BACKGROUND
enzymatic browning is a phenomenon frequently observed in fruits and vegetables upon physical damage.
the tissue discoloration results from the oxidation of endogenous phenolic compounds, which is catalyzed by oxidoreductases, notably polyphenol oxidases  or tyrosinases in the presence of oxygen  <cit> .
besides the unappealing tissue pigmentation, enzymatic browning affects nutritional properties, flavour and texture of food and feed during storage or processing and is therefore detrimental to food quality.
mechanical impact during harvest, transport and storage of potato tubers initiates the development of an internal tissue discoloration .
blackspot bruising leads to the rejection of the crop by consumers, the retail and the processing industry, which results in considerable economic losses.
'after cooking darkening' is another problematic consequence of enzymatic browning.
when cells are damaged, phenolic compounds present in the vacuoles are oxidized to quinones by ppos located in the plastids .
the quinones polymerize non-enzymatically further into dark pigments , attributing a dark gray to black appearance to the normally white to yellowish tissue  <cit>  .
bruising symptoms emerge  <dig> to  <dig> hours after the impact and the damaged tissue area is only seen after removal of the tuber skin.
previous studies report that the discoloration reaction strongly depends on ppo enzyme activity  <cit> .
potato ppos are encoded by a small gene family of at least six members, namely potp <dig> and potp <dig>  <cit> , pot <dig>  pot <dig>  pot <dig>  and pot <dig>  <cit> , which show tissue specific expression.
in contrast to potp <dig> and potp <dig>  which are mainly expressed in leaves and flowers  <cit> , pot <dig> is considered to be the major form expressed in tubers besides pot <dig> and pot <dig>  silencing ppo gene expression resulted in transgenic potato lines that exhibited a significantly reduced discoloration reaction  <cit> .
this observation further points to the important role of ppo enzyme activity for bruising.
in addition, it has been shown that molecular markers derived from the pot <dig> sequence co-localise with a quantitative trait locus  for enzymatic discoloration on potato chromosome viii in a diploid potato mapping population  <cit> .
taken together, functional and genetic studies suggest ppos as major candidate genes for influencing tissue discoloration and tuber bruising.
the role of the cellular levels of the ppo substrates is less clear.
contrasting findings do not allow a clear statement on the impact on discoloration and tuber bruising of chlorogenic acid contents, the main phenolic compound in potato tubers, of its precursor tyrosine and the partitioning of tyrosine between tuber protein and the free amino acid pool  <cit> .
in addition to ppo and its substrates, reducing agents such as ascorbate and carotenoids as well as redox homeostasis influence tuber bruising  <cit> ; figure  <dig>  besides the enzymatic component, structural factors like cellular membrane stability, cell number and architecture also affect tuber bruising  <cit>  .
the latter characters not only influence tuber bruising but are also involved in other tuber traits like tuber shape, starch and sugar content of tubers, and tuber yield  <cit> .
starch accounts for 10-25% of the tuber fresh weight and is the major storage compound of potato tubers  <cit> .
the amount and steric properties of starch granules influence tuber starch content, which correlates with specific gravity  <cit>  and thereby affect cellular stability and tuber bruising.
the susceptibility of potato tubers to blackspot bruising is a complex trait, which depends on multiple genetic factors, developmental stage and environment  <cit> .
potato breeding programs aim at the selection of cultivars with improved genetic resistance to bruising as an important quality trait.
however, the phenotypic evaluation of bruising susceptibility is difficult.
it requires large amounts of tubers, which become only available after several years of vegetative multiplication.
multi-year and -location trials are needed to separate genetic variability from the environmental one.
this prevents the selection of low bruising cultivars early in the breeding cycle.
an additional problem is the correlation between blackspot bruising and specific gravity, which makes the selection of superior alleles for resistance to blackspot bruising independent from specific gravity difficult.
identifying the molecular basis of the genetic variation of bruising susceptibility will therefore facilitate the early selection of low bruising cultivars by means of molecular markers.
such markers can be used to diagnose and select for or against superior or inferior alleles of the genes themselves that control bruising in parents and progeny of breeding programs.
besides application in breeding, knowledge of which and how gene variants influence the bruising phenotype will contribute to understanding the complex molecular and cellular networks involved in enzymatic browning in plants.
the identification of quantitative trait loci  underlying complex phenotypic traits became feasible with the introduction of dna-based markers  <cit> .
since then, a number of genes and their allelic variants underlying plant qtl have been identified by positional cloning .
positional qtl cloning is a laborious and time-intensive process, which is feasible in inbreeding species such as arabidopsis and rice but is severely handicapped in polyploid, non-inbred species such as potato  <cit> .
moreover, the bruising phenotype itself is rather prohibitive for positional cloning due to the difficulties of the phenotypic evaluation as outlined above.
an alternative to positional qtl cloning is the candidate gene approach, which is based on the knowledge of a gene's function in controlling a character of interest on the one hand, and genetic co-localization of a functional candidate gene with qtl for the character of interest on the other  <cit> .
the investigation of dna variation at loci fulfilling these criteria in natural populations of individuals related by descent  <cit>  can lead to the detection of associations with positive or negative character values  <cit> .
finding such associations indicates that dna variation, either at the candidate locus itself or at a linked locus is causal for the phenotypic variation.
the extent of linkage disequilibrium  present in the population under study determines the amount of indirect associations due to physical linkage.
validation of a direct association between a candidate gene allele and a qtl effect can be achieved by quantitative complementation analysis  <cit> .
whether the association is direct or indirect, association mapping can produce diagnostic markers for superior alleles of genes influencing phenotypic variation, thereby empowering plant breeding.
to dissect the molecular basis of tuber bruising susceptibility and to identify diagnostic markers for "precision breeding", we conducted an association mapping study in a population of tetraploid genotypes used for potato variety development.
in addition to bruising, the population was phenotyped for specific gravity, yield, tuber shape and plant maturity, in order to determine the correlation of these traits with bruising susceptibility.
we genotyped the population with a series of dna-based markers developed from candidate gene sequences.
candidate genes  were selected from the scientific literature and de novo from comparative proteomics of bruising resistant versus bruising susceptible cultivars .
highly significant marker-trait associations were detected for bruising susceptibility and other traits, which are discussed with respect to the mechanisms underlying natural variation of enzymatic discoloration and tuber bruising.
