Sugarcane is of important economic value for producing sugar and bioethanol. Tripidium arundinaceum (old name: Erianthus arundinaceum) is an intergeneric wild species of sugarcane that has desirable resistance traits for improving sugarcane varieties. However, the scarcity of chromosome markers has hindered the cytogenetic study of T. arundinaceum. Here we applied maize chromosome painting probes (MCPs) to identify chromosomes in sorghum and T. arundinaceum using a repeated fluorescence in situ hybridization (FISH) system. Sequential FISH revealed that these MCPs can be used as reliable chromosome markers for T. arundinaceum, even though T. arundinaceum has diverged from maize over 18 MYs (million years). Using these MCPs, we identified T. arundinaceum chromosomes based on their sequence similarity compared to sorghum and labeled them 1 through 10. Then, the karyotype of T. arundinaceum was established by multiple oligo-FISH. Furthermore, FISH results revealed that 5S rDNA and 35S rDNA are localized on chromosomes 5 and 6, respectively, in T. arundinaceum. Altogether, these results represent an essential step for further cytogenetic research of T. arundinaceum in sugarcane breeding.

Sugarcane is an important sugar and energy crop in the world. Germplasm innovation is a significant way to breed breakthrough sugarcane varieties. Modern sugarcane varieties all contain the blood relationship of Saccharum officinarum and Saccharum spontaneum. High sugar results from S. officinarum and the resistance genes from S. spontaneum. In order to improve the sugarcane quality, breeders use S. officinarum and S. spontaneum to cross and obtain hybrid offspring with high sugar and high resistance. Therefore, the authenticity of S. officinarum and S. spontaneum progeny materials directly affects the efficiency of sugarcane breeding. In this study, the tetra-primer amplification hindered mutation system (ARMS PCR) was used to identify ten suspected S. officinarum and eleven suspected S. spontaneum germplasm materials, then further validated by chromosome counting and genome in situ hybridization (GISH). Among the ten suspected S. officinarum materials to be identified, three were real S. officinarum materials, they were 14NG124, 51NG103, and Guan A. Nine of the eleven suspected S. spontaneum to be identified were fake S. spontaneum materials, these were Yunge 2007-12-165, Guangxi 87-20, Yunnan 82-16, Yunge 2007-11, YNLC 16, Laos No. 2, Yunnan 82-29, 2015-83, and 2013-20. The ARMS PCR results were the same as the GISH results. The three real S. officinarum materials had 80 chromosomes. Using ARMS PCR and GISH, three S. officinarum and nine S. spontaneum materials were proven to be authentic. Through chromosome number statistics, it was found that the three real S. officinarum had 80 chromosomes. Authentic materials were identified and selected to enrich the genetic background of sugarcane through hybridization and reduce the influence on the breeding process of the misuse of fake S. officinarum and S. spontaneum.

The barcode probe is a convenient and efficient tool for molecular cytogenetics. Tripidium arundinaceum, as a polyploid wild allied genus of Saccharum, is a useful genetic resource that confers biotic and abiotic stress resistance for sugarcane breeding. Unfortunately, the basic cytogenetic information is still unclear due to the complex genome. We constructed the Cot-20 library for screening moderately and highly repetitive sequences from T. arundinaceum, and the chromosomal distribution of these repetitive sequences was explored. We used the barcode of repetitive sequence probes to distinguish the ten chromosome types of T. arundinaceum by fluorescence in situ hybridization (FISH) with Ea-0907, Ea-0098, and 45S rDNA. Furthermore, the distinction among homology chromosomes based on repetitive sequences was constructed in T. arundinaceum by the repeated FISH using the barcode probes including Ea-0663, Ea-0267, EaCent, 5S rDNA, Ea-0265, Ea-0070, and 45S rDNA. We combined these probes to distinguish 37 different chromosome types, suggesting that the repetitive sequences may have different distributions on homologous chromosomes of T. arundinaceum. In summary, this method provide a basis for the development of similar applications for cytogenetic analysis in other species.