Agronomic and quality characteristics of triticale (X Triticosecale Wittmack) with HMW glutenin subunits 5+10

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Journal of Cereal Science 47 (2008) 68–78 www.elsevier.com/locate/jcs

Agronomic and quality characteristics of triticale (X Triticosecale Wittmack) with HMW glutenin subunits 5+10 P. Martineka,b,, M. Vinterova´c, I. Buresˇ ova´a,b, T. Vyhna´nekc a

Agricultural Research Institute Kromeˇrˇı´zˇ Ltd., Havlı´cˇkova 2787, 767 01 Kromeˇrˇı´zˇ, Czech Republic b Agrotest Fyto, Ltd., Havlı´cˇkova 2787, 767 01 Kromeˇrˇı´zˇ, Czech Republic c Mendel University of Agriculture and Forestry Brno, Zemeˇdeˇlska´ 1, 613 00 Brno, Czech Republic Received 5 April 2006; received in revised form 20 October 2006; accepted 1 February 2007

Abstract Sets of triticale (X Triticosecale Wittmack) lines derived from the cv. Presto with HMW glutenin allele Glu-D1d (subunits 5+10) translocated from bread wheat (Triticum aestivum L.) chromosome 1D to chromosome 1R were evaluated for agronomic and grain quality characteristics in 2002–2005. Two different translocation types were used: (a) single translocation 1R.1D5+10-2 where the long arm of 1R carries the wheat segment from 1DL with the Glu-D1d replacing a secalin locus Sec-3, (b) double translocation Valdy where the long arm of 1R has the translocation 1R.1D5+10-2 and the short arm has a segment from 1DS carrying wheat loci Gli-D1 and GluD3. The presence of Glu-D1d was determined by polyacrylamide gel electrophoresis (PAGE-ISTA) and DNA markers. The tested lines of triticale were compared with the check triticale cv. Presto and with wheat cultivars of different bread making quality (E-C quality classes). Single translocation 1R.1D5+10-2 reduced grain yield by 16% and Valdy translocation by 24% as compared with cv. Presto. The Valdy translocation had substantially shortened spike length and reduced specific weight in comparison with check cv. Presto. Wet gluten content (according to the Perten method) was 12% in both translocation types, 8% in check Presto and on average 24% in wheat. Translocations increased the Zeleny sedimentation value (Valdy — 27 ml, 1R.1D5+10-2 – 25 ml, cv. Presto — 23 ml). Triticale had a very low Hagberg falling number (FN) of 62–70 s without significant differences, while wheat had on average 301 s. The translocations did not significantly increase loaf volume; however, they improved loaf shape (height/width ratio): Valdy — 0.61, 1R.1D5+10-2 – 0.56, cv. Presto 0.44, wheat on average 0.70. The dough was non-sticky in Valdy, slightly sticky in 1R.1D5+10-2 and sticky in cv. Presto. Problems with a low FN for improving bread making quality of triticale are discussed. Higher bread making quality can be influenced by appropriate combination with donors of low a-amylase activity. r 2007 Elsevier Ltd. All rights reserved. Keywords: Triticale; Bread making quality; HMW glutenin subunit; Glu-D1d

1. Introduction The triticale (X Triticosecale Wittmack) shows some agronomic advantages relative to wheat; it has the ability Abbreviations: ARI, Agricultural Research Institute Kromeˇrˇ ı´ zˇ, Ltd.; HMW, high molecular weight; LMW, low molecular weight; CNS, Czech national standard; FN, Hagberg falling number; BU, Brabender unit; WG, wet gluten content; GI, gluten index Corresponding author. Agricultural Research Institute Kromeˇrˇ ı´ zˇ Ltd., Havlı´ cˇkova 2787, 767 01 Kromeˇrˇ ı´ zˇ, Czech Republic. Tel.: +420 573 317 158; fax: +420 573 339 725. E-mail addresses: [email protected], [email protected] (P. Martinek). 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.02.003

of producing higher amounts of the above-ground dry matter per unit area than wheat (Giunta and Motzo, 2004). Since current triticale cultivars have lower harvest index than wheat, breeding for a shorter stem is likely to increase triticale’s yield potential more rapidly than in wheat where the stem length might have already reached optimum values. Furthermore, triticale is able to resist some unfavourable biotic and abiotic environmental factors and thus produce good yield in marginal regions. Commonly grown triticales are hexaploids (2n ¼ 6x ¼ 42, AABBRR). They differ from bread wheat (Triticum aestivum L.) (2n ¼ 6x ¼ 42, AABBDD) by the presence of the R genome of rye that replaces wheat genome D,

