This Article Abstract Full Text (PDF) All Versions of this Article: 142/1/343    most recent pp.106.083279v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (10) Citing Articles via Google Scholar Google Scholar Articles by Davey, M. W. Articles by Keulemans, J. Search for Related Content PubMed PubMed Citation Articles by Davey, M. W. Articles by Keulemans, J. Agricola Articles by Davey, M. W. Articles by Keulemans, J.

GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Genetic Control of Fruit Vitamin C Contents¹

Laboratory for Fruit Breeding and Biotechnology, Department of Biosystems, Faculty of Applied Biosciences and Bioengineering, Catholic University of Leuven, B–3001 Heverlee, Belgium

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
An F₁ progeny derived from a cross between the apple (Malus x domestica) cultivars Telamon and Braeburn was used to identify quantitative trait loci (QTL) linked to the vitamin C (L-ascorbate [L-AA]) contents of fruit skin and flesh (cortex) tissues. We identified up to three highly significant QTLs for both the mean L-AA and the mean total L-AA contents of fruit flesh on both parental genetic linkage maps, confirming the quantitative nature of these traits. These QTLs account for up to a maximum of 60% of the total population variation observed in the progeny, and with a maximal individual contribution of 31% per QTL. QTLs common to both parents were identified on linkage groups (LGs) 6, 10, and 11 of the Malus reference map, while each parent also had additional unique QTLs on other LGs. Interestingly, one strong QTL on LG-17 of the Telamon linkage map colocalized with a highly significant QTL associated with flesh browning, and a minor QTL for dehydroascorbate content, supporting earlier work that links fruit L-AA contents with the susceptibility of hardfruit to postharvest browning. We also found significant minor QTLs for skin L-AA and total L-AA (L-AA + dehydroascorbate) contents in Telamon. Currently, little is known about the genetic determinants underlying tissue L-AA homeostasis, but the presence of major, highly significant QTL in both these apple genotypes under field conditions suggests the existence of common control mechanisms, allelic heterozygosity, and helps outline strategies and the potential for the molecular breeding of these traits.

Because of these important functional and nutritional properties, there is much interest in understanding the mechanisms underlying the regulation of tissue L-AA concentrations (Demmig-Adams and Adams, 2002; Fernie et al., 2006). However, it is only relatively recently that the pathway(s) for L-AA biosynthesis in plants have been identified, and while most of the genes proposed to be involved in these pathways have been cloned and expressed in various plant species, transformation strategies to increase L-AA concentrations have had only limited success (Ishikawa et al., 2006). There is thus a need for alternative approaches to identify the genetic determinants underlying whole plant and (fruit) tissue L-AA homeostasis. Many traits are determined by more than a single gene, and the quantitative contributions of each of the separate genes to that trait can be estimated using the statistical techniques of quantitative trait loci (QTL) analysis and QTL mapping (Asins, 2002; Collard et al., 2005). Several QTL analyses in apple have been published, including results for such traits as flowering time, growth, and certain fruit qualities (Lawson et al., 1995; Conner et al., 1998; King et al., 2000, 2001; Liebhard et al., 2003a, 2003b, 2003c; Kenis and Keulemans, 2005; K. Kenis and J. Keulemans, unpublished data). An overview of the location of known fruit quality traits on an integrated Malus reference linkage map is provided by Liebhard et al. (2003a). Currently however, we are unaware of any published information on QTL identification for L-AA contents in hard fruit species. Indeed, to date there have been only two reports of the identification of QTLs associated with tissue L-AA contents, to our knowledge, and both of these were in fruit of a library of tomato (Lycopersicon esculentum) introgression lines (ILs; Rousseaux et al., 2005; Schauer et al., 2006).

The aim of this work was therefore to localize genomic regions involved in the regulation of fruit L-AA and total L-AA (L-AA + dehydroascorbate [DHA]) concentrations using our laboratory's existing segregating F₁ Telamon x Braeburn mapping population, and updated versions of genetic linkage maps of these two varieties (Kenis and Keulemans, 2005). Although this population was initially established to investigate the inheritance of tree architecture traits, the characteristics of the fruit of these two varieties are highly divergent, making them well suited to the study of certain fruit quality traits. For example, Braeburn is a medium-sized, high-quality, late-maturing commercial eating apple with excellent storage properties and high L-AA contents. It also has good eating characteristics being crispy, juicy, and firm. By comparison, Telamon has a relatively small fruit that matures in the middle of the season and that has generally poor sensory qualities, and is particularly prone to mealiness and postharvest storage disorders. Telamon is an ornamental variety that is not grown for commercial production.

Our results have allowed us to identify surprisingly strong QTLs for both L-AA and total L-AA contents in the cortex of both varieties, as well as QTLs for skin L-AA and total L-AA contents in the Telamon parent, despite the presumed strong influence of environmental conditions on skin L-AA and antioxidant contents. Results are discussed in terms of the development of molecular breeding strategies for increased L-AA content and crop nutritional enhancement.

