1 Centre for Plant Conservation Genetics, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia
2 Australian Tropical Crops and Forages Centre, Queensland Department of Primary Industries and Fisheries, Biloela, QLD 4715, Australia
An open access article from Annals of Botany 2007 100(5):975-989.
Background: Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum)are members of the Andropogoneae tribe in the Poaceae and areeach other's closest relatives amongst cultivated plants. Bothare relatively recent domesticates and comparatively littleof the genetic potential of these taxa and their wild relativeshas been captured by breeding programmes to date. This reviewassesses the genetic gains made by plant breeders since domesticationand the progress in the characterization of genetic resourcesand their utilization in crop improvement for these two relatedspecies.
Genetic Resources: The genome of sorghum has recently been sequenced providinga great boost to our knowledge of the evolution of grass genomesand the wealth of diversity within S. bicolor taxa. Molecularanalysis of the Sorghum genus has identified close relativesof S. bicolor with novel traits, endosperm structure and compositionthat may be used to expand the cultivated gene pool. Mutantpopulations (including TILLING populations) provide a usefuladdition to genetic resources for this species. Sugarcane isa complex polyploid with a large and variable number of copiesof each gene. The wild relatives of sugarcane represent a reservoirof genetic diversity for use in sugarcane improvement. Techniquesfor quantitative molecular analysis of gene or allele copy numberin this genetically complex crop have been developed. SNP discoveryand mapping in sugarcane has been advanced by the developmentof high-throughput techniques for ecoTILLING in sugarcane. Geneticlinkage maps of the sugarcane genome are being improved foruse in breeding selection. The improvement of both sorghum andsugarcane will be accelerated by the incorporation of more diversegermplasm into the domesticated gene pools using molecular toolsand the improved knowledge of these genomes.
Key words: Genomics, sorghum, Sorghum bicolor, sugarcane, Saccharum officinarum, crop improvement, domestication
Arthropological evidence suggests that hunter-gatherers consumedsorghum as early as 8000 BC (Smith and Frederiksen, 2000). Thedomestication of sorghum has its origins in Ethiopia and surroundingcountries, commencing around 4000–3000 BC. Numerous varietiesof sorghum were created through the practice of disruptive selection,whereby selection for more than one level of a particular characterwithin a population occurs (Doggett, 1970). This results froma balance of farmer selection for cultivated traits and naturalselection for wild characteristics, generating both improvedsorghum types, wild types and intermediate types (Doggett, 1970).These improved sorghum types were spread via the movement ofpeople and trade routes into other regions of Africa, India(approx. 1500–1000 BC), the Middle East (approx. 900–700BC) and eventually into the Far East (approx. AD 400). By thetime sorghum was transported to America during the late 1800sto early 1900s, the diversity of new sorghum types, varietiesand races created through the movement of people, disruptiveselection, geographic isolation and recombination of these typesin different environments would have been large (Wright, 1931;Doggett, 1970). Initial domestication of sorghum would have focused primarilyon converting wild types with small, shattering (dehiscent)seed to improved types with larger, non-shattering seed. Disruptiveselection resulted in sorghum types with vastly different characteristicsin height, inflorescence type, and of course, end use (food,fodder, fibre, building materials, etc). Over time, sorghumhas been described and redescribed by numerous taxonomists (Fig. 1),and is now described under the family Poaceae, tribe Andropogoneae,subtribe Sorghinae and genus Sorghum Moench (Clayton and Renvoize, 1986).
The Sorghum genus as currently proscribed consists of 25 species(USDA ARS, 2007), although this varies in different scientificpublications confirming the dynamic nature of the classificationof cultivated sorghum and its wild relatives. The genus is separatedinto five taxonomic subgenera or sections: Eu-Sorghum, Chaetosorghum,Heterosorghum, Para-Sorghum and Stiposorghum (Garber, 1950).Section Eu-Sorghum contains all domesticated/cultivated sorghumraces and varieties as Sorghum bicolor subsp. bicolor, as wellas the wild and weed species S. halepense (L.) Pers. (Johnsonsgrass), S. propinquum (Kunth) Hitchc, S. x almum Parodi, S.x drummondii (Steud.) Millsp. & Chase, and S. arundinaceum(Desv.) Stapf. (the known progenitor of S. bicolor) (Harlan and de Wet, 1971;Doggett, 1988). All S. bicolor subsp. bicolor have 2n = 2x =20 chromosomes, and are described as annual, with thick culmsup to 5 m in height, often branched with many tillers. Theyhave been classified into five basic races: bicolor, guinea,caudatum, kafir and durra, with ten intermediate races of thesealso recognized (Harlan and de Wet, 1972). These 15 races ofcultivated sorghum are recognizable on spikelet/panicle morphologyalone, and can be linked back to their specific environmentsand the nomadic peoples that first cultivated them (Smith and Frederiksen, 2000).
A comprehensive analysis of genetic diversity in sorghum landracesand core collections based on race, latitude of origin, photoperiod,seed quality, agronomic traits and DNA markers has demonstratedsorghum has considerable polymorphism that has been poorly exploitedin terms of crop improvement (Wu et al., 2004; Abu Assar et al., 2005;Deu et al., 2006; Kayode et al., 2006). At the DNA level, twohigh-density maps have been completed, one intraspecific andanother from an interspecific cross (between S. bicolor andS. propinquum). These maps showed a high colinearity from whichthe divergence between Sorghum species and the diversity withincultivated S. bicolor has been indicated (Feltus et al., 2006).
