A primer set for identifying pumpkin varieties and a kit and application thereof
By providing primer sets and kits for pumpkin varieties, the problem of high cost of gene chips has been solved, enabling low-cost, high-throughput identification of pumpkin varieties and evaluation of genetic diversity. The results are accurate and guide breeding research.
Patent Information
- Application Number
- CN202610451724.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-08
- Publication Date
- 2026-06-23
AI Technical Summary
Existing gene chip testing for pumpkin varieties is costly and cannot meet personalized needs, making it difficult to apply on a large scale.
A primer set and kit for identifying pumpkin varieties are provided, comprising 500 primer pairs for PCR amplification and sequencing analysis, and for constructing a genetic diversity and phylogenetic tree of pumpkin.
It enables low-cost, high-throughput identification of pumpkin varieties and evaluation of genetic diversity, with accurate results that can distinguish different variety types and guide breeding research.
Smart Images
Figure CN122256550A_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of gene detection, and in particular to a primer set for identifying pumpkin varieties, a kit thereof, and its application. Background Technology
[0002] Pumpkins mainly include: Chinese pumpkin, Indian pumpkin, American pumpkin, gray-seeded pumpkin, and black-seeded pumpkin, etc. They are eaten for their fruit or seeds.
[0003] Currently used gene chips for detection are expensive, cannot be applied on a large scale, and cannot meet personalized needs. Developing a low-cost, high-throughput molecular marker detection system covering the entire pumpkin genome is of great significance for pumpkin molecular breeding.
[0004] Public content
[0005] To address the problems of existing technologies, this disclosure provides a primer set, a kit, and its application for identifying pumpkin varieties. The technical solution is as follows:
[0006] On one hand, this disclosure provides a primer set for identifying pumpkin varieties, the primer set comprising: primer pair 1 to primer pair 500, each primer pair comprising a forward primer and a reverse primer, the forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 500 and the reverse primer of primer pair 500 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 1000 in the sequence listing.
[0007] On the other hand, this disclosure provides a kit for identifying pumpkin varieties, the kit comprising the aforementioned primer set.
[0008] In another aspect, this disclosure provides an application of a primer set for identifying pumpkin varieties, the application including: using the primer set to identify pumpkin varieties.
[0009] Specifically, the application includes using the primer set to evaluate the genetic diversity of different varieties of pumpkin.
[0010] Specifically, the applications include: using the primer set for fingerprinting of the core germplasm resources of the pumpkin, molecular marker-assisted breeding, and whole-genome breeding.
[0011] Specifically, the applications include using the primer set for genotyping of the pumpkin germplasm resources, species evolution analysis, seed purity detection, and variety authenticity analysis.
[0012] The beneficial effects of the technical solution provided in this disclosure are as follows: This disclosure provides a primer set for identifying pumpkin varieties, a kit and its application. The primer set is used to identify pumpkin varieties and evaluate the genetic diversity of different varieties of pumpkins, and the results are accurate. Attached Figure Description
[0013] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0014] Figure 1 This is a schematic diagram of the distribution of 500 SNP loci on the chromosome provided in Embodiment 1 of this disclosure;
[0015] Figure 2 This is a schematic diagram of the phylogenetic tree of 94 pumpkins provided in Embodiment 3 of this disclosure, wherein purple represents Chinese pumpkin, red represents American pumpkin, and green represents Indian pumpkin;
[0016] Figure 3 This is the fingerprint spectrum of 33 pumpkin core germplasm samples provided in Embodiment 4 of this disclosure. The horizontal axis represents 32 SNP loci, and the vertical axis represents 33 varieties. Detailed Implementation
[0017] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings.
[0018] Example 1
[0019] This embodiment provides a primer set for identifying pumpkin varieties. The primer set includes primer pairs 1 to 500, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 500 and the reverse primer of primer pair 500 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 1000 in the sequence listing.
