三种兰科植物的保护遗传学研究初探

采用随机扩增多态DNA（RAPD）分析研究了中国3种珍稀濒危兰科植物硬叶兜兰（Paphiopedilum micranthum Tang et Wang),麻栗坡兜兰（P.malipoense S.C.Chen et Tsi)和独花兰（Changnienia amoena Chien)的遗传多样性与群体遗传结构,12个RAPD引物在2种兜兰中共扩增出131条带,对4个硬叶兜兰群体的检测表明其物种水平的多态条带百分率（PPB）为71.6%,Nei的基因多样度（h)为0.2171,Shannon多样性指数（I）为0.3301;4个群体的平均多样性水平为PPB=45.2%,h=0.1457,I=0.2204,低于远交兰花的平均水平,在总遗传变异中,群体间遗传变异占20.31%,略高于远交物种的平均水平,在物种水平上,麻栗坡兜兰的PPB为49.5%,h为0.1174,I为0.1764,均大大低于硬叶兜兰,对11个独花兰群体采用16个RAPD引物共扩增出119条带,物种水平PPB-76.5%,h=0.1941,I=0.3058;在群体水平上,上述3个指标的平均值则分别为37.2%,0.1197和0.1810。均低于远交兰花的平均水平,群体间的遗传变异占45.27%,遗传分化明显高于远交物种的平均水平,导致3个物种遗传多样性偏低而群体间遗传分化较高的主要原因在于人为的过度采挖和生境的片断化,研究结果为兰花保护策略和措施的制定提供了理论基础。     

采用空间自相关分析研究两种兰科植物的群体遗传结构

采用空间自相关分析方法对硬叶兜兰（Paphiopedilum micranthum)和独花兰（Changnienia amoena)4个天然群体的小尺度空间遗传结构进行了研究,以探讨两种兰科植物群体内遗传变异的分布特征及其形成机制。根据来自12个（硬叶兜兰）和16个（独花兰）RAPD引物所提供的多态位点,计算出每个群体的空间自相关系数Moran I值。结果表明,在2个硬叶兜兰群体中,遗传变异在短距离（3－4m)内表现出显著的正相关,在较大的距离内表现出显著的负相关,说明其遗传变异在群体内形成一定的空间结构。而对独花兰的空间自相关分析则显示,其遗传变异在参与计算的2个群体内不存在明显的空间结构。造成上述两种兰科植物具有不同空间分布特性的原因可能与其不同的繁殖方式有关。上述研究结果有助于进一步了解物种的进化历程和濒危机制,并为制定有效的保护策略和措施提供科学依据。     

基于RAPD标记的皖南山核桃野生居群遗传多样性分析

应用随机扩增多态性DNA(RAPD)分子标记方法分析了皖南山区4个山核桃(Carya cathayensis)野生居群的遗传多样性及其分化程度。由POPGENE软件分析表明,15条随机引物共检测出139个扩增条带,其中多态性条带87个,多态性位点百分比(PPB)为62.59%,各居群平均多态性位点百分比(PPB)为43.35%。物种水平Shannon信息多样性指数(I)为0.208,Nei基因多样性指数(H)为0.124,多样性水平较低。由于受到地理隔离的影响,居群间的基因流较低(Nm=0.700),遗传分化程度相对较高(Gst=0.417)。     

濒危植物毛瓣金花茶遗传多样性的ISSR分析

采用ISSR分子标记对毛瓣金花茶6个自然居群的遗传多样性进行了分析。利用11个引物对150个个体进行了扩增,共扩增出92条条带,其中多态性条带74条。毛瓣金花茶在物种水平和居群水平都表现出相对较高的遗传多样性,在物种水平上,多态位点百分率(PPB)为80.43%,Nei’s基因多样性指数(h)为0.245 1,Shannon多样性指数(I)为0.377 6;在居群水平上,PPB为58.70%~66.30%,h为0.199 7~0.229 3,I为0.300 9~0.343 8。Nei’s遗传多样性分析和AMOVA分析表明,毛瓣金花茶的遗传变异主要存在于居群内,居群间的遗传分化程度较低(Gst=0.126 6,Φst=11.37%),基因流(Nm)为3.448 0。Mantel检测表明,居群间的遗传距离和地理距离之间存在显著的相关关系(r=0.755 1,P0.05)。研究认为,毛瓣金花茶较高的遗传多样性和较低的遗传分化可能与其异交型繁育系统和鸟类传粉有关。     