Changing characteristics/traits of domesticated sorghum and effects on yield
Early domestication of sorghum was associated with changingthe small-seeded, shattering open panicles towards larger, non-shatteringseeds and more compact panicles. This involved several factors:significantly increasing the number of branches within the inflorescence;decreasing the internode length of the rachis; and an increasein seed size so it protruded out of the glumes (House, 1985).These changes contributed to an increase in yield over the originalsorghum landrace varieties.
Stable, high-yielding sorghum varieties have been recently developedthrough breeding/improvement programmes utilizing sorghum landracevarieties from Africa, India and China. This has involved selectingtraits such as photoperiod insensitivity, reduced height (toreduce lodging), drought tolerance, and pest and disease resistance(Reddy et al., 2006).
Plant height and photoperiod insensitivity were the focus ofconversion programmes that developed sorghum lines with desirableplant height and maturity that were usable in breeding programmesin both tropical, short-day environments and in long-day, temperateand subtropical environments. As sorghum originated in north-easternAfrica, the many landraces and early varieties were photoperiodsensitive, with a critical photoperiod of 12 h: once the daylength is shorter than 12 h, the sorghum plant changes fromvegetative to reproductive growth (Reddy et al., 2006). Growingthese photoperiod-sensitive landraces/lines as a summer cropin temperate zones of America and Australia where the day lengthis longer than 13 h was difficult, especially as many growth-relatedcharacteristics are poorly expressed under these long-day conditions(Reddy et al., 2006). This made breeding improved varietiesin semi-arid temperate and subtropical climates difficult. Cultivarsand landraces were identified in India that had higher criticalphotoperiods, with no delay in flowering observed when grownin day lengths up to 17 h. These photoperiod-insensitive sorghumcultivars have since been widely adopted in breeding programmesthroughout the world (Rai et al., 1999; Reddy et al., 2006).
Plant height and grain yield are highly correlated in some populationsof sorghum, with maximum productivity achieved at heights ofaround 1·75–1·80 m and flowering at 68–70d (Miller, 1982; Rao and Rana, 1982). However, plants of theseheights easily lodge, and are not easily cultivated under modernfarming practices. A selection of high-yielding, tall sorghumlandraces/lines were crossed to shorter, photoperiod-insensitivesorghum lines to develop improved high-yielding cultivars witha shorter stature (Miller, 1980; Rosenow and Dahlberg, 2000).
Sorghum is grown predominantly in low-rainfall, arid to semi-aridenvironments. The occurrence of drought stress is a major constraintto sorghum production globally. Two forms of drought stresshave been identified in sorghum: ‘pre-anthesis’where plants are stressed during panicle differentiation priorto flowering; and ‘post-anthesis’ when moisturestress occurs during the grain fill stage (Rosenow and Clark, 1995).The identification of varieties and lines with naturally highlevels of pre-anthesis drought tolerance and the selection ofthese for higher yields has developed sorghum varieties withstable, high yields (Ellis et al., 1997). Post-anthesis droughtstress can result in significant yield loss due to small grainsize, premature plant death and susceptibility to diseases.Post-flowering drought tolerance is referred to as stay-green,with plants maintaining green leaf area and photosynthetic capabilityunder severe moisture stress, which results in higher grainyields compared with senescent varieties (Borrell and Douglas, 1997;Borrell et al., 1999). The physiological components of stay-green(green leaf area at flowering; time of onset of senescence;rate of senescence) are independently inherited and easily combinedthrough breeding, resulting in new sorghum varieties exhibitinghigh levels of stay-green with stable high yields and good levelsof insect resistance (Borrell et al., 2000).
Sorghum production is affected by many pests and diseases globally.Some of the major pests include midge (Stenodiplosis sorghicolaCoquillett), green bug (Schizaphis graminum Rondani), variousaphids, shootfly (Atherigona soccata Rondani) and stem borer(Chilo partellus Swinhoe) (Sharma, 1993). Major diseases includedowny mildew, anthracnose, sorghum rust, leaf blight, ergotand head and kernel smut (House, 1985). Success in breedingfor insect resistance in sorghum varieties has been varied.Resistance to some pests is quantitatively inherited and thereforedifficult to transfer into high-yielding cultivars (Tao et al., 2003).The exception to this is midge resistance, where high levelsof midge immunity have been incorporated from Indian, Americanand Australian breeding lines into elite, high-yielding sorghumvarieties in Australia, with greater than 80 % of the plantedarea utilizing these resistant varieties (Jordan et al., 1998;Tao et al., 2003).
Development of disease-resistant sorghum varieties has reliedon identifying sorghum varieties/landraces with natural geneticresistance to the particular disease. To date, commercial sorghumvarieties have been developed with resistance to grain mouldsand anthracnose (Reddy et al., 2006).
The development of photoperiod-insensitive, dwarfed sorghumvarieties with some levels of pest/disease resistance has improvedthe yields of cultivated sorghum varieties. However, the developmentof a hybrid cropping system is responsible for increases inyields of more than 300 % since the 1950s (Rooney and Smith, 2000).Hybrid cultivars make use of male sterility to enhance the combiningabilities of the parental lines, resulting in heterosis andsignificant increases in phenotypic traits such as yield, plantheight and days to flowering (Reddy et al., 2006).
Although the domestication and resulting super-domesticationof sorghum has relied on principally S. bicolor subsp. bicolorvarieties/landraces/lines for significant gains in agriculturalproduction, the undomesticated Sorghum species offer an untappedwealth of novel traits for both biotic and abiotic stress resistanceand yield.
