Molecular markers for detecting L-series resistance genes of pepper mild mottle virus and their applications

Abstract

This invention provides a molecular marker for detecting L-series resistance genes to pepper mild mottle virus and its application, belonging to the field of genetic engineering technology. The molecular marker includes SNP1 and SNP2, where SNP1 is the T base at position 2574 of the gene sequence with NCBI accession number AB523370, and SNP2 is the T base at position 2832 of the gene sequence with NCBI accession number AB523370. The specific gene sequence is a specific gene sequence for the L1c type of the L-series resistance gene, specifically the CA bases at positions 2872-2873, the TT bases at positions 2877-2878, the TG bases at positions 2882-2883, and the AGA bases at positions 2885-2887 of the gene sequence with NCBI accession number AB523374. This invention utilizes the aforementioned molecular markers and specific sequences to rapidly and accurately detect L-series resistance gene types in yellow lantern peppers and annual peppers, and can be used for marker-assisted selection breeding.

Description

Technical Field

[0001] This invention relates to a molecular marker and its application, and more particularly to a molecular marker for detecting L-series resistance genes of pepper mild mottle virus and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] Yellow lantern chili (scientific name: *Capsicum chinense*), also known as yellow emperor chili, yellow pepper, or Chinese chili, is a plant belonging to the genus *Capsicum* of the subtribe of the Solanaceae family. It is one of the five cultivated species of chili peppers, originating from Cuba, Brazil, Peru, Bolivia, Paraguay, and other countries. It is named after the golden-yellow color of the cultivar cultivated in Hainan Province, China. Yellow lantern chili peppers are rich in capsaicin and have a high spiciness level, reaching up to 170,000 SHU. They are generally not eaten fresh and are mainly used to make yellow lantern chili sauce in Hainan Province. Large-scale cultivation of yellow lantern chili peppers in China is mainly concentrated in Wenchang, Qionghai, Wanning, and Lingshui in Hainan Province, with the main cultivated varieties being the local variety and the hybrid "Hot and Spicy No. 2".

[0003] Pepper mild mottle virus (PMMoV) belongs to the genus Tobacco Mosaic Virus. In recent years, PMMoV has been detected in many major pepper-producing areas in my country, and the detection rate is constantly increasing, seriously threatening the development of the pepper industry. Early symptoms of PMMoV infection are not obvious, mostly manifesting as mild chlorosis of the leaves. Later symptoms become more pronounced, causing leaf wrinkling, deformation, mottling, or yellow-green mosaic symptoms. This leads to smaller, deformed, mottled, and even necrotic fruits that are prone to falling off, severely affecting the yield and quality of peppers.

[0004] The L-series resistance genes are important resistance genes for pepper mild mottle virus, currently classified into L1, L1a, L1c, L2, L2b, L3, and L4 genes, with L1c and L3 genes originating from the yellow lantern pepper. The highly resistant gene L3 has been used for resistance breeding in annual peppers. Markers have been developed for use in annual pepper breeding based on sequences adjacent to L3. These markers are based on a SNP site linked to the L3 gene. Genotypes without the L3 gene are designated A, while those containing it are designated C. Genotypes containing A have a SspI restriction enzyme site, while those containing C do not. Therefore, previous researchers developed a method for identifying the L3 gene based on SspI restriction enzyme digestion. Researchers at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, have also developed the 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 variety of yellow lantern pepper. Regardless of whether yellow lantern pepper contains the L3 gene, the SNPs linked to the L gene are all C, which means that L3 and other markers cannot be used for molecular breeding of intraspecific or interspecific hybridization of yellow lantern pepper. Therefore, it is necessary to develop new markers for molecular breeding of disease resistance in yellow lantern pepper. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a molecular marker for detecting L-series resistance genes of pepper mild mottle virus and its application, in order to accelerate the breeding process of new disease-resistant varieties of yellow lantern pepper.