[0020] Primer pairs 1 through 500 each correspond to 500 SNP markers, such as Figure 1As shown, the distribution of these 500 SNP markers on the chromosomes is as follows: chromosome C01: 28 SNPs; chromosome C02: 25 SNPs; chromosome C03: 19 SNPs; chromosome C04: 42 SNPs; chromosome C05: 26 SNPs; chromosome C06: 28 SNPs; chromosome C07: 21 SNPs; chromosome C08: 22 SNPs; chromosome C09: 21 SNPs; chromosome C10: 22 SNPs. NPs: 27 SNPs on chromosome C11; 26 SNPs on chromosome C12; 23 SNPs on chromosome C13; 30 SNPs on chromosome C14; 30 SNPs on chromosome C15; 13 SNPs on chromosome C16; 24 SNPs on chromosome C17; 25 SNPs on chromosome C18; 19 SNPs on chromosome C19; 29 SNPs on chromosome C20; approximately 25 SNPs per chromosome on average. The primer pairs in the primer set provided in this example, when mixed in equimolar concentrations, can be amplified in the same PCR reaction.
[0021] The primer set provided in this embodiment is shown in Table 1.
[0022] Table 1 shows the information of 500 SNP sites and their corresponding primer pair sequences.
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
[0034] In Table 1, base 1 and base 2 are two single nucleotide polymorphisms (SNPs) to be detected.
[0035] Example 2
[0036] This embodiment provides a kit for identifying pumpkin varieties, which includes the primer set provided in Example 1.
[0037] Example 3
[0038] This embodiment provides an application of a primer set for identifying pumpkin varieties, which includes using the primer set provided in Embodiment 1 to identify pumpkin varieties.
[0039] Specifically, the application includes using the above primer set to evaluate the genetic diversity of different varieties of pumpkin.
[0040] Furthermore, the application also includes:
[0041] Genomic DNA was extracted from the sample to be tested, and young tissues of the sample were preferred.
[0042] The genomic DNA of the sample to be tested was amplified by primer set in the first round of PCR to obtain the first round of amplification products;
[0043] Using the first-round amplification product as a template, a second-round PCR amplification was performed using a primer set to obtain the second-round amplification product;
[0044] The second-round amplification products were mixed to obtain a mixed amplification product;
[0045] The mixed amplification products were purified and then sequenced to obtain the raw data.
[0046] The raw data was analyzed, and then the MEGA software was used to calculate the pairwise genetic distance matrix to construct the phylogenetic tree.
[0047] iTOL was used to visualize the phylogenetic tree to obtain the differentiation order branching tree and evolutionary relationship between at least two samples to be tested, which was used to evaluate the genetic diversity of different types of pumpkin and to identify pumpkin varieties.
[0048] Young pumpkin leaves were selected as the test samples. In this embodiment, 94 different types of pumpkin varieties were selected, including 5 Chinese pumpkins, 31 American pumpkins, and 58 Indian pumpkins. All of these pumpkin varieties were obtained from the laboratory of the Shanghai Academy of Agricultural Sciences. The specific details of these 94 different types of pumpkins are shown in Table 2.
[0049] Table 2 is a statistical table of 94 different types of pumpkins.
[0050]
[0051] Genomic DNA was extracted from young pumpkin leaves using a modified CTAB (hexadecyltrimethylammonium bromide) method. The specific extraction method is as follows: A thumb-sized sample of young leaf was placed in a 2 mL centrifuge tube and labeled. Two steel balls were added to the tube, followed by 100 μL of CTAB. The sample was then thoroughly ground using a grinder. After grinding, the tube was lightly centrifuged, the steel balls were discarded, and 500 μL of CTAB was added to the tube. The mixture was then inverted and mixed. The tube was placed in a 65°C water bath for 40 min, inverting and mixing every 10 min. An equal volume of 24:1 (chloroform:isoamyl alcohol) was added to the tube, and the mixture was thoroughly shaken and mixed. The tube was then centrifuged at 12,000 r / min at 4°C for 15 min, and the supernatant was collected. Take a new 1.5 mL centrifuge tube, add 400 μL of isopropanol and then 400 μL of supernatant, mix thoroughly by inverting, and incubate at -20°C for 10 min. Centrifuge at 12,000 r / min at 4°C for 5 min, remove the tube, discard the supernatant, retain the precipitate, and wash twice with 800 μL of 75% ethanol. After drying in a fume hood, add 50 μL of 0.1% RNase to dissolve the genomic DNA, and incubate in a water bath at 37°C for 30 min to fully dissolve the genomic DNA. After cooling, store at -20°C for later use.
