Application of haplotype combination related to goat weight and application method

Abstract

The present invention belongs to the technical field of animal molecular breeding, and specifically relates to the application and application method of a haplotype combination related to goat weight. The present invention provides a haplotype combination related to goat weight, the haplotype combination is located inside the ROBO1 gene, and the haplotype combination is any one of the haplotype combinations shown in SEQ ID NO.24, SEQ ID NO.16 or SEQ ID NO.19. The haplotype combination can be used to screen goat strains with large weight, thereby improving the accuracy of early seed selection.

Description

Technical Field

[0001] The invention belongs to the technical field of animal molecular breeding, and in particular relates to the application of a haplotype combination related to goat weight and an application method. Background Art

[0002] Zhongwei goat, also known as sand-haired goat, is a fur-type local breed of goat and the only goat breed in the world that produces white fur. In 2006, Zhongwei goat was included in the national list of livestock and poultry genetic resources for protection. As an excellent local breed in Ningxia, Zhongwei goat has the characteristics of tolerance to roughage, strong adaptability, stable genetic performance, etc., which has played an important role in increasing the income of local farmers and herdsmen. The fur and meat characteristics have high protection value. Therefore, it is of great significance to carry out research, protection and development and utilization of Zhongwei goat. The weight and other traits of livestock are important body size indicators reflecting the growth of livestock. The weight data can reflect the growth and development of the livestock and the relative development relationship between various parts, reflecting the production performance of individual or group livestock. With the increasing maturity of whole genome resequencing technology, SNPs significantly associated with weight traits can be mined from the genome level. SNPs tend to be globally inherited, and SNPs located in a haplotype have strong linkage disequilibrium. Therefore, it is necessary to study the role of SNP combination and growth traits of Zhongwei goat. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides the application of the haplotype combination related to goat weight and the application method.

[0004] The application of a haplotype combination associated with goat weight in screening or identifying goat weight, wherein the haplotype combination is located inside the ROBO1 gene, and the haplotype combination is any one of the haplotype combinations shown in SEQ ID NO.24, SEQ ID NO.16 or SEQ ID NO.19.

[0005] Preferably, when the haplotype combination at 25677801, 25677876, 25678239, 25678251, 25678324, 25678335, 25678368, 25678372, 25678427, 25678429, 25678495 and 25678511 on chromosome 1 of goat ROBO1 gene is as shown in SEQ ID When the haplotype combination shown in NO.24 is used, the haplotype combination at 25670191, 25670217, 25670232, 25670243, 25670262, 25670275, 25670279, 25670303, 25670387, 25670393, 25670460, 25670593, 25670744, 25670756, 25670768, 25671054, 25671329, 25671372, 25671652, 25671896 and 25671919 on chromosome 1 of the goat ROBO1 gene is as shown in SEQ ID When the haplotype combination is shown in NO.19 or the haplotype combination at 25668836, 25668883, 25668886, 25668903, 25668911, 25668938, 25668952, 25669255, 25669288, 25669326, and 25669344 on chromosome 1 of the goat ROBO1 gene is the haplotype combination shown in SEQ ID NO.16, the weight of the goat is greater than that of the goat with any of the above haplotype combinations.

[0006] Application of the reagent for detecting the haplotype combination related to goat weight in preparing a kit for identifying goat weight.

[0007] The application of the haplotype combination in goat genetic breeding.

[0008] Preferably, individuals with haplotype combinations such as the haplotype combination shown in SEQ ID NO.24, the haplotype combination shown in SEQ ID NO.16 or the haplotype combination shown in SEQ ID NO.19 are selected as parents, and group subgeneration is carried out to select goat strains with heavy weight and fast growth rate.

[0009] Preferably, the goat is a Zhongwei goat.

