CN117385046B - Application of SNP molecular marker rs669481944 related to goat growth traits - Google Patents
Application of SNP molecular marker rs669481944 related to goat growth traits Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to application of SNP molecular marker rs669481944 related to goat growth traits. The goat SNP molecular marker locus is shown as SEQ ID NO.1, and a T/C base mutation exists at the 51 st base locus of the sequence fragment. According to the invention, the dominant allele type of the SNP molecular marker is optimized, so that the weight of the offspring of goats at the age of 12 months can be increased, the offspring of goats can be used for genetic improvement, and the economic benefit of the mutton sheep breeding industry can be effectively improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of SNP molecular marker rs669481944 related to growth traits on goat chromosome 3.
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. At present, a large amount of mutton products such as frozen bone-in sheep and frozen head-in sheep are required to be imported from New Zealand and Australia every year in China, and the mutton products become the largest sheep imported country in the world from 2012. Dongbao black bellwether is a local variety improved meat goat, which has delicious meat quality and light mutton smell, and simultaneously maintains the high fertility level of the female parent Macheng black goat, but the annual meat yield is to be improved. The growth characteristics of the goat comprise weight, body size, daily gain, feed conversion ratio and the like, and have important influence on the meat production capacity of the goat.
Single nucleotide polymorphism (Singlenucleotidepolymorphism, SNP) refers to a polymorphism of a DNA sequence caused by variation of single nucleotides (A, T, C and G) at the same position in a genome between individuals, and mainly comprises four forms of base transition, transversion, insertion or deletion. SNP has the characteristics of large quantity, wide distribution, low heterozygosity, good genetic stability, suitability for high-throughput automatic detection and the like. Therefore, SNP can be used as a first choice tool for researches such as molecular breeding, gene localization, population evolution and the like. By means of modern selective breeding technology, the genetic improvement progress of the goat growth character can be remarkably improved by adding the molecular marker with remarkable effect into molecular marker auxiliary selection (marker associated selection, MAS) and genome selection (Genomic selection, GS), so that the meat yield of offspring goats is improved, and the efficient development of the goat industry in China is promoted. The Genome-wide association analysis (Genome-wide association studies, GWAS) technique facilitates efficient screening of molecular markers that have a significant impact on phenotype. However, although the GWAS technology has been significantly advanced, there are challenges such as low quality of data standardization, high cost in large-population sequencing, and the like, which need to be solved in the GWAS study. 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 growth traits reported so far include c.454C > G locus in goat NFAT5 gene, c.1103G > A locus in goat RSAD2 gene, and g.7919G > A locus in goat ZBP1 gene. Screening new SNP loci related to goat growth 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 rs669481944 related to goat growth traits, and the application of the molecular marker in goat growth trait detection or goat breeding.
The technical scheme of the invention is as follows:
the invention aims to provide an application of a goat SNP molecular marker in goat growth trait detection or goat breeding, wherein the SNP molecular marker is rs669481944 on a goat chromosome 3, and the nucleotide sequence of 50bp upstream and downstream of the SNP locus is shown as follows (SEQ ID NO: 1):
GGAAACCTATCAGGGACTCCCTCCTGCTGCTGCTGCTAAGTTGCTTCAGTN(T/C)GTGTCCGACTCTGTGCAACTCCATAGACAGCAGCTCACCAGACGCTCCCG.
n at base 51 of the above sequence is an allelic mutation of T51-C51, which makes the SEQ ID NO. 1 sequence nucleotide polymorphic. The molecular marker can be used for detecting the molecular marker related to the goat growth character, and is favorable for the goat to have higher weight of 12 months of age when the 51 st nucleotide on the sequence shown in SEQ ID NO. 1 is C.
The invention does not limit the goat breeds, and the goat breeds such as Dongbao black bellwether, macheng black goats, boer goats, yichang white goats, horse goats and the like can be selected.
Another object of the present invention is to provide a reagent or kit comprising a primer for detecting the above SNP molecular marker. 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 growth trait, thereby predicting the goat growth trait, especially the weight of 12 months old.
The reagent or the kit can be applied to goat growth trait detection or goat breeding. The goat growth character is the weight of the goat at 12 months of age.
Another object of the present invention is to provide a method for detecting the growth trait of goat, wherein the N-labeled mononucleotide in the sequence of SEQ ID NO. 1 of goat is T or C. The goat growth character is the weight of the goat at 12 months of age.
