Abstract
Sugarcane (Saccharum officinarum L.) is an important crop for sugar production and bioenergy worldwide. In this study, we performed transcriptome sequencing for six contrasting sugarcane genotypes involved in leaf abscission, tolerance to pokkah boeng disease and drought stress. More than 465 million high-quality reads were generated, which were de novo assembled into 93,115 unigenes. Based on a similarity search, 43,526 (46.74%) unigenes were annotated against at least one of the public databases. Functional classification analyses showed that these unigenes are involved in a wide range of metabolic pathways. Comparative transcriptome analysis revealed that many unigenes involved in response to abscisic acid and ethylene were up-regulated in the easy leaf abscission genotype, and unigenes associated with response to jasmonic acid and salicylic acid were up-regulated in response to the pokkah boeng disease in the tolerance genotype. Moreover, unigenes related to peroxidase, antioxidant activity and signal transduction were up-regulated in response to drought stress in the tolerant genotype. Finally, we identified a number of putative markers, including 8,630 simple sequence repeats (SSRs) and 442,152 single-nucleotide polymorphisms (SNPs). Our data will be important resources for future gene discovery, molecular marker development, and genome studies in sugarcane.
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Introduction
Sugarcane produces more than 70% of the sugar worldwide and is also one of the most important crops for biofuels1. As an alternative energy source, many countries have implemented plans to produce alcohol from sugarcane2,3,4. China produced 10.556 million tons of sugarcane during the 2014/2015 harvest, which was 2.762 million tons less than in 2013/14. Production reduced to 9.3 million tons in 2015/16, with particularly sharp decreases in Guangxi, where the major constraints in sugar production were the increased labor and production cost, decreased sugar price and plantation area, and the more serious drought stress and disease incidence.
The cost for sugarcane production has quickly risen in China over the past several years, especially with regard to labor costs for manual harvest as well as production costs for over fertilization, pesticides and herbicides. Currently, more than 95% of sugarcane is manually harvested in China, in contrast to several other countries where mechanical harvesting dominates. Removal of dead leaves from cane stalks (defoliation) is a major task during harvesting, which contributes greatly to increased costs. One way to address these problems and facilitate green-cane harvesting is the development of easy-defoliating cultivars5. Biotic and abiotic stresses are becoming more serious problems due to the severe singleness of the sugarcane variety in the main sugarcane growing areas, where ROC22 has occupied almost 70% of the total sugarcane area for more than 10 years and more than 80% of sugarcane grown in the upland areas where irrigation is not available6. In recent years, the incidence of sugarcane pokkah boeng disease in China showed a trend of gradually increasing, and has become the main disease during the early growth of sugarcane. Therefore, the release and extension of new cultivars with strong resistance to disease and drought and higher ratooning ability is urgently needed for sugarcane production in China.
Modern sugarcane cultivars are derived from the interspecific hybridizations between S. officinarum, S. spontaneum, and other species in order to obtain disease resistance, high sucrose content, and high yield7. The chromosome number of these modern cultivars ranges from 100 to 130, indicating high levels of polyploidy and aneuploidy8. The genome size of sugarcane cultivar R570 was estimated to be approximately 10 Gb, and average monoploid genome sizes of S. officinarum and S. spontaneum were estimated to be 985 Mb and 843 Mb, respectively9,10. To date, no complete sugarcane genome sequence has been reported, which restricts the development of functional genomics and modern breeding.
Expressed sequence tags (ESTs) provide an important resource for discovering novel genes and assisting in the genome annotation of sugarcane. The first EST database for sugarcane was constructed from leaf roll tissue (meristematic region)11. The Brazilian SUCEST project, the largest EST database (about 238,000 ESTs), was carried out to construct a genetic linkage map and to identify cell wall-related genes in 26 cDNA libraries from many tissues and sugarcane cultivars12,13,14. All of the ESTs are deposited in the Sugarcane Gene Index (version 3.0), which contains 282,683 ESTs and 499 complete cDNA sequences, resulting in 121,342 unigenes. However, more than 10,000 sugarcane coding genes have yet to be identified, highlighting the necessity for further studies on the sugarcane transcriptome15.
