Quality Effects of Rye (Secale cerealeL.) Chromosome Arm 1RL Transferred to Wheat (Triticum aestivumL.)

Journal of Cereal Science 29 (1999) 211–216 Article No. jcrs.1998.0237, available online at http://www.idealibrary.com on

Quality Effects of Rye (Secale cereale L.) Chromosome Arm 1RL Transferred to Wheat (Triticum aestivum L.)∗ R. A. Graybosch†, C. J. Peterson† and O. K. Chung‡ †University of Nebraska, USDA-ARS, 344 Keim, Lincoln, NE 68583, U.S.A.; ‡Grain Marketing and Production Research Center, USDA-ARS, Manhattan, KS 66502, U.S.A. Received 23 November 1998

ABSTRACT Quality effects of rye (Secale cereale L.) chromosome arm 1RL transferred to wheat (Triticum aestivum L.) were characterised by comparison of a group 1R(1B) substitution lines and 1BL.1RS translocation lines. The experimental materials were sister lines derived from the mating Mironovskaya10/ NE7060//NE80413. 1R(1B) substitution lines were identified by the presence of rye omega- and gamma-secalins among 70% ethanol soluble proteins, combined with the presence of high molecular weight (HMW) secalin proteins in total grain protein extracts. Genes on 1RL reduced grain weight, grain hardness, Mixograph time, Mixograph tolerance and SDS sedimentation volumes. 1RL had no effect on flour yield or grain and flour protein concentrations. The HMW secalin proteins encoded by genes on 1RL most probably caused the decline in dough strength seen in 1R(1B) lines relative to that of 1BL.1RS sister lines. Reduced grain hardness might also be related to the presence of HMW secalins, although a role for additional, unidentified genes on 1RL could not be discounted.  1999 Academic Press Keywords: wheat (Triticum aestivum), rye (Secale cereale), chromosomal substitution, chromosome arm 1RL, quality effects, grain hardness, dough strength.

INTRODUCTION Rye (Secale cereale L.) has served as a valuable donor of genes for the agronomic improvement of wheat (Triticum aestivum L.)1. Rye chromosome arm 1RS, transferred to wheat in the form of 1AL.1RS, 1BL.1RS or 1DL.1RS wheat-rye chromosomal translocations, has been the most successfully deployed piece of rye chromatin1–3. In wheat, 1RS, from a variety of rye sources, has been used to ∗ Joint contribution of the United States Department of Agriculture, Agricultural Research Service and the Department of Agronomy, University of Nebraska–Lincoln as Journal Series Paper No. 12123. Mention of firm names or trade products does not imply that they are endorsed or recommended by the USDA of the University of Nebraska over other firms or products not mentioned. 0733–5210/99/030211+06 $30.00/0

confer resistance to numerous pests and pathogens, and may boost grain yields in some environments1–4. Unfortunately, 1RS has a negative impact on wheat processing quality, largely as a consequence of the loss of genes encoding low molecular weight glutenin proteins, and the gain of genes producing rye secalin proteins5–7. Many 1BL.1RS cultivars trace their origin to 1R(1B) substitution lines1. In such lines, the entire 1R chromosome has replaced chromosome 1B. Several 1R(1B) substitution lines have been released as cultivars in Europe1,8. While reports on the quality effects of 1RS are common5–7,9, little information is available on the effects of 1R or 1RL. Intermatings of 1R(1B) lines with typical 1B wheat lines, followed by subsequent derivation and characterisation of sister lines with and without 1R, could be used to characterise 1R. Abadie et  1999 Academic Press

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al.10 used such an approach to study agronomic effects of 1R substituted for 1D. This method does not, however, allow one to separate the effects of 1RL from those of 1RS. 1RL.1DS translocations have been produced11, and could be used to characterise the quality effects of an isolated 1RL arm in wheat. In this report, an alternative method to the characterisation of 1RL was used. A population was developed from an intermating involving a 1R(1B) substitution line, and a 1BL.1RS translocation line. Analysis of seed storage protein composition12,13 was used to classify progeny lines as 1R(1B) or 1BL.1RS. The quality effects of 1RL, relative to 1BL, were measured as the difference in mean performances of 1R(1B) lines vs 1BL.1RS sister lines.

