QTL Mapping for Dough Mixing Characteristics in a Recombinant Inbred Population Derived from a Waxy × Strong Gluten Wheat (Triticum aestivum L.)

Journal of Integrative Agriculture

June 2013

2013, 12(6): 951-961

RESEARCH ARTICLE

QTL Mapping for Dough Mixing Characteristics in a Recombinant Inbred Population Derived from a Waxy × Strong Gluten Wheat (Triticum aestivum L.) ZHENG Fei-fei*, DENG Zhi-ying*, SHI Cui-lan, ZHANG Xin-ye and TIAN Ji-chun State Key Laboratory of Crop Biology, /Key Laborator y of Crop Biology of Shandong Province, Education Department of Shandong Province/Agronomy College, Shandong Agricultural University, Tai’an 271018, P.R.China

Abstract Protein and starch are the most important traits in determining processing quality in wheat. In order to understand the genetic basis of the influence of Waxy protein (Wx) and high molecular weight gluten subunit (HMW-GS) on processing quality, 256 recombinant inbred lines (RILs) derived from the cross of waxy wheat Nuomai 1 and Gaocheng 8901 were used as mapping population. DArT (diversity arrays technology), SSR (simple sequence repeat), HMW-GS, and Wx markers were used to construct the molecular genetic linkage map. QTLs for mixing peak time (MPT), mixing peak value (MPV), mixing peak width (MPW), and mixing peak integral (MPI) of Mixograph parameters were evaluated in three different environments. The genetic map comprised 498 markers, including 479 DArT, 14 SSR, 2 HMW-GS, and 3 Wx protein markers, covering 4 229.7 cM with an average distance of 9.77 cM. These markers were identified on 21 chromosomes. Eighteen additive QTLs were detected in three different environments, which were distributed on chromosomes 1A, 1B, 1D, 4A, 6A, and 7D. QMPT-1D.1 and QMPT-1D.2 were close to the Glu-D1 marker accounting for 35.2, 22.22 and 36.57% of the phenotypic variance in three environments, respectively. QMPV-1D and QMPV-4A were detected in all environments, and QMPV-4A was the nearest to Wx-B1. One minor QTL, QMPI-1A, was detected under three environments with the genetic distances of 0.9 cM from the nearest marker Glu-A1, explaining from 5.31 to 6.67% of the phenotypic variance. Three pairs of epistatic QTLs were identified on chromosomes 2D and 4A. Therefore, this genetic map is very important and useful for quality trait related QTL mapping in wheat. In addition, the finding of several major QTLs, based on the genetic analyses, further suggested the importance of Glu-1 loci on dough mixing characteristics. Key words: bread wheat, RIL population, genetic map, mixograph, QTL

INTRODUCTION The wheat genetic map has become more and more important for understanding genetic variation, QTL mapping, gene clone, and molecular maker-assisted selection since the 1980s. The first influential genetic map that consists of 230 SSR was constructed by Röder et al. in 1998. In 2004, a highest-density public

microsatellite map of wheat was constructed using three DH and one RIL populations with mapping 1 235 microsatellite loci, covering 2 569 cM and giving an average interval distance of 2.2 cM (Somers et al. 2004). Similarly, significant progress in constructing wheat genetic maps in China using different populations has also been made over the past decade or so (Guo 2003; Zhang et al. 2008; Cui et al. 2011). RFLPs, RAPD, STS, SSRs, and AFLPs are the most

Received 8 September, 2012 Accepted 7 February, 2013 ZHENG Fei-fei, E-mail: [email protected]; Correspondence TIAN Ji-chun, Tel/Fax: +86-538-8242040, E-mail: [email protected], [email protected] *

These authors contributed equally to this study. © 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60315-9

