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Acknowledgments We would like to thank Jorge Dominguez for supplying the plant material and Juan Antonio Martín for supplying the fungal strains. This research was funded by the Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA), and by the Spanish National Research Plan (AGL2012-35580). We would also like to express our gratitude to the Spanish Elm Breeding and Conservation Programme., Elms, especially Ulmus minor and U. americana, are carrying out a hard battle against Dutch elm disease (DED). This vascular wilt disease, caused by Ophiostoma ulmi and O. novo-ulmi, appeared in the twentieth century and killed millions of elms across North America and Europe. Elm breeding and conservation programmes have identified a reduced number of DED tolerant genotypes. In this study, three U. minor genotypes with contrasted levels of tolerance to DED were exposed to several biotic and abiotic stresses in order to (i) obtain a de novo assembled transcriptome of U. minor using 454 pyrosequencing, (ii) perform a functional annotation of the assembled transcriptome, (iii) identify genes potentially involved in the molecular response to environmental stress, and (iv) develop gene-based markers to support breeding programmes. A total of 58,429 putative unigenes were identified after assembly and filtering of the transcriptome. 32,152 of these unigenes showed homology with proteins identified in the genome from the most common plant model species. Well-known family proteins and transcription factors involved in abiotic, biotic or both stresses were identified after functional annotation. A total of 30,693 polymorphisms were identified in 7,125 isotigs, a large number of them corresponding to single nucleotide polymorphisms (SNPs; 27,359). In a subset randomly selected for validation, 87% of the SNPs were confirmed. The material generated may be valuable for future Ulmus gene expression, population genomics and association genetics studies, especially taking into account the scarce molecular information available for this genus and the great impact that DED has on elm populations, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The adaptive capacity of a species and its population is determined by both genetic and epigenetic variation, which defines the potential for adaptive evolution and plastic response to environmental changes. In this study, we used Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism (AFLP), similar genome-wide profiling techniques, to analyze the epigenetic and genetic variability in European beech provenances from Germany (DE), Spain (ES) and Sweden (SE), representing the latitudinal distribution of the species. In addition, we evaluated the effect of moderate water stress on cytosine methylation dynamics by comparing two latitudinal contrasting provenances. Our analysis revealed that trees from ES showed lower values of epigenetic and genetic diversity than those from DE and SE. Analyses of molecular variance for MSAPs and AFLPs showed that 16% and 15% of the among population variations were associated with epigenetic and genetic variation, respectively. The study of the effect of water stress on cytosine methylation dynamics in seedlings from ES and SE revealed no significant levels of epigenetic differentiation between well-watered and stressed plants. Only 2% of the epigenetic variation was explained by the watering regime in ES without changes in SE. The results support that DNA methylation may play a role in the local adaptation of Fagus sylvatica to environmental variation.

Increasing temperatures along with severe droughts are factors that may jeopardize the survival of the forests in the Mediterranean basin. In this region, Pinus pinaster is a common conifer species, that has been used as a model species in evolutionary studies due to its adaptive response to changing environments. Although its drought tolerance mechanisms are already known, knowledge about the dynamics of its root microbiota is still scarce. We aimed to decipher the structural (bacterial abundance), compositional, functional and associative changes of the P. pinaster rhizosphere bacterial communities in spring and summer, at DNA and RNA level (environmental DNA, live and dead cells, and those synthesizing proteins). A fundamental aspect of root microbiome-based approaches is to guarantee the correct origin of the samples. Thus, we assessed the genotype of host needles and roots from which rhizosphere samples were obtained. For more than 50% of the selected trees, genotype discrepancies were found and in three cases the plant species could not be determined. Rhizosphere bacterial communities were homogeneous with respect to diversity and structural levels regardless of the host genotype in both seasons. Nonetheless, significant changes were seen in the taxonomic profiles depending on the season. Seasonal changes were also evident in the bacterial co-occurrence patterns, both in DNA and RNA libraries. While spring communities switched to more complex networks, summer populations resulted in more compartmentalized networks, suggesting that these communities were facing a disturbance. These results may mirror the future status of bacterial communities in a context of climate change. A keystone hub was ascribed to the genus Phenylobacterium in the functional network calculated for summer. Overall, it is important to validate the origin and identity of plant samples in any plant-microbiota study so that more reliable ecological analyses are performed.

