LtuCAD1 Is a Cinnamyl Alcohol Dehydrogenase Ortholog Involved in Lignin Biosynthesis in Liriodendron tulipifera L., a Basal Angiosperm Timber Species

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and catalyzes the final step in the synthesis of monolignols. Seven CAD homologs (LtuCAD1 to LtuCAD7) have been previously identified from a basal angiosperm species Liriodendron tulipifera L., which is an important timber tree species with significant ecological and economic values. The phylogenetic analysis indicates that LtuCAD1 is the only Liriodendron CAD grouped with the bona fide CADs, the primary CAD genes involved in lignification. In this study, the predicted protein sequence of LtuCAD1 was found to have conserved domains and the same key determinant site with the bona fide CADs in other plant species. Additionally, LtuCAD1 had the highest expression level in xylem as revealed by quantitative RT-PCR analysis. The expression of beta-glucuronidase (GUS) driven by the LtuCAD1 promoter was largely localized in vascular tissues in Arabidopsis. In stem cross sections, GUS staining was found exclusively in xylem and phloem. When expressed in the Arabidopsis cad4 cad5 double mutant, LtuCAD1 was able to restore the total lignin content and decrease the S/G lignin ratio. Our data indicate that LtuCAD1 is a CAD ortholog involved in lignin biosynthesis in Liriodendron.     

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.     

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

Background

Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.

Results

Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.

Conclusions

When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.     

Evolution of the Cinnamyl/Sinapyl Alcohol Dehydrogenase (CAD/SAD) Gene Family: The Emergence of Real Lignin is Associated with the Origin of Bona Fide CAD

Lignin plays a vital role in plant adaptation to terrestrial environments. The cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and might have contributed to the lignin diversity in plants. To investigate the evolutionary history and functional differentiation of the CAD gene family, we made a comprehensive evolutionary analysis of this gene family from 52 species, including bacteria, early eukaryotes and green plants. The phylogenetic analysis, together with gene structure and function, indicates that all members of land plants, except two of moss, could be divided into three classes. Members of Class I (bona fide CAD), generally accepted as the primary genes involved in the monolignol biosynthesis, are all from vascular plants, and form a robustly supported monophyletic group with the lycophyte CADs at the basal position. This class is also conserved in the predicted three-dimensional structure and the residues constituting the substrate-binding pocket of the proteins. Given that Selaginella has real lignin, the above evidence strongly suggests that the earliest occurrence of the bona fide CAD in the lycophyte could be directly correlated with the origin of lignin. Class II comprises members more similar to the aspen sinapyl alcohol dehydrogenase gene, and includes three groups corresponding to lycophyte, gymnosperm, and angiosperm. Class III is conserved in land plants. The three classes differ in patterns of evolution and expression, implying that functional divergence has occurred among them. Our study also supports the hypothesis of convergent evolution of lignin biosynthesis between red algae and vascular plants.     

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.     

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle‐scale barcodes. Next‐generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high‐quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long‐range PCR and sequenced using next‐generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early‐diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome‐scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.     

Complete chloroplast genome sequence of Elodea canadensis and comparative analyses with other monocot plastid genomes

Elodea canadensis is an aquatic angiosperm native to North America. It has attracted great attention due to its invasive nature when transported to new areas in its non-native range. We have determined the complete nucleotide sequence of the chloroplast (cp) genome of Elodea. Taxonomically Elodea is a basal monocot, and only few monocot cp genomes representing early lineages of monocots have been sequenced so far. The genome is a circular double-stranded DNA molecule 156,700bp in length, and has a typical structure with large (LSC 86,194bp) and small (SSC 17,810bp) single-copy regions separated by a pair of inverted repeats (IRs 26,348bp each). The Elodea cp genome contains 113 unique genes and 16 duplicated genes in the IR regions. A comparative analysis showed that the gene order and organization of the Elodea cp genome is almost identical to that of Amborella trichopoda, a basal angiosperm. The structure of IRs in Elodea is unique among monocot species with the whole cp genome sequenced. In Elodea and another monocot Lemna minor the borders between IRs and LSC are located upstream of rps19 gene and downstream of trnH-GUG gene, while in most monocots, IR has extended to include both trnH and rps19 genes. A phylogenetic analysis conducted using Bayesian method, based on the DNA sequences of 81 chloroplast genes from 17 monocot taxa provided support for the placement of Elodea together with Lemna as a basal monocot and the next diverging lineage of monocots after Acorales. In comparison with other monocots, the Elodea cp genome has gone through only few rearrangements or gene losses. IR of Elodea has a unique structure among the monocot species studied so far as its structure is similar to that of a basal angiosperm Amborella. This result together with phylogenetic analyses supports the placement of Elodea as a basal monocot to the next diverging lineage of monocots after Acorales. So far, only few cp genomes representing early lineages of monocots have been sequenced and, therefore, this study provides valuable information about the course of evolution in divergence of monocot lineages.     

