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was determined by making a scatter plot using the principle components of papaya male, female and
hermaphrodite plants leaves. This analysis showed a little orientation from central axis of principal components in
male while, in female and hermaphrodite leaf shape was found to be distant away from the central axis of first two
principal components. Another test, Kruskal-Wall was failed to show much change in the leaf shape distribution
of papaya different sex types.
5.2 Cytological identification
Several cytological studies have also been done to identify either any chromatin body or presence of a
heteromorphic pair of chromosome in papaya which could help in identifying the different sex forms. However,
these studies failed due to none of the above have been identified (Datta, 1971).
5.3 Isozymes markers
Isozymes are isoforms of single enzyme that differs in amino acid sequences yet catalyzed the same chemical
reaction (Markert and Moller, 1959). Alteration in amino acid occurs due to mutation in DNA which changes the
net electric charge of the protein. Electrophoresis techniques can be used to detect such differences in ionic charge
and size of the protein and resolved using enzyme specific stains which results in a small number of specific
bands. Isozyme markers are co-dominant in nature. Cationic peroxidase isozyme was used for gender
determination in papaya; males could be differentiated from females on the basis of banding pattern. However,
females failed to differentiate from hermaphrodites (Sriprasertsak et al., 1988). Isozyme has some limitations for
sex type identification such as post transcriptional modification, affected by environmental conditions; their
expression varies from tissue to tissue. The failure of morphological traits study, cytological evidences (Parasnis et
al., 1999, 2000; Magdalitan et al., 2003; Gangopadhyay et al., 2007) and isozyme markers to identify or
differentiating the different sex forms of papaya at the early juvenile seedling stage has encouraged for the
development and utilization of PCR-based molecular markers.
5.4 PCR-based gender-linked markers
Mullis and Faloona (1987) discovered polymerase chain reaction (PCR) technology that leads to the development
of many novel fingerprinting techniques. In PCR-based methods, only one primer or a primer pairs are used for
DNA amplification reaction. These PCR based markers which are also termed second generation markers like
RAPD, SCARs, ISSRs, AFLPs, SSRs and third generation marker like SNPs are advantageous over the
first-generation markers [hybridization-based markers; e.g. restriction fragment length polymorphism (RFLP)], as
they require much less DNA (10-100 ng) of relatively lower quality, avoid DNA blotting and use of radioactivity,
amenable to automation and are much more user-friendly. RAPD (Welsh and McClelland, 1990;William et al.,
1990),AFLP (Vos et al.,1995), ISSR (Zietkiewicz et al., 1994), SSR (Akkaya et al., 1992) and SNP (Jordan and
Humphries, 1994) are most commonly used PCR based DNA marker techniques that have been used to develop
gender/sex-linked markers in papaya and in various other dioecious taxa.
5.4.1 Random amplified DNA polymorphism (RAPD)
Welsh and McClelland, (1990) and William et al., (1990) independently developed a new PCR based marker
technique called arbitrarily primed polymerase chain reaction (AP-PCR) or random amplified polymorphic DNA
(RAPD) technique, respectively. This technique utilizes short synthetic oligonucleotides (usually 10 bases long)
primer of random sequence. It is simple, cheap and no prior sequence information of template DNA is required. It
is dominant marker (scored as either ‘present’ or ‘absent’); showing high levels of polymorphism and required a
small quantity of DNA (Jiang, 2013). RAPD is most popular marker system for sex determination in papaya.
Several RAPD based sex-specific markers have been generated in
C. papaya
(Table 2). Magdalita and Mercado
(2003) used two 20mer primer pairs to predict the sex type in three papaya varieties (‘Cavite’, ‘Cariflora’ and
‘Sinta’ hybrid). Females produced a single band of 0.8 kb; hermaphrodites produced two distinct bands of 1.3 kb
and 0.8 kb, while males had no band. The frequency of males, hermaphrodites and females identified as such both
by field observation and PCR, showed 100% accuracy in the prediction. Bedoya and Nuenz (2007) developed
