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In species with large and complex genomes such as conifers, dense linkage maps are a useful resource for supporting genome assembly and laying the genomic groundwork at the structural, populational, and functional levels. However, most of the 600+ extant conifer species still lack extensive genotyping resources, which hampers the development of high-density linkage maps. In this study, we developed a linkage map relying on 21,570 single nucleotide polymorphism (SNP) markers in Sitka spruce (Picea sitchensis [Bong.] Carr.), a long-lived conifer from western North America that is widely planted for productive forestry in the British Isles. We used a single-step mapping approach to efficiently combine RAD-seq and genotyping array SNP data for 528 individuals from 2 full-sib families. As expected for spruce taxa, the saturated map contained 12 linkages groups with a total length of 2,142 cM. The positioning of 5,414 unique gene coding sequences allowed us to compare our map with that of other Pinaceae species, which provided evidence for high levels of synteny and gene order conservation in this family. We then developed an integrated map for P. sitchensis and Picea glauca based on 27,052 markers and 11,609 gene sequences. Altogether, these 2 linkage maps, the accompanying catalog of 286,159 SNPs and the genotyping chip developed, herein, open new perspectives for a variety of fundamental and more applied research objectives, such as for the improvement of spruce genome assemblies, or for marker-assisted sustainable management of genetic resources in Sitka spruce and related species. [ABSTRACT FROM AUTHOR]

In an attempt to comprehensively study the dynamics of non-structural carbon compounds (NCCs), we measured the seasonal changes of soluble sugars, starch, lipids and sugar alcohols in the leaves, branches, stem and roots of the fast-growing Pinus contorta (Loudon) (pine) and slow-growing Picea glauca (Moench) Voss (spruce) trees growing in a boreal climate. In addition to measuring the seasonal concentrations of these compounds, the relative contribution of these compounds to the total NCC pool within the organs of trees (~8 m tall) was estimated and compared across different phenological and growth stages. Both species showed large seasonal shifts from starch to sugars from spring to fall in nearly all organs and tissues; most likely an adaptation to the cold winters. For both species, the total fluctuation of sugar + starch across the year (i.e. the difference between the minimum and maximum observed across collection times) was estimated to be between 1.6 and 1.8 kg for all NCCs. The fluctuation, however, was 1.40 times greater than the minimum reserves in pine, while only 0.72 times the minimum reserves in spruce. By tissue type, NCC fluctuations were greatest in the roots of both species. Roots showed a large build-up of reserves in late spring, but these reserves were depleted over summer and fall. Storage reserves in needles and branches declined over the summer, and this decline may be linked to the sink strength of the stem during diameter growth. Some notable highlights of this holistic study: a late winter build-up of sugars in the stem xylem of both species, but especially spruce; and an increase in sugar alcohols in the bark of spruce in very late winter, which could indicate mobilization to support early growth in spring and high lipid reserves in the bark of pine, which appeared not to be impacted by seasonal changes between summer and winter. Collectively, these observations point toward a more conservative NCC reserve strategy in spruce compared with pine, which is consistent with its stress tolerance and greater longevity. [ABSTRACT FROM AUTHOR]

