Amplified DNA Fingerprinting Research Articles

GENETIC DIVERSITY BETWEEN THREE SPECIES OF DAVIDSONIA - THE AUSTRALIAN NATIVE PLUM

Davidson's plum (Davidsonia spp) is an endangered Australian native plum with high commercial potential due to its wide range of product uses. Molecular marker analysis using randomly amplified DNA fingerprinting (RAF), provided a preliminary view of genetic relationships between three species, D. pruriens, D. jerseyana and D. johnsonii. Using five accessions of each species, 47 dominant markers were identified, with only 13% of markers observed in all accessions. Considerable diversity was detected within species (HS=0.126) though differences were well resolved (GST=0.57). The tropical north Queensland species, D. pruriens, was the least diverse. Several markers could be classified as diagnostic of this species, However, the sympatric species D. jerseyana and D. johnsonii, found in subtropical coastal northern New South Wales, were not particularly closely related, and shared only one marker not found in D. pruriens. Genetic diversity was relatively high within these two species. A more comprehensive analysis is underway for all three species with the useful RAF marker system, which will have implications for the selection, improvement, and conservation of these threatened and endangered species.

DEVELOPMENT OF A CO-DOMINANT SCAR MARKER LINKED TO A PUTATIVE PRSV-P RESISTANCE LOCUS IN WILD “PAPAYA”

Molecular genetic analysis of interspecific hybrids of V. cundinamarcensis and V. parviflora using Randomly Amplified DNA Fingerprinting (RAF) has permitted the construction of genetic maps of these species. The maps presented provided a framework for the development of marker systems for detection of valuable trait loci. Subsequently, several DNA markers linked to a PRSV-P resistance gene locus were identified and mapped in V. cundinamarcensis. One of these markers (OPK41R) resulted from a Single Nucleotide Polymorphism (SNP) in close proximity to the PRSV-P resistance gene locus. The Sequence Characterised Amplified Region (SCAR) was shown to be present in both parents, and 4 additional Vasconcellea species including V. cauliflora and V. quercifolia, but not in papaya. Sequence analysis revealed several polymorphisms, one resulting in the creation of a restriction site within the OPK4IR region, which was used to convert the SCAR into an easily detected co-dominant marker. This marker presents a simple yet useful tool that may be applied as a diagnostic test for selection of homozygous PRSV-P resistant hybrids of V. cundinamarcensis. With the development of this marker there is new potential to bridge hybridisation between Hybrids of V. cundinamarcensis and C. papaya via marker assisted selection.

ASSESSMENT OF GENETIC DIVERSITY IN AUSTRALIAN-GROWN MANGOSTEEN (GARCINIA MANGOSTANA L.) AND ITS WILD RELATIVES

Mangosteen (Garcinia mangostana L.) is a popular dioecious tropical fruit tree cultivated throughout South East Asia and Northern Australia. Dioecious species are generally characterised by high levels of genetic diversity, however mangosteen is unusual in that phenotypic differences are uncommon. Almost all trees are female with only a few anecdotal reports of male trees reported. Furthermore, seeds from mangosteen are apomictic and consequently produce trees that are clones of the female parent. It has been suggested by some mangosteen growers in North Queensland that the occasional phenotypic variants observed in their plantations are a result of cross-pollination from wild related species. Whether these differences can be attributed to genetic variation or are purely a consequence of being cultivated at different geographical locations is unclear. To address these questions, we conducted a preliminary survey using a new high-efficiency DNA marker technique, Randomly Amplified DNA Fingerprinting (RAF), to examine the level of genetic diversity in a population of G. mangostana trees collected from orchards in Northern Queensland. Additionally, we examined the level of variation among a number of other Garcinia species including mangosteen's closest relative, G. hombroniana, as well as native Australian Garcinia species.

