Study On DNA Methylation Patterns Of Early And Late Satsuma With MSAP Analysis

Citrus is one of the most important fruits in tropical and subtropical areas. Satsumas have important economical value, which have the highest cultivated area among mandarins in China or even in the whole eastern Asia. Most of satsuma cultivars derived from bud mutation, the differences on DNA level are too small to be identified using traditional molecular markers even among those cultivars, whose ripening period is obviously different. Abundant bud mutation cultivars and the consistency of genetic background make satsumas the ideal material to study the development and ripening of fruit. Analysis of DNA methylation of20early and late satsuma varieties in the same time and2satsuma varieties(Ooita wase, Aoshima unshiu) in different development stages were performed in this study. Based on the methylation-sensitive fragment length polymorphism (MSAP) technique, the differences of DNA methylation between early and late satsuma varieties were estimated. The obviously differential bands were sequenced to find the changed genes by methylation modification involving in the fruit ripening process of satsuma. The aim of this study is to study the differences of DNA methylation between ripening early and late satsuma (Citrus unshiu Macf.), and explore the changes of methyaltion during the fruit ripening process of satsuma. Furthermore, this study provide us insight into the mechanism of the fruit ripening process of satsuma.The main research results obtained in this study are as follow:1.According to the MSAP analysis on ten ripening early satsumas cultivars (Ooita wase, Iwasaki Unshiu wase, Nichinan No.1, Satusama8-76, Long Yuan Zao,2003-2Satsuma, Hashimoto Wase,2003-5Satsuma, Miyamoto Wase, Ooura Wase) and ten ripening late satsumas cultivars (Judaro unshiu, Owari unshiu, Aoe unshiu, Ootsu No.4unshiu, Ning Hong73-19, Ikiriki unshiu, Aoshima Unshiu, Sugiyama Unshiu, Hayashi Unshiu, Ishikawa Unshiu), The extent and pattern of cytosine methylation in the CCGG sequences of genomes between early and late satsuma varieties are obviously different. The external methylation of CCGG sequences in ripening early satsuma (8.47%) is more than its in ripening late satsuma (6.74%), while the internal methylation of CCGG sequences in ripening early satsuma (24.98%) is less than its in ripening late satsuam (27.27%).2003-2Satsuma and Hashimoto Wase are the earliest cultivars among early satsumas, whose external methylation of CCGG sequences are respectively15.28%,15.46%. The late satsuma cultivars Ootsu No.4unshiu have high external methylation of CCGG sequences (13.77%), While the external methylation of CCGG sequences in other late satsuma cultivars are much lower than it. Ikiriki unshiu have the highest internal methylation of CCGG sequences among late satsuma cultivars, while the other cultivars are much lower than it. It indicated that the internal cytosine methylation tend to reduce and the external cytosine methylation tend to increase when satsuma fruits approach ripening. The total DNA methylation in early satsuma cultivars(33.45%) is slightly less than that one in late satsuma cultivars(34.01%). Furthermore, all of twenty satsumas displayed lower external cytosine methylation than internal cytosine methylation.The total average methylation in external cytosine is7.62%,while the total average methylation in internal cytosine is26.11%.2. According to analysis of DNA methylation of20early and late satsuma varieties in the same time, the methylation pattern of G-protein alpha subunit gene and EST sequence CX052976.1has obvious differences between late and early satsumas; Analysis of DNA methylation of Ooita wase and Aoshima unshiu in different development stages showed that the methylation pattern of Zinc-binding protein of the histidine triad (HIT) family gene and EST sequence CX071135.1changed significantly during the ripening process. Therefore, G-protein alpha subunit gene, Zinc-binding protein of the histidine triad (HIT) family gene and two EST sequences(CX052976.1and CX071135.1) might play an important role during the ripening process of satsumas.3. This study showed that MSAP is an effective method to detect the DNA methylation modification in satsumas. In this study, thirteen satsuma cultivars showed different DNA methylation pattern in all twenty satsuma cultivars.4. Testing of fruit quality characteristic of20satsuma cultivars were performed to compare the difference of physiological characteristic between late and early satsumas. The results indicated that the physiological characteristics (acid content, Vc content and fruit color) between late and early satsumas have significant differences. It also showed that the acid content were reduced and the Vc content were increased companied with the fruit color change during the fruit ripening process; while total soluble solid and fruit weight have no significant correlation with color change and acid content.