水稻黄绿叶基因ygl2（t）的分子定位及其育种利用研究
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摘要
水稻植株90-95％的干物质来自光合作用，其生物产量和经济产量主要是依靠光合产物。叶片是最主要的光合器官，因此定位和克隆与水稻光合作用相关的新基因、研究其功能，对阐明光合调控机理、提高水稻的光合生产能力和增加水稻产量具有重要的理论价值与实践意义。叶色突变是一类明显的性状突变，不仅在高等植物叶绿素生物合成、叶绿体结构和功能、遗传和分化以及发育等基础研究中具有重要意义，并可作为标记性状在杂交稻种子纯度鉴定中加以利用。
本研究以粳稻(Oryza sativa L．ssp．japonica)品种武运粳7号自然突变的黄绿叶突变体ygl2为材料，分别与武运粳7号和江西1587正交、反交，获得杂交F_1及自交F_2。对后代进行系统观察发现：F_1代叶片均表现为正常绿色，F_2代叶色性状发生分离，不同的杂交组合F_2代群体中的叶片都表现出黄绿叶色和正常绿色两种类型，正常绿色植株与黄绿叶植株的比例符合3:1的分离比，表明黄绿叶突变性状受1对隐性核基因控制。
利用微卫星标记(SSR)和ygl2／江西1587杂交组合的F_2群体对控制该黄绿叶性状的隐性基因初步定位，确定该基因在水稻第6染色体长臂上，位于RM541和RM30两个标记之间，遗传距离分别是13.13cM和9.09cM。文献检索发现，第6染色体在此位置上没有类似的基因报道，表明该基因为一新的叶色基因。由于在水稻中已有一个黄绿叶基因ygl，位于水稻第10染色体，因此将本研究中所发现的黄绿叶突变体基因暂命名为ygl2(t)(yellow-green，leaf 2)。
进一步利用已经公布的水稻基因组序列，在ygl2(t)基因附近区域寻找微卫星序列并发展新的SSR标记，在RM541和RM30之间找到8个连锁多态性分子标记。用这8个分子标记对ygl2／江西1587 F_2群体中的429株黄绿叶单体进行群体分析，将黄绿叶基因ygl2(t)定位在SSR6-16和RM7434之间，与SSR6-16和RM7434的遗传距离分别为0.62 cM和0.74 cM，为进一步克隆该基因奠定基础。
以自然突变体ygl2为黄绿叶供体、103S为光温敏不育基因供体，通过杂交、回交和不同生态条件下育性跟踪鉴定，选育出带有黄绿叶标记的新光温敏不育系3290S，并与绿叶恢复系测交筛选优势组合，所配优势组合杂种优势在主要经济性状上与正常叶色不育系103S所配组合无明显差异，表明该黄绿叶性状对杂种优势无显著负效应，同时黄绿叶性状在秧田期易识别，表明了黄绿叶突变体在育种实践中具备可行性。
Crop biomass and economic yield mainly depend on the photosynthetic assimilate ofleaves and other photosynthetic organs. 90-95% dry matter accumulation in rice is resultedfrom photosynthetic assimilate of leaves, the main photosynthetic organ. Therefore it isvery important in the theoretical research of photosynthesis regulatory mechanism to mapand clone new rice photosynthetic-related genes and investigate their function. Through thegene engineering approach, the photosynthesis ability and economic efficiency of rice maybe significantly increased. As an obvious trait mutation, leaf color mutation could not onlyused in the research of photosynthesis, chlorophyll biosynthesis, structure, function,inheritance, differentiation and growth of chloroplast, but also in the identification of seedpurity for hybrid rice.
Rice cultivar Wuyunjing 7, Jiangxi 1587 (upland rice) and a yellow-green leaf mutantygl2, origined from a Wuyunjing 7, was used in this study.
Two reciprocals (Wuyunjing 7×ygl2, Jiangxi1587×ygl2) were made to obtain F_1 andF_2 populations. The observation of leaf color showed that all the F_1 plants were normalgreen, but the leaf color trait in the all F_2 populations was segregated into yellow green andnormal green with a segregation ratio of 3: 1. It indicated that the leaf color of mutant ygl2was controlled by a single recessive nuclear gene.
The plants of F2 population, generated from the cross ygl2×Jiangxi 1587, were usedfor molecular mapping of this recessive gene. The preliminary results showed that themutant gene was located on the rice chromosome 6 between two SSR markers, RM541 andRM30, with the genetic distances of 13.13cM and 9.09cM respectively. Since there is nosimilar gene locus reported on this region, it indicates that this is new gene identified andtentatively named as ygl2(t) (yellow-green leaf 2). In order to establish the fine location ofthe ygl2(t) gene, the public rice genome sequence in the region containing ygl2(t) gene wasused to search new SSR markers. Then eight new SSR markers between RM541 and RM30 were developed. 429 yellow-green leaf plants of F_2 population generated from the crossygl2×Jiangxi 1587 were analyzed with above eight SSR markers, and the gene ygl2(t) wasmapped between SSR6-16 and RM7434 markers, with genetic distances of 0.62cM and0.74cM respectively.
