Molecular marker combination for analyzing goat variety and application

Abstract

The invention discloses a molecular marker combination for analyzing goat varieties and application, relates to the technical field of biology, and provides 13947 molecular markers capable of analyzing goats, and the physical position information of the molecular markers is determined based on genome sequence comparison of a goat reference genome ARS1.2. A molecular probe combination, a gene chip and a kit prepared from 13947 SNP molecular markers can be used for carrying out genetic evaluation on goat individuals, carrying out individual selection on goat varieties which are difficult to determine in the early stage, shortening the generation interval and accelerating the breeding process, so that a large amount of breeding cost is saved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the field of biological detection technology, and more particularly to the analysis of SNP site combinations of goat breeds and applications thereof. Background Art

[0002] Goats (Capra hricus, goat) were domesticated by humans in the Fertile Crescent about 10,000 years ago, becoming one of the earliest domesticated animals. They spread to all parts of the world with the migration of humans, providing humans with abundant meat, milk, wool, leather and other important living materials, playing an important role in human production and life. In the process of domestication and improvement, with the selection of environment and human beings, people have long cultivated rich goat breed resources. According to statistics from the Food and Agriculture Organization of the United Nations (FAO, https: / / www.fao.org / livestock-systems / en / ), there are more than 570 goat breeds in the world, with a stock of more than 1 billion. The identification of goat breed resources is crucial for the protection of local goat breeds, sustainable breeding strategies, traceability of mutton and registration of resource breeds. As an important genetic resource, local goat breeds have great genetic specificity in the genome, but most local breeds are at risk of extinction due to multiple adverse challenges including emerging diseases, climate change and competition from commercial breeds. Accurate identification of goat breed resources is one of the most important measures to deal with these risks. Summary of the invention

[0003] To meet the needs of goat breed research, the present invention provides a SNPs and molecular probe combination, gene chip, kit and application for analyzing goat breeds. By utilizing the site information provided by the present invention, goat breed screening, identification, tracing and breeding can be achieved quickly and accurately, which is beneficial to the protection and improvement of germplasm resources, with short time consumption, low cost and broad market benefits.

[0004] In order to achieve the technical purpose of the present invention, the present invention provides the following technical solutions: In a first aspect, the present invention provides an application of a 13947 SNP locus combination in analyzing goat breeds, and the physical locations of the 13947 SNP locus combinations are shown in Table 1: Table 1 Position information of 13947 loci combinations

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[0046] Its physical position is determined based on the genomic sequence alignment of the goat reference genome ARS1.2.

[0047] In a second aspect, the present invention provides a method for analyzing goat breeds, which compares the genotypes of 13,947 SNP loci of the genomic DNA of the goat to be tested with the genotypes of the 13,947 SNP loci of the genomic DNA of the control goat; Among them, the 13,947 SNP loci are the above-mentioned 13,947 SNP loci.

[0048] In a third aspect, the present invention provides a molecular probe combination for analyzing goat breeds, and the molecular probe combination detects the SNP locus combination shown in Table 1 in the sample to be tested, and the physical position information of the locus combination in Table 1 is determined based on the genomic sequence alignment of the goat reference genome ARS1.2.

[0049] In a fourth aspect, the present invention provides a gene chip for analyzing goat breeds, and the gene chip is loaded with the above-mentioned molecular probe combination.

[0050] In a fifth aspect, the present invention provides a kit for analyzing goat breeds, which has the above-mentioned molecular probe combination or gene chip.

[0051] In a sixth aspect, the present invention provides a method for analyzing goat breeds, which uses the above-mentioned molecular probe combination, gene chip or kit to detect the sample to be tested.

[0052] In a seventh aspect, the present invention provides the use of the above-mentioned molecular probe combination, gene chip or kit in the following aspects: (1) Application in screening goat breeds; (2) Application in identifying goat breeds; (3) Application in tracing the origin of goat breeds; (4) Application in goat breeding; (5) Application in the protection of germplasm resources; (6) Application in the improvement of germplasm resources.

[0053] Beneficial effects 1. An SNPs set is obtained through sufficient breed and sample data, containing more sufficient and representative information, meeting the requirements for identifying global goat breed resources.

[0054] 2. The present invention selects an effective SNPs set by using the PCA and NJ tree methods to clarify the genetic relationships among goat breeds, enabling the chip to be directly applied to the identification of goat breeds.

