Heredity66 (1991) 105—115 Received 16 May 1990 Genetical Society of Great Britain

The cyanogenic polymorphism in Trifolium repens L. (white clover)

M. A. HUGHES Department of Biochemistry and Genetics, The Medical School, The University, Newcastle upon Tyne NE2 4HH

Thecyanogenic polymorphism in white clover is controlled by alleles of two independently segregating loci. Biochemical studies have shown that non-functional alleles of the Ac locus, which controls the level of cyanoglucoside produced in leaf tissue, result in the loss of several steps in the biosynthetic pathway. Alleles of the Li locus control the synthesis of the hydrolytic enzyme, linamarase, which is responsible for HCN release following tissue damage. Studies on the selective forces and the distribution of the cyanogenic morphs of white clover are discussed in relation to the quantitative variation in cyanogenesis revealed by biochemical studies. Molecular studies reveal considerable restriction fragment length polymorphism for linamarase homologous genes.

Keywords:cyanogenesis,polymorphism, Trifolium repen, white clover.

genetics to plant taxomony. This review discusses the Introduction extensive and diverse ecological genetic studies in rela- Theterm cyanogenesis describes the release of hydro- tion to the more recent biochemical and molecular cyanic acid (HCN), which occurs when the tissues of studies of cyanogenesis in white clover. some plant species are damaged. The first report of cyanogenesis in Trifolium repens (white clover) was by Mirande (1912) and this was shortly followed by a Biochemistry paper which demonstrated that the species was poly- Theproduction of HCN by higher plants depends morphic for the character, with both cyanogenic and upon the co-occurrence of a cyanogenic glycoside and acyanogenic plants occurring in the same population catabolic enzymes. In white clover, two related cyano- (Armstrong et at., 1913). Since these reports, this poly- glucosides are produced, 1 -cyano- 1 -methylethyl fl-D- morphism has been the subject of a large number of glucopyranoside (linamarin) and R-1-cyano-1-methyl- ecological studies. Field studies, dating back to 1954, propyl /3-D-glucopyranoside(lotaustralin). These have investigated the distribution of the two morphs cyanoglucosides are also found in cassava (Manihot and both field and experimental studies have investi- esculenta Cranz), flax (Linum usitatissimum L.), rubber gated the nature of the selective forces responsible for (Hevea braziliensis L. Muell.-Arg.), lima bean maintaining the polymorphism in this species. This (Phaseolus lunatus L.) and bird's-foot trefoil (Lotus experimental system provides one of the few examples corniculatus L.). of a simply inherited, biochemical difference that is The primary precursors of all plant cyanoglycosides known in higher plants. Modified dihybrid Mendelian investigated are restricted to the five hydrophobic segregation ratios, in progeny scored for the produc- protein amino acids and one non-protein amino acid. tion of HCN, were demonstrated in the 1940s. More In white clover, the amino acids, valine and isoleucine, recently the morphs have been characterized bio- are precursors of the two related cyanoglucosides. The chemically and molecular studies, which will elucidate biosynthetic pathway (Fig. 1) follows the general the nature of the allelic differences responsible for the pattern found for all cyanoglucosides studied (Hughes polymorphism, are in progress. These studies provide & Conn, 1976; Collinge & Hughes, 1982a). The steps information at the molecular level about genetic differ- from amino acid to hydroxynitrile are carried out by a ences which have been subject to selection and which microsomal system and are metabolically channelled, may be complex. whereas the last step is carried out by a soluble The cyanogenic polymorphism in white clover is enzyme, UDP-glucosyltransferase (Hahlbrock & Conn, thus the subject of studies that range from molecular 1971). Recent work by Halkier eta!. (1989) has shown

105 106 M. A. HUGHES that the pathway shown in Fig. 1 must be modified for R' Hydroxynitrile R'

