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