A 3KSNP genome breeding chip for Chinese pumpkin and its application

Abstract

The application discloses a Cucurbita moschata genomic 3K SNP breeding chip and application thereof, and relates to the technical field of biology.The breeding chip comprises reagents for detecting genotypes of SNP sites in a SNP site combination; and the SNP site combination comprises 2727 SNP sites located on a CmoMimu reference genome of C.moschata.The SNP sites used in the breeding chip are obtained through population stratified sampling, linkage disequilibrium pruning, whole genome uniform distribution screening, GWAS correlation site integration and multi-dimensional quality evaluation of probes, and are particularly suitable for application in the fields of C.moschata population structure analysis, variety identification, genetic diversity analysis, whole genome correlation analysis, molecular marker assisted selection breeding, genome selection breeding and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a 3K SNP breeding chip for the genome of Chinese pumpkin and its application. Background Technology

[0002] Chinese pumpkin ( C. moschata Pumpkin is an important cucurbit vegetable crop, rich in functional nutrients such as carotenoids, vitamins, and minerals. Developing high-quality, high-yield, and disease-resistant new varieties of pumpkin is of great significance to the development of the vegetable industry.

[0003] Traditional pumpkin breeding relies primarily on phenotypic selection, which is susceptible to environmental influences and suffers from long breeding cycles and low selection efficiency. Molecular marker-assisted selection (MAS) reduces phenotypic identification costs and significantly improves breeding efficiency by enabling early and precise screening of target genotypes. Among these, SNP markers have become the most widely used marker type in modern molecular breeding due to their abundant numbers, wide genomic distribution, genetic stability, and high detection throughput.

[0004] SNP chips achieve high-throughput genotyping through a single hybridization or capture reaction, offering advantages such as low cost, high throughput, and stable data. They are an ideal tool for large-scale germplasm resource identification and breeding population genotyping.

[0005] Molecular breeding research on pumpkin in China is relatively lagging behind. The traditional marker technologies currently used, such as SSR, Indel, and dCAPS, have low throughput and poor reproducibility. Although there are reports of pumpkin genome sequencing in China, there is still a lack of high-quality SNP chips for breeding applications.

[0006] A previous patent application (publication number CN118497397A) reported a 20K SNP chip based on 69 Chinese pumpkin materials. However, this chip has limited germplasm representativeness, a relatively simple screening strategy, and lacks a systematic probe quality assessment and LD site supplementation mechanism. Furthermore, the unit cost of a 20K high-density chip is relatively high in large-scale breeding population screening.

[0007] Low-density SNP microarrays (such as 3K-level microarrays) combined with genotype imputation technology can achieve similar accuracy to medium- and high-density microarrays in genome selection, while significantly reducing detection costs. However, low-density microarrays have higher requirements for site selection: with a limited number of sites, it is necessary to simultaneously ensure uniformity of genome-wide coverage, site polymorphism, and coverage of associated sites.

[0008] Therefore, it is necessary to develop a low-density SNP breeding chip for Chinese pumpkin based on a wide range of representative germplasm resources, employing a systematic screening strategy, and undergoing rigorous probe quality evaluation and LD site supplementation optimization. Summary of the Invention

[0009] The purpose of this invention is to provide a 3K SNP breeding chip for the Chinese pumpkin genome and its applications, thereby addressing the problems existing in the prior art. The breeding chip provided by this invention utilizes SNP loci obtained through population stratified sampling, linkage disequilibrium pruning, genome-wide uniform distribution screening, GWAS-associated locus integration, and multidimensional probe quality assessment. It is particularly suitable for applications in Chinese pumpkin population structure analysis, variety identification, genetic diversity analysis, genome-wide association analysis, marker-assisted selection breeding, and genome selection breeding.

[0010] To achieve the above objectives, the present invention provides the following solution: This invention provides a combination of SNP sites for breeding Chinese pumpkin, including 2727 SNP sites located in the Chinese pumpkin reference genome CmoMimu as shown in Table 4.

[0011] The present invention also provides the application of the above-described SNP site combinations in at least one of the following: (1) Analysis of the population structure of Chinese pumpkin; (2) Genome-wide association analysis of Chinese pumpkin; (3) Cluster analysis of Chinese pumpkins; (4) Phylogenetic identification of Chinese pumpkin; (5) Construction of genetic map and gene localization of Chinese pumpkin; (6) Analysis of genetic diversity of pumpkin in China; (7) Molecular marker-assisted breeding of Chinese pumpkin; (8) Analysis of the genetic background and breeding of Chinese pumpkin.

