黄瓜白粉病抗性基因定位及候选基因分析

doi:10.3864/j.issn.0578-1752.2018.17.017

摘要/Abstract

摘要： 【目的】通过对黄瓜白粉病抗性基因的精细定位，明确抗性基因的候选区段，为进一步克隆基因及解析基因功能奠定基础。【方法】本研究选用高抗白粉病资源（自交系‘74’）和感白粉病资源（自交系‘80’）配制杂交组合，构建F₁、F₂群体，利用单囊壳白粉菌鉴定亲本及群体白粉病抗性并进行遗传分析；采用BSA-seq的方法对黄瓜白粉病抗性基因进行初步定位，在此基础上在候选区段开发分子标记，进一步精细定位白粉病抗性基因。【结果】分离群体单株表型鉴定结果表明，自交系74的抗性是由不完全隐性基因控制的。BSA-seq技术初步将抗性基因定位于5号染色体15—25 Mb位置，命名为PM⁷⁴。最终，利用分子标记将抗性基因定位于SSR15321和SSR07531之间，遗传距离为3.06 cM，物理距离为238 444 bp，可解释41.95%的表型变异。生物信息学分析表明在候选区段内包含有17个候选基因，其中Cucsa.275630为TIR-NBS-LRR类基因。【结论】本研究将黄瓜白粉病抗性基因精细定位到约238 kb的候选区段内，区段内包含有一个TIR-NBS-LRR基因，该基因将是今后研究的重点候选基因。

关键词: 黄瓜, 白粉病, 分离群体分组分析法（BSA）, NBS基因, 分子标记

Abstract: 【Objective】Identification and fine mapping of the candidate region and the gene associated with powdery mildew resistance is significant for gene cloning and functional genomics research in cucumber breeding. 【Method】Powdery mildew resistant cucumber line was identified using single capsule inoculation. Bulked segregant analysis sequencing (BSA-seq) technology was intended to complete the primary mapping of the PMR gene. 【Result】 Resistance identification showed inbred lines 74 exhibited excellent resistance to powdery mildew, in contrast, the inbred lines 80 showed high susceptibility. These two genotypes were used to construct F₁ segregation population which was then extended to F₂ population for an inheritance study. Our results indicated the resistance gene from inbred line 74 was partial-recessive inherited. BSA-seq localized the major resistance gene, named PM⁷⁴, to a 15-25 Mb genomic region in cucumber on chromosome 5. The resistance gene was further located with a 23 844 bp physical distance between SSR15321 and SSR07531 at the genetic distance of 3.06 cM and could explain 41.95% phenotypic variation. 17 annotated genes were found within the predicted candidate region, including one gene belongs to TIR-NBS-LRR gene family, named Cucsa.275630. 【Conclusion】This study mapped a PMR gene within the 238 kb physical interval on Chromosome 5, a TIR-NBS-LRR type gene was identified and would be focused in the future study.

Key words: cucumber, powdery mildew, Bulked Segregant Analysis (BSA), NBS gene, molecular marker

郝俊杰，李磊，王波，秦玉红，崔健，王瑛，王佩圣，江志训，孙吉禄，王珍青，岳欢，张守才. 黄瓜白粉病抗性基因定位及候选基因分析[J]. 中国农业科学, 2018, 51(17): 3427-3434.

HAO JunJie, LI Lei, WANG Bo, QIN YuHong, CUI Jian, WANG Ying, WANG PeiSheng, JIANG ZhiXun, SUN JiLu, WANG ZhenQing, YUE Huan, ZHANG ShouCai . Fine Mapping and Analysis Candidate Gene to Powdery Mildew in Cucumber (Cucumis sativus L.)[J]. Scientia Agricultura Sinica, 2018, 51(17): 3427-3434.

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.0578-1752.2018.17.017

https://www.chinaagrisci.com/CN/Y2018/V51/I17/3427

参考文献

[1] MORISHITA M, SUGIYAM A K, SAITO T, SAKATA Y. Powdery mildew resistance in cucumber. Japan Agricultural Research Quarterly, 2003, 37(1): 7-14.

