Molecular markers for tracking variation in lipoxygenase activity in wheat breeding
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- Geng, H., Zhang, Y., He, Z. et al. Mol Breeding (2011) 28: 117. doi:10.1007/s11032-010-9466-5
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Lipoxygenase (LOX) activity is an important factor determining the color of flour and end-use products of wheat. In the present study, quantitative trait loci (QTL) for LOX activity in common wheat were mapped using 71 doubled haploid (DH) lines derived from a Zhongyou 9507 × CA9632 cross, and SSR markers. Two QTL, QLpx.caas.1AL and QLpx.caas-4B, were identified on chromosomes 1AL and 4B, closely associated with LOX activity. The SSR loci Xwmc312 and Xgwm251 proved to be diagnostic and explained 13.4–25.2% of the phenotypic variance for the 1AL locus and 14.3–27.0% for the 4B locus across four environments. The SSR markers Xgwm251 and Xwmc312 were validated across 198 Chinese wheat cultivars and advanced lines and showed highly significant (P < 0.01) association with LOX activity. We further established a multiplexed PCR with SSR marker combination Xwmc312/Xgwm251 to test these wheat cultivars and advanced lines. The results suggested that the marker combination Xwmc312/Xgwm251 is efficient and reliable for evaluating LOX activity and can be used in marker-assisted selection (MAS) for targeting flour color attributes to noodle and other wheat-based products.
KeywordsTriticum aestivum L.Lipoxygenase activityMolecular markerQTLMultiplex PCR
Lipoxygenase (LOX) exerts significant effects on wheat flour color and the rheological properties of doughs (Shiiba et al. 1991; Pastore et al. 2000; Carrera et al. 2007). It is a non-haem dioxygenase, containing iron at the active site, and is a widely distributed enzyme in plant species, particularly abundant in seeds (Allen et al. 1999). The enzymes catalyze the introduction of molecular oxygen into a 1,4-cis-cis-pentadiene structure of polyunsaturated fatty acids, to form conjugated unsaturated hydroperoxydienoic or trienoic acids (Siedow 1991). During the intermediate steps of substrate peroxidation, fatty acid radicals are produced, which cause oxidative degradation of pigments and reduce the final color of wheat flour and products (Irvine and Anderson 1953; Siedow 1991; Borrelli et al. 1999). Although other enzymes such as peroxidase (POD) and polyphenol oxidases (PPO) can contribute to semolina bleaching, a major role appears to be played by lipoxygenase (Leenhardt et al. 2006). The concentration of yellow carotenoid pigments in wheat grain is an important quality trait and is determined both by the collection of yellow carotenoid pigments and by lipoxygenase-catalyzed degradation. In addition, lipoxygenase is capable of oxidizing the sulfhydryl groups of gluten proteins to form intra and intermolecular disulphide bonds, thus improving rheological properties of wheat flour doughs and the physical properties of the gluten protein complex (Shiiba et al. 1990; 1991).
Flour color is an important trait in the assessment of flour quality and exerts significant influence on noodles and other related products (Parker et al. 1998). A significant positive correlation between flour color and noodle quality was reported previously (Miskelly 1984). White-salted udon noodles, yellow alkaline (ramen), and Chinese white noodles are the most popular types in Asian countries (Liu et al. 2003; He et al. 2004). A bright yellow color is desirable for yellow alkaline noodles, consumed in Japan and southeastern Asia, and thus low LOX activity is desirable in wheat cultivars for use in these regions (Huang and Morrison 1988). In China, however, a bright white to creamy color is required for Chinese style foods such as steamed bread and Chinese white salted noodles, which account for more than 80% of wheat consumption (He et al. 2004; Fu 2008). Therefore, a high LOX activity is desirable in wheat cultivars for these products. In contrast, a bright yellow color is required for durum wheat products, such as pasta (Hessler et al. 2002; Carrera et al. 2007), and thus a low LOX activity is desirable in breeding durum wheat cultivars.
The objectives of this study were to map QTL for LOX activity in a wheat population of Zhongyou 9507 × CA9632, and validate SSR markers closely linked to these QTL in Chinese wheat cultivars and advanced lines.
