Molecular Plant Breeding 2016, Vol.7, No.28, 1
-
18
12
No AFLP marker is still available for sex determination in
C. papaya
, but it has been utilized in several other
plant species (Table 2). Wang et al. (2011) utilized 64 pairs of AFLP primer combinations to develop sex-specific
AFLP markers in
Eucommia ulmoides
Oliv. One male-specific marker (350 bp) was generated from
E-ACA/M-CTT primer combination. Further, a 247 bp SCAR marker was developed by utilizing this 350 bp
male-specific AFLP marker. Male-specific AFLP markers have also been identified in other plants species
including hemp (Flachowsky et al., 2001),
Broussonetia papyrifera
(Lianjun et al., 2012), whereas two
male-specific markers of 525 bp and 325 bp and a female-specific marker of 270 bp have been identified in
Simmondsia chinensis
(Agarwal et al., 2011). The AFLP method is rarely used for early sex diagnosis of seedlings
among plants due to some drawbacks such as high cost, more time consuming and laborious analysis method.
5.5 Sequence-based molecular markers
5.5.1 Simple Sequence Repeat (SSR)
SSRs are consisting of one to six (bp) tandem repeats (mono-, di-, tri-, tetra and penta-, hexanucleotides), and are
found throughout all genomes including prokaryotes (Li et al., 2004; Thiel et al., 2003). They are also termed as
simple sequence length polymorphisms (SSLPs; Tautz, 1989), microsatellite (Litt and latty, 1989), short tandem
repeats (STRs; Edwards et al., 1991).They are found in both coding and non-coding regions (Toth et al., 2000).
They are more valuable molecular marker than other PCR-based markers like RAPD, ISSR and AFLP due to their
sequence-specificity, multiallelic nature, co-dominant inheritance, abundance in the genome, high rate of
transferability, high level of polymorphism and reproducibility (Powell et al., 1996; Zane et al., 2002; Theil et al.,
2003). In addition, it does not required high quality of DNA and performs well with low quantity of template
DNA (10-100ng/reaction). The polymorphic nature of SSR was observed by Litt and Luty (1989). The length
polymorphism of SSR is generated due to variation in repeats number (Ellegren, 2004).The variations in these
repeats occur due to slippage of strand which creates mispairing (Levinson and Gutman, 1987) and repetitive
errors generated at the period of replication of DNA (Schlotterer and Tautz, 1992; Kattiet al., 2001), or unequal
crossing-over between sister chromatids during meiosis (Innan et al., 1997). The principle of polymorphism
detection involves the designing of primers from flanking sequences near the portion of microsatellite repeat motif.
Amplification is performed using PCR and running agarose or denaturing polyacrylamide gel for visualization of
variations in alleles. There are two types of SSRs on the basis of their location: (1) SSRs that are distributed
throughout the genome are called genomic-SSRs, (2) SSRs that are found only within genes (i.e. inside exons,
exon-intron junctions or introns) are called as genic-SSRs or Expressed Sequence Tags-SSRs (EST-SSRs).
With the advancement of functional genomics a large numbers of ESTs and other DNA sequences of various
organisms are available in various data banks. Availability of these large amounts of freely accessible data led to
the de
velopment of EST-based SSR markers through data mining. Development of EST-SSRs or genic-SSRs
in
silico
has become a fast, efficient, and relatively inexpensive method compared with the development of
genomic-SSRs (Gupta et al., 2003; Senan et al., 2014). Genic-SSRs act as functional markers owing to their origin
from expressed portion of genome. They possess several advantages such as ease of use, less time consuming,
cheapest to develop, occurrence in expressed portion, sequence-specificity and high rate of transferability i.e. the
ability to effectively transfer SSR markers across species and genera so they provide the better estimate of
polymorphism (Gupta et al., 2003). Genic-SSRs can also be used for comparative genomics study. EST-SSRs
developed for one species can be utilized for the related plant species for which small amount of data on ESTs and
SSRs is available in public databases by identifying rate of transferability in these species. It is believed that
EST-SSRs in the genetic maps revealed about the distribution of genes along the genetic map. They can also be
used for comparative mapping study (comparing the gene order of identical genes) in related plant species owing
to their origin from conserved region of the genome (Varshney et al., 2005).
One report is available for sex determination using microsatellite system in papaya. Chiu et al. (2015) analyzed
the sex characters in all hermaphrodite cultivar (Taichun Sunrise; TS) and typical hermaphrodite cultivar (Taiwan
Seed Station No.7; T7) of papaya and their F1 progeny using SSR markers. They performed SSR analysis of three
