阅读说明：本技术 一种筛选牛体高变异相关分子标记的方法及其应用 (Method for screening related molecular markers of bovine body hypervariability and application thereof ) 是由 张亚冉 黄金明 王金鹏 姜强 刘文浩 鞠志花 王秀革 魏晓超 高亚平 杨春红 肖 于 2021-10-26 设计创作，主要内容包括：本发明属于动物分子育种技术领域,涉及一种筛选牛体高变异相关分子标记的方法及其应用,所述分子标记包括位于GALNT8基因上的单核苷酸多态位点,分别为：rs133878668A﹥C、rs135659917A﹥G、rs210135533G﹥A和rs110000229A﹥G。利用全基因组关联分析方法结合基因型效应、环境效应及基因型与环境互作效应分析,准确高效地筛选体高变异相关的重要新候选基因和分子标记,方法科学合理,依据候选基因和标记设计的检测方法,具有操作简单快捷、结果准确性高和成本低的特点。(The invention belongs to the technical field of animal molecular breeding, and relates to a method for screening related molecular markers of bovine hypervariability and application thereof, wherein the molecular markers comprise single nucleotide polymorphic sites on GALNT8 gene, which are respectively as follows: rs133878668A > C, rs135659917A > G, rs210135533G > A and rs110000229A > G. The whole genome correlation analysis method is combined with genotype effect, environmental effect and genotype and environmental interaction effect analysis, important new candidate genes and molecular markers related to somatic hypermutation are accurately and efficiently screened, the method is scientific and reasonable, and the detection method designed according to the candidate genes and the markers has the characteristics of simplicity and rapidness in operation, high result accuracy and low cost.)

1. A molecular marker related to the screening of bovine hypervariation is characterized by comprising single nucleotide polymorphic sites on GALNT8 gene, which are respectively: rs133878668A > C, rs135659917A > G, rs210135533G > A and rs110000229A > G.

2. The molecular marker associated with the screening of bovine body height variation according to claim 1, wherein said molecular marker is rs110000229A > G.

3. A kit for detecting a mononucleotide polymorphic site rs110000229 is characterized by at least comprising the following primers and HhaI restriction endonuclease;

F:5‘-GGCAGGTTTGGACTTGGT-3’(SEQ ID NO.1)

R:5‘-GGAAGGGATTGGAGGTTG-3’(SEQ ID NO.2)。

4. a primer combination for detecting the molecular marker related to the bovine body hypervariability screening of claim 1 or 2.

5. Use of the primer combination of claim 4 for the preparation of a kit for identifying individuals with a high body height specific molecular marker in bovines.

6. The use of the molecular marker related to the bovine hypervariation screening in the assisted selection breeding according to claim 1 or 2, is characterized in that the application mode specifically comprises the following steps:

extracting blood DNA of different cattle individuals;

identifying the genotype of the marker by using a kit for detecting the molecular marker, and identifying individuals with high body height specific molecular markers;

further, the molecular marker is a single nucleotide polymorphic site rs 110000229.

7. A method for screening a molecular marker related to bovine somatic hypervariation, the method at least comprising:

carrying out genotyping on a DNA sample of each individual in a cattle group by using the SNP chip;

calculating the body height genome estimated breeding value of each cow by using the genotyping result;

carrying out whole genome correlation analysis on the body height genome breeding value and the genotype by utilizing GEMMA software; combining gene annotation to screen single nucleotide polymorphic SNPs molecular markers and candidate genes which are obviously related to the somatic height variation;

carrying out genotyping on specific SNP sites of the cattle with body height data by utilizing a PCR product direct sequencing method; defining high-body high-dominance allelic gene types;

further, the method specifically comprises:

s1, collecting a cattle group sample and extracting DNA;

s2, SNP chip detection analysis and body height genome breeding value calculation;

s3, carrying out GEMMA whole genome correlation analysis;

s4, annotating genes at the SNP sites and screening candidate genes;

s5, identifying SNPs (single nucleotide polymorphisms) which are positioned at the candidate gene and are obviously related to the somatic height variation and the specific SNP with the strongest correlation;

