0000000000976882

AUTHOR

Isabel Arrillaga

Regeneration of herbicide-tolerant black locust transgenic plants by SAAT

A protocol based on SAAT (sonication-assisted Agrobacterium-mediated transformation) has been developed to obtain herbicide-resistant transgenic black locust (Robinia pseudoacacia L.) plants. Cotyledon explants were co-cultivated with Agrobacterium AGL1 strain carrying the pTAB16 plasmid (bar and gusA genes). The effects of bacterial concentration (OD550 of 0.3, 0.6, 0.8) and method of infection (sonication vs immersion) on bacterial delivery were determined by assaying cotyledons for transient beta-glucuronidase expression 3 days after infection. SAAT increases transient expression efficiency especially at an OD550 of 0.6. After determining bacterial concentration and infection method, oth…

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Additional file 12: Table S5. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Primers used in PCR for validation of positive BAC clones in the BAC library screening. (DOCX 39 kb)

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Plant hormones and Agrobacterium tumefaciens strain 82.139 induce efficient plant regeneration in the cardenolide-producing plant Digitalis minor

Summary Shoot formation in explants of Digitalis minor was achieved, through axillary bud proliferation and adventitious bud differentiation, by varying the amount and source of plant hormones. Shoot regeneration was also obtained after infection of D. minor with the wild-type Agrobacterium tumefaciens strain 82.139, which induced shooty tumours. These shoots were not transgenic, as revealed by nopaline assays and the use of a C58pMP90/T139GUS-INT strain harbouring the intron inactivated gusA gene. Plants were easily rooted and transplanted into the greenhouse. Shoot cultures of D. minor accumulated up to 226 μg cardenolides per g dry mass when cultured on agar-solidified medium. Cardenolid…

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Induction of radiata pine somatic embryogenesis at high temperatures provokes a long-term decrease in dna methylation/hydroxymethylation and differential expression of stress-related genes

Based on the hypothesis that embryo development is a crucial stage for the formation of stable epigenetic marks that could modulate the behaviour of the resulting plants, in this study, radiata pine somatic embryogenesis was induced at high temperatures (23 &deg

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Additional file 14: of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Detailed description of the GeneAssembler pipeline (DOCX 124 kb)

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Maritime Pine Pinus Pinaster Aiton

Maritime pine (Pinus pinaster Aiton) is the most abundant conifer in the Mediterranean basin. Currently, maritime pine is considered to be a model conifer species for study of the adaption responses to drought stress from a genomics approach. In this context, the availability of protocols that allow not only mass vegetative propagation of selected families or genotypes, but also facilitate the functional analyses needed to verify and further to study the effects of candidate genes are necessary. Here we describe an improved protocol to generate maritime pine plants through somatic embryogenesis from immature megagametophytes. Accurate procedures for explant preparation, somatic embryo induc…

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Somatic embryogenesis from hypocotyl callus cultures of Digitalis obscura L.

Hypocotyl-derived calli obtained in agar solidified medium with several growth regulator combinations gave rise to proembryonal masses and globular embryos when transferred to liquid media with lower growth regulator and higher NH4HO3 levels. By transferring cultures from liquid media to different solidified media, new embryo formation took place, but further development of these embryos or those previously induced depended on the characteristics of these media. Normal development was only achieved on 8 g/l agar solidified medium without growth regulators. Typical cotyledonary embryos developed into whole plants when transferred to this same medium.

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Biotechnological Approaches to Increase Essential Oil Yield and Quality in Aromatic Plants: The Lavandula latifolia (Spike Lavender) Example. Past and Recommendations for the Future

Increasing knowledge about isoprenoid biosynthesis pathways has provided new tools for aromatic plant breeding using biotechnological approaches. Notably, there are possibilities to modify essential oil profiles and enhance production of valuable monoterpenes. This attains a particular significance in Lavandula latifolia Medik. (spike lavender), one of the most important essential oil crops in Spain. This chapter summarizes work done to improve essential oil yield and quality by engineering: (1) the enzymes controlling regulatory steps of methyl-D-erythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways to increase C5 units employed for monoterpene biosynthesis and (2) the monoterpene…

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Effects of environmental and temporal factors on Glomeromycotina spores in sand dunes along the Gulf of Valencia (Spain)