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commonly used markers for wheat map construction. Of these, the SSR markers are widely used in map construction because they are highly polymorphic, widely distributed in the genome, required a small amount of DNA and can be easily automated (Zhu and Jia 2003). Recently, a microarray hybridization technique known as DArT developed by Jaccoud et al. (2001) has been used in genetic mapping and fingerprinting studies in barley (Wenzl et al. 2004), Arabidopsis (Wittenberg et al. 2005), cassava (Xia et al. 2005), and rice (Hong et al. 2009). The bread wheat genetic map containing the first 339 DArT markers was created in 2006 (Akabari et al. 2006), and several more have been documented since then. Both Mantovani et al. (2008) and Peleg et al. (2008) have further suggested the benefit of DArT use in wheat genetic research. Success in genetic mapping based on various marker analyses can also be evidenced by Yao et al. (2010) who developed a genetic map consisting of 187 SSR and 291 DArT, covering the total length of 2 733.7 cM with the average distance of 7.14 cM, and by Cui (2011) who constructed a highdensity integrative genetic map that includes 575 DArT and 536 PCR-based marker loci, covering a total length of 2 946.98 cM with an average density of 2.65 cM. Based on the information of the RIL population containing 256 lines resulted from a waxy wheat × a strong gluten wheat cross, we report the use of DArT, SSR, HMW-GS, and Wx protein markers to develop our genetic linkage map. The waxy wheat 1 has three deletions of Wx loci, and the strong gluten Gaocheng 8901 has 1, 7+8 and 5+10 HMW-GS compositions. The QTLs of mixograph properties were analyzed based on this map. Traditionally, the Mixograph is used to determine the rheological characteristics and bread-making quality in the milling and baking industries (Kunerth and D’Appolonia 1985). In general, the dough rheological properties are believed to be controlled by multi-genes, and thus cannot be fully explained by storage proteins loci. Prior to this study, limited information on QTL mapping for mixographic characteristics is available. QTLs of mixing peak time and eight times band width have been mapped on chromosomes 1B and 1D (Wu et al. 2008) using 240 RIL lines resulted from the cross of PH82-2 and Neixiang 188. Zhang et al. (2009) reported the mixing peak time, peak width and eight times width on chromosomes 1A, 1B and 1D, respectively,

ZHENG Fei-fei et al.

while studies by several other researchers have suggested that QTLs of mixograph traits are located on different chromosomes, such as 1A, 2A, 3A, 1B, 2B, 3B, 1D, 4D, 5B, 5D, 6B, 6D, 7A, and 7D (Huang et al. 2006; Nelson et al. 2006; Elangovan et al. 2008; Sun et al. 2008; Kerfal et al. 2010; Tsilo et al. 2011). These differences may be the results of the use of different genetic populations and different genetic maps. The objective of this study was to evaluate the genetic effects of QTL on mixograph traits by using the genetic map generated from this RIL population. The results presented here will provide the important information for quantitative genetic studies related to wheat quality, and the major QTLs can be used in markerassisted quality breeding programs.

RESULTS Construction of genetic map Molecular marker distribution Among the 916 DArT marker provided by Triticarte Pty. Ltd., Australia, 479 primers showed polymorphism in the RIL population. According to provisional construction of linkage map, we found few markers located on 3D, 5D and 6D chromosomes. Therefore, 131 SSR markers were found from Grain Gene 2.0 to test the polymorphism between the two parental varieties. Of these, 31 SSR markers revealed polymorphism between the parents of the mapping population, 14 markers were used to construct the genetic map. The markers were not evenly distributed (Table 1, Fig.) on chromosomes 1A, 2A, 3B, 6A, 4D, 5A, 5D, and 6D. SSR markers were mainly located on 1D, 3A, 3B, 3D, 4D, 5D, 6A, 6B, and 6D. Two HMW-GS loci were mapped on chromosomes 1A and 1D, and three Waxy protein subunit loci were located on chromosomes 7A, 4A and 7D, respectively. Chi-squared test indicated that, among the 498 markers, 168 markers showed the genetics distortion segregation (P<0.05), accounting for 33.7% (Table 1). 62 DArT markers and one SSR marker (37.5%) exhibited distortion in favor of the female parent WN1 alleles, whereas 104 DArT markers and one HMW-GS marker (62.5%) were in favor of male parent Gc8901 alleles. The distortion loci were unevenly distributed (Table 1).

