Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum).

Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter-specific, and inter-ploidy admixture of its genome and lack of in depth genome-wide inheritance and comparative structural studies. Here we describe a new high-quality, phased, chromosome-scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high-density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species. Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr-6 and one of chr-10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter-crossable wild diploid species. These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this crop.

Unraveling the Complex Hybrid Ancestry and Domestication History of Cultivated Strawberry.

Cultivated strawberry (Fragaria × ananassa) is one of our youngest domesticates, originating in early eighteenth-century Europe from spontaneous hybrids between wild allo-octoploid species (Fragaria chiloensis and Fragaria virginiana). The improvement of horticultural traits by 300 years of breeding has enabled the global expansion of strawberry production. Here, we describe the genomic history of strawberry domestication from the earliest hybrids to modern cultivars. We observed a significant increase in heterozygosity among interspecific hybrids and a decrease in heterozygosity among domesticated descendants of those hybrids. Selective sweeps were found across the genome in early and modern phases of domestication-59-76% of the selectively swept genes originated in the three less dominant ancestral subgenomes. Contrary to the tenet that genetic diversity is limited in cultivated strawberry, we found that the octoploid species harbor massive allelic diversity and that F. × ananassa harbors as much allelic diversity as either wild founder. We identified 41.8 M subgenome-specific DNA variants among resequenced wild and domesticated individuals. Strikingly, 98% of common alleles and 73% of total alleles were shared between wild and domesticated populations. Moreover, genome-wide estimates of nucleotide diversity were virtually identical in F. chiloensis,F. virginiana, and F. × ananassa (π = 0.0059-0.0060). We found, however, that nucleotide diversity and heterozygosity were significantly lower in modern F. × ananassa populations that have experienced significant genetic gains and have produced numerous agriculturally important cultivars.

Mapping the black spot resistance locus Rdr3 in the shrub rose 'George Vancouver' allows for the development of improved diagnostic markers for DNA-informed breeding.

KEY MESSAGE: Rdr3 is a novel resistance gene of black spot in roses that maps to a chromosome 6 homolog. A new DNA test was developed and can be used to pyramid black spot resistance in roses. Diplocarpon rosae, the cause of rose black spot, is one of the most devastating foliar pathogens of cultivated roses (Rosa spp.). The primary method of disease control is fungicides, and they are viewed unfavorably by home gardeners due to potential environmental and health impacts. Planting rose cultivars with genetic resistance to black spot can reduce many of the fungicide applications needed in an integrated pest management system. To date, four resistance genes have been identified in roses (Rdr1, Rdr2, Rdr3, and Rdr4). Rdr3 was never mapped and is thought to be unique from Rdr1 and Rdr2. It is unknown whether it is an allele of Rdr4. To assess the novelty of Rdr3, a mapping population was created by crossing the Rdr3 containing cultivar George Vancouver with the susceptible cultivar Morden Blush. The mapping population was genotyped with the WagRhSNP 68 K Axiom array and mapped using the 'polymapR' package. Rdr3 was mapped to a chromosome 6 homolog confirming it is different from Rdr1 and Rdr2, found on chromosome 1, and from Rdr4, found on chromosome 5. The mapping information was used in conjunction with the Rosa chinensis genome assembly to develop new tightly linked SSRs for marker-assisted breeding. Three markers were able to predict the presence of Rdr3 in a 63-cultivar validation set. Additionally, 12 cultivars appear to have resistance genes other than Rdr3. The improved diagnostic markers will be a great asset to the rose-breeding community toward developing new black spot-resistant cultivars.

Dissecting Genetic Resistance to Fire Blight in Three Pear Populations.

Fire blight, caused by the bacterial pathogen Erwinia amylovora, is a persistent problem for pear (Pyrus spp.) growers in the United States. Growing resistant cultivars is one of the best options for managing fire blight. The cultivars Potomac and Old Home and the selection NJA2R59T69 display resistance to fire blight. As such, three mapping populations (El Dorado × Potomac, Old Home × Bartlett, and NJA2R59T69 × Bartlett) were developed to identify genomic regions associated with resistance to fire blight. Progeny were phenotyped during 2017 and 2018 by inoculating multiple actively growing shoots of field-grown seedling trees with E. amylovora isolate E153n via the cut-leaf method. Genotyping was conducted using the recently developed Axiom Pear 70 K Genotyping Array and chromosomal linkage groups were created for each population. An integrated two-way pseudo-testcross approach was used to map quantitative trait loci (QTLs). Resistance QTLs were identified on chromosome 2 for each population. The QTLs identified in the El Dorado × Potomac and Old Home × Bartlett populations are in the same region as QTLs that were previously identified in Harrow Sweet and Moonglow. The QTL in NJA2R59T69 mapped proximally to the previously identified QTLs and originated from an unknown Asian or occidental source. Future research will focus on further characterizing the resistance regions and developing tools for DNA-informed breeding.

