약배양이용벼멸구, 흰잎마름병및줄무늬잎마름병저항성복합내병충성벼계통육성 79 레스에저항성인유전자를확보하는데제한적이다 (Jeung et al. 2005). 자포니카는인디카에비해상대적으로약한벼멸구저항성을나타내며, 벼멸구저항성주동유전자중대부분인 26개가인디카와야생벼로부터발견되었

Transcription

1 Korean J. Breed. Sci. 46(1):78-89( ) Online ISSN: Print ISSN: 약배양이용벼멸구, 흰잎마름병및줄무늬잎마름병저항성복합내병충성벼계통육성 박현수 1 * 백소현 1 김우재 1 정지웅 2 이종희 3 하기용 1 박종호 1 남정권 1 백만기 1 유재수 1 백채훈 2 노태환 1 김기영 1 조영찬 1 김보경 2 이점호 1 1 농촌진흥청국립식량과학원벼맥류부, 2 농촌진흥청국립식량과학원, 3 농촌진흥청연구정책국 Development of Multi-resistant Lines to Brown Planthopper, Bacterial Blight, and Rice Stripe Virus using Anther Culture in Rice Hyun-Su Park 1 *, So-Hyeon Baek 1, Woo-Jae Kim 1, Ji-Ung Jeung 2, Jong-Hee Lee 3, Ki-Yong Ha 1, Jong-Ho Park 1, Jeong-Kwon Nam 1, Man-Kee Baek 1, Jae-Soo Yoo 1, Chae-Hoon Paik 2, Tae-Hwan Noh 1, Ki-Young Kim 1, Young-Chan Cho 1, Bo-Kyeong Kim 2, and Jeom-Ho Lee 1 1 Department of Rice and Winter Cereal Crop, NICS, RDA, Iksan , Korea 2 National Institute of Crop Science, RDA, Suweon , Korea 3 Research Policy Bureau, RDA, Suweon , Korea Abstract : This study was conducted to develop multi-resistant lines to brown planthopper, bacterial blight, and rice stripe virus using anther culture in rice. A total of 213 double haploid lines were developed the cross between HR conferring resistant to bacterial blight and rice stripe virus and SR conferring resistant to brown planthopper, bacterial bight, and rice stripe virus. Using DNA molecular marker, HR26234 and SR30071 were confirmed to have Xa3+xa5+Stvb-i and Bph18+Xa4+Stvb-i, respectively. All double haploid lines carried Stvb-i, and Bph18+Xa3, Bph18+Xa4, Bph18+Xa3+xa5, Bph18+Xa4+xa5, bph18+xa3, bph18+xa4, bph18+xa3+xa5, and bph18+xa4+xa5 combinations were identified. Segregation distortions such as no combinations carrying Bph18(or bph18)+xa5+stvb-i and fewer lines carrying Bph18 than bph18 were occurred in DH population. Brown planthopper resistant lines carrying Bph18 showed longer culm length than susceptible lines. Selected Bph18+Xa4+xa5+Stvb-i combination lines with short culm conferred resistant to brown planthopper, bacterial blight, and rice stripe virus, while showed deleterious effects such as spikelet sterility, lower yield, and vulnerable to lodging than standard and comparative varieties. Using anther culture, we rapidly developed multi-resistant lines to brown planthopper, bacterial blight, and rice stripe virus. However, distorted segregation in DH population and linkage drag with Bph18 were obstacles to develop practical multi-resistant cultivars. Keywords : Rice, Multi-resistant, Anther culture, Segregation distortion, Linkage drag 서 지속가능하고안정적인쌀생산을위해서는병해충에대한관리가필수적이다 (Savary et al. 2006). 벼의병해충에의한피해를가장적극적으로대처하는방법은저항성품종을 *Corresponding author ( mayoe@korea.kr, Tel: , Fax: ) (Received on February 3, Revised on February 27, Accepted on March 3, 2014.) 언 개발하고이를재배하는것이다 (Khush 1989). 세계각국에서벼병해충에대한저항성품종육종사업이수행되고있다. 우리나라자포니카벼품종에대해가장피해를많이주는병은도열병, 벼흰잎마름병, 벼줄무늬잎마름병이며, 해충은벼멸구에의한피해가가장많다 (Lee et al. 2011). 저항성육종사업은해당병해충에대해효과적인저항성원을확보하고이를우량품종으로도입하는일련의과정을거친다. 