Primer pair for identifying rice bacterial streak resistance major gene and application thereof

Abstract

The invention relates to the field of plant molecular biology, in particular to a primer pair for identifying a rice bacterial streak resistance major gene and application of the primer pair. The invention provides a primer pair for identifying the resistance of rice bacterial streak. The primer pair comprises an upstream primer as shown in SEQ ID NO. 1 and a downstream primer as shown in SEQ ID NO. 2. The primer pair provided by the invention can predict the resistance of rice plants to bacterial streak. The primer pair can be used for genotype detection of rice varieties or strains, whether the rice varieties or strains have bacterial streak resistance or not is judged, and then disease-resistant varieties or strains are rapidly screened. Therefore, the primer pair provided by the invention can be used for rice breeding.

Description

Technical Field

[0001] The invention relates to the field of plant molecular biology, in particular to a primer pair for identifying a major gene of resistance to rice bacterial leaf streak disease and an application thereof. Background Art

[0002] Rice (scientific name: Oryza sativa L.) belongs to the herbaceous genus Oryza. As one of the world's three major food crops (wheat, rice and corn), it feeds nearly two-thirds of the world's population. In my country, more than half of the people also rely on it as their staple food. In recent decades, the scope of various rice diseases has continued to expand and frequently break out, and rice yield and quality have also been seriously affected, and food security issues have become increasingly serious. Rice bacterial leaf streak (BLS), also known as rice leaf streak, is caused by infection with the Gram-negative bacterium Xanthomonas oryzae pv.oryzicola (XooC). Rice leaf streak is one of the dangerous diseases that should be quarantined for domestic transport plants. At present, some countries in Asia and Oceania have listed it as an important quarantine rice disease. Bacterial leaf streak of rice is also one of the four major rice diseases in my country (rice sheath blight, white leaf blight, rice blast and rice stripe disease). The factors causing the spread of rice leaf streak disease mainly include pathogens, susceptible varieties and environmental conditions suitable for the disease.

[0003] In recent years, the onset of rice streak disease has become earlier and earlier, the scope of the disease has continued to expand, and the severity of the disease has become more and more severe. According to research and investigation, under natural conditions, rice streak disease will cause a 15-25% yield reduction for susceptible varieties, and in severe cases, the yield reduction can reach 40-60%. The occurrence of this disease will reduce the yield by 10%-20% in mildly diseased fields, and up to 50%-60% in severely diseased fields. People often use pesticides such as thiophanate-methyl, thiazole zinc, chlorothiocarb, gray frost, ammonium, and agricultural streptomycin to prevent and control the occurrence of rice streak disease. However, the abuse of pesticides will cause great harm to the ecological environment. Therefore, as people's awareness of food safety and ecological environment protection continues to increase, the use of molecular biology and other means to cultivate pollution-free, pollution-free and disease-resistant varieties and improve the disease resistance of plants has become the most economical, effective and environmentally friendly way to control the disease. Summary of the invention

[0004] The purpose of the present invention is to provide a primer pair for identifying a major gene of resistance to rice bacterial leaf streak and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention explores and utilizes a major gene of resistance to rice bacterial leaf streak (named BLS3) to develop a primer pair that can be used to assist breeding, and the primer pair can effectively distinguish rice varieties resistant to bacterial leaf streak from rice varieties susceptible to bacterial leaf streak.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a primer pair for identifying resistance to rice bacterial leaf streak disease. The primer pair comprises an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2.

[0007] Further preferably, the molecular marker amplified by the primer pair is on the first chromosome of rice.

[0008] The invention provides a product for identifying resistance to rice bacterial leaf streak disease, and the product comprises the above primer pair.

[0009] Preferably, the product includes reagents, kits and chips.

[0010] The present invention provides the use of the primer pair or the product in identifying resistance to rice bacterial leaf streak disease.

[0011] The present invention provides a method for identifying resistance to rice bacterial leaf streak disease, comprising the following steps:

[0012] The genome of the sample to be tested is used as the sample template DNA, the genome of rice RL6 is used as the positive template DNA, and the genome of rice 9311 is used as the negative template DNA;

[0013] After the sample template DNA, positive template DNA and negative template DNA are mixed with the above primer pairs respectively, PCR amplification is performed to obtain the band pattern of the amplified product, and the resistance of the sample to be tested to rice bacterial leaf streak disease is judged according to the band pattern.

[0014] Preferably, when the band pattern of the sample template DNA is the same as the band pattern of the positive template DNA, the sample to be tested is judged to be a rice variety resistant to bacterial leaf streak disease; when the band pattern of the sample template DNA is the same as the band pattern of the negative template DNA, the sample to be tested is judged to be a rice variety susceptible to bacterial leaf streak disease.

[0015] Preferably, the PCR reaction system is: 1 μL of template DNA, 0.8 μL of upstream primer, 0.8 μL of downstream primer, 1 μL of Bffuer, 0.2 μL of dNTP and 0.1 μL of Taq enzyme.

