An InDel molecular marker for assisting in selecting the rice bacterial leaf streak resistance locus BLS3 and its application

Abstract

The present invention discloses an InDel molecular marker for assisting in the selection of the rice bacterial leaf streak resistance locus BLS3 and its application, belonging to the technical field of molecular markers. The present invention uses the whole-genome resequencing BSA technology to preliminarily identify the candidate region related to rice bacterial leaf streak resistance, and then uses the developed InDel markers to analyze the candidate region. Combining with the phenotypic data, finally, the rice bacterial leaf streak resistance locus BLS3 is finely mapped within a range of about 658 kb between the InDel markers M238-4 and M245-1. Using the InDel molecular markers M238-4 and M245-1 designed by the present invention to amplify the rice plant genome, it can be judged whether the tested plant contains the rice bacterial leaf streak resistance gene locus through the denaturing polyacrylamide gel electrophoresis map, and the resistance level of its rice bacterial leaf streak can be predicted, greatly improving the breeding efficiency of disease-resistant rice breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers, and particularly relates to an InDel molecular marker for assisting in selecting the rice bacterial leaf streak resistance locus BLS3 and its application. Background Art

[0002] Bacterial leaf streak (BLS), also known as bacterial leaf streak disease, is the fourth major disease of rice after rice blast, sheath blight, and bacterial blight. This disease is the main disease in the rice production areas of southern China and Southeast Asia. When the disease occurs, it can cause a 40%-60% loss in rice yield, posing a serious threat to the high and stable yield of rice. In recent years, due to the large-scale promotion and planting of susceptible hybrid indica rice and the southward propagation and transportation of rice seeds, the disease has shown a trend of spreading and intensifying. According to the prediction of the occurrence trend of crop pests and diseases across the country, the harm of bacterial leaf streak in the southern rice region will gradually become serious and there is a trend of advancing northward, and its occurrence in the Yangtze River Basin will expand. However, currently, the excellent varieties and combinations being promoted, especially indica rice, which accounts for most of the rice area in China, are basically susceptible to bacterial leaf streak. Practice has proved that breeding disease-resistant varieties is the most economical and effective method for controlling this disease.

[0003] The main reason for the slow progress in breeding rice varieties resistant to bacterial leaf streak is the low efficiency of resource utilization. There are relatively rich resources of rice resistant to bacterial leaf streak, especially in rice germplasm resources from tropical regions. However, most of these varieties have poor agronomic traits and are difficult to directly utilize in breeding. Improving them solely by conventional breeding methods has a long cycle and low resource utilization efficiency. Compared with conventional breeding methods, molecular breeding techniques mainly based on molecular marker-assisted selection can greatly improve breeding efficiency and shorten the breeding period. However, the current development of bacterial leaf streak resistance resources is insufficient, and there are few studies on the molecular mapping of major genes resistant to bacterial leaf streak at home and abroad. Therefore, in order to improve breeding efficiency and solve the problem that most of the main cultivated rice varieties are susceptible to bacterial leaf streak in production, it is very necessary to conduct molecular mapping and cloning of excellent resistance gene resources, develop markers for molecular marker-assisted selection, and formulate a scientific and reasonable breeding strategy, so as to better improve breeding efficiency, shorten the breeding period of new bacterial leaf streak-resistant lines (varieties), and fundamentally solve the problem of bacterial leaf streak harm in rice production. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an InDel molecular marker for assisting in selecting the rice bacterial leaf streak resistance locus BLS3 and its application. The present invention uses the whole-genome resequencing BSA technology to preliminarily identify the candidate region related to rice bacterial leaf streak resistance, then uses the developed InDel markers to analyze the candidate region, and combines the phenotypic data to finally finely map the rice bacterial leaf streak resistance locus BLS3 within a range of about 658 kb between the InDel markers M238-4 and M245-1. Using the InDel molecular markers M238-4 and M245-1 designed by the present invention to amplify the rice plant genome, it can be judged whether the tested plant contains the rice bacterial leaf streak resistance gene locus through the denaturing polyacrylamide gel electrophoresis map, and the resistance level of its rice bacterial leaf streak can be predicted, greatly improving the breeding efficiency of disease-resistant rice breeding.

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

[0006] The present invention provides a specific primer combination for detecting the rice bacterial leaf streak resistance locus BLS3, and its nucleotide sequence is shown in SEQ ID No.1 to SEQ ID No.2 or shown in SEQ ID No.3 to SEQ ID No.4.

