基于低拷贝核基因GBBSI的禺毛茛复合体系统进化研究

禺毛茛多倍体复合体及其近缘种系统进化关系复杂,杂交与多倍化现象同时存在。该复合体内高倍性植物的形成及扩散过程仍需进一步研究。首次克隆了毛茛属植物低拷贝核基因颗粒结合型淀粉合成酶 I (GBBSI )基因,并利用其构建禺毛茛多倍体复合体及其近缘种的系统进化树和网状进化关系,进而证明其适合于研究毛茛属植物种下系统发育研究。结果表明：匍枝毛茛与多倍体复合体关系密切,参与了该多倍体复合体的起源和进化;禺毛茛起源于茴茴蒜和卷喙毛茛,扬子毛茛起源于茴茴蒜和匍枝毛茛;在该类群中茴茴蒜是个关键种,它在多倍体复合体中可能起着枢纽基因组的重要作用。     

单拷贝核基因在昆虫分子系统学中的应用

单拷贝核基因(single-copy nuclear gene,scnDNA)是指基因组中拷贝数目少,只有1个或几个拷贝的核基因,大多数是属于生物体内组成型表达的持家基因。单拷贝核基因是分子系统学中的一种极有价值的分子标记,在构建生命树的主干及主干和末梢之间的中间分枝中将起极其重要的作用。文章主要介绍了单拷贝核基因在昆虫分子系统学中的应用情况、一些单拷贝核基因的特点以及应用单拷贝核基因研究昆虫分子系统学时存在的问题及注意事项。     

DNA序列在悬钩子属植物分子系统学研究中的应用进展

悬钩子属植物种类繁多,类群复杂,而且多为多倍体和杂种。该文就近年来国内外有关DNA序列在悬钩子属植物分子系统学研究中的应用现状和进展进行了综述,并对中国悬钩子属植物系统发育研究进行了展望。研究认为:叶绿体DNA序列多应用非编码区,且多与ITS序列联合分析;核基因组中ITS序列应用最为广泛,主要用于研究悬钩子属空心莓组与木莓组的进化关系、栽培品种间亲缘关系及部分杂种和多倍体的起源等;在该属植物中发现了ITS个体内多态性,但未进行ITS假基因检测,其系统学应用价值需重新评价;低拷贝核基因只有GBSSI和LEAFY有相关应用。同时认为,悬钩子属植物系统学研究中应用的DNA序列及研究类群均较少,缺乏对整个悬钩子属全面而系统的研究。指出应进一步选择具有代表性的样本、筛选合适的DNA片段,并结合形态学、孢粉学和细胞学等手段对中国悬钩子属植物系统关系进行深入研究。     

植物多倍体化中基因组和基因表达的变化

多倍体化在植物进化的历史过程中频繁发生, 对新物种的形成产生了很大影响。伴随着多倍体化, 植物在基因组和基因表达上发生了复杂的变化, 包括染色体数目变化、染色体重组、基因沉默、基因的非加性表达和表观遗传等变化。该文对多倍体化引起的这些变化及其相应的机理进行了综述, 以期为了解多倍体化中植物新表型的产生机理和在进化中的意义提供参考。     

种子植物抗细胞凋亡DAD基因的演化

抗细胞凋亡基因(DAD)是一个高度保守的细胞凋亡抑制基因,在植物生长发育中承担重要功能。为全面了解DAD基因在种子植物中的分布和演化规律,该文利用31种植物的全基因组数据,通过生物信息学手段,深入探讨和分析了不同植物类群中DAD基因的拷贝数目、基因结构和染色体定位,并综合另外7种裸子植物的转录组数据探讨了其在种子植物中的演化趋势。结果表明,DAD基因属于低拷贝基因,在不同种子植物中只具有1–3个拷贝;不同DAD基因编码的氨基酸长度在108–170 aa之间变动。同线性和系统发育分析进一步表明,种子植物DAD基因的演化具有明显的谱系特异性。随机复制和染色体大片段复制及其随后的基因丢失可能是其维持低拷贝的重要方式。     

