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Resistance of sugarcane (Saccharum officinarum L.) to smut disease (caused by Sporisorium scitamineum) is driven by two separate mechanisms, external and internal resistance. Two progenies generated from an introgression cross, with contrasting responses to smut infection were used to investigate this interaction. Histopathological screening at different stages of the plant growth was used to determine the extent of mycelium growth within sugarcane tissues. Ten disease resistance-related genes were selected, and the relative expression determined using quantitative real-time reverse transcription PCR (real-time RT-qPCR). The results revealed that PR10, HCT1 and ScChi were down-regulated in the susceptible progeny and up-regulated in the resistant progeny early infection process. This may reflect an early attempt to halt pathogen development by increasing the lignin deposition at the infection site. At 8 weeks post-inoculation, they were highly up-regulated in the susceptible progeny coincided with whip development. This reveals a major role for these genes to whip development in the susceptible progeny and indicates that while PR10 is involved in the resistance mechanism of resistant progeny early infection process it also has a role in susceptibility. These results on genetically related progeny with different responses to smut infection reveal a complex interaction of genes and gene networks being induced in response to fungal invasion.

Sugarcane smut caused by the fungus Sporisorium scitamineum is one of the major diseases of sugarcane worldwide, causing significant losses in productivity and profitability of this perennial crop. Teliospores of this fungus are airborne, can travel long distances, and remain viable in hot and dry conditions for >6 months. The disease is easily recognized by its long whiplike sorus produced on the apex or side shoots of sugarcane stalks. Each sorus can release ≤100 million teliospores in a day; the spores are small (≤7.5 µ) and light and can survive in harsh environmental conditions. The airborne teliospores are the primary mode of smut spread around the world and across cane-growing regions. The most effective method of managing this disease is via resistant varieties. Because of the complex genomic makeup of sugarcane, selection for resistant traits is difficult in sugarcane breeding programs. In recent times, the application of molecular markers as a rapid tool of discarding susceptible genotypes early in the selection program has been investigated. Large effect resistance loci have been identified and have the potential to be used for marker-assisted selection to increase the frequency of resistant breeding lines in breeding programs. Recent developments in omics technologies (genomics, transcriptomics, proteomics, and metabolomics) have contributed to our understanding and provided insights into the mechanism of resistance and susceptibility. This knowledge will further our understanding of smut and its interactions with sugarcane genotypes and aid in the development of durable resistant varieties.

Mate-allocation in breeding programs can improve progeny performance by exploiting non-additive effects. Breeding decisions in the mate-allocation approach are based on predicted progeny merit rather than parental breeding value. This is particularly attractive when non-additive effects are significant, and the best-predicted progeny can be clonally propagated, for example sugarcane. We compared mate-allocation strategies that leverage non-additive and heterozygosity effects to maximise sugarcane clonal performance to schemes that use only additive effects to maximise breeding value. We used phenotypes and genotypes from a population of 2,909 clones phenotyped in Australia’s sugarcane breeding program’s final assessment trials for three traits: tonnes of cane per hectare (TCH), commercial cane sugar (CCS), and fibre. The clones from the last generation of this data set were used as parents to simulate families from all possible crosses (1,225), each with 50 progenies. The breeding and clonal values of progeny were predicted using GBLUP (considering only additive effects) and the e-GBLUP model (incorporating additive, non-additive and heterozygosity effects). Integer linear programming was used to identify the optimal mate-allocation among the selected parents. Compared to the breeding value, the predicted progeny value of allocated crossing pairs based on clonal performance (e-GBLUP) increased by 57%, 12%, and 16% for TCH, CCS, and fibre, respectively. In our study, the mate-allocation strategy exploiting non-additive and heterozygosity effects resulted in better clonal performance. However, there was a noticeable decline in additive gain, particularly for TCH, which might have been caused by the presence of large epistatic effects. When crosses were chosen based on clonal performance for TCH, progenies’ inbreeding coefficients were found significantly lower than for random mating, indicating that utilising non-additive and heterozygosity effects has advantages for controlling inbreeding depression. Therefore, mate-allocation is recommended in clonal crops to improve clonal performance and reduce inbreeding.

Key message Non-additive genetic effects seem to play a substantial role in the expression of complex traits in sugarcane. Including non-additive effects in genomic prediction models significantly improves the prediction accuracy of clonal performance. Abstract In the recent decade, genetic progress has been slow in sugarcane. One reason might be that non-additive genetic effects contribute substantially to complex traits. Dense marker information provides the opportunity to exploit non-additive effects in genomic prediction. In this study, a series of genomic best linear unbiased prediction (GBLUP) models that account for additive and non-additive effects were assessed to improve the accuracy of clonal prediction. The reproducible kernel Hilbert space model, which captures non-additive genetic effects, was also tested. The models were compared using 3,006 genotyped elite clones measured for cane per hectare (TCH), commercial cane sugar (CCS), and Fibre content. Three forward prediction scenarios were considered to investigate the robustness of genomic prediction. By using a pseudo-diploid parameterization, we found significant non-additive effects that accounted for almost two-thirds of the total genetic variance for TCH. Average heterozygosity also had a major impact on TCH, indicating that directional dominance may be an important source of phenotypic variation for this trait. The extended-GBLUP model improved the prediction accuracies by at least 17% for TCH, but no improvement was observed for CCS and Fibre. Our results imply that non-additive genetic variance is important for complex traits in sugarcane, although further work is required to better understand the variance component partitioning in a highly polyploid context. Genomics-based breeding will likely benefit from exploiting non-additive genetic effects, especially in designing crossing schemes. These findings can help to improve clonal prediction, enabling a more accurate identification of variety candidates for the sugarcane industry.

Simulations highlight the potential of genomic selection to substantially increase genetic gain for complex traits in sugarcane. The success rate depends on the trait genetic architecture and the implementation strategy. Genomic selection (GS) has the potential to increase the rate of genetic gain in sugarcane beyond the levels achieved by conventional phenotypic selection (PS). To assess different implementation strategies, we simulated two different GS-based breeding strategies and compared genetic gain and genetic variance over five breeding cycles to standard PS. GS scheme 1 followed similar routines like conventional PS but included three rapid recurrent genomic selection (RRGS) steps. GS scheme 2 also included three RRGS steps but did not include a progeny assessment stage and therefore differed more fundamentally from PS. Under an additive trait model, both simulated GS schemes achieved annual genetic gains of 2.6-2.7% which were 1.9 times higher compared to standard phenotypic selection (1.4%). For a complex non-additive trait model, the expected annual rates of genetic gain were lower for all breeding schemes; however, the rates for the GS schemes (1.5-1.6%) were still greater than PS (1.1%). Investigating cost-benefit ratios with regard to numbers of genotyped clones showed that substantial benefits could be achieved when only 1500 clones were genotyped per 10-year breeding cycle for the additive genetic model. Our results show that under a complex non-additive genetic model, the success rate of GS depends on the implementation strategy, the number of genotyped clones and the stage of the breeding program, likely reflecting how changes in QTL allele frequencies change additive genetic variance and therefore the efficiency of selection. These results are encouraging and motivate further work to facilitate the adoption of GS in sugarcane breeding.

