Capsicum SNP molecular marker combination, SNP chip and use thereof

Abstract

Provided are a capsicum SNP molecular marker combination, an SNP chip, and a use thereof. The SNP molecular marker combination is composed of 51,172 SNP molecular markers having high polymorphism and a high detection rate, the SNP molecular markers being composed of 47,589 SNP sites evenly distributed on chromosomes, 3,194 MNP marker sites, and 389 sites related to important agronomic traits / genes such as capsicum disease resistance, capsicum fruit color, capsicum spiciness and capsicum male sterility. A 50K liquid-phase breeding chip for capsicum is also provided, in combination with the molecular markers. The chip has high detection throughput, a high target site detection rate, and accurate and reliable typing results. It can be applied to the identification of new capsicum varieties, molecular marker-assisted selective breeding, gene location, whole genome selection, population genetics, and population evolution analysis.

Description

A pepper SNP molecular marker combination, SNP chip and its application Technical Field

[0001] The present invention belongs to the technical field of plant molecular genetic breeding, and in particular relates to a pepper SNP molecular marker combination, a SNP chip and applications thereof. Background Art

[0002] Pepper (Capsicum annuum L.), a member of the Solanaceae family, is an important condiment and colorant. The pepper industry is the largest vegetable sector in my country and boasts significant economic value. Pepper cultivation has a long history, with a rich and diverse variety resource. Conventional pepper breeding has achieved significant success in recent years, but with rising consumer demand, the demand for diverse pepper varieties, including taste, color, and health benefits, is increasing. Providing high-quality varieties is the current development direction of pepper breeding.

[0003] Conventional pepper breeding relies primarily on morphological identification of field plants, but this method is limited by low accuracy, long identification time, high investment costs, and susceptibility to environmental influences. Furthermore, as the number of pepper varieties increases, phenotypic differences between varieties are becoming increasingly limited, making morphological identification even more difficult. China, not the origin of pepper, has a relatively limited germplasm resource base. While expanding its innovative varieties, China also needs to import high-quality germplasm from abroad, especially considering the scarcity of wild and specialized resources. Therefore, there is an increasing demand for the breeding of high-quality pepper varieties using modern molecular breeding techniques.

[0004] Currently, the commonly used molecular marker identification methods include RFLP, RAPD and SSR technologies, which have the following disadvantages: RFLP (restriction endonuclease fragment length polymorphism) technology is complex to operate, has high requirements on DNA quality, is expensive, and radioactive isotopes are harmful to the human body; RAPD technology (random amplified polymorphic DNA) results are unstable and have poor repeatability; SSR primer development is expensive and is not suitable for large-scale commercial identification; none of them meet the requirements of an ideal detection method.

[0005] Compared with other detection methods, the liquid-phase breeding chip based on SNP markers has the characteristics of high marker density, strong automation, and high detection throughput. It can establish a modern breeding system and promote the development of pepper breeding.

[0006] Summary of the Invention

[0007] In order to overcome the problems in the prior art, the present invention provides a pepper SNP molecular marker combination, a SNP chip and its application, which can be used for identification of new pepper varieties, molecular marker-assisted selection breeding, gene mapping, whole genome selection, population genetics, population evolution, etc.

[0008] In order to solve the above technical problems, the technical solutions proposed by the present invention are as follows:

[0009] The first aspect of the present invention provides a pepper SNP molecular marker combination, which consists of 51,172 SNP molecular markers. The 51,172 SNP molecular markers are shown in Table 1. The position and variation information of the SNP site are expressed in the form of chromosome: physical position: reference genotype / variant allele type. The physical position of the SNP molecular marker combination is based on the positioning analysis of the Zhangshugang pepper reference genome.

[0010] The source of the Zhangshugang pepper reference genome can be found in the paper "Genomes of cultivated and wild Capsicum species provide insights into pepper domestication and population differentiation."

[0011] The detailed information of 51,172 SNP molecular markers is shown in Table 1.

[0012] Table 1: SNP molecular marker information

[0013] Based on the same technical concept, the second aspect of the present invention further provides a pepper SNP chip, which includes probes or primers for detecting the above-mentioned pepper SNP molecular marker combination.

[0014] The present invention also provides the use of the pepper SNP molecular marker combination or pepper SNP chip in pepper genetic diversity analysis.

