Tightly linked molecular markers for resistance to rice bacterial leaf streak and their application

Abstract

The present invention discloses a molecular marker tightly linked to a resistance locus to bacterial leaf streak disease in rice and its application. The inventors hybridized HD10 (♂), a source resistant to rice leaf streak disease throughout the entire growth period, with 9311 (♀), a variety highly susceptible to rice leaf streak disease throughout the entire growth period. Using HD10 as the donor parent and 9311 as the recurrent parent, they obtained BC1F1 and self-pollinated the resulting BC1F2 population. The population was then subjected to resistance identification, phenotypic and resistance gene linkage analysis, and fine mapping of the resistance locus. The tightly linked molecular marker P1 was obtained. It is located at the physical position 26.45M on chromosome 2 of rice, and its amplified fragment has the base sequence of SEQ.ID.No.1 in the sequence listing. Studies have shown that the molecular marker P1 of the present invention has a population selection accuracy rate exceeding 96%, and can effectively and simply detect whether breeding rice materials contain gene loci that confer resistance to rice leaf streak disease strains in breeding. Therefore, the present invention can be applied to rice disease resistance breeding, enabling simple, rapid, and accurate screening of rice leaf streak disease-resistant materials, thereby improving breeding efficiency.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a molecular marker with tightly linked rice bacterial leaf streak resistance loci and an application thereof. Background Art

[0002] Bacterial leaf streak (BLS), also known as fine stripe, is caused by the Gram-negative bacterium Xanthomonas oryzae pv. oryzicola (XooC). BLS is a dangerous disease of rice that requires quarantine for transported plants in China, and some countries in Asia and Oceania have designated it as a major quarantine rice disease. BLS is one of the four major rice diseases in my country (rice sheath blight, bacterial leaf blight, rice blast, and fine stripe), and has become the fourth most common rice disease. The main factors contributing to the spread of BLS include the pathogen, susceptible varieties, and favorable environmental conditions. In recent years, the onset of BLS has been increasing, its scope has been expanding, and its severity has been increasing. Research indicates that under natural conditions, BLS can cause a 15-25% yield reduction in susceptible varieties, and in severe cases, the yield reduction can reach 40-60%. The disease can reduce yields by 10%-20% in mildly affected fields and by as much as 50%-60% in severely affected fields. To reduce the damage caused by fine stripe disease to rice, pesticides such as thiophanate-methyl, thiabendazole zinc, chlorothiazolinone, chloranil, mancozeb, and agricultural streptomycin are commonly used to control it.

[0003] At present, although there have been reports on the precise positioning of rice rice stripe resistance genes or QTLs, only one non-host gene for rice stripe resistance has been cloned, and its disease resistance mechanism has not yet been revealed. Resistance to rice leaf streak disease is controlled by different genes and has different modes of inheritance. Currently, it has been reported that it is controlled by two different types of genes: one is a qualitative trait controlled by major effect genes, which are controlled by single genes, two genes, or multiple genes with dominant or recessive effects. For example, Zhang Hongsheng et al. (1996) concluded that the resistance to leaf streak disease in IR36 is controlled by a pair of recessive genes, Xu Jianlong et al. (1997) concluded that the resistance to leaf streak disease in rice varieties Dular and Hashikalmi is controlled by two pairs of recessive genes, and Zhou Minghua et al. (1999) and He Yueqiu et al. (1994) concluded that the resistance to leaf streak disease in BJ1 is controlled by one to two pairs of dominant genes. The other is a quantitative trait controlled by multiple genes, whose disease resistance is affected by multiple factors such as the environment. For example, Tang Dingzhong et al. (1998), Zheng Jingsheng et al. (2005), and Chen Zhiwei et al. (2006) concluded that the resistance to leaf streak disease in rice is a quantitative trait controlled by multiple genes with minor effects. Despite this, there are currently very few bar streak resistance genes / QTLs that can be truly applied in breeding practice.

[0004] The misuse of pesticides can cause great harm to the ecological environment. Therefore, as people's awareness of food safety and ecological environment protection continues to increase, exploring and utilizing new resistance resources, using molecular biology and other means to cultivate pollution-free and harmless disease-resistant varieties, and improving the disease resistance of plants to achieve the purpose of prevention and control are the most economical, effective and environmentally friendly ways to control the disease, thereby achieving high-quality, stable and high-yield rice.

