Domestication to crop improvement: Genetic resources for sorghum and saccharum (Andropogoneae)

Background Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum) are members of the Andropogoneae tribe in the Poaceae and are each other's closest relatives amongst cultivated plants. Both are relatively recent domesticates and comparatively little of the genetic potential of these taxa and their wild relatives has been captured by breeding programmes to date. This review assesses the genetic gains made by plant breeders since domestication and the progress in the characterization of genetic resources and their utilization in crop improvement for these two related species. Genetic Resources The genome of sorghum has recently been sequenced providing a great boost to our knowledge of the evolution of grass genomes and the wealth of diversity within S. bicolor taxa. Molecular analysis of the Sorghum genus has identified close relatives of S. bicolor with novel traits, endosperm structure and composition that may be used to expand the cultivated gene pool. Mutant populations (including TILLING populations) provide a useful addition to genetic resources for this species. Sugarcane is a complex polyploid with a large and variable number of copies of each gene. The wild relatives of sugarcane represent a reservoir of genetic diversity for use in sugarcane improvement. Techniques for quantitative molecular analysis of gene or allele copy number in this genetically complex crop have been developed. SNP discovery and mapping in sugarcane has been advanced by the development of high-throughput techniques for ecoTILLING in sugarcane. Genetic linkage maps of the sugarcane genome are being improved for use in breeding selection. The improvement of both sorghum and sugarcane will be accelerated by the incorporation of more diverse germplasm into the domesticated gene pools using molecular tools and the improved knowledge of these genomes.

Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide from Sorghum bicolor L. Moench

Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

Development of a transformation system for sorghum (Sorghum bicolor L.)

Sorghum (Sorghum bicolor (L.) Moench) is the world’s fifth major cereal crop and holds importance as a construction material, food and fodder source. More recently, the potential of this plant as a biofuel source has been noted. Despite its agronomic importance, the use of sorghum production is being constrained by both biotic and abiotic factors. These challenges could be addressed by the use of genetic engineering strategies to complement conventional breeding techniques. However, sorghum is one of the most recalcitrant crops for genetic modification with the lack of an efficient tissue culture system being amongst the chief reasons. Therefore, the aim of this study was to develop an efficient tissue culture system for establishing regenerable embryogenic cell lines, micropropagation and acclimatisation for Sorghum bicolor and use this to optimise parameters for genetic transformation via Agrobacterium-mediated transformation and microprojectile bombardment. Using five different sorghum cultivars, SA281, 296B, SC49, Wray and Rio, numerous parameters were investigated in an attempt to establish an efficient and reproducible tissue culture and transformation system. Using immature embryos (IEs) as explants, regenerable embryogenic cell lines (ECLs) could only be established from cultivars SA281 and 296B. Large amounts of phenolics were produced from IEs of cultivars, SC49, Wary and Rio, and these compounds severely hindered callus formation and development. Cultivar SA281 also produced phenolics during regeneration. Attempts to suppress the production of these compounds in cultivars SA281 and SC49 using activated charcoal, PVP, ascorbic acid, citric acid and liquid filter paper bridge methods were either ineffective or had a detrimental effect on embryogenic callus formation, development and regeneration. Immature embryos sourced during summer were found to be far more responsive in vitro than those sourced during winter. In an attempt to overcome this problem, IEs were sourced from sorghum grown under summer conditions in either a temperature controlled glasshouse or a growth chamber. However, the performance of these explants was still inferior to that of natural summer-sourced explants. Leaf whorls, mature embryos, shoot tips and leaf primordia were found to be unsuitable as explants for establishing ECLs in sorghum cultivars SA281 and 296B. Using the florets of immature inflorescences (IFs) as explants, however, ECLs were established and regenerated for these cultivars, as well as for cultivar Tx430, using callus induction media, SCIM, and regeneration media, VWRM. The best in vitro responses, from the largest possible sized IFs, were obtained using plants at the FL-2 stage (where the last fully opened leaf was two leaves away from the flag leaf). Immature inflorescences could be stored at 25oC for up to three days without affecting their in vitro responses. Compared to IEs, the IFs were more robust in tissue culture and showed responses which were season and growth condition independent. A micropropagation protocol for sorghum was developed in this study. The optimum plant growth regulator (PGR) combination for the micropropagation of in vitro regenerated plantlets was found to be 1.0 mg/L BAP in combination with 0.5 mg/L NAA. With this protocol, cultivars 296B and SA281 produced an average of 57 and 13 off-shoots per plantlet, respectively. The plantlets were successfully acclimatised and developed into phenotypically normal plants that set seeds. A simplified acclimatisation protocol for in vitro regenerated plantlets was also developed. This protocol involved deflasking in vitro plantlets with at least 2 fully-opened healthy leaves and at least 3 roots longer than 1.5 cm, washing the media from the roots with running tap water, planting in 100 mm pots and placing in plastic trays covered with a clear plastic bag in a plant growth chamber. After seven days, the corners of the plastic cover were opened and the bags were completely removed after 10 days. All plantlets were successfully acclimatised regardless of whether 1:1 perlite:potting mix, potting mix, UC mix or vermiculite were used as potting substrates. Parameters were optimised for Agrobacterium-mediated transformation (AMT) of cultivars SA281, 296B and Tx430. The optimal conditions were the use of Agrobacterium strain LBA4404 at an inoculum density of 0.5 OD600nm, heat shock at 43oC for 3 min, use of the surfactant Pluronic F-68 (0.02% w/v) in the inoculation media with a pH of 5.2 and a 3 day co-cultivation period in dark at 22oC. Using these parameters, high frequencies of transient GFP expression was observed in IEs precultured on callus initiation media for 1-7 days as well as in four weeks old IE- and IF-derived callus. Cultivar SA281 appeared very sensitive to Agrobacterium since all tissue turned necrotic within two weeks post-exposure. For cultivar 296B, GFP expression was observed up to 20 days post co-cultivation but no stably transformed plants were regenerated. Using cultivar Tx430, GFP was expressed for up to 50 days post co-cultivation. Although no stably transformed plants of this cultivar were regenerated, this was most likely due to the use of unsuitable regeneration media. Parameters were optimised for transformation by particle bombardment (PB) of cultivars SA281, 296B and Tx430. The optimal conditions were use of 3-7 days old IEs and 4 weeks old IF callus, 4 hour pre- and post-bombardment osmoticum treatment, use of 0.6 µm gold microparticles, helium pressure of 1500 kPa and target distance of 15 cm. Using these parameters for PB, transient GFP expression was observed for up to 14, 30 and 50 days for cultivars SA281, 296B and Tx430, respectively. Further, the use of PB resulted in less tissue necrosis compared to AMT for the respective cultivars. Despite the presence of transient GFP expression, no stably transformed plants were regenerated. The establishment of regenerable ECLs and the optimization of AMT and PB parameters in this study provides a platform for future efforts to develop an efficient transformation protocol for sorghum. The development of GM sorghum will be an important step towards improving its agronomic properties as well as its exploitation for biofuel production.

