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Eukaryotic Cell, April 2003, p. 362-379, Vol. 2, No. 2
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.2.362-379.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Map of the Chlamydomonas reinhardtii Nuclear Genome

Pushpa Kathir, Matthew LaVoie, William J. Brazelton, Nancy A. Haas, Paul A. Lefebvre, and Carolyn D. Silflow*

Department of Genetics, Cell Biology, and Development and Department of Plant Biology, University of Minnesota, St. Paul, Minnesota

Received 12 August 2002/ Accepted 10 December 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Addendum in proof
 References
 
We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Addendum in proof
 References
 
Studies using the unicellular eukaryotic alga Chlamydomonas reinhardtii have yielded important insights into many cellular processes including photosynthesis (45, 123), flagellar assembly and motility (24, 28, 86, 112, 114, 124, 137, 140), basal body assembly and positioning (115), gametogenesis and fertilization (35, 42, 167), DNA repair (109), phototaxis (49, 139), cell wall assembly (1), circadian rhythms (91, 162), and the regulation of metabolic pathways (3, 14, 31, 50).

A major strength of C. reinhardtii as an experimental system is its usefulness for genetic experiments (45, 47, 74). Vegetative cells are haploid, facilitating the analysis of mutant phenotypes, but stable diploid strains can be easily produced for dominance and complementation tests. Gametes can be crossed to yield diploid zygotes that sporulate to produce four products of meiosis, allowing routine tetrad analysis. Over the past 50 years, hundreds of mutations have been isolated; more than 200 genetic loci have been mapped to 17 linkage groups (28, 29, 45, 46, 52). Mutations induced by chemical or UV mutagenesis have been supplemented recently by mutations induced by transposition of one of several transposable element families in the genome (17, 34, 130, 133) or by insertional mutagenesis (13, 101, 149).

Insertional mutagenesis has become the favored method for generating mutations since the development of procedures for efficient transformation of the nuclear genome (59, 132). Upon transformation, plasmid DNA inserts in random positions into the nuclear genome, facilitating cloning of affected genes by using the transforming plasmid as a hybridization probe. This method of gene tagging has led to the isolation of numerous genes identified by mutation over the past several years. Despite its usefulness, the insertional-mutagenesis approach has drawbacks, including the inability to clone essential genes, difficulty in analyzing the large deletions that occur in some cases, and a limitation in the types of phenotypes that can be found by using a method that generates mostly null mutations.

To increase the power of molecular genetic approaches using C. reinhardtii, we have developed a molecular map aligned with the genetic map. In this paper, we present a detailed map of the C. reinhardtii nuclear genome based on the analysis of restriction fragment length polymorphism (RFLP) and sequence-tagged site (STS) markers. The availability of such a physical map will facilitate the cloning of genes identified by any type of mutation in C. reinhardtii.

(A preliminary version of the molecular map of C. reinhardtii was published previously [133].)


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Addendum in proof
 References
 
C. reinhardtii strains, growth conditions, and genetic crosses. The C. reinhardtii standard laboratory wild-type strain 21gr mt+ (CC-1690) and the interfertile field isolate strain S1-C5 mt- (CC-1952) were used as parental strains. The 21gr strain and the other commonly used laboratory strain, 137c, are very closely related (64); almost all PCR amplifications of genomic DNA using primers predicted from the sequence of one of the strains amplify DNA from both strains. The S1-C5 strain is identical to the S1-D2 mt- strain (CC-2290) (44); the two strains were isolated from the same soil sample. The 21gr and S1-C5 strains were crossed as described previously (75). Tetrad progeny from the resulting zygotes were separated; a total of 136 random progeny from 136 complete tetrads were used in the mapping experiments. Cells were grown in TAP medium (43) or M medium (125) by using the modification described by Schnell and Lefebvre (130).

Molecular markers. Several types of molecular markers were mapped in this study. Markers designated GP were obtained by digesting C. reinhardtii genomic DNA (strain 137c) with the restriction enzyme PstI, size fractionating the DNA on an agarose gel, and preparing minilibraries of cloned fragments (0.5 to 6.0 kb) in plasmid vector pUC119 (119). Random cDNA clones constituting the CNA, CNB, and CNC series of markers were obtained from a C. reinhardtii cDNA library (143). Additional markers consisted of genomic DNA clones or cDNA clones provided by other laboratories and genomic DNA, cDNA, or expressed sequence tag (EST) sequences obtained from the GenBank database.

