Molecular marker and method for detecting gray mold of Chinese rose

Abstract

Rosa chinensis gray mold is a main disease in the cultivation process of rosa chinensis, and seriously threatens the quality and yield of cut-flower rosa chinensis. In order to effectively cope with the disaster stress which influences the yield and quality of the Chinese rose, 192 pairs of SSR primers are used in the research to carry out PCR amplification on DNA of parents' Jinmai '' Beijing red 'and 6 randomly selected offspring. 35 pairs of primers show genetic difference between male and female parents. The 35 pairs of primers are further used for carrying out PCR amplification on filial generation DNA, detection is carried out through PAGE electrophoresis, and an electrophoretic band is converted into a 0, 1 matrix for genetic typing. The method comprises the following steps: selecting an optimal subset through correlation regression analysis, and finally successfully screening out three primers, namely, Geno-BC-1, Geno-BC-2 and Geno-BC-3, which are related to gray mold resistance. The discovery provides an important theoretical basis for molecular marker-assisted Chinese rose disease-resistant breeding, and is expected to promote early identification of disease-resistant plants, so that the breeding period is shortened, and the breeding efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular breeding, and particularly to molecular markers and methods for detecting Botrytis cinerea of roses. Background Art

[0002] Rosa chinensis Jacq. belongs to the genus Rosa L. Roses have high demand in the fields of cut flowers, garden decoration, and flower beds due to their colorful and diverse flower colors and ever-changing flower shapes. In recent years, with the improvement of people's living standards, the market demand for cut roses has been increasing year by year and has become the largest category in the cut flower market.

[0003] During the growth, preservation, and transportation of roses, affected by factors such as humidity and temperature, roses are extremely prone to diseases caused by the invasion of pathogens such as Botrytis cinerea, Peronospora sparsa, and Podosphaera pannosa, which affect their production, circulation, and sales. Among these diseases, Botrytis cinerea is one of the most serious diseases in the current greenhouse rose cultivation and long-distance transportation processes.

[0004] Gray mold of rose mainly harms leaves, buds, flower buds and flowers, and can also harm young stems. When it occurs on the leaf margins and leaf tips, it is initially a water-soaked light brown spot, which is smooth and slightly sunken, and then expands and rots. When the flower buds are infected, the lesions are gray-black, which can prevent the flowers from opening, and the diseased buds turn brown and die. When the flowers are attacked, some petals turn brown, shrink and rot. Gray mold of rose is caused by Botrytis cinerea Pers. of the genus Botrytis of the subphylum Ascomycota. The gray mold pathogen infiltrates into tissues such as flowers and leaves in the form of mycelium and sclerotia to overwinter. In spring, when conditions are suitable, conidia invade the interior and cause damage to plant tissues. After the conidia mature and fall off, they are spread and reinfected through operations such as airflow, raindrops, and cultivated land. However, plants have formed a complex signal transduction network including local and systemic signal transduction networks during evolution to improve their own defense capabilities against pathogens. The plant immune system against pathogen infection mainly includes the immune response triggered by the effective components of pathogenic microorganisms (PAMP-Triggered-Immunity, PTI) and the immune response triggered by effector factors (Effector-Triggered-Immunity, ETI). Pathogens secrete some special conservative components (Pathogen Associated Molecular Patterns, PAMPs) that can be recognized by certain receptors on the surface of plant cells, thereby activating the PTI pathway. After the pathogen specifically infects the host, it can secrete virulence effectors to inhibit the PTI pathway. At this time, the nucleotide binding receptors (NB-LRR) in the cell recognize the effectors, thereby activating the ETI pathway to produce an immune response. Botrytis cinerea is a necrotrophic fungus. The OGs secreted when degrading the host cell wall are recognized by the receptor WAK1 and activate downstream resistance reactions. PAMPs and DAMPs induce immune responses, and there is a great degree of overlap in the activation of protein kinases, the synthesis of protective factors, and the regulation of hormones. The defense response formed by the combination of the two can effectively inhibit the infection of Botrytis cinerea.

[0005] Gray mold of rose not only reduces the ornamental value of cut rose, but also greatly restricts the development and rise of rose industry. In the prior art, the control method of gray mold of rose includes removing diseased flowers, reducing air humidity, and spraying the medicine for curing the disease when the disease occurs (for example, 600 times of 65% mancozeb wettable powder, 700 times of 75% thiophanate-methyl wettable powder or 1500 times of 70% thiophanate-methyl wettable powder). However, the control method relying on chemical agents is easy to affect the ecological environment and human health. The traditional screening and identification method for gray mold-resistant plants includes inoculating gray mold fungi into the identification host plant, and after a certain fungal spore reproduction cycle, recording and counting the changes in the susceptible area of ​​the host plant, so as to identify plant materials with different gray mold resistance grades.

