Random Amplified Polymorphic DNA Products Research Articles

Morphology, Biochemical, and Molecular Characterization of Pasteurella multocida Causing Hemorrhagic Septicemia in Indonesia.

Pasteurella multocida is a Gram-negative bacterium that causes hemorrhagic septicemia (HS) in buffaloes and cattle. The disease causes serious problems in Indonesian livestock and is classified as a serious transmissible animal disease. Previous research has determined the diversity of P. multocida using a serotyping method based on the antigenic properties of capsule polysaccharides. An alternative method for analysis utilizes sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and random amplified polymorphic DNA (RAPD). This study aimed to characterize and determine P. multocida diversity in several regions of Indonesia based on phenotypic character, protein profile, and the band pattern of RAPD results. Bacterial identification was performed using traditional biochemical techniques and API® 20NE systems and then confirmed molecularly using polymerase chain reaction (PCR). The freeze-thawing technique was performed to obtain the bacterial protein extract, and DNA extraction was executed using DNAzol. The extracted protein and RAPD product were then electrophoresed on 12% polyacrylamide gel and 1.5% agarose gel, respectively. The results indicate that the molecular weight range of the protein bands is 12-209 kDa, and the band pattern of the RAPD results ranged from 307-3,100 bp. Based on phenotypical analysis, P. multocida from South Sulawesi Province exhibited a variety of growth characteristics in MacConkey agar media. Using the hierarchical clustering analysis of the band patterns of RAPD and the whole-cell protein profiles, four and five clusters were formed, respectively. These results indicate molecular diversity among P. multocida from several regions of Indonesia.

English

Stemphylium solani isolates infect two varieties of eggplant, Solanum aethiopicum and Solanum melongena grown in Senegal (West Africa) and are morphologically quite similar. Using the random amplified polymorphic DNA (RAPD) procedure with arbitrary 10-mer primers, we were able to differentiate these S. solani isolates into two groups directly related to their plant host origin. A RAPD product of approximately 480 bases pairs obtained with OPF-20 primer were polymorphic between the two groups. Four new primers (F20F1, F20F2, F20R1 and F20R2) based on nucleotide sequence analysis of this 480 bases pairs RAPD fragment were developed. Such primers used pairwise amplified a single fragment from the DNA of S. solani isolates whatever their host origin. However, DNA extracted from Fusarium oxysporum (f. sp. vasinfectum, f. sp. elaeidis), Verticillium dahliae and Phyllosticta sp. isolates did not amplify using these primers. Our results indicate that these primers sets were good tools for specific identification of these two eggplants S. solani isolates by polymerase chain reaction (PCR). Key words: Stemphylium solani isolates, Solanum aethiopicum, Solanum melongena, random amplified polymorphic DNA (RAPD) markers, identification.

Development of SCAR Markers Linked to Cold Resistance for Marker-assisted Selection of Grapevines

포도 신품종 육성에서 내한성 계통의 조기 선발을 위하여, 다양한 품종을 대상으로 randomly amplified polymorphic DNA(RAPD)를 수행하고, 그 결과를 바탕으로‘Campbell Early’x ‘Kaiji’, ‘Golden Muscat’x‘Tamnara’,‘Campbell Early’x‘Neo Muscat’와‘Alden’x‘Kaiji’의 포도교배조합 실생계통을 이용하여 내한성 관련 sequence characterized amplified region(SCAR) 분자표지를 개발하였다. 400여개의 primer를 사용하여 bulk집단에서의 RAPD 분석을 통해서 6개의 UBC random primer를 최종적으로 선발하였고, RAPD 산물을 클로닝한 후 염기서열을 분석하였다. 6개의 SCAR primer (UBC123-428, UBC196-353, UBC221-503, UBC317-800, UBC342-500, UBC344-451)를 제작하고 교배조합 실생에 재검정한 결과 내한성과 관련한 특이밴드를 확인할 수 있었다. 포도나무의 내한성과 관련한 특이적인 밴드출현양상은 수피의 탄수화물함량, 전해질전도도, 포장에서의 신초생존율과 유사한 양상을 보였다. 본 연구에서 개발한 SCAR marker는 포도의 염색체상에서 위치를 확인할 수 있었으며, 그 주변에는 주로 cytochrome P450, glutathione S-transferase, leucine-rich repeat, pleiotropic drug resistance 12, ribosomal protein 등의 유전자가 분포하였다. 본 연구에서 개발된 포도 내한성 관련 SCAR marker는 내한성 포도 품종의 조기선발과 육종효율 증진에 기여할 수 있을 것으로 기대된다.

