Throughout their meiotic maturation in most mammals, oocytes are arrested twice, prophase I and metaphase II. Being released from these arrests, transient or oscillation of intracellular Ca2+ concentration is observed in the ooplasm, which is not answered in relation to the specific role in the resumption of meiotic arrest. Recently, Ca2+/calmodulin-dependent protein kinase II (CaM KII) has been known as a Ca2+ oscillation decoder from the in vitro experiment. CaM KII is multifunctional serine/threonine kinase observed in most cells. Present studies were performed to investigate the role of CaM KII during resumption of meiotic arrest and activation in vitro of mouse oocytes. It was questioned whether CaM KII might be involved in the meiotic resumption of mouse oocytes. Compared to the control, both of CaM KII inhibitors, KN-93 and KN-62, significantly inhibited germinal vesicle breakdown (GVBD) of mouse oocytes in a dose-dependent manner. As the concentration of KN-93 increased, concomitant decrease of intracellular Ca2+ concentration ([Ca2+]i) was also observed using confocal laser scanning microscope (CLSM) and an intracellular Ca2+ indicator, fluo 3-AM. When GVBD oocytes were treated with 6% ethanol, small [Ca2+]i transient was observed in oocytes bathed with Ca2+-free medium and large increase was observed in oocytes bathed with Ca2+-containing medium, suggesting that [Ca2+]i transient could happen from intracellular Ca2+ store as well as Ca2+ influx through Ca2+-channel on the oolemma. However, KN-93 inhibited the [Ca2+]i transient of GVBD oocytes in both cases. Using monoclonal antibodies against α-subunit of CaM KII, tubulin and microtubule-assocaited proteins (MAPs), CaM KII has been colocalized on the spindle with tubulin and MAPs. The present study also demonstrated the presence of α-subunit of CaM KII in heart, kidney, testes, ovary as well as in brain of the mouse. In ovarian follicles, CaM KII was expressed in granulosa cells and oocytes. Based on overall the above results, followings are suggested. First, CaM KII might be involved in the regulatory mechanism of meiotic resumption. Second, CaM KII might play a regulatory role in the stabilization of microtubule.

The last decade has witnessed tremendous progress in genome sequencing and explosion of genome sequence information. This remarkable advancement in genomics provides unprecedented opportunities for crop improvement. Pepper (Capsicum spp.) is an important vegetable crop worldwide. Pepper production is constantly challenged by various pathogens and developing cultivars harnessing multiple disease resistance genes are ever increasing. Molecular markers linked to disease resistance genes will expedite the gene pyramiding. Here, I introduce genome-assisted development of molecular markers linked to resistance genes, in pepper. Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of Capsicum annuum against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F2 population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 x 103 /mL). Resistance phenotype segregation ratios for the populations were close to 3:1 and 1:1 (resistant:susceptible), respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 were used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to Phytophthora resistance. Markers linked to these genes were developed and validated by testing 100 F1 commercial cultivars. Among the markers, Phyto5NBS1 showed about 90% accuracy in predicting resistance phenotypes to a low-virulence Phytophthora isolate. These results suggest that Phyto5NBS1 is a reliable marker for Phytophthora resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5

Genotyping-by-sequencing (GBS) is a robust and rapid tool to develop SNP markers. Reduced sequencing complexity and multiplex sequencing of GBS has reduced genotyping cost for complex genome. However multiplex sequencing brings low sequencing depth which can lead to reduced number of markers. Therefore to find the appropriate condition for GBS is needed. In this research we demonstrated the use of ApeKI and selective primers for GBS of pepper (Capsicum spp.). Selective primers which amplify the GBS library with one or two flanking sequences to ApeKI site were used to increase sequencing depth. By in silico digestion, we developed six different selective primers amplify 4,000-400,000 regions. We made the GBS library with eight pepper accessions in four species using six selective primers and sequenced. Proper selective primers and pooling rate for each species will be determined. This approach will be useful for genotyping Capsicum breeding lines or populations by developing high quality SNP markers.