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originating from Aegilops tauschii (Coss.) Schmal. (2n ¼ 2x ¼ 14, DD). Bread wheat is the only species among the other cereals that can be used for making leavened baking products for which the raw material with suitable viscoelastic dough properties is required. The genes conferring such properties are located in group 1 and 6 chromosomes of wheat. D genome is of great importance for bread making quality; however, it is absent in common cultivars of hexaploid triticale. The effect of such limitation is well illustrated by the difference in dough characteristics of bread wheat (2n ¼ 6x ¼ 42, AABBDD) and durum wheat, Triticum durum Desf. (2n ¼ 4x ¼ 28, AABB) (Fabriani and Lintas, 1988). For the same reason, triticale (AABBRR) has bread making quality inferior to that of bread wheat (AABBDD) and rye (RR) (Zeller and Hsam, 1984). Since there are substantial genetic distinctions between wheat and rye, different processing methods are required for bread making. The absence of D genome in triticale results in difficulties during the baking process. Therefore, experiments with additions of triticale to wheat flour were conducted. When 18.3% of triticale flour was blended with wheat flour, the greatest bread height (mm) and specific volume (cm3 g1) were obtained (Varughese et al., 1996). In T. aestivum, the long arms of homoeologous group 1 carry loci encoding high molecular weight (HMW) glutenins (Glu-1), the short arms — gliadins (Gli-1) and low molecular weight (LMW) glutenins (Glu-3), while the short arms of homoeologous group 6 carry loci encoding gliadins (Gli-2). Removal of the D genome in triticale has excluded the Glu-D1 (on 1DL), Gli-D1 and Glu-D3 (on 1DS) and Gli-D2 (on 6DS). Rye R genome has brought the loci Sec-3 (on 1RL), Sec-1 (on 1RS) and Sec-2 on 2RL to triticale (Lukaszewski, 2001; Shewry et al., 1984). These changes lead to the decrease in bread making quality of triticale (Zeller and Hsam, 1984). In wheat, the contribution to bread making quality of the HMW glutenins has been examined in the greatest detail and can even be expressed by numerical scores (Payne, 1987). Bread making quality in A genome is positively influenced by the glutenin allele Glu-A1b with HMW subunit 2* and in B genome by the alleles Glu-1Bb (with HMW subunits 7+8), Glu-1Bc (with HMW subunits 7+9) and Glu-1Bf (with HMW subunits 13+16). In D genome, bread making quality is positively affected by the glutenin allele Glu-D1d encoding subunits 5+10 present in almost all wheat cultivars exhibiting the best bread making quality classes E and A.1 1 In the Czech Republic and many EU countries, wheat cultivars are divided into four quality classes: E — elite class (very good, usable as an improving component in mixture for bread making), A — good-quality (optimal for bread making), B — baking (acceptable parameters for mixing with elite class), C (unacceptable for production of fermented dough). This classification takes into account minimal requirements of the processing industry and variation in absolute values of quality parameters in check cultivars and in individual years. The absolute values for selected quality parameters are converted into a 9-score scale. For E (or A) quality

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Some differences in dough viscoelastic characteristics and bread making quality are present in the currently available triticale cultivars. They depend on the composition of HMW alleles in A and B genomes (Tohver et al., 2005). Considerable improvement of bread making quality necessitates using positive effects of some HMW subunits on 1D that are absent in common triticale cultivars. The effect of chromosome 1D was first studied using the substitutions 1D(1A), 1D(1B) and 1D(1R) that led towards significant increase in Zeleny sedimentation value. The difference between the two glutenin alleles Glu-D1a (with HMW subunits 2+12) and Glu-D1d (with HMW subunits 5+10) was not as obvious as in wheat. Owing to its high cytological stability and minimal effect on agronomic performance, substitution 1D(1A) appears to be the most desirable one to use in triticale breeding, while the translocations 1D(1B) and 1D(1R) showed considerable yield loss (Lafferty and Lelley, 2001). The yield loss in the latter due to lower spike fertility was also confirmed by Lukaszewski (1990). Baking tests of the substitution 1D(1R), in the case Glu-D1d was used, showed a loaf volume, which was comparable with that in the wheat check cultivar with baking quality (Wos´ , pers. comm., 2002). The substitution 1D(1A) in cv. Presto slightly reduced fertility and markedly improved bread making quality characteristics. However, replacement of 1A or 1B by 1D does not eliminate the effect of chromosome 1R and rye secalins in triticale (Brezinski and Lukaszewski, 1998; Gustafson and Zilinsky, 1973). For these reasons, an improvement of its usability for bread making purposes by incorporation of the Glu-D1 locus from wheat chromosome 1D into rye chromosome 1R (Lukaszewski, 1994; Lukaszewski and Curtis, 1994; Wos´ et al., 2002) needs careful examination. This article presents results of agronomic important traits and grain quality evaluation in triticale using criteria for bread making quality in wheat.

2. Experimental 2.1. Materials The experiments involved triticale forms derived from cv. Presto that carried translocations of segments of wheat chromosome 1D to rye chromosome 1R. The lines used in this study were derived from original samples produced and supplied by Dr. A.J. Lukaszewski, University of California, Riverside, USA. These lines included the following two types of translocations: Presto 1R.1D5+10-2. Chromosome 1R.1D5+10-2 replaces a normal chromosome 1R. This translocation is a (footnote continued) class, required minimal loaf yield is 8 (or 6) scores, crude protein content 6 (or 4) scores, Zeleny sedimentation value 7 (or 5) scores, Hagberg falling number 6 (or 4) scores, specific weight 7 (or 6) scores and water absorption capacity 7 (or 5) scores.

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rye chromosome 1R with an intercalary segment of 1DL on its long arm. The 1DL segment introduces allele Glu-D1d encoding HMW glutenin subunits 5+10 and removes the corresponding locus Sec-3 encoding rye secalins (Lukaszewski and Curtis, 1992). Presto Valdy. This genetic material has a double translocation 1R.1D5+10-2/WR4, designated Valdy, replacing a normal rye chromosome 1R. Translocation Valdy was constructed from chromosome 1R.1D5+10-2, the same as present in the line above, and a wheat-rye translocation chromosome WR4 in the Australian triticale cultivar Rhino. In essence, WR4 introduces wheat loci Gli-D1 and Glu-D3 into 1RS, retaining the Sec-1 locus (Lukaszewski, 1998). The schemes of the presented translocated chromosomes are in Fig. 1. In addition to the segment of 1DL on the long arm as described above, Valdy also has a terminal segment of 1DS on its short arm that introduces wheat loci Gli-D1 and GluD3, encoding gliadins and LMW glutenins from wheat chromosome 1D, respectively. Rye secalin locus Sec-1 is also present. The cv. Presto carries the Glu-A1b allele (subunit 2*), known for its positive effect on bread making quality in wheat, and the allele Glu-B1b (subunits 7+8). The presence of Glu-A1b was likely to improve loaf shape in Presto compared with other tested genotypes that possessed also other glutenin alleles on 1A. Five triticale lines (designated 1–5) possessing the translocation Presto 1R.1D5+10-2, derived from the initial sample CT775-81, and 16 lines with the translocation Presto Valdy derived from three different original sources 26-97, 186-00 and 3915-01 were evaluated. From the sample 26-97 three lines (nos. 7, 8, 10), sample 186-00 eight lines (nos. 1–8), and sample 3915-01 five lines (nos. 1–5) were evaluated. The samples are owned by the University of California and were legally provided for research purposes. A number of them were genetically

