of chlamydomonas reznhardtzz segregation …aggregated zygotes form a pellicle at the meniscus and...

GENETICS OF CHLAMYDOMONAS REZNHARDTZZ DIPLOIDS I. ISOLATION AND CHARACTERIZATION AND MEIOTIC SEGREGATION PATTERN OF A HOMOZYGOUS DIPLOID

EVA M. E V E S AND KWEN-SHENG CHIANG182

Committee on Genetics1 and Department of Biophysics and Theoretical Biologyz, The Uniuersity of Chicago, Chicago, Illinois 60637

Manuscript received November 17,1980 Revised copy accepted October 12, 1981

ABSTRACT

A strain of Chlamydomonas reinhardtii has been investigated which, when mated with known wild-types, produces very few viable germination products and transmits its Mendelian markers to more than half of those products. Cyto- genetic observations, fluorometric measurements of DNA and genetic data all suggest that the strain, d mrery-M3a sr-u-l is a stable homozygous diploid. This strain has twice as many nuclear chromatin bodies at metaphase and twice as much DNA as its haploid progenitor, and the phenotypes of its meiotic progeny are consistent with predictions based on triploid meiosis. Data from crosses involving d mrery-M3a sr-u-I and from crosses involving hybrid diploids indicate that the frequency of second division segregation increases in triploid zygotes and that mitotic segregation following triploid meiosis is a frequent event which may more often result from mitotic recombination than from chromosome loss.

IPLOIDS of ChZamydomonas reinhardtii have previously been produced in several ways. WETHERELL and KRAUSS (1956) used colchicine to induce an

apparently stable diploid strain. The nuclei contained twice as many chromosomes as the haploid strain from which they were derived and the cells were 70% larger in dry mass and in volume. There are no reports of any further studies with this strain. BUFFALOE (1958), in a study of the vegetative and sexual life cycles of several species of Chlamydomonas, found that temporary diploidy or greater polyploidy could be induced in C. eugametos and C. moewusii, but not in C. reinhardtii, by increasing growth-light intensity from 1 00-foot to 800-foot candles (1075 to 8600 lux). However, these polyploids always divided to produce haploid daughter cells. MAGUIRE (1976) reported that the octet strain of C . reinhardtii (SUEOKA, CHIANG and KATES 1967) had twice as many chromosomes grown at 450 foot candles (4800 lux) than at 1500 foot candles (16,150 lux), but haploid daughter cells were produced by cell division at either light intensity.

EBERSOLD found that a few (< 1%, EBERSOLD 1963; 3 4 % , EBERSOLD 1967) paired gametes fuse and divide mitotically rather than developing into meiosis- competent zygotes. These strains appeared to be stable diploids although some mitotic recombination probably occurred (EBERSOLD 1967). These hybrid diploids, which were heterozygous for mating type, were capable of mating only with mt+ Genetics 100: 35-60 Janualy, 1981.

36 E. M. EVES A N D K.-S. CHIANG

haploid strains and 80% or more of the meiotic progeny from the triploid zygotes of such crosses were inviable.

Mendelian marker expression in hybrid diploids and polyploids of Chlamy- domoms reinhardtii has been studied in complementation and dominance tests (EBERSOLD 1967; HUDOCK and ROSEN 1976; MATAGNE and VINCENZOTTO 1979), in a comparison of Mecdelian and non-Mendelian gene mutation frequencies in haploid and diploids (LEE et al. 1973), and in searches for mitotic recombinants (LEE, WHITEWAY and YORK 1976; MATAGNE and ORBANS 1980). However, the transmission. of Mendelian markers in haploid X hybrid diploid crosses has been largely ignored except as a means of determining whether a diploid retains its input markers (EBERSOLD 1967; GILLHAM 1969; MATAGNE and ORBANS 1980).

Another hybrid diploid of C . reinhardtii has recently been formed by a poly- ethylene glycol (PEG) mediated cell fusion method (MATAGNE, DELTOUR and LEDOUX 1979). These hybrids are also selected for on the basis of complementation. Fusion of two mt+ cells has been accomplished by this method and presumably diploids of any of the three mating type combinations can be formed by this technique. In addition, those hybrid diploids can be fused with haploids to produce mitotic triploids (MATAGNE and VINCENZOTTO 1979). In contrast to the foregoing hybrid diploids, which are selected on the basis of complementation in either sexual or asexual cell fusions, spontaneous, stable, completely homozygous diploids of C. reinhardtii have not been previously characterized. Such strains, which have never been subjected to any artificial selective conditions, could be valuable for studies of non-Mendelian marker transmission as well as Mendelian marker segregation in triploid meiosis and for studies of possible mitotic recombination and/or chromosome segregation in the aneuploid progeny. We wish to report the isolation and characterization of what appears to be a spontaneous, stable mt-/mt- homozygous diploid strain whose gametes mate with mt+ haploid gametes.

MATERIALS A N D METHODS

Stra:ns: The strains and markers employed in thess studies are listed in Table 1. A small “d”, “hd” or “an” preceding the strain designation indicates that the strain is a

diploid, a hybrid diploid, or an aneuploid. Chlamydomonas reinhardtii mutant strains tradi- tionally have been named by the markers they carry. This usage has been conformed to for strains produced by others except f o r the linkage tester strains (%uYTH, MARTINEK and EBER- SOLD 1975), which are designated simply mtf and mt- LTS. Likewise, the full genotype de- scriptions of the hybrid diploids and the aneuploids produced in the course of this study are cumbersome and such strains have been given numerical designations. The linkage group positions and the phenotypes of the markers studied are presented in Table 2. The uniparental markers are followed immediately by a U, signifying that they are uniparentally transmitted in normal haploid x haploid crosses.

The original strain mter .yZy was obtained from Dr. N. GILLHAM in 1971 in response to our request for a non-Mendelian erythromycin resistant marker that was originally isolated in a mt wild-type strain. As designated at that time, the ery-Zy marker behaved as a typical non- Mendelian uniparentally transmitting marker carried by a m t haploid strain ( CHU-DER and CHIANG 1974). The strain was transferred to fresh slants several times between 1973 and 1975 but was not crossed. The results of crosses in 1975 showed that all recoverable erythromycin- resistant clones of the orig:nal nztery-2y strain had transmitted the erythromycin-resistant

GENETICS OF C. reinhardtii DIPLOIDS 37


M

GENETICS OF C . reinhardtii DIPLOIDS

TABLE 2

Markers employed

39

Marker Linkage group

(Dosition) * Phenmype

ery- M3a (er y -2y) t msr

ac-17 PY' m t f , mt- nic-7

ac-29a

act

can-la sr-la nic-13

Pf-2 sr-u-1 spr-U-1-27-3

I11 (R < 0.1) IV (R9) VI (L 30) VI (L 30)

VI (L33)

VI (R45)

VI1 (R 11) IX (L 12) x (L 12)

XI (L8) non-Mendelian non-Mendelian

resistant to 200 pg/ml erythromycin resistant to 300 ,pg/ml L-methionine-DL-

requires 2 mg/ml sodium-acetate resistant to 1 pg/ml pyrithiamine mating types requires 0.75 pg/ml nicotinamide, sensi-

tive to 75 pg/ml 3'-acetylpyridine pale green to yellow on acetate-containing

medium resistant to 10 ,pg/ml actidione

(cycloheximide) resistant to 1.0 mg/ml canavanine resistant to 50 pg/ml streptomycin requires 0.75 pg/ml nicotinamide, sensi-

tive t o 75 pg/ml3'-acetylpyridine paralyzed flagella resistant to 500 cg/ml streptomycin resistant to 100 ,pg/ml spectinomycin

sulf oximine

* Map units to right (R) or left (L) of centromere. -'r See footnote $ to Table 1.

phenotype as a Mendelian marker. Other investigators had also found that, in the ery-2y obtained from Dr. N. GILLHAM in 1974 and 1975, ery-resistance behaved as a Mendelian rather than non- Mendelian marker. The strain was then renamed ery-M3a (DAVIDSON, HANSON and BOGORAD 1978). The reason for this apparent switch is not understood.

