Inheritance of mitochondrial and chloroplast genome markers in

9 Springer-Verlag 1990

Inheritance of mitochondrial and chloroplast genome markers in backcrosses of Chlamydomonas eugametos X Chlamydomonas moewusii hybrids Robert W. Lee 1, Bonny Langille 1, Claude Lemieux 1, and Poppo H. Boer 3. Departments of IBiologyand 3Biochemistry,Dalhousie University,Halifax,Nova Scotia B3H 4J1, Canada 2 D@artement de biochimie(Sciences),Universit6 Laval, Qu6bec, Qu6bec G1K 7P4, Canada

Summary. Southern blot analysis of AvaI-digested total cellular DNA from the interfertile species Chlamydomonas eugametos and Chlamydomonas moewusii with a coxI mitochondrial gene probe from Chlamydomonas reinhardtii revealed single hybridizing fragments of 5.0 and 3.5 kb, respectively. The transmission of these mitochondrial DNA physical markers along with that of chloroplast genetic markers for resistance to streptomycin and resistance to erythromycin was studied in the fourth backcrosses of Fl hybrids to one or the other parent. Viability in these backcrosses is high in contrast to the cross C. eugametos )< C. moewusii and its reciprocal which are associated with considerable meiotic product lethality. The resulting zygospores were found to transmit the mitochondrial and chloroplast genome markers uniparentally or preferentially from the mating-type-plus parent. Thus the species pair C. eugametos and C. moewusii differs from the pair Chlamydomonas reinhardtii and Chlamydomonas smithii in which mitochondrial genome markers are transmitted uniparentally by the mating-type minus parent, while the chloroplast genome markers are transmitted uniparentally by the opposite parental mating-type (Boynton et al. 1987).

Key words: Chlamydomonas eugametos - Chlamydomonas moewusii - Hybrid backcrosses - Mitochondrial DNA polymorphism - Chloroplast genetic markers - Inheritance

Introduction Mating-type control over the sexual transmission of chloroplast DNA (cpDNA) appears to be a fundamental feature of the genus Chlamydomonas. Despite employing

* Present address: Department of Medicine, Universityof Ottawa, Ottawa K1H 8M5, Canada Offprint requests to: R. W. Lee

an isogamous mode of sexual reproduction, the heterothallic species Chlamydomonas reinhardtii, Chlamydomonas moewusii and Chlamydomonas eugametos all transmit chloroplast genetic and/or cpDNA physical markers uniparentally or preferentially from the mating-type-plus (mt +) parent (Sager 1954; Gillham 1978; McBride and McBride 1975; Grant et al. 1980; Lee and Lemieux 1986). These observations correlate with the selective loss in zygospores of cpDNA derived from the mating-typeminus (mt-) parent, in the case of C. reinhardtii (Kuroiwa et al. 1982), and from one parent, presumably the m t parent, in the case of C. moewusii (Coleman and Maguire 1983). Although mating-type designations in C. moewusii and C. eugametos are consistent, they are defined arbitrarily relative to those of C. reinhardtii (Gowans 1976). Hence, the mt + strain of C. reinhardtii is not necessarily equivalent to the mt + strain of C. moewusii and C. eugametos. Chloroplast genetic markers in Chlamydomohas monoica, an isogamous homothallic (self-mating) species, are also transmitted uniparentally but, due to the proposed switching of mating-types in the parental strains, two opposing or bidirectional patterns of uniparental transmission are observed (VanWinkle-Swift and Aubert 1983). Again, these genetic results are associated with a loss in zygospores of cpDNA derived from one parent (VanWinkle-Swift and Salinger 1988). Studies with C. reinhardtii aimed at determining the inheritance of the mitochondrial genome using putative mitochondrial mutations gave conflicting results (Alexander et al. 1974; Wiseman et al. 1977). Boynton et al. (1987), however, were able to identify physical differences between the mitochondrial DNA (mtDNA) of C. reinhardtii (Ryan et al. 1978; Grant and Chiang 1980; Boer et al. 1985) and its interfertile relative Chlamydomonas smithii and to follow the inheritance of these differences along with that of chloroplast genetic markers in reciprocal crosses. They demonstrated that hybrid zygospores transmit the mtDNA markers uniparentally from the m t - parent, which is the opposite parent from the one that transmits chloroplast genetic markers uniparentally.

74 Table 1. Backcross programs used to generate hybrids B-4-A and B4-a mt+ Parent A. Cm (er-nMl) F-1-H (er-nM1) B-1-A (er-nM1) B-2-A (er-nM1) B-3-A (er-nM1) B. Ce (sr-2) F-l-a pab-I (sr-2) B-l-b (st-2) B-2-a (sr-2) B-3-a (sr-2)

X X X X X X X X X X

mr- Parent

Progenya

Ce (st-2) Ce (st-2) Ce (sr-2) Ce (sr-2) Ce (sr-2) Cm pab-1 Cm Cm (er-nM1) Cm (er-nM1) Cm (er-nM1)

F-1-H (er-nM1) B-1-A (er-nM1) B-2-A (er-nM1) B-3-A (er-nM1) B-4-A (er-nM1) F-l-apab-1 (sr-2) B-l-b (sr-2) B-2-a (sr-2) B-3-a (sr-2) B-4-a (sr-2)

