Sep 1, 1991 - (RFLP), and gene markers. This project is a major undertaking for the maize community, with the rallying cry â20,000 loci by the year 2000!
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Maize Mutants for the 2Ist Century Mutants provide the primary means of Knl defines the first homeobox-conunderstanding gene function in higher taining plant gene (Vollbrecht et al., plants. At the 1991 Maize Genetics 1991). Using the Knl homeobox as a Meeting, which was held in Delavan, hybridization probe, Brenda Lowe and Wisconsin, from March 21 to March coworkers in Sarah Hake’s laboratory 24, a practical new strategy for stream- (Plant Gene Expression Center, Allining the identification and mapping of bany, CA) and Michael Freeling’s labomutants was presented. The “inte- ratory (University of California, Berkegrated mapping project” will produce a ley) have cloned additional homeoboxsingle high-resolution map of the maize containing genes. Phil Becraft and cogenome that combines physical, restric- workers in the Freeling laboratory tion fragment length polymorphism showed that one of these homeobox (RFLP), and gene markers. This project sequences maps to the same position is a major undertaking for the maize as Rough sheafh-1 (Rsl),a locus community, with the rallying cry “20,000 defined by an EMS-induced dominant loci by the year 2000!” In this report, I leaf mutation. They have cloned and will explain the integrated mapping partially sequenced the gene that project and highlight presentations maps to Rsl. As expected, it is very concerned with the characterization similar to Knl in the homeodomain and cloning of new genes. Detailed region, but in addition, regions of simidescriptions of many of the mutants larity are found outside of this motif. mentioned here are found in the chap- An additional half dozen fragments ters on genetics and cytogenetics in cross-hybridize to the homeodomain Sprague and Dudley (1988). Photo- region of Knl on DNA gel blots, and graphs of phenotypes are found in it is expected that at least severa1 of Neuffer et al. (1968). Another useful these will define developmentally inreference is Freeling and Walbot teresting genes. The challenge now is (1992). to define how Knl and Rsl control cell fates and whether the homeoboxcontaining genes of maize function differentially over developmental time Dominant Morphological (or position in the leaf) to program Mutants-The Homeobox normal organ development. Connection Dominant morphological mutations in maize typically show a “gain of func- Auxin Biosynthesis-Not from tion” phenotype in which the position Tryptophan after All? or size of normal structures is altered. The largest set of such mutants affects Maize geneticists have begun to benleaf morphology, particularly the struc- efit from gene cloning projects in other ture and position of the ligule, a fringe higher plants. An important example of tissue found at the junction of the is provided by the cloning of a gene sheath and blade. A number of these encoding the p subunit of tryptophan mutants were defined by ethylmethane synthase from Arabidopsis (Berlyn et sulfonate (EMS) mutagenesis (more on al., 1989). Karen Cone and her cothis later); however, the first dominant workers at the University of Missouri, leaf mutation cloned, Knotted-1(Knl), Columbia used the Arabidopsis gene was recovered by transposon tagging. to confirm that the orange pericarp
phenotype of maize, conditioned when two genes are nonfunctional (orpl orp2 double recessive), results from a deficiency in the tryptophan synthase p subunit. Tryptophan has been postulated to be the precursor of auxin in higher plants, but Allen Wright and coworkers in Gerry Neuffer’s laboratory (University of Missouri) showed that orange pericarp mutants contain 50-fold more auxin than do wild-type plants. Using feeding studies with radioactive precursors combined with chemical analysis, Wright and coworkers demonstrated that the primary pathway for auxin biosynthesis in maize does not involve tryptophan.
