Testing candidate plant barcode regions in the Myristicaceae

The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon - UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH-psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a more variable region (trnH-psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis.     

Phylogenetic analysis of the acetyl-CoA carboxylase and 3-phosphoglycerate kinase loci in wheat and other grasses

We have applied a two-gene system based on the sequences of nuclear genes encoding multi-domain plastid acetyl-CoA carboxylase (ACCase) and plastid 3-phosphoglycerate kinase (PGK) to study grass evolution. Our analysis revealed that these genes are single-copy in most of the grass species studied, allowing the establishment of orthologous relationships between them. These relationships are consistent with the known facts of their evolution: the eukaryotic origin of the plastid ACCase, created by duplication of a gene encoding the cytosolic multi-domain ACCase gene early in grass evolution, and the prokaryotic (endosymbiont) origin of the plastid PGK. The major phylogenetic relationships among grasses deduced from the nucleotide sequence comparisons of ACCase and PGK genes are consistent with each other and with the milestones of grass evolution revealed by other methods. Nucleotide substitution rates were calculated based on multiple pairwise sequence comparisons. On a relative basis, with the divergence of the Pooideae and Panicoideae subfamilies set at 60 million years ago (MYA), events leading to the Triticum/Aegilops complex occurred at the following intervals: divergence of Lolium (Lolium rigidum) at 35 MYA, divergence of Hordeum (Hordeum vulgare) at 11 MYA and divergence of Secale (Secale cereale) at 7 MYA. On the same scale, gene duplication leading to the multi-domain plastid ACCase in grasses occurred at 129 MYA, divergence of grass and dicot plastid PGK genes at 137 MYA, and divergence of grass and dicot cytosolic PGK genes at 155 MYA. The ACCase and PGK genes provide a well-understood two-locus system to study grass phylogeny, evolution and systematics.     

The non-photosynthetic plastid in malarial parasites and other apicomplexans is derived from outside the green plastid lineage.

The discovery of a non-photosynthetic plastid genome in Plasmodium falciparum and other apicomplexans has provided a new drug target, but the evolutionary origin of the plastid has been muddled by the lack of characters, that typically define major plastid lineages. To clarify the ancestry of the plastid, we undertook a comprehensive analysis of all genomic characters shared by completely sequenced plastid genomes. Cladistic analysis of the pattern of plastid gene loss and gene rearrangements suggests that the apicomplexan plastid is derived from an ancestor outside of the green plastid lineage. Phylogenetic analysis of primary sequence data (DNA and amino acid characters) produces results that are generally independent of the analytical method, but similar genes (i.e., rpoB and rpoC) give similar topologies. The conflicting phylogenies in primary sequence data sets make it difficult to determine the the exact origin of the apicomplexan plastid and the apparent artifactual association of apicomplexan and euglenoid sequences suggests that DNA sequence data may be an inappropriate set of characters to address this phylogenetic question. At present we cannot reject our null hypothesis that the apicomplexan plastid is derived from a shared common ancestor between apicomplexans and dinoflagellates. During the analysis, we noticed that the Plasmodium tRNA-Met is probably tRNA-fMet and the tRNA-fMet is probably tRNA-Ile. We suggest that P. falciparum has lost the elongator type tRNA-Met and that similar to metazoan mitochondria there is only one species of methionine tRNA. In P. falciparum, this has been accomplished by recruiting the fMet-type tRNA to dually function in initiation and elongation. The tRNA-Ile has an unusual stem-loop in the variable region. The insertion in this region appears to have occurred after the primary origin of the plastid and further supports the monophyletic ancestory of plastids.     

Maize chloroplast RNA polymerase: the 78-kilodalton polypeptide is encoded by the plastid rpoC1 gene.

