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Genetic Analysis on Short Culm and the Rice Blast Resistance of Namil(SA)-bl5, 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 12, 2014   • Accepted: September 16, 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 not only one of major crops grown in the world but also the staple food in Asia. However, in modern agriculture, the genetic diversity in rice gene pool is diminishing by selection breeding. Hence, rice is readily threatened by biotic and abiotic stresses. Rice blast is one of the major threats to stable rice production. The best approach to prevent rice blast is development of rice cultivars harboring resistance gene against blast disease. National Institute of Crop Science (NICS) in Korea developed a mutant line, Namil(SA)-bl5, which has inherited short culm and rice blast resistance by treatment of sodium azide (SA). To localize favorable mutated allele types, two mapping populations were constructed by using F2 progenies derives crosses between Namil(SA)-bl5 and Milyang23 and Namil (wild type) and Milyang23. Each F2 progenies were evaluated in terms of culm length and resistance level against a rice blast isolate, 94-254 along with their parental lines. Association analyses between marker genotype on 68 SSR anchor markers and evaluated phenotype of progeny lines were adopted to localize the putative chromosomal locations involved to culm length and blast resistance. Association analyses localized two major loci on the chromosomes of middle-low region of 6 (R2=15.8%) and the distal region of 7 (R2=20.0%), and a major locus on the middle of chromosome 12 (R2=46.6%) for the reduced culm length and increased resistance level against rice blast, respectively.
  • Keywords: Rice; Short culm; Rice blast; Sodium azide (SA); Association analysis
쌀은 인류에게 있어서 주요 곡물 중 하나이며, 특히 동남아 시아에서는 주곡작물로서 중요한 식량공급원이다. 우리나라 에서도 쌀은 주곡작물일뿐만 아니라 국내 농업소득의 70%에 달하는 농가의 주요 소득원이다(Kim, 2011). 하지만 현대 농 업에 있어서 선택육종을 통한 양질 벼 품종들은 유전적 변이 가 협소한 편이어서 유전적 다양성의 한계를 보이고 있다 (Jeung et al. 2005, Kang et al. 2007). 이러한 유전적 다양성 의 한계는 불량한 환경이나 특정 병해충의 발생과 같은 생물 학적 및 비생물학적 스트레스는 벼의 안정적 생산에 영향을 미칠 뿐만 아니라 심각한 피해를 줄 수 있다(Jena et al. 2006, Jeung et al. 2006).
심각한 병해 중 하나인 벼 도열병은 벼의 육묘단계부터 최 고분얼기까지 잎을 침해하고 출수기 이후에는 이삭목, 이삭가 지 뿐만 아니라 벼 알까지 침해하여 벼의 수량과 품질을 저해 한다(Shim et al. 2005). 또한, 벼 도열병은 수화제나 유제를 이용한 방제에도 기상조건에 따라 출수기 이후에 빠르게 증 식하기 때문에 방제효과가 낮다(Lee et al. 2006). 이와 같은 이유로 병해충 방제와 재해 예방에 있어서 가장 경제적이며 친환경적인 방법은 환경저항성 벼 품종을 육성하는 것이 가 장 효과적이라고 할 수 있다(Wang et al. 1994, Kim et al. 2011).
작물학적 특성을 신속히 보완하고 확충할 수 있는 이점을 가지고 있는 돌연변이 육종을 통해 육성된 돌연변이 계통의 유용형질에 대한 유전좌위를 파악하고 이를 분자마커로 표지 하는 작업은 원품종에 존재하는 기존의 유전자들과 돌연변이 계통에서 유래한 유전자들간의 상동성을 확인하거나, 목표형 질이 발현되는지를 확인하는데 있어서 매우 중요한 요소이다 (Jeung et al. 2011). 벼 유전체학의 발전은 저항성유전자들의 염색체상 위치와 유전기작을 파악하는데 분자마커의 활용성 을 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 조생 다수성 품종인 남일에 돌연변이원인 아지 드화나트륨(Sodium azide)을 활용하여 육성된 남일 돌연변이 계통인 Namil(SA)-bl5의 특성인 단간 및 도열병 저항성 유전 자의 유전양상을 구명하고, 유전좌위를 표지 할 수 있는 분자 마커를 확보하여 국내 자포니카 벼 품종의 작물학적 특성의 향상과 도열병 저항성 증진을 위한 육종소재를 확립하고자 수행하였다.
식물재료 및 DNA 추출
자포니카(Oryza sativa L. ssp. Japonica)의 조생 다수성 품종인 남일(Kim et al. 2003)을 아지드화나트륨(Sodium azide)을 이용하여 돌연변이 계통을 육성하였다(Shin et al. 2009). Namil(SA)-bl5는 단간이면서 도열병 저항성 돌연변이 후대계통으로써 계보는 Namil(SA)M2-1063-11-1-1-1-1-1-1 에 해당한다. 다양한 국내수집 도열병 균주에 대한 친화성 검 정을 위해서 주남, 온누리, 및 칠보가 대비품종으로 함께 공시 되었다. Namil(SA)-bl5의 단간 및 도열병 저항성 관련 유전 좌위에 대한 유전분석을 위해 Namil(SA)-bl5와 통일계 품종 인 밀양23호간의 F2 94개체를 사용하였으며, 돌연변이 계통 의 유전좌위 추정의 신뢰성을 높이기 위하여 남일과 밀양23 호간의 F2 103개체도 함께 사용하였다.
육성된 각 집단의 F2 식물체들과 모본, 부본의 DNA는 변 형된 CTAB방법을 이용하여 추출하였다(Murray & Thompson 1980). 추출된 DNA는 0.8% agarose gel에 전기영동하여 확 인하였으며, NanoDrop spectrophotometer (Thermo Fisher Scientific, USA)를 이용하여 정량 후 5 ng/ul로 희석하여 PCR에 이용하였다.
작물학적 특성 평가
Namil(SA)M2-1063-11-1-1-1-1-1-1의 계보를 지니며, Namil (SA)-bl5로 명명된 단간이며 도열병 저항성 돌연변이 계통을 원품종인 남일과 함께 2011년 국립식량과학원 답작과(수원 소재) 포장에 공시하여 작물학적 특성을 관찰하였다. 파종은 조기재배에 준하여 4월 7일 실시하여 약 30일정도 육묘한 후 5월 12일에 이앙하였다. 연관지도 작성 및 목표유전자위 표 지를 위해 공시된 두 개의 F2 집단들에 대해서는 포장에서 각 개체들을 표지 한 후 이들로부터 DNA 추출을 위한 시료를 취하였으며, 각 개체들의 간장을 측정한 후 F2:3 종자를 수확 하였다.
도열병 균주 선정 및 공시재료에 대한 친화성 판정
국내에서 수집된 도열병 균주 중 원품종인 남일과 친화성 을 발현하는 7개 균주를 선정하여, 공시재료들의 도열병 저항 성 정도를 평가하였다(Ryu et al. 1987, Han et al. 1994, Han et al. 1995, Shim et al. 2012). 도열병 균주의 접종 및 발병유도는 Kim et al. (2004)의 방법에 준하여 수행하였다. 공시재료들을 파종하여 3~4엽기까지 온실에서 육묘한 후, 포자농도가 1.5 × 105 spores/ml로 조정된 각각의 도열병 균 주를 분무접종하고, 26°C 암조건의 습윤상(상대습도 90% 이 상)에서 24시간 처리한 후 온실로 옮겨 2주간 발병을 유도하 였다. 공시재료들의 도열병 저항성 정도는 발병 정도에 따라 0~5단계로 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 중도 저항성 및 4~5는 감수성 등으로 접종된 도열병 균주에 대한 저항성과 감수성 정도를 구분하였다(Kim et al. 2011). 도열 병 저항성에 대한 유전분석을 위하여 남일과 밀양23호에 친 화성이며 Namil(SA)-bl5에는 비친화성인 도열병 균주 94-254를 사용하였다(Table 2, Fig. 1B). Namil(SA)-bl5와 밀양23호간의 F2 집단 내에서 도열병 저항성 유전자의 분리 양상을 평가하기 위해 각 F2 개체로부터 세대 진전된 F2:3 계 통의 저항성 수준을 평가하였다. 각 F2 계통 당 임의로 선택 된 15여립의 F2:3 종자를 전개하여 도열병 균주, 94-254를 접 종하였다. 계통 내 모든 식물체들의 병반응 정도는 접종 후 2 주일 경에 개체별로 병징을 조사하였으며, 그 평균값을 취하 여 유전분석을 위한 각 계통에 대응하는 F2 개체의 도열병 저 항성 표현형으로 사용하였다.
Table 2.
Reactions of the mutant line, Namil(SA)-bl5 and cultivars against selected Korean virulent blast isolates.
Table 2.
Lines Rice blast isolate (race)z
90-002 (KI215) 90-008 (KI1113) 93-093 (KI197) 94-254 (ND)y 97-260 (KJ103) 02-319 (KJ105) 02-039 (KJ201)
Namil 4 4 5 4 4 5 5
Milyang23 4 3 4 4 2 4 2
Junam 5 5 5 5 5 5 3
Onnuri 4 4 5 5 4 1 4
Chilbo 2 5 5 5 5 4 4
Namil(SA)-bl5 0 1 1 0 1 1 1

