Target Region Amplification Polymorphism (TRAP) for Assessing Genetic Diversity in Sugarcane Germplasm Collections

Target region amplification polymorphism (TRAP) is a fairly new PCR-based molecular marker technique which uses gene-based information for primer design. The objective of this study was to evaluate the utility of TRAP markers for assessing genetic diversity and interrelationships in sugarcane germplasm collections. Thirty genotypes from the genera Saccharum, Miscanthus, and Erianthus were used in the study. Among the genus Saccharum were the species, S. officinarum L., S. barberi Jesw., S. sinenseRoxb., S. spontaneum L., S. robustum Brandes and Jeswiet ex Grassl, cultivars, cultivar-derived mutants and interspecific hybrids between S. officinarum and S. spontaneum. Six fixed primers, designed from sucroseand cold tolerance-related EST (expressed sequence tags) sequences, paired with three arbitrary primers, were used to characterize the germplasm. Both the cluster and principal coordinate (PCoA) analyses placed the Erianthus spp. and Miscanthus spp. genotypes distinctly from each other and from the Saccharum species, thus, supporting their taxonomic classification as separate genera. Genotypes of the low sucrose and cold tolerant species, S. spontaneum, formed one distinct group, while the rest of the Saccharum species formed one interrelated cluster with no distinct subgroups. Sequence analysis of TRAP bands derived from a S. spontaneum genotype revealed homology with known gene sequences from other grass species including Sorghum. A BLASTn search using the homologous sequences from Sorghum matched with the S. officinarum GenBank accession from which the fixed TRAP primer was designed. These results ratify TRAPas a potentially useful marker technique for genetic diversity studies in sugarcane. THE GENUS Saccharum is composed of six species, namely S. officinarum L., S. barberi Jesw., S. sinense Roxb., S. spontaneum L., S. robustum Brandes and Jeswiet ex Grassl, and S. edule Hassk. (Brandes, 1958). The modern Saccharum spp. (cultivated sugarcane) is believed to have originated from complex hybridization events (termed ‘‘nobilization’’) between Saccharum officinarum, S. barberi, S. sinense, and the wild related species S. spontaneum (Sreenivasan et al., 1987). Until the end of the 19th century, cultivated sugarcane was comprised mainly of the vegetatively propagated S. officinarum (the main sugar producing cane) together with S. barberi and S. sinense (Jannoo et al., 1999). Saccharum officinarum, however, is believed to have evolved through hybridization of species such as Erianthus arundinaceus (Retz.) Jeswiet, S. spontaneum, and S. robustum (Daniels et al., 1975), whereas S. barberi and S. sinense are believed to be natural hybrids between S. officinarum and S. spontaneum (Daniels and Roach, 1987). Mukherjee (1957) coined the term Saccharum complex to encompass four closely related interbreeding genera viz., Saccharum, Erianthus (5 sect. Ripidium), Narenga, and Sclerostachya, all of which are supposedly implicated in the origin of sugarcane. Daniels et al. (1975) revised this grouping to include Miscanthus sect. Diandra Keng, but the phylogenetic relationship between members of the group remains unclear (Irvine, 1999). A better understanding of the genetic diversity and interrelationships among members of the Saccharum complex will facilitate exploitation of this germplasm in improving sugarcane. Traditional methods which combined agronomic and morphological characteristics have been useful in identifying and describing differences between members of the Saccharum complex (Artschwager and Brandes, 1958; Skinner, 1972; Skinner et al., 1987). However, members of the Saccharum complex are predominantly outcrossing and are maintained by vegetative propagation. As such, they are highly heterozygous and display enormous plasticity in the phenotypic expression of traits. Although morphological traits can be used to identify and classify clones, most of the traits are influenced by the environment under which the clones are grown or selected. Variability caused by genotype 3 environment interactions and inadvertent mislabeling of clones can adversely influence data derived from phenotypic evaluation and clonal records. With the advent of molecular markers, it is now possible to make direct inferences about genetic diversity and interrelationships among organisms at the DNA level without the confounding effects of the environment and/or faulty pedigree records. Indeed, a vast number of molecular marker techniques such as isoenzymes (Glaszmann et al., 1989), RFLP (restriction fragment length polymorphism) (D’Hont et al., 1994; Jannoo et al., 1999; Coto et al., 2002), ribosomal DNA (Glaszmann et al., 1990; Pan et al., 2000), microsatellites (Piperidis et al., 2001; Cordeiro et al., 2003), AFLP (amplified fragment length polymorphism) (Besse et al., 1998; Lima et al., 2002), and molecular cytogenetics (D’Hont et al., 1996) have been instrumental in explaining genetic diversity and interrelationships among accessions in sugarcane germplasm collections. The underlying goal for studying genetic diversity and interrelationships among germplasm collections is to eventually use that information to facilitate the development of better performing varieties of the cultivated species. The results from genetic diversity studies may, S. Alwala, A. Suman, J.A. Arro, and C.A. Kimbeng, Dep. of Agronomy and Environmental Management, Louisiana State University Agricultural Center, BatonRouge, LA 70803; J.C. Veremis, USDA-ARS, SRRC, Sugarcane Research Unit, 5883 USDA Road, Houma, LA 70360. This research was supported under USDA-NRI Project Award No. 200335300-13115 and by the American Sugarcane League of the U.S.A., Inc. Received 5Apr. 2005. *Corresponding author ([email protected]). Published in Crop Sci. 46:448–455 (2006). Plant Genetic Resources doi:10.2135/cropsci2005.0274 a Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 448 Published online January 24, 2006