大山雀两亚种基因指纹图的比较研究

寡核苷酸基因指纹探针LZF-I对大山雀青藏亚种(Parus major tibetanus)和华北亚种(P.m.artatus)随机个体进行了基因指纹图的比较检测,结果如下：(1)P.m.tibetanus个体在2.3 kb处均有一条杂交谱带,而P.m.artatus个体的共有谱带则位于4.8 kb处。不同的共有等位基因,是区别两亚种的遗传标记之一。(2)P.m.tibetanus和P.m.artatus随机个体的平均谱带数分别是14.65条和19.30条;个体间基因指纹图的相似系数分别是0.18和0.13;P.m.tibetanus和P.m.artatus个体之间基因指纹图的相似系数是0.09。结果表明：P.m.artatus群体内的遗传变异性高于P.m.tibetanus群体,且两亚种之间的变异性高于两亚种内部的变异性。(3)LZF-I探针在P.m.tibetanus随机个体检测中的鉴别几率为2.04×10^-12,在P.m.artatus则高达9.8×10^-17。表明两亚种的基因指纹图均具有个体特异性,且P.m.artatus群体基因组的多态性较P.m.tibetanus强。 该结果为开展对大山雀种内分化的基因多样性和近缘种之间的遗传差异性,以及P.m.artatus分类学地位的最终确立的研究,提供了分子遗传学资料。     

小熊猫圈养种群遗传多样性及其影响因子

利用寡核苷酸指纹探针ＬＺＦ－Ｉ检测了成都大熊猫繁育研究基地仿生态圈养场小熊猫种群全部２２只个体的ＤＮＡ指纹,检测结果表明其遗传多样性参数如下：⑴各体间ＤＡＮ指纹图的相似性为０．１７２;个体Ａ的指纹谱带在个体Ｂ中出现的概率为０．１８０６。⑵圈养种群的等位基因频率为０．０９４８,杂合率为９０．５２％,⑶种群中指名亚种亲本个体的引进是影响该种群遗传多样性的主要因子。     

基于线粒体细胞色素b基因序列探讨红喉姬鶲两亚种的分类地位

以线粒体DNA细胞色素b基因的全序列作为遗传标记,探讨红喉姬鶲普通亚种(Ficedula parva al—bicilla)和指名亚种(F．P．parva)的分类地位。应用Kimura 2—parameter法计算出红喉姬鶲普通亚种个体问的遗传距离为0．1％～0．2％,而与指名亚种之间的遗传距离为6．4％,两亚种之间的遗传距离远远大于种间的遗传距离。结合形态学的特征我们认为：红喉姬鶲指名亚种和普通亚种应为两个独立种,分别为红喉姬鶲(F．albicilla)和红胸姬鶲(F．parva),两者的分化时间为3．15～3．25百万年。     

新疆阜康荒漠地区叉毛蓬居群的遗传结构和分化

应用RAPD技术,对新疆阜康荒漠地区叉毛蓬进行了居群遗传分析。结果表明：①用14个随机引物对5个叉毛蓬亚居群的98个个体进行了RAPD扩增,共检出3919条扩增片段,多态带3868条,总的多态位点百分率为98．7％;②Shannon多样性指数(HPOP／HSP＝0．6933)和Nei基因多样性指数(HS／HT＝0．6948)显示出大部分的遗传变异存在于叉毛蓬亚居群内,遗传分化系数(GST＝o．3052)表明亚居群问的分子变异占居群总遗传变异的30％以上;②5个叉毛蓬亚居群间的平均遗传距离为0．1817,变异范围从0．1258到0．2445,与同一物种亚种间遗传距离的变幅较一致(0．02—0．20),表明5个亚居群间产生了遗传分化;④叉毛蓬亚居群的基因流Nm＝1．138,低于一般广布种的基因流水平(Nm＝1．881),且远低于毛乌素沙地柠条的基因流(Nm＝5．9529),相对有限的基因流可能在叉毛蓬居群遗传分化的维持中起着作用。以上分析表明,尽管大部分的遗传多样性存在于叉毛蓬亚居群内,但5个亚居群间已有明显遗传分化的趋势。     