zRice 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 cultivars, Junam, Onuri, and Chilbo were included as check varieties

yNot determined

Fig. 1.
Agronomic performances of the wild type, Namil (left) and the mutant line of Namil, Namil(SA)-bl5 (right) (A) and reaction patterns against virulent rice blast isolate, 94-254 of Namil, Milyang23, and Namil(SA)-bl5, sequentially from left to right (B).
KJBS-46-238_F1.gif
분리후대집단을 이용한 연관지도 작성
기존에 보고된 정보에 의거하여(McCouch et al. 2002) 벼 염색체상의 균등배치에 유의하면서 돌연변이원인 남일과 밀 양23호간에 다형성을 보이는 SSR 마커를 선정하였다. 이 를 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 혹은 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 마커에 대하여 F2 식물체들로부터 조사된 유전자형의 분리비가 기대치에 부합하는지 평가하기 위하여 적합성검정 (χ2 test)을 실시하였으며(Jeung et al. 2011), 각 개체의 유전 자형과 단간 및 도열병 저항성에 대한 표현형간의 연관분석 (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 μl로 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 염색한 후 각 F2 개체 들의 유전자형을 판정하였다(Jeung et al. 2011).
도열병 저항성 유전좌위 표지를 위한 유전통계분석
단간 및 도열병 저항성 관련 유전좌위의 벼 염색체상 위치 를 파악하기 위해 남일과 밀양23호 및 Namil(SA)-bl5와 밀 양23호간의 F2 개체들로부터 확보된 SSR 마커 유전자형과 단간에 대한 표현형 및 도열병 저항성에 대한 F2:3 계통들간의 연관성을 평가하였다(association analysis).
수집된 자료들에 대한 유전통계분석에는 SAS (Statistical Analysis System) 프로그램(SAS Institute, 2000)을 이용하 였다. SAS 프로그램의 PROC MEANS를 이용하여 기술통계 량을 산출하였고, 목표 유전좌위 표지를 위해 육성된 F2 집단 에 대한 연관분석(association analysis)은 PROC GLM을 이 용하여 각 SSR 마커들에 의해 표지되는 유전좌위가 단간 및 저항성에 관여되는 정도를 판정하였다(Single Locus ANOVA). 각 유전좌위에 대한 F 분석 결과 P 값이 0.05보다 작으면 유 의하다고 판정하고, F2 분리집단에서 관찰된 전체 단간 및 도 열병저항성 분산에 대한 해당 유전좌위의 기여도(R2 value; %)와 유전적 작용가(additive effect) 및 기작양상(degree of dominance)등을 추정하였다(Jeung et al. 2007).
남일과 돌연변이 계통 Namil(SA)-bl5의 작물학적 특성 평가
단간이며 도열병 저항성 계통인 Namil(SA)-bl5와 원품종 인 남일을 함께 공시하여 작물학적 특성을 평가하였다(Table 1, Fig. 1A). 돌연변이 계통인 Namil(SA)-bl5은 조생종인 원 품종(남일)에 비해 빠른 출수기를 보였으며, 간장 또한 남일 에 비해 평균 10 cm정도 단축된 단간형이었다. 이삭의 길이 와 수당립수는 원품종인 남일에 비해 감소하였으나 수량성은 현미를 기준으로 크게 감소하지는 않았다. 이는 Namil(SA)-bl5에서 수수가 유의하게 증가한 것에서 기인하는 것으로 판 단된다.
Table 1.
Comparisons between Namil, the wild type and Namil(SA)-bl5, 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 ± 0.6 11 ± 1.0 147 ± 7.8 74.4 ± 7.1 24.8 ± 0.0 599 ± 24.0
Namil(SA)-bl5 7.25 ± 0.6 65 ± 1.7 19 ± 0.0 17 ± 2.1 95 ± 0.6 74.2 ± 7.5 20.5 ± 0.2 578 ± 21.4

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. All numerical values were mean ± standard deviation.