Genetic mapping of a PRSV-P resistance gene in “highland papaya” based on inheritance of RAF markers

Papaya ringspot virus type P (PRSV-P) is a significant disease of Carica papaya. A major gene for PRSV-P resistance has been mapped in Vasconcellea cundinamarcensis, a distant relative of C. papaya. This was achieved by genetic mapping of the resistance phenotype and inherited, dominant, polymorphic randomly amplified DNA fingerprint (RAF) markers in F2 progenies of V. parviflora and V. cundinamarcensis. The parents of this cross confer resistance to several major diseases that affect C. papaya including PRSV-P in V. cundinamarcensis. Heredity of DNA markers and PRSV-P resistance was studied in the intrageneric population presented due to intergeneric fertility barriers between Carica and Vasconcellea. Genetic polymorphism between parents, based on RAF markers, was 75% with more than 70% of markers generated showing mendelian segregation for the expected ratios 1:3 or 1:1 (p < 0.05). Preferential inheritance of markers from either parent was not detected in the F2, indicating stable transfer of the genetic material. Discrete V. parviflora and V. cundinamarcensis linkage maps were compiled from 79 and 83 framework markers, delineating to 10 and 11 groups respectively. F1 and F2 progeny were screened for resistance to PRSV-P under controlled conditions. The resistant phenotype segregated 3:1 in the F2 and mapped to V. cundinamarcensis linkage group 7 with adjacent RAF markers within 4 cM. The framework maps of V. parviflora and V. cundinamarcensis presented cover 630.2 and 745.4 cM respectively, accounting for between 47–52 and 49–55 percent of the predicted genome lengths. These maps provide a platform for further genetic study of disease resistance characteristics identified in these species and the development of DNA markers tightly linked to these traits, which could be applied to the breeding of resistant C. papaya cultivars.

DNA amplification fingerprinting as a tool for checking genetic purity of strains in the cyanobacterial inoculum

The electrophoretic patterns for 17 different cyanobacterial cultures derived from 6 different decamer primers were analysed to provide diagnostic fingerprints for each culture and their genetic distances based on RAPD markers.The primer OPB 09 produced a maximum of 24 amplified products. The primers OPB 09, OPG 04 and OPAH 02 generated markers specific for Nostoc cultures. Westiellopsis was found to be distinct from other cyanobacterial cultures in the RAPD profile obtained with the primer OPAH 02. The primer OPF 03 generated specific markers for Tolypothrix tenuis. Fischerella cultures could be identified with the primers OPB 09, OPAG 03 and OPF 05. The study revealed that these RAPD markers could be further used to identify and establish the genetic purity of the strains in the cyanobacterial inoculum. There was a similarity of 60–90% within Westiellopsis cultures. Nostoc cultures shared 50–80% similarity with Westiellopsis cultures. Anabaenacultures were similar to Westiellopsiscultures by 60–70. The markers produced for each culture were also applied to phylogenetic analysis to infer genetic relatedness in this group of prokaryotes. The dendrogram analysis clearly revealed that free-living cyanobacterial cultures are closely related to each other and are distinct from the symbiotic forms.

DNA amplification fingerprinting in coconut: protocol optimization and analysis of resistance to root (wilt) disease

Root (wilt) disease is a serious malady in coconut causing a loss of approximately 968 million nuts a year in India. Integrated management practices are the only suggested methods to reduce its effect to some extent, while the development of resistant varieties will offer a permanent solution for which concrete breeding efforts are required. Considering the long life cycle of coconut, selection of resistant varieties through conventional methods will be time consuming and laborious. Molecular markers offer numerous advantages over markers traditionally used in plant mapping and selective breeding. Here, we present the optimization of PCR conditions for DNA amplification fingerprinting (DAF) of coconut using arbitrary oligonucleotide primers. Three of the primers could detect variations between root (wilt) resistant and susceptible coconut palms. This study constitutes the basis for future efforts to tag the root (wilt) resistant gene(s) in coconut.

