木林子国家级自然保护区苔藓植物物种与区系研究

基于野外调查、采集标本和查阅文献,对木林子国家级自然保护区苔藓植物物种及区系特点进行研究。结果显示:该保护区苔藓植物共有64科121属292种,其中,苔类28科38属88种,藓类36科83属204种。该保护区共有湖北新记录科2科、新记录属9属、新记录种73种。该保护区苔藓植物区系成分多样,有11个区系类型,以温带成分为主(占30.89%)、东亚成分次之(占18.92%)。相比清凉峰、大巴山、十万大山和佛坪4个国家级自然保护区,木林子呈现出科多种少的特点,物种分化程度更高。木林子保护区与清凉峰的苔藓植物组成相似度最高,与佛坪相似度最低。该研究结果从苔藓植物多样性的角度佐证了清江流域物种分化程度较高的观点,为丰富木林子国家级自然保护区苔藓植物和湖北省苔藓植物提供了基础资料。     

黄山短尾猴mtDNA控制区序列变异及种群的遗传多样性

短尾猴属灵长目(Primates)猴科(Cercopithecidae)猕猴属(Macaca),是我国特有的国家二级保护动物。为了更有效地保护其野生种群,本文研究了黄山短尾猴种群内的遗传多样性,并对黄山短尾猴与四川短尾猴种群间的遗传差异进行了分析。共测定了黄山短尾猴7个群体中的30个样本的mtDNA控制区5′端493bp的序列,只发现了7个变异位点,定义了3种单倍型,单倍型序列之间缺乏变异,种群中的核苷酸多样性很低(0.006);3种单倍型相应地将黄山种群分为了3个亚群,不同亚群之间呈现出一定的片断化分布,从分子水平上初步揭示了短尾猴黄山种群的遗传多样性。与四川短尾猴的相应序列比较,黄山短尾猴控制区序列存在很大差异,共有59个变异位点,而且存在大片段的碱基插入/缺失,有78%的遗传变异发生在两个种群之间,两个种群间的核苷酸歧异度已达8.21%。进一步分析表明,黄山短尾猴与四川短尾猴之间存在着极显著的遗传分化(FST=0.399,P<0.001),基于最大似然法和邻接法构建的系统发生树均将两者聚为不同的类群,支持将它们归入各自的管理单元。     

基于基因组重测序的藏羚Y染色体多态性遗传位点筛选

藏羚Pantholops hodgsonii是藏羚属Pantholops现存的唯一物种,由于Y染色体基因较保守,基于近缘物种的Y染色体多态性遗传位点筛选受到了很大的限制。本研究对15份藏羚新鲜组织样品进行基因组重测序,生物信息分析筛选出Y染色体雄性特异区（MSY）对应的scaffolds,对部分SNP及SSR位点进行多态性验证。共比对出44个scaffolds,以其中序列最长、候选变异位点最多且最完整的KE113803.1为参考,对其中190个SNP变异位点设计引物进行验证,共获得45条MSY DNA序列,其中15对引物扩增到的11 898 bp DNA序列中检测到27个SNP位点;同时对KE113803.1中除单核苷酸重复以外的134个SSR位点设计引物并验证多态性,筛选出56个Y-SSR位点,其中5个具有多态性。本研究结果为后续分析藏羚Y染色体遗传多样性及父系遗传奠定了良好基础。     

四川裂腹鱼乌江种群mtDNA控制区序列的遗传多样性分析

采用PCR扩增和直接测序法分析了四川裂腹鱼乌江种群32个个体mtDNA控制区的序列差异和遗传多样性.在该种群mtDNA控制区长度为464 bp的同源序列中,共计有9个变异位点,占总位点数的1.94%.32个个体共定义了9种单倍型,单倍型间平均遗传距离(P)为0.0060,单倍型多样度(Hd)为0.768.核苷酸多样性(π)为0.00324,平均核苷酸差异数(K)为1.500.用单倍型间遗传距离构建的NJ分子系统树聚为两个分支.结果提示四川裂腹鱼乌江种群遗传多样性匮乏,保护四川裂腹鱼乌江种群刻不容缓.     

