摘要
黄瓜植株茎、叶、卷须、花萼、子房表面均覆有短刚毛,果实均有刺,通过组织学观察,发现黄瓜叶片上的刚毛和果实上的果刺形态结构一样,均为多细胞无腺体的表皮毛。果实是黄瓜经济性状最重要的部分,而果刺是果实外观品质重要的性状。
黄瓜EST序列数量的迅速增加为开发新的SSR标记提供了宝贵的数据资源。本论文从GenBank上公布的黄瓜EST序列中检索到902条来自果实的序列,利用SSRIT软件分析得到63条包含SSR的EST序列,设计了37对EST-SSR引物,结果显示有9对引物在7份黄瓜自交系上扩增有多态,占所设计引物总数的24.3%,平均等位变异数为2.1个,7份自交系间C8和S06遗传相似系数最小为0.22。有27对引物在3份甜瓜上有扩增产物,其中1对引物扩增有多态。这为今后黄瓜基因组研究提供了新的标记。
利用中国华北类型的黄瓜自交系S94构建了一个BAC文库,该文库包含约19200个克隆,平均插入片段为105 kb,大约覆盖黄瓜基因组的5倍。为了使黄瓜的遗传连锁群锚定其染色体,从黄瓜的7个连锁群共选择了22个标记,其中15个SCAR标记和7个SSR标记。利用这些标记用PCR的方法筛选BAC文库的DNA池,15个标记筛选到至少2个克隆,共筛选到60个BAC克隆,最后确定了22个BAC克隆作为连锁群特异克隆,这个BAC文库的构建为今后黄瓜基因组研究奠定了基础。本论文利用欧洲温室类型自交系S06(母本)与黄瓜无毛(果刺)突变体gl(父本)构建了分离群体。通过F2和BC1群体的遗传分析,结果表明有毛(Gl)为显性,无毛(gl)为隐性,并且果实、茎、叶、等表面的表皮毛性状为一对核基因控制。同时控制果实、茎、叶、表皮毛性状的无毛基因(gl)对控制果瘤性状的果瘤基因(Tu)存在隐性上位作用。用SRAP标记结合BSA法进行黄瓜果刺形成基因的定位,找到两个与Gl连锁的SRAP标记,其中包括一个与Gl基因紧密连锁的标记ME4EM3,连锁距离为3.2cM,并将该标记转化为一个SCAR标记。用这个SCAR标记筛选黄瓜BAC文库,进行了BAC末端测序,为进一步精细定位与分离果刺形成基因奠定基础。
为了克隆控制黄瓜果刺形成相关的基因,本论文利用棉花和拟南芥TTG1基因进行同源克隆,得到一个CsTTG1基因。半定量RT-PCR分析表明,该基因在重要的分生组织及器官中都有表达。Southern blot实验表明,该基因在黄瓜中为单基因。功能互补实验表明CsTTG1能互补拟南芥ttg1突变体的表型,这个结果表明,黄瓜TTG1-like基因与拟南芥的TTG1基因功能同源。这有助于我们理解多细胞表皮毛形成的分子机制,也有助于认识黄瓜果刺这个外观品质性状。
The wild-type cucumber has trichomes on foliage and spines on fruit. The leaf trichomes and fruit spines have similar shape and structure with histology analysis in cucumber, and both structures are multicellular, non-glandular trichomes. Cucumber fruit is most important part of economic character, and breeding objectives for improvement are often focused on fruit yield and quality. The fruit spine is important trait of fruit appearance quality.
Simple sequence repeat markers derived from expressed sequence tags (EST-SSR) are potentially valuable tools for plant breeding and germplasm collection conservation, and increasingly, efforts have been made for developing this type of marker. In this study, we have identified 9 polymorphic SSR markers from cucumber fruit EST deposited in public sequence database. The average allele number was 2.1 per locus, ranging from two to three alleles during screening 7 cucumber genotypes. Genetic similarity coefficient ranged from 0.22-1, with an average of 0.66 among 7 cucumber genotypes. Amplification products were also detected by 27 pairs of primer in 3 melon genotypes. These informative EST-SSR markers can be used in cucumber genome research.
