Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s,respectively,and in part owing to their f lavors and health-promoting benefits are now cultivated and consumed worldwide.The industry continues to face a wide variety of production challenges(e.g.disease pressures),as well as a demand for higher-yielding cultivars with improved fruit quality characteristics.Unfortunately,molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops.Here,we describe the construction and analysis of the first pangenome for both blueberry and cranberry.Our analysis of these pangenomes revealed both crops exhibit great genetic diversity,including the presence-absence variation of 48.4%genes in highbush blueberry and 47.0%genes in cranberry.Auxiliary genes,those not shared by all cultivars,are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites,including compounds previously associated with improving fruit quality traits.The discovery of thousands of genes,not present in the previous reference genomes for blueberry and cranberry,will serve as the basis of future research and as potential targets for future breeding efforts.The pangenome,as a multiple-sequence alignment,as well as individual annotated genomes,are publicly available for analysis on the Genome Database for Vaccinium-a curated and integrated web-based relational database.Lastly,the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

Differential regulation of the anthocyanin profile in purple kiwifruit(Actinidia species)

Anthocyanins are a group of secondary metabolites that colour fruit and flowers orange,red,purple or blue depending on a number of factors,such as the basic structure,co-pigmentation,metal ion complexation and vacuolar pH.The biosynthesis of anthocyanin is regulated at the transcriptional level by a group of transcription factors,the MYB–bHLH–WD40(MBW)complex.In this study,the purple colouration in several kiwifruit(Actinidia)species was identified and characterised as red cyanidin-based and blue delphinidin-based anthocyanins.The differential pigmentation in the skin and flesh can be attributed to the differential ratio of cyanidin and delphinidin derivatives accumulated in the total anthocyanin profile.The expression of anthocyanin biosynthetic genes chalcone synthase(CHS),flavonoid 3-O-glucosyltransferase(F3GT),flavonoid 3′-hydroxylase(F3′H)and flavonoid 3′5′-hydroxylase(F3′5′H)is crucial for anthocyanin accumulation.However,the balance of expression of the F3′H and F3′5′H genes appears responsible for the ratio of cyanidin and delphinidin derivatives,while a lack of CHS,F3GT and MYB110 expression is responsible for a lack of total anthocyanins.The transcriptional regulation of the F3′H and F3′5′H promoters by the R2R3 MYB transcription factor MYB110 is markedly different in tobacco transient assays.When kiwifruit MYB10 or MYB110 are over-expressed in Actinidia chinensis both cyanidin-based and delphinidin-based anthocyanins are elevated,but F3′H and F3′5′H genes are not strongly correlated with MYB expression.These results suggest that the core kiwifruit anthocyanin pathway genes are dependent on characterised MYB transcription factors,while other regulatory proteins are more directly responsible for the expression of the F3′H and F3′5′H genes.

Genomic analysis uncovers functional variation in the C-terminus of anthocyanin-activating MYB transcription factors

MYB transcription factors regulate diverse aspects of plant development and secondary metabolism,often by partnering in transcriptional regulatory complexes.Here,we harness genomic resources to identify novel MYBs,thereby producing an updated eudicot MYB phylogeny with revised relationships among subgroups as well as new information on sequence variation in the disordered C-terminus of anthocyanin-activating MYBs.BLAST®and hidden Markov model scans of gene annotations identified a total of 714 MYB transcription factors across the genomes of four crops that span the eudicots:apple,grape,kiwifruit and tomato.Codon model-based phylogenetic inference identified novel members of previously defined subgroups,and the function of specific anthocyanin-activating subgroup 6 members was assayed transiently in tobacco leaves.Sequence conservation within subgroup 6 highlighted one previously described and two novel short linear motifs in the disordered C-terminal region.The novel motifs have a mix of hydrophobic and acidic residues and are predicted to be relatively ordered compared with flanking protein sequences.Comparison of motifs with the Eukaryotic Linear Motif database suggests roles in protein–protein interaction.Engineering of motifs and their flanking regions from strong anthocyanin activators into weak activators,and vice versa,affected function.We conclude that,although the MYB C-terminal sequence diverges greatly even within MYB clades,variation within the C-terminus at and near relatively ordered regions offers opportunities for exploring MYB function and developing superior alleles for plant breeding.

Differential regulation of triterpene biosynthesis induced by an early failure in cuticle formation in apple

Waxy apple cuticles predominantly accumulate ursane-type triterpenes,but the profile shifts with the induction of skin russeting towards lupane-type triterpenes.We previously characterised several key enzymes in the ursane-type and lupane-type triterpene pathways,but this switch in triterpene metabolism associated with loss of cuticle integrity is not fully understood.To analyse the relationship between triterpene biosynthesis and russeting,we used microscopy,RNA-sequencing and metabolite profiling during apple fruit development.We compared the skin of three genetically-close clones of‘Golden Delicious’(with waxy,partially russeted and fully russeted skin).We identified a unique molecular profile for the russet clone,including low transcript abundance of multiple cuticle-specific metabolic pathways in the early stages of fruit development.Using correlation analyses between gene transcription and metabolite concentration we found MYB transcription factors strongly associated with lupane-type triterpene biosynthesis.We showed how their transcription changed with the onset of cuticle cracking followed by russeting and that one factor,MYB66,was able to bind the promoter of the oxidosqualene cyclase OSC5,to drive the production of lupeol derivatives.These results provide insights into the breakdown of cuticle integrity leading to russet and how this drives MYB-regulated changes to triterpene biosynthesis.

A MYB-INDUCED MECHANISM FOR ENHANCED ASCORBATE IN RED-FLESHED APPLES

The results presented in"The anthocyanin biosynthetic regulator MdMYBl positively regulates ascorbic acid biosynthesis in apple"(An et al.#this issue)provide evidence for a new mechanism for the elevation of ascorbate concentration in apple.Using a red-fleshed apple breeding population,the authors show how the anthocyanin-regulating MYB transcription factor,MdMYBl,also increases ascorbate concentrations by directly activating transcription of the dehydroascorbate reductase gene MdDHAR.This gene recycles oxidized ascorbate back to ascorbate,leading to elevated concentrations of vitamin C.These red-fleshed apples have enhanced concentrations of both anthocyanins and ascorbate,both of which are appealing traits for the development of healthier apples.