SNP molecular marker combination for buffalo genotyping and its application

Abstract

The present invention relates to the field of molecular marker technology, and specifically discloses a SNP molecular marker combination for buffalo genotyping and its application. The SNP molecular marker combination for buffalo genotyping of the present invention is composed of 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are respectively shown as SEQ ID NOs: 1-731. The SNP molecular marker combination of the present invention can be applied to the localization of trait-related genes in buffalo, the improvement of the main economic traits of animals, the protection and development and utilization of buffalo germplasm resources, genome-wide association analysis, genomic selection breeding, and many other aspects, helping to accelerate molecular research and breeding work related to buffalo.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, in particular to a SNP molecular marker combination for buffalo genotyping and an application thereof. Background Art

[0002] Loss-of-function variants (LoFs) are a type of mutation that severely affects protein function. These mutations primarily include start codon mutations, stop codon mutations, and splice site mutations. They severely disrupt gene structure, affecting transcription and translation, rendering the protein inoperable and ultimately altering the individual's phenotype.

[0003] Gene chips are an ideal method for SNP detection. Currently, the main gene chip technologies used for SNP site typing are solid-phase chips and liquid-phase chips. Compared with traditional solid-phase chips, cGPS (Genotyping by Pinpoint Sequencing of liquid captured targets, cGPS) liquid-phase chips are based on targeted sequencing technology. They use synthetic specific probes to capture and enrich multiple target sequences located at different genomic locations through liquid-phase hybridization. The captured and enriched target intervals are then subjected to second-generation sequencing to obtain the genotypes of all sites within the target interval. They have the advantages of high detection accuracy, high throughput, flexible site deletion and addition, and lower cost, and are increasingly favored by the industry.

[0004] The commercial chip currently used in buffalo is The Buffalo Genotyping Array 90K (hereafter referred to as Axiom 90K) is a solid-phase array designed for selected river buffalo populations, with gene frequency and uniform distribution as key considerations. However, this array exhibits suboptimal polymorphism in swamp buffalo, underrepresents functional sites and regions, and lacks technical advantages over liquid-phase arrays. Therefore, designing a new liquid-phase array suitable for rapid and efficient detection of SNPs affecting protein function in diverse populations is crucial for molecular research and breeding efforts. Summary of the Invention

[0005] The purpose of the present invention is to provide a new SNP molecular marker combination for buffalo genotyping and its application.

[0006] To achieve this goal, the present invention used a reference population of 564 buffaloes from two different types of buffaloes from around the world: 294 river buffaloes and 270 swamp buffaloes. Using whole-genome sequencing technology, a process involving alignment of raw short reads, genomic variant detection, and functional annotation of variant sites was used to identify and screen 731 highly polymorphic and annotated SNPs in both types of buffaloes. Based on these findings, a buffalo cGPS liquid phase microarray was designed to detect these SNPs.

[0007] The present invention first screened 731 SNPs annotated as high-impact sites that exhibited good polymorphism in both types of buffalo. Specifically, based on the buffalo reference genome UOA_WB_1, 20,586,171 qualified SNP variants were detected through genome sequencing raw read alignment and genomic variation detection. The variant sites were then functionally annotated using SNPEFF software. Finally, SNPs annotated as high-impact sites that exhibited good polymorphism in both types of buffalo were selected. Based on these findings, the present invention provides a collection of 731 SNPs that affect protein function in buffalo.

[0008] The present invention also provides a molecular probe combination for detecting the SNP site set, which can be effectively detected by targeted capture sequencing technology.

[0009] Based on the above findings, the present invention has designed a cGPS liquid phase chip for detecting the above 731 SNP sites. The cGPS liquid phase chip is a reagent or kit containing the above molecular probe combination.

[0010] Specifically, the technical solutions of the present invention are as follows:

[0011] In a first aspect, the present invention provides a SNP molecular marker combination for buffalo genotyping, which consists of 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731, respectively.

