A cloud black goat 10K liquid phase chip and its application

Abstract

The present invention relates to a Yunshang black goat 10K liquid phase chip and an application thereof, wherein the chip contains probes for detecting 3872 foreground SNP sites, and the chip also contains probes for detecting 6000-10000 background SNP sites. The present invention provides a SNP marker combination of a Yunshang black goat 10K liquid phase chip, which can realize the variety identification and kinship analysis of Yunshang black goats, and contains SNP sites that have grown and reproduced with Yunshang black goats, providing technical support for the germplasm breeding and lambing trait and growth trait breeding of Yunshang black goats.

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 10K 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 chip is a new type of molecular detection technology after solid 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, it is figuratively called liquid chip. When preparing liquid chip, the whole genome sequencing results of the tested variety are usually compared to the reference genome for SNP site screening, and synthetic probes are designed based on the screened SNP sites. Since the liquid chip contains a large number of SNP sites, as long as the SNP site information of a specific trait is determined, a liquid chip with a corresponding detection effect can be designed. Compared with traditional genotyping detection technology and solid chip, liquid chip has the advantages of platform adaptability, labeling flexibility, detection efficiency, information additivity, support convenience and broad application spectrum, and has broad application prospects. At present, there is no research report on liquid chip applied to genotyping, kinship identification and subdivided trait identification of Yunshang black goats. Summary of the invention

[0006] In order to solve the above problems, the present invention proposes a Yunshang Black Goat 10K liquid phase chip and its application.

[0007] The present invention proposes a Yunshang black goat 10K liquid phase chip, wherein the chip contains probes for detecting 3872 foreground SNP sites, wherein the 3872 foreground SNP sites include 3829 SNP sites associated with growth and 43 SNP sites associated with lambing, wherein the information of the SNP sites associated with growth is shown in Table 1 of the specification, and the information of the SNP sites associated with lambing is shown in Table 2 of the specification, wherein the chip further contains probes for detecting 6000-10000 background SNP sites, wherein the background SNP sites contain 26 SNP sites for identifying Yunshang black goats, wherein the information of the SNP sites for identifying Yunshang black goats is shown in Table 4 of the specification, and the version number of the reference genome is ARS1.

[0008] Furthermore, the method for selecting the foreground SNP sites and the background SNP sites is:

[0009] S1. Site quality control: The selected data set is 140 resequencing data of Yunshang black goats. After data filtering, a total of 12,122,041 sites were obtained; 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;

[0010] S2. Selection of prospect sites: Based on the whole genome sequencing data of 140 Yunshang black goats obtained in S1, VCFtools-0.1.17 software was used to perform selection signal analysis, and the FST and θπ values ​​of fast-growing black goats and slow-growing black goats, high-breeding black goats and low-breeding black goats were calculated respectively. Sites that appeared in both the FST analysis Top1% window and the θπRatio analysis Top1% window were screened. After removing linkage disequilibrium sites, 3829 sites related to growth traits and 43 sites related to lambing traits were finally screened;

[0011] S3. Determine the background SNP sites according to the following priority principles:

[0012] 1) The site where the amino acid or start or stop codon changes;

[0013] 2) Uniform coverage of sites across the genome;

[0014] S4. Submit the proposed background 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 120 bp in length.

[0015] S5. Based on the above scoring results, a total of 6000-10000 background SNP sites were selected and used together with the 3872 foreground sites obtained in S2 to design 10K liquid phase chip probes.

[0016] Furthermore, the chip has 6128 probes for detecting background SNP sites, and information of the 6128 background SNP sites is shown in Table 3 of the specification.

[0017] The Yunshang black goat 10K liquid phase chip provided by the present invention is used in Yunshang black goat genotype detection, whole genome association analysis, variety identification, kinship analysis, and breeding of new strains of Yunshang black goats for growth or reproductive traits.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The present invention provides a SNP marker combination of the Yunshang Black Goat 10K liquid phase chip, which can realize the variety identification and kinship analysis of the Yunshang Black Goat, and contains SNP sites for the growth and reproduction of the Yunshang Black Goat, providing technical support for the germplasm breeding and lambing trait and growth trait breeding of the Yunshang Black Goat.

