Genetic Control of Fruit Vitamin C Contents1 Mark W. Davey*, Katrien Kenis, and Johan Keulemans Laboratory for Fruit Breeding and Biotechnology, Department of Biosystems, Faculty of Applied Biosciences and Bioengineering, Catholic University of Leuven, B–3001 Heverlee, Belgium
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 1 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.
Vitamin C (L-ascorbic acid [L-AA]) is essential for all living plant tissues. Apart from well-known functions in oxidative stress defense, associated with its antioxidant properties and its abilities to detoxify reactive oxygen species, it also has important roles in the regulation of plant cell growth and expansion, photosynthesis, as well as hormone functions (for review, see Davey et al., 2000; Smirnoff, 2000). Even though nutritional deficiencies are rare in modern western cultures, it is generally recognized that dietary L-AA also has important health benefits for the consumer, and an increased intake of vitamin C has been associated with a decreased incidence of several important human diseases and disorders (Carr and Frei, 1999; DemmigAdams and Adams, 2002; Hancock and Viola, 2005). Furthermore, in meat-poor diets, dietary L-AA can contribute to the improved uptake of iron and zinc, which are the major micronutrient deficiencies worldwide (Frossard et al., 2000). In apple (Malus x domestica) and other fruit species, there are indications that increased antioxidant contents and in particular higher L-AA levels may be associated with improved fruit postharvest properties (Barden and Bramlage, 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. * Corresponding author; e-mail
[email protected]; fax 32–16–322966. 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 (
[email protected]). www.plantphysiol.org/cgi/doi/10.1104/pp.106.083279
1994; Veltman et al., 1999, 2000; Muckenschnabel et al., 2001; Venisse et al., 2001; Franck et al., 2003a, 2003b; Hodges et al., 2004; Hancock and Viola, 2005). In addition, L-AA has been implicated in resistance to a wide range of biotic and abiotic stresses (Davey et al., 2000; Conklin and Barth, 2004). 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
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Davey et al. 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 1 dehydroascorbate [DHA]) concentrations using our laboratory’s existing segregating F1 Telamon 3 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 mediumsized, 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 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).
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). An overview of the population’s mean fruit L-AA/ total L-AA contents is provided in Table I. Mean fruit flesh total L-AA contents varied between 84 and 910 nmol/g fresh weight, while corresponding values for the skin were approximately 4-fold higher and varied between 465 and 3,377 nmol/g fresh weight. Interestingly, the range of total L-AA concentrations encountered within this subset of the mapping population, for skin and flesh tissues, respectively, are significantly higher at 7.3- and 10.8-fold, than the range previously reported for the whole apple L-AA/total L-AA contents in fruit from our collection of breeding parents (Davey and Keulemans, 2004). This might suggest that in the commercial collection there has already been some selection for genotypes with generally higher fruit L-AA concentrations, presumably via association with other favorable fruit quality traits. The data also show that there are linear correlations between the L-AA and total L-AA contents of fruit skin, and that of the underlying flesh (R2 5 0.5489 and 0.4766, respectively), and that fruit with a generally higher skin L-AA content also have a generally higher flesh L-AA content (Fig. 2), although outliers with, for example, unexpectedly higher flesh/cortex contents, might represent interesting targets for study and/or development.
Fruit Physiology
Fruit from each genotype were harvested when commercially ripe, which under our growth conditions occurred between September 5, 2005 and October 31, 344
QTLs Identified
Fruit flesh L-AA and total L-AA values can be considered to be the most important traits examined in this Plant Physiol. Vol. 142, 2006
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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 3 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.’’
