CN106636335B

CN106636335B - Molecular marking method of rice panicle blast resistance gene

Info

Publication number: CN106636335B
Application number: CN201610882281.7A
Authority: CN
Inventors: 鲍永美; 张红生; 方能炎; 王建飞; 黄骥; 王州飞; 程金平
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2016-10-09
Filing date: 2016-10-09
Publication date: 2020-05-08
Anticipated expiration: 2036-10-09
Also published as: CN106636335A

Abstract

The invention belongs to the field of crop molecular genetic breeding science, and discloses a molecular marking method of a rice panicle blast resistance gene. The method comprises the following steps: (1) taking a rice sample, and extracting the genomic DNA of the rice sample; (2) using any pair of molecular marker primers of RM27187 and RM27381 to perform PCR amplification on the rice sample genome DNA, performing electrophoresis detection on the PCR amplification product, and if a molecular marker DNA fragment with a corresponding size is amplified, marking the existence of the Pb-hk1 gene. The molecular marker of the rice panicle blast resistance gene Pb-hk1 provided by the invention can detect whether the gene is contained in the rice black hull japonica rice and the filial generation, backcross and backcross generations of the rice black hull japonica rice, can predict the resistance level of the rice blast, greatly improve the selection efficiency of the rice panicle blast resistance material and accelerate the disease-resistant breeding process.

Description

Molecular marking method of rice panicle blast resistance gene

Technical Field

The invention belongs to the field of crop molecular genetic breeding science, and relates to a molecular marking method of a rice panicle blast resistance gene.

Background

The rice is one of the most important grain crops in China, and has important significance for guaranteeing the grain safety and the economic growth of China. The rice blast is the most serious disease in rice production in China, and has the characteristics of quick transmission, wide occurrence, serious harm and the like (Lingzhi speciality, 1989, physiological race research of rice blast germs in rice areas in northern China, Chinese agricultural science, 22(3): 7-13). Further exploring and utilizing the rice blast resistance gene resources in China, and cultivating and planting disease-resistant varieties are the most economic and effective way for controlling the occurrence of rice blast and reducing the yield loss of rice.

Until now, scientists of various countries have identified more than 100 rice blast resistance genes from rice, and 24 of them have been successfully cloned. These disease-resistant genes can be introduced or polymerized into modern varieties to breed high-resistance and broad-spectrum varieties. However, the traditional breeding method is time-consuming and labor-consuming, the phenotype identification is difficult, the breeding efficiency is low, and the polymerization of disease-resistant genes is more difficult because the disease-resistant genes are dominant and the episomal interaction often exists among the genes. The problem can be effectively solved by molecular marker-assisted breeding.

China Taihu river basin rice has a long history, is regarded as one of the origins of japonica rice, contains rich rice seed resources, and is reported to have the characteristics of broad spectrum and high resistance of black hull japonica rice of local japonica rice varieties in Taihu river basins to seedling plague in China, such as Li Pekui (2007, genetic analysis and heredity of rice blast resistance of 4 local japonica rice varieties in Taihu river basins, 2007 10 th). In our study, it also showed very high resistance to panicle blast. The panicle blast resistance gene is identified and cloned from the specific germplasm resources of China, so that the gene with independent intellectual property rights can be obtained, and the method has great significance for improving the rice blast resistance breeding level of rice in China, particularly rice blast resistance breeding of japonica rice.

Disclosure of Invention

The purpose of the invention is: provides a molecular marking method of a rice variety black-shell japonica panicled blast resistance gene Pb-hk 1. By detecting the molecular marker which is closely linked with the disease-resistant gene Pb-hk1, the existence of the disease-resistant gene Pb-hk1 can be determined, the rice blast resistance of rice plants can be predicted, and the breeding progress of new rice varieties with the rice blast resistance can be accelerated.

