Abstract
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QTL mapping using NGS-assisted BSA was successfully applied to an F 2 population for downy mildew resistance in cucumber. QTLs detected by NGS-assisted BSA were confirmed by conventional QTL analysis.
Abstract
Downy mildew (DM), caused by Pseudoperonospora cubensis, is one of the most destructive foliar diseases in cucumber. QTL mapping is a fundamental approach for understanding the genetic inheritance of DM resistance in cucumber. Recently, many studies have reported that a combination of bulked segregant analysis (BSA) and next-generation sequencing (NGS) can be a rapid and cost-effective way of mapping QTLs. In this study, we applied NGS-assisted BSA to QTL mapping of DM resistance in cucumber and confirmed the results by conventional QTL analysis. By sequencing two DNA pools each consisting of ten individuals showing high resistance and susceptibility to DM from a F2 population, we identified single nucleotide polymorphisms (SNPs) between the two pools. We employed a statistical method for QTL mapping based on these SNPs. Five QTLs, dm2.2, dm4.1, dm5.1, dm5.2, and dm6.1, were detected and dm2.2 showed the largest effect on DM resistance. Conventional QTL analysis using the F2 confirmed dm2.2 (R 2 = 10.8–24 %) and dm5.2 (R 2 = 14–27.2 %) as major QTLs and dm4.1 (R 2 = 8 %) as two minor QTLs, but could not detect dm5.1 and dm6.1. A new QTL on chromosome 2, dm2.1 (R 2 = 28.2 %) was detected by the conventional QTL method using an F3 population. This study demonstrated the effectiveness of NGS-assisted BSA for mapping QTLs conferring DM resistance in cucumber and revealed the unique genetic inheritance of DM resistance in this population through two distinct major QTLs on chromosome 2 that mainly harbor DM resistance.
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References
Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H et al (2012) Genome sequencing reveals agronomically important loci in rice using MutMap. Nat Biotechnol 30:174–178
Arends D, Prins P, Jansen RC, Broman KW (2010) R/qtl: high-throughput multiple QTL mapping. Bioinformatics 26:2990–2992
Becker A, Chao DY, Zhang X, Salt DE, Baxter I (2011) Bulk segregant analysis using single nucleotide polymorphism microarrays. PLoS One 6:e15993
Broman KW, Wu H, Sen S, Churchill GA (2003) R/qtl: QTL mapping in experimental crosses. Bioinformatics 19:889–890
Call AD, Criswell AD, Wehner TC et al (2012a) Resistance of cucumber cultivars to a new strain of cucurbit downy mildew. HortSci 47:171–178
Call AD, Criswell AD, Wehner TC et al (2012b) Screening cucumber for resistance to downy mildew caused by Pseudoperonospora cubensis (Berk. and Curt.) Rostov. Crop Sci 52:577–592
Catchen J, Amores A, Hohenlohe P, Cresko W, Postlethwait J (2011) Stacks: building and genotyping loci de novo from short-read sequences. G3-Genes Genom Genet 1:171–182
Catchen J, Hohenlohe P, Bassham S, Amores A, Cresko W (2013) Stacks: an analysis tool set for population genomics. Mol Ecol 22(11):3124–3140
Chen JF, Kirkbride JH Jr (2000) A new synthetic species Cucumis (Cucurbitaceae) from interspecific hybridization and chromosome doubling. Brittonia 52:315–319
Chen JF, Staub JE, Qian ChT, Jiang JM, Luo XD, Zhuang FY (2003) Reproduction and cytogenetic characterization of interspecific hybrids derived from Cucumis hystrix Chakr. × Cucumis sativus L. Theor Appl Genet 106:688–695
Dhillon NPS, Pushpinder PS, Ishiki K (1999) Evaluation of landraces of cucumber (Cucumis sativus L.) for resistance to downy mildew (Pseudoperonospora cubensis). Plant Genet Resour Newsl 119:59–61
Dijkhuizen A (1994) Application of restriction fragment length polymorphism for the assessment of genetic variation and study of quantitatively inherited traits in cucumber (Cucumis sativus L.). Ph.D. Dissertation, University of Wisconsin-Madison, p 123
Ding G, Qin Z, Zhou X, Fan J (2007) RAPD and SCAR markers linked to downy mildew resistance genes in cucumber. Acta Bot Boreali-Occident Sin 27:1747–1751 (in Chinese)
