A study on the dna methylation and heterosis relations in maize (Zea mays L.)

Epigenetics is defined as heritable variations in gene expression and functions without the sequence variation in DNA. DNA methylation is the most studied DNA methylation mechanism among the epigenetic regulations. Due to very high occurrence of DNA methylation in cytosine bases, methyl cytosine is called as the fifth base in plants. Cytosine methylation is related with many epigenetic functions in organisms. It is known that epigenetic regulations related with internal and external factors and play important roles on heterosis, bio-defense, vernalization, flowering and phenotypic plasticity. Aims of this dissertation study were (i) investigation of DNA cytosine methylation levels among two maize inbred lines (Zea mays L.) along with their hybrid, (ii) comparison of DNA cytosine level alterations among parents and the hybrid treated with salt (NaCl) and 5-azacytidine (5-azaC), (iii) investigation of the effects of DNA cytosine methylation on heterosis. In this study, DNA cytosine methylation levels (in terms of hypo- and hyper-methylation compared to water control) of 6 day-old seedlings obtained from mature maize inbred lines [Mo17 (♀) and B73 (♂)] and their F1 hybrid [Mo17xB73] treated with 150 mM NaCl and 1.5 mM 5-azaC were determined. In the study, a total of 36 loci consisting of five loci using bisulfite sequencing (BiSeq), 18 loci using uniplex, duplex and multiplex Gene Specific-Methylation Sensetive Amplification Polymorphism (GS-MSAP), 16 loci using Methylation Sensitive Cleaved Universal Lenght Polymorphism (MS-CULP) and 4 loci using Methylation Sensitive Cleaved Random Amplified Lenght Polymorphism (MS-CRALP) were utilized. In the present study BiSeq studies clearly revealed that most of the loci studied did not show hypo- or hyper-methylation between mature seeds of inbreed maize parental lines. One locus produced allelic variation between parental lines. Due to low level of methylation differences among loci utilized in BiSeq studies, 6 day-old seedlings were not investigated using BiSeq method. Based on results we obtained, we beleived that further studies using BiSeq method would reveal much more information regarding the relationships between DNA cytosine methylation and heterosis. GS-MSAP method determined epialleles that showed methylation differences among Mo17, B73 and Mo17xB73 treated with salt and demethylating agent 5-azaC. Multiplexed GS-MSAP loci presented some valuable information on DNA cytosine methylation. Also our results indicated that some of loci stuided with MS-CULP method presented methylation differences among Mo17, B73 ve Mo17xB73. On the other hand, none of MS-CRALP loci revealed methylation differences among plant samples studied. Heterosis is an extremely important phenomenon in agriculture and plant breeding. In this study crosses between Mo17 and B73 inbreed lines created F1 hibrid (Mo17xB73) individuals. One hundered seed weight of hybrid individuals was much above (38.18%) the both parents, indicating heterosis. The effects of 5-azaC and NaCl differed between hybrid and parental inbred lines. Result revealed that NaCl administration on the in hybrid individuals affected growth and development positively while 5-azaC application affected negatively. On the other hand, findings indicated that the 5-azaC application had a greater negative effect on heterosis than NaCl administration. This was accepted as an indication of DNA cytosine methylation on heterosis. As a result, in the present study we observed that there were certain levels of DNA methylation differences in mature seeds and 6 day-old seedlings treated with NaCl and 5-azaC. Due to some limitations encountered during this study some objectives of this dissertation could not be effectively accomplished. Especially genetic mixture in one of the maize parents resulted in measurement differences among hybrids and among seedlings. Furthermore, the number of loci used in BiSeq method were very low. In order to reveal the methylation differences between parents and hybrid and the effects of DNA cytosine methylation on heterosis, more number of loci with several methods and different doses of NaCl and 5-azaC applications method should be used. Whole Genome Bisulfite Sequencing (WGBS) method was suggested for better and more scientific results. KEYWORDS: Cytosine, heterosis, methylation, salt, 5-azaC