[2] SAKATA Y, KUBO N, MORISHITA M, KITADANI E, SUGIYAMA M, HIRAI M. QTL analysis of powdery mildew resistance in cucumber (Cucumis sativus L.). Theoretical and Applied Genetics, 2006, 112: 243-250.

[3] 张桂华, 杜胜利, 王鸣, 马德华. 与黄瓜抗白粉病相关基因连锁的AFLP标记的获得. 园艺学报, 2004, 31(2): 189-192.

ZHANG G H, DU S L, WANG M, MA D H. AFLP markers of cucumber powdery mildew resistance-related gene. Acta Horticulturae Sinica, 2004, 31(2): 189-192. (in Chinese)

[4] 刘龙洲, 何欢乐, 潘俊松, 杜辉, 蔡润. 黄瓜种质R17对白粉病抗性的遗传测验. 种子, 2008, 27(3): 46-48.

LIU L Z, HE H L, PAN J S, DU H, CAI R. Genetic test of resistance to powdery mildew for cucumber germplasm R17. Seed, 2008, 27(3): 46-48. (in Chinese)

[5] SHANMUGASUNDARAM S, WILLIAMS P H, PETERSON C E. Inheritance of resistance to powdery mildew in cucumber. Phytopathology, 1971, 61(10): 1218-1221.

[6] SHANMUGASUNDARAM S, WILLIAMS P H, PETERSON C E. A recessive cotyledon marker gene in cucumber with pleiotropic effects. HortScience, 1972, 7: 555-556.

[7] 简德明. 黄瓜白粉病抗性基因紧密连锁的 AFLP 分子标记研究[D]. 北京: 首都师范大学, 2007.

JIAN D M. Study on linked AFLP markers with resistant gene of powdery mildew in cucumber (Cucumis sativus L.) [D]. Beijing: Capital Normal University, 2007. (in Chinese)

[8] 王振国. 黄瓜白粉病抗性基因遗传规律和相关分子标记的研究[D]. 哈尔滨: 东北农业大学, 2007.

WANG Z G. Study on genetic and molecular markers of resistant gene to powdery mildew in cucumber (Cucumis sativus L.) [D]. Harbin: Northeast Agricultural University, 2007. (in Chinese)

[9] MUNGER H M, MORALES A, OMARA S. Dominant genes for resistance to powdery mildew in cucumber. Cucurbit Genetics Cooperative Report, 1979, 2(10): 10-15.

[10] 沈丽平. 黄瓜白粉病抗性遗传分析及相关QTL初步定位[D]. 扬州: 扬州大学, 2009.

SHEN L P. Genetic analysis and preliminary mapping of QTL associated with powdery mildew resistance in cucumber [D]. Yangzhou: Yangzhou University, 2009. (in Chinese)

[11] 刘盼娜. 黄瓜茎蔓抗白粉病基因的定位研究[D]. 北京: 中国农业科学院, 2016.

LIU P N. Study on genetic mapping of resistance to powdery mildew in cucumber stem [D]. Beijing: Chinese Academy of Agricultural Sciences, 2016. (in Chinese)

[12] 刘龙洲, 蔡润, 袁晓君, 何欢乐, 潘俊松. 黄瓜抗白粉病QTL分子标记定位. 中国科学(C辑:生命科学), 2008, 38(9): 851-856.

LIU L Z, CAI R, YUAN X J, HE H L, PAN J S. QTL mapping of resistance markers to powdery mildew in cucumber (Cucumis sativus L.). Science in China Series (C: Life Sciences), 2008, 38(9): 851-856. (in Chinese)

[13] 张圣平, 刘苗苗, 苗晗, 张素勤, 杨宇红, 谢丙炎, 顾兴芳. 黄瓜白粉病抗性基因的QTL定位. 中国农业科学, 2011, 44(17): 3584-3593.

