Molecular marker and method for detecting powdery mildew of rosa chinensis

Abstract

Botrytis blight is a major disease in the cultivation of Rosa hybrida, which seriously threatens the quality and yield of cut rose. In order to effectively deal with this disaster stress affecting the yield and quality of rose, 192 pairs of SSR primers were used to amplify the DNA of parents 'Jinmali' and 'Beijinghong' and six randomly selected offspring. Among the parents, 35 pairs of primers showed genetic differences. Further, the DNA of the offspring was amplified using the 35 pairs of primers, and the electrophoresis bands were detected by PAGE electrophoresis and converted into '0, 1 matrix' for genotyping. Through the correlation regression analysis, the optimal subset selection was carried out, and finally three primers related to the resistance to Botrytis blight were successfully screened: Geno-BC-1, Geno-BC-2, Geno-BC-3. The above findings provide an important theoretical basis for molecular marker-assisted breeding of disease resistance in rose, and are expected to promote the early identification of disease-resistant plants, thereby shortening the breeding cycle and improving the breeding efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular breeding, and particularly relates to a molecular marker and method for detecting rose gray mold. BACKGROUND

[0002] Rosa chinensis Jacq. belongs to Rosa L. Rosa has a high demand in the field of fresh-cut flowers, yard decoration and flower bed due to its colorful and varied flower color and shape. In recent years, with the improvement of people's living standards, the market demand for Rosa fresh-cut flowers increases year by year, and Rosa has become the first category in the cut flower market.

[0003] In the growth, preservation and transportation process of Rosa, due to the influence of humidity, temperature and other factors, Rosa is prone to diseases caused by the invasion of Botrytis, Peronospora, powdery mildew and other pathogens, which affect its production, circulation and sales. Among these diseases, gray mold is one of the most serious diseases in the current greenhouse Rosa cultivation and long-distance transportation process.

[0004] The rose gray mold mainly harms leaves, buds, flower buds and flowers, and can also harm young stems. When it occurs on leaf edges and tips, it initially appears as water-stained light brown spots that are smooth and slightly sunken, and then expand and rot. When flower buds are infected, the disease spots are gray-black and can prevent flowers from opening, and the diseased buds turn brown and die. Rose gray mold is caused by Botrytis cinerea Pers. of the Deuteromycotina Botrytis genus. The pathogen of gray mold overwinters in the form of mycelium and sclerotia, and invades the interior of flowers, leaves, etc. when the conditions are suitable in the spring, causing harm to plant tissues. After the conidia mature and fall off, they are spread and re-infected through air currents, raindrops, and plowing operations. However, plants have evolved a complex signal transduction network that includes local and systemic components to improve their defense against pathogens. The immune system of plants against pathogen infection mainly includes PAMP-Triggered-Immunity (PTI) and Effector-Triggered-Immunity (ETI). Pathogen-associated molecular patterns (PAMPs) secreted by pathogenic bacteria can be recognized by certain receptors on the surface of plant cells, thereby activating the PTI pathway. After specific infection of the host, the pathogen can secrete virulence effectors to inhibit the PTI pathway. At this time, the nucleotide-binding receptor (NB-LRR) in the cell recognizes the effectors and activates the ETI pathway to produce an immune response. Gray mold fungus is a dead body nutrient type fungus, and the OGs secreted during the degradation of the host cell wall are recognized by the receptor WAK1 and activate the downstream resistance response. PAMPs and DAMPs induce an immune response, and there is a great degree of overlap in protein kinase activation, synapse synthesis, hormone regulation, etc. The defense response formed by the combination of the two can effectively inhibit the infection of gray mold.

[0005] The rose gray mold not only reduces the ornamental value of cut roses, but also greatly restricts the development and rise of the rose industry. In the prior art, the methods for preventing and treating rose gray mold include removing diseased flowers, reducing air humidity, and spraying disease treatment agents (such as 65% zinc omadine wettable powder 600 times liquid, 75% chlorothalonil wettable powder 700 times liquid, or 70% methylthiophanate wettable powder 1500 times liquid) when the disease occurs. However, the prevention and treatment method relying on chemical agents can easily affect the ecological environment and human health. The traditional screening and identification method for gray mold-resistant plants includes inoculating gray mold into the identification host plant, recording and counting the changes in the diseased area of the host plant after a certain fungal spore reproduction period, and identifying plants with different levels of resistance to gray mold.