s6, carrying out gene typing on the specific SNP locus by a PCR product direct sequencing method;

s7, defining the high-body high-advantage genotype of the specific SNP locus;

further, the specific method of S1 includes:

s1.1, collecting a blood or hair follicle sample of a cattle group;

s1.2, extracting DNA in blood or hair follicle tissues of each cow;

further, the specific method of S2 includes:

s2.1, carrying out genotyping on the DNA sample of each cow by using the SNP chip;

s2.2, estimating the body height genome breeding value of each individual according to the genotype;

further, the specific method of S3 includes:

s3.1, filtering the SNP data, and analyzing the SNP which meets the requirements;

s3.2, performing whole genome correlation analysis by using GEMMA, wherein an analysis model is a univariate linear mixed model, and covariates are the ages of the pasture and the cattle in months;

s3.3, correcting the P value by adopting an FDR (fully drawn receptor) multiple detection method;

s3.4, drawing a Manhattan graph of the GWAS result by using an R language;

s3.5, searching and annotating SNPs by using a UCSC database;

further, the specific method of S4 includes:

s4.1, selecting SNPs with a P value less than 0.05, namely SNPs remarkably related to body height variation;

s4.2, searching the annotated reference gene of the position where each SNP is located by using a UCSC genome browser;

s4.3, screening candidate genes;

further, the specific method of S5 includes:

s5.1, screening SNPs with the P value less than 0.05 at the position of the candidate gene, namely, screening the SNPs, namely, screening new molecular markers which are obviously related to the body height variation;

s5.2, screening the sites with the strongest correlation with the high variation of the cattle body in the molecular markers, wherein the sites are specific SNP markers with high cattle body characters;

further, the specific method of S6 includes:

s6.1, measuring the body height of a cattle group in a large-scale breeding pasture and collecting a blood sample;

s6.2, extracting blood genome DNA;

s6.3, designing a PCR primer covering the specific SNP locus, and carrying out PCR amplification;

s6.4, directly sequencing the PCR product, and genotyping the specific SNP locus of each cow according to a sequencing peak map;

further, the specific method of S7 includes:

s7.1, calculating the genotype effect and the environmental effect of the specific SNP locus on the body height, and the interaction effect of the genotype and the environment;

s7.2, determining dominant allelic gene types, and screening out high-body high-character cattle individuals or groups.

8. Use of the molecular marker related to the screening of bovine body hypervariability of claim 1 or 2 or the kit of claim 3 or the primer combination of claim 4 in dairy cow breeding, genetic improvement or marker-assisted selection.

The application of GALNT8 gene in screening cow body high variation cow.

10. The molecular marker related to the screening of the bovine body height variation as claimed in claim 11 or 2 is used for identifying and assisting in identifying the individuals with high body height superiority of the dairy cows or preparing products for identifying and assisting in identifying the individuals with high body height superiority of the dairy cows.

Technical Field

The invention belongs to the technical field of animal molecular breeding, and relates to a method for screening a molecular marker related to bovine hypervariation and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Body height is one of the important traits for genetic evaluation of Holstein cattle. The body height of a cow is closely related to the health level and the age of the cow, and determines the productivity and potential of the cow. For example, cow body height is positively correlated with properties such as milk yield (correlation coefficient 0.42), average body weight (0.63), number of breeds (0.67) (Berry et al, 2004). In modern dairy breeding, people prefer to select individuals with higher heights (Ajmone-Marsan et al, 2010). The average height of holstein cattle has increased by about 2mm annually over the last decade (Bouwman et al, 2018). In a domestic large-scale Holstein cattle farm, the first mating is carried out when the height of a cow reaches 132 cm.

