Studies Of Conifers Research Articles

Isolation and Expression of Transcription Factors Involved in Somatic Embryo Development by Transcriptome Analysis of Embryogenic Callus of Thuja koraiensis

Thuja koraiensis Nakai (Cupressaceae) is an endangered and ecologically important conifer endemic to Korea. Previously, we established a protocol for micropropagation in T. koraiensis, which involved somatic embryogenesis from embryogenic callus of T. koraiensis. However, the molecular mechanisms underlying somatic embryogenesis remain unclear. Herein, we performed transcriptomic analysis to identify somatic embryogenesis-related genes of T. koraiensis via Illumina RNA sequencing. We conducted de novo transcriptome assembly using a Trinity assembler, which produced 274,077 transcript contigs clustered into 205,843 transcripts (unigenes), with an average length of 825 base pairs. Of all the unigenes, 14.69%, 18.62%, and 7.4% had homologs in the Gene Ontology, NCBI Non-redundant Protein, and NCBI Nucleotide databases, respectively. Among these mRNA sequences, expression of putative embryogenesis-associated transcription factors, namely BABYBOOM (BBM), WUSCHEL-RELATED HOMEOBOX (WOX), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK), was analyzed during somatic embryogenesis. RT-PCR analysis revealed that TkBBM, TkWOX, and TkSERK were highly expressed in embryogenic callus and seedling roots, whereas their expression was reduced in seedling leaves. Our findings provide new insights into the roles of BBM, WOX, and SERK in somatic embryogenesis. Our results may serve as a reference for comparative transcriptome analysis in related species and further aid functional genomics studies in conifers.

A revised view on the evolution of glutamine synthetase isoenzymes in plants.

SUMMARYGlutamine synthetase (GS) is a key enzyme responsible for the incorporation of inorganic nitrogen in the form of ammonium into the amino acid glutamine. In plants, two groups of functional GS enzymes are found: eubacterial GSIIb (GLN2) and eukaryotic GSIIe (GLN1/GS). Only GLN1/GS genes are found in vascular plants, which suggests that they are involved in the final adaptation of plants to terrestrial life. The present phylogenetic study reclassifies the different GS genes of seed plants into three clusters: GS1a, GS1b and GS2. The presence of genes encoding GS2 has been expanded to Cycadopsida gymnosperms, which suggests the origin of this gene in a common ancestor of Cycadopsida, Ginkgoopsida and angiosperms. GS1a genes have been identified in all gymnosperms, basal angiosperms and some Magnoliidae species. Previous studies in conifers and the gene expression profiles obtained in ginkgo and magnolia in the present work could explain the absence of GS1a in more recent angiosperm species (e.g. monocots and eudicots) as a result of the redundant roles of GS1a and GS2 in photosynthetic cells. Altogether, the results provide a better understanding of the evolution of plant GS isoenzymes and their physiological roles, which is valuable for improving crop nitrogen use efficiency and productivity. This new view of GS evolution in plants, including a new cytosolic GS group (GS1a), has important functional implications in the context of plant metabolism adaptation to global changes.

Siberian larch (Larix sibirica Ledeb.) chloroplast genome and development of polymorphic chloroplast markers

BackgroundThe main objectives of this study were sequencing, assembling, and annotation of chloroplast genome of one of the main Siberian boreal forest tree conifer species Siberian larch (Larix sibirica Ledeb.) and detection of polymorphic genetic markers – microsatellite loci or simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs).ResultsWe used the data of the whole genome sequencing of three Siberian larch trees from different regions - the Urals, Krasnoyarsk, and Khakassia, respectively. Sequence reads were obtained using the Illumina HiSeq2000 in the Laboratory of Forest Genomics at the Genome Research and Education Center of the Siberian Federal University. The assembling was done using the Bowtie2 mapping program and the SPAdes genomic assembler. The genome annotation was performed using the RAST service. We used the GMATo program for the SSRs search, and the Bowtie2 and UGENE programs for the SNPs detection. Length of the assembled chloroplast genome was 122,561 bp, which is similar to 122,474 bp in the closely related European larch (Larix decidua Mill.). As a result of annotation and comparison of the data with the existing data available only for three larch species - L. decidua, L. potaninii var. chinensis (complete genome 122,492 bp), and L. occidentalis (partial genome of 119,680 bp), we identified 110 genes, 34 of which represented tRNA, 4 rRNA, and 72 protein-coding genes. In total, 13 SNPs were detected; two of them were in the tRNA-Arg and Cell division protein FtsH genes, respectively. In addition, 23 SSR loci were identified.ConclusionsThe complete chloroplast genome sequence was obtained for Siberian larch for the first time. The reference complete chloroplast genomes, such as one described here, would greatly help in the chloroplast resequencing and search for additional genetic markers using population samples. The results of this research will be useful for further phylogenetic and gene flow studies in conifers.

