A reference floral transcriptome of sexual and apomictic Paspalum notatum

Abstract

Background: Paspalum notatum Flügge is a subtropical grass native to South America, which includes sexual diploid and apomictic polyploid biotypes. In the past decade, a number of apomixis-associated genes were discovered in this species through genetic mapping and differential expression surveys. However, the scarce information on Paspalum sequences available in public databanks limited annotations and functional predictions for these candidates.

Results: We used a long-read 454/Roche FLX+ sequencing strategy to produce robust reference transcriptome datasets from florets of sexual and apomictic Paspalum notatum genotypes and delivered a list of transcripts showing differential representation in both reproductive types. Raw data originated from floral samples collected from premeiosis to anthesis was assembled in three libraries: i) sexual (SEX), ii) apomictic (APO) and iii) global (SEX + APO). A group of physically-supported Paspalum mRNA and EST sequences matched with high level of confidence to both sexual and apomictic libraries. A preliminary trial allowed discovery of the whole set of putative alleles/paralogs corresponding to 23 previously identified apomixis-associated candidate genes. Moreover, a list of 3,732 transcripts and several co-expression and protein -protein interaction networks associated with apomixis were identified.

Conclusions: The use of the 454/Roche FLX+ transcriptome database will allow the detailed characterization of floral alleles/paralogs of apomixis candidate genes identified in prior and future work. Moreover, it was used to reveal additional candidate genes differentially represented in apomictic and sexual flowers. Gene ontology (GO) analyses of this set of transcripts indicated that the main molecular pathways altered in the apomictic genotype correspond to specific biological processes, like biotic and abiotic stress responses, growth, development, cell death and senescence. This data collection will be of interest to the plant reproduction research community and, particularly, to Paspalum breeding projects.

Keywords: Apomixis; Next generation sequencing; Plant reproduction; Sexual reproduction; Transcriptomics.

Fig. 1

Assemblies derived from apomictic and sexual raw sequence data. The graphic shows the number of isogroups (genes), isotigs (alleles/splice variants), contigs (exons), isogroups with a single isotig (IsoSI) and isogroups with a single contig (IsoSC) obtained after assembly of the sexual, the apomictic and the apomictic + sexual (global) raw sequence data

Fig. 2

Validation of 454 libraries using BLAST search P. notatum mRNA (n = 80) and EST (n24) sequences deposited at NCBI were used as BLASTN queries against the 43,888 and 47,569 assembled isotigs from sexual (a, c) and apomictic (b, d) 454/Roche transcriptome libraries. a-b: Distribution of similarities values for the BLAST hits. c-d: E-value distribution of BLAST hits at a threshold E-value ≤ 10e^-5

Fig. 3

Cell components represented in the P. notatum floral transcriptome global assembly. The number of transcript units for the 30 most represented ontology terms is displayed

Fig. 4

Molecular functions represented in the P. notatum floral transcriptome global assembly. The number of transcript units corresponding to the 30 most represented ontology terms is displayed

Fig. 5

Biological processes represented in the P. notatum floral transcriptome floral assembly. The number of transcript units corresponding to the 30 most represented ontology terms is displayed

Fig. 6

Gene expression pairwise comparison between sexual and apomictic Paspalum notatum samples. For each isogroup, the log fold change (logFC) was plotted against the log of counts per million mapped reads (logCPM). Black dots: non-significant differential expression. Red dots: significant differential expression (p value < 0.01). Positive logFC: upregulation in apomictic samples. Negative logFC: upregulation in sexual samples

Fig. 7

Graphical representation of candidate protein-protein interaction networks involved in apomictic reproduction. Networks were generated with the BAR Arabidopsis interaction viewer (Geisler-Lee et al. 2007), by loading the putative Arabidopsis orthologs of the 200 top ranked candidates differentially expressed between the sexual and apomictic 454 libraries. Nodes represent proteins with the following localizations: green: chloroplast; pink: cytoplasm; yellow: vacuole; orange: plasma membrane; blue: nucleus; grey: unknown. Edges indicate the interactions. Light blue bold lines mark the experimentally determined interactions