黄瓜成熟瓜网纹基因H遗传定位及候选基因分析

doi:10.3864/j.issn.0578-1752.2014.08.011

摘要/Abstract

摘要： 【目的】有无网纹是黄瓜果实生理成熟后的重要表型性状之一，对其控制基因进行遗传定位和候选基因分析，为黄瓜果实性状改良提供理论依据和技术支撑，同时也可为网纹基因的精细定位及克隆奠定基础。【方法】利用成熟瓜无网纹黄瓜自交系 PI205996（P1）和成熟瓜有网纹自交系 PI263079（P2）为亲本构建不同遗传群体，进行网纹性状遗传分析。以包含230个单株的F2分离群体为试材，应用分离群体分组分析（BSA）法和2 112对SSR引物进行SSR分析，采用 JoinMap 4.0 作图软件和 MapInspect 软件构建成熟瓜网纹基因的SSR连锁群，并完成其染色体的初步定位。结合9 930黄瓜全基因组序列信息和115份核心种质重测序结果，利用Primer6.0 软件开发设计新标记，对初步定位区域进行标记加密，利用生物信息学的相关信息对定位区域进行候选基因分析。【结果】研究表明，黄瓜成熟瓜有网纹自交系 PI263079的有网纹性状是由显性单基因（H）控制的，有网纹对无网纹为显性。从2 112对SSR引物中筛选出255对在亲本间表现出多态性的引物，多态率为12.1%。利用亲本间具有多态性的引物对有网纹和无网纹各7个单株DNA进行分析，筛选获得了9对与H基因连锁的SSR标记，将H初步定位在黄瓜5号染色体（Chr.5）上，侧翼标记分别为SSR13006和CSWCT-17，遗传距离分别为3.6 cM 和8.2 cM。根据初步定位区域的序列信息，设计合成了97对新的SSR引物，其中4对引物在亲本间表现出多态，多态率为4.1%。利用这4对新的多态性SSR引物对亲本和F2群体的DNA 进行分析，最终构建了一张包含13个SSR标记的H分子标记连锁群，获得了与H最近的侧翼标记SSR13006和SSRH-90，遗传距离分别为3.6 cM 和1.7 cM。利用黄瓜全基因组测序提供的基因预测和注释结果，发现基因H所在区段的物理距离为297.7 kb，存在29个候选基因。根据前人研究结果，推测Csa5G591790是与成熟瓜网纹形成相关性较大的候选基因。【结论】黄瓜自交系PI263079的成熟瓜有网纹性状由显性单基因H控制，该基因位于黄瓜第5号染色体长臂的297.7 kb区段内，侧翼标记分别为SSR13006 和SSRH-90，遗传距离分别为3.6 cM 和1.7 cM。本研究为H 基因的精细定位和克隆奠定了良好基础，也为黄瓜成熟瓜网纹性状的分子标记辅助选择育种提供了理论参考。

关键词: 黄瓜 , 成熟瓜网纹 , SSR标记 , 基因定位 , 候选基因

Abstract: 【Objective】 Heavy netting of mature fruit is one of the important phenotypes of cucumber. Molecular mapping and candidate gene analysis for this trait will provide a theoretical basis for improvement of fruit traits. It also can lay a good foundation for fine mapping and gene cloning. 【Method】 Cucumber inbred lines PI205996(P1) without heavy netting mature fruit and PI263079 (P2) with heavy netting mature fruit were used as the experiment materials for genetic analysis and gene mapping in this study. Bulked segregation analysis (BSA) was performed in the F2 population with 230 individuals using 2 112 SSR markers. The sequence and re-sequencing information of 9 930 and 115 core germplasms were used to develop new SSR markers in the primary mapping region of the heavy netting gene by Primer 6.0. JoinMap 4.0 and MapInspect softwares were employed to construct a SSR linkage map for the gene. Bioinformatics analysis was conducted to detect candidate genes. 【Result】 Genetic analysis showed that a single dominant nuclear gene (H) controlled the heavy netting of mature fruit trait in PI263079. There were 255 of 2 112 SSR primers showed polymorphism in the parent lines and the polymorphic rate was 12.1%. Using the 255 polymorphic markers, the DNA of seven individuals with heavy netting fruit and seven individuals without heavy netting fruit was analyzed. Nine SSR markers on cucumber chromosome 5 were identified to be linked with the H gene. Flanking markers SSR13006 and CSWCT-17 were 3.6 and 8.2 cM away from the H gene, respectively. A total of 97 pairs of new SSR primers were developed based on the sequence information in the primary mapping region of the H gene. And only four polymorphic markers were detected between the parent lines with the polymorphic rate of 4.1%. Finally, 13 SSR markers were identified to be linked with the H gene after analysis of the F2 mapping population using the four new developed molecular markers. Flanking markers SSR13006 and SSR-90 were 3.6 and 1.7 cM away from the H gene, respectively. The physical distance between SSR13006 and SSRH-90 was 297.7 kb containing 29 predicted genes. Csa5G591790 was speculated as a possible candidate gene. 【Conclusion】 The heavy netting of mature fruit trait of PI263079 was controlled by one dominant nuclear gene (H). It was located on the Chr.5 of cucumber delimited in a physical distance of 297.7 Kb. The results in this study will be of great benefit to fine mapping and gene cloning for the H gene and MAS of heavy netting of mature fruit.