[0010] An application method, the application method is a method for screening or detecting goat weight, comprising the following steps:

[0011] Extract genomic DNA from goat ear tissue;

[0012] The genomic DNA is broken into DNA fragments, and then a library is constructed using the DNA fragments, and the library is sequenced to obtain sequencing data;

[0013] The sequencing data is identified and screened through the variant sites, and the haplotype combination on chromosome 1 of the ROBO1 gene in the genomic DNA of the ear tissue is detected. When the haplotype combination at 25677801, 25677876, 25678239, 25678251, 25678324, 25678335, 25678368, 25678372, 25678427, 25678429, 25678495 and 25678511 on chromosome 1 of the goat ROBO1 gene is the haplotype combination shown in SEQ ID NO.24, the goat is a heavy-weight goat;

[0014] When the haplotype combination at 25670191, 25670217, 25670232, 25670243, 25670262, 25670275, 25670279, 25670303, 25670387, 25670393, 25670460, 25670593, 25670744, 25670756, 25670768, 25671054, 25671329, 25671372, 25671652, 25671896 and 25671919 on chromosome 1 of goat ROBO1 gene is the haplotype combination shown in SEQ ID NO.19, the goat is of large weight;

[0015] When the haplotype combination at 25668836, 25668883, 25668886, 25668903, 25668911, 25668938, 25668952, 25669255, 25669288, 25669326, and 25669344 on chromosome 1 of the goat ROBO1 gene is the haplotype combination shown in SEQ ID NO.16, the goat is a heavy weight goat.

[0016] Preferably, the length of the DNA fragment is 320 bp to 380 bp.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a haplotype combination related to goat weight, which can be used to screen goat strains with large weight, thereby improving the accuracy of early seed selection, having good economic benefits and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the distribution diagram of SNPs in the 1Mb window of chromosome after quality control. The left Y-axis represents the chromosome name and the upper X-axis represents the window size.

[0020] Figure 2 Manhattan Plots and QQ-plots show the GWAS results of the body weight traits of Zhongwei goats, where A is the Manhattan Plots graph and B is the QQ-plots graph.

[0021] Figure 3 These are the results of haplotype analysis of ROBO1 gene related to weight in Zhongwei goats, where A is the D' value graph of the block formed by 12 SNPs in ROBO1 gene, and B is the r² value graph of the block formed by 12 SNPs in ROBO1 gene. In the figure, D' and R² measure the degree of linkage disequilibrium between two loci, D'=1 or r²=1 indicates complete linkage, D'=0 or r²=0 indicates no linkage or linkage equilibrium, D'>0.80 and r²>0.33 indicate strong linkage, and the numbers in the red squares in the figure represent the percentage between the two loci. The percentage without numbers in the red squares is close to 100 and is not displayed. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0023] Example

[0024] 1. Experimental animals and phenotype sources:

[0025] The experimental goats in this study were all from the Zhongwei goat breeding farm in Ningxia Hui Autonomous Region. The phenotypic records of body weight at 40 days of age in 2022 were measured, and the results are shown in Table 1. Ear tissue samples were collected from 240 Zhongwei goats, and all samples were immediately stored in liquid nitrogen after collection and transported to the laboratory for long-term storage at -80°C.

[0026] Table 1 Determination method of body weight traits of Zhongwei goats

[0027]

[0028] 2. Genomic DNA Extraction and Quality Inspection

[0029] DNA was extracted from ear tissue samples using the phenol-chloroform method, and the purity and integrity of DNA were detected using 1% agarose gel electrophoresis. The purity of DNA and the presence of protein or RNA contamination were detected using a NanoDrop2000 spectrophotometer at 260nm / 280nm, with an acceptance standard of 1.80-2.00.

[0030] 3. Library construction and sequencing

[0031] After the qualified genomic DNA samples were processed, the genomic DNA was randomly sheared into 350 bp fragments using a Covaris ultrasonic crusher. The DNA fragments were repaired at the end, poly(A) was added, sequencing adapters were added, purified, and PCR amplified to complete the entire library preparation. After the library construction was completed, Qubit2.0 was used for preliminary quantification, and the effective concentration of the library was accurately quantified using the qPCR method to ensure the quality of the library. After the library quality test was qualified, the sequencing was performed using the BGI-T7 sequencing platform, and the sequencing mode was PE150 mode.

