Combined Random Amplified Polymorphic DNA Research Articles

Phylogeny Study of 20 Selected Species of Zingiberaceae from Ex situ Collections in Peninsular Malaysia

Zingiberaceae is widely distributed in Malaysia, with 750 species and 31 genera. This family comprises a different number of subfamilies and genera according to different taxonomic classification methods — classical taxonomy: one subfamily and four tribes vs molecular taxonomy: four subfamilies and six tribes. However, the taxonomic classification of Zingiberaceae is still debated, especially the classical taxonomy. It is due to some Zingiberaceae species showing cryptic morphologies that make it difficult to classify them through classical taxonomy, which refers to the unique morphological characteristics of a tribe/species. Therefore, accurate taxonomic classification is required by using a molecular approach. In this study, 20 selected species of Zingiberaceae collected from the Agricultural Conservatory Park, Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM) were taxonomically classified using a molecular method with the help of three random amplified polymorphic DNA (RAPD) and three inter simple sequence repeat (ISSR) markers until the tribe level. The combined RAPD and ISSR unweighted pair group method with arithmetic mean (UPGMA) phylogenetic tree was comparable to Zingiberaceae’s current molecular and classical taxonomy. The 20 selected species were grouped into three tribes (Alpinieae, Zingiberaceae, and Globbeae). This finding has contributed additional biological information to better manage the 20 Zingiberaceae species in the Agricultural Conservatory Park, IBS, UPM. Further studies are needed to explore the genetic diversities and properties of Zingiberaceae species.

Genetic diversity of avocado (Persea americana Mill.) germplasm in Vietnam using RAPD and ISSR molecular markers

Avocado is a significant cash crop in Vietnam, while little is known about its genetic diversification. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were performed to investigate the genetic diversity of twenty-eight imported and domestic avocado cultivars being maintained at Western Highlands Agriculture and Forestry Science Institute, Dak Lak, Vietnam. 18 RAPD and 15 ISSR primers produced 3183 and 2807 scorable bands, of which 83.92% and 71.72% were polymorphic, respectively. The RAPD markers exhibited an average PIC of 0.27 and Rp index of 12.63 whereas the mean PIC and Rp values of ISSR primers were 0.21 and 13.37, respectively. The correlation between RAPD and ISSR markers was low (r = 0.338), suggesting that the ability to resolve genetic variation among varieties may relate to the number of detected polymorphisms rather than the marker employed. Nevertheless, the correlation between ISSR and combined data was high (r = 0.791) and the correlation between RAPD and combined matrices was even higher (r = 0.897). This indicates that RAPD markers have slightly higher efficiency over ISSR to resolve genetic variation among 28 avocado cultivars. High genetic diversity among 28 avocado cultivars was revealed with the Jaccard’s similarity coefficient ranging from 0.623 to 0.913 based on combined data analysis. The UPGMA dendrogram generated from combined RAPD and ISSR data grouped Vietnamese domestic and imported avocado cultivars into three different groups at 72% similarity. These results could be applied to the avocado conservation and breeding programs

Sequence-characterized amplified region markers and multiplex-polymerase chain reaction assays for kiwifruit cultivar identification

A simple and reliable multiplex polymerase chain reaction (PCR) method was developed using sequence-characterized amplified region (SCAR) markers to identify kiwifruit (Actinidia spp.) cultivars. We observed 183 different polymorphic random amplified polymorphic DNA (RAPD) markers using 23 arbitrary primers in 30 kiwifruit cultivars. The number of polymorphic bands per primer varied from five (OPM-15, OPN-05, and OPN-19) to 12 (OPG-01), with an average of eight. Fifty-eight cultivar-specific polymorphic RAPD fragments were converted into 16 SCAR markers after validating their specificity. Among them, 15 SCAR markers retained the dominant behavior of the original RAPD fragment, whereas the KA11_774 SCAR marker showed a co-dominant polymorphism. Based on RAPD and SCAR data sets, cluster analyses were performed with the unweighted pair-group method of arithmetic averages. The mean genetic similarities were estimated as 0.703 and 0.677 using the RAPD and SCAR markers, respectively. Thirty kiwifruit cultivars were divided into three clusters for RAPD analysis and into two clusters for SCAR analysis, based on a similarity value of 0.67. Overall, the kiwifruit cultivars with a similar breeding background tended to cluster together. Therefore, RAPD and SCAR markers could be successfully applied to assay genetic relationships for kiwifruit cultivar identification. Cluster analysis of combined RAPD and SCAR markers showed very similar results when compared to the RAPD analysis in dendrogram form. The kiwifruit cultivars tested in this study could be identified using only 10 different SCAR markers (KB08_182, KK08_287, KG19_295, KN15_316, KG01_411, KF07_433, KG09_468, KG09_477, KL01_764, and KA11_774). Furthermore, we optimized SCAR markers for simultaneous identification of the 30 kiwifruit cultivars by multiplex PCR, using two sets of five SCAR primer pairs. The developed SCAR markers and multiplex PCR approach facilitate efficient identification of kiwifruit cultivars.

