A method for rapid screening of rose germplasm resources resistant to gray mold

Abstract

The application discloses a method for rapidly screening rose germplasm resources with resistance to botrytis cinerea, which comprises the following steps of using a special primer RcWRKY22 for resistance to botrytis cinerea, inoculating botrytis cinerea, performing a fluorescent quantitative RTFQ-PCR test, and grading materials with resistance to botrytis cinerea. The method can accurately and efficiently identify rose germplasm resources with resistance to botrytis cinerea in various regions of China in less than 1 day and with a small amount of plant materials, thereby saving cost, time and labor.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of woody flower disease resistance breeding, and particularly relates to resistance identification and screening of different Rosa hybrida germplasm resources to gray mold. BACKGROUND

[0002] Rosa hybrida is a plant of Rosaceae and Rosa, and is favored by consumers and is the largest cut flower in the world. Yunnan Province is one of the main production areas of cut rose in the world.

[0003] Gray mold of Rosa hybrida is a fungal disease caused by Botrytis cinerea, and is the most serious fungal disease in the post-harvest transportation process of cut rose. The disease is most harmful to flowers, and the outbreak of the disease causes direct economic losses of more than 70% to producers and distributors. At present, the production mainly prevents and controls the occurrence of gray mold of Rosa hybrida by repeatedly applying chemical agents, which not only increases the production cost, but also causes pollution and damage to the environment, and also seriously reduces the ornamental value. Therefore, breeding and planting new varieties of Rosa hybrida resistant to gray mold is the key to green production in the future.

[0004] The prerequisite for breeding new varieties of Rosa hybrida resistant to gray mold is to apply accurate and efficient disease resistance identification and screening methods to a large number of Rosa hybrida parent materials and a large number of Rosa hybrida hybrid offspring for disease resistance identification and screening, so as to shorten the breeding cycle of resistance to gray mold. So far, there is no research report on screening of Rosa hybrida germplasm resources resistant to gray mold by using molecular marker technology. At present, the traditional screening and identification method for plants resistant to gray mold at home and abroad is mainly to inoculate gray mold on the identification host plant, record and count the change of the disease area of the host plant after a certain fungal spore propagation period, so as to identify different levels of plants resistant to gray mold.

[0005] Based on our previous research on the damage characteristics of gray mold on Rosa hybrida resources at different stages, cloning and sequencing of Rosa hybrida gray mold resistance genes, and development of Rosa hybrida gray mold resistance gene molecular markers, we found that the identification and screening of disease-resistant materials by combining modern molecular biological techniques can not only accurately identify and screen disease-resistant materials, but also shorten the identification cycle, and has the advantages of easy operation, saving labor and materials. SUMMARY

[0006] The purpose of the present application is to provide a method for quickly screening Rosa hybrida germplasm resources resistant to gray mold, so as to solve the defects of the traditional screening and identification method for plants resistant to gray mold, such as time-consuming, laborious and inaccurate.

[0007] The terms used in this paper are:

[0008] Fluorescence quantitative RTFQ-PCR technology: Real-time fluorescence quantitative polymerase chain reaction, English Real-Time Fluorescence Quantitative Polymerase Chain Reaction, abbreviated as: RTFQ-PCR.

[0009] UBC gene: Ubiquitin Conjugating protein gene.

[0010] RcWRKY22 gene: Rosa chinensis probable WRKY transcription factor 40 Locus Chromosome 2 (RcWRKY22)

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a special primer RcWRKY22 for rapidly screening gray mold-resistant rose germplasm resources using a fluorescent quantitative RTFQ-PCR technique. The special primer RcWRKY22 consists of an RcWRKY22 upstream primer and an RcWRKY22 downstream primer. The nucleotide sequence of the RcWRKY22 upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the RcWRKY22 downstream primer is shown in SEQ ID NO: 2.

[0013] The present invention also provides a method for rapidly screening rose germplasm resources resistant to gray mold using fluorescent quantitative RTFQ-PCR technology, comprising: washing petal samples of a rose germplasm material to be identified and a petal sample of a control rose germplasm material, respectively, and then flattening the petal samples in a culture dish containing a culture medium; using the above-mentioned special primer RcWRKY22, respectively, to determine the relative expression levels of rose gray mold resistance marker genes in the petal samples of the rose germplasm material to be identified that has been inoculated with rose gray mold for 12 hours and then washed, and the petal samples of the control rose germplasm material that has been cultured for 12 hours; and identifying the resistance level of the rose germplasm material to rose gray mold according to the resistance level of the rose gray mold marker gene.

