Botany and Improvement 27
an example of the earliest stage of sex chromosome evolution. In the second stage,
recombination is suppressed at and around the mutations, a crucial step in sex chro-
mosome evolution, leading to degeneration of the Y chromosome, though the YY
genotype is still viable, as seen in asparagus. In the third stage, the suppression of
recombination spreads to other loci, forming a male-specific region on the Y chro-
mosome, and though the chromosomes appear to be homomorphic at this stage,
some genes on the Y chromosome are lost through transposable element insertions,
deleterious mutations and chromosomal rearrangements causing an inviable YY
genotype. Papaya is an example of stage three. During the fourth stage, the MSY
accumulates transposable elements and duplications, causing a DNA expansion.
The non-recombining region spreads to majority of the Y chromosome and the sex
chromosomes are heteromorphic, with the Y chromosome often becoming consider-
ably larger than the X chromosome. Silene is a good example of an angiosperm in
stage 4. During stage five, though a small portion of the sex chromosomes contin-
ues to recombine, keeping the pair together, severe degeneration of the Y chromo-
some occurs and many genes lose function, leading to the loss of the non-functional
sequences, causing the Y chromosome to shrink. There are no current examples of
angiosperm sex chromosomes in this stage. Finally, in stage six, the suppression of
recombination spreads to the entire Y chromosome, causing the Y chromosome to
be completely lost. Sex is then determined by an X to the autosome ratio, as is seen
in Rumex.
Though many hypotheses had been made about the sex determination system of
papaya, little concrete molecular data had been generated to verify which hypothesis
accurately described what was occurring in papaya that led to these three sex types.
Genetic cross data, phenotypic data and some early cytological observations were
the only evidence used to form these early hypotheses. Scientists had no means to
tackle the question of papaya sex determination until the applications of molecu-
lar techniques and biotechnology. The first method explored to detect papaya sex
was through the use of sex-linked molecular markers. Microsatellite and sequence-
characterised amplified region (SCAR) markers, which showed different banding
patterns between the sex types, were successfully developed by different papaya
research groups. This allows papaya sex to be determined during the vegetative state,
but for commercial use, it is too costly to test thousands of seedlings and relocate
them to the field (Parasnis et al. 2000; Deputy et al. 2002; Urasaki et al. 2002).
Molecular markers were also used in producing multiple genetic linkage maps for
papaya. The first map was constructed by Hofmeyr (1939), consisting of three mor-
phological markers, namely sex, flower colour and stem colour. The second genetic
linkage map consisted of 62 randomly amplified polymorphic DNA (RAPD) mark-
ers and mapped sex onto linkage Group 1 (Sondur et al. 1996). The third map incor-
porated 1498 amplified fragment length polymorphism (AFLP) markers, the papaya
ringspot virus coat protein marker, sex and fruit flesh colour, totalling 1501 markers
which were mapped onto 12 linkage groups (Ma et al. 2004). Most recently a high
density genetic map using 712 simple sequence repeat (SSR) markers, designed from
BAC end sequences, whole-genome shot gun sequences and a morphological marker
resulted in nine large linkage groups and three small linkage groups (Chen et al.
2007). Sex was mapped onto linkage Group 1, one of the nine large linkage groups.