羊草遗传多样性分析和赖草属的分子系统发育

羊草（Leymus chinensis（Trin.）Tzvel.或Aneurolepidium chinense Trin.），隶属禾本科（Gramineae），大麦族（Hordeae），小麦亚族（Triticieae），赖草属（Leymus），兼具重要的生态价值和经济价值。面向国家重大需求，本论文选择羊草作为主要研究对象，开展了两方面的研究工作，即我国羊草种质资源的遗传多样性评价和赖草属物种的系统发育分析。 羊草是一种多年生根茎型禾草，根据叶色可以划分为黄绿型和灰绿型两种生态型。其分布范围横穿亚欧草原的东部，包括朝鲜和蒙古的西部，以及西伯利亚的西北部，集中分布于我国的东北部。羊草是我国典型草原植物群落中的优势种。它可以在多种土壤和气候条件下正常生长，如松嫩平原、内蒙古草原和黄土高原。从另一个角度讲，不同类型的生境也造就了羊草丰富的遗传多样性。 自上世纪90年代中期开始，我们陆续从我国东北部6个省市收集了293份羊草种质，包括205份灰绿型羊草和88份黄绿型羊草。经过连续3年的观测记录，通过37个重要农艺性状，对293份羊草的遗传多样性进行了评估。依据10个质量性状和27个数量性状，统计了羊草不同性状和不同地域羊草的Shannon遗传多样性指数，同时还使用了主成分分析和通径分析做了相关统计。结果显示：（1）与黄绿型羊草相比，灰绿型羊草具有更高的遗传变异（P<0.05）。结合这两个趋异型羊草的分布范围，可以得出两种类型羊草之间存在稳定的遗传差异;（2）通径分析显示，羊草营养生长性状和遗传多样性两者的组合效应可以解释羊草生殖特性中20.6％的遗传变异;（3）在124-128ºE这一区域，羊草的遗传多样性指数最高（H＝2.252），表明这一地区具有最丰富的羊草种质资源。 赖草属（Leymus Hochst.）是一个异源多倍体属，约有34个物种，该属未知基因组的起源一直争论不休。其倍性范围从四倍体（2n=4x=28）、八倍体（2n=8x=56）一直到十二倍体（2n=12x=84）。新麦草属（Psathyrostachys Nevski）只含有Ns一个基因组，约有9个物种。这两个属都是多年生牧草，具有抗旱、抗病和耐盐碱等生物学特性。 应用核糖体ITS（Internal Transcribed Spacer）序列和叶绿体trnL-F序列，我们对13个赖草属物种、小麦族18个属（40份二倍体材料）、以及Elymus californicus和Bromus catharticus，共计57份材料进行了系统发生分析。ITS序列分析表明，赖草属分别与新麦草属和小麦族中一个未知属在进化上具有紧密的关系。ITS谱系树表明赖草属内部存在大量的分化，以及赖草属物种具有多次起源的特征。trnL-F序列分析表明，赖草属物种的母本，部分来自Ns基因组，部分来自Xm基因组，这可能与赖草属物种的地理分布有关，分布于亚欧大陆的物种其母本是新麦草属，而分布于北美的大部分物种其母本是Xm基因组。trnL-F序列分析还表明，E. californicus和赖草属未知的基因组具有紧密的关系。以上研究结果表明：（1）从分子层面证明，赖草属的未知基因组并非来自薄冰草属，或是一个修正的新麦草基因组，其基因组组成应是NsNsXmXm;（2）赖草属物种的母本，部分来自Ns基因组，部分来自Xm基因组，这可能与赖草属物种的地理分布有关;（3）E. californicus的母本是Xm基因组，父本是Ns基因组，该物种应从披碱草属转移至赖草属。