[0006] CN115820918A discloses a method for rapidly screening rose germplasm resources resistant to Botrytis cinerea, which includes obtaining the relative expression level of the Botrytis cinerea resistance marker gene in rose by using the specific primer RcWRKY22 for rose germplasm resources resistant to Botrytis cinerea, so as to identify the resistance of rose to Botrytis cinerea.

[0007] At present, there is relatively little research on the genetic characteristics of rose genes resistant to Botrytis cinerea and related molecular markers in China.

[0008] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, although the applicant has studied a large number of documents and patents when making this invention, due to space limitations, all details and content are not listed in detail. However, this does not mean that this invention does not possess the features of these prior arts. On the contrary, this invention already possesses all the features of the prior arts, and the applicant reserves the right to add relevant prior arts in the background art. Summary of the Invention

[0009] The development of Simple Sequence Repeats (SSR) markers generally adopts various strategies, including traditional techniques, enrichment strategies, database construction, and high-throughput sequencing technologies. Compared with other types of molecular markers, SSR markers are well-known for their high polymorphism, rich information content, and good repeatability. Therefore, they are more widely used in the construction of genetic linkage maps and molecular marker-assisted breeding. Roses have a rich genetic background and a wide variety of cultivated varieties. The trait segregation of their hybrid offspring is significant, and they show high genetic diversity at the molecular level, which provides convenient conditions for the genomic research of roses. In this application, relevant research results show three molecular markers that are closely related to the resistance of roses to Botrytis cinerea. The purpose of this invention is to expand the application scope of SSR molecular markers and optimize the resistance of rose varieties, so as to provide more comprehensive means and solutions for the genetic improvement of roses.

[0010] One of the purposes of this invention is to provide the SSR molecular marker primers involved in this application, so as to be able to more accurately and quickly identify the disease-resistant traits of rose varieties and provide important molecular tools for rose breeding.

[0011] One of the purposes of this invention is to develop related kits and detection methods by using the SSR molecular marker primers involved in this application, which is convenient for screening and identifying rose varieties resistant to Botrytis cinerea in the actual production and breeding processes.

[0012] One of the purposes of this invention is to provide a method for rose breeding resistant to Botrytis cinerea. By combining the SSR molecular marker primers involved in this application with traditional breeding techniques, the disease resistance of new rose varieties can be significantly improved, the breeding process can be accelerated, and the breeding efficiency can be enhanced.

[0013] One of the objectives of the present invention is to provide a Marker Assisted Selection (MAS) method using the SSR molecular markers involved in this application to identify and select rose materials resistant to Botrytis cinerea at the early breeding stage, thereby optimizing the breeding process and reducing the workload and time cost of field screening.

[0014] One of the objectives of the present invention is to provide primer pairs for amplifying SSR molecular markers for detecting the resistance of roses to Botrytis cinerea, and the primer pairs are selected from one or more of the following primer pairs:

[0015] 1) The forward primer sequence is as shown in SEQ NO.65, and the reverse primer sequence is as shown in SEQ NO.66;

[0016] 2) The forward primer sequence is as shown in SEQ NO.67, and the reverse primer sequence is as shown in SEQ NO.68;

[0017] 3) The forward primer sequence is as shown in SEQ NO.69, and the reverse primer sequence is as shown in SEQ NO.70.

[0018] According to a preferred embodiment, the above molecular markers can be used in the identification or assisted identification of homozygous Botrytis cinerea-resistant and heterozygous Botrytis cinerea-susceptible traits in roses.

[0019] According to a preferred embodiment, for genetic linkage identification, if any specific band corresponding to the above 3 pairs of SSR primers can be amplified from the rose to be tested, it indicates that the genetic material contained in the rose germplasm to be tested is located in the corresponding genetic linkage group; otherwise, the genetic material contained in the rose germplasm to be tested does not have the corresponding genetic linkage group.

[0020] One of the objectives of the present invention is to provide the application of the SSR molecular marker primers for detecting the resistance of roses to Botrytis cinerea involved in this application in any one of the following 1) to 4):

[0021] 1) Identifying or assisting in the identification of Botrytis cinerea-resistant / susceptible rose materials;

[0022] 2) Identifying or assisting in the identification of Botrytis cinerea-resistant / susceptible genes in roses;

[0023] 3) Screening or assisting in the screening of Botrytis cinerea-resistant rose varieties;

[0024] 4) Rose breeding.

