Transcript profiles of wild and domesticated sorghum under water-stressed conditions and the differential impact on dhurrin metabolism.

MAIN CONCLUSION: Australian native species of sorghum contain negligible amounts of dhurrin in their leaves and the cyanogenesis process is regulated differently under water-stress in comparison to domesticated sorghum species. Cyanogenesis in forage sorghum is a major concern in agriculture as the leaves of domesticated sorghum are potentially toxic to livestock, especially at times of drought which induces increased production of the cyanogenic glucoside dhurrin. The wild sorghum species endemic to Australia have a negligible content of dhurrin in the above ground tissues and thus represent a potential resource for key agricultural traits like low toxicity. In this study we investigated the differential expression of cyanogenesis related genes in the leaf tissue of the domesticated species Sorghum bicolor and the Australian native wild species Sorghum macrospermum grown in glasshouse-controlled water-stress conditions using RNA-Seq analysis to analyse gene expression. The study identified genes, including those in the cyanogenesis pathway, that were differentially regulated in response to water-stress in domesticated and wild sorghum. In the domesticated sorghum, dhurrin content was significantly higher compared to that in the wild sorghum and increased with stress and decreased with age whereas in wild sorghum the dhurrin content remained negligible. The key genes in dhurrin biosynthesis, CYP79A1, CYP71E1 and UGT85B1, were shown to be highly expressed in S. bicolor. DHR and HNL encoding the dhurrinase and α-hydroxynitrilase catalysing bio-activation of dhurrin were also highly expressed in S. bicolor. Analysis of the differences in expression of cyanogenesis related genes between domesticated and wild sorghum species may allow the use of these genetic resources to produce more acyanogenic varieties in the future.

Multivariate selection mediated by aridity predicts divergence of drought-resistant traits along natural aridity gradients of an invasive weed.

Geographical variation in the environment underpins selection for local adaptation and evolutionary divergence among populations. Because many environmental conditions vary across species' ranges, identifying the specific environmental variables underlying local adaptation is profoundly challenging. We tested whether natural selection mediated by aridity predicts clinal divergence among invasive populations of capeweed (Arctotheca calendula) that established and spread across southern Australia during the last two centuries. Using common garden experiments with two environmental treatments (wet and dry) that mimic aridity conditions across capeweed's invasive range, we estimated clinal divergence and effects of aridity on fitness and multivariate phenotypic selection in populations sampled along aridity gradients in Australia. We show that: (1) capeweed populations have relatively high fitness in aridity environments similar to their sampling locations; (2) the magnitude and direction of selection strongly differs between wet and dry treatments, with drought stress increasing the strength of selection; and (3) differences in directional selection between wet and dry treatments predict patterns of clinal divergence across the aridity gradient, particularly for traits affecting biomass, flowering phenology and putative antioxidant expression. Our results suggest that aridity-mediated selection contributes to trait diversification among invasive capeweed populations, possibly facilitating the expansion of capeweed across southern Australia.

Acclimation to water stress improves tolerance to heat and freezing in a common alpine grass.

Alpine plants in Australia are increasingly exposed to more frequent drought and heatwaves, with significant consequences for physiological stress responses. Acclimation is a critical feature that allows plants to improve tolerance to environmental extremes by directly altering their physiology or morphology. Yet it is unclear how plant performance, tolerance, and recovery are affected when heat and water stress co-occur, and whether prior exposure affects responses to subsequent climate extremes. We grew a common alpine grass species under high or low watering treatments for three weeks before exposure to either none, one, or two heat stress events. We determined photosynthetic heat and freezing tolerance (LT50, mean temperature causing 50% irreversible damage to photosystem II) and growth. Physiological adjustments to low watering, including more negative water potentials and reduced growth, were also characterised by improved tolerance to high and low-temperature extremes. Shifts to higher heat tolerance were also evident with increasing exposure to heat stress events, though freezing tolerance was not affected. Acclimation effects were mostly short-term, however; prior exposure to heat and/or water stress had little to no effect on growth and thermal tolerance following the six-week recovery period. We conclude that rapid acclimation to water and heat stress that co-occur during summer enhances the capacity of alpine plants to tolerate increasingly frequent temperature extremes.

Regulation of dhurrin pathway gene expression during Sorghum bicolor development.

