叶绿体DNA片段的RFLP分析在黄精族系统学研究中的应用

对广义百合科黄精族6属23种及铃兰族1属1种的叶绿体基因组trnK和rpl16两个基因片段进 行了PCR-RFLP分析,结果表明：trnK基因的PCR产物在各类群间几乎不存在长度变异,均约2600bp,而rpl16基因则在各属之间及黄精属内表现出长度变异,变异范围在1140~1320bp之间;限制性酶切位点的同源性分析显示,黄精属、竹根七属、鹿药属和舞鹤草属构成的狭义黄精族与铃兰族中的铃兰属有较近的亲缘关系,并支持将扭柄花属和万寿竹属从广义百合科黄精族中分出的观点;在狭义黄精族内,黄精属与竹根七属聚成一支,鹿药属与舞鹤草属聚成另一支,为探讨族内属间的系统演化关系提供了分子生物学方面的证据。另外,本研究结果支持将金佛山黄精从鹿药属转隶至黄精属的观点。     

天门冬科黄精族细胞学研究进展

在全面收集和整理黄精族染色体数据的基础上,对国内外有关黄精族各类群间的染色体数目和倍性的变化规律进行了总结,并从染色体的多倍化和非整倍化与系统发育关系和地理分布方面探讨了黄精族内各属的起源和演化关系问题。黄精族包括黄精属、舞鹤草属、异黄精属和竹根七属,共约100余种,其中舞鹤草属(x=18)、异黄精属(x=16)和竹根七属(x=20)的染色体基数稳定,而黄精属染色体基数波动较大,主要为x=8~16,既有多倍化也有非整倍化现象。染色体数据表明黄精族4个属的染色体进化模式各不相同,揭示了黄精族内染色体从高基数向低基数演化的规律;各属内染色体的演化主要是体现在二倍体水平上的核型变异,多倍化在本族中不占主导地位;仅黄精属内伴有非常强烈的非整倍化现象;细胞学证据与分子系统发育的结果比较吻合,为黄精族内属间以及属下的系统发育与进化提供了重要的参考资料。     

Phylogeny and biogeographic diversification of Maianthemum (Ruscaceae: Polygonatae)

Maianthemum (Ruscaceae) comprises 28-38 species and includes the two traditionally recognized genera: Maianthemum sensu stricto and Smilacina. Thirty-seven samples representing 22 species of Maianthemum and six closely related outgroup taxa were sequenced for eight chloroplast and nuclear markers (trnL-F, rps16, rpl16, psbA-trnH, rbcL, ndhF, trnK, and ITS) with a total length of nearly 10,000 base pairs. Phylogenetic analyses supported the monophyly of Maianthemum with Maianthemum sensu stricto nested within Smilacina. Almost all species from the eastern Himalayan region in SW China except for Maianthemum tatsienense and M. stenolobum form a well supported clade. This clade is characterized morphologically by short filaments and large anthers, relatively large flowers, and pubescent stems and leaves. Maianthemum tatsienense and M. stenolobum from SW to central China form another clade. The other species from eastern Asia (central to NE China and Japan) and the New World fall into several clades. The intercontinental disjunction between eastern Asia and North America in Maianthemum sensu stricto is estimated to be at 1.68 million years ago (mya) with the Bayesian relaxed clock relying on uncorrelated rates. A recent radiation at about 2.04mya is suggested in the high mountains of SW China, corresponding to the geographical heterogeneity in that region after the uplift of the Himalayas. Long distance dispersal by birds may have facilitated the evolution of their intercontinental disjunction and their biogeographic diversifications in SW China.     

安徽黄精属(Polygonatum)植物的分支分析

以形态学为依据 ,结合细胞分类学、叶表皮和花粉形态的研究成果 ,用分支分析的方法探讨了安徽产黄精属 11种植物的种间演化关系。在分支分析中 ,选择万寿竹属、舞鹤草属和竹根七属作为复合外类群。根据复合外类群比较原则和一般演化规律 ,确定性状极性。结果表明 :( 1)琅琊黄精与长苞黄精为姊妹群 ,并与其余 9种黄精明显分为 2大支 ;( 2 )另一支 9种黄精中 ,玉竹、长梗黄精和金塞黄精亲缘关系密切并与其余 6种再分为 2支 ;( 3)剩下 6种黄精中 ,距药黄精和多花黄精 2种互生叶黄精极为密切并与 4种轮生叶黄精分为 2组 ;( 4 ) 4种轮生叶黄精中 ,黄精、轮叶黄精和安徽黄精亲缘关系最为密切     