大石鸡亚种分化及一新亚种描述(鸡形目,雉科)

通过地理分布、形态差异、分子进化证明了我国特有种大石鸡Alectoris magna分化为两个亚种,指名亚种A.magna magna和兰州亚种新亚种A.magna lanzhouensis subsp.nov..对新亚种与指名亚种的形态进行了比较.测新亚种12个和指名亚种7个样本的mtDNA控制区486个碱基,两个亚种间无共享单倍型,其间基因交流受到限制;新亚种各取样种群间共享一种单倍型,说明它们来自共同祖先;新亚种序列变异0.27%,而指名亚种为0.91%,其间差异显著(t=1.77,p=0.046＜0.05);两个亚种间的遗传距离为0.0103,约50万年前它们分歧进化.     

几种鼢鼠染色体组型和血清LDH同工酶电泳的比较研究

本文报道了中华鼢鼠指名亚种、甘肃亚种、四川亚种、罗氏鼢鼠的染色体组型和LDH同工酶电泳。发现中华鼢鼠3个亚种染色体数2n=60,罗氏鼢鼠2n=58;中华鼢鼠指名亚种和甘肃亚种LDH同工酶酶谱带数为3条,而中华鼢鼠四川亚种和罗氏鼢鼠为2条。同时,对分类学上争论的问题进行了讨论。     

横断山区复杂地形与气候对川西白腹鼠种群地理分化的影响

横断山区地质地貌复杂,历史上经历了剧烈的抬升和循环的冰期,这些地质和气候变化对物种的分化产生了重要的影响.川西白腹鼠Niviventer excelsior是广泛分布于横断山区的鼠类物种.本研究基于线粒体cyt b基因探讨来自四川、云南和西藏三地川西白腹鼠的种群分化.利用最大简约法(Maximum Parsimony)、最大似然法(Maximum Likelihood)和贝叶斯推断(Bayesian Inference)构建系统发育树.系统发育树显示川西白腹鼠和安氏白腹鼠Niviventer andersoni没有形成交互单系,二者可能有不完全谱系分拣或基因渐渗现象.川西白腹鼠种群存在亲缘地理结构,大体按理塘-稻城一线分为两个进化支Clade A和Clade B.sub-clade A1来自川西地区(包括九龙、康定、丹巴和二郎山)和云南泸水;sub-clade A2来自滇西地区的哀牢山.在Clade B中,sub-clade B1全都是来自西藏察瓦龙;sub-clade B2则来自川西北地区,包括炉霍卡沙湖、雅江和巴塘.支持滇西亚种N. e. tengchongnsis和指名亚种N. e. excelsior的分化.川西白腹鼠的种群分化可能与横断山区地形的复杂性和在历史上该地区多次的抬升和气候的波动有关.     

中国貉线粒体DNA多态性及其与亚种分化的关系

本文以８种限制性内切酶对来自７个地区的１４只中国貉进行了ｍｔＤＮＡ的ＲＦＬＰ分析,并构建了貉ｍｔＤＮＡ的限制酶物理图谱。根据各群体间的遗传距离构建ＵＰＧ分子系统树,结合形态、地理分布资料对中国貉的亚种分化进行了探讨。结果表明,中国貉的指名亚种已发生明显的遗传分化;对于分类地位没有确定的华北和陕西群体,建议将它们各自单独定为一个亚种;中国貉最先发生南北分歧,然后发生东西分歧。     

大紫蛱蝶三个地理种群的RAPD遗传多样性分析

采用RAPD技术分析了大紫蛱蝶朝鲜亚种Sasakia charonda coreana 3个地理种群的遗传多样性,并以同属的黑紫蛱蝶指名亚种Sasakia funebris funebris(Leech)及近缘属种黑脉蛱蝶指名亚种Hestina assimilis assimilis(Linnaeus)为外群,探讨了它们之间的亲缘关系.经筛选的10个随机引物对供试的58只蝶类标本共产生200条带,扩增谱带具有明显的属、种间多态性.     

