Molecular marker combinations and their applications in analyzing goat breeds

Abstract

The present invention discloses a molecular marker combination and application for analyzing goat breeds, relating to the field of biotechnology. The present invention provides 13,947 molecular markers capable of analyzing goats, wherein the physical location information of the molecular markers is determined based on genome sequence alignment of the goat reference genome ARS1.2. A molecular probe combination, a gene chip, and a kit prepared using the 13,947 SNP molecular markers can be used to perform genetic evaluation on individual goats, perform individual selection on goat breeds that were difficult to determine in the early stages, shorten the generation interval, accelerate the breeding process, and thus save a large amount of breeding costs.

Description

Technical Field

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

[0002] Goats (Capra hricus) were domesticated by humans in the Fertile Crescent approximately 10,000 years ago, becoming one of the earliest domesticated animals. Following human migration, they spread throughout the world, providing humans with a rich supply of essential necessities such as meat, milk, wool, and hides, playing a vital role in human production and daily life. Through the process of domestication and improvement, environmental and human selection have fostered a rich resource of goat breeds. According to the Food and Agriculture Organization of the United Nations (FAO, https: / / www.fao.org / livestock-systems / en / ), there are over 570 goat breeds worldwide, with a population exceeding 1 billion. Identification of goat breed resources is crucial for the conservation of local goat breeds, sustainable breeding strategies, goat meat traceability, and the registration of local goat breeds. Local goat breeds, as important genetic resources, possess significant genetic heterogeneity across their genomes. However, due to multiple challenges, including emerging diseases, climate change, and competition from commercial breeds, most local goat breeds are at risk of extinction. Accurate identification of goat breed resources is one of the most important measures to address 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. Utilizing the site information provided by the present invention, goat breed screening, identification, traceability 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:

[0005] In a first aspect, the present invention provides an application of a 13,947 SNP locus combination in analyzing goat breeds. The physical locations of the 13,947 SNP locus combinations are shown in Table 1:

[0006] Table 1 Position information of 13947 loci combinations

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[0049] Its physical location was determined based on genome sequence alignment with the goat reference genome ARS1.2.

[0050] In a second aspect, the present invention provides a method for analyzing goat breeds, comprising comparing the genotypes of 13,947 SNP sites in the genomic DNA of a test goat with the genotypes of the 13,947 SNP sites in the genomic DNA of a control goat;

[0051] Among them, the 13947 SNP sites are the 13947 SNP sites mentioned above.

[0052] In a third aspect, the present invention provides a molecular probe combination for analyzing goat breeds, wherein the molecular probe combination detects the SNP site combination shown in Table 1 in the sample to be tested, and the physical position information of the site combination in Table 1 is determined based on the genome sequence comparison of the goat reference genome ARS1.2.

[0053] In a fourth aspect, the present invention provides a gene chip for analyzing goat breeds, wherein the gene chip is loaded with the above-mentioned molecular probe combination.

[0054] In a fifth aspect, the present invention provides a kit for analyzing goat breeds, which comprises the above-mentioned molecular probe combination or gene chip.

[0055] In a sixth aspect, the present invention provides a method for analyzing goat breeds, using the above-mentioned molecular probe combination or gene chip or kit to detect a sample to be tested.

[0056] In a seventh aspect, the present invention provides uses of the above-mentioned molecular probe combination, gene chip, or kit in the following aspects:

[0057] (1) Application in goat breed screening;

[0058] (2) Application in goat breed identification;

[0059] (3) Application in goat breed traceability;

[0060] (4) Application in goat breeding;

[0061] (5) Application in germplasm resource conservation;

[0062] (6) Application in germplasm resource improvement.

[0063] Beneficial effects

[0064] 1. Obtain SNPs collections through sufficient breed and sample data, which contain more sufficient and representative information to meet the requirements for breed resource identification of global goats.

[0065] 2. The present invention uses PCA and NJ tree methods to select effective SNPs sets, clarify the genetic relationship between goat breeds, and enable the chip to be directly applied to goat breed identification.