Key words: Cucumis sativus L. , heavy netting of mature fruit , SSR marker , gene mapping , candidate gene

王敏1, 顾兴芳1, 苗晗1, 刘书林1, 王烨1, ToddC.Wehner2, 张圣平1. 黄瓜成熟瓜网纹基因H遗传定位及候选基因分析[J]. 中国农业科学, 2014, 47(8): 1550-1557.

WANG Min-1, GU Xing-Fang-1, MIAO Han-1, LIU Shu-Lin-1, WANG Ye-1, ToddC.Wehner2 , ZHANG Sheng-Ping-1. Molecular Mapping and Candidate Gene Analysis for Heavy Netting Gene (H) of Mature Fruit of Cucumber (Cucumis sativus L.)[J]. Scientia Agricultura Sinica, 2014, 47(8): 1550-1557.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.08.011

https://www.chinaagrisci.com/CN/Y2014/V47/I8/1550

参考文献

[1]孙晓丹, 商庆梅, 秦智伟. 黄瓜嫩果白色果皮颜色遗传规律及其AFLP标记研究. 北方园艺, 2011, 3: 135-140.

Sun X D, Shang Q M, Qin Z W. Inheredity of white skin color cucumber immature fruit and AFLP analysis. North Horticulture, 2011, 3: 135-140. (in Chinese)

[2]董邵云, 苗晗, 张圣平, 刘苗苗, 王烨, 顾兴芳. 黄瓜白色果皮基因遗传规律及基因定位.西北植物学报, 2012, 32 (11): 2177-2181.

Dong S Y, Miao H, Zhang S P, Liu M M, Wang Y, Gu X F. Genetic analysis and gene mapping of white fruit skin in cucumber(Cucumis sativus L.). Acta Botanica Boreali-Occidentalia Sinica, 2012, 32(11): 2177-2181. (in Chinese)

[3]Miao H, Zhang S P, Wang X W, Zhang Z H, Li M, Mu S Q, Cheng Z C, Zhang R W, Huang S W, Xie B Y, Fang Z Y, Zhang Z X, Weng Y Q, Gu X F. A linkage map of cultivated cucumber (Cucumis sativus L.) with 248 microsatellite marker loci and seven genes for horticulturally important traits. Euphytica , 2011, 182: 167-176.

[4]董邵云, 苗晗, 张圣平, 王烨, 王敏, 刘书林, 顾兴芳. 黄瓜果皮光泽性状的遗传分析及基因定位研究. 园艺学报, 2013, 40 (2): 247-254.

Dong S Y, Miao H, Zhang S P, Wang Y, Wang M, Liu S L, Gu X F. Genetic analysis and gene mapping of glossy fruit skin in cucumber. Acta Horticulturae Sinica, 2013, 40 (2): 247-254. (in Chinese)

[5]Wei-Wei Zhang, Jun-Song Pan, Huan-Le He , Chi Zhang, Zheng Li, Jun-Long Zhao, Xiao- Jun Yuan, Li-Huang Zhu, San-Wen Huang, Run Cai. Construction of a high density integrated genetic map for cucumber(Cucumis sativus L.). Theoretical and Applied Genetics, 2012, 124: 249-259.

[6]Xuqin Yang, Yue Li , Huanle He , Beibei Bie , Guoliang Ren , Junlong Zhao, Yunli Wang, Jingtao Nie, Junsong Pan, Run Cai. Fine mapping of the dull fruit skin gene D in cucumber (Cucumis sativus L.). Molecular Breeding, 2013, Online: DOI 10.1007/ s11032-013-9927-8

[7]Zhang S P, Miao H, Sun R F, Wang X W, Huang S W, Wehner T C, and Gu X F. Localization of a new gene for bitterness in cucumber. Journal of Heredity, 2013, 104(1): 134-139 .

[8]Bo K L, Song H, Shen J, Qian C T, Staub J E, Simon P W, Lou Q F, Chen J F. Inheritance and mapping of the ore gene controlling the quantity of b-carotene in cucumber (Cucumis sativus L.). Molecular Breeding, 2012, 30: 335-344.

[9]Li Y, Wen C, Weng Y. Fine mapping of the pleiotropic locus B for black spine and orange mature fruit color in cucumber identifies a 50 kb region containing a R2R3-MYB transcription factor. Theoretical and Applied Genetics, 2013, 126: 2187-2196.

[10]郁映君,司龙亭. 黄瓜瓜刺颜色与性型遗传规律的研究. 沈阳农业大学学报, 2010, 41 (2): 152-155.

Yu Y J, Si L T. 2010. Inheritance of the spine color and sex expression in cucumber. Journal of Shenyang Agricultural University, 2010, 41(2): 152-155. (in Chinese)

[11]S. Alan Walters, Nischit V. Shetty, Todd C. Wehner. Segregation and linkage of several genes in cucumber. Journal of the American Society for Horticultural Science. 2001, 126(4): 442-450.

