Molecular Map of the Chlamydomonas reinhardtii Nuclear Genome

Abstract

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.

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

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, 5× SSPE [1× SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA {pH 7.7}], 10× 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 2× SSPE-1% SDS for 20 min, followed by three washes in 0.2× 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 5× 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, 1× 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 λExlox 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 (T_m) 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.

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

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).

TABLE 1.

Description of markers

Linkage group	Locus	Gene or marker description	Source or reference(s)	Accession no.
I	CYP1	Cyclophilin 1	145	AF052206
I	OPSIN1	Chlamyopsin 1	21	Z48968
I	TCTEX1	14-kDa dynein light chain	48	AF039437
I	LF3	LF3/ULF1 gene	L.-W. Tam and P. Lefebvre, unpublished data
I	GP387	Random genomic fragment	This study	AY219891
I	CNA13	cDNA fragment	This study
I	GP123	Random genomic fragment	This study	AF525919
I	GP396B	Random genomic fragment	This study
I	PC1	NADPH: protochlorophyllide oxidoreductase	76	U36752
I	ARG7	Argininosuccinate lyase	19	X16619
I	PBT302	Genomic fragment	B. Tailon and J. Jarvik, unpublished data
I	CNC41	cDNA corresponding to EST 1031062D08	This study	BI722495
I	CNA79	cDNA corresponding to EST 1031013A09	This study	BI994521
I	GBP1	G-strand binding protein	110	U10442
I	RB47	Poly(A) binding protein	173	AF043297
I	CNA73	cDNA corresponding to EST 894090B12	This study	BE726259
I	GAS96	Gene expressed during cell differentiation	C. F. Beck, unpublished data
I	COX2B	Cytochrome c oxidase subunit II	104	AF305540
I	LC4	LC4, 17-kDa dynein light chain	60	U34345
II	PKA	Protein kinase A	N. Wilson and P. Lefebvre, unpublished data	AV390434
II	KLP1	Kinesin-like protein	6	X78589
II	GP15	Random genomic fragment	This study
II	GP36	Random genomic fragment	This study
II	GP130	Random genomic fragment	This study
II	CNC17	cDNA corresponding to EST 1031071E11	This study	B1724441
II	S641	pcf 6-41 cDNA, upregulated after deflagellation	129
II	CPX1	Coproporphyrinogen III oxidase precursor	117	AF133671
II	ALAD	Porphobilinogen synthase	84	U19876
II	DIC8	Genomic fragment	B. Williams and J. Rosenbaum, unpublished data
II	RB60	Protein disulfide isomerase	153	AF036939
II	GP226	Random genomic fragment	This study
II	S6175	pcf 6-175 cDNA, upregulated after deflagellation	129
II	LC1	LC1, 22-kDa dynein light chain	4	AF112476
II	CIA5	Regulator of carbon concentrating mechanism	170	AF317732
II	S6135	pcf 6-135 cDNA, upregulated after deflagellation	129
II	CNB8	cDNA corresponding to EST 1024021G10	This study	BG848462
II	GP366	Random genomic fragment	This study	AY220530
II	GP225	Random genomic fragment	This study	AY220531
II	DHC4	Dynein heavy chain 4	113	U81367
II	GSP1	Gamete specific protein 1	66	AF108140
II	CNA72	cDNA fragment	This study
II	CNA45	cDNA corresponding to EST BI724982	This study	BI724982
II	ZYMC16	Gene expressed in zygotes	161
II	RBCS2	Ribulose biphosphate carboxylase small subunit 2	41	X04472
II	DHC2	Dynein heavy chain 2	113	U61365
II	GAS18	Gene expressed during sexual differentiation	157
II	GP383	Random genomic fragment	This study
II	S321	pcf 3-21 cDNA, upregulated after deflagellation	129
II	YPTC4	Small G protein	26	U13167
II	CNA43	cDNA fragment	This study
III	CNC24	cDNA fragment	This study	AF525918
III	DHC α	Dynein heavy chain alpha	87, 88	L26049
III	EST 925021G06	EST	132a	BE442506
III	LRG5	Gene involved in blue light signaling	40	U73818
III	COX2A	Cytochrome c oxidase subunit II	104	AF305080