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

[0007] The primer set used to amplify the above-mentioned molecular markers and specific gene sequences includes three pairs of primers: an L1c primer pair specifically amplifying the specific gene sequence, wherein 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; an L3-1 primer pair specifically amplifying the SNP1 site, wherein 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; and an L3-2 primer pair specifically amplifying the SNP2 site, wherein 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 L-series genes of pepper mild mottle virus, the kit comprising the primer set described above.

[0009] The application of the above-mentioned molecular markers and specific gene sequences, primer sets, and reagent kits in the breeding process of disease-resistant pepper varieties.

[0010] A method for detecting L-series resistance genes to pepper mild mottle virus includes the following steps:

[0011] Step S1: Extract genomic DNA from chili peppers;

[0012] Step S2: Use the primer set or the kit described above to perform PCR amplification on the genomic DNA extracted in step S1, and then determine the type of L-series resistance gene based on the electrophoresis diagram of the amplification product.

[0013] Furthermore, the chili pepper mentioned in step S1 is a yellow lantern chili pepper or an annual chili pepper variety.

[0014] Furthermore, the specific criteria for determining the type of L-series resistance gene based on the electrophoresis pattern of the amplified products in step S2 are as follows: If the electrophoresis pattern of the specific primers for L1c can amplify a band, and the specific primers for the SNP1 site and 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 of the specific primers for L1c does not amplify a band, but the specific primers for the SNP1 site of L1c amplify a band... If a band is obtained, and the specific primer for the SNP2 site of L3 does not amplify a band, it indicates that the pepper is a disease-resistant variety, but with slightly weaker resistance, and the gene type is L2. If the specific primer for L1c does not amplify a band, but 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 pepper is a disease-resistant variety with relatively strong resistance, and the gene type is L3. Other electrophoresis results are unknown, and the disease-resistant gene type cannot be confirmed at this time.

[0015] The beneficial effects of the molecular markers for detecting L-series resistance genes of pepper mild mottle virus of the present invention and their applications are as follows:

[0016] This invention utilizes the aforementioned molecular markers and specific sequences to rapidly and accurately detect the L-series resistance gene types of yellow lantern peppers. It can be used for marker-assisted selection breeding, enabling rapid, accurate, and efficient screening of resistant varieties and accelerating the breeding process of new disease-resistant yellow lantern pepper varieties.

[0017] Specifically, SNP1 in this invention is the T base at position 2574 of the L3 gene, and SNP2 is the T base at position 2832 of the L3 gene. The TTG at positions 2801-2803 is a specific base sequence for the L gene. The bases at positions 2872-2873, 2877-2878, 2882-2883, and 2885-2887 of the L1c gene are specific sequences for L1c. Using specific primers designed based on these specific sites and gene sequences, PCR amplification of pepper plants can be performed, and the L-series genotype of the pepper variety can be quickly determined by analyzing the electrophoretic bands. The specific criteria are: if the electrophoretic image of the L1c-specific primers amplifies a band, and the specific primers for both the L3 SNP1 and L3 SNP2 sites also amplify bands, it indicates that the pepper is a susceptible variety, and the gene type... The gene type is L1c. If the specific primer electrophoresis of L1c does not amplify a band, but the specific primer for SNP1 of L1c amplifies a band, and the specific primer for SNP2 of L3 does not amplify a band, then the pepper is a disease-resistant variety, but with slightly weaker resistance, and the gene type is L2. If the specific primer electrophoresis of L1c does not amplify a band, but the specific primer for SNP1 amplifies a band, and the specific primer for SNP2 amplifies a band, then the pepper is a disease-resistant variety with relatively strong resistance, and the gene type is L3. Other electrophoresis results are unknown, and the disease resistance cannot be confirmed at this time. Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 This is a comparison of the amino acid sequences of the L-series genes and the positions of the amino acids corresponding to their SNPs in the embodiments of the present invention;

[0020] Figure 2 This refers to the alignment results of the L-series gene amino nucleotide sequences and the locations of their SNPs, L-gene-specific sequences, and L1c-specific sequences in the embodiments of this invention.

