앙골라에 잘 적응하는 다수성 옥수수 품종 육성을 위한 기초지식을 얻기 위해 앙골라 옥수수 재래종 자원 89점을 대상으로 24개의 SSR 마커를 이용하여 유전적 다양성 분석을 실시한 결과는 다음과 같다. 총 254개의 allele가 탐지되었고 마커당 평균 allele 수는 10.6이었다. 마커의 PIC 값은 0.23 ~ 0.92 범위에 있었으며 평균값이 0.64이었다. 앙골라 옥수수 재래종 유전자원들은 매우 높은 유전적 다양성을 보여 계통간 비유사성(dissimilarity) 평균값이 0.643 이었고, 뚜렷하게 소수의 그룹들로 구분되지 않았다. 이러한 고도의 다양성을 가진 재래종 옥수수 유전자원을 이용하여 앙골라에 잘 적응하는 다양한 다수성 품종을 육성할 수 있을 것으로 기대된다.

As a first step of mapping genes conferring resistance to the brown planthopper, Nilaparvata lugens Stål, in Gayabyeo using a population derived from a cross between Gayabyeo and Taebaegbyeo, we performed the whole genome resequencing of these two Tongil-type rice varieties. The amount of raw sequence data was about 18.5X109 bp and 17.9X109 bp in Gayabyeo and Taebaegbyeo, respectively. After quality trimming and read mapping onto Nipponbare reference genome sequence, 9.3X109 bp was mapped in Gayabyeo with mapping depth of 25.0X, and 9.5X109 bp was mapped in Taebaegbyeo with mapping depth of 25.5X. Between Gayabyeo and Nipponbare, 1,585,880 SNPs were detected, while 1,416,898 SNPs were detected between Taebaegbyeo and Nipponbare. Between Gayabyeo and Taebaegbyeo, 284,501 SNPs were detected. Among the SNPs between Gayabyeo and Taebaegbyeo, 21.2% were in genic region and 78.8% were in intergenic region. In CDS region, 15,924 SNPs were detected, among which synonymous SNPs covered 47.3% and non-synonymous SNPs covered 52.7%. We designed Cleaved Amplified Polymorphic Sequences (CAPS) markers with SNPs in the restriction enzyme recognition sites, and 20 CAPS markers were tested. Of the 20 markers, 19 markers showed polymorphism and one marker showed monomorphism between Gayabyeo and Taebaegbyeo. It is expected that sufficient DNA markers for mapping genes with a population derived from a cross between Gayabyeo and Taebaegbyeo can be developed based on the results of the study.

The rice recombinant inbred lines derived from Milyang23 and Gihobyeo cross were used in genetic mapping and QTL analysis studies. In this study, we developed a new 101 CAPS markers based on the SNPs in the whole genome region between these varieties. As a result, the total genetic distance and average distances were 1,696.97 cM and 3.64 cM, respectively. In comparison to the distance of the previous genetic map constructed based on 365 DNA markers, the new genetic map was found to have a decreased distance. The map was applied for the detection of QTLs on all seven traits relevant to diameter of stem internode, length of culms, length of panicles and the number of panicles including the correlation analysis between each trait. The QTLs results were similar to the report in previous studies, whereas the distance between the markers was narrowed and accuracy increased with the addition of 101 CAPS markers. A total of 9 new QTLs were detected for stem internode traits. Among them, qI1D-6 had higher LOD of 5.1 and phenotype variation of 50.92%. In this experiment, a molecular map was constructed with CAPS markers using next generation sequencing showing high accuracy for markers and QTLs. In the future, developing more accurate QTL information on stem internode diameters with various agriculturally important traits will be possible for further rice breeding.

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice, which causes severe yield loss annually. Gayabyeo, a Tongil type rice variety, is known to have broad spectrum resistance to BPH. Before, it was estimated that Gayabyeo has at least two BPH resistance genes. We started a research for mapping resistance genes of Gayabyeo. We did a cross between Taebaekbyeo, a BPH susceptible Tongil type rice variety, and Gayabyeo, We grew F1 plants in winter season of 2014-2015, and planted F2 population in this year. About 100 DNA markers (SSR and InDel markers) showing polymorphism between Gayabyeo and Tabaekbyeo were selected. In addition, we are going to do resequencing Gayabyeo and Taebaekbyeo using Illumina Hiseq2000 to find much more DNA polymorphisms between the two varieties and develop new markers for mapping. The BPH response data will be acquired using F3 plants from the cross between Gayabyeo and Taebaekbyeo next year. In a while, crosses between Gayabyeo and high quality japonica rice varieties are being carried out to introduce BPH resistance genes of Gayabyeo into japonica high quality rice varieties. We expect to develop new DNA markers for BPH resistance genes of Gayabyeo through mapping and produce several japonica high quality rice lines harboring those genes at the end of this project.