단간 특성을 보이는 Namil(SA)-bl5의 유전분석을 위한 F2 후대집단간의 작물학적 평가에서 간장은 Namil(SA)-bl5 x 밀양23호간의 분리후대집단에서 평균 72.5 cm였으며 남일과 밀양23호간의 분리후대집단에서는 평균 84.7로 Namil(SA)-bl5에 의해 간장이 단축되었다. 두 집단의 분산분포를 비교한 결과, 유사한 분산분포를 보였으나 Namil(SA)-bl5 × 밀양23 호간의 분리후대집단에서 다소 Namil(SA)-bl5쪽으로 편기되 는 양상을 보였다(Fig. 2A).
Fig. 2.
Graphical comparisons of F2 mapping populations derived from two crosses between Namil (wild type) or Namil(SA)-bl5 and Milyang23 on the agronomic trait, culm length (A) and virulent rice blast isolate, 94-254 (B).
To measure the agronomic trait, culm length of F2 progeny lines, harvested seeds from each F2 plant (F2:3 seeds) were planted in a raw. The descriptive statistics on the agronomic trait, culm length of 103 F2:3 lines of Namil x Milyang23, over-all mean, standard deviation, skewness, and kurtosis are 84.7, 7.4, 0.0, and -0.7, respectively. In 94 F2:3 lines of Namil(SA)-bl5 x Milyang23 on the agronomic trait, culm length, over-all mean, standard deviation, skewness, and kurtosis are 72.5, 6.7, 0.0, and -0.5, respectively. To estimate the resistant levels of F2 progeny lines, harvested seeds from each F2 plant (F2:3 seeds) were planted in a raw. Fourteen days after inoculation of rice blast isolate (94-254), individuals were evaluated. On virulent rice blast, 94-254, the descriptive statistics 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 94 F2:3 lines of Namil(SA)-bl5 x Milyang23 on virulent rice blast isolate, 94-254, over-all mean, standard deviation, skewness, and kurtosis are 2.3, 1.5, 0.2, and -1.2, respectively.
KJBS-46-238_F2.gif
Namil(SA)-bl5의 도열병 저항성 평가 및 검정 균주 선정
균주 선정에 있어서 원품종인 남일과 교배친인 밀양23호를 모두 침해하는 균주를 선정하기 위하여 국내수집 균주 7개를 접종하여 이병반응을 검정하였다(Table 2). 도열병 저항성 검 정에서 남일은 7개 균주 모두에서 감수성을 보였으며, 밀양23 호는 4개 균주에 대해 감수성 반응을 3개 균주에 대해서는 중 도저항성을 보였다. 근래에는 통일계 품종들이 거의 재배되지 않기 때문에 도열병 균주들의 밀양23호에 대한 친화성이 자 포니카계인 남일에 비해 낮은 것으로 사료된다. 대비품종들인 주남, 온누리, 및 칠보의 경우, 균주들에 대하여 서로 다른 이 병성을 보였다.
반면에 Namil(SA)-bl5는 선정된 7개 균주들에 대해서는 매우 안정적인 저항성을 보였다. 대비품종들 중 주남은 Pib, 교배친인 밀양23호는 Pib, Pita 및 Pik-p 등의 도열병 저항성 유전자를 보유하는 것으로 알려져 있다(Cho et al. 2007, Jeung et al. 2007). 도열병 저항성 검정결과로 보아, 도열병 에 대해 저항성 돌연변이 계통인 Namil(SA)-bl5는 대비품종 들이 보유하고 있는 저항성 유전자들과는 다른 유전자의 작 용에 의해 도열병 저항성이 발현되는 것으로 추정된다.
균주 검정을 통해 얻어진 결과를 바탕으로 도열병 저항성 이 분리되는 교잡후대에서 변별력 높은 표현형 결과를 확보 하기 위해 Namil(SA)-bl5는 비친화성이며 원품종인 남일과 교배친인 밀양23호에 친화성인 도열병 균주 94-254를 선정 하여 유전분석에 이용하였다.
Namil(SA)-bl5 x 밀양23호간 후대 F2 집단의 도열병 저항성 평가
연관분석(association analysis)을 위한 도열병 저항성 검정 에서 분리후대집단의 표현형 조사를 위해 남일과 밀양23호간 103개 F2 집단과 Namil(SA)-bl5와 밀양23호간의 94개 F2 집 단에서 각 F2 계통당 15립정도의 F2:3 종자를 전개하여 도열 병 균주 94-254를 접종하여 병반응 정도에 따라 집단 내에서 의 표현형 분리양상을 조사하였다(Fig. 2B). 도열병 저항성 검정에서 원품종인 남일과 교배친인 밀양23호는 각각 저항성 이 4.3과 3.7로 낮게 평가되었다. 반면에 Namil(SA)-bl5에서 는 0.3으로 저항성이 높게 평가되었으며 강한 저항성을 보였 다. 각각의 분리후대집단에서는 남일과 밀양23호간 집단에서 는 평균 4.0으로 낮게 평가되었으며, Namil(SA)-bl5와 밀양 23호간 집단에서는 평균 2.3으로 남일과 밀양23호간 집단에 비하여 높게 평가되었다.
도열병 저항성에 대한 두 분리후대집단의 분산양상을 비교 한 결과, 남일과 밀양23호간 집단은 오른쪽(감수성)으로 크게 편향된 양상(skewness=-0.8)을 보이는 반면, Namil(SA)-bl5 와 밀양23호간 분리후대집단은 왼쪽(저항성)으로 약간 편향 되어(skewness=0.2) 도열병 저항성에 관여하는 유전인자가 Namil(SA)-bl5에 존재하고 있음을 확인할 수 있었다.
Namil(SA)-bl5와 밀양23호간 후대 F2 집단을 이용한 연관 지도 작성
단간이며 도열병 저항성 돌연변이 계통 Namil(SA)-bl5와 밀양23호간 교잡을 통해 총 94개의 F2 식물체로 구성된 유전 분석 집단을 구축하였다. 각 F2 개체에서 DNA를 추출하여 SSR 마커를 이용한 연관지도를 작성하였다. 연관지도 작성에 있어서는 각 염색체마다 최소한의 수의 SSR 마커를 균등하게 배치하여 연관지도 초안(linkage map skeleton)을 작성하였다. 각각의 염색체상에 SSR 마커를 배치하기 위하여 e-Landing기 법에 의해 각 SSR 마커에 대응하는 벼 유전체정보를 파악하고 mirror map을 구축함으로써 분리집단의 유전자형에 대한 부가 적인 유전통계분석 절차를 생략하였다. 연관지도 초안을 위해 활용된 유전자형에 대한 분리비 검정을 수행하여(segregation test), 각 F2 집단에서 분리비 이상(segregation distortion)이 일어나고 있는 부위를 확인하였다(Table 3).
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(SA)-bl5 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 53 22 0 1.4
RM0600 1 9,461,346 9,461,566 221 21.7 AP001081 27.3 49.6 21 49 24 0 0.2
RM0449 1 15,305,619 15,305,758 140 35.1 AP008247 40.0 72.8 29 44 21 0 1.4
RM1349 1 25,398,082 25,398,300 219 58.3 AP002744 56.7 103.1 24 46 24 0 0.0
RM1003 1 33,803,800 33,803,927 128 77.5 AP003345 75.1 136.6 28 42 24 0 1.1
RM6321 1 43,251,019 43,251,186 168 99.2 AP003277 100.0 181.8 26 42 26 0 0.8
RM0154 2 1,083,895 1,084,077 183 3.0 AP005851 3.0 4.7 16 62 16 0 8.6 *
RM5699 2 8,981,409 8,981,575 167 25.0 AP005803 26.7 42.1 19 53 22 0 1.4
RM1211 2 18,450,427 18,450,588 162 51.4 AP005299 37.7 59.5 19 55 20 0 2.3
