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Advanced DNA, Identification and Forensic Facility ADIFF

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Advanced DNA, Identification and Forensic Facility ADIFF 23/08/2018 Plant pollination networks DNA barcoding applications Kor-Jent van Dijk, Elly Dormont, Andrew Lowe @ProfALowe linkedin.com/in/profandylowe/ [email protected] www.andylowe.org Advanced DNA, Identification and Forensic Facility ADIFF • Illegal wildlife trade • Species identification • Timber Tracking • Human identification • Crime scene investigation • Degraded remains • Forensic odontology • Environmental Analysis • Soil Analysis • Diet identification • Conservation management • Human health 1 23/08/2018 DNA – To monitor environmental, agricultural and public health outcomes Bioinformatics and analysis Next Generation Sequencing DNA extraction & library preparation Environmental samples • Soil • Leaves • Pollen • Air • Human skin & nose (Thomsen & Willerslev 2015) What we may want to know? Who is present in a sample? How do samples compare? What is the role of these taxa? 2 23/08/2018 Secure crop pollination through revegetation Apple, Canola, Almonds, Lucerne • Pollinator assessment • What bees pollinate crops? Honey-Native • What native plants support them? • When should they flower? Competition? • DNA Barcoding • Develop barcodes to identify pollen and bees • DNA Metabarcoding • Harvest pollen to identify host plants and ID bees 3 23/08/2018 Hybrid Capture Approach to generate Genomic Reference database Adaptor Ligation & Capture Pool DNA Sequence Voucher DNA Fragment Probe Hybridization of targets Library Deposit barcodes in database Dramatic increase in data we can gain from specimens… Last run: 200 million sequences 30.11 billion bases 80 specimens 380 million average For same cost (~$100 p sample) : • We could do 2-3000 bp with Sanger sequencing Now we get: • 10s of thousands of bp • 70% of Chloroplast • All traditional barcoding loci • Exon and Intron data for dozens of nuclear UCE’s (Ultra Conserved Elements) 4 23/08/2018 Why Hybrid Capture • Works on degraded samples • 180 y old sample worked ( Inga umbellifera, 1835) • 6ng DNA as input has been used • 100s to 1000s of loci can be sequenced at a relatively low cost • Can sequence more than a max of 500bp if paired end reads are used • Can deal with hybrids and or contaminations Eucalyptus leucoxylon transcriptome MyBait probe design • Used available transcriptomic and genomic resources • Mapped Eucalyptus leucoxylon transcriptome sequence to the Arabidopsis thaliana genome 85% & 1x • Genes with >85% mapping similarity Arabidopsis thaliana • Select only single copy loci in reference genome genomes (cf. 60 spp on Phytozome) • 760 genes found • 30 nuclear gene regions selected for first trial with exons and intron regions in ‘~200 bp vicinity’ 5 23/08/2018 herbarium specimens 1.00E+07 silica material 1.00E+06 2010 1890 herbarium material 1991 1.00E+05 1.00E+04 1.00E+03 1.00E+02 number of reads (log scale) (log of reads number 1.00E+01 1.00E+00 1880 1900 1920 1940 1960 1980 2000 2020 year of collection (herbarium) mean - herbarium : 800,000 reads/sample silica : 1,000,000 reads/sample 6 23/08/2018 basal eudicots conifers Dilleniales basal angiosperms rosids asterids monocots Plant Sampling Process State Herbarium Exemplar Project • Vouchers and photos from field • High resolution image • Pollen images • Leaf venation • Seed collection (image and storage) • DNA sample > DNA sequence • Develop bait based DNA reference library toolkit • Currently in final stages of pilot testing Allow use of wider range of vouchers including older specimen 7 23/08/2018 Hybrid Capture Approach to barcode environmental DNA (Pollen) against Genomic Reference database DNA Adaptor Ligation & Capture Sequence & compare Pollen mix Fragment Probe Hybridization of targets to reference database Apply to examine pollination networks in and around crops • Apples, pears, cherries, strawberries 8 23/08/2018 For further information: Email: [email protected] Blog: www.andylowe.org Twitter: @profalowe Bio and papers: www.adelaide.edu.au/directory/andrew.lowe Thanks •Plant and Ecosystem adaptation and restoration • Dr Martin Breed, Associate Professor Zdravko Baruch, Dr Nick Gellie, Dr Matt Christmas, Mr John McDonald, Mr Stefan Caddy-Retallic, Mr Jacob Mills, Ms Dona Kireta, Ms Kym McCallum •DNA timber tracking and genetic resources • Dr Elly Dormontt, Dr Kor Jent van Dijk, Dr Jen Young, Dr Bianca Dunker, Dr Rainbo Belton, Dr Patti Fuentes-Cross, Dr Craig Costion, Mr Duncan Jardine, Ms Marlee Crawford, Dr Shane Tobe •Ecosystem monitoring • Associate Professor Nikki Thurgate, Associate Professor Ben Sparrow, Ms Michelle Rodrigo, Dr Greg Guerin, Dr Jeff Foulkes, Dr Andrew Tokmakof, Mr Andrew White, Mr, Ian Fox, Mr Jim Deed Mr Caleb Coish, Mr Finn Hutchings, Mr Craig Walker, Dr David Turner, Dr Anita Smyth, Mr Emrys Leitch, Mr Mosheh Eliyahu, Mr Matt Schneider, Mr Tom Saleeba, Ms Christina Pahl, Ms Sally O’Neill, Mr Rick Filton, This project is supported by funding from the Australian Government Department of Agriculture and Water Resources as part of its Rural R&D for Profit program. 9.
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