Scoring markers by RFLP detection. Genomic DNA was isolated by the method of Schnell and Lefebvre (130). DNA (1 µg per lane) was digested with restriction enzymes (PstI, PvuII, EcoRI plus XhoI, or HindIII). DNA fragments were separated by electrophoresis on a 1% agarose gel (12.7 by 20 by 0.5 cm) at 35 V for 18 to 20 h in TBE buffer (0.45 M Tris, 0.44 M boric acid, 0.01 M EDTA [pH 8.0]). The DNA was denatured and transferred to a MagnaGraph nylon membrane (Micron Separations Inc., Westborough, Mass.) by using the protocol of Sambrook et al. (126). DNA was cross-linked to the membrane by using a model 1800 UV Stratalinker (Stratagene, La Jolla, Calif.) at 1,200 µJ for 30 s. The membrane was baked at 80°C for 2 h in a vacuum oven. Membranes containing digested DNA from each of the 136 random progeny mapping strains were incubated with hybridization solution (50% formamide, 5x SSPE [1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA {pH 7.7}], 10x Denhardt's solution, 4% sodium dodecyl sulfate [SDS], and 300 µg of single-stranded salmon sperm DNA/ml) for 1 h at 42°C. Denatured, labeled probe (see below) was added, and the hybridization reaction mixture was incubated overnight at 42°C. Filters were washed with 2x SSPE-1% SDS for 20 min, followed by three washes in 0.2x SSPE-0.2% SDS for 20 min each at 68°C. Digoxigenin-labeled probes were detected according to the protocol from Roche Molecular Biochemicals (Indianapolis, Ind.) except that Tween 20 was increased to 0.3% in buffer A and 5% powdered milk (Carnation) was used in buffer B instead of Blocking Powder.

Preparation of hybridization probes. Hybridization probes were labeled using the digoxigenin nonradioactive system from Roche Molecular Biochemicals. Plasmids or purified plasmid inserts were labeled by random priming according to the manufacturer's instructions with the following modifications. Probe DNA in 13 µl of H2O (200 to 500 ng of plasmid DNA or 50 to 75 ng of purified insert DNA) was denatured by boiling, and 4 µl of 5x OLB (0.225 M Tris-HCl [pH 8.0], 0.025 M MgCl2, 0.02 M dithiothreitol, 1.36 A260 units of hexanucleotides [Pharmacia Biotech, Piscataway, N.J.]), 2 µl of DIG DNA labeling mixture (Roche), and 1 µl (2 U) of Klenow enzyme (Roche) were added. The reaction mixture was incubated overnight at 37°C, and the reaction was stopped with 2 µl of 0.2 M EDTA. The probe was mixed with 20 µl of 5% Blue Dextran and column purified by using Bio-Gel P-60 agarose (Bio-Rad Laboratories, Hercules, Calif.). PCR labeling of cloned DNA fragments utilized sets of plasmid-specific primers and the PCR DIG Probe Synthesis kit (Roche). The PCR mixture (50 µl) contained 5 ng of plasmid DNA, 200 mM digoxigenin deoxynucleoside triphosphates, 1x PCR Mg2+ buffer, 25 pmol of primers, 9% dimethyl sulfoxide, and 2.5 U of Taq polymerase. PCR program steps were as follows: (i) 94°C for 5 min, (ii) 29 cycles of 94°C for 1 min, 56°C for 45 s, and 72°C for 3 min, and (iii) 94°C for 1 min, 56°C for 45 s, and 72°C for 10 min. For amplification of cDNA clones in the {lambda}Exlox vector, phage DNA was prepared by resuspending a plaque in 200 µl of distilled water, freezing in liquid nitrogen, and thawing at room temperature. The freeze-thaw cycle was repeated, and the sample was boiled for 5 min. The crude DNA (25 µl) was used as template in a 100-µl PCR mixture containing PCR buffer with Mg2+ (Roche), 2 µl of DIG DNA labeling mixture, 20 pmol of primers, 2.5% dimethyl sulfoxide, and 2.5 U of Taq polymerase (Roche). PCR program steps were as follows: (i) 94°C for 4 min, 55°C for 2 min, and 72°C for 3 min, (ii) 35 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min, and (iii) 94°C for 1 min, 55°C for 2 min, and 72°C for 10 min.

