Abstract
BackgroundThe investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection. In contrast, long-reads sequencing techniques revealed their potential to generate complete transcript assemblies even when a reference genome is lacking. Cynara cardunculus var. altilis (DC) (cultivated cardoon) is a perennial hardy crop adapted to dry environments with many industrial and nutraceutical applications due to the richness of secondary metabolites mostly produced in flower heads. The investigation of this species benefited from the recent release of a draft genome, but the transcriptome profile during the capitula formation still remains unexplored. In the present study we show a transcriptome analysis of vegetative and inflorescence organs of cultivated cardoon through a novel hybrid RNA-seq assembly approach utilizing both long and short RNA-seq reads.ResultsThe inclusion of a single Nanopore flow-cell output in a hybrid sequencing approach determined an increase of 15% complete assembled genes and 18% transcript isoforms respect to short reads alone. Among 25,463 assembled unigenes, we identified 578 new genes and updated 13,039 gene models, 11,169 of which were alternatively spliced isoforms. During capitulum development, 3424 genes were differentially expressed and approximately two-thirds were identified as transcription factors including bHLH, MYB, NAC, C2H2 and MADS-box which were highly expressed especially after capitulum opening. We also show the expression dynamics of key genes involved in the production of valuable secondary metabolites of which capitulum is rich such as phenylpropanoids, flavonoids and sesquiterpene lactones. Most of their biosynthetic genes were strongly transcribed in the flower heads with alternative isoforms exhibiting differentially expression levels across the tissues.ConclusionsThis novel hybrid sequencing approach allowed to improve the transcriptome assembly, to update more than half of annotated genes and to identify many novel genes and different alternatively spliced isoforms. This study provides new insights on the flowering cycle in an Asteraceae plant, a valuable resource for plant biology and breeding in Cynara and an effective method for improving gene annotation.
Highlights
The investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection
RNA sequencing We obtained a total of 1,023,768,646 read pairs from 34 samples for Illumina with a total read coverage of 280x with a mean Q always above 33; while from Nanopore sequencing, 1, 445,444 reads were obtained from a 48-h run (Table S2 and Fig. 1) with a read coverage of 2x
Hybrid RNA de novo assembly The reads from both sequencing platforms were combined to perform a hybrid transcriptome assembly to be compared to the assembly using only short reads
Summary
The investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection. Altilis (DC) (cultivated cardoon) is a perennial hardy crop adapted to dry environments with many industrial and nutraceutical applications due to the richness of secondary metabolites mostly produced in flower heads The investigation of this species benefited from the recent release of a draft genome, but the transcriptome profile during the capitula formation still remains unexplored. More work has been done on phenolic acid composition in globe artichoke flower heads [9,10,11], which shows significant variation in quality and quantity, depending on the harvesting stage [12] These phenol compounds are mostly mono- and di-caffeoylquinic acids (CQAs) with a relevant role as structural components of plant cell walls during flowering [13], and flavonoids, mostly represented by apigenin, luteonin and their conjugates; they protect the plant from solar irradiation acting as reactive oxygen species (ROS) scavenger [14]. The isolation of some genes involved in the phenylpropanoid biosynthesis [17,18,19] and in the regulation of flavonoid pathway [20, 21] provided insights on the molecular mechanisms controlling polyphenol accumulation in this plant
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