DNA Fingerprinting of Seeded Bermudagrass Cultivars

Bermudagrasses (Cynodon spp.) are important for turf and forage in temperate and tropical climates, with cultivars historically propagated clonally. Over the past two decades the number of seed‐propagated commercial cultivars has dramatically increased, but information is lacking on the extent of the genetic diversity among these new cultivars. Accordingly, this research was undertaken to assess the genetic relatedness of 17 seed‐propagated turf‐bermudagrass cultivars using DNA amplification fingerprinting (DAF). Four DAF and four Minihairpin‐DAF (MHP‐DAF) primers were used in this study. The DAF and MHP‐DAF primers amplified 90 and 131 amplicons, respectively. A total of 13 out of the 17 cultivars were practically indistinguishable using the DAF primers with an average similarity [similarity coefficient (SC)] of 0.982 while the MHP‐DAF primers distinguished all cultivars readily. Results from the DAF and MHP‐DAF analysis indicated that 14 out of the 17 cultivars were related to Arizona common germplasm with average SC of 0.833 in the MHP‐DAF analysis. Arizona common germplasm is naturalized to the Colorado River Valley production areas of Arizona and California. The three most distinct cultivars—‘Princess 77’, ‘Yukon’, and ‘SWI‐11’—had an average SC of 0.668. The most distinct cultivar was Yukon with an average SC of 0.604. Yukon showed 59 DNA signatures not observed in the other cultivars studied with DAF and MHP‐DAF. These results indicated that a majority of seeded‐type bermudagrasses developed over the past two decades depend on a narrow genetic base and that several recent cultivars are markedly genetically distinct, indicating a recent and significant broadening of the germplasm.

A combination of AFLP and SSR markers provides extensive map coverage and identification of homo(eo)logous linkage groups in a sugarcane cultivar

Sugarcane varieties are complex polyploids carrying in excess of 100 chromosomes and are derived from interspecific hybridisation between the domesticated Saccharum officinarum and the wild relative S. spontaneum. A map was constructed in Denotes variety covered by Australian plant breeding rights., an Australian cultivar, from a segregating F1 population, using 40 amplified fragment length polymorphism (AFLP) primer combinations, five randomly amplified DNA fingerprints (RAF) primers and 72 simple sequence repeat (SSR) primers. Using these PCR-based marker systems, we generated 1,365 polymorphic markers, of which 967 (71%) were single-dose (SD) markers. Of these SD 967 markers, 910 were distributed on 116 linkage groups (LGs) with a total map length of 9,058.3 cM. Genome organisation was significantly greater than observed in previously reported maps for Saccharum spp. With the addition of 123 double-dose markers, 36 (3:1) segregating markers and a further five SD markers, 1,074 markers were mapped onto 136 LGs. Repulsion phase linkage detected preferential pairing for 40 LGs, which formed 11 LG pairs and three multi-chromosome pairing groups. Using SSRs, double-dose markers and repulsion phase linkage, we succeeded in forming 127 of the 136 LGs into eight homo(eo)logy groups (HG). Two HGs were each represented by two sets of LGs. These sets of LGs potentially correspond to S. officinarum chromosomes, with each set aligning to either end of one or two larger LGs. The larger chromosomes in the two HGs potentially correspond to S. spontaneum chromosomes. This suggestion is consistent with the different basic chromosome number of the two species that are hybridised to form sugarcane cultivars, S. spontaneum (x=8) and S. officinarum (x=10), and illustrates the structural relationship between the genomes of these two species. The discrepancy of coverage between HGs highlights the difficulty in mapping large parts of the genome.

Genetic diversity revealed in the apomictic fruit species Garcinia mangostana L. (mangosteen)

The novel molecular marker technique Randomly Amplified DNA Fingerprinting (RAF) was used to survey genetic relationships between 37 accessions of the tropical fruit G. mangostana (mangosteen) and among 11 accessions from eight other Garcinia species. Although mangosteen is believed to reproduce exclusively through apomixis, our results show that considerable genetic diversity exists within G. mangostana and between other Garcinia species. Among the 37 G. mangostana accessions examined, nine different genotypes were identified which clustered into three distinct groups based on correspondence analysis (reciprocal averaging). For 26 (70%) of the accessions no marker variation was detected over 530 loci screened. A further eight (22%) accessions exhibited very low levels of variation (0.2-1%) suggesting at least one well conservedm angosteen genotype. The remaining three accessions (8%) showed extensive variation (22-31%) compared with the majority of accessions. The three mangosteen groups were 63-70% dissimilar to the other Garcinia species investigated. The genetic diversity identified in this research will assist in the conservation of Garcinia germplasm and provides a valuable framework for the genetic improvement of mangosteen.

