Silkworm SNP (Single Nucleotide Polymorphism) molecular marker combination, chip and application thereof

Abstract

The invention discloses a bombyx mori SNP molecular marker combination, a chip and application of the bombyx mori SNP molecular marker combination. Comprising 5,381 SNP molecular markers, and the physical positions of the 5,381 SNP molecular markers are determined by sequence alignment based on a bombyx mori reference genome GCA030269925.2. The SNP loci in the silkworm SNP molecular marker combination are screened based on 120 collected varieties of Henan province silkworms with rich diversity, are strong in representativeness, high in polymorphism, good in universality, high in coverage rate on genome and uniform in distribution, can effectively distinguish different silkworm varieties, and are more suitable for local varieties of Henan province silkworms.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a combination of SNP molecular markers for silkworms, a chip, and their applications. Background Technology

[0002] my country holds a leading position globally in silkworm genetic resources, possessing an extremely rich and diverse array of silkworm varieties. These include numerous unique national-level local varieties, high-quality cultivated varieties bred through long-term selection, and introduced specialty varieties. Furthermore, China has accumulated extensive and diverse resources in breeding material design. These resources have preserved the genetic diversity of my country's silkworm resources to the greatest extent possible, serving as the primary source of parents for silkworm genetic breeding and the most important material foundation for biological research fields such as genetic engineering and cell engineering.

[0003] Henan is a province that emphasizes both mulberry and tussah silkworm production. It is not only a suitable habitat for sericulture but also its birthplace, making it a traditional and advantageous industry with significant historical and strategic importance. Located in the transitional climate zone between North and South China, Henan possesses uniquely favorable conditions for developing sericulture. Vigorously developing silkworm breed improvement is a crucial step in raising the level of Henan's sericulture industry.

[0004] Precise genotyping is a crucial component of precise identification of animal germplasm resources. Conventional genotyping methods include molecular markers, SNP microarrays, and genome sequencing. However, the number of molecular markers available for PCR amplification is limited, and the cost increases with the number of markers detected. For species with large genomes, genome sequencing is also expensive and complex to analyze. Liquid-phase microarray technology based on single nucleotide polymorphisms (SNPs), as an innovative method enabling efficient, rapid, and large-scale SNP genotyping, has demonstrated great potential in genetic research for livestock and poultry breeding. Compared to traditional solid-phase microarray technology, liquid-phase microarrays are not only simpler to manufacture but also significantly improve cost control and detection accuracy. In particular, liquid-phase microarray technologies based on multiplex amplification capture sequencing and probe hybridization capture sequencing, with their advantages of high throughput, high accuracy, and low cost, have become indispensable tools for biological germplasm resource assessment and breeding research. Based on the characteristics of high labeling density, high automation, and high detection throughput of liquid phase chips, the integration and application of candidate sites that affect important economic traits of silkworms and their adaptation to the local climate and environment of Henan can accelerate the breeding of superior silkworm varieties and increase the yield of high-quality cocoons and silk. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a combination of SNP molecular markers for silkworms.

[0006] The present invention also proposes a primer set and / or probe for detecting the above-mentioned SNP molecular marker combination of silkworm.

[0007] The present invention also proposes a chip.

[0008] The present invention also proposes a reagent kit.

[0009] This invention also proposes a method for screening the above-mentioned combinations of SNP molecular markers in silkworms.

[0010] This invention also proposes an application of the above-mentioned silkworm SNP molecular marker combination, primer set and / or probe, chip or kit.

[0011] This invention also proposes a method for breeding silkworms.

[0012] According to one aspect of the present invention, a combination of SNP molecular markers for silkworms is proposed, comprising 5,381 SNP molecular markers. The physical locations of the 5,381 SNP molecular markers are determined by sequence alignment based on the silkworm reference genome GCA_030269925.2, and the specific site information is shown in Table 1 below.

[0013] Table 1

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[0031] In some embodiments of the present invention, the genotype information of the SNP molecular marker is represented in the form of "ref / alt (reference base / variant base)".

[0032] In some embodiments of the present invention, the chromosome in the SNP molecular marker location information is specifically “NC_” + the corresponding chr information in the table.

[0033] In a second aspect of the invention, primer sets and / or probes for detecting the above-mentioned combinations of SNP molecular markers in silkworms are proposed.

[0034] In some embodiments of the present invention, the probe has a length of 80-120 bp.

[0035] In some embodiments of the present invention, the probe is approximately 100 bp in length.

[0036] In some embodiments of the present invention, the GC content of the probe is between 20% and 80%.

[0037] In some embodiments of the present invention, the number of homologous regions of the probe is less than 5.