Caustic potash pulping study of water efficient Arudno donax and Sorghum bicolor; a comparison to sugarcane bagasse

Numerous crops grow in sugar regions that have the potential to increase the amount of biomass available to a small bagasse-based pulp factory. Arundo donax and Sorghum offer unique advantages to farmers compared to other agricultural crops. Sorghum bicolour requires only 1/3 of the water of sugarcane. Arundo donax is a very high yield crop, it can also grow with little water but it has the further advantage in that it is also highly stress tolerant, making it suitable for land which is unsuited to other crops. Pulps produced from these crops were benchmarked against sugarcane bagasse pulp. Arundo, sorghum and bagasse were pulped using KOH and anthraquinone to 20 Kappa number so as to produce a bleachable pulp. The unbleached sorghum pulp has better tensile strength properties than the unbleached Arundo pulp (43.8 Nm/g compared to 21.4 Nm/g) and the bleached sorghum pulp tensile strength was similar to bagasse (28.4 Nm/g). At 20 Kappa number, sorghum pulp had acceptable yield for a non-wood fibre (45% c.f. 55% for bagasse), Arundo donax pulp had low tensile strength, and relatively low yield (38.7%), even for an agricultural fibre and required severe cooking conditions to achieve similar delignification to sugarcane bagasse or sorghum. Sorghum and Arundo donax produced thicker handsheets than bagasse (>160 μm c.f. 122 μm for bagasse). In preliminary experiments sorghum and bagasse responded slightly better to Totally Chlorine Free bleaching (QPP), although none achieved a satisfactory brightness level and more optimisation is needed.

Whole-genome sequencing reveals untapped genetic potential in Africa’s indigenous cereal crop sorghum

Sorghum is a food and feed cereal crop adapted to heat and drought and a staple for 500 million of the world’s poorest people. Its small diploid genome and phenotypic diversity make it an ideal C4 grass model as a complement to C3 rice. Here we present high coverage (16–45 × ) resequenced genomes of 44 sorghum lines representing the primary gene pool and spanning dimensions of geographic origin, end-use and taxonomic group. We also report the first resequenced genome of S. propinquum, identifying 8 M high-quality SNPs, 1.9 M indels and specific gene loss and gain events in S. bicolor. We observe strong racial structure and a complex domestication history involving at least two distinct domestication events. These assembled genomes enable the leveraging of existing cereal functional genomics data against the novel diversity available in sorghum, providing an unmatched resource for the genetic improvement of sorghum and other grass species.

