珍稀濒危飘带兜兰叶绿体全基因组种内变异研究(附录)

飘带兜兰(Paphiopedilum parishii)分布范围狭窄,仅在中国、缅甸、泰国以及老挝有少量分布。近年来,因生境破坏和人为滥采而导致飘带兜兰野生种群极度缩减。为开发种内多态性的分子标记用于保护生物学研究,该研究对飘带兜兰4个野生个体经测序、组装、注释获得的叶绿体基因组序列,与已公布的飘带兜兰2个个体的叶绿体全基因组序列进行比对,分析飘带兜兰叶绿体基因组的种内差异。结果表明:(1)飘带兜兰叶绿体基因组具有典型被子植物叶绿体基因组环状四分体结构,基因组长度为154 403～154 809 bp,共编码129个基因,包括78个蛋白质编码基因、39个tRNA基因、8个rRNA基因,以及4个假基因。(2)在飘带兜兰6个个体叶绿体基因组中检测到103～107个SSRs(simple sequence repeats)位点,其中21个SSR位点具有多态性。此外,在6个个体叶绿体基因组中还检测到60个长序列重复,包括17～21个正向重复、18～29个反向重复、9～16个回文重复、4～9个互补重复。(3)通过比较6个个体叶绿体基因组序列的核苷酸多样性,共发现70处变异,包括10个SNPs(single nucleotide polymorphism)、60个插入缺失(InDels)。其中,有3个SNP位点发生了非同义替换,导致编码功能基因的氨基酸发生改变; 19个插入缺失多态性较高,具有开发为分子标记的潜力。(4)通过计算核苷酸多样性值(P_i)共发现8个有变异的区域,P_i值为0～0.006 32,其中变异度较大的是rps3-rpl22、trnL-UAC-rpl32、rpoB-trnC-GCA以及ycf4,这些高变区可开发为分子标记用于评估飘带兜兰遗传多样性。(5)系统发生分析结果表明,飘带兜兰6个个体叶绿体基因组序列聚在一起,与长瓣兜兰互为姐妹群。综上表明,飘带兜兰叶绿体基因组的SSRs、长序列重复、SNPs、InDels以及核苷酸序列呈现了足够的种内多样性,可开发成分子标记用于该种的系统演化及保护生物学研究。     

三种兜兰属植物种子发育过程及其与无菌萌发的关系

某些兰科植物的种子成熟前,在无菌萌发过程中有较高的萌发率,而成熟后萌发率急剧下降。本研究针对这一现象,对三种兜兰属植物种子的发育过程进行了解剖学观察,并对授粉后不同时期的种子进行无菌播种,80d后统计其萌发率,以探究种子发育过程中的解剖学特征与萌发率之间的关系。研究结果表明种胚刚发育到椭球形胚的时候,胚柄尚存,种皮细胞尚未皱缩,此时三种兜兰的萌发率都能达到较高水平,表明这是适合兜兰属植物萌发的最佳时期;此后,种皮开始皱缩,木质素类物质积累导致种皮透水性下降,推测这可能是导致其后期萌发率下降的主要因素之一。     

基于灰色关联分析方法评价13种野生兜兰的迁地保护适应性

由于过度收集和对原生境地的破坏,我国野生兜兰已濒临灭绝,对其进行迁地保护十分必要。通过对引种的13种40份野生兜兰资源进行迁地栽培,并观测兜兰种质在迁移地的各种生长性状指标等,本研究利用灰色关联分析方法评价13种兜兰的迁地适应性,分析影响其迁地保护的关键因素。结果表明:(1)带叶兜兰、同色兜兰、长瓣兜兰、文山兜兰适应性较强,与理想种的关联度均在0.8以上;巨瓣兜兰、白花兜兰、小叶兜兰、紫纹兜兰、紫毛兜兰、硬叶兜兰适应性一般,与理想种的关联度在0.7~0.8之间;麻栗坡兜兰、杏黄兜兰、红旗兜兰适应性较差,与理想种的关联度均在0.6以下;(2)不同兜兰种类、同一种类不同产地兜兰种质的迁地生长适应性不同,来源地和栽培温度可能是影响其迁地保护的关键因子。     

国产兜兰属植物观赏价值评价及其在华南地区的应用前景分析

以植株的生长状况、整株姿态、叶片观赏性、开花性、花形、花色和花朵观赏期为评价指标,利用灰色关联分析,对我国原产的27种兜兰属植物的观赏价值进行了综合评价并分析了它们在华南地区的应用前景。结果表明:多花长瓣型的飘带兜兰与理想种的关联度最高,达到0.7304,是最值得在华南地区栽培的兜兰种类,其次为白旗兜兰。而紫纹兜兰、汉氏兜兰;文山兜兰、格利兜兰、带叶兜兰、亨利兜兰、同色兜兰,它们的关联度系数超过0.6;紫毛兜兰、巨瓣兜兰、红旗兜兰、天伦兜兰、长瓣兜兰、根茎兜兰的关联度均超过0.5;也是值得推广兜兰种类;杏黄兜兰、硬叶兜兰、白花兜兰由于生长状况差、开花性差等原因与理想种的关联度分别只有0.1900、0.1921、0.2917,不适于华南地区栽培;其它9种兜兰的关联度虽然介于0.35~0.50之间,但其中有些种类如虎斑兜兰、麻栗坡兜兰、波瓣兜兰、德氏兜兰等由于具有独特的观赏价值和育种潜力,也是值得在华南地区栽培的兜兰种类。     