Recently, E. HARRIS tested the ery-2y stock from our laboratory (personal communication) and DAVIDSON, HANSON and BOGORAD (1978) tested ery-2y stocks from several laboratories. All their data support the designation of ery-2y as ery-IM3a and its allelism with another erythromycin-resistant mutant, ery-21, which DAVIDSON, HANSON and BOGORAD (1978) designated ery- M3b.

Media: The three basic media used were high-aalt medium HSM ( SUEOKA 1960), HSM plus 2.0 g/l sodium-acetate.3H20 (HSA medium), and HSM with no source of nitrogen (N-free medium). HSlM was employed for growth, normal light maintenance, zygote maturation, germination and scoring. HSA was used for dark growth, acetate-requiring strains, and dim light slants. N-free medium was used for gametogenesis and zygote maturation.

Nicotinamide (Baker grade) and sodium acetate.3H20 (reagent grade) were obtained from The Baker Company. All other components of the basic media were reagent grade from either Baker or Mallinckrodt. Actidione (cycloheximide), canavanine.H,SO,, L-methionine-D, L-sul- foximine, pyrithiamine.HBr and streptomycin-SO, were obtained from the Sigma Chemical Co.; 3'acetylpyridine from the Aldrich Chemical Co. and Erythrocin (erythromycin lactobionate) from Abbott Laboratories. Trobicin (spectinomycin.ZHC1) was purchased from Upjohn Company and spectinomycinS0, was obtained courtesy of Dr. GEORGE B. WHITFIELD of the Upjohn Com- pany. These selective agents were added to media at the concentrations specified in Table 2.

Maintenance and culture: Strains were routinely grown or maintained on solid HSM plates in the light (28", 200 foot candles [2150 lux]). HSA slants and dim light (22", 15 foot candles [I60 lux]) were used for long term maintenance. Those maintained in the light were transferred to fresh plates every 10-14 days.

40 E. 1M. EVES A N D K.-S. CHIANG

Liquid growth cultures were inoculated at 0.5-1.0 X 105 cells/ml. Synchronous growth was obtained on a 12 hr light-I2 hr dark cycle (KATES and JONES 1964). Liquid growth cultures were bubbled with 1.5% CO2 and were either stirred or shaken continuously, exczpt €or very small- volume cultures in which bubbling alone provided sufficient agitation.

Gametogenesis and mating: Cells were scraped from seven- to ten-day-old plates and resuspended at approximately 10G/ml in N-free medium in 60 mm Petri dishes, which were then placed in the light for gametogenesis, Approximately 12 hr were required for gametogenesis of haploids and up to 18 hr for aneuploids and diploids. Plus and m;nus mating type gametes were then mixed in approximately equal numbers in Petri dishes, which were returned to the light for a 30-60 min mating period. The time required for mating was cross-dependent; diploids, aneuploids and paralyzed flagella strains required more time to mate than did wild-type haploids. Aliquots of the mating mixture (0.5 to 0.75 ml) were transferred t o maturation plates when numerous fused gamete pairs could be observed, but before zygote aggregates could form.

Tests to determine mating type were performed similarly. Cells were scraped from a plate and resuspended in two tubes each containing 1.0 ml N-free medium. Following overnight incubation in the light, gametes of the mt+ tester strain (137c) were added to one of the tubes and mt- tester strain (137c) gametes to the other. Mating was allowed to proceed t o completion. Aggregated zygotes form a pellicle at the meniscus and the presence o r absence of mat’ng could usually be determined by simple inspection (SAGER 1955). Where no pellicle was visible in either tube, the contents oE both were plated out on N-free sdid medium. Unmated gametes would not survive a three- to seven-day incubation period under continuous illumination, whereas maturation of zygotes proceeded normally as they became enlarged and darkened. Since these large dark zygotes were readily visible under a dissecting microscope (50X) this system made it possible to assign mating types to strains in which only a small proportion of the cells was capable of mating.

Zygote Maturation and Germination: Two different protocols were employed for zygote maturation. Mating mixtures were plated either onto HSM plates and incubated for two days in the light (100 foot-candles [lo75 lux]) and five days in the dark (protocol A, based on SUEOKA, CHIANG and KATES 1967), o r onto N-free medium and incubated in the light (200 foot-candles [a150 lux]) for three days (protocol B, VAN WINKLE-SWIFT 1977).

After maturation, zyg3tes were scraped from the maturation plates, deposited on fresh solid medium (supplemented as required for any input markers) and positioned with a glass loop for ana1ysi.s. Prior to germination in the light, these plates were inverted over chloroform for 35 sec to kill any vegetative cells. Diploid zygotes matured by the A protocol germinate in 12-16 hr. The B maturation protocol increased the germination time by 10-1000,:,, depending on the cross.

Zygote colony isolation, tetrad dissection, replica plating and scoring: The germinated zygotes were allowed to form colonies approximately 1-2 mm in diameter before they were transferred to master plates. On tetrad dissection plates the germination products (usually four or eight) were separated within areas defined by circles drawn on the bottoms of the plates. Zoospore clones were allowed to grow up for several days or until a diameter of 1-2 mm was attained. Then all viable clones were transferred to fresh plates. For pedigree analysis the four (or rarely eight) zoospores of a zygote were widely separated to lie in sectors drawn on a plate. When the zoospores released the first set of daughter cells these were separated within the sector; time of this “first division” and the number of daughters were nosted. In some crosses the zoospores of a single zygote “divided” at different times. Thus, the number of “division” products varied from two to sixteen. The clones were allowed to grow to 1-2 mm and were then transferred to master plates.

Zygote colonies, progeny clones and streaks of the parental strains were allowed to grow up on master plates until they attained a size sufficient for replica plating, The master plates were inverted onto a sheet of Whatman # 1 sterile filter paper which had been fastened over a replica plating block. Replicas were made on the desired media and the replica plates were placed in the light.

The growth interval required between replicating and scoring depended on the strains and markers involved. Products from haploid X haploid crosses could usually be scored at seven to

GENETICS OF C . reinhardtii DIPLOIDS 41 ten days. Haploid x diploid and haploid x aneuploid cross products required up to 20 days due to the slow growth of some aneuploids. Paralyzed flagella (pf-2) were scored by resuspending cells in liquid medium and observing their swimming characteristics at a magnification of 400X. Mating types were scored as described above.

Hybrid diploid production: In most crosses a few percent of the mated pairs divide mitotically rather than developing into zygotes (EBERSOLD 1963, 1967). Such diploids are most readily isolated when the parent strains carry complementing mutations in tightly linked genes and the mating mixture is plated onto medium which selects against both parental mutants. The plates are placed in the light and diploid clones can be isolated within three to four days, usually before zygote maturation is complete.

