DNA extraction and amplification from Pinaceae dry wood

Wood constitutes the unique source of DNA in dead trees, but extraction of adequate quality DNA from dry wood is usually challenging. However, many different molecular studies require the use of such DNA. We have standardized and validated a modified CTAB protocol to isolate DNA from dry wood from Abies pinsapo and Cedrus atlantica species. Due to the degradation and very little DNA that is normally present in the wood from dead trees we have developed a PCR based test to certify the quality of the extracted samples. In the present study, we have proved too the effectiveness of this methodology to isolate DNA from conifer dry wood samples of sufficient quality to perform further molecular genetic experiments

De novo transcriptome sequencing and gene co-expression reveal a genomic basis for drought sensitivity and evidence of a rapid local adaptation on Atlas cedar (Cedrus atlantica)

IntroductionUnderstanding the adaptive capacity to current climate change of drought-sensitive tree species is mandatory, given their limited prospect of migration and adaptation as long-lived, sessile organisms. Knowledge about the molecular and eco-physiological mechanisms that control drought resilience is thus key, since water shortage appears as one of the main abiotic factors threatening forests ecosystems. However, our current background is scarce, especially in conifers, due to their huge and complex genomes.MethodsHere we investigated the eco-physiological and transcriptomic basis of drought response of the climate change-threatened conifer Cedrus atlantica. We studied C. atlantica seedlings from two locations with contrasting drought conditions to investigate a local adaptation. Seedlings were subjected to experimental drought conditions, and were monitored at immediate (24 hours) and extended (20 days) times. In addition, post-drought recovery was investigated, depicting two contrasting responses in both locations (drought resilient and non-resilient). Single nucleotide polymorphisms (SNPs) were also studied to characterize the genomic basis of drought resilience and investigate a rapid local adaptation of C. atlantica.ResultsDe novo transcriptome assembly was performed for the first time in this species, providing differences in gene expression between the immediate and extended treatments, as well as among the post-drought recovery phenotypes. Weighted gene co-expression network analysis showed a regulation of stomatal closing and photosynthetic activity during the immediate drought, consistent with an isohydric dynamic. During the extended drought, growth and flavonoid biosynthesis inhibition mechanisms prevailed, probably to increase root-to-shoot ratio and to limit the energy-intensive biosynthesis of secondary metabolites. Drought sensitive individuals failed in metabolism and photosynthesis regulation under drought stress, and in limiting secondary metabolite production. Moreover, genomic differences (SNPs) were found between drought resilient and sensitive seedlings, and between the two studied locations, which were mostly related to transposable elements.DiscussionThis work provides novel insights into the transcriptomic basis of drought response of C. atlantica, a set of candidate genes mechanistically involved in its drought sensitivity and evidence of a rapid local adaptation. Our results may help guide conservation programs for this threatened conifer, contribute to advance drought-resilience research and shed light on trees’ adaptive potential to current climate change

Understanding genetic diversity of relict forests. Linking long-term isolation legacies and current habitat fragmentation in Abies pinsapo Boiss

Increasing variability and uncertainty regarding future climate provide new challenges for the conservation of endangered tree species. For example, threat status can be impacted by genetic diversity, where forest trees show reduced geographic range size, isolated populations and fragmented distribution. We place the conservation insights of population genetic structure in a climate change context, using as experimental system a relict drought-sensitive fir (Abies pinsapo Boiss.). Nuclear (nSSR, ISSR) and chloroplast (cpSSR) markers were analysed to investigate the extent to that A. pinsapo evidences ongoing genetic erosion, isolation and divergent genetic diversity, among populations, elevations and cohorts (young, adult and old trees). We obtained contrasting patterns among chloroplast and nuclear markers. Based on cpSSRs, the highest genetic distances were found in the western portion of the distribution, while based on both nSSRs and ISSRs, differentiation appeared in the eastern portion of the distribution. Evidence for bottlenecks and genetic drift were found in all the studied populations, as well as low among-population genetic differentiation. Land use legacies e.g. impacting current forest structural diversity might be related to observed genetic diversity. No evidence of demographic genetic erosion among cohorts was found. Conservation efforts should focus on reducing the probability of occurrence of stochastic events such as fires and habitat loss due to human impacts or climate change to maximise A. pinsapo population sizes. Further research on adaptive potential should focus on identifying active genetic management strategies that might improve adaptation to future climates in such endangered relict species

Gene Frequency Shift in Relict Abies pinsapo Forests Associated with Drought-Induced Mortality: Preliminary Evidence of Local-Scale Divergent Selection