	RESULTS AND DISCUSSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Experimental Setup

Although relatively little is known about the regulation of fruit L-AA concentrations, available evidence indicates that L-AA and total L-AA levels will be responsive to the growing environment and in particular to conditions such as high-light exposure. This means that the local microclimate and/or the placement of the trees within the field could potentially influence final fruit L-AA or total L-AA contents. Examining the distribution of the individuals with both the 10% highest and 10% lowest fruit L-AA/total L-AA values within the population, clearly demonstrated that there was no influence of tree location on mean fruit L-AA, and total L-AA concentrations, or indeed on fruit fresh weights (data not shown).

Fruit Physiology

Fruit from each genotype were harvested when commercially ripe, which under our growth conditions occurred between September 5, 2005 and October 31, 2005. In contrast to previous results on 32 different apple breeding varieties, however (Davey and Keulemans, 2004), we did not observe any significant correlations between harvest dates and mean fruit L-AA and total L-AA concentrations (data not shown). All fruit were individually weighed at harvest. Within the group of 138 F₁ progeny analyzed, the mean weights varied between 54.5 and 265.7 g, with an overall population average of 150.6 g. The distribution of fruit mean fresh weights was normal. The rate genotype of fruit flesh (cortex) browning was determined on 10 individual, randomly selected fruit directly after harvest, as well as after postharvest storage (shelf life [SL]). Rates of flesh browning varied from less than 15 min to over 240 min under laboratory conditions. Again, these data were normally distributed.

Mean Vitamin C Contents and Distribution

Mean values of fruit L-AA and total L-AA contents in both the skin and flesh tissues were normally distributed (Fig. 1, A and B ), while the values for DHA, calculated as the difference between total L-AA and L-AA, were highly skewed (Fig. 1C). The highly distorted distribution of DHA contents means that the QTL analysis for DHA contents was carried out using the nonparametric Kruskal-Wallis function in the MapQTL v4.0 software, rather than using interval mapping and the restricted multiple QTL model (rMQM) mapping function, which is suitable for normally distributed data (Van Ooijen et al., 2002).

View larger version (16K): [in this window] [in a new window]   Figure 1. Distribution of skin total L-AA concentrations (A), flesh (cortex) total L-AA concentrations (B), and flesh (cortex) DHA contents (C) within the 138 analyzed F1 progeny of the Telamon x Braeburn mapping population. All concentrations expressed in nmol/g fresh weight and represent the means determined from the analysis of 10 randomly chosen fruit per individual as described in "Materials and Methods."

View larger version (17K): [in this window] [in a new window]   Figure 2. Correlations between mean fruit total L-AA contents of skin and flesh (cortex) in 138 individuals of a Telamon x Braeburn mapping population.

Fruit flesh L-AA and total L-AA values can be considered to be the most important traits examined in this work, since the flesh constitutes the bulk of the consumed apple (population mean of approximately 85% of whole fruit fresh weight), and because many people prefer to peel their fruit before consumption. By analyzing the flesh and skin tissues separately we also hoped to minimize the influence of environmental parameters such as incident sunlight on tissue L-AA concentrations. A summary of the QTL identified for all the traits analyzed here is provided in Table II and in Figure 3 .

View larger version (22K): [in this window] [in a new window]   Figure 3. Locations of QTLs for fruit mean L-AA, total L-AA, and DHA contents, for rate of flesh browning and for mean fruit fresh weights at harvest, on the genetic linkage maps of the apple varieties Telamon (Tel) and Braeburn (Br). Only those LGs on which a QTL for L-AA/total L-AA content has been localized have been shown. Thick bars to the side of each LG indicate significant QTLs (LOD > 3.5) with the length approximating to the 1.0 LOD support level, and the thin error bars to the 1.5 LOD support level, corresponding to a QTL coverage probability of 95%. Minor QTLs with LOD values between 3.0 and 3.5 are shown in italics.