The two mutant strains used for diploid production in these studies were mt+ nic-7 (nicotinamide-requiring, 3'-acetylpyridine sensitive) and mt- ac-29a (pale green to yellow flat clones). The nic-7 and ac-29a markers are virtually inseparable from their respective mating types (GILL- HAM 1969; SMYTH, MARTINEK and EBERSOLD 1975).

Counts of metaphase nuclear chromatin bodies: Aliquots of exponentially-growing liquid cultures were harvested and washed once with distilled H,O. The cells were resuspended in 8 0 ml of 3: l methanol: acetic acid and fixed at room temperature for 30 min. This was followed by two rapid changes of a fixative (5.0 ml each) and the cells were finally resuspended in 0.5-1.0 ml fixative. This suspension was dropped onto water-cooled slides, which were then heated briefly over a flame, air-dried and rinsed once with McIlvaine's Buffer pH 6.8.

The slides were stained with Gurr's Giemsa (1:lO in McIlvaine's Buffer pH 6.8 at room temperature for 4-5 min or with azure B (.0125% in McIlvaine's Buffer p H 4.0) for one hr at 60". Metaphase nuclear chromatin bodies were counted at a magnification of 1 0 0 0 ~ using both phase and bright field optics. Direct counting was more efficient than counting from photographs since the chromatin bodies were poorly separated and very rarely lay in a single focal plane. The nuclear chromatin bodies were counted only when their arrangement in a cell conformed to BUFFALOE'S (1958) description of the arrangement (i.e , a ring) of chromosomes at metaphase.

Determination of cellular D N A content: The diaminobenzoic acid (DABA) fluorometric pro- cedure used was a modification of that developed by CATTOLICO and GIBBS (1975). Preliminary cell counts were done early in the light period (Lo-L,) of 32 hr light-I2 hr dark synchronized cultures. Appropriate volumes of the cultures were harvested by centrifugation, washed with distilled H,O and killed with 5% formalin. Precise cell counts were made at this time. Triplicate samples of 5 x 106 or 1 x IO7 cells each were transferred with a nonwettable pipettor into 1.5 ml conical plastic centrifuge tubes. The cells were pelleted by centrifugation for 30 sec in an Eppendorf Model 3200/30 microcentrifuge. All but approximately 50 pl of the supernatant was aspirated off and the tubes were vortexed until the cells were completely resuspended. Then 0.5 ml of 80% acetone in H,O was added to each tube and the tubes were vortexed for mixing. This first acetone extraction was allowed to proceed for 15 min at room temperature. The samples were pelleted. resuspended by vortexing, and repeatedly extracted with 0.5 ml volumes of SO'% acetone until both pellet and supernatant were colorless. The samples were then resuspended and treated twice with 0.15 ml 0.6N TCA at 5" for 5 min, twice with 0.15 ml ethano1:water (2:l) at 5" for 5 min and twice with ethano1:water (2:l) at 60" for 5 min. Pelleting was always accomplished by 30-sec spins in the microcentrifuge and resuspension by vortexing. After the final centrifugation, the supernatant was removed and the uncapped tubes were placed in a 60" oven overnight to dry the pellets.

Standards were prepared by adding equal volumes of 2.0 N NH,OH to known amounts of DNA in H,O. The standards were made up in triplicate in 1.5 ml plastic conical tubes and dried overnight in the 60" oven. The DABA stock solution was prepared by adding 6.2 ml distilled H,O to 25 g DABA 2HC1 (Aldrich Chemical Co.); 1.5 g charcoal (Sigma Chemical Co. #C-4386) was added and mixed with the stock. The mixture was then filtered (Millipore 0.22~) to remove the charcoal and its adsorbed impurities. Charcoal addition and filtration were repeated until the DABA solution was straw yellow to colorless. Then, 100 a1 of the DABA stock was added to each of the dried standards and samples. The tubes were capped and incubated for 45 min at 60". All samples were allowed to cool to room temperature, then 1 .O N HC1 was added to increase the

42 E. M. EVES A N D K.-S. C H I A N G

sample volume to that required by the fluorometer cuvette employed. The samples were read in an Aminco Bowman Fluorometer Model (#J4-7439) using a GE F415-B bulb. The primary filter used was a Turner #405 (Kodak 110-812) for excitation at 405 nm and the secondary filters were Turner #’s 8 and 65A (Kodak 110-817, 110-825) for emission greater than 5% nm.

RESULTS

Znitial indications of diploidy: A combination of reduced viability of meiotic products and the preponderance of paternal Mendelian alleles expressed in the viable progeny of crosses involving the d mt- ery-M3a sr-u-l* strain of C. reinhardtii were the initial indications that this strain might be diploid. Crosses of mt+ haploid x d mt- ery-M3a sr-u1 produced 13- to 20% viable progeny and very few complete tetrads. Crosses involving mt- ery-M3a, the haploid progenitor of d mt- ery-M3a sr-u-1, produced 60- to 98% meiotic products capable of forming colonies. In crosses with several mt+ strains the dominant Mendelian alleles, i.e., m t and ery-M3a, canied by d mt- ery-M3a sr-ul were expressed by 63- 84% and 93%, respectively, of the progeny (Table 3) . These crosses yielded only a few progeny, but similarly small samples of progeny from haploid x haploid crosses yielded equal numbers of m t f and m t progeny and of ery-M3a and ery-M3a+ progeny.

We considered the possibility, however unlikely, that d mt- ery-M3a sr-u-l could be disomic for only its two marked linkage groups. To test this hypothesis the best linkage tester strain (LTS) available (SMYTH, MARTINEK and EBERSOLD 1975) was crossed to the putative diploid. In control crosses, the m t f LTS was crossed to known haploids, m t ery-M3a and mt- spr-U-1-27-3 in order to demonstrate normal Mendelian segregation for the LTS markers. The Mendelian markers did segregate 2:2 in all complete tetrads recovered from these crosses, and in small samples of single clones which survived from incomplete tetrads no sitlgle allele was expressed in less than 47% or more than 55% of the clones (Table 4).

The complete tetrads from these LTS x haploid crosses were ordered using ac-17 which has been mapped at less than 0.1 map unit from the centromere of linkage group I11 (EBERSOLD et al., 1962). The agreement of the second division segregation (SDS) frequencies and the resultant centromere to marker distances with the previously published map distances is satisfactory (Table 5 ) . These crosses also revealed the linkage of ery-M3a to msr, which had been assigned to linkage group I. The parental ditype: nonparental ditype: tetratype (P: N: T) ratio for these two markers is 12: 0: 6 and, upon employing the formula

x 100 = map distance T + 6 N

2 ( P + N + T ) -

(PERKINS 1949), the interval between ery-M3a and msr is found to be 16.7 map units. Thus, ery-M3a may be provisionally assigned to linkage group I. The fact that both ery-M3a and msr map to distances greater than 33 units from the centromere makes accurate ordering of these markers relative to the centromere

* For thc sake r r f L o x i s t e m ) , the diploid daigndtion “d” piecdes this strain, the evldence that t l u s IS indeed the caw I , pierented bel IW