同域分布垂穗披碱草和达乌力披碱草遗传多样性和遗传结构分析

披碱草属(Elymus L.)是禾本科(Poaceae)小麦族(Triticeae)中的一个多年生属,该属在青藏高原地区有广泛分布,多数物种是草原和草甸的组成成分,许多种类为品质优良的牧草。垂穗披碱草(E.nutans)和达乌力披碱草(E.dahuricus)同为禾本科小麦族披碱草属异源六倍体物种,染色体组组成皆为StYH。为探究垂穗披碱草遗传多样性形成的内在机制,该研究利用微卫星(SSR)分子标记,对采自青藏高原地区同域分布的垂穗披碱草和达乌力披碱草两个居群共58个个体进行遗传多样性和遗传结构分析。结果表明:8对引物在垂穗披碱草和达乌力披碱草扩增条带分别为163条和124条,多态性位点百分率(PPB)分别为89.71%和76.07%,多态性信息含量(PIC)分别介于0.583~0.929和0.524~0.830之间。垂穗披碱草遗传多样性(H_e=0.69,I=1.34,P_p=100%)高于达乌力披碱草(H_e=0.53,I=0.80,P_p=93.75%);同域分布的垂穗披碱草和达乌力披碱草居群,垂穗披碱草呈现出更高的遗传多样性。AMOVA分子变异显示,两个物种居群内遗传变异分别80.92%和63.62%,但居群间遗传分化水平较低。遗传结构分析揭示两个物种间有基因流存在。综合分析结果认为,该地区种间杂交基因渗透引起的种内遗传分化,在这两个物种多样性形成中可能起着重要作用。     

云南边境地区果实蝇属种类DNA条形码鉴定

【目的】果实蝇属Bactrocera中有国际上重要的检疫性害虫, 基于形态的物种鉴定有一定的局限性。另一方面, 云南边境地区为东南亚地区实蝇入侵我国的重要通道。因此, 对该地区实蝇分子鉴定方法的研究对于该属物种的快速准确鉴定具有重要意义。本研究旨在探讨DNA条形码技术在果实蝇属物种鉴定中的有效性。【方法】使用线粒体基因COI和COII序列的通用引物对果实蝇属20个物种60份样品进行PCR扩增、测序和序列分析; 采取距离方法和建树方法评价2种序列的鉴别能力。【结果】COI和COII序列平均长度分别为682 bp和339 bp, 种内和种间遗传差异较大, 有较明显的遗传距离间隔（barcoding gap）, 鉴定成功率分别为91.2%和90.7%。另外, 分子系统树表明华实蝇亚属Sinodacus不是单系群。【结论】COI和COII序列均能够将绝大多数果实蝇属物种进行准确鉴别, 应用COI或COII序列进行果实蝇属物种鉴定具有一定的可行性。     

箭筈豌豆两个肌动蛋白基因片段的克隆及序列分析

通过同源克隆获得了箭筈豌豆两个不同的Actin基因片段,为研究该箭筈豌豆其它基因的表达状况提供了内标参照。根据近缘物种Actin基因序列设计一对引物,通过RT-PCR技术分别从叶片和果实材料中克隆获得了两条不同的Actin基因片段。生物信息学分析表明,叶片中克隆的片段长度604 bp,编码201个氨基酸;果实中克隆的片段长度677 bp,编码225个氨基酸。两条序列与其它物种Actin基因的碱基序列相似度高于80%,氨基酸序列相似度高于93%。将来自叶片和果实的两条序列分别命名为VsACT7和VsACT11,并在GenBank注册,登录号分别为HM004434和GU946218。     