[0005] Therefore, it is of great significance to the long-term prevention and control of rice stripe disease to explore and utilize new molecular markers closely linked to rice stripe resistance loci to realize the use of molecular marker assisted selection (MAS) technology to screen rice stripe resistance germplasm resources and cultivate rice stripe resistance varieties, promote the rapid and effective application of valuable rice stripe resistance genes, and promote the rapid and effective application of valuable rice stripe resistance genes. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a molecular marker tightly linked to a rice bacterial leaf streak resistance locus. The molecular marker has a high degree of linkage with the rice fine stripe resistance locus and can be used for the positioning of fine stripe resistance genes and rice genetic breeding research.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] The molecular marker closely linked to the rice bacterial leaf streak resistance locus is located at the physical position of 26.45M on the second chromosome of rice, and its amplified fragment has the base sequence of SEQ.ID.No.1 in the sequence list.

[0009] The application of the above molecular markers in breeding rice varieties resistant to bacterial leaf streak disease and screening of resistance gene resources.

[0010] The primers for amplifying the above molecular markers include base sequences having the sequence list SEQ.ID.No.2 and SEQ.ID.No.3.

[0011] The primers are used in breeding rice varieties resistant to bacterial leaf streak disease and screening of resistance gene resources.

[0012] The PCR identification method of the above molecular marker is characterized by:

[0013] The reaction system is as follows: DNA template 1 μl, 10× PCR buffer 1 μl, dNTP (10 mM) 0.2 μl, forward primer (10 μM) 0.3 μl, reverse primer (10 μM) 0.3 μl, Taq DNA polymerase (5 U / μl) 0.1 μl, ddH2O 7.1 μl; the forward primer and reverse primer have the base sequences of SEQ.ID.No.2 and SEQ.ID.No.3 in the sequence listing, respectively;

[0014] The reaction procedure was as follows: pre-denaturation at 94°C for 5 min; followed by denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 45 sec, for a total of 34 cycles; and finally full extension at 72°C for 5 min, followed by storage at 4°C.

[0015] To address the current challenges in the practical application of rice bacterial leaf streak resistance genes and loci, the inventors hybridized HD10 (♂), a full-growing-period resistant strain, with 9311 (♀), a full-growing-period highly susceptible strain. Using HD10 as the donor parent and 9311 as the recurrent parent, they generated a BC1F1 population, which was then selfed to generate a BC1F2 population. This population was then subjected to resistance identification, phenotypic linkage analysis with resistance genes, and fine mapping of resistance loci. The inventors identified a tightly linked molecular marker, P1, located at position 26.45M on rice chromosome 2. The amplified fragment (175 bp in length) possessed the base sequence of SEQ.ID.No.1 in the sequence listing. Studies have shown that the molecular marker P1 has a population selection accuracy exceeding 96%, and can effectively and simply detect whether rice materials contain gene loci that confer resistance to bacterial leaf streak strains. Therefore, the present invention can be applied to rice disease resistance breeding, enabling simple, rapid, and accurate screening of rice material resistant to bacterial leaf streak, thereby improving breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a comparison of the lesion lengths of the parents HD10 and 9311 (20 days after inoculation).

[0017] Figure 2 This is an analysis of the resistance of F2 generation plants to stripe disease at the seedling stage.

[0018] Figure 3 The results of the positioning of the resistance gene to fine streak disease are shown in Figure a. The positioning results of the resistance gene to fine streak disease in the F2 population at the seedling stage are shown in Figure b. 2:3 Family group, mapping results of the streak disease resistance gene after adding P1 between B03021 and B03029.

[0019] Figure 4 This is a polymorphism test of the product amplified from linked marker P1 in a subset of individual plants in a BC1F2 population. Figures 1-14 are BC1F2 plants. Plants 1, 2, 5, 6, 7, and 9 have the same homozygous resistance pattern as the resistant parent, HD10; 3, 4, and 13 have the same homozygous susceptible pattern as the susceptible parent, 9311; and 8, 10, 11, 12, and 14 have heterozygous patterns for both parents. DETAILED DESCRIPTION

[0020] Example 1 Screening of molecular markers

[0021] (1) Construction and genetic analysis of the F2 segregating population of HD10 / 9311