GENETICS OF C. reinhardtii DIPLOIDS 43 TABLE 3

Marker transmission b y m t ery-M3a and related strains

Cross mt+ X mi-

90 x ery-M3a Lo x ery-M3a sr-u-l spr-U-1-27-3 (c!one 3) x ery M3a nic-7 ery-M3a sr-u-I (clone 3 ) x spr-U-1-27-3 nic-7 ery-M3a sr-u-l (clone 16) x spr-U-1-27-3

nic-7-ery-M3a sr-u-l (clone 16) x 89

nic-7 ery-M3a sr-u-1 spr-U-1-27-3 (clone 4) x 89

nic-7 ery-M3a sr-u-1 spr-U-1-27-3 (clone 4) x Lo

nic-7 x ery-M3a sr-u-I spr-U-1-27-3 (clone 14)

90 x ery-M3a sr-u-l spr-U-1-27-3 (clone 14)

137c x ery-M3a sr-u-I spr-U-1-27-3 (clone 14)

90 x ery-M3a sr-u-I spr-U-1-27-3 (clone 16)

LO x ery-M3a sr-u-I spr-U-1-27-3 (clone 16)

nic-7 ery-M3a sr-u-I spr-U-1-27-3 (clone 4) x ery-M3a sr-u-I spr-U-1-27-3 (clone 14)

LO x d ery-M3a sr-u-1 90 x d ery-M3a sr-u-1 137c x d ery-M3a sr-u-I

spr-U-1-27-3 x d ery-M3a sr-u-I

Clones from incomplete Tetrads shaving tetrads

Marker 2:2 segregation Mutant allele mi allele

erY 28 erY 29 erY 18 9 7 erY 13 9 7 ery 16 mt- 16 erY 20 mr 20 erY 24 mt- 24, erY 26 mt- 26 erY 4 mt- 44 ery 12 mt- 6 erY 19 m r 5 ery 4

19 4 23 14 27 6 31 2

* The phenotypes of all clones from these 11 tetrads were ery as expected.

impossible without additional data from crosses involving ery-IM3a and at least one more linkage group I marker. No other pair of markers segregating in the cross demonstrated linkage.

LTS X d mt- ery-M3a sr-u-1 crosses: In a haploid x diploid cross a dominant or codominant allele carried by the diploid is expected to be expressed among the progeny at a frequency of 0.833 - 0 . 0 2 8 ~ + 0 . 0 0 1 ~ ~ (where x = the frequency of SDS), while a recessive allele carried by the diploid should be expressed by 0.500 + 0 . 0 2 8 ~ - 0 . 0 0 1 ~ ~ of the progeny (FINCHAM and DAY 1965). These formulae are based on several assumptions:

1. The chromosomes associate as trivalents at synapsis, In any single region the homologous chromosomes associate in pairs, but pairing partners may change from region to region.

44 E. M. EVES AND K.-S. CHIANG

TABLE 4 Marker transmission in crosses with the linkage tester strain

Clones from inconiplete Cross Tetrads showing tetrads

m2+ X mt- Marker 2:2 segregation Mutant allele wt allele

LTS x ery-M3a erY msr ac-17 PYr mt- act can-la sr-la nic-13 P f - 2

LTS x spr-U-1-27-3 msr ac-I7 PYr mi- act sr-la nic-I3 Pf-2

18 20 18 18 19 19 18 18 20 18 20 18

18 21 17 18 18 19 19 18 19 19 18

15 19 19 15 21 1 7 15 18 20 15 15 19 19 15 21 17 15 20 18 15

la

~ ~~ ~ ~~

TABLE 5 Second diuision segregation frequencies

Centromere-locus Published centromere l n ~ u s hIarker rrcquency of SDS map distances map distance3

ery-M?n msr ac-I7 PY‘ m t act can-ln sr-la nic-I3 Pf-2

0.722 0.697 0 0.182 0.600 0.879 0.222 0.242 0.242 0.156

> 33.0 > 33.0

0 9.1

30.0 > 33.0

11.1 12.1 12.1 7.8

32”; 31b approx. 51c

< O.ld 8e 2 4 C ; 36e 41.5e 6e; 6-8c 10e 4c; 3e

< 1-5d; 0.5c

DAVIDSON, HANSON and BOCORAD 1978.

HASTINGS et al. 1965. EBERSOLD et al. 1962.

b HARRIS. E. (personal communication).

e SMYTH, MARTINEK and EBERSOLD 1975.

2. At the first meiotic anaphase the chromosomes move to the spindle poles at random; exchange partners do not necessarily go to opposite poles.

3. Although nullosomics undoubtedly cause much of the inviability that results from triploid meiosis, here each meiotic product is assumed to receive one or two, but not three, copies of each linkage group.

GENETICS OF C. reinhurdtii DIPLOIDS 45

Although these assumptions underlie the derivation of the above formulae, the first and second assumptions have almost no effect on the calculated expected frequencies. From the formulae it is clear that, only when the dominant or CQ-

dominant alleles are contributed by the diploid parent and are present in a ratio of 2 : l dominant:recessive in the zygote, will the allelic frequencies in a rela- tively small sample of random clones be diagnostic of diploidy.

Mutations that confer resistance to antibiotics or to analogs of normal me- tabolites were assumed to act in a dominant or codominant fashion (cf. HANSON and BOGORAD, 1977). The exception to this assumption among our markers is actidione-resistance (act). In lower eukaryotes this phenotype is found to be recessive (~ 'BRIEN 1980). mt- has been shown to be dominant to mt+ in hybrid diploids (EBERSOLD 1963, 1967) and to mt+/mt+ in hybrid triploids (MATAGNE and VINCENZOTTO 1979). Mutations that specified nutrients were assumed to be recessive (i.e., ac-17 and nic-13) ; Pf-2, which determines structurally normal but furxtionally abnormal flagella, (RANDALL and STARLING 1976) was assumed to be recessive to the wild-type. At least one other paralyzed flagella mutation has been shown to be recessive to the wild-type in hybrid diploids (STARLING 1969).

Table 6 presents three sets of expected phenotype frequencies. Column I1 values were calculated using the raw SDS frequencies obtained from the LTS x haploid crosses. The figures in Column I11 result when Assumptions 1 and 2 above are ignored, that is, the homologs are assumed to associate as a bivalent and a univalent. Under these conditions the members of the bivalent would move to opposite poles and the univalent would be free to move to either pole. DUCK

TABLE 6

mt+ LTS x d mt- ery-M3a sr-u-1 progeny phenotypes

Phenotype Observed frequency* I I1 111 Expected frequencies?

ery-M3a msr ac-17 PYr mt- act can-la sr-la nic-l3 Pf-2

0.712 0.483 0.136 0.545 0.809 0.254 0.545 0.441$ 0.193 0.172

0.806 0.474 0.167 0.492 0.807 0.194 0.491 0.490 0.177 0.174

0.813 0.482 0.167 0.495 0.816 0.191 0.496 0.494 0.1 74 0.171

0.833 0.500 0.167 0.500 0.833 0.167 0.500 0.500 0.167 0.167

* Sample size is shown in the parentheses. t See text for explanation of expected frequency calculation. $ The actual frequency of sr-la clones may have been slightly higher. All clones were scored

on 50 pg/ml streptomycin and on 500 pg/ml streptomycin. Only clones expressing the non- Mendelian marker sr-u-I could grow on the latter medium. 0.441 is the frequency of clones resistant to 50 pg/ml of the antibiotic but sensitive to the higher concentration. Approximately 9% of all the clones expressed sr-u-I. Therefore, the actual frequency of sr-la can be estimated as 0.441 (apparent frequency) f 0.044 (expected frequency x 0.09) or 0.485; since this can only he an estimate the frequency of known sr-Za clones is presented here.


and JAMES (1976) reported that SDS frequencies for markers on trisomic chromosomes in yeast are significantly higher than those for disomic members. The figures in Column I are derived when the raw SDS frequencies for the Chlamy- domonas markers are adjusted in accordance with their findings.

