쌀 가공산업을 활성화하고 소비를 촉진하여 국내 쌀 생산기반을 유지하기 위해서는 쌀가루 제분적성을 보유한 가공용벼 품종 개발이 시급하다. 농촌진흥청 국립식량과학원에서는 아지드화나트륨을 돌연변이원으로 활용하여 건식제분적합성을 보유한 분질배유 돌연변이 후대계통인 ‘Namil(SA)-flo1’를 육성한 바 있다. 본 연구는 염색체 상에서 ‘Namil(SA)-flo1’의 분질배유 특성을 지배하는 유전자위를 탐색하고자수행하였다. 주요 결과는 아래와 같다.1. ‘Namil(SA)-flo1’ × ‘밀양23호’로부터 유래한 F2 94 개체로부터 종자 분질립 비율을 검정하고 54개 SSR 마커의 유전자형을 검정하여 연관성분석(association analysis)을 수행한 결과 목표 유전자위는 5번 염색체 중하단부위로 추정되었다.2. 목표 부위의 SSR 마커 밀도를 높여 추가 연관성분석을 실시하였고, F2:3 종자 분질립 변이의 79.7%가 5번염색체 상의 RM164의 유전자형 변이에 의하여 설명된다는 것을 확인하였다.3. 이를 통하여 분질배유 지배 유전자위를 5번 염색체17.7~20.7 Mbp 부위로 추정하였으며, 추후 추가 분리집단을 이용하여 목표 유전자를 동정하고 쌀가루용품종 개발에 활용할 수 있는 핵산정밀표지인자를 개발할 계획이다.

Rice (Oryza sativa) is an excellent model monocot with a known genome sequence for studying developmental seeds. In the study, the seeds of 10th day after flowering (DAF) were conducted RNA-Seq of the variety Shindongjin and Sugary mutant using RNA-seq technique. Approximately 202 and 214 million high-quality paired-end reads (101-bp in size) were generated in Shindongjin and Sugary mutant, respectively. Comprehensive analysis on the transcript levels of genes which encode starch-synthesis enzymes is fundamental for the assessment of the function of each enzyme and the regulatory mechanism of starch biosynthesis in seeds. Quantitative real-time PCR was also used to validate the expression profiles of 28 rice genes encoding six classes of enzymes, viz., ADPglucose pyrophosphorylase (AGPase), starch synthase, starch branching enzyme, starch debranching enzyme, starch phosphorylase, and disproportionating enzyme at different developmental grain- filling stages (DAF 1-14) between Shindongjin and Sugary mutant. The results showed that the expression of most of starch synthesis genes were up-regulated except the cytosolic AGPase small subunit2b (AGPS2b), which sharply decreased at grain-filling stages in Sugary mutant. These results will expand our understanding of the complex molecular and cellular events in rice grain-filling stages and provide a fundamental understanding of future studies on developmental endosperm in rice and other cereal crops.

Rice hulls remain closed throughout the ripening period to maintain internal humidity of the grains. An Open-hull sterile mutant was induced by N-methyl-N-nitrosourea(MNU) treatment on Sinsunchalbyeo rice, a japonica type. This mutant showed open hulls even in the ripening stages and fully mature grains. In addition, several altered characteristics were observed, including of narrowed palea, decreased grain size, partial pollen sterility and erect panicle. Microscopic analysis showed that the palea was positioned slightly inside the lemma, and the size of palea decreased in the mutant. Genetic analysis of F2 and F3 segregation populations derived from the cross between the Open-hull sterile mutant (Oryza sativa ssp. japonica) and Milyang23 (O. sativa ssp. indica) indicated that the Open-hull trait was controlled by a single recessive allele. The fine-mapping with STS (sequence tagged site) markers revealed that the mutant gene was located on the short arm of chromosome 3. We were able to narrow it down until 30.6Kb where three candidate genes were found.

