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

Authors: Pan, Yong-Bao; Todd, James; Lomax, Lionel; White, Paul; Simpson, Sheron; Scheffler, Brian

Submitted to: Agronomy Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/27/2023
Publication Date: 10/29/2023
Citation: Pan, Y., Todd, J.R., Lomax, L.E., White Jr, P.M., Simpson, S.A., Scheffler, B.E. 2023. Molecular Dissection of the 5S Ribosomal RNA-Intergenic Transcribed Spacers in Saccharum spp. and Tripidium spp.. Agronomy Journal. https://doi.org/10.3390/agronomy13112728.
DOI: https://doi.org/10.3390/agronomy13112728

Interpretive Summary: Due to multiple sets of chromosomes, 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. A specific part of the plant’s DNA, the 5S nuclear ribosomal RNA-intergenic spacer (ITS), was investigated using a method called PCR with two special molecules, called primers PI and PII. In this study, a total of 171 plant specimens were collected, including various types of sugarcane and some other related plants of the wild Tripidium genus. DNA were extracted from the leaf tissue of the plants and the ITS spacers were amplified by PI/PII-primed PCR to produce different pieces of DNA, called amplicons. The amplicons were separated out through a process called electrophoresis, either on an agarose gel or in a capillary machine. The gel method showed five different banding patterns, but the CE method detected 42 different amplicons, ranging from 61 to 506 base pairs (bp) long. Three amplicons, 234-, 235-, and 236-bp in size, were amplified from all sugarcane plants, except for three plants that lacked the 236-bp amplicon. The 237-bp amplicon was occasionally produced in sugarcane and the 238-bp amplicon was rarely observed in sugarcane. Some wild sugarcane plants from China and Thailand also amplified additional 224- and 225-bp amplicons specific to the wild sugarcane plants. The 234-, 235-, 236-bp amplicons was less consistent in the wild sugarcane plants (S. spontaneum), sometimes missing a few but not all the amplicons in this region. An amplicon of 61-bp was amplified only from sugarcane hybrid varieties. The related wild plants of Tripidium had completely different 405-bp and 406-bp amplicons and lacked all sugarcane-specific amplicons. One Tripidium plant Kalimpong showed a unique CE-banding pattern different from all other plants. In conclusion, this method helped identify nine plants that had been wrongly classified. It could be a useful tool for sugarcane breeders to quickly check and differentiate between different types of sugarcane in their collections.

Technical 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., and 2 of unknown. The ITS spacer was amplified from 10 ng of leaf DNA of each accession with universal PCR primers PI [5’TGGGAAGTCCT(C/T)GTGTTGCA] and PII [5’(T/G)T(A/C)G(T/C)GCTGGTATGATCGCA]. 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 237-bp amplicon was occasionally produced in S. barberi, S. edule, S. officinarum, S. robustum, S. sinense and Saccharum spp. hybrids. However, the 238-bp amplicon was rarely observed and only in S. officinarum, S. robustum, and S. spontaneum. Some S. spontaneum accessions from China and Thailand also produced additional S. spontaneum-specific 224- and 225-bp amplicons. 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 sugarcane hybrid varieties. Moreover, all Saccharum-specific amplicons were absent in accessions of Tripidium spp., which produced 405-bp and 406-bp amplicons. The T. bengalense accession Kalimpong had a unique CE-banding pattern different from all other accessions. Although the clustering pattern of the 42 amplicons only discriminated at the genus level, but these amplicons helped identify nine mis-classified accessions. This study further demonstrates that the PI/PII could be a very useful marker for sugarcane breeders at the field locations to quickly confirm and discriminate among accessions of germplasm collections.