Doruchowski RW, Łazkowska-Ryk E (1992) Inheritance of resistance to downy mildew (Pseudoperonospora cubensis Berk. and Curt.) in Cucumis sativus. In: Doruchowski RW (ed) Proceedings of the V Eucarpia Cucurbitaceae Symposium. Skierniewice-Warszawa, Poland, pp 132–138
Ehrenreich IM, Torabi N, Jia Y, Kent J, Martis S et al (2010) Dissection of genetically complex traits with extremely large pools of yeast segregants. Nature 464:1039–1042
Epinat C, Pitrat M (1994) Inheritance of resistance to downy mildew (Pseudoperonospora cubensis) in muskmelon (Cucumis melo). I. Analysis of a 8 × 8 diallel table. Agronomie 14:239–248
Fanourakis NE (1984) Inheritance and linkage studies of the fruit epidermis structure and investigation of linkage relations of several traits and of meiosis in cucumber. Ph.D. Dissertation, University of Wisconsin-Madison
Han Y, Lv P, Hou S, Li S, Ji G, Ma X et al (2015) Combining Next Generation Sequencing with Bulked Segregant Analysis to Fine Map a Stem Moisture Locus in Sorghum (Sorghum bicolor L. Moench). PLoS One 10(5):e0127065. doi:10.1371/journal.pone.0127065
He X, Li YH, Pandey S, Yandell BS, Pathak M, Weng Y (2013) QTL mapping of powdery mildew resistance in WI 2757 cucumber (Cucumis sativus L.). Theor Appl Genet 126:2149–2161
Huang S, Li R, Zhang Z et al (2009) The genome of the cucumber, Cucumis sativus L. Nat Genet 41:1275–1281
Kaminski KP, Korup K, Andersen MN, Sonderkaer M, Andersen MS, Kirk HG, Nielsen KL (2016) Next generation sequencing bulk segregant analysis of potato support that differential flux into the cholesterol and stigmasterol metabolite pools is important for steroidal glycoalkaloid content. Potato Res 59:81–97
Kozik EU, Klosin´ska U, Call AD, Wehner TC (2013) Heritability and genetic variance estimates for resistance to downy mildew in cucumber accession Ames 2354. Crop Sci 53:177–182
Lander ES, Green P (1987) Construction of multilocus genetic linkage maps in humans. Proc Natl Sci USA 84:2363–2367
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359
Lebeda A, Prasil J (1994) Susceptibility of Cucumis sativus cultivars to Pseudoperonospora cubensis. Acta Phytopathol Entomol Hung 29:89–94
Lee J-S, Han K-S, Lee S-C, Soh J-W (2013) Screening for resistance to downy mildew among major commercial cucumber varieties. Res Dis 19(3):188–195 (in Korean)
Li H, Durbin R (2009) Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25:1754–1760
Li Z, Zhang Z, Yan P, Huang S, Fei Z, Lin K (2011) RNA-Seq improves annotation of protein-coding genes in the cucumber genome. BMC Genom 12:540
Magwene PM, Willis JH, Kelly JK (2011) The statistics of bulk segregant analysis using next generation sequencing. PLoS Comput Biol 7:e1002255
Mayer C (2006–2010) Phobos 3.3.11. Available at http://www.rub.de/spezzoo/cm/cm_phobos.htm
Michelmore RW, Paran I, Kesseli RV (1991) 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 USA 88:9828–9832
Nelson JC (1997) QGene: software for marker-based genomic analysis and breeding. Mol Breed 3:239–245
Oerke EC, Steiner U, Dehne HW, Lindenthal M (2006) Thermal imaging of cucumber leaves affected by downy mildew and environmental conditions. J Exp Bot 57:2121–2132
Olczak-Woltman H, Marcinkowska J, Niemirowicz-Szczytt K (2011) The genetic basis of resistance to downy mildew in Cucumis spp.—latest developments and prospects. J Appl Genetics 52:249–255
Palti J, Cohen Y (1980) Downy mildew of cucurbits (Pseudoperonospora cubensis). The fungus and its hosts, distribution, epidemiology and control. Phytoparasitica 8:109–147
Pang X, Zhou X, Wan H, Chen J (2013) QTL mapping of downy mildew resistance in an introgression line derived from interspecific hybridization between cucumber and Cucumis hystrix. J Phytopathol 161:536–543
Ren Y, Zhang Z, Liu J et al (2009) An integrated genetic and cytogenetic map of the cucumber genome. PLoS One 4:e5795
Rosen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Misener S, Krawetz SA (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386