[0007] The present invention also provides a molecular marker for detecting the rice bacterial leaf streak resistance locus BLS3, and the molecular marker is M238-4 or M245-1; the primer sequence of the molecular marker M238-4 is shown in SEQ ID No.1 to 2; the primer sequence of the molecular marker M245-1 is shown in SEQ ID No.3 to 4.

[0008] The present invention also provides a kit containing the above primer combination.

[0009] The present invention also provides the application of the above primer combination or the above molecular marker or the above kit in rice breeding.

[0010] The present invention also provides the application of the above primer combination or the above molecular marker or the above kit in rapidly screening bacterial leaf streak-resistant varieties or lines.

[0011] The present invention also provides a molecular marker method for the rice bacterial leaf streak resistance locus BLS3. Amplify the DNA of rice varieties or breeding materials with the molecular marker M238-4 primer or the molecular marker M245-1 primer. If a 173bp amplification fragment can be amplified with the molecular marker M238-4 primer or a 225bp amplification fragment can be amplified with the molecular marker M245-1 primer, it indicates that the rice variety or breeding material contains the bacterial leaf streak resistance locus BLS3; the primer sequences of the molecular marker M238-4 are shown in SEQ ID No.1-2; the primer sequences of the molecular marker M245-1 are shown in SEQ ID No.3-4.

[0012] The present invention also provides a screening method for the said molecular marker, which is characterized by including the following steps:

[0013] (1) Using the disease-resistant material African new rice Z3 as the male parent and the disease-susceptible material 9311 as the female parent, hybridize to obtain F 1 , and after harvesting the F 1 generation seeds, self-cross for multiple generations to construct a set of rice F 7:8 segregation population;

[0014] (2) Conduct artificial inoculation resistance identification of bacterial leaf streak on the two parents and their derived F 7:8 population;

[0015] (3) According to the resistance identification results of the F 7：8 population, select extremely resistant and extremely susceptible samples in the F 7 population, extract DNA, and use the bulked segregant analysis based on whole genome resequencing to conduct preliminary gene mapping of the gene against rice bacterial leaf streak;

[0016] (4) In the interval of preliminary gene mapping, develop InDel markers using the data obtained from the whole genome resequencing of the parents, and combine the phenotypic and genotypic analysis of the F 8 population to delimit the target gene between two molecular markers M238-4 and M245-1;

[0017] (5) Obtain one bacterial leaf streak resistance locus BLS3, and use the said molecular markers M238-4 and M245-1 to detect whether the varieties (lines) derived from the resistant material Z3 contain this major gene locus and predict their bacterial leaf streak resistance levels.

[0018] Preferably, in the step (4), the gene is mapped on chromosome 2.

[0019] Compared with the prior art, the present invention has the following technical effects:

[0020] 1. The present invention has discovered an InDel molecular marker for the rice bacterial leaf streak resistance gene BLS3 developed based on resequencing technology. BLS3 is different from the loci of previously reported rice bacterial leaf streak resistance genes such as BLS1, qBlsr5a, etc., which increases people's understanding of the molecular regulatory network of bacterial leaf streak resistance and enriches the diversity of bacterial leaf streak resistance gene resources, providing more choices for breeding rice varieties resistant to bacterial leaf streak.

[0021] 2. The specific InDel molecular marker of the present invention can make the auxiliary breeding goal more clear and the efficiency higher. In the past traditional breeding process, to improve the resistance of varieties, usually a donor parent containing a disease-resistant gene is crossed with a recipient parent, or multiple backcrosses or pyramiding crosses are carried out. During this process, the resistance of the materials needs to be identified multiple times; however, since the field resistance identification of rice bacterial leaf streak requires complex identification processes such as bacterial culture, artificial inoculation, and field investigation, the identification difficulty is very high, and the results of phenotypic identification are also easily affected by environmental conditions, requiring multiple repeated tests at multiple locations; this has caused the traditional resistance identification method to be difficult, costly, time-consuming, and laborious. By using the molecular marker in the present invention to detect the gene locus of rice bacterial leaf streak, resistant single plants can be accurately and quickly identified at the seedling stage, and other plants can be eliminated, which not only saves costs but also greatly improves the efficiency of disease-resistant breeding.