Complex traits in sugarcane can be accurately predicted using genome-wide DNA markers. Genomic single-step prediction is an attractive method for genomic selection in commercial breeding programs. Sugarcane breeding programs have achieved up to 1% genetic gain in key traits such as tonnes of cane per hectare (TCH), commercial cane sugar (CCS) and Fibre content over the past decades. Here, we assess the potential of genomic selection to increase the rate of genetic gain for these traits by deriving genomic estimated breeding values (GEBVs) from a reference population of 3984 clones genotyped for 26 K SNP. We evaluated the three different genomic prediction approaches GBLUP, genomic single step (GenomicSS), and BayesR. GenomicSS combining pedigree and SNP information from historic and recent breeding programs achieved the most accurate predictions for most traits (0.3–0.44). This method is attractive for routine genetic evaluation because it requires relatively little modification to the existing evaluation and results in breeding value estimates for all individuals, not only those genotyped. Adding information from early-stage trials added up to 5% accuracy for CCS and Fibre, but 0% for TCH, reflecting the importance of competition effects for TCH. These GEBV accuracies are sufficiently high that, combined with the right breeding strategy, a doubling of the rate of genetic gain could be achieved. We also assessed the flowering traits days to flowering, gender and pollen viability and found high heritabilities of 0.57, 0.78 and 0.72, respectively. The GEBV accuracies indicated that genomic selection could be used to improve these traits. This could open new avenues for breeders to manage their breeding programs, for example, by synchronising flowering time and selecting males with high pollen viability.

Polyploidy may contribute to invasive ability as it can lead to high survival and fitness during establishment and enhance the processes of adaptation to novel environments by increasing genetic diversity in invading propagules. Many grasses are polyploid and many are aggressive invaders, making them persistent problems in disturbed environments worldwide. Today, vast areas of central Panama are dominated bySaccharum spontaneum, a perennial grass that originates from Asia. While widely regarded as invasive, it is not known when or how it arrived in Panama. We explore hypotheses regarding the timing and origins of this invasion through literature review and comparisons of genetic diversity in Panama with accessions from available sugarcane germplasm collections, highlighting historical accessions that were likely brought to Panama in 1939 as part of a USDA sugarcane germplasm collection. Samples were haplotyped at two chloroplast loci and genotyped using eight microsatellite markers. All sequenced individuals from Panama belong to a single chloroplast lineage which is common worldwide and was common in the Historic germplasm collection. Although genotypic diversity was extremely high in all samples due to high ploidy, samples from Panama had reduced diversity and clustered with several accessions in the Historic collection which had the same haplotype and high ploidy levels. Our results suggest that accidental escape from the historical sugarcane germplasm collection is the likely origin of theS. spontaneuminvasion in Panama. Intraspecific hybridization among several historical accessions and pre-adaptation to local conditions may have facilitated its rapid spread and persistence. We discuss the implications of our findings for biosecurity of germplasm collections.

Sugarcane is a C4 and agro-industry-based crop with a high potential for biomass production. It serves as raw material for the production of sugar, ethanol, and electricity. Modern sugarcane varieties are derived from the interspecific and intergeneric hybridization between Saccharum officinarum, Saccharum spontaneum, and other wild relatives. Sugarcane breeding programmes are broadly categorized into germplasm collection and characterization, pre-breeding and genetic base-broadening, and varietal development programmes. The varietal identification through the classic breeding programme requires a minimum of 12–14 years. The precise phenotyping in sugarcane is extremely tedious due to the high propensity of lodging and suckering owing to the influence of environmental factors and crop management practices. This kind of phenotyping requires data from both plant crop and ratoon experiments conducted over locations and seasons. In this review, we explored the feasibility of genomic selection schemes for various breeding programmes in sugarcane. The genetic diversity analysis using genome-wide markers helps in the formation of core set germplasm representing the total genomic diversity present in the Saccharum gene bank. The genome-wide association studies and genomic prediction in the Saccharum gene bank are helpful to identify the complete genomic resources for cane yield, commercial cane sugar, tolerances to biotic and abiotic stresses, and other agronomic traits. The implementation of genomic selection in pre-breeding, genetic base-broadening programmes assist in precise introgression of specific genes and recurrent selection schemes enhance the higher frequency of favorable alleles in the population with a considerable reduction in breeding cycles and population size. The integration of environmental covariates and genomic prediction in multi-environment trials assists in the prediction of varietal performance for different agro-climatic zones. This review also directed its focus on enhancing the genetic gain over time, cost, and resource allocation at various stages of breeding programmes.

The large complex genomes of many crops constrain the use of new technologies for genome-assisted selection and genetic improvement. One method to simplify a genome is to break it into individual chromosomes by flow cytometry, however, in many crop species most chromosomes cannot be isolated individually. Flow sorting of a single copy of a chromosome has been developed in wheat and here we demonstrate its use to identify markers of interest in an Erianthus/Sacchurum hybrid. Erianthus/Saccharum hybrids are of interest because Erianthus is known to be highly resistant to soil borne diseases which cause extensive sugarcane yield losses in Australia. Sugarcane (Saccharum) cultivars are autopolyploids with a highly complex genome and over 100 chromosomes. Flow cytometry for sugarcane, as in most crops, does not resolve individual chromosomes to a karyotype peak for sorting. To isolate a single chromosome, we used genomic in situ hybridisation (GISH) to identify the flow karyotype region containing the Erianthus chromosomes, flow sorted single chromosomes from this region, PCR screened for the Erianthus chromosomes and sequenced them. One Erianthus chromosome amplified and sequenced well, and from this data we could identify 57 resistant type genes and SNPs in nearly half of these genes. We developed KASP SNP assays and demonstrated that the identified SNP markers segregated as expected in a small introgression population. The pipeline we developed here to flow sort and sequence single chromosomes could be used in any crop with a large complex genome to rapidly discover and develop markers to important loci.

Simulations highlight the potential of genomic selection to substantially increase genetic gain for complex traits in sugarcane. The success rate depends on the trait genetic architecture and the implementation strategy. Genomic selection (GS) has the potential to increase the rate of genetic gain in sugarcane beyond the levels achieved by conventional phenotypic selection (PS). To assess different implementation strategies, we simulated two different GS-based breeding strategies and compared genetic gain and genetic variance over five breeding cycles to standard PS. GS scheme 1 followed similar routines like conventional PS but included three rapid recurrent genomic selection (RRGS) steps. GS scheme 2 also included three RRGS steps but did not include a progeny assessment stage and therefore differed more fundamentally from PS. Under an additive trait model, both simulated GS schemes achieved annual genetic gains of 2.6-2.7% which were 1.9 times higher compared to standard phenotypic selection (1.4%). For a complex non-additive trait model, the expected annual rates of genetic gain were lower for all breeding schemes; however, the rates for the GS schemes (1.5-1.6%) were still greater than PS (1.1%). Investigating cost-benefit ratios with regard to numbers of genotyped clones showed that substantial benefits could be achieved when only 1500 clones were genotyped per 10-year breeding cycle for the additive genetic model. Our results show that under a complex non-additive genetic model, the success rate of GS depends on the implementation strategy, the number of genotyped clones and the stage of the breeding program, likely reflecting how changes in QTL allele frequencies change additive genetic variance and therefore the efficiency of selection. These results are encouraging and motivate further work to facilitate the adoption of GS in sugarcane breeding.