Sweet sorghum : opportunities for a new, renewable fuel and food industry in Australia

Sweet sorghum is receiving significant global interest as an agro-industrial crop because of its capacity to co-produce energy, food, and feed products in integrated biorefineries. This report assesses the opportunities to develop a sweet sorghum industry in Australia, reports on research demonstrating the production of energy, food, and feed products, and assesses the potential economic and sustainability benefits of sweet sorghum biorefineries in the Australian context.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

The effect of row configuration on yield reliability in grain sorghum: II. Modelling the effects of row configuration

In recent years many sorghum producers in the more marginal (<600 mm annual rainfall) cropping areas of Qld and northern NSW have utilised skip row configurations in an attempt to improve yield reliability and reduce sorghum production risk. But will this work in the long run? What are the trade-offs between productivity and risk of crop failure? This paper describes a modelling and simulation approach to study the long-term effects of skip row configurations. Detailed measurements of light interception and water extraction from sorghum crops grown in solid, single and double skip row configurations were collected from three on-farm participatory research trials established in southern Qld and northern NSW. These measurements resulted in changes to the model that accounted for the elliptical water uptake pattern below the crop row and reduced total light interception associated with the leaf area reduction of the skip configuration. Following validation of the model, long-term simulation runs using historical weather data were used to determine the value of skip row sorghum production as a means of maintaining yield reliability in the dryland cropping regions of southern Qld and northern NSW.

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

Response By Dairy Cows Grazing Tropical Grass Pasture To Barley or Sorghum Grain Based Concentrates and Lucerne Hay

This study investigated the responses by dairy cows grazing Callide Rhodes grass (Chloris gayana cv. Callide) pasture to supplementation with barley or sorghum based concentrates (5 grain:1 cotton seed meal) or barley concentrate plus lucerne (Medicago sativa) hay. It was conducted in summer - autumn 1999 with 20 spring calved cows in 4 treatments in 3 consecutive periods of 4 weeks. Rain grown pastures, heavily stocked at 4.4 cows/ha, provided 22 to 35 kg green DM and 14 to 16 kg green leaf DM/cow.day in periods 1 to 3. Supplements were fed individually twice daily after milking. Cows received 6 kg concentrate/day in period 1, increased by 1 kg/day as barley, sorghum or lucerne chaff in each of periods 2 and 3. The Control treatment received 6 kg barley concentrate in all 3 periods. Milk yields by cows fed sorghum were lower than for cows fed equivalent levels of barley-based concentrate (P<0.05). Faecal starch levels (14, 18 and 17%) for cows fed sorghum concentrate were much higher (P<0.01) than those of cows fed similar levels of barley (2.1, 1.2 and 1.7%) in each period respectively. Additional supplementation as lucerne chaff did not increase milk production (P>0.05). Increased concentrate supplementation did not alleviate the problem of low protein in milk produced by freshly calved Holstein-Friesian cows grazing tropical grass pasture in summer. Animal production for a consuming world : proceedings of 9th Congress of the Asian-Australasian Association of Animal Production Societies [AAAP] and 23rd Biennial Conference of the Australian Society of Animal Production [ASAP] and 17th Annual Symposium of the University of Sydney, Dairy Research Foundation, [DRF]. 2-7 July 2000, Sydney, Australia.

Feeding sorghum ergot (Claviceps africana) to sows before farrowing inhibits milk production

Objective: To assess the impact of feeding different amounts of sorghum ergot to sows before farrowing. Design: Fifty-one pregnant sows from a continually farrowing piggery were sequentially inducted into the experiment each week in groups of four to seven, as they approached within 14 days of farrowing. Diets containing sorghum ergot sclerotia within the range of 0 (control) up to 1.5% w/w (1.5% ergot provided 7 mg alkaloids/kg, including 6 mg dihydroergosine/kg) were randomly allocated and individually fed to sows. Ergot concentrations were varied with each subsequent group until an acceptable level of tolerance was achieved. Diets with ergot were replaced with control diets after farrowing. Post-farrowing milk production was assessed by direct palpation and observation of udders, and by piglet responses and growth. Blood samples were taken from sows on three days each week, for prolactin estimation. Results: Three sows fed 1.5% ergot for 6 to 10 days preceding farrowing produced no milk, and 87% of their piglets died despite supplementary feeding of natural and artificial colostrums, milk replacer, and attempts to foster them onto normally lactating sows. Ergot inclusions of 0.6% to 1.2% caused lesser problems in milk release and neo-natal piglet mortality. Of 23 sows fed either 0.3% or 0.6% ergot, lactation of only two first-litter sows were affected. Ergot caused pronounced reductions in blood prolactin, and first-litter sows had lower plasma prolactin than multiparous sows, increasing their susceptibility to ergot. Conclusion: Sorghum ergot should not exceed 0.3% (1 mg alkaloid/kg) in diets of multiparous sows fed before farrowing, and should be limited to 0.1 % for primiparous sows, or avoided completely.

