behavior of chloroplast genes during the early zygotic divisions of chlamydomonas reinhardtii

Current Genetics 1,137-153 (1980) Current Genetics © by Springer-Verlag 1980

Behavior of Chloroplast Genes during the Early Zygotic Divisions of Chlamydomonas reinhardtii

Jean L. Forster*, Constance T. Grabowy**, Elizabeth H. Harris, John E. Boynton, and Nicholas W. Gillham

Departments of Zoology and Botany, Duke University, Durham, NC 27706, USA

Summary. Chloroplast mutations in the green alga Chlamydomonas reinhardtii exhibit a predominantly maternal pattern of inheritance and this pattern can be perturbed by UV irradiation of the maternal gametes prior to mating. In a series of crosses over a range of UV doses, the transmission, segregation, and recombination of mutations at three closely linked chloroplast loci have been examined by pedigree analysis of products arising from the first three post-zygotic divisions. Stocks used in these crosses were constructed to permit identification of the nuclear products of each of the two meiotic divisions and the first post-meiotic mitotic division.

A bias toward maternal alleles at all three chloroplast loci was observed in all pedigrees and in zygote clones analyzed from the same crosses many generations after meiosis. This bias decreased with increasing UV dose and with each subsequent division. Segregation of chloroplast genes was rapid during the first three post-zygotic divisions. The type of segregation event from which a given heteroplasmic cell arose had a significant effect on its most likely segregation pattern in the subsequent division. The results presented here have been discussed in terms of published models of chloroplast gene segregation.

Key words: Chlamydomonas - Chloroplast genes - Segregation - Recombination.

Offprint requests to: T. E. Boynton at the above address

* Present address: Department of Health Education, University of North Carolina, Chapel Hill, NC 27514, USA

** Present address: Sidney Farber Cancer Center, 44 Binney Street, Boston, MA 02115, USA

Introduction

Physical evidence suggests that the single chloroplast of the green alga Chlamydomonas reinhardtii contains multiple copies of its genome, since the absolute amount of chloroplast DNA per cell is ~75 times the molecular weight of an individual chloroplast DNA molecule (cf. Adams, 1978). The precise manner in which this genome is replicated and transmitted in crosses is still uncertain. When gametes of opposite mating type are mixed, cell fusion occurs to form a diploid zygote, and fusion of the chloroplasts of the parent cells ensues within 3 h (Cavalier-Smith, 1970). Chloroplast DNA molecules con- tributed by the maternal and paternal gametes appear to undergo different fates during the subsequent zygote maturation period (Chiang, 1976; Sager and Lane, 1972; Burton et al., 1979), the ultimate result being a predominantly maternal pattern of inheritance of chloroplast mutations in progeny from the germinated zygote (cf. Gillham, 1969; Sager, 1972). That is, all meiotic products of >90% of the zygotes from a cross transmit only the alleles of chloroplast genes carried by the maternal (rot +) parent. The remaining exceptional zygotes transmit alleles of chloroplast genes from both parents (biparental zygotes) or, very infrequently, only chloroplast alleles from the paternal (mt - ) parent (paternal zygotes).

UV irradiation of maternal gametes of C. reinhardtii immediately prior to mating increases many fold the frequency of exceptional zygotes (Sager and Ramanis, 1967; Gillham et at., 1974) and has made the detailed study of transmission, segregation and recombination of chloroplast genes possible. Sager and Ramanis (1965, 1968, 1976a) reported that alleles of the chloroplast genes carried by the maternal and paternal parents appeared in a 1:1 ratio among the progeny of biparental zygotes. Both zygote clones and pedigrees showed this 1 : 1 allelic ratio regardless of UV dose given the maternal gametes

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138 J.L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in Chlarnydomonas

prior to mating (Sager and Ramanis, 1976a). Variations from this 1:1 allelic ratio were ascribed to allele- and locus-specific effects (Sager and Ramanis, 1976a).

In contrast we have found a consistent bias favoring the chloroplast alleles o f the maternal parent among the progeny of both spontaneous and UV-induced biparental zygotes (Gillham et al., 1974; Boynton et al., 1976; Conde et al., 1975; Harris et al., 1977; Adams et al., 1976; Adams, 1978). This maternal bias decreased when the maternal gametes were treated with increasing doses o f UV prior to mating. A similar maternal bias has also been reported in a limited number o f meiotic tetrads (Gillham, 1963, 1969; Boynton et al., 1976)i

The discrepancies in genetic results between our laboratory and Sager's may have several origins. First, crosses made in the two laboratories have usually involved different markers and different genetic backgrounds. Sec- ond, the gametogenesis and mating regimes used in the two laboratories are dissimilar (Sears et al., 1978). Third, Sager has looked at segregation and recombination chief- ly by pedigree analysis o f individual zygotes during meiosis and the two subsequent mitotic divisions at a time when many progeny are still heteroplasmic. In contrast, we have examined allelic ratios and recombination of chloroplast markers among a large number o f randomly sampled zygote progeny after many generations o f growth at a time when fewer than 1% of the cells are heteroplasmic for any chloroplast marker.

To establish whether the results we have reported in zygote clones are reflected in pedigrees under our laboratory conditions, we have now compared transmission and segregation of mutations at three chloroplast loci in

biparental zygotes from the same cross, using both zygote clone and pedigree analysis (cf. Fig. 1). These three chloroplast loci have been studied extensively in our laboratory and appear to be closely linked (Gillham et al., 1974; Boynton et al., 1976; Harris et al., 1977). Parental strains carrying these mutations were purposely constructed to facilitate pedigree analysis and to minimize potential marker effects (see Results). By treating the maternal gametes wi th a range o f UV doses, we have been able to address the following questions: 1) Is there a bias in favor of maternal chloroplast alleles among progeny analyzed both by pedigree and zygote clone analysis and does this bias change as a function o f UV dose and division? 2) At what rate do homoplasmic cells appear for the chloroplast alleles at each locus? 3) Does the segregation pattern vary as a function of the locus being studied? 4) What is the pattern of segregation o f individual chloroplast alleles in pedigrees and is this pattern affected by UV dose, division, and genotype of the parent cell? 5) Are all heteroplasmic cells alike in terms of the progeny they produce? 6) Do chloroplast markers that are closely linked by recombination analysis (Harris

et al., 1977) tend to segregate together more frequently than those that are farther apart?

Materials and Methods

All data presented in this paper are from crosses between the following two stocks of Chlamydomonas reinhardtii, strain 137c: nr-u-2-1 mt + (GB-86) and er-u-37 spr-u-l-6-2 st-u-2-60 ac-17 act nic-13 rot- (GB-412). The markers present in these stocks are as follows:

Chloroplast mutations

Genotype Phenotype Reference

er-u-37

nr-u-2-1

spr-u-l-6-2

st-u-2-60

erythromycin resistant GiUham et al., 1974

neamine and kanamycin Gillham, 1965; resistant Harris et al., 1977

spectinomycin resistant Gillham et al., 1974

streptomycin resistant Gillham and Levine, 1962; Gillham et al., 1974

Nuclear mutations

Genotype Phenotype Linkage Reference Group

ac-17 acetate-requiting III Levine and Goodenough, 1970

act actidione (cyclo- VI Smyth et al., 1975 heximide) resistant

nic-13 nicotinamide- X Levine and requiring Goodenough, 1970

The GB-412 stock was synthesized as follows: GB-29, a mr- stock containing several Mendelian mutations including ac-17, act and nie-13, was crossed to GB-147 which is a mt + stock carrying the spr-u-l-6-2 mutation. A rot- isolate was obtained among the progeny with the genotype ae-17act nit-13 spr-u-l-6-2 m t - . This isolate was crossed to GB-227 which has the genotype er-u-37 sr-u-2.60 spr-u-l-6-2 mt + and the GB-412 stock was derived from among the progeny.