Linamarase lyase I cyanoglucoside biosynthesis in sorghum, and this R—C—CN R—C—CN R—C demonstration of further steps in the sorghum pathway indicates the existence of similar steps in other species. 0—glucose OH 0 In white clover, one set of microsomal enzymes is (cyanoglucoside) (hydroxynitrile) (ketone) responsible for the biosynthesis of both hydroxynitrile + + intermediates from the amino acid precursors, valine glucose HCN and isoleucine (Collinge & Hughes, 1984). It has also 2Thehydrolytic release of HCN from the cyano- been demonstrated that, in flax, one soluble glycosyl- Fig. glucosides; R =CH3,R' =CH3linamarin; R =CH3, transferase is responsible for the production of both R' =C2H5lotaustralin. glucosides (Hahibrock & Conn, 1971). In general, cyanogenic glucosides are broken down by sequential hydrolysis (Poulton, 1988). Firstly, a (Fig. 2). There is one report of hydroxynitrilase activity 3-glucosidase (linamarase) cleaves off the glucose in white clover (Carvaiho, 1981) but the enzyme has residue, the two hydroxynitriles produced in white not been studied in detail. The importance of hydroxy- clover are unstable at high pH, but an a-hydroxynitrile nitrilase activity for rapid HCN production has, how- lyase has been characterized from cassava which ever, been shown in Hevea brasiliensis (Selmar et aL, breaks the hydroxynitriles down to HCN and a ketone 1989). The cyanogenic j3-glucosidase (linamarase) of white clover is a homodimer with a subunit molecular mass of 62,000 Mr. It is the major soluble high-mannose asparagine-linked glycoprotein in young leaves, where R—CH—CH—COOH Amino acid it can represent up to 5 per cent of the total soluble protein (Hughes & Dunn, 1982). The antibiotic, tuni- NH2 camycin, prevents linamarase synthesis but no pre- Microsomal cursor polypeptides have been found in vivo. In vitro translation of young leaf mRNA produces a major R—CH—CH—COOH N-hydroxyamino acid 59,000 Mr polypeptide, which is recognized by affinity-purified linamarase antibodies and processed NHOH by dog pancreas microsomes to a 62,000 Mr protein Microsomal (Dunn et al., 1988). Although the glycosylation of linamarase is clearly demonstrated by these results, the possibility of proteolytic processing remains unresolved. R—CH—CH Aldoxime Kinetic studies show that linamarase will hydrolyse a NOH number of synthetic glycosides and that the carbo- Microsomal hydrate moiety of each substrate attaches to the same R' binding site at the active centre (Pocsi eta!., 1989). It is the aglycone and the angular arrangement around the R—CH—CN Nitrile glycosidic linkage which are the major determinants in Microsomal substrate specificity.

R—C—CN Hydroxynitrile Developmental physiology In white clover both the cyanogenic glucosides and Soluble linamarase have been shown to be produced during shoot growth (Hughes, 1968; Collinge & Hughes, 1982b; Dunn et al., 1988). The roots, flowers, seeds R—C—CN Cyanoglucoside and seedlings before shoot emergence are not cyano- 0—glucose genic (Ware, 1925; Collinge & Hughes, 1982b). The Fig.1 The pathway for the biosynthesis of the cyanogenic components of cyanogenesis are synthesized during glucosides, linamarin and lotaustralin (after Hughes & Conn, leaf development and then stored in the mature leaf. Without tissue damage, the cyanoglucosides are only 1976); R =CH3,R' =CH3valine and linamarin; R =CH3, R' =C2H5isoleucine and lotaustralin. broken down during leaf senescence (Collinge & THE CYANOGENIC POLYMORPHISM IN WHITE CLOVER 107

