Botanic Gardens Trust, Sydney, Australia

Molecular Laboratory report 2010-2011

Carolyn Connelly - Molecular Laboratory Coordinator

The Laboratory currently supports approximately 20 researchers in Plant Diversity and Evolutionary Ecology research. The molecular data obtained in the laboratory is used for taxonomic projects and describing broader geographic patterns from plant population studies.

Carolyn Connelly took on a new role as the acting Laboratory Manager in September. Two current focus areas are rationalising storage and safety. Storage, curation and retrieval of plant tissue and genomic DNA samples held in the laboratory freezers are being reviewed.

Scientific volunteers - curation of the frozen tissue collection: January saw the commencement of a new project for scientific volunteers; to curate the valuable frozen preserved leaf material collected for DNA research. Principally this involves sorting dried leaf material from silica gel. Initially, samples were collected directly into plastic bags containing silica to dry them in the field. Meticulous sorting is required to separate the samples from the silica as the silica needs regular replacement. Samples are repackaged into envelopes then stored over fresh silica gel. The first volunteer, Daria Sosna, a student at USYD repackaged samples collected by Niko Strieber during her PhD studies in 2001. Curated samples are planned to be databased, linked in the Herbarium database to voucher specimens.

New Research Projects

Climatic gradients in Waratahs (Telopea; Proteaceae): From August 2010, Paul Rymer with Cathy Offord, Chris Allen, Katie Thurlby, Peter Weston and Maurizio Rossetto worked to determine the plastic and adaptive response to climatic gradients in Waratahs. This work integrates field, glasshouse and laboratory experiments with molecular analyses (nuclear microsatellites and RNA sequencing). The relative importance of plastic and adaptive responses has direct implications for population resilience to future climate scenarios. In February, Paul (now at University of Western Sydney, Hawkesbury) continued the collaborative research as an Honorary Research Associate (HRA).

Evolutionary history of Helicia (Proteaceae): Sarah Fayed (UTAS) visited from January to March, learning molecular laboratory techniques whilst commencing the molecular systematics component of her PhD project on the interesting evolutionary history of Helicia, the only genus of tropical Proteaceae to speciate and spread extensively and rapidly.

Systematics of the Australian Utricularia (Lentibulariaceae): Richard Jobson commenced work in the laboratory in February. He will be sequencing chloroplast markers trnL-F and rps16, with particular emphasis on subgenus Polypompholyx. A robust phylogenetic hypothesis will provide a framework for understanding the evolutionary and biogeographic history of this lineage. In addition, using sequence and microsatellite data, Richard will also examine phylogeographic patterns and taxonomic limits in the diverse Utricularia dichotoma complex.

Matt Renner commenced a three year ABRS funded Post Doctorate looking at the systematics and phylogeny of bryophytes: Australasian Radula (Radulaceae; Jungermanniopsida).

Completed Projects

A phylogeny of the Australian Lauraceae: Marlien van der Merwe, Paul Rymer and Katie Thurlby worked on assembling a nuclear gene phylogeny of the Australian Lauraceae with focus on the Cryptocarya group. The resultant partial RPBii gene phylogeny includes over 130 sequences representing over 75 species. Katie and Marlien also finalised the cpDNA phylogeny of the Lauraceae. Over 90% of the Australian woody Lauraceae are now included in the cpDNA phylogeny.

Australian Lomatia (Proteaceae): Emma McIntosh (USYD) carried out an Honours project titled 'Promiscuous Proteaceae: Natural hybridisation between Australian Lomatia species'. The research work will be presented in a poster at the IBC in Melbourne.

Technical Officer, Katie Thurlby departed in May after three and a half years in the molecular laboratory. Initially her research work was on Syzygium for an Honours Degree and employed part-time on Telopea population studies. On completion of Honours she assisted with various projects in Evolutionary Ecology.

Ongoing Projects

Australian Restionaceae: Barbara Briggs and Adam Marchant continued studies, developing a more comprehensively sampled data-set of chloroplast gene sequences for subfamily Leptocarpoideae. Analyses of the data-set will assist in determining an appropriate classification of the genera.

Project Camellia (Theaceae): George Orel (Honorary Research Associate), Anthony Curry (Richmond College of TAFE), Adam Marchant and Peter Wilson continue to conduct a multi-disciplinary study on material collected in Viet Nam, Cambodia and Laos including morphological analyses, molecular assessment and environmental appraisals to facilitate the publication of taxonomic papers and conference presentations.

Bicentenary Plant Diversity Program: In July 2010 Hannah McPherson, Paul Rymer and Katie Thurlby prepared DNA samples of 14 rainforest species from 11 different plant families to send to Southern Cross University for sequencing on the Solexa Illumina Next Generation Sequencing (NGS) platform. Thirteen samples were successfully sequenced in either multiplexed or individual runs. For eight of these there is data from the Sydney region and Northern NSW.

Hannah McPherson, Marlien van der Merwe and Paul Rymer have been analysing the data using CLC Genomics Workbench on the new high end PCs. They have constructed whole chloroplast sequences for the eight rainforest species occurring in two different geographic areas and are beginning to explore the data for areas of variation for use in a landscape-level study of rainforest diversity and phylogeography.

Hannah McPherson, Katie Thurlby, Carolyn Connelly and two students on student placement from the Australian Catholic University, Nick Weston and Juelian Siow, prepared approximately 2500 leaf samples for DNA extraction at AGRF in South Australia. The samples comprise population samples from more than 100 rainforest plant species from three distinct geographic areas: Dorrigo, Nightcap and the Border Ranges in Northern NSW. DNA will be used to explore phylogeographic patterns and the signal of landscape-level responses to the climatic cycles of the Quaternary.

Continuing Students

Endymion Cooper has conducted research on the phylogeny and biogeography of family Lepidoziaceae subfamily Lepidozioideae and a revision of Lepidozia and Telaranea. He has completed the sequencing component of his PhD project and will be submitting his thesis later this year. The first chapter of the thesis has been published in Molecular Phylogenetics and Evolution.

Rohan Mellick has conducted research comparing and contrasting molecular and fossil data in Podocarpus elatus, and is currently writing a PhD thesis.

Margaret Heslewood has conducted research on the historical biogeography and phylogeography of the family Cunoniaceae (Oxalidales) in Australasia, and is also currently writing a PhD thesis.

New Equipment

  • Two new high end computers are now housed on level 3 of the Herbarium. Each machine has a 64 bit dual processor and 48GB of RAM. CLC Genomics Workbench and Geneious have been installed on one machine and will primarily be for processing information from sequence data generated by next generation sequencing. The other has various analysis software installed and is available for analysis of large or complex datasets that would be time-consuming on the regular PCs. (IT budget)
  • May - replacement of the laboratory refrigerator, due to ongoing equipment breakdown.
  • June - purchase of a dedicated PCR workstation for RNA research work. Equipment used for RNA work has to be completely free of (contaminating) DNA.
  • Also new into the laboratory is a microplate mixer, purchased to ensure the solutions prepared for DNA amplification in a PCR plate are mixed effectively, increasing the success rate of these reactions.

 

 

 

 

Molecular Laboratory frozen tissue collection
Molecular Laboratory frozen tissue collection has started to be curated by scientific volunteers. Photo: C Connelly

MolLab3
New PCR cabinet for RNA research.
Photo: C. Connelly

Katie Thurlby Laboratory Technician
Katie Thurlby Laboratory Technician has swapped green pastures.
Copyright: Luke Thurlby Photography 2011