A new photo-thermo sensitive genic male sterile line 3290S with the yellow-green leaftag was developed in BC_1F_7 from the yellow-green leaf mutant and the photo-thermosensitive male sterile line 103S via cross, backcross and fertility identification. Goodhybrids were developed by identifying crosses from 3290S and 103S with a series of greenleaf restorer lines, and there were no obvious differences in all the main economiccharacteristics investigated, indicating that the yellow-green leaf trait had no significantnegative effect on the main economic characteristics. Meanwhile the seedlings withyellow-green leaves could be easily distinguished from the normal seedlings. Thus, it wasreasonable to breed japonica sterile lines with the yellow-green leaf tag.
本研究以粳稻(Oryza sativa L．ssp．japonica)品种武运粳7号自然突变的黄绿叶突变体ygl2为材料，分别与武运粳7号和江西1587正交、反交，获得杂交F_1及自交F_2。对后代进行系统观察发现：F_1代叶片均表现为正常绿色，F_2代叶色性状发生分离，不同的杂交组合F_2代群体中的叶片都表现出黄绿叶色和正常绿色两种类型，正常绿色植株与黄绿叶植株的比例符合3:1的分离比，表明黄绿叶突变性状受1对隐性核基因控制。
利用微卫星标记(SSR)和ygl2／江西1587杂交组合的F_2群体对控制该黄绿叶性状的隐性基因初步定位，确定该基因在水稻第6染色体长臂上，位于RM541和RM30两个标记之间，遗传距离分别是13.13cM和9.09cM。文献检索发现，第6染色体在此位置上没有类似的基因报道，表明该基因为一新的叶色基因。由于在水稻中已有一个黄绿叶基因ygl，位于水稻第10染色体，因此将本研究中所发现的黄绿叶突变体基因暂命名为ygl2(t)(yellow-green，leaf 2)。
进一步利用已经公布的水稻基因组序列，在ygl2(t)基因附近区域寻找微卫星序列并发展新的SSR标记，在RM541和RM30之间找到8个连锁多态性分子标记。用这8个分子标记对ygl2／江西1587 F_2群体中的429株黄绿叶单体进行群体分析，将黄绿叶基因ygl2(t)定位在SSR6-16和RM7434之间，与SSR6-16和RM7434的遗传距离分别为0.62 cM和0.74 cM，为进一步克隆该基因奠定基础。
以自然突变体ygl2为黄绿叶供体、103S为光温敏不育基因供体，通过杂交、回交和不同生态条件下育性跟踪鉴定，选育出带有黄绿叶标记的新光温敏不育系3290S，并与绿叶恢复系测交筛选优势组合，所配优势组合杂种优势在主要经济性状上与正常叶色不育系103S所配组合无明显差异，表明该黄绿叶性状对杂种优势无显著负效应，同时黄绿叶性状在秧田期易识别，表明了黄绿叶突变体在育种实践中具备可行性。
Crop biomass and economic yield mainly depend on the photosynthetic assimilate ofleaves and other photosynthetic organs. 90-95% dry matter accumulation in rice is resultedfrom photosynthetic assimilate of leaves, the main photosynthetic organ. Therefore it isvery important in the theoretical research of photosynthesis regulatory mechanism to mapand clone new rice photosynthetic-related genes and investigate their function. Through thegene engineering approach, the photosynthesis ability and economic efficiency of rice maybe significantly increased. As an obvious trait mutation, leaf color mutation could not onlyused in the research of photosynthesis, chlorophyll biosynthesis, structure, function,inheritance, differentiation and growth of chloroplast, but also in the identification of seedpurity for hybrid rice.
Rice cultivar Wuyunjing 7, Jiangxi 1587 (upland rice) and a yellow-green leaf mutantygl2, origined from a Wuyunjing 7, was used in this study.
Two reciprocals (Wuyunjing 7×ygl2, Jiangxi1587×ygl2) were made to obtain F_1 andF_2 populations. The observation of leaf color showed that all the F_1 plants were normalgreen, but the leaf color trait in the all F_2 populations was segregated into yellow green andnormal green with a segregation ratio of 3: 1. It indicated that the leaf color of mutant ygl2was controlled by a single recessive nuclear gene.
The plants of F2 population, generated from the cross ygl2×Jiangxi 1587, were usedfor molecular mapping of this recessive gene. The preliminary results showed that themutant gene was located on the rice chromosome 6 between two SSR markers, RM541 andRM30, with the genetic distances of 13.13cM and 9.09cM respectively. Since there is nosimilar gene locus reported on this region, it indicates that this is new gene identified andtentatively named as ygl2(t) (yellow-green leaf 2). In order to establish the fine location ofthe ygl2(t) gene, the public rice genome sequence in the region containing ygl2(t) gene wasused to search new SSR markers. Then eight new SSR markers between RM541 and RM30 were developed. 429 yellow-green leaf plants of F_2 population generated from the crossygl2×Jiangxi 1587 were analyzed with above eight SSR markers, and the gene ygl2(t) wasmapped between SSR6-16 and RM7434 markers, with genetic distances of 0.62cM and0.74cM respectively.
