Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Isolation and characterization of plastid terminal oxidase gene from carrot and its relation to carotenoid accumulation

Carrot (Daucus carota L.) is a biennial plant that accumulates considerable amounts of carotenoid pigments in the storage root. To better understand the molecular mechanisms for carotenoid accumulation in developing storage roots, plastid terminal oxidase (PTOX) cDNA was isolated and selected for reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Present in photosynthetic species, PTOX is a plastid-located, nucleus encoded plastoquinone (PQ)-O2 oxidoreductase (plastioquinol oxidase). The enzyme is known to play a role as a cofactor for phytoene desaturase, and consequently plays a key role in the carotenoid biosynthesis pathway. A single PTOX gene was identified (DcPTOX) in carrot. DcPTOX encodes a putative protein with 366 amino acids that contains the typical structural features of PTOXs from higher plants. The expression of DcPTOX was analysed during the development of white, yellow, orange, red, and purple carrot roots, along with five genes known to be involved in the carotenoid biosynthesis pathway, PSY2, PDS, ZDS1, LCYB1, and LCYE. Expression analysis revealed the presence of DcPTOX transcripts in all cultivars, and an increase of transcripts during the time course of the experiment, with differential expression among cultivars in early stages of root growth. Our results demonstrated that DcPTOX showed a similar profile to that of other carotenoid biosynthetic genes with high correlation to all of them. The preponderant role of PSY in the biosynthesis of carotenoid pigments was also confirmed.