RESULTS
phenotypic analysis
balanced phenotypic data from two years were obtained for  <dig> genotypes , except for yield  and maturity .
the phenotypic and genotypic data of the six data sets eu, sa, no, bs, ne and be, corresponding to location and breeder, were combined and constituted the data set all, which was used for the association analysis.
heritability  was high for all traits except yield.
apart from the ne data set, bruising index  as well as starch corrected bruising  showed a high overall heritability.
specific gravity  was characterized by high h <dig> values in every single data set.
low h <dig> values were identified for tuber shape  and plant maturity  in the no data set and in all single data sets concerning tuber yield  .
the phenotypic distributions  of bi, sg, scb, ty ts and pm in the all population are given in figure  <dig>  the all population showed a trend to bi below  <dig> points and sg between  <dig> and  <dig> percent .
in addition, genotypes were characterized by a mid plant maturity of about four or lower on the scale ranging from zero to nine .
correlation analyses of the phenotypic data pinpointed to a highly significant, positive correlation between bi and sg .
accordingly, the majority of the genotypes highly susceptible to bruising had a specific gravity of 15% and more .
scb did not correlate with sg, and genotypes with positive scb scores, indicating bruising susceptibility, were distributed evenly over all sg classes .
significant correlations were also observed between some other traits .
scb clearly correlated positively with bi and negatively with ts.
genotypes highly susceptible to bruising were characterized by elevated scb values and a trend of circular to ovoid tuber shape that also correlated with increasing sg.
pm correlated slightly but significantly with bi as well as with sg.
later maturing genotypes showed on average a higher specific gravity and elevated bruising susceptibility.
a very weak positive correlation between yield and tuber shape and a negative one between yield and sg were also identified.
na = not available; bs = bavaria saat; no = norika; eu = europlant; be = berding; sa = saka; ne = niehoff; all = combined data set
values were obtained using collected data in a population of  <dig> tetraploid potato genotypes in a two year trial at multiple environments.
* significant at α =  <dig> ; *** significant at α =  <dig> , ns = not significant
association analysis
first, population structure was analysed by principal coordinate analysis using the genotypic data of  <dig> ssr alleles scored at  <dig> loci  distributed on all twelve potato chromosomes.
no distinct subgroups were observed .
a total of  <dig> polymorphic dna fragments derived from  <dig> candidate genes and  <dig> ssr loci were scored in the  <dig> individuals .
dna polymorphisms were tested individually for association with all traits using a mixed model approach, which takes into account population structure and kinship, as well as by stepwise forward regression.
twenty one and eighty six marker-trait associations were found that were significant at p <  <dig>   and p <  <dig>  , respectively.
at p <  <dig> ,  <dig> polymorphic fragments that originated from five candidate and two ssr loci were associated with tuber bruising ).
interestingly, markers derived from ppo isoforms influenced tuber bruising in opposite directions, either decreasing or increasing bruising susceptibility compared to the population mean.
for example, the sscp marker pot32ps1-f with a frequency of only ten percent in the all population, decreased bi and scb by  <dig>  and  <dig>  points, respectively.
in contrast, the poloxa marker with a frequency of  <dig> percent increased bi and scb by  <dig>  and  <dig>  points, respectively .
markers from the starch phosphorylase pho1a were all associated with increased bruising susceptibility, whereas markers from lipase class iii, 4-coumarate coa ligase and hqt on average lowered bruising .
in addition, the ssr alleles sti024-e and sti013-a showed association with bi and sg but not with scb.
in contrast to the highly frequent sti024-e allele , which decreased bruising susceptibility and specific gravity , the sti013-a allele increased trait values when compared to population mean scores.
<dig> bi: bruising index; sg: specific gravity; scb: starch corrected bruising; ty: tuber yield.
<dig> frequency of presence of the marker fragment in the all population.
<dig> map position not known.
<dig> direction of effect either positive  or negative .
the candidate gene markers that showed highly significant association with sg were all derived from the starch phosphorylase genes pho1a and pho1b .
the pho1a-b fragment increased, whereas the pho1b-1a and pho1b-1b fragments decreased specific gravity.
in general, markers that were associated with both bi and sg showed a concerted allele effect, either increasing or decreasing bruising susceptibility and specific gravity .
this corresponds to the phenotypic correlation observed between these traits.
all pho1b markers associated with both bi and sg did not show association with scb.
markers specifically associated with tuber bruising , independent from specific gravity, originated from the candidate genes ppo , lipase class iii , hydroxycinnamoyl coa quinate transferase , starch phosphorylase l-type  and the ssr locus m <dig>  .
the ssr locus m <dig> is located in a cluster of protease inhibitor genes on potato chromosome iii  <cit> .
compared with the traits bi and sg, a lower number of markers were associated with tuber yield, shape and plant maturity.
for ts and pm, no markers associated at p <  <dig>  were identified.
the markers associated at p <  <dig>  are included in additional file  <dig>  seven markers that were associated with bi, sg and/or scb were also associated with ts .
in all cases, the direction of the effect on ts was opposite to the effect on bi, sg and scb, in concordance with the correlation of round tuber shape with increased bruising susceptibility and specific gravity.
two unique and highly significant associations were found for tuber yield.. a sscp fragment originating from l-galactono- <dig> -lactone dehydrogenase  was associated with a decrease in yield of  <dig>  dt/ha compared to the population mean.
this marker had a high allele frequency of 81% in the population .
