The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Tomato is a major crop in the Mediterranean basin, where the cultivation in the open field is often vulnerable to drought. In order to adapt and survive to naturally occurring cycles of drought stress and recovery, plants employ a coordinated array of physiological, biochemical and molecular responses. Transcriptomic studies on tomato responses to drought and subsequent recovery are few in number. As the search for novel traits to improve the genetic tolerance to drought increases, a better understanding of these responses is required. To address this need we designed a study in which we induced two cycles of prolonged drought stress and a single recovery by rewatering in tomato. In order to dissect the complexity of plant responses to drought, we analyzed the physiological responses (stomatal conductance, CO2 assimilation and chlorophyll fluorescence), abscisic acid (ABA) and proline contents. In addition to the physiological and metabolite assays, we generated transcriptomes for multiple points during the stress and recovery cycles. Cluster analysis of differentially expressed genes between the conditions has revealed potential novel components in stress response. The observed reduction in leaf gas exchanges and efficiency of the photosystem PSII was concomitant with a general down-regulation of genes belonging to the photosynthesis, light harvesting and photosystem I and II category induced by drought stress. Gene ontology (GO) categories such as cell proliferation and cell cycle were also significantly enriched in the down-regulated fraction of genes upon drought stress, which may contribute to explain the observed growth reduction. Several histone variants were also repressed during drought stress, indicating that chromatin associated processes are also affected by drought. As expected, ABA accumulated after prolonged water deficit, driving the observed enrichment of stress related GOs in the up-regulated gene fractions, which included transcripts putatively involved in stomatal movements. This transcriptomic study has yielded promising candidate genes that merit further functional studies to confirm their involvement in drought tolerance and recovery. Together, our results contribute to a better understanding of the coordinated responses taking place under drought stress and recovery in adult plants of tomato.

Purple carrots can accumulate large quantities of anthocyanins in their roots and –in some genetic backgrounds- petioles, and therefore they represent an excellent dietary source of antioxidant phytonutrients. In a previous study, using linkage analysis in a carrot F₂ mapping population segregating for root and petiole anthocyanin pigmentation, we identified a region in chromosome 3 with co-localized QTL for all anthocyanin pigments of the carrot root, whereas petiole pigmentation segregated as a single dominant gene and mapped to one of these “root pigmentation” regions conditioning anthocyanin biosynthesis. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify candidate genes controlling anthocyanin pigmentation in the carrot root and petiole. Fine mapping was performed in four carrot populations with different genetic backgrounds and patterns of pigmentation. The regions controlling root and petiole pigmentation in chromosome 3 were delimited to 541 and 535 kb, respectively. Genome wide prediction of transcription factor families known to regulate the anthocyanin biosynthetic pathway coupled with orthologous and phylogenetic analyses enabled the identification of a cluster of six MYB transcription factors, denominated DcMYB6 to DcMYB11, associated with the regulation of anthocyanin biosynthesis. No anthocyanin biosynthetic genes were present in this region. Comparative transcriptome analysis indicated that upregulation of DcMYB7 was always associated with anthocyanin pigmentation in both root and petiole tissues, whereas DcMYB11 was only upregulated with pigmentation in petioles. In the petiole, the level of expression of DcMYB11 was higher than DcMYB7. DcMYB6, a gene previously suggested as a key regulator of carrot anthocyanin biosynthesis, was not consistently associated with pigmentation in either tissue. These results strongly suggest that DcMYB7 is a candidate gene for root anthocyanin pigmentation in all the genetic backgrounds included in this study. DcMYB11 is a candidate gene for petiole pigmentation in all the purple carrot sources in this study. Since DcMYB7 is co-expressed with DcMYB11 in purple petioles, the latter gene may act also as a co-regulator of anthocyanin pigmentation in the petioles. This study provides linkage-mapping and functional evidence for the candidacy of these genes for the regulation of carrot anthocyanin biosynthesis.

