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Cytochrome P450s of the CYP79 family catalyze two N-hydroxylation reactions, converting a selected number of amino acids into the corresponding oximes. The sorghum genome (Sorghum bicolor) harbours nine CYP79A encoding genes, and here sequence comparisons of the CYP79As along with their substrate recognition sites (SRSs) are provided. The substrate specificity of previously uncharacterized CYP79As was investigated by transient expression in Nicotiana benthamiana and subsequent transformation of the oximes formed into the corresponding stable oxime glucosides catalyzed by endogenous UDPG-glucosyltransferases (UGTs). CYP79A61 uses phenylalanine as a substrate, whereas CYP79A91, CYP79A93, and CYP79A95 use valine and isoleucine as substrates, with CYP79A93 showing the ability also to use phenylalanine. CYP79A94 uses isoleucine as a substrate. Analysis of 249 sorghum transcriptomes from two different sorghum cultivars showed the expression levels and tissue-specific expression of the CYP79As. CYP79A1 is the committed gene in dhurrin formation and was the highest expressed gene in most tissues/organs. CYP79A61 was primarily expressed in fully developed leaf blades and leaf sheaths. CYP79A91 and CYP79A92 were expressed mainly in roots >200 cm below ground, while CYP79A93 and CYP79A94 were most highly expressed in the leaf collar and leaf sheath, respectively. The possible signalling effects of the oximes and their metabolites produced in different sorghum tissues are discussed., (© 2025 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Bioenergy sorghum is a drought-tolerant high-biomass C4 grass targeted for production on annual cropland marginal for food crops due primarily to abiotic constraints. To better understand the overall contribution of stem wax to bioenergy sorghum's resilience, the current study characterized sorghum stem cuticular wax loads, composition, morphometrics, wax pathway gene expression and regulation using vegetative phase Wray, R07020, and TX08001 genotypes. Wax loads on sorghum stems (~103-215 µg/cm 2 ) were much higher than Arabidopsis stem and leaf wax loads. Wax on developing sorghum stem internodes was enriched in C28/30 primary alcohols (~65%) while stem wax on fully developed stems was enriched in C28/30 aldehydes (~80%). Scanning Electron Microscopy showed minimal wax on internodes prior to the onset of elongation and that wax tubules first appear associated with cork-silica cell complexes when internode cell elongation is complete. Sorghum homologs of genes involved in wax biosynthesis/transport were differentially expressed in the stem epidermis. Expression of many wax pathway genes (i.e., SbKCS6, SbCER3-1, SbWSD1, SbABCG12, SbABCG11 ) is low in immature apical internodes then increases at the onset of stem wax accumulation. SbCER4 is expressed relatively early in stem development consistent with accumulation of C28/30 primary alcohols on developing apical internodes. High expression of two SbCER3 homologs in fully elongated internodes is consistent with a role in production of C28/30 aldehydes. Gene regulatory network analysis aided the identification of sorghum homologs of transcription factors that regulate wax biosynthesis (i.e., SbSHN1, SbWRI1/3, SbMYB94/96/30/60, MYS1 ) and other transcription factors that could regulate and specify expression of the wax pathway in epidermal cells during cuticle development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chemelewski, McKinley, Finlayson and Mullet.)

Bioenergy sorghum hybrids are being developed with enhanced drought tolerance and high levels of stem sugars. Raffinose family oligosaccharides (RFOs) contribute to plant environmental stress tolerance, sugar storage, transport, and signaling. To better understand the role of RFOs in sorghum, genes involved in myo -inositol and RFO metabolism were identified and relative transcript abundance analyzed during development. Genes involved in RFO biosynthesis ( SbMIPS1, SbInsPase, SbGolS1, SbRS ) were more highly expressed in leaves compared to stems and roots, with peak expression early in the morning in leaves. SbGolS, SbRS, SbAGA1 and SbAGA2 were also expressed at high levels in the leaf collar and leaf sheath. In leaf blades, genes involved in myo -inositol biosynthesis ( SbMIPS1, SbInsPase ) were expressed in bundle sheath cells, whereas genes involved in galactinol and raffinose synthesis ( SbGolS1, SbRS ) were expressed in mesophyll cells. Furthermore, SbAGA1 and SbAGA2 , genes that encode neutral-alkaline alpha-galactosidases that hydrolyze raffinose, were differentially expressed in minor vein bundle sheath cells and major vein and mid-rib vascular and xylem parenchyma. This suggests that raffinose synthesized from sucrose and galactinol in mesophyll cells diffuses into vascular bundles where hydrolysis releases sucrose for long distance phloem transport. Increased expression (>20-fold) of SbAGA1 and SbAGA2 in stem storage pith parenchyma of sweet sorghum between floral initiation and grain maturity, and higher expression in sweet sorghum compared to grain sorghum, indicates these genes may play a key role in non-structural carbohydrate accumulation in stems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McKinley, Thakran, Zemelis-Durfee, Huang, Brandizzi, Rooney, Mansfield and Mullet.)

The stay-green trait is recognized as a key drought adaptation mechanism in cereals worldwide. Stay-green sorghum plants exhibit delayed senescence of leaves and stems, leading to prolonged growth, a reduced risk of lodging, and higher grain yield under end-of-season drought stress. More than 45 quantitative trait loci (QTL) associated with stay-green have been identified, including two major QTL (Stg1 and Stg2). However, the contributing genes that regulate functional stay-green are not known. Here we show that the PIN FORMED family of auxin efflux carrier genes induce some of the causal mechanisms driving the stay-green phenotype in sorghum, with SbPIN4 and SbPIN2 located in Stg1 and Stg2, respectively. We found that nine of 11 sorghum PIN genes aligned with known stay-green QTL. In transgenic studies, we demonstrated that PIN genes located within the Stg1 (SbPIN4), Stg2 (SbPIN2), and Stg3b (SbPIN1) QTL regions acted pleiotropically to modulate canopy development, root architecture, and panicle growth in sorghum, with SbPIN1, SbPIN2, and SbPIN4 differentially expressed in various organs relative to the non-stay-green control. The emergent consequence of such modifications in canopy and root architecture is a stay-green phenotype. Crop simulation modelling shows that the SbPIN2 phenotype can increase grain yield under drought., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

A field-ready, fiber-based high spatial sampling snapshot imaging spectrometer was developed for applications such as environmental monitoring and smart farming. The system achieves video rate frame transfer and exposure times down to a few hundred microseconds in typical daylight conditions with ∼63,000 spatial points and 32 spectral channels across the 470nm to 700nm wavelength range. We designed portable, ruggedized opto-mechanics to allow for imaging from an airborne platform. To ensure successful data collection prior to flight, imaging speed and signal-to-noise ratio was characterized for imaging a variety of land covers from the air. The system was validated by performing a series of observations including: Liriope Muscari plants under a range of water-stress conditions in a controlled laboratory experiment and field observations of sorghum plants in a variety of soil conditions. Finally, we collected data from a series of engineering flights and present reassembled images and spectral sampling of rural and urban landscapes.

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., (© 2021. The Author(s).)

