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Genetic Analysis on the Rice Blast and Brown planthopper Resistance of Namil(EMS)-bl10,bph1, a Japonica Rice Mutant Line

Seong-Woo Cho, Ji-Ung Jeung*, Young-Seop Shin, Kyung-Ho Kang, Sang-Bok Lee, Bo-Kyeong Kim

Published online: August 31, 2014

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*Corresponding Author (E-mail: jrnj@korea.kr Tel: +82-31-290-6728, Fax: +82-31-290-6730)
• Received: July 29, 2014   • Revised: September 18, 2014   • Accepted: September 18, 2014

© 2014 by the Korean Society of Breeding Science

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Rice is a source of food supply as the staple food. The narrow genetic diversity of Korean Japonica cultivars is prone to be susceptible to abiotic and biotic stresses such as rice blast, bacterial blight, and brown planthopper (BPH). The best approach for prevention of the breeding and extermination of disease and insect pest would be the development of rice cultivars harboring multiple disease and insect pest resistance genes. National Institute of Crop Science (NICS) in Korea developed a mutant line, Namil(EMS)-bl10,bph1, by using ethyl-methane-sulfonate (EMS) as the chemical mutagen on the wild type, Namil. The mutant line exhibits strong resistance levels against rice blast and brown planthopper. To localize favorable mutated allele types, two mapping populations were constructed by using F2 progenies derives crosses between Namil(EMS)-bl10,bph1 and Milyang23 and Namil (wild type) and Milyang23. Each F2 progenies were evaluated in terms of DNA marker genotype as well as agronomic traits measured, and for rice blast and BPH resistance, virulent blast isolate 94-254 and Korean biotype of BPH were used to screen F2:3 seedlings to conduct association analysis between marker genotype and evaluated phenotype of progeny lines. Through comparisons on major loci between two mapping populations, the putative major loci for rice blast resistance (RM1337, R2=54.4%) and BPH (RM0277, R2=55.1%) were primarily localized on chromosome 12.
  • Keywords: Rice; Mutant line; Rice blast; Brown planthopper; Ethyl-methane-sulfonate; Association analysis
쌀은 우리나라의 주곡작물로서 중요한 식량공급원이나 (Kim, 2011), 고품질 벼 위주의 육종사업의 결과로 품종들간 의 유전적 다양성이 협소한 것으로 보고되어 왔다(Jeung et al. 2005, Kang et al. 2007). 이는 특정 병충해의 대발생과 불량환경 등에 효과적으로 대응할 수 있는 유용한 대립인자 (allele)를 확보하는데 큰 제한요인으로 작용하여 벼의 안정적 생산을 위협할 수 있다(Jena et al. 2006, Jeung et al. 2006). 우리나라의 대표적인 벼 병해충으로 도열병과 벼멸구를 들 수 있다. 벼 도열병은 벼의 육묘단계부터 최고분얼기까지 잎 을 침해하고 심지어 출수기 이후에도 이삭목, 이삭가지 뿐만 아니라 벼 알까지도 침해하여 벼의 수량과 품질을 저해한다 (shim et al. 2005, Jeung et al. 2014). 벼멸구는 해외에서 우 리나라로 건너오는 비래해충으로(Uhm et al. 1988) 쌀의 안 정적 생산을 크게 위협할 수 있다(Yeo & Sohn, 2001).
병해충 방제에 있어서 가장 경제적이며 친환경적인 방법은 복합내병충성 벼 품종을 육성하는 것이다(Wang et al. 1994, Kim et al. 2011, Shin et al. 2011). 국내외에서는 재배벼의 내병충성을 강화하기 위하여 야생벼나 원연종의 유용한 유전 인자들을 활용하려는 연구들이 지속되어왔다(Brar & Khush 1997, Jena et al. 2006, Jeung et al. 2007, Kang et al. 2007, Rahman et al. 2007, Kim et al. 2011, Jeung et al. 2014). 재배벼의 유전적 다양성을 확충하기 위한 또 다른 수단으로 돌연변이 육종을 들 수 있다(Shin et al. 2009). 국내에서는 돌연변이 육종이 주로 벼의 배유전분 특성을 다양화 하기 위 해 활용되어 왔으나(Mo et al. 2014), 근래에는 돌연변이 후 대계통들로부터 새로운 병해충저항성 유전인자를 확보하기 위한 노력이 진행 중이다.
돌연변이 계통의 유용형질에 대한 유전좌위를 파악하고 이 를 분자마커로 표지하는 작업은 원품종에 존재하는 기존의 유전자들과 돌연변이 계통에서 유래한 유전자들간의 상동성 을 확인하거나, 목표형질이 발현되는지를 확인하는데 있어서 매우 중요한 요소이다(Jeung et al. 2011). 벼 유전체학의 발 전은 저항성유전자들의 염색체상 위치와 유전기작을 파악하 는데 분자마커의 활용성을 크게 제고시켰다(Huang et al. 2008, Jeung et al. 2007, Kim et al. 2011, Rahman et al. 2007, Sallaud et al. 2003, Yang et al. 2009).
본 연구는 국내육성 벼 품종 남일에 화학적 돌연변이제인 에틸메탄설포네이트(ethyl-metane-sulfonate ;EMS)를 처리하 여 육성된 후대계통들 중, 도열병과 벼멸구에 복합저항성을 발현하는 것으로 판명된 Namil(EMS)-bl10,bph1 의 유전양 상을 밝히고 저항성에 관여하는 유전좌위에 대한 분자마커를 확보함으로써 우리나라 벼 품종의 병해충저항성 증진을 위한 육종모재로 활용코자 수행되었다.
돌연변이 집단육성 및 저항성 계통 Namil(EMS)-bl10, bph1의 선발
자포니카(Oryza sativa L. ssp. Japonica) 벼로서 숙기가 빠르고(8월4일 출수: 수원기준) 다수성(662kg/10a: 백미기준) 인 남일벼(Kim et al., 2003)에 대해 Choi (2006)의 방법에 의거하여 EMS가 처리된 돌연변이 후대계통들이 확보되었다. 간략하게는, M1 식물체들로부터 1개 이삭씩을 수확하였으며 (M2 종자), 이후부터는 M7세대까지 계통육종법에 의거하여 세대를 진전시키면서 고정계통을 확립하였다. 매 세대마다 각 계통 당 임성이 높은 1개 식물체를 무작위 선발하고 1수-1열 법에 의거하여 후대계통을 전개하였다. M7 식물체 세대에서 각 계통내의 개체들이 균일한 표현형을 보여 유전적으로 고 정되었다고 판단하고 총 2,830여개의 계통을 확정하였다. 원 품종(남일)에 비해 병해충 저항성이 향상된 돌연변이 계통을 확보하기 위한 생물검정을 통해 도열병과 벼멸구에 저항성을 발현하는 Namil(EMS)-bl10,bph1을 확보하였는데(Fig. 1 참 조), 계보는 Namil(EMS)M2-1463-1-1-1-1에 해당한다.
Fig. 1.
Agronomic performances of the wild type, Namil (left) and the mutant line of Namil, Namil(EMS)-bl10,bph1 (right) (A), reaction patterns against brown planthopper, Korean Biotype (B) of Namil (left) and Namil(EMS)-bl10,bph1 (right), and virulent rice blast isolate, 94-254 of Namil, Milyang23, and Namil(EMS)-bl10,bph1, sequentially from left to right (C).
KJBS-46-226_F1.gif
식물재료 및 DNA 추출
돌연변이 계통 Namil(EMS)-bl10,bph1에 대한 국내수집 도열병 균주들의 친화성 검정을 위해서 주남, 온누리, 칠보 등 이, 벼멸구 검정은 태백, 화삼, 삼강 등이 대비품종으로 공시 되었다. Namil(EMS)-bl10,bph1의 도열병 및 벼멸구 저항성 관련 유전좌위에 대한 유전분석을 위해 통일계 품종인 밀양 23호와의 교배를 통해 육성된 F2 97개체를 사용하였으며, 돌 연변이 계통의 유전좌위 추정의 신뢰성을 높이기 위하여 남 일과 밀양23호간의 F2 103개체도 함께 사용하였다.
