Signatures of piRNA biogenesis in the dysgenic germline show only modest defects.

<p>(A) Size distributions of small RNAs are similar between dysgenic and non-dysgenic germlines. Distribution of all small RNAs (not normalized) from four germline libraries (2 dysgenic, 2 non-dysgenic) filtered for tRNA, rRNA and snoRNA. (B) piRNA biogenesis signature heatmaps. TEs upregulated in the dysgenic germline (a difference of 5 RPKM or higher) are indicated with red bars. TEs upregulated in the non-dysgenic germline (a difference of 5 RPKM or higher) are indicated with purple. On the left are heatmaps for raw measures of abundance, the density of ping-pong pairs and percent ping-pong. On the right are heatmaps for the same metrics, but by row z-score. For raw measures, there are no globally discernible effects of dysgenesis on piRNA biogenesis. Row z-scores in dysgenesis do show lower values for abundance measures (abundance and ping-pong pair density), but not percent ping-pong (see text). (C) Fold excess in expression in dysgenesis vs. the difference in percent ping-pong Z-score between dysgenic and non-dysgenic germline. Of the top eight that are most differently expressed in dysgenesis, all have lower ping-pong z-scores in dysgenesis.</p

TE expression as a function of piRNA and siRNA abundance in parental strains and progeny.

<p>(A). Normalized (per 1 million mappers) piRNA abundance +0.1 (log 10) in the strain 9 germline vs. the strain 160 germline. A large number of TEs show increased piRNA expression in the strain 160 germline, especially TART and others enriched in abundance in strain 160. Diagonal lines indicate 10-fold levels of difference (B) Normalized (per 1 million mappers) piRNA abundance +0.1 (log 10) in the dysgenic germline vs. the non-dysgenic germline. piRNA abundances for many TEs with greater excess in strain 160 become similar in the dysgenic germline. A significant exception to this is the <i>Helena</i> element. Diagonal lines indicate 10-fold levels of difference. (C) TE piRNA excess in strain 160 vs. relative expression level in dysgenesis. TE piRNA asymmetry between 160 and 9 is not the sole determinant of increased expression in dysgenesis. Some elements, such as 750, are increased in expression in dysgenesis, despite similar piRNA abundances in 9 and 160. (D) TE piRNA excess in the non-dysgenic germline vs. relative TE expression level in dysgenesis. Elements such as <i>Skippy</i> and <i>Slicemaster</i> show equilibrated piRNA abundances, but excess expression in the dysgenic germline. (E) TE siRNA excess in strain 160 vs. relative TE expression level in dysgenesis. (F) TE siRNA excess in the non-dysgenic germline vs. relative TE expression level in dysgenesis.</p

Multiple transposable elements are associated with induction of hybrid dysgenesis.

<p>(A) Relative mapping abundance of single-end, 100 bp reads from strain 9 and strain 160 (normalized by reads mapping to the genome), to a consolidated repeat library. Eleven elements are in 3-fold excess in strain 160 and are indicated here and throughout with red. TART elements are about 1.7-fold in excess and are indicated here and throughout with blue. No apparent TEs were found in excess in strain 9. (B) Using piledriver (<a href="https://github.com/arq5x/piledriver" target="_blank">https://github.com/arq5x/piledriver</a>) we assessed homogeneity within reads mapping to the TE library by determining the average frequency of the major variant in both strains. TEs in excess in strain 160 are either more homogenous in strain 160 or similarly aged between strains, with the exception of element 1069 which shows slightly more homogeneity in strain 9.</p

Increased TE expression in the dysgenic germline persists through adulthood.