Stems of bioenergy sorghum (Sorghum bicolor L. Moench.), a drought-tolerant C4 grass, contain up to 50 nodes and internodes of varying length that span 4-5 m and account for approximately 84% of harvested biomass. Stem internode growth impacts plant height and biomass accumulation and is regulated by brassinosteroid signaling, auxin transport, and gibberellin biosynthesis. In addition, an AGCVIII kinase (Dw2) regulates sorghum stem internode growth, but the underlying mechanism and signaling network are unknown. Here we provide evidence that mutation of Dw2 reduces cell proliferation in internode intercalary meristems, inhibits endocytosis, and alters the distribution of heteroxylan and mixed linkage glucan in cell walls. Phosphoproteomic analysis showed that Dw2 signaling influences the phosphorylation of proteins involved in lipid signaling (PLDδ), endomembrane trafficking, hormone, light, and receptor signaling, and photosynthesis. Together, our results show that Dw2 modulates endomembrane function and cell division during sorghum internode growth, providing insight into the regulation of monocot stem development., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The stems of bioenergy sorghum hybrids at harvest are > 4 m long, contain > 40 internodes and account for ~ 80% of harvested biomass. In this study, bioenergy sorghum hybrids were grown at four planting densities (~ 20,000 to 132,000 plants/ha) under field conditions for 60 days to investigate the impact shading has on stem growth and biomass accumulation. Increased planting density induced a > 2-fold increase in sorghum internode length and a ~ 22% decrease in stem diameter, a typical shade avoidance response. Shade-induced internode elongation was due to an increase in cell length and number of cells spanning the length of internodes. SbGA3ox2 (Sobic.003G045900), a gene encoding the last step in GA biosynthesis, was expressed ~ 20-fold higher in leaf collar tissue of developing phytomers in plants grown at high vs. low density. Application of GA3 to bioenergy sorghum increased plant height, stem internode length, cell length and the number of cells spanning internodes. Prior research showed that sorghum plants lacking phytochrome B, a key photoreceptor involved in shade signaling, accumulated more GA1 and displayed shade avoidance phenotypes. These results are consistent with the hypothesis that increasing planting density induces expression of GA3-oxidase in leaf collar tissue, increasing synthesis of GA that stimulates internode elongation.

The development of a robust method to non-invasively visualize root morphology in natural soils has been hampered by the opaque, physical, and structural properties of soils. In this work we describe a novel technology, low field magnetic resonance imaging (LF-MRI), for imaging energy sorghum ( Sorghum bicolor (L.) Moench) root morphology and architecture in intact soils. The use of magnetic fields much weaker than those used with traditional MRI experiments reduces the distortion due to magnetic material naturally present in agricultural soils. A laboratory based LF-MRI operating at 47 mT magnetic field strength was evaluated using two sets of soil cores: 1) soil/root cores of Weswood silt loam (Udifluventic Haplustept) and a Belk clay (Entic Hapluderts) from a conventionally tilled field, and 2) soil/root cores from rhizotrons filled with either a Houston Black (Udic Haplusterts) clay or a sandy loam purchased from a turf company. The maximum soil water nuclear magnetic resonance (NMR) relaxation time T 2 (4 ms) and the typical root water relaxation time T 2 (100 ms) are far enough apart to provide a unique contrast mechanism such that the soil water signal has decayed to the point of no longer being detectable during the data collection time period. 2-D MRI projection images were produced of roots with a diameter range of 1.5-2.0 mm using an image acquisition time of 15 min with a pixel resolution of 1.74 mm in four soil types. Additionally, we demonstrate the use of a data-driven machine learning reconstruction approach, Automated Transform by Manifold Approximation (AUTOMAP) to reconstruct raw data and improve the quality of the final images. The application of AUTOMAP showed a SNR (Signal to Noise Ratio) improvement of two fold on average. The use of low field MRI presented here demonstrates the possibility of applying low field MRI through intact soils to root phenotyping and agronomy to aid in understanding of root morphology and the spatial arrangement of roots in situ .

Stem internodes of bioenergy sorghum inbred R.07020 are longer at high plant density (shade) than at low plant density (control). Initially, the youngest newly-formed subapical stem internodes of shade-treated and control plants are comparable in length. However, full-length internodes of shade-treated plants are three times longer than the internodes of the control plants. To identify the early molecular events associated with internode elongation in response to shade, we analyzed the transcriptome of the newly-formed internodes of shade-treated and control plants sampled between 4 and 6 hr after the start of the light period (14 hr light/10 hr dark). Sorghum genes homologous to the Arabidopsis shade marker genes ATHB2 and PIL1 were not differentially expressed. The results indicate that shade signals promote internode elongation indirectly because sorghum internodes are not illuminated and grow while enclosed with leaf sheaths. Sorghum genes homologous to the Arabidopsis morning-phased circadian clock genes LHY , RVE , and LNK were downregulated and evening-phased genes such as TOC1 , PRR5, and GI were upregulated in young internodes in response to shade. We hypothesize that a change in the function or patterns of expression of the circadian clock genes is the earliest molecular event associated with internode elongation in response to shade in bioenergy sorghum. Increased expression of CycD1 , which promotes cell division, and decreased expression of cell wall-loosening and MBF1 -like genes, which promote cell expansion, suggest that shade signals promote internode elongation in bioenergy sorghum in part through increasing cell number by delaying transition from cell division to cell expansion., Competing Interests: The authors declare that they have no conflict of interest., (© 2020 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.)

Sorghum bicolor is a drought-resilient facultative short-day C4 grass that is grown for grain, forage, and biomass. Adaptation of sorghum for grain production in temperate regions resulted in the selection of mutations in Maturity loci (Ma1 -Ma6) that reduced photoperiod sensitivity and resulted in earlier flowering in long days. Prior studies identified the genes associated with Ma1 (PRR37), Ma3 (PHYB), Ma5 (PHYC) and Ma6 (GHD7) and characterized their role in the flowering time regulatory pathway. The current study focused on understanding the function and identity of Ma2. Ma2 delayed flowering in long days by selectively enhancing the expression of SbPRR37 (Ma1) and SbCO, genes that co-repress the expression of SbCN12, a source of florigen. Genetic analysis identified epistatic interactions between Ma2 and Ma4 and located QTL corresponding to Ma2 on SBI02 and Ma4 on SBI10. Positional cloning and whole genome sequencing identified a candidate gene for Ma2, Sobic.002G302700, which encodes a SET and MYND (SYMD) domain lysine methyltransferase. Eight sorghum genotypes previously identified as recessive for Ma2 contained the mutated version of Sobic.002G302700 present in 80M (ma2) and one additional putative recessive ma2 allele was identified in diverse sorghum accessions., Competing Interests: The authors have declared that no competing interests exist.

Sorghum bicolor is a drought-resilient C4 grass used for production of grain, forage, sugar, and biomass. Sorghum genotypes capable of accumulating high levels of stem sucrose have solid stems that contain low levels of aerenchyma. The D -locus on SBI06 modulates the extent of aerenchyma formation in sorghum stems and leaf midribs. A QTL aligned with this locus was identified and fine-mapped in populations derived from BTx623*IS320c, BTx623*R07007, and BTx623*Standard broomcorn. Analysis of coding polymorphisms in the fine-mapped D -locus showed that genotypes that accumulate low levels of aerenchyma encode a truncated NAC transcription factor (Sobic.006G147400, SbNAC_d1 ), whereas parental lines that accumulate higher levels of stem aerenchyma encode full-length NAC TFs ( SbNAC-D ). During vegetative stem development, aerenchyma levels are low in nonelongated stem internodes, internode growing zones, and nodes. Aerenchyma levels increase in recently elongated internodes starting at the top of the internode near the center of the stem. SbNAC_D was expressed at low levels in nonelongated internodes and internode growing zones and at higher levels in regions of stem internodes that form aerenchyma. SbXCP1 , a gene encoding a cysteine protease involved in programmed cell death, was induced in SbNAC_D genotypes in parallel with aerenchyma formation in sorghum stems but not in SbNAC_d1 genotypes. Several sweet sorghum genotypes encode the recessive SbNAC_d1 allele and have low levels of stem aerenchyma. Based on these results, we propose that SbNAC_D is the D -gene identified by Hilton (1916) and that allelic variation in SbNAC_D modulates the extent of aerenchyma formation in sorghum stems., Competing Interests: The authors have no conflict of interests to declare.

