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.

Sequencing a Juglans regia×J.microcarpa hybrid yields high-quality genome assemblies of parental species

Members of the genus Juglans are monecious wind-pollinated trees in the family Juglandaceae with highly heterozygous genomes,which greatly complicates genome sequence assembly.The genomes of interspecific hybrids are usually comprised of haploid genomes of parental species.We exploited this attribute of interspecific hybrids to avoid heterozygosity and sequenced an interspecific hybrid Juglans microcarpa×J.regia using a novel combination of single-molecule sequencing and optical genome mapping technologies.The resulting assemblies of both genomes were remarkably complete including chromosome termini and centromere regions.Chromosome termini consisted of arrays of telomeric repeats about 8 kb long and heterochromatic subtelomeric regions about 10 kb long.The centromeres consisted of arrays of a centromere-specific Gypsy retrotransposon and most contained genes,many of them transcribed.Juglans genomes evolved by a whole-genome-duplication dating back to the Cretaceous-Paleogene boundary and consist of two subgenomes,which were fractionated by numerous short gene deletions evenly distributed along the length of the chromosomes.Fractionation was shown to be asymmetric with one subgenome exhibiting greater gene loss than the other.The asymmetry of the process is ongoing and mirrors an asymmetry in gene expression between the subgenomes.Given the importance of J.microcarpa×J.regia hybrids as potential walnut rootstocks,we catalogued disease resistance genes in the parental genomes and studied their chromosomal distribution.We also estimated the molecular clock rates for woody perennials and deployed them in estimating divergence times of Juglans genomes and those of other woody perennials.

Clarifying sub-genomic positions of QTLs for flowering habit and fruit quality in U.S. strawberry (Fragaria×ananassa) breeding populations using pedigree-based QTL analysis

The cultivated strawberry(Fragaria×ananassa)is consumed worldwide for its flavor and nutritional benefits.Genetic analysis of commercially important traits in strawberry are important for the development of breeding methods and tools for this species.Although several quantitative trait loci(QTL)have been previously detected for fruit quality and flowering traits using low-density genetic maps,clarity on the sub-genomic locations of these QTLs was missing.Recent discoveries in allo-octoploid strawberry genomics led to the development of the IStraw90 single-nucleotide polymorphism(SNP)array,enabling high-density genetic maps and finer resolution QTL analysis.In this study,breeder-specified traits were evaluated in the Eastern(Michigan)and Western(Oregon)United States for a common set of breeding populations during 2 years.Several QTLs were validated for soluble solids content(SSC),fruit weight(FWT),pH and titratable acidity(TA)using a pedigree-based QTL analysis approach.For fruit quality,a QTL for SSC on linkage group(LG)6A,a QTL for FWT on LG 2BII,a QTL for pH on LG 4CII and two QTLs for TA on LGs 2A and 5B were detected.In addition,a large-effect QTL for flowering was detected at the distal end of LG 4A,coinciding with the FaPFRU locus.Marker haplotype analysis in the FaPFRU region indicated that the homozygous recessive genotype was highly predictive of seasonal flowering.SNP probes in the FaPFRU region may help facilitate marker-assisted selection for this trait.

Phoenix phylogeny, and analysis of genetic variation in a diverse collection of date palm(Phoenix dactylifera) and related species

Date palm(Phoenix dactylifera),one of the most ancient crops,is grown commercially in>30 countries.Using whole plastome assemblies,phylogenetic analyses revealed that cultivated date palm accessions share the same clade with Phoenix sylvestris,Phoenix pusilla and Phoenix acaulis,which are native to the Indian subcontinent,and Phoenix caespitosa that is native to the Arabian Peninsula and the deserts of Somalia.Analysis of genetic diversity and genetic relationships among date palm accessions from 13 producing countries involved 195 date palm accessions that were genotyped at 19 microsatellite loci.Extensive genetic diversity was observed,with many accessions heterozygous for most markers in this clonally propagated crop.The average number of alleles per locus(42.1),expected heterozygosity(0.8),observed heterozygosity(0.47)and fixation indices(FST=0.42)demonstrated substantial genetic diversity and population structure.Iraqi accessions were found to have the richest allelic diversity,and the most private alleles.The model-based Bayesian method indicated that these accessions could be broadly divided into two structure groups,one group with predominantly African accessions and another predominantly Asian.Some germplasm,especially from Tunisia and Iraq,deviated from this generalization.Many accessions in the STRUCTURE-derived groups were found to be genetic admixtures,with gene flow between Asian and African groups.Indian and Pakistani date palms were found to be most closely related to North African germplasm.

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.

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.