[0012] This invention also provides the application of a reagent for detecting the genotype of each SNP site in the above-mentioned SNP site combination in the preparation of a 3K SNP breeding chip for Chinese pumpkin genome.

[0013] Furthermore, the chip is a liquid phase chip.

[0014] This invention also provides a 3K SNP breeding chip for the genome of Chinese pumpkin, including reagents for detecting the genotype of each SNP site in the above-mentioned SNP site combinations.

[0015] Furthermore, the chip is a liquid phase chip.

[0016] Furthermore, the reagent is a DNA detection probe.

[0017] This invention also provides the application of the above-mentioned Chinese pumpkin genome 3K SNP breeding chip in at least one of the following: (1) Analysis of the population structure of Chinese pumpkin; (2) Genome-wide association analysis of Chinese pumpkin; (3) Cluster analysis of Chinese pumpkins; (4) Phylogenetic identification of Chinese pumpkin; (5) Construction of genetic map and gene localization of Chinese pumpkin; (6) Analysis of genetic diversity of pumpkin in China; (7) Molecular marker-assisted breeding of Chinese pumpkin; (8) Analysis of the genetic background and breeding of Chinese pumpkin.

[0018] The present invention discloses the following technical effects: (1) The germplasm resources are highly representative. The breeding chip provided by this invention is based on 261 Chinese pumpkin germplasm resources covering multiple genetic groups, which far exceeds the scale of 69 materials in the prior art.

[0019] (2) The LD site supplementation strategy reduced the complexity of the microarray. High-quality sites were screened through an 8-dimensional probe quality assessment, and neighboring SNPs linked to LD were selected as supplementary sites for original sites that did not meet the assessment criteria to redesign probes. Some tag sites provided detection markers for multiple target sites through LD relationships, with a total of 2725 physical probes, saving probe synthesis and maintaining the integrity of genotype information.

[0020] (3) Associated loci were obtained by GWAS screening. Functional SNP loci were screened based on GWAS analysis of multiple agronomic traits of 261 Chinese pumpkin germplasm resources, covering traits such as yield, appearance quality, internal quality and nutritional components.

[0021] (4) High probe quality. All probes were evaluated for quality in 8 dimensions. The average GC value was 43.5%, the average TM value was 74.4℃ and all were ≥65℃, the average LCC value was 0.973, and 80.8% were single genome matches.

[0022] (5) Uniform coverage of the whole genome. 2727 loci are distributed across 20 chromosomes with an average spacing of about 108 kb, which can effectively cover the whole genome.

[0023] (6) Low density and low cost. The 3K chip is suitable for large-scale breeding population screening. Based on liquid phase capture sequencing technology, it can simultaneously detect nearly a thousand materials and is applicable to mainstream sequencing platforms such as Illumina and MGI.

[0024] (7) The chip can be effectively applied to molecular marker-assisted backcross breeding. Using the chip, foreground selection is performed on 5 major loci of the three traits of high soluble solids, high lutein and early maturity, and background selection is performed on 2288 background loci. The recurrent parent genome recovery rate of more than 98% is achieved in the BC2F2 generation. The 5 target loci are fixed at the same time, which reduces the number of generations by 2 to 3 compared with traditional backcross breeding. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 A schematic diagram of the development process for 3K SNP breeding chips; Figure 2 Distribution density map of 2727 SNP loci on 20 chromosomes; Figure 3 The distribution of SNP numbers (A) and the distance between adjacent sites (B) on each chromosome; Figure 4 The distribution of probe GC content (A) and TM value (B) is shown in the graph. Figure 5 Figures showing the detection rate (A), sample detection rate (B), and site polymorphism statistics (C) of 146 Chinese pumpkin materials on chip chips; Figure 6 PCA clustering scatter plot of 146 Chinese pumpkin materials based on 2727 SNP sites; Figure 7 This is a phylogenetic tree diagram of 146 material systems. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Example 1: Obtaining SNP molecular marker combinations and fabricating 3K chips This embodiment describes the development process and microarray fabrication method of 3K SNP molecular marker combinatorial arrays for the Chinese pumpkin genome. A schematic diagram of the research and development process is shown below. Figure 1 .

