利用低拷贝核基因重建菊科紫菀亚科族间系统发育关系

紫菀亚科(Asteroideae)是菊科最大的一个亚科, 包含的种数多于被子植物的绝大多数科。目前, 紫菀亚科族间的系统发育关系主要依赖于叶绿体基因信息, 但是叶绿体基因为单亲遗传, 并不能完整反映进化历史。鉴于杂交现象在菊科普遍存在, 故利用核基因可以反映更完整的紫菀亚科进化历史。该研究首次使用从转录组数据(20个新测+11个从NCBI数据库下载)中筛选出的47个直系同源低拷贝核基因来研究紫菀亚科的系统发育关系, 共选取了29个物种, 代表了紫菀亚科20个族中的13个族。用超矩阵分析方法和溯祖推测分析方法各获得了1个稳定的紫菀亚科系统树, 每个树上绝大多数分支都得到了高度支持, 且2个树之间没有明显的冲突。新的紫菀亚科族间系统发育关系揭示了千里光超族应并入紫菀超族, 春黄菊族可能是千里光族与紫菀族杂交起源的, 金鸡菊族很可能也是杂交起源的。该研究结果显示低拷贝核基因可以更好地解决科以下分类阶元的系统发育关系, 对菊科乃至被子植物其它科的系统发育研究具有重要的借鉴意义。     

Selecting Single-Copy Nuclear Genes for Plant Phylogenetics: A Preliminary Analysis for the Senecioneae (Asteraceae)

Compared to organelle genomes, the nuclear genome comprises a vast reservoir of genes that potentially harbor phylogenetic signal. Despite the valuable data that sequencing projects of model systems offer, relatively few single-copy nuclear genes are being used in systematics. In part this is due to the challenges inherent in generating orthologous sequences, a problem that is ameliorated when the gene family in question has been characterized in related organisms. Here we illustrate the utility of diverse sequence databases within the Asteraceae as a framework for developing single-copy nuclear genes useful for inferring phylogenies in the tribe Senecioneae. We highlight the process of searching for informative genes by using data from Helianthus annuus, Lactuca sativa, Stevia rebaudiana, Zinnia elegans, and Gerbera cultivar. Emerging from this process were several candidate genes; two of these were used for a phylogenetic assessment of the Senecioneae and were compared to other genes previously used in Asteraceae phylogenies. Based on the preliminary sampling used, one of the genes selected during the searching process was more useful than the two previously used in Asteraceae. The search strategy described is valid for any group of plants but its efficiency is dependent on the phylogenetic proximity of the study group to the species represented in sequence databases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

低拷贝核基因在异源多倍体植物中的进化与表达

异源多倍体植物在自然界中广泛分布。在这类植物谱系中, 低拷贝核基因具有特殊的进化特点和丰富的植物系统发生信息, 在转录水平存在基因沉默、基因激活和不均等表达等特点。以低拷贝核基因为主线, 概述了其在多倍体植物系统发生中的应用和相关注意事项, 并对其在多倍体植物中的表达变化及其分子机制进行了探讨, 系统地介绍了国际上相关领域的研究成果和最新动向。     

Allotetraploid origin and divergence in Eleusine (Chloridoideae, Poaceae): evidence from low-copy nuclear gene phylogenies and a plastid gene chronogram

Background and Aims Eleusine

(Poaceae) is a small genus of the subfamily Chloridoideae exhibiting considerable morphological and ecological diversity in East Africa and the Americas. The interspecific phylogenetic relationships of Eleusine are investigated in order to identify its allotetraploid origin, and a chronogram is estimated to infer temporal relationships between palaeoenvironment changes and divergence of Eleusine in East Africa.

Methods

Two low-copy nuclear (LCN) markers, Pepc4 and EF-1α, were analysed using parsimony, likelihood and Bayesian approaches. A chronogram of Eleusine was inferred from a combined data set of six plastid DNA markers (ndhA intron, ndhF, rps16-trnK, rps16 intron, rps3, and rpl32-trnL) using the Bayesian dating method.

