Abstract
Sponge gourd is a versatile vegetable that can be used as a household cleaning product, industrial material and medicine, and possesses anti-human immunodeficiency virus activity. In the current study, a large dataset composed of 77,858 unigenes derived from the sponge gourd transcriptome was assembled. Of all unigenes, 52,113 (66.93 %) had significant similarity to known proteins from NCBI non-redundant, Swiss-Prot, Clusters of Orthologous Group and Kyoto Encyclopedia of Genes and Genomes databases. Based on these generated sequences, we identified 12,932 putative simple sequence repeats (SSRs) and successfully designed 8,523 high-quality SSR primer pairs. Six hundred and forty-one primer pairs were randomly selected to be verified among S1174 [Luffa acutangula (L.) Roxb.], 93075 [L. cylindrica (L.) Roem.] and their hybrid F1. The result showed that 494 (77.07 %) exhibited successful amplification, of which 201 (40.69 %) revealed polymorphism between S1174 and 93075. Fifty polymorphic expressed sequence tag (EST)-SSRs were used to genotype 60 sponge gourd accessions. Cluster analysis revealed two major clusters, one comprising 42 accessions of L. acutangula and the other 18 accessions belonging to L. cylindrica. Transferability of the 77 polymorphic EST-SSR markers was investigated in six other cucurbits: pumpkin, cucumber, wax gourd, bitter gourd, bottle gourd and chieh-qua, and the percentage of cross-genus amplification was 85.90, 83.33, 83.33, 79.49, 75.64 and 74.36 %, respectively. This indicated that EST-SSRs in sponge gourd could well be applied to other cucurbits. These transcriptome shotgun assembly sequences and EST-SSR data are of great value for the discovery of novel genes and for marker-assisted selection in sponge gourd.
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References
Aggarwal RK, Hendre PS, Varshney RK, Bhat PR, Krishnakumar V, Singh L (2007) Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species. Theor Appl Genet 114:359–372
Ali AM, Karuppusamy S, Al-Hemaid FM (2010) Molecular phylogenetic study of Luffa tuberosa Roxb. (Cucurbitaceae) based on internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA and its systematic implication. Int J Bioinform Res 2:42–60. http://www.bioinfo.in/uploadfiles/13238557152_2_6_IJBR.pdf
Ashkani S, Rafii MY, Rusli I, Sariah M, Abdullah SNA, Rahim HA, Latif MA (2012) SSRs for marker-assisted selection for blast resistance in rice (Oryza sativa L.). Plant Mol Biol Rep 30:79–86
Barkley NA, Roose ML, Krueger RR, Federici CT (2006) Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). Theor Appl Genet 112:1519–1531
Bassam BJ, Caetano-Anolles G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196:80–83
Blanca J, Canizares J, Roig C, Ziarsolo P, Nuez F, Pico B (2011) Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genom 12:104
Boualem A, Fergany M, Fernandez R, Troadec C, Martin A, Morin H, Sari MA, Collin F, Flowers JM, Pitrat M, Purugganan MD, Dogimont C, Bendahmane A (2008) A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons. Science 321:836–838
Bushman BS, Larson SR, Mott IW, Cliften PF, Wang RR, Chatterton NJ, Hernandez AG, Ali S, Kim RW, Thimmapuram J, Gong G, Liu L, Mikel MA (2008) Development and annotation of perennial Triticeae ESTs and SSR markers. Genome 51:779–788
Cavagnaro PF, Senalik DA, Yang L, Simon PW, Harkins TT, Kodira CD, Huang S, Weng Y (2010) Genome-wide characterization of simple sequence repeats in cucumber (Cucumis sativus L.). BMC Genom 11:569
