Exploring the genetic basis of resistance to Neopestalotiopsis species in strawberry.

Aggressive strains of Neopestalotiopsis sp. have recently emerged as devastating pathogens of strawberry (Fragaria × ananassa Duchesne ex Rozier), infecting nearly all plant parts and causing severe outbreaks of leaf spot and fruit rot in Florida and globally. The development of host resistance is imperative due to the absence of fungicides that effectively inhibit Neopestalotiopsis sp. growth on an infected strawberry crop. Here, we analyzed 1578 individuals from the University of Florida's (UF) strawberry breeding program to identify and dissect genetic variation for resistance to Neopestalotiopsis sp. and to explore the feasibility of genomic selection. We found that less than 12% of elite UF germplasm exhibited resistance, with narrow-sense heritability estimates ranging from 0.28 to 0.69. Through genome-wide association studies (GWAS), we identified two loci accounting for 7%-16% of phenotypic variance across four trials and 3 years. Several candidate genes encoding pattern recognition receptors, intra-cellular nucleotide-binding leucine-rich repeats, and downstream components of plant defense pathways co-localized with the Neopestalotiopsis sp. resistance loci. Interestingly, favorable alleles at the largest-effect locus were rare in elite UF material and had previously been unintentionally introduced from an exotic cultivar. The array-based markers and candidate genes described herein provide the foundation for targeting this locus through marker-assisted selection. The predictive abilities of genomic selection models, with and without explicitly modeling peak GWAS markers as fixed effects, ranged between 0.25 and 0.59, suggesting that genomic selection holds promise for enhancing resistance to Neopestalotiopsis sp. in strawberry.

Strawberry soluble solids QTL with inverse effects on yield.

Sugars are the main drivers of strawberry sweetness, and understanding their genetic control is of critical importance for breeding. Large-scale genome-wide association studies were performed in two populations totaling 3399 individuals evaluated for soluble solids content (SSC) and fruit yield. Two stable quantitative trait loci (QTL) on chromosome 3B and 6A for SSC were identified. Favorable haplotypes at both QTL for SSC decreased yield, though optimal allelic combinations were identified with reduced impacts on yield. Metabolites in the starch and sucrose metabolism pathway were characterized and quantified for 23 contrasting genotypes in leaves, white fruit, and red fruit. Variations in sucrose concentrations/efflux indicated genetic variation underlying sucrose accumulation and transportation during fruit ripening. Integration of genome-wide association studies and expression quantitative locus mapping identified starch synthase 4 (FxaC_10g00830) and sugar transporter 2-like candidate genes (FxaC_21g51570) within the respective QTL intervals. These results will enable immediate applications in genomics-assisted breeding for flavor and further study of candidate genes underlying genetic variation of sugar accumulation in strawberry fruit.

Construction of a genome-wide genetic linkage map and identification of quantitative trait loci for powdery mildew resistance in Gerbera daisy.

Powdery mildew (PM) is a common fungal disease in many important crops. The PM caused by Podosphaera xanthii has been the most challenging problem in commercial Gerbera (Gerbera hybrida) production globally, often leading to severe losses of crop yield and quality. A small number of PM-resistant breeding lines and cultivars have been reported in Gerbera, but the underlying genetics for PM resistance in Gerbera is largely unknown. Scarcity of genomic resources such as genetic linkage maps and molecular markers has severely hindered the effort to understand the genetic basis and locate loci controlling PM resistance in Gerbera. This study aimed to construct a genome-wide genetic linkage map, identify quantitative trait loci (QTL), and molecular markers for PM resistance in Gerbera. A segregating mapping population was developed by crossing PM-resistant and -susceptible Gerbera breeding lines, genotyped by sequencing, and phenotyped for PM resistance. A genome-wide genetic linkage map constructed with 791 single polymorphic site (SNP) markers spans 1912.30 cM across 27 linkage groups (LG) and reaches a density of 1 marker per 2.42 cM. One major consistent QTL was discovered in LG16, explaining more than 16.6% of the phenotypic variance for PM resistance. The QTL was tagged with two flanking SNP markers. The availability of this genetic linkage map will be very useful for locating and tagging QTLs for other important traits in Gerbera, and the newly discovered QTL and SNP markers will enable development of molecular markers for improving Gerbera for resistance to PM.

