Genetic Diversity and QTL Mapping of Thermostability of Limit

School of Science and Health, University of Western Sydney, Penrith, NSW 2751, Australia. J. Agric. Food Chem. , 2015, 63 (14), pp 3778–3783. DOI: 1...
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Genetic Diversity and QTL Mapping of Thermostability of Limit Dextrinase in Barley Xiaolei Wang,† Xuelei Zhang,† Shengguan Cai,† Lingzhen Ye,† Meixue Zhou,§ Zhonghua Chen,‡ Guoping Zhang,† and Fei Dai*,† †

Department of Agronomy, Zhejiang Key Lab of Crop Germplasm, Zhejiang University, Hangzhou 310058, China Tasmanian Institute of Agriculture, University of Tasmania, P.O. Box 46, Kings Meadows, TAS 7249, Australia ‡ School of Science and Health, University of Western Sydney, Penrith, NSW 2751, Australia §

ABSTRACT: Limit dextrinase (LD) is an essential amylolytic enzyme for the complete degradation of starch, and it is closely associated with malt quality. A survey of 51 cultivated barley and 40 Tibetan wild barley genotypes showed a wide genetic diversity of LD activity and LD thermostability. Compared with cultivated barley, Tibetan wild barley showed lower LD activity and higher LD thermostability. A doubled haploid population composed of 496 DArT and 28 microsatellite markers was used for mapping Quantitative Trait Loci (QTLs). Parental line Yerong showed low LD activity and high LD thermostability, but Franklin exhibited high LD activity and low LD thermostability. Three QTLs associated with thermostable LD were identified. The major QTL is close to the LD gene on chromosome 7H. The two minor QTLs colocalized with previously reported QTLs determining malt-extract and diastatic power on chromosomes 1H and 2H, respectively. These QTLs may be useful for a better understanding of the genetic control of LD activity and LD thermostability in barley. KEYWORDS: barley (Hordeum vulgare L.), Tibetan wild barley, limit dextrinase, thermostability, quantitative trait locus

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INTRODUCTION Limit dextrinase (LD) is the only endogenous hydrolase that can cleave α-1−6 linkages amylopectin and β-limit dextrin;1,2 hence it is also a key enzyme determining malting quality. There are three forms of LDs in barley: active free, soluble inactive, and insoluble bound, of which only the active free form of LD (free LD) participates in starch degradation.3 According to Stenholm and Home (1999),4 free LD activity in barley is positively correlated with wort fermentability. The major industrial process of brewing includes malting (steeping, germination, kilning), mashing, and fermentation. Free LD is more heat labile than bound or latent LD and will quickly lose its activity in a mash at temperature higher than 63 °C.5 The loss of LD activity can reach up to 70% during kilning.6−9 Lower LD activity leads to lower levels of fermentable sugar production and higher α-dextrin level, thus lower alcohol levels and worse beer quality.10 Some brewers do not want a fully attenuated beer, so they are looking to retain LD activity to provide body and mouth feel to the beer. From a commercial point of view, maintaining high LD activity is desirable during malting, which will enhance the hydrolysis of wort dextrins to fermentable sugars, consequently increasing the yield of alcohol.7 Therefore, it is imperative to select and develop the barley cultivars with high activity and thermostability of LD so as to improve malt quality. In the barley genome, only one LD gene has been reported up to date, which encodes a 97 kDa protein.11,12 It was mapped to the short arm of chromosome 7H, using 150 doubled haploid (DH) lines derived from a cross of Steptoe/Morex, and cosegregated with a RFLP marker ABC154A.13 Large genetic variation of LD activity has been reported in cultivated barley14−16 and in wild barley.17 Similarly, thermostability of © 2015 American Chemical Society

