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Amplified Fragment Length Polymorphism (AFLP) Markers for Barley Malt Fingerprinting
Journal of Cereal Science 29 (1999) 257–260 Article No. jcrs.1999.0242, available online at http://www.idealibrary.com on
Amplified Fragment Length Polymorphism (AFLP) Markers for Barley Malt Fingerprinting P. Faccioli, N. Pecchioni, A. M. Stanca and V. Terzi Experimental Institute for Cereal Research, Section of Fiorenzuola d’Arda, Via S. Protaso 302 I-29017 Fiorenzuola d’Arda (PC), Italy Received 17 July 1998
ABSTRACT Barley is the cereal par excellence in the production of malt for beer and other alcoholic beverages. In this work, biochemical (hordein A-PAGE) and molecular (RAPDs, AFLPs) markers have been compared for their efficiency in malt fingerprinting. To overcome the difficulties related to the quality of the DNA extracted from grain malt, malted coleoptiles are proposed as useful tissue for the extraction. On the basis of our data, we exclude a fingerprinting system based on A-PAGE hordein analysis because of the degradation of these proteins during the malting process. Comparing molecular markers for their efficiency and repeatability, we indicate AFLP analysis based on template DNA extracted from the tissue of grain malt coleoptile as an excellent tool for malt fingerprinting. 1999 Academic press Keywords: barley, malting quality, molecular markers, fingerprinting.
INTRODUCTION Malt is the result of controlled germination of cereal kernels, which are initially steeped in cold aerated water, followed by 3–5 days of germination. The properties and flavour of the malt are then fixed by kilning, which dries the grains. At the end of these technological processes, the kernels become malt: a very rich source of sugars, aminoacids and enzymes that is predominantly used for beer preparation, but also for distilling and as an integrator in food products1. The malting aptitude of barley, the most important cereal for it, depends on a host of mainly gene-related quality traits, this means that specific varieties are used by brewers2,3. Acid polyacrylamide gel electrophoresis (APAGE) of barley hordeins (prolamins), together with a complex of morpho-physiological descriptors, has been adopted for varietal classification by the International Union for the
Corresponding author: P. Faccioli. 0733–5210/99/030257+04 $30.00/0
Protection of New Varieties of Plants (UPOV), is also listed in the official International Seed Testing Association (ISTA) methodology4 and is accepted as the standard procedure by the European Brewery Convention. Molecular markers have become, over the last few years, a new and promising method for barley fingerprinting, which is based not only on restriction fragment length polymorphisms (RFLPs) but particularly on polymerase chain reaction (PCR)generated markers5,6 such as random amplified polymorphic DNAs (RAPDs)7,8, sequence tagged sites (STSs)9, microsatellites10 and, more recently, amplified fragment length polymorphisms (AFLPs)11,12. The success of these approaches is related to the fact that molecular markers are theoretically available in unlimited number, are environment independent, often quick and easy to use and explore a higher portion of variability with a consequently more consistently detectable level of polymorphism compared to morpho-physiological descriptors or biochemical markers9,13. The present study reports for the first time the characterisation of barley malt using different approaches so as to 1999 Academic Press
P. Faccioli et al.
258
identify the best method for checking the corresponding barley variety with the minimum time required and the maximum reliability.
Micromalting
Amplification was performed in a Perkin Elmer Cetus Gene Amp PCR System 9600 programmed as follows: 1 cycle of 2 min at 94 °C, 1 min at 36 °C, 2 min at 72 °C; 46 cycles of 1 min at 94 °C, 1 min at 36 °C, 2 min at 72 °C. The amplified products were separated by 1·5% agarose gel electrophoresis in TBE buffer and stained with ethidium bromide. AFLP analysis was performed using Gibco BRL AFLPTM Analysis System I and the corresponding protocol with minor modifications. The primer combinations used were the following: E37 (5GACTGCGTACCAATTCACG)/M40 (5-GATGAGTCCTGAGTAAAGC) and E37/M33 (5GATGAGTCCTGAGTAAAAG). The PCR products were separated onto a 4·5% polyacrylamide gel in TBE buffer at 40 W for 1 h. The gel was then dried for 1 h at 80 °C and autoradiographed for 2 days on Kodak X-ray film.