Current climate change constitutes a challenge for the survival of several drought-sensitive forests. The study of the genetic basis of adaptation offers a suitable way to understand how tree species may respond to future climatic conditions, as well as to design suitable conservation and management strategies. Here, we focus on selected genetic signatures of the drought-sensitive relict fir, Abies pinsapo Boiss. Field sampling of 156 individuals was performed in two elevation ecotones, characterized by widespread A. pinsapo decline and mortality. The DNA from dead trees was investigated and compared to living individuals, accounting for different ages and elevations. We studied the genes gated outwardly-rectifying K+ (GORK) channel and Plasma membrane Intrinsic Protein (PIP1) aquaporin, previously related to drought response in plant model species, to test whether drought was the main abiotic factor driving the decline of A. pinsapo forests. A combination of linear regression and factor models were used to test these selection signatures, as well as a fixation index (Fst), used here to analyze the genetic structure. The results were consistent among these approaches, supporting a statistically significant association of the GORK gene with survival in one of the A. pinsapo populations. These results provide preliminary evidence for the potential role of the GORK gene in the resilience to drought of A. pinsapo

Comparative Stem Transcriptome Analysis Reveals Pathways Associated with Drought Tolerance in Maritime Pine Grafts

The maritime pine (Pinus pinaster Ait.) is a highly valuable Mediterranean conifer. However, recurrent drought events threaten its propagation and conservation. P. pinaster populations exhibit remarkable differences in drought tolerance. To explore these differences, we analyzed stem transcriptional profiles of grafts combining genotypes with contrasting drought responses under well-watered and water-stress regimes. Our analysis underscored that P. pinaster drought tolerance is mainly associated with constitutively expressed genes, which vary based on genotype provenance. However, we identified key genes encoding proteins involved in water stress response, abscisic acid signaling, and growth control including a PHD chromatin regulator, a histone deubiquitinase, the ABI5-binding protein 3, and transcription factors from Myb-related, DOF NAC and LHY families. Additionally, we identified that drought-tolerant rootstock could enhance the drought tolerance of sensitive scions by regulating the accumulation of transcripts involved in carbon mobilization, osmolyte biosynthesis, flavonoid and terpenoid metabolism, and reactive oxygen species scavenging. These included genes encoding galactinol synthase, CBL-interacting serine/threonine protein kinase 5, BEL1-like homeodomain protein, dihydroflavonol 4-reductase, and 1-deoxy-D-xylulose-5-phosphate. Our results revealed several hub genes that could help us to understand the molecular and physiological response to drought of conifers. Based on all the above, grafting with selected drought-tolerant rootstocks is a promising method for propagating elite recalcitrant conifer species, such as P. pinaster

Genotype and phenotype data standardization, utilization and integration in the big data era for agricultural sciences

17 Pág.Large-scale genotype and phenotype data have been increasingly generated to identify genetic markers, understand gene function and evolution and facilitate genomic selection. These datasets hold immense value for both current and future studies, as they are vital for crop breeding, yield improvement and overall agricultural sustainability. However, integrating these datasets from heterogeneous sources presents significant challenges and hinders their effective utilization. We established the Genotype-Phenotype Working Group in November 2021 as a part of the AgBioData Consortium (https://www.agbiodata.org) to review current data types and resources that support archiving, analysis and visualization of genotype and phenotype data to understand the needs and challenges of the plant genomic research community. For 2021-22, we identified different types of datasets and examined metadata annotations related to experimental design/methods/sample collection, etc. Furthermore, we thoroughly reviewed publicly funded repositories for raw and processed data as well as secondary databases and knowledgebases that enable the integration of heterogeneous data in the context of the genome browser, pathway networks and tissue-specific gene expression. Based on our survey, we recommend a need for (i) additional infrastructural support for archiving many new data types, (ii) development of community standards for data annotation and formatting, (iii) resources for biocuration and (iv) analysis and visualization tools to connect genotype data with phenotype data to enhance knowledge synthesis and to foster translational research. Although this paper only covers the data and resources relevant to the plant research community, we expect that similar issues and needs are shared by researchers working on animals. Database URL: https://www.agbiodata.org.We acknowledge the funding to AgBioData Consortium through the National Science Foundation (16) for the Research Coordination Network (RCN) project (award abstract #2126334), USDA National Institute of Food and Agriculture Specialty Crop Research Initiative project [2022-51181-38449], and USDA National Institute of Food and Agriculture National Research Support Project 10.Peer reviewe

Tripal, a community update after 10 years of supporting open source, standards-based genetic, genomic and breeding databases

Online, open access databases for biological knowledge serve as central repositories for research communities to store, find and analyze integrated, multi-disciplinary datasets. With increasing volumes, complexity and the need to integrate genomic, transcriptomic, metabolomic, proteomic, phenomic and environmental data, community databases face tremendous challenges in ongoing maintenance, expansion and upgrades. A common infrastructure framework using community standards shared by many databases can reduce development burden, provide interoperability, ensure use of common standards and support long-term sustainability. Tripal is a mature, open source platform built to meet this need. With ongoing improvement since its first release in 2009, Tripal provides full functionality for searching, browsing, loading and curating numerous types of data and is a primary technology powering at least 31 publicly available databases spanning plants, animals and human data, primarily storing genomics, genetics and breeding data. Tripal software development is managed by a shared, inclusive governance structure including both project management and advisory teams. Here, we report on the most important and innovative aspects of Tripal after 11 years development, including integration of diverse types of biological data, successful collaborative projects across member databases, and support for implementing FAIR principles

