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Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same

A gourd and variety technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long identification time, crops are easy to change with environmental conditions, etc., to improve efficiency and standardization, and stable detection results Reliable, cost-effective detection of results

Inactive Publication Date: 2011-11-23
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a kind of early-stage, high-efficiency and high-efficiency method for the identification of the purity of the seeds of gourd varieties, which mostly rely on the field phenotype observation method, which has a long identification time, seasonal restrictions, and crop phenotypes that are easy to change with environmental conditions. Year-round, rapid and accurate detection method for identifying the authenticity and purity of gourd 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 quickly detecting purity of seeds of bottle gourd varieties and kit used by same
  • Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same
  • Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: (the application kit is to the detection of one of the tested samples of the main cultivars required to identify the target species)

[0034] Follow these steps:

[0035] (1) DNA extraction: spread the seeds to be tested in the seedling trays equipped with vermiculite: peat = 1:2 substrate, place the seedlings at 25-30 ° C, and when the first pair of true leaves are flat, take them out as per plant 0.1 g of leaves, ground into powder with liquid nitrogen, extracted DNA by conventional CTAB method, and set aside;

[0036] (2) SSR primer amplification and electrophoresis detection: Perform PCR analysis with 8 pairs of SSR diagnostic primers provided in the kit, the reaction volume is 12.5 μL; each PCR component is 10×buffer 1.25 μL, 25 mM MgCl 2 0.75μL, 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 fo...

Embodiment 2

[0038] Embodiment 2: (the application kit is required to identify the target variety as one of the samples to be tested for non-main varieties)

[0039] Follow these steps:

[0040] (1) DNA extraction: the seeds to be tested and the standard seeds required to identify the target species are scattered in the seedling trays equipped with vermiculite: peat=1:2 substrate respectively, placed at 25-30 ° C for seedling cultivation, and wait for the first When the true leaves are flattened, take 0.1 g of leaves per plant, add liquid nitrogen and grind them into powder, and use the CTAB method to extract DNA for future use;

[0041] (2) SSR primer amplification and electrophoresis detection: same as Example 1;

[0042] (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;

[0043] (4) DNA ...

Embodiment 3

[0044] Embodiment 3: (contrast test of application tradition and the inventive method detection gourd seed purity)

[0045] It is required to identify that the target variety is one of the 5 main varieties, then carry out according to the method of embodiment 1;

[0046] It is required to identify the target variety as one of the non-main varieties, then carry out according to the method of embodiment 2;

[0047] The traditional detection method is carried out according to the following steps:

[0048] 1. Get the standard seeds of the species to be detected and the standard seeds that require identification of the target species to be sown in vermiculite: peat=1: 2 matrix in the 8 × 8cm nutrient bowl, sow 2 seeds in each nutrient bowl, and thin the seedlings when the cotyledons are flattened. Leave 1 plant in the nutrient pot; plant in the greenhouse at the stage of 2 leaves and 1 heart, with a row spacing of 70cm and a plant spacing of 40cm, more than 100 plants for each var...

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Abstract

The invention discloses a method for quickly detecting the purity of the seeds of bottle gourd varieties and a kit used by the same. The method comprises the following steps: (I) partial sequencing of bottle gourd genomic DNA and simple sequence repeat (SSR) primer development and design; (II) calculation of the polymorphism information content (PIC) of SSR primers and screening of combination ofdiagnostic SSR primers; (III) preparation of standardized DNA fingerprint of the five main cultivated bottle gourd varieties; and (IV) detection of bottle gourd samples to be detected. In the method,because of the use of the 8 pairs of diagnostic SSR primers which are designed independently, the capacity of identifying similar varieties is increased, the efficiency of detection is improved, the detection time is reduced to 4 to 5 hours from 50 to 60 hours which are required by traditional phenotype identification, the detection cost is lowered to about 1 / 6 of that required by the conventional method, and the accuracy of identification is improved. The method can be promoted and used in seed breeding and seed enterprises.

Description

technical field [0001] The invention belongs to the field of crop biotechnology, in particular to a method for quickly detecting the purity of seeds of main gourd varieties by using a combination of diagnostic SSR molecular markers and a kit thereof. Background technique [0002] Gourd is an important summer melon vegetable in my country and is deeply loved by the people. There are many scientific research institutes and enterprises engaged in gourd breeding, seed production and management in my country, and new gourd varieties are bred and launched every year. There are many types of gourds currently used in production, 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 intellectual property rights and quality of seeds occur from time to time. It is necessary to research and develop a kind of gourd. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 徐沛李国景吴晓花汪宝根刘永华鲁忠富王莎潘榆樱
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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