Genetic and genomic resources for Rubus breeding:a roadmap for the future

Rubus fruits are high-value crops that are sought after by consumers for their flavor,visual appeal,and health benefits.To meet this demand,production of red and black raspberries(R.idaeus L.and R.occidentalis L.),blackberries(R.subgenus Rubus),and hybrids,such as Boysenberry and marionberry,is growing worldwide.Rubus breeding programmes are continually striving to improve flavor,texture,machine harvestability,and yield,provide pest and disease resistance,improve storage and processing properties,and optimize fruits and plants for different production and harvest systems.Breeders face numerous challenges,such as polyploidy,the lack of genetic diversity in many of the elite cultivars,and until recently,the relative shortage of genetic and genomic resources available for Rubus.This review will highlight the development of continually improving genetic maps,the identification of Quantitative Trait Loci(QTL)s controlling key traits,draft genomes for red and black raspberry,and efforts to improve gene models.The development of genetic maps and markers,the molecular characterization of wild species and germplasm,and highthroughput genotyping platforms will expedite breeding of improved cultivars.Fully sequenced genomes and accurate gene models facilitate identification of genes underlying traits of interest and enable gene editing technologies such as CRISPR/Cas9.

Patterns of genomic and phenomic diversity in wine and table grapes

Grapes are one of the most economically and culturally important crops worldwide,and they have been bred for both winemaking and fresh consumption.Here we evaluate patterns of diversity across 33 phenotypes collected over a 17-year period from 580 table and wine grape accessions that belong to one of the world’s largest grape gene banks,the grape germplasm collection of the United States Department of Agriculture.We find that phenological events throughout the growing season are correlated,and quantify the marked difference in size between table and wine grapes.By pairing publicly available historical phenotype data with genome-wide polymorphism data,we identify large effect loci controlling traits that have been targeted during domestication and breeding,including hermaphroditism,lighter skin pigmentation and muscat aroma.Breeding for larger berries in table grapes was traditionally concentrated in geographic regions where Islam predominates and alcohol was prohibited,whereas wine grapes retained the ancestral smaller size that is more desirable for winemaking in predominantly Christian regions.We uncover a novel locus with a suggestive association with berry size that harbors a signature of positive selection for larger berries.Our results suggest that religious rules concerning alcohol consumption have had a marked impact on patterns of phenomic and genomic diversity in grapes.

A roadmap for research in octoploid strawberry

The cultivated strawberry(Fragaria×ananassa)is an allo-octoploid species,originating nearly 300 years ago from wild progenitors from the Americas.Since that time the strawberry has become the most widely cultivated fruit crop in the world,universally appealing due to its sensory qualities and health benefits.The recent publication of the first highquality chromosome-scale octoploid strawberry genome(cv.Camarosa)is enabling rapid advances in genetics,stimulating scientific debate and provoking new research questions.In this forward-looking review we propose avenues of research toward new biological insights and applications to agriculture.Among these are the origins of the genome,characterization of genetic variants,and big data approaches to breeding.Key areas of research in molecular biology will include the control of flowering,fruit development,fruit quality,and plant–pathogen interactions.In order to realize this potential as a global community,investments in genome resources must be continually augmented.

Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi,and P.pini in Juglans microcarpa×J.regia hybrids

Soil-borne plant pathogens represent a serious threat that undermines commercial walnut(Juglans regia)production worldwide.Crown gall,caused by Agrobacterium tumefaciens,and Phytophthora root and crown rots,caused by various Phytophthora spp.,are among the most devastating walnut soil-borne diseases.A recognized strategy to combat soil-borne diseases is adoption of resistant rootstocks.Here,resistance to A.tumefaciens,P.cinnamomi,and P.pini is mapped in the genome of Juglans microcarpa,a North American wild relative of cultivated walnut.Half-sib J.microcarpa mother trees DJUG 31.01 and DJUG 31.09 were crossed with J.regia cv.Serr,producing 353 and 400 hybrids,respectively.Clonally propagated hybrids were genotyped by sequencing to construct genetic maps for the two populations and challenged with the three pathogens.Resistance to each of the three pathogens was mapped as a major QTL on the long arm of J.microcarpa chromosome 4D and was associated with the same haplotype,designated as haplotype b,raising the possibility that the two mother trees were heterozygous for a single Mendelian gene conferring resistance to all three pathogens.The deployment of this haplotype in rootstock breeding will facilitate breeding of a walnut rootstock resistant to both crown gall and Phytophthora root and crown rots.

Huanglongbing:An overview of a complex pathosystem ravaging the world's citrus

Citrus huanglongbing（HLB） has become a major disease and limiting factor of production in citrus areas that have become infected. The destruction to the affected citrus industries has resulted in a tremendous increase to support research that in return has resulted in significant information on both applied and basic knowledge concerning this important disease to the global citrus industry. Recent research indicates the relationship between citrus and the causal agent of HLB is shaped by multiple elements, in which host defense responses may also play an important role. This review is intended to provide an overview of the importance of HLB to a wider audience of plant biologists. Recent advances on host-pathogen interactions, population genetics and vectoring of the causal agent are discussed.