Margarida Rocheta

Polyploidization as a retraction force in plant genome evolution: sequence rearrangements in triticale.

Background Polyploidization is a major evolutionary process in plants where hybridization and chromosome doubling induce enormous genomic stress and can generate genetic and epigenetic modifications. However, proper evaluation of DNA sequence restructuring events and the precise characterization of sequences involved are still sparse. Methodology/Principal Findings Inter Retrotransposons Amplified Polymorphism (IRAP), Retrotransposons Microsatellite Amplified Polymorphism (REMAP) and Inter Simple Sequence Repeat (ISSR) largely confirmed the absence of any intraspecific variation in wheat, rye and triticale. The comparative analysis of banding profiles between wheat and rye inbred lines revealed 34% of monomorphic (common to both parental species) bands for the ten different primer combinations used. The analysis of triticale plants uncovered nearly 51% of rearranged bands in the polyploid, being the majority of these modifications, due to the loss of rye bands (83%). Sequence analysis of rye fragments absent in triticale revealed for instance homology with hydroxyproline-rich glycoproteins (HRGP), a protein that belongs to a major family of inducible defence response proteins. Conversely, a wheat-specific band absent in triticale comprises a nested structure of copia-like retrotransposons elements, namely Claudia and Barbara. Sequencing of a polyploid-specific band (absent in both parents) revealed a microsatellite related sequence. Cytological studies using Fluorescent In Situ Hybridization (FISH) with REMAP products revealed a widespread distribution of retrotransposon and/or microsatellite flanking sequences on rye chromosomes, with a preferential accumulation in heterochromatic sub-telomeric domains. Conclusions/Significance Here, we used PCR-based molecular marker techniques involving retrotransposons and microsatellites to uncover polyploidization induced genetic restructuring in triticale. Sequence analysis of rearranged genomic fragments either from rye or wheat origin showed these to be retrotransposon-related as well as coding sequences. Further FISH analysis revealed possible chromosome hotspots for sequence rearrangements. The role of chromatin condensation on the origin of genomic rearrangements mediated by polyploidization in triticale is also discussed.

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.

Microarray-based uncovering reference genes for quantitative real time PCR in grapevine under abiotic stress

BackgroundQuantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA and transcription data analysis as it shows to have advantages over other more commonly used techniques. Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation, usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was submitted to water stress and heat stress as well as a combination of both to test the stability of the possible reference genes.ResultsUsing the analysis of microarray data available for grapevine, six possible reference genes were selected for RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was analyzed with the softwares GeNorm, NormFinder and the ΔCq method resulting in several combinations of reference genes suitable for data normalisation. In order to assess the best combination, the reference genes were tested in putative stress marker genes (PCO, Galsynt, BKCoAS and HSP17) also chosen from the same microarray, in water stress, heat stress and the combination of both.ConclusionsEach method selected different gene combinations (PADCP + act, TIF + TIF-GTP and ubiq + act). However, as none of the combinations diverged significantly from the others used to normalize the expression of the putative stress marker genes, then any combination is suitable for data normalisation under the conditions tested. Here we prove the accuracy of choosing grapevine reference genes for RT-qPCR through a microarray analysis.

Heat and water stress induce unique transcriptional signatures of heat-shock proteins and transcription factors in grapevine

Grapevine is an extremely important crop worldwide. In southern Europe, post-flowering phases of the growth cycle can occur under high temperatures, excessive light, and drought conditions at soil and/or atmospheric level. In this study, we subjected greenhouse grown grapevine, variety Aragonez, to two individual abiotic stresses, water deficit stress (WDS), and heat stress (HS). The adaptation of plants to stress is a complex response triggered by cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Approaches such as array-based transcript profiling allow assessing the expression of thousands of genes in control and stress tissues. Using microarrays, we analyzed the leaf transcriptomic profile of the grapevine plants. Photosynthesis measurements verified that the plants were significantly affected by the stresses applied. Leaf gene expression was obtained using a high-throughput transcriptomic grapevine array, the 23K custom-made Affymetrix Vitis GeneChip. We identified 1,594 genes as differentially expressed between control and treatments and grouped them into ten major functional categories using MapMan software. The transcriptome of Aragonez was more significantly affected by HS when compared with WDS. The number of genes coding for heat-shock proteins and transcription factors expressed solely in response to HS suggesting their expression as unique signatures of HS. However, a cross-talk between the response pathways to both stresses was observed at the level of AP2/ERF transcription factors.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

BackgroundCork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management.ResultsWe generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org.ConclusionsThis genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber.