Culture and Mating Conditions

Media, general procedures for stock maintenance and mating conditions described by Gillham et al. (1974), Conde et al. (1975) and Harris et al. (1977) were used with the following modifications. Cells from 7-day YA plates were suspended for gametogenesis for one hour in nitrogen-free liquid medium. Cells from plates of that age are gametes already and suspension in liquid medium merely induces flagellar regeneration (Martin and Goodenough, 1975). mt + gametes were irradiated immediately before mating as described by Gillham et al. (1974) for times ranging from 7.5 to 45 s at 11,000 ergs cm -2 s -1 and mated. Separate batches of gametes prepared under similar physiological conditions were used for each irradiation dose. After two hours dark incubation, the mating mixture was plated on HS medium lacking ammonium chloride and containing 4% agar. The plates

J. L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in Chlarnydomonas 139

Pedigree Anolysis Zygole Clone Analysis

Fig. 1. Comparison of the pedigree and zygote clone analysis methods used to study the inheritance of chloroplast genes in Chlamydomonas. A chloroplast marker for streptomycin resistance (st) is shown for example

C r o s s : s r x s r +

+

I I I I I I I I ! I I I I I

Mitosis

I I I I I

• • • • • • •

Test Each Clone for sr or sr + Genotype

Mitosis I

P r ~ cells)

0 0 0 0 0 0 0 0 ......... 0 64 I I I I I I I I I [ I I I I Mitosis I

O 0 0 0 0 0 0 0 . . . . . . . . . • 64

Test Eoch Clone for sr or sr + Genotype

were then incubated in the light (~6,000 lux, cool white fluor- escent) for 18-24 h, and placed in the dark for 6 or 7 days for zygote maturation prior to analysis.

Genetic Analysis

Procedures of Conde et al. (1975) and Harris et al. (1977) were followed with these exceptions: For clone analysis zygotes were allowed to germinate and grow into colonies on non-selective medium (HSA containing 1 ~zg/mi nicotinamide added before autoclaving). The colonies were transferred to a 10 x 10 matrix on the same medium. Biparental zygote colonies were identified by replica-plating the matrix to individual selective media containing nicotinamide and one of the following antibiotics: 100 #g/ml neamine (or 100/~g/ml kanamycin to which nr-u cells are cross-resistant), 200/~g/ml erythromycin and 100 gg/ml streptomycin. Sources of antibiotics and preparation of antibiotic-containing plates were as described by Harris et al. (1977). Once recognized, 25 zygote colonies biparental for all four chloroplast markers were picked from a non-selective replica and individually suspended, diluted and plated onto non-selective medium. Six- ty-four to 100 progeny clones from each original biparental zygote were randomly chosen to be tested for antibiotic resistance phenotype by replica-plating as described by Conde et at. (1975) (also see Table 2).

Zygotes for pedigree analysis, taken at the same time from the same maturation plates as those for zygote clone analysis, were germinated and separated into zoospores using the procedures of Ebersold and Levine (1959). Under our conditions, the majority of the zygotes germinated into 8 zoospores, i.e. they had already completed the first postmeiotic, mitotic division. The 8 zoospores from each zygote were separated from one another and permitted to grow into clones of approximately 106 cells. These zoospore clones were transferred to a nonselec- tive plate and replica-plated to appropriate media. Nuclear markers were scored by replica plating to the following media: HS + nicotinamide [ac-17J, HSA + nicotinamide + 1 /gg/ml cyclohexi-

mide added in ethanol after autoclaving (act), HSA (nic-13). The media used to score the chloroplast markers were identical to those used for the zygote clones described above. The eight zoospore clones from any zygote not biparental for at least one of the three chloroplast loci were discarded. To maximize the amount of information obtainable from these pedigrees, we have included progeny from zygotes biparental for any of the three chloroplast markers in the data summarized in Tables 4 and 8-11. Only progeny from zygotes biparental for all three chloroplast markers were included in Tables 5 - 7 , 12 and 14. The numbers of biparental zygotes analyzed as well as the markers for which they were biparental are summarized in Table 3. Zoospore clones from biparental zygotes which contained chloroplast antibiotic resistance markers were individually diluted, spread and replica- plated to antibiotic-containing media as above. In this way colonies could be positively identified as pure resistant, pure sensitive or heteroplasmic for alleles at each of the chloroplast loci.

Statistical Analysis: Where critical tests of hypotheses were needed, data were analyzed for heterogeneity using G 2 statistics. These are analogous to ×2 statistics but permit the estimation of three way, as well as two way, interactions in contingency tables (Bishop et at., 1975).

R e s u l t s

All da ta p r e sen t ed were o b t a i n e d f r o m crosses b e t w e e n a

single pair o f s tocks, nr-u-2-1 m t ÷ (GB-86) x er-u-37 spr-u-l-6-2 st-u-2-60 ac-17 nic-13 act ro t - (GB-412) in

w h i c h the four p h e n o t y p i c a l l y d i s t inc t ch lo rop las t mark-

ers can be d i s t ingu ished in all poss ible c o m b i n a t i o n s . Sager

a n d R a m a n i s ( 1 9 7 6 a ) r e p o r t e d for t he ch lo rop las t mark-

ers t h e y s tud ied t h a t w h e n alleles confe r r ing res is tance

to e r y t h r o m y c i n , s t r e p t o m y c i n a n d s p e c t i n o m y c i n

140 J.L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in Chlamydomonas

Table 1. Pedigree of a typical biparental zygote from the cross: nr-u.2-1 mt+ x er-u-37 spr-u-l-6-2 st-u-2-60 ac-17 nic-13 act rot-. The nr-u-2.1 marker is a phenotypicaUy distinct allele of spr-u-l-6-2 which is included in the cross only for the purpose of determining which progeny cells are heteroplasmic for that locus (see Materials and Methods)

Product Nuclear genotype Chloroplast genotype Division

Meiosis I Meiosis II Mitosis I

1 ac-17 nic-13 2 ac-17 nic-13 3 ac-17 + 4 ac-17 + 5 + nic-13 6 + nic-13 7 + + 8 + +

+ er/+ spr/+ sr/+ ] ]] 1 + er + + 2 act + spr sr ] 3 act + + + 4 + + + + ] ] 5 + + + + J 6 act er spr + ] ] 7 act er + sr J 8

Division Products

Meiosis,I Meiosis II

Mitosis I

Segregation pattern

er spr sr

1, 2, 3, 4, 5, 6, 7, 8 I I I 1, 2 & 3, 4 IIa I I 5, 6 & 7, 8 III IIa IIa 1 & 2 IIb IIa IIa 3 & 4 - III III 5&6 - - - 7 & 8 - III III

Type I: Both sister cells are heteroplasmic for the allelic pair

Type IIa:. One cell is heteroplasmic, the other homoplasmic for the maternal allele

Type IIb: One cell is heteroplasmic, the other homoplasmic for the paternal allele

Type III: One cell is homoplasmie for the maternal allele, the other homoplasmic for the paternal allele

Since Type llI segregation events give equal numbers of maternal and paternal segregants, maternal bias can be produced only by an excess of Type IIa events over Type lib events.

entered a cross f rom the maternal parent, a bias in favor of these maternal resistance alleles was seen (i.e. allele specific effects). To eliminate any possibility o f this being responsible for a maternal bias in our experiments, we constructed strains in which the resistance alleles of er-u-37, spr-u-l-6-2, and sr-u-2-60 all entered the cross from 'the paternal parent. This design also allowed us to detect any locus specific effects, i.e., any difference in the extent o f bias between markers at the three loci seen among the progeny. The very nature o f pedigree analysis minimizes the effects of -differential growth rates of specific genotypes during the divisions being examined. Any differential growth effects that occur during forma- tion o f colonies f rom individual zoospores will have little consequence for establishing qualitatively which chloroplast genotypes are present in the colony. Colonies heteroplasmic at the spr-u locus were detected by inclusion in the two parents o f the cross of nr-u-2-1 and spr-u4-6-2, which are phenotypically distinguishable mutations at the same locus (Harris et al., 1977). Colonies heteroplasmic at the er-u and sr-u loci were detected by respread- ing any colonies containing resistant cells.

