QTL Mapping of Polyphenol Oxidase _PPO_ Activity and Yellow

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                               Thai Journal of Agricultural Science 2010, 43(2): 109-118

                        QTL Mapping of Polyphenol Oxidase (PPO) Activity
                       and Yellow Alkaline Noodle (YAN) Color Components
                           in an Australian Hexaploid Wheat Population

                                      A. Sadeque1,* and M.A. Turner2

                      Department of Industry and Investment, Narrabri, NSW, Australia
                    The University of Sydney, Plant Breeding Institute, Cobbitty, Australia

                       *Corresponding author. Email:

        Polyphenol oxidase (PPO) catalyses the undesirable darkening of wheat products such as Asian
     noodles. Genetic variation for PPO activity is present in breadwheat and low PPO activity is a
     current target of Australian wheat breeding programs. An improved understanding of the genetic
     control of PPO activity in wheat backgrounds that are relevant to yellow alkaline noodle (YAN)
     wheat production in northern NSW and QLD would expedite this process. QTL (Quantitative Trait
     Loci) mapping of polyphenol oxidase (PPO) activity was investigated in a doubled haploid (DH)
     population that was derived from a hybrid between a low PPO line DM5637*B8 and high PPO
     Australian wheat variety H45. These trials were conducted at The University of Sydney Plant
     Breeding Institute, Narrabri, NSW during the 2005 and 2006 growing seasons. In both seasons
     PPO activity was significantly (P<0.001) correlated with changes in brightness (∆L*) and
     yellowness (∆b*) of YAN. . QTLs on chromosome 2A (QPPO.dmh45-2AL, Q∆L*.dmh45-2AL,
     Q∆b*.dmh45-2AL) were associated with the characters at a high level of significance (P>0.001)
     and had LRS values of 59.9, 47.7 and 53.8, respectively. In each case the identified QTL explained
     more than 50% of the phenotypic variation of the traits. A highly significant (P<0.001) association
     was identified between marker locus wPt-7024 (chromosome 2A), and PPO activity, ∆L* and
     ∆b*. This information may be applied to increase the efficiency of breeding efforts that aim to
     generate improved YAN wheat.

     Keywords: polyphenol oxidase (PPO), doubled haploid (DH), QTL mapping, Asian noodle,
     YAN, colour

                   Introduction                          contributor to browning of noodles, chapattis and
                                                         other wheat products (Demeke et al., 2001a).
   Wheat flour noodles are an important part of the      Polyphenol oxidase (PPO) catalyses the oxidation
diet in many Asian countries. Noodles that are           of phenolic compounds to quinones, which undergo
bright and have stable color are preferred by            further rearrangement, non enzymatic oxidation,
consumers. Those that are commonly sold fresh are        and polymerization to produce dark or brown
sensitive to time dependent darkening which is           colored melanin (Whitaker and Lee, 1995; Demeke
undesirable (Baik et al., 1995; Hou and Kruk, 1998;      et al., 2001). Due to oxidation, this action causes
Hatcher et al., 1999; Jimenez and Dubcovsky,             discoloration of some wheat products including
1999). Oxidative enzymes such as polyphenol              Asian noodles (Kruger et al., 1992; Baik et al.,
oxidase (PPO), lipoxygenase (LOX), peroxidase            1995, Fuerst et al., 2006), pasta (Simeone et al.,
and catalase cause time dependant darkening of           2002), pan breads and steam breads (Dexter et al.,
wheat products (Feillet et al., 2000). PPO is a major    1984). Cultivars with low PPO activity are
110                                        A. Sadeque and M. A. Turner          Thai Journal of Agricultural Science

