CN118726617B

CN118726617B - A SNP marker affecting early growth traits of cashmere goats and its application

Info

Publication number: CN118726617B
Application number: CN202411223404.7A
Authority: CN
Inventors: 张燕军; 戎友俊; 郭富荣; 尚方正; 敖晓芳; 夏钦澄; 马荣; 王瑞军; 冀北平
Original assignee: Inner Mongolia Beiping Textile Co ltd; Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Beiping Textile Co ltd; Inner Mongolia Agricultural University
Priority date: 2024-09-03
Filing date: 2024-09-03
Publication date: 2024-12-10
Anticipated expiration: 2044-09-03
Also published as: CN118726617A

Abstract

The invention relates to the technical field of genetic breeding, in particular to an SNP marker affecting early growth traits of a down producing goat and application thereof. The SNP marker comprises a first marker and/or a second marker, wherein the nucleotide sequence of the first marker is shown as SEQ ID NO.1, the 101bp position site has the mutation from A to G, the nucleotide sequence of the second marker is shown as SEQ ID NO.2, the 101bp position site has the mutation from T to G, the first marker affects the primary growth character of the cashmere goat, and the second marker affects the broken milk growth character of the cashmere goat. The invention improves the birth weight of offspring by selecting AA and TT genotype individuals as male parents or female parents.

Description

SNP marker affecting early growth traits of down producing goats and application thereof

Technical Field

The invention relates to the technical field of genetic breeding, in particular to an SNP marker affecting early growth traits of a down producing goat and application thereof.

Background

The inner Mongolia goat is an excellent local animal species obtained by long-term natural selection and artificial system breeding, and has important economic value. The growth and development of the lambs after birth have great influence on reproduction and production performance, and along with the change of feeding modes and environments, how to improve the growth speed of the cashmere goats has become one of the problems to be solved urgently. The birth weight and the weaning weight are two important indexes for measuring the growth speed of the cashmere goats. The birth weight refers to the weight of the cashmere goat at birth, and the weaning weight refers to the weight of the cashmere goat at weaning. Both of these indicators have a significant impact on the growth rate of the down producing goat. The prior art does not consider the primary weight and the weaning weight of the cashmere goats at the same time, so that the molecular markers affecting the primary weight and the weaning weight of the inner Mongolian cashmere goats are very necessary to be mined in a genome level.

Disclosure of Invention

In order to solve the problems, the invention provides an SNP marker affecting the early growth characteristics of the cashmere goats and application thereof, so as to improve the growth speed of the cashmere goats.

The invention is realized by the following technical scheme:

a SNP marker affecting the early growth trait of a down producing goat, the SNP marker comprising a first marker and/or a second marker;

The nucleotide sequence of the mark I is shown as SEQ ID NO.1, and the site at the 101 th bp is mutated from A to G;

The nucleotide sequence of the mark II is shown as SEQ ID NO.2, and the site at the 101 th bp is mutated from T to G;

The nucleotide sequence of the molecular marker of the marker II is shown as SEQ ID NO.1, specifically ：AACGTCATGAACACTGCGACAGAGGGAAGGAACTCCAAGCCAGGGAGAACAGTGAGCCCTGGGTGCTGCCCCCTGAGTAAGGCAGGCAGGCAGGGGCACCAGCTCTCCTTGGGGACCACACAGAGGGGCTGAGTACTTTACTATTTAGCGCTCTGGAGGCTTGAGGCCACCCTGGAGGGTTAATGTGTACAAAGAGCTGCC, shows the sequence of 100bp upstream and downstream of the mutation site, and the site 101 has the mutation from A to G.

The nucleotide sequence of the molecular marker of the marker II is shown as SEQ ID NO.2, specifically ：TTAGTCTTAATTAAGTATCTTAATCTTCCCAGGTTAAGGGCAGAAAAGTAAGAGAAAGCAGGCAGCCACGGGTGGACCCTTCGTAAGAGTGTGTCCTGGATGCCCTGGAGCCCTGCATCGCGGCTGACCCGGGTGTGTACATCCGGGGTTTGATATAGAAGGAAGATGAACGTGGAGAAGAAACACGTCTGACACCTCTTTC, shows the sequence of 100bp upstream and downstream of the mutation site, and the site 101 has the mutation from T to G.

The SNP marker is applied to identification of the primary weight and the weaning weight of the down producing goat, and comprises the first marker and/or the second marker.

The first base is a primary heavy cashmere goat when A, and the second base is a secondary heavy cashmere goat when T.

