SNP (Single Nucleotide Polymorphism) marker combination for pepper genotyping and application of SNP marker combination

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker combination for pepper genotyping, the SNP molecular marker combination comprises 30462 SNP molecular markers, the physical positions of the 30462 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 number of liquid phase probe capture sites corresponding to the SNP marker is large, the capture rate is high, the capture efficiency of a CC group is 90% or above, the capture efficiency of a non-CC group CA and a non-CC group CF is 79% or above, and the capture efficiency of a CP group is about 51%. The genetic background difference between the CP group and the sequencing group is large, but the CP group can capture about 15k markers and can also complete genetic background evaluation, and the sequencing depth is more than 7X. Overall, genetic background evaluation can be completed by capturing the marker number.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and particularly relates to a SNP marker combination for pepper genotyping 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 most cultivated in Asia, accounting for about 74% of the world's total production; Europe is second, accounting for about 14%. It is cultivated in various parts of China and is one of the main vegetables in summer. Hybridization between different pepper populations is the main way to create new varieties. Detecting the genetic background of different populations of breeding offspring helps to screen breeding offspring and shorten the breeding cycle. Capture sequencing can detect a large number of SNP markers at the same time, with low detection costs, which helps to screen the genetic background of breeding offspring.

[0003] Therefore, in order to improve the efficiency of hybrid breeding and shorten the breeding cycle, it is necessary to propose a method based on molecular markers to quickly identify pepper populations. Summary of the invention

[0004] In view of this, the present invention screened out 30462 polymorphic SNP marker combinations by resequencing data of 500 pepper varieties, and successfully distinguished pepper CA group, CC group, CF group and CP group. 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 genotyping, the SNP molecular marker combination comprises 30462 SNP molecular markers, the physical positions of the 30462 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 genotyping, wherein the liquid phase probe combination includes probes for detecting the above-mentioned SNP molecular marker combination.

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

[0009] A fourth object of the present invention is to provide a chili chip, wherein the chip comprises the above-mentioned liquid phase probe combination.

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

[0011] The fifth object of the present invention is to provide the application of the above-mentioned SNP molecular marker combination, the above-mentioned liquid phase probe combination, the above-mentioned kit or the above-mentioned chip in any one of the identification of new pepper varieties, pepper breeding, pepper population genetic diversity analysis, and pepper population structure analysis.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention provides a set of SNP markers for pepper genotyping, totaling 30,462. The 30,462 markers are evenly distributed on each chromosome of the whole genome. The target SNP markers can be accurately detected by detecting pepper samples using liquid phase capture technology. In terms of detection rate, the capture efficiency of the CC group is above 90%, the non-CC group CA and CF group is above 79%, and the CP group is about 51%. The CP group has a large difference in genetic background with the sequencing group, but it can capture about 15k markers and complete the genetic background assessment. The sequencing depth is above 7X; overall, the number of captured markers can complete the genetic background assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a diagram showing the distribution of full chromosome markers of pepper of the present invention. DETAILED DESCRIPTION

[0015] 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.

[0016] Example 1

[0017] This example screened out 30,462 polymorphic SNP marker combinations based on the collected whole genome resequencing data of 500 pepper varieties. The specific steps are as follows:

[0018] (1) We used 500 whole genome resequencing data published on NCBI, whose BioProject is PRJNA800056.

[0019] (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 to obtain 100111632 high-quality SNP sites.

[0020] (3) The SNP sites screened out in step (2) were further filtered using vcftools software according to the condition “minDP 3min alleles2max alleles 2max missing 0.8maf0.05” to obtain 90,100,468 SNP sites.

[0021] (4) The SNP sites screened out in step (3) were used to screen SNP molecular combinations using a perl program script, and ultimately 2,002,232 SNP markers were obtained.

[0022] The marker screening principles are:

[0023] The bedtools software was used to extract the SNP sites and the sequences 100 bp upstream and downstream. The SNP sites without simple repeat sequences were retained;

[0024] The bedtools software was used to extract the SNP site and its 100 bp upstream and downstream sequences. The SNP sites without InDels greater than 5 bp were retained;

[0025] 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;

[0026] The extracted sequences were aligned to the pepper "Hot1" genome using blastn software, and only single-copy SNP sites were retained.