锥栗种群在鼎湖山三个群落中的遗传分化研究

采用扩增片段长度多态性（AFLP,Amplified fragmen length polymorphism)方法对锥栗（Castanopsis chinensis)在3个不同群落亚种群的遗传分化进行了研究。研究地点位于广东省鼎湖山,3个群落分别是：针叶林群落,针阔叶混交林群落,常绿阔叶林群落。采集的叶要进行DNA提取,酶切、连接、PCR扩增后进行变异性聚丙烯酰胺的凝脉电泳,银染后把所得带进行1／0标记。结果表明,四组引物对分别扩增出27,20,33,39条带,其中分别有15,15,18,26条是多态性带,说明其多样性较高。用AMOVA（Analysis of molecular variance) 分析表明75．36％的遗传变异表现在种群内,24．64％的遗传变异表现在种群间,极显著（P＜0．001）。使用Nei遗传距离进行了算术平均数的非加权成组配对法(UPGMA）。其聚类结果表明常绿阔叶林群落亚种群和针阔叶混交林群落亚种群的种群聚合在一起。上述结果说明锥栗不同亚种群有不同的遗传多样性,种群间遗传分化较大。这是种群本身遗传特性的反映,并与群落演替密切相关,即不同群落微环境对种群遗传的影响。     

Mitochondrial phylogeography and subspecific variation in the red panda (Ailurus fulgens): implications for conservation

The red panda (Ailurus fulgens) is an endangered species and its present distribution is restricted to isolated mountain ranges in western China (Sichuan, Yunnan, and Tibet provinces) and the Himalayan Mountains chain of Nepal, India, Bhutan, and Burma. To examine the evolutionary history across its current range, and to assess the genetic divergence among current subspecies and population structure among different geographic locations, we sequenced mitochondrial DNA from the control region (CR) and cytochrome (cyt) b gene for 41 individuals in Sichuan, Yunnan, Tibet of China, and Burma. 25 CR haplotypes (10 for cyt b) were identified from 11 geographic locations. Only three haplotypes were shared among sample localities, including one among current subspecies. Nine haplotypes were shared with the study of Su et al. [Mol. Biol. Evol. 18 (2001) 1070]. CR haplotype diversity was high (0.95+/-0.02) and nucleotide diversity among all haplotypes was relatively low (0.018+/-0.009). Phylogenetic confirmed trees show a shallow pattern with very little structure or statistical robustness. The application of two coalescent-based tests for population growth allowed us to interpret this phylogeny as the result of a recent population expansion. Analysis of molecular variance and nested clade analysis failed to detect significant geographic structure in both data sets. The lack of significant differentiation between subspecies does not indicate the presence of evolutionary significant units. We suggest that the present population structure has resulted from habitat fragmentation and expansion from glacial refugia. Due to its habitat requirements it is likely that the red panda has undergone bottlenecks and population expansions several times in the recent past. The present population may exhibit a pattern reminiscent of a relatively recent population expansion.     

比较染色体涂色显示小熊猫(Ailurus fulgens)具有高度保守的核型

以狗的整条染色体特异探针,通过比较染色体涂色（Comparative Chromosome Painting）,建立了小熊猫和狗的比较染色体图谱。狗的38条常染色体探针在小熊猫染色体上共检出71个同源片段。其中狗的18条常染色体每一条在小熊猫染色全上各有1个同源片段,其余的20条常染色体每一条在小熊猫染色体上各有2至5个同源片段。广泛的染色体结构重排造成了小熊猫与狗的核型差异：至少需要经过28次断裂,49次融合,4次倒位才能将狗的核型（2n=78）“转变”为小熊猫的核型（2n=36）。结合已发表的狗与家猫的比较染色体图谱,我们推测：小熊猫与家猫之间共存在26个同源片段,二者的核型之间显示了较高的同源性。通过比较分析狗的染色体同源片段在小熊猫与家猫染色体上的分布和排列,可以看出：4次染色体易拉,2次倒位造成了小熊猫与家猫的核型差异。我们的工作进一步证实了利用基因组高度重排的物种（如：狗）的染色体特异探针与核型保守的物种（如：家猫、水貂、小熊猫）进行比较染色体涂色研究,不但可以准确快速地鉴别物种进化过程中所发生的染色体间的结构重排,而且还可揭示染色体内的结构重组。     

