Il Sheob Shin

Development of AFLP and CAPS markers linked to the scab resistance gene, Rvn2, in an inter-specific hybrid pear (Pyrus spp.)

Summary Scab, caused by Venturia nashicola, is one of the most damaging diseases in pear. The development of varieties resistant to scab has long been an aim of Asian pear breeding programmes. The application of molecular markers to select scab-resistant plants will increase the efficiency of breeding programmes. To identify DNA markers linked to the scab-resistance gene named Rvn2 in PS2-93-3-98, the progeny of a cross between PS2-93-3-98 and ‘Yali’ pear was studied. As scab-resistance clearly segregated 1:1 in the F1 progeny, Rvn2 is a single dominant gene. Bulked segregant analysis and amplified fragment length polymorphism (AFLP) analysis were then applied to identify markers associated with Rvn2. 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 previously identified scab-resistance gene loci. For marker-assisted selection of scab resistance, two of the AFLP markers (E-AGT/M-CCA234 and E-GGT/M-TCT217) were converted into cleaved amplified polymorphic sequence (CAPS) markers. These CAPS markers, designated PSC217-Xho I and PSC234-Hae III could distinguish between resistant and susceptible individuals.These CAPS markers therefore have the potential to increase the efficiency of selection for scab-resistance in pear breeding programmes.

Production System of Virus-free Apple Plants Using Heat Treatment and Shoot Tip Culture

Gunsup Lee*, Jeong Hee Kim*, Hyun Ran Kim, Il Sheob Shin, Kang Hee Cho, Se Hee Kim, Juhee Shin and Dae Hyun Kim** Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 440-706, Korea Apple Research Station, National Institute of Horticultural and Herbal Science, Rural Development Administration, Gunwi 716-812, Korea Future & Creation Strategy Team, Rural Development Administration, Suwon 441-707, Korea (Received on November 12, 2013; Revised on November 21, 2013; Accepted on November 25, 2013)

Identification of Korean pear cultivars using combinations of SCAR markers

The conventional method to identify pear tree cultivars is to evaluate the morphological characteristics of the trunk, leaves, flowers, and fruit. However, it is difficult to distinguish between the closely related pear cultivars using only morphological traits, particularly if the cultivars to be compared share the same pedigree. Thus, we developed more reliable DNA markers to identify 39 pear cultivars bred in Korea and Japan. In total, 147 random amplified polymorphic DNA (RAPD) markers were developed from 40 different random primers. The resulting 51 RAPD fragments were selected, and their sequences were determined to convert them into sequence-characterized amplified region (SCAR) markers. As a result, 19 of 51 RAPD fragments were successfully converted to SCAR markers. A single polymorphic band of the same size as the RAPD fragments or smaller DNA fragments were amplified depending on primer combinations in the 17 SCAR markers, and co-dominant polymorphisms were observed using two of the SCAR markers (named PK10_514 and PK15_616). A combination of eight SCAR markers (P561_331, P561_372, PH02_405, PH15_ 452, PT16_472, PK10_514, P270_593, and PK15_616) provided sufficient polymorphisms to identify 25 Korean pear cultivars among the 39 pear cultivars tested. A combination of nine SCAR markers (P561_331, P561_372, PH02_ 405, PH15_452, PT16_472, PT14_578, P270_593, PK10_514, and PK15_616) could be used to sufficiently distinguish among the 39 pear cultivars examined in this study. These newly developed markers will be useful as a fast and reliable tool to identify Korean pear cultivars.

Influence of temperature during transport on shelf-life quality of highbush blueberries (Vaccinium corymbosum L. cvs. Bluetta, Duke)

Quality changes of highbush blueberry cultivars (Vaccinium corymbosum L. cvs. Bluetta, Duke) were evaluated as influenced by transport temperature. Neither cultivar remained marketable after 6 days or 16 days at room temperature or 10°C. The ‘Duke’ cultivar had slightly lower weight loss than ‘Bluetta,’ but difference between the cultivars was not significant after 12 days of storage. Decrease of fruit firmness was delayed by storage at 10°C for both cultivars, with ‘Duke’ blueberries being firmer than ‘Bluetta’. Soluble solids content was 9.9 ± 0.3 °Brix for both cultivars, which was lower than previous reports, probably as a result of weather conditions before harvest. Organic acid content declined in both cultivars during storage. Hue value of the ‘Bluetta’ cultivar was higher (more purple-blue) than the ‘Duke’ cultivar, but Hunter L and hue angle did not change during storage. Fruit characteristics at harvest and postharvest maintenance of low temperature are clearly important factors affecting the post-harvest fruit quality during transportation and storage.

