30 Papaya
resulted in 12 genetic linkage groups, including nine major and three minor link-
age groups (Chen et al. 2007). The three minor linkage groups 10, 11 and 12 were
merged with major linkage groups 8, 9 and 7, respectively, using molecular cytoge-
netic approaches (Wai et al. 2010). To determine which chromosome corresponds
to which genetic linkage group, linkage group-specific BACs were used as probes
for fluorescence in situ hybridisation (FISH) in papaya meiotic pachytene chromo-
somes (Zhang et al. 2010). The X and Y chromosomes were identified as the second
longest chromosome pair and were designated as chromosome 1 in the karyotype.
The remaining papaya chromosomes were numbered according to length, chromo-
some 2 being the longest and chromosome 9 being the shortest. To locate the HSY
region on the sex chromosomes, two confirmed BACs in this area were directly
hybridised to interphase, prometaphase, metaphase and anaphase chromosomes
(Yu et al. 2007). These BACs hybridised near the centromere of the Yh. One BAC
had a weaker signal on the X, which suggested the sequences on the HSY and X in
this region were still relatively conserved. Since the second BAC only hybridised to
the Yh, it was likely that BAC sequence had diverged considerably. Pachytene FISH
was also utilised to map one of these HSY-specific BACs, along with its neighbour-
ing BACs and a non-HSY BAC. The HSY BACs showed strong signals only on the
Yh, whereas the non-HSY BAC showed a strong signal on a different homologous
pair of chromosomes. This study verified the identity of the X and Yh chromosomes
and located the HSY near the centromere on the Yh. To further explore the structure
of the MSY and X regions, hermaphrodite meiotic pachytene chromosomes were
stained with 4,6-diamidino-2-phenylindole (DAPI), which stains heterochromatic
regions of chromosomes (Zhang et al. 2008). Based on the staining, the arms of the
chromosomes were mostly euchromatic, but clusters of heterochromatin were found
around the centromere. Specifically, the XYh bivalent was mostly euchromatic, with
the X chromosome being the most euchromatic chromosome in the papaya genome,
but five knob-like regions of heterochromatin, numbered K1 through K5, were
found in the HSY (Zhang et al. 2010). The largest knob, K1, was shared between
the HSY and the X, but K 2 , K 3 , K 4 and K 5 were only found on the HSY. The knobs
were also found to be highly methylated. The heterochromatic knobs were likely
the result of transposable elements and the high DNA methylation in these regions
could be a defence mechanism against transposable element invasion. The HSY
contained two small regions of 5S rDNA, which is an element of the large subunit of
the ribosome involved in translation (Zhang et al. 2010). These regions were associ-
ated with K2 and K4. The X chromosome did not exhibit 5S rDNA. The accumula-
tion of 5S rDNA in the HSY likely led to the materialisation of heterochromatin and
assisted in the differentiation of the sex chromosomes (Zhang et al. 2010). During
X and Yh chromosomal pairing, a slight curve in the Yh chromosome occurred
to allow for pairing (Zhang et al. 2008). The region around K4 had accumulated
considerably more DNA then its X corresponding region, causing the curving of
the Yh chromosome during pairing. By implementing meiotic metaphase I-based
FISH using Yh-specific BACs, the centromere of the Yh chromosome was found
to be located in the HSY, specifically associating the centromere with K4 (Zhang
et al. 2008). The area around K4 showed more sequence divergence from the X than
other regions of the HSY.