Monoecious species provide a comprehensive system to study the developmental programs underlying the establishment of female and male organs in unisexual flowers. However, molecular resources for most monoecious non-model species are limited, hampering our ability to study the molecular mechanisms involved in flower development of these species. The objective of this study was to identify differentially expressed genes during the development of male and female flowers of the monoecious species Quercus suber, an economically important Mediterranean tree. Total RNA was extracted from different developmental stages of Q. suber flowers. Non-normalized cDNA libraries of male and female flowers were generated using 454 pyrosequencing technology producing a total of 962,172 high-quality reads with an average length of 264 nucleotides. The assembly of the reads resulted in 14,488 contigs for female libraries and 10,438 contigs for male libraries. Comparative analysis of the transcriptomes revealed genes differentially expressed in early and late stages of development of female and male flowers, some of which have been shown to be involved in pollen development, in ovule formation and in flower development of other species with a monoecious, dioecious, or hermaphroditic sexual system. Moreover, we found differentially expressed genes that have not yet been characterized and others that have not been previously shown to be implicated in flower development. This transcriptomic analysis constitutes a major step toward the characterization of the molecular mechanisms involved in flower development in a monoecious tree with a potential contribution toward the knowledge of conserved developmental mechanisms in other species.

Genetic variation, mating patterns and gene flow in a Pinus pinaster Aiton clonal seed orchard

Abstract• Relatedness among parents, variation in clonal fertility and background pollination deviate the realized genetic gain and the gene diversity of open pollinated seed orchard from expectation, in particular in wind pollinated species such as Pinus pinaster Aiton.• This work investigates the genetic variation, the mating system and the pollen contamination in a P. pinaster clonal seed orchard (CSO), by screening the 60 clones from the CSO and the seeds collected from 21 mother-trees with three nuclear microsatellites.• The expected diversity was similar, but the observed heterozygosity decreased 20% in the progenies compared with the parental trees. The outcrossing rate was 90.1%, the biparental inbreeding 21.7% computed through a multilocus approach, and the observed selfing 3.9%. The observed gene flow from outside the CSO was 52.4%.• From the results we concluded that the observed gene flow and the biparental inbreeding were high, and care should be taken in the implementation and management of future CSO, in particular clones should be checked for relatedness and the ramet number could be directly proportional to their breeding value.Résumé• L’apparentement entre parents, la variation de fertilité entre clones et la pollution pollinique font dévier le gain génétique réalisé et la diversité génétique des valeurs attendues en verger à graines de clones à pollinisation libre, en particulier pour une espèce à pollinisation anémophile comme Pinus pinaster Aiton.• Ce travail étudie la variation génétique, le système de reproduction et la contamination du pollen d’un verger à graines de clones (VGC) de P. pinaster, en génotypant les 60 clones du VGC et les graines récoltées sur 21 arbres-mères pour trois marqueurs microsatellites nucléaires.• La diversité attendue s’est révélée similaire mais l’hétérozygotie observée a diminué de 20 % chez les descendants par rapport aux parents. Le taux d’allofécondation était de 90,1 %, la consanguinité biparentale calculée grâce à une approche multilocus était de 21,7 % et le taux d’autofécondation observé était de 52,4 %.• D’après ces résultats, nous avons conclu que les flux de gènes observés et la consanguinité biparentale étaient élevés et que des précautions devaient être prises dans la mise en place et la gestion de futurs VGC, l’apparentement entre clones devrait en particulier être vérifié et le nombre de copies d’un individu pourrait être directement proportionnel à sa valeur génétique.

