A pair of sv molecular marker primers, a kit and application for constructing a molecular identity card of capsicum

Abstract

The application discloses an SV molecular marker primer pair for constructing a pepper molecular identity card, a kit and application. Based on large fragment structural variation of a pepper genome, multiple molecular markers distributed in 12 chromosomes of the pepper are developed. The pepper germplasm genotype can be conveniently and quickly identified by agarose gel electrophoresis detection, and the method is safe, non-toxic and simple to operate, so that a representative pepper molecular identity card is constructed. The application can realize identification of purity and genetic relationship of different pepper varieties, and has important application value for pepper germplasm resource management and utilization, variety protection and seed purity identification.

Description

Technical Field

[0001] This invention belongs to the field of chili genetic engineering technology, specifically relating to an SV molecular marker primer pair, kit, and application for constructing chili molecular identity cards. Background Technology

[0002] Chili pepper (Capsicum spp.) is an important vegetable, condiment, and industrial raw material. It is also the most widely cultivated vegetable crop in my country, with a stable annual planting area of ​​32 million mu (approximately 2.8 million hectares) and a yield exceeding 40 million tons. Currently, there are five main cultivated varieties of chili peppers: annual chili pepper, Chinese chili pepper, shrub chili pepper, drooping chili pepper, and hairy chili pepper. Chili pepper germplasm resources are rich and diverse, with numerous varieties, and consumer demands vary significantly across different regional markets. Due to the large number of chili pepper varieties and the high demand for hybrids, varietal homogenization in chili pepper production is severe, harming the legitimate rights and interests of breeders and producers, hindering the stimulation of breeders' innovation, and causing chaos in germplasm resource protection, market management, variety selection and approval, and agricultural production. Therefore, identifying the phylogenetic relationships between different chili pepper varieties and constructing molecular marker identification for chili peppers is a prerequisite for seed production and sales.

[0003] Common molecular markers mainly include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple repeat sequence (SSR), insertion / deletion marker (InDel), single nucleotide polymorphism (SNP), and structural variation (SV). SV markers are third-generation molecular marker technologies with the advantages of large variation range, large information content, and high polymorphism. They can be directly genotyped by ordinary agarose gel electrophoresis and are especially suitable for trait analysis, breeding selection, and population evolution research, making them an ideal choice for molecular breeding.

[0004] This invention constructs a molecular identity card for chili peppers based on SV molecular markers. Its application facilitates the accurate identification, management, and development of chili pepper germplasm resources, and establishes a technical tool for the protection of new varieties. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an SV molecular marker primer pair, kit, and application for constructing a molecular identity card for chili peppers.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide an SV molecular marker primer pair for constructing a molecular identity card for chili peppers. The primer pair includes 12 pairs of SV molecular marker primers, each pair corresponding to chromosomes 1 to 12 of the chili pepper, with each chromosome corresponding to one SV molecular marker.

[0008] The 12 primer pairs are named P01-201, P02-149, P03-124, P04-111, P05-182, P06-193, P07-125, P08-140, P09-60, P10-166, P11-101, and P12-112, respectively.

[0009] The nucleic acid sequences of the P01-201 primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0010] The nucleic acid sequences of the P02-149 primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0011] The nucleic acid sequences of the P03-124 primer pair are shown in SEQ ID NO.5 and SEQ ID NO.6;

[0012] The nucleic acid sequences of the P04-111 primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8;

[0013] The nucleic acid sequences of the P05-182 primer pair are shown in SEQ ID NO.9 and SEQ ID NO.10;

[0014] The nucleic acid sequences of the P06-193 primer pair are shown in SEQ ID NO.11 and SEQ ID NO.12;

[0015] The nucleic acid sequences of the P07-125 primer pair are shown in SEQ ID NO.13 and SEQ ID NO.14;

[0016] The nucleic acid sequences of the P08-140 primer pair are shown in SEQ ID NO.15 and SEQ ID NO.16;

[0017] The nucleic acid sequences of the P09-60 primer pair are shown in SEQ ID NO.17 and SEQ ID NO.18;

[0018] The nucleic acid sequences of the P10-166 primer pair are shown in SEQ ID NO.19 and SEQ ID NO.20;

[0019] The nucleic acid sequences of the P11-101 primer pair are shown in SEQ ID NO.21 and SEQ ID NO.22;

[0020] The nucleic acid sequences of the P12-112 primer pair are shown in SEQ ID NO.23 and SEQ ID NO.24.

[0021] The second objective of this invention is to provide a method for constructing a chili molecular identity card using SV molecular marker primer pairs for constructing a chili molecular identity card, comprising the following steps:

[0022] Step 1: Extract genomic DNA from the pepper sample to be tested;

[0023] Step 2: Using the genomic DNA of the pepper sample to be tested as a template, PCR amplification was performed sequentially using the 12 pairs of SV molecular marker primers described in claim 1 to obtain PCR amplification products, and the amplification products were detected by agarose gel electrophoresis.