Hughes, 1982b). Not all species have the same Stirling, 1982; Maher & Hughes, 1973), thus hetero- developmental profile, for example, in cassava the zygotes have intermediate levels of linamarase and roots are cyanogenic and in flax and rubber cyano- cyanoglucosides. glycosides are stored as disaccharides in the seed Antibodies raised to purified linamarase have been (Poulton, 1988). used to quantify linamarase protein produced in plants Under normal growth conditions the tissues of a of different genotype (Hughes et a!., 1985). Plants cyanogenic plant do not contain detectable HCN. This homozygous for the recessive Ii allele contain no strongly suggests compartmentalization of the cyano- linamarase antigen, in addition liii plants produce no glucosides and the hydrolytic enzymes. Linamarase of translatable linamarase mRNA (Dunn et aL, 1988). white clover (Kakes, 1985) has been shown to be Variant forms of white clover which produce reduced apoplastic and may be located in the epidermis. The levels of linamarase activity have also been studied tissue and subcellular localization of the cyanogluco- (Maher & Hughes, 1973; Hughes et aL,1985).The sides in white clover are not known. reduced levels of linamarase activity are due to Some variation in the test reaction for HCN within a reduced rates of synthesis of the enzyme in developing single plant was noted by Rogers & Frykoim in 1937; leaves and have been shown to be determined by a the levels of HCN depending on the age of the leaf, the genetic element which lies within 4 map units of the Li time of year and the size of the plant. The effect of the locus (Hughes et a!., 1985). Thus all the available environment on cyanogenesis was studied by Collinge evidence indicates that non-functional alleles at the Li & Hughes (198 2b) who demonstrated a major effect of locus result in reduced or zero synthesis of linamarase temperature on cyanoglucoside biosynthesis. This and have characteristics of mutations in a cis-acting effect was not simple, plants differed both in their regulatory region. sensitivity to temperature changes and in the optimum White clover plants possessing only the non- temperature for synthesis. One plant varied from 0.85 functional ac alleles, are unable to synthesize either p moles of glucoside per milligram of protein at 18°C to linamarin or lotaustralin from radiolabelled valine and undetected levels at 27°C. Variation in the levels of isoleucine (Hughes & Conn, 1976). In vivo and in vitro HCN released during the growing season has been labelling experiments have shown that ac ac plants recorded by Askew (1933), Fraser & Nowak (1988) have at least two steps in the converison of amino acid and Vickery et al. (1987), who showed that meristem to hydroxynitrile missing from microsomal prepara- stress, low light intensity, cool temperature and tions (Hughes & Conn, 1976; Collinge & Hughes, inadequate phosphorus all favour high HCN levels. 1982a). In vivo studies have suggested that the The mean effect of wilting was to increase HCN by 23 predicted soluble fi-glucosyltransferase is also missing per cent on a dry weight basis. in ac ac plants (Hughes & Conn, 1976). There is considerable inherited variation in the level of cyanoglucoside produced in different plants. Analy- Genetics sis of cyanoglucoside levels in progeny from a four- Theinheritance of cyanogenesis in white clover was generation backcross experiment has shown that most elucidated by a group working in New Zealand (Coop, of the inherited variation is attributable to the existence 1940; Corkill, 1940; Melville and Doak, 1940; Corkill, of different functional Ac alleles in the parent plants 1942). Corkill (1942) showed that the inheritance of (Hughes et a!., 1984). No evidence for the presence in cyanogenesis in white clover is diploid. Acyanogenic these plants of microsomes with different qualitative white clover plants fall into three phenotypic classes; properties was found and the results were consistent those which lack the cyanoglucosides, those which lack with the production of different quantities of micro- linamarase and those which lack both the cyano- somes (Hughes eta!., 1984). glucosides and linamarase. The presence or absence of There are several models for the nature of the Ac the two cyanoglucosides is determined by alleles at the locus which would give rise to non-functional alleles locus, Ac, whereas the presence or absence of lina- with these characteristics. Thus the Ac locus may marase is determined by alleles at the locus, Li. The represent a number of linked structural genes for the cyanogenic phenotype requires the presence of a func- cyanoglucoside biosynthetic pathway and the ac allele tional allele at both loci in the plant. The Ac and Li loci include mutations in at least three of these. An alterna- segregate independently of each other. tive model for the null ac alleles is that they represent A number of biochemical studies have been carried mutations in a sequence which controls the synthesis of out to characterize the non-functional alleles at these the enzymes for cyanoglucoside biosynthesis. The loci. Incomplete dominance at the biochemical level existence of Ac alleles, which results in reduced levels has been demonstrated for both Ac and Li (Hughes & of cyanoglucoside, suggests a controlling role for the 108 M. A. HUGHES locus. Furthermore, the intermediate levels in hetero- liii leavesandLi Li roots conflicts with the zero levels zygous Ac ac plants imply a cis-acting control function. ofenzyme activity in these tissues. There are two By carefully standardizing the Guiguard Picrate