Body height is a complex quantitative trait controlled by multiple genes and is influenced by the environment. The body height of mammals is highly genetic and easy to measure (Guo et al, 2017). The high heritability of cattle is 25% -85%, belongs to the characters of middle and high heritability (Nelsen et al, 1986; Northcutt and Wilson, 1993), and can obtain the effect by direct selection according to phenotype. However, the generation interval of the dairy cows is long, the breeding investment is large, the genetic progress can be remarkably accelerated by early selection, and the method has great value in breeding practice. However, in order to realize early genetic evaluation and selection, it is necessary to identify functional genes and markers related to body height for molecular genetic evaluation. In order to identify major genes affecting body height, studies on the genetic structure and molecular basis of body height have been extensively conducted in various species. Especially, over the last 10 years, with the development of high-throughput genotyping and DNA sequencing technologies, especially the application of Genome-wide association assays (GWAS), the research progress of basic analysis of somatotropic variant molecules has been promoted.

Two major candidate gene regions associated with hypervariable outphasing in cattle have been found to be the NCAPG-LCORL and PLAG 1-CHCHCHD 7 regions (Randhawa et al, 2015; Takasuga, 2016) located on cattle chromosome 6 and 14, respectively, which exhibit strong selection signals in multiple cattle breeds (Druet et al, 2013; Kemper et al, 2014). For example, Karim et al (2011) identified QTNs between PLAG1 and chchchhd 7 that were associated with body height in the population of hybrids of holstein and jersey F2. The QTNs consist of a (CCG) n trinucleotide repeat and an adjacent SNP, which affect the transcriptional activity of the bidirectional promoter by affecting the binding of transcription factors. Increased body height increased expression of PLAG1, chchhd 7 and other adjacent genes in tissues corresponding to the relevant alleles. Recently, it was found by haplotype analysis that the mutant form (Q) of bovine PLAG1 plays an important role in the body size change of modern cattle (Utsunomiya et al, 2017).

In the population of beef cattle breeds belonging to both common and tumor cattle, several major QTL regions and SNPs loci, including the SNPs of the PLAG 1-CHCHHD 7 and NCAPG-LCORL regions, account for variations in body height greater than 1/3 of cattle (Saatchi et al, 2014). However, within the milk holstein cow population, the major SNPs known can account only for the 9.3% of the body height variation of holstein cows (Bouwman et al, 2018). At present, a large number of molecular markers related to the high variation of holstein cattle still remain to be mined.

Disclosure of Invention

Aiming at the problems, the invention provides a method for screening a bovine hypervariation related molecular marker and application thereof. A cattle 50K SNP gene chip is utilized, genome-wide association analysis is carried out based on GEMMA software, new candidate genes and molecular markers thereof which influence the body height variation of Holstein cattle are screened out, the genotype effect and the environmental effect on the body height are analyzed according to specific molecular markers, the high body height advantage genotype is determined, and a corresponding detection method is provided. Provides scientific basis for screening and cultivating Holstein cattle with higher body height and better body shape and appearance by using a molecular breeding method, and establishes a feasible technical method. In addition, the research can also provide a new reference for the related research of analyzing the somatotropic molecular mechanism of other livestock and poultry and human beings.

Specifically, the invention is realized by the following technical scheme:

in a first aspect of the invention, a molecular marker associated with screening for bovine somatotropy, comprising a single nucleotide polymorphic site located on the GALNT8 gene, which is: rs133878668A > C (chr5:105800017, UMD3.1), rs135659917A > G (chr5:105838237, UMD3.1), rs210135533G > A (chr5:105837539, UMD3.1) and rs110000229A > G (chr5:105804923, UMD 3.1). Wherein the rs110000229A & gt G site has the highest relevance with the high variation of cattle body (genome breeding value). The high genome breeding value corresponding to the GG genotype individual at the rs110000229 locus is the largest, which indicates that the genotype GG is a candidate dominant genotype with high body height, and the genotype is selected to obtain the largest genetic progress, so that preferably, the molecular marker is rs110000229A & gt G.

In the second aspect of the invention, the primer combination is used for detecting the molecular marker related to the bovine body hypervariation screening. Researchers can design appropriate specific primers based on specific site information, and can realize the detection of the SNP molecular markers, so that any primer combination for detecting the SNP molecular markers belongs to the protection scope of the invention.

In a third aspect of the invention, the use of the claimed primer combination for the preparation of a kit for identifying individuals with a high body height specific molecular marker in a bovine body. Researchers can utilize the primer combination to realize the screening of high-body high-specificity cows, and the kit for designing and identifying the high-body high-specificity cows aiming at the primer combination is also within the protection scope of the invention.

