Gakushuin University led group determine complete sequence of W chromosome of silk moth family

A research group led by Assistant Professor Jung Lee and Professor Toru Shimada of the Department of Life Science, Faculty of Science at Gakushuin University, Distinguished Assistant Professor Toshiaki Fujimoto (currently at Kyushu University Graduate School), Professor Ken Sawara of Iwate University, Chief Technician Katsushi Yamaguchi and Professor Shuji Shigenobu of the National Institute for Basic Biology, and Professor Atsushi Toyoda of the National Institute of Genetics has succeeded in constructing a female genome assembly of Bombyx mori, including the W chromosome, which had been considered impossible to sequence, and a female genome assembly of Trilocha varians, a closely related species in the same family (Bombycidae) as B. mori, using the latest long-read sequencing technology. This is an important milestone for understanding the evolution of sex chromosomes in the Bombycidae family. The work was published in the online edition of Molecular Ecology.

Insects of the order Lepidoptera, such as B. mori, are female heterogametic, in which sex chromosomes are two Z chromosomes in males and one Z chromosome and one W chromosome in females. The W chromosome of lepidopterans is an enigmatic chromosome, and little is known about its origin or the number of genes it holds. In particular, for the W chromosome of B. mori, a model organism, only a female sex-determining gene called Fem has been isolated. Other than that, it remains unknown whether the W chromosome contains any other genes, and its sequence and structure have not been determined. Because meiotic recombination does not occur in females of lepidopterans, mutations tend to accumulate on the W chromosome. Furthermore, lepidopteran W chromosomes tend to accumulate repetitive sequences such as transposons, meaning that W chromosomes are difficult to sequence.

The research group sequenced female genomes in B. mori and, as a control for comparison, T. varians (Bombycidae family). As a result, they successfully scaffolded all of the chromosomes, including the W chromosome, into a single sequence for both species, and registered them in the U.S. NCBI database. The registered genome assemblies of B. mori and T. varians have been certified as reference sequences and are rated as one of the most accurate genome assemblies currently available. For B. mori, nearly 90% of the W chromosome was occupied by repetitive sequences, as previously predicted. The proportions of various repetitive sequences in the B. mori W chromosome differed substantially from those of the autosomes and Z chromosome. LTR retrotransposons accounted for large proportions of the W chromosome.

Although not as extreme as the results for B. mori W chromosome, the proportions of repetitive sequences of the T. varians W chromosome also differed from those in the Z chromosome and autosomes. LTR retrotransposons accounted for large proportions of the T. varians W chromosome. The B. mori and T. varians W chromosomes did not exhibit sequence homology to the respective Z chromosomes. Furthermore, the W chromosomes of both species showed no sequence homology with each other, although they belong to the same Bombycidae family. However, a careful examination of the genes expressed on the W chromosomes revealed that they are homologous to Z-linked genes.

This finding indicates that the W chromosome of both species originated from the Z chromosome. New facts were also discovered about the female sex-determining gene Fem. The group found that there are 129 copies of the entirely conserved Fem sequence on the B. mori W chromosome, and many more if trace elements are included. Furthermore, Fem was absent in the T. varians W chromosome, indicating that the Fem-dependent female sex-determination mechanism is a rare one observed only in a limited number of species of the Bombycidae family closely related to B. mori (e.g., B. mandarina). Fem has been speculated to be an allele of Masc, a male sex-determining gene expressed on the Z chromosome, based on their sequence homology. However, a sequence comparison of the W chromosomes of T. varians and B. mori revealed that the Fem sequence is most likely originated from the accidental fusion of multiple transposons.

This deduction underpins the claim "the Fem-dependent sex-determination mechanisms are found only in B. mori and very closely related species," as stated in reports by lepidopteran researchers around the world over the past 10 years since the discovery of Fem in 2014. As the W chromosome sequences have been clarified in this study, female-specific genome editing using gene knock-in and other techniques has become possible. The W chromosome sequences constructed by the research group are also expected to be useful as a basis for the determination and comparative analysis of the W chromosome sequences of various B. mori strains, particularly those with intersex traits.

Journal Information
Publication: Molecular Ecology
Title: W chromosome sequences of two bombycid moths provide an insight into the origin of Fem
DOI: 10.1111/mec.17434