Genetics

Barley Mutagenesis

Anders Falk, Alan H. Schulman, S0ren K. Rasmussen, and Christer Jansson

1 Introduction

The construction and utilization of mutants have a long history in plant breeding. In this review, we discuss two novel approaches to barley mutagenesis. First, we describe the powerful approach of neutron radia­tion for the production of deletion libraries in barley. With this method, deletions in the range of 100-10,000 bp can be generated. Based on the known number of neutron-induced mutations in barley, one can expect that between 10,000 and 20,000 mutagenized plants will be required in order to achieve a reasonable (>90%) probability of identifying at least one deletion mutant per gene.

The second approach deals with transposon mutagenesis. Since the rediscovery of Mendel's laws, genetic linkage, the genetic code and the molecular nature of the gene, three fundamental findings have changed our views of genetics: genome imprinting and other epigenetic phenom­ena, the existence of transposable elements, and the presence of repeti­tive DNA as the major component of the genome. During the past 20 years, it has become apparent that the last two discoveries are inextri­cably linked; much of the repetitive DNA in eukaryotes is composed of transposable elements and their transpositionally inert relics.

In the final section of the review, we give an example of ongoing mu­tational breeding in barley.

2 Construction and Utilization of Barley Mutant Libraries

a) Barley Mutants Induced by Radiation or Chemicals

When plant breeding was in its infancy, barley (Hordeum vulgare 1.) and other crop plants were mutagenized with the intention of generating plants with more agronomically favorable traits. The mutagenic treat­ments involved radiation such as X-rays, y rays, neutrons and chemicals, such as ethyl methane sulfonate and sodium azide. The mutagenized seed was grown for one generation, called the M1 generation. The screen

Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001

Barley Mutagenesis 35

for mutants was performed during the next generation, called the M2 generation, in which mutations occurred in the homozygous condition and mutated plants could be identified by their phenotype. While rela­tively few mutants of importance for plant breeding were isolated, it was realized that the mutant approach could reveal the functions of individ­ual genes of the plant. Since then, the analysis of mutants has been a popular method in plant biology. A large collection of mutants, espe­cially in barley, has therefore been collected during recent decades. The total number of barley mutants in these banks is estimated to be ap­proximately 10,000 (Lundqvist 1992), although not all are at different loci. Several different classes of mutants have been isolated.

Examples of viable mutations are the eceriferum (waxiess), erectoides and praematurum (early-heading) mutants. The eceriferum mutants were shown to be localized to 79 differ­ent loci (Lundqvist and Lundqvist 1988). Mutants whose biosynthesis of various bio­molecules (such as the anthocyanin-deficient mutants; Kristiansen and Rohde 1991; Olsen et al. 1993) or level of resistance against pathogens (Freialdenhoven et al. 1994, 1996; Jorgensen 1996) are affected have also been identified. Of the lethal mutants, many have affected pigment synthesis, as in the albina, xantha, viridis and tigrina mutants (Henningsen et al. 1993; Hansson et al. 1997), which define more than 100 different loci.

Although they are powerful techniques for the induction of mutations, chemicals and radiation have the drawback that they do not easily lend themselves to the cloning of the genes that have been mutated. Taking into account the availability of large mutant banks, the development of techniques that facilitate the cloning of the mutated genes is highly de­sirable and would make the existing mutant banks a valuable source for basic plant-biology research. This review will concentrate on methods that can be used to clone the genes mutated in these mutant banks and on the progress achieved to date. Basically, the techniques are of two different kinds: forward and reverse genetics.

b) Molecular Analysis of Barley Mutants Induced by Radiation or Chemicals

One option available for the cloning of a mutated gene is the method of map-based cloning (or chromosome landing as it is now popularly called; Tanksley et al. 1995). The technique has been most successfully applied to Arabidopsis, mainly due to the favorable relationship between genetic and physical distance in this species. However, two barley genes identified from mutant screens were recently cloned via the chromo­some-landing approach (Buschges et al. 1997; Lahaye et al. 1998a,b).

When mutated, the Mlo gene confers a durable, non-race-specific resistance to the pow­dery mildew fungus (Erysiphe graminis f. sp. hordei) in barley (Jorgensen 1992). Ampli­fied fragment-length polymorphism (AFLP) analysis of bulked segregants identified a set

36 Genetics

of AFLP markers that were polymorphic between the resistant and susceptible pools. Using a mapping population of 2000 resistant F2 plants, one AFLP marker was found to co-segregate with the Mlo gene (Simons et aI. 1997). Eventually, a 30,000-bp region was sequenced, and a likely candidate gene was identified. The final proof of cloning was found by analyzing rare intragenic recombinants in Mlo (Biischges et aI. 1997).

Similar procedures were applied to the cloning of the RARI gene al­though, in this case, fewer AFLP primer combinations were screened due to the identification of a co-segregating marker at an early stage during the mapping process (Lahaye et al. 1998a).

These clonings represent milestones in the analysis of barley mutants. Obviously, chromosome landing is still a tedious and labor-intensive process that needs to be much refined to make it applicable to large­scale cloning of the genes mutated in the existing mutant banks. One advantage of chromosome landing is that the method is applicable to all identified mutants.

Other cloning possibilities include extensive analysis of the pheno­type of the mutant, with the hope of thereby finding clues to the identity of the mutated gene. In such cases, similarities to mutants isolated from other organisms can prove useful.

In this way, the xantha-f, -g and -h genes were shown to encode Mg-chelatase (Jensen et al. 1996), and the ant-I8 gene was shown to encode dihydroflavonol-4-reductase (Kristiansen and Rohde 1991; Olsen et aI. 1993).

