GrainGenes

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UNIVERSITY OF AGRICULTURAL SCIENCES, VIENNA

Department of Plant Breeding, Gregor Mendel Str. 33, A-1180 Vienna, Austria.

 

Isolation of high-quality DNA from grains. [p. 24-25]

G. Stift and T. Lelley.

In this communication, we report on a fast and simple method for isolation of DNA from one-half of a wheat grain. The procedure gives high-quality DNA and good yield that is useful not only for RAPD analysis, but also for amplifications with specific primers. A chloroform extraction is not needed. The protocol described here is a modification of a method published by Benito et al. (1993).

The method.

• Cut one grain into two pieces. The half that contains the embryo can be kept for later germination.
• Crush the other half with pliers and put into a 1.5-ml centrifuge tube. Add 60 µl of extraction buffer (Dellaporta et al. 1983), 6 µl of 20 % SDS, and 1 µl of proteinase K (10 mg/ml). Mix thoroughly and incubate in a 65°C water bath for 10 min.
• Remove from water bath and add 1 µl RNaseA (10 mg/ml) and leave for 10 min at room temperature.
• Add 20 µl of a 4°C 5 M potassium-acetate (KC2H3O2) solution (pH 5.3) and shake vigorously (or vortex).
• Incubate for 20 min in an ice-cold water bath. Centrifuge for 20 min at 20,000 x g.
• Transfer the supernatant to a new 0.2 ml tube and add 50 µl of ice cold 96 % ethanol that contains 2 M ammonium-acetate (CH3COONH4). Incubate for 10 min in the ice-water bath, then centrifuge for 10 min at 15,000 x g.
• Wash the pellet once with 50 µl of 70 % ethanol, and centrifuge for 10 min at 15,000 x g. Pour off the alcohol. Dry the pellet for 10-20 min on the bench or for 10 min in a desiccator with application of vacuum.
• Dissolve the pellet in 20 µl TE, pH 8.0.

Discussion. For breeders, obtaining marker-based information as soon as possible, i.e., before beginning large experiments (crossing or selfing) is often of great advantage. With this method of DNA extraction for a PCR-based analyses, this information can be obtained from the seed before beginning an experiment. Judging from the results of the marker study, an experiment can be stopped or rearranged, avoiding loss of time. The part of the grain with the embryo is saved, thus after analyzing the DNA of a special grain, it is possible to use the embryo for further breeding purposes. The amount of DNA that we obtained depended on the size of the seed and varied between 200­2,400 ng. If the 65°C incubation time is extended from 10 min to 1 hr, twice the yield of DNA is possible. Comparing this protocol to the original method of Benito et al. (1993), we found no significant difference in the yield of DNA. However, the results from PCR with the method of Benito et al. were unsatisfactory in our laboratory, possibly due to the high salt content in the resulting DNA. Therefore, the following modifications were used: proteinase K was added at the beginning of the isolation procedure to help destroy cell walls, RNaseA as added tp eliminate RNA, and two alcohol washes were added at the end of the procedure. This protocol has been used successfully in several experiments by Stachel et al. (2000).

References.

• Benito C, Figueiras AM, Zaragoza C, Gallego FJ, and de la Pena A. 1993. Rapid identification of Triticeae genotypes from single seeds using the polymerase chain reaction. Plant Mol Bio 21:181-183.
• Dellaporta SL, Wood J, and Hicks JB. 1983. A plant DNA minipreparation: Version II. Plant Mol Bio 1:19-21.
• Stachel M, Lelley T, Grausgruber H, and Vollmann J. 2000. Application of microsatellites in wheat (Triticum aestivum L.) for studying genetic differentiation caused by selection for adaptation and use. Theor Appl Genet 100: 242-248.

 

The effect of the T1BL·1RS translocation in wheat: agronomic performance and quality. [p. 25-28]

Introduction. Bread wheat varieties with the T1BL·1RS wheat-rye translocation are grown world wide and have been the subject of numerous scientific investigations over the past two decades (Graybosch 2001). The translocated rye chromatin (1RS) provides disease and pest resistance to the host wheat variety (McIntosh 1983, Sebesta et al. 1995) and improves yield potential and contributes wide adaptation to the crop (Rajaram et al. 1983, Villareal et al. 1995). At the same time, however, it reduces bread-making quality (Zeller et al. 1982, Dhaliwal et al. 1987, Graybosch et al. 1993, Fenn et al. 1994).