[0012] In a second aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination in the preparation of a buffalo whole-genome SNP chip, or a buffalo genome detection reagent or kit.

[0013] In a third aspect, the present invention provides a buffalo whole-genome SNP chip comprising 731 SNP molecular markers, wherein the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731, respectively.

[0014] The buffalo whole genome SNP chip of the present invention adopts liquid phase capture technology.

[0015] In a fourth aspect, the present invention provides a buffalo whole genome detection reagent or kit, which comprises 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731, respectively.

[0016] In the above-mentioned SNP molecular marker combination for buffalo genotyping or buffalo whole genome SNP chip or buffalo whole genome detection reagent or kit of the present invention, the SNP sites of the SNP molecular markers are located at the sites represented by n in the nucleotide sequences shown in SEQ ID NO.1 to 731. The specific positions and polymorphism information of each SNP site can be found in the sequence listing.

[0017] In a fifth aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination for buffalo genotyping, or buffalo whole genome SNP chip, or buffalo whole genome detection reagent or kit in any of the following aspects:

[0018] (1) In the conservation and development and utilization of buffalo germplasm resources, or in the identification or improvement of economic traits;

[0019] (2) in genetic diversity analysis or genome-wide association study (GWAS) of buffalo;

[0020] (3) in genomic selection breeding (GS) of buffalo;

[0021] (4) In the construction of genetic maps of buffalo or the localization of trait-related genes.

[0022] In a sixth aspect, the present invention provides a buffalo genotyping method, which is a SNP site typing method based on a cGPS liquid phase chip, wherein the cGPS liquid phase chip comprises 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731, respectively;

[0023] Preferably, the method comprises: constructing a library using the genomic DNA of the buffalo to be tested; using the SNP molecular marker to hybridize and capture the target sequence; then performing PCR amplification and second-generation sequencing on the captured target sequence; and finally using the GATK process to perform variation detection to obtain the genotyping results of the target site.

[0024] Specifically, the method for applying the above-mentioned liquid phase chip in whole-genome association analysis is as follows: animal blood samples are collected, and genotyping is performed using the cGPS liquid phase chip produced based on the SNP molecular marker combination of the present invention and the variation detection process (see Example 2 for detailed process and steps), and then whole-genome association analysis is performed using the screened SNP sites and the phenotypic data of buffaloes.

[0025] The liquid phase array described above was applied to genomic selection breeding (GS) of buffalo. The specific method involved constructing a reference population and candidate population for genomic selection of buffalo, and then genotyping the buffalo using the liquid phase array and variant detection process described above (see Example 2 for detailed process and steps). The reference population was used to estimate the genetic effect of each molecular marker. The estimated breeding value of each individual was then predicted based on the genotype of the candidate population. Finally, individual breeding values ​​were selected based on their breeding values.

[0026] The above-mentioned liquid phase chip is applied to the protection and development and utilization of buffalo germplasm resources, or the identification or improvement of economic traits. The specific method is: using the above-mentioned liquid phase chip and variation detection process to perform genotyping on buffalo (see Example 2 for detailed process and steps), and locating genes and genotypes related to important economic traits in buffalo, such as meat quality, milk quality, growth rate, etc., through whole-genome association analysis, selection signal, linkage analysis and other methods, to provide a basis for breeding efficient and high-quality livestock and poultry breeds.

[0027] The method for applying the above-mentioned liquid phase chip to construct a genetic map of buffalo is as follows: a population for genetic map construction is constructed, and the population is genotyped using the above-mentioned liquid phase chip and variation detection process (for detailed process and steps, see Example 2). The screened SNP sites are used as molecular markers, and the order and genetic distance between the molecular markers are determined to obtain a genetic map of the buffalo.