[0066] 3. The present invention includes a rich variety of Chinese local goat breeds, which greatly improves the applicability of the chip to Chinese native goat breeds and is beneficial to the protection and utilization of Chinese goat germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 To analyze the location of 13,947 SNPs on various chromosomes in goat breeds;

[0068] Figure 2 PCA analysis results for 13,947 SNPs in goat breeds

[0069] Figure 3 A phylogenetic tree was constructed for analyzing 13,947 SNPs in goat breeds. DETAILED DESCRIPTION

[0070] The present invention is further illustrated below with reference to the detailed description of specific embodiments, but these embodiments are merely illustrative and should not be construed as limiting the present invention. If not otherwise specified, the technical means adopted in the embodiments are conventional means well known to those skilled in the art, and can be carried out with reference to the third edition of the original work "Bioinformatics and Functional Genomics" or related books, and the bioinformatics software and products adopted are also commercially available. Various processes and methods not described in detail are conventional methods well known in the art, and the materials used, trade names, and those that are necessary to list their components are all indicated when first occurring, and thereafter, same reagents used, if not otherwise specified, are identical with the content indicated for the first time.

[0071] In addition, it should be noted that the site combinations and applications provided by the present invention were completed by the inventors through arduous creative labor and optimization work.

[0072] The features and advantages described in the site combination section above are also applicable to the molecular probe combination, gene chip, kit and their applications formed based on the site combination, and will not be repeated here.

[0073] The SNP referred to in the present invention refers to single nucleotide polymorphism (SNP), which mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. The variation of the single nucleotide includes variation caused by conversion, transversion, insertion or deletion of a single base.

[0074] 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 and Minisatellite DNA; 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; analytical marker technology based on EST database development, etc. The molecular markers provided by the present invention can be used for genome mapping, gene localization research, map-based gene cloning, species relationship and systematic classification, etc.

[0075] 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 and a known sequence that is complementary to the target gene, such as a Taqman-MGB probe.

[0076] 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 experimentation, which can relieve the operator of the tedious process of reagent preparation and optimization. In one embodiment of the present invention, it contains primers for amplifying the site information provided by the present invention, molecular markers or probes or gene chips for detecting the site information provided by the present invention, enzymes and buffers for amplification, and optionally fluorescent markers for detection.

[0077] Example 1 Analysis of SNPs locus combinations in goat breeds

[0078] 1. Obtaining the total SNP set of goat genome

[0079] To meet the current needs of goat germplasm resources and breed identification, this study, based on high-depth whole-genome resequencing data from 360 domestic sheep individuals from 72 breeds worldwide, used the goat ARS1.2 genome as a reference and combined with existing research on goat breed resources, designed a 10K goat SNP array for global goat breed identification. The specific method is as follows:

[0080] This study collected whole-genome resequencing data for Capra capra from around the world 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 / ) databases, with an average sequencing depth of 20.83×. Sample-based filtering was performed to retain only paired-end sequencing samples. Ultimately, resequencing data for 360 domestic goat individuals from 72 breeds were collected, primarily distributed across Asia, Africa, and Europe. A single single-nucleotide polymorphism (SNP) set was generated by combining the results of SAMtools and GATK alignment. The goat breeds involved in this study are shown in Table 2:

[0081] Table 2 Goat breeds

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[0084] It should be noted that some of the above-mentioned goat breed names do not yet have official Chinese translations and are still using English names.