After studying the HMW-glutenin composition of a large number of breeding lines under registration in the Hungarian and Austrian Varietal Testing Authorities, we detected a number of lines that were heterogeneous for T1BL·1RS. From these advanced breeding lines, sister lines with and without the translocation were selected, which we consider to be nearly isogenic, and we tested them in field trials under different climatic and edaphic conditions.

Materials and methods. Field trials were made with six pairs of NILs, with and without the T1BL·1RS translocation. The material was sown in 10-m2 plots in two replications. In 1997­98, the lines were grown at five locations representing the widest possible range of environmental conditions for growing wheat in Austria. In 1999­00, they were grown at the same five locations as before and at one additional location in the southeastern Hungary, which represented a very dry, hot environment.

Results. A combined analysis of the data yielded the following results. In this material, the translocation significantly increased grain yield, especially through increased spikelet number and 1,000-kernel weight (Table 1). The effect of the translocation on plant height between the NILs also proved to be significant. The height reduction of the T1BL·1RS translocation plants (~5 cm) was connected with better resistance to lodging. Lines with the translocation had higher hectoliter weights, but the difference was not significant. A significant difference also was found in all quality traits, protein content, sedimentation value, wet-gluten content, and SDS gel protein. Lines with the translocation proved to be inferior for these traits (Table 1).

 

For all traits except the number of seeds/spike, a highly significant translocation­background interaction was found (Table 2). The interaction of the translocation with the environment was not significant for yield. In nine out of eleven environments, the translocation lines surpassed in yield isogenic lines lacking the translocation.

 

 

Discussion. Table 3 lists a summary of the data on the T1BL·1RS translocation from relevant papers over the last decade. Only those traits are listed that were investigated in our experiment. First, the table shows whether or not the studies were made with the use of NILs or with lines/varieties that differed only in the presence or absence of the translocation. The results are presented only by indicating whether or not the translocation lines were superior or inferior when compared to their respective lines lacking T1BL·1RS. For example, Villareal et al. (1991) found that the translocation lines had a higher 1,000-kernel weight when compared to genotypes lacking T1BL·1RS. The difference was highly significant and is indicated by +***. The list has two exceptions where obviously no difference was found in most studies similar results were obtained. Accordingly, 1,000-kernel weight is probably the main cause for the higher yield of the translocation lines. Another interesting result was obtained for protein content. No correlation seems to exist among the uniform inferiority of the translocation lines for Zeleny sedimentation value, SDS sedimentation, and SDS-gel protein. Obviously, the reduced bread-making quality of the translocation lines is not correlated with protein content.

Our results on the six pairs of NILs are very much in line with previously published data on the contrasting effect of the T1BL·1RS translocation on the yield and quality of wheat. The highly significant 'genotype x translocation' interaction (Table 2) also agrees with the observation of background effects on the phenotypic expression of 1RS (Carver and Rayburn 1994, Villareal et al. 1995). In addition, the higher environmental stability of the translocation lines in comparison to those where it is lacking is underlined by a nonsignificant interaction for yield of the translocation lines.

Schlegel and Korzun (1997) pointed out that because of the single origin the 1RS arm of most T1BL·1RS translocation lines in the world possess no allelic variation. At present, the advantage of this rye chromosome arm can be exploited only by selecting the appropriate wheat background. Methods have been suggested by Lelley et al. (2000) for the introduction of new allelic variation into 1RS by homologous recombination.

Acknowledgments. We gratefully acknowledge the kind cooperation of the following organizations for their help with the field experiments: Versuchsstation Univerität Bodenkultur, Großenzersdorf; Saatbau Linz, Reichersberg; Saatzucht Edelhof; Bundesamt und Forschungszentrum für Landwirtschaft, Wien Aussenstelle Grabenegg und Petzenkirchen; and the Cereal Research Non-Profit Company, Szeged Hungary.

References.