The phenotypic frequencies of the LTS x mt- ery-M3a sr-u-2 progeny are in good agreement with the predicted frequencies (Table 6) . The several sets of expected frequencies never differ by more than 0.027, or 7 clones. Thus, the precise SDS frequencies and the manner in which the chromosomes associate at meiosis are not significant factors for individual marker analysis of this type.

The genetic data are evidence for the presence of two copies of linkage groups I ( e r y - M h ) , 111 (ac-17), VI (mt- and act+), X (nic-23f) and XI ( p f - 2 + ) in d mt- ery-M?a sr-u-2. The probability that the strain is disomic for the linkage groups carrying these dominant alleles and monosomic for the others is almost negligible.

Further evidence of diploidy has been acquired from counts of nuclear chromatin bodies and from DABA- fluorometric determinations of whole cell DNA content. There is disagreement regarding the haploid number of chromosomes in C. rehhardtii. Some investigators have reported eight chromosomes ( BUF- FALOE 1958; LEVINE and FOLSOME 1959; MAGUIRE 1976) while others find 16 or more ( WETHERELL and KRAUSS 1956; MCVITTIE and DAVIES 1971 j STORMS and HASTINGS 1977). BUFFALOE’S (1958) observation that 16 or more densely staining bodies condense to form eight chromosomes at metaphase and MAGUIRE’S (1 976 j demonstration of temporary diploidy under particular culture conditions suggest that eight is the haploid number but the most recent published genetic map for C. reinhardtii (HARRIS, 1980) lists 16 (and possibly more) linkage groups. We have counted Giemsa-staining bodies (see MATERIALS AND METHODS)

which, under our conditions, are in a ring or a collapsed ring similar to those described or pictured by BUFFALOE (1958). We cannot be certain that these are individual chromosomes. Since these counts are presented for comparison of the putative diploids to the haploid strains, to avoid controversy we will refer to the stained bodies as metaphase nuclear chromatin bodies. The average number cf nuclear chromatin bodies visible at metaphase in haploid mt- ery-M?a cells is 7.7 +- 0.7; in d mt- ery-M3a sr-u-1 the average is 15.5 2 1.2 (Figure 1 j . DABA fluorometric determinations of cellular DNA content were variable over several experiments (Table 7). However, the average amount of DNA in d mt- ery-M3a sr-u-1 is 1.93 times the average of five haploid strains and 2.05 times that of mt-ery-M?a.

Thus, the genetic, cytogenetic, and biochemical data all suggest that d mt- ery-Mja sr-u-2 is a diploid. This diploid is capable of mating with mt+ haploid strains to form triploid zygotes, which in turn produce meiotic progeny that ex- press the input markers with the predicted frequencies.

Stability of the diploid: Six crosses between LTS and d mt- ery-M3a sr-u-2 over a 13-month period allowed us to assess the stability of the diploid. Chi-square values for the observed versus the expected number of clones expressing each marker in each cross are presented in Table 8. These data reveal no correlation

GENETICS OF C. rinhardiii DIPLOIDS

0 b

10

12 (D J - r 8 w 0

4

6 7 8

47

I 8

C

CHROMATIN BODIES

d

CHROMATIN BODIES FIGURE 1.-Nuclear Chromatin Body Counts: a ) Metaphase cell of m r ery-M3a. b) Chromatin

body counts from 25 mt- e r y - M k metaphase cells; mean number of chromatin bodies = 7.7; standard deviation -C 0.7; standard deviation as percent of mean = 9.5%. c) Metaphase cell of d mr ery-M3a a - U - 1 . d) Chromatin body counts from 26 d mr ery-M3a sr-u-1 metaphase cells; mean number of chromatin bodies = 15.5; standard deviation = f 1.2; standard deviation as percent of mean = 7.6%.

between deviations from predictions and strain age as represented by temporal order of the crosses or any correlations with maturation protocol or a particular marker or markers. This lack of correlation between the deviations from expectations and the known variables indicates that neither the strain nor any marker in the strain (as revealed by phenotype analysis) changed perceptibly over the course of these studies. LEE, WHITEWAY and YORKE (1976) reported that diploids originally produced

by gamete fusion can produce some haploids and aneuploids under culture conditions resembling those for gametogenesis. MATAGNE and ORBANS (1980) report


TABLE 7

DABA fluorometric determinations of cellular D N A content

Strain EXD. I

Cellular DNA ( X 10.' pg)

rnt+ 137c 1.71 k0.03 mt+ spr-U-1-27-3 m t ery-M3a mt- ery-M3a sr-u-1

m t + LTS d mi- ery-M3a sr-u-1 3.20t0.58

spr-U-1-27-3 (clone 14)

Exp. I1 Exp. I11

2.04t0.09 2.21 t0 .16 1.63 * 0.19 1.68 i 0.24 1.78 20.10 1.66k0.27

1 . 6 2 t 0.1 0 1.922 0.03 1.98t0.24

2.95t0.03* 4.41 t0 .33

Cellular DNA

average nf ( X lO-'pg/cell) all haploids

Average DNA content v ~ .

1.99 1.09 1.65 0.91 1.72 0.94

1.77 0.97 1.98 1.09 3.52 1.931

* In this experiment the d m t ery-M3a sr-u-l samples contained the most DNA. The resultant

-t We found it impossible to synchronize the hybrid diploids; thus. their cellular DNA content fluorometer readings were at the limit of linearity for the standards, so this value may be low.

could not be determined by this method.

the recovery of a small proportion (0.16%) of apparent aneuploids from hybrid diploid strains. A putative mitotic recombinant was also recovered in that study. MARTINEK, EBERSOLD and NAKAMURA (1970) have also reported on mitotic recombination in C. reinhardtii. Haploidization and/or aneuploidization do not seem to occur at any appreciable frequency in our spontaneous diploid. Haploidi- zation would result in the recovery of complete tetrads with 2: 2 segregation for all markers. No such tetrads were found.

Since our experiments were not designed to select against aneuploids, we cannot rule out the possibility that some aneuploids are being produced during the vegetative growth of d mt-ery-M3a sr-u-l.

Presumably. the production of aneuploids before or during d mt- ery-M3a sr-u-I gametogenesis would produce higher than predicted frequencies of progeny clones expressing LTS alleles. Indeed, several of the LTS alleles are expressed more frequently than expected (Table 6) : ery-M3a+, pyr, act and can-la have deviations of + 0.09, + 0.06, + 0.06, and + 0.05 respectively. Only the chi- square (x2) values for act ( 2 = 5.07) and ery-M3a+ ( 2 = 13.7) are significant at the p = 0.10 level and only that of ery-M3a+ is significant at the p = 0.05 level.

Several facts suggest that these deviations are not due primarily to chromosome loss or gain (aneuploidy). First, i f loss or gain were random with respect to linkage group all LTS alleles should be expressed more frequently than predicted. This was not observed. Perhaps more convincing is the finding that the unlinked markers ery-M3a (linkage group I) and act (linkage group VI) are expressed at elevated levels, whereas other markers on these linkage groups (msr and mt+ respectively) are not. Finally, there is some evidence that post-meiotic segregation (PMS) occurs in the progeny of these triploid zygotes and that the PMS frequencies at the ery-M3a and act loci and among the highest.