[0006] CN115820918A discloses a method for rapidly screening rose germplasm resources resistant to gray mold, which comprises obtaining the relative expression amount of the marker gene resistant to rose gray mold by using the special primer RcWRKY22 of rose germplasm resources resistant to gray mold, so as to identify the resistance of rose to gray mold.

[0007] At present, the genetic characteristics of rose gray mold resistance genes and related molecular markers are still less studied 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, a large number of literatures and patents have been studied by the applicant when making the present application, but due to the limitation of space, all the details and contents are not listed in detail, but this does not mean that the present application does not have these characteristics of the prior art, on the contrary, the present application has all the characteristics of the prior art, and the applicant reserves the right to add related prior art in the background art. SUMMARY

[0009] The development of simple sequence repeats (SSR) markers generally adopts various strategies, including traditional techniques, enrichment strategies, database construction, and high-throughput sequencing techniques. Compared with other types of molecular markers, SSR markers are known for their high polymorphism, rich information content, and good reproducibility, so they are more widely used in genetic linkage map construction and molecular marker-assisted breeding. Roses have a rich genetic background and a wide variety of cultivars, with significant trait segregation in their hybrid offspring and high genetic diversity at the molecular level, which provides convenient conditions for rose genome research. In this application, the relevant research results demonstrate three molecular markers closely related to rose gray mold resistance. The present application aims to expand the application range of SSR molecular markers, optimize the resistance of rose varieties, and thus provide more comprehensive means and schemes for genetic improvement of roses.

[0010] One of the purposes of the present application is to provide the SSR molecular marker primers involved in the present application to more accurately and quickly identify the disease resistance traits of rose varieties, providing important molecular tools for rose breeding.

[0011] One of the purposes of the present application is to develop related kits and detection methods using the SSR molecular marker primers involved in the present application, which facilitates the screening and identification of rose varieties resistant to gray mold in actual production and breeding processes.

[0012] One of the purposes of the present application is to provide a method for rose breeding resistant to gray mold, which significantly improves the disease resistance of new rose varieties, speeds up the breeding process, and improves the breeding efficiency by combining the SSR molecular marker primers involved in the present application with traditional breeding techniques.

[0013] One of the objectives of the present application is to provide a molecular marker assisted selection (MAS) method using the SSR molecular marker involved in the present application, so as to identify and select the rose material resistant to gray mold at an 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 application is to provide a primer pair for amplifying the SSR molecular marker for detecting the resistance of rose to gray mold, which is selected from one or more of the following primer pairs:

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

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

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

[0018] According to a preferred embodiment, the above-mentioned molecular marker can be used in the identification or assisted identification of rose homozygous resistance to gray mold and heterozygous susceptibility to gray mold.

[0019] According to a preferred embodiment, when used for genetic linkage identification, if the rose to be tested can amplify any specific band corresponding to the above-mentioned 3 pairs of SSR primers, 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 exist in the corresponding genetic linkage group.

[0020] One of the objectives of the present application is to provide the application of the SSR molecular marker primer for detecting the resistance of rose to gray mold in any one of the following 1) to 4):

[0021] 1) identification or assisted identification of rose resistant / susceptible to gray mold material;

[0022] 2) identification or assisted identification of rose resistant / susceptible to gray mold gene;

[0023] 3) screening or assisted screening of rose varieties resistant to gray mold;

[0024] 4) rose breeding.

[0025] One of the objectives of the present application is to provide a kit comprising the SSR molecular marker primer for detecting the resistance of rose to gray mold.

[0026] One of the purposes of the present application is to provide a gene chip comprising the SSR molecular marker primer as involved in the present application, which is used for high-throughput detection of the resistance-related genotype of gray mold in Rosa populations.

[0027] One of the purposes of the present application is to provide a method for identifying the resistance of Rosa to gray mold, which comprises 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 using the SSR molecular marker primer as involved in the present application to perform a PCR amplification reaction; and detecting the PCR amplification product, wherein the plant to be tested can amplify any one of the specific bands corresponding to the primers of 1), 2) and / or 3) above, indicating that the plant to be tested is susceptible to gray mold, and if no specific band is amplified, the plant to be tested is resistant to gray mold.

