Haizi water buffalo whole genome snp molecular marker combination and application

Abstract

The application discloses a Haizi buffalo whole-genome SNP molecular marker combination and application. The SNP molecular marker combination is shown in Table 1 and is composed of 47943 sequencing-derived high-heritage-information SNPs, 868 trait-related SNPs and 76 defect-related SNPs, and a total of 48887 sites. The Haizi buffalo whole-genome SNP molecular marker combination provided by the application can be used for design of a Haizi buffalo whole-genome chip and protection and breeding of Haizi buffalo genetic resources, can realize targeted gene detection work on a Haizi buffalo population, can meet various requirements in genetic resource protection and breeding, can effectively improve molecular breeding efficiency, and has important significance for protection, evaluation and utilization of Haizi buffalo genetic resources.

Description

TECHNICAL FIELD

[0001] The present application relates to Haizi buffalo genetic breeding, and in particular to a Haizi buffalo whole genome SNP molecular marker combination and application. BACKGROUND

[0002] Haizi buffalo is a unique swamp type buffalo breed in China, with excellent characteristics such as strong adaptability, roughage tolerance, and strong stress resistance, and is an important local livestock and poultry resource and cultural heritage. However, due to the accelerated development of modern agriculture and the adjustment of agricultural structure, its population has decreased sharply, and its genetic resources are in danger of loss. In 1990, the number of Jiangsu Province was 42,500, while the 2022 national third census data showed that only 4,051 remained, and it was basically 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 genetic resource diversity protection and breed improvement to avoid the ecological and cultural loss caused by its extinction.

[0003] Traditional breeding methods evaluate the value of cattle for breeding through appearance identification or phenotype determination, but have great limitations and low accuracy, and the long generation interval of cattle 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 target. As one of the commonly used technologies for molecular breeding, gene chips are the main tool for genomic selection breeding of livestock and poultry. However, the chips currently used on the market, such as Illumina Bovine SNP50, Illumina Bovine HD, Illumina Multi-Species Genotyping Bead Chips, etc., usually select single nucleotide polymorphism sites (Single nucleotide polymorphisms, SNPs) widely distributed among multiple breeds as their design sites, which lack specificity for Haizi buffalo populations.

[0004] Therefore, in order to protect and improve Haizi buffalo genetic resources, accelerate the process of molecular breeding, improve the efficiency of genetic diversity protection and precise breeding, and help the sustainable development of the population, it is crucial to design a Haizi buffalo whole genome SNP molecular marker combination for genetic resource protection and breeding. SUMMARY

[0005] The purpose of the present application is to provide a Haizi buffalo whole genome SNP molecular marker combination for genetic resource protection and breeding. The second purpose is to provide the application of the SNP site combination in Haizi buffalo whole genome chip and molecular breeding.

[0006] Technical scheme: The sea son buffalo whole genome SNP molecular marker combination of the application is composed of 48887 SNP sites, including 47943 sequencing source high genetic information amount SNP sites, 868 trait related sites and 76 defect related sites, as shown in Table 1.

[0007] Table 1 site information of sea son buffalo whole genome SNP molecular marker combination

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[0067] Preferably, the method for sequencing and obtaining high genetic information SNP sites in the Haizi water buffalo whole genome SNP molecular marker combination comprises the following steps:

[0068] (1) Raw filtering and index establishment based on the reference genome

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

[0070] (3) Optimizing the sequencing results;

[0071] (4) Filtering and screening high genetic information SNP sites using software.

[0072] Preferably, the software in step (4) of the above method is a software SNPSelection (v1.0) that can be used for selecting high genetic information sites in the process of designing livestock and poultry genome chips, and the software registration number is 2024SR1581643.

[0073] The Haizi water buffalo whole genome SNP molecular marker combination can be used for designing a Haizi water buffalo SNP chip, and the chip can detect one or more sites in the aforementioned Haizi water buffalo whole genome SNP molecular marker combination.

