Jpn. J. Genet. (1982) 57, pp. 349-360

D genome donors for crassa (DDMCrMCr, DDD2D2McrMcr) and Ac. vavilovii (DDMCrMCrSPSP)deduced from esterase analysis by isoelectric focusing1)

BY Yasuo NAKAI Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Kyoto 606

(Received February 15, 1982) ABSTRACT Putative D genome donors to Aegilops crassa (2n = 4x = 28, DDMCrMCrand 2n=6x=42, DDD2D2MCrMcr)and Ae. vavilovii (2n=6x=42, DDMCrMCrSPSP) were studied for esterase isozyme similarities. Esterase isozymes examined by gel isoelectrof ocusing reveal no inter- or intraspecific variation in Ae. crassa and Ae, vavilovii. The putative parents, Ae. squarrosa (2n=2x=14, DD) and Ae, comosa (2n=2x=14, MM), each exhibit three isozyme phenotypes. The natural tetraploid form of Ae. crassa has an isozyme pattern correspond- ing to a mixture of esterases from type 2 squarrosa and type 1 comosa. We conclude from this that the D genome donor to Ae. crassa 4x is type 2 squar- rosa. The hexaploid form of Ae. crassa possesses the same D genome as the tetraploid form, suggesting a duplication of the type 2 squarrosa D genome. Moreover, the D genome of the hexaploid form is the same as the D genome of Triticum aestivum cv. Chinese Spring (bread wheat, 2n=6x=42, AABBDD). It was also found that the putative D genome donor to Ae. vavilovii corres- ponds to type 2 squarrosa.

1. INTRODUCTION Aegilops crassa Boiss. is both tetraploid (2n = 28, genome constitution DDM~rM~r)and hexaploid (2n=42, DDD2D2MCrMCr) This species is assumed to have originated as hybrids between the two diploid species Ae. squarrosa L. (2n =14, DD) and Ae, comosa Sibth et Sm. (2n =14, MM), or Ae, uniaristata Vis. (2n =14, MUMU)(Kihara 1954, 1963). The tetraploid are widely distributed from Central Asia to the Near East through Western Asia, while the hexaploid plants are distributed in Tadjik, Uzbek, and Turkmenian SSR of USSR (Zhukovskyi 1928; Jaaska 1981) and the northern province of Afgha- nistan (Kihara et al. 1965) . Drs. H. Kihara and K. Yamashita collected hexa- ploid forms in Pul-i-Khumri, Maimana, and Laman, , in an expedi- tion to the Karakoram and Hindukush in 1955, but they did not find hexaploids in the southern part of Afghanistan. Ae. vavilovii (Zhuk.) Chen. is a hexaploid. This species formerly was clas- 1) Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Kyoto No. 440. 350 Y. NAKAI

Fig. 1. Spikes of Ae. crassa 4x (1), Ac. crassa 6x (2), and Ac. vavilovii (3). sified as a member of the Ae. crassa complex (Ae, crassa Boiss. var. palestiana Eig), but recently it has been reclassified as Ae. vavilovii (Zhuk.) Chen., and separated from the crassa complex, since its geographical distribution and cytogenetic characteristics differ from those of the hexaploid Ae. crassa of Afghanistan (Chennaveeraiah 1966). Kihara (1963) has suggested that Ae. vavilovii has a genome constitution of DDM~rM~rS1S1(DDMCrMCrSPSP, after Kihara and Tanaka 1970). The plants are distributed intermittently from Northern to Jordan through . The putative parents, Ae. squarrosa and Ae, comosa, or Ae. uniaristata, are not found in these areas, but another putative parent, Ae. longissima (2n =14, S1S1),is found in the Palestinian area, that overlaps Ae. vavilovii. The morphological characteristics of the tetraploid and hexaploid forms of Ae. crassa, and Ae. vavilovii are recognizably unique for each species (Fig. 1). The apical upper margin of the empty glume of Ae. crassa 4x has been sug- gested to have its origin in the M genome, while the truncate upper margin of the empty glume can be traced to the D genome of Ae. squarrosa (Kihara 1954; Kihara et al. 1965). Both species, Ae. crassa and Ae. vavilovii, have the D genome commonly found in Ae. cylindrica Host (2n=28, CCDD) and Triticum aestivum L. (2n=42, AABBDD). However, zymogram patterns of Esterase isozymes, Ae. crassa, Ae. vavilovii 351 esterase isozymes among the Ae. squarrosa lines show that T. aestivum (bread wheat) has the D genome found in type 2 squarrosa (Nakai 1979), and that Ae. cylindrica has the D genome found in type 3 squarrosa (Nakai 1981). In the present study, strains of Ae. crassa and Ae. vavilovii from different localities were investigated for esterase isozymes. Based on the results, pos- sible donors of the D genomes and other genomes to Ae, crassa and Ae. vavilovii are discussed.

