Genomics to Genetics

A BC1 progeny (PxF) obtained crossing two peach accessions, the selection IF7310828 used as female recurrent parent and the oriental accession Ferganensis (formerly P. ferganensis) used as male donor parent was genotyped with the IPSC 9K SNP array. Out of 8144 SNPs available on the chip 3326 (40.8%) resulted polymorphic. We mapped 1660 SNPs and 15 SSR loci, all informative for the F1 parent and segregating in a 1:1 ratio in the PxF population, using JoinMap 4.1 and the Maximum Likelihood mapping algorithm. Grouping was performed at a LOD higher than 10. After grouping, identical loci were eliminated , with the exception of SSRs that were always retained. The final map consisted of 242 loci distributed in 8 groups, covering 607 Haldane cM. This map was used to scan the genome for QTLs using MapQTL6. Interval mapping and MQM mapping were used, both with the mixture Model Algorithm. Traits related to plant structure (internode length, plant height, trunk perimeter), plant physiology (blooming and maturity date),fruit quality (fruit weight, fruit juice pH, titratable acidity, soluble solid content , fruit red overcolor ) were analyzed for at least two years. Several QTLs were identified, the major ones for blooming and maturity date, soluble solid content, fruit red overcolor.

MicroRNAs (miRNAs), interfering RNAs (siRNA, phase-based siRNAs (phasiRNA) and trans-acting siRNAs (tasiRNAs) are recently emerged as powerful regulators in plants. However, how they operate and function, in context of fruit productivity, in fruit crops remain largely unknown. Recently, we took advantage of a deep sequencing approach, in combination with computation and molecular analyses, to thoroughly characterize the profile of miRNASs, tasiRNAs, phasiRNAs and other types of siRNAs as well as their interaction in apple and peach, respectively. Our work showed that both apple and peach shared the conserved miRNAs with model species as well as evolved a large number of unique ones. We also demonstrated that a multitude of apple and peach miRNAs were capable of triggering phasiRNA production in a few gene families, and many of the produced phasiRNAs were able to target genes inside and outside the families. These miRNA-triggered, phasiRNA-cascaded robust networks represent novel regulatory mechanisms that have not been elucidated in other species. The potential function of the sRNA-based regulatory networks in regulation of plant growth, development, productivity and defense will be discussed.

While the utilization of next generation sequencing technologies is rapidly growing, the application of this approach to phylogenetic analysis has been fairly limited to date. We are conducting large scale phylogenetic analyses of Fragaria (strawberry) species using the Fluidigm Access Array system and 454 sequencing platform. Our aim is to clarify phylogenetic relationships among Fragaria species and to elucidate the sub-genome composition of the polyploid species. Twenty-four single-copy or low-copy nuclear genes distributed across the genome were selected, and amplicons were sequenced from ninety six genomic DNA samples representing species from diploid to decaploid. Individual gene trees and species trees were reconstructed by different tree-building methods. Our results support the monophyly of Fragaria, and illuminate phylogenetic relationships among diploid species. Our findings also suggest the presence of three types of sub-genomes within the octoploid strawberry genome, and support the prior implications of ancestry for three diploid species: F. vesca and/or F. mandshurica, and F. iinumae. We developed a bioinformatics pipeline that is useful for large scale phylogenetic analysis of other polyploid species, and demonstrate the power of high throughput sequencing technology to enable robust phylogeny estimation from multiple nuclear genes.

Until recently, tomato (Solanum lycopersicum) was regarded as a model for plastid development for all fruits. However, recent studies of plastid morphology and development in apple (Malus x domestica) have revealed that not only are there extreme differences between apple and tomato, but there are also significant differences between apple cultivars as well throughout fruit development. The recent sequencing of the ‘Golden Delicious’ genome has provided a wealth of sequence information that has shed some light on why such a stark difference exists; the apple genome is predicted to contain nearly 5,000 unique plastid-targeted proteins which are not represented in the genomes of Arabidopsis, grape, peach, pear, poplar, or tomato. These unique genes represent over 40% of the total plastid proteome in apples, which is currently predicted to have 10,000 proteins. Because these proteins have not been documented in plastids before now, each may have functions that are entirely new to plant biology. Of the 5,000 unique genes, we have narrowed our focus to genes which may function in crosstalk between the plastid and nucleus or in modulation of photosynthesis. The gene set was enriched with GO terms for electron transfer, chromatin-binding, transcription, translation, and proteins containing transmembrane domains; members of these categories are undergoing expression analysis, localization, and photosynthesis phenotyping experiments. This work will shed light on the plastid morphodevelopment in non-model fruit systems such as apple, and may unveil proteins with novel functions that can be utilized in the future for development of improved varieties.