The three unlinked nuclear markers carried by GB412 allow identification of the nuclear products o f each of the two meiotic divisions and the first post-meiotic, mitotic division. The nuclear products of meiosis I can

be identified as those having the same genotype with respect to ac-17 which is very closely linked to its own centromere. Tetratype tetrads for the unlinked act and nic-13 mutants are obtained with high frequency. In these tetrads, the cells produced by the first post-meiotic, mitotic division can be identified with respect to the second meiotic division products from which they arose. For example (Table 1) the two cells with the genotype ac-17 nic-13 act + are derived from one meiotic product whereas those with the genotype ac-17 nic-13 ÷ act are derived from a second meiotic product. Both of these products come from the same event at the first meiotic division since they both carry the ac-17 marker. Thus the nuclear genetic events at the first three post-zygotic divisions can be reconstructed. If one assumes that these events are accompanied by corresponding events involving partitioning or actual division o f the zygotic chloroplast, they can be used to indicate when segregation events involving maternal and paternal chloroplast alleles occur during the early divisions of the zygote. Since clear differences are observed in the behavior o f chloroplast markers when pedigrees are reconstructed according to the sequence o f nuclear divisions, we feel reasonably secure in concluding that segregation events are occurring in the chloroplast genomes in parallel to the nuclear genetic events.

J. L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in Chlamydomonas 141

Table 2. Fraction of homoplasmic progeny carrying the maternal chloroplast alleles at the er-u, spr-u and sr-u loci in zygote clone analysis as a function of UV dose given the maternal gametes prior to mating. All data are from crosses of nr-u-2-1 rat ÷ x er-u.37

spr-u-l-6-2 st-u-2-60 ac-17 nic-13 act ra t - , in which zygotes from a given mating were analyzed by both pedigree analysis (Table 3) and zygote clone analysis. Less than 1% of the zygote clone progeny are heteroplasmic for any marker at the time of analysis

UV dose % Exceptional Total progeny (s) zygotes analyzed

Fraction of homoplasmic progeny carrying the maternal allele

er-u spr-u sr-u Average for 3 loci

7.5 6.6 676 0.70 0.71 0.71 0.71 15 35.2 1,472 0.65 0.65 0.68 0.66 30 63.5 1,397 0.60 0.59 0.62 0.60 45 42.8 1,850 0.53 0.56 0.57 0.55

Fig. 2. Rates of segregation of chloroplast genes determined in pedigrees during the first three post- zygotic divisions in the crosses shown in Table 3. (A) Segregation rate as a function of dose of UV irradiation given the maternal gametes prior to mating. Data are averaged for the er-u, spr-u and sr-u markers. (B) Segregation rates of the three individual chloroplast markers. Data are averaged for the four UV doses

0,7

0,5

g 0,3 g

o

0,i

A Q

~- 30 SEC I-- 45 SEE

IB O

O-sr

I I 1 I I I

1 2 3 1 2 3 DIVISION

A maternal bias was observed for markers at all three chloroplast loci when these crosses, which were designed for pedigree analysis, were also subjected to zygote clone analysis (Table 2). This bias decreased with UV dose. These results are identical to those we have observed pre- viously with zygote clone analysis of reciprocal crosses involving these same three chloroplast markers (Giltham et al., 1974; Adams et al., 1976; Boynton et al., 1976). Pedigree analysis reveals that the three chloroplast markers studied in these crosses have already undergone a substantial amount of segregation by the end of the first meiotic division (Fig. 2, Table 3). Rapid segregation continues during the next two post-zygotic divisions and the majority of zoospore clones are homoplasmic for at least one of the three loci after the first mitotic division. Segregation appears to be delayed by increasing the UV dose from 7.5 to 15 s, but is not markedly affected further by higher UV doses (Fig. 2). The rate of segregation for all three markers-is approximately linear over the first three post-zygotic divisions and does not vary with UV dose (Fig. 2). However, the three markers differ with respect to the time at which they begin to segregate, with er-u-37 and spr-u-l-6-2 segregating early and st-u-2- 60 segregating later.

Cells homoplasmic for one or more loci show a very strong maternal bias for markers at all three chloroplast loci at all three post-zygotic divisions (Table 4A, B).

However the extent of the maternal bias decreases with successive divisions (Table 4A). This decrease must con- tinue, but at a reduced rate, during subsequent divisions, since the maternal bias seen in zygote clones (Table 2) is somewhat less than that seen after the third post-zygotic division (Table 4A, B). Thus chloroplast markers show a high degree of maternal bias among the segregated progeny during the early post-zygotic divisions when many of the cells are still heteroplasmic and a maternal bias continues to persist in zygote clone progeny where segregation is almost complete (cf. Harris et al., 1977). Increasing UV dose reduces the maternal bias seen at all three divisions (Table 4A). This same UV effect can be seen in zygote clones (Table 2).

So far our data on pedigree analysis have been presented without regard to cell lineage, but they already indicate that heteroplasmic cells segregate maternal chloroplast markers preferentially. One would not expect this result if there were only two copies of the chloroplast genome as proposed by Sager (of. Sager, 1972, 1977). Instead, our results imply that there are at least several copies of the chloroplast genome in heteroplasrnic cells, that these cells are biased in favor of the chloroplast genome derived from the maternal parent and that UV irradiation of the maternal parent prior to mating reduces this maternal bias. To provide a more direct test of this supposition, we determined the extent to which


Table 3. Transmission and segregation of chloroplast genes, analyzed over the first three post-zygotic divisions, of crosses of nr-u-2-1 mt÷ x er.u-37 spr-u-l-6-2 ~r-u-2-60 ac-17 nie-13 act ro t - , as a function of UV dose given the maternal gametes prior to mating

Table 4. Proportion of chloroplast alleles from the maternal parent among the homoplasmic progeny from the crosses shown in Table 3

A. Data averaged for markers at the three chloroplast loci analyzed.

A. Number of zygotes analyzed in pedigrees

UV dose:

Number of zygotes

7.5 s 15 s 30 s 45 s

maternal 402 215 77 163

paternal 0 1 1 0

biparental for 21 108 124 113 all 3 markers

biparental only for indicated markers er 0 0 1 0 sr 4 4 4 2 spr 0 1 1 0 er sr 2 0 3 2 er spr 0 1 0 0 sr spr 3 2 1 0

total 432 332 211 280

B. Number of biparental zygote progeny homoplasmic for at least one chloroplast marker at each of the first three post-zygotic divisions

Meiosis I

Number analyzed 58 203 188 190

Homoplasmic for at least one locus Number 23 39 37 28 % 40 19 20 15

UV dose (s)

Fraction of homoplasmic progeny carrying maternal chloroplast alleles at one or more of the three loci studied. (data averaged for the three loci)

at Meiosis I at Meiosis II at Mitosis I

7.5 1.0 1.0 0.95 15 0.95 0.93 0.88 30 0.83 0.74 0.69 45 0.73 0.76 0.69

B. Data presented for the three individual loci following Mitosis I

UVdose Number of (s) homoplasmic

progenyanm lyzed

Fraction of homoplasmic progeny carrying maternal chloroplast alleles at one or more of the three loci studied (data presented for the three individual loci)

er-u sr-u spr-u

7.5 225 0.96 0.92 0.97 15 788 0.90 0.82 0.92 30 740 0.72 0.64 0.70 45 720 0.70 0.66 0.72

Meiosis II



Mitosis I



zygotes originally heteroplasmic for all three chloroplast loci became homoplasmic for one, two or three loci at meiosis I and the extent to which triply heteroplasmic segregants became homoplasmic at the two subsequent

divisions. The pedigree data for each marker at every UV

dose and division are presented in Table 5 and are summarized in Tables 6 and 7. Progeny derived from triply heteroplasmic ceils are divided into four groups in Table 6 depending on whether they were homoplasmic for all three markers in maternal or paternal configuration, heteroplasmic for all three markers, or partial segregants, i.e. homoplasmic for one or two markers and heteroplasmic for the remaining marker(s). While most progeny cells derived from triply heteroplasmic cells are them- selves heteroplasmic for all three markers, there is a bias among the segregated progeny towards cells homoplasmic for the genotype of the maternal parent (Table 5). Al- though the maternal genotype predominates among parental genotypes segregated, a statistically significant increase in segregants with the paternal genotype occurs as a function of increasing UV dose (G 2 = 35.9, 2 df,

p ~ 0.001) and number of divisions (G 2 = 31.4, 3 df, p ~ 0.001). The total frequency of progeny segregated for one or two markers increases with successive divisions (G 2 = 42.2, 2 dr, p ~ 0.001). Partial segregants expressing paternal chloroplast alleles segregate later than those expressing maternal chloroplast alleles (Table 7). UV dose does not appear to have any consistent effect on the total frequency of the partial segregants or the ratio


