Sujeet Verma

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 Ca2+-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 Ca2+-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 (krel) 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 krel 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 (kdf) following stretch at both SLs 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.

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.

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.

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.

Genome-Assisted Breeding in the Octoploid Strawberry

The application of genomic information to the breeding of allo-octoploid (2n = 8x = 56) cultivated strawberry (Fragaria × ananassa) has increased rapidly in the last five years. These advances have been fueled by technological improvements in high-throughput genotyping and genome sequencing, as well as concerted efforts to develop DNA tests for routine use in breeding. Genome-wide and subgenome-specific markers have advanced availability of DNA tests for major loci, as well as the development and validation of genomic selection methodology for complex traits in strawberry. Eight DNA tests for fruit quality and disease resistance loci are fully or partially in the public sphere. Genome-wide predictions have delivered genetic gain efficiencies for parent selection larger than 50% of conventional methods but without the need for phenotypic information. Meanwhile, the construction of haploblocks and haplotypes allows increased understanding of genome structure as it relates to breeding applications. With octoploid sequence assemblies merely months away and the development of gene editing technologies, precision manipulation of genes may shape the future of strawberry genetic improvement.

High-throughput marker assays for FaRPc2-mediated resistance to Phytophthora crown rot in octoploid strawberry

Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is a destructive disease of the allo-octoploid cultivated strawberry (Fragaria ×ananassa Duch). Many major strawberry cultivars grown worldwide are susceptible to PhCR. Resistance is conferred by the recently-discovered FaRPc2 locus, but high-throughput markers are not yet available for marker-assisted breeding. In the current study, we developed DNA markers for two haplotypes at the FaRPc2 locus associated with resistance, H2 and H3. Marker validation and marker-assisted selection were performed in University of Florida (UF) breeding population. Seven single nucleotide polymorphism-based high resolution melting (HRM) markers linked to H2 and four HRM markers for H3 were developed. One HRM marker, RPCHRM3 linked to H3, was converted to a Kompetitive Allele Specific PCR (KASP) marker. To further examine the utility of the markers, they were screened in University of California Davis cultivars with known phenotypes as well as in 20 diverse accessions with phenotypes that are reported in the literature and that are preserved at the USDA-ARS National Clonal Germplasm Repository, in Corvallis, Oregon. The most informative markers for FaRPc2 resistance are being implemented in the UF strawberry breeding program to improve PhCR resistance.