The utilization of several genetic resources is the most important for developing rice, such as high yield seeds or various stresses. We used an efficient system to create rice mutant by Ac/Ds transposon insertion mutagenesis, such as selected homozygous mutant in dwarf phenotypes. We reported here the identification of function of dwarf OsGASD gene (Oryzasativa Gibberellin Acid Sensitive Dwarf). OsGASD gene encodes a 344 amino acid polypeptide and nohomology proteins in GeneBank. The osgasd mutnat was sensitive to exogenous gibberellic acid (GA) level. We performed experiment to controlled expression the OsGASD gene, its role in plant development, a quantitative analysis of endogenous GA content and sensitivity to GA. The osgasd mutant includes smaller amount of active GAs than wild-type. osgasd mutant plant of GA biosynthesis pathway causes GA deficiency and dwarf plants ,and endogenous GA suppliance can restore the wildtype phenotype in this mutant. The result indicated that OsGASD gene regulated the elongation of shoot, stem and plant height. The increased expression of OsGASD gene dramatically induces expression of the factors associated with GA biosynthesis such as CPS, KO, KAO, GA20ox and GA2ox, whereas osgasd mutant suppression of the factors associated with GA biosynthesis, loading to dwarf phenotypes. That applied GA3 at the plant development stage to survey the response of OsGASD gene to GA3. We suggest that OsGASD gene is related to factors of GA biosynthesis pathway regulating rice internodes development.

Rice is not only a model plant of monocots but also one of the most important crops all over the world. Despite the importance of leaf shape for achieving effective plant architecture for photosynthesis, little is known about the genetic mechanisms that determine leaf morphological characteristics. Explanation of the genetic basis of the control of leaf shape could be of use in the manipulation of crop traits, leading to increased crop production. Many mutants related to leaf morphology have been identified and classified according to their function in determining leaf morphology. search on the genetics of leaf development has used mutagensis to create loss-of-function mutations that change leaf shape. To understand the molecular mechanism of leaf morphogenesis, we identified a rice mutant gene, which was characterized by a phenotype of narrow leaves. While the mutation resulted in reduced leaf width, no significant morphological changes at the cellular level in leaves were observed, except in bulliform cells. The gene locus guess that it encodes a adenosine kinase, which displays sequence homology with ribokinase pfkB like superfamily. To test function of gene, we cloned gene which have 1140 nucleotides and 379 amino acids. This gene was transcribed in various tissues and was mainly expressed in panicles and leaves. NAL7, NAL1 and SLL1 were found to be downregulated, whereas OsAGO7 and NRL1 were upregulated in the mutant. These findings suggested that there might be a functional association between these genes in regulating leaf development.

Understanding salt tolerance mechanisms is important for the increase of crop yields, and so, several screening approaches were developed to identify plant genes which are involved in salt tolerance of plants. Here, we transformed the Arabidopsis cDNA library into a salt-sensitive calcineurin (CaN)-deficient (cnbD) yeast mutant and isolated the colonies which can suppress salt-sensitive phenotype of cnbD mutant. Through this functional complementation screen, a total of 34 colonies functionally suppressed the salt-sensitive phenotype of cnbD yeast cells, and sequencing analysis revealed that these are 9 genes, including CaS, AtSUMO1 and AtHB-12. Among these genes, the ectopic expression of CaS gene increased salt tolerance in yeast, and CaS transcript was up-regulated under high salinity conditions. CaS-antisense transgenic plants showed reduced root elongation under 100 mM NaCl treatment compared to the wild type plant, which survived under 150 mM NaCl treatment, whereas CaS-antisense transgenic plant leaves turned yellow under 150 mM NaCl treatment. These results indicate that the expression of CaS gene is important for stress tolerance in yeast and plants.

