A molecular marker combination for analyzing cashmere goat breeds and its application

Abstract

The present invention discloses a molecular marker combination for analyzing cashmere goat breeds and an application thereof, relating to the field of biotechnology. The present invention provides 2085 molecular markers capable of analyzing cashmere goat breeds, 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 2085 SNP molecular markers can perform genetic evaluation on individual goats, perform individual selection for cashmere goat phenotypic characteristics that are difficult to measure in 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 a SNP site combination of cashmere goat varieties and an application thereof. Background Art

[0002] Species gradually develop unique physical characteristics and morphologies through natural selection or artificial breeding. According to statistics, there are over 30 goat breeds worldwide primarily used for cashmere production, with over 60% of these goats being produced in China. Their total cashmere production is 9,000 to 10,000 tons, accounting for approximately 50% of the world's total. In complex environments and under precise breeding practices, the rapid and efficient selection of goat breeds with stable cashmere production is crucial. Given the current diversity of goat breeds and their varying traits, the rapid selection of cashmere goat breeds has become an urgent challenge. Summary of the Invention

[0003] In order to meet the current research on cashmere goat varieties in my country and the demand for identification and screening of cashmere goat varieties in agricultural production, the present invention provides a molecular probe combination, gene chip, kit and application for analyzing cashmere goat varieties. By utilizing the site information provided by the present invention, the screening, identification, traceability and goat breeding of cashmere goat varieties 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 2085 SNP locus combination in analyzing cashmere goat breeds. The physical locations of the 2085 SNP locus combinations are shown in Table 1:

[0006] Table 1 Position information of 2085 loci combinations

[0007]

[0008]

[0009]

[0010]

[0011]

[0012]

[0013] Its physical location was determined based on genome sequence alignment with the goat reference genome ARS1.2.

[0014] A second aspect provides a method for analyzing cashmere goat breeds, wherein the genotypes of 2085 SNP sites of the genomic DNA of a test goat are compared with the genotypes of the 2085 SNP sites of the genomic DNA of a control goat;

[0015] Among them, the 2085 SNP sites are the 2085 SNP sites described in Table 1.

[0016] The third aspect provides a molecular probe combination for analyzing cashmere goat breeds, which 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.

[0017] In the fourth aspect, a gene chip for analyzing cashmere goat breeds is provided, wherein the gene chip is loaded with the molecular probe combination described in the third aspect.

[0018] A fifth aspect is a kit for analyzing cashmere goat breeds, which comprises the molecular probe combination described in the third aspect or the gene chip described in the fourth aspect.

[0019] In a sixth aspect, a method for analyzing cashmere goat breeds is provided, wherein the sample to be tested is detected using the molecular probe combination described in the third aspect, the gene chip described in the fourth aspect, or the kit described in the fifth aspect.

[0020] The seventh aspect, the molecular probe combination described in the third aspect, the gene chip described in the fourth aspect, or the kit described in the fifth aspect has any of the following uses:

[0021] (1) Application in goat breed screening;

[0022] (2) Application in goat breed identification;

[0023] (3) Application in goat breed traceability;

[0024] (4) Application in goat breeding;

[0025] (5) Application in germplasm resource conservation;

[0026] (6) Application in germplasm resource improvement.

[0027] Beneficial effects

[0028] 1. Based on research on the genetic resources of numerous cashmere goats at home and abroad, the present invention provides a SNP locus combination consisting of 2085 SNP loci for analyzing cashmere goat breeds. The SNP locus combination provided by the present invention is not only widely applicable both domestically and internationally, but also can quickly evaluate cashmere goat breeds that are not visible in the early stages at the genetic level, obtain more accurate breeding assessment information, and control the breeding process. The above locus combination can also be used to screen, identify, and trace goat breeds, providing technical support for the protection and improvement of germplasm resources.