In the fourth aspect of the present invention, the application of the molecular marker related to the bovine hypervariation screening in assisted selective breeding specifically comprises:

extracting blood DNA of different cattle individuals;

and identifying the genotype of the marker by using a kit for detecting the molecular marker, and identifying individuals with high body height specific molecular markers.

In a fifth aspect of the present invention, a method for screening a bovine hypervariable associated molecular marker, the method at least comprises:

carrying out genotyping on a DNA sample of each individual in a cattle group by using the SNP chip;

calculating the body height genome estimated breeding value of each cow by using the genotyping result;

carrying out whole genome correlation analysis on the body height genome breeding value and the genotype by utilizing GEMMA software; combining gene annotation to screen single nucleotide polymorphic SNPs molecular markers and candidate genes which are obviously related to the somatic height variation;

carrying out genotyping on specific SNP sites of the cattle with body height data by utilizing a PCR product direct sequencing method; defining high-body high-dominance allelic type.

Further, rs110000229 locus of the cattle with body height data is subjected to genotyping by a PCR product direct sequencing method, the genotype effect and the environmental effect of the locus on the body height and the interaction effect of the genotype and the environment are analyzed, the genotype GG is determined to be a high-body high-dominance allele type, and the PCR product direct sequencing method can be directly used for screening the body height of the cattle.

In the sixth aspect of the invention, the molecular marker related to the bovine hypervariation screening or the kit or the primer combination is applied to breeding, genetic improvement or marker-assisted selection of cows.

In a seventh aspect of the invention, the use of the GALNT8 gene for screening of highly variant cows.

In the eighth aspect of the invention, the molecular marker related to the screening of the cow body height variation is used for identifying and assisting in identifying the cow body height dominant individual or preparing a product for identifying and assisting in identifying the cow body height dominant individual.

One or more embodiments of the present invention have the following advantageous effects:

the detection method is original, aiming at the genetic characteristic of the height of the cattle, in a cattle group, a whole genome correlation analysis method is utilized to combine genotype effect, environmental effect and genotype and environment interaction effect analysis, important new candidate genes and molecular markers related to the height variation of the cattle are accurately and efficiently screened, the method is scientific and reasonable, and the detection method designed according to the candidate genes and the markers has the characteristics of simple and rapid operation, high result accuracy and low cost;

by adopting the method, individuals or groups with higher body height can be effectively screened, and the method has important significance for genetic improvement work of the body appearance and related production traits of the Holstein cattle variety. The invention is a good application of molecular breeding technology in production practice, can greatly reduce breeding cost and shorten breeding time, and generates good economic and social benefits.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a flowchart of the method for screening Hostan cattle somatotropic mutation-related molecular markers in example 1.

FIG. 2 shows the new candidate gene GALNT8 for somatic hypermutation and 4 SNPs (rs133878668, rs135659917, rs210135533, and rs110000229) on the gene, which were screened by GEMMA whole genome association analysis in example 1. All 4 SNPs are significantly related to the hypervariability of the Holstein cow body (P < 0.01).

FIG. 3 is a schematic diagram showing the structure of GALNT8 gene and the positions of SNPs selected from the gene. Wherein, 3 SNPs are located in GALNT8 gene, and 1 SNP is located in 5' regulatory region of GALNT8 gene.

FIG. 4 shows the direct sequencing of the PCR product at the SNP (rs110000229, A > G) site of the variant candidate gene GALNT8 in example 1.

FIG. 5 shows the interaction pattern between the genotype and the environment at the SNP (rs110000229, A > G) site of the variant candidate gene GALNT8 in example 1.

FIG. 6 shows the result of PCR-RFLP detection of SNP (rs110000229, A > G) of the variant candidate gene GALNT8 in example 1.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Explanation:

according to the theory of quantitative genetics: the phenotype of a quantitative trait is largely dependent on genotype and environment, and there is an interaction between genotype and environment. The genotype in the invention is two pure combinations of two alleles aiming at one locus of the genome, the phenotype is the height of the cow, and the environment is different cow farms.