A comparison of molecular markers for genetic analysis of macadamia

SummaryVarious marker systems exist for genetic analysis of horticultural species. Isozymes were first applied to the woody perennial nut crop, macadamia, in the early 1990s. The advent of DNA markers saw the development, for macadamia, of STMS (sequence-tagged microsatellite site), RAPD (randomly amplified polymorphic DNA), and RAF (randomly amplified DNA fingerprinting). The RAF technique typically generates dominant markers, but within the dominant marker profiles, certain primers also amplify multi-allelic co-dominant markers that are suspected to be microsatellites. In this paper, we confirm this for one such marker, and describe how RAF primers can be chosen that amplify one or more putative microsatellites. This approach of genotyping anonymous microsatellite markers via RAF is designated RAMiFi (randomly amplified microsatellite fingerprinting). Several marker systems were compared for the type, amount, and cost-efficiency of the information generated, using data from published studies on macadamia. The markers were also compared for the way they clustered a common set of accessions. The RAMiFi approach was identified as the most efficient and economical. The availability of such a versatile tool offers many advantages for the genetic characterisation of horticultural species.

Population genetics and spatial structure of the fairy ring fungus Marasmius oreades in a Norwegian sand dune ecosystem

The population genetics and spatial structure of the fairy ring fungus Marasmius oreades (Bolt. : Fr.) Fr. was studied by DNA amplification fingerprinting (DAF). Basidiocarp samples were collected from fairy rings from two separate sand dune systems of about 560 m2 and 1750 m2, respectively, on the Lista Peninsula in southwestern Norway in 1996. Samples were collected after a careful mapping of fairy rings and a vegetation survey of the composition and spatial structure of vascular plants, bryophytes and lichens. DAF with standard arbitrary oligonucleotide primers was used to examine the genetic relationship between basidiocarp samples. The study showed that the fungal population contained a high number of genotypes and that about 90% of the fairy rings represented a separate genet. Both cluster and phylogenetic analyses of DAF amplification products established relationships between fairy rings and showed that genetically similar basidiocarps were found close to each other. Overall results showed a weak correspondence between genotype and spatial distribution and no correspondence between genotype and composition of the surrounding vegetation. Furthermore, the occurrence of the four dominant sand dune grass species was randomly distributed among the localities housing the various fungal genotypes, indicating that the fungus did not exhibit genotypic specialization to the various grass species that could host it as a pathogen. Results show that establishment of new individuals generally was mediated by basidiospore dispersal and not by fragmenting dikaryotic, vegetative mycelium, as previously proposed.

Sources of Bacillus cereus contamination in a pasteurized zucchini purée processing line, differentiated by two PCR-based methods

In previous work, raw materials used for processing pasteurized zucchini purée (zucchini, milk proteins and starch) and cultivation soil of zucchinis were found to be potential sources of stored product contamination with Bacillus cereus. 134 B. cereus strains originating from these sources and from the stored product were typed using coliphage M13 sequence-based polymerase chain reaction (M13-PCR) and DNA amplification fingerprinting. Combined patterns from the two methods were compared using Dice's coefficient and the unweighted pair group method with average cluster analysis. 16 combined profile groups and six unclustered strains were formed at 75% similarity level. Representative strains of each group were subsequently examined for growth at low temperature (4 degrees C, 7 degrees C and 10 degrees C). Based on strain relatedness, five major groups and two minor groups among the nine groups containing zucchini strains were common to both zucchini and soil origins, indicating that the soil was the main initial source of contamination for zucchinis. Strains from zucchinis and soil were heterogeneous compared with strains from dehydrated ingredients (milk proteins and starch), and were mainly composed of psychrotrophic strains. Convergent results from molecular typing and growth at low temperature showed that psychrotrophic contaminants of the stored product originated from zucchinis, whereas non-psychrotrophic contaminants originated from milk proteins. The number of genetic groups decreased during the purée processing, suggesting selection of strains during zucchini washing operations, heat treatment and storage.