MAIN CONCLUSION: Developmental and organ-specific expression of genes in dhurrin biosynthesis, bio-activation, and recycling offers dynamic metabolic responses optimizing growth and defence responses in Sorghum. Plant defence models evaluate the costs and benefits of resource investments at different stages in the life cycle. Poor understanding of the molecular regulation of defence deployment and remobilization hampers accuracy of the predictions. Cyanogenic glucosides, such as dhurrin are phytoanticipins that release hydrogen cyanide upon bio-activation. In this study, RNA-seq was used to investigate the expression of genes involved in the biosynthesis, bio-activation and recycling of dhurrin in Sorghum bicolor. Genes involved in dhurrin biosynthesis were highly expressed in all young developing vegetative tissues (leaves, leaf sheath, roots, stems), tiller buds and imbibing seeds and showed gene specific peaks of expression in leaves during diel cycles. Genes involved in dhurrin bio-activation were expressed early in organ development with organ-specific expression patterns. Genes involved in recycling were expressed at similar levels in the different organ during development, although post-floral initiation when nutrients are remobilized for grain filling, expression of GSTL1 decreased > tenfold in leaves and NITB2 increased > tenfold in stems. Results are consistent with the establishment of a pre-emptive defence in young tissues and regulated recycling related to organ senescence and increased demand for nitrogen during grain filling. This detailed characterization of the transcriptional regulation of dhurrin biosynthesis, bioactivation and remobilization genes during organ and plant development will aid elucidation of gene regulatory networks and signalling pathways that modulate gene expression and dhurrin levels. In-depth knowledge of dhurrin metabolism could improve the yield, nitrogen use efficiency and stress resilience of Sorghum.

Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Metabolites during Sorghum Germination.

Dhurrin is the most abundant cyanogenic glucoside found in sorghum (Sorghum bicolor) where it plays a key role in chemical defense by releasing toxic hydrogen cyanide upon tissue disruption. Besides this well-established function, there is strong evidence that dhurrin plays additional roles, e.g. as a transport and storage form of nitrogen, released via endogenous recycling pathways. However, knowledge about how, when and why dhurrin is endogenously metabolized is limited. We combined targeted metabolite profiling with matrix-assisted laser desorption/ionization-mass spectrometry imaging to investigate accumulation of dhurrin, its recycling products and key general metabolites in four different sorghum lines during 72 h of grain imbibition, germination and early seedling development, as well as the spatial distribution of these metabolites in two of the lines. Little or no dhurrin or recycling products were present in the dry grain, but their de novo biosynthesis started immediately after water uptake. Dhurrin accumulation increased rapidly within the first 24 h in parallel with an increase in free amino acids, a key event in seed germination. The trajectories and final concentrations of dhurrin, the recycling products and free amino acids reached within the experimental period were dependent on genotype. Matrix-assisted laser desorption/ionization-mass spectrometry imaging demonstrated that dhurrin primarily accumulated in the germinating embryo, confirming its function in protecting the emerging tissue against herbivory. The dhurrin recycling products, however, were mainly located in the scutellum and/or pericarp/seed coat region, suggesting unknown key functions in germination.

High sink strength prevents photosynthetic down-regulation in cassava grown at elevated CO2 concentration.

Cassava has the potential to alleviate food insecurity in many tropical regions, yet few breeding efforts to increase yield have been made. Improved photosynthetic efficiency in cassava has the potential to increase yields, but cassava roots must have sufficient sink strength to prevent carbohydrates from accumulating in leaf tissue and suppressing photosynthesis. Here, we grew eight farmer-preferred African cassava cultivars under free-air CO2 enrichment (FACE) to evaluate the sink strength of cassava roots when photosynthesis increases due to elevated CO2 concentrations ([CO2]). Relative to the ambient treatments, elevated [CO2] treatments increased fresh (+27%) and dry (+37%) root biomass, which was driven by an increase in photosynthesis (+31%) and the absence of photosynthetic down-regulation over the growing season. Moreover, intrinsic water use efficiency improved under elevated [CO2] conditions, while leaf protein content and leaf and root cyanide concentrations were not affected. Overall, these results suggest that higher cassava yields can be expected as atmospheric [CO2] increases over the coming decades. However, there were cultivar differences in the partitioning of resources to roots versus above-grown biomass; thus, the particular responses of each cultivar must be considered when selecting candidates for improvement.

Genome-wide association study of cyanogenic glycosides, proline, sugars, and pigments in Eucalyptus cladocalyx after 18 consecutive dry summers.