As one implementation mode, the primer for amplifying the sequence shown in SEQ ID NO. 1 is utilized to genotype the material of the goat to be tested, and the weight of the CC type goat at 12 months is better than that of the TT type goat. Preferably, the detection is performed using the above-described kit.
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 Performing quality control on the original sequencing data, comparing the original sequencing data with a goat reference genome, and performing genetic variation detection and genotype filling on all autosomes of a sample by adopting a Sentieon +Beagle strategy to obtain high-quality SNP locus data;
③ And carrying out GWAS analysis on the SNP locus and the weight of the goat at 12 months of age by using FarmCPU model through rMVP software to obtain the SNP molecular marker related to the goat growth trait.
As one embodiment, the low depth is 1-2X and the high depth is 15-20X; 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 the growth 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 51 th base in the SNP marker as TC type and/or CC type by subculture breeding of the breeding sheep, eliminating TT type individuals, and increasing the frequency of the gene C at the site by generations, thereby improving the weight performance of the offspring goats at 12 months of age.
The invention has the beneficial effects that:
According to the invention, through combination of low-depth resequencing and genotype filling, and the obvious SNP molecular marker influencing the goat growth trait is screened by utilizing a GWAS analysis strategy, and is used in molecular marker auxiliary selection and genome selection to select the genotype favorable for improving the goat growth trait for seed reservation, so that the gene frequency of dominant alleles is improved generation by generation, the progress of breeding improvement of the breeding sheep can be accelerated, and huge economic benefits are brought for goat breeding.
The invention verifies the influence effect of the SNP molecular marker on the weight of the goats at 12 months of age, and can be applied to the genetic improvement of the goats at 12 months of age, thereby improving the weight of offspring at 12 months of age and further increasing the market competitiveness of breeding enterprises.
Drawings
Fig. 1: manhattan plot of goat growth trait (12 month old body weight), black circles and arrows point to molecular markers selected by the invention, which are located on chromosome 3 of 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 a low depth, and the depth of 34 head sequencing is 15X at a high depth, the purpose is to fill genotypes of the low depth sequencing results (more) with the high depth sequencing results (less), and the sequencing cost is reduced. Then, the 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 a GWAS study on the 12 month old body weight of goats, and finally SNPs (rs 669481944) associated with the 12 month old body weight of goats were selected, the locus of the SNP marker being the 115682971 nucleotide locus on chromosome # 3 of caprine reference genome Capra hircus ARS1.2 version 3, the base of which is T or C. 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 C at the 51 st base is nucleotide after allelic mutation, and the specific nucleotide sequence is as follows:
GGAAACCTATCAGGGACTCCCTCCTGCTGCTGCTGCTAAGTTGCTTCAGTN(T/C)GTGTCCGACTCTGTGCAACTCCATAGACAGCAGCTCACCAGACGCTCCCG,
N at base 51 of the above sequence is an allelic mutation of T51-C51, which makes the SEQ ID NO. 1 sequence nucleotide polymorphic. The GWAS analysis results show that rs669481944 is significantly related to goat growth trait (12 month old body weight), and that the 12 month old body weight of individuals with genotype TC or CC is significantly higher than TT individuals, indicating that C is an allele favorable for growth trait improvement. The molecular marker can be used for detecting the molecular marker related to the goat growth character, and is beneficial to the goat to have higher weight of 12 months of age when the 51 st nucleotide on the sequence shown in SEQ ID NO. 1 is C, and has important significance for the breeding of the goat.
The molecular marker screened by the invention can be applied to the genotype of the goat growth trait related genes or the correlation analysis of the goat growth trait, and provides a new molecular marker resource for the auxiliary selection of the molecular marker of the goat growth 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) sending the genome DNA qualified in quality inspection to Beijing Nodejingyuan science and technology Co., ltd for secondary quality inspection and library establishment, and carrying out full genome re-sequencing of PE150 on a Huada gene platform. And obtaining the original downloading data, wherein the original data format is FASTQ. The 34 samples were subjected to high depth whole genome re-sequencing with an average sequencing depth of about 19.72X and a total data size of 1.4T;466 samples were subjected to low depth whole genome resequencing with an average sequencing depth of about 1.65X 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 with the following criteria: rejecting reads having a base matrix value of less than 20 to more than 30%; n bases are greater than 5% reads. Quality control is carried out by the steps to obtain cleanreads.