desirable for consumers and food manufacturers.            (AUS1408/Sunco), a line with low PPO activity,
The understanding of the influence of PPO activity         sprouting tolerance and low blackpoint incidence
on yellow alkaline noodle color is required by             (Howes pers. comm.) and Australian cultivar H45
breeders that are developing wheat cultivars that          (Ciano67/2*Olympic/3/WW80/3*Anza//Kalyonson
may be used as an input of yellow alkaline noodle          a/Bluebird) was used in this study. The doubled
manufacture. The influence of PPO activity on              haploid population was developed using the wheat
YAN darkening is known but mapping approaches              x maize system (Ahmed pers. comm.; Johnson et
concerned with YAN color and color stability have          al., 2005). For the PPO experiments, plants were
rarely been used in Australian wheats.                     sown in 3m rows and a sub set of 48 lines and
    Variation in PPO activity is reported between          parents were grown in 2 m x 6 m plots for the
wheat cultivars in different locations (Baik et al.,       yellow alkaline noodles (YAN) testing. A RCBD
1994; Park et al., 1997). The growing environment          design with 2 replications was employed for both
has an influence on PPO activity in wheat (Baik et         experiments. Trials were conducted at the Plant
al., 1994; Park 1997) and negatively impacts on the        Breeding Institute, Narrabri during the 2005 and
efficiency of selection for low PPO genotypes              2006 growing seasons.
(Demeke et al., 2001). Furthermore, PPO activity is
expressed in maternal tissue and it is difficult to        PPO Assay
select for low PPO activity early in the breeding              The grain PPO activity was determined by using
cycle using standard substrate assays in segregating       tyrosine as substrate for the assay procedure as
material. Because it might has the consequence of          described by Bernier and Howes (1994) and
reducing population size. Identification of                McCaig et al. (1999) except that the incubation
molecular markers that can select wheat lines with         period was increased from 2.5 to 3.5 h and the
low PPO phenotypes will enhance the efficiency of          optical density was determined at 450 nm by a
plant breeding programmes that have low PPO                Bench Mark Plus-Microplate Spectrophotometer
activity as a target. Loci on the long arm of              (BIORAD laboratories 2000, Alfred Nobel Drive,
chromosomes 2AL (Raman et al., 2005; Sun et al.,           Hercules, CA 94547). Twenty whole seeds for each
2005; Zang et al., 2005; He et al., 2007) and 2DL          line were incubated in 0.01 M disodium tyrosine
(Zang et al., 2005; He et al., 2007) in hexaploid          solution with 0.2% Tween 20 at 37oC for 3.5 h, and,
wheat have a major genetic effect on PPO activity          after leaving the seeds, the absorbance of the
in some genetic populations. Recently, He et al.           solution was measured at 450 nm.
(2007) found that a combination of markers
(PPO33/PPO16) for chromosomes 2A and 2D loci               Yellow Alkaline Noodles (YAN)
was an efficient and reliable means of selecting low          The yellow alkaline noodle evaluation
PPO genotypes. Hence, the present investigation            experiments for the crop grown in 2005 and 2006
aimed to identify the associations between PPO             were conducted in Western Wheat Quality
activity and yellow alkaline noodle color in a             Laboratory      (WWQL),     Washington     State
doubled haploid (DH) population derived from               University, USA and the Plant Breeding Institute,
DM5637*B8 (low PPO line) x H45 (high PPO                   University of Sydney (PBIN), Narrabri, NSW,
line). It also aimed to identify genomic regions that      Australia, respectively.
control PPO activity and color stability of YAN and
molecular markers that will be useful to select            Flour Milling
wheat lines with high color stability of yellow               Grains of the lines were milled using a
alkaline noodles.                                          Quadrumat Junior Mill (Brabender® OHG
                                                           DUISBURG, Germany). The flour extraction rate
             Materials and Methods                         was adjusted to 60 percent. Moisture and protein
                                                           contents were measured using Near Infrared
Plant Materials                                            Reflectance (NIR; Perten Instruments, Type
   A doubled haploid population (n = 187) that was         9100/01, Germany). The samples were conditioned
generated from a hybrid between DM5637*B8                  to 15% moisture overnight by adding the
Vol. 43, No.2, 2010        QTL mapping of PPO activity and YAN color in wheat population                        111