[0015] The invention also provides the use of the above-mentioned pepper SNP molecular marker combination or pepper SNP chip in pepper population structure analysis.

[0016] The invention also provides the use of the above-mentioned pepper SNP molecular marker combination or pepper SNP chip in pepper genetic background breeding analysis.

[0017] The invention also provides the use of the pepper SNP molecular marker combination or pepper SNP chip in identifying the genetic relationship of peppers.

[0018] The invention also provides the use of the above-mentioned pepper SNP molecular marker combination or pepper SNP chip in pepper whole genome association analysis.

[0019] The invention also provides the use of the above-mentioned pepper SNP molecular marker combination or pepper SNP chip in pepper variety identification.

[0020] The invention also provides the use of the above-mentioned pepper SNP molecular marker combination or pepper SNP chip in molecular marker-assisted selection breeding.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This method utilizes resequencing data from pepper germplasm resources, including major domestic and international pepper cultivars and improved local varieties from Hunan, to screen for genome-wide SNPs with high polymorphism (mean PIC value of 0.30), high detection rate (mean 99.5%), and uniform distribution. It also identifies MNP marker sites for plant variety identification. Furthermore, sites associated with 26 important pepper agronomic trait genes, including those for pungency, disease resistance, and fruit color and development, were added to develop a set of 51,172 SNP molecular markers, forming a high-density liquid-phase breeding chip for pepper. This chip boasts high detection throughput, a high target site detection rate, and accurate and reliable typing results. It can be applied to identifying new pepper varieties, molecular marker-assisted selection breeding, gene mapping, whole-genome selection, population genetics, and population evolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] FIG1 is a flow chart of site screening in Example 1 of the present invention;

[0025] FIG2 is a distribution diagram of PIC values ​​of 51,172 SNP sites in Example 1 of the present invention;

[0026] FIG3 is a distribution diagram of 51,172 SNP sites on chromosomes in Example 1 of the present invention;

[0027] FIG4 is a population structure analysis diagram of 139 pepper materials in Example 2 of the present invention;

[0028] Figure 5 is a PCA scatter plot of 139 pepper materials in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0030] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0032] Example 1

[0033] This embodiment provides a pepper SNP molecular marker combination, which consists of 51,172 SNP molecular markers. The 51,172 SNP molecular markers are shown in Table 1 in the Summary of the Invention section. The screening process is shown in FIG1 and is specifically as follows:

[0034] 1. Collection of pepper germplasm resources

[0035] In order to ensure the representativeness of pepper materials and the universality of liquid phase arrays, we collected 176 pepper germplasm resource data provided by Hunan Agricultural University and performed resequencing.

[0036] At the same time, resequencing data of more than 300 pepper varieties, including cultivated varieties, wild varieties, breeding varieties of different origins and diverse fruit shapes, were collected from gene banks in Asia, America, Africa and Europe.

[0037] 2. Pepper Whole Genome Resequencing

[0038] The 176 pepper samples collected were subjected to whole genome resequencing. The specific steps included:

[0039] (1) Extract DNA from pepper material using magnetic bead method.

[0040] (2) The MGI standard library construction method was used, and those that passed the quality inspection were used for DNA-seq sequencing library construction.

[0041] (3) The libraries that passed the quality inspection were sequenced using the BGI sequencing platform (MGI) with a sequencing strategy of PE150 and a sequencing depth of 10×, with 30 Gb sequenced for each strain.

[0042] The 176 resequencing data sets were aligned and variants were detected. The analysis process is as follows:

[0043] (1) Use Sentieon to align reads to the pepper reference genome (Zhangshugang pepper), positionally sort, and mark duplicate reads.

[0044] (2) Perform mutation site detection on each sample to obtain the mutation information of each sample.

[0045] (3) Perform a joint analysis of the gVCF of all samples to obtain the variation results of each individual in the population.

[0046] 3. Development of a Liquid-Phase Breeding Chip for Pepper

[0047] (1) Whole-genome loci

[0048] a. Resequencing variant sites: 176 pepper germplasm resources provided by Hunan Agricultural University were resequenced and approximately 130 million SNP variant sites were extracted. Resequencing data from over 300 pepper varieties collected domestically and internationally were used to extract approximately 76 million SNP variant sites. A total of 66 million shared SNP variant sites were screened.