Characterization of Xanthomonas citri subsp. CITRI isolated from grapefruit in Iran

Xanthomonas citri subsp. citri (Xcc) strains, obtained from cankers of grapefruit trees, were characterized by phenotypic, genotypic and pathogenicity features. In addition, some strains from Mexican lime were isolated to compare with Xcc strains from grapefruit. Specific polymerase chain reaction (PCR) analyses with three primer pairs and phylogenetic analysis based on sequencing of two housekeeping genes identified all Iranian isolates as Xcc-A*. The host range of the pathogen was assessed on four Citrus spp. Based on pathogenicity test, Iranian strains were divided into two pathogenic groups. The first group, corresponding to strains from Mexican lime, was only pathogenic to Mexican lime, whereas the second group, corresponding to strains from Mexican lime and grapefruit, was pathogenic to Mexican lime and grapefruit. However, reference strain was pathogenic to Mexican lime, grapefruit, tangerine and sweet lime. An insight into the genetic population features was attempted by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis. The combined RAPD and ERIC-PCR fingerprints divided the strains into four clusters/fifteen haplotypes. Strains isolated from grapefruit and some strains isolated from Mexican lime were grouped in cluster П. Based on our results, we determined that the sources of contamination of grapefruit trees were probably neighboring infected Mexican lime trees. To our knowledge, this is the first report of occurrence of citrus canker of grapefruit in the Fars province of Iran.

Genetic Diversity Analysis of Hedychium Species Based on RAPD and ISSR Markers

The present study reports for the first time the genetic diversity assessment of Hedychium species collected from different parts of Manipur using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers. Eleven RAPD and five ISSR markers produced 271 polymorphic fragments with an average polymorphism of 96.95% between the eleven Hedychium species and similarity coefficients ranged from 0.19 to 0.44 with an average of 0.30, which in turn, indicates high genetic diversity. Cophenetic correlation coefficients between the dendrogram and the combined similarity matrices were moderately significant (r = 0.67). UPGMA clustering based on Jaccard’s similarity coefficient using the combined RAPD and ISSR markers placed the accessions into three distinct clusters with respect to their morphological characteristics and geographical location. Both RAPD and ISSR markers were efficient in distinguishing the eleven Hedychium species, however, ISSR markers were observed to be better in displaying average polymorphism (99%) than RAPD (96.03%). The study has contributed in understanding the genetic diversity and gene pool conservation of Hedychium species.

Species delimitation in the genus Tamarix: Morphological and molecular data<br />

Tamarix (Tamaricaceae) is an ancient genus in Asia that contains about 54 to 90 species with unresolved taxonomy. Tamarix has major center of speciation in the Pakistan-Afghanistan-Iran-Turkmenistan-S-Kazakhstan-W-China area and in the eastern Mediterranean area. Tamarix species are highly plastic and tolerant to adversity being able to occupy different habitats, thus showing a wide range of ecological variations. Tamarix species have commercial value in the landscape and horticultural trade and are planted for erosion control and as wind breaks. In the genus Tamarix, many species are morphologically very similar probably due, in part, inter-specific hybridization. Tamarix species live in sympatry in Sistan and Baloochestan province of Iran and we have no report on the number of species and their genetic structure. Therefore, we performed morphometric study to delimit the species growing in these two provinces and also used combined ISSR (Inter simple sequence repeat) and RAPD (Random amplified polymorphic DNA) molecular markers to study their genetic structure. Morphological analyses separated plants of 6 studied species in different groups, indicating that morphological characters used can delimit Tamarix species. We also encountered some plants having intermediate morphological characters. AMOVA and Hickory test showed significant genetic differences of the studied species and Mantel test showed significant correlation between genetic distance and geographical distance. STRUCTURE analysis and population assignment test revealed some degree of genetic admixture among the studied species. Assignment tests showed that gene flow mainly occurred among the species that were growing in neighborhood regions. This genetic admixture may be the reason for intermediate morphological characters observed in some the plants.

