Turkish Journal of Agriculture and Forestry Turk J Agric For (2013) 37: 527-533 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1207-75

Marker-assisted breeding of a wheat cultivar for γ-gliadin and LMW-glutenin proteins affecting quality

1, 1 2 Ahmet YILDIRIM *, Özlem ATEŞ SÖNMEZOĞLU , Abdulvahit SAYASLAN , 2 1 3 Mehmet KOYUNCU , Tuğba GÜLEÇ , Nejdet KANDEMİR 1 Department of Biology, Karamanoğlu Mehmetbey University, Karaman, Turkey 2 Department of Food Engineering, Karamanoğlu Mehmetbey University, Karaman, Turkey 3 Department of Field Crops, Gaziosmanpaşa University, Tokat, Turkey

Received: 03.08.2012 Accepted: 14.12.2012 Published Online: 28.08.2013 Printed: 25.09.2013

Abstract: The pasta quality of durum wheat is one of the most important properties for the industry and consumers. Therefore, breeding for improved grain quality without yield penalties, using modern breeding methods, has been a primary objective in durum wheat breeding programs in recent years. In this study, 2 important gene regions that encode pasta-quality associated proteins (γ-gliadin 45 and LMW-2 glutenin) were transferred to a registered Turkish durum wheat variety, Sarıçanak-98, from a high-quality Canadian durum wheat cultivar, Kyle, through a marker-assisted backcross breeding method. Each of the F1 and backcross (BC) plants were backcrossed 4 times to the recurrent parent, and the backcrossed plants carrying the targeted gene regions in all generations were selected by marker- assisted selection (MAS). DNA markers in combination with A-PAGE were used for tracking the introgression of the targeted gene regions. Transfer of Gli-B1 locus encoding γ-gliadin 45 and Glu-B3 locus encoding LMW-2 glutenin to the wheat variety Sarıçanak-98 led to a considerable increase in protein content and gluten quality.

Key words: Durum wheat, Triticum durum, γ-gliadin 45, LMW-2 glutenin, backcross breeding, MAS, SSR