Sweet sorghums were identified that accumulate up to ~9% of their total stem dry weight as starch. Starch accumulated preferentially in stem pith parenchyma in close proximity to vascular bundles. Stem starch accumulated slowly between floral initiation and anthesis and more rapidly between anthesis and 43 days post-anthesis before declining in parallel with tiller outgrowth. Genes involved in stem starch metabolism were identified through phylogenetic approaches and RNA-seq analysis of Della stem gene expression during the starch accumulation phase of development. Genes differentially expressed in stems were identified that are involved in starch biosynthesis (i.e., AGPase SS/LS, starch synthases, starch-branching enzymes), degradation (i.e., glucan-water dikinase, β-amylase, disproportionating enzyme, alpha-glucan phosphorylase) and amyloplast sugar transport (glucose-6-P translocator). Transcripts encoding AGPase SS and LS subunits with plastid localization were differentially induced during stem starch accumulation indicating that ADP-glucose for starch biosynthesis is primarily generated in stem plastids. Cytosolic heteroglucan metabolism may play a role in stem sucrose/starch accumulation because genes encoding cytosolic forms of the disproportionating enzyme and alpha-glucan phosphorylase were induced in parallel with stem sucrose/starch accumulation. Information on the stem starch pathway obtained in this study will be useful for engineering sorghum stems with elevated starch thereby improving forage quality and the efficiency of biomass conversion to biofuels and bio-products.

This study was conducted to document the extent and basis of compositional variation of shoot biomass of the energy Sorghum bicolor hybrid TX08001 during development under field conditions. TX08001 is capable of accumulating ~40 Mg/ha of dry biomass under good growing conditions and this genotype allocates ~80% of its shoot biomass to stems. After 150 days of growth TX08001 stems had a fresh/dry weight ratio of ~3:1 and soluble biomass accounted for ~30% of stem biomass. A panel of diverse energy sorghum genotypes varied ~6-fold in the ratio of stem structural to soluble biomass after 150 days of growth. Near-infrared spectroscopic analysis (NIRS) showed that TX08001 leaves accumulated higher levels of protein, water extractives and ash compared to stems, which have higher sugar, cellulose, and lignin contents. TX08001 stem sucrose content varied during development, whereas the composition of TX08001 stem cell walls, which consisted of ~45-49% cellulose, ~27-30% xylan, and ~15-18% lignin, remained constant after 90 days post emergence until the end of the growing season (180 days). TX08001 and Della stem syringyl (S)/guaiacyl (G) (0.53-0.58) and ferulic acid (FA)/para-coumaric acid (pCA) ratios were similar whereas ratios of pCA/(S+G) differed between these genotypes. Additionally, an analysis of irrigated versus non-irrigated TX08001 revealed that non-irrigated hybrids exhibited a 50% reduction in total cell wall biomass, an ~2-fold increase in stem sugars, and an ~25% increase in water extractives relative to irrigated hybrids. This study provides a baseline of information to help guide further optimization of energy sorghum composition for various end-uses.

Sorghum bicolor is a drought tolerant C4 grass used for the production of grain, forage, sugar, and lignocellulosic biomass and a genetic model for C4 grasses due to its relatively small genome (approximately 800 Mbp), diploid genetics, diverse germplasm, and colinearity with other C4 grass genomes. In this study, deep sequencing, genetic linkage analysis, and transcriptome data were used to produce and annotate a high-quality reference genome sequence. Reference genome sequence order was improved, 29.6 Mbp of additional sequence was incorporated, the number of genes annotated increased 24% to 34 211, average gene length and N50 increased, and error frequency was reduced 10-fold to 1 per 100 kbp. Subtelomeric repeats with characteristics of Tandem Repeats in Miniature (TRIM) elements were identified at the termini of most chromosomes. Nucleosome occupancy predictions identified nucleosomes positioned immediately downstream of transcription start sites and at different densities across chromosomes. Alignment of more than 50 resequenced genomes from diverse sorghum genotypes to the reference genome identified approximately 7.4 M single nucleotide polymorphisms (SNPs) and 1.9 M indels. Large-scale variant features in euchromatin were identified with periodicities of approximately 25 kbp. A transcriptome atlas of gene expression was constructed from 47 RNA-seq profiles of growing and developed tissues of the major plant organs (roots, leaves, stems, panicles, and seed) collected during the juvenile, vegetative and reproductive phases. Analysis of the transcriptome data indicated that tissue type and protein kinase expression had large influences on transcriptional profile clustering. The updated assembly, annotation, and transcriptome data represent a resource for C4 grass research and crop improvement., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

A significant increase in agricultural productivity will be required by 2050 to meet the needs of an expanding and rapidly developing world population, without allocating more land and water resources to agriculture, and despite slowing rates of grain yield improvement. This review examines the proposition that high-biomass C 4 grasses could help fill the yield gap. High-biomass C 4 grasses exhibit high yield due to C 4 photosynthesis, long growth duration, and efficient capture and utilization of light, water, and nutrients. These C 4 grasses exhibit high levels of drought tolerance during their long vegetative growth phase ideal for crops grown in water-limited regions of agricultural production. The stems of some high-biomass C 4 grasses can accumulate high levels of non-structural carbohydrates that could be engineered to enhance biomass yield and utility as feedstocks for animals and biofuels production. The regulatory pathway that delays flowering of high-biomass C 4 grasses in long days has been elucidated enabling production and deployment of hybrids. Crop and landscape-scale modeling predict that utilization of high-biomass C 4 grass crops on land and in regions where water resources limit grain crop yield could increase agricultural productivity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sorghum is an important C4 grass crop grown for grain, forage, sugar, and bioenergy production. While tall, late flowering landraces are commonly grown in Africa, short early flowering varieties were selected in US grain sorghum breeding programs to reduce lodging and to facilitate machine harvesting. Four loci have been identified that affect stem length (Dw1-Dw4). Subsequent research showed that Dw3 encodes an ABCB1 auxin transporter and Dw1 encodes a highly conserved protein involved in the regulation of cell proliferation. In this study, Dw2 was identified by fine-mapping and further confirmed by sequencing the Dw2 alleles in Dwarf Yellow Milo and Double Dwarf Yellow Milo, the progenitor genotypes where the recessive allele of dw2 originated. The Dw2 locus was determined to correspond to Sobic.006G067700, a gene that encodes a protein kinase that is homologous to KIPK, a member of the AGCVIII subgroup of the AGC protein kinase family in Arabidopsis.

