Patterns of nucleotide substitution in angiosperm cpDNA trnL (UAA)-trnF (GAA) regions

Patterns of substitution in chloroplast encoded trnL_F regions were compared between species of Actaea (Ranunculales), Digitalis (Scrophulariales), Drosera (Caryophyllales), Panicoideae (Poales), the small chromosome species clade of Pelargonium (Geraniales), each representing a different order of flowering plants, and Huperzia (Lycopodiales). In total, the study included 265 taxa, each with > 900-bp sequences, totaling 0.24 Mb. Both pairwise and phylogeny-based comparisons were used to assess nucleotide substitution patterns. In all six groups, we found that transition/transversion ratios, as estimated by maximum likelihood on most-parsimonious trees, ranged between 0.8 and 1.0 for ingroups. These values occurred both at low sequence divergences, where substitutional saturation, i.e., multiple substitutions having occurred at the same (homologous) nucleotide position, was not expected, and at higher levels of divergence. This suggests that the angiosperm trnL-F regions evolve in a pattern different from that generally observed for nuclear and animal mtDNA (transitional/transversion ratio > or = 2). Transition/transversion ratios in the intron and the spacer region differed in all alignments compared, yet base compositions between the regions were highly similar in all six groups. A>-C transversions were significantly less frequent than the other four substitution types. This correlates with results from studies on fidelity mechanisms in DNA replication that predict A<->T and G<->C transversions to be least likely to occur. It therefore strengthens confidence in the link between mutation bias at the polymerase level and the actual fixation of substitutions as recorded on evolutionary trees, and concomitantly, in the neutrality of nucleotide substitutions as phylogenetic markers.

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences

Background and Aims The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences.Methods Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae.Key Results The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya.Conclusions The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.

A molecular marker that is specific to medicinal rhubarb based on chloroplast trnL/trnF sequences

"Da-Huang" (Radix et Rhizoma Rhei, medicinal rhubarb), a famous and important Traditional Chinese Medicine, has often been confused with the adulterant species in the same genus, Rheum. Through sequencing the trnL (UAA)/trnF (GAA) regions of chloroplast DNA of thirteen species of Rheum (three medicinal rhubarb species and ten adulterant ones), a molecular marker of the medicinal species was found. A pair of PCR primers based on the sequences, was thus designed, which amplified a highly specific DNA fragment in medicinal rhubarb exclusively, and absent in the adulterants at all under an optimized PCR condition.

Mitochondrial and chloroplast DNA-based phylogeny of Pelargonium (Geraniaceae)

Overall phylogenetic relationships within the genus Pelargonium (Geraniaceae) were inferred based on DNA sequences from mitochondrial(mt)-encoded nad1 b/c exons and from chloroplast(cp)-encoded trnL (UAA) 5' exon-trnF (GAA) exon regions using two species of Geranium and Sarcocaulon vanderetiae as outgroups. The group II intron between nad1 exons b and c was found to be absent from the Pelargonium, Geranium, and Sarcocaulon sequences presented here as well as from Erodium, which is the first recorded loss of this intron in angiosperms. Separate phylogenetic analyses of the mtDNA and cpDNA data sets produced largely congruent topologies, indicating linkage between mitochondrial and chloroplast genome inheritance. Simultaneous analysis of the combined data sets yielded a well-resolved topology with high clade support exhibiting a basic split into small and large chromosome species, the first group containing two lineages and the latter three. One large chromosome lineage (x = 11) comprises species from sections Myrrhidium and Chorisma and is sister to a lineage comprising P. mutans (x = 11) and species from section Jenkinsonia (x = 9). Sister to these two lineages is a lineage comprising species from sections Ciconium (x = 9) and Subsucculentia (x = 10). Cladistic evaluation of this pattern suggests that x = 11 is the ancestral basic chromosome number for the genus.

