Probe combination of racoon ussuriensis dog breeding chip, detection system and application

Abstract

The invention provides a probe combination of a racoon ussuriensis dog breeding chip, a detection system and application. Belongs to the technical field of molecular biology and genomics breeding. On the basis of 92 raccoon ussuriensis dog sample re-sequencing results, 40128 SNP loci are screened out, a molecular probe combination and an SNP gene chip are constructed, the molecular probe combination and the SNP gene chip are used for carrying out raccoon ussuriensis dog genome selective breeding, the purposes of early seed selection, breeding period shortening, breeding progress accelerating and raccoon dog production performance improving are achieved, and the application prospect is wide. And technical support is provided for breeding new varieties of large-scale high-quality racoon ussuriensis dogs.

Description

Technical Field

[0001] The invention relates to the technical fields of molecular biology and genomics breeding, and more specifically to a probe combination, a detection system and an application of a Ussuri raccoon dog breeding chip. Background Art

[0002] The Ussuri raccoon dog is native to China, which is the world's largest raccoon dog farmer. In 2020, the Ministry of Agriculture and Rural Affairs clearly listed raccoon dogs as special livestock and poultry in the "National Livestock and Poultry Genetic Resources Catalogue" for the first time, greatly promoting the development of my country's raccoon dog farming industry. Raccoon dog skin is a kind of large hairy skin, known for its lightness and warmth. In recent years, the popularity of parkas with raccoon dog skin as the lining has led to a sharp increase in the market demand for raccoon dog skin, which has greatly promoted the development of my country's modern agricultural economy.

[0003] my country's Ussuri raccoon breeding started late, and currently the selection of high-quality raccoon breeds is mainly based on phenotypes, which has the following problems: ① The breeding cycle is long and the cost is high; ② The phenotype is the result of the interaction between the genotype and the environment. Different phenotypes may be caused by different genotypes or environments, and the selection accuracy is not high; ③ The phenotypic information of traits with medium heritability (body length, fur color and fur quality) and low heritability (fertility) contains limited genetic information, making it difficult to make accurate genetic assessments of individuals. In order to improve the selection intensity and the accuracy of genetic assessments, it is necessary to develop Ussuri raccoon breeding chips and carry out Ussuri raccoon genomic selection breeding.

[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.

[0005] At present, cattle 90K chip, sheep Illumina 50K chip, goat Illumina 50K chip, pig high-density chip, chicken 600KAffymetrix high-density chip have been widely used in large-scale commercial genomic selection breeding. Among fur animals, genomic selection breeding has also been carried out for water raccoon dogs. However, the development of genomic selection breeding for Ussuri raccoon dogs has been slow, especially the related research on Ussuri raccoon dog SNP breeding chips, which are rarely recorded in existing technologies.

[0006] Therefore, how to develop a liquid phase molecular breeding chip for Ussuri raccoon dog genotyping suitable for fur quality and body size screening is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0007] In view of this, the present invention provides a probe combination, detection system and application of a Ussuri raccoon dog breeding chip, adopts a raccoon dog genome with a genome size of 2.39 Gb and scaffoldN5054 Mb (genome accession number: CNA0046198) as the raccoon dog reference genome of this study, and based on the resequencing results of 92 Ussuri raccoon dog samples, screens out 40128 SNP sites, constructs a molecular probe combination and a SNP liquid phase gene chip, which are used for carrying out Ussuri raccoon dog genome selection breeding, helps to achieve early selection, shorten the breeding cycle, speed up the breeding process, and improve the production performance of raccoon dogs, and provides technical support for the breeding of large-scale and high-quality new Ussuri raccoon dog varieties.

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

[0009] The application of 40128 SNP loci combination in the identification and breeding of fur quality and body size of Ussuri raccoon dog, the loci physical information of the 40128 SNP loci combination is shown in Table 6.

[0010] Preferably, the 40128 SNP site combinations are obtained by the following method:

[0011] (1) Based on the resequencing results of 92 Ussuri raccoon dog samples, a collection of variant sites of all samples was screened;

[0012] (2) Basic filtering conditions for SNP sites: ① minimum sequencing depth ≥ 5X; ② heterozygosity rate < 0.5, ③ deletion rate < 10%; ④ maf > 0.1; ⑤ removal of non-binary SNP sites;

[0013] (3) SNP site selection: ① het_rate < 50%; ② miss_rate < 10%; ③ maf ≥ 0.35; ④ uniform distribution; ⑤ conform to the site evaluation principles;

[0014] (4) Before the first round of testing: sort and select background sites based on the principles of high MAF, low deletion rate, and low heterozygosity rate for the first round of testing;

[0015] (5) After the second round of testing: Based on the results of the second round of testing, sites with high capture stability and site detection rate are selected as the final site combination.