The calculation methods of the genotype effect, the environmental effect and the interaction effect of the genotype and the environment on the body height of two genotypes of a certain genome site under two different environments (dairy farms) are as follows (tables 1 and 2):

in table μ_ijRepresents the average phenotypic value of the ith genotype under the jth environment. G₁And G₂Being a genotype effect of genotype 1 and genotype 2, E₁And E₂The environmental effects of environment 1 and environment 2, respectively. GE₁₁For the interaction of genotype 1 and Environment 1, GE₁₂For the interaction of genotype 1 and Environment 2, GE₂₁For the interaction of genotype 2 and Environment 1, GE₂₂Is the interaction effect of genotype 2 and environment 2.

TABLE 1 average expression of two genotypes under two environments and calculation of genotype Effect and environmental Effect

TABLE 2 calculation of genotype and environmental interaction effects for two genotypes under two environments

Therefore, the genotype effect in the invention refers to the contribution of different genotypes at a certain position to the variation of the body height of the cow, the environmental effect refers to the contribution of different environments (cow farms) to the variation of the body height of the cow, and the genotype and environment interaction effect refers to the contribution of the interaction of the genotypes and the environments (cow farms) to the variation of the body height of the cow.

The currently known major SNPs can only explain the variation of the Holstein cattle body by 9.3 percent, and a large number of molecular markers related to the Holstein cattle body height variation still need to be mined. To this end, a specific embodiment is provided, which is a method for screening a molecular marker associated with bovine hypervariability, the method at least comprising:

carrying out genotyping on a DNA sample of each individual in a cattle group by using the SNP chip;

calculating the body height genome estimated breeding value of each cow by using the genotyping result;

carrying out whole genome correlation analysis on the body height genome breeding value and the genotype by utilizing GEMMA software; combining gene annotation to screen single nucleotide polymorphic SNPs molecular markers and candidate genes which are obviously related to the somatic height variation;

carrying out genotyping on specific SNP sites of the cattle with body height data by utilizing a PCR product direct sequencing method; calculating the genotype effect and the environmental effect of the specific SNP locus on the height of the body and the interaction effect of the genotype and the environment, and determining the allele type with high body height superiority.

Further, the method specifically comprises:

s1, collecting a cattle group sample and extracting DNA;

s2, SNP chip detection analysis and body height genome breeding value calculation;

s3, carrying out GEMMA whole genome correlation analysis;

s4, annotating genes at the SNP sites and screening candidate genes;

s5, identifying SNPs (single nucleotide polymorphisms) which are positioned at the candidate gene and are obviously related to the somatic height variation and the specific SNP with the strongest correlation;

s6, carrying out gene typing on the specific SNP locus by a PCR product direct sequencing method;

s7, analyzing the genotype effect, the environmental effect and the interaction effect between the genotype and the environment of the specific SNP locus on the body height, and determining the high-body-height dominant genotype of the specific SNP locus;

further, the specific method of S1 includes:

s1.1, collecting a blood or hair follicle sample of a cattle group;

s1.2, extracting DNA in blood or hair follicle tissues of each cow;

further, the specific method of S2 includes:

s2.1, carrying out genotyping on the DNA sample of each cow by using the SNP chip;

s2.2, estimating the body height genome breeding value of each individual according to the genotype;

further, the specific method of S3 includes:

s3.1, filtering the SNP data, and analyzing the SNP which meets the requirements;

s3.2, performing whole genome correlation analysis by using GEMMA, wherein an analysis model is a univariate linear mixed model, and covariates are the ages of the pasture and the cattle in months;

s3.2, correcting the P value by adopting an FDR (fully drawn receptor) multiple detection method;

s3.3, drawing a Manhattan graph of the GWAS result by using an R language;

s3.4, searching and annotating SNPs by using a UCSC database;

further, the specific method of S4 includes:

s4.1, selecting SNPs with a P value less than 0.05, namely SNPs remarkably related to body height variation;

s4.2, searching the annotated reference gene of the position where each SNP is located by using a UCSC genome browser;

s4.3, screening candidate genes;

further, the specific method of S5 includes:

