A method for constructing beef cattle core germplasm and its application

Abstract

The present invention relates to the field of animal breeding technology, and in particular to a method for constructing a core germplasm of beef cattle and its application. The construction method provided by the present invention comprises: 1) obtaining genotype data of a beef cattle breed for which a core germplasm is to be constructed; 2) performing quality control and filling on the obtained genotype data, and screening common SNP sites; 3) using common SNP sites to calculate the genetic distance of an individual to a group, establishing a standard for eliminating outliers, and constructing a primary core germplasm of a beef cattle breed; 4) using the Core Hunter 3 algorithm to search for the core germplasm of the best proportion, and constructing a core germplasm of a beef cattle breed. The construction method provided by the present invention can represent the genetic diversity of each germplasm resource to the maximum extent with a minimum number of germplasms, greatly reducing the workload of preserving beef cattle germplasm resources, and has a certain promoting effect on the purity analysis of population pedigree.

Description

Technical Field

[0001] The present invention relates to the technical field of animal breeding, and in particular to a method for constructing a beef cattle core germplasm and an application thereof. Background Art

[0002] Germplasm resources refer to genetic materials and their carriers that carry biological information and have actual or potential utilization value. They are indispensable for the survival and sustainable development of human society, and are the material basis for original innovation in life sciences and the development of the bio-industry. my country's beef cattle have excellent characteristics such as tolerance to roughage, strong disease resistance, gentle temperament, good adaptability, stable genetics, and good meat quality. They are one of the important germplasm resources. However, due to changes in agricultural production methods and the advancement of hybridization and improvement, the population size and number of my country's beef cattle breeds are shrinking, and the excellent genes in some breeds have been lost. At the same time, gene exchange between breeds has led to a decrease in genetic diversity, a decrease in the adaptability and disease resistance of the population, and many genetic resources are in danger of losing genetic diversity. Therefore, the collection and preservation of beef cattle germplasm genetic resources plays an important role in the genetic diversity of beef cattle.

[0003] my country has a rich variety of beef cattle breeds. As of now, there are 85 beef cattle breeds, including 56 local breeds, 12 cultivated breeds, and 17 introduced breeds. The rich beef cattle germplasm resources provide a large amount of materials for breeding work, but the expansion of the scale of genetic materials makes the evaluation of germplasm resources relatively difficult. Core germplasm resources, that is, a core subset of preserved germplasm resources, are a core subset extracted from the preserved germplasm resources of a certain species using a certain method, with the least amount of genetic resource samples to maximize the representation of the genetic diversity of the entire resource group including geographical distribution. Therefore, the construction of beef cattle core germplasm can improve the management and utilization level of the entire germplasm bank. However, in the animal field, reports on core germplasm are relatively scarce, especially the construction process of beef cattle core germplasm, which is not yet very clear. Therefore, clarifying the methods and processes for the construction of core germplasm in beef cattle groups can greatly reduce the workload of breeders and efficiently complete the preservation of germplasm resources. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for constructing a core germplasm of beef cattle and its application. The construction method provided by the present invention can represent the genetic diversity of each germplasm resource to the maximum extent with a minimum number of germplasms, greatly reducing the workload of beef cattle germplasm resource preservation, and has a certain promoting effect on the purity analysis of population pedigree.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing a beef cattle core germplasm, comprising the following steps:

[0007] 1) Obtaining genotype data of the core germplasm beef cattle breed to be constructed; the genotype data is genotyping data of genomic SNP sites;

[0008] 2) Performing quality control on the genotype data in step 1) and filling in the missing SNP sites to obtain a set of beef cattle individuals with common SNP sites, which are recorded as original germplasm;

[0009] 3) using the common SNP loci described in step 2), calculating the average genetic distance between individuals in each breed and the breed population in the original germplasm according to formula I, using the quartile test method to establish a standard for eliminating outliers, eliminating individuals with large genetic differences in each breed, and obtaining the primary core germplasm of beef cattle breeds;

[0010] Formula I;

[0011] in, In is the average genetic distance, N is the number of individuals in the breed, unit: piece, d ij is the genetic distance between individuals;

[0012] The exclusion criteria are: In >UQ+(1.5~2)IQR; IQR=UQ-LQ, UQ is the upper quartile, LQ is the lower quartile;

[0013] 4) The Core Hunter 3 algorithm was used to perform a global search on the genotype data corresponding to the primary core germplasm obtained in step 3). The evaluation parameters were EN (Average entry-to-nearest-entry distance) and SH (Shannon's allelic diversity index). The weight ratio of EN and SH was 1:1. The evaluation parameters of EN and SH were combined using Formula II to screen the core germplasm and obtain the core germplasm of beef cattle breeds.