[0028] According to a preferred embodiment, the method for identifying the resistance of Rosa to gray mold comprises detecting the genotype of the corresponding SSR site by real-time fluorescent quantitative PCR or digital PCR method.

[0029] According to a preferred embodiment, the Rosa variety can be 'Beijing Red', 'Jinmali' or the hybrid offspring of the two.

[0030] One of the purposes of the present application is to provide a breeding method for a Rosa variety, which comprises the following steps: genotyping the Rosa material using the SSR molecular marker primer as involved in the present application; selecting the Rosa material resistant to gray mold for hybridization according to the genotyping results; and screening the hybrid offspring to obtain a new Rosa variety resistant to gray mold.

[0031] One of the purposes of the present application is to provide a breeding method for improving the resistance of Rosa to gray mold, which is performed by introducing the disease-resistant gene associated with the SSR molecular marker primer as involved in the present application for gene introduction or directional breeding.

[0032] One of the purposes of the present application is to provide the use of the genes with Geno-MK112843, Geno-MK141053 or Geno-MK17618 in identifying the resistance of Rosa to gray mold.

[0033] The technical solution of the present application has the following beneficial effects:

[0034] The article "Resistance Identification and Correlation Analysis of Different Rose Cultivars to Botrytis Blight" proposes that the resistance identification and breeding research progress of rose Botrytis blight shows that screening disease-resistant varieties is an effective way to improve rose resistance. For example, through two years of field investigation and petal artificial inoculation, it is found that the resistance of different rose resources varies greatly, and some highly resistant varieties such as Zhu Hong Nv Wang and Hei Ba Ke are selected as excellent optional materials for disease-resistant breeding. Therefore, resistance identification based on molecular markers is an effective means for breeding disease-resistant rose varieties that can be widely used.

[0035] As shown in Figure 4 , the present application uses the petal disc infection method to determine the resistance of common rose varieties 'Jinmali (JML)' and 'Beijinghong (BJH)' and their hybrid offspring to Botrytis blight. The primer with polymorphism is screened by using SSR molecular marker technology, and the rose hybrid population is subjected to genotyping analysis. In addition, the present application also carries out correlation analysis between the phenotype of rose Botrytis blight resistance and molecular markers, aiming to identify molecular markers related to rose Botrytis blight resistance. Through correlation screening, three molecular markers closely related to rose resistance / susceptibility to Botrytis blight are obtained.

[0036] Through further verification and analysis of the three molecular markers, the present application preliminarily determines their potential application value in Botrytis blight resistance breeding. These molecular markers can not only be used for early screening of resistant rose varieties, but also can improve the selection efficiency and shorten the breeding cycle in actual breeding process. At the same time, the correlation between resistance markers and phenotypic resistance is high, which indicates that these markers can effectively reflect the resistance phenotype of Botrytis blight. Therefore, these molecular markers can be used as a reliable tool for rose Botrytis blight resistance breeding, which helps to realize the large-scale application of disease-resistant varieties.

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

[0038] Figure 1 The phenotypic figure of rose varieties 'Beijinghong' (1A) and 'Jinmali' (1B) provided by the present application when they are not diseased;

[0039] Figure 2 The partial amplification results of No. 54 primer (Geno-MK116428) provided by the present application are shown in the figure, and the bands of DNA marker are 250 bp and 100 bp from top to bottom;

[0040] Figure 3The partial amplification results of the 59th primer (Geno-MK117353) provided by the application are shown in the figure, and the bands of the DNA marker are 250 bp and 100 bp from top to bottom;

[0041] Figure 4 The phenotype figures of the Rosa varieties 'Beijinghong', 'Jinmali' and the F1 population of the cross between 'Jinmali' and 'Beijinghong' infected by Botrytis cinerea are provided by the application. DETAILED DESCRIPTION

[0042] The application will be described in detail below with reference to the accompanying drawings.