RM3220 2 28,458,108 28,458,248 141 79.2 AP004118 74.8 118.1 13 52 29 0 5.8
RM3850 2 35,425,798 35,425,907 110 98.6 AP003989 100.0 157.9 13 55 26 0 5.6
RM6301 3 2,631,471 2,631,613 143 7.2 AC090485 8.1 13.3 25 48 21 0 0.2
RM0007 3 9,808,540 9,808,710 171 27.0 AC134232 27.0 44.4 15 47 31 1 4.8
RM1164 3 14,840,558 14,840,757 200 40.8 AC084766 41.8 68.7 21 44 29 0 1.4
RM6266 3 23,778,063 23,778,222 160 65.4 AC091246 57.7 94.9 24 45 25 0 0.1
RM1350 3 28,632,514 28,632,682 169 78.8 AC087181 77.1 126.8 28 44 22 0 0.9
RM3585 3 36,080,616 36,080,784 169 99.3 AC128647 98.4 161.7 27 42 25 0 0.9
RM0551 4 168.620 168,811 192 0.5 AL606442 2.4 3.1 22 48 24 0 0.0
RM5633 4 13,059,370 13,059,580 211 37.1 AL731595 15.4 19.9 13 54 27 0 5.5
RM1155 4 20,328,759 20,328,906 148 57.7 AL606453 45.4 58.9 24 35 35 0 8.0 *
RM3839 4 23,870,755 23,870,972 218 67.7 AL606614 56.2 72.8 19 38 37 0 9.4 **
RM3217 4 30,083,469 30,083,662 194 85.4 AL606683 77.7 100.7 23 47 18 6 0.7
RM0559 4 35,117,645 35,117,804 160 99.6 AL606637 100.0 129.6 22 56 16 0 3.7
RM5693 5 441,872 442,071 200 1.5 AC129716 3.8 4.6 26 51 17 0 2.0
RM5844 5 9,128,802 9,128,996 195 30.6 AC120989 43.7 53.5 27 46 18 3 1.4
RM5558 5 21,168,727 21,168,899 173 70.9 AC105769 70.3 86.0 22 48 24 0 0.0
RM1054 5 29,144,035 29,144,184 150 97.6 AC98598 99.8 122.0 25 44 25 0 0.2
RM3353 6 435,582 435,697 116 1.4 AP001129 1.1 1.4 18 50 26 0 1.4
RM3370 6 6,434,836 6,435,012 177 20.6 AP005545 27.6 34.3 18 49 27 0 1.5
RM0539 6 8,170,600 NA 26.1 AP005619 41.0 51.3 15 49 26 4 2.9
RM0527 6 9,863,290 9,863,522 233 31.6 AP005695 45.3 56.3 16 50 26 2 2.4
RM6818 6 16,581,413 16,581,542 130 53.1 AP004012 52.9 65.8 18 51 25 0 1.4
RM3628 6 23,737,032 23,737,157 126 76.0 AP003612 68.6 85.4 16 51 27 0 2.7
RM6274 6 26,319,462 26,319,530 69 84.2 AP003568 80.6 100.3 23 40 29 2 2.0
RM5604 6 29,048,075 29,048,152 78 93.0 AP003772 92.0 113.4 22 46 26 0 0.2
RM5753 6 30,966,850 30,967,050 201 99.1 AP004685 100.0 124.4 18 52 23 1 1.5
RM1093 7 668,161 668,310 150 2.3 AP003746 2.1 2.5 22 53 19 0 1.4
RM0481 7 2,876,165 2,876,333 169 9.7 AP005632 14.8 11.0 20 53 20 1 1.4
RM1243 7 3,555,641 3,555,797 157 12.0 AP004267 21.4 25.4 21 55 17 1 2.9
RM1377 7 12,782,829 12,783,009 181 43.1 AP004305 41.9 49.7 33 40 21 0 4.5
RM3743 7 19,342,334 19,342,513 180 65.2 AP003815 56.5 67.0 30 44 20 0 2.0
RM5623 7 23,110,498 23,110,681 184 77.8 AP005908 68.4 81.1 30 46 17 1 3.1
RM3555 7 27,891,580 27,891,733 154 93.9 AP004378 89.1 105.7 25 45 21 3 0.2
RM0172 7 29,560,592 29,560,750 159 99.6 AP005199 100.0 118.6 23 45 26 0 0.2
RM0408 8 119,935 120,063 129 0.4 AP005406 0.4 0.5 24 50 20 0 0.5
RM0547 8 5,586,058 5,586,291 234 19.7 AP004746 33.2 40.2 20 51 23 0 0.6
RM3262 8 22,248,334 22,248,500 167 78.6 AP005483 71.5 86.7 24 51 19 0 0.9
RM5545 8 28,141,927 28,142,083 157 99.4 AP004623 100 121.2 19 48 27 0 1.1
RM23654 9 151,453 151,639 187 0.7 AP006059 0.0 0.0 28 55 11 0 8.0 *
RM0316 9 1,074,933 1,075,126 194 4.7 AP005860 0.9 0.8 28 55 11 0 8.0 *
RM0219 9 7,887,585 NA 34.3 AP005912 22.1 20.7 27 53 14 0 4.5
RM0566 9 14,704,798 14,705,036 239 63.9 AP005397 54.2 50.7 24 48 22 0 0.0
RM0205 9 22,720,646 22,720,801 156 98.7 AP005546 100.0 93.5 20 44 30 0 2.0
RM7492 10 39,037 39,181 145 0.2 AC166065 0.0 0.0 21 55 18 0 2.4
RM0311 10 9,487,243 9,487,406 164 41.5 AC090482 20.0 16.8 22 50 22 0 0.2
RM1375 10 16,386,764 16,386,943 180 71.6 AC025905 51.0 42.7 27 44 23 0 0.5
RM0590 10 22,784,993 22,785,130 138 99.6 AC018727 100.0 83.8 16 56 22 0 3.7
RM0286 11 383,839 383,960 122 1.3 BX000497 1.2 1.4 15 54 24 1 3.6
RM6901 11 1,494,196 NA 5.2 AC123516 5.5 6.5 14 47 27 6 3.6
RM3428 11 13,445,211 13,445,367 157 47.2 AC128642 47.7 56.2 15 46 33 0 6.2 *
RM5349 11 19,148,807 19,148,929 123 67.3 AC134925 67.1 79.1 21 42 30 1 2.2
RM0144 11 28,246,930 28,247,154 225 99.2 AC134045 98.6 116.2 20 48 26 0 0.6
RM8215 12 1,585,781 1,586,001 221 5.8 BX000498 8.9 9.7 10 37 47 0 31.6 ***
RM3472 12 3,520,117 3,520,331 215 12.8 AL713902 24.8 27.1 15 31 48 0 32.3 ***
RM1337 12 11,935,984 11,936,165 182 43.4 BX000556 47.2 51.5 17 34 42 1 18.8 ***
RM0277 12 18,290,458 NA 66.5 AL831799 57.0 62.2 14 39 41 0 16.9 ***
RM1300 12 25,998,975 25,999,139 165 94.4 AL713940 93.4 100.9 8 42 44 0 27.0 ***
RM0017 12 26,954,668 26,954,835 168 98.0 AC027133 98.4 107.4 7 44 43 0 26.4 ***

