Raccoon ussuriensis dog low-density liquid-phase SNP chip and application thereof

Abstract

The invention discloses a racoon ussuriensis dog low-density liquid-phase SNP chip and application thereof. Belongs to the technical field of molecular detection. The SNP chip is characterized by comprising 4580 SNP loci, and the number of the SNP loci The invention discloses a low-density liquid-phase SNP (Single Nucleotide Polymorphism) chip for Raccoon ussuriensis dogs, which has specificity for the Raccoon ussuriensis dogs and can be used for efficiently and quickly identifying the genetic relationship of Raccoon ussuriensis dogs groups at low cost, avoiding inbreeding mating, guiding the breeding work of the Raccoon ussuriensis dogs and improving the economic value of the Raccoon ussuriensis dogs.

Description

Technical Field

[0001] The invention relates to the technical field of molecular detection, and more particularly to a Ussuri raccoon dog low-density liquid-phase SNP chip and application thereof. Background Art

[0003] Pedigree information plays a very important role in Ussuri raccoon breeding. Pedigree errors will greatly reduce the accuracy of genetic evaluation, affect the effect of selection and matching, reduce the genetic progress of the population, and bring huge economic impact to the company. However, in actual production, pedigree errors are inevitable, which are affected by many factors, such as breeding records, birth records, pedigree entry and human errors in the process of sorting, etc. Therefore, the development of SNP chips is crucial to improve the accuracy and efficiency of Ussuri raccoon breeding, and help promote its genetic improvement.

[0004] At present, in the gene chip technology used for SNP site typing, the traditional solid-phase chip is based on the complementary hybridization of the probe and the DNA sequence, and typing is performed through the fluorescent color development signal of the marker. Liquid-phase chips are based on resequencing technology to specifically capture each target site and perform high-depth resequencing, which has the advantages of high detection accuracy and high throughput. Liquid-phase chips generally include a biotin-labeled probe covering the target SNP designed for each site to be tested based on the principle of DNA complementarity. These probes hybridize with the target region of the genome in the liquid to form a double strand. The adsorption of streptavidin-coated magnetic beads and biotin-containing molecules can be used. After elution, amplification, and library construction, second-generation sequencing is performed to finally restore the genotype status of the target site and its surrounding SNPs. Liquid-phase chips currently have relatively mature applications in species evolution analysis, germplasm resource evaluation and DNA fingerprint identification, molecular genetic map construction, gene / QTL positioning and gene cloning, molecular marker-assisted selection, and whole genome selection. At present, liquid phase SNP chips have been developed to establish the genetic pedigree and kinship of livestock and poultry: low-density liquid phase SNP chips for dairy goats, targeted capture sequencing chips for kinship identification of broiler chickens, black pig SNP chips, etc. It can be seen that SNP chips have been widely used in livestock and poultry kinship work.

[0005] However, there is no method for using SNP markers to identify the kinship of Ussuri raccoon dogs. Other typing technologies are prone to problems such as poor polymorphism and low efficiency of DNA fragment capture. In addition, other typing technologies are rigid and require a certain amount of sample to be tested, which is not convenient for testing. Therefore, using the whole genome SNP information of Ussuri raccoon dogs to develop liquid phase molecular breeding chips can be used to efficiently, quickly and cost-effectively identify the kinship of Ussuri raccoon dog populations, avoid inbreeding, guide the breeding of Ussuri raccoon dogs, realize the variety identification and kinship analysis of Ussuri raccoon dogs, scientifically guide the hybridization and improvement of Ussuri raccoon dogs, improve the economic value of Ussuri raccoon dogs, and have high application value in many fields of Ussuri raccoon dog breeding.

[0006] In summary, how to provide a low-density liquid-phase SNP chip for identification of kinship of Ussuri raccoon dogs is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a Ussuri raccoon dog low-density liquid-phase SNP chip and application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A low-density liquid-phase SNP chip of Ussuri raccoon dog, comprising 4580 SNP sites; the physical positions of the 4580 SNP sites are determined based on the positive chain bases of the raccoon dog reference genome CNA0046198; the information of the 4580 SNP sites is shown in Table 1:

[0010] Table 1 Information of 4580 SNP loci

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] Chr4-94974680C>T Chr10-81287758G>T Chr18-40242648T>C Chr26-41600747C>T