MATERIALS AND METHODS Materials used in the study were F4-derived F8 sister lines from the cross Mironovskaya10/NE7060// NE80413. Mironovskaya10 (Bezostaya1/Erythrospermum2107) is a 1R(1B) substitution line12. NE80413 (Lovrin13/2∗Centurk78) is an experimental breeding line that carries 1BL.1RS. The 1BL.1RS translocation in NE80413 was derived from Lovrin13, and is the same translocation found in the Russian cultivars Kavkaz and Aurora1. NE7060, descended from Favorit/5/ Cirpiz/4/Jang Kwang/2/Atlas66/Comanche/3/ Velvet, carries no rye chromatin. A subset of these sister lines has been deposited in the USDA-ARS National Small Grains Collection at Aberdeen, ID, as Plant Introduction numbers 598313-598324. The presence of rye secalins, encoded by genes on 1RS, was verified by sodium dodecyl sulphate polyacrylamide gel electrophoretic (SDS-PAGE)13 separation of unreduced 70% ethanol soluble grain proteins using 12% (w/v) acrylamide gels. Lines were characterised as 1R(1B) or 1BL.1RS by SDSPAGE separations of glutenin proteins. Glutenins were reduced and alkylated14 before separation on 8·5% (w/v) acrylamide gels12 and 1R(1B) lines were identified by the presence of 1RL-encoded high molecular weight (HMW) secalins13. C-banding of select lines (K. S. Gill, University of Nebraska, pers. comm.) confirmed the presence of 1RL. Sixteen 1R(1B) and 15 1BL.1RS lines were planted in randomised complete block designs with three replications at Lincoln, Sidney, and

McCook, NE, in 1996. Four-row, 3 m plots were used. The study included four non-rye bearing wheat lines, 94L10124, ‘Arapahoe’, ‘Redland’ and ‘Vista’, and the 1BL.1RS cultivar ‘Siouxland’, as controls. A 300-seed sample from each harvested plot was analysed with a SKCS 4100 Single Kernel Classification System (Perten Instruments, Reno, NV) to determine kernel weight and kernel hardness, as well as the standard deviation of each characteristic within each sample. Grain samples (100 g) were milled to flour using a Brabender (Hackensack, NJ) Quadrumat Senior experimental mill. Percentage flour yield was determined. Grain and flour protein concentrations (14% mb) were measured by near infrared reflectance (NIR) as per AACC Method (39-70A)15. A National Manufacturing (Lincoln, NE) Mixograph was used to measure dough strength, using a 10 g flour sample (AACC Method 54-40). Mixograph time was recorded as time, in minutes, to peak dough resistance. Mixograph tolerance, or resistance to overmixing, was rated on a 0–6 scale, with 0 indicating no tolerance. Mixograph tolerance was rated by comparing mixograms to a set of standard Mixograph curves. SDS sedimentation volumes of flour samples were determined using a 2 g modification of AACC Method 56-61A. SAS16 programs and procedures were used for statistical computations. Analysis of variance was used to partition variation to location, replication within locations, entries, and the interaction of entry and location. Variation within entries was further partitioned to differences within three classes: check cultivars, 1R(1B) lines, and 1BL.1RS lines. Statistical contrasts16 were used to compare mean values of 1R(1B) lines vs 1BL.1RS sister lines. Mean squares from the entry X location interaction term were used to compute F statistics for entries, lines within classes, and contrasts. Mean comparisons among all entries were conducted by computation of least significant differences (l.s.d.)16.

RESULTS AND DISCUSSION All non-control lines were found to produce 1RSencoded omega and gamma secalins (not shown). 1R(1B) lines were identified by the presence of HMW secalins after separation of total grain proteins by SDS-PAGE (Fig. 1). HMW secalins migrated more slowly than the wheat 1B encoded

Effects of rye chromosome transferred to wheat

Figure 1 SDS-PAGE separation of glutenin proteins from parental line Mironovskaya 10 (lanes 1 and 6), two 1BL.1RS sister lines (lanes 2 and 3) and two 1R(1B) sister lines (lanes 4 and 5). HMW glutenins appear between anchored arrows; unanchored arrow denotes HMW secalin proteins.

HMW glutenin subunits13. All 1BL.1RS lines carried the 1B encoded HMW glutenin subunits 7+9. Identical HMW secalins were observed in the parental line Mironovskaya10. Significant differences in kernel weight, kernel weight standard deviations, kernel hardness and kernel hardness standard deviations were observed when 1R(1B) lines were compared to 1BL.1RS lines (contrasts, Table I). No differences were observed in grain and flour protein concentration or in flour yield. Significant differences between 1R(1B) and 1BL.1RS lines also were detected for