Extensive phenotypic analysis can be successful for the cloning of some structural genes but is less efficient for the cloning of regulatory genes, such as transcription factors. For instance, extensive analysis of the ti­grina mutants was not successful for the cloning of these genes (Hansson et al. 1998). Unfortunately, it is usually not possible to tell from the phe­notype of a mutant whether the mutated gene is a structural gene or a regulatory gene.

c) Fast Forward Genetics; Chromosome Landing Refined

Large-scale AFLP analysis of mutants can be expected to improve the process of chromosome landing. It is now possible to analyze AFLPs on automated sequencers with fluorescently labeled primers, thereby sig­nificantly speeding the screening of AFLP markers. A new method for the rapid AFLP-based mapping of mutants was recently described (Castiglione et al. 1998).

A number of AFLP markers were placed on the barley linkage map using di-haploid F2 lines from the Proctor x Nudinka cross. Linkage to these markers can be tested by means of AFLP analysis of F2 segregants from crosses of the type "mutant x Proctor" and "mutant x Nudinka".

Barley Mutagenesis 37

When linkage to a mapped AFLP marker has been established, further mapping can be done using known cleaved, amplified polymorphic se­quence (CAPS) or restriction fragment-length polymorphism markers. AFLP bands identified from bulked segregant analysis can also be quickly located on the right barley chromosome arm using a set of di­telosomic wheat-barley addition lines (Cannell et al. 1992). Again, fur­ther mapping should be done using known markers from this chromo­some arm. AFLP markers could alternatively be converted into more convenient markers, such as CAPS markers. The polymorphic AFLP band is then sequenced from both parent cultivars used in the mapping. However, because AFLPs are not more than a few hundred base pairs long, the probability of finding polymorphic sites that can be used for CAPS construction is quite low.

A more attractive solution would be to detect single-nucleotide poly­morphisms using denaturing high-performance liquid chromatography (DHPLC). DHPLC is a recently developed method of detecting small deletions or single-nucleotide polymorphisms (Liu et al. 1998; Giordano et al. 1999). In this way, a single-nucleotide polymorphism detected within a sequenced AFLP band could be used as a co-dominant marker. DHPLC can be fully automated and is therefore suitable for screening large mapping populations.

A further attractive approach is the utilization of deletion mutants for chromosome landing. For instance, one can land directly on deletions closely linked to a mutated gene using the AFLP analysis of bulked seg­regants. If the bulked segregants are mutant and wild-type F2 plants from a back-cross of the mutant to the wild type, all polymorphisms between the bulks are expected to be consequences of the mutation process. If methylation-sensitive enzymes (such as PstI or TaqI) are used, expressed regions of the genome would be preferentially screened in the AFLP analysis. In this way, one might even land on the very dele­tion that caused the mutant phenotype. Obviously, many AFLP markers would have to be screened for this approach, but the utilization of auto­matic sequencers would probably make this possible.

Obtained messenger RNA or complementary DNA (cDNA) from a deletion mutant could also be hybridized to a cDNA micro-array to identify the transcript lacking in the mutant. Obviously, the cloning of regulatory genes may be difficult with this approach, because several genes can be expected to be down-regulated as a consequence of a mu­tation in a regulatory gene.

The preferred method of inducing deletion mutants in plants is neu­tron radiation. The sizes of neutron-induced deletions have been exten­sively investigated only in Arabidopsis, where the size distribution indi­cated that most deletions are more than 8 kb in size (Bruggeman et al. 1996). However, a fraction of neutron mutants do not seem to be large deletions; thus, it is safest to have a set of independent mutants for each

38 Genetics

investigated locus. For many loci, such series of independent mutants do exist.

d) Barley Reverse Genetics

The advent of large-scale genomic sequencing and expressed sequence tags (ESTs) provides the basis of reverse genetics. The use of radiation­induced deletion mutants to obtain a reverse-genetics system in barley is intriguing. To detect deletion mutants, DNA fragments would be ampli­fied from DNA pools of M2 plants, and pools with amplified fragments smaller than the expected wild-type fragment would be further analyzed to identify the M2 plant that contains the deletion. Similar techniques were used to detect deletions in the nematode Caenorhabditis elegans, although the deletions were induced by transposons (Zwaal et al. 1993). Hybridization-based methods may also be used to detect deletions in reverse genetics. Individual preparations of M2-plant DNA could be immobilized on a (genomic) DNA micro-array, and probes for genes of interest would then be hybridized to the array. The generation of large numbers of plant DNA preparations can be done using various auto­mated techniques. Obviously, it will be important to optimize the signal­to-noise ratio in hybridizations to a genomic DNA micro-array.

The DHPLC (explained above) method may be used to detect small deletions or point mutations in polymerase chain reaction (PCR) frag­ments in M2-plant DNA pools. This method may be especially applicable in the detection of point mutations in essential genes, because deletions may not yield viable mutants in these cases. Also, point mutations can be more informative (for instance, about the contributions of individual amino acids to protein function). Therefore, a reverse-genetics system based on point mutations is desirable. In barley, point mutations are most effectively induced by sodium azide (Olsen et al. 1993).

For all PCR-based methods, the maximum pool size that can be used for the efficient detection of mutants is an important factor to consider. An attractive pool size would be 96 plants per pool, because this is the maximum number of samples accepted by most PCR machines. Then 96x96=9216 M2 plants can be grown in a square, and DNA preparations from all rows and columns in the square can be made, yielding 2x96 DNA preparations. Mutations would be identified by a row coordinate and a column coordinate. Before embarking on these adventures, it is necessary to consider the expected mutation frequency per gene, which varies among different genes and different mutagens. The mutation fre­quency in barley is calculated according to the spike-progeny method, which is the fraction of spike progenies that segregate for a certain phe­notype or a certain gene. Generally the mutation frequency is less than one mutant per gene per 15,000 spike progenies.

