CN101643790B

CN101643790B - Specific molecular marker of rice blast resistant gene Pi 25 for rice and special primer thereof

Info

Publication number: CN101643790B
Application number: CN2009101526174A
Authority: CN
Inventors: 陈洁; 吴建利; 庄杰云; 施勇烽
Original assignee: China National Rice Research Institute
Current assignee: China National Rice Research Institute
Priority date: 2009-09-07
Filing date: 2009-09-07
Publication date: 2012-03-07
Anticipated expiration: 2029-09-07
Also published as: CN101643790A

Abstract

The invention discloses a molecular marker P25-1 of rice blast resistant gene Pi 25 which is a nucleotide sequence amplified from SEQ ID NO:1 and SEQ ID NO:2 in the total DNA of rice by primer and applied to the Pi 25 gene molecular marker assisted selection in the rice blast resistant breeding to improve the efficiency of molecular marker assisted selection and culture of new species. The invention further discloses a primer obtaining P25-1 to SEQ ID NO:1 and SEQ ID NO:2, the nucleotide sequence of SEQ ID NO:1 is GGACAGAGCAGGAACTTCAGATG and the nucleotide sequence of SEQ ID NO:2 is TTCTGGGTCTCAGGCGTACT. The primer is capable of identifying the marker of DNA extracted during the seedling stage of rice to detect whether the rice blast resistant gene Pi 25 is existent with high detection accuracy.

Description

The specific molecular marker of rice blast resistant gene Pi 25 and primer special thereof

Technical field

The invention belongs to the biology field of identifying the paddy disease-resistant gene, particularly detect the molecule marker and the primer special thereof of rice blast resistant gene.

Background technology

Paddy rice is one of most important food crop in the world, also is the first food crop of China.Rice blast is by ascomycetes Pyricularia grisea (asexual generation synonymous Pyriculariaoryzae Cav.; What sexual generation teleomorph Magnaporthe oryzae) cause extensively occurs in one of each topmost rice disease in rice district of the world.According to statistics, from 1975 to nineteen ninety 16 in the period of, the global grain loss that is caused by rice blast is up to 1.57 hundred million tons, nearly every year on average 10000000 tons; Since the nineties in 20th century, China's rice blast year onset area is all more than 3,000,000 hectares, and year is lost the rice yield few hundred thousand tonnes of.

At present, adopt chemical prevention and plantation disease-resistant variety to control this disease usually.Chemical prevention was not only expensive but also lack permanently effective sterilant, also because contaminate environment constitutes the potential threat with pesticide residue to human health.The application of blast resisting kind is most economical effective prophylactic measures, but because the variety of rice blast microspecies heredity and pathogenic quick development, the new rice variety that carries monoclonal antibody property key-gene is often in promoting the back once to the several years, losing its resistance against diseases.It is generally acknowledged that multiple gene polymerization helps the rice varieties that seed selection has lasting anti-pest property.In the evaluation of variety resources of rice at home and abroad, evaluation and the utilization research; Find that also the only a few rice material all shows the good resistance to rice blast for a long time under artificial inoculation and natural evaluation condition, this excavation and the utilization that is referred to as lasting anti-pest property paddy rice resource more and more obtains paying attention to.Classical genetic analysis and assignment of genes gene mapping result of study show; The rice blast resistance of lasting anti-pest property rice material is generally by a plurality of major gene controls; And minor gene also has certain effect to its resistance, is difficult to directly apply to breeding because of economical character is bad but forefathers study resistance resources such as applied Moroberekan and Tetep.

Gumei2 is for there being the rice varieties of wide spectrum and stable resistance to domestic and international rice blast microspecies; Be the unique a semi-short-stalked long-grained nonglutinous rice that national rice blast research coorporative network screens from 38000 parts of paddy rice resources, have excellent agronomic characters and resistance breeding utility value widely.Utilize 156 preparing hybrids combination in Gumei2 and the susceptible variety; Adopt single seed descent to make up 304 RILs that individual plant is formed; Utilize this recombinant inbred lines that deep research has been carried out in the rice blast resistance heredity of Gumei2; Identify and located Pi 24 that Pi 25, Pi 26 and Pi b ^GmFour rice blast resistance key-genes and more than ten QTL.Find that Gumei2 is stablized and persistent anti-pest property is by a plurality of key-genes and the coefficient result of minor gene, work by different key-genes and minor gene, in particular cases, exist mutually between the minor gene and do to different rice blast microspecies.