Undomesticated Sorghum species as genetic resources for sorghum improvement
All cultivated sorghum varieties and landraces are S. bicolorsubsp. bicolor of the Eu-Sorghum subgeneric section of the Sorghumgenus. The other four sections, Chaetosorghum, Heterosorghum,Para-Sorghum and Stiposorghum contain 19, wild species nativeto Africa, Asia and Australia (Garber, 1950; Lazarides et al., 1991).These species are briefly outlined below, and contain new sourcesof genetic diversity for agronomic traits affecting yield, survivabilityand novel traits that may create new markets for sorghum products.
The monotypic sections Chaetosorghum and Heterosorghum containthe octaploid (2n = 40) Australian species S. macrospermum E.D.Garber and S. laxiflorum F.M. Bailey, respectively. SectionPara-sorghum contains the five Australian species S. grandeLazarides, S. leiocladum (Hack.) C.E. Hubb., S. matarankenseE.D. Garber & Snyder, S. nitidum (Vahl) Pers., S. timorense(Kunth) Buse, and the two African/Asian species S. purpureo-sericeum(Hochst. ex A. Rich.) Asch. & Schweinf. and S. versicolorAndersson. These species range in ploidy from 2n = 10 to 2n= 40, with S. grande, S. nitidum and S. timorense showing varyingploidy within species. Ten Australian endemic species form sectionStiposorghum: Sorghum amplum Lazarides, S. angustum S.T. Blake,S. brachypodum Lazarides, S. bulbosum Lazarides, S. ecarinatumLazarides, S. exstans Lazarides, S. interjectum Lazarides, S.intrans F. Muell. ex Benth., S. plumosum (R. Br.) P. Beauv.,and S. stipoideum (Ewart & Jean White) C.A. Gardner &C.E. Hubb. (Garber, 1950; Lazarides et al., 1991). Most of thesespecies are diploid with 2n = 10 chromosomes, while S. interjectumhas 2n = 30, 40 and S. plumosum has 2n = 10, 20, 30 (Garber, 1950;Lazarides et al., 1991).
The adaptability of these undomesticated Sorghum species tocolonize a wide range of soil and moisture conditions acrossa wide range of microenvironments is shown through their abilityto survive very hot, dry, nutrient-limited environments. Dueto their adaptability, many of the undomesticated Sorghum specieshave developed resistances to the many pests and diseases thataffect sorghum grain production globally. Interestingly, manyAustralian undomesticated species contain resistances to themajor pest/diseases of Africa and America, which are not yetpresent within Australia (Bapat and Mote, 1982; Karunakar et al., 1994;Franzmann and Hardy, 1996; Sharma and Franzmann, 2001; Kamala et al., 2002;Komolong et al., 2002).
Recent controlled-environment glasshouse trials have shown thatthe undomesticated Sorghum species, though adapted to specificabiotic conditions in the wild, showed prolific growth undermoderate temperature in a standard potting mix and watered regularly(Table 1). These data show useful variations to germinationtimes and time to flowering. Representatives of the undomesticatedHeterosorghum, Para-Sorghum, Stiposorghum and a Eu-Sorghum weregrown concurrently to compare their development under controlledconditions (Fig. 2). Cultivated S. bicolor takes 3–10d to germinate depending on soil temperatures, with the first30–35 d post-germination undergoing lower leaf growthfollowed by a rapid elongation in non-dwarf varieties. Floweringin S. bicolor occurs 55–70 d post-germination and seedsreach physiological maturity 30–40 d post-anthesis. Itthen takes 20–25 d to reduce the moisture content to the12 % required for post-harvest storage (House et al., 1995).There appears to be limited differences between undomesticatedspecies and S. bicolor for these traits (Table 1).
Undomesticated Sorghum species: grain attributes
The morphology of seed size and shape within the Sorghum genusvaries greatly. Figure 3 shows the morphology of the domesticatedS. bicolor subsp. bicolor and undomesticated Eu-Sorghum, Chaetosorghum,Heterosorghum, Para-Sorghum and Stiposorghum species. Variationin the grain morphology of representatives of the undomesticatedHeterosorghum, Para-Sorghum and Stiposorghum species have alsobeen evaluated at the microscopic level. Mature caryopses of13 species were critically point dried, snap fractured and examinedusing a Leostereoscan 440 scanning electron microscope to determineif novel variations existed in the undomesticated species (Shapter et al., 2007).
The endosperm of cultivated S. bicolor is described as havingtwo distinct regions or layers. The floury central endosperm(Fig. 4A) contains simple round or lenticellar starch granulesin a discontinuous protein matrix with few if any protein bodiespresent. The vitreous or corneous outer endosperm (Fig. 4B)is characterized by polygonal starch granules, 4–25 µmin diameter, the surface of which is typically indented fromthe protein bodies that are part of the continuous protein matrixsurrounding the granules. Variations to the distribution andconfiguration of these two regions have been shown to alterthe functional and putatively the nutritional value of sorghumflours and other foods (Serna-Saldivar and Rooney, 1995; Lindeboom et al., 2004;Tesso et al., 2006).
The undomesticated Sorghum species showed varied distributionof protein bodies throughout the endosperm (Fig. 4C–H).Similarly, variation in the starch granule size and shape wasalso noted (Shapter et al., 2007). Some of the undomesticatedspecies had distinctly smaller, more spherical granules throughoutthe endosperm (Fig. 4D). Importantly, several species showednative channelling of the starch granules and pores on theirsurface (Fig. 4C) which have been shown to improve thedigestion of sorghum starches (Fannon et al., 2003, 2004; Benmoussa et al., 2006).One species appeared to have sections of the endosperm withsmall rice-like starch granules, usually only seen in the sub-aleuronelayer in S. bicolor (Shapter et al., 2007). Several wild speciesalso maintained a single morphology across the entire endosperm,rather than the two layers seen in S. bicolor. Amongst thesedifferences some species retained the characteristic morphologyof the S. bicolor vitreous layer (Fig. 4F). The sub-aleurone of S. bicolor is described as being 15–30µm wide and is an area of very small starch granules anddenser protein matrix, the endosperm proper (Fig. 5A andB).
