RNA-Seq analysis of gene expression for floral development in crested wheatgrass (Agropyron cristatum L.)

Abstract

Crested wheatgrass [Agropyron cristatum L. (Gaertn.)] is widely used for early spring grazing in western Canada and the development of late maturing cultivars which maintain forage quality for a longer period is desired. However, it is difficult to manipulate the timing of floral transition, as little is known about molecular mechanism of plant maturity in this species. In this study, RNA-Seq and differential gene expression analysis were performed to investigate gene expression for floral initiation and development in crested wheatgrass. Three cDNA libraries were generated and sequenced to represent three successive growth stages by sampling leaves at the stem elongation stage, spikes at boot and anthesis stages. The sequencing generated 25,568,846; 25,144,688 and 25,714,194 qualified Illumina reads for the three successive stages, respectively. De novo assembly of all the reads generated 311,671 transcripts with a mean length of 487 bp, and 152,849 genes with an average sequence length of 669 bp. A total of 48,574 (31.8%) and 105,222 (68.8%) genes were annotated in the Swiss-Prot and NCBI non-redundant (nr) protein databases, respectively. Based on the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database, 9,723 annotated sequences were mapped onto 298 pathways, including plant circadian clock pathway. Specifically, 113 flowering time-associated genes, 123 MADS-box genes and 22 CONSTANS-LIKE (COL) genes were identified. A COL homolog DN52048-c0-g4 which was clustered with the flowering time genes AtCO and OsHd1 in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.), respectively, showed specific expression in leaves and could be a CONSTANS (CO) candidate gene. Taken together, this study has generated a new set of genomic resources for identifying and characterizing genes and pathways involved in floral transition and development in crested wheatgrass. These findings are significant for further understanding of the molecular basis for late maturity in this grass species.

Fig 1. Sequence length distributions of transcripts/genes assembled from Illumina sequence reads from three floral development stages of crested wheatgrass.

Fig 2

Statistics of sequence-homology search against NCBI nr protein database with respect to similarity (A) and species (B).

Fig 3. The plant circadian rhythm pathway revealed by KEGG annotation in three floral development stages of crested wheatgrass.

The genes highlighted in blue are found in our transcriptome data.

Fig 4. Characterization of the putative CONSTAN-like proteins in crested wheatgrass.

Fig 5. Differential expressed genes (DEGs) in two pairs of the floral developmental stages of crested wheatgrass [stem elongation stage (VS) vs. boot stage (BS) and boot stage (BS) vs. anthesis stage (AS)].

Fig 6. The qRT-PCR analysis of gene expression in the floral developmental stages of crested wheatgrass [stem elongation stage (VS) vs. boot stage (BS)].