SNP (Single Nucleotide Polymorphism) molecular marker combination for capsicum population structure analysis and application of SNP molecular marker combination

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker combination for pepper population structure analysis, the SNP molecular marker combination comprises 1068 SNP molecular markers, the physical positions of the 1068 SNP molecular markers are determined based on pepper Hot1 genome sequence comparison, and the specific SNP molecular marker condition is shown in the specification table 1. The marker combination is uniformly distributed on 12 chromosomes of capsicum and has high polymorphism. A liquid-phase probe chip based on the SNP marker has the characteristics of high marking density, strong automation, high detection flux and the like, and is more beneficial to establishment of a pepper population grouping system; the marker combination can provide theoretical and data support for new pepper strain identification, molecular marker-assisted selective breeding, whole genome selection, population genetic diversity analysis and the like, and has relatively important theoretical value and application significance.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and in particular relates to a SNP molecular marker combination for pepper population structure analysis and an application thereof. Background Art

[0002] Pepper (Capsicum spp) is one of the earliest cultivated crops, including five domesticated varieties: annual pepper (C. annuum L. CA), bush pepper (C. fiutescens L. CF), Chinese pepper (C. chinense Jacg. CC), drooping pepper (C. baccatum L. CB) and hairy pepper (C. pubescens Ruiz & Pav. CP). Pepper is native to tropical Asia and is distributed all over the world. It is cultivated most in Asia, accounting for about 74% of the world's total production; Europe is second, accounting for about 14%. As an important industrial strategic material, high-spiciness industrial pepper has a wide range of uses in food, sterilization, medicine, military and other industries due to its high capsaicin content and strong spiciness. China, as the world's major producer of peppers, mainly relies on international imports in the production and application of industrial peppers. Domestic industrial pepper hybrid varieties with high capsaicin content are scarce, which restricts the industrialization development of industrial peppers. The resources of the Chinese pepper CC cultivar are widely used in the combination of industrial pepper hybrids due to its high capsaicin content and high spiciness. At the same time, other high-quality and multi-resistant cultivars have also been introduced to improve the disease resistance and agronomic traits of the varieties. Therefore, the use of modern biotechnology to analyze the genetic background, genetic diversity and correlation differences between the Chinese pepper CC cultivar and other cultivars will help the development, utilization and variety breeding of germplasm resources, and accelerate the research and development of high-quality, high-yield and multi-functional industrial pepper varieties.

[0003] Chinese patent 202311662361.8 discloses a pepper SNP molecular marker combination, which is composed of loci related to important agronomic traits / genes such as pepper disease resistance, pepper fruit color, pepper spiciness and pepper male sterility, but does not involve the application of pepper population grouping. Therefore, in order to improve the efficiency of hybrid breeding and shorten the breeding cycle, it is necessary to propose a method for rapid identification of pepper populations based on molecular markers. Summary of the invention

[0004] In view of this, the present invention screened out 1068 polymorphic SNP marker combinations by resequencing the whole genome of 500 pepper varieties, and successfully distinguished the pepper CC strain from the non-CC strain. This marker combination can be used for the identification of new pepper varieties, molecular marker-assisted selection breeding, whole genome selection, population genetic diversity analysis, etc., and has the advantages of a wide variety of applicable materials, low cost, and high efficiency.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] One of the purposes of the present invention is to provide a SNP molecular marker combination for pepper population structure analysis, the SNP molecular marker combination comprises 1068 SNP molecular markers, the physical positions of the 1068 SNP sites are determined based on the comparison of the pepper "Hot1" genome sequence, and the specific SNP molecular markers are shown in Table 1 of the specification.

[0007] The second object of the present invention is to provide a liquid phase probe combination for pepper population structure analysis, the liquid phase probe combination includes probes for detecting the above-mentioned SNP molecular marker combination, and the specific situation of the probe is shown in Table 1 of the specification.

[0008] A third object of the present invention is to provide a kit for analyzing pepper population structure, the kit comprising the above-mentioned liquid phase probe combination.

[0009] The fourth object of the present invention is to provide the application of the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in the analysis of pepper population structure.

[0010] A fifth object of the present invention is to provide the use of the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in the identification of new pepper varieties.

