CN107130019B

CN107130019B - Molecular marker primer of rice local variety thin rice panicle blast resistance gene and application thereof

Info

Publication number: CN107130019B
Application number: CN201710278281.0A
Authority: CN
Inventors: 鲍永美; 张红生; 方能炎; 黄骥; 王建飞; 王州飞; 程金平
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2017-04-25
Filing date: 2017-04-25
Publication date: 2020-07-17
Anticipated expiration: 2037-04-25
Also published as: CN107130019A

Abstract

The invention belongs to the field of crop molecular genetic breeding science, and discloses a molecular marker primer of a rice local variety thin rice panicle blast resistance gene and application thereof. The invention relates to a molecular marker of a rice thin rice panicle blast resistance gene Pb-bd1, wherein an upstream primer of a molecular marker BS23 is BS 23-F: SEQ ID NO.1, the downstream primer is BS 23-R: SEQ ID NO.2, the size of the amplification product is 114 bp; the upstream primer of the molecular marker RM7654 is RM 7654-F: SEQ ID NO.3, the downstream primer is RM 7654-R: SEQ ID NO.4, the amplification product size is 155 bp. The molecular marker of the rice panicle blast resistance gene Pb-bd1 provided by the invention can detect whether the gene is contained in thin rice and hybrid, backcross and backcross progeny of the thin rice, and can predict the resistance level of the thin rice to rice blast, greatly improve the selection efficiency of rice panicle blast resistant materials, and accelerate the disease-resistant breeding process.

Description

Molecular marker primer of rice local variety thin rice panicle blast resistance gene and application thereof

Technical Field

The invention belongs to the field of crop molecular genetic breeding science, and relates to a molecular marker primer of a rice local variety thin rice panicle blast resistance gene and application thereof.

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 considered 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 local japonica rice varieties in Taihu river basins to seedling plague in China, such as Li Pekui and the like (2007, genetic analysis and heredity of 4 local japonica rice varieties in Taihu river basins for rice blast resistance, 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 thin rice panicle blast resistance gene Pb-bd 1. By detecting the molecular marker which is closely linked with the disease-resistant gene Pb-bd1, the existence of the disease-resistant gene Pb-bd1 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 marker of the rice thin rice panicle blast resistance gene Pb-bd1 is selected from any one of BS23 and RM 7654; the upstream primer of the molecular marker BS23 is BS 23-F: SEQ ID NO.1, the downstream primer is BS 23-R: SEQ ID NO.2, the size of the amplification product is 114 bp; the upstream primer of the molecular marker RM7654 is RM 7654-F: SEQ ID NO.3, the downstream primer is RM 7654-R: SEQ ID NO.4, the amplification product size is 155 bp.

The molecular marker primer of the rice thin rice panicle blast resistance gene Pb-bd1 is characterized in that an upstream primer of a molecular marker BS23 is BS 23-F: SEQ ID NO.1, the downstream primer is BS 23-R: SEQ ID NO.2, the size of the amplification product is 114 bp; the upstream primer of the molecular marker RM7654 is RM 7654-F: SEQ ID NO.3, the downstream primer is RM 7654-R: SEQ ID NO.4, the amplification product size is 155 bp.

The molecular marker disclosed by the invention is applied to identification of the rice panicle blast resistance gene Pb-bd 1.

The molecular marker primer disclosed by the invention is applied to identification of the rice panicle blast resistance gene Pb-bd 1.

The molecular marker primer disclosed by the invention is applied to identifying rice blast resistance rice.

A method for rapidly identifying the existence of a rice panicle blast resistance gene Pb-bd1 comprises the following steps:

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

(2) any one pair or two pairs of the molecular marker primers are utilized to carry out PCR amplification on the rice sample genome DNA, the PCR amplification product is subjected to electrophoresis detection on 8% non-denaturing polyacrylamide gel, and if a molecular marker fragment with a corresponding size is amplified, the existence of the Pb-bd1 gene is marked.

