Cytosine methylation polymorphisms in cotton using TD-MS-RAPD-PCR

Methylation in DNA and chemical modification in histone proteins are the two most studied epigenetic mechanisms in plants. There exist low-throughput and high-throughput DNA methylation detection techniques in epigenetic research. In this study, touch-down polymerase chain reactions methylation sensitive-random amplified polymorphic DNA (TD-MS-RAPD) technique was used to investigate cytosine methylation differences among three cotton varieties; Texas Marker 1 (TM-1), Pima 3-79 (3-79) and Maydos Yerlisi (MY), belonging to Gossypium hirsutum L., G. barbadense L., and G. herbaceum L., respectively. Genomic DNA samples extracted from the mature seeds of these varieties were treated with MspI, a relative methylation-insensitive restriction enzyme and HpaII, a methylation-sensitive restriction enzyme before touch-down polymerase chain reactions. Among 16 oligonucleotide primers used, three primers (AT03, W15, and C08) resulted in methylation polymorphisms among three varieties. TD-MS-RAPD-PCR method was cost-effective, required a simple method and basic instrumentation, and could easily be performed in our laboratory with basic setup using a regular DNA thermal cycler and DNA gel electrophoresis system, however, the level of methylation polymorphisms detected with this method were very low in cotton. We concluded that the low level of polymorphisms among the three cotton species were probably due to low occurrences of CCGG sites within the cotton genome. We also noted that TD-MS-RAPD-PCR method could be used in primary scanning studies in epigenetic research.