[0043] The rose disease-resistant identification method and the molecular marker related thereto of the application are illustrated by the following examples, but the application is not limited to the following examples, that is, it does not mean that the application must rely on the following examples to be implemented. It should be understood by those skilled in the art that any improvement of the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific modes and the like all fall within the protection scope and disclosure scope of the application.

[0044] Molecular marker refers to a means for specifically detecting genetic material at the DNA or RNA level. With the development of molecular biology, more and more molecular markers are widely used in the research of horticultural plant genomes, and molecular marker-assisted breeding has become an important means for improving the genetic traits of horticultural plants. Compared with morphological markers, cytological markers and biochemical markers, molecular markers are a more microscopic and root-oriented technical means, and are widely used in plant genome research. SSR molecular markers have the advantages of low requirements for DNA quantity and quality, easy operation, stable results, repeatability, etc., and can involve 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 been widely used in genetic diversity research, genetic mapping, important functional gene positioning, molecular-assisted breeding and other fields in recent years. SSR is a type of 1-6 bp nucleotide motif repeat sequence, which is widely distributed in the coding region and non-coding region of eukaryotic genomes. SSR markers use the flanking conserved sequence of SSR to design primers, and through PCR amplification, the polymorphism of DNA sequence is reflected according to the size of the band.

[0045] Example 1

[0046] 1. Experimental materials

[0047] As Figure 1The Rosa chinensis cultivars 'Beijing Red', 'Jinmali', and 'Jinmali' and 'Beijing Red' F1 hybrid population of 170 individual plants were all from a Rosa chinensis nursery production base in Xiaobeipu Village, Yanqing District, Beijing.

[0048] 2. Experimental procedure

[0049] (1) DNA extraction

[0050] DNA was extracted from the two parent plants 'Jinmali' and 'Beijing Red' and 6 randomly selected offspring plants. A suitable amount of tender shoot leaves was taken from each plant, sealed with tin foil paper, and labeled with a number before being quickly frozen in liquid nitrogen and then transferred to a -80°C refrigerator for storage. CTAB method was used to extract DNA from the tender leaf tissue in this experiment. The sample DNA was detected for concentration and purity by Nanodrop. The sample DNA with higher concentration and better purity was diluted to 50 ng / μL as the working solution for the next step of PCR amplification.

[0051] (2) Primer screening

[0052] 192 pairs of SSR primers were used for PCR amplification of the DNA samples of the parent plants 'Jinmali' and 'Beijing Red' and 6 randomly selected offspring plants. Figure 2 The amplification bands of Geno-MK116428 in the sample individual plants labeled as 1-1-5, 1-2-2, 3-1-1, etc. are shown. Figure 3 The amplification bands of Geno-MK117353 in the sample individual plants labeled as 15-2-3, 19-1-4, etc. are shown. Figures 2-3 The electrophoretogram of the amplification bands of some of the parent plants 'Jinmali', 'Beijing Red', and 6 randomly selected offspring DNA samples under amplification by 192 pairs of SSR primers is 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 refer to the labels of the individual sample plants. Figure 3 15-2-3, 19-1-4, 15-2-6 in refer to the labels of the individual sample plants.

[0053] The sample DNA was extracted using the CTAB Plant Genomic DNA Quick Extraction Kit from Beijing Aidley Biological Technology Co., Ltd. The PCR reaction system (20 μL) was as follows:

[0054] Table 1. PCR amplification reaction system

[0055]

[0056] The reaction program was as follows:

[0057] Table 2. PCR amplification program

[0058]

[0059] In the laboratory of the early development of SSR primer library randomly selected 192 pairs of primers, using PCR instrument to amplify the 192 pairs of primers.

[0060] The amplification results were detected by 7% polyacrylamide gel electrophoresis, and the primers without amplification product, unclear band and polymorphism were eliminated. The primers with clear band and polymorphism were selected for F1 population genotyping. Due to the complex genetic background of Chinese rose, PCR amplification is easy to produce more bands, so only the clear bands with similar predicted product size were selected when reading the bands. By comparing the amplification results, 62 pairs of primers with good polymorphism were finally selected.

[0061] Among the 62 pairs of primers screened, further screening was carried out to select the primers showing differences between 'Jinmali' and 'Beijinghong' parents, and 35 pairs of different primers were obtained.