[0024] Step 3: Based on the agarose gel electrophoresis results, assign genotype values ​​to each SV molecular marker site: homozygous fragments are assigned "0" or "1", heterozygous fragments are assigned "2", and no amplified bands are assigned "×". All marker assignment results are concatenated and sorted according to the order of chromosomes 1 to 12 of pepper to form a unique variety code, thus completing the construction of the pepper molecular ID card.

[0025] Preferably, in step 2, the total volume of the PCR amplification reaction system is 20.0 μL, including: 1.0 μL of 50 ng / μL genomic DNA, 10.0 μL of 2×Taq Master Mix, 1.5 μL of 10 μmol / L primer mixture, and the remainder is ddH2O.

[0026] Preferably, in step 2, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 1 min 30 s; 94℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 30 s, cycled 30 times; 72℃ final extension for 5 min, and storage at 16℃ for 5 min.

[0027] A third objective of this invention is to provide a kit comprising SV molecular marker primer pairs for constructing a molecular identity card for chili peppers.

[0028] Preferably, the kit also includes Taq enzyme, PCR amplification buffer, dNTPs, DNA marker, and reagents required for agarose gel electrophoresis.

[0029] The fourth objective of this invention is to provide an SV molecular marker primer pair or kit for constructing chili molecular identity cards, and its application in constructing chili molecular identity cards, identifying the purity and phylogenetic relationships of different chili varieties, managing and developing chili germplasm resources, and protecting and evaluating chili varieties.

[0030] The fifth objective of this invention is to provide a method for identifying chili germplasm and varieties, which uses the uniqueness of the molecular identification code constructed by the method or the kit to determine the corresponding chili germplasm or variety.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The SV molecular marker for identifying chili pepper molecules provided by this invention is developed based on large-segment structural variations between different chili pepper genomes. These sites have high polymorphism. Each chromosome of chili pepper contains one molecular marker, and each site has three possibilities (AA, Aa, and aa), for a total of 3. 12 =531,441 genotypes, which can theoretically meet the basic requirements for identifying complex and diverse chili germplasm resources, and can construct a representative chili molecular identity card.

[0033] (2) The SV molecular marker for identifying chili pepper molecules provided by the present invention can be detected by ordinary agarose gel electrophoresis. The operation is simple and can quickly and accurately identify different tissues of chili pepper at the molecular level.

[0034] (3) The chili molecular identity card based on SV molecular markers developed in this invention can identify the identity and kinship of different chili germplasm, which has important application value for chili germplasm resource management, development and utilization, variety protection and seed production. Attached Figure Description

[0035] Figure 1 The image shows the electrophoretic images of 24 chili pepper materials screened by the P01-201 marker in Example 3.

[0036] Figure 2 The image shows the electrophoretic images of 24 chili pepper materials screened using the P02-149 marker in Example 3.

[0037] Figure 3 The image shows the electrophoretic images of 24 chili pepper materials screened by the P03-124 marker in Example 3.

[0038] Figure 4 The image shows the electrophoretic images of 24 chili pepper materials screened using the P04-111 marker in Example 3.

[0039] Figure 5 The image shows the electrophoretic images of 24 chili pepper materials screened using the P05-182 marker in Example 3.

[0040] Figure 6 The image shows the electrophoretic images of 24 chili pepper materials screened using the P06-193 marker in Example 3.

[0041] Figure 7 The image shows the electrophoretic images of 24 chili pepper materials screened using the P07-125 marker in Example 3.

[0042] Figure 8The image shows the electrophoretic images of 24 chili pepper materials screened using the P08-140 marker in Example 3.

[0043] Figure 9 The image shows the electrophoretic images of 24 chili pepper materials screened using the P09-60 marker in Example 3.

[0044] Figure 10 The image shows the electrophoretic images of 24 chili pepper materials screened using the P10-166 marker in Example 3.

[0045] Figure 11 The image shows the electrophoretic images of 24 chili pepper materials screened by the P11-101 marker in Example 3.

[0046] Figure 12 The image shows the electrophoretic images of 24 chili pepper materials screened by the P12-112 markers in Example 3.

[0047] Figure 13 The results of the kinship analysis of 24 chili pepper materials in Example 3 based on the chili pepper molecular identification card are shown.

[0048] in, Figures 1-12 Lane 13 is a 2000bp DNA marker. Lanes 1-12 and 14-25 are lanes of the selected chili pepper materials. Lanes 1-12 are numbered L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, respectively. Lanes 14-25 are numbered L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, respectively. Detailed Implementation

[0049] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the instruments, materials and reagents used are commercially available unless otherwise specified.

