초록 ▼

Ⅳ. 연구개발 결과
제1세부연구과제 : 벼줄무늬잎마름병 저항성 유전자 다양화를 위한 유전자 탐색
국내 통일형 품종 및 국외 도입 유전자원을 중심으로 총 155 품종에 대한 저항성 생물검정과 기존에 개발된 Stv-bi 유전자 영역을 분자마커를 이용한 유전자형 검정을 통해 신규 유전자를 포함하는 저항성 자원을 선발함을 목적으로 수행한 결과 Daw dam 등 6품종은 Stv-bi 유전자가 아닌 다른 유전자에 의해 저항성을 나타낼 가능성이 높은 것으로 추정된다. 벼줄무늬 잎마름병 저항성 후보 유전자 탐색을 위해 각 유전자에 대한

Ⅳ. 연구개발 결과
제1세부연구과제 : 벼줄무늬잎마름병 저항성 유전자 다양화를 위한 유전자 탐색
국내 통일형 품종 및 국외 도입 유전자원을 중심으로 총 155 품종에 대한 저항성 생물검정과 기존에 개발된 Stv-bi 유전자 영역을 분자마커를 이용한 유전자형 검정을 통해 신규 유전자를 포함하는 저항성 자원을 선발함을 목적으로 수행한 결과 Daw dam 등 6품종은 Stv-bi 유전자가 아닌 다른 유전자에 의해 저항성을 나타낼 가능성이 높은 것으로 추정된다. 벼줄무늬 잎마름병 저항성 후보 유전자 탐색을 위해 각 유전자에 대한 발현분석 결과 Os11g31430 등 3종의 유전자가 저항성 모본인 신광벼에서 그 발현양이 현저히 감소하는 것을 발견하고 이들 유전자에 대한 형질전환체를 제작하였다. 제작된 형질전환체 중 확보된 과별현 순계 계통을 중심으로 유전자별 기능확인에 이용할 계획이다.

제2세부연구과제 : 끝동매미충 저항성 유전자 탐색 및 기능분석
끝동매미충 저항성 증진을 위한 유전자 탐색에서는 일품/신광 NIL 유래의 끝동매미충 (Grh) 저항성 관련 유전자를 탐색하고 저항성 분자마카를 개발하고자 하였다. 끝동매미충 저항성 유전자의 영역이 축소된 유전자 지도작성을 위해 염색체 내 위치분석을 일품/YR24353-60-1-1 후대인 F₃ 및 F₄ 계통을 이용하였다. 저항성 구분마커인 RM516, RM18161, RM18166, RM18171 및 Indel15040, Indel15510 등을 개발하여 저항성 영역을 축소한 결과 저항성 영역은 염색체 5번의 8.10Mbp-8.30Mbp(0.20Mbp)사이였고 저항성 영역 내에 존재하는 주요 저항성 후보 유전자는 10종 이었다. 저항성 후보 유전자 10종에 대한 발현분석 결과 8종은 발현을 확인하였고 2종(LOC_Os05g14470.1, LOC_Os05g14570.1)은 발현되지 않았다. 8종의 유전자 중 3종(LOC_Os05g14540, LOC_Os05g14480, LOC_Os05g14550)이 끝동매미충 저항성에 관련된다고 판단하기에 유의한 발현차이를 보였다. 저항성 후보 유전자 3종에 대한 기능구명을 위해 과발현 형질전환 벡터를 제작하였다. 끝동매미충 저항성 유전자 Grh1을 보유하는 11종의 우량계통 선발하여 차년도에 생산력검정을 시행 할 예정이다.