The 180-, 120- and 38-kDa polypeptides found in highly purified maize plastid RNA polymerase preparations are encoded by the maize plastid genes rpoC2, rpoB, and rpoA, respectively [Hu, J. and Bogorad, L. (1990) Proc. Natl. Acad. Sci. USA. 87, pp. 1531-1535]. These genes have segments that specify amino acid sequences homologous to those of E. coli RNA polymerase subunits. The plastid gene products are designated b", b and a, respectively. We report here that the amino-terminal amino acid sequence of a 78-kDa polypeptide also found in highly purified maize plastid RNA polymerase preparations matches precisely the sequence deduced from the maize plastid rpoC1 gene which has segments homologous to the 5' end of the E. coli rpoC gene. Thus, the 78-kDa polypeptide is likely to be a functional component of maize plastid DNA-dependent RNA polymerase. This polypeptide is designated subunit b'. Three polypeptides unrelated to RNA polymerase have also been identified in this preparation.     

Molecular analysis of a 21.1-kb fragment of wheat chloroplast DNA bearing RNA polymerase subunit (rpo) genes.

The entire nucleotide sequence of a 21.1-kb fragment of wheat chloroplast (ct) DNA was determined. This fragment carries 18 intact genes and parts of two additional genes, including the three RNA polymerase genes rpoB, rpoC1, and rpoC2. The gene arrangement of this region is conserved in wheat, rice, and maize, but not in non-grass species. Comparison of these 20 genes in wheat, rice, and maize showed that tRNA genes evolved more slowly than protein-coding genes in the chloroplast genome. Intergenic regions evolved much faster than both types of genes. Although the 19 genes of wheat, except for orf42, showed high identity to those of other plants, there were three novel structural features in the wheat rpoC2 gene; a deletion of 81 bp in the middle region, a variable insertion (408 bp), and a nonsense mutation in the 3' terminal region, resulting in truncation of a sequence of ca. 10 amino acids. An intermolecular recombination between the stretches of CTTAT and CTTTT was suggested as the mechanism of the 81-bp deletion in the wheat rpoC2 gene. Evolutionary distance between the chloroplast genomes of wheat and maize was larger than those between wheat and rice and between rice and maize.     

Molecular phylogenetics of Maxillaria and related genera (Orchidaceae: Cymbidieae) based on combined molecular data sets

The orchid genus Maxillaria is one of the largest and most common of neotropical orchid genera, but its current generic boundaries and relationships have long been regarded as artificial. Phylogenetic relationships within subtribe Maxillariinae sensu Dressler (1993) with emphasis on Maxillaria s.l. were inferred using parsimony analyses of individual and combined DNA sequence data. We analyzed a combined matrix of nrITS DNA, the plastid matK gene and flanking trnK intron, and the plastid atpB-rbcL intergenic spacer for 619 individuals representing ca. 354 species. The plastid rpoC1 gene (ca. 2600 bp) was sequenced for 84 selected species and combined in a more limited analysis with the other data sets to provide greater resolution. In a well-resolved, supported consensus, most clades were present in more than one individual analysis. All the currently recognized minor genera of "core" Maxillariinae (Anthosiphon, Chrysocycnis, Cryptocentrum, Cyrtidiorchis, Mormolyca, Pityphyllum, and Trigonidium) are embedded within a polyphyletic Maxillaria s.l. Our results support the recognition of a more restricted Maxillaria, of some previously published segregate genera (Brasiliorchis, Camaridium, Christensonella, Heterotaxis, Ornithidium, Sauvetrea), and of several novel clades at the generic level. These revised monophyletic generic concepts should minimize further nomenclatural changes, encourage monographic studies, and facilitate more focused analyses of character evolution within Maxillariinae.     