Development of a highly efficient Axiom™ 70 K SNP array for Pyrus and evaluation for high-density mapping and germplasm characterization.

BACKGROUND: Both a source of diversity and the development of genomic tools, such as reference genomes and molecular markers, are equally important to enable faster progress in plant breeding. Pear (Pyrus spp.) lags far behind other fruit and nut crops in terms of employment of available genetic resources for new cultivar development. To address this gap, we designed a high-density, high-efficiency and robust single nucleotide polymorphism (SNP) array for pear, with the main objectives of conducting genetic diversity and genome-wide association studies. RESULTS: By applying a two-step design process, which consisted of the construction of a first 'draft' array for the screening of a small subset of samples, we were able to identify the most robust and informative SNPs to include in the Applied Biosystems™ Axiom™ Pear 70 K Genotyping Array, currently the densest SNP array for pear. Preliminary evaluation of this 70 K array in 1416 diverse pear accessions from the USDA National Clonal Germplasm Repository (NCGR) in Corvallis, OR identified 66,616 SNPs (93% of all the tiled SNPs) as high quality and polymorphic (PolyHighResolution). We further used the Axiom Pear 70 K Genotyping Array to construct high-density linkage maps in a bi-parental population, and to make a direct comparison with available genotyping-by-sequencing (GBS) data, which suggested that the SNP array is a more robust method of screening for SNPs than restriction enzyme reduced representation sequence-based genotyping. CONCLUSIONS: The Axiom Pear 70 K Genotyping Array, with its high efficiency in a widely diverse panel of Pyrus species and cultivars, represents a valuable resource for a multitude of molecular studies in pear. The characterization of the USDA-NCGR collection with this array will provide important information for pear geneticists and breeders, as well as for the optimization of conservation strategies for Pyrus.

Development of a reliable Corylus sp. reference database through the implementation of a DNA fingerprinting test.

MAIN CONCLUSION: This DNA fingerprinting test confirmed 195 unique Corylus sp. accessions that were used to build a reference database for identity verification of unknown hazelnut trees from three locations in Ontario. Hazelnut is one of the most profitable tree nuts worldwide. Development of a hazelnut industry in Ontario is urgently required, but economically important cultivars must be genetically verified first in order to meet industry standards. Traditional methods for cultivar identification are largely trait-based and unreliable. In this study, a multiplexed fingerprinting test was modified to allow for hazelnut cultivar discrimination at the DNA level. Fourteen highly polymorphic SSR markers covering the 11 linkage groups of Corylus genome were PCR amplified in multiplex using fluorescent-labelled primers. PCR conditions and primer physical properties were optimized to generate a clear signal for each locus. The 14 SSRs were used to fingerprint 195 unique Corylus accessions collected from the USDA-NCGR. Fragment sizes were subjected to a UPGMA clustering analysis which separated Corylus accessions based on species and geographic origin. For validation purposes, hazelnut leaves from three locations in Ontario were collected for identity verification using this DNA fingerprinting test. As a result, 33.3% of the unknown trees were duplicates of seven distinct genotypes and a small percentage (8.3%) of these were identical to reference Corylus hybrids. These results reflect common mislabelling issues and genotype duplications that can prevent a uniform plant propagation system. Implementation of this test together with the addition of more unique accessions to the reference database will help verification of trueness-to-type of economically important cultivars for the hazelnut industry.

The genome of black raspberry (Rubus occidentalis).