2. MATERIALSAND METHODS Materials: The strains of Aegilops crassa Boiss. of the tetraploid form (2n=28, genome constitution DDMCrMCr),that of the hexaploid form (2n=42, DDD2D2McrM~r),and Ae. vavilovii (Zhuk.) Chen. (2n=42, DDMcrM~rSPSP)used in the present study are shown in Table 1. All of these strains were kindly provided by Dr. M. Tanaka, the Germplasm Institute of Kyoto Univer- sity, Kyoto. For comparison, strains of their putative parental species, Ae. squarrosa L. (2n=14, DD), Ae. comosa Sibth et Sm. (2n=14, MM), Ae. heldrei- chii Holzm. (2n=14, MM), Ae. uniaristata Vis. (2n=14, M°MU), Ae. longissima Schw. et Musch (2n=14, S1S1), Ae, sharonensis Eig (2n=14, S'S'), Ae. searsii Kislev et Feld. (2n =14, SSSS),Ae. bicornis (Forsk.) Jaub. et Sp. (2n =14, S"S"), were also used. Some of the strains were provided by Dr. Tanaka, and five strains of the M genome group were provided by Dr. J. G. Waines, from the University of California, Riverside, California, USA. The seeds of Ae. longis- sima and Ae, searsii were a gift from Dr. M. Feldman of the Weizman Insti-

Table 1. Number of strains of Aegilops crassa (4x and 6x) and Ae. vavilovii collected from different countries 352 Y. NAKAI

Fig. 2. Isoelectric focusing of esterase isozymes of Ae. crassa and Ae. vavilovii: (1) Ac. crassa var. macrothera 4x (KUSE 2316), (2) Ae. crassa var, glumiaristata 6x (KUSE 2309), and (3) Ae. vavilovii (BEM 2435). tote of Science in Rehovot, Israel. Ae. searsii is a new species, morpholo- gically similar to Ae. longissima. Methods: For esterase isozyme analysis, the disc (5WX 80h mm) and vertical (160WX 120h X 2d mm) isoelectric focusing method were used. Seeds were placed in a germination chamber for 24 hr at 23° C. Enzyme extracts were prepared by homogenizing the germinated seeds in a glass mortar in 1 ml (per one grain about 15-20mg) of 0.05M cold potassium phosphate buffer (p117.0). The extracts were centrifuged at about 20,000 X g for 20 min at 0°C. The supernatant was placed on a polyacrylamide gel containing a carrier ampholite (KLB producter AB) with a pH range of 6.0 to 8.0. The anode vessel on the top and the cathode vessel on the bottom were filled with 0.02 M hydrochloric acid and 0.02 M ethylenediamine, respectively. The electric current was stabilized at 250 V and was maintained for 3 hr. Gels were removed from the tubes or the glass board, and stained with 0.2/ Fast Blue RR Solt and 0.02% a-naphthyl acetate (w/v) in a phosphate buffer (pH 7.0,1/15 M) for 20-30 min. The homo- logy of the esterase isozymes was determined by using a mixture (1:1 by weight) of esterase extracts. Marker proteins were also used for estimation of pH values at different points on the gels. Esterase isozymes, Ae. crassa, Ae. vavilovii 353

Fig. 3. Photograph of esterase zymograms and a schematic drawing of the isozymes for Ae. squarrosa: (1) type 1 (Ae, squarrosa var. strangulata, KUSE 2135), (2) type 2 (Ae, squarrosa var, typica No. 1), and (3) type 3 (Ae. squarrosa var. typica No. 2).

