CN116970713A - SNP molecular marker related to reproduction traits on goat chromosome 26 and application - Google Patents
SNP molecular marker related to reproduction traits on goat chromosome 26 and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a SNP molecular marker related to reproduction traits on a goat chromosome 26 and application thereof. The goat SNP molecular marker locus is shown as SEQ ID NO.1, and a base mutation of A/G exists at the 51 st base locus of the sequence fragment. According to the invention, through optimizing the dominant allele type of the SNP molecular marker, the average number of the offspring of the goats per birth can be increased, and the progress of genetic improvement of the goats is accelerated, so that the economic benefit of goat breeding is effectively improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a SNP molecular marker related to reproduction traits on a goat chromosome 26 and application thereof.
Background
The goat is one of domestic animals domesticated earliest in ancient times in China, and provides a plurality of animal products such as meat, milk, hair and the like for the life of people. The Chinese is a large country for mutton consumption, and the mutton consumption is in a situation of rising year by year. In view of the large population in China, the demand for mutton is extremely large, the genetic improvement of mutton sheep is carried out, and the improvement of the technical level of mutton sheep industry is an unprecedented important task of the current breeding work.
The Hubei black head sheep is a new black head white body meat goat variety formed by carrying out cross fixation and multi-generation group subculture breeding by using Boer goats as male parents and Boer black goats as female parents through more than ten years of breeding by the national institute of livestock and veterinarian of the national academy of agricultural sciences of Hubei province. Has the characteristics of large body size, high propagation speed, delicious meat quality, light mutton smell and the like. The breeding determines one of the decisive characters of the breeding benefit, and in the small-embryo animals such as sheep, the breeding benefit of the ewes is larger in the total benefit. However, the propagation characteristics have low genetic transmission, long period and slow breeding progress, and the complex relationship with the propagation characteristics is a main factor for restricting the economic benefits of mutton sheep industry. Therefore, by means of modern selective breeding technology, the genetic improvement progress of the goat reproduction character can be remarkably improved by adding the molecular marker with remarkable effect into the molecular marker auxiliary selection (marker associated selection, MAS) and the genome selection (Genomic selection, GS), so that the number of the offspring goats to produce the lambs is improved, and the efficient development of the goat industry in China is facilitated.
The goat reproductive trait is a complex trait controlled by multiple genes, the major genes of the goat reproductive trait are difficult to accurately identify by adopting a conventional genetic means, and a Genome-wide association analysis (Genome-wide association studies, GWAS) based on SNP chips or sequencing provides an effective method for screening out molecular markers with obvious correlation with target traits. Although GWAS technology has progressed significantly, there are challenges such as low quality of data standardization, high cost in large population sequencing, etc., which need to be addressed in GWAS research. In modern livestock breeding, because the livestock population is generally large, higher cost is generated if high-depth sequencing is used for base information acquisition, so that economic benefit is reduced, and the quality of low-depth sequencing data is under investigation. Therefore, to meet the requirement of large-population sequencing, providing low-cost, high-quality SNP site information for GWAS analysis is an important factor in improving SNP screening efficiency.
SNP loci related to goat reproductive traits reported so far comprise a T > C mutation at 951bp (calculated by taking the first base of the coding region sequence as +1) of the coding region sequence of a goat BMP6 gene, and a 36491960 base of a goat chromosome 26, which is located in a 5' regulatory region of an RBP4 gene, and the base is a G > C mutation. Screening new SNP loci related to goat reproduction traits, providing new molecular marker resources for auxiliary selection of molecular markers of goats, and accelerating the progress of breeding improvement of breeding goats.
Disclosure of Invention
The invention aims to screen out a molecular marker related to goat reproduction traits and use the molecular marker in goat reproduction trait detection or goat breeding.