The preparation of malt by using 20 g of clean, sieved barley seeds was performed as summarized below14:
Data analysis
EXPERIMENTAL Genetic material Fifteen barley varieties for malting (Alexis, Magda, Gitane, Apex, Aura, Carina, Kaskade, Krona, Orchidea, Prisma, Tremois, Arco) and feed (Baraka, Arda, Kelibia) were randomly chosen. Young shoots from pot-grown plants and coleoptiles (removed from malted seeds) were harvested, frozen in liquid nitrogen and stored at −70 °C if not used immediately.
• 53 h steeping with air rests using mesh bottomed containers • 93 h modification in 150×24 mm test-tubes • 48 h kilning (heat regime of 50 and 65 °C) Hordein analysis and DNA extraction Hordein extraction from barley seeds and from the corresponding malt and gel electrophoresis separation, at acidic pH, were carried out according to the International Seed Testing Association (ISTA) procedure4. DNA was extracted from leaflets (4 g), whole malt (five seeds) and coleoptiles after the CTAB15 and sarkosyl16 procedure with minor modifications. RAPD amplification and AFLP analysis RAPD amplification reaction volume was 20 lL, each containing 20 m Tris-HCl pH 8·4, 50 m KCl, 1·5 m MgCl2, 100 l each of dATP, dCTP, dGTP, dTTP (Boehringer), 0·2 l primer (listed below), 20 ng genomic DNA and one unit of Taq polymerase (Gibco, BRL). The primers used in this study were the following: OPA16, OPE19, OPAL18, OPD02, OPAI09, OPG08, OPAB07, OPG06, OPQ19, OPY14, OPY10, OPAE19, OPR08, OPR10, OPX10, OPQ17, OPA04, OPQ12, OPQ11 (Operon, Alameda, California).
Data were scored for presence (1) or absence (0) of amplified fragments. Dendrograms based on distance matrix data were generated using the UPGMA analysis of the computer package PHYLIP (Phylogeny Inference Package) version 3·5, kindly provided by Dr J. Felsenstein (University of Washington, Seattle, U.S.A.). RESULTS AND DISCUSSION Fifteen commercial barley varieties were subjected to A-PAGE analysis of hordeins extracted both from seeds and the corresponding malted kernels after the ISTA protocol. As expected, the A-PAGE hordein pattern of grain malt did not match that of the respective barley grain17 (data not shown). In fact the hordein proteins are transformed into soluble peptides and amino acids by the action of proteolytic enzymes (endopeptidases, exopeptidases, dipeptidases) during the malting process. This hordein degradation changes the electrophoretic pattern, i.e., some bands are greatly reduced in intensity and others completely absent, which makes A-PAGE analysis for malt characterisation useless. Hence the reliability of RAPD and AFLP markers for malt fingerprinting was tested. DNA was extracted by two different methods from dry whole malt, barley grain and young leaflets of the same variety and its quality (i.e.,
Molecular markers for malt fingerprinting
integrity, suitability for PCR analysis) compared. Regardless of the extraction protocol, we found DNA from the former source partially degraded and unsuitable for fingerprinting. To overcome this problem, DNA was extracted from coleoptiles taken up from malted seeds which resulted comparable in quality to that from fresh young leaves. The extraction yield was also quite satisfactory, i.e., an average of a mere 10 coleoptiles yielded 10 lg of genomic DNA, enough for 40 AFLP combinations or 500 RAPD amplifications. RAPD analysis was then conducted on the set of the 15 barley varieties and their malts using the 20 different decamers listed above. Using DNA extracted from malted grain coleoptiles, the RAPD pattern was closer to the one produced both by young leaflets and whole barley grain than from that observed when whole grain malt was used. Yet the poor identity of RAPD patterns between a variety and the corresponding malt make these markers unsuitable for malt characterisation. In addition, some RAPD fragments, common to all the malts tested and absent in the corresponding varieties, were also found in the patterns generated by several primers. Because AFLP technique has proven to be a valuable tool for fingerprinting in several species given the wide coverage of the genome and the reproducibility of the assays, this approach was also tested for malt fingerprinting. Within our set of varieties, AFLP analysis starting from DNA extracted from whole barley grain and grain malt evinced contradictory results, including very poor amplification, which was likely caused by the low, unsuitable quality of DNA for complete enzyme digestion. These problems may be ascribable either to the presence of a protein matrix that is difficult to disrupt during DNA extraction in the former case or to the partial degradation of DNA in the latter. In the work of William et al.18 the authors found that DNA extracted from single seeds gave SSR patterns similar to those obtained starting from DNA extracted from leaf tissue. By contrast, we found that only DNA patterns obtained from grain malt coleoptiles were reliable and identical to those observed from barley leaflets DNA. For example, Figure 1(a) shows the patterns revealed by AFLP markers for the primer combination E37/M40 on a set of five barley varieties and of the corresponding malts. The pattern identity between a malt and corresponding variety is evident excluding a few fragments common to all the malt samples and absent in all the varieties
259
Figure 1 (a) AFLP analysis of five out of the 15 cultivars analysed (Baraka, Arda, Magda, Alexis, Apex) with E37/M40 primer combination. Each pair of patterns, corresponding to the same variety, represents the barley profile (left) and the malt profile (right). Arrows indicate two malt-specific fragments. (b) Phenogram of the same samples (1 barley, 2 malt) generated by UPGMA analysis; AFLP data were obtained with E37/M40 and E37/M33 primer combinations. The scale represents Jaccard’s coefficient of similarity.