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. In total we identified three highly significant QTLs (likelihood of odds [LOD] . 3.5) for fruit flesh mean L-AA contents, located on linkage groups (LGs) 6, 10, and 11 of the Braeburn linkage map (numbered and orientated as according to the Malus reference map of Liebhard et al. [2003b]). Two of these QTLs were classified as major QTLs, as they individually account for over 20% of the total analyzed population variance, and together these three QTLs explain a surprisingly high proportion (60%) of the total population variance Plant Physiol. Vol. 142, 2006
in mean fruit flesh L-AA contents. In Telamon we also identified three minor (percent population variance ,20%) QTLs for fruit flesh mean L-AA concentrations, and these were again localized in the same regions on the same three LGs (6, 10, and 11) as in the Braeburn parent. Together these account for 38% of the analyzed population variance. The fact that these QTLs are located on equivalent regions of the homologous LGs in both parents, suggests that they have a significant role in the regulation of flesh L-AA contents in both parents, and that different genetic backgrounds share similar regulatory mechanisms for this trait. Since L-AA and total L-AA (L-AA 1 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 345
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Table I. Overview of the distribution of fruit mean L-AA, total L-AA, and DHA contents in the flesh and skin tissues of 138 individuals of the F1 progeny of a Telamon 3 Braeburn population Lower/upper 10% represents the mean fruit concentrations of the 14 individuals with the lowest and highest mean trait concentrations, respectively. Skin Tissue
L-AA
DHA
%DHA Total L-AA
Flesh (Cortex) Range Total L-AA
Mean content in 1,640.8 160.6 8.9% 1,801.4 465.4 – 3,377.2 nmol/g fresh weight Lower 10% 888.1 22.4 14.8% 1,042.8 Upper 10% 2,465.9 625.8 7.9% 2,678.3
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 5 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
Figure 2. Correlations between mean fruit total L-AA contents of skin and flesh (cortex) in 138 individuals of a Telamon 3 Braeburn mapping population. 346
Total L-AA
Fold Variation
L-AA
7.3
448.4
2.8
183.2 2.4 14.9% 215.3 767.6 243.4 8.4% 838.2
DHA
%DHA
Range Total L-AA
53.3 10.6% 501.7 83.6 – 909.6
Fold Variation
10.8
3.9
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 singletrait 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 Plant Physiol. Vol. 142, 2006
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Table II. Overview of the QTLs identified for fruit mean L-AA, DHA, and total L-AA contents, fresh weights, and susceptibility to flesh browning on the genetic linkage maps of Telamon and Braeburn QTLs with a significance level of LOD . 3.0 but less than 3.5 are shown in italics and were not used to calculate the total explained percentage of population variance per trait (SUM). ****, QTL for DHA localized using the Kruskall-Wallace nonparametric mapping function, with significance level of P 5 0.05. Braeburn
Parent Trait
Telamon
QTL
LG
LOD
% Variation
Marker
QTL
LG
LOD
% Variation
flesh
1 2 3
10 11 6
1 2 3
10 11 6
–
–
28.3 22.0 9.9 60.2 –
CH03d11 ECATMCAT310 EAAGMCGG87
DHA flesh
8.4 4.1 3.9 SUM –
–
1
17
Total L-AA flesh
1 2 3
10 6 11
CH03d11 EAAGMCGG87 ECAAMCGA174
1 2
17 10
skin
–
–
20.9 11.3 8.9 32.2 –
17.2 11.1 9.3 37.6 2% 2.0 30.6 25.5
L-AA
7.5 4.4 3.0 SUM –
7.0 4.3 3.8 SUM **** SUM 3.7 8.8
–
1
9
DHA skin Total L-AA skin
– –
– –
– –
– –
– –
– 1
– 10
SUM 3.5 SUM – 3.1
56.1 11.9 11.9 – 10.2
Fruit grams fresh weight at harvest
1
14
3.5
12.1
EAATMCCA61
1
10
3.3
11.5
CH03d11
2 3
17 10
– –
– –
3
EAATMCCT160
1
17
– – SUM 14.8
– – 11.5 66.2
– –
1
15.2 9.7 12.1 76.9
GD96 CHO3d11
Rate of browning at harvest
3.4 3.1 SUM 7.8
ECAAMCGG123
Rate of browning after SL
–
–
SUM –
76.9 –
1
17
SUM 5.7
66.2 37.2
ECAAMCGG123
2
4
3.6 SUM
11.0 48.2
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 Plant Physiol. Vol. 142, 2006
Marker
CH03d11 CH04g07 ECAMCGA303 EAAGMCAC216 ECAGGMCCG73 EACAMCCA319 – EATTMCCT331 – EACAMCCA319
EAATMCCT301
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 3 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 6 347
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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.
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 3 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). 348
These positions have also been confirmed in the Telamon 3 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 Plant Physiol. Vol. 142, 2006
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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 (RoessnerTunali 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
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 bePlant Physiol. Vol. 142, 2006
tween 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 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 lab-
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oratory 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 1 3 1 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; published July 14, 2006.
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