We have generated 383 independent transgenic lines for genetically modified (GM) rice that contained PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes over-expression constructs under the control of the constitutive (CaMV 35S) promoter. TaqMan copy number assay was determined inserted T-DNA copy number. Also flanking sequence tags (FSTs) analysis was isolated from 203 single copy T-DNA lines of transgenic plants and sequence mapped to the rice chromosomes. In analyzing single copy lines, we identified 157 flanking sequence tags (FSTs), among which 58 (36%) were integrated into genic regions and 97 (62%) into intergenic regions. About 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assay. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNAs from leaf tissue of single copy, intergenic region of T-DNA insertion and homozygous T2 plants. The mRNA expression levels of the examined transgenic rice were significantly increased in all of the transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants were displayed higher levels of transgene protein. These results may be useful for producing of large-scale transgenic plants or T-DNA inserted mutants in rice.

Crops are exposed to various environmental stresses. These have been affecting the growth of crops, resulting in the severe loss of agronomic production in many countries. Therefore, development of new varieties of resistant crops is required to assure the desired productivity of crops in stress conditions. In this study, a putatively stress-related gene BrTSR53 was isolated from Brassica rapa. The BrTSR53 is 481 bp long and contains ORF region of 234 bp. The expression of BrTSR53 was determined by quantitative real-time PCR analysis. After 3 hr, the highest quantities of mRNA were revealed in cold and salt stress treatments. In drought stress treatments, there was the highest expression after 36 hr. Therefore, it was confirmed that the ORF in BrTSR53 should be a gene that confer increased resistance to B. rapa growing in different stress conditions. The ORF region of BrTSR53 gene was cloned into an expression vector, pYES-DEST52, and a new protein with molecular weight of 13 kDa was detected by western blot analysis. Also, stress tolerance tests showed that BrTSR53-ORF transgenic yeast exhibited increased resistance to the salt stresses compared with the control. In conclusion, the present data predicts that novel ORF in BrTSR53 can serve as an important genetic resource for abiotic stress resistance.

The next-generation sequencing (NGS) technology is being used for more effective genetic mapping and genome analysis. In this study, we performed whole-genome sequencing on the genomic DNA of Milyang23 and Gihobyeo using NGS and developed new cleaved amplified polymorphic sequence (CAPS) markers based on the single nucleotide polymorphisms (SNPs) in coding sequence between these varieties. Approximately, sequences of 60x coverage of the Nipponbare reference genome on average were obtained following Illumina sequencing. Totally, 1,726,798 SNPs between Milyang23 and Gihobyeo were detected. Among them, 149 SNP were selected for CAPS markers and located on genetic map with previously reported 219 PCR-based DNA markers. This map was applied to the detection of quantitative trait loci (QTLs) for stem internode diameters, culm length and panicle length in rice with MGRIL population. Newly 6 QTLs were detected for culm length (CL) and stem diameter (ID) traits including the first internode diameter (I1D), third internode diameter (I3D), and fourth internode diameter (I4D). Among those QTLs, qI1D5 and qCL5 had relatively higher LOD score and explained 8.99% and 4.24% of total variation. This study showed that the NGS allowed the rapid discovery of a large number of SNPs for CAPS marker. Only very small portion of SNPs through re-sequencing were used in this study. Furthermore, the results of QTL analysis described above shows relevance of molecular markers in mapping genes for useful traits.

Internode elongation is an important agronomic trait in rice that is associated with lodging, yield, and flooding adaptation. We identified a novel rice mutant line showing shortened uppermost internode among the rice Ac/Ds insertional mutant population and named it shortened uppermost internode 4 (sui4). The phenotypes of F1 plants and F2 plants from the cross of sui4 with its original variety, Dongjin, indicated that the SUI4 gene shows incomplete dominance or semidominance. Because the Ds genotypes did not co-segregate with the sui4 phenotypes, we performed mapping of this gene with 273 F2 plants from a cross between sui4 and Milyang23. Primary mapping revealed that the SUI4 locus was located between the S07012 and S07015 markers on rice chromosome 7. Further fine mapping narrowed down the location of SUI4 to the 1.1-Mbp interval of RM1253-S07015. Using re-sequencing data of this mutant along with its original variety, Dongjin, and five other varieties, we found six sui4 specific SNPs occurred within the genic region of five genes in the fine-mapped interval. Among them, one SNP is in exon, while the other five SNPs are in intron. This SNP in exon occurred in the miR172 binding site of a gene encoding AP2 domain transcription factor, which seems to interrupt suppression of this gene activity by miR172. We isolated the genomic region of this gene from sui4 and transformed the wild type variety, Dongjin. The transgenic plants showed remarkably shortened internodes, which indicates that this AP2 domain transcription factor gene is the SUI4 gene.