采用RAPD标记探讨瓣蕊唐松草(毛茛科唐松草属)的遗传多样性及其地理分布格局

采用随机扩增多态DNA(RAPD)标记对瓣蕊唐松草(Thalictrum petaloideum L.)(毛莨科)11个居群246个体的遗传多样性及居群遗传结构进行了检测.20个随机引物扩增出125个用于分析的条带.结果表明:瓣蕊唐松草在物种水平上具有极高的遗传多样性,多态条带比率(PPB)为96%,Nei的基因多样度(h)为0.3502,Shannon多样性指数(I)为0.519 9;居群间分化比较明显,基因分化系数(GsT)为0.351 1,分子方差分析(AMOVA)表明居群间遗传变异占总遗传变异的38.88%;根据条带频率与平均分类距离和Nei遗传距离分别进行UPGMA聚类,结果基本一致,并且反映出与该种地理分布有一定相关性;长江流域分布的两个居群表现出较高的相似性,并与北方分布的各居群构成独立的两支,显示出这两个居群的特殊性,从而对该物种独特分布区成因是由于冰退回迁造成的观点提供了一定的支持.     

基于RAPD标记的南苍术居群遗传多样性分析

采用RAPD标记技术对分布于江苏小九华山、小汤山和湖山,安徽金寨和芜湖以及湖北保康和英山的7个南苍术〔Atractylodes lancea(Thunb.)DC.〕野生居群的28个单株基因组总DNA进行PCR扩增,在此基础上分析居群的遗传多样性及遗传分化,并采用聚类分析法对居群的遗传关系进行分析。结果表明:用18条RAPD引物共扩增出193条带,其中多态性条带111条,多态性条带百分率(PPB)为57.51%;平均每条引物扩增出10.72条带,其中多态性条带6.17条。从省级水平看,安徽居群的PPB、有效等位基因数(Ne)、Nei’s基因多样性指数(H)和Shannon信息指数(I)均最低,而湖北居群的Ne、H和I均最高,但江苏居群的PPB最高;从居群水平看,湖北保康居群的PPB、Ne、H和I均最高,而安徽金寨居群均最低。7个居群的基因分化系数和基因流分别为0.206 5和1.921 5,说明7个居群总遗传变异的20.65%存在于居群间、79.35%存在于居群内。7个居群间的遗传距离为0.150 7～0.252 1,其中,安徽金寨和芜湖居群间最小(0.150 7),江苏湖山和安徽芜湖居群间最大(0.252 1)。基于遗传距离的聚类分析结果表明:7个居群可分为2组,湖北保康居群单独成组,其他6个居群聚为另一组;来自同一居群的单株均聚在一起。研究结果提示:南苍术居群间的遗传多样性较低,居群间无明显的遗传分化。     

基于ISSR的硬叶兜兰居群遗传多样性研究

利用ISSR标记对7个硬叶兜兰居群160个体扩增,10对引物共扩增出101个条带,其中多态性条带97个,种水平上的多态性条带比率(PPB)达96.02%,香侬指数(I)为0.488 9,Neis基因多样性指数(H)为0.332 4。居群水平的遗传参数,多态性条带比率(PPB)为33.51%,香侬指数(I)为0.194 3,Neis基因多样性指数(H)为0.133 2。总的遗传多样性(H_T)为0.323 9,个体遗传多样性(Hs)达到0.133 2。AMOVA结果揭示居群间遗传分化大,居群间基因流N_m为0.349 2。居群间的Neis遗传距离0.148 1~0.4385。基于UPGMA聚类,在遗传距离为0.327时,7个居群聚为两支,第一支由古林箐和马固、田坝、夹寒箐和杨柳井居群组成,第二支由斗咀和小坝子居群组成,聚类关系反映居群间遗传分化与地理距离无显著相关性。     

