Molecular marker for detecting L-series resistance gene of pepper mild mottle virus and application of molecular marker

Abstract

The invention provides a molecular marker for detecting pepper light mottle virus L-series resistance genes and application of the molecular marker, and belongs to the technical field of genetic engineering, the molecular marker comprises SNP1 and SNP2, the SNP1 is a base T at the 2574th site of a yellow lantern pepper genome sequence with the NCBI login number of AB523370, and the SNP2 is a base T at the 2832nd site of the yellow lantern pepper genome sequence with the NCBI login number of AB523370; the specific gene sequence is a specific gene sequence of an L-series resistance gene L1c type gene, and the specific gene sequence is a basic group CA at the 2872 site to the 2873 site, a basic group TT at the 2877 site to the 2878 site, a basic group TG at the 2882 site to the 2883 site and a basic group AGA at the 2885 site to the 2887 site of the yellow lantern pepper with the NCBI accession number being AB523374. By utilizing the molecular marker and the specific sequence, the L-series resistance gene types of the yellow lantern pepper and the annual pepper can be rapidly and accurately detected, and the molecular marker can be used for molecular marker-assisted selective breeding.

Description

Technical Field

[0001] The present invention relates to a molecular marker and its application, in particular to a molecular marker for detecting the L series resistance genes of Pepper mild mottle virus and its application, belonging to the technical field of genetic engineering. Background Art

[0002] Yellow lantern pepper (scientific name: Capsicum chinense), also known as Huangdijiao, yellow pepper, and Chinese pepper, is a plant of the genus Capsicum in the subfamily Solanaceae of the Solanaceae family. It is one of the five cultivated species of peppers. It is native to countries such as Cuba, Brazil, Peru, Bolivia, and Paraguay. It is named because the cultivated varieties in Hainan Province, China, are golden yellow in color. Yellow lantern pepper is rich in capsaicinoids and has a high spiciness, up to 170,000 SHU. It is generally not used for fresh consumption and is mainly made into yellow lantern pepper sauce in Hainan Province. The large-scale cultivation of yellow lantern pepper in China is mainly in Wenchang, Qionghai, Wanning, Lingshui and other places in Hainan Province. The main cultivated varieties are local varieties and the hybrid variety "Relala No. 2".

[0003] Pepper mild mottle virus (PMMoV) belongs to the genus Tobamovirus. In recent years, PMMoV has been detected in the main pepper-producing areas in many parts of China, and the detection rate has been increasing continuously, seriously threatening the development of the pepper industry. The early symptoms of PMMoV infection in plants are not obvious, mostly mild chlorosis of leaves. In the later stage, obvious symptoms can occur, which can cause leaf curling, deformation, mottling or mosaic symptoms of yellow and green alternation, resulting in smaller, deformed, convex and concave mottled or even necrotic and easily shed fruits, seriously affecting the yield and quality of peppers.

[0004] The L series resistance genes are important resistance genes of Pepper mild mottle virus. At present, they are divided into L1, L1a, L1c, L2, L2b, L3 and L4 genes. Among them, the L1c and L3 genes are derived from yellow lantern pepper. Among them, the highly resistant gene L3 has been used for the resistance breeding of annual peppers. For the adjacent sequence of L3, corresponding markers have been developed for the utilization of annual pepper breeding. This marker is based on a SNP locus linked to the L3 gene. The genotype without the L3 gene is A, the genotype with the L3 gene is C, the genotype with A has a restriction enzyme site SspI, and the genotype with C does not have SspI. Therefore, the predecessors developed a method for identifying the L3 gene based on SspI digestion. Relevant researchers from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences also developed a KASP marker using this SNP for identifying the L3 gene. However, we found that the markers developed based on this SNP are not applicable to the cultivated species of yellow lantern pepper. Whether yellow lantern pepper contains the L3 gene or not, the SNP linked to the L gene is C, resulting in these markers of L3 being unable to be used for molecular breeding of intraspecific or interspecific hybridization of yellow lantern pepper. Therefore, new markers need to be developed for the utilization of disease-resistant molecular breeding of yellow lantern pepper. Summary of the Invention

[0005] In view of the above problems, the present invention provides a molecular marker for detecting the resistance gene of the L series of pepper mild mottle virus and its application, so as to accelerate the breeding process of new disease-resistant varieties of Capsicum chinense Jacq..