LD also showed a wide genetic diversity in cultivated barley. The genetic variation of LD thermostability might be attributed to the amino acid substitution in the LD gene.2 In addition, LD activity in barley grains or malt was also affected by the activity of limit dextrinase inhibitor (LDI).18 Yang et al. (2009)2 aligned the amino acid sequences of LD in two barley varieties, Galleon and Maud, with significantly different LD thermostability. The Thr/Ala-233, Ala/Ser-885, and Gly/Cys-888 substitutions were identified to be associated with LD thermostability based on single-strand conformation polymorphism (SSCP) analysis (P < 0.0001). Furthermore, amino acid substitutions of Thr/Ala-233 and Ala/Ser-885 showed that additive effect on LD thermostability and Gly/ Cys-888 substitution may increase the binding ability between LD and LDI.2 LD binding to its inhibitor is protected and stabilized against heat inactivation, so the bound and latent LD showed relatively higher thermostability than the free form.19 However, relatively heat-stable LD in the inactive form can be released steadily during mashing,20 and this process can be promoted by dithiothreitol and low pH.21 Wild barley (Hordeum spontaneum L.), the progenitor of cultivated barley (H. vulgare L.), is an important genetic resource for barley improvement.22 It is rich in abiotic and biotic stress tolerance, 23,24 grain protein, and malting quality.16,17,25 Tibetan wild barley is one of the genome donators to modern cultivated barley,26,27 and is particularly rich in genetic diversity, as reported previously.16,17,25,28−31 In Received: Revised: Accepted: Published: 3778

January 14, 2015 March 29, 2015 March 29, 2015 March 30, 2015 DOI: 10.1021/acs.jafc.5b00190 J. Agric. Food Chem. 2015, 63, 3778−3783

Article

Journal of Agricultural and Food Chemistry

Figure 1. Limit dextrinase activity variation after thermo treatment at different temperatures for 0−20 min. A, B, C, D, and E: Limit dextrinase activity variation after thermo treatment at 54.5, 55.5, 56.5, 57.5, and 58.5 °C, respectively. Control: without thermo treatment.

following stages: 5 h steep and 8 h air-rest at 17 °C, 8 h steep and 12 h air-rest at 16 °C, 4 h steep, 5 h air-rest and 2 h steep at 15 °C followed by 96 h germination at 16 °C. Malting of all the genotypes and DH lines were milled to pass through a 0.5 mm screen for analysis. Thermo Treatment and LD Activity Assay. LD activity was determined using the Limit-Dextrinase assay kit (Megazyme, Ireland). According to McCleary (1992)34 and the Megazyme manual, LD was extracted from 0.25 g sample with 4.0 mL extracting solution, 0.1 mM sodium malate buffer (pH 5.5) containing 25 mM dithiothreitol, for 5 h at 40 °C. Thermo treatment in the current study referred to the method described by Evans et al. (2003)14 and was conducted using a Memmert WNE450 water bath (Memmert, Germany). Specifically, 0.5 mL of supernatant was added to a plastic tube after centrifuging at 1000g and incubated in a water bath for thermo treatment. Thermo treatment was terminated with an ice−water bath for 10 min. LD activity of thermo-treated and nonthermo-treated (control) supernatants was determined using Limit-Dextrizyme tablets at 40 °C. One unit of LD activity is the amount of enzyme required to release one μmol of glucose reducing-sugar equivalents per min from pullulan under the defined assay conditions. QTL Analysis. A genetic linkage map of the Yerong/ Franklin DH population was composed of 496 DArT and 28 microsatellite markers. 32 QTLs were analyzed using MapQTL5.0 software package,35 as described by Dai et al. (2011).36 Briefly, QTLs were analyzed by interval mapping (IM) and approximate multiple QTLs model (MQM) of MapQTL5.0. Logarithm of the odds (LOD) threshold values (set as 3.0) applied to declare the presence of QTLs were

the current study, a genetic diversity analysis was conducted for the identification of genotypes with high LD thermostability. Moreover, QTLs analysis of LD thermostability was carried out to identify relevant novel genes or genetic factors.