appropriate amount of water before milling. Flour           extraction buffer was prepared with 2% (w/v)
and bran yield was recorded for each sample.                CTAB (sigma), 1.4 M NaCl, 0.2% mM EDTA, 100
                                                            mM Tris-HCl, (pH 8.0).
Yellow Alkaline Noodle (YAN) Sheet
Preparation                                                 DArT Marker Analyses
    At WWQL, The noodle sheet was produced as                   2.5 µg of restriction quality DNA of each DH
described by Morris et al. (2000). The stock                genotype (n=92) and the parents of the population
solution was prepared by mixing 200 g NaCl with             DM5637*B8 x H45 was sent to Triticarte, Canberra
50 g of Na2CO3 in 1000 mL of H2O in a volumetric            (Australia; for whole
flask. Five mL of this stock solution containing 1 g        genome profiling using Diversity Array Technology.
of NaCl and 0.25 g of Na2CO3 was added to 50 g of           The 92 lines that were the subject of this study were
flour in this test. At PBIN, the stock solution was         randomly selected from the population. The loci were
prepared according to the method of Mares and               scored as present (1) or absent (0) and the locus
Campbell (2001) by mixing 25 g NaCl, 15 g KCO3              designations were as used by Triticarte Pty. Ltd. The
and 10 g Na2CO3 with 875 mL water. The yellow               P value reflects how well the two phases (present=1
alkaline noodle sheets were prepared by adding 10           vs absent=0) of the marker are separated in the
g of flour with 3.7 mL of alkaline noodles solution.        sample and P is based on ANOVA which is an
                                                            estimate of marker quality. Markers with P values 80,
Yellow Alkaline Noodle (YAN) Sheet and Flour                77-80, and 75-77 are termed as extremely reliable,
Color Measurement                                           usually scored, and provide useful information,
    Wheat noodle sheet color (CIE L*, a*, b*                respectively (Wenzl et al., 2004; Huttner et al., 2006).
denotes lightness which is white-black, red-green,
and yellow-blue scales, respectively) were measured         Linkage Map Construction and QTL Mapping
with a chromameter (Model-300, Minolta Camera                   The initial linkage mapping was performed with
Co., Ltd., Osaka Japan) with a 50 mm diameter               Cartablanche software, version 1.5.0(111), Keygene
measuring tube using a white tile background. The           Products B.V. Linkage groups were further
average of three readings, that were taken after            reassessed and reconstructed with Map Manager
moving the head each time, was employed in                  (QTXb20). The assignment of markers in linkage
analysis. Four color readings at 0, 2, 24and 48 h (2        groups for individual chromosome relied on the
h reading was not taken at WWQL) were taken.                consensus map of the Triticarte Ltd. and ongoing
Noodles sheets were stored in plastic bags at 21oC          construction of consensus map for the DArT assay
between color readings. The change in color values          (Akbari et al., 2006; Howes pers. comm.; http://
(CIE ∆L*,∆a* and ∆b*) was calculated by           
subtracting readings at 2, 24 and 48 h from those               Interval mapping was performed at P=0.01 for
immediately taken after noodle sheets were made             marker-trait association using Map Manager
(time zero). Values from duplicate noodle sheets            QTXb20 (Manly et al., 2001). Single marker
were used in all of the analyses.                           regression and simple interval mapping tools were
                                                            used for this purpose. The marker regression function
DNA Isolation                                               (P=0.01) was performed to find single marker loci
   Genomic DNA extractions were performed                   linked with the quantitative data. The analysis was
using a modified CTAB protocol according to                 carried out for each of the two individual
Doyle et al. (1990). 2.5µg of restriction quality           environments/years and for the pooled environments.
DNA of each DH genotype and the parents of the              The LRS thresholds in the regression analyses
DM5637*B8 x H45 population were prepared.                   P<0.01 and P<0.001 were used for declaring
Genomic DNA was extracted from fresh leaf tissue            significant and highly significant levels, respectively.
(0.5 to 1g) from a bulk of four plants. Leaves from         The Map Manager QTXb20 (Manly et al., 2001) and
three weeks old plants were taken. Leaves that were         MapChart version 2.2 (Voorrips 2002) were used to
dry, disease free and free from sticking soil               generate QTL map and chromosome map figures for
particles were selected for DNA extraction. CTAB            presentation.
112                                                                  A. Sadeque and M. A. Turner                                                   Thai Journal of Agricultural Science