烟草NtFAD2基因的沉默及功能研究

油酰磷脂酰胆碱去饱和酶（FAD2）是一种重要的植物脂肪酸去饱和酶，位于内质网中，催化油酸生成亚油酸。亚油酸等多不饱和脂肪酸含量过高的植物油脂不稳定，易氧化，不易贮藏，而且氧化产物对人体有害。因此油脂改良的一个重要目的是提高种子油脂的油酸含量，降低多不饱和脂肪酸含量。另外，关于FAD2的结构、功能和表达调控等问题目前还不清楚，有待深入研究。近年发展起来的RNAi技术可有效地沉默目标基因，研究植物FAD2基因沉默后膜脂和油脂的变化及温度对基因沉默的影响，有助于深入理解FAD2酶的功能、表达调控方式和有效地利用基因沉默技术改变膜脂组成、提高植物油脂品质。本研究的主要目的是，首先采用RNAi技术抑制烟草FAD2基因（NtFAD2）的表达以降低NtFAD2酶活性，得到膜脂不饱和度改变的烟草转基因株系。然后研究NtFAD2基因沉默后脂肪酸去饱和过程的变化及其机理，以及温度对NtFAD2基因沉默的影响。研究中首先用PCR方法克隆烟草NtFAD2基因，用其编码区的一个片段构建表达发卡RNA的沉默结构，用农杆菌介导的方法转化烟草，从第一代转基因植株中筛选出油酸含量高的突变体。然后对其中一个转基因株系S61进行脂肪酸分析，发现叶片总脂和质体外单脂PC和PE中的油酸含量大幅度增加。此外，NtFAD2基因沉默对叶片甘油脂的影响有多效性，即质体内的单脂油酸含量也有显著增加，有些单脂中软脂酸含量有所下降。烟草NtFAD2基因沉默后，在叶片和种子中油酸含量变化最大，说明NtFAD2基因在不同器官中的沉默效果不同：沉默结构对NtFAD2酶活性较高的器官抑制程度更大，内源基因没有表现均一的沉默效果。沉默植株的多种组织中NtFAD2基因的mRNA降解程度相似，说明油酸含量与NtFAD2转录本的丰度没有直接关系。 植物脂肪酸的不饱和程度受温度调控，因此研究FAD2基因沉默株系的油酸含量受温度影响情况及其调控规律对RNAi技术在油脂改良方面的实际应用具有十分重要的意义。研究结果表明，随着生长温度的下降，野生烟草叶片膜脂油酸含量降低，多不饱和脂肪酸含量随之增加;转基因烟草中油酸和多不饱和脂肪酸有相似的变化趋势，但是变化幅度远远大于野生烟草。低温下转基因烟草叶片油酸含量之所以大幅度下降，其中一个重要原因是被抑制的NtFAD2酶受低温的调节而活性升高。然而研究发现，与常温条件下相比，低温下转基因烟草NtFAD2基因的mRNA丰度不变，说明低温没有影响沉默结构对NtFAD2基因的mRNA的降解。低温环境下NtFAD2基因沉默植株的多不饱和脂肪酸含量有所回升可能是植物对低温的一种适应性响应。转基因烟草种子油脂的油酸含量也受温度影响：常温下表现高油酸性状，低温下油酸含量下降。因此种植这种高油酸品系时，需要考虑温度的影响。如果在适宜的地域和季节种植，避免转基因植株开花结实时遭遇持续低温，就可以利用转基因植株的遗传优势，生产富含油酸的优质植物油。

山茶属茶组植物分子系统学初步研究

山茶科植物的叶绿体基因和nrDNA基因分别为母系和双亲遗传，二者的联合分析为系统发育重建，特别是揭示网状进化过程提供了便利条件。茶组(Sect. Thea (L.) Dyer )属山茶科(Theaceae)山茶属(Camellia)，除茶（C. sinensis (L.) O. Kuntze）为广布种外，其余种主要分布在我国云南、广西、贵州和四川等地。自从W. T. Dyer（1874）建立茶组以来，该组作为一个自然类群，不存在大的争议，但是组下系统分类至今在国内外学术界中争议颇多。 本文选择来自于叶绿体的2个DNA片段，通过序列分析对茶组的28个种进行了系统发育重建。主要结果如下： (1) 叶绿体DNA rpS12-rpL20和trnS-G的序列分析 2个叶绿体DNA片段联合分析，将茶组植物分为两大支，部分种之间的系统发育关系得到了分辨。该叶绿体基因的分析结果支持茶组植物为一个单系类群，但与以往将茶组分为子房五室类群和三室类群的观点不相吻合。 (2) 茶组nrDNA ITS区研究结果 选用14个种茶组植物，对其nrDNA ITS区进行序列分析。茶组植物nrDNA ITS区扩增比较困难，且存在种内多态性，因而在测序之前需要对PCR产物进行克隆。

Evolution of genome organizations of squirrels (Sciuridae) revealed by cross-species chromosome painting

With complete sets of chromosome-specific painting probes derived from flow-sorted chromosomes of human and grey squirrel (Sciurus carolinensis), the whole genome homologies between human and representatives of tree squirrels (Sciurus carolinensis, Callosciurus erythraeus), flying squirrels (Petaurista albiventer) and chipmunks (Tamias sibiricus) have been defined by cross-species chromosome painting. The results show that, unlike the highly rearranged karyotypes of mouse and rat, the karyotypes of squirrels are highly conserved. Two methods have been used to reconstruct the genome phylogeny of squirrels with the laboratory rabbit (Oryctolagus cuniculus) as the out-group: ( 1) phylogenetic analysis by parsimony using chromosomal characters identified by comparative cytogenetic approaches; ( 2) mapping the genome rearrangements onto recently published sequence-based molecular trees. Our chromosome painting results, in combination with molecular data, show that flying squirrels are phylogenetically close to New World tree squirrels. Chromosome painting and G-banding comparisons place chipmunks ( Tamias sibiricus), with a derived karyotype, outside the clade comprising tree and flying squirrels. The superorder Glires (order Rodentia + order Lagomorpha) is firmly supported by two conserved syntenic associations between human chromosomes 1 and 10p homologues, and between 9 and 11 homologues.