[0006] To achieve the above object, the technical solution of the present invention is: a molecular marker and a specific gene sequence for detecting the resistance gene of the L series of pepper mild mottle virus, the molecular marker includes SNP1 and SNP2, the SNP1 is the base T at the 2574th position of the Capsicum chinense Jacq. genome sequence with the NCBI accession number AB523370, and the SNP2 is the base T at the 2832nd position of the Capsicum chinense Jacq. genome sequence with the NCBI accession number AB523370; the specific gene sequence is the specific gene sequence of the L1c type gene of the L series resistance gene, and the specific gene sequence is the bases CA at the 2872-2873rd positions, the bases TT at the 2877-2878th positions, the bases TG at the 2882-2883rd positions, and the bases AGA at the 2885-2887th positions of Capsicum chinense Jacq. with the NCBI accession number AB523374.

[0007] A primer set for amplifying the above molecular marker and specific gene sequence, the primer set includes three pairs of primers, specifically, the L1c primer pair for specifically amplifying the specific gene sequence, the upstream primer sequence of the L1c primer pair is as shown in SEQ ID NO.1, and the downstream primer sequence is as shown in SEQ ID NO.2; the L3-1 primer pair for specifically amplifying the SNP1 site, the upstream primer sequence of the L3-1 primer pair is as shown in SEQ ID NO.3, and the downstream primer sequence is as shown in SEQ ID NO.4; the L3-2 primer pair for specifically amplifying the SNP2 site, the upstream primer sequence of the L3-2 primer pair is as shown in SEQ ID NO.5, and the downstream primer sequence is as shown in SEQ ID NO.6.

[0008] A kit for detecting the L series genes of pepper mild mottle virus, the kit includes the above primer set.

[0009] The application of the above molecular marker and specific gene sequence, the above primer set, and the above kit in the breeding process of disease-resistant pepper varieties.

[0010] A method for detecting the resistance gene of the L series of pepper mild mottle virus, including the following steps: Step S1, extracting genomic DNA from pepper; Step S2, using the above primer set or the above kit to perform PCR amplification on the genomic DNA extracted in Step S1, and then judging the type of the L series resistance gene according to the electrophoresis pattern of the amplification product.

[0011] Further, the chili pepper described in step S1 is Capsicum chinense Jacq. or an annual chili pepper variety.

[0012] Further, the specific judgment criteria for judging the type of L series resistance genes based on the electrophoresis pattern of the amplification product in step S2 are as follows: If the electrophoresis pattern verification of the specific primer for L1c can amplify a band, and the specific primers for the SNP1 site of L3 and the specific primers for the SNP2 site of L3 can also amplify bands, it indicates that the chili pepper is a disease-susceptible variety, and the gene type is L1c; If the electrophoresis pattern verification of the specific primer for L1c fails to amplify a band, while the specific primer for the SNP1 site of L1c amplifies a band, and the specific primer for the SNP2 site of L3 fails to amplify a band, it indicates that the chili pepper is a disease-resistant variety, but the disease resistance is slightly weak, and the gene type is L2; If the electrophoresis pattern verification of the specific primer for L1c fails to amplify a band, the specific primer for the SNP1 site amplifies a band, and the specific primer for the SNP2 site also amplifies a band, it indicates that the chili pepper is a disease-resistant variety, and the disease resistance is strong, and the gene type is L3; Other electrophoresis results belong to unknown situations, and the disease resistance gene type cannot be confirmed temporarily.