DAF marker tightly linked to a major locus for Ascochyta blight resistance in chickpea (Cicer arietinum L.)

Resistance of chickpea against the disease caused by the ascomycete Ascochyta rabiei is encoded by two or three quantitative trait loci, QTL1, QTL2 and QTL 3. A total of 94 recombinant inbred lines developed from a wide cross between a resistant chickpea line and a susceptible accession of Cicer reticulatum, a close relative of cultivated chickpea, was used to identify markers closely linked to QTL1 by DNA amplification fingerprinting in combination with bulked segregant analysis. Of 312 random 10mer oligonucleotides, 3 produced five polymorphic bands between the parents and bulks. Two of them were transferred to the population on which the recent genetic map of chickpea is based, and mapped to linkage group 4. These markers, OPS06-1 and OPS03-1, were linked at LOD-scores above 5 to markers UBC733B and UBC181A flanking the major ascochyta resistance locus. OPS06-1 mapped at the peak of the QTL between markers UBC733B (distance 4.1 cM) and UBC181A (distance 9.6 cM), while OPS03-1 mapped 25.1 cM away from marker UBC733B on the other flank of the resistance locus. STMS markers localised on this linkage group were transferred to the population segregating for ascochyta resistance. Three of these markers were closely linked to QTL1. Twelve of 14 STMS markers could be used in both populations. The order of STMS markers was essentially similar in both populations, with differences in map distances between them. The availability of flanking STMS markers for the major resistance locus QTL1 will help to elucidate the complex resistance against different Ascochyta pathotypes in future.

A genetic map of macadamia based on randomly amplified DNA fingerprinting (RAF) markers

The first genetic linkage map of macadamia (Macadamia integrifolia and M. tetraphylla) is presented. The map is based on 56 F-1 progeny of cultivars 'Keauhou' and 'A16'. Eighty-four percent of the 382 markers analysed segregated as Mendelian loci. The two-way pseudo-testcross mapping strategy allowed construction of separate parental cultivar maps. Ninety bridging loci enabled merging of these maps to produce a detailed genetic map of macadamia, 1100 cM in length and spanning 70-80% of the genome. The combined map comprised 24 linkage groups with 265 framework markers: 259 markers from randomly amplified DNA fingerprinting (RAF), five random amplified polymorphic DNA (RAPD), and one sequence-tagged microsatellite site (STMS). The RAF marker system unexpectedly revealed 16 codominant markers, one of them a putative microsatellite locus and exhibiting four distinct alleles in the cross. This molecular study is the most comprehensive examination to date of genetic loci of macadamia, and is a major step towards developing marker-assisted selection for this crop.

Sources of Bacillus cereus contamination in a pasteurized zucchini purée processing line, differentiated by two PCR-based methods

In previous work, raw materials used for processing pasteurized zucchini purée (zucchini, milk proteins and starch) and cultivation soil of zucchinis were found to be potential sources of stored product contamination with Bacillus cereus. 134 B. cereus strains originating from these sources and from the stored product were typed using coliphage M13 sequence-based polymerase chain reaction (M13-PCR) and DNA amplification fingerprinting. Combined patterns from the two methods were compared using Dice’s coefficient and the unweighted pair group method with average cluster analysis. 16 combined profile groups and six unclustered strains were formed at 75% similarity level. Representative strains of each group were subsequently examined for growth at low temperature (4°C, 7°C and 10°C). Based on strain relatedness, five major groups and two minor groups among the nine groups containing zucchini strains were common to both zucchini and soil origins, indicating that the soil was the main initial source of contamination for zucchinis. Strains from zucchinis and soil were heterogeneous compared with strains from dehydrated ingredients (milk proteins and starch), and were mainly composed of psychrotrophic strains. Convergent results from molecular typing and growth at low temperature showed that psychrotrophic contaminants of the stored product originated from zucchinis, whereas non-psychrotrophic contaminants originated from milk proteins. The number of genetic groups decreased during the purée processing, suggesting selection of strains during zucchini washing operations, heat treatment and storage.