Table 5. Numbers of progeny, derived from cells biparental for all three chloroplast markers, which become homoplasmic for maternal or paternal chloroplast alleles at the indicated division. Data are from the crosses shown in Table 3

UV dose Loci Meiosis I Meiosis II Mitosis I (s) homoplasmic

7.5

15

30

45

Maternal Paternal Maternal Paternal Maternal Paternal allele allele allele allele allele allele

er spr sr 9 0 8 0 17 3 er sr 0 0 1 0 0 3

er spr 3 0 3 0 2 0

spr sr 0 0 0 0 1 0

er 0 0 1 0 0 0 spr 1 0 1 1 2 1

sr 0 0 0 0 0 2

heteroplasmic 27 32 31 for all markers

total analyzed 40 47 62

er spr sr 12 0 43 3 45 17 ersr 1 0 0 1 0 7

er spr 12 0 14 0 28 1

spr sr 1 0 2 0 0 0

er 2 0 5 1 8 4

spr 1 0 1 1 3 0

sr 1 0 1 2 0 17



er spr sr 11 2 50 17 57 25 er sr 2 1 2 7 1 19 er spr 9 0 3 0 11 0

spr sr 2 2 3 1 3 2

er 5 0 4 4 7 10 spr 4 1 5 4 2 2

sr 0 1 1 5 1 18



er spr sr 15 7 39 15 45 42 er sr 0 0 0 0 0 2

er spr 8 1 12 0 35 1 spr sr 0 0 0 0 0 3

er 2 1 4 2 8 12 spr 4 0 2 0 4 3 sr 0 0 0 0 1 4



of partial segregants carrying maternal or paternal chloroplast alleles at any division.

The results summarized in Table 7 show that among the progeny of triply heteroplasmic cells, the maternal and paternal alleles at the three chloroplast loci have undergone very different segregation patterns by the end of the third post-zygotic division. All three chloroplast

alleles from the maternal parent most often segregate together irrespective of UV dose. Except at the highest UV dose, segregants homoplasmic for one or two of the three paternal alleles, which must arise by recombina- tional events, occur in about equal frequency with segregants homoplasmic for all three alleles from this parent. Striking differences are apparent in the frequencies with

144 I.L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in Chlamydomonas

Table6. Fraction of progeny arising at each division from triply heteroplasmic cells (Table 5), which have maternal or paternal genotypes, or are homoplasmic for one or two of the three loci

UV dose Chloroplast genotype Meiosis Meiosis Mitosis (s) I II I

7.5

15

30

45

Maternal parent

Paternal parent

Heteroplasmic for all markers

Homoplasmic for one or two of three loci, heteroplasmic for remainder

total analyzed

Maternal parent

Paternal parent



total analyzed

Maternal parent

Paternal parent


Homoplasmic for one or two of three loci, heteroplasmic for remainder

total analyzed

Maternal parent

Paternal parent



total analyzed

0.22 0.17 0.27

0 0 0.05

0.68 0.68 0.50

0.10 0.14 0.18

40 47 62

0.07 0.16 0.12

0 0.01 0.04

0.83 0.73 0.66

0.10 0.10 0.18

177 273 382

0.06 0.16 0.15

0 0.06 0.07

0.78 0.65 0.58

0.14 0.13 0.20

186 304 378

0.08 0.14 0.11

0.04 0.05 0.11

0.80 0.73 0.59

0.08 0.07 0.18

188 278 394

which cells have become homoplasmic for maternal and paternal alleles at individual loci singly or in various pairwise combinations. For example, cells most frequently become homoplasmic for maternal chloroplast alleles at the er-u and spr-u loci together and for paternal chloroplast alleles at the er-u and sr-u loci together. Cells frequently become homoplasmic for the paternal allele at the sr-u locus by itself, whereas only rarely do they become homoplasmic for the maternal allele at this locus by itself. In fact, maternal and paternal alleles at given

locus, or pair of loci, show similar segregation frequencies in only a few cases.

Using pedigree analysis, Sager and Ramanis (1968) have described three distinct types of segregation events possible for any given pair of alleles at any chloroplast locus upon division of a heteroplasmic cell to form two progeny cells: Type I: Both cells are heteroplasmic for the alleles of

the locus in question. Type IIa: One cell is heteroplasmic, the other homplas-

mic for the allele carried by the maternal par-

ent. Type IIb: One cell is heteroplasmic, the other homoplas-

mic for the allele carried by the paternal par-

ent. Type III: Both cells are homoplasmic for the alleles at

the locus in question.

These events can occur at any meiotic or mitotic division and may be of different types for different markers in the same heteroplasmic cell at the same division (Sager and Ramanis, 1970b). An excess of Type IIa events over Type IIb events will result in a maternal bias in both pedigrees and zygote clones. By definition neither Type I nor Type III events can cause a bias.

Sager and Ramanis have reported that all chloroplast markers examined show the same frequency of Type II segregations in zoospore clones (Table 2 of Sager and Ramanis, 1976b) and that the ratio of Type IIa to Type IIb events is 1 : 1. We have examined the proportions of the different types of segregation events which occur at each division in pedigrees from zygotes biparental for one or more of the three chloroplast loci (Tables 8, 9). An example of a pedigree generated by a zygote biparental for all 3 chloroplast loci which has been analyzed in this way is shown in Table 3. This analysis of events

contrasts to our analysis of the frequencies of progeny genotypes occurring at each generation in the same pedigrees (Tables 4-7) . The effect o f successive divisions on the segregation process is especially evident (Table 8). At the first meiotic division, Type I segregation is the most common event at all UV doses. Type IIa events segregating maternal alleles are also frequent, but Type lib and Type III events segregating paternal alleles are rare or non-existent. At the second meiotic division, the proportion of Type I events decreases and the proportion of Type IIa events increases. At the first mitotic division, the proportion of Type I events continues to decline, while Type IIa events stabilize and Type IIb events increase. Type III events are rare at every division. At each division, increasing UV dose reduces the ratio of Type IIa events relative to Type IIb events for the three markers and thus reduces the bias toward segregation of maternal chloroplast alleles (Table 9). The increase in the frequencies of both Type I and Type IIb events at the expense of Type IIa events as a function of UV dose

J. L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in C h l a m y d o m o n a s 145

Table 7. Distribution of progeny homoplasmic for one or more chloroplast alleles, arising from zygotes and zygote progeny which were heteroplasmie for all three markers. Data summarized from Table 5

7.5 s UV 15 s UV 30 s UV 45 s UV Summed for all UV doses

Maternal Paternal Maternal Paternal Maternal Paternal Maternal Paternal Maternal Paternal Combined allele allele allele allele allele allele allele allele allele allele

total progeny homoplasmic for at least one locus 49 10 180 53 183 121 179 93 591 277 869

proportion of these progeny with indicated loci homoplasmic er spr sr 0.69 0.30 0.56 0.38 0.64 0.36 0.55 0.69 0.59 0.47 0.55 e r s p r 0.16 0 0.30 0.02 0.13 0 0.31 0.02 0.24 0.01 0.16 er sr 0.02 0.30 0.01 0.15 0.03 0.22 0 0.02 0.01 0.16 0.05 spr sr 0.02 0 0.02 0 0.04 0.04 0 0.03 0.02 0.03 0.02 er 0.02 0 0.08 0.09 0.09 0.12 0.08 0.16 0.08 0.12 0.09 spr 0.08 0.20 0.03 0.02 0.06 0.06 0.03 0.05 0.05 0.05 0.05 sr 0 0.20 0.01 0.36 0.01 0.20 0.01 0.04 0.01 0.18 0.06

Table 8. Proportion of different segregation events occurring at each of the first three post-zygotic divisions as function of UV dose. Data averaged for all three chloroplast loci in the crosses shown in Table 3

UV dose Type I Type IIa Type IIb Type III Average number of (s) events scored at

given division

Meiosis I 7.5 0.55 0.45 0 0 24 15 0.66 0.33 0.01 0 87 30 0.71 0.24 0.05 0 83 45 0.72 0.20 0.09 0 90

avg 0.66 0.31 0.04 0

Meiosis II 7.5 0.45 0.55 0 0 32 15 0.50 0.47 0.03 0.004 155 30 0.41 0.41 0.16 0.01 139 45 0.50 0.38 0.12 0 147

avg 0.47 0.45 0.08 0.004

Mitosis I 7.5 0.20 0.66 0.10 0.05 44 15 0.43 0.40 0.14 0.03 216 30 0.35 0.38 0.21 0.06 185 45 0.36 0.35 0.26 0.03 201

avg 0.34 0.45 0.18 0.04

(Tables 8, 9) results in the increase in the transmission of

paternal chloroplast markers seen for pedigrees in Tables

4 and 6 as well as for zygote clones in Table 2. While there is clearly a strong bias towards Type IIa segrega-

tions for all three markers, we find that the total frequen-

cy of Type IIa + l ib events is approximately the same

for each marker (Table 9) as was also reported by Sager

and Ramanis (1976b). Furthermore, this frequency does

not change markedly as a function of division or UV dose.