Transposon Mutagenesis and Transposon Effects
Tagging experiments are underway for maize loci involved in many developmental pathways. Members of the laboratory of Steve Dellaporta (Yale University, New Haven, CT) presented a strategy to exploit the local hopping behavior of Ds elements by creating a chromosome translocation that moves a known Ds element near the target gene prior to screening for mutants. Mutable alleles can then be recovered at 10-4 in the local region. As there are relatively few mobile copies of Ds, cloning is relatively straightforward. A 10-4 frequency of mutation, but throughout the genome, can be achieved using Robertson’s Mutator lines (Walbot, 1991). This transposon family, comprising at least nine distinct element types, is present in many copies per genome. Cloning is becoming routine, however, as a variety of strategies have been developed to simplify the identification of which Mu element among many has caused the mutation of interest. One powerful
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method for isolating genes whose 10cation is known is to clone sequences flanking the population of Mu elements and then determine where each flanking sequence is on the RFLP map. Another method is to recover several independent mutants, prepare libraries, and look for Mu-containing phage that cross-hybridize from the independent libraries, i.e., to identify Mu elements inserted in the same gene. For example, Guri Johal and Steve Briggs (Pioneer Hi-Bred, Johnston, IA) cloned Hm7, a gene conditioning resistance to Helminthosporium carbonum, after they analyzed several independent insertion mutants. Hml is the first plant disease resistance gene to be cloned. C. Han and colleagues (Cold Spring Harbor Laboratory, NY, and the University of Missouri) reported the cloning of a mutable allele of the nuclear gene iojap, which is required for normal chloroplast maturation or maintenance. Alice Barkan (University of Oregon, Eugene) reported the isolation of Mu-tagged alleles of additional genes involved in plastid biogenesis. Three laboratories reported the identification of candidates for the elusive regulatory element of Robertson’s Mutator. Using a mutable allele of the A1 gene of the anthocyanin pathway (al-mumZ), Paul Chomet and colleagues in the Freeling laboratory identified the single segregating “master” Mu element both genetically and on DNA gel blots probed with a new Mu element probe supplied by Kris Hardeman and Vicki Chandler (University of Oregon) (Chomet et al., 1991). Min-Min Qin and Albert Ellingboe (University of Wisconsin) cloned an element with a similar restriction map from a related stock. Independently, Janie Hershberger and colleagues in the Walbot laboratory (Stanford University, Stanford, CA) cloned and sequenced a nove1 5.0-kb Mu element that had transposed into 822, which encodes the enzyme that catalyzes the last step of anthocyanin biosynthesis (Hershberger et al.,
1991). This new Mu9 element shares the same restriction map as the two genetically defined master elements. Mu9 encodes two very abundant transcripts in active Mutator lines; these transcripts are absent in standard inbred lines and in lines that ,have epigenetically lost Mutator activity. Provided that the candidate “master” Mu elements move in heterologous hosts, very efficient transposon tagging may soon be available in other plant species. Drew Schwartz (Indiana University, Bloomington) reported a provocative effect between Ac and the regulation of a supposedly independent gene, C7. C7 encodes a myb-like DNA-binding protein that regulates transcription of the anthocyanin pathway structural genes A7, A2, Bz7, 6z2, and C2 (reviewed in Goff et al., 1990). Schwartz observed that expression of the standard C7 allele, whose promoter region contains multiple copies of the hexamei binding site of the Ac-encoded protein, is strongly repressed by Ac. In contrast, expression of the C1-l allele is independent of Ac. The major difference between the two promoters is that the C7-l upstream region has an insertion of more than 3 kb between the transcription start site and the hexamer binding sites. Based on this difference, Schwartz hypothesized that the Cl allele is suppressed by binding of the Ac product near the transcription initiation site, whereas the binding sites in the C1-/ allele are too distant to affect promoter function. This report suggests that the DNA-binding proteins encoded by transposons may have unexpected effects on host genes.