유전분석을 위해 육성된 각 F2 집단의 식물체들과 각각의 모본, 부본으로부터 변형된 CTAB 방법(Murray & Thompson 1980)으로 DNA를 추출하였다. 추출된 DNA는 0.8% agarose gel에 전기영동하여 확인하였으며, NanoDrop spectrophotometer (Thermo Fisher Scientific, USA)를 이용하여 정량 후 5 ng/ul 로 희석하여 polymerase chain reaction (PCR)에 이용하였다.
작물학적 특성 평가
도열병과 벼멸구에 대한 저항성 돌연변이 계통 Namil(EMS)-bl10,bph1과 야생종인 남일을 2011년 국립식량과학원 답작 과(수원 소재)포장에 공시하여 작물학적 특성을 관찰하였다. 조기재배에 준하여 파종은 4월 7일 실시하여 약 30일정도 육 묘한 후 5월 12일에 이앙하였다. 연관지도 작성 및 목표유전 자위 표지를 위해 공시된 두 개의 F2 집단들에 대해서는 포장 에 공시된 각 개체들을 표지하고 이들로부터 DNA를 추출하 기 위한 시료를 취하였으며, 저항성 평가를 위한 F2:3 종자를 수확하였다.
도열병 검정 및 균주 선정
국내에서 수집된 도열병 균주 중 원품종인 남일과 친화성 을 보이는 균주를 선정하여, 공시재료들의 도열병 저항성 검 정을 실시하였다(Ryu et al. 1987, Han et al. 1994, Han et al. 1995, Shim et al. 2012). 도열병 균주의 접종 및 발병유 도는 Kim et al. (2004)의 방법에 준하여 수행하였다. 공시재 료를 파종 후 3~4엽기까지 온실에서 육묘한 후, 포자농도가 1.5 × 105 spores/ml로 조정된 각각의 도열병 균주를 분무접 종하고, 26°C 암조건의 상대습도 90% 이상인 습윤상에서 24 시간 처리한 후 온실로 옮겨 2주간 발병을 유도하였다. 공시 재료들의 도열병 저항성 정도는 발병 정도에 따라 0~5 단계 로 판정하였는데(0: 병반 없음, 1 : 0.5 mm 이하의 갈색반점, 2 : 1 mm 이하의 원형반점, 3 : 1.5 mm 이하의 진갈색 병반, 4 : 2 mm 이하의 병반 중심으로부터 고사, 5 : 2 mm 이상의 병반 및 고사), 0~1은 저항성, 2~3은 중도저항성 및 4~5 는 감수성 등으로 접종된 도열병 균주에 대한 저항성과 감수 성 정도를 구분하였다(Kim et al. 2011). 도열병 저항성에 대 한 유전분석을 위하여 남일과 밀양23호에 친화성이며, Namil (EMS)-bl10,bph1에는 비친화성인 도열병 균주 94-254를 사 용하였다. Namil(EMS)-bl10,bph1 x 밀양23호 F2 집단 내에 서 도열병 저항성 유전자의 분리양상을 평가하기 위해 각 F2 개체로부터 세대 진전된 F2:3 계통의 저항성 정도를 평가하였 다. 각 F2 계통 당 임의로 선택된 15여립의 F2:3 종자를 전개 하여 도열병 균주, 94-254를 접종하였다. 계통 내 모든 식물 체들의 병반응 정도는 접종 후 약 2주 후에 개체별로 병징을 조사하였으며, 그 평균값을 취하여 유전분석을 위한 각 계통 에 대응하는 F2 개체의 도열병 저항성 표현형으로 사용하였다.
벼멸구 저항성 검정
벼멸구 저항성에 대한 평가를 위하여 국립식량과학원 답작 과에서 유지하고 있는 벼멸구 집단(Korean Biotype)을 사용 하였다(Jena et al. 2006). 벼멸구 저항성 정도를 비교하기 위 하여 감수성으로는 원품종인 남일과 통일계 품종인 태백을, 중도저항성으로는 화삼(Oh et al. 1997), 그리고 저항성으로 는 통일계인 삼강(Ha et al. 2000) 등을 대비품종으로 공시하 였다. 벼멸구 접종은 검정용 박스에 파종한 식물체가 4~5엽 기에 도달하였을 때 유묘당 5~7 마리의 밀도로 2령기의 벼 멸구를 접종하였다. 벼멸구 저항성 평가는 대비품종들의 반응 과 비교하여 저항성 정도를 판정하였다.
Namil(EMS)-bl10,bph1계통이 발현하는 벼멸구 저항성 지 배 유전좌위를 표지하기 위한 수단으로 남일 및 돌연변이 계 통과 밀양23호간의 교잡을 통해 육성된 F2:3 종자들을 이용하 여 벼멸구 저항성 생물검정을 수행하였으며, 각 F2 집단 내에 서의 표현형 분리양상을 조사하였다. 벼멸구 검정용 박스에 각 계통별당 약 20립 정도를 파종하고 육묘하여 접종하였으 며, 감수성계통으로 공시된 남일과 태백이 고사되는 시점을 기준으로 생존 비율(생존한 개체들의 비율, %)의 평균값을 구하여 유전분석을 위한 각 계통의 벼멸구 저항성에 대한 표 현형으로 사용하였다.
분리후대집단을 이용한 연관지도 작성
기존에 보고된 정보를 참조하여(McCouch et al. 2002) 야 생종인 남일과 교배친인 밀양23호간에 다형성을 보이며 벼 염색체상에 균등배치 된 SSR 마커를 선정하였다. 이를 위해 각 SSR 마커에 의해 표지되는 벼 유전체정보(Rice Pseudomolecules Release 6; http://rice.plantbiology.msu.edu/)에 대응하는 물리적 위치를 e-Landing 기법으로 파악하였다 (Jeung et al. 2007). 확인된 물리적 위치에 대응하는 BAC 혹 은 PAC 클론들에 대한 정보와 유전자지도상의 위치들(cM) 은 Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/)에서 제공하는 Rice Pseudomolecule Information 을 참조하였다. 벼 염색체상에서 균일분포에 유의하면서 SSR 마커를 이용하여 F2 식물체들의 유전자형(genotype)을 조사 하고 연관지도 초안(linkage map skeleton)을 작성하였다.
각 마커들에 대하여 F2 식물체들로부터 조사된 유전자형의 분리비가 기대치에 부합하는지 평가하기 위하여 적합성검정 (χ2 test)을 실시하였으며(Jeung et al. 2011), 각 개체의 유전 자형과 도열병 및 벼멸구 저항성에 대한 표현형간의 연관분 석(association analysis)을 수행하였다.
PCR은 10 ng의 DNA와 5 pmole의 forward 및 reverse primer, 0.2 mM dNTP mix, 1X PCR buffer [50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl2]및 1 unit의 Taq polymerase (Nurotics, Korea)를 이 용하여 총량 20 μl 로 실시하였다. PTC-100® thermocycler (Waltham, USA)를 사용하여, 95°C에서 3분간 초기변성 후 95°C 20초, 55°C 30초, 72°C 60초로 총 35회 반복하고, 7 2°C에서 5분간 반응하였다. PCR산물은 Sequencing gel [5 % polyacrylamide (19 : 1, acrylamide : bis-acrylamaide), 6 M Urea, 1X TBE, 80 W]을 이용하여 전기영동 한 후 Silver Sequence™ (Promega, USA)로 염색하여 각 F2 개체 들의 유전자형을 판정하였다(Jeung et al. 2011).
도열병 및 벼멸구 저항성 유전좌위 표지를 위한 유전통계분석
도열병 및 벼멸구 저항성 관련 유전좌위의 벼 염색체상 위 치를 파악하기 위해 남일과 밀양23호 및 Namil(EMS)-bl10, bph1와 밀양23호간의 F2 개체들로부터 확보된 마커 유전자 형과 도열병 균주, 94-254 및 벼멸구, 생태형 I(Korean Biotype) 에 대한 F2:3 계통들의 저항성 간의 연관성을 평가하기 위해 연관분석(association analysis)을 수행하였다.
수집된 자료들을 SAS (Statistical Analysis System)프로 그램(SAS Institute, 2000)을 이용하여 유전통계분석을 수행 하였다. SAS 프로그램의 PROC MEANS를 이용하여 기술통 계량을 산출하였으며, 목표유전좌위 표지를 위해 육성된 F2 집단에 대한 연관분석은 PROC GLM을 이용하여 각 SSR 마 커들에 의해 표지되는 유전좌위가 저항성에 관여되는 정도를 판정하였다(Single Locus ANOVA). 각 유전좌위에 대한 F 분석 결과 P 값이 0.05보다 작으면 유의하다고 판정하고, F2 분리집단에서 관찰된 도열병 및 벼멸구 저항성 분산에 대한 해 당 유전좌위의 기여도(R2 value; %)와 유전적 작용가(additive effect) 및 기작양상(degree of dominance)등을 추정하였다 (Jeung et al. 2007).
남일과 돌연변이 계통 Namil(EMS)-bl10,bph1의 작물학적 특성 평가
Namil(EMS)-bl10,bph1로 명명된 도열병 및 벼멸구 저항 성 계통을 야생종인 남일과 함께 공시하여 작물학적 특성을 평가하였다(Fig. 1A, Table 1). 도열병 및 벼멸구 저항성 돌 연변이 계통인 Namil(EMS)-bl10,bph1의 출수기는 조생종인 남일에 비해 빠른 출수기를 보였으며, 간장 또한 남일에 비해 돌연변이 계통인 Namil(EMS)-bl10,bph1에서 단간이었다. 벼멸구 저항성은 도복과 관련된 형질(3절간장)과 상관관계가 있으며, 저항성 유전자(bph2)를 가진 자포니카 계통은 긴 간 장을 가지고 있는 것으로 보고 되었다(Shin 1990, Yeo & Sohn 2001, Lee et al. 2011). 반면에 자포니카계 돌연변이 계통 Namil(ESM)-bl10,bph1은 간장이 단간 품종인 주남 (Yeo & Sohn 2001)과 유사한 크기의 간장이 관찰되었다. Namil(EMS)-bl10,bph1의 빠른 출수기와 단간은 유용한 형 질로 작용될 수 있으나 수량은 현미를 기준으로 남일에 비해 유의하게 감소하였다. 수량 감소의 원인으로는 수량구성요소 인 수당립수와 등숙비율이 남일에 비해 Namil(EMS)-bl10, bph1에서 감소하였기 때문인 것으로 사료된다.
Table 1.
Comparisons between Namil, the wild type and Namil(EMS)-bl10,bph1, a mutant line in terms of important agronomic traits.z
Table 1.
Lines HD (month.day) CL (cm) PL (cm) TN (No.) SN (No.) RGP (%) TWD (g) YDB (Kg/10a)
Namil 7.29 ± 0.0 77 ± 2.0 24.0 ± 0.6 11.0 ± 1.0 147.0 ± 7.8 74.4 ± 7.1 24.8 ± 0.0 599.0 ± 24.0
Namil(EMS)-bl10,bph1 7.24 ± 1.2 74 ± 1.0 21.0 ± 0.6 12.0 ± 1.0 128.0 ± 3.5 55.0 ± 1.7 22.2 ± 0.2 471.0 ± 7.0