[0014] Furthermore, in the above method, the petal samples of the rose germplasm resource material to be identified and the petal samples of the control rose germplasm resource material are both collected from the petals of the rose germplasm resource material to be identified that are free of diseases and insect pests.

[0015] Furthermore, in the above method, the petal samples collected are the petals in the first and second layers from the inside to the outside of the rose germplasm resource material to be identified, which are free of pests and diseases.

[0016] Furthermore, in the above method, the rose gray mold resistance grades divided according to the relative expression levels of the rose gray mold resistance marker genes are the following 4 grades:

[0017] Level 0: immune, relative expression level ≥ 8;

[0018] Level 1: high resistance, relative expression level ≥ 6 and < 8;

[0019] Level 2: moderate resistance, relative expression level ≥ 4 and < 6;

[0020] Level 3: low resistance, relative expression level ≥ 2 and < 4.

[0021] Furthermore, in the above method, the petal samples of the rose germplasm resource materials to be identified and the petal samples of the control rose germplasm resource materials are washed respectively with 70% v / v ethanol and sterile water, and the collected petal samples are first soaked in 70% v / v ethanol for 45-60s, and then washed with sterile water for 3 to 5 times.

[0022] Furthermore, in the above method, the rose germplasm resource material to be identified that has been inoculated with gray mold of rose is cultured for 12 hours and the cleaning comprises rinsing its petals with sterile water for 3 to 5 times.

[0023] Furthermore, in the above method, the inoculation is carried out by dripping the concentration of 1×10 5 / mL rose gray mold spore suspension was inoculated onto each petal.

[0024] Furthermore, in the above method, the culture is carried out under the conditions of indoor light intensity of 2000-3200 lx, indoor air relative humidity of 80-90%, and room temperature of 22°C-25°C.

[0025] Furthermore, in the above method, the culture medium is: MS+benzimidazole 0.4 g / L+agar 8.0 g / L.

[0026] Table 1 Primer sequences

[0027]

[0028] Compared with the prior art, the main innovations and beneficial effects of the present invention are:

[0029] 1. Through a large number of experiments in advance, 445 collected Rosa germplasm resources are screened for resistance to Rosa gray mold by the method of the application, and the results show that the Rosa germplasm resources identified by the method of the application and the traditional method are completely consistent, which shows that the method of the application can efficiently and accurately identify the Rosa germplasm resources resistant to gray mold in various regions of China, and is cost-saving, time-saving and labor-saving, and is especially suitable for early identification and screening of a large number of Rosa hybrid offspring resistant to gray mold, and is a method suitable for efficient identification and screening of Rosa germplasm resources resistant to gray mold in various regions of China.

[0030] 2. The core of the method of the application is to use the special primer RcWRKY22 of the Rosa gray mold resistance marker gene developed in the early stage, combined with the fluorescence quantitative RTFQ-PCR technology, to obtain the relative expression amount of the gray mold resistance marker gene (RcWRKY22 gene) in the petals of different Rosa germplasm resources inoculated, and finally to divide the gray mold resistance grades of each Rosa germplasm resource according to the range of the relative expression amount.

[0031] 3. The identification time is short. From the inoculation of the Rosa gray mold fungus to the completion of the identification of the resistance to gray mold, it takes less than 1 day, while the traditional method takes more than 3 days. The traditional identification method requires manual recording and statistics of the diseased area on the host material every day, which is time-consuming and labor-intensive, and is prone to statistical errors.

[0032] In summary, the method and the special primer provided by the application for identifying and screening Rosa germplasm resources resistant to gray mold in various regions of China make it possible to accurately and quickly identify and screen a large number of Rosa hybrid offspring resistant to gray mold in China. The method of the application shortens the identification and screening period of the Rosa germplasm resources resistant to gray mold to less than 1 day, uses less identification material (only 3-5 petals), has a wide range of practicality, is simple to operate, occupies less space, and is accurate in identification and fast in screening.

[0033] The nucleotide sequence of the RcWRKY22 upstream primer is shown in SEQ ID NO: 1 in the sequence listing.

[0034] The nucleotide sequence of the RcWRKY22 downstream primer is shown in SEQ ID NO: 2 in the sequence listing.

