Current Genetics 2, 1 8(1980)
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© by Springer-Verlag 1980
Changes in Chloroplast Genome Composition and Recombination during the Maturation of Zygospores of Chlamydomonas reinhardtii Barbara B. Sears*
Department of Botany, Duke University,Durham, North Carolina 27706, USA.
Summary. In crosses of the unicellular green alga Chlarnydomonas reinhardtii, the chloroplast genes are normally transmitted exclusively by the maternal parent to zygospore progeny. However, transmission of the paternal chloroplast alleles can be increased markedly by certain pretreatments of the maternal parent prior to mating. As zygospores age prior to induction of meiosis, they display decreased biparental transmission of chloroplast alleles and increased transmission of chloroplast alleles from only the maternal or paternal parent. In this report, chloroplast genome composition of biparental zygospores is shown to change in several ways during zygospore maturation. Allelic ratios of chloroplast genes within biparental zygospore clones become maternally or paternally skewed as the zygospores age, cotransmission of chloroplast alleles is reduced, and recombination increases, resulting in an expansion of genetic map distances between chloroplast markers used in this cross. The recovery of unequal frequencies of zygospore progeny expressing reciprocal recombinant genotypes confirms and extends other reports of the predominance of nonreciprocal recombination in organelle genetic systems. Key words: Chlamydomonas genetics.
Chloroplast - Organelle
Introduction
In most zygospores derived from crosses of Chlarnyclomonas reinhardtff, chloroplast genes are transmitted exclusively from the mating type plus (rot +) or maternal Current address: BotanischesInstitut, Universit~itDiisseldorf, Universitfitsstrasse 1, D-4000 Diisseldorf,FederalP,epublic of Germany
parent to zygospore progeny (reviewed by Gillham, 1978). Spontaneous transmission of chloroplast alleles from both parents (biparental transmission) or exclusively from the mating type minus (rnt-) or paternal parent occurs rarely, but the frequencies can be enhanced markedly by UV-irradiation of the mt+ gametes prior to mating (Sager and Ramanis, 1967). Although biparental zygospores of C. reinhardtii transmit chloroplast genes from both parents, the relative frequencies of maternal and paternal markers among progeny of biparental zygospores have been disputed. Sager and Ramanis have reported that maternal and paternal chloroplast alleles segregate to progeny of biparental zygospores in a 1 : 1 ratio during the first four postzygotic divisions (Sager and Ramanis, 1976) and Sager has therefore proposed that the chloroplast of Chlamydornonas is genetically diploid (Sager 1977a; 1977b). Sampling the progeny from biparental zygospore clones, Sager and Ramanis (1976) have also found that most chloroplast loci are represented by approximately equal frequencies of the maternal and paternal alleles, with some deviations appearing to be marker specific. In contrast, Gillham, Boynton, and colleagues have reported that when biparental zygospore colonies are subcloned, the progeny express predominantly the chloroplast alleles of the rnt + parent, although this maternal skew may be reduced by increasing the UV exposure of the mt+ gametes prior to mating (Gillham et al., 1974; Adams, 1978). Pedigree studies following the same markers during the first three postzygotic dMsions have affirmed the predominance of maternal chloroplast markers among meiotic products (Gillham, 1963; Boynton et al., 1976; Forster et al., 1980). Other analyses of biparental zygospore progeny have dealt with recombination and mapping (Gillham, 1965; Boynton et al., 1976; Singer et al., 1976;Harris et al., 1977) and cotransmission of chloroplast allele s (Adams et al., 1976; Birky, 0172-8083/80/0002/0001/S 01.60
B. B. Sears: Chloroplast Genome Composition C. W. Jr., VanWinkle-Swift, K. P., Sears, B. B., Boynton, J. E., Glllham, N. W. in preparation). When Chlamydomonas zygospores are sampled during an e x t e n d e d period o f zygospore i n c u b a t i o n or "maturat i o n " , changes in the inheritance pattern o f chloroplast alleles occur (Sears, 1980). Biparental transmission o f chloroplast alleles decreases, and exclusive transmission o f maternal or paternal chloroplast genes to zygospore progeny increases. Since the disappearance o f the biparental zygospore class occurs w i t h o u t a decrease in zygospore viability, heteroplasmic zygospores m u s t be losing chloroplast markers f r o m one or the o t h e r parent in the absence o f cell division. A l t h o u g h the loss o f the heteroplasmic state could involve a one step change, elim i n a t i o n o f the heteroplasmic state might also result f r o m gradual changes in chloroplast alMic c o n t e n t occurring within biparental zygospores during maturation. The experiments r e p o r t e d here were designed to determine w h e t h e r the duration o f zygospore m a t u r a t i o n affects r e c o m b i n a t i o n , cotransmission and the ratio o f maternal and paternal chloroplast alleles within biparental zygospores. My results show that among the progeny o f biparental zygospores obtained following U V irradiation o f the m t + parent, a11elic ratios b e c o m e m o r e uniparentally skewed and cotransmission o f chloroplast genes is reduced during an e x t e n d e d period o f zygospore m a t u ration. In addition, I demonstrate that r e c o m b i n a t i o n continues to occur b e t w e e n chloroplast genomes as zygospores age, resulting in an expansion o f genetic map distances b e t w e e n chloroplast markers. As previously d e m o n s t r a t e d by Gillham (1965) and VanWinkle-Swift and Birky (1978), r e c o m b i n a t i o n b e t w e e n chloroplast genomes was f o u n d to be primarily nonreciprocal.
Materials and Methods Strains, Media, Growth Conditions and Crosses. A single cross between mt + and m t - strains of Chlamydomonas reinhardtii respectively designated GB-104 and GB-198 provided the zygospore population which was analyzed in this investigation. GB-104 carries spr-u-l-27-3 (Spr), a chloroplast gene mutation which confers spectinomycin resistance (Harris et al., 1977); GB-198 contains two chloroplast gene mutations, st-u-2-60 (S r) and er-u-37 (Er), which confer resistance to streptomycin and erythromycin respectively. HSA medium has been described by Sueoka et al. (1967). Stocks were grown for 7 days on YA medium (HSA medium solidified with 15 g/1 agar and containing 4 g/1 Difco yeast extract) under 75 85 microeinsteins/m2s of photosynthetically active radiation (PAR) from cool white fluorescent light fixtures at 25-28 °C. Gametic differentiation was induced by suspending the cells in HSA medium lacking both NH4C1 and acetate ( - N medium) and by placing the suspensions on a shaker under high light intensity (about 200 uE/m2s PAR) for 5 hours. The cell density of the mt + gametes was adjusted to 5 x 106/ml, and the suspension was exposed to about 1.1 x 104 ergs/cm2s irradiation from a G8T5 germicidal lamp at a fixed distance for 30 s, while being stirred continuously, as described by Gillham et al. (1974).
The mt + gametes were then added to a dense suspension of m r gametes and they were allowed to mate for 2 h in the dark. 0.5 ml aliquots of the mating suspension were spread on plates of - N medium solidified with 4% Difco agar and left in the light for about 18 h before beingmoved to a dark incubator at 24 °C. To prevent dehydration of the agar, the zygospore maturation plates were wrapped in aluminum foil. Genetic Analysis. Zygospores were sampled from the maturation plates 2, 7, 15, and 23 clays after mating. To remove unmated cells, the agar surface of each maturation plate was scraped with a sterile razor blade. The mature zygospores have thickened rough cell wails and cling to the 4% agar, while the vegetative cells are easily removed by this technique. Zygospores were transferred to 1.5% Difco agar HSA plates and individually manipulated into 12 x 12 matrices under a dissecting microscope. These HSA plates were place d under continuous illumination (75 - 85 uE/m 2 s PAR) to speed zygospore germination and colony growth. After 4 - 6 days, zygospore viability was determined, and the zygospore clones were transferred to fresh HSA plates. These plates were used as masters for replica-plating to selective media containing spectinomycin, erythromycin, or streptomycin as described in detail by Harris et al. (1977). The initial replica plating allowed identification of biparental zygospores for further analysis. From the 2, !5 and 23 day old zygospore samptes, at least 100 biparental zygospore colonies scored as resistant on all three antibiotic plates were suspended individually in 8 ml - N medium. A loopful of each cell suspension was transferred to a 0.1 ml drop of liquid (H20 or - N medium) on a fresh HSA plate and spread. After about 6 days, 64 subclones (if available) from each biparental zygospore were transferred to fresh HSA plates and these plates were used as masters for a second round of replica-plating. Colonies resistant to two or three antibiotics in non-parental configurations were streaked from nonselective plates onto medium containing the appropriate combination of antibiotics. Although most chloroplast allele segregation has been completed by this stage (Harris et al., 1977), the multiple antibiotic plates allow unambiguous discrimination between clones arising from a heteroplasmon and those derived from a true recombinant cell. Computations and Statistieal Analysis. All biparental zygospore colonies producing 20 or more distinct clones following suspension and replating provided data used for determination of recombination frequencies and mapping (Figs 2, 3,4). Only those biparental zygospores yielding 50 64 subclones were used in determining the frequency of recombinants per biparental zygospore (Tables 2, 3, 4, Fig. 1). Statistical comparisons utilized two-way or multiway contingency table analysis with the log likelihood test described by Sokal and Rohlf (1969). For measuring the extent of cotransmission of chloroplast alleles, arcsine transformation of the square roots of the frequencies was necessary because the points do not represent a normal distribution. The nonparametric Spearman rank correlation coefficient was used to determine whether two recombinant genotypes are correlated in their occurrence within individual biparental zygospore clones. This analysis has been described in detail by VanWinkle-Swift and Birky (1978).