3. RESULTS Zymogram phenotypes of Ae. crassa and Ae. vavilovii

(1) Ae. crassa 4x: All 23 strains show identical zymogram phenotypes which consist of four major (3, 5, 6 and 8B), two moderate (2 and 4), and two minor (1 and 17) isozymes (Fig. 2-1). The isoelectric points of isozymes 4 and 5 are close.

(2) Ae. crassa 6x: All 12 strains show identical zymogram phenotypes which include the tetraploid phenotype (Fig. 2-2). (3) Ae, vavilovii: All three strains show zymogram phenotypes identical with those of the hexaploid Ae. crassa. A minor difference is seen in the intensity of isozymes 4 and 5 between Ae. vavilovii and Ae. crassa 6x: The intensity of isozymes 4 and 5 of Ae. vavilovii is higher than that of the corresponding isozymes of Ae. crassa 6x (Fig. 2-3).

Zymogram phenotypes of the putative parental species

(1) Ae. squarrosa: The isozyme patterns of each of the three zymogram phenotypes (types 1, 2 and 3, Nakai 1979, 1981, Fig. 3) show that type 1 has three major isozymes, 3, 5 and 6 (Fig. 3-1), and that type 2 has, in addition to these, one extra major isozyme, 8B (Fig. 3-2), and that type 3 has four major 354 Y. NAKAI

Fig. 4. Photograph of esterase zymograms and a schematic drawing of the isozymes for Ac. comosa; (1) type 1 (Ac. comosa var. typica; Greece), (2) type 2 (Ac. comosa var, typica; Greece), and (3) type 3 (Ac. comosa var. typica; ). 6, 8',10 and 12), three moderate (4, 5 and 6'), and two minor (1 and 3) isozymes (Fig. 3-3). Type 1 is found only in one strain. Factorial analysis of these three zymogram phenotypes reveal that one pair of allelic genes is responsible for the difference between each pair of types. (2) Ae. comosa, Ae. heldreichii and Ae. uniaristata: As shown in Fig. 4 and Table 2, three zymogram phenotypes are found in the samples of the Ae. comosa complex. Type 1 has two major (3 and 5) and one minor (2) isozymes (Fig. 4-1). Type 2 has one extra major isozyme, 6'E (Fig. 4-2), and type 3 has three major isozymes, 5, 6 and 8 (Fig. 4-3). The type 3 zymogram is similar to the type 2 zymogram of Ae. squarrosa. (3) Ae. longissima, Ae. sharonensis, Ae, searsii and Ae. bicornis: The zymo-

Table 2. Esterase zymograms for Ae. comosa, Ae. heldreichii and Ac. uniaristata Esterase isozymes, Ae. crassa, Ae. vavi lovii 355

Fig. 5. Photograph of esterase zymograms and a schematic drawing of the isozymes for Ae. longissima and its relatives; (1) type 1(Ae, searsii, TE 14; Golan Heights), (2) type 2 (Ae. longissima, TL 07; Hadera), (3) type 3 (Ae. longissima, TL 09; Dead Sea), and (4) type 4 (Ae. longissima, TL 03; Rehovot). gram phenotypes of the S genome group (except for Ae. speltoides) are classi- fied into four types. Type 1 is found only in Ae. searsii, and has three major isozymes, 2, 3 and 5 (Fig. 5-1). All tested strains of this species have the same phenotype (Table 3). Type 2 has one additional major isozyme, 6 (Fig. 5-2). Type 3 has one additional major isozyme, 8, plus isozymes of type 2 (Fig. 5-3) ; and, type 4 has, in addition to the isozymes of type 3, one major isozyme, 9' (Fig. 5-4). Of the strains studied, phenotype 1 occurs in 31.2%, phenotype 2 in 12.5%, phenotype 3 in 37.5%, and phenotype 4 in 18.8%, (Table 3).