[0016] Another object of the present invention is to provide: a molecular probe combination for identifying the fur quality and body size of Ussuri raccoon dog, wherein the molecular probe combination is designed according to the SNP site combination shown in Table 6.

[0017] Preferably, the design principles of the molecular probe combination are as follows: probe length is 110 bp, probe GC content is between 30-70%, and the number of homology regions is <5.

[0018] Another object of the present invention is to provide: a gene chip for identifying the quality of the fur and the size of the Ussuri raccoon dog, wherein the gene chip is loaded with the above-mentioned molecular probe combination.

[0019] Another object of the present invention is to provide: a detection system for identifying the quality and body size of the fur of Ussuri raccoon dogs, wherein the detection system comprises the above-mentioned molecular probe combination or the above-mentioned gene chip.

[0020] Another object of the present invention is to provide: a method for identifying the quality and size of the fur of Ussuri raccoon dog, using the above molecular probe combination or the above gene chip or the above detection system to detect the Ussuri raccoon dog sample.

[0021] Another object of the present invention is to provide: an application of the above molecular probe combination or the above gene chip or the above detection system, wherein the application is any one of the following:

[0022] A: Evaluation of the quality and size of the Ussuri raccoon dog's fur;

[0023] B: Screening of Ussuri raccoon dog varieties;

[0024] C: Genotyping of Ussuri raccoon dog;

[0025] D: Identification of species of Ussuri raccoon dog;

[0026] E: The origin of the Ussuri raccoon dog species;

[0027] F: Genetic diversity analysis of Ussuri raccoon dog;

[0028] G: Functional genomics study of Ussuri raccoon dog;

[0029] H: Construction of molecular genetic map of Ussuri raccoon dog;

[0030] I: Whole genome selection breeding of Ussuri raccoon dog.

[0031] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) Based on the resequencing results of 92 samples of Ussuri raccoon dogs, the present invention screened out 40,128 SNP sites, constructed a molecular probe combination and a SNP gene chip, which were used to carry out genome selection breeding of Ussuri raccoon dogs, which helped to achieve the goal of early selection, shorten the breeding cycle, speed up the breeding process, and improve the production performance of raccoon dogs, and provided technical support for the breeding of new varieties of large Ussuri raccoon dogs with excellent fur quality;

[0033] (2) The Ussuri raccoon dog whole genome 40K SNP breeding chip of the present invention performs well in uniformity and detection rate, and can meet the detection needs of a large number of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 For: SNP type statistics;

[0036] Figure 2 For: SNP functional annotation statistics in CDS region;

[0037] Figure 3 Figure 2: Uniform distribution of 40,128 SNP sites on chromosomes after two rounds of testing. DETAILED DESCRIPTION

[0038] The technical solution of the present invention will be clearly and completely described below in conjunction with 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 making creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] Screening of SNP sites

[0041] (1) Selection of experimental materials: 92 individuals with large body size and good wool quality were collected from three representative Ussuri raccoon dog breeding bases in China (Jiahe Raccoon Breeding Center in Changli, Hebei Province, Yuehe Raccoon Farm in Wangtan Town, Tangshan, Hebei Province, and Changbaishan Wildlife Resources Key Field Scientific Observation and Experimental Station of Zuojia Agriculture and Rural Affairs, Jilin Province) (Table 1). Blood was collected from the heart when the skin was taken and stored in frozen state.

[0042] Table 1 Number of raccoon dog samples

[0043]

[0044] (2) Extraction of genomic DNA

[0045] The DNA extraction kit was used to extract the sample DNA; the 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. The Agilent5400 fragment analyzer system was used to test the DNA integrity, and 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;

[0046] (3) Construction of cDNA library

[0047] use The DNA LibraryPrep Kit is used to construct a resequencing library for DNA that has passed the quality inspection. The steps are as follows:

[0048] ① Take 200ng of DNA that has passed the quality inspection and place it in a 0.2mL PCR tube. Add 4uL EndRepair Buffer and 2.6uL End Repar Enzvme, add water to 20uL, put it into ABL 9700PCR instrument, incubate at 37℃ for 20min, denature at 72℃ for 20min, complete DNA fragmentation, end repair and A-tailing process;

[0049] ②Add 2uL Ultra DNA Lioase, 8uL Ultra DNA Lioase Bute, 4uL GenoBaits Adapiet, add water to 40uL, put it in ABI 9700PCR at 20℃ for 60min to complete the connection of the primary linker. Then add 48uL GenoPrep DNA Cean 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 product with an insert fragment of 300-350bp;

[0050] ③ Add 10uL of PCR Master Mix and add water to 20uL; ABI 9700 PCR instrument was used for amplification, and the amplification program was as follows: 98℃ pre-denaturation for 2min, 98℃ denaturation for 30s, 65℃ annealing for 30s, 72℃ extension for 40s, 5 cycles, and 72℃ extension for 4min;

