Barley B-amylase and B-glucanase activities at germination in vulgare-type lines from backcrosses of wild, spontaneum strains with cv. Adorra Hannu Ahokas and Maria J. Erkkilä Ahokas, H. & Erkkilä, M.J, 1992. Barley B-amylase and B-glucanase activities at germination in vulgare-type lines from backcrosses of wild, spontaneum strains with cv. Adorra. Agric. Sei. Eini. 1: 339-350. (Agric. Res. Centre of Finland, Inst. Plant Breed., SF-31600 Jokioinen and Univ. Helsinki, Dept. Genetics, Arkadiankatu 7, SF-00100 Helsinki, Finland.) Two hundred and nineteen lines derived from the backcross progenies of eight differ- ent Hordeum spontaneum strains were evaluated for B-amylase and B-glucanase activ- ity five days after germination under aseptic conditions. The activities were deter- mined on the basis of soluble protein or grain mass. The recurrent parent cultivar, Adorra, served as the standard. Putative recombinants with high B-amylase activity were relatively easily achieved from high-activity strains ofH. spontaneum. Recombi- nants with high 6-glucanase were rare. They appear to be eliminated, possibly due to the strong selection for the domesticated phenotype during the derivation process. Key words: barley breeding, carbohydrate hydrolases, gene linkage, genetic resources, Hordeum spontaneum, malting barley Introduction The earliest recognition of the wild progenitor of barley (Hordeum vulgare ssp. spontaneum , or H. spontaneum) as a source ofgenetic variation of the grain constituents dates back to the 19705. Breeders have largely ignored the use ofwild barley as breed- ing material, supposedly because of the following reasons: highly shattering spikelets, need of manual cleaning of grains, vernalization requirement for growing under long-day conditions, apparent low grain yield and limited availability of material from the original habitats. Biologically H. spontaneum belongs to the same species as the domesticated barley. Shattering of spikes and firmness of awns appear predominantly in the Fj’s and subsequent generations after crossing with the domesticated barley, necessating extra careful manual work. The chemical composition of the wild barley grain attracted attention when high protein contents were observed in material from in original habitats in Israel (Ladizinsky 1975) and cultivated sources (Ahokas 1977). Variations were described in the protein and lysine contents (Ahokas 1982); the lysine content was later reported to be principally under polygenic control (Ahokas 1990). Corke and Atsmon (1988) observed significant positive correlation of lysine with most of the amino acids in wild barley genotypes. Many different hordein patterns were shown to be present (Doll and Brown 1979, Nevo et al. 1983) and to correlate with environmental parameters in Israel (Nevo et al. 1983). The grain mineral contents were mostly higher in a wild barley than in a standard cultivar 339 Agric. Sei. Fint. 1 (1992) https://www.c-info.fi/en/info/?token=w0gMRZtFG_AUJbeM.ITvak_4nkdaIoZHC4THmzw.RKIInFZaRwXmFSv81WWlHI5-vaJxbvGOzy58eAXmZeXu202_EqU2EHLgO1uuWYtGTPL1z78aW3gIVHG1N7TQ6iUsNtknCRwHltki9lBP7pEAdUyRueGSQtz5woW9H1Ji0Jb1QnQdHbsuflqG0WHUJAdIbz6nH0T-HAI4EmPWzHlGvk9ymzVkbe92HB213TFNC5BMjmEERIHA98oL3qxWLYQxyJPbe1nQxwTkZIyluPMaAzPcCyN-7Z7kdFgC_qO54pa7qH6YEBCgJd6wWp2maY3E0FWgWaDfbdUfGv4 (Friedman and Atsmon 1988). Wild barley was shown to be a rich genetic source of variation of trypsin inhibitor in endosperms (Kirsi and Ahokas 1983), while 22 Finnish cultivars or breeding lines of barley were relatively uniform (Kirsi 1973, Kirsi, personal communication). Of the enzymes of the germinating grains interest- ing to brewing or malting, unique a-amylase iso- enzyme patterns, especially at the a-amylase 2 locus, have been detected in wild barley in addition to the normal ones (Brown and Jacobsen 1982). Of the six enzyme activities studied in vitro among 175 strains of wild barley R-amylase and B-glu- canase varied most widely in extracts of wild bar- leys germinated for five days (Ahokas and Nas- kali 1990 a). The activity estimates for five en- zymes of 242 strains mostly correlated with the moisture parameters of the territory of origin in the Near East (Ahokas and Naskali 1990 b). So far, little has been published on the transfer of genes responsible for grain quality from wild bar- ley to domesticated backgrounds (Brown et al. 1988,Ahokas 1990,Ellis et al. 1991, Macleod et al. 1991), while the crossing programmes to trans- fer disease resistance genes to domesticated barley are older. Nevo (1992) has recently published a review on disease resistance and other useful sources in H. spontaneum. We report here the recombination of genotypes responsible for high activities of two enzymes, 6-amylase and 6-glu- canase, in some of the backcrossed progenies. The selection of genotypes for domesticated barley morphology had been done for five generations. Material and methods The grain material was derivedfrom the crossing of H. spontaneum/2*Adorra by Hannu Ahokas since 1978. The grain material used here was produced in single row plots of a length of about 1 m, average plant distance about 4 cm and row distance 20 cm, planted with a Plot Spider (Wintersteiger) planting machine. Cv. Adorra was grown as the standard usually two rows per 120 rows, the nearest Adorra sample was used as the standard for the selected lines the yield of which represented the BC-F6 generation. The material was grown in 1985 on the field of Anttila Experimental Farm on gyttja clay soil under the standard field practice of the Hank- kija Plant Breeding Institute. The rows ofmorphologically uniform and accept- able lines were harvested in binds by hand, dried under air stream at room temperature, and threshed with a Saatmeister (Kurt Pelz) seed cleaner. After storage for about one year at room temperature the grains were stored at about -15°C for four years. Germination was good, with no postharvest dor- mancy. Undamaged grains were husked manually, sterilized and rehusked with a treatment of 50% H2 S04, and germinated aseptically for five days at 15°C in the dark as described elsewhere (Ahokas and Naskali 1990 a). The extracts of the homoge- nates of the germinating grains in toto were used for the enzyme assays. The activity of B-amylase was assayed using a Betamyl kit (Biocon) which is an equivalent of the Testomar-Amylase Mono kit we used previously (Ahokas and Naskali 1990 a). The activity of 6-glucanase was assayed as describ- ed by Ahokas and Naskali (1990 a) with azo-bar- leyglucan (Biocon). The samples were worked in groups of twelve, each including an Adorra stand- ard from the nearest row. The protein content of extracts was determined with the BCA assay (Smith et al. 1985) using a reagent purchased from Sigma and bovine serum albumin (Sigma A-4503) as the standard. The ‘BS’ strains have previously been referred as ‘79BS’ (Ahokas 1981), but for simplicity appear here in the short form. Results The number of plants obtained from the BC-F 2 population of H. spontaneum/2*Adorra lies in the range of 1 to 4%. The selection criteria were rachis toughness, grain abscission from rachis, softness of awns and spring growth habit under long-day con- ditions. Selection took place at harvest by feeling each non-shattered mature spike. The same criteria were applied in the subsequent generations, where 340 Agric. Sei. Finl. 1 (1992) Agric. Sei. Finl. 1 (1992) increasing importance was also given to straw char- acteristics. The selected lines used here would in most cases need a single additional crossing step with a current elite cultivar to breed a competitive cultivar with properties of the current high-enzyme lines. The strict selection for morphology usually tends to reduce drastically the final number of F 6 lines obtained. The present material would therefore not show recognizable genes in any expected propor- tion. Without selection the allele frequency of the cultivar Adorra would be 0.75, and that of the wild partner 0.25. We consider bimodality of the activity distribu- tion with another mode at the high-activity side to represent a recombination of a high-activity allele from the wild partner of the cross. The distribution is probably affected also by modifying genes, or inhibitors or activators in the extracts would inter- fere with the activity measurement. The determina- tions based on grain mass are more meaningful for breeders and farmers, while those based on protein content may be interesting to biochemists and industrial applications (Table 1, Figs. 1-8). The high protein contents of wild barleys seemingly lower the activities when calculated for protein and compared with domesticated barley which has some 50% of the protein content of wild barley (Table 1). When the protein content is lowered as a consequence of the segregation in the offspring, segregants with high enzyme activities for protein are unmasked. The activities available from the original acces- sions ofwild barley (Table 1) are not fully compar- able with the present determinations, because of the somewhat different