Results Changes in the Pattern o f Chloroplast Gene Inheritance as a Function o f Time. Zygospores derived f r o m a cross involving UV-irradiated mt+ gametes were sampled 2, 7, 15, and 23 days after mating (Table 1). The decrease in
B. B. Sears: Chloroplast Genome Composition Table 1. The inheritance pattern of chloroplast genes as a function of time in the cross GB-104 x GB-198, The mt+ gametes were irradiated with UV for 30 s prior to mating. Zygospores sampled 2, 7, 15, and 23 days after mating were induced to germinate by transfer to HSA Difco agar medium in the light. Viability of individual zygospores was determined by their ability to form a colony of 16 or more cells 4 - 6 days after the induction of germination. A minimum sample size of 288 was used for the determination of zygospore viability Age of zygospores
% Viability
% Maternal
% Biparental
% Paternal
Zygospore sample size
2 days 7 days 15 days 23 days
38.9 53.8 90.4 57.1
7.9 14.6 45.3 65.5
80.9 83.1 41.3 16.2
11.2 2.3 13.4 18.3
215 957 1,010 1,179
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Fig. 1. Cotransmission of chloroplast alleles as a function of zygospore age. Zygospores were sampled and induced to germinate 2, 15, and 23 days after mating. Following the initial replica-plating and scoring of zygospore clones, biparental zygospore colonies were subcloned and subjected to a second round of replica-plating. The frequency of the paternal er-u-37 (E r) allele is plotted as a function of the frequency of the paternal sr.u-2-60 (Sr) allele within each biparental zygospore clone, for the three time points
biparental transmission of chloroplast alleles and the increase in maternal and paternal transmission of chloroplast alleles seen here agrees with previous reports (Sears et al., 1977; Sears, 1979; 1980). Because the frequency of biparental zygospores had not changed markedly between 2 and 7 days, only biparental zygospore clones from the 2, 15, and 23 day sample points were subcloned and analyzed further. Cotransmission o f Chloroplast Alleles as a Function o f Zygospore Age. When the transmission of one chloroplast marker is plotted against the transmission of the second marker to the progeny of biparental zygospores, perfect coordinate transmission of the two markers from one parent will produce a line with a slope of 1.0 and described by a correlation coefficient (r) equal to 1.0 (Adams et al., 1976). A reduction in the coordinate transmission of the two alleles results in a reduction of the correlation coefficient. Negatively correlated trans-
mission would be described by correlation coefficients approaching - 1 and would produce aline with a negative slope. Cotransmission of the paternal er-u-3 7 and sr-u-2-60 alleles in 2 and 15 day old biparental zygospore clones closely matches the theoretical line (Fig. 1); the points have a correlation coefficient of r(2 days) = 0.989 and r(15 days) = 0.974. However, among the older zygospores, cotransmission is reduced; the points become more scattered; and the correlation coefficient drops somewhat (ro3 days) = 0.918). Tests for homogeneity among these correlation coefficients demonstrate that they differ significantly (X 2 = 18.2, 2d.f., p < 0.001). Allelic Ratio as a Function o f Time. The frequency of cells expressing paternal chloroplast alleles within biparental zygospore clones varies with UV dose (Gillham et al., 1974; Adams, 1978; Forster et al., 1980). In addition, Sager and Ramanis (1976) have reported that the frequency varies with map position of the alleles under
4
B.B. Sears: Chloroplast Genome Composition
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consideration. For three closely linked chloroplast loci, I have found changes in paternal alMic ratios as zygospores age (Fig. 2). Analysis of the data in Fig. 2 with 3 x 5 contingency tables using the log likelihood test shows that paternal allelic ratios among biparental zygospore progeny change significantly during zygospore maturation: for the er-u-37 locus, G = 20.0; for the sr-u-2-60 locus, G = 26.1; for the spr-u-l-2 7-3 locus, G = 20.9 (with 8 degrees of freedom, 0.001 < p < 0.025). Although the mean paternal aUelic ratio is approximately the same for all three samples, the distribution becomes increasingly biased as the zygospores age, resulting in a U- or L-shaped distribution, with most biparental zygospore clones having either high or low frequencies of paternal chloroplast alleles. As a consequence, the variance increases as the biparental zygospores age (e.g., for the transformed values of the er-u-37 locus, 2 days: a z = 388; 15 days: a 2 = 514; 23 days: ~2 = 697). No significant differences were found when comparisons were made between the allelic ratio distributions for each of the three markers at each time point (for the 2, 15, and 23 day samples, G = 0.8, 1.0, 4.1, with 8d.f., p > 0.75).
Changes in Recombination Frequency as a Function of Zygospore Age. Recombination frequency increases as zygospores age (Table 2, Table 3, Fig. 3). The overall number of recombinant progeny from 2 day old zygospores is significantly lower than the number of recombinant progeny recovered from either 15 or 23 day old zygospores as determined from analysis of 2 x 2 con-
°
Sp*
Fig. 2. Changes in ratios of chloroplast alleles as a function of time. The frequencies of paternal alleles in biparental zygospore clones were determined by subcloning and replica-plating colonies of 2, !5, and 23 day old biparental zygospores. The frequencies of the paternal alleles for the three chloroplast loci have been grouped into five classes on the abscissa and are shown together with the relative numbers of zygospores transmitting only maternal (34) or paternal (P) chloroplast alleles
tingency tables (G = 53.5; G = 88.0; p < < 0.001). The overall frequency of recombinant progeny is also significantly lower within clones from 15 day old zygospores in comparison to 23 day old zygospore clones (G = 4.2; p < 0.05). At 2 days, on the average, 3% of the progeny of biparental zygospores are recombinant; at 15 days, 6% of the progeny are recombinant; and at 23 days, 7% of the progeny are recombinant (means derived from data of Fig. 3). This increased recovery of recombinant progeny does not simply reflect an amplification of specific recombinant genotypes within individual biparental zygospores, since Table 2 shows that more different types of recombinants are found within individual clones of the two older biparental zygospore samples than within clones of the 2 day old biparental zygospores (G = 18.9; 5d.f., 0.001 < p < 0.005, with the null class of zygosp ore clone s exclu de d from the analysis). The increased occurrence of recombination results in an expansion of the genetic map for these three chloroplast markers, yet the gene order remains the same (Fig. 4). Boynton et al. (1976) observed that more recombinant progeny were recovered from biparental zygospore clones with nearly equal ratios of maternal and paternal chloroplast alleles than in either the maternally or paternally biased clones. These findings may also be time-dependent since the highest frequency of recombinant progeny in the 2 day old biparental zygospore sample occurs in the most maternally-skewed class (Table 3) whereas the highest frequency of recombinants is observed in the unbiased classes from 15 and 23 day old zygospores.
