The use of nuclear DNA molecular markers for studying speciation and systematics as exemplified by the “Lacerta agilis complex” (Sauria: Lacertidae)

Four types of nuclear DNA markers identified by the taxonprint, RAPD, and IMP (Inter-MIR-PCR) methods, and the nucleotide sequences of satellite DNA monomers have been used to analyze the molecular genetic similarity between some populations, subspecies, and species of lizards combined into the group Lacerta s. str., as well as representatives of some other genera. The notions on the systematics and phylogeny of this group based on morphological and zoogeographic criteria have been compared to the conclusions based on molecular genetic data. The genus and species subdivisions of populations based on nuclear molecular markers and morphological characters generally agree with each other, the degree of genetic differences being correlated with the taxonomy suggested by zoomorphologists. The degree of differences between the subspecies of one of the species studied, Lacerta agilis, varies depending on the molecular markers used: according to the results of RAPD analysis, all subspecies substantially differ from one another, the variation within populations being small; with respect to other markers, the differences are smaller and not equivalent. The existence of the so-called eastern and western clades of this species earlier assumed by other researchers on the basis of mtDNA and morphological data has been confirmed. There are no distinct gradations exceeding individual variation in 14 populations of L. agilis exigua (the eastern clade) with respect to IMP markers, although these populations inhabit a vast area from the Ural Mountains to the Kabardino-Balkar Republic (the Caucasus). These data suggest that the subspecies has been rapidly spreading northwards since the Pleistocene glaciation (about 15,000 years ago).     

RAPD-marking of genomes of representative Hordeum species

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum. High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgare sensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between the Hordeum species, between the H. spontaneum populations, and between regional H. vulgare cultivars and a dendrogram was constructed.     

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。     

RAPD Analysis of the Genome in Species of the Genus Hordeum

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum.High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgaresensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between theHordeumspecies, between the H. spontaneumpopulations, and between regional H. vulgarecultivars and a dendrogram was constructed.     

Comparative molecular and phytochemical investigation of Leontodon autumnalis (Asteraceae, Lactuceae) populations from Central Europe

Previous analyses of Leontodon autumnalis L. revealed the existence of two chemotypes. In the current study molecular and phytochemical methods were combined to investigate 24 Central European populations of L. autumnalis. The focus of this study was the correlation of molecular and phytochemical characters at the intraspecific level. DNA fingerprint profiles of 183 individuals were obtained by random amplified polymorphic DNA (RAPD) providing 77 molecular markers. Contents of phenolics and sesquiterpenoids of flowering heads and sub-aerial parts were quantified by HPLC-DAD analyses. HPLC results were evaluated by principal component analysis. Geographic distribution of the two detected chemotypes partially overlapped. Phylogenetic groupings displayed in an unrooted neighbor-joining tree calculated from the RAPD data matrix were correlated with the geographical origin of the plant material. However, genetic profiles neither correlated with the two chemotypes nor with the morphologically based subspecies of L. autumnalis recognized by some authors. The presented data imply that the morphotypes are of multiple origins or due to different ecological growing conditions rather than genetically determined and that phytochemical races are induced by a limited number of genetical differences, which might have occurred independently in different lineages of the L. autumnalis group.     

Molecular characterization and RAPD analysis of Juniperus species from Iran

The genus Juniperus L. (Cupressaceae), an aromatic evergreen plant, consists of up to 68 species around the world. We classified five species of Juniperus found in Iran using molecular markers to provide a means for molecular identification of Iranian species. Plants were collected (three samples of each species) from two different provinces of Iran (Golestan and East Azarbayejan). The DNA was extracted from the leaves using a Qiagen Dneasy Plant Mini Kit. Amplification was performed using 18 ten-mer RAPD primers. Genetic distances were estimated based on 187 RAPD bands to construct a dendrogram by means of unweighted pair group method of arithmetic means. It was found that J. communis and J. oblonga were differentiated from the other species. Genetic distance values ranged from 0.19 (J. communis and J. oblonga) to 0.68 (J. communis and J. excelsa). Juniperus foetidissima was found to be most similar to J. sabina. Juniperus excelsa subspecies excelsa and J. excelsa subspecies polycarpos formed a distinct group.     

Identification of metalliferous ecotypes of Cistus ladanifer L. using RAPD markers

The genetic diversity of Cistus ladanifer ssp. ladanifer (Cistaceae) growing on ultramafic and non-ultramafic (basic and schists) soils in the NE of Portugal was studied in order to identify molecular markers that could distinguish the metal-tolerant ecotypes of this species. Random Amplified Polymorphic DNA (RAPD) markers were used in order to estimate genetic variation and differences between populations. The RAPD dataset was analysed by means of a cluster analysis and an analysis of molecular variance (AMOVA). Our results indicate a significant partitioning of molecular variance between ultramafic and non-ultramafic populations of Cistus ladanifer, although the highest percentage of this variance was found at the intra-population level. Mantel's test showed no relationship between inter-population genetic and geographic distances. A series of RAPD bands that could be related to heavy metal tolerance were observed. The identification of such markers will enable the use of Cistus ladanifer in phytoremediation procedures.     