[0025] One of the objectives of the present invention is to provide a kit containing the SSR molecular marker primers for detecting the resistance of roses to Botrytis cinerea involved in this application.

[0026] One of the objectives of the present invention is to provide a gene chip containing SSR molecular marker primers as involved in the present application, and the gene chip is used for high-throughput detection of genotypes related to Botrytis cinerea resistance in rose populations.

[0027] One of the objectives of the present invention is to provide a method for identifying Botrytis cinerea resistance in roses, and the method includes the following steps: extracting genomic DNA of the plant to be tested; using the genomic DNA of the plant to be tested as a template, and performing a PCR amplification reaction by using the SSR molecular marker primers involved in the present application; detecting the PCR amplification product, wherein if any specific band corresponding to any one of the above-mentioned 1), 2) and / or 3) pairs of primers can be amplified from the plant to be tested, it indicates that the plant to be tested is susceptible to Botrytis cinerea, and if no specific band is amplified, the plant to be tested is resistant to Botrytis cinerea.

[0028] According to a preferred embodiment, the method for identifying Botrytis cinerea resistance in roses includes detecting the genotype of the corresponding SSR locus by real-time fluorescence quantitative PCR or digital PCR method.

[0029] According to a preferred embodiment, the rose variety can be 'Beijing Red', 'Golden Mary' or the hybrid offspring of both.

[0030] One of the objectives of the present invention is to provide a method for cultivating rose varieties, which includes the following steps: performing genotyping on rose materials by using the SSR molecular marker primers involved in the present application; selecting rose materials resistant to Botrytis cinerea for hybridization according to the genotyping results; screening the hybrid offspring to obtain new rose varieties resistant to Botrytis cinerea.

[0031] One of the objectives of the present invention is to provide a breeding method for improving Botrytis cinerea resistance in roses, and the method is to introduce the disease-resistant genes associated with the SSR molecular marker primers involved in the present application for gene introduction or directional breeding.

[0032] One of the objectives of the present invention is to provide the use of the genes with Geno-MK112843, Geno-MK141053 or Geno-MK17618 in identifying Botrytis cinerea in roses.

[0033] Advantages of the present technical solution:

[0034] In the article "Identification of Botrytis cinerea Resistance and Analysis of Its Correlation with Phenotypes in Different Rose Cultivars", the research progress on the identification of Botrytis cinerea resistance and breeding in roses shows that screening disease-resistant cultivars is an effective way to improve rose resistance. For example, through two consecutive years of field surveys and artificial inoculation on petals, it was found that there were significant differences in disease resistance among different rose resources, and some highly resistant cultivars such as Zhuhong Queen and Black Baccara were screened out, which can be used as excellent alternative materials for disease-resistant breeding. Therefore, resistance identification based on molecular markers is an effective means for breeding commercially available disease-resistant rose cultivars on a large scale.

[0035] As Figure 4 shown, in this application, the in vitro petal disc infection method was used to measure the resistance of common rose cultivars 'Golden Mary (JML)' and 'Beijing Red (BJH)' and their hybrid offspring to Botrytis cinerea. Polymorphic primers were screened using SSR molecular marker technology, and genotyping analysis was performed on the rose hybrid population. In addition, this application also carried out an association analysis between the Botrytis cinerea resistance phenotype and molecular markers in roses, aiming to identify molecular markers related to Botrytis cinerea resistance in roses. Through relevant screening, three molecular markers closely associated with rose resistance / susceptibility to Botrytis cinerea were obtained.

[0036] Through further verification and analysis of these three molecular markers in this application, the potential application value of them in Botrytis cinerea resistance breeding was preliminarily determined. These molecular markers can not only be used for early screening of disease-resistant rose cultivars, but also improve the selection efficiency and shorten the breeding cycle in the actual breeding process. At the same time, the correlation between the resistance markers and phenotypic resistance is relatively high, indicating that these markers can effectively reflect the resistance phenotype of Botrytis cinerea. Therefore, these molecular markers can be used as reliable tools for Botrytis cinerea resistance breeding in roses, which helps to achieve the large-scale popularization and application of disease-resistant cultivars.

[0037] In summary, the three molecular markers proposed in this application not only provide a scientific and effective method for screening rose materials with disease resistance, but also help to accelerate the process of rose disease-resistant breeding, and at the same time provide a theoretical basis for future molecular marker-assisted selection breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a phenotypic diagram of the rose cultivars 'Beijing Red' (1A) and 'Golden Mary' (1B) provided by the present invention when not infected with the disease;

[0039] Figure 2 It is a partial amplification result of primer No. 54 (Geno-MK116428) provided by the present invention. As shown in the figure, the bands of the DNA marker are 250bp and 100bp from top to bottom in sequence;

[0040] Figure 3Partial amplification results of primer No. 59 (Geno-MK117353) provided by the present invention are shown in the figure. The bands of DNA marker are 250bp and 100bp from top to bottom in sequence;

[0041] Figure 4 Phenotype diagrams of rose cultivars 'Beijing Red', 'Golden Mary' and the F1 population hybridized from 'Golden Mary' and 'Beijing Red' infected with Botrytis cinerea provided by the present invention. Detailed implementation manners

[0042] The following is a detailed description with reference to the accompanying drawings.