A new photo-thermo sensitive genic male sterile line 3290S with the yellow-green leaftag was developed in BC_1F_7 from the yellow-green leaf mutant and the photo-thermosensitive male sterile line 103S via cross, backcross and fertility identification. Goodhybrids were developed by identifying crosses from 3290S and 103S with a series of greenleaf restorer lines, and there were no obvious differences in all the main economiccharacteristics investigated, indicating that the yellow-green leaf trait had no significantnegative effect on the main economic characteristics. Meanwhile the seedlings withyellow-green leaves could be easily distinguished from the normal seedlings. Thus, it wasreasonable to breed japonica sterile lines with the yellow-green leaf tag.
引文
[1] 崔海瑞，夏英武，高明尉，等．温度对水稻突变体W1叶色及叶绿素生物合成的影响[J]．核学报，2001，15(5)：269-273．
[2] Jung KH, Hur J, Ryu CH, et al. Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system [J]. Plant Cell Physiol,2003,44: 463-472.
[3] 胡忠，梁汉兴，彭丽萍．一个黄绿色水稻突变体的光合作用特性[J]．云南植物研究，1981，4：449-456．
[4] 夏英武，吴殿星，舒庆尧，等．水稻辐射白色转绿突变系的遗传及叶绿体超微结构的分析[J]．核农学报，1997，17(1)：1-4．
[5] 何瑞锋，丁毅，余金洪，等．水稻温敏叶绿素突变体叶片超微结构的研究[J]．武汉大学学报(自然科学版)，2001，19(1)：1-5．
[6] 林钰琼，刘松，傅亚萍，等．T-DNA插入水稻突变体库的叶绿素和净光合速率变化[J]．中国水稻科学，2003，17(4)：369-372．
[7] 胡忠，彭丽萍，蔡永华．一个黄绿色的水稻细胞核突变体[J]．遗传学报，1981，4：256-261．
[8] Ghirardi ML, Melis A. Chlorophyll b deficiency in soybean mutants effects on photosystem stoichiometry and chlorophyll antenna size [J]. Biochim Biophys Acta, 1988, 932:130-137.
[9] Ben A, Greene DR, Allred DT, et al. Hierarchical response of light harvesting chlorophyll-proteins in a light-sensitive chlorophyll b-deficient mutant of maize [J]. Plant Physiol, 1998, 87:357-364.
[10] Krol M, Spangfort MD, Huner NP. Chlorophyll a/b -binding proteins pigment conversions and early light-induced proteins in a chlorophyll b less barley mutant [J]. Plant Physiol,1995,107:873-883.
[11] Falbel TG, Meehl JB, Staehelin LA.Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis [J]. Plant Physiol, 1996, 12:821-832.
[12] Falbel TG, Staehelin LA. Partial block in the early steps of the chlorophyll synthesis pathway Pacommon feature of chlorophyll b-deficient mutants [J]. Physiol Plant, 1996, 97:311-320.
[13] Zhao Y, Du LF, Yang SH, et al. Chloroplast composition and structure differences in a chlorophyll-reduced mutant of oilseed rape seedlings [J]. Acta Boanicat Sinica, 2001, 43(8):877-880.
[14] Bae CH, Abe T, Matsuyama AT, et al. Regulation of chloroplast gene expression is affected in aliKa novel tobacco albino mutant [J]. Ann Bot, 2001, 88, 545-553.
[15] Kusum IK, Inada H, Kawabata SI, et al. Chlorophyll Deficiency Caused by a Specific Blockage of the C_5-Pathway in Seeding of Virescent Mutant [J].Rice Plant Cell physiol, 1994, 35:445-449.
[16] 陈善福，舒庆尧，吴殿星，等．利用γ射线辐照诱发水稻龙特甫B叶色突变[J]．浙江大学学报(农业与生命科学版)，1999，25(6)：569-572．
[17] Abe T, Matsuyama T, Sekido S, et al. Chlorophyll-deficient mutants of rice demonstrated the deletion of a DNA fragment by heavy-ionirradiation[J]. J. Radiat. Res, 2002, 43:157-161.
[18] Iwata N, Omura T, Satoh H. Linkage studies in rice (Oryza sativa L.) on some mutants for physiological leaf spots [J]. Fac.Agr. Kushu Univ, 1978, 22:243-251.
[19] 吴殿星，舒庆尧，夏英武，等．~(60)Co-γ射线诱发的籼型温敏核不育水稻叶色突变系变异分析[J]．作物学报，1999，25(1)：64-69．
[20] 吴关庭，王贤裕，周志远，等．辐射诱发水稻叶绿素突变及其遗传研究[J]．核农学通报， 1995，16(3)：156-158．
[21] 龚红兵，陈亮明，刁立平，等．水稻叶绿素b减少突变体的遗传分析及其相关特性[J]．中国农业科学，2001，34(6)：686-689．
[22] Li GQ, Li M, Wu DW, et al. The induced mutation of chlorophyll deletion of soybean through radiation and its genetic analysis [J]. Acta Agriculturae Universitatis Jilinensis, 1987, 9(3):46-51.