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MATERIALS AND METHODS Plant Materials and Sample Preparation. Fifty-one cultivated barley, 40 Tibetan wild barley genotypes, and a DH population consisted of 177 lines from a cross between Yerong and Franklin was used in this study.32 Franklin is an Australian two-rowed malting barley cultivar, and Yerong is an Australian six-rowed feed barley variety. Seeds of Tibetan wild barley, collected from the Qinghai-Tibet Plateau of China, were kindly provided by Professor Dongfa Sun of Huazhong Agricultural University, China. All materials were grown in the field of the experimental farm, Zhejiang University, Huajiachi campus, Hangzhou, China, in 2008−2009 and 2009−2010 winter barley growing seasons, except that the DH population was only cultivated in 2009− 2010 growing season. Each genotype or DH line was sown in a 2 m row with 0.25 m between rows, with three replications. Field management, including fertilization, weed, and disease control, was the same as applied locally. At maturity, the plants were harvested and stored in a cool room at 4 °C. Grains were mixed and used for micromalting and LD activity assay after milled to pass through a 0.5 mm screen. Grain samples (200 g) were micromalted in a Phoenix System Micromalting Apparatus (Adelaide, Australia) in the order of steeping, germination and kilning according to the method described by Dai et al. (2007),33 with some modification in the steeping stage. Briefly, there were the 3779

DOI: 10.1021/acs.jafc.5b00190 J. Agric. Food Chem. 2015, 63, 3778−3783

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Journal of Agricultural and Food Chemistry

thermostability of Franklin and Yerong were 68.5% and 71.8%, 73.1% and 80.2%, 59.2% and 64.3%, 62.7% and 65.1%, 47.9% and 58.5%, 35.2% and 49.9%, 33.4% and 43.0%, 21.4% and 33.2%, 19.4% and 30.5%, 10.8% and 21.0%, 7.9% and 17.2% after 5 min thermo treatments at 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59 °C, respectively. According to the method of Arora et al. (1992),37 we estimated the LT50 (lethal temperature of 50%), which is defined as the temperature at which 50% of LD activity lost after thermo treatment. In the current study, LT50 of Franklin and Yerong were 54.4 and 56.4 °C, respectively (Figure 2). In order to obtain a relative higher dose of thermo treatment, we chose 56.5 °C as the optimum temperature for thermo treatment in the current study rather than 55.5 °C, which was close to LT50 and similar to the results of Evans et al. (2003)14 and Yang et al. (2009).2 Difference in LD Thermostability in Cultivated and Wild Barley. In 2009, LD activity for the 40 wild barley (WB) genotypes ranged from 352.3 U kg−1 to 562.8 U kg−1, with a mean of 448.0 U kg−1, whereas LD activity for the 51 cultivated barley (CB) genotypes ranged from 366.2 U kg−1 to 787.6 U kg−1, with a mean of 539.6 U kg−1 (Table 1). Thermostable LD activity of WB ranged from 229.7 U kg−1 to 457.4 U kg−1, with a mean of 327.5 U kg−1, and CB ranged from 177.4 U kg−1 to 488.8 U kg−1, with a mean of 318.3 U kg−1. Obviously, WB had lower average value of LD activity than CB. As for the mean of thermostable LD activity, both cultivated and wild barley were basically the same. The LD thermostability of WB ranged from 59.9% to 91.3%, with a mean of 73.3%, and CB ranged from 27.1% to 85.4%, with a mean of 59.8%. It is clear that WB had much larger LD thermostability value than CB. In addition, the coefficient of variation (CV) for LD, thermostable LD, and LD thermostability were slightly smaller in WB than those in CB (Table 1). In 2010, the LD activity of WB and CB ranged from 285.6 U kg−1 to 604.8 U kg−1 and from 315.6 U kg−1 to 741.6 U kg−1, with means of 405.4 U kg−1 and 514.8 U kg−1, respectively. (Table 1). Thermostable LD activity of WB and CB ranged from 181.1 U kg−1 to 486.0 U kg−1 and from 172.8 U kg−1 to 555.6 U kg−1, with means of 275.9 U kg−1 and 319.8 U kg−1, respectively. However, for LD thermostability, WB and CB ranged from 50.5% to 80.4% and from 41.5% to 75.3%, with means of 67.8% and 61.3%, respectively. All the parameters in 2010 are very consistent to those in 2009, for instance, the maximum and mean LD thermostability of WB were higher than those of CB. Meanwhile, the CV values of WB were smaller uniformly. Identification of QTL Associated with Thermostable LD. In view of the significant differences in LD activity and