ZHANG S P, LIU M M, MIAO H, ZHANG S Q, YANG Y H, XIE B Y, GU X F. QTL mapping of resistance genes to powdery mildew in cucumber (Cucumis sativus L.). Scientia Agricultura Sinica, 2011, 44(17): 3584-3593. (in Chinese)

[14] SINGH V K, KHAN A W, JAGANATHAN D, THUDI M, ROORKIWAL M, TAKAGI H, GARG V, KUMAR V, CHITIKINENI A, GAUR P M, SUTTON T, TERAUCHI R, VARSHNEY R K. QTL-seq for rapid identification of candidate genes for 100-seed weight and root/total plant dry weight ratio under rainfed conditions in chickpea. Plant Biotechnology Journal, 2016, 14: 2110-2119.

[15] JARVIS D E, HO Y S, LIGHTFOOT D J, SCHMÖCKEL S M, LI B, BORM T J A, OHYANAGI H, MINETA K, MICHELL C T, SABER N, KHARBATIA N M, RUPPER R R, SHARP A R, DALLY N, BOUGHTON B A, WOO Y H, GAO G, SCHIJLEN EGWM, GUO X, MOMIN A A, et al. Corrigendum: The genome of Chenopodium quinoa. Nature, 2017, 542: 307-312.

[16] TAKAGI H, ABE A, YOSHIDA K, KOSUGI S, NATSUME S, MITSUOKA C, UEMURA A, UTSUSHI H, TAMIRU M, TAKUNO S, INNAN H, CANO L M, KAMOUN S, TERAUCHI R. QTL-seq: Rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. The Plant Journal, 2013, 74: 174-183.

[17] HUANG S, LI R, ZHANG Z, LI L, GU X, FAN W, LUCAS W J, WANG X, XIE B, NI P, REN Y, ZHU H, LI J, LIN K, JIN W, FEI Z, LI G, STAUB J, KILIAN A, VAN DER VOSSEN E A, et al. The genome of the cucumber, Cucumis sativus L. Nature Genetics, 2009, 41: 1275-1281.

[18] NY/T 1857.2—2010. 中华人民共和国农业行业标准-黄瓜主要病害抗病性鉴定技术规程第2部分: 黄瓜抗白粉病鉴定技术规程[S]. 北京: 中国农业出版社, 2010: 3-4.

NY/T. 1857.2-2010. The agriculture industry standard of China-Rules for evaluation of cucumber for resistance to diseases Part2: Rule for evaluation of cucumber for resistance to powdery mildew[S]. Beijing: China Agriculture Press, 2010: 3-4. (in Chinese)

[19] 安瑞生, 谭声江, 陈晓峰. 微卫星DNA在分子遗传标记研究中的应用. 昆虫知识, 2002, 39(03): 165-172.

AN R S, TAN S J, CHEN X F. Application of microsatellite DNA as molecular genetic marker. Entomological Knowledge, 2002, 39(03): 165-172. (in Chinese)

[20] ABE A, KOSUGI S, YOSHIDA K, NATSUME S, TAKAGI H, KANZAKI H, MATSUMURA H, YOSHIDA K, MITSUOKA C, TAMIRU M, INNAN H, CANO L, KAMOUN S, TERAUCHI R. Genome sequencing reveals agronomically important loci in rice using MutMap. Nature Biotechnology, 2013, 30: 174-178.

[21] REN Y, ZHANG Z, LIU J, STAUB J E, HAN Y, CHENG Z, LI X, LU J, MIAO H, KANG H, XIE B, GU X, WANG X, DU Y, JIN W, HUANG S. An integrated genetic and cytogenetic map of the cucumber genome. PLoS ONE, 2009, 4(6): e5795.

[22] Singh V K, Mangalam A K, Dwivedi S, Naik S. Primer Premier program for design of degenerate primers from a protein sequence. BioTechniques, 1998, 24: 318-319.

[23] MENG L, LI H H, ZHANG L Y, WANG J K. QTL IciMapping: Integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental populations. The Crop Journal, 2015, 3: 269-283.

[24] MICHELNORE R W, PARAN I, KESSELI R V. Identification of markers linked to disease resistance genes by bulked segregate analysis: a rapid method to detect markers in specific genomic regions by using segregation populations. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88: 9828-9832.

[25] VIKRAM P, SWAMY B M, DIXIT S, AHMED H, CRUZ M S, SINGH A K, YE G, KUMAR A. Bulk segregate analysis: “an effective approach for mapping consistent-effect drought grain yield QTLs in rice”. Field Crops Research, 2012, 134: 185-192.