[0014] Formula II;

[0015] in, F ( C ) is the comprehensive evaluation value of core germplasm, F i ( c ) is the i The evaluation function of the parameters is normalized to [0,1] by default; α i is the weight of the parameter;

[0016] The screening criteria include: selecting a set with a maximum number of core germplasm not exceeding 60% of the breed and a gene coverage of ≥99% of the beef cattle core germplasm, and selecting a subset with the maximum expected heterozygosity within the set; if the sampling ratio reaches 60% of the original germplasm and the gene coverage is still less than 99%, selecting the subset with the maximum sampling ratio as the beef cattle core germplasm.

[0017] Preferably, before performing a global search on the genotype data corresponding to the primary core germplasm obtained in step 3), the genotype data is quality-controlled again according to the variety.

[0018] Preferably, the quality control criteria include: retaining only autosomal loci, eliminating loci with typing success rate <90% and minimum allele frequency <0.05, and eliminating loci with detection rate <90%, Hardy-Weinberg equilibrium test P <1×10 -6 of individuals.

[0019] Preferably, the quality control tool includes PLINK software; the filling tool includes Beagle software.

[0020] Preferably, the construction method further comprises: after obtaining the core germplasm of beef cattle breeds, performing representative evaluation on the core germplasm of beef cattle breeds; the parameters of the representative evaluation include: one or more of the number of alleles, the number of allele sites, the number of effective allele sites, the Shannon information index, the observed heterozygosity, the expected heterozygosity and the polymorphism information content.

[0021] Preferably, the representatively evaluated control group is the primary core germplasm obtained in step 3).

[0022] Preferably, the method for obtaining in step 1) comprises: extracting DNA of beef cattle with core germplasm to be constructed, and performing genotyping on the DNA using a SNP chip to obtain the genotype data.

[0023] Preferably, the calculation tool for the genetic distance between individuals includes PLINK software.

[0024] The present invention provides application of the construction method described in the above technical solution in the preservation of beef cattle germplasm resources.

[0025] Preferably, the application comprises the analysis of the pedigree purity of beef cattle breeds.

[0026] Beneficial effects:

[0027] The construction method provided by the present invention is a screening method for beef cattle core germplasm established for the first time in beef cattle, which has guiding significance and can improve the efficiency of beef cattle germplasm resource preservation and utilization; the construction method provided by the present invention first constructs a primary core germplasm, and then selects the primary core germplasm by using the average genetic distance ( In ), and In >UQ+(1.5~2)IQR is used as a specific standard for eliminating outliers, thereby reducing the risk of false positives; in addition, compared with the fuzzy ratio selection of conventional studies, the present invention uses the Core Hunter 3 algorithm to establish the screening standard for the core germplasm of beef cattle, and according to this standard, compares the core germplasm subsets of different sampling ratios of each variety, and selects the best-scale subset that meets the standard as the core germplasm of the variety. After representative evaluation, it is found that the core germplasm screened by the present invention can well represent the original germplasm. It can be seen that the construction method provided by the present invention can maximize the representation of the genetic diversity of each germplasm resource with the minimum number of germplasms, greatly reducing the workload of beef cattle germplasm resource preservation, and has a certain promoting effect on the purity analysis of population pedigree. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 A flow chart of the beef cattle core germplasm construction method and application provided by the present invention. DETAILED DESCRIPTION

[0030] The present invention provides a method for constructing a beef cattle core germplasm, comprising the following steps:

[0031] 1) Obtaining genotype data of the core germplasm beef cattle breed to be constructed; the genotype data is genotyping data of genomic SNP sites;

