Genetic diversity analysis and RAPD marker

to construct a genetic maps (John et al., 1990, Soniski et al. , 1996, Mudge et al.,

1996)whichisnecessaryforthegeneticstudyofaplant.RAPDtechniqueissimple,

faster and cost effective in comparisons to RFLP (Restriction Fragment Length

Polymorphism) and does not involve the use of radioactivity. RAPD markers can

be generated in large numbers and can complement traditional characters that may

be limited in availability. They provide a valuable new resource for phylogenetic

studies (Hoey et al., 1996). So these molecular techniques are increasingly used

in DNA fingerprinting for cultivar identification (Kollar et al. , 1993) diversity

estimation (Chalmers et al., 1992), population genetic studies (Huff et al., 1993)

and marker aided selection in breeding (Barua et al., 1993). The present study

was carried out to the studies the inheritance of RAPD markers and to know the

molecular diversity of parents as well as progenies. The work will also provide

the DNA profiles of these two economically important mulberry varieties and the

progenies derived from the cross of Mysore Local x V-1.

materials and methods

Plant material

The study was carried out with two mulberry varieties namely, Mysore Local

and V-1. The variety Mysore Local has been maintained in gene bank of Central

Sericultural Research and Training Institute (CSR and TI), Mysore. V-1 was

cultivated in the number of plots for evaluation trials and leaf production. The

F ₁ progenies of Mysore local (♀) x V-1 (♂) was maintained in small plots of

molecular biology section following standard package of practices. Morphological

data of both parents and progenies were recorded following the norms of mulberry

germplasm characterization.

Genomic DNA isolation and quantification

Fresh young leaves from the plants were collected for DNA extraction using

the standard Cetyl Trimethyl Ammonium Bromide (CTAB) method (Doyle and

Doyle, 1990). To obtain high molecular weight and pure DNA from mulberry,

tissue (0.5g) was ground in liquid nitrogen with the help of mortar and pestle.

The powder was transferred to the oak-ridge tubes containing 10ml of extraction

buffer [1M Tris-HCI (pH-8.0); 5M NaCl; 0.5M EDTA; 10% SDS (w/v); 10%

PVP (w/v) and 2% β-mercaptoethanol (w/v)]. The slurry was incubated at 65°C

for 30 minutes and then at room temperature for 15 minutes. The mixture was

extracted with equal volume of SEVEG Chloroform: Isoamylalcohol (24:1) and

centrifuged at 7500 rpm for 10 minutes. The supernatant was transferred to a

fresh tube and repeated the SEVEG Chloroform: Isoamylalcohol (24:1) until the

white interphase disappeared. The aqueous phase was extracted once with Phenol

: Chloroform : Isoamylalcohol (25 : 24 :1) mixture and centrifuged at 7500 rpm

Int. J. Bioinformatics and Biological Sci.: v.2 n.1&2, p. 23-39. March & June 2014

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