III	GSAT	Glutamate-1-semialdehyde aminotransferase	83	UC3632
III	SAC1	Sulfur limitation gene	15	U47541
III	PF15	Component of the central pair microtubule apparatus	E. F. Smith and P. Lefebvre, unpublished data
III	MAA7	Tryptophan synthase beta-subunit	97	AF047024
III	CNB4	cDNA corresponding to EST 833013E07	This study	AW721386
III	GP108	Random genomic fragment	This study
III	GP219	Random genomic fragment	This study
III	EF12E	Genomic fragment	E. Fernandez, unpublished data
III	GAR1	Gamete activation-regulated cobalamin-independent methionine synthase	67	U36197
III	NAP	Novel actin-like protein	57, 72	U68060
III	NIT2	NIT2 gene	130
III	RIB43A	Microtubule ribbon protein	96	AF196576
III	FLA14	LC8, 10-kDa dynein light chain	61, 102	U19490
III	AC208	Apoplastocyanin	116	L07282
III	CPC1	Central pair-associated complex 1 protein	90
III	CNC21	Random cDNA fragment	This study
III	PTX2	Phototaxis-deficient gene	101
III	YPTC6	YPTC6, small G protein	26	U13169
III	LC6	LC6, 13-kDa dynein light chain	61	U19484
III	UNI3	δ-Tubulin	30	AF013108
III	TUA1	α-Tubulin	134	M11447
III	GP201	Random genomic fragment	This study
III	GP317	Random genomic fragment	This study
III	GP32	Random genomic fragment	This study
III	CNA34	cDNA corresponding to EST 1031065B08	This study	BI722955
III	GP437	Random genomic fragment	This study
III	RSB1	Radial spokehead polypeptide 1; corresponds to EST 1031064E02	165; this study	B1722838
III	PHOT1	Phototropin-like protein	52a	AJ416557
IV	TUA2	α2-Tubulin	134	M11448
IV	GP54	Random genomic fragment	This study
IV	BLD1	Intraflagellar transport protein 52	9, 18	AF397450
IV	COX3	Cytochrome c oxidase subunit III	105	AF233515
IV	PF20	Protein required for flagellar central pair microtubule assembly	144	U78547
V	GP7	Random genomic fragment	This study	AF467707
V	GP228	Genomic fragment; corresponds to EST 1024061H08	This study	BG859190
V	MUT6	DEAH Box RNA helicase involved in gene silencing	168	AF305070
V	ALD	Plastid fructose-1,6-bisphosphate aldolase	103	X85495
V	DHC6	Dynein heavy chain 6	113	U61369
V	ODA16	Genomic fragment	D. Mitchell, unpublished data
V	CNC19	cDNA corresponding to EST AV621283	This study	AV621283
V	PF26 (S6187)	pcf 6-187 cDNA, upregulated after deflagellation; radial spoke protein 6	12, 120, 129	M87526
VI	HSP70B	Chloroplast-localized heat shock protein	27	X96502
VI	TPX	Thioredoxin peroxidase	Y. Lee, S. H. Miller, and L. Keller, unpublished data	AF312025
VI	FA1	Flagellar autotomy protein	36, 37	AF246990
VI	ATPC	Chloroplast ATP synthase gamma subunit	142	M73493
VI	VFL6	VFL6 gene	K. Iyadurai and C. Silflow unpublished data
VI	DHC3	Dynein heavy chain 3	113	U61366
VI	CNA80	cDNA corresponding to EST 1031002F08	This study	B1816487
VI	CNA46	cDNA fragment	This study
VI	S813	G protein beta subunit-like protein	127	X53574
VI	VFL3	VFL3 gene	Iyadurai and Silflow, unpublished
VI	CNC30	cDNA corresponding to cab II-1	54; this study	M24072
VI	EST 925003F02	EST	132a	BE441254
VI	ICL	Isocitrate lyase	111	U18765
VI	CABI-1	Light harvesting complex protein I-20	53	X65119
VI	PBT201	Genomic fragment	B. Tailon and J. Jarvik, unpublished data
VI	PF14	Radial spoke polypeptide 3	166	X14549
VI	CRY1	DNA photolyase/blue light photoreceptor	141	L07561
VI	PP1	Axonemal type-1 phosphatase	172	AF156101
VI	TUG	γ-Tubulin	138	U31545
VI	CNA9	cDNA corresponding to EST 1024003B10	This study	BG843541
VI	GAS3	Gene expressed during sexual differentiation	157
VI	RPL41	Ribosomal protein L41 (ACT2 locus)	146	AF130727
VII	GP205	Random genomic fragment	This study	AF467706
VII	AV396486	EST	2	AV396486
VII	CHI H	Magnesium chelatase H subunit	10	AJ307055
VII	F25	Class IV zygote-specific cDNA	33
VII	SFA	SF-assemblin	70	U56982
VII	VFL5	VFL5 gene	Iyadurai and Silflow, unpublished data
VII	IFT88	Intraflagellar transport protein 88	100	AF298884