Complex traits in sugarcane can be accurately predicted using genome-wide DNA markers. Genomic single-step prediction is an attractive method for genomic selection in commercial breeding programs. Sugarcane breeding programs have achieved up to 1% genetic gain in key traits such as tonnes of cane per hectare (TCH), commercial cane sugar (CCS) and Fibre content over the past decades. Here, we assess the potential of genomic selection to increase the rate of genetic gain for these traits by deriving genomic estimated breeding values (GEBVs) from a reference population of 3984 clones genotyped for 26 K SNP. We evaluated the three different genomic prediction approaches GBLUP, genomic single step (GenomicSS), and BayesR. GenomicSS combining pedigree and SNP information from historic and recent breeding programs achieved the most accurate predictions for most traits (0.3-0.44). This method is attractive for routine genetic evaluation because it requires relatively little modification to the existing evaluation and results in breeding value estimates for all individuals, not only those genotyped. Adding information from early-stage trials added up to 5% accuracy for CCS and Fibre, but 0% for TCH, reflecting the importance of competition effects for TCH. These GEBV accuracies are sufficiently high that, combined with the right breeding strategy, a doubling of the rate of genetic gain could be achieved. We also assessed the flowering traits days to flowering, gender and pollen viability and found high heritabilities of 0.57, 0.78 and 0.72, respectively. The GEBV accuracies indicated that genomic selection could be used to improve these traits. This could open new avenues for breeders to manage their breeding programs, for example, by synchronising flowering time and selecting males with high pollen viability.

Sugarcane is a major industrial crop cultivated in tropical and subtropical regions of the world. It is the primary source of sugar worldwide, accounting for more than 70% of world sugar consumption. Additionally, sugarcane is emerging as a source of sustainable bioenergy. However, the increase in productivity from sugarcane has been small compared to other major crops, and the rate of genetic gains from current breeding programs tends to be plateauing. In this review, some of the main contributors for the relatively slow rates of genetic gain are discussed, including (i) breeding cycle length and (ii) low narrow-sense heritability for major commercial traits, possibly reflecting strong non-additive genetic effects involved in quantitative trait expression. A general overview of genomic selection (GS), a modern breeding tool that has been very successfully applied in animal and plant breeding, is given. This review discusses key elements of GS and its potential to significantly increase the rate of genetic gain in sugarcane, mainly by (i) reducing the breeding cycle length, (ii) increasing the prediction accuracy for clonal performance, and (iii) increasing the accuracy of breeding values for parent selection. GS approaches that can accurately capture non-additive genetic effects and potentially improve the accuracy of genomic estimated breeding values are particularly promising for the adoption of GS in sugarcane breeding. Finally, different strategies for the efficient incorporation of GS in a practical sugarcane breeding context are presented. These proposed strategies hold the potential to substantially increase the rate of genetic gain in future sugarcane breeding.

Sugarcane (Saccharum spp.) is grown in tropical and subtropical regions of the world and produces 80% of the world’s sucrose primarily for human consumption but is increasingly used as a feedstock for biofuel. Commercial cultivars are hybrids between two species, Saccharum officinarum L. and S. spontaneum L. Saccharum spontaneum is the wild species with generally low sucrose levels and is used to introgress traits such as high yield, increased disease resistance, and ratooning. Molecular markers generated across the Saccharum genome were used to analyze diversity across 430 S. spontaneum accessions collected from 21 different countries and 255 cultivars from two breeding programs. A total of 724 markers were used for principle coordinate and cluster analysis, which revealed two main clusters that corresponded to accessions collected north or south of the Tropic of Cancer. The majority of the accessions clustered according to country of origin. Genetic similarity ranged from 0.25 to 0.54, with the highest diversity in accessions collected in Indonesia, followed by China, India, and Thailand, and the lowest in the Philippines. The diversity in the cultivar collection was lower than in the S. spontaneum accessions, as expected. Despite the low number of S. spontaneum clones used for breeding varieties, 51 to 80% of markers were captured in the breeding populations, although many at a low frequency. This is due to the high heterozygosity of sugarcane related to high ploidy and the introgression programs that have been performed in a number of sugarcane breeding programs. The genetic similarity was highest within the breeding program, and there remains large amounts of unexplored diversity in the wild germplasm. Diversity analysis could be used to guide the selection of wild S. spontaneum accessions, along with phenotypic data for introgression into breeding programs to increase genetic diversity.

High cane yield and commercially extractable sucrose (CCS) content are two of the key sugarcane commercial traits selected in sugarcane breeding programs. Advancements in genomic prediction may provide opportunities to speed up gains for these traits in breeding programs by combining accurate prediction of breeding values in candidate parent clones shortening generation intervals. Selection trials in commercial breeding programs may provide training populations for developing genomic predictions. In this study, three different populations of clones in early and advanced stage selection trials in an established commercial sugarcane breeding program were used to assess genomic prediction accuracy. The clones (genotypes) were evaluated for cane yield and sugar content in field trials and genotyped using a SNP array developed for sugarcane cultivars and parents. Five models (Bayes A, Bayes B, Bayesian LASSO, Bayesian GBLUP and RKHS) were tested using pedigree and/or marker data. Prediction models that included marker information had higher prediction accuracies than models with pedigree data only. For CCS, the prediction accuracies for genotypes in advanced stage trials using DNA markers were superior compared with prediction accuracies for early-stage trials, suggesting that prior intensive selection for CCS did not diminish accuracy of genomic prediction. However, by contrast, for cane yield, the prediction accuracies were much less for the population in the advanced stages of selection. The levels of prediction accuracy obtained in most datasets (0.25–0.45) are encouraging for developing applications of genomic prediction to predict breeding values of yield and sugar content in sugarcane breeding programs.

Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.

Fusarium pseudograminearum is an important pathogen of wheat and barley, particularly in semi-arid environments. Previous genome assemblies for this organism were based entirely on short read data and are highly fragmented. In this work, a genetic map of F. pseudograminearum has been constructed for the first time based on a mapping population of 178 individuals. The genetic map, together with long read scaffolding of a short read-based genome assembly, was used to give a near-complete assembly of the four F. pseudograminearum chromosomes. Large regions of synteny between F. pseudograminearum and F. graminearum, the related pathogen that is the primary causal agent of cereal head blight disease, were previously proposed in the core conserved genome, but the construction of a genetic map to order and orient contigs is critical to the validation of synteny and the placing of species-specific regions. Indeed, our comparative analyses of the genomes of these two related pathogens suggest that rearrangements in the F. pseudograminearum genome have occurred in the chromosome ends. One of these rearrangements includes the transposition of an entire gene cluster involved in the detoxification of the benzoxazolinone (BOA) class of plant phytoalexins. This work provides an important genomic and genetic resource for F. pseudograminearum, which is less well characterized than F. graminearum. In addition, this study provides new insights into a better understanding of the sexual reproduction process in F. pseudograminearum, which informs us of the potential of this pathogen to evolve.

Smut caused by biotrophic fungus Sporisorium scitamineum is a major disease of cultivated sugarcane that can cause considerable yield losses. It has been suggested in literature that there are at least two types of resistance mechanisms in sugarcane plants: an external resistance, due to chemical or physical barriers in the sugarcane bud, and an internal resistance governed by the interaction of plant and fungus within the plant tissue. Detailed molecular studies interrogating these two different resistance mechanisms in sugarcane are scarce. Here, we use light microscopy and global expression profiling with RNA-seq to investigate these mechanisms in sugarcane cultivar CP74-2005, a cultivar that possibly possesses both internal and external defence mechanisms. A total of 861 differentially expressed genes (DEGs) were identified in a comparison between infected and non-infected buds at 48 hours post-inoculation (hpi), with 457 (53%) genes successfully annotated using BLAST2GO software. This includes genes involved in the phenylpropanoid pathway, cell wall biosynthesis, plant hormone signal transduction and disease resistance genes. Finally, the expression of 13 DEGs with putative roles in S. scitamineum resistance were confirmed by quantitative real-time reverse transcription PCR (qRT-PCR) analysis, and the results were consistent with the RNA-seq data. These results highlight that the early sugarcane response to S. scitamineum infection is complex and many of the disease response genes are attenuated in sugarcane cultivar CP74-2005, while others, like genes involved in the phenylpropanoid pathway, are induced. This may point to the role of the different disease resistance mechanisms that operate in cultivars such as CP74-2005, whereby the early response is dominated by external mechanisms and then as the infection progresses, the internal mechanisms are switched on. Identification of genes underlying resistance in sugarcane will increase our knowledge of the sugarcane-S. scitamineum interaction and facilitate the introgression of new resistance genes into commercial sugarcane cultivars.