Features of Maize That Aid Genetic Analysis
Maize genetics currently provides the most powerful tools for conducting genetics in plants. First, mutants are readily made and characterized because both selfing and crossing are
easy. Already, thousands of variants (new segregating phenotypes) and hundreds of mutants (loci mapped by a three-point cross) of maize have been identified. Second, maize is unrivaled for the study of many genetic phenomena, including chromosome mechanics, transposable elements, gene imprinting, and meiosis. The study of these phenomena depends on the large store of preexisting stocks and mutants as well as the visibility of maize chromosomes for physical analysis. Third, many genes have multiple alleles, which are a tremendous asset in unraveling the function of particular maize genes; point mutations, deletions, transposon-tagged alleles, alleles with altered biochemical properties, and alleles with altered regulatory properties often exist. Fourth, a combined genetic and biochemical approach is available to study gene regulation. For a number of pathways, regulatory mutants define transacting loci and a hierarchy of gene functions that are part of the signal transduction chain between environmental and developmental cues and changes in gene expression. For example, the DNA-binding protein encoded by Opaque-2 regulates one class of zeins (Schmidt et al., 1990); also, R (or B ) and C7 (or P1) encode proteins (likely transcription factors) required together for activation of target promoters in the structural genes of the anthocyanin biosynthetic pathway (Goff et al., 1990). Fifth, the translocation mapping stocks of maize, in which scoring is reduced to colored versus not colored (purple iodine staining of Waxy versus wx or anthocyanin versus colorless), facilitate placement of new mutants on chromosome arms and define the recombinational distance between a new gene and the translocation break point. Despite the long life cycle, it is both quicker and easier to map a gene in maize than in other plants. Finally, new methods of chromosome manipulation allow transmission of chromosomes otherwise
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lethal in the haploid gametophyte, opening the path to recovery of gametophyte-specific genes in maize (Birchler and Levin, 1991). With the advent of cloning, reverse genetics-that is, identification of genes as nucleotide sequences, followed by mutation of the presumptive gene and determination of its phenotype by reintroducing the mutant allele into the host-has become possible. Reverse genetics is very successful in haploid organisms in which gene replacement is the predominant fate of introduced genes. Geneticists working with higher plants would be quick to adopt these timesaving methods if they were feasible. In higher eukaryotes, however, reverse genetics is hampered by many features, including diploidy, the presence of functionally redundant gene families, the predominance of gene integration at nonhomologous sites, and the difficulty of achieving high frequency transformation in most species. Because of these limitations, it is likely that traditional genetics, in which mutant phenotypes initially define gene function, will remain for the foreseeable future the primary means of understanding the function of genes in higher plants.
Chemical Mutagenesis of Pollen
Because the maize genome is enormous, the integrated mapping project in maize is focused on genes, not the entire genome. For the project to succeed, thousands of mutants must be generated and mapped. Both transposon tagging and chemical mutagenesis of pollen are effective strategies for producing new maize mutants. For generating large numbers of new mutants, chemical mutagenesis of pollen is the preferred method. Because individual gametes are the target, all recovered mutants are independent and M1 progeny plants are simple heterozygotes; i.e., they are not chimeras. This method is easy to apply
Table 1.
Recovery of Mutants after EMS Mutagenesisa Mutantsb Number Screened
Verified
Located and Named
M1 individuals screened for dominant mutants
23,672
120
54
M2 families screened for recessive mutants
7,450
2,524
706
a
Types
Chromosomal Arms
124
19
Data supplied by M. G. Neuffer.
Verification refers to transmission to the next generation and Mendelian behavior. Mapping to a chromosomal arm and, as appropriate, allelism testing are the next steps. Loci assigned to a map position are named.