[0021] Figure 3 This is the alignment result of the sequence near SNP1 with other similar sequences in the genome; the red box indicates the location of SNP1.

[0022] Figure 4 This is the alignment result of the L gene-specific sequence and the sequence near SPN2 with other similar sequences in the genome. The red box indicates the location of the L gene-specific sequence and SPN2.

[0023] Figure 5 This is an electrophoresis diagram of the L-series resistance gene detection results in an embodiment of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Example 1

[0025] The development of molecular markers for detecting L-series resistance genes of pepper mild mottle virus and the design of corresponding detection primers are as follows:

[0026] The L-series resistance genes are important resistance genes for pepper mild mottle virus. Currently, they are divided into L1, L1a, L1c, L2, L2b, L3, and L4 genes. Among them, L1c and L3 genes are derived from yellow lantern pepper. Therefore, this invention mainly detects whether the germplasm resources of various yellow lantern pepper varieties contain L1c and L3 resistance genes. The focus is on detecting whether the L3 resistance gene is present and distinguishing it from other resistance genes in order to detect its resistance ability and provide a screening basis for yellow lantern pepper breeding.

[0027] This invention, by downloading and comparing the gene sequences of L1, L1a, L1c, L2, L2b, L3, and L4 from the NCBI gene database, discovered differences in base and amino acid types at several positions in these gene sequences. See details below. Figure 1 and Figure 2 The comparison results. The NCBI accession numbers for the L1 resistance gene (corresponding to the plant Capsicum annuum) are AB523372, L1a resistance gene (corresponding to the plant Capsicum annuum) is AB523373, L1c resistance gene (corresponding to the plant Capsicum chinense) is AB523374, L2 resistance gene (corresponding to the plant Capsicum frutescens) is AB523375, L2b resistance gene (corresponding to the plant Capsicum baccatum) is AB523376, L3 resistance gene (corresponding to the plant Capsicum chinense) is AB523370, and L4 resistance gene (corresponding to the plant Capsicum chacoense) is AB523377.

[0028] Based on the above comparison results, the present invention selected Figure 1 and Figure 2 The two bases marked with a specific color are designated as SNP sites: the T at position 2574 of the L3 resistance gene is SNP1, and the T at position 2832 is SNP2. The L1c, L2, and L3 resistance genes can be distinguished from other types of resistance genes based on the different bases at SNP1. The L1c and L3 resistance genes can be distinguished from the L2 resistance gene based on the different bases at SNP2, but the L1c and L3 resistance genes cannot be distinguished individually. The number after the L indicates the strength of the resistance; a larger number indicates stronger resistance. Varieties containing the L1c gene are susceptible varieties; L1c is a non-resistance gene, and L3 is a resistance gene.

[0029] Based on the above findings, this invention, according to the disclosed yellow lantern pepper gene sequence, and combined with the different mutation sites of the L-series genes ( Figure 3 , Figure 4 In the figure, the L at the beginning of each sequence is an arbitrary label. Searching for repetitive sequences near the mutation site revealed more than 50 homologous repetitive sequences near the L gene mutation site. However, relative to these homologous repetitive sequences, this invention found that three bases, TTG, near the mutation site are relatively specific sequences of the L gene. Figure 2 The L-series genes are disease-resistant R genes, and their genomes contain a large number of repetitive sequences. This can lead to the amplification of non-L genes, resulting in false positives. Therefore, the determination of L-gene-specific sequences is crucial. Existing genomes can be downloaded from the following address: http: / / www.bioinformaticslab.cn / files / genomes / pepper_pan / genome_v2 / Cchi.genome.v2.fa.gz. This invention utilizes these three bases to develop L-gene-specific primers and designs SNP primers based on the differences in SNPs at different L-gene variant sites to specifically detect L-series genes.

[0030] Based on the above comparison results, the present invention also found that the bases at positions 2877 and 2878 downstream of the SNP2 site in the L1c gene sequence are specific to other L genes, and there are several bases upstream and downstream of this site that are significantly different from other L gene sequences.