estimated by performing the genome wide permutation tests with at least 1000 permutations of the original data set for the trait. The percentage of variance explained by each QTL (R2) was obtained using restricted MQM mapping implemented with MapQTL5.0. Statistical Analysis. Each measurement was carried out with at least three replications. Statistical analysis was carried out using SPSS v16.0 for windows (SPSS Inc., Chicago, U.S.A.).

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RESULTS Effect of Thermo Treatment on LD Activity. In order to determine the optimal time and temperature for LD activities of the parental lines Franklin and Yerong, time- and temperaturedependent experiments were conducted. The LD activities of Franklin and Yerong after thermo treatments for 0−20 min at a set of temperature gradients are shown in Figure 1A−E, respectively. Under the control condition (without thermo treatment), LD activity of Franklin (521.9 U kg−1) was significantly higher than that of Yerong (250.9 U kg−1). LD activity of Franklin and Yerong declined by 26.9% and 19.8%, 37.3% and 34.9%, 64.8% and 50.1%, 78.6% and 66.8%, 89.2% and 79.0%, after 5 min thermo treatments at 54.5, 55.5, 56.5, 57.5, and 58.5 °C, respectively (Figure 1). The LD activity decreased sharply during the first 5 min of the thermo treatment, and thereafter, the rate of decline became much slower. Hence, the optimum treatment time at 5 min was chosen for comparing the difference in LD thermostability (expressed as a ratio of the remaining LD activities after 5 min thermo treatment to those in the control) between Franklin and Yerong. LD thermostability of Franklin and Yerong responded differently to increasing temperatures (Figure 2). The LD

Figure 2. Thermostability variation of limit dextrinase after 5 min thermo treatment at different temperatures from 54 to 59 °C.

Table 1. Genetic Diversity of Limit Dextrinase and Its Thermostability in Wild and Cultivated Barley Grown in 2008−2009 and 2009−2010 2009 LDa (U kg−1) min max mean CVd(%)

2010

thermostable LD (U kg−1)

LD thermostability (%)

LDa (U kg−1)

thermostable LD (U kg−1)

LD thermostability (%)

WBb

CBc

WB

CB

WB

CB

WB

CB

WB

CB

WB

CB

352.3 562.8 448.0 14.1

366.2 787.6 539.6 21.1

229.7 457.4 327.5 17.7

177.4 488.8 318.3 21.9

59.9 91.3 73.3 12.2

27.1 85.4 59.8 17.4

285.6 604.8 405.4 16.6

315.6 741.6 514.8 18.6

181.1 486.0 275.9 22.6

172.8 555.6 319.8 29.7

50.5 80.4 67.8 11.1

41.5 75.3 61.3 15.1

a

LD, limit dextrinase. bWB, wild barley (n = 40). cCB, cultivated barley (n = 51). dCV, coefficient of variation. Thermo treatment: 0.5 mL supernatant of each genotype was treated in a water bath at 56.5 °C for 5 min. 3780

DOI: 10.1021/acs.jafc.5b00190 J. Agric. Food Chem. 2015, 63, 3778−3783

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Figure 3. Frequency distribution for limit dextrinase and thermostable limit dextrinase activity in a DH population of Yerong/Franklin.