Fig. 1. Schema of translocations in chromosome 1R.

heterogeneous in Glu-D1d. For further investigations, we selected homozygous lines from plant progenies by the single plant selection method only that were multiplied at the Agricultural Research Institute Kromeˇrˇ ı´ zˇ, Ltd. (ARI). Heterogeneity of the initial samples was confirmed by the fact that some of the derived lines did not contain Glu-D1d. For further work, the lines possessing Glu-1Dd were used only. (These were also used in the breeding programme, which is not the topic of this article.) The sets of lines Presto 1R.1D5+10-2 and Presto Valdy with Glu-D1d were compared with the check cv. Presto (without translocations). It enabled us to evaluate the importance of individual types of translocations. It shall be stressed that observed differences in evaluated traits were directly affected by products of the translocations. Presence of the Glu-D1d was determined by polyacrylamide gel electrophoresis (PAGE) according to the International Seed Testing Association (ISTA) (ISTA, 1999) and by a modified PCR protocol of D’Ovidio and Anderson (1994) using the (50 ) GCC TAG CAA CCT TCA CAA TC (30 ), (50 ) GAA ACC TGC TGC CGA CAA G (30 ) primers at Mendel University of Agriculture and Forestry Brno. DNA was separated by horizontal electrophoresis (Biometra — agagel standard) in 1% agarose gel and stained with ethidium bromide (Vinterova´ et al., 2003). The final product of the 450 bp-size verifies the presence of the Glu-D1d allele, thus the translocation 1R.1D5+10-2 (Fig. 2). Surprisingly, Glu-D1d was not present in all plants in some original samples from the USA. The Valdy lines only had four backcrosses following the original cross of Presto 1R.1D5+10-2 to the Australian translocation WR4. They are not very homogeneous materials. Therefore, verified lines with translocations at a homozygous state, derived from original sources, were used for analyses only. The lines with translocations were also compared in the experiments with a group of other five genotypes of triticale without translocations: Saka 3005/98 (Glu-A1a; Glu-B1b), Moreno (heterogeneous Glu-A1a, Glu-A1a/Glu-A1b, GluA1b; Glu-B1b), SG-U 137 (heterogeneous Glu-A1b, GluA1a/Glu-A1b; Glu-B1b), SG-U 204 (Glu-A1c; Glu-B1b), and SG-U 237 (Glu-A1c; Glu-B1b). Though these lines are in no relation to the translocated forms, they were involved

Fig. 2. Results of electrophoretic separations: (a) size marker pBR322 Hae III, (b) missing product, and (c) product of the 450 bp-size verifying the presence of the allele Glu-D1d.

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in the experiment in order to have similar numbers of triticales with and without translocations. Presto itself and wheat cultivars with various grain quality/Ebi (elite quality class — E), Sulamit (E), Samanta (good quality — A), Sˇa´rka (baking quality — B), Rialto (B) and Mladka (unacceptable for bread making — C)/ were the checks. 2.2. Field experiments The triticale and wheat genotypes were cultivated at the ARI. The location is 235 m above sea level, with average daily temperature of 8.7 1C and annual precipitation of 599 mm. The experiments were conducted in the crop years 2001/2002, 2002/2003, 2003/2004 and 2004/2005 following rapeseed in rotation. All plots were fertilised in the autumn with 30 kg ha1 N, 36 kg ha1 P2O5, 36 kg ha1 K2O; and in spring with 40 kg ha1 N (in ammonium nitrate with lime — ANL). Fungicides and growth regulators were not applied. The plot area was 10 m2. In the 2001/2002 season, the genotypes were sown without replications, in subsequent years four replications were used. Standard methods for evaluation of field tests were used. The yields were corrected to 14% grain moisture. Average grain weight per spike was calculated from yield and spike number per square metre. The harvested grain was subjected to quality analyses. 2.3. Basic grain quality characteristics Grain quality was evaluated by the accredited laboratory of the ARI using the methods for grain quality assessment in wheat. Specific weight was assessed according to ISO 7971-2:1995, Determination of bulk density, called ‘mass per hectolitre’. Dry matter nitrogen content was assessed by the Dumas combustion method according to ICC (Standard Methods of the International Association for Cereal Science and Technology Vienna, Austria) no. 167. Crude protein in dry matter of grain was calculated as N  5.7. Ash content was assessed using the method ISO 2171. The sedimentation value was assessed either by the Zeleny sedimentation value according to ISO 5529: 1992, or by the SDS-sedimentation test of Axford according to CNS 46 1021 (Czech National Standard). Similarly, wet gluten content (WG) was measured by two different methods: In the Perten method, ICC standard no. 155, gluten is washed out from flour in 1% aqueous NaCl solution for 10 min. The use of NaCl solution helps glutenaggregation and emphasises differences among examined samples. Using this method for samples with poor grain quality, gluten protein is less tenacious during the washing process and there are rather low values of gluten content due to washing part of the sample away during the washing out. This is a standard method for the assessment of gluten content in wheat. For comparison, an older method using a six-place washing device by Sˇpidla-Hy´zˇa where gluten is washed out from wholemeal by water for 20 min was used.

