Assessing population genetic structure and variability with RAPD data: Application to Vaccinium macrocarpon (American Cranberry)

A method for estimating and comparing population genetic variation using random amplified polymorphic DNA (RAPD) profiling is presented. An analysis of molecular variance (AMOVA) is extended to accomodate phenotypic molecular data in diploid populations in Hardy-Weinberg equilibrium or with an assumed degree of selfing. We present a two step strategy: 1) Estimate RAPD site frequencies without preliminary assumptions on the unknown population structure, then perform significance testing for population substructuring. 2) If population structure is evident from the first step, use this data to calculate better estimates for RAPD site frequencies and sub-population variance components. A nonparametric test for the homogeneity of molecular variance (HOMOVA) is also presented. This test was designed to statistically test for differences in intrapopulational molecular variances (heteroscedasticity among populations). These theoretical developments are applied to a RAPD data set in Vaccinium macrocarpon (American cranberry) using small sample sizes, where a gradient of molecular diversity is found between central and marginal populations. The AMOVA and HOMOVA methods provide flexible population analysis tools when using data from RAPD or other DNA methods that provide many polymorphic markers with or without direct allelic data.     

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA(RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China.A captive population has been established in the Beijing Zoo since 1999.In order to determine the kinship of the offsprings in 2001,randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo.To amplify the genomic DNA of each individual,53 arbitrary primers were selected.The results of amplifica tions showed that 14 primers had clear and distinct RAPD patterns.Totally,226 amplified fragments were generated by RAPD in this study.Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male).This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds.     

Allozyme and RAPD analysis of the genetic diversity and geographic variation in wild populations of the American chestnut (Fagaceae)

Genetic variation among 12 populations of the American chestnut (Castanea dentata) was investigated. Population genetic parameters estimated from allozyme variation suggest that C. dentata at both the population and species level has narrow genetic diversity as compared to other species in the genus. Average expected heterozygosity was relatively low for the population collected in the Black Rock Mountain State Park, Georgia (He = 0.096 +/- 0.035), and high for the population in east central Alabama (He = 0.196 +/- 0.048). Partitioning of the genetic diversity based on 18 isozyme loci showed that ~10% of the allozyme diversity resided among populations. Cluster analysis using unweighted pair-group method using arithmetric averages of Rogers' genetic distance and principal components analysis based on allele frequencies of both isozyme and RAPD loci revealed four groups: the southernmost population, south-central Appalachian populations, north-central Appalachian populations, and northern Appalachian populations. Based on results presented in this study, a conservation strategy and several recommendations related to the backcross breeding aimed at restoring C. dentata are discussed.     

利用RAPD对稻蝗属昆虫亲缘关系的研究

通过20个随机引物的PCR扩增,得到了日本主要稻蝗的随机扩增多态性DNA（RAPD）图谱,根据扩增结果,计算了种间相似系数和遗传距离,建立了UPGMA系统树。结果表明,分布没有重叠、种间容易交配、能产生杂种的中华稻蝗台湾亚种与小翅稻蝗的亲缘关系最近;分布重叠的日本稻蝗与中华稻蝗台湾亚种、日本稻蝗和小翅稻蝗的亲缘关系较近。小稻蝗与其它3种稻蝗的亲缘关系较远。     

A preliminary examination of genetic variation in a peripheral population of Blanding's turtle, Emydoidea blandingii

Random amplified polymorphic DNA (RAPD) was used to compare the Nova Scotia population of Blanding's turtle (Emydoidea blandingii) with several populations from the species' main range. The Nova Scotia population is believed to have been isolated from the main range for 4000-8000 years. Cluster analysis using a neighbour-joining algorithm produced a dendrogram showing the Nova Scotia population clustering separately from those populations in the main range. Analysis of molecular variance shows 34.28% of total variance to be accounted for between the Nova Scotia population and populations in the main range. While this study is preliminary, the results suggest that the Nova Scotia population of Blanding's turtle may be important to the maintenance of genetic diversity in the species.     

Molecular differentiation of two morphological variants of Gracilaria salicornia

Xia in 1986 combined Gracilaria salicornia, G. canaliculata (G. crassa), G. cacalia and G. minor into one species: G.salicornia. Two morphological variants of G. salicornia were collectedfrom different localities in Malaysia. Variant A collected from Morib,Selangor grew on the roots of Avicennia. The samples showed absenceof main axis; segmented constrictions throughout; cylindrical or slightlycompressed thalli. Variant B was collected from the mudflats of TanjungTuan, growing on rocks, coral or forming mats on the mud. Plants showedabsence of main axis; segments were not constricted throughout the plant(if present only slightly articulated at the upper part), branching wasdichotomous or irregular; cylindrical or slightly compressed thalli. Thetechnique of Random Amplified Polymorphic DNA analysis (RAPD) wasused to investigate molecular characteristics of the two variants. Out ofsixty Operon primers that were screened, four primers, OPA 1, OPA 10,OPA 11 and OPK 7 were able to give polymorphism. The fingerprintsgenerated were stable and reproducible on repeated analysis. The DNAfingerprints generated were visually analysed and clustering analysis wascarried out using GelCompar 4.0. The matrix of similarities was based onthe Dice coefficients (S_D) and the cluster analysis was carried outusing the unweighted pair group method using arithmetic averages(UPGMA). DNA analysis showed that two primers (OPA 01, CAGGCCCTTC and OPK 07, AGCGAGCAAG) were able to differentiate the two variants.     

