Establishment of cell lines derived from skin biopsies of crested ibis (<I>Nipponia nippon</I>) and their biological characteristics

Abstract

The crested ibis is among the rarest and most endangered species worldwide. To preserve its genetic resources and conveniently provide materials for biological research, we successfully established two cell lines from biopsies of a male and female adult crested ibis. The cultured cells from both specimens had typical fibroblast morphology. Immunofluorescence staining revealed that the cultured cells strongly expressed the marker of smooth muscle specific α-actin, clearly indicating the cells were from the smooth muscle tissue. Growth property analysis showed that the cells grew well past the first 10 passages and continued growing with reduced proliferation after 15 passages, but ceased by passage 25 as the cells could not grow to form a confluent monolayer. From these two cell lines, we harvested mitotic metaphase chromosomes and conducted different staining, banding, and fluorescent in situ hybridization. Throughout the process, cells maintained normal diploidy, with the karyotypes of these two cell lines being 2n=68, ZZ in the male and 2n=68, ZW in the female. Patterns of Ag staining, C- and G-bands of the crested ibis chromosomes were also studied. Banding analyses and fluorescent in situ hybridization also allowed identification of the sex chromosomes. We suggest that the external implants method for establishing primary cell lines used in this study may also be applicable to other birds, especially similarly endangered avian species.

Comparative chromosome maps between the stone curlew and three ciconiiform species (the grey heron, little egret and crested ibis)

“…The diploid number of NNI is 68 (Fig. 1 d), as reported previously [ 12 , 15 , 32 ]. The karyotypes of ACI (Fig.…”

“…The karyotype of ACI and EGA were arranged according to the relative length of the chromosomes from the largest to the smallest. The chromosomes of NNI were numbered according to previously published karyotypes with a minor revision [ 12 , 15 , 32 ].…”

Evolution and expression patterns of the neo-sex chromosomes of the crested ibis

“…The sex differentiation region (SDR) of the W chromosome is 37.7 Mb in size, being one of the largest assembled bird W chromosomes (Supplementary Data 1). Such a large size for the assembled W chromosome is consistent with the cytogenetically estimated size 51 that falls between the sizes of chromosomes 9 and 10. We annotated 414 protein-coding genes on the sexually differentiated region (SDR) of chromosome W, making it the most gene-rich SDR of bird W chromosomes, suggesting that only 54.6% of the original gene content was lost from the W chromosome, in contrast to the greater that 90% found in most land birds 52 .…”

“…1b). The assembled Z chromosome is the sixth largest chromosome, also consistent with a cytogenetic study 51 , contains 1112 genes (Supplementary Table 3). Chromosome Z uniformly exhibits a 2-fold elevation of sequencing coverage in males relative to females (Fig.…”

“…Bird genomes usually possess a conserved karyotype with typically ~10 pairs of macrochromosomes and ~30 pairs of microchromosomes (2n = 80) in the course of over 100 million years of evolution 36,53 . Core waterbirds, however, frequently have a smaller number of chromosomes, with many species having a diploid chromosome number of 68, including crested ibis 51 . To investigate the karyotypical evolution of the crested ibis, we conducted a multi-way comparison of genome synteny using the chromosome-level genomes of the crested ibis (NNI) and several representative bird species, including the zebra finch (Taeniopygia guttata, TGU, Neoaves), California condor (Gymnogyps californianus, GCA, Neoaves), great bustard (Otis tarda, OTA, Neoaves), mallard (Aanas platyrhynchos, APL, Galloanserae), chicken (Gallus gallus, GGA, Galloanserae), and emu (Dromaius novaehollandiae, DNO, Palaeognathae), which cover all three major bird clades.…”