hqt-f, another sscp fragment originating from hydroxycinnamoyl quinate coa transferase, increased tuber yield on average by  <dig>  dt/ha.
markers associated with bi, scb, sg, ts or pm had no effect on tuber yield with the exception of lipiii-27-a .
stepwise linear regression at p <  <dig>   resulted in the identification of three markers associated with bi, which originated from starch phosphorylase pho1a , 4-coumarate coa ligase  and the non coding ssr allele sti013-e. the three markers explained together  <dig> % of the genetic variation of this trait.
the ssr allele sti013-a in combination with two markers from the pho1b locus  and the lipiii-27-a marker derived from a putative class iii lipase explained  <dig> % of the genetic variation of sg.
in the case of scb, stepwise forward regression resulted in a single significant marker derived from the candidate gene hydroxycinnamoyl quinate coa transferase .
concerning yield, the marker fragment gldh-h was the only one significant at p <  <dig> .
at this significance threshold no markers were identified for the traits tuber shape and plant maturity.
<dig> direction of effect, either trait value increasing  or decreasing
<dig> bruising index , specific gravity , starch corrected bruising , tuber yield
<dig> associated marker fragment derived from corresponding candidate gene
<dig> amount of genetic variance explained by the sum of significant markers at p <  <dig>
discussion
this study aimed at elucidating the genetic basis of blackspot bruising in potato tubers in combination with several other important agronomical traits.
the approach was to associate dna polymorphisms at multiple functional candidate loci with natural phenotypic variation in advanced tetraploid varieties and breeding materials.
we identified highly significant  associations between dna variants at candidate as well as non-candidate  loci for tuber bruising, specific gravity and yield.
to the best of our knowledge, the association mapping experiment described in this paper is amongst most comprehensive and first that investigates the molecular basis of the natural variation of enzymatic browning in plants.
association genetics in model and non-model plants substantially improved the understanding of the molecular basis of various complex plant phenotypes and their impact on plant performance and economically essential traits  <cit> .
the human genetics field provides the models how association studies influence the development of novel strategies in medicine  <cit> .
in crop genetics, the combination of genomic tools with phenotypic evaluation as used in the traditional breeding process can identify molecular markers useful in "marker-assisted selection"  and at the same time genetic factors that influence complex traits such as pathogen resistance and crop quality  <cit> .
the experimental design  comprised the phenotypic evaluation of tuber bruising, specific gravity, yield and shape as well as plant maturity with standard methods used for variety selection at six different locations in two consecutive years.
similar to a previous association mapping experiment  <cit> , proprietary breeding clones were evaluated at the corresponding singular breeding station, whereas a common set of potato varieties was evaluated at all six breeding stations, which facilitated joint data analysis.
this experimental design makes possible to perform studies including proprietary breeding materials that otherwise would not be available, insuring that the associations found are relevant for elite germplasm.
marker-trait associations were detected using a mixed model approach, which took into account field design, environment, genotype by environment interaction, population structure and kinship.
an ssr marker based genetic similarity matrix was included in the mixed model to correct for population substructure that was not detectable by principal coordinate analysis.
stepwise forward regression led to a combination of associated markers that collectively explained between  <dig>  and  <dig>  percent of the genetic variation of the evaluated traits.
the phenotypic data analysis during this study led to several correlations that were observed between the investigated traits.
in this respect, the strong positive correlation between bruising susceptibility and specific gravity which is well known in breeding practice was confirmed.
in addition, the effects of all markers associated with both traits always had the same direction.
a comparable connection between tuber traits exists in the case of specific gravity and chip color, the latter being determined by the amount of reducing sugars in the tubers  <cit> .
one explanation for the correlation between tuber bruising susceptibility and sg is close genetic linkage between different genes that control both traits independently.
this might be the case for some qtl but is rather unlikely to be the case for all qtl with concerted effects on both traits that were tagged by the same marker.
an alternative explanation concerning the impact of specific gravity upon bruising might be the interaction of starch grains with the surrounding internal membrane ultrastructure.
thus, a high amyloplastic load with starch grains provokes a faster and easier rupture of amyloplastic membranes upon mechanical stress.
the loss of membrane integrity results on the one hand in the release of ppo and on the other hand in a mix-up of cytoplasmic and vacuolar compounds leading to the encounter of ppo with phenolic substrates  <cit> .
indeed, associations with tuber bruising and specific gravity were identified with markers derived from ppo genes as well as starch phosphorylase and lipase genes in concordance with this hypothesis.
l-type starch phosphorylases  are starch degrading enzymes present in plastids.
the gene pho1a  on potato chromosome iii is expressed in the amyloplasts, whereas pho1b  on chromosome v is expressed mainly in the chloroplasts  <cit> .
the net amount of starch in the plastids is the result of anabolic and katabolic enzymatic reactions, and carbon flux from source leaves to sink tubers.
markers originating from pho1a increased mainly bruising susceptibility and to less extent specific gravity.
vice versa, markers derived from pho1b were associated primarily with specific gravity and secondary with bruising.
the effects of pho1a and pho1b allelic variants on sg verify associations found previously at these two loci in an independent association mapping experiment  <cit> .
the observed concerted allele effects on bruising susceptibility and specific gravity also fit the hypothesis outlined above, which predicts that alleles increasing sg will indirectly increase bruising susceptibility due to a higher load of starch grains and thereby higher mechanical stress susceptibility.
sequence differences both at dna and/or amino acid level could alter biochemical or molecular characteristics of starch phosphorylases and in consequence lead to variation in specific gravity of genotypes characterized by a given allelic composition.
polyphenol oxidases are the major functional candidate genes for the enzymatic browning reaction.
accordingly, markers derived from at least three paralogous ppo genes clustered at the ppo locus on potato chromosome viii were associated with tuber bruising.
the markers poloxa and pot32ps1-f were highly significantly associated with scb, which makes them particularly interesting for mas aiming at increased bruising resistance not compromised by low specific gravity of tubers.
the association of the poloxa marker with increased bruising susceptibility  is especially intriguing, regarding the reported tissue specific expression of the potp <dig> gene that is high in leaf tissue but restricted in tubers  <cit> .
sequencing the poloxa amplicon revealed that the corresponding primer pair tags more than one ppo gene or allele .
the observed association might therefore result from a ppo gene other than potp <dig>  which is expressed in tubers.
selection against the rather frequent  poloxa marker should improve the average bruising resistance in a breeding population.
the single strand conformation polymorphism  fragment pot32ps1-f, was derived from the pot <dig> gene by subjecting the caps marker for allele  <dig> described by werijet al.
