Analysis of genetic fidelity in micropropagated plants of sugarcane using SSR-SSCP assay

Abstract

Effective utilization of biotechnological approaches relies on efficient and reliable regeneration systems in vitro. Sugarcane micropropagation from shoot meristems is very useful in sugarcane breeding programmes, as it saves time in multiplying the promising varieties and clones. Despite the advantages of in vitro propagation, phenotypic instability has been observed in micropropagated plants, including sugarcane [1]. Parameters employed for assessing the effect of in vitro culture include morphological, physiological and cytogenetical studies, field assessment, and molecular studies [2-5]. Peroxidase isozymes detected gross genetic changes in tissue culture raised cultivars of sugarcane [6] Isozyme markers have limitations because DNA regions coding for soluble proteins can only be sampled which describe the genetic changes only partially. Several PCR based markers have been developed which are generally highly species-specific because recognition of specific sequences by the PCR primers allows for very little sequence redundancy [7]. Among these, RAPD has been used successfully to assess genetic stability [8,9]. Alternatively Simple Sequence Repeats (SSRs) or microsatellites have become a method of choice to determine the clonal fidelity and somaclonal variation, as they are more stable abundant, reproducibile and co-dominant [10-12]. The SSR amplified products when visualized for their single strand conformations, are expected to unravel the variations if any due to the changes in nucleotide sequences because single-strand conformation polymorphisms (SSCP) depends on differential mobilities of single stranded DNA molecules due to conformational differences [13]. SSCP-PCR analysis of simple sequence repeats has proved useful to differentiate closely related genotypes of sugarcane [14]. Hence it is presumed that by subjecting the sugarcane plantlets derived in vitro to SSR-SSCP assay, it would be possible to detect genetical changes more efficiently. The present study was therefore undertaken to assess the utility of SSR-SSCP markers in determining the genetic fidelity of micropropagated plantlets of sugarcane derived from shoot tip cultures.