Physicochemical evolution and positive selection of the gymnosperm matK proteins

It is not clear whether matK evolves under Darwinian selection. In this study, the gymnosperm Taxaceae, Cephalotaxaceae and Pinaceae were used to illustrate the physicochemical evolution, molecular adaptation and evolutionary dynamics of gene divergence in matKs. matK sequences were amplified from 27 Taxaceae and 12 Cephalotaxaceae species. matK sequences of 19 Pinaceae species were retrieved from GenBank. The phylogenetic tree was generated using conceptual-translated amino acid sequences. Selective influences were investigated using standard d _N/d _S ratio methods and more sensitive techniques investigating the amino acid property changes resulting from nonsynonymous replacements in a phylogenetic context. Analyses revealed the presence of positive selection in matKs (N-terminal region, RT domain and domain X) of Taxaceae and Pinaceae, and found positive destabilizing selection in N-terminal region and RT domain of Cephalotaxaceae matK. Moreover, various amino acid properties were found to be influenced by destabilizing positive selection. Amino acid sites relating to these properties and to different secondary structures were found and have the potential to affect group II intron maturase function. Despite the evolutionary constraint on the rapidly evolving matK, this protein evolves under positive selection in gymnosperm. Several regions of matK have experienced molecular adaptation which fine-tunes maturase performance.     

阴地蕨属药用植物matK基因编码区全序列测定及编码产物研究

以阴地蕨属(Botrychium)5种药用植物的matK基因为对象,分析matK基因编码区全序列和其编码产物MATK蛋白的氨基酸序列特征,并比较他们在用于阴地蕨属药用植物系统发育关系研究中的差异。结果显示,阴地蕨属5种植物matK基因全长为1500~1503 bp,共有153个变异位点,其编码产物均为不稳定的亲水性蛋白,无跨膜结构,二级结构以α-螺旋和无规则卷曲为主。系统发育分析结果表明,基于matK基因序列的系统发育分析更适合于阴地蕨属种间亲缘关系的鉴定,说明matK基因在阴地蕨属植物的鉴定中具有一定的应用价值。     

New universal matK primers for DNA barcoding angiosperms

The chloroplast maturase K gene (matK) is one of the most variable coding genes of angiosperms and has been suggested to be a "barcode" for land plants. However, matK exhibits low amplification and sequencing rates due to low universality of currently available primers and mononucleotide repeats. To resolve these technical problems, we evaluated the entire matK region to find a region of 600-800 bp that is highly variable, represents the best of all matK regions with priming sites conservative enough to design universal primers, and avoids the mononucleotide repeats. After careful evaluation, a region in the middle was chosen and a pair of primers named natK472F and matK1248R was designed to amplify and sequence the matK fragment of approximately 776 bp. This region encompasses the most variable sites, represents the entire matK region best, and also exhibits high amplification rates and quality of sequences. The universality of this primer pair was tested using 58 species from 47 families of angiosperm plants. The primers showed a strong amplification (93.1%) and sequencing (92.6%)successes in the species tested. We propose that the new primers will solve, in part, the problems encountered when using matK and promote the adoption of matK as a DNA barcode for angiosperms.     

Molecular Phylogeny of Nicotiana (Solanaceae) Based on the Nucleotide Sequence of the matK Gene

Abstract: Intrageneric relationships in the genus Nicotiana were investigated by comparison of DNA sequences of the matK gene of the chloroplast genome. A total of 40 taxa were examined in this study, representing 39 of the approximately 70 wild species of this genus. We obtained the full sequences of the 1530 bp matK ORFs; no variations in length due to insertions or deletions were found. The phylogenetic trees obtained from maximum parsimony (MP) and neighbour-joining (NJ) methods were fundamentally consistent. The genus Nicotiana formed a clade in these trees. The traditional classification of this genus was mostly in agreement with the molecular phylogeny. However, all three subgenera and some sections did not form a monophyletic group. Character-state mappings were used to infer a centre of origin, biogeographic history, and transition of chromosome number. The results support the previous hypothesis that this genus originated in South America and subsequently spread to other continents. The suggestion that the ancestral basic chromosome number is x = 12 and that polyploidy and aneuploidy have occurred independently several times during the evolution of Nicotiana species is also discussed.     