[12]Kennard W C, Poetter K, Dijkhuizen A, Meglic V, Staub J E, Havev J. Linkage among RFLP, RAPD, isozyme, disease-resistance and morphological markers in narrow and wide crosses of cucumber. Theoretical and Applied Genetics, 1994, 89: 42-48

[13]Heang D, Sato H, Sassa H, Koba T. Detection of two QTLs for fruit weight in cucumber(Cucumis sativus L.). Proceedings of the IX th EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), 2008, 5: 511-514.

[14]杨双娟, 苗晗, 张圣平, 程周超, 周健, 董邵云, Wehner T C, 顾兴芳. 黄瓜无毛基因gl-2的遗传分析和定位.园艺学报, 2011, 38 (9): 1685-1692.

Yang S J, Miao H, Zhang S P, Cheng Z C, Zhou J, Dong S Y, Wehner T C, Gu X F. Genetic analysis and mapping of gl-2 gene in cucumber(Cucumis sativus L.). Acta Horticulturae Sinica, 2011, 38(9): 1685-1692. (in Chinese)

[15]Zhang W W, He H, Yuan G, Du H, Yuan L H, Li Z, Yao D Q, Pan J S, Cai R. Identification and mapping of molecular markers linked to the tuberculate fruit gene in the cucumber (Cucumis sativus L.). Theoretical and Applied Genetics, 2010, 120(3): 645-654.

[16]Tkachenko, N. N. Preliminary results of a genetic investigation of the cucumber, Cucumis sativus L. Bull. Applied Plant Breeding, Ser. 2, 1935(9): 311-356.

[17]Hutchins, A. E. Inheritance in the cucumber. Journal of Agricultural Research, 1940, 60: 117-128.

[18]苗晗, 顾兴芳, 张圣平, 张忠华, 黄三文, 王烨, 程周超, 张若纬, 穆生奇, 李曼, 张振贤, 方智远. 黄瓜果实相关性状QTL定位分析.中国农业科学, 2011, 44(24): 5031-5040.

Miao H, Gu X F, Zhang S P, Zhang Z H, Huang S W, Wang Y, Cheng Z C, Zhang R W, Mu S Q, Li M, Zhang Z X, Fang Z Y. Mapping QTLs for fruit-associated traits in Cucumis sativus L.. Scientia Aguricultura Sinica, 2011, 44(24): 5031-5040. (in Chinese)

[19]王志坤, 秦智伟, 周秀艳. 黄瓜果实成熟衰老过程中几种物质的变化. 中国蔬菜, 2010, 12: 41-45.

Wang Z K,Qin Z W, Zhou X Y. Changes of several substances during ripening and senescence process of cucumber fruit. Chinese Vegetables, 2010, 12: 41-45. (in Chinese)

[20]李艳秋, 王志坤, 秦智伟, 周秀艳.活体黄瓜成熟衰老过程中的几种生理生化指标变化. 植物生理学通讯, 2006, 42(4): 671-673.

Li YQ, Wang Z K, Qin Z W, Zhou XYan. Changes in several physiological and biochemical indexes of cucumber fruit in vivo during senescence. Chinese Bulletin of Botany, 2006, 42(4): 671-673.(in Chinese)

[21]宋德颖, 秦智伟, 王志坤, 卢京国. 两品种黄瓜果实发育过程中脂氧合酶的活性变化比较.植物生理学通讯, 2007, 43(4): 716.

Song D J, Qin Z W, Wang Z K, Lu J G. The comparison of lipoxygenase activity in the process of fruit growth for two varieties of cucumber. Plant Physiology Communications, 2007, 43(4): 716. (in Chinese)

[22]Ren Y, Zhang Z H, Liu J H, Staub J E, Han Y H, Cheng Z H, Li X F, Miao H, Kang H X, Xie B Y, Gu X F, Wang X W, Du Y C, Jin W W, Huang S W. An integrated genetic and cytogenetic map of the cucumber genome. Plos One, 2009, 4(6): 5795.

[23]Webster B D, Graig M E.Characteristics of the surface of muskmelon. Journal of the American Society for Horticultural Science, 1976, 101: 412-415.

[24]Keren-Keiserman A, Tanami Z, Shoseyov O, Ginzberg I. Peroxidase activity associated with suberization process of the muskmelon (Cucumis melon) rind. Physiology Plant, 2004, 121: 141-148.

[25]Bernards M A, Fleming W D, Llewellyn D B, Priefer R, Yang Xiao-long, Sabatino A, Plourde G L. Biochemical characterization of the suberization-associated anionic peroxidase of potato. Plant Physiology, 1999, 121: 135-145.

[26]Adie B, Chico J M, Rubio-Somoza I, Solano R. Modulation of plant defenses by ethylene. Journal of Plant Growth Regulation, 2007, 26(2): 160-177.

[27]Gerchikov N, Keren-Keiserman A, Per-Treves R, Ginzberg I. Wounding of melon as a model system to study rind netting. Horticulture Science, 2008, 117: 115-122.