Molecular marker combination for analyzing variety of milk goats and application of molecular marker combination

Abstract

The invention discloses a molecular marker combination for analyzing varieties of milk goats and application of the molecular marker combination, relates to the technical field of biology, and provides 2014 molecular markers capable of analyzing the features of the varieties of the milk goats, and the physical position information of the molecular markers is compared and determined based on the genome sequence of a goat reference genome ARS1. A molecular probe combination, a gene chip and a kit prepared from the 2014 SNP molecular markers can be used for carrying out genetic evaluation on goat individuals, individual selection is carried out on milk goat varieties which are difficult to measure in the early stage, the generation interval is shortened, the breeding process is accelerated, and therefore a large amount of breeding cost is saved.

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 SNP locus combinations of dairy goat breeds and their applications. Background Art

[0002] Goats (Capra hircus) are one of the earliest domesticated animals (ZEDER, M A, 2006). In animal taxonomy, the domestic goat (Capra hircus) belongs to the class Mammalia, order Artiodactyla, suborder Ruminantia, family Bovidae, subfamily Caprinae, and genus Capra. It is estimated that the total number of domestic goats in the world has exceeded one billion (AMILLS, M, et al., 2017), and includes more than 700 different breeds (FAO, https: / / www.fao.org / home / zh). This extensive distribution and large population size benefit from the natural adaptability of domestic goats to harsh environments, low nutritional requirements, and easy management characteristics (NADERI, S, et al., 2007). They can adapt to various environments such as tropical and temperate, arid and humid, cold and hot, steep and mountainous. Goats play an important role in human survival and economic development as multi-purpose producers of dairy products, meat, and wool in many regions (CLUTTON-BROCK, J, 1990; COLLI, L, et al., 2018). The total number of sheep and goats in the world is close to 2.2 billion, and about one-fifth of them are involved in the production of dairy products (PULINA, G, et al., 2018). The dairy products provided by goats are an important source of nutrition for the human diet, and the milk production performance of goats has genetic polymorphism (there are genetic variations between dairy goats and non-dairy goats). For the current diverse and variable goat breeds, how to quickly screen goat breeds and distinguish whether they are dairy goats has become an urgent problem to be solved. Therefore, it is of great significance to design an SNP chip applicable to the Chinese goat population and capable of quickly and effectively detecting milk production performance. Summary of the Invention

[0003] To meet the current needs of goat breed research in China and the detection of dairy goat breeds in agricultural production, the present invention provides a molecular probe combination, gene chip, kit, and application for analyzing dairy goat breeds. Using the locus information provided by the present invention, it is possible to quickly and accurately screen, identify, trace the origin of dairy goat breeds, and conduct goat breeding, which is beneficial to germplasm resource protection and germplasm resource improvement, with short time consumption, low cost, and broad market benefits.

[0004] To achieve the technical object of the present invention, the present invention provides the following technical solutions: In a first aspect, the present invention provides an application of a combination of 2014 SNP loci in analyzing dairy goat breeds. The physical positions of the combination of the 2014 SNP loci are shown in Table 1: Table 1 Position information of the combination of 2014 loci

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[0010] Its physical position is determined based on the alignment of the genomic sequence of the goat reference genome ARS1.

[0011] In a second aspect, a method for analyzing dairy goat breeds is provided, wherein the genotypes of 2014 SNP loci of the genomic DNA of the goat to be tested are compared with the genotypes of the 2014 SNP loci of the genomic DNA of the control goat; Among them, the 2014 SNP loci are the 2014 SNP loci described in Table 1.

[0012] In a third aspect, a molecular probe combination for analyzing dairy goat breeds is provided. 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 alignment of the genomic sequence of the goat reference genome ARS1.

[0013] In a fourth aspect, a gene chip for analyzing dairy goat breeds is provided, and the gene chip is loaded with the molecular probe combination described in the third aspect.

[0014] In a fifth aspect, a kit for analyzing dairy goat breeds is provided, which has the molecular probe combination described in the third aspect or the gene chip described in the fourth aspect.

[0015] In a sixth aspect, a method for analyzing dairy goat breeds is provided, and the sample to be tested is detected by 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.

[0016] In a 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 one of the following uses: (1) Application in the screening of goat breeds; (2) Application in the identification of goat breeds; (3) Application in the traceability of goat breeds; (4) Application in goat breeding; (5) Application in the protection of germplasm resources; (6) Application in the improvement of germplasm resources.

[0017] Advantageous effects: 1. Based on the research of genetic resources of numerous goats at home and abroad, the present invention provides an SNP locus combination for analyzing dairy goat breeds, which consists of 2014 SNP loci. The SNP locus combination provided by the present invention not only has good universality at home and abroad, but also can quickly evaluate dairy goat breeds that cannot be manifested at the gene level in the early stage, obtain more accurate breeding evaluation information, control the breeding process, and can also screen, identify and trace goat breeds by using the above locus combination, providing technical support for the protection and improvement of germplasm resources.