[23] 吴殿星，夏英武，舒庆尧，等．植物叶绿素突变体的研究及其应用探讨[J]．中国农学通报，1995，11(4)：36-39．
[24] http://www.shigen.nig.ac.jp/rice/rgn/vol15/mutantgenestable.html
[25] 吴殿星，舒庆尧，夏英武，等．一个新的水稻转绿型白化突变系W25的叶色特征及遗传[J]．浙江农业学报，1996，8(6)：372-374．
[26] Sanchez AC, Khush GS. A new gene for virescence character in rice [J]. Rice Genetics Newsletter, 1998, 15:109-109.
[27] 夏英武，吴殿星，舒庆尧．人工诱发的植物叶绿素突变体的遗传及其应用[J]．核农学报，1996，1：41-44．
[28] Hsieh SC. Cytogenetic studies and genetic analysis in rice[J]. P H. D. Thesis. Hokkaid o.Univ, 1961:1-212.
[29] Wang C, He B, Xun M, et al. Tagging and mapping of a gene controlling yellowish-green leaf [J]. Rice Genetics Newsletter, 2003, 20:35-35.
[30] http://www.gramene.Org/newsletters/rice-genetics/rgn12/v12p93.htm
[31] 袁隆平．杂交水稻的育种战略设想[J]．杂交水稻，1987，(1)：1-3．
[32] 张自国，卢兴桂，袁隆平．光敏核不育育性转换的临界温度选择与鉴定的思考[J]．杂交水稻，1992，6：29-32．
[33] 程式华，孙宗修，闵绍楷，等．光敏核不育水稻的光温反应研究Ⅰ．光敏核不育水稻在杭州自然条件下的育性表现[J]．中国水稻科学，1990，4(4)：157-162．
[34] 孙宗修，熊振民，闵绍楷，等．温度敏感型雄性不育水稻的鉴定[J]．中国水稻科学，1989，3(2)：49-55．
[35] 董凤高，朱旭东，能振民，等．以淡绿叶为标记的籼型光-温敏核不育系M2S的选育[J]．中国水稻科学，1995，9(2)：65-70．
[36] 牟同敏，李春海，扬国才，等．紫叶水稻苗期叶色的遗传研究[J]．中国水稻科学，1995，9(1)：45-48．
[37] 王才林，汤玉庚，刘云松，等．BT型粳稻不育系六千辛A的自交结实原因的探讨[J]．江苏农业学报，1991，7(2)：1-7．
[38] 顾根宝，王海洋，顾闽峰，等．龙特甫A变异株类型及其影响因素[J]．江苏农业科学，1998，4．14-16．
[39] Cause M, et al. [J] Genetics, 1994,138:1251-1274.
[40] Kurata N, Nagamura Y, Yamamo to, et al. A 300 kilobase interval genetic map of rice including 883 expressed sequences [J]. Nature Genetics, 1994, 8: 365-372.
[41] 熊立仲，王石平，刘克德，等．微卫星DNA和AFLP标记在水稻分子标记连锁图上的分布[J]．植物学报，1998，40(7)：605-614．
[42] 沈利爽，朱立煌．应用PCR进行水稻染色体末端区域作图[J]．遗传学报，1998，25(6)：508-516．
[43] Williams JG, Kubelik AR, Livak KJ, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers [J]. Nucleic Acids Res, 1990, 18 (22):6531-6535.
[44] Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers [J]. Nucleic Acids Res, 1990, 18(24):7213-7218.
[45] Ronald PC, Albano B, Tabien R et al. Genetic and physical analysis of the rice bacterial blight resistance locus, Xa21. Mol Gen Genet, 1992, 236:113-120.
[46] Murayama S, Yamagishi H, Terachi T. Identification of RAPD and SCAR markers linked to a restorer gene for Ogura cytoplasmic male sterility in radish (Raphanus sativus L) by bulked segregant anlysis [J].Breeding Science, 1999, 49:115-121.
[47] Paran I, Michelmore RW. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce [J]. Theor Appl Genet, 1993, 85: 985-993.
[48] Garcia G M, Stalker H T, Shroeder E, et al. Identification of RAPD, SCAR, and RFLP markers tightly linked to nematode resistance genes introgressed from Arachis cardenasii into Arachis hypogaea [J]. Genome, 1996, 39(5): 836-845.
[49] Wang JX, Yang GS, Fu TD, et al. Development of PCR- based markers linked to the fertility restorer gene for the polima cytoplasmic malesterility in rapeseed (Brassica napus L) [J]. Journal of Genetics, 2000, 27(11): 1012-1016.
[50] Lu ZY, Sun QX, Li HJ, et al. Molecular identification and marker-assisted selection of Pm21 gene conferring resistance to powdery mildew in wheat [J]. Acta Genetica Sinica, 1999, 26(6):673-682.