[0032] 2) Performing quality control on the genotype data in step 1) and filling in the missing SNP sites to obtain a set of beef cattle individuals with common SNP sites, which are recorded as original germplasm;

[0033] 3) using the common SNP loci described in step 2), calculating the average genetic distance between individuals in each breed and the population in the original germplasm according to formula I, using the quartile test method to establish a standard for eliminating outliers, eliminating individuals with large genetic differences in each breed, and obtaining the primary core germplasm of beef cattle breeds;

[0034] Formula I;

[0035] in, In is the average genetic distance; N The number of individuals in the breed, unit: piece; d ij is the genetic distance between individuals;

[0036] The exclusion criteria are: In >UQ+(1.5~2)IQR; IQR=UQ-LQ, UQ is the upper quartile, LQ is the lower quartile;

[0037] 4) Using the Core Hunter 3 algorithm to perform a global search on the genotype data corresponding to the primary core germplasm obtained in step 3), the evaluation parameters selected were EN and SH, the weight ratio of EN and SH was 1:1, and the evaluation parameters of EN and SH were combined using Formula II to screen the core germplasm to obtain the core germplasm of beef cattle breeds;

[0038] Formula II;

[0039] in, F ( C ) is the comprehensive evaluation value of the core collection, which is the evaluation function of each parameter F i ( c ) and the corresponding weights α i Combination F i ( c ) is the i The evaluation function of the parameters is normalized to [0,1] by default; α i is the weight of the parameter;

[0040] The screening criteria include: selecting a set with a maximum number of core germplasm not exceeding 60% of the breed and a gene coverage of ≥99% of the beef cattle core germplasm, and selecting a subset with the maximum expected heterozygosity within the set; if the sampling ratio reaches 60% of the original germplasm and the gene coverage is still less than 99%, selecting the subset with the maximum sampling ratio as the beef cattle core germplasm.

[0041] The present invention obtains genotype data of a core germplasm beef cattle breed to be constructed; the genotype data is genotyping data of a genomic SNP site. As an embodiment, the method for obtaining comprises: extracting DNA of a core germplasm beef cattle breed to be constructed, and genotyping the DNA using a SNP chip to obtain the genotype data. As another embodiment, the sample source of the DNA comprises blood and / or semen.

[0042] After obtaining the genotype data, the present invention performs quality control on the genotype data and fills in the missing SNP sites to obtain a set of beef cattle individuals with common SNP sites, which are recorded as original germplasm. As an embodiment, the quality control standards include: retaining only autosomal sites, eliminating sites with typing success rate <90% and minimum allele frequency <0.05, and eliminating sites with detection rate <90%, Hardy-Weinberg equilibrium testP <1×10 -6 As an implementation, the quality control tool may be PLINK software; the filling tool may be Beagle software.

[0043] After obtaining the original germplasm, the present invention uses the public SNP loci to calculate the average genetic distance of the beef cattle individual to the group according to Formula I, uses the quartile test method to establish the elimination criteria for outliers, eliminates individuals with large genetic differences in each breed of beef cattle group, and obtains the primary core germplasm of the beef cattle breed. As an embodiment, the calculation tool for the genetic distance between the individuals can be PLINK software. As an embodiment, the elimination criteria are: In >UQ+1.5IQR. The elimination criteria selected by the present invention can retain more beef cattle individuals while minimizing the risk of false positives.

[0044] After obtaining the primary core germplasm of beef cattle breeds, the present invention uses the Core Hunter 3 algorithm to perform a global search on the genotype data corresponding to the primary core germplasm, selects EN and SH as evaluation parameters, and the weight ratio of EN and SH is 1:1. The evaluation parameters of EN and SH are combined together using Formula II, and the core germplasm is screened to obtain the core germplasm of beef cattle breeds.

[0045] As an implementation method, before the global search of the genotype data corresponding to the primary core germplasm is performed, the genotype data is quality controlled again according to the variety. The secondary quality control can provide a purer and more satisfactory data environment for the algorithm, so that the algorithm can run accurately as expected, thereby improving the reliability and effectiveness of the entire core germplasm screening process. The quality control standards are the same as above and will not be repeated here.

