Identification and Gene Mapping of a Rolled Leaf Mutant rl(t) in Rice

doi:10.16819/j.1001-7216.2017.6137

Abstract

Abstract:

【Objective】Rice leaf is an important factor for ideal plant architecture in rice, and moderate rolling of the leaves can improve light acceptance and photosynthetic rate. Genetic analysis and primary mapping of the target genes are useful in map-based cloning and function analysis.【Method】A stable inherited mutant, derived from an indica maintainer line Yixiang 1B by EMS treatment, exhibited significantly inward-rolling, more erect and green leaves. At the maturity stage, 10 plants of rl(t) and its wild type(WT) were randomly selected to measure the main agronomic traits including leaf rolling index and leaf erect index. At the tillering stage, the same part of leaf of rl16(t) and WT was taken and fixed by FAA for paraffin sectioning. The chlorophyll content in rl16(t) and WT was tested at the same stage. At the heading stage, the photosynthetic rate, intercellular carbon dioxide concentration, stamatal conductance,and transpiration rate of flag leaves in rl15(t) and WT were measured by using the portable gas exchange system Li-6400.【Result】Compared to wide type, rl(t) displayed reduced plant height, shortened panicle length as well as other agronomic traits. Leaf photosynthetic pigment contents were significantly higher than those of the wild type. However, no difference is observed in photosynthetic efficiency. Cytological analysis showed that the rolled leaf phenotype was possibly caused by the reduced size of bulliform cells. Genetic analysis indicated that rolled leaf phenotype in rl(t) was controlled by a single recessive nuclear gene. In an F₂ population derived from a cross between the mutant and Nipponbare, RL(t) was mapped to a 610 kb region between Ind3 and Ind4 on the long arm of chromosome 7. In target region, there are 102 genes altogether.【Conclusion】The rolled leaf of rl(t) was due to increased area of bulliform cells in adaxial layer and no gene related to roll-leaf was reported in the target region. So, RL (t) gene would be a putative novel rolled leaf gene.

Key words: rice, rolled leaf, gene mapping

摘要：

目的叶片是水稻理想株型的重要内容,叶片适度卷曲可以提高光合效率。对卷叶相关基因进行遗传分析和初步定位,为下一步的基因克隆与功能分析提供研究基础。方法利用EMS诱变雄性不育保持系宜香1B获得一份稳定遗传的叶片向内卷曲突变体,暂命名为rl(t)。在成熟期,测定野生型和rl(t) 的主要农艺性状;在分蘖期,取野生型和rl(t) 叶片用FAA固定液固定进行石蜡切片,同时,用野生型和rl(t)剑叶测定叶绿素含量;在抽穗期,利用Li-6400便携式光合仪测定10株抽穗期的野生型和rl(t)的光合参数;将rl(t)与野生型及日本晴杂交,观察 F₁植株表型,对F₂表型分离进行χ²测验,对突变体进行遗传分析。以 rl(t)/日本晴的 F₂ 群体为材料,利用 BSA 法进行定位。结果与野生型相比,突变体叶片向内卷曲明显,叶片更加直立,叶色变深,其他主要农艺性状均有不同程度降低。光合特性分析表明,突变体比野生型具有更高的光合色素含量,但光合效率没有明显差异。叶片组织切片观察表明,突变体中泡状细胞变小可能是导致叶片卷曲的主要原因。遗传分析表明,该突变体受一对隐性核基因控制,利用突变体与日本晴的F₂群体进行基因定位,最终将该基因定位在第7染色体长臂InDel标记Ind3和Ind4间610 kb的物理区间。结论 rl(t)叶片内卷是由于近轴面泡状细胞面积减小。RL(t)定位区间内未见卷叶相关基因报道,推测RL(t)可能是一对新基因。

关键词: 水稻, 卷叶, 基因定位

Baolin HAN, Yu TAO, Hongkai ZHANG, Chaojian GU, Yongxiang LIAO, Yongbin PENG, Hongyu ZHANG, Peizhou XU, Xiaoqiong CHEN, Xianjun WU. Identification and Gene Mapping of a Rolled Leaf Mutant rl(t) in Rice[J]. Chinese Journal OF Rice Science, 2017, 31(2): 149-156.

韩保林, 张洪凯, 顾朝剑, 廖泳祥, 彭永彬, 张红宇, 徐培洲, 陈晓琼, 吴先军. 水稻叶片内卷突变体rl(t)的鉴定与基因定位[J]. 中国水稻科学, 2017, 31(2): 149-156.