자포니카유전자원은협소한유전적배경으로생물적스트 Copyright c 2014 by the Korean 78 Society of Breeding Science This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 약배양이용벼멸구, 흰잎마름병및줄무늬잎마름병저항성복합내병충성벼계통육성 79 레스에저항성인유전자를확보하는데제한적이다 (Jeung et al. 2005). 자포니카는인디카에비해상대적으로약한벼멸구저항성을나타내며, 벼멸구저항성주동유전자중대부분인 26개가인디카와야생벼로부터발견되었다 (Huang et al. 2013). Bph18 저항성유전자는야생벼 O.australiensis로부터인디카계통에이전되었고마커활용선발법 (marker-assisted selection; MAS) 과전게놈의배경분석 (genome-wide background analysis) 을통해자포니카주남벼배경에도입되었다 (Jena et al. 2006, Suh et al. 2011). Bph18 도입계통들은벼멸구유묘및성체검정에서강한저항성반응을나타냈으며, 선발된우량계통이 2010년에품종명안미로등록되었다. 벼흰잎마름병저항성유전자 Xa2 (Tetep), Xa4 (TKM6), Xa11 (IR8), Xa18 (IR24) 등은인디카품종에서유래하였고, 야생벼로부터 Xa21 (O. longistaminata), Xa23 (O. rufipogon), Xa27(t) 와 Xa35(t) (O. minuta), Xa38 (O. nivara) 이도입되었다. 우리나라벼흰잎마름병저항성원으로많이활용되고있는 Xa3 는저항성이자포니카일본품종인 Wase Aikoku 3로부터유래하였고 1991년에저항성품종인화영벼와안중벼가개발된이후로이를이용하여많은우량품종이개발되었다 (Kim et al. 2007, Shin et al. 2011). 여교배를통해방글라데시 Aus 품종 DV85의 xa5를자포니카수원345호배경으로도입한근동질유전자계통이육성되었고실질적인품종개발로이어져 2006년에강백이육성되었고, 이를활용하여 Xa3와 xa5 가결합된진백 (2008) 이개발되어변이균인 K3a에대응할방안을마련하였다 (Shin et al. 2011). 우리나라자포니카벼품종의벼줄무늬잎마름병저항성은인디카 Modan 유래저항성유전자 Stvb-i가도입되어안정적으로이용되고있다 (Kwon et al. 2012). 1975년에최초의저항성품종인낙동벼가개발 (Chung et al. 1975) 된이후로저항성품종개발에 Stvb-i이지속적으로이용되고있다. 여러육종방법중에서약배양은육종기간을단축시키고분리집단에서고정계통을개발하는데유용하다 (Grewal et al. 2011). 농촌진흥청에서는약배양을통해 1985년에화성벼가개발된이후로 2013년까지 27개품종이개발되었다. 병해충저항성육종사업에서표현형정보만을이용할경우생물검정의어려움과하나의저항성유전자가다른저항성유전자의효과를숨길수있기때문에여러개의저항성유전자를집적시키거나다양한병해충에대한저항성품종개발에어려움이따른다 (Lee et al. 2011, Yoshimura et al. 1995). 해당저항성유전자를표지하는분자마커가개발되어있을경우이 를활용하여저항성유전자도입여부를초기세대부터확인할수있으며여러저항성유전자를하나의품종에집적시킬수있다 (Dokku et al. 2013, Suh et al. 2013, Xu 2013). 본연구는벼멸구, 벼흰잎마름병, 벼줄무늬잎마름병에저항성인자포니카복합내병충성품종을조기에개발하고자약배양을수행하여고정계통을육성하고, DNA 분자마커를활용하여저항성유전자를확인함과동시에병해충에대한생물검정을통해목표저항성유전자가집적된복합내병충성계통을선발하고자수행되었다. 또한육성과정중에발생할수있는문제점을파악하여저항성육종사업에반영하고자하였다. 재료및방법시험재료및재배방법벼흰잎마름병과벼줄무늬잎마름병에저항성인계통 HR 을자방친으로하고벼멸구, 벼흰잎마름병및벼줄무늬잎마름병에저항성인계통 SR 을화분친으로하여인공교배한 F 1 을약배양하여얻은 double haploid (DH) 213계통을이용하였다. 이들계통을 2011년국립식량과학원벼맥류부시험포장에재식거리 cm 로주당 1본씩이앙하였다. 주요농업형질조사와벼흰잎마름병균계검정을수행하였고벼멸구저항성검정은유묘검정법을이용하였다. DNA 분자표지를이용하여이들계통에대한저항성유전자를확인하였다. 2011년에선발된저항성유전자가집적된 7개계통을표준품종남평벼, 비교품종진백, 안미와함께 2012년에재식거리 cm로주당 3본식, 구당 180 주가량을 3반복으로이앙하여생산력검정시험을수행하였다. 벼흰잎마름병접종에따른수량성및품질변이를조사하기위해서생산력검정시험에공시된동일한재료를재식거리 cm로주당 3본식, 구당 180 주가량을무접종구, 접종구로나누어이앙하였다. 접종시험구는대조품종의최고분얼기에 K3a 균계 (HB0109 균주 ) 를이용하여시험구전개체를가위절엽접종하였다. 시비량은 N-P 2 O 5 - K 2O를 kg/ha으로질소는기비 : 분얼비 : 수비를 50 : 20 : 30 비율로분시하였고, 인산은전량기비로, 칼륨은기비 : 수비를 70 : 30 비율로분시하였다. 그밖의재배방법은농촌진흥청표준재배법을따랐다. 병해충저항성검정벼멸구저항성검정에이용된벼멸구는국립식량과학원벼

3 80 韓育誌 (Korean J. Breed. Sci.) 46(1), 2014 맥류부간척지농업과해충사육실에서동진벼를식이품종으로사육된벼멸구를이용하였다. 벼멸구유묘검정은 cm 기계이앙상자를 20칸으로구획한상자에계통당 20립정도씩파종하여유리온실베드에치상하였다. 파종후 15 20일경식물체가 4 5엽까지자랐을때개체당벼멸구유충이 7 10 마리씩되게접종하였고, 감수성인남평벼가완전히고사되었을때저항성여부를판정하였다 (Shin et al. 2011). 벼멸구성체검정은포장에재식된공시재료를최고분얼기에 3주씩플라스틱상자 ( cm) 에이식후벼멸구를접종하여감수성인남평벼가완전히고사되었을때저항성여부를판정하였다. 벼흰잎마름병검정은공시된약배양 213계통에대해서 HB01013 (K1 레이스 ), HB01009 (K3a) 균주를이용하여균계별로 3주씩엽선단약 3 cm 부위를가위절엽접종 (Kauffman et al. 1973) 하여접종후 3주후에가장긴병반을가진 3개엽의병반장을측정하여평균한값을이용하였다. 