Black raspberry (Rubus occidentalis) is an important specialty fruit crop in the US Pacific Northwest that can hybridize with the globally commercialized red raspberry (R. idaeus). Here we report a 243 Mb draft genome of black raspberry that will serve as a useful reference for the Rosaceae and Rubus fruit crops (raspberry, blackberry, and their hybrids). The black raspberry genome is largely collinear to the diploid woodland strawberry (Fragaria vesca) with a conserved karyotype and few notable structural rearrangements. Centromeric satellite repeats are widely dispersed across the black raspberry genome, in contrast to the tight association with the centromere observed in most plants. Among the 28 005 predicted protein-coding genes, we identified 290 very recent small-scale gene duplicates enriched for sugar metabolism, fruit development, and anthocyanin related genes which may be related to key agronomic traits during black raspberry domestication. This contrasts patterns of recent duplications in the wild woodland strawberry F. vesca, which show no patterns of enrichment, suggesting gene duplications contributed to domestication traits. Expression profiles from a fruit ripening series and roots exposed to Verticillium dahliae shed insight into fruit development and disease response, respectively. The resources presented here will expedite the development of improved black and red raspberry, blackberry and other Rubus cultivars.

FaRXf1: a locus conferring resistance to angular leaf spot caused by Xanthomonas fragariae in octoploid strawberry.

KEY MESSAGE: Angular leaf spot is a devastating bacterial disease of strawberry. Resistance from two wild accessions is highly heritable and controlled by a major locus on linkage group 6D. Angular leaf spot caused by Xanthomonas fragariae is the only major bacterial disease of cultivated strawberry (Fragaria ×ananassa). While this disease may cause reductions of up to 8 % of marketable yield in Florida winter annual production, no resistant cultivars have been commercialized. Wild accessions US4808 and US4809 were previously identified as resistant to the four genetic clades of X. fragariae, and introgression of the trait into commercial quality perennial-type germplasm was initiated. Previous reports indicated high heritability for the trait but proposed both single-locus and multi-locus inheritance models. The objective of this study was to determine the mode of inheritance of resistance, to identify causal loci, and to begin introgression of resistance into Florida-adapted germplasm. Resistance was observed in two years of field trials with inoculated plants that assayed four full-sib families descended from US4808 to US4809. Resistance segregated 1:1 in all families indicating control by a dominant allele at a single locus. Using a selective genotyping approach with the IStraw90 Axiom(®) SNP array and pedigree-based QTL detection, a single major-effect QTL was identified in two full-sib families, one descended from each resistant accession. High-resolution melt curve analysis validated the presence of the QTL in separate populations. The QTL was delimited to the 33.1-33.6 Mbp (F. vesca vesca v1.1 reference) and 34.8-35.3 Mbp (F. vesca bracteata v2.0 reference) regions of linkage group 6D for both resistance sources and was designated FaRXf1. Characterization of this locus will facilitate marker-assisted selection toward the development of resistant cultivars.

Development and preliminary evaluation of a 90 K Axiom® SNP array for the allo-octoploid cultivated strawberry Fragaria × ananassa.

BACKGROUND: A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca 'Hawaii 4' reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array. RESULTS: About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing "haploSNPs" (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative "codon-based" SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix's "SNPolisher" R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family 'Holiday' × 'Korona' with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM. CONCLUSIONS: The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array's high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

A genetic linkage map of black raspberry (Rubus occidentalis) and the mapping of Ag(4) conferring resistance to the aphid Amphorophora agathonica.

KEY MESSAGE: We have constructed a densely populated, saturated genetic linkage map of black raspberry and successfully placed a locus for aphid resistance. Black raspberry (Rubus occidentalis L.) is a high-value crop in the Pacific Northwest of North America with an international marketplace. Few genetic resources are readily available and little improvement has been achieved through breeding efforts to address production challenges involved in growing this crop. Contributing to its lack of improvement is low genetic diversity in elite cultivars and an untapped reservoir of genetic diversity from wild germplasm. In the Pacific Northwest, where most production is centered, the current standard commercial cultivar is highly susceptible to the aphid Amphorophora agathonica Hottes, which is a vector for the Raspberry mosaic virus complex. Infection with the virus complex leads to a rapid decline in plant health resulting in field replacement after only 3-4 growing seasons. Sources of aphid resistance have been identified in wild germplasm and are used to develop mapping populations to study the inheritance of these valuable traits. We have constructed a genetic linkage map using single-nucleotide polymorphism and transferable (primarily simple sequence repeat) markers for F1 population ORUS 4305 consisting of 115 progeny that segregate for aphid resistance. Our linkage map of seven linkage groups representing the seven haploid chromosomes of black raspberry consists of 274 markers on the maternal map and 292 markers on the paternal map including a morphological locus for aphid resistance. This is the first linkage map of black raspberry and will aid in developing markers for marker-assisted breeding, comparative mapping with other Rubus species, and enhancing the black raspberry genome assembly.