• Bullrich L, Tranquilli G, Pfluger LA, Suárez EY, and Barneix AJ. 1998. Bread-making quality and yield performance of 1BL/1RS wheat isogenic lines. Plant Breed 117:119-122.
  Burnett CJ, Lorenz KJ, and Carver BF. 1995. Effects of the 1B/1R translocation in wheat on composition and properties of grain flour. Euphytica 86:159-166.
• Carver BF and Rayburn AL. 1994. Comparison of related wheat stocks possessing 1B or 1RS.1BL chromosomes: Agronomic performance. Crop Sci 34:1505-1510.
• Carver BF and Rayburn AL. 1995. Comparison of related wheat stocks possessing 1B or T1BL.1RS chromosomes: Grain and flour quality. Crop Sci 35:1316-1321.
• Dhaliwal AS, Mares DJ, and Marshall DR. 1987. Effect of 1B/1R chromosome translocation on milling and quality characteristics of bread wheats. Cereal Chem 64:72-76.
• Fenn D, Lukow OM, Bushuk W, and Depauw RM. 1994. Milling and baking quality of 1BL/1RS translocation wheats. I. Effects of genotype and environment. Cereal Chem 71:189-195.
• Graybosch RA, Peterson CJ, Hansen LE, Worral D, Shelton DR, and Lukaszewski AJ. 1993. Comparative flour quality and protein characteristics of 1BL/1RS and 1AL/1RS wheat-rye translocations. J Cereal Sci 17:95-106.
• Graybosch RA. 2001. Uneasy unions: quality effects of rye chromatin transfers to wheat. J Cereal Sci 33:3-16.
• Hussain A, Lukow OM, Watts BM, and McKenzie RIH. 1997. Rheological properties of full-formula doughs derived from near-isogenic 1BL/1RS translocation lines. Cereal Chem 74:242-248.
• Lee JH, Graybosch RA, and Peterson CJ. 1995. Quality and biochemical effects of a 1BL/1RS wheat-rye translocation in wheat. Theor Appl Genet 90:105-112.
• Lelley T, Eder Ch, Nagy ED, and Molnár-Láng M. 2000. Effect of the 1BL/1RS translocation in wheat; induction of new genetic variability in the translocated chromosome. In: 50th Anniv Agric Res Inst, Martonvásár, Hungary (Bedö Z ed). pp. 81-88.
• Martin P and Carillo JM. 1999. Cumulative and interaction effects of prolamin allelic variation and of 1BL/1RS translocation on flour quality in bread wheat. Euphytica 108:29-39.
• McIntosh RA. 1983. A catalogue of gene symbols for wheat. In: Proc 6th Internat Wheat Genet Symp (Sakamoto S ed). Plant Germ-Plasm Inst, Kyoto, Japan. pp. 1197-1255.
• McKendry AL, Tague DN, and Miskin KE. 1996. Effect of 1BL·1RS on agronomic performance of soft red winter wheat. Crop Sci 36:844-847.
• McKendry AL, Tague DN, Finney PL, and Miskin KE. 1996. Effect of 1BL·1RS on milling and baking quality of soft red winter wheat. Crop Sci 36:848-851.
• Moreno-Sevilla B, Baenziger PS, Peterson CJ, Graybosch RA, and McVey DV. 1995a. The 1BL/1RS translocation: Agronomic performance of F3-derived lines from a winter wheat cross. Crop Sci 35:1051-1055.
• Moreno-Sevilla B, Baenziger PS, Shelton DR, Graybosch RA, and Peterson CJ. 1995b. Agronomic performance and end-use quality of 1B vs. 1BL/1RS genotypes derived from winter wheat 'Rawhide'. Crop Sci 35:1607-1612.
• Pena RJ, Amaya A, Rajaram S, and Mujeeb-Kazi A. 1990. Variation in quality characteristics associated with some spring 1B/1R translocation wheats. J Cereal Sci 12:105-112.
• Rajaram S, Mann CE, Ortis Ferrara G, and Mujeeb-Kazi A. 1983. Adaption, stability and high yield potential of certain 1B/1R CIMMYT wheats. In: Proc 6th Internat Wheat Genet Symp (Sakamoto S ed). Plant Germ-Plasm Inst, Kyoto, Japan. pp. 613-621.
• Schlegel R and Meinel A. 1994. A quantitative trait locus (QTL) on chromosome arm 1RS of rye and its effect on yield performance of hexaploid wheat. Cereal Res Commun 22:7-13.
• Sebesta EE, Wood EA, Porter DR, Webster JA, and Smith EL. 1995. Registration of Amigo wheat germplasm resistant to greenbug. Crop Sci 35:293.
• Singh RP, Huerta-Espino J, Rajaram S, and Crossa J. 1998. Agronomic effects from chromosome translocations 7DL·7Ag and 1BL·1RS in spring wheat. Crop Sci 38:27-33.
• Schlegel R and Korzun V. 1997. About the origin of 1RS·1BL wheat-rye chromosome translocations from Germany. Plant Breed 116:537-540.
• Villareal RL, Rajaram S, Mujeeb-Kazi A, and del Toro E. 1991. The effect of chromosome 1B/1R translocation on the yield potential of certain spring wheats (Triticum aestivum L.). Plant Breed 106:77-81.
• Villareal RL, Mujeeb-Kazi A, Rajaram S, and del Toro E. 1994. Associated effects of chromosome 1B/1R translocation on agronomic traits in hexaploid wheat. Breed Sci 44:7-11.
• Villareal RL, del Toro E, Mujeeb-Kazi A, and Rajaram S. 1995. The 1BL/1RS chromosome translocation effect on yield characteristics in a Triticum aestivum L. cross. Plant Breed 114:497-500.
• Villareal RL, Banuelos O, Mujeeb-Kazi A, and Rajaram S. 1998. Agronomic performance of chromosome 1B and T1BL·1RS near-isolines in the spring bread wheat Seri M82. Euphytica 103:195-202.
• Zeller FJ, Günzel G, Fischbeck G, Gerstenkorn P, and Weipert D. 1982. Veränderung der Backeigenschaften der Weizen-Roggen-Chromosomen-Translokation 1B/1R. Getreide Mehl Brot 36:141-143 (In German).