질적저항성은평균병반장이 5 cm 이하는저항성 (R; resistant), 5 10 cm는중도저항성 (MR; moderately resistant), 10 cm이상은이병성 (S; susceptible) 으로구분하였다 (Shin et al. 2011). 선발된우량고정계통에대해서는 HB01013 (K1), HB01014 (K2), HB01015 (K3), HB01009 (K3a 레이스 ) 균주를접종하여저항성에대해좀더면밀히검정하였다. 벼줄무늬잎마름병검정은망실을이용한대량검정법으로바이러스보독충의방사및계대사육으로보독충이충분히유지된망실에서계통당 1.5g을조파하였으며, 파종후 30일경이병성대비품종일품벼가병징을나타낼때저항성과감수성으로판정하였다 (Kwak et al. 2007). 수량및품질관련형질조사생산력검정시험에공시된재료의출수기를조사하고성숙기에시험구별 10개체에대해서간장, 수장, 수수를측정하였다. 출수후 45일에시험구별로 3주를예취하여등숙률및수당립수를측정하고, 수량조사는 100주를예취하여정조수량을측정한다음이중 1.5 kg에대해정현기로제영하여정현비율을측정하고, 100주정조수량에정현비율을곱하여현미수량을구하고 10a당수량으로환산하였다. 정현된현미를가지고현미천립중을측정하였다. 벼흰잎마름병접종에따른시험에서대조구와접종구에서각각 3주를예취하여등숙률을측정하고, 수량조사는 20주를 3반복으로예취하여정조수량을측정한다음제영하여현미수량을측정하였다. 현미외관품위조사는 RN300 (Kett Co., Japan) 을이용하여정립율을측정하였고, 단백질함량은 Infratec 1241 Grain Analyzer (Foss Co., Sweden) 를이용하여측정하였다. 저항성유전자확인 Genomic DNA 추출은 BioSprint 96 (Qiagen Co., Germany) 을이용하였다. 샘플을 TissueLyserⅡ (Qiagen Co., Germany) 를이용하여마쇄한후 BioSprint 96 DNA Plant Kit (Qiagen Co., Germany) 를이용하여 DNA를추출하였다. 벼줄무늬잎마름병저항성유전자 Stvb-i, 벼멸구저항성유전자 Bph18, 벼흰잎마름병저항성유전자 Xa3, Xa4, xa5 를확인하기위해대상유전자와밀접하게연관된 DNA 분자마커인 ST10 (Hayano-Saito et al. 2000), 7312.T4A (Jena et al. 2006), 9643.T4, T14, T10Dw을이용하였다 (Table 1). PCR은 10 ng의 DNA와 AccuPower PCR PreMix (Bioneer Table 1. Gene-specific PCR primers used for the identification of resistance genes. R-genes Chr. No. Marker name Stvb-i 11 ST10 Bph T4A Xa T4 Xa T14 xa T.10Dw Primer sequences Forward (5-3 ), Reverse (5-3 ) CGAAAGATGGTTTCTCCACC GACCAAGCAACTAATGACGC ACGGCGGTGAGCATTGG TACAGCGAAAAGCATAAAGAGTC AGCCGAGCAATGATACCGACA ACAACTGGGATCGAACCGACA TGTTGGAGGATTGGCAAGGAA TTCGTTGCGGCGTTGTTAATC GCACTGCAACCATCAATGAATC CCTAGGAGAAACTAGCCGTCCA Expected size (bp) Reference Hayano-Saito et. al. (2000) Jena et al. (2006) Jeung et al. (unpublished) Jeung et al. (unpublished) Jeung et al. (unpublished)

4 약배양이용벼멸구, 흰잎마름병및줄무늬잎마름병저항성복합내병충성벼계통육성 81 Co., Korea) 를이용하여 My-Genie 96 Thermal block (Bioneer Co., Korea) 에서수행되었다. PCR 반응은 ST10은 94 에서 4분간초기변성후 94 40초, 62 30초, 72 1분간총 35회반복하였고, 7312.T4A는 94 에서 5분간초기변성후 94 30초, 57 30초, 72 40초간총 45회반복하였고 72 에서 5분간반응하였다 T4와 T14는 94 에서 5분간초기변성후 94 40초, 63 또는 60 (10571.T14) 40초, 72 1분간총 40회반복하였고, T10Dw은 95 에서 4분간초기변성후 95 30초, 초, 72 1분간총 35회반복하고 72 에서 5분간반응하였다. 증폭된 PCR 산물 4 μl를 7312T4A는 3U Hinf I, 9643.T4는 2U Taqα Ⅰ와 65 에서 3시간, T14는 3U Tsp509 I와 65 에 2시간, T10Dw는 Rsa Ⅰ와 37 에서 3시간제한효소처리하였다. ST10에의한증폭산물은제한효소처리없이전기영동에이용하였다. EtBr이포함된 2% agarose gel에서전기영동후 UV transilluminator 를이용하여유전자형을판정하였다. 통계분석통계분석은 SAS 프로그램 (Version 9.2, SAS Institute Inc, Cary, North Carolina) 을이용하였다. 저항성유전자조합들의농업형질에대한평균과표준편차를기술통계법으로구하였고, 평균간비교는 PROC ANOVA로분산분석후유 의성이있을경우 5% 유의수준에서 Duncan s Multiple Range test (DMRT) 로분석하였다. PROC t-test를이용하여 K3a 균계접종에따른대조구와접종구의형질변이를비교하였다. 결과약배양계통육성약배양에모부본으로이용한 HR ( 이하 HR26234) 과 SR ( 이하 SR30071) 계통의농업형질특성과병해충저항성은 Table 2와같다. SR30071이 HR26234보다간장이 11 cm 컸고수장이길었으며수수는적었다. HR26234는벼흰잎마름병 K1과 K3a 레이스와벼줄무늬잎마름병에저항성을보였고, SR30071은벼멸구, 벼흰잎마름병 K1과 K3a 레이스및벼줄무늬잎마름병에저항성을나타냈다. HR26234를모본으로하고 SR30071을부본으로인공교배하여 F 1 29개체를육성하였다. 