Pi 25 is genes that seedling leaf pest and fringe pest are held concurrently and resisted, and is positioned on paddy rice the 6th karyomit(e).Be this major gene of Fine Mapping; Therefrom selecting two strains in the 156/ Gumei2 recombinant inbred lines and be G19 (disease-resistant) and G153 (susceptible) has made up a new RIL (containing 1487 strains systems) Pi 25 has been carried out Fine Mapping; Utilize figure position method and information biology method, Pi 25 is positioned between A7 and the RG456 the most at last, and confirms that the gene of a NB-ARC of coding is a candidate gene; Utilize agrobacterium mediation method; It is fine to change candidate gene over to susceptible japonica rice variety Japan, finds that 61% transfer-gen plant has rice blast resistance, explains that this candidate gene is major gene Pi 25.Because Gumei2 is the semi-dwarf mutant rice variety of having improved, and has wide spectrum and stable resistance, meaning is very special, and the blast resistant gene Pi 25 that it clones can directly use in the rice blast resistance breeding.

Conventional blast resisting breeding need be carried out resistance and identifies, arrange in design inoculation condition, season etc., waste time and energy, and the result is not very reliable that most breeding work persons do not possess the ability of own arrangement.And through modern dna molecular marker assisted Selection means, the man laboratory of most of breedings all satisfies the requirements, and carries out the genotype identification of critical material.Therefore develop Pi 25 gene specific molecule markers; Cost is low, method of use is simply effective; Can improve breeding man and transfer to recovery system to resistant gene; Speed in maintenance line or the conventional variety can reduce the breeding cost, shortens the breed of variety cycle, improves the gene utilising efficiency and create higher social economic effect.

Summary of the invention

The purpose of this invention is to provide the molecule marker P25-1 that detects rice blast resistant gene Pi 25, with the efficient of raising molecular marker assisted selection and the efficient of rearing new variety.

The molecule marker of rice blast resistant gene Pi 25, it is the nucleotide sequence that SEQ ID NO:1 and SEQID NO:2 is increased and from rice total dna with primer.

The primer of the molecule marker of the said rice blast resistant gene Pi 25 of acquisition claim 1 is to SEQ IDNO:1 and SEQ ID NO:2; SEQ ID NO:1 nucleotides sequence is classified GGACAGAGCAGGAACTTCAGATG as, and the nucleotides sequence of SEQ ID NO:2 is classified TTCTGGGTCTCAGGCGTACT as.

Specific PCR primer centering of the present invention; SEQ ID NO:1 is a upstream extremity; Classify GGACAGAGCAGGAACTTCAGATG from 5 ' end as to the nucleotides sequence of 3 ' end, SEQ ID NO:2 is a downstream end, classifies TTCTGGGTCTCAGGCGTACT as from 5 ' end to the nucleotides sequence of 3 ' end.This primer to carrying out mark to the DNA that rice seedling extracted is identified detect whether have blast resistant gene Pi 25, and the accuracy height of detection is simple to operate.Because above-mentioned primer designs being based on inner sense SNP (SNP) difference that resists of gene, in theory, rate of accuracy reached to 100%.Therefore, this molecule marker can be used as the application of Pi 25 gene molecule marker assisted Selection in the rice blast resistance breeding.

Description of drawings

Fig. 1 is molecule marker P25-1 amplified production gel electrophoresis figure between allelotrope;

Wherein, M:100bp molecular weight marker (Toyobo, Lot No:53240A3), 200ng;

1: Gumei2; 2: in 156; 3:G19; 4:G153; 5: Japan is fine; 6: in spend 11; The R representative is disease-resistant, and the S representative is susceptible.(the amplified production clip size is about 776bp.)

Fig. 2 is the amplified production gel electrophoresis figure of molecule marker P25-1 at the fine transfer-gen plant of Japan;

Wherein, M:100bp molecular weight marker (Toyobo, Lot No:53240A3), 200ng;

1: Gumei2; 2: in 156; 3:G19; 4:G153; 5: Japan is fine; 6: in spend 11; 7-25 represent respectively 19 fine with Japan be the transgenic individual plant of background.The R representative is disease-resistant, and the S representative is susceptible.

Fig. 3 is molecule marker P25-1 amplified production gel electrophoresis figure in the RIL individual plant;

Wherein, M:100bp molecular weight marker (Toyobo, Lot No:53240A3), 200ng;

1-21 representes the individual plant of 21 recombinant inbred lines respectively.

Fig. 4 be molecule marker P25-1 in spend the amplified production gel electrophoresis figure of 11 transfer-gen plants.

Wherein, M:100bp molecular weight marker (Toyobo, Lot No:53240A3), 200ng;

1: Gumei2; 2: in spend 11; 3-21 representes the individual plant of 19 transfer-gen plants respectively.