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.     

Mitochondrial phylogeny of hedgehogs and monophyly of Eulipotyphla

We sequenced the complete mitochondrial (mt) genomes of three insectivores: the long-eared hedgehog Hemiechinus auritus, the Japanese mole Mogera wogura, and the greater Japanese shrew-mole Urotrichus talpoides. These mtDNA data together with other previously sequenced mtDNAs were analyzed using a maximum likelihood method to infer their phylogenetic relationships among eutherians. Previous mitochondrial protein analyses used a simple model that did not consider site-heterogeneity, and Erinaceoidea (hedgehogs and moonrats) was placed at the basal eutherian position that is separated from Soricoidea (shrews) and Talpoidea (moles), suggesting the exclusion of the Erinaceoidea-Eulipotyphla tree. By including the new mtDNA sequences and introducing site-heterogeneity into the model, the Erinaceoidea-Eulipotyphla tree emerges as the best tree or as a tree with a log-likelihood score indistinguishable from that of the best tree. However, this conclusion depends on species sampling in Erinaceoidea, demonstrating the importance of both species sampling and use of an appropriate substitution model when inferring phylogenetic relationships.     

Multigene analyses identify the three earliest lineages of extant flowering plants

Flowering plants (angiosperms) are by far the largest, most diverse, and most important group of land plants, with over 250,000 species and a dominating presence in most terrestrial ecosystems. Understanding the origin and early diversification of angiosperms has posed a long-standing botanical challenge [1]. Numerous morphological and molecular systematic studies have attempted to reconstruct the early history of this group, including identifying the root of the angiosperm tree. There is considerable disagreement among these studies, with various groups of putatively basal angiosperms from the subclass Magnoliidae having been placed at the root of the angiosperm tree (reviewed in [2-4]). We investigated the early evolution of angiosperms by conducting combined phylogenetic analyses of five genes that represent all three plant genomes from a broad sampling of angiosperms. Amborella, a monotypic, vessel-less dioecious shrub from New Caledonia, was clearly identified as the first branch of angiosperm evolution, followed by the Nymphaeales (water lillies), and then a clade of woody vines comprising Schisandraceae and Austrobaileyaceae. These findings are remarkably congruent with those from several concurrent molecular studies [5-7] and have important implications for whether or not the first angiosperms were woody and contained vessels, for interpreting the evolution of other key characteristics of basal angiosperms, and for understanding the timing and pattern of angiosperm origin and diversification.     

SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C

Background

The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.

Results

The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.

Conclusions

The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.     

The 19 genomes of Drosophila: a BAC library resource for genus-wide and genome-scale comparative evolutionary research

The genus Drosophila has been the subject of intense comparative phylogenomics characterization to provide insights into genome evolution under diverse biological and ecological contexts and to functionally annotate the Drosophila melanogaster genome, a model system for animal and insect genetics. Recent sequencing of 11 additional Drosophila species from various divergence points of the genus is a first step in this direction. However, to fully reap the benefits of this resource, the Drosophila community is faced with two critical needs: i.e., the expansion of genomic resources from a much broader range of phylogenetic diversity and the development of additional resources to aid in finishing the existing draft genomes. To address these needs, we report the first synthesis of a comprehensive set of bacterial artificial chromosome (BAC) resources for 19 Drosophila species from all three subgenera. Ten libraries were derived from the exact source used to generate 10 of the 12 draft genomes, while the rest were generated from a strategically selected set of species on the basis of salient ecological and life history features and their phylogenetic positions. The majority of the new species have at least one sequenced reference genome for immediate comparative benefit. This 19-BAC library set was rigorously characterized and shown to have large insert sizes (125-168 kb), low nonrecombinant clone content (0.3-5.3%), and deep coverage (9.1-42.9×). Further, we demonstrated the utility of this BAC resource for generating physical maps of targeted loci, refining draft sequence assemblies and identifying potential genomic rearrangements across the phylogeny.     

