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Molecular Laboratory report 2011-2013
Carolyn Connelly - Molecular Laboratory Coordinator
The Molecular Laboratory is a critical resource located in the National Herbarium of NSW. It is utilised by researchers in Plant Diversity and Evolutionary Ecology. Molecular techniques are taught to new students and staff.
Chemicals and materials stored in the Science and Conservation chemical storeroom are under review. In some cases techniques have changed so that less toxic chemicals are now used or the technology has been superceded. Several pails of solid waste and drums of liquid waste have been safely disposed of via a contracted company.
New Techniques - RNA
Protocols for studying RNA were explored and developed. The plant tissue chosen was found to be critical to success; rapidly growing tissue was the best source of RNA. In addition the correct preservation of plant tissue prior to extraction is important if immediate extraction is unavailable. Several commercial kits were trialled for quality and quantity of yield. The Biodiversity adaptation across an environmental gradient project utilised these techniques. Seeds from Isopogon anemonifolius and Petrophile pulchella (Proteaceae) were collected from four populations in the Sydney sandstone region, each population representing a specific rainfall and altitude regime. Seed were extracted from eight mother plants for each species from each population and germinated at 20 degrees Celsius. Seed collected from one population of Petrophile canescens were also germinated. RNA was extracted from 25 seedlings (two cotyledon stage) for each population for both species and also the one P. canescens population. RNA from each population was pooled and samples sent for gene function analysis for RNA-seq on the Illumina Hiseq 2000. Nine libraries were multiplexed in one lane and over 26 Gbp of data were obtained. Data analysis is currently underway.
New Equipment - Qubit fluorometer
The Biodiversity adaptation project and another project studying rainforest species required accurate readings of DNA and RNA. No commercial companies offered a service for these precise measurements. The existing spectrophotometer in the laboratory was unable to provide the same sensitivity as measurement using fluorometry. Purchase of the Qubit fluorometer (pictured) has provided an accurate means for quantification of DNA and RNA, which is essential for genomics work. The costs of incorrect concentrations are significant, for example, the next generation sequencing (NGS) process as currently 24 to 48 samples can be loaded and run in a lane for NGS, where each lane costs nearly $4000. The Qubit instrument has no ongoing cost to run; the consumable cost is about $0.94 per sample.
Laboratory volunteer Gaye Wingett continued curation of plant tissues stored in the freezers this year. After barcoding, envelopes are scanned to reduce the time envelopes are at room temperature. The databasing information is keyed in from the image files. Aurelia Webster-Hawes and Brendan Molloy assisted Hannah McPherson and Marlien van der Merwe for 1 day per week.
Dr Adam Marchant resigned from the Royal Botanic Gardens and Domain Trust (the Trust). He was involved with the Laboratory for nearly 18 years.
New Research Projects
Many of the projects underway in the Molecular Laboratory are reported in more detail elsewhere on this website.
Two new ABRS Post Doctoral Fellows commenced molecular work. Trevor Wilson commenced Post Doctoral studies on systematics of the Prostantheroideae (Lamiaceae); and Yola Metti on phylogeny of the Laurencia complex (Rhodophyta, Rhodomelaceae).
Marco Duretto and Margaret Heslewood commenced molecular research on Rutaceae focusing on Boronia sensu lato and the Phebalium Group of genera.
Peter Wilson and Margaret Heslewood continued molecular research within the Myrtaceae, clarifying generic relationships within the Leptospermeae and Chamelaucieae which will lead to the recognition of several new genera, and new projects investigating several relationships within the tribe Myrteae. Molecular research also commenced on the Haloragis exalata group (Haloragaceae), clarifying the species status of a disjunct population at Geehi.
Student supervision is an important role of the Royal Botanic Gardens and Domain Trust (the Trust), and this year several new students started with us. Susan Rutherford commenced a PhD investigating speciation mechanisms in a group of closely related eucalypts called the green ashes. The project title is 'Adaptive variation vs vicariance: what drives speciation in eucalypts?' Susan will be utilising Diversity Arrays Technology (DArT, a recently developed technique that sequences across the genome). A phylogeny will be constructed initially from recognised species in the group; then focusing on only a few species for a population study. From that part of the project, molecular data will be used in combination with distributional, environmental, morphological and ecological data to study speciation and adaptation of each taxa across an altitudinal gradient in the Sydney region, from the coast to the upper Blue Mountains. Both high quality and quantity (50-100 ng/uL) of DNA is required for DArT analysis. Susan’s PhD supervisors are Maurizio Rossetto and Peter Wilson and Stephen Bonser from University of New South Wales.
Oliver Paul commenced a Master of Science at the University of Sydney (USYD). Oliver is from the Papua New Guinea National Herbarium, Papua New Guinea Forest Research Institute, Lae, Papua New Guinea is undertaking a study of the systematics of Polyosma (Escalloniaceae) in Australia and Papua New Guinea using variable nucleotide sequences so that he can reconcile genetic variation with morphological variation. He will also evaluate the genetic and morphological diversity within the geographically wide-ranging species Polyosma cunninghamii (in eastern Australia) and P. integrifolia (from Papua New Guinea). Oliver is co-supervised by Barry Conn and Murray Henwood (USYD).
Charles Foster undertook molecular training in the Laboratory in order to study Logania towards an Honours degree at USYD. He is supervised by Barry Conn and Murray Henwood (USYD) and Simon Ho (USYD).