Genetic Analysis of Methyl Anthranilate, Mesifurane, Linalool, and Other Flavor Compounds in Cultivated Strawberry (Fragaria × ananassa).

The cultivated strawberry (Fragaria × ananassa) is an economically important fruit crop that is intensively bred for improved sensory qualities. The diversity of fruit flavors and aromas in strawberry results mainly from the interactions of sugars, acids, and volatile organic compounds (VOCs) that are derived from diverse biochemical pathways influenced by the expression of many genes. This study integrates multiomic analyses to identify QTL and candidate genes for multiple aroma compounds in a complex strawberry breeding population. Novel fruit volatile QTL was discovered for methyl anthranilate, methyl 2-hexenoate, methyl 2-methylbutyrate, mesifurane, and a shared QTL on Chr 3 was found for nine monoterpene and sesquiterpene compounds, including linalool, 3-carene, ß-phellandrene, α-limonene, linalool oxide, nerolidol, α-caryophellene, α-farnesene, and ß-farnesene. Fruit transcriptomes from a subset of 64 individuals were used to support candidate gene identification. For methyl esters including the grape-like methyl anthranilate, a novel ANTHANILIC ACID METHYL TRANSFERASE-like gene was identified. Two mesifurane QTL correspond with the known biosynthesis gene O-METHYL TRANSFERASE 1 and a novel FURANEOL GLUCOSYLTRANSFERASE. The shared terpene QTL contains multiple fruit-expressed terpenoid pathway-related genes including NEROLIDOL SYNTHASE 1 (FanNES1). The abundance of linalool and other monoterpenes is partially governed by a co-segregating expression-QTL (eQTL) for FanNES1 transcript variation, and there is additional evidence for quantitative effects from other terpenoid-pathway genes in this narrow genomic region. These QTLs present new opportunities in breeding for improved flavor in commercial strawberry.

Discovery of three loci increasing resistance to charcoal rot caused by Macrophomina phaseolina in octoploid strawberry.

Charcoal rot caused by Macrophomina phaseolinais an increasing economic problem in annualized strawberry production systems around the world. Currently there are no effective postfumigation chemical controls for managing charcoal rot, and no information is available on the genetic architecture of resistance to M. phaseolina in strawberry (Fragaria ×ananassa). In this study, three multiparental discovery populations and two validation populations were inoculated at planting and evaluated for mortality in three consecutive growing seasons. Genome-wide SNP genotyping and pedigree-based analysis with FlexQTL™ software were performed. Two large-effect quantitative trait loci (QTL) increasing charcoal rot resistance were discovered and validated in cultivated germplasm. FaRMp1 was located on linkage group 2A in the interval 20.4to 24.9 cM, while FaRMp2 was located on linkage group 4B in the interval 41.1to 61.2 cM. Together these QTLs explained 27% and 17% of the phenotypic variance in two discovery populations consisting of elite breeding germplasm. For both QTLs, the resistant allele showed some evidence of partial dominance, but no significant interaction was detected between the two loci. As the dosage of resistant alleles increased from 0 to 4 across the two QTLs, mortality decreased regardless of the combination of alleles.A third locus, FaRMp3 on 4D, was discovered in FVC 11-58, a reconstituted F.×ananassa originating from diverse F. virginiana and F. chiloensis accessions. This locus accounted for 44% of phenotypic variation in four segregating crosses. These findings will form the basis for DNA-informed breeding for resistance to charcoal rot in cultivated strawberry.

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.

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 high-quality 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.

Independent Validation of Genomic Prediction in Strawberry Over Multiple Cycles.

