Evolution of the Genus Camellia Based on the Biological Interaction and the Historical Background

departmental bulletin pape

Isolation and characterization of 52 polymorphic EST-SSR markers for Callitris columellaris (Cupressaceae)

Premise of the study: We developed simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) for Callitris columellaris sensu lato (s.l.) to elucidate population genetic structure and detect outlier loci by genome scan. Methods and Results: mRNA from an individual seedling was subjected to cDNA synthesis and then de novo pyrosequencing. Two hundred and nineteen primer pairs bordering sequence regions were designed from the obtained sequence data. In total, 52 showed polymorphism within 16 individuals representative of the species ' entire range, with the number of alleles per locus and expected heterozygosity ranging from two to 10 and 0.06 to 0.84, respectively. Conclusions: The EST-SSR markers developed in this study will be useful for evaluating the range-wide genetic structure of C. columellaris s.l. and detecting outlier loci under selection, as well as providing useful markers to investigate the conservation genetics and reproductive ecology of the species

Molecular database for classifying Shorea species (Dipterocarpaceae) and techniques for checking the legitimacy of timber and wood products

The extent of tropical forest has been declining, due to over-exploitation and illegal logging activities. Large quantities of unlawfully extracted timber and other wood products have been exported, mainly to developed countries. As part of the export monitoring effort, we have developed methods for extracting and analyzing DNA from wood products, such as veneers and sawn timbers made from dipterocarps, in order to identify the species from which they originated. We have also developed a chloroplast DNA database for classifying Shorea species, which are both ecologically and commercially important canopy tree species in the forests of Southeast Asia. We are able to determine the candidate species of wood samples, based on DNA sequences and anatomical data. The methods for analyzing DNA from dipterocarp wood products may have strong deterrent effects on international trade of illegitimate dipterocarp products. However, the method for analyzing DNA from wood is not perfect for all wood products and need for more improvement, especially for plywood sample. Consequently, there may be benefits for the conservation of tropical forests in Southeast Asia. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10265-010-0348-z) contains supplementary material, which is available to authorized users

Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak

<p>Abstract</p> <p>Background</p> <p>The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the <it>Quercus </it>family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity.</p> <p>Results</p> <p>We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser <url>http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html</url>.</p> <p>Conclusions</p> <p>This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.</p

Application of a Simplified Method of Chloroplast Enrichment to Small Amounts of Tissue for Chloroplast Genome Sequencing

Premise of the study: High-throughput sequencing of genomic DNA can recover complete chloroplast genome sequences, but the sequence data are usually dominated by sequences from nuclear/mitochondrial genomes. To overcome this deficiency, a simple enrichment method for chloroplast DNA from small amounts of plant tissue was tested for eight plant species including a gymnosperm and various angiosperms. Methods: Chloroplasts were enriched using a high-salt isolation buffer without any step gradient procedures, and enriched chloroplast DNA was sequenced by multiplexed high-throughput sequencing. Results: Using this simple method, significant enrichment of chloroplast DNA-derived reads was attained, allowing deep sequencing of chloroplast genomes. As an example, the chloroplast genome of the conifer Callitris sulcata was assembled, from which polymorphic microsatellite loci were isolated successfully. Discussion: This chloroplast enrichment method from small amounts of plant tissue will be particularly useful for studies that use sequencers with relatively small throughput and that cannot use large amounts of tissue (e.g., for endangered species)

Morphology_Climate_data

Study sites, Morphological data and climatic conditions of the study sites

Migrate_infile_Weevil

The weevil mt COI data for the Migrate-n analysis

Generation of Expressed Sequence Tags and development of microsatellite markers for Castanopsis sieboldii var. sieboldii (Fagaceae)

• Castanopsis sieboldii var. sieboldii is an evergreen broadleaved canopy tree that grows on Honshu, Shikoku and Kyushu Islands in Japan. A closely-related, hybridizing, congener species, C. cuspidata var. cuspidata, has different leaf epidermis structure and different seed nuts morphology compared to C. sieboldii var. sieboldii. Furthermore, the habitats in which the two species grow often indicate different water requirements. • Analysis of genetic diversity, using transcribed sequences, will provide a sound basis for the management of this species, allowing successful reforestation, incorporating phylogeographic conservation. In order to obtain transcripts sequences for the species and to develop transcript-based markers, we constructed and analyzed a cDNA library, surveyed microsatellite sequences within it and designed PCR primers for the microsatellites. • The cDNA library was constructed using tissue from the inner bark of C. sieboldii var. sieboldii; 3 354 Expressed Sequence Tags (ESTs) were identified. We constructed 2 417 putative unigenes and assigned putative functions to 1 856 of them. Three hundred and fourteen microsatellites were found within the putative unigenes and 16 EST-SSR (Simple Sequence Repeat) markers were developed. The EST-SSR markers developpe in this study should facilitate future analysis of the genetic diversity of this and related species.Generation de marqueurs de séquences exprimées et développement de marqueurs microsatellites pour Castanopsis sieboldii var. sieboldii (Fagaceae). • Castanopsis sieboldii var. sieboldii est un feuillu sempervirent qui pousse sur les îles japonaises de Honshu, Shikoku et Kyushu. C. cuspidata var. cuspidata une espèce congénère, étroitement liée, hybridée, a une structure de l'épiderme des feuilles différente et une morphologie différente des noix semences par rapport à C. sieboldii var. sieboldii. En outre, les habitats dans lesquels les deux espèces poussent indiquent souvent des besoins en eau différents. • L'analyse de la diversité génétique, en utilisant des séquences transcrites, fournira une base solide pour la gestion de cette espèce, ce qui permettra le succès des reboisements, en intégrant une conservation phylogéographique. Afin d'obtenir les transcriptions des séquences de l'espèce et de mettre au point des marqueurs à base de transcription, nous avons construit et analysé une banque d'ADNc, étudié des séquences microsatellites et conçu des amorces PCR pour les microsatellites. • La banque d'ADNc a été construite en utilisant les tissus de l'écorce interne de C. sieboldii var. sieboldii, 3 354 marqueurs de séquences exprimées (EST) ont été identifiés. Nous avons construit 2 417 unigènes putatifs et assigné des fonctions putatives à 1 856 d'entre eux. Trois cents quatorze microsatellites ont été trouvés à l'intérieur des unigènes putatifs et 16 EST-SSR (Simple Sequence Repeat) marqueurs ont été développés. Les marqueurs EST-SSR développés dans cette étude devraient faciliter l'analyse future de la diversité génétique de cette espèce et des espèces apparentées