VII	CPN60B2	Chloroplast chaperonin beta-like subunit	152	L27473
VII	ODA5	Outer dynein arm protein	M. Blomberg-Wirschell and G. Witman unpub- lished data
VII	HEMA	Glutamyl-tRNA reductase	R. D. Willows et al.; unpublished data	AF305613
VII	GP332	Random genomic fragment	This study
VII	CNC43	cDNA corresponding to EST 1024039D03	This study	BG854228
VII	CRD1	Copper response target 1 protein	92	AF226628
VII	FA2	Flagellar autotomy protein	81	AF479588
VII	SULP	Chloroplast sulfate transport system permease	H.-C. Chen, K. Yokthongwattana, and A. Melis, unpublished data	AF481828
VIII	CNB2	cDNA corresponding to EST AV626610	This study	AV626610
VIII	LC7	LC7, 11-kDa dynein light chain	8	AF140239
VIII	GP337	Random genomic fragment	This study
VIII	HSP70A	70-kDa heat shock protein	93	M76725
VIII	PSBQ	OEE3 protein of photosystem II	85	X13832
VIII	VFL1	VFL1 gene	136	AF154916
VIII	LI818	Polypeptide related to CAB proteins	121	X95326
VIII	CNA50	cDNA corresponding to EST 833007A09	This study	AW676510
VIII	CNC66	cDNA fragment	This study
VIII	MCA1	RNA stability factor	A. Watson et al., unpublished data	AF330231
IX	ANT	Mitochondrial ADP/ATP translocator	131	X65194
IX	CNA47	cDNA corresponding to EST 1031051H10	This study	BI997794
IX	CNA38B	cDNA fragment	This study
IX	PPX1	Protoporphyrinogen oxidase precursor	118	AF068635
IX	HSP70C	68-kDa heat shock protein	158
IX	UNI2	UNI2 gene	W.-C. Wu and C. Silflow, unpublished data
IX	PF16	Protein in C1 microtubule of flagellar central apparatus	143	U40057
IX	PSBO	OEE1 protein of photosystem II	85	X13826
IX	GP35	Random genomic fragment	This study	AF467704
IX	GP209	Random genomic fragment	This study
IX	CNB6	cDNA corresponding to EST 1024027D07	This study	BG849900
IX	CNA36	cDNA fragment	This study
IX	NIT1	Nitrate reductase	32	AH001336
IX	CAH3	Carbonic anhydrase, alpha type	39	U73856
IX	MBO2	MBO2 gene	150	AF394181
IX	MBB1	Required for expression of psbB/psbT/psbH	155	AJ296291
IX	SC2	Genomic fragment	A. Nguyen and W. Dentler, unpublished data
X	GP359	Random genomic fragment	This study	AY219892
X	GP39	Random genomic fragment	This study
X	KAT	p60 katanin subunit	80	AF205377
X	PF24	PF24 gene	P. Yang and W. Sale, unpublished data
X	GP441	Random genomic fragment	This study
X	GP204	Random genomic fragment	This study
X	CNC72	cDNA fragment	This study
X	PF6	PF6 gene	124a	AF327876
X	GP220	Random genomic fragment	This study	AF525922
X	GP350	Random genomic fragment	This study	AF525921
X	GP145	Random genomic fragment	This study	AF525920
X	CNA83	cDNA corresponding to EST 1031072D11	This study	BI724522
X	CNA26	cDNA corresponding to EST AV634482	2; this study	AV634482
X	GP52	Random genomic fragment	This study
XI	PETC	Chloroplast Rieske Fe-S precursor protein	22, 23	X76299
XI	VFL2	Centrin/caltractin	73	X57973
XI	CNA19	cDNA fragment	This study	AF503637
XI	GP49	Random genomic fragment	This study
XI	SD165	Genomic fragment	S. Dutcher, unpublished data
XI	PSBW	Core subunit of photosystem II	7	AF170026
XI	ODA2	Dynein heavy chain gamma	163	U15303
XI	RPS14	Ribosomal protein S14 (CRY1 locus)	95	U06937
XI	GP40	Random genomic fragment	This study	AF525923
XII/XIII	GAPC	Glyceraldehyde-3-phosphate dehydrogenase, (NAD) cytosolic, subunit C	58	L27669
XII/XIII	PF9/IDA1	Dynein heavy chain 1	94	U61364
XII/XIII	CNC54	cDNA fragment	This study	AF174532
XII/XIII	CNB20	cDNA fragment	This study	AF486824
XII/XIII	CNA10	cDNA fragment	This study
XII/XIII	PSR1	Phosphorus metabolism regulatory protein	169	AF174532
XII/XIII	IDA4	p28, dynein inner arm light chain	71	Z48059
XII/XIII	LF2	LF2/ULF2 gene	C. Amundsen and P. Lefebvre, unpublished data
XII/XIII	CNC8	cDNA corresponding to EST 963109F12	This study	B1873562
XII/XIII	CNC53	cDNA corresponding to EST 894081D12	This study	BE725171
XII/XIII	M63	Gene involved in cytokinesis	J. Larsen, L.-W. Tam and C. Silflow, unpublished data