通过二代测序技术开发25个小熊猫的微卫星位点

采用Roche 454基因组测序平台在小熊猫Ailurus fulgens的基因组中进行测序拼装,得到了大量含有微卫星片段的序列。本文选择了其中467条序列设计引物,筛选出了115个微卫星位点,最终选出25个微卫星位点,重复单元包含TAT、GATA、AAACA、AAAACA等,并应用到42只小熊猫个体上进行了分型。观察到的等位基因数为3～10;观察杂合度和期望杂合度分别为0. 143～0. 929和0. 136～0. 869;多态信息含量为0. 128～0. 843,经检测所有位点均未偏离哈迪-温伯格平衡。这些微卫星位点可应用于小熊猫圈养种群的遗传管理,在未来还可进一步作为小熊猫的种群遗传学等研究的有力工具。     

大熊猫、小熊猫和黑熊血红蛋白的组份分离、结晶及氨基酸组成分析

 聚丙烯酰胺凝胶电泳和羧甲基纤维素柱层析都表明大熊猫(Ailuropodmelanoleuca)、小熊猫(Ailurus fulgens)和黑熊(Selenarctos thibetanus)的血红蛋白各有两个组份。在近似的条件下得到了它们的晶体。三种动物血红蛋白的氨基酸组成相似,而且N-末端氨基酸都是Val。有待进一步测定全部氨基酸顺序,以揭示它们之间的异同和亲缘关系。     

Genetic diversity and population history of the red panda (Ailurus fulgens) as inferred from mitochondrial DNA sequence variations

The red panda (Ailurus fulgens) is one of the flagship species in worldwide conservation and is of special interest in evolutionary studies due to its taxonomic uniqueness. We sequenced a 236-bp fragment of the mitochondrial D-loop region in a sample of 53 red pandas from two populations in southwestern China. Seventeen polymorphic sites were found, together with a total of 25 haplotypes, indicating a high level of genetic diversity in the red panda. However, no obvious genetic divergence was detected between the Sichuan and Yunnan populations. The consensus phylogenetic tree of the 25 haplotypes was starlike. The pairwise mismatch distribution fitted into a pattern of populations undergoing expansion. Furthermore, Fu's F(S) test of neutrality was significant for the total population (F(S) = -7.573), which also suggests a recent population expansion. Interestingly, the effective population size in the Sichuan population was both larger and more stable than that in the Yunnan population, implying a southward expansion from Sichuan to Yunnan.     

The long and the short of gene flow and reproductive isolation: Inter-Simple Sequence Repeat (ISSR) markers support the recognition of two floral forms in Pelargonium reniforme (Geraniaceae)

The current taxonomy of Pelargonium reniforme recognises two subspecies on the basis of habit and vegetative characters, but excludes floral characters. However, populations of P. reniforme in the wild tend to belong to easily discernable floral groups based on floral colour and hypanthium length. The aim of this study was to determine the genetic diversity within and between populations of both subspecies using the Inter-Simple Sequence Repeat (ISSR) method of DNA fingerprinting. Ninety individuals from eight populations were sampled. Both phenetic and Neighbor-Net analyses reveal that populations are genetically discernable, but that there is no genetic evidence to support the recognition of the two currently defined subspecies of P. reniforme. Instead, the analyses resolved all individuals that possess long hypanthia into a single group, and the role of different pollinators in driving reproductive isolation of this long-tubed form is suggested.     