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(SA)-bl5 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.

단간이며 도열병 저항성 돌연변이 계통인 Namil(SA)-bl5 와 밀양23호간에 다형성을 보이며 염색체상에 균등하게 위치 한 총 68개의 SSR 마커들을 이용하여 연관지도 초안을 작성 하였다(Table 3). 유전자형의 분리비를 검정한 결과, Namil (SA)-bl5와 밀양23호 F2 집단에서는 밀양23호의 대립인자에 대해 호모인 개체들의 빈도가 높은 데서 기인하는 염색체 12 번 전반에서 분리비이상(segregation distortion)이 매우 유의 하게 관찰되었다(Table 3).
연관성 평가에 의한 단간 및 도열병 저항성 유전좌위 표지
연관지도 초안에 배치된 총 68개 SSR 마커들에 대응하는 단간 및 도열병 저항성에 대한 F2 개체들의 유전자형과 F2:3 계통의 단간에 대한 표현형 및 도열병 검정을 통해 확보된 표 현형간의 연관분석(association analysis)을 수행하였다. 각 SSR 마커에 대해 94개 F2 개체들을 3개의 유전자형 군집으 로 나눈 후(No. of genotype), 각 군집의 표현형 또는 저항 정 도의 평균(genotype mean)에 대해 분산분석(Single-locus ANOVA)을 실시하였다(Table 4). 분산분석 결과 F 값이 유 의한 경우 해당 유전좌위 근접부위에 표현형 변이에 영향을 끼치는 유전자가 존재하는 것으로 판단하고 R2 값(전체 표현 형 분산에서 해당 유전좌위에 대한 기여도)을 제시하고 유전 적 작용력(Genetic effect)을 추정하였다. 연관지도 초안에 배 치된 SSR 마커들 중 연관분석을 통하여 단간 및 도열병 저항 성에 관여하는 통계적으로 유의한 F 값을 지니는 마커들의 유전좌위를 표지하였다(Fig. 3).
Table 4.
Summary of association analysis between DNA marker genotypes and phenotype performances of F2 progenies derived from the cross between Namil(SA)-bl5 and Milyang23z for culm length (CL) and rice blast resistance (BL).
Table 4.
Trait Evaluated Mirror Mapy
 No. of Genotypesx
 Genotype meanw
 Single-locus ANOVAv
 Genetic Effectu
Locus Ch cM cM% A H B A H B SSM SSE F-value R2 Add Dom DeD
CL (cm) N=94 Mean=72.6 RM1003 1 136.6 77.5 28 42 24 73.2 74.5 68.4 576.9 3640.4 7.21 ** 0.137 -2.38 3.68 -1.55
RM0154 2 4.7 2.2 16 62 16 77.8 71.2 72.4 554.7 3662.6 6.89 ** 0.132 -2.69 -3.92 1.46
RM0007 3 44.4 27.0 15 47 31 74.3 73.6 70.1 279.4 3935.5 3.19 * 0.066 -2.07 1.42 -0.69
RM1350 3 126.8 78.8 28 44 22 70.9 72.0 75.8 327.5 3889.9 3.83 * 0.078 2.46 -1.40 -0.57
RM0551 4 3.1 0.5 22 48 24 75.1 72.5 70.2 278.8 3938.5 3.22 * 0.066 -2.46 -0.15 0.06
RM5633 4 19.9 37.1 13 54 27 73.5 73.7 69.8 289.8 3927.5 3.36 * 0.069 -1.88 2.03 -1.08
RM3628 6 85.4 76.0 16 51 27 68.1 73.1 74.1 392.4 3824.9 4.67 * 0.093 2.97 2.02 0.68
RM6274 6 100.3 84.2 23 40 29 68.6 72.6 75.8 663.7 3544.6 8.33 *** 0.158 3.60 0.44 0.12
RM1093 7 2.5 2.3 22 53 19 69.2 73.5 73.7 325.1 3892.2 3.80 * 0.077 2.28 2.05 0.90
RM5623 7 81.1 77.8 30 46 17 69.5 73.9 74.4 426.9 3788.0 5.07 ** 0.101 2.47 1.95 0.79
RM0172 7 118.6 99.6 23 45 26 71.3 71.1 76.2 489.5 3727.9 5.97 ** 0.116 2.48 -2.68 -1.08
RM3555 7 105.7 93.9 25 45 21 71.1 70.8 78.1 841.6 3366.3 11.00 *** 0.200 3.53 -3.77 -1.07
BL (Degree: 0-5) N=86 Mean=2.3 RM1054 5 122.0 97.6 22 42 22 2.8 1.9 2.5 14.1 172.2 3.40 * 0.076 -0.12 -0.79 6.71
RM3370 6 34.3 21.2 18 41 27 1.5 2.3 2.7 17.7 168.6 4.35 * 0.095 0.64 0.21 0.33
RM0539 6 51.0 26.2 15 42 25 1.3 2.2 3.0 25.2 152.1 6.55 ** 0.142 0.82 0.09 0.12
RM6818 6 65.8 .53.1 18 45 23 1.4 2.2 3.1 27.3 159.0 7.13 ** 0.147 0.82 -0.03 -0.03
RM0527 6 56.3 31.6 16 44 24 1.4 2.2 3.1 31.3 149.2 8.50 *** 0.173 0.89 -0.06 -0.06
RM8215 12 9.7 5.8 9 34 43 1.3 2.1 2.6 13.9 172.3 3.36 * 0.075 0.64 0.14 0.21
RM3472 12 27.1 12.8 14 28 44 1.1 2.1 2.8 29.8 156.5 7.91 *** 0.160 0.81 0.11 0.13
RM0277 12 62.2 66.5 13 36 37 1.1 2.1 2.9 31.2 155.1 8.35 *** 0.168 0.87 0.09 0.11
RM1337 12 51.5 43.4 15 32 38 0.5 2.0 3.2 85.3 97.8 35.74 *** 0.466 1.37 0.12 0.09

zOnly significant loci to each trait tested are presented. 58 DNA markers were tested in 94 F2 progenies derived from the cross between Namil(SA)-bl5 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(SA)-bl5 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<0.001

**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(SA)-bl5 and Milyang23, H is heterozygous individuals at the tested locus.