Capsicum diversity is getting lower in modern crops because of the genetic erosion. In Capsicum, breeders have been mainly focused on agriculturally important traits such as disease resistances, high yield and pungency. This narrow breeding pool hampered to develop improved cultivars. It has become a hot issue to conservation of genetic diversity and exploitation of wild germplasm in Capsicum. However, although a large number of accessions are maintained in Capsicum germplasm collections, their use for crop improvement is limited by the scarcity of information on genetic diversity, population structure and proper phenotypic assessment. The identification of representative and manageable subset of accessions would facilitate access to the diversity available in large collections. A genome wide germplasm characterization using molecular markers can offer reliable tools for adjusting the quality and representativeness of core samples. We investigated patterns of molecular diversity at 48 single nucleotide polymorphisms (SNPs) in 4056 accessions from 11 Capsicum species from 89 different countries. Using these genetic variations and 32 different morphological traits, 250 core set was selected in whole Capsicum germplasm. The core collection could be a primary source for distributing germplasm to pepper breeders and other national programs as well as for evaluation

Radiation technologies have been widely used in mutation breeding of crops. Although gamma-radiation has been predominantly applied in radiation breeding, ion beam irradiation is recently emerging as a methodology highly effective in induction of mutations that are useful for plant breeding. Therefore, we investigated the biological effects caused by irradiation of two types of ion beams, which were proton and ion beams, respectively, in pepper. In the evaluation of survival rate, LD50 values were between 300 and 350 Gy, 30 and 40 Gy in irradiation of proton and carbon beams, respectively, while LD50 for gamma-ray was determined to be between 50 and 100 Gy. Growth traits including shoot length, root length, and root width were also examined in pepper seedlings according to does of proton and carbon ion beams to estimate biological effectiveness of each radiation. The result was applied in the construction of pepper mutant population which will be used to develop pepper breeding materials containing novel characteristics.

Capsinoids, low-pungent compounds, have the same biological effects as capsaicinoids such as anticancer and anti-obesity. A precursor of capsinoids, vanillyl alcohol, is known to be produced by mutations in the p-aminotransferase (p-AMT) gene. In the previous study, SNU11-001 (C. chinense) containing high levels of capsinoids was found in germplasm collections of Seoul National University. We found that this collection has a unique mutation in the p-AMT gene. In order to develop a cultivar containing high capsinoids contents, marker-assisted foreground and background selection were performed in this study. Backcrossing is an effective breeding method for introducing useful traits to an elite cultivar. Compared to conventional backcrossing, marker-assisted backcrossing (MABC) is extremely useful for recovery of a recurrent parent’s genetic background. To obtain background selection markers, a total of 412 single nucleotide polymorphism (SNP) markers was screened to obtain polymorphic SNP markers between ‘Takanotsume (C. annuum)’ and ‘SNU11-001’. Of the 412 SNP markers, 96 polymorphic SNP markers evenly distributed in pepper genome were finally selected. Plants carrying the pAmt/pamt genotype were selected from a BC1F1 population using SCAR markers derived from the unique p-AMT mutation of SNU11-001. BC1F1 plants carrying the pAmt/pamt genotype were subjected to background selection. Multiple genotype analysis was done using Fluidigm platform (BioMark). Once we obtain plants carrying most similar genetic background to recurrent parent, capsinoids contents will be measured and another round of MABC be done to obtain plants containing high levels of capsinoids.

In plants, eukaryotic translation elongation factor 1B (eEF1B) is composed of three subunits, eEF1Bα, eEF1Bβ and eEF1B γ. Two subunits are nucleotide exchange subunits (eEF1Bα and eEF1Bβ) and one is a structural protein (eEF1Bγ). In the previous study, eEF1B was identified as a common host factor for several RNA viruses. To test which subunit of eEF1B is essential for Potato virus X (PVX) replication, the virus-induced gene silencing (VIGS) for eEF1Bα, β or γ was performed in Nicotiana benthamiana and green fluorescent protein (GFP)-tagged PVX was inoculated. PVX-GFP accumulation was decreased when eEF1Bβ or γ subunit was silenced, whereas eEF1Bα had no effect on PVX-GFP accumulation in inoculated leaves. Targeting induced local lesions in genome (TILLING) was performed using a Capsicum annuum EMS population to test whether mutations in eEF1Bβ subunit affect virus infection in pepper. We obtained 81 eEF1Bβ mutant lines consisted of 16,759 individuals. These mutant lines are being tested to validate the function of eEF1B β in PVX replication.

Amplified fragment length polymorphism (AFLP) is one of molecular marker technique based on DNA and is extremely useful in detection of high polymorphism between closely related genotypes like Korean wheat cultivars. Six Korean wheat cultivar specific marker sets have been developed from inter simple sequence repeat (ISSR) analysis and we can identify the 13 Koran wheat cultivars form other cultivars using six that (Son et al., 2013). We used four combinations of primer sets in our AFLP analysis for developing additional cultivar specific markers in Korean wheat. Twenty-one of the AFLP bands were isolated from ACG/M-CAC primer combination and 19 bands were isolated from E-AGC/M-CTG primer combination, respectively. We used forty bands to design sequence characterized amplified region (SCAR) primer pairs for Korean wheat cultivar identification. Only one of 40 amplified primer pairs, C2, were able to use for wheat cultivar identification. The DNA band of 215bp length was amplified by C2 primer pairs in ten cultivars, Eunpa, Olgeuru, Gobun, Saeol, Milsung, Sinmichal, Jokyung, Sugang, Goso, and Joah. Then C2 primer was applied to these primer sets as newly SCAR marker, six cultivars are identifying from other cultivars, additionally. Finally, to use the C2 and six primer sets, 19 Korean wheat cultivars are identified.