<p>(A) RPKM+0.01 (log 10, average across both ages) for TEs, Dysgenic vs. Non-dysgenic germline. TEs that are in excess in 160 are more highly expressed, as well as many TEs that are not in excess. (B) Fold excess in expression (RPKM+0.01, log 2, average across both ages) vs. fold excess in abundance in strain 160. Nearly all TEs that are in excess in 160 show increased expression in the dysgenic germline (11/12). But multiple TEs that are equivalent in abundance between strains are also increased in expression. (C,D) Increased expression in the dysgenic germline is maintained as flies age. Note: Log scale obscures magnitude of difference for some TEs that demonstrate significant differences in expression identified due to low variation across replicates.</p

Properties of TEs significantly more expressed in the dysgenic germline by at last 2-fold (FDR<0.1).

<p>160:9 Abundance, 160:9 total piRNA, Expression: D (Dysgenic, RPKM), Expression: ND (Non-dysgenic, RPKM), Ping-pong pair density: D (Dysgenic, per Million piRNAs mapped), Ping-pong pair density: ND (Non-Dysgenic, per Million piRNAs mapped). Type I: Higher in copy number in 160, higher in piRNA abundance in 160, F1 piRNA ping-pong pair density defined by maternal loading. Type II: Higher in copy number in 160, higher in piRNA abundance in 160, F1 piRNA ping-pong density equilibrated. Type III. Higher in copy number in 160, higher in piRNA abundance in 160, F1 piRNA ping-pong density higher in dysgenic. Type IV: Higher in copy number in 9, higher in piRNA abundance in 160, F1 piRNA ping-pong pair density defined by maternal loading. Type V: Higher in copy number in 9, higher in piRNA abundance in 160, no ping-pong pairs detected. Type VI: Higher in copy number in 9, equivalent piRNA abundance in 9 and 160, F1 piRNA ping-pong density higher in non-dysgenic.</p><p>Properties of TEs significantly more expressed in the dysgenic germline by at last 2-fold (FDR<0.1).</p

Genic piRNA targeting is increased in the dysgenic germline.

<p>(A) log10 Z-score heat map of genic (CDS) piRNA density for <i>D</i>. <i>melanogaster</i> orthologs (above a threshold of 5 piRNAs per CDS per 1 million mapped in at least one of four columns). Of these 105 genes, there is an excess of genic piRNAs in the dysgenic germline (89 genes with greatest genic targeting in dysgenesis, P<0.001) (B) Sense vs. Anti-sense abundance for piRNAs in genic piRNA class for one library (Sample 1). Some CDS regions are predominantly the source of anti-sense piRNAs, but the majority are biased as a source of sense strand piRNA (C) Distribution of expression levels (log 10 RPKM+0.01) for all genes in the genome and piRNA target genes (expression levels from non-dysgenic germline). Genic piRNA targets are derived from more highly expressed genes (p < 0.001). (D) Of 105 genes, the 89 that show excess genic piRNA in dysgenesis are also more lowly expressed in dysgenesis. Shown is the distribution of expression ratios (dysgenic:non-dysgenic) for all genes and genes that are increased as a source of genic piRNAs in dysgenesis (p < 0.001).</p

Germline and ovary genic cluster behavior across generations for <i>D</i>. <i>virilis</i> orthologs of <i>center divider</i> and <i>oysgedart</i> from <i>D</i>. <i>melanogaster</i>.

<p>piRNA mapping densities are indicated. mRNA-seq RPKM for germline (0–2 H embryo) is also indicated. Allelism was determined by counting mRNA-seq reads based on SNPs that distinguish strain 9 and 160. Strain 160 cluster identity is maintained for <i>cdi</i> in non-dysgenic progeny in which strain 160 is the mother. This is correlated with silencing of both alleles in the non-dysgenic germline. In contrast, the cluster is not maintained in the dysgenic germline and both alleles are expressed. Somatic expression is not affected. Germline cluster identity for <i>oysgedart</i> (which in the germline is predominantly sense) is lost in progeny. In this case, expression is even between reciprocal progeny, but germline expression is lower from the 9 allele in both directions of the cross. For cluster behavior in F3 backcrosses, heterozygosity or homozygosity of the respective allele is indicated. Notice how cluster identity is maintained for <i>cdi</i> to varying degrees in individuals homozygous for the 9 allele. In contrast, cluster activity is absent in all progeny for <i>oysgedart</i>.</p

Genetic analysis of zygotic induction and maternal repression of gonadal atrophy.

