Alexandra Olarte/Edwin Pang/Nitin Mantri, RMIT University
Manufacture protocol
Plant material and genomic DNA extraction: In order to obtain a genomic DNA (gDNA) representation for the subtraction, a total of 151 species including angiosperms and non-angiosperms were sourced. Non-angiosperms were collected from Toolangi State Park, Victoria (Australia) and identified (Duncan and Isaac, 1994). Angiosperms were obtained only from verified nursery species; a total of 126 species were sourced to represent all angiosperm clades. In addition, a total of ten Salvia species (42 plants) were sourced to represent the different centres of diversity around the world. Salvia miltiorrhiza and S. sinica Migo plants were obtained from seeds which were used in a previous study (Li et al., 2009). The other Salvia species were obtained from verified specimens from various plant nurseries. Total DNA was extracted from fresh leaves using a modification of the standard CTAB procedure (Doyle and Doyle, 1987) and subsequent cleanup was performed by using the DNeasy® column of the DNeasy® Plant Mini Kit (Qiagen) following the protocol in the user manual.The quantity of DNA was measured using Eppendorf spectrophotometer whilst the quality/integrity was assessed by 1.5% agarose gel electrophoresis.
Genomic DNA subtraction and library construction: All DNA samples were pooled based on the Angiosperm Phylogeny Group (2009) classification (Bremer et al., 2009) in order to obtain representations of the following seven groups: Salvia (Tester pool), non-angiosperms, Monocots, Magnoliids, Rosids, Asterids (excluding Lamiaceae), and Eudicots not placed in either Rosids or Asterids subclades (Eudicots and Core Eudicots). Subtraction was performed using the PCR-Select™ cDNA Subtraction Kit (Clontech). The Salvia pool (tester) was represented by equal amounts of DNA extracted from 42 different plants of which 25 were Salvia miltiorrhiza (5 plants from 5 different lines). The driver pool was obtained by bulking 700 ng of each representation with the exception of the Salvia pool. Subsequently, 4 μg of pooled gDNA from each group was restriction digested in a 50 μL reaction using 5U of HaeIII and AluI (New England Biolabs). As previously described (Mantri et al., 2012), the digested diver gDNA pool was subtracted from digested tester gDNA pool to isolate Salvia-specific DNA. The Salvia-specific DNA fragments were cloned into pGEM-T® Easy Vector (Promega, Madison, WI) and transformed into Escherichia coli JM109 competent cells (Promega, Madison, WI).
Microarray construction and printing: The cloned inserts were PCR amplified from the corresponding plasmid using nested primers 1 and 2R (Clontech). After that, PCR products were precipitated in 96% ethanol and 3 M sodium acetate (pH 5.2). The precipitation was carried out at -20 °C overnight. The pellets obtained were washed with 70% ethanol, air dried and resuspended in 10 µl of 50% DMSO. Finally, a total of 285 clones with inserts ranging from 250 bp to 1000 bp, were transferred individually into a 384-well plate (Genetix, Hampshire, UK) together with positive and negative controls. Among the positive controls were three housekeeping genes (ribulose-1,5-bisphosphate carboxylase/oxygenase, ribosomal RNA, and chlorophyll a/b binding protein) sourced from Cicer arietinum (Coram and Pang, 2005). The 285 clones together with the controls were printed on aminosilane-coated slides using a BioRobotics® MicroGrid II Compact arrayer (Genomic Solutions) at RMIT University, Australia. Eight subarrays were gridded on a Corning® GAPS II coated slide (Corning Incorporated Life Sciences, Acton, MA). Each subarray was composed of 285 samples and 15 controls.
Coating
GAPS II
Description
Specific for all species belonging to the Salvia genus.
Only 10 subtracted fragments (clones) were sequenced.
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