Authentication of Akebia quinata DECNE. from its common adulterant medicinal plant species based on the RAPD-derived SCAR markers and multiplex-PCR

Akebiae Caulis, ‘Mutōng’ in Chinese, refers to the stem of Akebia quinata in the Korean modern pharmacopoeia. The stems of Aristolochia manshuriensis and Clematis armandii have also been traditionally used for clinical purposes, and are often marketed as ‘Mutōng’ under the names ‘Guān-Mutōng and Chuān-Mutōng’, respectively. Furthermore, due to the morphological similarity of the herbal medicines, their correct identification by traditional subjective methods is difficult. Therefore, to develop a reliable method for discriminating these three herbal medicines, we applied the tools of molecular genetics, including random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) marker development using samples of Akebia quinata, Akebia trifoliata, Aristolochia manshuriensis, and Clematis armandii. By comparative analysis of RAPD polymorphisms, we identified several potential PCR products that can be used to distinguish the herbal materials of each species. Using these RAPD products, we developed unique sequence characterized amplified region (SCAR) markers for determination of the species of plant materials; the products were 590 bp for A. quinata, 300 bp for A. trifoliata, 373 bp for A. manshuriensis, and 432 bp for C. armandii. Furthermore, we optimized SCAR markers for simultaneous discrimination of the four species by multiplex-PCR. These markers allow efficient identification of closely related herbal medicines originating from Akebia, Aristolochia, and Clematis species, and have potential uses in preventing the distribution of adulterants, as they can be used to identify each species and distinguish them from inauthentic substitutes.

Coupling of Random Amplified Polymorphic DNA Profiles Analysis and High Resolution Capillary Electrophoresis System for the Assessment of Chemical Genotoxicity

Cadmium (Cd) can be toxic to terrestrial snails, but few data are available about its genotoxic effects on early life stages (ELS). The aim of this study was to investigate the genotoxic potential of Cd in embryos of Helix aspersa using a new approach that couples Random Amplified Polymorphic DNA (RAPD) and a high-resolution capillary electrophoresis system (HRS). Clutches of H. aspersa were exposed to Cd solutions (2, 4, and 6 mg/L) from the beginning of their embryonic development. In addition to a dose-dependent effect of Cd on hatching rate, DNA fragmentation was observed in embryos that were exposed to 6 mg Cd/L. The analysis of RAPD products with HRS showed differences between the profiles of exposed and nonexposed embryos, starting at 2 mg Cd/L. In comparison to the profiles of the control samples, all profiles from the exposed snails exhibited an additional 270 bp DNA fragment and lacked a 450 bp DNA fragment. These profile modifications are related to the genotoxic effect of Cd on the ELS of H. aspersa . Our study demonstrates the efficacy of coupling RAPD and HRS for a rapid and efficient screening of the effects of chemicals on DNA.

Involvement of DNA polymerase beta overexpression in the malignant transformation induced by benzo[a]pyrene

ObjectiveTo explore the relationship between DNA polymerase β (pol β) overexpression and benzo[a]pyrene (BaP) carcinogenesis. MethodsFirstly, mouse embryonic fibroblasts that express wild-type level of DNA polymerase β (pol β cell) and high level of pol β (pol β oe cell) were treated by various concentrations of BaP to determine genetic instability induced by BaP under differential expression levels of pol β. Secondly, malignant transformation of pol β cells by low concentration of BaP (20μM) was determined by soft agar colony formation assay and transformation focus assay. Thirdly, the mRNA and protein levels of BaP-transformed pol β cells (named pol β-T cells) was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, and the genetic instability of these cells were examined by HPRT gene mutation assay and random amplified polymorphic DNA (RAPD) assay. ResultsPol β cells were successfully transformed into malignant pol β-T cells by an exposure to low concentration of BaP for 6 months. Pol β-T cells exhibited increased levels of pol β gene expression, HPRT gene mutation frequency and polymorphisms of RAPD products that were comparable to those of pol β oe cells. ConclusionPol β overexpression and its-associated genetic instability may play a key role in BaP carcinogenesis.