Discordance in the distribution of markers of different inheritance systems (nDNA,mtDNA, and Chromosomes) in the superspecies complex <Emphasis Type="Italic">Mus musculus</Emphasis> as a result of extensive hybridization in primorye

The genetic structure of eight Mus musculus L. populations in Primorskii krai was studied with the use of taxon-specific markers of different inheritance systems: nDNA (RAPD), mtDNA (D-loop), and chromosomes. The results obtained demonstrate that although the compared nuclear marker characteristics (nDNA and chromosomes) have the same basis they are not linke with each other and, moreover, are often mutually inconsistent. Discordance in the inheritance of the marker characteristics in most of the animals studied is a result of extensive hybridization involving two to four house mouse subspecies. To identify taxonspecific nuclear markers revealed by RAPD, some RAPD PCR products were cloned, and their localization on chromosomes was determined. It was found that some fragments similar in size consist of two different comigrating sequences that are localized on different chromosomes and belong to different subspecies. All sequenced anonymous markers are localized in protein-coding genes. The functions of genes containing the marker sequences have been established. Differences in the taxon-specific RAPD fragments are associated with changes in the structure of important functional genes, and this can be considered as a significant genetic marker.     

Genetic diversity of Mexican brook lamprey <Emphasis Type="Italic">Lampetra (Tetrapleurodon) geminis</Emphasis> (Alvarez del Villar, 1966)

Lampreys are the only surviving representatives of the oldest known vertebrates. The Mexican lamprey L. geminis (nonparasitic), is particularly interesting, because it is an endemic, biogeographical relict, and a threatened species. RAPD markers were used to describe genetic diversity in L. geminis A total of 77 specimens were collected from five populations, three in the R'o Grande de Morelia-Cuitzeo basin and two in the R'o Duero-Lerma-Chapala basin, Mexico. Eighty-eight RAPD markers were obtained from eight primers. Genetic diversity within each population was estimated using Shannon's index (S), heterozygosity (H) and gene diversity (h). These estimates revealed significant variation within populations, although a variance homogeneity test (HOMOVA) showed no significant differences among populations or between basins. Nei genetic distance values indicate a low genetic differentiation among populations. Analysis of molecular variance (AMOVA) indicates that most of the genetic diversity occurs within populations (91.4%), but that a statistically significant amount is found among populations (P0.001). Principal coordinates and cluster analyses of RAPD phenotypes show that specimens are not grouped by geographical origin. The genetic diversity found within L. geminispopulations may be explained by its breeding system and an overlapping of generations. The scarce genetic differentiation among populations is likely to the low rate of DNA change that characterizes the lamprey group.     

国产甘草属植物的RAPD分析及其分类学研究

应用RAPD技术,探讨甘草属(G ly cy rrh iza L.)13种1变种30个植物类群的遗传差异和几个争议种的分类地位。从60个随机引物中筛选出14个多态性好的引物进行RAPD实验,DNA片段的二态数据用U PGM A聚类法构建系统发育树。共扩增出250条带,多态性带204条,约占总数的81.7%。聚类结果显示RAPD分子标记构建的系统发育树与经典分类系统一致。甘草属植物具有丰富的遗传多样性,同种内不同居群间的遗传分化较大。黄甘草、胀果甘草、乌拉尔甘草三者亲缘关系较近,平卧甘草与粗毛甘草存在很大的遗传差异,作为独立种较合理。RAPD标记可为甘草属植物的系统分类研究提供分子生物学依据。     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Variability of multilocus DNA markers in populations of the Siberian (Capreolus pygargus Pall.) and European (C. capreolus L.) roe deer

Two types of nuclear DNA markers, M13 minisatellites and RAPD, were used to examine intraspecific and interspecific variation in closely related roe deer species, Capreolus capreolus L. and C. pygargus Pall. The roe deer populations studied were highly polymorphic for minisatellite DNA markers (S = 0.12-0.36). Heterozygosities of the RAPD loci were 0.185 (Russian Far East), 0.145, 0.131, 0.088 (Cis-Ural), and 0.06 (France). They correlated with karyotypic variation of B chromosomes (r = 0.975, P < 0.02; Spearman's correlation coefficient r = 1, P = 0.1 x 10(-5)), which indicated a contribution of microchromosomes to genetic variation of the species. The genetic distance D between the closely related species C. capreolus and C. pygargus was 50 times greater than the distance between populations within a species. The estimates of heterozygosity and genetic distance between local populations of Cis-Ural and the Far East suggest their specific spatial organization within this geographical range and reveal features of their historical development.     

