Insights into Conifer Giga-Genomes

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world’s forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20–30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes.Conifers are the most widely distributed group of gymnosperms, with 600 to 630 species in 69 genera, including 220 to 250 species of the Pinaceae family (Wang and Ran, 2014). Coniferous forests cover an estimated 39% of the world’s forests (Armenise et al., 2012). Conifers dominate many natural and planted forests in the northern hemisphere and are also planted as exotics for commercial forestry in the southern hemisphere. The importance of conifers for global ecosystem services, their value for forestry-dependent economies, and their contrasting biology with angiosperms are major drivers behind efforts to understand the complex structure, functions, and evolution of their genomes. However, owing to their nonmodel system attributes (i.e. slow-growing and long-lived life history traits), extremely large genome size (Fig. 1), and repeat-rich genome sequence with repeats mostly in the form of transposable elements, no reports of a conifer genome assembly, or any gymnosperm genome for that matter (Soltis and Soltis, 2013), were published until recently. Following early releases of the white spruce (Picea glauca) and loblolly pine (Pinus taeda) genome sequences in public databases (e.g. National Center for Biotechnology Information and http://dendrome.ucdavis.edu/treegenes/), a series of articles described the first conifer genome assemblies for Norway spruce (Picea abies; Nystedt et al., 2013) and interior white spruce, a genetic admix of white spruce (Birol et al., 2013) and loblolly pine (Neale et al., 2014; Zimin et al., 2014). Norway spruce is a prominent forest tree in northern Europe. White spruce is a dominant tree species across the large Canadian forest landscape. Loblolly pine dominates commercial forestry in the southeastern United States. White spruce, Norway spruce, and loblolly pine represent some of the most economically important conifers worldwide, and they are the subjects of important tree improvement/breeding programs (Mullin et al., 2011). This Update highlights significant insights obtained from these genomes as well as some ongoing challenges and recent developments in conifer genomics.Open in a separate windowFigure 1.Size and assembly of conifer genomes compared with other plant genomes. Genome size is plotted against the number of scaffolds divided by the haploid chromosome number for a range of plant species. As such, an assembly that reconstructs a genome with perfect contiguity will have a value of 1, and values greater than 1 represent increasing genome fragmentation. Genome assemblies that utilized Sanger sequencing either in full or in part are represented as white circles. Assemblies constructed using only next generation sequencing technologies are represented as black circles. Both axes are plotted on a log₁₀ scale. With the exception of Populus tremula, Hordeum vulgare, and the three conifer genomes, all genomes were obtained from the Phytozome resource (version 10; http://phytozome.jgi.doe.gov/). The early release draft assembly of P. tremula was obtained from the PopGenIE.org FTP resource (ftp://popgenie.org/popgenie/UPSC_genomes/UPSC_Draft_Assemblies/Current/Genome/) and H. vulgare ‘Morex’ from the Munich Information Center for Protein Sequences barley genome database FTP resource (ftp://ftpmips.helmholtz-muenchen.de/plants/barley/public_data/sequences/). The conifer genomes are detailed by Birol et al. (2013), Nystedt et al. (2013), and Zimin et al. (2014).     

Genome-wide SNP associations with rubella-specific cytokine responses in measles-mumps-rubella vaccine recipients

Genetic polymorphisms are known to affect responses to both viral infection and vaccination. Our previous work has described genetic polymorphisms significantly associated with variations in immune response to rubella vaccine from multiple gene families with known immune function, including HLA, cytokine and cytokine receptor genes, and in genes controlling innate and adaptive immunity. In this study, we assessed cellular immune responses (IFNγ and IL-6) in a cohort of healthy younger individuals and performed genome-wide SNP analysis on these same individuals. Here, we report the first genome-wide association study focused on immune responses following rubella vaccination. Our results indicate that rs16928280 in protein tyrosine phosphatase delta (PTPRD) and a collection of SNPs in ACO1 (encoding an iron regulatory protein) are associated with interindividual variations in IFNγ response to rubella virus stimulation. In contrast, we did not identify any significant genetic associations with rubella-specific IL-6 response. These genetic regions may influence rubella vaccine-induced IFNγ responses and warrant further studies in additional cohorts in order to confirm these findings.     