                                    Results                                          brightness (L*) at 24 hours was clearly influenced
                                                                                     by PPO activity (i.e. lower PPO activity = YAN
PPO Assay                                                                            brightness) of the genotypes.
    The PPO activities determined for each of the
lines were consistent across years and were                                          Noodle Sheet Color Trait b*
significantly correlated (r2=0.38, P<0.001; Figure                                       The ∆b* values were significantly correlated
1). The low PPO parent, DM5637*B8 had                                                (r2=0.53 and 0.67, P<0.001) with PPO activity in
consistently low PPO activities in 2005 and 2006                                     2005 and 2006 (Figure 3). Results indicate that low
(A450 of 0.229 and 0.274, respectively). The PPO                                     PPO activity is associated with elevated ∆b* in this
activity of H45 was high in both seasons (A450 of                                    population.
0.400 and 0.559, respectively). The PPO activity
(A450) of lines ranged between 0.144 to 0.603 with a
population mean of 0.314 in 2005. In 2006, the A450
ranged from 0.164 to 0.698 and the mean for the                                                                                                                                    y = 0.78x + 0.12
population was 0.367. In both seasons the frequency                                                                0.70                                                                R2 = 0.38
distributions of PPO activity were near normal (data

                                                                                      PPO activty (A ) Year-2006

not shown). Transgressive segregation was evident                                                                  0.50
in this population. However, sixty nine (37%) lines

in the population possessed a significantly higher
PPO activity than the low PPO parent DM5637*B8                                                                     0.30

across seasons (LSD0.05=0.137).                                                                                    0.20

Noodle Sheet Color Trait L*                                                                                            0.10             0.20          0.30         0.40           0.50          0.60

   The correlation between PPO activity (A450) and                                                                                                     PPO activity (A 450) Year-2005

YAN (∆L* [0-24h]) was significant r2=0.52 and
                                                                                     Figure 1 Relationship between PPO activity of lines
0.78 (P<0.001) in 2005 and 2006, respectively                                        from DM5637*B8 x H45 DH population in 2005 and
(Figure 2). Results of both years revealed that YAN                                  2006 seasons.

                                                     (a) Year 2005                                                                    (b) Year 2006
                                    20                                                                              11
                                             y = 36.651 + 2.5528                                                            y = 27.549x - 0.1039
                                                  R 2 = 0.5217                                                      10          R 2 = 0.7785

                                                                                                 YAN L* (0-24h)
                   YAN L* (0-24h)






                                     6                                                                              3
                                      0.10             0.20         0.30      0.40                                   0.10              0.20         0.30          0.40

                                                     PPO activity (A 450)                                                            PPO activity (A 450)

                Figure 2 Relationship between PPO activities and YAN ∆L* (0-24h) in a set of lines from
                the DM5637*B8 x H45 DH population in (a) 2005 and (b) 2006 (n=50).
Vol. 43, No.2, 2010                                  QTL mapping of PPO activity and YAN color in wheat population                                            113

                                                            (a) Year 2005                                                        (b) Year 2006
                                                                                                                  0.10           0.20          0.30    0.40
                                             0.10             0.20           0.30    0.40
                                                    y = 29.367x - 12.791                                      1          y=17.748x - 6.7903
                                       -2               R 2 = 0.5277                                                       R 2 = 0.6697


                                                                                            YAN b* (0-24h)

                      YAN b* (0-24h)
                                       -6                                                                    -2



                                       -12                                                                   -6

                                                              PPO activity (A 450)                                              PPO activity (A 450)

               Figure 3. Relationship between PPO activity and YAN ∆b* (0-24h) in a set of lines
               derived from the DM5637*B8 x H45 DH population in (a) 2005 and (b) 2006 (n=50).