considered. However, these ‘malt specific’ fragments, sometimes appearing as a difference in band intensity, were common to all the varieties tested, so their presence did not vitiate the validity of the malt fingerprinting. In fact, Figure 1(b) gives the phenogram obtained after scoring AFLP markers for the primer combinations E37/M40
P. Faccioli et al.
260
and E37/M33 on the five varieties and on the corresponding malts. Each variety and related malt are coupled together with a genetic similarity level of 0·84. In the matrix construction we have even computed the so-called ‘malt specific’ fragments. Their presence lowers the level of genetic similarity, without changing the final results of finding correspondence between a variety and its malt. Experiments are currently in progress to individuate whether or not this phenomenon may be related to a different DNA configuration in the organs examined (the variable level of methylation in the DNA hypothesized by Donini et al.19, could be a good explanation for AFLPs, but not for RAPDs). In conclusion, the overall results indicate that A-PAGE analysis of hordeins is to be ruled out because of protein degradation during germination, RAPD analysis has poor reproducibility, but AFLP analysis, based on template DNA extracted from grain malt coleoptiles, is an excellent tool for malt fingerprinting. AFLP is a marker system able to detect high levels of polymorphism and has high repeatability and speed of analysis. Only a few days were needed to verify the origin of a malt and to determine that a specific malt derives from a given malting variety. It is also possible to determine the raw variety of an unknown malt, by simply referring to a collection of malt AFLP patterns that includes the most common feeding and malting varieties.
4. 5. 6. 7.
8. 9.
10.
11.
12. 13. 14.
Acknowledgements This work has been partly supported by the ‘MiCIA’ Project and partly by P.O.M. Project, Measure 2 (Technological Innovation and its Diffusion) of the Italian Ministry of Agriculture.
15.
16.
REFERENCES 1. Briggs, D.E., Hough, J.S., Stevens, R. and Young, T.W. ‘Malts and Malting’, Thomson Science, London, Blackie Academic & Professional (1998). 2. Thomas, W.T.B., Powell, W., Swanston, J.S., Ellis, R.P., Chalmers, K.J., Barua, U.M., Jack, P., Lea, V., Forster, B.P., Waugh, R. and Smith, D.B. Quantitative trait loci for germination and malting quality characters in a spring barley cross. Crop Science 36 (1996) 265–273. 3. Han, F., Ulrich, S.E., Kleinhofs, A., Jones, B.L., Hayes, P.M. and Wesenberg, D.M. Fine structure mapping of
17. 18.