Pedigree analysis also permits a comparison of the

pattern of segregation events in consecutive divisions.

Table 10 summarizes the types of segregation events

undergone by cells heteroplasmic for any of the three chloroplast markers as a function of the type of segrega-

tion event from which they arose. Thus, heteroplasmic

cells derived from Type I, Type IIa, or Type IIb events

at either meiotic division can be followed in terms of the

segregation pattern shown for each marker at the next

division. In this analysis heteroplasmic cells derived from

146 J .L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in C h l a m y d o m o n a s

Table 9. Frequency of Type IIa plus Type IIb segregation events for the three chloroplast markers in the crosses shown in Table 3. Average number of events scored in each division as in Table 8

UV dose Division (s)

Frequency of cells undergoing Type II segregation events

Percent of Type IIa events of total Type II events

er-u spr-u sr-u er-u spr-u sr-u

7.5 Meiosis I 0.52 0.68 0.45 100 100 100 Meiosis II 0.61 0.54 0.50 100 100 100 Mitosis I 0.74 0.84 0.68 84 88 90

15 Meiosis I 0.64 0.36 0.27 97 97 96 Meiosis II 0.54 0.52 0.43 94 95 93 Mitosis I 0.58 0.53 0.52 77 82 64



each UV dose are treated separately. A number of con- clusions regarding the behavior of the three chloroplast markers can be drawn from the data in Table 10 following analysis using G 2 statistics: 1) Increasing UV dose has a highly significant effect (p < 0.001) on the segregation pattern shown by heteroplasmic cells at both meiotic divisions; 2) When heteroplasmic cells are summed regardless of the meiotic division at which they arose, the type of segregation event yielding tile heteroplasmic cell (i.e. Type I, 2Ia or 12b) had a highly significant effect (p < 0.001) on the segregation pattern which the heteroplasmic cell showed at the next division regardless of UV dose; 3) Lrv dose had a highly significant effect (p < 0.002) on the relative frequencies of heteroplasmic cells arising from Type I, 2Ia, and IIb events at both meiotic divisions when the data are summed regardless of the subsequent segregation pattern shown by these cells at each meiotic division; 4) The three way interaction between UV dose, origin of the heteroplasrnic cell and subsequent segregation pattern was not significant for any marker in either meiosis I or II (p < 0.2 to < 1).

Heterogeneity G 2 tests of the data in Table 10 show, for all three markers, that heteroplasmic cells derived from Type I, IIa, and IIb segregations at either meiotic division differ significantly (p ~ 0.002 to <0.01)in their segregation pattern at the next division. Examination of the percent deviations from expected (Table 11) reveals that heteroplasmic cells from Type 2Ia events yield more than the expected number of Type 2Ia events and less than the expected number of Type 2Ib events at the next division. Likewise heteroplasmic cells derived from Type IIb events undergo more than the expected number of Type 2Ib events and less than the expected number of Type 2Ia events. Heteroplasmic cells derived from Type

IIa and 22b events show less marked deviations from the expected number of Type I events at the next division. Heteroplasmic cells arising from Type I segregation events deviate least from the expected pattern of Type I, IIa and 22b events.

Alleles at the three chloroplast loci we have studied show marked differences in the frequency with which they segregate together in the three possible pairwise parental combinations (cosegregation) during the first three post-zygotic divisions (Table 7). However, for two of the three possible locus pairs, the frequencies of segregants homoplasmic for maternal alleles are vastly different from the frequencies of segregants homoplasmic for paternal alleles. Cosegregants containing alleles at the e r - u and s p r - u loci in the homoplasmic condition constitute the rarest class of paternal cosegregants, but the most frequent class of maternal cosegregants. The converse is true for cosegregants homoplasmic for maternal or paternal alleles at the e r - u and s r - u loci. Both maternal and paternal cosegregants for the s p r - u and s r - u loci are rare.

Pairwise cosegregation frequencies of the maternal chloroplast alleles generate the map order e r - u - s p r - u -

s r - u , based on the assumption that markers closest together will show the highest cosegregation frequencies and markers farthest apart, the lowest cosegregation frequencies (Table 7). This map order is identical to that obtained by recombination analysis in zygote clones (Harris et al., 1977). On the other hand, the pairwise cosegregation frequencies of the paternal chloroplast alleles yield a different map order: e r - u - s r - u - s p r - u . Inspec- tion of the data in Table 5 reveals that the two map orders are supported by the relative frequencies of maternal and paternal cosegregants that occurred at each divi.

J. L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in C h l a r n y d o r n o n a s 147

Table 10. Segregation patterns shown by heteroplasmie cells following division, as a function of the origin of the heteroplasmic cell and the UV dose

UV dose (s)

Segre- Heteroplasmic cell arose at Meiosis I from Heteroplasmic cell arose at Meiosis II from

gation at sub- I IIa IIb I IIa

sequent divi- er sr spr er sr spr er sr spr er sr sp r er

sion

IIb

sr spr er sr spr

7.5 I 7 15 IIa 9 12 IIb 0 0 III 0 0 Number analyzed 16 27

15 I 48 89 IIa 60 51 lib 5 6 III 0 1

Number analyzed 113 147

30 I 43 47 IIa 39 50 IIb 30 21 III 3 2

Number aria, lyzed 115 120

45 I 49 73 IIa 51 54 IIb 20 17 III 0 0

Number analyzed 120 144

6 5 4 5 0 0 0 4 11 3 3 4 3 0 0 0 6 9 8 9 0 0 0 11 22 17 12 14 10 0 0 0 0 0 0 0 0 0 0 2 3 1 2 1 2 0 0 0 0 0 0 0 0 0 0 2 2 0 0 3 2 0 0 0

12 14 12 14 0 0 0 19 38 21 17 22 15 0 0 0

54 15 11 19 1 1 1 53 87 65 17 18 25 2 2 3 57 15 14 14 0 0 0 45 46 53 42 30 38 0 1 0

4 0 0 0 0 0 0 18 33 14 6 9 3 2 2 1 0 0 0 0 0 0 0 1 4 1 3 4 2 1 1 0

115 30 25 33 1 1 1 117 170 133 68 61 68 5 6 4

57 9 6 6 4 1 2 30 39 58 11 16 19 11 7 11 47 10 11 9 4 1 1 27 26 42 32 32 32 6 3 1 23 1 1 0 2 9 4 35 32 19 6 7 1 10 16 10

1 0 0 0 0 0 0 13 10 4 3 6 5 4 3 1

128 20 18 15 10 11 7 105 107 123 52 61 57 31 29 23

57 9 8 9 8 9 7 39 76 48 15 15 17 4 7 7 53 10 6 8 1 0 1 44 43 48 25 26 29 4 l 4 15 0 0 1 1 0 0 32 42 37 11 10 7 9 6 4