[0046] The construction method provided by the present invention first constructs the primary core germplasm, and then uses the average genetic distance ( In ), and In >UQ+(1.5~2)IQR is used as the specific standard for eliminating outliers, thereby reducing the risk of false positives. In addition, compared with the fuzzy ratio selection in conventional studies, the present invention uses the Core Hunter 3 algorithm to establish the screening standard for the core germplasm of beef cattle, and according to this standard, compares the core germplasm subsets with different sampling ratios of each breed, and selects the subset with the best size that meets the standard as the core germplasm of the breed. Representative evaluation shows that the core germplasm screened by the present invention can well represent the original germplasm.

[0047] As an implementation mode, after obtaining the core germplasm of beef cattle breeds, the present invention performs a representative evaluation on the core germplasm of beef cattle breeds; the parameters of the representative evaluation include: one or more of the number of alleles, the number of allele sites, the number of effective allele sites, the Shannon information index, the observed heterozygosity, the expected heterozygosity and the polymorphism information content.

[0048] As an embodiment, the representative evaluation control group is the primary core germplasm. As an embodiment, if the genetic parameters of the beef cattle breed core germplasm and the control group are substantially the same, it means that the beef cattle breed core germplasm can well represent the original germplasm.

[0049] The present invention provides application of the construction method described in the above technical solution in the preservation of beef cattle germplasm resources.

[0050] As an embodiment, the application can be used as an analysis of the pedigree purity of beef cattle breeds. Using the construction method provided by the present invention, the core germplasm set of beef cattle breeds can be accurately determined. These core germplasm individuals are genetically representative and can cover most of the genetic diversity of the entire beef cattle breed. Focusing resources on the preservation of these core germplasms can achieve effective protection of key genetic information of beef cattle breeds under limited resource conditions (such as funds, sites, etc. for germplasm preservation). For example, the semen, embryos or cell lines of core germplasm individuals are preferentially preserved in the germplasm bank to ensure that the core genetic material of the beef cattle breed is retained when facing various risks (such as diseases, natural disasters, etc. that may cause a sharp decline in population size), providing basic materials for subsequent breed recovery and improvement.

[0051] The purpose of the present invention is to provide a method for constructing a core germplasm of beef cattle, and to apply the method to establish a core germplasm resource bank of beef cattle to preserve and manage my country's beef cattle germplasm resources, so as to achieve the maximum representation of the genetic diversity of the population with the minimum number of germplasms, greatly reduce the workload of preserving beef cattle germplasm resources, and have a certain promoting effect on the purity analysis and protection of the population pedigree.

[0052] In order to further illustrate the present invention, a method for constructing a beef cattle core germplasm and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1 Construction of core germplasm of different beef cattle breeds

[0054] Experimental materials: A total of 1193 individuals of 31 beef cattle breeds from 17 provinces across the country were selected as the research subjects, including 23 local cattle breeds, 4 cultivated breeds and 4 introduced breeds (see Table 1).

[0055] Table 1 Sample information of 31 beef cattle breeds

[0056]

[0057] The specific steps are as follows:

[0058] (1) DNA was extracted from the collected blood or frozen sperm samples according to the instructions of the blood or frozen sperm DNA extraction kit, and the DNA purity and concentration were tested by ultraviolet spectrophotometer and agarose gel electrophoresis. The samples with a DNA concentration >50 ng / μL and an A260 / A280 ratio between 1.9 and 2.1 were qualified. Genotyping was then performed to obtain the genotype information of genomic SNPs;

[0059] (2) Before the analysis, the genotyping data of 1193 cattle from 31 breeds were quality controlled using PLINK v 1.9 software. Only autosomal loci were retained, and loci with typing success rate < 90% and minimum allele frequency < 0.05 were eliminated. At the same time, loci with detection rate < 90% were eliminated. Hardy-Weinberg equilibrium test P <1×10 -6The specific operations are as follows: first, merge the genotype data of all varieties and perform quality control. The running code is: plink --bfile filename1 --merge filename2 --make-bed --out filename, plink --cow --bfile filename --geno 0.1 --make-bed --out filename; then perform quality control again for each variety. The running code is: plink --cow --bfile filename --geno 0.1 --mind 0.1 --maf 0.05 --hwe 0.000001 --make-bed --out filename; after quality control, use Beagle 4.0 to fill in the missing SNP sites for each variety. The running command of Beagle software is: java -Xmx1000m -jar unphased=file.bgl out=outputniterations=100; merge the genotype data of each variety after filling again, perform quality control, and eliminate SNPs with a detection rate of <0.9. The running code is: plink --bfile filename1 --merge filename2 --make-bed --out filename, plink --cow --bfile filename --geno 0.1 --make-bed --outfilename, and finally 19256 autosomal public SNPs of 1066 individuals were obtained for subsequent analysis (Table 1).

