Banks PM

Christopher MJ

Jennings R

English

Sydney eScholarship

 1A comparison of methods for assessing polyphenol oxidase status of wheat  Jennings R, Christopher MJ and Banks PM Queensland Department of Primary Industries and Fisheries (DPI&F), Leslie Research Centre, Toowoomba, QLD 4350, Australia  ABSTRACT  Polyphenol oxidases are involved in the time-dependent darkening and discolouration of Asian noodles and other end products of wheat.  It is desirable to select low PPO wheats.  In this study we compared a catechol and an L-tyrosine method for assessing PPO activity in seed, using single or dual filter optical density readings or visual scores.  We compared these results with marker profiles for the QTL on chromosome 2AL previously shown by Raman et al to explain a large percentage of variation for this trait.  We observed lower absorbance readings using catechol compared with tyrosine. Dual filter readings gave a lower reading than single filter.  Catechol readings were generally more consistent than tyrosine between replicates. Correlations were generally high between single and dual reads.  Correlation between the catechol and tyrosine methods was high for visual scoring, but poorer for optical density readings.  In general visual assessment was better correlated with optical densities for the tyrosine method than with the catechol method.  The ranking of our three standards differed between tyrosine and catechol methods.  There was some suggestion that there may be some environmental influence on PPO levels expressed in the seed.  There was a near perfect correlation between visual scores after catechol treatment and marker scores, and a high correlation after tyrosine treatment, with only a few discrepancies in middle rated lines.  Our conclusion is that although each of these methods can be useful for selecting lines for low PPO in selection programs, where molecular markers are already being applied for selection, the application of markers is likely to be the most cost effective strategy for enriching a population for low PPO.     MATERIALS AND METHODS  Plant Materials:  Advanced breeding material from the EGA northern node breeding program was tested for PPO levels.  Two sets of lines, 134 lines drawn from an early maturing gene pool (‘earlies’) and 131 from a later maturing gene pool (‘mains’), were planted 24/05/06.  Two replicates of each set were planted at the Gatton Research Station, Qld.  Standard lines Sunco (low PPO), Giles (medium PPO) and QT12302 (high PPO) were selected based on consistency on ranking for PPO in preliminary experiments.  They were planted in each replicate at Gatton, and were also grown at the Leslie Research Centre (LRC), Toowoomba, Queensland, Australia.    PPO detection methods:  Two methods of seed treatment were used to visualise the PPO level of seed.  One method uses catechol as the PPO substrate, the other tyrosine (Mares pers. comm.).  Briefly, the catechol method involved incubating seed in a 10mM solution of catechol with Tween for 45 minutes.  The tyrosine method involved incubating seed in a 10mM solution of disodium tyrosine with 100mM Trizma base, pH adjusted to 9.0, with Tween for three hours.  The former method has been reported to be non-destructive. The latter is destructive, in that it reduces germination post-testing.  In each case testing was undertaken in microtitre plates, including blanks, standards and replicates.  To each well was added one seed and 200µl of catechol or tyrosine solution, which was incubated at 37°C, then 100µl of solution was transferred to a separate reading plate.  For each treatment three methods of measuring seed colouration were applied:  visual scoring (scored 1, 2 or 3 with increasing colour); a single optical density (OD) reading at 415nm; or a dual read with an OD at 415 with a 655nm reference filter. A Bio-Rad Microplate Reader Model 680 was used.  In total, each line was tested using eight seeds from two field replicates x two chemical treatments x three discoloration scoring methods.   