Current Advances in Seed Orchard Layouts: Two Case Studies in Conifers

Currently, there has been an increasing demand on seed orchard designs with respect to mitigating inbreeding in advanced generations or facing severe mortality at sites. Optimum Neighborhood Algorithm design (ONA) excels in solving these issues, as shown in two case studies. The first demonstrates the application of the ONA for populating empty positions in an existing orchard. Following this concept, the ONA can be used for upgrading existing orchards, where only high-value parents are retained and new parents are introduced, thus avoiding the genetic penalty associated with the establishment of new-generation seed orchards. The second illustrates benefits of combining the ONA with the Minimum Inbreeding (MI) seed orchard design. Utilizing the MI, distances among clonal rows of selected clones were maximized on the orchard grid while the remaining single-tree positions were populated by ONA.

Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos.

Somatic embryogenesis (SE) provides us a potent biotechnological tool to manipulate the physical and chemical conditions (water availability) along the process and to study their effect in the final success in terms of quantity of somatic embryos produced. In the last years, our research team has been focused on the study of different aspects of the SE in Pinus spp. One of the main aspects affecting SE is the composition of culture media; in this sense, phytohormones play one of the most crucial roles in this propagation system. Many studies in conifers have shown that different stages of SE and somatic embryo development are correlated with distinct endogenous phytohormone profiles under the stress conditions needed for the process (i.e., cytokinins play a regulatory role in stress signaling, which it is essential for radiata pine SE). Based on this knowledge, the aim of this study was to test the effect of different temperatures (18, 23, and 28°C) and gelling agent concentrations (8, 9, and 10 gL-1) during the maturation stage of Pinus radiata SE in maturation and germination rates. Parallel, phytohormone profile of somatic embryos developed was evaluated. In this sense, the highest gellan gum concentration led to significantly lower water availability. At this gellan gum concentration and 23°C a significantly higher number of somatic embryos was obtained and the overall success of the process increased with respect to other treatments assayed. The somatic embryos produced in these conditions showed the highest concentration of iP-type cytokinins and total ribosides. Although, the different conditions applied during maturation of somatic embryos led to different hormonal profiles, they did not affect the ex vitro survival of the resulting somatic plants, where no significant differences were observed.

Wood formation in Norway spruce on a lowland site in Slovenia in 2015 and comparison with other conifers all over Europe

We present xylem formation in Norway spruce (Picea abies (L.) Karst.) in 2015 at Panška reka, near Ljubljana, Slovenia (ca. 46°00’N, 14°40’E, 400 m a.s.l.) with a temperate climate. The research was a part of a long-term project, which involves different sites and tree species. We measured the widths of cambium and the formation of xylem growth rings with differentiating cells in postcambial (PC) and secondary wall formation (SW) phases, and mature cells (MT). The results for 2015 were compared with those for previous years (2009–2011) in the same species and site, and with the published data from wood formation studies in conifers from all over Europe. The latter were used in two-factor regression models which confirmed the effects of latitude and altitude on the critical dates of onset, end, and duration of xylem production. The models thus helped us to predict/reconstruct phenology of wood formation in conifers in Europe.

Genetic Adaptation to Climate in White Spruce Involves Small to Moderate Allele Frequency Shifts in Functionally Diverse Genes.

