A cloud black goat 40K liquid phase chip and its application

Abstract

The present invention relates to a 40K liquid phase chip of Yunshang black goat and its application, wherein the chip contains probes for detecting 204 prospect SNP sites, and the 204 prospect SNP sites are functional SNP sites related to reproduction, growth, breast development, milk quality, coat color and Yunshang black goat gene ID card; the chip also contains probes for detecting 35000-45000 background SNP sites, and the selection principle of the background SNP sites is: SNP sites with changes in amino acids or start and stop codons selected based on the whole genome sequencing data of Yunshang black goat and SNP sites uniformly covered by the whole genome, and the version number of the reference genome is ARS1. The 40K liquid phase chip of Yunshang black goat provided by the present invention is applied in genotype detection, whole genome association analysis, variety identification, kinship analysis, and cultivation of new strains of Yunshang black goat, which can effectively improve the precision breeding level of Yunshang black goat.

Description

Technical Field

[0001] The present invention relates to the technical field of whole genome gene chips, and in particular to a Yunshang black goat 40K liquid phase chip and applications thereof. Background Art

[0002] Yunshang Black Goat has excellent characteristics such as black fur, fast growth and development, year-round estrus, high fertility, good meat production performance, strong adaptability and tolerance to roughage. It is suitable for feeding in stalls, grazing + supplementary feeding or full grazing, and can be widely adapted to cold and cool areas from low-altitude river valley areas (below 1000m) to higher altitudes (about 2000m), and has broad application prospects. With the continuous expansion of the scale of the Yunshang Black Goat industry and the gradual increase in breeding companies, in order to ensure the breed characteristics of Yunshang Black Goat, distinguish between true and false individuals, establish a market operation system with high quality and high price, protect the germplasm resources and excellent traits of Yunshang Black Goat, and enhance the brand value of this breed, it is necessary to carry out the breed identification of Yunshang Black Goat at the genome level and deeply explore the unique genomic loci information of this breed.

[0003] DNA molecular markers are of various types, and their detection methods are simple and rapid. They are widely used in animal and plant breeding. Single nucleotide polymorphism (SNP) is formed by the conversion and transversion of a single nucleotide on the genome. There are a large number of SNPs distributed on the genome, and SNP loci are often typed through whole genome resequencing and gene chip detection.

[0004] Chinese patent 202210375487.6 discloses a Yunshang black goat SNP marker and its application in identifying the Yunshang black goat variety, which discloses 26 SNP sites that can be used to identify Yunshang black goats. Using these 26 SNP sites and identification technology, the identification accuracy of Yunshang black goats reached 89.58%. However, with the expansion of the Yunshang black goat breeding population and market development, faster, more accurate and more functional gene identification technology is needed.

[0005] Liquid-phase chip is a new type of molecular detection technology after solid-phase chip. This technology is based on targeted sequencing genotyping detection (GBTS) technology. Because it can make the target probe and the target sequence complementary to each other for fixed-point capture, and can quickly complete thousands of probe hybridization reactions in the liquid phase at the same time, the kit is figuratively called liquid-phase chip. Compared with traditional genotyping detection technology and solid-phase chip, liquid-phase chip has the advantages of wide platform adaptability, labeling flexibility, detection efficiency, information additivity, support convenience and broad application spectrum, and has broad application prospects. The development of liquid-phase chip requires accurate SNP site information. At present, there is no research report on Yunshang Black Goat liquid-phase chip. Summary of the invention

[0006] In order to solve the above problems, the present invention proposes a Yunshang Black Goat 40K liquid phase chip and its application.

[0007] The present invention proposes a Yunshang black goat 40K liquid phase chip, wherein the chip contains probes for detecting 204 foreground SNP sites, wherein the 204 foreground SNP sites are functional SNP sites related to reproduction, growth, breast development, milk quality, coat color and a Yunshang black goat gene ID card, and information of the 204 foreground SNP sites is shown in Table 1; the chip also contains probes for detecting 35,000-45,000 background SNP sites, and the selection principle of the background SNP sites is: SNP sites with changes in amino acids or start and stop codons selected based on the whole genome sequencing data of the Yunshang black goat and SNP sites uniformly covered in the whole genome, and the version number of the reference genome is ARS1.