[0055] 3. The present invention includes a rich variety of Chinese local goat breeds, greatly improving the applicability of the chip to Chinese native goat breeds, which is beneficial to the protection and utilization of Chinese goat germplasm resources. Description of the drawings

[0056] Figure 1 To analyze the positions of 13,947 SNPs of goat breeds on each chromosome; Figure 2 Result diagram of PCA analysis for 13,947 SNPs of goat breeds Figure 3 To construct a phylogenetic tree for 13,947 SNPs of goat breeds. Detailed implementation manners

[0057] The present invention will be further clarified below with reference to the detailed description of specific implementation manners. However, these examples are merely illustrative and should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and can be referred to the third edition of the original book "Bioinformatics and Functional Genomics" or related books. The bioinformatics software and products used are also commercially available. The various processes and methods not described in detail are conventional methods well-known in the art. The sources of the materials used, the trade names, and those for which it is necessary to list their components are indicated at the first appearance. For the same reagents used later, unless otherwise specified, they are the same as those indicated at the first appearance.

[0058] In addition, it should be noted that the locus combinations and applications provided by the present invention are all achieved through the arduous creative labor and optimization work of the inventors.

[0059] The features and advantages described in the foregoing locus combination part of this article are equally applicable to the molecular probe combinations, gene chips, kits and their applications formed based on the locus combinations, and will not be elaborated herein.

[0060] The SNP referred to in the present invention means Single Nucleotide Polymorphism, which mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. The variation of the single nucleotide includes the variation caused by the conversion, transversion, insertion or deletion of a single base.

[0061] It should be noted that the molecular markers referred to in the present invention are all heritable and detectable DNA sequences or proteins, including but not limited to molecular markers based on molecular hybridization, such as RFLP, MinisatelliteDNA; molecular markers based on PCR technology, such as RAPD, STS, SSR and SCAR; DNA markers based on restriction enzyme digestion and PCR technology; molecular markers based on DNA chip technology, such as SNP; analysis marker technology developed based on EST database, etc. The molecular markers provided by the present invention can be used for genome mapping, gene mapping research, map-based gene cloning, species genetic relationship and system classification, etc.

[0062] It should be noted that the probe referred to in the present invention is a nucleic acid sequence (DNA or RNA) with a detection label, known sequence, and complementary to the target gene, such as Taqman-MGB probe.

[0063] It should be noted that the kit referred to in the present invention is any box commonly used in the art that contains reagents for detection or experiments, which facilitates the operator to get rid of the heavy reagent preparation and optimization process. In an embodiment of the present invention, it contains primers for amplifying the locus information provided by the present invention, molecular markers or probes or gene chips for detecting the locus information provided by the present invention, and also includes enzymes and buffers for amplification, or fluorescent labels for detection.

[0064] Example 1 Obtaining of SNP Locus Combinations for Analyzing Goat Breeds 1. Obtaining of the Total SNP Set of Goat Whole Genome To meet the current needs of goat germplasm resources and breed identification, relying on the high-depth whole-genome resequencing data of 360 domestic sheep individuals from 72 breeds worldwide, using the goat ARS1.2 genome as a reference, and combining existing research related to goat breed resources, a 10K goat SNP chip for global goat breed resource identification was designed. The specific method is as follows: The present invention collected the whole-genome resequencing sample information of Capra species worldwide from the National Center for Biotechnology Information-Sequence Read Archive (NCBI-SRA, https: / / www.ncbi.nlm.nih.gov / sra / ) and the European Bioinformatics Institute-European Nucleotide Archive (EBI-ENA, https: / / www.ebi.ac.uk / ena / browser / view / ). The average sequencing depth was 20.83×, and the samples were filtered based on the sample information, only retaining the paired-end sequenced samples. Finally, the resequencing data of 360 domestic goat individuals from 72 breeds were collected. The geographical distribution of all individuals was mainly in Asia, Africa, and Europe. Through the common results of alignment by SAMtools and GATK, a SNP set was obtained. The goat breeds involved in the present invention are shown in Table 2: Table 2 Goat Breeds

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[0066] It should be noted that some of the above goat breed names do not have official Chinese translations for the time being and the English names are still used.