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2.4. Physical grain quality characteristics Grain hardness in Brabender units (BU) was measured on the Hardness and Structure Tester (manufactured by Brabender GmbH & Co. KG, 47055 Duisburg, Germany). It is additional equipment to the farinograph for testing. The samples were milled in a conical mill while the torque and milling time were recorded. The resistance during milling the 10 g grain sample at 60 r  min1 is measured and recorded. Grain hardness corresponds to the curve area. Hagberg falling number (FN) was determined according to ISO 3093. Wholemeal was prepared using a mill FN LM 3100. The method is based upon the rapid gelatinisation of a suspension of flour or meal using a boiling water bath and the subsequent measurement of the liquefaction, by aamylase, of the starch contained in the sample. FN values bear a complex inverse relationship with the quantity of aamylase in the sample. This relationship is known as the Perten liquefaction equation. Dough mixing properties (water absorption, development time, degree of softening and stability) were determined using the Brabender Farinograph. Flour milling was performed in a Brabender Junior mill using grain samples previously conditioned to 14.0% moisture, to generate flours with low ash contents (0.5–0.7%). Water absorption was determined according to the method: ISO 5530-1 Wheat flour: Physical characteristics of doughs. Part 1: Determination of water absorption and rheological properties using farinograph. Development time, degree of softening and stability were determined by the same method. Dough stickiness was measured during the baking test according to ICC standard no. 131. To be considered as ‘non-sticky and machinable’ at the end of mixing, the dough should form a coherent mass, which hardly adheres to the sides of the bowl and spindle of the mixer. It should be possible to collect the dough by hand and remove it from the mixing bowl in a single motion without noticeable loss. Bread making properties were determined on 300 g flour samples using a straight-dough baking formula and short fermentation time (ICC standard no. 131). The baking method calls for high-speed dough mixing and a short fermentation time. A dough is made in a specified mixer from flour, water, dry yeast, salt, sucrose, ascorbic acid and, if necessary, malt flour. Dough pieces are scaled, rounded, rested 30 min, sheeted and moulded, placed in tins, proofed 50 min and baked. Dough handling properties are noted. Bread loaves were evaluated in relation to loaf yield (loaf weight as percent of flour used), volume, shape (loaf height/width radio), crust, and crumb characteristics. The data were analysed using a single factorial analysis of variance by the StatSoft software. Differences among groups of tested genotypes and among crop years were determined. Statistical differences were calculated using

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Tukey test at P495%. The statistically analysed set included purposely the two distinct crops (triticale and wheat) in order to define differences between the examined triticale forms and widely grown wheat cultivars. Such analyses better match the long-term research and breeding goals when these differences should be gradually eliminated and a level of triticale quality characteristics should approach to wheat bread making quality. 3. Results 3.1. Productivity of tested genotypes The average yields of lines carrying the two tested translocations of chromosome 1R were significantly lower than that of the check cv. Presto (Table 1). The group of lines derived from Presto 1R.1D5+10-2 yielded 6.60 t ha1 ( ¼ 84% of the check), while lines with the translocation Valdy yielded 5.99 t ha1 ( ¼ 76% of the check). The average yield of the group of wheat checks was of 9.25 t ha1 ( ¼ 117% of Presto). The yield results suggest a linkage between the examined translocations and reduced yield. In Presto Valdy, the yield reduction is apparently related to a shorter spike length and lower grain weight per spike (Fig. 3). The shortening of spike length was apparent particularly in the translocation Valdy, while this trait in the translocation 1R.1D5+10-2 was almost the same as in the check cv. Presto. The lines of Presto 1R.1D5+10-2 had the average spike weight 14% lower and Valdy translocation even 24% lower as compared with cv. Presto (1.39 g ¼ 100%). In triticale, the differences in a spike number per square metre were not statistically significant. 3.2. Grain quality characteristics The specific weights of Presto and Presto 1R.1D5+10-2 lines were, on average, 78.5 and 77.1 kg h l1, respectively, and not statistically significant, the average specific weight in Presto Valdy lines was 74.1 kg h l1. This value in check wheats averaged 80.9 kg h l1. Similarly, the crude protein content was significantly higher in Presto Valdy (14.9%) than in Presto 1R.1D5+10-2 (14.6%) and Presto itself (14.2%). The Zeleny sedimentation values were higher in the translocation lines than in the typical triticales, but they were not higher than in the E-B quality-type wheats. The average value was 23 ml in Presto, 25 ml in Presto 1R.1D5+10-2 and 27 ml in Presto Valdy. In wheat checks, the Zeleny sedimentation value averaged 31 ml, ranging from 40 ml (E quality class wheats) to 17 ml (C quality class wheats). The SDS-sedimentation value measured according to Axford showed the same pattern: 42 ml in Presto, 49 ml in Presto 1R.1D5+10-2 and 52 ml in Presto Valdy. The check wheat cultivars had the sedimentation value of 63 ml. No significant differences were observed in SDS-sedimentation value between the two translocation types (Table 1).