s5.1, screening SNPs with the P value less than 0.05 at the position of the candidate gene, namely, screening the SNPs, namely, screening new molecular markers which are obviously related to the body height variation;

s5.2, screening the sites with the strongest correlation with the high variation of the cattle body in the molecular markers, wherein the sites are specific SNP markers with high cattle body characters;

further, the specific method of S6 includes:

s6.1, measuring the body height of a cattle group in a large-scale breeding pasture and collecting a blood sample;

s6.2, extracting blood genome DNA;

s6.3, designing a PCR primer covering the specific SNP locus, and carrying out PCR amplification;

s6.4, directly sequencing the PCR product, and genotyping the specific SNP locus of each cow according to a sequencing peak map;

further, the specific method of S7 includes:

s7.1, calculating the genotype effect and the environmental effect of the specific SNP locus on the body height, and the interaction effect of the genotype and the environment;

s7.2, determining dominant allelic gene types, and screening out high-body high-character cattle individuals or groups.

The new molecular marker of the high variation of the cattle body screened based on the method, in particular to a GALNT8 gene which is a new candidate gene influencing the high variation of the cattle body, and the genetic variation of the GALNT8 gene can be used as the molecular marker of the high character of the holstein cattle body; meanwhile, the bovine hypervariable variant associated molecular marker comprises 4 single nucleotide polymorphic sites on the gene: rs133878668, rs135659917, rs210135533 and rs110000229, wherein rs110000229 has the strongest correlation with the high variation of holstein cattle, so the gene is most suitable to be used as a molecular marker of the high character of cattle; meanwhile, the genotype GG of the rs110000229 locus is a high-body high-dominance genotype, and can be used for selecting high-body Gaussian cattle individuals or groups.

In one or more embodiments, a kit for detecting a single nucleotide polymorphic site rs110000229, the kit at least comprising the following primers and a hhal restriction enzyme;

F:5‘-GGCAGGTTTGGACTTGGT-3’(SEQ ID NO.1)

R:5‘-GGAAGGGATTGGAGGTTG-3’(SEQ ID NO.2)。

the primer is designed at the upstream and downstream of the mononucleotide polymorphic site rs110000229, when the allele of the site is G, the HhaI restriction endonuclease site can be generated at the position close to the site, and when the allele is A, the HhaI restriction endonuclease site can not be generated. Accordingly, a fragment containing the polymorphic site can be amplified by PCR, and PCR products having different mutations can be digested into fragments of different lengths by digestion of the amplified fragment with the restriction enzyme HhaI; thereby distinguishing wild homozygous (AA) individuals, heterozygous individuals (AG) and homozygous mutant individuals (GG).

According to the invention, through carrying out whole genome association analysis on the cattle, a new specific SNP site related to cattle body height variation is discovered, genotype effect, environmental effect and genotype and environmental effect interaction analysis are carried out on the specific SNP site, and the height of the individual (population) body height of the Holstein cattle can be definitely judged by verifying a single SNP site in the same pasture. The method has important significance for molecular breeding of the Holstein cattle body type character.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

Example 1

1. Collection of Holstein cattle samples

In 11-scale breeding farms in China, blood or hair follicle samples of 2709 Holstein cattle are collected together, and the genome DNA of each cattle is extracted. The names of the 11 pastures, the locations and the number of samples collected are shown in table 1.

TABLE 1 Holstein cattle sampling pasture, site and number of samples

Pasture name	Location of a site	Sample number (head)
			Pasture A	Hebei chenchen platform	279
B pasture	Shandong Taian medicine	96
			C pasture	Inner Mongolia Tongliao	399
D pasture	Persistent branches of Jiangsu	285
			E pasture	Xinjiang tower city	200
F pasture	Gansu Jinchang	294
			G pasture	Guangdong province of Guangdong province	196
H pasture	Shandong Jinan	94
			I pasture	Shandong Dezhou	200
J pasture	Shandong Linyi	398
			K pasture	Shandong Ying	268

2. Genotyping Using Illumina bovis 50K SNP chip

The chip has 51,386 SNP markers. SNP data was filtered using Plink1.9 software, and after filtering, the remaining 47,843 SNPs were subsequently analyzed.