제3세부연구과제 : 영양성분 흡수 증진 기능성 형질 전환 벼 품종화
OsNAS2-D1, OsNAS3-D1 activation tagging line의 농업특성을 검정한 결과 두 계통 모두 화영벼에 비해 등숙율이 낮았으며 수량또한 적은 경향이였다. 특히 OsNAS3의 경우 화영벼에 비해 수량이 약 200 kg 낮아 농업특성 개량이 필요한 것으로 사료된다. 마커프리 형질전환벼 개발을 위해 형질전환을 실시하여 재분화식물체를 확보하였고 이를 이용해 PCR 검정을 통해 OsNASs 마커프리 벡터가 삽입된 개체를 선발하였다. 이들 형질전환벼의 현미 무기성분함량을 분석하여 철분 및 아연 함량이 동진벼에 비해 증가함을 확인하였고 세대진전 후 하이그로마이신 저항성 유전자가 제거되고 마커프리 형질전환용 벡터만 삽입된 개체를 선발하여 식물체 특허를 출원하였다.

제4세부연구과제 : 환경스트레스 저항성 형질 도입 벼 품종화
가뭄 처리에 의한 품종별 쌀수량 감소율을 분석하여 새일미, 일미, 일품, 신동진 품종이 무관수 처리에 의한 쌀수량 감소율이 약 12%로 다른 품종보다 재배 안정성이 높음을 확인하고 영농활용 기관제출을 하였다. 내건성 대량검정을 위한 벼 잎을 이용한 간이 검정법을 개발하고 산업재산권을 출원하였다. 이를 이용하여 유전자원 815 종에 대한 내건성 검정 수행하여 삼강, Apo, Gumei 4 등 내건성 품종을 선발하였다. 또한 삼강/낙동 유전분석용 집단을 이용하여 삼강 유래 내건성 QTL을 탐색하고 저항성 유전자가 11 염색체의 RM26755-RM287에 위치함을 발견하였다. 2단계 연구를 위하여 저항성 정밀지도 작성용 RM16990 등의 신규 분자마커를 10종 탐색하고 유전분석용 후대집단을 새로이 양성하였다.

제5세부연구과제 : 기후변화 대응 기능성 특수미 한발 저항성 증진을 위한 유전자좌 탐색
신규 유용 국외 유전자원의 표현형 분석을 통해 Handao10, Ningjing39 등 신규 내건성 유전자원을 15종, Xiangzaoxian7, Jiayu948 등 신규 고온 저항성 유전자원을 4종 선별하였다. 고온 저항성 형질 도입을 위하여 인공교배를 통해 후대집단을 양성 중에 있다. 또한 내건성 QTL 분석을 위한 표현형 분석과 잎 rolling, 엽록소 함량을 분석하여 집단의 분포도를 작성하였다. 2단계 연구로 유전형 분석을 통해 QTL 위치를 탐색할 계획이다.

Abstract ▼

Rice stripe disease, caused by rice stripe virus (RSV) is a serious constraint to rice production in subtropical regions of East Asia. We performed fine mapping of a RSV resistance QTL on chromosome 11, qSTV11SG, using nearisogenic lines (NILs, BC6F4) derived from a cross between the highly resistan