The technical scheme of the invention is as follows:
the invention aims at providing an SNP molecular marker related to goat reproduction traits, which is 45603473 nucleotide site on goat reference genome (Caprahircus ARS 1.2) chromosome 26, and the base of the site is A or G. The nucleotide sequence of 50bp upstream and downstream of the SNP locus is shown as follows (SEQ ID NO: 1):
TCTCAAGAGTCTTCTCCAACATCACACTTCAAAAGCATCAATTCTT CAGCN(A/G)CTCAGCTTTCTTCACAGTCCAACTATCACATCCATACATGA CCACTGGAA。
n at base 51 of the above sequence is an A51-G51 allele mutation which gives rise to a nucleotide polymorphism in the SEQ ID NO.1 sequence. The molecular marker can be used for detecting the molecular marker related to the reproduction traits of goats, and is favorable for goats to have higher average lambing number when the 51 st nucleotide on the sequence shown in SEQ ID NO.1 is A.
The goat breeds of the present invention are preferably black goats.
Another object of the present invention is to provide a substance for detecting the above SNP molecular markers, PCR primers for amplifying genomic DNA fragments including the SNP molecular markers, or a kit containing the primers. The primer capable of amplifying the sequence shown in SEQ ID NO.1 can be designed by a person skilled in the art according to the primer design principle so as to detect the SNP marker genotype related to the goat reproduction trait, thereby predicting the goat reproduction trait, particularly the average number of the born goats.
The SNP molecular marker or substance can be applied to goat reproduction trait detection or goat breeding. The goat reproductive trait is the average number of the goats born via birth.
Another object of the present invention is to provide a method for detecting the reproductive trait of goats, wherein the sequence of SEQ ID NO.1 of goats is whether the N-labeled mononucleotide is A or G. The goat reproduction character is the average number of the goats born by the warp, and the number of the goats born by the AA type is superior to that of the GG type goats.
As one embodiment, the primer for amplifying the sequence shown in SEQ ID NO.1 is used for genotyping the material of the goat to be tested. Preferably, the detection is performed using the above-described reagents or kits.
The invention also provides application of the single nucleotide polymorphism of the goat SNP locus or a substance for detecting the single nucleotide polymorphism of the goat SNP locus in detecting or assisting in detecting reproductive traits or goat breeding, wherein the goat SNP locus is positioned at 45603473 th nucleotide locus on a No. 26 chromosome of a goat reference genome, and the base of the locus is A or G.
The invention also provides a method for screening the SNP molecular markers, which comprises the following steps:
(1) extracting goat genome DNA, and performing low-depth and high-depth resequencing on the whole genome to obtain original sequencing data;
(2) quality control is carried out on the original sequencing data, the original sequencing data is compared with a goat reference genome, and genetic variation detection and genotype filling are carried out on all autosomes of a sample by adopting a Sentieon+Beagle strategy, so that high-quality SNP locus data are obtained;
(3) and carrying out GWAS analysis on the SNP locus and the average number of the born goats by rMVP software through a FarmCPU model to obtain the SNP molecular marker related to the goat reproductive traits.
As one embodiment, the low depth is 1-2×, and the high depth is 15-20×; preferably, the low depth number is higher than the high depth, and more low depth sequencing results are genotype filled with fewer high depth sequencing results, reducing sequencing costs.
Another object of the present invention is to provide a genetic breeding method for improving reproductive traits of goats, which determines the above SNP molecular markers of goats in a goat core group, and makes corresponding selections according to the goat SNP molecular markers: and (3) selecting individuals with the 51 st base in the SNP marker as the AA type by subculture breeding of the breeding sheep, eliminating AG or GG type individuals, and increasing the frequency of the gene A at the site by generations, thereby improving the average lamb number performance of the offspring goats.
The invention has the beneficial effects that:
according to the invention, through combination of low-depth resequencing and genotype filling, and the use of a GWAS analysis strategy to screen out significant SNP molecular markers affecting goat reproduction traits, the method is used in molecular marker auxiliary selection and genome selection to select genotypes which are favorable for improving goat reproduction traits for seed reservation, so that the gene frequency of dominant alleles is improved generation by generation, the progress of breeding improvement of breeding goats can be accelerated, and huge economic benefits are brought for goat cultivation.