Abstract AMF symbiosis in sand dunes is the key for maintenance of stable vegetation. The main goal of this work was to determine the effects of environmental and temporal factors on AMF living in sand dunes (Gulf of Valencia, Spain). Soil samples were collected seasonally at 6 sites, during 2 yrs, from three habitats and four plant species and the frequency and relative abundance of AMF was examined. AMF were more frequent in mobile than in embryonic dunes, in spring and in sites with old vegetation. Ten AMF species were identified, their distribution depending mainly on the anthropogenic disturbance of the site. Gigasporaceae Cetraspora sp. and Dentiscutata sp. preferred undisturbed soil …

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Effect of methyl jasmonate in gene expression, and in hormonal and phenolic profiles of holm oak embryogenic lines before and after infection with Phytophthora Cinnamomi

The dieback syndrome affecting Quercus ilex and other oak species impels the search for tolerant plant genotypes, as well as methods of plant immunization against such infections. Elicitation treatments can be an effective strategy to activate plant defense response and embryogenic lines represent a promising tool to generate new tolerant genotypes and also to study early markers involved in defense response. The aim of the presented work was to investigate changes in gene expression, and in hormonal and phenolic profiles induced in three holm oak embryogenic lines (ELs) elicited with methyl jasmonate (MeJA) before and after infection with the oomycete Phytophthora cinnamomi, which is the m…

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Agrobacterium tumefaciens-mediated genetic transformation of the cardenolide-producing plant Digitalis minor L.

A repeatable transformation system has been established for Digitalis minor using Agrobacterium tumefaciens. Leaf explants from 30-day-old seedlings were inoculated with either EHA105 (carrying the nptII and gusA genes) or AGL1 (with the bar and gusA genes) strains. Among the tested factors influencing T-DNA transfer to plants, the EHA105 strain and the addition of acetosyringone to the co-culture medium increased transformation. The highest transformation efficiency (8.4%) was obtained when freshly isolated explants, soaked in a bacterial suspension with an OD 550 of 0.9, were subcultured on selection medium after a 4-day co-culture with the bacteria. Evidence of stable transgene integrati…

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Essential oil variation within and among natural populations of Lavandula latifolia and its relation to their ecological areas

Essential oil yield and composition in seven natural populations of Lavandula latifolia from the eastern Iberian Peninsula were determined by GC/MS. Twenty-eight constituents were identified, accounting for 92.0e95.4% of the total oils. These oils were dominated by the monoterpene fraction and three of them (linalool, cineole and camphor) constituted 79.5e86.9% of the oil from flowers. Essential oil yield in leaves and flowers varied among and within populations, but hierarchic analyses of variance showed that the proportion of variation attributable to individuals was significantly higher than that attributable to population differences. Principal component and cluster analyses allowed thr…

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<p><strong><em>Scutellospora deformata</em></strong><strong> (Scutellosporaceae), a new species of Gigasporales from the Mediterranean sand dunes of Spain</strong></p>

A new species S. deformata, that occurs in six locations of marine sand dunes along the eastern Mediterranean coast of Spain is described and illustrated from spores. In the field, the species occurred in the rhizosphere of Ammophila arenaria (Poaceae), Elymus farctus (Poaceae), Otanthus maritimus (Asteraceae), and Echinophora spinosa (Apiaceae). Morphological characters related with outer, middle and inner wall of the glomerospores as well as phylogenetic analysis (partial SSU, ITS1-5.8S region and the partial LSU nrDNA) support the hypothesis that the fungus is a new species of the Scutellosporaceae.

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Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Background In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. Results In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were design…

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Additional file 4: Table S3. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Exon length comparison between the XET BAC clone from P. pinaster and two XET genes from Arabidopsis thaliana. The two gene capture models closest to the BAC clone are also included. (DOCX 18 kb)

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In vitroplant regeneration and gene transfer in the wild tomatoLycopersicon cheesmanii

SummaryThis paper reports on protocols for in vitro plant regeneration, gene transfer and on shoot salt tolerance from two accessions of L. cheesmanii (Riley). Shoot organogenesis was achieved on media with zeatin or BA/IAA combinations. The two accessions tested, LA1401 and LA530, showed differences in both their aptitude to regenerate and their salt tolerance in vitro. The accession LA530 had a higher organogenic potential than LA1401 (97% vs. 80% of organogenic explants). In contrast, none of the LA530 isolated shoots rooted when cultured in full strength MS-modified medium supplemented with 200.mM NaCl while 93% of shoots from LA1401 formed roots. Finally, Agrobacterium-mediated transfo…