[0022] 3. The present invention has established an identification method for the InDel molecular marker of the rice bacterial leaf streak resistance gene BLS3, which can accurately and quickly determine whether a rice material contains the gene locus BLS3 of rice bacterial leaf streak resistance, thereby predicting its resistance level to rice bacterial leaf streak, and then quickly screening disease-resistant varieties or lines for rice breeding, improving the selection efficiency of this trait, and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the denaturing polyacrylamide gel electrophoresis pattern of the molecular marker M238 - 4 in Example 2 of the present invention;

[0024] Figure 2 It is the denaturing polyacrylamide gel electrophoresis pattern of the molecular marker M245 - 1 in Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, or conditions of the present invention belongs to the scope of the present invention. The reagents, reagent kits, and instruments used in the following examples can all be obtained commercially. The methods used in the examples are the same as the commonly used methods unless otherwise specified.

[0026] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0027] Example 1

[0028] (1) Artificial inoculation identification method

[0029] Artificial inoculation identification was carried out using the needle puncture method. The pathogen of bacterial leaf streak was the dominant pathogenic strain JZ-8 in Guangxi, and the concentration of the bacterial solution was 6×10 8 CFU / mL, inoculated with a special pin at the rice booting stage, inoculated the flag leaf of each tiller for each plant, and investigated the length of lesions on three leaves 20 days later, and took the average value as the phenotypic value of the single plant. Three plants were inoculated in each line, and the phenotype was investigated on a single plant basis, and the average value of the three plants was taken as the phenotypic value of the line.

[0030] (2) Grading of Rice Bacterial Leaf Streak Disease

[0031] Immunity: no symptoms or only brown spots at the inoculation point; High resistance: lesion length 0.1~0.5cm; Disease resistance: lesion length 0.6~1.0cm; Moderate resistance: lesion length 1.1~1.5cm; Susceptible: lesion length 1.6~2.5cm; High sensitivity: lesion length >2.5cm.

[0032] (3) Resistance performance of parents

[0033] A. The African New Rice Z3 was identified in diseased areas for three consecutive years, and the results showed that it had good resistance to bacterial leaf streak disease. The African New Rice Z3 was identified by artificial inoculation using the above method, and the results showed that the material was highly resistant to the dominant pathogenic bacterial leaf streak disease strain JZ-8 in Guangxi, with a lesion length of 0.3 cm.

[0034] B. The above method was also used to conduct artificial inoculation identification on 9311. The results showed that the material was highly susceptible to Guangxi's dominant pathogenic bacterial leaf streak strain JZ-8, with a lesion length of 2.9 cm.

[0035] (4) Initial positioning

[0036] The F 1 Hybrid, Harvest F 1 After multiple generations of self-pollination, a set of recombinant inbred lines F containing 289 families was obtained through single seed descent. 7:8 The same method is used to calculate F 7:8 The population was artificially inoculated and identified according to F 7:8Based on the resistance identification results of the population, 30 recombinant inbred lines with extremely high resistance (lesion lengths are all less than 0.78 cm) and 30 recombinant inbred lines with extremely high susceptibility (lesion lengths are all greater than 2.75 cm) to bacterial leaf streak were selected to form two mixed pools of extreme traits, namely the resistant pool and the susceptible pool. DNA of each line was extracted by the CTAB method and purified by the magnetic bead method, and then equal amounts were mixed to form the resistant pool and the susceptible pool. Gene primary mapping of the gene against bacterial leaf streak of rice was carried out using trait mapping of the bulked segregant analysis (BSA) based on whole-genome resequencing.

[0037] ① A total of 66.90 Gbp of data was obtained by sequencing. After filtering, the Clean Bases obtained were 66.32 Gbp, and Q30 reached more than 90%. The average sequencing depths were 36× (Z3), 35× (9311), 39× (resistant pool) and 39× (susceptible pool) respectively. The clean reads of the 4 samples were aligned with the Nipponbare reference genome (Rice Genome Annotation Project (uga.edu)), and the results are shown in Table 1.

[0038] Table 1 Statistics of sequencing results

[0039]

[0040]

[0041] Among them: Raw_Read: The number of original sequencing reads; Clean_Reads: The number of remaining reads after filtering; Clean_Base: The number of remaining bases after filtering; GC(%) : The GC content of the sample, that is, the percentage of bases of G and C types in the total bases; Q20(%) : The percentage of bases with quality value greater than or equal to 20 in the total number of bases; CycleQ20(%) : CycleQ20 value; Q30(%) : The percentage of bases with quality value greater than or equal to 30 in the total number of bases; Ave_depth: The average coverage depth of the sample; Cov_ratio_1X(%) : The genomic coverage ratio corresponding to a depth of 1X; Cov_ratio_5X(%) : The genomic coverage ratio corresponding to a depth of 5X; Cov_ratio_10X(%) : The genomic coverage ratio corresponding to a depth of 10X; Total_reads: The number of Clean Reads, counted separately at both ends, that is, read1 and read2 are counted as 2 reads; Mapped(%) : The percentage of Clean Reads mapped to the reference genome in all Clean Reads; Properly_mapped(%) : Both ends of the sequencing sequence are mapped to the reference genome and the distance conforms to the length distribution of the sequencing fragment.