[51] Vos P, Hogers R, Bleeker M, et al. A FL P: a new technique for DNA finger printing [J]. Nucleic Acids Research, 1995, 23 (21): 4407-4414.
[52] Dib C, Faure S, Fizames C, et al. A comprehensive genetic map of the human genome based on 5264 microsatellites [J]. Nature, 1996, 380:152-154.
[53] Dietrich WE, Miller JC, Steer RG, et al. A comprehensive genetic map of mouse genome[J]. Nature, 1996, 380:149-152.
[54] Bell CJ, Echer JR. Assignment of 30 microsatellite loci to the linkage map of Arabidopsis [J]. Genomics, 1994, 19:137-144.
[55] Henry J, Robert M, Sterle S, et al. Application of molecular techniques to rice improvement in Austrilia. Sixth International Symposium on Rice Molecular biology[J].Program&Abstract, 1998, 23: 8-16.
[56] Roder M, Plashke J, Koening SU, et al. Abundance, variability and chromosomal location of microsatellites in wheat[J]. Mol Gen Genet, 1995, 246:327-333.
[57] Akkaya MS, Shoemaker RC, Specht JE, et al. Integration of simple sequence repeat DNA markers into a soybean linkage map [J]. Crop Sci, 1995, 35: 1439-1445.
[58] Zietkiewicz E. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification[J]. Genomic, 1994, 20: 176-183.
[59] Olson M, Hood L, Cantor C, et al. A common language for physical mapping of the human genome [J]. Science, 1989, 245:1434-1435.
[60] 龙跃生，陈良碧，夏快飞，等．分子标记技术及其在分离和克隆水稻基因中的应用[J]．生物学杂志，2001，18(2)：29-31．
[61] 徐吉臣，朱立煌，陈英，等．用双单倍体群体构建水稻的分子连锁图[J]．遗传学报，1994，21(3)：205-214．
[62] Michelmore RW, Paran I, Kesseli RV. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations [J]. Proc Natl Acad Sci, 1991, 88(21): 9828-9832.
[63] Paterson AH, DeVerna JW, Lanini B, et al. Fine mapping of quantitative trait lociusing selected overlapping recombinant chromosomes in an interspecies cross of tomato[J]. Genetics, 1990, 124: 735-742.
[64] 李敏，李焕秀，李靖．植物基因克隆技术研究进展[J]．生命科学研究，2004，8(2)：116-120．
[65] 易小平．可供利用的植物基因克隆技术[J]．广西农业生物科学，1999，18(3)：221-224．
[66] Huang N, Angeles ER, Domingo J,et al. Pyramiding of bacterial blight resistance genes in rice: marker-assisted select ion using RFL P and PCR. Theor Appl Genet, 1997, 95:313-320.
[67] 邹琦主编．植物生理学实验指导．中国农业出版社，2000，72-75．
[68] McCouch SR, Kotcket G, Yu ZH, et al. Molecular mapping of rice chromosome. Theror Appl Genet, 1988, 76:815-829.
[69] Lander ES, Green P, Abrahamson J, et al. Mapmaker: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1987, 1:174-181.
[70] Librojo AL, Khush GS. Chromosomal location of some mutant genes through the use of primary trisomics in rice. Rice Genetics. IRRI, Manila, Philippines.1986, 249-255.
[71] 周长海，王宝和，白和盛．粳型光敏核不育系103S的育性表现及其繁殖研究[J]．安徽农业科学，2001，29(2)：133-134．
[72] 王宝和，周长海，白和盛，等．浅谈光敏核不育系选育策略[J]．安徽农业科学，1999，27(4)：310-311．
[73] Wu KS, Tanksley SD. Abundance polymorphism and genetic mapping of microsatellites in rice [J]. Mol Gen Genet, 1993, 241: 225-235.
[74] Temnykh S, Park WD, et al. Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.) [J]. Thero Appl Genet, 2000, 100(5):697-712.
[75] Temnykh S, DeClerck G, Lukashova A, et al. Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential [J]. Genome Res, 2001, 11(8): 1441-1452.
[76] 石明松．对光照长度敏感的隐性雄性不育水稻的发现及初步研究[J]．中国农业科学，1985，(1)：44-48．
[77] 李泽炳，毕春群，万经猛，等．盛夏低温对光敏核不育水稻育性稳定性的干扰及其克服的预见性对策[J]．华中农业大学学报，1990，9(4)：343-347．
[78] 袁隆平．选育光-温敏不育系的技术策略[J]．杂交水稻，1992，(1)：1-5．
[79] 舒庆尧，夏英武，左晓旭，等．二系杂交水稻制繁种中利用标记辅助去杂技术[J]．浙江农业大学学报，1996，22(1)：56-60．
[80] 曹立勇，钱前，朱旭东，等．紫叶标记籼型光-温敏核不育系中紫S的选育及其配组的杂种优势[J]．作物学报，1999，25(1)：44-49．
[81] Wang B, Lan T, Wu WR, et al. Mapping of QTLs Controlling Chlorophyll Content in Rice[J]. Acta Cenetica Sinica, 2003, 30(12): 127-1132.