[0043] The present invention illustrates the rose disease resistance identification method and related molecular markers of the present invention through the following embodiments. However, the present invention is not limited to the following embodiments, that is, it does not mean that the present invention must rely on the following embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and public scope of the present invention.

[0044] Molecular Marker refers to a means for specifically detecting genetic materials at the DNA or RNA level. With the development of molecular biology, more and more molecular markers have been widely used in the research of horticultural plant genomes, and molecular marker-assisted breeding has become an important means to improve the genetic traits of horticultural plants. Compared with detection indexes such as morphological markers, cytological markers, and biochemical markers, molecular markers are a more microscopic and root-directed technical means, and are now widely used in the research of plant genomes. SSR molecular markers have the advantages of low requirements for the quantity and quality of DNA, easy operation, stable results, and repeatability, and can cover the entire genome and have high polymorphism. Compared with other molecular markers such as RFLP, AFLP, and ISSR, SSR markers have the characteristics of high polymorphism, co-dominant inheritance, good repeatability, and strong specificity, and have become the most widely used marker in the fields of genetic diversity research, genetic mapping, important functional gene mapping, and molecular-assisted breeding in recent years. SSR is a class of repetitive sequences composed of 1-6bp nucleotide motifs, which are widely distributed in the coding regions and non-coding regions of eukaryotic genomes. SSR markers design primers using the flanking conserved sequences of SSRs, and after PCR amplification, the polymorphism of DNA sequences is reflected according to the size of the bands.

[0045] Example 1

[0046] 1. Experimental materials

[0047] As Figure 1As shown, 170 individual plants of the rose varieties 'Beijing Red', 'Golden Mary' and the F1 population offspring of the cross between 'Golden Mary' and 'Beijing Red' all come from the rose seedling production base in Xiaobeibao Village, Yanqing District, Beijing.

[0048] 2. Experimental steps

[0049] (1) DNA extraction

[0050] DNA was extracted from the two parents, 'Golden Mary' and 'Beijing Red', and 6 randomly selected offspring plants. An appropriate amount of tender shoot leaves were picked from each plant, sealed with tin foil, numbered, frozen in liquid nitrogen, and then transferred to an -80°C refrigerator for storage. In this experiment, the CTAB method was used to extract DNA from young leaf tissues of plants. The concentration and purity of the extracted sample DNA were detected by Nanodrop. Samples of DNA with relatively high concentration and good purity were selected and diluted to 50 ng / μL as the working solution for the next PCR amplification.

[0051] (2) Primer screening

[0052] 192 pairs of SSR primers were used for PCR amplification of the parents 'Golden Mary' and 'Beijing Red' and 6 randomly selected offspring DNA samples. Figure 2 The amplified bands of Geno-MK116428 in sample individual plants numbered 1-1-5, 1-2-2, 3-1-1, etc. are shown. Figure 3 The amplified bands of Geno-MK117353 in sample individual plants numbered 15-2-3, 19-1-4, etc. are shown. Figures 2 - 3 The electrophoresis diagrams of the amplified bands of some of the parents 'Golden Mary', 'Beijing Red' and 6 randomly selected offspring DNA samples under the amplification of 192 pairs of SSR primers are shown. Figure 2 1-1-5, 1-2-2, 3-1-1, 3-1-4, 3-2-7, 3-1-10, 3-2-8, 3-2-9, 5-2-3 and 5-1-6 in [reference] refer to the numbers of individual plant samples. Figure 3 15-2-3, 19-1-4, 15-2-6 in [reference] refer to the numbers of individual plant samples.

[0053] The CTAB Plant Genomic DNA Rapid Extraction Kit was used to extract sample DNA, and the kit was from Beijing Aidlab Biotechnologies Co., Ltd. The PCR reaction system (20 μL) is as follows:

[0054] Table 1. PCR amplification reaction system

[0055]

[0056] The reaction program is as follows:

[0057] Table 2. PCR amplification program

[0058]

[0059] A total of 192 pairs of primers were randomly selected from the SSR primer library developed in the early stage of this laboratory, and these 192 pairs of primers were amplified using a PCR instrument.