The Mediterranean basin is drastically affected by intense and frequent droughts, which jeopardize the diversity and survival of its forest, for example, Pinus pinaster forests. The dynamics of the bacterial communities inhabiting the rhizosphere of Pinus pinaster and other plants from a pine dominated forest under contrasting hydric conditions was monitored. The forest was located in Sierra de Oria (southeast Spain), and it was mainly composed by P. pinaster, P. halepensis, woody shrub species and herbaceous plants. 18 trees visually belonging to P. pinaster located along the perimeter and across the forest were selected for the analysis. All the trees were separated at least 50 m each other. Although all of them belonged to P. pinaster morphologically according to visual identification, the genotyping of the roots confirmed that they corresponded to P. pinaster, P. halepensis, and other plant species different from genus Pinus, although in the last case it was not possible to identify the plant species. At a distance less than 50 cm from the trunk, the litter and topsoil were removed, and the soil closely attached to non-suberified roots (rhizosphere soil) was collected (depth of 5-25 cm). Sampling was carried out in two seasons with contrasting temperature and rainfall patterns: on July 18, 2017 (summer) and April 24, 2018 (spring). After rhizosphere soil DNA and RNA extraction (and cDNA synthesis), a metabarcoding approach was followed by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene and its derived transcripts by Illumina MiSeq platform. Sequencing reads were bioinformatically processed; specifically, they were filtered, trimmed, clustered into ASV (Amplicon Sequence Variants), and taxonomically identified. As a result, a total of 1,123,209 and 1,089,359 quality sequences were obtained from DNA and RNA-derived libraries, which resulted in 5,241 and 5,231 ASVs, respectively. Total communities (DNA) were mainly dominated by phyla Proteobacteria

Intraspecific grafting with selected scions and rootstocks was used to study drought tolerance in the recalcitrant Mediterranean conifer P. pinaster. Conifers store different terpene types in needles, stems and roots, which can be modified by drought, but most studies have focused on a single organ and over selected terpenes which limits understanding of the overall plant response. In this work, terpene profile (160 components among mono-, sesqui-, di-terpenes and resin acids) as well as eco-physiological traits were assessed in needles, roots, and scion and rootstock stems to analyze the drought sensibility of scions and rootstocks on grafted P. pinaster trees. We used two scions from wet (Galicia) and dry (Oria) provenances grafted onto rootstocks showing differences in the efficiency of water use, grown under two different water regimes, well-watered and water stressed. Constitutively, organs showed clearly different qualitative and quantitative terpene profiles, such as a long-term optimized resources allocation, being neutral and acid diterpenes those more discriminant. Environmental factors may selectively affect the regulation of diterpene biosynthesis in needles, since scion provenances determined the variation of their profiles, mainly regarding again neutral and acid diterpenes. Different scion-conferred terpene phenotypes were observed in each organ, having a relevant role isoabienol content. Drought triggered needle/root resin acids redistribution, which may provide higher protection to roots, correlated to the effective water stress endured by plants. Drought sensitivity of scion-rootstock combinations determined the intensity of drought-triggered changes in terpene levels in all organs, suggesting that chemical defense and drought tolerance are somehow interconnected.

Understanding the consequences of local adaptation at the genomic diversity is a central goal in evolutionary genetics of natural populations. In species with large continuous geographical distributions the phenotypic signal of local adaptation is frequently clear, but the genetic basis often remains elusive. We examined the patterns of genetic diversity in Pinus sylvestris, a keystone species in many Eurasian ecosystems with a huge distribution range and decades of forestry research showing that it is locally adapted to the vast range of environmental conditions. Making P. sylvestris an even more attractive subject of local adaptation study, population structure has been shown to be weak previously and in this study. However, little is known about the molecular genetic basis of adaptation, as the massive size of gymnosperm genomes has prevented large scale genomic surveys. We generated a both geographically and genomically extensive dataset using a targeted sequencing approach. By applying divergence-based and landscape genomics methods we identified several loci contributing to local adaptation, but only few with large allele frequency changes across latitude. We also discovered a very large (ca. 300 Mbp) putative inversion potentially under selection, which to our knowledge is the first such discovery in conifers. Our results call for more detailed analysis of structural variation in relation to genomic basis of local adaptation, emphasize the lack of large effect loci contributing to local adaptation in the coding regions and thus point out the need for more attention toward multi-locus analysis of polygenic adaptation.