Construction of high-resolution linkage map of the Ms locus, a restorer-of-fertility gene in onion (Allium cepa L.)

For the purpose of developing closely-linked molecular markers to the Ms locus, a restorer-of-fertility gene in onions (Allium cepa L.), bulked segregant analysis and randomly amplified polymorphic DNA (RAPD) analyses were utilized. Five RAPD markers polymorphic between male-fertile and male-sterile bulks were identified. These RAPD markers were converted into a simple PCR marker or cleaved amplified polymorphic sequence (CAPS) markers after sequencing the RAPD products and obtaining flanking sequences of the RAPD markers by genome walking. A linkage map was constructed with the Ms locus and flanking markers using a F2 population. There was no recombinant between the Ms locus and two CAPS markers, jnurf05 and jnurf17. To increase resolution among these closely linked molecular markers and the Ms locus, a total of 1,346 F2:3 and 2,927 F2:4 plants were analyzed with two flanking markers for detection of recombinants. Segregation of male-fertility phenotypes in large-sized populations confirmed allelic segregation distortion in favor of the recessive Ms allele. Analysis of the recombinants with closely linked markers revealed only two recombinants between the Ms locus and the jnurf05 markers among 4,273 segregating plants, showing very tight linkage between the two loci. However, linkage disequilibrium between the two loci was not too strong among the breeding lines. Despite weak linkage disequilibrium, these tightly linked markers are useful in accurate marker-assisted selection of the Ms alleles and ultimate isolation of the Ms gene by map-based cloning approach.

Genetic Characterization ofStreptococcus iniaein Diseased Farmed Rainbow Trout (Onchorhynchus mykiss) in Iran

Genetic characterization of strains of Streptococcus iniae recovered from morbidity and mortality of farmed rainbow trout in different provinces of Iran were studied. The Gram-positive cocci isolates were obtained from the kidney tissues of diseased rainbow trout on blood agar at 25°C for 72 h. The grown bacteria were then characterized using biochemical and molecular works. The identified 26 isolates of S. iniae producing a 513 bp in PCR procedure were then compared using random amplified polymorphic DNA (RAPD) analysis using 9 random primers. The phylogenetic tree of the RAPD product using UPMGA software included these strains in one genetic group but into two clusters. The results of this study show that S. iniae strains from the diseased rainbow trout in the north part of Iran are genetically similar to those strains in the south and west parts of the country.

Molecular and biochemical characterization of Trichoderma isolates inhibiting a phytopathogenic fungi Aspergillus niger Van Tieghem

Random Amplified Polymorphic DNA (RAPD) was used to examine the genetic variability among twelve isolates of Trichoderma representing three species and their ability to antagonize Aspergillus niger Van Tieghem causing collar rot in peanut using dual culture assay for correlation among RAPD products and their hardness to A. niger. One hundred and three of the 108 bands, using random decamer fungal primers, were polymorphic with an average frequency of 11.4 bands. The calculated Polymorphism Information Content (PIC) values for RAPD markers ranged from 0.172 to 0.401 and RAPD primer index (RPI) ranged from 0.99 to 6.01. RPI showed that RFu C-5 gave best results of polymorphism among the primer used in the experiment. RAPD analysis showed 10 marker loci for diagnosis of Trichoderma viride 60 and/or Trichoderma harzianum 2J, first two highest inhibitory acting antagonists. A UPGMA dendrogram constructed on the basis of Jaccard’s similarity coefficient using NTSYS 2.2 program which illustrated two distinct clusters of 12 isolates of Trichoderma and A. niger pathogen, and shared only 19% similarity. However, the in vitro highest A. niger growth inhibitory Trichoderma isolates – T. viride 60 (86.2%) and T. harzianum 2J (80.4%) were in same out group and shared 63% similarity. Relationship was found between the polymorphism showed by the Trichoderma isolates and their hardness to A. niger, in terms of in vitro production of cell wall degrading enzymes- chitinase, β-1,3 glucanase and protease, during antagonism.