我国东北三省自然保护区物种保护价值评估

自然保护区作为保护生物多样性最主要的手段之一,得到了世界各国的重视。如何科学客观地评价自然保护区的保护价值,并依据保护价值对其进行分类管理是当前我国自然保护区建设与管理的重要课题之一。以我国东北地区40个国家级自然保护区为研究对象,分别对其物种多样性保护价值和遗传种质资源保护价值进行了评估。通过选取物种的濒危性、特有性和保护等级等指标来计算野生动植物的多样性保护价值;选取分类独特性、近缘程度和濒危性等指标来计算遗传种质资源保护价值,进而计算出各自然保护区的综合物种保护价值。研究结果显示:该评价方法能够很好的反映自然保护区生物多样性及其各个层次和类群的保护价值,能够较准确地识别其物种保护优先性。不同自然保护区其保护价值存在一定差异;同一自然保护区中野生动物与植物之间的物种多样性保护价值没有显著差异,但其野生动物与植物之间的遗传种质资源保护价值存在一定差异性;绝大多数自然保护区其植物遗传种质资源保护价值大于动物遗传种质资源保护价值;虽同为国家级自然保护区,其综合物种多样性保护价值差异很大。吉林长白山国家级自然保护区、吉林珲春东北虎国家级自然保护区、吉林松花江三湖国家级自然保护区的综合保护价值显著高于同类型的其他自然保护区,而辽宁章古台国家级自然保护区、黑龙江双河国家级自然保护区和黑龙江三环泡国家级自然保护区的综合物种保护价值较低,不同类型自然保护区之间其综合保护价值则没有明显差异。该评价方法能较好地进行自然保护区物种保护价值评价,并用于进行自然保护区之间的比较,并不会因自然保护区所处生境、所分布物种不同而产生评价结果上的偏差;该方法在对自然保护区遗传种质资源部分的计算方面需要进一步完善;总体上来说该评价方法不会因自然保护区类型的不同产生差异;今后在对东北地区自然保护区管理分类研究中可将此评价结果作为参考,并作为评价该地区自然保护区能否晋升为国家级自然保护区的辅助工具。这在一定程度上减少了人为主观性,具有较大可行性。     

中国卵叶海桑遗传多样性的ISSR研究

卵叶海桑 (Sonneratiaovata)是海桑科濒危红树植物 ,在我国仅分布于海南文昌清澜自然保护区内。采用简单序列重复区间扩增 (ISSR)分子标记技术对该天然居群和东寨港红树林自然保护区引种的人工居群共 3个居群 3 9个个体进行了遗传变异分析。 1 1个引物共扩增出 1 85条带 ,其中 1 2 7条具多态性 ,多态位点百分率为 68.65 %。在居群水平上相对较低 ,多态位点百分率 3 6.76%～ 5 4.5 9% ,平均值为 47.2 1 %。Nei的基因多样性、Shannon信息指数在物种水平上分别为 0 .1 41 1和 0 .2 2 92 ;在居群水平上平均值分别为 0 .1 2 0 9和0 .1 91 0。Nei的遗传分化系数Gst表明 :87.5 8%遗传变异分布在居群内 ,1 2 .42 %的遗传变异分布在居群间。居群间的遗传一致度达 0 .970 7。东寨港迁地保护的人工居群有效地保护了卵叶海桑的遗传多样性。     

从D环和细胞色素b基因序列探讨懒猴属的分子系统发育关系(英文)

本研究测定了懒猴属(Nycticebus)D环的部分序列和细胞色素b基因的全序列(1140bp),分析了该属物种之间的系统发育进化关系。在DNA水平上,序列分析结果一致地提供了新的分类学证据：支持Rataiszczak和Groves的观点,即N．intermedus只是N．pygmaeus的成体(Ratajszczak,1998;Groves,1971)。对两种序列的数据做了联合及个别分析,获得相似的系统树,支持懒猴属由两个单系群组成：第一群由N．pygmaeus聚成,第二群由N．coucang聚成。该结果也提供了新的分子遗传证据,支持懒猴属由N．coucang和N．pygmaeus两物种组成。     

Autecology and conservation status of Magnolia sargentiana Rehder ＆ Wilson （Magnoliaceae） in the Dafengding region,southern Sichuan Province,China

This paper reports the first population ecology study of the endangered Magnolia sargentiana Rehder ＆ Wilson （Magnoliaceae）. Magnolia sargentiana is a protected species in China, but little is known about its present status in the field. In 2007 and 2008, we surveyed the population and conservation status ofM. sargentiana in the Provincial Mamize Nature Reserve and the National Meigu Dafengding Nature Reserve, Sichuan Province, southwestern China. Natural regeneration is poor because of unfavorable environmental conditions and anthropogenic disturbances. Flower buds and bark ofM. sargentiana are used in traditional Chinese medicine and their collection by local people over the period 1983-1994 has led to marked population declines. The collection of flower buds and bark is now banned, but hewing branches for firewood and grazing continues to have a negative impact on the recovery of M. sargentiana populations. To protect the species, we require a ban on hewing branches, closure of primary forests to reduce the impact of humans and ungulates, better education of local people, and increased awareness of wildlife conservation.     