The early senescence mutant was isolated from the japonica rice Koshihikari through Ethyl-methane-sulfonate (EMS) mutagenesis. The early senescence phenotype was controlled by a single recessive gene, tentatively symbolized as es-k. Using an F2 population derived from a cross between the mutant and Milyang23 and molecular markers, we mapped the es-k locus to the long arm of chromosome 7 between STS markers 147-1 and 147-2 with a physical distance of 66-kb. The symptom of early senescence appeared even before heading, while appeared during ripening in wild-type. Physiological characteristics of the es-k mutant before initiation of senescence was similar to the wild-type. However, after heading, es-k mutants started to exhibit a significant decrease in chlorophyll and soluble protein content compared to the wild-type. The wild-type leaf color appeared normal irrespective of temperature treatment, while the leaf of es-k mutant appeared pale-green at the low temperature and dark-green at the high temperature. During dark-induced senescence, mutant did not show significant differences compared to normal type. The results show that es-k is sensitive to temperature but not to light.

VitE (tocotrienols and tocopherols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. A new mutant line, T1001-1, was isolated from in vitro mutagenized population by ionizing radiation and shown to have increased VitE contents. The total VitE content was 26% increased in the T1001-1 mutant seeds compare with cv. Dongan (wild-type). In addition, we showed that the mutant confers retarded seedling growth during the early seedling growth stage in rice. To study the molecular mechanism of VitE biosynthesis, we used the rice microarray to identify genes that are upor down-regulated in T1001-1 mutant. In addition, we identified differentially regulated pathway using MapMan analysis, which provides deep insight into changes in transcript and metabolites. Our results enhanced the transcription of genes involved in starch and lipid metabolism in T1001-1 mutant. To identify the molecular mechanisms of the events involving transcription factors in tocopherol accumulation, we compared the expression patterns of transcription factors. The AP2-EREBP, WRKY, C2H2 transcription factor were up-regulated, whereas the MYB family was down-regulated in T1001-1 mutant. Our results demonstrate change of important transcript in high level of VitE accumulating rice mutant.

Brachypodium has been focused as new model plant for grass species. Like small size, small room requirement, and fast growth, Brachypodium shows numerous advantages as a model plant. Brachypodium is a typical grass at the genome level, which also exhibits an overall similarity of gene content and gene families when compared with rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes. Brachypodium is an excellent material for structural and functional genomic studies in grass species. Targeting-Induced Local Lesions IN Genomes (TILLING) is a high-throughput technique and an approach for reverse genetics study. Moreover, it has been wildly utilized to find induced mutation. Bradi3g45515 is orthologue of the cellulose synthase-like HvCslF8 in barley. For TILLING library construction, 384 M2 Brachypodium mutants induced by chronic-gamma irradiation were used. Single nucleotide polymorphysm (SNP) and small deletion in Bradi3g45515 were searched through TILLING analysis. Template DNA for PCR reaction were prepared according to two dimensional pooling (eightfold) strategy. Heteroduplex DNA was digested by SURVEYOR nuclease (TRANSGENOMIC) and the DNA fragment was detected using polyacrylamide gel electrophoresis. Positive signal appeared at polyacrylamide gel from more than 4 lines and their Bradi3g45515 region were sequenced. SNP(s) were identified in 509-2 and 677-3 mutant line. Cellulose content and/or cell wall materials content will be measured using these mutants.