The invention verifies the influence effect of the SNP molecular marker on the average number of the goats born by the goats, and can be applied to the genetic improvement of raising the average number of the goats born by the goats, thereby raising the average number of the offspring born by the goats and further raising the market competitiveness of breeding enterprises.
Drawings
Fig. 1: manhattan plot of goat reproductive trait (average number of lambings), black circles and arrows point to molecular markers selected for the invention, which are located on chromosome 26 of the goat.
Detailed Description
According to the embodiment of the invention, through carrying out whole genome re-sequencing on 500 Dongbao black head sheep, wherein the depth of 466 head sequencing is 1X at low depth, and the depth of 34 head sequencing is 15X at high depth, the purpose is to fill genotypes of the low depth sequencing result (more) with the high depth sequencing result (less), so that the accuracy of the low depth sequencing result is improved, and the purposes of low cost and improvement of accuracy are achieved. Then, resequencing data were aligned to a goat reference genome (genome version ARS 1.2), genetic variation detection and genotype filling were performed on all autosomes of 500 samples using the sentieon+beagle strategy, SNP locus data were obtained to develop GWAS study relating to average number of goats born lambs, and finally SNPs (SNP 26-45603473) relating to average number of goats born lambs was selected. Referring to Ensembl, a nucleotide sequence of 50bp at the upstream and downstream of the SNP locus is obtained, the nucleotide sequence of the fragment is shown as SEQ ID NO.1, wherein A at the 51 st base is nucleotide after allelic mutation, and the specific nucleotide sequence is as follows:
TCTCAAGAGTCTTCTCCAACATCACACTTCAAAAGCATCAATTCTT CAGCN(A/G)CTCAGCTTTCTTCACAGTCCAACTATCACATCCATACATGA CCACTGGAA，
n at base 51 of the above sequence is an A51-G51 allele mutation which gives rise to a nucleotide polymorphism in the SEQ ID NO.1 sequence. The results of the GWAS analysis showed that SNP 26-45603473 was significantly correlated with goat reproductive traits (average number of lambs born), the average number of lambs born in individuals with genotype AA was significantly higher than AG or GG individuals, indicating that a is an allele favorable for reproductive trait improvement. The molecular marker can be used for detecting the molecular marker related to the goat reproduction character, and is beneficial to the goat to have higher average number of the born goats when the 51 st nucleotide on the sequence shown in SEQ ID NO.1 is A, and has important significance for the breeding of the goat.
The molecular marker screened by the invention can be applied to genotype of goat reproduction trait related genes or correlation analysis of goat reproduction trait related genes, and provides a new molecular marker resource for auxiliary selection of the molecular marker of goat reproduction trait.
The present invention will be described in detail below with reference to examples for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but they should not be construed as limiting the scope of the present invention.
Example 1
Whole genome resequencing
1. Blood sample collection and leukocyte separation
A veterinary blood collection needle was used to collect 5mL of blood from the jugular vein of a goat in EDTA anticoagulation tube, the anticoagulation tube was put in an ice box with a large amount of ice bags and brought back to the laboratory, and these samples were stored in a refrigerator at 4 ℃ for leukocyte extraction, and the specific steps were as follows:
(1) 2-3 mL blood samples were taken in 10mL EP tubes.
(2) Ultrapure water was added to the EP so that the total volume of the liquid became 9mL.
(3) The EP tube was slowly turned upside down 20 times and left to stand for 10min.
(4) The EP tube was placed in a centrifuge and centrifuged at 5000rpm for 10min.
(5) The EP tube supernatant was slowly decanted.
(6) Ultrapure water was again added to make the total volume of the liquid 9mL.
(7) Repeating the steps (3), (4) and (5).
(8) Numbering the separated white blood cells, and placing the white blood cells in a refrigerator at the temperature of minus 80 ℃.