Zymogram phenotypes of extract mixtures and of synthetic Ae, crassa

Table 3. Esterase zymograms for the S genome group (except for Ae, speltoides and Ae. aucheri) 356 Y. NAKAI

Fig. 6. Isoelectric focusing patterns and diagrams of the esterase isozymes for Ae. crassa 4x and its parental species: (1) Ae. squarrosa var. typica No. 1 (type 2), (2) the 1:1 mixture by weight of extracts of Ac. squarrosa (type 2) and Ae. comosa (type 1), (3) natural Ae, crassa 4x, RUSE 2316, and (4) Ae. comosa, KU 17-5 (type 1).

Phenotype 1 of Ae. squarrosa lacks one major isozyme, 8, which is present in Ae. crassa. Moreover, phenotype 3 of Ae. squarrosa has two extra major isozymes (10 and 12) which are not present in Ae, crassa. The 6' isozyme of phenotype 2, found in Ae. comosa, is not found in Ae. crassa. Phenotype 3 of the comosa complex is similar to type 2 of Ae. squarrosa. The zymogram of an extract mixture between type 1 or 3 of Ae. squarrosa and any type of Ae. comosa differs greatly from the zymogram phenotype of natural Ae. crassa 4x. However, the zymogram of a mixture of type 2 squarrosa and type 1 comosa, in equal amounts by weight, resembles that of Ae. crassa 4x (Fig. 6). For example, the two major isozymes of type 2 squarrosa (6 and 8B) corre- spond to the two isozymes of Ae. crassa; and the three major isozymes of type 1 comosa (2, 3 and 5) correspond to the three isozymes of Ae, crassa. KU 47, a hexaploid strain synthesized by colchicine treatment from a hybrid between Ae. crassa 4x and A. squarrosa (type 2), shows the same phenotype as that of the 1:1 extract mixture between type 2 squarrosa and type 1 comosa (Fig. 6). The intensity of isozyme 5 is higher than that of the natural hexaploid form of Ae. crassa. Esterase isozymes, Ae. crassa, Ae. vavilovii 357

Fig. 7. Photograph of esterase zymograms and a schematic drawing of the isozymes for normal and aneuploid lines of bread wheat and Ae. crassa 6x: (1) Triticum aes- tivum cv. Chinese Spring (normal), (2) ditelocentric 3DS of Chinese Spring, and (3) Ac. crassa 6x (KUSE 2309). (Note: Arrows indicate the homology of each isozyme which was identified by a protein mixture of the extracts of Ac. crassa 6x and ditelo- 3DS in equal weight.)

4. DISCUSSION Origin of the tetraploid form of Ae, crassa

Ae. crassa Boiss. has been divided into two ploidy groups, tetraploid and hexaploid, since being suggested by Eig (1929). The tetraploid form exists in two varieties, typica and macrothera, and the hexaploid form in two varieties, typica and glumiaristata. A new awnless type of Ae. crassa 4x has been found in Afghanistan (Kihara et al. 1965) and in (Tanaka, personal com- munication). These facts indicate that Ae. crassa is polymorphic. Never- theless, all of the tested strains show the same and uniform zymogram phenotypes, which is interpreted to mean that similar genetic factors are responsible for their esterase isozyme profiles. Accordingly, the genes for esterase isozymes located on both of the D and M genome chromosomes of Ae. crassa 4x seem to have remained unaltered since the origin of the amphi- diploid. The D and M genome donors to Ae. crassa 4x in polymorphic popula- tions of Ae. squarrosa and the Ae. comosa complex are phenotypes 2 of Ae. squarrosa, and 1 of Ae. comosa. From a geographical viewpoint, type 2 squarrosa and type 1 comosa have no common distribution area. Type 2 squarrosa varieties are found in the 358 Y. NAKAI areas from the Transcaucasus to Western Iran, but type 1 comosa varieties are found only in Greece. More samples possessing the M genomes must be tested to clarify the parentage of Ae. crassa 4x.

