The complex polyploid genome architecture of sugarcane.

Sugarcane, the world's most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype ('monoploid') representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

Characterization of silicon transporter gene family in Saccharum and functional analysis of the ShLsi6 gene in biotic stress.

Silicon, one of the most prevalent elements in the soil, is beneficial for plant growth and defense against different stresses. The silicon transporter gene (Lsi) plays an important role in the uptake and transport of silicon in higher plants. In this study, a total of 32 Lsi genes, including 20 SsLsi in sugarcane wild species Saccharum spontaneum, 5 ShLsi in Saccharum hybrid cultivar R570 and 7 SbLsi in sugarcane related species Sorghum bicolor, were identified and classified into three groups. Bioinformatics analysis showed that instability, hydrophobicity, localization of cell membranes and vacuoles were the main features of the Lsi proteins. Whole genome and segmental duplication contributed to the main expansion of Lsi gene family. Collinearity analysis of the Lsi genes showed that S. spontanum and R570 had a collinear relationship with monocotyledonous plants S. bicolor and Oryza sativa, but not with dicotyledonous plants Arabidopsis thaliana and Vitis vinifera. The replicated Lsi genes were mainly subjected to strong selection pressure for purification. The diverse cis-regulatory elements in the promoter of SsLsi, ShLsi and SbLsi genes suggested that they were widely involved in the response of plants to various stresses and the regulation of the growth and development. Transcriptome data and real time quantitative PCR analysis showed that the Lsi genes exhibited different expression profiles in sugarcane tissues and under Sporisorium scitamineum, drought and cold stresses. In addition, the cDNA and genomic DNA sequences of ShLsi6 that was homologous to SsLsi1b gene was cloned from Saccharum hybrid cultivar ROC22. Transient expression analysis showed that, compared with the control, Nicotiana benthamiana leaves which overexpressed the ShLsi6 gene showed a high sensitivity after inoculation with tobacco pathogens Ralstonia solanacearum and Fusarium solani var. coeruleum. This study provides important information for further functional analysis of Lsi genes and resistant breeding in sugarcane.

Genome-Wide Identification of LRR-RLK Family in Saccharum and Expression Analysis in Response to Biotic and Abiotic Stress.

The leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene family is the largest family of the receptor-like protein kinases (RLKs) superfamily in higher plants, which is involved in regulating the plant growth and development, stress responses, signal transduction and so on. However, no comprehensive analyses of LRR-RLKs have been reported in sugarcane. Here, we performed a comprehensive analysis of the LRR-RLK gene family in sugarcane ancestor species Saccharum spontaneum. A total of 437 LRR-RLK genes were identified and categorized into 14 groups based on a maximum likelihood phylogenetic tree. The chromosome location showed an uneven distribution on all 32 chromosomes in sugarcane. Subsequently, the exon-intron organization structure and conserved motif arrangement were relatively conserved among the same groups or subgroups and between Arabidopsis and S. spontaneum genomes. Furthermore, the promoter sequences analyses showed that sugarcane LRR-RLK genes (SsLRR-RLKs) were strongly regulated by various environmental stimuli, phytohormonal factors and transcription factors (TFs). Eventually, the expression profiles of SsLRR-RLK genes at different stresses were analyzed based on RNA-seq data, suggesting their potential roles in the regulation of sugarcane responses to diverse abiotic and biotic stress. Overall, the findings provide insight into the potential functional roles and lay the foundation for further functional study.

Genome-wide analysis of general phenylpropanoid and monolignol-specific metabolism genes in sugarcane.

Lignin is the main component of secondary cell walls and is essential for plant development and defense. However, lignin is recognized as a major recalcitrant factor for efficiency of industrial biomass processing. Genes involved in general phenylpropanoid and monolignol-specific metabolism in sugarcane have been previously analyzed at the transcriptomic level. Nevertheless, the number of genes identified in this species is still very low. The recently released sugarcane genome sequence has allowed the genome-wide characterization of the 11 gene families involved in the monolignol biosynthesis branch of the phenylpropanoid pathway. After an exhaustive analysis of sugarcane genomes, 438 haplotypes derived from 175 candidate genes from Saccharum spontaneum and 144 from Saccharum hybrid R570 were identified as associated with this biosynthetic route. The phylogenetic analyses, combined with the search for protein conserved residues involved in the catalytic activity of the encoded enzymes, were employed to identify the family members potentially involved in developmental lignification. Accordingly, 15 candidates were identified as bona fide lignin biosynthesis genes: PTAL1, PAL2, C4H4, 4CL1, HCT1, HCT2, C3'H1, C3'H2, CCoAOMT1, COMT1, F5H1, CCR1, CCR2, CAD2, and CAD7. For this core set of lignin biosynthetic genes, we searched for the chromosomal location, the gene expression pattern, the promoter cis-acting elements, and microRNA targets. Altogether, our results present a comprehensive characterization of sugarcane general phenylpropanoid and monolignol-specific genes, providing the basis for further functional studies focusing on lignin biosynthesis manipulation and biotechnological strategies to improve sugarcane biomass utilization.

