Figure 1.
Computational validation of qPCR primers directed at AACAC satellite.
(A) Representation of homogenous block of AACAC repeat denatured and annealing to AACAC Forward and AACAC Reverse. Vertical lines indicate base pairs. (B) AACAC Forward hypothetically annealing to each of the five “phases”of AACAC (i.e., AACAC, ACACA, CACAA, ACAAC, and CAACA) and it’s reverse-complement. Bars indicate the number of base pairs possible in that phase, and line indicates ad hoc “melting” analysis. AACAC Forward has a strong preference for one phase and no affinity for the reverse-complement strand of the repeat. (C) as in (B), but with AACAC Reverse. (D) Shows the best possible pairing between AACAC Forward and Reverse. (E) AACAC Forward and AACAC Reverse pairing with every possible degree of overlap, from completely (at “0 offset”) to single 3′-most nucleotides pairing (at “37 offset”). Bars and lines are as in (B–C). In no case is AACAC Forward/Reverse dimer preferred over annealing to genomic targets (A–C) or the product of previous amplification.
Figure 2.
Experimental validation of qPCR assay to measure satellite copy number.
(A) Quantification cycle (Cq) of duplicate qPCR reactions plotted as a function of template DNA per reaction. X-axis represents log10 of an approximately 100-fold dilution series. (B) Quantification of satellite copy number in X/Y males and to X/Y/Y males, relative to X/Y (defined as 100%). Error bars represent standard error of the mean (S.E.M.) derived from triplicate reactions.
Figure 3.
Quantification of Y-linked satellite copy number variation in geographically divergent lines.
DNA and tissue samples obtained from males bearing Y chromosomes originally isolated from wild-caught flies. The genetic background of these males was otherwise isogenic. (A) Relative quantification of satellite copy number using qPCR. Percentages are relative to Y, Ohio (defined as 100%). Error bars represent standard error of the mean (S.E.M.) of triplicate qPCR reactions. (B) Fluorescence in situ hybridization to detect AACAC repeats (red) in squashed neuroblast cells derived from Y, Ohio larvae. DAPI stains DNA blue. (C) Quantification of in situ hybridization signals. Percentages calculated relative to Y, Ohio (defined as 100%). Error bars represent standard deviation (S.D.) of nuclei from thirty neuroblasts from each of three separate preparations per genotype (N = 90).
Figure 4.
Quantification of satellite copy number variation after a exposure to Su(var)20505/+ mutation.
(A) Relative satellite copy number on Y, 10Bt205 compared to Y, 10B (defined as 100%). The chromosomes are originally from a single progenitor, but the former was maintained for 6 years in a Su(var)2055/CyO mutant background. Error bars represent standard error of the mean (S.E.M.) of quadruplicate qPCR reactions. (B) DNA from four individual males bearing Y, 10Bt205 were separately prepared and used as template for qPCR. Graphs show population average, error bars represent standard deviation (S.D.) of individuals within populations pooled with standard error of the mean (S.E.M.) of replicate reactions. Average AACAC copy number (in (A)) are comparable to the average of the population (in (B)), and population distributions remain detectably different (10B vs. 10Bt205 in (B), P = 0.0336). (C) Images of female flies of genotype C(1)DX/Y, 10B (top) and C(1)DX/Y, 10Bt205 (bottom). The fly with 10Bt205 as sole source of rDNA exhibits a strong bobbed phenotype, indicating significant rDNA loss. (D) qPCR determination of rDNA copy number in the flies from (C). Error bars report standard error of the mean of replicate qPCR reactions from pooled siblings.