Rice stripe disease, caused by rice stripe virus (RSV) is a serious constraint to rice production in subtropical regions of East Asia. We performed fine mapping of a RSV resistance QTL on chromosome 11, qSTV11SG, using nearisogenic lines (NILs, BC6F4) derived from a cross between the highly resistant variety, Shingwang, and the highly susceptible variety, Ilpum, using 11 insertion and deletion (InDel) markers. qSTV11SG was localized to a 150-kb region between InDel 11 (17.86 Mbp) and InDel 5 (18.01 Mbp). Among the twomarkers in this region, InDel 7 is diagnostic of RSV resistance in 55 Korean japonica and indica rice varieties. InDel 7 could also distinguish the allele type of Nagdong, Shingwang, Mudgo, and Pe-bi-hun from Zenith harboring the Stv-bi allele. As a result, qSTV11SG is likely to be the Stv-bi allele. There were 21 genes in the 150-kb region harboring the qSTV11SG locus. Three of these genes, LOC_Os11g31430, LOC_Os11g31450, and LOC_Os11g31470, were exclusively expressed in the susceptible variety. These expression profiles were consistent with the quantitative nature along with incomplete dominance of RSV resistance. Sequencing of these genes showed that there were several amino acid substitutions between susceptible and resistant varieties. Putative functions of these candidate genes for qSTV11SG are discussed. Genetic diversity of 155 rice cultivars was evaluated using 9 co-dominant InDel markers and 1 STS marker, ST10, near Stv-bi allele. These cultivars were classified into two groups by cluster analysis based on Nei’s genetic distances. In comparison of bioassay for RSV resistance and genotyping using SSR markers showed that recombination value of RSV resistance and InDel7 marker type was 3.8%. Some RSV resistant varieties including umarat, Pervomajszkij, and Tung Ting Wan Hien 1 showed susceptable allele type in InDel7 and ST-10 marker. These results indicate those cultivars can be able to get the different gene resistance with Stv-bi gene. Further genetic studies on selected varieties in this study will be use for improving RSV resistance in japonica rice.
Green rice leafhopper (GRH), Nephotettix cinctieps Uhler, is a major insect pest of rice, and is found mostly in temperature regions of East Asia (Fujita et al, 2013). Populations of this insect, which multiply rapidly around the rice heading stage, sucks sap from xylem and phloem of susceptible rice varieties. and GRH also injure rice indirectly, by transmitting serious viruses such as rice tungro virus and also causes damage to rice indirectly by transmitting viral and phytoplasmal diseases such as rice dwarf disease, waika disease, and yellow dwarf disease (Fukushi, 1934; Shinkai, 1962; Hirao et al., 1974). The mortality of GRH nymphs is high in rice cultivar Shingwang, indicating that Shingwang is resistant to GRH. In this study, we mapped Grh1 locus in rice using F₃ and F₄ individual lines derived from a cross between the parents Ilpum(susceptible) and Shingwang(resistant). After antibiosis test for 5 days, biological assay was compared with genotyping result with SSR markers. Resistance to GRH in Shingwang was found to be controlled by a single dominant gene (Grh1) mapped within an approximately 200-kb region between 8.10 and 8.30 Mb on the short arm of chromosome 5. Genotypes with three simple sequence repeat markers (RM18166, RM516, and RM18171) and one indel marker (Indel 15040) co-segregated with GRH resistance controlled by the Grh1 locus. The 200-kb region of Grh1 contains at least 10 gene models, excluding transposon and retrotransposon genes (www.gramene.org/). Three candidate genes (LOC_Os05g14540, LOC_Os05g14480, LOC_Os05g14550) based on their expression patterns were selected. complementation tests for selected candidate genes would enable us to identify the gene conferring GRH resistance in Shingwang.
Even rice is one of the most important food crops in the world, its micronutrient contents including iron is not enough to solve mineral malnutrition which is a significant public health issue in most developing countries (Fitzgerald et al., 2009; Zhu et al., 2007). OsNASs activation tagging lines was developed to increased Zn content in rice (Sichul et al., 2009). Even though those OsNASs activation tagging lines have higher Zn and Fe content than wild-type rice, they contained antibiotic resistant gene as selective marker, here we tried to investgate the agronomic character of OsNASs activation tagging lines then develop selective marker free transgenic rice with over-expression of OsNASs. OsNAS1, OsNAS2, OsNAS3. The heading date of OsNAS2 activation tagging lines was five days delayed than hwayeong. The culm and panicle length of OsNAS2 was similar with hwayeong, however those of OsNAS3 were shorter than hwayeong. Yield of milled rice of OsNASs activation tagging lines were seriously reduced than hwayeong. After generation of transgenic rice by co-infection with OsNASs inserted selective marker free transformation vector and HPT inserted vector, 22, 24 and 20 