growing conditions and the dif- ferent kit used for determination of 6-amylase. However, these determinations are valuable when assessing the usefulness of screening of the mater- ial before assuming a breeding programme. The activity of the wild barley accession BS 06-4, the seed ofwhich was originally collected in bulk with BS 06-2 by Prof. Moshe Feldman in the same site in Israel has not been determined. BS 06-4 is also considered to be interesting, since BS 06-2 and BS 06-4 are phenotypically similar. The enzyme activities are presented in Figures 1- 8. The range of 0.3 to 1.7 for relative activity is expected to include practically all the segregants with an activity of the Adorra level, if distributed normally. Activities 1.7 times or more that of cv. Adorra were measured for 6-amylase in lines ofBS 05-1 (for protein and grain mass), those ofBS 06-2 (for grain mass), those of BS 06-4 (for protein and grain mass), those ofBS 13-3 (for protein and grain mass), those ofBS 16-2 (for grain mass) and in those ofR 1163-07 (for protein and grain mass). A second mode is evident at the high-activity ends of the Table 1. Determinations ofB-amylase and B-glucanase activity in the seed parent accessions ofwild barley used for the deri- vation of the present material. B-Amylase was determined with a Testomar-Amylase Mono kit and B-glucanase by the present method. (Ahokas and Naskali, unpublished data.) Accession 6-Amylase (U)* B-Glucanase (mil)* For protein For grain mass For protein For grain mass (mg) (g) (mg) (g) BS 05-1 14.6 [0.92] 613 [1.65] 17.5 [0.79] 734 [1.40] BS 06-2 16.4 [1.05] 766 [2.28] 32.9 [1.53] 1539 [3.31] BS 13-3 9.9 [0.35] 518 [0.83] 23.7 [1.45] 1240 [2,92] BS 16-2 16.1 [o.Bl] 665 [1.73] 21.4 [0.84] 880 [1.78] PI 391105 8.5 [0.63] 205 [0.73] 32.4 [1.40] 786 [1.64] R 1161-06 16.2 [0.67] 906 [2.02] 34.5 [1.79] 2100 [5.85] R 1163-07 16.2 [0.60] 992 [1,57] 25.2 [1.29] 1544 [3.23] * Values in brackets are the calculated equivalents to units in Figs. 1-8. 341 Agric. Sei. Finl. 1 (1992) 342 Figs. 1-8.Distribution of the activities ofB-amylase (a and b) and B-glucanase (c and d) among the selected lines in relation to the recurrent parent, cv. Adorra as the standard sample, whose value equals 1.0. Plots (a) and (c) have been calculated for protein and plots (b) and (d) for grain mass. The ordinate indicates the sample size. - Fig. 1. Progeny of the cross BS 05- I/2*Adorra. Several high-activity lines for B-amylase appear in the mode at about 1.6 in (a) and 1.75 in (b). distributions of 13-amylaseactivities for the derivati- ves ofBS 05-1, BS 13-3,BS 16-2, R 1161-06 and R 1163-07 (Figs, lb, 4a, b, sb, 7a,b and Ba, b). For 13-glucanase, the activity level 1.7 times that of cv. Adorra was reached only in the offspring ofBS 06-2 (Fig. 2c, d). Strains BS 05-1 and R 1161-6 might also be useful cross partners in breeding an increased 13-glucanase activity (Figs. Ic, d, and 7c, d). Fig. 2. Progeny of the cross BS 06-2/2*Adorra. High values observed especially for B-glucanase activity with an apparent high-activity mode at 1.45 in (c) and 1.4 in (d). Fig. 3. Progeny ofBS 06-4/2*Adorra. High-activity lines ofB-amylase in (a) and (b) 343 Agric. Sei. Finl. 1 (1992) Agric. Sei. Finl. 1 (1992) 344 Fig. 4. Progeny ofBS 13-3/2*Adorra. An apparent high-activity mode ofB-amylase at 1.6 in (a) and 1.55 in (b). Fig. 5. Progeny of BS 16-2/2*Adorra. High-activity lines ofB-amylase in (b), Fig. 6. Progeny ofPI 391105/2*Adorra. No great deviations from normality. The accession PI 391105 was selected because of its relatively high B-glucanase activity (Table 1). 345 Agric. Sei. Finl. 1 (1992) Fig. 7. Progeny ofR 1161-06/2*Adorra. R 1161-06 is evidently a promising parent for increasing B-amylase and B-glucanase activities. Fig. 8. Progeny ofR 1163-07/2*Adorra. R 1163-07 is evidently a promising parent for breeding high activity of B-amylase. 