<p>(A) Induction of sterility by 160 is broadly distributed across the genome, with the exception of chromosome 6 (the dot chromosome). Log odds ratios for probability of induction were estimated by crossing F1 males to strain 9, determining whether F2s had male gonadal atrophy and genotyping F2s to determine the chromosomes inherited from the father. Estimates were determined using a generalized linear model for logistic regression (binomial family with a logit link). Values in red are actual odds ratios. Whiskers are 95% confidence intervals. Chromosome 5 is significant at 0.1 level only. X chromosome is not scored because dysgenesis is scored in males and males do not inherit the X from their fathers (N = 92). (B) Scatterplot showing proportion of dysgenic testes (<i>y</i> axis) observed in the progeny of each F3 female individual (<i>x</i> axis). Red dots indicate F3 females that were selected for whole genome sequencing. (C) Single marker QTL analysis identified 3 putative QTLs: one flanking the centromeres of the 5^th and X chromosomes and one of the tested euchromatic regions of the 4^th chromosome. (D) Top row: Results from the genotyping assay. Colored rectangles represent the presence of strain 160 SNPs in individuals, ranked from top to bottom (most protective individuals on top). Scatterplots: sequencing results. Each dot represents the average number of base pairs that uniquely mapped to every 10kb of the 160 genome. Valleys indicate regions of strain 9 homozygosity. Black dots above scatterplots show the location of each SNP used for our genotyping assay. Grey background demonstrates that no region of the genome from 160 is necessary to protect against dysgenesis. Right-most columns: Number of piRNAs mapped to TART sequences, per million reads, for each F3 female individual. Color intensity is representative of TART piRNA abundance. Number of 21 nt endo-siRNAs mapped to <i>Penelope</i> sequences, per million 21 nt reads, for each F3 female individual. Color intensity is representative of <i>Penelope</i> endo-siRNA abundance. Red bar indicates position of one of several <i>Penelope</i> endo-siRNA loci on the X.</p

Comparative analysis of a set of HD-implicated TEs in the ovaries of dysgenic and reciprocal hybrids.

<p>A) Expression levels of <i>Penelope</i>, <i>Paris</i>, <i>Polyphemus</i>, <i>Helena</i> and <i>Ulysses</i> among the studied <i>P</i>-like, <i>M</i>-like and neutral strain. B) mRNA and piRNAs expression levels in the ovaries of the progeny from dysgenic and reciprocal hybrids. At the left—expression levels of indicated TEs relative to the level in <i>P</i>-strain <i>160</i>. At the right–normalized piRNAs expression levels. The dotted line indicates level in <i>P</i>-strain <i>160</i>. <i>P</i>-values were calculated using <i>t</i>-test. C) Expression level of <i>Penelope</i>, <i>Paris</i>, <i>Polyphemus</i>, <i>Helena</i> and <i>Ulysses</i> in the ovaries of dysgenic crosses compared to reciprocal ones. <i>P</i>-values were calculated using <i>t</i>-test.</p

Characterization of <i>Penelope</i> activity in the ovaries of <i>Penelope</i>-transformed strains.

<p>A) Expression levels of <i>Penelope</i> in <i>9(w3)</i>, <i>Tf1</i>, <i>Tf2</i> strains relative to <i>P</i>-strain <i>160</i>. B) (1) The coverage of normalized <i>Penelope</i>-piRNA reads (23–29 nt) on the entire body of the element, across transformed strains. Sense reads are shown as [+], antisense as [–]. (2) The ping-pong signature of <i>Penelope</i>-derived piRNAs. C) Mapping proportions of <i>Penelope</i>-piRNAs to canonical sequence of the element with the perfect match and with the assumption of up to 3 mismatches.</p