Diversity analysis of green gram (Vigna radiata (L.) Wilczek)through morphological and molecular markers

Twenty-three genotypes of green gram (Vigna radiata (L.) Wilczek) were collected to determine the extent of genetic diversity through morphological characters as well as random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and simple sequence repeat (SSR) fingerprinting results. Analysis of variance revealed significant mean square values that indicated a substantial degree of genetic variability among the genotypes. Significant genetic variance was observed for days to 50% maturity, days to maturity, plant height (cm), number of branches/plant, number of pods/plant, pod length (cm), 1000-seed weight (g), seed yield/plant (g), biological yield (g), and harvest index (%) during cultivation in three different environments. Hierarchical Euclidean and cluster analysis based on 10 morphological characteristics grouped all 23 genotypes into three divergent clusters. RAPD (15), ISSR (13), and SSR (10) markers produced a total of 216 bands, of which 190 exhibited polymorphism. The similarity coefficients were significant for all markers; however, they were higher in ISSR than in RAPD or SSR. In the UPGMA dendrogram based on the combined morphological, RAPD, ISSR, and SSR results, the 23 genotypes were divided into three main clusters. The present study revealed that morphological and molecular markers may be successfully utilized for determining genetic diversity and relationships in green gram genotypes and could be used in green gram breeding programs.

Assessment of genetic diversity in Pithecellobium dulce (Roxb.) Benth. germplasm using RAPD and ISSR markers

Pithecellobium dulce has a huge repository of morphological and genetic diversity within the species which has been documented for further tree research and breeding programmes. A total of 40 accessions of a multipurpose leguminous tree, Pithecellobium dulce, differing in morphological and phenological characteristics was collected from different locations in Rajasthan and genetic diversity assessed using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. Out of 60 RAPD primers, 43 decamer primers produced 311 scorable bands of which 223 bands were polymorphic. Polymorphic banding patterns with the number of amplified fragments varied from 1 (OPA-12 and OPF-17) to 12 (OPF-04, OPT-07, OPT-17, OPT-20). Percent polymorphism ranged from 33.3 % (OPF-07, OPA-20, OPT-05) to 89 % (OPA-13) with an average of 64.34 %. Out of the 20 ISSR primers screened, 8 primers produced 77 amplification products of which 55 were polymorphic. The number of bands amplified per primer varied between 6 (UBC-820) and 13 (UBC-808 and UBC-816) with average band size between 300 and 4,500 bp. Percent polymorphism ranged from 38 % (UBC-817) to 100 % (UBC-811) with an average of 71 %. Dendrogram constructed on the basis of combined RAPD and ISSR markers separated the accessions into five distinct clusters at 53 % variation with Jaccard’s similarity coefficient ranging from minimum similarity (0.35) between Karoli (K) and Malviya Nagar, Jaipur (MN1) to maximum similarity (0.81) between two populations of Kota (K1 and K2). The matrices for two markers, RAPD and ISSR, were also compared using Mantel’s test. The correlation value between the matrices is high (r = 0.83) indicating good correlation between the two molecular marker systems. The study indicated a high level of morphological and genetic diversity in P. dulce.

Two clonal lineages of Phytophthora citrophthora from citrus in South Africa represent a single phylogenetic species

