Jill M. Bushakra

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

Key messageWe have constructed a densely populated, saturated genetic linkage map of black raspberry and successfully placed a locus for aphid resistance.AbstractBlack 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.

Identification of QTL associated with flower and runner production in octoploid strawberry (Fragaria×ananassa)

BACKGROUND: Understanding the genetics of flowering in the strawberry (Fragaria × ananassa) will aid in the development of breeding strategies. OBJECTIVE: To search for quantitative trait loci (QTL) associated with remontancy and weeks of flowering in the strawberry. METHODS: Previously collected phenotypic data from two non-remontant ‘Honeoye’ × remontant ‘Tribute’ strawberry populations and simple sequence repeats (SSR) markers were used to search for QTL associated with repeat flowering, weeks of flowering and runner production, as well as the ability to produce flowers and runners at 17, 20 and 23 ◦ C. RESULTS: As was discovered in other studies, we found a major QTL that regulated remontancy and weeks of flowering on homeologous linkage group IV of ‘Tribute’. This QTL also had a negative effect on runner production and a positive influence on flower production under high temperatures. A number of additional QTL were discovered that significantly (LOD >3.0) influenced flower and runner production. CONCLUSIONS: Remontancy/non-remontancy is controlled by a major gene/locus and several minor modifying ones.

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.Plant genomics: Improved genome for the black raspberryAssembly of a high-quality reference genome for the black raspberry plant will inform future crop improvements. Sequencing the genomes of widely-grown, profitable fruit crops can help researchers identify the DNA markers linked to desirable traits, potentially improving crop health and yields. David Chagné at the New Zealand Institute for Plant and Food Research Limited and co-workers combined new techniques to significantly improve on an existing genome for the black raspberry (Rubus occidentalis L.). The researchers used Hi-C analysis to create a map identifying the interactions between chromatin fragments – macromolecules made from DNA, proteins and RNA – and the three-dimensional structure of chromosomes inside the cell nucleus. Then, they used a bioinformatics assembly method to construct the genome from this data. The new genome showed high accuracy when compared to another genome from the same family.

Sequence and Analysis of the Black Raspberry ( Rubus occidentalis ) Genome

The US Pacific Northwest is the primary production region of black raspberry, and this high-value specialty crop has been underutilized for several decades. Black raspberries contain high levels of anthocyanins and other bioactive compounds, which has sparked a renewed interest in breeding programs and cultivation. Despite this potential, black raspberry stands have seen a marked decline that many attribute to disease pressures and only three new cultivars have been released over the last 20 years. Here we discuss the available genomic resources for black raspberry, including the recently released draft genome. These resources will expedite marker-assisted improvement of raspberry with applications across the Rosaceae family. The 243 Mb black raspberry genome was sequenced using an Illumina-based whole genome shotgun sequencing approach, and a chromosome-scale assembly was generated using a high-density genetic map. Black raspberry is the sixth genome to be sequenced in the Rosaceae facilitating in-depth comparative genomics across the family. Black raspberry and the diploid wild strawberry (Fragaria vesca) are largely collinear with some lineage-specific structural rearrangements. The genome has 28,005 genes which is comparable to other Rosaceae species and includes a number of recently duplicated genes which may be related to domestication. Gene expression atlases during fruit ripening and Verticillium inoculation provide insights into ripening and disease resistance, respectively. Together the resources discussed here will provide tools for the improved understanding and breeding of Rosaceae crops.

Characterization of aphid resistance loci in black raspberry (Rubus occidentalis L.)

Viruses vectored by the aphid Amphorophora agathonica cause decline in black raspberry plant health resulting in a shortened life and poor fruit quality of the infected plantings. New aphid resistant cultivars could increase the longevity of plantings providing growers and processors with consistent fruit production. Recent exploration of the native range of black raspberry identified three sources of aphid resistance: Ag4 from Ontario (ON), Canada, Ag5 from Maine (ME), and a third source from Michigan (MI) with no formal designation. The objectives of this study were to assess segregation of these three sources of aphid resistance in populations with single and combined sources and develop markers that can identify each source of resistance. A genetic linkage map constructed for ORUS 4305 placed the ON aphid resistance locus on Rubus linkage group (RLG) 6. Segregation ratios in populations with single and combined sources, and linkage mapping in two populations (ORUS 4304 and ORUS 4812) segregating for the Ag5 and MI sources, respectively, indicated that these three sources of resistance are each conferred by single dominant genes/alleles that are linked on RLG6. Confirmation of marker association in 16 validation populations identified four markers that could be used to predict resistance; however, none could distinguish between the ON and MI sources. These four markers may be useful for screening populations to enrich the field-planted progeny for aphid resistance. Fine mapping of the resistance loci is needed to develop functional markers at each of the resistance loci to enable pyramiding and durable aphid resistance.

A near complete, chromosome-scale assembly of the black raspberry (Rubus occidentalis) genome

Abstract Background The fragmented nature of most draft plant genomes has hindered downstream gene discovery, trait mapping for breeding, and other functional genomics applications. There is a pressing need to improve or finish draft plant genome assemblies. Findings Here, we present a chromosome-scale assembly of the black raspberry genome using single-molecule real-time Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated V3 assembly has a contig N50 of 5.1 Mb, representing an ∼200-fold improvement over the previous Illumina-based version. Each of the 235 contigs was anchored and oriented into seven chromosomes, correcting several major misassemblies. Black raspberry V3 contains 47 Mb of new sequences including large pericentromeric regions and thousands of previously unannotated protein-coding genes. Among the new genes are hundreds of expanded tandem gene arrays that were collapsed in the Illumina-based assembly. Detailed comparative genomics with the high-quality V4 woodland strawberry genome (Fragaria vesca) revealed near-perfect 1:1 synteny with dramatic divergence in tandem gene array composition. Lineage-specific tandem gene arrays in black raspberry are related to agronomic traits such as disease resistance and secondary metabolite biosynthesis. Conclusions The improved resolution of tandem gene arrays highlights the need to reassemble these highly complex and biologically important regions in draft plant genomes. The updated, high-quality black raspberry reference genome will be useful for comparative genomics across the horticulturally important Rosaceae family and enable the development of marker assisted breeding in Rubus.