Natural variation of cyanogenic glycosides, soluble sugars, proline, and nondestructive optical sensing of pigments (chlorophyll, flavonols, and anthocyanins) was examined in ex situ natural populations of Eucalyptus cladocalyx F. Muell. grown under dry environmental conditions in the southern Atacama Desert, Chile. After 18 consecutive dry seasons, considerable plant-to-plant phenotypic variation for all the traits was observed in the field. For example, leaf hydrogen cyanide (HCN) concentrations varied from 0 (two acyanogenic individuals) to 1.54 mg cyanide g-1 DW. Subsequent genome-wide association study revealed associations with several genes with a known function in plants. HCN content was associated robustly with genes encoding Cytochrome P450 proteins, and with genes involved in the detoxification mechanism of HCN in cells (ß-cyanoalanine synthase and cyanoalanine nitrilase). Another important finding was that sugars, proline, and pigment content were linked to genes involved in transport, biosynthesis, and/or catabolism. Estimates of genomic heritability (based on haplotypes) ranged between 0.46 and 0.84 (HCN and proline content, respectively). Proline and soluble sugars had the highest predictive ability of genomic prediction models (PA = 0.65 and PA = 0.71, respectively). PA values for HCN content and flavonols were relatively moderate, with estimates ranging from 0.44 to 0.50. These findings provide new understanding on the genetic architecture of cyanogenic capacity, and other key complex traits in cyanogenic E. cladocalyx.

The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways.

The biosynthetic pathway for the cyanogenic glucoside dhurrin in sorghum has previously been shown to involve the sequential production of (E)- and (Z)-p-hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild-type and mutant sorghum or transiently transformed Nicotiana benthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)-p-hydroxyphenylacetaldoxime into the corresponding geometrical Z-isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme, but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsis thaliana cannot convert the (E)-p-hydroxyphenylacetaldoxime to the (Z)-isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the (E)-p-hydroxyphenylacetaldoxime into an S-alkyl-thiohydroximate with retention of the configuration of the E-oxime intermediate in the final glucosinolate core structure. Numerous microbial plant pathogens are able to detoxify Z-oximes but not E-oximes. The CYP79-derived E-oximes may play an important role in plant defense.

Metabolic consequences of knocking out UGT85B1, the gene encoding the glucosyltransferase required for synthesis of dhurrin in Sorghum bicolor (L. Moench).

Many important food crops produce cyanogenic glucosides as natural defense compounds to protect against herbivory or pathogen attack. It has also been suggested that these nitrogen-based secondary metabolites act as storage reserves of nitrogen. In sorghum, three key genes, CYP79A1, CYP71E1 and UGT85B1, encode two Cytochrome P450s and a glycosyltransferase, respectively, the enzymes essential for synthesis of the cyanogenic glucoside dhurrin. Here, we report the use of targeted induced local lesions in genomes (TILLING) to identify a line with a mutation resulting in a premature stop codon in the N-terminal region of UGT85B1. Plants homozygous for this mutation do not produce dhurrin and are designated tcd2 (totally cyanide deficient 2) mutants. They have reduced vigor, being dwarfed, with poor root development and low fertility. Analysis using liquid chromatography-mass spectrometry (LC-MS) shows that tcd2 mutants accumulate numerous dhurrin pathway-derived metabolites, some of which are similar to those observed in transgenic Arabidopsis expressing the CYP79A1 and CYP71E1 genes. Our results demonstrate that UGT85B1 is essential for formation of dhurrin in sorghum with no co-expressed endogenous UDP-glucosyltransferases able to replace it. The tcd2 mutant suffers from self-intoxication because sorghum does not have a feedback mechanism to inhibit the initial steps of dhurrin biosynthesis when the glucosyltransferase activity required to complete the synthesis of dhurrin is lacking. The LC-MS analyses also revealed the presence of metabolites in the tcd2 mutant which have been suggested to be derived from dhurrin via endogenous pathways for nitrogen recovery, thus indicating which enzymes may be involved in such pathways.

Age versus stage: does ontogeny modify the effect of phosphorus and arbuscular mycorrhizas on above- and below-ground defence in forage sorghum?

