Solving Ohno’s puzzle — resolving an ancient sex chromosome system

In the 1960s, prominent cytogeneticist Susumu Ohno described an atypical sex chromosome system in the creeping vole (Microtus Oregoni). He proposed that females had an XO and males had an XY karyotype; with males contributing either the Y chromosome or no sex chromosome during gamete development. Although sparking much debate at the time, the mechanisms behind this puzzle remained unresolved for almost 60 years, until the recent work of Couger et al. (2021)^1,2.

An initial dive into this fascinating system saw the use of short-read sequencing of male and female vole genomic DNA and pointed to a male-specific X chromosome. Intriguingly, amplicon sequencing of conserved Y chromosome genes revealed their presence in both male and female voles. No such Y chromosome genes were detected in females of closely related Microtus species, indicating that this system had been evolving independently for approximately 150 million years.

The placement and order of these Y genes was, however, still unclear, and so the team performed genome assembly with long reads. Despite the initial long-read assembly being 'quite excellent', sex chromosome contigs were shorter than autosomal contigs, likely due to the highly repetitive nature of the sex chromosomes¹. To resolve this, the team turned to ultra-long nanopore sequencing reads. Using the recently released Ultra-Long DNA Sequencing Kit, they got an 'amazing turnaround time for data' from two PromethION Flow Cells, with an N50 of 91 kb, and a significant proportion of their reads being ultra-long (>50 kb)¹.

The high-quality, ultra-long nanopore data aligned 'extremely well' to the genome, and further supported accurate SNP calling and phasing². Highlighting a 410 kb read that aligned to a region of the sex chromosome, lead researcher Matthew Brian Couger commented: 'that's a really solid contig for most peoples' assemblies, not their actual read generation'. With these long reads, unassembled regions of the repeat-rich sex chromosomes were connected (Figure 1).

Figure 1. Ultra-long nanopore reads closed the gaps in the initial assembly of the repetitive vole sex chromosome regions of interest. Figure courtesy of Matthew Brian Couger, Brigham and Women's Hospital, USA².

The position of genes relative to each other, both within a chromosome, and between the paternal X and maternal X chromosomes, was revealed. And so Ohno's puzzle was resolved, revealing a unique sex chromosome system whereby male gametes contain either a paternal X chromosome, generating male offspring, or no X chromosome, giving rise to female offspring (Figure 2).

Figure 2. Accurate mapping of the maternal and paternal X chromosomes supported a new model of sex determination in the creeping vole. Males have both maternal (X^M) and paternal (X^P) X chromosomes, with Xist-based silencing of the paternal copy, while females inherit just one X chromosome (X^M) from the mother. Figure adapted from Couger et al. (2021)¹.

At the Garvan Institute, Australia, Jillian Hammond is also using ultra-long nanopore sequencing reads to resolve previously uncharacterised genomic regions, including the highly repetitive, highly homologous sex chromosomes observed in Australian reptiles³. Interestingly, the sex of many reptiles is determined by both genotype and the environment, in particular the temperature. Typically, a ZZ genotype becomes a male and a ZW genotype becomes a female; however, at higher egg incubation temperatures this is overridden, and both become female.

According to Jillian, over a third of the reads obtained using the Ultra-Long DNA Sequencing Kit consistently exceeded 100 kb, with a significant number exceeding 500 kb. Furthermore, read N50s doubled from around 35 kb to over 70 kb when compared to their previous workflow. Draft genome assemblies for the bearded dragon, the Shingleback lizard, and two highly venomous sea snakes have now been generated using FLYE, with the team planning to further enhance these assemblies using Hi-C data and cDNA-based gene annotation. Using the completed assembles, they aim to elucidate the mechanisms that drive sex determination and better understand aquatic adaptations in sea snakes.