[0028] Those skilled in the art can also apply the above-mentioned liquid phase chip to identify recessive deleterious mutations. The specific method is as follows: collect blood samples from animals and perform genotyping on the population using the above-mentioned liquid phase chip and mutation detection process (for detailed process and steps, see Example 2). The number of different genotypes at each locus is calculated. Using the Hardy-Weinberg equilibrium test, SNP sites that deviate from Hardy-Weinberg equilibrium and lack recessive homozygotes are screened as potential recessive homozygous lethal mutations.

[0029] The beneficial effects of the present invention are at least:

[0030] 1. The SNP site set involved in the present invention includes 731 SNP sites that have good polymorphism and affect protein function in buffaloes in two types of buffaloes. The minimum allele frequency (MAF) of all sites is greater than 0.05, and the sites with MAF greater than or equal to 0.1 account for 70.59%. These sites can be used as candidate molecular markers to be applied to many aspects such as the positioning of animal major economic traits improvement, buffalo germplasm resource protection and development and utilization, whole genome association analysis (GWAS), genomic selective breeding (GS), and buffalo trait-related genes, and help to accelerate the molecular research and breeding work related to buffaloes.

[0031] 2. The buffalo liquid-phase array of the present invention is based on targeted capture sequencing technology, offering advantages such as low cost, ease of analysis, high accuracy, flexible site deletion, and the ability to capture more SNPs in target regions. This array has broad applicability and promising market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a density statistical diagram of the 731 SNP sites of the present invention.

[0033] Figure 2 This is the frequency distribution diagram of the 731 SNP sites of the present invention.

[0034] Figure 3 This is the evolutionary tree constructed in Example 3 of the present invention. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0036] The following examples illustrate the present invention but are not intended to limit its scope. Unless otherwise specified, the equipment and reagents used in each example are commercially available. Unless otherwise specified, water buffalo samples were obtained from China Agricultural University using methods known in the art.

[0037] Example 1 Screening of SNP sites affecting protein function in two types of buffalo

[0038] 1. Sample collection and genomic DNA extraction

[0039] The present invention collected genomic DNA from 564 buffaloes of two types from around the world: 294 river buffaloes and 270 swamp buffaloes. Sample information is shown in Table 1. Venous blood was collected for genomic DNA extraction using the phenol-chloroform method.

[0040] Table 1 Sample information

[0041]

[0042] Note: The sampling countries in Table 1 are named using the three-letter ISO standard country codes (for details, please see: https: / / baike.baidu.com / item / ISO%203166-1 / 5269555); River represents river buffalo, and Swamp represents swamp buffalo.

[0043] 2. Genome sequencing and SNP variation detection

[0044] The 564 buffaloes were sequenced using a high-throughput second-generation sequencing platform. The raw sequencing data were aligned to the buffalo reference genome version UOA_WB_1 for variant detection. The SNP variant detection process is as follows: (1) Use fastQC and Trimmomatic to perform quality control and statistics on reads; (2) Use BWA-MEM to align reads to the reference genome to obtain a sam file; (3) Use samtools and picard to sort and index the sam file and remove read duplication; (5) Use GATK to perform variant detection; (6) Use the GATK VariantFiltration module to filter variants with the following filtering parameters: "QD < 2.0", "QUAL < 30.0", "SOR > 3.0", "FS > 60.0", "MQ < 40.0", "MQRankSum < -12.5", "ReadPosRankSum < -8.0", and "HaplotypeScore > 13.0".

[0045] Initial filtering was performed using vcftools (https: / / vcftools.github.io / man_latest.html). The filtering criteria were a minimum allele frequency greater than 0.05 (--maf 0.05) and a site missingness rate less than 0.1 (--max-missing 0.9). A total of 20,586,171 SNP variants were identified through the GATK pipeline and initial filtering for further analysis.

[0046] 3. Functional annotation of SNPs

[0047] The 20,586,171 SNP variants were annotated using SNPEFF software (http: / / pcingola.github.io / SnpEff / ) and the SNPs were divided into high 、 middle 、 Low-impact and modifier SNPs. High-impact SNPs primarily include variants that cause protein truncation or loss or gain of gene function. Statistics of the number of different types of SNPs annotated by SNPEFF are shown in Table 2. A total of 2,046 high-impact SNPs were annotated.