Genetic variation and polymorphism in the Himalayan nettle plant Urtica dioica based on RAPD marker

Urtica dioica is a medicinal plant and is distributed throughout the ranges of Himachal Pradesh. The Genetic variation was found in U. dioica plant samples, with respect to plant distribution in middle and lower Himalayas. Genetic variability was based on change in altitude. Examination of random amplified polymorphic DNA (RAPD) markers from six plant samples collected at different heights from sea level indicated that genetic variation was appreciable, as samples from lower altitudes showed low genetic similarity with samples collected from higher altitudes. Eight Random decamer primers were used for RAPD amplification. The RAPD products ranged from 500 - 2800 bp. A total number of 134 RAPD markers were obtained from 8 primers, out of which 27 were polymorphic (20.2%) and the rest were monoporphic (79.8%). The polymorphism ranged from 4 - 50%.

Detection of genetic diversity among Indian strains of Xanthomonas campestris pv. mangiferaeindicae using PCR-RAPD

AbstractThe randomly amplified polymorphic DNA (RAPD) technique was used to investigate the genetic diversity in 6 strains of Xanthomonas campestris pv. mangiferaeindicae (Xcmi), the causal pathogen of mango bacterial canker disease (MBCD). The RAPD analysis was also intended to identify molecular markers, specific to the species to develop PCR-based markers for detection of Xcmi in mango field and planting materials. Twenty RAPD primers (CP 1-CP 20) were evaluated to establish molecular characters and genetic variability in the genome of Xcmi. Among these, only 4 were found efficient for development of reproducible banding pattern. It has been observed that the largest and smallest amplified RAPD products were of 2.036 and 0.201 kbp. A total of 136 bands were scored against 6 strains of Xcmi. There was 7.66 per cent polymorphism in individual isolates which indicates significant polymorphism among the evaluated strains, with mean difference of 0.33 (Xcmi 2 vs. Xcmi 8) and 0.29 (Xcmi 10 vs. Xcmi 12). However, the single linkage euclidean distances were statistically significant (P>0.05), i.e., 0.58. The markers CP 5, 10, 16 and 19 were amplified in all the strains with polymorphic alleles, which indicates that these markers could be used for rapid detection of genetic variability in Xcmi strains.

Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus.

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

Development of SCAR markers linked to powdery mildew (Uncinula necator) resistance in grapevine (Vitis vinifera L. and Vitis sp.)

Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.

Studies on genome relationship and species-specific PCR marker for Dasypyrum breviaristatum in Triticeae

Dasypyrum breviaristatum and nine related species in Triticeae were analyzed using the random amplified polymorphic DNA (RAPD) technique, in order to understand the genetic relationship and to develop species specific markers. The genome relationship dendrogram shows that D. breviaristatum and D. villosum could not be grouped together, indicating that D. breviaristatum was unlikely to be directly derived from D. villosum, while D. breviaristatum was closest to Thinopyrum intermedium, which implied that they might have similar breeding behaviors when introducing their chromatins into wheat. A D. breviaristatum genome specific RAPD product of 1182bp, was cloned and designated as pDb12H. Sequence analysis revealed that pDb12H was strongly homologuos to a long terminal repeat (LTR) Sabrina retrotransposon newly reported in Hordeum. The pDb12H was converted into a PCR based marker, which allows effectively monitoring the D. breviaristatum chromatin introgression into wheat. Fluorescence in situ hybridization (FISH) suggested that pDb12H was specifically hybridized throughout all D. breviaristatum chromosomes arms except for the terminal and centromeric regions, which can be used to characterize wheat -D. breviaristatum chromosome translocation. The genomes repetitive element will also be useful to study gene interactions between the wheat and alien genomes after the polyploidization.

Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer

Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T. rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5'-d[GGTGCGGGAA]-3' and 5'-d[CCCGTCAGCA]-3', as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPD products showed a high degree of similarity (>90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60 % similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPD analysis is especially suitable for molecular typing in T. rubrum.

Peracetic acid stress-induced genetic rearrangements in Escherichia coli H10407 detected by RAPD and RFLP analyses

The discriminatory powers of random amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP) were assessed for the detection and comparison of DNA modifications caused by an oxidative stress. DNA extracted from peracetic acid (PAA)-treated Escherichia coli H10407 was randomly amplified with the 10-mer primer OPZ14, which generated one stress-induced fragment. RFLP and RAPD profiles were hybridized by Southern blotting with the digoxigenin-labelled RAPD product. Untreated and PAA-treated cells had difference band profiles. The results indicate that RAPD analysis could be used as a discriminatory tool for investigating genetic rearrangements in E. coli caused by oxidative stress and that RFLP analysis could be used to confirm the rearrangements.