[0074] The Haizi buffalo whole genome SNP molecular marker combination can be used for Haizi buffalo genetic resource protection and breeding.

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

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

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

[0078] Beneficial effects: compared with the prior art, the present application has the following obvious advantages: 1, the Haizi buffalo whole genome SNP molecular marker combination is based on the reference genome and combined with the whole genome sequencing information of Haizi buffalo, and is especially suitable for genetic resource protection and breeding of Haizi buffalo population; 2, the Haizi buffalo whole genome SNP molecular marker combination can meet the various needs in genetic resource protection and breeding, the analysis result is clear and reliable, and can accurately reflect the richness degree of genetic diversity, inbreeding condition and establish the pedigree structure in the population, and can effectively promote the molecular breeding efficiency of Haizi buffalo to improve. BRIEF DESCRIPTION OF DRAWINGS

[0079] Figure 1 Figure 1 is a site density diagram of Haizi buffalo whole genome 50K SNP chip;

[0080] Figure 2 Figure 2 is a minimum allele frequency distribution diagram of Haizi buffalo;

[0081] Figure 2 Figure 3 is a SNP site polymorphism information content distribution diagram of Haizi buffalo;

[0082] Figure 2 Figure 4 is a heterozygosity analysis result diagram of Haizi buffalo;

[0083] Figure 3 Figure 5 is a ROH length distribution diagram of Haizi buffalo population;

[0084] Figure 3 Figure 6 is a chromosome ROH distribution diagram of Haizi buffalo;

[0085] Figure 3 Figure 7 is a inbreeding coefficient distribution diagram of Haizi buffalo individual;

[0086] Figure 4 Figure 8 is a Haizi buffalo IBS distance matrix visualization result diagram;

[0087] Figure 5 Figure 9 is a Haizi buffalo G matrix visualization result diagram;

[0088] Figure 6 Figure 10 is an evolutionary tree diagram of 46 Haizi buffalo population;

[0089] Figure 7 Figure 2 is a principal component analysis diagram of Haizi buffalo population;

[0090] Figure 8 Figure 3 is a parallel coordinate axis analysis diagram of Haizi buffalo population. DETAILED DESCRIPTION

[0091] The technical solutions of the present application are further described below.

[0092] Example 1: Establishment of Haizi buffalo whole genome SNP molecular marker combination

[0093] 1. 176 Haizi buffaloes were selected, and tail vein blood was collected to extract genomic DNA. The purity of the DNA was detected by NanoDrop2000, the concentration of the DNA was accurately quantified by Qubit2.0, and the integrity of the genomic DNA was detected by agarose gel electrophoresis. The DNA that passed the detection was subjected to whole genome sequencing;

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

[0095] 3. A software SNPSelection (v1.0, software registration number 2024SR1581643) that can be used for high genetic information site selection in the design process of livestock and poultry genome chips was used to select 47943 high genetic information SNP sites from the SNPs of 30x depth sequencing data, combined with 868 SNP sites related to Haizi buffalo meat quality, reproduction, health, development, etc. and 76 defect-related sites, to complete the establishment of Haizi buffalo whole genome SNP molecular marker combination.

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

[0097] According to the whole genome SNP molecular marker combination of Haizi water buffalo established in embodiment 1, probes were designed for all 48887 loci 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℃; avoiding dimers and hairpin structures. The designed probes were synthesized by Shijiazhuang Boruitai Biotechnology Co., Ltd., and loaded on appropriate solid supports, i.e. Haizi water buffalo 50K SNP chip.

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

[0099] 1. Haizi water buffalo breeding population near Yancheng City in Jiangsu Province was used as the test object, and 46 Haizi water buffalo tail vein blood was collected using blood collection tubes containing EDTA anticoagulant, and the number was recorded and stored at-80℃.

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

[0101] 3. The qualified DNA was sent to Shijiazhuang Boruitai Biotechnology Co., Ltd. for whole genome sequencing.