Aims Black spruce (Picea mariana [Mill.] B.S.P.) and white spruce (Picea glauca [Moench] Voss.) are congeneric species. Both are moderately shade tolerant and widely distributed across North American boreal forests. Methods To understand light effects on their ecophysiological responses to elevated CO2, 1-year-old seedlings were exposed to 360 µmol mol−1 and 720 µmol mol−1 CO2 at three light conditions (100%, 50% and 30% of full light in the greenhouse). Foliar gas exchanges were measured in the mid- and late-growing season. Important Findings Elevated CO2 increased net photosynthesis (P n) and photosynthetic water use efficiency, but it reduced stomatal conductance and transpiration. The stimulation of photosynthesis by elevated CO2 was greatest at 50% light and smallest at 100%. Photosynthesis, maximum carboxylation rate (V cmax) and light-saturated rate of electron transport (J max) all decreased with decreasing light. Elevated CO2 significantly reduced V cmax across all light treatments and both species in mid-growing season. However, the effect of elevated CO2 became insignificant at 30% light later in the growing season, with the response being greater in black spruce than in white spruce. Elevated CO2 also reduced J max in white spruce in both measurements while the effect became insignificant at 30% light later in the growing season. However, the effect on black spruce varied with time. Elevated CO2 reduced J max in black spruce in mid-growing season in all light treatments and the effect became insignificant at 30% light later in the growing season, while it increased J max later in the season at 100% and 50% light. These results suggest that both species benefited from elevated CO2, and that the responses varied with light supply, such that the response was primarily physiological at 100% and 50% light, while it was primarily morphological at 30% light. [ABSTRACT FROM AUTHOR]

Plant mitochondrial genomes vary widely in size. Although many plant mitochondrial genomes have been sequenced and assembled, the vast majority are of angiosperms, and few are of gymnosperms. Most plant mitochondrial genomes are smaller than a megabase, with a few notable exceptions. We have sequenced and assembled the complete 5.5-Mb mitochondrial genome of Sitka spruce (Picea sitchensis), to date, one of the largest mitochondrial genomes of a gymnosperm. We sequenced the whole genome using Oxford Nanopore MinION, and then identified contigs of mitochondrial origin assembled from these long reads based on sequence homology to the white spruce mitochondrial genome. The assembly graph shows a multipartite genome structure, composed of one smaller 168-kb circular segment of DNA, and a larger 5.4-Mb single component with a branching structure. The assembly graph gives insight into a putative complex physical genome structure, and its branching points may represent active sites of recombination. [ABSTRACT FROM AUTHOR]

Conifers' exceptionally large genome (20-30 Gb) is scattered with 60% retrotransposon (RT) components and we have little knowledge on their origin and evolutionary implications. RTs may impede the expression of flanking genes and provide sources of the formation of novel small RNA (sRNAs) populations to constrain events of transposon (TE) proliferation/transposition. Here we show a declining expression of 24-nt-long sRNAs and low expression levels of their key processing gene, pgRTL2 (RNASE THREE LIKE 2) at seed set in Picea glauca. The sRNAs in 24-nt size class are significantly less enriched in type and read number than 21-nt sRNAs and have not been documented in other species. The architecture of MIR loci generating highly expressed 24-/21-nt sRNAs is featured by long terminal repeat-retrotransposons (LTR-RTs) in families of Ty3/Gypsy and Ty1/Copia elements. This implies that the production of sRNAs may be predominantly originated from TE fragments on chromosomes. Furthermore, a large proportion of highly expressed 24-nt sRNAs does not have predictable targets against unique genes in Picea, suggestive of their potential pathway in DNA methylation modifications on, for instance, TEs. Additionally, the classification of computationally predicted sRNAs suggests that 24-nt sRNA targets may bear particular functions inmetabolic processes while 21-nt sRNAs target genes involved inmany different biological processes. This study, therefore, directs our attention to a possible extrapolation that lacking of 24-nt sRNAs at the late conifer seed developmental phase may result in less constraints in TE activities, thus contributing to the massive expansion of genome size. [ABSTRACT FROM AUTHOR]