Phylogenetic relationships among genera in theLiliaceae-Asparagoideae-Polygonatae s.l. inferred fromrbcL gene sequence data

The chloroplast gene encoding ribulose-1,5-bisphosphate-carboxylase (rbcL) was sequenced for phylogenetic analysis of 13 species (10 genera) in the tribePolygonatae s.l. of theLiliaceae-Asparagoideae. The data were analysed using maximum parsimony and neighbour-joining methods. There were 233 phylogenetically informative sites out of 1368 base pairs compared. The results suggest that there are three monophyletic groups withinPolygonatae s.l. with high bootstrap confidence values. Group A representsPolygonatae s.str., with generaMaianthemum, Smilacina, Convallaria, Disporopsis, andPolygonatum. Group B containsUvularia andDisporum and group C includesStreptopus, Tricyrtis, Clintonia, andProsartes. The study suggests thatPolygonatae s.l. are not a monophyletic group, including at least three groups of different phylogenetic origin. Monophyly of the taxa within groups A, B, and C is supported by the high bootstrap confidence values (85–100%) of the bootstrap replications for both parsimony and neighbour-joining methods. The differences between each group (calculated as 100x base substitutions per site) were 6.99–9.03 for group A and B, 4.92–7.35 for A and C, and 6.66–7.57 for B and C.     

Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III

We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'.     

油菜叶绿体16SrRNA基因的一级结构分析

本文报道了油菜叶绿体16S rRNA基因的全顺序及其5′端上游的156bp和3′端下游的101bp的核苷酸顺序。油菜叶绿体16s rRNA基因长为1491bp,和烟草、玉米相比,同源程度分别为98.5％、96.1％。油菜叶绿体16S rRNA基因5′端上游及3′端下游的顺序能互补而形成一个较大的茎环结构,但与烟草相比,由于3′端下游顺序有79bp的缺失,因此,该结构中的茎部分大小仅为烟草的二分之一。     

Molecular phylogenetics of Ruscaceae sensu lato and related families (Asparagales) based on plastid and nuclear DNA sequences

Background

Previous phylogenetics studies of Asparagales, although extensive and generally well supported, have left several sets of taxa unclearly placed and have not addressed all relationships within certain clades thoroughly (some clades were relatively sparsely sampled). One of the most important of these is sampling within and placement of Nolinoideae (Ruscaceae s.l.) of Asparagaceae sensu Angiosperm Phylogeny Group (APG) III, which subfamily includes taxa previously referred to Convallariaceae, Dracaenaaceae, Eriospermaceae, Nolinaceae and Ruscaceae.

Methods

A phylogenetic analysis of a combined data set for 126 taxa of Ruscaceae s.l. and related groups in Asparagales based on three nuclear and plastid DNA coding genes, 18S rDNA (1796 bp), rbcL (1338 bp) and matK (1668 bp), representing a total of approx. 4·8 kb is presented. Parsimony and Bayesian inference analyses were conducted to elucidate relationships of Ruscaceae s.l. and related groups, and parsimony bootstrap analysis was performed to assess support of clades.

Key Results

The combination of the three genes results in the most highly resolved and strongly supported topology yet obtained for Asparagales including Ruscaceae s.l. Asparagales relationships are nearly congruent with previous combined gene analyses, which were reflected in the APG III classification. Parsimony and Bayesian analyses yield identical relationships except for some slight variation among the core asparagoid families, which nevertheless form a strongly supported group in both types of analyses. In core asparagoids, five major clades are identified: (1) Alliaceae s.l. (sensu APG III, Amarylidaceae–Agapanthaceae–Alliaceae); (2) Asparagaceae–Laxmanniaceae–Ruscaceae s.l.; (3) Themidaceae; (4) Hyacinthaceae; (5) Anemarrhenaceae–Behniaceae–Herreriaceae–Agavaceae (clades 2–5 collectively Asparagaceae s.l. sensu APG III). The position of Aphyllanthes is labile, but it is sister to Themidaceae in the combined maximum-parsimony tree and sister to Anemarrhenaceae in the Bayesian analysis. The highly supported clade of Xanthorrhoeaceae s.l. (sensu APG III, including Asphodelaceae and Hemerocallidaceae) is sister to the core asparagoids. Ruscaceae s.l. are a well-supported group. Asparagaceae s.s. are sister to Ruscaceae s.l., even though the clade of the two families is weakly supported; Laxmanniaceae are strongly supported as sister to Ruscaceae s.l. and Asparagaceae. Ruscaceae s.l. include six principal clades that often reflect previously named groups: (1) tribe Polygonateae (excluding Disporopsis); (2) tribe Ophiopogoneae; (3) tribe Convallarieae (excluding Theropogon); (4) Ruscaceae s.s. + Dracaenaceae + Theropogon + Disporopsis + Comospermum; (5) Nolinaceae, (6) Eriospermum.