In the Para-Sorghum and Stiposorghum species examined, areasof the sub-aleurone have a striated appearance (Fig. 5C)not previously reported in microscopy studies (F. M. Shapteret al., unpubl. res.). Investigation of these areas under highmagnification showed what appeared to be a much denser proteinmatrix, embedded with spherical-shaped bodies reminiscent ofprotein bodies. Within this layer, small starch granules typicalof sub-aleurone starch granules are interspersed (Fig. 5Dand E). More investigation is needed to confirm if this layeris proteinaceous. From an adaptive point of view, the developmentof a highly proteinaceous layer directly below the aleuronewould provide a rich nitrogen source for the germinating seedlingwhen establishing itself in low nitrogen soils, typical of northernAustralia where many of these undomesticated species are endemic.Protein/starch interactions in sorghum have been shown to decreasestarch digestibility, especially after cooking (Duodo et al., 2003).The occurrence of increased protein content in the endospermmay therefore result in a further decrease in starch digestibilitywhich has utility for raising the glycaemic index of foods forWestern diets.
Hybridizing potential of undomesticated sorghum species
Modern sorghum breeding programmes have not used species outsideof section Eu-Sorghum as sources of genetic diversity due toa lack of information regarding the genetic relationships betweenthe species. Recent phylogenetic analysis of all 25 Sorghumspecies based on the three gene sequences ITS1, ndhF and Adh1has identified S. macrospermum and S. laxiflorum as the undomesticatedspecies outside of Eu-Sorghum most closely related to cultivatedsorghum varieties (Dillon et al., 2007). The relationships identifiedcan now act as a guide for plant breeders.
Most of the undomesticated Sorghum species fall within the tertiarygenepool, making gene transfer to domesticated species verydifficult due to strong sterility barriers (Harlan and de Wet, 1971).The nature of the sterility barriers in Sorghum have recentlybeen identified as pollen–pistil incompatibilities wherebythe pollen of undomesticated species behaves abnormally in thepistils of S. bicolor, resulting in no hybrid embryo formation(Hodnett et al., 2005). As a result, pollen rarely grew beyondthe stigma of S. bicolor; however, a single embryo was formedusing S. macrospermum pollen. The embryo of this S. bicolorx S. macrospermum cross was rescued and raised through tissueculture, with the seedling verified as a hybrid based upon cytologicaland morphological characteristics (Price et al., 2005b).
Although a hybrid embryo was formed and able to be rescued viatissue culture, pollen–pistil incompatibilities make thisan extremely rare occurrence. Methods of increasing the frequencyof hybridization are required to successfully utilize the undomesticatedSorghum species. An S. bicolor accession was discovered containinga recessive gene (inhibition of alien pollen = iap) that allowedmaize (Zea mays L.) pollen tubes to grow through S. bicolorpistils (Laurie and Bennett, 1989). This S. bicolor accessioncan successfully override the pollen–pistil incompatibilitiesbetween S. bicolor and undomesticated Sorghum species and leadto the production of hybrid embryos and plants (Price et al., 2006).Hybrids between S. bicolor x S. macrospermum were obtained fromgerminated seeds, while the hybrids between S. bicolor x S.angustum and S. bicolor x S. nitidum were recovered throughembryo rescue and tissue culture. The hybrid nature of theseseedlings was again confirmed by the presence of genomes fromboth parental species that could be readily identified basedupon chromosome size and number (Price et al., 2006).
Introgression of the undomesticated S. macrospermum genome withcultivated S. bicolor has been tracked using FISH (fluorescentin situ hybridization) (Kuhlman et al., 2006). FISH discriminatedbetween the chromosomes of the two parent species, and confirmedthrough bivalent formation and allosyndetic pairing that recombinationwas occurring. Progeny of this novel hybrid when backcrossedto S. bicolor expressed altered fertility, again confirmingthat introgression from the undomesticated parent has occurred(Kuhlman et al., 2006). The analysis of the amount of DNA introgressedfrom the undomesticated S. macrospermum is currently being undertakenusing AFLPs (L. C. Kuhlman et al., unpubl. res.).
The identification and use of the iap S. bicolor accession hasenabled the successful introgression of genes from undomesticatedSorghum species into cultivated sorghum, and is the first steptowards accessing these unique unexploited genes for both bioticand abiotic stresses and agronomic traits. The potential forimproving the yield productivity through these traits in commercialsorghum varieties is now a reality.
The role of genomics in improving domesticated S. bicolor
Sorghum bicolor, a diploid, has a relatively small genome (735Mbp), which although larger than rice (389 Mbp) is smaller thanthe other important cereals (wheat 16 900 Mbp, maize 2600 Mbp).The last genome duplication event for the S. bicolor genomeseems to have occurred much earlier than the divergence of themajor cereal crops from a common ancestor (Paterson et al., 2004).Completion of the whole genome sequencing project in 2007 willexponentially increase the sequence data available for Sorghumand will provide valuable information on cereal domesticationin the African continent, an event that appears to have occurredindependently of other continents though by similar reinforcedselective pressures (Paterson et al., 2004). In a way, the sorghumgenome sequencing will close a biographic triangle into theknowledge of the polymorphism shared before the divergence ofthese important grasses and ultimately in the understandingof the evolution in cereals crops between Africa, America andAsia (Kresovich et al., 2005). The tenets of colinearity andmicrolinearity of grass genomes mean that our knowledge of othercereals and their evolutionary ties will also greatly improve.Due to their economic and scientific value, cereal genomes havebeen studied over the last 15 years using highly advanced technologies.The similarity at the DNA level makes it possible to use comparativegenetics to look for particular genes of unknown sequence betweenthe genomes with the aim of using that information to developnew varieties or discovering new genes that could have a potentialimpact on traits that are of global importance (e.g. food quality,drought resistance).
