The sugarcane mitochondrial genome: assembly, phylogenetics and transcriptomics.

BACKGROUND: Chloroplast genomes provide insufficient phylogenetic information to distinguish between closely related sugarcane cultivars, due to the recent origin of many cultivars and the conserved sequence of the chloroplast. In comparison, the mitochondrial genome of plants is much larger and more plastic and could contain increased phylogenetic signals. We assembled a consensus reference mitochondrion with Illumina TruSeq synthetic long reads and Oxford Nanopore Technologies MinION long reads. Based on this assembly we also analyzed the mitochondrial transcriptomes of sugarcane and sorghum and improved the annotation of the sugarcane mitochondrion as compared with other species. METHODS: Mitochondrial genomes were assembled from genomic read pools using a bait and assemble methodology. The mitogenome was exhaustively annotated using BLAST and transcript datasets were mapped with HISAT2 prior to analysis with the Integrated Genome Viewer. RESULTS: The sugarcane mitochondrion is comprised of two independent chromosomes, for which there is no evidence of recombination. Based on the reference assembly from the sugarcane cultivar SP80-3280 the mitogenomes of four additional cultivars (R570, LCP85-384, RB72343 and SP70-1143) were assembled (with the SP70-1143 assembly utilizing both genomic and transcriptomic data). We demonstrate that the sugarcane plastome is completely transcribed and we assembled the chloroplast genome of SP80-3280 using transcriptomic data only. Phylogenomic analysis using mitogenomes allow closely related sugarcane cultivars to be distinguished and supports the discrimination between Saccharum officinarum and Saccharum cultum as modern sugarcane's female parent. From whole chloroplast comparisons, we demonstrate that modern sugarcane arose from a limited number of Saccharum cultum female founders. Transcriptomic and spliceosomal analyses reveal that the two chromosomes of the sugarcane mitochondrion are combined at the transcript level and that splice sites occur more frequently within gene coding regions than without. We reveal one confirmed and one potential cytoplasmic male sterility (CMS) factor in the sugarcane mitochondrion, both of which are transcribed. CONCLUSION: Transcript processing in the sugarcane mitochondrion is highly complex with diverse splice events, the majority of which span the two chromosomes. PolyA baited transcripts are consistent with the use of polyadenylation for transcript degradation. For the first time we annotate two CMS factors within the sugarcane mitochondrion and demonstrate that sugarcane possesses all the molecular machinery required for CMS and rescue. A mechanism of cross-chromosomal splicing based on guide RNAs is proposed. We also demonstrate that mitogenomes can be used to perform phylogenomic studies on sugarcane cultivars.

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.

Agronomic evaluation and molecular characterisation of the acetolactate synthase gene in imazapyr tolerant sugarcane (Saccharum hybrid) genotypes.

KEY MESSAGE: Mutagenesis had no effect on number of stalks/plot, stalk height, fibre and sucrose content of mutants. Imazapyr tolerance is likely due to a S622N mutation in the acetolactate synthase gene. The herbicidal compound imazapyr is effective against weeds such as Cynodon and Rottboellia species that constrain sugarcane production. This study aimed to compare agronomic characteristics of three imazapyr tolerant mutants (Mut 1, Mut 6 and Mut 7) with the non-mutated N12 control after 18 months of growth, and to sequence the acetolactate synthase (ALS) gene to identify any point mutations conferring imazapyr tolerance. There were no significant differences in the number of stalks/plot, stalk height, fibre and sucrose contents of the mutants compared with the N12 control. However, Mut 1 genotype was more susceptible to the Lepidopteran stalk borer, Eldana saccharina when compared with the non-mutated N12 (11.14 ± 1.37 and 3.89 ± 0.52% internodes bored, respectively), making Mut 1 less desirable for commercial cultivation. Molecular characterisation of the ALS gene revealed non-synonymous mutations in Mut 6. An A to G change at nucleotide position 1857 resulted in a N513D mutation, while a G to A change at nucleotide position 2184 imposed a S622N mutation. Molecular dynamics simulations revealed that the S622N mutation renders an asparagine side chain clash with imazapyr, hence this mutation is effective in conferring imazapyr tolerance.

Assessing the Likelihood of Gene Flow From Sugarcane (Saccharum Hybrids) to Wild Relatives in South Africa.

