Sea buffalo whole genome SNP molecular marker combination and application

Abstract

The invention discloses a marine buffalo whole genome SNP (Single Nucleotide Polymorphism) molecular marker combination and application. The SNP molecular marker combination is shown in a table 1 and is composed of 47943 sequencing source SNP sites with high genetic information content, 868 character related sites and 76 defect related sites, and the total number of the sites is 48887. The marine buffalo whole genome SNP molecular marker combination can be used for designing marine buffalo whole genome chips and protecting and breeding marine buffalo genetic resources, targeted gene detection work can be achieved for marine buffalo populations, various requirements in genetic resource protection and breeding are met, the molecular breeding efficiency is effectively improved, and the breeding cost is reduced. The method is of great significance to protection, evaluation and utilization of genetic resources of the marine buffalo.

Description

Technical Field

[0001] The invention relates to genetic breeding of Haizi water buffalo, and in particular to a whole genome SNP molecular marker combination and application of Haizi water buffalo. Background Art

[0002] Haizi buffalo is a swamp buffalo breed unique to my country. It has excellent characteristics such as strong adaptability, tolerance to roughage, and strong resistance to adversity. It is an important local livestock and poultry resource and cultural heritage. However, affected by the accelerated development of modern agriculture and the adjustment of agricultural structure, its population has sharply decreased and its genetic resources are on the verge of loss. In 1990, the number of stocks in Jiangsu Province reached 42,500, while the data of the third national census in 2022 showed that there were only 4,051 left, and they had basically been converted from service to meat. The extreme shrinkage of the population has seriously weakened its genetic diversity and increased the risk of extinction. In view of its unique value in the agricultural ecosystem, it is urgent to carry out the protection of genetic resource diversity and variety improvement to avoid the ecological and cultural losses caused by its demise.

[0003] Traditional breeding methods evaluate the breeding value of cattle through appearance identification or phenotypic determination, but they have great limitations and low accuracy, and the generation interval of cattle is long, which further leads to slow genetic progress. Molecular breeding is a method of selecting and improving biological genetic traits at the molecular level using genomics, molecular marker technology, etc. Compared with traditional breeding methods, it has many advantages such as high efficiency, accuracy, and strong targeting. As one of the commonly used technologies in molecular breeding, gene chips are the main tools in livestock and poultry genomic selection breeding. However, the chips currently used in the market, such as Illumina Bovine SNP50, Illumina Bovine HD, Illumina Multi-Species Genotyping Bead Chips, etc., usually select single nucleotide polymorphisms (SNPs) that are widely distributed among multiple breeds as their design sites, which lack the targeting of Haizi buffalo populations.

[0004] Therefore, in order to protect and improve the genetic resources of Haizi buffalo, accelerate the process of molecular breeding, enhance the efficiency of genetic diversity protection and precision breeding, and assist the sustainable development of the population, it is crucial to design a whole-genome SNP molecular marker combination for Haizi buffalo that can be used for genetic resource protection and breeding. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a whole genome SNP molecular marker combination of Haizi buffalo that can be used for genetic resource protection and breeding; the second purpose is to provide the application of this SNP site combination in the whole genome chip and molecular breeding of Haizi buffalo.

[0006] Technical solution: The whole genome SNP molecular marker combination of Haizi buffalo described in the present invention, as shown in Table 1, consists of 48,887 SNP sites, including 47,943 sequencing-derived high genetic information SNP sites, 868 trait-related sites and 76 defect-related sites.

[0007] Table 1 SNP molecular marker combination loci information for the whole genome of Haizi buffalo

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[0067] Preferably, in the Haizi buffalo genome-wide SNP molecular marker combination of the present invention, the method for establishing the sequencing-derived high genetic information content SNP loci comprises:

[0068] (1) Original filtering and indexing based on reference genome

[0069] (2) Screening suitable Haizi buffalo for whole genome sequencing;

[0070] (3) Optimizing the sequencing results;

[0071] (4) Use software to filter and screen to obtain SNP sites with high genetic information.

