Keywords

1 Introduction

Mutations, either natural or induced, provide raw material for evolution and plant breeding. Using modern molecular genetics techniques, mutational analyses play a crucial role in elucidation of gene function, metabolic processes, signaling, growth, and development. Mutational analyses fall into two broad categories: forward and reverse genetics. In forward genetics, informative mutant phenotypes are identified first. Then, the mutant with the desired trait is crossed to another ecotype that has extensive DNA polymorphism and the genomic region attributable to the phenotype is identified through genetic mapping. The mutated gene responsible for the phenotype is identified through traditional positional cloning or through sequencing candidate genes if sufficient information is available to infer the processes leading to the phenotype. In reverse genetics, a series of mutants for a gene is first isolated based on sequence differences from the wild-type sequence. Then, the mutant phenotypes are analyzed to deduce the function of the gene. Reverse genetics techniques are now available to enable high-throughput analysis of gene function on a genome-wide scale. Sorghum genome sequencing from a leading inbred BTx623 has been completed (Paterson et al. 2009b). The focus now is to establish the function of majority of the genes in the sorghum genome. A well-characterized mutant population and reverse genetics techniques will likely play an important role in establishing the function of genes, especially those for which a functional assay is not available.

2 Annotated Individually Pedigreed Mutagenized Sorghum Library

Mutagenesis has long been applied to sorghum to isolate novel phenotypes that may have potential application in breeding (Gaul 1964; Quinby and Karper 1942). Many mutants with unique phenotypes that have not been observed in natural sorghum collections have been selected from populations treated with various mutagens, such as X-ray and γ-irradiation, ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), diethyl sulfate (DES), N-nitroso methyl urea (NMU), N-nitroso ethyl urea (NEU), or combinations of chemical and irradiation mutagens (Quinby and Karper 1942; Sree Ramulu 1970a, b; Sree Ramulu and Sree Rangasamy 1972). Many beneficial mutations, including dwarfing, early flowering, high protein digestibility, high lysine, and others, have been widely used in sorghum breeding (Ejeta and Axtell 1985; Oria et al. 2000; Quinby 1975; Singh and Axtell 1973). The late Dr. Keith Schertz, a former sorghum geneticist with USDA-ARS, collected and preserved more than 400 natural and induced mutant lines from various genetic backgrounds. This collection, now under the curation of the Plant Stress and Germplasm Development Unit (USDA-ARS, Lubbock, TX), provides a starting point to study the functions of sorghum genes.

2.1 Development of an Annotated Individually Pedigreed Mutagenized Sorghum Library

The sorghum genome sequence and the identification of its genes have made it possible to study gene function on a genome-wide scale, and to compare gene function with other plants (Paterson 2008; Paterson et al. 2009b). A systematic mutant library that contains multiple mutations for all genes in the sorghum genome is urgently needed to deduce the functions of sorghum genes. Xin et al. (2008) reported a modest population of 768 pedigreed EMS-mutagenized lines of BTx623, a leading inbred used for sorghum genome sequencing. The mutant library was developed by single-seed-descent from individual mutagenized seeds (M1) to M3 generation. Genomic DNA was prepared with leaf samples collected from the M2 plants used to produce M3 seeds. Phenotypes are annotated at the M3 generation to ensure that any phenotype observed in a family is descended from a single mutagenesis event (a single germ cell), represented by an M2 plant used to prepare genome DNA. Following phenotype annotation, ten M3 panicles are bulked as M4 seeds, which are deposited in the library and will be distributed to end users on request. The mutant library is named the Annotated Individually pedigreed Mutagenized Sorghum (AIMS) library. Since the mutant library is pedigreed, recessive lethal mutations can be preserved in heterozygous state. A pilot study shows that the library has a mutation rate about 1/526 kb (Xin et al. 2008). Given the ∼730 Mb genome size of sorghum and the finding that about ¼ of the DNA is euchromatin (Paterson et al. 2009b), each mutant line is expected to harbor about 340 mutations in the euchromatin. A mutant library with 6,400 lines would contain more than two million independent mutations, i.e., about 80 mutations per gene. This level of coverage, although far from saturation mutagenesis, should provide an adequate resource for genome-wide identification of mutant series for most genes in the genome and to screen for mutants that can be used for sorghum improvement or biological studies. The library has now expanded to over 5,000 lines and will be expanded to 6,400 lines in the next 2 years. The mutant library can be accessed online at http://www.lbk.ars.usda.gov/psgd/index-sorghum.aspx.