B. B. Sears: Chloroplast Genome Composition
5
Table 2. Frequency of different types of recombinants in biparental zygospore clones. Biparental zygospores were selected from the zygospore population described in Table 1. Each zygospore clone was respread, and 50-64 subclones were tested for allelJc content by replica-plating. The three factor cross of GB-104 x GB-198 can yield 6 different recombinant genotypes (see Fig. 1) Age of zygospores
2 days 15 days 23 days
Numbei of zygospore clones with different types of recombinant products 0
1
2
3
4
5
6
average
Zygospore colonies analyzed (N)
3i 23 28
26 23 25
16 22 21
6 13 17
0 8 3
0 4 2
0 0 0
0.96 1.71 !.46
79 93 96
Table 3. Recombination frequency as a function of age and allelic ratio. Zygospores from the cross shown in Table 1 were sampled 2, 15, and 23 days after mating. After subcloning, the biparental zygospores were grouped into one of five divisions based on their paternal allelic ratio for the er-u-37locus. The frequency of recombinant progeny was determined by dividing the number of subclones which expressed recombinant phenotypes by the total number of subclones analyzed for each paternal allelic ratio class. Only biparental zygospores giving rise to 50-64 subclones were used; those zygospores which had been initially scored as biparental but which produced only maternal or paternal colonies upon subcloning, were excluded from this analysis Paternal allelic ratio
0.01-0.21 0.21-0.40 0.41-0,60 0.61-0.80 0.81-0.99
% of biparental zygospore progeny expressing recombinant phenotypes
Number of zygospores in each alMic ratio class
2 days
15 days
23 days
2 days
15 days
23 days
3.6 2.5 2.1 2.! 1.2
2.8 6.2 5.6 4.6 2.6
5.2 4.7 10.1 3.2 3.0
I2 12 19 15 19
26 17 18 11 16
17 14 17 10 30
77
88
88
Thus as zygospores age, the highest f r e q u e n c y o f recombinant genotypes appears to shift f r o m the maternally biased to the relatively unbiased zygospore clones. I have applied the n o n p a r a m e t r i c Spearman rank correlation coefficient as described by VanWinkle-Swift and Birky (1978) in m y statistical analysis to determine if any two r e c o m b i n a n t genotypes demonstrate a correlated occurrence in individual zygospore clones. Table 4 gives the correlation coefficient values for the recovery o f reciprocal and nonreciprocal r e c o m b i n a n t pairs f r o m biparental zygospore clones, o m i t t i n g clones which lack either m e m b e r o f a r e c o m b i n a n t pair. This class is e x c l u d e d for each pair of r e c o m b i n a n t s because the lack o f either r e c o m b i n a n t type w o u l d be scored as a positive correlation y e t p r o b a b l y reflects simply a low rate o f rec o m b i n a t i o n (VanWinkle-Swift and Birky, 1978). Statistically significant positive correlations were n o t e d o n l y three times and were for n o n r e c i p r o c a l r e c o m b i n a n t pairs f r o m the 2 day old zygospore clones (Table 4). Most significant correlations were negative and involved b o t h reciprocal and nonreciprocal r e c o m b i n a n t pairs. F o r one reciprocal and one nonreciprocal pair, negative correla-
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Fig. 3. Frequencies of recombinant progeny in clones of 2, 15, and 23 day old biparental zygospores, Biparental zygospores of the three ages were subcloned and replica-plated. Colonies resistant to two or three antibiotics in non-parental configurations were streaked onto medium containing those antibiotics in order to determine if the clones had arisen from a heteroplasmon or a true recombinant cell. The number of progeny expressing recombinant genotypes was divided by the total number of progeny analyzed to determine the frequency of each recombinant class
6
B.B. Sears: Chloroplast Genome Composition
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Table 4. Spearman rank correlation coefficient for the recovery of two recombinant genotypes from the same zygospore clone. From the three factor cross EsSssp r x Ersrsp s (abbreviated SSR x RRS), all possible recombinant genotype pairs are listed below. The frequency of recovery of each recombinant pair from individual zygospore clones was analyzed for 2, 15, and 23 day old biparental zygospores, excluding from each comparison those zygospore clones producing neither of the two recombinants. Values of the nonparametric Spearman rank correlation coefficients were tested for departure from 0 using the t-statistic: significant positive correlations are indicated by (a), and significant negative correlations by (b). Correlation coefficient
Fig. 4. Map distance as a function of time. The recombination frequencies of 2, 15, and 23 day old zygospores from Fig. 3 were used to determine map distance between the three chloroplast loci at each time point
tions were consistently found in all three zygospore age groups.
Discussion
Previously, I have reported that biparental transmission of chloroplast genes decreases, and uniparental transmission increases, during the aging of zygospores of C. reinhardtii, suggesting that the period commonly referred to as "dormancy" is, in fact, a period of dynamic changes in chloroplast genome composition (Sears, 1980). The present report supports and extends this view o f zygospore maturation by demonstrating the following additional changes in the composition of chloroplast genomes of biparental zygospores held in the non-dividing state for prolonged periods: (i) Populations of older biparental zygospores show decreased cotransmission of linked chloroplast alleles; (ii) Allele frequency distributions for populations of biparental zygospores become increasingly biased as the zygospores age, resulting in a shift from a nearly flat distribution to one with a U or L shape. While the variance in allele frequencies increases with the age of the population, the mean allele frequency of the paternal chloroplast alleles remains approximately the same; 0ii) Recombination frequencies for chloroplast genes increase with increasing zygospore age, resulting in an expansion of the genetic map without change in gene order. The increased recombination is detected both in terms of the frequencies of individual recombinant genotypes, and in the total number of different recombinant classes recovered; and (iv) The increases in recombination frequencies with increasing zygospore age are unequal for reciprocal classes; frequencies of reciprocal recombinants show no positive correlations within any age group of biparental zygospores. Separation of initially linked markers by multiple rounds of reciprocal recombination in conjunction with
2 days
15 days
23 days
-0.006 -0.528 b 0.619
-0.146 0.309 b -0.564 b
-0.212 -0.421 b -0.539 b
-0.518 b -0.318 -0.514 b 0.563 -0.264 -0.515 b 0.622 a -0.119 -0.228 0.648 a 0.039 0.528 a
-0.129 -0.374 b -0.409 b -0.213 0.023 0.099 0.037 -0.181 -0.454 b -0.288 0.178 0.137
-0.366 b 0.050 -0.601 b -0.336 -0.063 -0.288 -0.243 -0.178 -0.385 b -0.157 0.262 0.078
Reciprocal pairs RSR/SRS SRR/RSS SSS/RRR Nonreciprocal pairs RSR/SRR RSR/RSS RSR/SSS RSR/RRR SRR/SRS SRR/SSS SRR/RRR RSS/SRS RSS/SSS RSS/RRR SRS/SSS SRS/RRR
variability in sampling subclones of individual biparental zygospores could be offered as an explanation for the reduced cotransmission of linked chloroplast alleles observed in older populations of biparental zygospores (Fig. 1). However, observed deviations from the theoretical line of perfect cotransmission indicate a slight preferential transmission of the paternal sr-u-2-60 allele in comparison to the paternal er-u-37 allele. This observation is inconsistent with a continuing process of reciprocal recombination which should have no directional effect on allele frequencies. A recent study of genetic changes during zygospore maturation of C reinhardtii has demonstrated that the initially maternally-biased segregation of homoplasmic meiotic progeny changes as zygospores age (Sears, 1980). The time-dependent changes in allelic ratios within biparental zygospore clones reported here (Fig. 2), extend the earlier observations and suggest that the different allelic ratios reported by Sager and Ramanis, on the one hand, and Gillham, Boynton, and colleagues, on the other, may result from differences in the physiological ages of the zygospores occurring in response to variations
B. B. Sears: ChloroplastGenome Composition in the conditions usedfor zygospore maturation.Although the allele frequency distributions change significantly as zygospores age, the mean allele frequency for the population of biparental zygospores does not. Similar U- or Lshaped frequency distributions have been noted by other investigators for clones from 7 day old zygospores of Chlamydomonas (Gillham et al., 1974; Adams, 1978; Birky et al., in preparation 1980). Comparable data for allele frequencies from individual zygospores have not been published by Sager and colleagues, although extensive summaries of the results for different crosses and their alMic ratio means have been provided (Sager and Ramanis, 1976). As demonstrated here by inspection of Fig, 2, mean values indicating equal frequencies of the maternal and paternal chloroplast alleles in the population of zygospore progeny can be obtained by summation of counterbalancing differences, and reveal nothing about the extent of allelic bias in individual zygospores. Previous reports have shown that progeny reciprocally recombinant for chloroplast alleles are recovered in unequal frequencies from meiotic zygospore clones (Gillham, 1965) and vegetative zygote clones (VanWinkleSwift and Birky, 1978) of C. reinhardtii. Intercellular selection among zygospore progeny or intracellular selection within the zygospores could alter the frequencies of reciprocal recombinant genotypes. Intercellular selection against cells of a single Chlamydomonas stock resistant to streptomycin, neamine, erythromycin, and spectinomycin has been reported previously by Boynton et al. (1976). The growth disadvantage was shown to be due to a negative interaction of the linked streptomycin and neamine markers (VanWinkle-Swift, 1976). Among subclones of the population of individual zygospores from the cross reported here, I have identified all combinations of recombinant progeny, with the exception that all six recombinant types were never found in the same biparental zygospore clone (Table 2). Among these genotypes, the two reciprocal recombinants E+S+Sp + and Ersrsp r are both consistently the least frequent, which would be expected from classical recombination mechanisms since these two recombinants would be produced by double cross-over events (based on the map order established by Harris et al., 1977). However, if the recombinants result from gene conversion, the number of conversion events required to generate a given recombinant would depend on the parental origin of each genome. None of the other four recombinant genotypes is consistently in excess within individual zygospores containing several genotypes (data not shown) as would be expected if that recombinant phenotype conferred a growth advantage. Hence, differential growth is probably not responsible for the unequal recovery of these recombinant classes. The same evidence renders directional intracellular selection unlikely. A random intracellular selec-
7 tion mechanism could alter genotype frequencies within individual zygospores and hence, among their progeny. However, if the mechanism is truly random, equal frequencies of reciprocal recombinants should be recovered at the population level (VanWinkle-Swift and Birky, 1978), and this has not been found. VanWinkle-Swift and Birky (1978) have also pointed out that multiple rounds of pairing and exchange between organelle DNA molecules could alter reciprocal recombinant genotype frequencies in three factor crosses "if one recombinant genome from an early reciprocal exchange undergoes a second exchange while its reciprocal recombinant does not". However, multiple rounds of reciprocal recombination should not alter allele frequencies within individual ceils or within the population in the manner reported here (Figs 1, 2, 3). Multiple rounds of nonreciprocal recombination or "stochastic matching and conversion" could lead to genetic drift within each intracellular population of chloroplast genomes, as suggested by Birky and colleagues (Birky and Skavaril, 1976; Birky, 1978; Birky et al. in preparation, 1980). If equal numbers of chloroplast genomes are contributed by both parents, random genetic drift should result in equal frequencies of reciprocal recombinant progeny in the population of zygospores. Only with a directional mechanism superimposed on the recombination apparatus of the organelle genetic system, can one account for changes in cotransmission, allelic content, and frequencies of recombinant classes observed within this single population of zygospores sampled after different lenghts of maturation. Boynton et al. (1976) have compared recombination frequencies in progeny of meiotic zygospores and vegetative zygotes, and have found that map order is the same, although map distance is expanded in meiotic zygospores. Because vegetative zygotes of C. reinhardtii divide within 30 h after gamete fusion (VanWinkle-Swift, 1978), while meiotic zygospores are usually maintained in the nondividing state for 6-8 days before germination is induced (Levine and Ebersold, 1960; Hudock and Rosen, 1976), the differences noted by Boynton et al. (1976) may simply result from differences in the duration of the non-dividing state for the two types of zygotes. From this perspective, these observations are completely analogous to the increases in recombination frequency reported here for meiotic zygospores held dormant for prolonged periods of time. Collectively, these results are reminiscent of the increased recombination which occurs between phage DNA molecules within infected bacteria when cell lysis is postponed (reviewed by Stent, 1963). Alternatively, the increased recombination frequencies observed for meiotic zygospores as compared to spontaneously-occurring biparental vegetative zygotes, might be a consequence of the use of UV irradiation to increase biparental transmission in meiotic zygospores.