Towards the first linkage map of theDidymella rabiei genome

A genetic map was developed for the ascomyceteDidymella rabiei (Kovachevski) v. Arx (anamorph:Ascochyta rabiei Pass. Labr.), the causal agent of Ascochyta blight in chickpea (Cicer arietinum L.). The map was generated with 77 F1 progeny derived from crossing an isolate from the U.S.A. and an isolate from Syria. A total of 232 DAF (DNA Amplification Fingerprinting) primers and 37 STMS (Sequence-Tagged Microsatellite Site) primer pairs were tested for polymorphism between the parental isolates; 50 markers were mapped, 36 DAFs and 14 STMSs. These markers cover 261.4cM in ten linkage groups. Nineteen markers remained unlinked. Significant deviation from the expected 1:1 segregation ratios was observed for only two markers (Prob. of χ2<0.05). The implications of our results on ploidy level of the asexual spores are discussed.

Molecular Markers in Plant Breeding-I: Concepts and Characterization

Plant breeding is a combination of principles and methods of changing the genetic constitution of a plant to make it more suitable for human needs. Conventional plant breeding has evolved with the passage of time from simple seed saving of the best harvest to the selection of seeds according to the laws of Mendel. With the advent of morphological and biochemical markers, the selection process has hastened and the scope of conventional plant breeding increased many folds during the recent years. However, the process that actually revolutionized the plant breeding in the 20 century was the realization that th there exist a widespread polymorphism in natural populations, the degree of which can be assessed by sequencing or making restriction maps: an application of new tools of molecular biology. The first and the foremost molecular markers system i.e., restriction fragment length polymorphism (RFLP) was developed in early 1980. These are co-dominant markers and are available in unlimited number. Another breakthrough was the emergence of polymerase chain reaction (PCR) in 1990. With this technology, a new generation of DNA markers such as randomly amplified polymorphic DNA (RAPDs), sequence characterized amplified regions (SCARs), sequence tagged sites (STS), single polymorphic amplification test (SPLAT), variable number of tendom repeats (VNTRs), amplified fragment length polymorphism (AFLP), DNA amplification fingerprinting (DFA), single strand conformational polymorphism (SSCP), single nucleotide polymorphism (SNPs), micro-satellites or short tandem repeats (STRs), cDNA, DNA micro arrays and rDNA-ITS were introduced into the modern plant breeding systems. The concept of DNA based markers has increased our ability many folds, to follow minute regions of chromosome through opportunities such as map based cloning and Marker-assisted Plant (MAP) Breeding. In MAP breeding, the new ideas and concepts have been introduced which need to be understood thoroughly, before applying these ideas in practical breeding programmes particular in country like Pakistan where application of molecular biological approaches are still in its infancy. In order to achieve this objective, efforts were made to write a series of review articles in which concept of MAP breeding is being described thoroughly yet in a simple way so that student and researchers can build their understanding of plant molecular breeding and application of DNA based markers for the genetic dissection of qualitative and quantitative traits. In the present paper, concepts of MAP breeding and the type and characteristics of DNA markers is presented so that choice of the marker(s) can be made rational and for the defined purposes.

Genetic linkage analysis of the lesser grain borer Rhyzopertha dominica identifies two loci that confer high-level resistance to the fumigant phosphine.

High levels of inheritable resistance to phosphine in Rhyzopertha dominica have recently been detected in Australia and in an effort to isolate the genes responsible for resistance we have used random amplified DNA fingerprinting (RAF) to produce a genetic linkage map of R. dominica. The map consists of 94 dominant DNA markers with an average distance between markers of 4.6 cM and defines nine linkage groups with a total recombination distance of 390.1 cM. We have identified two loci that are responsible for high-level resistance. One provides approximately 50x resistance to phosphine while the other provides 12.5x resistance and in combination, the two genes act synergistically to provide a resistance level 250x greater than that of fully susceptible beetles. The haploid genome size has been determined to be 4.76 x 10(8) bp, resulting in an average physical distance of 1.2 Mbp per map unit. No recombination has been observed between either of the two resistance loci and their adjacent DNA markers in a population of 44 fully resistant F5 individuals, which indicates that the genes are likely to reside within 0.91 cM (1.1 Mbp) of the DNA markers.