[0060] The amplification results were detected by 7% polyacrylamide gel electrophoresis. Primers without amplification products, unclear bands, and no polymorphism were eliminated, and primers with clear bands and polymorphism were selected for the next step of F1 population genotyping. Due to the complex genetic background of roses and the easy generation of more miscellaneous bands in PCR amplification, only clear bands similar in size to the predicted products were selected when reading the bands in this experiment. By comparing the amplification results, 62 pairs of primers with good polymorphism were finally screened out.

[0061] Among the 62 pairs of primers screened out, primers showing differences between the two parents 'Golden Mary' and 'Beijing Red' were further screened out, and a total of 35 pairs of differential primers were obtained.

[0062] (3) Offspring DNA amplification

[0063] The 35 pairs of differential primers were used to perform PCR amplification on the offspring DNA samples, and the amplification reaction was carried out under the same PCR conditions.

[0064] The amplification products were separated by polyacrylamide gel electrophoresis (PAGE), and the electrophoresis bands were observed and recorded. Ensure the clarity of the electrophoresis results for subsequent data analysis.

[0065] (4) Data conversion and genotyping

[0066] According to the electrophoresis gel image, the electrophoresis results were statistically analyzed. The results were encoded in binary traits, that is, the state of each gene locus was represented by "present" and "absent", "1" represented the presence of the band (i.e., the gene locus), and "0" represented the absence of the band. The results (presence or absence of the band) of amplifying each single plant with each pair of primers (each locus) were converted into "1" "0" data. The electrophoresis bands were converted into a "0, 1 matrix", that is, the positions with bands were marked as "1", and the positions without bands were marked as "0", forming a binary matrix for genotyping analysis (when recording the bands, the following three principles were followed: excluding the bands that could not be clearly identified in the lanes being counted; excluding overlapping and blurred bands and recording only the clearly distinguishable bands; when the migration distances were the same and the intensity difference between the bands was less than twice, they were treated as the same band).

[0067] (5) Association regression analysis

[0068] For all the phenotypic data and DNA banding data of the individual plants amplified by 35 pairs of primers, one-way ANOVA was performed respectively to preliminarily screen out the loci that were significant at the 0.05 level. Then, multiple loci that were significant at the 0.05 level in the one-way ANOVA results of the phenotypic data of Botrytis cinerea resistance were used as a subset. The optimal subset selection was performed on the obtained genotype data using the association regression analysis method to screen out the SSR markers related to Botrytis cinerea resistance. Finally, through analysis, 3 SSR primers significantly related to the Botrytis cinerea resistance trait were screened out for subsequent research and application.

[0069] 3. Experimental Results

[0070] a. PCR Amplification Results

[0071] Table 3 shows the amplification statistical results of 192 pairs of primers. Among the 192 pairs of primers, 10 pairs of primers had no amplification products. After removing the primers with no amplification products, unclear bands, and no polymorphism, 62 pairs of primers with clear bands and polymorphism were obtained. The polymorphism ratio of the 62 pairs of primers was 32.29%. The primer polymorphism ratio can be used to quantify the genetic diversity between different individuals or populations. A high primer polymorphism ratio (the number of polymorphic samples 62 / total sample size 192 = polymorphism ratio 32.29%) indicates that there is rich genetic variation in this genetic region, and it has the potential to be used as a molecular marker.

[0072] As shown in Table 4, 35 pairs of primers showed differences between the parents.

[0073] Table 3. Statistics of PCR Amplification of 192 Pairs of Primers

[0074]

[0075] Table 4. Information of 35 Pairs of Polymorphic Primers

[0076]

[0077]

[0078] b. Molecular Marker Analysis Related to Botrytis cinerea Resistance

[0079] Finally, three primers related to Botrytis cinerea resistance were screened out through variance analysis and association analysis: Geno - BC - 1, Geno - BC - 2, Geno - BC - 3.

[0080] Table 5 shows the parameter estimation results of three predictor variables (Marker24, Marker99, Marker109) and their related primers in the statistical analysis.

[0081] The F value of Marker24 (Geno-BC-1) is 11.44173, which indicates that the primer Geno-BC-1 corresponding to Marker24 has strong significance in the regression model; the P value of Marker24 is 2.87*10 -5 , which is much less than 0.05, indicating that the primer Geno-BC-1 is statistically significant; the model-MS of Marker24 is 47.50838, and the results show that the primer Geno-BC-1 makes a greater contribution to the model. Geno-BC-1 is extremely significantly correlated (P<0.01), and the sequence near its marker site is closely associated with the Botrytis cinerea resistance gene and has the function of regulating RNA metabolism.