각개체에서이삭을채취하여약배양을수행하였고총 213개의고정계통을육성하였다 (Table 3). 저항성유전자확인목표유전자와연관된 DNA 분자마커를이용하여저항성유전자를확인하였다. HR26234는 Xa3+xa5, Stvb-i를 SR30071 은 Bph18, Xa4, Stvb-i를가지고있는것으로나타났다. 이들 Table 2. Characteristics of the parents using anther culture. Parents Heading date (DAS z ) Culm length (cm) Panicle length (cm) No. of panicles per hill rice stripe virus Reaction to brown plant-hopper bacterial blight K1 race K3a race Resistance genes HR R y S R R Xa3+xa5+Stvb-i SR R R R R Xa4+Bph18+Stvb-i z Day after seedling y R and S mean resistant and susceptible to insect and disease Table 3. Anther culture ability and double haploid lines derived from the cross of HR /SR Cali induced Plant regeneration Green plants Albino (no. and %) (no. and %) z (no. and %) y (no. and %) x 5,300 1,898 (35.8) 399 (21.0) 232 (58.1) 167 (41.9) 213 Anther cultured (no.) z Percent plant regeneration is the number of plants regenerated over number of cali plated y Percent green is the number of green plants regenerated over total plants regenerated x Percent albino is the number of albino plants regenerated over total plants regenerated w Double haploid lines are harvested plants having fertile grain Double haploid lines (no.) w

5 82 韓育誌 (Korean J. Breed. Sci.) 46(1), 2014 유래약배양계통들은모두 Stvb-i를가지고있었고, Bph18 +Xa3, Bph18+Xa4, Bph18+Xa3+xa5, Bph18+Xa4+xa5, bph18+xa3, bph18+xa4, bph18+xa3+xa5과 bph18+xa4+ xa5 등총 8개의유전자조합이확인되었다 (Fig. 1). Bph18을가지고있는계통은 42계통으로 bph18 계통에비해 4배가량 적게발생하였고, 작성가능한벼흰잎마름병저항성유전자조합중에서 xa5 단독계통은발생하지않았다 (Table 4). 약배양계통의농업형질특성및병해충저항성반응 Bph18 보유계통은 bph18에비해서출수기, 수장, 수수는 Fig. 1. PCR analysis to confirm the resistance genes using the gene specific DNA markers. Stvb-i, Bph18, Xa3, Xa4, and xa5 was confirmed by PCR product amplified with primer, ST10 (A), 7312.T4A(cleaved by HinfⅠ, B), 9643.T4 (cleaved by TaqαⅠ, C), T14 (cleaved by Tsp509 Ⅰ, D), and T10Dw (cleaved by Rsa Ⅰ, E), respectively. The white stars represent the lines carrying the resistance genes. Table 4. Agronomic characteristics of the groups classified by resistance genes combination and their resistance reaction to brown planthopper and bacterial blight. Resistance gene RSV z BPH BB No. of lines Reaction to BPH Reaction to BB K1 K3a Heading date (DAS) Culm length (cm) Panicle length (cm) No. of panicles per hill Remark Xa3 10 R S 112 ± 2.4ab y 77.1 ± 3.5bc 19.3 ± 1.7c 9.7 ± 1.3c sterility Xa4 9 R R 106 ± 1.9abc 80.4 ± 5.0ab 22.0 ± 0.7a 12.1 ± 1.0a R Bph18 Xa3+xa5 13 R R 113 ± 13.0a 83.7 ± 10.8a 20.6 ± 1.2b 11.9 ± 1.9a sterility Xa4+xa5 10 R R 101 ± 1.8c 76.8 ± 4.2bc 21.0 ± 0.8b 11.9 ± 1.4a Stvb-i Total ± 8.9A 79.8 ± 7.4A 20.7 ± 1.5A 11.4 ± 1.7A Xa3 53 R S 107 ± 12.5abc 68.4 ± 6.9d 20.4 ± 1.1b 11.8 ± 1.6ab Xa4 60 R R 104 ± 8.0bc 70.1 ± 7.9d 20.7 ± 1.1b 11.4 ± 1.5ab S bph18 Xa3+xa5 51 R R 109 ± 10.abc 70.4 ± 6.2d 20.8 ± 1.4b 11.7 ± 1.6ab Xa4+xa5 7 R R 103 ± 7.1c 71.8 ± 4.6cd 20.9 ± 0.9b 10.5 ± 1.5bc Total ± 10.3A 69.7 ± 7.0B 20.7 ± 1.2A 11.6 ± 1.6A z RSV: rice stripe virus, BPH: brown planthopper, BB: bacterial blight y Means with same letter are not significantly different at P< 0.05 (ANOVA followed by DMRT)