The results of the WG according to Perten differed significantly from the results obtained using internal water washing in a six-place washer. It was caused above all by differences in the two assessment methods. In WG measured according to the standard, apparently low amounts of gluten are washed out in samples with poor grain quality with less tenacity of gluten protein. It was common in evaluated triticale lines with very low values. WG for the Presto was 9% only and for groups of lines with Glu-D1d the values ranged between 9% and 15%. This range of WG was rather wide; however, the differences among the groups of lines with the translocation Valdy were not statistically significant. Considerable differences in WG content were found between wheat (24%), check cv. Presto (9%) and common triticale lines without translocation (8%). These differences were apparently induced by the crop effect and stressed by the method according to Perten that provides different results from those obtained using the internal method for WG assessment. To wash out gluten, water was used in this method and the differences in gluten content were not as distinct: wheat (29%), cv. Presto (18%) and common triticale lines without translocation (19%). Based on this internal method, the triticale forms with translocations exhibited statistically significant increase in gluten content (26%) as compared with triticales without translocation (19%). Internal gluten washing method produced data indicating that translocation-type groups have higher WG values than conventional triticale. In addition, some triticale translocation-groups had wet gluten values as high as those in the B- and C-wheat quality classes. A level of gluten strength and weakness is described by a gluten index (GI) that expresses a percentage of gluten that did not pass through a mesh with defined openings under defined conditions. GI was a characteristic strongly influenced by the environment and crop years. It was on average slightly higher in triticale with Glu-D1d than in Presto and higher than in wheat. On average, the tested triticale genotypes showed lower grain hardness than wheat (Table 2). Grain hardness in the set of check wheat cultivars averaged 614 BU, in Presto 475 BU, in Presto with one translocation 506 BU and in Presto Valdy 509 BU. Grain hardness is a strong varietal trait. For instance, cvs Sulamit and Ebi (E quality class) had significantly higher grain hardness than Samanta (A quality class). Average water absorption evaluated in the Farinograph was higher in Presto Valdy (55.0%) than in Presto without translocation (53.1%) and Presto 1R.1D5+10-2 (54.0%). Very large differences in FN were found between wheat (301 s) and triticale (65 s). The low values of this character in triticale could have adversely affected the other grain quality characteristics. In triticale, FN was not significantly influenced by the presence of translocations and ranged from 70 to 62 s. The differences between triticale and wheat in dough characteristics (development, stability and softening) were

Table 1 Basic characteristics of productivity and grain quality in examined samples of triticale and wheat (means of 4 years 2002–2005) Crop

Weighted means Wheat Check Triticale Triticale Influences of year 2001/02 2002/03 2003/04 2004/05

Sulamit, Ebi (E quality) Samanta (A quality) Sˇa´rka, Rialto (B quality) Mladka (C quality) Presto ( ¼ 100 %) Standard lines of triticalea Presto 1R.1D5+10-2 (CT775-81): line no. 1,2,3,4,5 Presto Valdy (26-97): line no. 7,8,10 Presto Valdy (186-00): line no. 1,2,3,4,5,6,7,8 Presto Valdy (3915-01): line nos. 1,2,3,4,5

(Standard lines and Presto) Presto Valdy

Yield (% of cv. Presto)

Number of spikes per 1 m2 (% of cv. Presto)

(kg ha1)

(%)

2 1 2 1 1 5 5 3 8 5

9.10 8.85 9.48 9.55 7.89 8.20 6.60 5.49 6.11 6.10

115 112 120 121 100 104 84 70 77 77

606 642 633 670 591 569 546 542 571 575

6 6 16

9.26 8.15 5.99

117 103 76

632 573 567

33 33 33 33

7.03 6.19 7.51 7.55

a ab a a b ab c d cd cd

ab b a a

594 574 588 548

bc ab ab ab b-d cd d d cd cd

a a a b

Grain weight per spike (% of cv. Presto)

(%)

(g)

102 109 107 113 100 96 92 92 97 97

1.50 1.38 1.50 1.43 1.39 1.38 1.21 1.02 1.07 1.07

107 97 96

1.47 1.38 1.06

1.08 1.08 1.28 1.37

a ab a ab ab a b c c c

bc c ab a

Specific weight

Zeleny sediment value

(%)

Crude protein content in dry matter (N  5.7) (kg h l1) (%)

(ml)

108 99 108 103 100 100 87 73 77 77

82.1 81.2 81.5 76.9 78.5 76.1 77.1 72.6 74.2 74.8

40 32 27 17 23 18 25 27 27 28

105 100 76

80.9 76.5 74.1

76.0 75.7 75.6 78.0

a ab a b–d a–c cd cd e de de

14.3 14.5 12.7 12.3 14.2 13.0 14.6 15.1 14.9 14.8

b b d d bc c ab a a a

13.5 13.2 14.9

ab b b a

14.0 14.1 14.6 14.4

ab b bc d cd d c bc c bc

31 18 27

a a a a

26 26 27 24

Wet gluten contentb

Wet gluten contentc

Gluten index

(ml)

(%)

(%)

(%)

74 66 57 47 42 34 49 53 51 52

27 27 19 24 9 8 12 9 12 15

30 36 26 27 18 19 26 26 26 26

66 61 66 50 61 55 76 76 77 67

SDSsediment value (Axford)

a ab bc de e f de cd de cd

63 35 52

a a a a

55 50 51 46

a a a–c ab c–e e c–e de c–e b–d

24 8 12

a ab ab b

13 15 13 13

ab a bc bc d d c c c bc

29 21 26

b a b b

26 26 24 25

a–c a–c a–c bc a–c c a ab a a–c

63 56 73

a a a a

58 84 63 67

c a bc bc

Homogeneity at P495%. a Five lines: Saka 3005/98, Moreno, SG-U 137, SG-U 204, and SG-U 237. b Method: ICC-standard no. 155 determination of wet gluten quantity and quality (gluten index according to Perten). c Method: internal (according to Sˇpidla-Hy´zˇa).

