Method for identifying barley varieties by SSR (simple sequence repeat) primers and applications of method

A variety of barley technology, applied in the field of agricultural barley breeding and application, to achieve the effects of convenient operation, improved test efficiency, and less environmental impact

Active Publication Date: 2012-11-21
JIANGSU ACAD OF AGRI SCI
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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 new identification method for barley varieties that can only be carried out in the field, and the workload is large, and a series of problems. Method for identifying barley varieties using SSR primers and its application

Method used

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  • Method for identifying barley varieties by SSR (simple sequence repeat) primers and applications of method
  • Method for identifying barley varieties by SSR (simple sequence repeat) primers and applications of method
  • Method for identifying barley varieties by SSR (simple sequence repeat) primers and applications of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Through analysis, 14 pairs of basic core SSR primers and 14 pairs of extended core SSR primers were identified among 153 pairs of SSR primers with polymorphism in barley varieties that have been published sequences:

[0028] The 14 pairs of basic core SSRs are Bmag0211, Bmag0378, Bmag0711, Bmac0209, Bmag0877, EBmac0701, EBmac0635, Bmag0353, Scssr10148, Scssr03907, Bmag0009, Bmag0867, HVCMA and EBmac0603.

[0029] The 14 pairs of extended core SSRs are Scssr02748, HVM54, Scssr07759, HvXan, HVM27, HVM60, HVM40, Bmac0181, Scssr07106, Bmag0387, Bmac0018, Scssr09398, Bmac0064 and Scssr15864.

[0030] For 14 pairs of basic core SSRs and 14 pairs of extended core SSRs, obtain the upstream primers and downstream primers corresponding to the SSRs through http: / / www.graingenes.org website (see Table 1):

[0031] Table 1 The primer sequence list is as follows:

[0032]

[0033] The primer sequences were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0035] The samples included two tested barley varieties, one of which was an unknown barley variety numbered B1 (hereinafter referred to as B1), and the other was a reference variety Supi No. 5. The two tested barley varieties were both produced in Jiangsu Province.

[0036] Reagent:

[0037] SuperTaq? DNA Polymerase (5U / μL), dNTP (2.5mmol / L), 10×PCR Buffer (containing Mg 2+ ); I set of SSR basic core primers synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0038] Sampling and DNA extraction:

[0039] Random sampling of incoming samples. Take 5 individual plants for each sample, take 0.5 g of seedling leaves and mix them into one sample, cut them into pieces, put them into a 2 mL centrifuge tube, add liquid nitrogen to the centrifuge tube to freeze, put them into a grinder, and grind them into powder immediately Add about 800 μL of preheated DNA extraction solution (15 g SDS, 0.5 M EDTA (pH8.0), 1 M Tris-HCl (pH8.0), 5 M NaCl), shake vigorously to mix, and pla...

Embodiment 3

[0048] The steps and methods of this embodiment are the same as in Example 2, and the only difference between it and Example 2 is that one part of the sample is the unknown barley variety numbered B2 (hereinafter referred to as B2), and the other is the reference variety Su beer No. 3 (all places of origin are Jiangsu Province).

[0049] In the PCR reaction, 14 pairs of basic core SSR primers and 14 pairs of extended core SSR primers described in Table 1 were used to amplify the template DNA of the unknown barley variety B2 and the reference variety Supi No. 3.

[0050] Denaturing polyacrylamide gel electrophoresis and silver staining electrophoresis figure 2 ,exist figure 2 Among them, the odd-numbered lanes are barley variety B2, and the even-numbered lanes are barley variety Supi No. 3. At the same time, lanes 1 and 2 are Bmag0211 primers, lanes 3 and 4 are Bmag0378, lanes 5 and 6 are Bmag0711, lanes 7 and 8 are Bmac0209 , lanes 9 and 10 are Bmag0877, lanes 11 and 12 ar...

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Abstract

The invention relates to a method for identifying barley varieties by SSR (simple sequence repeat) primers. The method comprises steps as follows: extracting DNA of samples to be tested; using 14 pairs of basic core SSR primers and 14 pairs of extended core SSR primers to perform PCR (polymerase chain reaction) amplification; performing gel electrophoresis, modified polyacrylamide gel electrophoresis and silver staining on the PCR amplified product; analyzing an SSR band, wherein the samples to be tested are at least two barley varieties; when the number of different sites of the varieties is larger than or equal to 3, the varieties are judged as different varieties; when the number of the different sites of the varieties is 1 or 2, the varieties are judged as similar varieties; and when the number of the different sites of the varieties is 0, the varieties are judged as suspected identical varieties. The method provided by the invention can be used for identifying the barley varieties to be tested and the known barley varieties, or establishing a barley variety database; the method has advantages of high efficiency, accuracy, low cost and convenient operation; and with the adoption of the method, the authenticity of the barley varieties is effectively monitored, crop varieties are protected, and fake varieties are prevented from entering the market.

Description

technical field [0001] The invention relates to a barley variety identification method and its application, in particular to a barley variety identification method using SSR primers and its application, and belongs to the technical field of agricultural barley breeding and application. [0002] Background technique [0003] 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 fine varieties is an important direction for the development of barley breeding in my country. Therefore, the accurate identification of new varieties is not only the prerequisite for the approval and protection of new varieties, but also to identify fake and inferior seeds, protect their own intellectual property rights, and safeguard the vital interests of breeders. Provide assurance and scientific basis. Therefore, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王艳平沈奇张继红李华勇吴燕王显生
Owner JIANGSU ACAD OF AGRI SCI
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