Since L-AA and total L-AA (L-AA + DHA) contents are closely related, we expected to find QTLs for the mean flesh total L-AA contents on the same LGs as those for flesh L-AA. In Braeburn this was indeed the case, where one major QTL and one minor QTL were found on LGs 10 and 6, respectively. Together these were responsible for 32% of the total analyzed population variance in this trait. There was also an additional minor QTL, again localized on LG 11. Although strictly speaking this latter QTL was not statistically significant (LOD = 3.0), its localization in the same region where a strong QTL for flesh L-AA content is also found in Braeburn, prompted us to include this data. Telamon by comparison shared only one QTL on LG 10, but interestingly we identified a new, highly significant major QTL, accounting for 31% of the analyzed population variance in mean flesh total L-AA contents on LG 17. Since the difference between tissue L-AA and total L-AA values is the amount of DHA present, the fact that Telamon has an important additional QTL for this trait indicates that the locus is either homozygous in Braeburn and will thus not show segregation, or that the Telamon genome contains a specific locus associated with higher levels of oxidized L-AA (DHA). In the latter case, this might indicate that the QTL on LG 17 is associated with mechanisms that are either involved in the turnover and/or recycling of DHA, or with those that promote L-AA oxidation. Interestingly, highly significant, major QTLs for flesh browning both at harvest (LOD 14.8, explained population variance 66%) as well as after SL storage (LOD 5.7, explained population variance 37%), were also localized to exactly the same region of the Telamon LG 17, together with the single weak QTL for cortex DHA contents that were found (Fig. 3). In previous work, we and others have shown that the occurrence of internal browning during the postharvest storage of hard fruit is related to the L-AA content of the flesh and other aspects of antioxidant metabolism (Veltman et al., 1999, 2003; Larrigaudiere et al., 1999, 2001; Franck et al., 2003a, 2003b; Ko et al., 2004). Taken together, therefore, these results suggest that the major QTL for flesh browning located on LG 17 of the Telamon map is associated with the presence of a more oxidized L-AA pool. This could be due either to a less efficient L-AA recycling system (for example enzymes of the ascorbate-glutathione cycle; Noctor and Foyer, 1998), or the presence of factors that promote oxidation of the L-AA pool such as (apoplastic) peroxidases, polyphenol oxidases (PPOs), or ascorbate oxidase itself, all of which are able to use L-AA as a substrate or a cosubstrate. The fact that L-AA (and derivatives) are used commercially to prevent browning in sliced fruit and that fruit PPOs are able to oxidize L-AA either directly (Espin et al., 1998, 2000) or indirectly via reduction of the o-quinones that are produced as the primary oxidation products of the PPO reaction, leads us to speculate that this locus could be associated with an enhanced PPO activity. Apple has at least two PPO genes that are differentially regulated in response to development and wounding (Kim et al., 2001), but they have yet to be mapped. However, a second QTL for browning susceptibility at harvest (LOD 7.8, explained population variance 77%), found on LG 3 of the Braeburn map, was not associated with any of our L-AA or total L-AA QTLs.

A group of QTLs for flesh mean L-AA and total L-AA contents was located to the same region of LG 10 on both parental maps (Fig. 3). This region also contains minor QTLs for fruit fresh weight, and maps to exactly the same region as the locus that is responsible for the columnar tree form of Telamon (the Co gene; Kenis and Keulemans, 2006). Together with additional unpublished data on fruit quality traits (K. Kenis and J. Keulemans, unpublished data), we suggest that this is a region that governs several aspects of general plant and fruit growth and size. Interestingly, Schauer et al. (2006) recently reported that nearly 50% of 889 single-trait QTLs identified in the tomato IL library of Eshed and Zamir (1995) were associated with at least one QTL modifying whole plant yields. Although in contrast to our results, according to these authors, L-AA contents were not correlated with any plant morphological traits (Schauer et al., 2006).

Finally, we identified a significant, minor QTL for skin L-AA content (LOD 3.5, percent variance 10%) on LG 9, and a QTL for skin total L-AA content just below the significance level (LOD 3.3, percent variance 12%) on LG 10 of the Telamon map. These results were surprising since we had expected that with the known sensitivity of fruit (and tissue) L-AA contents to environmental conditions and especially to light (Gatzek et al., 2002; Davey et al., 2004; Golan et al., 2006), environmental factors would largely override the genetic determinants of this trait. It remains to be seen whether these QTLs remain stable from year to year, but the localization of a QTL once again on LG 10 on the maps of both parents, in the same region as QTLs for flesh L-AA content, would again be indicative of a common mechanism in both fruit tissue types. The fact that some of the markers associated with these QTLs are codominant microsatellites, will allow transference and confirmation of these results in other varieties.

An overview of the location of published QTLs for mapped apple traits is provided by Liebhard et al. (2003a), King et al. (2000), and Kenis and Keulemans (2006). In this brief discussion we will focus mainly on traits located on LGs 6, 10, 11, 16, and 17, where the main QTL for tissue L-AA and total L-AA conents have been located. Of the general plant growth parameters studied to date, a few minor QTLs (for seedling leaf size, stem diameter, and height increment), explaining up to 8% of the population variance have been localized to a region at the very top of LG 17, but that is clearly distinct from the major QTLs for flesh total L-AA located on the same LG. Our own work on the QTL analysis of growth traits in the Telamon x Braeburn population (axis height, growth rate, internode length, etc.), indicates that major QTLs for all these traits are clustered in the same region of LG 10 along with QTLs for both L-AA and total L-AA contents of flesh in both parents and that these localize to the region where the Co gene maps (Kenis and Keulemans, 2006). QTLs for other plant architectural/growth traits do not, however, colocalize with those for fruit (total) L-AA contents. A number of apple fruit quality traits have also been mapped on the reference map of Liebhard et al. (2003a; King et al., 2001), and several minor QTLs for fruit weight, flesh firmness, and sugar content, accounting for up to a maximum of 12% of the population variance, have been found on LGs 6, 10, and 11, but of these it is only the QTLs for fruit weight on LG 6 that partially overlap with one for flesh L-AA contents in both of our parental maps. We have also carried out a comprehensive analysis of the fruit quality traits in the Telamon x Braeburn population, which again highlights a major clustering of traits (soluble sugar, hardness, and acidity) around the same region of LG 10 in both parental linkage maps (K. Kenis, personal communication), supporting our assertion that this is a region having many pleitropic effects on general plant growth characteristics, and that this region impacts directly or indirectly on fruit (total) L-AA contents.