[0022] The F1 hybrids were obtained by crossing HD10, a strain with high resistance to rice bacterial leaf streak (Reference 1: Hong Dengwei, Zhao Yan, Luo Dengjie, et al. 2017, Screening of broad-spectrum resistance resources to rice bacterial leaf streak, Southern Journal of Agriculture, 48(2):272-276) with 9311, a strain with high sensitivity to rice bacterial leaf streak (Reference 2: Zhao Yan, Luo Dengjie, He Shengxian, et al. Comparison of four inoculation methods for rice bacterial leaf streak [J]. Subtropical Agricultural Research, 2018, 14(4):242-246). The F1 hybrids were self-pollinated to obtain F2 segregating populations for genetic analysis. A pot experiment was conducted, with individual transplants of the F2 populations. The highly resistant and susceptible control varieties IR26 and King Kong 30, provided by International Rice, were used as resistant and susceptible controls, respectively. The Guangxi highly pathogenic rice streak disease strain GX01 (reference 2, this strain in the present invention was kindly donated by Professor He Yongqiang of the State Key Laboratory of Conservation and Utilization of Subtropical Agricultural Biological Resources) was shaken to the logarithmic phase and then diluted to 3×10 8 The cfu / mL concentration was used as the inoculum.

[0023] At the seedling stage, 244 individual plants of the F2 population were inoculated with needles and identified for resistance. According to the survey statistics of the mean value of lesions, and using the standards provided by the International Rice Research Institute (IRRI), the length of lesions was 1.5 cm as the dividing line between resistance and sensitivity, and 6 resistance levels [immune (I), asymptomatic wounds or only brown spots; high resistance (HR), lesion length 0.1-0.5 cm; resistant (R), lesion length 0.6-1.0 cm; medium resistance (MR), lesion length 1.1-1.5 cm; susceptible (S), lesion length 1.6-2.5 cm; highly susceptible (HS), lesion length greater than 2.5 cm] were used as identification standards for analysis. It was found that the average value of lesions in the F1 seedling stage was 3.98 cm, indicating high sensitivity; the mean length of lesions in the F2 segregating population at the seedling stage was continuously distributed, indicating that the material resistance is a quantitative trait controlled by recessive polygenes ( Figure 2 ).

[0024] (2) Molecular marker linkage analysis and preliminary positioning of resistance loci

[0025] The conventional CTAB method was used to extract the genomic DNA of each individual strain of HD10, 9311, F1 and F2 populations, and PCR was performed. The reaction system was: DNA template 1 μl, 10× PCR buffer 1 μl, dNTP (10 mM) 0.2 μl, forward primer (10 μM) 0.3 μl, reverse primer (10 μM) 0.3 μl, TaqDNA polymerase (5 U / μl) 0.1 μl, ddH2O 7.1 μl.

[0026] Reaction procedure: pre-denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 45 sec, for a total of 34 cycles; finally full extension at 72°C for 5 min, and storage at 4°C.

[0027] DNA amplification products were detected by polyacrylamide gel electrophoresis and silver staining followed by colorimetric analysis. Results were statistically analyzed: Bands identical to the maternal parent 9311 were designated "A," bands identical to the paternal parent HD10 were designated "B," and heterozygous bands were designated "H."

[0028] Based on seedling phenotypes, 10 highly resistant and 10 highly susceptible F2 plants were selected from the population. DNA from these 10 resistant and 10 susceptible plants was mixed at equal concentrations and volumes to construct disease-resistant and susceptible pools. PCR analysis of the polymorphisms between the resistant and susceptible pools was performed using 440 pairs of molecular markers (Table 1) that are relatively evenly distributed across the 12 rice chromosomes and are polymorphic between the two parents revealed seven polymorphic markers: RM6, B03059, RM03045, B03029, B03021, B0301, and B02065. Based on the genotypes and corresponding phenotypic values ​​of these markers in the F2 population, a genetic linkage map was constructed using JoinMap3.0 software, and the composite interval mapping method of MapQTL5.0 software was used to scan the target chromosome segment for resistance QTL loci. The preliminary positioning of the resistance gene was completed. The main effect QTL was located between B03029 and B03021 with an LOD value of 8.68, and the explained phenotypic variation rates were 13.1% ( Figure 3 a). Using HD10 as the donor parent and 9311 as the recurrent parent, BC1F1 was obtained and self-pollinated to obtain a BC1F2 population. Resistance was identified in 106 BC1F2:3 families, and a molecular marker P1 was developed between B03029 and B03021 (polymorphism see Figure 4 ), and further located the resistance locus between P1 and B03029, with an LOD value of 6.84, and the explained phenotypic variation rate was 27.2% ( Figure 3 b).