[0027] (5) The SNPs screened in step (4) were used to design probes based on a script written in perl program, and one marker was saved within 100 kb. Finally, 30,462 SNP markers were screened. The marker details are shown in Table 1, and the marker distribution is shown in Figure 1 .

[0028] Table 1 Details of 30462 SNP markers

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[0108] Example 2

[0109] This embodiment provides a method for detecting pepper using the 30462 SNP corresponding liquid phase probes described in Example 1, which specifically includes the following steps:

[0110] (1) Genomic DNA extraction

[0111] 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.

[0112] (2) Genomic DNA fragmentation and end repair

[0113] 1. Prepare the following reaction system in a sterile PCR tube on ice.

[0114] Table 2 List of whole genome DNA fragmentation systems

[0115] Reagents volume InputDNA XμL(250ng) Buffer 4.5μL Enzymes 5μL 1×TEBuffer Up to 30 μL Total volume 30μL

[0116] Here, 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.

[0117] 2. Use a pipette to pipette up and down or shake to ensure that the system is fully mixed, and then centrifuge briefly.

[0118] 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.

[0119] Table 3 Fragmentation and end repair reaction procedures

[0120] temperature time 32℃ 20min 65℃ 30min 4℃ Cool to 4℃ and proceed to the next step immediately

[0121] (3) Connector

[0122] 1. Prepare the reaction system as shown in Table 4.

[0123] Table 4 Reaction system for connecting linkers

[0124] Reagents volume EndPrepReactionMix (step (2) reaction product) 30μL LigationBuffer 15μL <![CDATA[ddH2O]]> 2.5μL Ligation Enzymes 5μL TruncatedAdaptor 2.5μL Total volume 55μL

[0125] Among them, all operations were performed on ice.

[0126] 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℃).

[0127] 3. DNA sample purification.

[0128] 3.1 Add 44 μL (0.8×) DNA Clean Beads to each sample and mix well; incubate at room temperature for 5 minutes;

[0129] 3.2 Place the PCR tube on the magnetic rack for 3 minutes, and remove the supernatant after the solution becomes clear;

[0130] 3.3 Add 180 μL 80% ethanol to rinse the beads, incubate for 30 seconds, remove the supernatant, and repeat the operation once;

[0131] 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;

[0132] 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;

[0133] 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.

[0134] (4) Library amplification and purification

[0135] 1. Prepare the PCR reaction system as shown in Table 5 below, mix by pipetting and centrifuge briefly.

[0136] Table 5 PCR reaction system

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[0139] 2. The prepared reaction system is placed in the PCR amplification program shown in Table 6 for amplification;

[0140] Table 6 PCR amplification program

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[0142] 3. Add 35 μL (1×) DNA Clean Beads to the amplified product, mix well, and incubate at room temperature for 5 minutes;

[0143] 4. Place the sample on the magnetic rack for 2 minutes. After the solution becomes clear, remove the supernatant.

[0144] 5. Add 200 μL 80% ethanol to rinse the magnetic beads, incubate for 30 seconds, remove the supernatant, and repeat this step once;

[0145] 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;

[0146] 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;

[0147] 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.

[0148] (5) Library and probe hybridization

[0149] 1. Take 750 ng of the library constructed in step (4) and add it to a PCR tube and mark it;

[0150] 2. Add purified magnetic beads to the library and mix gently with a pipette;

[0151] 3. Incubate at room temperature for 5 minutes and place the PCR tube on a magnetic rack for 3 minutes to clarify the solution;

[0152] 4. Remove the supernatant, place the PCR tube on the magnetic stand, add 180 μL 80% ethanol, and let stand for 30 seconds;

[0153] 5. Remove the supernatant, add 180 μL 80% ethanol to the PCR tube, let stand for 30 seconds, and then completely remove the supernatant;

[0154] 6. Leave at room temperature for 5 minutes to allow the residual ethanol to evaporate completely;

[0155] 7. Add the hybridization reaction system to the PCR tube according to Table 9 below;

[0156] Table 7 Hybridization reaction system

[0157] Reagents volume HybBuffer 13μL HybHumanBlock 5μL AdapterBlocker 2μL RnaseBlock 5μL NucleaseFreeWater 3μL Total volume 28μL

[0158] 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;

[0159] 9. Pipette 28 μL of supernatant into a new PCR tube, add 2 μL of Target Probe, gently pipette to mix, and centrifuge briefly;

[0160] 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.