[0011] The sixth object of the present invention is to provide the application of the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in pepper breeding.

[0012] The seventh object of the present invention is to provide the application of the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in the genetic diversity analysis of pepper population.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention provides a set of SNP molecular marker combinations for pepper population structure analysis, wherein the SNP molecular marker combination comprises 1068 SNP molecular markers. The marker combination is evenly distributed on the 12 chromosomes of pepper and has high polymorphism. The liquid phase probe chip based on the SNP marker has the characteristics of high labeling density, strong automation, and high detection throughput, which is more conducive to establishing a pepper population clustering system; the marker combination can provide theoretical and data support for the identification of new pepper varieties, molecular marker-assisted selection breeding, whole genome selection, population genetic diversity analysis, etc., and has relatively important theoretical value and application significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the density distribution diagram of 1068 SNP markers screened by the present invention.

[0016] Figure 2 This is the PCA result diagram of the whole genome SNP markers.

[0017] Figure 3 This is the result diagram of the whole genome SNP marker evolutionary tree.

[0018] Figure 4 This is the PCA result diagram of 1068 SNP markers screened by the present invention.

[0019] Figure 5 This is the evolutionary tree result diagram of 1068 SNP markers screened by the present invention.

[0020] Figure 6 This is the PCA result diagram of 1068 SNP markers corresponding to 19 samples in Example 3.

[0021] Figure 7 This is the evolutionary tree result diagram of 1068 SNP markers corresponding to 19 samples in Example 3. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0023] Example 1

[0024] This example screened out 1068 polymorphic SNP marker combinations based on the collected whole genome resequencing data of 500 pepper varieties. The specific steps are as follows:

[0025] (1) We used 500 whole genome resequencing data published on NCBI, whose BioProject is PRJNA800056.

[0026] (2) The clean data obtained by sequencing in step (1) was used as the reference genome of pepper "Hot1" (NCBI>BioProject: PRJNA1175375), and the clean data was aligned to the genome using bwa, and GATK mutation detection was performed to obtain high-quality SNP sites. The SNP sites were split according to 115 pepper CC groups and 385 pepper non-CC groups, and 85081260 SNP sites and 90086040 SNP sites were obtained respectively;

[0027] (3) The SNP sites selected in step (2) were further filtered using vcftools software under the condition “minDP 3min alleles2max alleles 2max missing 0.8maf 0.05”. 31,308,905 high-quality SNP sites and 67,736,907 SNP sites were obtained from the 115 pepper CC groups and 385 pepper non-CC groups, respectively. Population structure analysis was performed. The results are shown in Figure 2 (Genome-wide SNP marker PCA results), Figure 3 (Results of genome-wide SNP marker evolutionary tree).

[0028] (4) The SNP sites screened out in step (3) were used to screen the polymorphic markers of pepper CC group and non-CC group respectively using perl program script, and then the common SNP marker combination was combined to finally obtain 1068 SNP marker combinations (the density distribution of 1068 SNP markers is shown in Figure 1 ), the population structure SNP molecular markers were successfully constructed, and the marker information is shown in Table 1.

[0029] The screening principles are:

[0030] The chromosomes are evenly distributed, and the distance between two markers is greater than 500 kb;

[0031] The bedtools software was used to extract the SNP site and the 100 bp sequence upstream and downstream. The SNP sites with a GC content between 40-60% and no N base in the sequence were retained;

[0032] The extracted sequences were aligned to the pepper "Hot1" genome using blastn software, and only single-copy SNP sites were retained.

[0033] (5) The 1068 SNPs screened in step (4) were subjected to population structure analysis. The results are shown in Figure 4 (SNP marker PCA results corresponding to 1k), Figure 5 (1k corresponding SNP marker evolution tree results) As can be seen from the figure, the analysis results are basically consistent with the whole genome SNP results, and it is concluded that the 1068 SNP marker combination can be used for pepper population structure analysis.

[0034] (6) Design liquid phase probe sequences for the 1068 SNPs screened in step (5) for subsequent capture sequencing. The probe information is shown in Table 1.

