Rediprime 2 random prime labeling system

DNA hybridization was performed with 10 μg of total DNA digested with EcoR I (Invitrogen, Carlsbad, CA). The digested DNA fragments were separated by electrophoresis on a 1.0% (w/v) agarose gel at 55 v for 11 h and then transferred to a nylon membrane (GE Healthcare Life Sciences, Pittsburgh, PA). The LTRs of three retrotransposons were used to design primers to amplify DNA from Nipponbare. The primers used were as follows: CRR1 (Forward, 5′-GCAAGGACCAATGACTAGAG-3′; Reverse, 5′-CAAGCAAGAACAAGTTGACA-3′); RIRE3 (Forward, 5′- GTGCATGGTTTTGATAGTAGC-3′; Reverse, 5′-GGTGTACATCTTTACCCACAA -3′) and Hopi (Forward, 5′-TAGAGACTTGAGGCAGACACG -3′; Reverse, 5′- GTCACAAATCGGTCATTCTTG-3′). The PCR products were labeled with [α−³²P]-dCTP using the rediprime II random prime labeling system (GE Healthcare Life Sciences, Pittsburgh, PA) according to the manufactures instructions. Blots were hybridized at 58.5°C for overnight and washed with 1.5× SSC solution for 30 min and 1× SSC for 30 min. The membrane was exposed on a Fuji-image plate and the hybridization signals were captured using a Fujifilm FLA-5100 multifunctional scanner.