Materials and methods

Átírás

1 Research background and aims of the study Similar to the landraces and to other old Hungarian varieties, Bánkúti 1201 is a genetically heterogeneous population. They may possess special storage protein composition with alleles or allele combinations occurring rarely in modern wheat cultivars. The development of biochemical and molecular biological methods and fractionation techniques offer several possibilities to the breeders to use the old varieties as gene resources. Due to these facilities the detailed biochemical and molecular genetic analyses of the landraces and old cultivars maintained in gene banks is an important research task. Bánkúti 1201 was one of the most popular winter wheat varieties in the first part of the 20th century and due to its excellent functional properties variants of this variety were also used as breeding material in the neighbouring countries. In the early seventies the old Hungarian varieties were gradually removed from cultivation and their populations are only preserved in gene banks. In 1993 experiments were started in the Agricultural Research Institute of the Hungarian Academy of Sciences in order to test the old Hungarian wheat varieties and more detailed the Bánkúti 1201 population. The experiments were primary aimed at examining the HMW-glutenin subunit composition of the variety and to reveal the relationship with the technological properties. The good technological parameters detected in the different Bánkúti 1201 lines could explain partly by the heterogeneous HMW-GS composition (Bed ő et al., 1995). Based on the preliminary results the aim of our study was to determine the qualitative and quantitative storage protein components which play a role in the development of the good technological properties of the Bánkúti 1201 variety. 1. HMW-, LMW-glutenin subunit and gliadin allele composition were studied using polyacrylamide gelelectrophoresis. 2. Genespecific PCR primers were used to discover whether there is any explanation at the molecular level for the good technological properties of Bánkúti 1201 despite its 2+12 subunit composition on chromosome 1D. 3. Quantitative storage protein composition were analysed using different HPLC techniques. 4. Technological parameters of the different lines were determined based on the Hungarian wheat standard and small-scale rheology. 5. Correlation were determined between qualitative and quantitative storage protein composition and functional properties. 6. Based on the results storage protein alleles important for gene transformation and for breeding were identified. Plant material Materials and methods 1

2 52 spike progeny lines of the Bánkúti 1201 population developed by the Agricultural Research Institute of the Hungarian Academy of Sciences were involved in the experiments. Grain material harvested in were used to the detailed analysis of storage protein composition, to the molecular genetic studies and to the quality testing. Quantitative analyses and rheological parameters were carried out on 21 lines representing the population harvested in Storage protein composition HMW-, LMW-glutenin and gliadin allele composition were determined using Jackson s method (Jackson et al., 1996) and SDS- and acid polyacrylamide gelelectrophoresis. Analysis of HMW-glutenin genes using molecular markers A primerpair specific to the N-terminal region of the 1Dx5 HMW-glutenin gene were used to PCR testing. The amplified fragments were cloned and sequenced using an ABI 310 automatic sequencer. To evaluate the sequence data the EMBL and Genbank data basis and GCG software package were used. An another primer pair specific to 1Ax-type genes, published by D Ovidio et al. (1995) were used to amplify the mutant 1Ax2*B gene. Quantitative composition Total protein samples were prepared using Batey s method (Batey et al., 1991) to determine the glutenin and gliadin contents using SE-HPLC. Unextractable polymeric protein (UPP) content were measured based on the method of Gupta and MacRitchie (1994). HMW- and LMW-glutenin contents as well as relative amounts of individual HMW-GSs were determined using Marchylo s method (Marchylo et al., 1989). Technological parameters Gluten quality parameters were characterised by measuring protein-, and gluten contents, calculating gluten indexes and determining SDS-sedimentation values. Rheologycal properties were characterised using small-scale dough testing. Correlation between storage protein composition and functional properties were analysed using different statistical methods and the STATISTICA 6.0 program package (StatSoft Inc., USA). Results Genetic variability in the storage protein composition of Bánkúti 1201 wheat variety All HMW-, LMW-glutenin and gliadin loci showed high variability in the allelic composition. Similar to other European and Asianic landraces Bánkúti 1201 is characterised by 2+12 allele on locus Glu-D1. Concerning on Glu-A1 locus about 70% of the analysed lines possess a specific allele. The allele Glu-A1x2* B identified by molecular markers is characteristic to the variety Bánkúti 1201 and has an extra cysteine in its amino acid sequence. There is a C-G point mutation in the middle of the repetitive region of the encoding gene and it is resulting in a serine to cysteine aminoacid 2