1 1 0 0 0 0 0 5 3 2 5 0 4 2 0 0

126 20 14 18 10 9 7 120 164 135 56 51 57 19 14 15

Table 11. Percent deviation from expected segregation frequency shown by heteroplasmic cells in the next generation. Data used in this analysis are summarized in Table 10. Data were summed with respect to UV dose since the three way interaction between UV dose, origin of heteroplasmic cells, and subsequent segregation pattern was not significant (see text). Percent deviation from expected

al bl segregation frequency was calculated for each marker individually as follows: expected number for a 1 = ~- x ~- x T, where al , a2, a 3

are the sums for Type I, Type IIa, and Type IIb segregation frequencies respectively, and b l , b2, b 3 are the sums for frequencies of origin of heteroplasmic cells from Type I, Type IIa and Type IIb events at the preceding division. T is the sum of a 1 to a 3 plus b 1 to b 3. The percent deviation from expected = [(observed - expected)/expected] x 100

Segregation pattern at subsequent division

Heteroplasmic cell arose at Meiosis I from

er sr

Heteroplasmic cell arose at Meiosis II from

IIa IIb I IIa

spr er sr spr er sr spr er sr sp r er sr

IIb

spr er sr sp r

I -4 +2 -2 +8 -16 +4 +45 +4 +33 +12 +13 +5 -24 -29 -16 +7 -13 +25 IIa -2 -3 0 +19 +43 +16 -47 -88 -71 -14 -16 -9 +40 +58 +35 -52 -69 -72 IIb +20 -2 +12 -91 -86 -87 +13 +317 +153 +10 +2 +12 -41 -37 -55 +88 +128 +134

148 J.L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in C h l a m y d o m o n a s

Table 12. Numbers of progeny homoplasmic for all three chloroplast markers in parental and recombinant configurations at the end of the third post-zygotic division. Percent recombination for each of the three intervals is calculated as (the number of progeny recombinant for that interval/total number of progeny) x 100, for the overall data

Genotype Number of progeny

7.5sUV 15sUV 30sUV 45sUV Overall

+ + + (maternal) 134 er spr sr

(paternal) 4 e r + + 0

+ spr sr 0

+ + s r 1

er spr + 0

+ spr + 0

er + sr 0

333 274 236 977

34 90 113 241 0 4 0 4 0 5 0 5

15 6 3 25 1 0 0 1 0 0 0 0 0 1 0 1

total 139 383

Map interval % recombination

er -u-spr -u O. 8

er-u - s r - u 2.8

sr -u-spr -u 2.2

380 352 1,254

sion and UV dose. If one ignores the foregoing differences and computes the total frequency of cosegregants for each marker pair averaged over all divisions and UV doses (Table 7), a third map order sr-u - e r -u - s p r - u

results, which is identical to the order reported by Sager and Ramartis (1976b) from their cosegregation analysis. Our calculations of cosegregation frequency differ from those of Sager and Ramanis in that they exclude from the numerator the contribution of the genotypes of the maternal and paternal parents, which obviously contain these marker pairs, and use the total products analyzed rather than the total number of doublings as the denom- inator. However, neither of these changes should alter the o r d e r arrived at for the three markers, since both will have similar effects on the cosegregation frequencies of all three marker pairs.

Recombination frequencies which can be used for mapping purposes can also be obtained for the three chloroplast markers among those zoospore progeny homoplasmic for all three markers at the end of the first post-meiotic mitotic division (Table 12). The map order obtained for the three chloroplast markers among the "-60% homoplasmic progeny (1254/2473) is identical to that observed in zygote clones by Harris et al. (1977) and to the order established by pairwise cosegregation of the maternal chloroplast alleles. Therefore the remaining 50% heteroplasmic zoospores must undergo similar frequencies of recombination for the markers in question during subsequent mitotic divisions, since zoospores

following mitosis I show the same map order and map distances reasonably comparable to those observed in zygote clones.

One can also ask what genotypes will be present in lineages derived from cells homoplasmic for only one or two of the three markers. This is a relevant question because it attempts to relate cosegregation events to the genotypes actually scored as recombinant or parental in zygote clone analysis. Obviously clones homoplasmic for the three maternal or three paternal chloroplast markers in parental configuration will yield cell lineages which are of the two parental types. However a cell homoplasmic in parental configuration for two markers and heteroplasmic for the third will yield a lineage containing cells of one parental genotype and cells of recombinant genotype for the heteroplasmic locus with respect to the two homoplasmic loci (Table 13). For example, a cosegregant homoplasmic for the maternal alleles at the er -u and s p r - u loci and heteroplasmic at the sr-u locus (i.e. er + s p r + s r + / e r ÷ s p r ÷ sr ) is recognized by the fact that among its progeny are cells of the parental genotype er ÷ s p r ÷ sr ÷ and the recombinant genotype er ÷ s p r ÷

sr. If further secondary recombination events take place within this clone, they will not be recognized because the same two genotypes will be produced. Similarly a cosegregant for the paternal alleles at the er -u and s p r - u

loci will be heteroplasmic at the sr-u locus (i.e. er s p r

s r / e r s p r sr ÷) and can be recognized by the fact that among its progeny are cells of the parental genotype er

s p r sr and the recombinant genotype er s p r sr ÷. Thus each of the two cell lineages yields only one type of recombinant. The recombinants produced by the two lineages are of reciprocal genotype, depending on whether the cosegregant was homoplasmic for the maternal or paternal pair of alleles. By similar reasoning, cells which become homoplasmic at the er-u and sr-u loci by cosegregation yield recombinants involving the s p r - u locus and cells which become homoplasmic for the s p r - u and sr-u

loci yield reeombinants involving the er -u locus. In each case only one recombinant is obtained per lineage and the recombinant obtained when two maternal alleles become homoplasmic is reciprocal to that found when two paternal alleles at the same pair of loci become homoplasmic.

Cells homoplasmic for alleles at one locus can, the- oretically, yield as many as four different genotypes among their progeny. For example, a cell homoplasmic for the maternal allele at the er -u locus will be heteroplasmic at the s p r - u and sr-u loci (i.e. er + s p r ÷ s r+/er ÷

s p r s t ) . Such a cell can produce progeny of the parental genotype er ÷ s p r + sr + as well as three of the six theoreti- cally possible recombinants for this cross (i.e. e r * s p r sr,

er ÷ s p r ÷ sr and er ÷ s p r sr+). None of these recombinants are reciprocal for all three markers. Similarly, cells homoplasmic for the paternal er -u allele can yield progeny

J. L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in C h l a m y d o m o n a s

Table 13. Possible segregants that can be produced in lineages derived from ceUs homoplasmic for one or more chloroplast markers as determined by pedigree analysis. M = maternal, P = paternal

149

Chloroplastalleles

Homoplasmic Heteroplasmic

Possible segregants

Parental genotype Recombinant genotypes

er spr sr none er + s p r + sr + (M) none er spr sr (P) none

er s p r sr er + s p r + sr + (M) + er + spr + sr

er spr sr (P) + er spr sr +

e r sr spr er + s p r + sr + (M) + er + spr sr +

e r s p r sr (P) + er spr + sr

s p r sr e r e r + s p r + sr + (M) + er spr + sr +

e r s p r sr (P) + er + s p r sr

er spr sr e r + s p r + sr + (M) + er + spr sr / e r + spr sr + / e r + s p r ÷ sr

e r s p r sr (P) + er spr + sr + / e r s p r + sr / e r s p r sr +

spr er sr e r + s p r + sr + (M) + er spr + sr / e r spr + sr + / e r + s p r + sr

e r s p r sr (P) + er + s p r sr + / e r + s p r sr / e r s p r sr +

sr spr e r e r + s p r + sr + (M) + er spr sr + / e r + s p r sr + / e r spr+ sr +

e r s p r sr (P) + er + s p r + sr / e r s p r + sr / e r + s p r sr

Table 14. Pattern of segregation at the subsequent division of progeny arising at Meiosis I and Meiosis II which are heteroplasmic for one or two of the three chloroplast markers segregating in the cross. M = homoplasmic for any maternal allele, P = homoplasmic for any paternal allele and H = heteroplasmic for any allele. Data are pooled for all UV doses and for all three markers. A product designated MHH can be heteroplasmic for any two of the three markers and is homoplasmic for the maternal allele of the other marker

Nature of heteroplasmic cell

Progeny segregated by heteroplasmic cell

Type Number % of Total progeny

MHH MHH + MMM 39 64 MHH + MHH 1 2 MHH + MMH 17 28 MHM + MMH 2 3 MMM + MPH 2 3

PHH PHH + PPP 12 55 PHH + PHH 4 18 PHH + PPH 3 14 PPH + PMM 1 5 PHH + PMM 1 5 PPP + PMM 1 5

MMH MMH + MMM 115 77 MMH + MMH 27 18 MMP + MMM 8 5

PPH PPH + PPP 8 73 PPH + PPH 3 27

MPH MPH + MPH 1 -

of the paternal parental genotype and of the other three possible recombinants, which will be reciprocal to those generated by the cells homoplasmic for the maternal e r - u allele.