DArT Marker Screening                                                                           Three QTLs were detected in 2006, together
    The DNA of 92 lines and the two parents of the                                          accounting for 70% of variation in PPO activity
DM5637*B8 x H45 population were analysed by                                                 (Table 1). A highly significant (P<0.001) QTL on
DArT. Three hundred and eighty four polymorphic                                             chromosome 2AL was also associated with PPO
DArT markers were obtained with an overall call                                             activity in 2006. Marker locus wPt-7024, which
rate of 94.28%. The overall marker P values were                                            was located in the 2A QTL (QPPO.dmh45-2A) had
extremely reliable and ranged between 73.87 and                                             a highly significant association with PPO activity
98.84 with a mean of 89.63. Only 17 markers had P                                           (LRS = 59.9; P>0.001) and accounted for 50% of
values below 77. Of the 384 polymorphic markers                                             the observed variation. The QTL on 7BL
that were identified 317 markers were assembled                                             (QPPO.dmh45-7B) explained 10% of the
into 30 linkage groups using Cartablanche software,                                         phenotypic variation (LRS value of 9.5) and
version 1.5.0(111), Keygene Products B.V. and                                               corresponded with a QTL identified in 2005. In the
QTXb20. The remaining markers were unassigned.                                              2006 growing season, another QTL (QPPO.dmh45-
                                                                                            7A) was identified on chromosome 7A. It was not
QTL Analyses                                                                                detected in 2005. Marker locus wPt-0004 within
Polyphenol oxidase activity                                                                 this QTL was closely associated with PPO activity.
   Interval mapping analysis identified three                                               This marker had an LRS value of 8.9 (P>0.01) and
genomic regions that controlled PPO activity in the                                         explained 10% of the variation in PPO activity in
2005 trial (Table 1). A highly significant (P<0.001)                                        2006. The 4A QTL (QPPO.dmh45-7A) that was
(QTL (QPPO.dmh45-2AL)) was located on chromo-                                               identified in 2005 was not detected in 2006.
some 2AL (LRS=37.2, Figure 4) and explained                                                     The three QTLs that were identified in 2006
35% of phenotypic variation. The marker loci wPt-                                           (QPPO.dmh45-2AL; QPPO.dmh45-7A and QPPO.-
5865 and wPt-7024 in the 2AL QTL (QPPO.dmh45-                                               dmh45-7B) were also detected when analysing
2AL) were both significantly associated with PPO                                            pooled data across both seasons (Table 1). The
activity in this season. In 2005, significant QTLs                                          three QTLs together accounted for 71% of the
for PPO activity were also identified on chromo-                                            variation in PPO activity. The association of PPO
somes 4A (QPPO.dmh45-4A) and 7B (QPPO.dmh                                                   activity with markers wPt-5865 and wPt-7024 in
45-7B). The three QTLs that were identified                                                 the 2AL QTL was highly significant (P<0.001;
collectively explained 53% of the observed                                                  LRS of 63.7) and contributed 52% of the observed
phenotypic variation in this season (Table 1).                                              phenotypic variation. A suggestive QTL that was
114                                                   A. Sadeque and M. A. Turner                   Thai Journal of Agricultural Science

Table 1 Results of interval mapping for PPO activities (A450) in the DM5637*B8 x H45 DH population.

                     Chromosome                                                                                                Parent
       Site-year                                   QTLs                Closest marker      LRS value1/ PVE (%)2/
                       location                                                                                              (+ve PPO)
     Narrabri-2005        2AL               QPPO.dmh45-2AL               wPt-7024             37.2**            35               H45
                          4A                QPPO.dmh45-4A                wPt-7919              8.9*             10               H45
                          7B                QPPO.dmh45-7B                wPt-9925              7.1*              8               H45
     Narrabri-2006        2AL               QPPO.dmh45-2AL               wPt-7024             59.9**            50               H45
                          4A                QPPO.dmh45-4A                wPt-0004              8.9*             10               H45
                          7B                QPPO.dmh45-7B                wPt-9925              9.5*             10               H45
     Pooled               2AL               QPPO.dmh45-2AL               wPt-7024             63.7**            52               H45
                          4A                QPPO.dmh45-4A                wPt-7919              7.4*              8               H45
                          7B                QPPO.dmh45-7B                wPt-9925             10.2*             11               H45
   Likelihood statistic ratio; * and ** indicate significant and highly significant level at P<0.01 and P<0.001, respectively.
   Non asterisk LRS values indicate the suggestive level (P>0.05).
   Phenotypic variation explained.

      2AL                QPPO.dmh45-2AL                             Q∆L*.dmh45-2AL                           Q∆b*.dmh45-2AL

                            63.7                                     47.7                                      53.8

Figure 4 Likelihood Ratio Statistic (LRS) and additive effect plots of the QILs on chromosome 2AL for PPO activity,
stability of YAN brightness and yellowness, respectively. LRS scores and additive effect curves (solid and broken
respectively) are shown on the partial maps of chromosome. Vertical lines (green) from left to right indicate suggestive,
significant and highly significant levels of LRS values, respectively.