[0051] ④ Add 20uL GenoPrep DNA Clean Beads to the second round of PCR products, place on a magnetic stand until the solution is clear, discard the supernatant and add 100uL 80% ethanol to wash the beads, and add 35uL 10 mM Tris:HCL to obtain the purified cDNA library;

[0052] (4) Raw data preprocessing

[0053] 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: -n10-q20-u40; the data processing steps were:

[0054] ①Remove the adapter sequence;

[0055] ② When the N content in the sequencing read exceeds 10 bases, the paired reads need to be removed;

[0056] ③ When the number of low-quality (Q<20) bases in the sequencing read exceeds 40% of the read length, the paired reads need to be removed;

[0057] ④ After completing the quality control of the sequencing data, use BWAMEM to align the clean reads obtained by quality control with the reference genome of raccoon dog. Use samtools to sort the obtained bam files, use the default parameters of sambamba software to remove duplicates, remove redundancy and create indexes, so as to obtain the preliminarily processed bam files; then use the SortSam and MarkDuplicates modules of Picard software (http: / / broadinstitute.github.io / picard / ) to sort the alignment results and remove PCR duplicate sequences, and finally use Qualimap software to perform statistical analysis on the aligned and sorted bam files;

[0058] (5) Whole-genome SNP detection and annotation of Ussuri raccoon dog

[0059] 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 VariantFilteration tool was used for site quality filtering, finally obtaining a collection of variant sites of all SNP samples.

[0060] Result analysis:

[0061] The present invention performs whole genome resequencing with a sequencing depth of 10× on 77 Wusuli raccoon dogs and 15 Jilin white raccoon dogs. The two raccoon dog populations respectively generate 2101.33G and 403.01G of raw data, and obtain 2097.08G and 402.14G of clean data after quality control filtering; each sample of Wusuli raccoon dog and Jilin white raccoon dog measures 27.29G and 26.87G of raw data on average; Wusuli raccoon dog CleanData Q20 and Q30 are 97.03% and 92.10% respectively, and the average GC content is 42.31%; Jilin white raccoon dog CleanData Q20 and Q30 are 96.78% and 91.55% respectively, and the average GC content is 42.49% (Table 2).

[0062] 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.

[0063] Table 2 Summary of sequencing data

[0064]

[0065] By conducting whole genome resequencing, a total of 16,864,452 SNP variant sites were detected in 92 samples of Ussuri 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 166.67% ( Figure 1 ).

[0066] The Ussuri raccoon SNPs were annotated using ANNOVAR software, and it was found that there were 11,831,118 SNP variants located in the intergenic region; 229,180 SNP variants in the 2kb region upstream of the gene; 204,494 SNP variants in the 2kb region downstream of the gene; 4,461,858 SNP variants in the exon region; 7,083 SNP variants located within 2Kbp upstream of the gene and within 2Kbp downstream of the gene; 844 SNP variants in the splice site region; 1 SNP variant located in the CDS region and at the same time located in the splice site (2bp near the exon / intron boundary in the intron); and 129,874 SNP variants in the CDS region. The detailed information of the Ussuri raccoon population SNP annotation is shown in Table 3; the statistical diagram of the SNP functional annotation in the CDS region is shown in Figure 2 shown.

[0067] Table 3 SNP position annotation structure statistics

[0068]

[0069] Example 2

[0070] Development of a 40K SNP breeding chip for the whole genome of Ussuri raccoon dog

[0071] 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 using indicators such as site detection rate, site loss rate, and minimum allele frequency, and removed SNP sites that fell in the SV hotspot area to improve the accuracy of the sites. Next, we classified each breed 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. After scoring and re-correction, we finally screened out the suitable candidate sites for the 40KSNP chip. The specific process is as follows:

[0072] (1) The basic filtering conditions for resequencing VCF file processing are as follows: ① minimum sequencing depth ≥ 5X; ② missing rate < 10%; ③ maf > 0.1; ④ removal of non-biallelic SNP sites.

[0073] The further selection principles of SNP sites were as follows: ① het_rate < 50%; ② miss_rate < 10%; ③ maf ≥ 0.35; ④ uniform distribution; ⑤ compliance with site evaluation principles. Through the screening of steps (1) and (2), 45,425 SNP sites were successfully screened out.

[0074] (2) Based on the sites screened in step (1) and step (2), two rounds of testing were conducted to further screen suitable candidate sites for the 40K SNP chip: The test materials for the two rounds of testing were 29 blood samples of Ussuri raccoon dogs and Jilin white raccoon dogs collected in Changli in December 2023 and mailed to Reddy Company:

[0075] (3) Before the first round of testing: The 45,425 SNP sites screened out previously were sorted according to the principles of high maf, low deletion rate, and low heterozygosity rate to screen out a total of 40,128 background sites. The 40,128 sites screened out were used to design a chip, namely the 40K liquid phase chip, and a round of testing was performed. The results are shown in Table 4.

