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EST-STS marking primer 2 with differential rye1RS chromosome, screening method and use

A technology of EST-STS and labeled primers, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve problems such as inability to identify and distinguish, and broaden the scope of use and experimental conditions loose, binding effect

Inactive Publication Date: 2008-04-23
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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Problems solved by technology

However, these markers are amplified for the whole genome of rye and are not specific for 1R or 1RS chromosomes, so it is impossible to distinguish 1R chromosomes from 2R-7R chromosomes in rye
Koebner et al. synthesized the rye 1R chromosome-specific primer NOR-R1 based on the sequence difference between rye and wheat rRNA intergenic regions, which can identify the 1RS chromosome, but the primer NOR-R1 is used for recombinant materials that do not contain the nucleolus tissue region on the 1R chromosome. Will not be identified (reference: Koebner R.M.D. 1995. Generation of PCR-based markers for the detection of rye chromatin in wheat background. Theor ApplGenet, 90: 740-745)

Method used

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  • EST-STS marking primer 2 with differential rye1RS chromosome, screening method and use
  • EST-STS marking primer 2 with differential rye1RS chromosome, screening method and use
  • EST-STS marking primer 2 with differential rye1RS chromosome, screening method and use

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Embodiment 1

[0029] Embodiment 1, design and screening of primers

[0030] 1. Acquisition of EST sequence: According to the wheat expressed sequence tag (EST) database released by the United States Department of Agriculture website (http: / / wheat.pw.usda.gov / GG2 / index.shtml), search for the homology located in the first part of wheat ESTs and sequences of different physical segments of the group, select 35 EST sequences with strong conservation and base numbers greater than 300bp for download. Figure 1 is the base sequence of one of the EST-BE443401.

[0031] 2. Design and synthesis of primers: The EST sequences downloaded in step 1 were used to design primers using PrimerPremier 5.0 software, and a total of 35 pairs of primers were designed. The conditions for primer design are annealing temperature 50-60°C, primer length 18-22bp, GC% content (the ratio of Guanine and Cytosine to the total number of bases) is 40-60%, and the expected amplification product is 300-600bp . The designed pri...

Embodiment 2

[0039] Example 2. Detection application of the labeled primers of the present invention in 1RS translocation materials

[0040] Using the rye 1RS chromosome-specific marker primer STS screened in Example 1 WE3 , to amplify part of the progeny material of the cross between Xiaoyan 6 and "German white grain" rye (S.cereale L.cv german white), the reaction conditions and the amplification procedure are the same as step 4, and the amplified product is carried out after the amplification reaction Detection, the detection method is the same as step 4. As a result, among the 11 materials tested, 4 materials amplified specific bands with a size of 1680bp and 1750bp, which can be confirmed to contain the rye 1RS chromosome. The other 7 materials did not amplify the two corresponding specific bands, so it can be determined that they do not contain the rye 1RS chromosome, as shown in Figure 5 . This result was consistent with the identification result of genome in situ hybridization.

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Abstract

The present invention provides one kind of specific EST-STS marking primer-2 of rye 1RS chromosome. The marking primer-2 is designed with the EST sequence, BE443401, of the first partial wheat homologous group as the template, and has the sequences including STSWE3F of 5'-GCATCTGCCAACACTCTCAA-3' and STSWE3R of 5'-ACGGCAGCATGTAGAGACAA-3'. The primer STSWE3 has two amplified specific segments of 1680 bp and 1750 bp size separately located in rye 1RS chromosome. The marking primer-2 of the present invention expands the use range of wheat EST, provides one new tool for deep research of distant hybrid material for common wheat. The marking primer-2 may be applied in identifying rye 1RS chromosome simply and fast by means of common PCR technology.

Description

technical field [0001] The invention relates to a rye 1RS chromosome-specific EST-STS marker primer 2, a screening method and application, and belongs to the field of gene molecular marker research. Background technique [0002] Wheat is one of the most important food crops in the world. It is often threatened by various diseases during production, resulting in a decline in yield or quality. Rye, a close relative of wheat, has many excellent traits such as disease resistance, high yield, and barren tolerance. Studies have shown that there are many resistance genes such as powdery mildew resistance, stripe rust resistance, leaf rust resistance, stem rust resistance, and aphid resistance on the rye 1RS chromosome, as well as genes that improve yield and adaptability. These beneficial genes on rye 1RS chromosome can be introduced into common wheat through distant hybridization and chromosome manipulation techniques. [0003] After the rye 1RS chromosome or chromosome fragment...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 王春梅安调过高强
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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