Arbuscular mycorrhizas (AM) can increase plant acquisition of P and N. No published studies have investigated the impact of P and AM on the allocation of N to the plant defence, cyanogenic glucosides. We investigated the effects of soil P and AM on cyanogenic glucoside (dhurrin) concentration in roots and shoots of two forage sorghum lines differing in cyanogenic potential (HCNp). Two harvest times allowed plants grown at high and low P to be compared at the same age and the same size, to take account of known ontogenetic changes in shoot HCNp. P responses were dependent on ontogeny and tissue type. At the same age, P-limited plants were smaller and had higher shoot HCNp but lower root HCNp. Ontogenetically controlled comparisons showed a P effect of lesser magnitude, and that there was also an increase in the allocation of N to dhurrin in shoots of P-limited plants. Colonization by AM had little effect on shoot HCNp, but increased root HCNp and the allocation of N to dhurrin in roots. Divergent responses of roots and shoots to P, AM and with ontogeny demonstrate the importance of broadening the predominantly foliar focus of plant defence studies/theory, and of ontogenetically controlled comparisons.

The Putative GATA Transcription Factor SbGATA22 as a Novel Regulator of Dhurrin Biosynthesis.

Cyanogenic glucosides are specialized metabolites produced by over 3000 species of higher plants from more than 130 families. The deployment of cyanogenic glucosides is influenced by biotic and abiotic factors in addition to being developmentally regulated, consistent with their roles in plant defense and stress mitigation. Despite their ubiquity, very little is known regarding the molecular mechanisms that regulate their biosynthesis. The biosynthetic pathway of dhurrin, the cyanogenic glucoside found in the important cereal crop sorghum (Sorghum bicolor (L.) Moench), was described over 20 years ago, and yet no direct regulator of the biosynthetic genes has been identified. To isolate regulatory proteins that bind to the promoter region of the key dhurrin biosynthetic gene of sorghum, SbCYP79A1, yeast one-hybrid screens were performed. A bait fragment containing 1204 base pairs of the SbCYP79A1 5' regulatory region was cloned upstream of a reporter gene and introduced into Saccharomyces cerevisiae. Subsequently, the yeast was transformed with library cDNA representing RNA from two different sorghum developmental stages. From these screens, we identified SbGATA22, an LLM domain B-GATA transcription factor that binds to the putative GATA transcription factor binding motifs in the SbCYP79A1 promoter region. Transient assays in Nicotiana benthamiana show that SbGATA22 localizes to the nucleus. The expression of SbGATA22, in comparison with SbCYP79A1 expression and dhurrin concentration, was analyzed over 14 days of sorghum development and in response to nitrogen application, as these conditions are known to affect dhurrin levels. Collectively, these findings suggest that SbGATA22 may act as a negative regulator of SbCYP79A1 expression and provide a preliminary insight into the molecular regulation of dhurrin biosynthesis in sorghum.

Cassava.

Gleadow et al. introduce the food crop cassava.

A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production.

Cyanogenic glucosides are present in several crop plants and can pose a significant problem for human and animal consumption, because of their ability to release toxic hydrogen cyanide. Sorghum bicolor L. contains the cyanogenic glucoside dhurrin. A qualitative biochemical screen of the M2 population derived from EMS treatment of sorghum seeds, followed by the reverse genetic technique of Targeted Induced Local Lesions in Genomes (TILLING), was employed to identify mutants with altered hydrogen cyanide potential (HCNp). Characterization of these plants identified mutations affecting the function or expression of dhurrin biosynthesis enzymes, and the ability of plants to catabolise dhurrin. The main focus in this study is on acyanogenic or low cyanide releasing lines that contain mutations in CYP79A1, the cytochrome P450 enzyme catalysing the first committed step in dhurrin synthesis. Molecular modelling supports the measured effects on CYP79A1 activity in the mutant lines. Plants harbouring a P414L mutation in CYP79A1 are acyanogenic when homozygous for this mutation and are phenotypically normal, except for slightly slower growth at early seedling stage. Detailed biochemical analyses demonstrate that the enzyme is present in wild-type amounts but is catalytically inactive. Additional mutants capable of producing dhurrin at normal levels in young seedlings but with negligible leaf dhurrin levels in mature plants were also identified. No mutations were detected in the coding sequence of dhurrin biosynthetic genes in this second group of mutants, which are as tall or taller, and leafier than nonmutated lines. These sorghum mutants with reduced or negligible dhurrin content may be ideally suited for forage production.

Drying and processing protocols affect the quantification of cyanogenic glucosides in forage sorghum.