Rubinstein M, Katzenellenbogen M, Eshed R, Rozen A, Katzir N, Colle M et al (2015) Ultra high-density linkage map for cultivated cucumber using a single nucleotide polymorphism genotyping array. PLoS One 10:e0124101
Schneeberger K, Ossowski S, Lanz C, Juul T, Petersen AH, Nielsen KL, Jorgensen J-E, Weigel D, Andersen SU (2009) SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nat Methods 6:550–551
Shimizu S, Kanazawa K, Kato A, Yokota Y, Koyama T (1963) Studies on the breeding of cucumber for resistance to downy mildew. Part 3. Genetic observations for the resistance to downy mildew and other fruit characters. Bull Hort Res Sta A 2:65–81 (in Japanese with English abstract)
Sun JY, Zhang ZH, Zong X, Huang SW, Li ZY, Han YH (2013) A high-resolution cucumber cytogenetic map integrated with the genome assembly. BMC Genom 14:461
Szczechura W, Staniaszek M, Klosinska U, Kozik EU (2015) Molecular analysis of new sources of resistance to Pseudoperonospora cubensis (Berk. et Curt.) Rostov. in cucumber. Russian J Genet 51:974–979
Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S et al (2013) QTL-seq: Rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J (on line first). doi:10.1111/tpj.12105
Trick M, Adamski NM, Mugford SG, Jiang C-C, Febrer M, Uauy C (2012) Combining SNP discovery from next-generation sequencing data with bulked segregant analysis (BSA) to fine-map genes in polyploidy wheat. BMC Plant Biol 12:14
Vandenlangenberg KM (2015) Studies on downy mildew resistance in cucumber (Cucumis sativus L.). Ph.D. Dissertation, University of North Carolina State University
Wang Y, VandenLangenberg K, Wehner TC, Kraan PAG, Suelmann J, Zheng X, Owens K, Weng Y (2016) QTL mapping for downy mildew resistance in cucumber inbreed line W17120 (PI 330628). Theor Appl Genet. doi:10.1007/s00122-016-2719-x
Wei Q, Wang Y, Qin X, Zhang Y, Zhang Z, Wang J et al (2014) An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment (SLAF) sequencing. BMC Genom 15:1158. doi:10.1186/1471-2164-15-1158
Wenger JW, Schwartz K, Sherlock G (2010) Bulk segregant analysis by high-throughput sequencing reveals a novel xylose utilization gene from Saccharomyces cerevisiae. PLoS Genet 6(5):e1000942
Wolyn DJ, Borevitz JO, Loudet O, Schwartz C, Maloof J et al (2004) Light-response quantitative trait loci identified with composite interval and eXtreme array mapping in Arabidopsis thaliana. Genetics 167:907–917
Yang Z, Huang D, Tang W, Zheng Y, Liang K et al (2013) Mapping of quantitative trait loci underlying cold tolerance in rice seedlings via high-throughput sequencing of pooled extremes. PLoS One 8(7):e68433. doi:10.1371/journal.pone.0068433
Yoshioka Y, Sakata Y, Sugiyama M, Fukino N (2014) Identification of quantitative trait loci for downy mildew resistance in cucumber (Cucumis sativus L.). Euphytica 198:265–276
Zhang WW, Pan JS, He HL, Zhang C, Li Z, Zhao JL, Yuan XJ, Zhu LH, Huang SW, Cai R (2012) Construction of a high density integrated genetic map for cucumber (Cucumis sativus L.). Theor Appl Genet 124:249–259
Zhang S, Liu MM, Miao H et al (2013) Chromosomal mapping and QTL analysis of resistance to downy mildew in Cucumis sativus. Plant Dis 97:245–251
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This work was supported by grants from the Bio-industry Technology Development Program (111057-5) of iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry).
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This work was supported by grants from the Bio-industry Technology Development Program (111057-5) of iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry).
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Communicated by H. J. van Eck.
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Win, K.T., Vegas, J., Zhang, C. et al. QTL mapping for downy mildew resistance in cucumber via bulked segregant analysis using next-generation sequencing and conventional methods. Theor Appl Genet 130, 199–211 (2017). https://doi.org/10.1007/s00122-016-2806-z
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DOI: https://doi.org/10.1007/s00122-016-2806-z