Scoring markers by use of PCR. The PRIMER program (79) was used to design primers for amplification of fragments from 3' untranslated regions (3' UTR) of gene, cDNA, or EST sequences. The primer criteria included an optimal length of 22 nucleotides (range, 20 to 24), an optimal melting temperature (Tm) of 73°C (range, 71 to 75°C), and a GC content of 60 to 65%. The default settings of the program were used for other criteria. PCR mixtures (25 µl) contained buffer A (Fisher Biotech), 10% glycerol, 5% formamide, 200 µM each deoxynucleoside triphosphate, 12.5 pmol of each primer, 25 ng of genomic DNA, and 0.55 U of Taq DNA polymerase (Fisher Biotech). PCR program steps were as follows: (i) 94°C for 1 min, (ii) 30 cycles of 94°C for 1 min, 55°C (annealing temperature) for 1 min, and 72°C for 1 min, and (iii) 72°C for 10 min, followed by a 10°C hold. Amplified DNA fragments obtained from template DNA from the 21gr and the S1-C5 strain were sequenced by the Advanced Genetics Analysis Center, University of Minnesota. Nucleotide sequence polymorphisms between 21gr DNA and S1-C5 DNA provided the basis for manual design of allele-specific primers by using principles described by Dieffenbach et al. (25) and Kwok et al. (68). For some markers, ESTs obtained from strain S1-D2 (CC-2290) were available in the database; these sequences provided a source of nucleotide polymorphisms used in allele-specific primer design. The annealing temperature for PCRs using the allele-specific primers was optimized by using a gradient thermal cycler (DNA Engine Dyad; MJ Research, Waltham, Mass.) to amplify DNA from the 21gr and S1-C5 parent strains. DNA from progeny strains was amplified in 96-well format by using the optimal annealing temperature. Reaction products (7 µl) were fractionated on 1.5% agarose gels by using the Sunrise 96 apparatus (Life Technologies, Rockville, Md.) and visualized by ethidium bromide staining.

Linkage analysis. For all loci scored in this study, the data consisted of a scorable hybridization fragment or PCR product derived from each of the two parental strains. No plus-minus data sets were used in the analysis. Data were entered and annotated by using the Map Manager QTX program (version b12) (82) and were then exported to the mapping Mapmaker/QTL program (version 3.0) (69, 78) for linkage analysis and map construction. The F2 backcross function of the program was used for map construction in this haploid organism by classifying one genotype as "homozygous" and the other as "heterozygous." The "group" command, at a Lod score threshold of 4.0, was used to place the markers in linkage groups. All loci mapped to one of the 17 known linkage groups in the C. reinhardtii genome. Map order within linkage groups was determined by using the multipoint mapping functions of the "order" and "build" commands. Markers placed with a confidence of at least Lod 2.0 are presented on the map (Fig. 1). For markers that could not be placed on the map at a Lod score greater than or equal to 2.0, the "try" command was used to establish the most likely position of the marker on the map. The Kosambi function was used to assign map distances in centimorgans.








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  		FIG. 1. Chlamydomonas molecular and genetic maps. For each of the 17 linkage groups, the genetic map (adapted from the work of Harris [46]) is shown on the right, with the centromere represented by the black oval, and the molecular map is shown on the left. For genetic maps, spaces between markers represent recombination units (percent recombination); the scale bar is to the right of the maps for linkage group XIX. For molecular maps, numbers to the left of the vertical line indicate centimorgans (Kosambi units). The order of molecular markers placed on the map is predicted to be accurate with a Lod score of at least 2.0 by use of MAPMAKER/QTL 3.0 (78). For markers that could not be ordered with a Lod score of at least 2.0, the "try" command was used to determine the most likely map position. Such markers are enclosed in parentheses. For markers separated by commas on the same line, the order was indistinguishable by recombination. Dashed lines connecting the genetic and molecular maps indicate a molecular marker corresponding directly to a previously mapped phenotypic marker. The orientation of the molecular map with respect to the genetic map has not been confirmed for linkage groups VIII, XV, and XVI/XVII. Further information on the markers is available at http://www.biology.duke.edu/chlamydb/.