Estimates of conserved microsynteny among the genomes of <Emphasis Type="Italic">Glycine max</Emphasis>, <Emphasis Type="Italic">Medicago truncatula</Emphasis> and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A growing body of research indicates that microsynteny is common among dicot genomes. However, most studies focus on just one or a few genomic regions, so the extent of microsynteny across entire genomes remains poorly characterized. To estimate the level of microsynteny between Medicago truncatula (Mt) and Glycine max (soybean), and also among homoeologous segments of soybean, we used a hybridization strategy involving bacterial artificial chromosome (BAC) contigs. A Mt BAC library consisting of 30,720 clones was screened with a total of 187 soybean BAC subclones and restriction fragment length polymorphism (RFLP) probes. These probes came from 50 soybean contig groups, defined as one or more related BAC contigs anchored by the same low-copy probe. In addition, 92 whole soybean BAC clones were hybridized to filters of HindIII-digested Mt BAC DNA to identify additional cases of cross-hybridization after removal of those soybean BACs found to be repetitive in Mt. Microsynteny was inferred when at least two low-copy probes from a single soybean contig hybridized to the same Mt BAC or when a soybean BAC clone hybridized to three or more low-copy fragments from a single Mt BAC. Of the 50 soybean contig groups examined, 54% showed microsynteny to Mt. The degree of conservation among 37 groups of soybean contigs was also investigated. The results indicated substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. One contig group was examined in detail by a combination of physical mapping and comparative sequencing of homoeologous segments. A TBLASTX similarity search was performed between 1,085 soybean sequences on the 50 BAC contig groups and the entire Arabidopsis genome. Based on a criterion of sequence homologues <100 kb apart, each with an expected value of < or =1e-07, seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.     

Phylogenomics reveal a robust fungal tree of life

Our understanding of the tree of life (TOL) is still fragmentary. Until recently, molecular phylogeneticists have built trees based on ribosomal RNA sequences and selected protein sequences, which, however, usually suffered from lack of support for the deeper branches and inconsistencies probably due to limited subsampling of the entire genome. Now, phylogenetic hypotheses can be based on the analysis of full genomes. We used available complete genome data as well as the eukaryote orthologous group (KOG) proteins to reconstruct with confidence basal branches of the fungal TOL. Phylogenetic analysis of a core of 531 KOGs shared among 21 fungal genomes, three animal genomes and one plant genome showed a single tree with high support resulting from four different methods of phylogenetic reconstruction. The single tree that we inferred from our dataset showed excellent nodal support for each branch, suggesting that it reflects the true phylogenetic relationships of the species involved.     

Molecular evolution and phylogenetic utility of the petD group II intron: a case study in basal angiosperms

Sequences of spacers and group I introns in plant chloroplast genomes have recently been shown to be very effective in phylogenetic reconstruction at higher taxonomic levels and not only for inferring relationships among species. Group II introns, being more frequent in those genomes than group I introns, may be further promising markers. Because group II introns are structurally constrained, we assumed that sequences of a group II intron should be alignable across seed plants. We designed universal amplification primers for the petD intron and sequenced this intron in a representative selection of 47 angiosperms and three gymnosperms. Our sampling of taxa is the most representative of major seed plant lineages to date for group II introns. Through differential analysis of structural partitions, we studied patterns of molecular evolution and their contribution to phylogenetic signal. Nonpairing stretches (loops, bulges, and interhelical nucleotides) were considerably more variable in both substitutions and indels than in helical elements. Differences among the domains are basically a function of their structural composition. After the exclusion of four mutational hotspots accounting for less than 18% of sequence length, which are located in loops of domains I and IV, all sequences could be aligned unambiguously across seed plants. Microstructural changes predominantly occurred in loop regions and are mostly simple sequence repeats. An indel matrix comprising 241 characters revealed microstructural changes to be of lower homoplasy than are substitutions. In showing Amborella first branching and providing support for a magnoliid clade through a synapomorphic indel, the petD data set proved effective in testing between alternative hypotheses on the basal nodes of the angiosperm tree. Within angiosperms, group II introns offer phylogenetic signal that is intermediate in information content between that of spacers and group I introns on the one hand and coding sequences on the other.     