Isolation and characterization of 24 microsatellite loci in Paphiopedilum rothschildianum, an endangered slipper orchid

This paper describes the characterization of 24 polymorphic microsatellite loci in Paphiopedilum rothschildianum, an endangered slipper orchid endemic to Sabah, Malaysia. A total of 36 clones containing microsatellite repeat motifs yielded 41 dinucleotide, 20 tetranucleotide and 30 cryptic simple repeat sequences. Thirty microsatellite loci were randomly selected to characterize the population, of which 24 were found to be polymorphic. The expected heterozygosity ranged from 0.0813 to 0.7279 (mean 0.4533) and the observed heterozygosity ranged from 0 to 1 (mean 0.3800). The results indicated that there was limited migration between the three subpopulations and a significant degree of genetic differentiation (F_ST = 0.5098) between populations implying that gene flow has been disrupted as a result of habitat fragmentation. The molecular markers have a potential application in population management, CITES enforcement and the forensic detection of specimens in international trade.     

兜兰属宽瓣亚属（广义）的被充研究

对新种小囊兜兰Paphiopedilum globulosum与新变种扁球兜兰P.micranthum var.oblatum作了描述与绘图。两者均为采自云南东南部的栽培植物,与硬叶兜兰P.micranthum近似。但小囊兜兰具有宽得多的叶（宽2．8－3．4cm),先端多少呈钩状、宽1．1－1．2cm的辱瓣,以及与唇瓣囊口几乎同样大小的退化雄蕊,而扁球兜兰则叶宽2．2－3cm,唇瓣扁球形,囊深不超过1cm,可区别于硬叶兜兰。上述种类均属于宽瓣亚属（广义）。该亚属有16个种产中国、越南或两地共有。本文提供了分种检索表。     

濒危植物紫纹兜兰群落特征及其保护

对广东省河源市紫金县白溪自然保护区紫纹兜兰（Paphiopedilum purpuratum（Lindl.）Stein）所在的2个群落进行了样方调查,分析了其生境、物种组成、群落区系特点及群落结构特征。结果显示,调查样地内共有维管束植物47科79属94种。属的区系成分中泛热带和热带亚洲成分占主要地位,占非世界分布总属数的84.3%,具有热带向亚热带过渡的性质。群落1中扇叶铁线蕨（Adiantum flabellulatum Linn.）和中华苔草（Carex chinensis Retz.）为优势种,紫纹兜兰仅为伴生种,群落2中紫纹兜兰为优势种。两群落均以小高位芽植物为主,但缺乏大高位芽植物、地面芽植物及一年生草本,而藤本植物的丰富则进一步加大了林下的郁闭度。综合生境因素比较两个群落表明,紫纹兜兰对环境具有极强的依赖性,对坡度坡向及环境湿度有一定的要求,对伴生植物并无明显的选择性。建议加强对紫纹兜兰的就地保护,同时加大对其栽培繁殖技术的研究。     

On Paphiopedilum malipoense sp. nov.—An Intermediate Form Between Paphiopedilum and Cypripedium,with a Discussion on the Origin of the genus

Paphiopedilum malipoense S. C. Chen et Tsi is a very interesting new species with its flower similar to that of Cypripedium, especially section Cypripedium. It belongs to subgenus Brachypetalum, the most primitive group of Paphiopedilum, but differs from its allied species in hgniva elliptic-lanceolate sepal with cuspidately acuminate apex, rather narrow petals and horizontal lip, which are of common occurrence in many cypripediums, but very rare in paphiopedilums. Apparently, this is an intermediate form, or a link, between Paphiopedilum and Cypripedium, but it does not seem to arise from hybridization between them, because no Cypripedium has been found wherever Paphiopedilum occurs. The new species is distributed in southeastern Yunnan of China. In this area, as well as in river valleys of western Yunnan or the Hengduan Mountains, there have been four species of the same genus reported before. As we know, the Hengduan Mountains and their adjacent areas are rich in Cypripedium. The differentiation of the genus there is remarkable. All five sections it contains occur there and three of them are quite distinctive. For example, the general appearance of the section Bracleosa is dissimilar to that of any other cypripediums, but closely resembles that of Listera. It appears that the difference between sect. Bracteosa of Cypripedium and sect. Brachypetalum of Paphiopedilum is not necessarily wider than that between sect. Bracteosa and sect. Cypripedium of the same genus. Apparently, it is reasonable to consider Paphiopedilum to be an evolutional branch of Cypripedium extending into tropical area, with its primitive group (subgenus Brachypetalum) still remaining in its northern fringe area. This primitive subgenus has eight species, distributed from western Yunnan to the Malay Peninsula. Five of them, including the intermediate and primitive form published here, are found in the hilly land of southeastern Yunnan and the river valleys of western Yunnan. All these facts suggest its area of origin: the river valleys of the Hengduan Mountains and the lower hilly land contiguous to the southof them.