찰옥수수, 단옥수수, 초당옥수수 등은 종실에 전분이 저장되는 과정에서 돌연변이가 발생하여 전분의 구성에 차이를 일으키며 이들의 유전자들 상호간에는 상위성이 존재한다. 배유의 저장전분에 관여하는 유전자 중에서 brittle1(bt1), brittle2(bt2), shrunken1(sh1), shrunken2(sh2), sugary1(su1), sugary2(su2), sugary enhancer(se)들은 전분 합성을 억제하고 amylose extender(ae), dull(du), floury(fl), opaque2(o2), waxy(wx)들은 배유 내에 전분의 구성 및 구조 등을 변경한다. 전분합성을 억제하는 유전자들은 전분의 구성을 변경하는 유전자에 상위성을 가지며 그들의 작용을 피복한다. 이들의 사실을 기초로 본 시험은 단옥수수(se) 유전자와 찰옥수수(wx) 유전자를 함께 가지는 자식계통을 만들어 육종 소재로 활용하고자 수행하였다. 2011년에 se 유전자를 가진 자식계통과 wx 유전자를 가진 자식계통을 상호 교배하여 F1 종자를 만들고 2012년 이들을 자식(selfing)시켰다. 멘델에 유전법칙에 따라 유전자형은 Se_Wx_:Sesewxwx:seseWx_:sesewxwx가 9:3:3:1로 분리되고 표현형 분리비는 이들 유전자 간의 상위성에 따라 일반옥수수, 단옥수수, 찰옥수수가 9:4:3으로 표현된다. 이들 분리1세대(S1) 종자를 White light transilluminator (LCF-900-470V, Ultra Violet Products)를 이용하여 찰옥수수의 표현형을 나타내는 종자를 골라 2013년 4월 중순에 파종하였다. 앞으로 이들을 자식(selfing)시켜 분리2세대 종자를 수확하고 단옥수수의 형질을 나타내는 것을 분리하면 이들은 찰옥수수(wx)와 단옥수수(se)의 유전자를 동시에 가지는 double mutant일 것으로 사료되며 분자마커 및 검정교배를 통해 유전자들을 확인할 수 있을 것으로 생각된다.

To determine the expression levels of genes related to the salt stress response in rice, gene expression profiles were investigated through microarray analysis using the rice mutant line Till-II-877. There were no significant changes in physiological response under salt stress of the mutant increased less than that in the WT. The intensity of gene expression was analyzed and compared between the wild type and mutant lines using a microarray. Among the most significantly affected pathways, α-linolenic acid metabolism and linoleic acid metabolism (in lipid metabolism), fructose and mannose metabolism and glycolysis-gluconeogenesis (in carbohydrate metabolism), cysteine and methionine metabolism (in amino acid metabolism), and carbon fixation (in the energy metabolism of photosynthetic organisms) showed changes in gene expression levels under salt stress. These results further our understanding of the effects of salt stress in rice and may aid in the development of salt-tolerant rice cultivars.

Soluble sugar content in soybean seed is an important quality attribute for soyfood and feed. Usually, soluble sugars comprise 6 to 17% of total dry wt. in mature soybean seeds. In this study, 414 soybean mutant lines induced by gamma-ray were screened by colormetric assay, FACE (Fluorophore-assisted carbohydrate electrophoresis), and GC-MS to identify the change of soluble sugar contents. Among 414 soybean mutant lines, 12 mutant lines derived from three different soybean cultivars (Hwanggum, Paldal, and Bangsa) showed higher level of soluble sugar content compared to their original cultivars. However, 5 mutant lines derived from soybean landrace KAS 636-15 showed lower level in the colormetric assay. In FACE, 17 soybean mutant lines selected by colormetric assay also showed different band intensity compared with their original cultivars. However, there were no different soluble sugar patterns between soybean original cultivars and mutant lines. Finally, the variations of soluble sugar content in 17 soybean mutant lines were confirmed by using GC-MS. These mutant lines will be used for genetic study to find mutations of genes related soluble sugar biosynthesis.

Mutant lines induced by ethyl methane sulfonate (EMS) have been used for crop improvement and functional genomics. Since pepper is very recalcitrant to be transformed, EMS mutagenesis could be an alternative method to generate useful mutant lines and to characterize the function of genes. We have developed mutant lines consisting of about 3,938 M2 mutant lines using Korea local landrace, C. annuum ‘Yuwolcho’. Yuwolcho has suitable traits for mutagenesis such as early flowering and maturation, large number of seeds per fruit, and susceptibility to various diseases. Up to now, 917 M2 mutant lines were evaluated to confirm the effect of EMS. M2 mutant lines have shown variations in plant stature (small size, dwarfism, and early death), leaf development (light color, variegation and morphological change) and flower (inflorescence, morphological change) and fruit (size and color). We observed the largest morphological variation in leaf development. Most of these mutant phenotypes were inherited recessively. In addition, we are applying cel1-based TILLING to identify useful mutant lines. We will apply cel1-based TILLING to identify useful mutant lines. We are expecting that these mutant lines will be very useful to study the function of genes in C. annuum.