346 Agric. Sei. Finl. 1 (1992) Discussion The germination timeof the sterilized grains under axenic conditions was 120 hours or five days and was chosen as a compromise. The activity ofB-glu- canase is known to increase substantially still at a later stage of germination (Preece and Hoggan 1956, Bourne and Pierce 1970). Inversely, 6-amy- lase occurs at the same level in resting grains as in theirmalt (Visuri and Nummi 1972), and another 6- amylase gene may be present in non-grain tissues (Kreis et al. 1988). Of the stored B-amylase, about 60% is bound and mainly released between days 1 and 3 of germination (Sopanen and Lauriere 1989). The 6-amylase appears in proteolytically processed forms in barley (Lundgard and Svens- son 1987). The extent of release and processing of B-amylase after five days may be the sources of variation of activity other than that caused by the alleles themselves. Two (Woodward and Fincher 1982), or pos- sibly three, (Stuart et al. 1986) isoenzymes of 1,3;1,4-B-endoglucanases are present in germinat- ing barley grain, two of thembeing codedby differ- ent gene loci (Loi et al. 1988, Slakeski et al. 1990, Macleod et al. 1991). The activity level in vitro and possibly also in vivo is, however, often modified by specific or un- specific inhibitors or activators, making the segre- gation of the enzyme genes less clear. A high concentration of an endogenous inhibitor may also mask the product of a high-activity enzyme allele in the original wildbarley line. The level of B-glu- canase mRNA in barley aleurones is controlled by natural phytohormones - gibberellin increasing and abscisic acid decreasing it - during germination (Letts et al. 1990). The protein 6-amylase posses- ses an enzyme inhibitor capability in sweet potato (Pan et al. 1988). After five generations ofselling of the back-cross- ed derivatives, one line in 16 is expected to carry without selection both the parentally different alle- les as well as their heterozygotes in the ear-to-row model. This frequency of heterogeneity is not expected to abolish the value of the activity deter- minations for a distributionpattern. The genes for brittlerachis, probably concerning also H. spontaneum, have been allocated to chro- mosome 3 (Takahashi and Hayashi 1964). Thus, the strong selection pressure for the domesticated non-brittle rachis type in these cross derivatives is not assumed to affect the distribution of the loci for 6-amylase and 6-glucanase known to reside in other chromosomes. A difficulty might be expected to arise from the tight linkage of the winter habit gene and the B-amylase gene on chromosome 4 (Chojecki et al. 1989). The Israeli H. spontaneum barleys require vernalization when grown in Fin- land, but most proveniences set spikes without ver- nalization in original habitats in Israel (Prof. Dan Atsmon, personal commununication). The reason for this difference must be the response to the day length. In Israel, H.spontaneum is a short-day plant with spike initiation in winter or early spring, while under the almost continuous daylight in the early season in Finland the plants behave as long-day plants. The switch from short-day to long-day res- ponse is probably caused by the vernalization. It is not known, whether the linked winter growth habit locus on chromosome 4 is associated withphotope- riodic responses among the three loci which deter- mine the spring/winter growth habit on chromoso- mes 4, 5 and 7 (Takahashi and Yasuda 1971). With some wild barley genotypes, the recombina- tion of the 6-amylase gene into spring barley back- ground may be problematic. When there is pollen carrying different alleles of 6-amylase available at pollination, one of the geno- types can be at a relative advantage as compared with the other pollen carrying the different B-amy- lase allele, as shown for a backcross in barley, resulting in an unequal distribution (Pedersen 1988). Such a distortion of segregation may occur in heterozygotes of the 6-amylase gene in the pre- sent material. Bimodality of activity distribution with high- activity variants was observed in most crosses where the original wild barley used was known to have a high activity in 6-amylase, but more rarely in 6-glucanase. This may mean that the 6-glucanase 347 Agric. Sei. Finl. 1 (1992) gene is linked with genes discriminated when selecting for the domesticated phenotype, but such an association is not present in B-amylase genes in- troduced from the wildbarley to the derivative. The high-activity lines should be repeatedly studied after regrowing in order to verify their usefulness. Especially with B-amylase we can assume seasonal variation of activity, since it is a storage protein. With another wild barley strain studied later, we have direct evidence at the DNA level using restriction endonuclease fragmentation and probing with a barley B-amylase specific probe, the large Accl fragment of the plasmid pcBCSI (Kreis et al. 1987), that a different B-amylase allele has been recombined in a backcross derivative, also showing increased enzyme activity (unpublished data). Among the Fj’s of 248 Israeli H. spontaneum accessions crossed with cv. Adorra by Ahokas (1981) there were 