[26] ZOU C, WANG P, XU Y. Bulked sample analysis in genetics, genomics and crop improvement. Plant Biotechnology Journal, 2006, 14: 1941-1955.

[27] 张海英, 王振国, 毛爱军, 张峰, 王永健, 许勇. 与黄瓜白粉病抗病基因紧密连锁的SSR分子标记. 华北农学报, 2008, 23(6): 77-80.

ZHANG H Y, WANG Z G, MAO A J, ZHANG F, WANG Y J, XU Y. SSR markers linked to the resistant gene of cucumber powdery mildew. Acta Agriculturae Boreali-Sinica, 2008, 23(6): 77-80. (in Chinese)

[28] 聂京涛, 潘俊松, 何欢乐, 司龙亭, 蔡润. 黄瓜白粉病抗性遗传分析与连锁标记筛选. 中国蔬菜, 2011(10): 45-49.

NIE J T, PAN J S, HE H L, SI L T, CAI R. SSR inheritance analysis and screening for linked marker of powdery mildew resistance in cucumber (Cucumis sativus L.). China Vegetables, 2011(10): 45-49. (in Chinese)

[29] XU X W, YU T, XU R X, SHI Y, LIU X J, XU Q, QI X H, WENG Y Q, CHEN X B. Fine mapping of a dominantly inherited powdery mildew resistance major?effect QTL, Pm1.1, in cucumber identifies a 41.1 kb region containing two tandemly arrayed cysteine?rich receptor-like protein kinase genes. Theoretical and Applied Genetics, 2016, 129: 507-516.

[30] XU Q, XU X W, SHI Y, QI X H, CHEN X B. Elucidation of the molecular responses of a cucumber segment substitution line carrying Pm5.1 and its recurrent parent triggered by powdery mildew by comparative transcriptome profiling. BMC Genomics, 2017, 18: 21.

[31] LIU W, FRICK M, HUEL R, NYKIFORUK C L, WANG X, GAUDET DA, EUDES F, CONNER R L, KUZYK A, CHEN Q, KANG Z, LAROCHE A. The stripe rust resistance gene Yr10 encodes an evolutionary-conserved and unique CC-NBS-LRR sequence in wheat. Molecular Plant, 2014, 7: 1740-1755.

[32] PERIYANNAN S, MOORE J, AYLIFFE M, BANSAL U, WANG X, HUANG L, DEAL K, LUO M, KONG X, BARIANA H, MAGO R, MCINTOSH R, DODDS P, DVORAK J, LAGUDAH E. The gene Sr33, an ortholog of barley Mla genes, encodes resistance to wheat stem rust race Ug99. Science, 2013, 341: 786-788.

[33] ZHOU F, KURTH J, WEI F, ELLIOTT C, VALE G, YAHIAOUI N, KELLER B, SOMERVILLE S, WISE R, SCHULZE-LEFERT P. Cell-autonomous expression of barley Mla1 confers race-specific resistance to the powdery mildew fungus via a Rar1-independent signaling pathway. The Plant Cell, 2001, 13: 337-350.

[34] LAWRENCE G J, FINNEGAN E J, AYLIFFE M A, ELLIS J G. The L6 gene for flax rust resistance is related to the Arabidopsis bacterial resistance gene RPS2 and the tobacco viral resistance gene N. The Plant Cell, 1995, 7: 1195-1206.

[35] ANDERSON P A, LAWRENCE G J, MORRISH B C, AYLIFFE M A, FINNEGAN E J, ELLIS J G. Inactivation of the flax rust resistance gene associated with loss of a repeated unit within the leucine-rich repeat coding region. The Plant Cell,1997, 9: 641-651.

[36] Nie J T, He H L, Peng J L, Yang X Q, Bie B B, Zhao J L, Wang Y L, Si L T, Pan J S, Cai R. Identification and fine mapping of pm5.1: A recessive gene for powdery mildew resistance in cucumber (Cucumis sativus L.). Molecular Breeding, 2015, 35: 7. Doi: 10.1007/s11032-015-0206-8.