Comparison of transcriptome analysis between red flash peach cultivar and white flash peach cultivar using next generation sequencing

Differences of gene expression between red flash peach cultivar and white flash peach cultivar were investigated by Nest-generation sequencing (NGS). EST from the red flash peach cultivar and white flash peach cultivar were selected for nucleotide sequence determination and homology searches. The levels of transcripts coding for proteins involved in pathogenesis related proteins, temperature stress, ethylene signal pathway were significantly higher in white flash peach cultivar than in red flash peach cultivar. On the other hand, the up-regulation of proteins involved in anthocyanin and flavonol biosynthesis and protein degradation and sorbitol metabolism were observed in red flash peach cultivar. Chalcone synthase was preferentially expressed in the red flesh peach cultivar, agreeing with the accumulation of anthocyanin and expression of other previously identified genes for anthocyanin biosynthesis. Anthocyanin pathway related genes CHS, F3H, DFR, LDOX, UFGT differentially expressed between red flash peach cultivar and white flash peach cultivar. These results suggest that red flash peach cultivar and white flash peach cultivar have different anthocyanin biosynthesis regulatory mechanisms.

Identification of genes induced by Venturia nashicola in indigenous Korean pear ‘Hwangsilri’

Indigenous Korean pear ‘Hwangsilri’ leaves were inoculated with Venturia nashicola to build expressed sequence tags (ESTs) database as resources for transcripts induced in the inoculated leaves. After performing subtractive suppression hybridization using cDNA of the inoculated and uninoculated leaves harvested at 1, 48, and 96 hours after inoculation, 159 (1 hour), 384 (48 hours), and 110 clones (96 hours) were selected and sequenced. BLASTX searches each cDNA library against Genbank revealed 15, 50, and 22 unique sequences at 1, 48, and 96 hours after inoculation, respectively. Most highly represented ESTs at 1 hour after inoculation were photosynthesis- and senescence-related genes such as light harvesting chlorophyll a/b binding protein, ribulose-bishosphate carboxylase/oxyganase, and senescence-associated protein. Cytochrome P450-like TATA box binding proteins and manganese superoxide dismutase associated with the defense response, biotic and abiotic stresses were differentially expressed at 1 hour after inoculation. Although more than 50% of ESTs at 1 and 48 hours after inoculation were also associated with photosynthesis- and carbon fixation-related genes, defense-related genes were annotated as 14 clones at 48 hours after inoculation. Six ESTs associated with pathogen-defense and biotic and abiotic stresses were expressed only at 48 hours after inoculation. At 96 hours after inoculation, seven ESTs were involved in defense-response and biotic and abiotic stresses, and three out of the seven ESTs such as cyclophilin, F-box family, and leucine-rich repeat receptor-like kinase (LRR-RLK) were uniquely expressed. Quantitative real-time polymerase chain reaction analysis revealed that the transcripts for aldo-keto reductase (AKR) and LRR-RLK were highly expressed in the incompatible interaction. AKR gene was more highly expressed in resistant cultivar ‘Hwangsilri’ and moderately susceptible one ‘Gamcheonbae’ than in other cultivars. LRR-RLK showed differentially higher expression pattern in inoculated than in uninoculated leaves of susceptible cultivars including ‘Gamcheonbae’ and ‘Hwangkeumbae’. The ARK and LRRRLK genes were found to be associated with plant defense mechanism. However, more detailed further study using transformants introgressed these genes is required to understand how these genes are expressed and regulated by infection with V. nashicola.

Plant regeneration and transformation of grape (Vitis labrusca L.) via direct regeneration method

Abstract Efficient regeneration methods and transformation system are a priority for successful application of genetic engineering to vegetative propagated plants such as grape ( Vitis labrusca L.). This research is to establish shoot regeneration system from plant explants for ‘Campbell Early’, ‘Tamnara’, ‘Heukgoosul’, ‘Heukbosek’ using two types of plant growth regulators supplemented to medium. The highest adventitious shoot regeneration rate of 5% was achieved on a medium containing of Murashige and Skoog (MS) inorganic salts and Linsmaier and Skoog (LS) vitamins, 2 mg/L of TDZ and 0.1 mg/L of IBA. Leaf tissue of ‘Campbell Early’, was co-cultivated with Agrobacterium strains, LBA4404 containing the vector pBI121 carrying with CaMV 35S promoter, gus gene as reporter gene and resistance to kanamycin as selective agent, the other Agrobacterium strains, GV3101 containing the vector pB7 WG2D carrying with mPAP1-D gene. mPAP1-D is a regulatory genes of the anthocyanin biosynthetic pathway. ‘Campbell Early’ harboring