6 약배양이용벼멸구, 흰잎마름병및줄무늬잎마름병저항성복합내병충성벼계통육성 83 차이가없었으나간장이 10.1 cm 컸다. Bph18 보유계통중벼흰잎마름병 Xa3와 Xa3+xa5 조합의계통은출수가늦고불임이발생하는등열악형질이발현되었다. 자포니카형 Bph18 계통은벼멸구유묘저항성에서 Bph1을가지는통일형다산2 호와 Bph3를가지는인디카 IR72와비슷한수준의강한저항성반응을나타냈다 (Fig. 2). Xa4, Xa3+xa5와 Xa4 +xa5는벼흰잎마름병 K1과 K3a레이스에저항성반응을나타냈다. 내병충성계통선발및농업형질특성벼멸구저항성유전자 Bph18, 벼흰잎마름병저항성유전자 Xa4+xa5, 벼줄무늬잎마름병저항성유전자 Stvb-i가집적된 7 계통을선발하여 2012년에생산력검정시험을수행하였다 (Table 5). 출수일수는 96, 97일로진백 (110일), 남평벼 (106), 안미 (102) 보다빠른중생종이었고, 간장은 cm 로진백 (71 cm), 남평벼 (78), 안미 (77) 보다짧으며, 수당립수는 개로진백 (93개), 남평벼 (95) 보다는많고안미 (119) 보다는적었다. 등숙률이 % 로안미 (84.0%), Fig. 2. Resistance reaction of SR (A) and double haploid lines (B) derived from the combination of HR /SR to brown planthopper at seedling stage. Table 5. Yield-related traits of varieties and DH lines at yield trial. Var./lines Heading date (DAS z ) Culm length (cm) Panicle length (cm) No. of panicles per hill No. of spikelets per panicle Ratio of ripened grain (%) 1,000 grain weight of brown rice (g) Rough rice yield (kg/10a) Brown/rough rice ratio (%) Brown rice yield (kg/10a) Index of brown rice yield y (%) Nampyeongbyeo 106b x 78.3a 20.0bc 14 95b 90.1a 20.9ab 647a a 100 Jinbaek 110a 71.4b 20.8b 15 93b 87.6a 21.3a 643a a 100 Anmi 102c 76.5a 22.9a a 84.0ab 20.3b 674a a 104 HR28869-AC65 96d 65.6d 19.8c ab 77.8bcd 20.6ab 528b bc 82 HR28869-AC68 96d 67.4bcd 19.9c a 78.5bcd 20.3b 517b bc 80 HR28869-AC75 96d 67.5bcd 20.3bc a 80.7bc 21.2a 508b bc 79 HR28869-AC117 97d 70.6bc 20.1bc ab 78.7bc 20.9ab 527b bc 82 HR28869-AC157 96d 67.0cd 20.0bc a 74.8cd 21.1a 505b c 78 HR28869-AC158 96d 67.1bcd 20.3bc ab 79.4bc 21.2a 537b b 84 HR28869-AC159 96d 68.5bcd 20.4bc ab 72.0d 20.9ab 508b bc 79 z Day after seedling y Brwon rice yield of Nampyeongbyeo was a standard x Means with the same letter are not significantly different at P< 0.05 (ANOVA followed by DMRT)

7 84 韓育誌 (Korean J. Breed. Sci.) 46(1), 2014 진백 (87.6), 남평벼 (90.1) 에비해낮았고수량성은표준품종인남평벼에비해 79 84% 수준으로낮았다. 선발계통의병해충저항성성능검정선발된내병충성계통들은유묘및성체에서벼멸구에강한저항성을나타냈으며벼흰잎마름병 K1, K2, K3, K3a 균 계에저항성이고벼줄무늬잎마름병에강하였다 (Table 6, Fig 3, 4). 벼흰잎마름병에대한저항성성능검정을위해 K3a 균계접종한결과이병성인남평벼와안미는수량, 등숙률, 현미정상립률이대조구에비해유의하게감소한반면이들계통들은차이가나지않는경향이었다 (Table 7). Fig. 3. Resistance reaction to brown planthopper. At seedling stage (A). At maximum tillering stage (B). 1: Nampyeongbyeo, 2: Jinbaek, 3: Anmi, 4: HR28869-AC65, 5: HR28869-AC68, 6: HR28869-AC75, 7: HR28869-AC117, 8: HR28869-AC157, 9: HR28869-AC158, 10: HR28869-AC159. Check variety is Nampyeongbyeo. Fig. 4. Resistance reaction to bacterial blight, K3a (HB1009 isolate) race. Lesion length at 2 weeks after inoculation (A). Field scenery at maturing stage after inoculation (B). 1: Nampyeongbyeo, 2: Jinbaek, 3: Anmi, 4: HR28869-AC65, 5: HR28869-AC68, 6: HR28869-AC75, 7: HR28869-AC117, 8: HR28869-AC157, 9: HR28869-AC158, 10: HR28869-AC159. White bar indicates 10 cm.