zHeading date (HD), Culm length (CL), Panicle length (PL), Tiller number (TN), Spikelet number (SN), Ripened grain percentage (RGP), 1,000-grain weight of hulled grain (TWD), and Yield of brown rice (YDB). Note that the agronomic traits were evaluated by harvesting 100 plant for each replication plot (n=3), which were planted as three seedlings per plant during transplanting.

Namil(EMS)-bl10,bph1과 F2 집단의 도열병 저항성 평가
균주 선정에 있어서 야생종인 남일과 교배친인 밀양23호를 모두 침해하는 균주를 선정하기 위하여 국내수집 균주 2개를 접종하여 이병반응을 검정한 결과(Table 2), 남일과 밀양23호 는 두 균주에 대해 감수성을 보였으며, 대비품종들도 두 균주 에 대해 감수성을 보였다. 돌연변이 계통인 Namil(EMS)-bl10,bph1은 도열병 균주 94-254에 대해 중도저항성을 보인 반면에 균주 93-093에 대해서는 감수성을 보였다. 균주 검정 을 통해 얻어진 결과를 바탕으로 도열병 저항성이 분리되는 교잡후대에서 변별력 높은 표현형 결과를 확보하기 위해 Namil(EMS)-bl10,bph1는 비친화성이며 야생종인 남일과 교 배친인 밀양23호에 친화성인 도열병 균주 94-254가 유전분 석에 이용되었다(Fig. 1C).
Table 2.
Reactions of the mutant line, Namil(EMS)-bl10,bph1 and cultivars against selected Korean disease and insect pest.
Table 2.
Linesz Rice Blast isolate (race)y
 Linesx Brown planthopperw
94-254 (ND)z 93-093 (KI197) Korean Biotype
Namil 4 5 Namil S
Milyang23 4 4 Milyang23 S
Junam 5 5 Taebaek S
Onnuri 5 5 Hwasam M
Chilbo 5 5 Samgang R
Namil(EMS)-bl10,bph1 2 5 Namil(EMS)-bl10,bph1 R

zKorean rice cultivars, Junam, Onuri, and Chilbo were included as a check varieties on virulent rice blast isolates

yRice blast isolates were collected from Korean famers’ fields. Resistance degrees of virulent rice blast were classified five levels from 1 (resistance) to 5 (susceptibility)

xKorean rice cultivar, Taebaek, Hwasam, and Samkang were included as a check variety on brown planthopper

wResistance degrees of brown planthopper were classified into three levels susceptibility as susceptibility (S), moderate (M), and resistance (R)