Sugarcane cystatins: From discovery to biotechnological applications.

Phytocystatins are tight-binding cysteine protease inhibitors produced by plants. The first phytocystatin described was isolated from Oryza sativa and, since then, cystatins from several plant species were reported, including from sugarcane. Sugarcane cystatins were unraveled in Sugarcane EST project database, after sequencing of cDNA libraries from various sugarcane tissues at different developmental stages and six sugarcane cystatins were cloned, expressed and characterized (CaneCPI-1 to CaneCPI-6). These recombinant proteins were produced in different expression systems and inhibited several cysteine proteases, including human cathepsins B and L, which can be involved in pathologies, such as cancer. In this review, we summarize a comprehensive history of all sugarcane cystatins, presenting an updated phylogenetic analysis; chromosomal localization, and genomic organization. We also present protein docking of CaneCPI-5 in the active site of human cathepsin B, insights about canecystatins structures; recombinant expression in different systems, comparison of their inhibitory activities against human cysteine cathepsins B, K, L, S, V, falcipains from Plasmodium falciparum and a cathepsin L-like from the sugarcane weevil Sphenophorus levis; and enlighten their potential and current applications in agriculture and health.

Identification of genes from the general phenylpropanoid and monolignol-specific metabolism in two sugarcane lignin-contrasting genotypes.

The phenylpropanoid pathway is an important route of secondary metabolism involved in the synthesis of different phenolic compounds such as phenylpropenes, anthocyanins, stilbenoids, flavonoids, and monolignols. The flux toward monolignol biosynthesis through the phenylpropanoid pathway is controlled by specific genes from at least ten families. Lignin polymer is one of the major components of the plant cell wall and is mainly responsible for recalcitrance to saccharification in ethanol production from lignocellulosic biomass. Here, we identified and characterized sugarcane candidate genes from the general phenylpropanoid and monolignol-specific metabolism through a search of the sugarcane EST databases, phylogenetic analysis, a search for conserved amino acid residues important for enzymatic function, and analysis of expression patterns during culm development in two lignin-contrasting genotypes. Of these genes, 15 were cloned and, when available, their loci were identified using the recently released sugarcane genomes from Saccharum hybrid R570 and Saccharum spontaneum cultivars. Our analysis points out that ShPAL1, ShPAL2, ShC4H4, Sh4CL1, ShHCT1, ShC3H1, ShC3H2, ShCCoAOMT1, ShCOMT1, ShF5H1, ShCCR1, ShCAD2, and ShCAD7 are strong candidates to be bona fide lignin biosynthesis genes. Together, the results provide information about the candidate genes involved in monolignol biosynthesis in sugarcane and may provide useful information for further molecular genetic studies in sugarcane.

Saccharum spontaneum, a Novel Source of Resistance to Root-Lesion and Root-Knot Nematodes in Sugarcane.

Root-lesion nematode (Pratylenchus zeae) and root-knot nematode (Meloidogyne javanica) are two important pathogens of sugarcane (Saccharum hybrid). No commercial cultivars are resistant to these nematodes in Australia. Twenty accession lines of S. spontaneum, a wild relative of sugarcane, were tested against these two nematode species. S. spontaneum lines were tested twice for resistance to root-lesion nematode and three times for root-knot nematode. Reproduction (final population/starting population) of root-lesion nematodes was significantly lower in 17 of the 20 S. spontaneum accession lines tested in two experiments compared with two commercial cultivars. Four S. spontaneum lines supported a significantly lower number of root-lesion nematodes per gram of root than that of two commercial sugarcane cultivars. Reproduction of root-knot nematodes was significantly lower in 16 S. spontaneum lines compared with two commercial cultivars. Fourteen of the S. spontaneum lines tested supported significantly fewer eggs per gram of root compared with two commercial cultivars. This study showed that S. spontaneum lines possessed resistance for root-lesion and root-knot nematodes. Targeted crossing with commercial hybrid parental lines should be conducted to introduce nematode resistance into sugarcane cultivars for the Australian sugar industry.