Sugarcane (Saccharum spp.) is a crop of substantial international significance for both food and fuel, however its highly polyploid nature challenges investigation of its genetic composition. Efforts to generate the full sugarcane genome sequence are underway, however in the meantime crop improvement efforts are somewhat limited by the lack of genome sequence resources available for physiological characterization. Low-coverage survey sequence data was generated and assembled for six sugarcane genotypes representing a range of significant S. spontaneum, S. officinarum, and S. hybrid cultivar accessions from around the world. These data were explored to investigate the composition of repetitive sequences and variations in chloroplast genome sequence, as well as assembled into a conglomerate monoploid genome sequence for polymorphism comparison between the genotypes. Almost half (47 %) of the inter-genomic polymorphisms analysed in these data represented poly-allelic variations which cannot be applied in traditional present/absent marker analysis, suggesting that new approaches are required to better understand and access genetic diversity within the Saccharum genus. These results support previous assertions that S. spontaneum is both less repetitive (62 % repetitive k-mers in Mandalay vs. 65 % in IJ76-514) and more highly polymorphic (17 % poly-alleles in Mandalay vs. 10 % poly-alleles in IJ76-514) than S. officinarum, with S. hybrids being intermediate between the two. However, contrary to previous analysis the monoploid genome size of S. spontaneum does not appear to differ significantly from that of S. officinarum as had been expected. This genomic survey assembly will be a very useful resource for sugarcane genomics in the absence of a monoploid or polyploid genome sequence, and will be made available upon request.

Sugarcane smut disease is caused by the biotrophic fungus Sporisorium scitamineum. The disease is characterized by the development of a whip-like structure from the primary meristems, where billions of teliospores are produced. Sugarcane smut also causes tillering and low sucrose and high fiber contents, reducing cane productivity. We investigated the biological events contributing to disease symptoms in a smut intermediate-resistant sugarcane genotype by examining the transcriptional profiles (RNAseq) shortly after inoculating the plants and immediately after whip emission. The overall picture of disease progression suggests that premature transcriptional reprogramming of the shoot meristem functions continues until the emergence of the whip. The guidance of this altered pattern is potentially primarily related to auxin mobilization in addition to the involvement of other hormonal imbalances. The consequences associated with whip emission are the modulation of typical meristematic functions toward reproductive organ differentiation, requiring strong changes in carbon partitioning and energy production. These changes include the overexpression of genes coding for invertases and trehalose-6P synthase, as well as other enzymes from key metabolic pathways, such as from lignin biosynthesis. This is the first report describing changes in the transcriptional profiles following whip development, providing a hypothetical model and candidate genes to further study sugarcane smut disease progression.

Chromosome doubling in megaspores of sugarcane was first reported over 70 years ago and was seen primarily in interspecific crosses between Saccharum officinarum and Saccharum spontaneum. Since then, there has been robust debate on the mechanism(s) responsible for this doubling with hypotheses ranging from second division restitution (SDR), post-meiotic restitution to fusion of the two innermost cells of the megaspore tetrad. In the present study, we sought to answer this question using molecular marker data from a cross which demonstrated 2n + n chromosome transmission between an S. officinarum clone (IJ76-514; 2n transmission) and a commercial hybrid (Q165 ; n transmission). Molecular marker data from four other bi-parental crosses were used to form a “base-line” for expected segregation ratios (n + n transmission). Comparison of segregation ratios showed that 2n transmission from the S. officinarum clone could be identified by the increased segregation ratios, a characteristic of both SDR and tetrad cell fusion. Simulation was then undertaken based on current understandings of sugarcane meiosis, to determine if the observed segregation ratios matched those simulated for SDR/tetrad cell fusion. Analysis of the simulation confirmed that most and perhaps all progeny appear to have been formed from a mechanism akin to SDR/tetrad cell fusion. The genetic consequences of this are discussed.

Erianthus arundinaceus, a wild relative of sugarcane has a number of traits including tolerance to environmental stresses desired by sugarcane breeders. The value of genetic diversity studies in providing useful information for introgression breeding and germplasm conservation is well known. A previous study reported little genetic diversity in a number of Indonesian E. arundinaceus while Indian E . arundinaceus was more diverse. A small number of Chinese E. arundinaceus was observed as being relatively diverse. However, an assessment of the genetic diversity of E. arundinaceus from the major geographical areas in the world has not been reported before. In this study, 207 E. arundinaceus accessions from China, Indonesia, India, New Guinea, Philippines and Vietnam were sampled for genetic diversity analysis using AFLP markers. Three AFLP primer combinations generated 286 markers in total, with the percentage of polymorphism observed being 99.3%. A dendrogram and PCA diagram were constructed based on the matrix of similarity analyzed with UPGMA method by NTSYS software. The results showed that: (1) a high level of diversity found within the Chinese collection compared with the Indonesian collection and (2) there was a general division amongst clones from China based on those collected from eastern versus western parts of the country, and clones from Yunnan province (western China) were the most diverse.

Diversity Arrays Technology (DArT) provides whole genome profiling for hundreds to thousands of polymorphic markers in a single assay using a high-throughput microarray platform. The presented work aimed to establish DArT genotyping for the genetically challenging genome of sugarcane. Due to the genome complexity of this sugar-producing crop of high economic importance, an application of DArT genotyping to this species required extensive testing and optimization. As the method of genome complexity reduction determines the efficiency of polymorphism identification in DArT, various approaches and several methods were tested, in order to establish the most optimal. The sugarcane DArT markers generated with these established methods identified high genetic differentiation of sugarcane ancestral species from modern cultivars, in agreement with the data available for other types of molecular markers for this crop. The majority of sugarcane DArT markers segregated in a Mendelian fashion and were readily incorporated into the framework genetic map. As the DArT markers are sequence-ready genomic clones, we sequenced 384 clones and found that one-third of sequenced markers came from the transcribed portion of the sugarcane genome. The presented results further validate the potential of DArT technology in providing cost-effective genetic profiles for plants, irrespective of their genome complexity, for effective applications in molecular-assisted breeding, diversity analysis or genetic identity testing.