indicate that EMS-induced mutations to recessive loss of function are much more likely than gain of function alterations. Interestingly, many regulatory loci are defined by both recessive and dominant mutations. There is one puzzling exception concerning the frequency of dominant mutants: Dominant Oil yellow (Oy) mutants occur in 1/1000 M1 plants, the same frequency as new recessives at other loci. Neuffer showed that in Oy mutants, chlorophyll b fails to accumulate in the leaves, and embryos die during extended seed storage. The precise function of Oy is unknown in com, but similar genes have been reported in soybean, pea, and Arabidopsis. Classes of Dominant Mutations after Chemical Treatment The largest class of dominant mutants in maize are those with diseaseGerry Neuffer (University of Missouri) like symptoms (Les mutants). Each reported that since the early 198Os, Les mutant develops chlorotic and/or he has recovered and mapped a total necrotic leaf lesions that mimic speof 760 new dominant and recessive cific diseases of maize. Symptoms in maize mutants, as shown in Table 1. each Les mutant follow a specific Based on the number of EMS-treated developmental pattern with lesions gametes, the recovery of new domi- appearing in a relatively fixed pattern nant mutants is about 1/200,000 per within the tissue. In addition, lesion Iocus, whereas recovery of new re- development is strongly influenced by cessive mutants is much more fre- sunlight, wounding, temperature, and quent, 1/900 per locus. These results the genetic background of the plant.
in maize because millions of pollen grains can be collected in minutes; after mutagenesis it is trivial to pollinate receptive ears. With an expected yield of greater than 250 kernels per ear, more than 10,000 M1 progeny can be generated in a single day. New dominant mutants are visible as individual plants in the M1 population; after selfing each M1 plant, segregation for recessives is observed in the M2 families. Mutagen dosage is regulated to insure that a large fraction of the M2 families display a naked eye polymorphism (NEP).
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Thus far, 23 different phenotypic classes have been discovered, and 14 loci have been defined by mapping. Extrapolating from the lack of confirmed allelic pairs, it is likely that more than 200 Les loci exist in maize. The primary hypothesis to explain Les is that the plant begins to overproduce a defense compound in response to developmental or environmental rather than pathogen signals (Walbot et al., 1983). Dominant Les mutants are not restricted to maize; in a survey of mutagenized Arabidopsis, John Mulligan and Ron Davis (Stanford University) recovered four dominant Les mutations from 20,000 M1 individuals screened. Interestingly, transgenic tobacco plants that contain additional copies of the phenylalanine ammonialyase gene also develop lesions (Elkind et al., 1990).
Recessive Mutants from EMS Mutagenesis
Among the recessive mutants, the most common types include failure to green (albino, yellow), male sterile (sporophytic failure), defective kernel (dek), and semi-sterile (50% pollen function, gametophytic failure). Members of a type are tested for allelism by reciprocal crosses, and the unique mutants are located and named. The 706 recently located recessive mutants are found on 19 of the 20 arms of the maize chromosome set. There are, for example, now 207 dek mutants, and Neuffer has mapped 139 of these to 18 arms. Bill Sheridan (University of North Dakota, Grand Forks) reported that five of the 150 dek mutants tested in embryo culture experiments require proline for growth, and 22 of the mutants arrest at specific developmental stages. Janice Clark (Montana State University, Bozeman) and Bill Sheridan described another set of recessive mutants, the emb mutants, which result in defective embryos (see Clark and Sheridan [1991], in this issue).
How Many Different Loci Are There?
