A zucchini whole genome gene chip and application thereof

Abstract

The application provides a SNP site combination, a molecular probe combination, a zucchini whole genome gene chip and an application thereof. The SNP site combination comprises at least one SNP site in Table 1, and the physical position information of the SNP site in Table 1 is determined based on the alignment of the genomic sequence of the zucchini reference genome 22S20 or an updated version thereof. Specifically, the application provides a zucchini whole genome 10K liquid phase gene chip and an application thereof. The zucchini whole genome 10K liquid phase gene chip can be used for high-depth resequencing of a zucchini target site, and accurate detection of the genotype of the SNP site. Meanwhile, the zucchini whole genome 10K liquid phase gene chip has the advantages of low cost and high efficiency, which helps to speed up the zucchini breeding process and provides strong technical support for the development and utilization of zucchini germplasm resources.

Description

Technical Field

[0001] This application belongs to the field of gene chip technology, specifically, it relates to a zucchini whole genome gene chip and its application; more specifically, it relates to a zucchini whole genome 10K liquid phase gene chip and its application. Background Technology

[0002] Zucchini (Cucurbita pepo L.) is an annual vine-like herbaceous plant belonging to the genus Cucurbita in the family Cucurbitaceae, and is a type of cucurbit vegetable. The utilization of hybrid vigor has played a significant role in the healthy development of my country's zucchini industry. However, the development of first-generation zucchini hybrids relies on the breeding process of their inbred lines. Therefore, traditional breeding methods face problems such as long breeding cycles and high degree of uncertainty, resulting in low efficiency in the selection of new varieties.

[0003] Modern biotechnology has provided new directions for zucchini breeding. Currently, published patents involve molecular markers for genes related to traits such as seed hull absence, seed length, pericarp color, leaf notch gene Cpdll, hypocotyl color, dark green stem, number of lateral branches, and short vines, as well as molecular markers for resistance genes against zucchini ZYMV virus, PRSV-W virus, and powdery mildew. However, these markers are still severely insufficient and far from supporting the construction of a molecular design breeding technology system for zucchini. Therefore, developing a simple and efficient whole-genome typing tool has become a key problem that urgently needs to be solved in current zucchini molecular design breeding.

[0004] Single nucleotide polymorphisms (SNPs) refer to variations in a single nucleotide (A, T, C, G) in the genome, with a frequency >1% in the population. Compared to traditional molecular markers, SNPs are characterized by their large number, wide distribution, ease of rapid large-scale screening, and ease of genotyping, and are widely used in biology and agricultural breeding. For species with existing reference genomes, whole-genome resequencing and solid-state gene chip technology are two methods for rapid genotyping. However, whole-genome resequencing technology is costly, time-consuming to acquire data, produces large amounts of data, and is difficult to analyze, requiring specialized data platforms and bioinformatics personnel, making it difficult for general breeders to utilize. Furthermore, while solid-phase gene chips are low-cost, their initial research and development costs are high, and they lack flexibility and have slow detection rates. Compared to solid-phase gene chips, liquid-phase gene chip technology offers advantages such as high flexibility, low cost, and high-density labeling, demonstrating enormous application potential in agricultural breeding.

[0005] Liquid-phase gene chip technology has been applied in various species for germplasm resource identification, kinship identification, genetic mapping, gene localization, genome-wide selection breeding, genome-wide association analysis, and gene data mining. However, there are currently no liquid-phase gene chip products specifically for zucchini on the market. Therefore, developing a liquid-phase gene chip suitable for zucchini is of great significance for accelerating the molecular breeding process of zucchini. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this application is to provide a whole genome gene chip of zucchini and its application.

[0007] Specifically, this application relates to the following aspects:

[0008] 1. A combination of SNP sites, wherein the combination of SNP sites comprises at least one SNP site from Table 1;

[0009] The physical location information of the SNP sites in Table 1 was determined based on the genome sequence alignment of the zucchini reference genome 22S20 or its updated version.

[0010] 2. A molecular probe assembly, wherein the molecular probe assembly is used to detect SNP site assemblies as described in item 1.

[0011] 3. The application of the SNP site combination described in item 1 and / or the molecular probe combination described in item 2 in the preparation of a whole genome gene chip of zucchini;

[0012] Preferably, the gene chip is a liquid-phase gene chip.