飞蝗总DNA的抽提及其RAPD分析条件的摸索

通过试验寻求得到一种快速、简便抽提飞蝗（Ｌocusta sp.)总DNA方法,使每头雄性和雌性成虫分别可以得以５０和１００μg的总ＤＮＡ。所得到的总ＤＮＡ ＯＤ２６０／ＯＤ２８０为１．５－２．２,分子量４５kb。为了获得高分子量的ＤＮＡ产品,使ＲＰＡＤ结果具重复性,酚氯仿抽提后的ＤＮＡ沉淀用灭菌Ｔip头挑出,而不用离心收集。对各种分析条件如摸板、Ｔaq酶、dNTP及引物的浓度、不同的ＰＣＲ仪、反应管进行了比较试验,发现在一定的范围内,它们对ＲＡＰＤ结果影响。用优化的试验条件对我国３个飞蝗亚种５个地理种群进行ＲＡＰＤ分析。结果在３个亚种ＵＰＧＭＡ聚类图中,东亚飞蝗和西藏飞蝗珠２个种群以１００％Ｂootstrap分别聚类在一起,亚洲飞蝗与东亚飞蝗的２个种群以６６％的Ｂootstrap聚类在一起,在３个亚种所有个体的ＵＰＧＭＡ聚类图中,亚种内的所有个体都聚类在一起,各自形成独立分支,说明３个飞蝗亚种有明显的区别。西藏飞蝗的２个种群之间,群居型与散居型东亚飞蝗之间在聚类图中混合聚类,说明它们之间存在基因交流。     

Phylogeography, genetic structure and population divergence time of cheetahs in Africa and Asia: evidence for long-term geographic isolates

The cheetah (Acinonyx jubatus) has been described as a species with low levels of genetic variation. This has been suggested to be the consequence of a demographic bottleneck 10 000–12 000 years ago (ya) and also led to the assumption that only small genetic differences exist between the described subspecies. However, analysing mitochondrial DNA and microsatellites in cheetah samples from most of the historic range of the species we found relatively deep phylogeographic breaks between some of the investigated populations, and most of the methods assessed divergence time estimates predating the postulated bottleneck. Mitochondrial DNA monophyly and overall levels of genetic differentiation support the distinctiveness of Northern‐East African cheetahs (Acinonyx jubatus soemmeringii). Moreover, combining archaeozoological and contemporary samples, we show that Asiatic cheetahs (Acinonyx jubatus venaticus) are unambiguously separated from African subspecies. Divergence time estimates from mitochondrial and nuclear data place the split between Asiatic and Southern African cheetahs (Acinonyx jubatus jubatus) at 32 000–67 000 ya using an average mammalian microsatellite mutation rate and at 4700–44 000 ya employing human microsatellite mutation rates. Cheetahs are vulnerable to extinction globally and critically endangered in their Asiatic range, where the last 70–110 individuals survive only in Iran. We demonstrate that these extant Iranian cheetahs are an autochthonous monophyletic population and the last representatives of the Asiatic subspecies A. j. venaticus. We advocate that conservation strategies should consider the uncovered independent evolutionary histories of Asiatic and African cheetahs, as well as among some African subspecies. This would facilitate the dual conservation priorities of maintaining locally adapted ecotypes and genetic diversity.     

陈旧皮张中DNA提取的新方法

对传统的馆藏陈旧皮张标本DNA提取方法进行了改进,所提DNA分子量可达1kb,而且具有样品用量少（约0.01g),消化时间短（约14h)和操作步骤简单等优点,利用所提DNA,对小熊猫等珍稀动物线粒体DNA的细胞色素b和控制区序列的部分片段进行了PCR扩增,序列测定和比较分析,证实所提DNA合格而无污染,完全可以用于珍稀动物保护遗传学研究。     

Isolation and characterization of microsatellite loci for the red panda,Ailurus fulgens

We report on the isolation and characterization of 10 microsatellites in the red panda Ailurus fulgens from genomic DNA‐enriched libraries. Twenty‐one microsatellites were screened from the libraries, and 10 of the screened microsatellites were polymorphic. The number of observed alleles for each locus in 24 individuals ranged from three to 12, and the expected and observed heterozygosity was 0.60–0.90 and 0.50–1.00, respectively. These markers should prove a useful tool for the study of genetic variation in red panda in the future.