XII/XIII	GP330	Random genomic fragment	This study
XII/XIII	GP31	Random genomic fragment	This study
XII/XIII	ODA6	IC70 dynein intermediate chain	89	X55382
XII/XIII	PTX4	Gene involved in phototaxis	G. Pazour, unpublished data
XII/XIII	EYE2	Gene required for eyespot assembly	122	AF233430
XII/XIII	SAC3	Kinase regulating response to sulfur limitation	16	AF100162
XII/XIII	GP399	Random genomic fragment	This study
XII/XIII	GP346D	Random genomic fragment	This study
XII/XIII	IC138	Inner arm dynein I1 subunit	P. Yang and W. Sale, unpublished data
XII/XIII	GP227	Random genomic fragment	This study
XII/XIII	ODA12	LC2, 19-kDa outer arm dynein light chain	102	U89649
XII/XIII	LC3	LC3, 17-kDa dynein light chain	99	U43610
XII/XIII	GS2	Glutamine synthetase 2 (GS2)	11	U46208
XII/XIII	ALK	Chlamydomonas aurora-like kinase	98	AF199021
XII/XIII	CNA38A	cDNA fragment	This study
XII/XIII	ODA9	IC78 dynein intermediate chain	164	U19120
XII/XIII	14-3-3	14-3-3 protein	77	X79445
XII/XIII	LAO1	L-Amino acid oxidase catalytic subunit	156	U78797
XII/XIII	TUB2	β2-Tubulin	174	K03281
XII/XIII	RDB	Roadblock protein, EST 1024060G02	S. King, unpublished data	BG858985
XII/XIII	GP114	Random genomic fragment	This study
XII/XIII	CNC63	cDNA fragment	This study
XII/XIII	TUB1	β1-Tubulin	174	M10064
XIV	ACYLC	EST 832001D08	132a	AW676021
XIV	LF4	Genomic fragment	S. Berman and P. Lefebvre, unpublished data
XIV	AC206	Ccs1, required for chloroplast C-type holocytochrome formation	55	U70999
XIV	GP336	Random genomic fragment	This study	AF467702
XIV	GP221	Genomic fragment; corresponds to EST 1031037H01	132a	BI996414
XIV	PCMA1	Genomic fragment	C. Asleson and P. Lefebvre, unpublished data
XIV	GP324	Random genomic fragment	This study	AF467703
XIV	IDA5	Actin	147	D50838
XV	IDA2	Dynein heavy chain 1-β of I1 complex	108	AJ242525
XV	EST 894011B10	EST	132a	BE056715
XV	F146	Class VI zygote-specific cDNA	33
XVI/XVII	ZYS3	Zygote-specific cDNA	65	AB004043
XVI/XVII	F4	Class I zygote-specific cDNA	33
XVI/XVII	CYTC1	Mitochondrial cytochrome c1	A. Atteia et al., unpublished data	AF245393
XVI/XVII	GP13	Random genomic fragment	This study	AF467701
XVI/XVII	DHC9	Dynein heavy chain 9	113	U61372
XVIII	EST 925020F05	EST	132a	BE442454
XVIII	CYTC6	Cytochrome c6	51	M67448
XVIII	S68	pcf6-8 cDNA, upregulated after deflagellation	129
XVIII	GP384	Random genomic fragment	This study
XVIII	CNA35	cDNA corresponding to EST AV622556	This study	AV622556
XVIII	CNC28	cDNA fragment	This study
XVIII	CLPK1	Cyclic nucleotide-dependent protein kinase	U. Kawabata et al., unpublished data	AB042714
XVIII	RAA2	Trans-splicing factor	106	AJ243394
XVIII	ODA1	cDNA corresponding to EST BG854636	148	AY039618
XVIII	ARS	Arylsulfatase	20	X52304
XVIII	DHC8	Dynein heavy chain 8	113	U61371
XVIII	CNB21	cDNA corresponding to EST 833007B10	This study	AW676519
XVIII	J134	Genomic fragment	J. Jarvik, unpublished data
XVIII	IDA7	140-kDa inner arm dynein	107, 171	AF159260
XVIII	GP431	Random genomic fragment	This study
XVIII	GP223	Random genomic fragment	This study	AF467708
XIX	GP230	Random genomic fragment	This study
XIX	ATP2	ATP synthase mitochondrial F1 beta subunit	38	X61624
XIX	BAC 27e16	BAC end sequence	This study
XIX	C4	Genomic fragment	W. Dentler, unpublished data
XIX	ODA3	Genomic fragment	63	AF001309
XIX	GP37	Random genomic fragment	This study
XIX	LC5	LC5, 14-kDa dynein light chain	99	U43609
XIX	ZYS 1B	Protein expressed during zygote formation	154	X76117
XIX	GLE	Gamete lytic enzyme	62	D90503
XIX	CNA37	cDNA corresponding to Lhcb4	151; this study	AB051211
XIX	S926	cDNA, upregulated after deflagellation	128	X62135
XIX	UND7	Genomic fragment	P. J. Ferris and U. W. Goodenough, unpublished data
XIX	FLA10	Kinesin-homologous protein	160	L33697
XIX	EF3A	Genomic fragment	E. Fernandez, unpublished data
XIX	CPN60B1	Chloroplast chaperonin beta-like subunit	152	L27471