[0013] 4. A whole genome gene chip for zucchini, wherein the genotyping sites of the gene chip include at least one SNP site listed in Table 1;

[0014] The physical location information of the SNP sites in Table 1 was determined based on the genome sequence alignment of the zucchini reference genome 22S20 or its updated version.

[0015] 5. The zucchini whole genome gene chip according to item 4, wherein the genotyping sites of the gene chip include 10,667 SNP sites as shown in Table 1.

[0016] 6. A whole genome gene chip for zucchini, wherein the gene chip is loaded with the molecular probe combination as described in item 2.

[0017] 7. A zucchini whole genome gene chip according to any one of items 4-6, wherein the gene chip is a liquid phase gene chip.

[0018] 8. A kit comprising a molecular probe combination as described in item 2, a zucchini whole genome gene chip as described in any one of items 4-7, or probes and / or primers for detecting a combination of SNP sites as described in item 1.

[0019] 9. The application of the SNP site combination according to item 1, the molecular probe combination according to item 2, the whole genome gene chip of zucchini according to any one of items 4-7, or the kit according to item 8 in the localization of color genes in the main stem of zucchini.

[0020] 10. The application of the SNP site combination according to item 1, the molecular probe combination according to item 2, the zucchini whole genome gene chip according to any one of items 4-7, or the kit according to item 8 in the genome-wide association analysis of the longitudinal diameter of zucchini fruit.

[0021] 11. The application of the SNP site combination described in item 1, the molecular probe combination described in item 2, the zucchini whole genome gene chip described in any one of items 4-7, or the kit described in item 8 in the identification of zucchini germplasm resources, identification of kinship, construction of genetic maps, gene mapping, molecular marker-assisted selection, assessment of genetic diversity, whole genome breeding or assisted breeding, whole genome selection, whole genome association analysis and gene data mining.

[0022] 12. The application of the SNP site combination according to item 1, the molecular probe combination according to item 2, the zucchini whole genome gene chip according to any one of items 4-7, or the kit according to item 8 in zucchini genotyping.

[0023] Beneficial effects:

[0024] (1) The 10K liquid phase gene chip of the whole genome of zucchini provided in this application contains 10,667 SNP markers, which can fully cover the key regions of the zucchini genome and provide rich genetic information for molecular breeding of zucchini.

[0025] (2) The 10K liquid phase gene chip of zucchini whole genome provided in this application ensures the accuracy and reliability of the chip detection results through strict SNP site screening and quality control, as well as scientific verification methods. Compared with solid phase chips, it has the advantages of low cost, large detection volume and fast detection speed.

[0026] (3) The 10K liquid phase gene chip of zucchini whole genome provided in this application can also be applied to multiple fields such as germplasm resource identification, molecular marker-assisted breeding, and whole genome selection of Cucurbita plants. Attached Figure Description

[0027] Figure 1 This is a distribution map of 10,667 SNP loci on the chromosome of the whole genome of zucchini using a 10K liquid phase gene chip, with a window size of 10 kb.

[0028] Figure 2 A statistical diagram of 10,667 SNP loci from the whole genome of zucchini on different chromosomes using a 10K liquid phase gene chip.

[0029] Figure 3 Map showing the gene loci regulating the "dark green to light green" color of zucchini main stem.

[0030] Figures 4A-4B show the results of genome-wide association analysis of the longitudinal diameter trait of zucchini fruit. Figure 4A is a Manhattan plot of the longitudinal diameter trait of zucchini fruit. Figure 4B A QQ image showing the longitudinal diameter of zucchini fruit. Detailed Implementation

[0031] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0032] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0033] definition

[0034] As used in this article, the terms “comprising,” “including,” “having,” “containing,” etc., are all open-ended terms, meaning that they include, but are not limited to, those included in.

[0035] As used in this article, the term "SNP" stands for Single Nucleotide Polymorphism, which refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level. An SNP site is the specific location in the DNA sequence where a single nucleotide variation occurs.

[0036] As used herein, unless otherwise stated, the term "whole genome" encompasses not only the entire genome sequence of an organism but also the genomes of organelles (such as the mitochondrial genome).

[0037] In a first aspect, this application provides a combination of SNP sites, which includes at least one SNP site from Table 1, such as at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least one hundred, at least one thousand, at least one hundred thousand, at least one hundred thousand, or all 10,667 SNP sites.