test possible explanations: firstly, both types of tissue for HCN, Corkill (1941) was able to identify high and contain low levels of an immunologically distinct non- low HCN-producing plants. The results of crossing cyanogenic /3-glucosidase (Hughes & Dunn, 1982; plants giving different picrate reactions (Table 1) Collinge & Hughes, 1982a; Hughes etat.,1985; Kakes indicated that the level of HCN produced was & Eettink, 1985).Becausesmall changes in the protein inherited. Corkill interpreted these results to indicate primary structure may result in major differences in the the presence of modifying genes, however, the exist- antigenicproperties, itis possible for linamarase cDNA ence of different functional Ac alleles, the probable toshow homology to a non-cyanogenic /3-glucosidase existence of different functional Li alleles, and the mRNA produced at low levels in these tissues. An intermediate HCN levels of heterozygotes, may alternative explanation is that linamarase mRNA is account for these results. produced at a low level in these tissues, which is detect- Although the simple modified Mend elian diliybrid able by Northern blot analysis but not at the protein segregation ratios for alleles of the Ac and Li loci are level. widely observed, some authors report aberrant ratios The cDNA clones have also been used to analyse (e.g. Till, 1987). These are often observed when work- genomic DNA using Southern blot analysis (Hughes et ing with plants from wild populations rather than at., 1990). Considerable restriction fragment length commercial cultivars and can sometimes be explained polymorphismexists in white clover for genomic by the very low levels of HCN produced by some wild sequenceshomologous to linamarase cDNA (Hughes plants and the limits of detection of the tests used. etaL, 1988)(Fig. 3). This polymorphism has been used toanalyse the genomic organizationof these sequences inthe Li Li plant used as a source of mRNA for Moleculargenetics cloning (Hughes et at., 1990). The studies show that Thebiochemical difference between Li Li and liii the white clover genome contains three genes with plants has been used to select linamarase clones from homology to linamarase cDNA and that at least two of white clover developing leaf cDNA libraries (Hughes these genes segregate independently. Analysis of the et at., 1990). The identity of the clones was established co-segregation of linamarase activity and the presence using hybrid select translation and immunoprecipita- of genomic restriction fragments identifies the genomic tion of the polypeptide product. The linamarase cDNA sequence specifying linamarase structure and indicates clones have been used in a number of studies. Northern either a structural or cis-acting control function for the blot analysis of mRNA shows that high levels of a 2.1 Li locus. kb molecule are produced in Li Li young leaves and The existence of a small multigenefamily for lina- very reduced levels seen in liii leaves and Li Li roots. marase is a paradox given the single diploid locus for Heterozygotes, (Li Ii) produced intermediate niRNA linamarase activity reported widely in the literature. levels in young leaf tissue. The presence of low but Onemodel for the Lilocus,which would be consistent measurable levels of linamarase homologous mRNA in with the data, is close linkageof the three structural genes.This is, however,refuted by the independent segregationof at least two of the genes. Two possible Table 1 The quantitative inheritance of HCN production explanationsfor the paradox are, firstly, there may be (after Corkil, 1941) inactive pseudogenes in the genome, and secondly, there may be homology between the linamarase gene Test reaction grade Parents picrate and other /3-glucosidase genes. grade 6 5 4 3 2 1 0 Distribution 2x0 0 0 0 3 8 1 17 2x0 0 0 4 32 18 2 44 Dadaypublished two papers in1954(Daday, 2x3 0 2 51434 23 0 1954a,b), which demonstrated a clear association 3x0 Numberof 0 0 472 221 0 between the frequency of cyanogenic morphs in 3x0 plantsin 0 0 3 16 29 2 0 natural populations of white clover and the mean 3x6 the progeny 11 6 3 4 9 0 0 6x0 01822 9 0 0 1 January isotherm. The association is such that popula- 6x0 738 31 0 0 0 tions at higheraltitudes and higher latitudes have lower 6x3 21128 8 1 0 0 frequenciesof cyanogenic plants. The relationship between altitude and frequency of cyanogenic plants THE CYANOGENIC POLYMORPHISM IN WHITE CLOVER 109 has been confirmed by Daday (1958) and a number of means that it is difficult to observe changes in gene other workers (Table 2). In addition, Foulds & Grime frequencies in natural populations. In addition there is (1972) showed a decreasing frequency of Ac with con- evidence that T. repens can show strong local speciali- ditions of soil moisture stress (drought). Linkage dis- zation (Gliddon & Trathan, 1985) and large differences equilibrium has been demonstrated for the Ac and Li in the local frequencies of Ac and Li have been loci (Table 2), indicating that it is the production of detected in The Netherlands (Kakes, 1987) which HCN which is the important character in determining could not be attributed to January temperature differ- the distribution of the alleles of these loci and, in fact, ences. most workers have assumed that the release of HCN is the selectively important phenotype. Although the forces pattern of distribution demonstrated by Daday has Selective been confirmed, the low rate of seedling recruitment in Afundamental question concerning the cyanogenic undisturbed populations (Turkington et al., 1979) polymorphism is whether the observed variation is