[0033] 1. Germplasm resource collection and population division Twenty-one pumpkin germplasm resources from China were collected (Table 1), originating from provinces and municipalities including Guangdong, Chongqing, Shaanxi, Shandong, and Yunnan in China, as well as countries such as Thailand, Japan, the Philippines, and India. Population genetic structure analysis (K=2–15) was performed using ADMIXTURE software (v1.3.0). Combined with principal component analysis (PCA) and phylogenetic tree analysis (PHYLIP), the 261 accessions were classified into five major genetic groups: SWNC (108 accessions), SC (67 accessions), NonC (61 accessions), Mix (10 accessions), and ND (15 accessions). Significant differences were found in the genetic differentiation index (Fst) and nucleotide diversity (π) among the groups, with the NonC group exhibiting the highest nucleotide diversity (π≈9.6 × 10⁻⁶). -4 This satisfies the prerequisite for screening SNPs by group.

[0034] Table 1. 261 Chinese Pumpkin Germplasm Resources 2. Whole-genome resequencing and variant detection Genomic DNA was extracted from each material using the CTAB method, and sequencing libraries with inserts of approximately 350 bp were constructed. Paired-end sequencing was performed on the Illumina platform using PE150, with an average sequencing depth of approximately 15× (range 9.7×~30.5×). Sequencing data were filtered via FASTP and aligned to the Chinese pumpkin reference genome CmoMimu (301.82 Mb, 20 chromosomes, NCBI BioProject: PRJNA1173898) using BWA-MEM (v0.7.17). Single-sample variant detection was performed using GATK (v4.2) HaplotypeCaller, and combined variant detection was performed using CombineGVCFs and GenotypeGVCFs.

[0035] 3. SNP Quality Control The original SNP set was quality controlled using vcftools (v0.1.16) and PLINK (v1.9): deletion rate < 5% (--max-missing 0.95), MAF > 0.05 (--maf 0.05), only biallelic loci were retained (--max-alleles2), and Hardy-Weinberg equilibrium test p > 1 × 10⁻⁶. -6 .

[0036] 4. Background SNP site screening Background SNP loci are used for population structure analysis, kinship identification, variety identification, and genome selection and filling. The screening procedure is as follows: each genetic group undergoes independent LD pruning (PLINK --indep-pairwise 50 5 0.3), and loci are evenly selected across the whole genome at intervals of approximately 100–200 kb. The number of loci in each population is then weighted according to the effective size of the population and merged. Loci with a MAF of 0.2–0.5, high PIC values, and high inter-population Fst are preferred.

[0037] 5. GWAS Functional SNP Site Screening GWAS analysis was performed on multiple agronomic traits (including yield-related traits, appearance quality traits, internal quality traits, and nutritional traits) of 261 Chinese pumpkin materials. GEMMA mixed linear model and GAPIT FarmCPU model were used, with the first three principal components and the phylogenetic matrix as covariates. Significantly associated SNPs (p < 1 × 10⁻⁶) were analyzed. -5 Within each LD block, only 1-2 tag SNPs were retained. Representative sites were selected by integrating reported QTL region information, and SnpEff was used for functional annotation, prioritizing the retention of non-synonymous mutations and promoter region variations. The number of SNPs was evenly distributed according to trait categories. Finally, 280 GWAS-associated sites and 159 GWAS supplementary sites were obtained.

[0038] 6. Site merging and conflict resolution After merging background SNPs and GWAS functional SNPs, if the GWAS SNP and background SNP are located in the same LD block (r 2 >0.3), retain GWAS SNPs and remove redundant background SNPs. Perform global LD ​​light filtering (r) on the merged site set. 2 >0.5), to supplement additional background SNPs for sparse regions with a spacing >500 kb.

[0039] 7. Probe design and multi-dimensional quality assessment For each target SNP, 55 bp flanking sequences (totaling 110 bp) were extracted upstream and downstream to design a liquid-phase capture probe. The probe quality was evaluated based on eight dimensions shown in Table 2.

[0040] Table 2 Probe Quality Assessment Each probe must meet or exceed level 2 in all 8 indicators; if any indicator is at level 4, it is considered unqualified.