Many factors affect the quality of mutant libraries. The first important factor is the choice of mutagens. EMS was used to generate the AIMS library because of its high rate of success in sorghum and many other plant species (Greene et al. 2003). In a comparative study of multiple mutagens, EMS is shown to induce ten times more chlorophyll mutations than NUE and MMS (Sree Ramulu 1970b). It has been used extensively to create sorghum mutants with useful traits such as early flowering, dwarfing, and a series of mutants with no or sparse epicuticular wax layers (Jenks et al. 1994; Peters et al. 2009; Singh and Drolsom 1974; Sree Ramulu 1970b). The second factor is the dosage of the mutagen used. The concentration of EMS used to generate the mutant library must be evaluated carefully to balance seed setting with adequate mutation frequency. This will involve trial and error and may vary for different varieties or even different batches of EMS (Henikoff and Comai 2003). BTx623 is very sensitive to EMS treatment. At 0.1% (v/v) EMS, only 40% of M1 plants set seeds (Xin et al. 2008). This concentration is much lower than the 0.3% (v/v) EMS which is frequently used in Arabidopsis and many other organisms (Greene et al. 2003). The highest concentration of EMS that can be tolerated by BTx623 is 0.25%, at which less than 10% of the plants produced seeds (Xin et al. 2008). Thus, the mutant library is generated with a series of EMS concentration ranging from 0.1 to 0.25%, to balance mutation frequency with survival of mutants. Other factors also impact the establishment of useful TILLING populations in sorghum. For example, cross-pollination must be vigorously controlled to produce a high-quality mutant library. Under normal growth conditions, sorghum is predominantly self-fertilized with a cross-fertilization rate ranging from 5 to 10% (Ellstrand and Foster 1983). After EMS-mutagenesis, cross-fertilization increased dramatically. A previous sorghum mutagenesis attempt was unsuccessful when cloth bags (Lawson Bags, Northfield, IL) failed to prevent cross-pollination. An examination of resulting M2 plants using four hyperpolymorphic sorghum simple sequence repeat (SSR) markers, Xtxp287, Xtxp270, Xtxp51, and Xtxp295 [publicly available (Menz et al. 2002)], showed that over 30% of the M2 plants were the result of cross-pollination from unknown sources. Cross-pollination can be effectively controlled by covering the panicles at each generation with rainproof paper pollination bags (Lawson Bags, Northfield, IL) before anthesis. Corn earworms and birds also pose serious threats to the limited seed set in M1 plants during the grain-filling period. The pollination bags must be injected with pesticide to control corn earworm. Despite these challenges, a sizable mutant library has been established and ready for distribution.

Ongoing phenotype annotation shows that this mutant library displays a variety of phenotypes, potentially serving as both a forward genetic resource for identifying useful traits and their genes for sorghum improvement, and as a reverse genetic resource for identifying mutant series in specific genes to deduce their functions. A selection of phenotypes is presented in Fig. 8.1. Here, we discuss two traits that may be useful for improving conversion efficiency of sorghum stover to ethanol and biomass production. Readers are referred to the online database for a complete compilation of phenotypes observed in the mutant library.

Fig. 8.1
figure 1

A selection of mutant phenotypes that may have potential to improve bioenergy conversion efficiency and biomass production in sorghum. A complete collection of mutant phenotypes can be found online (http://www.lbk.ars.usda.gov/psgd/index-sorghum.aspx)

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2.2 Brown Midrib Mutations

Brown midrib (bmr) mutants have been isolated from C4 cereals such as maize, sorghum, and millet through natural or induced mutations (Sattler et al. 2010). The mutant phenotype is typified by a distinctive brownish colored mid veins of leaves, which can be easily identified in the field. A typical bmr mutant is shown in Fig. 8.1. Some mutants also accumulate reddish brown to yellow pigment in the stalk, root, and stem pith. The bmr mutation is associated with reduced lignin content, increased digestibility for livestock, and increased conversion efficiency of sorghum stover to ethanol (Vermerris et al. 2007).