[0082] The F value of Marker99 (Geno-BC-2) is 5.3232, which indicates that the primer Geno-BC-2 corresponding to Marker99 has strong significance in the regression model; the P value of Marker99 is 0.00612, and its significance level is less than 0.05, indicating that the primer Geno-BC-2 is statistically significant in the model; the model-MS of Marker99 is 26.44743, and the results show that the primer Geno-BC-2 has a certain explanatory power in the model. Geno-BC-2 has an obvious correlation (P<0.05), and its corresponding gene has the function of galactose oxidase.

[0083] The F value of Marker109 (Geno-BC-3) is 4.25392, which indicates that the primer Geno-BC-3 corresponding to Marker109 has statistical significance; the P value of Marker109 is 0.01645, and the P value is less than 0.05, indicating that the primer Geno-BC-3 is statistically significant; the model-MS of Marker109 is 22.34853, and the results show that the primer Geno-BC-3 has a significant effect. Geno-BC-3 has an obvious correlation (P<0.05), and its corresponding gene can regulate related proteins to help plants cope with external stresses.

[0084] Table 5. Parameter Estimation

[0085]

[0086] Find the corresponding genes according to the SSR molecular markers significantly related to Botrytis cinerea resistance. Table 6 shows the corresponding gene function annotation information. Searching for multiple disease-resistant candidate genes by molecular marker means is of great significance for the screening of rose resistance materials, the cloning of disease-resistant genes, and disease-resistant directional breeding.

[0087] Table 6. Table of Primer-Related Functional Genes

[0088]

[0089] Among the candidate genes of the screened primers, considering the NCBI annotation and the gene expression level in petals, the above three candidate genes have effective anti - fungal functions in the process of rose resistance to Botrytis cinerea.

[0090] The gene corresponding to the Botrytis cinerea resistance - associated primer Geno - BC - 1 is Geno - MK112843. The annotation of this gene is chloroplast stem - loop binding protein of 41kDa, that is, a chloroplast stem - loop structure - binding protein with a size of 41kDa. This protein is named CSP41. CSP41 is a bifunctional protein with ribonuclease and ribonucleic acid - binding activities. CSP41 can cleave both single - stranded and double - stranded RNA, but not DNA, and it has a preference for cleaving RNA with stem - loops. Some studies have shown that when the 3’ - untranslated region of petD pre - mRNA is used as a substrate, CSP41 specifically cleaves it within the stem - loop region, which means that CSP41 plays an important role in controlling the stability of petD mRNA; the sequence - specific RNA - binding activity of CSP41 affects the rate of its RNase activity, but does not affect its specificity, indicating that in addition to participating in RNA degradation, CSP41 may also be involved in other reaction processes of chloroplast RNA metabolism.

[0091] Botrytis cinerea (caused by Botrytis cinerea) is a common plant disease. CSP41 enhances plant resistance to Botrytis cinerea by regulating the stability of RNA molecules related to plant immune responses. By binding to and regulating disease - resistant - related RNA, CSP41 helps to enhance the expression of defense - related genes, thereby improving the resistance of plants to pathogens. By participating in the RNA metabolism process, CSP41 regulates disease - resistant - related metabolic pathways, such as the synthesis of secondary metabolites, which play an important role in resisting pathogen invasion. CSP41 is involved in the regulation of plant defense signal transduction pathways, such as enhancing plant immune responses by regulating RNA splicing or translation efficiency.

[0092] The candidate gene of primer Geno-BC-2 is Geno-MK141053, and the annotation of this gene is: kelch repeat-containing protein At3g27220. Kelch repeat protein is a galactose oxidase, which is a copper enzyme that can reduce O2 to H2O2, thereby oxidizing primary alcohols including d-galactose and its constituent polysaccharides into aldehydes. Related research shows that Kelch repeat protein was first identified as related to galactose oxidase in Drosophila kelch protein, and its structure was analyzed, and then it was considered to be widely distributed in prokaryotes and eukaryotes. Although the functions of Kelch repeat proteins in bacteria and fungi have been relatively well studied, the functions of plant Kelch repeat proteins are still unknown.

[0093] H2O2 is an important signaling molecule in plant defense responses, which can promote plants to produce oxidative bursts and fight against pathogens. Kelch repeat protein enhances the resistance of plants to Botrytis cinerea by regulating the production of H2O2. At the same time, Kelch repeat protein is involved in the oxidation process of polysaccharides, which has an important impact on the stability of cell wall structure. A strong cell wall is the first line of defense for plants to resist pathogen invasion. Therefore, this protein may enhance the disease resistance of plants by regulating the strength and integrity of the cell wall.