Candidate Genes

Several genes involved in the inheritance of monogenic traits have already been mapped onto genetic linkage maps of other apple varieties (Maliepaard et al., 1998). This aids in comparative mapping studies and allows us to identify the LGs that are homologous across the various parents. The reported association of fruit tissue L-AA contents with quality characteristics such as storage potential suggests an obvious link to ripening and to the ethylene responses that are so critical for ripening in climacteric fruit like apple. 1-Aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO), two genes of the ethylene biosynthetic pathway, have recently been positioned on LGs 15 and 10, respectively, of two genetic linkage maps (Costa et al., 2005). These positions have also been confirmed in the Telamon x Braeburn population (R. Dreesen, personal communication). The ACO gene on LG 10 once again falls within the region containing a large cluster of quality trait QTLs, and also borders a fruit firmness QTL identified by King et al. (2001) and Maliepaard et al. (2001), but little additional information is available about other ripening-related genes in apple. In tomato however, recent work from Alba et al. (2005) on the Never-ripe mutant, which contains a mutation in an ethylene receptor and that consequently has a reduced ethylene sensitivity and fails to ripen, clearly outlines a role for ethylene metabolism in the accumulation of L-AA in the pericarp during tomato fruit ripening (Alba et al., 2005). Other apple genes that have been mapped but that do not colocalize with our L-AA QTLs include those for malic acid (Ma) content and scab resistance (Vf; Hemmat et al., 1998; Liebhard et al., 2003c; Baldi et al., 2004; Gygax et al., 2004; Freslon et al., 2006).

Although there has been no work published on the mapping of candidate genes involved in L-AA metabolism in apple, this is not the case in tomato, where 14 genes involved in L-AA metabolism (Zou et al., 2006) and 63 genes involved in carbon metabolism (Causse et al., 2004) have recently been mapped using the IL library of Eshed and Zamir (1995). Interestingly, QTLs for whole fruit antioxidants (and L-AA) in the same IL library have also just been published (Rousseaux et al., 2005). Comparing these publications, we see that Rousseaux et al. (2005) identified three QTLs on ILs 3-3, 3-4, and 10-1, for whole fruit L-AA contents that were stable over 2 years. Of these, however, only one is in the region of the map position of an enzyme of L-AA metabolism, and this is GDP-Man pyrophosphorylase, which was localized to the overlap between IL 3-2 and IL 3-3 (Zou et al., 2006). Interestingly, the reduced L-AA content of the low-ascorbate and ozone-sensitive Arabidopsis (Arabidopsis thaliana) mutant vtc1 has a reduced GDP-Man pyrophosphorylase activity (Conklin et al., 1999). Zou et al. (2006) also identified one QTL on IL 12-4 that increased L-AA contents by 44% in 1 year only, but again this did not colocalize with any of the 15 mapped L-AA metabolic genes localized by Rousseaux et al. (2005).

More recently Schauer et al. (2006) identified a total of four QTLs for tomato fruit L-AA contents, including one on IL 12-4, but they also found a total of 25 QTLs for DHA content. It is not clear why so many DHA QTLs were identified, but in our experience this could be related to the analytical methodology used by these authors, where the need for partitioning, drying down, and derivatisation of plant metabolite extracts in a basic environment (pyridine) for subsequent gas chromatography-mass spectrometry analysis (Roessner-Tunali et al., 2003) leads to significant losses and breakdown of L-AA. Obviously caution has to be exercised when drawing conclusions across different experiments, and as Rousseaux et al. (2005) themselves noted, they also experienced significant problems associated with the tomato fruit L-AA analyses, with the tissue values measured doubling according to the extraction procedure used (Rousseaux et al., 2005). In this work, however, we were careful to use validated methods developed especially for the analysis of L-AA contents in apple fruit tissues (Davey et al., 2003).

Despite these results in tomato, it is clear that it will be important to map the apple gene orthologs of known L-AA metabolic enzymes onto our genetic linkage maps. However, these collective results suggest that the genetic control of fruit L-AA contents will include other, as yet unsuspected mechanisms outside the studied enzymes of L-AA biosynthesis or turnover. A similar precedent for this has recently been shown in tomato, where fruit-specific suppression of DET1, a negative transcriptional regulator of photomorphogenesis, unexpectedly lead to significant increases in carotenoid and flavonoid levels (Davuluri et al., 2005).

	CONCLUSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
It has become increasingly clear that the regulation of plant L-AA metabolism is complex, presumably due to the multiple cellular functions of this ubiquitous, small molecule. We consider that working in fruit, rather than in foliar systems (for example using model systems such as Arabidopsis), may offer significant advantages by reducing the complexity of the system being analyzed, and thus the degree of interaction between control mechanisms. Using different fruit tissues of apple, we have identified a number of highly significant, major QTLs regulating fruit mean L-AA and total L-AA concentrations on the genetic linkage maps of both the Telamon and Braeburn cultivars. In both parents, common QTLs were localized to the same region of LGs 6, 10, and 11, which together accounted for up to 60% of the total observed population variance. Markers for some of these QTL alleles could be used to select for elevated L-AA/total L-AA contents. We consider that these positive results are in part due to the stringent sampling and analytical conditions developed for these analyses.