[0032] Chr4-95369669T>C Chr10-81421849T>C Chr18-40578673G>A Chr26-41789049A>G

[0033] Chr4-95579340A>G Chr10-81462778A>T Chr18-41065813C>T Chr27-171911T>C

[0034] Chr4-96676242T>C Chr10-81772051C>A Chr18-41786098G>C Chr27-615056A>G

[0035] Chr4-98877152A>G Chr10-81876675G>A Chr18-42288998G>A Chr27-665809A>C

[0036] Chr4-99015898C>T Chr10-81989006G>A Chr18-43538716G>A Chr27-746413G>C

[0037] Chr4-99162214G>A Chr10-82339430A>T Chr18-44807007C>T Chr27-775689G>A

[0038] Chr4-99691105G>C Chr10-82868799C>T Chr18-44938273A>G Chr27-909071T>G

[0039] Chr4-100606648C>A Chr10-83101451T>C Chr18-45674474A>C Chr27-1183298T>A

[0040] Chr4-101211584G>T Chr11-40443A>T Chr18-46498615A>G Chr27-1388108C>T

[0041] Chr4-101410206A>C Chr11-295394C>T Chr18-47038745A>G Chr27-1534698G>A

[0042] Chr4-102493894T>C Chr11-327540C>T Chr18-47435855G>A Chr27-1939697A>G

[0043] Chr4-103333512A>G Chr11-624108T>C Chr18-47634584A>G Chr27-2931913T>C

[0044] Chr4-103560715T>C Chr11-714242C>G Chr18-47808711A>G Chr27-2979150G>T

[0045] Chr4-103909396C>T Chr11-914048T>A Chr18-47954506G>A Chr27-3375731T>C

[0046] Chr4-104450792C>T Chr11-999419C>G Chr18-48109518A>G Chr27-3550016C>T

[0047] Chr4-105282142A>G Chr11-1064340A>G Chr18-48115013T>G Chr27-3605876G>C

[0048] Chr4-105529745G>A Chr11-1128428A>G Chr18-48253798A>G Chr27-4164634T>C

[0049] Chr4-105586774A>T Chr11-1395177C>T Chr18-48953533C>T Chr27-4276855C>T

[0050] Chr4-105915243G>C Chr11-1507778T>C Chr18-51378914G>A Chr27-5161189G>T

[0051] Chr4-106018722A>T Chr11-2192864T>A Chr18-51516337A>G Chr27-5821048C>T

[0052] Chr4-106071384A>G Chr11-2345392T>G Chr18-51886599T>C Chr27-6254157G>A

[0053] Chr4-106324456G>C Chr11-2518231T>C Chr18-52147115C>T Chr27-48600665T>G

[0054] Chr4-106521840G>A Chr11-2576772G>C Chr18-52301908T>G Chr27-48617240T>C

[0055] Chr4-107500033G>T Chr11-2758168C>G Chr18-52777642G>A Chr27-83537551C>GChr4-108275486A>G Chr11-2875933T>A Chr18-53052955C>T Chr27-121947997C>G

[0056] Among them, Chr1-133754A>G means that the 133754th position on chromosome 1 changes from A to G.

[0057] The application of the above-mentioned SNP chip in the breeding of Ussuri raccoon dog.

[0058] Application of the above-mentioned SNP chip in the analysis of kinship of Ussuri raccoon dog.

[0059] Application of the above-mentioned SNP chip in genotyping of Ussuri raccoon dog.

[0060] Application of the above-mentioned SNP chip in the genetic diversity analysis of Ussuri raccoon dog.

[0061] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0062] The invention discloses a Ussuri raccoon dog low-density liquid-phase SNP chip, which is specific to the Ussuri raccoon dog and can be used to efficiently, quickly and at low cost identify the kinship of a Ussuri raccoon dog population, avoid inbreeding, guide the breeding work of the Ussuri raccoon dog and improve the economic value of the Ussuri raccoon dog. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0064] Figure 1 This is a statistical diagram of SNP types in Example 1 of the present invention;

[0065] Figure 2 This is a statistical diagram of SNP function annotation in the CDS region in Example 1 of the present invention;

[0066] Figure 3 This is a diagram showing the uniform distribution of SNP sites on autosomes of the Ussuri raccoon dog liquid phase chip in Example 1 of the present invention;

[0067] Figure 4 A sample cluster diagram drawn according to the PCA result in Example 1 of the present invention;