Table I

Mixograph time, Mixograph tolerance and SDS sedimentation volume (Table II). Statistical comparisons of mean values of 1R(1B) and 1BL.1RS are given in Table III. Mean values for control lines are given only for reference. The 1BL.1RS lines had higher mean values for kernel weight, kernel weight standard deviations, kernel hardness, Mixograph mix time, Mixograph mixing tolerance and SDS sedimentation volume. The 1R(1B) lines exceeded their 1BL.1RS sister lines only in kernel hardness standard deviation. Significant differences among both 1R(1B) lines and 1BL.1RS lines were detected for all attributes except grain protein concentration, flour yield and flour protein concentration (Tables I and II). SDS sedimentation volumes, plotted as a function of protein contents (Fig. 2), showed that the highest 1BL.1RS lines exceeded the 1BL.1RS cultivar Siouxland, and equalled the ryeless cultivar Arapahoe. Arapahoe is considered to be of acceptable quality to the U.S. baking industry while Siouxland is not. 1R(1B) lines with the highest mean SDS sedimentation volumes were not significantly different than Siouxland; however, SDS sedimentation volumes of all 1R(1B) lines were significantly lower than that of Arapahoe. Thus, while variation exists within each class for improvement in dough strength through successive rounds of selection, intermating and repeated selection, progress likely will be more difficult with 1R(1B) lines. As both sets of sister lines carry 1RS, observed differences in quality characteristics of 1R(1B) and 1BL.1RS lines must arise as a consequence of genes on either 1RL or 1BL. Loss of dough

Mean square from analysis of variance of grain and milling properties of 1R(1B) and 1BL.1RS sister lines, and controls

Source of variation Location Rep (location) Entry Among controls Among 1BL.1RS lines Among 1R(1B) lines 1BL.1RS vs 1R(1B) Entry X location Error a

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df

Grain weight

Grain weight ..

Grain hardness

Hardness ..

Grain protein concentration

Flour yield

2 6 35 4 14 15 1 70 203

434·71a 3·30 38·86a 6·07 10·96a 62·17a 238·33a 3·08a 0·42

20·25a 0·47 2·78a 4·60a 1·88a 1·20a 34·49a 0·49 0·42

2108·98a 10·06 185·56a 484·06a 85·37a 78·96a 1455·63a 29·62a 4·55

33·54a 1·93 9·16a 5·68a 5·37a 2·51a 8·83a 1·27 0·98

279·77a 1·58a 2·20a 8·10a 0·90 1·05 2·33 0·86a 0·30

495·13a 10·05a 8·67a 22·10a 1·93 2·57 0·06 2·08a 0·89

Significant F-value at p=0·05.

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Table II

Mean squares from analysis of variance of flour properties of 1R(1B) and 1BL.1RS sister lines, and controls

Source of variation Location Rep (location) Entry Among controls Among 1BL.1RS lines Among 1R(1B) lines 1R(1B) vs 1BL.1RS Entry X location Error a

Flour protein concentration

Mixograph time

Mixograph tolerance

SDS sediment

2 6 35 4 14 15 70 203

254·26a 1·28 2·99a 6·15a 10·70a 1·14 0·98 0·89a 0·32

35·34a 0·19 4·56a 1·75a 1·35a 1·93a 18·31a 0·27a 0·13

60·09a 1·12 16·04a 3·69a 4·67a 6·51a 215·04a 0·53a 0·25

1855·72a 4·79 246·74a 155·37a 98·67a 113·82a 2041·79a 11·22a 3·59

Significant F-value at p=0·05.

Table III

Mean quality characteristics of 1R(1B) (n=16) and 1BL.1RS (n=15) sister lines, and controls

Attribute Grain weight (mg) Grain weight .. (mg) Grain hardness (U) Grain hardness .. (U) Flour yield (%) Grain protein conc. (%) Flour protein conc. (%) Mixograph time (min) Mixograph tolerance (0–6) SDS sediment volume (mL) a

df

1BL.1RS 34·46 8·62 70·53 14·82 71·01 13·98 12·96 2·75 3·09 33·26

1R(1B) a

32·64 7·91a 65·77a 15·18a 71·04 14·15 13·26 2·21a 1·29a 27·66a

Arapahoe

Redland

Siouxland

Vista

94L10124

32·44 7·98 65·44 14·67 71·80 13·70 12·46 3·95 4·33 36·83

32·49 7·87 52·11 14·78 73·89 12·49 11·33 4·21 4·23 39·44

33·11 7·42 72·22 14·00 73·64 13·38 12·18 3·21 3·22 33·33

34·48 8·27 62·78 14·67 72·98 12·98 11·70 4·32 4·33 42·44

33·10 9·33 65·78 16·11 70·08 15·00 14·16 4·11 5·00 43·50

Denotes significantly different mean (p=0·05) based on 1R(1B) vs 1BL.1RS contrast.