Plastid genome evolution in mycoheterotrophic Ericaceae

Unlike parasitic plants, which are linked to their hosts directly through haustoria, mycoheterotrophic (MHT) plants derive all or part of their water and nutrients from autothrophs via fungal mycorrhizal intermediaries. Ericaceae, the heather family, are a large and diverse group of plants known to form elaborate symbiotic relationships with mycorrhizal fungi. Using PHYA sequence data, we first investigated relationships among mycoheterotrophic Ericaceae and their close autotrophic relatives. Phylogenetic results suggest a minimum of two independent origins of MHT within this family. Additionally, a comparative investigation of plastid genomes (plastomes) grounded within this phylogenetic framework was conducted using a slot-blot Southern hybridization approach. This survey encompassed numerous lineages of Ericaceae with different life histories and trophic levels, including multiple representatives from mixotrophic Pyroleae and fully heterotrophic Monotropeae and Pterosporeae. Fifty-four probes derived from all categories of protein coding genes typically found within the plastomes of flowering plants were used. Our results indicate that the holo-mycoheterotrophic Ericaceae exhibit extensive loss of genes relating to photosynthetic function and expression of the plastome but retain genes with possible functions outside photosynthesis. Mixotrophic taxa tend to retain most genes relating to photosynthetic functions but are varied regarding the plastid ndh gene content. This investigation extends previous inferences that the loss of the NDH complex occurs prior to becoming holo-heterotrophic and it shows that the pattern of gene losses among mycoheterotrophic Ericaceae is similar to that of haustorial parasites. Additionally, we identify the most desirable candidate species for entire plastome sequencing.     

Phylogeny, rate variation, and genome size evolution of Pelargonium (Geraniaceae)

The phylogeny of 58 Pelargonium species was estimated using five plastid markers (rbcL, matK, ndhF, rpoC1, trnL-F) and one mitochondrial gene (nad5). The results confirmed the monophyly of three major clades and four subclades within Pelargonium but also indicate the need to revise some sectional classifications. This phylogeny was used to examine karyotype evolution in the genus: plotting chromosome sizes, numbers and 2C-values indicates that genome size is significantly correlated with chromosome size but not number. Accelerated rates of nucleotide substitution have been previously detected in both plastid and mitochondrial genes in Pelargonium, but sparse taxon sampling did not enable identification of the phylogenetic distribution of these elevated rates. Using the multigene phylogeny as a constraint, we investigated lineage- and locus-specific heterogeneity of substitution rates in Pelargonium for an expanded number of taxa and demonstrated that both plastid and mitochondrial genes have had accelerated substitution rates but with markedly disparate patterns. In the plastid, the exons of rpoC1 have significantly accelerated substitution rates compared to its intron and the acceleration was mainly due to nonsynonymous substitutions. In contrast, the mitochondrial gene, nad5, experienced substantial acceleration of synonymous substitution rates in three internal branches of Pelargonium, but this acceleration ceased in all terminal branches. Several lineages also have dN/dS ratios significantly greater than one for rpoC1, indicating that positive selection is acting on this gene, whereas the accelerated synonymous substitutions in the mitochondrial gene are the result of elevated mutation rates.     

A widespread occurrence of extra open reading frames in plant Ty3/gypsy retrotransposons

Long terminal repeat (LTR) retrotransposons make up substantial parts of most higher plant genomes where they accumulate due to their replicative mode of transposition. Although the transposition is facilitated by proteins encoded within the gag-pol region which is common to all autonomous elements, some LTR retrotransposons were found to potentially carry an additional protein coding capacity represented by extra open reading frames located upstream or downstream of gag-pol. In this study, we performed a comprehensive in silico survey and comparative analysis of these extra open reading frames (ORFs) in the group of Ty3/gypsy LTR retrotransposons as the first step towards our understanding of their origin and function. We found that extra ORFs occur in all three major lineages of plant Ty3/gypsy elements, being the most frequent in the Tat lineage where most (77?%) of identified elements contained extra ORFs. This lineage was also characterized by the highest diversity of extra ORF arrangement (position and orientation) within the elements. On the other hand, all of these ORFs could be classified into only two broad groups based on their mutual similarities or the presence of short conserved motifs in their inferred protein sequences. In the Athila lineage, the extra ORFs were confined to the element 3' regions but they displayed much higher sequence diversity compared to those found in Tat. In the lineage of Chromoviruses the extra ORFs were relatively rare, occurring only in 5' regions of a group of elements present in a single plant family (Poaceae). In all three lineages, most extra ORFs lacked sequence similarities to characterized gene sequences or functional protein domains, except for two Athila-like elements with similarities to LOGL4 gene and part of the Chromoviruses extra ORFs that displayed partial similarity to histone H3 gene. Thus, in these cases the extra ORFs most likely originated by transduction or recombination of cellular gene sequences. In addition, the protein domain which is otherwise associated with DNA transposons have been detected in part of the Tat-like extra ORFs, pointing to their origin from an insertion event of a mobile element.     