Background: Bioenergy sorghum accumulates 75% of shoot biomass in stem internodes. Grass stem internodes are formed during vegetative growth and elongate in response to developmental and environmental signals. To identify genes and molecular mechanisms that modulate the extent of internode growth, we conducted microscopic and transcriptomic analyses of four successive sub-apical vegetative internodes representing different stages of internode development of the bioenergy sorghum genotype R.07020., Results: Stem internodes of sorghum genotype R.07020 are formed during the vegetative phase and their length is enhanced by environmental signals such as shade and floral induction in short days. During vegetative growth, the first visible and youngest sub-apical internode was ~0.7 cm in length, whereas the fourth fully expanded internode was ~5 cm in length. Microscopic analyses revealed that all internode tissue types including pith parenchyma and vascular bundles are present in the four successive internodes. Growth in the first two sub-apical internodes occurred primarily through an increase in cell number consistent with expression of genes involved in the cell cycle and DNA replication. Growth of the 3rd internode was associated with an increase in cell length and growth cessation in the 4th internode was associated with up-regulation of genes involved in secondary cell wall deposition. The expression of genes involved in hormone metabolism and signaling indicates that GA, BR, and CK activity decreased while ethylene, ABA, and JA increased in the 3rd/4th internodes. While the level of auxin appears to be increasing as indicated by the up-regulation of ARFs, down-regulation of TIR during development indicates that auxin signaling is also modified. The expression patterns of transcription factors are closely associated with their role during the development of the vegetative internodes., Conclusions: Microscopic and transcriptome analyses of four successive sub-apical internodes characterized the developmental progression of vegetative stem internodes from initiation through full elongation in the sorghum genotype R.07020. Transcriptome profiling indicates that dynamic variation in the levels and action of GA, CK, IAA, BR, ethylene, ABA, and JA modulate gene expression and growth during internode growth and development. This study provides detailed microscopic and transcriptomic data useful for identifying genes and molecular pathways regulating internode elongation in response to various developmental and environmental signals.

Bioenergy sorghum is targeted for production in water-limited annual cropland therefore traits that improve plant water capture, water use efficiency, and resilience to water deficit are necessary to maximize productivity. A crop modeling framework, APSIM, was adapted to predict the growth and biomass yield of energy sorghum and to identify potentially useful traits for crop improvement. APSIM simulations of energy sorghum development and biomass accumulation replicated results from field experiments across multiple years, patterns of rainfall, and irrigation schemes. Modeling showed that energy sorghum's long duration of vegetative growth increased water capture and biomass yield by ~30% compared to short season crops in a water-limited production region. Additionally, APSIM was extended to enable modeling of VPD-limited transpiration traits that reduce crop water use under high vapor pressure deficits (VPDs). The response of transpiration rate to increasing VPD was modeled as a linear response until a VPD threshold was reached, at which the slope of the response decreases, representing a range of responses to VPD observed in sorghum germplasm. Simulation results indicated that the VPD-limited transpiration trait is most beneficial in hot and dry regions of production where crops are exposed to extended periods without rainfall during the season or to a terminal drought. In these environments, slower but more efficient transpiration increases biomass yield and prevents or delays the exhaustion of soil water and onset of leaf senescence. The VPD-limited transpiration responses observed in sorghum germplasm increased biomass accumulation by 20% in years with lower summer rainfall, and the ability to drastically reduce transpiration under high VPD conditions could increase biomass by 6% on average across all years. This work indicates that the productivity and resilience of bioenergy sorghum grown in water-limited environments could be further enhanced by development of genotypes with optimized VPD-limited transpiration traits and deployment of these crops in water limited growing environments. The energy sorghum model and VPD-limited transpiration trait implementation are made available to simulate performance in other target environments.

Dissecting the genetic basis of complex traits is aided by frequent and nondestructive measurements. Advances in range imaging technologies enable the rapid acquisition of three-dimensional (3D) data from an imaged scene. A depth camera was used to acquire images of sorghum (Sorghum bicolor), an important grain, forage, and bioenergy crop, at multiple developmental time points from a greenhouse-grown recombinant inbred line population. A semiautomated software pipeline was developed and used to generate segmented, 3D plant reconstructions from the images. Automated measurements made from 3D plant reconstructions identified quantitative trait loci for standard measures of shoot architecture, such as shoot height, leaf angle, and leaf length, and for novel composite traits, such as shoot compactness. The phenotypic variability associated with some of the quantitative trait loci displayed differences in temporal prevalence; for example, alleles closely linked with the sorghum Dwarf3 gene, an auxin transporter and pleiotropic regulator of both leaf inclination angle and shoot height, influence leaf angle prior to an effect on shoot height. Furthermore, variability in composite phenotypes that measure overall shoot architecture, such as shoot compactness, is regulated by loci underlying component phenotypes like leaf angle. As such, depth imaging is an economical and rapid method to acquire shoot architecture phenotypes in agriculturally important plants like sorghum to study the genetic basis of complex traits., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Phytochrome B (phyB) enables plants to modify shoot branching or tillering in response to varying light intensities and ratios of red and far-red light caused by shading and neighbor proximity. Tillering is inhibited in sorghum genotypes that lack phytochrome B (58M, phyB-1) until after floral initiation. The growth of tiller buds in the first leaf axil of wild-type (100M, PHYB) and phyB-1 sorghum genotypes is similar until 6 d after planting when buds of phyB-1 arrest growth, while wild-type buds continue growing and develop into tillers. Transcriptome analysis at this early stage of bud development identified numerous genes that were up to 50-fold differentially expressed in wild-type/phyB-1 buds. Up-regulation of terminal flower1, GA2oxidase, and TPPI could protect axillary meristems in phyB-1 from precocious floral induction and decrease bud sensitivity to sugar signals. After bud growth arrest in phyB-1, expression of dormancy-associated genes such as DRM1, GT1, AF1, and CKX1 increased and ENOD93, ACCoxidase, ARR3/6/9, CGA1, and SHY2 decreased. Continued bud outgrowth in wild-type was correlated with increased expression of genes encoding a SWEET transporter and cell wall invertases. The SWEET transporter may facilitate Suc unloading from the phloem to the apoplast where cell wall invertases generate monosaccharides for uptake and utilization to sustain bud outgrowth. Elevated expression of these genes was correlated with higher levels of cytokinin/sugar signaling in growing buds of wild-type plants., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The efficiency with which a plant intercepts solar radiation is determined primarily by its architecture. Understanding the genetic regulation of plant architecture and how changes in architecture affect performance can be used to improve plant productivity. Leaf inclination angle, the angle at which a leaf emerges with respect to the stem, is a feature of plant architecture that influences how a plant canopy intercepts solar radiation. Here we identify extensive genetic variation for leaf inclination angle in the crop plant Sorghum bicolor, a C4 grass species used for the production of grain, forage, and bioenergy. Multiple genetic loci that regulate leaf inclination angle were identified in recombinant inbred line populations of grain and bioenergy sorghum. Alleles of sorghum dwarf-3, a gene encoding a P-glycoprotein involved in polar auxin transport, are shown to change leaf inclination angle by up to 34° (0.59 rad). The impact of heritable variation in leaf inclination angle on light interception in sorghum canopies was assessed using functional-structural plant models and field experiments. Smaller leaf inclination angles caused solar radiation to penetrate deeper into the canopy, and the resulting redistribution of light is predicted to increase the biomass yield potential of bioenergy sorghum by at least 3%. These results show that sorghum leaf angle is a heritable trait regulated by multiple loci and that genetic variation in leaf angle can be used to modify plant architecture to improve sorghum crop performance., (Copyright © 2015 by the Genetics Society of America.)