Materials and methods
A total of 71 doubled haploid (DH) lines derived from the cross between Zhongyou 9507 and CA9632 were used for QTL mapping of LOX activity. The DH lines were developed at the Chinese Academy of Agricultural Sciences, Beijing, China. The 198 Chinese wheat cultivars and advanced lines from four major wheat-growing regions were employed for the validation of SSR markers closely linked to the QTL. Chinese Spring (CS) nullisomic 4A-tetrasomic 4D (CS N4A-T4D), CS N4D-T4B and ditelosomic line 4BS (CS DT4BS), provided by Prof. R. A. McIntosh, University of Sydney, and LDN 4D (4B) chromosome substitution line from Dr. Steven Xu, USDA-ARS, Northern Crop Science Laboratory, Fargo, USA, were employed to verify the chromosomal location of QTL QLpx.caas-4B. The LDN 4D (4B) chromosome substitution line was developed from the cross of durum (cv. Langdon) and common wheat (cv. Chinese Spring; Li et al. 2006).
During the 2006–2007 and 2007–2008 cropping seasons, the DH lines and their parents were sown at Beijing and Anyang in Henan province, providing data for four environments. For the validation of SSR markers, 198 Chinese wheat cultivars and advanced lines were sown at Anyang and Jinan in Shandong province in the 2006–2007 cropping season. The LOX activities averaged from two locations for the cultivars and lines were used for association analysis. All the field trials were conducted in randomized complete blocks with two replicates. Each plot comprised two 2-m rows spaced 25 cm apart, with 75 plants in each row. Test plots were managed according to local practices.
Determination of LOX activity
Wheat seeds (10 g) were milled to whole wheat meal with a Cyclotec 1093 sample mill (0.5 mm mesh screen), and used for testing LOX activity within a week.
For LOX activity determination, an extract was prepared according to the descriptions of McDonald (1979), Pastore et al. (2000) and Gokmen et al. (2007) with minor modifications. Enzyme was extracted by mixing 0.2 g of whole wheat meal with 1 ml of 0.1 mM sodium phosphate buffer (pH 6.5) at 4°C in a homogenizer for 10 min. The mixture was placed at 4°C for 1 h, stirred four times and then centrifuged (Eppendorf model 5417R, Germany) at 12,000 rpm for 10 min at 4°C. The supernatant containing LOX was used for subsequent assay of LOX activity. Pastore et al. (2000) suggested that the crude enzyme activity was almost constant for 24 h, and then decreased with time, so the extract was stored in an ice-water bath, and used the same day.
Linoleic acid substrate (7.5 mM) was prepared as described by Hessler et al. (2002) and Carrera et al. (2007), with minor modifications. Substrate solution was prepared as follows: 0.25 ml of Tween 20 was dissolved in 10 ml of 0.1 M sodium phosphate buffer (pH 9.0) and 0.25 ml of linoleic acid (99%) was added drop by drop. Then 0.5 ml of 1 N NaOH was added and the mixture once again agitated until a clear transparent solution was obtained, and the final volume made up to 100 ml with sodium phosphate buffer (pH 6.5). The substrate was divided into small volumes, sealed under N2 and maintained at −20°C. The same batch of substrate was used with all samples to ensure the same level of auto-oxidation. Each sample was assayed in duplicate.
Lipoxygenase activity was determined by measuring the conjugated diene absorption at 234 nm with a UV–visible spectrophotometer (TU-1800PC, Beijing Purkinje, China; Gokmen et al. 2007; Carrera et al. 2007). A quartz cuvette of 1 cm thickness was used in the solution measurement. For each data point, LOX activity was determined in duplicate extracts of whole wheat meal with parallel spectrophotometric measurements and the mean values were reported. If the coefficient of variation was more than 10% for the spectrophotometric assay of LOX activity in the duplicates, the test was repeated. One unit of LOX activity (A234) was defined as an increase in absorbance at 234 nm per minute per gram of whole wheat meal under assay conditions.
Analysis of variance (ANOVA) and computation of correlation coefficients were performed with the SAS System for Windows version 9.0 (SAS Institute Inc, Cary, NC, USA). The contribution of the genotype and environment was evaluated by two-way ANOVA. Broad-sense heritability (h2) for LOX activity was calculated using the formula h2 = σg2/(σg2 + σge2/e + σε2/re), where σg2, σge2, and σε2 were estimates of genotypic, genotype × environment interaction and error variances, respectively, and e and r were the numbers of environments and replicates per environment, respectively. For the 198 cultivars and advanced lines, the LOX activity of each genotype was measured in two environments, and averaged to verify the association between LOX activity and SSR markers Xgwm251 and Xwmc312. The differences in LOX activities in the genotypes with different PCR products were tested using Fischer’s protected LSD.