[0013] The beneficial effects of the molecular marker for detecting the L series resistance genes of chili pepper mild mottle virus and its application in the present invention are as follows: By using the said molecular marker and specific sequence, the present invention can quickly and accurately detect the type of L series resistance genes of Capsicum chinense Jacq., which can be used for molecular marker-assisted selection breeding, quickly, accurately and efficiently screen resistant varieties, and accelerate the breeding process of new disease-resistant varieties of Capsicum chinense Jacq.

[0014] Specific SNP1 of the present invention is the base T at the 2574th position of the L3 gene, SNP2 is the base T at the 2832nd position of the L3 gene, and TTG at positions 2801 - 2803 is the specific base sequence of the L gene. The bases at positions 2872 - 2873, 2877 - 2878, 2882 - 2883, and 2885 - 2887 of the L1c gene are the specific sequences of L1c. By using the specific primers designed based on the above specific sites and specific gene sequences to perform PCR amplification on pepper plants and then judging according to the electrophoresis band situation, the L series genotypes of pepper varieties can be quickly obtained. The specific judgment criteria are as follows: If the electrophoresis diagram verification of the specific primers for L1c can amplify a band, and the specific primers for the SNP1 site of L3 and the specific primers for the SNP2 site of L3 can also amplify bands, it indicates that the pepper is a disease-susceptible variety, and the gene type is L1c; if the electrophoresis diagram verification of the specific primers for L1c fails to amplify a band, while the specific primers for the SNP1 site of L1c can amplify a band and the specific primers for the SNP2 site of L3 fail to amplify a band, it indicates that the pepper is a disease-resistant variety, but the disease resistance is slightly weak, and the gene type is L2; if the electrophoresis diagram verification of the specific primers for L1c fails to amplify a band, the specific primers for the SNP1 site can amplify a band, and the specific primers for the SNP2 site can also amplify a band, it indicates that the pepper is a disease-resistant variety, and the disease resistance is strong, and the gene type is L3; other electrophoresis results belong to unknown situations, and the disease resistance situation cannot be confirmed for the time being. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0016] Figure 1 is the comparison of the amino acid sequences of the L series genes and the positions of the amino acids corresponding to the SNPs in the embodiments of the present invention; Figure 2 is the alignment result of the nucleotide sequences of the L series genes and the positions of the SNPs, the specific sequence of the L gene, and the specific sequence of L1c in the embodiments of the present invention; Figure 3 is the alignment result of the sequence near SNP1 and other similar sequences in the genome, and the red box is the position of SNP1; Figure 4 is the alignment result of the specific sequence of the L gene and the sequence near SPN2 and other similar sequences in the genome, and the red box is the position of the specific sequence of the L gene and SPN2; Figure 5 is the electrophoresis diagram of the detection results of the L series resistance genes in the embodiments of the present invention. SPECIFIC EMBODIMENTS

[0017] The following clearly and completely describes the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below. Example 1

[0018] The development of molecular markers for detecting the resistance genes of the L series of pepper mild mottle virus and the design of corresponding detection primers are as follows: The L series of resistance genes are important resistance genes of pepper mild mottle virus. Currently, they are divided into L1, L1a, L1c, L2, L2b, L3 and L4 genes. Among them, the L1c and L3 genes are derived from Capsicum chinense. Therefore, the present invention mainly detects whether the germplasm resources of various varieties of Capsicum chinense contain the L1c and L3 resistance genes, with the focus on detecting whether they contain the L3 resistance gene and distinguishing it from other resistance genes to detect its resistance ability, providing a screening basis for Capsicum chinense breeding.

