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1 作物学报 ACTA AGRONOMICA SINICA 2012, 38(3): ISSN ; CODEN TSHPA9 DOI: /SP.J 水稻卷叶基因 RL 13 的遗传分析和分子定位 * 田晓庆桑贤春赵芳明李云峰凌英华杨正林何光华 / /, :,, EMS 10,,,, rl 13 (rolled leaf 13) rl 13 a b 10,, rl 13, rl 13,, 2,, 1A F 2,, F , RL 13 6 RM276 SWU6-1, 1.1 cm 0.2 cm : (Oryza sativa L.); rl 13 ; ; Genetic Analysis and Molecular Mapping of a Rolled Leaf Gene RL 13 in Rice (Oryza sativa L.) TIAN Xiao-Qing, SANG Xian-Chun, ZHAO Fang-Ming, LI Yun-Feng, LING Ying-Hua, YANG Zheng-Lin, and HE Guang-Hua * Rice Research Institute of Southwest University / Chongqing Key Laboratroy of Application and Safety Control of Genetically Modified Crops / Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing , China Abstract: Rolled 1eaf mutants are good materials rice in breeding for leaf and plant type. A rolled 1eaf mutant rl 13 was derived from an EMS-treated mutation in an indica line (Jinhui 10). The chlorophyll content of rl 13 was significantly higher than that of wild type Jinhui 10. There was a highly significant difference in the curl of the three functional mutant leaves compared with those with wild-type. However, there were no significant differences between the three functional mutants. Through the analysis of paraffin, mesophyll cells per layer in rl 13 thinned, wild-type cells contain a large bubble into two similar size vesicular cells of mutants, which resulted in the character of rolled leaf. We made the combination of Xinong 1A/rl 13 to establish genetic population for genetic analysis, the results confirmed that the mutant trait was controlled by a single recessive nuclear gene. In the F 2, the gene of the rolled leaf trait was mapped between two SSR markers, RM276 and SWU6-1, with distances of 1.1 cm and 0.2 cm, respectively. Keywords: Rice (Oryza sativa L.); Rolled leaf mutant; Genetic analysis; Molecular mapping,,, [1],,, [2-4],, ;,,, (CSTC, 2010AA1019) (2009ZX B) * (Corresponding author):, hegh@swu.edu.cn Received( ): ; Accepted( ): ; Published online( ): URL:

00423 水稻卷叶基因 RL 13 的遗传分析和分子定位 * 田晓庆桑贤春赵芳明李云峰凌英华杨正林何光华 / /, 400715 :,, EMS 10,,,, rl 13 (rolled leaf 13) rl 13 a b 10,, rl 13, rl 13,, 2,, 1A F 2,, F 2 1 215, RL 13 6

2 424 38,,, [5] 6 rl1 rl2 rl3 rl4 rl5 rl6, 9, 2 [6-7], /, HD-ZIPⅢ / [8] Luo [9] RL9 MYB, KANADI Yan [10] RL9, GARP, KANADI Zhang [7] SLL1 SHAQKYF MYB-DNA,, Shi [6] T-DNA 11, R05, OSAG07, 1 048, PAZ PIWI, OSAG07 AG07, AGO, OSAG07A mirna, / [5], rl(t) AGO, mirna EMS 10 rl 13 (rolled leaf 13),,, SSR, 1 材料与方法 1.1 EMS 10 rl 13,, 1A, A/rl 13, 2007 F 1, F F 2, 1.3 Reich [11] , 10, (RLI) [12], RLI = (Lw Ln)/Lw 100%,, Lw, Ln 1.5 rl 13, 70% 2 1 7,,, 10~15 μm, NIKON E6000 [7] 1.6 DNA PCR CTAB DNA [13], DNA [14] Panaud [15],, PCR 25 μl, 10 PCR buffer 2.5 μl, 25 mmol L 1 MgC μl, 2.5 mmol L 1 dntps 1.0 μl, ddh 2 O 16.0 μl, 10 μmol L μl, DNA 2.0 μl, 5 U μl 1 Taq DNA 0.2 μl PCR 94 5 min; s, s, 72 1 min, 35 ; min PCR 10%, 1.7 rl 13 B, 1A A, F 1 H, Mapmaker3.0 [16] 2 结果与分析 2.1 rl 13 rl 13,, ( 1)