[0060] (3) PLINK v1.9 software was used to calculate the genetic distance between individuals. The running code was: plink --bfilefilename --distance-matrix --out filename. For each variety, the average genetic distance between the individual and the rest of the individuals calculated according to Formula I was used as the individual i The average genetic distance of the population ( In ).

[0061] Formula I;

[0062] in, In is the average genetic distance; N The number of individuals in the breed, unit: piece; d ij is the genetic distance between individuals. D iThe larger the value, the more individual i The less similar they are to the rest of the individuals in the group.

[0063] Then, the quartile test method (IQR=UQ-LQ, UQ is the upper quartile, LQ is the lower quartile) was used to eliminate individuals with large genetic differences within the breed. The elimination criteria were: In >UQ+1.5IQR, the screening thresholds for each breed are shown in Table 2. In addition, all individuals of Leiqiong cattle and Tibetan cattle In The values ​​did not meet the standards and the genetic differences within the varieties were large, indicating that the bloodline of the variety was impure and the variety representativeness was poor. Therefore, it was eliminated in the construction of the primary core germplasm, and finally 939 primary core germplasms of 29 varieties were established (Table 2).

[0064] Table 2 Primary core collection

[0065]

[0066] (4) For the primary core germplasm of 939 individuals from 29 varieties, quality control was performed on each population individually. The quality control standard was consistent with step (2). The running code was: plink --cow --bfile filename --geno 0.1 --mind 0.1 --maf 0.05 --hwe 0.000001 --make-bed --recode A --out filename. The number of SNP loci after quality control for each variety is shown in Table 2.

[0067] (5) Input the genotype files of each beef cattle breed obtained in step (4) into the corehunter software package, and search for the optimal sampling ratio through the sampleCore function. The evaluation parameters of sampleCore are EN and SH, and the weight parameters are set as: objective ("EN", "MR", weight = 0.5), objective ("SH", weight = 0.5), and the germplasm sets with different sampling ratios are calculated according to formula II.

[0068] Formula II;

[0069] F ( C ) is the comprehensive evaluation value of core germplasm, F i ( c ) is the i The evaluation function of the parameters is normalized to [0,1] by default to ensure the balance between parameters; α i is the weight of this parameter, and the weight ratio of EN and SH is 1:1.

[0070] Then, the genetic parameters of various germplasm sets were calculated by R language, and the beef cattle breeds that met the screening criteria were selected as core germplasms. The screening criteria were: the maximum number of core germplasms did not exceed 60% of the breed and the gene coverage (CV) of the beef cattle core germplasm was ≥ 99%. The subset with the largest expected heterozygosity (He) was selected from the set. If the sampling ratio of the core germplasm reached 60% of the original germplasm and the CV was still less than 99%, the subset with the largest sampling ratio was selected as the beef cattle core germplasm. After calculation, 29 beef cattle breed core germplasms were finally obtained (Table 3).

[0071] (6) In order to further illustrate the representativeness of the core germplasm of beef cattle constructed by the present invention, the genetic parameters of the core germplasm and the primary core germplasm of each beef cattle breed were calculated using R language, including the number of alleles (A), the number of allele sites (a), the number of effective allele sites (Ne), the Shannon information index (I), the observed heterozygosity (Ho), the expected heterozygosity (He) and the polymorphism information content (PIC). The genetic parameters of the two germplasm sets were compared, and the results showed that the genetic parameters of the core germplasm were basically consistent with the genetic parameters of the primary core germplasm, and the core germplasm screened by the present invention can well represent the original germplasm (Table 3 and Table 4).