Shoot branches or tillers develop from axillary buds. The dormancy versus outgrowth fates of buds depends on genetic, environmental and hormonal signals. Defoliation inhibits bud outgrowth indicating the role of leaf-derived metabolic factors such as sucrose in bud outgrowth. In this study, the sensitivity of bud outgrowth to selective defoliation was investigated. At 6 d after planting (6 DAP), the first two leaves of sorghum were fully expanded and the third was partially emerged. Therefore, the leaves were selectively defoliated at 6 DAP and the length of the bud in the first leaf axil was measured at 8 DAP. Bud outgrowth was inhibited by defoliation of only 2 cm from the tip of the second leaf blade. The expression of dormancy and sucrose-starvation marker genes was up-regulated and cell cycle and sucrose-inducible genes was down-regulated during the first 24 h post-defoliation of the second leaf. At 48 h, the expression of these genes was similar to controls as the defoliated plant recovers. Our results demonstrate that small changes in photosynthetic leaf area affect the propensity of tiller buds for outgrowth. Therefore, variation in leaf area and photosynthetic activity should be included when integrating sucrose into models of shoot branching., (© 2014 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Recent advances in variant calling made available in the Genome Analysis Toolkit (GATK) enable the use of validated single-nucleotide polymorphisms and indels to improve variant calling. However, large collections of variants for this purpose often are unavailable to research communities. We introduce a workflow to generate reliable collections of single-nucleotide polymorphisms and indels by leveraging available genomic resources to inform variant calling using the GATK. The workflow is demonstrated for the crop plant Sorghum bicolor by (i) generating an initial set of variants using reduced representation sequence data from an experimental cross and association panels, (ii) using the initial variants to inform variant calling from whole-genome sequence data of resequenced individuals, and (iii) using variants identified from whole-genome sequence data for recalibration of the reduced representation sequence data. The reliability of variants called with the workflow is verified by comparison with genetically mappable variants from an independent sorghum experimental cross. Comparison with a recent sorghum resequencing study shows that the workflow identifies an additional 1.62 million high-confidence variants from the same sequence data. Finally, the workflow's performance is validated using Arabidopsis sequence data, yielding variant call sets with 95% sensitivity and 99% positive predictive value. The Recalibration and Interrelation of genomic sequence data with the GATK (RIG) workflow enables the GATK to accurately identify genetic variation in organisms lacking validated variant resources., (Copyright © 2015 McCormick et al.)

Stay-green sorghum plants exhibit greener leaves and stems during the grain-filling period under water-limited conditions compared with their senescent counterparts, resulting in increased grain yield, grain mass, and lodging resistance. Stay-green has been mapped to a number of key chromosomal regions, including Stg1, Stg2, Stg3, and Stg4, but the functions of these individual quantitative trait loci (QTLs) remain unclear. The objective of this study was to show how positive effects of Stg QTLs on grain yield under drought can be explained as emergent consequences of their effects on temporal and spatial water-use patterns that result from changes in leaf-area dynamics. A set of four Stg near-isogenic lines (NILs) and their recurrent parent were grown in a range of field and semicontrolled experiments in southeast Queensland, Australia. These studies showed that the four Stg QTLs regulate canopy size by: (1) reducing tillering via increased size of lower leaves, (2) constraining the size of the upper leaves; and (3) in some cases, decreasing the number of leaves per culm. In addition, they variously affect leaf anatomy and root growth. The multiple pathways by which Stg QTLs modulate canopy development can result in considerable developmental plasticity. The reduction in canopy size associated with Stg QTLs reduced pre-flowering water demand, thereby increasing water availability during grain filling and, ultimately, grain yield. The generic physiological mechanisms underlying the stay-green trait suggest that similar Stg QTLs could enhance post-anthesis drought adaptation in other major cereals such as maize, wheat, and rice., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

To meet future food needs, grain production must increase despite reduced water availability, so waterproductivity must rise. One way to do this is to raise the ratio of biomass produced to water transpired, which is controlled by the ratio of CO2 assimilation (A) to transpiration (E) (i.e. the transpiration ratio, A : E divided by vapour pressure deficit) or anything affecting stomatal movement.. We describe the genetic variation and basis of A, E and A : E among 70 recombinant inbred lines (RILs) of sorghum (Sorghum bicolor (L.) Moench), using greenhouse experiments. Experiment 1 used 40% and 80% of field capacity (FC) as water regimes; Experiment 2 used 80% FC. Genotype had a significant effect on A, E and A : E. In Experiment 1, mean values for A : E were 1.2-4.4 mmol CO2 mol-1 H2O kPa-1 and 1.6-3.1 mmol CO2 mol-1 H2O kPa-1 under 40% and 80% FC, respectively. In Experiment 2, values were 5.6-9.8 mmol CO2 mol-1 H2O kPa-1. Pooled data for A : E and A : E VPD-1 from Experiment 1 indicate that A : E fell quickly at temperatures >32.3°C. A : E distributions were skewed. Mean heritabilities for A : E were 0.9 (40% FC) and 0.8 (80% FC). Three significant quantitative trait loci (QTLs) associated with A:E, two on SBI-09 and one on SBI-10, accounted for 17-21% of the phenotypic variation. Subsequent experiments identified 38 QTLs controlling variation in height, flowering, biomass, leaf area, greenness and stomatal density. Colocalisation of A : E QTLs with agronomic traits indicated that these QTLs can be used for improving sorghum performance through marker assisted selection (MAS) under preflowering drought stress.

Recombinant inbred populations of many plant species exhibit more heterozygosity than expected under the Mendelian model of segregation. This segregation distortion causes the overestimation of recombination frequencies and consequent genetic map expansion. Here we build upon existing genetic models of differential zygotic viability to model a heterozygote fitness term and calculate expected genotypic proportions in recombinant inbred populations propagated by selfing. We implement this model using the existing open-source genetic map construction code base for R/qtl to estimate recombination fractions. Finally, we show that accounting for excess heterozygosity in a sorghum recombinant inbred mapping population shrinks the genetic map by 213 cM (a 13% decrease corresponding to 4.26 fewer recombinations per meiosis). More accurate estimates of linkage benefit linkage-based analyses used in the identification and utilization of causal genetic variation., (Copyright © 2014 Truong et al.)

Light signaling by phytochrome B in long days inhibits flowering in sorghum by increasing expression of the long day floral repressors PSEUDORESPONSE REGULATOR PROTEIN (SbPRR37, Ma1) and GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (SbGHD7, Ma6). SbPRR37 and SbGHD7 RNA abundance peaks in the morning and in the evening of long days through coordinate regulation by light and output from the circadian clock. 58 M, a phytochrome B deficient (phyB-1, ma3R) genotype, flowered ∼60 days earlier than 100 M (PHYB, Ma3) in long days and ∼11 days earlier in short days. Populations derived from 58 M (Ma1, ma3R, Ma5, ma6) and R.07007 (Ma1, Ma3, ma5, Ma6) varied in flowering time due to QTL aligned to PHYB/phyB-1 (Ma3), Ma5, and GHD7/ghd7-1 (Ma6). PHYC was proposed as a candidate gene for Ma5 based on alignment and allelic variation. PHYB and Ma5 (PHYC) were epistatic to Ma1 and Ma6 and progeny recessive for either gene flowered early in long days. Light signaling mediated by PhyB was required for high expression of the floral repressors SbPRR37 and SbGHD7 during the evening of long days. In 100 M (PHYB) the floral activators SbEHD1, SbCN8 and SbCN12 were repressed in long days and de-repressed in short days. In 58 M (phyB-1) these genes were highly expressed in long and short days. Furthermore, SbCN15, the ortholog of rice Hd3a (FT), is expressed at low levels in 100 M but at high levels in 58 M (phyB-1) regardless of day length, indicating that PhyB regulation of SbCN15 expression may modify flowering time in a photoperiod-insensitive manner.