The University of Florida strawberry (Fragaria × ananassa) breeding program has implemented genomic prediction (GP) as a tool for choosing outstanding parents for crosses over the last five seasons. This has allowed the use of some parents 1 year earlier than with traditional methods, thus reducing the duration of the breeding cycle. However, as the number of breeding cycles increases over time, greater knowledge is needed on how multiple cycles can be used in the practical implementation of GP in strawberry breeding. Advanced selections and cultivars totaling 1,558 unique individuals were tested in field trials for yield and fruit quality traits over five consecutive years and genotyped for 9,908 SNP markers. Prediction of breeding values was carried out using Bayes B models. Independent validation was carried out using separate trials/years as training (TRN) and testing (TST) populations. Single-trial predictive abilities for five polygenic traits averaged 0.35, which was reduced to 0.24 when individuals common across trials were excluded, emphasizing the importance of relatedness among training and testing populations. Training populations including up to four previous breeding cycles increased predictive abilities, likely due to increases in both training population size and relatedness. Predictive ability was also strongly influenced by heritability, but less so by changes in linkage disequilibrium and effective population size. Genotype by year interactions were minimal. A strategy for practical implementation of GP in strawberry breeding is outlined that uses multiple cycles to predict parental performance and accounts for traits not included in GP models when constructing crosses. Given the importance of relatedness to the success of GP in strawberry, future work could focus on the optimization of relatedness in the design of TRN and TST populations to increase predictive ability in the short-term without compromising long-term genetic gains.

Disease Resistance Genetics and Genomics in Octoploid Strawberry.

Octoploid strawberry (Fragaria ×ananassa) is a valuable specialty crop, but profitable production and availability are threatened by many pathogens. Efforts to identify and introgress useful disease resistance genes (R-genes) in breeding programs are complicated by strawberry's complex octoploid genome. Recently-developed resources in strawberry, including a complete octoploid reference genome and high-resolution octoploid genotyping, enable new analyses in strawberry disease resistance genetics. This study characterizes the complete R-gene collection in the genomes of commercial octoploid strawberry and two diploid ancestral relatives, and introduces several new technological and data resources for strawberry disease resistance research. These include octoploid R-gene transcription profiling, dN/dS analysis, expression quantitative trait loci (eQTL) analysis and RenSeq analysis in cultivars. Octoploid fruit eQTL were identified for 76 putative R-genes. R-genes from the ancestral diploids Fragaria vesca and Fragaria iinumae were compared, revealing differential inheritance and retention of various octoploid R-gene subtypes. The mode and magnitude of natural selection of individual F. ×ananassa R-genes was also determined via dN/dS analysis. R-gene sequencing using enriched libraries (RenSeq) has been used recently for R-gene discovery in many crops, however this technique somewhat relies upon a priori knowledge of desired sequences. An octoploid strawberry capture-probe panel, derived from the results of this study, is validated in a RenSeq experiment and is presented for community use. These results give unprecedented insight into crop disease resistance genetics, and represent an advance toward exploiting variation for strawberry cultivar improvement.

The Genetics of Differential Gene Expression Related to Fruit Traits in Strawberry (Fragaria ×ananassa).

Octoploid strawberry (Fragaria ×ananassa) is a major specialty crop under intense annual selection for traits relating to plant vigor and fruit quality. Most functional validation experiments rely on transgenic or transient gene expression assays in the mature receptacle. These findings are not typically translatable to breeding without identifying a natural genetic source of transcript level variation, and developing reliable markers for selection in octoploids. Expression QTL (eQTL) analysis is a genetic/transcriptomic association approach for identifying sequence variants predicting differential expression. This eQTL study analyzed a wide array of mature receptacle-expressed genes, encompassing the majority of total mature receptacle transcript accumulation and almost all strawberry genes described in the literature. These results identified segregating genetic variants associated with the differential expression of hundreds of strawberry genes, many with known interest to breeders. Several of these eQTL pertain to published genes whose expression levels have been demonstrated to influence mature receptacle phenotypes. Many include key genes of the phenylpropanoid pathway, vitamin C, carotenoid, pectin, and receptacle carbohydrate/sugar metabolism. These subgenome-specific genetic markers may allow breeders to select for desired ranges of target gene expression. These results may also guide basic research efforts and facilitate the identification of causal genes underlying trait QTL.