Preferably, the identification of the primary weight and the weaning weight of the cashmere goats comprises the following steps:

extracting the genomic DNA of the ear tissue of the cashmere goat;

crushing genome DNA to obtain DNA fragments, constructing a library by using the DNA fragments, and sequencing the library to obtain sequencing data;

identifying and screening mutation sites on sequencing data, and detecting the first marker on the chromosome 14 of the cashmere goat by using whole genome association analysis, wherein the base of the first marker is a cashmere goat with primary importance when A;

Detecting the second marker on the chromosome 23 of the cashmere goat, wherein the base of the second marker is the cashmere goat with heavy milk breaking when the base is T.

A genetic method for the cashmere goats with high early growth character phenotype value features that AA and TT genotype individuals are chosen as parents to increase the birth weight of offspring.

Preferably, the cashmere goats are inner mongolian cashmere goats.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides SNP markers affecting the early growth traits of a cashmere goat and application thereof, wherein two SNP markers affecting the early growth traits of the cashmere goat are screened out through methods such as genome sequencing, mutation site identification, whole genome association analysis and the like, namely a first marker and a second marker. Is used for identifying the initial weight and the weaning weight of the early growth characters of the down producing goat, and the birth weight of offspring is improved by selecting AA and TT genotype individuals as male parents or female parents.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing distribution of SNPs after quality control over a chromosome 1Mb window;

FIG. 2 is a principal component analysis chart;

FIG. 3 is a Manhattan diagram showing the results of a whole genome association analysis of the birth weight trait of an inner Mongolian cashmere goat;

FIG. 4 is a quantile-quantile diagram of the primary weight trait;

FIG. 5 is a Manhattan diagram showing the results of whole genome association analysis of the weaning weight trait of an inner Mongolian down goat;

fig. 6 is a score-score plot of the weaning weight trait.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention, and preferred embodiments of the present invention are set forth. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The technical scheme of the invention will be further described with reference to specific embodiments.

Examples

The first part, experimental animals and phenotypic sources.

The experimental sheep are all from two-wolf mountain pastures of North China textile Limited liability company, and the two-wolf mountain national grade seed protection farms of the internal Mongolian cashmere goats, and the raised cashmere goats are two-wolf mountain internal Mongolian cashmere goats. The Birth weight, english, birth weight and weaning weight were determined according to the methods described in Table 1, english was WEANING WEIGHT trait phenotypes recorded. Ear tissue samples of 212 inner Mongolian goat individuals are collected, all samples are immediately stored in liquid nitrogen after being collected, and are stored for a long time at-80 ℃ after being transported to a laboratory.

Table 1 determination method for early growth traits of inner Mongolian cashmere goats

And the second part, genome DNA extraction and quality inspection.

DNA extraction:

1. the tissue sample with the size of bean grains is cut and put into a 1.5ml disinfection EP centrifuge tube, and is cut and broken as much as possible, and then tissue lysate STE 0.5ml,10% SD S50 μl and proteinase K10 μl are added, and are fully and uniformly mixed, and then digested in a 55 ℃ water bath overnight.

2. To each EP centrifuge tube was added an equal volume of about 500. Mu.l of Tris saturated phenol, mixed well by shaking for 5min, and centrifuged at 12000rpm for 5min.

3. The second step is repeated.

4. The supernatant was placed in a fresh EP centrifuge tube, 500. Mu.l of 25:24:1 phenol/chloroform/isoamyl alcohol was added, and after thorough mixing, it was centrifuged at 12000rpm for 5min.

5. The supernatant was placed in a fresh EP centrifuge tube, and about 1mL of ice isopropyl alcohol was added in a volume twice that of the supernatant, and the supernatant was shaken horizontally to give a white flocculent DNA precipitate.

6. The iced isopropanol was discarded, and the DNA was washed with 75% ethanol, and after shaking washing, it was centrifuged at 12000rpm for 10min.

7. After discarding 75% ethanol and naturally drying, 100. Mu.l of sterilized ddH ₂ O was added for dissolution.

DNA quality inspection:

The detection of the DNA concentration, the absorption nanowavelength ratio of the highest absorption peak of nucleic acid to protein and phenols was carried out using a spectrophotometer model NanoDrop2000 at 260:280 and the absorption nanowavelength ratio of the highest absorption peak of carbohydrates at 260:230, and the DNA quality detection evaluation was carried out using a 1% agarose gel.

Third, library construction and on-machine sequencing.