Storage proteins of wheat their effect on bread-making quality and their use in breeding. [p. 29]

Sabine Grausgruber-Gröger and Tamas Lelley.

Crosses between Austrian, Hungarian, and Croatian winter wheat genotypes were made to study the influence of the Glu-1, Glu-3, and Gli-1 loci on bread-making quality. Double haploid lines were developed using the 'wheat x maize' system. Protein alleles were identified by using SDS- and A-PAGE. A selection of lines was used to study the effect of the protein-coding genes on bread-making quality in an orthogonal fashion. Quality was determined by grain-protein content and the Zeleny sedimentation volume. Analysis of variance revealed the influence of the different loci on bread-making quality.

Among 1,110 DH lines, no recombination between the Gli-1 and Glu-3 alleles was found. In two crosses between genotypes with and without the T1BL·1RS translocation, lines were detected containing neither Gli-B1/Glu-B3 proteins nor secalins. In one cross, it could be shown that this was due to a deletion in the distal region of chromosome 1RS. The presence of T1B·1RS had a negative effect on the sedimentation volume in some crosses. The deletion lines revealed that in these lines the presence of the secalins had a more negative effect than the loss of the Gli-B1/Glu-B3 proteins. The effect of T1BL·1RS seems to depend on the genetic background.

The greatest effect on sedimentation volume was observed for Glu-D1. In some crosses, the effects of Gli-A1/Glu-A3 and Gli-B1/Glu-B3 were of the same order as those of Glu-A1 and Glu-B1. From the results, we ranked the loci with respects to their effect on Zeleny sedimentation value. To name the gliadin and low molecular weight glutenin alleles in the loci Gli-1 and Glu-3, respectively, the nomenclature of Jackson et al. (1996) was followed.

Glu-A1:	2* > 1
Glu-B1:	7+8 > 7+9
Glu-D1:	5+10 > 2+12, 5+10 > 5*+12
Gli-A1/Glu-A3:	o/2 > l/1, b/3 > 5/a, a/6 > 8/9
Gli-B1/Glu-B3:	f/fg > 4/d
Gli-D1/Glu-D3:	k/c > b/a

Significant effects of alleles at the locus Gli-1/Glu-3 were found, although the D-zone gliadins could not be differentiated. The D-zone gliadins alone do not seem to be sufficient for differentiating quality. Although some alleles were present in several crosses, their effects on sedimentation volume and protein content were not constant. Therefore, interactions appear to exist between the diverse loci and alleles and the genetic background. Although the separation of gliadins using A-PAGE was simple, the separation of LMW glutenins required a laborious extraction procedure.