Conclusions

The analyses here were largely conducted with new data collected for the same loci as in previous studies, but in this case from different species/DNA accessions and greater sampling in many cases than in previously published analyses; nonetheless, the results largely mirror those of previously conducted studies. This demonstrates the robustness of these results and answers questions often raised about reproducibility of DNA results, given the often sparse sampling of taxa in some studies, particularly the earliest ones. The results also provide a clear set of patterns on which to base a new classification of the subfamilies of Asparagaceae s.l., particularly Ruscaceae s.l. (= Nolinoideae of Asparagaceae s.l.), and examine other putatively important characters of Asparagales.     

A three-step model for the rearrangement of the chloroplast trnK-psbA region of the gymnosperm Pinus contorta.

A region of the Pinus contorta chloroplast genome which contains a duplication of the psbA gene was characterized. From previous experiments it was known that the two copies of the psbA gene were located approximately 3.3 kilobase pairs (kbp) apart, that they had the same orientation and that one endpoint of the duplication was 19 base pairs (bp) downstream of the psbA stop codon. In order to determine the size and additional genetic content of the duplicated segment, both copies as well as the intervening DNA were sequenced completely. It was found that the duplicated segment was 1969 bp long, that the two copies were completely identical and were separated by 2431 bp. The duplicated segment carried, in addition to psbA, the 3' exon of the trnK gene, which was partially included in a 124 bp direct repeat. The translocated copy of the duplicated segment was found to be inserted upstream of the trnK(UUU) gene and was immediately followed by a repeated 41 bp stretch from the psbA coding region. The trnK gene was split by a 2509 bp intron which contained an open reading frame of 515 codons. Sequence comparisons of the duplicated segment and its flanking DNA to the corresponding regions of P. sylvestris, a species which lacks the rearrangements found in P. contorta, made it possible to identify 3-9 bp homologies within which recombinations had occurred. A model was derived which would accommodate the conversion of a trnK-psbA locus of the ancestral P. sylvestris-like organization into the rearranged structure found in P. contorta.     

Nucleotide sequence variation of the chloroplast trnK/matK region in two wild Fagopyrum (Polygonaceae) species, F. leptopodum and F. statice

Nucleotide sequence polymorphisms of the intron of the chloroplast trnK (UUU) gene, including a matK gene, were investigated within two wild Fagopyrum species, F. leptopodum and F. statice, to assess the degree and pattern of the inter- and intraspecific differences in coding and noncoding chloroplast DNA regions in higher plants. Ten and five accessions were used for F. leptopodum and F. statice, respectively. The length of the trnK intron region in these species ranged from 2494 to 2506 bp. In the trnK intron, the net nucleotide substitution number per site (Da) between the two species was 0.00109, lower than the nucleotide diversity (pi), 0.00195 for F. leptopodum and 0.00144 for F. statice, suggesting a low level of interspecific divergence. This result seems to be due to the phylogenetic pattern that both species are interspersed with each other, which was revealed by the phylogenetic analyses based on the nucleotide substitutions and indels. In the matK gene region (1524 bp), seven and two nucleotide substitutions were found within F. leptopodum and F. statice, respectively. All of the nine nucleotide substitutions (eight of which were nonsynonymous) within and between F. leptopodum and F. statice were clustered in the 5' part of the matK gene region, and no variation was found in the 3' part. This suggests that most of the 3' part is occupied by the conserved domains that are important for the binding activity of the gene product to the precursor mRNA, and therefore implies that the 3' part is more functionally constrained than the 5' part.     