Figures/Tables 8

References 33

[1]	陈温福, 徐正进, 张龙步. 水稻理想株型的研究. 沈阳农业大学学报,1989(4): 417-420.
	Chen W F, Xu Z J, Zhang B L.Rice ideal pant-type. JShenyang Agric Univ, 1989(4): 417-420. (in Chinese)
[2]	程式华, 曹立勇, 陈深广, 朱德峰, 王熹, 闵绍楷, 翟虎渠. 后期功能型超级杂交稻的概念及生物学意义. 中国水稻科学, 2005, 19(3): 280-284.
	Cheng S H, Cao L Y, Chen S G, Zhu D F, Wang X, Min S K, Zhai H Q.Conception of late-stage vigor super hybrid rice and its biological Significance.Chin J Rice Sci, 2005,19(3): 280-284. (in Chinese with English abstract)
[3]	Khush G S, Kinoshita T.Rice karyotype, marker genes, and linkage groups//Khush G S, Toenniessen G H. Rice Biotechnology. Wallingford: CAB International and IRRI, 1991: 83-108.
[4]	李仕贵, 马玉清, 何平, 黎汉云, 陈英, 周开达, 朱立煌. 一种未知的卷叶基因的识别和定位. 四川农业大学学报, 1998, 16(4): 391-393.
	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): 391-393. (in Chinese with English abstract)
[5]	严长杰, 严松, 张正球, 梁国华, 陆驹飞, 顾铭洪. 一个新的水稻卷叶突变体rl9(t)的遗传分析和基因定位. 科学通报, 2005, 50(24): 2757-2762.
	Yan C J, Yan S, Zhang Z Q, Liang G H, Lu J F, Gu M H.Genetic analysis and gene fine mapping for a rice novel mutant rl9(t) with rolling leaf character. Chin Sci Bull, 2005, 50(24): 2757-2762. (in Chinese with English abstract)
[6]	Yi J C, Zhuang C X, Wang X J, Cao Y P, Liu Y G, Mei M T.Genetic analysis and molecular mapping of a rolling leaf mutation gene in rice.J Integr Plant Biol, 2007, 49: 1746-1753.
[7]	施勇烽, 陈洁, 刘文强, 黄奇娜, 沈波, Hei Leung, 吴建利. 一个新的水稻卷叶突变体的遗传分析与基因定位. 中国科学: C 辑, 2009, 39(4): 407-412.
	Shi Y F, Chen J, Liu W Q, Huang Q N, Shen B, Leung H, Wu J L.A new rice leaf mutant of genetic analysis and gene location.Sci China Series C: Life Sci, 2009, 39(4): 407-412. (in Chinese with English abstract)
[8]	余东, 吴海滨, 杨文韬, 巩鹏涛, 李有志, 赵德刚. 水稻单侧卷叶突变体 B157 遗传分析及基因初步定位. 分子植物育种, 2008, 6(2): 220-226.
	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 Breeding, 2008, 6(2): 220-226. (in Chinese with English abstract)
[9]	Wang D K, Liu H Q, Li K L, Li S J, Tao Y Z.Genetic analysis and gene mapping of a narrow leaf mutant in rice (Oryza sativa L.). Chin Sci Bull, 2009, 54(5): 752-758.
[10]	罗远章, 赵芳明, 桑贤春, 凌英华, 杨正林, 何光华. 水稻新型卷叶突变体 rl12(t)的遗传分析和基因定位. 作物学报, 2009, 35(11): 1967-1972.
	Luo Y Z, Zhao F M, Sang X C, Ling Y H, Yang Z L, He G H.Genetic analysis and gene mapping of a novel rolled leaf mutantrl12(t) in rice. Acta Agron Sin, 2009, 35(11): 1967-1972. (in Chinese with English abstract)
[11]	顾兴友, 顾铭洪. 一种水稻卷叶性状的遗传分析. 遗传, 1995, 17(5): 20-23.
	Gu X Y, Gu M H.A genetic analysis of rice leaf traits.Hereditas, 1995, 17(5): 20-23. (in Chinese with English abstract)
[12]	Fang L K, Zhao F M, Cong Y F, Sang X C, Du Q, Wang D Z, Li Y F, Ling Y H, Yang Z L, He G H.Rolling-leaf14 is a 2OG-Fe(Ⅱ) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves.Plant Biotechnol J, 2012, 10(5): 524-532.
[13]	张礼霞, 刘合芹, 于新, 王林友, 范宏环, 金庆生, 王建军. 水稻卷叶突变体 rl15(t)的生理学分析及基因定位. 中国农业科学, 2014,47(14): 2881-2888.
	Zhang L X, Liu H Q, Yu X, Wang L Y, Fan H H, Jin Q S, Wang J J.Molecular mapping and physiological characterization of a novel mutantrl15(t) in rice. Sci Agric Sin, 2014, 47(14): 2881-2888. (in Chinese with English abstract)
[14]	刘晨, 孔维一, 尤世民, 钟秀娟, 江玲, 赵志刚, 万建民. 一个水稻卷叶基因的遗传分析和精细定位. 中国农业科学, 2015, 48(13): 2487-2496.
	Liu C, Kong W Y, You S M, Zhong X J, Jiang L, Zhao Z G, Wang J M.Genetic analysis and fine mapping of a novel rolled leaf gene in rice.Sci Agric Sin, 2015, 48(13): 2487-2496. (in Chinese with English abstract)
[15]	Hibara K I, Obara M, Hayashida E, Abe M, Ishimaru T, Satoh H, Itoh J I, Nagato Y.TheADAXIALIZED LEAF1 gene functions in leaf and embryonic pattern formation in rice. Dev Biol, 2009, 334: 345-354.
