Method for authenticating rice indica subspecies and special primer composition thereof

A primer composition and rice technology, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of difficult repeatability, high DNA purity, low specificity, etc., and achieve accurate and reliable results. Good resolution and high sensitivity

Inactive Publication Date: 2011-01-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, most of the classification standards used in traditional classification methods are quantitative traits, which require not only experienced appraisers with accurate observation ability, but also the correct choice of appraisal period, and the use of standards is difficult to grasp
RFLP method: Southern hybridization is required, the operation is complicated and the workload is heavy; single-copy probes are generally used, the number of markers is relatively small, and the polymorphism is low; the amount of DNA is large, and relatively complete DNA is required; enzyme digestion reaction requires DNA higher purity
RAPD method: the template DNA is very sensitive to the tem

Method used

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  • Method for authenticating rice indica subspecies and special primer composition thereof
  • Method for authenticating rice indica subspecies and special primer composition thereof
  • Method for authenticating rice indica subspecies and special primer composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0036] Example 1, 3 pairs of SSR primers

[0037] Primer pair RM130:

[0038] Upstream primer: 5'-TGTTGCTTGCCCTCACGCGAAG-3' (sequence 1 in the sequence list);

[0039] Downstream primer: 5'-GTCGCGTGCTTGGTTTGGTTC-3' (sequence 2 in the sequence listing).

[0040] The size of the target sequence: 84bp or 90bp.

[0041] Primer pair RM7009:

[0042] Upstream primer: 5'-GGGATTTATTGGTCGGACTG-3' (sequence 3 in the sequence list);

[0043] Downstream primer: 5'-GTAAGGCGGCACAAAGAATC-3' (sequence 4 in the sequence listing).

[0044] The size of the target sequence: 96bp or 99bp.

[0045] Primer pair RM7337:

[0046] Upstream primer: 5'-TTCTTCCCAGTTGGGTTGAC-3' (sequence 5 in the sequence listing);

[0047] Downstream primer: 5'-CATCTTGTTGATGGTGGTGG-3' (sequence 6 in the sequence listing).

[0048] The size of the target sequence: 122bp or 134bp.

Example Embodiment

[0049] Example 2. Using the primer composition of Example 1 to identify subspecies of rice indica and japonica

[0050] 1. CTAB method to extract genomic DNA of rice

[0051] The total DNA was extracted from each rice sample using the following methods:

[0052] 1. Weigh 0.4g-0.5g fresh leaves, add liquid nitrogen in a mortar and quickly grind and crush them, then transfer them to a 1.5ml sterile centrifuge tube.

[0053] 2. Add 700μl of CTAB extraction buffer (pH 8.0) preheated at 65°C to each tube, in a water bath at 65°C for 30 minutes, and shake it every 5 minutes.

[0054] 3. Add 400 μl chloroform / isoamyl alcohol (24:1) and gently mix for 10 minutes, and then centrifuge at 12,000 rpm for 10 minutes.

[0055] 4. Transfer the supernatant to another 1.5ml sterile centrifuge tube, add 300μl chloroform / isoamyl alcohol (24:1) and gently mix for 10 minutes, and then centrifuge at 12,000 rpm for 10 minutes;

[0056] 5. Transfer the supernatant to a new 1.5ml sterile centrifuge tube again, ad...

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Abstract

The invention discloses a method for authenticating rice indica subspecies and a special primer composition thereof. The primer composition provided by the invention comprises a primer pair RM130 which is formed by DNA shown as a sequence 1 of a sequence table and DNA shown as a sequence 2 of the sequence table, a primer pair RM7009 which is formed by DNA shown as a sequence 3 of the sequence table and DNA shown as a sequence 4 of the sequence table and a primer pair RM7337 which is formed by DNA shown as a sequence 5 of the sequence table and DNA shown as a sequence 6 of the sequence table. The primer composition can be used for assisting to authenticate the rice indica subspecies, has the advantages of high speed, high efficiency, high sensitivity, high resolution, accurate and reliableresult, and the like and is suitable for application in indica attribute authentication, rice breeding, plant evolution research, and the like of rice species.

Description

technical field [0001] The invention relates to a method for identifying rice indica and japonica subspecies and a special primer composition thereof. Background technique [0002] Indica-japonica differentiation is the mainstream of genetic differentiation in Asian cultivated rice, and indica-japonica subspecies show great differences in many aspects. Therefore, the utilization of heterosis between indica and japonica subspecies is a very important means for super-high-yielding rice breeding, and the direct use of heterosis between indica and japonica has become the main direction of hybrid breeding. In order to carry out super-high-yield breeding of heterosis between indica and japonica subspecies, the first step is to correctly identify indica and japonica. Indica-japonica intermediate type or atypical indica-japonica may originate from natural hybridization or artificial hybridization of indica-japonica, and may also originate from primitive transitional varieties befor...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 李自超张冬玲张洪亮王美兴王凤梅宋立营马占峰
Owner CHINA AGRI UNIV
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