The application of DNA markers linked to traits of commercial value in sugarcane may increase the efficiency of sugarcane breeding. The majority of markers generated for quantitative trait locus mapping in sugarcane have been single sequence repeats or AFLPs (amplified fragment length polymorphisms). Since AFLP markers are not adapted for large-scale implementation in plant breeding, our objective was to assess the feasibility of converting AFLP markers to fast, cheap and reliable PCR-based assays in a complex polyploid, sugarcane. Three AFLP markers were selected on the basis of an association to resistance to the fungal pathogen Ustilago scitaminea, the causal agent of smut in sugarcane. We developed an approach which enabled the identification of polymorphisms in these AFLP markers. Towards this goal, we employed GenomeWalking and 454 sequencing to isolate sequences adjacent to the linked AFLP markers and identify SNP (single nucleotide polymorphisms) haplotypes present in the homo(eo)logous chromosomes of sugarcane. One AFLP marker was converted to a cleavage amplified polymorphic sequence marker, another to a SCAR (sequence characteristered amplified region) marker and the final AFLP marker to a SNP PCR-based assay. However, validation of each of the markers in 240 genotypes resulted in 99, 90 and 60% correspondence with the original AFLP marker. These experiments indicate that even in a complex polyploid such as sugarcane, polymorphisms identified by AFLP can be converted to high-throughput marker systems, but due to the complexity this would only be carried out for high-value markers. In some cases, the polymorphisms identified are not transferable to more sequence-specific PCR applications.

In polyploids, the copy number (or marker dosage) of a dominant marker is the number of copies of a dominant allele carried by a parent. It is estimated using a hypothesis testing procedure. This procedure, however, suffers from issues associated with multiple testing and assumes all chromosomes pair uniformly. In this paper, a new Bayesian approach is presented for estimating the copy number of a dominant marker in polyploids. By using a probability model that explicitly accounts for preferentially paired chromosomes, the Bayesian approach more closely reflects reality. Its superiority over the hypothesis testing procedure is evidenced through the analysis of simulated and sugarcane data. The Bayesian methodology described in this paper is implemented in a C program, bdose, which is freely available (see Supplementary Materials).

Sugarcane is a major crop of tropical and sub-tropical climates. Modern cultivars are hybrids of several Saccharum species. Many attempts have been made to make intergeneric crosses to increase the diversity of germplasm available for breeding. Currently methods of incorporating new traits through genetic manipulation are also being researched. A review of the attempts by breeders to make hybridisations with sugarcane has been performed to determine the likelihood of spontaneous transfer of a transgene to a range of other species. When combined with the list of sexually compatible species growing outside of cultivation in Australia, Saccharum spontaneum L. was considered to be the most likely to spontaneously hybridise with commercial sugarcane. Using a combination of local knowledge and herbarium samples, S. spontaneum has been documented at five previously unpublished locations in Australia. Analysis of the DNA of plants growing at each of these locations, by molecular markers, showed vegetative propagation at three sites, predominantly vegetative propagation at a fourth and the presence of a more diverse set of plants at the fifth. At the latter location there may have been multiple introductions, interbreeding between clones or both. An analysis of both pollen and seed viability from these plants would discriminate between these options and determine whether S. spontaneum is interbreeding with commercial sugarcane.

International audience; As sugarcane is a complex polyaneuploid with many chromosomes, large numbers of markers are required to generate genetic maps with reasonable levels of genome coverage. Comparative mapping was investigated as an approach for both quantitative trait loci (QTL) validation and genetic map enhancement in sugarcane. More than 1000 SSR and AFLP markers were scored in a bi-parental Australian sugarcane population (Q3) that was segregating widely for sugar content-related traits. Two maps were constructed, one for each parent. The Q117 (female) and MQ77-340 (male) maps each contained almost 400 markers distributed onto approximately 100 linkage groups (LGs), of which nearly half could be assigned to homology groups (HGs) on the basis of SSRs. Then, using common SSR and AFLP markers, the two Q3 parental maps were aligned with the maps of the French cultivar, R570, and of the Australian cultivar, Q165A (A denotes variety covered by Australian plant breeding rights). As a result of comparative mapping, all ten HGs in the Q117 map, and all eleven HGs in the MQ77-340 map could be re-assigned to seven of the expected eight sugarcane HGs, revealing that one sugarcane HG was not covered at all in either Q3 parental map, and that other HGs were poorly represented. QTL analysis in the Q3 population identified approximately 75 marker-trait associations (MTAs) from approximately 18 chromosomal regions or putative QTL in each map for three sugar content-related traits. QTL location appeared to be consistent between the 4 maps; two of the eight HGs were observed to contain MTAs for brix in two or three maps, strongly suggesting the location of sugar content-related trait loci in these HGs. © 2007 Springer Science+Business Media B.V.

Saccharum officinarum L. is an octoploid with 80 chromosomes and a basic chromosome number of x = 10. It has high stem sucrose and contributes 80% of the chromosomes to the interspecific sugarcane cultivars that are grown commercially for sucrose. A genetic linkage map was developed for S. officinarum (clone IJ76-514) using a segregating population generated from a cross between Q165 (a commercial sugarcane cultivar) and IJ76-514. In total, 40 AFLP and 72 SSR primer pairs were screened across the population, revealing 595 polymorphic bands inherited from IJ76-514. These 595 markers displayed a frequency distribution different from all other sugarcane genetic maps produced, with only 40% being simplex markers (segregated 1:1). Of these 240 simplex markers, 178 were distributed on 47 linkage groups (LGs) and 62 remained unlinked. With the addition of 234 duplex markers and 80 biparental simplex markers (segregating 3:1), 534 markers formed 123 LGs. Using the multi-allelic SSR markers, repulsion phase linkage, and alignment with the Q165 linkage map, 105 of the 123 LGs could be grouped into 10 homology groups (HGs). These 10 HGs were further assigned to the 8 HGs observed in cultivated sugarcane and S. spontaneum . Analysis of repulsion phase linkage indicated that IJ76-514 is neither a complete autopolyploid nor an allopolyploid. Detection of 28 repulsion linkages that occurred between 6 pairs of LGs located in 4 HGs suggested the occurrence of limited preferential chromosome pairing in this species.

Anthracnose, caused by Colletotrichum trifolii, is one of the most serious diseases of lucerne worldwide. The disease is managed through deployment of resistant cultivars, but new pathotypes present a challenge to the successful implementation of this strategy. This paper reports the genetic map locations of quantitative trait loci (QTL) for reaction to races 1, 2 and 4 of C. trifolii in a single autotetraploid lucerne clone, designated W126 from the Australian cv. Trifecta. Resistance was mapped in a backcross population of 145 individuals, and reaction was assessed both by spray and injection inoculation of stems. Resistance to injection inoculation with races 1 and 4 was incompletely dominant and closely linked (phenotypic markers 2.2 cM apart); these resistances mapped to a linkage group homologous to Medicago truncatula linkage group 8. When the spray inoculation data were subjected to QTL analysis, the strongest QTL for resistance was located on linkage group 8; six QTL were identified for race 1 and four for race 4. Resistance to race 2 was incompletely recessive; four QTL were identified and these include one QTL on linkage group 4 that was also identified for race 1. Modelling of the interactions between individual QTL and marker effects allowed a total of 52-63% of the phenotypic variation to be described for each of the different races. These markers will have value in breeding lucerne, carrying multiple sources of resistance to the three known races of C. trifolii.

