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

Supplementary Material for: Extraordinary Diversity in the Origins of Sex Chromosomes in Anurans Inferred from Comparative Gene Mapping

Sex determination in frogs (anurans) is genetic and includes both male and female heterogamety. However, the origins of the sex chromosomes and their differentiation processes are poorly known. To investigate diversity in the origins of anuran sex chromosomes, we compared the chromosomal locations of sex-linked genes in 4 species: the African clawed frog<i> (Xenopus laevis)</i>, the Western clawed frog <i>(Silurana/X. tropicalis)</i>, the Japanese bell-ring frog <i>(Buergeria buergeri)</i>, and the Japanese wrinkled frog<i> (Rana rugosa)</i>. Comparative mapping data revealed that the sex chromosomes of <i>X. laevis</i>, <i>X. tropicalis</i> and <i>R. rugosa </i>are different chromosome pairs; however, the sex chromosomes of <i>X. tropicalis</i> and <i>B. buergeri</i> are homologous, although this may represent distinct evolutionary origins. We also examined the status of sex chromosomal differentiation in <i>B. buergeri</i>, which possesses heteromorphic ZW sex chromosomes, using comparative genomic hybridization and chromosome painting with DNA probes from the microdissected W chromosome. At least 3 rearrangement events have occurred in the proto-W chromosome: deletion of the nucleolus organizer region and a paracentric inversion followed by amplification of non-W-specific repetitive sequences

Supplementary Material for: Karyotype Reorganization with Conserved Genomic Compartmentalization in Dot-Shaped Microchromosomes in the Japanese Mountain Hawk-Eagle (<i>Nisaetus nipalensis orientalis</i>, Accipitridae)

The karyotype of the Japanese mountain hawk-eagle <i>(Nisaetus nipalensis</i><b> </b><i>orientalis)</i> (2n = 66) consists of a large number of medium-sized and small chromosomes but only 4 pairs of dot-shaped microchromosomes, in contrast to the typical avian karyotype with a small number of macrochromosomes and many indistinguishable microchromosomes. To investigate the drastic karyotype reorganization in this species, we performed a molecular cytogenetic characterization employing chromosome in situ hybridization and molecular cloning of centromeric heterochromatin. Cross-species chromosome painting with chicken chromosome-specific probes 1-9 and Z and a paint pool of 20 microchromosome pairs revealed that the <i>N. n. orientalis</i> karyotype differs from chicken by at least 13 fissions of macrochromosomes and 15 fusions between microchromosomes and between micro- and macrochromosomes. A novel family of satellite DNA sequences (NNO-<i>Apa</i>I) was isolated, consisting of a GC-rich 173-bp repeated sequence element. The NNO-<i>Apa</i>I sequence was localized to the C-positive centromeric heterochromatin of 4 pairs of microchromosomes, which evolved concertedly by homogenization between the microchromosomes. These results suggest that the 4 pairs of dot-shaped microchromosomes have retained their genomic compartmentalization from other middle-sized and small chromosomes