Karyotypic conservatism in the suborder Feliformia (Order Carnivora)

Multidirectional comparative chromosome painting was used to investigate the karyotypic relationships among representative species from three Feliformia families of the order Carnivora ( Viverridae, Hyaenidae and Felidae). Complete sets of painting probes derived from flow-sorted chromosomes of the domestic dog, American mink, and human were hybridized onto metaphases of the spotted hyena (Crocuta crocuta, 2n = 40) and masked palm civet (Paguma larvata, 2n = 44). Extensive chromosomal conservation is evident in these two species when compared with the cat karyotype, and only a few events of chromosome fusion, fission and inversion differentiate the karyotypes of these Feliformia species. The comparative chromosome painting data have enabled the integration of the hyena and palm civet chromosomes into the previously established comparative map among the domestic cat, domestic dog, American mink and human and improved our understanding on the karyotype phylogeny of Feliformia species. Copyright (C) 2005 S. Karger AG, Basel.

Cross-species chromosome painting unveils cytogenetic signatures for the Eulipotyphla and evidence for the polyphyly of Insectivora

Insectivore-like animals are traditionally believed among the first eutherian mammals that have appeared on the earth. The modern insectivores are thus crucial for understanding the systematics and phylogeny of eutherian mammals as a whole. Here cross-spe

人类线粒体DNA世系的系统发育关系研究

本文以人类线粒体DNA为例,回顾了其系统发育关系的重建的研究历史,进而总结介绍了该分析方法在人类进化历史研究、线粒体DNA数据质量评估以及疾病相关线粒体DNA突变的甄别等方面的应用,以期对该方法在国内的推广应用有所裨益.

系统发育研究中"长枝吸引"假象概述

下载PDF阅读器系统发育研究(phylogeny)不仅有助于重建地球所有生物体的进化历史,而且还可以揭示进化生物学领域中的一些基本问题.清晰了解各生物物种进化历程及不同物种之间的进化关系,是进一步研究和探索生物学其他学科的基础.但是现今广泛应用的所有系统发育分析方法都存在一定的局限性,在一定程度上不能有效消除各种误差,从而不能客观地处理和分析数据,也就不能成功重建生物进化历程,真实反映物种进化关系.系统发育研究中,"长枝吸引"(Long-branch Attraction,LBA)假象是最为困扰研究者的问题.文章从"长枝吸引"问题的产生原由、检测方法以及消除策略等多个方面进行详尽概述,并通过列举典型实例,阐述了解决"长枝吸引"问题的途径.

食肉目哺乳动物的系统发育学研究概述

Carnivora; Caniformia; Feliformia; Phylogenetic tree; Mitochondrial DNA; Nuclear genes; 【摘要】 追溯生物界不同生物类型的起源及进化关系,即重建生物类群的系统发育树是进化生物学领域中一个十分重要的内容。食肉目哺乳动物位于食物链顶端,很多成员不仅在我国野生动物保护工作中占有重要地位,而且还是研究动物适应性进化遗传机制的重要模式生物。因而,食肉目物种作为物种资源中的一个重要类群,其系统发育学一直是国内外研究的热门课题。构建可靠的食肉目分子系统树,无疑将具有重要的进化理论意义和保护生物学价值。鉴于目前食肉目各科间系统发育关系仍然处于“广泛争论”的状态,本文将针对食肉目科水平上的系统发育学研究进展,包括来自于形态学特征、细胞学及分子生物学方面的证据,做简要概述,并提出目前研究中存在的问题。这对今后食肉目系统发育方面的进一步研究工作具有指导意义,并为以该类群作为模式生物开展适应性进化研究奠定基础

基于磷酸丙糖异构酶基因序列的蓝氏贾第鞭毛虫分子系统发育的研究

[目的 ]探讨蓝氏贾第鞭毛虫种内系统发育及遗传多样性。 [方法 ]对不同来源虫株的磷酸丙糖异构酶(tim)基因进行 PCR扩增、序列测定后 ,用简约法和 NJ法构建分子系统树进行系统学分析。 [结果 ]在所测序列中共有 12 4个位点存在变异 (2 3% ) ,且大多数为发生在第三密码子的同义突变 ,两种构树方法所得两树的分枝结构相似 ,均将受试的 16株蓝氏贾第鞭毛虫分为明显的两组。 [结论 ]tim基因可作为研究蓝氏贾第鞭毛虫群体遗传结构一个有效的遗传标记