[0018] 2. The probe combination, gene chip and kit for analyzing dairy goat breeds provided by the present invention also have the characteristics of small throughput, low cost and easier analysis, wide universality and broad market prospects. Description of the drawings

[0019] Figure 1 is the identification map of potential selection regions of FST and π ratio dairy goat populations; Figure 2 is the Manhattan plot of Tajima's D of dairy goats and non-dairy goats; Figure 3 is the Manhattan plot of ZHP of dairy goat populations and non-dairy goat populations; Figure 4 is the comparison map of selected genes by different methods; Figure 5 is the result map of the significance test of the determination result of the population threshold analysis in the present application. Detailed implementation manners

[0020] The present invention will be further clarified below with reference to the detailed description of specific embodiments. However, these embodiments are merely illustrative and should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments 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 occurrence. For the same reagents used later, unless otherwise specified, they are the same as those indicated at the first occurrence.

[0021] In addition, it should be noted that the locus combinations and applications provided by the present invention are all completed by the inventors through arduous creative labor and optimization work.

[0022] The features and advantages described in the foregoing locus combination part of this article also apply to the molecular probe combinations, gene chips, kits, and their applications formed based on the locus combinations, and will not be elaborated herein.

[0023] 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 transition, transversion, insertion, or deletion of a single base.

[0024] 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 databases, 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.

[0025] 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.

[0026] It should be noted that the kit referred to in the present invention is any kind of box that is commonly used in the art and contains reagents for detection or experiments, which facilitates operators to get rid of the heavy reagent preparation and optimization processes. 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, and also includes enzymes and buffers for amplification, or fluorescent labels for detection.

[0027] Example 1 Obtaining the SNP Locus Combination for Milk Production Performance To meet the current requirements for chip locus function detection and function research in breeding production and reflect the genetic characteristics of dairy goats worldwide, this study relied on the high-depth whole-genome re-sequencing data of 147 dairy goats and 53 non-dairy goats from around the world. Using the goat ARS1.2 genome as a reference and combining existing research related to goat functions, a 5K goat SNP chip targeting the milk production performance of goats was designed. The specific method is as follows.

[0028] 1. Obtaining the Total SNP Set A total of 5.5 T of re-sequencing data from 200 goats was collected, including 147 dairy goats of 9 breeds and 53 non-dairy goats of 5 breeds. The sample information of goat breeds is shown in Table 2. The sequencing data was all downloaded from three databases, namely NCBI (https: / / www.ncbi.nlm.nih.gov / ), EBI (https: / / www.ebi.ac.uk / ), and NGDC (https: / / ngdc.cncb.ac.cn / ?lang=zh). The average sequencing depth of the samples was 22.99× (8.80–42.64×). By filtering the Raw data of the whole-genome re-sequencing of all dairy goats and non-dairy goats, adapter-containing and low-quality reads were removed to obtain Clean data. The effective sequencing data was aligned to the reference genome using the BWA software. The alignment results were de-duplicated using SAMTOOLS, and the GATK was used to mark and remove the duplicate sequences in the BAM file. SAMTOOLS was used to build an index for the processed BAM file. The GATK was used to detect the variant sites in each sample, generate gVCF files, perform variant typing, generate variant call files in VCF format, filter out the unfiltered variants, and perform further quality control. In one embodiment of the present invention, the plink tool was used to perform quality control on the data, setting the minimum allele frequency to 0.01, the maximum missing rate of genotypes and individuals to 0.1, and allowing non-standard chromosome numbers, generating the final VCF file for analysis, and a total of 28,651,586 SNP variant sites were detected.

[0029] Table 2 Information Table of Goat Breed Samples

[0030] 2. Screening of Candidate Genes and Their Functional Regions Grouping was carried out according to the significant differences in milk performance among goat breeds worldwide. All breed individuals of dairy goats and non - dairy goats were summarized and divided into two groups, dairy goats and non - dairy goats, for selective sweep analysis. That is, dairy goat breeds (Alpine goat, Ampere goat, Guanzhong dairy goat, Laoshan dairy goat, Nubian goat, Peacock goat, Saanen goat, Toggenburg goat, Tangshan dairy goat) and non - dairy goat breeds (Boots goat, Gerson striped goat, Guishan goat, Longlin goat, Yunshang black goat) were taken as a group. Through F ST , π ratio, H P and Tajima’s D four methods, the intersection was taken to screen out the functional regions related to milk production traits, and then the genes in this region ( Figures 1-4 ) were found.

[0031] Finally, 37 candidate genes related to milk performance with very definite functions were determined, as shown in Table 3. Furthermore, the functional regions corresponding to the above - mentioned candidate genes were determined through perl scripts.

[0032] Table 3

[0033] 3. Obtaining of SNP Locus Combinations for Milk Production Using snpEff to search for SNP loci corresponding to the functional regions of the candidate genes determined in step 2 in the total SNP set, a milk performance locus combination containing 2014 SNP loci was obtained.

[0034] Example 2 Using the SNP Locus Combinations of Dairy Goats for Preparing Primer Combinations and Probe Combinations Those skilled in the art designed primers according to the sequence information of each locus in the SNP locus combinations of dairy goats provided by the present invention, and evaluated the secondary structure and Tm value of the designed primers. Finally, primers with good specificity, high sensitivity and capable of achieving the detection purpose under the same reaction conditions were obtained.

