DNA分子标记技术在濒危物种保护中的应用

近20年来,随着分子生物学技术的迅猛发展,涌现出一批高效、可靠的DNA分子标记技术.本文论述了限制性片段长度多态性、微卫星DNA、随机扩增多态性DNA、扩增片段长度多态性等DNA分子标记技术的基本原理及技术特点;同时,介绍了DNA分子标记在濒危物种种群遗传学研究、致危因素分析及保护策略的制定等保护生物学方面的应用.     

濒危石斛兰野生原种的迁地保护与引种驯化

总结了连续几年的石斛兰引种与驯化实践,探讨了石斛兰生长习性与栽培措施的特殊性,对其抗寒、耐旱及繁殖特性及对应的管理方法进行了阐述。     

DNA分子标记在药用植物中的应用

对DNA分子标记技术在药用植物鉴定、中药质量标准化、遗传图谱构建和近缘物种进化关系等方面的研究进展进行了综述,并展望了分子标记技术在药用植物研究中的发展前景。     

分子标记在烟粉虱蚜小蜂分类中的应用

本对分子标记技术在烟粉虱寄生蜂——蚜小蜂分类中的应用进行了概述,包括利用随机扩增多态性DNA、限制性片段长度多态性和DNA测序等技术对蚜小蜂进行分子鉴定;利用核糖体28s rDNA D2与D3扩展区、线粒体细胞色素氧化酶、核糖体内转录间隔区等DNA序列分析研究蚜小蜂的系统进化等。对分子标记在蚜小蜂分子鉴定和系统进化研究中取得的进展进行了简述。     

重要物种优先保护种群的确定

由于同一物种不同种群的重要性不同、用于物种保护的资金有限以及保护与发展经济之间的矛盾,因此对于重要物种（尤其是濒危种类以及农作物和驯化动物的野生近缘种）需要确定保护什么以及保护哪儿。目前确定优先保护种群的方法主要有3类,分别为基于遗传变异、基于遗传差异性和基于遗传贡献率的方法。基于遗传变异的方法主要是根据遗传变异程度（尤其是等位基因多样性）来确定优先保护的顺序,但忽略了种群之间的遗传差异性,这容易使得存在于遗传变异程度较低的种群中的特有等位基因得不到有效保护。而基于遗传差异性的方法（如确定进化显著单元）则是从遗传分化程度的角度考虑优先性,即独特性越强的种群越具有保护价值。基于遗传贡献率的方法由于综合考虑了遗传多样性和差异性,最适合于确定哪些种群需要优先保护。我国开展此类研究十分必要。     

分子系统学在生物保护中的意义

本文综述了近年来分子系统学的原理和方法及其在生物多样性保护中的应用和发展。分子系统学方法可以很好地确定物种保护的基本单元——进化显著性单元,并可用于推测群体的发展状态,从而为物种的保护提供了一项新的具很强操作性的科学手段。     

稀有濒危植物坡垒的迁地保护

国家二级保护树种坡垒（ＨｏｐｅａｈａｉｎａｎｅｎｓｉｓＭｅｒｒ．ｅｔＣｈｕｎ）原产海南岛。在西双版纳勐仑热带植物园迁地栽培,年平均增高０．４４～０．９ｍ,胸径加粗０．５４～１．３１ｃｍ,比原产地野生或其他迁地保护地区的植株生长快。花期８～１０月,果熟期翌年３～５月。种子发芽率达９６．７％。经多年观察,未见寒害和严重的病虫害。已繁衍了３代并扩大种植２８４株,说明坡垒在西双版纳热带植物园迁地保护获得成功。     

DNA分子标记在柑桔中的应用

ＤＮＡ分子标记是最为理想的遗传标记 ,依其多态性检出所用的分子生物学技术 ,大致可分为Ｓｏｕｔｈｅｍ杂交技术为核心的分子标记和ＰＣＲ技术为核心的分子标记。前者的代表性技术有ＲＥＬＰ(ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ)和ＤＮＡ指纹技术 (ＤＮＡｆｉｎｇｅｒｐｒｉｎｔｉｎｇｔｅｃｈｎｉｑｕｅｓ)。后者的代表性技术有ＲＡＰＤ(ｒａｎｄｏｍａｍｐｌｉｆｉｅｄｐｏｌｙｍｏｒｐｈｉｃＤＮＡ)、ＳＣＡＲ(ｓｅｑｕｅｎｃｅｃｈａｒａｃ ｔｅｒｉｓｔｉｃａｍｐｌｉｆｉｅｄｒｅｇｉｏｎ)…     

单核苷酸多态性及其在油菜分子标记辅助育种中的应用

油菜是一种重要的油料作物,油菜的基因组功能研究已引起了各国政府和科研工作者的高度重视。SNP是目前为止分布最广泛,存在数量最多且标记密度最高的一种遗传多态性标记。对油菜等作物的基因组功能研究提供了丰富的信息资源。综述了近来单核苷酸多态性的研究进展以及其在油菜的分子标记辅助品质育种中的可能用途。     