外源基因在转基因油菜染色体上的定位及分析

原位杂交技术 ( in situ hybridization,ISH)是基因定位的主要技术之一。近来 ,随着植物细胞染色体制片技术的发展 ,以及酶联放大检测系统的采用 ,在植物中已有低拷贝和单拷贝甚至小于 1 kb的 DNA序列定位的成功报道 [1 ,2 ]。染色体原位杂交技术不仅可以用于基因的物理作图 ,而且可以用来对转基因植物中的外源基因进行染色体定位 [3 5] 。研究表明 ,外源目的基因在转基因植物中的表达与整合位点有关 [6] 。因而 ,进行外源基因在转基因植物染色体上的定位以及研究外源基因的整合位点与表达之间的关系 ,对于开发和利用转基因植物具有重要…     

植物异源多倍体进化中基因表达的变化

多倍化是植物物种进化的主要动力,异源多倍体植物在形成早期发生着快速的基因表达变化。本文概述了异源多倍体植物中基因表达变化的特点,包括基因的沉默、激活和部分同源基因表达水平的变化,探讨了基因表达变化的分子机制和生物学意义,并对研究中的问题进行了分析和展望。     

植物异源多倍体进化中基因表达的变化

多倍化是植物物种进化的主要动力, 异源多倍体植物在形成早期发生着快速的基因表达变化。本文概述了异源多倍体植物中基因表达变化的特点, 包括基因的沉默、激活和部分同源基因表达水平的变化, 探讨了基因表达变化的分子机制和生物学意义, 并对研究中的问题进行了分析和展望。     

荧光原位杂交技术在植物多倍体起源与进化研究中的应用

多倍化是植物物种形成与多样化的重要原动力。研究植物特别是一些重要经济作物和园艺植物多倍体的起源与进化,不仅对于揭示多倍体形成过程中性状变异的分子机制具有重要意义,而且可为植物遗传资源的保护与利用提供理论和技术支持。作为连接基因组序列片段到染色体组的桥梁,荧光原位杂交技术长期被广泛用来研究多倍体形成与进化过程中相关特异基因或序列的表达定位、外源染色体检测和鉴定、基因组结构变异等科学问题。因此,在简单介绍荧光原位杂交技术发展历史和植物多倍体主要类型的基础上,主要总结了荧光原位杂交技术在植物多倍体起源与进化相关研究上的应用。     

一种新的植物miRNA体内下调表达系统

MicroRNA(miRNA)是一种内源性的21到24个核苷酸长度的小分子RNA,在植物发育过程中起重要作用。旨在建立一种新的植物miRNA下调表达系统,为miRNA的功能研究提供有力的工具。不同于原有target mimicry的构建方法,以miRNA核心序列为基础,通过设计特定DNA引物,利用聚合酶链式反应(PCR)获得多拷贝的靶基因类似序列(Target mimicry or target mimics),构建target mimicry载体,转化拟南芥。通过qRT-PCR验证miRNA靶基因在转基因植株中的表达。以拟南芥6个保守miRNA为基础,获得了各个miRNA下调表达转基因株系,在转基因植株中检测miRNA靶基因的表达,与野生型相比差异明显,均发生了明显的上调。我们基于一种新的target mimicry的载体构建方法,成功建立了一种在植物中具有普遍适用性的高效miRNA低表达系统。     

Utility of Low-Copy Nuclear Gene Sequences in Plant Phylogenetics

Low-copy nuclear genes in plants are a rich source of phylogenetic information. They hold a great potential to improve the robustness of phylogenetic reconstruction at all taxonomic levels, especially where universal markers such as cpDNA and nrDNA are unable to generate strong phylogenetic hypotheses. Low-copy nuclear genes, however, remain underused in plant phylogenetic studies due to practical and theoretical complications in unraveling the evolutionary dynamics of nuclear gene families. The lack of the universal markers or universal PCR primers of low-copy nuclear genes has also hampered their phylogenetic utility. It has recently become clear that low-copy nuclear genes are particularly helpful in resolving close interspecific relationships and in reconstructing allopolyploidization in plants. Gene markers that are widely, if not universally, useful have begun to emerge. Although utilizing low-copy nuclear genes usually requires extra lab work such as designing PCR primers, PCR-cloning, and/or Southern blotting, rapid accumulation of gene sequences in the databases and advances in cloning techniques have continued to make such studies more feasible. With the growing number of theoretical studies devoted to the gene tree and species tree problem, a solid foundation for reconstructing complex plant phylogenies based on multiple gene trees began to build. It is also realized increasingly that fast evolving introns of the low-copy nuclear genes will provide much needed phylogenetic information around the species boundary and allow us to address fundamental questions concerning processes of plant speciation. Phylogenetic and molecular evolutionary analyses of developmentally important genes will add a new dimension to systematic and evolutionary studies of plant diversity.     

