Molecular Plant Breeding 2016, Vol.7, No.28, 1
-
18
11
Despite these, RAPD have some limitations such as high sensitivity to variations in PCR amplification conditions
resulting low reproducibility and reveals only homology (Mishra et al., 2014).
5.4.2 Sequence characterized amplified region (SCAR)
Due to high sensitivity of RAPD to variations in PCR amplification conditions, these markers are converted into
more reliable and stable marker termed specific sequence characterized amplified region (SCAR; Paran and
Michelmore, 1993). SCAR markers are sequence specific, highly reproducible and simple to use. These are
developed by cloning the amplified bands of RAPD, then sequencing their ends. The sequence information is used
to design forward and reverse SCAR primers (22-24 nucleotides long) and amplified using PCR followed by
bands visualized on agarose gel. Polymorphisms either detected as length polymorphisms (co-dominant) or as
presence or absence of amplified band (dominant; Singh and Singh, 2015).
Several male-hermaphrodite specific RAPD markers were developed into SCAR markers (Deputy et al., 2002;
Lemos et al., 2002). Urasaki et al., (2002) identified a 450 bp fragment, named PSDM (Papaya Sex Determination
Marker) in all male but not in the female plants. From this RAPD marker a SCAR was developed that amplified
fragments from the genomes of male and hermaphrodite plants, but not the female ones. A SCAR marker was also
developed from RAPD marker OPY7 (900 bp) to differentiate plants of hermaphrodite and male from female
plants in Colombian papaya genotype (Bedoya and Nuenz, 2007). Results indicated that sequences utilized for the
development of SCAR marker is present on Y chromosome. Chaturvedi et al. (2014) have validated the SCAR
marker W11 among different cultivars of dioecious and gynodioecious papaya genotypes (Table 2).
5.4.3 Inter Simple Sequence Repeat (ISSR)
ISSR (Zietkiewicz et al., 1994) is PCR based DNA fingerprinting technique utilizes single primer that contains
microsatellite sequences, usually 15-30 nucleotide long and amplifies regions between adjacent, inversely oriented
SSR-microsatellites (Gupta et al., 1996). There are two types of primer, non-anchored primer consists of
microsatellite sequences and anchored primer containing microsatellite sequences in addition usually, two
nucleotides long) arbitrary sequence either at the 3′ or 5′end of the primer. ISSR marker offers several advantages
such as no prior sequence data is required for primer synthesis, dominant marker and low quantity of DNA
(5-50ng/reaction) are needed (Singh and Singh, 2015).
ISSR markers have been employed for sex identification in various dioecious plants (Table 2). A 500 bp band was
observed in solo group (SS72/12) particularly in hermaphrodite plants of papaya and further investigations were
done to validate these results. A marker around 500 bp was found co-segregating with sex in three genotypes
viz
.,
Solo group (SS72/12), hybrid (Tainung H), and Formosa group (Tailândia) (Da Costa et al., 2011). Gangopdhayay
et al. (2007) utilized three microsatellite probes (CAG)
5
, (GACA)
4
and (CAA)
5
for sex-identification in papaya.
Out of three primers, only primer (GACA)
4
generated one female-specific band which was detected in all female
and hermaphrodite plants. Parasnis et al. (1999) utilized (GATA)
4
microsatellite probe which generated a 5 kb
male-specific band. Papaya Y chromosome is morphologically identical to the X chromosome. To study this,
probes, such as (GAA)
6
and (GATA)
4
were utilized. But, the differences were observed in the plants of male and
female at molecular level. This indicates that the way of divergence occurs between the genetic material of papaya
chromosomes X and Y is sex-specific. Due to some disadvantages of ISSR such as low reproducibility and limited
number of bands generated and labour involved during analysis process makes this marker system less interesting
among researchers for sex-identification in plants.
5.4.4 Amplified fragment length polymorphism (AFLP)
A novel PCR based technique was developed by Vos et al. (1995) which involves PCR amplification of restriction
fragment of sample DNA. This technique is used for generating fingerprints of DNA of any origin or complexity.
It is highly efficient marker offers many advantages such as high reproducibility, high level of polymorphism,
high genomic abundance, detects multiple loci and no prior knowledge of sequence information is required.
Polymorphism is analyzed on the basis of presence and absence of restriction fragments (Mishra et al., 2014).