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Sowing date, transplanting, plant density and nitrogen fertilization affect indigo production from Isatis species in a Mediterranean region of Spain

Abstract The increasing interest in natural products from a renewable source has encouraged growers to reintroduce indigo-producing crops into the European agriculture. We studied agronomic conditions (sowing date, plant density, nitrogen fertilization, irrigation rate, seedling transplanting) influencing production of the blue pigment indigo, from Isatis tinctoria and I. indigotica crops in a Mediterranean region of Spain (Valencia). I. tinctoria was more suitable for cultivation in our climate conditions than I. indigotica . Indigo yield from Spanish I. tinctoria trials was greater than in Northern and Central Europe. Furthermore, indigo production was maintained when water and nitrogen s…

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Additional file 4: Table S3. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Exon length comparison between the XET BAC clone from P. pinaster and two XET genes from Arabidopsis thaliana. The two gene capture models closest to the BAC clone are also included. (DOCX 18 kb)

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Cryopreservation of Digitalis obscura selected genotypes by encapsulation-dehydration

Shoot-tips from several genotypes of the cardenolide-producing perennial shrub Digitalis obscura L. were successfully cryopreserved using the encapsulation-dehydration technique. Precultivation on MS medium containing 0.5 M sucrose, followed by 2.5 h dehydration (final weight 30 %) induced shoot regrowth in 42 % of cryopreserved shoot-tips. Cold-hardening of the in vitro cultures before sucrose treatment dramatically increased shoot recovery up to 86 %. The optimized cryopreservation protocol was then employed using different shoot cultures from five D. obscura genotypes. Responses to cryopreservation depended mainly on the genotype, best results being obtained when shoot tips from HU3 and …

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New Approaches to Optimize Somatic Embryogenesis in Maritime Pine

Maritime pine (Pinus pinaster Aiton) is a coniferous native of the Mediterranean basin. Because of its adaptability to a wide range of environmental conditions, the species have become a model for studies in coniferous forest management and functional genomics. Somatic embryogenesis (SE) has been so far, the preferred biotechnological strategy for maritime pine breeding programs initiated at the middle-end of the 20th century. To overcome the limitations of the induction and maturation phases in maritime pine SE, we analyzed the possible maternal influence on the embryogenic capability of megagametophytes from controlled crosses, as well as the effect of the temperature and water availabili…

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Somatic Embryogenesis and Plant Regeneration from Hypocotyl Cultures of Digitalis obscura L.

Summary Hypocotyl sections from 20-day-old seedlings of Digitalis obscura L. were used to examine the effect of IAA on somatic embryogenesis. Although embryogenesis was obtained with all IAA concentrations (0.1, 0.5 and 1 ppm) and under both illumination conditions tested (16 h photoperiod and darkness), incubation in photoperiod increased yield of embryos and shortened the embryo induction time. Optimum results were obtained with 1 ppm IAA. About 30 % of the embryos obtained developed into complete plantlets when transferred to basal medium without growth regulators.

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Priming maritime pine megagametophytes during somatic embryogenesis improved plant adaptation to heat stress

In the context of global climate change, forest tree research should be addressed to provide genotypes with increased resilience to high temperature events. These improved plants can be obtained by heat priming during somatic embryogenesis (SE), which would produce an epigenetic-mediated transgenerational memory. Thereby, we applied 37 °C or 50 °C to maritime pine (Pinus pinaster) megagametophytes and the obtained embryogenic masses went through the subsequent SE phases to produce plants that were further subjected to heat stress conditions. A putative transcription factor WRKY11 was upregulated in priming-derived embryonal masses, and also in the regenerated P37 and P50 plants, suggesting …

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Additional file 3: Table S2. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Intron length comparisons between Susy BAC clone from P. pinaster and Susy from two angiosperm plants. The intron I8 in the BAC clone contains a gap. The gene capture model is also included (DOCX 20 kb)

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Dynamics of Monoterpene Formation in Spike Lavender Plants

The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways was analyzed in spike lavender (Lavandula latifolia Med) on the basis of 13CO2-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first and key enzyme of the MVA pathway. The plants were labelled in the presence of 13CO2 in a gas chamber for controlled pulse and chase periods of time. GC/MS and NMR analysis of 1,8-cineole and camphor, the major monoterpenes present in their essential oil, indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes …

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Additional file 5: Table S4. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Intron length comparison between the XET BAC clone from P. pinaster and two XET genes from Arabidopsis thaliana. The two gene capture models closest to the BAC clone are also included. (DOCX 17 kb)

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Adventitious shoot regeneration from hypocotyl cultures of service tree(Sorbus domesticaL.)