<cit>  to single strand conformation analysis.
pot <dig> allele  <dig> co-localized with a large effect qtl for enzymatic discoloration on chromosome viii and its presence increased enzymatic discoloration  <cit> .
the fact that scb is virtually uncoupled from the structural components of tuber bruising makes it more comparable to enzymatic discoloration catalyzed by ppo.
the pot32ps1-f marker decreased scb and therefore likely represents a novel ppo allele that was detected due to the higher resolution of sscp analysis and the use of tetraploid breeding material instead of the diploid mapping population employed by werij et al.
<cit> .
furthermore, a particularly interesting candidate gene is a putative class iii lipase, which has been discovered de novo by comparing the tuber proteome of bruising resistant and susceptible cultivars .
sscp markers derived from this gene showed significant associations with either bi, scb and ts  or bi and sg  or sg and ty  .
the marker lipiii-27-1h, present in 80% of the tetraploid individuals, was associated with reduced scb and a trend towards round shaped tubers.
a functional role of lipases in tuber bruising, specific gravity or shape is not obvious, apart from membrane modification by altering lipid composition.
recent work on the closest homolog of potato class iii lipases, the capla <dig> gene encoding a hot pepper phospholipase a <dig>  points to a possible connection with tuber bruising.
seo et al.
<cit>  proposed that capla <dig> is involved in the regulation of cell shape and number as well as the control of carbon flux through gluconeogenesis and β-oxidation.
in addition, it was postulated that the enzyme is involved in lipid signalling and thereby regulates cellular and biochemical functions in heterotrophic plant tissue.
the capla <dig> homologous potato unigene lipiii- <dig>  might fulfil analogous functions in heterotrophic tuber tissue, thereby indirectly influencing tuber bruising susceptibility and specific gravity.
a putative role in the regulation of cell shape and number could explain why the marker lipiii-27-1h associates with tuber shape as well.
the remaining candidate markers associated with tuber bruising and specific gravity can be classified as genes involved in polyphenol synthesis , redox homeostasis  and general carbohydrate and energy metabolism .
populations of cultivated, tetraploid potato are characterized by large haplotype blocs, resulting from a limited number of meiotic recombination events separating the individuals and/or selection  <cit> .
this is demonstrated by the finding of highly significant associations with ssr markers that did not encode any candidate gene.
large haplotype blocs are on the one hand favourable for the identification of diagnostic markers for breeding purposes but on the other hand prevent the verification of candidate genes by association genetics.
by using linear regression for the identification of significant marker trait association, marker redundancy could be limited.
the results concerning bruising validate the main candidate associations for polymorphic fragments derived from starch phosphorylase and 4-coumarate coa ligase genes as well as the non-coding ssr locus sti <dig> previously identified by the mixed model approach .
ppo markers significantly associated with bi using mixed model statistics were also tagged by linear regression when the probability threshold was increased to p <  <dig>  .
in the case of sg, markers with a significant impact on this trait by linear regression originate either from the pho1b gene or the non coding ssr locus sti <dig>  which also influenced bi.
regarding tuber yield and scb, linear regression resulted in the repeated selection of significantly associated marker fragments from the l-galactono- <dig> -lactone dehydrogenase and hydroxycinnamoyl coa quinate transferase genes, respectively.
these results strongly support the impact of candidate genes identified by both association analysis and linear regression on the analyzed traits.
the ambiguity whether a candidate gene variant is causal for the trait effect or whether the association observed is due to ld with the causal gene has to be resolved by further functional analysis of candidate gene alleles.
candidate gene alleles associated with positive or negative trait values can be isolated and functionally characterized in heterologous model systems such as yeast, arabidopsis or tobacco  <cit> .
multiple correlations between traits were evident in the investigated population.
most of these correlations can be explained on a physiological or genetic basis, as described above for tuber bruising susceptibility and specific gravity.
plant maturity was correlated with specific gravity, yield and bruising susceptibility.
this might be due to a longer vegetation period  that results consequently in higher tuber yield and specific gravity, and indirectly in higher bruising.
the reason for the negative correlation between tuber shape and bruising is probably physical.
the surface of round shaped tubers is more exposed to mechanical impact than oblong tubers.
the interconnection of the traits investigated and the associated marker fragments identified are a valuable resource for breeders and molecular biologists dealing with enzymatic tissue discoloration and in particular potato tuber bruising.
functional studies will have to provide further evidence on whether and how sequence diversity of candidate genes results in functional differences and thereby explains phenotypic variation.
the present work is the result of a collaboration between the breeding industry and basic research and accentuates the efforts to transfer the genetic and molecular knowledge gained during such studies into the field.
CONCLUSIONS
in conclusion, our study demonstrates that association genetics based on candidate genes in advanced breeding populations of potato allows the identification of diagnostic markers that are valuable for mas, especially for complex traits such as tuber bruising.
twenty one highly significant  and robust marker-trait associations were identified that will facilitate the diagnosis and combination of superior alleles in breeding programs.
for example, selection against the rather frequent poloxa allele and enrichment of the rare pot <dig> allele  associated with higher and lower bruising susceptibility, respectively, should improve tuber quality with respect to blackspot bruising.
the associations found with certain candidate genes such as lipase class iii point to new structural and biochemical properties that play a role in bruising.
the identified candidate genes and their associations with enzymatic discoloration and bruising are a basis to elucidate the underlying molecular networks and the sequence diversity at the given loci, not only in potato but also in other fruits and vegetables that suffer from mechanical damage.