Carotenoids are tetraterpenoid lipophilic pigments found in most plant organs. In leaves, carotenoids serve as light harvesting complexes, and they are responsible for protection against reactive oxygen species generated in the chloroplast. Carotenoids are also precursors for the production of signaling compounds such as abscisic acid and strigalactone. In flowers and fruits, the colors of carotenoids play roles as visual attractants for pollinators and seed dispersers, and roles as antioxidants in these organs are also probable. Strawberry production depends on successful development of reproductive structures and fertilization, processes highly susceptible to abiotic stresses. Insight into the functions of carotenoids in reproductive organs could provide the groundwork for breeding or genetically engineering more stress tolerant strawberries and other horticultural crops. Flowers of the diploid woodland strawberry, F. vesca, have yellow-orange anthers, yellow carpels, and yellow pollen due to the presence of carotenoids, but the precise role(s) of these pigments have not been defined. We are assessing the potential for these compounds to protect reproductive organs against elevated temperature stress using biochemical analysis, genomics, and genetics. Unlike in leaves, HPLC analysis showed that developing F. vesca carpels and stamens have no detectable beta-carotene. Candidate genes involved in carotenoid biosynthesis were identified, revealing the existence of small gene families. Gene expression patterns throughout the plant, and expression in response to heat stress in the reproductive structures were analyzed by QRTPCR. Mass spectrometric analyses of carotenoids in control and heat stressed tissues, and in a male sterile mutant with pale anthers are underway.

In recent years, phylogenetic, and other evidence has accumulated implicating diploid (2n=2x=14) F. iinumae as a second subgenome contributor to the octoploid (2n=8x=56) strawberries, including the cultivated strawberry, Fragaria ×ananassa and its ancestors F. chiloensis and F. virginiana. Previously, diploid F. vesca had been identified as an ancestral subgenome donor, and the representative ‘Hawaii4’ genome was published in 2011 as the first Fragaria reference genome. We have developed germplasm and genomic resources for F. iinumae, including high throughput genomic sequence data and a linkage mapping population. We now report construction of two high density linkage maps, constructed using GBS markers and SNPs from the IStraw90 Axiom ® Strawberry array. The maps, each of seven linkage groups, are based on segregation data from an F2 population (F2Ds) derived from a cross between two F. iinumae accessions (CFRA1955 and CFRA1849) collected in Hokkaido, Japan by Tom Davis and Kim Hummer in 2004. The maps differ with regard to the numbers of genotyped individuals and the markers employed. A GBS-only map based on 85 F2 plants defines 220 loci comprising 972 markers, with a map density of 2.1 cM and a map length 457.4 cM. A combined GBS-SNP map of only 21 individuals defines 158 loci comprising 3,181 SNP and 994 GBS markers. We plan to expand the SNP genotyping to include all 85 F2 individuals. The F. iinumae linkage maps will be used to anchor an F. iinumae genome assembly, thereby providing an important new genomic resource for Fragaria.

The RosBREED project aimed to enhance the infrastructure for marker-assisted breeding, including platform development for high throughput genome scans. For the allo-octoploid, cultivated strawberry, this goal was met by the public release of the International Strawberry 90K (IStraw90TM) Axiom® genotyping array developed in collaboration with Affymetrix. Array design mainly exploited polymorphisms identified in the cultivated strawberry. The challenges of allo-octoploidy were addressed by maximizing the number of SNP targets to compensate for potential low conversion of candidate to functional SNPs, and sub-genome-specific probe targeting. Additionally, some reduced ploidy genomic sites were targeted via fortuitous probe placement. The array was evaluated on four mapping populations, numerous pedigreed breeding populations, various cultivars and selections, and several genetically unusual strawberry genotypes. New filters were developed and octoploid-specific thresholds were used in the genotyping procedure to identify potentially well-resolved and more complex SNP markers. A total of 12,609 well-resolved SNPs were obtained and evaluated through the generation of a genetic linkage map for ‘Holiday’ × ‘Korona’ (HK), where 6,689 of the 8,084 polymorphic HK-SNPs were successfully mapped. These mapped SNPs were also checked for marker consistency in the pedigreed cultivars and breeding selections using FlexQTLTM. Results of the SNP development strategies, improved gridding algorithm and automated genotype scoring will be presented. This array is the first high throughput genotyping platform in an octoploid organism and we expect it to enable genome-wide scanning in the cultivated octoploid strawberry and facilitate QTL discovery for many traits of economic significance in this important Rosaceous fruit crop.