横断山区马先蒿属的形态分化与分子进化

半寄生植物马先蒿属（Pedicularis）是列当科（Orobanchaceae）中最大的属，也是北温带被子植物最大的属之一。该属至少有500种植物，主要分布在北半球的高山、亚高山地区或高纬度地区，其中超过一半的种类分布在东喜马拉雅至横断山区，构成该地区高山植物区系的主要成分。马先蒿属花部器官的强烈分化程度在被子植物中极为罕见，导致这种分化发生的机制仍是难解之谜。马先蒿属下系统非常混乱，迄今为止该属属下分类系统不下10个。关于该属的起源时间、地点及迁移散布过程只是基于一些间接证据的推测。针对以上问题，本文通过大量的标本查阅、野外考察、传粉生物学观察以及分子系统学研究，得出了一些初步的结果。 　　1．形态学 通过大量的野外考察及标本观察，发现马先蒿属花部器官变异非常复杂，是区分近缘种的主要性状依据，但是花部器官存在明显的平行进化现象，不适合作为划分群、组等属下高级分类单元的主要依据;而营养性状比较保守，可作为划分群、组的主要依据。通过考证，发现直管群万叶系的德钦马先蒿（P. deqinensis）实属轮枝群纤细系多枝马先蒿（P. ramosissima）的异名。同时发现一个新种，即折喙马先蒿（P. inflexirostris），该种属于直管群的万叶系。 　　2.传粉生物学 对27种马先蒿的昆虫传粉行为进行了初步的观察。发现横断山区的马先蒿主要靠熊蜂进行有效的传粉。昆虫的传粉方式有两种，即背触式（Nototribic）和腹触式（Sternotribic）。不同花冠类型的马先蒿属植物中，昆虫的传粉方式也有所区别。对短管、无喙、无花蜜的马先蒿，昆虫主要以腹触式完成传粉;对短管、无喙、具花蜜的马先蒿，昆虫既可以通过背触式也可以通过腹触式完成传粉;而对短管、具喙和长管、具喙的马先蒿，昆虫都以腹触式完成传粉。没有发现鳞翅目的昆虫访问长管类型的马先蒿。不同花冠类型传粉方式的不同说明马先蒿花部形态结构和传粉媒介的行为之间存在协同进化关系。 3.核rDNA ITS分析 对12个群的42种马先蒿的核rDNA ITS序列进行了分析。基于ITS序列构建的基因树和经典的属下分类系统很不一致，基因树上的大部分分支和经典系统中的高级分类单元不相吻合，原因可能是马先蒿属花部器官发生了平行进化，而经典的分类系统过于权重这些花部形态性状。此外，发现在横断山区这一相对狭小的地域范围内，nrDNA ITS序列在马先蒿种间存在很大差异。造成此差异的原因可能有两个方面：一方面是马先蒿属的起源和分化的时间可能较早，不同的支系从其他地域先后多次迁入横断山区;另一方面可能是由于半寄生植物马先蒿中快速的分子进化造成的。 4.叶绿体基因组trnT-F区序列分析 对8个群的11种马先蒿的trnT-F区序列进行了分析，发现种间存在大量的插入/缺失序列，其中甘肃马先蒿（P. kansuensis）和大王马先蒿（P. rex）分别在trnT-trnL（UAA）和trnL–trnF基因间区发生了长达228bp和303bp碱基序列缺失，说明半寄生植物的叶绿体基因组也可能存在大量基因丢失现象。 5. GLOBOSA-like MADS-box基因的研究 对11种马先蒿属植物（8个群）中控制花瓣发育的GLOBOSA（PGLO）基因的部分片段进行了分离、克隆和测序，发现该基因在种间发生了明显的分化，但是碱基的变异主要发生在非编码区或非结构域，基因的同义突变率远高于非同义突变率，说明PGLO基因的进化受到强烈的功能制约。PGLO基因在马先蒿种间的明显分化表明：在辐射分化类群中，调节基因也可能发生了快速分化。对11种马先蒿属植物的PGLO基因树、nrDNA ITS基因树以及trnT-F基因树的比较发现：三个树图在结构上既有一致、也有相互矛盾之处，推测可能是因为这些基因具有不同的遗传体系或经历了不同的进化历史所致，另一方面说明GLOBOSA基因在探讨近缘类群系统发育关系方面的价值有待进一步验证。

Phylogenetic relationships withinPelargonium sect. Peristera (Geraniaceae) inferred from nrDNA and cpDNA sequence comparisons

Phylogenetic analysis of nrDNA ITS and trnL (UAA) 5′ exon-trnF (GAA) chloroplast DNA sequences from 17 species ofPelargonium sect.Peristera, together with nine putative outgroups, suggests paraphyly for the section and a close relationship between the highly disjunct South African and Australian species of sect.Peristera. Representatives fromPelargonium sectt.Reniformia, Ligularia s. l. andIsopetalum (the St. Helena endemicP. cotyledonis) appear to be nested within thePeristera clade. The close relationship between the South African and AustralianPeristera is interpreted as being caused by long-range dispersal to Australia, probably as recent as the late Pliocene.

Association study of a functional variant in intron 8 of the dopamine transporter gene and migraine susceptibility

Migraine is a common, genetically influenced neurovascular disorder. The dopamine transporter gene is a candidate for migraine association studies. This study tested a functionally linked variable number tandem repeat (VNTR) in intron 8 of the dopamine transporter gene (DATInt8) in 550 migraine cases (401 with aura, 149 without aura) and 550 non-migraine controls. Chi-squared analysis of the DATInt8 revealed that the allele and genotype frequency distributions for migraine cases (including subtype analysis) and controls were not different (P > 0.1). These findings offer no evidence for an association of the DATInt8 with migraine with and without aura and therefore do not implicate the dopamine transporter gene as a modifier of migraine risk.