The genomic DNA sample with qualified quality detection is randomly broken into fragments with the length of 350bp by a covarias ultrasonic breaker. The DNA fragment is subjected to the steps of terminal repair, polyA addition, sequencing joint addition, purification, PCR amplification and the like to complete the preparation of the whole library. After the library construction is completed, the qubit2.0 is used for preliminary quantification, and the qPCR method is used for accurately quantifying the effective concentration of the library so as to ensure the quality of the library. And after the quality of the library is detected to be qualified, sequencing by utilizing a Huada MGI-T7 sequencing platform, wherein the sequencing mode is a PE150 mode, and original sequencing data are obtained.

Fourth, identification, screening and annotation of mutation sites.

The method comprises the steps of performing quality control and pretreatment on original sequencing data by using V0.20.0 version fastp software to obtain sequencing data CLEAN READS DATA, establishing a genome index for a reference genome, comparing the quality-controlled CLEAN READS DATA with goat reference genome ARS1 and GCF_001704415.1 by using V0.7.17 version Burows-WHEELER ALIGNER software, converting a compared SAMtools file into a bam file by using V1.8-20 version SAMtools software and sequencing the bam file, removing repeated data from the sequenced bam file by using MarkDapplicates program in V3.8 version Genome Analysis Toolkit software to obtain a final bam file, establishing an index for the final bam file, performing SNP mutation detection by using HaplotypeCaller module in GATK software, and filtering by using VariantFiltration module after obtaining a vcf file. The detected genetic variation is functionally annotated using the ANNOVAR software package, and the region where the variation site occurs in the genome, such as the intergenic region, the intronic region or the CDS region, and the influence of the variation, such as the synonymous mutation, are obtained based on the position of the variation site on the reference genome and the genetic position information on the reference genome.

And fifthly, data quality control and population layering correction.

The detection rate, english name call rate, minimum allele frequency, english abbreviation MAF, hardy-Wenberg equilibrium, english abbreviation HWE.

The obtained genotyping data were quality controlled using Plink software version V1.90, eliminating individuals with genotype detection rate <90%, SNPs with detection rate <95%, SNPs with minimum allele frequency <5%, SNPs with hastelloy balance test P value <10 ^-6. A total of 34,248,064 SNPs participate in quality control, the detection rate of 460822 SNPs is less than 95%, the rest sites are subjected to Hardy-Wenberg equilibrium filtration, minimum allele frequency filtration and individual detection rate filtration, and 17258261 SNPs sites are obtained for subsequent analysis, and are uniformly distributed on 29 pairs of autosomes of goats, as shown in figure 1. The former three main components are calculated by using the 'pca 3' parameter of Plink software of V1.90 version, and the former 3 main components are used as covariates to correct the population layering phenomenon of the two-wolf mountain type inner Mongolian down goat. The PCA diagram is drawn by using R software of V3.6.0 version, and the result is shown in figure 2, the test sample has population stratification, the genetic correlation degree among individuals is higher, and the first 3 main components are required to be used as covariates to correct the population stratification phenomenon of the inner Mongolian goat.

And part six, whole genome association analysis.

Correlation analysis between SNPs and birth weight traits was performed using the fastGWA-mlm model in GCTA software version v1.94.0beta. The formula is as follows:

y=Xα+Zβ+Wu+e

Where y is a phenotype vector, alpha is a vector of fixed effects including gender, field, birth type, beta is a genotype vector, u is a random effect, X is an incidence matrix of alpha, Z is an incidence matrix of beta, W is an incidence matrix of u, and e is a residual vector.

The significance threshold for the whole genome correlation analysis was too stringent as determined using the Bonferroni correction method. The present study adjusts the threshold for significant whole genome association to p=1×10 ^-6. The statistical genome expansion factor, λ, was examined and calculated by the slope of the linear regression between the quantiles observed in the V3.6.0 version of R software and the theoretical quantiles. As shown in FIG. 4, the lambda value of the birth weight trait is 0.952, indicating no genome expansion, and the results are reliable, and as shown in FIG. 6, the lambda value of the weaning weight trait is 1.025, indicating no genome expansion. Based on the resequencing data of 212 white goats from two wolf mountain, 17 significant SNP sites associated with primary weight traits were detected, which were located on chromosome 3, 10, 14, 19, respectively, as shown in Table 2 and FIG. 3, and 4 significant SNP sites associated with weaning weight traits were detected, which were located on chromosome 19, 20, 23, respectively, as shown in Table 3 and FIG. 5.

TABLE 2 significant SNP loci associated with Primary weight traits

TABLE 3 significant SNP loci associated with weaning weight traits

Seventh, SNP affecting early growth traits.

Further research on SNPs reaching the whole genome significance level shows that the A-G mutation at 54137103 on chromosome 14 of the goat genome can significantly influence the primary weight character of the cashmere goat.