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Check Check Check Check Check

Number of genotypes in the set

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Wheat Wheat Wheat Wheat Triticale Triticale Triticale Triticale Triticale Triticale

Cultivar

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Fig. 3. Differences in the spike shape in the tested lines: left Presto; middle Presto 1R.1D5+10-2; right Presto Valdy.

significant in many cases. It is most apparent in the degree of softening. As compared to the check cv. Presto, the lines Presto 1R.1D5+10-2 and Presto Valdy showed a slight increase in the values of development time and stability, and substantial improvement of the degree of softening (cv. Presto — 203 FU, Presto 1R.1D5+10-2 — 150 FU and Presto Valdy — 118 FU, mean of wheat checks — 70 FU). The dough made from Presto was sticky, that from Presto 1R.1D5+10-2 was slightly sticky and the dough from Presto Valdy and from wheats of E-B quality classes was not sticky. The cultivar Mladka with C quality class (unacceptable for bread making) was slightly sticky. It is in correspondence with characteristics of degree of softening. The effect of the translocations on loaf volume was insignificant (338 cm3 in Presto, 364 cm3 in Presto 1R.1D5+10-2 and 345 cm3 in Presto Valdy). In contrast, the appearance, shape and structure of loaves were markedly better in the translocation lines than in Presto itself. The translocations strongly influenced loaf shape (loaf h/w ratio). The check cv. Presto (0.44) and group of other triticale cultivars without translocation (0.30) had flat topped shape of breads. Presto 1R.1D5+10-2 (0.56) and Presto Valdy (0.61) had medium and well round topped shape of breads. Loaf shape in the translocation Valdy was higher than in the check wheat cultivar with quality C (Mladka) 0.58, but lower than in wheat cultivars with bread making quality B (Sˇa´rka, Rialto) 0.65, A

(Samanta) 0.72 and E (Sulamit, Ebi) 0.81. Results of the baking test in selected samples harvested are illustrated in Fig. 4. Statistical differences were found for loaf shape (h/w ratio) only in line no. 1 derived from the sample CT775-81 and in line no. 7 derived from the sample 26-97 that exhibited a flat loaf. Both these samples had also sticky dough. These two lines had worse values of loaf shape than expected. Therefore, they were analysed for their storage proteins using the PAGE-ISTA. Though the heterogeneous proportions of Glu-D1d were not documented in them, heterogeneity in other HMW prolamins was found. In line no. 1 three different spectra were found. Based on the calculation of identity index (ii), they can be considered sister prolamin sublines (ii ¼ 0.64–0.74) (Vyhna´nek and Bedna´rˇ , 2003) (Fig. 5). Similarly, two sister gliadin sublines were detected in line no. 7 (one of Presto Valdy sister lines). These findings can elucidate why the loaf quality was worse in these genotypes. It needs to be pointed out that the line of Presto Valdy originating from plant 186-00 has looked suspicious and its progenies have never been used for any tests but the samples for this research were conveyed to the authors before the developer become aware of the problem (Lukaszewski, pers. comm., 2005). Except the loaf shape, no significant differences were found among samples within the groups of tested genotypes during statistical analysis of individual lines. 4. Discussion The results documented yield decrease by 26% in Presto Valdy and by 14% in the Presto 1R.1D5+10-2. In Presto Valdy, this yield reduction must have been caused by the reduction of spike length, resulting in a lower grain number per spike. The gene(s) encoding the shortened ear length is (are) are obviously located on 1RS where the wheat segment with loci Gli-D1 and Glu-D3 is introduced. This problem was first observed in 2000 and its nature and genetics are still unclear. Apparently, the character manifests itself only under certain, so far undefined environmental conditions and has never been noticed during line development in the greenhouse in California. It was first observed in Poland in field planted materials, and later in Oregon, in fall planting but not in winter planting. Several steps have been taken to remedy this problem, so far unsuccessfully. For this reason, a series of new translocation chromosomes have been produced from different initial stocks of 1RS and 1DS than in Valdy, and so far no spike length reduction has been observed (Lukaszewski, pers. comm., 2006). The reduction in yield of the Presto 1R.1D5+10-2 line is somewhat more disturbing. While the Valdy chromosomes are three-point translocations with different segments originating from two different wheats and the rye segments originating from at least three different ryes, and they never had sufficient backcrosses to Presto to guarantee elimination of any background effects, chromosome 1R.1D5+10-2 is a two

Table 2 Physical characteristics of grain quality in examined samples of triticale and wheat (means of 4 years 2002–2005) Crop

Triticale

Sulamit, Ebi (E quality) Samanta (A quality) Sˇa´rka, Rialto (B quality) Mladka (C quality) Presto Standard lines of triticalea Presto 1R.1D5+10-2 (CT775-81): line nos. 1,2,3,4,5 Presto Valdy (26-97): line nos. 7,8,10 Presto Valdy (186-00): line nos. 1,2,3,4,5,6,7,8 Presto Valdy (3915-01): line nos. 1,2,3,4,5

Weighted means Wheat Check Triticale (Standard lines and Presto) Triticale Presto Valdy Influences of year 2001/ 02 2002/ 03 2003/ 04 2004/ 05

Development Degree of time (min) softening (FU)

Stability (min)

Prevailing stickiness

Loaf yield (%)

Loaf volume (cm3)

Loaf shape (h:w ratio)

