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bioRxiv · 10.64898/2026.10.09.757075

Microscale bulk segregant analysis

Bruni, G. N.·Crisman, L. C.·Alamo, L. A.·Marquez, H.·Boocock, J.·Bloom, J. S.·Kruglyak, L.
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Abstract

The traits of an organism depend on its genetic background and environment. Bulk segregant analysis (BSA) maps quantitative trait loci (QTL) by measuring parental allele frequencies in pools of genetically recombined progeny (segregants). Conventional BSA requires laborious separate sample handling for each cross and environmental condition. Here, we describe a 96-well liquid format for extreme QTL mapping (X-QTL) in Saccharomyces cerevisiae. We crossed 40 isolates to a common parent carrying mating-type-specific drug-resistance markers at HO, which enabled us to select diploids and haploid segregants in 1 mL cultures. Each well held one segregant pool, which we then grew in deep wells in four conditions: the standard glucose medium, sodium chloride or caffeine added to the standard medium, or acetate replacing glucose as a carbon source. We barcoded DNA samples from each of 384 wells and sequenced them in one Illumina Nova-seq X run. Sequencing data for 181 segregant pools passed quality control steps and was used to map 1,472 QTL in 35 crosses. We replicated a subset of QTL previously mapped in these growth conditions by conventional X-QTL. The 96-well approach increases the throughput of every step of X-QTL, enabling genetic analysis of many more cross and conditions.

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Bruni, G. N., Crisman, L. C., Alamo, L. A., Marquez, H., Boocock, J., Bloom, J. S., Kruglyak, L.. 2026-10-15. Microscale bulk segregant analysis. https://doi.org/10.64898/2026.10.09.757075

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