Detection and characterization of a plastid envelope DNA-binding protein which may anchor plastid nucleoids.

Chloroplast DNA (cpDNA) binds to the envelope membrane of actively dividing chloroplasts (plastids) in young pea leaves. South-western blotting was used to identify and characterize the protein involved in the binding of cpDNA to the envelope membrane. A 130 kDa protein in the inner chloroplast (plastid) envelope membrane binds specific sequences within the cpDNA. These included a 0.41 kbp sequence located upstream of the psaAB gene, a 0.57 kbp sequence located downstream of the petA gene and a 1.2 kbp sequence located within the rpoC2 gene. The protein was detected in the envelope membrane of young pea leaves in which the cpDNA had been located by fluorescence microscopy at the chloroplast periphery, whereas it was undetectable in mature leaves. We therefore propose that the 130 kDa protein is involved in the binding of cpDNA to the envelope membrane, and named it plastid envelope DNA-binding protein.     

Rice chloroplast RNA polymerase genes: The absence of an intron in rpoC1 and the presence of an extra sequence in rpoC2

Summary The chloroplast genome contains sequences homologous to the Escherichia coli rpoA, rpoB and rpoC genes. The Choroplast rpoC gene is divided into rpoC1 and rpoC2, of which rpoC1 contains an intron. Comparison of the rice rpo genes with those from tobacco, spinach and liverwort revealed unique features of the rice genes; the lack of an intron in rpoC1 and the presence of an extra sequence of 381 by in rpoC2. The intron in rpoC1 is thus optional, and possible intron boundary sites in split rpoC1 genes can be estimated by comparison with rice rpoC1. The extra sequence is located in the middle of rpoC2 and has repeated structures. The amino acid sequence deduced from this sequence is extremely hydrophilic and anionic. The origin and function of this sequence are discussed.     

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10–60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, “secondary” recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of candidate DNA barcoding loci for economically important timber species of the mahogany family (Meliaceae)

There has been considerable debate regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from proposed candidate loci on a common set of samples. In this study, we evaluated main candidate plastid regions (rpoC1, rpoB, accD) and additional plastid markers (psbB, psbN, psbT exons and the trnS-trnG spacer) as well as the nuclear ribosomal spacer region (ITS1-5.8S-ITS2) in a group of land plants belonging to the mahogany family, Meliaceae. Across these samples, only ITS showed high levels of resolvability. Interspecific sharing of sequences from individual plastid loci was common. The combination of multiple loci did not improve performance. DNA barcoding with ITS alone revealed cryptic species and proved useful in identifying species listed in Convention on International Trade of Endangered Species appendixes.     

Implications of the Plastid Genome Sequence of Typha (Typhaceae, Poales) for Understanding Genome Evolution in Poaceae

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.     

A phylogeny of the flowering plant family Apiaceae based on chloroplast DNA rpl16 and rpoC1 intron sequences: towards a suprageneric classification of subfamily Apioideae

The higher level relationships within Apiaceae (Umbelliferae) subfamily Apioideae are controversial, with no widely acceptable modern classification available. Comparative sequencing of the intron in chloroplast ribosomal protein gene rpl16 was carried out in order to examine evolutionary relationships among 119 species (99 genera) of subfamily Apioideae and 28 species from Apiaceae subfamilies Saniculoideae and Hydrocotyloideae, and putatively allied families Araliaceae and Pittosporaceae. Phylogenetic analyses of these intron sequences alone, or in conjunction with plastid rpoC1 intron sequences for a subset of the taxa, using maximum parsimony and neighbor-joining methods, reveal a pattern of relationships within Apioideae consistent with previously published chloroplast DNA and nuclear ribosomal DNA ITS based phylogenies. Based on consensus of relationship, seven major lineages within the subfamily are recognized at the tribal level. These are referred to as tribes Heteromorpheae M. F. Watson & S. R. Downie Trib. Nov., Bupleureae Spreng. (1820), Oenantheae Dumort. (1827), Pleurospermeae M. F. Watson & S. R. Downie Trib. Nov., Smyrnieae Spreng. (1820), Aciphylleae M. F. Watson & S. R. Downie Trib. Nov., and Scandiceae Spreng. (1820). Scandiceae comprises subtribes Daucinae Dumort. (1827), Scandicinae Tausch (1834), and Torilidinae Dumort. (1827). Rpl16 intron sequences provide valuable characters for inferring high-level relationships within Apiaceae but, like the rpoC1 intron, are insufficient to resolve relationships among closely related taxa.     