Gene Frequency Shift in Relict Abies pinsapo Forests Associated with Drought-Induced Mortality: Preliminary Evidence of Local-Scale Divergent Selection

Current climate change constitutes a challenge for the survival of several drought-sensitive forests. The study of the genetic basis of adaptation offers a suitable way to understand how tree species may respond to future climatic conditions, as well as to design suitable conservation and management strategies. Here, we focus on selected genetic signatures of the drought-sensitive relict fir, Abies pinsapo Boiss. Field sampling of 156 individuals was performed in two elevation ecotones, characterized by widespread A. pinsapo decline and mortality. The DNA from dead trees was investigated and compared to living individuals, accounting for different ages and elevations. We studied the genes gated outwardly-rectifying K+ (GORK) channel and Plasma membrane Intrinsic Protein (PIP1) aquaporin, previously related to drought response in plant model species, to test whether drought was the main abiotic factor driving the decline of A. pinsapo forests. A combination of linear regression and factor models were used to test these selection signatures, as well as a fixation index (Fst), used here to analyze the genetic structure. The results were consistent among these approaches, supporting a statistically significant association of the GORK gene with survival in one of the A. pinsapo populations. These results provide preliminary evidence for the potential role of the GORK gene in the resilience to drought of A. pinsapo

Presiones selectivas del cambio climático sobre bosques relictos sensibles a la sequía. Respuestas genéticas de Abies pinsapo y Cedrus atlantica a un entorno cambiante

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 15/10/2020. Tesis restringida por expreso deseo de la autora.Increasing variability and uncertainty regarding future climate provide new challenges for understanding species adaptive capacity. Trees are especially concerning given their lower evolutionary rates and ecological importance. Thus, the knowledge of their intraspecific genetic diversity is crucial to predict the possible evolutionary consequences of global climate change and its long-term effects on biodiversity. Furthermore, since drought stress is among the first limitations for plants facing a changing climate, understanding the underlying mechanisms involved in drought stress response/resilience in trees constitutes one of the most valuable research objectives in evolutionary ecology. Relict species constitute reliable models given their reduced geographic range size, isolation and usually fragmented distribution, which subject them to many shifting genetic processes. Moreover, despite these theoretically limitant genetic characteristics for adaptation, empirical evidence frequently shows a lack of decreased fitness (Hampe and Jump 2011). A possible explanation can be that many of them originated from populations that overcome the cold stages of the Quaternary. Therefore, they might contain key genetic variants in ecologically relevant traits (Crawford 2008, Kawecki 2008, Hampe and Jump 2011). A particularly interesting group are those that now inhabit at southern locations since they can be potentially preadapted to environmental conditions expected in future in more northerly central areas in the future (Hampe and Petit, 2005). Here, we attempted to test the effect of these processes in the genetic diversity and genetic structure of trees, as well as determining their evolutionary potential under a climate change scenario, by using as experimental models two southern relict and climate changethreatened conifers, located in altitudinal ecotones and sharing a biogeographic origin: the Southernomost european fir (Abies pinsapo Boiss.), endemic to Southern Spain, and the Atlas cedar (Cedrus atlantica), endemic to Northern Morocco...Los cambios en las condiciones medioambientes como consequencia del cambio climático y la incertidumbre ante ellos suponen nuevos retos para la capacidad adaptativa de las especies. Los árboles son particularmente preocupantes, dadas su menor tasa evolutiva y su importancia ecológica. Por ello, el conocimiento de su diversidad genética es crucial para predecir las posibles consecuencias evolutivas frente al cambio climático y su efecto en la biodiversidad a largo plazo. Considerando que la sequía es uno de los principales factores limitantes para las especies vegetales afectadas por el cambio climático, el conocimiento de los mecanismos implicados en la respuesta y resiliencia a la sequía en árboles constituye uno de los objetivos de investigación más importantes en ecología evolutiva. Las especies relictas son excelentes modelos debido a sus reducidas distribuciones geográficas, su aislamiento y su distribución frecuentemente fragmentada, que las somete a diversos procesos de cambio genético. A pesar de que, en teoría, estas características dificultarían su adaptación, la evidencia empírica, en muchos casos, no muestra una menor eficacia biológica en estas especies (Hampe and Jump 2011). Una posible explicación sería que muchas de ellas tienen su origen en poblaciones que sobrevivieron las frías etapas del Cuaternario. Por lo tanto, podrían contener variantes genéticas clave en rasgos ecológicamente importantes (Crawford 2008, Kawecki 2008, Hampe and Jump 2011). Las especies relictas más meridionales son de especial interés, ya que podrían estar preadaptadas a las condiciones ambientales esperadas en el futuro en áreas más septentrionales como consecuencia del cambio climático (Hampe and Petit, 2005). En la presente tesis se ha probado el efecto de estos procesos en la diversidad y estructura genética de los árboles, así como su potencial adaptativo en un escenario de cambio climático, usando como modelos experimentales dos coníferas relictas y amenazadas por el cambio climático: El pinsapo (Abies pinsapo) y el cedro del Altas (Cedrus atlantica)...Fac. de Ciencias BiológicasTRUEunpu