Deposition of lignin in four species of Saccharum.

We used primers designed on conserved gene regions of several species to isolate the most expressed genes of the lignin pathway in four Saccharum species. S. officinarum and S. barberi have more sucrose in the culms than S. spontaneum and S. robustum, but less polysaccharides and lignin in the cell wall. S. spontaneum, and S. robustum had the lowest S/G ratio and a lower rate of saccharification in mature internodes. Surprisingly, except for CAD, 4CL, and CCoAOMT for which we found three, two, and two genes, respectively, only one gene was found for the other enzymes and their sequences were highly similar among the species. S. spontaneum had the highest expression for most genes. CCR and CCoAOMT B presented the highest expression; 4CL and F5H showed increased expression in mature tissues; C3H and CCR had higher expression in S. spontaneum, and one of the CADs isolated (CAD B) had higher expression in S. officinarum. The similarity among the most expressed genes isolated from these species was unexpected and indicated that lignin biosynthesis is conserved in Saccharum including commercial varieties Thus the lignin biosynthesis control in sugarcane may be only fully understood with the knowledge of the promotor region of each gene.

Early prediction of sugarcane genotypes susceptible and resistant to Diatraea saccharalis using spectroscopies and classification techniques.

The aim of this work was to use spectroscopic methods and partial least squares discriminant analysis (PLS-DA) for the early prediction of genotype resistance or susceptibility to sugarcane borer. The sugarcane leaf +1 was directly analyzed with no sample preparation by ultraviolet-visible-near-infrared (UV-VIS-NIR), middle-infrared (MID), and near-infrared (NIR) spectroscopies. Also, laser-induced breakdown spectroscopy (LIBS) was used to analyze pellets of dried and ground leaves and stalks of sugarcane. Classification models were built using PLS-DA. The models built using UV-VIS-NIR, MID or NIR spectra exhibited ideal sensitivity, specificity, and classification errors, i.e., 1 for both sensitivity and specificity and 0 for classification errors. Regarding the models built using LIBS spectra, those using spectra of pellets made from dried and ground leaves also presented ideal sensitivity, specificity, and classification errors; on the other hand, models built using the spectra of pellets made of dried and ground stalks did not present ideal values for these parameters. Thus, the models built, except for the one using LIBS of pellets made of stalks, showed excellent predictive capacity, making them suitable for predicting the resistance or susceptibility of sugarcane genotypes in the early stages of a plant's life.

Comparative analysis of glyoxalase pathway genes in Erianthus arundinaceus and commercial sugarcane hybrid under salinity and drought conditions.

BACKGROUND: Glyoxalase pathway is a reactive carbonyl species (RCS) scavenging mechanism involved in the detoxification of methylglyoxal (MG), which is a reactive α-ketoaldehyde. In plants under abiotic stress, the cellular toxicity is reduced through glyoxalase pathway genes, i.e. Glyoxalase I (Gly I), Glyoxalase II (Gly II) and Glyoxalase III (Gly III). Salinity and water deficit stresses produce higher amounts of endogenous MG resulting in severe tissue damage. Thus, characterizing glyoxalase pathway genes that govern the MG metabolism should provide new insights on abiotic stress tolerance in Erianthus arundinaceus, a wild relative of sugarcane and commercial sugarcane hybrid (Co 86032). RESULTS: In this study, three glyoxalase genes (Glyoxalase I, II and III) from E. arundinaceus (a wild relative of sugarcane) and commercial sugarcane hybrid (Co 86032) were characterized. Comparative gene expression profiles (qRT-PCR) of Glyoxalase I, II and III under salinity and water deficit stress conditions revealed differential transcript expression with higher levels of Glyoxalase III in both the stress conditions. Significantly, E. arundinaceus had a higher expression level of glyoxalase genes compared to commercial sugarcane hybrid. On the other hand, gas exchange parameters like stomatal conductance and transpiration rate were declined to very low levels under both salt and drought induced stresses in commercial sugarcane hybrid when compared to E. arundinaceus. E. arundinaceus maintained better net photosynthetic rate compared to commercial sugarcane hybrid. The phylogenetic analysis of glyoxalase proteins showed its close evolutionary relationship with Sorghum bicolor and Zea mays. Glyoxalase I and II were predicted to possess 9 and 7 isoforms respectively whereas, Glyoxalase III couldn't be identified as it comes under uncharacterized protein identified in recent past. Chromosomal mapping is also carried out for glyoxalase pathway genes and its isoforms. Docking studies revealed the binding affinities of glyoxalase proteins in both E. arundinaceus and commercial sugarcane hybrid with their substrate molecules. CONCLUSIONS: This study emphasizes the role of Glyoxalase pathway genes in stress defensive mechanism which route to benefit in progressive plant adaptations and serves as potential candidates for development of salt and drought tolerant crops.

