G. C. Graham

Random amplified polymorphic DNA analysis of Australian rice (Oryza sativa L.) varieties

SummaryThe genetic relationships between rice varieties were analysed by using the polymerase chain reaction (PCR), with arbitrary oligonucleotide primers in the random amplified polymorphic DNA (RAPD) method. PCR with 22 arbitrary primers applied to 37 varieties produced 144 useful markers, of which 67% were polymorphic. Thus, with selected primers sufficient polymorphism could be detected to allow identification of individual varieties. Visual examination of electrophoresis gels and analysis of banding patterns confirmed that commercial Australian and USA lines and their relatives were very closely related, with similarity indices of 88–97%. Three varieties originating from more distant geographical centres were easily distinguished, producing variety-specific amplification profiles and expressing a lower similarity index of 80% to all other varieties tested. PCR offers a potentially simple, rapid and reliable method for rice genotype identification and recognition of lines that could contribute genetic diversity to new commercial varieties.

RAPD and isozyme analysis of genetic relationships between Carica papaya and wild relatives

The genetic origin of cultivated papaya is not clear. Wild relatives of papaya (Carica papaya) from central southern America were investigated using isozyme and RAPD analysis. Seven other species (including six from the genus Carica) were found to be relatively distant from papaya providing no indication of the genetic origin of papaya. Isozyme and RAPD data gave similar measures of genetic similarity within this group. C. papaya was about 70% dissimilar to the other Carica species by both methods. The other Carica species had average dissimilarities around 50%. Two species, C. pubescens and C. stipulata were much closer to each other with similarities of 87% by isozyme analysis and 82% by RAPD analysis. Although both methods gave similar measures for genetic distance the large number of RAPD markers available made RAPD analysis more reliable for analysis of the extremes (e.g. closely related taxa may show no isozyme differences and distant taxa may show no isozyme similarities).

Genetic shifts in Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) over a year in the Dawson/Callide Valleys

Microsatellites were used to analyse 68 collections of Helicoverpa armigera in the Dawson/Callide Valleys in central Queensland. The study aimed to evaluate the genetic structure in this region over a 12-month period (September 2000-August 2001). The results detected genetic shifts in H. armigera collections, with genetic changes occurring month by month. Collections in any month were genetically distant from the preceding months collections. There was no observed difference between collections of H. armigera from the Biloela region and those found in the Theodore region of central Queensland. The data support the current area-wide management strategies for H. armigera by reinforcing the importance and contribution of local management practices. The study also indicates a need for the continuation of regional or Australia-wide approaches to management of the low levels of immigration that are occurring, and for future high pest pressure years.

Sugarcane moth borers (Lepidoptera: Noctuidae and Pyraloidea): phylogenetics constructed using COII and 16S mitochondrial partial gene sequences

Sugarcane moth borers are a diverse group of species occurring in several genera, but predominately within the Noctuidae and Pyraloidea. They cause economic loss in sugarcane and other crops through damage to stems and stalks by larval boring. Partial sequence data from two mitochondrial genes, COII and 16S, were used to construct a molecular phylogeny based on 26 species from ten genera and six tribes. The Noctuidae were found to be monophyletic, providing molecular support for the taxonomy within this subfamily. However, the Pyraloidea are paraphyletic, with the noctuids splitting Galleriinae and Schoenobiinae from the Crambinae. This supports the separation of the Pyralidae and Crambinae, but does not support the concept of the incorporation of the Schoenobiinae in the Crambidae. Of the three crambine genera examined, Diatraea was monophyletic, Chilo paraphyletic, and Eoreuma was basal to the other two genera. Within the Noctuidae, Sesamia and Bathytricha were monophyletic, with Busseola basal to Bathytricha. Many species in this study (both noctuids and pyraloids) had different biotypes within collection localities and across their distribution; however the individual biotypes were not phylogenetically informative. These data highlight the need for taxonomic revisions at all taxon levels and provide a basis for the development of DNA-based diagnostics for rapidly identifying many species at any developmental stage. This ability is vital, as the species are an incursion threat to Australia and have the potential to cause significant losses to the sugar industry.

Gene-flow between populations of cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) is highly variable between years.

Both large and small scale migrations of Helicoverpa armigera Hübner in Australia were investigated using AMOVA analysis and genetic assignment tests. Five microsatellite loci were screened across 3142 individuals from 16 localities in eight major cotton and grain growing regions within Australia, over a 38-month period (November 1999 to January 2003). From November 1999 to March 2001 relatively low levels of migration were characterized between growing regions. Substantially higher than average gene-flow rates and limited differentiation between cropping regions characterized the period from April 2001 to March 2002. A reduced migration rate in the year from April 2002 to March 2003 resulted in significant genetic structuring between cropping regions. This differentiation was established within two or three generations. Genetic drift alone is unlikely to drive genetic differentiation over such a small number of generations, unless it is accompanied by extreme bottlenecks and/or selection. Helicoverpa armigera in Australia demonstrated isolation by distance, so immigration into cropping regions is more likely to come from nearby regions than from afar. This effect was most pronounced in years with limited migration. However, there is evidence of long distance dispersal events in periods of high migration (April 2001-March 2002). The implications of highly variable migration patterns for resistance management are considered.