Understanding the genetic basis of adaptation to climate is of paramount importance for preserving and managing genetic diversity in plants in a context of climate change. Yet, this objective has been addressed mainly in short-lived model species. Thus, expanding knowledge to nonmodel species with contrasting life histories, such as forest trees, appears necessary. To uncover the genetic basis of adaptation to climate in the widely distributed boreal conifer white spruce (Picea glauca), an environmental association study was conducted using 11,085 single nucleotide polymorphisms representing 7,819 genes, that is, approximately a quarter of the transcriptome.Linear and quadratic regressions controlling for isolation-by-distance, and the Random Forest algorithm, identified several dozen genes putatively under selection, among which 43 showed strongest signals along temperature and precipitation gradients. Most of them were related to temperature. Small to moderate shifts in allele frequencies were observed. Genes involved encompassed a wide variety of functions and processes, some of them being likely important for plant survival under biotic and abiotic environmental stresses according to expression data. Literature mining and sequence comparison also highlighted conserved sequences and functions with angiosperm homologs.Our results are consistent with theoretical predictions that local adaptation involves genes with small frequency shifts when selection is recent and gene flow among populations is high. Accordingly, genetic adaptation to climate in P. glauca appears to be complex, involving many independent and interacting gene functions, biochemical pathways, and processes. From an applied perspective, these results shall lead to specific functional/association studies in conifers and to the development of markers useful for the conservation of genetic resources.

Sequenced genomes and rapidly emerging technologies pave the way for conifer evolutionary developmental biology.

Conifers, Ginkgo, cycads and gnetophytes comprise the four groups of extant gymnosperms holding a unique position of sharing common ancestry with the angiosperms. Comparative studies of gymnosperms and angiosperms are the key to a better understanding of ancient seed plant morphologies, how they have shifted over evolution to shape modern day species, and how the genes governing these morphologies have evolved. However, conifers and other gymnosperms have been notoriously difficult to study due to their long generation times, inaccessibility to genetic experimentation and unavailable genome sequences. Now, with three draft genomes from spruces and pines, rapid advances in next generation sequencing methods for genome wide expression analyses, and enhanced methods for genetic transformation, we are much better equipped to address a number of key evolutionary questions relating to seed plant evolution. In this mini-review we highlight recent progress in conifer developmental biology relevant to evo-devo questions. We discuss how genome sequence data and novel techniques might allow us to explore genetic variation and naturally occurring conifer mutants, approaches to reduce long generation times to allow for genetic studies in conifers, and other potential upcoming research avenues utilizing current and emergent techniques. Results from developmental studies of conifers and other gymnosperms in comparison to those in angiosperms will provide information to trace core molecular developmental control tool kits of ancestral seed plants, but foremost they will greatly improve our understanding of the biology of conifers and other gymnosperms in their own right.

Identification and expression profiles of sRNAs and their biogenesis and action-related genes in male and female cones of Pinus tabuliformis.

BackgroundSmall RNA (sRNA) play pivotal roles in reproductive development, and their biogenesis and action mechanisms are well characterised in angiosperm plants; however, corresponding studies in conifers are very limited. To improve our understanding of the roles of sRNA pathways in the reproductive development of conifers, the genes associated with sRNA biogenesis and action pathways were identified and analysed, and sRNA sequencing and parallel analysis of RNA ends (PARE) were performed in male and female cones of the Chinese pine (Pinus tabuliformis).ResultsBased on high-quality reference transcriptomic sequences, 21 high-confidence homologues involved in sRNA biogenesis and action in P. tabuliformis were identified, including two different DCL3 genes and one AGO4 gene. More than 75 % of genes involved in sRNA biogenesis and action have higher expression levels in female than in male cones. Twenty-six microRNA (miRNA) families and 74 targets, including 46 24-nt sRNAs with a 5’ A, which are specifically expressed in male cones or female cones and probably bind to AGO4, were identified.ConclusionsThe sRNA pathways have higher activity in female than in male cones, and the miRNA pathways are the main sRNA pathways in P. tabuliformis. The low level of 24-nt short-interfering RNAs in conifers is not caused by the absence of biogenesis-related genes or AGO-binding proteins, but most likely caused by the low accumulation of these key components. The identification of sRNAs and their targets, as well as genes associated with sRNA biogenesis and action, will provide a good starting point for investigations into the roles of sRNA pathways in cone development in conifers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1885-6) contains supplementary material, which is available to authorized users.