Population genetic variation in rare and endangered Iliamna (Malvaceae) in Virginia

Random amplified polymorphic DNA (RAPD) markers were used as input for an analysis of molecular variance (AMOVA), homogeneity of molecular variance analysis (HOMOVA), and cluster analysis to describe the population genetic structure of Iliamna corei, a federally endangered plant located only in Virginia, and I. remota , a rare plant in Virginia, Indiana, and Illinois. The analysis was performed to help clarify the taxonomic relationship between the two closely related species. We analysed four clones in the only known population of I. corei , breeding stock derived from seeds originating from the population site, and three I. remota populations in Virginia. Eighty-five percent of screened primers revealed DNA polymorphisms in Iliamna. Ninety-nine informative markers were generated using seven primers. No significant statistical differences (at P = 0.05) in RAPD variation was found between species (24% of variance) using the AMOVA procedure. However, within species/among populations (31 % of the variance) and within populations (45% of the variance) there were significant differences (P < 0.002). An unweighted paired group method using arithmetic averages (UPGMA) cluster analysis showed the federally endangered I. corei population to be genetically distinct from the apparently recently introduced (in Virginia: ∼ 100 ybp) I. remota. The lack of significant differences from the AMOVA and the high number shared bands between I. corei and I. remota suggest that I. corei may be more appropriately classified as a subspecies of I. remota. Iliamna corei plants in the natural population were genetically similar to one another while the I. corei breeding stock plants and I. remota plants were genetically relatively diverse.     

Inter- and intraspecific genetic variation inHippophae (Elaeagnaceae) investigated by RAPD markers

Genetic diversity has been investigated by the application of molecular markers in, for the first time, all the taxa recognised in recent treatises of the genusHippophae. RAPD (random amplified polymorphic DNA) analyses were conducted with 9 decamer primers, which together yielded 219 polymorphic markers. We found 16 fixed RAPD markers, i.e. markers that either occurred in all plants of a population or were absent from all plants. Several of these markers were useful for analysis of interspecific relationships, whereas others can be considered as taxon-specific markers. Clustering of taxa and populations in our neighbour-joining based dendrogram was in good agreement with some recently suggested taxonomic treatises ofHippophae. Amount and distribution of genetic variability varied considerably between species. Partitioning of molecular variance withinH. rhamnoides supported earlier findings that a considerable part of the total variance resides among subspecies (59.6%) Within-population variability also differed considerably. Percentage polymorphic RAPD loci and Lynch and Milligan within-population gene diversity estimates showed relatively high values for some species close to the geographic centre of origin in Central Asia, e.g.H. tibetana and the putatively hybridogenousH. goniocarpa. Spatial autocorrelation analyses performed on 12 populations ofH. rhamnoides revealed positive autocorrelation of allele frequencies when geographic distances ranged from 0 to 700 km, and no or negative autocorrelation at higher distances. At distances between 700 and 1900 km, we observed deviations from the expected values with strongly negative autocorrelation of allele frequencies. A corresponding relationship between geographic and genetic distances could not be found when the analysis instead was based on one population from each of 8 species.     

Discordance in the distribution of markers of different inheritance systems (nDNA, mtDNA, and chromosomes) in superspecies complex Mus musculus as a result of extensive hybridization in Primor'e

The genetic structure of eight Mus musculus L. populations in Primorskii krai was studied with the use of taxon-specific markers of different inheritance systems: nDNA (RAPD), mtDNA (D-loop), and chromosomes. The results obtained demonstrate that although the compared nuclear marker characteristics (nDNA and chromosomes) have the same basis they are not linke with each other and, moreover, are often mutually inconsistent. Discordance in the inheritance of the marker characteristics in most of the animals studied is a result of extensive hybridization involving two to four house mouse subspecies. To identify taxon-specific nuclear markers revealed by RAPD, they were cloned and sequenced, and their localization on chromosomes was determined. It was found that some fragments similar in size consist of two different comigrating sequences that are localized on different chromosomes and belong to different subspecies. All sequenced anonymous markers are localized in protein-coding genes. The functions of genes containing the marker sequences have been established. Differences in the taxon-specific RAPD fragments are associated with changes in the structure of important functional genes, and this can be considered as a significant genetic marker.     

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson''s correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.     

