Internal transcribed spacer or ITS region of nuclear ribosomal DNA (rDNA) has been used to evaluate genetic assortment and phylogenetic relationship in nine sugarcane genotypes including Saccharum species and another related genus as Erianthus, Narenga and hybrid. DNA was extracted from selected genotypes and ITS (ITS-1 and ITS-2) regions were amplified using specific primers. The sequence lengths ITS-1 showed 205-207 bp, while ITS2 was ranged from 211-218 bp. However, G+C content (%) 65.2% -67% in ITS-1 and in ITS-2 68.4% -99.7%. The sequence lengths of fragment and GC content of ITS-1 and ITS-2 regions showed variable. To evaluate the phylogenetic association of both the region of ITS (ITS-1 and ITS-2) neighbor-joining (NJ) method was employed. The cluster A of ITS-1 and cluster B for ITS-2 and cluster C combined between ITS1+ ITS2 sequences gave two distinct groups A and B. The group A represented the ITS1 sequences which showed two subgroups I and II. The A-I subgroup consisted of wild species of sugarcane; Erianthus, Narenga and S. robustum, whereas the A-II subgroup consisted of the Saccharum species and hybrid. The ITS2 sequences in the group B showed better correlation amongst each other. The sequences ITS-1 & ITS-2 combined and compared with some selected sequences from NCBI database using NJ method. The results have confirmed that ITS region can be used for evaluating the genetic assortment in Saccharum and its closely related genes.

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