Although cultivated barley Hordeum vulgare, is ... the Cerealia (Hordeum) section and other groups ..... the species H. arizonicum, brachyantherum, bul-.
Hereditas 90: 251-256 (1979)
Comparison of flavonoid patterns in wild and cultivated Hordeum species SUNE FROST,I JEFFREY B. HARBORNE,, SVEN ASKER’ and NABIEL SALEHZ* I
Institute of Genetics, University of Lund, Sweden Phytochemical Unit, Plant Science Laboratories, University of Reading, U . K .
FROST.S., HARBORNE,J. B.. ASKER, S. and SALEH,N. 1979. Comparison of flavonoid patterns in wild and cultivated Hordeum species. - Hereditas 90: 251-256. Lund, Sweden. ISSN 0018-0661. Received December 4, 1978 Seven flavonoids have been identified variously in leaf extracts of 18 wild Hordeurn species. Six are C-glycosylflavones. namely isovitexin. iso-orientin and iso-orientin 3’-methyl ether and the respective 74-glucosides. The seventh is the flavone 0-glucoside. tricin 5-glucoside. Several other C-glycosylflavones based on iso-orientin 3’-methyl ether are present in trace amounts. The pattern is essentially a simple one, with little variation between species. It contrasts markedly with the much more complex pattern of flavonoids found in different races of cultivated barley. The results indicate that the process of cultivation has had a noticeable effect on flavonoid synthesis in barley leaves and they confirm the taxonomic separation of the H . vulgare complex from all the other species. Sune Frost, Institute of Genetics, University of Lund, S-223 62 Lund, Sweden
Although cultivated barley Hordeum vulgare, is one of the most intensely studied crops, there is still some controversy regarding its origin. Together with the closely related H . spontaneum and H . agriocrithon, it has been placed in the section Cerealia (or Hordeum). These three taxa are wholly interfertile and obviously belong to the same species (cf. BOWDEN1959).** Most phylogenetic discussions on cultivated barley deal only with this group. Based on archaeological, genetical, morphological and phytogeographical data, several authors propose a monophyletic origin from the wild and weedy H . spontaneum (BAKHTEYEV 1964; HARLAN 1971; ZOHARY 1971b). Others maintain that wild six-row types were involved in the origin of cultivated barley (TAKAHASHI and TOMISHISA 1971; KAMM1977). We return to this matter in a later section of the paper. On the other hand, the relationships between the Cerealia (Hordeum) section and other groups in the genus Hordeum remain obscure. The cytogenetic studies so far published, including a multitude of interspecific and intergeneric crosses, suggest only a limited degree of homology
between the H . vulgare genome and those of other species. Studies of flavonoid patterns revealed a striking polymorphism in cultivated barley, where five “chemical races” A, B1,B,, C, and S could be discerned. These races were also rec0gnized.h H . agriocrithon (only A and B) and H . spontaneurn (FROST et al. 1975; FROSTand HOLM1975). The geographical and taxonomic distribution of these chemical races is of some significance for discussions on the origin in, and migration from primary and secondary gene centers of cultivated barley. The genetic differences between the races have been analysed, and the results will be published in a forthcoming paper. The chemical composition of the principal flavonoids of H . vulgare has now et al. 1977). been determined (FROST * Permanent address: National Research Centre, Dokki, Cairo, Egypt
** According to BOWDEN (1959). the correct description of the taxa in the Cerealia group should be H . vulgare L. emend. subsp. sponfaneum (C. Koch) Thellung; H . vulgare L. emend. subsp. vulgare var. vulgare and H . vulgare L. emend. subsp. vulgare var. vulgare f. agriocrithon (Aberg) Bowden.
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A preliminary investigation of wild species outand ASKER1977) side the Cerealia group (FROST showed the occurrence of flavonoid polymorphisms even here, but also patterns different from those observed within Cerealia. To study the degree of similarity between this and other groups of the genus, we decided to analyse the chemical structure of the flavonoids from several collections of wild Hordeum species.
Materials and methods Wild species were analysed from material in the collection of the Institute of Genetics, University of Lund. Plants were also grown from seed at Reading. Analyses were carried out on leaves collected from plants in flower, in order to avoid variations in flavonoid pattern due to ontogenetic effects. Voucher specimens of all species have been retained for inspection. Flavonoid identifications were carried out essentially by methods described in our earlier paper (FROSTet al. 1977). In the species survey, 2D paper chromatography was carried out on both direct and acid hydrolysed extracts and identities were established by co-chromatography with markers. In the case of H . bulbosum, more detailed analyses were carried out, using spectral and hydrolytic procedures.