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QTL Mapping for Dough Mixing Characteristics in a Recombinant Inbred Population Derived from a Waxy × Strong Gluten

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Table 1 Genetic distance and marker distribution as well as distorted loci among different linkage groups Linkage group 1A1 1A2 1B 1D 2A 2B 2D 3A 3B 3D 4A 4B 4D 5A 5B 5D 6A1 6A2 6B 6D 7A 7B 7D1 7D2 Total Average

Length of linkage group

Average distance

DArT

Number of marker SSR

HMW-GS/Wx loci

Total

(cM)

(cM)

DArT

SSR

HMW-GS/Wx loci

Total

23 33 18 20 28 15 30 18 41 18 35 11 5 5 24 2 35 17 34 1 10 20 20 16 479

0 0 0 1 0 0 0 1 1 2 0 0 1 0 0 1 1 2 2 2 0 0 0 0 14

1 HMW-GS 0 0 1 HMW-GS 0 0 0 0 0 0 1 Wx 0 0 0 0 0 0 0 0 0 1 Wx 0 0 1 Wx 5 20.75

24 33 18 22 28 15 30 19 42 20 36 11 6 5 24 3 36 19 36 3 11 20 20 17 498

155.9 262.3 234.8 240.5 316.5 90.3 221.3 195.9 411.2 169.9 156.6 121.8 88.5 70.1 239.6 34.4 137.3 160.3 339.1 35.0 162 245.4 97.8 42.8 4 229.7 176.2

7.80 11.40 16.77 13.36 12.17 6.45 8.85 10.88 10.54 12.13 4.89 12.18 14.75 17.52 10.41 11.47 4.29 9.42 10.60 11.67 16.2 12.91 6.11 2.85

2 15 6 3 20 11 7 5 14 5 1 3 4 5 15 0 5 1 19 1 3 2 4 16 166

0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Wx 1

2 15 6 3 20 11 7 5 14 5 1 3 4 5 15 0 5 1 19 1 3 2 4 17 168

Genetic map Four hundred ninety eight markers (479 DArTs, 14 SSRs, two HMW-GS loci and three Waxy protein subunit loci) were mapped on the 24 linkage groups (Fig.). The genetic map spanned about 4 229.7 cM in wheat genome and the average distance of markers was 9.77 cM (including 65 overlapping sites). The A genome contained 211 markers (including 29 overlapping sites), with total length of 1 617.3 cM, and the average distance of markers was 8.89 cM, while the B genome had 166 markers (including 15 overlapping sites), covering 1 682.2 cM with an average distance of 11.14 cM. The D genome contained 121 (including 21 overlapping sites) markers, the average distance of markers was 9.30 cM, and spanning about 930.2 cM. The average length of each linkage group is 176.2 cM. The 3B is the longest chromosome (411.2 cM), and 5D is the shortest, with only 34.4 cM. Each linkage group possesses three (e.g., the 5D and 6D chromosomes) to 42 markers (e.g., the 3B chromosome). However, large gaps were found on chromosomes 1A, 6A and 7D, respectively. In addition, 55 new markers were mapped on the 18 linkage groups without the involvement of chromosomes 1B, 2A, 2B, 3A, 4B, 5B, and 6A (Table 2). The

9.77

Distorted locus

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 7

genomes of A, B and D consist of 10, 13 and 32 markers, respectively.

QTLs for dough mixing characteristics Phenotypic variation for RIL lines and parents Data listed in Table 3 indicated that, mixing peak time (MPT), mixing peak width (MPW), mixing peak integral (MPI), and 8 min width (MTxW) of Gc8901 are higher than those of WN1 under all environmental conditions and the phenotypic variations among the RIL lines are observed. Mixing peak time (MPT), mixing peak value (MPV), mixing peak width (MPW), and mixing peak integral (MPI) of RIL population segregated continuously and fit normal distribution with both absolute values of skewness and kurtosis less than 1.0. Therefore, the data can be used for QTL analysis. QTLs for mixograph parameters When the P<0.005, 18 additive QTLs and three pairs of epistatic QTLs were detected (Tables 4 and 5). Seven additive QTLs for MPT parameter were identified on chromosomes 1A, 1B, 1D, and 6A. Gc8901 allele had positive additive effects on the QTLs except for QMPT-1B. The QMPT-1D.1 was only detected in

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(Continued on next page)

Fig. Genetic map of 498 loci constructed by a RIL population derived from the cross of a waxy wheat and a strong gluten wheat (WN1 × , the position of additive QTLs; , the position of epistatic QTLs; ---, epistatic Gc8901) and all QTLs for mixograph parameters. effect. MPT, mixing peak time; MPV, mixing peak value; MPW, mixing peak width; MPI, mixing peak integral; MTxW, 8 min width.

