Alexandra Olarte/Nitin Mantri/Edwin Pang, RMIT University
Manufacture protocol
In order to develop a gDNA representation for the subtraction, DNA from a total of 143 species including angiosperms and non-angiosperms were soured. 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 118 species were sourced to represent all angiosperm clades. Additionally, a total of 24 lines were used to represent the Echinacea genus. Five Echinacea species, as mentioned by McGregor (E. angustifolia, E. paradoxa, E. pallida, E. purpurea and E. tennesseensis), were obtained from three different sources. The other four species (E. atrorubens, E. laevigata, E. sanguinea, E. simulata) could not be obtained as quarantine restrictions prevented importation into Australia. Nineteen of the 24 lines were selected from the germplasm collection of the U.S. National Plant Germplasm System maintained by the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS). The other remaining lines were obtained from Botanical Resources Australia (Tasmania) and from verified specimens from a specialized plant nursery (The Diggers Club, Dromana Victoria). 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: all Echinacea species (subtraction pool), Asterids (excluding Asteraceae), non-angiosperms, Monocots, Magnoliids, Rosids, and Eudicots not belonging to the Rosids or Asterids (Eudicots and Core Eudicots). Subtraction was performed using the PCR-Select™ Bacterial Genome Subtraction Kit (Clontech), following the manufacturer's protocol. The Echinacea pool (tester) was prepared by mixing equal amounts of DNA extracted from the 24 genotypes mentioned above. The driver pool was formed by bulking 700 ng of each non-Echinacea representation [Asterids (excluding Asteraceae), non-angiosperms, Monocots, Magnoliids, Rosids, and Eudicots not belonging to the Rosids or Asterids]. 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 Echinacea-specific DNA. The Echinacea-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 283 Echinacea-specific DNA clones were amplified in 100 µl PCR reactions using nested primers 1 and 2R (Clontech). The template used for the amplification was obtained by mixing 10 µl of bacterial cell culture with 10 µl of MilliQ water and then heated at 100°C for 10 min to disrupt the cells and release the plasmid DNA. Then, 1.5 µl of this sample was used as template. 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 283 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 283 clones, together with the controls, were printed on aminosilane-coated slides using a BioRobotics® MicroGrid II Compact arrayer (Genomic Solutions) at RMIT University, Australia. Ten subarrays were gridded on a Corning® GAPS II-coated slide (Corning Incorporated Life Sciences, Acton, MA). Each subarray was composed of 283 samples and 17 controls.
Coating
GAPS II
Description
Specific for all species belonging to the Echinacea genus.
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