Sequence of dasypyrum villosum 6VS DNA permeating into powdery mildew resistant near-isogenic line of wheat and application

A near-isogenic, powdery mildew-resistant technology, applied in the field of agricultural biotechnology engineering, can solve the problems of tracking, that is, the donor gene detection report is less, the cultivation time is long, and it is easily affected by development, environmental conditions and human factors.

Inactive Publication Date: 2015-01-14
TIANJIN NORMAL UNIVERSITY
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  • Claims
  • Application Information

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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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  • Sequence of dasypyrum villosum 6VS DNA permeating into powdery mildew resistant near-isogenic line of wheat and application
  • Sequence of dasypyrum villosum 6VS DNA permeating into powdery mildew resistant near-isogenic line of wheat and application
  • Sequence of dasypyrum villosum 6VS DNA permeating into powdery mildew resistant near-isogenic line of wheat and application

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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 test material, the common cultivated wheat Jing 411 ( Triticum aestivum , 2N=6X=42, AABBDD), excellent agronomic traits, but susceptible to powdery mildew; powdery mildew-resistant wheat-Triticum aestivum chromosome translocation line 6VS.6AL (T6VS.6AL), 6VS in the translocation line comes from the cluster Wool wheat ( Haynaldia villosa 2N=2X=14, VV). The near-isogenic line Jing411 / 6VS.6AL / 京411 prepared with 6VS.6AL as the disease-resistant donor parent and Jing411 as the reincarnation parent 7 (Near-isogenic Lines, NIL). The process is T6VS.6AL×京411 hybridization, and F 1 All are resistant to powdery mildew, backcrossed with Jing 411 for 7 generations, and self-bred for the first generation to produce powdery mildew resistant near-isogenic lines 411 / 6VS.6AL / 京411 7 ; For each cross and backcross, disease-resistant s...

Embodiment 2

[0071] AFLP detection of NIL derived from 6VS powdery mildew resistance gene

[0072] The wheat DNA was extracted using the CTAB method, with a slight modification: Weigh 0.2g of young leaves and cut them into a mortar, add liquid nitrogen to quickly grind them into powder and transfer to a 5ml centrifuge tube, add 1ml DNA extraction buffer (0.1M Tris· Cl, pH8.0; 0.02mM EDTA, pH8.0; 1.4M NaCl; 4%CTAB; 1%PVP), water bath at 65℃ for 90min, shaking every 10min during this period; centrifugation at 10000rpm, 4℃ for 10min, take the supernatant In a new 5ml centrifuge tube, add an equal volume of phenol-chloroform-isoamyl alcohol (25? 24 ?1) and gently mix upside down. Centrifuge at 10000 rpm for 10 min. Pipette the supernatant into a new 5ml centrifuge tube and add again. Equal volume of phenol-chloroform-isoamyl alcohol extraction, centrifugation at 10,000 rpm for 10 minutes, suck the upper water phase into a 2ml centrifuge tube; add an equal volume of cold isopropanol, and place it ...

Embodiment 3

[0075] Obtained molecular evidence from 6VS in NIL

[0076] Using the AFLP method, molecular marker screening was carried out in V. vulgaris (VV), translocation line T6VS.6AL, NIL, and recurrent parent common wheat Jing 411. In this experiment, 15 AFLP profiles ( Figure 5 ), of which type I is the AFLP pattern that exists in T. vulgaris, T6VS.6AL, NIL and reincarnation parents. The frequency of this AFLP pattern is 3.8%. Triticum aestivum is a wild relative of common wheat, which proves that they have certain homology in the type Ⅰ AFLP genome, but have no correlation with the resistance to powdery mildew; type II: Triticum aestivum does not exist in T6VS.6AL Compared with common wheat, T6VS.6AL and NIL have the 6VS chromosomes derived from Triticum acuminata. Obviously this type of AFLP profile has nothing to do with 6VS, but is derived from common wheat. Wheat genome; type Ⅲ: only exists in P. tutus, not in T6VS.6AL, NIL, and common wheat. Since T6VS.6AL and NIL do not exis...

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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] This invention was funded by the National Natural Science Foundation of China (No. 31071671) and Tianjin Science and Technology Support Key (11ZCKFNC00700). Technical field [0002] The present invention belongs to agricultural biotechnology engineering, and relates to the preparation of powdery mildew resistant near-isogenic lines, and the sequence evidence and application of the infiltration of 6VS DNA of P. tutus into wheat powdery mildew-resistant near isogenic lines. Background technique [0003] Near isogenic line (NIL) is a cultivar that has excellent agronomic traits but is not ideal for a certain trait as a recurrent parent (PR) and crosses or backcrosses a variety or line with the target gene. After -8 generations, they were bred by selfing one generation. Therefore, NIL and PR are a pair of varieties with the same genetic background but different target genes. Since NIL can be used for the isolation of target genes, fine mapping and cloning of genes, and gene pl...

Claims

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Application Information

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Patent Type & Authority Applications(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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