The association analysis of the SNP locus at 54137103 on chromosome 14 of the goat genome with the primary weight trait is as follows:

TABLE 4 polymorphism at 54137103 on chromosome 14 of goat genome

Note that the different letters represent significant differences, p <0.05, the same letter represents insignificant differences, p >0.05

As is clear from Table 4, the individuals with genotype AA had the greatest birth weight, and the individuals with genotype GG had the smallest birth weight.

In the whole genome association analysis adopting the mixed linear model, SNP molecular markers at 54137103 on chromosome 14 of the goat genome reach a whole genome significant level, which indicates that the markers are significantly related to the birth weight character of the cashmere goat, and when the genotypes of the markers are AA, the large birth weight of the cashmere goat is facilitated.

TABLE 5 SNP Gene frequency and genotype frequency at 54137103 on chromosome 14 of goat genome

As can be seen from Table 5, A has a higher gene frequency than G, indicating A is the dominant allele, and AA and GA have higher genotype frequencies than GG, indicating AA is the dominant allele.

Further research on SNPs reaching the whole genome significance level shows that the T-G mutation at 16477721 on chromosome 23 of the goat genome can significantly influence the weight-loss character of the cashmere goat.

The association analysis of the SNP locus at 16477721 on chromosome 23 of the goat genome with the weaning weight trait was as follows:

Table 6 polymorphism at 16477721 on goat genome chromosome 23

As is clear from Table 6, the individuals with genotype TT had the greatest weaning weights, and the individuals with genotype GG had the smallest weaning weights.

In the whole genome association analysis adopting the mixed linear model, the SNP molecular marker at 16477721 on chromosome 23 of the goat genome reaches the whole genome significant level, which indicates that the marker is significantly related to the birth weight character of the cashmere goat, and when the genotype of the marker is TT, the marker is favorable for the cashmere goat to have larger weaning weight.

TABLE 7 SNP Gene frequency and genotype frequency at 16477721 on chromosome 23 of goat genome

As can be seen from Table 7, the gene frequency of T is greater than G, indicating that T is the dominant allele, and the genotype frequencies of TT and TG are both greater than GG, indicating that TT is the dominant allele.

It can be seen that the primary heavy cashmere goat breeds can be bred through the A-G mutation at 54137103 th on chromosome 14 of the goat genome, the heavy cashmere goat breeds can be bred through the T-G mutation at 16477721 th on chromosome 23 of the goat genome, and the offspring birth weight can be increased by selecting AA and TT genotype individuals as male parents or female parents.

It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

The application of SNP markers in the identification of the primary weight and the weaning weight of the down producing goat is characterized in that the SNP markers comprise a first marker and/or a second marker;

the nucleotide sequence of the mark I is shown as SEQ ID NO.1, and the site at the 101bp position has A to G mutation;

The nucleotide sequence of the mark II is shown as SEQ ID NO.2, and the 101bp locus has T-G mutation;

The mark I affects the primary growth and the secondary growth characteristics of the cashmere goats, and the mark II affects the broken milk and the secondary growth characteristics of the cashmere goats;

The base of the 101bp position of the mark A is a primary heavy cashmere goat, and the base of the 101bp position of the mark B is a T is a broken large cashmere goat;

The cashmere goats are inner Mongolia cashmere goats.
2. The use of the SNP marker of claim 1 for identifying the primary weight and the weaning weight of a down producing goat, wherein the identification of the primary weight and the weaning weight of the down producing goat comprises the steps of:

extracting the genomic DNA of the ear tissue of the cashmere goat;

crushing genome DNA to obtain DNA fragments, constructing a library by using the DNA fragments, and sequencing the library to obtain sequencing data;

The sequencing data is identified and screened by mutation sites, and the whole genome correlation analysis is used for detecting the first marker on the No. 14 chromosome of the down producing goat, wherein the first marker is a heavy down producing goat when the base of the 101bp site is A;

Detecting the second marker on the chromosome 23 of the cashmere goat, wherein the base at the 101bp position of the second marker is the cashmere goat with great weaning when the base is T.
The application of SNP markers in the genetic breeding of the down producing goats is characterized in that the SNP markers comprise a first marker and/or a second marker;

the nucleotide sequence of the mark I is shown as SEQ ID NO.1, and the site at the 101bp position has A to G mutation;

The nucleotide sequence of the mark II is shown as SEQ ID NO.2, and the 101bp locus has T-G mutation;

The mark I affects the primary growth and the secondary growth characteristics of the cashmere goats, and the mark II affects the broken milk and the secondary growth characteristics of the cashmere goats;

The offspring birth weight is increased by selecting individuals with genotypes AA and TT as parents;

The cashmere goats are inner Mongolia cashmere goats.