Results A survey of 18 species of Hordeum, represented by 38 accessions, showed that only seven major flavonoids were regularly present (Table 1). Six of these are C-glycosylflavones, namely isovitexin, iso-orientin and iso-orientin 3’methyl ether (isoscoparin) and their respective 7 4 glucosides. The seventh is tricin 5-glucoside. Many of these compounds, or related structures, have been identified in cultivated barley, so that there is no sharp distinction in chemistry between H . vulgare s.1. and the other species. Nevertheless, it is quite apparent that most of the major constituents found in different races of cultivated barley are not readily detectable in any of the wild species. Furthermore, three major compounds of the wild species (substances 28-30, Table 1) are not recorded in cultivated barley, at least in any significant concentration.
Hereditas 90 (1979)
All seven flavonoids of the wild species are widely present. However, free isovitexin could not be detected in H . glaucum, H . stenostachys, H . pusillum and H . secalinum. This is not always a significant difference, since the related 7glucoside occurs in the first two of these species. Only in H . pusillum and H . secalinum are both isovitexin and its 7-glucoside absent. In order to confirm the results of the survey, a more detailed study was carried out in H . bulbosum leaves. All the seven flavonoids reported above were isolated and their structures confirmed. Some additional minor constituents were also detected and partially characterized. They proved to be derivatives of iso-orientin 3’methyl ether with additional glycosylation at the 6-C-glucose residue, and also at the 7-position. None of these minor components corresponded directly with any of the similar C-glycosylflavones of cultivated barley. In general, therefore, the results show a marked contrast in flavonoid patterns between the wild and cultivated Hordeum species, with a significant increase in glycosidic complexity and in acylation as a result of cultivation. These contrasting patterns might simply indicate a marked difference in chemistry between the Cereulia section and the rest of the genus. However, it would seem more likely that the complexities of glycosylations are indirectly the result of selective pressures during the development by man of the improved cultivated forms. An increasing complexity in flavonoid glycosylation has been noted before in cultivated crops as compared to the related wild species, most notably in Pisum and Lathyrus (See HARBORNE. 1971).
Discussion In view of the continuing controversy about the origin of cultivated barley (,see Introduction), it wbuld seem useful here to first discuss relationships within the Cerealia group, before considering relationships within the genus as a whole. The relationships within Cereulici -or within H . vulgare L. emend BOWDEN(1959) now seem obvious, although still disputed by some authors. The wild, two-rowed spontaneurii with brittle rachis has a wide distribution, mainly in the Near East. Cultivated forms have tough rachis and are generally either two- or six-rowed. For a long time, the
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254
s. FROST ET AL.
Hcroditci.s YO f IY7Y)
oldest known barleys were the six-rowed ones spontaneurn. The’oldest archaeological sites with found in the tombs within the Egyptian pyramids. cultivated two-rowed barley are located within the Six-rowed, brittle-eared barley, named H . present area of distribution of H . spontaneum agriocrithon, was first found in Tibet ERG (HARLAN 1971). The development of cultivated barley obviously 1938). At that time, it was thought to bea“missing link”; such wild six-rowed forms could have given took place without assistance from species outside rise to cultivated six-row barley, whereas two- the Cerealia group. Or - to cite HARLAN (1971) rowed cultivated barley might have originated “We need not look for exotic contributions from from H . spontaneum or spontaneum-like forms. alien species to account for the diversity within However, more recent archaeological evidence the crop”. But one question remains: what is the from still older sites, around 7,000 B.C., shows relationship between subsp. spontaneurn and with considerable certainty that two-rowed barley other wild species? was cultivated first (HELBAEK 1965; KIRKBRIDE The genus Hordeum has been variously sub(1941) distinguished four sections, 1966). Further, the difference between brittle ver- divided. NEVSKI sus tough rachis is controlled by two pairs of viz. Bulbohordeum Nevski, Cerealia Ands., alleles, Btlbt and BtJbt,. Brittle barley carries Hordeastrum Doll. and Stenostachys Nevski. (1959) described seven groups, partly both dominant alleles in the homozygous condi- MORRISON tion, whereas cultivated barley (tough rachis) is based upon