Fig. 3.
Linkage map skeleton of the agronomic trait, culm length and rice blast resistance on rice chromosomes. In Namil x Milyang23, wild type, preliminary gene locus concerned with culm length is on rice chromosome 1, and putative gene loci of rice blast resistance are on rice chromosome 5 and 11 (A). In Namil(SA)-bl5, a mutant line, preliminary gene loci concerned with culm length are on rice chromosome 6 and 7, and putative gene loci of rice blast resistance are on the same rice chromosome 6 and 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-46-238_F3.gif
단간에 관련된 유전좌위는 12개의 SSR 마커에 의해 표지 되었다(Table 4). 유전좌위 표지에 사용된 SSR 마커는 1번과 2번 염색체상에 각각 1개씩과 3번, 4번, 및 6번 염색체상에서 각각 2개씩, 그리고 7번 염색체상에 4개가 확인되었다. 1번 염색체에서 RM1003 (R2=13.7%)에 대응하는 유전좌위가 표 지되었으나 추정된 상가적 효과(additive effect)는 2.38로 밀 양23호의 대립인자에 의해 간장이 단축되었으며, 남일과 밀 양23호 후대에서 표지된 유의한 유전좌위와 비교한 결과에 따라 1번 염색체 하단에 RM1003은 제외되었다. 6번 염색체 에서 RM6274에 대응하는 유전좌위는 15.8% 기여도를 보이며, 7번 염색체에서는 20.0%의 기여도를 보이는 RM3555에 대 응하는 유전좌위가 표지되었다(Fig. 3B). 이들 유전좌위의 추 정된 상가적 효과는 6번 염색체에서 3.60과 7번 염색체에서 3.53으로써 Namil(SA)-bl5의 대립인자에 의해 간장이 단축 되었다(Table 4).
대부분의 벼 품종들의 반왜성은 단일 유전자에 의해 조절 된다고 보고되고 있는데, 반왜성 유전좌들 중 가장 널리 활용 되는 sd1은 1번 염색체 하단에 위치한다(Foster & Rutger 1978, Cho et al. 1994, Tanisaka et al. 1994). 상기 2개 분자 마커들에 대응하는 유전좌위는 반왜성 유전좌위와 다른 염색 체상에 표지되었으며, 남일과 밀양23호간의 유전분석 집단에 서도 표지되지 않았으므로 Namil(SA)-bl5의 고유 유전좌위 임이 확인되었다. 또한 간장은 주동유전자적 조절에 의해 단 축되는 것으로 추정된다. 도복저항성의 향상에 있어서 반왜성 은 중요한 특성이며(Miura et al. 2009), 실중의 감소 없이 수 수의 증가 또한 매우 중요하다(Chen et al. 2013). 돌연변이 계통인 Namil(SA)-bl5는 이삭의 길이와 수당립수는 원품종 인 남일에 비해 감소하였으나 수량에 있어서 원품종인 남일 과 큰 차이를 보이지 않았다. 이는 Namil(SA)-bl5에서 수수 가 유의하게 증가한 것에서 기인하는 것으로 판단된다.
도열병에 관련되어서는 총 68개의 마커들 중 9개의 마커들 이 유의한 F 값을 지니는 것으로 확인되었다. 9개의 마커들은 5번, 6번, 및 12번 염색체에서 각각 1개, 4개, 및 4개가 확인 되었다. 연관분석(association analysis)결과로 미루어 보아 도열병 균주 94-254에 저항성을 발현하는 것으로 추정되는 유전좌위들은 6번 염색체 중단 부위(RM0527) 및 12번 염색 체 중단 부위(RM1337)에 표지되었다. 이들 유전좌위들의 기 여도는 6번 염색체에서 16.8% (RM0527) 및 7번 염색체에서 46.4% (RM1337)을 보였으며, 추정된 상가적 효과는 0.89 (RM0527)와 1.37 (RM1337)로써 모두 Namil(SA)-bl5의 대 립인자에 의해 도열병에 대한 저항성이 증가하였다. RM1337 에 대응하는 유전좌위는 46.4%의 기여도를 보이는 주동 유전 좌위로 추정된다. 이 유전좌위는 Namil(SA)-bl5의 고유 유전 좌위임이 확인되었다.
본 연구에서 돌연변이 계통인 Namil(SA)-bl5의 단간 및 도열병 저항성에 대한 e-Landing 기법을 이용하여 연관지도 초안을 작성하여 유전좌위를 표기하였다. 하지만 단간에 대한 좀 더 명확한 유전좌위 표지가 필요하며, 도열병 저항성에 대 한 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 염색 체 중단에 위치하는 것으로 보고된(Sallaud et al. 2003, Yang et al. 2009) 적어도 9개의 저항성 유전자들과의 유전적 관련 성에 대한 판정이 필요할 것으로 사료된다. 따라서 앞으로 고 밀도 연관지도 작성을 위해 마커밀도를 선택적으로 높여주어 좀 더 정밀한 유전좌위 표기 및 e-Landing 기법을 이용하여 BAC/PAC의 전사부위(Open reading frame; ORF) 정보를 활용하여 기존에 보고된 도열병 저항성 유전자들과의 유전적 관련성을 분석할 예정이다.
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, 다양한 작물학적 특성 및 병해충 저항성에 대한 돌연변이 계통의 유용형질들을 교 배를 통해 고품질 벼의 보완을 위해 이전하는 것이 용이한 이 점을 가지고 있다.
농촌진흥청 국립식량과학원에서는 국내 육성 자포니카 벼 품종인 남일에 아지드화나트륨을 돌연변이원으로 활용하여 단간이며 도열병 저항성 돌연변이 후대계통인 Namil(SA)-bl5를 육성하였다. 본 연구는 염색체상에서 Namil(SA)-bl5의 단간 특성과 도열병 저항성에 관련된 유전좌위를 탐색하고자 수행하였다.
주요 연구 결과로 Namil(SA)-bl5와 밀양23호간에서 유래 한 F2 94 개체로부터 단간에 대한 표현형을 조사하고 68개 SSR 마커의 유전자형을 검정하여 연관분석(association analysis)을 수행한 결과 목표 유전좌위는 6번 염색체 중하단 부위와 7번 염색체 하단 부위로 간장의 단축은 주동유전자적 조절에 의 한 것으로 추정되었다. 또한, 분자마커의 유전자형과 도열병 저항성 간의 연관성평가를 수행한 결과를 보면, Namil(SA)-bl5의 도열병 저항성에 관여하는 주동 유전좌위는 12번 염색 체 중단의 RM1337 좌위에 위치하는 것으로 추정되었다. 추 후 목표 유전좌위에 대한 마커밀도를 선택적으로 높여 고밀 도 유전자지도 작성함으로 초정밀 분자표지인자를 개발할 계 획이다.
본 연구는 농촌진흥청 바이오그린21사업(과제번호 PJ00816) 의 지원으로 수행된 결과이며, 이에 감사 드립니다.
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Genetic Analysis on Short Culm and the Rice Blast Resistance of Namil(SA)-bl5, a Japonica Rice Mutant Line
Korean. J. Breed. Sci.. ;46(3):238-249.   Published online September 30, 2014
DOI: https://doi.org/10.9787/KJBS.2014.46.3.238
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Genetic Analysis on Short Culm and the Rice Blast Resistance of Namil(SA)-bl5, a Japonica Rice Mutant Line
Korean. J. Breed. Sci.. ;46(3):238-249.   Published online September 30, 2014
DOI: https://doi.org/10.9787/KJBS.2014.46.3.238
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Genetic Analysis on Short Culm and the Rice Blast Resistance of Namil(SA)-bl5, 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(SA)-bl5 (right) (A) and reaction patterns against virulent rice blast isolate, 94-254 of Namil, Milyang23, and Namil(SA)-bl5, sequentially from left to right (B).
Fig. 2. Graphical comparisons of F2 mapping populations derived from two crosses between Namil (wild type) or Namil(SA)-bl5 and Milyang23 on the agronomic trait, culm length (A) and virulent rice blast isolate, 94-254 (B). To measure the agronomic trait, culm length of F2 progeny lines, harvested seeds from each F2 plant (F2:3 seeds) were planted in a raw. The descriptive statistics on the agronomic trait, culm length of 103 F2:3 lines of Namil x Milyang23, over-all mean, standard deviation, skewness, and kurtosis are 84.7, 7.4, 0.0, and -0.7, respectively. In 94 F2:3 lines of Namil(SA)-bl5 x Milyang23 on the agronomic trait, culm length, over-all mean, standard deviation, skewness, and kurtosis are 72.5, 6.7, 0.0, and -0.5, respectively. To estimate the resistant levels of F2 progeny lines, harvested seeds from each F2 plant (F2:3 seeds) were planted in a raw. Fourteen days after inoculation of rice blast isolate (94-254), individuals were evaluated. On virulent rice blast, 94-254, the descriptive statistics 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 94 F2:3 lines of Namil(SA)-bl5 x Milyang23 on virulent rice blast isolate, 94-254, over-all mean, standard deviation, skewness, and kurtosis are 2.3, 1.5, 0.2, and -1.2, respectively.
Fig. 3. Linkage map skeleton of the agronomic trait, culm length and rice blast resistance on rice chromosomes. In Namil x Milyang23, wild type, preliminary gene locus concerned with culm length is on rice chromosome 1, and putative gene loci of rice blast resistance are on rice chromosome 5 and 11 (A). In Namil(SA)-bl5, a mutant line, preliminary gene loci concerned with culm length are on rice chromosome 6 and 7, and putative gene loci of rice blast resistance are on the same rice chromosome 6 and 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 Short Culm and the Rice Blast Resistance of Namil(SA)-bl5, a Japonica Rice Mutant Line

Reactions of the mutant line, Namil(SA)-bl5 and cultivars against selected Korean virulent blast isolates.