SSR analysis and QTL detection
The DH population used for QTL analysis was previously genotyped with 143 SSR, 4 STS (sequence-tagged-site) and 26 AFLP (amplified fragment length polymorphism) markers (Zhang et al. 2005). The linkage groups were established with the software Mapmaker 3.0b (Lincoln et al. 1992). The map distances between markers were calculated using Kosambi’s (1944) mapping function. QTL analysis was performed with the software IciMapping 2.2 by inclusive composite interval mapping (ICIM; Li et al. 2007), and a LOD score of 2.5 was used as the threshold for declaration of linkage and QTL detection. The percentages of phenotypic variance explained (PVE) by individual QTL and additive effect at the LOD peak were obtained through stepwise regression (Li et al. 2007). Each QTL is represented by a 20-centimorgan (cM) interval with the local LOD maximum as center. Adjacent QTL on the same chromosome were considered different when the distances between the curve peaks are over 20 cM or when the support intervals were non-overlapping.
Multiplex PCR assay
Two SSR markers, Xwmc312 and Xgwm251, closely linked to the QTL QLpx.caas-1AL and QLpx.caas-4B, respectively, were multiplexed to investigate 198 Chinese wheat cultivars and advanced lines, for evaluating the correlation between SSR alleles and LOX activities. The multiplex PCR conditions were performed in a total volume of 20 μl including 20 mM of Tris–HCl (pH 8.4), 20 mM of KCl, 150 μM of each of the dNTPs, 1.5 mM of MgCl2, 1 unit of Taq DNA polymerase (Tiangen Biotech, Beijing) and 50 ng of genomic DNA, and 2 pmol of each primer of Xwmc312 and 3 pmol of each primer of Xgwm251. The PCR was carried out in an MJ Research PTC-200 thermal cycler and the reaction conditions were 95°C for 5 min, followed by 36 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension of 72°C for 10 min. The PCR products were separated by electrophoresis on a 6% polyacrylamide gel and resolved by silver staining (Bassam et al. 1991). The multiplex PCR was repeated twice. The PCR products amplified from the Xwmc312/Xgwm251 markers were excised from dried gels and reclaimed using a Poly-Gel DNA Extraction Kit (Biomiga, USA). The targeted fragments were cloned into pGEM-T easy vector (Tiangen Biotech, Beijing), transformed into DH5α, and sequenced by Shanghai Sangon Biological Engineering & Technology and Service (Shanghai, China, http://www.sangon.com) for determining the sizes of the fragments.
Analysis of variance of LOX activity values on the DH population derived from the cross Zhongyou 9507 × CA9632
Source of variation
Mean of squares
Line × environment
QTL for lipoxygenase activity
Quantitative trait loci (QTL) detected for LOX activity in the DH population derived from the cross Zhongyou 9507 × CA9632
Location and yeara
Total PVE (%)
Statistical analysis of the association between the genotypes of SSR loci Xwmc312 and Xgwm251 and LOX activity in 198 Chinese wheat cultivars and advanced lines
Accession numbers tested
Mean LOX activityb,c
QLpx.caas-1AL was mapped closely to Xwmc312, explaining 25.2, 13.4, 21.7, 15.0 and 26.8% of the phenotypic variance for LOX activity in Anyang 2007, Beijing 2007, Anyang 2008, Beijing 2008 and the averaged data, respectively. The averaged LOD scores ranged from 2.5 to 5.6 (Table 2).
The QTL QLpx.caas-4B closely linked to Xgwm251 was identified in Anyang 2007, Beijing 2007, Anyang 2008 and the averaged data, accounting for 14.3, 27.0, 15.4, and 28.4% of the phenotypic variance, respectively (Table 2).
Validation of the SSR markers Xwmc312 and Xgwm251
The LOX activities of 198 Chinese cultivars and advanced lines were tested with two SSR markers, Xwmc312 and Xgwm251, and the multiplex combination of Xwmc312/Xgwm251. The results indicated that Xwmc312 amplified 227, 235, and 247-bp fragments in various accessions (Table 3). Of 198 accessions tested, the mean LOX activity of 60 accessions with the 227-bp PCR fragment (Xwmc312-227) was 59.3 A234 min−1 g−1, whereas the mean LOX activity of 72 accessions with the 235-bp fragment (Xwmc312-235) was 73.0 A234 min−1 g−1, and the averaged LOX activity of 66 accessions with the 247-bp fragment (Xwmc312-247) was 64.3 A234 min−1 g−1. ANOVA showed that the genotype Xwmc312-235 had significantly higher mean LOX activity than the genotypes Xwmc312-227 and Xwmc312-247. The genotypes Xwmc312-227 and Xwmc312-247 displayed lower LOX activity, but no significant differences were found between them.