Cm = C. moewusii and Ce = C. eugametos; pab-1 is a nuclear marker and sr-2 and er-nM1 chloroplast markers

a Progeny chosen to continue a backcross lineage were subclones of meiotic product clones

Given these observations, it is of interest to know the inheritance pattern of m t D N A in C. eugarnetos and C. rnoewusii, two interfertile species that have diverged considerably from C. reinhardtii and C. srnithii (Gowans 1976; Hoshaw and Ettl 1966). Boer et al. (1985) identified the putative mitochondrial genome of C. eugarnetos and C. rnoewusii by Southern blot analysis of undigested and E c o R I - and SalI-digested preparations of total cellular D N A from these algae using a C. reinhardtii probe containing only coding sequences of the mitochondrial gene for subunit I of cytochrome oxidase (coxl). In this paper the C. reinhardtii c o x I probe was used to detect single hybridizing fragments of 5.0 and 3.5 kb in Southern blots of A v a I digests of total cellular D N A from C. eugarnetos and C. rnoewusii, respectively. We have previously verified by restriction mapping that the hybridizing E c o R I and S a l I fragments identified by Boer et al. (1985) and the A v a I fragments identified here are indeed part of the mitochondrial genome of each species (Lee et al., unpublished). The inheritance of the A v a I fragment markers was studied along with that of chloroplast genetic markers for resistance to streptomycin (sr-2) and erythromycin (er-nM1) in the fourth backcross of FI hybrids to one or the other parent. These backcrosses were chosen over the cross C. eugarnetos X C. rnoewusii and its

reciprocal which show considerable lethality (Gowans 1963; Cain 1979) that interferes with genetic analysis. The results show that the A v a I m t D N A difference is transmitted uniparentally from the rnt + parent, the same parent which transmits chloroplast genetic markers uniparentally or preferentially. A portion of this work has already been published in preliminary form (Lee et al. 1985).

Materials and methods Strains, culture and genetic analysis. The origin and characterization of the C. moewusii (er-nM1) and C. eugametos (sr-2) strains has been described previously, as have the procedures used for culture and genetic analysis (Lemieux et al. 1980, 1981; Lee and Lemieux 1986; Lemieux and Lee 1987). Strains B-4-A and B-4-a are subclones (Lemieux et al. 1981)of meiotic products derived from the backcross programs summarizd in Table 1. DNA isolation. Total cellular DNA was isolated as described previously (Lemieux et al. 1980) except that after extraction with phenol, nucleic acids were precipitated twice with ethanol in the presence of 2.5 M ammonium acetate and redissolved in 10 mM TrisHCI, pH 8.0/1 mM EDTA. Southern blot hybridization. AvaI digests of total cellular DNA were fractionated by 0.8 % agarose gel electrophoresis and transferred to nitrocellulose according to the method of Southern (1975). Hybridization experiments were performed using 32p-labelled M 13 clone 11e as described by Boer et al. 1985. Clone lle contains a 360 base fragment derived from coding sequences of the C. reinhardtii mitochondrial coxI gene (Boer et al. 1985).

Results

Table 2 presents data on the meiotic product survival and the inheritance of the er-nM1 and sr-2 chloroplast genetic markers in the backcrosses B-4-A rnt + ( e r - n M 1 ) ) < C. eugarnetos rnt- (st-2) and B-4-a rnt + (sr-2) X C. moewusii rnt- (er-nM1). For both backcrosses meiotic product survival was about 85%, a value equivalent to or better than that observed for intraspecific crosses with wild-type C. eugarnetos and C. moewusii strains (Cain 1979). In the B-4-A backcross, roughly equal numbers of zygospores transmitted the chloroplast markers either uniparentally from the m t + parent (UP + zygospores) or biparentally (BP zygospores), while in the B-4-a backcross, significantly more zygospores were UP + than BP. A m o n g the progeny

Table 2. Meiotic product survival and transmission of chloroplast genetic markers (sr-2 and er-nM1) in hybrid backcrosses Chloroplast gene transmission pattern (frequency of zygospore clones)

Meiotic product survival

UP+

BP

UP-

0.54 (51) 0.73 (111)

0.46 (44) 0.27 (42)

0.00 (0) 0.00 (0)

1

B-4-A mt + (er~nM1) )< C. eugametos mr- (sr-2) B-4-a mt+ (sr-2) N C. moewusii rot- (er-nM1)

86% (388) 85 % (632)

UP+, inheritance of resistance marker from the mt+ parent, no inheritance of resistance marker from the mt- parent; BP, biparental inheritance of the resistance markers; UP-, no inheritance of resistance marker from the mt+ parent, inheritance of resistance marker from the mt- parent. The number of meiotic products scored and the number of zygospore clones in each class is indicated in parenthesis

75 transmitted uniparentally from the m t + parent, the parent which transmits chloroplast genetic markers uniparentally or preferentially in the same crosses. Moreover, the fact that hybrids B-4-A and B-4-a are each homoplasmic for the mtDNA marker of their original C. rnoewusii or C. eugarnetos mt+ parent, despite five backcrosses to the other species, lends further support to the UP + transmission of mtDNA in C. eugarnetos and C. Fig. 1. Inheritance of mtDNA Ava I fragment length polymorphisms in the B4-hybrid backcrosses B-4-A mU (er-nM1))< C. eugametos mt- (sr-2) and B-4-a mU (sr-2) )