[0016] Preferably, the PCR reaction procedure is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C-62°C for 30 s, extension at 72°C for 45 s, and extension at 72°C for another 5 min, for 35 cycles.

[0017] The present invention also provides the application of the primer pair or the product in rice breeding.

[0018] The present invention discloses the following technical effects:

[0019] 1. The primer pair provided by the present invention can locate the major gene of resistance to rice stripe disease in rice varieties.

[0020] 2. The primer pair provided by the present invention can predict the resistance of rice plants to bacterial leaf streak disease. Therefore, the primer pair can be used for genotype detection of rice varieties or strains, to determine whether the rice varieties or strains have resistance to bacterial leaf streak disease, and then quickly screen disease-resistant varieties or strains for rice breeding.

[0021] 3. Auxiliary breeding selection has clear goals and saves costs. In traditional breeding methods, parents with disease-resistant genes are generally collected first for a series of hybridization with cultivated varieties, and rice varieties must be identified for resistance to bacterial leaf streak and selected for breeding. The breeding work is cumbersome and time-consuming, and is also greatly affected by the environment. However, by detecting the major gene loci for resistance to bacterial leaf streak, highly disease-resistant individual plants can be identified at the seedling stage, and other plants can be eliminated, which can not only save production costs, but also greatly improve the selection efficiency of rice resistant to leaf streak, greatly shortening the breeding cycle of rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The polymorphism test result diagram of primer pairs used for identifying resistance to rice bacterial leaf streak disease in parents, resistant and susceptible pools, and individual plants; wherein, M is a marker (size is 250bp and 100bp from top to bottom), 1 is the susceptible parent 9311, 2 is the resistant parent RL6, 3-4 is the susceptible gene pool, 5-6 is the resistant gene pool, 7-12 is the offspring susceptible individual plants, 13-18 is the offspring resistant individual plants, and 19-22 is the offspring heterozygous individual plants;

[0024] Figure 2 This is a comparison chart of the disease resistance of parents 9311 and RL6, and a picture of the lesions 20 days after inoculation; A is the susceptible parent 9311, and B is the resistant parent RL6. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Unless otherwise specified, the instruments, reagents and kits used in the present invention are all purchased by those skilled in the art.

[0031] Example 1

[0032] 1. Construction and phenotypic identification of BC1F2 population:

[0033] (1) The resistant line RL6 provided by our laboratory (published in "Screening of Broad-spectrum Resistance Resources to Rice Bacterial Leaf Streak Disease", promised to be released to the public for 20 years), and the susceptible material 9311 is a well-known rice restorer line. The disease-resistant material RL6 (male parent) and the susceptible material 9311 (female parent) were hybridized to obtain F1, and then self-pollinated to obtain F2 population. The extremely resistant individual plants (lesion length less than 0.5 cm) were selected through phenotypic identification, backcrossed with 9311 as the recurrent parent, and self-pollinated once more to obtain the BC1F2 population.

[0034] (2) The inoculation strain (the strain GX01 used was provided by Professor He Yongqiang of the College of Agriculture of Guangxi University and published in "Study on Mutation of the Main Pathogenic System Genes Predicted by the GX01 Strain of Rice Bacterial Leaf Streak Pathogen", which is committed to be released to the public for 20 years) was activated on NA medium and cultured at 28°C for 48 h. A single colony with normal morphology was picked and inoculated into NB medium. The culture was cultured on a shaker (28°C, 200 rpm) until the logarithmic phase. The suspension was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were made into a suspension with sterile water and diluted to 3×10 8 CFU / mL is used for inoculation, and the inoculum solution should be prepared and used immediately.

[0035] The rice was inoculated when it grew to the tillering stage, and the needle puncture method was used for inoculation; leaves growing at the same period were selected, and the middle of the leaves were placed flat on the sponge. After piercing the rice leaves with a large needle, the sponge was gently pressed to squeeze out the suspension. Three leaves were inoculated per plant, and six needle puncture points were made on each leaf. During this period, the suspension was replenished. 20 days after inoculation, the length of the lesions on the rice leaves was measured, and the disease was graded according to the following identification standards: Grade 0, immune (I), the wound was asymptomatic or only had brown spots; Grade 1, high resistance (HR), the lesion length was 0.1-0.5cm; Grade 3, resistant (R), the lesion length was 0.6-1.0cm; Grade 5, moderate resistance (MR), the lesion length was 1.1-1.5cm; Grade 7, susceptible (S), the lesion length was 1.6-2.5cm; Grade 9, highly susceptible (HS), the lesion length was greater than 2.5cm, among which the disease resistance of the resistant line RL6 and the susceptible material 9311 were as follows: Figure 2 shown.