[0032] 4. Identification, screening and annotation of variant sites

[0033] The fastp software version V0.20.0 was used to filter the Raw reads data into Clean reads data, and a genome index was established for the reference genome. The Burrows-Wheeler Aligner software version V0.7.17 was used to align the quality-controlled Clean reads data with the goat reference genome, where the goat reference genome is ARS1, GCF_001704415.1, and the Burrows-Wheeler Aligner is abbreviated as BWA. The SAMtools software version V1.8-20 was used to convert the aligned sam files into bam files and sort the bam files. The MarkDuplicates program in the GenomeAnalysis Toolkit software version V3.8 was used to remove duplicate data from the sorted bam files to obtain the final bam files. Genome Analysis Toolkit is abbreviated as GATK; the final bam file is indexed, and the HaplotypeCaller module in the GATK software is used to detect SNP variations. After obtaining the vcf file, the VariantFiltration module is used for filtering. The ANNOVAR software package is used to perform functional annotations on the detected gene variants. According to the location of the variant site on the reference genome and the gene location information on the reference genome, the region where the variant site occurs in the genome and the impact of the variant can be obtained. The region where the variant site occurs in the genome is the intergenic region, intron region or CDS region, and the impact of the variant is synonymous or non-synonymous mutation.

[0034] After whole genome resequencing, variant detection and strict quality control of 240 individuals of Zhongwei goat, all detected variants of Zhongwei goat were annotated using gene annotation files downloaded from the Ensembl database, and it was found that the most variants were found in intergenic regions and intronic regions, and only a few of them were located in coding regions. These potential functional variants provide valuable genetic resources for exploring the genetic structure and functional genes of Zhongwei goat.

[0035] 5. Data Quality Control

[0036] The obtained genotyping data were quality controlled using Plink software version V1.90, and individuals with genotype detection rate <95%, SNPs with detection rate <95%, SNPs with minimum allele frequency <5%, and Hardy-Weinberg equilibrium test P value <10 were excluded. -6 For SNPs, the detection rate is recorded as call rate, the minimum allele frequency is recorded as MAF, and the Hardy-Weinberg equilibrium is recorded as HWE.

[0037] A total of 34,248,064 SNPs were involved in quality control. After genotype detection rate filtering, Hardy-Weinberg equilibrium filtering, minimum allele frequency filtering, and individual detection rate filtering, a total of 15,958,716 SNPs sites were obtained for subsequent analysis. These sites are evenly distributed on the 29 pairs of autosomes of goats, such as Figure 1 .

[0038] VI. Genome-wide association analysis and gene annotation

[0039] The association analysis between SNP and body weight traits was performed using the fastGWA-mlm model in the GCTA software version V1.94.0beta.

[0040] y=X snp β snp +X c β c +g+e

[0041] where y is an n×1 phenotype vector; X snp is a genotype vector with effects β snp ;X c is the correlation matrix of the fixed covariates, and its corresponding coefficient is β c , the fixed covariates are sex and the first three PCAs; g is the vector of total genetic effects captured by the SNP-derived genetic relationship matrix, denoted as GRM.

[0042] Because the Bonferroni correction method is too strict to determine the significance threshold of GWAS, this study adjusted the threshold of genome-wide significant association to P = 1 × 10 -6 . The genomic expansion factor of the test statistic was calculated by the slope of the linear regression between the observed quantile and the theoretical quantile in R software version V3.6.0, and the genomic expansion factor was recorded as λ. The λ value of the 40-day-old body weight trait was calculated to be 1.063, indicating that there was no genomic expansion. Based on the resequencing data of 240 Zhongwei, 147 significant SNP sites associated with the 40-day-old body weight trait were detected, which were located on chromosomes 1, 5, 13, 17, 19, 20, and 26, as shown in Tables 2 to 5 and Figure 2 shown.