基于trnK基因的葱属植物分子系统研究

在形态和细胞分类研究的基础上选择产于中国的9组47种葱属植物(含外类群5种),运用PCR方法扩增叶绿体trnK基因,选择26种限制性内切酶对PCR扩增片段进行了RFLP分析.结果表明:trnK基因的PCR产物在各分类群间几乎不存在长度变异,约为2 520 bp,PCR扩增片段酶切后,共得到247个变异位点,其中信息位点201个.运用PAU P 4.0 B 10.0和M EGA 3.1软件进行分析,构建葱属系统发育的D o llo和W agner最简约(M P)树及邻接(N J)树.分析表明:(1)宽叶组类群组成比较自然的单系群,洋葱组和葱组也各自形成独立分枝,表明这3个组的划分是比较自然的.多籽组和合被组在本次分析中形成1个单系群,表明这2个组具有较密切的亲缘关系.而粗根组、根茎组和单生组的划分是不自然的,需进一步研究后作适当的调整.粗根组的类群在trnK基因的RFLP分析中,得到很好的分辨,可按其染色体基数分为3个大的类群.(2)中国葱属植物可以划分为6个亚属的新等级,在各亚属下可以再分组.(3)本文还对葱属的种间亲缘、进化关系等问题进行了讨论.     

Comparative plastome data analysis of Dendrosicyos socotranus and Corallocarpus boehmii

Comparison of the Dendrosicyos socotranus and Corallocarpus boehmii (tribe Coniandreae, family Cucurbitaceae) plastome data was of interest. Data on RNA, tRNA, GC%, plastome size, CDS and pseudogene were tabulated for the two species. The total length of 1,57,380 bp and 1,58,744 bp which includes LSC, SSC, IRa, and IRb, while their GC content was 37.1% and 37% respectively. The variation in the length of genes e.g. ndhD, ndhI, rpl22, rpoC2, rps16, rps19, rps8, ycf1and ycf2 noted. Data help to document the genetic differences between usual (climber) with those of tree cucurbits.     

水稻叶绿体16S启动子克隆改造、载体构建及转化研究

利用PCR方法从水稻叶绿体基因组DNA中分离16S启动子,并在其下游加入rbcL基因SD序列,以增强该启动子的翻译能力;序列分析表明,除加入的SD序列外,扩增片段与水稻(Oryza sativa)叶绿体基因组DNA序列16S启动子相应区域同源性为100％。将16S启动子与bar基因和gfp基因的融合基因连接,以psbA基因的3′序列为终止子,并以烟草叶绿体trnH—psbA和trnK为同源片段构建了烟草叶绿体表达载体pRl6S。用基因枪转化烟草,转化植株经Southern、Northern检测及后代遗传学分析,发现：16S启动子具有启动活性,融合基因已在烟草叶绿体中稳定整合并遵循母系遗传规律。     

Cloning and characterization of chloroplast ribosomal protein-encoding genes, rpl16 and rps3, of the marine macro-algae, Gracilaria tenuistipitata

In Gracilaria tenuistipitata, a highly differentiated multicellular member of the marine red algae, Rhodophyta, chloroplast (cp) DNA can be separated as a satellite band from the nuclear DNA in a CsCl gradient. Using a heterologous probe from Chlamydomonas, the ribosomal protein-encoding gene, rpl16, was located on a 4.5-kb EcoRI fragment of cp DNA. The fragment was cloned and a 1365-bp region around rpl16 was sequenced. The gene order around rpl16, 5′ rpl22-rps3-rpl16, is identical to that detected in the chloroplast DNA of liverwort, tobacco and maize. Both the nucleotide sequence and the amino-acid sequence of rpl16 are more conserved than that of rps3. The rpl16 gene contains no intron, a feature which shows more similarity to the unicellular green algae, Chlamydomonas, than the other land plants. Sequences that may form a stable stem-loop structure were detected within the coding sequence of rpl16.     

Using restriction-site variation of PCR-amplified cpDNA genes for phylogenetic analysis of tribe Cheloneae (Scrophulariaceae)

Data from restriction-site variation of three PCR-amplified chloroplast genic regions (trnK, rps2, and rbcL) were used to assess the utility of PCR-based methodology for phylogenetic reconstruction. Seventeen genera from tribe Cheloneae s.l. (Scrophulariaceae), and one genus each from Solanaceae, Acanthaceae, and Bignoniaceae, representing 32 taxa, were sampled. Phylogenetic reconstruction, based on a combined data set of 138 variable restriction sites, revealed a monophyletic clade of North American Cheloneae, which were not inconsistent with a polyphyletic Scrophulariaceae. Separate analyses of individual genie regions were unable to completely resolve the phylogeny, but were adequate for resolving relationships of major clades among the taxa sampled. We suggest that analysis of PCR-product restriction-site variation is useful for phylogenetic reconstruction above the species level.