[0036] 2. Molecular marker analysis of BC1F2 population

[0037] (1) DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method; the genomic DNA of the parents (susceptible parent 9311, resistant parent RL6) and all the individuals in the BC1F2 population used in this study was subjected to molecular marker analysis. The PCR reaction system was 10 μL in total, specifically: DNA template 1 μL, Y1 upstream and downstream primers 0.8 μL each (Y1 upstream primer is shown in SEQ ID NO.1, specifically: GCTGAGTTAGGGTTTGGG; Y1 downstream primer is shown in SEQ ID NO.2, specifically: GGTAATGGTTGAGGCTGT), Bffuer 1 μL, dNTP (10 mM) 0.2 μL, Taq enzyme (5 U / L) 0.1 μL. The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C-62°C (adjusted according to the actual Tm value of the primers) for 30 s, extension at 72°C for 45 s, and extension at 72°C for another 5 min, for 35 cycles.

[0038] After PCR amplification, the samples were placed in a refrigerator at 4°C. DNA amplification products were detected by polyacrylamide gel electrophoresis and silver staining and color analysis. Results: The same band pattern as the maternal parent 9311 was recorded as "A" (171 bp), the same band pattern as the paternal parent RL6 was recorded as "B", and the heterozygous band pattern was recorded as "H".

[0039] (2) Refer to the BSA (Bulked Segregation Analysis) method of Michelmore et al. (1991), i.e., the Bulk Segregation Analysis method, to screen SSR markers linked to the major effect QTL. Through resistance identification of the BC1F2 population, 15 high-resistant plants were randomly selected, and their genomic DNA was mixed in equal amounts to construct a disease-resistant gene pool. At the same time, 15 high-susceptible plants were randomly selected, and their genomic DNA was mixed in equal amounts to construct a disease-susceptible gene pool. The SSR markers that were screened out with co-dominant polymorphism between the parents (RL6 and 9311) were used to screen markers that still showed polymorphism between the resistance and susceptible pools.

[0040] 3. Results

[0041] The test results of parents, some offspring plants, susceptible gene pool and resistant gene pool are as follows: Figure 1 As shown. Figure 1 It can be seen that the primer pair provided by the present invention can effectively distinguish between rice varieties resistant to bacterial leaf streak disease and rice varieties susceptible to bacterial leaf streak disease.

[0042] At the same time, the coincidence rate between the genotype and phenotype of the F2 generation population detected by the Y1 primer pair (SEQ ID NO.1 and SEQ ID NO.2) was counted, and the results are shown in Table 1.

[0043] Table 1 The coincidence rate between genotype and phenotype of F2 population detected by primer pair Y1

[0044]

[0045] There is a linkage relationship between Y1 and the dominant major effect gene of resistance to rice streak disease carried by DL6. Comparing with the published rice molecular genetic linkage map, it can be seen that the molecular marker amplified by primer pair Y1 is located at about 23cM on chromosome 1.

[0046] In summary, this primer pair can be used to identify and screen rice varieties resistant to bacterial leaf streak disease, providing a new approach for breeding rice varieties resistant to bacterial leaf streak disease.

[0047] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer pair for identifying resistance to rice bacterial leaf streak disease, It is characterized in that The primer pair includes an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.

2.

2. A product for identifying resistance to rice bacterial leaf streak disease, It is characterized in that The product comprises the primer pair described in claim 1.

3. The product according to claim 2, It is characterized in that The products include reagents, kits and chips.

4. Use of the primer pair according to claim 1 or the product according to claim 2 or 3 in identifying resistance to rice bacterial leaf streak disease.

5. A method for identifying resistance to rice bacterial leaf streak disease, It is characterized in that The following steps are involved: The genome of the sample to be tested is used as the sample template DNA, the genome of rice RL6 is used as the positive template DNA, and the genome of rice 9311 is used as the negative template DNA; After the sample template DNA, positive template DNA and negative template DNA are mixed with the primer pair of claim 1 respectively, PCR amplification is performed to obtain the band pattern of the amplified product, and the resistance of the sample to be tested to rice bacterial leaf streak disease is judged according to the band pattern.

6. The method according to claim 5, It is characterized in that When the band pattern of the sample template DNA is the same as the band pattern of the positive template DNA, the sample to be tested is judged to be a rice variety resistant to bacterial leaf streak disease; when the band pattern of the sample template DNA is the same as the band pattern of the negative template DNA, the sample to be tested is judged to be a rice variety susceptible to bacterial leaf streak disease.

7. The method according to claim 5, It is characterized in that The PCR reaction system is: 1 μL of template DNA, 0.8 μL of upstream primer, 0.8 μL of downstream primer, 1 μL of Bffuer, 0.2 μL of dNTP and 0.1 μL of Taq enzyme.

8. The method according to claim 5, It is characterized in that The PCR reaction program is: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C-62°C annealing for 30 s, 72°C extension for 45 s, 72°C extension for another 5 min, 35 cycles.

9. Use of the primer pair according to claim 1 or the product according to claim 2 or 3 in rice breeding.

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