Selection of haploid cell lines from megagametophyte cultures of maritime pine as a DNA source for massive sequencing of the species

The potential of haploid tissues for genetic studies in conifers is hampered by the lack of abundant and homogeneous plant material suitable for DNA isolation. In this work we have determined factors promoting haploid callus induction and proliferation from megagametophytes of Oria 6, a genotype of Pinus pinaster Aiton (maritime pine) from the natural population Sierra de Oria (Almeria, Spain), selected based on its response to extreme drought conditions. The generation of haploid callus was restricted to megagametophytes isolated from light-brown cones with no dehydrated seeds collected in September. Culture medium composition did not significantly affect callus induction, but a modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine favored further multiplication. The ploidy status of the callus lines was determined by flow cytometry and seven polymorphic microsatellites. A total of sixteen haploid callus lines were established and one of these is being used as a source of DNA for massive sequencing of maritime pine genome.

Soil water stress affects both cuticular wax content and cuticle-related gene expression in young saplings of maritime pine (Pinus pinaster Ait)

BackgroundThe cuticle is a hydrophobic barrier located at the aerial surface of all terrestrial plants. Recent studies performed on model plants, such as Arabidopsis thaliana, have suggested that the cuticle may be involved in drought stress adaptation, preventing non-stomatal water loss. Although forest trees will face more intense drought stresses (in duration and intensity) with global warming, very few studies on the role of the cuticle in drought stress adaptation in these long-lived organisms have been so far reported.ResultsThis aspect was investigated in a conifer, maritime pine (Pinus pinaster Ait.), in a factorial design with two genetic units (two half-sib families with different growth rates) and two treatments (irrigated vs non-irrigated), in field conditions. Saplings were grown in an open-sided greenhouse and half were irrigated three times per week for two growing seasons. Needles were sampled three times per year for cuticular wax (composition and content) and transcriptome (of 11 genes involved in cuticle biosynthesis) analysis. Non-irrigated saplings (i) had a higher cuticular wax content than irrigated saplings and (ii) overexpressed most of the genes studied. Both these trends were more marked in the faster growing family.ConclusionsThe higher cuticular wax content observed in the non-irrigated treatment associated with strong modifications in products from the decarbonylation pathway suggest that cuticular wax may be involved in drought stress adaptation in maritime pine. This study provides also a set of promising candidate genes for future forward genetic studies in conifers.

Canopy uptake of atmospheric N deposition at a conifer forest: Part II- response of chlorophyll fluorescence and gas exchange parameters

Spruce foliage was sprayed with NH4+ NO3- and control solutions to investigate the effect of canopy nitrogen (N) uptake on chlorophyll fluorescence and gas exchange at a RockyMountain subalpine forest (Niwot Forest) at the Niwot Ridge Long-Term Ecological Research site. N-treated branches received NH4+NO3- in an ion-matrix solution that was representative of mean precipitation ion concentrations. Branches were sprayed with NH4+NO3- to increase the wet N deposition to experimental branches 60% above ambient. Control branches received only the ion-matrix solution (no N), while background branches received only natural precipitation. N content of N-treated new growth and old growth shoots was 2 and 8% greater, respectively, than the background and control shoots N content. N-treatment enhanced photosynthetic efficiency (Fv ‘/Fm’) of old growth spruce shoots (≥1 yr old); N-treated shoots (Fv’/Fm’) was 11.12% greater than control and background shoots Fv’/Fm’ (p ≥ 0.05). Greater maximum carboxylation rates accompanied this increased photosynthetic efficiency with the N-treated old growth shoots Vcmax 14.15% greater than background and control shoots Vcmax (p ≥ 0.05). New growth gas exchange and fluorescence results were similar (although p > 0.05). Both inorganic N assimilation and the incorporation of applied N into the photosynthetic apparatus likely account for the above results of this conifer study.

