Increased concentrations of the most abundant phenolics such as chichoric acid, quercetin derivatives Q-3MG and Q-3G, CQA, CTA, DCQA, L-7G and K-3MG were observed in the HAO lines, though out of these metabolites only CQA and K-3MG were found to be significantly positively correlated with AO potential. The lack of significant correlation between AO potential and total phenolics , despite the positive relationship observed for the extreme HAO and LAO RILs , is likely to reflect the wide genetic background of the RIL population.For the first time to our knowledge, we have linked genomic regions in lettuce underlying AO status to candidate genes, using genomic resources developed for lettuce.In the present investigation, two QTL for total carotenoid content were identified on LG3 and LG7, which based on current literature are the only QTL so far determined for carotenoid content in a leafy vegetable. Although QTL for chlorophyll have previously been determined to vary depending on growing environment,37 QTL for chlorophyll content on LG3, 7 and 9 measured from the glasshouse trial confirm those previously identified from a UK field trial,providing strong evidence for consistency in these QTL. The four QTL identified for AO potential mapped to LG3, 4, 7 and 9,strawberry grow pots with the largest effect QTL on LG3 accounting for almost one-third of the PV for this trait, thus a large-effect QTL. Alleles inherited from L. serriola increased AO potential for all QTLs excluding alleles at the QTL on LG7, which was inherited from L. sativa in the majority of RILs with a higher AO potential. This was to be expected given that the wild parent was measured to possess an overall greater AO potential than cultivated lettuce.

It is perhaps unsurprising that QTL for total chlorophyll and carotenoids were found to co-locate on LG3 and 7, given their coordinated synthesis and intimate relationship in the chloroplasts as part of photosynthetic complexes and as the biosynthetic pathways are commonly linked through the precursor GGPP.GGPP was one of the candidates identified in the LG3 hotspot region which was found to be more highly expressed in the wild parent and could explain the co-location of QTL for chlorophyll and carotenoids observed. The co-locating QTL on LG7 for total chlorophyll and carotenoids explained 16–18% of the PV and possession of the L. serriola allele was found to increase trait values, which was expected as the wild parent had significantly increased concentrations of both pigments in comparison with L. sativa. QTL for total chlorophyll and carotenoids on LG3 had effects in the opposite direction as would be predicted with respect to the phenotype and measured gene expression, with the L. sativa allele increasing trait value. Trans arrangement of positive alleles has previously been linked with transgressive segregation of traits from an inter specific cross of tomato;a phenomenon which was observed for both chlorophyll and carotenoids in the present investigation of lettuce. Co-location of QTL for total chlorophyll and carotenoids on LG3 and LG7 with total AO potential supports the findings of Hayashi et al.Despite this, although chlorophyll and carotenoid were positively correlated with each other, they were measured to be negatively correlated with AO potential in the present investigation. The large effect QTL for AO on LG3 also co-located with a QTL for Q-3MG, which explained 17.8% of the PV. QTL for AO on LG9 also co-located with Q-3MG , as well as the quercetin metabolite Q-3MG-7G . As quercetin metabolites act as powerful AOs,it is therefore likely that fluctuations can notably affect total AO potential, suggesting that we have identified an important metabolic trait underpinning AO potential in this lettuce mapping population. On each LG there was evidence of co-location of QTL for metabolites and in some cases, QTL for the same trait mapped to the same position in both environments.

For example, QTL consistent across environments included CTA and on another LG, QTL for CTA1 and chicoric acid measured from the field trial colocated with a QTL for the kaempferol derivative K-3MG measured from the glasshouse . Total phenolics measured from the field grown RILs co-located with 5-CoQA2 measured from the glasshouse, which has a key role in the phenylpropanoid pathway for secondary metabolism biosynthesis.It has been known for some time that genes with a related function often cluster into operons in bacteria and there is growing evidence for the clustering of genes encoding secondary metabolites in plants.For example, metabolic gene clusters for terpenoid biosynthesis have now been found in oat and Arabidopsis and more recently in the wild legume Lotus japonicas,which may explain the many instances of QTL for different metabolites co-locating to the same region. QTL which have a consistent effect across different growing environments are considered more stable, thus are valued for use in breeding,but this was not observed for all traits. Given that only a subset of the total RIL population was used for this study, the ability to detect small effect QTL was likely to be limited as population size has been demonstrated to limit the sensitivity of QTL detection.Another possible explanation is that significant genotype 3 environment interactions are occurring, which is perhaps unsurprising given that environmental factors are known to have an impact on secondary metabolism.Indeed, significant G 3 E interactions have been detected for AO and CHL QTL; however, similar analyses of data collected from the glasshouse and field environments in present investigations would not be appropriate due to differences in experimental design. Interestingly, a cluster of QTL co-located to the centre of LG3 for dry weight following nutrient limitation and drought recovery using this population,indicating a potential link between abiotic stress and AO potential, though direct comparisons of QTL were not possible due to differences in linkage maps utilised. Future work to analyse phenotype data using compatible mapping resources could reveal co-locations of QTL for abiotic stress tolerance and nutritional quality to the same genomic position, highlighting a strong target for marker-assisted breeding.Using the Lettuce Genome Resource, along with the previously sequenced genomes of A. thaliana and S. lycopersicum, severalpromising gene candidates explaining variation in AO potential in the lettuce RILs were identified on LG3, including two MYB transcription factors thought to regulate anthocyanin biosynthesis . Expression analysis revealed that both PAP2 and MYB114 genes were differentially expressed,growing strawberries in pots with expression increasing and decreasing, in the wild and cultivated parents, respectively.