karyotype studies: the vulgare, either BtBtbt,bt,, btbtBt,Bt, or, rarely btbtbtzbtz marinum, murinum, californicurn, stenostachysl (TAKAHASHI 1955). It now appears that all primitive pusillum, bulbosum, and jubatum groups. H . vulChinese barleys are BtBtbt,bt,, whereas all Indian gare is diploid (2n = 14), whereas both tetraploid barleys are btbtBt,Bt,. Any cross between barleys and hexaploid forms are known from other from these two regions - which is likely to take species. Both allo- and autoploidy probably occur. place in intermediate areas like Tibet - would thus The flavonoid results (Table I ) confirm the disproduce a type with a brittle rachis. The kernels of tinctiveness of H . vulgare and the section CereH . agriocrithon were found among seed samples alia but do not help in separating the other species into groups. of cultivated barley and wheat, and H . agriocritSeveral authors have tried interspecific and hon was never found to grow wild in Tibet. intergeneric crosses with Hordeum species. The KAMM (1954) reported the occurrence of wild most extensive work in this field was performed in brittle six-rowed barley in Israel. In this case, H . MORRISON and SYMKO (1964). agriocrithon obviously arose from spontaneous Ottawa by RAJHATHY, Reproductive barriers between species are strong crosses between H . spontaneum and cultivated in Hordeum. Embryo culture techniques have to ( I 964) presented experisix-rowed barley. ZOHARY be applied, or else the embryo degenerates at an mental evidence for the hybrid nature of H . agriocrithon in Israel by means of progeny testing early stage. Most mature hybrid plants are totally of naturally occuring H . agriocrithon plants and sterile, like the hybrids between H . vulgare and production of H . agriocrithon types from control- the species H . arizonicum, brachyantherum, bulbosum (4x). californicum, compressum, depresled crosses between typical H . spontaneum plants and local cultivated six-row variants. Plants of sum, jubatum, lechleri, leporinurn, rnurinum and agriocrithon type are probably not able to main- stenostachys. Due to the-peculiar behaviour of chromosomes tain themselves in nature (ZOHARY 1971a). Thus, as far as is known, all “agriocrithon” finds are the in certain hybrids, it is not so easy to draw products of crosses including cultivated six-row conclusions on the degree of relationship between species from cytogenetic data. H . bulbosum has barley. Still another piece of evidence for the mono- sometimes been thought to be the closest relative phyletic origin of cultivated barley is the fact that of H . vulgare. However, both in 2 x x 2 x and six-rowed barley is rather easily produced from 4x x 4x crosses between these species, preferentwo-rowed, but not vice versa. Several two-rowed tial elimination of bulbosum chromosomes takes to six-rowed recessive mutations have been de- place, and the progeny includes a large proportion scribed, whereas six-rowed barley cannot give of haploid and dihaploid plants, respectively (LANGE 1968; LANGE and JOCHE.MSEN 1976; BENNETT rise directly to two-rowed - only to intermediumet al. 1976). Based upon the degree of elimination like forms (HARLAN 1968). In fact, most evidence available today points to of vulgare or bulbosum chromosomes in interspecific crosses, other species may be grouped in a a monophyletic origin of cultivated barley from
Hrrc,ditas 90 (1979)
hierarchy
of chromosome elimination
RAHMANYAM
1977, 1978).
FLAVONOID PATTERNS I N HORDEUM SPECIES
(S UB-
Further studies of chromosomes pairing in hybrids indicate that meiotic pairing between fully homologous chromosomes is suppressed in ceret al. 1964). Thus, tain combinations (RAJHATHY genome homologies cannot be assessed by studies of chromosome pairing only. Anyway, there is no proof of homology between the genomes of H. vulgare and other species. Thus, a transfer of useful characters from wild species in barley breeding is not easy to obtain. Evolutionary relationships between species should be further studied by collections of wild material and the use of suitable crossing programs and JACOBSEN 1978). Giemsa (cf. VON BOTHMER C-banding techniques (L INDE -L AURS E N 1976, 1978; NODAand KASHA1976; VOSA1976) should be used in karyotype studies. Studies of isozyme variation within H . vulgare and H . spontaneum by starch gel electrophoresis have been performed by BROWNet al. 1978. Using other techniques, we have initiated such studies in several species. Acknowledgments. - These investigations were supported by grants from the Swedish Natural Science Council, C. Trygger's Fund, Magn. Bergvall's Fund, E. Philip Sorensens Fund and the Nilsson-Ehle Fund. Dr. N. A. M. Saleh is grateful to the von Humboldt Foundation for financial support for his visit to Reading.
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