8 Previously, the highest recombination rates for chloroplast markers have been found in the biparental zygospore class with nearly equal maternal and paternal chloroplast allelic ratios (Boynton et al., 1976). By analogy with the theory of phage genetics proposed by Visconti and Delbrack (1953), Boynton and colleagues equated chloroplast allelic output (paternal and maternal allelic ratios) with the alMic input from the two parents and suggested that more recombinant progeny are observed when approximately equal numbers o f the two parental chloroplast DNA molecules are transmitted to the zygote. I have now shown that in the youngest zygospores - which have the lowest overall number o f recombinant progeny - more recombinant progeny are recovered from the most maternally-skewed class ofbiparental zygospores than in those with more equal allelic ratios (Table 3). As the zygospores age, the class with nearly equal maternal andpaternal alMic composition yields the highest frequency of recombinant progeny. Although a precise molecular explanation for these changes cannot yet be offered, the data do suggest that the opportunity for recombinational exchange m a y be limited in young zygospores. If the chloroplast DNA is organized into nucleoids as suggested by VanWinkle-Swift (1976; 1980) and Birky (1978), perhaps little interaction has occurred between nucleoids from the different parents by the time that zygospore germination is induced two days after mating. These studies have demonstrated that changes in chloroplast genome composition and recombination occur within biparental zygospores o f Chlamydomonas as a consequence of length o f zygospore maturation. Delay of the mitotic cell divisions o f two species o f yeast results in similar changes in mitochondrial gene composition (Thrailkill, K. M., Birky, C. W., Jr., Wolf, K., Ltickemann, G. in preparation). Thus, genetic drift seems to occur within intracellular populations of organelle DNA molecules. Acknowledgments. For their encouragement, helpful discussions, and constructive criticisms in the preparation of this manuscript, I am very grateful to Drs. K. P. VanWinkle-Swift, N. W. Gillham, J. E. Boynton, and C. W. Birky, Jr. I am indebted to Drs. C. W. Birky, Jr., and J. Antonovics for their assistance and advice on many of the statistical analyses. This work utilized facilities in the laboratory of Drs. N. W. Gillham and J. E. Boynton, supported by N. I. H. Grant GM-19427. I also wish to acknowledge a traineeship from Public Health Service Training Grant GM-02007.
B.B. Sears: Chloroplast Genome Composition hardtii. In: The genetics of algae (R. A. Lewin, ed.), pp. 69118. Oxford: Blackwell Scientific Publication 1976 Birky, C. W., Jr,: Annu. Rev. Gen. 12,471-512 0978) Birky, C. W., Jr., Skavarii, R. W.: Genet. Res. 27, 249-265 (1976) Boynton, J. E., Gillham, N. W., Harris, E. H., Tingle, C. L., VanWinkle-Swift, K., Adams, G. M. W.: Transmission, segregation, and recombination of chloroplast genes in Chlamydomonas. In: Genetics and biogenesis of chloroplasts and mitochondria (Th. Biicher, W. Neupert, W. Sebald, S. Werner, eds.), pp. 313 to 322. Amsterdam: North-Holland Biomedical Press 1976 Forster, J. L., Grabowy, C. T., Harris, E. H., Boynton, J. E., Gillham, N. W.: Curr. Genet. 1,137 153 (1980) Gillham, N. W.: Genetics 48,431-439 (1963) Gillham, N. W.: Proc. Nat. Acad. Sci. USA 54, 1560-1567 (1965) Gillham, N. W.: Organelle heredity, 602 pp. New York: Raven Press 1978 Gillham, N. W.,Boynton, J.E.,Lee, R.W.: Genetics 78,439 457 (1974) Harris, E. H., Boynton, J. E., Gillham, N. W., Tingle, C. L., Fox, S. B.: Mol. Gen. Genet. 155,249 266 (1977) Hudock, G. A., Rosen, H.: Formal genetics of Chlamydomonas reinhardtii. In: The genetics of algae (R. A. Lewin, ed.), pp. 29-48. Oxford: Blackwell Scientific Publications 1976 Levine, R. P., Ebersold, W. T.: Annu. Rev. Microbiol. 14, 197 to 216 (1960) Sager, R.: Adv. Genet. 19,287-340 (1977a) Sager, R.: Cytoplasmic inheritance. In: Cell biology: a comprehensive treatise (L. Goldstein, D. Prescott, eds.), pp. 279-317. New York: Academic Press 1977b Sager, R., Ramanis, Z.: Proc. Nat. Acad. Sci. USA. 58,931 935 (1967) Sager, R., Ramanis, Z.: Genetics 83,303 321 (1976) Sears, B. B.: Plasmid 2, 300 (Abstract) (1979) Sears, B. B.: Plasmid, 3, 18 34 (1980) Sears, B. B., Boynton, J. E., Gillham, N. W.: Genetics 86, s56 to 57 (Abstract) (1977) Singer, B., Sager, R., Ramanis, Z.: Genetics 83,341-354 (1976) Sokal, R. R., Rohlf, F. J.: Biometry 776, pp. San Francisco: W. H. Freeman 1969 Stent, G. S.: Molecular biology of bacterial viruses, 474 pp. San Francisco: W. H. Freeman 1963 Sueoka, N., Chiang, K.-S., Kates, J. R.: J. Mol. Biol. 25, 47-66 (1967) VanWinkle-Swift, K. P.: The transmission, segregation, and recombination of chloroplast genes in diploid strains of Chlamydomonas reinhardtii. Ph.D. thesis, Duke University, Durham, N. C., 445 pp. (1976) VanWinkle-Swift, K. P.: Nature 275,749-751 (1978) VanWinkte-Swift, K. P.: Current Genet. 1,113-125 (1980) VanWinkle-Swift, K. P., Birky, C. W., Jr.: Mol. Gen. Genet. 166, 193-209 (1978) Visconti, N., Delbrtick, M.: Genetics 38, 5-33 (1953)
References Adams, G. M. W.: Plasmid 1,522-535 (1978) Adams, G. M. W., VanWinkle-Swift, K. P., Gillham, N. W., Boynton, J. E.: Plastid inheritance in Chlamydornonas rein-
Communicated by K. P. VanWinkle-Swift Received December 12, 1979/January 10, 1980
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© by Springer-Verlag 1980
Changes in Chloroplast Genome Composition and Recombination during the Maturation of Zygospores of Chlamydomonas reinhardtii Barbara B. Sears*
Department of Botany, Duke University,Durham, North Carolina 27706, USA.