Embodiment

The concrete application of molecule marker P25-1 of the present invention comprises pure laboratory technique part and to the transgenic line that contains Pi 25 genes and the detection of breeding population rice material.Its application is not limited to following instance.

DNA extraction, pcr amplification and electrophoretic analysis when embodiment 1:P25-1 detects:

One, DNA extraction:

1. cut the long blade tip of 2cm in the 1.5-ml centrifuge tube, cover tight lid, put on number, place on ice.

2. be cut into the long segment of 0.5cm to blade tip, put into mortar.

3. add 400 μ, 1 extracting solution (Tris-Hcl 50mM, pH 8.0; EDTA 25mM, pH 8.0; NaCl300mM; SDS 1%), grind.

4. add 400 μ l extracting solutions again, mixing is drawn 400 μ l in original 1.5-ml centrifuge tube.

5. add 400 μ l chloroforms, mixing, centrifugal 1 minute of 12000rpm.

6. carefully draw in the new 1.5-ml centrifuge tube of supernatant to, put on number.

7. add 800 μ l absolute ethyl alcohols, mixing, centrifugal 3 minutes of 12000rpm abandons supernatant.

8. precipitate with after 75% washing with alcohol natural air drying.

With 50 μ lTE (Tris-Hcl 10mM, pH 8.0; EDTA 1mM, pH 8.0) dissolution precipitation.Be stored in-20 ℃.

10. get 1 μ l and be used for pcr amplification reaction.

Two, pcr amplification:

Pcr amplification reaction carries out on the pcr amplification appearance.

1. reaction system is following:

1 μ l, 10 * PCR damping fluid (ancient cooking vessel state DH332-1)/10 μ l; 0.5 μ M SEQ ID NO:1 upstream primer/10 μ l; 0.5 μ M SEQ ID NO:2 downstream primer/10 μ l; 0.5 the Taq of unit archaeal dna polymerase (ancient cooking vessel state DH332-1)/10 μ l; 1 μ l DNA/10 μ l.

2. the temperature cycle condition is following:

Reaction conditions: 94 ℃ 2 minutes; 94 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 45 minutes, 30 circulations; 72 ℃ 5 minutes; 4 ℃ 10 minutes.

Three, the PCR product detects:

Connect electrode, electrophoresis under the 100V constant voltage, when tetrabromophenol sulfonphthalein migrates to enough DNA isolation fragments (60min), powered-down.

Take off gel, with 2 * Gel redTM (Biotium, cat:41003-0.5ml) dyeing is after 30 minutes, (Bio-red, Cat:76S/08184) the record result is as shown in Figure 1 to use gel images analytical system Bio-red Universal Hood II.

Embodiment 2: the P25-1 marker detection of changeing the fine plant of Pi 25 genes Japan

Altogether 25 fine plant of transgenic Japan are extracted DNA, carry out 1 described testing process like embodiment.The result shows in 25 materials that totally 16 amplify Gumei2 allelotrope banding pattern; And rice blast inoculation experiments result shows, in these 16 materials, 15 individuals show rice blast toxicity microspecies resistance, have only 1 to show as susceptible (as shown in Figure 2).The goodness of fit of mark and phenotype is 93.7%.

Embodiment 3: from the detection of RIL Pi 25 genes of middle mirror 100/ Gumei2

195 strains systems to from the RIL (F8) of middle mirror 100/ Gumei2 extract DNA, carry out above-mentioned testing process.In 195 plant that detect, 86 banding patterns that Gumei2 occurs are arranged, with theoretical value 97.5 differences not significantly (P＜0.05) (as shown in Figure 3).

Embodiment 4: change in Pi 25 genes and spend 11 genotype detection.

Centering spends 11 to change 82 individual plants extractions of Pi 25 genes DNA, carries out above-mentioned testing process.In 82 plant that detect, 56 banding patterns that Gumei2 occurs are arranged, (as shown in Figure 4).

Sequence table

< 110>China Paddy Rice Inst

< 120>specific molecular marker of rice blast resistant gene Pi 25 for rice and primer special thereof

<130>

<160>2

<170>PatentIn?version?3.3

<210>1

<211>23

<212>DNA

< 213>artificial sequence

<400>1

ggacagagca?ggaacttcag?atg 23

<210>2

<211>20

<212>DNA

< 213>artificial sequence

<400>2

ttctgggtct?caggcgtact 20

Claims

1. the primer of a molecule marker that obtains rice blast resistant gene Pi 25 is to SEQ ID NO:1 and SEQ ID NO:2; It is characterized in that: SEQ ID NO:1 nucleotides sequence is classified GGACAGAGCAGGAACTTCAGATG as, and the nucleotides sequence of SEQ ID NO:2 is classified TTCTGGGTCTCAGGCGTACT as.