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Method for identifying varieties of Chinese cabbage by using composite expressed sequence tag-simple sequence repeat (EST-SSR) marker

A variety identification and Chinese cabbage technology, applied in the field of agricultural vegetable breeding and application, can solve problems such as restricting development and application, and achieve the effects of convenient operation, protection of crop varieties, and prevention of entering the market.

Inactive Publication Date: 2013-07-17
CENT LAB TIANJIN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The development of conventional SSR molecular markers requires the construction of genomic libraries, preparation of probes and molecular hybridization, which greatly restricts its development and application.

Method used

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  • Method for identifying varieties of Chinese cabbage by using composite expressed sequence tag-simple sequence repeat (EST-SSR) marker
  • Method for identifying varieties of Chinese cabbage by using composite expressed sequence tag-simple sequence repeat (EST-SSR) marker
  • Method for identifying varieties of Chinese cabbage by using composite expressed sequence tag-simple sequence repeat (EST-SSR) marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] (1) The samples included 4 test Chinese cabbage hybrids No. 1 to No. 4, produced in Tianjin.

[0055] (2) Reagents: GoTaq? Master Mixes solution produced by Promega; Ⅰ or Ⅱ sets of composite EST-SSR primers synthesized by Shanghai Shenggong;

[0056] (3) The sequence table of EST-SSR primers of set I or set II is as follows:

[0057]

[0058] (4) Sampling and DNA extraction

[0059] Random samples were taken from farmers in the base. 10 plants of each variety were taken, 50-100 mg of tender leaves near the roots were taken and mixed as a sample, frozen ground with liquid nitrogen (Retsch MM400 bio-pulverizer), and CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) 500 μL, lysed in a constant temperature water bath (Neslab) at 65°C for 30 minutes, and subsequent DNA extraction and purification are performed according to the resin-type genomic DNA purification kit (Syberson). After DNA extraction, the concentration is uniformly diluted to 50±1 ng / μL, Nano...

Embodiment 2

[0067] (1) The samples included 4 test Chinese cabbage hybrids No. 5-8, produced in Tianjin.

[0068] (2) Reagents: GoTaq? Master Mixes solution produced by Promega; Ⅰ or Ⅱ sets of composite EST-SSR primers synthesized by Shanghai Shenggong;

[0069] (3) The sequence table of EST-SSR primers of set I or set II is as follows:

[0070]

[0071] (4) Sampling and DNA extraction

[0072] Random samples were taken from farmers in the base. 10 plants of each variety were taken, 50-100 mg of tender leaves near the roots were taken and mixed as a sample, frozen ground with liquid nitrogen (Retsch MM400 bio-pulverizer), and CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) 500 μL, lysed in a constant temperature water bath (Neslab) at 65°C for 30 minutes, and subsequent DNA extraction and purification are performed according to the resin-type genomic DNA purification kit (Syberson). After DNA extraction, the concentration is uniformly diluted to 50±1 ng / μL, Nanodrop ND...

Embodiment 3

[0080] (1) The samples included 4 test Chinese cabbage varieties Hybrid No. 9-12, produced in Tianjin.

[0081] (2) Reagents: GoTaq? Master Mixes solution produced by Promega; Ⅰ or Ⅱ sets of composite EST-SSR primers synthesized by Shanghai Shenggong;

[0082] (3) The sequence table of EST-SSR primers of set I or set II is as follows:

[0083]

[0084] (4) Sampling and DNA extraction

[0085] Random samples were taken from farmers in the base. 10 plants of each variety were taken, 50-100 mg of tender leaves near the roots were taken and mixed as a sample, frozen ground with liquid nitrogen (Retsch MM400 bio-pulverizer), and CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) 500 μL, lysed in a constant temperature water bath (Neslab) at 65°C for 30 minutes, and subsequent DNA extraction and purification are performed according to the resin-type genomic DNA purification kit (Syberson). After DNA extraction, the concentration is uniformly diluted to 50±1 ng / μL, N...

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Abstract

The invention discloses a method for identifying varieties of Chinese cabbage by using a composite expressed sequence tag-simple sequence repeat (EST-SSR) marker. According to the method, 30 pairs of core EST-SSR primers are synthesized by using the genome DNA of 12 parts of Chinese cabbage hybrid varieties as templates and analyzing the EST sequence of the Chinese cabbage. 2 sets of marker composition are obtained through screening to identify the difference of 12 parts of Chinese cabbage varieties. According to the field verification test, the primers have high stability and are fit with the result of the field test, so that the technology and the composition can express the polymorphism of the Chinese cabbage varieties and can identify the Chinese cabbage varieties efficiently and accurately. The detection method can identify the seed varieties within 4 hours and has the advantages of high efficiency, accuracy, low cost, convenience for operation and the like. According to the method, authenticity of the Chinese cabbage seeds can be effectively monitored, the crop varieties are protected, fake varieties are prevented from entering the market, and the powerful technological means can be provided for new variety registration test and resource germplasm genetic analysis.

Description

technical field [0001] The invention belongs to the technical field of vegetable breeding and application in agriculture, and specifically relates to a method for identifying 12 Chinese cabbage hybrid varieties, which is a method for efficiently identifying Chinese cabbage varieties based on composite EST-SSR markers. Background technique [0002] With the development of plant breeding and seed industry, Protection of New Varieties of Plant (PVP), as one of the 10 contents of intellectual property rights, is becoming more and more important. Cultivating and promoting new and excellent varieties is an important direction for the development of vegetable breeding in my country. Therefore, accurate identification of new varieties is not only a prerequisite for the approval and protection of new varieties, but also for identifying fake and inferior seeds, protecting their own intellectual property rights, and safeguarding the vital interests of breeders. Provide assurance and sci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 赵新王永兰青阔陈锐朱珠郭永泽程奕
Owner CENT LAB TIANJIN ACADEMY OF AGRI SCI
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