Linkage mapping vs Association: A comparison of two RADseq-based
approaches to identify markers for homomorphic sex chromosomes in large
genomes.
Abstract
Reliable tools for the identification of genetic sex are invaluable in
many fields of biology, but their design requires knowledge of
sex-linked sequences, which is lacking in many taxa. Restriction-site
associated DNA sequencing (RADseq) is widely used to identify sex-linked
markers, but multiple distinct strategies are employed, and it is often
not obvious which is most suitable. In this study we compare two
approaches for using RADseq to identify sex-linked markers. We use the
common newt, Lissotriton vulgaris, as our study system, providing a
challenging combination of homomorphic sex chromosomes and an
exceptionally large genome. We attempt an associative approach,
sequencing 60 adult newts of known-sex individuals, and compare this to
a linkage mapping approach utilizing a family of 146 offspring with
unknown sex. After optimization for a highly paralogous genome, the
associative approach identifies five Y-chromosome linked markers in L.
vulgaris and we design a robust PCR protocol for molecular sexing of
four more related species. Via the linkage approach we construct a
high-density map featuring 10,763 markers, matching the observed
karyotype of L. vulgaris and showing broad synteny with the Iberian
ribbed newt (Pleurodeles waltl). However, without incorporating the
markers identified via the association-based approach, we cannot
confidently distinguish a sex-determining region in the linkage map,
either by analyzing marker density or by identifying clusters of
paternal markers. We conclude that linkage mapping alone is unlikely to
yield sex-linked markers in organisms with very small sex-determining
regions, however association-based RADseq can still be effective under
these conditions.