[0072] Preferably, in the Haizi buffalo whole genome SNP molecular marker combination described in the present invention, the software described in step (4) in the above establishment method is a software SNPSelection (v1.0) that can be used for selecting high genetic information sites in the livestock and poultry genome chip design process, and the software registration number is 2024SR1581643.

[0073] The Haizi buffalo genome-wide SNP molecular marker combination of the present invention can be used for the Haizi buffalo SNP chip design, and the chip can detect one or more sites in the aforementioned Haizi buffalo genome-wide SNP molecular marker combination.

[0074] The whole genome SNP molecular marker combination of Haizi water buffalo described in the present invention can be used for the protection and breeding of Haizi water buffalo genetic resources.

[0075] Preferably, the application is genetic diversity analysis of Haizi buffalo.

[0076] Preferably, the application is analysis of individual kinship of Haizi buffalo.

[0077] Preferably, the application is the pedigree structure analysis of the Haizi buffalo population.

[0078] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The whole genome SNP molecular marker combination of Haizi buffalo is based on the reference genome and combined with the whole genome sequencing information of Haizi buffalo, which is particularly suitable for the genetic resource protection and breeding of Haizi buffalo population; 2. The whole genome SNP molecular marker combination of Haizi buffalo can meet various needs in genetic resource protection and breeding, the analysis results are clear and reliable, and can accurately reflect the richness of genetic diversity and inbreeding in the population and establish family structure, which can effectively promote the improvement of molecular breeding efficiency of Haizi buffalo. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 This is the site density map of the whole genome 50K SNP chip of Haizi buffalo;

[0080] Figure 2 A is the frequency distribution diagram of the smallest allele of Haizi buffalo;

[0081] Figure 2 B is the distribution map of polymorphic information content of SNP loci in Haizi buffalo;

[0082] Figure 2 C is the result of heterozygosity analysis of Haizi buffalo;

[0083] Figure 3 A is the ROH length distribution diagram of Haizi buffalo population;

[0084] Figure 3 B is the chromosome ROH distribution map of Haizi buffalo;

[0085] Figure 3 C is the distribution diagram of inbreeding coefficient of Haizi buffalo individuals;

[0086] Figure 4 This is the visualization result of the IBS distance matrix of Haizi buffalo;

[0087] Figure 5 This is the visualization result diagram of Haizi buffalo G matrix;

[0088] Figure 6 This is the evolutionary tree of the 46 Haizi buffalo population;

[0089] Figure 7 This is the principal component analysis diagram of the Haizi buffalo population;

[0090] Figure 8 This is the parallel coordinate axis analysis diagram of the Haizi buffalo population. DETAILED DESCRIPTION

[0091] The technical solution of the present invention is further described below.

[0092] Example 1: Establishment of a genome-wide SNP molecular marker combination for Haizi buffalo

[0093] 1. 176 Haizi buffaloes were selected, and genomic DNA was extracted after collecting tail venous blood. The purity of DNA was tested using NanoDrop2000, the concentration of DNA was accurately quantified using Qubit2.0, and the integrity of genomic DNA was tested using agarose gel electrophoresis. The qualified DNA was subjected to whole genome sequencing;

[0094] 2. Based on the swamp buffalo reference genome GCF_029407905.1, NGSQCToolkit (v2.3) and Burrows-Wheeler Aligner (v0.7.17) were used for raw filtering and reference genome index establishment; Samtools (v1.21) was used for file format conversion, and Picard software was used for reordering, and identical reads were marked, and a new index was generated; Genome Analysis Toolkit (v4.3) was used to generate the original .vcf file; then SNP and index selection was performed; finally, SNP filtering was performed to obtain SNPs of 30× deep sequencing data. The filtering requirements are as follows: the minimum allele frequency is greater than 0.05, the P value of the Hardy-Weinberg equilibrium test is greater than 10-6, the detection rate is greater than 90% of the SNP sites, and the R2 with the tag SNP is greater than 0.1, and it is marked as a dimorphic site;