No role for estrogen receptor 1 gene intron 1 Pvu II and exon 4 C325G polymorphisms in migraine susceptibility

Background We have previously reported an association between the estrogen receptor 1 (ESR1) gene exon 8 G594A polymorphism and migraine susceptibility in two independent Australian cohorts. In this paper we report results of analysis of two further single nucleotide polymorphisms (SNPs) in the ESR1 gene in the same study group, the T/C Pvu II SNP in intron 1 and the C325G SNP in exon 4, as well as results of linkage disequilibrium (LD) analysis on these markers. Methods We investigated these variants by case-control association analysis in a cohort of 240 migraineurs and 240 matched controls. The SNPs were genotyped using specific restriction enzyme assays. Results were analysed using contingency table methods incorporating the chi-squared statistic. LD results are presented as D' statistics with associated P values. Results We found no evidence for association of the Pvu II T/C polymorphism and the C325G polymorphism and migraine susceptibility and no evidence for LD between these two SNPs and the previously implicated exon 8 G594A marker. Conclusion We have found no role for the polymorphisms in intron 1 and exon 4 with migraine susceptibility. To further investigate our previously implicated exon 8 marker, we suggest the need for studies with a high density of polymorphisms be undertaken, with particular focus on markers in LD with the exon 8 marker.

Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens

In binary vectors, the antibiotic resistance gene used for selection of transformed plant cells is also usually expressed in the transforming Agrobacterium cells. This expression gives the bacterium antibiotic resistance, an unnecessary advantage on selective medium containing the antibiotic. Insertion of a castor bean catalase-1 (CAT-1) gene intron or a Parasponia andersonii haemoglobin gene intron into the coding region of the selectable marker gene, hph, completely abolished the expression of the gene in Agrobacterium, rendering it susceptible to hygromycin B. Use of these modified binary vectors minimized the overgrowth of Agrobacterium during plant transformation. Both of the introns were correctly spliced in plant cells and significantly enhanced hph gene expression in transformed rice tissue. The presence of these introns in the hph coding sequence not only maintained the selection efficiency of the hph gene, but with the CAT-1 intron also substantially increased the frequency of rice transformation. Transgenic lines with an intron-hph gene generally contained fewer gene copies and produced substantially more mRNA of the predicted size. Our results also indicate that transgenic plants with many copies of the transgene were more likely to show gene silencing than plants with 1-3 copies.

Effect of an estrogen receptor-α intron 4 polymorphism on fat mass in 11-year-old children

Context: in the ESR1 gene encoding estrogen receptor (ER)-α may be associated with fat mass in adults. Objectives: The objective of the study was to establish whether ESR1 polymorphisms influence fat mass in childhood. Design: This was a cross-sectional analysis after genotyping of rs9340799, rs2234693, and rs7757956 ESR1 polymorphisms. Setting: The Avon Longitudinal Study of Parents and Children (ALSPAC) was a population-based prospective study. Participants: Participants included 3097 11-yr-old children with results for ESR1 genotyping, puberty measures, and dual-energy x-ray absorptiometry results. Outcomes: Relationships between ESR1 polymorphisms and indices of body composition were measured. Results: The rs7757956 polymorphism was associated with fat mass (P = 0.002). Total body fat mass (adjusted for height) was reduced by 6% in children with TA/AA genotypes, and risk of being overweight (≥85th centile of fat mass) was decreased by 20%. This genetic effect appeared to interact with puberty in girls (P = 0.05 for interaction): in those with the TT genotype, total body fat mass (adjusted for height) was 18% higher in Tanner stages 3-5 vs. stages 1-2; the equivalent difference was 7% in those with TA/AA genotypes. Furthermore, the risk of being overweight was 36% lower in girls with TA/AA genotypes in Tanner stages 3-5, but no reduction was seen in those in stages 1-2. Neither rs9340799 nor rs2234693 polymorphisms were associated with body composition measures. Conclusions: Fat mass in 11-yr-old children was related to the rs7757956 ESR1 polymorphism. This association was strongest in girls in more advanced puberty, in whom the risk of being overweight was reduced by 36% in those with the TA/AA genotype.

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light.

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA-treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had no detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These data indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA- treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had not detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These date indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.

