Metabolite-based genome-wide association studies enable the dissection of the genetic bases of flavonoids, betaine and spermidine in wolfberry (Lycium).

Wolfberry is a plant with medicinal and food values. However, its bioactive ingredients and the corresponding genetic bases have not been determined. Here, we de novo generated a chromosome-level genome assembly for wolfberry, yielding a genome sequence of ~1.77 Gb with contig N50 of 50.55 Mb and 39 224 predicted gene models. A variation map, using 307 re-sequenced accessions, was called based on this genome assembly. Furthermore, the fruit metabolome of these accessions was profiled using 563 annotated metabolites, which separated Lycium barbarum L. and non-L. barbarum L. The flavonoids, coumarins, alkaloids and nicotinic acid contents were higher in the former than in the latter. A metabolite-based genome-wide association study mapped 156 164 significant single nucleotide polymorphisms corresponding to 340 metabolites. This included 19 219 unique lead single nucleotide polymorphisms in 1517 significant association loci, of which three metabolites, flavonoids, betaine and spermidine, were highlighted. Two candidate genes, LbUGT (evm.TU.chr07.2692) and LbCHS (evm.TU.chr07.2738), with non-synonymous mutations, were associated with the flavonoids content. LbCHS is a structural gene that interacts with a nearby MYB transcription factor (evm.TU.chr07.2726) both in L. barbarum and L. ruthenicum. Thus, these three genes might be involved in the biosynthesis/metabolism of flavonoids. LbSSADH (evm.TU.chr09.627) was identified as possibly participating in betaine biosynthesis/metabolism. Four lycibarbarspermidines (E-G and O) were identified, and only the lycibarbarspermidines O content was higher in L. barbarum varieties than in non-L. barbarum varieties. The evm.TU.chr07.2680 gene associated with lycibarbarspermidines O was annotated as an acetyl-CoA-benzylalcohol acetyltransferase, suggesting that it is a candidate gene for spermidine biosynthesis. These results provide novel insights into the specific metabolite profile of non-L. barbarum L. and the genetic bases of flavonoids, betaine and spermidine biosynthesis/metabolism.

ERF5.1 modulates carotenoid accumulation by interacting with CCD4.1 in Lycium.

Carotenoids are important natural pigments and have medical and health functions for humans. Carotenoid cleavage dioxygenase 4 (CCD4) and ethylene responsive factor (ERF) participate in carotenoid metabolism, but their roles in Lycium have not been discovered. Here, we annotated LbCCDs from the Lycium reference genome and found that LbCCD4.1 expression was significantly correlated with the carotenoid metabolites during Lycium five fruit developmental stages. Over-expression of LbCCD4.1 in NQ's leaves resulted in a series of significantly lower contents of carotenoid metabolites, including ß-carotene and ß-cryptoxanthin. Moreover, LbERF5.1, a transcription factor belonging to the ERF family that was located in the nucleus, was isolated. Significant reductions in the carotenoids, especially lutein, violaxanthin and their derivatives, were observed in over-expressing ERF5.1 transgenic NQ's leaves. Over-expression or virus-induced gene silencing of LbERF5.1 in NQ's leaves induced a consistent up- or down-expression, respectively, of LbCCD4.1. Furthermore, yeast one-hybrid and dual-luciferase reporter assays showed that ERF5.1 interacted with the promoter of CCD4.1 to increase its expression, and LbERF5.1 could bind to any one of the three predicted binding sites in the promoter of LbCCD4.1. A transcriptome analysis of LbERF5.1 and LbCCD4.1 over-expressed lines showed similar global transcript expression, and geranylgeranyl diphosphate synthase, phytoene synthase, lycopene δ-cyclase cytochrome, cytochrome P450-type monooxygenase 97A, cytochrome P450-type monooxygenase 97C, and zeaxanthin epoxidase in the carotenoid biosynthesis pathway were differentially expressed. In summary, we uncovered a novel molecular mechanism of carotenoid accumulation that involved an interaction between ERF5.1 and CCD4.1, which may be used to enhance carotenoid in Lycium.