Multiplex PCR amplification with Xwmc312/Xgwm251
PCR amplification with the combination Xwmc312/Xgwm251 produced nine genotypes and showed three significantly different levels of mean LOX activity in 198 wheat cultivars and advanced lines (Table 3; Electronic Supplementary Material: Supplementary Table 1). The LOX activities of five genotypes Xwmc312-235/Xgwm251-117, Xwmc312-247/Xgwm251-117, Xwmc312-235/Xgwm251-113, Xwmc312-227/Xgwm251-117 and Xwmc312-235/Xgwm251-125 were 75.1, 69.8, 69.3 69.2 and 67.0 A234 min−1 g−1, respectively, which were significantly higher than those of the other four genotypes Xwmc312-247/Xgwm251-113, Xwmc312-247/Xgwm251-125, Xwmc312-227/Xgwm251-113, and Xwmc312-227/Xgwm251-125, with mean LOX activities of 59.5, 58.1, 57.6 and 55.1 A234 min−1 g−1, respectively (Table 3; Fig. 3). The genotypes Xwmc312-235/Xgwm251-117 and Xwmc312-227/Xgwm251-125 showed the highest (75.1 A234 min−1 g−1 on average) and the lowest LOX activity (55.1 A234 min−1 g−1 on average), respectively (Table 3).
A high level of LOX activity is one of the major determinants for the appearance of wheat-based products, particularly for flour color natural whitening (Siedow 1991; Borrelli et al. 1999). In the present study, two major QTL, QLpx.caas-1AL and QLpx.caas-4B, were found in the DH population. The QTL QLpx.caas.1AL, located on chromosome 1AL closely linked to SSR marker Xwmc312, has not been reported previously, and is likely to represent a new QTL for LOX activity in wheat. Zhao et al. (2009) found a QTL (QTspr.sdau-1A) associated with springiness of Chinese dry noodle in the recombinant inbred line (RIL) population Chuan 35050× Shannong 483. The QTL (QTspr.sdau-1A) for the textural property of springiness was detected on chromosome 1A, being flanked by SSR markers Xwmc93 and Xgwm135, at a similar position to QLpx.caas.1AL based on the wheat consensus map (Somers et al. 2004; http://wheat.pw.usda.gov/GG2/index.shtml). High LOX activity could enhance gluten strength and improve noodle quality, since the improvement of rheological properties is one of the functions of lipoxygenase (Shiiba et al. 1991; Trufanov et al. 2007). Therefore, the QTL QTspr.sdau-1A for the textural property of springiness and QLpx.caas.1AL for LOX activity could be at the same locus with pleiotropic effects or closely linked genes.
Previous studies indicated that LOX activity was significantly affected by genotype (G), environment (E) and genotype × environment (G × E; Borrelli et al. 1999). In the present study, LOX activity varies among wheat genotypes and is also significantly affected by environments (Table 1). However, the LOX activities of the DH lines across four environments have a high heritability (h2 = 0.94), indicating that it is mainly affected by genetic factors. Therefore, the selection for LOX activity can be effectively applied at the earlier generation of the breeding process. Paterson et al. (1991) and Mackill and Ni (2001) indicated that the QTL would be more stable if the trait had high heritability and large variation (>25%). Veldboom and Lee (1996) suggested that the QTL identified in more than one environment or those identified using data pooled over environments are useful from the point of view of marker-assisted selection (MAS). In this study, two major QTL for LOX activity, QLpx.caas-1AL and QLpx.caas-4B, were consistently detected in four and three environments, respectively, indicating that they were stable in different environments, suggesting a significant effect on LOX activity, and that their linked molecular markers can be used for MAS in wheat breeding.
The authors are very grateful to Prof. Robert McIntosh, University of Sydney, for reviewing this manuscript. This study was supported by the National Science Foundation of China (30871516 and 30830072), National Basic Research Program (2009CB118300), National 863 Program (2006AA10Z1A7 and 2006AA100102), international collaboration project from Ministry of Agriculture (2006-G2), and earmarked fund for the Modern Agro-industry Technology Research System.