RNA sequencing (RNA-seq) is an effective tool for deciphering the transcriptome and is particularly useful for species lacking a sequenced genome. The large quantity of reads obtained can be assembled for gene annotation, gene discovery, gene expression, and identification of regulation patterns in organisms. RNA-seq has also been used for discovery of putative molecular markers (SNPs, SSRs) to facilitate trait map** and marker-assisted selection without the requirement for a reference genome16. RNA-seq technology has been used for transcriptome analysis in many species, such as rice, maize, soybean, and sugarcane17,18,19,20.
In this study, the transcriptomic characterization of six contrasting sugarcane genotypes was compared in response to pokkah boeng, drought and leaf abscission. The sequenced data from the Illumina Hiseq. 2500 were de novo assembled and annotated against several public databases. The putative markers (SSRs, SNPs) were identified, which will be useful for screening variation among the contrasting sugarcane genotypes.
Results
Sequencing and assembly
Each RNA sample was extracted and sequenced using the Illumina paired-end sequencing technology. After quality assessment and data filtering, more than 465 million high-quality reads were used for de novo assembly (Supplementary Table S1). The raw reads were deposited in the Sequence Read Archive (SRA) at GenBank databases ID: SRP127762. A total of 471,654 transcripts were assembled with a mean length of 1,450 bp and an N50 length of 2,067 bp using Trinity software (Table 1). These transcripts represented a total of 93,115 unigenes, with a mean length of 910 bp and an N50 of 1,774 bp. The average length and N50 of the assembled unigenes was higher than those observed for S. spontaneum (801 bp and 1,337 bp) and sugarcane variety GT35 (460 bp and 640 bp) using similar sequencing technologies21,22, demonstrating the high quality of our sugarcane transcriptomic sequences. In total, more than 43,308 unigenes (46.51%) were over 500 bp in length, 25,486 (27.37%) over 1,000 bp, and 12,278 (13.19%) longer than 2,000 bp (Table 1).
Functional annotation
The lack of a reference sugarcane genome is a challenge for gene function prediction and utilization of the transcriptome dataset. Overall, 43,526 (46.74%) unigenes were annotated against at least one of the public databases (Table 2 and Supplementary Table S2). A total of 42,042 (45.15%) unigenes showed homologs in the NR database, while 22,660 (24.34%) unigenes had similarity to proteins in the Swiss-Prot database. However, a total of 49,589 (53.26%) unigenes could not be annotated, suggesting that these unannotated unigenes might be novel genes, although some of these unigenes may represent non-coding RNAs. Among the BLASTx top hits, 19,632 (46.71%) were matched to Sorghum bicolor proteins, followed by Zea mays (9,272; 22.06%), Setaria italica (3,812; 9.07%), and Oryza sativa Japonica Group (1,804; 4.29%) (Fig. 1). These results were consistent with previous reports due to the higher collinearity in the genic regions between sugarcane and sorghum genomes23,24. Interestingly, only 857 (2.04%) unigenes showed significant homology with those of the Saccharum hybrid cultivar R570, which was consistent with a previous report20. This result might be due to the lack of reference genome sequence and limited public data in sugarcane, and might also point to the high genetic variation among different sugarcane genotypes. A total of 20,738 (81.37%) of the unigenes with sizes over 1,000 bp showed homologous matches, whereas only 7,705 (27.67%) of the unigenes shorter than 300 bp were annotated.
Cluster of Orthologous Group (COG)
All assembled sugarcane unigenes were searched against the COG database for functional prediction and classification. A total of 10,575 (11.36%) unigenes were assigned and classified into 25 COG categories (Fig. 2). The cluster for ‘general function prediction only’ (2,833; 18.22%) represented the largest group, followed by ‘replication, recombination, and repair’ (2,047; 13.16%), ‘transcription’ (1,500; 9.64%), ‘translation, ribosomal structure and biogenesis’ (1,429; 9.19%) and ‘signal transduction mechanisms’ (1,333; 8.57%). Only a few unigenes were assigned to ‘cell motility’ and ‘nuclear structure’ (8 and 7 unigenes, respectively). In addition, 330 unigenes were assigned to a class representing unknown function. Unigenes in categories representing ‘energy production and conversion’ (745; 4.79%), ‘carbohydrate transport and metabolism’ (712; 4.58%), ‘signal transduction mechanisms’ (1,295; 8.53%), and ‘defense mechanisms’ (178; 1.14%) may be used to develop molecular markers of agronomic traits, such as biomass, sugar content, and abiotic and/or disease resistance.