Stay-green is an integrated drought adaptation trait characterized by a distinct green leaf phenotype during grain filling under terminal drought. We used sorghum (Sorghum bicolor), a repository of drought adaptation mechanisms, to elucidate the physiological and genetic mechanisms underpinning stay-green. Near-isogenic sorghum lines (cv RTx7000) were characterized in a series of field and managed-environment trials (seven experiments and 14 environments) to determine the influence of four individual stay-green (Stg1-4) quantitative trait loci (QTLs) on canopy development, water use and grain yield under post-anthesis drought. The Stg QTL decreased tillering and the size of upper leaves, which reduced canopy size at anthesis. This reduction in transpirational leaf area conserved soil water before anthesis for use during grain filling. Increased water uptake during grain filling of Stg near-isogenic lines (NILs) relative to RTx7000 resulted in higher post-anthesis biomass production, grain number and yield. Importantly, there was no consistent yield penalty associated with the Stg QTL in the irrigated control. These results establish a link between the role of the Stg QTL in modifying canopy development and the subsequent impact on crop water use patterns and grain yield under terminal drought., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Background: Sorghum genotypes used for grain production in temperate regions are photoperiod insensitive and flower early avoiding adverse environments during the reproductive phase. In contrast, energy sorghum hybrids are highly photoperiod sensitive with extended vegetative phases in long days, resulting in enhanced biomass accumulation. SbPRR37 and SbGHD7 contribute to photoperiod sensitivity in sorghum by repressing expression of SbEHD1 and FT-like genes, thereby delaying flowering in long days with minimal influence in short days (PNAS_108:16469-16474, 2011; Plant Genome_in press, 2014). The GIGANTEA (GI)-CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway regulates flowering time in Arabidopsis and the grasses (J Exp Bot_62:2453-2463, 2011). In long day flowering plants, such as Arabidopsis and barley, CONSTANS activates FT expression and flowering in long days. In rice, a short day flowering plant, Hd1, the ortholog of CONSTANS, activates flowering in short days and represses flowering in long days., Results: Quantitative trait loci (QTL) that modify flowering time in sorghum were identified by screening Recombinant Inbred Lines (RILs) derived from BTx642 and Tx7000 in long days, short days, and under field conditions. Analysis of the flowering time QTL on SBI-10 revealed that BTx642 encodes a recessive CONSTANS allele containing a His106Tyr substitution in B-box 2 known to inactivate CONSTANS in Arabidopsis thaliana. Genetic analysis characterized sorghum CONSTANS as a floral activator that promotes flowering by inducing the expression of EARLY HEADING DATE 1 (SbEHD1) and sorghum orthologs of the maize FT genes ZCN8 (SbCN8) and ZCN12 (SbCN12). The floral repressor PSEUDORESPONSE REGULATOR PROTEIN 37 (PRR37) inhibits sorghum CONSTANS activity and flowering in long days., Conclusion: Sorghum CONSTANS is an activator of flowering that is repressed post-transcriptionally in long days by the floral inhibitor PRR37, contributing to photoperiod sensitive flowering in Sorghum bicolor, a short day plant.

Background: Rapid acquisition of accurate genotyping information is essential for all genetic marker-based studies. For species with relatively small genomes, complete genome resequencing is a feasible approach for genotyping; however, for species with large and highly repetitive genomes, the acquisition of whole genome sequences for the purpose of genotyping is still relatively inefficient and too expensive to be carried out on a high-throughput basis. Sorghum bicolor is a C4 grass with a sequenced genome size of ~730 Mb, of which ~80% is highly repetitive. We have developed a restriction enzyme targeted genome resequencing method for genetic analysis, termed Digital Genotyping (DG), to be applied to sorghum and other grass species with large repeat-rich genomes., Results: DG templates are generated using one of three methylation sensitive restriction enzymes that recognize a nested set of 4, 6 or 8 bp GC-rich sequences, enabling varying depth of analysis and integration of results among assays. Variation in sequencing efficiency among DG markers was correlated with template GC-content and length. The expected DG allele sequence was obtained 97.3% of the time with a ratio of expected to alternative allele sequence acquisition of >20:1. A genetic map aligned to the sorghum genome sequence with an average resolution of 1.47 cM was constructed using 1,772 DG markers from 137 recombinant inbred lines. The DG map enhanced the detection of QTL for variation in plant height and precisely aligned QTL such as Dw3 to underlying genes/alleles. Higher-resolution NgoMIV-based DG haplotypes were used to trace the origin of DNA on SBI-06, spanning Ma1 and Dw2 from progenitors to BTx623 and IS3620C. DG marker analysis identified the correct location of two miss-assembled regions and located seven super contigs in the sorghum reference genome sequence., Conclusion: DG technology provides a cost-effective approach to rapidly generate accurate genotyping data in sorghum. Currently, data derived from DG are used for many marker-based analyses, including marker-assisted breeding, pedigree and QTL analysis, genetic map construction, map-based gene cloning and association studies. DG in combination with whole genome resequencing is dramatically accelerating all aspects of genetic analysis of sorghum, an important genetic reference for C4 grass species.

Optimal flowering time is critical to the success of modern agriculture. Sorghum is a short-day tropical species that exhibits substantial photoperiod sensitivity and delayed flowering in long days. Genotypes with reduced photoperiod sensitivity enabled sorghum's utilization as a grain crop in temperate zones worldwide. In the present study, Ma(1), the major repressor of sorghum flowering in long days, was identified as the pseudoresponse regulator protein 37 (PRR37) through positional cloning and analysis of SbPRR37 alleles that modulate flowering time in grain and energy sorghum. Several allelic variants of SbPRR37 were identified in early flowering grain sorghum germplasm that contain unique loss-of-function mutations. We show that in long days SbPRR37 activates expression of the floral inhibitor CONSTANS and represses expression of the floral activators Early Heading Date 1, FLOWERING LOCUS T, Zea mays CENTRORADIALIS 8, and floral induction. Expression of SbPRR37 is light dependent and regulated by the circadian clock, with peaks of RNA abundance in the morning and evening in long days. In short days, the evening-phase expression of SbPRR37 does not occur due to darkness, allowing sorghum to flower in this photoperiod. This study provides insight into an external coincidence mechanism of photoperiodic regulation of flowering time mediated by PRR37 in the short-day grass sorghum and identifies important alleles of SbPRR37 that are critical for the utilization of this tropical grass in temperate zone grain and bioenergy production.

Apomixis, asexual reproduction through seed, is widespread among angiosperm families. Gametophytic apomixis in Pennisetum squamulatum and Cenchrus ciliaris is controlled by the apospory-specific genomic region (ASGR), which is highly conserved and macrosyntenic between these species. Thirty-two ASGR bacterial artificial chromosomes (BACs) isolated from both species and one ASGR-recombining BAC from P. squamulatum, which together cover approximately 2.7 Mb of DNA, were used to investigate the genomic structure of this region. Phrap assembly of 4,521 high-quality reads generated 1,341 contiguous sequences (contigs; 730 from the ASGR and 30 from the ASGR-recombining BAC in P. squamulatum, plus 580 from the C. ciliaris ASGR). Contigs containing putative protein-coding regions unrelated to transposable elements were identified based on protein similarity after Basic Local Alignment Search Tool X analysis. These putative coding regions were further analyzed in silico with reference to the rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes using the resources at Gramene (www.gramene.org) and Phytozome (www.phytozome.net) and by hybridization against sorghum BAC filters. The ASGR sequences reveal that the ASGR (1) contains both gene-rich and gene-poor segments, (2) contains several genes that may play a role in apomictic development, (3) has many classes of transposable elements, and (4) does not exhibit large-scale synteny with either rice or sorghum genomes but does contain multiple regions of microsynteny with these species.

Background: Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps., Results: A total of 110,592 maize BAC clones (approximately 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize 12., Conclusion: This BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information.

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.