Effect of soil burial depth and wetting on mortality of diapausing larvae and patterns of post-diapause adult emergence of sorghum midge, Stenodiplosis sorghicola (Coquillett) (Diptera: Cecidomyiidae)

In south-eastern Queensland, Australia, sorghum planted in early spring usually escapes sorghum midge, Stenodiplosis sorghicola, attack. Experiments were conducted to better understand the role of winter diapause in the population dynamics of this pest. Emergence patterns of adult midge from diapausing larvae on the soil surface and at various depths were investigated during spring to autumn of 1987/88–1989/90. From 1987/88 to 1989/90, 89%, 65% and 98% of adult emergence, respectively, occurred during November and December. Adult emergence from larvae diapausing on the soil surface was severely reduced due to high mortality attributed to surface soil temperatures in excess of 40°C, with much of this mortality occurring between mid-September and mid-October. Emergence of adults from the soil surface was considerably delayed in the 1988/89 season compared with larvae buried at 5 or 10 cm which had similar emergence patterns for all three seasons. In 1989/90, when a 1-cm-deep treatment was included, there was a 392% increase in adult emergence from this treatment compared with deeper treatments. Some diapausing larvae on the surface did not emerge at the end of summer in only 1 year (1989/90), when 28.0% of the larvae on the surface remained in diapause, whereas only 0.8% of the buried larvae remained in diapause. We conclude that the pattern of emergence explains why spring plantings of sorghum in south-eastern Queensland usually escape sorghum midge attack.

Domestication to crop improvement: Genetic resources for Sorghum and Saccharum (Andropogoneae).

Background: Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum) are members of the Andropogoneae tribe in the Poaceae and are each other's closest relatives amongst cultivated plants. Both are relatively recent domesticates and comparatively little of the genetic potential of these taxa and their wild relatives has been captured by breeding programmes to date. This review assesses the genetic gains made by plant breeders since domestication and the progress in the characterization of genetic resources and their utilization in crop improvement for these two related species. Genetic Resources: The genome of sorghum has recently been sequenced providing a great boost to our knowledge of the evolution of grass genomes and the wealth of diversity within S. bicolor taxa. Molecular analysis of the Sorghum genus has identified close relatives of S. bicolor with novel traits, endosperm structure and composition that may be used to expand the cultivated gene pool. Mutant populations (including TILLING populations) provide a useful addition to genetic resources for this species. Sugarcane is a complex polyploid with a large and variable number of copies of each gene. The wild relatives of sugarcane represent a reservoir of genetic diversity for use in sugarcane improvement. Techniques for quantitative molecular analysis of gene or allele copy number in this genetically complex crop have been developed. SNP discovery and mapping in sugarcane has been advanced by the development of high-throughput techniques for ecoTILLING in sugarcane. Genetic linkage maps of the sugarcane genome are being improved for use in breeding selection. The improvement of both sorghum and sugarcane will be accelerated by the incorporation of more diverse germplasm into the domesticated gene pools using molecular tools and the improved knowledge of these genomes.

QTL analysis of ergot resistance in sorghum

Sorghum ergot, caused predominantly by Claviceps africana Frederickson, Mantle, de Milliano, is a significant threat to the sorghum industry worldwide. The objectives of this study were firstly, to identify molecular markers linked to ergot resistance and to two pollen traits, pollen quantity (PQ) and pollen viability (PV), and secondly, to assess the relationship between the two pollen traits and ergot resistance in sorghum. A genetic linkage map of sorghum RIL population R931945-2-2 x IS 8525 (resistance source) was constructed using 303 markers including 36 SSR, 117 AFLP™, 148 DArT™ and two morphological trait loci. Composite interval mapping identified nine, five, and four QTL linked to molecular markers for percentage ergot infection (PCERGOT), PQ and PV, respectively, at a LOD >2.0. Co-location/linkage of QTL were identified on four chromosomes while other QTL for the three traits mapped independently, indicating that both pollen and non pollen-based mechanisms of ergot resistance were operating in this sorghum population. Of the nine QTL identified for PCERGOT, five were identified using the overall data set while four were specific to the group data sets defined by temperature and humidity. QTL identified on SBI-02 and SBI-06 were further validated in additional populations. This is the first report of QTL associated with ergot resistance in sorghum. The markers reported herein could be used for marker-assisted selection for this important disease of sorghum.