The commonality between parents of the localized QTLs and the relatively high degree of population percent variance explained, not only confirms the quantitative nature of these traits, but also indicates the existence of similar mechanisms of regulation of fruit L-AA/total L-AA contents in both genetic backgrounds, and even in one case between tissue types. We consider that the QTLs on LG 10 are related to a locus that governs general plant and fruit growth characteristics, while the major QTLs found on LGs 6 and 11 do not appear to coincide with other known fruit quality traits. Of particular interest to us, however, was a major, highly significant QTL for flesh total L-AA contents on LG 17 of the Telamon map, which colocalized not only with a QTL for DHA content, but also with very strong QTLs for flesh browning. We speculate that this QTL maybe involved in regulating the redox status of the fruit flesh L-AA pool, possibly via the activity of PPOs or peroxidases.

The presence of major QTLs for fruit L-AA and total L-AA contents under field conditions offers the promise of new targets for investigating the molecular basis of the control of this important trait, and several of the QTLs we identified are associated with codominant microsatellite markers that will allow their transference to other mapping populations. Of course confirming QTL stability and the further fine mapping of each region are required before direct experiments can be carried out.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Mapping Population

An F₁ mapping population consisting of 257 individuals was created from a cross between the apple (Malus x domestica) cultivars Telamon and Braeburn using Telamon as the female parent (Kenis and Keulemans, 2005). This population was originally established to study aspects of the inheritance and genetics of tree architecture, as the Telamon parent contains the dominant Columnar (Co gene) locus, but the large differences in fruit quality between these parents mean that they are also ideal to study various fruit quality traits. For this work a population of single copies of 4-year-old progeny grown on M9 rootstock on the laboratory's experimental field station at Rillaar, Aarschot, Belgium in 2005 was used.

Trait Analysis

Ten randomly chosen, healthy fruit were harvested when commercially ripe from each individual, and then directly transferred in a cool box to the laboratory where they were weighed, decored, sliced, and separated into skin and flesh (cortex) portions. The individual skin and flesh fractions were pooled into three groups per genotype (3 + 3 + 4), and immediately extracted by blending in 6% metaphosphoric acid/EDTA/polyvinylpolypyrrolidone for L-AA analysis using especially developed methods (Davey et al., 2003). L-AA analysis usually occurred on the same day within a few hours of harvest, although at peak harvest time it was sometimes necessary to store apples for a maximum of up to 48 h in a cool cell at 2°C before extraction. Previous work demonstrated that this had no detectable influence on fruit L-AA and total L-AA contents within this storage period (Davey et al., 2004). L-AA and total L-AA values were determined by HPLC analysis using standardized, validated protocols (Davey et al., 2003). In total, the fruit from 138 individual genotypes were analyzed for L-AA and total L-AA contents in the season of 2005.

The rate of browning of the apple cortex (flesh) was determined using a copy of the same mapping population grown on the same site in Rillaar, and analyzed in the same season. In brief, the methodology adopted was as follows: A small strip of skin was peeled from the sunny (red) and shaded (green) sides of each of 10 randomly selected apples from each genotype. The apples were then allowed to stand at room temperature under normal laboratory conditions, and the color of the flesh periodically evaluated at regular time intervals by two independent investigators. Apples were classified as brown when the flesh located below the skin was uniformly colored in all or the majority of apples of any one genotype and the time taken to reach this point was noted. In total, 150 individual genotypes were analyzed directly at harvest, and 130 individuals after SL storage. Rates of browning varied from between less than 15 min to over 240 min at room temperature. Full details will be published in another article (K. Kenis and J. Keulemans, unpublished data).

QTL Analysis

QTLs for the mean L-AA and total L-AA values of fruit skin and flesh tissues as well as fruit fresh weight at harvest and browning at harvest and after SL, were calculated using updated published genetic linkage maps for both parents, on which an additional 17 microsatellite markers have been added (Kenis and Keulemans, 2005), and the MapQTL v4.0 software (Van Ooijen et al., 2002). This software package allows QTL analysis to be carried out on a full-sib family of a cross-pollinating species such as apple, generating four QTL alleles per segregating QTL. The updated map of Telamon consists of 17 LGs with an average length of 63.6 cm and 16.8 markers per LG, while that of Braeburn had an average length of 75.4 cm, and an average density of 17.2 markers per LG (Kenis and Keulemans, 2005; K. Kenis and J. Keulemans, unpublished data).