Gene Ontology (GO)
GO was used to classify the unigene function predictions according to three main categories: molecular function, biological process, and cellular component. A total of 30,677 (32.95%) unigenes were classified into 55 GO functional sub-categories (Fig. 3). Cellular components represented the majority of the functional terms (77,191; 40.19%), followed by biological processes (76,711; 39.94%) and molecular functions (38,159; 19.87%). Within the cellular components category, ‘cell’ and ‘cell part’ (19,953; 10.39%) was the most dominant group, followed by ‘organelle’ (17,728; 9.23%), ‘membrane’ (7,600; 3.96%), and ‘organelle part’ (3,559; 1.85%). Within the molecular function category, ‘binding’ (17,434; 9.08%) and ‘catalytic activity’ (15,430; 8.03%) were prominently represented, and within the biological process category, ‘metabolic process’ (19,385; 10.09%) and ‘cellular process’ (17,026; 8.86%) were the most enriched. The unigenes involved in the categories representing ‘signaling’ (1,235), ‘receptor activity’ (229), ‘response to stimulus’ (5,937), and ‘antioxidant activity’ (276), might be closely related to drought or/and disease response and provided valuable information for further studies.
KEGG pathway
To further understand the biology and intricate metabolic pathways of sugarcane, all assembled unigenes were annotated using the KEGG pathway database. In total, 12,367 (13.28%) unigenes were annotated to 126 pathways (Supplementary Table S3). The most highly represented pathways were ‘ribosome’ (1,009; 8.16%), followed by ‘carbon metabolism’ (452; 3.66%), ‘biosynthesis of amino acids’ (393; 3.18%) and ‘protein processing in endoplasmic reticulum’ (388; 3.14%). Unigenes in the ‘plant-pathogen interaction’ pathway (358; 2.90%) may be useful for studying the resistance mechanisms to pokkah boeng disease in sugarcane. Pathways associated with leaf abscission and drought stress, such as ‘plant hormone signal transduction’ (276; 2.23%), ‘glutathione metabolism’ (177; 1.43%) and ‘ubiquitin mediated proteolysis’ (189; 1.53%) will be the focus of future studies.
Comparative Transcriptomic Characterization of Contrasting Sugarcane Cultivars
Based on the RNA-seq data, the unique and shared unigenes were determined among the contrasting sugarcane cultivars (Fig. 4A). GXU-34140 and GXU-34176 shared a total of 55,461 (75.82%) unigenes, and GXU-34176 exhibited a higher number of unique unigenes (9,987) than those of GXU-34140 (7,703). A total of 72,562 (87.29%) unigenes were co-expressed in GN18 and FN95–1702 under drought stress, but the number of uniquely expressed unigenes in GN18 (6,855) was larger than that in FN95–1702 (3,709). GUC2 and GUC10 shared 36,854 (72.65%) unigenes, while the pokkah boeng disease susceptible GUC10 exhibited a higher number of unique unigenes (7,573) than the resistant cultivar GUC2 (6,301).
A total of 7,838 differentially expressed genes (DEGs) were identified between GXU-34140 and GXU-34176, with 4,582 up-regulated and 3,062 down-regulated in GXU-34176 (both +1 and +3 leaf sheath samples) when compared with GXU-34140. The remaining 194 DEGs showed the opposite expression pattern. According to GO functional enrichment analysis, GO terms, including ‘abscisic acid-activated signaling pathway’ (22 unigenes) and ‘jasmonic acid mediated signaling pathway’ (24 unigenes), were enriched in the up-regulated DEGs, while ‘cellulose synthase (UDP-forming) activity’ (26 unigenes), ‘cellulose biosynthetic process’ (30 unigenes), ‘plant-type primary cell wall biogenesis’ (7 unigenes), and ‘cell wall’ (56 unigenes) were enriched in the down-regulated DEGs (Supplementary Table S4). These results suggested that those genes might play an important role in responsive to leaf abscission for the easy defoliation GXU-34176.