Distribution and phylogenetic relationships of Australian glow-worms Arachnocampa (Diptera, Keroplatidae)

Glow-worms are bioluminescent fly larvae (Order Diptera, genus Arachnocampa) found only in Australia and New Zealand. Their core habitat is rainforest gullies and wet caves. Eight species are present in Australia; five of them have been recently described. The geographic distribution of species in Australia encompasses the montane regions of the eastern Australian coastline from the Wet Tropics region of northern Queensland to the cool temperate and montane rainforests of southern Australia and Tasmania. Phylogenetic trees based upon partial sequences of the mitochondrial genes cytochrome oxidase II and 16S mtDNA show that populations tend to be clustered into allopatric geographic groups showing overall concordance with the known species distributions. The deepest division is between the cool-adapted southern subgenus, Lucifera, and the more widespread subgenus, Campara. Lucifera comprises the sister groups, A. tasmaniensis, from Tasmania and the newly described species, A. buffaloensis, found in a high-altitude cave at Mt Buffalo in the Australian Alps in Victoria. The remaining Australian glow-worms in subgenus Campara are distributed in a swathe of geographic clusters that extend from the Wet Tropics in northern Queensland to the temperate forests of southern Victoria. Samples from caves and rainforests within any one geographic location tended to cluster together within a clade. We suggest that the morphological differences between hypogean (cave) and epigean (surface) glow-worm larvae are facultative adaptations to local microclimatic conditions rather than due to the presence of cryptic species in caves.

Isolation and characterization of nine microsatellite loci from the Hawaiian grouper Epinephelus quernus (Serranidae) for population genetic analyses.

The availability of variable genetic markers for groupers (Serranidae) has generally been limited to mitochondrial DNA. For studies of population genetic structure, more loci are usually required; particularly useful are those that are nuclear in origin such as microsatellites. Here, we isolated and characterized 9 microsatellite loci from the endemic Hawaiian grouper Epinephelus quernus using a biotin-labeled oligonucleotide-streptavidin–coated magnetic bead approach. Of the 20 repeat-containing fragments isolated, 15 had sufficient flanking region in which to design primers. Among these, 9 produced consistent polymerase chain reaction product, and 6 were highly variable. These 6 loci were all composed of dinucleotide repeats, with the number of alleles ranging from 6 to 18, and heterozygosities from 33.3% to 91.7%. The high levels of variability observed should make these markers useful for population genetic studies of E. quernus, and potentially other epinephelines.

Population dynamics and gene flow of Helicoverpa armigera (Lepidoptera : Noctuidae) on cotton and grain crops in the Murrumbidgee Valley, Australia

Abstract The population dynamics of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in the Murrumbidgee Valley, Australia, has been characterized using five highly variable microsatellite loci. In the 2001–2002 growing season, there were very high levels of migration into the Murrumbidgee Valley with no detectable genetic structuring, consistent with previous analyses on a national scale. By contrast, there was significant genetic structuring over the 2002–2003 growing season, with three distinct genetic types detected. The first type corresponded to the first two generations and was derived from local individuals emerging from diapause and their progeny. The second genetic type corresponded to generation 3 and resulted from substantial immigration into the region. There was another genetic shift in generation 4, which accounts for the third genetic type of the season. This genetic shift occurred despite low levels of immigration. During the third generation of the 2002–2003 growing season, different population dynamics was characterized for H. armigera on maize, Zea mays L., and cotton Gossipium hirsutum L. Populations on cotton tended to cycle independently with very little immigration from outside the region or from maize within the region. Maize acted as a major sink for immigrants from cotton and from outside the region. If resistance were to develop on cotton under these circumstances, susceptible individuals from maize or from other regions would not dilute this resistance. In addition, resistance is likely to be transferred to maize and be perpetuated until diapause, from where it may reemerge next season. If low levels of immigration were to occur on transgenic cotton, this may undermine the effectiveness of refugia, especially noncotton refugia.

A protocol for the efficient screening of putatively transformed plants for the bar, the selectable marker gene, using the polymerase chain reaction

Amplification of thebar gene usingTaq DNA polymerase in PCR is often not successful, possibly due tobars high GC content. We describe a PCR protocol in which reliable amplification at a sensitivity of one gene copy per genome (in this study, barley) present in the reaction was achieved using a novel pair of primers and Expandtm High Fidelity DNA polymerase mix (Boehringer Mannheim). This method should allow for rapid screening of plants putatively transformed withbar.

A method for extraction of total RNA fromPinus radiata and other conifers

Carbohydrates, polyphenolic compounds, terpenoids and tannins interfere with the extraction of intact, uncontaminated total RNA from conifers. A method for extraction of total RNA fromPinus radiata is described. This method uses cesium trifluoroacetate in the ultracentrifugal separation of RNA to overcome the problems of co-purification of contaminating secondary metabolites.