Discussion

When the behavior of chloroplast genes in crosses is studied by zygote clone analysis, relatively few progeny are sampled from each biparental zygote colony after many divisions, when almost all of the cells have become homoplasmic for the chloroplast genes they carry (Fig. 1).

Because many biparental zygotes can be analyzed by this method at a t ime when heteroplasmic cells are very rare, zygote clone analysis is ideal for measuring output bias o f specific chloroplast alleles (cf. Gillham et al., 1974) and for mapping chloroplast genes using conven- tional recombinat ion analysis (cf. Harris et al., 1977). However, zygote clone analysis is subject to the problem of differential growth rates o f certain genotypes (Van- Winkle-Swift, 1976). Furthermore, one cannot measure the rates or patterns of chloroplast gene segregation in zygote clones. To assess these, one must resort to pedigree analysis, which is possible in C h l a m y d o r n o n a s during the first three or four post-zygotic divisions (Fig. 1, Ta- ble 1). However, since many cellS are still heteroplasmic for the chloroplast genes that they carry at the end of

this t ime, pedigrees do not constitute the most satisfac- tory method for analyzing recombinat ion frequencies. Nonetheless, Sager and Ramanis (cf. 1976b; Singer et al., 1976) have developed two novel methods for mapping in

150 J .L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in Chlamydomonas

pedigrees based on segregation patterns seen for individual chloroplast genes and for groups of chloroplast genes.

The impetus for the experiments reported here came from the disagreement between the observation by Sager and Ramanis (1965, 1968) that chloroplast alleles segregated 1 : 1 both in pedigrees and among the progeny of zygote clones (Sager and Ramanis, 1976a), and our own ob- servations that segregants homoplasmic for maternal chloroplast alleles predominated among the progeny of zygote clones (GilLham et al., 1974) and among the individual meiotic products (Boynton et al., 1976). Whereas we observed that the extent of maternal bias in zygote clones was reduced by pretreatment of the maternal gametes with increasing UV doses, Sager and Ramanis (1976a) reported no such effect of UV on the 1:1 allelic ratio for chloroplast genes which they observed. In this paper we demonstrate that, using our stocks, conditions of gametogenesis, and mating regimes, maternal chloroplast alleles predominate during each of the first three post- zygotic divisions, and also in zygote clones analyzed after many divisions. In both zygote clones and pedigrees, this maternal bias is reduced by increasing UV dose administered to the maternal parent prior to mating. We assume that the behavior of the three closely linked chloroplast markers used in the experiments is representative of the chloroplast genome as a whole, but have no way of substantiating this at present.

The observation of Sager and Rarnanis (1968, 1976a, b; Singer et al., 1976) that chloroplast genes segregate rapidly during the first three post-zygotic divisions has been confirmed in our pedigree analysis. More impor- tantly, we have found that heteroplasmic cells differ from one another in terms of their propensity to segregate maternal or paternal chloroplast alleles. Heteroplas- mic cells derived from Type IIa segregations are more likely than expected to undergo a Type IIa segregation at the next division, and less likely to undergo a Type IIb segregation (Tables 10 and 11). The converse is true for heteroplasmic cells derived from Type IIb events. Thus the origin of a heteroplasmic cell determines the type of segregation it will undergo at the next division.

The results we have just summarized can be related to the existing models of chloroplast gene segregation in C. reinhardtii. Since these models have been discussed in detail in the recent review by Birky (1978), they will be restated only briefly here. The diploid model of Sager and Ramanis (cf. Sager, 1977) assumes that the chloroplast markers being studied genetically are present in two copies of the chloroplast genome. This model con- flicts with the physical evidence discussed earlier that the chloroplast genome appears to be present in many copies in C. reinhardtii. According to Sager's model, a biparental zygote contains one copy of the chloroplast genome from the maternal parent and one from the paternal parent. Each genome replicates prior to meiosis

and two copies are segregated to each daughter cell by an equational (mitotic) mechanism involving a centromere-like attachment point. Therefore, a cell will remain heteroptasmic for a given marker unless recombination occurs .

In Sager's model most recombination is thought to result from a nonreciprocal (gene conversion) mechanism yielding a Type II segregation in which one daughter cell is homoplasmic for the marker in question and the second remains heteroplasmic for that marker. Whether a Type IIa or Type lib segregation event occurs depends on whether a paternal allele is converted to a maternal allele or the converse. Cosegregation of two or more markers results when these markers have been co- converted in the same direction in a given cell prior to division. By this model, a ceil in our crosses would segregate the parental genotypes, i.e. become homoplasmic for the er-u, spr-u, and sr-u markers, at a given division as a result of co-conversion for all three markers in the same direction. Type III segregations would result when reciprocal recombination occurs for a marker pair. Be- cause of the equational mode of segregation of chloroplast genomes, genes furthest from the attachment point will show the highest frequency of Type III segregations and those closest to the attachment point, the lowest. Thus markers can be ordered by the frequency of Type III segregations.

Unless paternal chloroplast alleles are preferentially converted to maternal alleles, the bias in marker output we see in zygote clones and pedigrees cannot be accounted for by this model. To explain the effect of UV in de- creasing maternal bias, such preferential conversion would have to depend on the UV dose administered to the maternal gametes prior to mating. The propensity of heteroplasmic cells derived from Type IIa segregations to segregate maternal markers by the same mechanism at the next division is Consistent with a preferential gene conversion mechanism whereby paternal chloroplast alleles are converted to their maternal counterparts. To explain the strong tendency of heteroplasmic cells derived from Type IIb segregations to yield paternal markers as a result of Type IIb segregations at the next division, one would have to assume that gene conversion in this case is preferentially maternal to paternal. Assum- ing multiple copies of the chloroplast genome (see below) instead of two copies, an initial bias in either the maternal or paternal direction in a heteroplasmic cell could be amplified by gene conversion during subsequent rounds of segregation.

A multicopy model was proposed by Gillham et al. (1974) who felt that the chloroplast markers studied genetically were most logically located in the ca. 75 copies of chloroplast DNA known to exist in the single chloroplast of C. reinhardtii (of. Gillham, 1978). Mater- hal bias among the progeny of biparental zygotes seen in

J. L. Forster et al.: Behavior of Chloroplast Genes during Zygotic Divisions in Chlarnydomonas 151

zygote clone analysis was assumed to result from a bias towards maternal chloroplast genomes in the original zygote. UV irradiation of the maternal parent prior to mating was thought to inactivate maternal chloroplast genomes, thus facilitating the survival and transmission of paternal chloroplast genomes to the meiotic progeny. This model assumed that there were a fixed number of attachment sites in the zygotic chloroplast required for replication and segregation of chloroplast genomes, and that under normal cirumstances these sites were preferentially occupied by maternal chloroplast genomes. Segregation was thought to occur by the equational mechanism proposed by Sager and Ramanis (cf. Sager, 1972). The precise number of copies of chloroplast genomes per gamete or zygote was assumed to be variable.

Both Type II and Type III segregations were explained in terms of reciprocal recombination, with Type II segregations occurring as a result of recombination in cells with more than two copies and Type III segregations taking place in cells with only two copies. Clearly, the assumption of reciprocal recombination to explain Type II segregation in the original model is incorrect since adjacent markers can undergo Type II and Type III segregations respectively in the same cell at the same division (Sager and Ramanis, 1970b). Evidence showing that recombination of chloroplast genes is nonreciprocal is also to be found in the data published by Gillham (1965) for meiotic zygotes and those of VanWinkle- Swift and Birky (1978) for mitotic zygotes. The original multicopy model also does not adequately explain why chloroplast gene segregation rates are so rapid.

