Chinese pepper SNP (Single Nucleotide Polymorphism) molecular marker combination, liquid-phase probe combination and application thereof

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker combination for distinguishing Chinese pepper materials, the SNP molecular marker combination comprises 181 SNP molecular markers, the physical positions of 181 SNP loci are determined based on Chinese pepper Hot1 genome sequence comparison, and the specific SNP molecular marker condition is shown in the specification table 1. 181 SNP markers are obtained by utilizing the liquid-phase probe group, the detection rate is 100%, the minimum allele frequency is greater than 0.05, any two Chinese pepper materials can be distinguished, and experiments prove that the Chinese pepper materials can be efficiently and quickly identified by utilizing the liquid-phase probe; in addition, due to the adoption of a new-generation sequencing technology, the procedure is simple and standard, the error of manual operation is reduced, the unit price of batch identification analysis is extremely low, and the method has the characteristics of economy and high efficiency, is particularly suitable for large sample analysis and identification, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker breeding, and specifically relates to a Chinese pepper SNP molecular marker combination, a liquid phase probe combination and applications thereof. Background Art

[0002] Pepper (Capsicum spp.) is an annual or perennial herbaceous plant of the genus Capsicum in the Solanaceae family. It is a global vegetable and spice crop. Pepper cultivation has a long history, and its variety resources are rich and complex, including five domesticated varieties, namely: annual pepper ( C. annuum L. CA), bush pepper ( C. fiutescens L. CF), Chinese pepper ( C.chinense Jacg. CC), drooping peppers ( C.baccatum L. CB) and Capsicum annuum ( C. pubescens Ruiz&Pav. That is, CP). Among them, Chinese pepper has a high content of capsaicin and a strong spiciness. In addition to its diversified functions as a condiment and flavor food, it also has a wide range of uses in the military and medical industries, and the demand for it is increasing. At present, there is an urgent need to develop Chinese pepper varieties with high capsaicin content, high yield and multifunctionality to meet market demand. However, there are few research reports on Chinese pepper at home and abroad. The collection of Chinese pepper germplasm resources and the mining of genetic diversity are of great significance to the utilization of the dominant traits of Chinese pepper germplasm resources and the breeding of diversified new varieties.

[0003] Conventional pepper breeding is mainly based on field plant morphological identification, but this method is limited by factors such as low accuracy, long identification time, high investment cost and susceptibility to environmental influences. Therefore, the demand for breeding high-quality pepper varieties with high capsaicin content in combination with modern molecular breeding technology is increasing. Existing molecular marker detection technologies, such as KASP, Taqman, SNaPshot, etc., can only detect one marker at a time. For marker sets composed of multiple SNPs, the detection cycle is long and the throughput is low. Summary of the invention

[0004] In view of this, the present invention uses 115 parts of Chinese pepper materials ( C.chinense Jacg. Based on the genome resequencing data of C. cerevisiae CC, 181 SNP loci were developed that can completely distinguish these 115 pepper germplasms. The SNP marker combination can be used for the authenticity and identity identification of Chinese pepper, 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: One of the purposes of the present invention is to provide a SNP molecular marker combination for distinguishing Chinese pepper materials, the SNP molecular marker combination comprises 181 SNP molecular markers, the physical positions of the 181 SNP sites are determined based on the comparison of the Chinese pepper "Hot1" genome sequence, and the specific SNP molecular markers are shown in Table 1 below.

[0006] Table 1 Detailed information of 181 SNP molecular markers

[0007] The second object of the present invention is to provide a liquid phase probe combination for distinguishing Chinese pepper materials, wherein the liquid phase probe combination includes probes for detecting the above-mentioned SNP molecular marker combination, and the specific situation of the probes is shown in Table 2 below.

[0008] Table 2 Detailed information of liquid phase probes corresponding to 181 SNP molecular marker combinations

[0009] A third object of the present invention is to provide a kit for distinguishing Chinese pepper materials, the kit comprising the above-mentioned liquid phase probe combination.

[0010] The fourth object of the present invention is to use the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in the core germplasm screening of Chinese pepper materials.

[0011] The fifth object of the present invention is to use the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in the genetic diversity analysis of Chinese pepper materials.

[0012] The sixth object of the present invention is to use the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in the construction of SNP fingerprint map of Chinese pepper materials.

[0013] The seventh object of the present invention is to use the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in the identification of Chinese pepper materials or purity.

[0014] The eighth object of the present invention is to use the above-mentioned SNP molecular marker combination, liquid phase probe combination or kit in molecular marker-assisted breeding of Chinese pepper materials.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a liquid phase probe set for identifying Chinese pepper, which is composed of 181 liquid phase probes developed based on a Chinese pepper genome SNP data set. 181 SNP markers were obtained using the liquid phase probe set of the present invention, with a detection rate of 100%, a minimum allele frequency greater than 0.05, and any two Chinese pepper materials can be distinguished. Experimental verification shows that the liquid phase probe of the present invention can efficiently and quickly complete the identification of pepper materials.

