A goat SNP molecular marker combinatorial system, its preparation of a whole-genome liquid-phase chip, and its applications.

Abstract

This invention discloses a goat SNP molecular marker combinatorial system, its preparation of a whole-genome liquid-phase microarray, and its applications. The goat SNP molecular marker combinatorial system comprises 50,508 SNP molecular markers, the physical locations of which are determined by sequence alignment based on the goat reference genome ARS1. The goat SNP molecular marker combinatorial system of this invention can be used for specific identification of goat breeds. Its application in preparing a goat whole-genome liquid-phase microarray allows for low-cost and large-scale genotyping of goats. Based on the principle of liquid-phase microarrays, the loci are flexible, and new functional marker loci can be added at any time. This invention provides a goat whole-genome liquid-phase microarray that can be used for goat whole-genome selection breeding, genome-wide association analysis, phylogenetic analysis, and germplasm resource mining and utilization. It provides an important tool for goat germplasm resource genotyping and molecular breeding, playing a significant role in related research.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a goat SNP molecular marker combinatorial array, its preparation of a whole-genome liquid-phase chip, and its applications. Background Technology

[0002] The diversity of livestock and poultry genetic resources is a precious legacy bestowed upon us by nature. It is not only the cornerstone of the sustainable prosperity and innovative development of animal husbandry, but also greatly enriches the variety of food and cultural diversity in human society. Currently, the existing goat germplasm resources are particularly rich and unique, containing numerous excellent traits that offer limitless possibilities for the transformation and upgrading of animal husbandry and breed improvement. However, the current challenges cannot be ignored: inappropriate crossbreeding practices are quietly threatening the survival of local goat breeds, with many breeds possessing unique genetic characteristics experiencing a sharp decline in numbers, even facing the crisis of extinction. This situation not only weakens the diversity of goat genetic resources but also limits the potential for sustainable development of animal husbandry. Therefore, it is crucial to utilize efficient modern molecular breeding technologies and tools to develop and utilize superior goat germplasm resources in a multi-dimensional and multi-level manner, accurately identify and explore the genetic traits of goats, and ultimately create new high-yielding, disease-resistant, and highly reproductive high-quality goat breeds.

[0003] DNA microarrays based on single nucleotide polymorphisms (SNPs) are a high-throughput, automated, and batch-based technology for SNP genotyping, and have been applied in many species. Traditional solid-phase microarrays are difficult to produce and have high detection costs, and have been gradually replaced by emerging liquid-phase microarrays. Liquid-phase microarray technologies based on multiplex amplification capture sequencing or probe hybridization capture sequencing offer advantages such as high throughput, high accuracy, and low detection costs, and have been widely used in various fields of biological germplasm resource and breeding research. This invention fully utilizes domestic and international goat germplasm resource genomic sequence information, screens high-quality SNP sites to design probes, and develops a universal liquid-phase microarray for goats. This can accelerate the accurate identification of goat germplasm resources and the precise screening of breeding populations, and is of great significance for promoting the sustainable development of the goat industry. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a goat SNP molecular marker combinatorial approach.

[0005] The present invention also proposes a whole-genome liquid-phase chip for detecting the above-mentioned goat SNP molecular marker combinations.

[0006] The present invention also proposes a kit for detecting the above-mentioned goat SNP molecular marker combinations.

[0007] This invention also proposes a method for screening goat SNP molecular marker combinations.

[0008] This invention also proposes an application of the above-mentioned goat SNP molecular marker combination, whole genome liquid chip, or kit.

[0009] This invention also proposes a method for breeding goats.

[0010] According to one aspect of the present invention, a goat SNP molecular marker combinatorial method is proposed, comprising 50,508 SNP molecular markers, wherein the physical locations of the 50,508 SNP molecular markers are determined by sequence alignment based on the goat reference genome ARS1, and the specific site information is shown in Table 1 below.

[0011] Table 1

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[0057]

[0058] In some embodiments of the present invention, the ID represents chromosome number chr: physical location.

[0059] In some embodiments of the present invention, the information of the SNP site is represented in the form of chromosome number: physical location REF / ALT.

[0060] In some embodiments of the present invention, the SNP sites are numbered from top to bottom and from left to right, as SNP1-SNP50508.

[0061] In some embodiments of the present invention, SNP48039-SNP50508 are named according to the reference genome, and the specific chromosomes are sex chromosomes.

[0062] In a second aspect of the invention, a whole-genome liquid-phase chip is provided, the whole-genome liquid-phase chip comprising a primer set and / or probes for detecting the above-described goat SNP molecular marker combinations.

[0063] In a third aspect of the invention, a kit is provided comprising a primer set and / or probes for detecting the above-described goat SNP molecular marker combinations.

