Genetic variation segregates as linked sets of variants or haplotypes. Haplotypes and linkage are central to genetics and underpin virtually all genetic and selection analysis. Yet, genomic data often omit haplotype information due to constraints in sequencing technologies. Here, we present “haplotagging,” a simple, low-cost linked-read sequencing technique that allows sequencing of hundreds of individuals while retaining linkage information. We apply haplotagging to construct megabase-size haplotypes for over 600 individual butterflies (Heliconius erato and H. melpomene), which form overlapping hybrid zones across an elevational gradient in Ecuador. Haplotagging identifies loci controlling distinctive high- and lowland wing color patterns. Divergent haplotypes are found at the same major loci in both species, while chromosome rearrangements show no parallelism. Remarkably, in both species, the geographic clines for the major wing-pattern loci are displaced by 18 km, leading to the rise of a novel hybrid morph in the center of the hybrid zone. We propose that shared warning signaling (Müllerian mimicry) may couple the cline shifts seen in both species and facilitate the parallel coemergence of a novel hybrid morph in both comimetic species. Our results show the power of efficient haplotyping methods when combined with large-scale sequencing data from natural populations.

One-sentence summary:Haplotagging, a novel linked-read sequencing technique that enables whole genome haplotyping in large populations, reveals the formation of a novel hybrid race in parallel hybrid zones of two co-mimicking Heliconius butterfly species through strikingly parallel divergences in their genomes. Short title:Haplotagging reveals parallel formation of hybrid races Abstract Genetic variation segregates as linked sets of variants, or haplotypes. Haplotypes and linkage are central to genetics and underpin virtually all genetic and selection analysis. And yet, genomic data often lack haplotype information, due to constraints in sequencing technologies. Here we present "haplotagging", a simple, low-cost linked-read sequencing technique that allows sequencing of hundreds of individuals while retaining linkage information. We apply haplotagging to construct megabasesize haplotypes for over 600 individual butterflies (Heliconius erato and H. melpomene), which form overlapping hybrid zones across an elevational gradient in Ecuador. Haplotagging identifies loci controlling distinctive high-and lowland wing color patterns. Divergent haplotypes are found at the same major loci in both species, while chromosome rearrangements show no parallelism. Remarkably, in both species the geographic clines for the major wing pattern loci are displaced by 18 km, leading to the rise of a novel hybrid morph in the centre of the hybrid zone.We propose that shared warning signalling (Müllerian mimicry) may couple the cline shifts seen in both species, and facilitate the parallel co-emergence of a novel hybrid morph in both co-mimetic species. Our results show the power of efficient haplotyping methods when combined with large-scale sequencing data from natural populations.

Cell migration is an important process occurring during normal animal development but also in the early stages of metastatic cancer when cells invasively migrate out of the primary tumor. It is therefore likely that some of the same genes function in both migrations. Although transcriptional profiling has identified many genes differentially expressed between metastases and primary tumors, we do not understand the function of most of these genes in either normal or disease states. Our goal has been to identify conserved genes used in migration of mammalian metastasis and normal C. elegans development to further characterize their function in both systems. From two published transcriptional profile datasets1,2, we selected genes that were upregulated in metastases compared to primary tumors, and then identified the C. elegans orthologs of those genes to generate a list of 107 genes. We further narrowed our selection to 87 genes based on the gene's expression in the linker cell, a migratory C. elegans cell that we had previously transcriptionally profiled3. We used RNAi by feeding to reduce the function of these genes in three migratory cell types in C. elegans. The male linker cell and hermaphrodite distal tip cell are both somatic gonadal cells that migrate long distances while pulling along attached, non-motile gonadal cells, yet differ in their gender and in their migratory path. The sex myoblast migrates individually and for a short distance. We scored defects in migratory path, cell shape, and speed. 19 genes affected the migration of the linker cell and 16 genes that of the distal tip cell. Six genes overlapped between these two cell migrations, including SNTB2/syntrophin, an adaptor protein in the dystrophin pathway, and ATP6V0A1, a vacuolar proton-translocating ATPase. The sex myoblast screen is still ongoing, but only one of 25 genes affects its migration, showing even fewer overlapping genes with the other two cells. Among the genes that have penetrant but cell-specific defects are UTRN/utrophin, CAP1/ adenylate cyclase-associated protein, STXBP2/syntaxin binding protein, and MYL12A/myosin light chain regulatory subunit. Our results indicate that different cells use different genes for their migration, and suggest that different cancer cell types may also. This underscores the importance of characterizing the function of diverse genes to both understand their role in metastasis and identify cell type-specific drug targets. 1. Alonso, S.R. et al. (2007) A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis. Cancer Res. 67:3450-3460. 2. Patsialou, A. et al. (2012) Selective gene-expression profiling of migratory tumor cells in vivo predicts clinical outcome in breast cancer patients. Breast Cancer Res. 14:R139. 3. Schwarz, E.M.*, Kato, M*. Sternberg, P.W. (2012) Functional transcriptomics of a migrating cell in Caenorhabditis elegans. Proc. Natl. Acad. Sci.109: 16246-51. Citation Format: Mihoko Kato, Jonathan Liu, Olivia Box Power, Anand Upadhyaya, John Yim, Paul Sternberg. Comparison of genes upregulated in metastasis with three C. elegans cell migrations. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A46.

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