[0052] The quality of genomic DNA was assessed using agarose gel electrophoresis and ultraviolet spectrophotometry. Specifically, agarose gel electrophoresis showed a clear and intact main band of genomic DNA, indicating no degradation or RNA contamination. Ultraviolet spectrophotometry showed an OD260 / 280 ratio between 1.8 and 2.2, indicating the absence of protein and visible contaminants. The genomic DNA quality assessment results provided in this example all met the relevant quality requirements and can be used for subsequent experiments.
[0053] Using genomic DNA from the samples to be tested (94 different varieties of pumpkin) as templates, the target sites were amplified using VAHTS Multi-PCR Mix to obtain a large number of first-round amplification products (fragments).
[0054] Specifically, the reaction system for each 10 μL first round of PCR amplification includes: 1.0 μL of genomic DNA from the sample to be tested, 2.5 μL of 4×VAHTS Multi-PCR Mix, 1.0 μL of primer set (both forward and reverse primers are 0.2 μM), and 5.5 μL of water.
[0055] The reaction procedure for the first round of PCR amplification is as follows:
[0056]
[0057] Specifically, each 10 μL reaction system for the second round of PCR amplification includes: 1.0 μL of diluted first-round amplification product (in this embodiment, the dilution concentration is 0.2 μM for each forward primer and each reverse primer), 2.5 μL of 4×VAHTSMulti-PCR Mix, 1.5 μL of primer set, 0.5 μL of barcode, and 4.5 μL of water.
[0058] The reaction procedure for the second round of PCR amplification is as follows:
[0059]
[0060] After two rounds of PCR amplification, the second-round amplification products were mixed to obtain a mixed amplification product. This mixed amplification product was then purified using AMPure XP Beads to obtain a purified mixed amplification product, which was used for library construction. After library construction, quality control was performed using agarose gel electrophoresis, real-time quantitative PCR, and Agilent 2100 for quality control and quantification. Once the quality control was satisfactory, the library was subjected to Target-seq sequencing using the Illumina HiSeq next-generation sequencing platform to obtain raw data. (The text repeats itself here.)
[0061] Data quality control was performed on the raw sequencing data to obtain high-quality clean data. Then, BWA software was used to break down the clean data into target loci, resulting in SAM alignment results. The SAM files were then converted to BAM format using samtools software. Finally, the reads in the BAM files were sorted using SortSam in Picard, yielding the final BAM file. GATK was then used to determine the genotype of each target locus. A total of 7,345,007 high-quality SNP variants were identified. Among them, there were 4,591,668 transitions (Ts: A / G and C / T) and 2,753,339 transversions (Tv: A / C, A / T, C / G, and G / T), with a transition / transversion ratio (Ts / Tv) of 1.67. The genome-wide average SNP density was 21,281.50 SNPs / Mb. The distribution of SNPs was uneven across different chromosomes, with the highest density on chromosome 4 (Chr04) (31,406.24 SNPs / Mb) and the lowest density on chromosome 3 (Chr03) (12,440.09 SNPs / Mb). All variant sites were widely distributed across the 20 chromosomes of pumpkin (Chr01–Chr20). As can be seen, the primer set provided in this embodiment demonstrates the high efficiency and accuracy of SNP-paneling.
