Salt stress is one unfavorable factor of global climate change that adversely affects rice plant growth and yield. To identify novel salt-tolerant genes and new varieties of salt-tolerant rice, a better understanding of the molecular regulation mechanism of salt tolerance in rice is needed. In this study we used transcriptome analyses to examine changes in gene expression of salt-tolerant and salt-sensitive rice plants. The salt-tolerant cultivar HH11 and salt-sensitive cultivar IR29 were treated with 200 mM NaCl solution for 0 h, 6 h, 24 h and 48 h at the three leaf stage. Physiological parameters and transcriptome were measured and analyzed after each treatment. Activity of SOD and POD, as well as the MDA and protein content of the two rice cultivars generally increased with increasing time of exposure to NaCl. Meanwhile, the APX activity first increased, then decreased in both cultivars, with maximum values seen at 6 h for IR29 and at 24 h for HH11. The GR and GPX activity of HH11 were stronger than that of IR29 in response to salt stress. The H₂O₂ content first increased at 0–6 h, then decreased at 6–24 h, and then increased again at 24–48 h under salt stress. Compared with IR29, SOD, POD and APX activity of HH11 was more sluggish in response to salt stress, reaching the maximum at 24 h or 48 h. The MDA, H₂O₂ and proline content of HH11 was lower than that of IR29 under salt stress. Relative to untreated HH11 plants (0 h) and those exposed to salt for 6 h, 24 h, and 48 h (H0-H6, H0-H24 and H0-H48), 7462, 6363 and 6636, differentially expressed genes (DEGs), respectively, were identified. For IR29, the respective total DEGs were 7566, 6075 and 6136. GO and KEGG enrichment analysis showed that metabolic pathways related to antioxidative responses and osmotic balance played vital roles in salt stress tolerance. Sucrose and starch metabolism, in addition to flavonoid biosynthesis and glutathione metabolism, showed positive responses to salt stress. Expression of two SPS genes (LOC_Os01g69030 and LOC_Os08g20660) and two GST genes (LOC_Os06g12290 and LOC_Os10g38740) was up-regulated in both HH11 and IR29, whereas expression of LOC_Os09g12660, a glucose-1-phosphate adenylyltransferase gene, and two SS genes (LOC_Os04g17650 and LOC_Os04g24430) was up-regulated differential expression in HH11. The results showed that HH11 had more favorable adjustment in antioxidant and osmotic activity than IR29 upon exposure to salt stress, and highlighted candidate genes that could play roles in the function and regulation mechanism of salt tolerance in rice.

Sugarcane (Saccharum spp. hybrid) is an important crop for sugar and biofuels, and often suffers from water shortages during growth. Currently, there is limited knowledge concerning the molecular mechanism involved in sugarcane response to drought stress (DS) and whether chitooligosaccharide could alleviate DS. Here, we carried out a combined transcriptome and metabolome of sugarcane in three different treatment groups: control group (CG), DS group, and DS + chitooligosaccharide group (COS). A total of 12,275 (6404 up-regulated and 5871 down-regulated) differentially expressed genes (DEGs) were identified when comparing the CG and DS transcriptomes (T_CG/DS), and 2525 (1261 up-regulated and 1264 down-regulated) DEGs were identified in comparing the DS and COS transcriptomes (T_DS/COS). GO and KEGG analysis showed that DEGs associated with photosynthesis were significantly enriched and had down-regulated expression. For T_DS/COS, photosynthesis DEGs were also significantly enriched but had up-regulated expression. Together, these results indicate that DS of sugarcane has a significantly negative influence on photosynthesis, and that COS can alleviate these negative effects. In metabolome analysis, lipids, others, amino acids and derivatives and alkaloids were the main significantly different metabolites (SDMs) observed in sugarcane response to DS, and COS treatment reduced the content of these metabolites. KEGG analysis of the metabolome showed that 2-oxocarboxylic acid metabolism, ABC transporters, biosynthesis of amino acids, glucosinolate biosynthesis and valine, leucine and isoleucine biosynthesis were the top-5 KEGG enriched pathways when comparing the CG and DS metabolome (M_CG/DS). Comparing DS with COS (M_DS/COS) showed that purine metabolism and phenylalanine metabolism were enriched. Combined transcriptome and metabolome analysis revealed that pyruvate and phenylalanine metabolism were KEGG-enriched pathways for CG/DS and DS/COS, respectively. For pyruvate metabolism, 87 DEGs (47 up-regulated and 40 down-regulated) and five SDMs (1 up-regulated and 4 down-regulated) were enriched. Pyruvate was closely related with 14 DEGs (|r| > 0.99) after Pearson’s correlation analysis, and only 1 DEG (Sspon.02G0043670-1B) was positively correlated. For phenylalanine metabolism, 13 DEGs (7 up-regulated and 6 down-regulated) and 6 SDMs (1 up-regulated and 5 down-regulated) were identified. Five PAL genes were closely related with 6 SDMs through Pearson’s correlation analysis, and the novel.31257 gene had significantly up-regulated expression. Collectively, our results showed that DS has significant adverse effects on the physiology, transcriptome, and metabolome of sugarcane, particularly genes involved in photosynthesis. We further show that COS treatment can alleviate these negative effects.