2. Genomic DNA extraction and whole genome resequencing
The DNA extraction of the leucocytes was carried out using a small extraction kit for genomic DNA of the desert organism (cat# d 3024), the specific method being as described in the specification. And (3) delivering the genome DNA qualified in quality inspection to Beijing NodeB source technology and technology Co., ltd for secondary quality inspection and library establishment, and carrying out whole genome re-sequencing by utilizing the MGISEQ-200 platform of Huada genes. The 34 samples were subjected to high depth whole genome re-sequencing with an average sequencing depth of about 19.72×, and total data size of 1.4T;466 samples were subjected to low depth whole genome resequencing with an average sequencing depth of about 1.65×, and total data size of 1.6T.
Example 2
Genome alignment, genetic variation detection and genotype filling
1. Original sequencing data analysis and genome alignment
The high depth sequencing data and the low depth sequencing data were quality controlled using the same procedure.
(1) The raw data was filtered using Fastp software, the filter criteria were as follows: rejecting reads having a base matrix value of less than 20 to more than 30%; n bases are greater than 5% reads. And (3) performing quality control through the steps to obtain clearready.
(2) Clearready was aligned to goat reference genome (capra_hircus.ars1.2) using BWA software.
(3) The aligned BAM files were ranked using Samtools software.
(4) Reads were repeated using the Picard mark.
(5) Samtools software constructs the index.
2. Mutation site detection and genotype filling
(1) The GATKBAPloytypeCaller generates a gvcf file for each sample separately by autosomal number.
(2) The GATKCombineGCFs pooled individual chromosome samples gvcf files.
(3) GATKGGenotypeGCFs group SNPcallig by chromosome.
(4) The GATKMeVcfs pooled autosomal population vcf file.
(5) The GATKSelectVariants screen for group vcf file SNPs.
(6) GATKVariant filtration marks false positive SNP sites.
(7) grep command filters the labeled SNP sites
(8) The SNP sites were filtered by the Plink software (geno 0.1-maf 0.05-hwe 1 e-06).
(9) Beagle software fills in the deletion sites.
(10) Group genomic genetic variation detection and typing was performed using Sentieon Haplotyper and gvctyper modules.
(11) Genotyping was performed using Beagle, resulting in 26131221 high quality SNPs.
Example 3
Application of SNP 26-45603473 molecular marker type method in goat reproduction trait association analysis
Analysis of association of SNP 26-45603473 molecular markers with goat reproductive traits (average number of lambs born).
(1) Phenotypes for genotype and reproductive trait association analysis were measured by skilled artisans strictly according to measurement specifications, counting the average number of born lambs, totaling 408 samples.
(2) The SNP loci were analyzed by rvmp software for GWAS with average number of merits using the farm cpu model. The FarmCPU model iterates using a fixed effect model and a random effect model. The fixed effect analysis model is as follows:
y＝Xb+Z t u t +S i d i +e
wherein y is the observer vector of the trait; b is an individual fixation effector vector comprising the first three primary components of SNPs, birth season, birth parity and birth weight; u (u) t T pseudo quantitative trait nucleotide genotype matrixes are used as a fixed effect; x and Z t B and u, respectively t Is a correlation matrix of (a); s is S i Is the ith SNP marker, d i Is the corresponding effect value; e is a random residual effect vector, which accords with normal distribution e-N (0,I sigma) e 2 )。
The results of the GWAS analysis show that SNP 26-45603473 is obviously related to goat reproduction traits (average number of lambs born), the influence of different genotypes of the marker on the goat reproduction traits is shown in Table 1, and the difference analysis of the average number of lambs born of three genotypes of individuals in a group is shown in Table 2.
TABLE 1 Effect of different genotypes of SNP 26-45603473 on goat reproductive traits
Note that: a significant marker was found when the P-value of the marker was <0.05/26131221 ≡1.91E-09 (Bonferroni correction).
Table 2 analysis of average number difference between the born goats of different genotypes of SNP 26-45603473
Note that: * P <0.05, < P <0.001.
As can be seen from tables 1 and 2, for the goat average number of merits trait, the average number of merits of individuals with genotype AA is significantly higher than those of AG or GG individuals, indicating that A is an allele that contributes to improved reproductive traits.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A SNP molecular marker related to goat reproductive traits is characterized in that the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and N in the sequence is an allele mutation of A or G.