3 change. Similar to the subunit 5 coded on chromosome 1D, 2* B contains 5 cysteine residues, in contradiction with the x-type HMW-GSs which contain in generally 4 cysteine residues. There is an important difference between the two subunits in the localization of the extra cysteine. While in the subunit 5 it is located at the beginning of the repetitive region, by subunit 2* B the extra cyteine is to find in the middle of this region. This difference may affect the intra- and intermolecular disulfide bridges and the development and characteristics of the gluten. There was high heterogeneity in the allelic composition of the LMW-glutenin subunits and gliadin alleles. We identified some alleles, which were not characteristic to modern European wheat varieties but more frequent in Canadian cultivars. This similarity were confirmed by pedigreeanalysis. Together eight HMW-, 5 LMW-GS and eight Gliadin allele compositions were identified in the 52 analysed Bánkúti 1201 lines. 20 different allele combination were found and three of them covered 61,5 % of the lines. The most frequent 3 combinations: 2* B , f-i-c, m-m-a (40,4%), a 2* B , a-i-c, a-m-a (11,5%) and 2* B , f-i-c, m-m-a (9,6%). Next to the allelic composition the quantitative storage protein composition also revealed high variability in Bánkúti The variety can be characterised by an average glutenin content and by high amount of Gliadin, resulting in a relative low glutenin to gliadin ratio. The Bánkúti 1201 lines contain high amounts of UPP in contradiction to other varieties possessing allele 2+12 on Glu-D1 locus. In generally amounts of Bx-type subunits were detected above the average. About 1/3 of the studied lines can be characterised by higher Glu/Gli values and higher UPP content. In three cases the detected UPP values were near that of the extra strong Canadian wheat variety Glenlea (57,8%). In the same lines an overexpression in subunit Bx7 could be observed. Regarding to both allelic and quantitative storage protein composition the lines possessing the subunits 7+8 resulted in higher glutenin%, UPP% and Bx% values. Based on the homogeneity found in locus Glu-D1 we could not evaluate the effect of the 2+12 allele on qualitative and quantitative parameters. The most characteristic LMW-glutenin (fic) and Gli-1 (mma) allelic composition correlated positively with higher monomeric and lower polymeric protein content. The effect of qualitative and quantitative composition on functional properties Both gluten quality testing and small-scale testing confirmed the good bread-making quality of Bánkúti Based on the Hungarian wheat standard the Bánkúti 1201 has improving quality. The rheological parameters indicate medium strong or strong and well extensible dough. The statistical analyses highlight the effect of the most frequent LMW-glutenin and gliadin alleles (Glu-A3f, Glu-B3i, Gli-A1m, Gli-B1m, Gli-D1a) on extensibility. Other alleles occurring in lower frequency, like Glu-B1 7+8, Glu-A3d, Glu-B3b have improving effect on dough strength, dough stability and maximal resistance by extension. 3

