Transcriptional Profiling of Early Defense Response to White Pine Blister Rust Infection in Pinus albicaulis (Whitebark Pine).

Pathogen perception generates the activation of signal transduction cascades to host defense. White pine blister rust (WPBR) is caused by Cronartium ribicola J.C. Fisch and affects a number of species of Pinus. One of the most severely affected species is Pinus albicaulis Engelm (whitebark pine). WPBR resistance in the species is a polygenic and complex trait that requires an optimized immune response. We identified early responses in 2-year-old seedlings after four days of fungal inoculation and compared the underlying transcriptomic response with that of healthy non-inoculated individuals. A de novo transcriptome assembly was constructed with 56,796 high quality-annotations derived from the needles of susceptible and resistant individuals in a resistant half-sib family. Differential expression analysis identified 599 differentially expressed transcripts, from which 375 were upregulated and 224 were downregulated in the inoculated seedlings. These included components of the initial phase of active responses to abiotic factors and stress regulators, such as those involved in the first steps of flavonoid biosynthesis. Four days after the inoculation, infected individuals showed an overexpression of chitinases, reactive oxygen species (ROS) regulation signaling, and flavonoid intermediates. Our research sheds light on the first stage of infection and emergence of disease symptoms among whitebark pine seedlings. RNA sequencing (RNA-seq) data encoding hypersensitive response, cell wall modification, oxidative regulation signaling, programmed cell death, and plant innate immunity were differentially expressed during the defense response against C. ribicola.

A genome sequence for the threatened whitebark pine.

Whitebark pine (WBP, Pinus albicaulis) is a white pine of subalpine regions in the Western contiguous United States and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola) and additional threats from mountain pine beetle (Dendroctonus ponderosae), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short reads of haploid megagametophyte tissue and Oxford Nanopore long reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gb of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gb). Approximately 87.2% (24.0 Gb) of total sequence was placed on the 12 WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the 3 subclasses of NLRs. Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo-assembled transcriptomes.

Changes in demography and geographic distribution in the weeping pinyon pine (Pinus pinceana) during the Pleistocene.

Climate changes, together with geographical barriers imposed by the Sierra Madre Oriental and the Chihuahuan Desert, have shaped the genetic diversity and spatial distribution of different species in northern Mexico. Pinus pinceana Gordon & Glend. tolerates extremely arid conditions. Northern Mexico became more arid during the Quaternary, modifying ecological communities. Here, we try to identify the processes underlying the demographic history of P. pinceana and characterize its genetic diversity using 3100 SNPs from genotyping by sequencing 90 adult individuals from 10 natural populations covering the species' entire geographic distribution. We inferred its population history and contrasted possible demographic scenarios of divergence that modeled the genetic diversity present in this restricted pinyon pine; in support, the past distribution was reconstructed using climate from the Last Glacial Maximum (LGM, 22 kya). We inferred that P. pinceana diverged into two lineages ~2.49 Ma (95% CI 3.28-1.62), colonizing two regions: the Sierra Madre Oriental (SMO) and the Chihuahuan Desert (ChD). Our results of population genomic analyses reveal the presence of heterozygous SNPs in all populations. In addition, low migration rates across regions are probably related to glacial-interglacial cycles, followed by the gradual aridification of the Chihuahuan Desert during the Holocene.

Comparative analysis of differential gene expression indicates divergence in ontogenetic strategies of leaves in two conifer genera.

In land plants, heteroblasty broadly refers to a drastic change in morphology during growth through ontogeny. Juniperus flaccida and Pinus cembroides are conifers of independent lineages known to exhibit leaf heteroblasty between the juvenile and adult life stage of development. Juvenile leaves of P. cembroides develop spirally on the main stem and appear decurrent, flattened, and needle-like; whereas adult photosynthetic leaves are triangular or semi-circular needle-like, and grow in whorls on secondary or tertiary compact dwarf shoots. By comparison, J. flaccida juvenile leaves are decurrent and needle-like, and adult leaves are compact, short, and scale-like. Comparative analyses were performed to evaluate differences in anatomy and gene expression patterns between developmental phases in both species. RNA from 12 samples was sequenced and analyzed with available software. They were assembled de novo from the RNA-Seq reads. Following assembly, 63,741 high-quality transcripts were functionally annotated in P. cembroides and 69,448 in J. flaccida. Evaluation of the orthologous groups yielded 4140 shared gene families among the four references (adult and juvenile from each species). Activities related to cell division and development were more abundant in juveniles than adults in P. cembroides, and more abundant in adults than juveniles in J. flaccida. Overall, there were 509 up-regulated and 81 down-regulated genes in the juvenile condition of P. cembroides and 14 up-regulated and 22 down-regulated genes in J. flaccida. Gene interaction network analysis showed evidence of co-expression and co-localization of up-regulated genes involved in cell wall and cuticle formation, development, and phenylpropanoid pathway, in juvenile P. cembroides leaves. Whereas in J. flaccida, differential expression and gene interaction patterns were detected in genes involved in photosynthesis and chloroplast biogenesis. Although J. flaccida and P. cembroides both exhibit leaf heteroblastic development, little overlap was detected, and unique genes and pathways were highlighted in this study.

The Relevance of a Physiological-Stage Approach Study of the Molecular and Environmental Factors Regulating Seed Germination in Wild Plants.

Germination represents the culmination of the seed developmental program and is affected by the conditions prevailing during seed maturation in the mother plant. During maturation, the dormancy condition and tolerance to dehydration are established. These characteristics are modulated by the environment to which they are subjected, having an important impact on wild species. In this work, a review was made of the molecular bases of the maturation, the processes of dormancy imposition and loss, as well as the germination process in different wild species with different life histories, and from diverse habitats. It is also specified which of these species present a certain type of management. The impact that the domestication process has had on certain characteristics of the seed is discussed, as well as the importance of determining physiological stages based on morphological characteristics, to face the complexities of the study of these species and preserve their genetic diversity and physiological responses.

Population structure, diversifying selection, and local adaptation in Pinus patula.

PREMISE: Climate change is predicted to affect natural and plantation forests. The responses of conifers to overcome changing environments will depend on their adaptation to local conditions; however, intraspecific adaptive genetic variation is unknown for most gymnosperms. Studying genetic diversity associated with phenotypic variability along environmental gradients will enhance our understanding of adaptation and may reveal genetic pools important for conservation and management. METHODS: We used target enrichment and genome skimming to obtain single nucleotide polymorphisms (SNPs) from 61 individuals of Pinus patula, a pine tree native to Mexico widely used in plantation forestry. We investigated the adaptive genetic variation of two varieties with morphological and distributional differences potentially related to genetic and adaptive divergence. RESULTS: Population structure and haplotype network analyses revealed that genetic diversity between P. patula var. patula and P. patula var. longipedunculata was structured, even within populations of P. patula var. longipedunculata. We observed high genetic diversity, low inbreeding rate, and rapid linkage disequilibrium (LD) decay in the varieties. Based on outlier tests, loci showing signatures of natural selection were detected in geographically distant P. patula var. longipedunculata populations. For both varieties, we found significant correlations between climate-related environmental variation and SNP diversity at loci involved in abiotic stress, cell transport, defense, and cell wall biogenesis, pointing to local adaptation. CONCLUSIONS: Overall, significant intraspecific adaptive genetic variation in P. patula was detected, highlighting the presence of different genetic pools and signs of local adaptation that should be considered in forestry and conservation.