[0008] Furthermore, the background SNP sites are 39796, which together with the 204 foreground SNP sites constitute 40,000 SNP sites. The Yunshang Black Goat 40K liquid phase chip contains probes of the 40,000 SNO sites. The information of the 40,000 SNP sites is shown in Table 2.

[0009] The Yunshang black goat 40K liquid phase chip provided by the present invention is used in genotype detection, whole genome association analysis, variety identification, kinship analysis, and breeding of new strains of Yunshang black goats.

[0010] The present invention also provides a method for preparing a Yunshang Black Goat 40K liquid phase chip, comprising the following steps:

[0011] S1. SNP site quality control: The selected data set is 153 Yunshang black goat resequencing data for data filtering; the data filtering criteria are as follows: site and sample missing rate threshold <0.2, site heterozygosity threshold <0.2, MAF filtering threshold >0.05;

[0012] S2. Selection of prospective SNP sites: Based on the data filtered in S1 and the progress of selection signal analysis, 204 prospective sites were obtained, including SNP sites for reproduction, growth, breast development, milk quality, coat color, and the genetic ID card of Yunshang black goat;

[0013] S3. Sliding window site selection and probe design: Determine candidate SNP sites based on the following priority principles:

[0014] 1) Prospect SNP sites;

[0015] 2) SNP sites where amino acids or start and stop codons change;

[0016] 3) SNP sites with uniform coverage across the entire genome;

[0017] S4. Submit the proposed candidate SNP site set to the probe design system for scoring, and design the probe based on the evaluation results of the upstream and downstream sequences of the target site. The specificity, complexity, GC content, etc. of the upstream and downstream sequences of the target site are mainly evaluated. The target site is placed in the middle of the probe first, and the designed probe is 120bp in length.

[0018] S5. Based on the above scoring results, a total of 35,000-45,000 SNP sites including 204 prospective SNP sites were selected for designing 40K liquid phase chip probes.

[0019] The beneficial effects of the present invention are as follows:

[0020] The present invention provides a combination of SNP markers and targeted capture sequencing of a 40K liquid phase chip of a Yunshang black goat, which can realize the identification of Yunshang black goat varieties and kinship analysis, and includes 204 functional SNP sites related to the reproduction, growth, breast development, milk quality, hair color and gene ID card of Yunshang black goat, of which 178 SNP sites are significantly associated with important economic traits such as reproduction, growth, breast development, milk quality, hair color, etc., which can be used for the whole genome selection breeding of Yunshang black goat; 26 SNP sites are purebred Yunshang black goat-specific gene markers, which can be used for the identification of Yunshang black goat varieties. Using the Yunshang black goat 40K liquid phase chip of the present invention, a reference database analysis model can be constructed, and the individual whole genome estimated breeding value (GEBV) can be calculated as the basis for the next generation breeding value estimation and individual selection, and finally conventional breeding technology and MOET technology are combined to accelerate the genetic progress and accuracy of each generation of Yunshang black goat breeding, effectively improve the precision breeding level of Yunshang black goat, and provide technical support for the application of genomic selection technology in the continuous breeding and improvement of Yunshang black goat. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The distribution of 40,000 SNP sites used for black goat genotyping in the present invention in the whole genome;

[0022] Figure 2 This is a schematic diagram of the process of using the Yunshang Black Goat 40K liquid phase chip for detection in Example 2;

[0023] Figure 3 The PCA analysis results of 73 goats in Example 4 are as follows;

[0024] Figure 4 This is the result of the phylogenetic tree analysis of 73 goats in Example 4. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the embodiments.

[0026] Example 1 Development process of black goat whole genome breeding chip

[0027] S1. Site quality control: The selected data set is 153 black goat resequencing data, and the data filtering criteria are as follows: 1) site and sample missing rate, threshold <0.2; 2) site heterozygosity, threshold <0.2; 3) MAF filtering, threshold >0.05. After filtering, a total of 12,122,041 sites were obtained.