不同天然居群小蓬竹的遗传多样性

从分子水平探讨不同居群小蓬竹的遗传多样性以及与环境因子的相关性,揭示其濒危原因,为小蓬竹的保护和后续开发利用提供理论支撑,助力实施极危物种最佳保护策略。运用RAPD标记技术和POPGENE32对16个小蓬竹天然居群进行遗传多样性研究和遗传变异分析。结果表明,8个RAPD随机引物共扩增出105条清晰、重复性高的条带,其中多态性条带有98条,分子量300~2000bp;物种水平多态性位点百分率PPL=93.33%,有效等位基因数Ne=1.4942,Nei’s基因多样性H=0.3005,Shannon多样性指数I=0.4586;落湾(ZY1)居群的遗传多样性水平最高(PPL=60.95%,H=0.2329,I=0.3451),[JP3]桃坡(PT1)居群的最低(PPL=44.76%,H=0.1700,[JP]I=0.2523);16个天然居群的遗传分化系数Gst=0.3231,基因流Nm=1.0478,基于Shannon’s多样性指数的分化系数[(HSP-HPOP)/HSP]为0.3429。小蓬竹居群内存在丰富的遗传多样性,各个天然居群间具有一定的遗传分化但分化水平并不高,主要的遗传变异存在于居群内部。     

大麻品种遗传多样性的AFLP分析

利用POPGENE 3.2软件对13个不同来源的大麻群体进行遗传多样性分析。结果显示:云南地区的大麻群体具有最高的遗传多样性水平(PPB=88.82%,He=0.3000,I=0.4571),其次为黑龙江群体(PPB=75.66%,He=0.2572,I=0.3897)。13个大麻群体的多态位点百分率(PPB)为92.11%,Nei’s总遗传多样性(Ht)为0.3837,Shannon’s信息指数I=0.5374。群体内遗传多样性(Hs)为0.1640,群体间的遗传分化系数(Gst)为0.5725,总的遗传变异中有57.25%发生在群体间,42.75%发生在群体内。根据Nei’s(1978)的方法计算了13个大麻群体间的遗传距离和遗传一致度。结果显示:各群体间的遗传一致度在0.6556～0.9258之间,其中四川群体和广西群体间具有最高的遗传一致度(0.9258);云南群体与贵州群体和四川群体间遗传一致度分别为0.9196、0.9173。所有群体中甘肃群体和山西群体遗传一致度最低为0.6556,说明大麻种内具有较大的遗传变异。     

栲树天然群体遗传结构的RAPD分析

利用RAPD分子标记对5个栲树(Castanopsis fargesii Franch.) 天然群体共计188个个体的遗传多样性和群体遗传结构进行了分析.41个随机寡核苷酸引物共检测到385个位点,其中多态位点157个,占40.78%.物种水平的Shannon多样性指数I=0.459 7,Nei基因多样度h=0.296.遗传变异分析表明,栲树群体的遗传变异主要存在于群体内,利用Shannon多样性指数估算的分化(Hsp-Hpop)/Hsp=0.047 6,遗传分化系数Gst =0.042 9,分子方差分析(AMOVA)也证实了这一结论,群体内的变异组分占了94.97%,群体间变异只占5.03%.AMOVA分析结果的显著性检验也表明,群体间及群体内个体间均呈现出显著分化(P<0.001).     

