Complete plastid genome sequences of Drimys, Liriodendron, and Piper : implications for the phylogenetic relationships of magnoliids

Background  

The magnoliids with four orders, 19 families, and 8,500 species represent one of the largest clades of early diverging angiosperms. Although several recent angiosperm phylogenetic analyses supported the monophyly of magnoliids and suggested relationships among the orders, the limited number of genes examined resulted in only weak support, and these issues remain controversial. Furthermore, considerable incongruence resulted in phylogenetic reconstructions supporting three different sets of relationships among magnoliids and the two large angiosperm clades, monocots and eudicots. We sequenced the plastid genomes of three magnoliids, Drimys (Canellales), Liriodendron (Magnoliales), and Piper (Piperales), and used these data in combination with 32 other angiosperm plastid genomes to assess phylogenetic relationships among magnoliids and to examine patterns of variation of GC content.     

Development of a BAC library for yellow-poplar (Liriodendron tulipifera) and the identification of genes associated with flower development and lignin biosynthesis

Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited. In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802 Mbp per haploid genome (±16 SD). The BAC library contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117 kb, a chloroplast DNA content of 0.2%, and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches, we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun genomic clones and BAC clones are under accession DU169330–DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429–DQ223434. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Generation of a large-scale genomic resource for functional and comparative genomics in Liriodendron tulipifera L.

Liriodendron tulipifera L., a member of Magnoliaceae in the order Magnoliales, has been used extensively as a reference species in studies on plant evolution. However, genomic resources for this tree species are limited. We constructed cDNA libraries from ten different types of tissues: premeiotic flower buds, postmeiotic flower buds, open flowers, developing fruit, terminal buds, leaves, cambium, xylem, roots, and seedlings. EST sequences were generated either by 454 GS FLX or Sanger methods. Assembly of almost 2.4 million sequencing reads from all libraries resulted in 137,923 unigenes (132,905 contigs and 4,599 singletons). About 50% of the unigenes had significant matches to publically available plant protein sequences, representing a wide variety of putative functions. Approximately 30,000 simple sequence repeats were identified. More than 97% of the cell wall formation genes in the Cell Wall Navigator and the MAIZEWALL databases are represented. The cinnamyl alcohol dehydrogenase (CAD) homologs identified in the L. tulipifera EST dataset showed different expression levels in the ten tissue types included in this study. In particular, the LtuCAD1 was found to partially recover the stiffness of the floral stems in the Arabidopsis thaliana CAD4 and CAD5 double mutant plants, of the LtuCAD1 in lignin biosynthesis. L. tulipifera genes have greater sequence similarity to homologs from other woody angiosperm species than to non-woody model plants. This large-scale genomic resour"HistryDatesce will be instrumental for gene discovery, cDNA microarray production, and marker-assisted breeding in L. tulipifera, and strengthen this species' role in comparative studies.     

Organization of the chromosome region harboring a FLORICAULA/LEAFY gene in Liriodendron

FLORICAULA/LEAFY (FLO/LFY) plays an important role in the reproductive transition and controls flower spatial patterning by inducing the expression of the ABC floral organ identity genes. In this study, we sequenced two bacterial artificial chromosomes harboring a FLO/LFY and three other genes from yellow-poplar (Liriodendron tulipifera L.) and compared the gene order in this locus between several species. Besides the conserved terminal domains, key residues involved in interactions with DNA bases, backbone, and in dimerization were also conserved in the yellow-poplar FLO/LFY. Phylogenetic analysis of the FLO/LFY amino acid sequences placed yellow-poplar closer to eudicots than to monocotyledonous species. We found that gene content and order in this region of the yellow-poplar genome was more similar to corresponding regions in Vitis vinifera L., Carica papaya L., Populus trichocarpa Torr. & Gray, and Ricinus communis L., regardless of the evolutionary relationship. In addition, evidence for transposition, large insertions, and duplications were found, suggesting multiple and complex mechanisms of basal angiosperm genome evolution.     

The chloroplast genome of the “basal” angiosperm Calycanthus fertilis – structural and phylogenetic analyses

The nucleotide sequence of the complete chloroplast genome of a basal angiosperm, Calycanthus fertilis, has been determined. The circular 153337 bp long cpDNA is colinear with those of tobacco, Arabidopsis and spinach. A total of 133 predicted genes (115 individual gene species, 18 genes duplicated in the inverted repeats) including 88 potential protein-coding genes (81 gene species), 8 ribosomal RNA genes (4 gene species) and 37 tRNA genes (30 gene species) representing 20 amino acids were identified based on similarity to their homologs from other chloroplast genomes. This is the highest gene number ever registered in an angiosperm plastome. Calycanthus fertilis cpDNA also contains a homolog of the recently discovered mitochondrial ACRS gene. Since no gene transfer from mitochondria to the chloroplast has ever been documented, we investigated the evolutionary affinity of this gene in detail. Phylogenetic analysis of the protein-coding subset of the plastome suggests that the ancient line of Laurales emerged after the split of the angiosperms into monocots and dicots. Calycanthus fertilis Walter var. ferax (Michy.) Rehder is a synonym of C. floridus L. var. glaucus (Willd.) Torr. & A. Gray.Data deposition: The sequence reported in this paper has been deposited in the EMBL database (accession no. AJ428413).     