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.

TABLE 2.

Mapping primers^a

Linkage group	Gene (3′ UTR)	Accession no.b	Primersc	C. reinhardtii allele (bp)	S1-C5 allele (bp)	Annealing temp (°C)
I	CYP1	AF052206	cyp1-R, GCATGCATTCCACAACGCACGC	232	378	56.0
			cyp1-F2, GGCGCGTACGCTCCGCGCd
			cyp1-F3, GGCTAATGGCTGTGCGGCTGGe
I	ARG7	X16619	ARG7-R, CGTCCCACACCTCCAAACGCCA	367	236	56.0
			ARG7-F2, GCCTTGACGTGAGGCTGCGCTGe
			ARG7-F3, TGGGGTACAAGGCGCTGTGAGAGAd
I	CNA73	BE726259	CNA73-R, CTTCTGCAGCCGTAGAAACCCGGC	436	379	63.9
			CNA73-F2, GCATAGGGGCTGTGCGGCGe
			CNA73-F3, TCTGTATGTGCCCCATGCGCACAd
I	COX2B	AF305540	cox2B-R, ATGGCTACGCCACCGCCGGTTT	386	547	63.9
cox2BDNA-F3, TTGCTGGGCTGTGGCCGCGd
			cox2BDNA-F4, CAGCGGTCCCTCAGGGACGTTACAe
I	LC4	U34345	LC4-F, GCCGCGCGAGCTGGAAGAGTTT	565	∼590	62.7
			LC4-R, GTGCCGCCCACGAAACGTTCTG
II	CPX1	AF133671	Cpx1-F, TTGCGTGCTAGCAGGCGTGGTG	329	393	61.0
			Cpx1-R2, GCTCCAAACCTGCTGCGGTCAGTCe
			Cpx1-R3, GCACGCACACACGCACACGAd
II	RB60	AF036939	RB60-R, GCGTTCGGAACCACGCACATCC	229	433	55.0
			RB60-F2, ACCAGCAGCAGCGCGTGATCCGGd
			RB60-F3, GCCAAAGAGGGACGCTGTCCACAGe
II	CIA5	AF317732	CIA5-F, TGGCTGCGTGCCACGACCGT	354	314	55.0
			CIA5-R2, GCTGAAGGTGAGTGCGGCAGCGd
			CIA5-R3, CCAGCTGCTGTTGGCGCCAGCe
II	CNA45	B1724982	CNA45-R, CGTGGTTCTTACATCACCCCAGCG	244	328	58.9
			CNA45-F2, TGTGGTGGGTGTTGATGGAGGAATGd
			CNA45-F3, TTGCGCGCATTGACAGATGTACAGe
II	YPTC4	U13167	YptC4-R, CGCCGTGATCAGCAGCAACAAGC	360	269	56.4
			YptC4-F2, TCCACATGATGGCTAGTGCGGACGe
			YptC4-F3, CCGTCAGCTACTGGGAAGGCCCGd
III	DHCa	L26049	DHC-alpha-F, AGGACATGCCCGCCAAGTGGGT	309	∼290	58.9
			DHC-alpha-R2, GCGGCACCTGGCTACTGCTGTACA
III	COX2A	AF305080	cox2A-F, TGCGGAGAAGGCGCTGGTCAAG	522	∼490	55.0
			cox2A-R, GGCGTCTTGCGCCATTGCTGAA
III	GSAT	U03632	GSAT-R, GAGGGTGCAATCAGAGCCCCCTTG	561	389	55.0
			GSAT-F2, CGCGTGCACAGCTTGCAGCAAAe
			GSAT-F3, CGGGCGGTGCCTGGTTCTTCGd
III	MAA7	AF047024	MAA7-F, CGGCGACAAGGACGTCAACAACG	114f	349	55.0
			MAA7-F2, TGTGGGAGCGGGAGTGACTGCA	349
			MAA7-R, TGCAACCATCTCCCTTCGGCCC	473
			MAA7-R2, TAATCCGCCTCAGCCCCAACCG
III	GAR1	U36197	MethSyn-R, GCAATGCGTTGGGTTACAAGCAGC	339	179	61.0
			MethSyn-F2, GCGAGCGGTACCGACTAGGCAGAe
			MethSyn-F3, GCTGAATTGTGTACGGTGCACACGGd
III	FLA14	U19490	LC8-F, TTCAAGTCGGGCTAAGCGGCCG	189	97	55.0
			LC8-R2, CATCCCTCCCCGCTATGTCCCGd
			LC8-R3, CCAGAGACCGCGCTCCGCCe
III	CNA34	B1722955	CNA34-F, GCAGCTGCCTGTCAATGCGCCT	376	∼350	55.0
			CNA34-R, GTCTGCGTAGCCGTACACGCGTCA
III	RSB1	B1722838	RSB1-R3, CCGCCACCCATGTCACGGCd	109	146	55.0
			RSB1-F2, TGTTCCCGCCGGAGGAGG
			RSB1-R4, CACACCACACGCTGCCTACAGGe
III	PHOT1	AJ416557	PHOT1-F, CGCGAGGAAGGGTTTGAGGTGCTGd	338	407	55.0
			PHOT1-R, CGGATAACAGCTGCGTCCTTCCCC
			PHOT1-F2, CCGCCCCGGCTGCAGCTAAe
IV	TUA2	M11448	a2-tub-R, GCCAATAGAGGCACGGTCGTGGA	130	150	55.0
			a2-tub-F2, GGCGTGATCTGAGGCTTCGTTGG