[0029] 2. The probe combination, gene chip, and kit for analyzing cashmere goat breeds provided by the present invention also have the characteristics of low throughput, low cost, and easier analysis. They are widely applicable and have broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Manhattan plot of elimination analysis for cashmere and non-cashmere goat breeds. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] It should be noted that the cashmere goat breed referred to in the present invention refers to a goat breed with cashmere.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 1 Analysis of SNP site combinations in cashmere goat breeds

[0040] To meet the current demand for chip site functional detection and functional research in breeding production and to reflect the genetic characteristics of domestic cashmere goats, this study relies on high-depth whole-genome resequencing data from 85 cashmere goats and 72 non-cashmere goats in China. With the goat ARS1.2 genome as a reference, combined with existing research related to goat functions, a 5K goat SNP chip targeting cashmere performance was designed.

[0041] 1. Acquisition of the total SNP set

[0042] Resequencing data from 157 goats were collected, including 85 cashmere goats from seven breeds and 72 non-cashmere goats from seven breeds. Detailed sample information is provided in Table 2, where all breed names are in English. Sequencing data were downloaded from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) and EBI (https: / / www.ebi.ac.uk / ) databases. The average sequencing depth for each sample was 13.11×. Raw data from the whole-genome resequencing of all cashmere and non-cashmere goats were filtered to remove adapter-containing and low-quality reads to generate clean data. Valid sequencing data were aligned to the reference genome using BWA software. Alignment results were deduplicated using SAMTOOLS. Repeat sequences in the BAM files were marked and removed using GATK, and the processed BAM files were indexed using SAMTOOLS. GATK was used to detect variants in each sample, generate gVCF files, perform variant typing, generate VCF-formatted variant call files, filter out unfiltered variants, and perform further quality control. The Plink tool was used to perform quality control on the data, and finally 12,716,092 SNPs were obtained.

[0043] Table 2 Sample information

[0044]

[0045] 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.

[0046] 2. Screening of candidate genes and their functional regions

[0047] Liaoning cashmere goat, Arbus cashmere goat, Alashan cashmere goat, Erlangshan cashmere goat, Chaidamu cashmere goat, Ujumqin cashmere goat, Tibetan cashmere goat, Huai goat, Leizhou goat, Tangshan dairy goat, Guishan goat, Longling goat, Yunshang black goat, Yunnan black goat bone), according to their significant differences in cashmere traits, they were grouped (cashmere goats: Liaoning cashmere goats, Arbas cashmere goats, Alashan cashmere goats, Erlangshan cashmere goats, Qaidam cashmere goats, Ujumqin cashmere goats, Tibetan cashmere goats; non-cashmere goats: Huanghuai goats, Leizhou goats, Tangshan dairy goats, Guishan goats, Longlin goats, Yunshang black goats, Yunnan black bone black goats). The functional regions related to cashmere production traits were screened out by taking the intersection of FST, π ratio, and XP-EHH methods, and then the genes in this region were found. Then, the literature was consulted and the scope was further narrowed down through existing related studies. Finally, 322 candidate genes related to cashmere production were identified, some of which are as follows. Figure 1 As shown in Table 3:

[0048] Table 3 322 candidate genes related to velvet production

[0049]

[0050] 3. Analysis of SNP site combinations in villi

[0051] The final results of this study were obtained by using SnpEff to search for SNP sites corresponding to the functional regions of these candidate genes in the total SNP set. Ultimately, 2085 SNP sites were selected as the core sites of the cashmere goat breed analysis chip.

[0052] Example 2: Using SNP sites for cashmere goat breed analysis to prepare primer and probe combinations

[0053] Those skilled in the art design primers based on the sequence information of each site in the hair performance SNP site combination provided by the present invention, and perform secondary structure evaluation and Tm value evaluation on the designed primers, and finally obtain primers with good specificity and high sensitivity that can achieve the detection purpose under the same reaction conditions.

[0054] 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.

[0055] The above methods are all conventional methods. The site information in the SNP site combination of the cashmere goat breed provided in this application can be obtained without any creative labor. Therefore, the primers obtained according to the SNP site combination provided by the present invention also fall within the scope of protection of the present invention.

[0056] Similarly, the use of the SNP site combination provided by the present invention to prepare probes, such as tanqman probes, also falls within the scope of protection of the present invention.

[0057] Example 3: Combining SNPs for Cashmere Goat Breed Analysis to Prepare Gene Chips

[0058] 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.