RAPD分子标记在食用菌研究中的应用

RAPD分子标记以其简单、快速、经济等优点正在被广泛应用于食用菌的研究中。RAPD分子标记克服了传统的标记手段和形态分类学的缺点,在食用菌种质的鉴定和遗传多样性研究,在亲本和杂交种的鉴别以及在基因克隆与分离和遗传图谱的建立等研究中都起着重要的作用。RAPD分子标记为食用菌的深入研究提供了强有力的工具和手段,其应用和发展前景广阔。     

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

Cypripedium japonicum Thunb. (Orchidaceae), once a common perennial herb, is now designated as endangered throughout most of its distribution due to habitat destruction and fragmentation, and the impacts of horticultural collection. We investigated the genetic characteristics of this species for conservation purposes, using microsatellite markers to examine the genetic diversity and structure of 15 native and 5 ex situ populations in Japan. The results imply that although allelic variation is low in Japanese C. japonicum, sexual reproduction by seed, as well as clonal propagation, may occur in some populations. Both native and ex situ populations were found to be genetically differentiated, indicating that some populations may have experienced recent population declines, genetic fragmentation, or bottlenecks. The degree of genetic drift from the putative ancestral population, inferred through STRUCTURE analysis, was more pronounced in northern populations than in southern populations. Some of the ex situ conserved populations exhibited a low degree of differentiation from ancestral native populations. Our results imply that conservation of C. japonicum in Japan is best supported by maintaining individual populations and their unique genetic characteristics.     

Applications of mitochondrial DNA analysis in conservation: a critical review

Patterns of variation in mitochondrial DNA (mtDNA) increasingly are being investigated in threatened or managed species, but not always with clearly defined goals for conservation. In this review I identify uses of mtDNA analysis which fall into two different areas: (i) 'gene conservation' - the identification and management of genetic diversity, and (ii) 'molecular ecology' - the use of mtDNA variation to guide and assist demographic studies of populations. These two classes of application have different conceptual bases, conservation goals and time-frames. Gene conservation makes extensive use of phylogenetic information and is, in general, most relevant to long-term planning. Appropriate uses here include identification of Evolutionarily Significant Units and assessment of conservation priority of taxa or areas from an evolutionary perspective. Less appropriate are inferences about fitness from within-population diversity and about species boundaries. Molecular ecology makes more use of allele frequencies and provides information useful for short-term management of populations. Powerful applications are to identify Management Units and to define and use naturally occurring genetic tags. Estimating demographic parameters, e.g migration rate and population size, from patterns of mtDNA diversity is fraught with difficulty, particularly where populations are fluctuating, and is unlikely to produce quantitative estimates sufficiently accurate to be useful for practical management of contemporary populations. However, through comparative studies, mtDNA analysis can provide qualitative signals of population changes, allowing efficient targeting of resource-intensive ecological studies. Thus, there are some relatively straightforward uses of mtDNA, preferably in conjunction with assays of nuclear variation, that can make a significant contribution to the long-term planning and short-term execution of species recovery plans.     

Identifying evolutionarily significant units for conservation of the endangered Malus sieversii using genome‐wide RADseq data

Malus sieversii, a wild progenitor of the domesticated apple, is an endangered species and is assigned second conservation priority by the China Plant Red Data Book. It is urgent to carry out in situ conservation of this species, but previous studies have not identified evolutionarily significant units (ESUs) for conservation management. In this study, we investigated the genetic diversity and relationships of six M. sieversii populations from China using integrated analysis of microsatellite (nSSR) data, genome‐wide SNPs and previous results in order to propose a reasonable conservation management. The results showed that levels of genetic diversity were inconsistently reflected by our nSSR and previous studies, suggesting that indices of genetic diversity are not effective to identify priority conservation areas for M. sieversii. Based on the selection criteria of ESUs for endangered species conservation, ESUs should reflect lineage divergence, geographical separation and different adaptive variation. Our phylogenetic tree based on genome‐wide SNPs yielded a clear relationship of divergent lineages among M. sieversii populations, leading to new different from those of previous studies. Three independent lineages, including the pairs of populations Huocheng‐Yining, Gongliu‐Xinyuan and Tuoli‐Emin, were identified. The geographic distances between populations among the different phylogenetic lineages were much greater than those within the same phylogenetic lineage. A cluster analysis on environmental variables showed that the three independent lineages inhabit different environmental conditions, suggesting that they may have adapted to different environments. Based on the results, we propose that three independent ESUs should be recognized as conservation units for M. sieversii in China.     

An early evaluation of translocation actions for endangered plant species on Mediterranean islands

In situ conservation is widely considered a primary conservation strategy. Plant translocation, specifically, represents an important tool for reducing the extinction risk of threatened species. However, thus far, few documented translocations have been carried out in the Mediterranean islands. The CareMediflora project, carried out on six Mediterranean islands, tackles both short-and long-term needs for the insular endangered plants through in situ and ex situ conservation actions. The project approach is based on using ex situ activities as a tool to improve in situ conservation of threatened plant species.Fifty island plants(representing 45 taxa) were selected for translocations using common criteria.During the translocations, several approaches were used, which differed in site selection method, origin of genetic material, type of propagative material, planting method, and more. Although only preliminary data are available, some general lessons can be learned from the experience of the CareMediflora project. Among the factors restricting the implementation of translocations, limited financial resources appear to be the most important. Specific preliminary management actions, sometimes to be reiterated after translocation, increase the overall cost, but often are necessary for translocation success. Translocation using juvenile/reproductive plants produces better results over the short term,although seeds may provide good results over the long run(to be assessed in the future). Regardless,plant translocation success can only be detected over long periods; therefore, proper evaluation of plant translocations requires a long-term monitoring protocol. Care-Mediflora project represents the first attempt to combine the existing approaches in a common plant conservation strategy specifically focusing on the Mediterranean islands.     