identified on chromosome 7A (QPPO.dmh45-7A)                             2005 (Table 2). The major QTL was located on
(LRS=7.4) explained 8% of phenotypic variation.                         chromosome 2AL (LRS=48.5) and explained 64%
Analysis of pooled data revealed that the 7B QTL                        of phenotypic variation. The QTL (Q∆L*.dmh45-
was significantly associated (P>0.01) with PPO                          1AS) on chromosome 1AS explained 27% of
activity (LRS=10.2) and explained 11% of                                phenotypic variation (LRS=13.1). QTL on
phenotype variation. In these QTLs the closest                          chromosome 1B (Q∆L*.dmh45-1B) and 7AL
identified markers were consistently identified                         (Q∆L*.dmh45-7A) were also detected. These QTL
across seasons. Interval mapping analysis revealed                      explained 14% (LRS=7) and 20% (LRS=10.7), of
that H45 contributed alleles of the three QTLs that                     phenotypic variation, respectively.
conferred elevated PPO activity.                                            In 2006, Q∆L*.dmh45-2AL was the only QTL
                                                                        detected (LRS value of 30.2) and it explained 47%
Yellow Alkaline Noodle Color Components                                 of variation. Analysis of pooled data detected all of
   Interval mapping analysis identified four                            the four QTLs that were identified in 2005 (Table
genomic regions that controlled ∆L* for YAN in                          2). Each was of a similar significance level to that
Vol. 43, No.2, 2010                  QTL mapping of PPO activity and YAN color in wheat population                                   115

Table 2 Results of interval mapping of yellow alkaline noodle (YAN) color stability CIE ∆L* and ∆b* at 0-24 hours in
the DM5637*B8 x H45 DH population.

                         Chromosome                                                                                        Contribution
        Site-year                                   QTLs               Closest marker       LRS value1/ PVE (%)2/
                           location                                                                                           parent
       YAN ∆L*           1AS               Q∆L*.dmh45-1AS              wPt-6455               13.1**           27          H45
                         1B                Q∆L*.dmh45-1B               wPt-0705                7*              14          DM5637*B8
                         2AL               Q∆L*.dmh45-2AL              wPt-7024               48.5**           64          DM5637*B8
                         7AL               Q∆L*.dmh45-7AL              wPt-0004               10.7*            20          H45
                         7BL               Q∆a*.dmh45-7BL              wPt-4140                6.8*            13          H45
         YAN ∆b*         1AS               Q∆b*.dmh45-1AS              wPt-6455                7.6*            17          H45
                         2AL               Q∆b*.dmh45-2AL              wPt-7024               47.1**           63          DM5637*B8
                         7AL               Q∆b*.dmh45-7AL              wPt-0004               12.1*            22          H45
       YAN ∆L*           2AL               Q∆L*.dmh45-2AL              wPt-7024               30.2**           47          DM5637*B8
       YAN ∆b*           2AL               Q∆b*.dmh45-2AL              wPt-7024               39.2**           57          DM5637*B8
                         7AL               Q∆b*.dmh45-7AL              wPt-0004                8.9*            17          H45
       YAN ∆L*           1AS               Q∆L*.dmh45-1AS              wPt-6455               12.5**           26          H45
                         1B                Q∆L*.dmh45-1B               wPt-0705                7.1*            14          DM5637*B8
                         2AL               Q∆L*.dmh45-2AL              wPt-7024               47.7**           64          DM5637*B8
                         7AL               Q∆L.dmh45-7AL               wPt-3226                8.8*            17          H45
         YAN ∆b*         1AS               Q∆b*.dmh45-1AS              wPt-6455                7.7*            17          H45
                         2AL               Q∆b*.dmh45-2AL              wPt-7024               53.8**           68          DM5637*B8
                         7AL               Q∆b*.dmh45-7AL              wPt-0004               12.3*            23          H45
     Likelihood statistic ratio; * and ** indicate significant and highly significant level at P<0.01 and P<0.001, respectively.
     Non asterisk LRS values indicate the suggestive level (P>0.05).
     Phenotypic variation explained.