[0038] The physical location information of the SNP sites in Table 1 (i.e., the specific location of the SNP on the chromosome) was determined based on the genome sequence alignment of the zucchini reference genome 22S20 or its updated version.

[0039] It should be noted that the SNP site combinations in this application are not limited to including all the SNP sites listed in Table 1. As long as the SNP combination includes the physical location information of one or more SNP sites determined based on the zucchini reference genome 22S20 (or its updated version) in Table 1, it falls within the protection scope of this application.

[0040] In some embodiments, the SNP locus combination includes 10,667 SNP loci listed in Table 1, and the physical location information of the SNP loci is determined based on the genome sequence alignment of the zucchini reference genome 22S20.

[0041] In some implementations, the SNP sites are combined into 10,667 SNP sites as shown in Table 1, and the physical location information of the SNP sites is determined based on the 22S20 sequence alignment of the zucchini reference genome.

[0042] Specifically, this application uses whole-genome resequencing to determine the SNP site combination of the zucchini, and probes can be designed for each SNP site.

[0043] Secondly, this application provides a molecular probe assembly for detecting the SNP site combination described in the first aspect of this application.

[0044] In some embodiments, the molecular probe assembly includes probes for detecting the 10,667 SNP sites in Table 1.

[0045] In some embodiments, the molecular probe array is used to detect the 10,667 SNP sites in Table 1.

[0046] Thirdly, this application provides a primer set for detecting the SNP site combination described in the first aspect of this application.

[0047] Fourthly, this application provides the application of the SNP site combination described in the first aspect of this application, the molecular probe combination described in the second aspect of this application, and / or the primer set described in the third aspect of this application in the preparation of zucchini whole genome gene chip.

[0048] It should be understood that the SNP site combinations provided in this application are universal and adaptable to various high-throughput genotyping platforms. Those skilled in the art can design probes suitable for different detection systems using the SNP site combinations according to actual needs, including but not limited to liquid-phase gene chips, solid-phase gene chips, and hybridization-capture-based targeted sequencing platforms. In some embodiments, the gene chip is a liquid-phase gene chip.

[0049] Fifthly, this application provides a whole genome gene chip for zucchini, wherein the genotyping sites of the gene chip include at least one SNP site listed in Table 1, for example, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least one hundred, at least one thousand, at least one hundred thousand, at least one hundred thousand, or all 10,667 SNP sites.

[0050] The physical location information of the SNP sites in Table 1 (i.e., the specific location of the SNP on the chromosome) was determined based on the genome sequence alignment of the zucchini reference genome 22S20 or its updated version.

[0051] It should be noted that the genotyping sites in the gene chip of this application are not limited to all SNP sites listed in Table 1. As long as the genotyping site contains the physical location information of one or more SNP sites determined based on the zucchini reference genome 22S20 (or its updated version) in Table 1, it falls within the protection scope of this application.

[0052] In some embodiments, the gene chip is a liquid-phase gene chip.

[0053] Specifically, the gene chip is loaded with the molecular probe combination described in the second aspect of this application.

[0054] Specifically, the gene chip is a 10K liquid-phase gene chip of the whole genome of zucchini, and its genotyping sites include 10,667 SNP sites as shown in Table 1. The physical location information of the SNP sites is determined based on the 22S20 sequence alignment of the zucchini reference genome.

[0055] Preferably, the genotyping sites of the 10K liquid phase gene chip of the whole genome of zucchini are the 10667 SNP sites in Table 1, and the physical location information of the SNP sites is determined based on the 22S20 sequence alignment of the zucchini reference genome.

[0056] Those skilled in the art will understand that the 10K liquid-phase gene chip for the whole genome of zucchini in this application has greater flexibility than traditional solid-phase chips, and can adjust the SNP density of the gene chip according to actual needs, providing multiple versions such as 10K, 5K, and 1K.

[0057] In some embodiments, the zucchini whole genome 10K liquid phase gene chip contains primer sets and / or probes for detecting SNP sites (including 10,667 SNP sites in Table 1).

[0058] In some embodiments, the zucchini whole genome 10K liquid phase gene chip includes a 10K probe mixture for capturing SNP sites (including 10,667 SNP sites in Table 1) and a targeting capture reagent. Preferably, the zucchini whole genome 10K liquid phase gene chip is composed of individually packaged 10K probe mixture and targeting capture reagent.