transgenic rice were selected in OsNAS1, OsNAS2 and OsNAS3, respectively. After confirmed the over-expression of target gene in each transgenic rice, inorganic nutrient content in each transgenic brown rice were analyzed. In each transgenic rice, inorganic content such as Fe, Zn and K were increased. Selective marker free transgenic rice in OsNAS3 were selected after several generation progress and the increasement of Fe and Zn was confirmed. To develop efficient screening method of high Fe contained brown rice, leaf color and Fe content of brown rice were investigated. Leaf color cultivated on MS medium which contained various Fe content was significantly reduced according to decrement of Fe content in MS medium. In MS medium which contained 5% Fe content compare to control condition, the correlation of leaf chlorophyll content and Fe content in brown rice was the highest among several MS medium. Anthocyanin content, the main component of black rice, was easily changed according cultivation environment. Here we tried to investigate the change of leaf anthocyanin content in black rice according to different abiotic stress to easily anticipate the change of anthocyanin content in brown rice. By treating ABA, cold, drougth and NaCl, leaf anthocyanin content was significantly increased in black rice whose anthocyanin content in brown rice changed in different cultivation area. However heugjinju, heugnam whose anthocyanin content in brown rice did not changed according to different cultivation area did not show signficant different in leaf anthocyanin content in abiotic stress treatment.
Little progress has been made in the genetic analysis for drought tolerance, because it is a complex trait controlled by a number of genes and affected by various environmental factors. In here, we selected 11 rice varieties which are widely cultivated in Korea and we have compared rice yield between rain-fed and irrigated conditions on the field. Rice yield of these cultivar was reduced at about 10% to 30% because the panicle number was decreased. This result indicates that drought is also important environmental conditions for rice cultivation in Korea. Therefore, we have optimized leaf water loss rate assay with plastic ware for large screening and we examined leaf water loss rate of 815 rice varieties. The range of leaf water loss rate was between about 15% to 35%. With this leaf wate loss rate, we have also screened the visual drought phenotype on the bed in the greenhouse conditions. Among 400 varieties, 39 varieties showed drought tolerant phenotype, whereas most of the varieties were sensitive to drought stress in our experimental conditions. Especially, Samgang showed strong drought tolerant phenotype both experimental conditions. Furthermore, To identify drought QTL, we generated doubled-haploid population consist of 101 lines derived from a cross the drought tolerant cultivar ‘Samgang’ and the drought sensitive cultivar ‘Nagdong’ for QTL analysis. To score the drought phenotype degrees of this population, we withheld water for 6 weeks and treated the watering for 7 days. After watering visual phenotype was observed according to the standard evaluation system from IRRI. Main QTL was detected on chromosome 11 with flanking markers RM26755-RM287 and accounted for 24.4% phenotype variation with a LOD score of 4.9.
Rice germplasms are very important for developing new variety and QTL analysis. In here, to use rice germplasms which develpmed in China for developing new variety and QTL analysis, we have made collaboration with China Agricultural and Academy Science. We examined the phenotype of Samgang/Nagdong DH population as drought tolerant and sensitive in the rice field in China. With this visual drought phenotype, we also analyzed the leaf rolling index and chlorophyll content of Samgang/Nagdong DH population. And to develop elite line for drought, we examined agricultural traits such as panicle number. Among these traits, grain filling rate is most closed with visual phenotype and drought tolerant DH lines showed about 60% grain filling rate under drought stress treatment conditions, whereas drought sensitive DH lines with Nagdong showed about 10-30 of grain filling rate. We will develop intermediate drought tolerant elite line with these drought tolerant DH lines after backcrossing with Nagdong variety. To find new drought QTL, we screened 15 drought germplasms such as Ningjing39. We examined the leaf rolling index and chlorophyll content for two IAPAR-9 driven RIL populations. To dermine the location of drought tolerant QTLs, We will examine the genotype with SSR molecular marker. With drought tolerant QTL study, we also tried to develop heat tolerant variety with known China heat tolerant varieties. For this purpose, we crossed between Junam and heat tolerant varieties. To new heat tolerant varieties, we screened germplasms and found 4 new varieties such as Xiangzaoxian7. We have plan to develop intermediate elite line with these heat tolerant varieties.