농작물은 다양한 외부 환경스트레스에 노출되어 있다. 환경스트레스는 작물의 성장에 영향을 주어 세계 각 지역의 농업 생산량을 심각하게 감소시키고 있다. 따라서 작물의 생산성을 높이기 위해서 다양한 환경스트레스에 내성이 강한 새로운 품종의 개발이 요구된다. 최근의 연구 동향은 환경스트레스 저항성 유전자를 작물에 도입시켜 환경 변화에 대한 저항성이 강한 작물을 개발하는 것이다. 본 연구에서는 배추의 저온, 고농도의 염과 건조 등의 환경스트레스에 대한 저항성 유전자로 추정되는 BrTSR53의 염기서열을 분석하였다. BrTSR53의 유전자의 총 길이는 481 bp이며 이중에서 ORF 부위는 234 bp이었다. 이 ORF의 염기서열 상동성을 분석한 결과 Arabidopsis에서 보고된 유전자와 유사한 것으로 나타났다. BrTSR53의 발현을 분석하기 위하여 quantitative real-time PCR을 실시하였다. 그 결과 배추를 고염 처리, 저온 처리하고 3시간 후에 가장 높은 mRNA 양을 보였으며, 건조 처리에서는 36시간 후에 발현량이 최대치를 보였다. 따라서 이 ORF는 환경스트레스에 대한 배추의 저항성 유전자임을 확인하였다. 그리고 BrTSR53 유전자를 효모발현 벡터인 pYES-DEST52에 삽입하고 western blot 분석법을 통해 효모에서 분자량이 약 13 kDa인 저항성 단백질의 발현을 확인하였다. 또한 BrTSR53 형질전환 효모는 염분 스트레스에 대한 저항성이 증가한 것으로 나타났다. 따라서 BrTSR53 유전자는 농작물의 환경스트레스 저항성을 높여줄 수 있는 주요한 유전자원으로 이용될 수 있다고 사료된다.

최근 급속하게 발달한 차세대 유전체분석기술을 기반으로 밀양23호와 기호벼의 유전체 서열을 분석하고, 새로운 CAPS 마커를 개발하였다. NGS를 통해 Nipponbare 유전체 길이의 60 배수만큼 염기서열을 결정하였고, CDS 안에서 두 품종간 특이적으로 나타나는 SNP를 CAPS 마커로 이용하였다. 새롭게 개발된 146개 CAPS 마커와 기존의 보고된 219개 마커를 통합하여 총 365개의 마커로 밀양23호/기호벼의 재조합자식 유전집단에 대해 분자 유전지도를 작성하였다. 벼의 줄기굵기와 간장 그리고 수장에 관한 QTL을 탐색한 결과, 총 19개의 유의성이 있는 QTL을 찾을 수 있었다. 이 중에 4개 줄기굵기 형질 관련 QTL과 2개 간장 형질 관련 QTL이 기존에 보고되지 않은 새로운 QTL이었다. 그 줄기굵기 QTL 중 가장 큰 LOD값을 갖는 qI1D5는 5번 염색체에서 탐색되었으며, 1절굵기 표현형 변이는 8.99%였다. 또한, 간장관련 QTL 중 가장 큰 LOD 값을 갖는 qCL5은 5번 염색체에서 탐색되었고, 이 QTL의 간장 표현형 변이는 4.24%였다. 재염기서열을 통해 밝혀진 SNP 중 소수만이 본 연구에 사용되었다. 향후 본 연구에서 밝혀진 SNP 정보를 이용한다면 더 많은 마커를 개발하여, 고밀도 유전지도 작성이 가능할 것이다. 더 나아가 MGRIL을 이용하여 농업적으로 유용한 형질에 대해 더 정확한 QTL 분석과 유용유전자의 개발이 가능하게 될 것이다.

In order to find new genetic sources of rice salt tolerance, we did screening with about 10,000 rice mutant lines created by Ac/Ds insertional mutagenesis. First, we raised rice seedlings with media soil on 0.7% NaCl solution and selected 71 putative salt tolerant lines and analyzed their Ds insertion sites. We tested their salt tolerance by growing seedlings on MS medium containing, 0 mM, 150 mM, and 250 mM NaCl. Also, their seedling salt tolerance were evaluated by growing on Yoshida nutritional solution containing 0.6% NaCl. Finally, we selected eight mutant lines showing increased seedling salt tolerance compared with wild type variety, Dongjin, repeatedly. We grow them in rice field and investigated their agronomic traits such as heading time, culm length, panicle length, and panicles per hill. Among them two lines which were named Salt10 and Salt23 and showed favorable agronomic characteristics were crossed with Dongjin for further genetic analysis and mapping the causative gene variation.