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Fig. (Continued from preceding page)

two environments (Tai’an 2008, 2009), and it was close to the QMPT-1D.2 accounting for 35.2, 22.22, and 36.57% of the phenotypic variance in three environments, respectively. The genetic distance from Glu-D1 marker was 4-5.9 cM. Five additive QTLs for MPV parameter were mapped on 1A, 1D, 4A, and 7D chromosomes. The

positive additive effect alleles of QMPV-4A and QMPV7D were coming from WN1 in each environment. QMPV-1D and QMPV-4A were detected with stable effects explaining from 6.20 to 11.63% and 6.30 to 6.93% of the phenotypic variance, respectively. QMPV-4A was close to the Wx-B1 marker with genetic distance of 2.4-3.4 cM. QMPV-1A.1 was de-

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Table 2 Novel loci mapped on the linkage groups in the RIL population Chromosome 1A1 1A2 1D 2D 3B 3D 4A 4D 5A 5D 6A2 6B 6D 7A 7B 7D1 7D2

Marker

Number

wPt-665250, wPt-731190 wPt-731357, wPt-666885 wPt-729773, wPt-730589, wPt-666414, wPt-665480, wPt-669498, wPt-665037, wPt-665204, wPt-671415 wPt-669199, wPt-664438, wPt-666395, wPt-666987, wPt-667294, wPt-730613, wPt-666008 wPt-664393, wPt-731490, wPt-730146, wPt-666340, wPt-1871 wPt-730146, wPt-666340, wPt-1871, wPt-730115, wPt-731146, wPt-729808, wPt-669255 wPt-664948 wPt-666853, wPt-666601 wPt-3069 wPt-666937 wPt-664937, wPt-729920 wPt-0315, wPt-664174, wPt-672044, wPt-664719, wPt-666615, wPt730273 wPt-730912 wPt-667038, wPt-731311 wPt-669158, wPt-7009 wPt-730263, wPt-731416, wPt-667560, wPt-667098, wPt-664056 wPt-664252

2 2 8 7 5 7 1 2 1 1 2 6 1 2 2 5 1

Table 3 Distribution of mixograph in parent and population of wheat among different environments Environment

Trait1)

Tai’an 2008

MPT MPV MPW MPI MTxW MPT MPV MPW MPI MTxW MPT MPV MPW MPI MTxW

Tai’an 2009

Suzhou 2011

1)

Parent WN1 1.82 67.24 22.74 102.91 4.86 3.00 62.48 22.90 160.67 7.65 2.04 69.72 29.73 120.02 7.37

RIL line Gc8901 4.87 58.64 23.92 238.30 13.90 4.51 57.63 25.88 221.46 13.55 3.92 61.64 32.53 192.78 16.61

Range

Mean±SD

Skewness

Kurtosis

1.50-5.53 48.50-77.92 11.43-32.81 68.14-300.94 2.64-20.72 1.50-9.01 47.26-73.89 12.95-37.99 74.68-426.63 3.43-43.98 1.50-5.15 46.98-86.97 11.22-43.40 56.90-283.84 4.21-33.64

2.91±0.05 61.90±0.32 23.55±0.21 146.32±2.57 6.06±0.19 3.90±0.09 60.87±0.34 23.19±0.31 192.38±4.20 9.27±0.33 2.66±0.04 63.63±0.38 25.75±0.30 135.38±2.49 9.85±0.31

0.428 0.155 -0.103 0.526 2.378 0.790 0.038 0.368 0.709 2.972 0.717 0.588 0.526 0.779 1.595

-0.059 0.093 0.405 0.255 6.292 0.713 -0.580 0.246 0.571 12.604 0.527 0.885 0.899 0.910 3.031

MPT, mixing peak time; MPV, mixing peak value; MPW, mixing peak width; MPI, mixing peak integral; MTxW, 8 min width.