Intersectional Hybrids between Darrow's Blueberry (V. darrowii Camp) and Lingonberry (V. vitis-idaea L.).

An initial cross of V. darrowii 'Johnblue' (Darrow's blueberry) × V. vitis-idaea 'Red Sunset' (lingonberry) produced more than 30 true intersectional diploid hybrids as confirmed by molecular markers. The most vigorous of these hybrids was extensively evaluated. This hybrid, US 2535-A, was floriferous and morphologically intermediate to the respective parents. Examination of pollen suggested low male fertility. Numerous crosses using the hybrid as a female reflected similarly low fertility and potential crossing barriers. Stylar examination suggested blockage of pollen tube growth in self-pollinations and significantly retarded growth in backcross pollinations. Nonetheless, two confirmed hybrid offspring were produced using the F1 hybrid as a female in crosses with V. vitis-idaea and V. darrowii, respectively. In a second set of crosses utilizing additional V. darrowii and V. vitis-idaea genotypes, another 23 verified hybrids in seven parental combinations were produced. Hybrids such as the ones presented offer the potential for generating de novo interspecific fruit types in blueberry and/or broadening the adaptation of lingonberry.

Saturated linkage map construction in Rubus idaeus using genotyping by sequencing and genome-independent imputation.

BACKGROUND: Rapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Recently, robust genotyping methods based on high-throughput sequencing were developed, which provide high marker density, but result in some genotype errors and a large number of missing genotype values. Imputation can reduce the number of missing values and can correct genotyping errors, but current methods of imputation require a reference genome and thus are not an option for most species. RESULTS: Genotyping by Sequencing (GBS) was used to produce highly saturated maps for a R. idaeus pseudo-testcross progeny. While low coverage and high variance in sequencing resulted in a large number of missing values for some individuals, a novel method of imputation based on maximum likelihood marker ordering from initial marker segregation overcame the challenge of missing values, and made map construction computationally tractable. The two resulting parental maps contained 4521 and 2391 molecular markers spanning 462.7 and 376.6 cM respectively over seven linkage groups. Detection of precise genomic regions with segregation distortion was possible because of map saturation. Microsatellites (SSRs) linked these results to published maps for cross-validation and map comparison. CONCLUSIONS: GBS together with genome-independent imputation provides a rapid method for genetic map construction in any pseudo-testcross progeny. Our method of imputation estimates the correct genotype call of missing values and corrects genotyping errors that lead to inflated map size and reduced precision in marker placement. Comparison of SSRs to published R. idaeus maps showed that the linkage maps constructed with GBS and our method of imputation were robust, and marker positioning reliable. The high marker density allowed identification of genomic regions with segregation distortion in R. idaeus, which may help to identify deleterious alleles that are the basis of inbreeding depression in the species.

Insights into phylogeny, sex function and age of Fragaria based on whole chloroplast genome sequencing.

The cultivated strawberry is one of the youngest domesticated plants, developed in France in the 1700s from chance hybridization between two western hemisphere octoploid species. However, little is known about the evolution of the species that gave rise to this important fruit crop. Phylogenetic analysis of chloroplast genome sequences of 21 Fragaria species and subspecies resolves the western North American diploid F. vesca subsp. bracteata as sister to the clade of octoploid/decaploid species. No extant tetraploids or hexaploids are directly involved in the maternal ancestry of the octoploids. There is strong geographic segregation of chloroplast haplotypes in subsp. bracteata, and the gynodioecious Pacific Coast populations are implicated as both the maternal lineage and the source of male-sterility in the octoploid strawberries. Analysis of sexual system evolution in Fragaria provides evidence that the loss of male and female function can follow polyploidization, but does not seem to be associated with loss of self-incompatibility following genome doubling. Character-state mapping provided insight into sexual system evolution and its association with loss of self-incompatibility and genome doubling/merger. Fragaria attained its circumboreal and amphitropical distribution within the past one to four million years and the rise of the octoploid clade is dated at 0.372-2.05 million years ago.