[0072] Table 3 Core collections and their representative evaluation 1 (core collections / primary core collections)

[0073]

[0074] Table 4 Core collections and their representative evaluation 2 (core collections / primary core collections)

[0075]

[0076] In summary, the beef cattle core germplasm resource bank established by the beef cattle core germplasm construction method provided by the present invention can be used to preserve and manage my country's beef cattle germplasm resources, achieve the maximum representation of the genetic diversity of the population with the minimum number of germplasms, greatly reduce the workload of beef cattle germplasm resource preservation, and have a certain promoting effect on the purity analysis of the population pedigree.

[0077] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a beef cattle core germplasm, characterized in that: The following steps are involved: 1) Obtaining genotype data of the core germplasm beef cattle breed to be constructed; the genotype data is genotyping data of genomic SNP sites; 2) Performing quality control on the genotype data in step 1) and filling in the missing SNP sites to obtain a set of beef cattle individuals with common SNP sites, which are recorded as original germplasm; The quality control criteria include: retaining only autosomal loci, eliminating loci with typing success rate <90% and minimum allele frequency <0.05, and eliminating loci with detection rate <90%, Hardy-Weinberg equilibrium test P <1×10 -6 individuals; 3) using the common SNP loci described in step 2), calculating the average genetic distance between individuals in each breed and the breed population in the original germplasm according to formula I, using the quartile test method to establish a standard for eliminating outliers, eliminating individuals with large genetic differences in each breed, and obtaining the primary core germplasm of beef cattle breeds; Formula I; wherein, Di is the average genetic distance; N The number of individuals in the breed, unit: piece; d ij is the genetic distance between individuals; the elimination criteria are: Di >UQ+(1.5~2)IQR; IQR=UQ-LQ, UQ is the upper quartile, LQ is the lower quartile; 4) Using the Core Hunter 3 algorithm to perform a global search on the genotype data corresponding to the primary core germplasm obtained in step 3), the evaluation parameters selected were EN and SH, the weight ratio of EN and SH was 1:1, and the evaluation parameters of EN and SH were combined using Formula II to screen the core germplasm to obtain the core germplasm of beef cattle breeds; Formula II; in, F ( C ) is the comprehensive evaluation value of core germplasm; F i ( c ) is the i The evaluation function of the parameters is normalized to [0,1] by default; α i is the weight of the parameter; The screening criteria include: selecting a set with a maximum number of core germplasm not exceeding 60% of the original germplasm of the breed and a gene coverage of ≥99% of the core germplasm of beef cattle, and selecting a subset with the largest expected heterozygosity among the set; if the sampling ratio reaches 60% of the original germplasm and the gene coverage is still less than 99%, selecting the subset with the maximum sampling ratio as the core germplasm of beef cattle.

2. The construction method according to claim 1, characterized in that: Before conducting a global search on the genotype data corresponding to the primary core collection obtained in step 3), the genotype data is quality controlled again according to the variety.

3. The construction method according to claim 1 or 2, characterized in that: The quality control tool includes PLINK software; the filling tool includes Beagle software.

4. The construction method according to claim 1, characterized in that: The construction method further comprises: after obtaining the core germplasm of beef cattle breeds, performing representative evaluation on the core germplasm of beef cattle breeds; the parameters of the representative evaluation include: one or more of the number of alleles, the number of allele sites, the number of effective allele sites, the Shannon information index, the observed heterozygosity, the expected heterozygosity and the polymorphism information content.

5. The construction method according to claim 4, characterized in that: The control group for the representative evaluation is the primary core germplasm obtained in step 3).

6. The construction method according to claim 1, characterized in that: The method for obtaining in step 1) includes: extracting DNA of beef cattle with core germplasm to be constructed, and performing genotyping on the DNA using a SNP chip to obtain the genotype data.

7. The construction method according to claim 1, characterized in that: The calculation tool for the genetic distance between individuals includes PLINK software.

8. Application of the construction method according to any one of claims 1 to 7 in the conservation of beef cattle germplasm resources.

9. The use according to claim 8, characterized in that: The applications include analysis of the pedigree purity of beef cattle breeds.

Images