[0051] Example 1: Development of SV molecular markers for chili peppers.

[0052] Using the Genome Variant Scan plugin of the TBtools-II bioinformatics platform, a whole-genome alignment was performed between the pepper Ca59 genome and the Dempsey genome, with the VarRange parameter set to 30-500 bp. The alignment yielded information on large-segment structural variant insertions or deletions covering all 12 pepper chromosomes, including the location of SVs on the pepper chromosomes, the sequence information of inserted or deleted SVs, and the primer sequence information for amplifying SV sequences obtained through batch design.

[0053] Primer screening for SV was performed using the Primer check plugin. In the first round of screening, whole-genome e-PCR amplification was performed on the Ca59 reference genome to select primers that amplified only a single band, whose e-PCR fragment was in the 0-300 bp range, and which were located on the chromosome where the predicted large structural variation was found. In the second round of screening, whole-genome e-PCR amplification was performed on the *C. annuum* Ca59, Dempsey, Zhangshugang, and G1-36576 reference genomes to select primers that amplified only a single band, exhibited polymorphism among the four reference genomes, and were located on the chromosome where the predicted large structural variation was found.

[0054] Twelve pairs of polymorphic SV primers were selected, located on chromosomes 1-12 of the pepper plant. Each chromosome has one SV molecular marker, which were named P01-201, P02-149, P03-124, P04-111, P05-182, P06-193, P07-125, P08-140, P09-60, P10-166, P11-101, and P12-112, respectively. Specifically, primer P01-201 amplifies the amplification site on chromosome 1 (Chr01), primer P02-149 amplifies the amplification site on chromosome 2 (Chr02), primer P03-124 amplifies the amplification site on chromosome 3 (Chr03), primer P04-111 amplifies the amplification site on chromosome 4 (Chr04), primer P05-182 amplifies the amplification site on chromosome 5 (Chr05), and primer P06-193 amplifies the amplification site on chromosome 6 (Chr06). Primer P07-125 amplifies the location on chromosome 7 (Chr07), primer P08-140 amplifies the location on chromosome 8 (Chr08), primer P09-60 amplifies the location on chromosome 9 (Chr09), primer P10-166 amplifies the location on chromosome 10 (Chr10), primer P11-101 amplifies the location on chromosome 11 (Chr11), and primer P12-112 amplifies the location on chromosome 12 (Chr12).

[0055] Table 1: Specific primer sequences.

[0056]

[0057]

[0058] Example 2: A method for constructing a chili pepper molecular identity card using SV molecular markers.

[0059] Step 1: Extract genomic DNA from the peppers to be tested; extract genomic DNA from the peppers using the CTAB method.

[0060] Step 2: Using the genomic DNA of the pepper material to be tested as a template, PCR amplification was performed using the primers listed in Table 1. The PCR amplification system was as follows: 20.0 μL total volume, 1.0 μL 50 ng / μL DNA, 10.0 μL 2×Taq Master Mix, 1.5 μL 10 μmol mixed primers (i.e., forward and reverse primers), and the remainder ddH2O. The PCR amplification program was as follows: 94℃ pre-denaturation for 1 min 30 s, 94℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 30 s, 30 cycles, 72℃ extension for 5 min, and storage at 16℃ for 5 min. The PCR amplification products were detected by agarose gel electrophoresis.

[0061] Step 3: Analyze the SV locus genotype of the PCR amplification products. Based on the size of the fragment amplified by each pair of SV primers and whether it is heterozygous, digitize each molecular marker and assign it a value of "0" (only large fragment amplified, homozygous insertion AA), "1" (only small fragment amplified, homozygous deletion aa), or "2" (both large and small fragments appear simultaneously, heterozygous Aa). If no band is amplified, assign "×". String the markers together according to the chromosome order and sort them sequentially to form a variety code, constructing a molecular ID card for peppers.

[0062] Example 3: Accuracy verification or application.

[0063] Twenty-four chili pepper varieties or hybrid combinations preserved in the Hubei Provincial Key Laboratory of Vegetable Germplasm Innovation and Genetic Improvement were used for variety identification. The specific variety names are shown in Table 2. The 24 chili pepper materials were identified using the 12 molecular markers described in Example 1, following the method described in Example 2. The agarose gel electrophoresis results are shown in Table 2. Figures 1-12 The molecular identity cards of each chili germplasm are shown in Table 2.

[0064] Table 2: Statistical analysis of molecular identification codes for 24 chili pepper samples.