1 2 3 4 5 6 7 8 9101112131415161718

23.1 Fig. 3 Restriction fragment length polymorphism in white clover for 9.4 sequences homologous to linamarase 6.6 cDNA. Genotype Li Li track 1; geno- type Liii tracks, 2, 3,7, 9, 10, 11, 12, 4.4 16, 18; genotype liii tracks 4, 5, 6, 8, 13, 14,15,17. Genomic DNA digested with Hind III and fragments separated on 0.8 per cent agarose, A Hind III molecular weight markers in kilobases.

Table 2 Studies on distribution of cyanogenic phenotypes

Author Locality Phenotypes Factors studied Sample size*

Daday, 1954a Europe 4 Latitude (association with mean January isotherm) Daday, 1954b Europe 4 Altitude (association with mean January isotherm) Foulds & Grime, Derbyshire, UK 4 Soil moisture (decrease in Ac 50 1972 with drought) De Araujo, 1976 North Wales, UK 4 Altitude (30—48 m) (decrease in 25—64 Ac Li with increasing altitude) Ennos, 1982 Liverpool and 4 Linkage disequilibrium 53—104 Chester, UK Dirzo & Harper, North Wales, UK 2 Distribution of active slugs 1982b Boersma et ai., Southern France 4 Altitude (120—1170 m) (decrease 100 1983 in Li with increasing altitude) Kakes, 1987 Netherlands 4 Linkage disequilibrium 19—116 Till, 1987 Southern France 2 Altitude (120—830 m) (decrease in 50 Ac Li with increasing altitude) Till-Bottraud etai., Southern France 4 Altitude and time (120—1560 m, 12—151 1988 1978—1985) (decrease in Ac Li with increasing altitude)

*Per site. 110 M.A.HUGHES neutral or is subject to selection. The role of cyano- glucoside and hydrolytic enzyme are required for genesis in plants has been widely discussed (Jones, discriminatory feeding. However, the simple demon- 1981). The existence of the polymorphism argues stration of selective grazing does not reveal the com- against a role in primary metabolism and there is con- plexity of cyanogenesis as a plant defence reaction. siderable evidence that HCN, liberated during tissue Burgess & Ennos (1987) have demonstrated that slugs damage by small grazing animals, acts as a feeding taken from a site with a low frequency of cyanogenic deterrent (Table 3). In particular, there are eight morphs show a significantly greater degree of selective reports of selection against the acyanogenic morph by eating than slugs taken from a site with a high molluscs. In two studies (Dirzo & Harper, 1982a; frequency of cyanogenic morphs. These results Kakes, 1989) it has been directly shown that both indicate that the selective advantage of the cyanogenic

