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Trichophyllum 6vs chromosome-specific molecular marker 6vs-bh1 and its use

A 6VS-BH1 and molecular marker technology, applied in the fields of molecular biology and genetic breeding science, can solve the problems of limited EST quantity and molecular markers, and achieve wide annealing temperature, high resolution, and good stability of PCR amplification Effect

Inactive Publication Date: 2019-05-31
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of ESTs located in the target chromosomal region is limited, and the molecular markers that can be developed are also limited

Method used

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  • Trichophyllum 6vs chromosome-specific molecular marker 6vs-bh1 and its use
  • Trichophyllum 6vs chromosome-specific molecular marker 6vs-bh1 and its use
  • Trichophyllum 6vs chromosome-specific molecular marker 6vs-bh1 and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Design and synthesis of primers

[0024] powdery mildew resistance gene located on chromosome 6VS Stpk-V (GeneBank accession number: HQ864471) BLAST was performed to find the homologous gene Bradi3g04940 in the wheat model plant Brachypodium distachyon (GeneBank accession number: XM_003575353, GeneID: 100840649). Centering on the homologous gene Bradi3g04940 in Brachypodium distachyon, its upstream and downstream sequences were obtained from the NCBI website (http: / / www.ncbi.nlm.nih.gov), and in the wheat genome (http: / / wheat-urgi .versailles.inra.fr) to obtain sequences located on the short arm of the wheat homology group VI (6AS, 6BS, 6DS), and perform multiple sequence alignment analysis on these homologous sequences, according to Design 5 pairs of specific primers for the conserved sequences on both sides of the length polymorphic region in the comparison result (see figure 1 , Table 1), primers were synthesized by Shanghai Jierui Bioengineering Co., ...

Embodiment 2

[0027] Example 2: Screening of the 6VS chromosome-specific marker 6VS-BH1 of T. villosa

[0028] (1) PCR amplification: T6VS.6AL ( Pm21 gene), translocation line T6VS.6DL ( PmV Gene), hard cluster wheat, etc. as materials (the materials used are provided by Nanjing Agricultural University and Jiangsu Lixiahe Regional Agricultural Science Research Institute, all of which are well-known and public germplasm materials). After the genomic DNA is extracted, PCR amplification is performed. Contains about 10-20ng template DNA, 1×PCR buffer, 1.5mmol / L MgCl2, 200mmol / L dNTP, the final concentration of the two primers is 0.2µmol / L, 0.5U Taq DNA polymerase, supplement the reaction with sterile distilled water The PCR reaction program is: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 20 seconds, 60°C-66°C annealing for 30 seconds, 72°C extension for 60 seconds, 32 cycles; 72°C extension for 5 minutes; 4°C storage .

[0029] (2) Detection of PCR products: PCR amplificatio...

Embodiment 3

[0034] Example 3: Molecular marker 6VS-BH1 traces the translocation line T6VS.6AL ( Pm21 gene) on the 6VS chromosome

[0035] Powdery Mildew Resistant Wheat-T. villosa Translocation Line T6VS.6AL ( Pm21 gene) and powdery mildew-susceptible Yangmai No. 9 (provided by Jiangsu Lixiahe Regional Agricultural Science Research Institute, which is a known public germplasm material) to construct F 2 The genetic population was inoculated with powdery mildew at the second-leaf stage, and the resistance and susceptibility phenotypes were identified, and each individual plant was identified with the molecular marker 6VS-BH1. Only the bands corresponding to 6AS and 6DS were amplified in the susceptible individual plants, but no specific bands of 6VS-Pm21 were amplified. In disease-resistant homozygotes, only 6DS and 6VS-Pm21 specific bands are amplified, while disease-resistant heterozygotes can simultaneously amplify three bands of 6AS, 6DS, and 6VS-Pm21 (see figure 2 ). This result...

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Abstract

The invention relates to a haynaldia villosa's 6VS chromosome specific molecular marker 6VS-BH1 and an application thereof, and belongs to the technical fields of molecular biology and genetic breeding science. The labeled primer 6VS-BH1 is developed on the basis of wheat-brachypodium distachyon comparative genomics means, and the specific sequence is 6VS-BH1F, as shown in SEQ ID NO.1, and 6VS-BH1R, as shown in SEQ ID NO.2. The molecular marker 6VS-BH1 has the advantages of wide annealing temperature range (60-66 DEG C), good stability, strong product brightness, high resolution and the like. The molecular marker 6VS-BH1, as a co-dominant marker, not only can be used for effectively tracing haynaldia villosa's 6VS chromosome in wheat background, but also can be used for distinguishing a homozygote and heterozygote and for distinguishing a Pm21 gene carried translocation line T6VS.6AL and a PmV gene carried translocation line T6VS.6DL. Therefore, the molecular marker 6VS-BH1 disclosed by the invention has an important practical value in the molecular marker-assisted selection breeding of wheat powdery mildew and the pyramiding breeding of Pm21 gene and PmV gene.

Description

technical field [0001] The invention relates to a 6VS chromosome-specific molecular marker 6VS-BH1 and its use, belonging to the technical fields of molecular biology and genetic breeding. Background technique [0002] Wheat powdery mildew is caused by powdery mildew ( Blumeria graminis f.sp. tritici ) is one of the main diseases affecting wheat production. Breeding disease-resistant varieties is the most economical and effective way to avoid the damage of wheat powdery mildew, and the key is the discovery and utilization of powdery mildew resistance genes. As an important wild resource for wheat powdery mildew resistance breeding, wheat tufts ( Dasypyrum villosum ,VV,2 n = 2x = 14) Completely immune or highly resistant to known physiological races of Erysiphe miltiorrhiza, and its disease resistance genes have been successfully transferred to the common wheat background by multiple research groups. For example, Chen Peidu's research group carried the 6VS chromosome ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895
Inventor 何华纲朱姗颖岑波汪峰别同德
Owner JIANGSU UNIV
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