Effects of growth regulators and N03:NH4 ratio were investigated to determine the mor- phogenic capacity of hypocotyl explants of service tree (Sorbus domestica L.) Organogenic responses were preceded by an initial callus phase and depended mainly on the auxin type used. NAA induced root differentiation whereas IAA added to media containing BA promoted adventitious shoot regeneration. Best results were obtained with 2.5 μM each of BA and IAA (19% of caulogenic explants). This reponse was increased (up to 44%) by varying nitrate to ammonium ratio from 2:1 to 4:1.

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Holm Oak Quercus ilex L.

The holm oak is an evergreen tree species representative of Mediterranean forests. The largest populations of this species are in the Iberian Peninsula, forming the tree cover of an agro-silvo-pastoral system called “dehesa” in Spain and “montado” in Portugal. The high ecological and economic value of the species is of interest for rural development. The main current product are the acorns used for feeding Iberian black pigs, which are the basis of an important gastronomic industry. In addition, several bioactive compounds with new potential industrial applications in nutrition, pharmacology and cosmetics have been identified in acorns of oak species. Plantations with holm oak seedlings myc…

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Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves

Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the …

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Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia

Spike lavender (Lavandula latifolia) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T0 plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression …

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<p><strong><em>Diversispora valentina</em></strong><strong> (Diversisporaceae), a new species of arbuscular mycorrhizal fungi from the Mediterranean sand dunes of Spain</strong></p>

A new arbuscular mycorrhizal fungal species, Diversispora valentina, is described and illustrated. In the field, this species is associated with marine dunes located along the Mediterranean coast in eastern Spain. Spores of D. valentina occurred in sporocarps, in clusters, and singly in the soil or inside the roots of Ammophila arenaria (Poaceae), Elymus farctus (Poaceae), Otanthus maritimus (Asteraceae), and Echinophora spinosa (Apiaceae) in the six locations studied. A single-species culture of D. valentina was obtained using Trifolium repens as a host plant. The small subunit internal transcribed spacer and large subunit (SSU-ITS1-5.8S-ITS2-LSU) nrDNA sequences place the new species in t…

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Up-regulation of 1-deoxy-D-xylulose-5-phosphate synthase enhances production of essential oils in transgenic spike lavender.

Abstract Spike lavender (Lavandula latifolia) is an aromatic shrub cultivated worldwide for the production of essential oils. The major constituents of these oils are monoterpenes, which are obtained from isopentenyl diphosphate and dimethylallyl diphosphate precursors through the plastidial methylerythritol phosphate (MEP) pathway and/or the cytosolic mevalonate pathway. 1-Deoxy-d-xylulose-5-P synthase (DXS) catalyzes the first step of the MEP pathway. A cDNA coding for the Arabidopsis (Arabidopsis thaliana) DXS was constitutively expressed in spike lavender. Gas chromatography/mass spectrometry analyses revealed that transgenic plants accumulated significantly more essential oils compared…

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Additional file 2: Table S1. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Exon length comparison between SuSy BAC clone from P. pinaster and SuSy from two angiosperm plants. The first exon is lacking in the BAC clone. The gene capture model is also included (DOCX 19 kb)

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Additional file 3: Table S2. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Intron length comparisons between Susy BAC clone from P. pinaster and Susy from two angiosperm plants. The intron I8 in the BAC clone contains a gap. The gene capture model is also included (DOCX 20 kb)

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Holm Oak Somatic Embryogenesis: Current Status and Future Perspectives

Quercus ilex (holm oak) is one of the most representative trees in the Mediterranean basin, but now the sustainability of its ecosystems is at serious risk due to the lack of natural regeneration and to the presence of a severe disease called oak decline that has caused the death of thousands of trees. The application of biotechnological tools, such as somatic embryogenesis, allows programs of genetic improvement of the species to be speeded up and helps in the conservation of its ecosystems. Somatic embryogenesis is currently considered one of the main biotechnological techniques that has demonstrated significant benefits when has applied to forest tree species, providing significant advan…

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Micropropagation of juvenile and adult Sorbus domestica L.

Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.