[0028] S2. Selection of prospect loci: Based on the latest research progress of 153 whole genome sequencing data of Yunshang black goats and selection signal analysis, 204 prospect loci were obtained (as shown in Table 1), including SNP loci of reproduction, growth, breast development, coat color and 26 Yunshang black goat gene ID cards;

[0029] S3. Sliding window site selection and probe design: Determine candidate SNP sites based on the following priority principles:

[0030] 1) Foreground sites;

[0031] 2) The site where the amino acid or start or stop codon changes;

[0032] 3) Uniform coverage of sites across the genome;

[0033] S4. Submit the proposed candidate SNP site set to the probe design system for scoring, and design the probe based on the evaluation results of the upstream and downstream sequences of the target site. The specificity, complexity, GC content, etc. of the upstream and downstream sequences of the target site are mainly evaluated. The target site is placed in the middle of the probe first, and the designed probe is 120bp in length.

[0034] S5. Based on the above scoring results, a total of 40,000 SNP sites including 204 foreground SNP sites (Table 1) and 39,796 background SNP sites (Table 2) were selected for designing 40K liquid phase chip probes. The distribution of the 40,000 SNP sites on the genome is as follows: Figure 1 As shown, from Figure 1 It can be seen that the selected loci have the advantages of being evenly distributed on the chromosome and high reliability of typing results.

[0035] Table 1 Information on 204 prospective SNP loci used for the design of 40K liquid phase chip probes for Yunshang black goats

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[0038] Table 2 Information on 40,000 SNP loci used for the design of 40K liquid phase chip probes for Yunshang black goat

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[0226] Example 2: Process of testing black goat DNA samples using Yunshang black goat 40K liquid phase chip

[0227] For the 40,000 SNP sites obtained in Example 1, as shown in Table 1, 50 bp of each site flank was extracted, and probes for detecting the 40,000 SNP sites were designed for preparing the Yunshang Black Goat 40K liquid phase chip.

[0228] The schematic diagram of the method for using the above detection product for detection is as follows Figure 2 The details are as follows:

[0229] 1. Sample DNA extraction and detection:

[0230] Blood samples from Yunshang black goats were collected and the sample DNA was extracted using a magnetic bead DNA extraction kit (Kangwei Century CW2361S). The samples were stored at -20°C. The genomic DNA required an absorbance OD260 / 280 ratio between 1.7 and 2.0, a single genomic band, and a clear and complete main sample band without degradation or slight degradation.

[0231] 2. Liquid phase capture library construction:

[0232] 2.1. Genomic DNA fragmentation, end repair & addition of A: Take 300 ng of DNA sample, add 4 μL of Smearase Buffer, 2 μL of Smearase Enzymes, and the total volume is 24 μL. Then place the reaction plate in a PCR instrument and execute the program: 4℃1min→30℃10min→72℃20min→store at 4℃.

[0233] 2.2. Fragment screening: The fragment range is screened by magnetic beads to remove oversized and undersized fragments, so that the DNA fragments are concentrated in 200-300bp as much as possible. The final volume of the fragment screening is 25ul and stored in a 96-well PCR plate.

[0234] 2.3. Adapter ligation and enrichment: Add 5μL CAGT UniversalAdapters and 20μL Ligation Master Mix to the 96-well PCR plate in the above step, vortex and mix, centrifuge briefly to collect the reaction solution to the bottom of the tube, react in a PCR instrument at 20°C for 15min to complete the sequencing adapter ligation. After purifying the ligation product, perform PCR amplification and enrichment, take 15μL of the purified ligation product, add 10μL CAGT UDIPrimer, 25μL Equinox Library Amp Mix (2x), mix well, and draw 35μL of the reaction solution in a PCR instrument for library amplification. After the whole genome library is constructed, use the dsDNA HSAssay Kit for Qubit kit to quantify the library; at the same time, use electrophoresis to detect whether the main peak of the library fragment is in the range of 300-500bp.