The purpose of the invention can be realized by the following technical scheme:

the molecular marking method of the rice black-shell japonica panicled blast resistance gene Pb-hk1 comprises the following operation steps:

(1) taking a rice sample, and extracting the genomic DNA of the rice sample;

(2) utilizing any one pair or two pairs of molecular marker primers in the table 1 which are closely linked with the rice sheath blight resistance gene Pb-hk1 to perform PCR amplification on the rice sample genome DNA, performing electrophoresis detection on PCR amplification products on 8% non-denaturing polyacrylamide gel, and if a molecular marker DNA fragment with a corresponding size is amplified, marking the existence of the Pb-hk1 gene:

TABLE 1

Wherein, the reaction system of PCR amplification is as follows: 10 Xbuffer (containing Mg)²⁺) Mu.l of 1.0. mu.l of 4 pmol/. mu.l of the molecular marker primer pair described in Table 1, 0.2. mu.l of 2.5mM dNTPs, 0.1. mu.l of 5U/. mu.l of Taq enzyme, 1. mu.l of 10 ng/. mu.l of rice sample genomic template DNA, and water to 10. mu.l; the reaction procedure is as follows: DNA was pre-denatured at 94 ℃ for 5 minutes; denaturation at 94 ℃ for 40 seconds, annealing at 55 ℃ for 40 seconds, extension at 72 ℃ for 40 minutes, and circulation for 35 times; final extension at 72 ℃ for 10 min.

The molecular marker primer pair of the rice black-shell japonica panicle blast resistance gene Pb-hk1 is selected from any one of the following two molecular marker primer pairs: RM 27187-F: SEQ ID NO.1/RM 27187-R: SEQ ID No. 2; RM27381-F SEQ ID NO.3/RM 27381-R: SEQ ID NO. 4.

The molecular marker primer pair disclosed by the invention is applied to identifying the rice blast resistant gene Pb-hk 1.

The molecular marker primer pair disclosed by the invention is applied to screening of rice blast resistance rice.

The molecular marker primer pair disclosed by the invention is applied to molecular breeding of rice blast resistance rice.

Advantageous effects

The molecular marking method of the rice black-hull japonica panicle blast resistance gene provided by the invention has the following advantages:

(1) the black hull japonica rice is a local variety of japonica rice in Taihu lake basin and has the characteristic of high resistance to panicle blast, the main disease-resistant gene Pb-hk1 of the black hull japonica rice is a new panicle blast resistance gene, and molecular markers RM27187 and RM27381 which are closely linked with the new panicle blast resistance gene are obtained by screening, so that a foundation is laid for molecular marker-assisted selective breeding and Pb-hk1 gene cloning. Any pair of molecular markers which are closely linked with the rice sheath blight resistance gene Pb-hk1 are used for identifying the rice sheath blast resistance gene Pb-hk1, the selection efficiency is over 93 percent, and the selection efficiency of two pairs of molecular marker primers is 99.78 percent.

(2) The gene locus positioned by the molecular marker of the invention is accurate and convenient to identify. Because the recombination rate of the markers and the panicle blast resistance gene Pb-hk1 is low (less than or equal to 6.79 percent), the existence of the rice blast resistance gene Pb-hk1(t) can be determined by detecting the molecular markers which are closely linked with the disease-resistant gene Pb-hk1, the rice blast resistance of rice plants can be predicted, and the disease-resistant breeding process is rapidly realized.

(3) The auxiliary breeding selection target is clear, and the cost is saved. In the traditional disease-resistant breeding method, the panicle blast resistance of the breeding material is subjected to phenotypic identification, the influence of the inoculation environment is large, and the reliability of the phenotypic identification result is low. Therefore, the breeding for disease resistance is not only time-consuming, but also difficult and high in cost. By detecting the panicle blast resistance gene Pb-hk1, sampling can be carried out in the seedling stage, DNA is extracted, the single plant resistant to rice blast can be identified by using the marker, other plants are eliminated, the production cost is saved, the breeding population scale is controlled, and the individual selection efficiency of the rice blast resistance is greatly improved.