Identification ofporphyra lines (rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.

Characterization and mapping of Rpi1, a gene that confers dominant resistance to stalk rot in maize

The maize inbred lines 1145 (resistant) and Y331 (susceptible), and the F(1), F(2) and BC(1)F(1) populations derived from them were inoculated with the pathogen Pythium inflatum Matthews, which causes stalk rot in Zea mays. Field data revealed that the ratio of resistant to susceptible plants was 3:1 in the F(2) population, and 1:1 in the BC(1)F(1)population, indicating that the resistance to P. inflatum Matthews was controlled by a single dominant gene in the 1145xY331 cross. The gene that confers resistance to P. inflatum Matthews was designated Rpi1 for resistance to P. inflatum) according to the standard nomenclature for plant disease resistance genes. Fifty SSR markers from 10 chromosomes were first screened in the F(2) population to find markers linked to the Rpi1 gene. The results indicated that umc1702 and mmc0371 were both linked to Rpi1, placing the resistance gene on chromosome 4. RAPD (randomly amplified polymorphic DNA) markers were then tested in the F(2)population using bulked segregant analysis (BSA). Four RAPD products were found to show linkage to the Rpi1 gene. Then 27 SSR markers and 8 RFLP markers in the region encompassing Rpi1 were used for fine-scale mapping of the resistance gene. Two SSR markers and four RFLP markers were linked to the Rpi1 gene. Finally, the Rpi1 gene was mapped between the SSR markers bnlg1937 and agrr286 on chromosome 4, 1.6 cM away from the former and 4.1 cM distant from the latter. This is the first time that a dominant gene for resistance to maize stalk rot caused by P. inflatum Matthews has been mapped with molecular marker techniques.

An identification tool for the Australian weedy Sporobolus species based on random amplified polymorphic DNA (RAPD) profiles

Based on morphological features alone, there is considerable difficulty in identifying the 5 most economically damaging weed species of Sporobolus [viz. S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur and Schinz, S. fertilis (Steud.) Clayton, S. africanus (Poir.) Robyns and Tourney, and S. jacquemontii Kunth.] found in Australia. A polymerase chain reaction (PCR)-based random amplified of polymorphic DNA (RAPD) technique was used to create a series of genetic markers that could positively identify the 5 major weeds from the other less damaging weedy and native Sporobolus species. In the initial RAPD profiling experiment, using arbitrarily selected primers and involving 12 species of Sporobolus, 12 genetic markers were found that, when used in combination, could consistently identify the 5 weedy species from all others. Of these 12 markers, the most diagnostic were UBC51490 for S. pyramidalis and S. natalensis; UBC43310, 2000, 2100 for S. fertilis and S. africanus; and OPA20850 and UBC43470 for S. jacquemontii. Species-specific markers could be found only for S. jacquemontii. In an effort to understand why there was difficulty in obtaining species-specific markers for some of the weedy species, a RAPD data matrix was created using 40 RAPD products. These 40 products amplified by 6 random primers from 45 individuals belonging to 12 species, were then subjected to numerical taxonomy and multivariate system (NTSYS pc version 1.70) analysis. The RAPD similarity matrix generated from the analysis indicated that S. pyramidalis was genetically more similar to S. natalensis than to other species of the ‘S. indicus complex’. Similarly, S. jacquemontii was more similar to S. pyramidalis, and S. fertilis was more similar to S. africanus than to other species of the complex. Sporobolus pyramidalis, S. jacquemontii, S. africanus, and S. creber exhibited a low within-species genetic diversity, whereas high genetic diversity was observed within S. natalensis, S. fertilis, S. sessilis, S. elongates, and S. laxus. Cluster analysis placed all of the introduced species (major and minor weedy species) into one major cluster, with S. pyramidalis and S. natalensis in one distinct subcluster and S. fertilis and S. africanus in another. The native species formed separate clusters in the phenograms. The close genetic similarity of S. pyramidalis to S. natalensis, and S. fertilis to S. africanus may explain the difficulty in obtaining RAPD species-specific markers. The importance of these results will be within the Australian dairy and beef industries and will aid in the development of integrated management strategy for these weeds.

Identification of 27 Porphyra lines (Rhodophyta) by DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.