[0035] The nucleotide sequence of the UBC upstream primer of the universal primer of the internal reference is shown in SEQ ID NO: 3 in the sequence listing.

[0036] The nucleotide sequence of the UBC downstream primer of the universal primer of the internal reference is shown in SEQ ID NO: 4 in the sequence listing. DETAILED DESCRIPTION

[0037] The following examples are routine methods. All the materials involved in the examples, such as reagents, kits, Botrytis cinerea, 50 rose germplasm resources, etc. can be purchased from commercial channels.

[0038] Example 1 Identification of resistance to Botrytis cinerea of 50 rose germplasm resources collected from different regions in China

[0039] (1) Preparation of inoculation materials: Collect the petal of 50 rose germplasm resources (see Table 1) without pests and diseases, select the petal of the first and second layers of the flower without pests and diseases, and clean them with 70% v / v ethanol and sterile water, respectively. The cleaning process is as follows: soak the collected petals in 70% v / v ethanol for 1 min, then clean them with sterile water for 3-5 times, and then place the cleaned petals face up on a culture dish containing nutrient medium, 3 petals per culture dish. The formula of the nutrient medium is: MS + benzimidazole 0.4 g / L + agar 8.0 g / L.

[0040] (2) Inoculation test: Select the Botrytis cinerea cultured in the laboratory as the inoculation fungus, and use a sterilized dissecting needle to pick the cultured Botrytis cinerea into sterile distilled water containing 0.03% (v / v) Tween 20. Adjust the spore concentration of Botrytis cinerea to 1 x 10 5 / mL suspension with sterile distilled water containing 0.03% (v / v) Tween 20; then use a pipette to inoculate 200 μl of the suspension onto the petals to be identified (200 μl of the suspension per culture dish), 3 petals per material, 3 replicates per treatment. At the same time, reserve a corresponding portion of the same treatment without inoculation of the suspension as a control. After inoculation, incubate the culture dishes of the materials to be identified and the controls in the room under the conditions of indoor light intensity of 2000-3200 lx, indoor air relative humidity of 80-90%, and room temperature of 22-25°C for 12 h. The diagnosis of Botrytis cinerea can refer to the diagnosis method in the literature (Noack R. Breeding and selection strategies for disease and pest resistance. In: Encyclopedia of Rose Science. Roberts AV, Debener T, Gudin S eds., Oxford: Elsevier Academic Press, 2003, 1: 144-147) for the diagnosis of Botrytis cinerea.

[0041] (3) Fluorescent quantitative RTFQ-PCR test: The anti-gray mold marker gene specific primer RcWRKY22 and the internal reference universal primer UBC provided by the present application were synthesized by Shanghai Jeery Biotechnology Co., Ltd. for use. The anti-gray mold marker gene is RcWRKY22 (R. chinensis probable WRKY transcription factor 40 Locus Chromosome 2), which has a full gene sequence on GenBank with accession number XM_024320209.1. The internal reference gene is UBC (ubiquitin conjugating protein), which has a full gene sequence on GenBank with accession number JN399227. The specific primer RcWRKY22 is composed of the RcWRKY22 upstream primer shown in SEQ ID NO: 1 and the RcWRKY22 downstream primer shown in SEQ ID NO: 2. The internal reference universal primer UBC is composed of the internal reference universal primer UBC upstream primer shown in SEQ ID NO: 3 and the internal reference universal primer UBC downstream primer shown in SEQ ID NO: 4.

[0042] After the inoculation material was cultured for 12 h and then washed (at this point, the cultured petals were washed with sterile water for 3-5 times to remove the residual P. rosea in the inoculated spore suspension), the petal samples and the control petal samples cultured for 12 h were tested according to the instructions of the RNA extraction kit and the cDNA synthesis kit produced by Beijing TransGen Biotech Co., Ltd. After the cDNA of all the control petal samples and the inoculated petal samples was synthesized, the relative expression amount of the anti-gray mold marker gene of the inoculated material and the control was determined according to the steps of the fluorescent quantitative RTFQ-PCR kit produced by Beijing TransGen Biotech Co., Ltd. The fluorescent quantitative RTFQ-PCR reaction system was 20 μl, including ddH2O 6 μl, fluorescent quantitative mixed reagent 10 μl, upstream primer (0.25 nM) 1 μl, downstream primer (0.25 nM) 1 μl, inoculated material or control material cDNA 2 μl; the reaction program was 95°C pre-denaturation for 5 min, 95°C denaturation for 15 s, 58°C annealing for 15 s; +Plate Read (fluorescent signal collection, the collection of fluorescent signal was performed once in each cycle), a total of 45 cycles. The internal reference gene fluorescent quantitative RTFQ-PCR reaction system (same as above) and the reaction program (same as above) were used. The determination results were analyzed by ANOVA (analysis of variance) using SAS V9.0 software, and the average value and standard error of the relative expression amount of the anti-gray mold marker gene were calculated. The final data was recorded in the form of average value ± standard error of relative expression amount.