The official maize map contains 670 defined loci: 440 NEPs and 230 isozyme loci. There are about 100 sequenced genes; many of these have not yet been placed on the gene map. Approximately 1000 NEP genes have been located to a chromosome arm utilizing translocation stocks but are not yet precisely mapped, and another 6000 NEP mutants await initial mapping. If all of these represent different genes, a total of approximately 7500 maize loci will have been identified when all of the known mutants are mapped. It is likely, however, that there are many more genes than this. The major limitation to the mutation approach for counting loci is redundant function within gene families. In a few cases, duplicate loci have been identified. For example, two chalcone synthases were defined by mutation (c2 and whp) because the pollen is white in the double mutant. The orange pericarp trait is also a double recessive. Many proteins are likely encoded by more than two loci, however, including such obvious examples as tubulins, the small subunit of ribulose-1,5-bisphosphatecarboxylase/ oxygenase, and histones; multi-copy status makes it difficult to find a mutant that identifies the genes encoding these proteins. Furthermore, even within the well-studied anthocyanin biosynthetic pathway, genes are missing: Mutants have not been identified for either chalcone isomerase or the 3-hydroxylation step. Tom Peterson and colleagues (Cold Spring Harbor Laboratory) demonstrated that maize contains three or more genes that crosshybridize to heterologous chalcone isomerase probes; a multiplicity of genes would explain the failure to recover recessive mutants. Ed Coe (USDNARS, University of Missouri) reminded the Maize Genetics Meeting participants that the RFLP and physical maps of maize are actu-
ally more detailed than the current gene map. The various RFLP maps contain more than 1300 markers, more than one for each of the approximately 1200 centimorgans (cM) of the maize genetic map. Probe identification for RFLP mapping in maize is very efficient because most probes define polymorphisms in many pairwise combinations of lines (Shattuck-Eidens et al., 1990). RFLP placement is aided both by using monosomics (10 stocks each with 19 rather than 20 chromosomes; Weber and Helentjaris, 1989) to place markers on chromosomes and by recombinant inbred lines that provide permanent mapping materials (Burr and Burr, 1991). Both RFLPs and traditional gene markers are, of course, located by recombinational distance. The physical placement of genes requires a physical location on an arm. For this purpose, maize excels. There are 84 B-A translocation stocks in which a segment of a standard “ A chromosome has been translocated to a supernumerary “8” chromosome. The break points in each affected arm define the physical location, proximal or dista1 to the break point, of all the genes on the arm. More refined physical maps are being developed by mapping the 880 reciprocal A-A chromosome translocation break points against the RFLP map. These translocations define 1760 points on the 20 chromosome arms of maize, for an average density of more than one per cM .
The Goals of the lntegrated Mapping Project
During the 1990, maize geneticists are committed to developing a high resolution map in which the recombination and physical data are merged. The mapping project is being coordinated by Coe, David Weber (Illinois State University, Normal), and Tim Helentjaris (University of Arizona, Tucson), with
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active participation by many maize geneticists. Maize geneticists expect to identify most genes and their cDNAs through the merged map. Approximately 6500 more NEP mutants must be mapped. In addition, 15,000 to 20,000 cDNA clones, including cross-hybridizingcDNAs from other species, will be partially sequenced and mapped as RFLP markers. Because many cDNAs will represent small gene families, they are expected to yield multiple RFLP locations. Virtually all cDNAs are expected to yield useful RFLPs because of the high polymorphism of maize. Hence, the total number of cDNA-based RFLPs should reach 50,000. The NEP and RFLP maps will be assigned to approximate physical locations along the chromosome arms using the numerous translocation stocks to define loci proximal and dista1 to specific break points. The final goal is to divide the maize genome into intervals of approximately 1 cM, defined by physical markers on the chromosomes, that also contain well-mapped NEP and RFLP markers. An important refinement for matching mutants to cDNAs is the inclusion of RNA dot blots from a variety of tissue sources on each DNA gel blot used for RFLP mapping. The computer entry for each cDNA RFLP will contain not only a marker location(s) on the map but a survey of the expression pattern and abundance of the mRNA as well. The emphasis on organ and tissue localization fits with the renewed interest in developmental mutants of maize, particularly in loci responsible for differentiation of the ear, tassel, and leaves, and in mutants that shift developmental timing (heterochronic mutants). In the future, an enterprising geneticist interested in a particular property of maize could pick a mapped mutant that defines the trait and have available at least 1 0 to 20 partially sequenced cDNAs that map within the same interval as the gene of interest.
