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Rapid identification of the three homoeologues of the wheat dwarfing gene Rht using a novel PCR-based screen of three-dimensional BAC pools

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Abstract:

A high-throughput two-step PCR strategy for the identification of selected genes from a BAC library derived from hexaploid wheat (16 974 Mbp) is described. The screen is based on the pooling of DNA from BAC clones into 675 “superpools” arrayed in a three-dimensional configuration. Each BAC clone is represented in three superpools to allow the identification of candidate 384-well plates of clones after the first round of PCR; identification is facilitated by an associated Perl script. A second round of PCR detects the specific BAC clone within the candidate plate that corresponds to the gene of interest. Thus, a single copy of the target gene can be identified from the library of over 700 000 clones (approximately 5 genome equivalents) by assaying only three 384-well plates. The pooling strategy was validated by screening the library with primers specific for the reduced height (Rht-1a) gene. Using relatively stringent selection criteria, 13 Rht-containing clones were identified from 17 candidate plates, and sequence analysis of the amplified products showed that all three Rht homoeologues were represented. Furthermore, the method confirmed the estimated coverage of the BAC library. Thus, this methodology allows the rapid and cost-effective identification of genes, and their homoeologues, from large-insert libraries of complex genomes such as hexaploid wheat.

Une stratégie de criblage PCR à haut débit en deux étapes pour l’identification de gènes ciblés au sein d’une banque de clones BAC, issue du blé hexaploïde (16 974 Mb), est décrite. Le crible repose sur des ADN composites provenant de 675 « superpools » de clones BAC disposés dans une configuration en trois dimensions. Chaque clone BAC est représenté dans trois « superpools », ce qui permet d’identifier des plaques à 384 puits candidates après une première amplification PCR. Cette identification est facilitée par l’emploi d’un script Perl. Une seconde ronde d’amplification PCR permet de détecter un clone BAC spécifique qui contient le gène d’intérêt au sein de la plaque candidate. Ainsi, on peut identifier une copie unique du gène ciblé au sein d’une banque de plus de 700 000 clones (l’équivalent d’environ 5 génomes) en interrogeant seulement trois plaques à 384 puits. Cette stratégie a été validée en criblant la banque à l’aide d’amorces spécifiques pour le gène reduced height (Rht-1a). À l’aide de critères relativement stricts, 13 clones contenant Rht ont été identifiés parmi 17 plaques candidates et le séquençage des produits amplifiés a montré que les trois homéologues Rht étaient représentés. De plus, la méthode a permis de confirmer la couverture estimée de la banque de clones BAC. Ainsi, cette méthodologie permet d’identifier rapidement et à peu de frais les gènes et leurs homéologues au sein de banques d’inserts de grande taille pour des génomes complexes comme celui du blé hexaploïde.

Document Type: Research Article

Publication date: December 1, 2009

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  • From its inception in 1957, this international cytogenetics journal has catered to the research areas of the members of the Genetics Society of Canada; traditionally, these have included agriculture, entomology, genetics/cytogenetics, and evolutionary mechanisms. The contents of the journal have evolved as contributors developed new technologies and interests. A 20-member Editorial Board is composed of scientists from around the world. Reviews and commentary from respected experts are often featured.
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