8 약배양이용벼멸구, 흰잎마름병및줄무늬잎마름병저항성복합내병충성벼계통육성 85 고찰여러육종방법중에서약배양은육종기간을단축시키고분리집단에서고정계통을개발하는데유용하다 (Grewal et al. 2011). 벼흰잎마름병과벼줄무늬잎마름병에저항성우량계통 HR ( 이하 HR26234) 과벼멸구, 벼흰잎마름병및벼줄무늬잎마름병에저항성인 SR ( 이하 SR30071) 계통을인공교배한 F 1 을약배양하여총 213 개고정계통을조기에육성하였다 (Table 3). DNA 분자마커 는마커활용선발법 (marker-assisted selection; MAS) 을통해전통육종의효율성과정확성을개선할커다란가능성을가지고있다 (Collard and Mackill 2008). 저항성육종사업에서해당저항성유전자를표지하는분자마커가개발되어있을경우이를활용하여저항성유전자도입여부를초기세대부터확인할수있으며여러저항성유전자를하나의품종에집적시킬수있다 (Dokku et al. 2013, Suh et al. 2013, Xu 2013). 본연구에서는벼멸구저항성유전자 Bph18에 7312.T4A (Jena et al. 2006), 벼흰잎마름병저항성유전자 Xa3, Xa4, Table 6. Reaction of varieties and DH lines to brown planthopper, bacterial blight, and rice stripe virus. Var./lines Brown planthopper at seedling at maximum tillering Bacterial blight K1 K2 K3 K3a Rice stripe virus Resistance genes Nampyeongbyeo S z S S S S S R Stvb-i Jinbaek S S R R R R S Xa3+xa5 Anmi R R R R R S R Bph18+Xa3+Stvb-i HR28869-AC65 R R R R R R R Bph18+Xa4+xa5+Stvb-i HR28869-AC68 R R R R R R R Bph18+Xa4+xa5+Stvb-i HR28869-AC75 R R R R R R R Bph18+Xa4+xa5+Stvb-i HR28869-AC117 R R R R R R R Bph18+Xa4+xa5+Stvb-i HR28869-AC157 R R R R R R R Bph18+Xa4+xa5+Stvb-i HR28869-AC158 R R R R R R R Bph18+Xa4+xa5+Stvb-i HR28869-AC159 R R R R R R R Bph18+Xa4+xa5+Stvb-i z R and S mean resistant and susceptible to insect and disease Table 7. Comparison of the control and K3a inoculation about rice yield, ratio of ripened grain, and perfect kernel of brown rice. Var./lines Rough rice yield (g) Brown rice yield (g) Control Ratio of Perfect kernel ripened grain of brown rice (%) (%) Rough rice yield (g) K3a inoculation Brown rice yield (g) Ratio of Perfect kernel ripened grain of brown rice (%) (%) Nampyeongbyeo **z 378 ** 79.5 ** 63.9 ** Jinbaek ns 443 ns 85.0 ns 82.2 ns Anmi ** 399 ** 71.5 ** 72.1 * HR28869-AC ns 292 ns 81.7 ns 70.1 ns HR28869-AC ns 341 ns 77.7 ns 74.4 ns HR28869-AC ns 318 ns 65.6 ns 72.4 ns HR28869-AC * 322 * 74.6 ns 73.6 ns HR28869-AC ns 343 ns 70.8 ns 72.7 ns HR28869-AC ns 343 * 70.2 ns 70.9 ns HR28869-AC ns 332 * 71.7 ns 71.8 ns z ns, *, and ** mean no significant, significant at P< 0.05, and P< 0.01 by t-test, respectively