Sorghum bmr mutants were first isolated by Porter et al. (1978) from diethyl sulfate mutagenized population. Twenty-eight sorghum bmr mutants represented by four loci (bmr2, bmr6, bmr12, and bmr19) have been isolated from various sources including natural mutation (Sattler et al. 2010). Two of the loci have been cloned by candidate gene approaches. The bmr6 mutation encodes a cinnamyl alcohol dehydrogenase (CAD) and bmr12 encodes a caffeic O-methyl transferase (COMT) (Bout and Vermerris 2003; Saballos et al. 2009; Sattler et al. 2009). These two enzymes are involved in the last two steps of biosynthesis of monolignols, the precursors for lignin biosynthesis. Among these four loci, bmr2 and bmr19 are represented by a single locus, indicating that saturation mutagenesis has not been achieved (Sattler et al. 2010). Moreover, both bmr6 and bmr12, which are the main sources for commercial bmr forage sorghum, are complete knockout mutation. To identify additional bmr mutants and to isolate non-knockout alleles of bmr6 and bmr12, we initiated a systematic approach to isolate additional bmr mutants. A close inspection of about 3,000 individual M3 families identified over 100 independent mutants with the typical brownish midrib. Many mutants have been confirmed in the next generation (Pedersen JF, personal communication). Ongoing complementation study showed that in addition to many alleles of the previous known bmr loci, six novel mutants that could not complement the previously known loci were also identified. It is not clear how many new loci these six mutants represent. These novel mutants and new alleles of previously known loci provide new genetic resource to improve the digestibility of forage sorghum and the conversion efficiency of sorghum stover to ethanol while minimizing the effect of bmr mutation on biomass production and lodging.

2.3 Erect Leaf Mutants

Total biomass yield and efficient conversion of the biomass to bioenergy are two critical factors for sorghum to become a major bioenergy feedstock. Although sorghum has excellent tolerance to abiotic stresses such as drought and high temperature, and can thrive on poor soil with minimal fertilizer, sorghum biomass and grain yield are generally lower than maize across a range of environmental conditions (Mason et al. 2008). Moreover, the increase in potential yield of sorghum hybrids released in the several decades since the Green Revolution is only one-third of that of maize hybrids released in the same period of time (Dhugga 2007). Regardless of the pace of the increase, the improvement in genetic yield potential in both maize and sorghum is strongly correlated with increases in the number of ears (maize) or panicles (sorghum) per unit area. Over this period of time, the density of maize hybrids increased by an average of ∼1,000 plants·per hectare per year, corresponding to ∼1% annual increase in grain yield (Dhugga 2007). In the 36 maize hybrids released from 1936 to 1991, leaf angle score of new hybrids displayed an improvement of 122% over the old ones, the greatest change among all ten plant traits examined (Duvick and Cassman 1999). The modern maize hybrids have much more acute (erect) leaf angle than older hybrids, which allows the hybrids to be planted at higher density to capture more solar radiation per unit land area (Duvick and Cassman 1999). Erect leaf mutants in rice have also been shown to have increased biomass and grain yield (Sakamoto et al. 2006).

Compared with modern maize hybrids, sorghum exhibits an open canopy with wide leaf angles that almost parallel the ground. A sorghum mutant with erect leaf angle has been reported previously (Singh and Drolsom 1973). This mutant, associated with no leaf ligule and other undesirable traits, has not been used to improve leaf angle in sorghum breeding. Among the 3,000 M3 plots in the field, over 50 plots segregated for leaf angles that vary from the wild-type BTx623. Eleven of these mutants were confirmed at the next generation (M4). Several mutants have similar or slightly bigger panicles than wild type (Table 8.1). Although these erl mutants need to be confirmed in a segregating F2 population and homozygous F3 generation under different environments and plant densities, some may prove to be useful for improving sorghum biomass and grain production based on the yield improvement achieved in maize hybrids through improved leaf angle (Dhugga 2007). Other traits from the mutant library, such as monoculm, multiple tillers, and large panicle sizes (Fig. 8.1), may also help to improve biomass and grain production in sorghum. Furthermore, beneficial traits may be stacked to increase biomass yield and biomass conversion efficiency to develop feedstock genotypes tailored to bioenergy production (for example, crossing bmr mutants with mtl mutants and/or erl mutants to develop double or triple mutant plants).