A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen markers gave at least two positive clones. As a result, twenty-two BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.
To mapping gene of fruit spine formation, we was constructed segregate populations with inbred line S06 (Europe greenhouse type) and glabrous (gl) mutant. The trichoms characteristic of foliage surface was controlled by a pair of nuclear genes. The characteristic of trichomes (Gl) was dominant to that of glabrous foliage (gl) with heredity analysis. The gene of foliage trichomes also controlled fruit spines. The Gl gene together with Tu gene decided fruit surface, which showed three phenotypes on fruit: have warts and spines, only have spines, without warts and spines, with its proportion being 9:3:4. This result indicated the epistatic recessiveness of glgl gene to Tu_ gene. Combining the bulked segregant analysis (BSA) and the sequence-related amplified polymorphism (SRAP) technology, we found two markers linking to the Gl/gl locus. Among them, the one closely linked SRAP markers flanking the Gl/gl locus were marker ME4EM3 with 3.2cM, and then this marker transform to SCAR markers. A BAC library of cucumber will be PCR screened by this marker and get positive BAC clones with end sequence. This work is base for further fine mapping and isolated gene of fruit spine formation.
To identify genes involved in the molecular control of cucumber fruit spine formation, we isolated one putative homologues of the Arabidopsis trichome associated gene TRANSPARENT TESTA GLABRA1 (TTG1). CsTTG1 genes are expressed in many tissues throughout the plant with RT-PCR analysis, including shoot apices and floral buds. The gene used in turn to probe Southern blots of cucumber genomic DNA restricted with various endonucleases. This result showed that cucumber genome contains single copy of CsTTG1 gene.This cucumber TTG1-like gene was able to restore trichome formation in ttg1 mutant Arabidopsis plants. These results indicate that these cotton genes may be functional homologues of AtTTG1. These results helpful understand molecular mechanism about muticelluar trichome pattern.
引文
[1] FAO. Year book production 1992.Food and Agriculture Organization of the United Nations, Rome, Italy, 1993
[2] Malepszy S, Niemirowicz-Szczytt K. Sex determination in cucumber (Cucumis sativus) as a model system for molecular biology. Plant Sci, 1991, 80: 39-47
[3] Arumuganathan K, Earle E D. Nuclear DNA content of some important plant species. Plant Mol Biol Rep, 1991, 9:208-218
[4] Bostein D, White R I, Skolni X K M, et al. Construction of genetic linkage map in man using restriction fragment length polymorphism. Amer Genet, 1993, 32: 314-318
[5] Williams J G K, Kubelik A R, Livak K J, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl Acids Res, 1990, 18: 6531-6535
[6] Li G, Quiros C F. Sequence-related amplified polymorphisim (SRAP), a new maker system based on a simple RCR reaction: Its application to mapping and gene tagging in Brassica. Theor Appl Genet, 2001, 103:455-461
[7] Li G, Gao M, Yang B, Quiros CF. Gene for gene alignment between the Brassica and Arabidopsis genomes by direct transcriptome mapping. Theor Appl Genet, 2003, 107: 168-180
[8] Lin Z X, Zhang X L, Nie Y C, et al. Construction of a genetic Linkage map for cotton based on SRAP. Chinese Science Bulletin, 2003, 48(15): 1676 -1679
[9] Ferriol M, Pico B, Nuez F. Genetic diversity of a germplasm collection of Cucubita pepo using SRAP and AFLP markers. Theor Appl Genet, 2003, 107: 271-282