Utility of low-copy nuclear gene sequences in plant phylogenetics

Low-copy nuclear genes in plants are a rich source of phylogenetic information. They hold a great potential to improve the robustness of phylogenetic reconstruction at all taxonomic levels, especially where universal markers such as cpDNA and nrDNA are unable to generate strong phylogenetic hypotheses. Low-copy nuclear genes, however, remain underused in plant phylogenetic studies due to practical and theoretical complications in unraveling the evolutionary dynamics of nuclear gene families. The lack of the universal markers or universal PCR primers of low-copy nuclear genes has also hampered their phylogenetic utility. It has recently become clear that low-copy nuclear genes are particularly helpful in resolving close interspecific relationships and in reconstructing allopolyploidization in plants. Gene markers that are widely, if not universally, useful have begun to emerge. Although utilizing low-copy nuclear genes usually requires extra lab work such as designing PCR primers, PCR-cloning, and/or Southern blotting, rapid accumulation of gene sequences in the databases and advances in cloning techniques have continued to make such studies more feasible. With the growing number of theoretical studies devoted to the gene tree and species tree problem, a solid foundation for reconstructing complex plant phylogenies based on multiple gene trees began to build. It is also realized increasingly that fast evolving introns of the low-copy nuclear genes will provide much needed phylogenetic information around the species boundary and allow us to address fundamental questions concerning processes of plant speciation. Phylogenetic and molecular evolutionary analyses of developmentally important genes will add a new dimension to systematic and evolutionary studies of plant diversity.     

Phylogenetic utility of the glycerol-3-phosphate acyltransferase gene: evolution and implications in Paeonia (Paeoniaceae)

The nuclear-encoded chloroplast-expressed glycerol-3-phosphate acyltransferase (GPAT) gene has been found to be single-copy in a number of angiosperm families. In this study we investigated the phylogenetic utility of the GPAT gene at the interspecific level using the genus Paeonia (Paeoniaceae) as an example. An approximately 2.3- to 2.6-kb fragment of the GPAT gene, containing a large intron of more than 2 kb, was amplified, cloned, and sequenced from 19 accessions representing 13 Paeonia species. The GPAT gene phylogeny inferred by parsimony analysis supported interspecific relationships that were previously unresolved, suggesting that large introns of low-copy nuclear genes are particularly informative in the resolution of close relationships at low taxonomic levels. Whereas the GPAT phylogeny is largely congruent with the previous phylogenetic hypothesis of Paeonia, it shows a significant discordance involving the paraphyly of section Paeonia. Given evidence of an ancient duplication and the subsequent silencing of one GPAT locus in P. anomala, this discordance is most likely the result of paralogy. Two distinct genomic clones containing partial GPAT genes were isolated from P. anomala. The GPAT sequence from one clone corresponded to the functional copy of the gene, and the second genomic clone was determined to contain a GPAT pseudogene. The insertion of a retrotransposon in an intron of this pseudogene may have been responsible for the silencing of this GPAT locus in P. anomala. This study suggests that, although it is unlikely that universal nuclear gene markers free from paralogy are usually available, low-copy nuclear genes can be very useful in plant phylogenetic reconstruction, especially at low taxonomic levels, as long as the evolutionary dynamics of the genes are carefully examined.     

Phylogeny of Campanuloideae (Campanulaceae) with Emphasis on the Utility of Nuclear Pentatricopeptide Repeat (PPR) Genes

Background

The Campanuloideae (Campanulaceae) are a highly diverse clade of angiosperms found mostly in the Northern Hemisphere, with the highest diversity in temperate areas of the Old World. Chloroplast markers have greatly improved our understanding of this clade but many relationships remain unclear primarily due to low levels of molecular evolution and recent and rapid divergence. Furthermore, focusing solely on maternally inherited markers such as those from the chloroplast genome may obscure processes such as hybridization. In this study we explore the phylogenetic utility of two low-copy nuclear loci from the pentatricopeptide repeat gene family (PPR). Rapidly evolving nuclear loci may provide increased phylogenetic resolution in clades containing recently diverged or closely related taxa. We present results based on both chloroplast and low-copy nuclear loci and discuss the utility of such markers to resolve evolutionary relationships and infer hybridization events within the Campanuloideae clade.