Barley Mutagenesis 39

e) Extending the Mutant Banks of Barley: Contribution from Arabidopsis

Since the days of mutation breeding, the mutant approach has also been applied to the model plant Arabidopsis. The contribution of Arabidopsis research is clearly seen in the invention of a number of new screening techniques used to identify mutants, especially in the field of plant­pathogen interactions. Using very fine-tuned screens, many Arabidopsis mutants that are either compromised in their resistance to pathogens (Glazebrook et al. 1996) or exhibit an enhanced level of resistance to pathogens (Frye and Innes 1998) have now been identified. These new screening techniques are also readily applicable to barley and other crop plants. Using the new screening methods and the emerging methods for reverse genetics, the mutant banks of barley can be significantly in­creased. Eventually, when the barley genome has been completely se­quenced, all genes will be identified and their function analyzed by a combination of forward- and reverse genetics techniques. Many techni­cal problems remain to be solved in the outlined methods. Barley is still a difficult plant to transform, and many of the described methods need an efficient system for the complementation of mutants. It is not likely that the method of intragenic recombination, as seen in the case of Mlo, could be generally used to establish the identity of a gene. In some cases, such as the resistance genes against powdery mildew (Shirasu et al. 1999), a transient expression system may prove sufficient for comple­mentation tests.

3 Transposable Elements As Major Contributors and Tools in Genomic Mutagenesis

a) The Mutagenic Impact and Application of DNA Transposons

Transposable elements are, in contrast to the genes recognized by classi­cal methods, self-mobilizing, independent genetic units that comprise a dynamic, fluid, rapidly evolving fraction of the genome. Transposable elements comprise two classes: the class-I elements or retro­transposons, which replicate via an RNA intermediate, and the class-II or DNA transposons, which move as DNA via a cut-and-paste mecha­nism (Finnegan 1990).

The class-II elements or transposons were the first to be actively studied in plants. Work on chromosome breakage in maize by McClin­tock during the 1930s (McClintock 1939) led to her pioneering proposal of the existence of "controlling elements" (McClintock 1956). The genet­ics of controlling elements has perhaps been best characterized in maize (Fedoroff 1983). Characteristically, they are found in two states:

40 Genetics

autonomous and non-autonomous. Autonomous elements excise, fully or partially restoring gene function and giving rise to sectors if the exci­sion is somatic, then re-integrate at another locus. Non-autonomous elements are stably integrated unless mobilized by the presence of an autonomous element (Dellaporta and Chomet 1985). In addition, the autonomous elements may undergo a phase change in which they be­come "cryptic" or inactive.

During the 1980s, the molecular nature of controlling elements was determined to be that of mobile DNA transposons (Shure et al. 1983; Doring and Starlinger 1984), a conceptual revolution recognized when McClintock received the Nobel Prize (McClintock 1984). All DNA trans­posons share a similar organization: short, terminal, inverted repeats of approximately 10 bp and a central region encoding a transposase re­quired for the cutting and pasting of the transposon at its termini during mobilization. Autonomous versions are generally 4-10 kb, whereas non­autonomous forms are smaller deletion derivatives of the autonomous elements, ranging to less than 400 bp. The best-characterized DNA transposons have been the AciDs (paired as autonomous/non-auto­nomous), En or Spm/I or dSpm, Mutator of maize, and the Tam elements of snapdragon (Antirrhinum majus; Doring and Starlinger 1986; Gierl and Saedler 1986). Analysis of these systems in both species has been aided by the insertion of the elements into genes involved in the expres­sion of easily scored phenotypic traits, such as anthocyanin and amylose biosynthesis. This has enabled simultaneous exploration of the effects of transposon insertions on gene expression and regulation, and examina­tion of the mechanisms affecting transposon activity (Martin and Lister 1989; Weil and Wessler 1990; Fedoroff et al. 1995; Fedoroff 1999; Girard and Freeling 1999)

Given the spectacular phenotypes generated by transposon insertion and excision, perhaps it is not surprising that molecular characterization of the elements led to their development as tools for gene tagging and mutagenesis. The first successful transposon tagging was that of the bronze gene in maize, on the anthocyanin-biosynthesis pathway (Fedoroff et al. 1984). Combining the recombinational-genetic analysis of insertionally induced mutants and their excision-generated rever­tants, transposons were applied in their native hosts in many similar efforts thereafter. The real beginning of the widespread application of transposons for gene tagging and mutagenesis came with the transfer of well-characterized maize elements to other species (beginning with the easily transformed tobacco) and the demonstration of the elements' mobilities in those species (Baker et al. 1986).

As in maize, the successful tagging of genes in heterologous species has relied on easily scored and cell-autonomous phenotypes, such as a yellow-leaf phenotype in tomato (Peterson and Yoder 1993) or corolla color in petunia (Chuck et al. 1993). Due to the power of the genetics of

Barley Mutagenesis 41

Arabidopsis thaliana, it was natural to move maize transposons to this plant (Van Sluys et al. 1987). Successful gene tagging strategies for A. thaliana based both on Spm (Aarts et al. 1993) and on modified Ac vec­tors with increased transposase expression in trans with a Ds element (Bancroft et al. 1993) were developed. Two-element tagging strategies, particularly employing Ac transposase under a strong promoter and a Ds element modified to be selectable or screenable, have become increas­ingly popular (Osborne and Baker 1995; Fitzmaurice et al. 1999). Such systems have proven effective in the monocot rice (Izawa et al. 1997; Chin et al. 1999) and are being used in other monocots previously re­calcitrant to transformation, such as barley (McElroy et al. 1997) and wheat (Takumi et al. 1999).

b) The Nature of Retro-Transposons

Unlike the type-II DNA transposable elements, such as Ac and En, inte­grated copies of retro-transposons are not excised as a part of transposi­tion. Instead, transposition is a replicative process and would be better described as propagation. The retro-transposons may be divided into two main classes: long terminal repeat (LTR)-bearing retro-transposons (Grandbastien 1992; Bennetzen 1996; Kumar and Bennetzen 1999), and the long- and short-interspersed elements (LINEs and SINEs, respec­tively), which do not bear LTRs (Schmidt 1999). The LTR retro­transposons resemble retroviruses in their organization, encoded gene products and life cycle. The life cycle for both retro-transposons and the retroviruses involves successive transcription, reverse transcription and integration back into the genome (Boeke and Chapman 1991). These two groups are highly likely to have been in existence in the last common ancestor of the fungi, plants and animals, or were laterally transferred into each group shortly thereafter. Retro-transposons are functionally distinguished from retroviruses by their lack of infectivity in mammals, depending on the env or envelope gene. Both major groups of retro­transposons, the copia-like (Flavell et al. 1992; Voytas et al. 1992) and the gypsy-like (Suoniemi et al. 1998a), are ubiquitous in plants.

