Fig. 1: Existence of pachytene piRNAs in chickens.

a Roosters express pachytene piRNAs during spermatogenesis. (i) Key biological events during chicken spermatogenesis. (ii) Length distribution of total small RNAs. Ppm, parts per million. Blue, miRNAs. (iii) Abundance of piRNAs as measured by small RNA-seq. (iv) Expression of CIWI as measured by RNA-seq. Tpm, transcript per million. b Immunolabeling of squashed pachytene spermatocytes from adult chicken testes using anti-CIWI, anti-SYCP1, and DAPI. Scale bar, 10 µm. SYCP1, marker for synaptonemal complex formed during pachynema. We took at least 30 pictures and the representative pictures were shown. c Heatmap of normalized piRNA abundance per piRNA locus across the eight developmental stages of chicken testes. d Box plots of piRNA abundance at piRNA loci (n = 1321) in adult chicken testes and at their homolog regions (n = 637) in adult duck testes. Ppm: parts per million. Box plots show the 25th and 75th percentiles, whiskers represent the 5th and 95th percentiles, and midlines show median values. e Median value of (left) the mean phastCons score from 77 vertebrate genome alignments (probability that each nucleotide belongs to a conserved element) and (right) the mean phyloP score from 363 bird genome alignments (represent −log p-values under a null hypothesis of neutral evolution) of piRNA loci (red, n = 1321) and randomly shuffled control sequences (yellow, n = 10,000). Violin plots represent the medians of randomly shuffled control sequences that were computed 10,000 times.