strength, beyond that typically observed in 1RS lines, was the most obvious effect. Diminished dough strength in 1R(1B) lines most probably arises from the presence of genes on 1RL encoding HMW secalins. Reconstitution studies, in which purified HMW secalins were added to wheat doughs, have demonstrated a negative effect of these proteins on dough strength17. Kipp et al.17 suggested this was due to a higher frequency of cysteine residues. These perhaps confer a different type of polymerisation pattern to HMW secalins that might interrupt the formation of the large glutenin polymers necessary for strong doughs. Differences between surface hydrophobicities of HMW secalins and HMW glutenins also were detected. Field and Shewry18 had earlier reported HMW secalins, when present in triticale (X Triticosecale Wittmark), combined with HMW glu-

tenins to form mixed polymers of lower native molecular weight than those typical of HMW glutenins. It seems likely, then, that the HMW secalins act as ‘end-blockers’19 to terminate glutenin polymers at sizes less than optimal for adequate dough strength. 1R(1B) and 1BL.1RS wheats also differed in grain hardness, with the 1BL.1RS wheats being significantly harder. The standard deviations of hardness were higher in 1R(1B) lines, indicating more variation for hardness within each line of this class. Harder grains in the 1BL.1RS wheats could result from a factor on wheat chromosome arm 1BL that increases grain hardness, or there may be a gene or genes on 1RL that soften grains. Previous investigations20–22 on the chromosomal location of wheat genes influencing grain hardness have demonstrated little effect of chromosome 1B

Effects of rye chromosome transferred to wheat

Figure 2 Flour protein contents and SDS sedimentation volumes of 1BL.1RS and 1R(1B) lines compared to control wheats. Β, Controls; Φ, 1BL.1RS; Η, 1R(1B); ARA, Arapahoe; SX, Siouxland.

on this trait. Doekes and Belderok22 studied the effects of all 21 wheat chromosomes on grain hardness through the use of four sets of intervarietal substitution lines. 1B from three donor cultivars had no effect on grain hardness in the recipient cultivar ‘Chinese Spring’. 1B from ‘Hope’, however, did increase grain hardness. Thus, it is possible that some wheat lines carry a modifier gene on 1B that is not widespread. Similarly, the specific 1BL arm associated with 1RS in this cross might carry a modifier gene that increases hardness, or, a minor gene modifying hardness could occur on the specific 1RL arm used in this study. Previous studies on the chromosomal location20–22 of genes influencing hardness have indirectly measured this characteristic by determinations of flour yield, particle size after grinding or damaged starch content after milling. The SKCS 4100 measures hardness directly, as resistance to crushing. Despite differences in mean hardness between 1R(1B) and 1BL.1RS sister lines, no differences in flour yield were observed (Table III). Thus, the past lack of hardness effects of 1B might have resulted from procedural limitations at differentiating true hardness differences. In the present study, the control, Redland, had the lowest hardness score, yet had the highest flour yield. Flour yield, therefore, may be an unreliable indicator of grain hardness.

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In recent years, hardness in wheat has been attributed to the behaviour of a low molecular weight starch granule protein23 now known as friabilin, or to associated lipids24. However, it is also apparent that hardness is the result of the accumulated effects of major genes, perhaps those encoding friabilin, plus minor (modifier) genes20, and environmental effects25. Stenvert and Kingswood26 suggested grain hardness was the result of the continuity of the grain protein matrix and its interactions with starch granules. Anything that interrupted the continuity of the protein matrix, whether it be a major gene effecting hardness, a minor gene, or an environmental factor, could result in a ‘weakened endosperm structure’. Hence, if HMW secalins result in smaller glutenin polymers, with a consequently less-continuous protein matrix, the softer kernels of 1R(1B) lines might be an indirect effect of these ‘strangers in a strange land’. This would not explain, however, the observation that the 1BL.1RS sister lines, and the 1RS cultivar Siouxland, all having weaker doughs than typical hard wheats, had higher hardness values than the ryeless control lines. More recently27, direct effects of specific storage proteins on grain hardness have been suggested. Thus, it is possible the rye HMW secalins somehow influence the protein-starch matrix to the extent that hardness is reduced. Cultivars carrying 1R have not been released as frequently as those carrying only 1RS. The additional reduction in dough strength, most likely a consequence of HMW secalins, may have caused 1R experimental lines to be eliminated in early generation quality screens. Characterisations of 1R or 1RL lines from additional rye sources, coupled with complete amino acid sequences of the HMW secalins (not yet available) would, however, add valuable insight to the mechanism of protein polymerisation in wheat endosperm. Acknowledgements The authors wish to thank K. Gill, M. S. Caley, B. W. Seabourn, T. Bailey, K. Schemmerhorn, K. Ditch, D. Samson and J.-H. Lee for assistance during the course of this study.

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