[16]	Shi Z Y, Wang J, Wan X S, Shen G Z, Wang X Q, Zhang J L.Over-expression of riceOsAGO7 gene induces upward curling of the leaf blade that enhanced erect-leaf habit. Planta, 2007, 226(1): 99-108.
[17]	李磊,薛芗,左示敏,陈宗祥,张亚芳,李前前,朱俊凯,马玉银,潘学彪,潘存红.抑制OsAGO1a基因的表达导致水稻叶片近轴面卷曲. 中国水稻科学,2013, 27(3): 223-230.
	Li L, Xue X, Zou S M, Chen Z X, Zhang Y F, Li Q Q, Zhu J K, Ma Y Y, Pan X B, Pan C H.Suppressed expressed of AGO1a leads to adaxial leaf rolling in rice.Chin J Rice Sci, 2013, 27(3): 223-230.
[18]	Zou L P, Sun X H, Zhang Z G, Liu P, Wu J X, Tian C J, Qiu J L, Lu TG.Leaf rolling controlled by the homeodomain leucine zipper class IVgeneRoc5 in rice. Plant Physiol, 2011, 156: 1589-1602.
[19]	Li L, Shi Z Y, Li L, Shen G Z, Wang X Q, An L S, Zhang J L.Overexpression of ACL1 (abaxially curled leaf 1) increased bulliform cells and induced abaxial curling of leaf blades in rice. Mol Plant, 2010, 3: 807-817.
[20]	Hu J, Zhu L, Zeng D L, Gao Z Y, Guo L B, Fang Y X, Zhang G H, Dong G J, Yan M X, Liu J, Qian Q.Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice. Plant Mol Biol, 2010, 73(3): 283-292.
[21]	Fujino K, Matsuda Y, Ozawa K, Nishimura T, Koshiba T, Fraaije M W, Sekiguchi H.NARROW LEAF 7 controls leaf shape mediated by auxin in rice. Mol Genet Genom, 2008, 279: 499-507.
[22]	Hu J, Zhu L, Zeng D L, Gao Z Y, Guo L B, Fang Y X, Zhang G H, Dong G J, Yan M X, Liu J, Qian Q.Identification and characterization ofNARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice. Plant Mol Biol, 2010, 73(3): 283-292.
[23]	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(3): 719-735.
[24]	Liu X F, Li M, Liu K, Tang D, Sun M F, Li Y F, Shen Y, Du G J, Cheng Z K.Semi-Rolled Leaf2 modulates rice leaf rolling by regulating abaxial side cell differentiation. J Exp Bot, 2016, 67(8): 2139-2150.
[25]	Wu R H, Li B S, He S, Wabmann F, Yu C, Qin G J, Schreiber L, Qu LJ, Gu H Y.CFL1, a WW domain protein, regulates cuticle development by modulating the function of HDG1, a class IV homeodomain transcription factor, in rice and Arabidopsis. Plant Cell, 2011, 23: 3392-3411.
[26]	Lichtenthaler H K.Chlorophyll and carotenoids: Pigments of photosynthetic membranes.Methods Enzymol, 1987, 148: 350-382.
[27]	Rogers S O, Bendich A J.Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol, 1985, 5(2): 69-76.
[28]	赵芳明,魏霞, 马玲, 桑贤春, 王楠, 张长伟,凌英华,何光华.水稻生育后期卷叶突变体lrl1的鉴定及基因定位和候选基因预测. 科学通报, 2015(32): 3133-3143.
	Zhao F M, Wei X, Ma L, Sang X C, Wang N, Zhang C W, Ling Y H, He G H.Identification, gene mapping and candidate gene prediction of a late-stage rolled leaf mutantlrl1 in rice(Oryza sativa L.). Chin Sci Bull, 2015, 60: 3133-3143. (in Chinese with English abstract)
[29]	Sangster A G, Parry D W.Some factors in relation to bulliform cell silicification in the grass leaf.Annals Bot, 1969, 33(130): 315-323.
[30]	Zhao S Q, Hu J, Guo L B, Qian Q, Xue H W.Rice leaf inclination2, a VIN3-1ike protein, regulates leaf angle through modulating cell division of collar.Cell Res, 2010, 20(8): 935-947.
[31]	Li Y Y, Shen A, Xiong W, Sun Q L, Luo Q, Song T, Li Z L, Luan W J.Overexpression ofOsHox32 results in pleiotropic effects on plant type architecture and leaf development in rice. Rice, 2016, 9(1): 46.
[32]	Dai M Q, Zhao Y, Ma Q, Hu Y F, Hedden P, Zhang Q F, Zhou D X.The rice YABBY1 gene is involved in the feedback regulation of gibberellin metabolism.Plant Physiol, 2007, 5(144): 121-133.
[33]	Yang C H, Li D Y, Liu X, Ji C J, Hao L L, Zhao X F, Li X B, Chen C Y, Cheng Z K, Zhu L H.OsMYB103L, an R2R3-MYB transcription factor, influences leaf rolling and mechanical strength in rice (Oryza sativa L.). BMC Plant Biol, 2014, 14(1): 1-15.

