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Method for detecting X-type HMW-GS (High Molecular Weight Glutenin Subunit) gene in distant hybridization wheat crossbreed

A distant hybridization and gene technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the target gene tracking, identification and evaluation standards that are cumbersome to operate and cannot be transferred from outside sources It is not easy to accurately grasp the problems, so as to achieve the effect of simple identification method and reliable results

Active Publication Date: 2013-05-22
SICHUAN AGRI UNIV
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  • Abstract
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Problems solved by technology

[0007] Therefore, in the process of continuous cross breeding, it is necessary to establish a simple, efficient and rapid method for detecting the X-type glutenin subunit gene of A. However, the morphological and cytological observation and identification methods are cumbersome to operate, difficult to accurately grasp the evaluation criteria, and cannot quickly track and identify the target gene transferred from the foreign source. Accordingly, we based on the known high molecular weight X Type glutelin subunit gene sequence, developed specific primers, not only amplified and screened, but also identified the coding gene strips belonging to the X-type subunit of A. band, and can track and detect the transfer of the X-type glutenin subunit gene of A. sharongensis in successive hybrid generations. Accelerate the process of quality breeding by researching future generations

Method used

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  • Method for detecting X-type HMW-GS (High Molecular Weight Glutenin Subunit) gene in distant hybridization wheat crossbreed
  • Method for detecting X-type HMW-GS (High Molecular Weight Glutenin Subunit) gene in distant hybridization wheat crossbreed
  • Method for detecting X-type HMW-GS (High Molecular Weight Glutenin Subunit) gene in distant hybridization wheat crossbreed

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Determination of primers for detecting the X-type glutenin subunit gene of A. sharongensis

[0044] According to the literature (Jiang Q T, Ma J, Wei Y M, et al. Novel variants of HMW glutenin subunits from Aegilops section Sitopsis species in relation to evolution and wheat breeding. BMC Plant Biology, 2012, 12:73.) on Sharon goat grass According to the research results, the complete coding gene of A. sharongensis X-type HMW-GS was obtained from the NCBI database, and the gene sequence structure was analyzed. In the present invention, the coding gene of X-type glutenin subunit of Sharon goatgrass and the HMW-GS gene commonly existing in emmer wheat and common wheat (the accession number of the coded gene of X-type subunit of Sharong goat grass glutenin is divided into JN001485; Common subunits in wheat and common wheat are 1Ax, 1Bx7, 1By8, 1Dx2, 1Dx5, 1Dy10, 1Dy12, whose coding gene accession numbers are EF055262, BK006773, JN255519, BK006460, HM050419, EU287...

Embodiment 2

[0047] Example 2 Establishment of a method for detecting the X-type HMW-GS gene of A. sharongensis in wheat distant hybrid hybrids

[0048] 1. Obtaining and identification of wheat distant hybrids

[0049] With tetraploid wheat (Z636) as the female parent, artificial detasseling (cut off the old spikelets at the top and the spikelets at the base, leaving only 10-15 spikelets in the middle, bagged). After 2-3 days, fresh Sharong Goatgrass (R7) pollen was applied (cut off the flowering ears, shake the anthers and fall on the emasculated female florets), and then bagged until harvesting to obtain a small amount of seeds.

[0050] Take the above-mentioned distant hybridization seeds and cut each seed in half with a knife, take half of the seeds containing endosperm and grind them into fine powder, put them in a 1.5ml centrifuge tube, add 25 μl of protein per mg of fine powder for extraction The buffer solution was leached at room temperature for 2.5 hours, and vortexed and mixed ...

Embodiment 3

[0081] Example 3 Detection of the specificity of the primers for the X-type HMW-GS gene of A. sharongensis in wheat distant hybrid hybrids

[0082] In order to ensure that the primers developed by the present invention can truly detect the transfer and existence of the X-type HMW-GS gene of A. sharongensis, it is necessary to verify the specificity of the primers. According to the plant genomic DNA extraction method and PCR amplification method in Example 2, 5 tetraploid wheats (Z636, Z606, Z639, JR-11-48, S24, provided by the Wheat Research Institute of Sichuan Agricultural University) were extracted, 7 Genomic DNA of common wheat (Chuannong 16, Liangmai No. 2, Liangmai No. 4, Mianmai 37, Shumai 482, Chuanyu 12, and Zhongguochun, provided by the Wheat Research Institute of Sichuan Agricultural University) and using the designed Primer RX1 is used for PCR amplification detection, and the amplification detection method is the same as in Example 2;

[0083] For the results of t...

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Abstract

The invention provides a method for detecting an aegilops sharonensis X-type HMW-GS (High Molecular Weight Glutenin Subunit) gene in a distant hybridization wheat crossbreed. The invention further provides a primer for detecting the aegilops sharonensis X-type HMW-GS gene. The nucleotide sequence of the primer is shown in SEQ ID NO. 1-2. Whether the distant hybridization wheat crossbreed contains an aegilops sharonensis X-type HMW-GS genetic locus can be rapidly and accurately detected by carrying out PCR (Polymerase Chain Reaction) through using the primer. By utilizing the method, the transfer and existence of the aegilops sharonensis X-type HMW-GS gene can be accurately detected, the gene can be conveniently tracked, the working efficiency in breeding is greatly improved, and the progress in wheat quality breeding is accelerated, so that the method has good application prospects in the field of crossbreeding of wheat species.

Description

technical field [0001] The invention relates to the field of molecular genetics, in particular to primers for detecting X-type high molecular weight glutenin subunit (high molecular weight glutenin subunit, HMW-GS) gene of A. A method and a detection kit for detecting the X-type glutenin subunit gene in hybrids and offspring. Background technique [0002] The wild relatives of wheat have many excellent genetic resources that are lacking in the gene pool of common wheat, and are valuable resource pools available. So far, people have introduced beneficial genes of close relative germplasms such as rye, tufted wheat, echinopsis intermedia, elymus, tall ibex, barley and so on into wheat through chromosome engineering technology. For example, Goatwort species have good genes that are very important for wheat improvement, such as resistance to stripe rust, leaf rust, stem rust, powdery mildew, drought tolerance, salt-alkali tolerance, cold tolerance, and high protein, and are exo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 江千涛郑有良赵全志魏育明赵珊马建蒲至恩
Owner SICHUAN AGRI UNIV
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