Extending rnaSPAdes functionality for hybrid transcriptome assembly.

BACKGROUND: De novo RNA-Seq assembly is a powerful method for analysing transcriptomes when the reference genome is not available or poorly annotated. However, due to the short length of Illumina reads it is usually impossible to reconstruct complete sequences of complex genes and alternative isoforms. Recently emerged possibility to generate long RNA reads, such as PacBio and Oxford Nanopores, may dramatically improve the assembly quality, and thus the consecutive analysis. While reference-based tools for analysing long RNA reads were recently developed, there is no established pipeline for de novo assembly of such data. RESULTS: In this work we present a novel method that allows to perform high-quality de novo transcriptome assemblies by combining accuracy and reliability of short reads with exon structure information carried out from long error-prone reads. The algorithm is designed by incorporating existing hybridSPAdes approach into rnaSPAdes pipeline and adapting it for transcriptomic data. CONCLUSION: To evaluate the benefit of using long RNA reads we selected several datasets containing both Illumina and Iso-seq or Oxford Nanopore Technologies (ONT) reads. Using an existing quality assessment software, we show that hybrid assemblies performed with rnaSPAdes contain more full-length genes and alternative isoforms comparing to the case when only short-read data is used.

Hybrid transcriptome sequencing approach improved assembly and gene annotation in Cynara cardunculus (L.).

BACKGROUND: 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. 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. RESULTS: The 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. CONCLUSIONS: This 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.

Cystoseira compressa and Ericaria mediterranea: Effective Bioindicators for Heavy- and Semi-Metal Monitoring in Marine Environments with Rocky Substrates.

Marine environmental monitoring is essential to ensure that heavy-metal (HM) concentrations remain within safe limits. Most seawater analyses currently consider sediment or water samples, but this approach does not apply to rocky substrates, where water samples can only indicate immediate contamination. We used two common Mediterranean algae species, Cystoseira compressa and Ericaria mediterranea, as bioindicators living in the intertidal zone on rocky substrates along the seacoast. HM concentrations were assessed over a one-year period in the perennial base crust and in the seasonal frond, considering marine sites characterised by different contamination risks. Both algae showed that HMs accumulate mainly in the perennial base rather than in the seasonal frond. Furthermore, the algae species always showed a different order of bioaccumulation factors: Cd > Ni > Pb > Cr > Cu > Mn > Zn for the frond and Pb > Cr > Ni > Cd > Mn > Cu > Zn for the base. Our study shows that C. compressa and E. mediterranea accumulate HM consistently with the types of sites analysed and differentially with respect to the part of the thallus. These results demonstrate that these algae can be effectively used as reliable bioindicators to assess the presence of HM in marine environments with rocky substrates, providing both short- and long-term monitoring.

Seed Transcriptome Annotation Reveals Enhanced Expression of Genes Related to ROS Homeostasis and Ethylene Metabolism at Alternating Temperatures in Wild Cardoon.

The association among environmental cues, ethylene response, ABA signaling, and reactive oxygen species (ROS) homeostasis in the process of seed dormancy release is nowadays well-established in many species. Alternating temperatures are recognized as one of the main environmental signals determining dormancy release, but their underlying mechanisms are scarcely known. Dry after-ripened wild cardoon achenes germinated poorly at a constant temperature of 20, 15, or 10 °C, whereas germination was stimulated by 80% at alternating temperatures of 20/10 °C. Using an RNA-Seq approach, we identified 23,640 and annotated 14,078 gene transcripts expressed in dry achenes and achenes exposed to constant or alternating temperatures. Transcriptional patterns identified in dry condition included seed reserve and response to dehydration stress genes (i.e., HSPs, peroxidases, and LEAs). At a constant temperature, we observed an upregulation of ABA biosynthesis genes (i.e., NCED9), ABA-responsive genes (i.e., ABI5 and TAP), as well as other genes previously related to physiological dormancy and inhibition of germination. However, the alternating temperatures were associated with the upregulation of ethylene metabolism (i.e., ACO1, 4, and ACS10) and signaling (i.e., EXPs) genes and ROS homeostasis regulators genes (i.e., RBOH and CAT). Accordingly, the ethylene production was twice as high at alternating than at constant temperatures. The presence in the germination medium of ethylene or ROS synthesis and signaling inhibitors reduced significantly, but not completely, germination at 20/10 °C. Conversely, the presence of methyl viologen and salicylhydroxamic acid (SHAM), a peroxidase inhibitor, partially increased germination at constant temperature. Taken together, the present study provides the first insights into the gene expression patterns and physiological response associated with dormancy release at alternating temperatures in wild cardoon (Cynara cardunculus var. sylvestris).

MYB5-like and bHLH influence flavonoid composition in pomegranate.

The fruit of the pomegranate (Punica granatum L.) is an important nutraceutical food rich in polyphenolic compounds, including hydrolysable tannins, anthocyanins and flavonols. Their composition varies according to cultivar, tissue and fruit development stage and is probably regulated by a combination of MYB and bHLH type transcription factors (TFs). In this study, metabolomics analysis during fruit developmental stages in the main pomegranate cultivars, Wonderful and Valenciana with contrasting colour of their ripe fruits, showed that flavonols were mostly present in flowers while catechins were highest in unripe fruits and anthocyanins in late fruit maturation stages. A novel MYB TF, PgMYB5-like, was identified, which differs from previously isolated pomegranate TFs by unique C-terminal protein motifs and lack of the amino-acid residues conserved among anthocyanins promoting MYBs. In both pomegranate cultivars the expression of PgMYB5-like was high at flowering stage, while it decreased during fruit ripening. A previously identified bHLH-type TF, PgbHLH, also showed high transcript levels at flowering stage in both cultivars, while it showed a decrease in expression during fruit ripening in cv. Valenciana, but not in cv. Wonderful. Functional analysis of both TFs was performed by agro-infiltration into Nicotiana benthamiana leaves. Plants infiltrated with the PgMYB5-like+PgbHLH combined construct showed a specific and significant accumulation of intermediates of the flavonoid pathway, especially dihydroflavonols, while anthocyanins were not produced. Thus, we propose a role for PgMYB5-like and PgbHLH in the first steps of flavonoid production in flowers and in unripe fruits. The expression patterns of these two TFs may be key in determining the differential flavonoid composition in both flowers and fruits of the pomegranate varieties Wonderful and Valenciana.