[0019] In the present invention, by downloading the gene sequences of L1, L1a, L1c, L2, L2b, L3 and L4 from the NCBI gene database and comparing them, it is found that there are differences in the types of bases and amino acids at several positions in these gene sequences. For specific details, refer to Figure 1 and Figure 2 for the comparison results. Among them, the accession number of the L1 resistance gene (corresponding plant: Capsicum annuum) in NCBI is AB523372, the accession number of the L1a resistance gene (corresponding plant: Capsicum annuum) is AB523373, the accession number of the L1c resistance gene (corresponding plant: Capsicum chinense) is AB523374, the accession number of the L2 resistance gene (corresponding plant: Capsicum frutescens) is AB523375, the accession number of the L2b resistance gene (corresponding plant: Capsicum baccatum) is AB523376, the accession number of the L3 resistance gene (corresponding plant: Capsicum chinense) is AB523370, and the accession number of the L4 resistance gene (corresponding plant: Capsicum chacoense) is AB523377.

[0020] According to the above comparison results, the present invention selects Figure 1 and Figure 2The bases at the two positions of the color mark are used as SNP sites, namely the base T at the 2574th position of the L3 resistance gene as SNP1, and the base T at the 2832nd position as SNP2. According to the difference in the bases at the SNP1 site, the L1c, L2 and L3 resistance genes can be distinguished from other types of resistance genes. According to the difference in the bases at the SNP2 site, the L1c and L3 resistance genes can be distinguished from the L2 resistance gene, but the L1c and L3 resistance genes cannot be distinguished. The number after the above L indicates the strength of resistance. The larger the number, the stronger the resistance. Among them, the varieties containing the L1c gene are susceptible varieties, L1c is a non-resistance gene, and L3 is a resistance gene.

[0021] Based on the above findings, the present invention is based on the above disclosed yellow lantern pepper gene sequence, and combined with the different mutation sites of the L series genes ( Figure 3 , Figure 4 , the L at the beginning of each sequence in the figure is an arbitrary label for each sequence), and the repetitive sequence at the position near the mutation site is searched. It is found that there are more than 50 homologous repetitive sequences near the mutation site of the L gene. However, compared with its homologous repetitive sequences, the present invention finds that there are three bases TTG near the mutation site, which are relatively specific sequences of the L gene ( Figure 2 , L series genes are disease-resistant R genes. There are a large number of repeated sequences in the genome, which will amplify non-L genes during amplification, resulting in false positives. Therefore, it is very important to determine the specific sequence of the L gene. The existing genome can be downloaded from the following address: http: / / www.bioinformaticslab.cn / files / genomes / pepper_pan / genome_v2 / Cchi.genome.v2.fa.gz). The present invention uses these three bases to develop specific primers for detecting L genes, and at the same time uses the differences in SNPs at different L gene variation sites to design SNP primers to specifically detect L series genes.

[0022] Based on the above comparison results, the present invention also found that the bases at positions 2877 and 2878 of the L1c gene sequence downstream of the SNP2 site are specific to other L genes, and several bases upstream and downstream of this site are significantly different from other L gene sequences.

[0023] Based on the above comparison results, and through the amplification of DNA from different materials and the optimization of amplification conditions, a set of molecular markers was successfully obtained, which can accurately distinguish the resistance genes L2, L3, and L1c. Specifically, SNP1 is the base T at the 2574th position of the L3 resistance gene, SNP2 is the base T at the 2832nd position, the TTG at positions 2801 - 2803 is the specific base sequence of the L gene, and the bases at positions 2872 - 2873, 2877 - 2878, 2882 - 2883, and 2885 - 2887 of the L1c gene are the specific sequences of L1c. Based on the above mutation sites, the following primer sequences were designed. The primer sequences are shown in Table 1, and the PCR conditions for specifically amplifying each site using these primer sequences are shown in Table 2, and the amounts of each substance used in the PCR reaction are shown in Table 3.