19.
barley chromosome-1 centromere region containing malting-quality QTLs. Theoretical and Applied Genetics 95 (1997) 903–910. Cooper, S.R. Report of the Rules Committee 1983–1986. Seed Science and Technology 15 (1987) 555–575. White, T.J. The future of PCR technology: diversification of technologies and applications. Tibtech 14 (1996) 478– 483. Ko, H.L. and Henry, R.J. Identification of barley varieties using the polymerase chain reaction. Journal of the Institute of Brewing 100 (1994) 405–407. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18 (1990) 6531–6535. Dweikat, N., Mackenzie, S., Levy, M. and Ohm, H. Pedigree assessment using RAPD-DGGE in cereal crop species. Theoretical and Applied Genetics 85 (1993) 497–505. Faccioli, P., Terzi, V., Monetti, A., Nicola, J. and Pecchioni, N. B-hordein STS markers for barley genotype identification: comparison with RFLPs, hordein A-PAGE and morpho-physiological traits. Seed Science and Technology 23 (1995) 415–427. Russell, J., Fuller, J., Young, G., Thomas, B., Taramino, G., Macaulay, M., Waugh, R. and Powell, W. Discriminating between barley genotypes using microsatellite markers. Genome 40 (1997) 442–450. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23 (1995) 4407–4414. Becker, J., Vos, P., Kuiper, M., Salamini, F. and Heun, M. Combined mapping of AFLP and RFLP markers in barley. Molecular & General Genetics 249 (1995) 65–73. Pecchioni, N., Stanca, A.M., Terzi, V. and Cattivelli, L. RFLP analysis of highly polymorphic loci in barley. Theoretical and Applied Genetics 85 (1993) 926–930. Morgan, A.G. The relationship between barley extract viscosity curves and malting ability. Journal of the Institute of Brewing 83 (1977) 231–234. Saghai-Maroof, M.A., Soliman, K.A., Jorgensen, R.A. and Allard, R.W. Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location and population dynamics. Proceedings of the National Academy of Science USA 81 (1984) 8014–8018. Weining, S. and Langridge, P. Identification and mapping of polymorphisms in cereals based on polymerase chain reaction. Theoretical and Applied Genetics 82 (1991) 209–216. Baxter, E.D. Hordein in barley and malt: a review. Journal of the Institute of Brewing 87 (1981) 173–176. William, M., Dorocicz, I. and Kasha, K.J. Use of microsatellite DNA to distinguish malting and nonmalting barley cultivars. Journal of the American Society of Brewing Chemists 55 (1997) 107–111. Donini, P., Elias, M.L., Bougourd, S.M. and Koebner, R.M.D. AFLP fingerprinting reveals pattern differences between template DNA extracted from different plant organs. Genome 40 (1997) 521–526.
Amplified Fragment Length Polymorphism (AFLP) Markers for Barley Malt Fingerprinting P. Faccioli, N. Pecchioni, A. M. Stanca and V. Terzi Experimental Institute for Cereal Research, Section of Fiorenzuola d’Arda, Via S. Protaso 302 I-29017 Fiorenzuola d’Arda (PC), Italy Received 17 July 1998
ABSTRACT Barley is the cereal par excellence in the production of malt for beer and other alcoholic beverages. In this work, biochemical (hordein A-PAGE) and molecular (RAPDs, AFLPs) markers have been compared for their efficiency in malt fingerprinting. To overcome the difficulties related to the quality of the DNA extracted from grain malt, malted coleoptiles are proposed as useful tissue for the extraction. On the basis of our data, we exclude a fingerprinting system based on A-PAGE hordein analysis because of the degradation of these proteins during the malting process. Comparing molecular markers for their efficiency and repeatability, we indicate AFLP analysis based on template DNA extracted from the tissue of grain malt coleoptile as an excellent tool for malt fingerprinting. 1999 Academic press Keywords: barley, malting quality, molecular markers, fingerprinting.