[0062] (3) DNA amplification of offspring

[0063] The 35 pairs of different primers were used for PCR amplification of DNA samples of offspring, 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. The clarity of the electrophoresis results was ensured to facilitate subsequent data analysis.

[0065] (4) Data conversion and genotyping

[0066] According to the electrophoresis gel, the electrophoresis results were counted. The results were encoded as binary traits, that is, "yes" and "no" were used to represent the state of each genetic locus. "1" means the existence of band (genetic locus), and "0" means the absence of band. The results of each primer (each locus) amplification of each single plant (presence or absence of band) were converted into "1" and "0" data. The electrophoresis bands were converted into "0, 1 matrix", that is, the positions of existing bands were marked as "1", and the positions of missing bands were marked as "0", forming a binary matrix for genotyping analysis (when recording the bands, the following three principles were followed: remove the bands that cannot be clearly identified in the counted lanes; remove the overlapping and blurred bands, and record the clear bands; when the migration distance is the same, the intensity difference between the bands is less than two times, and they are treated as the same band).

[0067] (5) Correlation regression analysis

[0068] All phenotype data and DNA banding data of 35 pairs of primers amplified single-stranded were subjected to corresponding one-way ANOVA, and the significant sites at the level of 0.05 were preliminarily screened out, and then the multiple sites at the level of 0.05 in the one-way ANOVA results of the phenotype data of gray mold resistance were used as a subset. The genotype data obtained were subjected to optimal subset selection by using the correlation regression analysis method, and the SSR markers related to the resistance to gray mold were screened out. Finally, three SSR primers significantly related to the resistance to gray mold were screened out by analysis and were used for subsequent research and application.

[0069] 3. Experimental results

[0070] a. PCR amplification results

[0071] Table 3 shows the amplification statistics of 192 pairs of primers. Among the 192 pairs of primers, 10 pairs of primers have no amplification products, and after removing the primers without amplification products, 62 pairs of primers with clear bands and polymorphism are obtained. The polymorphism rate of the 62 pairs of primers is 32.29%. The primer polymorphism rate can be used to quantify the genetic diversity between different individuals or populations. A high primer polymorphism rate (62 polymorphic samples / total sample size 192 = polymorphism rate 32.29%) indicates that there is rich genetic variation in this genetic region, which 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. PCR amplification statistics of 192 pairs of primers

[0074]

[0075] Table 4. Information of 35 pairs of polymorphic primers

[0076]

[0077]

[0078] b. Gray mold resistance-related molecular marker analysis

[0079] Three primers (Geno-BC-1, Geno-BC-2, and Geno-BC-3) related to the resistance to gray mold were finally screened out by variance analysis and correlation analysis.

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

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

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

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

[0084] Table 5. Parameter estimation

[0085]

[0086] According to the SSR molecular marker significantly related to gray mold resistance, the corresponding gene is found, and Table 6 is the corresponding gene function annotation information. Finding multiple disease resistance candidate genes through molecular marker method has very important significance for rose resistance material screening, disease resistance gene cloning, and disease resistance directional breeding.

[0087] Table 6. Primer related functional gene table

[0088]

[0089] Among the screened primers of candidate genes, in combination with NCBI annotation and expression of genes in petals, the above three candidate genes have effective resistance functions in the process of resistance to gray mold in roses.

[0090] The gene corresponding to the primer Geno-BC-1 associated with resistance to gray mold is Geno-MK112843, and the annotation of the gene is chloroplast stem-loop binding protein of 41kDa, i.e., a 41kDa chloroplast stem-loop structure binding protein. The protein is named CSP41. CSP41 is a bifunctional protein with ribonuclease and ribonucleic acid binding activity, which can cut both single-stranded and double-stranded RNA, but not DNA, and it is more inclined to cut RNA with stem-loop. Studies have shown that when the 3'-untranslated region of petD pre-mRNA is used as a substrate, CSP41 will specifically cut it in 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 not its specificity, which indicates that in addition to participating in RNA degradation, CSP41 may also be involved in other processes of chloroplast RNA metabolism.