The first genetic map of the American cranberry: exploration of synteny conservation and quantitative trait loci.

The first genetic map of cranberry (Vaccinium macrocarpon) has been constructed, comprising 14 linkage groups totaling 879.9 cM with an estimated coverage of 82.2 %. This map, based on four mapping populations segregating for field fruit-rot resistance, contains 136 distinct loci. Mapped markers include blueberry-derived simple sequence repeat (SSR) and cranberry-derived sequence-characterized amplified region markers previously used for fingerprinting cranberry cultivars. In addition, SSR markers were developed near cranberry sequences resembling genes involved in flavonoid biosynthesis or defense against necrotrophic pathogens, or conserved orthologous set (COS) sequences. The cranberry SSRs were developed from next-generation cranberry genomic sequence assemblies; thus, the positions of these SSRs on the genomic map provide information about the genomic location of the sequence scaffold from which they were derived. The use of SSR markers near COS and other functional sequences, plus 33 SSR markers from blueberry, facilitates comparisons of this map with maps of other plant species. Regions of the cranberry map were identified that showed conservation of synteny with Vitis vinifera and Arabidopsis thaliana. Positioned on this map are quantitative trait loci (QTL) for field fruit-rot resistance (FFRR), fruit weight, titratable acidity, and sound fruit yield (SFY). The SFY QTL is adjacent to one of the fruit weight QTL and may reflect pleiotropy. Two of the FFRR QTL are in regions of conserved synteny with grape and span defense gene markers, and the third FFRR QTL spans a flavonoid biosynthetic gene.

A multiplexed plant-animal SNP array for selective breeding and species conservation applications.

Reliable and high-throughput genotyping platforms are of immense importance for identifying and dissecting genomic regions controlling important phenotypes, supporting selection processes in breeding programs, and managing wild populations and germplasm collections. Amongst available genotyping tools, single nucleotide polymorphism arrays have been shown to be comparatively easy to use and generate highly accurate genotypic data. Single-species arrays are the most commonly used type so far; however, some multi-species arrays have been developed for closely related species that share single nucleotide polymorphism markers, exploiting inter-species cross-amplification. In this study, the suitability of a multiplexed plant-animal single nucleotide polymorphism array, including both closely and distantly related species, was explored. The performance of the single nucleotide polymorphism array across species for diverse applications, ranging from intra-species diversity assessments to parentage analysis, was assessed. Moreover, the value of genotyping pooled DNA of distantly related species on the single nucleotide polymorphism array as a technique to further reduce costs was evaluated. Single nucleotide polymorphism performance was generally high, and species-specific single nucleotide polymorphisms proved suitable for diverse applications. The multi-species single nucleotide polymorphism array approach reported here could be transferred to other species to achieve cost savings resulting from the increased throughput when several projects use the same array, and the pooling technique adds another highly promising advancement to additionally decrease genotyping costs by half.

An affordable and convenient diagnostic marker to identify male and female hop plants.

Hop production utilizes exclusively female plants, whereas male plants only serve to generate novel variation within breeding programs through crossing. Currently, hop lacks a rapid and accurate diagnostic marker to determine whether plants are male or female. Without a diagnostic marker, breeding programs may take 1-2 years to determine the sex of new seedlings. Previous research on sex-linked markers was restricted to specific populations or breeding programs and therefore had limited transferability or suffered from low scalability. A large collection of 765 hop genotypes with known sex phenotypes, genotyping-by-sequencing, and genome-wide association mapping revealed a highly significant marker on the sex chromosome (LOD score = 208.7) that predicted sex within our population with 96.2% accuracy. In this study, we developed a PCR allele competitive extension (PACE) assay for the diagnostic SNP and tested three quick DNA extraction methodologies for rapid, high-throughput genotyping. Additionally, the marker was validated in a separate population of 94 individuals from 15 families from the USDA-ARS hop breeding program in Prosser, WA with 96% accuracy. This diagnostic marker is located in a gene predicted to encode the basic helix-loop-helix transcription factor protein, a family of proteins that have been previously implicated in male sterility in a variety of plant species, which may indicate a role in determining hop sex. The marker is diagnostic, accurate, affordable, and highly scalable and has the potential to improve efficiency in hop breeding.

A chromosome-length genome assembly and annotation of blackberry (Rubus argutus, cv. "Hillquist").