for year 2005. Interval mapping analysis revealed a                        This QTL had a positive additive effect which was
positive additive affect of the Q∆L*.dmh45-1B and                          significantly associated with brightness stability of
the major QTL (Q∆L*.dmh45-2A) on chromosome                                yellow alkaline noodles that was contributed by the
2AL which indicated that parent DM5637*B8                                  parent DM5637*B8. Regression analysis identified
contributed a positive allele at each of these QTLs.                       that marker wPt-7024 was significantly (P<0.001)
The additive effect for the other QTLs                                     associated with ∆b*. The other significant QTL on
(Q∆L*.dmh45-1AS and Q∆L*.dmh45-7AL) were                                   chromosome arm 7AL was detected across the
negative in both growing seasons, indicating that                          growing seasons and accounted for 22% and 17%
parent H45 contributed the positive allele for the                         of phenotypic variation (LRS=12.1 and 8.9), in
QTLs.                                                                      2005 and 2006, respectively. This QTL explained
   QTL for YAN trait ∆b* were detected on                                  23% of variation across seasons. The marker wPt-
chromosomes 1AS (Q∆b*.dmh45-1AS), 2AL                                      0004 was closely linked to locus controlling this
(Q∆b*.dmh45-2AL) and 7AL (Q∆b*.dmh45-7AL)                                  QTL. The negative additive effect recorded for
were detected (Table 2). Q∆b*.dmh45-1AS was                                QTLs located on chromosomes 1AS and 7AL
detected only in 2005 and explained 17% of                                 indicate that the high PPO parent (H45) contributed
observed variation in that season (LRS=7.6). The                           the positive allele for these QTLs. In pooled
QTL on 2AL (Figure 4) and 7AL were detected in                             environments, the three QTLs (Q∆b*.dmh45-1AS,
both years. The 2AL was highly significant                                 Q∆b*.dmh45-2AL and Q∆b*.dmh45-7AL) were
(P<0.001) in both years, accounting for 63% and                            significant (P<0.01) explaining 17%, 68% and 23%
57% of variation in ∆b* (with LRS values of 47.1                           of phenotypic variation, respectively.
and 39.2 in 2005 and 2006, respectively, Figure 4).
116                                       A. Sadeque and M. A. Turner           Thai Journal of Agricultural Science

                    Discussion                            using DArT markers. A large amount of phenotypic
                                                          variation was explained by the QTLs for changes in
    Both ∆L* and ∆b* were significantly (P<0.01)          brightness, Q∆L*.dmh45-2AL, and yellowness,
correlated with PPO activity across growing               Q∆b*.dmh45-2AL, (64% and 68% respectively) on
seasons. The findings of this study are in agreement      chromosome 2A. Low PPO wheat line
with the reports of the role of PPO on time               DM5637*B8 exhibited color stability in both years.
dependent darkening (darkening due to aging) on           Not many studies concerned with mapping of color
yellow alkaline noodles (Kruger et al., 1994; Morris      stability of YAN in Australian wheat have been
1995; Fuerst et al., 2006).                               conducted and little information is available. Mares
    The results of the current study reconfirm that       and Campbell (2001) found a QTL on chromosome
PPO activity in wheat is mainly controlled by a           2D that was clearly associated with these traits and
locus on the long arm of chromosome 2A with               another on 2A which had some association. These
strong genetic association with DArT marker wPt-          findings are partly in agreement with findings of
7024. Other studies have identified that PPO              the current study. The marker locus wPt-7024 was
activity is strongly associated with the SSR marker       identified as an important DArT marker for all of
loci Xgwm312, Xgwm294 and WMC170 which are                the color stability traits of YAN, and PPO activity.
located on chromosome 2A (Sun et al., 2005;                  Correlations between PPO activity and change
Raman et al., 2005; Watanabe et al., 2006). The           in brightness were observed in this population.
chromosome 2A location is consistent with                 QTL mapping detected that DArT marker locus
previous findings (Sun et al., 2005; Raman et al.,        wPt-7024 on chromosome 2AL had the closest
2007) however it is not certain if the identified         genetic association with PPO activity and color
QTLs are in precisely the same location as                stability of both ∆L* and ∆b* (52% and 68% PVE,
identified in previous studies. The association of        respectively). QTL mapping results identified
DArT marker wPt-9925 on chromosome 7BL with               common locations of QTLs for both characters.
PPO activity in wheat has not yet been reported in        Also the parental contribution was consistent in
the literature. This QTL (QPPO.dmh45-7BL)                 each case. The DArT marker locus wPt-7024 had
explained 8-11% phenotypic variation across               the highest contribution to YAN color components
environments in consecutive years. QTLs on                ∆L* and ∆b*. Therefore, it can be concluded that
chromosomes 4A and 7A were detected in one                PPO activity is the major factor that influences
season (Narrabri 2005 and 2006, respectively). The        YAN color stability. DArT marker wPt-7024 was
QTL on 7A, was also detected when data was                associated with both YAN brightness and
pooled across environments. A QTL on                      yellowness along with PPO activity. In addition,
chromosome 7A was also detected in other studies          two potential QTLs for YAN ∆L* and ∆b* have
involving hexaploid wheat that were grown at the          been identified on chromosomes 1AS and 7AL.
same site (Sadeque 2008). Therefore, it is possible       The QTL on 7AL was detected across
that the minor QTL that is located on                     environments. A QTL on chromosome 7A was also
chromosmome 7A is sensitive to environmental              identified for PPO activity strengthening the
influence. The QTL QPPO.dmh45-4A explained                hypothesis that it is an important determinant of the
10% of variation in PPO activity in one season            change in YAN brightness. It is interesting that a
(Narrabri 2005). There are no published reports of        locus on 7B for PPO activity was identified in both
the presence of QTL on chromosome 4A, thus the            seasons but a corresponding QTL for change in
existence of QTL on chromosome 4A needs to be             brightness was not detected in this analysis.
confirmed. The analyses of allelic effects of the            The identification and mapping of PPO genes in
QTLs revealed that selection using markers within         wheat is an important prerequisite for developing
QPPO.dmh45-2AL may be an efficient means to               low PPO wheat cultivars that exhibit high levels of
select low PPO activity genotypes in this DH              color stability in wheat end products, especially for
population.                                               yellow alkaline and white salted noodles. This
    In the present study, QTL mapping of stability        study confirms that PPO activity in wheat is under
of YAN color components were also performed               polygenic control. The DArT marker wPt-7024 that
Vol. 43, No.2, 2010            QTL mapping of PPO activity and YAN color in wheat population                         117