[0095] 3. A software called SNPSelection (v1.0, software registration number 2024SR1581643), which can be used for selecting high genetic information sites in the design of livestock and poultry genome chips, was used to select 47,943 SNP sites with high genetic information from the SNPs in the above 30× deep sequencing data. Combined with 868 SNP sites related to meat quality, reproduction, health, development, etc. and 76 defect-related sites, the whole genome SNP molecular marker combination of Haizi buffalo was established.

[0096] Example 2: Haizi buffalo genome-wide SNP molecular marker combination for Haizi buffalo SNP chip design

[0097] According to the whole genome SNP molecular marker combination of Haizi buffalo established in Example 1, probes were designed for all 48887 sites using Primer3 (v2.6.1), and the design principles were as follows: GC content between 40% and 60%; length of 18-30 base pairs; annealing temperature of 55-65°C; avoid dimers and hairpin structures. The designed probes were synthesized by Shijiazhuang Boredy Biotechnology Co., Ltd. and loaded on a suitable solid phase carrier, namely the Haizi buffalo 50K SNP chip.

[0098] Example 3: DNA extraction and genome sequencing for verification of whole genome SNP molecular marker combination of Haizi buffalo

[0099] 1. The Haizi buffalo conservation population in a ranch near Yancheng City, Jiangsu Province was used as the experimental subject. The tail venous blood of 46 Haizi buffaloes was collected in a blood collection tube containing EDTA anticoagulant, and the blood was stored at -80℃ after recording the number.

[0100] 2. Use RK02008 Biomarker Blood / Cell / Tissue DNA Kit from Biomarker to extract genomic DNA, use NanoDrop2000 to detect DNA purity, use Qubit2.0 to accurately quantify DNA concentration, and use agarose gel electrophoresis to detect the integrity of genomic DNA.

[0101] 3. Send qualified DNA to Shijiazhuang Boridi Biotechnology Co., Ltd. for whole genome sequencing.

[0102] Example 4: Application of whole genome SNP molecular marker combination of Haizi buffalo in genetic resource protection and breeding

[0103] 1. Used for genetic diversity analysis of Haizi buffalo:

[0104] Plink (v1.90) was used to analyze the whole genome sequencing results of Haizi buffalo in Example 2 based on the whole genome SNP molecular marker combination of Haizi buffalo. A total of 56.57 effective alleles were detected in 46 Haizi buffaloes, with an average effective allele number of 1.24. The minimum allele frequency was 0.011, and the distribution diagram is as follows Figure 2 As shown in A, its distribution is relatively uniform; the polymorphic information content of SNP sites is 0.021-0.375, and the average polymorphic information content is 0.159. Figure 2 As shown in B, it shows that the genetic diversity of this population is relatively rich; in addition, the average expected heterozygosity of this population is 0.198, and the average observed heterozygosity is slightly higher than the average expected heterozygosity, which is 0.201. Figure 2 As shown in C, it indicates that the conservation population may not have inbreeding, and a small amount of foreign blood has been introduced.

[0105] 2. Used for analysis of individual kinship of Haizi buffalo

[0106] Plink (v1.90) was used to analyze the runs of homozygosity (ROH) in the whole genome sequencing results of Haizi buffalo in Example 2 based on the whole genome SNP molecular marker combination of Haizi buffalo. The distribution, length and number of ROH of each cow were counted, and the ratio of the total length of ROH segments to the total length of the autosomal genome in each individual was calculated. This ratio is the inbreeding coefficient based on ROH. Figure 3 As shown in A, a total of 1764 ROHs were detected in 46 Haizi buffalo populations, and 96.94% of the ROHs were between 0 and 5 Mb in length. The shortest ROH was 1.00 Mb in length and located on chromosome 1; the longest ROH was 10.81 Mb in length and located on chromosome 7. Figure 3 As shown in B, the distribution of ROH is relatively uniform, with the largest number of ROH on chromosome 1 being 187, and the smallest number of ROH on chromosome 24 being 25. The total length of ROH of each Haizi buffalo ranges from 1.05 to 470.74 Mb, with an average total length of 71.03 Mb. The largest number of individuals have a total length of ROH between 10 and 50, with a total of 24. Figure 3 As shown in C, the average inbreeding coefficient of the Haizi buffalo conservation population based on ROH is 0.029, indicating a low level of inbreeding.