A phylogenetic hypothesis for the Aizoaceae (Caryophyllales) based on four plastid DNA regions

The Aizoaceae is the largest family of leaf succulent plants, and most of its species are endemic to southern Africa. To evaluate subfamilial, generic, and tribal relationships, we produced two plastid DNA data sets for 91 species of Aizoaceae and four outgroups: rps16 intron and the trnL-F gene region (both the trnL intron and the trnL-F intergenic spacer). In addition, we generated two further plastid data sets for 56 taxa restricted to members of the Ruschioideae using the atpB-rbcL and the psbA-trnH intergenic spacers. In the combined tree of the rps16 intron and trnL-F gene region, three of the currently recognized subfamilies (Sesuvioideae, Mesembryanthemoideae, and Ruschioideae) are each strongly supported monophyletic groups. The subfamily Tetragonioideae is polyphyletic, with Tribulocarpus as sister to the Sesuvioideae and Tetragonia embedded in the Aizooideae. Our study showed that the group consisting of the Sesuvioideae, Aizooideae, and Tetragonioideae does not form a monophyletic entity. Therefore, it cannot be recognized as a separate family in order to accommodate the frequently used concept of the Mesembryanthemaceae or "Mesembryanthema," in which the subfamilies Mesembryanthemoideae and Ruschioideae are included. We also found that several genera within the Mesembryanthemoideae (Mesembryanthemum, Phyllobolus) are not monophyletic. Within the Ruschioideae, our study retrieved four major clades. However, even in the combined analysis of all four plastid gene regions, relationships within the largest of these four clades remain unresolved. The few nucleotide substitutions that exist among taxa of this clade point to a rapid and recent diversification within the arid winter rainfall area of southern Africa. We propose a revised classification for the Aizoaceae.     

Structure of the bovine natural resistance associated macrophage protein (NRAMP 1) gene and identification of a novel polymorphism.

The NRAMP 1 gene is a major candidate gene influencing the outcome of infections with intracellular pathogens in numerous species. NRAMP 1 is highly conserved in many mammalian species and the NRAMP 1 gene shows considerable conservation in structure between mice and humans. The association of NRAMP 1 gene polymorphisms with disease in cattle has been limited to a single microsatellite located within the 3'-non coding region of the bovine NRAMP 1 gene. In order to facilitate further studies on this important gene, we now report the nearly complete structure of the bovine NRAMP 1 gene, including sizes and positions of 13 introns relative to the bovine NRAMP 1 gene coding sequence and the DNA sequence of intron-exon junctions. Comparison of the bovine, murine and human NRAMP 1 gene structures revealed a high degree of conservation in intron placement, though the lengths of several introns were less-well conserved. In general, the greatest divergence in intron lengths occurred in regions of the NRAMP 1 gene displaying the lowest coding sequence conservation. In addition, mutations near intron-exon junctions could account for 25 of the 75 total amino acid differences between murine and bovine NRAMP 1. Using information gained through this study, it was possible to rapidly identify a novel polymorphism within the bovine NRAMP 1 gene intron X. This polymorphism was shown by direct DNA sequence analysis to consist of insertion of three guanine nucleotides at positions 37,40 and 98 relative to the intron X start point. Initial scans of several cattle breeds suggest that the two intron X alleles identified here are stable and widespread in the Bos taurus population.