[0076] (4) After the second round of testing: Based on the capture stability and site detection rate of the second round of testing, the sites were selected as the final site set. The results are shown in Table 5.

[0077] Through the screening of the above steps (3)-(4), 40128 loci were finally screened out, and the locus information is shown in Table 6; the chromosome distribution map of the 40128 SNP loci finally screened out is shown in Table 6. Figure 3 As shown, the results showed that the SNP loci were widely and evenly distributed throughout the whole genome of Ussuri raccoon dog.

[0078] Table 4 SNP site capture statistics of the first round of test samples

[0079]

[0080] Table 5 SNP site capture statistics of the second round of test samples

[0081]

[0082]

[0083] In one round, 12 samples were tested, all of which were Ussuri raccoon dogs. The average number of detected sites in each sample was 38,047, with a detection rate between 94.498% and 95.126%, and an average detection rate of 94.815%. The total number of detected SNP sites was 40,128 (Table 4).

[0084] In the second round, 17 samples were tested, including 3 Ussuri raccoon dogs and 14 Jilin white raccoon dogs. The average number of detected loci per sample was 37,964, with a detection rate between 94.348% and 94.739%, and an average detection rate of 94.609% (Table 5). The above detection indicators and statistical information of the 17 samples also proved that the Ussuri raccoon dog whole genome 40K SNP breeding chip we developed performed well in uniformity, detection rate and typing consistency, and can meet the detection needs of a large number of samples.

[0085] Table 6 Physical information of 40128 SNP loci combinations

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] Note: Taking the data in the first cell in Table 6 as an example, Chr1-63164A>G means that the SNP site is located on the Chr1 chromosome, with a physical position of 63164, an A before mutation and a G after mutation.

[0206] The various embodiments in the 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.

[0207] 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. The application of 40128 SNP loci combination in the identification and breeding of Ussuri raccoon dog fur quality and body size, characterized in that: The site physical information of the 40128 SNP site combinations is shown in Table 6.

2. The use of the 40128 SNP loci combination according to claim 1 in the identification and breeding of the fur quality and body size of Ussuri raccoon dog, characterized in that: The 40128 SNP loci combination was obtained by the following method: (1) Based on the resequencing results of 92 Ussuri raccoon dog samples, a collection of variant sites in all samples was screened; (2) Basic filtering conditions for SNP sites: ① minimum sequencing depth ≥ 5X; ② heterozygosity rate < 0.5, ③ deletion rate < 10%; ④ maf > 0.1; ⑤ removal of non-binary SNP sites; (3) SNP site selection: ① het_rate < 50%; ② miss_rate < 10%; ③ maf ≥ 0.35; ④ uniform distribution; ⑤ conform to the site evaluation principles; (4) Before the first round of testing: sort and select background sites based on the principles of high MAF, low deletion rate, and low heterozygosity rate for the first round of testing; (5) After the second round of testing: Based on the results of the second round of testing, sites with high capture stability and site detection rate are selected as the final site combination.

3. A molecular probe combination for identifying the quality and size of the fur of Ussuri raccoon dogs, characterized in that: The molecular probe combination is designed according to the SNP site combination shown in Table 6 of claim 1.

4. A molecular probe combination for identifying the fur quality and body size of Ussuri raccoon dog according to claim 3, characterized in that: The design principles of the molecular probe combination are as follows: probe length is 110 bp, probe GC content is between 30-70%, and the number of homology regions is <5.

5. A gene chip for identifying the quality and size of the fur of Ussuri raccoon dogs, characterized in that: The gene chip is loaded with the molecular probe combination according to claim 3 or 4.

6. A detection system for identifying the quality and size of Ussuri raccoon dog fur, characterized in that: The detection system comprises the molecular probe combination according to claim 3 or 4 or the gene chip according to claim 5.

7. A method for identifying the quality and size of Ussuri raccoon dog fur, characterized in that: The Ussuri raccoon dog sample is detected by using the molecular probe combination described in claim 3 or 4, the gene chip described in claim 5, or the detection system described in claim 6.

8. Use of the molecular probe combination according to claim 3 or 4, the gene chip according to claim 5, or the detection system according to claim 6, characterized in that: The application is any of the following: A: Evaluation of the quality and size of the Ussuri raccoon dog's fur; B: Screening of Ussuri raccoon dog varieties; C: Genotyping of Ussuri raccoon dog; D: Identification of species of Ussuri raccoon dog; E: The origin of the Ussuri raccoon dog species; F: Genetic diversity analysis of Ussuri raccoon dog; G: Functional genomics study of Ussuri raccoon dog; H: Construction of molecular genetic map of Ussuri raccoon dog; I: Whole genome selection breeding of Ussuri raccoon dog.

Images