* Background and Aims Trees invest in both primary (e.g. height) and secondary (e.g. diameter) growth. The trade-off between these investments varies between species and changes with the tree growing environment. To better establish this trade-off, readily available allometric exponents relating height to diameter at breast height (γh,dbh) and stem volume to diameter at breast height (αvdbh) were simultaneously studied. * Methods Allometric exponents αv,dbh and γh,dbh were obtained from 8893 individual tree stem analyses from two broadleaved species (Betula papyrifera, Populus tremuloides) and four conifers (Picea glauca, Picea mariana, Pinus banksiana, Abies balsamea) in the temperate and boreal forests of the province of Quebec, Canada. αvdbh and γh,dbh were related to tree age, stand density index (SDI), and mean temperature (TGS) and total precipitation (PGS) of the growing season. * Key Results αv,jdbh and γh,dbh were found to be invariant with PGS and positively related to SDI and TGS for all species except Pinus banksiana. The parameter values associated with SDI and TGS were of higher value for conifers than for broadleaved species. * Conclusions This suggests that conifers and broadleaved species have different growth patterns. This could be explained by their different mode of development, the conifer species having a stronger apical dominance than broadleaved species. Such results could be further considered in allocation studies to quantify future carbon stocks in managed forests. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background and Aims MADS-box genes comprise a gene family coding for transcription factors. This gene family expanded greatly during land plant evolution such that the number of MADS-box genes ranges from one or two in green algae to around 100 in angiosperms. Given the crucial functions of MADS-box genes for nearly all aspects of plant development, the expansion of this gene family probably contributed to the increasing complexity of plants. However, the expansion of MADS-box genes during one important step of land plant evolution, namely the origin of seed plants, remains poorly understood due to the previous lack of whole-genome data for gymnosperms. Methods The newly available genome sequences of Picea abies, Picea glauca and Pinus taeda were used to identify the complete set of MADS-box genes in these conifers. In addition, MADS-box genes were identified in the growing number of transcriptomes available for gymnosperms. With these datasets, phylogenies were constructed to determine the ancestral set of MADS-box genes of seed plants and to infer the ancestral functions of these genes. Key Results Type I MADS-box genes are under-represented in gymnosperms and only a minimum of two Type I MADS-box genes have been present in the most recent common ancestor (MRCA) of seed plants. In contrast, a large number of Type II MADS-box genes were found in gymnosperms. The MRCA of extant seed plants probably possessed at least 11–14 Type II MADS-box genes. In gymnosperms two duplications of Type II MADS-box genes were found, such that the MRCA of extant gymnosperms had at least 14–16 Type II MADS-box genes. Conclusions The implied ancestral set of MADS-box genes for seed plants shows simplicity for Type I MADS-box genes and remarkable complexity for Type II MADS-box genes in terms of phylogeny and putative functions. The analysis of transcriptome data reveals that gymnosperm MADS-box genes are expressed in a great variety of tissues, indicating diverse roles of MADS-box genes for the development of gymnosperms. This study is the first that provides a comprehensive overview of MADS-box genes in conifers and thus will provide a framework for future work on MADS-box genes in seed plants. [ABSTRACT FROM AUTHOR]

Redundancy and competition between R2R3-MYB activators and repressors on common target genes has been proposed as a fine-tuning mechanism for the regulation of plant secondary metabolism. This hypothesis was tested in white spruce [Picea glauca (Moench) Voss] by investigating the effects of R2R3-MYBs from different subgroups on common targets from distinct metabolic pathways. Comparative analysis of transcript profiling data in spruces overexpressing R2R3-MYBs from loblolly pine (Pinus taeda L.), PtMYB1, PtMYB8, and PtMYB14, defined a set of common genes that display opposite regulation effects. The relationship between the closest MYB homologues and 33 putative target genes was explored by quantitative PCR expression profiling in wild-type P. glauca plants during the diurnal cycle. Significant Spearman’s correlation estimates were consistent with the proposed opposite effect of different R2R3-MYBs on several putative target genes in a time-related and tissue-preferential manner. Expression of sequences coding for 4CL, DHS2, COMT1, SHM4, and a lipase thio/esterase positively correlated with that of PgMYB1 and PgMYB8, but negatively with that of PgMYB14 and PgMYB15. Complementary electrophoretic mobility shift assay (EMSA) and transactivation assay provided experimental evidence that these different R2R3-MYBs are able to bind similar AC cis-elements in the promoter region of Pg4CL and PgDHS2 genes but have opposite effects on their expression. Competitive binding EMSA experiments showed that PgMYB8 competes more strongly than PgMYB15 for the AC-I MYB binding site in the Pg4CL promoter. Together, the results bring a new perspective to the action of R2R3-MYB proteins in the regulation of distinct but interconnecting metabolism pathways. [ABSTRACT FROM PUBLISHER]