The MapQTL 4.0 software package was used to identify and locate QTLs linked to markers using both interval mapping and the rMQM mapping functions. Using the permutation test option in the MapQTL software, and the tables and formula provided by Van Ooijen (1999), the presence of a QTL was considered to be significant in a single trait analysis when the LOD value was larger than 3.5. Markers covered by a QTL with a LOD of >3.5 from the interval mapping were used as cofactors in subsequent rounds of rMQM mapping. If the LOD value linked with a cofactor fell below 3.5 during subsequent rounds, the cofactor was removed and the analysis repeated. This process was continued until the cofactor list remained stable. The estimated additive effect and the percentage of variance explained by each QTL were obtained from the final rMQM mapping round. During analysis, we encountered several QTLs with LOD values just below the significance threshold of 3.5 (but still >3.0). Since these colocalized with other highly significant QTLs, we also included these in our results as supporting information. These results are shown in italics in Table II and in Figure 2. However, the percent variance values associated with these LOD < 3.5 QTLs were not included when assessing the overall contribution of each QTL to the total observed population variance for each trait (Table II). For the presentation of these results, the genetic LG numbering and alignment system of the Malus reference map of Liebhard et al. (2003b) was adopted.

	ACKNOWLEDGMENTS

 
The authors would like to acknowledge expert technical assistance from E. Stals and G. Vrebos, Dr. R. Dreesen for generously sharing results on the mapping of ACS and ACO, and Dr. S. Kushnir for critical reading of the manuscript. The authors also gratefully acknowledge stimulating discussions with Calvin, Lucas, and Eva, and the help of Otto Van Poeselaere and Katerine Kotsirkof in the preparation of the manuscript.

Received May 8, 2006; accepted July 3, 2006.

	FOOTNOTES

 
¹ This work was supported by a grant from the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (grant no. IWT 000125) and in collaboration with Better3Fruit.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mark W. Davey (mark.davey@biw.kuleuven.be).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.083279

^* Corresponding author; e-mail mark.davey{at}biw.kuleuven.be; fax 32–16–322966.

	LITERATURE CITED

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Alba R, Payton P, Fei ZJ, McQuinn R, Debbie P, Martin GB, Tanksley SD, Giovannoni JJ (2005) Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development. Plant Cell 17: 2954–2965[Abstract/Free Full Text]

Asins MJ (2002) Present and future of quantitative trait locus analysis in plant breeding. Plant Breed 121: 281–291[CrossRef]

Baldi P, Patocchi A, Zini E, Toller C, Velasco R, Komjanc M (2004) Cloning and linkage mapping of resistance gene homologues in apple. Theor Appl Genet 109: 231–239[CrossRef][Web of Science][Medline]

Barden CL, Bramlage WJ (1994) Accumulation of antioxidants in apple peel as related to preharvest factors and superficial scald susceptibility of the fruit. J Am Soc Hortic Sci 119: 264–269[Abstract/Free Full Text]

Carr AC, Frei B (1999) Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans. Am J Clin Nutr 69: 1086–1107[Abstract/Free Full Text]

Causse M, Duffe P, Gomez MC, Buret M, Damidaux R, Zamir D, Gur A, Chevalier C, Lemaire-Chamley M, Rothan C (2004) A genetic map of candidate genes and QTLs involved in tomato fruit size and composition. J Exp Bot 55: 1671–1685[Abstract/Free Full Text]

Collard BCY, Jahufer MZZ, Brouwer JB, Pang ECK (2005) An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: the basic concepts. Euphytica 142: 169–196[CrossRef][Web of Science]

Conklin PL, Barth C (2004) Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant Cell Environ 27: 959–970[CrossRef]

Conklin PL, Norris SR, Wheeler GL, Williams EH, Smirnoff N, Last RL (1999) Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci USA 96: 4198–4203[Abstract/Free Full Text]

Conner PJ, Brown SK, Weeden NF (1998) Molecular-marker analysis of quantitative traits for growth and development in juvenile apple trees. Theor Appl Genet 96: 1027–1035

Costa F, Stella S, Van de Weg WE, Guerra W, Cecchinel M, Dallavia J, Koller B, Sansavini S (2005) Role of the genes Md-ACO1 and Md-ACS1 in ethylene production and shelf life of apple (Malus domestica Borkh). Euphytica 141: 181–190

Davey MW, Dekempeneer E, Keulemans J (2003) Rocket-powered high-performance liquid chromatographic analysis of plant ascorbate and glutathione. Anal Biochem 316: 74–81[CrossRef][Web of Science][Medline]

Davey MW, Franck C, Keulemans J (2004) Distribution, developmental and stress responses of antioxidant metabolism in Malus. Plant Cell Environ 27: 1309–1320

Davey MW, Keulemans J (2004) Determining the potential to breed for enhanced antioxidant status in Malus: mean inter- and intravarietal fruit vitamin C and glutathione contents at harvest and their evolution during storage. J Agric Food Chem 52: 8031–8038[Medline]

Davey MW, Van Montagu M, Inze D, Sanmartin M, Kanellis A, Smirnoff N, Benzie IJJ, Strain JJ, Favell D, Fletcher J, et al (2000) Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J Sci Food Agric 80: 825–860[CrossRef][Web of Science]

Davuluri GR, van Tuinen A, Fraser PD, Manfredonia A, Newman R, Burgess D, Brummell DA, King SR, Palys J, Uhlig J, et al (2005) Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat Biotechnol 23: 890–895[CrossRef][Web of Science][Medline]

Demmig-Adams B, Adams WW (2002) Antioxidants in photosynthesis and human nutrition. Science 298: 2149–2153[Abstract/Free Full Text]

Eshed Y, Zamir D (1995) An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated qtl. Genetics 141: 1147–1162[Abstract]