sy-2 (Seychelles-2) is a temperature sensitive mutant of Capsicum chinense and native to Seychelles Island in Africa. Previously we showed that sy-2 leaves were irregularly shaped and defective in chlorophyll development at temperatures lower than 24℃. A segregation test revealed that the sy-2 gene is controlled by a single recessive gene. To identify the sy-2 gene, we performed a map-based cloning approach using a total of 1,010 F2 plants derived from crossing sy-2 and the wild type C. chinense ‘No.3341’. sy-2 gene is located on chromosome 1, 0.3 cM and 0.1cM away from cosII markers C2_At4g29120 and C2_At1g09070, respectively. The tomato genome sequence between those two markers was compared with pepper genome sequence. We found three of pepper scaffold sequences in this region. We developed seven ingle nucleotide polymorphism (SNP) markers on the pepper scaffold sequences, among which five SNP markers were co-segregated with sy-2. To fill the gap between the scaffolds which contains co-segregating markers, we screened a bacterial artificial chromosome (BAC) library, and end-sequences of total of 22 AC clones were i. We found that five clones were overlapped to cover the gap. We fully sequenced four AC clones and found that the physical distance between C2_At4g29120 and C2_At1g09070 is 343kb. This region contains 70 putative genes such as HSP90-like ATPase family proteins, lipid-transfer proteins, calmodulin-domain protein kinases, and zinc finger proteins (ZFPs). To identify the sy-2 gene, we performed RT-PCR and found that a ZFP-like gene is differentially expressed between WT and sy-2 leaves. This result suggests that the ZFP-like gene is a strong candidate for the sy-2 gene. We are currently characterizing this candidate gene.

Tomato yellow leaf curl disease is a devastating disease of tomato (Solanum lycopersicum), which is caused by begomoviruses generally referred to as tomato yellow leaf curl virus (TYLCV). The breeding for TYLCV resistance has been based on the introgression of the Ty-3 resistance locus. Knowledge about the exact location of the Ty-3 on tomato chromosome 6 is needed to understand the genomic organization of the Ty-3 locus. In this study, we conducted a genetic analysis using a segregating population derived from a cross between resistant accession S. lycopersicum “A45” and susceptible accession S. lycopersicum “A39”. The F1 plants showed resistance to TYLCV and F1 was self-pollinated to produce F2 progeny. To screen the TYLCV resistance in 145 F2 plants, a leaf agroinfiltration method was used. F2 plants showed a classical Mendelian seregation (106 resistance : 39 susceptibility) for resistance to TYLCV respectively. SCAR and CAPS markers linked to the Ty-3 were tested for genotyping F2 plants and .genotyping and agroinfiltration results were cosegregated in the newly developed F2 population.

Capsinoids, low-pungent compounds, have the same biological effects as capsaicinoids such as anticancer and anti-obesity. A precursor of capsinoids, vanillyl alcohol, is known to be produced by mutations in the p-aminotransferase (p-AMT) gene. In the previous study, SNU11-001 (C. chinense) containing high levels of capsinoids was found in germplasm collections of Seoul National University. We found that this collection has a unique mutation in the p-AMT gene. In order to develop a cultivar containing high capsinoids contents, marker-assisted foreground and background selection were performed in this study. Backcrossing is an effective breeding method for introducing useful traits to an elite cultivar. Compared to conventional backcrossing, marker-assisted backcrossing (MABC) is extremely useful for recovery of a recurrent parent’s genetic background. To obtain background selection markers, a total of 412 single nucleotide polymorphism (SNP) markers was screened to obtain polymorphic SNP markers between ‘Takanotsume (C. annuum)’ and ‘SNU11-001’. Of the 412 SNP markers, 96 polymorphic SNP markers evenly distributed in pepper genome were finally selected. Plants carrying the pAmt/pamt genotype were selected from a BC1F1 population using SCAR markers derived from the unique p-AMT mutation of SNU11-001. BC1F1 plants carrying the pAmt/pamt genotype were subjected to background selection. Multiple genotype analysis was done using Fluidigm platform (BioMark). Once we obtain plants carrying most similar genetic background to recurrent parent, capsinoids contents will be measured and another round of MABC be done to obtain plants containing high levels of capsinoids