Key Results

The monophyly of Eleusine is strongly supported by sequence data from two LCN markers. In the cpDNA phylogeny, three tetraploid species (E. africana, E. coracana and E. kigeziensis) share a common ancestor with the E. indica–E. tristachya clade, which is considered a source of maternal parents for allotetraploids. Two homoeologous loci are isolated from three tetraploid species in the Pepc4 phylogeny, and the maternal parents receive further support. The A-type EF-1α sequences possess three characters, i.e. a large number of variations of intron 2; clade E-A distantly diverged from clade E-B and other diploid species; and seven deletions in intron 2, implying a possible derivation through a gene duplication event. The crown age of Eleusine and the allotetraploid lineage are 3·89 million years ago (mya) and 1·40 mya, respectively.

Conclusions

The molecular data support independent allotetraploid origins for E. kigeziensis and the E. africana–E. coracana clade. Both events may have involved diploids E. indica and E. tristachya as the maternal parents, but the paternal parents remain unidentified. The habitat-specific hypothesis is proposed to explain the divergence of Eleusine and its allotetraploid lineage.     

Development of microsatellite markers for the endangered medicinal plant Launaea arborescens (Asteraceae)

• Premise of the study: Microsatellite loci were developed in Launaea arborescens, an endangered and medicinal Asteraceae species in North Africa, for further investigation of its conservation genetics. • Methods and Results: We isolated and characterized 10 polymorphic and nine monomorphic microsatellite loci from L. arborescens using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). For the 10 polymorphic loci, the number of alleles detected per locus varied from two to six, and the observed and expected heterozygosities ranged from 0.000 to 0.833 and 0.059 to 0.713, respectively. • Conclusions: The polymorphic markers provide a useful tool for conservation genetics studies of L. arborescens, including analysis of mating system, estimating gene flow, and identifying discrete genetic units within the species.     

Development and characterization of nine new microsatellite markers in Taraxacum (Asteraceae)

This study aims at developing and characterizing new microsatellite primer pairs in Taraxacum officinale auct. to produce polymorphic markers for genetical and evolutionary studies on apomixis in this sexual‐apomictic complex. A total of 24 diploid plants were tested for allelic polymorphism and heterozygosity. Out of nine loci three deviated significantly from the Hardy–Weinberg equilibrium expectations, probably due to the presence of null‐alleles. Successful cross‐species amplification was obtained for all markers in 29 Taraxacum microspecies from five sections.     

Phylogeny of the New World diploid cottons (Gossypium L., Malvaceae) based on sequences of three low-copy nuclear genes

American diploid cottons (Gossypium L., subgenus Houzingenia Fryxell) form a monophyletic group of 13 species distributed mainly in western Mexico, extending into Arizona, Baja California, and with one disjunct species each in the Galapagos Islands and Peru. Prior phylogenetic analyses based on an alcohol dehydrogenase gene (AdhA) and nuclear ribosomal DNA indicated the need for additional data from other molecular markers to resolve phylogenetic relationships within this subgenus. Toward this end, we sequenced three nuclear genes, the anonymous locus A1341, an alcohol dehydrogenase gene (AdhC), and a cellulose synthase gene (CesA1b). Independent and combined analyses resolved clades that are congruent with current taxonomy and previous phylogenies. Our analyses diagnose at least two long distance dispersal events from the Mexican mainland to Baja California, following a rapid radiation of the primary lineages early in the diversification of the subgenus. Molecular data support the proposed recognition of a new species closely related to Gossypium laxum that was recently collected in Mexico.     

Development of eight mitochondrial markers for Funariaceae (Musci) and their amplification success in other mosses

? Premise of the study: In comparison to the wide use of chloroplast markers, few mitochondrial markers are available for phylogenetic studies in bryophytes. We investigated the phylogenetic suitability of several mtDNA markers within the Funariaceae and across mosses. ? Methods and Results: By comparing mitochondrial genomes of two mosses, eight regions with higher substitution rates were identified and sequenced for three species in the Funariaceae and one outgroup taxon. Variations in the substitution rate of these new loci were compared to previously used markers. Thirty-four samples representing all major moss lineages were targeted to assess the universality of the newly designed primers. ? Conclusions: The new markers provided similar or more sequence variations in Funariaceae compared to previously developed mtDNA markers. Five out of eight loci were amplified in 70% of other taxa, indicating that these markers may be suitable for phylogenetic studies in other moss lineages.     