[0038] In some embodiments of the present invention, the number of SNP variant sites on the probe is ≤5.

[0039] In some embodiments of the present invention, the probe has no SSR region and N region.

[0040] In a third aspect of the invention, a chip is provided that includes the aforementioned primer set and / or probes.

[0041] In a fourth aspect of the invention, a kit is provided comprising the primer set and / or probes described above.

[0042] In a fifth aspect of the present invention, a method for screening the above-mentioned combinations of silkworm SNP molecular markers is proposed, the method comprising the following steps: A1) After comparing and detecting variants in the whole genome sequencing data of the silkworm samples using Sentieon software, preliminary hard filtering was performed to obtain a file containing SNP variant information of all samples. A2) The target SNPs are obtained by mining and screening the file containing SNP variant information of all samples; the mining and screening parameters are: Maf≥0.05, detection rate≥90%, heterozygosity≤20%, sequencing depth≥8×; A3) For the target site SNPs obtained in step A2), design probes and screen to obtain SNPs that meet the requirements of the probes; A4) Filter the file containing SNP variation information of all samples with a threshold of FST>0.8 and design probes to obtain variety-specific SNPs; A5) Perform GWAS analysis on the file containing SNP variation information of all samples to obtain associated sites; design probes for the associated sites, screen SNPs that meet the requirements of the probes, and obtain SNPs related to cocoon weight and total cocoon weight. A6) Integrating the probes obtained from A3) to obtain the required SNPs, the variety-specific SNPs obtained from A4) and the SNPs related to the cocoon layer weight trait and the total cocoon weight trait obtained from A5) yields the SNP molecular marker combination for silkworms.

[0043] In some embodiments of the present invention, the comparison and variation detection of whole-genome sequencing data of silkworm samples using Sentieon software specifically includes the following steps: B1) The whole genome sequencing data of the silkworm samples were aligned to the silkworm reference genome GCA_030269925.2 using Sentieon software, and the positions were sorted and duplicate reads were marked. B2) Use Sentieon software to detect variant sites for each sample and obtain the gVCF for each sample; B3) Use Sentieon to perform joint-calling and perform joint analysis of gVCF for all samples to obtain the variation results for each individual in the population.

[0044] In some embodiments of the present invention, the hard filtering criteria are as follows: QD<2.0 || FS>60.0 || MQ<40.0 || SOR>3.0 || MQRankSum<-12.5 || ReadPosRankSum<-8.0.

[0045] In a sixth aspect of the invention, the application of the above-described silkworm SNP molecular marker combinations, primer sets and / or probes, chips, or kits in any of the following: C1) Silkworm genotyping detection; C2) Identification of silkworm germplasm resources; C3) Assessment of genetic diversity in silkworms; C4) Construction of the genetic map of silkworm and QTL mapping; C5) Genome-wide association study of silkworms; C6) Molecular marker-assisted selection in silkworms; C7) Whole-genome selection breeding or intelligent design breeding of silkworms; C8) Cluster analysis and phylogenetic analysis of silkworms; C9) Gene identification and functional analysis of important economic traits in silkworms; C10) Analysis of silkworm population structure; C11) Germplasm resource exploration and utilization.

[0046] In some embodiments of the present invention, the application can be implemented through the following methods: S1. Genotyping of the samples to be tested is performed using at least one of the following: silkworm SNP molecular marker combination, primer set and / or probe, chip and kit, to obtain genotyping results; S2. Analyze the genotyping results obtained in step S1.

[0047] In a seventh aspect of the present invention, a method for breeding silkworms is proposed, comprising the following steps: detecting the DNA of the silkworm to be tested using one of the above-mentioned silkworm SNP molecular marker combination, primer set and / or probe, chip and kit, and selecting silkworms for subsequent breeding.

[0048] In some embodiments of the present invention, the breeding includes conducting relevant breeding by distinguishing different silkworm varieties in Henan Province.

[0049] In some embodiments of the present invention, the detection is performed based on liquid-phase probe capture sequencing genotyping technology.

[0050] The present invention has at least the following beneficial effects: The SNP sites in the SNP molecular marker combination of the present invention are selected from 120 diverse silkworm varieties from Henan Province. They are highly representative, highly polymorphic (mean MAF 0.34), have good universality, high genome coverage (average coverage 99%), and are evenly distributed. They can effectively distinguish different silkworm varieties and are more suitable for local silkworm varieties in Henan Province.