[10] Wu K S, Tanksley S D. Abundance polymorphism and genetic mapping of microsatellite in rice. MGG, 1993, 241: 226-235
[11] Frageau G J, Fourney R M. DNA typing with short tandem repeat: A sensitive and accurate approach to human identification. Bio Techniques, 1993, 15: 100-119
[12] Sharon D, Adato A, Mhameed S, et al. DNA fingerprint in plant using simple-sequence repeat and minisatellite probes. Hort Science, 1995, 30(1): 109-112
[13] Varshney R K, Graner A, Sorrells M E. Genic microsatellite markers in plants: features and applications. Trends in Biotechnology, 2005, 23: 48-55
[14] Thiel T, Michalek W, Varshney R K. et al. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet, 2003, 106: 411-422
[15] Gao L F, Jing R L, Huo N X, et al. One hundred and one new microsatellite loci derived from ESTs (EST-SSRs) in bread wheat. Theor Appl Genet, 2004, 108:1392-1400
[16] Zabeau M, Vos P. Selective restriction fragment amplification, a general method for DNA fingerprints. European Patent Application Pub, 1993
[17] Vos P , Hogers R , Bleeker M, et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research, 1995, 21(23): 4407-4414
[18] Konieczny A, Ausubel F M. A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. The Plant Journal, 1998, 4(2): 402-410
[19] Cho R J, Mindrinos M, Richards D R, et al. Genome-wide mapping with biallelic markers in Arabidopsis thaliana. Nat Genet,1999, 23: 203-207
[20] Dijkhuizen A, Kennard W C, Havey M J, et al. RFL P variation and genetic relationships in cultivated cucumber. Euphytica, 1996, 90(1): 97-87
[21] Horejsi T, Staub J E. Genetic variation in cucumber (Cucumis sativus L.) as assessed by random amplified polymorphic DNA. Genetic Resources and Crop Evolution, 1999, 46: 337-350
[22]李锡香,朱德蔚,杜永臣,等.黄瓜种质资源遗传多样性及其亲缘关系的AFLP分析.园艺学报, 2004, 31(3): 309-314
[23]李锡香,朱德蔚,杜永臣,等.黄瓜种质资源遗传多样性的RAPD鉴定与分类研究.植物遗传资源学报, 2004, 5(2): 147-152
[24] Katzir N,Danin-Poleg Y,Tzuri G,Karchi Z,Lavi U,Cregan P B. Length polymorphism and homologies of microsatellites in several Cucurbitaceae species. Theor Appl Genet, 1996, 93: 1282-1290
[25] Danin-Poleg Y,Reis N,Tzuri G,et al.Development and characterization of microsatellite markers in Cucumis.Theor Appl Genet,2001,102:61～72
[26]司旻星,关媛,潘俊松,何欢乐,蔡润.黄瓜(Cucumis sativus L.)种质资源遗传多样性及亲缘关系分析.上海交通大学学报(农业科学版),2007,25(2): 130-137
[27] Hjerdin A, Torbjorn S, Hallden C, et al. Isolation of RAPD markers linked to a beet cyst nematode resistance gene using pools of DNA from a segregating population. Plant Genome III Conference. San Diego, USA. 1995
[28]张海英,王永健.黄瓜种质资源遗传亲缘关系的RAPD分析.园艺学报, 1998, 25(4): 345-349
[29] Fazio G, Chung S M, Staub J E. Comparative analysis of response to phenotypic and marker-assisted selection for multiple lateral branching in cucumber (Cucumis sativus L.), Theor Appl Genet, 2003, 107:875-883
[30] Fan Z H, Robbins M D, Staub J E. Population development by phenotypic selection with subsequent marker-assisted selection for line extraction in cucumber (Cucumis sativus L.). 2006, 112(5): 843-855
[31] Staub J E., and Meglic V. Molecular genetic markers and their legal relevance for cultivar discrimination: a case study in cucumber. Hort. Technology, 1993, 3:291-300
[32]方宣钧,吴为人,唐纪良.作物DNA标记辅助育种.北京:科技出版社. 2001, 51
[33] Kennard W C, Poetter K, Dijkuizen A, Meglic V, Staub J E. Linkages among RFLP, RAPD, isozyme, disease, resistance, and morphological markers in narrow and wide crosses of cucumber. Theor Appl Genet, 1994, 89: 42-48
