Supplementary Material for: Divergence of Repetitive DNA Sequences in the Heterochromatin of Medaka Fishes: Molecular Cytogenetic Characterization of Constitutive Heterochromatin in Two Medaka Species: <b><i>Oryzias hubbsi</i></b> and <b><i>O. celebensis</i></b> (Adrianichthyidae, Beloniformes)

The large biarmed chromosomes of <i>Oryzias celebensis</i> [2n = 36, fundamental arm number (FN) = 48] are considered to have resulted from fusions of acrocentric chromosomes in the ancestral karyotype of <i>Oryzias</i>, which is retained in <i>O. hubbsi</i> (2n<i> = </i>48, FN = 48). To understand the molecular evolution of heterochromatin associated with karyotype reorganization in medaka fishes, we cloned 3 and 6 novel families of heterochromatin-related repetitive DNA sequences from <i>O</i>.<i> hubbsi</i> and <i>O. celebensis</i>, respectively, and characterized them using molecular cytogenetics. Two AT-rich repetitive sequences isolated from the genomic DNA of <i>O. hubbsi</i>, a 164-bp satellite DNA (OHU-<i>Rsa</i>I-Scen) and a 177-bp telomere-specific repeat (OHU-<i>Rsa</i>I-Stelo), were shown to be major components of the constitutive heterochromatin of centromeres and telomeres, respectively. A GC-rich 326-bp sequence, named OHU-<i>Alu</i>I-M1, was colocalized with the 18S-28S ribosomal RNA gene cluster to a single autosomal pair of chromosomes and the W chromosome. In <i>O. celebensis</i>, 2 major satellite DNA sequences (the AT-rich 157-bp OCE-<i>Alu</i>I-Scen sequence and the 186-bp OCE-<i>Hin</i>fI-Scen sequence) were identified in the centromeric regions of almost all chromosomes. The 197-bp OCE-<i>Hin</i>fI-S6 sequence was located in the centromeric and distal and/or interstitial heterochromatin of almost all chromosomes, and the 191-bp OCE-<i>Hin</i>fI-S8 sequence was located in 6 pairs of chromosomes. Constitutive heterochromatin on the short arm of large submetacentric chromosome 5 was composed of at least 3 different repetitive sequences: the 171-bp OCE-<i>Alu</i>I-S18 sequence, the 197-bp OCE-<i>Hin</i>fI-S6 sequence and the 172-bp OCE-<i>Hin</i>fI-S11 sequence. All families of repeated sequences showed no nucleotide sequence similarity with each other and high species-specificity among 7 different species. These results suggest that the heterochromatin of <i>O</i>. <i>hubbsi</i> and <i>O</i>. <i>celebensis </i>consists of various types of repetitive sequence and that the sequences evolved independently and were then amplified site-specifically in each lineage after karyotype reorganization occurred in the ancestral karyotype.