4 As summary of the results the population can be divided into two subpopulations based on their alleles on locus Glu-B1. These two groups differ from each other in both storage protein composition and technological properties. The differences emerging from the presence or absence of the subunits 7+8 have overstrong effects in contradiction with that of other loci (like the mutant 2* B subunit on chromosome 1A), or these secondary effects realise just in their interactions and coincidences. The first group, which contains about 60% of the lines possess next to the Glu-B3i and Gli-A1m alleles the 7+9 subunits on chromosome 1B. The cumulative effect of these alleles and their interactions results in high protein-, gluten content and good extensibility. The 7+8 group takes about 40% of the population and due to the overexpression of subunit Bx7 and the higher UPP content resulting in higher doughstrength and dough stability. Our opinion is that the complex good quality of the variety, that means the balance between the stabile, strength dough properties and the good extensibility, follows from the ratio (60% to 40%) of the two subpopulations manifested in the variety. New scientific results 1. Based on the HMW-, LMW-GS and gliadin composition Bánkúti 1201 is a heterogeneous population consisting of lines with different storage protein composition. 2. We identified an LMW glutenin subunit and with these closely joint gliadin allele (Glu-B3i / Gli-B1m) which show strong positive correlation with dough extensibility. 3. Using molecular markers we identified and sequenced the cysteine-mutant HMW-GS gene, the 1Ax2*B. The subunit 2*B has positive influence on gluten quality and doughstrength, although it is realised mainly through allelic interactions in the Bánkúti 1201 due to the specific storage protein composition. 4. The quantitative storage protein composition showed above average gliadin content in the population. In some lines we measured higher UPP content and detected over-expression of the subunit Bx7. 5. Based on the qualitative and quantitative storage protein composition and functional properties the population is a mixture of two subpopulation. The lines coding 7+9 subunits in Glu-B1 locus can be characterised by higher gluten and gliadin content and lower amounts of unextractable polimeric protein (UPP). These genotypes resulting extensible but less strong and less stabile dough. In the another type (group 7+8) the subunit Bx7 is expressed in above average amounts combined with higher UPP content. Due to the stronger gluten network the doughstrength and stability is higher and the dough is less extensible. 6. The complex good quality of Bánkúti 1201 (strong dough with high extensibility) is a result of the 60% group 7+9 to 40% group 7+8. Identification of agronomically important storage proteins and their use in the wheat breeding 4

5 Storage protein genes found in the old varieties and landraces may serve as genesources for breeding varieties with good breadmaking quality. Due to its good quality the Bánkúti 1201 population became an important material in isolation of storage protein genes. A detailed storage protein composition analysis were carried out to identify storage protein subunits correlating well with technological parameters. The identified LMW-GS Glu-B3i and the joint gliadin allele (Gli- B1m) are characteristic but not unique to the variety. The alleles show significant positive correlation to dough extensibility. Due to the conjugation between the two alleles it is enough to identify the LMW-GSs in the same gel as the HMW-GSs. After further experiments demonstrating this correlation in other genotypes too, the allele Glu-B3i is able to use for selecting genotypes with high gluten extensibility. We did not find any specific HMW-glutenin allele using SDS-PAGE. Using RP-HPLC some lines were identified with Bx7 subunit overexpression. Since this increased Bx content is combined with significantly higher dough strength it can be used to select these lines for quality purposes. The overexpession is thought to be due to the gene regulation apparatus. It is planned to analyse the promoter region in more detail. The isolation of the cysteine mutant gene 1Ax2* B may be important for molecular breeding. The effect of this subunit has only been studied in the special background of Bánkúti Further information can be obtained from in vitro experiments, where wheat storage proteins expressed in bacteria are built into the gluten network of genotypes with weak breadmaking quality. Although subunit 5 has a stronger effect on functional properties, the effect of subunit 2*B alone or together with subunit 5 may result in quality improvement. The 1Ax2*B gene has been isolated and it is ready for use in gene transformation. For the transformation we are using Genotypes with different HMW-GS compositions will be used for transformation in order to study the effect of this subunit in vivo. In conclusion the importance of identifying and isolating of storage protein genes can be emphasized. Old Hungarian wheat varieties may serve as an important basic material in experiments, aimed at developing genotypes for special quality purposes, such as extra strong dough or high extensibility. References Bed ő, Z., Kárpáti, M., Vida, Gy., Kramarik-Kissimon, J. and Láng, L.: Good breadmaking quality wheat (Triticum aestivum L.) genotypes with 2+12 subunit composition at the Glu-D1 locus. Cer. Res. Comm 23, 3: , 1995 D'Ovidio, R., Porceddu, E. and Lafiandra, D.: PCR analysis of genes encoding allelic variants of high-molecular-weight glutenin subunits at the Glu-D1 locus. Theor Appl Genet 88: ,

