Copyright Ó 2008 by the Genetics Society of America DOI: 10.1534/genetics.107.078618 Identification of the Minus-Dominance Gene Ortholog in the Mating-Type Locus of Gonium pectorale Takashi Hamaji,*,1 Patrick J. Ferris,† Annette W. Coleman,‡ Sabine Waffenschmidt,§ Fumio Takahashi,** Ichiro Nishii†† and Hisayoshi Nozaki* *Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan, †Plant Biology Laboratory, Salk Institute, La Jolla, California 92037, ‡Division of Biology and Medicine, Brown University, Providence, Rhode Island 02906, §Institute of Biochemistry, University of Cologne, Cologne 50674, Germany, **Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai-shi, Miyagi 980-8577, Japan and ††Frontier Research System, RIKEN, Wako-shi, Saitama 351-0198, Japan Manuscript received August 8, 2007 Accepted for publication November 6, 2007 ABSTRACT The evolution of anisogamy/oogamy in the colonial Volvocales might have occurred in an ancestral isogamous colonial organism like Gonium pectorale. The unicellular, close relative Chlamydomonas reinhardtii has a mating-type (MT) locus harboring several mating-type-specific genes, including one involved in mating-type determination and another involved in the function of the tubular mating structure in only one of the two isogametes. In this study, as the first step in identifying the G. pectorale MT locus, we isolated from G. pectorale the ortholog of the C. reinhardtii mating-type-determining minus-dominance (CrMID)gene, which is localized only in the MTÀ locus. 39-and59-RACE RT–PCR using degenerate primers identified a CrMID-orthologous 164-amino-acid coding gene (GpMID) containing a leucine-zipper RWP-RK domain near the C-terminal, as is the case with CrMID. Genomic Southern blot analysis showed that GpMID was coded only in the minus strain of G. pectorale. RT–PCR revealed that GpMID expression increased during nitrogen starvation. Analysis of F1 progeny suggested that GpMID and isopropylmalate dehydratase LEU1S are tightly linked, suggesting that they are harbored in a chromosomal region under recombinational suppression that is comparable to the C. reinhardtii MT locus. However, two other genes present in the C. reinhardtii MT locus are not linked to the G. pectorale LEU1S/MID, suggesting that the gene content of the volvocalean MT loci is not static over time. Inheritance of chloroplast and mitochondria genomes in G. pectorale is uniparental from the plus and minus parents, respectively, as is also the case in C. reinhardtii. OGAMOUS reproduction, which involves anisog- 1994; Nozaki et al. 2000); second, several mating-type- O amous fusion of distinctive sperm and egg cells, specific genes have been identified in the closely re- has apparently evolved from isogamous sexual repro- lated isogamous, unicellular alga Chlamydomonas rein- duction where gametes of different mating types are hardtii (Ferris et al. 1995; Ferris and Goodenough very similar in size and appearance. Although oogamy 1997). Therefore, the volvocine algae possess unrivaled is known in animals and land plants, the origins of features for studying the evolution of sex in terms of oogamy are so ancient that there seem to be no extant molecular biology (Kirk 2005). isogamous close relatives of them (Karol et al. 2001; Gonium pectorale has flattened 16-celled colonies and Rokas et al. 2005). The volvocine or colonial volvoca- produces isogametes in sexual reproduction. Hetero- lean algae are a model lineage for studying the evolution thallic sexuality in G. pectorale with two mating types, plus of sexual reproduction for two reasons: first, they have and minus, was studied by Schreiber (1925) and Stein both isogamous (Gonium, Pandorina, and Yamagishiella) (1958). Although only one (plus) of the two conjugating and anisogamous/oogamous (Eudorina, Pleodorina, isogametes of C. reinhardtii has a tubular mating struc- and Volvox) genera, the latter forming bundles of male ture (TMS), both isogametes of G. pectorale extend a gametes (sperm) and large female gametes (eggs), which TMS toward the other (Nozaki 1984; Nozaki and Itoh are phylogenetically well studied (Nozaki and Itoh 1994). Phylogenetic analyses imply that anisogamous/ oogamous species of the colonial Volvocales evolved from an ancestral colonial species that exhibits isogamy Sequence data from this article have been deposited with the DDBJ/ as in G. pectorale (Nozaki and Itoh 1994; Nozaki et al. EMBL/GenBank Data Libraries under accession nos. AB353340, 2000). The genus Gonium is phylogenetically impor- AB353887–AB353889, AY860423, and DQ068275. tant, as it represents the most basal lineage within the 1Corresponding author: Department of Biological Science, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan. relatively