[0064] In a fourth aspect of the present invention, a screening method for the above-mentioned goat SNP molecular marker combinations is proposed. The screening method includes the following steps: using goat whole genome sequencing data, mining and screening target site SNPs, designing probes for the target site SNPs, screening SNPs whose probes meet the requirements, and finally using them as goat SNP molecular markers.

[0065] In some embodiments of the present invention, the mining and screening of target SNPs includes the following steps: using Sentieon software to align and detect variants in goat resequencing data; performing preliminary hard filtering on the detected sites; and further screening the hard-filtered sites to obtain target SNPs; the screening parameters are: MAF ≥ 0.05, SNP detection rate ≥ 90%, heterozygosity ≤ 35%, and sequencing depth ≥ 5×.

[0066] In some embodiments of the present invention, the comparison and mutation detection of goat resequencing data using Sentieon software specifically includes the following steps:

[0067] (1) The goat whole genome sequencing data were aligned to the goat reference genome ARS1 using Sentieon software, and positional sorting and repeat read marking were performed;

[0068] (2) Use Sentieon software to detect variant sites for each sample and obtain the gVCF for each sample;

[0069] (3) Use Sentieon to perform joint-calling and perform joint analysis of gVCF for all samples to obtain the variation results of each individual in the population.

[0070] In some embodiments of the present invention, the mining and screening of target SNPs further includes aligning functional gene sequences to a reference genome ARS1 to obtain the corresponding positions in ARS1, extracting the genetic parameters of all sites within the interval in the resequencing data, including MAF, detection rate, heterozygosity, and sequencing depth, and screening SNPs as candidate sites according to the conditions of MAF≥0.05, SNP site detection rate≥90%, heterozygosity ≤50%, and sequencing depth≥5×.

[0071] In some embodiments of the present invention, the functional genes include growth trait-related genes, milk production trait-related genes, reproductive trait-related genes, adaptation-related genes, disease resistance trait-related genes, immune trait-related genes, and villous trait-related genes.

[0072] In some embodiments of the present invention, the hard filtering criteria are as follows: QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0 || MQRankSum < -12.5 ||

[0073] ReadPosRankSum < -8.0.

[0074] In some embodiments of the present invention, the probe has a length of 80-120 bp.

[0075] In some embodiments of the present invention, the probe is approximately 100 bp in length.

[0076] In some embodiments of the present invention, the GC content of the probe is between 20% and 80%.

[0077] In some embodiments of the present invention, screening for SNPs that meet the requirements includes the following steps: sequencing the probes and removing probes that cannot be uniquely aligned on the genome or whose flanking sequences contain repetitive sequences.

[0078] In a fifth aspect of the invention, an application of at least one of the above-mentioned goat SNP molecular marker combinations, whole genome liquid microarrays, and kits is proposed, wherein the application is in the detection of goat breeds.

[0079] In some embodiments of the present invention, the application is an application in whole-genome selection breeding of goats.

[0080] In some embodiments of the present invention, the application is an application in goat genome-wide association analysis.

[0081] In some embodiments of the present invention, the application is in the identification of goat germplasm resources.

[0082] In some embodiments of the present invention, the application is in goat cluster analysis and kinship identification.

[0083] In some embodiments of the present invention, the application is in the discovery and identification of important traits in goats.

[0084] In some embodiments of the present invention, the application is in goat genotyping detection.

[0085] In some embodiments of the present invention, the application is in the construction of goat genetic maps and gene localization.

[0086] In some embodiments of the present invention, the application is in the molecular design breeding of goats.

[0087] In some embodiments of the present invention, the application can be implemented through the following methods:

[0088] S1. Genotyping of the sample to be tested is performed using at least one of the above-mentioned goat SNP molecular marker combination, whole genome liquid chip and kit to obtain genotyping results;

[0089] S2. Analyze the genotyping results obtained in step S1.

[0090] In a sixth aspect of the present invention, a method for breeding goats is proposed, comprising the following steps: using at least one of the above-mentioned goat SNP molecular marker combination, whole genome liquid phase chip and kit to detect the DNA of the goat to be tested, and selecting suitable goats for subsequent breeding.

[0091] The present invention has at least the following beneficial effects: the goat SNP molecular marker combination of the present invention can be used to distinguish different goat breeds and classify different goat qualities. When used to prepare goat whole genome liquid microarrays, it can be used for low-cost and large-scale genotyping of goats. Based on the principle of liquid microarrays, the loci are flexible and new functional marker loci can be added at any time. It can be used for goat whole genome selection breeding, whole genome association analysis, kinship analysis, germplasm resource mining and utilization, providing an important tool for goat germplasm resource genotyping and molecular breeding, and playing an important role in related research.

