Kirsten D Scott

Analysis of SSRs derived from grape ESTs

Abstract One hundred and twenty four microsatellites were isolated from analysis of 5000 Vitis expressed sequence tags (ESTs). A diversity of dinucleotide and trinucleotide simple sequence repeat (SSR) motifs were present. Primers were designed for 16 of these SSRs and they were tested on seven accessions. Ten of the sixteen primer pairs resulted in PCR products of the expected size. All ten functional primers were polymorphic across the accessions studied. Polymorphisms were evident at the level of cultivars, Vitis species, and between related genera. SSRs that were from the 3′ untranslated region (3′UTR) were most polymorphic at the cultivar level, the 5′ untranslated region (5′ UTR) SSRs were most polymorphic between cultivars and species, and those SSRs within coding sequence were most polymorphic between species and genera. These results show that EST-derived SSRs in Vitis are useful as they are polymorphic and highly transferable. With EST SSRs being applicable to studies at several taxonomic levels, the large number of SSRs (approximately 1000) that will be available from an expanded EST database of 45 000 will have many potential applications in mapping and identity research.

Analysis of grape ESTs: global gene expression patterns in leaf and berry.

Analysis of 2479 ESTs from Vitis vinifera berry tissue and 2438 from leaf revealed that 1% of the ESTs match to known Vitis proteins, 72% to plant proteins, 11% to non-plant, and 16% had no match (P[N]>0.5). The levels of redundancy were similar in the leaf and berry libraries. Only 12% of the genes matched by the ESTs were common to both libraries indicating marked differences in the genes expressed in the two tissues. The abundance of transcripts with predicted cellular roles in leaf and berry were estimated by classifying the primary BLAST matches to known proteins (score >80) into functional categories. Thirty-six percent of the leaf transcripts were involved in photosynthesis, compared to 3% in the berry. This is a much higher proportion of transcripts involved with a function limited to specialized cells, than was found when transcripts of 33 human tissues were compared using a similar approach, suggesting plant cells may involve their cellular machinery to a greater extent in specialized activities than animal cells. Relatively enhanced expression of specific transcription factors, and genes involved in defense, detoxification, stress response, proteolysis, trafficing, and signal transduction, suggests berry tissue is actively engaged in responding to environmental stimuli.

AFLP markers distinguishing an early mutant of Flame Seedless grape

Molecular markers have been frequently used to differentiate grape species and cultivars. There are fewer cases where molecular markers have been used to differentiate grape clones within a cultivar, or for the demarcation of somatic mutants from parental clones. This study reports the first successful utility of AFLPs for the differentiation of somatic mutants from their parental grapevine line, and discusses the potential for similar AFLP applications. The somatic mutant analysed demonstrates earlier budburst characteristics than the Flame Seedless line from which is arose. Analysis of 64 AFLP primer combinations in silver stained polyacrylamide produced in excess of 3000 markers in Vitis vinifera, and provided two markers which differentiated the somatic mutant, from its parental line. One marker was 440 bpin length and was produced with primer combinationEcoR1-AT and Mse1-CTT. The second marker was 340 bp in length and generated with primer combination EcoR1-TC and Mse1-CAC.

Is the genus Cissus (Vitaceae) monophyletic: evidence from plastid and nuclear ribosomal DNA

Abstract The trnL (UAA) intron and ITS1 region were sequenced to investigate relationships among the five genera of Vitaceae present in Australia relative to Vitis. Congruent results were obtained between separate and combined data sets, with all major clades being shared among trees. All bootstrap consensus trees obtained from single sequences or combined analysis strongly suggest that Cissus is polyphyletic, corroborating the morphological inconsistencies reported previously. Cissus opaca and Clematicissus angustissima consistently grouped in a common clade. A further four taxa (C. antarctica, C. hypoglauca, C. oblonga, and C. sterculiifolia) also grouped within a clade disjunct from the main Cissus clade. Our results suggest that these five species currently classified as Cissus should be segregated from the genus. Of further interest is the close relationship between Cayratia and Tetrastigma. Overall, the results presented provide new insights into the relationships within a number of Vitaceae genera and suggest directions for future studies. Communicating Editor: Aaron Liston

Intergeneric relationships in the Australian Vitaceae: new evidence from cpDNA analysis

Taxa related to important agricultural species are likely to contain a considerable amount of potentially valuable genetic diversity. Nevertheless, before breeding programs or gene discovery projects can be initiated it is important to understand the phylogenetic relationships between the species involved. A component of a major gene discovery project in grapes at the Centre for Plant Conservation Genetics (Southern Cross University, Australia) is directed at the discovery of novel genes in native Vitaceae. As a result a study was conducted in order to assess the phylogenetic relationships between V. vinifera and the native members of the three major Australian genera: Cayratia, Cissus and Tetrastigma. CpDNA sequence analysis (from the trnL intron) adequately resolved intergeneric relationship between the majority of the species studied and provided some useful new information on the phylogenetic relationships within the Vitaceae. This preliminary project identified two species, C. hypoglauca and C. sterculiifolia, as being closely related to V. vinifera and worthy of further in-depth investigation.

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.

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.