Investigation of genome structure of a cinnamyl alcohol dehydrogenase locus in a basal angiosperm hardwood species, Liriodendron tulipifera L., reveals low synteny

Basal angiosperms contain a wide diversity of floral and growth forms and gave rise to the largest recent angiosperm lineages.As none of the basal angiosperm genomes has been sequenced,examining large bacterial artificial chromosome (BAC) inserts remains the main approach to providing a first glimpse of the structure and organization of their genomes.In this study,we sequenced a 126.9-kbp BAC contig harboring a cinnamyl alcohol dehydrogenase gene (LtuCAD1) in a basal angiosperm species,Liriodendron tulipifera L.,an important timber tree species with significant ecological and economic values.A key enzyme in lignin biosynthesis,CAD catalyzes the final step in the synthesis of monolignols.We carried out phylogenetic analyses of seven full-length CAD family genes (LtuCAD1-7) obtained from a comprehensive Liriodendron expressed sequence tag dataset.The phylogenetic tree suggests that LtuCAD1 is the primary CAD gene involved in lignifications as it is the only Liriodendron CAD grouped with the bona fide CADs class.As well as the LtuCAD1,the BAC contig contained fragmented sequences for one integrase,eight hypothetical proteins,two gag-pol polyproteins,one RNase H family protein,and one chromatin binding protein.Comparative analysis with other angiosperm species suggests that the genomic segment in this BAC has undergone frequent arrangement.This study is our initial step in identifying and understanding lignin biosynthesis genes from basal angiosperm species.Such knowledge can help bridge the information gap between hardwood (angiosperm) and softwood (gymnosperm) species and benefit potential breeding and biotechnology application for enhanced production ofbiomass and digestibility in L.tulipifera.     

The ABC model and its applicability to basal angiosperms

BACKGROUND: Although the flower is the central feature of the angiosperms, little is known of its origin and subsequent diversification. The ABC model has long been the unifying paradigm for floral developmental genetics, but it is based on phylogenetically derived eudicot models. Synergistic research involving phylogenetics, classical developmental studies, genomics and developmental genetics has afforded valuable new insights into floral evolution in general, and the early flower in particular. SCOPE AND CONCLUSIONS: Genomic studies indicate that basal angiosperms, and by inference the earliest angiosperms, had a rich tool kit of floral genes. Homologues of the ABCE floral organ identity genes are also present in basal angiosperm lineages; however, C-, E- and particularly B-function genes are more broadly expressed in basal lineages. There is no single model of floral organ identity that applies to all angiosperms; there are multiple models that apply depending on the phylogenetic position and floral structure of the group in question. The classic ABC (or ABCE) model may work well for most eudicots. However, modifications are needed for basal eudicots and, the focus of this paper, basal angiosperms. We offer 'fading borders' as a testable hypothesis for the basal-most angiosperms and, by inference, perhaps some of the earliest (now extinct) angiosperms.     

Expression of floral MADS-box genes in basal angiosperms: implications for the evolution of floral regulators

The ABC model of floral organ identity is based on studies of Arabidopsis and Antirrhinum, both of which are highly derived eudicots. Most of the genes required for the ABC functions in Arabidopsis and Antirrhinum are members of the MADS-box gene family, and their orthologs are present in all major angiosperm lineages. Although the eudicots comprise 75% of all angiosperms, most of the diversity in arrangement and number of floral parts is actually found among basal angiosperm lineages, for which little is known about the genes that control floral development. To investigate the conservation and divergence of expression patterns of floral MADS-box genes in basal angiosperms relative to eudicot model systems, we isolated several floral MADS-box genes and examined their expression patterns in representative species, including Amborella (Amborellaceae), Nuphar (Nymphaeaceae) and Illicium (Austrobaileyales), the successive sister groups to all other extant angiosperms, plus Magnolia and Asimina, members of the large magnoliid clade. Our results from multiple methods (relative-quantitative RT-PCR, real-time PCR and RNA in situ hybridization) revealed that expression patterns of floral MADS-box genes in basal angiosperms are broader than those of their counterparts in eudicots and monocots. In particular, (i) AP1 homologs are generally expressed in all floral organs and leaves, (ii) AP3/PI homologs are generally expressed in all floral organs and (iii) AG homologs are expressed in stamens and carpels of most basal angiosperms, in agreement with the expectations of the ABC model; however, an AG homolog is also expressed in the tepals of Illicium. The broader range of strong expression of AP3/PI homologs is inferred to be the ancestral pattern for all angiosperms and is also consistent with the gradual morphological intergradations often observed between adjacent floral organs in basal angiosperms.     