Summary. In crosses of the unicellular green alga Chlarnydomonas reinhardtii, the chloroplast genes are normally transmitted exclusively by the maternal parent to zygospore progeny. However, transmission of the paternal chloroplast alleles can be increased markedly by certain pretreatments of the maternal parent prior to mating. As zygospores age prior to induction of meiosis, they display decreased biparental transmission of chloroplast alleles and increased transmission of chloroplast alleles from only the maternal or paternal parent. In this report, chloroplast genome composition of biparental zygospores is shown to change in several ways during zygospore maturation. Allelic ratios of chloroplast genes within biparental zygospore clones become maternally or paternally skewed as the zygospores age, cotransmission of chloroplast alleles is reduced, and recombination increases, resulting in an expansion of genetic map distances between chloroplast markers used in this cross. The recovery of unequal frequencies of zygospore progeny expressing reciprocal recombinant genotypes confirms and extends other reports of the predominance of nonreciprocal recombination in organelle genetic systems. Key words: Chlamydomonas genetics.
Chloroplast - Organelle
Introduction
In most zygospores derived from crosses of Chlarnyclomonas reinhardtff, chloroplast genes are transmitted exclusively from the mating type plus (rot +) or maternal Current address: BotanischesInstitut, Universit~itDiisseldorf, Universitfitsstrasse 1, D-4000 Diisseldorf,FederalP,epublic of Germany
parent to zygospore progeny (reviewed by Gillham, 1978). Spontaneous transmission of chloroplast alleles from both parents (biparental transmission) or exclusively from the mating type minus (rnt-) or paternal parent occurs rarely, but the frequencies can be enhanced markedly by UV-irradiation of the mt+ gametes prior to mating (Sager and Ramanis, 1967). Although biparental zygospores of C. reinhardtii transmit chloroplast genes from both parents, the relative frequencies of maternal and paternal markers among progeny of biparental zygospores have been disputed. Sager and Ramanis have reported that maternal and paternal chloroplast alleles segregate to progeny of biparental zygospores in a 1 : 1 ratio during the first four postzygotic divisions (Sager and Ramanis, 1976) and Sager has therefore proposed that the chloroplast of Chlamydornonas is genetically diploid (Sager 1977a; 1977b). Sampling the progeny from biparental zygospore clones, Sager and Ramanis (1976) have also found that most chloroplast loci are represented by approximately equal frequencies of the maternal and paternal alleles, with some deviations appearing to be marker specific. In contrast, Gillham, Boynton, and colleagues have reported that when biparental zygospore colonies are subcloned, the progeny express predominantly the chloroplast alleles of the rnt + parent, although this maternal skew may be reduced by increasing the UV exposure of the mt+ gametes prior to mating (Gillham et al., 1974; Adams, 1978). Pedigree studies following the same markers during the first three postzygotic dMsions have affirmed the predominance of maternal chloroplast markers among meiotic products (Gillham, 1963; Boynton et al., 1976; Forster et al., 1980). Other analyses of biparental zygospore progeny have dealt with recombination and mapping (Gillham, 1965; Boynton et al., 1976; Singer et al., 1976;Harris et al., 1977) and cotransmission of chloroplast allele s (Adams et al., 1976; Birky, 0172-8083/80/0002/0001/S 01.60
B. B. Sears: Chloroplast Genome Composition C. W. Jr., VanWinkle-Swift, K. P., Sears, B. B., Boynton, J. E., Glllham, N. W. in preparation). When Chlamydomonas zygospores are sampled during an e x t e n d e d period o f zygospore i n c u b a t i o n or "maturat i o n " , changes in the inheritance pattern o f chloroplast alleles occur (Sears, 1980). Biparental transmission o f chloroplast alleles decreases, and exclusive transmission o f maternal or paternal chloroplast genes to zygospore progeny increases. Since the disappearance o f the biparental zygospore class occurs w i t h o u t a decrease in zygospore viability, heteroplasmic zygospores m u s t be losing chloroplast markers f r o m one or the o t h e r parent in the absence o f cell division. A l t h o u g h the loss o f the heteroplasmic state could involve a one step change, elim i n a t i o n o f the heteroplasmic state might also result f r o m gradual changes in chloroplast alMic c o n t e n t occurring within biparental zygospores during maturation. The experiments r e p o r t e d here were designed to determine w h e t h e r the duration o f zygospore m a t u r a t i o n affects r e c o m b i n a t i o n , cotransmission and the ratio o f maternal and paternal chloroplast alleles within biparental zygospores. My results show that among the progeny o f biparental zygospores obtained following U V irradiation o f the m t + parent, a11elic ratios b e c o m e m o r e uniparentally skewed and cotransmission o f chloroplast genes is reduced during an e x t e n d e d period o f zygospore m a t u ration. In addition, I demonstrate that r e c o m b i n a t i o n continues to occur b e t w e e n chloroplast genomes as zygospores age, resulting in an expansion o f genetic map distances b e t w e e n chloroplast markers. As previously d e m o n s t r a t e d by Gillham (1965) and VanWinkle-Swift and Birky (1978), r e c o m b i n a t i o n b e t w e e n chloroplast genomes was f o u n d to be primarily nonreciprocal.
Materials and Methods Strains, Media, Growth Conditions and Crosses. A single cross between mt + and m t - strains of Chlamydomonas reinhardtii respectively designated GB-104 and GB-198 provided the zygospore population which was analyzed in this investigation. GB-104 carries spr-u-l-27-3 (Spr), a chloroplast gene mutation which confers spectinomycin resistance (Harris et al., 1977); GB-198 contains two chloroplast gene mutations, st-u-2-60 (S r) and er-u-37 (Er), which confer resistance to streptomycin and erythromycin respectively. HSA medium has been described by Sueoka et al. (1967). Stocks were grown for 7 days on YA medium (HSA medium solidified with 15 g/1 agar and containing 4 g/1 Difco yeast extract) under 75 85 microeinsteins/m2s of photosynthetically active radiation (PAR) from cool white fluorescent light fixtures at 25-28 °C. Gametic differentiation was induced by suspending the cells in HSA medium lacking both NH4C1 and acetate ( - N medium) and by placing the suspensions on a shaker under high light intensity (about 200 uE/m2s PAR) for 5 hours. The cell density of the mt + gametes was adjusted to 5 x 106/ml, and the suspension was exposed to about 1.1 x 104 ergs/cm2s irradiation from a G8T5 germicidal lamp at a fixed distance for 30 s, while being stirred continuously, as described by Gillham et al. (1974).
The mt + gametes were then added to a dense suspension of m r gametes and they were allowed to mate for 2 h in the dark. 0.5 ml aliquots of the mating suspension were spread on plates of - N medium solidified with 4% Difco agar and left in the light for about 18 h before beingmoved to a dark incubator at 24 °C. To prevent dehydration of the agar, the zygospore maturation plates were wrapped in aluminum foil. Genetic Analysis. Zygospores were sampled from the maturation plates 2, 7, 15, and 23 clays after mating. To remove unmated cells, the agar surface of each maturation plate was scraped with a sterile razor blade. The mature zygospores have thickened rough cell wails and cling to the 4% agar, while the vegetative cells are easily removed by this technique. Zygospores were transferred to 1.5% Difco agar HSA plates and individually manipulated into 12 x 12 matrices under a dissecting microscope. These HSA plates were place d under continuous illumination (75 - 85 uE/m 2 s PAR) to speed zygospore germination and colony growth. After 4 - 6 days, zygospore viability was determined, and the zygospore clones were transferred to fresh HSA plates. These plates were used as masters for replica-plating to selective media containing spectinomycin, erythromycin, or streptomycin as described in detail by Harris et al. (1977). The initial replica plating allowed identification of biparental zygospores for further analysis. From the 2, !5 and 23 day old zygospore samptes, at least 100 biparental zygospore colonies scored as resistant on all three antibiotic plates were suspended individually in 8 ml - N medium. A loopful of each cell suspension was transferred to a 0.1 ml drop of liquid (H20 or - N medium) on a fresh HSA plate and spread. After about 6 days, 64 subclones (if available) from each biparental zygospore were transferred to fresh HSA plates and these plates were used as masters for a second round of replica-plating. Colonies resistant to two or three antibiotics in non-parental configurations were streaked from nonselective plates onto medium containing the appropriate combination of antibiotics. Although most chloroplast allele segregation has been completed by this stage (Harris et al., 1977), the multiple antibiotic plates allow unambiguous discrimination between clones arising from a heteroplasmon and those derived from a true recombinant cell. Computations and Statistieal Analysis. All biparental zygospore colonies producing 20 or more distinct clones following suspension and replating provided data used for determination of recombination frequencies and mapping (Figs 2, 3,4). Only those biparental zygospores yielding 50 64 subclones were used in determining the frequency of recombinants per biparental zygospore (Tables 2, 3, 4, Fig. 1). Statistical comparisons utilized two-way or multiway contingency table analysis with the log likelihood test described by Sokal and Rohlf (1969). For measuring the extent of cotransmission of chloroplast alleles, arcsine transformation of the square roots of the frequencies was necessary because the points do not represent a normal distribution. The nonparametric Spearman rank correlation coefficient was used to determine whether two recombinant genotypes are correlated in their occurrence within individual biparental zygospore clones. This analysis has been described in detail by VanWinkle-Swift and Birky (1978).