Origin of the hexaploid form of Ae. crassa

The D and Mgrgenomes of the hexaploid form of Ae. crassa are also common to the tetraploid form of Ae. crassa. Kihara et al. (1959) reported that the third putative genome is the D genome of Ae. squarrosa. They pointed out that the third D genome of the hexaploid form diners from the D genome of the tetraploid form. However, the D genome donor to the hexaploid form has been identified as phenotype 2 of Ae. squarrosa; that is, the esterase isozymes of the hexaploid form are identical to those of the tetraploid form. The pro- teins controlled by the genes of the D genome in the hexaploid form are iden- tical to those of the tetraploid form, suggesting that the genes in these two D genomes are identical. No modification of the D genome is observed in the hexaploid form. This observation is supported in the following way: The D genome of Ae. crassa 6x appears to be the same as that of Triticum aestivum cv. Chinese Spring (Fig. 7). The ditelocentric line, 3DS, lacks three major isozymes, 4, 6 and 8B (Fig. 7-2). Which is to say that the isozymes 4, 6 and 8B are controlled by a gene (s) on the long arm of chromosome 3D. These three isozymes are found in both of Ae. crassa 6x and T. aestivum (Fig. 7-3). This finding indicates that the proteins of Ae. crassa 6x derived from Ae. squarrosa (type 2) are controlled by the same gene(s) as exists in T. aestivum. As shown in Fig. 2, the zymogram phenotypes of the tetraploid and hexaploid Ae. crassa differ only slightly from one another. The intensity of isozymes 6 and 8B in the hexaploid form is higher than that of the corresponding isozymes in the tetraploid form. This suggests a dosage effect following the duplication of the D genome. The esterase isozymes are already known to show a dosage effect by autopolyploidization in the genus Aegilops (Nakai 1977). Kihara et al. (1965) suggested that the hexaploid form of Ae. crassa origi- nated as a hybrid between Ae. crassa 4x and Ae. squarrosa in the northern stretch of the Hindukush Range in Afghanistan. However, type 2 squarrosa that donated the D genome to present-day Ae. crassa 4x and 6x is not found in this area (Nakai 1979).

Origin of Ae. vavilovii `A ccording to Chennaveeraiah (1960), Ae. vavilovii shows many chromosomal irregularities during meiosis; for example, isochromosomes, irregular distribu- tion of bivalents, and chromosome bridges. Fl hybrids between Ae. crassa 6x and Ae. vavilovii do not set seed. These facts show the genetic differences Esterase isozymes, Ae. crassa, Ae. vavilovii 359

between these two hexaploid species, but the two genomes of Ae. vavilovii, D and Mgr, are common to Ac. crassa 4x and 6x. From these results and their morphological differences, Kihara (1963) and Kihara and Tanaka (1970) have proposed that the third putative genome is S1 (or SP). The zymogram phenotype of Ac. vavilovii is the same as that of Ac. crassa 6x (Fig. 2). Quite possibly, the D genome of this species was also derived from type 2 squarrosa. A minor difference is observed in the intensity of isozyme 5 which seems to be controlled by a gene of the M genome. The zymogram phenotype of type 1 comosa is most similar to that of Ac. searsii (Figs. 4 and 5). If an S genome species contributed the third genome, then isozymes 3, 4 and 5 of Ae. searsii must correspond to those of Ac. vavilovii. It is shown, however, that the intensity of isozymes 4 and 5 of Ac. vavilovii is higher than that of the corresponding isozymes of Ac. crassa 6x. This fact suggests a duplication of the M genome in Ac. vavilovii, that produces a dosage effect on isozymes 4 and 5 of Ac. vavilovii. Therefore, Ac. vavilovii probably originated by duplication of the M genome from the tetraploid form of Ac. crassa. Subsequently, of course, gene mutations have caused differences between M genomes carried in different species.

I am grateful to Dr. J. G. Waines, the University of California, Riverside, California, USA, Dr. M. Feldman, the Weizman Institute of Science, Rehovot, Israel and Dr. M. Tanaka, Kyoto University, Kyoto, for supplying seed stocks of the strains used in the present study. I also wish to thank Dr. K. Tsunewaki, Professor of the Laboratory of Genetics, Kyoto University, Kyoto, for his°kind guidance, and Dr. V. W. Woodward, the University of Minnesota, Minnesota, USA, and Dr. S. Tsuji, the Laboratory of Genetics, Kyoto University, Kyoto, for their help for revising the manuscript.

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