[0048] Table 2 Number of different types of SNPs annotated by SNPEFF

[0049]

[0050] 4. Screening high-quality sites that affect protein function

[0051] 2,046 high-impact SNPs required further screening before they could be used. They were retained only if they met the following conditions in both the river buffalo and swamp buffalo populations. Retention conditions: (1) Site detection rate ≥ 0.95; (2) The sequencing depth of the site met the following conditions: the average sequencing depth of the site in all samples was greater than 1 / 2 of the average depth of all sites, and less than 2 times the average sequencing depth of all sites; (3) Hardy-Weinberg equilibrium test p value ≥ 0.00001; (4) Minimum allele frequency (MAF) ≥ 0.01. At the same time, considering that some high-impact sites are population-specific, sites with MAF = 0 in one population and MAF ≥ 0.01 in another population were also included. Through the above screening, 936 high-quality sites that affect protein function were obtained.

[0052] Example 2 Design of cGPS liquid phase chip to detect the above-mentioned sites affecting protein function

[0053] 1. Design of specific molecular probes

[0054] The 936 high-quality SNP site sets that affect protein function obtained in Example 1 were sent to Huazhi Biotechnology Co., Ltd. (official website https: / / www.higentec.com / , Changsha City, Hunan Province) for probe design. Based on the physical position of the buffalo reference genome UOA_WB_1 and the SNP sites obtained in Example 1 on the genome, the company's probe design software was used to take 50bp before and after the variant site to preliminarily design specific molecular probes for each SNP site. The designed probes were then evaluated and scored based on the thermal stability, capture efficiency, and specificity of the molecular probes. The unqualified probes were redesigned by adjusting their front and back positions, and the SNP sites for which qualified probes could not be designed were discarded. Finally, 731 SNP sites were selected (see Table 3, which shows the physical position information of each SNP site). The designed probe information is shown in SEQ ID NO: 1-731, and the target site is located at the position represented by "n" inside the molecular probe.

[0055] The density statistics of the 731 SNP sites of the present invention are shown in Figure 1 For frequency distribution diagram, see Figure 2 .

[0056] Table 3 List of chip sites and their variant types

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] 2. Develop cGPS liquid phase chip for mutation detection

[0067] The reagents or kits for producing corresponding cGPS liquid phase chips based on the above molecular probes are as follows:

[0068] Extraction of buffalo genomic DNA: Blood samples were collected from the animals by tail vein bleeding method, and then genomic DNA was extracted from the blood of each sample using the standard phenol-chloroform method.

[0069] DNA sample quality testing: DNA concentration was determined using a NanoDrop assay; DNA integrity was assessed by agarose gel electrophoresis. Sample acceptance criteria included: total DNA volume ≥ 200 ng, concentration ≥ 10 ng / μL, volume > 20 μL; OD260:OD280 ≤ 1.8, OD260:OD230 ≥ 1.8; and a clear main band with minimal or no degradation.

[0070] cGPS liquid phase chip testing: Qualified DNA samples were sent to Higentec Biotechnology Co., Ltd. (official website: https: / / www.higentec.com / , Changsha, Hunan Province) and the company's standard liquid phase chip testing procedures were followed.

[0071] Bioinformatics Analysis: Raw sequencing data were filtered using fastQC software. Filtered reads were aligned to the buffalo reference genome UOA_WB_1 using BWA (Buffalo Genome Association) using default parameters. PCR duplicates were marked and removed using the Picard tool (version 1.54). Finally, variant detection was performed using the Genome Analysis Toolkit (GATK, version 3.8) to obtain genotype results for the target loci. GATK implemented the following "variant filtering" parameters for all SNPs: "QD < 2.0," "QUAL < 30.0," "SOR > 3.0," "FS > 60.0," "MQ < 40.0," "MQRankSum < -12.5," "ReadPosRankSum < -8.0," and "HaplotypeScore > 13.0."