[0042] It was found that the average alignment efficiency was above 97.32%, and the 1X genome coverage was 93.72% (at least one base coverage). The sequencing data was of qualified quality and could be used for subsequent variant detection and analysis.

[0043] ② Variant detection

[0044] SNP detection: A total of 1,228,881 SNPs were obtained between the parents, of which 113,479 were non-synonymous mutation SNPs; a total of 165,304 SNPs were obtained between the pooled samples, and 18,303 of them caused non-synonymous mutations. After data analysis and filtering of the SNPs, a total of 1,045,643 high-quality and reliable SNP sites were finally obtained.

[0045] InDel detection: A total of 293,090 Small InDels were obtained between the parents; a total of 43,625 Small InDels were obtained between the pooled samples. After data analysis and filtering of the InDels, a total of 230,810 high-quality and reliable InDel sites were finally obtained.

[0046] ③ Association analysis

[0047] The SNP-index association algorithm and the ED association algorithm were used for association analysis, and the results are shown in Table 2.

[0048] Table 2 Statistical table of association region information

[0049]

[0050]

[0051] From the SNP results in Table 2, it can be seen that: using the SNP-index association algorithm, 1 candidate region related to the trait was obtained on chromosome 2, with a total length of 4.43 Mb; while using the ED association algorithm, the result was close to the association region obtained by using the SNP-index association algorithm. The intersection of the two methods obtained 1 candidate region related to the trait on chromosome 2, located at 22730000 - 27160000 bp, with a total length of 4.43 Mb.

[0052] From the InDel results in Table 2, it can be seen that: using the SNP-index association algorithm, 1 candidate region related to the trait was obtained on chromosome 2, with a total length of 4.36 Mb; while using the ED association algorithm, the result was close to the association region obtained by using the SNP-index association algorithm. The intersection of the two methods obtained 1 candidate region related to the trait on chromosome 2, located at 22,780,000 - 27,140,000 bp, with a total length of 4.36 Mb.

[0053] From the results of the intersection of SNPs and InDels in Table 2, it can be seen that by taking the intersection of the intersection regions of SNPs and InDels associated regions, 1 candidate region related to the trait was obtained on chromosome 2, located at 22,780,000 - 27,140,000 bp, with a total length of 4.36 Mb. A total of 859 genes were annotated within the associated region.

[0054] (5) Gene analysis and verification

[0055] In the candidate region related to the trait, InDel markers were developed using the data obtained from the whole-genome resequencing of the parents. 123 pairs of InDel primers were designed on rice chromosome 2. After verification, a total of 68 pairs of InDel markers with good polymorphism between the two parents were screened out. Combining with the F 8 plants that recombined within this mapping interval in the population for phenotypic and genotypic analysis, finally, the major gene BLS3 resistant to rice bacterial leaf streak was finely mapped between two InDel molecular markers M238 - 4 and M245 - 1, that is, within the range of 658.43 kb between 23842863 - 24501292 bp on chromosome 2. The two InDel molecular markers M238 - 4 and M245 - 1 co-segregated with the major gene BLS3 resistant to rice bacterial leaf streak, and their corresponding nucleotide sequences are shown in Table 3. By detecting these two molecular markers, it can be judged whether the major gene locus is contained in the varieties (lines) derived from the resistant material Z3.

[0056] Table 3 Primers and their sequences

[0057]

[0058] Example 2

[0059] Materials: Using Z3 as the donor and 9311 as the recipient, the recipient and donor materials were hybridized to obtain hybrid F 1 generation seeds. After sowing the F 1 generation seeds, F 2 generation rice materials were obtained.

[0060] Methods: Using the fresh young shoots or young leaves of 10 F 2 generation segregating individual rice materials as samples, their DNA was extracted by the CTAB method. Then PCR was performed on it. The PCR amplification system was a total of 10 μl, including: DNA template (1.0 μl), 10×Buffer buffer (1.0 μl), dNTPs (10 mM) (0.2 μl), upstream and downstream primers (0.4 μl each), Taq enzyme (5 U / μl) (0.1 μl), water (6.9 μl).

