Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis

Nobuyuki Higashiguchi,Kiyomi Murakumo,Keiichi Sato,Shigehiro Kuraku,Yoshinobu Uno,Ryo Nozu,Itsuki Kiyatake,Shuji Sodeyama

doi:10.1038/s42003-020-01373-7

Abstract

Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Karyotype reports of chondrichthyans are scarce because of the difficulty in cell culture. Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. The utility of the cultured cells enabled the high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum). We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our protocols are applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level.

Highlights

Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods
We adopted culture medium supplemented with the combination of four mitogens, concanavalin A (Con A), LPS, PHA and phorbol 12-myristate 13-acetate (PMA), at the concentrations used in previous studies[24,35] (Supplementary Fig. 1 and Supplementary Data 2)
The long-term infeasibility of high-fidelity shark cell culture was thought to be attributable to insufficient adaptation of the culture medium to body fluid osmolality

Summary

Introduction

Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Among vertebrates, the karyotype of species within Chondrichthyes (cartilaginous fishes) is the least investigated because of the lack of a reliable protocol for chromosome preparation using cultured cells[5,6] This limitation has been observed for the elephant fish Callorhinchus milii ( called elephant shark), the first chondrichthyan species with a sequenced genome in the absence of karyotype information[7]. Orectolobiformes is the third most species-rich order of sharks and comprises 45 species that inhabit mainly temperate or tropical waters in the Pacific Ocean Within this order, the whale shark Rhincodon typus (Fig. 1) is unique as a pelagic species with the largest body size as a ‘fish’ The sequencing output cannot be validated without the goal of sequencing, namely karyotype information that provides the inherent number and size of chromosomes

Methods

Results

Conclusion