[0051] The SNP molecular marker combination of the present invention was used to prepare a whole-genome SNP liquid-phase breeding chip for local silkworm varieties in Henan Province. This can effectively reduce the cost of applications such as genetic diversity analysis, QTL mapping, and GWAS analysis in silkworm research, accelerate the process of basic silkworm research and breeding, achieve accurate genotyping of large-scale populations, and standardize the breeding process. In addition, the liquid-phase chip is flexible in site design. Through continuous optimization and adjustment of existing products, sites can be added or removed based on existing chips, thereby changing the chip density to meet the specific needs of different research or application scenarios. Attached Figure Description

[0052] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The silkworm 5K in this embodiment of the invention c GPS liquid-phase chip site chromosome density distribution map; Figure 2 As described in the embodiments of the present invention c Schematic diagram of GPS liquid phase chip process testing; Figure 3 This is a cluster analysis diagram of silkworms in the test examples of this invention. Detailed Implementation

[0053] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0054] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0055] Example 1 This embodiment provides a silkworm SNP molecular marker combinatorial system. The combinatorial system comprises 5,381 SNP loci, the physical locations of which were determined based on sequence alignment from the silkworm reference genome (GCA_030269925.2). Specific information about these 5,381 SNP loci is shown in Table 1 of the specification. The screening process is as follows: 1. Collection of silkworm resequencing samples To obtain genetically rich whole-genome loci, 120 samples from 12 varieties in Henan Province were collected for whole-genome resequencing. Specific sample information is shown in Table 2 below.

[0056] Table 2 Sample Variety Information

[0057] 2. Whole-genome resequencing and resequencing data analysis of silkworms Whole-genome resequencing was performed on 120 collected silkworm samples. The specific steps included: (1) DNA was extracted using the magnetic bead method. (2) The MGI library standard method was used to construct DNA-seq sequencing libraries that passed quality control. (3) After the libraries passed quality control, they were sequenced using the BGI sequencing platform with the PE150 sequencing strategy. Sentieon was used to align and detect variants in 120 resequencing data. The analysis process was as follows: 1) Sentieon was used to align reads to the corresponding silkworm reference genome, orient them, and mark duplicate reads. 2) Sentieon was used to detect variant sites in each sample to obtain the gVCF of each sample. 3) Sentieon was used for joint-calling to perform joint analysis of the gVCF of all samples to obtain the variant results of each individual in the population. To ensure SNP accuracy, a preliminary hard filter was performed on the SNP sites obtained after joint analysis (SNP hard filter criteria: "QD<2.0 || FS>60.0 || MQ<40.0 || SOR>3.0 || MQRankSum<-12.5 || ReadPosRankSum<-8.0"). After filtering, a .vcf file containing SNP variation information for all samples was obtained, totaling 18,888,555 SNP sites.

[0058] 3. Site screening (1) Whole genome loci The MAF value, detection rate, and heterozygosity rate of the loci were calculated and statistically analyzed from the VCF file containing SNP variant information of all samples. Target loci were selected based on MAF ≥ 0.05, SNP detection rate ≥ 90%, heterozygosity rate ≤ 20%, and sequencing depth ≥ 8×. Probe design was then performed on the selected target loci set. The probe design principles were: a. Design probes within 100 bp to the left and right of each target locus, with a probe length generally of 100 bp; b. Calculate the GC content of the target region; c. Calculate the number of homologous regions of the target probe. The probe selection criteria were: a. Probe GC content between 20% and 80%; b. Number of homologous regions < 5; c. SNP variant sites on the probe ≤ 5; d. No SSR or N regions on the probe; e. After selecting the probe sequences that finally met the requirements, 4,691 SNP loci were selected as whole-genome loci based on the principle of uniform distribution on the reference genome.

[0059] (2) Variety-specific loci The interpopulation genetic differentiation index (FST) is a measure of population differentiation and genetic distance; a higher FST indicates greater diversity. FST ranges from 0 to 1, with values ​​closer to 1 indicating greater differentiation and selection between populations, and values ​​closer to 0 indicating less differentiation and selection. For all samples, SNP variation information from the VCF file was analyzed, totaling 18,888,555 SNP loci (single-point calculation) using the parameter `--weir-fst-pop`. Finally, filtering and probe design were performed based on an FST threshold of >0.8, resulting in 668 variety-specific loci.

[0060] (3) GWAS (Genome-Wide Association Studies) loci GWAS analysis was performed using a linear mixture model (LMM) in GEMMA software. To reduce the influence of population stratification on GWAS, the first three principal components were used as covariates. The specific statistical model for GWAS is as follows: y = SNP + COV + Kin + e, where y is the phenotypic value, Kin is the kinship matrix, SNP is the SNP marker effect, COV is the covariate, and e is the residual effect. The significance threshold was calculated using the Bonferroni correction method, ultimately yielding 28 associated loci, including 6 cocoon weight traits and 22 total cocoon weight traits. After probe design, 22 SNP loci were retained.