[0094] The candidate gene of primer Geno-BC-3 is Geno-MK17618, which is a ribosomal protein (39S ribosomal protein L41, mitochondrial-like). Ribosomal proteins account for most of the chemical composition of ribosomes. Ribosomal proteins are very important for cell proliferation in eukaryotic cells. The normal function of ribosomes requires the participation of multiple ribosomal proteins at the same time. Dysfunction of ribosomes will occur after the deletion of ribosomal proteins. Some studies have shown that ribosomal proteins can also help plants cope with external stresses. Chloroplast RP2 is responsible for regulating seed germination under various stresses such as cold stress, drought stress and salt stress. Chloroplast RPS5 on the 30S small subunit of chloroplast ribosomes can regulate proteins involved in responding to cold stress and photosynthesis to affect plant growth and development; in tobacco, RPL12 and RPL19 can regulate in the same way to enable plants to obtain non-host disease resistance and can also resist the invasion of pathogens; in woody cotton, RPL18 can participate in the resistance to Verticillium wilt; RPL24A in Arabidopsis thaliana can regulate the proline level in zinc finger structure mutants to affect seed germination and the sensitivity of early seedlings to abscisic acid and osmotic stress.

[0095] The ribosomal proteins encoded by Geno-MK17618 can ensure that plants can rapidly synthesize defense proteins when attacked by Botrytis cinerea by maintaining the normal function of ribosomes. These defense proteins may include antibacterial proteins, cell wall-related enzymes, and other proteins involved in immune responses, thereby enhancing the resistance of plants to pathogenic bacteria. The translation process in which ribosomal proteins participate is closely related to the regulation of cell death and the expression of disease-resistant genes. In the face of pathogen attack, programmed cell death (such as the hypersensitive response) is an important mechanism for plants to resist pathogens. Geno-MK17618 can help plants effectively inhibit the spread of Botrytis cinerea by affecting the signal pathways related to cell death. Just like the performance of other ribosomal proteins under different stress conditions, Geno-MK17618 can support the adaptive response of plants by regulating ribosome function and translation efficiency when plants encounter the biotic stress of Botrytis cinerea infection, thereby reducing the impact of the disease.

[0096] The disease-resistant mechanism of plants is affected by various factors such as hormones, enzymes, and proteins regulated by disease-resistant genes. These factors cooperate and restrict each other to form a complete disease-resistant system against the invasion of pathogenic bacteria. By analyzing the functional sequences of resistance molecular markers, proteins and receptors related to disease resistance, as well as various functional sequences related to enzymes and hormones, are found near the two primer marker sites.

[0097] Example 2

[0098] This example is used to verify the resistance differences of the above-mentioned functional genes and their corresponding loci in plants. 24 individual plants from the offspring of the 'Golden Mary' × 'Beijing Red' hybrid F1 population were used to verify the resistance differences of the above-mentioned loci.

[0099] As Figure 4 shown in Table 7, the disease-resistant phenotype is represented by the lesion diameter. Figure 4 Images of the mycelial plaques of some materials of the female parent, male parent, and hybrid offspring after inoculation with Botrytis cinerea are shown. Table 7 is the statistics of the mycelial plaque diameter and genotype of all materials after inoculation with Botrytis cinerea. 24 materials were selected for each position. The molecular verification genotypes are represented by 0 and 1. According to the aforementioned genotyping rules, "0" indicates no band, and "1" indicates the presence of a band (single band or multiple bands). It should be noted that since the roses used in the experiment are tetraploids, there will be a situation where multiple bands appear for the same primer under the action of different primers in the electrophoresis bands.

[0100] According to Table 7, when no band appears at the spot17 position amplified based on the primer pair Geno-BC-1 (the primer pair sequences are SEQ NO.65 and SEQ NO.66), it indicates that the rose individual / species belongs to the Botrytis cinerea-resistant type. When a band is amplified at the spot17 position amplified based on the primer pair Geno-BC-1, it indicates that the rose individual / species belongs to the Botrytis cinerea-susceptible type. According to Table 7, when no band appears at the spot63 position amplified based on the primer pair Geno-BC-2 (the primer pair sequences are SEQ NO.67 and SEQ NO.68), it indicates that the rose individual / species belongs to the Botrytis cinerea-resistant type. When a band is amplified at the spot63 position amplified based on the primer pair Geno-BC-2, it indicates that the rose individual / species belongs to the Botrytis cinerea-susceptible type. According to Table 7, when no band appears at the spot65 position amplified based on the primer pair Geno-BC-3 (the primer pair sequences are SEQ NO.69 and SEQ NO.70), it indicates that the rose individual / species belongs to the Botrytis cinerea-resistant type. When a band is amplified at the spot65 position amplified based on the primer pair Geno-BC-3, it indicates that the rose individual / species belongs to the Botrytis cinerea-susceptible type.