2 1 2 1 1 5 5

683 525 618 548 475 493 506

4.3 3.8 2.0 1.2 1.9 1.4 1.8

6.5 6.8 4.0 3.5 2.7 2.1 3.9

Non-sticky Non-sticky Non-sticky Slightly sticky Sticky Sticky Slightly stickyb

137 154 131 121 104 100 107

683 636 513 408 338 275 364

0.81 0.72 0.65 0.58 0.44 0.30 0.56

3 8

523 c 503 c

62 b 63 b

55.8 b–d 54.8 b–d

2.0 b 1.9 b

115 c 119 c

2.6 bc 3.2 bc

Non-stickyc Non-sticky

101 de 106 cd

5

511 c

63 b

54.8 b–d

2.1 b

120 c

2.8 bc

Non-sticky

102 cde 352 cd

0.63 b

Non-sticky Sticky Non-sticky

135 104 104

573 327 345

0.70 0.32 0.61

a c b b c c c

297 298 298 316 70 69 63

a a a a b b b

59.5 57.6 56.3 52.4 53.1 52.4 54.0

a ab a–c de b–e e c–e

a A b d bc c bc

50 63 78 100 140 203 128

a ab ab bc c d c

a a b bc bc c b

ab a b bc c–e e cd

a a d c c–e e cd

322 d 350 cd

a ab b cd d e c

0.54 cd 0.63 b

6 6 16

614 478 509

301 70 63

56.9 53.0 55.0

2.9 1.8 1.9

70 150 118

5.2 2.6 2.9

33

501 b

112 a

55.8 a

2.1 a

106 a

3.1 c

107 b

373 ab

0.57 a

33

559 a

107 a

53.6 b

2.0 a

123 ab

4.8 a

112 a

422 a

0.60 a

33

513 b

102 a

54.9 ab

2.1 a

145 b

2.2 b

110 ab

339 b

0.55 a

33

524 b

107 a

54.7 ab

2.0 a

125 ab

3.4 c

110 ab

379 ab

0.55 a

Homogeneity at P495%. a Five lines: Saka 3005/98, Moreno, SG-U 137, SG-U 204, and SG-U 237. b Line no. 1 derived from CT775/81 was sticky. c Line no. 7 derived from 26/97 was sticky.

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Triticale Triticale

Check Check Check Check Check

Grain Hagberg Water hardness falling absorption (BU) number (%) (s)

Number of genotypes in the set

P. Martinek et al. / Journal of Cereal Science 47 (2008) 68–78

Wheat Wheat Wheat Wheat Triticale Triticale Triticale

Cultivar

75

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Fig. 4. Demonstration of a baking test: check cv. Presto without translocation (loaf height /h/:width /w/ ratio is 1:2.27; loaf volume is 338 ml), Presto 1R.1D5+10-2 (h:w ¼ 1:1.78; volume 364 ml), Presto Valdy (h:w ¼ 1:1.63; volume 345 ml).

Fig. 5. Electrophoreogram of prolamins in Presto 1R.1D5+10-2: line no. 1 derived from the sample CT775/81: sister prolamin sublines 1 (46.7%); 2 (40.0%); 3 (13.3%); identity index ii: 1-2: 0.74, 1-3: 0.69, 2-3: 0.64. P — check triticale cv. Presto; A — check wheat cv. Astella.

breakpoint translocation, its rye portions originate from triticale cv. Rhino and from Presto, while the wheat segment is from cv. Wheaton (Lukaszewski and Curtis, 1992). This chromosome had 10 backcrosses to Presto from Rhino. Consequently, it is assumed that apart from segments of 1R from Rhino in the vicinity of the translocation breakpoints, the line does not deviate from Presto in any substantial manner. It is not clear where the yield reduction comes from but it appears real. Literature concerning the bread making quality of triticale shows very promising results due to substitution of chromosome 1D for chromosome 1R. This substitution eliminates both secalin loci from chromosome 1R, Sec-3 and Sec-1 and introduces all storage protein loci from wheat chromosome 1D. Unfortunately, this substitution 1D(1R) is agronomically unacceptable for several reasons including severe fertility reduction (Lafferty and Lelley,

2001). It was the agronomic problem of the substitution that prompted the effort of generating chromosome translocations. Unfortunately, severe yield depression has also been observed in the translocation lines. A genetic mechanism of this yield reduction has not been studied yet. It is possible that genetic backgrounds suitable for the translocations will be identified. In the doubled haploid breeding programme at IHAR in Malyszyn and Borowo, Poland, a line with the parentage ‘(Krakowiak  Presto Valdy)  MAH 1596’ was derived that possesses the translocation Valdy and has normal spike length. This line yielded 7 t ha1 in tests carried out in Poland in 2004 (Wos´ , pers. comm., 2004). Within the hybridisation programme conducted by the ARI, where parental genotypes studied in this work were used, genotypes with the translocation Valdy and a longer spike were found among genotypes in F5 generation in 2005. We hope to stabilise these genotypes and reach satisfactory grain yields and improved bread making quality. Despite positive trends in grain quality improvement in the examined materials, it is obvious that it will be difficult to achieve in triticale the parameters of bread making quality that are comparable to bread wheat. There is little doubt that the main culprit was the high a-amylase activity producing low FN. It is imperative that any programme aiming at bread making quality improvement in triticale addresses at the same time the issue of premature sprouting resistance and low a-amylase activity particularly in humid environments (Sodkiewicz, 1999). A high activity of amylase enzymes produces a low FN. The cereals with a low FN have worse bread making quality (dough is sticky, badly machinable, loaf volume is low, bread crumb is less flexible). The differences observed in quality traits could have been the results of separate or combined effects caused by translocation type and FN values. In wheat, the genes conferring resistance to sprouting are localised on chromosome 6D (Gale et al., 1990; Rybka, 2003). The substitution 6D(6R) in Presto increased FN in comparison with the euploid Presto (Masojc, 1997). Resistance to sprouting is also affected by genes for higher seed dormancy (Trethowan et al., 1993). As some literature sources refer, substantial decrease in the a-amylase activity can be achieved by strong selection. A possibility of the development of triticale with improved resistance to sprouting naturally depends on knowledge of corresponding genes. Since the current breeding of triticale cultivars is based particularly on mutual crosses of parent cultivars and not on newly developed primary and secondary forms, no substantially rich gene pool is available. European triticale cultivars are typical for a low FN. In wheat, the value of this character less than 120 s indicates that samples are unacceptable for baking. Thus, all triticale samples described in this study can be considered unacceptable. Relatively higher values of this parameter have been found, for instance, in Polish cvs Krakowiak and Sekundo that reached the limit of 120 s in multiyear experiments. Based on results of the Central Institute for