In the comparison of GN18 with FN95–1702, a total of 5,314 DEGs were identified, of which 3,428 DEGs were up-regulated and 1,839 DEGs were down-regulated in GN18 responses to drought stress (both in mild drought and severe drought). The remaining 47 DEGs showed the opposite pattern. GO functional enrichment analysis indicated that ‘photosynthesis’ (23 unigenes), ‘chloroplast thylakoid membrane’ (36 unigenes), and ‘photosystem I’ (7 unigenes) were enriched in the up-regulated DEGs (Supplementary Table S4). These results suggested that interference of photosynthesis was less affected by drought stress in GN18 when compared with FN95–1702.
A total of 3,645 DEGs were identified in the comparison between GUC2 and GUC10, of which 2,175 DEGs were up-regulated and 1,454 DEGs were down-regulated in pokkah boeng resistant GUC2 (both in healthy and infected samples). Only 16 DEGs showed the opposite pattern. GO functional enrichment analysis indicated that ‘protein phosphorylation’ (162 unigenes), ‘protein serine/threonine kinase activity’ (139 unigenes), ‘transmembrane receptor protein serine/threonine kinase signaling pathway’ (16 unigenes), and ‘response to salicylic acid’ (19 unigenes) were enriched in the up-regulated DEGs (Supplementary Table S4), suggesting that they might represent special mechanisms in GUC2 responses to pokkah boeng disease.
Quantitative Real-time PCR (qRT-PCR) validation of target genes
To validate the reliability and reproducibility of the Illumina RNA-Seq results, fourteen unigenes based on their characterizations of contrasting sugarcane genotypes were validated via qRT-PCR analysis. The primers used for qRT-PCR are listed in Supplementary Table S5. In GXU-34176, c71654.graph_c0 (encoding lipase-like PAD4) and c65832.graph_c0 (encoding probable protein phosphatase 2 C) related to ‘abscisic acid-activated signaling pathway’, c67492.graph_c0 (encoding putative serine/threonine-protein kinase-like protein CCR3) associated with ‘response to ethylene’ and c64240.graph_c0 (encoding zinc finger CCCH domain-containing protein) related to ‘leaf senescence’ were up-regulated in +3 leaf sheath compared to GXU-34140 (Fig. 5). Interestingly, the c65986.graph_c0 (encoding COBRA-like protein) related to ‘cell wall modification involved in abscission’ was down-regulated in +3 leaf sheath. In GN18, c54647.graph_c0 (encoding putative leucine-rich repeat receptor-like protein kinase family protein), c56804.graph_c0 (encoding protein TIFY 9), and c61335.graph_c0 (encoding heat shock protein 90) associated with ‘response to water deprivation’ as well as c57471.graph_c0 (encoding peroxidase) related to ‘response to oxidative stress’ were up-regulated under mild or severe drought stress compared to FN95–1702 (Fig. 5). In resistant cultivar GUC2, c69746.graph_c0 (encoding U-box domain-containing protein) associated with ‘ubiquitin-protein transferase activity’, c72075.graph_c0 (encoding respiratory burst oxidase homolog protein) related to ‘defense response to fungus’, and c65355.graph_c0 (encoding allene oxide synthase 2) related to ‘response to jasmonic acid’ were up-regulated after infected with F. verticillioides compared to sensitive cultivar GUC10 (Fig. 5). Pearson correlation coefficient between RNA-Seq and qRT-PCR was 0.89 and the Significance (two-tailed t test) was 1.11 × 10−4. These results showed that the expression trend of these genes was consistent with the transcriptome data.
Putative marker discovery for marker-selection of important traits
SSR markers are important tools for studying genetic diversity, constructing genetic maps, and performing comparative genomics25. Potential SSR markers were detected from unigenes with lengths over 1,000 bp using MISA software. A total of 8,630 SSRs were mined from 6,883 unigenes, of which 1,413 sequences contained more than one SSR and 286 SSRs were present in a compound formation (Supplementary Table S6). On average, the distribution density of SSRs was 1/6.62 kb. The most abundant repeat motifs were mononucleotide (4,162; 48.23%) and trinucleotide (2,924; 33.88%), followed by dinucleotide (1,392; 16.13%) and tetranucleotide (120; 1.39%) (Fig. 6A). Pentanucleotide and hexanucleotide repeat motifs represented only 0.22% and 0.15% of the total SSRs, respectively. The identified proportion of trinucleotide repeats were similar to the survey in the sugarcane EST (SUCEST) database (30.5%), but the percentage of tetranucleotides was lower than that obtained in the previous report26. Taken together, 188 types of nucleotide motif repeats were detected among 8,630 SSR loci. The most abundant repeat type was A/T (4,084; 47.32%), followed by CCG/CGG (1,237; 14.33%), AG/CT (712; 8.25%) and AGC/CTG (460; 5.33%) (Fig. 6B). These results were similar to those of the SSR motif previously reported20. Based on the 8,630 SSRs, primer pairs were designed using Primer 3.0 and are listed in Supplementary Table S6. These data are valuable resources for further studies on marker-assisted selection in sugarcane breeding.