A harvest framework for a recovering American black bear population

Having reproducible and transparent science-based processes in wildlife management ensures the integrity of decision making. These processes are particularly important when establishing harvest frameworks, as guiding information in the peer-reviewed literature is limited. We provide an example using multiple data sets, whose products guided aspects of the development of a harvest framework for a population of recolonizing American black bears (Ursus americanus) in Missouri, USA. To characterize the spatial distribution of harvest, we used 10 years (2010–2019) of black bear global positioning system (GPS) location data and 30 years (1991–2020) of sightings data to assess spatial vulnerability to harvest as the intersection among information on bear occurrence, bear sightings, and hunter land-use tendencies (i.e., the avoidance of steep slopes, large distances from roads). We then used the spatial vulnerability assessment, information on the distribution of public and private lands, and easily discernable boundaries (i.e., major highways, rivers) to suggest boundaries for bear management zones. Additionally, to identify the timing of harvest that would limit female harvest bias, we assessed the temporal vulnerability of harvest using sex-based changes in average daily step lengths and monthly utilization distribution sizes during fall. Black bear occurrence and sighting propensity was greater in southwestern Missouri, and potential hunter land use appeared pervasive across the landscape given the lack of landscape features that would disincentivize use. Given the influence of black bear occurrence and sighting propensity, spatial harvest vulnerability diminished from southern and southeastern to central portions of Missouri, with areas north of the Missouri River not a part of the established black bear range. We consequently divided areas south of the Missouri River into 3 black bear management zones: a small southwestern zone with primarily private lands and high harvest vulnerability, a southeastern zone that encompassed considerable public lands and moderate amounts of vulnerability, and a central zone that was composed mainly of areas of low vulnerability. Temporally, males did not exhibit movement-based changes, but females became less active after the first week of October and used 63.9% less area through fall. Based on movements rates of males and females, a hunting season after the first week of October could reduce the likelihood of females being harvested. Harvests from the black bear harvest season in 2021 suggest that the proportion of bears harvested in each zone was similar in distribution to the proportion of permits allocated across zones with no harvest sex bias, which was aligned with agency goals. Animal movement and space use data products can guide harvest framework decision-making.     

Net ecosystem carbon exchange and the greenhouse gas balance of tidal marshes along an estuarine salinity gradient

Tidal wetlands are productive ecosystems with the capacity to sequester large amounts of carbon (C), but we know relatively little about the impact of climate change on wetland C cycling in lower salinity (oligohaline and tidal freshwater) coastal marshes. In this study we assessed plant production, C cycling and sequestration, and microbial organic matter mineralization at tidal freshwater, oligohaline, and salt marsh sites along the salinity gradient in the Delaware River Estuary over four years. We measured aboveground plant biomass, carbon dioxide (CO₂) and methane (CH₄) exchange between the marsh and atmosphere, microbial sulfate reduction and methanogenesis in marsh soils, soil biogeochemistry, and C sequestration with radiodating of soils. A simple model was constructed to estimate monthly and annually integrated rates of gross ecosystem production (GEP), ecosystem respiration (ER) to carbon dioxide ( \( {\text{ER}}_{{{\text{CO}}_{2} }} \) ) or methane ( \( {\text{ER}}_{{{\text{CH}}_{4} }} \) ), net ecosystem production (NEP), the contribution of sulfate reduction and methanogenesis to ER, and the greenhouse gas (GHG) source or sink status of the wetland for 2 years (2007 and 2008). All three marsh types were highly productive but evidenced different patterns of C sequestration and GHG source/sink status. The contribution of sulfate reduction to total ER increased along the salinity gradient from tidal freshwater to salt marsh. The Spartina alterniflora dominated salt marsh was a C sink as indicated by both NEP (~140 g C m^?2 year^?1) and ²¹⁰Pb radiodating (336 g C m^?2 year^?1), a minor sink for atmospheric CH₄, and a GHG sink (~620 g CO_2-eq m^?2 year^?1). The tidal freshwater marsh was a source of CH₄ to the atmosphere (~22 g C–CH₄ m^?2 year^?1). There were large interannual differences in plant production and therefore C and GHG source/sink status at the tidal freshwater marsh, though ²¹⁰Pb radiodating indicated modest C accretion (110 g C m^?2 year^?1). The oligohaline marsh site experienced seasonal saltwater intrusion in the late summer and fall (up to 10 mS cm^?1) and the Zizania aquatica monoculture at this site responded with sharp declines in biomass and GEP in late summer. Salinity intrusion was also linked to large effluxes of CH₄ at the oligohaline site (>80 g C–CH₄ m^?2 year^?1), making this site a significant GHG source (>2,000 g CO_2-eq m^?2 year^?1). The oligohaline site did not accumulate C over the 2 year study period, though ²¹⁰Pb dating indicated long term C accumulation (250 g C m^?2 year^?1), suggesting seasonal salt-water intrusion can significantly alter C cycling and GHG exchange dynamics in tidal marsh ecosystems.     

DNA methylation profiles in primary cutaneous melanomas are associated with clinically significant pathologic features

DNA methylation studies have elucidated a methylation signature distinguishing primary melanomas from benign nevi and provided new insights about genes that may be important in melanoma development. However, it is unclear whether methylation differences among primary melanomas are related to tumor pathologic features with known clinical significance. We utilized the Illumina GoldenGate Cancer Panel array to investigate the methylation profiles of 47 primary cutaneous melanomas. Arraywide methylation patterns revealed a positive association of methylation with Breslow thickness and mutated BRAF, a negative association with mitotic rate, and a weak association with ulceration. Hierarchical clustering on CpG sites exhibiting the most variable methylation (n = 235) divided the melanoma samples into three clusters, including a highly methylated cluster that was positively associated with Breslow thickness and an intermediately methylated cluster associated with Breslow thickness and mitotic rate. Our findings provide support for the existence of methylation‐defined subsets in melanomas with increased methylation associated with Breslow thickness.     