[0016] At the same time, the present invention also provides a method for efficiently distinguishing Chinese peppers. Due to the use of a new generation of sequencing technology, the procedure is simple and standardized, which reduces errors caused by human operation, makes the unit price of batch identification analysis extremely low, and has the characteristics of being economical and efficient. It is particularly suitable for large sample analysis and identification and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the SNP marker combination distribution diagram provided in Example 1 of the present invention.

[0018] Figure 2 This is a statistical graph of the number of SNP markers that differ between pepper material samples provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1 This example provides a method for screening SNP marker combinations and designing liquid phase probes based on the published 115 Chinese pepper whole genome resequencing data (DOI: 10.1038 / s41467-023-41251-4, NCBI>BioProject: PRJNA800056), which specifically includes the following process: (1) The genomic DNA of 115 pepper leaf tissues was extracted using the magnetic bead method. After the DNA quality inspection was qualified, the DNA library was prepared. After the library inspection was qualified, it was sequenced using BGI DNBSEQ-T7.

[0021] (2) The clean data obtained by sequencing in step (1) was used as the reference genome, with the Chinese pepper "Hot1" genome (NCBI>BioProject: PRJNA1175375) used for genome alignment using bwa and GATK for variant detection to obtain 85,081,260 high-quality SNP sites.

[0022] (3) The SNP sites screened out in step (2) were further filtered using the vcftools software according to the condition “minDP 3 minalleles 2 max alleles 2 max missing 0.8 maf 0.05” to obtain 31,308,905 SNP sites detected in all samples with high quality.

[0023] (4) The SNP sites screened out in step (3) were used to screen SNP marker combinations using a perl program script, and ultimately 181 SNP marker combinations were obtained, and the DNA fingerprint map was successfully constructed.

[0024] The principle is: the SNP sites with the most differences between any two pepper materials are evenly distributed on the chromosomes; the SNP sites and their upstream and downstream 100bp sequences are extracted, and the extracted 201bp fragments are separated by bedtools software. The SNP sites with GC content between 40-60% and no N bases in the sequence are retained. The extracted sequences are aligned to the pepper "Hot1" genome using blastn software, and only single-copy SNP sites are retained.

[0025] (5) The 181 SNPs screened in step (4) (for details, see Table 1) were used to design liquid phase probe sequences (for details, see Table 2) for subsequent capture sequencing.

[0026] There are 181 SNP marker combinations. The number of SNP markers that differ between any two Chinese pepper materials is shown in Table 3.

[0027] Table 3 Statistics of the number of SNPs with differences between pairs

[0028] The distribution diagram of the above selected SNP marker combinations is as follows Figure 1 shown.

[0029] The statistical diagram of the difference in the number of SNP markers between any two Chinese pepper materials is shown in the figure below: Figure 2 shown.

[0030] Example 2 This embodiment provides a method for detecting pepper using the liquid phase probes corresponding to the 181 SNPs obtained in Example 1, and the specific steps are as follows: (1) Genomic DNA extraction 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.

[0031] (2) Genomic DNA fragmentation and end repair 1. Prepare the following reaction system in a sterile PCR tube on ice.

[0032] Table 4 List of whole genome DNA fragmentation systems

[0033] Here, X represents any volume not exceeding 20 μL, which is added with 4.5 μL of Buffer and 5 μL of Enzymes. The part less than 30 μL is made up with 1×TE Buffer.

[0034] 2. Use a pipette to pipette up and down or shake to ensure that the system is fully mixed, and then centrifuge briefly.

[0035] 3. Immediately place the PCR tube on a PCR instrument preheated to 32°C, run the reaction program shown in Table 5, 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.

[0036] Table 5 Fragmentation and end repair reaction procedures

[0037] (3) Connector 1. Prepare the reaction system as shown in Table 6.

[0038] Table 6 Reaction system for connecting linkers

[0039] Among them, all operations were performed on ice.

[0040] 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℃).

[0041] 3. DNA sample purification.

[0042] 3.1 Add 44 μL (0.8×) DNA Clean Beads to each sample and mix well; incubate at room temperature for 5 minutes; 3.2 Place the PCR tube on the magnetic rack for 3 minutes. After the solution becomes clear, remove the supernatant. 3.3 Add 180 μL 80% ethanol to rinse the beads, incubate for 30 seconds, remove the supernatant, and repeat the operation once; 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; 3.5 in 21 μL ddHO 2 Resuspend the magnetic beads in 5% CO and let stand at room temperature for 1 min to fully release the DNA on the magnetic beads; 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.

[0043] (4) Library amplification and purification 1. Prepare the PCR reaction system as shown in Table 7 below, mix by pipetting and centrifuge briefly.

[0044] Table 7 PCR reaction system details

[0045] 2. The prepared reaction system is placed in the PCR amplification program shown in Table 8 for amplification; Table 8 PCR amplification procedure details

[0046] 3. Add 35 μL (1×) DNA Clean Beads to the amplified product, mix well, and incubate at room temperature for 5 minutes; 4. Place the sample on the magnetic rack for 2 minutes. After the solution becomes clear, remove the supernatant. 5. Add 200 μL 80% ethanol to rinse the magnetic beads, incubate for 30 seconds, remove the supernatant, and repeat this step once; 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; 7. Add 31 μL ddHO 2 Resuspend the magnetic beads in 5% CO and let stand at room temperature for 1 min to fully release the DNA on the magnetic beads; 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.