后河国家级自然保护区蝴蝶遗传多样性的RAPD分析

目的：利用随机扩增多态性DNA(RAPD)技术对湖北省五峰后河国家级自然保护区的蝶类遗传多样性分析。方法：采用RAPD技术,对湖北省五峰后河国家级自然保护医的碧风蝶(Pa;ilio bianor Cranmer)和曲纹蜘蛱蝶(Araschnia doris Leech)的遗传多样性进行研究,并应用RAPDistance软件及MEGA程序,计算Nei氏相似系数和遗传距离,建立了UPGMA和NJ聚类图谱。结果：在事先优化的反应条件下用11个随机引物扩增,共得到136条片段,片段长度为170-2000bp。结论：利用相似性系数及遗传距离进行的聚类分析结果,这两种蝴蝶的不同季节种群的遗传组成存在季节变化。     

引起蜘蛛流行病的紫色野村菌种群异质性

应用ISSR分子标记对安徽琅琊山自然保护区和牯牛降自然风景区引起蜘蛛流行病的紫色野村菌Nomuraea atypicola遗传多样性进行了研究。筛出的9个ISSR引物共扩增出117条带,多态性比率高达94%。ISSR扩增结果显示:22株紫色野村菌之间的遗传分化较大,Jaccard遗传相似系数为0.48-0.88,平均为0.71。UPGMA方法构建的系统树表明,在遗传相似系数0.60处,22株紫色野村菌按照两个自然保护区明显分为2大类群。第一大类是分离自琅琊山不同蜘蛛的10株菌株,其种群内平均遗传相似系数为0.74。第二大类是采自牯牛降不同蜘蛛的12株菌株,其种群内平均遗传相似系数为0.76。种群内的遗传相似性虽高于种群间的遗传相似性,但未见两株菌株的ISSR指纹图谱相同或高度相似。这表明,不管是在琅琊山还是在牯牛降,紫色野村菌的两个种群均无优势的流行菌株。两地的蜘蛛流行病都是由高度异质的紫色野村菌种群造成的。紫色野村菌的菌株遗传相似性和地理来源相关而和寄主无关。     

Species identification of Radix Astragali (Huangqi) by DNA sequence of its 5S-rRNA spacer domain

About 300 species and varieties of Astragalus are identified in China, making the identification of the origin of a particular Astragalus species on the consumer market difficult. A molecular genetic approach was developed to identify various species of Astragalus. Although the 5S-rRNA coding sequence is conserved in higher eukaryotes, the spacer domain of the 5S-rRNA gene has great diversity among different species. The 5S-rRNA spacer domain was amplified by polymerase chain reaction (PCR) from the isolated genomic DNA, and the PCR products (approximately 300 bp) covering the 5S-rRNA spacer domain were sequenced. The nucleotide sequences of Astragalus membranaceus, A. membranaceus var. mongholicus, A. lehmannianus, A. hoantchy, and of one closely related species Hedysarum polybotrys (Hongqi), were determined. Diversity in DNA sequence and restriction enzyme mapping among various species was found in their 5S-rRNA spacer domains. This is the first report on the detection of 5S-rRNA spacer region sequence of Astragalus, and the results could be used for genetic identification of Huangqi.     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

凉水、丰林国家自然保护区红松居群遗传多样性的RAPD分析

采用了RAPD分子标记技术分析了凉水和丰林国家自然保护区红松(Pinus koraiensis)居群的遗传多样性。10个随机引物检测到61个可重复的位点,多态位点比率为0.6935。Nei指数统计结果表明,红松的遗传多样性凉水居群(0.2634)大于丰林居群(0.2607)。对比分析了这两个天然红松居群的遗传多样性变化,红松种内的遗传变异主要存在于居群内,居群间存在一定的遗传分化。     

