title: RMIT University Strawberry-specific Array
status: Public on Feb 09 2017
contact.city: Bundoora
contact.first: Mian-Chee
contact.last: Gor
contact.email: mianchee.gor@rmit.edu.au
contact.state: Victoria
contact.address: Plenty Road
contact.department: School of Applied Sciences
contact.country: Australia
contact.institute: RMIT University
organism: Fragaria x ananassa
manufacture_protocol: In order to develop genomic DNA representation for the subtraction, DNA from five strawberry genotypes (Albion, Juliette, 07-102-41, 07-095-35, 04-069-91) and nine non-angiosperm species (Dryopteris kuratae, Dicksonia antarctica, Asplenium australasicum, Blechnum tabulare, Wollemia nobilis, Cupressus macrocarpa, Juniperus communis, Cycas revoluta and Ginkgo biloba) were sourced. gDNA was extracted using Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Prior to subtraction, genomic representations were prepared by pooling equal amount of DNA extracted from five strawberry genotypes and nine non-angiosperm species into tester and driver pools, respectively to a final amount of 4 µg. The pooled DNA samples were then fragmented overnight with 15 units of AluI and HaeIII restriction enzymes (NEB, Ipswich, MA) in 100 µL of digestion mixture. The subsequent gDNA subtraction was performed using the PCR-SelectTM cDNA Subtraction kit (Clontech, Mountain View, CA) based on Suppression Subtractive Hybridization (SSH) method with a few modifications as described in Olarte et al. (2013). Briefly, the digested tester pool was purified with phenol/chloroform extraction, divided into two portions and ligated with Adaptor 1 and Adaptor 2R, respectively. The ligated products were then subjected to two rounds of tester-driver hybridisation. To perform the hybridisation step, excess driver was added to obtain a tester to driver ratio of 1:60. In addition, the spike-in control was removed by adding approximately 20.4 ng of human skeletal muscle cDNA into the driver pool. Finally, subtracted DNA fragments (specific to strawberry) present in the tester but absent in the driver were selectively amplified by Suppression PCR and further enriched with nested PCR as described in the user manual. The nested PCR products were cloned into pGEM®-T Easy vector and transformed into E. coli JM109 competent cells (Promega, San Luis Obispo, CA) according to the manufacturer’s instructions. For microarray construction, 331 white colonies were randomly amplified using Nested Primer 1 and Nested Primer 2R provided in the PCR-SelectTM cDNA Subtraction kit. The PCR products of 290 strawberry-specific clones ranging from 200 - 1,000 bp were precipitated overnight by adding 0.1 volume of 3M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol. The resulting pellets after centrifugation were washed in 70 % ethanol, air dried and resuspended in 15 µL of 50 % DMSO printing buffer. DNA concentration of each PCR product was normalised to 250 ng/µL. Together with nine negative controls (50% DMSO, Nested Primer 1, Nested Primer 2R, and AluI and HaeIII digested pGEMT-Easy vector), six positive controls (beta-actin, pectate lyase B, alcohol acyltransferase, alcohol dehydrogenase, sesquiterpene synthase and the subtracted PCR product), two printing controls (Cy-3 and Cy-5) and one spike-in controls (aromatase gene derived from the ovary tissue of Murray River Rainbowfish), 10 µL of each strawberry-specific clone was arrayed onto Corning® GAPSTM II coated slides (Corning Incorporated, NY, USA) using a BioRobotics® MicroGrid II Compact array printing robot (Genomics Solutions, Ann Arbor, MI) at RMIT University, resulting in a 308-feature strawberry-specific SDA. Each slide contained two subarrays comprising of six technical replicates, where each technical replicate consisted of 308 samples.