Each transcript of a retro-transposon has the potential (as eDNA) to be re-integrated into the genome, thereby giving rise to additional tran­scripts following integration. These new copies are inherited if the inte­grations occur in cells ultimately giving rise to gametes. Therefore, per­haps it is not surprising that retro-transposons are highly prevalent in many plant genomes, where the germ line is formed only following many somatic divisions. Retro-transposons may even contribute half of the total DNA content in some plants (Pearce et al. 1996a; San Miguel et al. 1996) and comprise a major part of the repetitive DNA component of the genome. Their replicative dynamics appear, at least in some cases (Rai

42 Genetics

and Black 1999; Vicient et al. 1999), to be a major factor contributing to genome size variations in plants.

c) The Mutagenic Impact and Application of Retro-Transposons

Due to their invasiveness, promoter activity and sheer copy numbers, retro-transposons can have many effects on the genome and organism. Insertional gene inactivation or mutagenesis by SINEs, LINEs and retro­transposons has been documented (Varagona et al. 1992; Britten 1997; Hirochika 1997). Changes in promoter specificity or activation pattern by LTR retro-transposons have also been demonstrated in plants (Marillonnet and Wessler 1997). The sheer quantity of DNA comprising particularly prevalent retro-transposon families can affect the genome size (Vicient et al. 1999). The genome size, in turn, is thought have many physiological, ecological and developmental consequences through ef­fects on the size of the cell nucleus, cell-cycle time and the time to ma­turity.

For several reasons, genotypic change due to retro-transposon activ­ity can be much more rapid than change due to mutations of single-copy genes or small gene families. Retro-transposon insertions have great mutagenic potential, because they are kilobase-scale alterations in the surrounding DNA. Each element contains transcriptional control ele­ments that can cause major perturbations in the activity of adjacent genes. Furthermore, retro-transposons are known to be activated by stress in plants (Wessler 1996; Grandbastien 1998; Takeda et al. 1998). Retro-transposon insertions create joints between genomic DNA and their own conserved termini; therefore, they can also serve as convenient tools for tracking the changes they induce. Several techniques that pro­duce marker bands from retro-transposon insertion loci have been de­veloped (Waugh et al. 1997; Ellis et al. 1998; Flavell et al. 1998; Gribbon et al. 1999; Kalendar et al. 1999). Retro-transposon insertions are unidi­rectional, leading to progressive genome diversification, which can be subjected to pedigree analysis (Shimamura et al. 1997; Ellis et al. 1998; Flavell et al. 1998).

Mutagenesis by retro-transposons, due to the linkage of retro­transposon activity to experimentally induced stress, may also be con­nected to environmental stimuli. When such environmental stress fac­tors display eco-geographical variation, the genomic effects in natural populations may be detectable in the retro-transposon prevalence and insertion patterns. Moreover, retro-transposon replication is error prone, because it relies on reverse transcriptase; therefore, the retro­transposon fraction of the genome evolves comparatively rapidly. This may result in even greater variation in transposition rates between or-

Barley Mutagenesis 43

ganisms and may cause genome diversification both within populations and between them.

Thus, plant genomes may be viewed as being in dynamic flux, with retro-transposons playing a considerably greater role than DNA trans­posons in genomic modification. The development of these retro­transposons as gene-tagging tools, however, has not been widespread. Several reasons may be cited:

The high copy number of most retro-transposon families makes genetic analysis difficult. This problem is increased by the lack of reversion due to the absence of retro-transposon excision. Furthermore, many retro-transposon families, as demonstrated for maize and barley, exhibit a nested insertion pattern; they are found preferentially inserted into other retro-transposon families (San Miguel et al. 1996; Suoniemi et al. 1997). However, particularly low-copy-number families of retro-transposons appear to be far more mutagenic than those present in greater numbers; perhaps for this reason, they are found in fewer copies.

Nevertheless, gene tagging and mutagenesis are possible with retro­transposons. The first demonstrably active plant retro-transposon, Tnt-1, was isolated by selecting for insertion into a nitrate-reductase gene (Grandbastien et al. 1989), arguably a tagging procedure. The Tos17 retro-transposon of rice, highly active in tissue cultures, is used in a large-scale program that screens for mutants among regenerated plants (Hirochika 1997). The replicative nature of retro-transposition has se­lected for highly effective integration mechanisms catalyzed by the en­zyme integrase. Integrase is highly conserved, and plant integrase ap­pears to closely match retroviral integrases in structure (Suoniemi et al. 1998b). Hence, the applicability of retroviruses and retro-transposons as vectors for gene therapy and transformation has been well recognized (Kingsman et al. 1991; Ivics et al. 1993; Bushman 1994; Katz et al. 1996). Due to the increasing understanding of the life cycle of plant retro­transposons (JaaskeHiinen et al. 1999), there is no reason these cannot be developed for gene ablation or specific tagging using the same princi­ples.