Lines Rice blast isolate (race)z
90-002 (KI215) 90-008 (KI1113) 93-093 (KI197) 94-254 (ND)y 97-260 (KJ103) 02-319 (KJ105) 02-039 (KJ201)
Namil 4 4 5 4 4 5 5
Milyang23 4 3 4 4 2 4 2
Junam 5 5 5 5 5 5 3
Onnuri 4 4 5 5 4 1 4
Chilbo 2 5 5 5 5 4 4
Namil(SA)-bl5 0 1 1 0 1 1 1

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 cultivars, Junam, Onuri, and Chilbo were included as check varieties

Not determined

Comparisons between Namil, the wild type and Namil(SA)-bl5, 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 ± 0.6 11 ± 1.0 147 ± 7.8 74.4 ± 7.1 24.8 ± 0.0 599 ± 24.0
Namil(SA)-bl5 7.25 ± 0.6 65 ± 1.7 19 ± 0.0 17 ± 2.1 95 ± 0.6 74.2 ± 7.5 20.5 ± 0.2 578 ± 21.4

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. All numerical values were mean ± standard deviation.

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(SA)-bl5 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 53 22 0 1.4
RM0600 1 9,461,346 9,461,566 221 21.7 AP001081 27.3 49.6 21 49 24 0 0.2
RM0449 1 15,305,619 15,305,758 140 35.1 AP008247 40.0 72.8 29 44 21 0 1.4
RM1349 1 25,398,082 25,398,300 219 58.3 AP002744 56.7 103.1 24 46 24 0 0.0
RM1003 1 33,803,800 33,803,927 128 77.5 AP003345 75.1 136.6 28 42 24 0 1.1
RM6321 1 43,251,019 43,251,186 168 99.2 AP003277 100.0 181.8 26 42 26 0 0.8
RM0154 2 1,083,895 1,084,077 183 3.0 AP005851 3.0 4.7 16 62 16 0 8.6 *
RM5699 2 8,981,409 8,981,575 167 25.0 AP005803 26.7 42.1 19 53 22 0 1.4
RM1211 2 18,450,427 18,450,588 162 51.4 AP005299 37.7 59.5 19 55 20 0 2.3
RM3220 2 28,458,108 28,458,248 141 79.2 AP004118 74.8 118.1 13 52 29 0 5.8
RM3850 2 35,425,798 35,425,907 110 98.6 AP003989 100.0 157.9 13 55 26 0 5.6
RM6301 3 2,631,471 2,631,613 143 7.2 AC090485 8.1 13.3 25 48 21 0 0.2
RM0007 3 9,808,540 9,808,710 171 27.0 AC134232 27.0 44.4 15 47 31 1 4.8
RM1164 3 14,840,558 14,840,757 200 40.8 AC084766 41.8 68.7 21 44 29 0 1.4
RM6266 3 23,778,063 23,778,222 160 65.4 AC091246 57.7 94.9 24 45 25 0 0.1
RM1350 3 28,632,514 28,632,682 169 78.8 AC087181 77.1 126.8 28 44 22 0 0.9
RM3585 3 36,080,616 36,080,784 169 99.3 AC128647 98.4 161.7 27 42 25 0 0.9
RM0551 4 168.620 168,811 192 0.5 AL606442 2.4 3.1 22 48 24 0 0.0
RM5633 4 13,059,370 13,059,580 211 37.1 AL731595 15.4 19.9 13 54 27 0 5.5
RM1155 4 20,328,759 20,328,906 148 57.7 AL606453 45.4 58.9 24 35 35 0 8.0 *
RM3839 4 23,870,755 23,870,972 218 67.7 AL606614 56.2 72.8 19 38 37 0 9.4 **
RM3217 4 30,083,469 30,083,662 194 85.4 AL606683 77.7 100.7 23 47 18 6 0.7
RM0559 4 35,117,645 35,117,804 160 99.6 AL606637 100.0 129.6 22 56 16 0 3.7
RM5693 5 441,872 442,071 200 1.5 AC129716 3.8 4.6 26 51 17 0 2.0
RM5844 5 9,128,802 9,128,996 195 30.6 AC120989 43.7 53.5 27 46 18 3 1.4
RM5558 5 21,168,727 21,168,899 173 70.9 AC105769 70.3 86.0 22 48 24 0 0.0
RM1054 5 29,144,035 29,144,184 150 97.6 AC98598 99.8 122.0 25 44 25 0 0.2
RM3353 6 435,582 435,697 116 1.4 AP001129 1.1 1.4 18 50 26 0 1.4
RM3370 6 6,434,836 6,435,012 177 20.6 AP005545 27.6 34.3 18 49 27 0 1.5
RM0539 6 8,170,600 NA 26.1 AP005619 41.0 51.3 15 49 26 4 2.9
RM0527 6 9,863,290 9,863,522 233 31.6 AP005695 45.3 56.3 16 50 26 2 2.4
RM6818 6 16,581,413 16,581,542 130 53.1 AP004012 52.9 65.8 18 51 25 0 1.4
RM3628 6 23,737,032 23,737,157 126 76.0 AP003612 68.6 85.4 16 51 27 0 2.7
RM6274 6 26,319,462 26,319,530 69 84.2 AP003568 80.6 100.3 23 40 29 2 2.0
RM5604 6 29,048,075 29,048,152 78 93.0 AP003772 92.0 113.4 22 46 26 0 0.2
RM5753 6 30,966,850 30,967,050 201 99.1 AP004685 100.0 124.4 18 52 23 1 1.5
RM1093 7 668,161 668,310 150 2.3 AP003746 2.1 2.5 22 53 19 0 1.4
RM0481 7 2,876,165 2,876,333 169 9.7 AP005632 14.8 11.0 20 53 20 1 1.4
RM1243 7 3,555,641 3,555,797 157 12.0 AP004267 21.4 25.4 21 55 17 1 2.9
RM1377 7 12,782,829 12,783,009 181 43.1 AP004305 41.9 49.7 33 40 21 0 4.5
RM3743 7 19,342,334 19,342,513 180 65.2 AP003815 56.5 67.0 30 44 20 0 2.0
RM5623 7 23,110,498 23,110,681 184 77.8 AP005908 68.4 81.1 30 46 17 1 3.1
RM3555 7 27,891,580 27,891,733 154 93.9 AP004378 89.1 105.7 25 45 21 3 0.2
RM0172 7 29,560,592 29,560,750 159 99.6 AP005199 100.0 118.6 23 45 26 0 0.2
RM0408 8 119,935 120,063 129 0.4 AP005406 0.4 0.5 24 50 20 0 0.5
RM0547 8 5,586,058 5,586,291 234 19.7 AP004746 33.2 40.2 20 51 23 0 0.6
RM3262 8 22,248,334 22,248,500 167 78.6 AP005483 71.5 86.7 24 51 19 0 0.9
RM5545 8 28,141,927 28,142,083 157 99.4 AP004623 100 121.2 19 48 27 0 1.1
RM23654 9 151,453 151,639 187 0.7 AP006059 0.0 0.0 28 55 11 0 8.0 *
RM0316 9 1,074,933 1,075,126 194 4.7 AP005860 0.9 0.8 28 55 11 0 8.0 *
RM0219 9 7,887,585 NA 34.3 AP005912 22.1 20.7 27 53 14 0 4.5
RM0566 9 14,704,798 14,705,036 239 63.9 AP005397 54.2 50.7 24 48 22 0 0.0
RM0205 9 22,720,646 22,720,801 156 98.7 AP005546 100.0 93.5 20 44 30 0 2.0
RM7492 10 39,037 39,181 145 0.2 AC166065 0.0 0.0 21 55 18 0 2.4
RM0311 10 9,487,243 9,487,406 164 41.5 AC090482 20.0 16.8 22 50 22 0 0.2
RM1375 10 16,386,764 16,386,943 180 71.6 AC025905 51.0 42.7 27 44 23 0 0.5
RM0590 10 22,784,993 22,785,130 138 99.6 AC018727 100.0 83.8 16 56 22 0 3.7
RM0286 11 383,839 383,960 122 1.3 BX000497 1.2 1.4 15 54 24 1 3.6
RM6901 11 1,494,196 NA 5.2 AC123516 5.5 6.5 14 47 27 6 3.6
RM3428 11 13,445,211 13,445,367 157 47.2 AC128642 47.7 56.2 15 46 33 0 6.2 *
RM5349 11 19,148,807 19,148,929 123 67.3 AC134925 67.1 79.1 21 42 30 1 2.2
RM0144 11 28,246,930 28,247,154 225 99.2 AC134045 98.6 116.2 20 48 26 0 0.6
RM8215 12 1,585,781 1,586,001 221 5.8 BX000498 8.9 9.7 10 37 47 0 31.6 ***
RM3472 12 3,520,117 3,520,331 215 12.8 AL713902 24.8 27.1 15 31 48 0 32.3 ***
RM1337 12 11,935,984 11,936,165 182 43.4 BX000556 47.2 51.5 17 34 42 1 18.8 ***
RM0277 12 18,290,458 NA 66.5 AL831799 57.0 62.2 14 39 41 0 16.9 ***
RM1300 12 25,998,975 25,999,139 165 94.4 AL713940 93.4 100.9 8 42 44 0 27.0 ***
RM0017 12 26,954,668 26,954,835 168 98.0 AC027133 98.4 107.4 7 44 43 0 26.4 ***