[0065]

[0066]

[0067]

[0068] Using NTsys 2.10e software, phylogenetic analysis based on UPGMA was performed on the above results. It was found that L16, L17, L18, and L19 clustered into one group, which consisted of four bred chili pepper combinations of the "Chili King" type. These combinations shared the same paternal parent, while their maternal parents were phenotypically similar lines classified from different sources of Shizhu red chili peppers. L21, L22, and L23 were processing-type chili pepper combinations. These three combinations shared the same paternal parent, while their maternal parents were different lines isolated from 21HN504 (source: Shiliuhong F1), exhibiting significant phenotypic differences. Among them, 21HN504... HN504-1 is a medium-sized fruit with a blunt tip and a tall plant type; 21HN504-2 is a medium-sized fruit with a pointed tip, a medium plant type, and a low initial flowering node; 21HN504-7 is a small-sized fruit with a blunt tip, a short plant type, and a low initial flowering node. However, L21, L22, and L23 were all clustered into the processing type of chili pepper combination or variety group; L24 was clustered into a separate group, which is a short ram's horn type fresh-eating chili pepper, and its plant type, fruit shape, and size differed relatively greatly from the other chili peppers. It can be seen that the above SV molecular identity cards are reliable for identifying the kinship of chili peppers.

[0069] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An SV molecular marker primer pair for constructing a molecular identity card for chili peppers, characterized in that, The primer pairs include 12 pairs of SV molecular marker primers, each pair corresponding to chromosomes 1 to 12 of the pepper plant, with one SV molecular marker corresponding to each chromosome. The 12 primer pairs are named P01-201, P02-149, P03-124, P04-111, P05-182, P06-193, P07-125, P08-140, P09-60, P10-166, P11-101, and P12-112, respectively. The nucleic acid sequences of the P01-201 primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2; The nucleic acid sequences of the P02-149 primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4; The nucleic acid sequences of the P03-124 primer pair are shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleic acid sequences of the P04-111 primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleic acid sequences of the P05-182 primer pair are shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleic acid sequences of the P06-193 primer pair are shown in SEQ ID NO.11 and SEQ ID NO.12; The nucleic acid sequences of the P07-125 primer pair are shown in SEQ ID NO.13 and SEQ ID NO.14; The nucleic acid sequences of the P08-140 primer pair are shown in SEQ ID NO.15 and SEQ ID NO.16; The nucleic acid sequences of the P09-60 primer pair are shown in SEQ ID NO.17 and SEQ ID NO.18; The nucleic acid sequences of the P10-166 primer pair are shown in SEQ ID NO.19 and SEQ ID NO.20; The nucleic acid sequences of the P11-101 primer pair are shown in SEQ ID NO.21 and SEQ ID NO.22; The nucleic acid sequences of the P12-112 primer pair are shown in SEQ ID NO.23 and SEQ ID NO.

24.

2. A method for constructing a chili molecular identity card using the SV molecular marker primer pair for constructing a chili molecular identity card as described in claim 1, characterized in that, Includes the following steps: Step 1: Extract genomic DNA from the pepper sample to be tested; Step 2: Using the genomic DNA of the pepper sample to be tested as a template, PCR amplification was performed sequentially using the 12 pairs of SV molecular marker primers described in claim 1 to obtain PCR amplification products, and the amplification products were detected by agarose gel electrophoresis. Step 3: Based on the agarose gel electrophoresis results, assign genotype values ​​to each SV molecular marker site: homozygous fragments are assigned "0" or "1", heterozygous fragments are assigned "2", and no amplified bands are assigned "×". All marker assignment results are concatenated and sorted according to the order of chromosomes 1 to 12 of pepper to form a unique variety code, thus completing the construction of the pepper molecular ID card.

3. The method according to claim 2, characterized in that, In step 2, the total volume of the PCR amplification reaction system is 20.0 μL, including: 1.0 μL of 50 ng / μL genomic DNA, 10.0 μL of 2×Taq Master Mix, 1.5 μL of 10 μmol / L primer mixture, and the remainder is ddH2O.

4. The method according to claim 2, characterized in that, In step 2, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 1 min 30 s; 94℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 30 s, 30 cycles; 72℃ final extension for 5 min, and storage at 16℃ for 5 min.

5. A reagent kit, characterized in that, The kit contains the SV molecular marker primer pair for constructing chili molecular identity cards as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The kit also includes Taq enzyme, PCR amplification buffer, dNTPs, DNA marker, and reagents required for agarose gel electrophoresis.

7. The application of the SV molecular marker primer pair for constructing chili molecular identity cards as described in claim 1, or the kit as described in any one of claims 5-6, in constructing chili molecular identity cards, identifying the purity and phylogenetic relationships of different chili varieties, managing and developing chili germplasm resources, and protecting and evaluating chili varieties.

8. A method for identifying chili germplasm and varieties, characterized in that, The uniqueness of the molecular identification code constructed by the method as described in any one of claims 2-4 or the kit as described in any one of claims 5-6 is used to determine the corresponding germplasm or variety of chili pepper.

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