6 Jackson, E.A., Morel, M.H., Sontag-Strohm, T., Branlard, G., Metakovsky E.V. and Redaelli, R. Proposal for combining the classification systems of alleles of Gli-1 and Glu-3 loci in bread wheat (Triticum aestivum L.) J. Genet & Breed. 50: , 1996 D Ovidio, R., Masci, S. and Porceddu, E.: Development of a set of oligonucleotide primers specific for genes at the Glu-1 complex loci of wheat. Theor. Appl. Genet. 91: , 1995 Batey, I.L., Gupta, R.B. and MacRitchie, F.: Use of size-exclusion high-performance liquid chromatography in the study of wheat flour proteins: An improved chromatographic procedure. Cereal Chemistry 68: , 1991 Gupta, R.B. and MacRitchie, F.: Allelic variation at glutenin subunit and gliadin loci, Glu-1, Glu-3 and Gli-1, of common wheats. II. Biochemical basis of the allelic effects on dough properties. J. Cereal Sci. 19:19-29, 1994 Marchylo, B.A., Kruger, J.E. and Hatcher, D.W.: Quantitative reversed-phase high-performance liquid chromatographic analysis of wheat storage proteins as a potential quality prediction tool. J. Cereal Sci. 9: , 1989 Referált folyóiratok Bed ő Z, Karsai I, A témakörben megjelent publikációk Juhász A, Szű cs P, Mészáros K, Hayes P, Láng L.: Contribution of biotechnological methods to cereal breeding. Journal of Plant Biotechnlogy 1: Bed ő Z, Vida Gy, Láng L, Juhász A, Karsai I. Breeding a wheat variety with different lines for technological quality and HMW glutenin composition. Journal of Genetics and Breeding 53:57-62, 1999 Juhász A, Zeller F. J, Bed ő Z, Láng L. Die Backqualität ungarischer Winterweizensorten. Getreide, Mehl und Brot. 54: , 2000 Juhász A., Tamás L., Karsai I., Vida Gy., Láng L., Bed ő Z. Identification, cloning and characterisation of a HMW-Glutenin gene from an old Hungarian wheat variety, Bánkúti Euphytica 119:75-79, 2001 Vida Gy, Bed ő Z, Láng L, Juhász A.: Analysis of the quality traits of a Bánkúti 1201 population. Cereal Research Communications 26: , 1998 Bed ő Z., Láng L., Juhász A., Rakszegi M: Kemény endospermium szerkezet ű, jó malom- és sütőipari minőség ű búza kutatása Martonvásáron. A jó minőség ű, keményszem ű búza termesztése (Bed ő Z. szerk), Martonvásár Nádudvar - Szeged, p , 2001 ISBN

7 Juhász A., Tamás L., Láng L., Bed ő Z.: Mi a titka a Bánkúti 1201 jó minő ségének? Molnárok Lapja 106/4:3-4, 2001 Nemzetközi konferencia előadás és poszter Juhász A, Tamás L, Karsai I, Vida Gy, Láng L, Bed ő Z.: Identification, cloning and characterisation of a HMW-glutenin gene from an old Hungarian wheat variety, Bánkúti Proceedings of the 6th International Wheat Conference, Budapest ISBN Juhász A, Larroque OR, Tamás L, Vida Gy, Zeller FJ, Békés F, Bed ő Z: Biochemical and molecular genetic background of the traditional Bankut breadmaking quality. Cereals 2000, Proceedings of the 11th ICC Cereal and Bread Congress and of the 50th Australian Cereal Chemistry Conference (M. Wootton, I.L. Batey and C.W. Wrigley eds.), North Melbourne, Australia, pp , ISBN Bed ő Z, Vida Gy, Juhász A, Láng L.: Introduction of the traditional Bankut breadmaking quality type into modern wheat varieties. Proceedings of the 9th Int. Wheat Genet. Symposium (Ed. Slinkard AE), Saskatchewan, Canada pp , ISBN Juhász A, Békés F, Vida Gy, Láng L, Tamás L, Bed ő Z.: Quantitative analyses of storage proteins of an old Hungarian wheat population using the SE-HPLC method. Wheat Gluten (P.R. Shewry and A.S. Tatham eds.) Proceedings of the Gluten 2000 pp34-37, ISBN Juhász A, Vida Gy, Karsai I, Bed ő Z. Biochemical and molecular markers characterising wheat populations for breadmaking quality. EUCARPIA Section Cereals Meeting, Sept , Tulln, Austria, 56, 1997 Juhász A, Békés F, Larroque O. R, Vida Gy, Láng L, Tamás L, Bed ő Z. Quantitative analyses of the storage proteins of an old Hungarian wheat population using the SE-HPLC and RP-HPLC methods. 6th International Wheat Conference, June 5-9, 2000 Budapest, 137, 2000 Juhász A., Tamás L., Karsai I., Vida Gy., Láng L., Bed ő Z. Isolation of 1Ax2*B a new mutant HMW-glutenin subunit gene in common wheat (Triticum aestivum L.). Plant and Animal Genome Conference IX., 78, 2001 Magyar nyelv ű konferencia előadás és poszter Juhász A, Zeller FJ, Láng L, Bed ő Z. Magyar búzafajták vizsgálata a német minő ségi szabványok alapján V. Növénynemesítési Tudományos Napok március 9. Bp

