Y-derived genes localize to the nonrecombining and meiotically silenced X Chromosome in E. lutescens. (A) Localization of FISH probes for E. lutescens homologs of mouse, Usp9y and Zfy (green) on the single X (encircled) in combination with an E. lutescens X-Chromosomal BAC FISH probe (red) in E. lutescens meiotic spreads also immunostained for SYCP3 (purple). Enlargements of indicated areas are shown on the right. DAPI counterstaining of the DNA is shown in the top and middle images. (B) Immunostaining for H2AFX and SYCP3 (top), polymerase (RNA) II (DNA-directed) polypeptide A (POLR2A) and SYCP3 (middle), and MLH1 and SYCP3 (bottom) on spread pachytene E. lutescens spermatocytes. Phosphorylated H2AFX marks the single X chromatin (encircled) in pachytene. POLR2A is depleted from the X Chromosome region in comparison with the rest of the nucleus. MLH1 marks crossover sites along the synaptonemal complexes of all autosomes. Enlargements of indicated areas are shown on the right. (C) Expression level of X-Chromosomal genes compared with autosomal genes. E. lutescens testis RNA was assembled and annotated using mouse cDNA; transcript abundance was quantified and plotted using the mouse chromosomal map. Low expressed genes (genes below the 25th percentile of the data) were removed before plotting. Expression level is expressed in FPKM (log2 scale). (D) Female E. lutescens reads were aligned to the male E. lutescens reference genome, and SNVs were called that are homozygous within the female but differ from the assembled male genome. High-quality SNVs were plotted using the mouse chromosome annotation and normalized to the number of coding genes per chromosome. None were found for the X Chromosome.