INTRODUCTION Malt is the result of controlled germination of cereal kernels, which are initially steeped in cold aerated water, followed by 3–5 days of germination. The properties and flavour of the malt are then fixed by kilning, which dries the grains. At the end of these technological processes, the kernels become malt: a very rich source of sugars, aminoacids and enzymes that is predominantly used for beer preparation, but also for distilling and as an integrator in food products1. The malting aptitude of barley, the most important cereal for it, depends on a host of mainly gene-related quality traits, this means that specific varieties are used by brewers2,3. Acid polyacrylamide gel electrophoresis (APAGE) of barley hordeins (prolamins), together with a complex of morpho-physiological descriptors, has been adopted for varietal classification by the International Union for the
Corresponding author: P. Faccioli. 0733–5210/99/030257+04 $30.00/0
Protection of New Varieties of Plants (UPOV), is also listed in the official International Seed Testing Association (ISTA) methodology4 and is accepted as the standard procedure by the European Brewery Convention. Molecular markers have become, over the last few years, a new and promising method for barley fingerprinting, which is based not only on restriction fragment length polymorphisms (RFLPs) but particularly on polymerase chain reaction (PCR)generated markers5,6 such as random amplified polymorphic DNAs (RAPDs)7,8, sequence tagged sites (STSs)9, microsatellites10 and, more recently, amplified fragment length polymorphisms (AFLPs)11,12. The success of these approaches is related to the fact that molecular markers are theoretically available in unlimited number, are environment independent, often quick and easy to use and explore a higher portion of variability with a consequently more consistently detectable level of polymorphism compared to morpho-physiological descriptors or biochemical markers9,13. The present study reports for the first time the characterisation of barley malt using different approaches so as to 1999 Academic Press
P. Faccioli et al.
258
identify the best method for checking the corresponding barley variety with the minimum time required and the maximum reliability.
Micromalting
Amplification was performed in a Perkin Elmer Cetus Gene Amp PCR System 9600 programmed as follows: 1 cycle of 2 min at 94 °C, 1 min at 36 °C, 2 min at 72 °C; 46 cycles of 1 min at 94 °C, 1 min at 36 °C, 2 min at 72 °C. The amplified products were separated by 1·5% agarose gel electrophoresis in TBE buffer and stained with ethidium bromide. AFLP analysis was performed using Gibco BRL AFLPTM Analysis System I and the corresponding protocol with minor modifications. The primer combinations used were the following: E37 (5GACTGCGTACCAATTCACG)/M40 (5-GATGAGTCCTGAGTAAAGC) and E37/M33 (5GATGAGTCCTGAGTAAAAG). The PCR products were separated onto a 4·5% polyacrylamide gel in TBE buffer at 40 W for 1 h. The gel was then dried for 1 h at 80 °C and autoradiographed for 2 days on Kodak X-ray film.
The preparation of malt by using 20 g of clean, sieved barley seeds was performed as summarized below14:
Data analysis
EXPERIMENTAL Genetic material Fifteen barley varieties for malting (Alexis, Magda, Gitane, Apex, Aura, Carina, Kaskade, Krona, Orchidea, Prisma, Tremois, Arco) and feed (Baraka, Arda, Kelibia) were randomly chosen. Young shoots from pot-grown plants and coleoptiles (removed from malted seeds) were harvested, frozen in liquid nitrogen and stored at −70 °C if not used immediately.
• 53 h steeping with air rests using mesh bottomed containers • 93 h modification in 150×24 mm test-tubes • 48 h kilning (heat regime of 50 and 65 °C) Hordein analysis and DNA extraction Hordein extraction from barley seeds and from the corresponding malt and gel electrophoresis separation, at acidic pH, were carried out according to the International Seed Testing Association (ISTA) procedure4. DNA was extracted from leaflets (4 g), whole malt (five seeds) and coleoptiles after the CTAB15 and sarkosyl16 procedure with minor modifications. RAPD amplification and AFLP analysis RAPD amplification reaction volume was 20 lL, each containing 20 m Tris-HCl pH 8·4, 50 m KCl, 1·5 m MgCl2, 100 l each of dATP, dCTP, dGTP, dTTP (Boehringer), 0·2 l primer (listed below), 20 ng genomic DNA and one unit of Taq polymerase (Gibco, BRL). The primers used in this study were the following: OPA16, OPE19, OPAL18, OPD02, OPAI09, OPG08, OPAB07, OPG06, OPQ19, OPY14, OPY10, OPAE19, OPR08, OPR10, OPX10, OPQ17, OPA04, OPQ12, OPQ11 (Operon, Alameda, California).