[0085] 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. 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 the secondary alignment, -R adds ReadGroup header information to the output BAM file, and ${i} is a variable in the Shell script, usually the sample name, \t refers to the tab key space, ID refers to the unique identifier of the read group, SM refers to the sample name, PL:illumina refers to the sequencing platform as Illumina, LB refers to the library name, and PU refers to the sequencing unit; the obtained bam file was sorted using SAMtools (v1.6); PCR duplicates were removed from the bam file using the MarkDuplicate module in GATK (v4.1.2.0), and the obtained bam file was used for downstream variant detection; the GATK HaplotypeCaller module was used to detect variants in the sample to obtain GVCF files, and the GVCF files of each sample were merged using the GATK CombineGVCFs module. Finally, the GATK GenotypeGVCFs module was used to convert the GVCF files of each population into VCF files to obtain SNP sites; the GATK software SelectVariants was used to retain only SNP variant sites; the obtained VCF files were hard filtered using the gatk VariantFiltration with the specific parameters "QUAL < 30.0 | QD < 2.0 | MQ < 40.0 | FS > 60.0 | SOR > 3.0 | MQRankSum < -12.5 | ReadPosRankSum < -8.0. Advanced filtering was performed on the vcf file obtained by hard filtering using vcftools (v0.1.16) with the following parameters: remove polymorphic SNP sites, retain dimorphic SNP sites, --min-alleles 2 --max-alleles 2.

[0086] To avoid the impact of data quality on the analysis results, the study used PLINK v1.07 software to perform strict quality control on individuals and SNPs in the dataset. In the dataset, 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, resulting in 13,947 SNPs as shown in Table 1. The specific locations are shown in Tables 1 and Figure 1 As shown in the figure, the marker positions of SNPs on chromosomes 1 to 26, and the white vertical bars are SNPs.

[0087] It should be noted that the genetic information referred to in the present invention refers to the information that is passed from parent to offspring in order for an organism to replicate itself, or that is passed from cell to cell each time a cell divides.

[0088] 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 applied for high-depth sequencing.

[0089] 3. Genetic distance analysis

[0090] In this study, principal component analysis (PCA) and neighbor-joining tree (NJ tree) were used to analyze the genetic relationships among goat populations. PCA analysis was performed using the SmartPCA package in EIGENSOFT v6.0 software (Patterson et al., 2006), and the results were visualized 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 the figure. Figure 3 shown.

[0091] according to Figure 2The principal component analysis (PCA) of 360 individuals (representing 72 breeds) based on the screened molecular marker set shown in the figure showed that PC1 and PC2 could clearly distinguish all breeds. Figure 3 The phylogenetic tree constructed using the same molecular marker set further verified this result, and each variety formed an independent branch.

[0092] Example 2: Using SNPs from goat breeds for preparation of primer and probe combinations

[0093] Those skilled in the art design primers based on the sequence information of each site in the SNPs analyzed for goat breeds provided by the present invention, and perform secondary structure evaluation and Tm value evaluation on the designed primers, ultimately obtaining primers with good specificity and high sensitivity that can achieve the detection purpose under the same reaction conditions.

[0094] The secondary structure and Tm value evaluation can be performed using any method commonly used in the art, such as using a DNA folding form to evaluate the secondary structure, see (http: / / unafold.rna.albany.edu / ?q=mfold / DNA-Folding-Form) for details, and then using the software RaW-Probe to evaluate the Tm value.

[0095] The above methods are all conventional methods. According to the site information in the SNPs provided in this application, they can be obtained without any creative work. Therefore, the primers obtained according to the SNPs provided by the present invention also fall within the scope of protection of the present invention.

[0096] Likewise, the use of the SNPs provided by the present invention to prepare probes, such as tanqman probes, also falls within the scope of protection of the present invention.

[0097] Example 3 Analysis of gene chips for goat breeds

[0098] The SNP gene chip of the present invention is prepared by fixing the primers or probes obtained in Example 2 on a polymer substrate, such as a nylon membrane, nitrocellulose membrane, plastic, silica gel wafer, micro magnetic beads, etc., using conventional methods, 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 method of using the SNP gene chip of the present application is the same as the conventional method.

[0099] It should be noted that those skilled in the art can prepare SNP gene chips for detecting goat breeds in any manner, or can entrust a biological company to prepare them. However, SNP gene chips prepared based on the SNP site combinations for goat breed analysis provided in this application all fall within the scope of protection of the present invention.