[0043] (4) Identification of the grade of resistance to gray mold: According to the relative expression amount of the marker gene of resistance to gray mold in the petals of different resources to be identified, the grade of resistance to gray mold is divided into the following four grades:

[0044] Grade 0: Immune, the relative expression amount is >= 8;

[0045] Grade 1: High resistance, the relative expression amount is >= 6 and < 8;

[0046] Grade 2: Medium resistance, the relative expression amount is >= 4 and < 6;

[0047] Grade 3: Low resistance, the relative expression amount is >= 2 and < 4;

[0048] When the relative expression amount is < 2, it indicates that the germplasm resource is susceptible to gray mold and does not belong to the resource resistant to gray mold.

[0049] The results are shown in Table 1.

[0050] Table 2 Identification results of 50 Rosa chinensis germplasm resources for resistance to gray mold

[0051]

[0052]

[0053]

[0054] Note: In Table 2, “--” indicates that the germplasm resource is susceptible to gray mold and does not belong to the resource resistant to gray mold; the relative expression amount data of the gene resistant to gray mold is the average value of three repetitions.

Claims

1. A method for rapidly screening rose germplasm resources resistant to gray mold using fluorescent quantitative RTFQ-PCR technology, characterized by: After cleaning the petal samples of the rose germplasm resources to be identified and the rose germplasm resources of the control, the petal samples were spread flat on a culture dish containing culture medium and the special primers were used to identify the rose germplasm resources. RcWRKY22 The relative expression levels of rose gray mold resistance marker genes were determined for the petal samples of the rose germplasm resource materials to be identified after being inoculated with rose gray mold for 12 hours and the petal samples of the control rose germplasm resource materials that were not inoculated with rose gray mold for 12 hours. The resistance level of rose gray mold of the rose germplasm resource materials to rose gray mold was identified according to the relative expression levels of the resistance marker genes. The special primers RcWRKY22 Depend on RcWRKY22 Upstream primer and RcWRKY22 The downstream primer composition is RcWRKY22 The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, RcWRKY22 The nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2; the petal samples of the rose germplasm resource material to be identified and the petal samples of the control rose germplasm resource material are both collected from the rose germplasm resource material to be identified without disease or insect pests.

2. The method according to claim 1, wherein: The petal samples collected are the petals in the first and second layers from the inside to the outside of the flowers of the rose germplasm resources to be identified, which are free of diseases and insect pests.

3. The method according to claim 1, wherein: The rose gray mold resistance grades divided according to the relative expression levels of the rose gray mold resistance marker genes are as follows: Level 0: immune, relative expression level ≥ 8; Level 1: high resistance, relative expression level ≥ 6 and < 8; Level 2: moderate resistance, relative expression level ≥ 4 and < 6; Level 3: low resistance, relative expression level ≥ 2 and < 4.

4. The method according to claim 1, wherein: The petal samples of the rose germplasm resource materials to be identified and the petal samples of the control rose germplasm resource materials are washed respectively with 70% v / v ethanol and sterile water. The collected petal samples are first soaked in 70% v / v ethanol for 45-60 seconds, and then washed with sterile water for 3-5 times.

5. The method according to claim 1, wherein: The cleaning of the petals of the rose germplasm resource material to be identified that has been inoculated with gray mold of rose for 12 hours comprises rinsing the petals with sterile water for 3 to 5 times.

6. The method according to claim 1, wherein: The inoculation was carried out by dripping method with a concentration of 1×10 5 / mL rose gray mold spore suspension was inoculated onto each petal.

7. The method according to claim 1, wherein: The culture is carried out under the conditions of indoor light intensity of 2000-3200 lx, indoor air relative humidity of 80-90%, and room temperature of 22-25°C.

8. The method according to claim 1, wherein: The culture medium is: MS+benzimidazole 0.4 g / L+agar 8.0 g / L.

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