A Chinese pumpkin SNP molecular marker combination, SNP chip and its application

Abstract

The present invention discloses a Chinese pumpkin SNP molecular marker combination, a SNP chip, and their applications, relating to the field of biotechnology. The pumpkin SNP molecular marker combination includes 20,213 SNP molecular markers, whose loci are determined by sequence alignment of the pumpkin reference genome "Cmoschata_genome_v1.fa.gz," as shown in Table 1 of the specification. The combination can be used for molecular marker fingerprint analysis of pumpkin cultivar resources, genotype identification of hybrid population progeny, authenticity verification of varieties, genetic background analysis and screening of breeding materials, genome-wide association analysis, genetic diversity analysis of germplasm resources, and kinship identification, providing an indispensable tool for high-throughput pumpkin genotyping, gene mapping, fingerprinting, genome-wide selection, and other molecular breeding research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Chinese pumpkin SNP molecular marker combination, a SNP chip and applications thereof. Background Art

[0002] Pumpkin (Cucurbita moschata) is an important cucurbit vegetable. Basic breeding research lags far behind that of other major cucurbit vegetables in the Cucurbitaceae family, and faces challenges such as unstable quality, yield, and resistance. Cultivating high-quality, high-yielding new Chinese pumpkin varieties using modern biological methods, such as molecular breeding, is crucial for advancing pumpkin breeding technology and accelerating the development of superior new varieties.

[0003] Currently, molecular research on Chinese pumpkins mainly uses molecular marker technologies such as Simple Sequence Repeat (SSR) markers, Inter-Simple Sequence Repeat (ISSR) markers, Amplified Fragment Length Polymorphism (AFLP) markers, and Random Amplified Polymorphism DNA (RAPD) markers. SSR markers have low throughput and high R&D costs, making them unsuitable for large-scale commercial identification. RAPD markers are dominant markers and cannot distinguish between dominant homozygous and heterozygous genotypes, resulting in poor reproducibility of results. Although marker technologies such as ISSR and AFLP are simple and practical, their results are unstable and have poor reproducibility. Therefore, it is very important to provide a molecular marker detection method for Chinese pumpkin breeding. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a Chinese pumpkin SNP molecular marker combination.

[0005] The present invention also provides a SNP chip for detecting the pumpkin SNP molecular marker combination.

[0006] The present invention also provides a kit for detecting the above pumpkin SNP molecular marker combination.

[0007] The present invention also provides an application of the pumpkin SNP molecular marker combination, SNP chip or kit.

[0008] The invention also provides a pumpkin breeding method.

[0009] According to one aspect of the present invention, a pumpkin SNP molecular marker combination is proposed, including 20213 SNP molecular markers, the site information of the 20213 SNP molecular markers is determined based on sequence alignment of the pumpkin reference genome "Cmoschata_genome_v1.fa.gz", and the site information is shown in Table 1 of the specification.

[0010] In some embodiments of the present invention, in the base type "A / B" in Table 1, A refers to the base type corresponding to the reference genome, and B refers to the base type after mutation.

[0011] In a second aspect of the present invention, a pumpkin SNP chip is proposed, which includes a primer set and / or probe for detecting the above-mentioned pumpkin SNP molecular marker combination.

[0012] In some embodiments of the present invention, the pumpkin SNP chip is a solid phase chip or a liquid phase chip.

[0013] In the third aspect of the present invention, a kit is provided, which comprises the above-mentioned pumpkin SNP chip.

[0014] In a fourth aspect of the present invention, the use of at least one of the above-mentioned pumpkin SNP molecular marker combination, pumpkin SNP chip and kit is proposed.

[0015] In some embodiments of the present invention, the application can be implemented by the following method:

[0016] S1. Perform genotyping on the sample to be tested using at least one of the pumpkin SNP chip and kit to obtain a genotyping result;

[0017] S2. Analyze the genotyping results obtained in step S1.

[0018] In some embodiments of the present invention, the application is application in pumpkin population structure analysis.

[0019] In some embodiments of the present invention, the application is an application in genome-wide association analysis of pumpkin.

[0020] In some embodiments of the present invention, the application is application in cluster analysis and kinship identification of pumpkin.

