Molecular Plant Breeding 2016, Vol.7, No.10, 1-17
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RFLP is the primarily known type of hybridization-based molecular marker in plant genome and initially used during 1975
for the detection of DNA sequence polymorphism in gene mapping (Helentjaris et al., 1986). The methodology of
RFLP depends on restriction enzymes which show comparison among DNA sequences individually. Dissimilarity
in DNA sequences produce loss, gain or alteration of restriction site. Therefore, digestion of DNA with restriction
enzymes produce fragments having difference in number and size between populations and species. RFLP study is
an abundantly authentic technique for DNA profiling and for computing heredity. Many scientists produced
genome mapping of cotton by using RFLPs (Ulloa and Meredith, 2000). First molecular map of the cotton
genome was established by utilizing 705 RFLP loci and partitioned into 41 linkage groups (Reinsich et al., 1994).
The efficacy of RFLP markers in marker assisted selection (MAS) was described and RFLP resistance allele for
bacterial blight resistance germplasm was confirmed (Wright et al., 1998). In the science of omics, RFLPs have
played a significant part (Rahman et al., 2009). As RFLP technique includes costly chemicals and takes long time
for analysis which limits its use in marker assisted selection (Agarwal et al., 2008).
5AFLP (Amplified Fragment Length Polymorphism)
AFLP approach collaborate RFLP with the adoptability of PCR-based technology by ligating adaptors to the
restricted DNA (Lynch and Walsh, 1998). The focal point of AFLP is its ability for “genome representation”
evaluate the representative DNA regions dispersed throughout the genome at the same time. In plants AFLP
markers can be produced and there is no need for prior information and sequence analysis for development of
primer. For genetic mapping, AFLP is helpful owing to their high availability and randomly distribution all over
the genome (Vos et al, 1995). A linkage map of cotton was published using the AFLP and RAPD markers (Altaf
et al., 1997). Phylogenetic studies have been done by using AFLP to observe the genetic resemblance (Abdalla et
al., 2001; Lacape et al., 2003) and map saturation in cotton (Zhanget al., 2005). The benefits of AFLP include: 1)
Reliable and reproducible (Jones et al., 1997). 2) No need of DNA sequence for analysis 3) It is information-rich
due to having ability for analyzing a large number of polymorphic loci simultaneously with a single primer
combination on a single gel as compared to RFLPs and microsatellites (Russell et al., 1997). Both good quality
and partially degraded DNA can be used for digestion but the DNA should be free of restriction enzyme and PCR
inhibitors.
6 Randomly Amplified Polymorphic DNA (RAPD)
Random nucleotide sequence magnification of genomic DNA having one primer is established by using RAPDs
(Williams et al., 1990). DNA fragments having sequence of about 10 bp are amplified with artificial primers by
using PCR (Khanam et al., 2012). RAPDs are used for genotype profiling by using primers which shows
polymorphism about precise information of sequence. The primers used for this technique should be free from
palindromic sequences and should have minimum 40% GC contents in the fragments (William et al., 1990). The
methodology of RAPD has been studied by a number of researchers in cotton (Khan et al., 2000; Rahman et al.,
2002; Hussein et al., 2005). Sheidail et al., (2007) conducted phylogenetic studies in cotton and argued that this
procedure is helpful for introgression of desirable traits. RAPDs were used in cotton for comparing cotton
cultivars resistance to jassids, mites and aphids (Geng et al., 1995). In addition, linkage maps established and
genetic diversity was observed by using RAPD in cotton (Lu and Mayers, 2002). DNA finger printing, mapping
and genetic diversity has been observed in cotton through RAPDs (Zhang et al., 2008; Zahra et al., 2011). The
demerit of this methodology is that in order to obtain highly polymorphic bands rigorously follow the reaction conditions
but practically band profiles are hard to magnify.
7 Inter-Simple Sequence Repeat (ISSR)
DNA fragments which are amplified from an amplifiable location between two similar simple sequence, repeated
sequences are located at adjacent points are called ISSR (Sharma et al., 2012). Among simple sequence repeat
polymorphism is observed using primer (16-25bp) adjacent to a single SSR and annealing occur at either ends
(Sharma et al., 2012). ISSR marker methodology establishes the comprehensive use of RAPDs amalgamating the
merits of AFLPs and SSRs (Bornet and Branchrd, 2001). Usually ISSR primers have substantial fragments