[0062] Based on the genotyping results of these 94 pumpkin samples, pairwise genetic distance matrices were calculated. A phylogenetic tree was constructed using the neighbor-joining method in MEGA7 software (version 7.0). Finally, the phylogenetic tree was visualized using iTOL software (version 6.9) to show the phylogenetic relationships between the tested samples. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the 94 different pumpkins can be clearly divided into three types by the primer set provided in this embodiment of the invention: American pumpkin, Chinese pumpkin, and Indian pumpkin. Pumpkins of the same type can cluster together, indicating that they are more closely related. This classification result is completely consistent with the morphological classification of the samples to be tested, which is in line with expectations. The phylogenetic tree results not only identified the variety types of these 94 pumpkins, but also revealed their phylogenetic relationships, which will effectively guide the next step of pumpkin hybridization research.
[0063] Example 4
[0064] Specifically, the application may also include: using the primer set provided in Example 1 for fingerprinting, molecular marker-assisted breeding, and whole-genome breeding of pumpkin core germplasm resources.
[0065] Specifically, the application may also include: using the primer set provided in Example 1 for genotyping, species evolution analysis, seed purity detection, and variety authenticity analysis of pumpkin core germplasm resources.
[0066] To verify the application of the primer set provided in this embodiment in 33 pumpkin core germplasm resources, 32 SNP markers were selected for fingerprinting.
[0067] Specifically, the application may also include the screening of SNP loci. In this embodiment, candidate SNP loci are first retrieved from a whole-genome database. The screening criteria follow the principle of "whole-genome coverage," meaning that the physical distance between loci remains relatively equal, avoiding excessive clustering in local chromosomal regions. These loci constitute a "core marker set" that distinguishes different varieties or individuals.
[0068] Furthermore, the application may also include: standardized encoding of genotype data. Raw SNP detection results are typically present in base pairs (e.g., AA, AT, TT) or in a raw format (0 / 0, 0 / 1, 1 / 1). To facilitate computer processing and algorithm comparison, this invention implements the following unified encoding logic: 0 represents a homozygous reference genotype (0 / 0); 1 represents a heterozygous genotype (0 / 1); 2 represents a homozygous variant genotype (1 / 1); and 9 represents missing data.
[0069] Sequence construction of molecular identity cards: The numerical codes of each locus are obtained and arranged according to their physical location on the chromosome (from chromosome 1 to the terminal chromosome, from acrosome to distal telomere).
[0070] Visualization and Application: To enhance the practical value of molecular identification, this invention provides a visualization path, mapping digital sequences to bar charts of different colors or heights. By visually observing the bar chart patterns, it is possible to quickly determine whether two samples are closely related. Specifically, as shown below... Figure 3 As shown, by Figure 3 As can be seen, the 32 SNP loci provided in this embodiment offer sufficient resolution to distinguish all 33 varieties. No two varieties have the same fingerprint string, indicating 100% distinguishing efficiency in the test population.
[0071] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.
Claims
1. A primer set for identifying pumpkin varieties, characterized in that, The primer set includes primer pairs 1 to 500, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 500 and the reverse primer of primer pair 500 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 1000 in the sequence listing.
2. A kit for identifying pumpkin varieties, characterized in that, The kit includes the primer set as described in claim 1.
3. The application of a primer set for identifying pumpkin varieties, characterized in that, The application includes using the primer set as described in claim 1 to identify pumpkin varieties.
4. The application according to claim 3, characterized in that, The application includes using the primer set to evaluate the genetic diversity of different varieties of pumpkin.
5. The application according to claim 3, characterized in that, The applications include: using the primer set for fingerprinting the core germplasm resources of the pumpkin, molecular marker-assisted breeding, and whole-genome breeding.
6. The application according to claim 3, characterized in that, The applications include using the primer set for genotyping of the pumpkin germplasm resources, species evolution analysis, seed purity detection, and variety authenticity analysis.