The genetic diversity existing within and between AustralianSorghum species was recently evaluated using simple sequencerepeats (SSRs) (Dillon et al., 2005). SSRs were sourced fromthe cultivated S. bicolor (Brown et al., 1996; Taramino et al., 1997;Kong et al., 2000) to determine diversity in these closely relatedtaxa. This method has successfully evaluated diversity in therelated species of many crop groups (e.g. Peakall et al., 1998;Hernández et al., 2001; Chen et al., 2002; Scott et al., 2003;González-Martínez et al., 2004; Sudupak, 2004).This evaluation of the Australian species has shown significantlyhigher levels of genetic diversity both between (inter-) andwithin (intra-) species compared with the intra-specific diversityof S. bicolor varieties. The relatively high transfer rate ofS. bicolor-derived SSRs to the wild species and their high levelof diversity suggests that these SSRs are an efficient, highlyinformative source of molecular markers for the undomesticatedSorghum species.
Screening for novel genetic variation in S. bicolor
Mutations, both natural and artificially induced, provide analternate source of genetic diversity. Mutants have long beena valuable resource in plant breeding (van Harten, 1998) and,in recent times, in plant genomics research (Henikoff and Comai, 2003;Till et al., 2003; Henikoff et al., 2004). However, the methodemployed (irradiation or chemical) to induce a mutated populationwill affect its usefulness and application for genomics research.A review of the comprehensive International Atomic Energy Agency'sMutant Varieties Database (http://www-mvd.iaea.org/MVD/default.htm)shows only 15 induced sorghum mutant accessions amongst morethan 2500 registered mutants.
As a result of the random nature of mutation induction, by physicaland chemical means, each individual in a population will containa unique range of gene mutations. This provides a powerful resourcefor genome analysis employing recent molecular technologies.It is well established that the ultimate goal in DNA researchis to ascertain the DNA sequence of a gene. However, the existingtechnology for genotyping has become a powerful way to avoidthe sequencing step or at least for reducing dramatically thenumber of samples needed to be sequenced. Analysis of DNA polymorphismin natural and mutated populations is more efficient with theuse of capillary electrophoresis (Szantai et al., 2005; Davies et al., 2006)which has the advantages of improved efficiency, sensitivityand throughput (Tang et al., 2004) when compared with gel electrophoresis(Vouk et al., 2000; Cordeiro et al., 2006b). Additionally, theuse of capillary electrophoresis has the advantage of reducingcosts and time through multiplexing (Kan et al., 2004).
Gamma irradiation and EMS (ethyl-methane-sulfonate) mutationprotocols have been optimized for selected S. bicolor populationsto generate random changes in the sorghum genome. The secondgeneration of plants was screened to assess the amount of polymorphismthat has been generated and now mutations can be identifiedin candidate genes by utilizing an approach to genetic analysiscalled TILLING (Targeting Induced Local Lesions IN Genomes),which was first applied in plants by McCallum et al. (2000).A significant body of scientific literature is now availableon this technique (Comai and Henikoff, 2006).
TILLING allows for genotypic screening for allelic variationsprior to commencing with the more costly and labour-intensivephenotyping (Henikoff et al., 2004). EMS-induced TILLING populationshave been produced for the major cereal crops: wheat (Slade et al., 2005),rice (Wu et al., 2005), barley (Caldwell et al., 2004), maize(Till et al., 2004) and sorghum (in the authors' laboratory).TILLING is fast becoming a mainstream technology for mutationcharacterization (Comai and Henikoff, 2006) and for analysingsingle nucleotide polymorphisms (SNP) (Cordeiro et al., 2006a).A very sensitive high-throughput screening method based on capillaryelectrophoresis has been developed (Cross et al., 2007) usingEndonucleolytic Mutation Analysis by Internal Labelling (EMAIL)to greatly improve the effectiveness of this new reverse geneticsapproach to crop improvement.
Taxonomy of sugarcane
Sugarcane belongs to the genus Saccharum, first establishedby Linnaeus in Species Plantarum in 1753 with two species: S.officinarum and S. spicalum L. The original classification ofLinnaeus' has since been revised to contain six species: S.officinarum, known as the noble cane; S. spontaneum L., S. robustumE.W. Brandes & Jeswiet ex Grassl, and S. edule Hassk., classifiedas wild species; and S. sinense Roxb. and S. barberi Jeswiet,classified as ancient hybrids (Buzacott, 1965; Daniels and Roach, 1987;D'Hont and Layssac, 1998). The genus falls in the tribe Andropogoneaein the grass family, Poaceae, that includes other tropical grassessuch as Sorghum and Zea (maize). Closely related to Saccharumare another four genera (Erianthus section Ripidum, Miscanthussection Diandra, Narenga and Sclerostachya) that purportedlyreadily interbreed, forming the ‘Saccharum complex’(Daniels and Roach, 1987). They have in common a high levelof polyploidy and aneuploidy (unbalanced number of chromosomes)that creates a challenge for both the taxonomist and molecularbiologist (Daniels and Roach, 1987; Sreenivasan et al., 1987).