1. Introduction among specific gliadin and glutenin proteins and the gluten Wheat quality is simply defined as its suitability for a strength. The most important of those proteins are γ-gliadin given final product. For durum wheat, quality means 45 and LMW-2 glutenin, which are linked very tightly to its suitability for . The quality of pasta each other. γ-gliadin 45 is strongly correlated with high products is strongly correlated with the physical and gluten strength and pasta quality (Gupta et al. 1994; Kovacs chemical properties of durum wheat. Kernel vitreousness, et al. 1995; D’Ovidio and Porceddu 1996). It has been protein content and quality (proper gluten strength), reported that transfer of a Gli-B1 locus encoding γ-gliadin milling properties (semolina yield and ash content), yellow 45 and a Glu-B3 locus encoding LMW-2 glutenin together pigment content, and activities of lipoxygenase (LOX), into a variety leads to improvement in the pasta cooking peroxidase (POD), and polyphenol oxidase (PPO) enzymes quality of durum wheat (D’Ovidio et al. 1992; Kovacs et al. are among the well-documented quality parameters 1994; Clarke et al. 1998). (Clarke et al. 1998; Borrelli et al. 1999; Troccoli et al. 2000; Turkey is blessed with agro-climatic regions suitable for Morris 2004). Of these quality parameters, protein content the cultivation of high-quality durum wheat. It is among the and quality, yellow pigment content, and the activities of top 3 durum wheat producing countries in the world with 3 the oxidative enzymes are of vital importance for pasta Mt of production during 2009. However, only about 30%– quality, as they overwhelmingly determine the so-called 40% of the total durum wheat is able to meet the quality cooking characteristics and bright yellow color of criteria required by the pasta industry (IGC 2003), leading pasta products (Troccoli et al. 2000; Aalami et al. 2007). to a rise in importation of durum wheat with desirable pasta However, protein content and gluten quality (including qualities each year. Therefore, the pasta processing quality gliadin and glutenin proteins) are the 2 most important of Turkish durum wheat cultivars must be improved using variables in determining pasta cooking properties (D’Edigio modern breeding methods, without adversely affecting et al. 1990; Kovacs et al. 1995; Blanco et al. 1996; Troccoli grain yield. For this purpose, existing varieties may be et al. 2000). Moreover, there is an important correlation improved through introgression of genes/QTLs associated * Correspondence: [email protected] 527 YILDIRIM et al. / Turk J Agric For with pasta quality. Sarıçanak-98, a durum wheat cultivar, The PCR was run on a Thermo (Px2) thermal cycler as is widely grown in southeastern Turkey due to its high follows: an initial denaturation step of 3 min at 94 °C was yield potential, resistance to common wheat diseases in the followed by 32 cycles of denaturation for 1 min at 94 °C, region, and tolerance of poor soil conditions, particularly annealing for 1 min at 60 °C, extension for 1 min at 72 °C, in terms of microelement deficiencies (Özberk et al. 2010; and conclusion with a final extension step for 5 min at 72 CRIFC 2011). However, the cultivar Sarıçanak-98 suffers °C. PCR products were analyzed on 3% metaphore agarose from poor end-use quality, as it has the γ-gliadin 42 and gels or 1% agarose gels. Electrophoresis was conducted at LMW-1 proteins, which are related to poor pasta quality. 90 W of constant power for 3–4 h. Sarıçanak-98 may be further improved by transfer of 2.4. A-PAGE γ-gliadin 45 and LMW-2 glutenin proteins associated with The gliadins (γ-gliadin 42/45) of wheats were identified superior pasta-cooking quality. The aim of this study was using the A-PAGE method that was originally described to transfer 2 important gene regions encoding γ-gliadin by Bushuk and Zillman (1978) and modified by Khan et al. 45 and LMW-2 glutenin proteins from the high-quality (1985). In each generation, an average of 50–100 BC plants Canadian durum wheat cultivar Kyle to Sarıçanak-98 were screened by A-PAGE. through a marker-assisted backcross breeding method. 2.5. Embryo culture To speed up BC generations, some BC plants were 2. Materials and methods generated by immature embryo cultures following the 2.1. Plant materials process described by Arzani and Mirodjagh (1999). Sarıçanak-98, widely grown in Turkey and lacking the 2.6. Quality measurements on grain γ-gliadin 45 and LMW-2 glutenin encoding genes, was Selected grain properties associated with quality were used as the recurrent parent. The donor parent was a high- measured using the following methods in 3 replications: quality Canadian durum wheat cultivar, Kyle. In each kernel vitreousness by Köksel et al. (2000), thousand generation, F1 and BC plants were backcrossed 4 times kernel weight and kernel size distribution by Elgün et (BC4) to the recurrent parent, and progenies carrying al. (2002), and test weight, kernel color, yellow pigment the targeted gene regions were selected by MAS. In each content, SDS sedimentation volume, moisture content, generation, an average of 50–100 BC plants were screened protein content, and ash content by the AACC methods by molecular DNA markers, and all screening results were of 55-10, 14-22, 14-50, 56-70, 44-15A, 46-10, and 08-01, verified and tested with A-PAGE. Advanced breeding lines respectively (AACC 2000). (ABLs) were obtained by selfing BC4F2 plants twice. Seeds Lipoxygenase (LOX) activity was determined by of the lines were multiplied by growing in field conditions spectroscopic measurement of a conjugated diene during the 2009–2010 growing seasons. formation upon the reaction of wheat extracts with a The cultivars Marquis and Kyle were used as check linoleic acid substrate, as described by Rani et al. (2001) genotypes for the identification of γ-gliadin 42/45 bands and Aalami et al. (2007). One unit of LOX activity (EU) on A-PAGE. was described as 1.0 unit min–1 change in the absorbance 2.2. DNA screening under the assay conditions and reported as EU per g Seeds of F1 and backcross plants were cut into halves. One wheat. Polyphenol oxidase (PPO) activity was determined half was used for A-PAGE and the other half, including following the method of Coseteng and Lee (1987), as the embryo, was germinated and used for screening with modified by Aalami et al. (2007), where color changes DNA markers. DNA was extracted from the leaf of each in the mixture of wheat extract–catechol substrate were plant using a genomic DNA purification kit (Fermentas monitored at 420 nm. One unit of PPO activity was Life Sciences). described as 0.1 unit min–1 change in the absorbance under DNA samples were screened. For this purpose, the gene- the assay conditions and reported as EU per g wheat. based marker GAG5-6 (Von Büren et al. 2000), linked to Peroxidase (POD) activities were measured according to both Gli-B1 and Glu-B3 loci, was used for screening of the the method of Aparicio-Cuesta et al. (1992), modified by F1 and backcross population for the presence of γ-gliadin Aalami et al. (2007), where colored product formation 45 and for LMW-2 glutenin alleles. upon the reaction of wheat extracts with an o-dianisidin 2.3. PCR amplification substrate in the presence of hydrogen peroxide, was Polymerase chain reactions (PCRs) were performed under monitored at 460 nm. One unit of POD activity was –1 the conditions given elsewhere (Von Büren et al. 2000) described as 1.0 unit min change in the absorbance with some modifications. PCR reaction volume was 40 under the assay conditions and reported as EU/g wheat. µL. PCR reactions contained 50–100 ng of genomic DNA, 2.7. Quality measurements of semolina