Erianthus rockii, a wild relative of sugarcane, is drought and cold tolerant, and both are potentially important agronomic traits to the sugarcane industry worldwide. As such it is of interest as a source of parental germplasm to sugarcane breeders and is currently being used in sugarcane introgression programs in both China and Australia. To date morphological characters have been used to verify the putative hybrids produced. Two crosses were generated between two different Saccharum species and two E. rockii accessions. Over 400 AFLP markers were used to identify the intergeneric hybrid progeny as well as determine hybrid diversity. Both crosses generated hybrids but efficiency levels were very different and are probably related to the different Saccharum parent used in each cross. Cross 1 was between a Saccharum officinarum and E. rockii and generated 100% hybrid progeny. Cross 2, however, was between a sugarcane hybrid (S. officinarum × Saccharum spontaneum) and E. rockii and only 10% of the progeny were intergeneric hybrids. Inheritance of markers in the progeny was analysed and for both crosses there were equal numbers of markers from both parents indicating n + n transmission of chromosomes. This is the first verification of E. rockii hybrids with molecular markers. It may now be possible to exploit genes of value from E. rockii in sugarcane breeding programs.

Little is known about the extent of allelic diversity of genes in the complex polyploid, sugarcane. Using sucrose phosphate synthase (SPS) Gene (SPS) Family III as an example, we have amplified and sequenced a 400 nt region from this gene from two sugarcane lines that are parents of a mapping population. Ten single nucleotide polymorphisms (SNPs) were identified within the 400 nt region of which seven were present in both lines. In the elite commercial cultivar Q165(A), 10 sequence haplotypes were identified, with four haplotypes recovered at 9% or greater frequency. Based on SNP presence, two clusters of haplotypes were observed. In IJ76-514, a Saccharum officinarum accession, 8 haplotypes were identified with 4 haplotypes recovered at 13% or greater frequency. Again, two clusters of haplotypes were observed. The results suggest that there may be two SPS Gene Family III genes per genome in sugarcane, each with different numbers of different alleles. This suggestion is supported by sequencing results in an elite parental sorghum line, 403463-2-1, in which 4 haplotypes, corresponding to two broad types, were also identified. Primers were designed to the sugarcane SNPs and screened over bulked DNA from high and low Sucrose-containing progeny from a cross between Q165(A) and IJ76-514. The SNP frequency did not vary in the two bulked DNA samples, suggesting that these SNPs from this SPS gene family are not associated with variation in sucrose content. Using an ecotilling approach, two of the SPS Gene Family III haplotypes were mapped to two different linkage groups in homology group 1 in Q165(A). Both haplotypes mapped near QTLs for increased sucrose content but were not themselves associated with any sugar-related trait.

The identification of markers linked to quantitative trait loci (QTLs) for increased sugar accumulation could improve the effectiveness of current breeding strategies in sugarcane. Progeny from a cross between a high sucrose producing cultivar, Open image in new window (denotes Australian plant breeding rights), and a Saccharum officinarum clone, IJ76-514 were grown in two field experiments in different years, and evaluated in the early and mid-season phases of crop maturity, to identify robust QTLs in Open image in new window affecting sucrose content in cane. Using an extensive genetic map constructed for Open image in new window with over 1,000 AFLP and SSR markers, a total of 37 QTLs were identified for brix and pol of which, 16 were detected in both experiments. Of these 37 QTL, 30 were clustered into 12 genomic regions in six of the eight homo(eo)logous groups. Each QTL explained from 3 to 9% of the phenotypic variation observed. Both positive and negative effects were identified and the location of the QTLs on linkage groups belonging to the same homo(eo)logy group suggested that a number of the QTLs were allelic forms of the same genes. Of the 37 QTLs identified, the majority were significant in both early and mature cane, but 8 were identified as early specific QTLs and 9 as mature cane QTLs. In total, 97 interactions were significant (P

Erianthus rockii is a drought and cold tolerant wild relative of sugar cane from China that is currently being used in sugar cane introgression programs. It has not been described outside of China. Although classified as an Erianthus species using morphological characters, its relationship with other Erianthus species and other species in the Saccharum complex is not well understood. Genotypes representing seventeen species from five genera in the Saccharum complex were evaluated with approximately 200 microsatellite (SSR) and amplified fragment length polymorphism (AFLP) markers and similarity matrices constructed. Principal component analysis (PCA) was undertaken separately with the AFLP and SSR data. The results from both data sets were very similar and suggested that E. rockii was distinct from other Erianthus and Saccharum species. E. rockii clustered with E. fulvus and two Miscanthus species, mid-way between the major Saccharum and Erianthus clusters.

Erianthus arundinaceus has a number of important agronomic traits including good ratooning ability, tolerance to both drought and water logging, disease resistance and vigour and is of interest as a potential source of parental germplasm to sugarcane breeders. However, to date, attempts to produce fertile hybrids between sugarcane (Saccharum spp.) and E. arundinaceus have been unsuccessful. Microsatellite markers that generated genus-specific markers and were highly polymorphic within sugarcane were identified. The microsatellite markers and 5S rDNA PCR were used to screen intergeneric (F 1 ) clones from Saccharum officinarum x E. arundinaceus crosses, and two Saccharum backcross progeny (BC 1 ) populations derived from crosses between selected F 1 clones and sugarcane (Saccharum spp.), to identify genuine Saccharum spp. BC 1 progeny. The 5S rDNA PCR marker and highly polymorphic microsatellites with Erianthus-specific bands confirmed the F 1 parent of the two putative BC 1 populations was a S. officinarum x E. arundinaceus hybrid and allowed the identification of the genuine BC 1 progeny from selfs of the F 1 parent. This is the first verification of BC 1 progeny from an F 1 intergeneric hybrid x sugarcane (Saccharum spp.) clone with molecular markers and confirms the introgression of E. arundinaceus germplasm into sugareane. It should now be possible to exploit genes of value from E. arundinaceus in sugarcane breeding programmes.

Commercial sugarcane, belonging to the genus Saccharum (Poaceae), is an important industrial crop accounting for nearly 70% of sugar produced worldwide. Compared to other major crops, efforts to improve sugarcane are limited and relatively recent, with the first introduction of interspecific hybrids about 80 yr ago. Progress in traditional breeding of sugarcane, a highly polyploid and frequently aneuploid plant, is impeded by its narrow gene pool, complex genome, poor fertility, and the long breeding/selection cycle. These constraints, however, make sugarcane a good candidate for molecular breeding. In the past decade considerable progress has been made in understanding and manipulating the sugarcane genome using various biotechnological and cell biological approaches. Notable among them are the creation of transgenic plants with improved agronomic or other important traits, advances in genomics and molecular markers, and progress in understanding the molecular aspects of sucrose transport and accumulation. More recently, substantial effort has been directed towards developing sugarcane as a biofactory for high-value products. While these achievements are commendable, a greater understanding of the sugarcane genome, and cell and whole plant physiology, will accelerate the implementation of commercially significant biotechnology outcomes. We anticipate that the rapid advancements in molecular biology and emerging biotechnology innovations would play a significant role in the future sugarcane crop improvement programs and offer many new opportunities to develop it as a new-generation industrial crop.