Espin JC, Garcia-Ruiz PA, Tudela J, Garcia-Canovas F (1998) Study of stereospecificity in pear and strawberry polyphenol oxidases. J Agric Food Chem 46: 2469–2473

Espin JC, Veltman RH, Wichers HJ (2000) The oxidation of L-ascorbic acid catalysed by pear tyrosinase. Physiol Plant 190: 1–6

Fernie AR, Tadmor Y, Zamir D (2006) Natural genetic variation for improving crop quality. Curr Opin Plant Biol 9: 196–202[CrossRef][Web of Science][Medline]

Franck C, Baetens M, Lammertyn J, Scheerlinck N, Davey MW, Nikolai BM (2003a) Ascorbic acid mapping to study core breakdown development in "Conference" pears. Postharvest Biol Technol 30: 133–142

Franck C, Baetens M, Lammertyn J, Verboven P, Davey MW, Nicolai BM (2003b) Ascorbic acid concentration in cv. conference pears during fruit development and postharvest storage. J Agric Food Chem 51: 4757–4763[Medline]

Freslon V, Bus VGM, Gianfranceschi L, Patocchi A, Durel CE (2006) Identification and genetic characterisation of Cdr1, a new major scab resistance gene from the cultivar "Dulmer Rosenapfel." 3rd International Rosaceae Genomics Conference, Napier, New Zealand

Frossard E, Bucher M, Machler F, Mozafar A, Hurrell R (2000) Potential for increasing the content and bioavailability of Fe, Zn, and Ca in plants for human nutrition. J Sci Food Agric 80: 861–879[CrossRef][Web of Science]

Gatzek S, Wheeler GL, Smirnoff N (2002) Antisense suppression of l-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l-galactose synthesis. Plant J 30: 541–553[CrossRef][Web of Science][Medline]

Golan T, Muller-Moule P, Niyogi KK (2006) Photoprotection mutants of Arabidopsis thaliana acclimate to high light by increasing photosynthesis and specific antioxidants. Plant Cell Environ 29: 879–887

Gygax M, Gianfranceschi L, Liebhard R, Kellerhals M, Gessler C, Patocchi A (2004) Molecular markers linked to the apple scab resistance gene Vbj derived from Malus baccata jackii. Theor Appl Genet 109: 1702–1709[CrossRef][Web of Science][Medline]

Hancock RD, Viola R (2005) Improving the nutritional value of crops through enhancement of L-ascorbic acid (vitamin C) content: rationale and biotechnological opportunities. J Agric Food Chem 53: 5248–5257

Hemmat M, Weeden NF, Aldwinckle HS, Brown SK (1998) Molecular markers for the scab resistance (V-f) region in apple. J Am Soc Hortic Sci 123: 992–996[Abstract/Free Full Text]

Hodges DM, Lester GE, Munro KD, Toivonen PMA (2004) Oxidative stress: importance for postharvest quality. HortScience 39: 924–929

Ishikawa T, Dowdle J, Smirnoff N (2006) Progress in manipulating ascorbic acid biosynthesis and accumulation in plants. Physiol Plant 126: 343–355[CrossRef]

Kenis K, Keulemans J (2005) Genetic linkage maps of two apple cultivars (Malus x domestica Borkh.) based on AFLP and microsatellite markers. Mol Breed 15: 205–219

Kenis K, Keulemans J (2006) Study of tree architecture of apple (Malus x domestika Borkh.), by QTL analysis of growth traits. Mol Breed (in press)

Kim JY, Seo YS, Kim JE, Sung SK, Song KJ, An G, Kim WT (2001) Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple. Plant Sci 161: 1145–1152

King GJ, Lynn JR, Dover CJ, Evans KM, Seymour GB (2001) Resolution of quantitative trait loci for mechanical measures accounting for genetic variation in fruit texture of apple (Malus pumila Mill.). Theor Appl Genet 102: 1227–1235[CrossRef][Web of Science]

King GJ, Maliepaard C, Lynn JR, Alston FH, Durel CE, Evans KM, Griffon B, Laurens F, Manganaris AG, Schrevens T, et al (2000) Quantitative genetic analysis and comparison of physical and sensory descriptors relating to fruit flesh firmness in apple (Malus pumila Mill.). Theor Appl Genet 100: 1074–1084

Ko TS, Nelson RL, Korban SS (2004) Screening multiple soybean cultivars (MG 00 to MG VIII) for somatic embryogenesis following Agrobacterium-mediated transformation of immature cotyledons. Crop Sci 44: 1825–1831[Abstract/Free Full Text]

Larrigaudiere C, Lentheric I, Vendrell M (1999) Relationship between enzymatic browning and internal disorders in controlled-atmosphere stored pears. J Sci Food Agric 78: 232–236

Larrigaudiere C, Pinto E, Lentheric I, Vendrell M (2001) Involvement of oxidative processes in the development of core browning in controlled-atmosphere stored pears. J Hortic Sci Biotechnol 76: 157–162

