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A
novel approach of gene-enrichment of complex plant genome, well known as
Hypomethylated partial restriction (HMPR), has been applied for six diverse
genotypes of G. hirsutum to construct six specific HMPR librairies by using
independent partial digestions with methylation-sensitive restriction enzymes
HpaII (5′-CCGG-3′) and ClaI (5′-ATCGAT-3′). All six genotypic specific HMPR
libraries were sequenced using Genome Sequencer FLX 454 (Roche) and assembled
within genotypes and super- assembled among six genotypes to eliminate the
common sequences. All the assembled 40,95,129 unigenes (contigs and singletons)
were explored with high stringency criteria for identifying a total of 1,64,189
SSRs. Among all the unit size of different repeat type, DNR stood with maximum
frequency (1,16,982) followed by TNR (21,795) and MNR (18,706). Out of 1,45,483
SSRs (excluding MNRs), 56,142 SSRs were found to be successful in primer
designing in which 47,093 SSRs were found to be novel against Cotton Marker
Database (CMD).
All the Novel SSRs have been submitted to NCBI GenBank under GenBank accession numbers: JX576804-JX623896. |
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