[82] 冯双华．水稻叶绿素含量的简易测定[J]．福建农业科技，1997，4：7-8．
[83] 章志兴，徐开盛，陈俊涛，等．叶色标记技术在杂交水稻种子生产中的应用[J]．种子科技，2001，1：33-34．
[84] 潘学彪，陈宗祥，董桂春，等．一种紫色水稻的遗传及其在光敏不育系育种中应用的研究[J]．遗传，1995，17(3)：31-34．
[2] Jung KH, Hur J, Ryu CH, et al. Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system [J]. Plant Cell Physiol,2003,44: 463-472.
[3] 胡忠，梁汉兴，彭丽萍．一个黄绿色水稻突变体的光合作用特性[J]．云南植物研究，1981，4：449-456．
[4] 夏英武，吴殿星，舒庆尧，等．水稻辐射白色转绿突变系的遗传及叶绿体超微结构的分析[J]．核农学报，1997，17(1)：1-4．
[5] 何瑞锋，丁毅，余金洪，等．水稻温敏叶绿素突变体叶片超微结构的研究[J]．武汉大学学报(自然科学版)，2001，19(1)：1-5．
[6] 林钰琼，刘松，傅亚萍，等．T-DNA插入水稻突变体库的叶绿素和净光合速率变化[J]．中国水稻科学，2003，17(4)：369-372．
[7] 胡忠，彭丽萍，蔡永华．一个黄绿色的水稻细胞核突变体[J]．遗传学报，1981，4：256-261．
[8] Ghirardi ML, Melis A. Chlorophyll b deficiency in soybean mutants effects on photosystem stoichiometry and chlorophyll antenna size [J]. Biochim Biophys Acta, 1988, 932:130-137.
[9] Ben A, Greene DR, Allred DT, et al. Hierarchical response of light harvesting chlorophyll-proteins in a light-sensitive chlorophyll b-deficient mutant of maize [J]. Plant Physiol, 1998, 87:357-364.
[10] Krol M, Spangfort MD, Huner NP. Chlorophyll a/b -binding proteins pigment conversions and early light-induced proteins in a chlorophyll b less barley mutant [J]. Plant Physiol,1995,107:873-883.
[11] Falbel TG, Meehl JB, Staehelin LA.Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis [J]. Plant Physiol, 1996, 12:821-832.
[12] Falbel TG, Staehelin LA. Partial block in the early steps of the chlorophyll synthesis pathway Pacommon feature of chlorophyll b-deficient mutants [J]. Physiol Plant, 1996, 97:311-320.
[13] Zhao Y, Du LF, Yang SH, et al. Chloroplast composition and structure differences in a chlorophyll-reduced mutant of oilseed rape seedlings [J]. Acta Boanicat Sinica, 2001, 43(8):877-880.
[14] Bae CH, Abe T, Matsuyama AT, et al. Regulation of chloroplast gene expression is affected in aliKa novel tobacco albino mutant [J]. Ann Bot, 2001, 88, 545-553.
[15] Kusum IK, Inada H, Kawabata SI, et al. Chlorophyll Deficiency Caused by a Specific Blockage of the C_5-Pathway in Seeding of Virescent Mutant [J].Rice Plant Cell physiol, 1994, 35:445-449.
[16] 陈善福，舒庆尧，吴殿星，等．利用γ射线辐照诱发水稻龙特甫B叶色突变[J]．浙江大学学报(农业与生命科学版)，1999，25(6)：569-572．
[17] Abe T, Matsuyama T, Sekido S, et al. Chlorophyll-deficient mutants of rice demonstrated the deletion of a DNA fragment by heavy-ionirradiation[J]. J. Radiat. Res, 2002, 43:157-161.
[18] Iwata N, Omura T, Satoh H. Linkage studies in rice (Oryza sativa L.) on some mutants for physiological leaf spots [J]. Fac.Agr. Kushu Univ, 1978, 22:243-251.
[19] 吴殿星，舒庆尧，夏英武，等．~(60)Co-γ射线诱发的籼型温敏核不育水稻叶色突变系变异分析[J]．作物学报，1999，25(1)：64-69．
[20] 吴关庭，王贤裕，周志远，等．辐射诱发水稻叶绿素突变及其遗传研究[J]．核农学通报， 1995，16(3)：156-158．
[21] 龚红兵，陈亮明，刁立平，等．水稻叶绿素b减少突变体的遗传分析及其相关特性[J]．中国农业科学，2001，34(6)：686-689．
[22] Li GQ, Li M, Wu DW, et al. The induced mutation of chlorophyll deletion of soybean through radiation and its genetic analysis [J]. Acta Agriculturae Universitatis Jilinensis, 1987, 9(3):46-51.
[23] 吴殿星，夏英武，舒庆尧，等．植物叶绿素突变体的研究及其应用探讨[J]．中国农学通报，1995，11(4)：36-39．
[24] http://www.shigen.nig.ac.jp/rice/rgn/vol15/mutantgenestable.html
[25] 吴殿星，舒庆尧，夏英武，等．一个新的水稻转绿型白化突变系W25的叶色特征及遗传[J]．浙江农业学报，1996，8(6)：372-374．
[26] Sanchez AC, Khush GS. A new gene for virescence character in rice [J]. Rice Genetics Newsletter, 1998, 15:109-109.