Profile of a flower: How rates of morphological evolution drive floral diversification in Ericales and angiosperms

Premise

Recent studies of floral disparity in the asterid order Ericales have shown that flowers vary strongly among families and that disparity is unequally distributed between the three flower modules (perianth, androecium, gynoecium). However, it remains unknown whether these patterns are driven by heterogeneous rates of morphological evolution or other factors.

Methods

Here, we compiled a data set of 33 floral characters scored for 414 species of Ericales sampled from 346 genera and all 22 families. We conducted ancestral state reconstructions using an equal-rates Markov model for each character. We estimated rates of morphological evolution for Ericales and for a separate angiosperm-wide data set of 19 characters and 792 species, creating “rate profiles” for Ericales, angiosperms, and major angiosperm subclades. We compared morphological rates among flower modules within each data set separately and between data sets, and we compared rates among angiosperm subclades using the angiosperm data set.

Results

The androecium exhibits the highest evolutionary rates across most characters, whereas most perianth and gynoecium characters evolve more slowly in both Ericales and angiosperms. Both high and low rates of morphological evolution can result in high floral disparity in Ericales. Analyses of an angiosperm-wide floral data set reveal that this pattern appears to be conserved across most major angiosperm clades.

Conclusions

Elevated rates of morphological evolution in the androecium of Ericales may explain the higher disparity reported for this floral module. Comparing rates of morphological evolution through rate profiles proves to be a powerful tool in understanding floral evolution.     

Floral structure and evolution of primitive angiosperms: Recent advances

Concepts of primitive angiosperm flowers have changed in recent years due to new studies on relic archaic groups, new paleobotanical finds and the addition of molecular biological techniques to the study of angiosperm systematics and evolution.Magnoliidae are still the hot group, but emphasis is now on small primitive flowers with few organs and also on the great lability of organ number. Of the extant groups, a potential basal position of the paleoherbs has been discussed by some authors. Although some paleoherbs have a simple gynoecium with a single orthotropous ovule, anatropous ovules may still be seen as plesiomorphic in angiosperms. Anatropy is not necessarily a consequence of the advent of closed carpels. It may also exhibit biological advantages under other circumstances as is the case in podocarps among gymnosperms. Valvate anthers have now been found in most larger subgroups of theMagnoliidae (recently also in paleoherbs) and in some Cretaceous fossils. Nevertheless, as seen from its systematic distribution, valvate dehiscence is not necessarily plesiomorphic for the angiosperms, but may be a facultative by-product of the thick connectives and comparatively undifferentiated anther shape inMagnoliidae and lowerHamamelididae. A perianth is relatively simple in extantMagnoliidae or even wanting in some families. In groups with naked flowers the perianth may have been easily lost because integration in the floral architecture was less pronounced than in more advanced angiosperm groups. Problems with the comparison of paleoherb flowers with those ofGnetales are discussed. The rapid growth of information from paleobotany and molecular systematics requires an especially open attitude towards the evaluation of various hypotheses on early flower evolution in the coming years.     

Duplicate Genes and the Root of Angiosperms, with an Example Using Phytochrome Sequences

The root of the angiosperm tree has not yet been established. Major morphological and molecular differences between angiosperms and other seed plants have introduced ambiguities and possibly spurious results. Because it is unlikely that extant species more closely related to angiosperms will be discovered, and because relevant fossils will almost certainly not yield molecular data, the use of duplicate genes for rooting purposes may provide the best hope of a solution. Simultaneous analysis of the genes resulting from a gene duplication event along the branch subtending angiosperms would yield an unrooted network, wherein two congruent gene trees should be connected by a single branch. In these circumstances the best rooted species tree is the one that corresponds to the two gene trees when the network is rooted along the connecting branch. In general, this approach can be viewed as choosing among rooted species trees by minimizing hypothesized events such as gene duplication, gene loss, lineage sorting, and lateral transfer. Of those gene families that are potentially relevant to the angiosperm problem, phytochrome genes warrant special attention. Phylogenetic analysis of a sample of complete phytochrome (PHY) sequences implies that an initial duplication event preceded (or occurred early within) the radiation of seed plants and that each of the two resulting copies duplicated again. In one of these cases, leading to thePHYAandPHYClineages, duplication appears to have occurred before the diversification of angiosperms. Duplicate gene trees are congruent in these broad analyses, but the sample of sequences is too limited to provide much insight into the rooting question. Preliminary analyses of partialPHYAandPHYCsequences from several presumably basal angiosperm lineages are promising, but more data are needed to critically evaluate the power of these genes to resolve the angiosperm radiation.     

