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21 Pág. Instituto de Ciencias Forestales (ICIFOR), 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., This research was funded by the Spanish Ministry of Science and Innovation (AGL2014-57762-R and AGl2011-25365).

International audience; The advent of next-generation genome sequencing technologies has allowed approaching the sequencing and analysis of large and complex conifer genomes. Maritime pine (Pinus pinaster Ait.) is an economically and ecologically important conifer species widely distributed in South-West Europe, which shows a significant genetic and adaptive variability. This chapter takes on the task of reviewing the insights into the maritime pine genome sequencing breakthrough and its impact on downstream analysis. Maritime pine genome sequencing and assembly approaches are described along with the impact of related tools. A section of the state-of-the-art research on comparative, functional, structural, and translational genomics aimed at dissecting the genetic basis and the specific regulation of biological processes underlying the expression of traits of interest in maritime pine and other conifers is also described. Perspectives about the impact of these tools as well as additional research approaches are discussed.

16 Pág., 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., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness [AGL2015-66048-C2-1-R; RTI2018-098015-B-I00], and by the Spanish Ministry of Science and Innovation project SUMHAL [LIFEWATCH-2019-09-CSIC-13, POPE 2014-2020], both co-financed by the (ERDF)European Regional Development Fund

Pines are the dominant conifers in Mediterranean forests. As long-lived sessile organisms that seasonally have to cope with drought periods, they have developed a variety of adaptive responses. However, during last decades, highly intense and long-lasting drought events could have contributed to decay and mortality of the most susceptible trees. Among conifer species, Pinus pinaster Ait. shows remarkable ability to adapt to different environments. Previous molecular analysis of a full-sib family designed to study drought response led us to find active transcriptional activity of stress-responding genes even without water deprivation in tolerant genotypes. To improve our knowledge about communication between above- and below-ground organs of maritime pine, we have analyzed four graft-type constructions using two siblings as rootstocks and their progenitors, Gal 1056 and Oria 6, as scions. Transcriptomic profiles of needles from both scions were modified by the rootstock they were grafted on. However, the most significant differential gene expression was observed in drought-sensitive Gal 1056, while in drought-tolerant Oria 6, differential gene expression was very much lower. Furthermore, both scions grafted onto drought-tolerant rootstocks showed activation of genes involved in tolerance to abiotic stress, and is most remarkable in Oria 6 grafts where higher accumulation of transcripts involved in phytohormone action, transcriptional regulation, photosynthesis and signaling has been found. Additionally, processes, such as those related to secondary metabolism, were mainly associated with the scion genotype. This study provides pioneering information about rootstock effects on scion gene expression in conifers., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (AGL2015-66048-C2-1-R; RTI2018-098015-B-I00), and by University of Alcalá (UAH-AE 2017-2).

23 Pág. Centro de Investigación en Sanidad Animal (CISA), 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., 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

Eccrine porocarcinoma is a rare, malignant cutaneous adnexal tumor that arises from the ducts of sweat glands. Found mainly in patients of advanced age, this tumor has diverse clinical presentations. Histology confirms the diagnosis, detects features relevant to prognosis, and guides treatment. Growth is slow, but the prognosis is poor if the tumor metastasizes to lymph nodes or visceral organs. We report 7 cases of eccrine porocarcinoma, describing patient characteristics, the clinical and histopathologic features of the tumors, and treatments used. Our observations were similar to those of other published case series. Given the lack of therapeutic algorithms or protocols for this carcinoma, we propose a decision-making schema based on our review of the literature and our experience with this case series. The algorithm centers on sentinel lymph node biopsy and histologic features.

