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.

Molecular characterization of intergeneric hybrids between Malus and Pyrus

Apple(Malus)and pear(Pyrus)are economically important fruit crops well known for their unique textures,f lavours,and nutritional qualities.Both genera are characterised by a distinct pattern of secondary metabolites,which directly affect not only resistance to certain diseases,but also have significant impacts on the f lavour and nutritional value of the fruit.The identical chromosome numbers,similar genome size,and their recent divergence date,together with DNA markers have shown that apple and pear genomes are highly co-linear.This study utilized comparative genomic approaches,including simple sequence repeats,high resolution single nucleotide polymorphism melting analysis,and single nucleotide polymorphism chip analysis to identify genetic differences among hybrids of Malus and Pyrus,and F2 offspring.This research has demonstrated and validated that these three marker types,along with metabolomics analysis are very powerful tools to detect and confirm hybridity of progeny derived from crosses between apple and pear in both cross directions.Furthermore,this work analysed the genus-specific metabolite patterns and the resistance to fire blight(Erwinia amylovora)in progeny.The findings of this work will enhance and accelerate the breeding of novel tree fruit crops that benefit producers and consumers,by enabling marker assisted selection of desired traits introgressed between pear and apple.

Two quantitative trait loci, Dw1 and Dw2, are primarily responsible for rootstock-induced dwarfing in apple

The apple dwarfing rootstock‘Malling9’(‘M9’)has been used worldwide both to reduce scion vigour and as a genetic source for breeding new rootstocks.Progeny of‘M9’segregate for rootstock-induced dwarfing of the scion,indicating that this trait is controlled by one or more genetic factors.A quantitative trait locus(QTL)analysis of a rootstock population derived from the cross between‘M9’בRobusta5’(non-dwarfing)and grafted with‘Braeburn’scions identified a major QTL(Dw1)on linkage group(LG)5,which exhibits a significant influence on dwarfing of the scion.A smaller-effect QTL affecting dwarfing(Dw2)was identified on LG11,and four minor-effect QTLs were found on LG6,LG9,LG10 and LG12.Phenotypic analysis indicates that the combination of Dw1 and Dw2 has the strongest influence on rootstock-induced dwarfing,and that Dw1 has a stronger effect than Dw2.Genetic markers linked to Dw1 and Dw2 were screened over 41 rootstock accessions that confer a range of effects on scion growth.The majority of the dwarfing and semi-dwarfing rootstock accessions screened carried marker alleles linked to Dw1 and Dw2.This suggests that most apple dwarfing rootstocks have been derived from the same genetic source.

Chromosome-scale scaffolding of the black raspberry (Rubus occidentalis L.) genome based on chromatin interaction data

Black raspberry(Rubus occidentalis L.)is a niche fruit crop valued for its flavor and potential health benefits.The improvement of fruit and cane characteristics via molecular breeding technologies has been hindered by the lack of a high-quality reference genome.The recently released draft genome for black raspberry(ORUS 4115-3)lacks assembly of scaffolds to chromosome scale.We used high-throughput chromatin conformation capture(Hi-C)and Proximity-Guided Assembly(PGA)to cluster and order 9650 out of 11,936 contigs of this draft genome assembly into seven pseudo-chromosomes.The seven pseudo-chromosomes cover~97.2%of the total contig length(~223.8 Mb).Locating existing genetic markers on the physical map resolved multiple discrepancies in marker order on the genetic map.Centromeric regions were inferred from recombination frequencies of genetic markers,alignment of 303 bp centromeric sequence with the PGA,and heat map showing the physical contact matrix over the entire genome.We demonstrate a high degree of synteny between each of the seven chromosomes of black raspberry and a high-quality reference genome for strawberry(Fragaria vesca L.)assembled using only PacBio long-read sequences.We conclude that PGA is a cost-effective and rapid method of generating chromosome-scale assemblies from Illumina short-read sequencing data.

Apple whole genome sequences:recent advances and new prospects

In 2010,a major scientific milestone was achieved for tree fruit crops:publication of the first draft whole genome sequence(WGS)for apple(Malus domestica).This WGS,v1.0,was valuable as the initial reference for sequence information,fine mapping,gene discovery,variant discovery,and tool development.A new,high quality apple WGS,GDDH13 v1.1,was released in 2017 and now serves as the reference genome for apple.Over the past decade,these apple WGSs have had an enormous impact on our understanding of apple biological functioning,trait physiology and inheritance,leading to practical applications for improving this highly valued crop.Causal gene identities for phenotypes of fundamental and practical interest can today be discovered much more rapidly.Genome-wide polymorphisms at high genetic resolution are screened efficiently over hundreds to thousands of individuals with new insights into genetic relationships and pedigrees.High-density genetic maps are constructed efficiently and quantitative trait loci for valuable traits are readily associated with positional candidate genes and/or converted into diagnostic tests for breeders.We understand the species,geographical,and genomic origins of domesticated apple more precisely,as well as its relationship to wild relatives.The WGS has turbo-charged application of these classical research steps to crop improvement and drives innovative methods to achieve more durable,environmentally sound,productive,and consumer-desirable apple production.This review includes examples of basic and practical breakthroughs and challenges in using the apple WGSs.Recommendations for“what’s next”focus on necessary upgrades to the genome sequence data pool,as well as for use of the data,to reach new frontiers in genomics-based scientific understanding of apple.