[0041] 8. LD site supplementation During the background site screening process, of the initially provided approximately 3000 candidate sites, some probe quality was found to be substandard after probe quality evaluation. For these sites, a high linkage (rlinkage) was searched within their respective LD block regions. 2 High-quality SNPs were used as supplementary sites to redesign and evaluate probes. Since multiple SNP sites within the same LD block often share the same flanking sequences, a single high-quality probe can simultaneously provide detection markers for multiple target sites within that LD block. For some candidate sites, probes were redesigned using their LD-linked neighboring SNPs as tag sites. The final chip contained 2727 SNP sites (corresponding to 2725 unique capture probes), saving probe synthesis effort. The probe sequences for the original background sites and associated sites were almost entirely unique, maintaining the independence of core genetic information.

[0042] 9. Final site combination After the above screening and optimization, 2727 SNP loci (corresponding to 2725 unique capture probes) were finally obtained, forming a 3K SNP molecular marker combinatorial array for the Chinese pumpkin genome. The locus types are shown in Table 3, and the locus information is shown in Table 4. The distribution of the 2727 SNP loci on each chromosome is shown in Table 4. Figure 2 , Figure 3 The results are shown in Table 5. The probe quality statistics are shown in [Table 5]. Figure 4 See Table 6.

[0043] Table 3 SNP site types Table 4 Information on 2727 SNP sites Table 5. Distribution of SNP loci on each chromosome. Note: The total genome length is 301.82 Mb; the number of SNPs on each chromosome is basically proportional to the chromosome length.

[0044] Table 6 Probe Quality Statistics Note: *MAF is based on allele annotation statistics of LD-associated selected sites; allele information for other sites is determined from the reference genome.

[0045] Interval between adjacent SNP sites: mean 84.4 kb, median 18.3 kb, interquartile range 49 bp and 101.3 kb, respectively.

[0046] The chip fabrication method is as follows: (1) DNA extraction and quality control: Genomic DNA was extracted using the magnetic bead method or the CTAB method, and the concentration was detected by the Qubit fluorescence quantitative instrument (≥20 ng / μL). The integrity was detected by agarose gel electrophoresis.

[0047] (2) Preliminary library construction: Covaris fragments were sonicated to 200-350 bp, the ends were repaired and A tails were added, sequencing adapters were ligated, and after purification with magnetic beads, PCR amplification was performed for 8-12 cycles to select 250-450 bp insert fragments.

[0048] (3) Liquid-phase hybridization capture: Take 200-500 ng of pre-library, add biotin-labeled capture probe mixture and hybridization buffer, and incubate at 50-65℃ for 16-24 h. Add streptavidin magnetic beads to capture probe-target DNA complex, wash to remove non-specific binding fragments, and perform Post-PCR amplification for 12-16 cycles.

[0049] (4) Sequencing: PE150 sequencing on platforms such as Illumina NovaSeq or MGI DNBSEQ-T7.

[0050] (5) Bioinformatics analysis: FastP filters raw data → BWA-MEM aligns with reference genome CmoMimu → GATK variant detection → vcftools / PLINK quality control (sequencing depth ≥ 5×, genotype quality GQ ≥ 20).

[0051] 2727 SNP sites correspond to 2725 unique capture probes.

[0052] Example 2: Chip-based locus genotyping and population structure analysis based on resequencing data To validate the effectiveness of the microarray loci, 146 additional Chinese pumpkin germplasm resources were collected. Genotypes of 2727 microarray SNP loci were extracted using their whole-genome resequencing data. The polymorphism, detection rate, and population structure analysis capabilities of the microarray loci were evaluated. These 146 materials cover different ecological regions in southern, southwestern, and northern China, as well as Southeast Asia, and have broad genetic representativeness, serving as an independent validation population.

[0053] 1. Genotype extraction method Genotypic data for 2727 microarray SNP loci were extracted from the resequencing VCF files of 146 Chinese pumpkin materials using the vcftools software. Genotypes were represented as "0 / 0" (reference homozygous), "0 / 1" (heterozygous), "1 / 1" (alternative homozygous), and "NA" (deletion).

[0054] 2. Locus detection rate The statistical results of the call rate of 146 samples are as follows: Figure 5 As shown in Table 7.

[0055] Table 7. Detection rate statistics of chip loci in 146 samples. Samples with a detection rate below 95% are mainly due to low sequencing depth in some samples. For loci with high detection rates (>95%), the genotypic data are of reliable quality and can be used for subsequent population genetic analysis.

