Molecular Dissection of the 5S Ribosomal RNA-Intergenic Transcribed Spacers in Saccharum spp. and Tripidium spp.

Abstract

Due to complex polyploid, sugarcane whole genome sequencing and characterization lag far behind other crops. PCR-based DNA markers are a viable low-cost option to evaluate genetic diversity and verify genotypes. In this study, the 5S ribosomal RNA-intergenic spacer (ITS) of 171 accessions of Saccharum spp. and Tripidium spp. was dissected, including 30 accessions of S. officinarum, 71 of S. spontaneum, 17 of S. robustum, 25 of S. barberi, 13 of S. sinense, 2 of S. edule, 5 sugarcane cultivars (Saccharum spp. hybrids), 6 of Tripidium spp. (formally Erianthus spp.), and 2 of unknown species. The ITS spacers were amplified from 10 ng of the leaf DNA of each accession with the universal PCR primers PI and PII. The PCR-amplified spacers (amplicons) were analyzed by both agarose gel and capillary electrophoresis (CE). While agarose gel electrophoresis revealed five banding patterns, a total of 42 polymorphic amplicons, ranging from 60 to 506 bp, were detected by CE. Three amplicons, 234-, 235-, and 236-bp in size, were amplified from all accessions of six Saccharum species, except for three S. robustum accessions (Molokai 5573, NG 57-054, and NG 77-235) that lacked the 236-bp amplicon. The 234-, 235-, 236-bp banding pattern found in S. spontaneum was less consistent than other Saccharum species, sometimes missing a few but not all the bands in this region. An amplicon of 61-bp was amplified only from the sugarcane hybrid varieties. The PI/PII patterns indicated diversity and subpopulations within Saccharum, which could potentially be used in Breeding. Moreover, all Saccharum-specific amplicons were mostly absent in Tripidium spp. accessions, which produced 405-bp and 406-bp amplicons, and any pattern of the exceptions indicated misidentification. The T. bengalense accession Kalimpong had a unique CE-banding pattern that was different from all other accessions. Although the clustering pattern of the 42 amplicons only discriminated at the genus level, these amplicons helped identify nine misclassified accessions. This study further demonstrates that these PI/PII amplicons could be particularly useful markers for breeders at sugarcane field stations to quickly confirm and discriminate among the accessions of germplasm collections.