LTERN Data Portal

Leaf tissue was collected over the summer 2012-13, with additional sampling at Mt. Stirling and Mt. Howitt in October 2013. For each species, a minimum of 30 ~1 gram samples of fresh growth was collected within an area of approximately 100 m2, specifically avoiding adjacent individuals to reduce the possibility of sampling closely related individuals. This is particularly important for prostrate spreading species such as Pimelea alpina, where clonal/spreading individuals can be difficult to distinguish. GPS coordinates were logged at each site within. Individual samples with unique identifiers were preserved in paper coffee filters and desiccated with silica gel.

For each species at each location 30 mg leaf of tissue were weighed out and sent to the Australian Genome Research Facility Ltd (AGRF) Plant Genomics Centre, Adelaide. Extraction was performed as per the NucleoSpin® 96 Plant II protocol (Machery-Nagel Inc., Düren, KO, GER). DNA quantitation was performed as per the QuantiFluor® dsDNA System (Promega Inc,, Madison, NY, USA).

Genotyping By Sequencing (GBS) was chosen for the study. GBS facilitates genotyping across populations for tens of thousands to hundreds of thousands of anonymous genetic markers (Elshire et al. 2011; Thudi et al. 2012; Lu et al. 2013).

Due to financial limitations of the project and the number samples sequenced, samples were pooled at the population level for each species. Anderson et al. (2014) recently outlined potential issues associated with pooling samples for molecular ecology, in light of this, stringent DNA quantifications and standardisation steps, coupled with stringent SNP filtering criteria were implemented.

For each species ten individual DNA extractions from each site were pooled to a total of 500 ng. Subsequently volumes were standardised by evaporating the samples with a CentriVap® Centrifugal Concentrator (Labcono, Kansas City, MO, USA) at 45ºC for two hours, and re-suspending in nuclease-free water. A 0.75 ng/µl concentration of a PstI barcode adapter working stock was aliquoted at 3 µl per sample. Samples were digested with 0.2 µl of PstI-HF® (New England BioLabs, Ipswich, MA, USA) enzyme at 37ºC for two hours. The samples were then purified using a MinElute® PCR purification kit (QIAGEN, Redwood City, CA, USA). A volume of 20 µl of T4 DNA ligase (Bioline, Taunton, MA, USA) was used to ligate the PstI adaptors, incubated at 16ºC for two hours and 80ºC for 80 minutes. PCR amplification was performed on 10 µl of purified post-ligation product with 25 µl of MyTaq™ HS Red Mix (Bioline, Taunton, MA, USA) and 1 µl at 10 µM each of Illumina Dual Index Sequencing Primers 1 & 2 (Illumina Inc., San Diego, CA, USA). PCR conditions were; 72º for 5 min and 95º for 1 min, followed by; 95º for 30 s, 65º for 30 s, 72º for 30 s for 24 cycles, followed by; 72º 5 min.

Quantitation, quality checking and size fractionation was performed on a MCE®-202 MultiNA with a DNA-1000 kit (Shimadzu, Kyoto), see appendix 2 for virtual gel captures.

Libraries were subsequently pooled equimolar into single 0.6 µl microcentrifuge tube and sequenced on a single HiSeq™ 2000 lane at the Australian National University Biomolecular Resource Facility.

References

Anderson EC, Skaug HJ, Barshis DJ (2014) Next‐generation sequencing for molecular ecology: a caveat regarding pooled samples. Molecular ecology, 23, 502–512.

Elshire RJ, Glaubitz JC, Sun Q et al. (2011) A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species (L Orban, Ed,). PLoS ONE, 6, e19379.

Lu F, Lipka AE, Glaubitz J et al. (2013) Switchgrass Genomic Diversity, Ploidy, and Evolution: Novel Insights from a Network-Based SNP Discovery Protocol. PLoS Genet, 9, e1003215.

Thudi M, Li Y, Jackson SA, May GD, Varshney RK (2012) Current state-of-art of sequencing technologies for plant genomics research. Briefings in Functional Genomics, 11, 3–11.