Genotyping:  Genetic screening was conducted on a subset of 57 ‘earlies’ and 40 ‘mains’. DNA was extracted from 3 seeds per line, and each line assessed for two markers reported to flank the QTL on chromosome 2A associated with PPO levels, wmc170 and gwm312 (Raman et al., 2005).  Band sizing was conducted on a Beckman CEQ 880.    RESULTS AND DISCUSSION  Comparison of PPO staining methods Catechol gave a lower absorbance reading than tyrosine (Table 1.).  Catechol readings were more consistent within the eight individual grains tested per line and between field replicates.  Tyrosine gave a greater range of readings (data not shown).   The ranking of the standards differed between the two staining methods (Table 1).  Thus, there was a genotype by staining method interaction observed.    Comparison of PPO measurement methods Dual reads generally gave a lower reading than a single read (Table 1).  Single and dual read methods were highly correlated (Table 2).  Visual scores were only moderately correlated with either method (Table 2).  For each of the 4 staining by measurement method treatments, lines with a visual 1 rating had significantly  2 lower average scores, but differences between the average 2 and 3 visual ratings were small (Table 3).  Correlation between early and late genotype sets using different methods Table 2 shows the correlations between methods.  Catechol and tyrosine visual scores were highly correlated, but absorbance readings were only moderately correlated.  Within both the catechol and tyrosine staining methods, the two spectral measurement methods correlated well, as would be expected.  Correlations were higher amongst ‘earlies’ than ‘mains’ for all staining and scoring methods.  The two differences between the ‘earlies’ and ‘mains’ are the different conditions experienced during maturation of the grain and the different genepools from which these two sets have been drawn.  Either of these factors could be having an effect on the relationship between the underlying PPO content of the grain and accuracy with which this is measured using different methodologies.  For comparison between staining methods this may indicate a different relationship between the underlying PPO concentration and the accuracy with which the different methodologies measure this.  It is more difficult to explain the difference between ‘early’ and ‘main’ genotype sets in correlations between methods for measuring staining intensity.  This result suggests that there is either an environmental effect acting upon this trait, or that there are significant epistatic effects, perhaps involving maturity genes.   Table 1.  Mean PPO scores for the three standard genotypes were lower at LRC than at Gatton regardless of the staining or measurement method used.   Sunco rated “1”, Giles “2” and QT12302 was rated “3” on visual rating in each data set for each staining method.     Catechol - Single Catechol - Dual Tyrosine -Single Tyrosine - Dual Sunco:     LRC 0.072 0.059 0.261 0.167 Main Gatton 0.088 0.069 0.311 0.215 Early Gatton 0.084 0.070 0.337 0.232 Giles:     LRC 0.086 0.073 0.326 0.192 Main Gatton 0.095 0.112 0.408 0.260 Early Gatton 0.133 0.113 0.473 0.292 QT12302:     LRC 0.078 0.064 0.376 0.207 Main Gatton 0.107 0.094 0.497 0.277 Comparison of standards between environments  Standard lines, Sunco, Giles and QT12302 all showed lower PPO scores at the LRC site compared with the Gatton site regardless of staining or measurement method used (Table 1).  This supports the idea of an environmental effect on PPO.  The difference between PPO scores when harvested with the ‘mains’ or ‘earlies’ from Gatton was small.  This would be expected, since the individual standard lines would be maturing at the same time in the same environment, regardless of the set with which it was included.   Table 2.  ‘Mains’ showed a poorer correlation between methods of staining and between methods for measuring staining intensity compared with ‘earlies’.   Correlations Early Main Visual Scoring: Catechol v Tyrosine 0.80 0.78 Single Read Mode: Catechol v Tyrosine 0.46 0.32 Dual Read Mode: Catechol v Tyrosine 0.44 0.44 Catechol Method: Visual v Single Read 0.60 0.33 Catechol Method: Visual v Dual Read 0.62 0.45 Catechol Method: Single Read v Dual Read 0.98 0.77 Tyrosine Method: Visual v Single Read 0.70 0.56 Tyrosine Method: Visual v Dual Read 0.55 0.46 Tyrosine Method: Single Read v Dual Read 0.96 0.94  Comparison between molecular markers and staining methods A subset of 57 ‘earlies’ were screened with markers.  