IV	COX3	AF233515	cox3-F, ACGGCATCATCTACGTCGGCCAG	470	∼500	55.0
			cox3-R, ACATAAACCGTCCACGCGGCTGC
IV	PF20	U78547	PF20-F, TGTCTCTCCGTTCCCTTGCGCGe	328	223	59.3
			PF20-F4, GGACCCCGGTCCTCTGCTACCGd
			PF20-R2, ACACACCAAACCGCCCATGACCC
V	GP228	BG859190	GP228-F, CAACATGGTGGAGGAGCAGGAGGG	244	369	63.9
			GP228-R2, GGCAGCCATCACCTCACACCAe
			GP228-R3, GCTTCTCATCACCCCCTGCTCTTAAd
V	ALD	X85495	ald-F, CTGCCCAGGGCATGTACGAGAAGG	588	122	60.0
			ald-R, TCCCGACGCTCGATGGATAGGAGGe
			ald-R4, GTCGCAGGCTGCTGCGGCTGd
V	PF26	M87526	RSP6-R, AGATGAACCTCGTGCCTCAGCGGC	206	529	61.0
			RSP6-F2, GAAGAACGGATCAGAGCGGTGTGGGd
			RSP6-F3, GGAACGTGGGGGACATACCCGGe
VI	FA1	AF246990	FA1-F, ACGAGGAGGACATTCGGGAGCTGC	182	331	64.3
			FA1-R2, GCTCAGCCGTTCCAAGGAAGCAATG
			FA1-R3, GTCCAAGCAGGTCCAAGCCGTCAA
VI	ATPC	M73493	ATPC-R, TCGCCTCATGTCGGCACACAGG	578	411	55.0
			ATPC-F2, TAAATGCCTGGGCTCTTGGGCTCGe
			ATPC-F3, CGCCGTGAATTTGCGTGGCGd
VI	S8-13	X53574	S8-13-F, GCGCCCCGAGTTCAACATCACC	331	285	55.0
			S8-13-R2, CCTCAACACACCGCGACGCAGAd
			S8-13-R3, GCATCAACGCGTTACAGATCGCCAe
VI	CRY1	L07561	DNAph-R, GAGACCGAAAGGCAAGGCACAGGC	443	443	63.5
			DNAph-F3, AGCTAACCATGTCGGCCGGTCGd	147f
			DNAph-F4, GCTGCATTGGGCGCACATGGe
VI	TUG	U31545	g-tub-R, GTCGCCAGGAATTTTGCCCCTGG	281	443	61.0
			g-tub-F2, GCGCGCCTGGCGGTAGCACATAd
			g-tub-F3, AGCAGCGCTATGTTCGCTTCCCCe
VI	RPL41	AF130727	RPL41-R, TGCAACTTGCAATCCATCCGTTGC	262	107	55.0
			RPL41-F2, GCAACTAAACGTGGCGGCCTACCGe
			RPL41-F3, GGTAACCGATTCGAGCGTTCTGGAd
VII	CHLH	AJ307055	chlh-F, TTGGCGGGTTGTGGTTGGACTAGG	127	375	62.4
			chlh-R2, TCCTCGCGGAGCGCTCTCGe
			chlh-R3, CACAGCTCACACACACACGCACAAd
VII	SFA	U56982	SF-assem-R, ACAGCATGCCCTGCAAGCTCGC	330	211	65.0
			SF-assem-F2, TTGCATGGGCAGCACTGGTCGAe
			SF-assem-F3, GCCGTATAAATTCAGGGCAGGCGCd
VII	IFT88	AF298884	IFT88-F, TGCTGAGCTTTGGCTCGGCTGG	191	91	55.0
			IFT88-R2, ACATACACAAATGGCGGGCTGCAGd
			IFT88-R3, CTGGGGACCCCTGCAGCCAAAAe
VII	CPN60B2	L27473	cr2-R2, AGCTGCTTGGCAGCGGCTGTTG	260	621	61.0
			cr2-F4, TGGAATTGGCGGTGCGAGCGd
			cr2-F5, TGCAGCACAACTCCCGGCTGCe
VII	FA2	AF479588	fa2-F, GCACGTCGTACTACACCAGCGCCA	140	396	55.0
			fa2-R2, CCCCGTCAACCTGGGCCAATCAe
			fa2-R3, CCGTCAACACCTCGAGTGGACACGAd
VII	SULP	AF481828	SulP-R, TGCGTCCTTCGCTCAATCCCTGC	339	193	55.0
			SulP-F2, GTGGGAGGGGTGGGACTTTGGGe
			SulP-F3, GGTATGGGGATGTCCGCACGCTTCd
VIII	MCA1	AF330231	MCA1-F, CGCGGGCGAGTTTGCTGTTGCT	113	226	55.0
			MCA1-R2, CGGATCCCGAACAGCGGCAGe
			MCA1-R3, CCCCGTGACTCAATCAAGTCCCTGd
VIII	LI818	X95326	LI818-R, TCCGATGCACTCACGCTCACAGC	334	141	55.0
			LI818-F2, TGGGCATGCGGAAATGCGTGTCe
			LI818-F3, CTTGCTTGGCCGGCACGGGd
VIII	PSBQ	X13832	OEE3-R, CGTGCTGTTGCGAGCCACTCCA	347	535	65.0
OEE3-F2, GCCGAGTTCTCAACCCTCTCGGCd
			OEE3-F3, GGGTGCAACCTCCGGTGGCCTAe
IX	PPX1	AF068635	Ppx1-R, CATGGCACTTATGGGCGAAGCCG	526	181	55.0
			Ppx1-F2, GGGCAAGCGGAGTGGAGGCGAe