[0068] Figure 5 A cross-validation error rate line graph in Example 1 of the present invention;

[0069] Figure 6 The genetic composition bar graph of the sample in Example 1 of the present invention;

[0070] Figure 7 is the phylogenetic tree in Example 1 of the present invention;

[0071] Figure 8This is a heat map of kinship in Example 1 of the present invention. DETAILED DESCRIPTION

[0072] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0073] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0074] Example 1

[0075] 1 Materials and methods

[0076] 1.1 Experimental animals and samples

[0077] A total of 92 individuals were collected from three representative Ussuri raccoon dog breeding populations in China (Jiahe Raccoon Breeding Center in Changli, Hebei Province, Yuehe Raccoon Farm in Wangtan Town, Tangshan Haigang, Hebei Province, and Changbaishan Key Field Scientific Observation and Experimental Station of Wildlife Resources in Zuojia Agricultural Department, Jilin Province). Blood was collected from the heart when the skin was removed and stored in frozen form.

[0078] Table 2 Number of raccoon dog samples

[0079]

[0080] 1.2 Extraction of genomic DNA

[0081] A DNA extraction kit was used for extraction. NanoDrop2000 ultra-micro spectrophotometer and agarose gel electrophoresis were used to determine the concentration and quality of genomic DNA, respectively. The results showed that the OD260 / 280 of the extracted DNA was between 1.8 and 2.0, indicating that the DNA purity was good; the DNA agarose gel electrophoresis detected a single target band, and the DNA quality was qualified and could be used for subsequent cDNA library construction and sequencing.

[0082] 1.3 Construction of cDNA library

[0083] use The DNA Library Prep Kit constructs a resequencing library for DNA that has passed the quality inspection. The steps are as follows:

[0084] (1) Take 200 ng of DNA that has passed the quality inspection and place it in a 0.2 mL PCR tube. Add 4 uL EndRepair Buffer and 2.6uL End Repair Enzvme, add water to 20uL, put it into ABI 9700PCR instrument, incubate at 37℃ for 20min, denature at 72℃ for 20min, and complete the DNA fragmentation, end repair and A-tailing process.

[0085] (2) Add 2uL Ultra DNA Lioase, 8uL Ultra DNA LioaseBute, 4uL Adapiet, add water to 40uL, put it in ABI 9700PCR instrument at 20℃ for 60min to complete the connection of the primary linker. Then add 48uL of GenoPrep DNA Clean Beads to the connection product to purify the connection product. After purification, use 0.68+0.2 times magnetic beads to screen the fragments and retain the connection products with insert fragments of 300-350bp.

[0086] (3) Add 10uL to the PCR tube in the previous step PCR Master Mix was added and water was added to 20uL; amplification was performed using an ABI 9700 PCR instrument with the following program: pre-denaturation at 98°C for 2min, denaturation at 98°C for 30s, annealing at 65°C for 30s, extension at 72°C for 40s, 5 cycles, and extension at 72°C for 4min.

[0087] (4) Add 20uL GenoPrep DNA Clean Beads to the second-round PCR product, place on a magnetic rack until the solution is clear, discard the supernatant and add 100uL 80% ethanol to wash the magnetic beads, and add 35uL 10mM Tris:HCL to obtain a purified DNA library.

[0088] 1.4 Raw data preprocessing

[0089] Fastp software was used to perform quality control on the double-end sequencing file (rawdata (fastg format)) obtained from the sequencing machine to obtain effective reads (clean reads). The quality control parameters were: -n 10-q 20-u 40. The data processing steps were:

[0090] (1) Removal of adapter sequence;

[0091] (2) When the N content in the sequencing read exceeds 10 bases, the paired reads need to be removed;

[0092] (3) When the number of low-quality (Q<20) bases in a sequencing read exceeds 40% of the length of the read, the paired reads need to be removed.

[0093] After completing the quality control of the sequencing data, the clean reads obtained by quality control were aligned with the reference genome of raccoon dog using BWA MEM. The bam files obtained were sorted using samtools, and the default parameters of the sambamba software were used to remove duplicates, redundancies and create indexes to obtain the preliminarily processed bam files. Then, the SortSam and MarkDuplicates modules of the Picard software (http: / / broadinstitute.github.io / picard / ) were used to sort the alignment results and remove PCR duplicate sequences. Finally, the Qualimap software was used to perform statistical analysis on the aligned and sorted bam files.