Post-meiotic segregation: Approximately 200 single clones were isolated from each of five LTS x d mt- ery-M3a sr-u-l progeny colonies and scored for seven


- 151 I 8 0

8 % I $ I I 0 C . l

V

0 ? I s 1 I 0 0 0


TABLE 9

Mitotic segregation in aneuploid strains ~~ ~~~ ~

Clones expressing the marker Strain Sample size ery-ll.13a msr ac-17 pyr act sr- la nic-13

an 35 ,-ss-8-3 204 201 0 0 132 157 201 0 an 35,-ss-4-12 196 0 169 196 194 0 61 0 an 35,-ss-8-9 203 182 191 0 203 203 202 0 an 35 ,-3-4 207 20 7 0 0 207 14 52 207 an35 -6-1 203 203 0 0 203 152 203 203

Mendelian markers. All five colonies exhibited mitotic segregation for at least one marker (Table 9). In the absence of cross data for these strains it is impossible to determine exactly how many are heterozygous at any given locus. How- ever, we can designate possible heterozygotes and, from this number, calculate the expected number of heterozygotes for a dominant or codominant marker. The ratio of the number of strains segregating the marker to the number of expected heterozygotes can then be compared to the distance of that locus from its centromere (Table 10). Even in this small sample the frequency of mitotic segregation increases with distance from the centromere. This sort of analysis is less convincing for recessive markers since a heterozygote could be phenotypically scored as either f or -, depending on whether segregation takes place before scoring.

Pedigree analysis was used to demonstrate that early PMS was occurring in these crosses. Only 20 of the 228 zoospores dissected for pedigree analysis yielded more than one daughter cell capable of forming a clone. However. in these 20 sets eight PMS events were found for the seven markers scored. The three markers farthest from their centromeres (msr, act, and ery-IM3a) accounted for seven of those events. The eighth early PMS event occurred at nic-13, which is approximately 12 map units from its centromere. The remaining three loci, sr-la, pyr and ac-17, approximately 12, 9 and 0 map units from their respective cmtrorr_eres, exhibited no early PMS in this sample.

Although the correlations between frequency of PMS and the distance of the locus from its centromere are somewhat crude, such correlations cannot be ignored. They suggest that a large part of the mitotic segregation occurring in the progeny of triploid zygotes is due to mitotic recombination rather than to chro-

TABLE 10

Marker segregation and centromere-locus interual

Possible Expected Number of itrains n Centrornere- X'lailier heterozygotes heterozygotes segregating marker h Incus intcrv,,l

A B

-

msr 2 1.2 2 1.7 51 ery-M3a 4 1.4 2 1.4 32-36 sr-la 5 3.2 4 1.2 10-12 iwr 5 3.2 2 0.6 8- 9


mosome loss. This is not to imply that chromosome segregation is not taking place; it almost certainly is. Nuclear chromatin bodies were counted in metaphase cells of four related aneuploid strains from a mtf spr-u-1-27-3 X d mt- ery-M3a sr-u-1 cross (Figure 2). The aneuploids averaged 11 to 14 chromatin bodies and the standard deviations expressed as fractions of the means ranged from 0.13 to 0.22, compared to a maximum of 0.1 for the haploid and diploid lines in which nuclear chromatin bodies were counted at metaphase. Thus, chromosome complements did vary considerably from cell to cell within an aneuploid line. Whether chromosome segregation takes place throughout a strain's existence or only in the early post-meiotic division is not known.

Partial chromosome deletions or a series of mitotic nondisjunctions that would eliminate whole chromosomes are both possible mechanisms for PMS although neither of these mechanisms would be expected to yield a correlation between PMS frequency and centromere to marker map distance. Nondisjunction has previously been proposed as a mechanism for mitotic segregation in C. reinhardtii (MARTINEK, EBERSOLD and NAKAMURA 1970).

Hybrid diploids: Before the isolation of d mt- ery M3a sr-u-1, hybrid or heterozygous diploids were the only C. reinhardtii diploids known to maintain their diploidy through gametogenesis and mating. Hybrids are formed when a fused gamete pair proceeds to divide mitotically rather than forming a zygote capable of meiosis (EBERSOLD 1963) or when cells are fused by PEG treatment (MA- TAGNE, DELFOUR and LEDOUX 1979). Thus the modes of formation of hybrid diploids and of the homozygous diploid are presumed to be different. In addition, LEE, WHITEWAY and YORKE (1976) have reported that hybrid diploids are somewhat unstable under conditions resembling those used for gametogenesis and MATAGNE and ORBANS (1980) reported apparent marker or chromosome segregation from hybrid diploids in liquid culture. W e decided to examine the stability of hybrid diploids under conditions where d mt- ery-M3a sr-u-2 had proved to be quite stable.

Attempts to construct hybrid diploids involving an LTS strain were unsuccess- ful but several hybrid diploids were recovered from a mt+ nic-7 x m t ac-29a cross. The extremely tight linkage between these two nutritional markers and their respective mating types ( GILLHAM 1969) greatly facilitated isolation of hybrid diploids. Such colonies, in which the markers complement, are readily discriminated from haploid parental colonies on selective plates. Two hybrid diploids, hd 2-5-1 and hd 2-5-9, were each crossed three times to m t f nic-7 ery- M3a sr-u-2 spr-u-1-27-3 (clone 4) over six months. Progeny phenotypes were recovered at the expected frequencies for the hd 2-5-1 crosses and iterations 1 and 3 of the hd 2-5-9 cross also produced clones whose phenotypes conformed to predictions (Table 11 ) . Progeny viability in those five samples ranged from 18% to 20%. Apparently, however, a significant amount of self-mating occurred for hd 2-5-9 the second time it was crossed to m t f nic-7 ery-M3a sr-u-2 spr-u- 1-27-3 (clone 4). Not only are the maternal (mi+) alleles underexpressed among the progeny, but 51% of the progeny o€ this cross survived and a number of complete tetrads were recovered. In those complete tetrads nic-7 and ac-29a

52 E. M. EVES AND K . 4 . CHIANG

0 b

6 1

-I W 0 2

C

6

4

I

0 1 1 13 15

CHROMATIN BODIES

d

1

CHROMATIN BODIES FIGURE &.-Nuclear chromatin body counts in aneuploids: a) Metaphase cell of an 18A,-1-1.

b) Chromatin body counts from 16 an 18A,-1-1 metaphase cells; mean number of chromatin bodies = 13.8; standard deviation = f 1.7; standard deviation as percent of mean = 12.6%. c) Metaphase cell of an 18A2-1-2. d) Chromatin body counts from 22 an 18A2-1-2 metaphase cells; mean number of chromatin bodies = 10.9; standard deviation = f 2.4; standard deviation as

segregated 2: 2 and these were not erythromycin-resistant clones. Phenotypic segregation was not observed in the vegetative population of hd 2-5-9, nor was there formation of a significant number of zygotes in the hd 2-5-9 gamete culture before the mt+ gametes were introduced. Hence, the physical preseme of mt+ gametes appears to have been a prerequisite for self-mating of hd 2-5-9 gametes.

