Aim of the study
In our previous study [1] we mapped phenolic compounds in ripe fruits of a segregating F1 population derived from the cross between cultivars ‘Prima’ and ‘Fiesta’. There appeared to be a strong hotspot of mQTLs at the top of LG16. Annotation of the metabolites showed that the compounds that mapped on the LG16 hotspot belong to the phenylpropanoid and flavonoid pathways (Figure 5).
We wanted to discover which gene(s) controlled this mQTL hotspot. Therefore, in the present research, transcript abundances for the candidate genes in the mQTL region were measured in progeny genotypes that segregated for these mQTLs. In addition, structural genes of the phenylpropanoid and flavonoid pathways and putative transcription factor genes that are candidates for regulating these pathways and located elsewhere were evaluated as mentioned in the Methods section in detail.
MdLAR1 seems to be the only gene that can explain the mQTL hotspot on LG16
As shown in Figure 3, MdLAR1 was the only gene for which the expression was clearly correlated with the metabolite content, both in peel and flesh. None of the other genes showed a clear correlation with procyanidin dimer II content. Moreover, Figure 6 shows clearly that the expression of MdLAR1 was low for the genotypes that had inherited the recessive alleles (mm), and had low content of the representative metabolite procyanidin dimer II. The progeny that had inherited one or two dominant alleles (Mm, MM) had higher expression levels of MdLAR1 and higher content of procyanidin dimer II (Figure 6). This pattern was observed both in peel and in flesh. This was not the case for any of the other genes studied, which suggests that MdLAR1 was responsible for the hotspot of mQTLs on LG16. Furthermore, it indicates that MdLAR1 exerted its influence by means of its expression level. Recent findings in grape also showed a genetic association between a LAR gene and a procyanidin QTL [15].
The procyanidin content was higher in the flesh compared to the peel (Figure 1). However, the expression of MdLAR1 was lower in the flesh compared to the peel (Figure 6). A possible explanation is the fact that flavonols and anthocyanins are produced in the peel only. These may compete for the pool of available substrates, leading to relatively lower procyanidins level.
How can MdLAR1 explain the observed mQTLs?
The MdLAR1 gene clearly explains the mQTL for procyanidin content, as LAR from leguminosal species has been implicated in the synthesis of catechin, a building block for procyanidins [11]. Remarkably, we found several mQTLs in the same hotspot on LG16 for metabolites (kaempferol glycosides, phloridzin, phenolic esters) that are synthesized by different branches from the phenylpropanoid pathway [1] (Figure 5). Since LAR is not known to be involved in the biosynthesis of these other metabolites, the observed differential LAR expression does not provide a straightforward explanation for the presence of the mQTLs of these more upstream metabolites.
One could speculate about the effect that LAR overexpression may have effect on the total flux through the phenylpropanoid pathway. We note that the positively associated mQTLs (procyanidins, dihydrochalcones, phenolic esters and kaempferol glycosides) all map downstream of coumaroyl-CoA ligase (4CL) in the pathway (Figure 5). A metabolite that maps upstream of 4CL is coumaroyl hexoside, for which the level was negatively correlated with e.g. procyanidins. This appears also from Figure 3.
In apple, no 4CL-like gene is located at the mQTL hotspot [12]. Moreover, the expression of the tested 4CL gene did not correlate with the metabolites that mapped at the hotspot. One explanation may be that MdLAR1 overexpression relieves a feedback mechanism on the enzymatic activity of 4CL. 4CL is known to be feedback inhibited by metabolites from the phenylpropanoid pathway, such as naringenin [16]. Possi bly, the enhanced MdLAR1 activity will lead to depletion of pathway intermediates such as naringenin, which may thus activate 4CL activity and lead to a higher general flux, from coumaroyl glycoside towards the downstream metabolites. The support for such a mechanism needs extensive experimentation, which is outside the scope of this article.
An unlikely, but still possible alternative explanation for the mQTL hotspot could be that a transcription factor at the mQTL hotspot regulated the expression of MdLAR1. As we did not see any differencial expression of the transcription factor genes at the mQTL hotspot, the different alleles of that transcription factor gene would not differ in expression levels, but theoretically could differ in effect of the protein. Further, that transcription factor might have influenced 4CL paralogous that were not covered by the used primer pair. We do not regard this as a likely explanation, but it cannot be completely excluded.
Transcript abundances of several structural genes and transcription factor genes were correlated
MdANR also contributes to the synthesis of procyanidins (Figure 5). The expression level of this gene significantly correlated with expression of several structural genes such as PAL, CHS, DFR, and ANS (Figures 3 and 4). Moreover, there was a clear correlation between the expression of these structural genes, and the expression of the transcription factor genes MYB9 and MYB11 (Figures 3 and 4). Possibly, these transcription factors regulated the mentioned structural genes. However, the transcript abundances of none of these structural or transcription factor genes did correlate significantly with the metabolite abundances that mapped at the mQTL hotspot on LG16 (Figure 3). This indicates that these structural genes were not the bottleneck for the pathway, whereas probably MdLAR1 was the limiting factor in the progeny that had inherited both lowly expressed alleles of this gene (mm). Presumably, the bottleneck was (partly) removed in case of presence of one or two higher expressed alleles of MdLAR1 (MM, Mm).
Applications
The dominant allele of the MdLAR1 gene, causing increased content of metabolites that are potentially health beneficial, could be used in marker assisted selection of current apple breeding programs. This selection could be made at seedling stage. This would reduce the production costs for the breeders by discarding the undesired seedlings at earlier stage of growth, whereas in classical breeding only after six years, when trees start to bear fruits, selection on fruit content is possible. Another possibility is to clone the dominant allele or alleles for engineering increased content of metabolite(s) into existing apple cultivars by different transformation technologies including cisgenesis [17, 18].