			Ppx1-F3, TCGAAGTGCCTTCGAAAGTGGGCAd
IX	PSBO	X13826	OEE1-R, CGCATGCACGACGAGAAGCGAG	510	161	55.0
			OEE1-F2, GTCGACCGCTGCGAGGAGGAGAe
			OEE1-F3, CGAGCGCCGTATCATCCGGCTTAd
IX	MBO2	AF394181	MBO2-F, CGTTAACAGCCCTGAACTCGGCCG	516	406	65.0
			MBO2-R2, TCACGCCACACCTGTACGTGCAAd
			MBO2-R3, ATGCGCCAAACCCGGAGCTACCe
IX	CAH3	U73856	CAH3-F, CATGCAGCCCATCAAGGTGCCC	307	359	55.0
			CAH3-R2, TCCCCACCGTGGGCCAAACCe
			CAH3-R3, CGTGCAGGCGATGCCTCCAd
IX	MBB1	AJ296291	mbb1 F, GGATGAGGCGGTGGAGGCACTACA	382	192	55.0
			mbb1-R2, GCGTGCCGCGTGTCACCAAGTAd
			mbb1-R3, GGCCATACGCCATCATAACCGAGGe
X	KATANIN	AF205377	Katan-F, ACGAGGAGTGGCTCAGCGTGTTCG	394	∼410	55.0
			Katan-R, GGACGCCCAAGCTTCAAATCCACG
X	PF6	AF327876	PF6-R, GACAAACCCGTGTACCATCCGGCC	362	229	55.0
			PF6-F2, GCACAAGCAATGCATCGGTGTGCe
			PF6-F3, CGTTCCAGCGGCACTCACGGGd
X	CNA83	B1724522	CNA83-R, TGCACATACCTCTGCCGCTCCACC	278	324	55.0
			CNA83-F3, CGGATCGGGTATGCGGATGCCAd
			CNA83-F4, CGGCCGCTGAAGCTGCTGTGAe
XI	VFL2	X57973	vfl2-R, CCGCAGGCTGGCGATGGGAATA	463	283	58.9
			vfl2-F2, GACGCCGGGGCTTGCTTTCACCe
			vfl2-F4, TGCTGTGAAGGGTGGACACCCTGGd
XI	ODA2	U15303	ODA2-R, CACGCAGTGGCATCCTGCGC	298	197	55.0
			ODA2-F2, TTAGGGAGGCGGCACTGACGCAe
			ODA2-F3, AGCGTGCGATTGGCGTACGAGATTAd
XI	RPS14	U06937	CRY1-F, CATCCCCACCGACTCCACCCG	268	248	54.2 (C. reinhardtii), 58.9 (S1-C5)
CRY1-R2, CCCGCCGCCGCCACCTAd
			CRY1-R3, CCAGCCGCCAGGCGGGCe
XII/XIII	GAPC	L27669	gapC-F, CAGATTGCTTCAGGGCTTCGGCG	572	∼500	56.0
			gapC-R, TTCACGCACCGTGTGGCAGTCC
XII/XIII	PSR1	AF174532	Psr1-R, AGCACCCGTCCACACACCGCAA	344	189	62.0
			Psr1-F2, GCACCTGCGCATGCATCTGTTGe
			Psr1-F3, AGACAGCGGTTGGCCCTTGCTTGd
XII/XIII	EYE2	AF233430	EYE2-F, CGCGCGAGCTGACAGCTGAAGA	525	211	64.8
			EYE2-R2, TCACATACTGCGCAGCGCTCTCCd
			EYE2-R3, CGGGGTTGCCACAAGTTTCCTTCCe
XII/XIII	SAC3	AF100162	Sac3-R, ACTGCACAGCTCTGGGATGTCGCC	324	508	55.0
			Sac3-F2, ACGGAGCGCACTGGGTTCTTGCAAd
			Sac3-F3, TCGCGGTCCGGTCCCAGTATGe
XII/XIII	IC138		IC138-F, CGGGGCAGGCGTAGGACTGGAA	287	171	55.0
			IC138-R2, GCAAGCCTGGCCCCATCTGTTCd
			IC138-R3, CCTGGGCATCAGCACAGCACTTGe
XII/XIII	ODA12	U89649	LC2-F, GAGTAATGGTGCGGCCAAGCTGCC	426	∼450	55.0
			LC2-R, TTGCAACGGCAAGCCGCCAT
XII/XIII	14-3-3	X79445	14-3-3R, AGTGCGCTTCAACACGCCTCACG	317	205	55.0
			14-3-3F2, CGGCGTGAAGTGGCGTTACAGCTAe
			14-3-3F3, TGACATTGTGTTGGCCATCACGGAd
XII/XIII	TUB2	K03281	b2-tub-R, CACGTGCACGAGTGTGTGGCCA	113	283	55.0
			b2-tub-F2, GGAGGGGGGCCCATTGCCCd
			b2-tub-F3, CGGCAGGGGCAGGTAACTGCCe
XII/XIII	RDB	BG858985	CRB-R, CGTCAATTTGGCCGACCTGACCG	558	257	55.0
			CRB-F2, CCCGAAGCCATGGGCATCGAAe
			CRB-F3, GCGTCGACAACCATCTGCGACCAd
XIV	AC206	U70999	CCS1-R, ACGAATGCTGGGTGGGCCAAGC	483	156	64.7
			CCS1-F2, GGGGTCACGACAGGGGTAGGGTGe
			CCS1-F3, CGTGCGGCAAACAAGCACCCTTGd
XIV	IDA5	D50838	ACTIN-R, AAACCCCAGCGCTTTGGCGC	249	529	55.0
			ACTIN-F2, AAGCGCTTGTGAGTGCGCCAGAd
