Divergence and hypermethylation of knob-associated DNA sequences in papaya MSY. (a) BACs 95B12 (green) and 61H02 (red) were selected by a pair of conserved DNA sequences specific to the MSY and the X chromosome, respectively. These two BACs hybridized only to their respective chromosomes. (b) The same pachytene chromosome was converted into a black-white image. BAC 95B12 can be located on K3. (c) BACs 85B24 (green) and 53E18 (red) selected by a pair of conserved DNA sequences specific to the MSY and the X chromosome, respectively. These two BACs hybridized only to the respective chromosomes. (d) The same pachytene chromosome was converted into a black-white image. BAC 85B24 can be located on K5. (e) Detection of 5mC (green signals) on an interphase nucleus. The MSY region is identified by a MSY-specific BAC 99O03 (red arrow), which is adjacent to a major punctuated 5mC signal (white arrow). The nucleus was stained by DAPI (blue). (f) Digitally separated BAC (red arrow) and DAPI signals. (g) Digitally separated 5mC signals. (h) A partial pachytene cell stained with DAPI. The MSY region was identified by the MSY-specific BAC 99O03 (red arrow). (i) Immunofluorescence assay using an anti-5mC antibody on the same pachytene cell. Bright signals are clearly visible on K1, K2, K3, and K5. The signal associated with K4 is relatively weaker. The five arrowheads in images a, b, c, d, h, and i point to the five knobs.