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

[0092] The candidate gene of primer Geno-BC-2 is Geno-MK141053, and the annotation of the gene is kelch repeat-containing protein At3g27220. Kelch repeat-containing protein is a galactose oxidase, which is a copper enzyme that can reduce O2 to H2O2, thereby oxidizing primary alcohols including d-galactose and polysaccharides composed thereof to aldehydes. Related studies have shown that Kelch repeat-containing protein was first identified in Drosophila kelp protein as being associated with galactose oxidase, and its structure was resolved, and then it was believed to be widely distributed in prokaryotes and eukaryotes. Although the function of Kelch repeat-containing protein in bacteria and fungi has been relatively well studied, the function of plant Kelch repeat-containing protein is not yet known.

[0093] H2O2 is an important signal molecule in plant defense response, which can promote the plant to produce oxidative burst to resist pathogenic bacteria. Kelch repeat-containing protein enhances the resistance of plants to Botrytis cinerea by regulating the production of H2O2. At the same time, Kelch repeat-containing protein is involved in the oxidation process of polysaccharides, which has an important influence on the stability of cell wall structure. A solid cell wall is the first line of defense for plants against pathogenic bacteria, so the protein can 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 a large part of the chemical composition of ribosomes, and ribosomal proteins are very important for cell proliferation in eukaryotic cells. Normal function of ribosomes requires the participation of multiple ribosomal proteins, and ribosomal protein deficiency can cause dysfunction of ribosomes. Studies have shown that ribosomal proteins can also help plants cope with external stress. Chloroplast RP2 is responsible for regulating seed germination under various stresses such as cold stress, drought stress and salt stress, and chloroplast RPS5 on the 30S small subunit of chloroplast ribosome can regulate proteins involved in responding to cold stress and photosynthesis to affect plant growth and development; in tobacco, RPL12 and RPL19 can regulate plants to obtain non-host disease resistance in the same way, and also can resist the invasion of pathogens; RPL18 in woody cotton can participate in resistance to Verticillium wilt; RPL24A in Arabidopsis can regulate proline levels in zinc finger structure mutants to affect seed germination and the sensitivity of early seedlings to abscisic acid and osmotic stress.

[0095] The ribosomal protein 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 can include antibacterial proteins, cell wall-related enzymes, and other proteins involved in immune responses, thereby enhancing the plant's resistance to pathogenic bacteria. The translation process involving ribosomal proteins is closely related to cell death regulation and the expression of disease resistance genes. When facing pathogen attacks, programmed cell death (such as hypersensitivity) is an important mechanism for plants to resist pathogens. Geno-MK17618 can help plants effectively inhibit the spread of Botrytis cinerea by affecting cell death-related signaling pathways. Just as other ribosomal proteins perform under different stress conditions, Geno-MK17618 can support the adaptive response of plants when they encounter Botrytis cinerea infection by regulating ribosome function and translation efficiency, thereby reducing the impact of disease.

[0096] The disease resistance mechanism of plants is influenced by various factors such as hormones, enzymes, and proteins regulated by disease resistance genes. Each factor interacts and restricts each other, forming a complete disease resistance system against pathogen invasion. Through the analysis of functional sequences of resistance molecular markers, proteins and receptors related to disease resistance, as well as various enzyme and hormone-related functional sequences were found near the two primer marker sites.

[0097] Example 2

[0098] This example is used to verify the resistance differences of the above functional genes and their corresponding sites in plants. 24 single plants of the 'Jinmali' G 'Beijinghong' hybrid F1 population were used to verify the resistance differences of the above sites.

[0099] As Figure 4 As shown in Table 7, the disease resistance phenotype is represented by the diameter of the disease spot. Figure 4 The images of the disease spots of the maternal, paternal, and hybrid progeny after inoculation with Botrytis cinerea are shown. Table 7 is a statistical analysis of the spot diameter and genotype of all materials after inoculation with Botrytis cinerea. 24 materials were selected for each position. The molecular verification genotype is represented by 0 and 1, following the same rules as the aforementioned genotyping. "0" indicates no band, and "1" indicates the presence of a band (single band or multiple bands). It should be noted that the roses used in the experiment are tetraploid, so multiple bands may appear in the electrophoresis bands under the action of different primers.