The sugarcane genome
The complexity and size of the sugarcane genome is a major limitationin genetic improvement. Whilst continued selective breedingfor enhanced sucrose accumulation has been able to achieve overhalf of the yield increase in the past 50 years, it has beenreported as having reached a plateau due to limits to the genepool exploited in traditional breeding programmes (Mariotti,2002). Individual research programmes, however, have been shownto still be making significant annual genetic gains by maintaininga diverse gene pool (Edme et al., 2005). The employment of newtechnologies to assist in the association of traits with geneticmarkers and genetic maps can aid in achieving further yieldincreases in breeding programmes.
Most sugarcane cultivars contain more than 100 chromosomes whichcan be assigned to eight homology groups (Rossi et al., 2003;Aitken et al., 2005). Over the past two decades, studies utilizingvarious molecular techniques to unravel the complexity of thisimportant crop species have provided a greater understandingof its complex genetic make-up (Bonierbale et al., 1988; Wu et al., 1992;D'Hont, 1994; Sills et al., 1995; Grivet et al., 1996; Ming et al., 2001;Rossi et al., 2003). Significant achievements include milestonesthat demonstrate the use of single (markers present on one chromosomeonly) and double dose (marker present on two chromosomes) markersfor mapping and QTL analysis (Ming et al., 2001, 2002; Hoarau et al., 2002;Aitken et al., 2004), and large-scale EST sequencing projectsby SUCEST-Sugar Cane EST Genome Project (Vettore et al., 2001),SASRI-South African Sugar Research Institute (Carson and Botha, 2000),UGA-University of Georgia, USA (Ma et al., 2004), and CSIRO-Australia's Commonwealth Scientific and Industrial ResearchOrganization (Casu et al., 2004). Unfortunately, despite theseachievements, the pace of progress with sugarcane genomics haslagged behind that achieved with other agricultural crops (Ramsay et al., 2000;Delseny et al., 2001; Mullet et al., 2002).
Analysis of variation in the sugarcane genome
In 1997, an effort was made by the International Consortiumfor Sugarcane Biotechnology to develop and evaluate simple sequencerepeats (SSRs) or microsatellite sequences as a marker systemfor sugarcane. Markers were developed from an enriched microsatellitelibrary and were shown to have the capacity to distinguish betweensugarcane genotypes due to their ability to detect large numbersof alleles (Cordeiro et al., 2000). To date, this marker systemhas delivered a number of applications that have advanced bothsugarcane research and breeding. Published applications includethe mapping of alleles generated from 72 SSR primer pairs ontoa genetic map constructed on the Australian hybrid cultivar,Q165A (Aitken et al., 2005); validation of the introgressionof genes into F1 hybrids of crosses made between S. spontaneumand elite commercial clones (Pan et al., 2004); the confirmationof fertile intergeneric F1 hybrids of S. officinarum and E.arundinaceus as well as backcross (BC1) progeny from the F1to hybrid sugarcane (Cai et al., 2005); and the use of the markersto register and confirm sugarcane varieties by the United StatesDepartment of Agriculture (USDA) (Tew et al., 2003). SSR markershave also been used to draw useful information on the relationshipsbetween various members of the ‘Saccharum complex’(Cordeiro et al., 2003; Cai et al., 2005) as well as relationshipsbetween clonal cultivars of hybrid canes (Pan et al., 2003a).A fingerprint database of major Australian sugarcane cultivarshas been developed using these markers (Piperidis et al., 2001)as has molecular genotyping of elite clones produced by theUSDA (Pan et al., 2003a, b).
High-throughput SNP genotyping
High-throughput genotyping technologies based on single nucleotidepolymorphisms (SNPs) or small-scale insertion/deletions (indel)could become efficient alternative tools for traditional markersbecause of their greater abundance in the genome and ease ofmeasurement. SNPs are being identified and rapidly mapped toprovide a rich source of genetic information with the potentialfor allowing a greater insight into understanding the geneticcomplexity of many organisms. SNPs are present in high frequencyin any genome, amenable to high throughput analysis and havethe ability to reveal hidden polymorphisms where other methodsfail (Bhattramakki and Rafalski, 2001). In plants, a numberof studies have been able to link SNPs with phenotypic traitsof agronomic interest, such as the putative betaine aldehydedehydrogenase gene responsible for the fragrance trait in rice(Bradbury et al., 2005) and SNPs found in the starch synthaseIIa gene associated with starch gelatinization temperature inrice (Waters et al., 2005). These studies highlight the usefulnessof SNP markers, demonstrating both the abundance of this markertype and the potential causal association between a single nucleotidealteration and organism phenotype. A further major advantageof SNP markers is that they allow easy and unambiguous identificationof alleles or haplotypes.
Whilst numerous technical methods have been developed for theirdetection (Gut, 2001), the majority are applicable mainly todiploid genomes where a simple presence/absence of either oneor both of the alternative bases would indicate homozygosityor heterozygosity. Sugarcane, with its complex genome comprisingan estimated 8–14 copies of every chromosome (Rossi et al., 2003;Aitken et al., 2004), can have up to 14 different alleles present,with individual alleles in varying numbers. Thus, the frequencyof an SNP base at a gene locus will be determined by both thenumber of chromosomes carrying the gene, and the number of differentalleles (or haplotypes) and frequency of each allele possessingeach SNP base. Hence, any method used to detect SNPs at a particularlocus in sugarcane must be able to determine the frequency ofeach SNP base in different genotypes, rather than simply detectingthe presence and absence of SNPs. Such detection systems aregenerally more complex and expensive than simpler and more commonmethods used for detecting less complex genomes (Ross et al., 1998;Ahmadian et al., 2000; Alderborn et al., 2000; Nurmi et al., 2001;Storm et al., 2003).