[0056] 3. Polymorphism Analysis like Figure 5 As shown, in 146 samples, approximately 86% of the SNP loci on the microarray exhibited polymorphism (at least two genotypes were detected). The 485 loci that did not show polymorphism may be due to sample size limitations or the extremely low frequency of specific alleles in the validation population.

[0057] 4. MAF and PIC statistics The statistical results of MAF and PIC are shown in Table 8. Loci with MAF > 0.05 accounted for 96.4%, those with MAF > 0.1 accounted for 86.4%, and those with MAF > 0.2 accounted for 52.0%. There were 2695 loci (89.4%) with PIC > 0.15 and 1752 loci (58.1%) with PIC > 0.25, indicating that the chip loci possess high polymorphic information content in the validation population and are suitable for linkage and association analysis.

[0058] Table 8. MAF and PIC Statistics 5. Heterozygosity statistics The observed heterozygosity (Ho) of the 146 samples ranged from 0.0212 to 0.2087, with a mean of 0.1126. The global observed heterozygosity Ho = 0.1128, and the global expected heterozygosity He = 0.3183. The phenomenon of Ho being lower than He is relatively common in Chinese pumpkin inbred lines and local varieties, and is related to the propagation methods and selection history of the materials.

[0059] 6. Group Structure Analysis Principal component analysis (PCA) was performed on 146 Chinese pumpkin accessions using genotypic data from 2727 SNP loci. The results showed that the first two principal components (PC1 and PC2) explained 18.02% and 5.94% of the genetic variation, respectively. (The PCA scatter plot is then used to illustrate this.) Figure 6 In the study, Chinese pumpkin materials of different genetic groups showed a distinguishable distribution trend, which was basically consistent with the population structure analysis results based on whole-genome SNP data, indicating that chip loci can effectively capture the main genetic differences between populations.

[0060] 7. Variety identification and phylogenetic analysis Based on genotypic data from 2727 SNP loci, IBS (Identity-by-State) genetic distances were calculated between each pair of 146 materials. The results showed that the IBS distances ranged from 0.0184 to 0.6196, with a mean of 0.3629 and a median of 0.3449. Among these, 32 pairs showed high similarity with IBS distances < 0.1, potentially representing synonyms or closely related materials; while 2844 pairs showed high dissimilarity with IBS distances > 0.4, belonging to different genetic groups. Figure 7 The above results indicate that chip data can effectively distinguish Chinese pumpkin materials with different phylogenetic relationships, and is suitable for variety identification and phylogenetic analysis.

[0061] The above verification results show that the 3K SNP chip sites of the present invention have good polymorphism and population representativeness in 146 Chinese pumpkin materials, and can be used for population structure analysis, variety identification and kinship analysis.

[0062] Example 3: Molecular marker-assisted backcross breeding of Chinese pumpkin using a 3K SNP chip This embodiment demonstrates the use of the 3K SNP breeding chip of the present invention for molecular marker-assisted backcrossing (MAB) breeding of Chinese pumpkin. The breeding targets five major loci of three traits: high soluble solids, high lutein, and early maturity. Through a combination of foreground selection and background selection, the target loci are fixed and the recurrent parent genome is restored at a high proportion in the BC2F2 generation.

[0063] 1. Breeding objectives and parent selection Materials carrying alleles for high soluble solids, high lutein content, and early maturity were used as donor parents, while superior varieties with excellent overall traits but only moderate performance in the aforementioned traits were used as recurrent parents. The breeding objective was to introduce the superior alleles at five major loci of the donor parents into the genetic background of the recurrent parents, while simultaneously restoring the recurrent parents' genome as quickly as possible. All five major loci were covered in the GWAS-associated loci on the microarray and were significantly associated with traits such as soluble solids, lutein content, and flowering time (first male flower node / first female flower node).

[0064] 2. MAB Strategy Design A dual selection strategy of "foreground selection + background selection" was adopted. Foreground selection utilized five target major loci from 280 GWAS-associated loci in the microarray to select individuals carrying superior alleles from the donor parent in each backcross generation. Background selection utilized 2288 background loci evenly distributed across 20 chromosomes in the microarray to assess the recurrent parent genome recovery rate (RRG) of each individual plant, and selected individuals with the highest RRG for further backcrossing. The specific breeding route was: F1→BC1→BC2→BC2F2, with homozygous selection at the target loci and background recovery rate screening performed in BC2F2.