Adaptation of long-living forest trees to respond to environmental changes is essential to secure their performance under adverse conditions. Water deficit is one of the most significant stress factors determining tree growth and survival. Maritime pine (Pinus pinaster Ait.), the main source of softwood in southwestern Europe, is subjected to recurrent drought periods which, according to climate change predictions for the years to come, will progressively increase in the Mediterranean region. The mechanisms regulating pine adaptive responses to environment are still largely unknown. The aim of this work was to go a step further in understanding the molecular mechanisms underlying maritime pine response to water stress and drought tolerance at the whole plant level. A global transcriptomic profiling of roots, stems, and needles was conducted to analyze the performance of siblings showing contrasted responses to water deficit from an ad hoc designed full-sib family. Although P. pinaster is considered a recalcitrant species for vegetative propagation in adult phase, the analysis was conducted using vegetatively propagated trees exposed to two treatments: well-watered and moderate water stress. The comparative analyses led us to identify organ-specific genes, constitutively expressed as well as differentially expressed when comparing control versus water stress conditions, in drought-sensitive and drought-tolerant genotypes. Different response strategies can point out, with tolerant individuals being pre-adapted for coping with drought by constitutively expressing stress-related genes that are detected only in latter stages on sensitive individuals subjected to drought.

An increasing number of microRNAs (miRNAs) and miRNA-related sequences produced during miRNA biogenesis, comprising the isomiRome, have been recently highlighted in different species as critical mediators of environmental stress responses. Conifers have some of the largest known genomes but an extensive characterization of the isomiRome from any conifer species has been lacking. We provide here a comprehensive overview of the Pinus pinaster isomiRome expressed in roots, stem and needles under well-watered and drought conditions. From the 13,441 unique small RNA sequences identified, 2,980 were annotated as canonical miRNAs or miRNA* and the remaining were classified as isomiRNA or miRNA-like sequences. A survey of their expression patterns highlighted roots as the most responsive organ under drought, where specific sequences of which a 24-nt novel miRNA stood out, were strongly down-regulated. Given the putative roles of the miRNA-targeted transcripts validated specifically in root tissues, some of the miRNAs, conserved and novel, are shortlisted as potential regulators of drought response. These results provide a valuable resource for comparative studies between gymnosperms and angiosperms. Furthermore, it evidences high transferability of the isomiRome between pine species being a useful basis for further molecular regulation and physiological studies, and especially those focused on adaptation to drought conditions.

Funding information: FP7 People: Marie-Curie Actions, Grant/Award Number: PIEF-GA-2013-627761; Fundação para a Ciência e a Tecnologia, Grant/Award Numbers: IF/01168/2013, SFRH/BD/79779/2011; SFRH/BD/128827/2017; UID/Multi/04551/2013; UID/Multi/04046/2019; DL57/2016/CP1351/CT0003; Horizon 2020 Framework Programme, Grant/Award Number: 676559; Ministerio de Economía, Industria y Competitividad, Gobierno de España, Grant/Award Numbers: AGL2015-66048-C2-1-R, RTI2018-098015-B-I00; Seventh Framework Programme, Grant/Award Number: 289841, An increasing number of microRNAs (miRNAs) and miRNA-related sequences produced during miRNA biogenesis, comprising the isomiRome, have been recently highlighted in different species as critical mediators of environmental stress responses. Conifers have some of the largest known genomes but an extensive characterization of the isomiRome from any conifer species has been lacking. We provide here a comprehensive overview of the Pinus pinaster isomiRome expressed in roots, stem and needles under well-watered and drought conditions. From the 13,441 unique small RNA sequences identified, 2,980 were annotated as canonical miRNAs or miRNA* and the remaining were classified as isomiRNA or miRNA-like sequences. A survey of their expression patterns highlighted roots as the most responsive organ under drought, where specific sequences of which a 24-nt novel miRNA stood out, were strongly down-regulated. Given the putative roles of the miRNA-targeted transcripts validated specifically in root tissues, some of the miRNAs, conserved and novel, are shortlisted as potential regulators of drought response. These results provide a valuable resource for comparative studies between gymnosperms and angiosperms. Furthermore, it evidences high transferability of the isomiRome between pine species being a useful basis for further molecular regulation and physiological studies, and especially those focused on adaptation to drought conditions., European Commission, Ministerio de Economía y Competitividad (España), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