연관분석(association analysis)을 위한 도열병 저항성 검정 에서 분리후대집단의 표현형 조사를 위해 남일과 밀양23호간 103개 F2 집단과 Namil(EMS)-bl10,bph1과 밀양23호간의 97개 F2 집단에서 각 F2 계통당 15립정도의 F2:3 종자를 전개 하여 선정된 균주 94-254를 접종하여 병반응 정도에 따라 집 단 내에서의 표현형 분리양상을 조사하였다(Fig. 2A). 도열병 에 대한 저항성이 야생종인 남일과 교배친인 밀양23호에서 각각 4.3과 3.7로 낮게 평가되었으며, 돌연변이 계통인 Namil (EMS)-bl10,bph1에서는 2.1로 남일과 밀양23호에 비해 높게 평가되었으나 중도저항성을 보였다. 각각의 분리후대집단에 서는 남일과 밀양23호간 분리후대집단에서는 평균 4.0으로 낮게 평가되었으며, Namil(EMS)-bl10,bph1과 밀양23호간 분리후대집단에서는 평균 3.2로 남일과 밀양23호간 집단에 비하여 높게 평가되었으나 Namil(EMS)-bl10,bph1과 유사한 저항성 정도인 평균적으로 중도저항성을 보였다. 도열병 저항 성에 대한 두 분리후대집단의 분산분포를 비교한 결과, 두 집 단 모두 편향된 정도는 달랐으나 평균을 중심으로 감수성쪽 으로 편기되는 부적편포를 보였다. 남일과 밀양23호간 분리 후대집단에서는 완전 편향되는 양상을 보였으며(skewness=-0.8), Namil(EMS)-bl10,bph1간 분리후대집단에서는 대체적 으로 감수성쪽으로 편기되는 양상을 보였다(skewness =-0.4). 감 수성쪽으로 완전히 편향된 양상을 보인 남일과 밀양23호간의 분리후대집단에 비하여 Namil(EMS)-bl10,bph1과 밀양23호 간의 분리후대에서는 분포의 범위가 넓었으며, 저항성 범위 (0~1)에 관측수가 없는 남일과 밀양23호간 분리후대집단에 반하여 Namil(EMS)-bl10,bph1과 밀양23호간 분리후대집단 에서는 Namil(EMS)-bl10,bph1(중도저항성)을 중심으로 저 항성 범위(0~1)에 관측수가 분포하고 있음을 확인하였다. 이 러한 결과로 미루어 보아, 돌연변이 계통인 Namil (EMS)-bl10,bph1의 유전인자에 의해 도열병 저항성이 증가하는 것 으로 판단되었다.
Fig. 2.
Histograms of mean resistance of F2 mapping populations derived from two crosses between Namil (wild type), Namil (EMS)-bl10,bph1 and Milyang23 on virulent rice blast isolate, 94-254 (A) and brown planthopper, Koran Biotype (B).
To estimate the resistant levels of F2 progeny lines, harvested seeds from each F2 plants (F2:3 seeds) were planted in a row. Fourteen days after inoculation of rice blast isolate (94-254) or infestation of brown planthopper (Korean Biotype) to two week old seedlings, individuals were evaluated (rice blast) or over-all survival ratio was calculated (brown planthopper), respectively. The descriptive statistics on virulent rice blast isolate, 94-254 of 103 F2:3 lines of Namil x Milyang23, over-all mean, standard deviation, skewness, and kurtosis are 4.0, 0.6, -0.8, and 0.6, respectively. In 97 F2:3 lines of Namil(EMS)-bl10,bph1 x Milyang23 on virulent rice blast isolate, 94-254, over-all mean, standard deviation, skewness, and kurtosis are respectively 3.2, 1.2, -0.4, and -0.5. On brown planthopper, Korean Biotype, the descriptive statistics of 97 F2 population of Namil(EMS)-bl10,bph1 × Milyang23, over-all mean, standard deviation, skewness, and kurtosis are 63.9, 36.7, -0.9, and -0.8, respectively. All F2:3 lines of Namil x Milyang23 were excepted because of drying on brown planthopper, Korean Biotype.
KJBS-46-226_F2.gif
Namil(EMS)-bl10,bph1과 F2 집단의 벼멸구 저항성 평가
남일과 밀양23호는 벼멸구에 감수성을 보인 반면에 돌연변 이 계통인 Namil(EMS)-bl10,bph1은 매우 안정적인 저항성 을 발현하였다(Fig. 1B). 대비품종인 태백은 감수성, 화삼은 중도저항, 그리고 삼강은 저항성을 나타내었다(Table 2). 벼 멸구 저항성 유전분석을 위한 F2 후대집단의 벼멸구 저항성 평가에서는 남일과 밀양23호간의 F2 후대집단은 전부 고사하 여 평가에서 제외하였으며, Namil(EMS)-bl10,bph1 x 밀양 23호간의 F2 97개체 집단에서만 벼멸구 저항성 평가가 실시 되었다. 돌연변이 계통인 Namil(EMS)-bl10,bph1의 벼멸구 에 대한 저항성은 94.4%의 높은 생존율을 나타내었다. 벼멸 구 저항성 돌연변이 계통인 Namil(EMS)-bl10,bph1에 의해 남일과 밀양23호간의 F2 후대집단은 전부 고사했으나 Namil (EMS)-bl10,bph1과 밀양23호 F2 후대집단은 벼멸구에 대한 평균 생존율이 63.9%로 조사되었다. 벼멸구 저항성에 대한 분산분포를 보면, Namil(EMS)-bl10,bph1과 밀양23호 F2 후 대집단의 벼멸구 저항성 분포는 뚜렷하게 감수성과 저항성의 2개 군집으로 구분되는 양상을 보였다(Fig. 2B). 이러한 분산 분포로 보아 벼멸구에 대한 저항성은 Namil(EMS)-bl10,bph1 의 소수의 주동유전자에 의해 조절되는 것으로 판단되었다.
Namil(EMS)-bl10,bph1 x 밀양23호간 후대 F2 집단을 이 용한 연관지도 작성
도열병 및 벼멸구에 대해 저항성을 보이는 돌연변이 계통 Namil(EMS)-bl10,bph1과 밀양23호간 교잡을 통해 육성된 총 97개 F2 식물체로 구성된 유전분석 집단을 구축하였다. 각 F2 개체로부터 DNA를 추출하여 SSR 마커를 이용한 연관지 도를 작성하였다. 연관지도 작성은 각 염색체마다 균등하게 최소한의 수의 SSR 마커를 배치하여 연관지도 초안(linkage map skeleton)을 작성하였다. 각각의 염색체상에 SSR 마커 를 배치하기 위하여 e-Landing기법에 의해 각 SSR 마커에 대응하는 벼 유전체정보를 파악하고 mirror map을 구축함으 로써 분리집단의 유전자형에 대한 부가적인 유전통계분석 절 차를 생략하였다(Table 3). 연관지도 초안을 위해 활용된 유 전자형에 대한 분리비 검정을 수행하여(segregation test), 각 F2 집단에서 분리비 이상(segregation distortion)이 일어나고 있는 부위를 확인하였다(Table 3). 도열병 및 벼멸구에 대해 저항성을 보이는 돌연변이 계통 Namil(EMS)-bl10,bph1과 밀양23호간에 다형성을 보이며 염색체상에 균등하게 위치한 총 60개의 SSR 마커들을 이용하여 연관지도 초안을 작성하였 다. 유전자형의 분리비를 검정한 결과(Table 3), Namil(EMS)-bl10,bph1과 밀양23호간의 F2 집단에서는 염색체 7번 중단에 서 분리비이상이 매우 유의하게 관찰되었다. 염색체 7번에서 는 Namil(EMS)-bl10,bph1에 비해 밀양23호의 유전자형의 빈도가 매우 높게 관찰되었다.
Table 3.
List of SSR primer sets, their e-Landing mediated physical positions on rice Pseudomolecule 6, corresponding BAC/PAC clones with their determined cM positions, and the segregating (χ2 test) on the F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23.
Table 3.
Markerz Physical informationy
 Mirror mapx
 Segregation Testw
Ch Start Stop e-PCR e % BAC/PAC cM% cM A H B M χ2
RM3252 1 299,681 299,852 172 0.7 AP002818 0.2 0.3 19 28 50 0 1.4
RM0600 1 9,461,346 9,461,566 221 21.7 AP001081 27.3 49.6 19 30 48 0 2.1
RM0449 1 15,305,619 15,305,758 140 35.1 AP008247 40.0 72.8 21 27 49 0 0.5
RM1349 1 25,398,082 25,398,300 219 58.3 AP002744 56.7 103.1 19 25 53 0 1.3
RM1003 1 33,803,800 33,803,927 128 77.5 AP003345 75.1 136.6 20 29 48 0 1.3
RM6321 1 43,251,019 43,251,186 168 99.2 AP003277 100.0 181.8 31 18 48 0 3.0
RM0154 2 1,803,895 1,084,077 183 3.0 AP005851 3.0 4.7 19 29 49 0 1.7
RM5699 2 8,981,409 8,981,575 167 25.0 AP005803 26.7 42.1 20 26 50 1 0.7
RM1211 2 18,450,427 18,450,588 162 51.4 AP005299 37.7 59.5 22 28 47 0 0.6
RM3220 2 28,458,108 28,458,248 141 79.2 AP004118 74.8 118.1 25 22 50 0 0.1
RM3220 2 35,425,798 35,425,907 110 98.6 AP003989 100.0 157.9 25 25 47 0 0.0
RM4108 3 514,862 515,003 142 1.4 AC113930 0.7 1.1 23 30 44 0 1.5
RM5628 3 3,101,122 3,101,282 161 8.5 AC099401 9.0 14.8 19 35 43 0 5.8
RM0007 3 9,808,540 9,808,710 171 27.0 AC134232 27.0 44.4 15 40 42 0 13.5 ***
RM6266 3 23,778,063 23,778,222 160 65.4 AC091246 57.7 94.9 30 22 45 0 1.4
RM3525 3 30,344,279 30,344,457 179 83.5 AC117988 83.0 136.5 24 38 35 0 10.7 ***
RM3585 3 36,080,616 36,080,784 169 99.3 AC128647 98.4 161.7 18 28 51 0 1.9
RM8213 4 4,433,178 4,433,388 211 12.6 AL662959 8.7 11.3 22 26 49 0 0.2
RM5633 4 13,059,370 13,059,580 211 37.1 AL731595 15.4 19.9 26 28 43 0 1.0
RM1155 4 20,328,759 20,328,906 148 57.7 AL606453 45.4 58.9 29 30 38 0 3.9
RM3839 4 23,870,755 23,870,972 218 67.7 AL606614 56.2 72.8 32 30 35 0 6.8 *
RM3217 4 30,083,469 30,083,662 194 85.4 AL606683 77.7 100.7 30 20 45 2 1.9
RM0559 4 35,117,645 35,117,804 160 99.6 AL606637 100.0 129.6 29 14 54 0 5.2
RM5693 5 441,872 442,071 200 1.5 AC129716 3.8 4.6 24 24 49 0 0.0
RM5844 5 9,128,802 9,128,996 195 30.6 AC120989 43.7 53.5 34 14 49 0 7.4 *
RM0249 5 10,755,867 10,755,994 128 36.0 AC136522 44.4 54.3 34 14 49 0 7.4 *
RM3870 5 22,879,699 22,879,891 193 76.6 AC108503 77.9 95.3 24 21 52 0 0.5
RM1054 5 29,144,035 29,144,184 150 97.6 AC098598 99.8 122 19 31 47 0 2.6
RM3353 6 435,582 435,697 116 1.4 AP001129 1.1 1.4 23 25 49 0 0.0
RM0253 6 5,425,498 5,425,631 134 17.4 AP004754 14.0 17.4 13 32 52 0 7.1 *
RM0276 6 6,230,046 6,230,169 124 19.9 AP003488 26.9 33.5 13 33 51 0 7.7 *
RM6818 6 16,581,413 16,581,542 130 53.1 AP004012 52.9 65.8 18 26 53 0 1.8
RM3628 6 23,737,032 23,737,157 126 76.0 AP003612 68.6 85.4 19 21 57 0 2.6
RM5753 6 30,966,850 30,967,050 201 99.1 AP004685 100 124.4 27 23 47 0 0.3
RM1093 7 668,161 668,310 150 2.3 AP003746 2.1 2.5 25 22 50 0 0.1
RM1377 7 12,782,829 12,783,009 181 43.1 AP004305 41.9 49.7 15 38 44 0 10.7 ***
RM5793 7 17,488,937 17,489,063 127 58.9 AP004299 51.3 60.8 10 40 47 0 17.4 ***
RM0172 7 29,560,592 29,560,750 159 99.6 AP005199 100 118.6 22 24 51 0 0.2
RM0408 8 119,935 120,063 129 0.4 AP005406 0.4 0.5 21 36 40 0 6.9 *
RM0547 8 5,586,058 5,586,291 234 19.7 AP004746 33.2 40.2 17 24 56 0 2.9
RM3262 8 22,248,334 22,248,500 167 78.6 AP005483 71.5 86.7 17 27 53 0 2.4
RM5545 8 28,141,927 28,142,083 157 99.4 AP004623 100 121.2 22 27 48 0 0.3
RM23654 9 151,453 151,639 187 0.7 AP006059 0.0 0.0 24 24 47 2 0.0
RM0316 9 1,074,933 1,075,126 194 4.7 AP005860 0.9 0.8 25 25 47 0 0.0
RM0219 9 7,887,585 NA 34.3 AP005912 22.1 20.7 20 20 50 0 0.8
RM0566 9 14,704,798 14,705,036 239 63.9 AP005397 54.2 50.7 17 17 53 0 2.4
RM0205 9 22,720,646 22,720,801 156 98.7 AP005546 100 93.5 20 20 54 0 1.1
RM7492 10 39,037 39,181 145 0.2 AC166065 0.0 0.0 29 29 52 0 3.4
RM5689 10 13,223,351 13,223,453 103 57.8 AC021891 31.1 26.1 25 26 46 0 0.1
RM1375 10 16,386,764 16,386,943 180 71.6 AC025905 51.0 42.7 28 23 46 0 0.5
RM0590 10 22,784,993 22,785,130 138 99.6 AC018727 100 83.8 26 16 55 0 3.3
RM6901 11 1,494,196 NA 5.2 AC123516 5.5 6.5 23 19 55 0 1.7
RM0536 11 8,968,470 8,968,712 243 31.5 AC138197 41.6 49.1 19 28 50 0 1.4
RM3428 11 13,445,211 13,445,367 157 47.2 AC128642 47.9 56.5 18 30 49 0 2.5
RM5349 11 19,148,807 19,148,929 123 67.3 AC134925 67.1 79.1 21 28 48 0 0.7
RM0144 11 28,246,930 28,247,154 225 99.2 AC134045 98.6 116.2 26 22 49 0 0.2
RM3472 12 3,520,117 3,520,331 215 12.8 AL713902 24.8 27.1 23 29 45 0 1.0
RM1337 12 11,933,319 11,933,500 182 43.4 BX000556 46.2 50.4 22 28 47 0 0.6
RM0277 12 18,290,458 NA 66.5 AL831799 57.0 62.2 25 27 45 0 0.4
RM0017 12 26,954,668 26,954,835 168 98.0 AC027133 98.4 107.4 30 22 45 0 1.4

