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

Abstract

Background: 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway, and is the target of the herbicide glyphosate. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the allohexaploid wheat EPSPS gene were cloned and characterized.

Methods: Genomic and coding DNA sequences of EPSPS from the three related genomes of allohexaploid wheat were isolated using PCR and inverse PCR approaches from soft white spring "Louise'. 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. Sequence alignments of cDNA sequences from wheat and wheat relatives served as a basis for phylogenetic analysis.

Results: The three genomic copies of wheat EPSPS differed by insertion/deletion and single nucleotide polymorphisms (SNPs), largely in intron sequences. RT-PCR analysis and cDNA cloning revealed that EPSPS is expressed from all three genomic copies. However, TaEPSPS-4A1 is expressed at much lower levels than TaEPSPS-7A1 and TaEPSPS-7D1 in wheat seedlings. Phylogenetic analysis of 1190-bp cDNA clones from wheat and wheat relatives revealed that: 1) TaEPSPS-7A1 is most similar to EPSPS from the tetraploid AB genome donor, T. turgidum (99.7 % identity); 2) TaEPSPS-7D1 most resembles EPSPS from the diploid D genome donor, Aegilops tauschii (100 % identity); and 3) TaEPSPS-4A1 resembles EPSPS from the diploid B genome relative, Ae. speltoides (97.7 % identity). Thus, EPSPS sequences in allohexaploid wheat are preserved from the most two recent ancestors. The wheat EPSPS genes are more closely related to Lolium multiflorum and Brachypodium distachyon than to Oryza sativa (rice).

Conclusions: The three related EPSPS homoeologues of wheat exhibited conservation of the exon/intron structure and of coding region sequence, but contained significant sequence variation within intron regions. The genome-specific primers developed will enable future characterization of natural and induced variation in EPSPS sequence and expression. This can be useful in investigating new causes of glyphosate herbicide resistance.

Fig. 1

Nucleotide polymorphisms from an alignment of EPSPS cDNA sequences of allohexaploid wheat and wheat progenitors. Vertical lines in the diagram indicate the position of nucleotide polymorphisms identified in a ClustalW multiple sequence alignment of the 1190-bp cDNA clones from T. aestivum ‘Louise’ (T.a_cDNA#), with our 1190-bp cDNA consensus sequences for T. turgidum (AB progenitor) (T.t_cDNA), Ae. tauschii (D progenitor) (Ae.t_cDNA), T. monococcum (A-relative) (T.m_cDNA), and Ae. speltoides (B-relative) (Ae.s_cDNA). The equivalent 1190-bp sequence of the genomic DNA consensus sequences of “TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1” are shown with intron sequences removed. Only the TaEPSPS-7A1 and TaEPSPS-7D1 cDNAs were recovered by F3-R1 PCR amplification. The corresponding 1190-bp TaEPSPS-4A1 cDNA sequence (T.a_cDNA13) was derived from an independent experiment

Fig. 2

Exon-intron structure of the wheat EPSPS genes compared to OsEPSPS. The boxes and solid lines represent exons (E#) and introns (I#), respectively. Numbers indicate exon and intron size in bp. The cleavage site of the chloroplastic transit signal peptide (cTP) predicted by PredSL is shown. The positions of the primers F1.2, F3, and R1 (arrows) used for initial genomic and cDNA cloning are labeled

Fig. 3

Optimization of the GC-rich TaEPSPS-7A1 PCR amplification. Louise genomic DNA was amplified using the F1.2-R1 primer pair with the indicated buffers designed for amplification of difficult templates (GCI, GCII, D, E, F, G, H, and I), and the indicated concentrations of DMSO (0 %, 2.5 %, 5 %, and 7.5 %). ‘M’ stands for the 1 kb DNA ladder (Thermo Scientific)

Fig. 4

Mapping the chromosome locations of the wheat EPSPS genes using nulli-tetrasomic lines. PCR amplification was performed using indicated Chinese Spring nulli-tetrasomic genomic DNA. These lines contain no copy of one chromosome (nullisomic), and 4 doses (tetrasomic) of a homoeologous chromosome. The lines are referred to as nulli1A-tetra1D or N1A-T1D, N2A-T2D, etc. DNA was amplified with the indicated primer pairs for: a TaEPSPS-7D1 failed to amplify in N7D-T7B line with primers F18-D-R16-D indicating localization to chromosome 7D; (b and c) TaEPSPS-7A1 failed to amplify in N7A-T7D with indicated primers; (d and e) TaEPSPS-4A1 failed to amplify in N4A-T4D

Fig. 5

RT-qPCR analysis of wheat EPSPS gene expression in seedlings. The relative expression levels of TaEPSPS-7A1 (7A1), TaEPSPS-7D1 (7D1), and TaEPSPS-4A1 (4A1) were determined by RT-qPCR using genome-specific primers (Additional file 1). The fold change was calculated relative to the TaEPSPS-4A1 mRNA level (set to 1) using the comparative C_q method. Letters (a, b, and c) represent significant differences (P < 0.005, based on a one-way ANOVA with Tukey’s adjustment at the 99 % significance level). Bars indicate the standard error

Fig. 6

Phylogenetic tree of EPSPS genes of wheat and wheat relatives. The UPGMA method was used to analyze nucleotide sequence similarity based on the 1190-bp cDNA sequences. Numbers at each branch indicate the genetic distance

Fig. 7

Phylogenetic tree of EPSPS genes of grass species. The 1190-bp cDNA sequences of TaEPSPS-7A1, TaEPSPS-4A1, and TaEPSPS-7D1 from allohexaploid wheat ‘Louise’ were compared to the corresponding EPSPS sequences from other species using the Neighbor-Joining method with the dicot A. thaliana as the outgroup. Numbers at each branch represent the genetic distance. The EPSPS homologues include A. thaliana [GenBank:NM_130093], B. distachyon [XM_003557194], E. indica [HQ403647], S. halepense [HQ436353], Z. mays [X63374], O. sativa [AF413081], and L. multiflorum [DQ153168]