Wheat BSR-Seq gene locating method

A gene mapping and wheat technology, which is applied in the field of genetic breeding, can solve the problems of limited application, high sequencing cost, and errors caused by high copy characteristics of wheat genes, and achieve the effect of high precision and low cost

Active Publication Date: 2016-12-07
北京麦美瑞生物科技有限公司
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Problems solved by technology

However, the high-quality SNP markers obtained by this technology are limited, and the high-copy characteristics of...

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  • Wheat BSR-Seq gene locating method
  • Wheat BSR-Seq gene locating method
  • Wheat BSR-Seq gene locating method

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specific Embodiment approach

[0029] The method for wheat BSR-Seq gene mapping of the present invention, its preferred embodiment is:

[0030] Include steps:

[0031] A. Construction and sequencing of mixed pools:

[0032] Mapping populations based on recombinant inbred lines, doubled haploid (DH) populations, backcross introgression populations, F 2 or F 2:3 As a result of the phenotype identification of the segregated population, 15-30 or more homozygous extreme high-value individuals and 15-30 or more homozygous extreme low-value individuals were used to form a high-value mixed pool and a low-value mixed pool, respectively, and the phenotype did not show Differences, or after showing differences, equal amounts of leaf tissue were mixed to form a high-value pool and a low-value pool, and the mRNAs of the high-value pool and low-value pool were extracted and then transcriptome sequencing was performed to obtain the transcriptomes of the two mixed pools sequencing data.

[0033] B. High-quality variati...

Embodiment 1

[0057] Example 1: Analysis and Fine Mapping of Mixed Transcriptome Sequencing Data of Wheat Powdery Mildew Resistance Gene PmTm4

[0058] The specific method is:

[0059] (1) Construction and sequencing of mixed pools. In order to fine-tune the powdery mildew resistance gene PmTm4 in wheat, a F with 1,504 individuals was constructed using the resistant parent Tangmai 4 and the susceptible parent Nongda 015 as parental combinations. 2 Separate groups and get their F 2:3 Family, 60 homozygous disease-resistant F after phenotypic identification 2:3 Pedigree and 60 homozygous susceptible F 2:3 The families were sampled at the 3-leaf stage after being infected with powdery mildew, and the leaf tissue of 5 centimeters (cm) from the top of the third leaf of an individual in each family was collected and mixed, and the disease-resistant families were mixed into resistant families. Infected mixed pools, susceptible families were mixed into susceptible mixed pools, and RNA extractio...

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Abstract

The invention discloses a wheat BSR-Seq gene locating method. The wheat BSR-Seq gene locating method comprises the following steps of: constructing and sequencing mixing pools, performing quality variation mining, screening a transcript tightly linked to a target gene, developing and locating a molecular marker, etc. The next-generation transcriptome sequencing technology (transcriptome sequencing, RNA-Seq) and a mixing pool technology (Bulked Segregant Analysis BSA) are combined; a wheat sequencing draft sequence is used as a reference sequence at first; then, a lot of high-quality SNP heritable variations on the transcript is mined at high throughput by adopting the next-generation sequencing technology; the allele frequency is precisely calculated in combination with the mixing pool technology, so that the transcript tightly linked to the target gene possibly can be rapidly screened out; false positive is precisely checked and controlled through Fish; the method is independent of a reference genomic sequence, low in cost, rapid and high in precision; the wheat gene locating efficiency and precision are increased; the wheat polymorphic molecular marker developing cost is reduced; therefore, the wheat gene fine locating working time is reduced to several months from several years; the locating precision is reduced to a fraction or 0 cM from several cM; and the fine locating cost is reduced to several thousands from several ten thousands.

Description

technical field [0001] The invention relates to a genetic breeding technology, in particular to a wheat BSR-Seq (Bulked SegregantRNA-Seq, mixed pool transcriptome sequencing) gene positioning method. Background technique [0002] Wheat is one of the main ration crops for human beings. Under the background of decreasing arable land and increasing demand, its production is critical to food security. Breeding high-yielding, high-quality, stress-resistant wheat varieties and genetically improving important agronomic traits of wheat are important methods for stabilizing and increasing wheat yield. Improving the efficiency of traditional wheat breeding methods, continuous innovation and application of new breeding techniques are the only way to go. At present, molecular marker technology, marker-assisted selection breeding and genome selection are getting more and more attention. The use of closely linked molecular markers to identify and screen chromosome segments or genes relat...

Claims

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Application Information

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IPC IPC(8): G06F19/22
CPCG16B30/00
Inventor 谢菁忠刘志勇吴秋红
Owner 北京麦美瑞生物科技有限公司
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