[0035] Table 11068 SNP markers and their probe details

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[0136] Example 2

[0137] This embodiment provides a method for detecting pepper using the 1068 SNP corresponding liquid phase probes described in Example 1, which specifically includes the following steps:

[0138] (1) Genomic DNA extraction

[0139] Pepper leaves were collected, stored with ice packs and promptly transported back to the laboratory, and the total DNA of peppers was extracted using the magnetic bead method.

[0140] (2) Genomic DNA fragmentation and end repair

[0141] 1. Prepare the following reaction system in a sterile PCR tube on ice.

[0142] Table 2 List of whole genome DNA fragmentation systems

[0143] Reagents volume InputDNA XμL(250ng) Buffer 4.5μL Enzymes 5μL 1×TEBuffer Up to 30 μL Total volume 30μL

[0144] Wherein, X represents any volume not exceeding 20 μL, which is added with 4.5 μL of Buffer and 5 μL of Enzymes, and the part less than 30 uL is supplemented with 1×TE Buffer.

[0145] 2. Use a pipette to pipette up and down or shake to ensure that the system is fully mixed, and then centrifuge briefly.

[0146] 3. Immediately place the PCR tube on a PCR instrument preheated to 32°C, run the reaction program shown in Table 4, and set the thermal cover of the PCR instrument to 75°C; after the temperature drops to 4°C, immediately place it on ice for the next experiment, and do not keep it at 4°C.

[0147] Table 3 Fragmentation and end repair reaction procedures

[0148] temperature time 32℃ 20min 65℃ 30min 4℃ Cool to 4℃ and proceed to the next step immediately

[0149] (3) Connector

[0150] 1. Prepare the reaction system as shown in Table 4.

[0151] Table 4 Reaction system for connecting linkers

[0152]

[0153]

[0154] Among them, all operations were performed on ice.

[0155] 2. Place the PCR tube on the PCR instrument. The reaction program is 22℃, 15min. The PCR instrument does not have a hot cover. Then store it at 4℃ (proceed to the next step immediately after cooling to 4℃).

[0156] 3. DNA sample purification.

[0157] 3.1 Add 44 μL (0.8×) DNA Clean Beads to each sample and mix well; incubate at room temperature for 5 minutes;

[0158] 3.2 Place the PCR tube on the magnetic rack for 3 minutes. After the solution becomes clear, remove the supernatant.

[0159] 3.3 Add 180 μL 80% ethanol to rinse the beads, incubate for 30 seconds, remove the supernatant, and repeat the operation once;

[0160] 3.4 Keep the PCR tube on the magnetic rack, use a 10μL pipette to remove the ethanol remaining at the bottom of the tube, and dry until no ethanol remains;

[0161] 3.5 Resuspend the magnetic beads in 21 μL ddH2O and let stand at room temperature for 1 min to fully release the DNA on the magnetic beads;

[0162] 3.6 Place the PCR tube on the magnetic stand for 2 minutes and transfer 20 μL of supernatant to a new PCR tube for library amplification.

[0163] (4) Library amplification and purification

[0164] 1. Prepare the PCR reaction system as shown in Table 5 below, mix by pipetting and centrifuge briefly.

[0165] Table 5 PCR reaction system

[0166] Reagents volume Adapter-ligated DNA 20μL 2×PCRMix 12.5μL PCRIndexPrimer 2.5μL UniversalPCRPrimer 2.5μL Total volume 35μL

[0167] 2. The prepared reaction system is placed in the PCR amplification program shown in Table 6 for amplification;

[0168] Table 6 PCR amplification program

[0169]

[0170] 3. Add 35 μL (1×) DNA Clean Beads to the amplified product, mix well, and incubate at room temperature for 5 minutes;

[0171] 4. Place the sample on the magnetic rack for 2 minutes. After the solution becomes clear, remove the supernatant.

[0172] 5. Add 200 μL 80% ethanol to rinse the magnetic beads, incubate for 30 seconds, remove the supernatant, and repeat this step once;

[0173] 6. Keep the PCR tube on the magnetic rack, use a 10μL pipette to remove the ethanol remaining at the bottom of the tube, open the tube cap and dry until no ethanol remains;

[0174] 7. Resuspend the magnetic beads in 31 μL ddH2O and let stand at room temperature for 1 min to fully release the DNA on the magnetic beads;

[0175] 8. Place the sample on the magnetic stand for 2 minutes, transfer 30 μL of supernatant to a new PCR tube, and store the library at 20°C for subsequent library quality testing and sequencing.