[0101] In Table 7, the mean phenotypic values in the association population and the validation population represent the average plaque diameters of the two populations. In terms of phenotype, if the plaque diameter of a rose individual is greater than the mean, it indicates that the individual is Botrytis cinerea-susceptible. If the plaque diameter of a rose individual is less than the mean, it indicates that the individual is Botrytis cinerea-resistant. Taking the spot17 position as an example, among 24 materials, the number of resistant phenotypic materials (lesion diameter less than the mean) is 10, and the number of susceptible phenotypic materials (lesion diameter greater than the mean) is 14; the number of resistant genotype materials (0) is 6; the number of susceptible genotype materials (1) is 11.

[0102] Table 7. Genotyping Statistics

[0103]

[0104] It should be noted that the above specific embodiments are exemplary. Those skilled in the art can come up with various solutions inspired by the disclosure content of the present invention, and these solutions also fall within the disclosure scope of the present invention and within the protection scope of the present invention. Those skilled in the art should understand that the specification and drawings of the present invention are illustrative and do not constitute a limitation to the claims. The protection scope of the present invention is defined by the claims and their equivalents. The specification of the present invention contains multiple inventive concepts. For example, "preferably" and "according to a preferred embodiment" indicate that the corresponding paragraphs disclose an independent concept. The applicant reserves the right to file divisional applications according to each inventive concept. Throughout the text, the features guided by "preferably" are only optional ways and should not be understood as must be set. Therefore, the applicant reserves the right to waive or delete the relevant preferred features at any time.

Claims

1. SSR molecular marker primers for detecting the resistance of roses to Botrytis cinerea, characterized in that, The primer pair for amplifying the SSR molecular marker is selected from one or more of the following primer pairs: 1) The forward primer sequence is as shown in SEQ NO.65, and the reverse primer sequence is as shown in SEQ NO.66; 2) The forward primer sequence is as shown in SEQ NO.67, and the reverse primer sequence is as shown in SEQ NO.68; 3) The forward primer sequence is as shown in SEQ NO.69, and the reverse primer sequence is as shown in SEQ NO.

70.

2. The SSR molecular marker primer according to claim 1, characterized in that, The rose cultivar is 'Beijing Red', 'Golden Mary' or the hybrid offspring of both.

3. The application of the SSR molecular marker primer for detecting the resistance of rose to Botrytis cinerea as described in claim 1 or 2 in any one of the following 1) to 4): 1) Identifying or assisting in identifying rose materials resistant / susceptible to Botrytis cinerea; 2) Identifying or assisting in identifying genes resistant / susceptible to Botrytis cinerea in roses; 3) Screening or assisting in screening rose cultivars resistant to Botrytis cinerea; 4) Rose breeding.

4. A kit containing the SSR molecular marker primer for detecting the resistance of rose to Botrytis cinerea as described in claim 1 or 2.

5. A gene chip comprising the SSR molecular marker primers as described in claim 1 or 2, characterized in that, The gene chip is used for high-throughput detection of genotypes related to Botrytis cinerea resistance in rose populations.

6. The use of Gene ID Geno-MK112843, Geno-MK141053 or Geno-MK17618 in identifying Botrytis cinerea in roses.

7. A method for identifying the resistance of roses to Botrytis cinerea, characterized in that, Comprising the following steps: Extracting the genomic DNA of the plant to be tested; Using the genomic DNA of the plant to be tested as a template and performing a PCR amplification reaction with the SSR molecular marker primer as described in claim 1 or 2; Detecting the PCR amplification product, wherein, If the plant to be tested can amplify any specific band corresponding to the primer pairs 1), 2) and / or 3), it indicates that the plant to be tested is susceptible to Botrytis cinerea. If no specific band is amplified, the plant to be tested is resistant to Botrytis cinerea.

8. The method according to claim 7, wherein The rose cultivar is 'Beijing Red', 'Golden Mary' or the hybrid offspring of both.

9. A cultivation method for a rose variety, characterized in that, The method comprises the following steps: Genotyping rose materials using the SSR molecular marker primer as described in claim 1; Selecting rose materials resistant to Botrytis cinerea for hybridization according to the genotyping results; Screening the hybrid offspring to obtain a new rose cultivar resistant to Botrytis cinerea.

10. A breeding method for improving the gray mold resistance of roses, characterized in that, The method conducts gene introduction or directional breeding by introducing the disease-resistant genes associated with the SSR molecular marker primer as described in claim 1.

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