Results Changes in the Pattern o f Chloroplast Gene Inheritance as a Function o f Time. Zygospores derived f r o m a cross involving UV-irradiated mt+ gametes were sampled 2, 7, 15, and 23 days after mating (Table 1). The decrease in
B. B. Sears: Chloroplast Genome Composition Table 1. The inheritance pattern of chloroplast genes as a function of time in the cross GB-104 x GB-198, The mt+ gametes were irradiated with UV for 30 s prior to mating. Zygospores sampled 2, 7, 15, and 23 days after mating were induced to germinate by transfer to HSA Difco agar medium in the light. Viability of individual zygospores was determined by their ability to form a colony of 16 or more cells 4 - 6 days after the induction of germination. A minimum sample size of 288 was used for the determination of zygospore viability Age of zygospores
% Viability
% Maternal
% Biparental
% Paternal
Zygospore sample size
2 days 7 days 15 days 23 days
38.9 53.8 90.4 57.1
7.9 14.6 45.3 65.5
80.9 83.1 41.3 16.2
11.2 2.3 13.4 18.3
215 957 1,010 1,179
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Fig. 1. Cotransmission of chloroplast alleles as a function of zygospore age. Zygospores were sampled and induced to germinate 2, 15, and 23 days after mating. Following the initial replica-plating and scoring of zygospore clones, biparental zygospore colonies were subcloned and subjected to a second round of replica-plating. The frequency of the paternal er-u-37 (E r) allele is plotted as a function of the frequency of the paternal sr.u-2-60 (Sr) allele within each biparental zygospore clone, for the three time points
biparental transmission of chloroplast alleles and the increase in maternal and paternal transmission of chloroplast alleles seen here agrees with previous reports (Sears et al., 1977; Sears, 1979; 1980). Because the frequency of biparental zygospores had not changed markedly between 2 and 7 days, only biparental zygospore clones from the 2, 15, and 23 day sample points were subcloned and analyzed further. Cotransmission o f Chloroplast Alleles as a Function o f Zygospore Age. When the transmission of one chloroplast marker is plotted against the transmission of the second marker to the progeny of biparental zygospores, perfect coordinate transmission of the two markers from one parent will produce a line with a slope of 1.0 and described by a correlation coefficient (r) equal to 1.0 (Adams et al., 1976). A reduction in the coordinate transmission of the two alleles results in a reduction of the correlation coefficient. Negatively correlated trans-
mission would be described by correlation coefficients approaching - 1 and would produce aline with a negative slope. Cotransmission of the paternal er-u-3 7 and sr-u-2-60 alleles in 2 and 15 day old biparental zygospore clones closely matches the theoretical line (Fig. 1); the points have a correlation coefficient of r(2 days) = 0.989 and r(15 days) = 0.974. However, among the older zygospores, cotransmission is reduced; the points become more scattered; and the correlation coefficient drops somewhat (ro3 days) = 0.918). Tests for homogeneity among these correlation coefficients demonstrate that they differ significantly (X 2 = 18.2, 2d.f., p < 0.001). Allelic Ratio as a Function o f Time. The frequency of cells expressing paternal chloroplast alleles within biparental zygospore clones varies with UV dose (Gillham et al., 1974; Adams, 1978; Forster et al., 1980). In addition, Sager and Ramanis (1976) have reported that the frequency varies with map position of the alleles under
4
B.B. Sears: Chloroplast Genome Composition
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consideration. For three closely linked chloroplast loci, I have found changes in paternal alMic ratios as zygospores age (Fig. 2). Analysis of the data in Fig. 2 with 3 x 5 contingency tables using the log likelihood test shows that paternal allelic ratios among biparental zygospore progeny change significantly during zygospore maturation: for the er-u-37 locus, G = 20.0; for the sr-u-2-60 locus, G = 26.1; for the spr-u-l-2 7-3 locus, G = 20.9 (with 8 degrees of freedom, 0.001 < p < 0.025). Although the mean paternal aUelic ratio is approximately the same for all three samples, the distribution becomes increasingly biased as the zygospores age, resulting in a U- or L-shaped distribution, with most biparental zygospore clones having either high or low frequencies of paternal chloroplast alleles. As a consequence, the variance increases as the biparental zygospores age (e.g., for the transformed values of the er-u-37 locus, 2 days: a z = 388; 15 days: a 2 = 514; 23 days: ~2 = 697). No significant differences were found when comparisons were made between the allelic ratio distributions for each of the three markers at each time point (for the 2, 15, and 23 day samples, G = 0.8, 1.0, 4.1, with 8d.f., p > 0.75).
Changes in Recombination Frequency as a Function of Zygospore Age. Recombination frequency increases as zygospores age (Table 2, Table 3, Fig. 3). The overall number of recombinant progeny from 2 day old zygospores is significantly lower than the number of recombinant progeny recovered from either 15 or 23 day old zygospores as determined from analysis of 2 x 2 con-
°
Sp*
Fig. 2. Changes in ratios of chloroplast alleles as a function of time. The frequencies of paternal alleles in biparental zygospore clones were determined by subcloning and replica-plating colonies of 2, !5, and 23 day old biparental zygospores. The frequencies of the paternal alleles for the three chloroplast loci have been grouped into five classes on the abscissa and are shown together with the relative numbers of zygospores transmitting only maternal (34) or paternal (P) chloroplast alleles
tingency tables (G = 53.5; G = 88.0; p < < 0.001). The overall frequency of recombinant progeny is also significantly lower within clones from 15 day old zygospores in comparison to 23 day old zygospore clones (G = 4.2; p < 0.05). At 2 days, on the average, 3% of the progeny of biparental zygospores are recombinant; at 15 days, 6% of the progeny are recombinant; and at 23 days, 7% of the progeny are recombinant (means derived from data of Fig. 3). This increased recovery of recombinant progeny does not simply reflect an amplification of specific recombinant genotypes within individual biparental zygospores, since Table 2 shows that more different types of recombinants are found within individual clones of the two older biparental zygospore samples than within clones of the 2 day old biparental zygospores (G = 18.9; 5d.f., 0.001 < p < 0.005, with the null class of zygosp ore clone s exclu de d from the analysis). The increased occurrence of recombination results in an expansion of the genetic map for these three chloroplast markers, yet the gene order remains the same (Fig. 4). Boynton et al. (1976) observed that more recombinant progeny were recovered from biparental zygospore clones with nearly equal ratios of maternal and paternal chloroplast alleles than in either the maternally or paternally biased clones. These findings may also be time-dependent since the highest frequency of recombinant progeny in the 2 day old biparental zygospore sample occurs in the most maternally-skewed class (Table 3) whereas the highest frequency of recombinants is observed in the unbiased classes from 15 and 23 day old zygospores.