Tomato is a high value crop and the primary model for fleshy fruit development and ripening. Breeding priorities include increased fruit quality, shelf life and tolerance to stresses. To contribute towards this goal, we re-sequenced the genomes of Corbarino (COR) and Lucariello (LUC) landraces, which both possess the traits of plant adaptation to water deficit, prolonged fruit shelf-life and good fruit quality. Through the newly developed pipeline Reconstructor, we generated the genome sequences of COR and LUC using datasets of 65.8 M and 56.4 M of 30–150 bp paired-end reads, respectively. New contigs including reads that could not be mapped to the tomato reference genome were assembled, and a total of 43, 054 and 44, 579 gene loci were annotated in COR and LUC. Both genomes showed novel regions with similarity to Solanum pimpinellifolium and Solanum pennellii. In addition to small deletions and insertions, 2, 000 and 1, 700 single nucleotide polymorphisms (SNPs) could exert potentially disruptive effects on 1, 371 and 1, 201 genes in COR and LUC, respectively. A detailed survey of the SNPs occurring in fruit quality, shelf life and stress tolerance related-genes identified several candidates of potential relevance. Variations in ethylene response components may concur in determining peculiar phenotypes of COR and LUC.

Anthocyanins are natural health promoting pigments that can be produced in large quantities in some purple carrot cultivars. Decoration patterns of anthocyanins, such as acylation, can greatly influence their stability and biological properties and use in the food industry as nutraceuticals and natural colorants. Despite recent advances made toward understanding the genetic control of anthocyanin accumulation in purple carrot, the genetic mechanism controlling acylation of anthocyanin in carrot root have not been studied yet. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify the genetic factor conditioning the accumulation of non-acylated (Cy3XGG) versus acylated (Cy3XFGG and Cy3XSGG) cyanidin derivatives, in three carrot populations. Segregation and mapping analysis pointed to a single gene with dominant effect controlling anthocyanin acylation in the root, located in a 576kb region containing 29 predicted genes. Orthologous and phylogenetic analyses enabled the identification of a cluster of three SCPL-acyltransferases coding genes within this region. Comparative transcriptome analysis indicated that only one of these three genes, DcSCPL1, was always expressed in association with anthocyanin pigmentation in the root and was co-expressed with DcMYB7, a gene known to activate anthocyanin biosynthetic genes in carrot. DcSCPL1 sequence analysis, in root tissue containing a low level of acylated anthocyanins, demonstrated the presence of an insertion causing an abnormal splicing of the 3^rd exon during mRNA editing, likely resulting in the production of a non-functional acyltransferase and explaining the reduced acylation phenotype. This study provides strong linkage-mapping and functional evidences for the candidacy of DcSCPL1 as a primary regulator of anthocyanin acylation in carrot storage root.

Background: Venom is one of the most important sources of regulation factors used by parasitic Hymenoptera to redirect host physiology in favour of the developing offspring. This has stimulated a number of studies, both at functional and “omics” level, which, however, are still quite limited for ectophagous parasitoids that permanently paralyze and suppress their victims (i.e., idiobiont parasitoids).Results: Here we present a combined transcriptomic and proteomic study of the venom of the generalist idiobiont waspBracon nigricans, an ectophagous larval parasitoid of different lepidopteran species, for which we recently described the host regulation strategy and the functional role of the venom in the induction of physiological changes in parasitized hosts. The experimental approach used led to the identification of the main components ofB. nigricansvenom involved in host regulation. Enzymes degrading lipids, proteins and carbohydrates are likely involved in the mobilization of storage nutrients from the fat body and may concurrently be responsible for the release of neurotoxic fatty acids inducing paralysis, and for the modulation of host immune responses.Conclusion: The present work contributes to fill the gap of knowledge on venom composition in ectoparasitoid wasps, and, along with our previous physiological study on this species, provides the foundation on which to develop a functional model of host regulation, based both on physiological and molecular data. This paves the way towards a better understanding of parasitism evolution in the basal lineages of Hymenoptera and to the possible exploitation of venom as source of bioinsecticidal molecules.