[0067] Specifically, the above method uses the mem algorithm in the Bwa (v0.7.17) software to align the high-quality gene sequences of each population to the ARS1.2 (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_001704415.2 / ) reference genome. Specifically, the configuration tool is called in the operating system, and the configuration tool contains the code: -t 32 -k 32 -M –R"@RG\tID:'${i}'\tSM:'${i}'\tPL:illumina\tLB:'${i}'\tPU:'${i}'", where -t32 in the code specifies 32 threads, -k 32 sets the minimum seed length to 32bp, -M marks secondary alignments, -R adds ReadGroup (read group) header information to the output BAM file, ${i} is a variable in the Shell script, usually the sample name, \t refers to the tab key space, ID refers to the unique read group identifier, SM refers to the sample name, PL:illumina indicates that the sequencing platform is illumina, LB refers to the library name, and PU refers to the sequencing unit; the obtained bam file is sorted using SAMtools (v1.6); the PCR duplicates in the bam file are removed through the MarkDuplicate section in GATK (v4.1.2.0), and the obtained bam file is used for downstream variant detection; the GATK HaplotypeCaller module is used to detect the variants in the sample to obtain a GVCF file, and the GVCF files of each sample are merged through the GATK CombineGVCFs module. Finally, the gatk GenotypeGVCFs module is used to convert the GVCF files of each population into vcf files to obtain SNP sites; the SelectVariants of the GATK software is used to only retain SNP variant sites; gatk VariantFiltration is used to perform hard filtering on the obtained VCF file, and the specific parameters are "QUAL<30.0 | QD<2.0 | MQ<40.0 | FS>60.0 | SOR>3.0 | MQRankSum<-12.5 |ReadPosRankSum<-8.0; vcftools (v0.1.16) is used to perform advanced filtering on the vcf file obtained by hard filtering, and the specific parameters are as follows: remove polymorphic SNP sites and retain dimorphic SNP sites --min-alleles 2 --max-alleles 2.

[0068] In order to avoid the influence of data quality on the analysis results, the study used PLINK v1.07 software to conduct strict quality control of individuals and SNPs in the data set. In the data set, individuals or SNPs that did not meet the following criteria were deleted: missing chromosome number and physical location information; minimum allele frequency (MAF) < 0.05; individual detection rate < 1.0; SNP genotyping missing rate > 0.05; Hardy-Weinberg equilibrium shift probability P < 0.000001, and 13947 SNPs were obtained as shown in Table 1. The specific locations are shown in Table 1 and Figure 1 As shown, the SNPs are located on chromosomes 1 to 26, and the white vertical bars are SNPs.

[0069] It should be noted that the genetic information referred to in the present invention refers to information that is passed from parent to offspring in order for an organism to replicate something identical to itself, or that is passed from cell to cell each time each cell divides.

[0070] It should be noted that the extraction of genetic information (such as DNA) from samples for high-depth sequencing can be completed by biological companies, such as BGI, Illumina, etc. The high-depth sequencing method adopts conventional methods in the field or methods of biological companies. In one embodiment of the present invention, an average sequencing depth of ~25.7× is used, and a resequencing analysis process is used for high-depth sequencing.

[0071] 3. Genetic distance analysis In this study, the genetic relationship between goat populations was analyzed using principal component analysis (PCA) and neighbor-joining tree (NJ tree). PCA analysis was performed using the SmartPCA package in EIGENSOFT v6.0 software (Patterson et al., 2006), and the results were graphically displayed using R v3.3.0 software (http: / / www.r-project.org / ). Figure 2 The allele frequency of each locus was calculated using PEAS v.1.0 software, and then the population NJ tree was developed using the subroutine in PHYLIP v.3.695 software. Finally, the results were graphically displayed using FigTree v.1.4.2 software, as shown in Figure 1. Figure 3 shown.

[0072] according to Figure 2 The principal component analysis (PCA) of 360 individuals (representing 72 breeds) based on the screened molecular marker set shown was performed, and the results showed that PC1 and PC2 could clearly distinguish all breeds.Figure 3 The phylogenetic tree constructed using the same set of molecular markers further verified this result, and each variety formed an independent branch.

[0073] Example 2 Using SNPs for analyzing goat breeds to prepare primer combinations and probe combinations Those skilled in the art design primers according to the sequence information of each locus in the SNPs for analyzing goat breeds provided by the present invention, and perform secondary structure evaluation and Tm value evaluation on the designed primers. Finally, primers with good specificity, high sensitivity, and capable of achieving the detection purpose under the same reaction conditions are obtained.

[0074] Among them, the secondary structure evaluation and Tm value evaluation can be carried out in any common manner in the art. For example, the DNA folding form is used to evaluate its secondary structure (specifically refer to http: / / unafold.rna.albany.edu / ?q=mfold / DNA-Folding-Form), and then the software RaW-Probe is used to evaluate its Tm value.