[0102] Embodiment 4: Whole genome SNP molecular marker combination of Haizi water buffalo for genetic resource protection and breeding

[0103] 1. For genetic diversity analysis of Haizi water buffalo:

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

[0105] 2. Individual relationship analysis of Haizi water buffalo

[0106] Using Plink (v1.90), based on the combination of whole-genome SNP molecular markers of Haizi water buffalo, the Runs of homozygosity (ROH) in the whole-genome sequencing results of Haizi water buffalo in Example 2 were analyzed, the distribution, length and number of ROH of each water buffalo were counted, and the ratio of the total length of ROH fragments to the total length of the autosomal genome in each individual was calculated, which was the inbreeding coefficient based on ROH. As shown in Figure 3 A, a total of 1764 ROH fragments were detected in the 46 Haizi water buffalo population, and the length of 96.94% of the ROH was between 0-5 Mb, among which the shortest ROH was 1.00 Mb, located on chromosome 1; the longest ROH was 10.81 Mb, located on chromosome 7. As shown in Figure 3 B, the distribution of ROH was relatively uniform, and the number of ROH on chromosome 1 was the most, which was 187; the number of ROH on chromosome 24 was the least, which was 25. The total length of ROH of each Haizi water buffalo was between 1.05-470.74 Mb, and the average total length of ROH was 71.03 Mb, and the number of individuals with ROH total length between 10-50 was the most, which was 24. As shown in Figure 3 C, the average inbreeding coefficient based on ROH of the Haizi water buffalo conservation population was 0.029, indicating that there was a low level of inbreeding phenomenon.

[0107] Further using Gmatix (v2) software, identity by state (IBS) distance matrix and G matrix were constructed respectively, and heat map was drawn to analyze the relationship of the conservation population. As shown in Figure 4 、 5 The IBS genetic distance of the conservation population was between 0.092-0.172, and the average was 0.157. The IBS distance matrix result was consistent with the G matrix, most of the Haizi water buffalo individuals had moderate relationship, and a small part of the individuals had close relationship, indicating that the Haizi water buffalo conservation population had a low inbreeding risk.

[0108] 3. Pedigree structure analysis of Haizi water buffalo population

[0109] Using MegaX (v10.0), based on the combination of whole-genome SNP molecular markers of Haizi water buffalo, the SNP sites after quality control of the whole-genome sequencing results of Haizi water buffalo in Example 2 were analyzed and the population phylogenetic tree was drawn to determine the pedigree structure of the conservation Haizi water buffalo population. As shown in Figure 6As shown in Table 1, there were genetic distances of different sizes among 46 Haizi water buffaloes, but some individuals had strong genetic links, forming 7 family structures; the principal component analysis results are shown in Figure 7 As shown, it also indicates that the Haizi water buffalo population has a certain family structure, and 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 8 As shown, it is found that the first 4 principal components cannot well divide the population, which also indicates that the population has a weak family structure.

Claims

1. Application of a Haizi buffalo whole genome SNP molecular marker combination in Haizi buffalo genetic resource protection and breeding, characterized in that, The molecular marker combination consists of 48887 SNP sites, and the specific site information on the swamp type buffalo reference genome GCF_029407905.1 is shown in Table 1.

2. Use according to claim 1, characterized in that, The molecular marker combination includes 47943 sequencing-derived high genetic information SNP sites, 868 trait-related sites and 76 defect-related sites.

3. Use according to claim 2, characterized in that, The establishment method of the sequencing-derived high genetic information SNP site is: (1) original filtering and index establishment based on the reference genome; (2) screening suitable Haizi buffalo for whole genome sequencing; (3) optimizing the sequencing results; (4) filtering and screening high genetic information SNP sites using software.

4. The use according to claim 1, characterized in that, The application is the genetic diversity analysis of Haizi buffalo.

5. The use according to claim 1, characterized in that, The application is the kinship analysis of Haizi buffalo individuals.

6. The use according to claim 1, characterized in that, The application is the family structure analysis of Haizi buffalo population.

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