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thermostability between Franklin and Yerong found in our preliminary study, we further conducted the QTL analysis using a DH population consisted of 177 lines from a cross between Yerong and Franklin (Li et al., 2008) in order to identify novel QTLs associated with LD activity and thermostability. Normal distributions of LD and thermostable LD activity exhibited no significant skewness and kurtosis (Figure 3). Transgression beyond the parental values was also observed. Moreover, some favorable combinations, such as high LD activity under both control conditions and thermo treatments, were found in this DH population. A major QTL controlling LD activity in malt was identified on chromosome 7H under both control (qLD2) and thermo treatment (qTS-LD3) at the position of 58.7 cM on the genetic map with the nearest marker being bPb-2866 (Table 2). This Table 2. Quantitative Trait Locus (QTLs) for Limit Dextrinase in the DH Population of Yerong/Franklin Grown in 2009−2010 treatment control thermo treatment

QTL

chr.

nearest marker

position (cM)

LOD

R2 (%)

qLD1 qLD2 qTS-LD1 qTS-LD2 qTS-LD3

4H 7H 1H 2H 7H

bPb-0365 bPb-2866 HVHVA1 bPb-5440 bPb-2866

58.2 58.7 102.0 83.7 58.7

3.71 10.73 4.03 3.66 7.49

9.2 31.0 9.9 8.9 19.6

DISCUSSION

In this study, we first investigated LD activity and thermostability of parental lines Franklin and Yerong under different temperature and time treatments in order to determine the optimal time and temperature for further research. It was found that both cultivars showed a rapid reduction of LD activity during the first 5 min of thermo treatment, and then leveled off over the course of another 15 min (Figure 1). Hence, we chose 5 min as the optimal time for thermo treatment. However, the time duration is shorter than those used in previous studies.2,38,39 On the other hand, the optimal temperature was determined according to LT50. Based on the influence of different temperature on LD activity, we chose 56.5 °C as the optimal temperature for thermo treatment. The results are basically consistent with some previous reports.2,14 Under these thermo treatment conditions, we found Tibetan wild barley demonstrated much greater genotypic variation in LD thermostability than cultivated barley (Table 1), indicating that the wild barley is indeed an elite germplasm for barley breeders in developing cultivars with high LD thermostability. The wide genetic diversity of LD and thermostable LD activity in the DH population (Figure 3) has enabled us to identify novel QTLs. The Yerong/Franklin DH population has already been used successfully for QTL analysis of waterlogging tolerance32 and phytase activity.36 A major QTL associated with LD and thermostable LD activity in malt was identified on chromosome 7H with the nearest marker bPb-2866 and a position of 58.2 cM on genetic map. The QTL is close to the LD gene with a position of 46 cM on chromosome 7H reported by Li et al. (1999).13 Although there is no available sequence for bPb-2866, the sequence of a marker named bPb-8049 with available sequence has been found (http://www.diversityarrays. com/), which is close to bPb-2866 (2.36 cM) on the genetic map of Yerong/Franklin DH population.18 We further mapped the sequences of LD gene (AF022725) and bPb-8049 to a synthetic assembly of the barley genome based on the WGS contigs assembly of cv. Morex, Barke and Bowman,27 in an attempt to identify their physical locations. Interestingly, LD gene and bPb-8049 were mapped to contigs of barke_contig_281373 and barke_contig_275219, respectively. Their physical distance was 1.0 Mega base in the synthetic assembly of the barley genome. Thus, it may be suggested that the major QTL we identified on 7H is the LD gene in barley.

QTL was able to explain 19.6% of the phenotypic variation of LD activity under thermo treatments, in comparison to 31.0% of the phenotypic variation of LD activity under control condition. Both of these QTLs are contributed by Franklin. In addition, a minor QTL (qLD1) controlling LD activity was identified at the position of 58.2 cM on chromosome 4H, with the nearest marker being bPb-0365. This QTL was not detected for LD activity under thermo treatment. Instead, two other minor QTLs were found, one on chromosome 1H (qTSLD1) contributed by Franklin at 102.0 cM and the closest marker of HVHVA1, and another on chromosome 2H (qTSLD2) contributed by Yerong at 83.7 cM and the nearest marker of bPb-5440 (Table 2). No significant QTL was identified for LD thermostability. 3781