 enzymatic browning is a phenomenon frequently observed in fruits and vegetables upon physical damage. the tissue discoloration results from the oxidation of endogenous phenolic compounds, which is catalyzed by oxidoreductases, notably polyphenol oxidases or tyrosinases in the presence of oxygen. besides the unappealing tissue pigmentation, enzymatic browning affects nutritional properties, flavour and texture of food and feed during storage or processing and is therefore detrimental to food quality. mechanical impact during harvest, transport and storage of potato tubers initiates the development of an internal tissue discoloration. blackspot bruising leads to the rejection of the crop by consumers, the retail and the processing industry, which results in considerable economic losses.'after cooking darkening' is another problematic consequence of enzymatic browning. when cells are damaged, phenolic compounds present in the vacuoles are oxidized to quinones by ppos located in the plastids. the quinones polymerize non-enzymatically further into dark pigments, attributing a dark gray to black appearance to the normally white to yellowish tissue. bruising symptoms emerge to hours after the impact and the damaged tissue area is only seen after removal of the tuber skin. previous studies report that the discoloration reaction strongly depends on ppo enzyme activity. potato ppos are encoded by a small gene family of at least six members, namely potp and potp, pot pot pot and pot, which show tissue specific expression. in contrast to potp and potp which are mainly expressed in leaves and flowers, pot is considered to be the major form expressed in tubers besides pot and pot silencing ppo gene expression resulted in transgenic potato lines that exhibited a significantly reduced discoloration reaction. this observation further points to the important role of ppo enzyme activity for bruising. in addition, it has been shown that molecular markers derived from the pot sequence co-localise with a quantitative trait locus for enzymatic discoloration on potato chromosome viii in a diploid potato mapping population. taken together, functional and genetic studies suggest ppos as major candidate genes for influencing tissue discoloration and tuber bruising. the role of the cellular levels of the ppo substrates is less clear. contrasting findings do not allow a clear statement on the impact on discoloration and tuber bruising of chlorogenic acid contents, the main phenolic compound in potato tubers, of its precursor tyrosine and the partitioning of tyrosine between tuber protein and the free amino acid pool. in addition to ppo and its substrates, reducing agents such as ascorbate and carotenoids as well as redox homeostasis influence tuber bruising; figure besides the enzymatic component, structural factors like cellular membrane stability, cell number and architecture also affect tuber bruising. the latter characters not only influence tuber bruising but are also involved in other tuber traits like tuber shape, starch and sugar content of tubers, and tuber yield. starch accounts for 10-25% of the tuber fresh weight and is the major storage compound of potato tubers. the amount and steric properties of starch granules influence tuber starch content, which correlates with specific gravity and thereby affect cellular stability and tuber bruising. the susceptibility of potato tubers to blackspot bruising is a complex trait, which depends on multiple genetic factors, developmental stage and environment. potato breeding programs aim at the selection of cultivars with improved genetic resistance to bruising as an important quality trait. however, the phenotypic evaluation of bruising susceptibility is difficult. it requires large amounts of tubers, which become only available after several years of vegetative multiplication. multi-year and -location trials are needed to separate genetic variability from the environmental one. this prevents the selection of low bruising cultivars early in the breeding cycle. an additional problem is the correlation between blackspot bruising and specific gravity, which makes the selection of superior alleles for resistance to blackspot bruising independent from specific gravity difficult. identifying the molecular basis of the genetic variation of bruising susceptibility will therefore facilitate the early selection of low bruising cultivars by means of molecular markers. such markers can be used to diagnose and select for or against superior or inferior alleles of the genes themselves that control bruising in parents and progeny of breeding programs. besides application in breeding, knowledge of which and how gene variants influence the bruising phenotype will contribute to understanding the complex molecular and cellular networks involved in enzymatic browning in plants. the identification of quantitative trait loci underlying complex phenotypic traits became feasible with the introduction of dna-based markers. since then, a number of genes and their allelic variants underlying plant qtl have been identified by positional cloning. positional qtl cloning is a laborious and time-intensive process, which is feasible in inbreeding species such as arabidopsis and rice but is severely handicapped in polyploid, non-inbred species such as potato. moreover, the bruising phenotype itself is rather prohibitive for positional cloning due to the difficulties of the phenotypic evaluation as outlined above. an alternative to positional qtl cloning is the candidate gene approach, which is based on the knowledge of a gene's function in controlling a character of interest on the one hand, and genetic co-localization of a functional candidate gene with qtl for the character of interest on the other. the investigation of dna variation at loci fulfilling these criteria in natural populations of individuals related by descent can lead to the detection of associations with positive or negative character values. finding such associations indicates that dna variation, either at the candidate locus itself or at a linked locus is causal for the phenotypic variation. the extent of linkage disequilibrium present in the population under study determines the amount of indirect associations due to physical linkage. validation of a direct association between a candidate gene allele and a qtl effect can be achieved by quantitative complementation analysis. whether the association is direct or indirect, association mapping can produce diagnostic markers for superior alleles of genes influencing phenotypic variation, thereby empowering plant breeding. to dissect the molecular basis of tuber bruising susceptibility and to identify diagnostic markers for "precision breeding", we conducted an association mapping study in a population of tetraploid genotypes used for potato variety development. in addition to bruising, the population was phenotyped for specific gravity, yield, tuber shape and plant maturity, in order to determine the correlation of these traits with bruising susceptibility. we genotyped the population with a series of dna-based markers developed from candidate gene sequences. candidate genes were selected from the scientific literature and de novo from comparative proteomics of bruising resistant versus bruising susceptible cultivars. highly significant marker-trait associations were detected for bruising susceptibility and other traits, which are discussed with respect to the mechanisms underlying natural variation of enzymatic discoloration and tuber bruising.  