A high-throughput method for the detection of homologous gene deletions in hexaploid wheat.
BACKGROUND
Mutational inactivation of plant genes is an essential tool in gene function studies. Plants with inactivated or deleted genes may also be exploited for crop improvement if such mutations/deletions produce a desirable agronomical and/or quality phenotype. However, the use of mutational gene inactivation/deletion has been impeded in polyploid plant species by genetic redundancy, as polyploids contain multiple copies of the same genes (homologous genes) encoded by each of the ancestral genomes. Similar to many other crop plants, bread wheat (Triticum aestivum L.) is polyploid; specifically allohexaploid possessing three progenitor genomes designated as 'A', 'B', and 'D'. Recently modified TILLING protocols have been developed specifically for mutation detection in wheat. Whilst extremely powerful in detecting single nucleotide changes and small deletions, these methods are not suitable for detecting whole gene deletions. Therefore, high-throughput methods for screening of candidate homologous gene deletions are needed for application to wheat populations generated by the use of certain mutagenic agents (e.g. heavy ion irradiation) that frequently generate whole-gene deletions.


RESULTS
To facilitate the screening for specific homologous gene deletions in hexaploid wheat, we have developed a TaqMan qPCR-based method that allows high-throughput detection of deletions in homologous copies of any gene of interest, provided that sufficient polymorphism (as little as a single nucleotide difference) amongst homologues exists for specific probe design. We used this method to identify deletions of individual TaPFT1 homologues, a wheat orthologue of the disease susceptibility and flowering regulatory gene PFT1 in Arabidopsis. This method was applied to wheat nullisomic-tetrasomic lines as well as other chromosomal deletion lines to locate the TaPFT1 gene to the long arm of chromosome 5. By screening of individual DNA samples from 4500 M2 mutant wheat lines generated by heavy ion irradiation, we detected multiple mutants with deletions of each TaPFT1 homologue, and confirmed these deletions using a CAPS method. We have subsequently designed, optimized, and applied this method for the screening of homologous deletions of three additional wheat genes putatively involved in plant disease resistance.


CONCLUSIONS
We have developed a method for automated, high-throughput screening to identify deletions of individual homologues of a wheat gene. This method is also potentially applicable to other polyploidy plants.
CSIRO Plant Industry, 306 Carmody Road, St Lucia, QLD 4067, Australia. Timothy.Fitzgerald@csiro.au
Timothy L
TL
Fitzgerald
Kemal
K
Kazan
Zhongyi
Z
Li
Matthew K
MK
Morell
John M
JM
Manners
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics*; D011123:Polyploidy; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid; D014908:Triticum / Q000235:genetics*
D010940:Plant Proteins