FaRCa1: a major subgenome-specific locus conferring resistance to Colletotrichum acutatum in strawberry.

Optimal strategies for genetic improvement in crops depend on accurate assessments of the genetic architecture of traits. The overall objective of the present study was to determine the genetic architecture of anthracnose fruit rot (AFR) resistance caused by the fungus Colletotrichum acutatum in the University of Florida strawberry (Fragaria × ananassa) breeding germplasm. In 2016-2017, 33 full-sib families resulting from crosses between parents with varying levels of AFR resistance were tested. In 2017-2018, six full-sib families resulting from putative heterozygous resistant parents and homozygous susceptible parents were tested. Additionally, a validation population consisting of 77 advanced selections and ten cultivars was tested in the second season. Inoculation was performed using a mixture of three local isolates of the C. acutatum species complex. Phenotypes were scored weekly, and genotyping was performed using the IStraw35 Affymetrix Axiom® SNP array. A pedigree-based QTL analysis was performed using FlexQTL™ software. A major resistance locus, which we name FaRCa1, was detected in both seasons with a peak located at 55-56 cM on LG 6B and explaining at least 50% of the phenotypic variation across trials and seasons. The resistant allele exhibited partial dominance in all trials. The FaRCa1 locus is distinct from the previously discovered Rca2 locus, which mapped to LG 7B. While Rca2 is effective against European isolates from pathogenicity group 2, FaRCa1 appears to confer resistance to isolates of pathogenicity group 1.

Impact of the Myosin Modulator Mavacamten on Force Generation and Cross-Bridge Behavior in a Murine Model of Hypercontractility.

Background Recent studies suggest that mavacamten (Myk461), a small myosin-binding molecule, decreases hypercontractility in myocardium expressing hypertrophic cardiomyopathy-causing missense mutations in myosin heavy chain. However, the predominant feature of most mutations in cardiac myosin binding protein-C ( cMyBPC ) that cause hypertrophic cardiomyopathy is reduced total cMyBPC expression, and the impact of Myk461 on cMyBPC -deficient myocardium is currently unknown. Methods and Results We measured the impact of Myk461 on steady-state and dynamic cross-bridge ( XB ) behavior in detergent-skinned mouse wild-type myocardium and myocardium lacking cMyBPC (knockout (KO)). KO myocardium exhibited hypercontractile XB behavior as indicated by significant accelerations in rates of XB detachment (krel) and recruitment (kdf) at submaximal Ca2+ activations. Incubation of KO and wild-type myocardium with Myk461 resulted in a dose-dependent force depression, and this impact was more pronounced at low Ca2+ activations. Interestingly, Myk461-induced force depressions were less pronounced in KO myocardium, especially at low Ca2+ activations, which may be because of increased acto-myosin XB formation and potential disruption of super-relaxed XB s in KO myocardium. Additionally, Myk461 slowed krel in KO myocardium but not in wild-type myocardium, indicating increased XB " on" time. Furthermore, the greater degree of Myk461-induced slowing in kdf and reduction in XB recruitment magnitude in KO myocardium normalized the XB behavior back to wild-type levels. Conclusions This is the first study to demonstrate that Myk461-induced force depressions are modulated by cMyBPC expression levels in the sarcomere, and emphasizes that clinical use of Myk461 may need to be optimized based on the molecular trigger that underlies the hypertrophic cardiomyopathy phenotype.

FaRCg1: a quantitative trait locus conferring resistance to Colletotrichum crown rot caused by Colletotrichum gloeosporioides in octoploid strawberry.

