Schematic of D. virilis, D. americana (carrying the X–4 fusion), and F₁ hybrid chromosomes. For D. virilis and D. americana females, X chromosomes and autosomes are labeled (order is the same for males and hybrids shown below). The Y and dot chromosomes are represented by hooked bars and small squares, respectively. The X–4 and 2–3 chromosomal fusions of D. americana are represented by connected bars that form backward “L” shapes. AV refers to an F₁ hybrid with D. americana as the maternal parent, whereas VA refers to an F₁ hybrid with D. virilis as the maternal parent. Note that D. americana males carry one unfused chromosome 4. Only in one direction of the interspecific cross (VA) does the F₁ male inherit two unfused copies of chromosome 4, which allows the independent assortment of this chromosome in backcrosses.

Genetic mapping of D. virilis–D. americana F₂ hybrid male sterility. Likelihood ratio (LR) test statistic profiles from composite interval mapping (CIM) of male fertility in (VA)(VA) F₂ mapping populations. Horizontal lines mark LR significance thresholds and vertical double lines delineate unlinked regions. The genetic positions of molecular markers are indicated by triangles, and the corresponding physical locations along chromosomes X, 4, 2, 3, and 5 (based on the D. virilis genome assembly) are indicated below by vertical bars. Physical distance is shown scaled to genetic distance, and therefore, differs among chromosomes; to the left of the label for each chromosome is a 10-Mb scale bar. The shaded horizontal bars on chromosomes denote inverted regions, with lighter shading representing uncertainty in precise physical locations. To facilitate comparisons of QTL peak positions between mapping experiments, a subset of molecular markers genotyped in both F₂ mapping populations are indicated by asterisks. (A) Genomewide mapping of hybrid male sterility in (VA)(VA) F₂ males generated using the SB 02.06 D. americana strain, which carries the 5b inversion on chromosome 5 (N = 572). Note that markers flanking the X–4 chromosomal fusion should be more tightly linked than they appear here because genotypic variation via recombination events are confounded by independent assortment of the paternal fourth chromosome. As a result, the QTL associated with these markers (indicated by dotted lines and a black arrow) appears somewhat wider than it should. LR significance threshold (indicated by a horizontal line) is equal to 12.9. See Figure S1 for a slightly different analysis that excludes paternal fourth chromosome haplotypes, but still yields similar QTL mapping results. (B) Targeted mapping of hybrid male sterility on chromosomes 2 and 5 in (VA)(VA) F₂ males generated using the CB 05.14 D. americana strain, which carries the 5a inversion (but not 5b) on chromosome 5 (N = 459). LR significance threshold is equal to 11.3.

Genetic mapping of D. americana–D. virilis F₂ hybrid male sterility. Likelihood ratio (LR) test statistic profile from composite interval mapping (CIM) of male fertility in the (AV)(AV) F₂ mapping population (generated using the SB 02.06 D. americana strain, N = 347). A horizontal line marks the LR significance threshold of 14.3 and vertical double lines delineate unlinked regions. The genetic positions of molecular markers are indicated by triangles, and the corresponding physical locations along chromosomes X, 4, 2, 3, and 5 (based on the D. virilis genome assembly) are indicated below by vertical bars. Physical distance is shown scaled to genetic distance, and therefore, differs among chromosomes; to the right of the label for each chromosome is a 10-Mb scale bar. The shaded horizontal bars on chromosomes denote inverted regions, with lighter shading representing uncertainty in precise physical locations. Note that, in contrast to , markers flanking the X–4 chromosomal fusion are tightly linked. Because (AV)(AV) F₂ males must inherit the D. virilis Y and fourth chromosomes together, crossover events at the junction of the X–4 fusion can be easily detected. Also note that markers on the fourth chromosome are split into two linkage groups; additional markers will be required to resolve the genetic map in this region.