Mismatch Recognition Protein MutS�� Does Not Hijack (CAG)n Hairpin Repair in Vitro

CAG repeats form stable hairpin structures, which are believed to be responsible for CAG repeat expansions associated with certain human neurological diseases. Human cells possess an accurate DNA hairpin repair system that prevents expansion of disease-associated CAG repeats. Based on transgenic animal studies, it is suggested that (CAG)_n expansion is caused by abnormal binding of the MutSβ mismatch recognition protein to (CAG)_n hairpins, leading to hijacking mismatch repair function during (CAG)_n hairpin repair. We demonstrate here that MutSβ displays identical biochemical and biophysical activities (including ATP-provoked conformational change, ATPase, ATP binding, and ADP binding) when interacting with a (CAG)_n hairpin and a mismatch. More importantly, our in vitro functional hairpin repair assays reveal that excess MutSβ does not inhibit (CAG)_n hairpin repair in HeLa nuclear extracts. Evidence presented here provides a novel view as to whether or not MutSβ is involved in CAG repeat instability in humans.Expansion of trinucleotide repeats (TNRs)³ causes hereditary neurological disorders such as Huntington disease and myotonic dystrophy, whose clinical symptoms are directly linked to expansion of CAG and CTG repeats, respectively (1–3). The precise mechanisms by which TNR expansion occurs and the factors that promote it are not fully understood. It has been proposed that CAG and CTG repeats form thermostable hairpins that include A-A and T-T mispairs in the hairpin stem (4, 5). Therefore, cellular mechanisms that process DNA hairpin/loop structures and/or A-A or T-T mispairs may influence TNR stability.Recent studies have identified and characterized a DNA hairpin repair (HPR) system in human cells that promotes CAG/CTG repeat stability (6, 7). The mechanism of human HPR involves incision and removal of CAG/CTG repeat hairpins in a nick-directed and proliferating cell nuclear antigen-dependent manner, followed by DNA resynthesis using the continuous strand as a template (6). In addition to human HPR, the human mismatch repair (MMR) system is well known for its role in stabilizing simple repetitive sequences called microsatellites, which are prone to forming small loops or insertion/deletion (ID) mispairs. In human cells, MutSα (MSH2–MSH6) and MutSβ (MSH2–MSH3) both bind to 1–2-nt ID mispairs, but MutSβ has higher affinity for these small loops (8). Defects in MMR genes cause microsatellite instability and predisposition to cancer (9), demonstrating that MMR is essential for genetic stability in human cells. Surprisingly, genetic studies in mice suggest that MutSβ promotes (CAG)_n expansion and TNR instability. These studies show that expansion of a heterologous (CAG)_n tract occurs in wild type and MSH6^−/− mice but that expansion of the (CAG)_n tract is suppressed in MSH2^−/− and MSH3^−/− mice (10, 11). Recently, Owens et al. (11) reported that binding to a (CAG)_n hairpin influences the protein conformation, nucleotide binding, and hydrolysis activities of MutSβ so that they are different from what has been reported for MutSα during mismatch recognition. It is therefore hypothesized that (CAG)_n hairpins, through their ability to alter the biochemical properties of MutSβ, hijack the MMR process, leading to CAG repeat expansion instead of CAG hairpin removal (11). However, it is not clear why MMR, a major genome maintenance system, would promote TNR instability instead of TNR stability. We, therefore, have developed a novel functional assay and examined the validity of this hypothesis. Our results reveal that MutSβ displays normal biochemical activities when binding to CAG hairpins and does not inhibit (CAG)_n hairpin repair. The observations presented here provide novel thoughts on whether or not or how MutSβ is involved in CAG repeat instability in human cells.     

Combinatorial discovery of fluorophores and fluorescent probes

The properties of organic fluorophores are difficult to predict, even in simple cases. Fluorescent probes--which combine fluorescent properties with the equally challenging problem of molecular recognition--are even more difficult to develop. Combinatorial approaches to the development of such molecules are a new but promising endeavor, and reviewing the state of the art delineates the near-and long-term possibilities.     

The genetics and genomics of the drought response in Populus

The genetic nature of tree adaptation to drought stress was examined by utilizing variation in the drought response of a full-sib second generation (F(2)) mapping population from a cross between Populus trichocarpa (93-968) and P. deltoides Bart (ILL-129) and known to be highly divergent for a vast range of phenotypic traits. We combined phenotyping, quantitative trait loci (QTL) analysis and microarray experiments to demonstrate that 'genetical genomics' can be used to provide information on adaptation at the species level. The grandparents and F(2) population were subjected to soil drying, and contrasting responses to drought across genotypes, including leaf coloration, expansion and abscission, were observed, and QTL for these traits mapped. A subset of extreme genotypes exhibiting extreme sensitivity and insensitivity to drought on the basis of leaf abscission were defined, and microarray experiments conducted on these genotypes and the grandparent species. The extreme genotype groups induced a different set of genes: 215 and 125 genes differed in their expression response between groups in control and drought, respectively, suggesting species adaptation at the gene expression level. Co-location of differentially expressed genes with drought-specific and drought-responsive QTLs was examined, and these may represent candidate genes contributing to the variation in drought response.