[0021] In some embodiments of the present invention, the application is application in genetic diversity analysis of pumpkin.

[0022] In some embodiments of the present invention, the application is application in pumpkin breeding or assisted breeding.

[0023] In some embodiments of the present invention, the application is an application in pumpkin genetic background breeding analysis.

[0024] In a fifth aspect of the present invention, a pumpkin breeding method is proposed, comprising the following steps: using at least one of the pumpkin SNP chip and the kit to detect the DNA of the pumpkin to be tested, and selecting a suitable pumpkin for subsequent breeding.

[0025] In some embodiments of the present invention, the detection is performed based on liquid phase probe capture sequencing typing technology.

[0026] The present invention has at least the following beneficial effects:

[0027] The pumpkin SNP molecular marker combination of the embodiment is evenly distributed on the chromosome, has good site representation, strong specificity, and high polymorphism. By performing genotyping identification on the corresponding pumpkin SNP molecular marker combination in pumpkin, molecular marker fingerprint analysis of pumpkin variety resources, genotyping of hybrid population offspring, variety authenticity identification, genetic background analysis and screening of breeding materials, genome-wide association analysis, genetic diversity analysis of germplasm resources, and kinship identification can be performed. It can be widely used for the detection and application of different types of pumpkin materials, such as wild species, landraces, and cultivated species, and provides an indispensable and important tool for molecular breeding research such as high-throughput pumpkin genotyping, gene mapping, fingerprinting, and whole-genome selection.

[0028] Using high-throughput sequencing technology, we genotyped pumpkins using corresponding pumpkin SNP molecular marker combinations. This method offers high throughput, produces a large amount of data at once, and can simultaneously test nearly a thousand samples. It is also compatible with mainstream second-generation sequencing platforms such as Illumina and MGI, demonstrating broad platform adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the PIC value distribution diagram of SNP sites in the pumpkin SNP molecular marker combination;

[0030] Figure 2 This is the chromosome distribution map of the SNP sites in the pumpkin SNP molecular marker combination;

[0031] Figure 3 The results of annotation analysis of SNP sites in the pumpkin SNP molecular marker combination are shown;

[0032] Figure 4 This is a schematic diagram of the cGPS liquid chip process detection;

[0033] Figure 5 This is the phylogenetic tree obtained by analyzing 60 pumpkin materials using the pumpkin 20K liquid phase breeding chip in test example 1. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0035] Example 1

[0036] This example provides a pumpkin SNP molecular marker combination, including 20,213 SNP sites, the physical locations of which are determined based on sequence alignment of the pumpkin reference gene (Cmoschata_genome_v1.fa.gz). The 20,213 SNP molecular markers are shown in Table 1 below.

[0037] Table 1

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[0080]

[0081] The average polymorphic information content (PIC) of the SNP sites in the pumpkin SNP molecular marker combination was 0.31 (the highest PIC value was 0.375), indicating that the site polymorphism was high. Figure 1 As shown. The SNP sites are evenly distributed on the 20 chromosomes of pumpkin, with an average spacing of 11Kb. Figure 2 The snpeff tool was used to perform functional annotation of SNPs. Figure 3 shown.

[0082] The screening process is as follows:

[0083] 1. Collection of pumpkin germplasm resources:

[0084] In order to ensure the representativeness of pumpkin materials and the universality of liquid phase arrays, 69 Chinese pumpkin diversity materials (old edible type, young edible type, etc.) were collected from the Hunan Vegetable Research Institute as the source of resequencing data.

[0085] 2. Pumpkin Whole Genome Resequencing:

[0086] The 69 collected pumpkin materials were subjected to whole genome resequencing. The specific steps included:

[0087] (1) The DNA of pumpkin materials was extracted by magnetic bead method, and the concentration of DNA samples was detected by Qubit fluorescence quantification instrument; the integrity of DNA samples was detected by 1% agarose gel electrophoresis, and samples that passed quality control were used for library preparation.

[0088] (2) The sequencing library was constructed using the standard method of MGI library construction.

[0089] (3) High-throughput sequencing was performed using the BGI sequencing platform, with a sequencing strategy of PE150 and a sequencing depth of 10×, with 4 Gb sequenced for each pumpkin material.

