Researchers sequenced the genomes of three medakas, or Japanese rice fish, which are a common model organism used for biological research, nearly 10 and 20 years ago. While the sequencing of the genomes was relatively straightforward, the assembly of the genomes was anything but.
In the team's first study, the researchers used the traditional Sanger sequencing method to produce approximately 800 to 1,000 base pair sequencing reads, which resulted in low-quality sequences in some regions of the approximately 800 Mb genome and many sequencing gaps. Ten years later, in 2017, the research team performed PacBio SMRT (Single Molecule Real-Time) sequencing in two Japanese medaka strains (Hd-rR and HNI) and one Korean strain (HSOK). These longer reads spanned tens of kilobases, improving downstream genome assembly, but about 1,000 gaps still remained in the genome.
The team, led by scientists from The University of Tokyo and the National Institute of Genetics, part of the Research Organization of Information and Systems (ROIS) in Japan, published their paper on August 3 in the journal Genome Research.
"We set out to complete the genome sequence of the medaka fish, which previously contained about 1,000 gaps, and to uncover important biological information hidden in those gaps, including insights into chromosome stability, mobile DNA and sex determination," said Yoshihiko Suzuki, project lecturer at the Research Organization of Information and Systems (ROIS) and primary author of the research paper.
The medaka genome contains many repetitive regions that can be difficult to assemble using traditional DNA sequencing methods. Importantly, these repetitive regions often exist in centromeres, a specialized, constricted region of a chromosome that links sister chromatids together; telomeres, or protective DNA sequences located at the end of chromosomes; ribosomal DNA arrays made up of clusters of ribosomal RNA genes; XY Chromosomes; and the giant mobile element Teratorn, which can move to different places in the medaka genome.
"Genomic regions once considered too difficult to decode contain key biological insights into how chromosomes are accurately distributed to new cells, how mobile DNA survives in the genome and how biological sex is determined," said Suzuki.
The team used two state-of-the-art sequencing technologies to generate reads long and accurate enough to cover repetitive regions traditional methods couldn't adequately resolve through genome assembly. PacBio HiFi sequencing, a refined form of SMRT sequencing technology, yields highly accurate long reads, while Oxford Nanopore ultra-long sequencing produces reads hundreds of kilobases long, enough to span the most repetitive stretches.
By sequencing and assembling the Hd-rR medaka strain completely and nearly completing the HNI and HSOK strains, the researchers could compare centromeric sequences, giant transposons and sex chromosomes between strains and species, providing insight into how repetitive genomic regions can contribute to the phenotypic (observable) traits of different organisms.
The team observed centromeric sequences between medaka strains that differ from other eukaryotic organisms. In medaka, centromeric satellites, or highly repetitive, non-coding DNA sequences located at the centromere, are more conserved between strains than what has been observed in other eukaryotic species. These satellite sequences may facilitate specific DNA-protein interactions to stabilize kinetochore, a complex, disc-shaped protein structure assembled on the centromere of a chromosome, attachment.
The genetic element Teratorn, which is a full herpesvirus genome fused to a transposable element that allows it to move around the genome, also persists in the medaka genome. This genetic element is capable of changing the expression of nearby genes and has been shown to influence medaka fin shape. Further, the researchers observed that genes on X and Y (sex) chromosomes are retained in medaka as pairs except near the Y chromosome-specific region, which differs from the threespine stickleback, another fish, with a Y chromosome that is over 26 million years old.
While the identification and analysis of repetitive medaka genomic regions contributes to our genetic understanding of the species, the research team is excited to determine the function of specific elements within the genome, which are found in a variety of species.
"The next step is to experimentally test the functions of the newly identified regions. Our ultimate goal is to use complete genome information to understand how differences in DNA sequences shape traits in vertebrates and eventually to help clarify human genome function," said Suzuki.
Kazuki Ichikawa, Yusuke Inoue, Chie Owa, Haruka Kobayashi, Ryohei Nakamura, Hiroyuki Aburatani and Shinichi Morishita from The University of Tokyo; Kenji Morikami and Shigehiro Kuraku from the National Institute of Genetics; Takafumi Ikeda and Hiroyuki Takeda from Kyoto Sangyo University; Manabu Fujie, Mayumi Kawamitsu, Nana Arakaki and Eugene W. Myers from the Okinawa Institute of Science and Technology; Yusuke Takehana from the Nagahama Institute of Bio-Science and Technology; Masaru Matsuda from the Utsunomiya University, Kiyoshi Naruse from the National Institute for Basic Biology also contributed to this research.
This research was supported by the Japan Agency for Medical Research and Development (AMED) CREST Grant Number JP23gm1110007, AMED GRIFIN Grant Number 24tm0424219h0004, JSPS KAKENHI Grant Number JP22H04925 (PAGS), JSPS KAKENHI Grant Number JP23H02484, the Kyoto Sangyo University Research Grants #H2501 and JSPS KAKENHI Grant Number JP24K18091.
About National Institute of Genetics (NIG)
National Institute of Genetics (NIG) was established to carry out broad and comprehensive research in genetics. NIG contributes to the development of academic research as one of the inter-university research institutes constituting the Research Organization of Information and Systems (ROIS).
About the Research Organization of Information and Systems (ROIS)
ROIS is a parent organization of four national institutes (National Institute of Polar Research, National Institute of Informatics, the Institute of Statistical Mathematics and National Institute of Genetics) and the Joint Support-Center for Data Science Research. It is ROIS's mission to promote integrated, cutting-edge research that goes beyond the barriers of these institutions, in addition to facilitating their research activities, as members of inter-university research institutes.