Table 8.1 A list of erect leaf mutants confirmed at M4 generation
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2.4 A Sorghum Mutant Library as a Source to Identify Beneficial Traits for Other Saccharinae Species

Phenotype diversity and frequency after mutagenesis depend on the dosage of mutagen used, polyploidy level, and gene redundancy. As a minimally redundant genome that last experienced genome duplication ∼70 million years ago (MYA) (Paterson et al. 2004), sorghum is very sensitive to mutagenesis treatments and displays a wide spectrum and high frequency of mutant phenotypes even after treatment with low dosage of mutagens (Peters et al. 2009; Sree Ramulu 1970a, b; Xin et al. 2008). In hexaploid and tetraploid wheat, less than 0.5% of mutagenized lines display visible mutant phenotypes even after heavy mutagenesis (0.6–1.2% EMS for 18 h) that resulted in a mutation rate of one base substitution per 24–80 kb (Slade et al. 2005). Rice, a diploid plant, has experienced an evolutionary history very similar to that of sorghum, with no genome duplication in about 70 million years (Paterson et al. 2004; Yu et al. 2005). However, rice often requires high concentrations of mutagens to create mutant populations that have useful mutation frequencies (Till et al. 2007; Wu et al. 2005). After treatment with 0.8% EMS, about 3% of the M3 lines in rice segregated for albinism (Wu et al. 2005), while 17% of M3 lines in sorghum treated with 0.25% EMS segregated from albino seedlings (Xin et al. 2008). Other common mutant phenotypes occur at much lower frequency in mutagenized rice than in sorghum. The presence of a husk outside of the rice grain may account in part for the requirement of a high concentration of EMS but gamma radiation, which can penetrate husks with little impedance, results in only slightly higher albinism rates (Wu et al. 2005). To overcome the effect of husks, a mutagenesis protocol for treating individual zygotes in developing panicles has been developed (Suzuki et al. 2007). Mutation rates in populations generated with this new protocol appear to be higher at DNA level than those in previous populations generated by treating mature seeds (Suzuki et al. 2007; Till et al. 2007). The frequency of albinism or other visible mutant phenotypes was not reported for this mutagenized population. Maize, although a “diploid” plant based on chromosome pairing, is believed to have experienced a whole genome duplication just 11 MYA and thus has more gene redundancy than sorghum. TILLING populations have been developed in maize with adequate mutation frequency at DNA level (Till et al. 2004; Weil and Monde 2007); however, the frequency and diversity of visible mutant phenotypes have not been reported.

Many close relatives of sorghum are polyploids. Sugarcane (Saccharum) is an autopolyploid with variable number of chromosomes from 2n  =  80 to 140 (Dillon et al. 2007; Irvine 1999). Miscanthus × giganteus is a sterile allotriploid that is naturally crossed from diploid Miscanthus sinensis (2n  =  38) and tetraploid Miscanthus sacchariflorus (4n  =  76) (Rayburn et al. 2009). Due to the variable chromosome number and ploidy levels, no natural or induced mutant has been reported. It is likely to be challenging to develop mutant populations for these species. A sorghum mutant library with well-annotated phenotypes could identify useful traits for improving close relatives such as Saccharum and Miscanthus for biomass production and bioenergy conversion efficiency. For example, bmr mutations have shown to increase the digestibility and ethanol conversion efficiency of maize and sorghum stover (Sattler et al. 2010; Vermerris et al. 2007). Two genes in the lignin synthesis pathway, that lead to the bmr phenotype when mutated, CAD and COMT, have been cloned. RNAi techniques might be applied to other grasses such as Miscanthus × giganteus to down-regulate these two genes to improve the efficiency of converting biomass to ethanol. Due to recent genome duplications, the annotated sorghum mutant library may be also useful for functional studies of maize genes.