This study examines the long-term effects of experimental conifer monocultures on stem volume and soil chemistry. Replicated plots of black spruce (Picea mariana), white spruce (Picea glauca), and red pine (Pinus resinosa) were planted in 1950 at three spacings (1.8, 2.7, and 3.6 m) on a briefly cultivated agricultural field on glaciolacustrine sandy loam soil near Thunder Bay, Ontario, Canada. Stem volumes per hectare and per tree were measured in 2002 (52 years after planting). Surface organic (i.e., forest floor) and mineral soil fertility in terms of pH, total N and P, and extractable NH4-N and P were measured in 2000 (50 years after planting). Of the three species red pine had the highest volume per hectare at all the three spacing followed by white spruce and black spruce; volume for all three species peaked at the 1.8-m spacing. The effects of conifer species on soil physical and chemical properties were more pronounced than spacing effects and the changes were mainly confined to forest floor layer. Few changes in mineral soil properties occurred because of the species and spacing treatments. Per hectare forest floor nutrient pools were higher under white spruce and red pine than black spruce, a pattern likely driven by higher litterfall and forest floor accumulation. It appears that toward the end of first rotation the higher productivity of red pine compared with the other conifers did not come at the cost of reduced soil pools of available NH4, P04, or K, but it was associated with reduced Ca levels in the forest floor. [ABSTRACT FROM AUTHOR]

Conifers are among the most genetically diverse plants but show the lowest levels of genetic differentiation, even among geographically distant populations. High gene flow among populations may be one of the most important factors in maintaining these genetic patterns. Here, we provide empirical evidence for extensive pollen-mediated gene dispersal between natural stands of a widespread northern temperate/boreal conifer, Picea glauca. We used 6 polymorphic allozyme loci to quantify the proportion of seeds sired by pollen originating from different sources in a landscape fragmented by agriculture in North Central Ontario, Canada. In 7 stands, a small proportion of seeds were sired by self-pollen or neighboring trees but 87.1% (±1.7% standard error [SE]) of seeds were sired by pollen from at least 250 to 3000 m away. In 4 single isolated trees, self-fertilization rates were low and more than 96% (±1.3% SE) of seeds were sired by immigrant pollen. The average minimum pollen dispersal distance in outcrossed matings was 619 m. These results provide strong evidence that extensive long-distance pollen dispersal plays a primary role in maintaining low genetic differentiation among natural populations of P. glauca and helps maintain genetic diversity and minimize inbreeding in small stands in a fragmented landscape. [ABSTRACT FROM AUTHOR]

A new member of the Argonaute (AGO) family of proteins was isolated from conifers and designated as PgAGO (Gene Bank Accession No. DQ068741; protein ID AAY67884). The complete coding sequence of PgAGO was obtained through screening cDNA libraries generated from white spruce (Picea glauca (Moench) Voss) somatic embryos. The PgAGO gene has an open reading frame of 2880 bp and encodes a protein of 960 amino acids. The predicted protein has an isolectric point of 9.17, a molecular mass of 107 kD and lacks prominent hydrophobic domains, which makes its cellular location inconclusive. The PgAGO protein contains the two conserved regions (the PAZ and PIWI domains) typically found in all members of the AGO family. The PAZ domain of PgAGO comprises 117 amino acid residues and shares a low degree of homology with similar domains in other species. The C-terminal PIWI domain contains 86 amino acids and is more conserved. Localization and transformation studies suggest that PgAGO is required for embryo development, specifically for proper shoot and root apical meristem differentiation. Based on RNA-in situ hybridization, the PgAGO transcripts are preferentially localized in cells of the shoot and root apical meristems from the early phases of embryo development. The RNA-mediated suppression of PgAGO results in severe abnormalities during embryo development, including the formation of poorly organized apical meristems. The root meristems lack the group of large central cells that separate the procambial region from the root cap, whereas the shoot meristem fails to differentiate apical initials. These abnormalities result in poor post-embryonic performance, leading to meristem abortion and growth cessation. [ABSTRACT FROM AUTHOR]

