overall patterns of DNA sequence variation, reducing variability around centromeres and on sex chromosomes (e.g., ref. [62]), remains to be determined. Methods PCR-Screen for SD Chromosomes We used a molecular assay to screen for SD chromosomes in a collection of 452 isofemale lines from across sub-Saharan Africa, kindly provided by Drs. John Pool, Charles Aquadro and Andy Clark (Cornell University). We used a single-reaction PCR assay involving three primers, a forward primer (F) and two reverse primers (R1 and R2): F = TTTGGAGACTGCCTGATCAAAA- CTAATG; R1 = CAACGTCGCGGAGGAGACTGCCTATGT; R2 = CGTGTTCTGAGCGTTTCGCACAGTGTAT. One pri- mer pair (F-R1) amplifies a 463-bp fragment from the parent gene, RanGAP (a positive control), and the other (F-R2) amplifies a 353- bp SD-specific fragment that spans the breakpoint of the Sd- RanGAP-RanGAP junction (Figure 1B). Only one amplicon results from flies that lack SD chromosomes and two result from flies that carry SD (Figure 1B). Extracting SD Chromosomes Isofemale lines found to be SD-positive by PCR assay could be homozygous SD/SD or heterozygous SD/SD+. We therefore extracted SD chromosomes onto a common genetic background, then maintained homozygous viable SD chromosomes as homo- zygous stocks, and maintained homozygous lethal SD chromo- somes over the CyO balancer chromosome. To extract SD chromosomes, we crossed 3–5 w118; In(2LR)Gla, wgGla-1 Bc1/CyO (hereafter, w118; Gla/CyO) virgin females to 3–5 males from the SD- positive isofemale lines. We then collected 5 white-eyed CyO sons and individually backcrossed them to 5–10 w118; Gla/CyO females. Once larvae appeared in the backcross vials, we PCR-tested the 5 white-eyed CyO sons for SD (see above) and retained progeny from a single SD-positive male. We then crossed w118/w118; SD/CyO virgin daughters to w118; SD/CyO sons. If the SD chromosome was homozygous viable, we used the progeny to establish a w118; SD/ SD stock; if the SD chromosome was homozygous lethal, we maintained a w118; SD/CyO stock. Last, we confirmed that all of the final stocks carried the SD chromosome by PCR assay. Inversion-Typing SD Chromosomes Many SD chromosomes possess one or more inversions on chromosome 2 (reviewed in ref. [2]). To determine the inversion types of SD chromosomes, we examined polytene chromosomes from larval salivary gland squashes. We crossed virgin cn bw females to SD males to generate larvae; cn bw chromosomes have standard arrangement second chromosomes. Salivary glands were dissected from F1 larvae in 1% Na-citrate hypotonic solution on siliconized slides and then transferred and fixed for 10–15 seconds in 45% acetic acid. The dissections were stained with 1% lacto- aceto-orcein for 25–35 minutes. We determined inversion break- points by comparing photographs with the standard maps of chromosome 2. Complementation Tests among SD Chromosomes We performed complementation tests between all pairwise combinations of SD chromosomes. For all homozygous lethal SD chromosomes, we tested the viability of all SDi/SDj combinations by crossing five SDi/CyO virgin females to 3–5 SDj/CyO males. If CyO+ progeny appear, then the lethality of SDi and SDj chromosomes must map to different complementation groups. We also tested the male and female fertility of viable SDi/SDj Segregation Distorter Causes Sweep combinations. At least two replicates each of 3–5 SDi/SDj males and 3–5 virgin SDi/SDj females were crossed to OreR virgin females and males, respectively. SDi/SDj flies that produced larvae were considered fertile, whereas those that failed to produce any progeny over multiple replicates were considered sterile. Estimating the Strength of Segregation Distortion We estimated the strength of distortion for each SD chromo- some by measuring the rate of transmission, k, of the SD chromosome through heterozygous SD/Gla males. In preliminary work, we screened a series of balancer chromosomes (Bal) for sensitivity to distortion by assaying transmission from SD-5/Bal males. SD-5 is a well-characterized, non-African SD chromosome. These crosses revealed that the In(2LR)Gla chromosome (hereafter, Gla) carries a sensitive Rsps allele. Gla is an effective balancer of most of the second chromosome and carries a dominant eye- phenotype marker. We estimated k by individually crossing five SD/Gla males of each SD chromosome to five 3–5 day old cn bw virgin females each. After four days, each cross was transferred to a fresh food vial every fourth day. We then scored all progeny emerging until 20 days after the parents were removed from each of the four vials. The rate of transmission of SD to progeny depends both on the strength of distortion and on the relative viability of the SD chromosome. Therefore, to distinguish the strength of distortion from relative viability, we measured the rate of transmission of SD chromosomes through heterozygous SD/Gla females. As distortion is male-specific, the rate of transmission of SD through females allows estimation of SD relative viability. By using the Gla balancer to minimize recombination on the second chromosome in females, we could estimate the viability of intact SD chromosomes like those transmitted through males (which lack recombination in D. melanogaster). For each SD chromosome we set up three replicate crosses of five 3–5 day old SD/Gla virgin females with three 3–5 day old cn bw males. After four days, each cross was transferred to fresh vial every fourth day. We used our estimates of relatively viability to estimate a corrected strength of distortion, k*, following ref. [63]. Sequencing of Sd-RanGAP To sequence the new Sd-RanGAP duplicate gene, we first isolated SD chromosomes in heterozygous state over a chromo- somal deficiency, Df(2L)Sd77, which deletes the 37D1– 37D2;38C1–38C2 region including the RanGAP locus. After isolating genomic DNA from SD/Df(2L)Sd77 flies, we PCR amplified two fragments from the Sd-RanGAP region with two sets of primers. All PCR products therefore come from the SD chromosome. The first set amplifies a 2,994-bp fragment from the 59-half of Sd-RanGAP. The forward primer (F4) binds the distal intergenic region between Sd-RanGAP and the neighboring gene CG10237; the reverse primer (R4) binds in intron 1 of Sd-RanGAP (which, on the reverse strand, is exon 2 of Hs2st). The second primer set amplifies a 2,410-bp fragment from the 39-half of Sd- RanGAP with a 280-bp overlap with the first fragment. The forward primer (F6) binds in the first intron of Sd-RanGAP (which, on the reverse strand, is intron 2 of Hs2st); the reverse primer (R6) binds the intergenic region between Sd-RanGAP and RanGAP. Both the R4 and F6 primers bind two genomic locations in flies with SD chromosomes. First, R4 binds the first intron of Sd-RanGAP and the homologous sequence of the parent gene RanGAP. However, when the F4-R4 primer pair is used and PCR extension times are constrained, only product from the first R4 binding location results. Second, F6 binds the first intron of Sd-RanGAP and the homologous sequence of RanGAP. However, when the F6-R6 PLoS Genetics | www.plosgenetics.org 11 May 2009 | Volume 5 | Issue 5 | e1000463