4 Mutational Breeding in Barley: an Example. Improving Nutritional Qualities

Mutational breeding in barley was initiated to reduce the anti-nutrition­al effect of phytic acid [myoinositol 1,2,3,4,5,6-hexakisphosphate (IP6)] and to provide plant material for biochemical studies of the biosynthesis of IP6 via the sequential addition of phosphate to myoinositol. Barley, like other cereals, stores up to 80% of the total grain phosphate as phytin (Raboy and Gerbasi 1996). The remaining phosphate is free phosphate, phospholipid nucleic acids, etc. Most of the phytin is stored as electron-

44 Genetics

dense particles in protein bodies in the aleuron cell layer of barley, and the remaining (-10%) is stored in the embryo. This is in contrast to maize, which stores most of its phytin in the embryo (O'Dell et al. 1972). The anti-nutritional effect of phytic acid is due to the lack of phytase activity in monogastic animals, such as pigs, chicken, fish and humans; hence, phytin passes undegraded through the intestines and stomach and is released with the feces. In the soil, phytin is degraded and thus contributes to the pollution of freshwater streams, because phosphate leads to the eutrophic growth of algae. Phytate also forms salts with cations, such as zinc, calcium and iron, making these unavailable for uptake. Limited bio-availability of particulate iron is of great concern in human nutrition, as detailed in the World Food Summit held in Rome in 1996. Modern agriculture compensates for phytate effects by adding phosphate to fodder and supplementing it with minerals, or by adding microbial phytase as a feed enzyme.

The initial screening for low phytate grains was an indirect measure­ment based on the assumption that reduced phytate results in increased free phosphate. This allowed the use of a simple molybdate staining pro­cedure for free phosphate; grains staining blue were taken as indicative of reduced phytate content.

Initially, 2000 M2 half grains from sodium-azide-treated Pallas POI grains were screened; for those that scored positive, the embryo-containing part was germinated, and M3 spikes were harvested. To confirm that these actually were low in phytate, a thin-layer chromatography (TLC) system was used to detect and separate phytic acid from free phosphate and intermediates of phytate, and 18 mutants were confirmed to be low­phytate varieties (Rasmussen and Hatzack 1998). There was an unexpectedly high num­ber of mutants, which could indicate that phytate biosynthesis has a hot spot for muta­tions. The selected mutants were tentatively diveded into two classes: the A-type (with less than 10% phytate) and the B-type (with 50-60% phytate, compared with 100% for the non-mutated strain). Tests for allelism showed that the mutations are located in unlinked loci. DNA gel blot analyses using inositol 1,3,4-trisphosphate 5/6-kinase and myoinositol phosphate synthase as probes did not indicate a mutation at these loci (Rasmussen, unpublished).

The TLC system was further refined to detect myoinositol with less than six phosphate groups. The selected lines have been field grown in in­spection plots at several locations in Denmark for 2 years, and many exhibited normal vigor and normal or almost normal seed set. As ex­pected, in some cases, pleiotropic effects could be noted (such as shriv­eled kernels, particularly with the A type). These and other effects might be due to additional mutations in the raw mutants, which could be eliminated by cross-breeding. Several lines were also propagated in New Zealand during 1998-1999 and in Denmark during 1999 to increase the amount available for animal feed trails.

Barley Mutagenesis 45

The mutant lines have been investigated with respect to phosphorus nutrition in 201 pots, with rock wool as the growth medium. The plants were grown to maturity outdoors with an automated siphon airlift watering system.

The grain yield of the B-type low-phytate mutants was the same as for the parent varieties. The grain yield for the A-type was severely reduced at all phosphate levels. To test whether nutritional improvements had been achieved, tests were initiated with rats, because these were previ­ously successfully used in tests for high lysin and nitrogen nutrition by Eggum (1973). The tests showed that the apparent digestibility of phos­phate was improved in mutant lines and that more zinc was taken up from these lines by the rats (Poulsen et al. 2000). This indicated that mutational breeding is feasible and suggested that feeding trials should be repeated with piglets and broilers.

Several high-lysin barley mutants have been generated and analyzed genetically, biochemically, nutritionally and in breeding programs. Al­though much effort has been spent on attempts to breed barley for high lysin content, the yield has always been 5-10% lower than those used in national tests. This could be because of lower starch content and rela­tively higher amounts of non-starch carbohydrates in the mutants. It was generally accepted that it might not be possible to breed high­yielding barley with a high lysin content. Genetically, there is no simple relationship between improved lysin content and lower grain yield (Doll 1975). From the genetic analyses, it was evident that many loci on differ­ent chromosomes could be mutated to yield high-lysin barley. It was also known that high lysin content was obtained due to an increase in albu­min at the expense of the concentration of the storage protein hordein. Therefore, a new strategy was used by Jensen (1991): screening for low­hordein mutants with a minimal yield reduction as an indirect way of finding mutants with a high lysin content. The so-called turbidity test, which gives a simple reflection of the content of alcohol-soluble storage proteins, was used. Twenty low-hordein barley mutants were scored from 49,000 M2 grains. Several of these had improved lysin content, minimal yield depression and a kernel weight similar to that of the mother variety Sultan (Eggum et al. 1995). In balanced feeding trials with rats, the mutants resulted in an increase in biological value of up to 20%. Thus, it seems to be possible to develop high-lysin barley cultivars. Furthermore, the higher biological values of the mutant lines resulted in a significant reduction of nitrogen in the slurry and, therefore, a reduced impact on the environment.

46 Genetics

Baker B, Schell J, Lorz H, Fedoroff NY (1986) Transposition of maize controlling element Activator in tobacco. Proc Nat! Acad Sci USA 83:4844-4848

Bancroft I, Jones JDG, Dean C (1993) Heterologous transposon tagging of the DRLllocus in Arabidopsis. Plant Cell 5:631-638

Bennetzen JL (1996) The contributions of retroelements to plant genome organization, function and evolution. Trends MicrobioI4:347-353

Boeke JD, Chapman KB (1991) Retrotransposition mechanisms. Curr Opin Cell BioI 3:502...,507

Britten RJ (1997) Mobile elements inserted in the distant past have taken on important functions. Gene 205:177-182

Bruggemann E, Handwerger K, Essex C, Storz G (1996) Analysis of fast neutron­generated mutants at the Arabidopsis thaliana HY410cus. Plant J 10:755-760

Buschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, Van Daelen R, Van der Lee T, Diergaarde P, Groenendijk J, Topsch S, Vos P, Salamini F, Schulze­Lefert P (1997) The barley Mlo gene: a novel control element of plant pathogen resis­tance. Cell 88:695-705

Bushman FD (1994) Tethering human immunodeficiency virus 1 integrase to a DNA site directs integration to nearby sequences. Proc Nat! Acad Sci USA 91:9233-9237

Cannell M, Karp A, Isaac PG, Shewry P (1992) Chromosomal assignment of genes in barley using telosomic wheat-barley addition lines. Genome 35: 17 -23

Castiglioni P, Pozzi P, Heun M, Terzi V, Muller KJ, Rohde W, Salamini F (1998) An AFLP-based procedure for the efficient mapping of mutations and DNA probes in barley. Genetics 149:2039-2056

Chin HG, Choe MS, Lee SH, Park SH, Koo JC, Kim NY, Lee JJ, Oh BG, Yi GH, Kim SC, Choi HC, Cho MJ, Han CD (1999) Molecular analysis of rice plants harboring an AciDs transposable element-mediated gene trapping system. Plant J 19:615-623

Chuck GT, Robbins T, Nijar C, Ralston E, Courtney-Gutteerson N, Dooner H (1993) Tagging and cloning of a petunia flower color gene with the maize transposable ele­ment Activator. Plant Cell 5:371-378

Dellaporta SL, Chomet PS (1985) The activation of maize controlling elements. In: Hohn B, Dennis ES (eds) Genetic flux in plants. Springer, Berlin Heidelberg New York, pp 169-216

Doll H (1975) Genetic studies of high lysine mutants. In: Gaul HM (ed) Barley genetics III. Thieming, Munich, pp 542-546

Doring H-P, Starlinger P (1984) Barbara McClintock's controlling elements: now at the DNA level. Cell 39:253-259

Doring H-P, Starlinger P (1986) Molecular genetics of transposable elements in plants. Annu Rev Genet 20:175-200

Eggum BO (1973) A study of certain factors influencing protein utilization in rats and pigs (no. 406). National Institute of Animal Science, Copenhagen

Eggum BO, Brunsgaard G, Jensen J (1995) The nutritive value of new high-lysin barley mutants. J Cereal Sci 22:171-176

Ellis THN, Poyser SJ, Knox MR, Vershinin AV, Ambrose MJ (1998) Tyl-copia class retro­transposon insertion site polymorphism for linkage and diversity analysis in pea. Mol Gen Genet 260:9-19

Fedoroff N, Schlappi M, Raina R (1995) Epigenetic regulation of the maize Spm trans­poson. Bioessays 17:291-297

Fedoroff NY (1983) Controlling elements in maize. In: Shapiro JA (ed) Mobile genetic elements. Academic Press, New York, pp 1-63

Fedoroff NY (1999) The Supressor-Mutator element and the evolutionary riddle of transposons. Genes Cells 4: 11-19

Fedoroff NY, Furtek DB, Nelson OE (1984) Cloning of the bronze locus in maize by a simple and generalizable procedure using the transposable element Activator (Ac). Proc Nat! Acad Sci USA 81:3825-3829

Barley Mutagenesis 47

Finnegan DJ (1990) Transposable Elements and DNA Transposition in Eukaryotes. Curr Opin Cell Bioi 2:471-477

Fitzmaurice WP, Nguyen LV, Wernsman EA, Thompson WF, Conkling MA (1999) Transposon tagging of the sulfur gene of tobacco using engineered maize AcIDs ele­ments. Genetics 153:1919-1928

Flavell AJ, Dunbar E, Anderson R, Pearce SR, Hartley R, Kumar A (1992) Tyl-copia group retrotransposons are ubiquitous and heterogeneous in higher plants. Nucleic Acids Res 20:3639-3644

Flavell AJ, Knox MR, Pearce SR, Ellis THN (1998) Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis. Plant J 16:643-650

Freialdenhoven A, Scherag B, Hollricher K, Collinge DB, Thordal-Christensen H, Schulze-Lefert P (1994) Nar-l and Nar-2, two loci required for Mlal2-specified race­specific resistance to powdery mildew in barley. Plant Cell 6:983-994

Freialdenhoven A, Peterhiinsel C, Kurth J, Kreuzaler F, Schulze-Lefert P (1996) Identifi­cation of genes required for the function of non-race-specific mlo resistance to pow­dery mildew in barley. Plant Cell 8:5-14

Frye CA, Innes RW (1998) An Arabidopsis mutant with enhanced resistance to powdery mildew. Plant Cell 10:947-956

Gierl A, Saedler H (1986) The EnlSpm transposable element of Zea mays. Plant Mol Bioi 13:261-266

Giordano M, Oefner pJ, Underhill PA, Cavalli-Sforza LL, Tosi R, Momigliano-Richardi P (1999) Identification by denaturing high-performance liquid chromatography of nu­merous polymorphisms in a candidate region for multiple sclerosis susceptibility. Genomics 56:247-253

Girard L, Freeling M (1999) Regulatory changes as a consequence of transposon inser­tion. Dev Genet 25:291-296

Glazebrook J, Rogers EE, Ausubel FM (1996) Isolation of Arabidopsis mutants with en­hanced disease susceptibility by direct screening. Genetics 143:973-982

Grandbastien M-A (1992) Retroelements in higher plants. Trends Genet 8:103-108 Grandbastien M-A (1998) Activation of retrotransposons under stress conditions. Trends

Plant Sci 3:181-187 Grandbastien M-A, Spielmann A, Caboche M (1989) Tntl, a mobile retroviral-like trans­

posable element of tobacco isolated by plant cell genetics. Nature 337:376-380 Gribbon BM, Pearce SR, Kalendar R, Schulman AH, Jack P, Kumar A, Flavell AJ (1999)

Phylogeny and transpositional activity of Tyl-copia group retrotransposons in cereal genomes. Mol Gen Genet 261:883-891