[0059] The 10K probe mixture contains probes with high sequence specificity, which can effectively identify and detect SNP sites, and the targeting capture reagent can efficiently and stably capture SNP sites.

[0060] Furthermore, the design of the 10K probes for the 10K liquid-phase gene chip of the whole genome of zucchini follows the following principles: the probe length is 110bp, the GC content of the probe is 30%-70%, the number of homologous regions of the probe sequence on the whole genome is ≤5, the probe region does not contain repetitive sequences and NA regions, and it is evenly distributed on the chromosome.

[0061] Furthermore, the design of the 10K probes for the 10K liquid-phase gene chip of the whole genome of zucchini follows the following principle: with the SNP site as the center, nucleotide sequences with a GC content of about 45% on the left and right sides of the center are selected as probes.

[0062] Sixthly, this application provides a method for preparing the zucchini whole genome gene chip described in the fifth aspect of this application.

[0063] It should be understood that preparing gene chips (e.g., liquid-phase gene chips) using known SNP site information is routine in this field.

[0064] Specifically, this application provides a method for preparing a 10K liquid-phase gene chip of the whole genome of zucchini.

[0065] In some embodiments, the method for preparing a 10K liquid-phase gene chip of the whole genome of zucchini includes:

[0066] Second-generation resequencing data of 248 zucchini breeding inbred lines were aligned to the zucchini reference genome 22S20 using BWA-mem, and SNP sites were screened to obtain them.

[0067] SNP calling was performed using GATKV4.1 to further evaluate and filter SNP sites, and SNP sites with a deletion rate <0.1%, heterozygosity rate <0.15% and MAF ≥0.35 were selected as a preliminary SNP library.

[0068] Further screening was conducted by determining whether the flanking sequences of SNP sites were suitable for probe design. The screening criteria were: probe length of 110 bp, GC content of 30%-70%, number of homologous regions of the probe sequence in the whole genome ≤ 5, probe regions do not contain repetitive sequences and NA regions, and are evenly distributed on chromosomes.

[0069] The obtained 110bp nucleotide sequence was used to synthesize a single-stranded DNA with a biotinylated group at the 5' end to prepare a probe.

[0070] The prepared probes were coupled onto fluorescent microspheres, and a targeting capture reagent was added to obtain the gene chip.

[0071] The homology is calculated as follows: a) completely identical for more than 40 bp; b) 85% similarity, length 80 bp; c) 95% similarity, length 70 bp.

[0072] In some embodiments, the method for preparing a 10K liquid-phase gene chip of the whole genome of zucchini further includes:

[0073] During probe preparation, nucleotide sequences with approximately 45% GC content on either side of the SNP site are selected as probes.

[0074] In a seventh aspect, this application provides a kit comprising the molecular probe combination described in the second aspect of this application, the primer set described in the third aspect of this application, or the zucchini whole genome gene chip described in the fifth aspect of this application.

[0075] Eighthly, this application provides the application of the SNP site combination described in the first aspect of this application, the molecular probe combination described in the second aspect of this application, the primer set described in the third aspect of this application, the zucchini whole genome gene chip described in the fifth aspect of this application, or the kit described in the seventh aspect of this application in the localization of color genes in the main stem of zucchini.

[0076] Ninthly, this application provides the application of the SNP site combination described in the first aspect of this application, the molecular probe combination described in the second aspect of this application, the primer set described in the third aspect of this application, the zucchini whole genome gene chip described in the fifth aspect of this application, or the kit described in the seventh aspect of this application in the genome-wide association analysis of the longitudinal diameter of zucchini fruit.

[0077] Tenthly, this application provides the application of the SNP site combination described in the first aspect of this application, the molecular probe combination described in the second aspect of this application, the primer set described in the third aspect of this application, the zucchini whole genome gene chip described in the fifth aspect of this application, or the kit described in the seventh aspect of this application in the identification of zucchini germplasm resources, identification of kinship, construction of genetic maps, gene mapping, molecular marker-assisted selection, assessment of genetic diversity, whole genome breeding or assisted breeding, whole genome selection, whole genome association analysis and gene data mining.

[0078] In the eleventh aspect, this application provides the application of the SNP site combination described in the first aspect of this application, the molecular probe combination described in the second aspect of this application, the primer set described in the third aspect of this application, the zucchini whole genome gene chip described in the fifth aspect of this application, or the kit described in the seventh aspect of this application in zucchini genotyping.