[0061] All target sites (1)-(3) were summarized and finally used to construct a 5K liquid phase chip for probe synthesis in silkworms. The total number of sites was 5,381, with an average spacing of 8.5 Kb (e.g., ...). Figure 1As shown in the figure), the site information is shown in Table 1.

[0062] Example 2 This embodiment provides a 5K liquid phase chip for silkworms ( c GPS liquid phase chip).

[0063] The 5,381 SNP loci screened in Example 1 were used for genotyping by pinpoint sequencing of liquid probes, a technology independently developed by Huazhi. c aptured target, c GPS) was used to develop a 5K SNP liquid phase breeding chip for silkworms. c GPS is based on an optimized thermodynamic stability algorithm model. It designs specific probes for the target region sequence, and then uses the synthesized specific probes to capture and enrich multiple different target sequences located at different genomic positions through liquid phase hybridization. Finally, it constructs sequencing libraries and performs high-throughput sequencing on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel sites in the target region. c A schematic diagram of the GPS liquid phase chip testing process is shown below. Figure 2 As shown.

[0064] Example 3 This embodiment provides a method for genotyping silkworm samples using the 5K liquid chromatography chip from Embodiment 2. The steps are as follows: 1. Extraction and detection of gDNA from silkworms Silkworm pupae were collected, and gDNA was extracted from the tissue using the magnetic bead method. The concentration was accurately quantified using Qubit, and the integrity of the gDNA was analyzed using 1% agarose gel electrophoresis. Samples that passed quality control proceeded to the next step of the experiment.

[0065] 2. c GPS Experimental Procedure (1) Take 200ng of qualified gDNA, digest it into 100-500 bp fragments using enzyme digestion reagent, and then add Taq enzyme for end repair. (2) Use T4 ligase to ligate the adapter fragments to both ends of the DNA, and use fragment sorting magnetic beads to purify the ligation product and remove small fragments; (3) Use primers with tagged sequences to perform PCR amplification on the purified ligation product. The amplification product is purified using magnetic beads to remove impurity fragments from the PCR process and complete the library construction. (4) Use Qubit to detect library concentration and Agilent 2100 / 2200 Bioanalyzer to detect library quality. The library fragment size is between 250-500bp. (5) Mix the 12 qualified libraries into one hybridization library, concentrate the mixed library using a vacuum concentrator, and then add the genome blocking reagent, RNase inhibitor, and probe to the concentrated library. After mixing, place the sample on a PCR instrument for hybridization reaction and incubate overnight at 55°C (16 h-24 h). (6) Use streptavidin magnetic beads to capture the hybridization product. Add the capture magnetic beads to the hybridization product to adsorb the probe and target region binding complex and bind at room temperature for 30-60 min. Then wash the magnetic beads with washing solution to remove non-specific binding fragments. Finally, perform PCR amplification on the captured target region. Purify the amplification product with fragment sorting magnetic beads to remove excess non-target products. (7) Library quality inspection, same as step (4). After the library quality inspection is qualified, PE150 sequencing is performed using the BGI sequencing platform.

[0066] (8) The raw data after high-throughput sequencing were processed by quality control filtering, and reads containing adapter contamination and low-quality reads were removed using FASTP software. BWA software was used to compare with the target genome, and then GATK software was used to analyze the mutation sites of the sequencing results to obtain the genotyping results of the target sites.

[0067] Example 4 This embodiment demonstrates the application of the 5K liquid chromatography chip in silkworm genotyping as described in Embodiment 2. Genotyping was performed on 24 different silkworm samples (including 3 duplicate samples) using the 5K liquid chromatography chip (see Embodiment 3 for specific operating methods).

[0068] Table 3 Locus detection rate of 24 silkworm samples

[0069] Table 4

[0070] The results are shown in Tables 3 and 4. Table 3 shows that the locus detection rate of the 24 samples (including 3 replicates) was between 99.16% and 99.85%, with an average detection rate of 99.49%. Table 4 shows that the genotypic consistency rate of the 3 technical replicates was between 99.46% and 99.91%, with an average consistency rate of 99.96%. The evaluation of the genotyping effect of the 5K liquid phase chip in silkworm showed that the 5K liquid phase chip in silkworm had a high genotyping detection rate, a high average genotyping consistency rate, and accurate and reliable genotyping results.