Data were scored for presence (1) or absence (0) of amplified fragments. Dendrograms based on distance matrix data were generated using the UPGMA analysis of the computer package PHYLIP (Phylogeny Inference Package) version 3·5, kindly provided by Dr J. Felsenstein (University of Washington, Seattle, U.S.A.). RESULTS AND DISCUSSION Fifteen commercial barley varieties were subjected to A-PAGE analysis of hordeins extracted both from seeds and the corresponding malted kernels after the ISTA protocol. As expected, the A-PAGE hordein pattern of grain malt did not match that of the respective barley grain17 (data not shown). In fact the hordein proteins are transformed into soluble peptides and amino acids by the action of proteolytic enzymes (endopeptidases, exopeptidases, dipeptidases) during the malting process. This hordein degradation changes the electrophoretic pattern, i.e., some bands are greatly reduced in intensity and others completely absent, which makes A-PAGE analysis for malt characterisation useless. Hence the reliability of RAPD and AFLP markers for malt fingerprinting was tested. DNA was extracted by two different methods from dry whole malt, barley grain and young leaflets of the same variety and its quality (i.e.,
Molecular markers for malt fingerprinting
integrity, suitability for PCR analysis) compared. Regardless of the extraction protocol, we found DNA from the former source partially degraded and unsuitable for fingerprinting. To overcome this problem, DNA was extracted from coleoptiles taken up from malted seeds which resulted comparable in quality to that from fresh young leaves. The extraction yield was also quite satisfactory, i.e., an average of a mere 10 coleoptiles yielded 10 lg of genomic DNA, enough for 40 AFLP combinations or 500 RAPD amplifications. RAPD analysis was then conducted on the set of the 15 barley varieties and their malts using the 20 different decamers listed above. Using DNA extracted from malted grain coleoptiles, the RAPD pattern was closer to the one produced both by young leaflets and whole barley grain than from that observed when whole grain malt was used. Yet the poor identity of RAPD patterns between a variety and the corresponding malt make these markers unsuitable for malt characterisation. In addition, some RAPD fragments, common to all the malts tested and absent in the corresponding varieties, were also found in the patterns generated by several primers. Because AFLP technique has proven to be a valuable tool for fingerprinting in several species given the wide coverage of the genome and the reproducibility of the assays, this approach was also tested for malt fingerprinting. Within our set of varieties, AFLP analysis starting from DNA extracted from whole barley grain and grain malt evinced contradictory results, including very poor amplification, which was likely caused by the low, unsuitable quality of DNA for complete enzyme digestion. These problems may be ascribable either to the presence of a protein matrix that is difficult to disrupt during DNA extraction in the former case or to the partial degradation of DNA in the latter. In the work of William et al.18 the authors found that DNA extracted from single seeds gave SSR patterns similar to those obtained starting from DNA extracted from leaf tissue. By contrast, we found that only DNA patterns obtained from grain malt coleoptiles were reliable and identical to those observed from barley leaflets DNA. For example, Figure 1(a) shows the patterns revealed by AFLP markers for the primer combination E37/M40 on a set of five barley varieties and of the corresponding malts. The pattern identity between a malt and corresponding variety is evident excluding a few fragments common to all the malt samples and absent in all the varieties
259
Figure 1 (a) AFLP analysis of five out of the 15 cultivars analysed (Baraka, Arda, Magda, Alexis, Apex) with E37/M40 primer combination. Each pair of patterns, corresponding to the same variety, represents the barley profile (left) and the malt profile (right). Arrows indicate two malt-specific fragments. (b) Phenogram of the same samples (1 barley, 2 malt) generated by UPGMA analysis; AFLP data were obtained with E37/M40 and E37/M33 primer combinations. The scale represents Jaccard’s coefficient of similarity.
considered. However, these ‘malt specific’ fragments, sometimes appearing as a difference in band intensity, were common to all the varieties tested, so their presence did not vitiate the validity of the malt fingerprinting. In fact, Figure 1(b) gives the phenogram obtained after scoring AFLP markers for the primer combinations E37/M40
P. Faccioli et al.
260
and E37/M33 on the five varieties and on the corresponding malts. Each variety and related malt are coupled together with a genetic similarity level of 0·84. In the matrix construction we have even computed the so-called ‘malt specific’ fragments. Their presence lowers the level of genetic similarity, without changing the final results of finding correspondence between a variety and its malt. Experiments are currently in progress to individuate whether or not this phenomenon may be related to a different DNA configuration in the organs examined (the variable level of methylation in the DNA hypothesized by Donini et al.19, could be a good explanation for AFLPs, but not for RAPDs). In conclusion, the overall results indicate that A-PAGE analysis of hordeins is to be ruled out because of protein degradation during germination, RAPD analysis has poor reproducibility, but AFLP analysis, based on template DNA extracted from grain malt coleoptiles, is an excellent tool for malt fingerprinting. AFLP is a marker system able to detect high levels of polymorphism and has high repeatability and speed of analysis. Only a few days were needed to verify the origin of a malt and to determine that a specific malt derives from a given malting variety. It is also possible to determine the raw variety of an unknown malt, by simply referring to a collection of malt AFLP patterns that includes the most common feeding and malting varieties.