Cytogenetic maps of sorghum chromosomes 3-7, 9, and 10 were constructed on the basis of the fluorescence in situ hybridization (FISH) of approximately 18-30 BAC probes mapped across each of these chromosomes. Distal regions of euchromatin and pericentromeric regions of heterochromatin were delimited for all 10 sorghum chromosomes and their DNA content quantified. Euchromatic DNA spans approximately 50% of the sorghum genome, ranging from approximately 60% of chromosome 1 (SBI-01) to approximately 33% of chromosome 7 (SBI-07). This portion of the sorghum genome is predicted to encode approximately 70% of the sorghum genes ( approximately 1 gene model/12.3 kbp), assuming that rice and sorghum encode a similar number of genes. Heterochromatin spans approximately 411 Mbp of the sorghum genome, a region characterized by a approximately 34-fold lower rate of recombination and approximately 3-fold lower gene density compared to euchromatic DNA. The sorghum and rice genomes exhibit a high degree of macrocolinearity; however, the sorghum genome is approximately 2-fold larger than the rice genome. The distal euchromatic regions of sorghum chromosomes 3-7 and 10 are approximately 1.8-fold larger overall and exhibit an approximately 1.5-fold lower average rate of recombination than the colinear regions of the homeologous rice chromosomes. By contrast, the pericentromeric heterochromatic regions of these chromosomes are on average approximately 3.6-fold larger in sorghum and recombination is suppressed approximately 15-fold compared to the colinear regions of rice chromosomes.

Improved knowledge of the sorghum transcriptome will enhance basic understanding of how plants respond to stresses and serve as a source of genes of value to agriculture. Toward this goal, Sorghum bicolor L. Moench cDNA libraries were prepared from light- and dark-grown seedlings, drought-stressed plants, Colletotrichum-infected seedlings and plants, ovaries, embryos, and immature panicles. Other libraries were prepared with meristems from Sorghum propinquum (Kunth) Hitchc. that had been photoperiodically induced to flower, and with rhizomes from S. propinquum and johnsongrass (Sorghum halepense L. Pers.). A total of 117,682 expressed sequence tags (ESTs) were obtained representing both 3' and 5' sequences from about half that number of cDNA clones. A total of 16,801 unique transcripts, representing tentative UniScripts (TUs), were identified from 55,783 3' ESTs. Of these TUs, 9,032 are represented by two or more ESTs. Collectively, these libraries were predicted to contain a total of approximately 31,000 TUs. Individual libraries, however, were predicted to contain no more than about 6,000 to 9,000, with the exception of light-grown seedlings, which yielded an estimate of close to 13,000. In addition, each library exhibits about the same level of complexity with respect to both the number of TUs preferentially expressed in that library and the frequency with which two or more ESTs is found in only that library. These results indicate that the sorghum genome is expressed in highly selective fashion in the individual organs and in response to the environmental conditions surveyed here. Close to 2,000 differentially expressed TUs were identified among the cDNA libraries examined, of which 775 were differentially expressed at a confidence level of 98%. From these 775 TUs, signature genes were identified defining drought, Colletotrichum infection, skotomorphogenesis (etiolation), ovary, immature panicle, and embryo.

With an aim to clone the sorghum fertility restorer gene Rf1, a high-resolution genetic and physical map of the locus was constructed. The Rf1 locus was resolved to a 32-kb region spanning four open reading frames: a plasma membrane Ca(2+)-ATPase, a cyclin D-1, an unknown protein, and a pentatricopeptide repeat (PPR13) gene family member. An approximately 19-kb region spanning the cyclin D-1 and unknown protein genes was completely conserved between sterile and fertile plants as was the sequence spanning the coding region of the Ca(2+)-ATPase. In contrast, 19 sequence polymorphisms were located in an approximately 7-kb region spanning PPR13, and all markers cosegregated with the fertility restoration phenotype. PPR13 was predicted to encode a mitochondrial-targeted protein containing a single exon with 14 PPR repeats, and the protein is classified as an E-type PPR subfamily member. To permit sequence-based comparison of the sorghum and rice genomes in the Rf1 region, 0.53 Mb of sorghum chromosome 8 was sequenced and compared to the colinear region of rice chromosome 12. Genome comparison revealed a mosaic pattern of colinearity with an approximately 275-kb gene-poor region with little gene conservation and an adjacent, approximately 245-kb gene-rice region that is more highly conserved between rice and sorghum. Despite being located in a region of high gene conservation, sorghum PPR13 was not located in a colinear position on rice chromosome 12. The present results suggest that sorghum PPR13 represents a potential candidate for the sorghum Rf1 gene, and its presence in the sorghum genome indicates a single gene transposition event subsequent to the divergence of rice and sorghum ancestors.

Genome wide changes in gene expression were monitored in the drought tolerant C4 cereal Sorghum bicolor, following exposure of seedlings to high salinity (150 mM NaCl), osmotic stress (20% polyethylene glycol) or abscisic acid (125 microM ABA). A sorghum cDNA microarray providing data on 12,982 unique gene clusters was used to examine gene expression in roots and shoots at 3- and 27-h post-treatment. Expression of approximately 2200 genes, including 174 genes with currently unknown functions, of which a subset appear unique to monocots and/or sorghum, was altered in response to dehydration, high salinity or ABA. The modulated sorghum genes had homology to proteins involved in regulation, growth, transport, membrane/protein turnover/repair, metabolism, dehydration protection, reactive oxygen scavenging, and plant defense. Real-time PCR was used to quantify changes in relative mRNA abundance for 333 genes that responded to ABA, NaCl or osmotic stress. Osmotic stress inducible sorghum genes identified for the first time included a beta-expansin expressed in shoots, actin depolymerization factor, inositol-3-phosphate synthase, a non-C4 NADP-malic enzyme, oleosin, and three genes homologous to 9-cis-epoxycarotenoid dioxygenase that may be involved in ABA biosynthesis. Analysis of response profiles demonstrated the existence of a complex gene regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to ABA, high salinity and water deficit. Modulation of genes involved in signal transduction, chromatin structure, transcription, translation and RNA metabolism contributes to sorghum's overlapping but nonetheless distinct responses to ABA, high salinity, and osmotic stress. Overall, this study provides a foundation of information on sorghum's osmotic stress responsive gene complement that will accelerate follow up biochemical, QTL and comparative studies.

We have conducted a large-scale study of gene expression in the C4 monocot sorghum (Sorghum bicolor) L. Moench cv BTx623 in response to the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA), and the ethylene precursor aminocyclopropane carboxylic acid. Expression profiles were generated from seedling root and shoot tissue at 3 and 27 h, using a microarray containing 12,982 nonredundant elements. Data from 102 slides and quantitative reverse transcription-PCR data on mRNA abundance from 171 genes were collected and analyzed and are here made publicly available. Numerous gene clusters were identified in which expression was correlated with particular signaling compound and tissue combinations. Many genes previously implicated in defense responded to the treatments, including numerous pathogenesis-related genes and most members of the phenylpropanoid pathway, and several other genes that may represent novel activities or pathways. Genes of the octadecanoic acid pathway of jasmonic acid (JA) synthesis were induced by SA as well as by MeJA. The resulting hypothesis that increased SA could lead to increased endogenous JA production was confirmed by measurement of JA content. Comparison of responses to SA, MeJA, and combined SA+MeJA revealed patterns of one-way and mutual antagonisms, as well as synergistic effects on regulation of some genes. These experiments thus help further define the transcriptional results of cross talk between the SA and JA pathways and suggest that a subset of genes coregulated by SA and JA may comprise a uniquely evolved sector of plant signaling responsive cascades.