[0092] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0093] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0094] Figure 1 This is the MAF distribution histogram in Embodiment 1 of the present invention;

[0095] Figure 2 This is a chromosome density distribution map of the goat 50K cGPS liquid-phase chip site in Example 1 of the present invention;

[0096] Figure 3 This is a schematic diagram of the cGPS liquid phase chip process detection in Embodiment 1 of the present invention;

[0097] Figure 4 This is a clustering analysis diagram of goat liquid phase chip in Embodiment 4 of the present invention. Detailed Implementation

[0098] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0099] Example 1

[0100] This embodiment prepared a goat SNP molecular marker combination and a goat whole genome liquid phase chip designed based on the goat SNP molecular marker combination.

[0101] 1. The screening process for goat SNP molecular marker combinations is as follows:

[0102] (1) Collection of goat germplasm resources

[0103] To obtain a rich and diverse range of whole-genome SNP loci, whole-genome sequencing data of 294 goat germplasm resources from different regions and breeds (wild breeds, local breeds, and mainstream commercial breeds) were collected from the National Genome Database. These included Boer goats, Black goats, White goats, Saanen goats, Tugendhoven goats, Cashmere goats, Yunling goats, Nubian goats, and wild goats. Additionally, 110 goat samples were collected from Shandong Agricultural University, 134 from Nanjing Agricultural University, and 55 from Yunnan University for whole-genome resequencing.

[0104] (2) Goat whole genome resequencing

[0105] Whole genome resequencing was performed on 299 goat samples collected. The specific steps included: 1) DNA extraction using magnetic beads.

[0106] 2) Using the MGI library standard method, qualified libraries were used for DNA-seq sequencing library construction.

[0107] 3) After the library passed quality control, sequencing was performed using the MGI sequencing platform. The sequencing strategy was PE150, the sequencing depth was 10×, and 32Gb was sequenced for each strain.

[0108] Sentieon was used to align and detect variants in 593 resequencing data points. The analysis workflow is as follows:

[0109] 1) Use Sentieon to align reads to the corresponding goat reference genome (ARS1), perform positional sorting, and mark duplicate reads.

[0110] 2) Use Sentieon to detect variant sites for each sample and obtain the gVCF for each sample.

[0111] 3) Sentiion was used for joint-calling to perform joint analysis of gVCF for all samples, obtaining the variation results for each individual in the population. To ensure the accuracy of SNPs, preliminary hard filtering was performed on the SNP sites obtained after joint analysis (SNP hard filtering criteria: "QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0").

[0112] (3) Site screening

[0113] From the VCF file containing SNP variant information for all samples, the MAF value, detection rate, heterozygosity, and sequencing depth of the loci were calculated and statistically analyzed. Based on MAF ≥ 0.05, SNP detection rate ≥ 90%, heterozygosity ≤ 35%, and sequencing depth ≥ 10×, 7,704,446 target loci were identified. Probe design was performed on the selected target loci set, targeting each target locus within a 50 bp radius to its left and right. Probe lengths were approximately 100 bp, and GC content ranged from 20% to 80%. Based on the probe design results, probes that could not be uniquely aligned to the genome or contained repetitive sequences in their flanking sequences were removed. Based on the principle of uniform distribution, 44,377 highly polymorphic SNP loci were selected as genome-wide loci (MAF distribution map shown in Figure 1). Figure 1 (As shown); Simultaneously, functional genes of important economic traits reported in existing literature were collected, and these gene sequences were aligned to the reference genome ARS1 to obtain the corresponding positions in ARS1. Genetic parameters of all loci within the interval were extracted from 593 resequencing data, including MAF, detection rate, heterozygosity, and sequencing depth. SNPs were screened as candidate loci according to the conditions of MAF≥0.05, SNP detection rate≥90%, heterozygosity≤50%, and sequencing depth≥5×, totaling 6,131 SNP loci, including 1,502 loci related to growth traits, 1,298 loci related to milk production traits, 1,220 loci related to reproductive traits, 1,178 loci related to adaptation, 414 loci related to disease resistance traits, 408 loci related to immunity traits, and 111 loci related to wool traits. These were used to construct a sheep 50K liquid phase chip for probe synthesis, with a total of 50,508 loci and an average spacing of 59Kb (cGPS liquid phase chip locus chromosome density distribution diagram as shown). Figure 2 (As shown).

[0114] 2. Goat whole genome liquid chip

[0115] Based on the 50,508 SNP sites obtained through screening, a goat whole genome liquid phase chip was prepared by synthesizing liquid phase capture probes using Huazhi Biotechnology Co., Ltd. and using the precise localization sequencing and genotyping technology (cGPS) based on liquid phase capture of target region genome sequences to form a system.