Flower development and sex specification in wild grapevine

BackgroundWild plants of Vitis closely related to the cultivated grapevine (V. v. vinifera) are believed to have been first domesticated 10,000 years BC around the Caspian Sea. V. v. vinifera is hermaphrodite whereas V. v. sylvestris is a dioecious species. Male flowers show a reduced pistil without style or stigma and female flowers present reflexed stamens with infertile pollen. V. vinifera produce perfect flowers with all functional structures. The mechanism for flower sex determination and specification in grapevine is still unknown.ResultsTo understand which genes are involved during the establishment of male, female and complete flowers, we analysed and compared the transcription profiles of four developmental stages of the three genders. We showed that sex determination is a late event during flower development and that the expression of genes from the ABCDE model is not directly correlated with the establishment of sexual dimorphism. We propose a temporal comprehensive model in which two mutations in two linked genes could be players in sex determination and indirectly establish the Vitis domestication process. Additionally, we also found clusters of genes differentially expressed between genders and between developmental stages that suggest a role involved in sex differentiation. Also, the detection of differentially transcribed regions that extended existing gene models (intergenic regions) between sexes suggests that they may account for some of the variation between the subspecies.ConclusionsThere is no evidence of differences of expression levels in genes from the ABCDE model that could explain the shift from hermaphroditism to dioecy. We propose that sex specification occurs after floral organ identity has been established and therefore, sex determination genes might be having an effect downstream of the ABCDE model genes.For the first time a full transcriptomic analysis was performed in different flower developmental stages in the same individual. Our experimental approach enabled us to create a comprehensive catalogue of transcribed genes across developmental stages and genders that will contribute for future work in sex determination in seed plants.

Envelope -Like Retrotransposons in the Plant Kingdom: Evidence of Their Presence in Gymnosperms ( Pinus pinaster )

Retroviruses differ from retrotransposons due to their infective capacity, which depends critically on the encoded envelope. Some plant retroelements contain domains reminiscent of the env of animal retroviruses but the number of such elements described to date is restricted to angiosperms. We show here the first evidence of the presence of putative env-like gene sequences in a gymnosperm species, Pinus pinaster (maritime pine). Using a degenerate primer approach for conserved domains of RNaseH gene, three clones from putative envelope-like retrotransposons (PpRT2, PpRT3, and PpRT4) were identified. The env-like sequences of P. pinaster clones are predicted to encode proteins with transmembrane domains. These sequences showed identity scores of up to 30% with env-like sequences belonging to different organisms. A phylogenetic analysis based on protein alignment of deduced aminoacid sequences revealed that these clones clustered with env-containing plant retrotransposons, as well as with retrotransposons from invertebrate organisms. The differences found among the sequences of maritime pine clones isolated here suggest the existence of different putative classes of env-like retroelements. The identification for the first time of env-like genes in a gymnosperm species may support the ancestrality of retroviruses among plants shedding light on their role in plant evolution.

Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues

BackgroundTransposable elements (TEs) make up a large part of eukaryotic genomes. Due to their repetitive nature and to the fact that they harbour regulatory signals, TEs can be responsible for chromosomal rearrangements, movement of gene sequences and evolution of gene regulation and function. Retrotransposon ubiquity raises the question about their function in genomes and most are transcriptionally inactive due to rearrangements that compromise their activity. However, the activity of TEs is currently considered to have been one of the major processes in genome evolution.FindingsWe report on the characterization of a transcriptionally active gypsy-like retrotransposon (named Corky) from Quercus suber, in a comparative and quantitative study of expression levels in different tissues and distinct developmental stages through RT-qPCR. We observed Corky’s differential transcription levels in all the tissues analysed.ConclusionsThese results document that Corky’s transcription levels are not constant. Nevertheless, they depend upon the developmental stage, the tissue analysed and the potential occurring events during an individuals’ life span. This modulation brought upon by different developmental and environmental influences suggests an involvement of Corky in stress response and during development.