[34] Meglic V, Staub J E. Inheritance and linkage relationships of allozyme and morphological loci in cucumber (Cucumis sativus L.). Theor Appl Genet, 1996, 92: 865-872
[35] Serquen F C, Bacher J, Staub J E. Mapping and QTL analysis of horticultural traits in a narrow cross in cucumber (Cucumis sativus L.) using random amplified polymorphic DNA makers. Molecular Breeding, 1997, 3(4): 257-268
[36] Park Y H, Sensoy S, Wye C. A genetic map of cucumber composed of RAPDs, RFLPs, AFLPs, and loci conditioning resistance to papaya ringspot and zucchini yellow mosaic viruses. Genome, 2000, 43(6): 1003-1010
[37] Bradeen J M, Staub J E, Wye C, Antonise R, Peleman J. Towards an expanded and integrated linkage map of cucumber (Cucumis sativus L.). Genome, 2001, 44:111-119
[38] Fazio G, Staub J E, Stevens M R. Genetic mapping and QTL analysis of horticultural traits in cucumber (Cucumis sativus L.) using recombinant inbred lines. Theor Appl Genet, 2003, 107:864-874
[39]张海英,葛风伟,王永健,许勇,陈青君.黄瓜分子遗传图谱的构建.园艺学报.2004,31(5):617-622
[40] Li X Z,Pan J S,Wang G, et al. Localization of genes for lateral branch and female sex expression and construction of a molecular linkage map in cucumber (Cucumis sativus L.) with RAPD markers.PROGRESS IN NATURAL SCIENCE,2005,15(2):143-148
[41] Pan J S,Wang G,Li X Z, et al. Construction of a genetic map with SRAP markers and localization of the gene responsible for the first-flower-node trait in cucumber (Cucumis sativus L.).PROGRESS IN NATURAL SCIENCE,2005,15(5):407-413
[42] Yuan X J, Li X Z, Pan J S, et al. Genetic linkage map construction and location of QTLs for fruit-related traits in cucumber. Plant Breeding, 2008, 127: 180-188
[43] Wang G, Pan J, Li X, et al. Construction of a cucumber genetic linkage map with SRAP markers and location of the genes for lateral branch traits. Sci China C life Sci. 2005,48(3): 213-220
[44] Fanourakis N E, Simon P W. Analysis of genetic linkage in the cucumber. The Journal of Heredity, 1987, 78(4): 238-242
[45] Pierce L K, Wehner T C. Review of genes and linkage groups in cucumber. HortScience, 1990, 25(6): 605-615
[46] Walters S A, Shetty N V, Wehner T C. Segregation and linkage of several genes in cucumber. J. Amer. Soc. Hort. Sci., 2001, 126 (4): 442-450
[47] Knerr L D, Staub J E. Inheritance and linkage relationships of isozyme loci in cucumber (Cucumis sativus L). Theor Appl Genet, 1992, 84: 217-224
[48] Meglic V, Staub J E. Inheritance and linkage relationships of allozyme and morphological loci in cucumber (Cucumis sativus L). Theor Appl Genet, 1996, 92: 865-872
[49] Xie J H, Wehner T C. 2001 cucumber gene list. http://www.umresearch.umd.edu/CGC/genelist/cucumber.pdf, 2001
[50] Yuan X J, Pan J S, Cai R, et al. Genetic mapping and QTL analysis of fruit and flower related traits in cucumber (Cucumis sativus L.) using recombinant inbred lines. Euphytica, 2008 (under review)
[51] Muehlbauer G J, Specht J E, Thomas-Compton, et al. Near-isogenic lines- A potential resource in the integration of conventional and molecular marker linkage map. Crop Sci, 1988,28:729-735
[52] Michelmore R W, Paranand I, Kessali R V. 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. Proc. Natl. Acad. Sci., 1991, 88: 9828-9832
[53] Mohan M, Nair S, Bentur J S, et al. RFLP and RAPD mapping of the rice Gm2 gene that confers resisitance to biotype 1 of a gall midge (Orseolia oryzae). Theor Appl Genet, 1994, 87: 782-788
[54]朱立煌,徐吉臣,陈英,等.用分子标记定位一个未知的抗稻瘟病基因.中国科学(B辑),1994, 24: 1048-1052