[0031] 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 T at position 2574 of the L3 resistance gene, SNP2 is the T at position 2832, 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 variant sites, the following primer sequences were designed, as shown in Table 1. 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.

[0032] Table 1 Primer sequences

[0033]

[0034] Table 2 PCR reaction conditions

[0035]

[0036] Table 3 Amounts of each substance used in the PCR reaction

[0037]

[0038] In this embodiment, the above primer sequences and PCR reaction conditions were used to amplify genes in different pepper varieties with identified resistance types to verify the specificity of the molecular marker sites and designed primers of the present invention. Specifically, the following varieties of yellow lantern pepper and 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 pepper is a germplasm resource of yellow lantern pepper preserved by the Institute of Germplasm Resources of the Chinese Academy of Tropical Agricultural Sciences. Cc518 contains the L1c gene, and Cc506, Cc507, and Cc509 contain the L1c gene. Cc512, Cc513, and Cc545 are susceptible varieties, while Zhongjiao 106 and Zhongjiao 107 are one-year-old resistant pepper varieties infused with the L3 resistance gene. DNA from these pepper varieties was obtained using existing techniques. PCR amplification was performed 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 shown below. Figure 5 The electrophoresis diagram shows that 1-9 correspond to Cc518, Cc506, Cc507, Cc509, Cc512, Cc513, Cc545, Zhongjiao 106, and Zhongjiao 107, respectively, while 10-12 are blank controls.

[0039] Figure 5 A represents the band amplified using L1c-specific primers designed with specific sites on the L1c site. This band matches the traits of susceptible varieties Cc518, Cc506, Cc507, and Cc509. Cc512, Cc513, and Cc545 are resistant varieties, while Zhongjiao 106 and Zhongjiao 107 are resistant varieties introduced with the L3 resistance gene. These varieties of peppers did not amplify any bands using L1c-specific primers.

[0040] Figure 5 B is an electrophoresis diagram of the PCR amplification results obtained using specific primers designed for the SNP1 molecular marker of the L3 gene. Bands were amplified for Cc518, Cc506, Cc507, Cc509, Cc512, Cc513, Cc545, Zhongjiao 106 and Zhongjiao 107.

[0041] Figure 5 C is an electrophoresis diagram of the PCR amplification results obtained using specific primers designed for the SNP2 molecular marker of the L3 gene. Bands were amplified for Cc518, Cc506, Cc507, Cc509, Cc512, Cc513, Cc545, Zhongjiao 106 and Zhongjiao 107.

[0042] None of the blank controls produced any amplified bands.

[0043] Based on the specificity of the above loci, if the electrophoresis of specific primers for L1c can amplify a band, and the specific primers for SNP1 and SNP2 of L3 can also amplify bands, it indicates that the pepper is a susceptible variety with the gene type L1c. If the electrophoresis of specific primers for L1c does not amplify a band, but the specific primers for SNP1 and SNP2 amplify a band, it indicates that the pepper is a resistant variety with strong resistance and the gene type L3.

[0044] Furthermore, based on the above gene sequence alignment results and the correspondence between primer design and each SNP site and specific sequence, it can be seen that if the specific primer for L1c does not amplify a band in the electrophoresis verification, but the specific primer for the SNP1 site of L1c amplifies a band, and the specific primer for the SNP2 site of L3 does not amplify a band, it indicates that the pepper is a disease-resistant variety, but its disease resistance is slightly weak, and the gene type is L2; ​​other electrophoresis results are unknown, and the disease resistance cannot be confirmed at this time. Example 2

[0045] Using the SNP sites and specific sequences verified in Example 1, and employing the specific primers and PCR amplification conditions designed in Example 1, 44 core germplasm resources of yellow lantern pepper collected, maintained, and purified by the Chinese Academy of Tropical Agricultural Sciences were tested. The results showed that most of the core germplasm resources of yellow lantern pepper contained the L1c gene and were susceptible materials. Other yellow lantern pepper materials mostly contained L2 or L3 resistance genes and were resistant materials. In particular, the resistant materials containing L3, including Cc505, Cc512, and Cc530, were highly resistant materials previously identified in the field by the inventors of this invention. Therefore, this invention found that the resistance to pepper mild mottle virus in yellow lantern pepper is due to the resistance conferred by the presence of L2 or L3 genes. Specific results are shown in Table 4, where 1 represents the presence of a band and 0 represents the absence of a band.