zThe primer sequences were used as the query to localize on Rice Pseudomolecule 6

yWhen both primer sequences successfully recognized their physical locations to be annealed, the expected PCR product size (e-PCR:‘Stop’-‘Start’+1bp, NA=not available) was used to judge PCR products. If any primer failed for e-Landing, the other primer's result was adopted to estimate the corresponding point. The determined physical regions followed by e-Landing were used to match corresponding BAC/PAC clones depend on the ‘Rice Pseudomolcule Information and Gene Search (http://rice.plantbiology.msu.edu/)

xThe determined cM positions for each BAC/PAC clones was directly adopted from the ‘Rice Pseudomolcule Information and Gene Search’ which were estimated using the marker sequences obtained from the Cornell Rice Genes Database and the Japanese Rice Genome Program. To take some convenient in comparing the cM positions from various mapping populations having different total lengths, the percentage expressed relative genetic positions, ‘cM%’, was also calculated

wA and B are homozygous for the Namil(EMS)-bl10,bph1 and Milyang23 allele types, H is for heterozygous progenies at the tested locus, and M indicates the number of missing. Segregation distorted loci are indicated with asterisk; χ2 values for the significant levels are less than 0.05

***significant at P < 0.0001

**significant at P < 0.01

*significant at P < 0.05

연관성 평가에 의한 도열병 및 벼멸구 저항성 유전좌위 표지
연관지도 초안에 배치된 총 60개 SSR 마커들에 대응하는 도열병 및 벼멸구 저항성에 대한 F2 개체들의 유전자형과 F2:3 도열병 및 벼멸구 검정을 통해 확보된 표현형간의 연관분석 (association analysis)을 수행하였다(Table 4). 각 SSR 마커 에 대해 97개 F2 개체들을 3개의 유전자형 군집으로 나눈 후 (No. of genotype), 각 군집의 저항 정도의 평균(genotype mean)에 대해 분산분석(Single-locus ANOVA)을 실시하였다. 분산분석 결과 F 값이 유의한 경우 해당 유전좌위 근접부위 에 표현형 변이에 영향을 끼치는 유전자가 존재하는 것으로 판단하고 R2 값(전체 표현형 분산에서 해당 유전좌위의 기여 도)을 제시하고 유전적 작용력(Genetic effect)을 추정하였다.
Table 4.
Summary of association analysis between DNA marker genotypes and phenotype performances of F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23z for rice blast resistance and brown planthopper resistance.
Table 4.
Trait Evaluated Mirror Mapy
 No. of Genotypesx
 Mean of Genotypew
 Single-locus ANOVAv
 Genetic Effectu
Locus Ch cM cM% A H B A H B SSM SSE F-value R2 Add Dom DeD
BL (Degree: 0-5) Pop. No.=97 Pop. Mean=3.2 RM3850 2 158.0 100 25 47 25 3.0 3.0 3.7 10.2 138.4 3.5 * 0.068 0.37 -0.38 -1.0
RM1377 7 49.7 41.9 15 44 38 2.4 3.4 3.3 10.7 137.8 3.6 * 0.072 0.41 0.55 1.3
RM23654 9 0.0 0.0 24 47 24 3.0 3.0 3.8 11.8 134.5 4.0 * 0.081 0.43 -0.36 -0.9
RM0316 9 0.8 0.9 25 47 25 2.9 3.0 3.8 11.2 137.3 3.8 * 0.076 0.42 -0.32 -0.8
RM3472 12 27.1 24.8 23 45 29 2.7 3.1 3.7 15.4 133.1 5.4 ** 0.104 0.54 -0.11 -0.2
RM1337 12 50.4 46.2 22 47 28 1.9 3.1 4.4 80.9 67.7 56.0 **** 0.544 1.27 -0.05 0.0
RM0277 12 62.2 57.0 25 45 27 2.1 3.2 4.3 63.4 85.1 35.0 **** 0.427 1.1 -0.01 0.0
Bph (Ratio: %) Pop. No.=97 Pop. Mean=63.9 RM0547 8 40.2 33.2 17 56 24 39.6 70.2 66.6 12392 116980 5.0 ** 0.096 13.47 17.08 1.3
RM3262 8 86.7 71.5 17 53 27 63.8 56.6 78.4 8576 120796 3.3 * 0.066 7.34 -14.6 -2.0
RM3472 12 27.1 24.8 23 45 29 84.0 61.1 52.4 13541 115831 5.5 ** 0.105 -15.8 -7.13 0.5
RM1337 12 50.4 46.2 22 47 28 90.6 69.4 33.8 42504 86868 23.0 **** 0.329 -28.4 7.22 -0.3
RM0277 12 62.2 57.0 25 45 27 92.5 73.1 22.2 71258 58114 58.0 **** 0.551 -35.2 15.73 -0.4
RM0017 12 107.0 98.4 30 45 22 77.1 68.1 37.3 21651 107720 9.4 *** 0.167 -19.9 10.93 -0.5

zOnly significant loci to each trait tested are presented. 60 DNA markers were tested in 97 F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23.

yBoth cM positions and the percentage expressed relative genetic positions (cM%) are presented

xF2 progenies were split into three subgroups based on the revealed genotype by SSR markers. A and B are homozygous for the Namil(EMS)-bl10,bph1 and Milyang23 allele types and H indicates heterozygous progenies at the tested locus.

wThe means of each subpopulation for each locus are calculated.

vFor the F-test, SSR markers having less than significant levels (p) were declared as significant empirically. Explainable phenotypic variation portion at the tested locus (R2) was also calculated. The significant levels are less than 0.05

****significant at P < 0.0001

***significant at P < 0.001

**significant at P < 0.01

*significant at P < 0.05

uuAdditive effect (Add), domiant effect (Do) and degree of dominance (DeD) were then estimated at the declared loci: Add=(Bmean- Amean)/2, Do= Hmean-(Bmean+Amean)/2, and DeD=Do/Add. where A and B are homozygous F2 individuals for Namil(EMS)-bl10,bph1 and Milyang23, H is heterozygous individuals at the tested locus.