[0100] According to Table 7, when no band appears at spot 17 position amplified based on primer pair Geno-BC-1 (primer pair sequences are SEQ NO. 65, SEQ NO. 66), it indicates that the Rosa individual / species belongs to the resistant type to gray mold, and when a band appears at spot 17 position amplified based on primer pair Geno-BC-1, it indicates that the Rosa individual / species belongs to the susceptible type to gray mold. According to Table 7, when no band appears at spot 63 position amplified based on primer pair Geno-BC-2 (primer pair sequences are SEQ NO. 67, SEQ NO. 68), it indicates that the Rosa individual / species belongs to the resistant type to gray mold, and when a band appears at spot 63 position amplified based on primer pair Geno-BC-2, it indicates that the Rosa individual / species belongs to the susceptible type to gray mold. According to Table 7, when no band appears at spot 65 position amplified based on primer pair Geno-BC-3 (primer pair sequences are SEQ NO. 69, SEQ NO. 70), it indicates that the Rosa individual / species belongs to the resistant type to gray mold, and when a band appears at spot 65 position amplified based on primer pair Geno-BC-3, it indicates that the Rosa individual / species belongs to the susceptible type to gray mold.

[0101] The average value of the phenotype in the association population and the average value of the phenotype in the verification population in Table 7 represent the average values of the spot diameters of the two populations, and the spot diameter of the Rosa individual is greater than the average value in the aspect of phenotype, which indicates that the individual is susceptible to gray mold, and the spot diameter of the Rosa individual is less than the average value in the aspect of phenotype, which indicates that the individual is resistant to gray mold. Taking spot 17 position as an example, among the 24 materials, the number of resistant phenotype materials (disease spot diameter less than the average value) is 10, and the number of susceptible phenotype materials (disease spot diameter greater than the average value) is 14; the number of resistant genotype materials (0) is 6; and 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, and those skilled in the art can think of various solutions under the inspiration of the disclosure of the present application, and these solutions also belong to the disclosed range of the present application and fall within the protection scope of the present application. Those skilled in the art should understand that the present application specification and its drawings are illustrative and not constitute a limitation on the claims. The protection scope of the present application is defined by the claims and their equivalents. The present application specification contains multiple inventive concepts, such as “preferably”, “according to a preferred embodiment”, which all indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application according to each inventive concept. Throughout the text, the features introduced by “preferably” are only optional ways, and should not be understood as necessarily set, therefore the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

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

70.

2. The SSR molecular marker primer as described in claim 1, characterized in that, The rose varieties are 'Beijing Red', 'Golden Mary', or hybrids of both.

3. The application of the SSR molecular marker primers for detecting resistance to gray mold in roses as described in claim 1 or 2, in any of the following 1) to 4): 1) To identify or assist in the identification of rose varieties resistant to / susceptible to gray mold; 2) Identify or assist in the identification of genes that resist / suscept to gray mold in roses; 3) Screening or assisting in the screening of rose varieties resistant to gray mold; 4) Breeding roses resistant to gray mold; The rose variety is 'Beijing Red', 'Golden Mary', or a hybrid of both.

4. A kit containing the SSR molecular marker primers for detecting resistance to gray mold in roses as described in claim 1 or 2.

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

6. A method for identifying resistance to gray mold in roses, characterized in that, Includes the following steps: Genomic DNA was extracted from the plants to be tested; Using the genomic DNA of the plant to be tested as a template, PCR amplification reaction was performed using the SSR molecular marker primers described in claim 1 or 2; Detection of PCR amplification products, among which, If the tested plant can amplify any one of the specific bands corresponding to the primer pairs 1), 2) and / or 3), it indicates that the tested plant is susceptible to gray mold. If no specific band is amplified, the tested plant is resistant to gray mold. The rose variety is 'Beijing Red', 'Golden Mary', or a hybrid of both.

7. A method for cultivating a rose variety, characterized in that, The method includes the following steps: Genotyping of rose materials was performed using the SSR molecular marker primers as described in claim 1. Based on the genotyping results, rose materials resistant to gray mold were selected for hybridization; The hybrid offspring were screened to obtain new rose varieties resistant to gray mold; The rose variety is 'Beijing Red', 'Golden Mary', or a hybrid of both.

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