[0079] In a twelfth aspect, this application provides a method for genotyping, zucchini breeding, and / or genome-wide association analysis, the method comprising: using the zucchini whole genome gene chip (specifically, a zucchini whole genome 10K liquid phase gene chip) described in the fifth aspect of this application for detection or analysis.

[0080] In a thirteenth aspect, this application provides a method for locating the color gene of the main stem of zucchini, the method comprising: using the whole genome gene chip of zucchini described in the fifth aspect of this application (specifically, the whole genome 10K liquid phase gene chip of zucchini) for analysis.

[0081] In a fourteenth aspect, this application provides a method for genome-wide association analysis of the longitudinal diameter of zucchini fruit, the method comprising: using the zucchini whole genome gene chip (specifically, the zucchini whole genome 10K liquid phase gene chip) described in the fifth aspect of this application for analysis.

[0082] In this application, the inventors discovered a novel zucchini genome sequence (zucchini reference genome 22S20), which has significant advantages over currently published zucchini genomes, specifically in the following ways: higher genome integrity and longer sequence; significantly improved assembly quality; more comprehensive and accurate genome annotation; broader coverage of genetic variations; and the ability to be fabricated into high-density gene chip designs.

[0083] This application develops a whole-genome gene chip for zucchini based on the newly discovered zucchini reference genome 22S20. Specifically, this application develops a 10K liquid-phase gene chip for the whole genome of zucchini based on targeted gene capture technology using liquid-phase probe hybridization, which contains 10,667 SNP markers. Using the 10K liquid-phase gene chip for the whole genome of zucchini developed in this application, high-depth resequencing of target loci in zucchini can be performed, achieving accurate detection of SNP loci genotypes. At the same time, this 10K liquid-phase gene chip for the whole genome of zucchini has the advantages of low cost and high efficiency, which helps to accelerate the zucchini breeding process and provides strong technical support for the development and utilization of zucchini germplasm resources.

[0084] Example

[0085] The following description, in conjunction with specific embodiments, illustrates the content of this application, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following embodiments are all conventional reagents and instruments in the art and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described schemes and obtain corresponding results based on the embodiments.

[0086] Example 1: Development of a 10K liquid-phase gene chip for the whole genome of zucchini

[0087] The zucchini reference genome 22S20 used in this application (National Genome Science and Technology Center https: / / ngdc.cncb.ac.cn / gwh, Accession number GWHGPYM00000000.1) was obtained by the inventors through T2T genome sequencing in the early stage. The specific steps are as follows: Zucchini material with the number 22S20 (provided by the Zucchini Breeding Laboratory of the Cotton Research Institute of Shanxi Agricultural University) was planted and leaves were collected. After extracting genomic DNA, a sequencing library was constructed. Hifi sequencing and ONT sequencing were then performed, and finally the T2T genome was assembled.

[0088] This embodiment provides a 10K liquid phase chip for zucchini. The information of 10667 SNP sites in the 10K zucchini chip is shown in Table 1 below. In Table 1, the 10667 SNP sites are evenly distributed across 20 pairs of chromosomes: chromosome CpChr01 contains 441 SNP sites; chromosome CpChr02 contains 477 SNP sites; chromosome CpChr03 contains 483 SNP sites; chromosome CpChr04 contains 528 SNP sites; chromosome CpChr05 contains 441 SNP sites; chromosome CpChr06 contains 441 SNP sites; chromosome CpChr07 contains 700 SNP sites; chromosome CpChr08 contains 456 SNP sites; chromosome CpChr09 contains 520 SNP sites; chromosome CpChr... Chromosome CpChr10 contains 681 SNP sites; chromosome CpChr11 contains 466 SNP sites; chromosome CpChr12 contains 960 SNP sites; chromosome CpChr13 contains 729 SNP sites; chromosome CpChr14 contains 484 SNP sites; chromosome CpChr15 contains 423 SNP sites; chromosome CpChr16 contains 525 SNP sites; chromosome CpChr17 contains 579 SNP sites; chromosome CpChr18 contains 410 SNP sites; chromosome CpChr19 contains 523 SNP sites; and chromosome CpChr20 contains 400 SNP sites. The reference (Ref) indicates the base of the SNP site on the reference genome, and the position indicates the specific location of the SNP site on the chromosome.