BACKGROUND: Cyanogenic glucosides are common bioactive products that break down to release toxic hydrogen cyanide (HCN) when combined with specific ß-glucosidases. In forage sorghum, high concentrations of the cyanogenic glucoside dhurrin lead to reduced productivity and sometimes death of grazing animals, especially in times of drought, when the dhurrin content of stunted crops is often higher. The aim of this study was to develop harvesting protocols suitable for sampling in remote areas. RESULTS: Dhurrin concentration in air- and oven-dried leaves was the same as in fresh leaves, with no subsequent losses during storage. Dhurrin concentration was halved when leaves were freeze-dried, although activity of the endogenous dhurrinase was preserved. Direct measurement of dhurrin concentration in methanolic extracts using liquid chromatography/mass spectrometry (LC/MS) gave similar results to methods that captured evolved cyanide. A single freezing event was as effective as fine grinding in facilitating complete conversion of dhurrin to cyanide. CONCLUSION: Direct measurement of dhurrin using LC/MS is accurate but expensive and not appropriate for fieldwork. Air drying provides an accurate, low-cost method for preparing tissue for dhurrin analysis, so long as the specific ß-glucosidase is added. It is recommended that comparative studies like the one presented here be extended to other cyanogenic species.

Cyanide Content of Cassava Food Products Available in Australia.

In 2009, Food Standards Australia New Zealand set a total cyanide content limit of 10 ppm for ready-to-eat cassava products to address food safety concerns about cyanogenic glucosides in cassava. This study surveys a range of cassava food products available in Melbourne, Australia, ten years after the implementation of these regulations. Of all the products tested, the mean cyanide content was greatest in ready-to-eat cassava chips (48.4 ppm), although imported ready-to-eat products had a higher mean cyanide content (95.9 ppm) than those manufactured in Australia (1.0 ppm). Cyanide was detected in frozen cassava products (grated mean = 12.9 ppm; whole root mean = 19.8 ppm), but was significantly reduced through processing according to packet instructions in both product types. Three methods were used to quantify total cyanide content: the evolved cyanide method, the picrate absorbance method and the picrate chart method, with satisfactory agreement between methods. The picrate absorbance and chart methods reported mean cyanide contents 13.7 ppm and 23.1 ppm higher, respectively, than the evolved cyanide method. Our results reaffirm the need for the ongoing testing of cassava food products, especially ready-to-eat products whose cyanide content will not be reduced before consumption.

Cyanogenesis in the Sorghum Genus: From Genotype to Phenotype.

Domestication has resulted in a loss of genetic diversity in our major food crops, leading to susceptibility to biotic and abiotic stresses linked with climate change. Crop wild relatives (CWR) may provide a source of novel genes potentially important for re-gaining climate resilience. Sorghum bicolor is an important cereal crop with wild relatives that are endemic to Australia. Sorghum bicolor is cyanogenic, but the cyanogenic status of wild Sorghum species is not well known. In this study, leaves of wild species endemic in Australia are screened for the presence of the cyanogenic glucoside dhurrin. The direct measurement of dhurrin content and the potential for dhurrin-derived HCN release (HCNp) showed that all the tested Australian wild species were essentially phenotypically acyanogenic. The unexpected low dhurrin content may reflect the variable and generally nutrient-poor environments in which they are growing in nature. Genome sequencing of six CWR and PCR amplification of the CYP79A1 gene from additional species showed that a high conservation of key amino acids is required for correct protein function and dhurrin synthesis, pointing to the transcriptional regulation of the cyanogenic phenotype in wild sorghum as previously shown in elite sorghum.

Effects of Salinity on the Growth and Nutrition of Taro (Colocasia esculenta): Implications for Food Security.

Taro (Colocasia esculenta (L.) Schott) is a staple food crop in the Asia-Pacific region in areas where rising sea levels are threatening agricultural production. However, little is known about its response to salinity. In this study, we investigated the effects of salinity on the growth, morphology, physiology, and chemical traits of taro to predict the impacts of rising sea levels on taro production and nutritional value in the Pacific. We grew taro (approximately 4 months old) with a range of NaCl treatments (0-200 mM) for 12 weeks. Full nutrient, micronutrient, and secondary metabolite analyses were conducted, including measures of calcium oxalate (CaOx), an irritant that reduces palatability. Significant reductions in growth and biomass were observed at and above 100 mM NaCl. Concentrations of macro- and micronutrients, including sodium, were higher on a per mass basis in corms of plants experiencing salt stress. Foliar sodium concentrations remained stable, indicating that taro may utilize a salt exclusion mechanism. There was a large amount of individual variation in the concentrations of oxalate and phenolics, but overall, the concentrations were similar in the plants grown with different levels of salt. The total contents of CaOx and phenolics decreased in plants experiencing salt stress. Taro's ability to survive and produce corms when watered with a 200 mM NaCl solution places it among the salt-tolerant non-halophytes. The nutritional quality of the crop is only marginally affected by salt stress. Taro is, therefore, likely to remain a useful staple in the Pacific region in the future.