phenotypic analysis balanced phenotypic data from two years were obtained for genotypes, except for yield and maturity. the phenotypic and genotypic data of the six data sets eu, sa, no, bs, ne and be, corresponding to location and breeder, were combined and constituted the data set all, which was used for the association analysis. heritability was high for all traits except yield. apart from the ne data set, bruising index as well as starch corrected bruising showed a high overall heritability. specific gravity was characterized by high h values in every single data set. low h values were identified for tuber shape and plant maturity in the no data set and in all single data sets concerning tuber yield. the phenotypic distributions of bi, sg, scb, ty ts and pm in the all population are given in figure the all population showed a trend to bi below points and sg between and percent. in addition, genotypes were characterized by a mid plant maturity of about four or lower on the scale ranging from zero to nine. correlation analyses of the phenotypic data pinpointed to a highly significant, positive correlation between bi and sg. accordingly, the majority of the genotypes highly susceptible to bruising had a specific gravity of 15% and more. scb did not correlate with sg, and genotypes with positive scb scores, indicating bruising susceptibility, were distributed evenly over all sg classes. significant correlations were also observed between some other traits. scb clearly correlated positively with bi and negatively with ts. genotypes highly susceptible to bruising were characterized by elevated scb values and a trend of circular to ovoid tuber shape that also correlated with increasing sg. pm correlated slightly but significantly with bi as well as with sg. later maturing genotypes showed on average a higher specific gravity and elevated bruising susceptibility. a very weak positive correlation between yield and tuber shape and a negative one between yield and sg were also identified. na = not available; bs = bavaria saat; no = norika; eu = europlant; be = berding; sa = saka; ne = niehoff; all = combined data set values were obtained using collected data in a population of tetraploid potato genotypes in a two year trial at multiple environments. * significant at α =; *** significant at α =, ns = not significant association analysis first, population structure was analysed by principal coordinate analysis using the genotypic data of ssr alleles scored at loci distributed on all twelve potato chromosomes. no distinct subgroups were observed. a total of polymorphic dna fragments derived from candidate genes and ssr loci were scored in the individuals. dna polymorphisms were tested individually for association with all traits using a mixed model approach, which takes into account population structure and kinship, as well as by stepwise forward regression. twenty one and eighty six marker-trait associations were found that were significant at p < and p <, respectively. at p <, polymorphic fragments that originated from five candidate and two ssr loci were associated with tuber bruising ). interestingly, markers derived from ppo isoforms influenced tuber bruising in opposite directions, either decreasing or increasing bruising susceptibility compared to the population mean. for example, the sscp marker pot32ps1-f with a frequency of only ten percent in the all population, decreased bi and scb by and points, respectively. in contrast, the poloxa marker with a frequency of percent increased bi and scb by and points, respectively. markers from the starch phosphorylase pho1a were all associated with increased bruising susceptibility, whereas markers from lipase class iii, 4-coumarate coa ligase and hqt on average lowered bruising. in addition, the ssr alleles sti024-e and sti013-a showed association with bi and sg but not with scb. in contrast to the highly frequent sti024-e allele, which decreased bruising susceptibility and specific gravity, the sti013-a allele increased trait values when compared to population mean scores. bi: bruising index; sg: specific gravity; scb: starch corrected bruising; ty: tuber yield. frequency of presence of the marker fragment in the all population. map position not known. direction of effect either positive or negative. the candidate gene markers that showed highly significant association with sg were all derived from the starch phosphorylase genes pho1a and pho1b. the pho1a-b fragment increased, whereas the pho1b-1a and pho1b-1b fragments decreased specific gravity. in general, markers that were associated with both bi and sg showed a concerted allele effect, either increasing or decreasing bruising susceptibility and specific gravity. this corresponds to the phenotypic correlation observed between these traits. all pho1b markers associated with both bi and sg did not show association with scb. markers specifically associated with tuber bruising, independent from specific gravity, originated from the candidate genes ppo, lipase class iii, hydroxycinnamoyl coa quinate transferase, starch phosphorylase l-type and the ssr locus m. the ssr locus m is located in a cluster of protease inhibitor genes on potato chromosome iii. compared with the traits bi and sg, a lower number of markers were associated with tuber yield, shape and plant maturity. for ts and pm, no markers associated at p < were identified. the markers associated at p < are included in additional file seven markers that were associated with bi, sg and/or scb were also associated with ts. in all cases, the direction of the effect on ts was opposite to the effect on bi, sg and scb, in concordance with the correlation of round tuber shape with increased bruising susceptibility and specific gravity. two unique and highly significant associations were found for tuber yield.. a sscp fragment originating from l-galactono- -lactone dehydrogenase was associated with a decrease in yield of dt/ha compared to the population mean. this marker had a high allele frequency of 81% in the population. hqt-f, another sscp fragment originating from hydroxycinnamoyl quinate coa transferase, increased tuber yield on average by dt/ha. markers associated with bi, scb, sg, ts or pm had no effect on tuber yield with the exception of lipiii-27-a. stepwise linear regression at p < resulted in the identification of three markers associated with bi, which originated from starch phosphorylase pho1a, 4-coumarate coa ligase and the non coding ssr allele sti013-e. the three markers explained together % of the genetic variation of this trait. the ssr allele sti013-a in combination with two markers from the pho1b locus and the lipiii-27-a marker derived from a putative class iii lipase explained % of the genetic variation of sg. in the case of scb, stepwise forward regression resulted in a single significant marker derived from the candidate gene hydroxycinnamoyl quinate coa transferase. concerning yield, the marker fragment gldh-h was the only one significant at p <. at this significance threshold no markers were identified for the traits tuber shape and plant maturity. direction of effect, either trait value increasing or decreasing bruising index, specific gravity, starch corrected bruising, tuber yield associated marker fragment derived from corresponding candidate gene amount of genetic variance explained by the sum of significant markers at p < discussion this study aimed at elucidating the genetic basis of blackspot bruising in potato tubers in combination with several other important agronomical traits. the approach was to associate dna polymorphisms at multiple functional candidate loci with natural phenotypic variation in advanced tetraploid varieties and breeding materials. we identified highly significant associations between dna variants at candidate as well as non-candidate loci for tuber bruising, specific gravity and yield. to the best of our knowledge, the association mapping experiment described in this