Global Responses of Autopolyploid Sugarcane Badila (Saccharum officinarum L.) to Drought Stress Based on Comparative Transcriptome and Metabolome Profiling.

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.

Chromosome-specific painting unveils chromosomal fusions and distinct allopolyploid species in the Saccharum complex.

Karyotypes provide key cytogenetic information on the phylogenetic relationships and evolutionary origins in related eukaryotic species. Despite our knowledge of the chromosome numbers of sugarcane and its wild relatives, the chromosome composition and evolution among the species in the Saccharum complex have been elusive owing to the complex polyploidy and the large numbers of chromosomes of these species. Oligonucleotide-based chromosome painting has become a powerful tool of cytogenetic studies especially for plant species with large numbers of chromosomes. We developed oligo-based chromosome painting probes for all 10 chromosomes in Saccharum officinarum (2n = 8x = 80). The 10 painting probes generated robust fluorescence in situ hybridization signals in all plant species within the Saccharum complex, including species in the genera Saccharum, Miscanthus, Narenga and Erianthus. We conducted comparative chromosome analysis using the same set of probes among species from four different genera within the Saccharum complex. Excitingly, we discovered several novel cytotypes and chromosome rearrangements in these species. We discovered that fusion from two different chromosomes is a common type of chromosome rearrangement associated with the species in the Saccharum complex. Such fusion events changed the basic chromosome number and resulted in distinct allopolyploids in the Saccharum complex.

Repetitive Sequence Barcode Probe for Karyotype Analysis in Tripidium arundinaceum.

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.

Efficient Anchoring of Erianthus arundinaceus Chromatin Introgressed into Sugarcane by Specific Molecular Markers

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.

Integrated Analysis of Transcriptome and Metabolome Reveals the Regulation of Chitooligosaccharide on Drought Tolerance in Sugarcane (Saccharum spp. Hybrid) under Drought Stress.

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.

Gene Co-Expression Analysis Reveals Transcriptome Divergence between Wild and Cultivated Sugarcane under Drought Stress.

Drought is the main abiotic stress that constrains sugarcane growth and production. To understand the molecular mechanisms that govern drought stress, we performed a comprehensive comparative analysis of physiological changes and transcriptome dynamics related to drought stress of highly drought-resistant (ROC22, cultivated genotype) and weakly drought-resistant (Badila, wild genotype) sugarcane, in a time-course experiment (0 h, 4 h, 8 h, 16 h and 32 h). Physiological examination reviewed that ROC22, which shows superior drought tolerance relative to Badila, has high performance photosynthesis and better anti-oxidation defenses under drought conditions. The time series dataset enabled the identification of important hubs and connections of gene expression networks. We identified 36,956 differentially expressed genes (DEGs) in response to drought stress. Of these, 15,871 DEGs were shared by the two genotypes, and 16,662 and 4423 DEGs were unique to ROC22 and Badila, respectively. Abscisic acid (ABA)-activated signaling pathway, response to water deprivation, response to salt stress and photosynthesis-related processes showed significant enrichment in the two genotypes under drought stress. At 4 h of drought stress, ROC22 had earlier stress signal transduction and specific up-regulation of the processes response to ABA, L-proline biosynthesis and MAPK signaling pathway-plant than Badila. WGCNA analysis used to compile a gene regulatory network for ROC22 and Badila leaves exposed to drought stress revealed important candidate genes, including several classical transcription factors: NAC87, JAMYB, bHLH84, NAC21/22, HOX24 and MYB102, which are related to some antioxidants and trehalose, and other genes. These results provide new insights and resources for future research and cultivation of drought-tolerant sugarcane varieties.

Chromosome behavior during meiosis in pollen mother cells from Saccharum officinarum × Erianthus arundinaceus F1 hybrids.