Development and characterization of microsatellite markers for the invasive weed Mikania micrantha (Asteraceae)

Mikania micrantha is a successful invasive weed in many parts of the world. Twelve polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of this species. Twenty-eight individuals from one population in Dongguan were tested for polymorphism. The average allele number of these microsatellites was three per locus, ranging from two to five. The expected and observed heterozygosities ranged from 0.275 to 0.742, and from 0.250 to 1.000, respectively. These microsatellite markers can be applied to study the population genetics in the native and invasive ranges of this species, and to trace its invasion history.     

Reticulate evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes

Background and Aims

Here evidence for reticulation in the pantropical orchid genus Polystachya is presented, using gene trees from five nuclear and plastid DNA data sets, first among only diploid samples (homoploid hybridization) and then with the inclusion of cloned tetraploid sequences (allopolyploids). Two groups of tetraploids are compared with respect to their origins and phylogenetic relationships.

Methods

Sequences from plastid regions, three low-copy nuclear genes and ITS nuclear ribosomal DNA were analysed for 56 diploid and 17 tetraploid accessions using maximum parsimony and Bayesian inference. Reticulation was inferred from incongruence between gene trees using supernetwork and consensus network analyses and from cloning and sequencing duplicated loci in tetraploids.

Key Results

Diploid trees from individual loci showed considerable incongruity but little reticulation signal when support from more than one gene tree was required to infer reticulation. This was coupled with generally low support in the individual gene trees. Sequencing the duplicated gene copies in tetraploids showed clearer evidence of hybrid evolution, including multiple origins of one group of tetraploids included in the study.

Conclusions

A combination of cloning duplicate gene copies in allotetraploids and consensus network comparison of gene trees allowed a phylogenetic framework for reticulation in Polystachya to be built. There was little evidence for homoploid hybridization, but our knowledge of the origins and relationships of three groups of allotetraploids are greatly improved by this study. One group showed evidence of multiple long-distance dispersals to achieve a pantropical distribution; another showed no evidence of multiple origins or long-distance dispersal but had greater morphological variation, consistent with hybridization between more distantly related parents.     

New anonymous nuclear DNA markers for the pearl oyster Pinctada margaritifera and other Pinctada species

The black‐lipped oyster Pinctada margaritifera is highly exploited in French Polynesia where the pearl industry relies mostly on spat collection, and therefore on resources available in the wild. Little is known of these resources, and population genetic studies would be useful to improve management. We used two methods, direct amplification of length polymorphism (DALP) and exon primed intron crossing (EPIC) to develop five new nuclear markers presenting length polymorphism. Although these markers remained anonymous after using blast on GenBank, they are codominant and follow Hardy–Weinberg equilibrium. Tests on related species or subspecies of Pinctada gave encouraging results.     

微生物遗传转化筛选标记及其生物安全性的研究进展

在分子生物技术中,筛选标记基因是遗传转化载体所必备的基本元件之一,其主要功能是在基因操作中进行目标克隆的筛选,以及在应用过程中通过选择压力维持基因重组性状。抗药基因是微生物遗传转化中常用的筛选标记,大肠杆菌载体和一般穿梭载体中通常带有抗药基因。带有抗药基因的工程菌可以被广泛地应用于酶和有机化学品的发酵生产,因为工业发酵过程是在封闭系统中进行的,并且最终产品需要经过提炼。但是当人们需要用基因改良的菌株进行食品和饲料加工、环境修复、病虫害生物防治时,抗药基因类筛选标记应该被禁止使用。因此,发展生物安全性筛选标记成为遗传转化技术推广应用中的一个技术关键。本文介绍常用作筛选标记的抗药基因,以及针对抗药基因的安全性问题而发展的无选择标记的遗传转化技术及生物安全性筛选标记的基因工程技术。葡萄糖胺合成酶基因是近年发展起来的新型生物安全性筛选标记,它弥补了其他营养缺陷互补型和功能附加型筛选标记的缺陷,具有广阔的应用前景。     

Phylogenetic relationships among diploid species of Symphyotrichum (Asteraceae: Astereae) based on two nuclear markers, ITS and GAPDH