[0100] Example 4 Kit for analyzing goat breeds

[0101] The present application provides a SNP detection kit for analyzing goat breeds, including primers, probes, or gene chips based on the SNPs obtained in Example 1. Depending on the type of use, the kit also includes corresponding detection reagents. For example, when the SNPs obtained in Example 1 are prepared as TaqMan probes, the kit also includes buffers, ligases, AceQUniversal U+ Probe Master Mix V2, and TaqMan probes commonly used in fluorescent quantitative PCR reactions.

[0102] Those skilled in the art can configure different SNP kits for detecting goat breeds according to different usage modes, but all goat breed SNP detection kits configured for analyzing goat breed SNPs provided in this application fall within the scope of protection of the present invention.

[0103] Example 5 Detection of goat breeds

[0104] Based on the phylogenetic tree constructed based on the whole-genome SNP marker combination data of goats, it was found that 72 domestic and foreign goat breeds can be separated according to breed. The relevant numbers representing the breeds are shown in Table 2.

[0105] PCA analysis based on genome-wide SNP marker data revealed that PC1 and PC2 clearly separated 72 domestic and international goat breeds. The phylogenetic tree and PCA analysis results were consistent and mutually supportive, demonstrating the reliability of the breed identification results.

[0106] Industrial Applications

[0107] Based on the SNP site combination for analyzing goat breeds composed of 13,947 SNPs provided in this application, technicians in this field can prepare SNP probe combinations, gene chips, and kits for analyzing goat breeds, which can be used to screen and identify goat breeds at the genomic level, control the breeding process, and can also be applied to goat breed tracing, goat pedigree reconstruction, germplasm resource protection, and germplasm resource improvement.

[0108] The above description is only a preferred example to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Without violating the concept of the present invention, various changes or modifications made by those skilled in the art to the present invention on this basis should also fall within the scope of the present invention.

Claims

1. Application of 13947 SNP loci combination in analyzing goat breeds. The physical locations of the 13947 SNP loci combination are shown in Table 1: Table 1 ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； Its physical location was determined based on genome sequence alignment with the goat reference genome ARS1.2; in, The goat breeds are shown in Table 2: 。 2. A method for analyzing goat breeds, wherein the genotypes of 13,947 SNP sites in the genomic DNA of the test goat are compared with the genotypes of the 13,947 SNP sites in the genomic DNA of the control goat; in, The 13947 SNP sites are the 13947 SNP sites described in claim 1, and the goat breed is the goat breed shown in Table 2 in claim 1.

3. A molecular probe combination for analyzing goat breeds, wherein the molecular probe combination detects the SNP site combination shown in Table 1 in the test sample, wherein the physical location information of the site combination in Table 1 is determined based on the genome sequence alignment of the goat reference genome ARS1.2; in, The goat breed is the goat breed shown in Table 2 of claim 1.

4. A gene chip for analyzing goat breeds, wherein the gene chip is loaded with the molecular probe combination according to claim 3; in, The goat breed is the goat breed shown in Table 2 of claim 1.

5. A kit for analyzing goat breeds, comprising the molecular probe combination according to claim 3 or the gene chip according to claim 4; in, The goat breed is the goat breed shown in Table 2 of claim 1.

6. A method for analyzing goat breeds, comprising detecting a sample using the molecular probe combination of claim 3, the gene chip of claim 4, or the kit of claim 5; in, The goat breed is the goat breed shown in Table 2 of claim 1.

7. The molecular probe assembly according to claim 3, the gene chip according to claim 4, or the kit according to claim 5 has the following uses: (1) Application in goat breed screening; (2) Application in goat breed identification; (3) Application in goat breed traceability; (4) Application in goat breeding; (5) Application in goat germplasm resource protection; (6) Application in goat germplasm improvement; in, The goat breed is the goat breed shown in Table 2 of claim 1.

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