Colletotrichum crown rot (CCR) is an important disease of strawberry (Fragaria ×ananassa) throughout the Southeastern US and in subtropical climates around the world, where hot and humid conditions facilitate rapid disease development. Yet no resistance loci have been described to date, as genetic studies have been historically difficult in allo-octoploid (2n = 8x = 56) strawberry. In the present study, we investigate the genetic architecture of resistance to CCR. Four population sets from the University of Florida were inoculated in four different seasons from 2013-2014 to 2016-2017. Two large, multiparental discovery population sets were used for QTL discovery, and two validation sets of cultivars and advanced selections representing the parent pool of the breeding program were also assessed. Subgenome-specific single-nucleotide polymorphism (SNP) markers were mapped, and FlexQTL™ software was utilized to perform a Bayesian, pedigree-based QTL analysis. A quantitative trait locus on linkage group 6B, which we name FaRCg1, accounts for most of the genetic variation for resistance in the discovery sets (26.8-29.8% in 2013-2014 and 17% in 2015-2016). High-throughput marker assays were developed for the most significant SNPs which correlated with the mode of the QTL region. The discovery and characterization of the FaRCg1 locus and the molecular tools developed from it will be utilized to achieve increased genetic gains for resistance.

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.

Dose-Dependent Effects of the Myosin Activator Omecamtiv Mecarbil on Cross-Bridge Behavior and Force Generation in Failing Human Myocardium.

BACKGROUND: Omecamtiv mecarbil (OM) enhances systolic function in vivo by directly binding the myosin cross-bridges (XBs) in the sarcomere. However, the mechanistic details governing OM-induced modulation of XB behavior in failing human myocardium are unclear. METHODS AND RESULTS: The effects of OM on steady state and dynamic XB behavior were measured in chemically skinned myocardial preparations isolated from human donor and heart failure (HF) left ventricle. HF myocardium exhibited impaired contractile function as evidenced by reduced maximal force, magnitude of XB recruitment (Pdf), and a slowed rate of XB detachment (krel) at submaximal Ca2+ activations. Ca2+ sensitivity of force generation (pCa50) was higher in HF myocardium when compared with donor myocardium, both prior to and after OM incubations. OM incubation (0.5 and 1.0 µmol/L) enhanced force generation at submaximal Ca2+ activations in a dose-dependent manner. Notably, OM induced a slowing in krel with 1.0 µmol/L OM but not with 0.5 µmol/L OM in HF myocardium. Additionally, OM exerted other differential effects on XB behavior in HF myocardium as evidenced by a greater enhancement in Pdf and slowing in the time course of cooperative XB recruitment (Trec), which collectively prolonged achievement of peak force development (Tpk), compared with donor myocardium. CONCLUSIONS: Our findings demonstrate that OM augments force generation but also prolongs the time course of XB transitions to force-bearing states in remodeled HF myocardium, which may extend the systolic ejection time in vivo. Optimal OM dosing is critical for eliciting enhanced systolic function without excessive prolongation of systolic ejection time, which may compromise diastolic filling.

Pedigree-Based Analysis in a Multiparental Population of Octoploid Strawberry Reveals QTL Alleles Conferring Resistance to Phytophthora cactorum.

Understanding the genetic architecture of traits in breeding programs can be critical for making genetic progress. Important factors include the number of loci controlling a trait, allele frequencies at those loci, and allele effects in breeding germplasm. To this end, multiparental populations offer many advantages for quantitative trait locus (QTL) analyses compared to biparental populations. These include increased power for QTL detection, the ability to sample a larger number of segregating loci and alleles, and estimation of allele effects across diverse genetic backgrounds. Here, we investigate the genetic architecture of resistance to crown rot disease caused by Phytophthora cactorum in strawberry (Fragaria × ananassa), using connected full-sib families from a breeding population. Clonal replicates of > 1100 seedlings from 139 full-sib families arising from 61 parents were control-inoculated during two consecutive seasons. Subgenome-specific single nucleotide polymorphism (SNP) loci were mapped in allo-octoploid strawberry (2n = 8 × = 56), and FlexQTL software was utilized to perform a Bayesian, pedigree-based QTL analysis. A major locus on linkage group (LG) 7D, which we name FaRPc2, accounts for most of the genetic variation for resistance. Four predominant SNP haplotypes were detected in the FaRPc2 region, two of which are strongly associated with two different levels of resistance, suggesting the presence of multiple resistance alleles. The phenotypic effects of FaRPc2 alleles across trials and across numerous genetic backgrounds make this locus a highly desirable target for genetic improvement of resistance in cultivated strawberry.

