SSR primer group and method for malt variety identification by virtue of primer group

A variety identification and primer group technology, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve problems affecting saccharification, filtration and brewing performance, secondary germination, and difficulty in separation , to achieve the effect of preventing malt adulteration, maintaining brand image, and increasing efficiency

Active Publication Date: 2015-11-18
TSINGTAO BREWERY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Compared with the relatively mature technology of barley variety identification, there is no clear detection method for malt variety identification. Conventional malt indicators are indicators that reflect the mixed state of malt and cannot reflect changes in malt varieties.
However, the existing methods for identification of barley varieties are not suitable for the identification of malt varieties.
(1) The appearance characteristics of barley have changed after germination and drying, and the protein begins to decompose under the action of protease, so the traditional barley morphology identification method and protein electrophoresis identification method are completely unsuitable for the identification of malt varieties
(2) In terms of molecular marker technology, barley DNA is usually extracted from germinated fresh seedling leaves. Fresh leaves contain less impurities and DNA extraction is easy; Made after the malting process, it cannot be germinated again, so DNA can only be extracte

Method used

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  • SSR primer group and method for malt variety identification by virtue of primer group
  • SSR primer group and method for malt variety identification by virtue of primer group
  • SSR primer group and method for malt variety identification by virtue of primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Select 21 barley samples from different origins, germinate and dry them into malt.

[0036] Table 1.21 Malt samples

[0037]

[0038] (1) Extract the DNA of the test variety sample

[0039] Use CTAB method to extract DNA from samples of tested varieties: Take a single malt sample in a 1.5ml centrifuge tube, about 25-45mg, add 7-8mm steel balls, and use Thmorgan CK1000D high-throughput tissue grinder to crush. Add 600ul of CTAB extraction buffer preheated at 65°C to the sample, and vortex to mix the lysed sample. Add 4ul β-mercaptoethanol and 1% PVP (polyvinylpyrrolidone) to prevent polyphenols from oxidative browning and DNA degradation, effectively remove polyphenols and polysaccharides, and vortex to mix. Water bath at 65°C for 15 minutes, shaking gently during mixing. Add 600ul chloroform / isoamyl alcohol (24:1), vortex and mix vigorously for 20s to form an emulsion. Centrifuge at 10,000 rpm for 10 minutes at room temperature, pipette 300ul of the supernatant into a new ...

Embodiment 2

[0052] The sample includes 2 test malt varieties, one is the control variety Copeland with 2 grains, and the other is the malt variety A to be identified with 6 grains, to determine whether the variety A is Copeland.

[0053] (1) Prepare malt DNA for the sample, use 6 pairs of SSR primers for PCR amplification, polyacrylamide gel electrophoresis, silver staining, and map analysis. The specific implementation steps are as in Example 1 (1)-(4).

[0054] (2) Result analysis: by Figure 7 It can be seen that lanes 1-8 are the first pair of primers scssr10148, lanes 1 and 2 are the control species Copeland, lanes 3-8 are the species to be identified, and the electrophoretic bands in lanes 3, 5, 6, and 7 are compared with lanes 1, 2 The electrophoresis bands of the samples are completely the same, but the electrophoresis bands in lanes 4 and 8 are different from the control sample Copeland, so it can be determined that lanes 4 and 8 are other varieties. Lanes 9-16 are the second pair of ...

Embodiment 3

[0056] The samples included 2 test malt varieties, one was the control variety Metcalfe with 2 grains, and the other was the unidentified malt variety B with 6 grains.

[0057] (1) Prepare malt DNA for the sample, use 6 pairs of SSR primers to perform PCR amplification, polyacrylamide gel electrophoresis, silver staining, and map analysis. The specific implementation steps are as the steps (1)-(4) of Example 1.

[0058] (2) Result analysis: by Figure 8 It can be seen that lanes 1-8 are the first pair of primers scssr10148, lanes 1 and 2 are the control variety Metcalfe, and lanes 3-8 are the varieties to be identified. The electrophoresis bands in lanes 4-8 are the same as those of the control samples in lanes 1 and 2. The bands are completely the same, but the electrophoretic bands in lane 3 are different from the control sample Metcalfe, which can confirm that lane 3 is of other varieties. Lanes 9-16 are the second pair of primers Bmac0030, lanes 9, 10 are the control varieties ...

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PUM

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Abstract

The invention provides an SSR primer group, which comprises 6 pairs of SSR primers, namely scssr07759, HVM68, Bmac0030, scssr10148, scssr03907 and Bmag0120. The invention also provides a method for malt variety identification by virtue of the SSR primers, wherein the method comprises the following steps: extraction of DNA of a sample of experimental variety, PCR amplification by virtue of the SSR primers, polyacrylamide gel electrophoresis as well as silver staining and SSR spectrum band analysis. The method, through SSR spectrum band analysis after the PCR amplification of the 6 pairs of SSR primers, can be used for identifying experimental malt variety and known malt variety and can be used for constructing a malt variety database. Through malt variety identification by virtue of the specific SSR primer group, the method can be used for rapidly identifying various malt varieties, and the method has the advantages of being rapid and accurate, and convenient in operation.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for identifying malt varieties, in particular to a technique for identifying malt varieties using SSR primers. Background technique [0002] Beer is a low-alcohol alcohol full of carbon dioxide, which is brewed by yeast fermentation with malt, hops, and water as the main raw materials. As the main raw material of beer, malt is made by soaking, sprouting, drying and roasting barley. The quality of malt directly determines the quality of beer. Variety identification of barley and malt is the most important indicator for evaluating barley quality and malt quality, both of which are important indicators for ensuring beer quality. [0003] At present, the identification technology of barley varieties has been relatively mature, including traditional morphological identification methods, protein electrophoresis identification methods and DNA molecular marking methods. The co...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 董建军张志军林艳余俊红岳杰房莉周月南祁明霞张翠
Owner TSINGTAO BREWERY
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