The Summerland breeding program has a long history of producing unique sweet cherry cultivars with a variety of desirable characteristics. Recent successes include the licensing of Sentennial, Sovereign and Staccato, which are progeny of Sweetheart, an earlier Summerland cultivar. Plant Breeders Rights for these cultivars are currently held by the Okanagan Plant Improvement Corporation (PICO) with detailed descriptors of the cultivars as the defining characteristics. We are currently developing molecular techniques to discriminate between these very closely related cultivars. Previously, AFLP and SSR analysis have successfully differentiated most cherry cultivars. However, only one SSR and one AFLP marker distinguished Sweetheart from Staccato. The requirement for more unique markers led to development of our current SNP fingerprinting strategy. New single nucleotide polymorphisms (SNPs) were identified from analysis of Illumina RNA-Seq libraries generated for Sweetheart and its three progeny. High coverage Sweetheart SNPs were examined for segregation in the progeny and confirmed by High Resolution Melt (HRM) assays. A set of 16 SNPs was chosen for independent segregation across the genome that provided a unique fingerprint of Sweetheart and each of its progeny. The straightforward SNP HRM technique was further tested on a broader set of 35 cultivars of interest to PICO, providing unique fingerprints for each. Within this group of 35 cultivars, 16 were selected for genotyping-by-sequencing to identify additional SNP markers. The choice of multiple unlinked SNPs increases the probability that each pattern of inheritance will be unique and can be used to verify the identity of unknown cultivars.

Development of genomics-guided physiological solutions in biological systems lacking significant genomic resources is challenging. Utilization of controlled physiological models with time course gene expression analysis can address these challenges. Harboring unique ripening biology and responses to the ethylene signaling inhibitor 1-MCP, pear is an ideal model for investigation of the molecular underpinnings of System 2 ethylene induction. Induction of autocatalytic ethylene biosynthesis in climacteric fruits acts as a powerful indicator of – and trigger of –ripening. We identified differential expression of genes in a ripening-related pathway during postharvest conditioning in peel tissue of two pear varieties: Anjou and Bartlett. Anjou requires 60-day cold at 0-5°C to gain competency for ripening and System 2 ethylene biosynthesis, while Bartlett requires 15 days. Further, pear exhibits variable recovery from 1-MCP treatments, with some fruit ‘locked’ permanently in an unripe state. We developed ripening-stimulating compounds (RCs) thought to increase the activity of the pathway we previously identified in comparison of Anjou and Bartlett tissue. Initial tests of mature unconditioned, 1-MCP treated fruit have shown accelerated ethylene biosynthesis, respiration rate and development of fruit quality traits from RC exposure. Future work will seek to characterize global transcriptomic responses to exposure of these RCs in 1-MCP treated and control Anjou and Bartlett fruit, as well as identify means to enhance RC penetration into fruit interior. This work demonstrates a unique means of accelerating pear ripening, and can help facilitate the use of 1-MCP in the pear postharvest management chain.

In some genotypes of non-climacteric sweet cherry, ethylene induces the development of a clearly defined abscission zone between the fruit and pedicel. The aim of this study is to identify and analyze the genetic components of sweet cherry abscission in response to exogenous ethylene application. We performed a time course transcriptome analysis of the fruit-pedicel abscission zone treated with exogenously applied ethylene. Three unique genotypes were used, representing three distinct classes of phenotypes of fruit-pedicel abscission zone formation in sweet cherry. RNA-seq captured a global snapshot of the transcriptome at each time point sampled. The transcriptome data was assembled and relative expression values for each gene (RPKM) were calculated. Genes with at least a five-fold difference in expression between treatment and control were selected for annotation using Blast2GO and polymorphism analysis. Gene ontology and pathway information identified both known and cherry specific gene networks involved in the abscission process. In the future, the polymorphisms embedded in differentially expressed abscission related genes will be validated in populations segregating for ethylene-induced abscission. Additionally, they are expected to be used as gene-based markers for the development of varieties that exhibit desired traits for new and developing harvest technologies.