0.25 µM of each primer, 0.2 µM dNTP mix, 2.5 µM MgCl2, Selected quality characteristics of semolina were measured 10X PCR buffer, and 0.5 units of Taq DNA polymerase. in 3 replications as follows: a dark-colored speck count

528 YILDIRIM et al. / Turk J Agric For based on Elgün et al. (2002) and milling yield, color, alleles through molecular markers (Figure 1) and A-PAGE pigment content, gluten content/index, moisture content, (Figure 2) in each generation. protein content, and ash content according to the AACC A 1-to-1 segregation ratio was clearly seen in the methods of 26-41/42, 14-22, 14-50, 38-12A, 44-15A, 46- BC1F1 generation. Fifty percent of the backcross plants 10, and 08-01, respectively (AACC 2000). carried the recurrent parent allele (Sarıçanak-98), while the rest were heterozygous, as expected. 3. Results In each backcross generation, screening was carried out 3.1. Molecular and biochemical marker-assisted in combination with molecular DNA markers and A-PAGE. selection As a result of the screening, heterozygous backcross plants The genes encoding γ-gliadin 45 and LMW-2 glutenin having the targeted alleles were determined and the BC4F1 proteins, which affect end-use quality of durum wheat, seeds were obtained through hybridization of those plants. were transferred to the Turkish durum wheat cultivar The BC4F2 seeds were obtained by selfing of BC4F1 plants. Sarıçanak-98 following a marker-assisted backcross In the BC4F2 plants, homozygous plants (25%) were breeding program. The F1 and BC plants were backcrossed identified by molecular (Figure 3a) and A-PAGE screening 4 times to the recurrent parent, and the backcross plants (Figure 3b), and homozygous BC4F2 seeds were increased (from BC1F1 to BC4F1) were screened for the targeted by selfing to use for the yield trials later.

K S H H H A H A A H H B B B

-45 -42

K S A A H A A H A H H H H

-45 -42

Figure 1. Representative PCR profiles of parents and BC1F1 plants due to GAG 5-6 markers linked to Gli-B1 locus. (H: plants heterozygous for targeted gene regions.)

M K S 1 2 3 4 5 6 7 8 9 10 M

-42 -45 A HH AHA H AHH

Figure 2. Representative profiles of parents and BC1F1 plants screened with A-PAGE. (A: BC1F1 plants homozygous for the recurrent parent alleles, H: BC1F1 plants heterozygous for the targeted gene regions, K: cv. Kyle, S: cv. Sarıçanak-98).

