meat yield of the offspring.
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FIG. 1 is a genome-wide association analysis (GWAS) Manhattan plot on chromosome 21 for weight of Lysimachia tendon in China and cattle; wherein: the abscissa indicates the chromosome number (bp) of cattle; the ordinate represents the-logP value.
FIG. 2 shows the weight of the tendon of Christina loosestrife in China 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/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
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 sample Nushiqin Biotech (Shanghai) Ltd, according toThe bovine whole genome Illumina Bovine HD chip 770K SNP chip (Illumina, USA) genotype determination was performed by the standard procedure. 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. And determining the significance threshold of the correlation degree of the SNP and the weight traits of the desmodium tendon 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. From FIG. 1, it can be seen that there are loci in chromosome 22 of beef Simmental cattle that significantly affect the weight of the tendon of Lysimachia, and the most strongly associated SNP is g.97C > A (P < 0.05).
5. Association analysis of different genotypes with the tendon weighing phenotype
As can be seen from Table 1, the SNP site g.161T > C of the molecular marker is extremely significantly related to the weight of the Lysimachia christinae Hance (P <0.001), which indicates that the molecular marker significantly affects the weight of the Lysimachia christinae Hance of cattle, and the weight of the Lysimachia christinae Hance of the group can be increased by auxiliary selection of the SNP site of cattle, thereby accelerating the breeding process of the trait.
As can be seen from Table 1, TC-type and CC-type cattle are heavier than TT-type Lysimachia christinae Hance, indicating that TT-type cattle are disadvantageous to the selection of the weight of the Lysimachia christinae Hance, and TC-type and CC-type cattle are preferred to be kept. The weight of the tendril-leaved desmodium is an important index for measuring the beef yield, and the improvement of the weight of the tendril-leaved desmodium of the cattle is beneficial to improving the beef yield, thereby increasing the market competitiveness. Therefore, TT type cattle need to be gradually eliminated in the breeding process, TC and CC type cattle are preferentially reserved, and the frequency of the allele C of the locus is increased generation by generation.
TABLE 1 correlation of SNP site g.161T > C of molecular marker with weight of Lysimachia tendon
6. Amplification and sequencing of DNA sequences of interest
(1) Primer design
The DNA sequence of SEQ ID NO:1 on chromosome 21 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'-TGGCTCTTCCCTATACTCCAAG-3', respectively;
p002 reverses: 5'-TCCATATGCCTTAGAGCAAC-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 59 ℃ 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.161T > C effect analysis
By the auxiliary selection of the molecular marker, cattle with the gene type TT in the group are eliminated, the weight of the monetary tendon 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 161 th base mutation site in the SEQ ID NO. 1 sequence and primarily applying the correlation analysis between the genotype and the bovine tendinous tendon 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. A SNP molecular marker affecting the weight of the Chinese and bovine Lysimachia tendinae Hance, wherein the SNP marker locus corresponds to the 63471788 th T > C mutation on the chromosome 21 of the reference sequence of the international bovine genome UMD version 3.1.
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 molecular marker of claim 1 or 2, wherein the primer set has the following nucleic acid sequences:
p001 forward direction: 5'-TGGCTCTTCCCTATACTCCAAG-3', respectively;
p002 reverses: 5'-TCCATATGCCTTAGAGCAAC-3' are provided.
4. A kit for detecting the SNP molecular marker according to claim 1 or 2, wherein the kit comprises the primer set according to claim 3.
5. A method for increasing the weight of chinese and bovine tendrils of money, said method comprising the steps of: detecting the genotype of 63471788 th nucleotide site on the chromosome of UMD3.1 version 21 of the international reference genome of cattle, and selecting individuals of CA and CC type of 63471788 th nucleotide site as cattle.
6. The method of claim 5, wherein the method for detecting the genotype of the 63471788 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 molecular marker according to claim 1 or 2 for increasing the weight of bovine Lysimachia tendon.
9. Use of the primer pair of claim 3 or the kit of claim 4 for increasing the weight of bovine Lysimachia tendon.
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| CN110438237A (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 hind leg, shaven head weight and application on No. 6 chromosomes of meat Simmental |
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| CN110438237A (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 hind leg, shaven head weight and application on No. 6 chromosomes of meat Simmental |
Non-Patent Citations (2)
| Title |
|---|
| B.AN 等: "Genome-wide association study reveals candidate genes associated with body measurement traits in Chinese Wagyu beef cattle", 《ANIMAL GENETICS》 * |
| ZEZHAO WANG等: "Genome-Wide Scan Identifies Selection Signatures in Chinese Wagyu Cattle Using a High-Density SNP Array.", 《ANIMALS》 * |
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