A diagram of X–Y^h chromosomal pairing in the MSY.

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.

Heterochromatin formation in papaya MSY. (a) A complete pachytene cell stained with DAPI. The MSY is included in the square. (b) The same pachytene cell was converted into a black-white image. The five knobs in MSY are clearly visible. The diagram in the second square illustrates the (red) X and (green) Y^h chromosomes and the five (blue) knobs. (c) A complete pachytene XY^h bivalent stained by DAPI. The MSY is included in the square. (d) The DAPI image was converted into a black-white image. The diagram in the second square illustrates the X (red) and Y^h (green) chromosomes and the five knobs (blue). Bars, 10 μm.

Pachytene FISH mapping of the MSY and the centromere of the Y^h chromosome. (a) FISH mapping of seven BAC clones. BACs 69A15 and 86B15, which mark the boundary of MSY, were mapped outside of knobs K1 and K5, respectively. FISH signals pointed to by arrowheads 1–5 were derived from BACs 12I03, 99O03, 95B12, 52H15, and 85B24, respectively. Bar, 10 μm. (b) The same pachytene cell image was converted to a black-white image. The locations of the FISH signals derived from the five BACs matched the locations of the five knobs. (c) FISH mapping of K4-specific BAC 52H15 (red) and K5-specific 85B24 (green) on metaphase I (MI) XY^h bivalent chromosome. K4 is positioned more poleward than K5. (d) MI FISH mapping of K4-specific 52H15 (green) and K3-specific BAC 47B08 (red). K4 is positioned more poleward than K3. (e) MI FISH mapping of K3-specific BAC 47B08 (red) and K2-specific 99O03 (green). (f) MI FISH mapping of K2-specific 99O03 (red) and K1-specific BAC 12I03 (green). The white arrow points to the K1 on the X chromosome.