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Addendum in proof
 References
 
Using a combination of RFLP and PCR-based markers, we have placed 264 molecular markers on the 17 linkage groups of the C. reinhardtii genome (Fig. 1; Table 1). These markers were mapped on a panel of 136 random progeny from a cross of strain 21gr (mt+) with the field isolate S1-C5 (mt-) (44). All of the markers map to the 17 known linkage groups, indicating that if other linkage groups exist in the C. reinhardtii genome, they must be very small. The total length of the molecular map (in Kosambi units) is 1,025 centimorgans (cM). Any point on the C. reinhardtii genome is, on average, 2 cM from one of the 264 mapped molecular markers. Given that the size of the genome is approximately 108 bp (46), 1 cM in C. reinhardtii should correspond to about 100,000 bp. This number is consistent with the centimorgan-to-base pair ratio found during the positional cloning of the LF1 gene (R. Nguyen and P. A. Lefebvre, unpublished data).


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  		TABLE 1. Description of markers

 
Frequency of polymorphism. Previous efforts to develop an extensive molecular map for C. reinhardtii were hampered by the low frequency of DNA polymorphism observed for molecular markers by using C. reinhardtii and the interfertile strain Chlamydomonas smithii (120, 135). In this study, the laboratory strain 21gr and an interfertile field isolate strain (S1-C5) showed a high degree of polymorphism for many molecular markers. When DNA from the two strains was digested with PstI or PvuII, 94% of hybridization probes tested (n = 204) showed an RFLP with one or both of the enzymes. With three additional restriction enzymes, EcoRI plus XhoI and HindIII, the RFLP rate increased to 98%. Thus, it was possible to map almost all markers by using a set of standard filters prepared with genomic DNA digested with only a few different restriction enzymes.

The underlying variation in DNA sequence responsible for the high level of RFLP was confirmed by direct sequencing of a large number of 3' UTR sequences from S1-C5 DNA. We chose to examine 3' UTR sequences because they exhibit greater sequence variation than do coding sequences. In addition, 3' UTR sequences are likely to be unique, even among genes in a multigene family. To obtain 3' UTR sequences from S1-C5 genes, primers were designed to amplify 3' UTR fragments from C. reinhardtii genes available in the GenBank database. Of 100 reactions that produced products by using 21gr DNA as a template, 82 also produced products by using S1-C5 DNA. Among these products, 16% showed a length polymorphism with the product obtained from the 21gr template DNA. We sequenced the amplified 3' UTR regions from 62 S1-C5 genes, for a total of 29,053 bp. When these sequences were compared with the equivalent regions from strain 21gr or 137c (available in GenBank), single nucleotide substitutions were found at 793 positions (447 transitions and 346 transversions), for an average of 2.7 base substitutions per 100 bp of sequence. In addition, at 159 sites we found insertions or deletions of bases at a frequency of 0.54 per 100 bp.

The high level of sequence polymorphism in the S1-C5 gene sequences made it possible to design allele-specific primers based on single nucleotide polymorphisms (SNPs). We designed primers that yield PCR products of different lengths when template DNAs from the 21gr and S1-C5 parental strains are used (Table 2). These primer sets reproducibly generated reaction products when uniform reaction components and optimized annealing temperatures were used. The primer sets, corresponding to loci distributed over each of the linkage groups, were used to amplify PCR products ranging from 100 to 600 bp by using template DNA from the random progeny strains. The lengths of the resulting products were analyzed and scored by using agarose gel electrophoresis.