ACKNOWLEDGMENTS I. Aranda (INIA-CIFOR) is gratefully acknowledged for his valuable collaboration and support in obtaining and interpreting data on physiology. D. Sobral (Instituto Gulbenkian de Ciência, Portugal) is gratefully acknowledged for his advice in the bioinformatics analyses. Bartolomé Sabater is gratefully acknowledged for critical review of the manuscript. S. Ferrándiz (INIA-CIFOR) is gratefully acknowledged for helping with the preparation of the manuscript. This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (AGL2015-66048-C2-1-R; RTI2018-098015-B-I00; IMP2018-005)]; European Union's Seventh Framework Programme 2007-2013 (FP7-289841); EU H2020 Programme (H2020-676559); Fundação para a Ciência e a Tecnologia, FCT (IF/01168/2013; UID/Multi/04551/2013); and People Programme, Marie Curie Actions (PIEF-GA-2013-627761)., Adaptation of long-living forest trees to respond to environmental changes is essential to secure their performance under adverse conditions. Water deficit is one of the most significant stress factors determining tree growth and survival. Maritime pine (Pinus pinaster Ait.), the main source of softwood in southwestern Europe, is subjected to recurrent drought periods which, according to climate change predictions for the years to come, will progressively increase in the Mediterranean region. The mechanisms regulating pine adaptive responses to environment are still largely unknown. The aim of this work was to go a step further in understanding the molecular mechanisms underlying maritime pine response to water stress and drought tolerance at the whole plant level. A global transcriptomic profiling of roots, stems, and needles was conducted to analyze the performance of siblings showing contrasted responses to water deficit from an ad hoc designed full-sib family. Although P. pinaster is considered a recalcitrant species for vegetative propagation in adult phase, the analysis was conducted using vegetatively propagated trees exposed to two treatments: well-watered and moderate water stress. The comparative analyses led us to identify organ-specific genes, constitutively expressed as well as differentially expressed when comparing control versus water stress conditions, in drought-sensitive and drought-tolerant genotypes. Different response strategies can point out, with tolerant individuals being pre-adapted for coping with drought by constitutively expressing stress-related genes that are detected only in latter stages on sensitive individuals subjected to drought, Ministerio de Economía y Competitividad (España), European Commission, Portuguese Foundation for Science and Technology, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Pinus pimaster Aiton displays marked heteroblasty during its vegetative phase, showing complex interactions between developmental processes and environmental adaptation, with a patent differentiation in functional performance according to leaf type resulting from heteroblasty. In this work, untargeted quantitative metabolic profiling in adult and juvenile needles of four water-deficit stressed P. pinaster genotypes was investigated to know if metabolic responses to drought are genotype-dependent and vary according to the stage of needle ontogeny. Among the changes in metabolome, it highlights the increase of osmotically active substances, as well as the overproduction of antioxidant compounds by up-regulating ascorbate, shikimate/phenylalanine and phenylpropanoid metabolic pathways. Moreover, significant inter-clonal quantitative variations were also found at different levels, in both primary and secondary metabolism pathways, although terpenoids and specially flavonoids showed the highest significance. A surprising result was the ontogenetic changes in metabolic profiles. Juvenile needles contain significant higher levels of UV screener and antioxidant flavonoids. The level of drought tolerance of a genotype and the ontogenetic stage were important factors in determining the drought-induced level of flavonoid increase in needles of a given genotype. Regarding terpenoids, the significant drought-induced accumulation of neutral diterpenes in juvenile needles, and mono- and sesquiterpenes in adults, was genotype dependent regardless its degree of drought tolerance. These results point to flavonoids as very versatile metabolites in maritime pine needle, acting like important compounds modulating not only drought acclimation but also changes in needle metabolism associated with developmental processes such as heteroblasty. © 2017 Elsevier B.V.