2.5 TILLING

Heritable mutation induced by physical radiation, transposon insertion, or chemical mutagenesis with an ideal phenotype (such as early flowering) is occasionally but infrequently used directly for agricultural production, sometimes used as a source of variation in the breeding process, and very frequently used for biological studies. Large chromosomal rearrangements (such as insertions, deletions, inversions, or translocations) usually induced by radiation can be traced by cytogenetics. However, detection of small insertions or deletions (indels) and point mutations is far beyond the resolution of cytogenetic analysis. Targeting induced local lesions in genomes (TILLING) (McCallum et al. 2000a, b) is a new technique that can be efficiently used for detecting small indels and point mutations. TILLING is particularly suitable for genome-wide analysis with a large mutant population induced by chemical mutagenesis and can be used in a high-throughput format to make links between characterized mutant genotypes and the resulting phenotypes. TILLING mainly includes the following steps: development of a mutant population with a reasonable number of individuals by mutagenesis; collection of leaf tissue from M2 plants and extraction of DNA; pool of DNA samples from mutants by one or multiple dimensions; design of PCR primers covering regions of interest from sequence database; PCR amplification of targeted regions of interest; heteroduplex formation between wild-type and mutated amplicons; detection of mutation by dHPLC or other separation systems; dissection of individuals within a pool containing a mutation by further PCR; reconfirmation of an individual with a mutation by sequencing; and association of the confirmed mutant genotype with a resulting mutant phenotype. The details of TILLING procedures can be altered or modified from lab to lab. For example, the chemicals used for mutagenesis, the number of mutants within a population for covering the full spectrum, and the number of pooling dimensions and folds should be dependent on species. The detection platform can be dHPLC (Underhill et al. 1997), agarose gel system (Raghavan et al. 2007), and denaturing polyacrylamide gel or even capillary system depending on the equipment available in the lab. A subset of 768 lines from the AIMS mutant library were selected to conduct a pilot TILLING (Xin et al. 2008). Despite the sensitivity of sorghum to EMS treatment, a mutation rate of 1/526 kb was found with four amplicons, a mutation rate that is adequate for high-throughput TILLING to deduce the function of sorghum genes.

3 Transposon Mutagenesis

3.1 Sorghum Candystripe1 Transposon

In sorghum, a variegated line was originally collected by Dr. O. Webster from Gedaref, Sudan. Genetic analysis and mutable behavior of this allele indicated the presence of a transposon (Zanta et al. 1994). Further molecular characterization led to the isolation of the transposable element associated with the sorghum candystripe (variegated) phenotype (Chopra et al. 1999). The host sequence of the transposon was the yellow seed 1 (y1) gene which encodes an MYB domain protein that is closely related to P1 and homologous MYB proteins in maize and teosinte (Chopra et al. 2002; Jiang et al. 2004; Zhang et al. 2000). The variegated allele was designated as y1-candystripe (y1-cs). Based on the high frequency of somatic and germinal reversions of y1-cs to functional y1, it was obvious that the phenotype of the y1-cs allele results from the presence of a transposable element in the y1 gene (Hu et al. 1991; Zanta et al. 1994). Moreover, the y1-cs allele bears a marked resemblance to the maize p1-vv allele. In maize, the p1 gene regulates the production of the phlobaphenes in kernel pericarp and other plant tissues (Chopra et al. 1996; Lechelt et al. 1989). The y1-cs allele specifies variegated seed pericarp pigmentation (Fig. 8.2). By virtue of its phenotypic effect on the expression of the pericarp pigmentation, the transposon was named as Candystripe1 (Cs1) (Chopra et al. 1999). It is a member of the CACTA family of plant transposable elements, which has been suggested to be an important vector for reshuffling sorghum exons and genes, similar to PACKMULEs in rice and Helitrons in maize (Paterson et al. 2009b).

Fig. 8.2
figure 2

Panel A depicts the Sorghum candystripe phenotype and its similarity to the maize variegated phenotype conditioned by P1-vv. Left of panel A shows a sorghum head with variegated and red kernels next to a full red head (revertant). Panel B depicts the position of the Cs1 transposon in the y1 sequence. Loss of the Cs1 results in generation of a functional y1 allele. Star indicates the 2 bp footprint left upon excision of the Cs1 element

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Of several members of this family, the En/Spm element of maize has been best understood at the genetic and molecular levels. It was originally identified as Enhancer (En) (Peterson 1960) and was shown to be homologous to the Suppressor–Mutator system (Spm) (McClintock 1954) by genetic and molecular tests (Pereira et al. 1986; Peterson 1965). Besides maize, CACTA elements have been characterized from several other plant species.