Pitfalls in the analysis of ancient human mtDNA

The retrieval of DNA from ancient human specimens is not always successful owing to DNA deterioration and contamination although it is vital to provide new insights into the genetic structure of ancient people and to reconstruct the past history. Normally, only short DNA fragments can be retrieved from the ancient specimens. How to identify the authenticity of DNA obtained and to uncover the information it contained are difficult. We employed the ancient mtDNAs reported from Central Asia (including Xinjiang, China) as an example to discern potentially extraneous DNA contamination based on the updated mtDNA phylogeny derived from mtDNA control region, coding region, as well as complete sequence information. Our results demonstrated that many mtDNAs reported are more or less problematic. Starting from a reliable mtDNA phylogeney and combining the available modern data into analysis, one can ascertain the authenticity of the ancient DNA, distinguish the potential errors in a data set, and efficiently decipher the meager information it harbored. The reappraisal of the mtDNAs with the age of more than 2000 years from Central Asia gave support to the suggestion of extensively (pre)historical gene admixture in this region.

Mitochondrial cytochrome b sequences variation of Protura and molecular systematics of Apterygota

Mitochondrial cytochrome b genes of about 450 bp fragments from 3 proturan species, 5 collembolan species and 2 dipluran species have been sequenced. The number of nucleotide substitutions and Kimura 2-parameter distances have also been calculated, and a series of molecular phylogenetic trees reconstructed by using parsimony and distance methods. The proturan, collembolan and dipluran species have evolved monophyletic groups. The results suggest that Protura and Collembola are sister groups, while Diplura is more or less demonstrating a closer phylogenetic relationship to the pterygotan insects. The phylogeny and their systematic position of protura and other groups are also discussed.

Molecular systematics of Xenocyprinae (Teleostei : Cyprinidae): Taxonomy, biogeography, and coevolution of a special group restricted in east Asia

We surveyed mitochondrial DNA (mtDNA) sequence variation in the subfamily Xenocyprinae from China and used these data to estimate intraspecific, interspecific, and intergeneric phylogeny and assess biogeographic scenarios underlying the geographic structu

Episodic evolution of prolactin receptor gene in mammals: coevolution with its ligand

Divergence of proteins in signaling pathways requires ligand and receptor coevolution to maintain or improve binding affinity and/or specificity. In this paper we show a clear case of coevolution between the prolactin (PRL) gene and its receptor (prolactin receptor, PRLR) in mammals. First we observed episodic evolution of the extracellular and intracellular domains of the PRLR, which is closely consistent with that seen in PRL. Correlated evolution was demonstrated both between PRL and its receptor and between the two domains of the PRLR using Pearson's correlation coefficient. On comparing the ratio of the nonsynonymous substitution rate to synonymous substitution rate (omega=d(N)/d(S)) for each branch of the star phylogeny of mammalian PRLRs, separately for the extracellular domain (ECD) and the transmembrane domain/intracellular domain (TMD/ICD), we observed a lower omega ratio for ECD than TMD/ICD along those branches leading to pig, dog and rabbit but a higher ratio for ECD than TMD/ICD on the branches leading to primates, rodents and ruminants, on which bursts of rapid evolution were observed. These observations can be best explained by coevolution between PRL and its receptor and between the two domains of the PRLR.

Molecular phylogenetics and biogeography of Lepus in Eastern Asia based on mitochondrial DNA sequences

In spite of several classification attempts among taxa of the genus Lepus, phylogenetic relationships still remain poorly understood. Here, we present molecular genetic evidence that may resolve some of the current incongruities in the phylogeny of the leporids. The complete mitochondrial cytb, 12S genes, and parts of ND4 and control region fragments were sequenced to examine phylogenetic relationships among Chinese hare taxa and other leporids throughout the World using maximum parsimony, maximum likelihood, and Bayesian phylogenetic reconstruction approaches. Using reconstructed phylogenies, we observed that the Chinese hare is not a single monophyletic group as originally thought. Instead, the data infers that the genus Lepus is monophyletic with three unique species groups: North American, Eurasian, and African. Ancestral area analysis indicated that ancestral Lepus arose in North America and then dispersed into Eurasia via the Bering Land Bridge eventually extending to Africa. Brooks Parsimony analysis showed that dispersal events followed by subsequent speciation have occurred in other geographic areas as well and resulted in the rapid radiation and speciation of Lepus. A Bayesian relaxed molecular clock approach based on the continuous autocorrelation of evolutionary rates along branches estimated the divergence time between the three major groups within Lepus. The genus appears to have arisen approximately 10.76 MYA (+/- 0.86 MYA), with most speciation events occurring during the Pliocene epoch (5.65 +/- 1.15 MYA similar to 1.12 +/- 10.47 MYA). (c) 2005 Elsevier Inc. All rights reserved.