CN110178719A - A method of improvement Rice Resistance To Rice Blast and bacterial leaf spot resistance - Google Patents

Abstract

The invention discloses a kind of methods for improveing Rice Resistance To Rice Blast and bacterial leaf spot resistance, hybridize Bacterial blight resistance gene and blast resistant gene is carried simultaneously with the rice material for not carrying above-mentioned resistant gene, seedling stage foreground detection is carried out using SNP marker, selection carries the single plant of Bacterial blight resistance gene and blast resistant gene, and be selfed after using rice material of the resistance wait improveing carrying out continuous backcross as recurrent parent, obtain bacterial leaf-blight and rice blast resistance improvement and comprehensive agronomy character improved materials similar with recurrent parent.Pass through the high filial generation of the SNP marker screening blast resisting, Bacterial blight resistance gene and background response rate of close linkage, realize polymerization rice blast, bacterial leaf spot resistance gene not only to achieve the purpose that improve seed rice disease resistance, and improved materials are similar to recurrent parent, breeding cycle is short, saves cost.

Description

A method for improving rice blast resistance and bacterial blight resistance

technical field

The invention relates to the technical field of rice breeding, in particular to a method for simultaneously improving rice blast resistance and bacterial blight resistance.

Background technique

Rice is an important food crop in my country. Rice blast and bacterial blight are the main diseases that harm rice. When the damage is severe, rice production can be reduced by 30-50% or even crop failure. The strong and excellent restorer line R900 of hybrid rice is a major innovation result of super hybrid rice research, and its hybrid combination "Chaoyouqian" has set high-yield records in many places in recent years. However, the restorer line R900 and its combination are highly susceptible to bacterial blight and rice blast, and there is a risk of serious yield reduction due to outbreaks of bacterial blight or rice blast during popularization and application.

Breeding resistant rice varieties has been recognized by the breeding community as the most economical and effective way to control rice blast and bacterial blight. The invention proposes a molecular breeding method for rapid and directional improvement of the bacterial blight and rice blast resistance of the restorer line R900 based on the whole genome SNP molecular marker. Utilizing broad-spectrum bacterial blight resistance and rice blast resistance main genes, combined with the foreground selection of SNP molecular markers closely linked to the resistance target gene and the background selection of genome-wide SNP molecular markers, rapid and directional improvement of bacterial blight in the restorer line R900 Disease resistance and rice blast resistance, while maintaining its high yield, high quality and other characteristics, to achieve the coordination and unity of disease resistance of improved varieties and maintaining the original excellent characteristics. The achievements of the invention will provide guarantee for the further safe and full play of the advantages and large-scale application of the hybrid rice restorer line R900 and its hybrid combination.

Contents of the invention

The present invention uses SNP molecular markers linked with the bacterial blight resistance gene and the rice blast resistance gene to select the above-mentioned resistance gene present in the hybrid segregation population of the material to be improved and the bacterial blight and rice blast resistance material , and carry out genome-wide SNP molecular marker background selection on plants with resistance genes, combined with conventional breeding methods to select rice materials with significantly improved resistance to bacterial blight and rice blast, and comprehensive agronomic traits that are basically consistent with the materials to be improved Breeding methods.

In order to achieve the above object, the technical solution of the present invention is achieved through the following steps:

A method for improving rice blast resistance and bacterial blight resistance, comprising the following steps:

( ₁ ) The recurrent parent is crossed with the rice material carrying the bacterial blight resistance gene and the rice blast resistance gene to obtain hybrid F1 generation seeds;

(2) Plant hybrid F ₁ generation seeds, use SNP molecular markers to exclude false hybrid plants, and backcross true hybrid F ₁ generation with recurrent parents to obtain BC ₁ F ₁ generation seeds;