3.GEMMA genome-wide association analysis (GWAS)

The GWAS analysis employed a GEMMA univariate linear mixture model. The height of the body is taken as a dependent variable of GWAS analysis, and the cattle farm and the cattle month age are taken as covariates, so that the analysis strategy can obviously improve the accuracy of analysis. The P value was corrected using the FDR method. And drawing a Manhattan graph of the GWAS analysis result by using a qqman R software package. Annotated SNPs are retrieved in the UCSC database.

4. Screening of candidate genes and SNPs

(1) According to the results of GEMMA whole genome correlation analysis, 26 SNPs with significant differences (P <0.05) are screened, and 14 SNPs are located in 8 candidate genes or regulatory regions, as shown in Table 2.

TABLE 2 SNPs and candidate genes significantly associated with somatotype variation

(2) There are 4 SNPs (rs 110000229A > G, rs135659917A > G, rs133878668A > C, rs210135533G > a) which are significantly related to the body height variation (P <0.01) and are located in GALNT8 gene (fig. 2), and GALNT8 gene is a new candidate gene which is firstly screened to be related to the body height variation, wherein rs110000229 has the strongest correlation with the body height variation, and therefore, is most suitable as a molecular marker of the body height trait of holstein cattle.

(3) Further analyzing body height genome breeding values corresponding to different genotypes of the rs110000229 locus (table 3), finding that estimated breeding values corresponding to GG genotype individuals are all remarkably higher than AG and AA genotypes (P <0.01), the estimated breeding value corresponding to the AA genotype is the lowest, and prompting that the genotype GG is a candidate dominant genotype of high body height, and selecting the genotype can obtain the largest genetic progress.

TABLE 3 high genomic breeding values for individuals of different genotypes at the SNP (rs110000229, A > G) site

Note that: the upper right capital letter a \ B \ C of the number indicates a very significant level of difference (P < 0.01).

5. PCR direct sequencing method for analyzing genotype effect and environmental effect of SNP (rs110000229, A > G) site of highly new candidate gene GALNT8 and genotype and environmental interaction effect

(1) 70 holstein cows were selected to measure body height data (fat height) in the Shandong Jinan pasture 1 and the Tahan pasture 2, respectively, blood was collected and blood DNA was extracted.

(2) A pair of PCR primers was designed for amplifying a DNA fragment containing the SNP site of interest (rs.110000229).

F:5‘-TCGCTTTGGCATTTGTCT-3’(SEQ ID NO.3)

R:5‘-TTAGACACCCAGTCACCAT-3’(SEQ ID NO.4)

(3) The PCR reaction system is 20 μ L, and comprises 1.0 μ L (10 μmol/L) of the upstream primer, 1.0 μ L (10 μmol/L) of the downstream primer, 1 μ L (. about.50 μ g/L) of the DNA template, 10.0 μ L of 2 XTAQQ PCR Master Mix, and 7 μ L of ddH2O 7. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 59.3 ℃ for 30s, and extension at 72 ℃ for 30s, this step being performed for 35 cycles; extension at 72 ℃ for 10 min. The length of the target DNA fragment obtained by amplification is 1146 bp. The PCR product was detected by electrophoresis on a 1% agarose gel.

(4) The PCR product is directly sequenced, the sequencing result is compared and analyzed with a bovine GALNT8 gene sequence provided by an NCBI GenBank database, the SNP (rs110000229, A > G) locus of each Holstein cow is subjected to genotyping, and a sequencing peak image of individuals with wild homozygous AA genotype, heterozygous AG genotype and pure and mutant GG genotype is shown in figure 4.