Phytophthora citrophthora from citrus in eastern Corsica and Spain consists of distinct clonal lineages. In South Africa the extent of genetic variation among citrus-associated P. citrophthora isolates is unknown. This was investigated with isolates from South Africa (n =60), Spain (n =10) and six isolates representing three P. citrophthora groups CTR1, CTR2 and CTR3 previously identified with isozyme polymorphisms (Mchau and Coffey 1994). South African and Spanish isolates belonged to two lineages (G1, G2) based on an internal transcribed spacer (ITS) phylogeny, random amplified microsatellites (RAMS) and random amplified polymorphic DNA (RAPD) profiling. Although combined RAMS and RAPD data identified 14 genotypes, unweighted pair group method with arithmetic mean (UPGMA) analyses grouped the isolates into two clusters corresponding to lineages G1 and G2. Lineage G1 predominated among isolates from South Africa (92%) and Spain (100%). Phylogenetic analyses of the β-tubulin, cytochrome c oxidase subunit I (COX1) and ITS regions did not support the hypothesis that the two lineages represent distinct phylogenetic species but suggested that isozyme group CTR2 and possibly CTR3 are species distinct from P. citrophthora sensu stricto. Mating-type analyses, using tester strains from groups CTR2 and CTR3 revealed that most G1 lineage isolates (n =57) were sterile but that some were of the A1 mating type (n =8) whereas all G2 lineage isolates were A2 (n =5). The mating-type designation was confirmed with P. capsici tester strains. However, when A1 (G1 lineage) and A2 (G2 lineage including CTR1 reference isolates) mating-type isolates were paired in all possible combinations, no oogonia or antheridia were produced. This suggests that only tester strains P. capsici, CTR2 and CTR3 were able to produce sexual structures and that lineages G1 and G2 are sterile and reproductively isolated, which is supported by molecular data.

Genetic diversity of Vicia faba L. based on random amplified polymorphic DNA and simple sequence repeat markers

The genetic diversity of 13 Tunisian faba bean (Vicia faba L.) accessions, and the commercial variety “Aguadulce”, were studied using simple sequence repeats (SSRs) and random amplified polymorphic DNA (RAPD) markers. In comparison to RAPD markers, SSRs showed a higher level of polymorphism and greater information content, as assessed by the expected heterozygosity. One hundred and fourteen bands were amplified using six SSR primer combinations and 376 bands were observed using four RAPD primers. The percentages of polymorphic fragments were 100% and 60.63% for SSR and RAPD markers, respectively. The polymorphism information content, gene diversity and population differentiation test were 0.370, 0.490 and 0.746, respectively, for SSR markers and 0.319, 0.406 and 0.775 for RAPD markers. The correlation coefficients of similarity based on the Mantel test were statistically significant for the both marker systems used, but were higher for SSR data than for RAPD. Dendrogram topologies, conducted separately on polymorphic RAPD and SSR markers, showed minor differences in the grouping of accessions. The dendrogram based on combined RAPD and SSR data sets identified clustering of accessions according to their geographic diffusion. Collectively, these results will aid Tunisian faba bean germplasm management, conservation and breeding.

Genetic Diversity of Pleurotus pulmonarius Revealed by RAPD, ISSR, and SRAP Fingerprinting

Pleurotus pulmonarius is one of the most widely cultivated and popular edible fungi in the genus Pleurotus. Three molecular markers were used to analyze the genetic diversity of 15 Chinese P. pulmonarius cultivars. In total, 21 random amplified polymorphic DNA (RAPD), 20 inter-simple sequence repeat (ISSR), and 20 sequence-related amplified polymorphism (SRAP) primers or primer pairs were selected for generating data based on their clear banding profiles produced. With the use of these RAPD, ISSR, and SRAP primers or primer pairs, a total of 361 RAPD, 283 ISSR, and 131 SRAP fragments were detected, of which 287 (79.5%) RAPD, 211 (74.6%) ISSR, and 98 (74.8%) SRAP fragments were polymorphic. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of these three methods were structured similarly, grouping the 15 tested strains into four clades. Subsequently, visual DNA fingerprinting and cluster analysis were performed to evaluate the resolving power of the combined RAPD, ISSR, and SRAP markers in the differentiation among these strains. The results of this study demonstrated that each method above could efficiently differentiate P. pulmonarius cultivars and could thus be considered an efficient tool for surveying genetic diversity of P. pulmonarius.

Genetic diversity study of Chromobacterium violaceum isolated from Kolli hills by amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD)

Chromobacterium are saprophytes that cause highly fatal opportunistic infections. Identification and strain differentiation were performed to identify the strain variability among the environmental samples. We have evaluated the suitability of individual and combined methods to detect the strain variations of the samples collected in different seasons. Amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD) profiles were obtained using four different restriction enzyme digestions (AluI, HaeIII, MspI and RsaI) and five random primers. A matrix of dice similarity coefficients was calculated and used to compare these restriction patterns. ARDRA showed rapid differentiation of strains based on 16S rDNA, but the combined RAPD and ARDRA gave a more reliable differentiation than when either of them was analysed individually. A high level of genetic diversity was observed, which indicates that the Kolli Hills' C. violaceum isolates would fall into at least three new clusters. Results showed a noteworthy bacterial variation and genetic diversity of C. violaceum in the unexplored, virgin forest area.