[27] 夏英武，吴殿星，舒庆尧．人工诱发的植物叶绿素突变体的遗传及其应用[J]．核农学报，1996，1：41-44．
[28] Hsieh SC. Cytogenetic studies and genetic analysis in rice[J]. P H. D. Thesis. Hokkaid o.Univ, 1961:1-212.
[29] Wang C, He B, Xun M, et al. Tagging and mapping of a gene controlling yellowish-green leaf [J]. Rice Genetics Newsletter, 2003, 20:35-35.
[30] http://www.gramene.Org/newsletters/rice-genetics/rgn12/v12p93.htm
[31] 袁隆平．杂交水稻的育种战略设想[J]．杂交水稻，1987，(1)：1-3．
[32] 张自国，卢兴桂，袁隆平．光敏核不育育性转换的临界温度选择与鉴定的思考[J]．杂交水稻，1992，6：29-32．
[33] 程式华，孙宗修，闵绍楷，等．光敏核不育水稻的光温反应研究Ⅰ．光敏核不育水稻在杭州自然条件下的育性表现[J]．中国水稻科学，1990，4(4)：157-162．
[34] 孙宗修，熊振民，闵绍楷，等．温度敏感型雄性不育水稻的鉴定[J]．中国水稻科学，1989，3(2)：49-55．
[35] 董凤高，朱旭东，能振民，等．以淡绿叶为标记的籼型光-温敏核不育系M2S的选育[J]．中国水稻科学，1995，9(2)：65-70．
[36] 牟同敏，李春海，扬国才，等．紫叶水稻苗期叶色的遗传研究[J]．中国水稻科学，1995，9(1)：45-48．
[37] 王才林，汤玉庚，刘云松，等．BT型粳稻不育系六千辛A的自交结实原因的探讨[J]．江苏农业学报，1991，7(2)：1-7．
[38] 顾根宝，王海洋，顾闽峰，等．龙特甫A变异株类型及其影响因素[J]．江苏农业科学，1998，4．14-16．
[39] Cause M, et al. [J] Genetics, 1994,138:1251-1274.
[40] Kurata N, Nagamura Y, Yamamo to, et al. A 300 kilobase interval genetic map of rice including 883 expressed sequences [J]. Nature Genetics, 1994, 8: 365-372.
[41] 熊立仲，王石平，刘克德，等．微卫星DNA和AFLP标记在水稻分子标记连锁图上的分布[J]．植物学报，1998，40(7)：605-614．
[42] 沈利爽，朱立煌．应用PCR进行水稻染色体末端区域作图[J]．遗传学报，1998，25(6)：508-516．
[43] Williams JG, Kubelik AR, Livak KJ, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers [J]. Nucleic Acids Res, 1990, 18 (22):6531-6535.
[44] Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers [J]. Nucleic Acids Res, 1990, 18(24):7213-7218.
[45] Ronald PC, Albano B, Tabien R et al. Genetic and physical analysis of the rice bacterial blight resistance locus, Xa21. Mol Gen Genet, 1992, 236:113-120.
[46] Murayama S, Yamagishi H, Terachi T. Identification of RAPD and SCAR markers linked to a restorer gene for Ogura cytoplasmic male sterility in radish (Raphanus sativus L) by bulked segregant anlysis [J].Breeding Science, 1999, 49:115-121.
[47] Paran I, Michelmore RW. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce [J]. Theor Appl Genet, 1993, 85: 985-993.
[48] Garcia G M, Stalker H T, Shroeder E, et al. Identification of RAPD, SCAR, and RFLP markers tightly linked to nematode resistance genes introgressed from Arachis cardenasii into Arachis hypogaea [J]. Genome, 1996, 39(5): 836-845.
[49] Wang JX, Yang GS, Fu TD, et al. Development of PCR- based markers linked to the fertility restorer gene for the polima cytoplasmic malesterility in rapeseed (Brassica napus L) [J]. Journal of Genetics, 2000, 27(11): 1012-1016.
[50] Lu ZY, Sun QX, Li HJ, et al. Molecular identification and marker-assisted selection of Pm21 gene conferring resistance to powdery mildew in wheat [J]. Acta Genetica Sinica, 1999, 26(6):673-682.
[51] Vos P, Hogers R, Bleeker M, et al. A FL P: a new technique for DNA finger printing [J]. Nucleic Acids Research, 1995, 23 (21): 4407-4414.
[52] Dib C, Faure S, Fizames C, et al. A comprehensive genetic map of the human genome based on 5264 microsatellites [J]. Nature, 1996, 380:152-154.
[53] Dietrich WE, Miller JC, Steer RG, et al. A comprehensive genetic map of mouse genome[J]. Nature, 1996, 380:149-152.