标记 Marker	正向引物（5'-3') Forward primer(5'-3')	反向引物（5'-3') Reverse primer(5'-3')
RM3394	CCCTTACGTGCAGTACATTG	ATGCAGGCTACTTACTAGCG
Ind1	CCAGTCAACAGGAGCATA	TTAATTATTAGCCTGCTCGA
Ind2	TATAGATGAGCACGCAATCA	GTATGATCGGAAACGTCACT
Ind3	TTGCAGGAGTACTGAAATGA	GCCAGTATTACCAGAGGATG
Ind4	CAAGGTCATCAAGTTCGC	CTGACGGAGCCAGGATCT
Ind5	TGGAAAAGAACTTCAATGCT	TTGAATCACCACAATTTAGC
Ind6	GTCAACCGTCAAGAGATCAT	TGCTTTAGCAGAGTTGCATA
RM5752	TTGCAATTAATTCGATCTCC	GCAGATCGATTCGTTAGTTC
ACL1	CTGAAGCTGAACCTCTCGCTG	GGAGCATGACGTAGATGAAGCAG
RL14	CTCTTTCAGGCATTCCATTGATG	CAACACCTTGTCAGCTTTCAAGC
ROC5	CGCAAGAGGAAGAAGCGATAC	GCTCCAGTTGCGTCTTCATC
SRL1	TGCATCTTTTGACCCAGCTAC	ACAACAAGGGTGCCCAGATAA
SLL1	CAGGTGTCCAACCATGAGC	GCCTCTGTGATTGCCATCTAAT
CFL1	TCGAGCTCAACTCCGAGGTC	ACGGACGACACGGAGGTGTA
NRL1	TCAGTAGTGTAGTGGTGTCGAGTTCA	GCACTCCTTCATGTGAGCTTCA
NAL7	CAAGAACATCACCGGCAAGA	CGATTTGATCAAGGACCATGCT
OSAGO7	CCGCATCCCCTTGATGATT	GGCCAATTCATGCTTGCAA
SRL2	TCCATCTGCGCAGCATTTCA	CTACTGGGCACGATATGCAG

