P1. Syst. Evol. 161, 147-153 - Plant Systematics and Evolution © by Springer-Verlag 1988 Genome relationships in the Elytrigia group of the genus Agropyron (Poaceae) as indicated by seed protein electrophoresis M. MOUSTAKAS,L. SYMEONIDIS, and G. OUZOUMDOU Received October 31, 1986; in revised form February 10, 1988 Key words" Angiosperms, Poaceae, Agropyron, Thinopyrum, Lophopyrum. - Polyploid complex, isoelectric focusing, protein profile. Abstract: Agropyron bessarabicum (2n= 14), A. rechingeri (2n=28), A. junceiforme (2n=28), A. elongatum (2n= 14), A. flaccidifolium (2n=28) and A. scirpeum (2n=28) were studied by isoelectric focusing of seed soluble proteins. - The protein profiles obtained from the six taxa showed a striking degree of similarity; typically they consist of 40 bands. No qualitative but only quantitative differences (in the intensity of some bands) were found.- Combined with the cytological information available these protein data indicate that the two polyploid complexes must be placed in the recently erected genus Thinopyrum with the genome designations: T. bessarabicum jh jh, T. sartorii (= A. rechingeri) jh Jh jJ3 JJ3, T. junceiforme jJl y~ JJ2 jJ2, T. elongatum J el Jel, T. flaccidifolium J el Jel jel Jel and T. scirpeum J el J el je2 je2 The genus Agropyron (Poaceae), first defined by GAERTNER 1770 (in CAUDERON 1966), has attracted the attention of wheat breeders since the beginning of this century. Certain species of the genus, which cross readily with wheat, have been classified at times in Triticum, Agropyron, Elymus, Elytrigia, Thinopyrum and Lophopyrum. Some scientists (TZVELZV 1976, CAUDERON 1979, DVORAK 1981, DEWEY 1983) joined them together under the name Elytrigia, either as a group or section in the genus Agropyron or as a separate genus. This group includes only perennial species. CAUI~E~ON (1979) recognized in the Elytrigia group two polyploid complexes and four aggregate species. The two polyploid complexes are represented by Agropyronjunceum (L) P. B. (2n = 14, 28, 42, 56) and Agropyron elongatum (HosT) P. B. (2n= 14, 28, 56, 70). The taxa of both polyploid complexes were found to possess genes that can be used for the improvement of cold hardiness, drought tolerance, salt resistance, seed protein content or nutritive value in cultivated wheat (CAUDERON 1979, PIENNAR 1981, DEWEY 1984). In order to understand the species relationships in the two polyploid complexes a number of studies have been carried out (BRETON-SINTES & CAUDERON 1978, 148 M. MOUSTAKAS& al. CAUDERON 1958, 1966, 1979, CAUDERON & SAIGNE 1961, DVORAK 1981, ENDO & GrLL 1984, EVANS 1962, HENEEN 1962, 1977, HENEEN & RUNEMARK 1972a, b, HSIAO • al. 1986, JAASKA 1972, McGUxRE 1984, MOUSTAKAS& COUCOLI 1982, MOUSTAKAS & al. 1983, 1986, SCHULZ-SCHAEFFER& JURASITS 1962, WANO 1985, and others). However, present knowledge does not allow a precise estimation of the genome relations among the two poplyploid complexes (DEwv.Y & HSIAO 1983, DEWEY 1984). The applicability of seed protein electrophoresis to elucidate phylogenetic relationships between diploid and polyploid taxa is shown by the extensive literature on the subject (LADIZINSKY& HYMOWITZ 1979, MOUSTAKAS & al. 1986). In the present study isoelectric focusing was selected among the electrophoretic techniques since it affords the highest resolution. Recently, two classification systems of Tritieeae slightly differing from each other and both based on genomic relationships were published (LOVE 1984, DEWEY 1984). The taxa of the two complexes were placed in the same genus (Thinopyrum) by DEWEY (1984) but in two different genera (Thinopyrum and Lophopyrum) by LOvE (1984). Materials and methods The sources of the seed materials examined cytologically and subjected