Do mycorrhizae increase plant growth and pollutant removal in stormwater biofilters?

Mycorrhizae can improve plant growth and drought tolerance by enhancing plant uptake of nutrients and water, which are important targets for biofilters, a common stormwater treatment system. This study evaluated the role of mycorrhizal inoculation on plant growth, photosynthetic efficiency and pollutant removal in two Australian plant species grown in stormwater biofilters. During the establishment period and column study, Ficinia nodosa showed over 80% mycorrhizal colonization, leading to a doubling of shoot and root biomass compared to the control, while Carex appressa showed less than 26% mycorrhizal colonization and no effect on shoot and root biomass. Columns planted with mycorrhizal-inoculated F. nodosa had 5% higher removal of total phosphorus and 10% higher removal of total nitrogen (Figure 5), phosphate (Figure 6), and cadmium (Table 3). Mycorrhizal colonization did not appear to affect plant stress during drought as indicated by similar photosynthetic efficiencies within species. Our results indicate that mycorrhizal inoculation can be highly successful in biofilters while increasing plant growth and nutrient removal, opening opportunities to further study the role of mycorrhizae in enhancing plant drought tolerance and pollutant removal in existing biofiltration systems.

Nitrogen availability and allocation in sorghum and its wild relatives: Divergent roles for cyanogenic glucosides.

Crop plants are assumed to have become more susceptible to pests as a result of selection for high growth rates during the process of domestication, consistent with resource allocation theories. We compared the investment by domesticated sorghum into cyanogenic glucosides, nitrogen-based specialised metabolites that break down to release hydrogen cyanide, with five wild relatives native to Australia. Plants were grown in pots in a greenhouse and supplied with low and high concentrations of nitrogen and monitored for 9 weeks. The concentrations of nitrate, total phenolics and silicon were also measured. Domesticated Sorghum bicolor had the highest leaf and root cyanogenic glucoside concentrations, and among the lowest nitrate and silicon concentrations under both treatments. Despite partitioning a much higher proportion of its stored nitrogen to cyanogenic glucosides than the wild species, S. bicolor's nitrogen productivity levels were among the highest. Most of the wild sorghums had higher concentrations of silicon and phenolics, which may provide an alternative defence system. Cyanogenic glucosides appear to be integral to S. bicolor's physiology, having roles in both growth and defence. Sorghum macrospermum displayed consistently low cyanogenic glucoside concentrations, high growth rates and high nitrogen productivity and represents a particularly attractive genetic resource for sorghum improvement.

Phylogenetic relationships in the Sorghum genus based on sequencing of the chloroplast and nuclear genes.

Sorghum [Sorghum bicolor (L.) Moench] is an important food crop with a diverse gene pool residing in its wild relatives. A total of 15 sorghum accessions from the unexploited wild gene pool of the Sorghum genus, representing the five subgenera, were sequenced, and the complete chloroplast genomes and 99 common single-copy concatenated nuclear genes were assembled. Annotation of the chloroplast genomes identified a total of 81 protein-coding genes, 38 tRNA, and four rRNA genes. The gene content and gene order among the species was identical. A total of 153 nonsynonymous amino acid changes in 40 genes were identified across the species. Phylogenetic analysis of both the whole chloroplast genome and nuclear genes revealed a similar topology with two distinct clades within the genus. The species within the subgenera Eusorghum, Chaetosorghum, and Heterosorghum clustered in one clade, whereas the species within the subgenera Parasorghum and Stiposorghum clustered in a second clade. However, the subgenera Parasorghum and Stiposorghum were not monophyletic, suggesting the need for further research to resolve the relationships within this group. The close relationship between the two monotypic subgenera Chaetosorghum and Heterosorghum suggests that species within these subgenera could be considered as one group. This analysis provides an improved understanding of the genetic relationships within the Sorghum genus and defines diversity in wild sorghum species that may be useful for crop improvement.