Validation of SNP markers for fruit quality and disease resistance loci in apple (Malus × domestica Borkh.) using the OpenArray® platform

Genome mapping has promised much to tree fruit breeding during the last 10 years.Nevertheless,one of the greatest challenges remaining to tree fruit geneticists is the translation of trait loci and whole genome sequences into diagnostic genetic markers that are efficient and cost-effective for use by breeders,who must select genetically optimal parents and subsequently select genetically superior individuals among their progeny.To take this translational step,we designed the apple International RosBREED SNP Consortium OpenArray v1.0(IRSCOA v1.0)assay using a set of 128 apple single nucleotide polymorphisms(SNPs)linked to fruit quality and pest and disease resistance trait loci.The Thermo Fisher Scientific OpenArray®technology enables multiplexed screening of SNP markers using a real-time PCR instrument with fluorescent probe-based Taqman®assays.We validated the apple IRSCOA v1.0 multitrait assay by screening 240 phenotyped individuals from the Plant&Food Research apple cultivar breeding programme.This set of individuals comprised commercial and heritage cultivars,elite selections,and families segregating for traits of importance to breeders.In total,33 SNP markers of the IRSCOA v1.0 were validated for use in marker-assisted selection(MAS)for the scab resistances Rvi2/Vh2,Rvi4/Vh4,Rvi6/Vf,fire blight resistance MR5/RLP1,powdery mildew resistance Pl2,fruit firmness,skin colour,flavour intensity,and acidity.The availability of this set of validated trait-associated SNP markers,which can be used individually on multiple genotyping platforms available to various apple breeding programmes or re-designed using the flanking sequences,represents a large translational genetics step from genomics to crop improvement of apple.

Genome mapping of postzygotic hybrid necrosis in an interspecific pear population

Deleterious epistatic interactions in plant inter-and intraspecific hybrids can cause a phenomenon known as hybrid necrosis,characterized by a typical seedling phenotype whose main distinguishing features are dwarfism,tissue necrosis and in some cases lethality.Identification of the chromosome regions associated with this type of incompatibility is important not only to increase our understanding of the evolutionary diversification that led to speciation but also for breeding purposes.Development of molecular markers linked to the lethal genes will allow breeders to avoid incompatible inbred combinations that could affect the expression of important agronomic tratis co-segregating with these genes.Although hybrid necrosis has been reported in several plant taxa,including Rosaceae species,this phenomenon has not been described previously in pear.In the interspecific pear population resulting from a cross between PEAR3(Pyrus bretschneideri×Pyrus communis)and‘Moonglow’(P.communis),we observed two types of hybrid necrosis,expressed at different stages of plant development.Using a combination of previously mapped and newly developed genetic markers,we identified three chromosome regions associated with these two types of lethality,which were genetically independent.One type resulted from a negative epistatic interaction between a locus on linkage group 5(LG5)of PEAR3 and a locus on LG1 of‘Moonglow’,while the second type was due to a gene that maps to LG2 of PEAR3 and which either acts alone or more probably interacts with another gene of unknown location inherited from‘Moonglow’.

Multiple quantitative trait loci contribute to resistance to bacterial canker incited by Pseudomonas syringae pv.actinidiae in kiwifruit(Actinidia chinensis)

Pseudomonas syringae pv.actinidiae(Psa)biovar 3,a virulent,canker-inducing pathogen is an economic threat to the kiwifruit(Actinidia spp.)industry worldwide.The commercially grown diploid(2×)A.chinensis var.chinensis is more susceptible to Psa than tetraploid and hexaploid kiwifruit.However information on the genetic loci modulating Psa resistance in kiwifruit is not available.Here we report mapping of quantitative trait loci(QTLs)regulating resistance to Psa in a diploid kiwifruit population,derived from a cross between an elite Psa-susceptible‘Hort16A’and a resistant male breeding parent P1.Using high-density genetic maps and intensive phenotyping,we identified a single QTL for Psa resistance on Linkage Group(LG)27 of‘Hort16A’revealing 16–19%phenotypic variance and candidate alleles for susceptibility and resistance at this loci.In addition,six minor QTLs were identified in P1 on distinct LGs,exerting 4–9%variance.Resistance in the F1 population is improved by additive effects from‘Hort16A’and P1 QTLs providing evidence that divergent genetic pathways interact to combat the virulent Psa strain.Two different bioassays further identified new QTLs for tissue-specific responses to Psa.The genetic marker at LG27 QTL was further verified for association with Psa resistance in diploid Actinidia chinensis populations.Transcriptome analysis of Psa-resistant and susceptible genotypes in field revealed hallmarks of basal defense and provided candidate RNA-biomarkers for screening for Psa resistance in greenhouse conditions.

A high-throughput S-RNase genotyping method for apple

Knowledge of the genotypes for the self-incompatibility locus(S-locus)in apple varieties and in genotypes being used as parents is critical for breeding and commercial production.We present a high-throughput set of molecular markers for the identification of 13 common S-RNase alleles(S_(1),S_(2),S_(3),S_(5),S_(7),S_(8),S_(9),S_(10),S_(20),S_(2)3,S_(24),S_(25)and S_(28).This set is composed of seven allele-specific quantitative PCR-based High-Resolution Melting assays and four multi-allelic SSR markers.Validation of these markers was performed using 86 apple accessions,including cultivars with known S-genotypes and recent commercial varieties arising from the Plant&Food Research(PFR)cultivar breeding programme.We also characterized the S-genotypes of 183 genotypes representing some of the most valuable parents within PFR’s cultivar breeding programme.The results of this work demonstrate the practical usefulness of this marker set to provide accurate cross-compatibility information to optimise choice of pollenisers in commercial apple orchard design,and to identify compatible parents and guide parental selection when executing apple breeding programmes,to optimise fruit crop yield and quality.