paper is amongst most comprehensive and first that investigates the molecular basis of the natural variation of enzymatic browning in plants. association genetics in model and non-model plants substantially improved the understanding of the molecular basis of various complex plant phenotypes and their impact on plant performance and economically essential traits. the human genetics field provides the models how association studies influence the development of novel strategies in medicine. in crop genetics, the combination of genomic tools with phenotypic evaluation as used in the traditional breeding process can identify molecular markers useful in "marker-assisted selection" and at the same time genetic factors that influence complex traits such as pathogen resistance and crop quality. the experimental design comprised the phenotypic evaluation of tuber bruising, specific gravity, yield and shape as well as plant maturity with standard methods used for variety selection at six different locations in two consecutive years. similar to a previous association mapping experiment, proprietary breeding clones were evaluated at the corresponding singular breeding station, whereas a common set of potato varieties was evaluated at all six breeding stations, which facilitated joint data analysis. this experimental design makes possible to perform studies including proprietary breeding materials that otherwise would not be available, insuring that the associations found are relevant for elite germplasm. marker-trait associations were detected using a mixed model approach, which took into account field design, environment, genotype by environment interaction, population structure and kinship. an ssr marker based genetic similarity matrix was included in the mixed model to correct for population substructure that was not detectable by principal coordinate analysis. stepwise forward regression led to a combination of associated markers that collectively explained between and percent of the genetic variation of the evaluated traits. the phenotypic data analysis during this study led to several correlations that were observed between the investigated traits. in this respect, the strong positive correlation between bruising susceptibility and specific gravity which is well known in breeding practice was confirmed. in addition, the effects of all markers associated with both traits always had the same direction. a comparable connection between tuber traits exists in the case of specific gravity and chip color, the latter being determined by the amount of reducing sugars in the tubers. one explanation for the correlation between tuber bruising susceptibility and sg is close genetic linkage between different genes that control both traits independently. this might be the case for some qtl but is rather unlikely to be the case for all qtl with concerted effects on both traits that were tagged by the same marker. an alternative explanation concerning the impact of specific gravity upon bruising might be the interaction of starch grains with the surrounding internal membrane ultrastructure. thus, a high amyloplastic load with starch grains provokes a faster and easier rupture of amyloplastic membranes upon mechanical stress. the loss of membrane integrity results on the one hand in the release of ppo and on the other hand in a mix-up of cytoplasmic and vacuolar compounds leading to the encounter of ppo with phenolic substrates. indeed, associations with tuber bruising and specific gravity were identified with markers derived from ppo genes as well as starch phosphorylase and lipase genes in concordance with this hypothesis. l-type starch phosphorylases are starch degrading enzymes present in plastids. the gene pho1a on potato chromosome iii is expressed in the amyloplasts, whereas pho1b on chromosome v is expressed mainly in the chloroplasts. the net amount of starch in the plastids is the result of anabolic and katabolic enzymatic reactions, and carbon flux from source leaves to sink tubers. markers originating from pho1a increased mainly bruising susceptibility and to less extent specific gravity. vice versa, markers derived from pho1b were associated primarily with specific gravity and secondary with bruising. the effects of pho1a and pho1b allelic variants on sg verify associations found previously at these two loci in an independent association mapping experiment. the observed concerted allele effects on bruising susceptibility and specific gravity also fit the hypothesis outlined above, which predicts that alleles increasing sg will indirectly increase bruising susceptibility due to a higher load of starch grains and thereby higher mechanical stress susceptibility. sequence differences both at dna and/or amino acid level could alter biochemical or molecular characteristics of starch phosphorylases and in consequence lead to variation in specific gravity of genotypes characterized by a given allelic composition. polyphenol oxidases are the major functional candidate genes for the enzymatic browning reaction. accordingly, markers derived from at least three paralogous ppo genes clustered at the ppo locus on potato chromosome viii were associated with tuber bruising. the markers poloxa and pot32ps1-f were highly significantly associated with scb, which makes them particularly interesting for mas aiming at increased bruising resistance not compromised by low specific gravity of tubers. the association of the poloxa marker with increased bruising susceptibility is especially intriguing, regarding the reported tissue specific expression of the potp gene that is high in leaf tissue but restricted in tubers. sequencing the poloxa amplicon revealed that the corresponding primer pair tags more than one ppo gene or allele. the observed association might therefore result from a ppo gene other than potp which is expressed in tubers. selection against the rather frequent poloxa marker should improve the average bruising resistance in a breeding population. the single strand conformation polymorphism fragment pot32ps1-f, was derived from the pot gene by subjecting the caps marker for allele described by werijet al. to single strand conformation analysis. pot allele co-localized with a large effect qtl for enzymatic discoloration on chromosome viii and its presence increased enzymatic discoloration. the fact that scb is virtually uncoupled from the structural components of tuber bruising makes it more comparable to enzymatic discoloration catalyzed by ppo. the pot32ps1-f marker decreased scb and therefore likely represents a novel ppo allele that was detected due to the higher resolution of sscp analysis and the use of tetraploid breeding material instead of the diploid mapping population employed by werij et al.. furthermore, a particularly interesting candidate gene is a putative class iii lipase, which has been discovered de novo by comparing the tuber proteome of bruising resistant and susceptible cultivars. sscp markers derived from this gene showed significant associations with either bi, scb and ts or bi and sg or sg and ty. the marker lipiii-27-1h, present in 80% of the tetraploid individuals, was associated with reduced scb and a trend towards round shaped tubers. a functional role of lipases in tuber bruising, specific gravity or shape is not obvious, apart from membrane modification by altering lipid composition. recent work on the closest homolog of potato class iii lipases, the capla gene encoding a hot pepper phospholipase a points to a possible connection with tuber bruising. seo et al. proposed that capla is involved in the regulation of cell shape and number as well as the control of carbon flux through gluconeogenesis and β-oxidation. in addition, it was postulated that the