The hybrids averaged 15.4 k 0.8 and 15.5 f 0.7 nuclear chromatin bodies per cell a t metaphase (Figure 3) and the fewest chromatin bodies observed in any hybrid was 14. The genetic and cytogenetic data indicate that such hybrids tend

GENETICS OF C . reinhardtii DIPLOIDS 53

e f

18

d 6 0

CHROMATIN BODIES

9 h

9

b[ i6 3

4 p a , * 4 .

PI&

8 10 12 14 16 CHROMATIN BODIES

percent of mean = 21.9%. e) Metaphase cell of an 184-1-3. f ) Chromatin body counts from 44 an 1 SA,- 1-3 metaphase cells; mean number of chromatin bodies = 11.6; standard deviation = f 1.6; standard deviation as percent of mean = 13.4%. g) Metaphase cell of an 18A2-1-4. h) chromatin body counts from 30 an 18A2-1-4 metaphase cells; mean number of chromatin bodies = 13.0: standard devintion = f 2.2; standard deviation as percent of mean 16.6%.

to be stable complete diploids. However, under certain presently undefined conditions a hybrid can undergo intracellular zygote formation or haploidization followed by mating. Intracellular zygote formation is presumed impossible in a homozygous diploid, and no evidence for haploidization was found throughout a series of six crosses. On the basis of data presected above, d mr ery-M3a sr-u-2 appears to be more stable than hybrid diploids and therefore more useful, not only for the study of triploid meiosis but also for studies concerning the effects of diploidy on the transmission of non-Mendelian genes (manuscript in preparation).


TABLE 11

Phenotypes of progeny from constructed diploid crosses

Cross

Phenotypes Saniple ery-313a nic-7 ac-29n TTild~i)-pe

size Ohserved Expected Observed Expected Observed Expected Observed Expected

mt+ nic-7 ery-M3a sr-u-1 spr-u-1-27-3

(clone 4) X hd 2-5-1

34 14 22 21 1 75 36 36 39 40 25 25 23 28 12 10 18 15 2 53

3 27 13 13 8 14 6 5 13 8 mtf nic-7 ery-M3a sr-u-I spr-u-1-27-3

(clone 4) x hd 2-5-9

27 29 9 11 20 16 1 56 24 27 6 11 2 38 10 18 19 20 13 7

3 66 28 31 31 25 9 12 26 19

DISCUSSION

The results presented here suggest that a spontaneous homozygous diploid of Chlamydomonas reinhardtii has been isolated. We do not know how or exactly when d mt- ery-M3a sr-u-2 became a diploid. The most likely mechanism of diploidization was endoreduplication as seen by MAGUIRE (1976) ~ but in the case of d mt- ery-M3a sr-u-2 endoreduplication was not followed by a reduction division. Other mechanisms for the formation of a homozygous diploid are possible. Such mechanisms include (1) the fusion of two cells and (2) the occurrence of a diploidizing mutation. The latter seems unlikely in view of the number of nuclear chromatin bodies seen at metaphase in aneuploid progeny and the DNA content data. The spontaneous fusion of two cells of like mating type has never been observed although MATAGNE, DELTOUR and LEDOUX (1979) have presentel evidence for chemically induced fusion of cells of the same mating type.

The presumed diploidy of d mt- ery-M3a sr-u-2 has been supported genetically by crosses to a mt+ linkage tester strain as well as cytologically by counts of metaphase nuclear chromatin bodies and biochemically by DABA-fluorometric determinations of whole-cell DNA content. In aggregate, the data obtained by these different methods strongly suggest that the strain is a diploid. Transmission genetics results alone are not sufficient for such an identification in C . reinhardtii because of the paucity of mapped markers and because of the unavailability of one or a few strains carrying markers on all known linkage groups.

Ten markers were scored in the crosses between the linkage tester strain (LTS) and d mt- ery-M3a sr-u-1, but only six, ery-M3a, ac-17, mt, act, nic-13 and pf-2, can be regarded as diagnostic for diploidy (Table 6). The dominant or codominant alleles of the remaining four markers are carried by the LTS and their expected frequencies of expression among single-clone progeny differ by less than 3% for haploid X haploid and haploid X diploid crosses. Thus, the

GENETICS OF C. reinhardtii DIPLOIDS

0 b

C

55

CHROMATIN BODIES

d

6 cn W 0 i 4

2

CHROMATIN BODIES FIGURE 3.-Nuclear chromatin body counts in heterozygous diploids: a) Metaphase cell of

hd 2-5-1. b) Chromatin body counts from 19 hd 2-5-1 metaphase cells; mean number of chromatin bodies = 15.4; standard deviation = -C 0.8; standard deviation as percent of mean = 3.4%. c) Metaphase cell of hd 2-5-9. d) Chromatin body counts from 11 hd 2-5-9 metaphase cells; mean number of chromatin bodies = 155; standard deviation = k 0.68; standard deviation as percent of mean = 4.4%.

genetic studies demonstrate disomy for only five linkage groups (mt and act have both been mapped to linkage group VI). However, the phenotype frequencies among the triploid zygote progeny indicate that the chromosomes tend to associate as trivalents in meiosis (Table 6) and that increased SDS frequencies similar to those found by DUCK and JAMES (1976) in yeast trisomics can be applied to triploids of Chlamydomonas. Data showing that d m t ery-M3a sr-u-1 has twice as many nuclear chromatin bodies at metaphase (Figure I) and twice as much DNA per cell (Table 7) as its haploid progenitor, mt my-M3u, support the diploid condition suggested by the genetic analysis.


The biological characteristics of d mt- ery-M3a a-u-l and its progeny from crosses with mt+ haploids conform to expectations for a diploid and its aneuploid progeny. The diploid has approximately 70% more mass (as determined by optical density measurements) than mt- ery-M3a and requires a longer period of time for both gametogenesis and mating. Germination times for related diploid and triploid zygotes are similar. Viability among the germination products of triploid meiosis is low, i.e., 10-20%. This is also true for crosses involving EBER- SOLD’S (1967) hybrid diploids and the hybrid diploids isolated in the course of this study. Most triploid zygotes produce four or eight zoospores at germination but the majority of the zoospores never divide and many of those which do divide die during the first few post-meiotic mitotic divisions.

The aneuploid progeny are unstable with regard to phenotypic expression (Table 9) . The broad range in numbers of nuclear chromatin bodies at metaphase (Figure 2) in the aneuploids suggests that some of this instability is at- tributable to changes in chromosome number. However, the positive correlations between centromere-locus map distance and the frequency of post-meiotic segregation of phenotypes (Table 10) suggest that mitotic recombination may also play a major role in the segregation pattern documented in this study. Pedigree analysis has shown that phenotypic segregation can take place in the first few divisions following meiosis. The resulting intraclonal heterozygosity helps to explain the genetic data from haploid x aneuploid crosses (EVES, 1979). The phenotype frequencies among the progeny of such crosses often fall between the values expected if the aneuploid strains were monosomic o r homogeneous disomic for the relevant linkage groups.

Mendelian marker transmission from hybrid diploids was examined to confirm the diploidy of these strains and to compare their characteristics with the spontaneous homozygous diploid. The phenotype frequencies observed among the progeny of the haploid x hybrid diploid crosses agree with the expectations (Table 11) and the counts of nuclear chromatin bodies at metaphase confirm diplsidy (Figure 3 ) .