4, 10, (RLI) [12], RLI = (Lw Ln)/Lw 100%,, Lw, Ln 1.5 rl 13, 70% 2 1 7,,, 10~15 μm, NIKON E6000 [7] 1.6 DNA PCR CTAB DNA [13], DNA [14] Panaud [15],, PCR 25 μl, 10 PCR buffer 2.

3 3 : RL , 3,,, rl 13,, rl 13 2 ( 1-d, e ), 图 1 突变体和野生型叶片形态特征 Fig. 1 Morphological characteristics of mutant and WT a: ; b: ; c: (bar = 2 mm); d: ( 40, bar=25 μm); e: rl 13 ( 40, bar = 25 μm) a: whole plant; b: leaf; c: cross-section of leaf (bar=2 mm); d: cross-section of leaf of WT during tillering stage ( 40, bar=25 μm); e: cross-section of leaf of rl 13 during tillering stage ( 40, bar=25 μm). 2.2 rl 13, ( a b ),, ( 2) 2.3 1A/rl 13 F 1, F 2,, 3 705, 1 215, χ (χ 2 = 0.228< χ = 3.84), 2.4 rl 13 1A/rl 13 F 2, SSR, SSR, 6 RM587 RM5531, 10 10, RM587 RM , 6.3 cm 13.4 cm 50, RM19573 RM276 SWU6-1 (P1: 5'-CCCACAAGTCATTCTAAGGTCC-3', P2: 5'-CA ACGTCTCGTGACCACATT-3') ( 3),, , RM276 RM587, SWU6-1, RM276 SWU6-1, 1.1 cm 0.2 cm ( 4), 560 kb, 120 rl 13 3 讨论,,,,, [17],,,,,,,

cross-section of leaf of WT during tillering stage ( 40, bar=25 μm); e: cross-section of leaf of rl 13 during tillering stage ( 40, bar=25 μm). 2.2 rl 13, ( a b ),, ( 2) 2.

4 [9] [4],,, [18], 1.5, Fig. 2 图 2 不同生长时期野生型 (WT) 和卷叶突变体 (rl 13 ) 的叶绿素和类胡萝卜素含量 Chlorophyll and carotenoid contents between mutant and WT at different growth stage ** 1% ** Represent the 1% differential level. 图 3 SSR 标记 SWU6-1 与突变位点连锁验证 Fig. 3 Linkage analysis between SSR marker SWU6-1 and RL 13 locus P1: 1A; P2: rl 13 ; W1: ; W2: ; 1~10: F 2 ; 11~20: F 2 M: marker; P1: Xinong 1A; P2: rl 13 ; W1: the normal gene pool; W2: the mutant gene pool; 1 10: the normal plants in F 2 population; 11 20: the mutant plants in F 2 population. 图 4 RL 13 在第 6 染色体上定位结果 Fig. 4 Mapping of RL 13 gene on chromosome 6,,,,, 1.5,, 3% rl ,, rl1 rl2 rl3 rl4 rl5 rl6 6, [19,20], 11, url1(t) [21] rl7 [22] rl8 [5] rl9 [23] rl10 [9] nrl2(t) [24] RL12(t) [25]