Cytogenetic and agronomic characterization of intergeneric hybrids between Saccharum spp. hybrid and Erianthus arundinaceus.

In sugarcane (Saccharum spp. hybrid) breeding, introgression of useful genes via intergeneric hybridization is a powerful strategy for improving the crop productivity. Erianthus arundinaceus shows great potential in terms of useful traits; however, little is known about the cytogenetic and agronomic characteristics of intergeneric hybrids between these two species. Here, we examine the cytogenetic and agronomic characteristics, and relationships between the two in intergeneric F1 hybrids between modern sugarcane cultivar and E. arundinaceus identified by amplification of 5S rDNA markers and morphological characteristics. The nuclear DNA content of the hybrids varied from 6.07 to 8.94 pg/2C, with intra-clonal variation in DNA content and 5S rDNA sites. Genomic in situ hybridization revealed 53 to 82 chromosomes in the hybrids, with 53 to 56 derived from sugarcane and 1 to 29 from E. arundinaceus. There were significant positive correlations between the number of E. arundinaceus chromosomes and dry matter yield, millable stalk weight, single stalk weight, and stalk diameter, but not sucrose content, reducing sugar content, sucrose/reducing sugar ratio or fiber content. This detailed information on intergeneric F1 hybrids between modern sugarcane cultivar and E. arundinaceus will contribute to effective utilization of E. arundinaceus in sugarcane breeding.

Genetic diversity and population structure analysis of Saccharum and Erianthus genera using microsatellite (SSR) markers.

In order to understand the genetic diversity and structure within and between the genera of Saccharum and Erianthus, 79 accessions from five species (S. officinarum, S. spontaneum, S. robustum, S. barberi, S. sinense), six accessions of E. arundinaceus, and 30 Saccharum spp. hybrids were analyzed using 21 pairs of fluorescence-labeled highly poloymorphic SSR primers and a capillary electrophoresis (CE) detection system. A total of 167 polymorphic SSR alleles were identified by CE with a mean value of polymorphic information content (PIC) of 0.92. Genetic diversity parameters among these 115 accessions revealed that Saccharum spp. hybrids were more diverse than those of Saccharum and Erianthus species. Based on the SSR data, the 115 accessions were classified into seven main phylogenetic groups, which corresponded to the Saccharum and Erianthus genera through phylogenetic analysis and principle component analysis (PCA). We propose that seven core SSR primer pairs, namely, SMC31CUQ, SMC336BS, SMC597CS, SMC703BS, SMC24DUQ, mSSCIR3, and mSSCIR43, may have a wide appicability in genotype identification of Saccharum species and Saccharum spp. hybrids. Thus, the information from this study contibites to manage sugarcane genetic resources.

Whole chloroplast genome and gene locus phylogenies reveal the taxonomic placement and relationship of Tripidium (Panicoideae: Andropogoneae) to sugarcane.