Author for correspondence e-mail: lynne.mcintyre@csiro.au; International audience; Mandalay is an important Saccharum spontaneum clone used historically in Australian sugarcane breeding programs, and has given rise to many valuable cultivars. In order to better understand the genetic contribution of Mandalay to Australian varieties and elite parental material, a combined pedigree and quantitative trait loci (QTL) mapping approach was undertaken. A genetic map containing 400 single-dose markers was constructed for the Australian sugarcane clone MQ77–340, one parent of an Australian sugarcane population (Q117 · MQ77–340), using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. This cultivar was selected because it is a direct descendent of Mandalay; its grandparents are Korpi, a S. o.cinarum clone, and Mandalay. The 400 markers were scattered onto 101 linkage groups (LGs) with an estimated map length of 3582 cM. The ancestral origin of all of the markers was determined with approximately 25% of the markers shown to originate from Mandalay, and a similar percentage from Korpi. Of the 101 LGs, 65 contained markers originating from Mandalay and/or Korpi. QTL analysis was undertaken using the map and 3 years of field data for three sugar-related traits (pol, brix, and CCS) and using single year .eld data for .bre, stalk weight and cane and sugar yield. Markers from both Mandalay and Korpi were found to be associated with both positive and negative effects on all of the traits analysed

Sucrose phosphate synthase (SPS) is a key enzyme in the production of sucrose. Five SPS gene families have been identified in monocotyledonous plants including sugarcane. Using SPS family-specific primers to four of the five families (we had previously characterised the fifth gene family), an approximately 400-nt region was amplified from the parents of a sugarcane mapping population, namely the cultivar Q165 and a S. officinarum line IJ76-514. Alignment of the sequences from both parents suggested from one to three genes per SPS gene family, with variable numbers of alleles per gene. Single-dose (SD) single-nucleotide polymorphisms (SNPs) were identified in at least one allele from each SPS gene family and mapped in Q165. For gene families SPS I–IV, SNPs from different alleles in each gene family mapped to different linkage groups within the same homology group (HG), suggesting a single gene per gene family, or multiple genes at a single locus. These map locations were syntenic with SPS gene family locations in sorghum. Two SNPs from different alleles in gene family SPS V were mapped to two different HGs, suggesting two genes in this family; one of the map locations was syntenic with the location of SPS V in sorghum. QTL analysis for sugar-related traits was undertaken with the SD and double-dose SNP markers. SNPs from SPS gene family IV were strongly associated with sugar-related traits, while SNPs from other gene families were associated with agronomic traits, such as stalk weight, diameter, and number. This study provides insight into the evolution of this important polyploid crop as well as highlights the importance of this gene family to sugar production in sugarcane.

Highlight qPCR-RBIP was used to examine the dosage of particular markers and evolutionary history in Saccharum and the related genera, Erianthus and Miscanthus. It also differentiated between S. spontaneum and S. officinarum., Sugarcane is the main source of the world’s sugar and is becoming increasingly important as a source of biofuel. The highly polyploid and heterozygous nature of the sugarcane genome has meant that characterization of the genome has lagged behind that of other important crops. Here we developed a method using a combination of quantitative PCR with a transposable marker system to score the relative number of alleles with a transposable element (TE) present at a particular locus. We screened two genera closely related to Saccharum (Miscanthus and Erianthus), wild Saccharum, traditional cultivars, and 127 modern cultivars from Brazilian and Australian breeding programmes. We showed how this method could be used in various ways. First, we showed that the method could be extended to be used as part of a genotyping system. Secondly, the history of insertion and timing of the three TEs examined supports our current understanding of the evolution of the Saccharum complex. Thirdly, all three TEs were found in only one of the two main lineages leading to the modern sugarcane cultivars and are therefore the first TEs identified that could potentially be used as markers for Saccharum spontaneum.

Background The understanding of sugarcane genetics has lagged behind that of other members of the Poaceae family such as wheat, rice, barley and sorghum mainly due to the complexity, size and polyploidization of the genome. We have used the genetic map of a sugarcane cultivar to generate a consensus genetic map to increase genome coverage for comparison to the sorghum genome. We have utilized the recently developed sugarcane DArT array to increase the marker density within the genetic map. The sequence of these DArT markers plus SNP and EST-SSR markers was then used to form a bridge to the sorghum genomic sequence by BLAST alignment to start to unravel the complex genomic architecture of sugarcane. Results Comparative mapping revealed that certain sugarcane chromosomes show greater levels of synteny to sorghum than others. On a macrosyntenic level a good collinearity was observed between sugarcane and sorghum for 4 of the 8 homology groups (HGs). These 4 HGs were syntenic to four sorghum chromosomes with from 98% to 100% of these chromosomes covered by these linked markers. Four major chromosome rearrangements were identified between the other four sugarcane HGs and sorghum, two of which were condensations of chromosomes reducing the basic chromosome number of sugarcane from x = 10 to x = 8. This macro level of synteny was transferred to other members within the Poaceae family such as maize to uncover the important evolutionary relationships that exist between sugarcane and these species. Conclusions Comparative mapping of sugarcane to the sorghum genome has revealed new information on the genome structure of sugarcane which will help guide identification of important genes for use in sugarcane breeding. Furthermore of the four major chromosome rearrangements identified in this study, three were common to maize providing some evidence that chromosome reduction from a common paleo-ancestor of both maize and sugarcane was driven by the same translocation events seen in both species.

Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 x Q165). The sequencing yielded 96,755 (IJ76-514) and 86,241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP--an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent--with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.

A major focus of sugarcane variety improvement programs is to increase sugar yield, which can be accomplished by either increasing the sugar content of the cane or by increasing cane yield, as the correlation between these traits is low. We used a cross between an Australian sugarcane variety Q165, and a Saccharum officinarum accession, IJ76-514, to dissect the inheritance of yield-related traits in the complex polyploid sugarcane. A population of 227 individuals was grown in a replicated field trial and evaluated over 3 years for stalk weight, stalk diameter, stalk number, stalk length and total biomass. Over 1,000 AFLP and SSR markers were scored across the population and used to identify quantitative trait loci (QTL). In total, 27 regions were found that were significant at the 5% threshold using permutation tests with at least one trait; individually, they explained from 4 to 10% of the phenotypic variation and up to 46% were consistent across years. With the inclusion of digeneic interactions, from 28 to 60% of the variation was explained for these traits. The 27 genomic regions were located on 22 linkage groups (LGs) in six of the eight homology groups (HGs) indicating that a number of alleles or quantitative trait alleles (QTA) at each QTL contribute to the trait; from one to three alleles had an effect on the traits for each QTL identified. Alleles of a candidate gene, TEOSINTE BRANCHED 1 (TB1), the major gene controlling branching in maize, were mapped in this population using either an SSR or SNP markers. Two alleles showed some association with stalk number, but unlike maize, TB1 is not a major gene controlling branching in sugarcane but only has a minor and variable effect.

Sporisorium scitamineum is a biotrophic fungus responsible for the sugarcane smut, a worldwide spread disease. This study provides the complete sequence of individual chromosomes of S. scitamineum from telomere to telomere achieved by a combination of PacBio long reads and Illumina short reads sequence data, as well as a draft sequence of a second fungal strain. Comparative analysis to previous available sequences of another strain detected few polymorphisms among the three genomes. The novel complete sequence described herein allowed us to identify and annotate extended subtelomeric regions, repetitive elements and the mitochondrial DNA sequence. The genome comprises 19,979,571 bases, 6,677 genes encoding proteins, 111 tRNAs and 3 assembled copies of rDNA, out of our estimated number of copies as 130. Chromosomal reorganizations were detected when comparing to sequences of S. reilianum, the closest smut relative, potentially influenced by repeats of transposable elements. Repetitive elements may have also directed the linkage of the two mating-type loci. The fungal transcriptome profiling from in vitro and from interaction with sugarcane at two time points (early infection and whip emergence) revealed that 13.5% of the genes were differentially expressed in planta and particular to each developmental stage. Among them are plant cell wall degrading enzymes, proteases, lipases, chitin modification and lignin degradation enzymes, sugar transporters and transcriptional factors. The fungus also modulates transcription of genes related to surviving against reactive oxygen species and other toxic metabolites produced by the plant. Previously described effectors in smut/plant interactions were detected but some new candidates are proposed. Ten genomic islands harboring some of the candidate genes unique to S. scitamineum were expressed only in planta. RNAseq data was also used to reassure gene predictions.