Table 3 Studies on selection against acyanogenic phenotype by herbivores

Species Seedling Field vs. Number vs. adultNumber ofAble to discriminateUnable to discriminate Author experimentalof plants plants phenotypesbetween phenotypesbetween phenotypes

Bishop & Korn, E 50 A 2 Helix aspera (n.) 1969 Agriolimax reticulatus (s) Crawford-Sidebotham,E 2 A 2 Anon hortensis (s) Cepea nemoralis (n), 1972 Anon ater (s) A. reticulatus (s), Cepea hortensis (n) H. apara (n) Helicella virgata (n) Thebapisana (n) Whitman, 1973 F 1000 A 2 Unknown herbivores Angseesing, 1974 E 30 A 2 A. ater(s) Anon subfi.iseus (s) A. reticulatus (s) Miller etal., 1975 F NG S 2 Field crickets, Small grass-hoppers Dritschilo eta!., 1979 F NG A 2 Aphis craccivora (i),Four species, Therivaphis tnifolii (i) homoptera, Two species aphid Dirzo & Harper, F/E NG A/S 4 Agriolimax 1982a carnanae (s)* A. reticulatus (s)* A. ater(s)* H. aspera (n)* Dirzo & Harper, F 10 A 2 Moluscs 1982b Ennos, 1981b F 120—160/siteA 4 Unknown factors Ennos, 1985 F 640 S 4 Unknown factors Horrill & Richards, E 125/exp. S 2 A. hortensis(s) 1986 Burgess & Ennos, E NG A 2 Deroceras 1987 reticulatum (s) Kakes, 1989 F/E 342 JVS 4 H. aspera (n)* C. nemoralis (n)* Mowatt & Shakeel, F NG A 2 Weevil larvae 1989 (Sitona spp.) Leather jackets Slugs *Needs both glucoside and enzyme. F= field trials; E =experimentaldata; A= adult; S =seedling,n =snail;s =slug;i =insect;NG =notgiven. THE CYANOGENIC POLYMORPHISM IN WHITE CLOVER 111 morph under grazing by the slug Deroceras reticulatum ing selection and the results of these studies are is likely to be frequency dependent. equivocal (Paim & Dean, 1975) (Table 4). The only Three groups of Lepidoptera (the Zygaenidea, character which has been noted by more than one Heliconiini and Acraeinae) feed selectively on plants author, and which may provide a selective advantage to containing cyanoglucosides (Raubenheimer, 1989). the acyanogenic morph, is flowering. In fact Caradus et These insects release HCN when crushed and it has a!. (1989), in a classification of 109 white clover culti- been shown (Nahrstedt & Davis, 1986) that in Zygaena vars, also notes a significant trend for the highly cyano- trifolii the chemical basis of cyanogenesis is the cyano- genic cultivars to flower earlier but have a lower glucosides, linamarin and lotaustralin. Interestingly, Z. maximum number of flowers. Daday (1965), in an trzfolii can both sequester these cyanoglucosides from experimental study, shows increased flower production the host plant and synthesize them de novo. of the ac ii phenotype in cool conditions compared to Clearly understanding selection in herbivore/plant the cyanogenic (Ac Li) phenotype. Experimental data systems also requires a study of the effect of the plant which demonstrate a selective advantage of the acyano- on the herbivore before a complete understanding can genic morph, based on the 'metabolic costs' of cyano- be achieved. Another indication of the complexity of genesis, do not exist, neither is there any convincing cyanogenesis as a defence reaction is the demonstra- data to demonstrate a differential effect of frost tion by Lieberei et a!. (1989) that cyanogenesis in the damage on the two morphs, although both factors have rubber tree inhibits the active defence reaction of the been widely discussed in the literature. plant to a fungal pathogen (Microcyelus ulei). Despite the early demonstration of inherited quanti- The striking association between the frequency of tative variation in HCN production by Corkill (1942) cyanogenic plants and the mean January isotherm and the later biochemical studies (Hughes et a!., 1984, suggests that a balancing environmental factor selects 1985) very few of the ecological genetic studies of the against the cyanogenic morph thus maintaining the cyanogenic polymorphism in white clover have polymorphism in white clover. However, compared examined HCN production quantitatively. Figure 4 with selection against the acyanogenic morph, there are (M. A. Hughes & R. A. Ennos, unpublished) shows the fewer experimental and field studies of possible balanc- levels of cyanoglucoside present in young leaves of