[0024] Table 1 Primer Sequences

[0025] Table 2 PCR Reaction Conditions

[0026] Table 3 Amounts of Each Substance Used in the PCR Reaction

[0027] In this example, the above primer sequences and PCR reaction conditions were used to amplify the genes of different varieties of peppers with determined resistance types to verify the specificity of the above molecular marker sites and the designed primers of the present invention. Specifically, the following varieties of yellow lantern peppers and the positive controls Zhongjiao 106 and Zhongjiao 107 were selected: Cc518, Cc506, Cc507, Cc509, Cc512, Cc513, Cc545, Zhongjiao 106, and Zhongjiao 107. Among them, the yellow lantern peppers are the germplasm resources of yellow lantern peppers preserved by the Institute of Crop Resources, Chinese Academy of Tropical Agricultural Sciences. Cc518 contains the L1c gene, Cc506, Cc507, and Cc509 are susceptible varieties, Cc512, Cc513, and Cc545 are resistant varieties, and Zhongjiao 106 and Zhongjiao 107 are annual pepper resistant varieties into which the L3 resistance gene has been introduced. The DNA of the above varieties of peppers was obtained by using the method of extracting DNA in the prior art, and PCR reaction amplification was carried out using the specific primers in Table 1, the PCR conditions in Table 2, and the amounts of each substance in Table 3. The amplification results are as Figure 5 shown in the electrophoresis diagram, where 1 - 9 correspond to Cc518, Cc506, Cc507, Cc509, Cc512, Cc513, Cc545, Zhongjiao 106, and Zhongjiao 107 respectively, and 10 - 12 are blank controls. Among them, Figure 5Band A was amplified by the specific primers of L1c designed based on the specific sites of L1c, which was consistent with the traits of the susceptible varieties Cc518, Cc506, Cc507, and Cc509; Cc512, Cc513, and Cc545 were resistant varieties, and Zhongjiao 106 and Zhongjiao 107 were resistant varieties into which the L3 resistance gene was introduced. No bands were amplified from the peppers of these varieties using the specific primers of L1c. Figure 5 Band B was the electrophoresis pattern of the results obtained by PCR amplification using the specific primers designed for the SNP1 molecular marker of the L3 gene. Bands were amplified from Cc518, Cc506, Cc507, Cc509, Cc512, Cc513, Cc545, Zhongjiao 106, and Zhongjiao 107. Figure 5 Band C was the electrophoresis pattern of the results obtained by PCR amplification using the specific primers designed for the SNP2 molecular marker of the L3 gene. Bands were amplified from Cc518, Cc506, Cc507, Cc509, Cc512, Cc513, Cc545, Zhongjiao 106, and Zhongjiao 107. No bands were amplified from the above blank controls. Based on the specificity of the above sites, if the electrophoresis pattern verification of the specific primers for L1c can amplify bands, and the specific primers for the SNP1 site of L3 and the specific primers for the SNP2 site of L3 can also amplify bands, it indicates that the pepper is a susceptible variety, and the gene type is L1c; if the electrophoresis pattern verification of the specific primers for L1c does not amplify bands, the specific primers for the SNP1 site amplify bands, and the specific primers for the SNP2 site also amplify bands, it indicates that the pepper is a resistant variety with strong disease resistance, and the gene type is L3.

[0028] In addition, according to the above gene sequence alignment results and the corresponding relationship between primer design and each SNP site and specific sequence, if the electrophoresis pattern verification of the specific primers for L1c does not amplify bands, the specific primers for the SNP1 site of L1c amplify bands, and the specific primers for the SNP2 site of L3 do not amplify bands, it indicates that the pepper is a resistant variety, but the disease resistance is slightly weak, and the gene type is L2; other electrophoresis results are unknown, and the disease resistance situation cannot be confirmed for the time being. Example 2

[0029] Using the SNP loci and specific sequences verified in Example 1, and the specific primers and PCR amplification conditions designed in Example 1, 44 core germplasm resources of Capsicum chinense Jacq. collected, maintained and purified by the Chinese Academy of Tropical Agricultural Sciences were detected. The detection results showed that most of the core germplasm resources of Capsicum chinense Jacq. contained the L1c gene and were disease-susceptible materials. Most of the other Capsicum chinense Jacq. materials contained the L2 or L3 resistance genes and were resistant materials. In particular, the resistant materials containing L3, including Cc505, Cc512, and Cc530, were all highly resistant materials previously identified by the inventors of the present invention in the field. Therefore, the present invention found that the resistance sources of Capsicum chinense Jacq. to Pepper mild mottle virus were all conferred by the L2 or L3 gene. The specific results are shown in Table 4, where 1 represents the presence of a band and 0 represents the absence of a band.