B. B. Sears: Chloroplast Genome Composition
5
Table 2. Frequency of different types of recombinants in biparental zygospore clones. Biparental zygospores were selected from the zygospore population described in Table 1. Each zygospore clone was respread, and 50-64 subclones were tested for allelJc content by replica-plating. The three factor cross of GB-104 x GB-198 can yield 6 different recombinant genotypes (see Fig. 1) Age of zygospores
2 days 15 days 23 days
Numbei of zygospore clones with different types of recombinant products 0
1
2
3
4
5
6
average
Zygospore colonies analyzed (N)
3i 23 28
26 23 25
16 22 21
6 13 17
0 8 3
0 4 2
0 0 0
0.96 1.71 !.46
79 93 96
Table 3. Recombination frequency as a function of age and allelic ratio. Zygospores from the cross shown in Table 1 were sampled 2, 15, and 23 days after mating. After subcloning, the biparental zygospores were grouped into one of five divisions based on their paternal allelic ratio for the er-u-37locus. The frequency of recombinant progeny was determined by dividing the number of subclones which expressed recombinant phenotypes by the total number of subclones analyzed for each paternal allelic ratio class. Only biparental zygospores giving rise to 50-64 subclones were used; those zygospores which had been initially scored as biparental but which produced only maternal or paternal colonies upon subcloning, were excluded from this analysis Paternal allelic ratio
0.01-0.21 0.21-0.40 0.41-0,60 0.61-0.80 0.81-0.99
% of biparental zygospore progeny expressing recombinant phenotypes
Number of zygospores in each alMic ratio class
2 days
15 days
23 days
2 days
15 days
23 days
3.6 2.5 2.1 2.! 1.2
2.8 6.2 5.6 4.6 2.6
5.2 4.7 10.1 3.2 3.0
I2 12 19 15 19
26 17 18 11 16
17 14 17 10 30
77
88
88
Thus as zygospores age, the highest f r e q u e n c y o f recombinant genotypes appears to shift f r o m the maternally biased to the relatively unbiased zygospore clones. I have applied the n o n p a r a m e t r i c Spearman rank correlation coefficient as described by VanWinkle-Swift and Birky (1978) in m y statistical analysis to determine if any two r e c o m b i n a n t genotypes demonstrate a correlated occurrence in individual zygospore clones. Table 4 gives the correlation coefficient values for the recovery o f reciprocal and nonreciprocal r e c o m b i n a n t pairs f r o m biparental zygospore clones, o m i t t i n g clones which lack either m e m b e r o f a r e c o m b i n a n t pair. This class is e x c l u d e d for each pair of r e c o m b i n a n t s because the lack o f either r e c o m b i n a n t type w o u l d be scored as a positive correlation y e t p r o b a b l y reflects simply a low rate o f rec o m b i n a t i o n (VanWinkle-Swift and Birky, 1978). Statistically significant positive correlations were n o t e d o n l y three times and were for n o n r e c i p r o c a l r e c o m b i n a n t pairs f r o m the 2 day old zygospore clones (Table 4). Most significant correlations were negative and involved b o t h reciprocal and nonreciprocal r e c o m b i n a n t pairs. F o r one reciprocal and one nonreciprocal pair, negative correla-
o
1"3 2 day old zygo|pores; N= 5663 zygospore
x
[g 15 day old zygospore=; N=6180 zygospore progeny r~ 25 day old zygospores; N=6615 zygospore progeny
progeny
"1 i]
oE
o
cr
Ersrspr
E*S*Sp*
ErS*Sp *
Ers*sp r E*srsp r
E+SrSp *
Recombinant Class
Fig. 3. Frequencies of recombinant progeny in clones of 2, 15, and 23 day old biparental zygospores, Biparental zygospores of the three ages were subcloned and replica-plated. Colonies resistant to two or three antibiotics in non-parental configurations were streaked onto medium containing those antibiotics in order to determine if the clones had arisen from a heteroplasmon or a true recombinant cell. The number of progeny expressing recombinant genotypes was divided by the total number of progeny analyzed to determine the frequency of each recombinant class
6
B.B. Sears: Chloroplast Genome Composition
Mop
Distance
Related
er-u-37
to
spr-u-l-6-2
15
25
Age
sr-u-2-60
I
I Z.4
2 days
Zygospore
I
II
1.5
~
3.5
t I
3,3
uy~'~a -^
4.4
t
5.9
doys
I i
4.7
,s.7
I II
3.2
,
t
Table 4. Spearman rank correlation coefficient for the recovery of two recombinant genotypes from the same zygospore clone. From the three factor cross EsSssp r x Ersrsp s (abbreviated SSR x RRS), all possible recombinant genotype pairs are listed below. The frequency of recovery of each recombinant pair from individual zygospore clones was analyzed for 2, 15, and 23 day old biparental zygospores, excluding from each comparison those zygospore clones producing neither of the two recombinants. Values of the nonparametric Spearman rank correlation coefficients were tested for departure from 0 using the t-statistic: significant positive correlations are indicated by (a), and significant negative correlations by (b). Correlation coefficient
Fig. 4. Map distance as a function of time. The recombination frequencies of 2, 15, and 23 day old zygospores from Fig. 3 were used to determine map distance between the three chloroplast loci at each time point
tions were consistently found in all three zygospore age groups.
Discussion
Previously, I have reported that biparental transmission of chloroplast genes decreases, and uniparental transmission increases, during the aging of zygospores of C. reinhardtii, suggesting that the period commonly referred to as "dormancy" is, in fact, a period of dynamic changes in chloroplast genome composition (Sears, 1980). The present report supports and extends this view o f zygospore maturation by demonstrating the following additional changes in the composition of chloroplast genomes of biparental zygospores held in the non-dividing state for prolonged periods: (i) Populations of older biparental zygospores show decreased cotransmission of linked chloroplast alleles; (ii) Allele frequency distributions for populations of biparental zygospores become increasingly biased as the zygospores age, resulting in a shift from a nearly flat distribution to one with a U or L shape. While the variance in allele frequencies increases with the age of the population, the mean allele frequency of the paternal chloroplast alleles remains approximately the same; 0ii) Recombination frequencies for chloroplast genes increase with increasing zygospore age, resulting in an expansion of the genetic map without change in gene order. The increased recombination is detected both in terms of the frequencies of individual recombinant genotypes, and in the total number of different recombinant classes recovered; and (iv) The increases in recombination frequencies with increasing zygospore age are unequal for reciprocal classes; frequencies of reciprocal recombinants show no positive correlations within any age group of biparental zygospores. Separation of initially linked markers by multiple rounds of reciprocal recombination in conjunction with
2 days
15 days
23 days
-0.006 -0.528 b 0.619
-0.146 0.309 b -0.564 b
-0.212 -0.421 b -0.539 b
-0.518 b -0.318 -0.514 b 0.563 -0.264 -0.515 b 0.622 a -0.119 -0.228 0.648 a 0.039 0.528 a
-0.129 -0.374 b -0.409 b -0.213 0.023 0.099 0.037 -0.181 -0.454 b -0.288 0.178 0.137
-0.366 b 0.050 -0.601 b -0.336 -0.063 -0.288 -0.243 -0.178 -0.385 b -0.157 0.262 0.078
Reciprocal pairs RSR/SRS SRR/RSS SSS/RRR Nonreciprocal pairs RSR/SRR RSR/RSS RSR/SSS RSR/RRR SRR/SRS SRR/SSS SRR/RRR RSS/SRS RSS/SSS RSS/RRR SRS/SSS SRS/RRR
variability in sampling subclones of individual biparental zygospores could be offered as an explanation for the reduced cotransmission of linked chloroplast alleles observed in older populations of biparental zygospores (Fig. 1). However, observed deviations from the theoretical line of perfect cotransmission indicate a slight preferential transmission of the paternal sr-u-2-60 allele in comparison to the paternal er-u-37 allele. This observation is inconsistent with a continuing process of reciprocal recombination which should have no directional effect on allele frequencies. A recent study of genetic changes during zygospore maturation of C reinhardtii has demonstrated that the initially maternally-biased segregation of homoplasmic meiotic progeny changes as zygospores age (Sears, 1980). The time-dependent changes in allelic ratios within biparental zygospore clones reported here (Fig. 2), extend the earlier observations and suggest that the different allelic ratios reported by Sager and Ramanis, on the one hand, and Gillham, Boynton, and colleagues, on the other, may result from differences in the physiological ages of the zygospores occurring in response to variations