Genetic diversity analysis of ginger ( Zingiber officinale Rosc.) germplasm based on RAPD and ISSR markers

A global collection of ginger germplasm consisting of 46 accessions were characterized using two types of molecular markers, RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats). UPGMA dendrograms constructed based on three similarity coefficients, i.e., Jaccard's, Sorensen–Dice and Simple Matching using the combined RAPD and ISSR markers placed the accessions in four similar clusters in all the three dendrograms revealing the congruence of clustering patterns among the similarity coefficients and a rather less genetic distance among the accessions. Improved varieties/cultivars are grouped together with primitive types. Moreover, in the clustering pattern of the accessions, a geographical bias was also evident implying that germplasm collected from nearby locations especially with vernacular identity may not be genetically distinct. The clustering of the accessions was largely independent of its agronomic features.

Application of Molecular Markers in Predicting Production Quality of Cultivated Cistanche deserticola

We developed a set of molecular markers in Cistanche deserticola Y. C. MA to evaluate the production quality of cultivated C. deserticola individuals. This application utilizes the inter-simple-sequence repeat (ISSR) polymerase chain reaction (PCR) and random amplified polymorphic DNA (RAPD) PCR as molecular markers to determine the echinacoside content in cultivated C. deserticola individuals. The unweighted pair-group method using arithmetic average clustering (UPGMA) confirmed that the combined ISSR and RAPD data could categorize all C. deserticola individuals into three groups according to their respective echinacoside content. The stepwise multiple regression analysis (SMRA) revealed six potential markers associated with echinacoside accumulation in C. deserticola and produced 18 echinacoside-marker prediction models, four of which were successfully used to predict the quality of C. deserticola from Neimenggu. Both clustering and SMRA showed a correlation between the echinacoside content and molecular markers in cultivated C. deserticola. The relative average deviation of prediction (RADP) of the prediction models could be improved by simplifying and adjusting the model. It was found that the RADP value could reach 2.6% after adjustment and the simplified prediction models could successfully predict the quality of cultivated C. deserticola individuals.

Assessment of genetic diversity in cultivars and wild accessions of Luohanguo (Siraitia grosvenorii [Swingle] A. M. Lu et Z. Y. Zhang), a species with edible and medicinal sweet fruits endemic to southern China, using RAPD and AFLP markers

Genetic variation of wild populations and cultivars of Luohanguo (Siraitia grosvenorii), a plant species endemic to southern China, was assessed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. Based on the results for 130 individuals from seven populations, a high level of genetic diversity of Luohanguo was observed at the species level. The percentage of polymorphic loci (P) was 89.4%, Nei’s gene diversity (He) was 0.239, and Shannon’s information index (Ho) was 0.373 based on the combined AFLP and RAPD data. There was a high degree of genetic differentiation, with 45.1% of the genetic variation attributed to differences between the populations. The genetic diversity of the Luohanguo cultivars is much lower than that of wild populations (P = 41.8%, He = 0.141, Ho = 0.211), and a distinct genetic differentiation is observed between the cultivars and wild accessions. The pool of genetic variation in the wild populations provides an excellent gene resource for Luohanguo breeding.

Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology, RAPD and AFLP markers

Abstract Floral morphology, random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) were used to characterize and verify genetic diversity within a white sapote cultivar collection and to develop molecular markers for germplasm identification. On the basis of floral morphology, the cultivars were classified into three types: type I included 23 cultivars with large ovaries and small anthers; type II included 13 cultivars with small ovaries and large anthers; and type III included one cultivar, named ‘Maltby’, with a large ovary and large anthers. DNA was isolated from 39 cultivars of white sapote and subjected to RAPD and AFLP analysis using 24 and 7 primers, respectively. One hundred and sixty-eight RAPD and 286 AFLP bands were used to assess genetic characterization among white sapote. Sixty percent of the RAPD and 77% of the AFLP amplification products were polymorphic among accessions. RAPD or AFLP markers differentiated all white sapote cultivars effectively. Moreover, each flower type was characterized as specially associated with two RAPD bands. UPGMA dendrograms based on RAPD and AFLP data, showed the majority of the cultivars from flower type I and flower type II clustering together. Finally 101 RAPD markers and 220 AFLP markers were used to construct a neighbor-joining dendrogram. This showed that the 37 cultivars could be classified into six distinct clusters, between which the similarity coefficient was as low as 0.00–0.55, even though the cultivars were morphologically very similar. The remaining two cultivars namely ‘Smathers’ and ‘Maltby’ were found genetically very distant from the other cultivars in RAPD, AFLP or combined RAPD and AFLP based dendrograms. The results suggested that the level of genetic variation among white sapote cultivars is diverse and the morphological and molecular data may lead to representation of the cultivar relationships as well as flower type discrimination.