Shoot apical development during in vitro embryogenesisThis review is one of a selection of papers published on the Special Theme of Shoot Apical Meristems.

Formation of the shoot apical meristem (SAM) has been extensively investigated in zygotic embryos of flowering plants, where it follows a prolonged and dynamic developmental pattern underlined by precise temporal and spatial changes in gene expression. Studies conducted on the plant model system Arabidopsis have revealed that SAM formation is controlled by a genetic network and involves the participation of several regulatory genes expressed at different stages of development. As a general rule apical meristem development in vivo occurs very early; at the globular stage of development in flowering plants and in club-stage embryos of conifers. Once formed, meristems of zygotic embryos are stable structures that become reactivated at the onset of germination. Shoot apical meristem formation during in vitro embryogenesis is demarked by structural events similar to those described for zygotic embryos, although differences can be observed during the late phases of development, where cellular differentiation and formation of intercellular spaces disrupt the architecture of SAMs produced in culture. These events, which denote the “unstable” nature of SAMs of somatic embryos, often result in poor conversion frequency and reduced plant regeneration. By using Picea glauca (Moench) Voss (white spruce) somatic embryos and microspore-derived embryos of Brassica napus L. (canola) as model systems, this review provides methods for improving SAM formation through manipulations of the culture medium which alter the cellular redox status. Meristem marker genes from Arabidopsis, such as WUSCHEL (which is required for the acquisition of stem fate identity), represent a valuable tool for estimating the quality of SAM produced by microspore-derived embryos of canola. In spruce, the identification of two novel meristem marker genes, HBK1 and PgAGO, will allow similar studies in conifers.

Genetic transformation of conifers utilizing somatic embryogenesis.

Over the last 5 yr, the production of transgenic conifers has been greatly facilitated by the ability to transform somatic embryonal tissues (somatic embryos) via cocultivation with Agrobacterium tumefaciens. This has allowed us to develop protocols for the genetic transformation of several spruce species. Furthermore, these procedures can produce an average of 20 independent transgenic lines (translines) per gram fresh mass of embryonal tissue, providing for the first time the magnitude-of-scale required for implementing large-scale functional genomics studies in conifers. Combined with efficient regeneration of transgenic trees via somatic embryos, the potential for genetic engineering of conifers has been demonstrated by stable reporter gene expression (GUS or GFP) resulting from single insert T-DNA integration events.

Population and subspecific genetic differentiation in the foxtail pine (Pinus balfouriana).

We performed an allozyme survey of genetic differentiation in Pinus balfouriana, a subalpine conifer endemic to California that is comprised of two allopatric subspecies, one in the Klamath Mountains and the other in the southern Sierra Nevada. Although the two subspecies are morphologically distinct and gene flow between them is virtually nonexistent, we observed much higher levels of differentiation among populations within a subspecies than between the two subspecies. Differentiation is particularly strong in the Klamath populations (multilocus FST = 0.242), which are small, isolated, and ecologically marginal. We attribute this strong differentiation to the mountain island effect, in which populations restricted to high elevations become isolated from each other on different mountains separated by unsuitable intervening habitat, with consequent reduced gene flow allowing populations to evolve independently. Populations of P. balfouriana in the Klamath region only exist scattered on the few highest ridges and peaks that rise above 2,000 m, which defines the lower limit of the species elevational distribution. This pattern of distribution has allowed genetic drift and allelic sorting through historical events to produce strong population-level differentiation, which was likely in place before the two subspecies were geographically separated. Because P. balfouriana occurs on both serpentine soils and nonserpentine soils in the Klamath Mountains, we tested for genetic differentiation between populations growing on serpentine versus nonserpentine soils and our results were equivocal. Our data, combined with several other studies of conifers, show that the mountain island effect can produce significant genetic differentiation in conifers whose life-history traits of widely dispersed pollen, long generation times, and high outcrossing rates would lead us to predict a more homogenous population genetic structure.

