The draft genome of the C3 panicoid grass species Dichanthelium oligosanthes

Abstract

BackgroundComparisons between C3 and C4 grasses often utilize C3 species from the subfamilies Ehrhartoideae or Pooideae and C4 species from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the C3 panicoid grass Dichanthelium oligosanthes from the independent C4 lineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of C3 and C4 grasses.ResultsWe report the assembly of the nuclear and chloroplast genomes of D. oligosanthes, from high-throughput short read sequencing data and a comparative transcriptomics analysis of the developing leaf of D. oligosanthes, S. viridis, and S. bicolor. Physiological and anatomical characterizations verified that D. oligosanthes utilizes the C3 pathway for carbon fixation and lacks Kranz anatomy. Expression profiles of transcription factors along developing leaves of D. oligosanthes and S. viridis were compared with previously published data from S. bicolor, Zea mays, and Oryza sativa to identify a small suite of transcription factors that likely acquired functions specifically related to C4 photosynthesis.ConclusionsThe phylogenetic location of D. oligosanthes makes it an ideal C3 plant for comparative analysis of C4 evolution in the panicoid grasses. This genome will not only provide a better C3 species for comparisons with C4 panicoid grasses, but also highlights the power of using high-throughput sequencing to address questions in evolutionary biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1080-3) contains supplementary material, which is available to authorized users.

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis

“…The genes selected from this dataset included, 5 ′- ADENYLYLSULFATE REDUCTASE6 ( ASPR6 ; Sevir.3G358100 ), SERINE - THREONINE PROTEIN KINASE ( STK ; Sevir.1G021400 ) and ADIPOSE - REGULATORY PROTEIN ( SEIPIN )- RELATED ( SEIPIN; Sevir.2G298500 ). Four additional candidate reference genes were next selected from a second S. viridis derived RNA-Seq dataset generated from an expanding leaf, leaf 3 [5], and included DUAL SPECIFICITY PROTEIN PHOSPHATASE ( DUSP ; Sevir.4G179200 ), SERINE - THREONINE KINASE WNK1 - RELATED ( WNK1 ; Sevir.2G373600 ), and one member each from the GRAS DOMAIN ( GRAS ; Sevir.1G267700 ) and F - BOX DOMAIN ( FBoxD ; Sevir8G147200 ) gene families. A further four candidate reference genes previously reported as appropriate candidates for S. viridis [9] or sorghum [14] gene expression normalisation were also included in this analysis, including PROTEIN PHOSPHATASE2A ( PP2A ; Sevir.9G262700 ), PHOSPHOGLUCOMUTASE ( PGM ; Sevir.9G117100 ), CULLIN ( CUL ; Sevir.3G038900 ) and FOLYPOLYGLUTAMATE SYNTHASE ( FPGS ; Sevir.9G574400 ).…”

“…The first dataset interrogated, represented four developmentally distinct zones of an elongating internode, internode 5, and included the; MS, CEZ, TZ and MatZ zones [4]. The second dataset analysed, was derived from leaf 3 sampled from a 10 day old plant [5]. The remaining four candidate reference genes selected for detailed assessment in this study, namely CUL , FPGS , PGM and PP2A , were included based on their expression profiles reported in previous RT-qPCR studies performed in S. viridis [9] and sorghum [14].…”

“…The eleven normalisation candidates assessed were identified from two RNA-Seq datasets, namely an elongating internode dataset [4] and an expanding leaf dataset [5], in combination with those reported previously for RT-qPCR analyses in other C 4 species [9, 14]. NormFinder and geNorm analyses of candidate reference gene performance identified three candidates, SERINE / THERONINE - PROTEIN PHOSPHATASE 2A ( PP2A ), 5 ′- ADENYLYLSULFATE REDUCTASE 6 ( ASPR6 ) and DUAL SPECIFICITY PHOSPHATASE ( DUSP ), as the most suitable combination of reference genes for the accurate and reliable normalisation of target gene expression across S. viridis tissues.…”

IsoSeq transcriptome assembly of C3 panicoid grasses provides tools to study evolutionary change in the Panicoideae

“…However, every domesticated grass in the PACMAD clade with a sequenced genome utilizes one or more variants of the C 4 photosynthetic pathway (Bennetzen et al, ; Schnable et al, ). As a result, while published whole genome sequence assemblies exist for at least 14 grasses within the PACMAD clade (Table ), only one of these ( Dichanthelium oligosanthes , a wild species) (Studer et al, ) utilizes C 3 photosynthesis. Long‐read sequencing can effectively generate sequence for large numbers of full‐length cDNAs even in species lacking reference genome assemblies (An, Cao, Li, Humbeck, & Wang, ; Zhang et al, ).…”

“…The final species targeted for transcriptome sequences was Dichanthelium oligosanthes . D. oligosanthes is the only PACMAD species exclusively utilizing C 3 photosynthesis with a published genome sequence to date (Studer et al, ). It is a member of the grass tribe Paniceae, a group which also includes foxtail millet, proso millet, and switchgrass, but is an outgroup to the MPC C 4 subclade of exclusively C 4 ‐utilizing species within that tribe (Giussani, Cota‐Sánchez, Zuloaga, & Kellogg, ; GPWG II, ; Washburn et al, ; Washburn, Schnable, Davidse, & Pires, ).…”