Phylogenetic analyses reveal high levels of polyphyly among pleurocarpous lineages as well as novel clades

Phylogenetic analyses of the Hypnales usually show the same picture of poorly resolved trees with a large number of polyphyletic taxa and low support for the few reconstructed clades. One odd clade, however, consisting of three genera that are currently treated either within the Leskeaceae (Miyabea) or Neckeraceae (Homaliadelphus and Bissetia), was retrieved in a previously published phylogeny based on chloroplast rbcL. In order to elucidate the reliability of the observed Homaliadelphus - Miyabea - Bissetia - clade (HMB-clade) and to reveal its phylogenetic relationships a molecular study based on a representative set of hypnalean taxa was performed. Sequence data from all three genomes, namely the ITS1 and 2 (nuclear), the trnS-rps4-trnT-trnL-trnF cluster (plastid), the nad5 intron (mitochondrial), were analyzed. Although the phylogenetic reconstruction of the combined data set was not fully resolved regarding the backbone it clearly indicated the polyphyletic nature of various hypnalean families, such as the Leskeaceae, Hypnaceae, Hylocomiaceae, Neckeraceae, Leptodontaceae and Anomodontaceae with respect to the included taxa. In addition the results favor the inclusion of the Leptodontaceae and Thamnobryaceae in the Neckeraceae. The maximally supported HMB-clade consisting of the three genera Homaliadelphus (2-3 species), Miyabea (3 species) and Bissetia (1 species) is resolved sister to a so far unnamed clade comprising Taxiphyllum aomoriense, Glossadelphus ogatae and Leptopterigynandrum. The well-resolved and supported HMB-clade, here formally described as the Miyabeaceae, fam. nov. is additionally supported by morphological characters such as strongly incrassate, porose leaf cells, a relatively weak and diffuse costa and the presence of dwarf males. The latter are absent in the Neckeraceae and the Leskeaceae. It is essentially an East Asian family, with one species occurring in North America.

Evolution of the Neckeraceae (Bryophyta): resolving the backbone phylogeny

Earlier phylogenetic studies, including species belonging to the Neckeraceae, have indicated that this pleurocarpous moss family shares a strongly supported sister group relationship with the Lembophyllaceae, but the family delimitation of the former needs adjustment. To test the monophyly of the Neckeraceae, as well as to redefine the family circumscription and to pinpoint its phylogenetic position in a larger context, a phylogenetic study based on molecular data was carried out. Sequence data were compiled, combining data from all three genomes: nuclear ITS1 and 2, plastid trnS-rps4-trnT-trnL-trnF and rpl16, and mitochondrial nad5 intron. The Neckeraceae have sometimes been divided into the two families, Neckeraceae and Thamnobryaceae, a division rejected here. Both parsimony and Bayesian analyses of molecular data revealed that the family concept of the Neckeraceae needs several further adjustments, such as the exclusion of some individual species and smaller genera as well as the inclusion of the Leptodontaceae. Within the family three well-supported clades (A, B and C) can be distinguished. Members of clade A are mainly non-Asiatic and nontropical. Most species have a weak costa and immersed capsules with reduced peristomes (mainly Neckera spp.) and the teeth at the leaf margins are usually unicellular. Clade B members are also mainly non-Asiatic. They are typically fairly robust, distinctly stipilate, having a single, at least relatively strong costa, long setae (capsules exserted), and the peristomes are well developed or only somewhat reduced. Members of clade C are essentially Asiatic and tropical. The species of this clade usually have a strong costa and a long seta, the seta often being mammillose in its upper part. The peristome types in this clade are mixed, since both reduced and unreduced types are found. Several neckeraceous genera that were recognised on a morphological basis are polyphyletic (e.g. Neckera, Homalia, Thamnobryum, Porotrichum). Ancestral state reconstructions revealed that currently used diagnostic traits, such as the leaf asymmetry and costa strength are highly homoplastic. Similarly, the reconstructions revealed that the 'reduced' sporophyte features have evolved independently in each of the three clades.

An Improved molecular tool for distinguishing monoecious and dioedious Hydrilla

Two biotypes of hydrilla [Hydrilla verticillata(L.f.) Royle] occur in the United States, a dioecious type centered in the southeast and a monoecious type in the central Atlantic and northeastern states. Ecosystem managers need tools to distinguish the types as the ranges of each type expand and begin to overlap. A molecular tool using the randomly amplified polymorphic DNA (RAPD) procedure is available but its use is limited by a need for reference samples. We describe an alternative molecular tool which uses “universal primers” to sequence the trnL intron and trnL-F intergenic spacer of the chloroplast genome. This sequence yields three differences between the biotypes (two gaps and one single nucleotide polymorphism). A primer has been designed which ends in a gap that shows up only in the dioecious plant. A polymerase chain reaction (PCR) using this primer produces a product for the monoecious but not the dioecious plant.