POPULATION AND SUBSPECIFIC GENETIC DIFFERENTIATION IN THE FOXTAIL PINE (PINUS BALFOURIANA)

We performed an allozyme survey of genetic differentiation in Pinus balfouriana, a subalpine conifer endemic to California that is comprised of two allopatric subspecies, one in the Klamath Mountains and the other in the southern Sierra Nevada. Although the two subspecies are morphologically distinct and gene flow between them is virtually nonexistent, we observed much higher levels of differentiation among populations within a subspecies than between the two subspecies. Differentiation is particularly strong in the Klamath populations (multilocus FST = 0.242), which are small, isolated, and ecologically marginal. We attribute this strong differentiation to the mountain island effect, in which populations restricted to high elevations become isolated from each other on different mountains separated by unsuitable intervening habitat, with consequent reduced gene flow allowing populations to evolve independently. Populations of P. balfouriana in the Klamath region only exist scattered on the few highest ridges and peaks that rise above 2000 m, which defines the lower limit of the species elevational distribution. This pattern of distribution has allowed genetic drift and allelic sorting through historical events to produce strong population-level differentiation, which was likely in place before the two subspecies were geographically separated. Because P. balfouriana occurs on both serpentine soils and nonserpentine soils in the Klamath Mountains, we tested for genetic differentiation between populations growing on serpentine versus nonserpentine soils and our results were equivocal. Our data, combined with several other studies of conifers, show that the mountain island effect can produce significant genetic differentiation in conifers whose life-history traits of widely dispersed pollen, long generation times, and high outcrossing rates would lead us to predict a more homogenous population genetic structure.Corresponding Editor: M. Dudash

Genetics of allozyme variants in loblolly pine

' 4 Applications of isozymes in breeding programs also have been demonstrated' 9 . However, we must understand how isozyme variants are inherited to fully realize their value in genetic investigations. Furthermore, we must look at as many loci as possible if results are to be more representative of the entire genome' 0 To date, few reports have been published on the inheritance of isozymes in forest trees, and fewer still have involved more than two or three loci ' .1 8.22 The presence of the haploid (In) megagametophyte tissue in seeds has greatly facilitated isozyme studies in conifers. Each megagametophyte, representing a single meiotic product, has the same genetic constitution as the female gamete. Thus, the Mendelian genetics of allozyme (allelic isozyme) variants can be confirmed without making crosses because allozymes are expected to segregate in 1:1 ratios in the megagametophytes of heterozygous mother trees. In addition, when allozymes are expressed clearly in both embryo and megagametophyte tissues, the male contribution to the embryo' genotype can be separated from that of the female. This unique feature of conifers has proven useful for studying pollen migration 7 and mating systems in forest trees ' ' 6 In this paper, we describe allozyme variants of 10 enzyme systems occurring in megagametophyte and embryo tissues of loblolly pine (Pinus taeda L.) seeds and provide evidence for their inheritance. Linkage relationships among the loci coding these allozymes are reported elsewhere 2

Electrophoretic analysis of isozymes from seeds of Pinus, Abies, and Pseudotsuga

Isozyme patterns of L-leucine-amino-peptidase (LAP), esterase, and peroxidase in extracts from conifer seeds (eight species of Pinus, Abies concolor Hoopes, and Pseudotsuga taxifolia Britt.) indicated that certain specific isozyme patterns might be useful in systematic studies of conifers. The isozyme patterns of LAP from these seeds showed an apparent phylogenetic relationship. Eight different LAP bands were detected, and all eight species of Pinus displayed the same three LAP bands. The two slowest moving LAP bands were found to be common to all 10 species, whereas the mobility of the other LAP bands varied among species. Isozyme patterns of esterase from seeds of Pinus, Abies, and Pseudotsuga varied from species to species, and no species displayed the same number of bands. The number of esterase bands detected varied from 5 to 16, and no phylogenetic relationships were apparent. The banding patterns of peroxidase from these conifer seeds, which ranged from three to nine bands, also appeared to be species specific, and no common trends or homologies were apparent.

Simulated Fallout Studies in Conifers - A Preliminary Report

Simulated Fallout Studies in Conifers - A Preliminary Report