Using catechol staining and visual rating, 35 were rated 1 and 22 were rated either 2 or 3.  Marker scores for wmc170 correlated perfectly with visual catechol scores.  All lines expressing the A (low PPO) allele (wmc170227bp) had a visual rating of 1 and all lines expressing the B (high PPO) allele (wmc170214bp) had a visual rating of 2 or 3.  Marker scores for wmc170 also correlated well with visual tyrosine scores. Using tyrosine staining 29 lines were visually rated 1 and 28 were rated either 2 or 3.  Of the 35 lines expressing the A allele, 29 were rated 1 and six were rated 2. All lines expressing the B allele rated 2 or 3.   The lines followed the same pattern for the gwm312 marker, with all lines with a visual catechol rating of 1 expressing an A allele (either gwm312222bp or gwm312224bp), while those with a 2 or 3 rating expressed a B allele (either gwm312193bp or  gwm312191bp).  One heterozygous or mixed line rated 2.    A subset of 40 ‘mains’ were screened with markers of which 11 scored a visual rating of 1 and 29 has visual ratings of either 2 or 3.  All but one line expressing the A allele for wmc170 visually rated 1. All lines expressing the B allele visually rated 2 or 3.  For the gwm312 marker all but 2 lines with a visual rating of 1  3expressed an A allele, with all of those expressing a 2 or 3 rating expressed a B allele.    Table 3.  Average PPO scores for two staining methods and two visualisation methods showed that average rating was always lower for those rating a visual score of 1 than those rating a visual score of 2 or 3.   Visual Catecol-Visual Catecol-Single Catechol-Dual Tyrosine - Visual Tyrosine- SingleTyrosine- Dual Mains       1 0.088 0.076 0.318 0.222 0.088 0.076 2 0.116 0.101 0.447 0.274 0.116 0.101 3 0.116 0.101 0.446 0.269 0.116 0.101 Earlies       1 0.088 0.071 0.295 0.198 0.088 0.071 2 0.105 0.089 0.404 0.246 0.105 0.0893 0.111 0.094 0.415 0.251 0.111 0.094  For each of the 4 staining by measurement method treatments, lines carrying the A marker alleles on average had significantly lower scores when compared with those carrying the B marker alleles (Table 4).  In general then, markers correlated well with visual ratings for both staining methods.  Markers also correlated well with spectrally captured scores, identifying most of those rating low in PPO on two or more readings.  Table 4.  Average PPO scores for two staining methods and two visualisation methods showed that average rating was always lower for those expressing the A marker alleles than the B marker alleles.   Allele Catecol-Visual Catecol-Single Catechol- Dual Tyrosine - Visual Tyrosine - SingleTyrosine - Dual Mains       A 1.08 0.09 0.07 1.17 0.30 0.21 B 2.29 0.11 0.09 2.36 0.41 0.25 Earlies       A 1.00 0.08 0.07 1.17 0.35 0.23 B 2.32 0.12 0.10 2.09 0.47 0.28  Conclusions Each of these methods can be useful for selecting lines for low PPO in selection programs.  There was some indication that environment can affect PPO levels in seed.  The application of markers would overcome the influence of this on selection for lines with genes conferring low PPO.  Where molecular markers are already being applied for selection, the application of markers is likely to be the most cost effective strategy for enriching a population for low PPO.     ACKNOWLEDGEMENTS  Thanks to QDPI&F, Enterprise Grains Australia and GRDC for funding this work through the Australian Winter Cereals Molecular Marker Program.  REFERENCE  Raman, R., Raman, H., Johnstone, K., Lisle, C., Smith, A., Martin, P., Allen, H.  2005.  Genetic and in silico comparative mapping of the polyphenol oxidase gene in bread wheat (Triticum aestivum L.)  Funct. Integr. Genomics 5:  185-200. 

A comparison of methods for assessing polyphenol oxidase status of wheat

https://ses.library.usyd.edu.au/bitstream/2123/3170/1/P223.pdf

A comparison of methods for assessing polyphenol oxidase status of wheat

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