9 86 韓育誌 (Korean J. Breed. Sci.) 46(1), 2014 xa5에 9643.T4, T14, T.10Dw (Jeung et al. unpublished), 벼줄무늬잎마름병저항성유전자 Stvb-i에 ST10 (Hayano-Saito et al. 2000) 을이용하여저항성유전자도입여부를확인하였다 (Fig. 1). HR26234는 Xa3+xa5, Stvb-i을, SR30071은 Bph18, Xa4, Stvb-i를가지고있는것으로나타났다. 약배양유래계통중 Bph18을가지고있는계통은 42 계통으로 171개인 bph18 계통에비해 4배가량적게발생하였고작성가능한벼흰잎마름병저항성유전자조합중에서 xa5 단독계통은발생하지않았다. 또한 Bph18+Xa4+xa5+ Stvb-i 저항성유전자조합의선발된 7개내병충성계통들의출수기, 간장, 수장, 수당립수, 등숙률등농업형질이비슷한특성을나타내어 segregation distortion이발생한것으로생각된다 (Table 4, 5). Segregation distortion은분리집단에서멘델의분리법칙을따르지않고편의되어서후대가생성되는것으로모부본배우체의불임에기인한다든지특정배우체의유전자형이선택적으로수정이되어서생성되는것으로나눌수있다 (Xu et al. 1997). 약배양에서인디카는약의조기사멸, 낮은캘러스형성률과식물체재분화율및잦은백색체발생으로자포니카에비하여저조한효율을보이는것으로알려져있어생태형에따라다르며 (Grewal et al. 2011), 불임관련유전자들의작용에의해특정유전자형의소멸또는약배양효율이높은배우체유전자형의선택적수정으로인하여 segregation distortion이발생하는것으로보인다. 자포니카유전자원은협소한유전적배경으로생물적스트레스에저항성인유전자를확보하는데제한적이다. 이를보완하기위해인디카의유용한유전자를자포니카로도입하는데있어서전통적육종법을활용해서는높은불임, 불량한초형과열악형질수반 (linkage drag) 으로어려움이있다 (Jeung et al. 2005). Bph18 저항성유전자는야생벼 O.australiensis로부터인디카계통에이전되었고 MAS기법과전게놈의배경분석 (genome-wide background analysis) 을통해자포니카주남벼배경에도입되었다 (Jena et al. 2006, Suh et al. 2011). Bph18은 12번염색체장완에위치하고있으며이위치에는교잡불임과관련된대립유전자가존재한다. Kubo and Yoshimura (2005) 는 12번염색체장완에서인디카품종 IR24와자포니카품종 Asominori의교잡시불임과관련된대립유전자 hsa1-ir을보고하였고, Yara et al. (2010) 또한 Bph26(t) 와 hsa1부근에서교잡불임과관련된 hsy(t) 가존재한다고하였다. 2011년육성당시 Bph18 보유약배양계통중 Xa3와 Xa3+xa5 조합은심한불임을나타내었으나 SR30071 의 Xa4와 HR26234의 xa5가결합된유전자집적 Xa4+xa5 조합의계통은불임에문제가없는것으로판단되어선발하여생산력검정을수행하였다. 그러나 2012년생산력검정시험중에 Bph18+Xa4+xa5 조합의계통들에서불임의문제점이발견되었다 (data not shown). 이는선발과정에서면밀한검토가이루어지지않았고 2012년기상이불임발현에영향을미친것으로생각한다. 자포니카주남벼배경으로 Bph18 이도입된 SR30071 조합의계통들은 Bph18 표지마커를이용하여 MAS 후반복적인여교배를통해육성되어대부분의게놈이주남벼로회복이되었으나, 여교배후대계통들에대해서전게놈배경분석을실시한결과 1, 2, 10, 11, 12번염색체상에 Bph18 수여친인인디카계통 IR 의염색체절편이남아있고이부위는여교배세대진전에관계없이유지된다고하였다 (Shin et al. 2011, Suh et al. 2011). 인디카 IR24의불임관련대립유전자 hsa1-ir (Chr. 12) 는 Asominori의불임과관련된대립유전자 hsa2-as (Chr. 8) 와 hsa3-as (Chr. 9) 와한게놈상에존재할경우심한불임이발생한다 (Kubo and Yoshimura 2005). hsa1-ir 발현에 hsa2-as 와 hsa3-as가상위성 (epistasis) 을나타내고이로인한불임발생은특정대립유전자조합의선택적제거를가져오게된다. 따라서약배양집단에서 segregation distortion이발생한것이나일부계통들에서불임이발생한것은재조합과정중 Bph18 부근의불임관련대립유전자와상위성을나타내는다른대립유전자들간의조합에기인한것으로생각한다. Shin et al. (2011) 은자포니카벼에 Bph18 도입할경우출수일수는짧은쪽으로, 간장과수장은큰쪽으로작용하며등숙비율은낮은쪽으로영향을미치는것으로보인다하였다. 본연구에서도 Bph18 도입계통이 bph18 계통에비해간장이유의한수준에서 10.1 cm 컸고등숙비율은표준품종과비교품종들에비해낮았다 (Table 4, 5). Bph18이위치하고있는 12번염색체장완에는다른벼멸구저항성유전자 Bph1, bph2, Bph9, Bph10, Bph21, Bph26(t) 가위치하는것으로알려져있다 (Jena and Kim 2010, Murai et al. 2003, Yara et al. 2010). Bph1는간장을크게한다고하였고 bph2에의해간장과 3절간장이길어져도복지수가커진다하였다 (Lee et al. 2005, Yeo and Sohn 2001, Yeo et al. 2002). 이러한결과를종합하여 Shin et al. (2011) 은 12번염색체장완에존재하는 Bph1, bph2, Bph18은장간유전자와밀접히연관되어있어이들유전자를가지고있는벼품종들은재배과정에서도복되기쉬운특성을가진것으로보인다하였다. 본연구