The reaction system for PCR amplification is 10 × Mg-containing²⁺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, annealing at 60 ℃ for 40 seconds, extension at 72 ℃ for 40 seconds, and circulation for 30 times; final extension at 72 ℃ for 10 min.

Advantageous effects

The molecular marking method of the rice thin rice panicle blast resistance gene provided by the invention has the following advantages:

(1) the thin rice is a local variety of japonica rice in Taihu river basin and has the characteristic of high resistance to panicle blast, the main disease-resistant gene Pb-bd1 of the thin rice is a new panicle blast resistance gene, molecular markers BS23 and RM7654 which are closely linked with the new panicle blast resistance gene are obtained by screening, and a foundation is laid for molecular marker-assisted selective breeding and Pb-bd1 gene cloning. Any pair of molecular markers which are closely linked with the paddy rice thin rice panicle blast resistance gene Pb-bd1 are used for identifying the panicle blast resistance gene Pb-bd1, the selection efficiency is over 91 percent, and the selection efficiency of two pairs of molecular marker primers is 99.32 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-bd1 is low (less than or equal to 8.96 percent), the existence of the rice blast resistance gene Pb-bd1 can be determined by detecting the molecular markers which are closely linked with the disease resistance gene Pb-bd1, the rice blast resistance of rice plants can be predicted, and the disease-resistant breeding process is fast.

(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-bd1, sampling can be carried out in the seedling stage, DNA can be extracted, the single plant resistant to rice blast can be identified by using the marker, other plants can be 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 an 8% non-denaturing polyacrylamide gel electrophoresis diagram of a rice panicle blast resistance gene Pb-bd1 closely linked SSR marker BS 23. Wherein: m: a molecular weight Marker; b: thin rice; s: suyunuo; f: a heterozygote type; 1-5: a susceptible recombinant inbred line; 6-10: disease-resistant recombinant inbred line.

FIG. 2 shows an 8% non-denaturing polyacrylamide gel electrophoresis diagram of the rice panicle blast resistance gene Pb-bd1 closely linked SSR marker RM 7654.

Wherein: m: a molecular weight Marker; b: thin rice; s: suyunuo; f: a heterozygote type; 1-5: a susceptible recombinant inbred line; 6-10: disease-resistant recombinant inbred line.

Detailed Description

Example 1

Materials and methods:

1. lipefu and the like, F is obtained by hybridizing local rice varieties of rice blast resistant rice, namely thin rice (male parent) and susceptible rice varieties, namely Suyunuo (male parent)₁Selfing to obtain F₂The population was isolated for genetic analysis, and it was determined that the resistance of thin rice to the "north 1" strain was predominantly controlled by a single major gene. 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 212 lines_2:8And (4) recombining the inbred line population. The population is used to locate a major effective site related to resistance to panicle blast, the site is located between and closely linked with the 11 th chromosome markers BS23 and RM7654, the genetic distance is 2.7cM, and the major effective site provides resistance of not less than 51.56%.

2. The strain culture and inoculation identification method refers to plum Penfu et al (Chinese Rice science, 2007, 21: 579-584) and L iu et al (Tianjin Agr Sci, 2007, 13: 55-58).

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 thin rice and Suyunuo is used as a template, and rice SSR marker (http:// www.gramene.org) published on a Gramene website is 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.

PCR reaction System with a volume of 10. mu.l, 10 × buffer (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, SSR markers BS23 and RM7654 are closely linked with a disease-resistant gene Pb-bd1 (FIG. 1 and FIG. 2), and the amplification band sizes in parents are shown in Table 1. The selection efficiency calculation method among 212 family materials of the recombinant inbred line is as follows

19 interchange gametes appear between the SSR marker BS23 and the disease-resistant gene Pb-bd1, the recombination rate is only 8.96%, and the selection efficiency of the marker on resistance reaches 91.04%;

16 interchange gametes appear between the SSR marker RM7654 and the disease-resistant gene Pb-bd1, the recombination rate is only 7.55%, and the selection efficiency of the marker on resistance reaches 92.45%;

the selection efficiency of the double-marker screening of SSR markers BS23 and RM7654 is 1-8.96% by 7.55% to 99.32%;

the existence of the rice blast resistant gene Pb-bd1 can be efficiently identified by the 2 molecular markers, the single-marker selection efficiency reaches 91%, and the double-marker combination selection efficiency reaches 99.32%. 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 1.