[0072] Detection test: Following the above process, 58 buffalo samples with available second-generation whole-genome sequencing results were selected for detection testing. These included 6 river buffalo and 43 swamp buffalo from the original reference population, as well as 9 hybrid buffalo from a non-original reference population. Technical replicates were performed on one river buffalo, two swamp buffalo, and one hybrid buffalo. The results showed an average detection rate of 99.75% for all 58 samples, 96.85% concordance between the chip and next-generation sequencing (CHIP / NGS) genotyping results, and an average genotyping concordance of 99.69% for four pairs of technical replicates. This demonstrates that the cGPS liquid phase chip offers a high genotyping rate, high genotyping accuracy, good reproducibility, and excellent overall performance. Specific results are shown in Tables 4 and 5.

[0073] Table 4 Test results of 58 samples

[0074]

[0075]

[0076] The sample ID in Table 4 consists of three parts: country or region, sampling city or province, and number. The specific names of countries or regions refer to the ISO standard country codes, and the sampling cities or provinces are represented by their pinyin abbreviations. River represents river buffalo, Swamp represents swamp buffalo, and X represents hybrid buffalo.

[0077] Table 5 Genotype consistency rate of technical replicate samples

[0078]

[0079] Example 3 Application of buffalo liquid phase chip in population genetic structure analysis

[0080] The genotyping results of the 58 samples in Example 2 were used to construct a phylogenetic tree. The vcf format genotype file was converted to fasta format using the prepared Python script vcf2phylip (download address: https: / / github.com / edgardomortiz / vcf2phylip). The maximum likelihood method (FastTree software) was used to construct the phylogenetic tree. Finally, the results were visualized using the online website iTOL (https: / / itol.embl.de / tree / ). The constructed phylogenetic tree is shown in Figure 2. Figure 3 As shown, it is shown that the buffalo liquid phase chip of the present invention and the above method can cluster similar samples and can be used for the genetic structure analysis of buffalo populations.

[0081] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A SNP molecular marker combination for buffalo genotyping, characterized in that: It consists of 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731 respectively.

2. Use of the SNP molecular marker combination according to claim 1 in preparing a buffalo whole genome SNP chip.

3. Use of the SNP molecular marker combination according to claim 1 in the preparation of a buffalo genome detection reagent.

4. Use of the SNP molecular marker combination according to claim 1 in preparing a buffalo genome detection kit.

5. A buffalo whole genome SNP chip, characterized in that: It contains 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731 respectively.

6. A buffalo whole genome detection reagent, characterized in that: It contains 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731 respectively.

7. A buffalo whole genome detection kit, characterized in that: It contains 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731 respectively.

8. Use of the SNP molecular marker combination for buffalo genotyping according to claim 1, the buffalo whole-genome SNP chip according to claim 5, the buffalo whole-genome detection reagent according to claim 6, or the buffalo whole-genome detection kit according to claim 7 in buffalo genetic diversity analysis or genome-wide association analysis.

9. Use of the SNP molecular marker combination for buffalo genotyping according to claim 1, the buffalo whole-genome SNP chip according to claim 5, the buffalo whole-genome detection reagent according to claim 6, or the buffalo whole-genome detection kit according to claim 7 in constructing a buffalo genetic map.

10. A method for genotyping buffalo, characterized in that: The method is a SNP site typing method based on a cGPS liquid phase chip, wherein the cGPS liquid phase chip comprises 731 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers are shown in SEQ ID NOs: 1-731, respectively.

11. The method according to claim 10, characterized in that The method comprises: constructing a library using genomic DNA of a buffalo to be tested; performing hybridization capture on a target sequence using the SNP molecular marker; performing PCR amplification and second-generation sequencing on the captured target sequence; and finally performing variation detection using the GATK process to obtain a genotyping result of the target site.

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