3.2 Structural Features of Candystripe1 Transposon

Cs1 has features similar to those of other members of the CACTA family: a short terminal inverted repeat (TIR) sequence 5′-CACTATGTGAAAAAAGCTTA-3′, and these termini are flanked by 3-bp target site duplication. Subterminal regions, 250 bp interior to the TIR, contain multiple copies of direct and indirect repeats. A 12 bp partially conserved sequence motif [5′-TTATTACAGACG-3′] is repeated eight and six times, respectively, in the 5′- and 3′- subterminal regions. Sequences similar to the subterminal repeat motif are also present at seven sites in the central region of the Cs1 transposon. Interspersed within the Cs1 element are other tandem repeats as well as several copies of High Copy Short interspersed Repeats (HCSRs). Several of these repeat sequences have high similarity (up to 95%) to an Sb1 Tourist element of sorghum as well as to other Miniature Inverted Repeat Transposable Elements (MITEs) (Wessler et al. 1995). Transposable elements of the CACTA family are relatively large (En/Spm: 8.2 kb, Tam1: 15.1 kb) (Gierl 1996); the 23,018 bp Cs1 element is the largest known member of this family. The Cs1 copy present at the y1 locus is an autonomous element (Zanta et al. 1994). Recent sequence analysis of sorghum genome has shown the presence of deletion derivatives of CACTA elements that carry pieces of other genes (Paterson et al. 2009a).

3.3 Transposition and Reinsertion of Cs1

Cs1-Y1 offers a unique genetic system to study biology and function of Cs1 transposons in sorghum. The y1-cs allele can revert to a functional state (Y1-rr; red pericarp and red glumes; see Fig. 8.2) in both somatic and germinal tissues, resulting in the appearance of frequent red sectors and fully red seed heads, respectively. Germinal reversions lead to a heritable and functional y1 gene, while somatic reversions are not heritable and their frequencies vary within the progeny and depend on the transposition rates of the Cs1 element in a tissue and genetic background (Chopra et al. 2002; Zanta et al. 1994). Full red revertants (plants producing red panicles) appear at a frequency ranging between 12 and 20% in a growout of the homozygous y1-cs line or crosses involving y1-cs allele with sorghum inbreds having different backgrounds (Carvalho et al. 2005; Chopra et al. 1999; Zanta et al. 1994). Reinsertion of the excised Cs1 elsewhere in the genome was identified in full red head plants (Y1/Y1 or Y1/y1-cs) derived from excision of the Cs1 element from the y1-cs. Furthermore, PCR amplification of DNA from several independent red revertant plants using flanking primers showed that the Cs1 element excised from the y1 gene and left a 2 bp footprint (Chopra et al. 1999).

3.4 Cs1 Can Generate Large Deletions in the Flanking Sequences

When the Cs1 element excises from the host sequence, the majority of excision events leave a 2-bp footprint. For example, Cs1 is inserted into intron II of the y1 gene, and 2-bp footprints do not affect Y1-reading frame or intron splicing and thus lead to the normal function of the gene. These excisions do produce germinal red revertants with a frequency of about 20% (Carvalho et al. 2005). However, screens for germinal excision events resulted in identification of loss of y1 function alleles (Ibraheem et al. 2010). Through DNA gel blot analysis, these excision events were found to contain partial deletions in the y1 gene and of the Cs1. The mechanism of deletions in the Cs1 and the flanking y1 DNA sequences is not yet clear. In addition to finding deletion derivatives, collection of several y1-cs alleles with different degrees of variegation demonstrates alleles with differential excision activities during plant growth and development. These alleles can be attributed to somatic or germinal excision of the Cs1 element from the y1 locus. Characterization of these alleles may allow answering the question of excision mechanism(s) of the Cs1 transposon as has been demonstrated in the case of maize Ac at the p1 locus (Peterson 1990). Genetic and molecular analysis of these alleles will establish multiple copies of the element (because of linked or short-range transposition) that may be producing a negative dosage effect which in turn may be responsible for silencing leading to variable degrees of transposition (Dooner and Belachew 1991).