[0094] 1.5 Whole-genome SNP detection and annotation of Ussuri raccoon dog

[0095] To improve the quality of SNP detection, the BaseRecalibration method of GATK software was used for realignment, and then HaplotypeCaller was used to generate GVCF for each sample. VariantRecaliration (site realignment) was performed on Ussuri raccoon dog and Jilin white raccoon dog, and the Variant Filteration tool was used for site quality filtering, and finally a collection of variant sites of all samples was obtained.

[0096] 1.6 Development of a 5K SNP breeding chip for the whole genome of Ussuri raccoon dog

[0097] The mining of Ussuri raccoon dog-specific SNPs was performed using the resequencing data of the above 92 samples. The basic filtering conditions for resequencing VCF file processing were: (1) minimum sequencing depth ≥ 5X; (2) missing rate < 10%; (3) maf > 0.1; (4) removal of non-biallelic SNP sites.

[0098] Site selection principles: (1) het_rate < 50%; (2) miss_rate < 10%; (3) maf ≥ 0.35; (4) uniform distribution; (5) in accordance with the site evaluation principles, the probe was successfully designed.

[0099] The density distribution of all detected SNP sites on the chromosomes of the whole genome of Ussuri raccoon dog was counted. Based on the above research, we further controlled the quality of the obtained SNP sites by indicators such as site detection rate, site loss rate, and minimum allele frequency, and removed the SNP sites that fell in the SV hotspot area to improve the accuracy of the sites. Next, we classified each variety according to different geographical locations, and in each group, for the SNP sites with more than 10 individual support numbers for the detected SNPs, and gradually filtered the remaining SNP sites, and finally screened out the suitable candidate sites for the 5K SNP chip after scoring and re-correction.

[0100] 1.7 Bloodline Analysis

[0101] 1.7.1 Principal Component Analysis

[0102] PCA analysis was performed using gcta (v1.92.4) software based on the filtered SNP markers (5K SNPs were finally screened out) to obtain the variance explanation of each PC and the score matrix of the sample in each PC. The key information extracted from the SNP information was divided into PC1, PC2, and PC3 according to the effect from large to small.

[0103] 1.7.2 Group structure analysis

[0104] Based on the filtered SNP markers, Admixture software was used to infer the population structure, assuming that the number of clusters (K value) of the samples was 1 to 15, and then clustering was performed. The optimal number of clusters was determined based on the cross-validation error rate, and the K value with the smallest cross-validation error rate corresponded to the optimal number of clusters.

[0105] In order to simulate the classification of the population and the pedigree composition of each sample under different subgroup numbers (subgroup number K = 1 to 15), the genetic composition of each sample in each subgroup was plotted into a bar graph using pophelper software, and each color represented a Cluster within each K value.

[0106] 1.7.3 Phylogenetic tree construction

[0107] The neighbor-joining method was used to construct the phylogenetic tree. Based on the SNP markers obtained after screening, the NJ tree was constructed using MEGA-X software (model: p-distance; bootstrap: 1000 times).

[0108] 1.7.4 Kinship Analysis

[0109] Kinship is defined as the relative value of the genetic similarity between two specific materials and the genetic similarity between any materials. GCTA software was used to perform kinship analysis and obtain the kinship matrix between two samples.

[0110] 2 Results and analysis

[0111] 2.1 DNA quality detection and sequencing quality distribution

[0112] After the blood DNA was extracted, the DNA quality was tested immediately. The results of the NanoDrop 2000 nucleic acid protein concentration meter showed that OD260 / 280 was between 1.8 and 2.0, indicating that the DNA purity was good. The DNA integrity was tested using the Agilent 5400 fragment analyzer system. The results showed that the DNA was intact, without degradation or contamination, and the sample quality met the quality requirements for library construction and sequencing.