[0176] (5) Library and probe hybridization

[0177] 1. Take 750 ng of the library constructed in step (4) and add it to a PCR tube and mark it;

[0178] 2. Add purified magnetic beads to the library and mix gently with a pipette;

[0179] 3. Incubate at room temperature for 5 minutes and place the PCR tube on a magnetic rack for 3 minutes to clarify the solution;

[0180] 4. Remove the supernatant, place the PCR tube on the magnetic stand, add 180 μL 80% ethanol, and let stand for 30 seconds;

[0181] 5. Remove the supernatant, add 180 μL 80% ethanol to the PCR tube, let stand for 30 seconds, and then completely remove the supernatant;

[0182] 6. Leave at room temperature for 5 minutes to allow the residual ethanol to evaporate completely;

[0183] 7. Add the hybridization reaction system to the PCR tube according to Table 9 below;

[0184] Table 7 Hybridization reaction system

[0185] Reagents volume HybBuffer 13μL HybHumanBlock 5μL AdapterBlocker 2μL RnaseBlock 5μL NucleaseFreeWater 3μL Total volume 28μL

[0186] 8. Mix by gently pipetting, let stand at room temperature for 3 minutes, centrifuge briefly, place the PCR tube on a magnetic rack, and let stand for 3 minutes;

[0187] 9. Pipette 28 μL of supernatant into a new PCR tube, add 2 μL of Target Probe, gently pipette to mix, and centrifuge briefly;

[0188] 10. Set the PCR instrument parameters as follows: hot cover temperature: 85℃; 80℃ 5min; 50℃ hold; place the PCR tube on the PCR instrument, run the above program, and incubate overnight.

[0189] (6) Capture target region DNA library

[0190] 1. Magnetic Bead Pretreatment

[0191] 1.1 Remove the capture magnetic beads from 4°C, vortex and resuspend, and place at room temperature for 30 minutes;

[0192] 1.2 Take 50 μL of magnetic beads and add them to a new PCR tube. Place it on a magnetic rack for 1 minute to allow the solution to clarify and remove the supernatant.

[0193] 1.3 Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, gently pipette several times to mix, and resuspend the magnetic beads; place on the magnetic stand for 1 minute, remove the supernatant; repeat this step once;

[0194] 1.4 Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, gently pipette and resuspend the magnetic beads for later use;

[0195] 2. Capture target region DNA library

[0196] 2.1 Keep the hybridization product on the PCR instrument, add 180 μL of capture magnetic beads resuspended in step 1 to the hybridization product, mix with a pipette, and place on a rotating mixer at room temperature for 30 minutes;

[0197] 2.2 Place the PCR tube on a magnetic rack for 2 minutes to clarify the solution and remove the supernatant;

[0198] 2.3 Add 150 μL of 50℃ preheated Wash Buffer, gently pipette to mix, then centrifuge briefly, and incubate on a constant temperature oscillating mixer at 50℃ for 10 min;

[0199] 2.4 Centrifuge briefly, place the PCR tube on a magnetic rack for 2 minutes to clarify the solution, and remove the supernatant; repeat this step twice, washing the magnetic beads 3 times in total;

[0200] 2.5 Keep the sample on the magnetic rack, add 150 μL 80% ethanol to the PCR tube, let it stand for 30 seconds, then completely remove the ethanol solution and dry it at room temperature;

[0201] 2.6 Add 24 μL Nuclease-free Water to the PCR tube, remove the PCR tube from the magnetic stand, and use a pipette to gently resuspend and mix the magnetic beads for later use.

[0202] (7) Post-capture PCR amplification

[0203] 1. Take out Post PCR Master Mix and Post PCR Primer from the -20℃ refrigerator, place them on an ice box to melt, mix well and place on ice or at 4℃ for later use.