Anthocyanins are a subclass of flavonoids synthesised from dihydroflavonols known to be one of the major compounds controlling plant colour, particularly fruits and this is largely regulated by the MYB transcription factors.The presence of anthocyanins in the red lettuce cultivars investigated is likely to have resulted in a higher AO and phenolic content compared to the green cultivars and the high AO RILs, the latter of which are green-leaved, with no anthocyanin metabolites detected in this population. Mulabagal et al.investigated the phenolic contents of red and green lettuce types and although one anthocyanin was identified in red lettuce, none were detected in green types, which is also supported by phenolic composition analysis by Llorach et al.Enhancing the expression of PAP1 and the highly similar PAP2 using activationtagging in Arabidopsis resulted in increased expression of phenylpropanoid biosynthesis genes, including phenylalanine ammonia lyase, the enzyme which initiates the phenylpropanoid pathway and chalcone synthase, the first enzyme acting in flavonoid biosynthesis,which could explain how the increased expression of PAP2observed could contribute to AO status, despite the lack of anthocyanins detected. MYB114 also has a role in regulating anthocyanin biosynthesis that is similar to PAP2, through interaction with basic helix-loop-helix proteins, in a mechanism which is highly conserved throughout the plant kingdom.Given that effects of MYB114 overexpression are dependent on overexpression of a corresponding bHLH transcription factor, this could explain how L. sativa had reduced phenolic content and AO status compared to L. serriola, despite exhibiting increased expression of MYB114. Synchronised increases in the expression of MYB and bHLH transcription factors may result in the red leaf phenotype observed in commercial lettuce types. Another promising gene candidate identified within this region was F3H, which was more highly expressed in wild relative to cultivated lettuce. F3H is involved in the conversion of naringenin to the dihydroflavonols dihydrokaempferol and dihydroquercetin , which are the precursors for kaempferol and quercetin, respectively.Both kaempferol and quercetin were present in higher concentrations in the wild parent than the cultivated parent , likely to result from an increased abundance of dihydroflavnols caused by increased levels of F3H and consistent with the former having a greater AO potential. Dihydroquercetin is essential not only as a precursor for quercetin metabolites, but also for flavonoids such as catechin and the proanthocyadins,which may also contribute to AO potential. Derivatives of quercetin and kaempferol were found to be strongly positively correlated in the present investigation , indicating tightly coordinated regulation of the biosynthesis of these flavonoids. Ferulate-5-hydroxylase , an enzyme acting in the phenylpropanoid biosynthesis pathway,was also found to be more abundant in wild lettuce. Knocking out F5H in Arabidopsis has revealed a range of phenotypes, affecting lignin biosynthesis, UV protection and response to wounding.F5H mutants had increased expression of MYB4,a negative regulator of chalcone synthase,thus reducing flavonoid biosynthesis which is consistent with the reduced levels of flavonoids detected in cultivated lettuce in the present investigation.

Sustainable intensification and breeding for increased AO potential Enhanced food security requires that we achieve ‘more from less’ and that yield enhancements in future crops must be complemented by higher nutritional value . Many crop-breeding programmes are now dedicated to developing enhanced crop nutrition where wild progenitors of crops may be exploited for higher concentrations of target phytonutrients relative to those observed in their commercial counterparts.Indeed, such an approach has been successfully deployed for many food crops including tomatoes,berries carrots and potatoes.This can be a powerful approach – broccoli florets from cultivated varieties were found to have between 3 and 10 mmol/g of health-benefitting glucosinolates, whilst wild species can contain 50–100 mmol/g.Here, the AO potential of cultivated lettuce was significantly lower than that of the wild progenitor , with notable differences in phenolic composition. Past artificial selection of lettuce for improved yield traits is likely to be linked to indirect selection against characteristics such as AO status since phenolics are known to have a bitter taste.During evolution we have learned to reject bitter tastes and with bitter taste receptors characterised, aversion is likely to have been crucial to survival.A RIL which had comparable levels of AOs to the red varieties but a reduced phenolic content was identified in the present investigation , which could be utilised in future breeding programmes. Co-incidentally, improving leaf nutritional quality may also afford greater plant protection from pests and diseases, given that many secondary metabolites have roles in defence against herbivore and pathogen attack with mechanical wounding resulting in the accumulation of phenolic compounds in lettuce.Tomatoes genetically engineered to overexpress anthocyanins had an increased shelf life in comparison to wild-type-cultivated tomatoes, with the transgenic tomatoes demonstrating reduced susceptibility to the fungal pathogen Botrytis cinerea.Leaf AO potential and derivatives of the phenolics, quercetin and kaempferol, measured in present investigations were found to positively correlate with shelf life, measured on the same RIL subset by Zhang and colleagues,32 further supporting a link between phytochemicals and shelf life. A gene encoding a XTH involved in cell wall loosening and located within the QTL hotspot in LG3 has also been linked with shelf life, with the down-regulation of a XTH previously shown to increase the shelf life of lettuce,though the XTH identified was not determined to be differentially expressed between the wild and cultivated parents in present investigations . Clearly further investigations are required to dissect the link between nutritional quality and shelf life in lettuce. Improving the phytonutrient content of widely consumed yet relatively nutritionally poor vegetables, such as lettuce, is an important target for plant breeding and here we identified several candidates controlling flavonoid biosynthesis within the large-effect QTL for AO potential; a number of which were shown to be differentially expressed between wild and cultivated lettuce. The QTL region underpinning these traits is a strong target for future breeding and on-going research is focusing on introgressing this genomic region into commercial lettuce breeding lines. At the same time, further proof of functionality through genome editing and other molecular routes is also underway. Taken together, this study provides the first detailed insight into lettuce phytonutrient traits and how they may be deployed in the future for an enhanced food plant, consumed widely and of global significance.