[0046] Table 4. Identification of resistance genes in 44 core germplasm resources of yellow lantern pepper.

[0047]

[0048] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A primer set for amplifying molecular markers and specific gene sequences for detecting L-series resistance genes to pepper mild mottle virus, characterized in that, The molecular markers include SNP1 and SNP2, where SNP1 is the T base at position 2574 of the gene sequence with NCBI accession number AB523370, and SNP2 is the T base at position 2832 of the gene sequence with NCBI accession number AB523370; the specific gene sequence is the specific gene sequence of the L-series resistance gene L1c type gene, where the specific gene sequence is the CA base at positions 2872-2873, the TT base at positions 2877-2878, the TG base at positions 2882-2883, and the AGA base at positions 2885-2887 of the gene sequence with NCBI accession number AB523374; The primer set includes three pairs of primers, specifically L1c primer pairs that specifically amplify sequences containing specific genes. 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 containing the SNP1 site was specifically amplified, wherein the upstream primer sequence of the L3-1 primer pair is shown in SEQ ID NO.3 and the downstream primer sequence is shown in SEQ ID NO.4; The L3-2 primer pair containing the SNP2 site was specifically amplified. The upstream primer sequence of the L3-2 primer pair is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.

6.

2. A kit for detecting L-series genes of pepper mild mottle virus, characterized in that, The kit includes the primer set as described in claim 1.

3. The application of the primer set of claim 1 and the kit of claim 2 in the breeding of disease-resistant pepper varieties, characterized in that, The chili pepper is the yellow lantern chili pepper, and the disease-resistant variety is a chili pepper mild mottle virus resistant variety; If specific primers for L1c amplify a band in electrophoresis, and specific primers for both SNP1 and SNP2 sites of L3 also amplify bands, then the pepper is a susceptible variety with the L1c genotype. If specific primers for L1c do not amplify a band, but specific primers for SNP1 amplify a band while specific primers for SNP2 amplify a band, then the pepper is a resistant variety, but with slightly weaker resistance, and the genotype is L2. If specific primers for L1c do not amplify a band, but specific primers for both SNP1 and SNP2 amplify a band, then the pepper is a resistant variety with relatively strong resistance, and the genotype is L3. Other electrophoresis results are unknown, and the resistant genotype cannot be confirmed at this time.

4. A method for detecting L-series resistance genes of pepper mild mottle virus, characterized in that, Includes the following steps: Step S1: Extract genomic DNA from chili peppers; Step S2: PCR amplification of the genomic DNA extracted in step S1 is performed using the primer set as described in claim 1 or the kit as described in claim 2, and then the type of L-series resistance gene is determined based on the electrophoresis diagram of the amplification product. The chili pepper mentioned in step S1 is the yellow lantern chili pepper; The specific criteria for determining the type of L-series resistance gene based on the electrophoresis pattern of the amplified products in step S2 are as follows: If the electrophoresis pattern of the specific primers for L1c can amplify a band, and the specific primers for the SNP1 site and 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 of the specific primers for L1c does not amplify a band, but the specific primers for the SNP1 site of L1c amplify a band... If the specific primers for the SNP2 site of L3 do not amplify a band, it indicates that the pepper is a disease-resistant variety, but with slightly weaker resistance, and the gene type is L2. If the specific primers for L1c do not amplify a band, but the specific primers for the SNP1 site and the specific primers for the SNP2 site both amplify a band, it indicates that the pepper is a disease-resistant variety with relatively strong resistance, and the gene type is L3. Other electrophoresis results are unknown, and the disease-resistant gene type cannot be confirmed at this time.

Images