[55] Nam Y W, Lee J R, Song K H, et al. Construction of two BAC libraries from cucumber (Cucumis sativus L.) and identification of clones linked to yield component quantitative trait loci. Theor Appl Genet, 2005, 111: 150-161
[56] Gutman W, Pawe?kowicz M, Woycicki R, Piszczek E, Przybecki Z. The construction and characteristics of a BAC library for Cucumis sativus L.‘B10’. Cellular & Molecular Biology Letters, 2008, 13(1): 74-91
[57] Meyer J DF, Deleu W, Garcia-Mas J, et al.Construction of a fosmid library of cucumber ( Cucumis sativus ) and comparative analyses of the eIF4E and eIF(iso)4E regions from cucumber and melon (Cucumis melo). Molecular Genetics and Genomics, 2008, Doi:10.1007/s00438-008-0326-5
[58] Guan Y, Chen Q, Pan J S, et al. Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones. Progress in Natural Science, 2008, 18(2): 143-147
[59]候峰.黄瓜.天津科学技术出版社.天津:1999,78
[60] Poole C F. Genetics of cultivated cucurbits. J. Hered., 1944, 35:122-128
[61] Andeweg J M. The breeding of scab-resistant frame cucumbers in theNetherlands. Euphytica, 1956, 5:185-195
[62]曹辰兴,郭红芸.黄瓜突变新类型-无毛黄瓜.中国蔬菜,1999,(4):29
[63]曹辰兴,张松,郭红芸.黄瓜茎叶无毛性状与果实瘤刺性状的遗传关系.园艺学报,2001,28 (6):565-566
[64]曹辰兴,张松,郭红芸,郭延奎.黄瓜无毛突变体叶片叶绿体超微结构与光合特性.园艺学报,2002,29 (2):145-148
[65]马德华,庞金安,温晓刚,李淑菊,霍振荣,林世青.黄瓜无毛突变体的生理特性研究.园艺学报,2002,29(3):282-284
[66]戚春章,胡建平.软毛无卷须黄瓜突变株的性状研究.园艺学报, 1989, 16(2): 123-126
[67] Werker E. Trichome diversity and development. in: Advances in Botanical Research incorporating Advances in Plant Pathology, Vol 31 Academic Press, San Diego 2000, 1-35
[68] Wilkins T A, Rajasekaran K, Anderson D M. Cotton biotechnology. Crit. Rev. Plant Sci. 2000, 19: 511-550
[69] Larkin J C, Brown M L, Schiefelbein J. How do cells know what they want to be when they grow up? Lessons from epidermal patterning in Arabidopsis. Annu. Rev. Plant Biol., 2003, 54: 403-430
[70] Marks, M D, Feldmann K A. Trichome development in Arabidopsis thaliana. I. T-DNA tagging of the GLABROUS1 gene. Plant Cell, 1989, 1: 1043-1050
[71] Oppenheimer D G, Herman P L, Sivakumaran S, et al. A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules. Cell, 1991, 67: 483-493
[72] Koornneef M. The complex syndrome of ttg mutants. Arabidopsis Inform. Serv., 1981, 18: 45-51
[73] Marks M D, Feldmannb K A. Trichome Development in Arabidopsis thaliana.1. T-DNA Tagging of the GLABROUSI Gene. Plant Cell, 1989, 1: 1043-1050
[74] Galway M E, Masucci J D, Lloyd A M, et al. The TTG gene is required to specify epidermal cell fate and cell patterning in the Arabidopsis root. Dev. Biol., 1994, 166: 740-754
[75] Walker A R, Davison P A, Bolognesi-Winfield A C, et al. The TRANSPARENT TESTA GLABRA1 Locus, Which Regulates Trichome Differentiation and Anthocyanin Biosynthesis in Arabidopsis, Encodes a WD40 Repeat Protein. Plant Cell, 1999, 11: 1337-1349
[76] Payne C T, Zhang F, Lloyd A M. GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1. Genetics, 2000, 156: 1349-1362
[77] Kirik, V. Schnittger A, Radchuk V, et al. Ectopic expression of the Arabidopsis AtMYB23 gene induces differentiation of trichome cells. Development, 2001, 235, 366-377
[78] Kirik, V. Lee M M, Wester K, et al. Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation. Development, 2005, 132, 1477-1485
[79] Zhang F, Gonzalez A, Zhao M, et al. A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development, 2003, 130: 4859-4869
[80] Schnittger A, Jurgens G, Hulskamp M. Tissue layer and organ specificity of trichome formation are regulated by GLABRA1 and TRIPTYCHON in Arabidopsis. Development, 1998,125: 2283-2289