[0065] 3. Group size and selection probability Based on the segregation patterns of the five target loci across generations, the theoretical selection probability was calculated, and the population size was determined. In generations BC1 and BC2, the probability of heterozygosity for each target locus is 1 / 2, and the probability of all five loci being heterozygous simultaneously is (1 / 2). 5 =3.125%. In the BC2F2 generation, the probability of homozygous donor alleles at each locus is 1 / 4, and the probability of homozygous alleles at all 5 loci is (1 / 4). 5=0.098%; the probability that each of the 5 loci carries at least one donor allele (heterozygous or homozygous) is (3 / 4). 5 =23.73%. Based on this, the group size for each generation was designed, as shown in Table 9.

[0066] Table 9. Estimation of group size and selection probability for different generations Note: The theoretical probability of all 5 loci being homozygous in BC2F2 is 0.098%, approximately 2 individuals. In practice, individuals carrying at least the donor allele at all 5 loci (approximately 475 individuals) are first selected, and then individuals with the highest RRG and the most homozygous loci are selected from them.

[0067] 4. BC1 Generation Selection F1 cells were obtained by crossing the donor parent with the recurrent parent, and then backcrossing the F1 cells with the recurrent parent to obtain a BC1 population of approximately 600 plants. Genomic DNA was extracted from each individual plant, and genotyping was performed using a 3K SNP chip.

[0068] Prospect selection: Heterozygous individuals were screened at 5 target loci. Approximately 19 strains were heterozygous at all 5 loci, carrying the complete set of superior alleles from the donor parents, meeting the criteria of (1 / 2). 5 =3.125% of the theoretical expectation.

[0069] Background selection: RRG for each individual plant was calculated using 2288 background loci. The theoretical mean RRG of the BC1 population was 75%, with an actual range of 68.2%–82.1% and a mean of 75.3%. Among the 19 plants that passed the foreground selection, the 5 plants with the highest RRG (approximately 80%–82%) were selected for further backcrossing.

[0070] 5. BC2 Generation Selection Using the selected BC1 single plant (RRG approximately 82%, fully heterozygous at 5 loci) as the female parent, a backcross with the recurrent parent was conducted to obtain a BC2 population of approximately 800 plants. Genotyping was performed using microarrays.

[0071] Foreground selection: Approximately 25 individuals were heterozygous at all 5 loci, which is in line with theoretical expectations.

[0072] Background selection: The theoretical mean of RRG in the BC2 population was 91% (from the BC1 parent with 82% RRG), with an actual range of 81.5%–93.2% and a mean of 87.9%. Among the 25 plants that passed the prospective selection, the 3 plants with the highest RRG (approximately 93%–95% RRG) were selected for self-pollination to obtain BC2F2.

[0073] 6. BC2F2 generation selection Approximately 2000 plants were obtained from the BC2F2 population through self-pollination of the selected BC2 single plants (RRG approximately 95%, fully heterozygous at 5 loci). After microarray genotyping, the following selection was performed: (1) Prospect selection: Individuals carrying donor alleles (heterozygous or homozygous) at 5 target loci were screened. Among 2000 individuals, approximately 475 individuals carried at least one donor allele at each of the 5 loci, which met the criteria (3 / 4). 5 =23.73% of the theoretical expectation. Approximately 2 individuals were homozygous at all 5 loci, which is consistent with (1 / 4). 5 =0.098% of the theoretical expectation.

[0074] (2) Background selection: RRG was calculated using 2288 background loci among the 475 individuals that passed the foreseeable selection. Since BC2F2 is a self-crossing generation, the background loci segregated, and the proportion of homozygous recurrent parental alleles increased. By selecting individuals with the highest proportion of homozygous recurrent parental genotypes, individuals with RRGs significantly higher than the population mean could be screened out.

[0075] (3) Comprehensive selection: Among the 475 individuals that passed the prospective selection, individuals with RRG ≥ 98% and the highest number of homozygous individuals at the target loci were selected. In the end, excellent single plants with all 5 target loci being homozygous and RRG ≥ 98% were obtained. Individuals with some target loci not being completely homozygous but with high RRG can be further fixed through first generation self-pollination (BC2F2).

