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/

• 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

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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?

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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

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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)

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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’

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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

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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

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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

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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.