advanced volvocine algae composed of isoga- E-mail: [email protected] mous genera and anisogamous/oogamous members Genetics 178: 283–294 ( January 2008) 284 T. Hamaji et al. (Nozaki et al. 2000). In C. reinhardtii the mating-type were examined and compared with those in C. reinhardtii (MT ) loci—mating type plus (MT1) and mating type (Boynton et al. 1987) and in V. carteri (Adams et al. minus (MT À)—consist of a 200-kb region dimorphic be- 1990). The evolutionary significance of GpMID and the tween the two mating types, referred to as the rear- uniparental inheritance of chloroplast and mitochon- ranged (R) domain, and include genes involved in drial genomes are discussed in this article. mating-type determination and in the function of the TMS, as well as housekeeping genes with alleles present in both MT loci. The dimorphism results in recombi- MATERIALS AND METHODS national suppression over a region of 1Mb(Ferris oodenough erris Experimental organisms and culture and mating methods: and G 1994; F et al. 2002). The names of the two mating types of G. pectorale (plus and The minus-dominance (MID) gene is one of the minus- minus) were assigned arbitrarily (Stein 1958), and since we specific genes in the MTÀ locus of C. reinhardtii and was show here that the MTÀ loci of both G. pectorale and C. demonstrated to be the master regulator in mating-type reinhardtii carry the MID gene, we have chosen to continue minus determination (CrMID:Ferris and Goodenough with this usage. Four strains of G. pectorale were used here: Kaneko3 (minus) and Kaneko4 (plus), originating from Okinawa 1997). CrMID contains an RWP-RK domain, which is a Prefecture, Japan (Yamada et al. 2006), and Mongolia 4 characteristically conserved putative DNA-binding do- (minus) and Mongolia 1 (plus). The Kaneko strains have been main observed in plants, oomycetes, and cellular slime deposited in the Microbial Culture Collection at the National molds (Schauser et al. 1999, 2005; Nozaki et al. 2006). Institute for Environmental Studies (Tsukuba, Japan; Kasai Ferris et al. (1997) identified only a single MID ortholog, et al. 2004) as NIES-1710 and -1711; the Mongolia strains are deposited in the Culture Centre of Algae and Protozoa (CCAP, CiMID,fromChlamydomonas incerta, the closest known Ambleside, Scotland; Gachon et al. 2007) as CCAP32/13 and relative to C. reinhardtii, although they attempted to find CCAP32/14. The Mongolia strains were isolated as single cells MID orthologs from other Chlamydomonas species, G. from incubated petri dishes in which a small amount of dried pectorale, and Volvox carteri by means of low-stringency mud had been rewetted with distilled water, and they were ringsheim DNA gel blot hybridization. They concluded, from com- maintained in soil-water medium (P 1946). The mud samples were collected by R. A. Lewin (University of paring divergence of housekeeping genes and MID, that California, San Diego) in October 1997. The mud came from sex-related genes evolve very rapidly (Ferris et al. 1997; two small pools of freshwater adjacent to a large hypersaline P. J. Ferris, unpublished results). Recently, however, lake (Cha-gan-nur, Hao-tong-yin) in Inner Mongolia. Mating the Pleodorina starrii minus-dominance gene ortholog types of Mongolia 4 and 1 were determined by crossing with abry (PlestMID) was obtained by reverse transcribed (RT)– Alaska 1 and 2 strains (F et al. 1998), whereas those of Kaneko3 and -4 were tested against Mongolia 4 and 1. polymerase chain reaction (PCR) from nitrogen-starved, The cultures of Kaneko3 and -4 were grown in AF-6, VTAC, sexually induced males using degenerate primers de- or standard Volvox media (SVM) (Starr 1969; Kasai et al. signed from the RWP-RK domains of C. reinhardtii and 2004) at 20°–25°, with alternating periods of 14 hr light C. incerta (Nozaki et al. 2006). This result motivated us and 10 hr dark at a light intensity of 30–200 mmol photons Á À2 À1 to identify additional volvocine MID orthologs and, in m Á s provided by cool white fluorescent lamps. For crossing, cells of these strains were grown in liquid Tris– turn, the MT loci. The genomic changes accompanying acetate–phosphate (TAP) medium (Harris 1989) on a light the evolution of sex will be elucidated by a step-by-step shelf under constant illumination until growth appeared sat- analysis of the volvocine MT loci. urated (typically 4–5 days). The cells were then pelleted and The G. pectorale MT locus, by analogy with the C. resuspended in nitrogen-free SVM medium (Starr 1969). reinhardtii MT locus, is likely to be a complex, recombi- After sitting overnight, strains of opposite mating type were mixed. Flagellar agglutination could be observed just after