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Additional file 2: Table S1. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Exon length comparison between SuSy BAC clone from P. pinaster and SuSy from two angiosperm plants. The first exon is lacking in the BAC clone. The gene capture model is also included (DOCX 19 kb)

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Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender

[EN] Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants con…

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Glomus ibericum, Septoglomus mediterraneum, and Funneliformis pilosus, three new species of arbuscular mycorrhizal fungi

Three new arbuscular mycorrhizal fungal species—Glomus ibericum, Septoglomus mediterraneum, and Funneliformis pilosus—are described and illustrated. In the field, the three species were associated with roots of Ammophila arenaria (Poaceae), Elymus farctus (Poaceae), Otanthus maritimus (Asteraceae), and Echinophora spinosa (Apiaceae) colonizing maritime dunes located along the Mediterranean coast in eastern Spain. The novelty of these species is supported by morphological, molecular, and phylogenetic analyses. Single-species cultures of S. mediterraneum and F. pilosus were obtained using Trifolium repens as a host plant, both forming arbuscular mycorrhizae, whereas single-species cultures fr…

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Metabolic cross-talk between pathways of terpenoid backbone biosynthesis in spike lavender

Abstract The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in developing spike lavender ( Lavandula latifolia Med) was analyzed using specific inhibitors and on the basis of 13 C-labeling experiments. The presence of mevinolin (MEV), an inhibitor of the MVA pathway, at concentrations higher than 0.5 μM significantly reduced plant development, but not the synthesis of chlorophylls and carotenoids. On the other hand, fosmidomycin (FSM), an inhibitor of the MEP pathway, at concentrations higher than 20 μM blocked the synthesis of chlorophyll, carotenoids and essential oils, and significantly reduced stem development. Notably, 1.2 mM MVA cou…

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Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves.

We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase c…

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Factors influencing axillary shoot proliferation and adventitious budding in cedar.

We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior ch…

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Additional file 5: Table S4. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Intron length comparison between the XET BAC clone from P. pinaster and two XET genes from Arabidopsis thaliana. The two gene capture models closest to the BAC clone are also included. (DOCX 17 kb)

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Additional file 13: Figure S7. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Functional categories by GO terms for the full-length unigenes used in the capture approach (TIF 269 kb)

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Additional file 7: Figure S3. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Alignment of the PAL gene promoter [GenBank:HE866754], to the gene capture PAL gene 5´upstream region (TIF 389 kb)

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Additional file 11: Table S6. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Summary of AS contigs obtained in the gene capture and used to build the AS model. (XLS 138 kb)

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Additional file 9: Figure S5. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Nucleotide alignment of maritime pine AS cDNAs. (TIF 533 kb)

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Additional file 6: Figure S2. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Alignment of the GS1a gene promoter [GenBank:AJ225121], to the gene capture GS1a gene 5´upstream region (TIF 245 kb)

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Additional file 6: Figure S2. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Alignment of the GS1a gene promoter [GenBank:AJ225121], to the gene capture GS1a gene 5´upstream region (TIF 245 kb)

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Additional file 8: Figure S4. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Alignment of the PAT gene promoter [GenBank:HE866755], to the gene capture PAT gene 5´upstream region. (TIF 332 kb)

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Additional file 7: Figure S3. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Alignment of the PAL gene promoter [GenBank:HE866754], to the gene capture PAL gene 5´upstream region (TIF 389 kb)

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Additional file 8: Figure S4. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Alignment of the PAT gene promoter [GenBank:HE866755], to the gene capture PAT gene 5´upstream region. (TIF 332 kb)

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Additional file 10: Figure S6. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Amino acid alignment of maritime pine AS protein (TIF 460 kb)

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Additional file 9: Figure S5. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Nucleotide alignment of maritime pine AS cDNAs. (TIF 533 kb)

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Additional file 1: Figure S1. of Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing

Workflow overview followed to isolate and sequence maritime pine BAC clones. (A) Primary PCR screening of pools of the BAC library using specific oligonucleotides against XET cDNA. (B) Plating of cells of the positive pools on 24.2 X 24.2 cm plates. (C) The single clones of the pool were individualized in 36 X 384 plates using a QPix and grown orderly gridded in high-density filters. (D) The replica filters containing the ordered single clones of each pool were hybridized with cDNA 32P-labeled specific probes and exposed to a phosphorimaging screen. (E) Secondary PCR screening was performed on single clones to isolate positives. (F) BAC DNA isolation of positives to prepare a 454 library ei…

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