10 약배양이용벼멸구, 흰잎마름병및줄무늬잎마름병저항성복합내병충성벼계통육성 87 에서는이러한도복관련문제에대응하기위해단간위주로복합내병충성계통을선발하여생산력검정을수행하였다. 이들계통들은표준품종인남평벼와비교품종인진백, 안미에비해서간장이작았다 (Table 5). 하지만등숙후기에이들계통들의식물체기부가기울어서단간임에도도복에안정적이지못한특성이나타났는데 (data not shown). 간장은줄어들었으나상대적으로 3절간장의길이가길어도복에안정적이지못한것이아닌가생각한다. 우리나라벼흰잎마름병저항성원으로많이활용되고있는 Xa3는저항성이자포니카일본품종인 Wase Aikoku 3로부터유래하였고 1991년에저항성품종인화영벼와안중벼가개발된이후로이를이용하여많은우량품종이개발되었다 (Kim et al. 2007, Shin et al. 2011). 여교배를통해방글라데시 Aus 품종 DV85의 xa5를자포니카수원345호배경으로도입한근동질유전자계통이육성되었고실질적인품종개발로이어져 2006년에강백이육성되었고이를활용하여 Xa3와 xa5가결합된진백 (2008) 이개발되어변이균인 K3a 에대응할방안을마련하였다 (Shin et al. 2011). 현재우리나라자포니카벼흰잎마름병저항성육종사업의목표유전자조합은 Xa3와 xa5가결합된 Xa3+xa5 조합 (Park et al. 2013) 으로이들조합의품종으로진백 (2008), 신백 (2010), 해품 (2013) 이개발되었으며저항성유전자도입에따른열악형질수반문제는보고된바가없다. Xa4는인디카품종 TKM6 로부터도입되어인디카품종의벼흰잎마름병저항성원으로광범위하게이용되었다 (Mew et al. 1992). Suh et al. (2013) 는 DNA 분자마커활용여교배법 (marker-assisted backcrossing; MAB) 을이용하여 Xa4+xa5+Xa21을자포니카품종에도입하였고, 이들도입된여교배진전육성계통 (advanced backcross breeding line; ABL) 들은열악형질이수반되지않고반복친인자포니카품종과비슷한농업형질과품질특성을나타낸다하였다. 하지만 Xa4는아직까지우리나라에서실질적인품종개발에활용된적이없기때문에 Xa4 도입에따른파악되지않은문제점이발생할수있으므로면밀한검토가필요하다고생각한다. 우리나라자포니카벼품종의벼줄무늬잎마름병저항성은인디카 Modan 유래저항성유전자 Stvb-i가도입되어안정적으로이용되고있다 (Kwon et al. 2012). 1975년에최초의저항성품종인낙동벼가개발 (Chung et al. 1975) 된이후로저항성품종개발에 Stvb-i이지속적으로이용되고있으나 Stvb-i 도입에따른열악형질수반문제는보고된바가없다. 약배양을통해단기간에벼멸구, 벼흰잎마름병, 벼줄무늬잎마름병에저항성인복합내병충성계통을확보할수있었다. 하지만계통선발시파악되지않았던불임립의발생, 낮은수량성, 단간임에도도복에안정적이지못한점등열악형질이발견되었다. 병해충에대한저항성육종사업에서약배양을활용하여조기에육종목표를달성하고자할경우에 segregation distortion이발생하여편의된변이가발생할수있고, 저항성유전자도입시 linkage drag에의해예기치못한열악형질특성이나타날수있음을고려하여신중하게목표에접근하여야할것으로생각한다. 적요본연구는벼멸구, 벼흰잎마름병, 벼줄무늬잎마름병에저항성인자포니카복합내병충성품종을조기에확보하고자약배양을수행하여목표저항성유전자조합의우량고정계통을개발하고육성과정중에발생할수있는문제점을파악하여병해충저항성육종사업에반영하고자수행하였다. 벼흰잎마름병과벼줄무늬잎마름병에저항성인우량계통 HR 과벼멸구, 벼흰잎마름병, 벼줄무늬잎마름병에저항성인 SR 을인공교배한 F 1 을약배양하여 213개고정계통을육성하였다. HR26234는 Xa3+xa5, Stvb-i, SR30071은 Bph18, Xa4, Stvb-i를가지고있는것으로확인되었다. 이들유래약배양계통들은모두 Stvb-i를가지고있었고, Bph18+Xa3, Bph18+Xa4, Bph18+Xa3+xa5, Bph18+Xa4+xa5, bph18+xa3, bph18+xa4, bph18+xa3+ xa5과 bph18+xa4+xa5의조합이확인되었다. 약배양집단에서 xa5+bph18( 또는 bph18)+stvb-i 조합이발생하지않았고 Bph18 보유계통이 bph18 보다적게발생하는등 segregation distortion이발생하였다. Bhp18을보유하며벼멸구에저항성인계통들이감수성인계통들에비해간장이컸다. 선발된 Bph18+Xa4+xa5+Stvb-i 조합의계통은단간이면서벼멸구, 벼흰잎마름병, 벼줄무늬잎마름병에저항성을나타내었으나, 낮은수량성과일부불임의발생, 단간임에도도복에안정적이지못한특성을나타냈다. 병해충에대한저항성육종사업에서약배양을활용하여조기에육종목표를달성하고자할경우에 segregation distortion이발생하여편의된변이가발생할수있고, 저항성유전자도입시 linkage drag에의해예기치못한열악형질특성이나타날수있음을고려하여신중하게목표에접근하여야할것으로생각한다.

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