An experimental validation of genomic selection in octoploid strawberry.

The primary goal of genomic selection is to increase genetic gains for complex traits by predicting performance of individuals for which phenotypic data are not available. The objective of this study was to experimentally evaluate the potential of genomic selection in strawberry breeding and to define a strategy for its implementation. Four clonally replicated field trials, two in each of 2 years comprised of a total of 1628 individuals, were established in 2013-2014 and 2014-2015. Five complex yield and fruit quality traits with moderate to low heritability were assessed in each trial. High-density genotyping was performed with the Affymetrix Axiom IStraw90 single-nucleotide polymorphism array, and 17 479 polymorphic markers were chosen for analysis. Several methods were compared, including Genomic BLUP, Bayes B, Bayes C, Bayesian LASSO Regression, Bayesian Ridge Regression and Reproducing Kernel Hilbert Spaces. Cross-validation within training populations resulted in higher values than for true validations across trials. For true validations, Bayes B gave the highest predictive abilities on average and also the highest selection efficiencies, particularly for yield traits that were the lowest heritability traits. Selection efficiencies using Bayes B for parent selection ranged from 74% for average fruit weight to 34% for early marketable yield. A breeding strategy is proposed in which advanced selection trials are utilized as training populations and in which genomic selection can reduce the breeding cycle from 3 to 2 years for a subset of untested parents based on their predicted genomic breeding values.

Cardiac Myosin Binding Protein-C Phosphorylation Modulates Myofilament Length-Dependent Activation.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is an important regulator of contractile function, however, its contributions to length-dependent changes in cross-bridge (XB) kinetics is unknown. Therefore, we performed mechanical experiments to quantify contractile function in detergent-skinned ventricular preparations isolated from wild-type (WT) hearts, and hearts expressing non-phosphorylatable cMyBP-C [Ser to Ala substitutions at residues Ser273, Ser282, and Ser302 (i.e., 3SA)], at sarcomere length (SL) 1.9 µm or 2.1µm, prior and following protein kinase A (PKA) treatment. Steady-state force generation measurements revealed a blunting in the length-dependent increase in myofilament Ca(2+)-sensitivity of force generation (pCa50) following an increase in SL in 3SA skinned myocardium compared to WT skinned myocardium. Dynamic XB behavior was assessed at submaximal Ca(2+)-activations by imposing an acute rapid stretch of 2% of initial muscle length, and measuring both the magnitudes and rates of resultant phases of force decay due to strain-induced XB detachment and delayed force rise due to recruitment of additional XBs with increased SL (i.e., stretch activation). The magnitude (P2) and rate of XB detachment (k rel) following stretch was significantly reduced in 3SA skinned myocardium compared to WT skinned myocardium at short and long SL, and prior to and following PKA treatment. Furthermore, the length-dependent acceleration of k rel due to decreased SL that was observed in WT skinned myocardium was abolished in 3SA skinned myocardium. PKA treatment accelerated the rate of XB recruitment (k df) following stretch at both SL's in WT but not in 3SA skinned myocardium. The amplitude of the enhancement in force generation above initial pre-stretch steady-state levels (P3) was not different between WT and 3SA skinned myocardium at any condition measured. However, the magnitude of the entire delayed force phase which can dip below initial pre-stretch steady-state levels (Pdf) was significantly lower in 3SA skinned myocardium under all conditions, in part due to a reduced magnitude of XB detachment (P2) in 3SA skinned myocardium compared to WT skinned myocardium. These findings demonstrate that cMyBP-C phospho-ablation regulates SL- and PKA-mediated effects on XB kinetics in the myocardium, which would be expected to contribute to the regulation of the Frank-Starling mechanism.

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.