(5) Combining the body height data and genotype data of each cow, the AA and GG genotype effects at the SNP (rs110000229, A > G) site and the environmental effects of both pastures (Table 4), as well as genotype and environmental interaction effects (Table 5) were calculated. As shown in Table 4, GG genotype effect of SNP (rs110000229, A > G) site increases body height of cattle by 1cm, AA genotype effect decreases body height of cattle by 1cm, and environmental effect of pasture 1 (environment 1) increases body height of cattle by 2cm, and environmental effect of pasture 2 (environment 2) decreases body height of cattle by 2 cm. As shown in table 5, there is an interaction between two genotypes and two environments, the interaction effect of genotype GG with environment 1 and environment 2 leads to a decrease and increase in body height of 0.4cm, respectively, while the interaction effect of genotype AA with pasture 1 (environment 1) and pasture 2 (environment 2) leads to an increase and decrease in body height of 0.4cm, respectively. As shown in FIG. 5, although there is an interaction between genotype and environment, the interaction pattern between genotype and environment is non-cross interaction, and there is no change in the order of genotype superiority, i.e. GG genotype is superior to AA genotype in both environments for cow height. In conclusion, in two pastures, the GG genotype is the dominant genotype of the high body height of cattle, the AA is the disadvantaged genotype of the high body height, and the body height can be selected according to the genotype of the rs110000229 locus.

TABLE 4 calculation of genotype effect and environmental effect of SNP (rs110000229, A > G) site on Holstein high trait

	Pasture 1 (Environment 1)	Pasture 2 (Environment 2)	Line average	Genotype effects
					Genotype GG	146cm	142.8cm	144.4cm	1cm
Genotype AA	144.8cm	140cm	142.4cm	-1cm
					Column average	145.4cm	141.4cm	143.4cm
Environmental effects	2cm	-2cm

TABLE 5 calculation of genotype-environment interaction effects of two genotypes at SNP (rs110000229, A > G) sites under two environments

	Pasture 1 (Environment 1)	Pasture 2 (Environment 2)
			Genotype GG	-0.4cm	0.4cm
Genotype AA	0.4cm	-0.4cm

6. The SNP (rs110000229, A > G) gene enzyme digestion detection method of the high candidate gene GALNT 8:

(1) holstein cattle with different body heights are selected, and blood DNA is extracted.

(2) Designing PCR primers at the upstream and downstream of SNP (rs110000229, A > G) site:

F:5‘-GGCAGGTTTGGACTTGGT-3’(SEQ ID NO.1)

R:5‘-GGAAGGGATTGGAGGTTG-3’(SEQ ID NO.2)

ensuring that no other HhaI cutting sites exist in the amplified fragment of the primer. The HhaI cleavage site appears only when the SNP site is the G mutation. Thus, the hhal restriction enzyme can cut PCR products with different mutations into fragments of different lengths.

(3) PCR-RFLP genotype analysis

Firstly, the target fragment is PCR amplified, the PCR reaction system is 20 mu L, and comprises 1.0 mu L (10 mu mol/L) of each of the upstream primer and the downstream primer, 10.0 mu L of 2 xTaq PCR Master Mix, 1 mu L (50 mu g/L) of DNA template, and ddH₂O7.0. mu.L. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 63.5 ℃ for 30s, and extension at 72 ℃ for 15s, in 35 cycles; extension at 72 ℃ for 10 min. The PCR product is 209bp in length.

The PCR product was digested with the HhaI endonuclease at 37 ℃ and then subjected to electrophoresis using a 2.5% agarose gel. The wild type individual (AA) in the electrophoresis detection result has only one electrophoresis band of 209 bp; the heterozygote individual (AG) has three bands of 209bp, 148bp and 61 bp; homozygous mutant individuals (GG) can separate two bands of 148bp and 61 bp. The specific detection result is shown in FIG. 5.

Secondly, the embodiment also discloses a kit containing the primer and the enzyme.

The kit also comprises a PCR amplification reaction reagent and an enzyme digestion reaction reagent.

Specifically, the PCR amplification reaction reagent comprises dNTP (25mM each), MgCl₂(25mM)、PCR Bμffer、ddH₂O, etc.;

the enzyme cutting reagent comprises ddH₂O, HhaI enzyme Buffer, HhaI enzyme (1U/. mu.l).

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Dairy cow research center of Shandong province academy of agricultural sciences

<120> method for screening related molecular markers of bovine hypervariability and application thereof

<130> 202125283

<160> 2

<170> PatentIn version 3.3

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ggcaggtttg gacttggt 18

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