B. B. Sears: ChloroplastGenome Composition in the conditions usedfor zygospore maturation.Although the allele frequency distributions change significantly as zygospores age, the mean allele frequency for the population of biparental zygospores does not. Similar U- or Lshaped frequency distributions have been noted by other investigators for clones from 7 day old zygospores of Chlamydomonas (Gillham et al., 1974; Adams, 1978; Birky et al., in preparation 1980). Comparable data for allele frequencies from individual zygospores have not been published by Sager and colleagues, although extensive summaries of the results for different crosses and their alMic ratio means have been provided (Sager and Ramanis, 1976). As demonstrated here by inspection of Fig, 2, mean values indicating equal frequencies of the maternal and paternal chloroplast alleles in the population of zygospore progeny can be obtained by summation of counterbalancing differences, and reveal nothing about the extent of allelic bias in individual zygospores. Previous reports have shown that progeny reciprocally recombinant for chloroplast alleles are recovered in unequal frequencies from meiotic zygospore clones (Gillham, 1965) and vegetative zygote clones (VanWinkleSwift and Birky, 1978) of C. reinhardtii. Intercellular selection among zygospore progeny or intracellular selection within the zygospores could alter the frequencies of reciprocal recombinant genotypes. Intercellular selection against cells of a single Chlamydomonas stock resistant to streptomycin, neamine, erythromycin, and spectinomycin has been reported previously by Boynton et al. (1976). The growth disadvantage was shown to be due to a negative interaction of the linked streptomycin and neamine markers (VanWinkle-Swift, 1976). Among subclones of the population of individual zygospores from the cross reported here, I have identified all combinations of recombinant progeny, with the exception that all six recombinant types were never found in the same biparental zygospore clone (Table 2). Among these genotypes, the two reciprocal recombinants E+S+Sp + and Ersrsp r are both consistently the least frequent, which would be expected from classical recombination mechanisms since these two recombinants would be produced by double cross-over events (based on the map order established by Harris et al., 1977). However, if the recombinants result from gene conversion, the number of conversion events required to generate a given recombinant would depend on the parental origin of each genome. None of the other four recombinant genotypes is consistently in excess within individual zygospores containing several genotypes (data not shown) as would be expected if that recombinant phenotype conferred a growth advantage. Hence, differential growth is probably not responsible for the unequal recovery of these recombinant classes. The same evidence renders directional intracellular selection unlikely. A random intracellular selec-
7 tion mechanism could alter genotype frequencies within individual zygospores and hence, among their progeny. However, if the mechanism is truly random, equal frequencies of reciprocal recombinants should be recovered at the population level (VanWinkle-Swift and Birky, 1978), and this has not been found. VanWinkle-Swift and Birky (1978) have also pointed out that multiple rounds of pairing and exchange between organelle DNA molecules could alter reciprocal recombinant genotype frequencies in three factor crosses "if one recombinant genome from an early reciprocal exchange undergoes a second exchange while its reciprocal recombinant does not". However, multiple rounds of reciprocal recombination should not alter allele frequencies within individual ceils or within the population in the manner reported here (Figs 1, 2, 3). Multiple rounds of nonreciprocal recombination or "stochastic matching and conversion" could lead to genetic drift within each intracellular population of chloroplast genomes, as suggested by Birky and colleagues (Birky and Skavaril, 1976; Birky, 1978; Birky et al. in preparation, 1980). If equal numbers of chloroplast genomes are contributed by both parents, random genetic drift should result in equal frequencies of reciprocal recombinant progeny in the population of zygospores. Only with a directional mechanism superimposed on the recombination apparatus of the organelle genetic system, can one account for changes in cotransmission, allelic content, and frequencies of recombinant classes observed within this single population of zygospores sampled after different lenghts of maturation. Boynton et al. (1976) have compared recombination frequencies in progeny of meiotic zygospores and vegetative zygotes, and have found that map order is the same, although map distance is expanded in meiotic zygospores. Because vegetative zygotes of C. reinhardtii divide within 30 h after gamete fusion (VanWinkle-Swift, 1978), while meiotic zygospores are usually maintained in the nondividing state for 6-8 days before germination is induced (Levine and Ebersold, 1960; Hudock and Rosen, 1976), the differences noted by Boynton et al. (1976) may simply result from differences in the duration of the non-dividing state for the two types of zygotes. From this perspective, these observations are completely analogous to the increases in recombination frequency reported here for meiotic zygospores held dormant for prolonged periods of time. Collectively, these results are reminiscent of the increased recombination which occurs between phage DNA molecules within infected bacteria when cell lysis is postponed (reviewed by Stent, 1963). Alternatively, the increased recombination frequencies observed for meiotic zygospores as compared to spontaneously-occurring biparental vegetative zygotes, might be a consequence of the use of UV irradiation to increase biparental transmission in meiotic zygospores.
8 Previously, the highest recombination rates for chloroplast markers have been found in the biparental zygospore class with nearly equal maternal and paternal chloroplast allelic ratios (Boynton et al., 1976). By analogy with the theory of phage genetics proposed by Visconti and Delbrack (1953), Boynton and colleagues equated chloroplast allelic output (paternal and maternal allelic ratios) with the alMic input from the two parents and suggested that more recombinant progeny are observed when approximately equal numbers o f the two parental chloroplast DNA molecules are transmitted to the zygote. I have now shown that in the youngest zygospores - which have the lowest overall number o f recombinant progeny - more recombinant progeny are recovered from the most maternally-skewed class ofbiparental zygospores than in those with more equal allelic ratios (Table 3). As the zygospores age, the class with nearly equal maternal andpaternal alMic composition yields the highest frequency of recombinant progeny. Although a precise molecular explanation for these changes cannot yet be offered, the data do suggest that the opportunity for recombinational exchange m a y be limited in young zygospores. If the chloroplast DNA is organized into nucleoids as suggested by VanWinkle-Swift (1976; 1980) and Birky (1978), perhaps little interaction has occurred between nucleoids from the different parents by the time that zygospore germination is induced two days after mating. These studies have demonstrated that changes in chloroplast genome composition and recombination occur within biparental zygospores o f Chlamydomonas as a consequence of length o f zygospore maturation. Delay of the mitotic cell divisions o f two species o f yeast results in similar changes in mitochondrial gene composition (Thrailkill, K. M., Birky, C. W., Jr., Wolf, K., Ltickemann, G. in preparation). Thus, genetic drift seems to occur within intracellular populations of organelle DNA molecules. Acknowledgments. For their encouragement, helpful discussions, and constructive criticisms in the preparation of this manuscript, I am very grateful to Drs. K. P. VanWinkle-Swift, N. W. Gillham, J. E. Boynton, and C. W. Birky, Jr. I am indebted to Drs. C. W. Birky, Jr., and J. Antonovics for their assistance and advice on many of the statistical analyses. This work utilized facilities in the laboratory of Drs. N. W. Gillham and J. E. Boynton, supported by N. I. H. Grant GM-19427. I also wish to acknowledge a traineeship from Public Health Service Training Grant GM-02007.
B.B. Sears: Chloroplast Genome Composition hardtii. In: The genetics of algae (R. A. Lewin, ed.), pp. 69118. Oxford: Blackwell Scientific Publication 1976 Birky, C. W., Jr,: Annu. Rev. Gen. 12,471-512 0978) Birky, C. W., Jr., Skavarii, R. W.: Genet. Res. 27, 249-265 (1976) Boynton, J. E., Gillham, N. W., Harris, E. H., Tingle, C. L., VanWinkle-Swift, K., Adams, G. M. W.: Transmission, segregation, and recombination of chloroplast genes in Chlamydomonas. In: Genetics and biogenesis of chloroplasts and mitochondria (Th. Biicher, W. Neupert, W. Sebald, S. Werner, eds.), pp. 313 to 322. Amsterdam: North-Holland Biomedical Press 1976 Forster, J. L., Grabowy, C. T., Harris, E. H., Boynton, J. E., Gillham, N. W.: Curr. Genet. 1,137 153 (1980) Gillham, N. W.: Genetics 48,431-439 (1963) Gillham, N. W.: Proc. Nat. Acad. Sci. USA 54, 1560-1567 (1965) Gillham, N. W.: Organelle heredity, 602 pp. New York: Raven Press 1978 Gillham, N. W.,Boynton, J.E.,Lee, R.W.: Genetics 78,439 457 (1974) Harris, E. H., Boynton, J. E., Gillham, N. W., Tingle, C. L., Fox, S. B.: Mol. Gen. Genet. 155,249 266 (1977) Hudock, G. A., Rosen, H.: Formal genetics of Chlamydomonas reinhardtii. In: The genetics of algae (R. A. Lewin, ed.), pp. 29-48. Oxford: Blackwell Scientific Publications 1976 Levine, R. P., Ebersold, W. T.: Annu. Rev. Microbiol. 14, 197 to 216 (1960) Sager, R.: Adv. Genet. 19,287-340 (1977a) Sager, R.: Cytoplasmic inheritance. In: Cell biology: a comprehensive treatise (L. Goldstein, D. Prescott, eds.), pp. 279-317. New York: Academic Press 1977b Sager, R., Ramanis, Z.: Proc. Nat. Acad. Sci. USA. 58,931 935 (1967) Sager, R., Ramanis, Z.: Genetics 83,303 321 (1976) Sears, B. B.: Plasmid 2, 300 (Abstract) (1979) Sears, B. B.: Plasmid, 3, 18 34 (1980) Sears, B. B., Boynton, J. E., Gillham, N. W.: Genetics 86, s56 to 57 (Abstract) (1977) Singer, B., Sager, R., Ramanis, Z.: Genetics 83,341-354 (1976) Sokal, R. R., Rohlf, F. J.: Biometry 776, pp. San Francisco: W. H. Freeman 1969 Stent, G. S.: Molecular biology of bacterial viruses, 474 pp. San Francisco: W. H. Freeman 1963 Sueoka, N., Chiang, K.-S., Kates, J. R.: J. Mol. Biol. 25, 47-66 (1967) VanWinkle-Swift, K. P.: The transmission, segregation, and recombination of chloroplast genes in diploid strains of Chlamydomonas reinhardtii. Ph.D. thesis, Duke University, Durham, N. C., 445 pp. (1976) VanWinkle-Swift, K. P.: Nature 275,749-751 (1978) VanWinkte-Swift, K. P.: Current Genet. 1,113-125 (1980) VanWinkle-Swift, K. P., Birky, C. W., Jr.: Mol. Gen. Genet. 166, 193-209 (1978) Visconti, N., Delbrtick, M.: Genetics 38, 5-33 (1953)
References Adams, G. M. W.: Plasmid 1,522-535 (1978) Adams, G. M. W., VanWinkle-Swift, K. P., Gillham, N. W., Boynton, J. E.: Plastid inheritance in Chlamydornonas rein-
Communicated by K. P. VanWinkle-Swift Received December 12, 1979/January 10, 1980