Hansson M, Gough SP, Kannangara CG, Von Wettstein D (1997) Analysis of RNA and enzymes of potential importance for regulation of 5-aminolevulinic acid synthesis in the protochlorophyllide accumulating barley mutant tigrina-dI2. Plant Physiol Bio­chern 35:827-836

Hansson M, Gough SP, Kannangara CG, Von Wettstein D (1998) Chromosomal locations of six barley genes encoding enzymes of chlorophyll and heme biosynthesis and the sequence of the ferrochelatase gene identify two regulatory genes. Plant Physiol Bio­chern 36:545-554

Hatzack F, Rasmussen SK (1999) High-performance thin-layer chromatography method for inositol phosphate analysis. J Chromatogr B Biomed AppI736:221-229

Henningsen KW, Boynton JE, Von Wettstein D (1993) Mutants at xantha and albina loci in relation to chloroplast biogenesis in barley (Hordeum vulgare L.). Hereditas 129:107-113

Hirochika H (1997) Retrotransposons of rice: their regulation and use for genome analy­sis. Plant Mol Bioi 35:231-240

lvics Z, Izsvak Z, Hackett PB (1993) Enhanced incorporation of transgenic DNA into zebrafish chromosomes by a retroviral integration protein. Mol Mar Bioi Biotechnol 2:162-173

48 Genetics

Hirochika H (1997) Retrotransposons of rice: their regulation and use for genome analy­sis. Plant Mol Bioi 35:231-240

Ivics Z, Izsv<ik Z, Hackett PB (1993) Enhanced incorporation of transgenic DNA into zebrafish chromosomes by a retroviral integration protein. Mol Mar Bioi Biotechnol 2:162-173

Izawa T, Ohnishi T, Nakano T, Ishida N, Enoki H, Hashimoto H, !toh K, Terada R, Wu C, Miyazaki C, Endo T, !ida S, Shimamoto K (1997) Transposon tagging in rice. Plant Mol Bioi 35:219-229

Jaaskelainen M, Mykkanen A-H, Arna T, Vicient C, Suoniemi A, Kalendar R, Savilahti H, Schulman AH (1999) Retrotransposon BARE-I: expression of encoded proteins and formation of virus-like particles in barley cells. Plant J 20:413-422

Jensen J (1991) New high yielding, high lysine mutants in barley. In: Plant mutation breeding for crop improvement. STI/PUB International Atomic Energy Agency, Vienna. Vol. 842 pp 31-41

Jensen PE, Willows RD, Petersen BL, Vothknecht UC, Stummann BM, Kannangara CG, Von Wettstein D, Henningsen KW (1996) Structural genes for Mg-chelatase subunits in barley: xantha-f, -g and -h. Mol Gen Genet 250:383-394

Jorgensen JH (1992) Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley. Euphytica 63:141-152

Jorgensen JH (1996) Effect of three suppressors on the expression of powdery mildew resistance genes in barley. Genome 39:492-498

Kalendar R, Grob T, Regina M, Suoniemi A, Schulman AH (1999) IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. Theor Appl Genet 98:704-711

Katz RA, Merkel G, Skalka AM (1996) Targeting of retroviral integrase to a heterologous DNA binding domain: in vitro activities and incorporation of a fusion protein into vi­ral particles. Virology 217:178-190

Kingsman AJ, Adams SE, Burns NR, Kingsman SM (1991) Retroelement particles as purification, presentation and targeting vehicles. Trends Biotechnol 9:303-309

Kristiansen KN, Rohde W (1991) Structure of the Hordeum vulgare gene encoding dihy­droflavonol-4-reductase and molecular analysis of antI8 mutants blocked in fla­vonoid synthesis. Mol Gen Genet 230:49-59

Kumar A, Bennetzen J (1999) Plant retrotransposons. Annu Rev Genet 33:479-532 Lahaye T, Hartmann S, Topsch S, Freialdenhoven A, Schulze-Lefert P (1998a) High­

resolution genetic and physical mapping of the Rarl locus in barley. Theor Appl Genet 97:526-534

Lahaye T, Shirasu K, Schulze-Lefert P (1998b) Chromosome landing at the barley Rarl locus. Mol Gen Genet 260:92-101

Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, James CD (1998) Denaturing high per­formance liquid chromatography (DHPLC) used in the detection of germline and so­matic mutations. Nucleic Acids Res 26:1396-1400

Lundqvist U (1992) Mutation research in barley. Doctoral thesis, Swedish University of Agricultural Sciences, SvalOv, Sweden

Lundqvist U, Lundqvist A (1988) Mutagen specificity in barley for 1580 eceriferum mu­tants localized to 79 loci. Hereditas 108:1-12

Marillonnet S, Wessler SR (I997) Retrotransposon insertion into the maize waxy gene results in tissue-specific RNA processing. Plant Cell 9:967-978

Martin C, Lister C (1989) Genome juggling by transposons: Tam3-induced rearrange­ments in Antirrhinum majus. Dev Genet 10:438-451

McClintock B (1939) The behavior in successive nuclear divisions of a chromosome broken at meiosis. Proc Nat! Acad Sci USA 25:405-416

McClintock B (1956) Controlling elements and the gene. Cold Spring Harb Symp Quant Bioi 21:197-216

Barley Mutagenesis 49

McClintock B (1984) The significance of responses of the genome to challenge. Science 226:792-801

McElroy D, Louwerse JD, McElroy SM, Lemaux PG (1997) Development of a simple tran­sient assay for AciDs activity in cells of intact barley tissue. Plant J 11:157-165

O'Dell BL, De Boland AR, Koirtyohann SR (1972) Distribution of phytate and nutrition­ally important elements among the morphological components of cereal grains. J Ag­ric Food Chern 20:718-721

Olsen 0, Wang X, Von Wettstein D (1993) Sodium azide mutagenesis: preferential gen­eration of AT -GC transitions in the barley AnU8 gene. Proc Nat! Acad Sci USA 90:8043-8047