[81] Schnittger A, Folkers U, Schwab B, et al. Generation of a spacing pattern: the role of TRIPTYCHON in trichome patterning in Arabidopsis. Plant Cell, 1999, 11:1105-1116
[82] Schellmann S, Schnittger A, Kirik V, et al. TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis. EMBO J., 2002, 21: 5036-5046
[83] Kirik V, Simon M, Huelskamp M, et al. The ENHANCER OF TRY AND CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis. Dev. Biol., 2004, 268: 506-513
[84] Kirik V, Simon M, Wester K, et al. ENHANCER of TRY and CPC2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis. Plant Mol. Biol., 2004, 55: 389-398
[85] Szymanski D B, Jilk R A, Pollock S M, et al. Control of GL2 expression in Arabidopsis leaves and trichomes. Development, 1998, 125: 1161-1171
[86] Wang S, Wang J W, Yu N, et al. Control of plant trichome development by a cotton fiber MYB gene. Plant Cell, 2004, 16: 2323-2334
[87] Humphries J A, Walker A R, Timmis J N, Orford S J. Two WD-repeat genes from cotton are functional homologues of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (TTG1) gene. Plant Molecular Biology, 2005, 57: 67-81
[88] Serna L, Martin C. Trichomes: different regulatory networks lead to convergent structures. TRENDS in plant science, 2006, 11: 274-280
[89] Suo J, Liang X, Pu L, et al. Identification of GhMYB109 encoding a R2R3 MYB transcriptional factor that expresses specifically in fiber initials and elongating fibers of cotton (Gossypium hirsutum L.). Biochim. Biophys. Acta, 2003, 1630: 25–34
[90] Noda K, Glover B J, Linstead P, et al. Flower colour intensity depends on specialized cell shape controlled by a Myb-related transcription factor. Nature, 1994, 369: 661-664
[91] Glover B J, Perez-Rodriguez M, Martin C. Development of several epidermal cell types can be specified by the same MYB-related plant transcriptional factor. Development, 1998, 125: 3497-3508
[92] Martin C, Bhatt K, Baumann, K, et al. The mechanics of cell fate determinationin petals. Philos. Trans. R. Soc. Lond. B Biol. Sci., 2002, 357: 809-813
[93] Martin C, Paz-Ares J. MYB transcription factors in plants. Trends Genet., 1997, 13: 67-73
[94] Payne T, Clement J, Arnold D, Lloyd A. Heterologous myb genes distinct from GL1 enhance trichome production when overexpressed in Nicotiana tabacum. Development, 1999, 126: 671-682
[95] Glover B J, Bunnewell S, Martin C. Convergent evolution within the genus Solanum: the specialised anther cone develops through alternative pathways. Gene, 2004, 331: 1-7
[96] van Houwelingen A, Souer E, Spelt K, et al. Analysis of flower pigmentation mutants generated by random transposon mutagenesis in Petunia hybrida. Plant J., 1998, 13: 39–50
[97] Perez-Rodriguez M, Jaffe F W, Butelli E, et al. Development of three different cell types is associated with the activity of a specific MYB transcription factor in the ventral petal of Anthirrhinum majus flowers. Development, 2005, 132: 359-370
[98] Lloyd A M, Walbot V, Davis R W. Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science, 1992, 258: 1773-1775
[99] Spelt C, Quatrocchio F, Mol J N M, et al. anthocyanin1 of petunia encodes a basic-helix loop helix protein that directly activates structural anthocyanin genes. Plant Cell, 2000, 12: 1619-1631
[100] Mooney M, Desnos T, Harrison K, et al. Altered regulation of tomato and tobacco pigmentation genes caused by delila gene of Antirrhinum. Plant J., 1995, 7: 333-339
[101] Spelt C, Quattrocchio F, Mol J, et al. ANTHOCYANIN1 of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanism. Plant Cell, 2002, 14: 2121-2135
