DOI

10.30707/1783431597.456829

Document Type

Senior Thesis

Publication Date

Spring 2026

Committee Chair

Thomas Hammond

Committee Member

Wade Nichols

Abstract

Spore killer-3 (Sk-3) is a selfish genetic element on Chromosome III of Neurospora that biases its own transmission by killing meiotic products that fail to inherit the element. Although the resistance component of Sk-3 is known, the killer has not been identified. Previous deletion mapping confined the candidate killer region to a small block of DNA near interval i350 and suggested that the nearby 178 bp interval i398 might show different behavior in qualitative versus quantitative assays. The goal of this thesis was to construct and analyze an Sk-3 i398Δ strain and determine whether deletion of i398 measurably alters spore killing or gene drive. A deletion vector, v398, was assembled by Double-Joint PCR using Sk-3 genomic flanks and a hygromycin resistance cassette and then introduced into the Sk-3 strain. Quantitative transmission assays were then used to score the proportion of hygromycin-resistant offspring in MSUD-proficient and MSUD-deficient backgrounds. Archived Spring 2025 ascus dissections were used to define benchmark phenotypic categories for fully active versus absent Sk-3 killing. In the Fall 2025 MSUD-proficient assay, 38 of 40 progeny from Cross RTBHA21 were hygromycin resistant (95.0%), whereas only 2 of 40 were sensitive. In the Spring 2026 MSUD-deficient assay, 50 of 54 progeny from Cross RTBha30 were hygromycin resistant (92.3%), whereas 4 of 54 were sensitive. Both assays were strongly inconsistent with a 1:1 segregation ratio expected if i398 were essential for drive. Benchmark Spring 2025 dissections further established the morphological distinction between 4-black/4-clear killer asci and 8-black nonkiller asci, providing a calibrated framework for interpreting later imaging. Together, these data indicate that deletion of i398 does not stop Sk-3 drive and is most consistent with i398 being dispensable for full Sk-3 function.

Funding Source

This work was supported by the National Science Foundation (Award Number 200595, Elucidating the mechanism of meiotic drive by mRNA editing-mediated spore killing in Neurospora fungi).

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