Use of SNPs in sugarcane
Currently, whilst there are only a limited number of papersdescribing the use of SNPs to understand the sugarcane genome,they point to this marker system as a valuable means of mappingcandidate genes and for identifying the genetic basis of QTLsof agronomically important traits. These studies include a discussionon the ability of SNPs to: delineate a set of 64 ESTs into twogroups that are likely to represent two gene family membersof 6-phosphogluconate dehydrogenase (Grivet et al., 2001); delineationof 178 ESTs into three paralogous genes to reveal the expressionof an Adh2 and two Adh1 genes in sugarcane (Grivet et al., 2003);the development of co-dominant cleaved amplified polymorphicsequence (CAPS) markers (Quint et al., 2002); and to map severalcandidate genes and ESTs (McIntyre et al., 2005).
In sugarcane, the proportional frequencies of each SNP basewill vary depending on the number of alleles of the gene containingthe SNP locus. The ability to capture this information accuratelyacross several SNPs within a set of homo(eo)logous alleles cangive an indication of the number of allele haplotypes presentfor a gene and potentially provide the haplotype sequences.This information could have implications for sugarcane breeding.High yield potential may be due to the presence of, or differentnumber of copies of a specific allele(s) present at a gene locus,or possibly a combination of both. Knowledge of the sequenceunderlying each allele haplotype has the potential to allowallele-specific markers to be designed.
Quantitative methods to detect allele dosage in sugarcane arenow possible with such techniques as pyrophosphate sequencingusing the PyrosequencerTM platform (Cordeiro et al., 2006b)and mass-spectrometry using the SequenomTM platform (Cordeiro et al., 2006b).These methods have allowed the quantitative detection of frequenciesof consensus to alternate SNP bases at any particular SNP locus.Utilizing a group of SNP markers developed to the same EST orgene, it becomes possible to infer the likely copy number ofthe EST or gene. This information then allows for possible haplotypesof a gene present in hybrid cane to be determined through statisticalapproaches (Cordeiro et al., 2006b).
In theory, the association of SNP variations with either thepresence or absence of different phenotypes among individualsor among individuals from different populations appears straightforward.This simplistic view does not account for the majority of basepolymorphisms that do not result in any amino acid change. Determiningthe haplotypes is more important for predicting individual phenotypesthan are the underlying SNPs. Determining haplotypes also allowsthe ability to infer the evolutionary history of a DNA region(Templeton et al., 1988; Tishkoff et al., 1998). However, difficultiesare encountered in determining SNP haplotypes when inbred orhomozygous individuals are not available (Rafalski, 2002) asis usually the case with sugarcane.
The ability to determine SNP base frequencies provides the meansto determine the likely copy number of homo(eo)logous loci insugarcane. Where chromosome counts have been performed for agenotype, this information can be used to support the inferenceof the most likely copy number of homo(eo)logous loci. Knowledgeof the number of homo(eo)logous loci will assist in the deductionof the allelic composition of the locus in any particular sugarcanegenotype. The ability to determine haplotypes also opens possibilitiesin unraveling the complexities of the sugarcane genome. By defininghaplotypes in parents of crosses, it may be possible to deducetheir segregation in progeny; or to determine allele dosageand composition in any particular genotype in relation to phenotypicperformance. A further level of analysis is required to determinethe level of expression of each of the haplotypes in this complexgenome.
Genetic maps are widely used in plant breeding to identify genomicregions controlling traits of interest. Such information assistsin understanding the genetic basis of the target trait, as wellas providing DNA markers for use in marker-assisted breeding.In sugarcane, only markers that are present as a single copyin one parent and absent in the second [i.e. single-dose (SD)marker] can be incorporated into maps using populations of conventionalsize (approx. 250 progeny) (Wu et al., 1992). In these populations,SD markers segregate in a 1 : 1 ratio.
The first maps of a cultivar were initiated on the selfed progenyof SP70-1006 (D'Hont, 1994). This map was later transferredand further developed on the cultivar R570 (Grivet et al., 1996)using RFLP probes from maize and sugarcane. By 2001, the R570map, as it had become commonly known, contained some 600 RFLPmarkers derived from a number of grass (Poaceae) species (D'Hont and Glaszmann, 2001).The markers on this map distribute over 98 cosegregation groupscovering a total length of 2008 cM. A parallel mapping effortwas also carried out to place 939 single-dose AFLP markers onR570, of which 887 were distributed into 120 cosegregation orlinkage groups (Hoarau et al., 2001). A more recent map hasbeen developed on a cross between the Australian commercialvariety Q165A (2n = 115) with the S. officinarum clone IJ76-514(2n = 80) using a combination of AFLP and SSR markers. A totalof 967 single dose markers were generated from the two markersystems, and 910 were distributed across 116 linkage groupscovering a total map length of 9058·3 cM (Aitken et al., 2005).Markers on these maps have all been generated through anonymousmarker systems. However, the use of SNP markers are resultingin ESTs mapped onto the Q165A map.