[0020] 2. The present invention also provides 3829 growth-related SNP sites and 43 lambing-related SNP sites as prospect sites, providing technical support for subsequent whole-genome association analysis.

[0021] 3. The present invention also provides 3829 growth-related SNP sites, which are more suitable for whole genome selection breeding of growth traits.

[0022] 4. The present invention reduces the density of the chip and reduces the detection and analysis cost by 20%, thereby reducing the breeding cost for whole genome selection breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The distribution of 10,000 SNP sites in the whole genome of the Yunshang Black Goat 10K liquid phase chip in Example 1;

[0024] Figure 2 This is a schematic diagram of the process of using the black goat full gene breeding chip for detection in Example 2;

[0025] Figure 3 The PCA analysis results of 59 goats in Example 4;

[0026] Figure 4 This is the result of the phylogenetic tree analysis of 59 goats in Example 4. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the embodiments.

[0028] Example 1: Obtaining SNP site information of Yunshang Black Goat 10K liquid phase chip and designing probes

[0029] The SNP site selection method for Yunshang Black Goat 10K liquid phase chip is as follows:

[0030] S1. Site quality control: The selected data set is 140 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.

[0031] S2. Selection of prospect SNP sites: Based on the whole genome sequencing data of 140 black goats, VCFtools-0.1.17 software was used to perform selection signal analysis, and the FST and θπ values ​​of fast-growing black goats and slow-growing black goats, high-breeding black goats and low-breeding black goats were calculated respectively. The sites that appeared in both the FST analysis Top1% window and the θπRatio analysis Top1% window were screened. After removing linkage disequilibrium sites, 3829 SNP sites related to growth (as shown in Table 1) and 43 SNP sites related to lambing (as shown in Table 2) were screened.

[0032] S3. Determine the background SNP sites according to the following priority principles:

[0033] 1) The site where the amino acid or start or stop codon changes;

[0034] 2) Uniform coverage of sites across the genome;

[0035] S4. Submit the proposed background 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 120 bp in length.

[0036] S5. According to the above scoring results, a total of 6128 background SNP sites were selected, and the background SNP site information is shown in Table 3. The reference genome of the marker is ARS1. The 6128 background SNP sites and the 3872 foreground SNP sites obtained in S2, a total of 10,000 SNP sites, were used to design 10K liquid phase chip probes.

[0037] The 26 SNP sites disclosed in 202210375487.6 that can be used to identify Yunshang black goats (as shown in Table 4) are located in the background SNP sites.

[0038] The distribution of these 10,000 SNP sites on the genome is as follows: Figure 1 As shown. Figure 1 It can be seen that the selected loci have the advantage of being evenly distributed on the chromosome and the typing results are highly reliable.

[0039] Table 1. Information on 3829 SNP loci related to growth

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Table 2. Information on 43 SNP loci associated with lambing

[0065] Serial number Location Serial number Location Serial number Location Serial number Location 1 1:130122718 12 5:26321072 23 8:60367574 34 15:81855052 2 1:130199736 13 5:26340173 24 9:42015116 35 17:52463575 3 1:138927715 14 5:26482395 25 10:75900977 36 20:18545140 4 1:138993620 15 5:57984899 26 10:75928575 37 21:30806940 5 3:102230839 16 5:58076550 27 10:76009332 38 21:30887615 6 4:16307687 17 7:88930239 28 10:76027080 39 24:46226737 7 4:16361347 18 8:44093010 29 11:32106529 40 24:46300253 8 4:27414346 19 8:44179899 30 11:48222159 41 24:46328924 9 4:43301861 20 8:44208719 31 14:22709641 42 24:56814002 10 4:43325881 21 8:60296554 32 14:51802332 43 28:4301235 11 4:43395269 22 8:60337791 33 15:81681966

[0066] Table 3. Information of 6128 background SNP sites

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Table 4. 26 SNP loci in the background loci used for identification of Yunshang black goats

[0105] Serial number Location Serial number Location Serial number Location Serial number Location 1 3:116568146 8 7:67980454 15 15:35311828 22 24:7388496 2 4:27534255 9 9:4210890 16 21:9035839 23 24:37720766 3 5:27394846 10 9:6480114 17 21:26850780 24 24:60570385 4 5:96914121 11 9:19778462 18 23:33685044 25 28:14160630 5 5:112308839 12 11:52712411 19 23:34234088 26 28:43951753 6 7:480924 13 13:72160967 20 23:35769794 7 7:19019153 14 15:1341438 21 24:5617052