529 YILDIRIM et al. / Turk J Agric For

a b K S H H B A H H B 1 2 K S M 3 4 5 6 7 8 9 10 11 12

H H -45 -42 B H A B A B H H H H

K S A A H A A H A H H H H

Figure 3. Representative profiles of parents and BC4F2 plants screened with GAG 5-6 markers (a) and A-PAGE (b). (A: Backcross plants homozygous for the recurrent parent alleles, B: Backcross plants homozygous for the donor parent alleles, H: Heterozygous backcross plants.)

Immature embryo cultures were used to reduce the line and its parents were fairly comparable in terms of time required for seed generation after hybridization kernel physical properties, although the differences were and acceleration of the backcross processes. Half of the statistically significant (P < 0.05) (Table 1). However, BC seeds in each generation were germinated through the breeding line significantly differed from its parents immature embryo cultures and the rest were left on BC with respect to gluten quality and protein content (Table spikes for normal maturation. Thus, each generation was 2). Gluten quality of the breeding line was significantly produced approximately 40 days earlier. Consequently, improved, as indicated by its higher SDS sedimentation more than 2 generations were obtained in a year with the and specific sedimentation volumes, which was the aim help of the embryo culture approach. of this work. Additionally, protein content of the breeding 3.2. Quality improvement line was significantly higher (P < 0.05) than that of its Selected physical, chemical, and technological properties recurrent parent, Sarıçanak-98. Indeed, simultaneous of the advanced breeding line (BC4F4) and its parents improvement of gluten quality and protein content is a (cultivars Kyle and Sarıçanak-98) were investigated and significant achievement for the advanced breeding line. the findings summarized in Tables 1–3. The breeding Initial observations indicated that yield of the ABL did not

Table 1. Physical properties of the advanced breeding line and its parents.a,b

Parents /Advanced Thousand kernel Test weight Kernel vitreousness Kernel size distribution (%) –1 breeding line weight (g) (kg hL ) (%) >2.8 mm >2.5 mm >2.2 mm >2.0 mm Homogeneity

Kyle 36.7 a 79.2 c 99.8 a 23.6 37.3 29.1 10.2 Heterogeneous Sarıçanak-98 36.8 a 82.3 a 99.1 b 27.4 47.6 18.6 6.5 Homogeneous ABL (BC4F4) 35.5 b 80.6 b 99.5 ab 47.3 34.0 14.6 4.1 Homogeneous a14% moisture basis bDifferent letters in a column indicate significant difference (P < 0.05) ABL: Advanced breeding line, BC: Backcross

Table 2. Chemical and technological properties of the advanced breeding line and its parents.a,b

Parents / Advanced Moisture Ash content Protein content Sedimentation Specific sedimentation breeding line content (%) (%) (%) volume (mL) volume (mL)

Kyle 10.4 ns 1.54 b 14.8 a 22.1 a 1.49 a Sarıçanak-98 9.8 ns 1.48 b 13.8 b 17.9 b 1.30 b ABL (BC4F4) 10.3 ns 1.65 a 14.8 a 22.5 a 1.52 a a14% moisture basis bDifferent letters in a column indicate significant difference (P < 0.05) ns: Not significant ABL: Advanced breeding line, BC: Backcross