[0043] Table 2 Significant SNPs associated with body weight

[0044]

[0045] Table 3 Continuation of Table 2

[0046]

[0047] Table 4 Continuation of Table 3

[0048]

[0049] Table 5 Continuation of Table 4

[0050]

[0051] VII. Construction of ROBO1 gene haplotype combination

[0052] Data statistics and analysis were performed based on the genome-wide significant sites obtained from GWAS. According to the calculation principles of parameters such as allele frequency, genotype frequency, homozygosity and heterozygosity, Excel functions were written to calculate population genetics parameters and test whether the population conformed to the Hardy-Weinberg equilibrium principle, as shown in Tables 6 to 8.

[0053] Table 6 Population genetic parameters of SNPs in ROBO1 gene for body weight traits in goats

[0054]

[0055] Table 7 Continuation of Table 6

[0056]

[0057] Table 8 Continuation of Table 7

[0058]

[0059] Subsequently, the haplotype was constructed using Haploview software, and it was found that 12 SNPs at 25677801, 25677876, 25678239, 25678251, 25678324, 25678335, 25678368, 25678372, 25678427, 25678429, 25678495 and 25678511 on chromosome 1 constituted the haplotype. The block includes 4 haplotypes, named T1, T2, T3 and T4; 25670191, 25670217, 25670232, 25670243, 25670262, 25670275, 25670279, 25670303, 25670387, 25670393, 25670460, 2567059 3. 21 SNPs at 25670744, 25670756, 25670768, 25671054, 25671329, 25671372, 25671652, 25671896 and 25671919 constitute a block, which includes 7 haplotypes, named S1, S2, S3, S4, S5, S6 and S7 respectively; 836, 25668883, 25668886, 25668903, 25668911, 25668938, 25668952, 25669255, 25669288, 25669326, and 25669344, 11 SNPs constitute a block, which includes 4 haplotypes, named R1, R2, R3, and R4, as shown in Tables 9 and Figure 3 .

[0060] Table 9 Different haplotypes of ROBO1 gene

[0061]

[0062] 8. Association analysis between ROBO1 gene haplotype combination and body weight of Zhongwei goats

[0063] SAS9.2 software was used to analyze the relationship between haplotype combinations and body weight traits in individuals.

[0064] Table 10 lists the statistical data of the body weight of Zhongwei goats under the haplotype combination of the ROBO1 gene. Since the number of goats with haplotypes S3-S7, R3-R4 and T3-T4 was less than 3, the genotype was rare, and the proportion of individuals was too small, the variance analysis results did not meet the statistical significance, so they were eliminated and no multiple comparisons were performed; the body weight of the T2T2 and T1T2 haplotype combinations of the ROBO1 gene was significantly higher than that of the T1T1 combination (P<0.05), the body weight of the R2R2 haplotype combination was significantly higher than that of the R1R1 and R1R2 combinations (P<0.05), and the S2S The weight of the 2 haplotype combination was significantly higher than that of the S1S1 and S1S2 combinations (P<0.05). Since homozygotes tend to be stably inherited, it can be seen from Table 10 that the weight of T2T2 was 6.4389±0.3537, the weight of R2R2 was 6.438±0.354, and the weight of S2S2 was 6.475±0.399. The weights of T2T2, R2R2 and S2S2 were greater than those of other haplotype combinations. Therefore, individuals with the T2T2, R2R2 or S2S2 haplotype combination can be selected as the sire or dam to improve the weight trait of Zhongwei goats.

[0065] Table 10 Body weight of Zhongwei goats under different haplotype combinations of ROBO1 gene / kg

[0066]

[0067] Note: Different letters indicate significant differences, p < 0.05; the same letter indicates insignificant differences, p > 0.05.