[0161] (6) Capture target region DNA library

[0162] 1. Magnetic Bead Pretreatment

[0163] 1.1 Remove the capture magnetic beads from 4°C, vortex and resuspend, and place at room temperature for 30 minutes;

[0164] 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.

[0165] 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;

[0166] 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;

[0167] 2. Capture target region DNA library

[0168] 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;

[0169] 2.2 Place the PCR tube on a magnetic rack for 2 minutes to clarify the solution and remove the supernatant;

[0170] 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;

[0171] 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;

[0172] 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;

[0173] 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.

[0174] (7) Post-capture PCR amplification

[0175] 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.

[0176] 2. After capture, the DNA library needs to be amplified by PCR. Prepare the reaction system according to Table 10 below:

[0177] Table 8 PCR reaction system

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[0180] 3. Adjust the pipette to 40 μL, gently pipette and mix 6 times, and then immediately place on the PCR instrument;

[0181] 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;

[0182] 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;

[0183] 6. Briefly centrifuge and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clarify;

[0184] 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;

[0185] 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;

[0186] 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;

[0187] 10. Briefly centrifuge and place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify;

[0188] 11. Use a pipette to transfer 23 μL of supernatant to a 1.5 mL centrifuge tube and mark the sample information;

[0189] 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;

[0190] 13. Take 1 μL of sample and use Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit) to measure the fragment length.

[0191] (8) High-throughput sequencing

[0192] 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.

[0193] (9) Liquid phase probe capture rate evaluation

[0194] 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;

[0195] 2. Use SNP genotyping data to complete the population structure analysis of pepper varieties.

[0196] Example 3

[0197] 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:

[0198] (1) Experimental Materials

[0199] 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.

[0200] Table 9 Coverage depth and detection results of 19 pepper varieties

[0201] Sample_ID Depth of coverage Detection rate (%) CA-1 10 84.14 CA-2 6 79.08 CA-3 7 80.97 CA-4 9 83.98 CP-1 8 51.7 CP-2 7 51.2 CC-1 11 96.96 CC-11 12 95.27 CC-12 11 94.36 CC-13 10 94.41 CC-18 10 96.77 CC-2 7 90.51 CC-20 9 93.3 CC-21 9 92.89 CC-22 7 91.7 CC-23 14 95.28 CC-3 11 93.8 CC-8 10 93.98 CF-1 11 85.83

[0202] As shown in Table 9, from the detection rate, the capture efficiency of CC group is above 90%, that of non-CC group CA and CF group is above 79%, and that of CP group is about 51%. The genetic background of CP group is quite different from that of sequencing group, but it can capture about 15k markers and complete genetic background assessment, and the sequencing depth is above 7X. Overall, the number of captured markers can complete genetic background assessment.

[0203] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0204] 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 genotyping, characterized in that: The SNP molecular marker combination includes 30462 SNP molecular markers, and the physical positions of the 30462 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 genotyping, 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 pepper genotyping, characterized in that: The kit comprises the liquid phase probe combination according to claim 2.

4. A chili chip, characterized in that: The chip comprises the liquid phase probe combination according to claim 2.

5. Use of the SNP molecular marker combination according to claim 1, the liquid phase probe combination according to claim 2, the kit according to claim 3 or the chip according to claim 4 in pepper genotyping.

6. Use of the SNP molecular marker combination of claim 1, the liquid phase probe combination of claim 2, the kit of claim 3 or the chip of claim 4 in any one of identification of new pepper strains, pepper breeding, analysis of genetic diversity of pepper populations, and analysis of pepper population structure.

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