中国舞毒蛾六个地理种群的RAPD分析及SCAR标记构建

舞毒蛾Lymantria dispar L.是世界性农林害虫, 包含不同的亚种, 其中亚洲舞毒蛾的雌蛾具有较强的飞行能力, 已成为国际性的重要检疫性有害生物。然而, 不同舞毒蛾亚种及种群间形态难辨, 因此采用传统的手段鉴别舞毒蛾亚种种群是很困难的。本研究首先采用RAPD标记分析了中国舞毒蛾6个地理种群的遗传多态性。结果表明, 所检测的舞毒蛾种群的遗传分化系数G_st为0.7571, 由此推算出的平均有效迁移数（基因流参数）N_em为0.1604, 说明不同舞毒蛾种群间的遗传分化程度较高, 缺乏广泛的基因流动。本研究在RAPD遗传分析基础之上, 筛选出了4个舞毒蛾种群的特异性遗传位点, 然后对这些特异性位点进行了克隆测序、 序列分析和位点特异性引物设计。结果表明, 其中2个舞毒蛾种群的位点特异性引物可产生序列特征性扩增区域（SCAR）标记。经验证, 这些标记可被用来鉴别特定的舞毒蛾地理种群, 因此有助于对这些舞毒蛾地理种群的分布与扩散进行监测。     

Genetic (RAPD) Diversity between Oleria onega agarista and Oleria onega ssp. (Ithomiinae, Nymphalidae, Lepidoptera) in North-Eastern Peru

Oleria onega agarista Felder and Felder and Oleria onega ssp. nov. are two Ithomiinae subspecies from north-eastern Peru, that differ for some morphological and behavioural traits. Two contact zones are known near the town of Tarapoto: Ahuashiyacu, where both subspecies cohabit but do not seem to hybridise, and Estero (near the village of Shapaja), where they apparently hybridise. Genetic differences between the two subspecies and between populations were investigated with random amplified polymorphic DNA (RAPD) markers. Both Cluster and Principal Coordinates Analyses (CCoA and PCoA) performed using these data, provided a clear but weak discrimination between the two subspecies. Genetic diversity is much higher within the populations than between them. Moreover, the geographically more distant populations are grouped together by the genetic data. Morphological traits on the wing patterns of the hybrids are intermediary between the two butterflies subspecies, while RAPDs data place them closer to O. onega agarista than to O. onega ssp. The individuals of the Ahuashiyacu population are clearly separated into two groups, those of O. onega ssp. and O. onega agarista, by both morphology and RAPDs data. Moreover, none of those individuals show RAPD similarity with the hybrids, suggesting that hybridisation has not occurred in this population.     

Conservation genetics of the rare and endangered Leucopogon obtectus (Ericaceae)

Leucopogon obtectus Benth. is a declared rare species found in the kwongan vegetation in Western Australia. Plants on a mineral sand mine and the rehabilitation area are subject to disturbance. Genetic diversity was examined within and among all known populations using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs) for conservation. Both molecular markers revealed a high percentage (> 89%) of polymorphic markers and a high mean genetic distance among individuals (D = 0.3). Analysis of molecular variance showed that 86.7% (RAPD) and 89.7% (AFLP) of variability was partitioned among individuals within populations. Exact tests showed no significant population differentiation. The analyses indicated that L. obtectus exhibits high levels of genetic diversity despite small population sizes. The high levels of variability among individuals and the lack of clear population differentiation suggest that this species comprises a single, genetically diverse group. Conservation and management of L. obtectus should concentrate on maintaining the high levels of genetic variability through mixing genotypes and promoting outcrossing.     

Implications of ITS sequences and RAPD markers for the taxonomy and biogeography of the Oxytropis campestris and O. arctica (Fabaceae) complexes in Alaska

Taxonomic consensus is lacking on the Oxytropis arctica and O. campestris species complexes, two polyploid complexes found in the interior and arctic areas of Alaska. One classification has emphasized flower size, whereas flower color is considered a key diagnostic character in another classification. Our analyses of internal transcribed spacer (ITS) sequences and random amplified polymorphic DNA (RAPD) markers provided no support for either classification system. The trees generated from ITS sequences and the phenogram derived from RAPD markers suggest that most recognized taxa in the two complexes are probably polyphyletic, including O. arctica var. barnebyana, which is listed as threatened in Alaska. The only consistent pattern detected by both types of molecular markers was a geographic split dividing the northeastern arctic populations from most other populations (48.60-55.03% in AMOVA analyses). This genetic subdivision probably reflects a Pleistocene barrier formed by the northern coastal ice shield. Our molecular data, in conjunction with the previously reported variation of ploidy levels in these groups, suggest a scenario of recent and multiple origins of polyploidy. It is possible that most Alaskan populations of these two complexes are best referred to as a single taxonomic species despite morphological differentiation within the complexes.