연관지도 초안에 배치된 SSR 마커들 중 연관분석을 통하 여 도열병 및 벼멸구 저항성에 관여하는 통계적으로 유의한 F 값을 지니는 마커들에 대응하는 유전좌위를 표지하였다 (Fig. 3). 도열병 저항성에 관련된 유전좌위는 7개의 SSR 마 커에 의해 표지되었으며, 벼멸구 저항성에 관련된 유전좌위는 6개의 SSR 마커에 의해 표지되었다. 도열병 저항성 관련 유 전좌위 표지에 사용된 SSR 마커는 2번, 7번, 9번, 및 12번 염 색체상에서 각각 1개, 1개, 2개, 및 4개로 확인되었으며, 벼멸 구 저항성과 관련 유전좌위 표지에 사용된 SSR 마커는 8번과 12번 염색체상에 각각 2개와 4개로 확인되었다. 특이한 점은 Namil(EMS)-bl10,bph1의 도열병 저항성과 벼멸구 저항성에 관련하는 유전좌위가 모두 12번 염색체 중단 부위에 RM1337 과 RM0277에 의해 표지되었다는 점이다(Fig. 3B). RM1337 에 의해 표지 된 해당 유전좌위는 도열병 및 벼멸구에서 각각 전체분산에서 약 54.4%와 32.9%의 기여도를 보였으며, RM0277에 의해 표지 된 해당 유전좌위는 도열병 및 벼멸구 에서 각각 전체분산에 약 42.7%와 55.1%의 기여도를 보였 다. 도열병 저항성에 대해 RM1337에 의해 표지되는 유전좌 위는 전체분산의 약 54.4%를 설명하는 주동유전좌위였다. 도 열병 저항성에 대한 유전좌위는 기존의 Pi-6(t), Pi12(t), Pi-21(t), Pi31(t), Pi-32(t), Pi-41, Pi-157, Pita, Pi-ta2 및 Pi-tq6 등 12번 염색체 중단에 위치하는 것으로 보고된 (Sallaud et al. 2003, Yang et al. 2009) 적어도 9개의 저항 성 유전자들과의 유전적 관련성에 대한 확인이 필요하다. 벼 멸구 저항성에 대해 RM0277에 의해 표지되는 유전좌위는 전제분산의 약 55.1%를 설명하는 주동유전좌위였다. 12번 염색체에서 벼멸구 저항성과 관련된 유전자들은 Bph1, Bph2, Bph9, Bph10, Bph18, Bph12, 및 BPH26으로 알려져 있으 며, 이 유전자들은 12번 염색체의 긴 팔쪽에 유전좌위가 표지 되어있다(Murata et al. 2000, Lang & Buu 2003, Sharma et al. 2004, Kim & Sohn 2005, Su et al. 2006, Jena et al. 2006, Rahman et al. 2009, Shin et al. 2011, Myint et al. 2012). 따라서 RM0277에 의해 표지 된 유전좌위가 이미 보 고된 유전자들과 물리적 위치가 동일한지 판정이 필요할 것 으로 판단된다.
Fig. 3.
Linkage map skeleton of rice blast resistance and brown planthopper resistance on rice chromosomes. In Namil × Milyang23, wild type, preliminary gene loci of rice blast resistance are on rice chromosome 5 and 11 (A). In Namil(EMS)-bl10,bph1, a mutant line, preliminary gene loci of rice blast resistance and brown planthopper resistance are on the same rice chromosome 12 (B). SSR markers were applied based on F-statistics from single-locus ANOVA results to narrow done the putative location responsible for rice blast resistance and brown planthopper resistance. The genetic intervals are shown in centimorgan (cM).
KJBS-46-226_F3.gif
연관분석의 결과에 의하면, 도열병 및 벼멸구 저항성에 관 련된 유전자가 비록 같은 유전좌위에 표지되었다 하더라도 각 유전좌위에 의한 작용가가 도열병과 벼멸구 저항성에 대 해 서로 평행적으로 관찰되지 않았기 때문에 서로 독립적인 유전좌위들인 것으로 추정된다. Namil(EMS)-bl10,bph1의 도열병 및 벼멸구 저항성에 관여하는 유전좌위가 단일유전좌 위로서 다면발현효과를 발현하는 것인지, 염색체상에서 서로 근접된 독립적인 유전좌위인지에 대한 명확한 판정이 필요할 것이다. 따라서 고밀도 연관지도 작성을 위해 마커밀도를 선 택적으로 높여주어 좀 더 정밀한 유전좌위 표기 및 e-Landing 기법을 이용하여 BAC/PAC의 전사부위(Open reading frame; ORF) 정보를 활용하여 기존에 보고된 벼멸구 저항성 유전자 들과의 유전적 관련성 분석이 필요할 것으로 사료된다.
작물학적 특성을 신속히 보완하며 확충할 수 있는 이점을 가지고 있는 돌연변이 육종을 통해 육성된 유용형질발현 돌 연변이계통들의 다양한 작물학적 특성과 병해충 저항성은 유 용한 유전자원으로 평가되고 있으며, 병해충 저항성에 대한 돌연변이 계통의 유용형질들을 교배를 통해 고품질 벼의 보 완을 위해 이전하는 것이 용이하다. 농촌진흥청 국립식량과학 원에서는 국내 육성 자포니카 벼 품종인 남일에 EMS를 돌연 변이원으로 활용하여 도열병 및 벼멸구 저항성 돌연변이 후 대계통인 Namil(EMS)-bl10,bph1를 육성하였다. 염색체 상 에 Namil(EMS)-bl10,bph1의 도열병 및 벼멸구 저항성에 관 련된 유전자좌위를 표지하기 위해 Namil(EMS)-bl10,bph1 x 밀양23호로부터 유래한 F2 97 개체로부터 도열병과 벼멸구에 대한 저항성을 검정하고 60개 SSR 마커의 유전자형을 검정 하여 연관성분석(association analysis)을 수행하였다. 분자마 커의 유전자형과 저항성 간의 연관성분석 결과, Namil(EMS)-bl10,bph1 의 도열병 및 벼멸구 저항성에 관여하는 주동유전 자위는 12번 염색체 중단의 RM1337 및 RM0277에 각각 표 지 되었다.
본 연구는 농촌진흥청 바이오그린21사업(과제번호 PJ00816) 의 지원으로 수행된 결과이며, 이에 감사 드립니다.
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Genetic Analysis on the Rice Blast and Brown planthopper Resistance of Namil(EMS)-bl10,bph1, a Japonica Rice Mutant Line
Korean. J. Breed. Sci.. ;46(3):226-237.   Published online September 30, 2014
DOI: https://doi.org/10.9787/KJBS.2014.46.3.226
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Genetic Analysis on the Rice Blast and Brown planthopper Resistance of Namil(EMS)-bl10,bph1, a Japonica Rice Mutant Line
Korean. J. Breed. Sci.. ;46(3):226-237.   Published online September 30, 2014
DOI: https://doi.org/10.9787/KJBS.2014.46.3.226
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Genetic Analysis on the Rice Blast and Brown planthopper Resistance of Namil(EMS)-bl10,bph1, a Japonica Rice Mutant Line
Image Image Image
Fig. 1. Agronomic performances of the wild type, Namil (left) and the mutant line of Namil, Namil(EMS)-bl10,bph1 (right) (A), reaction patterns against brown planthopper, Korean Biotype (B) of Namil (left) and Namil(EMS)-bl10,bph1 (right), and virulent rice blast isolate, 94-254 of Namil, Milyang23, and Namil(EMS)-bl10,bph1, sequentially from left to right (C).
Fig. 2. Histograms of mean resistance of F2 mapping populations derived from two crosses between Namil (wild type), Namil (EMS)-bl10,bph1 and Milyang23 on virulent rice blast isolate, 94-254 (A) and brown planthopper, Koran Biotype (B). To estimate the resistant levels of F2 progeny lines, harvested seeds from each F2 plants (F2:3 seeds) were planted in a row. Fourteen days after inoculation of rice blast isolate (94-254) or infestation of brown planthopper (Korean Biotype) to two week old seedlings, individuals were evaluated (rice blast) or over-all survival ratio was calculated (brown planthopper), respectively. The descriptive statistics on virulent rice blast isolate, 94-254 of 103 F2:3 lines of Namil x Milyang23, over-all mean, standard deviation, skewness, and kurtosis are 4.0, 0.6, -0.8, and 0.6, respectively. In 97 F2:3 lines of Namil(EMS)-bl10,bph1 x Milyang23 on virulent rice blast isolate, 94-254, over-all mean, standard deviation, skewness, and kurtosis are respectively 3.2, 1.2, -0.4, and -0.5. On brown planthopper, Korean Biotype, the descriptive statistics of 97 F2 population of Namil(EMS)-bl10,bph1 × Milyang23, over-all mean, standard deviation, skewness, and kurtosis are 63.9, 36.7, -0.9, and -0.8, respectively. All F2:3 lines of Namil x Milyang23 were excepted because of drying on brown planthopper, Korean Biotype.
Fig. 3. Linkage map skeleton of rice blast resistance and brown planthopper resistance on rice chromosomes. In Namil × Milyang23, wild type, preliminary gene loci of rice blast resistance are on rice chromosome 5 and 11 (A). In Namil(EMS)-bl10,bph1, a mutant line, preliminary gene loci of rice blast resistance and brown planthopper resistance are on the same rice chromosome 12 (B). SSR markers were applied based on F-statistics from single-locus ANOVA results to narrow done the putative location responsible for rice blast resistance and brown planthopper resistance. The genetic intervals are shown in centimorgan (cM).

Fig. 1.

Fig. 2.

Fig. 3.

Genetic Analysis on the Rice Blast and Brown planthopper Resistance of Namil(EMS)-bl10,bph1, a Japonica Rice Mutant Line

Comparisons between Namil, the wild type and Namil(EMS)-bl10,bph1, a mutant line in terms of important agronomic traits.z

Lines HD (month.day) CL (cm) PL (cm) TN (No.) SN (No.) RGP (%) TWD (g) YDB (Kg/10a)
Namil 7.29 ± 0.0 77 ± 2.0 24.0 ± 0.6 11.0 ± 1.0 147.0 ± 7.8 74.4 ± 7.1 24.8 ± 0.0 599.0 ± 24.0
Namil(EMS)-bl10,bph1 7.24 ± 1.2 74 ± 1.0 21.0 ± 0.6 12.0 ± 1.0 128.0 ± 3.5 55.0 ± 1.7 22.2 ± 0.2 471.0 ± 7.0

Heading date (HD), Culm length (CL), Panicle length (PL), Tiller number (TN), Spikelet number (SN), Ripened grain percentage (RGP), 1,000-grain weight of hulled grain (TWD), and Yield of brown rice (YDB). Note that the agronomic traits were evaluated by harvesting 100 plant for each replication plot (n=3), which were planted as three seedlings per plant during transplanting.