[0068] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0069] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0070] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of a haplotype combination associated with goat weight in identifying goat weight, characterized in that: The haplotype combination is located inside the ROBO1 gene, the reference genome of the goat is ARS1, GCF_001704415.1, and the haplotype combination is any one of the haplotype combinations shown in SEQ ID NO.24, SEQ ID NO.16 or SEQ ID NO.19; (1) When the haplotype combination at 25677801, 25677876, 25678239, 25678251, 25678324, 25678335, 25678368, 25678372, 25678427, 25678429, 25678495 and 25678511 on chromosome 1 of the goat ROBO1 gene is the haplotype combination shown in SEQ ID NO.24, the weight of the goat is greater than the weight of the goat with the non-above haplotype combination, and the non-above haplotype combination is the haplotype combination shown in SEQ ID NO.22 and SEQ ID NO.23; (2) The haplotype combination at positions 25670191, 25670217, 25670232, 25670243, 25670262, 25670275, 25670279, 25670303, 25670387, 25670393, 25670460, 25670593, 25670744, 25670756, 25670768, 25671054, 25671329, 25671372, 25671652, 25671896 and 25671919 on chromosome 1 of the goat ROBO1 gene is as shown in SEQ ID When the haplotype combination shown in NO.19 is used, the weight of the goat is greater than the weight of the goat with the haplotype combination other than the above, and the haplotype combination other than the above is the haplotype combination shown in SEQ ID NO.20 and SEQ ID NO.21; or (3) when the haplotype combination at positions 25668836, 25668883, 25668886, 25668903, 25668911, 25668938, 25668952, 25669255, 25669288, 25669326, and 25669344 on chromosome 1 of the goat ROBO1 gene is the haplotype combination shown in SEQ ID NO.16, the weight of the goat is greater than the weight of the goat with a haplotype combination other than the above, and the haplotype combination other than the above is the haplotype combination shown in SEQ ID NO.17 or SEQ ID NO.18; The goats are Zhongwei goats.

2. The application of the haplotype combination described in claim 1 in goat genetic breeding, characterized in that: Individuals with haplotype combinations such as the haplotype combination shown in SEQ ID NO.24, the haplotype combination shown in SEQ ID NO.16 or the haplotype combination shown in SEQ ID NO.19 are selected as parents, and group succession is performed to select goat strains with heavy weight and fast growth rate; The goats are Zhongwei goats.

3. A method for detecting the weight of a goat, characterized in that: The steps include: Extract genomic DNA from goat ear tissue; The genomic DNA is broken into DNA fragments, and then a library is constructed using the DNA fragments, and the library is sequenced to obtain sequencing data; The sequencing data is subjected to identification and screening of variant sites, and the haplotype combination on chromosome 1 of the ROBO1 gene in the genomic DNA of the ear tissue is detected. When the haplotype combination at 25677801, 25677876, 25678239, 25678251, 25678324, 25678335, 25678368, 25678372, 25678427, 25678429, 25678495 and 25678511 on chromosome 1 of the goat ROBO1 gene is the haplotype combination shown in SEQ ID NO. 24 in claim 1, the goat is a heavy-weight goat; When the haplotype combination at 25670191, 25670217, 25670232, 25670243, 25670262, 25670275, 25670279, 25670303, 25670387, 25670393, 25670460, 25670593, 25670744, 25670756, 25670768, 25671054, 25671329, 25671372, 25671652, 25671896 and 25671919 on chromosome 1 of goat ROBO1 gene is the haplotype combination shown in SEQ ID NO.19 in claim 1, the goat is a heavy-weight goat; When the haplotype combination at positions 25668836, 25668883, 25668886, 25668903, 25668911, 25668938, 25668952, 25669255, 25669288, 25669326, and 25669344 on chromosome 1 of the goat ROBO1 gene is the haplotype combination shown in SEQ ID NO.16 in claim 1, the goat is of large weight; The reference genome of the goat is ARS1, GCF_001704415.1; The goats are Zhongwei goats.

4. The method according to claim 3, characterized in that The length of the DNA fragment is 320 bp to 380 bp.

5. Use of a reagent for detecting the haplotype combination associated with goat weight as claimed in claim 1 in preparing a kit for identifying goat weight.

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