[0035] Among them, the secondary structure evaluation and Tm value evaluation can be carried out by any common method in the art. For example, the DNA folding form can be 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 can be used to evaluate its Tm value.

[0036] The above methods are all conventional methods and can be obtained without creative labor according to the site information in the SNP locus combination for milk production provided in this application. Therefore, the primers obtained according to the SNP locus combination provided by the present invention also fall within the protection scope of the present invention.

[0037] Similarly, using the SNP locus combination provided by the present invention to prepare a probe, such as a tanqman probe, also falls within the protection scope of the present invention.

[0038] Example 3: Using the SNP locus combination for analyzing milk performance to prepare a gene chip 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 a nylon membrane, a nitrocellulose membrane, plastic, a silica wafer, a microbead, etc., or by fixing the probe on a glass plate, or directly synthesizing the primers or probes obtained in Example 2 on a hard surface such as glass. The use method of the SNP gene chip of the present application is the same as the conventional method.

[0039] It should be noted that those skilled in the art can prepare the SNP gene chip for detecting dairy 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 combination for milk performance analysis provided in this application all fall within the protection scope of the present invention.

[0040] Example 4: Analysis kit for dairy goat breeds The SNP detection kit for milk performance analysis provided in this application includes primers or probes or gene chips obtained based on the SNP locus combination obtained in Example 1. According to different usage types, it also includes corresponding detection reagents. For example, when the Taqman probe is obtained based on the SNP locus combination obtained in Example 1, it also includes buffers, ligases, AceQUniversal U+ Probe Master Mix V2, TaqMan Probe, etc. commonly used in fluorescence quantitative PCR reactions.

[0041] Those skilled in the art can configure SNP kits for detecting different dairy goat breeds according to different usage methods. However, SNP detection kits for dairy goat breeds configured based on the SNP locus combination for milk performance provided in this application all fall within the protection scope of the present invention.

[0042] Example 5 Detection of Dairy Goat Breeds Based on the SNP locus combination for analyzing dairy goat breeds provided in Example 1 of this application, the milk performance of known dairy and non-dairy goats was detected. According to the detection results and combined with the known phenotypes of dairy and non-dairy goats, the accuracy of the detection was judged. Specifically: Peripheral blood of goats was collected by conventional methods, and the genomic DNA therein was extracted to obtain a genomic DNA sample. By conventional methods, a gene chip was designed according to the locus information in the SNP locus combination provided by the present invention, and the genomic DNA sample of the goats was detected to obtain the genotyping results of each locus in the goats (that is, whether each locus is a homozygote, heterozygote, mutant homozygote or base deletion result), the frequency value of the genotyping results of each locus was calculated, and compared with the population threshold. The comparison results showed that the gene detection results were consistent with the corresponding dairy goat breed phenotypes.

[0043] It should be noted that the population threshold in this application was obtained by analyzing different populations of dairy goats and non-dairy goats, and the method is the same as above.

[0044] This application conducted a significance test (independent sample Mann-Whitney U test) on the determination results of the analysis of the dairy goat population and the non-dairy goat population. The results are as Figure 5 shown. It can be seen from the results in the figure that P < 0.01, and the difference is extremely significant. It can be seen that the results determined by the method of the present invention are accurate and effective.

[0045] Industrial Application Based on the SNP locus combination for analyzing dairy goat breeds consisting of only 2014 SNP loci provided in this application, those skilled in the art can make SNP probe combinations, gene chips and kits for analyzing dairy goat breeds, which can analyze the milk performance of goat individuals at the genomic level, evaluate genetic information, screen breeds, identify breeds, control the breeding process, and can also be applied to goat breed traceability, goat pedigree reconstruction, germplasm resource protection and germplasm resource improvement.

[0046] 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 on this basis shall also fall within the scope of the present invention.

Claims

1. Application of the combination of 2014 SNP loci in analyzing dairy goat breeds, and the physical positions of the combination of the 2014 SNP loci are shown in the following table: ； The physical positions are determined based on the genomic sequence alignment of the goat reference genome ARS1.

2. Method for analyzing dairy goat breeds, which comprises comparing the genotypes of the 2014 SNP loci of the genomic DNA of the goat to be tested with the genotypes of the 2014 SNP loci of the genomic DNA of the control goat; Among them, The 2014 SNP loci are the 2014 SNP loci described in claim 1.

3. Molecular probe combination for analyzing dairy goat breeds, 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.

4. Gene chip for analyzing dairy goat breeds, the gene chip is loaded with the molecular probe combination described in claim 3.

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

6. Method for analyzing dairy goat breeds, which uses the molecular probe combination described in claim 3, or the gene chip described in claim 4, or the kit described in claim 5 to detect the sample to be tested.

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 dairy goat breeds; (2) Application in identifying dairy goat breeds; (3) Application in tracing the origin of dairy goat breeds; (4) Application in dairy goat breeding; (5) Application in protecting dairy goat germplasm resources; (6) Application in improving dairy goat germplasm resources.

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