BACKGROUND: For over 50 years, attempts have been made to introgress agronomically useful traits from Erianthus sect. Ripidium (Tripidium) species into sugarcane based on both genera being part of the 'Saccharum Complex', an interbreeding group of species believed to be involved in the origins of sugarcane. However, recent low copy number gene studies indicate that Tripidium and Saccharum are more divergent than previously thought. The extent of genus Tripidium has not been fully explored and many species that should be included in Tripidium are still classified as Saccharum. Moreover, Tripidium is currently defined as incertae sedis within the Andropogoneae, though it has been suggested that members of this genus are related to the Germainiinae. RESULTS: Eight newly-sequenced chloroplasts from potential Tripidium species were combined in a phylogenetic study with 46 members of the Panicoideae, including seven Saccharum accessions, two Miscanthidium and three Miscanthus species. A robust chloroplast phylogeny was generated and comparison with a gene locus phylogeny clearly places a monophyletic Tripidium clade outside the bounds of the Saccharinae. A key to the currently identified Tripidium species is presented. CONCLUSION: For the first time, we have undertaken a large-scale whole plastid study of eight newly assembled Tripidium accessions and a gene locus study of five Tripidium accessions. Our findings show that Tripidium and Saccharum are 8 million years divergent, last sharing a common ancestor 12 million years ago. We demonstrate that four species should be removed from Saccharum/Erianthus and included in genus Tripidium. In a genome context, we show that Tripidium evolved from a common ancestor with and extended Germainiinae clade formed from Germainia, Eriochrysis, Apocopis, Pogonatherum and Imperata. We re-define the 'Saccharum complex' to a group of genera that can interbreed in the wild and extend the Saccharinae to include Sarga along with Sorghastrum, Microstegium vimineum and Polytrias (but excluding Sorghum). Monophyly of genus Tripidium is confirmed and the genus is expanded to include Tripidium arundinaceum, Tripidium procerum, Tripidium kanashiroi and Tripidium rufipilum. As a consequence, these species are excluded from genus Saccharum. Moreover, we demonstrate that genus Tripidium is distinct from the Germainiinae.

A comparative study of the biomass properties of Erianthus and sugarcane: lignocellulose structure, alkaline delignification rate, and enzymatic saccharification efficiency.

A comprehensive understanding of the structure and properties of gramineous lignocelluloses is needed to facilitate their uses in biorefinery. In this study, lignocelluloses from fractionated internode tissues of two taxonomically close species, Erianthus arundinaceus and sugarcane (Saccharum spp.), were characterized. Our analyses determined that syringyl (S) lignins were predominant over guaiacyl (G) or p-hydroxyphenyl (H) lignins in sugarcane tissues; on the other hand, S lignin levels were similar to those of G lignin in Erianthus tissues. In addition, tricin units were detected in sugarcane tissues, but not in Erianthus tissues. Distributions of lignin inter-monomeric linkage types were also different in Erianthus and sugarcane tissues. Alkaline treatment removed lignins from sugarcane tissues more efficiently than Erianthus tissues, resulting in a higher enzymatic digestibility of sugarcane tissues compared with Erianthus tissues. Our data indicate that Erianthus biomass displayed resistance to alkaline delignification and enzymatic digestion.

Transcriptomic characterization and potential marker development of contrasting sugarcane cultivars.

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.

Comparative genetic analysis of the 45S rDNA intergenic spacers from three Saccharum species.

The 45S ribosomal DNA (rDNA) units are separated by an intergenic spacer (IGS) containing the signals for transcription and processing of rRNAs. For the first time, we sequenced and analyzed the entire IGS region from three original species within the genus Saccharum, including S. spontaneum, S. robustum, and S. officinarum in this study. We have compared the IGS organization within three original species of the genus Saccharum. The IGS of these three original species showed similar overall organizations comprised of putative functional elements needed for rRNA gene activity as well as a non-transcribed spacer (NTS), a promoter region, and an external transcribed spacer (ETS). The variability in length of the IGS sequences was assessed at the individual, intraspecies, and interspecies levels of the genus Saccharum, including S. spontaneum, S. robustum, and S. officinarum. The ETS had greater similarity than the NTS across species, but nevertheless exhibited variation in length. Within the IGS of the Saccharum species, base substitutions and copy number variation of sub-repeat were causes of the divergence in IGS sequences. We also identified a significant number of methylation sites. Furthermore, fluorescent in situ hybridization (FISH) co-localization of IGS and pTa71 probes was detected on all representative species of the genus Saccharum tested. Taken together, the results of this study provide a better insight into the structure and organization of the IGS in the genus Saccharum.

Analysis of Three Sugarcane Homo/Homeologous Regions Suggests Independent Polyploidization Events of Saccharum officinarum and Saccharum spontaneum.