<110> Nanjing university of agriculture

<120> molecular marker primer of local variety thin rice panicle blast resistance gene of rice and application thereof

<160>4

<210>1

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> rice blast resistance gene Pb-bd1 molecular marker BS23 upstream primer BS23-F of thin rice

<400>1

cgcgactaat ctagactct 19

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> rice blast resistance gene Pb-bd1 molecular marker BS23 downstream primer BS23-R of thin rice

<400>2

tggacactta tgcaattagc 20

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> rice blast resistance gene Pb-bd1 molecular marker RM7654 upstream primer RM7654-F of thin rice

<400>3

ctcatggttg tgtcgtggtc 20

<210>4

<211>219

<212>DNA

<213> Artificial sequence

<220>

<223> rice blast resistance gene Pb-bd1 molecular marker RM7654 downstream primer RM7654-R of thin rice

<400>4

gtgcagtgcc agtggtacg 19

Sequence listing

2

Claims

1. The molecular marker of the rice thin rice panicle blast resistance gene Pb-bd1 is characterized in that the molecular marker is selected from any one of BS23 and RM 7654; the upstream primer of the molecular marker BS23 is BS 23-F: SEQ ID NO.1, the downstream primer is BS 23-R: SEQ ID NO.2, the size of the amplification product is 114 bp; the upstream primer of the molecular marker RM7654 is RM 7654-F: SEQ ID NO.3, the downstream primer is RM 7654-R: SEQ ID NO.4, the amplification product size is 155 bp.

2. The molecular marker primer of the rice panicle blast resistance gene Pb-bd1 as claimed in claim 1, wherein the upstream primer of the molecular marker BS23 is BS 23-F: SEQ ID NO.1, the downstream primer is BS 23-R: SEQ ID NO.2, the size of the amplification product is 114 bp; the upstream primer of the molecular marker RM7654 is RM 7654-F: SEQ ID NO.3, the downstream primer is RM 7654-R: SEQ ID NO.4, the amplification product size is 155 bp.

3. The application of the molecular marker of claim 1 in identifying the rice panicle blast resistance gene Pb-bd 1.

4. The application of the molecular marker primer of claim 2 in identifying the rice panicle blast resistance gene Pb-bd 1.

5. The use of the molecular marker primer of claim 2 for identifying rice blast resistance rice.

6. A method for rapidly identifying the existence of a rice panicle blast resistance gene Pb-bd1 is characterized by comprising the following steps:

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

(2) the method comprises the steps of utilizing any one pair or two pairs of the molecular marker primers as claimed in claim 2 to carry out PCR amplification on the genomic DNA of the rice sample, carrying out electrophoresis detection on the PCR amplification product on 8% non-denaturing polyacrylamide gel, and if a molecular marker fragment with a corresponding size is amplified, marking the existence of the Pb-bd1 gene.

7. The method of claim 6, wherein the PCR amplification reaction system is 10 × containing Mg²⁺1.0 mul, 4 pmol/mul of the molecular marker primer pair, 0.1 mul of Taq enzyme of 2.5mM dNTPs 0.2 mul, 5U/mul, 1 mul of 10 ng/mul of rice sample genome template DNA, and adding water to 10 mul; the reaction procedure is as follows: DNA was pre-denatured at 94 ℃ for 5 minutes; denaturation at 94 ℃ for 40 seconds, annealing at 60 ℃ for 40 seconds, extension at 72 ℃ for 40 seconds, and circulation for 30 times; final extension at 72 ℃ for 10 min.