[0235] 2.4. Liquid chip hybridization capture: The whole genome library of each sample is mixed, and the total amount of the final hybrid capture library is 4μg. The library is probe hybridized to capture the fragments of the target region from the whole genome library, and the excess probes, hybridization reagents and other reagent components are removed through the elution step. Finally, the target region is enriched through PCR amplification after hybridization to obtain the machine library.

[0236] 2.5. Library quality inspection and sequencing: After the hybridization capture library is constructed, the dsDNA HS Assay Kit for Qubit is used to quantify the library; at the same time, electrophoresis is used to detect whether the main peak size of the library is in the range of 300-500bp. The constructed library is sequenced on the DNBSEQ-T7 sequencer.

[0237] 3. Data quality control and genotyping: The raw data were quality controlled to remove reads with adapters or low quality, and the parameters used by Clean Reads Fastp software were obtained, namely -u 50-n 1-q 5-l 30. Clean Reads were then aligned to the reference genome using BWA0.7.17 software, sorted and indexed using samtools 1.7, and Bam files were deduplicated using the module provided by GATK 4.1.8.0 software. The variant site file was generated using the GenotypeGVCFs module in GATK 4.1.8.0 software, and the target interval variant site analysis of the samples to be tested was completed for genotyping.

[0238] Example 3: Quality Assessment of Yunshang Black Goat 40K Liquid Chip Genotyping

[0239] 100 Yunshang black goats were selected to perform genotyping detection on the Yunshang black goat 40K liquid phase chip using the detection process of Example 2, and the following quality evaluation was performed on the genotyping chip:

[0240] 1. Detection rate:

[0241] The SNP detection rate and individual detection rate are important indicators for measuring chip quality. The SNP detection rate of the Yunshang black goat 40K liquid phase chip prepared in Example 2 is very high, and the average detection rate of 100 test samples is 97.74%. It can be seen that the genotype detection quality of the black goat whole genome breeding chip described in the present invention is very good.

[0242] 2. Genotyping stability:

[0243] Stability is generally measured by the typing consistency and correlation coefficient of repeated samples. Three Yunshang black goats were used to detect the Yunshang black goat 40K liquid phase chip prepared in Example 2. The typing consistency results of the three repeated samples are as follows: the genotype consistency is 99.78%. It can be seen that the Yunshang black goat 40K liquid phase chip prepared by the present invention has good stability for genotype detection, and can significantly improve the accuracy and stability of large-scale genotyping detection of black goats.

[0244] Example 4: Application of Yunshang Black Goat 40K Liquid Phase Chip in Black Goat Genealogy Analysis

[0245] As in Example 1, 40,000 sites were screened from 153 copies of Yunshang black goat whole genome sequencing data and evenly distributed on each chromosome, such as Figure 1 The final site set screened was submitted to Beijing Compson Technology Co., Ltd. for preparation into the Yunshang Black Goat 40K liquid phase chip.

[0246] As shown in Table 3, a total of 73 samples of black goats of four local breeds were collected from Yunnan Province. After DNA extraction and chip testing, genotype data were obtained. The quality control standards for analysis were as follows: (1) autosomal loci were used; (2) SNP detection rate was greater than or equal to 90%; individual detection rate was greater than or equal to 90%; (3) MAF was greater than or equal to 0.01; (4) Hardy-Weinberg equilibrium test P < 10 -6 The qualified loci after quality control were used for subsequent kinship analysis.

[0247] Table 3 Sample collection information table

[0248]

[0249]

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[0251]

[0252] The genotype data of the above samples were analyzed by principal component analysis (PCA) using Plink (V1.9) software. The results were shown in the following figure using R language function programming. Figure 3 As shown, the 73 goats were unevenly distributed with obvious group stratification. The four goat breeds could be divided into three groups. The Yunshang black goats and Ninglang black-headed goats were in separate groups, while the Yunling goats and Fengqing hornless goats were in one group with no obvious group stratification. This may be because the Yunling goats and Fengqing hornless goats are distributed in similar areas, making blood exchange difficult to avoid.