[0089] Table 1. Information on 10,667 SNP sites in the 10K liquid-phase gene chip of the whole genome of zucchini.

[0090]

[0091] The specific screening process for 10K liquid phase chips for zucchini is as follows:

[0092] (1) Screening of zucchini germplasm resources

[0093] To ensure the representativeness of the zucchini data and the universality of the prepared liquid-phase gene chip, 248 zucchini breeding inbred lines provided by the Cotton Research Institute of Shanxi Agricultural University were used as the source of resequencing data.

[0094] (2) Zucchini whole genome resequencing

[0095] ① DNA was extracted from different varieties of zucchini using the magnetic bead method, and the concentration of the DNA samples was detected using a micro spectrophotometer; the integrity of the DNA samples was detected by 1% agarose gel electrophoresis, and samples that passed quality control were used for library preparation.

[0096] ② Sequencing libraries were constructed using the standard method developed by BGI (Shenzhen BGI Genomics Co., Ltd.);

[0097] ③ High-throughput sequencing was performed using the Shenzhen BGI sequencing platform (MGI). The sequencing strategy was PE150, the sequencing depth was 10×, and the data volume for each zucchini material was 10Gb.

[0098] ④ The resequencing data of different zucchini varieties were compared and variants were detected. The analysis process is as follows:

[0099] a. Use Sentieon to align reads to the zucchini reference gene 22S20, sort by position, and mark duplicate reads;

[0100] b. Use Sentieon to detect variant sites for each sample and obtain the gVCF file for each sample;

[0101] c. Use Sentieon to perform joint-calling analysis on the gVCF files of all samples to obtain the variation results for each individual in the population.

[0102] (3) Screening of SNP sites in 10K liquid phase microarray for zucchini

[0103] SNP loci were obtained by aligning the resequencing data of 248 zucchini breeding inbred lines to the zucchini reference genome 22S20 using BWA-mem. SNP calling (single nucleotide polymorphism identification) was then performed using GATK v4.1, and the SNP loci were further evaluated and filtered. Finally, 12,436 SNP loci with a deletion rate <0.1%, heterozygosity <0.15%, and minor allele frequency (MAF) ≥0.35 were selected as the preliminary SNP library.

[0104] (4) Design and prepare 10K liquid-phase gene chip SNP site probes for the whole genome of zucchini, as follows:

[0105] ① Probe design principles: The probe length is 110bp, the CG content of the probe is 30-70%, the number of homologous regions is ≤5 (homology calculation: 40bp or more is completely identical; similarity 85%, length 80bp; similarity 95%, length 70bp), the probe region does not contain repetitive sequences and NA regions; uniform distribution principle of chromosomes.

[0106] ② Using the SNP site obtained in step (3) as the center, select the sequence with GC content on the left and right sides of the center that is closer to 45% as the probe according to the probe design principle;

[0107] ③ Using the 110bp nucleotide sequence obtained from screening in step (4) ②, a single-stranded DNA with a biotinylate group modified at the 5' end is synthesized, which is called the zucchini 10K SNP site probe.

[0108] ④ The probe prepared in step (4) is coupled to fluorescent microspheres and a targeting capture reagent is added to obtain a 10K liquid phase gene chip of the whole genome of zucchini, which contains 10667 SNP sites.

[0109] The distribution map of 10667 SNP loci on chromosomes of the whole genome of zucchini using a 10K liquid phase gene chip is shown below. Figure 1 As shown, the statistical graphs on different chromosomes are as follows: Figure 2 As shown.

[0110] Example 2: Application of 10K liquid-phase gene chip in the localization of color genes in zucchini main stem

[0111] (1) Source of the experimental group

[0112] The dark green main stem variety P1 and the light green main stem variety P2 were crossed to obtain the F1 population. The F1 population was then self-crossed to obtain the F2 segregating population. The color of the main stem of 267 individual plants in the segregating population was visually counted, and the results showed a segregation ratio of 3:1.

[0113] (2) Detection of samples using a 10K liquid phase gene chip of the whole genome of zucchini.

[0114] ① DNA extraction and quality inspection

[0115] DNA was extracted from leaf samples of two parents and 100 randomly selected individual plants from the zucchini segregation population using a high-throughput DNA extraction kit. The concentration of DNA samples was detected using a micro spectrophotometer, and the purity, integrity, and contamination of the DNA were analyzed by 1% agarose gel electrophoresis.