[0090] (4) Comparison and variation detection were performed on the 69 resequencing data. The analysis process is as follows:

[0091] a. Use Sentieon to align reads to the pumpkin reference genome (Cmoschata_genome_v1.fa.gz) (http: / / cucurbitgenomics.org), sort by position, and mark duplicate reads.

[0092] b. Use Sentieon to detect variant sites for each sample and obtain a gVCF file for each sample.

[0093] c. Use Sentieon for joint variant calling, jointly analyze the gVCF files of all samples, and obtain the variant results of each individual in the population.

[0094] 3. Development of pumpkin SNP molecular marker combination:

[0095] (1) Whole-genome site screening:

[0096] a. Candidate site screening: Quality indicators of resequencing sites were calculated, and SNP polymorphic sites with a heterozygosity rate <0.3, site deletion rate <0.15, MAF minimum allele frequency ≥0.1, PIC site polymorphism >0.15, and sequencing depth >5x were selected as candidate sites. A total of 1,610,763 sites were obtained.

[0097] b. Candidate site probe design and screening: Extract 50bp of sequence upstream and downstream of the SNP site, analyze its specificity and GC content, and screen 687,872 SNP sites for chip development.

[0098] c. Site density screening: Based on the principle of uniform site distribution, SNP sites evenly distributed on pumpkin chromosomes were screened. The average spacing between sites was 11 Kb, and 19,903 SNP sites were included.

[0099] (2) Determination of important functional sites:

[0100] We collected literature reports on important pumpkin traits and genes related to disease resistance, stress tolerance, fruit quality, and short vine length, and screened these genes for polymorphic single-nucleotide polymorphisms (SNPs) as candidate loci. A total of 310 SNPs were identified for potential array development. Among these, 99 markers were associated with stress resistance, 77 with disease resistance, 82 with short vine length, 45 with pistillate / staminate flower differentiation, and 7 with fruit quality and weight.

[0101] This example also provides a pumpkin 20K liquid-phase breeding array, developed using liquid-phase array targeted sequencing and typing technology based on 20,213 SNP sites in the pumpkin SNP molecular marker panel. The steps involved are as follows:

[0102] 1) Sample DNA extraction and quality control:

[0103] DNA samples were extracted using a magnetic bead method. DNA concentration was determined using a Qubit fluorescence quantifier. DNA integrity was checked by 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0104] 2) Preliminary library construction and quality control:

[0105] a. Use fragmentase to digest the DNA sample, repair the digested ends, add an A base to the 3' end, and detect the fragment size by agarose gel electrophoresis.

[0106] b. Use T4 ligase to ligate the sequencing adapter to the DNA fragment and purify the ligation product using magnetic beads. Determine the concentration of the purified product using a Qubit fluorometer and the size of the fragment using agarose gel electrophoresis.

[0107] c. Perform PCR amplification on the purified ligation product and screen the amplified product using magnetic beads. Determine the concentration of the screened product using a Qubit fluorescence quantifier and the size of the fragments using agarose gel electrophoresis.

[0108] 3) Final library construction and quality control:

[0109] a. Take 200 ng of the constructed prelibrary, add the probe and hybridization reagent, and incubate at 50°C for 16-24 hours to complete the hybridization reaction.

[0110] b. Use magnetic beads to capture the target segment, use cleaning solution to wash the captured product to remove non-specific binding fragments, and then perform another round of PCR amplification.

[0111] c. Library concentration is measured using a Qubit fluorescence quantifier, and fragment size is measured by agarose gel electrophoresis. Once the concentration and fragment size are qualified, the final sequencing library is constructed.

[0112] 4) High-throughput sequencing:

[0113] The prepared final sequencing library was sequenced using a BGI sequencer with high throughput sequencing using the PE150 sequencing strategy. Figure 4 .

[0114] 5) Bioinformatics analysis:

[0115] a. Raw data filtering (sequencing data quality control): The raw sequencing sequences (Raw Reads) obtained by sequencing are filtered to obtain high-quality Clean Reads.

[0116] b. Contamination detection: Use Blastn to align the sequences to the NCBI NT database for contamination assessment.

[0117] c. Reference genome alignment: Use BWA software to align sequencing reads to the reference genome.