Identification of functional open reading frames in chloroplast genomes

We have used a rapid computer dot-matrix comparison method to identify all DNA regions which have been evolutionarily conserved between the completely sequenced chloroplast genomes of tobacco and a liverwort. Analysis of these regions reveals 74 homologous open reading frames (ORFs) which have been conserved as to length and amino acid sequence; these ORFs also have an excess of nucleotide substitutions at silent sites of codons. Since the nonfunctional parts of these genomes have become saturated with mutations and show no sequence similarity whatsoever, the homologous ORFs are almost certainly functional. A further four pairs of ORFs show homology limited to only a short part of their putative gene products. Amino acid sequence identities range between 50 and 99%; some chloroplast proteins are seen to be among the most slowly evolving of all known proteins. A search of the nucleotide and amino acid sequence databanks has revealed several previously unidentified genes in chloroplast sequences from other species, but no new homologies to prokaryotic genes.     

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.     

Evolution of alcohol dehydrogenase genes in peonies (Paeonia): phylogenetic relationships of putative nonhybrid species

Alcohol dehydrogenase genes were amplified by PCR, cloned, and sequenced from 11 putative nonhybrid species of the angiosperm genus Paeonia. Sequences of five exons and six intron regions of the Adh gene were used to reconstruct the phylogeny of these species. Two paralogous genes, Adh1A, and Adh2, were found; an additional gene, Adh1B, is also present in section Moutan. Phylogenetic analyses of exon sequences of the Adh genes of Paeonia and a variety of other angiosperms imply that duplication of Adh1 and Adh2 occurred prior to the divergence of Paeonia species and was followed by a duplication resulting in Adh1A and Adh1B. Concerted evolution appears to be absent between these paralogous loci. Phylogenetic analysis of only the Paeonia Adh exon sequences, positioning the root of the tree between the paralogous genes Adh1 and Adh2, suggests that the first evolutionary split within the genus occurred between the shrubby section Moutan and the other two herbaceous sections Oneapia and Paeonia. Restriction of Adh1B genes to section Moutan may have resulted from deletion of Adh1B from the common ancestor of sections Oneapia and Paeonia. A relative-rate test was designed to compare rates of molecular change among lineages based on the divergence of paralogous genes, and the results indicate a slower rate of evolution within the shrubby section Moutan than in section Oneapia. This may be responsible for the relatively long branch length of section Oneapia and the short branch length between section Moutan and the other two sections found on the Adh, ITS (nrDNA), and matK (cpDNA) phylogenies of the genus. Adh1 and Adh2 intron sequences cannot be aligned, and we therefore carried out separate analyses of Adh1A and Adh2 genes using exon and intron sequences together. The Templeton test suggested that there is not significant incongruence among Adh1A, ITS, and matK data sets, but that these three data sets conflict significantly with Adh2 sequence data. A combined analysis of Adh1A, ITS, and matK sequences produced a tree that is better resolved than that of any individual gene, and congruent with morphology and the results of artificial hybridization. It is therefore considered to be the current best estimate of the species phylogeny. Paraphyly of section Paeonia in the Adh2 gene tree may be caused by longer coalescence times and random sorting of ancestral alleles.      

Molecular evolution and positive Darwinian selection of the chloroplast maturase matK

It is not clear whether matK evolves under Darwinian selection. In this study, 70 plant groups, representing 2,279 species at various evolutionary levels, were used to illustrate the molecular adaptation and evolutionary dynamics of gene divergence in matKs. Selective influences were investigated using standard dN/dS ratio methods. Analyses revealed the presence of positive selection in matKs of 32 plant groups. More positively selected sites were detected in the N-terminal region than in the RT domain and domain X of matK. Moreover, removing amino acid sites that are under positive selection has a significant effect on the bootstrap values of phylogenetic reconstruction. Our results suggest that the rapidly evolving matK evolves under positive selection in some lineages of land plants. Several regions of matK have experienced molecular adaptation, which fine-tunes maturase performance.     

Fine structural features of the chloroplast genome: comparison of the sequenced chloroplast genomes.

The entire nucleotide sequences of the rice, tobacco and liverwort chloroplast genomes have been determined. We compared all the chloroplast genes, open reading frames and spacer regions in the plastid genomes of these three species in order to elucidate general structural features of the chloroplast genome. Analyses of homology, GC content and codon usage of the genes enabled us to classify them into two groups: photosynthesis genes and genetic system genes. Based on comparisons of homology, GC content and codon usage, unidentified ORFs can also be assigned to each of these groups such that it is possible to speculate about the functions of products which may be produced by these ORFs. The spacer regions and intron sequences were compared and found to have no obvious homology between rice and liverwort or between tobacco and liverwort.     

