Interspecies hybrid sterility has been extensively studied, especially in the genus Drosophila. Hybrid sterility is more often found in the heterogametic (XX or ZW) sex, a trend called Haldane’s rule. Although this phenomenon is pervasive, identification of a common genetic mechanism remains elusive, with modest support found for a range of potential theories. Here, we identify a single precise morphological phenotype, which we call “needle-eye sperm,” that is associated with hybrid sterility in three separate species pairs that span the Drosophila genus. The nature of the phenotype indicates a common point of meiotic failure in sterile hybrid males. We used ten generations of backcross selection paired with whole-genome pooled sequencing to genetically map the regions underlying the needle-eye sperm phenotype. Surprisingly, the sterility phenotype was present in ~50% of males even after ten generations of backcrossing, yet the genetic map showed multiple regions associated with sterility, indicating multiple regions may have the capacity to be sufficient to induce sterility in the F1. Due to the common phenotype among sterile male hybrids and the strong effect of individual loci, further exploration of the genes uncovered here may identify a universal mechanism for the evolution of hybrid sterility. 

DNA extraction for each phenotype was performed as a modified protocol of the QIAGEN Gel Extraction Kit (Qiagen, Toronto, ON, Canada). Each sample was held at 95⁰C for 5 mins in a buffer solution (1M Tris-HCl, 0.5M EDTA, and 5M NaCl) containing 200µg/mL Protinase K. Isopropanol was then added to each sample and incubated overnight at -20 ⁰C. DNA purification was performed as directed by the QIAGEN Gel Extraction Kit. Purified DNA samples were then sequenced using the Illumina HiSeq 2000 platform with paired-end 100-bp reads at Génome Québec Innovation Center (Montréal, QC, Canada) using a target insert size of 400 bp.

Assembly of the FASTQ files generated by Illumina sequencing technology was performed using two Linux-based de novo assemblers: SOAPdenovo 1.05 and its companion program, GapCloser 1.12, (Li et al., 2008), and Abyss 1.5.2 (Simpson et al., 2009) at Kmer values of 41 to 91. The assembly with the highest N50 value was chosen for further analysis.

These are text files in FASTA format.