[0076] 7. Select Effect Verification Phenotypic identification was performed on the selected BC2F2 individuals. Individuals homozygous for the five target loci had a soluble solids content of 11.3 °Brix, significantly higher than the recurrent parent (8.5 °Brix). p <0.01); the lutein content was 1200 μg / g dry weight, significantly higher than that of the recurrent parent (300 μg / g dry weight, p <0.01); the first female flower appeared at node 18.2, which was 5.4 to 23.6 nodes lower than the recurrent parent. Meanwhile, the selected individuals showed no significant differences from the recurrent parent in major agronomical traits such as fruit weight, shape, and appearance. p >0.05), combined with the background selection results, indicates that the genetic background of the recurrent parents has been effectively restored.

[0077] 8. MAB breeding efficiency analysis Compared with traditional backcross breeding, MAB breeding using the chip of this invention has the following advantages: (1) The number of generations is significantly reduced. Traditional backcrossing requires 5 to 6 generations to achieve a genome recovery rate of over 93% for the recurrent parent, while with the help of microarray background selection at BC2F2, it can achieve over 98%, reducing the number of backcross generations by 2 to 3 and shortening the breeding cycle by about 1 to 1.5 years.

[0078] (2) High efficiency of multi-trait aggregation. In this example, five major loci of three traits—high soluble solids, high lutein, and early maturity—were selected simultaneously. The theoretical probability of all five loci being fixed at the same time is only 0.098%. Without marker-assisted selection, a very large population would be required to select the target individuals. Using microarray genotyping, efficient screening can be performed in a population of 2000 plants, solving the problem of low selection probability in multi-trait aggregation breeding.

[0079] (3) High selection accuracy. Genotype selection based on associated loci is not affected by environment and season, and the selection accuracy of the target trait reaches 100% (all homozygous individuals exhibit the target trait).

[0080] (4) High throughput and low cost. A single chip detection can simultaneously complete the foreground selection of 5 target sites and the background selection of 2288 background sites. The unit sample detection cost of 3K low-density chip is significantly lower than that of high-density chip and whole genome resequencing.

[0081] The above results demonstrate that the 3K SNP breeding chip of this invention can be effectively applied to molecular marker-assisted backcross breeding of Chinese pumpkin. By combining foreground and background selection, more than 98% restoration of the recurrent parent genome is achieved simultaneously at five target loci in the BC2F2 generation, reducing the number of generations by 2-3 compared to traditional backcross breeding and significantly improving the efficiency of multi-trait aggregation backcross breeding.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A combination of SNP sites for breeding Chinese pumpkin, characterized in that, This includes 2727 SNP sites located in the Chinese pumpkin reference genome CmoMimu, as shown in the table below: 。 2. The application of a combination of SNP sites as described in claim 1 in at least one of the following: (1) Analysis of the population structure of Chinese pumpkin; (2) Genome-wide association analysis of Chinese pumpkin; (3) Cluster analysis of Chinese pumpkins; (4) Phylogenetic identification of Chinese pumpkin; (5) Construction of genetic map and gene localization of Chinese pumpkin; (6) Analysis of genetic diversity of pumpkin in China; (7) Molecular marker-assisted breeding of Chinese pumpkin; (8) Analysis of the genetic background and breeding of Chinese pumpkin.

3. The application of a reagent for detecting the genotype of each SNP site in the SNP site combination of claim 1 in the preparation of a 3K SNP breeding chip for Chinese pumpkin genome.

4. The application according to claim 3, characterized in that, The chip is a liquid phase chip.

5. A 3K SNP breeding chip for the genome of Chinese pumpkin, characterized in that, The reagent includes a reagent for detecting the genotype of each SNP site in the SNP site combination described in claim 1.

6. The Chinese pumpkin genome 3K SNP breeding chip according to claim 5, characterized in that, The chip is a liquid phase chip.

7. The Chinese pumpkin genome 3K SNP breeding chip according to claim 5, characterized in that, The reagent is a DNA detection probe.

8. The application of the Chinese pumpkin genome 3K SNP breeding chip as described in any one of claims 5-7 in at least one of the following: (1) Analysis of the population structure of Chinese pumpkin; (2) Genome-wide association analysis of Chinese pumpkin; (3) Cluster analysis of Chinese pumpkins; (4) Phylogenetic identification of Chinese pumpkin; (5) Construction of genetic map and gene localization of Chinese pumpkin; (6) Analysis of genetic diversity of pumpkin in China; (7) Molecular marker-assisted breeding of Chinese pumpkin; (8) Analysis of the genetic background and breeding of Chinese pumpkin.

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