Drawings

FIG. 1 is 8% non-denaturing polyacrylamide gel electrophoresis diagram of rice panicle blast resistance gene Pb-hk1 closely linked SSR marker RM 27187.

Wherein: m: a molecular weight Marker; h: black shell japonica; s: suyunuo; f: a heterozygote type; 1-5: disease-resistant recombinant inbred lines; 6-10: susceptible recombinant inbred line.

FIG. 2 is an 8% non-denaturing polyacrylamide gel electrophoresis diagram of the rice panicle blast resistance gene Pb-hk1 closely linked SSR marker RM 27381.

Detailed Description

Example 1

Materials and methods:

1. lipefu and the like, F is obtained by hybridizing local rice blast resistant rice variety Ningzi (male parent) with susceptible variety Suyunuo (male parent)₁Selfing to obtain F₂And separating the colony for genetic analysis, and determining that the resistance of the black hull japonica to the northern 1 bacterial line is controlled by the interaction of two pairs of inhibition genes. On the basis, the invention adopts a single-particle transmission method to construct F_2:8Recombination inbred line population, i.e. two parents crossing to obtain F₁Selfing to obtain F₂，F₂Selfing the single plant to generate lines, continuously selfing for 6 generations, and finally constructing F including 162 lines_2:8And (4) recombining the inbred line population. The colony is used for positioning a major effective site related to the resistance to the panicle blast, the site is positioned between the 11 th chromosome markers RM27187 and RM27381, and is closely linked with the two markers, and the genetic distance is 6.34cM., so that the resistance is not less than 34.00 percent.

2. The strain culture and inoculation identification method refers to plum culture and the like (Chinese rice science, 2007, 21: 579-584).

The DNA extraction method comprises the following steps: DNA of each individual plant of the isolated population was extracted by CTAB method.

4. Determination of closely linked molecular markers:

(1) and (3) screening marker polymorphism: the DNA of parent black-hull japonica rice and Suyunuo rice is used as a template, and rice SSR markers (http:// www.gramene.org) published on a Gramene website are used for carrying out polymorphism analysis through PCR reaction.

(2) And (3) screening the disease resistance pools of the polymorphic markers, namely randomly selecting 5 family materials (disease-resistant recombinant inbred lines) inoculated with the markers and identified as disease-resistant phenotypes and 5 family materials (disease-sensitive recombinant inbred lines) inoculated with the markers and identified as disease-sensitive phenotypes from the recombinant inbred line population, extracting DNA, mixing the DNA to form a disease resistance pool and a disease resistance pool, screening the polymorphic markers to analyze the relationship between the polymorphic markers and the disease resistance, and if the electrophoresis result of the disease resistance pool of a certain marker is consistent with that of a disease-resistant parent and the electrophoresis result of the disease resistance pool is consistent with that of a disease-sensitive parent, indicating that the markers are possibly closely linked with the disease-resistant genes.

(3) Verification of closely linked molecular markers: the molecular markers which are possibly closely linked with the disease-resistant genes are verified in 5 family materials forming the resistant pool respectively, if the linkage relation exists indeed, verification is carried out in all the family materials, the recombination frequency between the markers and the genes is analyzed according to the linked polymorphic markers and the corresponding resistant phenotypes, and the selection efficiency of the marker resistance is calculated.

5, PCR reaction system: the volume was 10. mu.l, 10 Xbuffer (containing Mg)²⁺) 1.0. mu.l, 1. mu.l of molecular marker primer pair (4 pmol/. mu.l), 0.2. mu.l of 2.5mM dNTPs, 0.1. mu.l of Taq enzyme (5U/. mu.l), 1. mu.l of template DNA (10 ng/. mu.l), and water to 10. mu.l. The reaction procedure was that after 5 minutes of pre-denaturation at 94 ℃ of DNA, 30 cycles of (denaturation at 94 ℃ for 40 seconds, annealing at 55 ℃ for 40 seconds, and extension at 72 ℃ for 40 minutes) and final extension at 72 ℃ for 10 minutes. PCR amplification was performed on a biometre amplifier, and the amplified products were electrophoretically separated on 8% non-denaturing polyacrylamide gel (100ml of polyacrylamide solution containing 7.6 g of acrylamide and 0.4 g of methylenebisacrylamide), followed by phase contrast on a UV transilluminator and recording of the results.