[0106] Example 2: Detection of black goat DNA samples using Yunshang black goat 10K liquid phase chip

[0107] For the 10,000 SNP sites obtained in Example 1, as shown in Tables 1-3, 60 bp of each site flank were extracted, and probes for detecting the 10,000 SNP sites were designed to prepare a Yunshang Black Goat whole genome breeding detection product based on targeted capture sequencing technology for detecting the 10,000 SNP sites, namely, the Yunshang Black Goat 10K liquid phase chip.

[0108] The schematic diagram of the method for using the above detection product for detection is as follows Figure 2 The details are as follows:

[0109] 1. Sample DNA extraction and detection:

[0110] 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 2.1, a single genomic band, and a clear and complete main sample band with no or slight degradation.

[0111] 2. Liquid phase capture library construction:

[0112] 2.1 Genomic DNA fragmentation, end repair, and 3' end A addition:

[0113] Take 300 ng of DNA sample, add 4 μL of Smearase Buffer and 2 μL of Smearase Enzymes, with a total volume of 24 μL, and then place the reaction plate in the PCR instrument to execute the program: 4℃1min→30℃10min→72℃20min→store at 4℃.

[0114] 2.2 Fragment screening:

[0115] The broken fragment range is screened by magnetic beads to remove overly large and undersized fragments, so that the DNA fragments are concentrated as much as possible in 200-300bp. The final volume of the fragment screening is 25ul and stored in a 96-well PCR plate.

[0116] 2.3 Adapter ligation and enrichment:

[0117] Add 5μL CAGT Universal Adapters 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 connection. After the connection product is purified, PCR amplification and enrichment are performed, 15μL of the connection purified product is taken, 10μL CAGT UDI Primer and 25μL Equinox Library Amp Mix (2x) are added, mixed, and 35μL of the reaction solution is aspirated in the PCR instrument for library amplification. After the whole genome library is constructed, the library is quantified using the dsDNA HS Assay Kit for Qubit kit; at the same time, the main peak of the library fragment is detected by electrophoresis to see if it is in the range of 300-500bp.

[0118] 2.4 Liquid chip hybridization capture:

[0119] The whole genome library of each sample was mixed, and the total amount of the final hybrid capture library was 4μg. The library was 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 were removed through the elution step. Finally, the target region was enriched through PCR amplification after hybridization to obtain the machine library.

[0120] 2.5 Library quality inspection and sequencing:

[0121] After the hybrid capture library was constructed, the dsDNA HS Assay Kit for Qubit was used to quantify the library; at the same time, electrophoresis was used to detect whether the main peak size of the library was in the range of 300-500bp. The constructed library was sequenced on a DNBSEQ-T7 sequencer.

[0122] 3. Data quality control and genotyping:

[0123] 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.

[0124] Example 3: Quality evaluation of the 10K liquid phase chip genotyping results of Yunshang black goats

[0125] 100 Yunshang black goats were selected for genotyping detection using the Yunshang black goat 10K liquid phase chip obtained in Example 2, and the following quality evaluation was performed.

[0126] 1. Detection rate:

[0127] SNP detection rate and individual detection rate are important indicators for measuring chip quality. The SNP detection rate of the Yunshang Black Goat 10K liquid phase chip obtained 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 Yunshang Black Goat 10K liquid phase chip described in the present invention is very good.

[0128] 2. Genotyping stability:

[0129] Stability is generally measured by the typing consistency and correlation coefficient of repeated samples. Three Yunshang black goats were used to detect the genotyping stability of the Yunshang black goat 10K liquid phase chip in Example 2. The typing consistency results of the three repeated samples are as follows: the number of consistent sites for typing of the three samples is 9893, 9882 and 9874, respectively, and the genotype consistency rates are 99.76%, 99.73% and 99.75%, respectively. It can be seen that the stability of the genotyping detection of the Yunshang black goat 10K liquid phase chip of the present invention is very good. From this result, it can be seen that the Yunshang black goat 10K liquid phase chip of the present invention can significantly improve the accuracy and stability of large-scale genotyping detection of Yunshang black goats.