(3) Plant BC ₁ F ₁ generation seeds, use the SNP molecular markers closely linked to bacterial blight resistance gene and rice blast resistance gene to detect the seedling stage prospect of BC ₁ F ₁ generation population plants, and select the plants that carry white blight at the same time. The BC ₁ F ₁ generation individual plants of the leaf blight resistance and rice blast resistance genes, and then use the genome-wide SNP molecular markers to detect the genetic background of the plants containing the resistance genes, and select the individual plants with the highest background recovery rate and recurrent parents Continuous backcrossing, each backcrossing generation selects the single plant carrying bacterial blight resistance and rice blast resistance genes and the highest background recovery rate to backcross with the recurrent parent, the number of backcrossing generations is m (m≧2), Obtain backcross offspring BC _m F _l ;

(4) Plant BC _m F ₁ generation seeds, and use SNP molecular marker detection to select BC _m F ₁ generation single plants that carry bacterial blight resistance genes and rice blast resistance genes and have the highest background recovery rate for selfing. The number is n (n≧2), to obtain BC _m F _n -generation seeds with stable comprehensive agronomic traits;

(5) Plant BC _m F _n generation seeds, use SNP molecular markers to detect, select BC _m F _n generation single plants that carry both bacterial blight resistance and rice blast resistance homozygous genes, and obtain bacterial blight and rice Improved materials with improved blast resistance and comprehensive agronomic traits similar to recurrent parents.

Further, the recurrent parent is the hybrid rice restorer line R900.

Further, the bacterial blight resistance gene includes at least one of Xa23, Xa21, and Xa7.

Further, the rice blast resistance gene includes at least one of Pi1, Pi2, Pi9, and Pigm.

Furthermore, in the step (3), step (4), and step (5), the single plant selected by the SNP molecular marker carries 1-3 bacterial blight resistance genes, and simultaneously carries rice blast resistance There are 1-2 genes.

Further, in the step (1), the rice material carrying both the bacterial blight resistance gene and the rice blast resistance gene is from any breeding material among indica rice, japonica rice, restorer line, and maintainer line.

Further, the step (2), step (3), step (4), and step (5) use SNP molecular markers to detect bacterial blight resistance genes and rice blast resistance genes for backcrossing or self-selection. The purpose of crossing is to ensure that the above-mentioned resistance genes always exist in the improved offspring.

Furthermore, in the steps (3) and (4), the SNP whole-genome molecular markers are used to detect the background recovery rate, and the purpose of continuous backcrossing between plants with the highest background recovery rate and recurrent parents is to obtain comprehensive Improved materials with similar agronomic traits to recurrent parents.

Further, in the step (2), step (3), step (4), and step (5), the SNP molecular markers for detecting bacterial blight resistance gene and rice blast resistance gene are self-developed.

Further, in the steps (3) and (4), the genome-wide SNP molecular markers for detecting the background recovery rate are self-developed.

Beneficial effects of the present invention:

(1) The breeding material finally obtained in the present invention aggregates rice blast resistance gene and bacterial blight resistance gene;

(2) When using closely linked SNP markers to detect resistance genes, the present invention simultaneously uses whole-genome SNP markers for background selection and comprehensive agronomic trait selection, which can significantly shorten the breeding cycle and improve breeding efficiency;

(3) The invention significantly improves the rice blast resistance and bacterial blight resistance of the finally obtained breeding material, and is basically consistent with the agronomic traits of the material to be improved.

Detailed ways

It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

A specific implementation example is used to describe in detail: using the bacterial blight resistance gene Xa23 and the rice blast resistance gene Pi9 carried by HZ02455 to directional improve the resistance to bacterial blight and rice blast of the hybrid rice restorer line R900, the inventors SNP molecular marker detection shows that the restorer line R900 does not carry Xa23 and Pi9 resistance genes, while HZ02455 carries Xa23 and Pi9 resistance genes. The specific implementation steps are as follows:

1. The restorer line R900 was crossed with HZ024550 to obtain 56 hybrid F1 seeds. When crossing, use the restorer line R900 as the female parent, HZ024550 as the male parent or HZ024550 as the female parent, and the restorer R900 as the male parent;

As a preference, in this embodiment, the restorer line R900 is used as the female parent and HZ024550 is used as the male parent for hybridization to obtain the F1 generation.