[0047] (5) Library and probe hybridization 1. Take 750 ng of the library constructed in step (4) and add it to a PCR tube and mark it; 2. Add purified magnetic beads to the library and mix gently with a pipette; 3. Incubate at room temperature for 5 minutes and place the PCR tube on a magnetic rack for 3 minutes to clarify the solution; 4. Remove the supernatant, place the PCR tube on the magnetic stand, add 180 μL 80% ethanol, and let stand for 30 seconds; 5. Remove the supernatant, add 180 μL 80% ethanol to the PCR tube, let stand for 30 seconds, and then completely remove the supernatant; 6. Leave at room temperature for 5 minutes to allow the residual ethanol to evaporate completely; 7. Add the hybridization reaction system to the PCR tube according to Table 9 below; Table 9 Hybridization reaction system

[0048] 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; 9. Pipette 28 μL of supernatant into a new PCR tube, add 2 μL of Target Probe, gently pipette to mix, and centrifuge briefly; 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.

[0049] (6) Capture target region DNA library 1. Magnetic Bead Pretreatment 1.1 Remove the capture magnetic beads from 4°C, vortex and resuspend, and place at room temperature for 30 minutes; 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. 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; 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; 2. Capture target region DNA library 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; 2.2 Place the PCR tube on a magnetic rack for 2 minutes to clarify the solution and remove the supernatant; 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; 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 2 times, washing the magnetic beads 3 times in total; 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; 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.

[0050] (7) Post-capture PCR amplification 1. Take out PostPCR MasterMix and PostPCR 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.

[0051] 2. After capture, the DNA library needs to be amplified by PCR. Prepare the reaction system according to Table 10 below: Table 10 PCR reaction system

[0052] 3. Adjust the pipette to 40 μL, gently pipette and mix 6 times, and then immediately place on the PCR instrument; 4. Run the PCR instrument program: Hot cover temperature: 105℃ Program: 95℃ 1min; 98℃ 20s; 60℃ 30s Ncycles; 72℃ 30s; 72℃ 5min; 4℃ hold; 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; 6. Briefly centrifuge and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clarify; 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; 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; 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; 10. Briefly centrifuge and place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify; 11. Use a pipette to transfer 23 μL of supernatant to a 1.5 mL centrifuge tube and mark the sample information; 12. Take 1 μL of library and use Qubit dsDNAHS Assay Kit for quantification and record the library concentration, which is about 1-20 ng / μL; 13. Take 1 μL of sample and use Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit) to measure the fragment length.

[0053] (8) High-throughput sequencing 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.

[0054] (9) Liquid phase probe capture rate evaluation 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 the pepper material; 2. Use SNP genotyping data to complete the fingerprint analysis of pepper materials.

[0055] Example 3 This example uses the method of Example 2 to evaluate the liquid phase probe capture efficiency of 12 Chinese pepper materials. The specific steps are as follows: (1) Experimental materials 12 Chinese pepper leaves were selected, refrigerated and transported to the laboratory in time, and the total DNA of each individual plant was extracted by magnetic bead method. The sample numbers are shown in Table 11.

[0056] Table 11 Names of 12 Chinese pepper samples

[0057] (2) Genotyping data analysis The method described in Example 2 was used to perform 181 SNP liquid phase probe hybridization and genotyping on each individual plant in Table 11, and the typing data of each individual plant was obtained, and the detection rate was 100%. Among them, the genotyping results of the 12 Chinese pepper material samples in this example are shown in Appendix 12.

[0058] Table 12 Genotyping results of 12 Chinese pepper samples

[0059] Continued from the table above

[0060] The liquid phase probe designed based on the 181 SNP marker combination detected 12 Chinese pepper samples, and the SNP genotyping number was obtained. From the results, it can be seen that the pepper material detection rate is 100%. It shows that the liquid phase probe group developed using the 181 SNP marker combination of the present invention can efficiently and quickly complete the identification of Chinese pepper materials.

[0061] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0062] 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 distinguishing Chinese pepper materials, characterized in that: The SNP molecular marker combination includes 181 SNP molecular markers, and the physical locations of the 181 SNP sites are determined based on the comparison of the Chinese pepper "Hot1" genome sequence. The specific SNP molecular markers are shown in Table 1 of the specification.

2. A liquid phase probe combination for distinguishing Chinese pepper materials, 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 distinguishing Chinese pepper materials, the kit 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 core germplasm screening of Chinese pepper materials.

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 the genetic diversity analysis of Chinese pepper materials.

6. Use of the SNP molecular marker combination described in claim 1, the liquid phase probe combination described in claim 2, or the kit described in claim 3 in the construction of SNP fingerprints of Chinese pepper materials.

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 identification of Chinese pepper materials or purity.

8. Use of the SNP molecular marker combination of claim 1, the liquid phase probe combination of claim 2 or the kit of claim 3 in molecular marker-assisted breeding of Chinese pepper materials.

Images