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  		TABLE 2. Mapping primersa

 
Anchoring the molecular map to the genetic map. For most linkage groups, the molecular map was anchored to the genetic map by using as mapping probes genes corresponding to mapped phenotypic markers. On linkage group I, for example, molecular probes for the LF3 gene (149) and for the ARG7 gene (19) were mapped, allowing the molecular map to be oriented relative to the genetically mapped lf3 and arg7 loci. Cloned genes corresponding to mapped mutations were not available for some linkage groups. For these six linkage groups, the molecular map was oriented relative to the preexisting genetic map by reference to earlier molecular mapping results that provided information about centromere linkage. The orientation of linkage group II, for example, is based on the observation that the centromere distances for markers S6175 and S6135 are 3 and 2 cM, respectively. For linkage group IV, the orientation of the map was supported by data from tetrads indicating that TUA2 ({alpha}2 tubulin) and PYR1 (the pyrithiamine resistance gene) are on opposite sides of the centromere and that TUA2 maps within 7 cM of its centromere (120). For linkage group V, the orientation of the map was determined by examining the data from tetrad progeny showing that DHC6 lies between the PF26 marker and the centromere (113). The correspondence of the molecular and genetic maps for linkage group XVI/XVII is based on the demonstration that DHC9 is linked to the phenotypic marker y1 (113). The orientation of the molecular map for linkage group XIX is supported by data showing that the EF3A marker maps within 3 cM of the centromere (120). For linkage groups VIII, XV, and XVI/XVII, the orientation of the anchored map relative to the genetic map is not known because there is only a single point of anchorage.

For most loci, cloned genes corresponding to previously mapped phenotypic loci were placed on the expected locations on the molecular map. The single exception was the ac21 locus on linkage group XI. The PETC gene (encoding the chloroplast Rieske iron-sulfur center protein) has been shown to be the gene affected by the ac21 mutation (5, 22, 23). When we mapped PETC, however, it was indistinguishable from VFL2 by recombination. The map location of ac21 was previously determined to be on the opposite arm of linkage group XI.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Addendum in proof
 References
 
The availability of the C. reinhardtii molecular map should enable researchers to take advantage of rapid advances in C. reinhardtii genomics to identify genes corresponding to mapped mutations. Sequences from more than 100,000 cDNA clones are publicly available (Chlamydomonas Genetics Center, Duke University [http://www.biology.duke.edu/chlamy_genome/]; Kazusa DNA Research Institute, Kazusa, Japan [http://www.kazusa.or.jp/en/plant/chlamy/EST/]). The ends of 15,000 bacterial artificial chromosome (BAC) clones have been sequenced by the Joint Genome Institute (JGI; Walnut Creek, Calif.) and are available for searching by use of BLAST algorithms (http://bahama.jgi-psf.org/prod/bin/chlamy/home.chlamy.cgi). The complete sequence of the nuclear genome is being completed by the JGI and should be available early in 2003.

All of these resources taken together should allow positional cloning and candidate gene approaches to be used to clone the genes identified by mapped mutations. BAC clone contigs anchored on each of the molecular markers have already been prepared. The total length of genomic DNA contained in these contigs represents more than 20% of the nuclear genome. By comparing the sequence of the nuclear genome to the sequences of the BAC clone ends, it will be possible to rapidly complete the coverage of the nuclear genome with mapped BAC clones.

For positional cloning, C. reinhardtii offers many technical advantages for efficient testing of whether a particular BAC clone corresponds to a mutation of interest. Transformation of C. reinhardtii involves simple and fast procedures such as vortexing with glass beads (59) or electroporation (132). The efficiency of cotransformation with BAC clones and a selectable marker gene on a separate plasmid is usually in the range of 1 to 2%, and it is easy to generate hundreds of transformants for screening. A BAC clone can be tested for rescue of a mutant phenotype in less than 2 weeks. Because screening of BAC clones for phenotypic rescue is so rapid and efficient, it should not be necessary to do extensive genetic mapping of new mutations in preparation for cloning. Numerous BAC clones, covering a large genetic interval, can be readily tested for phenotypic rescue of a mutation of interest upon transformation. As the sequence of the nuclear genome is completed and annotated, it should be possible to accelerate positional cloning and transformation using a candidate gene approach.