to electrophoresis are as follows: Agropyron bessarabicum: Greece: Euboea, littoral zone; A. rechingeri:Greece: Naxos, in schistose littoral rocks; A. flaccidifolium: Greece: Naxos. The seeds of all three above mentioned taxa were collected by MOUSTAKAS& SYMEONIDISand the voucher specimens are deposited in the Herbarium of the University of Thessaloniki (TAU). A. elongatum: cultivated at Versailles; A. junceiforme: French Atlantic coast; A. scirpeum: Italian coast; seeds of these three taxa were kindly made available by Y. CAUDERON;voucher specimens are deposited at I.N.R.A. Versailles, France. All materials, before being subjected to electrophoresis, were examined cytologically with the usual Feulgen technique of staining and squashing, as described by COUCOLI& SYMEONm~S(1980) to determine the chromosome number. Proteins were extracted from bulked seeds by grinding mature seeds with cold (4 °C) distilled water over an ice bath. The resulting mixture was centrifuged at 10 000 g for 15 rain and the supernatant was lyophylized (MouSTAKAS~: COUCOLI 1982). The proteins thus obtained are probably largely albumins. The electrophoretic procedure was carried out by applying the isoelectric focusing method (MouSTAKAS~: al. 1983). A polyacrylamide gel containing 2.2% w/v carrier ampholite with pH range 4.0-6.0 was used. Three gel replications were used to verify the reproducibility of the results and thus each extract was run an average of 8 times. Results and discussion The electrophoretic phenotypes obtained from the six taxa of the present study showed a striking degree of similarity. F r o m Fig. 1 it can be observed that the patterns of A. bessarabicum, A. rechingeri, A. junceiforme, A. elongatum, A. flaccidifolium, and A. seirpeum had the same 40pI bands. No qualitative protein phenotypic differences were found. Some quantitative differences were recognized as expected. Interpopulational and intrapopulational variation was checked by previously selecting A. bessarabicum as a representative taxon and examining proteins from the seeds of individual plants of 5 populations. In most populations 6 - 10 plants were studied. In all instances no qualitative differences were detected (MousTAKAS & COUCOLI 1982). Seed protein electrophoresis in Agropyron 149 The most conspicuous intensity differences in the patterns of the six taxa mostly concern the bands between 8 and 22. That is, bands 9, 15, and 16 were the most intense in the profile of the diploid A. bessarabicum. Bands 9, 14, 16, and 17 showed a higher degree of intensity in A. junceiforme, while bands 9, 10, 13, and 14 were more intense in A. rechingeri. In A. elongatum the most intense bands were 8, 16, 18, and 35. Finally, bands 9, 17, 18, 21, 22, 27, and 35 showed higher intensity in A. scirpeum while bands 14 and 16 were more intense in A. flaccidifolium. The occurrence of the same pI bands in diploid and polyploid taxa indicate an autopolyploid origin of the polyploids as has been mentioned by SYMEONIDIS al. (1985). According to LADIZINSKY& HYMOWITZ(1979) the difference in darkness of bands in seed protein profiles suggest that the formation of these bands is probably under the control of quantitative gene systems. From their cytological analyses, HZNZEN & RUNEMARK (1972a) and MOUSTAKAS & COLrCOLI (1982) conclude that the seven pairs of A. bessarabicurn show a striking similarity in chromosome size and position of centromeres with the seven largest pairs in tetraploid A. rechingeri and A. junceiforrne. Furthermore, the karyotype of the diploid A. bessarabicum is very similar to the karyotype of the diploid A. elongatum ( (EVANS 1962, HENEEN & RUNEMARK 1972a, b, MOUSTAKAS & COUCOLI 1982, WANG 1985, HSIAO & al. 1986). WANG (1985) successfully crossed A. bessarabicum