5 3 : RL sd-sl nal3(t) sd-sl, 6 InDel XL6-6 XL6-1 [26] nal3(t),, 12 SSR RM7018 RM3331 [27] [25] SSR RL12(t) 10 SWU-1 SWU-2, 1.5 cm 0.2 cm, RL12(t) 10 [24] 10 EMS 1 nrl2(t), SSR 3 SSR s3rm1 s3rm3, 114 kb, QTL QTL, [22] 8 / 17 DH rl7 5 RFLP RG13 RG573 [5] 5 RM6954 RM QTL, rl8, rl 13, 6 SSR RM276 SWU6-1, 560 kb, sd-sl, rl 13, sd-sl,, rl 13,,, sd-sl 6 XL626 XL621, PAC AP AP [26], RL 13 6 SSR RM276 SWU6-1, RL 13,,,, RM276 SWU6-1, 1.1 cm 0.2 cm, 560 kb, mirna, / HD-ZIPIII AGO, RL 13,,, 4 结论 EMS 10, ; ; ; RL 13 6 RM276 SWU6-1, 1.1 cm 0.2 cm, 560 kb References [1] Yuan L-P( ). Breeding for super for high-yielding in hybrid rice. Hybrid Rice ( ), 1997, 12(6): 1 3 (in Chinese with English [2] Chen W-F( ), Xu Z-J( ), Zhang W-Z( ), Zhang L-B( ), Yang S-R( ). Creation of new plant type and breeding rice for super high yield. Acta Agron Sin ( ), 2001, 27(5): (in Chinese with English [3] Chen Z-X( ), Chen G( ), Hu J( ), Dai L-C( ), Tao G-Y( ), Pan X-B( ). Genetic expression and effects of rolled leaf gene Rl(t) in hybrid rice (Oryza sativa L.). Acta Agron Sin ( ), 2002, 28(6): (in Chinese with English [4] Zhu D-F( ), Lin X-Q( ). Comparison of leaf photosynthetic characteristics among rice hybrids with different leaf rolling index. Acta Agron Sin ( ), 2001, 27(3): (in Chinese with English [5] Shao Y-J( ), Chen Z-X( ), Zhang Y-F( ), Chen E-H( ), Qi X-C( ), Miao J( ), Pan X-B( ). One major QTL mapping and physical map construction for rolled leaf in rice. Acta Genet Sin ( ), 2005, 32(5): (in Chinese with English [6] Shi Z Y, Wang J, Wan X, Shen G, Wang X, Zhang J. Overexpression of rice OsAGO7 gene induces upward curling of the leaf blade that enhanced erect-leaf habit. Planta, 2007, 226: [7] Zhang G H, Xu Q, Zhu X D, Qian Q, Xue H W. SHALLOT- LIKE1 is a KANADI transcription factor that modulates rice leaf rolling by regulating leaf abaxial cell development. Plant Cell, 2009, 21: [8] Vaucheret H, Vazquez F, Crete P, Bartel D P. The action of ARGONAUTE1 in the mirna pathway and its regulation by the

rl 13, sd-sl,, rl 13,,, sd-sl 6 XL626 XL621, PAC AP003490 AP005619 [26], RL 13 6 SSR RM276 SWU6-1, RL 13,,,, RM276 SWU6-1, 1.1 cm 0.

