CN1294278C

CN1294278C - Molecular marker SA7 for detecting rice blast resistant gene Pi25(t)

Info

Publication number: CN1294278C
Application number: CNB2003101008892A
Authority: CN
Inventors: 庄杰云; 郑康乐; 吴建利; 樊叶扬
Original assignee: China National Rice Research Institute
Current assignee: China National Rice Research Institute
Priority date: 2003-10-15
Filing date: 2003-10-15
Publication date: 2007-01-10
Anticipated expiration: 2023-10-15
Also published as: CN1607256A

Abstract

The present invention provides a molecular marker SA7 for detecting the spike pestilence resistance gene Pi25 (t) of paddy rice. A nucleotide sequence of a specific PCR primer at the upstream end from the 5' end to the 3' end is CGGGTGAGTAAAACTTATCTGG; the nucleotide sequence at the downstream end from the 5' end to the 3' end is TAGTGATTGAAACGGGTGCACT. The molecular marker SA7 of the present invention can carry out marking identification to DNA extracted in the seedling stage of paddy rice, and detect whether the gene Pi25 (t) resisting leaf pestilence and spike pestilence exists or not, namely whether the gene Pi25(t) has the capability of resisting leaf pestilence and spike pestilence. The molecular marker SA7 has the advantages of high detection accuracy and simple operation. Because Gumei No. 2 is a universally applied parent of hybrid varieties of resisting rice pestilence, the molecular marker also detects whether the universal significance of resisting leaf pestilence and spike pestilence exists or not. The molecular marker can be used as a detection tool of breeding materials using the Gumei No. 2 as a parent.

Description

Detect the molecule marker SA7 of paddy rice rice blast resistant gene Pi25 (t)

Technical field

The present invention relates to the detection technique field in the rice breeding, particularly detect the molecule marker of paddy rice rice blast resistant gene.

Background technology

Rice blast is one of most important rice disease that extensively betides in each rice district, the world.Wherein the fringe pest are particularly serious to the influence of rice yield.Plantation anti-rice blast rice kind is the most economical effective means that address this problem.In the back cross breeding of transformation disease-resistant gene, must before heading, finish the evaluation of fringe pest resistance, conventional fringe pest are identified and must just can be carried out after heading, can't satisfy this condition.

Summary of the invention

Technical problem to be solved by this invention provides the molecule marker SA7 that detects paddy rice rice blast resistant gene Pi25 (t), raises the efficiency.For this reason, the present invention is by the following technical solutions: its specific PCR primer, upstream extremity is classified CGGGTGAGTAAAACTTATCTGG from 5 ' end as to the nucleotides sequence of 3 ' end, and downstream end is classified TAGTGATTGAAACGGGTGCACT as from 5 ' end to the nucleotides sequence of 3 ' end.

Generally, often will resist rice blast kind and high-yield variety to hybridize and obtain the anti-pest kind of high yield.Gumei2 is that China paddy rice pathologist screens from 38000 parts of seed rice resources unique semi-dwarf mutant material with lasting anti-pest (Peng Shaoqiu, Scientia Agricultura Sinica, 1996,29:52-58).The contriver is under study for action in 3 years repeated inoculations 156 and the Gumei2 recombinant inbred lines, analyzed the dna marker genotype of each strain system, a gene Pi 25 (t) that has leaf blast resistance and fringe pest concurrently is positioned the 6th karyomit(e) of paddy rice, and its resistance allele derives from Gumei2.

In process of the present invention, the contriver has designed specific PCR primer, and it can be to the DNA that rice seedling extracted, carry out mark and identify, detect the gene Pi 25 (t) that whether has leaf blast resistance and fringe pest, promptly whether have leaf blast resistance and fringe pest ability, the accuracy height of its detection, simple to operate.Because Gumei2 is the parent of the anti-rice blast Hybrid of widespread usage, therefore, this molecule marker also has the universal significance whether detection has leaf blast resistance and fringe pest, can be used as other with the testing tool of Gumei2 as parent's breeding material.

Description of drawings

Fig. 1 is the result that molecule marker SA7 centering 156/ Gumei2 RIL of the present invention detects.Among the figure, in the P1 representative 156; P2 represents Gumei2; Other materials is a RIL; R is anti-fringe pest; S is sense fringe pest.

Embodiment

In rice blast lesion, Fujian plantation is above-mentioned 156 and Gumei2 reorganization inbreeding population.Adopt molecule marker provided by the present invention to carry out leaf blast resistance and the detection of fringe pest in seedling stage.The specific PCR primer of this molecule marker, upstream extremity is classified CGGGTGAGTAAAACTTATCTGG from 5 ' end as to the nucleotides sequence of 3 ' end, and downstream end is classified TAGTGATTGAAACGGGTGCACT as from 5 ' end to the nucleotides sequence of 3 ' end.

One DNA extraction:

1 long blade tip of 2cm is cut in the 1.5-ml centrifuge tube, covers tight lid, puts on number, places on ice.

2 blade tips are cut into the long segment of 0.5cm, put into mortar.

3 addings, 400 μ l extracting solutions (Tris-Hcl 50mM, pH 8.0; EDTA 25mM, pH 8.0; NaCl 300mM; SDS 1%), grind.

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

5 add 400 μ l chloroforms, mixing, centrifugal 30 seconds of 12000rpm.

6 careful absorptions in the new 1.5-ml centrifuge tube of supernatant to are put on number.

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

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

9 usefulness, 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 is used for pcr amplification reaction.

Two pcr amplifications:

Pcr amplification reaction carries out on the pcr amplification instrument.

1 reaction system is as follows:

67mM Tris-Hcl, pH 8.8; 16mM (NH ₄) ₂SO ₄1.5mM MgCl; 0.01%TWEEN-20; 200 μ M dNTPs; 0.5 μ M upstream primer; 0.5 μ M downstream primer; 1 Taq of unit archaeal dna polymerase/10 μ l; 1 μ l DNA/10 μ l

2 temperature cycle conditions are as follows:

94 ℃ 1 minute; 94 ℃ 45 seconds, 42 ℃ 45 seconds, 72 ℃ 1 minute, 35 the circulation; 72 ℃ 8 minutes

Three PCR products detect:

With amplified production, be splined on 2.5% sepharose, electrophoresis to tetrabromophenol sulfonphthalein is run out of bottom line, uses gel images analytical system GDS-7600 (UVP) record result behind ethidium bromide staining.

Extract DNA in 236 strain systems altogether, carry out above-mentioned testing process, the result shows that wherein 121 have Gumei2 resistance allele (Fig. 1).

Above-mentioned 236 strains system is carried out after blast resisting identifies, finding has 114 to show as resistance in above-mentioned 121 strains system, and 7 show as susceptible.The goodness of fit of mark and phenotype is 94.2%.

If in conjunction with another molecule marker SK17, its specific PCR primer, upstream extremity is classified CAGCTGTGGACTCAGACCTCTC as from 5 ' end to the nucleotides sequence of 3 ' end, downstream end is classified GCGAACAAGCGATGAATCTATC from 5 ' end as to the nucleotides sequence of 3 ' end, and then the goodness of fit of mark and phenotype can be up to 98.1%.

Claims

1, detects the molecule marker SA7 of paddy rice rice blast resistant gene Pi25 (t), the specific PCR primer that it is characterized in that it, upstream extremity is classified CGGGTGAGTAAAACTTATCTGG from 5 ' end as to the nucleotides sequence of 3 ' end, and downstream end is classified TAGTGATTGAAACGGGTGCACT as from 5 ' end to the nucleotides sequence of 3 ' end.