ISOLATION AND CHARACTERIZATION OF MICROSATELLITES IN MANGO (Mangifera indica L.)
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Date
2005
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ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY, RAJENDRANAGAR, HYDERABAD
Abstract
This study represents the first Indian effort in isolation of microsatellites
from Mango (Mangifera indica L.). Microsatellites were isolated by the selective
hybridization method in which DNA fragments containing repeat motifs specific to
repeat probe used in hybridization were captured. In the present study six
biotinylated oligos of the Di-, Tri- and Tetra-nucleotide repeat types were used as
probes in individual hybridization reactions.
Genomic DNA was isolalated from flushing tender leaves and digested with
Sau3A restriction enzyme. Digested DNA was dephosphorylated, size selected from
300-1000 bp to which Sau3A ds linkers were ligated. The linker ligated genomic
digest was hybridized against biotinylated repeat oligo and eluted microsatellite
enriched DNA fragments were enriched by PCR using sau3A primer. Enriched
fragments were cloned in pCR 2.1 TA cloning vector and transformed into INVF
competent cells. Totally 120 white colonies were screened and the clones having
insert size above 300 bp were selected and sequenced. Of the 75 clones sequenced,
25 were found to be positive for microsatellites.
Of the 25 microsatellites 16 were dinucleotide repeats, 6 were trinucleotide
repeats, only one tetra nucleotide repeat and 2 imperfect repeats were captured. To
the captured microsatellites primers were designed and standardized.
Description
Keywords
ISOLATION, CHARACTERIZATION, MICROSATELLITES, MANGO