Erianthus arundinaceus is a valuable gene reservoir for sugarcane improvement. However, insufficient molecular markers for high-accuracy identification and tracking of the introgression status of E. arundinaceus chromatin impede sugarcane breeding. Fortunately, suppression subtractive hybridization (SSH) technology provides an excellent opportunity for the development of high-throughput E. arundinaceus-specific molecular markers at a reasonable cost. In this study, we constructed a SSH library of E. arundinaceus. In total, 288 clones of E. arundinaceus-specific repetitive sequences were screened out and their distribution patterns on chromosomes were characterized by fluorescence in situ hybridization (FISH). A subtelomeric repetitive sequence Ea086 and a diffusive repetitive sequence Ea009, plus 45S rDNA-bearing E. arundinaceus chromosome repetitive sequence EaITS were developed as E. arundinaceus-specific molecular markers, namely, Ea086-128, Ea009-257, and EaITS-278, covering all the E. arundinaceus chromosomes for high-accuracy identification of putative progeny. Both Ea086-128 and Ea009-257 were successfully applied to identify the authenticity of F₁, BC₁, BC₂, BC₃, and BC₄ progeny between sugarcane and E. arundinaceus. In addition, EaITS-278 was a 45S rDNA-bearing E. arundinaceus chromosome-specific molecular marker for rapid tracking of the inherited status of this chromosome in a sugarcane background. Three BC₃ progeny had apparently lost the 45S rDNA-bearing E. arundinaceus chromosome. We reported herein a highly effective and reliable SSH-based technology for discovery of high-throughput E. arundinaceus-specific sequences bearing high potential as molecular markers. Given its reliability and savings in time and efforts, the method is also suitable for development of species-specific molecular markers for other important wild relatives to accelerate introgression of wild relatives into sugarcane.

Drought is a major abiotic constraint on sugarcane production, but systematic multitrait evaluation of drought tolerance remains limited in genetically complex advanced backcross progenies derived from sugarcane × Tripidium arundinaceum. In this study, 100 genotypes, including 93 BC₄F₁ or BC₅F₁ progenies and seven parental or control genotypes, were evaluated under a 20 d progressive drought treatment in pots at the seven-leaf stage. Sixteen traits related to root architecture, leaf water status, gas exchange, PSII photochemistry, antioxidant activity, and lipid peroxidation were measured. Drought significantly reduced TRL, RSA, RV, RWC, Pn, Gs, Tr, ΦPSII, and ETR and significantly increased SOD activity and MDA content. Twelve traits with relatively clear and biologically interpretable relationships with drought tolerance were integrated using membership functions and trait weights to calculate an integrated D value. D values ranged from 0.078 to 0.516, and 19, 45, and 36 genotypes were classified into groups with relatively high, moderate, and low drought tolerance within this germplasm collection, respectively. Genotypes 55-109, YCE07-65, 55-43, 211-172, and 91-74 had the highest integrated D values. Within the integrated evaluation framework, higher D values were most strongly associated with RSA, RV, TRL, Pn, ΦPSII, and ETR, while PC1 was strongly correlated with the D value (r = 0.893). Maintenance of root growth and photosynthetic function therefore represented major phenotypic features associated with overall drought tolerance under the present experimental conditions. These features may provide useful criteria for screening drought-tolerant germplasm, but their utility requires validation in independent drought experiments and field environments.