tected in Taian (2008) and Suzhou (2011) accounting for 6.67 and 6.24% of the phenotypic variance, respectively. This QTL was close to Glu-A1 marker. One additive QTL with increasing MPW by 1.45 from WN1 alleles detected on the 4A chromosome only expressed in one environment (Tai’an 2009). Five additive QTLs for MPI parameter were identified on 1A, 1B, 1D, and 6A chromosomes. Of these QTLs, Gc8901 alleles increased MPI with the exception of QMPI-1B. QMPI-1A was detected across three environments with genetic distances of 0.9 cM from the nearest marker Glu-A1 explaining from 5.31 to 6.67% of the phenotypic variance. QMPI-1D.1 (Tai’an 2008; Suzhou 2011), QMPI-6A (Tai’an 2008, 2009) and QMPI-1B (Tai’an 2008, 2009) were detected in two environments. QMPI-1D.2 was close

to QMPI-1D.1 explaining 30.94, 20.92 and 29.08% of the phenotypic variance, respectively. The genetic distance from Glu-D1 marker was 4-5.9 cM. Three pairs of epistatic QTLs were identified for MPW in Tai’an 2008, and they were located on chromosomes 2D and 4A (Table 5). QMPW-2D.1/ QMPW-4A.1 had the largest effect. These QTLs ranged from 1.86 to 10.96%.

DISCUSSION Features of the genetic map Segregation distortion is the deviation of observed genetic ratios from the expected Mendelian ratios of a given genotypic class within a segregating population

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Table 4 Additive effects for mixograph of RIL population in different environments Environment Tai’an 2008

Trait

QTL

MPT

MPV

MPI

Tai’an 2009

MPT

MPV

MPW MPI

Suzhou 2011

Chromosome

QMPT-1A.1 QMPT-1D.1 QMPT-6A.1 QMPV-1A.1 QMPV-1D QMPV-4A QMPV-7D QMPI-1A QMPI-1B QMPI-1D.1 QMPI-6A QMPT-1B QMPT-1D.2 QMPT-6A.2 QMPV-1A.2 QMPV-1D QMPV-4A QMPW-4A QMPI-1A QMPI-1B QMPI-1D.2 QMPI-6A QMPT-1A.2 QMPT-1D.1 QMPV-1A.1 QMPV-1D QMPV-4A QMPI-1A QMPI-1D.1

MPT MPV

MPI

Interval marker

1A 1D 6A 1A 1D 4A 7D 1A 1B 1D 6A 1B 1D 6A 1A 1D 4A 4A 1A 1B 1D 6A 1A 1D 1A 1D 4A 1A 1D

Position (cM)

wPt-9757-Glu-A1 Glu-D1-wPt-3743 wPt-729920-wPt-664792 wPt-664666-wPt-9757 cfd-183-wPt-729773 wPt-664948-Wx-B1 Wx-D1-wPt-664368 wPt-9757-Glu-A1 wPt-6642-wPt-3824 Glu-D1-wPt-3743 wPt-729920-wPt-664792 wPt-6442-wPt-3824 wPt-3743-wPt-666719 wPt-664792-wPt-730772 wPt-9757-Glu-A1 cfd-183-wPt-729773 wPt-664948-Wx-B1 Wx-B1-wPt-0105 wPt-9757-Glu-A1 wPt-6442 -wPt-3824 wPt-3743-wPt-666719 wPt-729920-wPt-664792 Glu-A1-wPt-665259 Glu-D1-wPt-3743 wPt-664666-wPt-9757 cfd-183-wPt-729773 wPt-664948-Wx-B1 wPt-9757-Glu-A1 Glu-D1-wPt-3743

87.7 111.4 132.7 82.3 0.0 100.4 0.0 87.7 169.5 111.4 130.7 168.5 113.3 143.8 84.7 0.0 99.4 107.8 87.7 167.5 113.3 131.7 88.6 112.4 81.3 0.0 99.4 87.7 112.4

A 1)

H2(A) (%)2)

-0.19 -0.51 -0.26 -1.29 -1.77 1.35 1.06 -11.30 7.64 -24.32 -14.49 0.39 -0.68 -0.33 -1.38 -1.72 1.33 1.45 -15.98 17.84 -31.73 -22.32 -0.16 -0.42 -1.64 -1.52 1.60 -9.77 -21.09