2. Plant the above-mentioned F1 generation seeds, use molecular markers to identify whether the F1 generation individual plants carry the bacterial blight resistance gene Xa23 and the rice blast resistance gene Pi9 at the seedling stage to exclude false hybrids, and obtain 24 F1 generation true hybrids; select F1 true hybrids The male parent of the hybrid was backcrossed with the restorer line R900, and 772 BC ₁ F ₁ seeds were obtained.

BC ₁ F ₁ generation seeds were planted, and a total of 562 heterozygous individual plants with simultaneous bacterial blight resistance gene Xa23 and rice blast resistance gene Pi9 were identified by SNP molecular markers at the seedling stage; after that, the selected individual plants were used Genome-wide SNP molecular markers were used to select the genetic background of the target gene heterozygous single plant, and the single plant with the highest background recovery rate and the most similar agronomic traits to the restorer line R900 at the heading stage was selected as the male parent, and backcrossed with the restorer line R900 , Obtained 1097 BC2F1 seeds.

The genetic background is detected. If the recipient genotype is AA, it is recorded as "0", the donor genotype is BB, which is recorded as "2", and the heterozygous genotype is AB, which is recorded as "1". The background recovery rate = ( a*2+b)/[(a+c+b)*2].

Where a represents the total number of "0", b represents the total number of "1", and c represents the total number of "2";

Plant BC2F1 seeds, and use SNP molecular markers to identify heterozygous individual plants containing both the bacterial blight resistance gene Xa23 and the rice blast resistance gene Pi9 at the seedling stage, a total of 747 plants; after that, use the genome-wide SNP for the selected individual plants Molecular markers were used to select the genetic background of the heterozygous individual plants for the target gene, and the three individual plants with the highest background recovery rate, that is, the most similar agronomic traits to the restorer line R900 at maturity, were selected, and the individual plants were harvested after selfing to obtain There are 2984 BC ₂ F ₂ seeds of the 3 lines.

5. Plant BC2F2 seeds, use SNP molecular markers to identify individual plants that are homozygous for bacterial blight resistance gene Xa23 and rice blast resistance gene Pi9 at the seedling stage, a total of 116 plants; Genomic SNP molecular markers were used to detect the genetic background of the homozygous individual plants for the target gene, and 9 individual plants with the highest background recovery rate and the most similar agronomic traits to the restorer line R900 at maturity were selected, and the individual plants were harvested after selfing. 5884 BC2F3 seeds of 9 strains were obtained.

6. Select 20 seeds for each line, plant 9 lines of BC2F3 seeds, and use SNP molecular markers to identify single plants that are homozygous for bacterial blight resistance gene Xa23 and rice blast resistance target gene Pi9 at the seedling stage , and selected 3 lines with uniform population and similar comprehensive traits to the restorer line R900, and selected 1 typical individual plant for each line, harvested separately by selfing, and obtained 3016 BC2F4 seeds of the 3 lines.

7. Plant 3 strains of BC2F4 seeds, carry out bacterial blight inoculation and rice blast disease resistance identification in Hunan at the same time, identify high resistance to bacterial blight and rice blast, stable and consistent population, comprehensive traits and restorer lines One strain most similar to R900.