[102] de Vetten N, Quattrocchio F, Mol J, et al. The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast, plants, and animals. Genes Dev., 1997, 11: 1422-1434
[103] Carey C C, Strahle J T, Selinger D A, et al. Mutations in the pale aleurone color1 regulatory gene of the Zea mays anthocyanin pathway have distinct phenotypes relative to the functionally similar TRANSPARENT TESTA GLABRA1 gene in Arabidopsis thaliana. Plant Cell, 2004, 16: 450-464
[104] Fazio G., Staub J E, Chung S M, et al. Development and characterization of PCR markers in cucumber. Amer. Soc. Hort. Sci., 2002, 127(4): 545-557
[105] Kong Q, Xiang C, Yu Z. Development of EST-SSRs in Cucumis sativus from sequence database. Molecular Ecology Notes, 2006, 6: 1234-1236
[106] Clark M S. Plant molecular Biology-A Laboratory Manual. Heidelberg: Springer-Verlag Berlin, 1997. 4～6
[107] Nei M, Li W H. Mathematical model for studying genetic variation in terms ofrestriction endonucleases. Proceeding of the National Academy of Science of USA, 1979, 76: 5269-5273
[108] Scott K D, Eggler P, Seaton G. Analysis of SSRs derived from grape ESTs, Theor Appl Genet, 2000, 100: 723-726
[109] Gonzalo M J, Oliver M, Garcia-Mas J, et al. Simple-sequence repeat markers used in merging linkage maps of melon (Cucumis melo L.), Theor Appl Genet, 2005, 110: 802-811
[110] Frary A, Nesbitt T C, Grandillo S, et al. fw2.2: a quantitative trait locus key to the evolution of tomato fruit size. Science, 2000, 289:85-88
[111] Li X Y, Qian Q, Fu Z M, et al. Control of tillering in rice. Nature, 2003, 422: 618-621
[112] Jiang J, Gill B S. Nonisotopic in situ hybridization and plant genome mapping: the first ten years. Genome, 1994, 37:717-725
[113] Jiang J, Gill B S, Wang G L, et al. Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes. Proc Natl Acad Sci USA, 1995, 92:4487-4491
[114] Lapitan N L V, Brown S E, Kennard W, et al. FISH physical mapping with barley BAC clones. Plant J, 1997, 11:149-156
[115] Song J, Dong F, Jiang J. Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research. Genome, 2000, 43:199-204
[116] Lee H R, Eom E M, Lim Y P, et al. Construction of a garlic BAC library and chromosomal assignment of BAC clones using the FISH technique. Genome, 2003, 46:514-520
[117] Hoshi Y, Plader W, Malepszy S. New C-banding pattern for chromosome identification in cucumber (Cucumis sativus L.). Plant Breeding, 1998, 117: 77-82
[118] Hoshi Y, Plader W, Malepszy S. Physical mapping of 45S rRNA gene loci in the cucumber (Cucumis sativus L.) using fluorescence in situ hybridization. Caryologia, 1999, 52: 49-57
[119] Chen J F, Staub J E, Jiang J. A reevaluation of karyotype in cucumber (Cucumis sativus L.). Genet. Resour. Crop. Evol., 1998, 45: 301-305
[120] Chen J F, Staub J E, Adelberg J W, et al. Physical mapping of 45S rRNA genes in Cucumis species by fluorescence in situ hybridization. Can. J. Bot, 1999, 77: 389-393
[121] Koo D H, Hur Y, Jin D C, et al. Karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) using C-banding and bicolor fluorescence in situ hybridization. Mol. Cells, 2002, 13: 413-418
[122] Koo D H, Choi H W, Cho J, et al. A high-resolution karyotype of cucumber (Cucumis sativus L.‘Winter Long’) revealed by C-banding, pachytene analysis, and RAPD-aided fluorescence in situ hybridization. Genome, 2005, 48: 534-540
[123] Zhang H B, Zhao X, Ding X, et al. Preparation of megabase-size DNA from plantnuclei. Plant J, 1995, 7: 175-184
[124] Luo M, Wing R A. An improved method for plant BAC library construction. Methods Mol Biol, 2003, 236:3-20
