Kit and method for identifying seed coat color of rape seed in very early development stage

A kit and very early technology, applied in the testa color rapid identification kit and its identification field at the very early stage of rapeseed development, achieving the effects of strong sensitivity and specificity, improving efficiency and saving time

Inactive Publication Date: 2011-07-27
HUNAN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Rapeseed yellow seeds have always been the most concerned issue for rapeseed breeders, especially the very early selection of the trait of yellow seeds (within 10 days after flowering), but the color of rapeseeds cannot be revealed until they are mature, which was difficult to achieve in the past at this point

Method used

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  • Kit and method for identifying seed coat color of rape seed in very early development stage

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Rapeseed seed coat color identification kit for early stage development consists of the following parts:

[0053] (1) RNA extraction solution A, containing Trizol solution, 2 tubes, 5ml / tube.

[0054] (2) RNA extraction solution B, containing phenol-chloroform, the volume ratio of phenol and chloroform is 25:24, 1 tube, 5ml / tube.

[0055] (3) RNA extraction solution C, filled with isopropanol, 2 tubes, 5ml / tube.

[0056] (4) RNA extraction solution D, filled with 75% ethanol, 2 tubes, 5ml / tube.

[0057] (5) RNA Extraction Solution E, filled with ultrapure water sterilized after DEPC treatment, 1 tube, 1ml / tube.

[0058] (6) RT reaction solution F, containing reaction buffer (5×Buffer), dNTP, oligo dT primer, RNase inhibitor, reverse transcriptase M-MLV and DEPC-treated ultrapure water, 1 tube, 0.2ml / tube

[0059] (7) RT-PCR reaction G1 solution, containing Mg 2+ reaction buffer (10×Buffer), dNTP, Taq enzyme, forward primer F, reverse primer R and ultrapure water, 1 tu...

Embodiment 2

[0086] The identification method of seed coat color in the early stage of rapeseed development uses the test kit in Example 1 to carry out according to the following steps:

[0087] (1) Take 0.05-0.1 g of the fresh seed coat after removing the hilum, add 500 μL of RNA extraction solution A, homogenate in an ice bath, and let stand at room temperature for 2-5 minutes.

[0088] (2) Add 250 μL of RNA extraction solution B to the above homogenate, mix well, and let stand for 2-5 minutes.

[0089] (3) Centrifuge at 12000g for 5min at 4-10°C.

[0090] (4) Take 500 μL of supernatant, add 500 μL of RNA extraction solution C, mix well, and let stand for 5-10 min.

[0091] (5) Centrifuge at 12000g for 5min at 4-10°C. Remove the supernatant, wash twice with 250 μL of RNA extraction solution D, dry at room temperature or blow dry in an ultra-clean bench, add 15-30 μL of RNA extraction solution E to dissolve, and obtain RNA solution.

[0092] (6) Take 1 μL of the above RNA solution, add...

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Abstract

The invention relates to an identifying kit, and particularly discloses a method for quickly identifying the seed coat color of a rape seed in the very early development stage. The kit and the identifying method disclosed by the invention are created on the basis that a primer pair designed according to the sequence of differential expression genes in the very early development stage of a yellow / black rape seed is used as the main body. By using the reverse transcription-polymerase chain reaction (RT-PCR) technology to carry out qualitative detection on specific RNA nucleic acid segments of the seed coat (with the hilum removed) of the rape in the early development stage (4-5 days after blooming), the invention has the advantages of convenient and fast process, good specificity and high sensitivity, and can be widely used for breeding selection based on the seed coat color in the very early stage, thereby improving the breeding efficiency, accelerating the breeding process, saving the breeding cost, improving the scientific management efficiency and having high use value.

Description

technical field [0001] The invention relates to the field of biology, in particular to a rapid identification kit for the color of the testa coat in the very early stage of rapeseed development and an identification method thereof. [0002] Background technique [0003] A large number of studies at home and abroad have shown that under the same genetic background, yellow seed coat rape (referred to as yellow seed rape) has thinner seed coat, higher oil content, less pigment and impurities in oil than black seed coat rape (referred to as black seed rape). , Low lignin content and other advantages. Rapeseed yellow seeds have always been the most concerned issue for rapeseed breeders, especially the very early selection of the trait of yellow seeds (within 10 days after flowering), but the color of rapeseeds cannot be revealed until they are mature, which was difficult to achieve in the past at this point. Whether it is yellow seeds or black seeds, the seed coat is green at t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 严明理刘丽莉向建华屠波陆赢谭勇俊
Owner HUNAN UNIV OF SCI & TECH
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