DNA methylation alterations and exchanges during in vitro cellular differentiation in rose (Rosa hyb

DNA methylation alterations and exchanges during in vitro cellular differentiation in rose (Rosa hyb

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Theor Appl Genet (2004) 109: 899 910 DOI 10.1007/s00122-004-1717-6 ORIGINAL PAPER Mingliang Xu . Xiangqian Li . Schuyler S. Korban DNA-methylation alterations and exchanges during in vitro cellular differentiation in rose ( Rosa hybrida L.) Received: 28 October 2003 / Accepted: 27 April 2004 / Published online: 19 June 2004 # Springer-Verlag 2004 Abstract DNA-methylation profiles of leaf tissues of Rosa hybrida cv. Carefree Beauty collected from in vivo- grown greenhouse plants, in vitro-grown proliferating shoots at different passages, regenerants of embryogenic callus, regenerants of organogenic callus, as well as calli from undifferentiated callus (UC), embryogenic callus, and organogenic callus were investigated using an amplified fragment-length polymorphism (AFLP)-based detection technique. Three types of AFLP bands were recovered. Type I bands were observed with both isoschizomers Msp and Hpa II, while type II and type III bands were observed only with Msp I and Hpa II, respectively. Sequence analysis of the three types of AFLP bands revealed that a nonmethylated Msp I/ Hpa II- recognition site 5 -CCGG-3 resulted in a type I band, while an inner 5-methylcytosine generated most type II and type III bands. About 40% of inner and 20% of outer cytosines in 5 -CCGG-3 sequences were fully methylated, and only a few hemimethylated outer cytosines were observed. Changes in types of AFLP bands among different tissues were frequently observed, including appearance and disappearance of type I, II, and III AFLP bands, as well as exchanges between either type I and type II or type I and type III AFLP bands. Methylation alterations of outer cytosines in 5 -CCGG-3 sequences triggered appearance and disappearance of type I and II AFLP bands. Methylation changes of both outer and inner cytosines resulted in either removal or generation of type III AFLP bands. Methylation alteration of an inner cytosine was responsible for exchange between type I and type II, while hemimethylation of an outer cytosine accounted for exchange between type I and type III AFLP bands. During UC induction, a significant DNA-methyl- ation alteration was detected in both inner and outer cytosines. Variations in methylation profiles significantly differed between somatic embryogenesis and in vitro organogenesis. Demethylation of outer cytosines occurred at a high frequency during somatic embryogenesis, and most altered AFLP bands in embryogenic callus were passed on to its regenerants. However, most methylation- altered AFLP bands during organogenesis were recovered in shoot regenerants derived via organogenic callus. Seven tissue-specific bands were isolated, cloned, and se- quenced. Blast search revealed that two of these might be derived from functional genes. Introduction DNA methylation in both plant and mammalian systems has long been associated with changes in gene expression, chromatin structure alterations, activation of transposable elements, genomic imprinting, and carcinogenesis (Fergu- son-Smith and Surani 2001 ; Finnegan 2001 ; Rossi et al.
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DNA methylation alterations and exchanges during in vitro cellular differentiation in rose (Rosa hyb

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