Evaluation of Microsatellites (simple Sequence Repeats) as Genetic Markers in Sugarcane

Cultivated sugarcane genotypes (Saccharum spp.) are derived from complex interspecific hybridisations between the species S. spontaneum and S. officinarum. To analyse this complex genome, we have investigated the potential of sugarcane microsatellite sequences as genetic markers in terms of their abundance, variability and ability to detect polymorphisms. From an enriched sugarcane genornic DNA microsatellite library, primer sets were designed and synthesised for over 200 microsatellite sequences. These were tested over five Saccharum genotypes. The number of alleles recorded per marker ranged between 2 and 12 (mean of 8). Markers that showed polymorphism had a Polymorphism Information Content (or PIC, where a value of 0 = monomorphic and 1 = highly polymorphic) value between 0.48 and 0.8 (mean of 0.72). Fluorescently labelled primers for eight of these microsatellite markers were tested on a population of 20 sugarcane cultivars. Results indicated the ability of these markers to accurately fingerprint genotypes, predict relationships, be used in genetic mapping and assist in selecting specific, genetically diverse parents for use in introgression. However, these genomic DNA derived microsatellites transferred poorly to related genera. By comparison, sugarcane microsatellites derived from EST sequences had a low PIC level when tested within cultivars, but a significantly higher level of polymorphism when applied to progenitor and related species. Characteristics of SSRs from different sources As part of an international collaborative effort, an enriched SSR library (i.e. of anonymous SSRs) was created from sugarcane genomic DNA (Edwards et al., 1996; Cordeiro et al., 1999, 2000): This library was characterised and compared with sugarcane SSRs mined from a sugarcane EST library (Cordeiro et al., 2001). The frequency of non-redundant SSRs of seven or more dinucleotide repeats and five or more trinucleotide repeats in the total population of cDNA clones in the EST database was 2.88% compared with 57.3% (Cordeiro et al.,. 2000) from enriched sugarcane genomic DNA libraries. Differences in the frequency of repeat unit lengths and motif types are compared in Table 1. The ability of the SSRs from the two sources to detect polymorphisms differed. Of 35 primer sets designed and synthesised for EST SSRs, 21 amplified products in both sugarcane cultivars and ancestral Saccharum species. Seventeen were polymorphic in two or more of five Saccharum genotypes tested, representing 6.8% of total EST SSRs isolated (Cordeiro et al., 2001). With anonymous SSRs, 254 primer pairs were designed and synthesised. 183 were polymorphic, representing 20% of total SSRs isolated (Cordeiro et al., 2000). Relatively few alleles were detected by the EST SSRs within five Saccharum genotypes tested [cv. Q124 from which the library was constructed (as a positive size control); cv. Q117 (both developed by the Bureau of Sugar Experiment Stations, Australia); cv. R570 (developed at the Centre d'Essai, de Recherche et de Formation (CERF), RCunion; and the ancestral species S. officinarum var. Chittan and S. spontaneum var. Saigon]. Allele numbers ranged from 0 to 5.0, with an average of 2.7. Anonymous SSRs detect a greater number of alleles, averaging 8.0 (Cordeiro et al., 2000). When the analysis was extended to related genera, the average number of alleles detected by EST SSRs increased to 7.5 while that for anonymous SSRs fell to 3.0. Cross transferability Of the polymorphic EST SSRs, five were randomly selected and found to be cross-transferable to the related genera, Sorghum and Erianthus (Figure 1). This is in contrast to anonymous SSRs where only 3 of 20 primer pairs tested transferred to closely related genera. The mean PIC value of EST SSRs tested on cultivated Saccharum cultivars was 0.23. With anonymous SSRs, the mean value was 0.72 (Cordeiro et al., 2000). However, the mean PIC value of EST SSRs increased to 0.62 when transferred to sugarcane ancestral species (S. oflicinarum and S, spontaneum) and to 0.80 when transferred across to closely related genera (Erianthus and Sorghum spp.). Conversely, the mean PIC value of anonymous SSR primers decreased to 0.66 when transferred to sugarcane ancestral species and to 0.00 when transferred to closely related genera. This is despite the fact that the same primer pairs have a PIC value of >0.5 when tested over the 5 Saccharum genotypes. These differences in the level of polymorphism within the