标记 Marker	正向引物（5'-3') Forward primer(5'-3')	反向引物（5'-3') Reverse primer(5'-3')
RM3394	CCCTTACGTGCAGTACATTG	ATGCAGGCTACTTACTAGCG
Ind1	CCAGTCAACAGGAGCATA	TTAATTATTAGCCTGCTCGA
Ind2	TATAGATGAGCACGCAATCA	GTATGATCGGAAACGTCACT
Ind3	TTGCAGGAGTACTGAAATGA	GCCAGTATTACCAGAGGATG
Ind4	CAAGGTCATCAAGTTCGC	CTGACGGAGCCAGGATCT
Ind5	TGGAAAAGAACTTCAATGCT	TTGAATCACCACAATTTAGC
Ind6	GTCAACCGTCAAGAGATCAT	TGCTTTAGCAGAGTTGCATA
RM5752	TTGCAATTAATTCGATCTCC	GCAGATCGATTCGTTAGTTC
ACL1	CTGAAGCTGAACCTCTCGCTG	GGAGCATGACGTAGATGAAGCAG
RL14	CTCTTTCAGGCATTCCATTGATG	CAACACCTTGTCAGCTTTCAAGC
ROC5	CGCAAGAGGAAGAAGCGATAC	GCTCCAGTTGCGTCTTCATC
SRL1	TGCATCTTTTGACCCAGCTAC	ACAACAAGGGTGCCCAGATAA
SLL1	CAGGTGTCCAACCATGAGC	GCCTCTGTGATTGCCATCTAAT
CFL1	TCGAGCTCAACTCCGAGGTC	ACGGACGACACGGAGGTGTA
NRL1	TCAGTAGTGTAGTGGTGTCGAGTTCA	GCACTCCTTCATGTGAGCTTCA
NAL7	CAAGAACATCACCGGCAAGA	CGATTTGATCAAGGACCATGCT
OSAGO7	CCGCATCCCCTTGATGATT	GGCCAATTCATGCTTGCAA
SRL2	TCCATCTGCGCAGCATTTCA	CTACTGGGCACGATATGCAG

性状 Trait	宜香1B Yixiang 1B	rl(t)
抽穗期 Days to heading / d	87.3±1.3	81.1	±1.4^**
株高 Plant height / cm	112.3±4.1	103.5	±2.8^**
分蘖数 No. of tillers	14.6±1.1	6.0	±0.8^**
穗长 Panicle length / cm	29.1±0.9	26.9	±0.9^**
每穗实粒数 No. of filled grains per panicle	196.7±5.2	134.3	±7.0^**
每穗粒数 No. of grains per panicle	212.4±7.2	173.6	±9.5^**
结实率 Seed-setting rate / %	93.0±1.0	77.0	±22.0^**
千粒重 1000-grain weight / g	30.1±0.2	21.9	±0.1^**
粒长 Grain length / mm	9.9±0.0	9.2	±0.0^**
粒宽 Grain width / mm	2.7±0.2	2.7	±0.0
长宽比 Grain length-width ratio	3.7±0.3	3.4	±0.0^*
数据表示为平均数±标准差;样本数量n=10;^*, ^分别表示差异达0.01和0.05显著水平(t检验)。 Values are listed as means ± SD (n=10). ^*,^ Significantly different at 0.01 and 0.05 levels, respectively(t test).

性状 Trait	宜香1B Yixiang 1B	rl(t)
抽穗期 Days to heading / d	87.3±1.3	81.1	±1.4^**
株高 Plant height / cm	112.3±4.1	103.5	±2.8^**
分蘖数 No. of tillers	14.6±1.1	6.0	±0.8^**
穗长 Panicle length / cm	29.1±0.9	26.9	±0.9^**
每穗实粒数 No. of filled grains per panicle	196.7±5.2	134.3	±7.0^**
每穗粒数 No. of grains per panicle	212.4±7.2	173.6	±9.5^**
结实率 Seed-setting rate / %	93.0±1.0	77.0	±22.0^**
千粒重 1000-grain weight / g	30.1±0.2	21.9	±0.1^**
粒长 Grain length / mm	9.9±0.0	9.2	±0.0^**
粒宽 Grain width / mm	2.7±0.2	2.7	±0.0
长宽比 Grain length-width ratio	3.7±0.3	3.4	±0.0^*
数据表示为平均数±标准差;样本数量n=10;^*, ^分别表示差异达0.01和0.05显著水平(t检验)。 Values are listed as means ± SD (n=10). ^*,^ Significantly different at 0.01 and 0.05 levels, respectively(t test).

材料 Material	净光合速率P_n /(µmol·m^-2 s^-1)	气孔导度G_s /(µmol·m^-2 s^-1)	胞间二氧化碳浓度C_i /(µmol·m^-2 s^-1)	蒸腾速率T_r /(µmol·m^-2 s^-1)
宜香1B Yixiang 1B	18.62±0.14	0.51±0.00	295.92±5.68	4.59±0.06
rl(t)	18.57±0.25	0.50±0.00	297.53±1.52	4.57±0.04
数据表示为平均数±标准差;样本数量n=10; ^*,^分别表示差异达0.01和0.05显著水平(t检验)。 Values are means ± SD (n=10). ^*,^ Significantly different at 0.01 and 0.05 levels, respectively(t test).