[0116] cGPS, based on an optimized thermodynamic stability algorithm model, designs probes for genomic sequences in different target regions. It then uses synthesized specific probes to capture and enrich multiple target sequences located at different genomic positions through liquid-phase hybridization. Following this, it constructs sequencing libraries and performs high-throughput sequencing on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel loci within the target region. Figure 3 As shown.

[0117] Example 2

[0118] This embodiment provides a method for using the goat whole genome liquid phase chip in Example 1 above for goat identification, including the following:

[0119] 1. Extraction and detection of goat genomic DNA

[0120] Twelve goat muscle tissue samples were selected as validation samples for the 50K liquid-phase microarray development system. The sample numbers are 20-21, 21-060, 21-29, 21092, 21704, 39, 64, A08, A09, A41, B1, and B24. Genomic DNA was extracted from the tissues using a magnetic bead method. The integrity and purity of the genomic DNA were analyzed by 1% agarose gel electrophoresis, and the concentration was accurately quantified using Qubit.

[0121] 2. cGPS Experiment Procedure

[0122] 1) Take 200ng of qualified genomic DNA, digest it into 100-500bp fragments using enzyme digestion reagents, and then add Taq enzyme for end repair.

[0123] 2) Using T4 ligase, the adapter fragments are ligated to both ends of the DNA. Fragment sorting magnetic beads are used to purify the ligation products and amplify the library to complete the library construction.

[0124] 3) Place the qualified library, blocking reagent, RNase inhibitor, and 50K liquid phase chip probe on a PCR instrument for hybridization reaction and incubate at 55℃ for 16-24 hours.

[0125] 4) The hybridization product was captured using streptavidin, and the captured library was amplified and enriched. PE150 sequencing was performed using the BGI sequencing DNBSEQ-T7 platform.

[0126] 5) The raw data after high-throughput sequencing undergoes quality control filtering and other processes. FastP software is used to remove reads containing adapter contamination and low-quality reads. BWA software is used to align the sequencing data with the target genome, and GATK software is then used to analyze variant sites to obtain genotyping results for the target loci. A flowchart is shown below. Figure 3 As shown.

[0127] Example 3: Evaluation of the Genotyping Effect of Goat Whole Genome Liquid Microarray

[0128] To verify the genotyping effect of the goat whole genome liquid chip, the goat whole genome liquid chip obtained in Example 1 was used to perform genotyping detection on 12 goat samples (including 4 duplicate samples) (see Example 2 for specific operation method).

[0129] Table 1. Locus detection rate of 16 sheep samples

[0130]

[0131] Table 2. Average Genotypic Consistency Rate of Technically Duplicate Samples

[0132]

[0133] Sequencing and data analysis revealed experimental results, as shown in Tables 1 and 2. Table 1 shows that the locus detection rate of the 16 samples ranged from 99.58% to 99.84%, with an average detection rate of 99.78%. Table 2 shows that the genotyping results of the four replicate samples showed a genotyping consistency rate between 99.18% and 99.62%, with an average consistency rate of 99.46%. Evaluation of the genotyping performance of the 50K liquid-phase chip (the goat whole-genome liquid-phase chip prepared in Example 1) indicates that the goat whole-genome liquid-phase chip prepared in this embodiment of the invention exhibits good detection stability, a high target locus detection rate, and accurate and reliable genotyping results.

[0134] Example 4: Population structure analysis of goat whole-genome liquid microarray

[0135] Genotyping of 580 goat accessions (of known origin) was performed using the goat whole-genome liquid-phase chip prepared in Example 1. Genetic distance matrices were calculated using the IBS method in Plink software, followed by cluster analysis and phylogenetic tree construction. Results are as follows: Figure 4 As shown in the figure, the goat whole genome liquid chip prepared in Example 1 can effectively classify goats from different sources, and the classification effect is consistent with the actual results.

[0136] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A whole-genome liquid-phase chip, characterized in that, The whole-genome liquid-phase chip includes probes for detecting goat SNP molecular marker combinations; the goat SNP molecular marker combinations consist of 50,508 SNP molecular markers, the physical locations of which are determined by sequence alignment based on the goat reference genome ARS1, and the specific site information is shown in Table 1 of the specification.

2. The application of the whole-genome liquid-phase chip according to claim 1 in any of the following: (1) Testing goat breeds; (2) Goat genome-wide selection breeding; (3) Genome-wide association analysis of goats; (4) Identification of goat germplasm resources; (5) Goat cluster analysis and kinship identification; (6) Construction of goat genetic maps and gene localization; (7) Molecular design breeding of goats.

3. A method for breeding goats, characterized in that, The process includes the following steps: using the whole-genome liquid phase chip as described in claim 1 to detect the DNA of the goat to be tested, and selecting goats for subsequent breeding.

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