Grain sorghum (Sorghum bicolor) is a major staple for a large portion of the world. The crop ranks fifth among the cereals world-wide with respect to its importance for food and feed applications. To this end, the grain harvested from sorghum, and the millets provides an important source for dietary calories and protein for approximately one billion people in the semi-arid regions of the world. However, grain sorghum products are known to have relatively poor digestibility, only approximately 50%–70%, in comparison with other grains, such as wheat and maize, which tend to have digestibility percentages over 80% and 70%, respectively. Protein with high digestibility is by definition nutritionally superior owing to the increased availability of amino acids. Digestibility can be impacted by both protein–protein and⁄or protein–nonprotein interactions. However, with respect to grain sorghum, it is thought that the major factor influencing digestibility is the former because of high protein cross-linking around the protein body. To understand the mechanism of seed storage proteins in the sorghum, the proteomic analysis was carried out between the wild(BTX623) and mutant(M271207) genotypes of sorghum. Proteins were separated from the mature seed using IEF in the first-dimension and SDS-PAGE in the second dimension along with hybrid LTQ-FTICR mass spectrometry. After image analysis using Progenesis SameSpot software, we identified the 62 differential expressed protein spots out of 293 protein spots. Out of total differential expressed spots, 35 differential expressed protein spots (more than2-fold) were analyzed by mass spectrometry. Out of 35 protein spots, we were identified 20 protein spots as up-regulated and 15 protein spots as downregulated, significantly. In our proteomic investigation, the candidate proteins may provide novel clues for better understanding the characteristics of seed proteins in Sorghum.

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.

Plant height is an important agronomic trait that affects grain yield. Previously, we reported a novel semi-dominant dwarf mutant, D-h, derived from chemical mutagenesis using N-methyl-N-nitrosourea(MNU) on a japonica rice cultivar, Hwacheongbyeo. In this study, we cloned the gene responsible for the dwarf mutant using the map-based approach. Fine mapping revealed that the mutant gene was located on the short arm of chromosome 1 in a 48 kb region. Sequencing of the candidate genes and rapid amplification of cDNA ends-polymerase chain reaction(RACE-PCR) analyses identified the gene, d-h, which encodes a protein of unknown function, but whose sequence is conserved in other cereal crops. Real-time (RT)-PCR analysis and promoter activity assay showed that the d-h gene was primarily expressed in the nodes and the panicle. In the D-h mutant plant, the gene was found to carry a 63-bp deletion in the ORF region, which was confirmed to be directly responsible for the mutant’s gain of a functional phenotype by subsequent transgenic experiments. Since the mutant plants exhibit a defect in the GA response, but not in the GA synthetic pathway, it appears that the d-h gene may be involved in a GA signaling pathway.

The architecture of rice panicle is primarily determined by the arrangement of branches and spikelets, and it directly affects grain yield. We identified a mutant for panicle apical abortion from a japonica cultivar Hwacheongbyeo treated with N-methyl-N-nitrosourea. Under normal growth conditions, the mutant had multiple abnormal phenotypes, such as a slight reduction in plant height, narrow and dark green leaf blades, and small erect panicles with clear panicle apical abortion compared to the wild-type plants. Genetic analysis revealed that the panicle apical abortion was controlled by a single recessive gene, which is tentatively designated as paa. The paa gene was fine mapped at an interval of 71 kb flanked by STS markers aptn3 and S6685-1 at the long arm of chromosome 4. Sequence analysis of the candidate genes within the delimited region showed a single base-pair change corresponding to an amino acid substitution from glycine to glutamic acid. We expect that the paa gene will be a clue to uncover the molecular mechanism of panicle apical abortion and to maintain the panicle identity for grain yield in rice breeding programs.