In eastern Australia and California, USA, one of the major lethal fungal diseases of lucerne (Medicago sativa) is Stagonospora root and crown rot, caused by Stagonospora meliloti. Quantitative trait loci (QTL) involved in resistance and susceptibility to S. meliloti were identified in an autotetraploid lucerne backcross population of 145 individuals. Using regression analysis and interval mapping, we detected one region each on linkage groups 2, 6 and 7 that were consistently associated with disease reaction to S. meliloti in two separate experiments. The largest QTL on linkage group 7, which is associated with resistance to S. meliloti, contributed up to 17% of the phenotypic variation. The QTL located on linkage group 2, which is potentially a resistance allele in repulsion to the markers for susceptibility to S. meliloti, contributed up to 8% of the phenotypic variation. The QTL located on linkage group 6, which is associated with susceptibility to S. meliloti, contributed up to 16% of the phenotypic variation. A further two unlinked markers contributed 5 and 8% of the phenotypic variation, and were detected in only one experiment. A total of 517 simple sequence repeat (SSR) markers from Medicago truncatula were screened on the parents of the mapping population. Only 27 (6%) SSR markers were polymorphic and could be incorporated into the autotetraploid map of M. sativa. This allowed alignment of our M. sativa linkage map with published M. truncatula maps. The markers linked to the QTL we have reported will be useful for marker assisted selection for partial resistance to S. meliloti in lucerne.

Association between markers and sugarcane diseases were investigated in a collection of 154 sugarcane clones, consisting of important ancestors or parents, and cultivars. 1,068 polymorphic AFLP and 141 SRR markers were scored across all clones. Data on the four most important diseases in the Australian sugarcane industry were obtained; these diseases being pachymetra root rot (Pachymetra chaunorhiza B.J. Croft & M.W. Dick), leaf scald (Xanthomonas albilineans Dowson), Fiji leaf gall (Fiji disease virus), and smut (Ustilago scitaminea H. & P. Sydow). By a simple regression analysis, association between markers and diseases could be readily detected. However, many of these associations were due to the effects of embedded population structure and random effects. After taking population structure into account, we found that 59% of the phenotypic variation in smut resistance ratings could be accounted for by 11 markers, 32% of variation for leaf scald and pachymetra root rot rating by 4 markers, and 26% of Fiji leaf gall by 5 markers. The results suggest that marker-trait associations can be readily detected in populations generated from modern sugarcane breeding programs. This may be due to special features of past sugarcane breeding programs leading to persistent linkage disequilibrium in modern parental populations.

Sugarcane varieties are complex polyploids carrying in excess of 100 chromosomes and are derived from interspecific hybridisation between the domesticated Saccharum officinarum and the wild relative S. spontaneum. A map was constructed in Denotes variety covered by Australian plant breeding rights., an Australian cultivar, from a segregating F1 population, using 40 amplified fragment length polymorphism (AFLP) primer combinations, five randomly amplified DNA fingerprints (RAF) primers and 72 simple sequence repeat (SSR) primers. Using these PCR-based marker systems, we generated 1,365 polymorphic markers, of which 967 (71%) were single-dose (SD) markers. Of these SD 967 markers, 910 were distributed on 116 linkage groups (LGs) with a total map length of 9,058.3 cM. Genome organisation was significantly greater than observed in previously reported maps for Saccharum spp. With the addition of 123 double-dose markers, 36 (3:1) segregating markers and a further five SD markers, 1,074 markers were mapped onto 136 LGs. Repulsion phase linkage detected preferential pairing for 40 LGs, which formed 11 LG pairs and three multi-chromosome pairing groups. Using SSRs, double-dose markers and repulsion phase linkage, we succeeded in forming 127 of the 136 LGs into eight homo(eo)logy groups (HG). Two HGs were each represented by two sets of LGs. These sets of LGs potentially correspond to S. officinarum chromosomes, with each set aligning to either end of one or two larger LGs. The larger chromosomes in the two HGs potentially correspond to S. spontaneum chromosomes. This suggestion is consistent with the different basic chromosome number of the two species that are hybridised to form sugarcane cultivars, S. spontaneum (x=8) and S. officinarum (x=10), and illustrates the structural relationship between the genomes of these two species. The discrepancy of coverage between HGs highlights the difficulty in mapping large parts of the genome.

Previous studies into the population structure of Melaleuca alternifolia by both isozyme and microsatellite analysis revealed little evidence for genetic structuring within genetic provenances. In contrast, analysis of the oil composition within these same regions showed distinct clustering of chemotypes within the provenances suggesting either that chemotype was not under genetic control, or that there is strong environmental selection for plant chemotypes. To investigate the level of genetic control of monoterpene composition in the essential oil of M. alternifolia, individuals representing the three extreme chemotypes of high terpinen-4-ol, high 1,8-cineole and high terpinolene were crossed with an individual with the commercially desirable high terpinen-4-ol oil profile. The progeny resulting from these crosses displayed oil profiles that were intermediate to that of the parent. Further analysis of the survey of oil chemotypes within the natural population also suggests that these intermediate chemotypes may arise naturally between regions containing high proportions of the extreme chemotypes. These results imply that there is a level of genetic structure for chemotype determination within the genetic provenance that is undetected by isozyme and microsatellite analysis. This information could play a vital role in the selection of appropriate genetic material to be used in future essential oil selection and breeding programs.

Background Sugarcane genetic mapping has lagged behind other crops due to its complex autopolyploid genome structure. Modern sugarcane cultivars have from 110-120 chromosomes and are in general interspecific hybrids between two species with different basic chromosome numbers: Saccharum officinarum (2n = 80) with a basic chromosome number of 10 and S. spontaneum (2n = 40-128) with a basic chromosome number of 8. The first maps that were constructed utilised the single dose (SD) markers generated using RFLP, more recent maps generated using AFLP and SSRs provided at most 60% genome coverage. Diversity Array Technology (DArT) markers are high throughput allowing greater numbers of markers to be generated. Results Progeny from a cross between a sugarcane variety Q165 and a S. officinarum accession IJ76-514 were used to generate 2467 SD markers. A genetic map of Q165 was generated containing 2267 markers, These markers formed 160 linkage groups (LGs) of which 147 could be placed using allelic information into the eight basic homology groups (HGs) of sugarcane. The HGs contained from 13 to 23 LGs and from 204 to 475 markers with a total map length of 9774.4 cM and an average density of one marker every 4.3 cM. Each homology group contained on average 280 markers of which 43% were DArT markers 31% AFLP, 16% SSRs and 6% SNP markers. The multi-allelic SSR and SNP markers were used to place the LGs into HGs. Conclusions The DArT array has allowed us to generate and map a larger number of markers than ever before and consequently to map a larger portion of the sugarcane genome. This larger number of markers has enabled 92% of the LGs to be placed into the 8 HGs that represent the basic chromosome number of the ancestral species, S. spontaneum. There were two HGs (HG2 and 8) that contained larger numbers of LGs verifying the alignment of two sets of S. officinarum chromosomes with one set of S. spontaneum chromosomes and explaining the difference in basic chromosome number between the two ancestral species. There was also evidence of more complex structural differences between the two ancestral species. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-152) contains supplementary material, which is available to authorized users.