Table 4 Studies on selection for acyanogenic phenotype

Factors investigated Seedling Field vs. Number vs. adult Number of No Author experimental of plants plants phenotypes Selection selection

Daday, 1965 F/E 325F A 4 Flowering in Vegetative 28—8 1E cool conditions growth Foulds & Young, E 20 A 2 Frost, drought 1977 (respiration and photosynthesis) Wilkinson & E 4 A 4 Stemphylium Millar, 1978 sarciniforme (pepper spot) Dommee et a!., E 72 A 4 Root growth 1980 Ennos, 1981a E 39x4 A 4 Competition! Leaf size interaction (Ii) Dirzo & Harper, F 10 A 2 Frost, flowering, 1982b Uromyces trifolii (rust) Dirzo, 1984 F NG A 2 Competition under artificial grazing Jarvis & Hatch E 90 S 2 Aluminium 1987 Kakes, 1989 F/E 342 A 4 Flowering

F= field trials; E =experimentaldata; NG, not given; A= adult; S =seedling. 112 M.A.HUGHES plants taken from two populations collected from picrate or Feigl—Anger tests, was weak in populations different altitudes and containing different proportions (600, 830 m) with low frequencies of cyanogenic of cyanogenic plants. The plants were grown in the plants. Furthermore, when different leaves of the same same environment for more than 12 months before leaf plant were tested, a proportion of the plants from these samples were taken. The number of heterozygous two populations showed both a positive and negative plants in each population is not known and can only be test reaction ('intraindividual heterogeneity'). This deduced from the Hardy—Weinberg equation. Thus the result is most easily explained by intraplant variation Cheviot population contains a higher proportion of (Collinge & Hughes, 1 982b) reducing levels of gluco- heterozygous (Ac ac) plants, and this will contribute to side or linamarase below the level of detection of the the marked difference in cyanoglucoside levels tests used. The phenomenon of 'Intraindividual hetero- between the two populations. Caradus eta!. (1989) geneity' was most marked with the cyanoglucoside-only also found that high HCN potential was clearly associ- phenotype, as would be expected from this explana- ated with cultivars which have a high frequency of Ac tion. The heterogeneic phenotype was also shown to be Li phenotypes. In contrast, Boersma et a!. (1983) inherited, consistent with the demonstration of inheri- examined levels of linamarase activity in plants taken tance of different levels of cyanoglucoside (Hughes et from populations at three altitudes in Southern France at., 1984). (800, 1,000 and 1,170 m). Although considerable differences between plants in enzyme activity were Evolution shown, no difference between the two populations con- taining cyanogenic morphs was seen. However, one Severalother species of Trifolium are cyanogenic sample only contained four linamarase-positive plants. (Table 5). These are all in the subsections Lotoidea and Till (1987) studied four French populations (120, Platystylium of section Lotoidea of the genus Trifolium 170, 600 and 830 m) where the frequency of cyano- (Zohary & Heller, 1984). In a survey of 31 species no genic plants varied from 80 to 4 per cent. She found other HCN-producing Tnfolium species have been that the cyanogenic reaction, as measured by either the found (M. A. Hughes, unpublished observations).

Fig. 4 Comparison of cyanogenic 6 glucoside content of plants from a 5 lowland (UTooting61 m) and an a4 upland population (0Cheviot610 m) 0 of white clover. The cyanoglucoside U 3 E 2 content of young leaves was measured colorimetrically using the method of 0 ______Hugheseta!. (1984). All plants had the I I I I I I I I I I I I I I I .I I Ii glucoside only phenotype (Ac ii) and o I 2 3 4 5 6 7 8 9 10 II12 13 4 5 1617 118 were selected using the picrate test Cyariogenic glucoside content (nmol mg protein) (Corkill, 1940). Table5 Classification of Trifolium species for cyanogenesis