The outer dense fiber 2 (ODF2) protein is an important component of sperm tail outer dense fiber and localizes at the centrosome. It has been reported that the RO072 ES cell derived homozygote knock out of ODF2 results in an embryonic lethal phenotype, and XL169 ES cell derived heterozygote knock out causes severe defects in sperm tail development. The ODF2s splicing variant, Cenexin1, possesses a C-terminal extension, and the phosphorylation of serine 796 residue in an extended C-terminal is responsible for Plk1 binding. Cenexin1 assembles ninein and causes ciliogenesis in early stages of the cell cycle in a Plk1-independent manner. Alternatively, in the late stages of the cell cycle, G2/M phase, Cenexin1 binds to Plk1 and results in proper mitotic progression. In this study, to identify the in vivo function of Plk1 binding to phosphorylated Cenexin1 S796 residue, and to understand the in vivo functional differences between ODF2 and Cenexin1, we generated ODF2/Cenexin1 S796A/Cenexin1 WT expressing transgenic mice in a RO072 ES cell derived knock out background. We observed a severe defect of sperm tail development by ectopic expression of Cenexin1 S796A mutant and no phenotypic differences between the ectopic expression of ODF2/Cenexin1 WT in background and in normal wild type mice.

In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. It was recently reported that ZEBRA2 encodes carotenoid isomerase (CRTISO) and that low levels of lutein, an essential carotenoid for non-photochemical quenching, cause leaf variegation in z2 mutants. However, we found that the z2 mutant phenotype was completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants under CL. In addition, three types of reactive oxygen species (ROS; superoxide [O2-], hydrogen peroxide [H2O2], and singlet oxygen [1O2]) accumulated to high levels in z2 mutants grown under short-day conditions (SD; alternate 10-h light/14-h dark; restrictive), but do not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than normal in both permissive CL and restrictive SD growth conditions, indicating that deficiency of these two carotenoids is not responsible for the leaf variegation phenotype. We found that the CRTISO substrate tetra-cis-lycopene accumulated during the dark periods under SD, but not under CL conditions. Its accumulation was also positively correlated with 1O2 levels generated during the light period, which consequently altered the expression of 1O2-responsive and cell death-related genes in the variegated z2 leaves. Taking these results together, we propose that the z2 leaf variegation can be largely attributed to photoperiodic accumulation of tetra-cis-lycopene and generation of excessive 1O2 under natural day-night conditions.

Narrow genetic diversity of Korean commercial rice lines have been a major limit factor in breeding new breeding lines having resistance and tolerance against biotic and abiotic stresses. Introducing novel favorable allele types could be possible through crossing with wild relatives, it demands additional tedious efforts to restore the unique genetic background of the recurrent parents, which determine commercial value in the market. Our study is preliminary based on the mutation breeding, by which agronomic traits could be acquired with the least impact on the unique haplotype of the wild type. Through screening and evaluating more than 7,000 mutant lines of Namil, a high yielding Korean japonica cultivar, several dozens of mutant lines expressing improved performances in terms of resistance or tolerance against biotic or abiotic stresses. One Sodium azide treated mutant line, Namil(SA)M2-1063-11-1-1-1-1-1-1, designated as ‘Namil(SA)-bl5’ performed high level of resistance against rice blast as well as reduced culm length. Two mapping populations, to dissect genetic basis of the blast resistance and short culm length, were constructed by using F2 progenies derives crosses between Namil(SA)-bl5 and Milyang23 and Namil(wild type) and Milyang23. Each progenies were evaluated in terms of DNA marker genotype as well as basal agronomic traits including blast resistance by using F2:3 seeds. Association analysis between marker genotype and evaluated phenotype of progeny lines were adopted to localize the putative chromosomal locations involved to culm length and blast resistance. The putative locations unique to Namil(SA)-bl5 were then elucidated through the comparisons with those of Namil x Milyang23 reference population. Tentatively, the genetic factors for reduced culm length and blast resistance were identified on chromosome 7 and chromosome 12, respectively.