DOI: 10.1021/acs.jafc.5b00190 J. Agric. Food Chem. 2015, 63, 3778−3783

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Journal of Agricultural and Food Chemistry

(3) Sissons, M. J.; Lance, R. C. M.; Wallace, W. Bound and free forms of barley limit dextrinase. Cereal Chem. 1994, 71, 520−521. (4) Stenholm, K.; Home, S. A new approach to limit dextrinase and its role in mashing. J. Inst. Brew. 1999, 105, 205−210. (5) Sissons, M. J.; Taylor, M.; Proudlove, M. Barley malt limit dextrinase: Its extraction, heat stability, and activity during malting and mashing. J. Am. Soc. Brew. Chem. 1995, 53, 104−10. (6) Manners, D. J.; Yellowlees, D. Studies on debranching enzymes. The limit dextrinase activity of extracts of certain higher plants and commercial malts. J. Inst. Brew. 1973, 79, 377−385. (7) Lee, W. J.; Pyler, R. E. Barley malt limit dextrinase: varietal, environmental and malting effects. J. Am. Soc. Brew. Chem. 1984, 42, 11−17. (8) Kristensen, M.; Svensson, B.; Larsen, J. Purification and characterization of barley limit dextrinase during malting. Proc. Congr. Eur. Brew. Conv. 1993, 24, 37−43. (9) Longstaff, M. A.; Bryce, J. H. Development of limit dextrinase in germinated barley (Hordeum vulgare L.). Plant Physiol 1993, 101, 881− 889. (10) Gomes, I.; Gomes, J.; Steiner, W. Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: production and partial characterization. Bioresour. Technol. 2003, 90, 207−214. (11) Burton, R. A.; Zhang, X. Q.; Hrmova, M.; Fincher, G. B. A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley. Plant Physiol 1999, 119, 859−887. (12) Kristensen, M.; Lok, F.; Planchot, V.; Svendsen, I.; Leah, R.; Svensson, B. Isolation and characterization of the gene encoding the starch debranching enzyme limit dextrinase from germinating barley. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1999, 1431, 538− 546. (13) Li, C. D.; Zhang, X. Q.; Eckstein, P.; Rossnagel, B. G.; Scoles, G. J. A polymorphic microsatellite in the limit dextrinase gene of barley (Hordeum vulgare L.). Mol. Breed. 1999, 5, 569−577. (14) Evans, D. E.; van Wegen, B.; Ma, Y.; Eglinton, J. K. The impact of the thermostability of α-amylase, β-amylase and limit dextrinase on potential wort fermentability. J. Am. Soc. Brew. Chem. 2003, 61, 210− 218. (15) Wang, X. D.; Yang, J.; Zhang, G. P. Genotypic and environmental variation in barley limit dextrinase activity and its relation to malt quality. J. Zhejiang Univ., Sci., B 2006, 7, 386−392. (16) Jin, X. L.; Cai, S. G.; Ye, L. Z.; Chen, Z. H.; Zhou, M. X.; Zhang, G. P. Association of HvLDI with limit dextrinase activity and malt quality in barley. Biotechnol. Lett. 2013, 35, 639−645. (17) Huang, Y. Q.; Cai, S. G.; Ye, L. Z.; Han, Y.; Wu, D. Z.; Dai, F.; Li, C. D.; Zhang, G. P. Genetic architecture of limit dextrinase inhibitor (LDI) activity in Tibetan wild barley. BMC Plant Biol. 2014, 14, 117. (18) Li, F.; Zhang, J. E.; Liu, H. M.; Tian, S. J.; Yang, X. G.; Ma, J. X.; Sun, M. X. Comparative study of activity and heat stability of limit dextrinase in 16 barley cultivars. Cereal Chem. 2008, 85, 271−275. (19) Walker, J. W.; Bringhurst, T. A.; Broadhead, A. L.; Brosnan, J. M.; Pearson, S. Y. The survival of limit dextrinase during fermentation in the production of Scotch whisky. J. Inst. Brew. 2001, 107, 99−106. (20) MacGregor, E. A. The proteinaceous inhibitor of limit dextrinase in barley and malt. Biochim. Biophys. Acta, Proteins Proteomics 2004, 1696, 165−170. (21) Heisner, C. B.; Bamforth, C. W. Thioredoxin in barley: could it have a role in releasing limit dextrinase in brewery mashes? J. Inst. Brew. 2008, 114, 122−126. (22) von Bothmer, R.; Seberg, O.; Jacobsen, N. Genetic resources in the Triticeae. Hereditas 1992, 116, 141−150. (23) Nevo, E.; Chen, G. Drought and salt tolerances in wild relatives for wheat and barley improvement. Plant, Cell Environ. 2010, 33, 670− 685. (24) March, T. J.; Richter, D.; Colby, T.; Harzen, A.; Schmidt, J.; Pillen, K. Identification of proteins associated with malting quality in a subset of wild barley introgression lines. Proteomics 2012, 12, 2843− 2851.

