title: Universal endogenous gene controls for bisulphite conversion in analysis of plant DNA methylation.
abstract: Accurate analysis of DNA methylation by bisulphite sequencing depends on the complete conversion of all cytosines into uracil. Until now there has been no standard or universal gene identified as an endogenous control to monitor the conversion frequency in plants. Here, we report the development of PCR based assays for one nuclear gene IND (INDEHISCENT) and two mitochondrial genes, NAD (NICOTINAMIDE ADENINE DINUCLEOTIDE) and ATP1 (ATPase SUBUNIT 1). We demonstrated their efficacy as bisulphite conversion controls in Brassica and other plant taxa. The target regions amplified by four primer pairs were found to be consistently free from DNA methylation. Primer pairs for IND.a and NAD were effective within Brassica species, whereas two primer pairs for ATP1 provided reliable controls across a representative range of dicot and monocot angiosperm species. These primer sets may therefore be adopted as controls in plant methylation analysis for a wide range of studies.
authors: National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, P, R, China. graham.king@scu.edu.au.
authors: Jing
authors: J
authors: Wang
authors: Chongnan
authors: C
authors: Wang
authors: Yan
authors: Y
authors: Long
authors: Clare
authors: C
authors: Hopkins
authors: Smita
authors: S
authors: Kurup
authors: Kede
authors: K
authors: Liu
authors: Graham J
authors: GJ
authors: King
authors: Jinling
authors: J
authors: Meng