The mostly North American subtribe Symphyotrichinae (Asteraceae: Astereae) comprises Canadanthus, Ampelaster, Psilactis, Almutaster, and Symphyotrichum. Intergeneric and interspecific relationships within the subtribe have been investigated in the past, particularly by Nesom [Nesom, G.L., 1994. Review of the taxonomy of Aster sensu lato (Asteraceae: Astereae), emphasizing the new world species, Phytologia 77, 141–297] and Semple [Semple, J.C., 2005. Classification of Symphyotrichum. Available from: <http://www.jcsemple.uwaterloo.ca/Symphyotrichumclassification.htm/>], using morphological and cytological approaches. Symphyotrichum is the largest and most complex genus within the subtribe and includes four subgenera: Symphyotrichum (x = 7, 8), Virgulus (x = 4, 5), Astropolium (x = 5), and Chapmaniani (x = 7). In this study we used two nuclear markers, the nrDNA internal transcribed spacer (ITS) and the low-copy nuclear gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), to resolve intergeneric and interspecific relationships within the subtribe at the diploid level, and to determine whether our phylogenies validate the classifications of Nesom or Semple. Our results confirm the distinct generic status of Canadanthus and Ampelaster, whereas Psilactis and Almutaster form a polytomy with Symphyotrichum. Within Symphyotrichum, subg. Virgulus is monophyletic based on ITS and appears polyphyletic based on GAPDH. Neither the ITS nor the GAPDH analyses support a distinct status for subg. Astropolium, which groups within subg. Symphyotrichum. In general, interspecific relationships within Symphyotrichum are unresolved. Lack of resolution may be interpreted as a case of recent and rapid evolutionary radiation.     

RAPD marker diversity within and divergence among species of Dendroseris (Asteraceae: Lactuceae)

Random Amplified Polymorphic DNA (RAPD) markers were used to measure genetic diversity within and divergence among species of Dendroseris (Asteraceae: Lactuceae), a genus endemic to the Juan Fernandez Islands, Chile. Results were compared to previous studies employing allozymes. For five of the species, RAPD banding patterns distinguished all individuals examined, and different mutilocus genotypes were found even in species exhibiting no allozyme diversity. RAPD band diversities ranged from 0.003 to 0.022 within species; >90% of total diversity was among species and <10% within them. Relative levels of allozyme and RAPD diversity were similar for some species, particularly those with highest and lowest diversities, but overall there was no significant correlation. Relationships inferred from a neighbor-joining tree generated from RAPD bands were similar to results obtained from morphology, chloroplast DNA (cpDNA) restriction site mutations, and sequences from the internal transcribed spacer regions of nuclear ribosomal DNA (ITS), but somewhat better resolution was achieved. Relationships shown by allozymes differed from trees based on other data; this ostensibly is a result of the sharing of ancestral alleles and the absence of alleles generated subsequent to speciation. Dendroseris represents an example where RAPD markers, because of their greater variability, provide a useful alternative to allozymes for assessing diversity in rare species endemic to oceanic islands and for resolving relationships among the species.     

Taxonomic problems in Carthamus (Asteraceae): RAPD markers and sectional classification

Recent molecular studies have clarified the problem of the generic limits of Carthamus . However, taxonomic problems remain in the sectional classification: section Carthamus was confirmed as a natural group, but molecular phylogenies based on DNA sequences did not resolve the other sections, resulting in a confusing conglomerate. A survey based on RAPD markers has been carried out in this conflicting group of sections, with the aim of evaluating the usefulness of these markers at the section and species levels. On the basis of the results, correlated with morphological and karyological characters, some recommendations are made in relation to the taxonomy of Carthamus . The number of sections is reduced from five to two: section Thamnacanthus is currently classified as a different genus, Phonus ; section Carthamus s.s. is retained in its present delimitation, and section Atractylis is redefined, including current sections Atractylis , Lepidopappus and Odontagnathius . The commonly accepted treatment of some taxa as subspecies is not well founded, and C. alexandrinus , C. creticus , C. tenuis and C. turkestanicus should be considered as independent species. Our results confirm that methodological problems posed by RAPD markers can be avoided by careful laboratory procedures and appropriate data analyses; they also suggest that this kind of marker is useful at low taxonomic levels and is, furthermore, complementary to DNA sequence analysis.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 147 , 375–383.     