Two minor QTLs associated with thermostable LD activity were found to be located on the position of 83.7 cM on chromosome 2H (qTS-LD2) contributed by Yerong and 102.0 cM on chromosome 1H (qTS-LD1) contributed by Franklin, indicating that it should be exist a gene related to thermostable LD activity in Franklin other than the major QTL of LD activity in 7H. Recently, Huang et al. (2014)17 identified two DArT markers related to LDI content, bPb-8347 on the 73.4 cM of chromosome 6H and bPb-8399 on the 25.7 cM of chromosome 2H. We compared the position of qTS-LD1, qTSLD2 and that of bPb-8347, bPb-8399. Although qTS-LD2 and bPb-8399 are located on the same chromosome, their chromosome positions differ largely according to the consensus maps.40 It was reported that the synergistic function of LD and beta-amylase caused increase of maltose in the mash liquor, and adding LD to mashes resulted in a substantial increase in levels of fermentable sugars.41 qTS-LD1 identified in this study coincides with a QTL determining malt-extract, which is located at the position of 91.9−106.5 cM on chromosome 1H.42 Diastatic power is another important malt quality trait and is also a measure of combined activity of four starchdegrading enzymes, including α-amylase, beta-amylase, LD and α-glucosidase.43 qTS-LD2 identified in this study is close to a QTL determining diastatic power.42 Recent study has shown that only increases in LD activity may increase the production of fermentable sugar.43 It might be because of the different amino acid substitution, considering the case of beta-amylase.44 One of a possible mechanism is a single amino acid substitution, which improves the persistence of beta-amylase during mashing to result in slightly higher fermentable sugar production during mashing. The other type of mechanism is protein refold after a period of heating. Hence, there is a long way to go and much to be done to figure out the mechanism of LD thermostability. In short, the current study identified the novel QTLs associated with LD thermostability and again proved the wide diversity of Tibetan wild barley in LD thermostability. Moreover, the detected QTLs should be useful for a better understanding of the genetic control of LD activity and thermostability in malt.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86 571 86971115. Fax: +86 571 86971117. Funding

This study was supported by the Natural Science Foundation of China (31201166 and 31471480) and the Fundamental Research Funds for the Central Universities. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor D. Sun (Huazhong Agricultural University, China) for providing the Tibetan wild barley.

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REFERENCES

(1) Manners, D. J.; Marshall, J. J.; Yellowlees, D. The specificity of cereal limit dextrinase. Biochem. J. 1970, 116, 539−541. (2) Yang, X. Q.; Westcott, S.; Gong, X.; Evans, E.; Zhang, X. Q.; Lance, R. C. M.; Li, C. D. Amino acid substitutions of the limit dextrinase gene in barley are associated with enzyme thermostability. Mol. Breed. 2009, 23, 61−74. 3782

DOI: 10.1021/acs.jafc.5b00190 J. Agric. Food Chem. 2015, 63, 3778−3783

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DOI: 10.1021/acs.jafc.5b00190 J. Agric. Food Chem. 2015, 63, 3778−3783