Presence of Presence of glucoside hydrolytic enzymes

Species 2n Gibson et al.* Gibson et al.* T.unifiorum 32 N + N 0 T. nigrescenst 16 + + + + T. isthmocarpum 16 + + P + Tmontanum 16 0 P 0 0 T.ambiguum 16,32,48 + P 0 0 T.repens 32 P P P P T. occidental4 16 + + 0 0

*Gibson eta!., 1972. tlncludes subspecies petrisavii and meneghinianium. tSpecies hybridized with T. repens. §Trace amounts; N =nottested; P= polymorphic. THE CYANOGENIC POLYMORPHISM IN WHITE CLOVER 113

Three species (T ambiguum, T. isthmocarpum, T siderable variation in the cyanogenic system of white montanum) have been shown to be polymorphic for clover which is not described by the simple test for the either the presence of the glucoside or the hydrolytic presence versus absence of HCN commonly used to enzyme. It is likely that all reports of the glucoside-only detect and study the polymorphism. It is unlikely that a phenotype in a species, for example in T. occidentale, complete understanding of the distribution and indicate polymorphism for the cyanogenic character, maintenance of the cyanogenic polymorphism will be particularly given the small number of plants tested by elucidated without consideration of the quantitative each author. All the species that have been hybridized variation in HCN production. In addition, the ability to with T repens (Chen & Gibson, 1972; Gibson et al., detect allelic differences at the DNA level, using 1972) are cyanogenic but there are three additional restriction fragment polymorphisms, will provide cyanogenic species not known to hybridize with T. further insight into the relationships of particular repens. The T. repens linamarase cDNA clone has been alleles and the ecogenetics of each cyanogenic morph. shown (M. A. Hughes & T. Carron, unpublished data) A combination of ecogenetic and molecular studies to have homology to genomic sequences in T nigres- may also resolve one unexplained feature of the genetic cens and T. isthmocarpum. It would be interesting to control of the cyanogenic polymorphism, namely the know the relationship of the linamarase genes in these independent assortment of the Li and Ac loci. species, as this may provide valuable information on speciation in the genus. The taxonomic significance of References cyanogenesis in the genus Lotononis (Leguminosae) has been discussed by Van Wyk (1989). In this genus ANGSEEING, J.A.P.1974. Selective eating of the acyanogenic basic groups are either cyanogenic or acyanogenic but form of Trifolium repens. Heredity, 32, 73—83. some groups contain both cyanogenic and acyanogenic ARMSTRONG, H. E., ARMSTRONG, E. F. AND HORTON, E. 1913. species. This feature of the distribution of cyanogenesis Herbage studies II —Variationin Lotus corn iculatus and in Lotononis is similar to the pattern seen in Trifolium. Trifolium repens (cyanophoric plants). Roy. Soc. (Lond.) As the ability to produce HCN is correlated with Proc. B, 86, 262—269. ASKEW, H. o. 1933. Determination of hydrocyanic acid in morphological variation, further genetic and molecular white clover. NZJ. Sci. Technol. B, 14, 359—365. information may provide evidence for the infrageneric BISHOP, J. A. AND KORN, M. E. 1969. Natural selection and cyano- classification of Trifolium. genesis in white clover, Trzfolium repens. Heredity, 24, 423—430. Conclusion BOERSMA, P., KAKES, P. AND SCHRAM, A. w. 1983. Linamarase and f3-glucosidase activity in natural populations of Trifolium Thegenetic units that control the cyanogenic poly- repens. Acta. Bot. Neerl., 32, 39—47. morphism in white clover are complex and provide a BURGESS, R. S. L. AND ENNOS, R. A. 1987. Selective grazing of model system for the study of the organization and acyanogenic white clover: Variation in behaviour among control of plant genes which determine this type of populations of the slug Deroceras reticulatum. Oecologia, metabolic process. The biochemical characterization 73, 432—435. of null alleles of both the Ac and Li locus is not com- CARADUS, J. R., MACKAY, A. C., WOODFIELD, D. R., VAN DEN BOSCH, J. AND WEWALA, S. 1989. 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