is located within a QTL on chromosome 2AL and                   Fuerst, E.P., J.V. Anderson and C.F. Morris. 2006.
and has a high level of significance with PPO                      Delineating the role of polyphenol oxidase in the
                                                                   darkening of alkaline wheat noodles. J. Agric. Food
activity (P<0.001). In this study a new QTL                        Chem. 54: 2378-2384.
location on chromosome 7B was identified. A                     He, X.Y., Z.H. He, L.P. Zhang, D.J. Sun, C.F. Morris,
possible QTL location on chromosome 7A was also                    E.P. Fuerst and X.C. Xia. 2007. Allelic variation of
revealed in this study. The identification of these                polyphenol oxidase (PPO) genes chromosome 2A
                                                                   and 2D and development of functional for the PPO
QTLs and markers associated with PPO activity in
                                                                   genes in common wheat. Theor. Appl. Genet. 115:
this study will enable application of marker assisted              47-58.
selection in plant breeding programs of Eastern                 Huttner, E., P. Wenzyl, J. Carling, L. Xia, S. Yang, D.
Australia which is an efficient approach to pyramid                Jaccoud, V. Caig, M. Evers, G. Uszynsi, N. Howes,
genes for low PPO activity. The results of the                     P. Sharp, P. Vaughan, B. Rathmell and A. Kilian.
                                                                   2006. Triticarte: whole-genome profiling service for
current study confirmed earlier findings about the                 wheat and barley using Diversity Arrays Technology
involvement of some genomic regions in                             (DArT). Proceedings 13th Australasian Plant Breeding
controlling the traits investigated. In addition, new              Conference. 18-21 April, 2006. Christchurch, New
genomic regions and associated DArT markers                        Zealand.
                                                                Jaccoud, D., K. Peng, D. Feinstein and A. Kilian. 2001.
were identified that were linked to loci that control
                                                                   Diversity Arrays: a solid state technology for
wheat PPO and YAN color stability components.                      sequence information independent genotyping. Nucl.
                                                                   Acids Res. 29: e25.
                      Acknowledgments                           Jimenez, M. and J. Dubcovsky. 1999. Chromosome
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Sydney Plant Breeding Institute, Cobbitty, NSW,                 Johnson, C., A. Khan, A. Kilian, E. Huttner and D. Falk.
Australia and financed by The Value Added Wheat                    2005. Fundamentals of plant breeding and early-stage
CRC Ltd.                                                           genetic testing, pp. 27-44. In G.B. Fincher and C.
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                                                             Manuscript received 3 March 2010, accepted 30 September 2010

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