基于粪便DNA的青藏高原雪豹种群调查和遗传多样性分析

基于非损伤性取样,利用mtDNA和微卫星DNA遗传标记,对来自青藏高原三江源国家级自然保护区、羌塘国家级自然保护区和甘肃党河南山地区等地的277 份疑似雪豹粪便样品进行了物种鉴定、个体识别和遗传多样性研究。结果显示：在190份成功扩增的mtDNA cyt b基因片段中,确定有89份属于雪豹;微卫星分析确定其属于48个不同的个体。在48个雪豹mtDNA cyt b基因片段中,共检测出13个多态性位点,定义了9个单倍型,单倍型多态性为0.776,核苷酸多态性为 1.50%, 9个单倍型的遗传距离为 0.009-0.058。研究表明在研究区域均存在雪豹分布,并具有一定的遗传多态性,地理距离分布较远的羌塘国家级自然保护区和甘肃党河南山地区的雪豹样品具有一定的遗传差异。     

Genetic relationships among fruiting-mei (Prunus mume Sieb. et Zucc.) cultivars evaluated with AFLP and SNP markers.

Genetic relationships among 50 fruiting-mei (Prunus mume Sieb. et Zucc.) cultivars from China and Japan were investigated, using 767 amplified fragment length polymorphism (AFLP) and 103 single nucleotide polymorphism (SNP) markers. The polymorphism among the cultivars was found to be 69.77%, based on EcoR I + Mse I AFLP primer pairs. The sequence alignment of 11 group sequences, derived from 50 samples, yielded 103 SNPs; the total length of genomic sequences was 3683 bp. Among these SNPs, 73 were heterozygous in the loci of different cultivars. The SNP distribution was 58% transition, 40% transversion, and 2% InDels. There was also 1 trinucleotide deletion. AFLP and SNP markers allowed us to evaluate the genetic diversity of these 50 fruiting-mei cultivars. The 2 derived cladograms did display some differences: all cultivars formed 2 subclusters (1A and 1B) in the cladogram based on AFLP polymorphisms, and formed 3 subclusters (2A, 2B, and 2C) in the cladogram based on SNP polymorphisms; and, in the cladogram based on AFLP polymorphisms, most cultivars from the Guangdong to Fujian provinces (G-F) in China, from the Yunnan, Hunan, and Sichuan provinces (Y-S-H) in China, and from Japan grouped in cluster 1A, and 18 (78.26%) of 23 cultivars from Jiangsu to Zhejiang provinces in China (J-Z) grouped in cluster 1B. The results demonstrate that mei cultivars from Japan are clustered with cultivars from China, and support the hypothesis that mei in Japan were introduced from China. Cultivars from the J-Z region of China have more genetic similarities. Cultivars from the G-F and Y-S-H regions have fewer genetic similarities and suggest more germplasm exchanges in the past.     

Single nucleotide polymorphism (SNP) discovery in mammals: a targeted-gene approach

Single nucleotide polymorphisms (SNPs) have rarely been exploited in nonhuman and nonmodel organism genetic studies. This is due partly to difficulties in finding SNPs in species where little DNA sequence data exist, as well as to a lack of robust and inexpensive genotyping methods. We have explored one SNP discovery method for molecular ecology, evolution, and conservation studies to evaluate the method and its limitations for population genetics in mammals. We made use of 'CATS' (or 'EPIC') primers to screen for novel SNPs in mammals. Most of these primer sets were designed from primates and/or rodents, for amplifying intron regions from conserved genes. We have screened 202 loci in 16 representatives of the major mammalian clades. Polymerase chain reaction (PCR) success correlated with phylogenetic distance from the human and mouse sequences used to design most primers; for example, specific PCR products from primates and the mouse amplified the most consistently and the marsupial and armadillo amplifications were least successful. Approximately 24% (opossum) to 65% (chimpanzee) of primers produced usable PCR product(s) in the mammals tested. Products produced generally high but variable levels of readable sequence and similarity to the expected genes. In a preliminary screen of chimpanzee DNA, 12 SNPs were identified from six (of 11) sequenced regions, yielding a SNP on average every 400 base pairs (bp). Given the progress in genome sequencing, and the large numbers of CATS-like primers published to date, this approach may yield sufficient SNPs per species for population and conservation genetic studies in nonmodel mammals and other organisms.