Microsatellite and RAPD polymorphisms in Ontario corn hybrids are related to the commercial sources and maturity ratings

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were used to estimate the genetic relationships among 37 Ontario corn hybrids. Almost all (95%) of the 160 RAPD fragments and all of the 79 microsatellite alleles were polymorphic across the 37 hybrids. Similarity values among the hybrids ranged from 31% to 86% when based on the RAPD data. The similarities based on microsatellite markers ranged from 12% to 77%. The genetic diversity revealed by microsatellite marker analysis was higher than that obtained from RAPD analysis. The similarity matrices for the microsatellite data and the RAPD data were moderately correlated (0.43). Cluster analyses based on either type of marker showed that most of the hybrids from the same company were closely related to each other. Both dendrograms clustered similar pairs or groups of hybrids. A principal component analysis, based on the combined RAPD and microsatellite data, yielded a good separation of the hybrids with Ontario Corn Heat Unit (OCHU) values 2800. Seventeen RAPD markers and 5 microsatellite markers were significantly associated with the OCHU ratings of the hybrids.

Genetic relatedness between oral and intestinal isolates of Porphyromonas endodontalis by analysis of random amplified polymorphic DNA

Genomic fingerprints from the DNA of 27 strains of Porphyromonas endodontalis from diverse clinical and geographic origins were generated as random amplified polymorphic DNA (RAPD) using the technique of PCR amplification with a single primer of arbitrary sequence. Cluster analysis of the combined RAPD data obtained with three selected 9- or 10-mer-long primers identified 25 distinct RAPD types which clustered as three main groups identifying three genogroups. Genogroups I and II included exclusively P. endodontalis isolates of oral origin, while 7/9 human intestinal strains of genogroup III which linked at a similarity level of 52% constituted the most homogeneous group in our study. Genotypic diversity within P. endodontalis, as shown by RAPD analysis, suggests that the taxon is composed of two oral genogroups and one intestinal genogroup. This hypothesis remains to be confirmed.

Discrimination of species and strains of basidiomycete genus Coprinus by random amplified polymorphic DNA (RAPD) analysis

All five examined strains ofCoprinus cinereus could be clearly discriminated from the strains of five otherCoprinus species by RAPD patterns with 12 of 13 primers. Also one specimen of unknownCoprinus strain was identified to beC. cinereus by this method. The RAPD patterns were similar among the strains in the same species; many common DNA fragments were recognized as well as some strain-specific DNA fragments. Thus all seven strains ofC. cinereus and all four strains ofC. angulatus examined could be distinguished individually. Diakryotic strains showed the combined RAPD patterns of the two monokaryotic strains constituting the dikaryon. The combined RAPD markers observed in the dikaryons were segregated in their basidiospore progeny. All 18 randomly picked progeny showed different combinations of RAPD markers from the parental strains.

Molecular Phylogeny of mangroves III Parentage analysis of a Rhizophora hybrid using random amplified polymorphic DNA and restriction fragment length polymorphism markers

In this study, random amplified polymorphic DNA (RAPD) markers of genomic DNA and restriction fragment length polymorphism (RFLP) of mitochondrial DNA using a mitochondrial genome specific probe, atp 6 (from maize), were used to identify the parentage of a Rhizophora hybrid. The combined RAPD profiles of two species, Rhizophora apiculata and R. mucronata had 96.5% similarity with that of the hybrid, indicating that these two species are the probable parents. RFLPs for the mitochondrial genome were readily observed, and the RFLP profile of R. apiculata was exactly the same as that of the hybrid, establishing its maternal status for the hybrid. This is the first report on characterising the parentage of a Rhizophora hybrid using molecular markers.