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Title: Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.)

Author
item ARAMAK, ATTAWAN - Washington State University
item KIDWELL, KIMBERLEE - Washington State University
item Steber, Camille
item BURKE, IAN - Washington State University

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/13/2015
Publication Date: 10/23/2015
Citation: Aramak, A., Kidwell, K., Steber, C.M., Burke, I. 2015. Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.). Biomed Central (BMC) Genomics. 16:844.

Interpretive Summary: Three wheat genes encoding the enzyme 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) were cloned and sequenced. The EPSPS enzyme is important because it is the target of the herbicide glyphosate, marketed as Roundup. Knowing the sequence of this gene will help investigators who may want to explore whether Roundup resistant wheat in farmers fields results from naturally occurring mutations. This study made use of the EPSPS sequence to examine the evolution of this gene in wheat and its relatives, and to show that all three copies of the gene are expressed in seedlings.

Technical Abstract: Background: 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the wheat EPSPS gene were cloned, investigated for molecular characteristics and expression, as well as examined for phylogenetic relationship to wheat relatives and other plant species. Results: Genomic and coding DNA sequences of EPSPS from the three related genomes of allohexaploid wheat were isolated by PCR and inverse PCR approaches. Insertion/deletion (In/Del) and single nucleotide polymorphisms (SNPs) allowed differentiation of the three genomic copies. Development of genome-specific primers allowed the mapping and expression analysis of TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1 on chromosomes 7A, 7D, and 4A, respectively. Comparative expression analysis revealed that EPSPS is expressed from all three genomic copies. However, TaEPSPS-4A1 is expressed at significantly lower levels than TaEPSPS-7A1 and TaEPSPS-7D1 in wheat seedlings. Alignment and phylogenetic analysis of the 1190-bp cDNA clones recovered from wheat and wheat relatives revealed that the TaEPSPS-7A1 gene is most similar to EPSPS from the tetraploid AB genome donor, T. turgidum (99.7% identity), TaEPSPS-7D1 resembles EPSPS from the diploid D genome donor, Aegilops tauschii (100% identity), and TaEPSPS-4A1 resembles EPSPS from the diploid B genome relative, Ae. speltoides (97.7% identity). The wheat EPSPS genes are more closely related to Lolium multiflorum and Brachypodium distachyon than to Oryza sativa (rice). However, the wheat EPSPS homoeologues resembled rice EPSPS in that they were comprised of 8 exons and 7 introns, that differed mainly in the size and nucleotide sequence of introns. Conclusions: The EPSPS homoeologues from three related genomes of allohexaploid wheat exhibited conservation of the exon/intron structure and of sequence in the coding region, but had significant genetic variation within the intron regions. Allohexaploid wheat contains three expressed copies of the EPSPS gene. Gene sequences corresponded to the AB and D genome ancestral donors rather than to the diploid relatives of the A and B genome donors. Thus, EPSPS sequences in allohexaploid wheat are preserved from the most two recent ancestors.