Effects of drying and cryopreservation on survival of spruce (Picea glauca (Moench) Voss and Picea glauca × engelmannii complex) somatic embryos (SEs) were investigated with the aim of developing simple and robust protocols for embryo storage. Somatic embryos dried over salt solutions of known water potential (Ψ) survived removal of virtually all free water, to a relative water content (RWC) of approximately 0.13, a value similar to that for spruce zygotic embryos from dry seed. Desiccated SEs also survived subsequent freezing in liquid nitrogen, without the addition of cryoprotectant or pre-culture steps. Highest survival (> 80%) after freezing in liquid nitrogen was in embryos pre-dried to Ψ of –15 to –20 MPa, which yielded RWC close to predicted bound (apoplastic) water values. Low (< 35%) or no survival after freezing was observed in embryos pretreated at higher Ψ (above –5 MPa) or at very low Ψ (–540 MPa, using silica gel), respectively. [ABSTRACT FROM PUBLISHER]

It has previously been shown that exogenous applications of ascorbic acid (AA) increase the conversion frequency of somatic embryos of white spruce (Picea glauca (Moench) Voss). To determine whether ascorbic acid alters purine metabolism during the early phases of embryo germination, the relative rates of purine salvage and degradation were investigated by following the metabolic fates of exogenously applied [8-14C]adenine, [8-14C]adenosine, and [8-14C]inosine, and the activities of several key enzymes. We demonstrated that both the salvage and the degradation pathways operate during germination. Specifically, adenine and adenosine were mainly salvaged to nucleotides and nucleic acids, whereas an appreciable amount of inosine was degraded to CO2 and ureides. Comparisons of purine metabolism between control and AA-treated embryos showed that exogenous applications of ascorbic acid enhanced the ability of the embryos to take up adenine and adenosine throughout the germination period. Furthermore, the higher enzymatic activities of adenosine kinase and adenine phosphoribosyltransferase were responsible for the larger proportion of adenine and adenosine being salvaged in AA-treated embryos compared with control embryos. Thus, there was a positive correlation between the ability to anabolize purine precursors and successful embryo conversion. [ABSTRACT FROM AUTHOR]

Significant reductions in needle water content were observed in white spruce (Picea glauca (Moench) Voss), black spruce (Picea mariana (Mill) B.S.P.), and jack pine (Pinus banksiana Lamb.) seedlings in response to a 10-day drought, although turgor was apparently maintained. When the seedlings were re-watered after the drought, jack pine needles regained their original saturated volume, whereas white spruce and black spruce needles did not. Significant drought-induced reductions in turgor-loss volume (i.e., tissue volume at the point of turgor loss) were observed in shoots of all three species, especially jack pine. Repeated exposure to 7 days of drought or treatment with the cytochrome P450 inhibitor, paclobutrazol ((2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-pentan-3-ol), reduced seedling height relative to that of untreated controls in all three species. The reductions in saturated and turgor-loss needle volumes in the paclobutrazol-treated seedlings were comparable with those of seedlings subjected to a 10-day drought. The treatment-induced reductions in shoot and needle water contents enabled seedlings to maintain turgor with tissue volumes close to, or below, the turgor-loss volume of untreated seedlings. Paclobutrazol-treated seedlings subsequently survived drought treatments that were lethal to untreated seedlings. [ABSTRACT FROM AUTHOR]