with A. elongatum, while ENDO & GILL (1984) found a distinction between the two species in C-banding patterns. However, WANG (1985) concluded that the interspecific hybrids that were capable of homologous pairing outweigh the C-banding differences. We believe, in agreement with HSIAO & al. (1986), that the C-banding differences of the two taxa are due to structural rearrangements. At first evaluation, our protein patterns tend to support the hypothesis that in the examined taxa of the A. junceum and the A. elongatum complex there exists only one genome, so that the polyploids should be considered as autopolyploids. However, from the cytological information (H~NEEN 1962, 1977, BRETON-SlNTES & CAUDERON 1978), chromosomal rearrangements must have taken place in most of the taxa. Such rearrangements obviously play an important role in speciation (STEBBINS 1971, AVISE 1976, NAGL 1978) and may cause rapid evolution (FERNANDEZ-PERALTA & al. 1983). Allopolyploidy has played an important role in the evolution of higher plants, whereas strict autopolyploidy is considered as a rather uncommon phenomenon (STEBBINS 1971). Nevertheless, recent studies have revealed some species which must be considered as autopolyploids (GONZALEZ-AGUILERA& FERNANDEZ-PERALTA 1983). Such polyploids with time can undergo further genetic changes and chromosome differentiation (GONZALEZ-AGUILERA& FERNANDEZ-PERALTA 1983, MOUSTAKAS & al. 1986). Such a situation might have taken place in most of the examined polyploids of the A. junceum and the A. elongatum complexes. Taking into consideration the close relationship between the genomes J and E (CAuDERON 1966, 1979), the ability of their corresponding chromosomes to pair with each other (WANG 1985), and our results of seed protein electrophoresis, we consider the J and E genomes as variations of the same genome. Moreover, we think that the two polyploid complexes must be placed in the genus Thinopyrurn as was proposed by DEWEY (1984). Thinopyrum is a genus erected recently by LOvE (1980) who included in it only 150 M. MOUSTAKAS& al. Table 1. Proposed genome designations and synonyms so far given for the examined taxa of Agropyron junceum and A. elongatum polyploid complexes proposed to be placed both in the genus Thinopyrum. Genomes differing by superscripts are structurally modified with respect to each other Taxon Ploidy level Genome designations Thinopyrum bessarabicum (SAvuL. • RAYSS) A. LOVE = Agropyron bessarabicum SAVUL. & RAYSS 2X JJl JJl 4x JJl yl y2JJ2 Thinopyrum sartorii (Bo~ss. & HELDR.) A. LOVE = Agropyron rechingeri RUNEMARK = Agropyron sartorii (Boxss. & HELDR.) GRECESCU = Elytrigia rechingeri (RuNEMARK) HOLUB = Elymus rechingeri (RuNEMARK) RUNEMARK = Elymus farctus subsp, rechingeri (RUNEMARK) MELDERIS = Triticum sartorii BoIss. & HELDR. 4x jJl j31 j33jJ3 Thinopyrum elongatum (HosT) D. R. DEWEY = Agropyron elongatum (HOST) P. BEAUV. = Elytrigia elongata (HosT) NEVSKI = Elymus elongatus (HosT) RUNEMARK = Triticum elongatum HOST = Lophopyrum elongatum (HosT) A. LOVE 2X jel jel Thinopyrum flnccith'folium (BoIss. & HELI)R.) MOUSTAKAS,comb. IIOVa based on Agropyron scirpeum var. flaccidifolium BoIss. & HELDR., 4x jel jel Jel Jel 4X jel Jel Je2Je2 = Agropyron striatulum (RUNEMARK)MOUSTAKAS& COUCOLI = Elytrigia bessarabica (SAvuL. t~ RAYSS) DUBOVlK = Elytrigia striatula (RuNEMARK) HOLUB = Elytrigia juncea subsp, bessarabica (SAVUL. & RAYSS) TZVELEV = Elymus striatulus RUNEMARK = Elymus farctus subsp, bessarabicus (SAvuL. & RAYSS) MELDERIS Thinopyrum junceiforme (A. LOVE & D. LOVE) A. LOvE = Agropyron junceiforme A. LOVE & D. LOVE = Agropyron junceum subsp, boreoatlanticum SIMONET& GUINOCHET = Elytrigiajuncea subsp, boreoatlantica ( S~MONET& GUINOCHETHYLANDER = Elytrigia junceiformis