A comparison of methods for the estimation of genetic diversity in strawberry cultivars

RAPD markers were used to examine the genetic relatedness of eight strawberry cultivars released from four breeding programmes around the world. Ten random primers successfully amplified DNA fragments from each cultivar and specific fingerprints were generated from the molecular marker data. The cultivars were traced back to founding clones and the relationships between the cultivars were examined from both the molecular and the pedigree data.     

Molecular separation of genera in Cassiinae (Leguminosae), and analysis of variation in the nodulating species of Chamaecrista

Randomly Amplified Polymorphic DNA (RAPD) methods have been adapted for use as a phenetic tool on the legume tribe Cassiinae. RAPD-generated polymorphism within local populations was lower than between populations from different geographic regions, between species and genera. Examination of three Cassia species, 12 Chamaecrista species and 13 Senna species using eight primers showed the potential for separation of the nodulated/nitrogen fixing genus Chamaecrista from the previously congeneric groups Cassia and Semis. Similarly, RAPD analysis of two groups of nine Ch. rotundifolia and nine Ch. mimosoides samples using 11 primers has given separation according to both species and to geographical location. Analysis of a small sample of five Chamaecrista species from Brazil with eight primers gave separation consistent with known variations in nodule structure.     

The RAPD technique fails to detect a male-specific genetic marker in Atlantic salmon

The RAPD technique failed to detect a male-specific genetic marker in Atlantic salmon Salmo salar .     

Clonality and recombination in the arctic plant Saxifraga cernua

The circumarctic clonal plant Saxifraga cernua reproduces efficiently via bulbils, largely depends on insects for pollination and appears to set seed very rarely. However, high levels of genotypic variation observed at small spatial scales in the arctic archipelago of Svalbard have been taken as evidence of occasional sexual reproduction. Here we assess the relative contributions of mutation and recombination to random amplified polymorphic DNA variation in four populations in East Greenland and re-analyse the Svalbard data. Greater variation due to recombination was predicted in Greenland than in Svalbard, because the higher summer temperatures and longer growing season likely increase the chances for sexual reproduction. Although we observed higher levels of genotypic diversity in Greenland than in Svalbard, matrix incompatibility and linkage disequilibrium measures provided no evidence of more sexuality, suggesting differences in glacial/postglacial history. The genetic structure and spatial distribution of clones suggest that clonal migration may increase variability in local populations, which is consistent with frequent large-scale migration in this species inferred from a circumarctic analysis of chloroplast DNA haplotypes. We conclude that a combination of somatic mutations and sexual reproduction has contributed to the observed patterns of genotypic diversity in the Greenland and Svalbard populations of S. cernua , and that sexual reproduction is important in maintaining genotypic diversity, despite the rarity of observations of seed setting.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 209–217.     

Phylogenetic study of bisexual Artemia using random amplified polymorphic DNA

Study of polymorphisms in the eukaryotic genome is an important way to discover the evolutionary relationships between species. Artemia (Crustacea, Anostraca) offers a very interesting model for evolutionary studies. In fact the genus, distributed all over the world in hundreds of known biotopes, comprises both bisexual sibling species and parthenogenetic populations easily available from the Artemia Reference Center of Ghent. In spite of great interest in it and its extensive use in aquaculture, little is known about relationships between the different species and intraspecific populations. Recently it has been demonstrated that polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains in many organisms. We have used this technique to estimate the phylogenetic relationships existing between 14 populations living in the American continent, in the Mediterranean area, and in China. The principal coordinate analysis (PCO) obtained from 86 random amplified polymorphic DNA (RAPD) markers indicates that the populations analyzed can be divided into homogeneous clusters representing the four known bisexual species—the American A. franciscana and A. persimilis, the Mediterranean A. salina, and the A. species from China.     