6 mirna pathway are crucial for plant. Genes Dev, 2004, 43: [9] Luo Z K, Yang Z L, Zhong B Q, Li Y F, Xie R, Zhao F M, Ling Y H, He G H. Genetic analysis and fine mapping of a dynamic rolled leaf gene, RL10(t), in rice (Oryza sativa L.). Genome, 2007, 50: [10] Yan C-J( ), Yan S( ), Zhang Z-Q( ), Liang G-H( ), Lu J-F( ), Gu M-H( ). Genetic analysis and gene mapping of a new rolled leaf mutant rl9(t) in rice. Chin Sci Bull ( ), 2006, 51(1): (in Chinese) [11] Reich P B. Effects of low concentrations of O 3 on net photosynthesis, dark respiration, and chlorophyll contents in aging hybrid popular leaves. Plant Physiol, 1983, 73: [12] Gao Y-H( ), Lü C-G( ), Wang M-Q( ), Wang P( ), Yan X-Y( ), Xie K( ), Wan J-M( ). QTL mapping for rolled leaf gene in rice. Jiangsu J Agric Sci ( ), 2007, 23(1): 5 10 (in Chinese with English [13] Wang G-L( ), Fang H-Y( ). Plant Gene Engineering ( ), 2nd edn. Beijing: Science Press, pp (in Chinese) [14] Sang X-C( ), He G-H( ), Zhang Y( ), Yang Z-L( ), Pei Y( ). The simple gain of templates of rice genomes DNA for PCR. Hereditas ( ), 2003, 25(6): (in Chinese with English [15] Panaud O, Chen X, McCouch S R. Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.). Mol Gen Genet, 1996, 259: [16] Xing G-N( ), Zhao T-J( ), Gai J-Y( ). Application technique of marker grouping and ordering in genetic linkage map construction using Mapmaker/Exp. Acta Agron Sin ( ), 2008, 34(2): (in Chinese with English [17] Lü C-G( ), Zou J-S( ). Comparative analysis on plant type of two super hybrid rice and Shanyou 63. Sci Agric Sin ( ), 2003, 36(6): (in Chinese with English [18] Lang Y-Z( ), Zhang F-J( ), Gu X-Y( ), Yang X-J( ), Zhu Q-S( ). Physiological and ecological effects of crimpy leaf character in rice (Oryza sativa L.): II. Photosynthetic character, dry mass production and yield forming. Acta Agron Sin ( ), 2004, 30(9): (in Chinese with English [19] Kinoshita T. Gene Analysis and Linkage Map. Tokyo: Japan Science Society Press, pp [20] Khush G S, Kinoshita T. Rice karyotype, marker genes, and linkage groups. In: Khush G S, Toenniessen G H, eds. Rice Biology. Wallingford: CAB International and IRRI, pp [21] Yu D( ), Wu H-B( ), Yang W-T( ), Gong P-T( ), Li Y-Z( ), Zhao D-G( ). Genetic analysis and mapping of the unilateral rolled leaf trait of rice mutant B157. Mol Plant Breed ( ), 2008, 6(2): (in Chinese with English [22] Li S-G( ), Ma Y-Q( ), He P( ), Li H-Y( ), Chen Y( ), Zhou K-D( ), Zhu L-H( ). Genetic analysis and mapping the flag leaf roll in rice (Oryza sativa L.). J Sichuan Agric Univ ( ), 1998, 16(4): (in Chinese with English [23] Yan S, Yan C J, Zeng X H, Yang Y, Fang Y, Tian C, Sun Y, Cheng Z, Gu M. ROLLED LEAF 9, encoding a GARP protein, regulates the leaf abaxial cell fate in rice. Plant Mol Biol, 2008, 68: [24] Wang D-Z( ), Sang X-C( ), You X-Q( ), Wang Z( ), Wang Q-S( ), Zhao F-M( ), Ling Y-H( ), Li Y-F( ), He G-H( ). Genetic analysis and gene mapping of a novel narrow and rolled leaf mutant nrl2(t) in rice (Oryza sativa L.). Acta Agron Sin ( ), 2011, 37(7): (in Chinese with English [25] Luo Y-Z( ), Zhao F-M( ), Sang X-C( ), Lin Y-H( ), Yang Z-L( ), He G-H( ). Genetic analysis and gene mapping of a novel rolled leaf mutant rl12(t) in rice. Acta Agron Sin ( ), 2009, 35(11): (in Chinese with English [26] Xia L( ), Chen L( ), Guo C-M( ), Zhang H-X( ), Zhao Z( ), Shen M-S( ). Genetic analysis and mapping of rice (Oryza sativa L.) sd-sl mutant. J Xiamen Univ ( ), 2007, 46(6): (in Chinese with English [27] Wang D K, Liu H Q, Li K L. Genetic analysis and gene mapping of a narrow leaf mutant in rice (Oryza sativa L.). Chin Sci Bull, 2009, 54:

Genetic analysis and gene mapping of a new rolled leaf mutant rl9(t) in rice. Chin Sci Bull ( ), 2006, 51(1): 2757 2762 (in Chinese) [11] Reich P B.

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