Molecular marker closely linked with rice blast Pi9 gene and application thereof

A rice blast, gene technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of difficulty in selecting the Pi9 gene, no polymorphism, etc.

Inactive Publication Date: 2015-04-08
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently used marker pB8 closely locked with the Pi9 gene has no polymorphism between the donor parent 75-1-127 and H02, Xieqingzao B and other parents, which brings great difficulties to the selection of the Pi9 gene by means of molecular markers. difficulty

Method used

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  • Molecular marker closely linked with rice blast Pi9 gene and application thereof
  • Molecular marker closely linked with rice blast Pi9 gene and application thereof
  • Molecular marker closely linked with rice blast Pi9 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Development of Molecular Markers Tightly Locked to Pi9 Gene

[0056] 1. Experimental method

[0057] 1.1 Genomic DNA extraction from rice by CTAB method

[0058] Cut Pi9 gene donor 75-1-127, Pi9 gene transgenic offspring N632S and N779S, other materials 93-11, Nipponbare, f232, f251, f254, 1892S materials young leaves 100-200mg, transfer to 2.0mL centrifuge tube , add liquid nitrogen to the centrifuge tube and use a glass rod to fully grind to powder, then add 700 μL of 65 °C preheated DNA extraction solution (81.7g NaCl and 20g CTAB fully dissolved in an appropriate amount of water, then add 1mol / L Tris-HCl 100mL , 0.5mol / L EDTA 40mL, dilute to 1000mL, store at 4°C), incubate at 65°C for 1 hour, mix by inverting every 15 minutes; add an equal volume of chloroform / isoamyl alcohol, mix gently, and let stand at room temperature for 15 minutes; Centrifuge at 12,000rpm for 15min, transfer the supernatant to another 1.5mL centrifuge tube, add an equal volume of i...

experiment example 1

[0106] Experimental Example 1 Using Pi9-AflIIF / Pi9-AflIIR markers to distinguish Pi9 gene heterozygotes

[0107] 1. Experimental method

[0108] Utilize PCR detection primer Pi9-AflIIF / Pi9-AflIIR (nucleotide sequence is shown in SEQ ID No.11 and SEQ ID No.12) to amplify rice variety Guangzhan 63S (do not contain Pi9 gene two-line male sterile line), 93-11 (without Pi9 gene restorer line), N632S (two-line sterile line with Pi9 gene), SE2 (with Pi9 gene restorer line); Guangzhan 63S / SE2F1 generation hybrid (Pi9 gene heterozygote); N632S / Genome of 93-11F1 generation hybrid (Pi9 gene heterozygote). The amplified PCR product was digested with AflII endonuclease (see Example 1 for the reaction system and procedure). Digested products were separated by 4% agarose electrophoresis.

[0109] 2. Experimental results

[0110] see results Figure 5 . Amplified by primers Pi9-AflIIF / Pi9-AflIIR and digested with AflII, only the 447BP band was found in the restriction bands of rice cul...

experiment example 2

[0112] Experimental Example 2 Assisted Breeding of Pi9 Gene Using Markers Pi9-AflIIF / Pi9-AflIIR

[0113] 1. Experimental method

[0114] 1.1 Breeding of Pi9-containing gene material

[0115] For the selection process of Pi9-containing gene material, see Figure 6 .

[0116] 1.2 Rice genomic DNA extraction

[0117] Rice materials used: Guangzhan 63S male sterile line is a single plant of the BC2F4 generation population selected by the female parent and rice blast resistance material 75-1-127.

[0118] Among them, Guangzhan 63S male sterile line does not contain Pi9 gene, which is the offspring of N422S / Guangzhan 63 hybrid; 75-1-127 is the donor material containing Pi9 gene.

[0119] Cut out 100-200 mg of rice young leaves at the adult plant stage, and extract genomic DNA by CTAB method.

[0120] 1.3 PCR amplification and digestion of rice genomic DNA

[0121] Use the primers Pi9-AflIIF / Pi9-AflIIR to amplify the genomic DNA of 75-1-127, widely occupied 63S and BC2F4 popula...

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Abstract

The invention discloses a molecular marker closely linked with rice blast Pi9 gene and an application thereof and belongs to the field of molecular marker-assisted breeding. The invention discloses primer pairs for detecting the marker closely linked with rice blast Pi9 gene and a method for detecting whether a rice variety or a plant line contains Pi9 gene or not by virtue of the primer pairs. The method comprises the following steps: (1) extracting genomic DNA of a to-be-detected rice sample; (2) carrying out PCR amplification through the primer pairs; (3) carrying out enzyme digestion on the PCR amplification product obtained in the step (2) with an endonuclease and carrying out electrophoresis; and (4) determining whether the to-be-detected rice sample contains the Pi9 gene or not according to the enzyme digestion results. By the method, whether the to-be-detected rice sample contains the Pi9 gene or not and the contained Pi9 gene is heterozygous or homozygous can be accurately determined and the purity of an F1 hybrid seed of one of the parents containing the Pi9 gene can also be identified and the method has application prospects in Pi9 gene-assisted breeding.

Description

technical field [0001] The invention relates to a molecular marker, in particular to a molecular marker closely linked with rice blast Pi9 gene, and belongs to the field of molecular marker assisted breeding. Background technique [0002] Rice (Oryaa sativa L.) is an important food crop in China, with an annual cultivation area of ​​more than 30 million hectares, and its output ranks first among food crops. Rice blast is one of the major diseases that seriously endanger rice production and often occurs in rice-growing areas. Rice blast is a fungal disease caused by Pyricularia Oryzae Cav. It occurs in rice planting areas all over the country. In popular years, the yield is generally reduced by 10% to 20%, and the serious one is more than 40% to 50% (Ou, 1985). Discovering new sources of resistance and breeding resistant varieties are still the most economical and effective ways to prevent rice blast. [0003] So far, more than 70 rice blast resistance genes have been ident...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 倪大虎杨剑波宋丰顺倪金龙李莉
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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