Genetic analysis of metabolites in apple fruits indicates an mQTL hotspot for phenolic compounds on linkage group 16

Abstract

Apple (Malus×domestica Borkh) is among the main sources of phenolic compounds in the human diet. The genetic basis of the quantitative variations of these potentially beneficial phenolic compounds was investigated. A segregating F₁ population was used to map metabolite quantitative trait loci (mQTLs). Untargeted metabolic profiling of peel and flesh tissues of ripe fruits was performed using liquid chromatography-mass spectrometry (LC-MS), resulting in the detection of 418 metabolites in peel and 254 in flesh. In mQTL mapping using MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 in the flesh. Four linkage groups (LGs), LG1, LG8, LG13, and LG16, were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. The genetics of annotated metabolites was studied in more detail using MapQTL®. A number of quercetin conjugates had mQTLs on LG1 or LG13. The most important mQTL hotspot with the largest number of metabolites was detected on LG16: mQTLs for 33 peel-related and 17 flesh-related phenolic compounds. Structural genes involved in the phenylpropanoid biosynthetic pathway were located, using the apple genome sequence. The structural gene leucoanthocyanidin reductase (LAR1) was in the mQTL hotspot on LG16, as were seven transcription factor genes. The authors believe that this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species.

Fig. 1.

Number of mQTLs over the apple genome. The linkage groups are separated by vertical dotted lines. In this figure, markers are ordered and positioned equidistantly, thus ignoring their genetic distances. (This figure is available in colour at JXB online.)

Fig. 2.

Significant mQTLs with range of log P-values over the apple genome. An mQTL was considered as significant if its -log (P)-value was >3.8.

Fig. 3.

Mapping of (+)-catechin, (–)-epicatechin, several procyanidins, and pH (Ma) on linkage group 16.

Fig. 4.

A scatter plot showing the distribution of F₁ progeny of ‘Prima’בFiesta’ over the four genotype classes for low/high pH and low/high procyanidin dimer II content, whereby procyanidin dimer II represents the metabolites that share the strong mQTL on LG16. The trait pH co-localizes to this hotspot. The dominant allele for a high metabolite level is denoted as M, and for a low pH (high acidity; presumably high level of malic acid) as Ma. As the dominant alleles M and Ma are in repulsion phase in both parents, giving as alleles in the gametes Mma and mMa, the progeny segregate into three genotypes, lacking the genotype mm mama. The horizontal dashed line represents the 3:1 clear segregation for the procyanidin dimer II that demonstrates that the two classes (i.e. mm and Mm+MM) show full dominance.

Fig. 5.

The phenylpropanoid pathway of phenolic compounds in two apple fruit tissues, peel (A) and flesh (B). The metabolites for which mQTLs were found are presented in coloured boxes. Colourless boxes show the metabolites that were not detected in the present analysis or have no mQTL. Boxes with green colour indicate mQTLs of which the + alleles are in the coupling phase. Boxes with yellow colour show mQTLs for metabolites other than on LG16A. The metabolites in the red box show a negative correlation with the metabolites in the green boxes, having an mQTL on LG16A. The linkage group (LG) where an mQTL was located is given. If different mQTLs were present on different regions of an LG, these regions are distinguished with the letters A, B, C, etc. The alleles ‘a’ and ‘b’ originate from the parent ‘Prima’, and the alleles ‘c’ and ‘d’ originate from the parent ‘Fiesta’, thus following JoinMap codes for outcrossers. As many metabolites in the phenylpropanoid pathway were mapped, for the purpose of simplicity, metabolites that belong to a similar group of compounds are shown as a group (e.g. phenolic esters is a group of several metabolites). Gene names are abbreviated as: phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumaroyl:CoA-ligase (4CL), chalcone isomerase (CHI), chalcone synthase (CHS), flavonone 3' hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), hydroxycinnamoyl-CoA quinate/shikimate hydroxycinnamoyl transferase (HCT), leucoanthocyanidin 4-reductase (LAR), UDP-glycosyltransferase (UGT), flavonol synthase (FLS), and anthocyanidin synthase (ANS).

Fig. 6.

Genetic linkage maps of ‘Prima’ and ‘Fiesta’ and a physical map of the apple cv. ‘Golden Delicious’ for the mQTL hotspot region on LG16. Procyanidin dimer II was used as a representative for the metabolites that mapped to the LG16 mQTL hotspot. The mQTL regions were genetically mapped as monogenic traits by means of graphical genotyping in both parents and are indicated as horizontal green arrows. The structural gene leucoanthocyanidin reductase (LAR) of the phenylpropanoid pathway appeared to be present in this region, according to the putative genes in the whole genome sequence of cv. ‘Golden Delicious’. Seven putative transcription factor genes including MYB and bHLH were also detected in this region. The structural gene LAR and transcription factor genes are presented in bold text.