[0049] b. Candidate site screening: Calculate site quality indicators and select SNP polymorphic sites with a heterozygosity rate <0.2, site deletion frequency <0.1, minimum allele frequency >0.1, site polymorphism >0.15, and sequencing depth >5x as candidate sites. A total of approximately 2 million SNP sites were obtained.

[0050] c. Site probe design and screening: Extract 50bp of sequence upstream and downstream of the SNP site and analyze its specificity, GC content, and other factors to determine whether the probe is suitable for design. Approximately 860,000 SNP sites that can be used for chip development were screened.

[0051] d. Site density screening: Based on the principle of uniform site distribution, SNP sites evenly distributed on pepper chromosomes were screened, including a total of 47,589 SNP sites with an average spacing of 60 Kb.

[0052] (2) Pepper MNP sites

[0053] According to the pepper MNP marker primer sequences published in "Plant Variety Identification—MNP Marker Method" (GB / T 38551-2020), they were aligned to the pepper reference genome, and a total of 831 target intervals were obtained. High-quality SNP sites in the intervals were extracted as candidate sites, totaling 3194 SNP sites.

[0054] (3) Identification of important genes / functional sites

[0055] We mined genes related to important agronomic traits of peppers, such as disease resistance, fruit color, spiciness, and male sterility, reported in the literature. We screened for high-quality SNPs within the gene region and the 1K interval upstream and downstream. A total of 389 SNPs were included. The selected gene types and marker numbers are shown in Table 2 below:

[0056] Table 2: Gene types and marker numbers

[0057] 4. Development of a 50K SNP liquid phase array for pepper

[0058] Resequencing analysis was performed on pepper germplasm resources from Hunan Agricultural University, combining a diverse collection of pepper materials (including major international cultivars, Hunan local varieties, and varieties with diverse origins and fruit shapes) with those from Hunan Agricultural University. SNPs were identified. Based on the quality indicators of these SNPs, 47,589 SNPs, 3,194 MNP markers, and 389 loci associated with important agronomic traits / genes, such as disease resistance, fruit color, pungency, and male sterility, were selected to form a 50K liquid-phase array for pepper.

[0059] Polymorphism analysis of the pepper 50K chip SNP loci: The polymorphism of the 50K SNP loci in the selected pepper was statistically analyzed, with an average polymorphism information content (PIC) of 0.30. The PIC value distribution is shown in Figure 2.

[0060] Chromosomal distribution of SNP sites in the pepper 50K chip: The distribution of the screened 50K chip on the 12 chromosomes was statistically analyzed, and it was found that the 50K SNP sites were evenly distributed on the 12 pepper chromosomes, and the average spacing of the SNP sites on the chromosomes was 60Kb. The site distribution map is shown in Figure 3.

[0061] The 51,172 selected SNPs were then mapped using Huazhi's proprietary liquid-phase probe pinpoint sequencing (cGPS) technology, developed into a Chili 50K liquid-phase array. This liquid-phase array technology uses an optimized thermodynamic stability algorithm to design specific probes for target interval sequences (3K-150K). Synthesized specific probes are then used to capture and enrich multiple target sequences at different genomic locations through liquid-phase hybridization. The captured and enriched target intervals are then subjected to library construction and next-generation sequencing to determine the genotype of the target SNP.

[0062] The technique involves the following steps:

[0063] (1) Sample DNA extraction and quality control: Sample DNA was extracted using the magnetic bead method. The concentration of the DNA sample was determined using a Qubit fluorescence quantifier. The integrity of the DNA sample was determined using 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0064] (2) Library construction and quality control: a. Use fragmentase to digest the DNA sample, repair the digested ends, and add A base to the 3' end, and use agarose gel electrophoresis to detect the fragment size. b. Use T4 ligase to connect the sequencing adapter and DNA fragments, and use magnetic beads to purify the ligation product. The concentration of the purified product is detected by Qubit fluorescence quantitative instrument, and the fragment size is detected by agarose gel electrophoresis. c. PCR amplify the purified ligation product, and use magnetic beads to screen the fragments of the amplified product. The concentration of the fragment-screened product is detected by Qubit fluorescence quantitative instrument, and the fragment size is detected by agarose gel electrophoresis. d. Take 200 ng of the constructed library, add probes and hybridization reagents, and incubate at 50°C for 16-24 hours to complete the hybridization reaction. Use streptavidin magnetic beads to capture the target segment, use washing solution to wash the captured product, remove non-specific binding fragments, and then perform another round of PCR amplification. Library concentration was measured using a Qubit fluorometer, and fragment size was determined by agarose gel electrophoresis. Once the concentration and fragment size were qualified, sequencing library construction was complete. The prepared library was subjected to high-throughput sequencing using a BGI sequencer, using the PE150 sequencing strategy.

