An Assessment of Amplified Fragment Length Polymorphism at the 5S Nuclear Ribosomal RNA and the Chloroplast NON-Coding Regions Among Saccharum and Erianthus Species Using Fluorescence-Labeled Universal PCR Primers and Capillary Electrophores

As complex polyploids, sugarcane genome sequencing and characterization lag far behind other crops. Fluorescence-labeled universal PCR primers were used to amplify DNA fragments from 173 accessions, including sugarcane cultivars (9), S. officinarum (29), S. robustum (19), S. spontaneum (70), S. barberi (25), S. sinense (16), S. edule (2), and Erianthus spp. (3). Ten nanograms of extracted leaf DNA were amplified from each accession with universal PCR primers PI/PII for the 5S nuclear ribosomal RNA-ITS or c/f for the chloroplast TrnL/TrnF non-coding region. Amplicons were separated by capillary electrophoresis (CE) on an ABI3730XL DNA analyzer using GeneScan^TM Size Standards GS500 (ROX) or GS1200 (LIZ). The <.fsa> files were processed by GeneMarker® software to visualize amplicons of different sizes. Thirty-two different PI/PII-amplicons ranging from 217 to 506 bp and four c/f -amplicons (934, 950, 955, and 996 bp) were observed. PI/PII-amplicons of 234, 235, or 236-bp appeared in 166 accessions (96%). The 950-bp c/f-amplicon was amplified in 162 accessions (93.6%). The 934 bp c/f-amplicon was noted in nine accessions (5.2%), eight of which belonged to S. spontaneum and one to Erianthus procerus. The 955-bp amplicon was observed only in S. spontaneum accession IN84-010, while the 996-bp amplicon was found only in E. bengalense accession IMP9751. The resolving power of CE was one base pair; however, the AFLP data failed to produce a discriminatory clustering pattern among different species. Nucleotide sequencing of all amplicons may be required prior to phylogenetic analysis among sugarcane and related Saccharum or Erianthus species.