[54] Bell CJ, Echer JR. Assignment of 30 microsatellite loci to the linkage map of Arabidopsis [J]. Genomics, 1994, 19:137-144.
[55] Henry J, Robert M, Sterle S, et al. Application of molecular techniques to rice improvement in Austrilia. Sixth International Symposium on Rice Molecular biology[J].Program&Abstract, 1998, 23: 8-16.
[56] Roder M, Plashke J, Koening SU, et al. Abundance, variability and chromosomal location of microsatellites in wheat[J]. Mol Gen Genet, 1995, 246:327-333.
[57] Akkaya MS, Shoemaker RC, Specht JE, et al. Integration of simple sequence repeat DNA markers into a soybean linkage map [J]. Crop Sci, 1995, 35: 1439-1445.
[58] Zietkiewicz E. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification[J]. Genomic, 1994, 20: 176-183.
[59] Olson M, Hood L, Cantor C, et al. A common language for physical mapping of the human genome [J]. Science, 1989, 245:1434-1435.
[60] 龙跃生，陈良碧，夏快飞，等．分子标记技术及其在分离和克隆水稻基因中的应用[J]．生物学杂志，2001，18(2)：29-31．
[61] 徐吉臣，朱立煌，陈英，等．用双单倍体群体构建水稻的分子连锁图[J]．遗传学报，1994，21(3)：205-214．
[62] Michelmore RW, Paran I, Kesseli RV. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations [J]. Proc Natl Acad Sci, 1991, 88(21): 9828-9832.
[63] Paterson AH, DeVerna JW, Lanini B, et al. Fine mapping of quantitative trait lociusing selected overlapping recombinant chromosomes in an interspecies cross of tomato[J]. Genetics, 1990, 124: 735-742.
[64] 李敏，李焕秀，李靖．植物基因克隆技术研究进展[J]．生命科学研究，2004，8(2)：116-120．
[65] 易小平．可供利用的植物基因克隆技术[J]．广西农业生物科学，1999，18(3)：221-224．
[66] Huang N, Angeles ER, Domingo J,et al. Pyramiding of bacterial blight resistance genes in rice: marker-assisted select ion using RFL P and PCR. Theor Appl Genet, 1997, 95:313-320.
[67] 邹琦主编．植物生理学实验指导．中国农业出版社，2000，72-75．
[68] McCouch SR, Kotcket G, Yu ZH, et al. Molecular mapping of rice chromosome. Theror Appl Genet, 1988, 76:815-829.
[69] Lander ES, Green P, Abrahamson J, et al. Mapmaker: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1987, 1:174-181.
[70] Librojo AL, Khush GS. Chromosomal location of some mutant genes through the use of primary trisomics in rice. Rice Genetics. IRRI, Manila, Philippines.1986, 249-255.
[71] 周长海，王宝和，白和盛．粳型光敏核不育系103S的育性表现及其繁殖研究[J]．安徽农业科学，2001，29(2)：133-134．
[72] 王宝和，周长海，白和盛，等．浅谈光敏核不育系选育策略[J]．安徽农业科学，1999，27(4)：310-311．
[73] Wu KS, Tanksley SD. Abundance polymorphism and genetic mapping of microsatellites in rice [J]. Mol Gen Genet, 1993, 241: 225-235.
[74] Temnykh S, Park WD, et al. Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.) [J]. Thero Appl Genet, 2000, 100(5):697-712.
[75] Temnykh S, DeClerck G, Lukashova A, et al. Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential [J]. Genome Res, 2001, 11(8): 1441-1452.
[76] 石明松．对光照长度敏感的隐性雄性不育水稻的发现及初步研究[J]．中国农业科学，1985，(1)：44-48．
[77] 李泽炳，毕春群，万经猛，等．盛夏低温对光敏核不育水稻育性稳定性的干扰及其克服的预见性对策[J]．华中农业大学学报，1990，9(4)：343-347．
[78] 袁隆平．选育光-温敏不育系的技术策略[J]．杂交水稻，1992，(1)：1-5．
[79] 舒庆尧，夏英武，左晓旭，等．二系杂交水稻制繁种中利用标记辅助去杂技术[J]．浙江农业大学学报，1996，22(1)：56-60．
[80] 曹立勇，钱前，朱旭东，等．紫叶标记籼型光-温敏核不育系中紫S的选育及其配组的杂种优势[J]．作物学报，1999，25(1)：44-49．
[81] Wang B, Lan T, Wu WR, et al. Mapping of QTLs Controlling Chlorophyll Content in Rice[J]. Acta Cenetica Sinica, 2003, 30(12): 127-1132.
[82] 冯双华．水稻叶绿素含量的简易测定[J]．福建农业科技，1997，4：7-8．
[83] 章志兴，徐开盛，陈俊涛，等．叶色标记技术在杂交水稻种子生产中的应用[J]．种子科技，2001，1：33-34．
[84] 潘学彪，陈宗祥，董桂春，等．一种紫色水稻的遗传及其在光敏不育系育种中应用的研究[J]．遗传，1995，17(3)：31-34．