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

Melanie Febrer, Ed Wilhelm, Nadia Al-Kaff, Jonathan Wright, Wayne Powell, Michael W Bevan, Margaret I Boulton

Research output: Contribution to journalArticleResearchpeer-review

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)993-1000
Number of pages8
JournalMammalian Genome
Volume52
Issue number12
DOIs
Publication statusPrint publication - Dec 2009
Externally publishedYes

Fingerprint

Triticum
Clone Cells
Polymerase Chain Reaction
Libraries
Genes
Genomic Library
Patient Selection
Sequence Analysis
Genome
Costs and Cost Analysis
DNA

Keywords

  • Base Sequence
  • Chromosomes, Artificial, Bacterial/genetics
  • Cloning, Molecular/methods
  • DNA, Plant/chemistry
  • Genes, Plant/genetics
  • Genomic Library
  • Molecular Sequence Data
  • Polymerase Chain Reaction/methods
  • Polyploidy
  • Reproducibility of Results
  • Sequence Analysis, DNA
  • Sequence Homology, Nucleic Acid
  • Triticum/genetics

Cite this

Febrer, Melanie ; Wilhelm, Ed ; Al-Kaff, Nadia ; Wright, Jonathan ; Powell, Wayne ; Bevan, Michael W ; Boulton, Margaret I. / Rapid identification of the three homoeologues of the wheat dwarfing gene Rht using a novel PCR-based screen of three-dimensional BAC pools. In: Mammalian Genome. 2009 ; Vol. 52, No. 12. pp. 993-1000.
@article{4ba04634a728410983fec092be26cd1b,
title = "Rapid identification of the three homoeologues of the wheat dwarfing gene Rht using a novel PCR-based screen of three-dimensional BAC pools",
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.",
keywords = "Base Sequence, Chromosomes, Artificial, Bacterial/genetics, Cloning, Molecular/methods, DNA, Plant/chemistry, Genes, Plant/genetics, Genomic Library, Molecular Sequence Data, Polymerase Chain Reaction/methods, Polyploidy, Reproducibility of Results, Sequence Analysis, DNA, Sequence Homology, Nucleic Acid, Triticum/genetics",
author = "Melanie Febrer and Ed Wilhelm and Nadia Al-Kaff and Jonathan Wright and Wayne Powell and Bevan, {Michael W} and Boulton, {Margaret I}",
year = "2009",
month = "12",
doi = "10.1139/G09-073",
language = "English",
volume = "52",
pages = "993--1000",
journal = "Mammalian Genome",
issn = "0938-8990",
publisher = "NRC Research Press",
number = "12",

}

Rapid identification of the three homoeologues of the wheat dwarfing gene Rht using a novel PCR-based screen of three-dimensional BAC pools. / Febrer, Melanie; Wilhelm, Ed; Al-Kaff, Nadia; Wright, Jonathan; Powell, Wayne; Bevan, Michael W; Boulton, Margaret I.

In: Mammalian Genome, Vol. 52, No. 12, 12.2009, p. 993-1000.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Rapid identification of the three homoeologues of the wheat dwarfing gene Rht using a novel PCR-based screen of three-dimensional BAC pools

AU - Febrer, Melanie

AU - Wilhelm, Ed

AU - Al-Kaff, Nadia

AU - Wright, Jonathan

AU - Powell, Wayne

AU - Bevan, Michael W

AU - Boulton, Margaret I

PY - 2009/12

Y1 - 2009/12

N2 - 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.

AB - 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.

KW - Base Sequence

KW - Chromosomes, Artificial, Bacterial/genetics

KW - Cloning, Molecular/methods

KW - DNA, Plant/chemistry

KW - Genes, Plant/genetics

KW - Genomic Library

KW - Molecular Sequence Data

KW - Polymerase Chain Reaction/methods

KW - Polyploidy

KW - Reproducibility of Results

KW - Sequence Analysis, DNA

KW - Sequence Homology, Nucleic Acid

KW - Triticum/genetics

U2 - 10.1139/G09-073

DO - 10.1139/G09-073

M3 - Article

VL - 52

SP - 993

EP - 1000

JO - Mammalian Genome

JF - Mammalian Genome

SN - 0938-8990

IS - 12

ER -