Microsatellite markers indicate genetic differences between cultivated and natural populations of endangered Taxus yunnanensis

Taxus yunnanensis (Taxaceae) is an endangered plant known for its effective natural anti‐cancer metabolite, taxol. To assess the effectiveness of T. yunnanensis ex situ conservation in China, the genetic diversity and genetic structure of nine cultivated and 14 natural populations were compared using 11 polymorphic microsatellite markers. The purpose of this comparison was to determine whether the cultivated populations were genetic representatives of natural types. Among the 14 natural populations studied, three were from Tibet and 11 were from the Hengduan Mountains. There were fewer genetic variations among cultivated populations than among natural populations, but this difference was not significant. However, 23 unique alleles were detected in cultivated populations and 14 were detected in natural populations. Striking differences in genetic structure (G_ST = 0.365) was observed between them. STRUCTURE, AMOVA and cluster analyses also consistently showed a clear genetic separation between the 14 natural and seven cultivated populations. Only five natural populations from the south‐central Hengduan Mountains were genetically represented in two cultivated collections. The reason for the occurrence of distinct genetic differences between cultivated and natural populations and the loss of natural population‐specific alleles in the cultivated populations can be attributed to uneven selection during establishment. These results suggest that the genetic structure of T. yunnanensis may not have been conserved effectively. Based on our study, practical conservation strategies for ex situ preservation of T. yunnanensis have been developed. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 450–461.     

Multiplication of the endangered Indian pitcher plant (Nepenthes khasiana) through enhanced axillary branching in vitro

Axenic seedling-derived two- to three-node stem segments of Nepenthes khasiana Hook.f. were successfully cultured on Woody Plant Medium containing 2.2 M benzyladenine to produce a 0.5–1.5 cm axillary shoot from each node in 7–8 weeks. The rapid growth along with the axillary branching of this shoot enabled amassing of 6–12 shoots during subculture. Excised shoots transferred to basal medium or rooted in medium containing 2.7 M naphthaleneacetic acid produced typical pitchers at leaf tips. Rooted plants were established in pots at 90–95% survival rate.Abbreviations AA ascorbic acid - AC activated charcoal - CA citric acid - BA 6-benzyladenine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - KC Knudson-C (1946) basal medium - MS Murashige & Skoog (1962) basal medium - WPM Woody Plant basal medium (Lloyd & McCown 1980)     

Cryptic divergent lineages of Pultenaea pauciflora M.B. Scott (Fabaceae: Mirbelieae) exhibit different evolutionary history

Genetic structure among disjunct population groups of Pultenaea pauciflora was assessed to determine the evolutionary history of this species as a basis for conservation management strategies. Analysis of individuals from all extant populations using 1737 amplified length polymorphism markers revealed two highly divergent genetic entities with strong geographical structuring. Populations located at Narrogin and Brookton clustered together in Bayesian assignment analysis with every individual optimally placed in a single cluster with complete membership. Genetic differentiation between populations in these two areas was very low. Populations at Boddington were highly divergent from those located at Narrogin and Brookton. All individuals from Boddington populations were optimally placed into a second cluster with complete membership. Populations located at Boddington maintain lower levels of allelic diversity, yet greater levels of mean heterozygosity than populations located at Narrogin and Brookton. The degree of genetic differentiation and different patterns of genetic diversity strongly suggest historical divergence and separate evolutionary influences on the two lineages that occur in different ecological habitat. These Evolutionary Significant Units are likely to represent two cryptic sister taxa in the extant populations currently recognized as P. pauciflora, and the reassessment of taxonomic and conservation status of both lineages is required. © 2013 State of Western Australia. Biological Journal of the Linnean Society © 2013 The Linnean Society of London     

分子标记技术在昆虫入侵研究中的应用

遗传变异与种群持续性及其进化潜力密切相关,而生物入侵导致种群遗传变异或遗传多样性的改变为研究自然界中各种生态和进化问题提供了理想模式。分子标记技术是调查种群遗传变异的重要工具,揭示了入侵种的入侵过程和结果,并预测未来的发生情况。本综述归纳了分子标记技术在昆虫入侵机制研究中的应用,以典型的研究个案为例,分别综述了分子标记技术在隐蔽入侵的监测应用,分子标记技术在重构入侵历史研究中的推算方式,分子标记技术在探索种群遗传变异与成功入侵机制方面取得的重要进展,并进一步介绍了高分辨率熔解曲线（high-resolution melting, HRM）分析在昆虫入侵研究中的应用前景。