Dissecting miRNAs in Wheat D Genome Progenitor, Aegilops tauschii

Abstract

As the post-transcriptional regulators of gene expression, microRNAs or miRNAs comprise an integral part of understanding how genomes function. Although miRNAs have been a major focus of recent efforts, miRNA research is still in its infancy in most plant species. Aegilops tauschii, the D genome progenitor of bread wheat, is a wild diploid grass exhibiting remarkable population diversity. Due to the direct ancestry and the diverse gene pool, A. tauschii is a promising source for bread wheat improvement. In this study, a total of 87 Aegilops miRNA families, including 51 previously unknown, were computationally identified both at the subgenomic level, using flow-sorted A. tauschii 5D chromosome, and at the whole genome level. Predictions at the genomic and subgenomic levels suggested A. tauschii 5D chromosome as rich in pre-miRNAs that are highly associated with Class II DNA transposons. In order to gain insights into miRNA evolution, putative 5D chromosome miRNAs were compared to its modern ortholog, Triticum aestivum 5D chromosome, revealing that 48 of the 58 A. tauschii 5D miRNAs were conserved in orthologous T. aestivum 5D chromosome. The expression profiles of selected miRNAs (miR167, miR5205, miR5175, miR5523) provided the first experimental evidence for miR5175, miR5205 and miR5523, and revealed differential expressional changes in response to drought in different genetic backgrounds for miR167 and miR5175. Interestingly, while miR5523 coding regions were present and expressed as pre-miR5523 in both T. aestivum and A. tauschii, the expression of mature miR5523 was observed only in A. tauschii under normal conditions, pointing out to an interference at the downstream processing of pre-miR5523 in T. aestivum. Overall, this study expands our knowledge on the miRNA catalog of A. tauschii, locating a subset specifically to the 5D chromosome, with ample functional and comparative insight which should contribute to and complement efforts to develop drought tolerant wheat varieties.

Keywords: Aegilops tauschii; D genome; Triticum aestivum; drought; microRNA; next generation sequencing.

FIGURE 1

Repetitive element distributions in miRNA stem–loops. (A) Class II DNA transposons, (B) Class I retroelements, and (C) other repeat elements.

FIGURE 2

Percent representations of A. tauschii 5D stem–loops. (A) Repetitive, and (B) non-repetitive. Low confidence miRNAs are denoted by diamonds, ‘◆.’

FIGURE 3

Representations of wheat 5D stem–loops. (A) Percent representations of bread wheat 5D miRNAs, and (B) Percent representations of miRNAs common to A. tauschii and Triticum aestivum 5D chromosomes. miRNAs with higher representations in A. tauschii or T. aestivum 5D chromosomes are emphasized with the green or red lines, respectively (Total miRNA repertoires of Aegilops and wheat 5D were accepted to be 577 and 748 units, respectively. miRNA representation values are expressed in units in the comparative bar graphs).

FIGURE 4

Representations of Aegilops miRNAs. (A) Aegilops miRNAs predicted from whole genome assembly, and (B) comparative representations of miRNAs in Aegilops 5D chromosome and whole genome (5D chromosome miRNA repertoire was accepted to constitute 14.32% of the overall Aegilops miRNA content. Comparative bar graphs show the percent representation on the 5D chromosome and Aegilops genome corresponding to each miRNA).

FIGURE 5

Expression levels of miR167, miR5175, and miR5205 in A. tauschii and T. aestivum seedlings in response to drought stress. Ata: A. tauschii, Tae: T. aestivum var. CS.

FIGURE 6

miR5523 expression and coding regions in A. tauschii and T. aestivum var. CS (A) real time amplification curve showing mature miR5523 expression in A. tauschii, and (B) pre-miR5523 PCR amplicons showing that 5D chromosome-located miR5523 coding regions are present in both species. (C) Pre-miR5523 expression in both species in control and 4 h shock drought stress conditions (CS-1: Chinese Spring 1 leaf stage control; CS-1D: Chinese Spring 1 leaf stage drought; Ae: A. tauschii 1 leaf stage control; AeD: A. tauschii 1 leaf stage drought; CS-2: Chinese Spring 2 leaf stage control; CS-2D: Chinese Spring 2 leaf stage drought; Neg: Negative control; -RT: No RT control; -RNA: No RNA control).