Characterization of candidate class A,B and E floral homeotic genes from the perianthless basal angiosperm <Emphasis Type="Italic">Chloranthus spicatus</Emphasis> (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus. Nucleotide sequences data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY316311, AY397762 and AY379963.     

Construction and characterization of a cDNA library from floral organs and fruitlets of <Emphasis Type="Italic">Citrus reticulata</Emphasis>

To explore and isolate genes related to flowering and fruit development, we constructed a cDNA library from floral organs and fruitlets of Ponkan mandarin (Citrus reticulata Blanco). A total of 661 high-quality expressed sequence tags (ESTs) were generated and submitted to GenBank with the accession numbers from GO343532 to GO344192. All these ESTs were assembled into 43 contigs and 296 singletons (totally 339 unigenes). The BLAST2GO software was employed to annotate the unigenes, among which 77 ones had no significant homology with the sequences in NCBI non-redundant proteins database by BLASTX analysis. Additionally, gene ontology (GO) analysis revealed an overview of sequences distribution, which implied some specially expressed genes related to flower and fruit development. Furthermore, some abundantly expressed unigenes involved in several crucial metabolic pathways related to fruit quality were highlighted and three types of homologues of miraculin-like protein2 were analyzed by both semiquantitative RT-PCR and real-time PCR. The results showed different expression profiles of these genes, which meant that they contribute distinctly to fruit development.     

Comparative floral anatomy and ontogeny in Magnoliaceae

Floral anatomy and ontogeny are described in six species of Magnoliaceae, representing the two subfamilies Liriodendroideae (Liriodendron chinese and L. tulipifera) and Magnolioideae, including species with terminal flowers (Magnolia championi, M. delavayi, M. grandiflora, M. paenetalauma) and axillary flowers (Michelia crassipes). The sequence of initiation of floral organs is from proximal to distal. The three distinct outermost organs are initiated in sequence, but ultimately form a single whorl; thus their ontogeny is consistent with a tepal interpretation. Tepals are initiated in whorls, and the stamens and carpels are spirally arranged, though the androecium shows some intermediacy between a spiral and whorled arrangement. Carpels are entirely free from each other both at primordial stages and maturity. Ventral closure of the style ranges from open in Magnolia species examined to partially closed in Michelia crassipes and completely closed in Liriodendron, resulting in a reduced stigma surface. Thick-walled cells and tannins are present in all species except Michelia crassipes. Oil cells are normally present. Floral structure is relatively homogeneous in this family, although Liriodendron differs from other Magnoliaceae in that the carpels are entirely closed at maturity, resulting in a relatively small stigma, in contrast to the elongate stigma of most species of Magnolia. The flower of Magnolia does not terminate in an organ or organ whorl but achieves determinacy by gradual diminution.     

Identification of Conserved and Novel microRNAs from Liriodendron chinense Floral Tissues

Background

Liriodendron chinense (L. chinense) is an endangered basal angiosperm plant in China because of its low reproductive efficiency. Recently, miRNAs have obtained great attention because they can play important roles. Through high throughput sequencing technique, large amount of miRNAs were identified from different plant species. But there were few studies about the miRNAs in the basal angiosperms especially in the sexual reproduction process.

Results

Deep sequencing technology was applied to discover miRNAs in L. chinense flowers at different stages. After bioinformatic analysis, 496 putative conserved miRNAs representing 97 families and 2 novel miRNAs were found. Among them, one is previously regarded as gymnosperm specific. Their expressions were further validated by Real-time PCR for 13 selected miRNAs. Putative targeting genes were predicted and categorized with gene ontology (GO) analysis. About ten percents of the targets are involved in the reproduction process. Further expressional analysis showed that many of these miRNAs were highly related to the reproductive growth.

Conclusions

This is the first comprehensive identification of conserved and novel miRNAs in L. chinense. The data presented here might not only help to fill the gap of miRNA registered about basal angiosperm plants but also contribute to understanding the evolution of miRNAs. The differential expression of some of the miRNAs and the prediction of their target genes are also helpful in understanding the regulation of L. chinense sexual reproduction.