			ACTIN-F3, ACGCAGGTGGCAGGCCGAGGe
XV	EST 894011B10	BE056715	BE056715-R, CCCCCAAAATCAGCATGGGGTCC	316	237	55.0
			BE056715-F2, TGGATGAGGTGGGGTCGTTTGTCGe
			BE056715-F3, ACTGGCGTCGCGTCTGCAGGd
XV	IDA2	AJ242525	dhc10-F, TGCTGCTGTCGCTGGCCACGTA	514	∼550	55.0
			dhc10-R, TCACGGCAACCTGAAAGGACGCC
XVI/XVII	CYTC1	AF245393	Cytc1-F, GCCCATCAAGTCGCAGCGCATC	131	436	66.0
			Cytc1-R3, CAGCTGAACAGCCTGTGCGGCAd
			Cytc1-R4, GCAAAGACACTCAGGCCGCGCTCe
XVI/XVII	ZYS3	AB004043	Zys3-F, AGCCGCCACGTGTTTGTGGAGG	344	∼530	55.0
			Zys3-R, ACTGCCTTCTGGCTCGTATGCGGG
XVIII	CYTC6	M67448	CytC6-R, AAGCGCGTTCATGGTTCGGCC	221	319	55.0
			CytC6-F2, GTGTAGCTAGCTTTTGCCCCGGCAd
			CytC6-F3, CATCACGCAAATGGACACGTTCCGe
XVIII	CLPK1	AB042714	cpk1-F, GGATGGCAGCGTACCAGCTGTCACd	314	252	62.0
			cpk1-R, CACCGCATGTGTATTGGAGGCGC
			cpk1-F2, ATGCAGCAACGGTAGGCGCTAGCGe
XVIII	RAA2	AJ243394	Maa2-F, ATTGACCACTGCGGCGCTAGCG	595	∼530	59.0
			Maa2-R, TAGTAGGGGCATCCGTGGCTCTCG
XVIII	ODA1	AY039618	CDS-022-F, GACGCGGCGGTGATGGGCd	160	277	55.0
			CDS-022-R, CCCCGAGCGGATTGAGGTAATGG
			CDS-022-F2, CAAGGGTTCAGGGGCAGAATACCGe
XVIII	IDA7	AF159260	IC140-F, TGCTTATGGAAGGGCTGGGCGG	174	395	65.0
			IC140-R2, CTTGCGCCCGCCTCAGACACGe
			IC140-R3, GCGCAAGGCTTCGTCAGGCTGTCd
XIX	ATP2	X61624	atpB-R, TCGCAGTCCGTACCCTTGACACCG	102	255	55.0
			atpB-F2, GGCAGGGCGGTGCAGGCTTAAd
			atpB-F3, CGGGGCCATGTCAGCATGGGAe
XIX	BAC 27e16	PTQ10139.x1	BAC27e16-F, GTCTGTGCAGCGCTGCGCCTTT	296	∼400	64.3
			BAC27e16-R, AATGGCCAGGATGTGCGGGTAGC
XIX	ZYS1B	X76117	zys1B-F, GCTTTGAGTGGAGCGAGGCGCA	466	303	61.0
			zys1B-R2, TAAATGCATCTCCGCAGTTTTCTCCGb
			zys1B-R3, GCATTGGGCATAACCAGTATGTGCCAe
XIX	FLA10	L33697	FLA10-F, CTGCGCGCCAGCAAGCTCAAGT	491	467	55.0
			FLA10-R, GGTAACAGCCCGTCTTCCAGGGCC
XIX	CNB60B1	L27471	cr40-F2, GATTGGGGGCAGTGGGCAGGe	388	350	56.0
			cr40-R2, CACCGCCATGCGAAAGTGCC
			cr40-F3, GCCTGTGCGGGATGGCGTGAGe

^aAll amplification reactions should be performed as described in Materials and Methods, at the annealing temperature given for each locus, and all of the primers listed should be mixed in a single tube for each amplification.

^bAccession numbers refers to entries in the GenBank database (http://www.ncbi.nlm.nih.gov/), except for marker BAC 27e16 on linkage group XIX, which is from the JGI Chlamydomonas BAC-end sequence database (http://bahama.jgi-psf.org/prod/bin/chlamy/home.chlamy.cqi).

^cWhen only two primers are listed they generate allele-specific product size differences.

^dS1-C5-specific primers.

^e21gr-specific primers.

^fThe predominant product when more than two amplification products were observed.

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 (α2 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

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

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.