4.91 35.29 9.05 6.19 11.63 6.78 4.23 6.67 3.05 30.94 10.98 7.37 22.22 5.26 6.74 10.57 6.30 8.92 5.31 6..61 20.92 11.30 5.38 36.57 7.28 6.20 6.93 6.24 29.08

Additive effects, positive values indicate that positive effect alleles are derived from WN1, whereas negative values indicate that the negative effect alleles are contributed by Gc8901. 2) Percentage of phenotypic variation explained by QTL with additive effect. 1)

Table 5 Epistatic QTLs for mixograph of RIL population in different environments Environment Tai’an 2008

Trait

QTL

MPW

QMPW-2D.1 QMWP-2D.1 QMPW-2D.2

Interval marker wPt-7901-wPt-6687 wPt-7901-wPt-6687 wPt-1301-wPt-6343

Position 120.3 120.3 130.4

QTL QMPW-4A.1 QMPW-4A.2 QMPW-4A.2

Interval marker

Position

AA 1)

H2(AA) (%)2)

wPt-671707-wPt-730913 wPt-5003-wPt-6440 wPt-5003-wPt-6440

127.3 136.2 136.2

-1.13 0.47 -0.61

10.69 1.86 3.14

Epistatic effects, positive values indicate that positive effect alleles are inherited from WN1, while negative values indicate that the negative effect alleles are derived from Gc8901. 2) Percentage of phenotypic variation explained by QTL with epistatic effect. 1)

(Song et al. 2006). In the present study, 168 (33.7%) segregation distortion loci were located on the genetic map, and many marker loci are in agreement with previous reports, especially Glu-A1, Glu-D1, Wx-A1, WxB1, and Wx-D1 (Clark et al. 1991; Ainsworth et al. 1993). Although many DArT markers mapped on the chromosomes are in agreement with those of published in Pty. Ltd., mapping of some markers have been realized for the first time (e.g., wPt-665250, wPt-731357 and wPt-666885). The unique feature of this study, compared with many others, is that there are some clear differences in

grain quality between the two parents: WN1 has special starch quality due to the deletions of its three Wx loci and Gc8901 shows good protein quality because of its strong gluten. This means the WN1’s starch quality (e.g., starch content and starch pasting properties) and the Gc8901’s dough rheological characteristics (e.g., dough stability time, mixograph and extensograph) can be analyzed successfully based on this genetic map. It is true that there is a large gap between makers on some chromosomes due to fewer markers available at this point. We believe that more markers needed be added in this map in the near future.

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Comparison with previous reports It is well known that glutenin proteins are the major factors affecting the dough viscoelastic properties and bread making quality (Huebner and Wall 1976; Zanetti et al. 2001). The relationship between Glu-1 alleles and bread making quality has been studied, especially the variation at the Glu-D1 locus. In general, Glu-D1d (5+10) plays a more important role in dough quality and bread making quality than Glu-D1a (2+12) (Hsam et al. 2001; Zheng et al. 2009). These conclusions are mainly based on statistical analysis using different wheat varieties that have different HMW-GS compositions. However, in this study, we investigated the effect of Glu-1 on dough mixograph parameters using RIL population which possesses special starch and protein quality. Nine major QTLs which showed more than 10% of the phenotypic variance were found to be associated with the four mixograph traits under three environmental conditions. Eight of them were identified on chromosome 1D, and the other was located on chromosome 6A. QTLs, located in between their flanking markers Glu-D1 and wPt-3743, were found to be responsible for MPT and MPI which showed more than 30% phenotypic variance under the three environments. All additive effects (5+10) were contributed by enhancing alleles located on 1D chromosome of Gc8901. These results indicate that Glu-D1 plays a major role in controlling dough mixograph, as supported by several other studies (Campbell et al. 2001; Arbelbide and Bernardo 2006; Huang et al. 2006; Zhang et al. 2009). In addition to Glu-D1, other loci on chromosome 1D that also affect dough mixograph include those that are located in between cfd-183 and wPt-729773 as well as between wPt-3743 and wPt-66719, further suggesting the importance of chromosome 1D on dough quality. Additional loci that contribute to dough mixograph quality, but with minor effect, were identified on chromosome 1A. These QTLs are located between markers of wPt-9757 and Glu-A1 and between those of wPt664666 and wPt-9757 on chromosome 1A. Genetic analysis indicated that they have much less effect on dough quality than Glu-D1. Interestingly, several novel QTLs that are related to starch quality and control MPV, which showed stable expressions under three environments, were detected on