[0118] d. Mutation detection: Use GATK software to detect mutation sites in each sample.

[0119] Test Example 1

[0120] In this example, based on the pumpkin 20K liquid-phase breeding chip provided in Example 1, genotyping detection was performed on 60 pumpkin materials from three populations (these 60 materials had different sources from the 69 pumpkin diversity materials in Example 1, and the 60 pumpkin materials were numbered N1 to N60, respectively) to analyze their genotyping and population structure.

[0121] The pumpkin 20K liquid-phase breeding array was used to test 60 pumpkin accessions, with four randomly selected biological replicates. Testing and verification showed that the site detection rates for the 60 accessions ranged from 98.75% to 99.99%, with an average site detection rate of 99.44%. The genotype concordance rates for the four replicates ranged from 99.44% to 99.90%, with an average concordance rate of 99.75%. This indicates that the selected pumpkin SNP molecular marker combination has good stability and high genotyping accuracy.

[0122] Plink software was used to calculate the genetic distance matrix and perform cluster analysis on pumpkin materials. The phylogenetic tree was constructed to determine the kinship, evolutionary relationship, and composition structure of different materials. Cluster analysis revealed that 60 pumpkin materials were divided into three subgroups, which was consistent with the actual clustering effect. This indicates that the selected pumpkin SNP molecular marker combination has a high representativeness and can be used for population structure analysis. Figure 5 shown.

[0123] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of primer sets and / or probes for detecting pumpkin SNP molecular marker combinations in pumpkin population structure analysis; The pumpkin SNP molecular marker combination is 20213 SNP molecular markers, and the site information of the 20213 SNP molecular markers is determined by sequence alignment based on the pumpkin reference genome "Cmoschata_genome_v1.fa.gz". The site information is shown in Table 1 of the specification.

2. Use of a pumpkin SNP chip comprising the primer set and / or probe for detecting pumpkin SNP molecular marker combination as claimed in claim 1 in pumpkin population structure analysis.

3. Use of a kit comprising the pumpkin SNP chip according to claim 2 in pumpkin population structure analysis.

4. Application of primer sets and / or probes for detecting pumpkin SNP molecular marker combinations in genome-wide association studies of pumpkin; The pumpkin SNP molecular marker combination is 20213 SNP molecular markers, and the site information of the 20213 SNP molecular markers is determined by sequence alignment based on the pumpkin reference genome "Cmoschata_genome_v1.fa.gz". The site information is shown in Table 1 of the specification.

5. Use of a pumpkin SNP chip comprising the primer set and / or probe for detecting pumpkin SNP molecular marker combination according to claim 4 in genome-wide association analysis of pumpkin.

6. Use of a kit comprising the pumpkin SNP chip according to claim 5 in genome-wide association analysis of pumpkin.

7. Application of primer sets and / or probes for detecting pumpkin SNP molecular marker combinations in cluster analysis and phylogenetic relationship identification of pumpkin; The pumpkin SNP molecular marker combination is 20213 SNP molecular markers, and the site information of the 20213 SNP molecular markers is determined by sequence alignment based on the pumpkin reference genome "Cmoschata_genome_v1.fa.gz". The site information is shown in Table 1 of the specification.

8. Use of a pumpkin SNP chip comprising the primer set and / or probe for detecting pumpkin SNP molecular marker combination according to claim 7 in cluster analysis and kinship identification of pumpkin.

9. Use of a kit comprising the pumpkin SNP chip according to claim 8 in cluster analysis and kinship identification of pumpkin.

10. Application of primer sets and / or probes for detecting pumpkin SNP molecular marker combinations in pumpkin genetic diversity analysis; The pumpkin SNP molecular marker combination is 20213 SNP molecular markers, and the site information of the 20213 SNP molecular markers is determined by sequence alignment based on the pumpkin reference genome "Cmoschata_genome_v1.fa.gz". The site information is shown in Table 1 of the specification.

11. Use of a pumpkin SNP chip comprising the primer set and / or probe for detecting pumpkin SNP molecular marker combination according to claim 10 in analysis of pumpkin genetic diversity.

12. Use of a kit comprising the pumpkin SNP chip according to claim 11 in analyzing the genetic diversity of pumpkin.

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