Blackberries (Rubus spp.) are the fourth most economically important berry crop worldwide. Genome assemblies and annotations have been developed for Rubus species in subgenus Idaeobatus, including black raspberry (R. occidentalis), red raspberry (R. idaeus), and R. chingii, but very few genomic resources exist for blackberries and their relatives in subgenus Rubus. Here we present a chromosome-length assembly and annotation of the diploid blackberry germplasm accession "Hillquist" (R. argutus). "Hillquist" is the only known source of primocane-fruiting (annual-fruiting) in tetraploid fresh-market blackberry breeding programs and is represented in the pedigree of many important cultivars worldwide. The "Hillquist" assembly, generated using Pacific Biosciences long reads scaffolded with high-throughput chromosome conformation capture sequencing, consisted of 298 Mb, of which 270 Mb (90%) was placed on 7 chromosome-length scaffolds with an average length of 38.6 Mb. Approximately 52.8% of the genome was composed of repetitive elements. The genome sequence was highly collinear with a novel maternal haplotype-resolved linkage map of the tetraploid blackberry selection A-2551TN and genome assemblies of R. chingii and red raspberry. A total of 38,503 protein-coding genes were predicted, of which 72% were functionally annotated. Eighteen flowering gene homologs within a previously mapped locus aligning to an 11.2 Mb region on chromosome Ra02 were identified as potential candidate genes for primocane-fruiting. The utility of the "Hillquist" genome has been demonstrated here by the development of the first genotyping-by-sequencing-based linkage map of tetraploid blackberry and the identification of possible candidate genes for primocane-fruiting. This chromosome-length assembly will facilitate future studies in Rubus biology, genetics, and genomics and strengthen applied breeding programs.

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 flavors 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.

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 flavors 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.

Generation and analysis of blueberry transcriptome sequences from leaves, developing fruit, and flower buds from cold acclimation through deacclimation.

BACKGROUND: There has been increased consumption of blueberries in recent years fueled in part because of their many recognized health benefits. Blueberry fruit is very high in anthocyanins, which have been linked to improved night vision, prevention of macular degeneration, anti-cancer activity, and reduced risk of heart disease. Very few genomic resources have been available for blueberry, however. Further development of genomic resources like expressed sequence tags (ESTs), molecular markers, and genetic linkage maps could lead to more rapid genetic improvement. Marker-assisted selection could be used to combine traits for climatic adaptation with fruit and nutritional quality traits. RESULTS: Efforts to sequence the transcriptome of the commercial highbush blueberry (Vaccinium corymbosum) cultivar Bluecrop and use the sequences to identify genes associated with cold acclimation and fruit development and develop SSR markers for mapping studies are presented here. Transcriptome sequences were generated from blueberry fruit at different stages of development, flower buds at different stages of cold acclimation, and leaves by next-generation Roche 454 sequencing. Over 600,000 reads were assembled into approximately 15,000 contigs and 124,000 singletons. The assembled sequences were annotated and functionally mapped to Gene Ontology (GO) terms. Frequency of the most abundant sequences in each of the libraries was compared across all libraries to identify genes that are potentially differentially expressed during cold acclimation and fruit development. Real-time PCR was performed to confirm their differential expression patterns. Overall, 14 out of 17 of the genes examined had differential expression patterns similar to what was predicted from their reads alone. The assembled sequences were also mined for SSRs. From these sequences, 15,886 blueberry EST-SSR loci were identified. Primers were designed from 7,705 of the SSR-containing sequences with adequate flanking sequence. One hundred primer pairs were tested for amplification and polymorphism among parents of two blueberry populations currently being used for genetic linkage map construction. The tetraploid mapping population was based on a cross between the highbush cultivars Draper and Jewel (V. darrowii is also in the background of 'Jewel'). The diploid mapping population was based on a cross between an F1 hybrid of V. darrowii and diploid V. corymbosum and another diploid V. corymbosum. The overall amplification rate of the SSR primers was 68% and the polymorphism rate was 43%. CONCLUSIONS: These results indicate that this large collection of 454 ESTs will be a valuable resource for identifying genes that are potentially differentially expressed and play important roles in flower bud development, cold acclimation, chilling unit accumulation, and fruit development in blueberry and related species. In addition, the ESTs have already proved useful for the development of SSR and EST-PCR markers, and are currently being used for construction of genetic linkage maps in blueberry.