ZHENG Fei-fei et al.

the 4A chromosome. They are located between markers wPt-664948 and Wx-B1. Our study also showed that additive effects were contributed by the WN1 parent. This perhaps indicated the Waxy protein was not expressed at Wx-B1 contributed to the dough MPV. Other new QTLs that are flanked by markers wPt-729920 and wPt-664792 are responsible for controlling MPT and MPI, and they were identified on chromosome 6A. Although the expressions of QTLs under different environments were not consistent, some major QTLs effect could be detected in all environments or at multiple sites. Thus, it is possible to use their corresponding markers to conduct molecular marker-assisted breeding.

MATERIALS AND METHODS Plant materials A population of 256 F10 RILs was generated by single seed descent from the cross of waxy wheat 1 and strong gluten Gaocheng 8901 (Zhai and Tian 2007). Waxy wheat 1 (WN1), a soft wheat, was produced from Jiangsu Baihuomai × Guandong 107 by China Agricultural University (Song et al. 2004). It has deletions of three Wx protein subunits. Gaocheng 8901 (Gc8901), a hard wheat, was produced from a 77546-2 × Lingzhang by Gaocheng Academy of Agricultural Sciences. WN1 and Gc8901 were registered in Beijing in 2005 and in Hebei Province in 1998, respectively. The rapid viscosity analysis (RVA) indicated that the pasting temperature and setback value of WN1 are lower than Gc8901, but peak viscosity value is higher than Gc8901. These two parents have different HMW-GS compositions. WN1 has null, 7+8, 2.2+12 at Glu-A1, Glu-B1 and Glu-D1 loci, respectively, whereas Gc8901 has 1, 7+8, 5+10 at these corresponding loci. In addition, Pertern SKCS4100 analysis showed the hardness index of Gc 8901 is 71 and WN 1 is 22, suggesting that the former is a hard wheat and the latter is a soft wheat. Three types (hard, medium and soft) of grain hardness have been observed in the RIL population. Therefore, there will be different water additions when tempering wheat before milling. Field trials were conducted under three environmental conditions: 2007-2008 and 2008-2009 in Tai’an (36°57´N, 116°36´E), Shandong Province, China; 2010-2011 in Suzhou (33°38´N, 116°58´E), Anhui Province, China. The experimental design followed a completely randomized block design with two replications at each location. All lines and parental lines were grown in 2 m long by four-row plots, spaced 26 cm apart in every environment. Suzhou and Taian differ in climate and soil conditions. Even in Tai’an,

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QTL Mapping for Dough Mixing Characteristics in a Recombinant Inbred Population Derived from a Waxy × Strong Gluten

the same location, there were temperature differences in 2007 and 2008. During the growing season, management was in accordance with the local practice, and there was no disease or insect issues.

Molecular marker detection DNA was extracted by approved method of CTAB as described by https://www.triticarte.com.au. Agar gel (0.8%) electrophoresis was used to detect the concentration and purity of extracted DNA. DArT markers were provided and detected by Triticarte Pty. Ltd. (https://www.triticarte.com.au) in Australia. Primer sequences for 33 GWM SSR markers were available from Röder et al. (1998) and Pestsova et al. (2000), whereas 62 GPW SSR markers were described in Grain Gene 2.0 (http://wheat.pw.usda.gov/GG2/index.shtml), and 36 CFD SSR markers were kindly provided by Dr. Xia Xianchun, Chinese Academy of Agricultural Sciences, Beijing, China. The amplification reaction mixture was 25 μL. It contained 3 μL 10× buffer (Tris-HCl 10 mmol L-1, pH 8. 0, MgCl2 22.0 mmol L-1), 2.5 μL dNTP (2.5 mmol L-1), 2 μL primer (25 μmol L-1), 0.2 μL Taq polymerase enzyme (5 U μL-1), 2 μL DNA (20 ng μL-1), and 15.3 μL water. DNA amplification was programmed at 94°C for 3 min, followed by 36 cycles of 94°C for 60 s, 55-58°C for 45 s, 72°C for 1 min, and a final extension of 10 min at 72°C before cooling to 4°C. The PCR products were separated on 6% (w/v) denatured polyacrylamide gel and detected by silver staining (Karakousis et al. 2003).

