The Sequence and Application of Introgression of 6vs DNA in Wheat Powdery Mildew Resistant Near-isogenic Lines

A near-isogenic, powdery mildew-resistant technology, applied in the field of agricultural biotechnology engineering, can solve the problems of tracking, i.e., donor gene detection, which is less reported, susceptible to development, environmental conditions and human factors, and long cultivation time.

Inactive Publication Date: 2016-10-19
TIANJIN NORMAL UNIVERSITY
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  • Abstract
  • Description
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Problems solved by technology

However, due to the long breeding time of NIL, it is easy to be affected by development, environmental conditions and human factors in the process of backcross breeding. Therefore, in order to ensure the quality of NIL breeding and selection effect, it is very important to evaluate and detect the genetic background of NIL
Early researchers used backcross generation and appearance to evaluate the genetic background of NIL, but this method mostly focused on the genetic similarity analysis between NIL and PR, tracking specific target genes, that is, the distribution of donor genes in NIL. Detection is less reported

Method used

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  • The Sequence and Application of Introgression of 6vs DNA in Wheat Powdery Mildew Resistant Near-isogenic Lines
  • The Sequence and Application of Introgression of 6vs DNA in Wheat Powdery Mildew Resistant Near-isogenic Lines
  • The Sequence and Application of Introgression of 6vs DNA in Wheat Powdery Mildew Resistant Near-isogenic Lines

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Preparation of near-isogenic lines and identification of disease resistance:

[0069] The preparation of the near-isogenic line of the present invention: the common cultivated wheat Jing 411 (Triticum aestivum, 2N=6X=42, AABBDD) as the test material has excellent agronomic properties, but is susceptible to powdery mildew; The translocation line 6VS.6AL (T6VS.6AL), the 6VS in the translocation line comes from Haynaldiavillosa (2N=2X=14, VV). The near-isogenic line Jing 411 / 6VS.6AL / Jing 411 prepared with 6VS.6AL as the disease-resistant donor parent and Jing 411 as the recurrent parent 7 (Near-isogenic Lines, NIL). The process is T6VS.6AL×Jing 411 hybridization, resulting in F 1 All are resistant to powdery mildew. Jing 411 was used for 7 generations of backcrossing, and 1 generation of self-crossing bred the near-isogenic line 411 / 6VS.6AL / Jing 411 resistant to powdery mildew. 7 ; For every hybridization and backcrossing, disease-resistant single plants were selected u...

Embodiment 2

[0071] Detection of AFLP Derived from 6VS Powdery Mildew Resistance Gene in NIL

[0072]Wheat DNA was extracted using the CTAB method with a slight modification: Weigh 0.2g of young leaves and cut them into pieces in a mortar, add liquid nitrogen and quickly grind them into powder, transfer them to a 5ml centrifuge tube, add 1ml of DNA extraction buffer (0.1M Tris· Cl, pH8.0; 0.02mM EDTA, pH8.0; 1.4M NaCl; 4%CTAB; 1%PVP), in a water bath at 65°C for 90min, shaking once every 10min during this period; at 10000rpm, centrifuge at 4°C for 10min, and take the supernatant in To a new 5ml centrifuge tube, add an equal volume of phenol-chloroform-isoamyl alcohol (25 ׃24׃1) and gently invert up and down to mix well, centrifuge at 10000rpm for 10min, draw the supernatant into a new 5ml centrifuge tube, and add again Extract by volume phenol-chloroform-isoamyl alcohol, centrifuge at 10,000rpm for 10min, absorb the upper aqueous phase into a 2ml centrifuge tube; add an equal volume of col...

Embodiment 3

[0075] Molecular evidence obtained from 6VS in NIL

[0076] Using the AFLP method, molecular marker screening was carried out in Trichosanthes villosa (VV), translocation lines T6VS.6AL, NIL, and the recurrent parent common wheat Jing 411. In this experiment, 15 screened AFLP profiles were listed ( Figure 5 ), among which type Ⅰ is an AFLP pattern that exists in T. villosa, T6VS.6AL, NIL and recurrent parents, and the frequency of this AFLP pattern is 3.8%. Trichophyllum is a close wild species of common wheat, which proves that they have certain homology in type I AFLP genome, but there is no correlation with powdery mildew resistance; , NIL and AFLP patterns in common wheat species, since T6VS.6AL and NIL all have 6VS chromosomes from T. Wheat genome; type III: present only in T. villosa, absent in T6VS.6AL, NIL, and common wheat. Since T6VS.6AL and NIL do not exist in the translocation line T6VS.6AL and NIL with T. villosa 6VS, this type of AFLP Bands are also not associ...

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Abstract

The invention discloses a preparation method of a powdery mildew resistant near-isogenic line Jing 411 / 6VS.6AL / Jing 411<7> and a DNA fragment sequence of a 6VS permeating into the near-isogenic line. The near-isogenic line is obtained by the following steps: by using a powdery mildew resistant translocation line T6VS.6AL as an anti-disease gene donor parent and cultured wheat Jing 411 which is excellent in agronomic character but is not powdery mildew resistant as a recurrent parent, after hybridization of T6VS.6AL and Jing 411, carrying out continuous back cross by using the obtained F1 and Jing 411 for 7 generations; selfing for 1 generation and breeding to obtain the near-isogenic line 411 / 6VS.6AL / Jing 411<7>; and selecting an anti-disease single plant under stress of powdery mildew for hybridization and backcross every time. The experimental result provided by the invention shows that EACG / M14-301 is a specific molecular marker on 6VS and can be used for screening of germplasm resources of powdery mildew resistance of wheat, mechanism study of characteristics of high resistance and broad-spectrum resistance of Pm21 and researches of wheat origin and evolution, parent selection of distant hybridization and the like.

Description

[0001] Supported by the National Natural Science Foundation of China (No. 31071671) and Tianjin Science and Technology Support Key (11ZCKFNC00700). technical field [0002] The invention belongs to agricultural biotechnology engineering, and relates to the preparation of powdery mildew-resistant near-isogenic lines, the sequence evidence and application of the infiltration of 6VS DNA of tufted wheat into powdery mildew-resistant near-isogenic lines of wheat. Background technique [0003] A near isogenic line (NIL) is a cultivar with excellent important agronomic traits but a certain trait is not ideal as a recurrent parent (Recurrent parent, PR) to cross and backcross with a variety or line with the target gene6 NIL and PR are a pair of varieties with the same genetic background but differences in target genes. Because NIL can be used for the isolation of target genes, the fine mapping and cloning of genes, and the analysis of gene pleiotropy, it has been widely used in gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 王振英刘晓颖彭永康范宝莉陈宏
Owner TIANJIN NORMAL UNIVERSITY
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