Cloning And Characterization Of Resistance Gene Candidate Sequences In Two Ornamental Plants And Application Of Molecular Markers For Powdery Mildew Resistance In Gerbera

The most common class of disease resistance genes (R genes) in plant genomes seems tobe the nucleotide-binding site leucine-rich repeat (NB-LRR) class genes. High levels ofsequence conservation in this class of R genes have enabled the designing of degenerateprimers for PCR amplification of resistance gene candidate (RGC) sequences from numerousplants. In this study, RGCs were cloned and charactered in two ornamental plants, gerbera andcaladium.84gerbera RGC sequences (GhRGCs) were identified from cloned PCR productsamplified from powdery mildew (PM)-resistant gerberas with degenerate oligonucleotideprimers from two conserved motifs of plant disease resistance genes (R-genes). NineteenGhRGC sequences could be translated into polypeptides with≤90%amino acid identity.Multiple sequence alignment analysis of the19representative polypeptide sequences suggeststwo major clusters of gerbera RGCs. Twelve GhRGCs in cluster1contain the typical motifsof the toll and interleukin receptor–nucleotide binding site–leucine-rich repeat (TIR–NB–LRR) class R-genes within the nucleotidebinding site (NB) domain, and the seven GhRGCsin cluster2possess the typical motifs of the coiled coil–nucleotide binding site–leucine-richrepeat (CC–NB–LRR) class R-genes in the NB domain. The19GhRGCs were further dividedinto nine sub-families that share15–50%amino acid identity. Thirty specific oligonucleotideprimers were designed from15GhRGCs and tested on gerbera breeding line UFGE4033and‘Sunburst Snow White’ that are PM-resistant and PM-susceptible, respectively, and areparents of two mapping populations for developing molecular markers for PM resistance. Onesequence characterized amplified polymorphism (SCAR) and11cleaved amplifiedpolymorphic site (CAPS) markers were developed and were polymorphic between the twoparents. When used with the target region amplification polymorphism (TRAP) markersystem, the30GhRGC-derived primers detected242additional DNA bands that werepolymorphic between UFGE4033and ‘Sunburst Snow White’. This study represents the first effort to sample and characterize R-gene candidate sequences in gerbera. The obtainedsequences may provide a valuable entry point to obtain full-length or additional sequences ofgerbera NB–LRR genes. The SCAR, CAPS and TRAP markers developed may be valuablefor mapping of genes or quantitative trait loci responsible for disease resistance in gerbera.Caladium is an important aroid widely used in the ornamental plant industry. Concernshave been raised about possible loss of genetic diversity due to a drastic decline in the numberof cultivars in the last century. This study used fourteenth combinations of reporteddegenerate primers to amplify RGCs from caladium. Out of224fragments sequenced,71were RGC sequences containing the typical motifs of the NB domain of the NB-LRR R genes.Eighteenth CRGC sequences could be translated into polypeptides with≤90%amino acididentity. Multiple sequence alignment analysis of the18representative polypeptide sequenceswere clustered into eight groups, they were all belong to the CC (coiled coil)-NB-LRRsubfamily of plant NB-LRR R genes.designated as CCNL1to CCNL8, respectively. To assessgenetic diversity and relationships among caladium cultivars, eight major cultivars wereanalyzed using the target-region amplification polymorphism marker system (TRAP). TwelveTRAP combinations of primers containing two arbitrary primers and six fixed primersgenerated260scorable DNA fragments,35.9%of which were polymorphic. Cultivars shareda small similarity at the molecular level with the Jaccard coefficient from0.281to0.731. Theeight cultivars were clustered into two groups and four subgroups by cluster analysis. Theseresults are beneficial for us to use various target variants to widen the genetic background ofcucumber cultivar。Gerbera (Gerbera hybrida) is an important floricultural crop in the United States andworldwide. Powdery mildew (PM) caused by Podosphaera fusca Braun and Shishkoff is themost common and destructive disease in gerbera production and landscape use. Previouslytwo sources of PM resistance were identified out of2,000+gerbera lines screened in centralFlorida. Gerbera breeding line UFGE31-19is one of the few sources of resistance to PM ingerbera and has contributed its resistance to new gerbera cultivars. To determine the mode ofinheritance for PM resistance in UFGE31-19, one of its PM-resistant progeny, UFGE4033,was crossed with PM-susceptible cultivar, Sunburst Snow White, and their progeny wereevaluated for PM severity. Distribution of PM severity ratings among the progeny wascontinuous but with two peaks, suggesting that the PM resistance in UFGE4033and UFGE31-19is a quantitative trait, likely controlled by major genes. Bulked segregant analysisidentified17molecular markers present in UFGE4033and the PM-resistant bulk but absentin ‘Sunburst Snow White’ and the PM-susceptible bulk. Eleven of the molecular markerswere mapped to one genetic linkage group, and two regions on this linkage group together explained71.1%of the phenotypic (PM severity rating) variance in the segregatingpopulation. It was proposed inoculated on the leaf surface of UFGE4033germinated, formedsecondary germ tubes, and formed appressoria at high percentages, similar to those on the leafsurface of ‘Sunburst Snow White’. However, P. fusca hyphae branched significantly less,were significantly shorter, and produced substantially fewer conidia on the leaf surface ofUFGE4033and its PM-resistant progeny than on the leaf surface of ‘Sunburst Snow White’.These results should provide a sound foundation for use of UFGE31-19and progeny UFGE4033in gerbera disease resistance breeding and facilitate further investigation andunderstanding of the genetic bases of PM resistance in gerbera.