[0204] 2. After capture, the DNA library needs to be amplified by PCR. Prepare the reaction system according to Table 10 below:

[0205] Table 8 PCR reaction system

[0206] Reagents volume Step (6) Capturing the DNA library of the target region 24μL PostPCRMasterMix 25μL PostPCRPrimer (select according to library type) 1μL Total volume 50μL

[0207] 3. Adjust the pipette to 40 μL, gently pipette and mix 6 times, and then immediately place on the PCR instrument;

[0208] 4. Run the PCR instrument program: Hot cover temperature: 105℃ Program: 95℃ 1min; 98℃ 20s; 60℃ 30s N cycles; 72℃ 30s; 72℃ 5min; 4℃ hold;

[0209] 5. After PCR, add 55 μL of purified magnetic beads to the sample, vortex or pipette to mix, and let stand at room temperature for 5 minutes;

[0210] 6. Briefly centrifuge and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clarify;

[0211] 7. Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL 80% ethanol solution into the PCR tube, and let it stand for 30 seconds;

[0212] 8. Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and then completely remove the supernatant; let it stand at room temperature for 5 minutes to allow the residual ethanol to evaporate completely;

[0213] 9. Add 25 μL of Nuclease-free water, remove the PCR tube from the magnetic rack, shake or pipette to mix 10 times, and let stand at room temperature for 2 minutes;

[0214] 10. Briefly centrifuge and place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify;

[0215] 11. Use a pipette to transfer 23 μL of supernatant to a 1.5 mL centrifuge tube and mark the sample information;

[0216] 12. Take 1 μL of library and use Qubit dsDNA HS Assay Kit for quantification and record the library concentration, which is about 1-20 ng / μL;

[0217] 13. Take 1 μL of sample and use Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit) to measure the fragment length.

[0218] (8) High-throughput sequencing

[0219] The library obtained after capture and amplification in step (7) was subjected to high-throughput sequencing using the MGI DNBSEQ-T7 sequencing platform to obtain the sequencing results of the tree species genomic DNA, and the obtained data was basically cleaned.

[0220] (9) Liquid phase probe capture rate evaluation

[0221] 1. Use bwa software to align the cleaned sequencing data to the pepper "Hot1" reference sequence, and use GATK software to obtain the SNP genotyping data of pepper varieties;

[0222] 2. Use SNP genotyping data to complete the population structure analysis of pepper varieties.

[0223] Example 3

[0224] This example provides a method for evaluating the capture efficiency of liquid probes for 19 pepper varieties using the method in Example 2. The specific steps are as follows:

[0225] (1) Experimental Materials

[0226] 19 pepper leaves were selected, refrigerated and promptly transported to the laboratory, and the total DNA of each individual plant was extracted by magnetic bead method. The sample numbers are shown in Table 9.

[0227] Table 9 Detailed information of 19 samples (2) Population genetic analysis

[0228]

[0229] The results of genetic clustering of pepper population based on 1068 SNPs (see Figure 6 , Figure 7 ): The actual grouping results of the 19 test samples were consistent with the theoretical grouping results. The 19 pepper materials could be divided into 4 groups, namely CC, CA, CF and CP, and the CC cultivars could be distinguished from other cultivars.

[0230] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0231] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A SNP molecular marker combination for pepper population structure analysis, characterized in that: The SNP molecular marker combination includes 1068 SNP molecular markers, and the physical positions of the 1068 SNP sites are determined based on the comparison of the pepper "Hot1" genome sequence. The specific SNP molecular markers are shown in Table 1 of the specification.

2. A liquid phase probe combination for pepper population structure analysis, characterized in that: The liquid phase probe combination includes probes for detecting the SNP molecular marker combination according to claim 1.

3. A kit for analyzing pepper population structure, comprising the liquid phase probe combination according to claim 2.

4. Use of the SNP molecular marker combination according to claim 1, the liquid phase probe combination according to claim 2 or the kit according to claim 3 in the analysis of pepper population structure.

5. Use of the SNP molecular marker combination according to claim 1, the liquid phase probe combination according to claim 2 or the kit according to claim 3 in identifying new pepper varieties.

6. Use of the SNP molecular marker combination according to claim 1, the liquid phase probe combination according to claim 2 or the kit according to claim 3 in pepper breeding.

7. Use of the SNP molecular marker combination according to claim 1, the liquid phase probe combination according to claim 2 or the kit according to claim 3 in the analysis of genetic diversity of pepper populations.

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