Specifically, identification of rice blast resistance: In rice areas where rice blast is severely damaged all year round, rice fields with high soil fertility and convenient irrigation and drainage are selected as disease nurseries, which are convenient for management and disease investigation. The amount of seeds to be tested and identified is 5-10g, each sowing row is 50cm long, 10cm wide, and the row spacing is 10cm. Three replicates are randomly set up, and the induced susceptible varieties are mixed and sown around the tested and identified varieties. When the seedlings grow to the stage of two leaves and one heart, collect the leaves infected by rice blast in other fields of the disease nursery, cut them into 2-3 cm, keep warm and moisturize for 24 hours, and sprinkle them on the induced varieties. Transplant at 30-35d seedling age, transplant 2 rows for each variety, 7 clumps in each row, 14 clumps in total for each variety; the row spacing between plants is 20cm; 1 copy of resistant variety is inserted between every 10 varieties. Protective rows were planted around the reference varieties, with the same row spacing as the reference varieties; susceptible varieties were used for the protection row varieties. Seedling blast was investigated at the 4-6 leaf stage before rice transplanting, and leaf blast was investigated at the tillering stage after rice transplanting. The 10 plants with the most severe disease of each variety were used as the investigation object, and the leaves with the most serious disease were investigated for each plant, and the average value of the 3 plants with the most serious disease was taken as the basis for variety evaluation. If the leaf blast level of the susceptible control seedlings does not reach level 4, the experiment of this group is invalid. Investigate panicle blast from rice heading to wax ripening stage. For each plot of identified variety, investigate the rice panicle with the most serious disease, no less than 100 ears, and count the incidence rate of panicle blast and the loss rate of single panicle blast. If the disease level of the susceptible control ear blast does not reach level 7, the experiment of this group is invalid.

Identification of resistance to bacterial blight: 20 days before heading of rice, representative strains of the dominant pathogenic type of bacterial blight of rice were selected. Cultivate the strain with Wakimoto's potato semi-synthetic medium at a constant temperature of 28°C for 72 hours, wash the bacterial lawn with sterile water, and dilute the bacterial suspension to 10 ⁸ to 10 ⁹ cells/ml by McNairney turbidimetry bacteria liquid. It is advisable to use the leaf-cut method to cut off the inoculum about 2 cm from the top of the fully developed leaves on the upper part of the rice plant in sunny or cloudy afternoons. 20 to 25 days after inoculation, the investigation will be carried out. Only when the disease level of the susceptible species reaches level 7 (sensitivity) and above can the current identification be considered valid.

Screening: According to the incidence of rice materials, the strains with high resistance to bacterial blight and rice blast were identified.

This line is the final improved line with two resistance genes of Pi9 and Xa23 introduced at the same time, and its agronomic traits are similar to the restorer line R900.

Similarly, using other breeding materials carrying bacterial blight resistance gene Xa21 or Xa7 and rice blast resistance gene Pi1 or Pi2 or Pigm can also obtain corresponding improved strains according to the above method.

The development method of SNP molecular markers for detecting the presence of bacterial blight resistance gene Xa23 and rice blast resistance target gene Pi9 is self-developed based on the reported DNA sequences of Xa23 and Pi9 resistance genes.

The method to detect whether a single rice plant carries the two resistance genes Xa23 and Pi9 is: use the 4 specific SNP markers developed by ourselves to detect the leaf DNA of the individual plant separately, if the 4 closely linked SNP markers can be detected simultaneously If it is a favorable (homozygous) genotype, the individual plant carries two resistance genes Xa23 and Pi9 at the same time.

The experimental technology such as SNP detection involved in the present invention is based on the principle of competitive allele PCR (Kompetitive AlleleSpecific PCR, KASP), and the reaction detection is carried out on the Array Tape genotyping platform. This method has been successively applied to molecular marker-assisted breeding, target traits Gene mapping, seed purity and authenticity identification, etc., have the advantages of low cost, high throughput and accurate fluorescence signal collection data. The supporting reagents and consumables were purchased from LGC, UK.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (10)