Phylogenetic relationships among seed plant taxa, especially within the gymnosperms, remain contested. In contrast to angiosperms, for which several genomic, transcriptomic and phylogenetic resources are available, there are few, if any,molecularmarkers that allow broad comparisons among gymnosperm species. With few gymnosperm genomes available, recently obtained transcriptomes in gymnosperms are a great addition to identifying single-copy gene families as molecular markers for phylogenomic analysis in seed plants. Taking advantage of an increasing number of available genomes and transcriptomes, we identified singlecopy genes in a broad collection of seed plants and used these to infer phylogenetic relationships between major seed plant taxa. This study aims at extending the current phylogenetic toolkit for seed plants, assessing its ability for resolving seed plant phylogeny, and discussing potential factors affecting phylogenetic reconstruction. In total, we identified 3,072 single-copy genes in 31 gymnosperms and 2,156 single-copy genes in 34 angiosperms. All studied seed plants shared 1,469 single-copy genes, which are generally involved in functions likeDNAmetabolism, cell cycle, and photosynthesis.Aselected set of 106 single-copy genes provided good resolution for the seed plant phylogeny except for gnetophytes. Although some of our analyses support a sister relationship between gnetophytes and other gymnosperms, phylogenetic trees from concatenated alignments without 3rd codon positions and amino acid alignments under theCAT+GTRmodel, support gnetophytes as a sister group to Pinaceae.Our phylogenomic analyses demonstrate that, in general, single-copy genes can uncover both recent and deep divergences of seed plant phylogeny. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Haploids are a valuable tool for genomic studies in higher plants, especially those with huge genome size and long juvenile periods, such as conifers. In these species, megagametophyte cultures have been widely used to obtain haploid callus and somatic embryogenic lines. One of the main problems associated with tissue culture is the potential genetic instability of the regenerants. Because of this, chromosomal stability of the callus and/or somatic embryos should also be assessed. To this end, chromosome counting, flow cytometry and genotyping using microsatellites have been reported. Here, we present an overview of the work done in conifers, with special emphasis on the production of a haploid cell line in maritime pine (Pinus pinaster L.) and the use of a set of molecular markers, which includes Single Nucleotide Polymorphisms (SNPs) and microsatellites or Single Sequence Repeats (SSRs), to validate chromosomal integrity confirming the presence of all chromosomic arms. © 2016 by the authors.

Maritime pine provides essential ecosystem services in the south-western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three-generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene-based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies. © 2016 John Wiley & Sons Ltd.

Drought is an important driver of plant survival, growth, and distribution. Water deficit affects different pathways of metabolism, depending on plant organ. While previous studies have mainly focused on the metabolic drought response of a single organ, analysis of metabolic differences between organs is essential to achieve an integrated understanding of the whole plant response. In this work, untargeted metabolic profiling was used to examine the response of roots, stems, adult and juvenile needles from Pinus pinaster Ait. full-sib individuals, subjected to a moderate and long lasting drought period. Cyclitols content showed a significant alteration, in response to drought in all organs examined, but other metabolites increased or decreased differentially depending on the analyzed organ. While a high number of flavonoids were only detected in aerial organs, an induction of the glutathione pathway was mainly detected in roots. This result may reflect different antioxidant mechanisms activated in aerial organs and roots. Metabolic changes were more remarkable in roots than in the other organs, highlighting its prominent role in the response to water stress. Significant changes in flavonoids and ascorbate metabolism were also observed between adult and juvenile needles, consistent with previously proven differential functional responses between the two developmental stages. Genetic polymorphisms in candidate genes coding for a Myb1 transcription factor and a malate dehydrogenase (EC 1.1.1.37) were associated with different concentration of phenylalanine, phenylpropanoids and malate, respectively. The results obtained will support further research on metabolites and genes potentially involved in functional mechanisms related to drought tolerance in trees. © 2016 Elsevier Masson SAS.

We have carried out a candidate-gene-based association genetic study in Pinus pinaster Aiton and evaluated the predictive performance for genetic merit gain of the most significantly associated genes and single nucleotide polymorphisms (SNPs). We used a second generation 384-SNP array enriched with candidate genes for growth and wood properties to genotype mother trees collected in 20 natural populations covering most of the European distribution of the species. Phenotypic data for total height, polycyclism, root-collar diameter and biomass were obtained from a replicated provenance-progeny trial located in two sites with contrasting environments (Atlantic vs Mediterranean climate). General linear models identified strong associations between growth traits (total height and polycyclism) and four SNPs from the korrigan candidate gene, after multiple testing corrections using false discovery rate. The combined genomic breeding value predictions assessed for the four associated korrigan SNPs by ridge regression-best linear unbiased prediction (RR-BLUP) and cross-validation accounted for up to 8 and 15% of the phenotypic variance for height and polycyclic growth, respectively, and did not improve adding SNPs from other growth-related candidate genes. For root-collar diameter and total biomass, they accounted for 1.6 and 1.1% of the phenotypic variance, respectively, but increased to 15 and 4.1% when other SNPs from lp3.1, lp3.3 and cad were included in RR-BLUP models. These results point towards a desirable integration of candidate-gene studies as a means to pre-select relevant markers, and aid genomic selection in maritime pine breeding programs. © 2015 The Author. Published by Oxford University Press. All rights reserved.