Phylogeny of the Malus (apple tree) species,inferred from its morphological traits and molecular DNA analysis

Some debated issues of the genus Malus (apple) taxonomy were examined using a variety of species from the collection of the Maikop Experimental Station, Vavilon Research Institute of Plant Industry (Krasnodar krai). Phylogenetic relationships among these species were studied using traditional analysis of morphological traits, RAPD, and complete sequencing of the 5'-internal transcribed spacer (ITS1), 5.8S rRNA, 3'-internal transcribed spacer (ITS2) (constituting a cluster of the rRNA genes), and the terminal fragment of the matK gene encoding chloroplast maturase. The results showed that the Sorbomalus section was polyphyletic; the American apple M. fusca was closely related to the species contributing to the East Asian center of the genus origin, and the American species M. angustifolia, M. coronaria, and M. ioensis were closely related to the M. trilobata relict species, whose assignment to the genus Malus is debated by some authors. Molecular analysis of the species relationships showed that the Middle Asian apple M. sieversii is the species from which apple domestication started.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L. (Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH–psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH–psbA, and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH–psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH–psbA as a candidate barcode sequence.     

Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing

In plants, calcium-dependent calmodulin-independent protein kinases (CDPKs) are the predominant calcium-regulated protein kinases and their genes are encoded by a multigene family. A CDPK gene was cloned from a liverwort, Marchantia polymorpha, which showed a high level of sequence similarities to other higher plant CDPK genes. The liverwort CDPK gene consisted of 9 exons and 8 introns. The 6th and 7th exons (Exon 6A and Exon 6B) were almost identical except for 4-amino acid substitutions, both of which coded for EF-hands in the calcium-binding domain. RT-PCR analysis revealed that two species of mature mRNA containing either Exon 6A or Exon 6B were generated from a single CDPK gene by mutually exclusive alternative splicing. Both histidine-tagged fusion proteins derived from cDNAs containing either Exon 6A or Exon 6B exhibited calcium-dependent protein kinase activity in vitro. Preferential accumulation of the mature mRNA with Exon 6A detected in male sexual organ implies possible sexual control of the ratio between the two CDPK isozymes through alternative splicing. Functions and evolution of CDPKs are discussed based on the structure and expression of the liverwort CDPK gene.     

DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.     

DNA-Barcoding of Some Medicinal Plant Species in Saudi Arabia Using <i>rbcL</i> and <i>matK</i> Genes

In the Kingdom of Saudi Arabia (KSA), thousands of plants are considered to have therapeutic value. The ambiguous use of identification mainly morphological characteristics of many plants has resulted in the adulteration and displacement of plant products which undermine their therapeutic value and weak documentation of plant resources. The aims of this study were therefore to evaluate genetic variability and explore the phylogeographic architecture for Saudi medicinal plant samples using rbcL and matK genes as barcodes for genomic identification. The matK and rbcL sequences collected for these samples were used as key markers for examining the relationship between Saudi medicinal plant species based on genetic diversity. During our study we were successful in identifying and documenting 4 different species (Foeniculum vulgare, Nitraria retusa, Dodonaea viscosa, and Rumex nervosus) located in Saudi Arabia using DNA barcoding technique. A total number of 8 sequences were obtained with a total sequence length of 6176 bp, where it ranged from 617 bp to 878 bp with an average length of 772 bp. The total number of rbcL sequences length is 2801 bp, where it ranges from 617 bp to 807 bp with an average length of 700.2 bp. Out of the 4 plant samples used, only three samples were identified correctly on the species level with an identity percentage higher than 95% using rbcL gene. Additionally, 4 matK sequences have been retrieved belong to 4 species. The total number of matK sequences length is 3375 bp, where it ranges from 819 bp to 878 bp with an average length of 843.8 bp. Out of the 4 plant samples used, only two samples were identified correctly on the species level with an identity percentage higher than 98% using matK gene. Both rbcL and matK have been able to identify most of our collected plant samples by genus, and some by species. Using only one DNA-barcoding technique was not reliable for plant identification, where matK and rbcL must be used as a dual DNA-barcoding procedure.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, marK, and trnH-psbA),were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%,respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.