Recently, the multicopy mode/has been modified by grouping the many copies of the chloroplast genome into multicopy transmissional units or nucleoids (Van- Winkle-Swift, 1976, 1978, 1980; Adams, 1978). The VanWinlde-Swift model postulates that the nucleoids often remain spatially separated and segregate by a reductional mechanism when the chloroplast divides. In the fused zygotic chloroplast, maternal and paternal nucleoids would segregate only maternal and paternal chloroplast genotypes by reductional division unless the maternal and paternal nucleoids come in contact and there is recombination between paternal and maternal chloroplast genomes or transfer of whole chloroplast genomes between nucleoids. This nucleoid segregation model has the great virtue of resolving the paradox of many physical copies and few genetic copies of the chloroplast genome. Furthermore, the model is flexible enough to accommodate the presence or absence of a maternal bias as well as the effect of UV on diminishing the degree of bias. The number of nucleoids per plastid and the number of chloroplast genomes per nucleoid may be variable and may depend on such parameters as conditions of gametogenesis and mating (Sears et al.,

1978, 1980), length of zygote maturation before germi- nation (Sears et al., 1977; Sears, 1979), and pretreat- ments of the maternal parent with agents that perturb the amount of chloroplast DNA and extent of maternal inheritance (cf. Wurtz et al., 1977).

A fundamental difference between the equational and reductional models of chloroplast gene segregation lies in how one interprets the events leading to the production of cells homoplasmic for one or more chloroplast markers. Sager's equational model supposes that a cell containing a paternal and a maternal chloroplast genome will remain heteroplasmic for all markers unless recombination occurs. Recombination , whether nonreciprocal or reciprocal, results in homoplasmicity for one or more markers. In contrast, the model of VanWinkle-Swift (1978, 1980), which involves reductional segregation of nucleoids, assumes that if spatial separation of maternal and paternal nucleoids is maintained in the fused chloroplast of the biparental zygote, mixing of alleles from the two parents will be minimized and rapid segregation will occur without recombination. Assuming minimal mixing of nucleoids and depending on the ratio of maternal to paternal nucleoids, this model predicts that cells heteroplasmic for the three markers in our crosses should yield a high frequency of Type III segregations for all three marker pairs, thus generating the two parental genotypes in equal frequency. Instead we see the maternal genotype appearing in high frequency as the result of Type IIa segregation for all three markers during the early divisions, while the paternal genotype segregates out by Type lib events only at later divisions and high UV doses. Type III segregations of any of the three markers are rare.

When the extent of mixing of separate maternal and paternal nucleoids is maximized and only rare recombi- national events are postulated to occur between genomes of separate nucleoids, reductional division will remit in a high frequency of Type I and Type II segregations for all markers in parental configuration (VanWinkle-Swift, 1980). Both the frequency of Type III events and the rate of segregation will be minimized by this mixing. When maternal nucleoids predominate in the chloroplast of the biparental zygote, the majority of the segregation events will be of Type IIa, whereas in the rare cases when the paternal nucleoids predominate, mostly Type IIb segregation will occur.

The recombinant genotypes we observe during the first three post-zygotic divisions and later in zygote clones can be accounted for by VanWinkle-Swift's assumption that certain chloroplast genomes of the maternal and paternal nucleoids recombine at a low frequency to produce one or more heteroptasmic nucleoids. However, such a heteroplasmic nucleoid would be perpetuated indefinitely if its constituent genomes did not segregate from one another or undergo non-recip-


rocal recombination to produce homoplasmic progeny. Without such intranucleoid segregation or non-reciprocal recombination, cells containing several maternal nucleoids together with a heteroplasmic nucleoid will initially undergo Type IIa segregations. Eventually the heteroplasmic cells produced by these Type IIa events will contain all heteroplasmic nucleoids and therefore will subsequently undergo only Type I segregation. Hence, if the nucleoid-reductional segregation model is to explain the types of progeny we observe in pedigree and zygote clone analysis, one must assume that there is extensive mixing of parental nucleoids to minimize the Type III segregations, and that there is a certain amount of intranucleoid segregation or non-reciprocal recombination within heteroplasmic nucleoids to prevent the establish- ment of perpetual heteroplasmic cells as suggested by VanWinkle-Swift (1980).

In the three factor cross analyzed here, cells homoplasmic for a maternally derived chloroplast allele (M) and heteroplasmic (H) for alleles of the two other markers (i.e. MHH) most often yield one cell of similar phenotype (MHH) and one of the phenotype of the maternal parent (MMM) at the next division (Table 14). Conversely, cells homoplasmic for a paternal chloroplast allele (P) and heteroplasmic for the other two markers (i.e. PHH) most often produce one cell of the same phenotype (PHH) and one of the phenotype of the paternal parent (PPP). Cells homoplasmic for two maternal chloroplast markers (MMH) usually yield progeny cells of MMH and MMM phenotypes at the next division, while most of those homoplasmic for two paternal chloroplast markers (PPH) produce cells of PPH and PPP phenotypes.

These results are consistent with the reductional model insofar as it predicts a high incidence of Type II segregations, and are not predicted by the equational model unless highly directed gene conversion occurs. For example, by the equational model, a ceil of the phenotype MHH should produce two daughter cells of the same phenotype at the next division. To obtain a cell of the MMM phenotype the two heteroplasmic markers would have to be co-converted simultaneously to maternal genotype prior to the next division. A cell of the phenotype PHH should produce two daughter cells of the phenotype PHH unless co-conversion of the "two heteroplasmic markers to paternal phenotype occurs. In short, directed gene conversion must be postulated in both cases with the direction being different in each case.

The polarity of Type III segregations reported by Sager and Ramanis (1970a, 1976b) and studied more extensively by Singer et al. (1976) using the liquid segregation method can be readily explained by assuming that the markers close to the attachment point recombine least frequently and therefore segregate most rapidly

(VanWinkle-Swift, 1980). This relationship between map position and segregation rate is the reverse of that predicted by the equational model of Sager and Ramanis. Whether polarity is in fact observed may depend on the arrangement of parental genomes within the nucleoid, as discussed by VanWinkle-Swift (1980). Since no clearcut polarity in segregation rates was seen for the three markers used in the experiments reported here, our data cannot be used in any case to establish a map order with respect to a hypothetical attachment point.

Finally we come to the question of how map orders obtained by frequency of cosegregation and by frequency of recombination relate to one another. In theory, the map order obtained for pairs of markers by cosegregation and by recombination analysis should be the same. If the order of three markers is a-b-c, then the least frequent cosegregant class should be the one carrying alleles of the a and c markers in parental combination and the most frequent recombinant class should involve the a and c markers. Unfortunately this relationship is not supported by the cosegregation data from our three factor cross for reasons we do not understand. Two different map orders are obtained depending on whether cosegregants homoplasmic for maternal or paternal chloroplast alleles are considered. A priori one would expect the two map orders to be the same. The third possible map order, spr-er-sr, is observed if one combines the two sets of cosegregation data and this is the same as that reported by Sager and Ramanis (1976b) for the total frequencies of cosegregation observed. The map order er-spr-sr established by recombination analysis in both zygote clones (cf. Harris et al., 1977) and in cells homoplasmic for all three markers at the end of the third post-zygotic division (Table 12) agrees with the order established by the frequencies of pairwise cosegregants homoplasmic for the maternal alleles of the three markers. The agreement between the map order based on recombination frequencies of the three markers seen in zygote clones and in pedigrees implies that differential growth of specific genotypes in the zygote clones does not yield an erroneous map order. At least for the three closely linked chloroplast markers studied here, we feel that map order obtained by recombination analysis is far more reliable than that established by cosegregation analysis.

Acknowledgements. We would like to thank Drs. Janis Antonovics and Thomas Meagher for the statistical analysis of multiway contingency tables. Drs. Barbara Sears and Karen VanWinkle-Swift have been the source for many illuminating discussions. Spectin- omycin and neamine were the gift of Dr. George Whitfield of the Upjohn Pharmaceutical Company. This work was supported by National Institutes of Health Grant GM-19427, and by Postdoc- toral Fellowship GM-05411 to J. L. F.


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Communicated by F. Kaudewitz

Received July 11, 1979

behavior of chloroplast genes during the early zygotic divisions of chlamydomonas reinhardtii

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