Molecular characterization of isolates of Metarhizium from locusts and grasshoppers

The relationships between 30 strains of Metarhizium were investigated by isoenzyme analysis, PCR-RAPDs, and protease production. The strains included representatives of M. anisopliae, M. flavoviride var. flavoviride and M. flavoviride var. minus. Thirteen isolates conforming to M. flavoviride from acridoid hosts and pathogenic to locusts and grasshoppers were shown to be a single, distinctive genotype with a pan-tropical distribution. In addition, the separation of M. flavoviride var. minus as a host specific taxon distinct from European isolates of M. flavoviride was also supported. The possibility of coevolution within some insect pathogenic populations is discussed.     

Genetic diversity of the narrow endemic Allium aaseae (Alliaceae)

Low levels of genetic variability are common for a number of geographically restricted plants: these data are consistent with theoretical expectations that small populations should be genetically depauperate. However, in some species, high levels of variability have been found in rare species. Allium aaseae is a rare, narrow endemic in the foothills of the Boise Front of southwestern Idaho. Genetic variation in the rare endemic A. aaseae, and nearby populations of the more common species, A. simillimum, was examined with randomly amplified polymorphic DNA (RAPD) data. Eight populations of A. aaseae and six populations of A. simillimum (three near the range of A. aaseae, and three distant populations) of 25 individuals each were examined for this analysis. Genetic diversity as determined with RAPD markers of both species examined in this study is largely found within and not among populations. Levels of genetic diversity are high, especially for a narrow endemic species such as A. aaseae. Proportion of RAPD loci polymorphic was high in both species, although slightly higher in the more common A. simillimum. Because these higher levels of genetic variability run counter to theory, alternative explanations beyond population size must be invoked to explain the levels of genetic diversity found in this study. Possible explanations are (1) A. aaseae is only recently derived from A. simillimum, (2) hybridization between A. aaseae and A. simillimum is occurring, (3) multiple origins of A. aaseae, (4) populations of A. simillimum included in the analysis are all A. aaseae, (5) A. aaseae and A. simillimum are conspecific, and (6) an artifact of RAPD data.     

Detection of genetic diversity and selective gene introgression in coffee using RAPD markers

RAPD (randomly amplified polymorphic DNA) markers generated by arbitary decamers have been successfully employed to detect genetic polymorphisms between coffee species and between Coffea arabica genotypes. The RAPD profiles were used to construct dendrograms and these were consistent with the known history and evolution of Coffea arabica. Material originating from Ethiopia and the arabica sub-groups — C. arabica var. typica and C. arabica var. bourbon — were clearly distinguished. RAPD analysis therefore reflects morphological differences between the sub-groups and the geographical origin of the coffee material. Species-specific amplification products were also identified, but, more importantly, amplification products specific to C. canephora were identified in two C. arabica genotypes, Rume Sudan and Catimor 5175. This diagnostic product is therefore indicative of interspecific gene flow in coffee and has biological implications for selective introgressive hybridisation in coffee. Our study demonstrates the power of the polymerase chain reaction technology for the generation of genetic markers for long-lived perennial tree and bush crops.On study leave from: Universidad de San Carlos de Guatemala, Facultad de Agronomia, Ciudad Universitaria, Zona 12, Apartado Postal No. 1545, Guatemala, Central America     

Pre-germination genotypic screening using PCR amplification of half-seeds

A simple and rapid PCR-based method has been developed for determining the genotype of seeds before germination. Single half-seeds of rice (Oryza sativa L.) and wheat (Triticum aestivum L. em. Thell.) were preincubated, without grinding, in an aqueous extraction buffer. The resulting supernatants were then used in polymerase chain reaction (PCR) with oligonucleotide primers corresponding to rice single-copy sequences or a wheat microsatellite repeat. PCR products of identical size were amplified using either the half-seed extract or DNA isolated from leaf tissue. The remnant half-seeds can be maintained in ordered arrays using microtiter plates allowing the recovery of selected genotypes. Pre-germination genotypic screening of seed populations as described in this report should be useful for a variety of applications in plant breeding and genetics studies.