[0029] Table 1 Molecular marker polymorphisms and average interval distances between HD10 and 9311

[0030]

[0031]

[0032] (3) Genotype of molecular marker P1 and its corresponding resistance to fine streak disease

[0033] P1 was used to detect 263 plants in the BC1F3 population (Table 2). Fifty-six plants were homozygous for the susceptible marker genotype (AA), with an average lesion length of 3.21 cm; 146 plants were heterozygous for the susceptible marker genotype (Aa), with an average lesion length of 3.07 cm; and 61 plants were homozygous for the resistant marker genotype (aa), with an average lesion length of 1.09 cm. The difference in lesion length between the homozygous susceptible marker genotypes (AA) and (Aa) was not significant, but the difference in lesion length between the susceptible marker genotypes (AA and Aa) and the resistant marker genotype (aa) was extremely significant. This shows that detecting the genotype of molecular marker P1 can effectively select disease-resistant and susceptible plants.

[0034] Table 2 Resistance performance of individual plants with molecular marker P1 genotype

[0035]

[0036] Note: A and a in the marker genotype represent the susceptible marker genotype and the disease-resistant genotype of P1 respectively.

[0037] Example 2 Validation of molecular markers

[0038] 1. Materials and Methods

[0039] 1.1 Materials

[0040] BC1F was constructed with the resistant HD10 as the donor parent and the susceptible cultivar 9311 as the recipient parent. 2:3 group.

[0041] Molecular marker: P1.

[0042] 1.2 Methods

[0043] The genomic DNA of disease-resistant HD10, susceptible 9311 and BC1F2 was extracted and PCR amplified using P1 primer and analyzed by electrophoresis in the same manner as in Example 1. The banding patterns of individual BC1F2 plants were statistically analyzed. 2：3 The lesion lengths of 10 individual plants in a family were measured, and the average value was taken as the resistance phenotype of the BC1F2 individual plant.

[0044] 2. Results

[0045] Table 3. Coincidence rate between molecular marker P1 detection and population resistance-susceptibility phenotype

[0046]

[0047] As shown in Table 3, using the P1 molecular marker to detect 106 BC1F2 plants, 13 plants identified as resistant by field inoculation (a resistant subpopulation) showed resistant bands in 12 individuals and susceptible bands in 1 individual, resulting in a genotype-phenotype concordance rate of 92.31%. Among the 93 plants identified as susceptible by phenotypic analysis (a susceptible subpopulation), this marker detected 90 susceptible bands and 3 resistant bands, resulting in a genotype-phenotype concordance rate of 96.77%. The concordance rate for molecular marker-assisted selection for the entire population was 96.23%. Therefore, the use of molecular marker P1 has a high degree of accuracy in selecting loci for resistance to fine streak disease.

[0048] The results demonstrate that the P1 molecular marker method provided by the present invention can accurately screen for bacterial leaf streak resistance loci at the seedling stage and predict rice plant resistance to bacterial leaf streak. Specifically, using the rice genomic DNA to be tested as a template, PCR amplification is performed using the labeled primer P1. The product is analyzed by polyacrylamide gel electrophoresis. If a 175-bp amplified fragment is amplified, the selection of resistant materials is effective. In summary, the widespread application of this invention can greatly accelerate the screening of rice materials resistant to bacterial leaf streak. Sequence Listing <110> Guangxi University <120> Tightly linked molecular markers for resistance to rice bacterial leaf streak and their application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 175 <212> DNA <213> Artificial Sequence <400> 1 tggctggcac tgtgtctgca tttctgtatg cgtggtgtgt gtgagtgtgt gagagagaga 60 gaatgagaga gagagagaga gagagagtct gagactgtat gaatatgtgt gcgagagt 120 gtgtaaatgc taagcggtta caggggcatt gaagaggggg ctcagtggct ctcat 175 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 tggctggcac tgtgtctg 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 atgagagcca ctgagccc 18

Claims

1. A molecular marker tightly linked to a rice bacterial leaf streak resistance locus, characterized in that The molecular marker is located on rice chromosome 2 and is shown in the sequence listing SEQ.ID.No.

1.

2. Use of the molecular marker according to claim 1 in breeding rice varieties resistant to bacterial leaf streak disease.

3. Use of the molecular marker according to claim 1 in screening for rice bacterial leaf streak resistance gene resources.

4. Use of primers for amplifying the molecular marker of claim 1 in breeding rice varieties resistant to bacterial leaf streak disease, characterized in that: The primers are shown in the sequence listing as SEQ.ID.No.2 and SEQ.ID.No.

3.

5. The use of the primers for amplifying the molecular marker of claim 1 in screening for rice bacterial leaf streak resistance gene resources is characterized by: The primers are shown in the sequence listing as SEQ.ID.No.2 and SEQ.ID.No.3.

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