The identification of germplasm resources is an important aspect of sugarcane breeding. The aim of this study was to introduce a new method for identifying Saccharum spontaneum and its progeny. First, we cloned and sequenced nuclear ribosomal DNA internal transcribed spacer (nrDNA-ITS) sequences from 20 Saccharum germplasms. Analysis of these nrDNA-ITS sequences showed a stable mutation at base 89. Primers (FO13, RO13, FI16, and RI16) were then designed for tetra-primer amplification refractory mutation system (ARMS) PCR based on mutations at base 89 of the nrDNA-ITS sequence. An additional 71 Saccharum germplasms were identified using this tetra-primer ARMS PCR method, which confirmed that the method using the described primers successfully identified Saccharum spontaneum and progeny. These results may help improve the efficiency of modern molecular breeding of sugarcane and lay a foundation for identification of sugarcane germplasms and the relationships among them.

Sugarcane (Saccharum spp. hybrid) is frequently affected by seasonal drought, which causes substantial declines in quality and yield. To understand the drought resistance mechanisms of S. officinarum, the main species of modern sugarcane, at a molecular level, we carried out a comparative analysis of transcriptome and metabolome profiling of the sugarcane variety Badila under drought stress (DS). Compared with control group (CG) plants, plants exposed to DS had 13,744 (6663 up-regulated and 7081 down-regulated) differentially expressed genes (DEGs). GO and KEGG analysis showed that the DEGs were enriched in photosynthesis-related pathways and most DEGs had down-regulated expression. Moreover, the chlorophyll content, photosynthesis (Photo), stomatal conductance (Cond), intercellular carbon dioxide concentration (Ci) and transpiration rate (Trmmol) were sharply decreased under DS. These results indicate that DS has a significant negative influence on photosynthesis in sugarcane. Metabolome analysis identified 166 (37 down-regulated and 129 up-regulated) significantly regulated metabolites (SRMs). Over 50% of SRMs were alkaloids, amino acids and their derivatives, and lipids. The five most significantly enriched KEGG pathways among SRMs were Aminoacyl-tRNA biosynthesis, 2-Oxocarboxylic acid metabolism, Biosynthesis of amino acids, Phenylalanine metabolism, and Arginine and proline metabolism (p < 0.05). Comparing CG with DS for transcriptome and metabolome profiling (T_CG/DS and M_CG/DS, respectively), we found three of the same KEGG-enriched pathways, namely Biosynthesis of amino acids, Phenylalanine metabolism and Arginine and proline metabolism. The potential importance of Phenylalanine metabolism and Arginine and proline metabolism was further analyzed for response to DS in sugarcane. Seven SRMs (five up-regulated and two down-regulated) and 60 DEGs (17 up-regulated and 43 down-regulated) were enriched in Phenylalanine metabolism under DS, of which novel.31261, Sspon.04G0008060-1A, Sspon.04G0008060-2B and Sspon.04G0008060-3C were significantly correlated with 7 SRMs. In Arginine and proline metabolism, eight SRMs (seven up-regulated and one down-regulated) and 63 DEGs (32 up-regulated and 31 down-regulated) were enriched, of which Sspon.01G0026110-1A (OAT) and Sspon.03G0002750-3D (P5CS) were strongly associated with proline (r > 0.99). These findings present the dynamic changes and possible molecular mechanisms of Phenylalanine metabolism as well as Arginine and proline metabolism under DS and provide a foundation for future research and sugarcane improvement.

Intergeneric hybrids between Saccharum spp. and Erianthus arundinaceus and clones derived from these hybrids and backcrosses to Saccharum spp. were used to study the transmission of E. arundinaceus chromosomes by genomic in situ hybridization (GISH). True hybrid progenies were precisely identified using PCR with a primer pair, AGRP52/53. The results showed that AGRP52/53 was an E. arundinaceus-specific primer pair and could be used as molecular marker to assist breeding. EaHN92, a 364 bp E. arundinaceus-specific tandem repeat satellite DNA sequence, was cloned from the E. arundinaceus clone HN92–105 with AGRP52/53, and was localized on sub-telomeric regions of all E. arundinaceus chromosomes. YCE06–61, a BC₃ progeny, had 7 E. arundinaceus chromosomes and its progenies had approximately 1–6 E. arundinaceus chromosomes. The number of E. arundinaceus chromosomes in true hybrids appeared as Gaussian distribution in 3 cross combinations. In addition, GISH detected intergeneric chromosome translocation in a few progenies. Hence, screening clones containing approximately 1–2 E. arundinaceus chromosomes without translocation could be used for sorting and sequencing E. arundinaceus chromosomes. This study provides a method for breeders to select true hybrid progenies between Saccharum spp. and E. arundinaceus, which will accelerate this intergeneric hybridization breeding.

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland’s Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.