[0061] The reaction procedure for PCR amplification was as follows:

[0062]

[0063] Among them, steps 2 to 4 are looped 32 times.

[0064] The PCR products were detected by 7% non-denaturing polyacrylamide gel electrophoresis (PAGE), followed by silver staining and color development, and the amplified DNA bands were recorded. The results are as Figure 1-2 shown. At the same time, artificial inoculation identification of bacterial leaf streak pathogen was carried out on the F 2 -generation segregating individuals, and the results are shown in Table 4.

[0065] Table 4 Artificial inoculation identification results of bacterial leaf streak pathogen for individual plants in the F 2 population

[0066]

[0067]

[0068] It was found that for segregating individual plants 1, 6, and 8, only a 173-bp DNA fragment could be amplified using molecular marker M238-4, and only a 225-bp DNA fragment could be amplified using molecular marker M245-1, indicating that this sample contains the rice bacterial leaf streak resistance gene BLS3. Moreover, the artificial inoculation identification results of these individual plants in Table 4 showed that the lesion lengths were significantly shorter than those of other individual plants, showing resistance to bacterial leaf streak. The identification results using the two methods were consistent.

[0069] In summary, the two molecular markers of the present invention can quickly and accurately screen out materials containing major resistance genes during the breeding process of disease-resistant materials, thereby greatly improving the breeding efficiency.

[0070] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting rice resistance to bacterial leaf streak disease BLS3 A specific primer combination, characterized in that Its nucleotide sequence is shown as SEQIDNo.1~SEQIDNo.2 or as SEQIDNo.3~SEQIDNo.

4.

2. A method for detecting rice resistance to bacterial leaf streak disease BLS3 A molecular marker characterized in that The molecular marker is M238-4 or M245-1; the primer sequence of the molecular marker M238-4 is shown in SEQIDNo.1~2, and its amplification product is a 173bp or 160bp amplification fragment; the primer sequence of the molecular marker M245-1 is shown in SEQIDNo.3~4, and its amplification product is a 225bp or 213bp amplification fragment; the molecular marker M238-4 primer or the molecular marker M245-1 primer is used to amplify the rice breeding material DNA, if the molecular marker M238-4 primer can amplify a 173bp amplification fragment or the molecular marker M245-1 primer can amplify a 225bp amplification fragment, it indicates that the bacterial leaf streak resistance site BLS3 exists in the rice breeding material; the rice breeding material is Z3, 9311 or the hybrid offspring of the two.

3. A kit comprising the primer combination according to claim 1.

4. Use of the primer combination according to claim 1, the molecular marker according to claim 2, or the kit according to claim 3 in rice breeding, characterized in that: The DNA of rice breeding materials is amplified using the molecular marker M238-4 primer or the molecular marker M245-1 primer. If the molecular marker M238-4 primer can amplify a 173bp amplified fragment or the molecular marker M245-1 primer can amplify a 225bp amplified fragment, it indicates that the bacterial leaf streak resistance site BLS3 exists in the rice breeding material; the rice breeding material is Z3, 9311 or the hybrid offspring of the two.

5. Use of the primer combination according to claim 1, the molecular marker according to claim 2, or the kit according to claim 3 in rapid screening of rice varieties or strains resistant to bacterial leaf streak disease, characterized in that: The DNA of rice breeding materials is amplified using the molecular marker M238-4 primer or the molecular marker M245-1 primer. If the molecular marker M238-4 primer can amplify a 173bp amplified fragment or the molecular marker M245-1 primer can amplify a 225bp amplified fragment, it indicates that the bacterial leaf streak resistance site BLS3 exists in the rice breeding material; the rice variety is Z3 or 9311, and the rice line is a hybrid offspring line of the two.

6. A rice bacterial leaf streak resistance site BLS3 The molecular labeling method is characterized in that The DNA of rice breeding materials is amplified using the molecular marker M238-4 primer or the molecular marker M245-1 primer. If the molecular marker M238-4 primer can amplify a 173 bp amplified fragment or the molecular marker M245-1 primer can amplify a 225 bp amplified fragment, it indicates that there is a bacterial leaf streak resistance site in the rice breeding material. BLS3 ; The primer sequence of the molecular marker M238-4 is shown in SEQIDNo.1~2; the primer sequence of the molecular marker M245-1 is shown in SEQIDNo.3~4; the rice breeding material is Z3, 9311 or the hybrid offspring of the two.

7. The molecular labeling method according to claim 6, characterized in that: Said BLS3 The gene is located on chromosome 2.

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