8 Juhász A, Bed ő Z, Vida Gy, Karsai I, Láng L.: A Bánkúti 1201 búzafajta felhasználása a minő ségi tulajdonságok genetikai diverzitásának növelésére. IV. Magyar Genetikai Kongreszzus április Siófok Juhász A, Békés F, Vida Gy, Láng L, Tamás L, Bed ő Z. Tartalékfehérjék mennyiségi meghatározása SE-HPLC technikával egy régi magyar búzafajta populációjában. VI. Növénynemesítési Tudományos Napok március 8-9. Bp., 22., 2000 Juhász A, Kárpáti M, Vida Gy, Rakszegi M, Láng L, Zeller FJ, Hsam S.L.K, Bed ő Z.: Régi magyar búzafajták populációinak elemzése új genetikai források elő állítására. Az Agrobiodiverzitás megő rzése és hasznosítása szimpózium Jánossy Andor emlékére május 4-5. Bp Juhász A., Tamás L., Karsai I., Vida Gy., Láng L., Bed ő Z. Egy mutáns HMW-glutenin gén azonosítása, klónozása és jellemzése egy régi magyar búzafajta, a Bánkúti 1201 populációjában. VII. Növénynemesítési Tudományos Napok január Bp., 24, 2001 Király I, Larroque, O., Juhász A, Tamás L, Békés F, Bed ő Z. Régi magyar búzafajták tartalékfehérjéinek vizsgálata HPCL kromatográfiás módszerekkel. VII. Növénynemesítési Tudományos Napok január Bp., 63, 2001 Juhász A, Kárpáti M, Vida Gy, Békés F, Bed ő Z. Régi magyar búzafajták glutenin spektrumának és sütőipari minő ségi paramétereinek vizsgálata. IV. Növénynemesítési Tudományos Napok Bp. p.94., 1998 Juhász A, Vida Gy, Karsai I, Bed ő Z. A Bánkúti 1201 búzafajta HMW-glutenin alegységének molekuláris vizsgálata az 1D kromoszómán. IV. Növénynemesítési Tudományos Napok január Bp. p Vida Gy, Bed ő Z, Láng L, Juhász A. Bánkúti 1201 típusú sütőipari minőség ű ő szi búzafajták nemesítése. IV. Növénynemesítési Tudományos Napok január Bp. p. 134., 1998 Juhász A, Kárpáti M, Vida Gy, Békés F, Bed ő Z. Régi magyar búzafajták glutenin spektrumának és sütőipari minő ségi paramétereinek vizsgálata. IV. Növénynemesítési Tudományos Napok Bp. p.94., 1998 Juhász A, Vida Gy, Karsai I, Bed ő Z. A Bánkúti 1201 búzafajta HMW-glutenin alegységének molekuláris vizsgálata az 1D kromoszómán. IV. Növénynemesítési Tudományos Napok január Bp. p

9 Vida Gy, Bed ő Z, Láng L, Juhász A. Bánkúti 1201 típusú sütőipari minőség ű ő szi búzafajták nemesítése. IV. Növénynemesítési Tudományos Napok január Bp. p. 134., 1998 Külföldi szabadalom vagy bejelentés, találmány: Identification, Isolation and use of a storage Protein Gene Variant (PCT/HU01/00045), benyújtás ideje: április Magyar szabadalom vagy bejelentés, találmány: Egy búzatartalékfehérje-génvariáns azonosítása, izolálása és alkalmazása Szabadalom (Ügyszám: P ), benyújtás ideje: április 9