Burr, B., and Burr, F.A. (1991). Recombinant inbreds for molecular mapping in maize. Trends Genet. 7, 55-60. Chomet, P.S., Lisch, D., Hardeman, K.J., Chandler, V.L., and Freeling, M. (1991). ldentification of a regulatory transposon that controls the Mufafor transposable element in maize. Genetics, in press. Clark, J.K., and Sheridan, W.F. (1991). lsolation and characterization of 51 embryo-specific mutations of maize. Plant Cell 3, 935-951. Elkind, Y., Edwards, R., Mavandad, M., Hedrick, S.A., Ribak, O., Dixon, R.A., and Lamb, C.J. (1990). Abnormal plant development and down-regulation oí phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. Proc. Natl. Acad. Sci. USA 87, 9057-9061. Freeling, M., and Walbot, V. (eds) (1992). The Maize Handbook. (New York: Springer-Verlag), in press. Goff, S.A., Klein, T.M., Roth, B.A., Fromm, M.E., Cone, K.C., Radicella, J.P., and Chandler, V.L. (1990). Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. EMBO J. 9, 2517-2522. Hershberger, R.J., Warren, C., and Walbot, V. (1991.) Mufafor activity in maize correlates with the presence and expression of Mu9, a new Mu transposable element. Proc. Natl. Acad. Sci. USA, in press. Virginia Walbot Department of Biological Sciences Neuffer, M.G., Jones, L., and Zubar, M.S. Stanford University (eds) (1968). The Mutants of Maize. (Madison, WI: Crop Science Society of Stanford, CA 94305-5020 America). Schmidt, R.J., Burr, F.A., Aukerman, M.J., and Burr, B. (1990). Maize regulatory gene opaque-2 encodes a protein REFERENCES with a "leucine-zipper" motif that binds to zein DNA. Proc. Natl. Acad. Sci. USA 87, 46-50. Berlyn, M.B., Last, R.L. and Fink, G.R. Shattuck-Eidens, D.M., Bell, R.N., Neu(1989). A gene encoding the tryptophan hausen S.L., and Helentjaris,T. (1990). synthase p subunit of Arabidopsis DNA sequence variation within maize fhaliana. Proc. Natl. Acad. Sci. USA 86, and melon: Observations from poly4604-4608. merase chain reaction amplification and Birchler, J.A., and Levin, D.M. (1991). direct sequencing. Genetics 126, 207-217. Directed synthesis of a segmenta1 chromosomal transposition: An approach to Sprague, G.F., and Dudley, J.W. (eds) (1988). Corn and Corn Improvement, 3rd the study of chromosomes lethal to the ed. (Madison, WI: American Society of gametophyte generation of maize. Agronomy). Genetics 127, 609-618.
Those cDNAs expressed in the appropriate tissues and developmental stages would be the most likely to encode or affect the trait of interest. Geneticists will also be able to match transposon insertion sites, many of which define mutable genes, with known cDNAs by generating inverse PCR probes flanking transposon insertion sites. Even in the absence of a specific mutant, the cDNAs expressed in particular patterns could well provide clues to the identification of new genes. In summary, the maize genetics community is rapidly generating new mutants using both EMS mutagenesis and transposon tagging. Transposon tagging allows access to genes that would be virtually impossible to obtain through other means because of the lack of biochemical characterization. Although mutants are interesting in their own right, the mapping of all new and existing mutants is the foundation for the integrated mapping project. Matching genes to proteins and to phenotypes will be greatly facilitated by the integrated map in which NEP, RFLP, and physical locations are merged into a guide to the maize genome.
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Vollbrecht, E., Veit, B., Sinha, N., and Hake, S. (1991). The developmental gene K ~ m d - 7is a member of a maize homeobox gene family. Nature 350,241-243. Walbot, V. (1991). The Mufator transposable element family of maize. In Genetic
Engineering, Vol. 13, J.K. Setlow, ed (New York: Plenum Press), pp. 1-37. Walbot, V., Hoisington, D.A, and Neuffer, M.G. (1983). Disease lesion mimic mutations. In Genetic Engineering of Plants, T. Kosuge, C.P. Meredith, and A .
Hollaender, eds (New York: Plenum Publishing Corp.), pp. 431-442. Weber, D., and Helentjaris, T. (1989). Mapping RFLP loci in maize using 6-A translocations. Genetics 121, 583-590.