[0065] (3) Bioinformatics analysis: ①. Raw data filtering: The raw sequencing sequences (Raw Reads) obtained by sequencing are filtered to obtain high-quality Clean Reads, and the data are quality controlled using FASTP software. ②. Contamination detection: The sequences are aligned to the NCBI NT database using BLAST software for contamination assessment. ③. Reference genome alignment: The sequencing reads are aligned to the reference genome using BWA software, and the positions are sorted to obtain the bam file after sample sorting. ④. Mutation detection: a. Use GATK software to detect the variant sites of each sample and obtain the variant result files of each sample and population. ⑤. Target site genotyping: The target site is judged based on the ratio of the number of support reads of different Alles at the site. When the mutation read support ratio is ≥0.8 or ≤0.2, the site is judged as a homozygous genotype. When the mutation read support ratio is between 0.2-0.8, it is judged as a heterozygous genotype. Finally, 28 converted variant result files are obtained, which can obtain the genotyping results of each target SNP in a specific individual with high throughput, realizing high-throughput SNP genotyping.

[0066] Example 2

[0067] Application of pepper SNP molecular marker combination in polymorphism and population structure analysis of pepper breeding materials.

[0068] Using the developed pepper 50K liquid-phase breeding chip, 139 pepper materials were tested and the target site genotypes were extracted. The average detection rate of the samples was 99.63%, and the repetition consistency rate of the same batch of materials was 99.99%, indicating that the liquid-phase chip target site detection rate of this scheme is high and the typing results are accurate and reliable.

[0069] Plink software was used to calculate the genetic distance matrix and perform cluster analysis on pepper breeding materials. A phylogenetic tree was constructed to determine the genetic relationships, evolutionary relationships, and compositional structure of different materials. Cluster analysis of 139 pepper materials from different populations using the present invention revealed that the pepper breeding materials were divided into three subgroups, consistent with the actual clustering. The analysis results, shown in Figure 4, indicate that the 51,172 SNP sites screened were highly representative.

[0070] Plink software was used to analyze the PCA (Principal Component Analysis) components of the test materials and construct a PCA scatter plot. Each locus in the scatter plot represents a sample. The greater the distance between two samples in the plot, the greater the difference in their genetic backgrounds. Individuals with similar genetic backgrounds are clustered together in the plot. It was found that the pepper breeding materials were divided into three distinct groups, as shown in Figure 5.

[0071] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

Claims

1. A pepper SNP molecular marker combination, characterized in that: The SNP molecular marker combination consists of 51,172 SNP molecular markers, as shown in Table 1. The position and variation information of the SNP site are expressed in the form of chromosome: physical position: reference genotype / variant allele type. The physical position of the SNP molecular marker combination is based on the positioning analysis of the Zhangshugang pepper reference genome.

2. A pepper SNP chip, characterized in that: The pepper SNP chip comprises probes or primers for detecting the pepper SNP molecular marker combination described in claim 1.

3. Use of the pepper SNP molecular marker combination as described in claim 1 or the pepper SNP chip as described in claim 2 in pepper genetic diversity analysis.

4. Use of the pepper SNP molecular marker combination as described in claim 1 or the pepper SNP chip as described in claim 2 in pepper population structure analysis.

5. Use of the pepper SNP molecular marker combination as described in claim 1 or the pepper SNP chip as described in claim 2 in pepper genetic background breeding analysis.

6. Use of the pepper SNP molecular marker combination as described in claim 1 or the pepper SNP chip as described in claim 2 in identifying the genetic relationship of pepper.

7. Use of the pepper SNP molecular marker combination as described in claim 1 or the pepper SNP chip as described in claim 2 in pepper genome-wide association analysis.

8. Use of the pepper SNP molecular marker combination according to claim 1 or the pepper SNP chip according to claim 2 in pepper variety identification.

9. Use of the pepper SNP molecular marker combination as described in claim 1 or the pepper SNP chip as described in claim 2 in molecular marker-assisted selection breeding.

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