Osborne BI, Baker B (1995) Movers and shakers: maize transposons as tools for analyz­ing other plant genomes. Curr Opin Cell BioI7:406-4l3

Pearce SR, Harrison G, Li D, Heslop-Harrison JS, Kumar A, Flavell AJ (1996a) The Tyl­copia group of retrotransposons in Vida species: copy number, sequence heteroge­neity and chromosomal localisation. Mol Gen Genet 205:305-315

Peterson PW, Yoder JI (1993) Ac-induced instability at the Xanthophyllic locus of to­mato. Genetics l34:931-942

Poulsen HD, Johansen KS, Hatzack F, Boisen S, Rasmussen SK (2000) The nutritional value oflow-phytate barley evaluated in rats. Acta Agric Scand A Anim Sci (in press)

Raboy V, Gerbasi P (1996) Genetics of myo-inositol phosphate synthesis and accumula­tion. In: Biswas BB, Biswas S (eds) Subcellular biochemistry: myoinositol phosphates, phosphoinositides, and signal transduction, vol 26. Plenum Press, New York, pp 257-285

Rai KS, Black WC IV (1999) Mosquito genomes: structure, organization, and evolution. Adv Genet 41:1-33

Rasmussen SK, Hatzack F (1998) Identification of two low-phytate barley (Hordeum vulgare L.) grain mutants by TLC and genetic analysis. Hereditas 129: 107 -113

San Miguel P, Tikhonov A, Jin YK, Motchoulskaia N, Zakharov D, Melake-Berhan A, Springer PS, Edwards KJ, Lee M, Avramova Z, Bennetzen JL (1996) Nested retrotrans­posons in the intergenic regions of the maize genome. Science 274:765-768

Schmidt T (1999) LINEs, SINEs and repetitive DNA: non-LTR retrotransposons in plant genomes. Plant Mol Bioi 40:903-910

Shimamura M, Yasue H, Ohshima K, Abe H, Kato H, Kishiro T, Goto M, Munechika I, Okada N (1997) Molecular evidence from retroposons that whales form a clade within even-toed ungulates. Nature 388:666-670

Shirasu K, Nielsen K, Piffanelli P, Oliver R, Schulze-Lefert P (1999) Cell-autonomous complementation of mlo resistance using a biolistic transient expression system. Plant J 17:293-299

Shure M, Wessler S, FedoroffN (1983) Molecular identification and isolation of the waxy locus in maize. Cell 35:25-233

Simons G, Van der Lee T, Diergaarde P, Van Daelen R, Groenendijk J, Frijters A, Blisch­ges R, Hollricher K, Topsch S, Schulze-Lefert P et al. (1997) AFLP-based fine mapping of the Mia gene to a 30-kb DNA segment of the barley genome. Genomics 44:61-70

Suoniemi A, Schmidt D, Schulman AH (1997) BARE-l insertion site preferences and evolutionary conservation of RNA and cDNA processing sites. Genetica 100:219-230

Suoniemi A, Tanskanen J, Schulman AH (1998a) Gypsy-like retrotransposons are wide­spread in the plant kingdom. Plant J 13:699-705

Suoniemi A, Tanskanen J, Pentikllinen 0, Johnson MS, Schulman AH (1998b) The core domain of retrotransposon integrase in Hordeum: predicted structure and evolution. Mol Bioi EvoI15:1135-1144

Takeda S, Sugimoto K, Otsuki H, Hirochika H (1998) Transcriptional activation of the tobacco retrotransposon Ttol by wounding and methyl jasmonate. Plant Mol Bioi 36:365-376

50 Genetics

Takumi S, Murai K, Mori N, Nakamura C (1999) Variations in the maize Ac transposase transcript level and the Ds excision frequency in transgenic wheat callus lines. Genome 42:1234-1241

Tanksley SD, Gana! MW, Martin GB (1995) Chromosome landing: a paradigm for map­based cloning in plants with large genomes. Trends Genet 11 :63-68

Van Sluys MA, Tempe J, Fedoroff N (1987) Studies on the introduction and mobility of the maize Activator element in Arabidopsis thaliana and Daucus carota. EMBO J 6:3881-3889

Varagona MJ, Purugganan M, Wessler SR (1992) Alternative splicing induced by inser­tion of retrotransposons into the maize waxy gene. Plant Cell 4:811-820

Vicient CM, Suoniemi A, Anamthawat-J6nsson K, Tanskanen J, Beharav A, Nevo E, Schulman AH (1999) Retrotransposon BARE-l and its role in genome evolution in the genus Hordeum. Plant Cell 11:1769-1784

Voytas DF, Cummings MP, Konieczny AK, Ausubel FM, Rodermel SR (1992) Copia-like retrotransposons are ubiquitous among plants. Proc Nat! Acad Sci USA 89:7124-7128

Waugh R, McLean K, Flavell AJ, Pearce SR, Kumar A, Thomas BBT, Powell W (1997) Genetic distribution of BARE-I-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol Gen Genet 253:687-694

Weil CF, Wessler SR (1990) The effects of plant transposable element insertion on tran­scription initiation and RNA processing. Annu Rev Plant Physiol Plant Mol BioI 41:527-552

Wessler SR (1996) Turned on by stress. Plant retrotransposons. Curr BioI 6:959-961 Zwaa! RR, Broeks A, Van Meurs J, Groenen JTM, Plasterk RHA (1993) Target-selected

gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank. Proc Nat! Acad Sci USA 90:7431-7435

Communicated by K. Esser

Dr. Anders Falk Dr. Christer Jansson Department of Plant Biology The Swedish University of Agricultural Sciences P.O. Box 7080 75007 Uppsala, Sweden

Dr. Alan H. Schulman Institute of Biotechnology University of Helsinki Plant Genomics Laboratory P.O. Box 56 (Viikinkaari 6) 00014 Helsinki, Finland

Dr. Seren K. Rasmussen Rise National Laboratory Roskilde, Denmark