Phylogenetic and evolutionary implications of complete chloroplast genome sequences of four early-diverging angiosperms: Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae)

We have determined the complete chloroplast genome sequences of four early-diverging lineages of angiosperms, Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae), to examine the organization and evolution of plastid genomes and to estimate phylogenetic relationships among angiosperms. For the most part, the organization of these plastid genomes is quite similar to the ancestral angiosperm plastid genome with a few notable exceptions. Dioscorea has lost one protein-coding gene, rps16; this gene loss has also happened independently in four other land plant lineages, liverworts, conifers, Populus, and legumes. There has also been a small expansion of the inverted repeat (IR) in Dioscorea that has duplicated trnH-GUG. This event has also occurred multiple times in angiosperms, including in monocots, and in the two basal angiosperms Nuphar and Drimys. The Illicium chloroplast genome is unusual by having a 10 kb contraction of the IR. The four taxa sequenced represent key groups in resolving phylogenetic relationships among angiosperms. Illicium is one of the basal angiosperms in the Austrobaileyales, Chloranthus (Chloranthales) remains unplaced in angiosperm classifications, and Buxus and Dioscorea are early-diverging eudicots and monocots, respectively. We have used sequences for 61 shared protein-coding genes from these four genomes and combined them with sequences from 35 other genomes to estimate phylogenetic relationships using parsimony, likelihood, and Bayesian methods. There is strong congruence among the trees generated by the three methods, and most nodes have high levels of support. The results indicate that Amborella alone is sister to the remaining angiosperms; the Nymphaeales represent the next-diverging clade followed by Illicium; Chloranthus is sister to the magnoliids and together this group is sister to a large clade that includes eudicots and monocots; and Dioscorea represents an early-diverging lineage of monocots just internal to Acorus.     

Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses

Background

Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change.

Methodology/Principal Findings

We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses.

Conclusions/Significance

Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects.     

DNA C-values in seven families fill phylogenetic gaps in the basal angiosperms

The evolutionary significance of the c . 1000-fold range of DNA C-values in angiosperms (1C =  c . 0.1–127.4 pg) has often attracted interest. A recent analysis, which superimposed available C-value data onto the angiosperm phylogeny, that placed Ceratophyllaceae as the most basal angiosperm family led to the conclusion that ancestral angiosperms were characterized by small genomes (defined as 1C £ 3.5 pg). However, with the recent increase in DNA sequence data and large-scale phylogenetic analyses, strong support is now provided for Amborellaceae and/or Nymphaeaceae as the most basal angiosperm families, followed by Austrobaileyales (comprising Schisandraceae, Trimeniaceae and Austrobaileyaceae). Together these five families comprise the ANITA grade. The remaining basal angiosperm families (Ceratophyllaceae, Chloranthaceae and magnoliids), together with monocotyledons and eudicotyledons, form a strongly supported clade. A survey showed that C-value data were scarce in the basal angiosperm families, especially the ANITA grade. The present paper addresses these phylogenetic gaps by providing C-value estimates for each family in ANITA, together with C-values for species in Chloranthaceae, Ceratophyllaceae and a previously unrepresented family in the magnoliids, the Winteraceae.  © The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 140 , 175–179.     

Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage

Cultivation-independent surveys of ribosomal RNA genes have revealed the existence of novel microbial lineages, many with no known cultivated representatives. Ribosomal RNA-based analyses, however, often do not provide significant information beyond phylogenetic affiliation. Analysis of large genome fragments recovered directly from microbial communities represents one promising approach for characterizing uncultivated microbial species better. To assess further the utility of this approach, we constructed large-insert bacterial artificial chromosome (BAC) libraries from the genomic DNA of planktonic marine microbial assemblages. The BAC libraries we prepared had average insert sizes of 80 kb, with maximal insert sizes > 150 kb. A rapid screening method assessing the phylogenetic diversity and representation in the library was developed and applied. In general, representation in the libraries agreed well with previous culture-independent surveys based on polymerase chain reaction (PCR)amplified rRNA fragments. A significant fraction of the genome fragments in the BAC libraries originated from as yet uncultivated microbial species, thought to be abundant and widely distributed in the marine environment. One entire BAC insert, derived from an uncultivated, surface-dwelling euryarchaeote, was sequenced completely. The planktonic euryarchaeal genome fragment contained some typical archaeal genes, as well as unique open reading frames (ORFs) suggesting novel function. In total, our results verify the utility of BAC libraries for providing access to the genomes of as yet uncultivated microbial species. Further analysis of these BAC libraries has the potential to provide significant insight into the genomic potential and ecological roles of many indigenous microbial species, cultivated or not.