Sesquiterpene lactones and their precursors as chemosystematic markers in the tribe Cichorieae of the Asteraceae

This review summarizes all reports on sesquiterpene lactones and their immediate precursors from the Cichorieae (Lactuceae) tribe of the Asteraceae. A total of 360 compounds have been reported from this tribe. The reported substances belong to three classes of sesquiterpenoids: guaianolides (243 compounds), eudesmanolides (73 compounds), and germacranolides (44 compounds). Sources of these compounds encompass 139 taxa from 31 different genera. The distribution of these lactones within the tribe Cichorieae is discussed in a chemosystematic context. Moreover, some general ideas about the interpretation of chemosystematic data are discussed.     

Development and characterisation of microsatellite loci in Farfugium japonicum (Asteraceae)

Eight microsatellite loci for the perennial herb Farfugium japonicum, including the rheophytic variety luchuense endemic to riparian areas of the Ryukyu Islands, Japan, were isolated and characterised. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.344–0.885 and 0.121–0.754, respectively, from 69 individuals in one population. Six loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). The primers amplifying microsatellite sequences in F. japonicum may provide a population genetics tool useful in the establishment of a conservation strategy.     

植物核DNA含量在全球尺度上的纬度变异式样及其气候适应意义——以菊科植物为例

核DNA含量是重要的生物学概念,涉及DNA C-值和基因组大小。前人有关植物核DNA含量在纬度梯度上的变异规律存在着矛盾的报道,而且多数将核DNA含量与纬度、海拔、气候等因素之间的关系描述成线性关系。核DNA含量是否具有环境适应上的意义,也还存在争议。先前有关核DNA含量与环境因素间关系的矛盾性报道可能与取样过小、地理范围过窄、研究对象遗传背景差异过大有关。如果对一个遗传背景相近的类群在全球范围内进行取样,核DNA含量会呈现有规律的纬度梯度变化,可能与大的气候因素之间存在非线性关系。菊科(Asteraceae)是被子植物的最大科,是一个广泛认可的自然分类群。为揭示全球空间尺度上植物核DNA含量在纬度梯度上的变异规律,以及这种变异是否具有环境适应意义,以菊科为对象开展了核DNA含量与纬度、生物气候因素关系的统计分析。从"植物DNA C-值数据库"检索到822种菊科植物的核DNA含量数据;在全球范围内,沿经度方向上设立10条样带,每条样带横跨15个经度,每条样带又均分成22个样块,每个样块纵跨7.5个纬度;其次,从"世界气候数据网站"下载1950—2000年时间段14个生物气候因子数据,应用Arc GIS 9.3获得每个样块14个生物气候因子的平均值;根据"全球生物多样性信息网站"记录,计算每个样块菊科植物平均的核DNA含量数据。为避免气候变量之间的多重共线性对数据分析的影响,应用主成分分析对数据进行了降维,发现最冷季度平均温度、最干季度雨量分别是第一、二主成分上荷载最大的因子,去除与它们相关性在-0.7至+0.7之间的其他气候因子后获得了最冷季度平均温度、最干季度雨量和最湿月份雨量三个变量用于进一步数据分析。结果发现,菊科植物在全球10个样带上的核DNA含量与纬度关系密切,与最冷季度平均温度、最干季度雨量和最湿月份雨量呈现极显著的单峰型的非线性关系,可以用二项式进行拟合。因此,全球空间尺度上植物核DNA含量沿着纬度梯度有规律性的非线性变化,这种变化具有很强的气候适应意义。     

Anonymous single‐copy nuclear DNA (scnDNA) markers for two endemic log‐dwelling beetles: Apasis puncticeps and Adelium calosomoides (Tenebrionidae: Lagriinae: Adeliini)

Three approaches — microsatellite library screening, consensus primer PCR (polymerase chain reaction) and sequencing with arbitrary primer pairs (SWAPP) — were used to develop single‐copy nuclear DNA (scnDNA) markers for log‐dwelling beetles Apasis puncticeps and Adelium calosomoides. We are unaware of other nuclear markers for Adeliini. We tested > 70 primer pairs per species, but despite exhaustive optimization, we obtained only five polymorphic markers. Nonetheless, the markers are valuable in detection of effects of habitat fragmentation.