The effects of regeneration by fragmentation upon clonal diversity in the tropical forest shrub Poikilacanthus macranthus: random amplified polymorphic DNA (RAPD) results

Poikilacanthus macranthus (Acanthaceae), like many shrubs in the neotropical cloud forest of Monteverde, Costa Rica, is capable of regeneration through stem and twig fragments. Our field sampling indicated that reproduction in P. macranthus is primarily through fragmentation in the Monteverde region. Random amplified polymorphic DNAs (RAPDs) were employed to examine clonal diversity in four populations of P. macranthus , and two primers yielded 11 variable markers that were used to identify genets. Sixty-eight multilocus genotypes were identified out of 277 plants sampled. Clonal diversity values were higher than the average for clonal plant species, as the number of genets per ramet was 0.25, while the mean D value was 0.87 and the mean E value was 0.84.     

Application of polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers in the molecular identification of selected Sargassum species (Phaeophyta,Fucales)

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used for the molecular characterisation and identification of Sargassum spp. A total of 17 samples of Sargassum (Sargassaceae, Fucales) was obtained from various localities around Peninsular Malaysia and Singapore. On the basis of morphological characteristics, the samples were tentatively grouped into five species: Sargassum baccularia, S. glaucescens, S. oligocystum, S. polycystum and S. siliquosum. By RAPD-PCR, five of 31 random primers tested generated reproducible amplification products, and polymorphic loci were detected by four of them (OPA02, OPA03, OPA04, OPA13). The RAPD-PCR profiles did not correlate with the morphological grouping into five species and extensive variation was detected between different isolates of the same species. A 450 base pair fragment generated using OPA13 was detected in 12 of 17 samples of Sargassum. This fragment was also present in profiles from Turbinaria (Sargassaceae). This study suggests that RAPD-PCR is useful in discriminating individual samples of the genus Sargassum and in developing fingerprints for them.     

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA (RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China. A captive population has been established in the Beijing Zoo since 1999. In order to determine the kinship of the offsprings in 2001, randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo. To amplify the genomic DNA of each individual, 53 arbitrary primers were selected. The results of amplifications showed that 14 primers had clear and distinct RAPD patterns. Totally, 226 amplified fragments were generated by RAPD in this study. Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male). This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds. __________ Translated from Journal of Beijing Normal University (Natural Science), 2005, 41(5): 507–509 [译自: 北京师范大学学报 (自然科学版), 2005, 41(5): 507–509]     

Ex situ cultivation affects genetic structure and diversity in arable plants

Worldwide, botanical gardens cultivate around 80,000 taxa, corresponding to approximately one‐quarter of all vascular plants. Most cultivated taxa are, however, held in a small number of collections, and mostly only in small populations. Lack of genetic exchange and stochastic processes in small populations make them susceptible to detrimental genetic effects, which should be most severe in annual species, as sowing cycles are often short. In order to assess whether ex situ cultivation affects genetic diversity of annuals, five annual arable species with similar breeding systems were assessed with 42 in situ populations being compared to 20 ex situ populations using a random amplified polymorphic DNA (RAPD) analysis approach. Population sizes tended to be lower under ex situ cultivation and levels of genetic diversity also tended to be lower in four of the five species, with differences being significant in only two. Ex situ populations showed incomplete representation of alleles found in the wild. The duration of cultivation did not indicate any effect on genetic diversity. This implies that cultivation strategies resulted in different genetic structures in the garden populations. Although not unequivocally pronounced, differences nonetheless imply that conservation strategies in the involved gardens may need improvement. One option is cold storage of seeds, a practice that is not currently followed in the studied ex situ collections. This may reflect that the respective gardens focus on displaying living plant populations.     

Genetic variation of Usnea filipendula (Parmeliaceae) populations in western Germany investigated by RAPDs suggests reinvasion from various sources

Random amplified polymorphic DNA (RAPD) markers were characterized for 25 specimens of Usnea filipendula to evaluate the genetic diversity of populations reinvading formerly uninhabited regions in Northrhine-Westphalia due to decreasing sulfur dioxide levels. With six 10-mer randomly amplified polymorphic DNA (RAPD) primers, a 66 character by 25 specimens matrix was generated. Phenetic analysis (UPGMA) showed no obvious groupings. The reinvading populations are distributed over the phenogram and are not genetically closely related. The results suggest that the reinvading populations of this usually sterile species are derived from different sources and do not consist of a particular clone capable of re-entering the area.     