(2) Cleanreads was aligned to the goat reference genome (capra_hircus.ars1.2) using BWA software.
(3) The post-comparison 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) GATKHaploytypeCaller generates gvcf files separately for each sample by autosomal numbering.
(2) GATKCombineGVCFs pool individual chromosome samples gvcf files.
(3) GATKGenotypeGVCFs chromosome-wise population SNPCALLING.
(4) GATKMERGEVCFS merge autosomal population vcf files.
(5) GATKSELECTVARIANTS screening of group vcf file SNPs.
(6) GATKVariantFiltration marks the false positive SNP site.
(7) Grep command filters the labeled SNP sites
(8) The Plink software filters the SNP sites (geno 0.1- -maf0.05- -hwe e-06).
(9) Beagle software fills in the deletion sites.
(10) Group genomic genetic variation detection and typing were performed using Sentieon Haplotyper and GVCFTYPER modules.
(11) Genotyping was performed using Beagle, resulting in 26131221 high quality SNPs.
Example 3
Application of rs669481944 molecular marker type method in goat growth trait correlation analysis
And (3) carrying out association analysis on rs669481944 molecular markers and goat growth traits (weight of 12 months):
(1) Phenotypes for genotype and growth trait association analysis are measured by a professional technician strictly according to measurement specifications, the day of age is 360+/-15 days, sheep only fasted for 12-16 hours, and the living weight weighed by 2 hours of forbidding is expressed in kilograms (kg), and total number of samples is 304.
(2) The SNP sites were analyzed by rMVP software for GWAS with 12 month old body weight using FarmCPU 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+Ztut+Sidi+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 t is t pseudo quantitative trait nucleotide genotype matrices as a fixed effect; x and Z t are the correlation matrices of b and u t, respectively; s i is the ith SNP marker, and d i is the corresponding effect value; e is a random residual effect vector, which conforms to normal distribution e-N (0,I sigma e 2).
The GWAS analysis results show that rs669481944 is obviously related to goat growth traits (12 month old body weight), the influence of different genotypes of the marker on the goat growth traits is shown in table 1, and the 12 month old body weight difference analysis of three genotype individuals in a group is shown in table 2.
TABLE 1 influence of different genotypes of rs669481944 on goat growth 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 12 month old weight differential analysis of goats of different genotypes of rs669481944
Note that: * P <0.01, P <0.001.
As can be seen from tables 1 and 2, for the 12 month old body weight trait of goat, the 12 month old body weight of individuals with genotype TC or CC was significantly higher than TT individuals, indicating that C is an allele that favors improvement in growth 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 (5)
1. The application of the SNP molecular marker of the goat in the detection of the growth traits of the goat or the breeding of the goat is characterized in that the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO. 1, the 51 st base in the sequence is T or C, the base is C which is favorable for improving the weight of Dongbao black bellwether months old, and the goat is Dongbao black sheep.
2. The application of the reagent or the kit for detecting the SNP molecular marker in the genetic breeding of the Dongbao black bellwether for detecting the body weight of the Dongbao black bellwether in the age of bellwether in the age of 3912 in the age of directly-coded Dongbao black is characterized in that the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, the 51 st base in the sequence is T or C, and the base is C which is favorable for improving the body weight of the Dongbao black bellwether in the age of directly-coded Dongbao black; the reagent or the kit comprises PCR primers for amplifying the sequence shown in SEQ ID NO. 1.
3. A method for detecting the weight of Dongbao black head sheep at 12 months of age is characterized by detecting whether the 51 st base is T or C in the sequence shown in SEQ ID NO. 1 of Dongbao black head sheep, wherein C is an allele which is favorable for improving the weight growth character of Dongbao black bellwether months of age.
4. The method according to claim 3, wherein the material to be tested Dongbao Black bellwether is genotyped by using the primer of the amplification sequence SEQ ID NO. 1, and the 12 month old body weight of CC type Dongbao Black bellwether is better than TT type.
5. A genetic breeding method for improving the growth trait of Dongbao black bellwether, which is characterized by determining the SNP molecular markers in claim 1 of sheep in a Dongbao black bellwether core group and making corresponding selection according to the Dongbao black bellwether SNP molecular markers: and (3) selecting individuals with 51 th base in the SNP marker as TC type and/or CC type by subculture breeding of the sheep, eliminating TT type individuals, and increasing the frequency of the gene C at the site by generations, thereby improving the 12 month old weight performance of the offspring Dongbao black bellwether.
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