2. A substance for detecting the SNP molecular marker according to claim 1, characterized by comprising PCR primers for amplifying the genomic DNA fragment including the SNP molecular marker or a kit containing the primers.
3. Use of the SNP molecular marker of claim 1 or the substance of claim 2 in goat reproductive trait detection or goat breeding.
4. The use according to claim 3, wherein the goat reproductive trait is average number of parity lambs.
5. The use according to claim 3, wherein the goat is a black-head sheep.
6. A method for detecting the reproduction character of goat is characterized by detecting whether N marked mononucleotide is A or G in the sequence shown in SEQ ID NO.1 of goat.
7. The method of claim 6, wherein the material of the goat to be tested is genotyped with the primer of the amplification sequence SEQ ID NO.1, and the number of the AA-sized goats born by the lambs is superior to that of the GG-sized goats.
8. The method according to claim 6 or 7, characterized in that the detection is performed with a substance according to claim 2.
9. The application of single nucleotide polymorphism of a goat SNP locus or a substance for detecting single nucleotide polymorphism of the goat SNP locus in detecting or assisting in detecting reproductive traits or goat breeding is characterized in that the goat SNP locus is positioned at 45603473 nucleotide locus on a No. 26 chromosome of a goat reference genome, and the base of the locus is A or G.
10. A genetic breeding method for improving goat reproductive traits, which is characterized by determining the SNP molecular markers of claim 1 of sheep in a goat core group, and making corresponding selections according to the goat SNP molecular markers: and (3) selecting an AA type individual at the 51 st base in the SNP marker in the subculture breeding selection of the breeding goats, and eliminating AG or GG type individuals to increase the frequency of the gene A at the site generation by generation, thereby improving the average lambing number performance of offspring goats.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531608A (en) * | 2018-01-24 | 2018-09-14 | 华南农业大学 | Molecular labeling of the BMP6 genes as black goat litter size character |
| CN112251518A (en) * | 2020-11-05 | 2021-01-22 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to lambing number and growth traits in goat RSAD2 gene and application thereof |
| CN114134236A (en) * | 2021-12-07 | 2022-03-04 | 中国农业科学院北京畜牧兽医研究所 | Application of reagent for detecting SNP molecular marker in goat RBP4 genotyping and/or goat molecular marker assisted breeding |
| WO2023277709A1 (en) * | 2021-07-01 | 2023-01-05 | Dairy Goat Co-Operative (N.Z.) Limited | Genetic markers and methods related thereto |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531608A (en) * | 2018-01-24 | 2018-09-14 | 华南农业大学 | Molecular labeling of the BMP6 genes as black goat litter size character |
| CN112251518A (en) * | 2020-11-05 | 2021-01-22 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to lambing number and growth traits in goat RSAD2 gene and application thereof |
| WO2023277709A1 (en) * | 2021-07-01 | 2023-01-05 | Dairy Goat Co-Operative (N.Z.) Limited | Genetic markers and methods related thereto |
| CN114134236A (en) * | 2021-12-07 | 2022-03-04 | 中国农业科学院北京畜牧兽医研究所 | Application of reagent for detecting SNP molecular marker in goat RBP4 genotyping and/or goat molecular marker assisted breeding |
Non-Patent Citations (3)
| Title |
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| UCSC: "SNP for 232 goat accessions (26:A45603473)", pages 1 - 2, Retrieved from the Internet <URL:http://animal.omics.pro/genomebrowser/cgi-bin/hgc?hgsid=262621_CSV53gPMipYdQzXbmGCXKU3AX8dd&c=26&l=45603472&r=45603473&o=45603472&t=45603473&g=SNPs&i=26%3A45603473> * |
| 刘泽林等: "山羊ZBP1基因多态性及与产羔性能的关联分析", 中国畜牧兽医, vol. 50, no. 1, 6 January 2023 (2023-01-06), pages 161 - 173 * |
| 张年等: "湖北黑头羊母羊繁殖性能测定", 黑龙江畜牧兽医, no. 12, 20 June 2018 (2018-06-20), pages 77 - 80 * |
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