Identification and Assessing the Cultivars of Laminaria Lamx.(Phaeophyceae) with Molecular Markers

Molecular markers were used to identify and assess cultivars ofLaminaria Lamx. and to delineate their phylogenetic relationships. Random amplified polymorphic DNA (RAPD) analysis was used for detection. After screening, 11 primers were selected and they yielded 133 bands in all, of which approximately 99.2% were polymorphic. The genetic distances between gametophytes ranged from 0.412 to 0.956.Two clusters were formed with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on the simple matching coefficient. All cultivars of Laminaria japonica Aresch. used for breed ing in China fell into one cluster. L. japonica from Japan, L. saccharina (L.) Lam., and L. angustata Kjellm.formed the other cluster and showed higher genetic variation than L. japonica from China. Nuclear ribosomal DNA (rDNA) sequences, including internal transcribed spacers (ITS1 and ITS2) were studied and aligned. The nucleotides of the sequences ranged from 634 to 668, with a total of 692 positions including ITS1, ITS2, and the 5.8S coding region. The phylogenetic tree obtained by the neighbor-joining method favored, to some extent, the results revealed by RAPD analysis. The present study indicates that RAPD and ITS analyses could be used to identify and assess Laminaria germplasm and to distinguish some species and, even intraspecies, in Laminaria.     

Micropropagation and genetic stability of a Dendrobium hybrid (Orchidaceae)

Summary Dendrobium hybrids have great economic importance in a number of countries. Asymbiotic seed germination and the conventional vegetative method have been commonly used by growers to propagate these plants. To overcome somaclonal variation, which is commonly exhibited by Dendrobium (Nobile group) when micropropagated from protocorm-like bodies, a protocol for propagating Dendrobium Second Love in vitro using axillary buds in the presence of thidiazuron was developed. Random amplified polymorphic DNA analysis was also carried out to check for possible genetic alterations in plants originating from six consecutive subcultures. The results revealed that the established protocol was efficient for the in vitro cloning of this orchid hybrid and the plants obtained from the six subcultures did not exhibit any type of polymorphism.     

Male-specific DNA in the dioecious species Atriplex garrettii (Chenopodiaceae)

The mechanism of sex determination in dioecious species of the genus Atriplex (Chenopodiaceae) has not been determined. This paper reports the discovery of a male-specific DNA fragment in the diploid dioecious species A. garrettii. DNA samples extracted individually from ten male and ten female plants were bulked by sex. Random amplified polymorphic DNA (RAPD) fragments were generated in the two bulks in order to identify markers that were polymorphic between male and female plants. A total of 158 decamer primers were tested. A 2075 base-pair (bp) male-specific DNA fragment generated with the OPAF-14 primer was identified. The fragment was cloned and partially sequenced and 24-mer primers that exclusively amplified this fragment were constructed. When 124 male plants, 126 female plants, and one hermaphroditic plant were tested individually, the male-specific 2075-bp DNA fragment was present in the hermaphrodite and all but one of the male plants, and was absent in all female plants. A smaller DNA fragment (~1800 bp) that was homologous to the 2075-bp fragment was amplified from the single male plant that lacked the 2075-bp fragment. Cytogenetic analysis revealed no apparent heteromorphic sex chromosomes. These observations suggest that sex determination in A. garrettii is genetic, with no evidence of heteromorphic sex chromosomes.     

Assessment of random amplified polymorphic DNA (RAPD) for determining the origin ofYoungia koidzumiana Kitamura (compositae)

We determined the parental species ofYoungia koidzumiana (a natural interspecific hybrid) using PCR and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. These markers, generated by three primers, were sufficient to distinguishYoungia sonchifolia, Youngia denticulata, Youngia chelidoniifolia, andY. koidzumiana. The electrophoresis profiles of the amplified products from each of the four species were then compared. Three primers produced a total of 42 scorable markers; nine were specific markers forY. denticulata andY. chelidoni-ifolia. The length of the amplified DNA fragments ranged from 370 to 2500 b p. The three primers revealed polymorphic bands, which were indicators of the parental species ofY. koidzumiana. These bands showed a combination of specific profiles forY. denticulata andY. chelidoniifolia. Our results also were comparable to the data obtained for flowering times, floret numbers, and chromosome numbers of the four species. Therefore, we suggest thatY. koidzumiana is a hybrid betweenY. denticulata andY. chelidoniifolia}, and that RAPD markers are well suited for assessing the origins of plant species.     

Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.