Determining the allele content of the Gli-1 and Glu-3 loci was only partly successful. The Gli-1 alleles could be easily identified if they occurred in the upper part of the gel. The T1BL·1RS translocation was clearly seen in A-PAGE. The analysis of Glu-3 alleles was hampered by the many bands of very similar molecular weight. The unsystematic appearance of the gliadin and LMW-glutenin bands suggested that they probably were coded for by loci other than Gli-1, Gli-2, and Glu-3.

For practical breeding, both the identification of HMW-glutenin alleles and the presence and/or absence of the T1BL·1RS translocation in the crossing partners can be recommended. Early testing for sedimentation value of bread-making quality seems to be the simplest and a most reliable approach. The knowledge of the effects of Gli-1/Glu-3 alleles on quality parameters gained in this study and available in the literature does not allow us to recommend their systematic selection in practical breeding,

References.

• Grausgruber-Gröger S. 2000. Weizenspeicherproteine - ihre Bedeutung für die Backqualität und ihre Verwendung in der Qualitätsweizenselektion. Doctoral Thesis, Universität für Bodenkultur, Vienna, Austria (in German).
• Jackson EA, Morel M-H, Sontag-Strohm T, Branlard G, Metakovsky EV, and Redaelli R. 1996. Proposal for combining the classification systems of alleles of Gli-1 and Glu-3 loci in bread wheat (Triticum aestivum L.). J Genet Breed 50:321-336.

 

 

UNIVERSITY OF AGRICULTURAL SCIENCES VIENNA

Department of Plant Breeding, Gregor Mendel Str. 33, A 1180 Vienna, Austria.

 

Evaluation of winter wheat genotypes for adaptation in Kosovo. [p. 30]

Salih Salihi, Heinrich Grausgruber, Roman Tumpold, and Peter Ruckenbauer.

A set of 75 winter wheats was grown in 1999­00 at the University of Prishtina (in cooperation with Prof. S. Fetahu) for evaluation under Kosovan growing conditions. The trial was initiated in order to establish knowledge on field experiments and to find out genotypes that could be used for biparental crosses at the beginning of a wheat breeding program.

The material consisted of genotypes from Austria, central and eastern Europe, the U.S.A., and CIMMYT. All but the Austrian wheats were donated by CIMMYT, Ankara, Tukey. Sowing was by hand in mid-October. Plot size was 15 sq m. The experiment was an RCBD with three replicates. However, the trial was analyzed as a row-column design in order to control spatial variation. Composite samples over the replicates were used for quality evaluations, which were estimations of the protein content by NIRS and dough extension tests using a SMS Texture Analyzer TA.XT2i® with the Kieffer dough and gluten extensibility rigs.

The highest yielding genotypes originated from Turkey, Ukraine, Hungary, and diverse CIMMYT programs. The Austrian cultivars performed only average. However, the Austrian cultivars showed the highest protein contents. The dough extension tests revealed several genotypes with very weak and/or very strong dough characteristics. This strenghtening and/or weakening effect was probably due to heat stress during grain filling.

 

Publications. [p. 30]

• Grausgruber H, Oberforster M, Werteker M, Ruckenbauer P, and Vollmann J. 2000. Stability of quality traits in Austrian-grown winter wheats. Field Crops Res 66:257-267.
• Grausgruber H, Kreuzmayr AE, and Ruckenbauer P. 2000. Selektion auf Backqualität bei Weizen. Bericht 50. Arbeitstagung Vereinigung österreichischer Pflanzenzüchter, 23-25 November, 1999. BAL Gumpenstein. pp. 69-72 (in German).
• Lelley T, Stachel M, Grausgruber H, and Vollmann J. 2000. Analysis of relationships between Aegilops tauschii and the D genome of wheat using microsatellites. Genome 43:661-668.
• Stachel M, Lelley T, Grausgruber H, and Vollmann J. 2000. Application of microsatellites in wheat (Triticum aestivum L.) for studying genetic differentiation caused by selection for adaptation and use. Theor Appl Genet 100:242-248.