4. 5. 6. 7.
8. 9.
10.
11.
12. 13. 14.
Acknowledgements This work has been partly supported by the ‘MiCIA’ Project and partly by P.O.M. Project, Measure 2 (Technological Innovation and its Diffusion) of the Italian Ministry of Agriculture.
15.
16.
REFERENCES 1. Briggs, D.E., Hough, J.S., Stevens, R. and Young, T.W. ‘Malts and Malting’, Thomson Science, London, Blackie Academic & Professional (1998). 2. Thomas, W.T.B., Powell, W., Swanston, J.S., Ellis, R.P., Chalmers, K.J., Barua, U.M., Jack, P., Lea, V., Forster, B.P., Waugh, R. and Smith, D.B. Quantitative trait loci for germination and malting quality characters in a spring barley cross. Crop Science 36 (1996) 265–273. 3. Han, F., Ulrich, S.E., Kleinhofs, A., Jones, B.L., Hayes, P.M. and Wesenberg, D.M. Fine structure mapping of
17. 18.
19.
barley chromosome-1 centromere region containing malting-quality QTLs. Theoretical and Applied Genetics 95 (1997) 903–910. Cooper, S.R. Report of the Rules Committee 1983–1986. Seed Science and Technology 15 (1987) 555–575. White, T.J. The future of PCR technology: diversification of technologies and applications. Tibtech 14 (1996) 478– 483. Ko, H.L. and Henry, R.J. Identification of barley varieties using the polymerase chain reaction. Journal of the Institute of Brewing 100 (1994) 405–407. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18 (1990) 6531–6535. Dweikat, N., Mackenzie, S., Levy, M. and Ohm, H. Pedigree assessment using RAPD-DGGE in cereal crop species. Theoretical and Applied Genetics 85 (1993) 497–505. Faccioli, P., Terzi, V., Monetti, A., Nicola, J. and Pecchioni, N. B-hordein STS markers for barley genotype identification: comparison with RFLPs, hordein A-PAGE and morpho-physiological traits. Seed Science and Technology 23 (1995) 415–427. Russell, J., Fuller, J., Young, G., Thomas, B., Taramino, G., Macaulay, M., Waugh, R. and Powell, W. Discriminating between barley genotypes using microsatellite markers. Genome 40 (1997) 442–450. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23 (1995) 4407–4414. Becker, J., Vos, P., Kuiper, M., Salamini, F. and Heun, M. Combined mapping of AFLP and RFLP markers in barley. Molecular & General Genetics 249 (1995) 65–73. Pecchioni, N., Stanca, A.M., Terzi, V. and Cattivelli, L. RFLP analysis of highly polymorphic loci in barley. Theoretical and Applied Genetics 85 (1993) 926–930. Morgan, A.G. The relationship between barley extract viscosity curves and malting ability. Journal of the Institute of Brewing 83 (1977) 231–234. Saghai-Maroof, M.A., Soliman, K.A., Jorgensen, R.A. and Allard, R.W. Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location and population dynamics. Proceedings of the National Academy of Science USA 81 (1984) 8014–8018. Weining, S. and Langridge, P. Identification and mapping of polymorphisms in cereals based on polymerase chain reaction. Theoretical and Applied Genetics 82 (1991) 209–216. Baxter, E.D. Hordein in barley and malt: a review. Journal of the Institute of Brewing 87 (1981) 173–176. William, M., Dorocicz, I. and Kasha, K.J. Use of microsatellite DNA to distinguish malting and nonmalting barley cultivars. Journal of the American Society of Brewing Chemists 55 (1997) 107–111. Donini, P., Elias, M.L., Bougourd, S.M. and Koebner, R.M.D. AFLP fingerprinting reveals pattern differences between template DNA extracted from different plant organs. Genome 40 (1997) 521–526.