Protein extraction and electrophoresis HMW-GS extraction and electrophoresis The extraction and electrophoresis of HMW glutenin subunits by SDSPAGE was conducted according to the method of Deng et al. (2005). Proteins extracted from Chinese Spring and Marquis were used as controls. HMW-GS was classified using the nomenclature of Payne and Lawrence (1983). Waxy protein extraction and electrophoresis Starch granules were isolated from one seed of each wheat line according to the procedure described by Zhao and Sharp (1996) with the following modifications. The seed was crushed and soaked overnight in 1 mL of water at 4°C. After shaking for 5 min, the samples were centrifuged for 3 min at 10 000×g. The pellet was added with 1 mL washing buffer containing 0.138 mol L-1 Tris-HCl, pH 6.8, 5.75% (w/v) SDS, 12.5% (v/v) 2-mercaptoethanol, 25% (v/v) glycerol, then centrifuged for 3 min at 10 000×g. The supernatant was discarded. The starch was then washed twice with water, twice with acetone, and finally air dried. The Wx protein was extracted by adding protein solution buffer (0.062 mol L-1 Tris-HCl, pH 6.8, 2.3% (w/v) SDS, 5% (v/v)

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2-mercaptoethanol, 10% (v/v) glycerol, and 0.005% (w/v) bromophenol blue), on a 100-μL for about 5 mg basis. The extracts were heated in a boiling water bath for 15 min, followed by being cooled for 5 min in ice water, and finally were centrifuged at 10 000×g for 10 min. A 30-μL sample of the supernatant was used to separate the waxy proteins using the SDS-PAGE system with 4.5% stacking gel and 15% separation gel, which was programmed for under 20 mA per gel at the stacking gel. After the samples had run into the separation gel, 22 mA per gel was applied for about 17 h. The protein bands were detected by silver staining.

Mill flour Seed samples obtained from the harvested population were normally stored for about 1 mon and then milled using Bühler experimental mill ((Buhler, Buhler-Miag Co., Germany) with a flour extraction yield of approximately 70%.

Mixograph parameters Mixograph analysis was carried out using a 10-g mixograph system (National Manufacturing Co., USA) according to AACC approved method 54-40A. Parameters were recorded at mixing peak time (MPT), mixing peak value (MPV), mixing peak width (MPW), mixing peak integral (MPI), 8 min width (MTxW).

Genetic map construction and QTLs mapping The molecular genetic map was constructed by MAPMAKER/EXP ver. 3.0b (Lincoln et al. 1993). The commands “group”, “sequence” and “map” were used to develop the linkage groups and the position of markers on each chromosome, and the command “try” and “compare” were located on the unlinked markers on the chromosomes. The Kosambi (1994) mapping function was used to convert recombination fraction into cM values as map distances. Finally, the linkage map was drawn by Mapchart ver. 2.1 (Voorips 2002).

Statistic analysis Analysis of variance was carried out using SPSS ver. 13.0 (SPSS, Chicago, USA). QTLs with additive effects and epistatic effects expressed in RIL population under three environments were detected by the software QTLNetwork ver. 2.0 (Yang and Zhu 2005) based on the mixed linear model (Wang et al. 1999). QTL was abbreviated with every parameter followed by its relevant chromosome number. If there were more than one QTL on the same chromosome,

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the serial number was added after the chromosomal number separated by a dot.

Acknowledgements This research was supported by the National Natural Science Foundation of China (31171554), the National Basic Research Program of China (2009CB118301), and the Natural Science Foundation of Shandong Province, China (ZR2009DQ009). We thank Dr. Zhao Jiping, Ball Horticultural Co., China, for his constructive review and improving the English.

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QTL Mapping for Dough Mixing Characteristics in a Recombinant Inbred Population Derived from a Waxy × Strong Gluten

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