[125]陈琪,邓一文,黄彬彬,等.抗稻瘟病水稻细菌人工染色体文库的构建与鉴定.上海大学学报(自然科学版), 2007, 13(3): 325-330
[126] Sakata Y. Kubo N. Morishita M. et al. QTL analysis of powdery mildew resistance in cucumber (Cucumis sativus L.). Theor Appl Genet, 2006,112(2): 243-250
[127] Ota T, Amemiya C T. A nonradioactive method for improved restriction analysis and fingerprinting of large P1 artificial chromosome clones. Genet Anal, 1996, 12: 173-178
[128] Shizuya H, Kouros-Mehr H. The development and applications of the bacterial artificial chromosome cloning system. Keio J Med, 2001, 50(1):26-30
[129] Luo M, Wang Y, Frisch D, et al. Melon bacterial artificial chromosome (BAC) library construction using an improved method and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2). Genome, 2001, 44:154-162
[130]王桂玲,秦智伟,周秀艳,等.黄瓜果瘤的遗传及SSR标记.植物学通讯,2007, 24(2): 168-172
[131]朱正歌,贾继增,孙宗修. AFLP指纹银染法显带研究.中国水稻科学,2002, 16(1): 71-73
[132] Lander E.S, Green P, Abrahamson J, et al. MAPMARKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. 1987
[133]刘仁虎,孟金陵. MapDraw,在Excel中绘制遗传连锁图的宏.遗传, 2003, 25(3): 317-321
[134] Robinson R W. Chlorosis induced in glabrous cucumber by high temperature. Cucurbit Genet. Coop. Rpt., 1987, 10: 7
[135] Rahman M, McVetty P B E, Li G. Development of SRAP, SNP and multiplexed SCAR molecular markers for the major seed coat color gene in Brassica rapa L.. Theor Appl Genet, 2007, 115: 1101-1107
[136] Neer E J, Schmidt C J, Nambudripad R, et al. The ancient regulatory-protein family of WD-repeat proteins. Nature, 1994, 371: 297-300
[137] Holton A T, Cornish E C. Genetics and Biochemistry of Anthocyanin Biosynthesis. Plant Cell, 1995, 7: 1071-1083
[138] Pelletier M K, Shirley B W. Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Plant Physiol., 1996, 111: 339-345
[139] Ludwig S R, Habera L F, Dellaporta S L, et al. Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcription activators and contains the myc-homology region. Proc. Natl.Acad. Sci. USA, 1989, 86: 7092:7096
[140] Goodrich J, Carpenter R, Coen E S. A common gene regulates pigmentation pattern in diverse plant species. Cell, 1992, 68: 955-964
[141] Goff S A, Cone K L, Chandler V L. Functional analysis of the transcriptional activator encoded by the maize B gene: Evidence for a direct functional interaction between two classes of regulatory proteins. Genes Dev., 1992, 6: 864-875
[142] Lesnick M L, Chandler V L. Activation of the maize anthocyanin gene a2 is mediated by an element conserved in many anthocyanin promoters. Plant Physiol. 1998, 117: 437-445
[143] Nicholas K B, Nicholas H B Jr., Deerfield D W. II. GeneDoc: Analysis and Visualization of Genetic Variation, EMBNEW.NEWS, 1997, 4:14
[144] Deleage G, Combet C, Blanchet C, et al. ANTHEPROT: An integrated protein sequence analysis software with client/server capabilities. COMPUTERS IN BIOLOGY AND MEDICINE, 2001, 31 (4): 259-267
[145]萨姆布鲁克J,弗里奇E F,曼尼阿蒂斯T.分子克隆实验指南第二版.北京:科学出版社, 1999, 474-490
[146] Clough S J, Bent A F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 1998, 16: 735-743
[147] Yu L H, Gaitatzes C, Neer, E, et al. Thirty-plus functional families from a single motif. Prot. Sci., 2000, 9: 2470-2476
[148] Li D, Roberts R. WD-repeat proteins: structure characteristics, biological function, and their involvement in human diseases. Cell Mol. Life Sci., 2001, 58: 2085-2097
[149] Sompornpailin K, Makita Y, Yamazaki M, et al. A WD-repeat-containing putative regulatory protein in anthocyanin biosynthesis in Perilla frutescens. Plant Mol. Biol., 2002, 50: 485-495