Elms, especially Ulmus minor and U. americana, are carrying out a hard battle against Dutch elm disease (DED). This vascular wilt disease, caused by Ophiostoma ulmi and O. novo-ulmi, appeared in the twentieth century and killed millions of elms across North America and Europe. Elm breeding and conservation programmes have identified a reduced number of DED tolerant genotypes. In this study, three U. minor genotypes with contrasted levels of tolerance to DED were exposed to several biotic and abiotic stresses in order to (i) obtain a de novo assembled transcriptome of U. minor using 454 pyrosequencing, (ii) perform a functional annotation of the assembled transcriptome, (iii) identify genes potentially involved in the molecular response to environmental stress, and (iv) develop gene-based markers to support breeding programmes. A total of 58,429 putative unigenes were identified after assembly and filtering of the transcriptome. 32,152 of these unigenes showed homology with proteins identified in the genome from the most common plant model species. Well-known family proteins and transcription factors involved in abiotic, biotic or both stresses were identified after functional annotation. A total of 30,693 polymorphisms were identified in 7,125 isotigs, a large number of them corresponding to single nucleotide polymorphisms (SNPs; 27,359). In a subset randomly selected for validation, 87% of the SNPs were confirmed. The material generated may be valuable for future Ulmus gene expression, population genomics and association genetics studies, especially taking into account the scarce molecular information available for this genus and the great impact that DED has on elm populations. © 2015 Perdiguero, Venturas, Cervera, Gil and Collada.

Although recent advances have been gained on genome evolution in angiosperm lineages, virtually nothing is known about karyotype evolution in the other group of seed plants, the gymnosperms. Here, we used high-density gene-based linkagemapping to compare the karyotype structure of two families of conifers (the most abundant group of gymnosperms) separated around 290 Ma Pinaceae and Cupressaceae.We propose for the first time amodel based on the fusion of 20 ancestral chromosomal blocks that may have shaped the modern karyotpes of Pinaceae (with n=12) and Cupressaceae (with n=11). The considerable difference in modern genome organization between these two lineages contrasts strongly with the remarkable level of synteny already reported within the Pinaceae. It also suggests a convergent evolutionary mechanism of chromosomal block shuffling that has shaped the genomes of the spermatophytes. ©The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

The potential of haploid tissues for genetic studies in conifers is hampered by the lack of abundant and homogeneous plant material suitable for DNA isolation. In this work we have determined factors promoting haploid callus induction and proliferation from megagametophytes of Oria 6, a genotype of Pinus pinaster Aiton (maritime pine) from the natural population Sierra de Oria (Almería, Spain), selected based on its response to extreme drought conditions. The generation of haploid callus was restricted to megagametophytes isolated from light-brown cones with no dehydrated seeds collected in September. Culture medium composition did not significantly affect callus induction, but a modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine favored further multiplication. The ploidy status of the callus lines was determined by flow cytometry and seven polymorphic microsatellites. A total of sixteen haploid callus lines were established and one of these is being used as a source of DNA for massive sequencing of maritime pine genome. © 2014 Springer Science+Business Media Dordrecht.

Background Understanding molecular mechanisms that control photosynthesis and water use efficiency in response to drought is crucial for plant species from dry areas. This study aimed to identify QTL for these traits in a Mediterranean conifer and tested their stability under drought.Results High density linkage maps for Pinus pinaster were used in the detection of QTL for photosynthesis and water use efficiency at three water irrigation regimes. A total of 28 significant and 27 suggestive QTL were found. QTL detected for photochemical traits accounted for the higher percentage of phenotypic variance. Functional annotation of genes within the QTL suggested 58 candidate genes for the analyzed traits. Allele association analysis in selected candidate genes showed three SNPs located in a MYB transcription factor that were significantly associated with efficiency of energy capture by open PSII reaction centers and specific leaf area.Conclusions The integration of QTL mapping of functional traits, genome annotation and allele association yielded several candidate genes involved with molecular control of photosynthesis and water use efficiency in response to drought in a conifer species. The results obtained highlight the importance of maintaining the integrity of the photochemical machinery in P. pinaster drought response. © 2014 de Miguel et al.; licensee BioMed Central Ltd.