The Molecular Basis of Genetic Diversity among Cytoplasms of Triticum and Aegilops Species. 11. on the Origin of Polyploid Wheat Cytoplasms as Suggested by Chloroplast Dna Restriction Fragment Patterns

In attempts to identify the phylogenetic donors of cytoplasm to EmmerDinkel and Timopheevi groups of wheat (Triticum), and the Aegilops kotschyiAe. variabilis complex, the restriction fragment patterns of chloroplast DNAs of representative species were compared with those of their putative diploid ancestors. The following seven restriction enzymes were used; BamHI, EcoRI, HindIII, KpnI, PstI, SmoI and XhoI. The restriction fragment patterns of an Emmer and a Dinkel (common) wheat were identical with those of Ae. longissima, and different from those of Ae. aucheri, Ae. bicornis, Ae. searsii, Ae. shoronensis, Ae. speltoides, and T. urartu by 4 to 12 fragments. The restriction fragment patterns of a Timopheevi wheat were identical with those of Ae. aucheri, and different from those of all other diploids by four to nine fragments. The restriction fragment patterns of Ae. variabilis were identical to those of Ae. bicornis and Ae. searsii, and different from those of all other species. Thus, we have concluded that Ae. longissima, Ae. aucheri and Ae. bicornis (or Ae. searsii) were the cytoplasm donors to the Emmer-Dinkel and the Timopheevi groups, and the Ae. kotschyi-Ae. variabilis complex, respectively. A diphyletic origin of Emmer and Timopheevi groups is supported by the present results. OLYPLOID wheats can be classified into the following three groups by P nuclear genome constitution: Emmer (AABB), Dinkel (AABBDD) and Timopheevi (AAGG and AAAAGG). The A and D genome have been postulated to have derived from Einkorn wheat (KIHARA 1924) and Aegilops squarrosa (KIHARA 1944; MCFADDEN and SEARS 1946), respectively, but there is diversity of opinion as to the donor of the B genome of Emmer and Dinkel, and the G genome of Timopheevi: Ae. speltoides (SARKAR and STEBBINS 1956; RILEY, UNRAU and CHAPMAN 1958), Ae. bicornis (SEARS 1956), T. urartu (JOHNSON 1972, 1975), Ae. searsii (FELDMAN 1978), and Ae. sharonensis (KUSHNIR and HALLORAN 1981) have been proposed to be the B genome donor, whereas Ae. speltoides (MAAN and LUCKEN 1971; SHANDS and KIMBER 1973) and Ae. aucheri (TSUNEWAKI 1980) have been suggested to be the G genome donor. All of these species, except T. urartu, belong to the section, Sitopsis, of Aegilops, and have an S or modified S genome. Two tetraploid Aegilops, namely Ae. kotschyi and Ae. variabilis, Genetics 104: 155-171 May, 1983. 156 K. TSUNEWAKI AND Y. OGIHARA with the same (CuCuSvSv) nuclear genome, received their cytoplasm from the S' genome donor (MUKAI and TSUNEWAKI 1975). It is still unknown, however, from which Sitopsis species this genome was derived. Restriction enzyme digestion of chloroplast DNA (hereafter ctDNA) has become a new means for studying interspecific, phylogenetic relationship in plants (VEDEL et al. 1978; KUNC, ZHU and SHEN 1982; GORDON et al. 1982). BOWMAN et al. (1981) constructed a physical map of wheat ctDNA by use of this technology, and was able to locate a few genes on it. OGIHARA and TSUNEWAKI (1982) compared ctDNA from 29 species (37 strains in total) of two related genera, Triticum and Aegilops, by the restriction enzyme technique, and demonstrated its usefulness for clarifying interspecific relationships. This work has been extended, now, to include ctDNAs of all Sitopsis species and T. urartu, which have been contrasted to ctDNAs of T. aestivum (a Dinkel wheat), T. dicoccoides (an Emmer wheat), T. timopheevi and Ae. variabilis, the aim being to identify the cytoplasm donors of these polyploid species. The results will be reported below. MATERIALS AND METHODS Species and varieties used as a source of ctDNA are shown in Table 1. CtDNAs of common wheat (Triticum aestivum cv. Chinese Spring), T. monococcum, T. urortu and Aegilops seorsii were prepared from their normal (euplasmic) lines. CtDNAs of the last two species were extracted from more than one strain and pooled before the restriction analysis, since only small amounts of ctDNA could be isolated from individual lines, CtDNAs of other species were isolated from fertile, alloplasmic lines of common wheat carrying their cytoplasms because large numbers of seeds were available for ctDNA extraction. Seeds of all lines, except T. urartu and Ae. searsii, were soaked 24 hr in running water, sown in wooden flats and kept at 20' during a 17-hr light period and at 15' during the remaining dark period. Seedling leaves were collected at the three-leaf stage, from which chloroplasts were isolated. Regenerated leaves of the same seedlings were also used for chloroplast isolation. Seedlings of T. urartu and Ae. seorsii were allowed to grow until the end of tillering stage, and the entire foliage was used for chloroplast isolation. Intact chloroplasts were isolated from seedling leaves or foliage using the method of KOLODNER and TEWARI (1975). Chloroplasts were purified from crude preparations using a discontinuous gradient made with 10, 40 and 75% Percoll solutions at pH 8.0, all of which ccntained 0.44 M mannitol, 50 mM Tris, 3 mM EDTA, 1 mM 2-mercaptoethanol and 0.1% bovine serum albumin. Purified chloroplasts recovered from the 40-7576 Percoll interface were gently diluted with 2 volumes of buffer containing 0.44 M mannitol, 50 mM Tris and 3 mM EDTA (pH 8.0), and pelleted at 1500 X g for 10 min. Pelleted chloroplasts were gently resuspended in TE buffer (50 mM Tris, 20 mM EDTA, pH 8.0) and lysed in a 2% sodium lauryl sarcosinate solution (at the final concentration) containing 200 pg/ ml proteinase K. DNA was isolated according to the method of KOLODNER and TEWARI (1975). The following seven restriction enzymes were used: BamHI, EcoRI, HindIII, KpnI, PstI, Smal and XhoI. Digestion of ctDNA with these enzymes was carried out according to the directions given by the supplier, Takara Shuzo Co. Ltd., Kyoto, Japan. The DNA fragments were separated by electrophoresis at 1 V/cm for 40-48 hr, using 0.8 or 1.2 % agarose slab gels containing 40 mM Tris, 20 mM sodium acetate and 2 mM EDTA. The DNA fragments were made visible by staining the gel with ethidium bromide (0.5 pg/ml) and were photographed under long wave UV light. From the photographs, the restriction fragment pattern of ctDNA with each enzyme was drawn by measuring the distance of each fragment from the origin. The molecular sizes of individual restriction fragments were estimated from their mobility, in comparison with restricted XDNA fragments of known molecular sizes. We used the molecular sizes estimated by BOWMAN et al. (1981) as referents for estimating the molecular sizes of our ctDNA fragments. CtDNA RESTRICTION PATTERNS OF WHEAT 157