Reactions of the mutant line, Namil(EMS)-bl10,bph1 and cultivars against selected Korean disease and insect pest.

Linesz Rice Blast isolate (race)y
 Linesx Brown planthopperw
94-254 (ND)z 93-093 (KI197) Korean Biotype
Namil 4 5 Namil S
Milyang23 4 4 Milyang23 S
Junam 5 5 Taebaek S
Onnuri 5 5 Hwasam M
Chilbo 5 5 Samgang R
Namil(EMS)-bl10,bph1 2 5 Namil(EMS)-bl10,bph1 R

Korean rice cultivars, Junam, Onuri, and Chilbo were included as a check varieties on virulent rice blast isolates

Rice blast isolates were collected from Korean famers’ fields. Resistance degrees of virulent rice blast were classified five levels from 1 (resistance) to 5 (susceptibility)

Korean rice cultivar, Taebaek, Hwasam, and Samkang were included as a check variety on brown planthopper

Resistance degrees of brown planthopper were classified into three levels susceptibility as susceptibility (S), moderate (M), and resistance (R)

List of SSR primer sets, their e-Landing mediated physical positions on rice Pseudomolecule 6, corresponding BAC/PAC clones with their determined cM positions, and the segregating (χ2 test) on the F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23.

Markerz Physical informationy
 Mirror mapx
 Segregation Testw
Ch Start Stop e-PCR e % BAC/PAC cM% cM A H B M χ2
RM3252 1 299,681 299,852 172 0.7 AP002818 0.2 0.3 19 28 50 0 1.4
RM0600 1 9,461,346 9,461,566 221 21.7 AP001081 27.3 49.6 19 30 48 0 2.1
RM0449 1 15,305,619 15,305,758 140 35.1 AP008247 40.0 72.8 21 27 49 0 0.5
RM1349 1 25,398,082 25,398,300 219 58.3 AP002744 56.7 103.1 19 25 53 0 1.3
RM1003 1 33,803,800 33,803,927 128 77.5 AP003345 75.1 136.6 20 29 48 0 1.3
RM6321 1 43,251,019 43,251,186 168 99.2 AP003277 100.0 181.8 31 18 48 0 3.0
RM0154 2 1,803,895 1,084,077 183 3.0 AP005851 3.0 4.7 19 29 49 0 1.7
RM5699 2 8,981,409 8,981,575 167 25.0 AP005803 26.7 42.1 20 26 50 1 0.7
RM1211 2 18,450,427 18,450,588 162 51.4 AP005299 37.7 59.5 22 28 47 0 0.6
RM3220 2 28,458,108 28,458,248 141 79.2 AP004118 74.8 118.1 25 22 50 0 0.1
RM3220 2 35,425,798 35,425,907 110 98.6 AP003989 100.0 157.9 25 25 47 0 0.0
RM4108 3 514,862 515,003 142 1.4 AC113930 0.7 1.1 23 30 44 0 1.5
RM5628 3 3,101,122 3,101,282 161 8.5 AC099401 9.0 14.8 19 35 43 0 5.8
RM0007 3 9,808,540 9,808,710 171 27.0 AC134232 27.0 44.4 15 40 42 0 13.5 ***
RM6266 3 23,778,063 23,778,222 160 65.4 AC091246 57.7 94.9 30 22 45 0 1.4
RM3525 3 30,344,279 30,344,457 179 83.5 AC117988 83.0 136.5 24 38 35 0 10.7 ***
RM3585 3 36,080,616 36,080,784 169 99.3 AC128647 98.4 161.7 18 28 51 0 1.9
RM8213 4 4,433,178 4,433,388 211 12.6 AL662959 8.7 11.3 22 26 49 0 0.2
RM5633 4 13,059,370 13,059,580 211 37.1 AL731595 15.4 19.9 26 28 43 0 1.0
RM1155 4 20,328,759 20,328,906 148 57.7 AL606453 45.4 58.9 29 30 38 0 3.9
RM3839 4 23,870,755 23,870,972 218 67.7 AL606614 56.2 72.8 32 30 35 0 6.8 *
RM3217 4 30,083,469 30,083,662 194 85.4 AL606683 77.7 100.7 30 20 45 2 1.9
RM0559 4 35,117,645 35,117,804 160 99.6 AL606637 100.0 129.6 29 14 54 0 5.2
RM5693 5 441,872 442,071 200 1.5 AC129716 3.8 4.6 24 24 49 0 0.0
RM5844 5 9,128,802 9,128,996 195 30.6 AC120989 43.7 53.5 34 14 49 0 7.4 *
RM0249 5 10,755,867 10,755,994 128 36.0 AC136522 44.4 54.3 34 14 49 0 7.4 *
RM3870 5 22,879,699 22,879,891 193 76.6 AC108503 77.9 95.3 24 21 52 0 0.5
RM1054 5 29,144,035 29,144,184 150 97.6 AC098598 99.8 122 19 31 47 0 2.6
RM3353 6 435,582 435,697 116 1.4 AP001129 1.1 1.4 23 25 49 0 0.0
RM0253 6 5,425,498 5,425,631 134 17.4 AP004754 14.0 17.4 13 32 52 0 7.1 *
RM0276 6 6,230,046 6,230,169 124 19.9 AP003488 26.9 33.5 13 33 51 0 7.7 *
RM6818 6 16,581,413 16,581,542 130 53.1 AP004012 52.9 65.8 18 26 53 0 1.8
RM3628 6 23,737,032 23,737,157 126 76.0 AP003612 68.6 85.4 19 21 57 0 2.6
RM5753 6 30,966,850 30,967,050 201 99.1 AP004685 100 124.4 27 23 47 0 0.3
RM1093 7 668,161 668,310 150 2.3 AP003746 2.1 2.5 25 22 50 0 0.1
RM1377 7 12,782,829 12,783,009 181 43.1 AP004305 41.9 49.7 15 38 44 0 10.7 ***
RM5793 7 17,488,937 17,489,063 127 58.9 AP004299 51.3 60.8 10 40 47 0 17.4 ***
RM0172 7 29,560,592 29,560,750 159 99.6 AP005199 100 118.6 22 24 51 0 0.2
RM0408 8 119,935 120,063 129 0.4 AP005406 0.4 0.5 21 36 40 0 6.9 *
RM0547 8 5,586,058 5,586,291 234 19.7 AP004746 33.2 40.2 17 24 56 0 2.9
RM3262 8 22,248,334 22,248,500 167 78.6 AP005483 71.5 86.7 17 27 53 0 2.4
RM5545 8 28,141,927 28,142,083 157 99.4 AP004623 100 121.2 22 27 48 0 0.3
RM23654 9 151,453 151,639 187 0.7 AP006059 0.0 0.0 24 24 47 2 0.0
RM0316 9 1,074,933 1,075,126 194 4.7 AP005860 0.9 0.8 25 25 47 0 0.0
RM0219 9 7,887,585 NA 34.3 AP005912 22.1 20.7 20 20 50 0 0.8
RM0566 9 14,704,798 14,705,036 239 63.9 AP005397 54.2 50.7 17 17 53 0 2.4
RM0205 9 22,720,646 22,720,801 156 98.7 AP005546 100 93.5 20 20 54 0 1.1
RM7492 10 39,037 39,181 145 0.2 AC166065 0.0 0.0 29 29 52 0 3.4
RM5689 10 13,223,351 13,223,453 103 57.8 AC021891 31.1 26.1 25 26 46 0 0.1
RM1375 10 16,386,764 16,386,943 180 71.6 AC025905 51.0 42.7 28 23 46 0 0.5
RM0590 10 22,784,993 22,785,130 138 99.6 AC018727 100 83.8 26 16 55 0 3.3
RM6901 11 1,494,196 NA 5.2 AC123516 5.5 6.5 23 19 55 0 1.7
RM0536 11 8,968,470 8,968,712 243 31.5 AC138197 41.6 49.1 19 28 50 0 1.4
RM3428 11 13,445,211 13,445,367 157 47.2 AC128642 47.9 56.5 18 30 49 0 2.5
RM5349 11 19,148,807 19,148,929 123 67.3 AC134925 67.1 79.1 21 28 48 0 0.7
RM0144 11 28,246,930 28,247,154 225 99.2 AC134045 98.6 116.2 26 22 49 0 0.2
RM3472 12 3,520,117 3,520,331 215 12.8 AL713902 24.8 27.1 23 29 45 0 1.0
RM1337 12 11,933,319 11,933,500 182 43.4 BX000556 46.2 50.4 22 28 47 0 0.6
RM0277 12 18,290,458 NA 66.5 AL831799 57.0 62.2 25 27 45 0 0.4
RM0017 12 26,954,668 26,954,835 168 98.0 AC027133 98.4 107.4 30 22 45 0 1.4