[0116] ② Library construction and sequencing library construction

[0117] The qualified DNA is randomly fragmented using an ultrasonic disruptor, and the DNA fragments of the required length are recovered by electrophoresis. Adapters are added to the ends of the fragments to form a library. The sample library is then amplified by LM-PCR and purified to form a sequencing library, which can be used for probe hybridization experiments.

[0118] ③ Construction and quality control of hybridization capture libraries

[0119] Take 300 ng of the constructed sequencing library, freeze-dry it, add it to the 10K liquid gene chip of the whole genome of zucchini and hybridization reagent, denature it and incubate it at 65℃ for 6 hours to complete the hybridization reaction; after washing the hybridization product with washing solution, perform another round of PCR to complete the construction of the hybridization capture library; use Qubit 2.0 for preliminary quantification, and use qPCR to accurately quantify the effective concentration of the library to ensure library quality.

[0120] ④ Sequencing

[0121] Sequencing was performed using an Illumina sequencer and related reagents to obtain the sequencing data. The raw sequencing reads were processed using FASTP software to remove low-quality reads and adapter sequences. BWA software was used to align the reads with the zucchini reference genome 22S20, and the alignment files for each sample were obtained. Bedtools software was then used to analyze the sequence coverage of each probe site. Samtools software was used to sort the alignment results in BAM files, remove PCR duplicates, and calculate alignment statistics. GATK software was used to generate the original population VCF file.

[0122] (3) Locating the color gene of the main stem of zucchini using the results of 10K liquid phase gene chip detection of the whole genome of zucchini.

[0123] The data obtained from the chip detection and the color data of the zucchini main stem were imported into JoinMap 4.0 software to obtain the genetic map. The results are as follows: Figure 3 As shown, the regulatory gene loci for the "dark green-light green" color of the zucchini main stem were located within a 1.02 Mb physical interval between two loci, CpChr05_1405215 and CpChr05_2425054.

[0124] Example 3: Application of 10K liquid-phase gene chip in whole genome analysis of zucchini fruit longitudinal diameter in genome-wide association study (GWAS)

[0125] (1) Source of the experimental group

[0126] The long cylindrical fruit variety P1 and the short round fruit variety P2 were crossed to obtain the F1 population. The F1 population was then self-crossed to obtain the segregating population. The longitudinal diameter of 100 randomly selected individual fruits in the segregating population was measured using a centimeter ruler.

[0127] (2) Detection of samples using a 10K liquid phase gene chip of the whole genome of zucchini.

[0128] The detection was performed according to the method described in Example 2.

[0129] (3) GWAS analysis of the longitudinal diameter of zucchini fruit was performed using the results of 10K liquid phase gene chip detection of the whole genome of zucchini.

[0130] Data quality control was performed using plink software to ensure accuracy and reliability. The quality-controlled data were then imported into R software, and phenotypic-genotypic association analysis under a mixed linear model (MLM) was performed using the gapit package. Finally, GWAS visualization was completed using the ggplot package. The results are shown in Figures 4A-4B. Through GWAS analysis, the significance threshold was set at log... 10 SNPs with a p value greater than 6 are considered to be SNPs that are significantly associated with the longitudinal diameter of zucchini fruit; based on this criterion, one gene associated with the longitudinal diameter of zucchini fruit is located on chromosome Cpchr06.

[0131] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A 10K liquid-phase gene chip for the whole genome of zucchini, wherein the 10K liquid-phase gene chip for the whole genome of zucchini is loaded with a combination of molecular probes for detecting SNP site combinations, wherein the SNP site combinations are 10667 SNP sites in Table 1, and the physical location information of the 10667 SNP sites is determined based on the 22S20 sequence alignment of the zucchini reference genome; Table 1 。 2. A kit comprising the 10K liquid-phase gene chip of the whole genome of zucchini as described in claim 1.

3. The application of the 10K liquid phase gene chip of zucchini whole genome according to claim 1, or the kit according to claim 2, in the localization of color genes in the main stem of zucchini.

4. The application of the 10K liquid phase gene chip of zucchini whole genome according to claim 1, or the kit according to claim 2, in the genome-wide association analysis of the longitudinal diameter of zucchini fruit.

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