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(SA)-bl5 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(SA)-bl5 and Milyang23z for culm length (CL) and rice blast resistance (BL).

Trait Evaluated Mirror Mapy
 No. of Genotypesx
 Genotype meanw
 Single-locus ANOVAv
 Genetic Effectu
Locus Ch cM cM% A H B A H B SSM SSE F-value R2 Add Dom DeD
CL (cm) N=94 Mean=72.6 RM1003 1 136.6 77.5 28 42 24 73.2 74.5 68.4 576.9 3640.4 7.21 ** 0.137 -2.38 3.68 -1.55
RM0154 2 4.7 2.2 16 62 16 77.8 71.2 72.4 554.7 3662.6 6.89 ** 0.132 -2.69 -3.92 1.46
RM0007 3 44.4 27.0 15 47 31 74.3 73.6 70.1 279.4 3935.5 3.19 * 0.066 -2.07 1.42 -0.69
RM1350 3 126.8 78.8 28 44 22 70.9 72.0 75.8 327.5 3889.9 3.83 * 0.078 2.46 -1.40 -0.57
RM0551 4 3.1 0.5 22 48 24 75.1 72.5 70.2 278.8 3938.5 3.22 * 0.066 -2.46 -0.15 0.06
RM5633 4 19.9 37.1 13 54 27 73.5 73.7 69.8 289.8 3927.5 3.36 * 0.069 -1.88 2.03 -1.08
RM3628 6 85.4 76.0 16 51 27 68.1 73.1 74.1 392.4 3824.9 4.67 * 0.093 2.97 2.02 0.68
RM6274 6 100.3 84.2 23 40 29 68.6 72.6 75.8 663.7 3544.6 8.33 *** 0.158 3.60 0.44 0.12
RM1093 7 2.5 2.3 22 53 19 69.2 73.5 73.7 325.1 3892.2 3.80 * 0.077 2.28 2.05 0.90
RM5623 7 81.1 77.8 30 46 17 69.5 73.9 74.4 426.9 3788.0 5.07 ** 0.101 2.47 1.95 0.79
RM0172 7 118.6 99.6 23 45 26 71.3 71.1 76.2 489.5 3727.9 5.97 ** 0.116 2.48 -2.68 -1.08
RM3555 7 105.7 93.9 25 45 21 71.1 70.8 78.1 841.6 3366.3 11.00 *** 0.200 3.53 -3.77 -1.07
BL (Degree: 0-5) N=86 Mean=2.3 RM1054 5 122.0 97.6 22 42 22 2.8 1.9 2.5 14.1 172.2 3.40 * 0.076 -0.12 -0.79 6.71
RM3370 6 34.3 21.2 18 41 27 1.5 2.3 2.7 17.7 168.6 4.35 * 0.095 0.64 0.21 0.33
RM0539 6 51.0 26.2 15 42 25 1.3 2.2 3.0 25.2 152.1 6.55 ** 0.142 0.82 0.09 0.12
RM6818 6 65.8 .53.1 18 45 23 1.4 2.2 3.1 27.3 159.0 7.13 ** 0.147 0.82 -0.03 -0.03
RM0527 6 56.3 31.6 16 44 24 1.4 2.2 3.1 31.3 149.2 8.50 *** 0.173 0.89 -0.06 -0.06
RM8215 12 9.7 5.8 9 34 43 1.3 2.1 2.6 13.9 172.3 3.36 * 0.075 0.64 0.14 0.21
RM3472 12 27.1 12.8 14 28 44 1.1 2.1 2.8 29.8 156.5 7.91 *** 0.160 0.81 0.11 0.13
RM0277 12 62.2 66.5 13 36 37 1.1 2.1 2.9 31.2 155.1 8.35 *** 0.168 0.87 0.09 0.11
RM1337 12 51.5 43.4 15 32 38 0.5 2.0 3.2 85.3 97.8 35.74 *** 0.466 1.37 0.12 0.09

Only significant loci to each trait tested are presented. 58 DNA markers were tested in 94 F2 progenies derived from the cross between Namil(SA)-bl5 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(SA)-bl5 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<0.001

significant at P < 0.01

significant at P < 0.05.

Additive 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(SA)-bl5 and Milyang23, H is heterozygous individuals at the tested locus.

Table 2. Reactions of the mutant line, Namil(SA)-bl5 and cultivars against selected Korean virulent 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 cultivars, Junam, Onuri, and Chilbo were included as check varieties

Not determined

Table 1. Comparisons between Namil, the wild type and Namil(SA)-bl5, 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. All numerical values were mean ± standard deviation.

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(SA)-bl5 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(SA)-bl5 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(SA)-bl5 and Milyang23z for culm length (CL) and rice blast resistance (BL).

Only significant loci to each trait tested are presented. 58 DNA markers were tested in 94 F2 progenies derived from the cross between Namil(SA)-bl5 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(SA)-bl5 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<0.001

significant at P < 0.01

significant at P < 0.05.

Additive 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(SA)-bl5 and Milyang23, H is heterozygous individuals at the tested locus.

Table 2.

Table 1.

Table 3.

Table 4.

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