[0075] The above methods are all conventional methods and can be obtained based on the locus information in the SNPs provided by this application without the need for creative labor. Therefore, the primers obtained based on the SNPs provided by the present invention also fall within the protection scope of the present invention.

[0076] Similarly, preparing probes using the SNPs provided by the present invention, such as tanqman probes, also falls within the protection scope of the present invention.

[0077] Example 3 SNP gene chip for analyzing goat breeds The SNP gene chip of the present invention is prepared by fixing the primers or probes obtained in Example 2 on a polymer substrate by a conventional method, such as nylon membrane, nitrocellulose membrane, plastic, silica wafer, microbeads, etc., or fixing the probes on a glass plate, or directly synthesizing the primers or probes obtained in Example 2 on a hard surface such as glass. The usage method of the SNP gene chip of this application is the same as the conventional method.

[0078] It should be noted that those skilled in the art can prepare the SNP gene chip for detecting goat breeds in any way, and can also entrust a biological company to prepare it. However, the SNP gene chips prepared based on the SNP locus combinations for goat breed analysis provided by this application all fall within the protection scope of the present invention.

[0079] Example 4 Kit for analyzing goat breeds The SNPs detection kit for analyzing goat breeds provided by this application includes primers, probes or gene chips obtained based on the SNPs obtained in Example 1. According to different usage types, corresponding detection reagents are also included. For example, when the SNPs obtained in Example 1 are prepared as Taqman probes, it also includes buffers, ligases, AceQUniversal U+ Probe Master Mix V2, TaqMan Probe, etc. commonly used in fluorescence quantitative PCR reactions.

[0080] Those skilled in the art configure different SNP kits for detecting goat breeds according to different usage methods, but the SNP kits for detecting goat breeds configured based on the SNPs for analyzing goat breeds provided by this application all fall within the protection scope of the present invention.

[0081] Example 5 Detection of Goat Breeds Based on the phylogenetic tree constructed from the goat whole-genome SNP marker combination data, it is found that 72 domestic and foreign goat breeds can be separated by breed, and the relevant numbers representing breeds are shown in Table 2.

[0082] Based on the PCA analysis of the goat whole-genome SNP marker combination data, it is found that PC1 and PC2 clearly separate 72 domestic and foreign goat breeds. The results of the phylogenetic tree and PCA analysis are consistent and support each other, indicating the reliability of the breed identification results.

[0083] Industrial Application Those skilled in the art can make SNP probe combinations, gene chips, and kits for analyzing goat breeds based on the SNP locus combination for analyzing goat breeds consisting of 13,947 SNPs provided by this application, which can be used for screening goat breeds, breed identification, controlling the breeding process at the genomic level, and can also be applied to goat breed traceability, goat pedigree reconstruction, germplasm resource protection, and germplasm resource improvement.

[0084] The above are only preferred examples to help understand the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Without departing from the idea of the present invention, various changes or modifications made by those skilled in the art based on this should also fall within the scope of the present invention.

Claims

1. Application of 13,947 SNP locus combinations in analyzing goat breeds, wherein the physical positions of the 13,947 SNP locus combinations are shown in Table 1: Its physical position is determined based on the genomic sequence alignment of the goat reference genome ARS1.

2.

2. Method for analyzing goat breeds, comparing the genotypes of 13,947 SNP loci of the genomic DNA of the goat to be tested with the genotypes of the 13,947 SNP loci of the genomic DNA of the control goat; Among them, The 13,947 SNP loci are the 13,947 SNP loci described in claim 1.

3. Molecular probe combination for analyzing goat breeds, the molecular probe combination detecting the SNP locus combination shown in Table 1 in the sample to be tested, and the physical position information of the locus combination in Table 1 is determined based on the genomic sequence alignment of the goat reference genome ARS1.

2.

4. Gene chip for analyzing goat breeds, the gene chip carrying the molecular probe combination described in claim 3.

5. Kit for analyzing goat breeds, having the molecular probe combination described in claim 3 or the gene chip described in claim 4.

6. Method for analyzing goat breeds, detecting the sample to be tested by applying the molecular probe combination described in claim 3 or the gene chip described in claim 4 or the kit described in claim 5.

7. The molecular probe combination described in claim 3 or the gene chip described in claim 4 or the kit described in claim 5 has the following uses: (1) Application in screening goat breeds; (2) Application in identifying goat breeds; (3) Application in tracing the origin of goat breeds; (4) Application in goat breeding; (5) Application in protecting goat germplasm resources; (6) Application in improving goat germplasm resources.

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