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Supervising and Testing in Agriculture (Czech Republic), FN in cv. Sekundo was 154 s as compared with the mean of other triticales (115 s) in 3-year trials in 2003–2005. Donors of resistance to sprouting can be developed using the breeding methods that involve new primary forms of triticale derived from crosses of wheat (Triticum spp.) and rye (Secale cereale L.), and other methods of wide hybridisation (Sodkiewicz, 1999). If there is a sufficiently heterogeneous breeding material, selection for the increased sedimentation value can be an efficient tool of improving bread making quality in triticale (Metzger and Lukaszewski, 1998). The first bread cultivar of winter triticale Valentin-90 was probably developed in this way at the KNIISH, Krasnodar (Russia), by breeder V.Y. Kovtunenko et al. (2007). Since the presence of GluD1d has not been confirmed in this cultivar, we assume that it does not possess chromosome 1R translocation. We suppose that bread making quality of cv. Valentin was achieved particularly by intensive selection for a high value of FN. The small differences in loaf volume between Presto and its translocation lines may be caused by a different baking process used here relative to the published results. The pan bread baking tests (Lafferty and Lelley, 2001; Wos´ et al., 2002) showed substantial improvements in loaf volumes in substitution and translocation triticale lines. Our hearth bread baking tests allowed us to assess even shape characteristics of test loaves but did not show much difference in loaf volume. Large differences are interesting in WG content in triticale determined according to Perten and by an internal method using a six-place washing device by Sˇpidla-Hy´zˇa. These differences might be induced by different gluten protein aggregation during washing out between wheat and triticale. If the standard gluten washing method is not able to generate acceptable, near-100% gluten mass, then this is not a suitable method to determine WG in triticale. It looks that the standard gluten washing method developed for wheat was not quite appropriate to measure gluten qualityrelated parameters in triticale. Due to poor dough viscoelastic properties in triticale as indicated by farinographic and bread making parameters, lower values were expected than in bread wheat cultivars. An optimum range of values for wheat is between 50% and 80%. High GI values show strong gluten that almost does not pass the mesh, by contrast, low values (less than 40%) characterise weak gluten. High GI values shown by the triticale lines suggest that the GI method used to assess gluten quality in wheat may not be applicable in triticale. Lukaszewski (2006) states that the translocation Presto 1R.1D5+10-2 increased SDS-sedimentation value to 173.974.9%, the translocation Valdy to 209.0710.1% and substitution 1D(1R) to 250.479.7% in comparison with cv. Presto (100%). There was a considerable effect of Gli-D1 and Glu-D3, in which both translocation types differ. In our study, such big differences were not found. It can be caused by a very low FN, which was confirmed for

77

cv. Presto and identical derived lines also in another laboratory, SELGEN Co. Ltd. It is interesting that this firm, affiliation Breeding Station U´hrˇ etice, derived the line M4/2 from the cross of Presto 1R.1D5+10-2 (line 1 derived from CT771-85)  Moreno with some good-quality parameters. It had loaf volume 1700 cm3 and FN 97 s. The wheat check cv. Samanta (A quality class) had loaf volume 1850 cm3 and FN 372 s and cv. Rheia (B quality class) had loaf volume 1610 cm3 and FN 357 s (Hroma´dko, pers. comm., 2005). Though the FN was also very low in M4/2, it was higher than in Table 2. It seems that even such a little increase can positively affect other grain quality parameters. Results of comprehensive tests of grain quality in both translocation types in comparison with the check Presto and wheat check cultivars were not published earlier. Apart from the negative agronomic effects of the translocations, 1R.1D5+10-2 and Valdy also carry the Sec-1 locus on the short arm. This locus is believed to negatively impact bread making quality of wheat, even though this may be an oversimplification. A new series of translocations are being developed that would not only carry the Gli-1 and Glu-3 loci on their short arms, but would also have the Sec-1 locus deleted. As some of these new translocations are based on chromosome 1RS of the Kavkaz source that was so successful in wheat worldwide (Braun et al., 1998), there is a chance that the agronomic effects of the translocations in triticale will also be mitigated (Lukaszewski, 2006). The lines of studied cereals are maintained at the ARI and can be used for further research based on the authors’ and Dr. A.J. Lukaszewski’s agreement. Acknowledgement We thank Dr. A. J. Lukaszewski for providing original samples of the translocation lines and cooperation. The research was supported by the Czech Scientific Foundation, project GA CR no. 521/03/0113 and project MSM 2532885901 (stage E) from the Ministry of Education of the Czech Republic. References Braun, H.J., Payne, T.S., Morgounov, A.I., Van Ginkel, M., Rajaram, S., 1998. The challenge: one billion tons of wheat by 2020. In: Slinkard, A.E. (Ed.), Proceedings of the Ninth International Wheat Genetics Symposium, Saskatoon, Canada, 2–7 August. University of Ext. Press, University of Saskatchewan, Saskatoon, SK, Canada, pp. 33–40. Brezinski, W.J., Lukaszewski, A.J., 1998. Allelic variation at the Glu-1, Sec-2 and Sec-3 loci in winter triticale. In: Proceedings of the Fourth International Triticale Symposium, Red Deer, Alberta, Canada, July 26–31, pp. 12–13. D’Ovidio, R., Anderson, O.D., 1994. PCR analysis to distinguish between alleles of member of a multigene family correlated with bread-making quality. Theoretical and Applied Genetics 88, 759–763. Fabriani, G., Lintas, C., 1988. Durum Wheat: Chemistry and Technology. AACC, St. Paul, 332pp.

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