[0130] Example 4: Application of Yunshang Black Goat 10K Liquid Phase Chip in Black Goat Genealogy Analysis

[0131] A total of 59 black goat samples were collected from various regions of Yunnan Province. After DNA extraction and testing, Yunshang black goat 10K liquid phase 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 markers. The qualified loci after quality control were used for subsequent kinship analysis.

[0132] Table 5 Sample collection information table

[0133]

[0134]

[0135] 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 59 goats were unevenly distributed and there was obvious group stratification. The three goat breeds could be divided into three groups, and individuals within the breeds clustered together, indicating that the individuals between breeds were distantly related, while the individuals within the breeds were closely related.

[0136] 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 three goat breed groups. This diagram can intuitively show the closeness of the relationship between each individual in the group. Figure 4 It can be seen that the 59 sheep are clearly divided into three large branches. All individuals of Yunshang black goats, Ninglang black-headed goats and Fengqing hornless goats are clustered into one large branch, indicating that the three black goat breeds are distantly related.

[0137] It can be seen that the results of using the Yunshang Black Goat 10K liquid phase chip to detect sample genotypes, perform principal component analysis and construct an evolutionary tree are consistent with the actual situation shown in Table 5, indicating that the Yunshang Black Goat 10K liquid phase chip of the present invention can be used for black goat kinship analysis.

[0138] Example 5: Application of Yunshang Black Goat 10K Liquid Phase Chip in Yunshang Black Goat Molecular ID Card Identification

[0139] A total of 59 black goat samples were collected from Yunnan Province. After DNA extraction and testing and Yunshang black goat 10K liquid phase chip testing, genotype data were obtained. The 26 sites in the chip used for Yunshang black goat molecular ID identification (as shown in Table 4 of Example 1) were extracted to identify the true and false Yunshang black goat varieties. The results are shown in Table 6, with an accuracy of 100%.

[0140] Table 6 Identification results of genuine and fake Yunshang Black Goat breeds using Yunshang Black Goat 10K liquid phase chip

[0141]

[0142]

[0143] Note: No - the individual is not the "Black Goat on the Cloud", Yes - the individual is the "Black Goat on the Cloud".

[0144] The Yunshang Black Goat 10K liquid phase chip of the present invention also contains 3829 SNP sites related to growth and 43 SNP sites related to lambing, indicating that the application of the Yunshang Black Goat 10K liquid phase chip of the present invention can not only identify the Yunshang Black Goat variety, but also explore the functional genes of important economic traits of the growth and reproduction of the Yunshang Black Goat, providing important technical support for breeding new strains of Yunshang Black Goats with fast growth and high reproduction rate.

[0145] In summary, the Yunshang Black Goat 10K 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 liquid phase chip is more suitable for research on whole genome selection breeding of Yunshang Black Goats, mining of functional genes of important economic traits, and kinship combing, providing a reference for the protection of genetic resources and continuous breeding and improvement of varieties of Yunshang Black Goats.

[0146] 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.

[0147] 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.

[0148] 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 10K liquid phase chip, characterized in that: The chip contains probes for detecting 3872 prospect SNP sites, the 3872 prospect SNP sites include 3829 SNP sites associated with growth and 43 SNP sites associated with lambing, the information of the SNP sites associated with growth is shown in Table 1, the information of the SNP sites associated with lambing is shown in Table 2, the chip also contains probes for detecting 6128 background SNP sites, the information of the 6128 background SNP sites is shown in Table 3, the background SNP sites contain 26 SNP sites for identifying Yunshang black goats, the information of the 26 SNP sites of the background SNP sites for identifying Yunshang black goats is shown in Table 4, and the version number of the reference genome is ARS1; The 6128 background SNP sites and 3872 foreground SNP sites are used together to design 10K liquid phase chip probes, and the target site is placed in the middle of the probe, and the probe length is 120 bp; The information of 43 SNP loci related to lambing in Table 2 is as follows: ； The information of the 26 SNP sites used to identify Yunshang black goats in Table 4 is as follows: 。 2. Application of the Yunshang Black Goat 10K 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 for growth or reproductive traits.

Images