The rb-bands and INTs differ from each other in gene density, expression and tissue specificity.

<p><b>(A, B)</b> Rb-bands in the three replication completion groups (LR0–1, LR2–3, and LR4–5) are significantly depleted in genes as compared with intervals between these bands (INTs). (A) Gene density (genes per 100 kb) according to FlyBase annotations and (B) 5'-UTR density (5'-UTRs per 100 kb). Two-sample permutation tests, 10,000 iterations, ***p < E-5. <b>(C)</b> The expression of genes lying entirely in rb-bands in various tissues is significantly different from that of genes lying entirely in the intervals between rb-bands. Boxplots of gene expression data for LR0–LR1 (green bars), LR2–LR3 (blue bars), and LR4–LR5 (purple bars) bands and intervening sequences (red bars) in different larval or adult tissues. Gene expression data in FPKM (Fragments Per Kilobase Million) were generated by modENCODE ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref043" target="_blank">43</a>], March 2016 data freeze). All groups of rb-bands are significantly different from INTs and from one another (the Mann–Whitney <i>U</i> test, p < E-26). <b>FB wl</b>: fat bodies, wandering larvae; <b>FB p</b>: fat bodies, 2-day prepupae; <b>CNS wl</b>: central nervous system, wandering larvae; <b>CNS p</b>: central nervous system, two-day old prepupae; <b>DS wl</b>: digestive system, wandering larvae; <b>DS a</b>: digestive system, one-day old mixed adults; <b>Head af</b>: heads, one-day old female adults; <b>ID wl</b>: imaginal discs, wandering larvae; <b>SG wl</b>: salivary glands, wandering larvae; <b>SG p</b>: salivary glands, prepupae; <b>AG am</b>: male accessory glands, male adults; <b>Testis am</b>: testes, male adults; <b>Ov af</b>: female gonads, female adults. <b>(D)</b> The later the bands are replicated, the higher the proportion of testis-specific genes they tend to have. The proportion of testis-specific genes (according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref055" target="_blank">55</a>]) in INTs and rb-bands grouped based on their replication completion time (LR0–1, LR2–3, and LR4–5). Two-sample permutation tests, 10,000 iterations, ***p < E-3. <b>(E)</b> The latest-replicating rb-bands are typically enriched with multigene clusters of testis-specific genes. The proportion of stand-alone testis-specific genes, two-item, and multiple-item clusters among all testis-specific genes (according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref055" target="_blank">55</a>]) on chromosome 2R (All), in intervals between rb-bands (INTs) and in rb-bands classified based on replication completion time in salivary glands (LR0–1, LR2–3, and LR4–5). Blue bars correspond to the proportion of stand-alone testis-specific genes among all testis-specific genes; red bars, to the proportion of two-item clusters; green bars, to the proportion of multiple-item clusters. The INTs are devoid of multigene clusters of testis-specific genes. This distribution is significantly different from random (resamples n = 10,000, goodness-of-fit test, p < E-5.). LR4–5 rb-bands are enriched with multigene clusters and depleted of stand-alone testis-specific genes. This distribution is significantly different from a random one (resamples n = 10,000, goodness-of-fit test, with provision for cluster size, p < E-5). <b>(F)</b> Genes in the rb-bands tend to be expressed in a limited number of tissues. By contrast, the intervals between rb-bands are enriched with genes that are highly expressed in most of the tissues under consideration. Boxplots of the numbers of tissues with above-threshold gene expression. Threshold values: 0; 0.1; 1; 10; 100. At 0–10, INTs significantly differ from any rb-band (***p < E-30). At 0 and 0.1, LR2-3 bands significantly differ from LR4–5 bands (**p < E-5; Mann–Whitney <i>U</i> test). Gene expression data were generated by modENCODE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref043" target="_blank">43</a>].