SNPs are also widely used as molecular markers for the identification of quantitative trait locus, evolutionary analysis, and development marker-assisted selection to accelerate plant breeding. A total of 442,152 putative SNP positions were identified in 55,659 different unigenes (Supplementary Table S7). The unique and shared SNPs between the contrasting sugarcane genotypes were evaluated in Fig. 4B. The number of shared SNPs were 313,666 (80.25%) for GXU-34140 and GXU-34176, 172,118 (68.59%) for GUC2 and GUC10, and 386,996 (91.42%) for GN18 and FN95–1702, respectively. More shared SNPs resulted from the same parent of GXU-34140 and GXU-34176 or GN18 derived from FN95–1702 mediated with the Ea-DREB2B gene. A total of 77,184 SNPs (in 30,171 unigenes), 78,807 SNPs (in 25,029 unigenes), and 36,332 SNPs (in 19,719 unigenes) were different between GXU-34140 and GXU-34176, GUC2 and GUC10, and GN18 and FN95–1702, respectively. According to the GO annotation of unigenes that contained the unique SNPs in each group (Supplementary Table S8), several important categories were found to be associated with the sugarcane genotypes. For GXU-34140 and GXU-34176, 138 unigenes with SNPs were confirmed in the ‘response to abscisic acid’ category, 34 unigenes with SNPs in the ‘response to ethylene’ category, 17 unigenes with SNPs in the ‘leaf senescence’ category, and 17 unigenes with SNPs in the ‘cell wall modification’ category. Only one unigene with SNP was identified in the ‘cell wall modification involved in abscission’. For GUC2 and GUC10, 58 unigenes with SNPs were observed in the ‘defense response to fungus’, 17 in the ‘defense response to fungus, incompatible interaction’, 25 in the ‘response to fungus’, one in the ‘regulation of defense response to fungus, incompatible interaction’, and four in the ‘jasmonic acid and ethylene-dependent systemic resistance’. For GN18 and FN95–1702, 164 unigenes with SNPs were observed in the ‘defense response’, 50 in the ‘response to stress’, five in the ‘response to desiccation’, 49 in the ‘response to oxidative stress’, 45 in the ‘response to water deprivation’, and seven in the ‘water transport’. The SNPs derived from transcriptome sequences (in the transcribed regions) may be directly linked to expressed genes. The SNPs unique to each genotype are useful for develo** markers associated with important agronomic traits of the contrasting genotypes.
Discussion
Biotic and abiotic stresses in nature seriously restrict the development of the sugarcane industry worldwide. Currently the major aims of sugarcane breeding programs are to develop cultivars with strong resistance to phytopathogens (particularly Ustilago scitaminea and Fusarium species complex) and abiotic stress (especially drought stress), high sucrose and yield, and suitable for mechanized harvest. In our experiments, six sugarcane genotypes with contrasting responses to stress (drought, pokkah boeng disease) and defoliation were sequenced using the Illumina sequencing platform and 93,115 unigenes were assembled with an N50 of 1,774 bp, of which 43,526 unigenes were annotated against at least one of the public databases. The more assembled and annotated sequences in our studies indicated a comprehensive reference transcriptome of sugarcane when compared with other reports20,21. Many unigenes were identified in the signal transduction (1,333) and response to stimulus (5,937) categories, which were conducive to understanding the resistance mechanism.