Pre-commercialization studies on environmental biosafety of genetically modified (GM) crops are necessary to evaluate the potential for sexual hybridization with related plant species that occur in the release area. The aim of the study was a preliminary assessment of factors that may contribute to gene flow from sugarcane (Saccharum hybrids) to indigenous relatives in the sugarcane production regions of Mpumalanga and KwaZulu-Natal provinces, South Africa. In the first instance, an assessment of Saccharum wild relatives was conducted based on existing phylogenies and literature surveys. The prevalence, spatial overlap, proximity, distribution potential, and flowering times of wild relatives in sugarcane production regions based on the above, and on herbaria records and field surveys were conducted for Imperata, Sorghum, Cleistachne, and Miscanthidium species. Eleven species were selected for spatial analyses based on their presence within the sugarcane cultivation region: four species in the Saccharinae and seven in the Sorghinae. Secondly, fragments of the nuclear internal transcribed spacer (ITS) regions of the 5.8s ribosomal gene and two chloroplast genes, ribulose-bisphosphate carboxylase (rbcL), and maturase K (matK) were sequenced or assembled from short read data to confirm relatedness between Saccharum hybrids and its wild relatives. Phylogenetic analyses of the ITS cassette showed that the closest wild relative species to commercial sugarcane were Miscanthidium capense, Miscanthidium junceum, and Narenga porphyrocoma. Sorghum was found to be more distantly related to Saccharum than previously described. Based on the phylogeny described in our study, the only species to highlight in terms of evolutionary divergence times from Saccharum are those within the genus Miscanthidium, most especially M. capense, and M. junceum which are only 3 million years divergent from Saccharum. Field assessment of pollen viability of 13 commercial sugarcane cultivars using two stains, iodine potassium iodide (IKI) and triphenyl tetrazolium chloride, showed decreasing pollen viability (from 85 to 0%) from the north to the south eastern regions of the study area. Future work will include other aspects influencing gene flow such as cytological compatibility and introgression between sugarcane and Miscanthidium species.

Herbicide targets and detoxification proteins in sugarcane: from gene assembly to structure modelling.

In a genome context, sugarcane is a classic orphan crop, in that no genome and only very few genes have been assembled. We have devised a novel exome assembly methodology that has allowed us to assemble and characterize 49 genes that serve as herbicide targets, safener interacting proteins, and members of herbicide detoxification pathways within the sugarcane genome. We have structurally modelled the products of each of these genes, as well as determining allelic, genomic, and RNA-Seq based polymorphisms for each gene. This study provides the largest collection of sugarcane structures modelled to date. We demonstrate that sugarcane genes are highly polymorphic, revealing that each genotype is evolving both uniquely and independently. In addition, we present an exome assembly system for orphan crops that can be executed on commodity infrastructure, making exome assembly practical for any group. In terms of knowledge about herbicide modes of action and detoxification, we have advanced sugarcane from a crop where no information about any herbicide-associated gene was available to the situation where sugarcane is now a species with the single largest collection of known and annotated herbicide-associated genes.

Elucidating modes of activation and herbicide resistance by sequence assembly and molecular modelling of the Acetolactate synthase complex in sugarcane.

Acetolactate synthase (ALS) catalyzes the first portion of the biosynthetic pathway leading to the generation of branched-chain amino acids. As such it is essential for plant health and is a major target for herbicides. ALS is a very poorly characterized molecule in sugarcane. The enzyme is activated and inhibited by a regulatory subunit (known as VAT1 in plants) whose mode of action is entirely unknown. Using Saccharum halepense as a template we have assembled the ALS gene of sugarcane (Saccharum hybrid) and have modelled the structure of ALS based on an Arabidopsis template (the first ALS model for a monocot). We have also assembled the ALS regulatory proteins (VAT1 and VAT2) from sugarcane and show that VAT2 is specific to true grasses. Employing a bacterial model, we have generated a structural model for VAT1, which explains why the separate domains of the proteins bind to either leucine or valine but not both. Using co-evolution studies we have determined molecular contacts by which we modelled the docking of VAT1 to ALS. In conclusion, we demonstrate how the binding of VAT1 to ALS activates ALS and show how VAT1 can also confer feedback inhibition to ALS. We validate our ALS model against biochemical data and employ this model to explain the function of a novel herbicide binding mutant in sugarcane.