EcoTILLING for mapping ESTs
Parallel to the development of quantitative SNP frequency scoringmethods has been the adaptation of the EcoTILLING method fordetecting and mapping sugarcane ESTs. TILLING utilizes the CelImismatch-cleavage enzyme on heteroduplexed DNA strands withdetection of end-labelled cleavage product (McCallum et al., 2000).A variant of this method utilizes natural populations for thediscovery of polymorphisms (SNPs, SSRs and indels), and is referredto as EcoTILLING (Comai et al., 2004). Both methods as publishedrely largely on electrophoretic gels to separate and visualizethe products. In sugarcane, this does not allow SNPs that occuron a single allele to be clearly detected. Modifying the protocoland moving the detection system to capillary electrophoresishas allowed the detection of single-dose SNPs in sugarcane tobe identified (Cordeiro et al., 2006b) and mapped (McIntyre et al., 2006).Our early experience with family members of the sucrose phosphatesynthase gene indicate straightforward detection of the presenceof 5–11 SNPs in fragment lengths of genomic DNA between300 bp and 400 bp in length. Neither prior knowledge of anySNP in the fragment nor the alignment of multiple ESTs are requiredto identify putative SNPs and their location. Whilst the methodis as yet unable to indicate the frequency at which an individualSNP base is present, it has been demonstrated that the detectedvariation in base composition segregates as expected in progenyof mapping populations. Using the SPS gene family members asan example, the mapping of the gene family members through theEcoTILLING approach supports sequence information that threeof the five gene family members may contain more than one gene,with each gene possessing from one to five alleles (McIntyre et al., 2006).This observation will in time allow further unravelling of thecomplexities of the sugarcane genome.
Sorghum genome information as a resource for sugarcane
Sorghum is the closest cultivated relative of sugarcane. Sugarcanehas a large genome that has duplicated at least twice sinceit diverged from sorghum, around 5 million years ago (Al-Janbi et al., 1997).The extensive similarity in the gene order between these twogenomes, where intercrosses are still possible (Ming et al., 1998),makes sorghum the best model crop for the Androponeae tribe(Price et al., 2005a) with the aim of understanding the extensivegene rearrangements and assisting the development of geneticmaps in sugarcane.
Sequencing of Sorghum provides another model genome within thegrasses, which particularly when utilized in conjunction withrice, will stimulate evolutionary understanding of the entirePoaceae. Sequencing will stimulate gene and allele discoveryand crop improvement in Sorghum as it did in rice. Sugarcanegenomics will be supported by the Sorghum sequence data. Thesequences of Sorghum genes and to a lesser extent the locationof genes in the genome should be useful in sugarcane.Genetic resources for sorghum and sugarcane improvement havebeen enhanced by the application of genomic tools to analysisof wild relatives in the Sorghum and Saccharum genera. Mutantpopulations (including TILLING populations) of Sorghum expandthe options for gene discovery and genetic manipulation. Protocolsfor EcoTILLING (Cordeiro et al., 2006a) and quantitative SNPanalysis in the complex sugarcane genome should be valuabletools for gene mapping, gene discovery and association geneticsin sugarcane. The availability of a Sorghum genome sequencewill further accelerate the potential to apply these techniquesin both Sorghum and sugarcane. Gene discovery in this germplasmwill also be supported by application of advances in expressionprofiling tools as has been applied to other crop species inthe Poaceae (McIntosh et al., 2007).
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* DTT, Days till transplant: seeds were germinated on damp filter paper; once a strong radicle and the first coleoptile had emerged they were transplanted to potting mix.
md, Missing data, (p), prostrate growth habit; fl, height was measured to the top of flag leaf or seed head.
A dash indicates that delayed onset of flowering caused the trial to be terminated before counts could be made.
Figure 1 Time-line displaying the changes in Sorghum nomenclature over time. 1House et al. (1995); 2Spangler (2003); 3Smith and Frederiksen (2000); 4Garber (1950); 5Lazarides et al. (1991); 6Hodnett et al. (2005), Price et al. (2005a); 7Dillon et al. (2007).
Figure 2 Growth trial of Sorghum species at seedling stage. Note the broader leaf (far left) of the Eu-sorghum, S. propinquum compared with the Para-Sorghum, Stiposorghum and Heterosorghum species.
Figure 3 Variation in Sorghum species seed and caryopsis morphology and size. Letters on the figure denote different species: a–e, S. bicolor caryopsis AusTRCF 322649, 322618, 322620, 322666 and 322611, respectively; f, S. propinquum; g, S. halepense; h, S. macrospermum 322277 seed and caryopsis; i, S. laxiflorum 302503 seed and caryopsis; j, S. grande 302580 seed; k, S. leiocladum 300170 seed and caryopsis; l, S. matarankense 302521 seed and caryopsis; m, S. nitidum 302539 seed; n, S. timorense 302660 seed and caryopsis; o, S. purpureo-sericeum 321134 seed and caryopsis; p, S. versicolor 321126 seed and caryopsis; q, S. amplum 302623 seed and caryopsis; r, S. angustum 302604 seed and caryopsis; s, S. brachypodium 302480 seed and caryopsis; t, S. bulbosum 302646 seed and caryopsis; u, S. ecarinatum 302661 seed; v, S. exstans 302577 seed and caryopsis; w, S. interjectum 302563 seed; x, S. intrans 302390 seed and caryopsis; y, S. plumosum 302489 seed and caryopsis; z, S. stipoideum 302644 seed.
Figure 4 The left-hand column shows the variation in the central endosperm and the right-hand column compares the outer layers. (A, B) Representative images of S. bicolor, showing the standard floury and vitreous endosperm, respectively. (C–H) Images from outside the Eu-Sorghums are representative of the variations observed across the species. PB, Protein bodies; M, matrix; S, starch granule; D, indentations left by protein bodies; C, channels; P, pores; CG, small polygonal starch granules forming compound granules.
Figure 5 Novel sub-aleurone morphology of the Para-Sorghums and Stiposorghums: (A) the characteristic S. bicolor outer endosperm and pericarp; (B) an increased magnification of the sub-aleurone layer itself; (C) a representative image of the novel morphology found in the undomesticated sorghums; (D) and (E) magnified features of the morphology shown in (C). SA, Sub-aleurone layer; A, aleurone; PB, protein bodies; M, matrix,; S, starch granule; D, indentations left by protein bodies; P, pericarp; CG, small polygonal starch granules forming compound granules.