Cloning and characterization of resistance gene candidate sequences and molecular marker development in gerbera (Gerbera hybrida)

Abstract

Improving disease resistance has become an important breeding objective in gerbera, one of the most important floricultural crops in the world. Development and application of molecular markers are expected to assist selection of gerberas with improved disease resistance. The availability of resistance gene candidate (RGC) sequences has accelerated the development of molecular markers for disease resistance traits in numerous plants. In this study, 84 gerbera RGC sequences (GhRGCs) were identified from cloned PCR products amplified from powdery mildew (PM)-resistant gerberas with degenerate oligonucleotide primers from two conserved motifs of plant disease resistance genes (R-genes). Nineteen GhRGC sequences could be translated into polypeptides with ≤90% amino acid identity. Multiple sequence alignment analysis of the 19 representative polypeptide sequences suggests two major clusters of gerbera RGCs. Twelve GhRGCs in cluster 1 contain the typical motifs of the toll and interleukin receptor–nucleotide binding site–leucine-rich repeat (TIR–NB–LRR) class R-genes within the nucleotide-binding site (NB) domain, and the seven GhRGCs in cluster 2 possess the typical motifs of the coiled coil–nucleotide binding site–leucine-rich repeat (CC–NB–LRR) class R-genes in the NB domain. The 19 GhRGCs were further divided into nine sub-families that share 15–50% amino acid identity. Thirty specific oligonucleotide primers were designed from 15 GhRGCs and tested on gerbera breeding line UFGE 4033 and ‘Sunburst Snow White’ that are PM-resistant and PM-susceptible, respectively, and are parents of two mapping populations for developing molecular markers for PM resistance. One sequence characterized amplified polymorphism (SCAR) and 11 cleaved amplified polymorphic site (CAPS) markers were developed and were polymorphic between the two parents. When used with the target region amplification polymorphism (TRAP) marker system, the 30 GhRGC-derived primers detected 242 additional DNA bands that were polymorphic between UFGE 4033 and ‘Sunburst Snow White’. This study represents the first effort to sample and characterize R-gene candidate sequences in gerbera. The obtained sequences may provide a valuable entry point to obtain full-length or additional sequences of gerbera NB–LRR genes. The SCAR, CAPS and TRAP markers developed may be valuable for mapping of genes or quantitative trait loci responsible for disease resistance in gerbera.