Results

The inclusion of low-copy nuclear genes into the analyses provides increased phylogenetic resolution in two species-rich clades containing recently diverged taxa. We also obtain support for the placement of two early diverging lineages (Jasione and Musschia-Gadellia clades) that have previously been unresolved. Furthermore, phylogenetic analyses of PPR loci revealed potential hybridization events for a number of taxa (e.g., Campanula pelviformis and Legousia species). These loci offer greater overall topological support than obtained with plastid DNA alone.

Conclusion

This study represents the first inclusion of low-copy nuclear genes for phylogenetic reconstruction in Campanuloideae. The two PPR loci were easy to sequence, required no cloning, and the sequence alignments were straightforward across the entire Campanuloideae clade. Although potentially complicated by incomplete lineage sorting, these markers proved useful for understanding the processes of reticulate evolution and resolving relationships at a wide range of phylogenetic levels. Our results stress the importance of including multiple, independent loci in phylogenetic analyses.     

Phylogenetic Properties of 50 Nuclear Loci in Medicago (Leguminosae) Generated Using Multiplexed Sequence Capture and Next-Generation Sequencing

Next-generation sequencing technology has increased the capacity to generate molecular data for plant biological research, including phylogenetics, and can potentially contribute to resolving complex phylogenetic problems. The evolutionary history of Medicago L. (Leguminosae: Trifoliae) remains unresolved due to incongruence between published phylogenies. Identification of the processes causing this genealogical incongruence is essential for the inference of a correct species phylogeny of the genus and requires that more molecular data, preferably from low-copy nuclear genes, are obtained across different species. Here we report the development of 50 novel LCN markers in Medicago and assess the phylogenetic properties of each marker. We used the genomic resources available for Medicago truncatula Gaertn., hybridisation-based gene enrichment (sequence capture) techniques and Next-Generation Sequencing to generate sequences. This alternative proves to be a cost-effective approach to amplicon sequencing in phylogenetic studies at the genus or tribe level and allows for an increase in number and size of targeted loci. Substitution rate estimates for each of the 50 loci are provided, and an overview of the variation in substitution rates among a large number of low-copy nuclear genes in plants is presented for the first time. Aligned sequences of major species lineages of Medicago and its sister genus are made available and can be used in further probe development for sequence-capture of the same markers.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Phylogenetic utility of nuclear nitrate reductase: A multi-locus comparison of nuclear and chloroplast sequence data for inference of relationships among American Lycieae (Solanaceae)

There has been considerable interest and research into the benefits of multiple low-copy nuclear regions for phylogenetic studies at low taxonomic levels. In this study, the phylogenetic utility of DNA sequence data from two low-copy nuclear genes, nitrate reductase (NIA) and granule-bound starch synthase I (GBSSI), was compared with data from nuclear ribosomal ITS and the cpDNA spacers trnT–trnF and trnD–trnT across 33 closely related taxa in tribe Lycieae (Solanaceae). The NIA data (introns 1–2) had the most parsimony-informative characters, with over twice the number provided by GBSSI, but NIA also had greater homoplasy. Although gene trees were generally concordant across the four regions, there were some notable areas of incongruence, suggesting both incomplete lineage sorting as well as possible reticulate origins.     

Allopolyploid evolution in Geinae (Colurieae: Rosaceae) – building reticulate species trees from bifurcating gene trees

A previous phylogenetic study of paralogous nuclear low-copy granule-bound starch synthase (GBSSI) gene sequences from polyploid and diploid species in Geinae indicated that the clade has experienced two major allopolyploid events in its history. These were estimated to have occurred several million years ago. In this extended study we test if the reticulate phylogenetic hypothesis for Geinae can be maintained when additional sequences are added. The results are compatible with the hypothesis and strengthen it in minor aspects. We also attempt to identify extant members of one of the inferred ancestral lineages of the allopolyploids. On the basis of previous molecular phylogenies, one specific group has been proposed to be the descendants of this taxon. However, none of the additional paralogues belong to this ancestral lineage. A general method is proposed for converting a bifurcating gene tree, with multiple paralogous low-copy gene sequences from allopolyploid taxa, into a reticulate species tree.     

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。