The homozygous and hybrid diploids are similar in many characteristics: length of time required for gametogenesis and mating, progeny viability, and chromosome numbers. However, in one cross some cells of one of the hybrid diploid strains apparently self-mated, either internally or by gamete fusion following haploidization. The factors inducing this self-mating are unknown but are presumed to be environmental since the strain mated as a diploid at times preceding and at times following that cross. Since no gamete clumps or zygotes were observed before the hybrid diploid gametes were mixed with the haploid mt+ gametes, one can speculate that some factor required for self-mating was suppressed and such mating could only proceed when this factor was introduced o r induced by the presence of mt + haploid gametes. If the intra-strain mating of the hybrid diploid was preceded by haploidization, such reduction did not occur in crosses involving the homozygous diploid. However, this is not sufficient evidence to prove that the spontaneous homozygous diploid is more stable than the hybrid diploids. While the phenotype frequencies of the haploid x hy-

GENETICS OF C . reinhardtii DIPLOIDS 57

brid diploid progeny show some increases in deviations from the expectations with strain aging (Table ll), the sample sizes from individual crosses are not large enough to assign much significance to this trend. If the increases in deviations are real, they may be due to mitotic recombination; similar events would be undetectable in the homozygote. I t is unlikely that chromosome loss produces significant heterogeneity in the hybrids since they exhibit the smallest range, in numbers, of chromatin bodies of all strains examined (Figures 1,2, and 3 ) . Thus, no clear differences in genetic stability between the homozygous and hybrid diploids have been observed aside from a single incidence of apparent self-mating of a hybrid diploid for which the inductive conditions are not understood at present.

The aneuploids, on the other hand, appear highly unstable during early post- meiotic cell divisions. A high incidence of morphologically atypical cells following gametogenesis suggests that genetic instability also affects gametic diff eren- tiation. For these reasons the aneuploids are not particularly useful. However, repeated subclonng might produce a library of stable disomics that would be valuable for genetic mapping and studies of mitotic recombination and gene con- version.

There have not been many studies concerning the transmission of Mendelian markers through triploid meiosis. With a few exceptions, triploids produce very few viable offspring (DARLINGTON and MATHER 1969) ; the more fertile trisomics are preferred for investigations of trivalent pairing, mitotic recombination and linkage studies (DUCK and JAMES 1976; MACKINNON and JOHNSTON 1972; DARLINGTON and MATHER 1969). In algae other than Chlamydomonas, where haploid X diploid matings have been achieved, the triploid zygotes are meiot- ically sterile (BIEBEL 1976). Triploid plants are often vigorous but the majority of these produce highly infertile gametophytes ( STURTEVANT and BEADLE 1962; DARLINGTON 1973). The lack of a proliferative gametophyte (haplophase) in higher plants limits genetic analysis of the meiotic products to a deductive process based on the viable plants produced following mating and germination. Early studies of triploid sporophytes in Bryophytes revealed little spore germination and even lower viability (< 1%) for the resultant gametophytes (ALLEN 1935). However, the comparison between Bryophyte gametophytes and those of Chlam- ydomonas has limited value since the former develop into complex plants while the latter are single cells. Chlamydomonas differs from all the plants mentioned above in that its predominant haplophase requires no further development or mating before it can be analyzed genetically. The findings that triploid Bryo- phytes and triploid higher plants tend to produce offspring which are euploid or. if aneuploid, usually trisomic diploids or disomic triploids (ALLEN 1935; DARL- INGTON and MATHER 1969; HARLAN afid DEWET 1975) are based on plants which have survived differentiation or mating and germination.

In order to compare the viability and aneuploidy tolerance of the progeny of triploid meiosis in Chlamydomonas, it is necessary to look at organisms with free-living and undifferentiated haplophases. Saccharomyces cerevisiae is clearly the relevant organism with the most intensively studied genetics. I n yeast the


number of genetic studies on triploids is limited since the higher viability of meiotic progeny obtained from trisomics (ROMAN, PHILLIPS and SANDS 1955; DUCK and JAMES 1976) makes sporulation of a number of trisomic strains more efficient than sporulation of a triploid strain. In their analysis of triploid S. cereuisiae, POMPER, DANIELS and MCKEE ( 1954) reported germination frequencies of 69% and 83% compared to 48-100% found for Chlamydomonas triploids in the present studies. In a more recent study of triploid yeast. MACKINNON and JOHNSTON ( 1972) reported that their triploids “sporulated well.” POMPER, DANIELS and MCKEE (1954) recovered six (12%) complete asci and confined their genetic analysis to those 24 ascospores. The segregation of three of their markers is as expected for such a small sample (see Table 1 in FOMPER, DANIELS and MCKEE 1954). However, the ratios for the other two markers are highly aberrant and suggest either misclassification of the parental strains or pre- sporulation homozygosis via mitotic recombination. The possibility of undetected pre-sporulation mitotic recombination is a drawback in the utilization of an organism with a proliferative sporophyte phase for triploid studies. MACKINNON and JOHPTSTON (1972) reported viabilities of 19% and 20% for triploid meiotic progeny and a sample of 50 spores from two crosses yielded phenotypic frequencies comparable to those for markers in C. reinhardtii.

The comparable viability of meiotic progeny and the extensively mapped linkage groups make S. cereuisiae a more useful organism than Chlamydomonas for the study of triploid or trisomic meiosis at this time. However, if many more mutants can be isolated and mapped in C. reinhardtii and if a library of disomics can be accumulated, then the alga will be even more useful than yeast for such studies since its sporophyte (zygote) does not normally divide mitotically and thus possible complications arising from mitotic recombination are eliminated.

The valuable discussions and suggestions of Drs. ROCHELLE ESPOSITO, ARNOLD RAVIN, JANICE SPOFF~RD and HEWSON SWIFT during the course of this work and in the organization of the initial report (E. M. EVES, Ph.D. Dissertation) are gratefully acknowledged. This work was supported by U. S. Public Health Service research grant H D 05804 and training grant GM 00090.

LITERATURE CITED

ALLEN, C. E., 1935 BIEBEL, P., 1976

BUFFALOE, N. D., 1958

CATTOLIC~, R. A. and S. P. GIBBS, 1975

CHU-DER, 0. M. Y. and K.-S. CHIANG, 1974

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A comparative cytological study of four species of Chlamydomonas. Bull. Torrey Botanical Soc. 85: 157-178.

Rapid filter method for the microfluorometric analysis of DNA. Anal. Biochem. 69: 572-582.

Interaction between Mendelian and non-Mendelian genes. Regulation of the transmission of non-Mendelian genes by a Mendelian gene in Chlamydomonas rehhardfii . Proc. Natl. Acad. Sci. U.S. 71: 153-157.

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The Elements of Genetics. 1st Schocken Edition. Schocken Books, New York.

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DARLINGTON, D. C. and K. MATHER, 1969


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GILLHAM, N. W., 1969 Uniparental inheritance in Chlamydomonas reinhardi. Amer. Naturalist 103 : 355-388.

HANSON, M. R. and L. BOGORAD, 1977 Complementation analysis at the ery-MI locus in Chlamy- domonas reinhardi. Molec. Gen. Genet. 153: 271-277.

HARL4N. J. R. and J. M. J. DEWET, 1975 On 0. Winge and a prayer: The origin of polyploids. Bot. Rev. 41 : 361-390.

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Corresponding Editor: J. E. BOYNTON

of chlamydomonas reznhardtzz segregation …aggregated zygotes form a pellicle at the meniscus and...

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