BACKGROUND: In recent years, sugarcane has attracted increasing attention as an energy crop. Wild resources are widely used to improve the narrow genetic base of sugarcane. However, the infertility of F1 hybrids between Saccharum officinarum (S. officinarum) and Erianthus arundinaceus (E. arundinaceus) has hindered sugarcane breeding efforts. To discover the cause of this infertility, we studied the hybridization process from a cytological perspective. RESULTS: We examined the meiotic process of pollen mother cells (PMCs) in three F1 hybrids between S. officinarum and E. arundinaceus. Cytological analysis showed that the male parents, Hainan 92-77 and Hainan 92-105, had normal meiosis. However, the meiosis process in F1 hybrids showed various abnormal phenomena, including lagging chromosomes, micronuclei, uneven segregation, chromosome bridges, and inability to form cell plates. Genomic in situ hybridization (GISH) showed unequal chromatin distribution during cell division. Interestingly, 96.70% of lagging chromosomes were from E. arundinaceus. Furthermore, fluorescence in situ hybridization (FISH) was performed using 45S rDNA and 5S rDNA as probes. Either 45S rDNA or 5S rDNA sites were lost during abnormal meiosis, and results of unequal chromosomal separation were also clearly observed in tetrads. CONCLUSIONS: Using cytogenetic analysis, a large number of meiotic abnormalities were observed in F1. GISH further confirmed that 96.70% of the lagging chromosomes were from E. arundinaceus. Chromosome loss was found by further investigation of repeat sequences. Our findings provide insight into sugarcane chromosome inheritance to aid innovation and utilization in sugarcane germplasm resources.

Preferential meiotic chromosome pairing among homologous chromosomes with cryptic sequence variation in tetraploid maize.

Meiotic chromosome pairing between homoeologous chromosomes was reported in many nascent allopolyploids. Homoeologous pairing is gradually eliminated and replaced by exclusive homologous pairing in well-established allopolyploids, an evolutionary process referred to as the diploidization of allopolyploids. A fundamental question of the diploidization of allopolyploids is whether and to what extent the DNA sequence variation among homoeologous chromosomes contribute to the establishment of exclusive homologous chromosome pairing. We developed aneuploid tetraploid maize lines that contain three copies of chromosome 10 derived from inbred lines B73 and H99. We were able to identify the parental origin of each copy of chromosome 10 in the materials using oligonucleotide-based haplotype-specific chromosome painting. We demonstrate that the two identical copies of chromosome 10 from H99 pair preferentially over chromosome 10 from B73 in different stages of prophase I and metaphase I during meiosis. Thus, homologous chromosome pairing is favored to partners with the most similar DNA sequences and can be discriminated based on cryptic sequence variation. We propose that innate preference of homologous chromosome pairing exists in nascent allopolyploids and serves as the first layer that would eventually block all homoeologous chromosome pairing in allopolyploids.

Chromosomal Characterization of Tripidium arundinaceum Revealed by Oligo-FISH.

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.

Genome-wide identification and expression analysis of AP2/ERF transcription factors in sugarcane (Saccharum spontaneum L.).

BACKGROUND: APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors play essential roles in plant growth, development, metabolism, and responses to biotic and abiotic stresses. However, few studies concerning AP2/ERF genes in sugarcane which are the most critical sugar and energy crops worldwide. RESULTS: A total of 218 AP2/ERF genes were identified in the Saccharum spontaneum genome. Phylogenetic analysis showed that these genes could be divided into four groups, including 43 AP2s, 160 ERFs and Dehydration-responsive element-binding (DREB) factors, 11 ABI3/VPs (RAV), and four Soloist genes. These genes were unevenly distributed on 32 chromosomes. The structural analysis of SsAP2/ERF genes showed that 91 SsAP2/ERFs lacked introns. Sugarcane and sorghum had a collinear relationship between 168 SsAP2/ERF genes and sorghum AP2/ERF genes that reflected their similarity. Multiple cis-regulatory elements (CREs) present in the SsAP2/ERF promoter were related to abiotic stresses, suggesting that SsAP2/ERF activity could contribute to sugarcane adaptation to environmental changes. The tissue-specific analysis showed spatiotemporal expression of SsAP2/ERF in the stems and leaves of sugarcane at different development stages. In ten sugarcane samples, 39 SsAP2/ERFs were not expressed, whereas 58 SsAP2/ERFs were expressed in all samples. Quantitative PCR experiments showed that SsERF52 expression was up-regulated under salt stress, but suppressed under dehydration stress. SsSoloist4 had the most considerable upregulation in response to treatment with the exogenous hormones ABA and GA. Within 3 h of ABA or PEG6000 treatment, SsSoloist4 expression was up-regulated, indicating that this gene could play a role in the responses to ABA and GA-associated dehydration stress. Analysis of AP2/ERF gene expression patterns under different treatments indicated that SsAP2/ERF genes played an essential role in dehydration and salt stress responses of S. spontaneum. CONCLUSIONS: In this study, a total of 218 members of the AP2 / ERF superfamily were identified in sugarcane, and their genetic structure, evolution characteristics, and expression patterns were studied and analyzed. The results of this study provide a foundation for future analyses to elucidate the importance of AP2/ERF transcription factors in the function and molecular breeding of sugarcane.