[0113] 2.2 Data quality control and SNP annotation analysis

[0114] In this study, 77 Ussuri raccoon dogs and 15 Jilin white raccoon dogs were subjected to whole genome resequencing with a sequencing depth of 10×. The two raccoon dog populations generated a total of 2101.33G and 403.01G of raw data, respectively. After quality control filtering, clean data of 2097.08G and 402.14G were obtained, respectively. The average raw data of each sample of Ussuri raccoon dog and Jilin white raccoon dog was 27.29G and 26.87G respectively. The Clean Data Q20 and Q30 of Ussuri raccoon dog were 97.03% and 92.10%, respectively, and the average GC content was 42.31%. The Clean Data Q20 and Q30 of Jilin white raccoon dog were 96.78% and 91.55%, respectively, and the average GC content was 42.49% (Table 3). Clean Data was compared with the reference genome, with an average comparison ratio of 99.54%, an average coverage depth of 10.3×, an average coverage of 98.71%, and an average genome coverage of sequencing data above 5× of 84.16%. The sample sequencing depth and coverage distribution were highly consistent and could be used for subsequent analysis.

[0115] Table 3 Summary of sequencing data

[0116]

[0117] By conducting whole genome resequencing, a total of 16,864,452 SNP variant sites were detected in 92 samples of Wusuli raccoon dogs and Jilin white raccoon dogs, of which 10,638,625 were transition types (C / T and G / A), 6,225,827 were transversion types (A / C, A / T, C / G and G / T), and the transition-transversion ratio was 170.88% ( Figure 1 ).

[0118] The SNPs of Ussuri raccoon were annotated by ANNOVAR software, and it was found that there were 11,831,118 SNP variations in the intergenic region; 229,180 SNP variations in the 2kb region upstream of the gene; 204,494 SNP variations in the 2kb region downstream of the gene; 4,461,858 SNP variations in the intron region; 844 SNP variations in the splicing site region; and 129,874 SNP variations in the CDS region. The detailed information of SNP annotation of the Ussuri raccoon population is shown in Table 4.

[0119] Table 4 SNP position annotation structure statistics

[0120]

[0121] The statistical results of SNP functional annotation in CDS region are as follows Figure 2 As shown, there are 66,806 synonymous variants, 61,606 non-synonymous variants, 1,410 premature protein terminations, 37 stop codon variants, and 16 unknown variants.

[0122] 2.3 Distribution characteristics of 5K SNP chip loci in the whole genome of Ussuri raccoon dog

[0123] like Figure 3 As shown, the results show that SNP sites are widely distributed and evenly distributed in the whole genome of Ussuri raccoon dog. We annotated all SNP chip sites, most of which fell in the functional element region, and screened out 4580 SNP sites that can be used for chip design (Table 1). Finally, the corresponding probes were designed according to these sites to make breeding chips. The probe length was 110bp and the probe GC content was 30-70%.

[0124] 3.1 Kinship

[0125] 3.1.1 PCA analysis

[0126] The sample clustering diagram drawn based on the PCA results is as follows Figure 4 As shown in the figure, it can be seen that the two principal components PC1 and PC2 of Ussuri raccoon dog and Jilin white raccoon dog are obviously clustered into one cluster, and some individuals are outliers.

[0127] 3.1.2 Group structure analysis

[0128] The cross-validation error rate line chart is as follows Figure 5 As shown, the optimal number of clusters determined in this analysis is K=1.

[0129] The genetic composition of the samples is shown in the histogram Figure 6 As shown in Figure 2, the optimal number of clusters K = 1, all samples are classified as one genetic component, and no genetic differences between samples are detected.

[0130] 3.1.3 Phylogenetic tree construction

[0131] The phylogenetic tree results are as follows Figure 7 As shown, the Ussuri raccoon dog and the Jilin white raccoon dog did not show obvious clustering results, indicating that their genetic backgrounds were similar.

[0132] 3.1.4 Kinship Analysis

[0133] Kinship heat map Figure 8 As shown, there is a significant genetic similarity between the Ussuri raccoon dog and the Jilin white raccoon dog, indicating that the Ussuri raccoon dog and the Jilin white raccoon dog may originate from a similar ancestral lineage.

[0134] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0135] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A low-density liquid phase SNP chip of Ussuri raccoon dog, characterized in that: Including 4580 SNP sites; the physical positions of the 4580 SNP sites are determined based on the positive chain bases of the raccoon dog reference genome CNA0046198; the information of the 4580 SNP sites is as follows:

2. Use of the SNP chip according to claim 1 in breeding of Ussuri raccoon dogs.

3. Use of the SNP chip according to claim 1 in kinship analysis of Ussuri raccoon dogs.

4. Use of the SNP chip according to claim 1 in genotyping of Ussuri raccoon dog.

5. Use of the SNP chip according to claim 1 in the analysis of genetic diversity of Ussuri raccoon dog.

Images