Resumen El porocarcinoma ecrino es un tumor anexial cutaneo maligno poco frecuente, que deriva de la porcion ductal de las glandulas sudoriparas. Predomina en pacientes de edad avanzada, pudiendo adoptar diversas presentaciones clinicas. El estudio histologico confirma el diagnostico y establece factores pronosticos relevantes a la hora de decidir el tratamiento. El curso evolutivo es lento, pero su pronostico es sombrio cuando aparecen metastasis linfaticas o viscerales. Presentamos 7 pacientes diagnosticados de porocarcinoma ecrino, y describimos los aspectos epidemiologicos, clinicos, histopatologicos y los datos relacionados con el tratamiento de cada uno de ellos. Comparamos los datos obtenidos con las mayores series de casos publicadas, obteniendo similares resultados. Ante la ausencia de algoritmos terapeuticos protocolizados se propone un esquema de tratamiento basado en la literatura revisada y la experiencia personal con nuestra serie, que tiene como ejes centrales los factores pronosticos histologicos y la biopsia selectiva de ganglio centinela.

Fibro-osseous pseudotumor of the digits is a benign neoplasm that originates in the soft tissue adjacent to the short bones of the hands and feet. We present a case in a 13-year-old girl that was initially misdiagnosed as pyogenic granuloma. Familiarity with this entity and imaging and histologic studies are necessary to avoid incorrect diagnoses and aggressive surgical interventions.

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

Central centrifugal cicatricial alopecia (CCCA) is included among the primary lymphocytic cicatricial alopecias. The current nomenclature of CCCA suggested by the North American Hair Research Society refers to the traditional clinical presentation pattern of this type of alopecia, which begins in the central area of the scalp and has a progressive and symmetric centrifuge evolution. However, some exceptions should be highlighted, and a new clinical variety of CCCA presenting with patches of hair loss affecting the lateral and posterior scalp has been recently described. Here, we report a new case of CCCA presenting with a patchy pattern. In addition to the fact that this new patchy presentation of CCCA is not contemplated in the current terminology, it has a difficult differential diagnosis with other alopecias that have patches as their presentation. In these cases, both the trichoscopy and the histopathology are indispensable for diagnosis that will allow a targeted treatment and avoid an unfortunate prognosis.

Wine production in most countries is based on the use of commercial strains leading to the colonisation of the wineries by these strains with the consequent reduction of autochthonous biodiversity. This implies that wine styles could therefore become standardised. The vineyard could be an important source of native yeasts of oenological interest. For this reason the objective of this study was to compare two agronomic conditions with the aim of preserving yeast biodiversity in the vineyard. A three year sampling plan was designed to evaluate the influence of different agronomic parameters on the biodiversity of fermentative grape yeasts. Thus two vineyards, one organic and one conventional, with three different grape varieties (Shiraz, Grenache and Barbera) were chosen. In total, 27 samples were collected from both vineyards. Of these, 1080 colonies were isolated and a total of 9 species were identified. The strains identified as Saccharomyces cerevisiae were genotyped by microsatellite analysis obtaining nine different electrophoretic patterns. Classical ecology indexes were used to obtain the richness (S), the biodiversity (H') and the dominance (D) of the species studied. The results indicated a clear influence on grape associated yeast diversity of the phytosanitary treatment used in the vineyard. This is the first time that classical ecology indexes have been used to study the ecology of the spontaneous fermentation of grape musts and the species Candida sorbosa and Pichia toletana have been described in vineyards of the Madrid winegrowing region.

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.

Eucalyptus globulus Labill. has been used in Spain for decades as a cellulose source for the paper and textile industry. Since 1997, Sniace group has tested new provenances and families from 29 different Eucalyptus species to explore their capacity for biomass and bioenergy production. Plus trees for growth, wood quality, rooting capacity and tolerance to Mycosphaerella sp have been identified by mass selection, and more than 300 controlled crosses among those trees have been carried out. However, the restrictions caused by the high susceptibility to Mycosphaerella leaf disease and the low rooting capacity of the species Eucalyptus globulus Labill. delay the application of the gains to a commercial scale. The objective of this project is to improve the production of Eucalyptus in the North of Spain focused on the improvement of two traits of economic importance: clonal/rooting capacity and tolerance to Mycosphaerella sp. To achieve this goal three partial objectives have been approached