3.5 Genome Mutagenesis Utilizing Cs1 in Sorghum

Cs1-homologous sequences are present at low copy number in sorghum (Chopra et al. 1999). The low copy number combined with high transposition frequency of Cs1 implies that this transposon could prove to be an efficient gene isolation tool in sorghum. Additionally, there are at least 12 copies of the defective Cs1 elements in sorghum. As opposed to the full-length sequence of the autonomous Cs1 elements, the defective elements range in size from 400 bp to 4.0 kbp. Sequence analysis of these deletion derivatives (dCs2, dCs3, and dCs4) showed that 200 bp on the 5′ end and up to 150 bp at the 3′ end are conserved, while each copy carries varying lengths of internal deletions (Carvalho et al. 2005). Another interesting feature of Cs1 is that its transposition appears to be sensitive to environmental conditions. Transposition of the Antirrhinum Tam3 element is sensitive to temperature, and this provides a means to control the frequency of transposition (Harrison and Fincham 1964). Further, effect and analysis of the flanking genomic sequences to investigate the genomic context of Cs1 elements is underway.

3.6 Characterization of Selected Cs1 Induced Mutations

A sorghum line (CS8110419) with moderate activity of Cs1 was crossed with an agronomically well-adapted line Tx2737 and this cross was characterized further. In the F1 generation, plants were selfed and their DNA was used to test for transposon excision and insertion activities. All randomly selected F1 plants showed polymorphic patterns for transposon insertions that were different from the either of the two parental lines. All F1 plants were maintained by selfing until the F7 generation (Carvalho et al. 2005). In each generation, red revertant as well as candystripe plants were saved. Red revertant plants were further used for molecular and genetic analysis to molecular and genetic analysis to follow excision and reinsertion of the Cs1 in the genome. Simultaneously, any mutant phenotypes were also saved and characterized in further generations. Segregating families showing mutant/wild-type phenotypes were further selected for gene identification and isolation work using Cs1 as a tag (Fig. 8.3). Isolation of gene(s) tagged with a Cs1 element will provide the ultimate proof of its ability as a transposon tag in sorghum. Sequences isolated from these mutants will be mapped onto the sorghum genetic map to enrich the map with respect to phenotypic markers. Additionally, genes identified through this approach will be of interest in sorghum improvement programs utilizing genetic information for lignin biosynthesis (bmr mutations), drought tolerance (bloomless mutations), and disease resistance (wilty and lesion mimics). Results based on the analysis of these putative mutants have further strengthened the use of the Cs1 element in generating and selecting non-targeted insertions. Developing a two-element system in sorghum also seems feasible and efforts have been focused on this aspect. Two-element systems have proven successful both as endogenous (Ac/Ds or En/dSpm in maize) and heterologous systems (En/dSpm in Arabidopsis). To find the reporter dCs element that functions in the presence of the autonomous Cs1, we have recently recovered an anthocyanin phenotype in the endosperm.

Fig. 8.3
figure 3

Candystripe tagged putative mutants. (a) Zebra crossbands; (b) oldgold; (c) bloomless—mutant indicated with a red arrow and compared with the w-t plant on the left; (d) wilty; (e) yellow green; (f) lession mimic; (g) brown midrib; (h) striate leaves; (i) dwarf; (j) iojap striping; (k) virescent; (l) third leaf yellow; (m) premature senescence

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4 Perspective

The sorghum genome sequence together with its rich genetic resources (see Kresovich et al. Chap. 2, this volume) and cost-effective sequencing technology makes us rethink how to use these resources and technology for research. TILLING and EcoTILLING can mine induced mutations and natural sequence variation, respectively, within a species (Barkley and Wang 2008; Barkley et al. 2008; Comai et al. 2004). As sequence technology becomes increasingly cost-effective, it may become reasonable to sequence hundreds or more sorghum germplasm accessions as references for mining natural sequence variation. These unmutagenized and sequenced germplasm accessions can also be used as references for TILLING. An EMS-induced sorghum mutant population has been generated and used in the sorghum community (Xin et al. 2008). To broaden the mutational spectrum available, more sorghum mutagenized populations need to be generated by using different kinds of mutagens including irradiation. In the near future, TILLING by sequencing pooled mutant lines, known as SequeTILLING, will become a reality as new high-throughput and cost-effective sequencing technologies are developed (Weil 2009). A high-quality mutant library with well-annotated phenotypes and efficient pooling strategies will make SequeTILLING a very powerful approach for mutation identification.