Leaf abscission, which is one of the important traits for the sugarcane breeding program, is beneficial for improving the efficiency of sugarcane harvest. Two contrasting sugarcane genotypes (GXU-34140 with difficult-defoliation and GXU-34176 with easy-defoliation) derived from the same cross Co1001 × ROC22 showed different defoliation ability from our previous studies. ABA and ethylene played a more important role in the abscission of organs27,28,29. Compared to the leaf packaging sugarcane varieties (Q2 and B), ten transcripts involved in ‘abscisic acid associated pathways’ were up-regulated in leaf abscission sugarcane varieties (Q1 and T)30. In this work, more unigenes involved in ‘abscisic acid-activated signaling pathway’ (22 unigenes) and ‘response to abscisic acid’ (38 unigenes) were up-regulated in easy-defoliation GXU-34176 when compared to difficult-defoliation GXU-34140. Interestingly, 15 unigenes involved in ‘response to ethylene’ were up-regulated in GXU-34176, which was not observed in leaf abscission sugarcane varieties (Q1 and T)30. The position of organ separation from the plant body is called abscission zones (AZs). In Phaseolus vulgaris petioles, ethylene may induced the formation of AZs31. A total of 10 unigenes related to ‘cell wall macromolecule catabolic processes’ were up-regulated and 30 unigenes involved in ‘cellulose biosynthetic processes’ were down-regulated in GXU-34176. It is well-known that the common feature of abscission processes is cell wall degradation32. Genes involved in cell-wall modification during the abscission period have also been identified in Citrus and Tomato33,34.
Sugarcane pokkah boeng disease, caused by Fusarium species complex, is one of the most serious and devastating diseases recorded in countries where sugarcane is grown35. It has reportedly caused a yield loss of 40.8–64.5% in infection-susceptible sugarcane cultivars36,37. Based on our field survey and inoculation test, GUC2 was a promising genotype resistant to pokkah boeng disease, whereas GUC10 was susceptible to pokkah boeng. Comparative transcriptomic analysis indicated that 12 and 19 unigenes involved in response to jasmonic acid and salicylic acid were up-regulated in resistant GUC2. Salicylic acid (SA)-dependent signaling pathways and jasmonic acid (JA)-dependent signaling pathways are thought to form the backbone of the plant defense system38. In general, SA-mediated defenses are activated to resist biotrophic pathogens, whereas JA-mediated defenses confer resistance against necrotrophic pathogens that kill host cells39,40. SA and JA has been reported as defense elicitors to enhance resistance to Dutch elm disease in the tolerant phenotype of American elm41. The ubiquitin-proteasome system plays important roles in the regulation of plant immunity, especially the E3 ubiquitin ligases42. The results presented here showed one and 14 unigenes encoding an ubiquitin conjugating enzyme and ubiquitin-protein transferase activity were up-regulated in GUC2, respectively. In addition, our data also revealed that more than 100 unigenes encoding protein serine/threonine kinases were up-regulated in response to Fusarium species infection in GUC2. Future studies should focus on how pathogens and elicitors are perceived by protein serine/threonine kinases and how protein serine/threonine kinases and their activated signaling pathways are regulated during the Fusarium infection process in GUC2.
Natural disasters, such as drought and low temperature, occur frequently and have caused serious loss to sugarcane production43. Therefore, the development of sugarcane cultivars tolerant to abiotic stresses is crucial to improve the profitability of sugarcane industries. Transgenic technology can improve the efficiency of genetic improvement in many crops44,59. Three biological and three technical replications were performed for each sample. The data were analyzed using LightCycler® 480 sofware version 1.5.1 provided by Roche. The relative fold change of the selected genes was calculated using 2−△△Ct algorithm60. The Pearson correlation test was performed by Origin 9.0 sofware.
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Acknowledgements
We greatly appreciate Bioscience Editing Solutions for critically reading this paper and providing helpful suggestions. Financial support was provided by Natural Science Foundation of Guangxi (2014GXNSFFA118002) and National Natural Science Foundation of China (31460374 and 31660420).
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Conceived and designed the experiments: Z.M.Q. and C.B.S. Performed the experiments: X.S.Q., W.J.H., S.H.Y., J.H.T. and H.Y.Z. Analyzed the data: X.S.Q., W.J.H., S.H.Y., Y.W. and Z.M.Q. Contributed reagents/materials/analysis tools: Z.M.Q. and C.B.S. Wrote the paper: X.S.Q. and Z.M.Q. All authors read and approved the final version of the paper.
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Xu, S., Wang, J., Shang, H. et al. Transcriptomic characterization and potential marker development of contrasting sugarcane cultivars. Sci Rep 8, 1683 (2018). https://doi.org/10.1038/s41598-018-19832-x
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DOI: https://doi.org/10.1038/s41598-018-19832-x
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