Method for rapidly detecting seed purity of asparagus bean cultivars and reagent kit thereof

A cowpea and seed technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of crops easily changing with environmental conditions, long identification time, etc., and achieve stable and reliable detection results, convenient operation, and easy implementation. normalized effect

Inactive Publication Date: 2012-07-04
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a kind of early-stage, high-efficiency, long-term identification of long cowpea variety seed purity identification mostly rely on field phenotype observation method existing identification time is long, subject to seasonal restrictions and crop phenotypes are easy to change with environmental conditions and other defects. Year-round, rapid and accurate detection method for identifying the authenticity and purity of long cowpea varieties; another purpose of the present invention is to provide a test kit for conveniently implementing the above method

Method used

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  • Method for rapidly detecting seed purity of asparagus bean cultivars and reagent kit thereof
  • Method for rapidly detecting seed purity of asparagus bean cultivars and reagent kit thereof
  • Method for rapidly detecting seed purity of asparagus bean cultivars and reagent kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: (application kit is required to identify target variety to be the detection of one of five main cultivars to be tested samples) carry out according to the following steps:

[0033] (1) DNA extraction: spread the seeds to be tested in a seedling tray equipped with a substrate (vermiculite:peat=1:2), place the seedlings at 25-30°C, and when the first pair of true leaves are flat, separate Take 0.1 g of leaves, grind them into powder with liquid nitrogen, and extract DNA by conventional CTAB method;

[0034] (2) SSR primer amplification and electrophoresis detection: use SSR10 provided in the kit for PCR analysis of diagnostic primers, the reaction volume is 12.5 μL; each PCR component is 1.25 μL of 10×buffer, 25 mM MgCl 20.75μL, 2.5mM dNTPs 1μL, Taq enzyme (5U / μL) 0.1μL, template DNA 10ng, add water to 12.5μL; SSR reaction program: 94°C pre-denaturation for 3min, 94°C denaturation for 30sec, 52°C annealing for 30sec, 72°C Extend for 50 sec, cycle 35 times, ...

Embodiment 2

[0036] Embodiment 2: (application kit is to the detection that requires identification target variety to be one of non-five main cultivars tested sample)

[0037] Follow these steps:

[0038] (1) DNA extraction: the seeds to be tested and the standard seeds required to identify the target species are spread respectively in seedling trays equipped with substrates (vermiculite: peat=1: 2), placed at 25-30°C for seedling cultivation, When the first pair of true leaves are flattened, take 0.1 g of leaves per plant, add liquid nitrogen and grind them into powder, and use CTAB method to extract DNA;

[0039] (2) SSR primer amplification and electrophoresis detection: same as Example 1;

[0040] (3) according to the PCR amplification and electrophoresis detection results of the seeds to be tested and the seeds of the standard variety respectively, construct the DNA fingerprints of the seeds to be tested and the seeds of the standard variety;

[0041] (4) DNA Fingerprint Comparison ...

Embodiment 3

[0042] Embodiment 3: (the contrast test of the present invention and traditional method identification seed purity)

[0043] The traditional identification method is carried out in the following steps:

[0044] 1. Sow the standard seeds of the variety to be tested and the target variety required to be identified in the field or greenhouse, with a row spacing of 70cm and a plant spacing of 30cm, 1 seed per hole, more than 100 holes for each variety, and the minimum environmental temperature requirement is higher than 15°C;

[0045] 2. Daily field management refers to conventional cultivation techniques.

[0046] 3, generally observe carefully the characteristic characteristic of this standard variety in the tender pod blooming period, compare the difference of the characteristic characteristic of the variety to be tested and the standard variety one by one, determine the authenticity of the seed, and use the formula after the detection is all completed: seed purity=(detected tr...

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Abstract

The invention discloses a method for rapidly detecting seed purity of asparagus bean cultivars and a reagent kit thereof, belonging to the field of crop biotechnology and comprising the following steps: (I) downloading a cowpea DNA database, treating sequences and designing SSR primers; (II) calculating polymorphism information content of the SSR primers and screening diagnostic SSR primers; (III) preparing standard fingerprints of five main cultivars of asparagus bean; and (IV) detecting asparagus bean samples to be detected and the like. To facilitate the popularization and application of the method, the invention simultaneously provides a reagent kit which can simultaneously detect hundreds of samples in 4-5 hours and has low detection cost which is about one sixtieth of that of the traditional phenotypic identification, therefore, the invention has the characteristics of rapidness, accuracy, stability and the like, and can be popularized and applied in units for bean research breeding, seed business and the like.

Description

technical field [0001] The invention belongs to the field of crop biotechnology, in particular to a method for rapidly detecting the purity of seeds of main varieties of long cowpea by using a combination of diagnostic SSR molecular markers and a kit thereof. Background technique [0002] Long cowpea is an important summer bean vegetable in my country and is deeply loved by the people. There are many scientific research institutes and enterprises engaged in long cowpea breeding, seed production and management in my country, and a batch of new long cowpea varieties are bred every year. At present, there are many varieties of long cowpea used in production, with a large amount of seeds used each year. The genetic similarity between commercial varieties is getting higher and higher. The phenomenon of different names of the same species and different species of the same name is becoming more and more serious, and disputes over seed quality often occur. Therefore, it is very nec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 李国景徐沛刘永华吴晓花汪宝根胡婷婷鲁忠富
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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