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Method for Purity Identification of Flower Bud Seeds of Melon Hybrids Based on est-ssr Markers

A purity identification and hybrid technology, applied in the field of agricultural vegetable breeding and application, can solve problems such as being susceptible to environmental and seasonal factors, reducing seed purity, and heavy workload, and achieving stable identification results, high repeatability, Easy-to-use effects

Active Publication Date: 2019-10-22
TIANJIN RES INST OF VEGETABLE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reduced seed purity will significantly reduce crop yield and product quality, causing farmers to suffer huge economic losses
The traditional identification method of seed purity is based on the observation of the field morphology of the plant after sowing, which has a long cycle, heavy workload, and is easily affected by environmental and seasonal factors, resulting in deviations in the results

Method used

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  • Method for Purity Identification of Flower Bud Seeds of Melon Hybrids Based on est-ssr Markers
  • Method for Purity Identification of Flower Bud Seeds of Melon Hybrids Based on est-ssr Markers
  • Method for Purity Identification of Flower Bud Seeds of Melon Hybrids Based on est-ssr Markers

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] (1) Reagents: GoTaq® Master Mix solution produced by Promega; EST-SSR primers synthesized by Shanghai Sangon;

[0051] (2) The sequence list of EST-SSR primers is as follows:

[0052]

[0053] (3) Sampling and DNA extraction

[0054] 90 seedlings of the tested variety were frozen and ground in liquid nitrogen (Retsch MM400 bio-shredder), and 500 μL of CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) was added, and the temperature was kept at 65 °C The water bath (Neslab) was lysed for 30 minutes, and the subsequent DNA extraction and purification were performed according to the resin-type genomic DNA purification kit (Saibaisheng). After DNA extraction, the concentration was uniformly diluted to 50±1 ng / μL, Nanodrop ND1000, Thermol). The DNA extraction method can also be carried out according to a commercially available kit.

[0055] (4) PCR reaction

[0056] Carry out PCR amplification on the DNA template of the tested seedlings and the...

Embodiment 2

[0064] (1) Reagents: GoTaq® Master Mix solution produced by Promega; EST-SSR primers synthesized by Shanghai Sangon;

[0065] (2) The sequence list of EST-SSR primers is as follows:

[0066]

[0067] (3) Sampling and DNA template extraction

[0068] 144 seedlings of the tested variety were frozen and ground in liquid nitrogen (Retsch MM400 bio-shredder), and 500 μL of CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) was added, and the temperature was kept at 65 °C The water bath (Neslab) was lysed for 30 minutes, and the subsequent DNA extraction and purification were performed according to the resin-type genomic DNA purification kit (Saibaisheng). After DNA extraction, the concentration was uniformly diluted to 50±1 ng / μL, Nanodrop ND1000, Thermol). The DNA extraction method can also be carried out according to a commercially available kit.

[0069] (4) PCR reaction

[0070] Carry out PCR amplification on the DNA template of the tested seedlin...

Embodiment 3

[0078] (1) Reagents: GoTaq® Master Mix solution produced by Promega; EST-SSR primers synthesized by Shanghai Sangon;

[0079] (2) The sequence list of EST-SSR primers is as follows:

[0080]

[0081] (3) Sampling and DNA extraction

[0082] 94 individual hybrid plants of the tested varieties were frozen and ground in liquid nitrogen (Retsch MM400 biocrusher), and 500 μL of CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) was added, Lysis was carried out in a constant temperature water bath (Neslab) at 65°C for 30 minutes, and the subsequent DNA extraction and purification was performed according to the resin-type genomic DNA purification kit (Saibaisheng). After DNA extraction, the concentration was uniformly diluted to 50±1 ng / μL, Nanodrop ND1000, Thermol). The DNA extraction method can also be carried out according to a commercially available kit.

[0083] (4) PCR reaction

[0084] According to the PCR amplification primers mentioned in the t...

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Abstract

The invention discloses a method for identifying the purity of 'bud' (muskmelon variety) hybrid seeds based on EST-SSR marks. According to the method, bud H (muskmelon variety) hybrids and biparental genomic DNA thereof are used as a template, through a muskmelon EST sequence published on a Gene Bank database, the design of Primer 3.0 on-line primers is utilized, and EST-SSR primers are designed. Through screening, a pair of primers of which hybrid banding patterns are biparental complementary banding patterns is obtained; through field proof tests, the primers are favorable in stability, are fit with field test results, and can perform purity identification on muskmelon hybrid varieties. According to the detection method disclosed by the invention, the work of identifying the purity of seeds can be completed within 3h, and the detection method has the advantages of being quick, low in cost, convenient to operate and the like.

Description

technical field [0001] The invention belongs to the technical field of agricultural vegetable breeding and application, and in particular relates to a method for identifying the purity of "buds" of melon hybrids, which is a method for identifying the purity of hybrid seeds based on EST-SSR molecular marker technology. Background technique [0002] "Hua Bui" is the first-generation hybrid variety of thin-skinned melon cultivated by Tianjin Kerun Vegetable Research Institute. It is famous for its good resistance, high yield, novel appearance and high quality. The plant grows vigorously and has good comprehensive resistance. Both sub-vines and grandchildren can bear fruit. A single plant can retain 4-5 melons, with an average weight of 500 grams. The ripening period of the fruit is 30 days, and the peel is yellow when ripe, covered with dark green spots. The pulp is green, with a sugar content of more than 15%. The flesh is crisp, with a good taste and strong aroma. Long she...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
Inventor 张若纬兰青阔彭冬秀武云鹏王成赵新李秀秀王永张国华王钦马淑燕
Owner TIANJIN RES INST OF VEGETABLE
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