Linkage group identities and homologies were determined for metaphase chromosomes of Sorghum bicolor (2n = 20) by FISH of landed BACs. Relative lengths of chromosomes in FISH-karyotyped metaphase spreads of the elite inbred BTx623 were used to estimate the molecular size of each chromosome and to establish a size-based nomenclature for sorghum chromosomes (SBI-01-SBI-10) and linkage groups (LG-01 to LG-10). Lengths of arms were determined to orient linkage groups relative to a standard karyotypic layout (short arms at top). The size-based nomenclature for BTx623 represents a reasonable choice as the standard for a unified chromosome nomenclature for use by the sorghum research community.

To integrate genetic, physical, and cytological perspectives of the Sorghum bicolor genome, we selected 40 landed bacterial artificial chromosome (BAC) clones that contain different linkage map markers, 21 from linkage group 2 (LG-02) and 19 from linkage group 8 (LG-08). Multi-BAC probe cocktails were constructed for each chromosome from the landed BACs, which were also preevaluated for FISH signal quality, relative position, and collective chromosome coverage. Comparison to the corresponding linkage map revealed full concordance of locus order between cytological and prior segregation analyses. The pericentromeric heterochromatin constituted a large quasi-uniform block in each bivalent and was especially large in the bivalent corresponding to LG-08. Centromere positions in LG-02 and LG-08 were progressively delimited using FISH to identify landed BACs for which the FISH signals visibly flanked the centromere. Alignment of linkage and cytological maps revealed that pericentromeric heterochromatin of these sorghum chromosomes is largely devoid of recombination, which is mostly relegated to the more distal regions, which are largely euchromatic. This suggests that the sorghum genome is thus even more amenable to physical mapping of genes and positional cloning than the C-value alone might suggest. As a prelude to positional cloning of the fertility restorer, Rf1, FISH of BAC clones flanking the Rf1 locus was used to delimit the chromosomal position of the gene. FISH of BACs that contain the most proximal linkage markers enabled localization of Rf1 to a approximately 0.4-Mbp euchromatic region of LG-08. Cytogenetic analyses of Rf1 and other trait loci will aid in assessing the feasibility of positional cloning and help formulate strategies required for cloning this and other agriculturally critical genes.

Phylogenetic analysis of sequences from gene families and homologous genes from species of varying divergence can be used to identify conserved noncoding regulatory elements. In this study, phylogenetic analysis of 5'-noncoding sequences was optimized using rab17, a well-characterized ABA-responsive gene from maize, and five additional rab16/17 homologs from sorghum and rice. Conserved 5'-noncoding sequences among the maize, sorghum, and rice rab16/17 homologs were identified with the aid of the software program FootPrinter and by screening for known transcription-factor-binding sites. Searches for 7 of 8 (7/8)bp sequence matches within aligned 5'-noncoding segments of the rab genes identified many of the cis-elements previously characterized by biochemical analysis in maize rab17 plus several additional putative regulatory elements. Differences in the composition of conserved noncoding sequences among rab16/17 genes were related to variation in rab gene mRNA levels in different tissues and to response to ABA treatment using qRT-PCR. Absence of a GRA-like element in the promoter of sorghum dhn2 relative to maize rab17 was correlated with an approximately 85-fold reduction of dhn2 RNA in sorghum shoots. Overall, we conclude that phylogenetic analysis of gene families among rice, sorghum, and maize will help identify regulatory sequences in the noncoding regions of genes and contribute to our understanding of grass gene regulatory networks.

Gametophytic apomixis is asexual reproduction as a consequence of parthenogenetic development of a chromosomally unreduced egg. The trait leads to the production of embryos with a maternal genotype, i.e. progeny are clones of the maternal plant. The application of the trait in agriculture could be a tremendous tool for crop improvement through conventional and nonconventional breeding methods. Unfortunately, there are no major crops that reproduce by apomixis, and interspecific hybridization with wild relatives has not yet resulted in commercially viable germplasm. Pennisetum squamulatum is an aposporous apomict from which the gene(s) for apomixis has been transferred to sexual pearl millet by backcrossing. Twelve molecular markers that are linked with apomixis coexist in a tight linkage block called the apospory-specific genomic region (ASGR), and several of these markers have been shown to be hemizygous in the polyploid genome of P. squamulatum. High resolution genetic mapping of these markers has not been possible because of low recombination in this region of the genome. We now show the physical arrangement of bacterial artificial chromosomes containing apomixis-linked molecular markers by high resolution fluorescence in situ hybridization on pachytene chromosomes. The size of the ASGR, currently defined as the entire hemizygous region that hybridizes with apomixis-linked bacterial artificial chromosomes, was estimated on pachytene and mitotic chromosomes to be approximately 50 Mbp (a quarter of the chromosome). The ASGR includes highly repetitive sequences from an Opie-2-like retrotransposon family that are particularly abundant in this region of the genome.

The completed rice genome sequence will accelerate progress on the identification and functional classification of biologically important genes and serve as an invaluable resource for the comparative analysis of grass genomes. In this study, methods were developed for sequence-based alignment of sorghum and rice chromosomes and for refining the sorghum genetic/physical map based on the rice genome sequence. A framework of 135 BAC contigs spanning approximately 33 Mbp was anchored to sorghum chromosome 3. A limited number of sequences were collected from 118 of the BACs and subjected to BLASTX analysis to identify putative genes and BLASTN analysis to identify sequence matches to the rice genome. Extensive conservation of gene content and order between sorghum chromosome 3 and the homeologous rice chromosome 1 was observed. One large-scale rearrangement was detected involving the inversion of an approximately 59 cM block of the short arm of sorghum chromosome 3. Several small-scale changes in gene collinearity were detected, indicating that single genes and/or small clusters of genes have moved since the divergence of sorghum and rice. Additionally, the alignment of the sorghum physical map to the rice genome sequence allowed sequence-assisted assembly of an approximately 1.6 Mbp sorghum BAC contig. This streamlined approach to high-resolution genome alignment and map building will yield important information about the relationships between rice and sorghum genes and genomic segments and ultimately enhance our understanding of cereal genome structure and evolution.

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.

A "gene-island" sequencing strategy has been developed that expedites the targeted acquisition of orthologous gene sequences from related species for comparative genome analysis. A 152-kb bacterial artificial chromosome (BAC) clone from sorghum (Sorghum bicolor) encoding phytochrome A (PHYA) was fully sequenced, revealing 16 open reading frames with a gene density similar to many regions of the rice (Oryza sativa) genome. The sequences of genes in the orthologous region of the maize (Zea mays) and rice genomes were obtained using the gene-island sequencing method. BAC clones containing the orthologous maize and rice PHYA genes were identified, sheared, subcloned, and probed with the sorghum PHYA-containing BAC DNA. Sequence analysis revealed that approximately 75% of the cross-hybridizing subclones contained sequences orthologous to those within the sorghum PHYA BAC and less than 25% contained repetitive and/or BAC vector DNA sequences. The complete sequence of four genes, including up to 1 kb of their promoter regions, was identified in the maize PHYA BAC. Nine orthologous gene sequences were identified in the rice PHYA BAC. Sequence comparison of the orthologous sorghum and maize genes aided in the identification of exons and conserved regulatory sequences flanking each open reading frame. Within genomic regions where micro-colinearity of genes is absolutely conserved, gene-island sequencing is a particularly useful tool for comparative analysis of genomes between related species.

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.

A high-resolution genetic, physical, and cytological map of the sorghum genome is being assembled using AFLP DNA marker technology, six-dimensional pooling of BAC libraries, cDNA mapping technology, and cytogenetic analysis. Recent advances in sorghum comparative genomics and gene-transfer technology are accelerating the discovery and utilization of valuable sorghum genes and alleles.