530 YILDIRIM et al. / Turk J Agric For show any difference from Sarıçanak-98. However, reliable 45 and LMW-2 glutenin is better than that of γ-gliadin 42 results can be obtained from replicated field trials in the and LMW-1 glutenin (Damidaux et al. 1978; Chihab 1990; wheat’s natural environment . Kaan et al. 1995; Nachit et al. 1995; Pena 2000; Edwards As seen in Table 3, yellow pigment content and color et al. 2007; Yüksel 2009). It is also well known that gluten properties (L* and b*) of the breeding line were significantly quality and the protein content of the durum are the main higher (P < 0.05) than those of the recurrent parent. On parameters that determine pasta quality (Bushuk 1998; the other hand, the breeding line and its recurrent parent Troccoli et al. 2000). were statistically comparable in terms of their oxidative During this study, important gene regions encoding enzymes (LOX, PPO, and POD) that contribute to the pasta-quality associated proteins (γ-gliadin 45 and LMW- bright yellow color of pasta products. 2 glutenin) were transferred from the cultivar Kyle to Semolina yields of the breeding line and its parents the cultivar Sarıçanak-98, leading to the development were quite similar; however, the breeding line had of an advanced breeding line with improved quality. As significantly higher levels of pigment and protein with expected, this line has increased gluten quality, as judged slightly improved gluten quality (Table 4). It is obvious by the sedimentation and gluten index tests. Additionally, that quality measurements recorded on the grain (Tables 2 the protein content of the breeding line also increased. and 3) were in close agreement with those recorded on the Besides the protein content and gluten quality, a yellow semolina (Table 4). pigment color of the grain also contributes to improved pasta quality. Pigment contents ranging from 4 to 8 mg 4. Discussion kg–1 in durum wheat have been reported by different Pasta products are appreciated worldwide as an end-product researchers. The pigment content (7.55 mg kg–1) of the of durum wheat. Considering increased competition in breeding line developed during the present study was the global pasta market, improving the end-use quality of higher than the pigment content (4 to 8 mg kg–1) reported durum wheat is essential. It has been reported that there is a in earlier durum wheat studies (Kaan et al. 1995; Köksel et positive relationship between γ-gliadin 45 protein (LMW- al. 2000; Troccoli et al. 2000; Sakin et al. 2011). Thus, the 2 glutenin) and the optimum gluten strength associated breeding lines developed should produce yellow-colored with the al dente cooking quality of pasta (Kovacs et al. pasta products, preferred by consumers. 1995; Troccoli et al. 2000). It has been widely reported that The results of this study confirm that marker-assisted the gluten quality of durum wheat containing γ-gliadin selection and backcross breeding can be successfully

Table 3. Color properties and oxidative enzyme activities of the advanced breeding line and its parents.a,b

Pigment LOX POD PPO Parents / Advanced Color Content Activity Activity Activity breeding line (mg/kg) L* a* b* (EU g–1) (EU g–1) (EU g–1)

Kyle 7.58 a 77.0 a 5.05 b 18.59 a 44.9 b 29.3 b 6.8 ns Sarıçanak-98 6.41 b 59.3 b 5.22 a 17.37 b 48.1 a 62.7 ab 6.9 ns ABL (BC4F4) 7.55 a 76.5 a 5.17 a 18.59 a 48.5 a 78.9 a 11.1 ns a14% moisture basis bDifferent letters in a column indicate significant difference (P < 0.05) ns: not significant ABL: Advanced breeding line, BC: Backcross

Table 4. Chemical and technological properties of semolina milled from the advanced breeding line and its parents.a,b

Parents / Semolina Pigment Speck count Ash Protein Wet gluten Dry gluten Gluten Advanced yield content (number 100 content content content content index breeding line (%) (mg kg–1) cm–2) (%) (%) (%) (%)

Kyle 54.7 7.31 a 110 0.59 a 13.8 a 44.0 ns 16.5 ns 80 b Sarıçanak-98 55.4 6.00 c 110 0.62 a 12.9 b 38.2 ns 14.6 ns 91 ab ABL (BC4F4) 54.1 6.85 b 75 0.55 b 13.8 a 39.4 ns 15.7 ns 98 a a14% moisture basis bDifferent letters in a column indicate significant difference (P < 0.05) ns: not significant ABL: Advanced breeding line, BC: Backcross

531 YILDIRIM et al. / Turk J Agric For employed in wheat breeding programs and that molecular the development of a durum wheat candidate with elevated markers can be used alone or in combination with the protein content and gluten quality. A-PAGE technique in each backcross generation. Quality- targeted breeding through marker-assisted backcross Acknowledgments breeding in this work was completed in about 3 years by This study was supported by the Scientific and raising 3 generations per year. With the help of marker- Technological Research Council of Turkey (TÜBİTAK; assisted selection, breeding time was reduced and the Project No. 107O004) within the framework of COST- efficiency of backcross breeding was increased, leading to FA-0604 TritiGen Action.

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