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 INVaF 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. |
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