Whole genome duplication has played an important role in plant evolution and diversification. Sugarcane is an important crop with a complex hybrid polyploid genome, for which the process of adaptation to polyploidy is still poorly understood. In order to improve our knowledge about sugarcane genome evolution and the homo/homeologous gene expression balance, we sequenced and analyzed 27 BACs (Bacterial Artificial Chromosome) of sugarcane R570 cultivar, containing the putative single-copy genes LFY (seven haplotypes), PHYC (four haplotypes), and TOR (seven haplotypes). Comparative genomic approaches showed that these sugarcane loci presented a high degree of conservation of gene content and collinearity (synteny) with sorghum and rice orthologous regions, but were invaded by transposable elements (TE). All the homo/homeologous haplotypes of LFY, PHYC, and TOR are likely to be functional, because they are all under purifying selection (dN/dS ≪ 1). However, they were found to participate in a nonequivalently manner to the overall expression of the corresponding gene. SNPs, indels, and amino acid substitutions allowed inferring the S. officinarum or S. spontaneum origin of the TOR haplotypes, which further led to the estimation that these two sugarcane ancestral species diverged between 2.5 and 3.5 Ma. In addition, analysis of shared TE insertions in TOR haplotypes suggested that two autopolyploidization may have occurred in the lineage that gave rise to S. officinarum, after its divergence from S. spontaneum.

Genetic divergence and admixture of ancestral genome groups in the sugarcane variety 'RB867515' (Saccharum spp).

We analyzed 80 plants of the sugarcane (Saccharum spp) variety 'RB867515' in order to investigate its diversity and genetic structure at the molecular level. Four simple sequence repeat (SSR) loci (UGSM51, SMC1237, SEGMS1069, and UGSM38) and five expressed sequence tag (EST)-SSR loci (ESTA68, ESTB92, ESTB145, ESTC66, and ESTC84) were used as molecular markers. The polymorphic loci rate was 66.6%. A total of 17 alleles and an average of 1.88 alleles/locus were detected. The number of alleles in the EST-SSR loci was lower than the number of alleles in the SSRs of non-expressed loci. The mean observed heterozygosity among the nine SSR loci was 0.3291. Genetic structure analysis showed that 'RB867515' contains alleles from three ancestral groups (K = 3), but there is little admixing of alleles in the same plant (from 0.8 to 17.3%); only 1.88% of the plants shared alleles from two or three groups. ESTB92, ESTC84, and UGSM38 were monomorphic, but there was evidence of polymorphism in ESTA68, ESTB145, ESTC66, UGSM51, SMC1237, and SEGMS1069, indicating that 'RB867515' has variability at the molecular level and the potential to be used as a parent in breeding programs. The molecular variability observed in 'RB867515' indicates that the clone terminology that is used to identify this cultivar is inconsistent with the original meaning of "clone", which is defined as a sample of genetically identical plants.

The two chromosomes of the mitochondrial genome of a sugarcane cultivar: assembly and recombination analysis using long PacBio reads.

Sugarcane accounts for a large portion of the worlds sugar production. Modern commercial cultivars are complex hybrids of S. officinarum and several other Saccharum species. Historical records identify New Guinea as the origin of S. officinarum and that a small number of plants originating from there were used to generate all modern commercial cultivars. The mitochondrial genome can be a useful way to identify the maternal origin of commercial cultivars. We have used the PacBio RSII to sequence and assemble the mitochondrial genome of a South East Asian commercial cultivar, known as Khon Kaen 3. The long read length of this sequencing technology allowed for the mitochondrial genome to be assembled into two distinct circular chromosomes with all repeat sequences spanned by individual reads. Comparison of five commercial hybrids, two S. officinarum and one S. spontaneum to our assembly reveals no structural rearrangements between our assembly, the commercial hybrids and an S. officinarum from New Guinea. The S. spontaneum, from India, and one sample of S. officinarum (unknown origin) are substantially rearranged and have a large number of homozygous variants. This supports the record that S. officinarum plants from New Guinea are the maternal source of all modern commercial hybrids.

Metabolite Profiling of Sugarcane Genotypes and Identification of Flavonoid Glycosides and Phenolic Acids.

Sugarcane is an important agricultural crop in the economy of tropical regions, and Brazil has the largest cultivated acreage in the world. Sugarcane accumulates high levels of sucrose in its stalks. Other compounds produced by sugarcane are currently not of economic importance. To explore potential coproducts, we have studied the chemical diversity of sugarcane genotypes, via metabolite profiling of leaves by NMR and LC-DAD-MS. Metabolites were identified via in-house and public databases. From the analysis of 60 HPLC-fractionated extracts, LC-DAD-MS detected 144 metabolites, of which 56 were identified (MS-MS and (1)H NMR), including 19 phenolics and 25 flavones, with a predominance of isomeric flavone C-glycosides. Multivariate analysis of the profiles from genotypes utilized in Brazilian breeding programs revealed clustering according to sugar, phenolic acid, and flavone contents.