Complete genome sequence of Indian Sugarcane mosaic virus and its genetic diversity with other country isolates

Abstract

For the first time we determined complete nucleotide sequence of SCMV-IND from infecting popular variety CoC 671 from India. From the same variety complete genome sequence of SCSMV has already been reported by Parameswari et al. (2013) which is also the first study in India. Nine overlapping primer pairs were designed from the reference sequences collected from the genbank covering the whole genome of SCMV. cDNA was synthesized from the total RNA extracted from the variety CoC 671 and PCR conditions were optimized for each primer sets. The products were purified using GenElute Gel Extraction kit and the resulting fragments were cloned into the pTZ57R/T vector After the recombinant DNA was introduced into E. coli DH5α by transformation, positive clones were identified by colony PCR and two clones from each of the nine amplified fragments were sequenced in both directions to eliminate potential sequence heterogeneity introduced by Taq DNA polymerase. The nt and amino acid sequences of other known SCMV isolates were retrieved from the GenBank and used for comparison The overlapping sequences of the nine fragments were assembled using the BioEdit software.The linearly assembled single strand positive sense RNA genome of SCMV-IND was about 9573 nucleotides in length excluding the poly (A) tail. The genome encodes a polyprotein of about 3064 amino acid residues which is processed to form ten mature proteins (P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP). Based on this whole genome study, the Indian isolate showed close identity to the Australian isolate Brisbane of about 98.2% and 94.9% at the protein and nucleotide level, respectively. The Indian isolate shares the least identity with the American isolate Ohio of about 88.2% and 78.9% at the protein and nucleotide sequences respectively. This study supports the work done by Viswanathan et al. (2009) on SCMV coat protein gene analysis and its genetic variability where the Indian isolates shared a very close similarity with the Australian isolate sharing 99% nucleotide identity unlike other country isolates. Further, phylogenetic tree drawn using the software Bioedit through Neighbor joining method with 1000 bootstrap replications clearly depicted its close similarity with Australian and American isolate sharing a common progenitor whereas the other country isolates diverged to a different cluster. Within the monocot specific potyvirus, the Indian SCMV isolate shares close identity with Sorghum mosaic virus with 69.1% identity followed by Maize dwarf virus with 67.9% identity at the nucleotide level. Further study on recombination event among the SCMV complete genome sequences is in progress which will provide a good insight on SCMV evolutionary biology.