Lawson DM, Hemmat M, Weeden NF (1995) The use of molecular markers to analyse the inheritance of morphological and developmental traits in apple. J Am Soc Hortic Sci 120: 532–537[Abstract/Free Full Text]

Liebhard R, Kellerhals M, Pfammatter W, Jertmini M, Gessler C (2003a) Mapping quantitative physiological traits in apple (Malus x domestica Borkh.). Plant Mol Biol 52: 511–526[CrossRef][Web of Science][Medline]

Liebhard R, Koller B, Gianfranceschi L, Gessler C (2003b) Creating a saturated reference map for the apple (Malus x domestica Borkh.) genome. Theor Appl Genet 106: 1497–1508[Medline]

Liebhard R, Koller B, Patocchi A, Kellerhals M, Pfammatter W, Jermini M, Gessler C (2003c) Mapping quantitative field resistance against apple scab in a "Fiesta" x "Discovery" progeny. Phytopathology 93: 493–501[Medline]

Maliepaard C, Alston FH, Van AG, Brown LM, Chevreau E, Dunemann F, Evans KM, Gardiner S, Guilford P, Van Heusden AW, et al (1998) Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers. Theor Appl Genet 97: 60–73

Maliepaard C, Sillanpaa MJ, van Ooijen JW, Jansen RC, Arjas E (2001) Bayesian versus frequentist analysis of multiple quantitative trait loci with an application to an outbred apple cross. Theor Appl Genet 103: 1243–1253[CrossRef]

Muckenschnabel I, Goodman BA, Deighton N, Lyon GD, Williamson B (2001) Botrytis cinerea induces the formation of free radicals in fruits of Capsicum annuum at positions remote from the site of infection. Protoplasma 218: 112–116[CrossRef][Web of Science][Medline]

Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49: 249–279[CrossRef][Web of Science]

Roessner-Tunali U, Hegemann B, Lytovchenko A, Carrari F, Bruedigam C, Granot D, Fernie AR (2003) Metabolic profiling of transgenic tomato plants overexpressing hexokinase reveals that the influence of hexose phosphorylation diminishes during fruit development. Plant Physiol 133: 84–99[Abstract/Free Full Text]

Rousseaux MC, Jones CM, Adams D, Chetelat R, Bennett A, Powell A (2005) QTL analysis of fruit antioxidants in tomato using Lycopersicon pennellii introgression lines. Theor Appl Genet 111: 1396–1408[CrossRef][Web of Science][Medline]

Schauer N, Semel Y, Roessner U, Gur A, Balbo I, Carrari F, Pleban T, Perez-Melis A, Bruedigam C, Kopka J, et al (2006) Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement. Nat Biotechnol 24: 447–454[CrossRef][Web of Science][Medline]

Smirnoff N (2000) Ascorbic acid: metabolism and functions of a multi-facetted molecule. Curr Opin Plant Biol 3: 229–235[Web of Science][Medline]

Van Ooijen JW (1999) LOD significance thresholds for QTL analysis in experimental populations of diploid species. Heredity 83: 613–624

Van Ooijen JW, Boer MP, Jansen RC, Maliepaard C (2002) MapQTL 4.0: Software for the Calculation of QTL Positions on Genetic Maps. Plant Research International B.V., Wageningen, The Netherlands

Veltman RH, Kho RM, van Schaik ACR, Sanders MG, Oosterhaven J (2000) Ascorbic acid and tissue browning in pears (Pyrus communis L. cvs Rocha and Conference) under controlled atmosphere conditions. Postharvest Biol Technol 19: 129–137[CrossRef]

Veltman RH, Lentheric I, Van der Plas LHW, Peppelenbos HW (2003) Internal browning in pear fruit (Pyrus communis L. cv Conference) may be a result of a limited availability of energy and antioxidants. Postharvest Biol Technol 28: 295–302

Veltman RH, Sanders MG, Persijn ST, Peppelenbos HW, Oosterhaven J (1999) Decreased ascorbic acid levels and brown core development in pears (Pyrus communis L-cv. Conference). Physiol Plant 107: 39–45[CrossRef]

Venisse JS, Gullner G, Brisset MN (2001) Evidence for the involvement of an oxidative stress in the initiation of infection of pear by Erwinia amylovora. Plant Physiol 125: 2164–2172[Abstract/Free Full Text]

Zou LP, Li HX, Ouyang B, Zhang JH, Ye ZB (2006) Cloning and mapping of genes involved in tomato ascorbic acid biosynthesis and metabolism. Plant Sci 170: 120–127[CrossRef]

This article has been cited by other articles:

				R. Stevens, M. Buret, P. Duffe, C. Garchery, P. Baldet, C. Rothan, and M. Causse Candidate Genes and Quantitative Trait Loci Affecting Fruit Ascorbic Acid Content in Three Tomato Populations Plant Physiology, April 1, 2007; 143(4): 1943 - 1953. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 142/1/343    most recent pp.106.083279v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (10) Citing Articles via Google Scholar Google Scholar Articles by Davey, M. W. Articles by Keulemans, J. Search for Related Content PubMed PubMed Citation Articles by Davey, M. W. Articles by Keulemans, J. Agricola Articles by Davey, M. W. Articles by Keulemans, J.