Development of Molecular Markers and Mining Genes Related to Pear Scab (Venturia nashicola) Resistance

Abstract

Scab, caused by Venturia nashicola, is one of the most damaging diseases in Asian pear, especially genotypes belonging to Pyrus pyrifolia. The development of cultivars resistant to scab has long been an aim of Asian pear breeding programs. Screening conditions were optimized for selecting scab-resistant sources. Pyrus accessions could be classified according to their susceptibilities around 6 weeks after inoculation with higher than 5 Ⅹ 103 conidia/mL and leaf ages of 10 days after unfolding were suitable to be inoculated. The degree of resistance of 172 accessions including 24 species collected from all temperate regions of the world, mainly P. pyrifolia, P. ussuriensis, P. communis, and P. bretschneideri were examined for 7 weeks after artificial inoculation with V. nashicola conidial suspension at 1 Ⅹ 105 conidia/mL. Scab resistance was classified into four groups. P. aromatica, P. betulaefolia, P. calleryana, P. dimorphophylla, P. lindleyi, P. pashia, P. phaeocarpa, P. sinkiangensis, P. sohayakinensis, and P. communis including its hybrid cultivars except Mustafabey were evaluated as highly resistant. Local cultivars such as Hwangsilri and Yeongmokri belonging to P. ussuriensis, and Hoengseongcheongri and Chanxixueli belonging to P. pyrifolia were resistant. However, most cultivars of P. bretschneideri such as Yali and Cili and P. pyrifolia such as Niitaka and Whangkeumbae, and P. ussuriensis such as Anli were susceptible or highly susceptible. DNA markers linked to the scab resistance gene named Rvn2 were identified from the progeny of a cross between PS2-93-3-98 and Yali. As scab resistance clearly segregated 1:1 in the F1 progeny, Rvn2 was found to be the single dominant gene. Through bulked segregant analysis and amplified fragment length polymorphism (AFLP) analysis, three AFLP markers linked to Rvn2 were identified. Mapping of the markers indicated that the three selected markers, E-AGT/M-CCA234, E-ATT/M-CCG328, and E-GGT/M-TCT217, were located 4.9, 3.2, and 0.8 cM from the Rvn2 locus, respectively. Rvn2 was found to be located in a different linkage group from the previously identified scab resistance gene loci. For marker-assisted selection of scab resistance, two of the AFLP markers (E-AGT/MCCA234 and E-GGT/M-TCT217) were converted into cleaved amplified polymorphic sequence (CAPS) markers. These CAPS markers, PSC217-XhoI and PSC234-HaeIII, could distinguish resistant and susceptible individuals, and thus have the potential to increase the selection efficiency for scab resistance in pear breeding programs. After performing subtractive suppression hybridization using cDNA of treated (tester) and control (driver) leaves harvested at 1, 48, and 96 h after inoculation, 159 (1 h forward), 242 (1 h reverse), 384 (48 h forward), 190 (48 h reverse), 110 (96 h forward), and 144 clones (96 h reverse) were randomly selected and their characteristics were analyzed through sequencing. Using BLASTX searches of GenBank for the each cDNA library, 15, 26, 50, 29, 22, and 34 unique sequences were isolated from 1 h forward, 1 h reverse, 48 h forward, 48 h reverse, 96 h forward, and 96 h reverse, respectively. Most of the highly represented expressed sequence tags (ESTs) obtained from 1 h forward and reverse were photosynthesis- and senescence-related genes such as light harvesting chlorophyll a/b binding protein, ribulose-bisphosphate carboxylase/oxyganase, and senescence-associated protein. Although more than 50% of ESTs expressed from 48 h forward and reverse were associated with photosynthesis- and carbon fixation-related genes, defense-related genes as major allergens were annotated in 48 h forward. Unlike expressed ESTs from 1 and 48 h forward, defense-related ESTs such as cytochrome P450, pathogenesis-related protein, Al-induced protein, metallothionein-like protein, allergen, and F-box family protein accounted for 57% of 96 h forward and 2% of 96 h reverse. Among 28 clones involved defense mechanism and expressed majority at each time, ten clones showed more than one relative expression rate in 96 h inoculated Hwangsilri leaf were chosen. Two clones out of ten were selected using six pear cultivars ranging from highly susceptible, Yali, to highly resistant Bartlett (non-host) leaves harvested at 96 h after inoculation by preliminary quantitative real-time polymerase chain reaction (PCR) analysis. The quantitative real-time PCR analysis indicated that expression levels of the transcripts for eight clones were similar between the incompatible and compatible interactions. On the other hand, one clone, aldo-keto reductase gene (96h_T_28) related to monomeric NADPH-dependent oxido- reductase showed higher expression level in resistant cultivars, Bartlett and Hwangsilri and lower susceptible cultivar, Gamcheonbae than in susceptible and highly susceptible cultivars. The other, hypothetical protein gene (96h_T_110) showed differentially high expression pattern in Gamchenbae, classified into lower susceptible cultivar. Aldo-keto reductase and hypothetical protein genes observed in the present study were found to be associated with plant defense mechanism. In order to transform these genes into target cultivar, more detailed functional study is required to understand how these genes are related to the defense system against V. nashicola in pears.