Molecular epidemiology of cystic fibrosis-linked Burkholderia cepacia complex isolates from three national referral centres in Ireland.

Burkholderia cepacia is a Gram-negative bacterium associated with increasing morbidity and mortality and is readily transmitted among infected cystic fibrosis (CF) patients. The B. cepacia complex consists of five distinct subgroups, termed genomovars. A collection of 17 presumptive B. cepacia isolates, obtained from three national CF referral centres located in different geographical regions in Ireland, was studied. The aim of this study was to investigate these isolates using molecular subtyping protocols for evidence of genetic relationships and for the presence of antibiotic resistance-encoding class 1 integron structures. Genomovar classifications were assigned to each isolate based on HaeIII enzyme profiles of their recA locus. Genetic relationships among this collection were also assessed after restriction fragment length polymorphism (RFLP)-mediated analysis of the 16S rDNA locus and DNA amplification fingerprinting (DAF). The surface expression of the cable pilus gene (cblA) may facilitate an early step in the infection process. All isolates were tested by amplification strategies for this marker. Burkholderia cepacia is known to be resistant to several antimicrobial agents. Resistance typing showed that the majority were resistant to three or more common antimicrobial agents. Five of the 17 isolates were resistant to sulphonamide, a characteristic linked with the presence of class 1 integrons. Gene cassettes containing beta-lactamase (oxa) and aminoglycoside acetyltransferase (aac(6')-1a) encoding genes were identified by polymerase chain reaction amplification. Most of the isolates in this study were classified as genomovar III and were indistinguishable based on their corresponding 16S rDNA-RFLP profiles, whilst DAF further subtyped the collection. The cblA marker was identified in 47% of the isolates, many of which clustered in the genomovar III group. Class 1 integrons with recombined gene cassettes containing bla-OXA and aac(6')-1a genes were identified. This study demonstrates the application of molecular methods to investigate B. cepacia, a well-recognized human pathogen, cultured from Irish CF patients. Genomovar III was the most common genomic type identified. DNA fingerprinting further subtyped the latter isolates, facilitating a more detailed description of the molecular epidemiology. Drug resistance in these organisms can be explained, at least in part, by the presence of class 1 integrons. Development of targeted infection control strategies could be facilitated using these applied methods.

CHARACTERISING MACADAMIA GERMPLASM WITH CODOMINANT RADIOLABELLED DNA AMPLIFICATION FINGERPRINTING (RAF) MARKERS

The cultivated subtropical nut crop macadamia is based on two Australian native species, Macadamia integrifolia and M. tetraphylla. The macadamia industry prefers M. integrifolia, although hybrid cultivars do occur. There are preliminary results to indicate that breeders may be able to exploit hybrid vigour, but to advance these studies, hybrid status must first be resolved. At present, species allocation is diagnosed on the basis of a few morphological traits, but this is inadequate to resolve proportional contributions of each species to each hybrid genotype. Neither can morphology be used to distinguish each cultivar or satisfactorily organise the complicated array of available cultivars. Such gaps in our knowledge may be filled by application of new DNA marker technologies. Radiolabelled DNA amplification fingerprinting (RAF) is a DNA marker technique recently applied to macadamia chromosome mapping. Here we demonstrate RAF to be a simple technique for identifying highly informative codominant markers in macadamia. We have discovered 19 new RAF codominant markers, which were used together with dominant markers to characterise 30 cultivars representative of the macadamia industry. Individual identity was assigned to each cultivar, and species proportions were calculated. Many cultivars contained a higher degree of M. tetraphylla than was previously thought. The markers were also used to assign cultivars to germplasm groups that reflected species status and breeding origins. Species composition was found to be a major determinant of group distinction. We now have a simple screening procedure for characterising macadamia germplasm.