[0030] Table 4 Identification of resistance genes in 44 core germplasm resources of Capsicum chinense Jacq.

[0031] Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A molecular marker and specific gene sequence for detecting the resistance gene of the L series of pepper mild mottle virus, characterized in that, The molecular markers include SNP1 and SNP2. SNP1 is the base T at the 2574th position of the genome sequence of Capsicum chinense with the NCBI accession number AB523370, and SNP2 is the base T at the 2832nd position of the genome sequence of Capsicum chinense with the NCBI accession number AB523370. The specific gene sequence is the specific gene sequence of the L1c type gene of the L series resistance gene, and the specific gene sequence is the bases CA at the 2872-2873rd positions, TT at the 2877-2878th positions, TG at the 2882-2883rd positions, and AGA at the 2885-2887th positions of Capsicum chinense with the NCBI accession number AB523374.

2. A primer set for amplifying the molecular marker and specific gene sequence described in claim 1, characterized in that The primer set includes three pairs of primers, specifically the L1c primer pair for specifically amplifying the sequence containing the specific gene sequence. The upstream primer sequence of the L1c primer pair is as described in SEQ ID NO.1, and the downstream primer sequence is as shown in SEQ ID NO.

2. The L3-1 primer pair for specifically amplifying the sequence containing the SNP1 site. The upstream primer sequence of the L3-1 primer pair is as shown in SEQ ID NO.3, and the downstream primer sequence is as described in SEQ ID NO.

4. The L3-2 primer pair for specifically amplifying the sequence containing the SNP2 site. The upstream primer sequence of the L3-2 primer pair is as shown in SEQ ID NO.5, and the downstream primer sequence is as described in SEQ ID NO.

6.

3. A kit for detecting the L series genes of pepper mild mottle virus, characterized in that, The kit includes the primer set as claimed in claim 2.

4. The application of the molecular markers and specific gene sequences as claimed in claim 1, the primer set as claimed in claim 2, and the kit as claimed in claim 3 in the breeding process of disease-resistant pepper varieties.

5. A method for detecting the resistance gene of the L series of pepper mild mottle virus, characterized in that, It includes the following steps: Step S1, extract genomic DNA from pepper. Step S2, perform PCR amplification on the genomic DNA extracted in step S1 using the primer set as claimed in claim 2 or the kit as claimed in claim 3, and then judge the type of the L series resistance gene based on the electrophoresis pattern of the amplification product.

6. The method according to claim 5, characterized in that, The pepper in step S1 is Capsicum chinense.

7. The method according to claim 5, wherein The specific judgment criteria for determining the type of L series resistance genes based on the electrophoresis pattern of the amplification product described in step S2 are as follows: If the electrophoresis pattern verification of the specific primer for L1c can amplify a band, and the specific primers for SNP1 locus of L3 and the specific primers for SNP2 locus of L3 can also amplify bands, it indicates that the pepper is a susceptible variety, and the gene type is L1c; If the electrophoresis pattern verification of the specific primer for L1c fails to amplify a band, while the specific primer for the SNP1 locus of L1c amplifies a band and the specific primer for the SNP2 locus of L3 fails to amplify a band, it indicates that the pepper is a disease-resistant variety, but the disease resistance is slightly weak, and the gene type is L2; If the electrophoresis pattern verification of the specific primer for L1c fails to amplify a band, the specific primer for the SNP1 locus amplifies a band, and the specific primer for the SNP2 locus also amplifies a band, it indicates that the pepper is a disease-resistant variety and has strong disease resistance, and the gene type is L3; Other electrophoresis results belong to unknown situations, and the disease resistance gene type cannot be confirmed temporarily.

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