[0059] It should be noted that those skilled in the art can prepare SNP gene chips for detecting cashmere goat breeds in any manner, or can entrust a biological company to prepare them. However, SNP gene chips prepared based on the SNP site combination for cashmere goat breed analysis provided in this application all fall within the scope of protection of the present invention.

[0060] Example 4 Analysis Kit for Cashmere Goat Breeds

[0061] The SNP detection kit for mountain cashmere goats provided in this application includes primers, probes, or gene chips obtained based on the SNP site combination obtained in Example 1. Depending on the type of use, corresponding detection reagents are also included. For example, when the SNP site combination obtained in Example 1 is a TaqMan probe, the kit also includes a buffer, ligase, AceQUniversal U+ Probe Master Mix V2, TaqMan Probe, etc. commonly used in fluorescent quantitative PCR reactions.

[0062] Those skilled in the art can configure different SNP kits for detecting cashmere goat breeds according to different usage modes, but all SNP detection kits for cashmere goat breeds configured based on the wool performance SNP site combination provided in this application fall within the scope of protection of the present invention.

[0063] Example 5 Detection of Cashmere Goat Breeds

[0064] Based on the SNP site combination for analyzing cashmere goat breeds provided in Example 1 of the present application, goats with known villi were tested. According to the test results, combined with the known presence or absence of villi, the accuracy of the test was judged, specifically:

[0065] The peripheral blood of goats was collected by conventional methods, and whole genomic DNA was extracted therefrom to obtain whole genomic DNA samples;

[0066] A gene chip was designed using conventional methods based on the site information in the SNP site combination provided by the present invention, and a whole-genome DNA sample of a goat was tested to obtain the typing results of each site in the goat (i.e., whether each site is homozygous, heterozygous, mutant homozygous, or has a base deletion). The frequency value of the typing result for each site was calculated and compared with the population threshold. The comparison results showed that the genetic test results were consistent with the corresponding goat villi phenotype.

[0067] It should be noted that the population threshold in the present application is obtained by analyzing whether there are different groups of villi, using the same method as above.

[0068] The present application conducted a significance test (independent sample Mann-Whitney U test) on the judgment results of the analysis of the large cashmere goat group and the small cashmere goat group. The results showed that P<0.01, and the difference was extremely significant. It can be seen that the results of the judgment using the method of the present invention are accurate and effective.

[0069] Industrial Applications

[0070] Based on the SNP site combination for analyzing goat hair consisting of only 2085 SNP sites provided in this application, technicians in this field can make SNP probe combinations, gene chips, and kits for analyzing cashmere goat breeds. These can realize the screening and variety identification of cashmere 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.

[0071] 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 2085 SNP loci combination in analyzing whether a goat is a cashmere goat breed. The physical locations of the 2085 SNP loci combination are shown in the following table: ； ； ； ； ； ； Its physical location was determined based on genome sequence alignment with the goat reference genome ARS1.

2.

2. A method for analyzing whether the goat is a cashmere goat breed is to compare the genotypes of 2085 SNP sites in the genomic DNA of the test goat with the genotypes of the 2085 SNP sites in the genomic DNA of the control goat; in, The 2085 SNP sites are the 2085 SNP sites described in claim 1.

3. A molecular probe combination for analyzing whether the sample is a cashmere goat breed, wherein the molecular probe combination detects the SNP site combination as described in claim 1 in the sample to be tested, and the physical location information of the SNP site combination is determined based on the goat reference genome ARS1.2 genome sequence comparison.

4. A gene chip for analyzing whether the goat is a cashmere breed, wherein the gene chip is loaded with the molecular probe combination according to claim 3.

5. A kit for analyzing whether a goat is a cashmere goat, comprising the molecular probe combination according to claim 3 or the gene chip according to claim 4.

6. A method for analyzing whether a goat is a cashmere goat, comprising detecting a sample using the molecular probe combination of claim 3, the gene chip of claim 4, or the kit of claim 5.

7. Use of the molecular probe combination according to claim 3, the gene chip according to claim 4, or the kit according to claim 5 in screening whether a goat is a cashmere goat.

8. Use of the molecular probe combination according to claim 3, the gene chip according to claim 4, or the kit according to claim 5 in identifying whether a goat is a cashmere breed.

Images