To use positional cloning or candidate gene approaches to clone genes corresponding to mutations, it is necessary to place these mutations on the genetic map. Hundreds of mutations have been mapped using phenotypic markers and multiply marked strains, but genetic mapping using phenotypic markers is tedious and is limited by the availability of useful mutations. A serious limitation is that many potentially useful genetic markers produce a similar phenotype, such as acetate auxotrophy or paralyzed flagella, so that only one mutant of each type can be used in an individual mapping cross. The molecular markers described in this report should greatly facilitate genetic mapping of new mutations in preparation for positional cloning.

To map the mutation in a new mutant strain, it should first be crossed to the S1-C5 strain, and 20 to 50 random progeny from different tetrads should be scored for the phenotype of interest. Small quantities of DNA from each progeny strain can then be used as template DNA for PCR-based mapping strategies. The primers for SNP scoring reported in this study (Table 2), because they produce PCR products of distinguishable sizes from the two parent strains, facilitate scoring of molecular markers in the progeny strains by using readily available thermal cycler and gel electrophoresis equipment. The marker set covers most of the genome, with multiple loci representing both arms of most linkage groups. Previously, primers for the scoring of SNPs using high-throughput methods were defined for 186 loci, including many of those mapped in this study (159).

A hierarchical approach to marker selection for mapping can be used to limit the number of PCRs to be performed. For each linkage group, the first marker to be tested for linkage analysis should be one that maps near the center of the group of markers. More than 50% of the molecular markers we developed map to only five linkage groups (I, II, III, VI, and XII/XIII). That the density of molecular markers corresponds roughly to the underlying gene density is suggested by the fact that slightly more than 50% of previously mapped mutations map to these five linkage groups as well. The first markers to be tested, therefore, should be from these linkage groups. If no linkage is detected between the mutant phenotype and the central marker on each linkage group, additional tests with markers spaced 20 to 30 cM from the first marker may be carried out until linkage is detected.

As soon as a new mutation is mapped to an arm of one of the linkage groups, other molecular markers mapping to that arm can be used to attempt to place the mutation between pairs of flanking markers. Consulting the overlapping BAC contig map will then allow the choice of BAC clones to be tested for phenotypic rescue using cotransformation with a selectablemarker gene (http://www.biology.duke.edu/chlamy_genome/BAC/index.html). A number of selectable markers are available for transformation of C. reinhardtii (47). If a project requires fine structure mapping of a genetic region, and multiple STS markers are not available, it is possible to generate additional STS loci. The sequences of all the BAC ends are available, and reference to the BAC contig map places these sequences at intervals of tens of thousands of bases along the genome. A BAC end sequence can be turned into an STS locus by sequencing the same region of genomic DNA from the S1-C5 strain and using sequence polymorphisms to design informative primers for PCR. The high degree of sequence polymorphism between the laboratory strains of C. reinhardtii and S1-C5 makes the task of finding useful sequence polymorphisms routine. One source of sequence polymorphisms is the set of ESTs derived from CC-2290 (strain S1-D2), available in the National Center for Biotechnology Information (NCBI) database. Many of these EST sequences were used to generate the markers reported in this study. Another possible source for additional molecular markers for mapping is microsatellite repeat sequences, which have been shown to be abundant in the C. reinhardtii genome (56).


   ADDENDUM IN PROOF
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Addendum in proof
References
 
The sequence of the Chlamydomonas genome obtained by the DOE Joint Genome Institute is posted on the Internet at http://genome.jgi-psf.org/chlre1/chlre1.home.html.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the NSF (MCB 9975765 and MCB 8819133), the NIH (GM 34437), and the University of Minnesota Experiment Station. W. J. Brazelton was supported by a Mabel and Arnold Beckman Foundation Award. C. reinhardtii strains were obtained from the Chlamydomonas Genetics Center at Duke University (supported by NSF grant DBI 9319941).

We thank Laura Ranum and Marilyn Kobayashi for contributions to the early stages of this work. We also thank our many colleagues in the Chlamydomonas community for providing molecular markers for mapping.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Plant Biology, 250 Bioscience Center, University of Minnesota, 1445 Gortner Ave., St. Paul, MN 55108. Phone: (612) 624-0729. Fax: (612) 625-1738. E-mail: carolyn{at}biosci.cbs.umn.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Addendum in proof
 References
 

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