(II) results and analysis:

as a result of research, the SSR markers RM27187 and RM28381 are closely linked with the disease-resistant gene Pb-hk1 (FIG. 1 and FIG. 2), and the amplification band sizes in the parents are shown in Table 2. The selection efficiency calculation method among the 162 family materials of the recombinant inbred line is as follows

11 interchange gametes appear between the SSR marker RM27187 and the disease-resistant gene Pb-hk1, the recombination rate is only 6.79 percent, and the selection efficiency of the marker to resistance reaches 93.21 percent;

7 interchange gametes appear between the SSR marker RM27381 and the disease-resistant gene Pb-hk1, the recombination rate is only 4.32%, and the selection efficiency of the marker to resistance reaches 95.68%;

the selection efficiency of the double-marker screening of the SSR markers RM27187 and RM27381 is 1-6.79% by 4.32% ═ 99.78%;

the existence of the rice blast resistant gene Pb-hk1 can be efficiently identified by the 2 molecular markers, the single-marker selection efficiency reaches 93 percent, and the double-marker combination selection efficiency reaches 99.78 percent. Can be used for molecular marker-assisted selection to effectively improve the breeding process of rice disease-resistant varieties and control the scale of breeding groups.

Table 2.

Claims

1. The molecular marking method of the rice black-shell japonica panicled blast resistance gene Pb-hk1 is characterized by comprising the following steps:

(1) taking a rice sample, and extracting the genomic DNA of the rice sample;

(2) performing PCR amplification on the genome DNA of the rice sample by using any one or two pairs of molecular marker primers in the table 1, which are closely linked with the rice variety Perilla frutescens paniculata resistance gene Pb-hk1, performing electrophoresis detection on a PCR amplification product on 8% non-denatured polyacrylamide gel, and marking the existence of the Pb-hk1 gene if a molecular marker fragment with a corresponding size is amplified;

TABLE 1

2. The molecular marking method of the rice sheath blight resistance gene Pb-hk1 as claimed in claim 1, wherein the reaction system of PCR amplification is as follows: 10 x containing Mg²⁺1.0. mu.l of the buffer solution of (1), 4 pmol/. mu.l of the molecular marker primer pair, 0.2. mu.l of 2.5mM dNTPs, 0.1. mu.l of 5U/. mu.l of Taq enzyme, 1. mu.l of 10 ng/. mu.l of rice sample genomic template DNA, and adding water to 10. mu.l; the reaction procedure is as follows: DNA was pre-denatured at 94 ℃ for 5 minutes; denaturation at 94 ℃ for 40 seconds, denaturation at 60 ℃ for 40 seconds, and denaturation at 72 ℃ for 40 secondsStretching at the temperature for 40 seconds and circulating for 30 times; final extension at 72 ℃ for 10 min.

3. The molecular marker primer pair of the rice black-hull japonica panicle blast resistance gene Pb-hk1 is characterized by being selected from any one of the following two molecular marker primer pairs: RM 27187-F: SEQ ID NO.1/RM 27187-R: SEQ ID No. 2; RM27381-F SEQ ID NO.3/RM 27381-R: SEQ ID NO. 4.

4. The use of the pair of molecular marker primers of claim 3 for identifying the rice blast resistance gene Pb-hk 1.

5. The use of the pair of molecular marker primers of claim 3 in screening rice blast resistance rice.

6. The use of the pair of molecular marker primers of claim 3 in molecular breeding of rice blast resistance rice.