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.

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.

Interspecific hybridization serves as a crucial strategy for innovating sugarcane germplasms. Currently, nearly all modern sugarcane varieties that incorporate genetic material are derived from Saccharum spontaneum. The number of chromosomes in S. spontaneum ranges from 40 to 128, contributing significantly to the diversity of its genetic resources. However, the genetic mechanisms driving chromosome number variation in S. spontaneum remain to be fully elucidated. Here, oligonucleotide fluorescence in situ hybridization (Oligo-FISH) was conducted to identify individual chromosomes and explore chromosome transmission during the intraspecific hybridization of S. spontaneum. The results indicate that from the progenies generated from S. spontaneum Yunnan2017-22 (2n = 8x = 64) and Yunnan82-1 (2n = 8x = 64) emerged two distinct karyotypes, 2n = 12x = 96 (A1) and 2n = 8x = 64 (A2, A33-1, A18). This implies that the chromosome inheritances were 2n + n and n + n in the progenies. However, self-pollinated samples of A1 (2n = 12x = 96) produced normal offspring C1 (2n = 94) and C2 (2n = 96). The 2n + n inheritance pattern did not continue. In another cross, the progenies derived from S. spontaneum Yunnan2017-41 (2n = 8x = 64) and Yunnan8 (2n = 10x = 80) carried a karyotype of 2n = 9x = 72, with n + n inheritance mode. These findings highlight the existence of two chromosome inheritance modes, 2n + n and n + n, in the context of the intraspecific hybridization of S. spontaneum. Additionally, hybridization between different ploidy S. spontaneum was also accompanied by chromosomal translocations (A1, A2, A18, A18) and loss (A2, A33-1, AA-4, and C2) that further resulted in the complexity of the S. spontaneum genome. Together, these findings highlight diverse chromosome inheritance in S. spontaneum hybridization, and provide a theoretical foundation for the further utilization of S. spontaneum germplasm in sugarcane breeding.

Saccharum spontaneum serves as an essential genetic resource for sugarcane improvement. Traditional breeding methods, characterized by slow selection and limited germplasm exploitation, often lead to suboptimal progeny performance. In this study, we revised the utilization strategy by initially hybridizing several S. spontaneum clones, followed by intercrossing their F1 progeny to establish a heterogeneous ‘polymeric’ population, which was then subjected to ‘nobilization’. A natural Saccharum spontaneum (S0) plant was used as the parent to create a hybrid (S1) containing two S. spontaneum bloodlines. The agronomic traits of S1 were compared, leading to the identification of three superior hybrids. These hybrids were then crossed in a complete diallel design, resulting in six crosses. Significant genetic variation was observed for the agronomic traits. Compared with S0, the plant height in S1 increased by 31.5%, and by 32.22% in S2. The stem diameter in S1 increased by 38.71%, and by 51.61% in S2. The single stem weight increased by 125% in S1 and 150% in S2. Other yield traits also showed varying degrees of improvement. A correlation analysis indicated that the plant height, stalk diameter, single stalk weight, and leaf width were significantly positively correlated with yield, and the leaf width with brix. There was no significant correlation between the millable stalks and yield. This study successfully developed a novel S. spontaneum hybrid with significantly improved agronomic traits, enhancing the genetic foundation of S. spontaneum germplasm for nobilization breeding programs. These findings provide a valuable germplasm base for developing high-performance sugarcane varieties, improving the utilization of S. spontaneum.