The primer sequences were used as the query to localize on Rice Pseudomolecule 6

When both primer sequences successfully recognized their physical locations to be annealed, the expected PCR product size (e-PCR:‘Stop’-‘Start’+1bp, NA=not available) was used to judge PCR products. If any primer failed for e-Landing, the other primer's result was adopted to estimate the corresponding point. The determined physical regions followed by e-Landing were used to match corresponding BAC/PAC clones depend on the ‘Rice Pseudomolcule Information and Gene Search (http://rice.plantbiology.msu.edu/)

The determined cM positions for each BAC/PAC clones was directly adopted from the ‘Rice Pseudomolcule Information and Gene Search’ which were estimated using the marker sequences obtained from the Cornell Rice Genes Database and the Japanese Rice Genome Program. To take some convenient in comparing the cM positions from various mapping populations having different total lengths, the percentage expressed relative genetic positions, ‘cM%’, was also calculated

A and B are homozygous for the Namil(EMS)-bl10,bph1 and Milyang23 allele types, H is for heterozygous progenies at the tested locus, and M indicates the number of missing. Segregation distorted loci are indicated with asterisk; χ2 values for the significant levels are less than 0.05

significant at P < 0.0001

significant at P < 0.01

significant at P < 0.05

Summary of association analysis between DNA marker genotypes and phenotype performances of F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23z for rice blast resistance and brown planthopper resistance.

Trait Evaluated Mirror Mapy
 No. of Genotypesx
 Mean of Genotypew
 Single-locus ANOVAv
 Genetic Effectu
Locus Ch cM cM% A H B A H B SSM SSE F-value R2 Add Dom DeD
BL (Degree: 0-5) Pop. No.=97 Pop. Mean=3.2 RM3850 2 158.0 100 25 47 25 3.0 3.0 3.7 10.2 138.4 3.5 * 0.068 0.37 -0.38 -1.0
RM1377 7 49.7 41.9 15 44 38 2.4 3.4 3.3 10.7 137.8 3.6 * 0.072 0.41 0.55 1.3
RM23654 9 0.0 0.0 24 47 24 3.0 3.0 3.8 11.8 134.5 4.0 * 0.081 0.43 -0.36 -0.9
RM0316 9 0.8 0.9 25 47 25 2.9 3.0 3.8 11.2 137.3 3.8 * 0.076 0.42 -0.32 -0.8
RM3472 12 27.1 24.8 23 45 29 2.7 3.1 3.7 15.4 133.1 5.4 ** 0.104 0.54 -0.11 -0.2
RM1337 12 50.4 46.2 22 47 28 1.9 3.1 4.4 80.9 67.7 56.0 **** 0.544 1.27 -0.05 0.0
RM0277 12 62.2 57.0 25 45 27 2.1 3.2 4.3 63.4 85.1 35.0 **** 0.427 1.1 -0.01 0.0
Bph (Ratio: %) Pop. No.=97 Pop. Mean=63.9 RM0547 8 40.2 33.2 17 56 24 39.6 70.2 66.6 12392 116980 5.0 ** 0.096 13.47 17.08 1.3
RM3262 8 86.7 71.5 17 53 27 63.8 56.6 78.4 8576 120796 3.3 * 0.066 7.34 -14.6 -2.0
RM3472 12 27.1 24.8 23 45 29 84.0 61.1 52.4 13541 115831 5.5 ** 0.105 -15.8 -7.13 0.5
RM1337 12 50.4 46.2 22 47 28 90.6 69.4 33.8 42504 86868 23.0 **** 0.329 -28.4 7.22 -0.3
RM0277 12 62.2 57.0 25 45 27 92.5 73.1 22.2 71258 58114 58.0 **** 0.551 -35.2 15.73 -0.4
RM0017 12 107.0 98.4 30 45 22 77.1 68.1 37.3 21651 107720 9.4 *** 0.167 -19.9 10.93 -0.5

Only significant loci to each trait tested are presented. 60 DNA markers were tested in 97 F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23.

Both cM positions and the percentage expressed relative genetic positions (cM%) are presented

F2 progenies were split into three subgroups based on the revealed genotype by SSR markers. A and B are homozygous for the Namil(EMS)-bl10,bph1 and Milyang23 allele types and H indicates heterozygous progenies at the tested locus.

The means of each subpopulation for each locus are calculated.

For the F-test, SSR markers having less than significant levels (p) were declared as significant empirically. Explainable phenotypic variation portion at the tested locus (R2) was also calculated. The significant levels are less than 0.05

significant at P < 0.0001

significant at P < 0.001

significant at P < 0.01

significant at P < 0.05

uAdditive effect (Add), domiant effect (Do) and degree of dominance (DeD) were then estimated at the declared loci: Add=(Bmean- Amean)/2, Do= Hmean-(Bmean+Amean)/2, and DeD=Do/Add. where A and B are homozygous F2 individuals for Namil(EMS)-bl10,bph1 and Milyang23, H is heterozygous individuals at the tested locus.

Table 1. Comparisons between Namil, the wild type and Namil(EMS)-bl10,bph1, a mutant line in terms of important agronomic traits.z

Heading date (HD), Culm length (CL), Panicle length (PL), Tiller number (TN), Spikelet number (SN), Ripened grain percentage (RGP), 1,000-grain weight of hulled grain (TWD), and Yield of brown rice (YDB). Note that the agronomic traits were evaluated by harvesting 100 plant for each replication plot (n=3), which were planted as three seedlings per plant during transplanting.

Table 2. Reactions of the mutant line, Namil(EMS)-bl10,bph1 and cultivars against selected Korean disease and insect pest.

Korean rice cultivars, Junam, Onuri, and Chilbo were included as a check varieties on virulent rice blast isolates

Rice blast isolates were collected from Korean famers’ fields. Resistance degrees of virulent rice blast were classified five levels from 1 (resistance) to 5 (susceptibility)

Korean rice cultivar, Taebaek, Hwasam, and Samkang were included as a check variety on brown planthopper

Resistance degrees of brown planthopper were classified into three levels susceptibility as susceptibility (S), moderate (M), and resistance (R)

Table 3. List of SSR primer sets, their e-Landing mediated physical positions on rice Pseudomolecule 6, corresponding BAC/PAC clones with their determined cM positions, and the segregating (χ2 test) on the F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23.

The primer sequences were used as the query to localize on Rice Pseudomolecule 6

When both primer sequences successfully recognized their physical locations to be annealed, the expected PCR product size (e-PCR:‘Stop’-‘Start’+1bp, NA=not available) was used to judge PCR products. If any primer failed for e-Landing, the other primer's result was adopted to estimate the corresponding point. The determined physical regions followed by e-Landing were used to match corresponding BAC/PAC clones depend on the ‘Rice Pseudomolcule Information and Gene Search (http://rice.plantbiology.msu.edu/)

The determined cM positions for each BAC/PAC clones was directly adopted from the ‘Rice Pseudomolcule Information and Gene Search’ which were estimated using the marker sequences obtained from the Cornell Rice Genes Database and the Japanese Rice Genome Program. To take some convenient in comparing the cM positions from various mapping populations having different total lengths, the percentage expressed relative genetic positions, ‘cM%’, was also calculated

A and B are homozygous for the Namil(EMS)-bl10,bph1 and Milyang23 allele types, H is for heterozygous progenies at the tested locus, and M indicates the number of missing. Segregation distorted loci are indicated with asterisk; χ2 values for the significant levels are less than 0.05

significant at P < 0.0001

significant at P < 0.01

significant at P < 0.05

Table 4. Summary of association analysis between DNA marker genotypes and phenotype performances of F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23z for rice blast resistance and brown planthopper resistance.

Only significant loci to each trait tested are presented. 60 DNA markers were tested in 97 F2 progenies derived from the cross between Namil(EMS)-bl10,bph1 and Milyang23.

Both cM positions and the percentage expressed relative genetic positions (cM%) are presented

F2 progenies were split into three subgroups based on the revealed genotype by SSR markers. A and B are homozygous for the Namil(EMS)-bl10,bph1 and Milyang23 allele types and H indicates heterozygous progenies at the tested locus.

The means of each subpopulation for each locus are calculated.

For the F-test, SSR markers having less than significant levels (p) were declared as significant empirically. Explainable phenotypic variation portion at the tested locus (R2) was also calculated. The significant levels are less than 0.05

significant at P < 0.0001

significant at P < 0.001

significant at P < 0.01

significant at P < 0.05

uAdditive effect (Add), domiant effect (Do) and degree of dominance (DeD) were then estimated at the declared loci: Add=(Bmean- Amean)/2, Do= Hmean-(Bmean+Amean)/2, and DeD=Do/Add. where A and B are homozygous F2 individuals for Namil(EMS)-bl10,bph1 and Milyang23, H is heterozygous individuals at the tested locus.

Table 1.

Table 2.

Table 3.

Table 4.

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