enzyme is involved in lipid signalling and thereby regulates cellular and biochemical functions in heterotrophic plant tissue. the capla homologous potato unigene lipiii- might fulfil analogous functions in heterotrophic tuber tissue, thereby indirectly influencing tuber bruising susceptibility and specific gravity. a putative role in the regulation of cell shape and number could explain why the marker lipiii-27-1h associates with tuber shape as well. the remaining candidate markers associated with tuber bruising and specific gravity can be classified as genes involved in polyphenol synthesis, redox homeostasis and general carbohydrate and energy metabolism. populations of cultivated, tetraploid potato are characterized by large haplotype blocs, resulting from a limited number of meiotic recombination events separating the individuals and/or selection. this is demonstrated by the finding of highly significant associations with ssr markers that did not encode any candidate gene. large haplotype blocs are on the one hand favourable for the identification of diagnostic markers for breeding purposes but on the other hand prevent the verification of candidate genes by association genetics. by using linear regression for the identification of significant marker trait association, marker redundancy could be limited. the results concerning bruising validate the main candidate associations for polymorphic fragments derived from starch phosphorylase and 4-coumarate coa ligase genes as well as the non-coding ssr locus sti previously identified by the mixed model approach. ppo markers significantly associated with bi using mixed model statistics were also tagged by linear regression when the probability threshold was increased to p <. in the case of sg, markers with a significant impact on this trait by linear regression originate either from the pho1b gene or the non coding ssr locus sti which also influenced bi. regarding tuber yield and scb, linear regression resulted in the repeated selection of significantly associated marker fragments from the l-galactono- -lactone dehydrogenase and hydroxycinnamoyl coa quinate transferase genes, respectively. these results strongly support the impact of candidate genes identified by both association analysis and linear regression on the analyzed traits. the ambiguity whether a candidate gene variant is causal for the trait effect or whether the association observed is due to ld with the causal gene has to be resolved by further functional analysis of candidate gene alleles. candidate gene alleles associated with positive or negative trait values can be isolated and functionally characterized in heterologous model systems such as yeast, arabidopsis or tobacco. multiple correlations between traits were evident in the investigated population. most of these correlations can be explained on a physiological or genetic basis, as described above for tuber bruising susceptibility and specific gravity. plant maturity was correlated with specific gravity, yield and bruising susceptibility. this might be due to a longer vegetation period that results consequently in higher tuber yield and specific gravity, and indirectly in higher bruising. the reason for the negative correlation between tuber shape and bruising is probably physical. the surface of round shaped tubers is more exposed to mechanical impact than oblong tubers. the interconnection of the traits investigated and the associated marker fragments identified are a valuable resource for breeders and molecular biologists dealing with enzymatic tissue discoloration and in particular potato tuber bruising. functional studies will have to provide further evidence on whether and how sequence diversity of candidate genes results in functional differences and thereby explains phenotypic variation. the present work is the result of a collaboration between the breeding industry and basic research and accentuates the efforts to transfer the genetic and molecular knowledge gained during such studies into the field.  in conclusion, our study demonstrates that association genetics based on candidate genes in advanced breeding populations of potato allows the identification of diagnostic markers that are valuable for mas, especially for complex traits such as tuber bruising. twenty one highly significant and robust marker-trait associations were identified that will facilitate the diagnosis and combination of superior alleles in breeding programs. for example, selection against the rather frequent poloxa allele and enrichment of the rare pot allele associated with higher and lower bruising susceptibility, respectively, should improve tuber quality with respect to blackspot bruising. the associations found with certain candidate genes such as lipase class iii point to new structural and biochemical properties that play a role in bruising. the identified candidate genes and their associations with enzymatic discoloration and bruising are a basis to elucidate the underlying molecular networks and the sequence diversity at the given loci, not only in potato but also in other fruits and vegetables that suffer from mechanical damage. 
association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. tuber susceptibility to bruising is a complex trait of the cultivated potato that is crucial for crop quality. twenty one highly significant and robust marker-trait associations were identified. the genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. in addition, this study presents novel candidates that might control enzymatic tissue discoloration and tuber bruising. one such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact. the markers diagnostic for increased or decreased bruising susceptibility will facilitate the combination of superior alleles in breeding programs. in total polymorphic dna fragments, derived from candidate genes and ssr loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare. most agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. the observed trait correlations and associated marker fragments provide new insight in the molecular basis of bruising susceptibility and its natural variation. as phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. the natural genetic diversity for bruising susceptibility was evaluated in elite middle european potato germplasm in order to elucidate its molecular basis.
 most agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. one such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact. tuber susceptibility to bruising is a complex trait of the cultivated potato that is crucial for crop quality. as phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. the genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare.  the natural genetic diversity for bruising susceptibility was evaluated in elite middle european potato germplasm in order to elucidate its molecular basis. association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. correlations were found between different traits. in total polymorphic dna fragments, derived from candidate genes and ssr loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. twenty one highly significant and robust marker-trait associations were identified.  the observed trait correlations and associated marker fragments provide new insight in the molecular basis of bruising susceptibility and its natural variation. the markers diagnostic for increased or decreased bruising susceptibility will facilitate the combination of superior alleles in breeding programs. in addition, this study presents novel candidates that might control enzymatic tissue discoloration and tuber bruising. their validation and characterization will increase the knowledge about the underlying biological processes. 