Characterization of a novel satellite DNA sequence from Flying Dragon (Poncirus trifoliata) Bruna De Felice 1 , Robert R.Wilson 2 , Loredana Ciarmiello 1 , Maria Teresa Scarano 3 ,& Sergio Ferrante 3 1 Department of Life Sciences, University of Naples II, Via Vivaldi 43, Caserta, Italy (Phone: +39-823- 274543; Fax: +39-823-274571; E-mail: bruna.defelice@unina2.it); 2 NOAA, 325 Broadway, Boulder, CO, USA; 3 CNR-IGV, Research Institute of Plant Genetics, Corso Calatafimi, 414, 90129, Palermo, Italy Received 9 April 2005; Accepted 25 August 2005 Key words: methylation, Poncirus trifoliata, repeated sequences Abstract Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and the molecular analysis and methylation status of a novel tan- demly organized repetitive DNA sequence from the genome of Poncirus trifoliata. Digestion of P. trifoliata DNA with Afa I produced a prominent fragment of approximately 400 bp. Southern blotting analysis of genomic DNA digested with the same enzyme revealed a ladder composed of DNA fragments that are multimers of the 400-bp Afa I band, indicating that the repetitive DNA is arrayed in tandem. This suggests that Afa I isolated a novel satellite that we have called Poncirus trifoliata satellite DNA 400 (PN400). This satellite composes 25% of the genome and it is also present in lemon, sour orange and kumquat. Analysis of the methylation status demonstrated that the cytosines in CCGG sequences in this satellite were methylated. Introduction Satellite DNA (stDNA) is a highly repetitive DNA organized in long tandem repeats in constitutive heterochromatin (Charlesworth, Sniegowski & Stephan, 1994). Satellite DNAs are highly abun- dant sequences repeated in tandem and mainly located in centromeric and telomeric regions of eukaryotic chromosomes (Charlesworth, Snie- gowski & Stephan, 1994). Its function is still unclear but many studies associate satellite DNA with heterochromatin organization and centromeric function (Ugarkovic & Plohl, 2002). Different motifs found in satellite repeats are thought to play an important role in those functions: phased A or T runs ‡3 producing DNA bending (Martinez-Balbas et al., 1990), cruciform configurations(Juan et al., 1993) or the presence of specific motifs binding centromeric proteins (Lorite et al., 2002). Concerted evolution generally leads to striking intra-specific sequence identity among repetitive units, which constitute a stDNA family (Elder & Turner, 1995). However, some stDNA families have divergent repetitive sequences that can be grouped into different sub- families due to the presence of diagnostic sites (Trick & Dover, 1984; Pons, Petitpierre & Juan, 2002a). These positions have a particular mutation across all sequences of one subfamily, while at the same sites all sequences of the other subfamily have a different mutation (homogenized sites). On the other hand, at the inter-specific level concerted evolution generally leads to species- specific stDNAs whose repeats are completely different in length and nucleotide sequence. The Genetica (2006) 127:45–53 Ó Springer 2006 DOI 10.1007/s10709-005-2479-z