[0071] Example 5 This embodiment provides the application of the 5K liquid chromatography-mass spectrometry (LC-MS) chip in the analysis of silkworm population structure. Using the 5K LC-MS chip from Example 2, genotyping was performed on 96 additional silkworm samples of different varieties (different local varieties from Nanzhao County, Nanyang City, Henan Province). The obtained genotyping results underwent quality control, and individuals with a MAF < 0.5, a genotype deletion rate > 0.1, or a sample deletion rate > 0.1 were removed. Polymorphic SNP loci were obtained, and the genetic distance matrix was calculated using the IBS method in Plink software, followed by cluster analysis and phylogenetic tree construction.

[0072] The results are as follows Figure 3 As shown in the figure, the 5K liquid phase chip for silkworms can effectively distinguish different silkworm varieties in Henan Province, and the classification effect is consistent with the actual classification.

[0073] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A combination of Bombyx mori SNP molecular markers, characterized by, The 5,381 SNP molecular markers comprise 5,381 SNP molecular markers, the physical position of which is determined by sequence alignment based on the reference genome of the domestic silkworm GCA_030269925.2, and the site information is specifically shown in Table 1 in the specification.

2. A primer set and / or probe for detecting the combination of the SNP molecular markers of the domestic silkworm according to claim 1; Preferably, the length of the probe is 80-120 bp; Preferably, the GC content of the probe is between 20%-80%; Preferably, the number of homologous regions of the probe is <5; Preferably, the number of SNP variation sites on the probe is ≤5; Preferably, the probe has no SSR region and N region.

3. A chip, characterized by The chip comprises the primer set and / or probe according to claim 2; Preferably, the chip comprises a liquid chip.

4. A kit characterized in that, The kit comprises the primer set and / or probe according to claim 2.

5. A method of screening the combination of the Bombyx mori SNP molecular markers according to claim 1, characterized in that, The method comprises the following steps: A1) After aligning and detecting the variation of the whole genome sequencing data of the domestic silkworm sample using the Sentieon software, preliminary hard filtering is performed to obtain a file containing SNP variation information of all samples; A2) The file containing SNP variation information of all samples is mined and filtered to obtain target site SNPs; the mining and filtering parameters are: Maf≥0.05, detection rate≥90%, heterozygosity≤20%, and sequencing depth≥8×; A3) The target site SNPs obtained in step A2) are subjected to probe design, and SNPs with probes meeting the requirements are screened; A4) The file containing SNP variation information of all samples is filtered and subjected to probe design with a threshold of FST>0.8 to obtain breed-specific SNPs; A5) The file containing SNP variation information of all samples is subjected to GWAS analysis to obtain associated sites; the associated sites are subjected to probe design, and SNPs with probes meeting the requirements are screened to obtain SNPs related to cocoon weight and whole cocoon weight traits; A6) The SNPs with probes meeting the requirements obtained in A3), the breed-specific SNPs obtained in A4), and the SNPs related to cocoon weight and whole cocoon weight traits obtained in A5) are integrated to obtain the combination of the SNP molecular markers of the domestic silkworm.

6. The method of claim 5, wherein, The aligning and detecting of the whole genome sequencing data of the domestic silkworm sample using the Sentieon software specifically comprises the following steps: B1) The whole genome sequencing data of the domestic silkworm sample is aligned to the reference genome of the domestic silkworm GCA_030269925.2 using the Sentieon software, and the position is sorted and the reads are marked for duplication; B2) The Sentieon software is used to detect the variation sites of each sample to obtain the gVCF of each sample; B3) Joint-calling is performed using Sentieon to jointly analyze the gVCF of all samples to obtain the variation results of each individual in the population.

7. The method of claim 5, wherein, The hard filtered criteria are as follows: QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.

0.

8. The use of the silkworm SNP molecular marker combination of claim 1, the primer set and / or probe of claim 2, the chip of claim 3 or the kit of claim 4 in any of the following: C1) silkworm genotyping detection; C2) silkworm germplasm identification; C3) silkworm genetic diversity evaluation; C4) silkworm genetic map construction and QTL positioning; C5) silkworm genome-wide association analysis; C6) silkworm molecular marker assisted selection; C7) silkworm whole genome selection breeding or intelligent design breeding; C8) silkworm cluster analysis and kinship analysis; C9) gene identification and functional analysis of important economic traits of silkworm; C10) silkworm population structure analysis; C11) silkworm germplasm mining and utilization.

9. A silkworm breeding method comprising the following steps: detecting the DNA of the silkworm to be tested using the silkworm SNP molecular marker combination of claim 1, the primer set and / or probe of claim 2, the chip of claim 3 or the kit of claim 4, and selecting silkworms for subsequent breeding.

10. The method of claim 9, wherein, The detection is based on liquid phase probe capture sequencing typing technology.

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