1. A method for improving rice blast resistance and bacterial blight resistance, characterized in that, comprising the following steps: ( ₁ ) The recurrent parent is crossed with the rice material carrying the bacterial blight resistance gene and the rice blast resistance gene to obtain hybrid F1 generation seeds; (2) Plant hybrid F ₁ generation seeds, use SNP molecular markers to exclude false hybrid plants, and backcross true hybrid F ₁ generation with recurrent parents to obtain BC ₁ F ₁ generation seeds; (3) Plant BC ₁ F ₁ generation seeds, use the SNP molecular markers closely linked to the bacterial blight resistance gene and the rice blast resistance gene to detect the prospects of the BC ₁ F ₁ generation population plants at the seedling stage, and select to carry bacterial blight The BC ₁ F ₁ generation individual plants with rice blast resistance and rice blast resistance genes, and then use the genome-wide SNP molecular markers to detect the genetic background of the plants containing the resistance genes, and select the individual plants with the highest background recovery rate to continuously regenerate with the recurrent parents. In each backcross generation, the single plant carrying bacterial blight resistance and rice blast resistance genes and the highest background recovery rate was selected for backcrossing with the recurrent parent. The number of backcrossing generations was m (m≧2), and the return Make offspring BC _m F _l ; (4) plant BCmF1 generation seeds, utilize SNP molecular marker detection, select to carry bacterial blight resistance gene and rice blast resistance gene and the BCmF1 generation single plant with the highest background recovery rate to carry out selfing, the number of selfing generations is n (n ≧2), to obtain BCmFn generation seeds with stable comprehensive agronomic traits; (5) Plant BCmFn generation seeds, detect with SNP molecular markers, select BCmFn generation single plants carrying bacterial blight resistance and rice blast resistance homozygous genes, and obtain bacterial blight and rice blast resistance improvement and integrated agronomic Improved material with traits similar to reincarnated parents. 2. The method for improving rice blast resistance and bacterial blight resistance according to claim 1, wherein the recurrent parent is a hybrid rice restorer line R900. 3. The method for improving rice blast resistance and bacterial blight resistance according to claim 1, wherein the bacterial blight resistance gene comprises at least one of Xa23, Xa21, and Xa7. 4. The method for improving rice blast resistance and bacterial blight resistance according to claim 1, wherein the rice blast resistance gene comprises at least one of Pi1, Pi2, Pi9, and Pigm. 5. the method for improving rice blast resistance and bacterial blight resistance according to claim 1, is characterized in that, in described step (3), step (4), step (5), by SNP molecular marker The selected single plant carries 1-3 genes for resistance to bacterial blight and 1-2 genes for resistance to rice blast. 6. The method for improving rice blast resistance and bacterial blight resistance according to claim 1, characterized in that, in the step (1), the bacterial blight resistance gene and the rice blast resistance gene are carried at the same time. The rice breeding materials with sex genes come from any one of indica rice, japonica rice, restorer line and maintainer line. 7. the method for improving paddy rice blast resistance and bacterial blight resistance according to claim 1, is characterized in that, described step (2), step (3), step (4), step (5) The purpose of backcrossing or selfing individual plants using SNP molecular markers to detect bacterial blight resistance genes and rice blast resistance genes is to ensure that the above resistance genes always exist in the improved offspring. 8. the method for improving rice blast resistance and bacterial blight resistance according to claim 1, is characterized in that, in described step (3), step (4), utilize SNP whole genome molecular marker to detect background Recovery rate, the purpose of continuous backcrossing of plants with the highest background recovery rate and the recurrent parent is to obtain improved materials with comprehensive agronomic characteristics similar to the recurrent parent more quickly. 9. the method for improving paddy rice blast resistance and bacterial blight resistance according to claim 1, is characterized in that, described step (2), step (3), step (4), step (5) Among them, SNP molecular markers for detecting bacterial blight resistance genes and rice blast resistance genes were developed by ourselves. 10. the method for improving paddy rice blast resistance and bacterial blight resistance according to claim 1, is characterized in that, in described step (3), step (4), detect the whole genome SNP of background recovery rate Molecular markers are self-developed.