[0253] Phylogenetic tree analysis, neighbor-joining (NJ) method was used to construct the phylogenetic tree. MegaX (V10.0) software was used to construct the phylogenetic tree. Figure 4 This is the evolutionary tree of the black goat population. This diagram can intuitively show the closeness of the relationship between individuals in the population. Figure 4 This is a population genetic structure analysis of four goat breeds. This graph can visually show the closeness of the relationship between each individual in the population. Figure 4 It can be seen that the Ninglang black-headed goats are independently clustered into a large branch, the Yunshang black goats, Yunling goats and Fengqing hornless goats are clustered into a large branch, among which the Yunshang black goats and Fengqing hornless goats are clustered into a small branch respectively, and most of the Yunling goats are clustered into a small branch, but 3 Yunling goats and Yunshang black goats are clustered together. These 3 goats may have mixed with the Yunshang black goat bloodline at the sampling site. At the same time, due to the special geographical location, the Ninglang black-headed goat has a very small chance of introducing foreign blood, so it shows a relatively far genetic distance from the other 3 breeds.

[0254] Example 4: Application of Yunshang Black Goat 40K Liquid Phase Chip in the Division of Yunshang Black Goat Pedigrees

[0255] In this embodiment, the above-mentioned Yunshang black goat 40K liquid phase chip is used to divide the 57 Yunshang black goat groups in the breeding farm of Yunnan Province Sheep Breeding and Promotion Center into families. The specific results are shown in Table 4. The 57 Yunshang black goat groups are divided into 22 families in total. The number of families is sufficient, the average inbreeding coefficient of the group is 0.041, and the inbreeding coefficient of the group is well controlled.

[0256] Table 4 Results of the division of individual families of the 57 Yunshang black goat population

[0257] Family line 1 YG1 YG52 YG93 Family 2 YG9 YG31 YG49 YG55 YG42 Family 3 YG14 YG36 Family 4 YG16 YG42 YG44 YG28 YG9 Family lineage 5 YG18 YG26 YG37 YG40 YG46 YG94 Family lineage 6 YG28 YG32 YG39 YG43 YG45 YG50 YG16 Family 7 YG11 YG13 YG23 Family 8 YG41 Family line 9 YG7 Family line 10 YG54 Family line 11 YG2 YG21 YG95 YG5 Family line 12 YG4 YG47 Family line 13 YG5 YG44 YG95 YG16 Family line 14 YG25 Family line 15 YG12 Family line 16 YG8 Family line 17 YG3 YG17 YG27 YG34 YG33 Family line 18 YG19 YG20 YG38 Family line 19 YG33 YG38 YG48 YG51 YG27 YG20 Family line 20 YG22 YG24 Family line 21 YG10 YG30 YG53 YG56 Family line 22 YG15 YG35

[0258] In summary, the Yunshang black goat 40K liquid phase chip of the present invention can be used for low-cost and large-scale genotyping of Yunshang black goats. The chip probe design is flexible, the sample size for amplification detection can be increased, and functional marker sites can be flexibly added. The breeding chip is more suitable for research such as Yunshang black goat breeding and kinship combing, and provides a reference for genetic resource protection and variety improvement of Yunshang black goats.

[0259] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0260] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0261] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A Yunshang Black Goat 40K liquid phase chip, characterized in that: The chip contains probes for detecting 204 prospect SNP sites, which are functional SNP sites related to reproduction, growth, breast development, milk quality, coat color and Yunshang black goat gene ID card. The information of the 204 prospect SNP sites is shown in the following table: The version number of the reference genome is ARS1; The background SNP sites are 39796, which together with the 204 foreground SNP sites constitute 40,000 SNP sites; the Yunshang Black Goat 40K liquid phase chip contains probes for the 40,000 SNP sites, and the information of the 40,000 SNP sites is shown in Table 2 of the specification. The target site is placed in the middle of the probe, and the designed probe is 120bp in length.

2. Application of the Yunshang Black Goat 40K liquid phase chip described in claim 1 in genotype detection, whole genome association analysis, variety identification, kinship analysis, and breeding of new strains of Yunshang Black Goats.

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