A. LOVE & D. LOvE = Elymusfarctus subsp, boreoatlanticus StMONET& GUINOCHETRUNEMARK =Elymusfarctus subsp, boreali-atlanticus (SIMONET& GUINOCHET)MELD- ERIS 1884, in BOISSIERE., Flora orientalis 5:666 = Agropyron flaccidifolium (Bolss. & HELDR.) CANDARGY = Elytrigia flaccidifolia (BoIss. & HELDR.) HOLUB = Elymus elongatus subsp, flaccidifolius (BoIss. & HELDR.) RUNEMARK = Elymus flaccidifolius (Bolss. & HELDR.) MELDERIS = Lophopyrumflaccidifolium (BoIss. & HELDR.) A. LOVE Thinopyrum scirpeurn (K. PRESL) D. R. DEWEY = Agropyron scirpeum K. PRESL = Agropyron elongatum subsp, scirpeum (K. PRESL) CIFERRI & GIACCOMINI = Elytrigia scirpea (K. PRESL) HOLUB = Lophopyrum scirpeum (K. PRESL) A. LOVE Seed protein electrophoresis in Agropyron 151 pH 4.8 pH 5.2 ® pH 5.9 ..... ..... - - a b ------:::::: :----2- . . . . . . . . . . . . . . . . . . . . . . . . _--EE--E EE-EEE E:E--~E--ZEE -_=---_----~_=_=_E =_=_-?.~ :::::-: ---s-~ -- . . . . . . . . . .__z • ~ .... - . . . . . . . . .:zzz:: ....... '~ ...... 5 78910 . . . . . . . . . . . . - - - - - d 1 2 3 4 8 _. . . . . . . . . . . . . . . . . . . . . . . ,::~, : : 11:: ~ - ..... ~ -2-~5F. I1121314 ~------~ 18 ...... - ..... ~ - . . . . . . . . . . . ::::--": :::::: ,--,-,,,-- ~ 15 1718 1 9 20 21 2223 24 25 26 27 -,-_7,Z::: 282930 . . . . . . . . . . . . ~-----Z _---_-Z~_ :::::-" ~ ----] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :::::: ZZ-2-_ZZ '- ....... ~ :::::: 1 :::::: 2 3 :::::: 4 :::-_:: 5 3!13233 3 4 3 5 383738 39 40 6 Fig. 1. Seed protein profiles of 1 Agropyron bessarabicum, 2 A. junceiforme, 3 A. rechingeri, 4 A. elongatum, 5 A.flaccidifolium, 6 A. scirpeum. Photograph and corresponding schematic representation with key to shading in order of increasing band intensities six species of the former Agropyronjunceum complex. DEWEY (1984) expanded the genus to about 20 species from Thinopyrum, Lophopyrum and part of Elytrigia. Thinopyrum sensu LOVEconsists of species based on the J genome while Lophopyrum sensu LOVE is a genus of about 20 species with L. elongatum as the type species and the E genome as the basic genome (LOVE 1982). DVORAK(1981) and McGUIRE (1984) favour combining the J and E genome designations under the letter E. We have decided to retain the designation J for both genomes, because it is the older. The genome designations of the six Thinopyrum taxa given in Table 1 are based on cytological information and the seed protein patterns of the present study combined with information provided by other workers, jh and Jel stand for the same but structurally differentiated genome. Our results of seed protein electrophoresis confirm the stability of seed protein profiles and support the statement that intrinsic changes in plants such as chromosomal rearrangements or even doubling of the chromosome number have no effects on the seed storage protein patterns and the structural genes responsible for their coding (LADIZINSKY• HYMOWITZ 1979, MOUSTAKAS & al. 1983, 1986). 152 M. MOUSTAKAS8¢ al. References ArisE, J. C., 1976: Genetic differentiation during speciation. - In AYALA, F. 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L., 1971: Chromosomal evolution in higher plants. - London: E. Arnold. SYMEONIDIS, L., MOUSTAKAS, M., COUCOLI, H., 1985: Karyotype and seed protein profile analysis of diploid and tetraploid Hordeum bulbosum L. - Phyton 2 5 : 3 1 - 38. TZVELEV,N. N., 1976: Tribe 3. Triticeae DUM. -- In: "Poaceae U.S.S.R.", pp. 105-206. Leningrad: Nauka. WANG, R.R.-C., 1985: Genome analysis of Thinopyrum bessarabicum and T. elongatum. - Canad. J. Genet. Cytol. 27: 722-728. - - - - Address of the authors: MICHAEL MOUSTAKAS,LAZAROSSYMEONIDIS,and GEORGIA OUZOtrNIDOU, Department of Botany, University of Thessaloniki, 540 06 Thessaloniki, Greece.