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

A distant hybridization and gene technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the complicated operation and cannot be transferred from foreign sources. Target gene tracking, identification, and evaluation standards It is difficult to accurately grasp and other problems, so as to achieve the effect of reliable results and simple identification methods.

Active Publication Date: 2014-06-25
SICHUAN AGRI UNIV
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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

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Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Determination of primers for X-type gluten subunit gene detection of Aegilops sharong

[0044] According to the literature (Jiang QT, Ma J, Wei YM, 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.) According to the results of the study, the complete coding gene of Aegilops sylvestris X-type HMW-GS was obtained from the NCBI database, and the gene sequence structure was analyzed. In the present invention, the X-type gluten subunit encoding gene of Aegilops sharong and the HMW-GS gene ubiquitous in wheat and common wheat (Aegilops sharong gluten X-type subunit encoding gene accession number is divided into JN001485; Ergrain) The common subunits in wheat and common wheat are 1Ax, 1Bx7, 1By8, 1Dx2, 1Dx5, 1Dy10, 1Dy12, and their coding gene accessions are EF055262, BK006773, JN255519, BK006460, HM050419, EU287437, BK006459). Through repeated observation ...

Embodiment 2

[0047] Example 2 Establishment of the method for detecting the X-type HMW-GS gene of Aegilops sylvestris in wheat distant hybrid hybrids

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

[0049] Using tetraploid wheat (Z636) as the female parent, perform artificial emasculation (cut off the old spikelets on the upper part and the spikelets that are too tender at the base, leaving only the middle 10-15 spikelets and bagging). After 2-3 days, they are given fresh Aegilops (R7) pollen (cutting the blooming ears, shaking the anthers to fall on the emasculated female parent florets), then bagging them until harvesting, and a few seeds are obtained.

[0050] Take each seed obtained from the above-mentioned distant hybridization and cut it in half with a knife. Take half of the seed containing endosperm and grind it into fine powder, place it in a 1.5ml centrifuge tube, add 25μl protein per milligram of fine powder to extract The buffer solution was leached at room temper...

Embodiment 3

[0081] Example 3 Detecting the specificity verification of the primers of the X-type HMW-GS gene of Aegilops sarongii in wheat distant cross hybrids

[0082] In order to ensure that the primer developed by the present invention can truly detect the transfer and existence of the Aegilops X-type HMW-GS gene, it is necessary to verify the specificity of the primer. 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 two common wheats (Channong 16, Liangmai 2, Liangmai 4, Mianmai 37, Shumai 482, Chuanyu 12, China Spring, provided by the Wheat Research Institute of Sichuan Agricultural University) and use the designed Primer RX1 is used for PCR amplification detection, and the amplification detection method is the same as that in Example 2;

[0083] See the amplification test resul...

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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 present invention relates to the field of molecular genetics, in particular to a primer for detecting Aegilops sylvestris X-type high molecular weight glutenin subunit (HMW-GS) gene. The present invention also relates to the use of the primer for wheat remote Method and kit for detecting X-type gluten subunit gene in hybrids and offspring. Background technique [0002] The wild relatives of wheat have many excellent gene resources that are lacking in the common wheat gene bank, and they are valuable resource banks that can be utilized. So far, through chromosome engineering technology, people have introduced beneficial genes from related germplasms such as rye, wheatgrass, Elytrigia intermedius, Elymus dahurica, Aegilops tall, and barley. For example, Aegilops species have resistance to stripe rust, leaf rust, stalk rust, powdery mildew, drought tolerance, salt-alkali tolerance, cold tolerance, high protein and other excellent genes that are important ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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