4.8% partially sterile whose pattern fitted with translocation heterozygosity. Translocation heterozygosity is known to cause a variable degree of floret sterility, in the 35 cases in barley on average 42% (Burnham et al. 1954). After a backcross, the level of 4.8% of rearrange- ments is not necessarily any significant problem for breeding, unless a specific gene is linked with the translocation breakpoint, or a selected translocation homozygosity is associated with an inferiorbehavi- our as a cultivar. In the original habitats of wild barley, 6-glu- canase and especially B-amylase show negative correlations between parameters describing the availability of moisture in the environment and enzyme activity (Ahokas and Naskali 1990 b). The reason for this must be adaptive. Domesticated barley does not necessarily show such a correla- tion, because its life span is different, involving i.a. storage of the grains sheltered and not in the soil. Acknowledgements. We are obliged to Prof. Erkki Kivi for the opportunity to grow material in the field of the Hankkija Plant Breeding Institute. Ms. Anneli Kaseva helped prepare the graphs. The study was partly supported with grants from the Foundation for Biotechnical and Industrial Fermentation Research and from the Ministry ofAgriculture and Forestry. H. Ahokas’ study was partly done under the auspices of the Academy ofFinland. References Ahokas, H. 1977. Lysine and tryptophan content of some high-protein stocks and species of Hordeum. Hereditas 86: 143-145. 1981. Cytoplasmic male sterility in barley. X. Distribu- tion ofmsml fertility restoration ability in the wild pro- genitor ofbarley in Israel. Ann. Bot. Fenn. 18: 313-320. 1982. Variation of kernel protein and lysine in the wild progenitor ofbarley. Hereditas 96: 29-37. 1990. Hordeum spontaneum as a source of variation of grain composition. Sver. Utsädesför. Tidskr. 100: 86-87. & Naskali, L. 1990 a. Variation of a-amylase, 6-amy- lase, B-glucanase, pullulanase, proteinase and chitinase activity in germinated samples of the wild progenitor of barley. J, Inst. Brew. 96: 27-31. & Naskali, L. 1990 b. 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Genetics of earliness and growth habit in barley. In: Nilan, R.A. (ed.). Barley Genetics 11. Washington State Univ. Press, Pullman, WA. p. 388- 408. Visuri, K. & Nummi, M. 1972. Purification and characteri- sation of chrystalline B-amylase from barley. Eur. J. Biochem. 28: 555-565. Woodward, J.R. & Fincher, G.B. 1982. Purification and chemical properties of two 1,3;1,4-6-glucan endohydro- lases from germinating barley. Eur. J. Biochem. 121: 663-669. Manuscript received June 1992 Hannu Ahokas Maria J. Erkkilä Agricultural Research Centre ofFinland Institute of Plant Breeding SF-31600 Jokioinen, Finland 349 Agric. Sei. Fint. 1 (1992) SELOSTUS Itämisaikaisen 1.1-amylaasin ja B-glukanaasin aktiviteettimuuntelu linjoissa, jotka on jalostettu spontaneum-\illiohrien takaisinristeytyksistä Adorra-lajikkeella Hannu Ahokas ja Maria J. Erkkilä Maatalouden tutkimuskeskus ja Helsingin yliopisto Villiohran (Hordeum spontaneum) takaisinristeytyksestä viljaohran Adorra-lajikkeella on valintajalostuksella kehi- tetty viljaohran kaltaisia linjoja, joiden perimästä laskennal- lisesti on 25 % villiohrasta. F 6-polvea edustavasta, idätetystä jyvämateriaalista on määritetty B-amylaasi- ja B-glukanaa- siaktiivisuudet in vitro yhteensä 219:sta linjasta jyvämassaa ja liukoista proteiinia kohti laskettuna. Linjat perustuvat kahdeksaan eri villiohrakantaan, joista seitsemässä on löy- detty korkea aktiivisuus jommastakummasta entsyymistä tai molemmista. Rekombinanttilinjoja, joihin on todennäköisesti siirtynyt korkea B-amylaasiaktiivisuus, saatiin suhteellisen usein lähes kaikista korkean B-amylaasin omaavien villiohrien ris- teytyksistä. Korkea B-glukanaasiaktiivisuus periytyi linjoi- hin harvinaisemmin. Ohran B-glukanaasigeeni saattaa olla kytkeytynyt johonkinvillinkasvin ominaisuutta määräävään geeniin, jonka voimakkaan karsimisen kautta on usein menetetty myös villiohran B-glukanaasialleeli valintapol- vien aikana. Siten B-glukanaasialleelia rekombinoitaessa villiohrasta viljaohramuotoihin on käytettävä varsin laajaa materiaalia. Kun aktiivisuudet lasketaan näytteen proteiinia kohti, villiohran korkea proteiinipitoisuus (tavallisesti yli 20 %) saattaa näennäisesti madaltaa suurenkin entsyymiaktiivi- suuden. Osassa rekombinanttilinjoja alentunut proteiinipi- toisuus sinänsä tuo esiin korkean, proteiiniin suhteutetun entsyymiaktiivisuuden. 350 Agric. Sei. Fint. 1(1992)