[0107] Gmatix (v2) software was further used to construct the identity by state (IBS) distance matrix and G matrix, and heat maps were drawn to analyze the genetic relationship of the conservation population. Figure 4 , 5 As shown in the figure, the IBS genetic distance of the conservation population ranges from 0.092 to 0.172, with an average of 0.157. The results of the IBS distance matrix are consistent with the G matrix. Most of the Haizi buffalo individuals have a moderate degree of genetic relationship, and a small number of individuals have a close genetic relationship, indicating that the conservation population of Haizi buffalo has a low inbreeding risk.

[0108] 3. Family structure analysis of Haizi buffalo population

[0109] Using MegaX (v10.0), based on the whole genome SNP molecular marker combination of Haizi buffalo, the SNP sites after quality control of the whole genome sequencing results of Haizi buffalo in Example 2 were analyzed and a population evolution tree was drawn to determine the family structure in the conservation Haizi buffalo population. The results are as follows Figure 6As shown in Table 1, there are different genetic distances among the 46 Haizi buffaloes, but some individuals have strong genetic connections, forming 7 family structures; the results of principal component analysis are shown in Table 1. Figure 7 As shown in Figure 2, it also shows that the Haizi buffalo population has a certain family structure. The first four principal components explain 3.2%, 3.1%, 3.1% and 3.0% of the population variation respectively; the results of spatial parallel coordinate axis analysis are shown in Figure 2. Figure 8 As shown in the figure, it was found that the first four principal components could not divide the population well, which also showed that the population had a weak family structure.

Claims

1. A genome-wide SNP molecular marker combination of Haizi buffalo, characterized in that: The molecular marker combination consists of 48887 SNP loci, and the specific loci information is as follows:

2. The whole genome SNP molecular marker combination of Haizi buffalo according to claim 1, characterized in that: The molecular marker combination includes: 47943 sequencing-derived SNP sites with high genetic information content, 868 trait-related sites and 76 defect-related sites.

3. The whole genome SNP molecular marker combination of Haizi buffalo according to claim 2, characterized in that: The method for establishing the sequencing-derived SNP loci with high genetic information content is as follows: (1) Original filtering and indexing based on reference genome (2) Screening suitable Haizi buffalo for whole genome sequencing; (3) Optimizing the sequencing results; (4) Use software to filter and screen to obtain SNP sites with high genetic information.

4. The whole genome SNP molecular marker combination of Haizi buffalo according to claim 3, characterized in that: In step (4), the software is SNPSelection (v1.0), a software that can be used for selecting high genetic information sites in the livestock and poultry genome chip design process, and the software registration number is 2024SR1581643.

5. Use of the whole genome SNP molecular marker combination of Haizi buffalo according to claim 1 in the design of Haizi buffalo SNP chip.

6. The use according to claim 5, characterized in that: The Haizi buffalo SNP chip designed in the application can detect one or more sites described in claim 1.

7. An application of the whole genome SNP molecular marker combination of Haizi buffalo according to claim 1 in the protection and breeding of Haizi buffalo genetic resources.

8. The use according to claim 7, characterized in that: The application is the genetic diversity analysis of Haizi buffalo.

9. The use according to claim 7, characterized in that: The application is the kinship analysis of Haizi water buffalo individuals.

10. The application according to claim 7, characterized in that: The application is the family structure analysis of the Haizi buffalo population.

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