Cavagnaro PF, Chung SM, Manin S, Yildiz M, Ali A, Alessandro MS, Iorizzo M, Senalik DA, Simon PW (2011) Microsatellite isolation and marker development in carrot—genomic distribution, linkage mapping, genetic diversity analysis and marker transferability across Apiaceae. BMC Genom 12:386
Choudhary S, Sethy NK, Shokeen B, Bhatia S (2009) Development of chickpea EST-SSR markers and analysis of allelic variation across related species. Theor Appl Genet 118:591–608
Cloutier S, Niu Z, Datla R, Duguid S (2009) Development and analysis of EST-SSRs for flax (Linum usitatissimum L.). Theor Appl Genet 119:53–63
Collins LJ, Biggs PJ, Voelckel C, Joly S (2008) An approach to transcriptome analysis of non-model organisms using short-read sequences. Genome Inform 21:3–14
Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676
Diaz A, Fergany M, Formisano G, Ziarsolo P, Blanca J, Fei Z, Staub JE, Zalapa JE, Cuevas HE, Dace G, Oliver M, Boissot N, Dogimont C, Pitrat M, Hofstede R, van Koert P, Harel-Beja R, Tzuri G, Portnoy V, Cohen S, Schaffer A, Katzir N, Xu Y, Zhang H, Fukino N, Matsumoto S, Garcia-Mas J, Monforte AJ (2011) A consensus linkage map for molecular markers and quantitative trait loci associated with economically important traits in melon (Cucumis melo L.). BMC Plant Biol 11:111
Ezura H, Owino WO (2008) Melon, an alternative model plant for elucidating fruit ripening. Plant Sci 175:121–129
Fernandez-Silva I, Eduardo I, Blanca J, Esteras C, Pico B, Nuez F, Arus P, Garcia-Mas J, Monforte AJ (2008) Bin mapping of genomic and EST-derived SSRs in melon (Cucumis melo L.). Theor Appl Genet 118:139–150
Gonzalez-Ibeas D, Blanca J, Roig C, Gonzalez-To M, Pico B, Truniger V, Gomez P, Deleu W, Cano-Delgado A, Arus P, Nuez F, Garcia-Mas J, Puigdomenech P, Aranda MA (2007) MELOGEN: an EST database for melon functional genomics. BMC Genom 8:306
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652
Guo S, Zheng Y, Joung JG, Liu S, Zhang Z, Crasta OR, Sobral BW, Xu Y, Huang S, Fei Z (2010) Transcriptome sequencing and comparative analysis of cucumber flowers with different sex types. BMC Genom 11:384
Hinchliffe DJ, Turley RB, Naoumkina M, Kim HJ, Tang Y, Yeater KM, Li P, Fang DD (2011) A combined functional and structural genomics approach identified an EST-SSR marker with complete linkage to the Ligon lintless-2 genetic locus in cotton (Gossypium hirsutum L.). BMC Genom 12:445
Hu J, Zhou X, Li J (2010) Development of novel EST-SSR markers for cucumber (Cucumis sativus) and their transferability to related species. Sci Hortic (Amsterdam) 125:534–538
Hu J, Wang L, Li J (2011) Comparison of genomic SSR and EST-SSR markers for estimating genetic diversity in cucumber. Biol Plant 55:577–580
Hwang JH, Ahn SG, Oh JY, Choi YW, Kang JS, Park YH (2011) Functional characterization of watermelon (Citrullus lanatus L.) EST-SSR by gel electrophoresis and high resolution melting analysis. Sci Hortic (Amsterdam) 130:715–724
Iseli C, Jongeneel CV, Bucher P (1999) ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. In: Proceedings of international conference on intelligent systems for molecular biology, pp 138–148
Jiang B, Xie D, Liu W, Peng Q, He X (2013) De novo assembly and characterization of the transcriptome, and development of SSR markers in wax gourd (Benicasa hispida). PLoS ONE 8:e71054
Joshi BK, Tiwari RK, Ghale M, Sthapit BR, Upadhyay MP (2004) Descriptors for sponge gourd (Luffa cylindrica). NARC, LIBIRD and IPGRI. Nepal Agricultural Research Council (NARC). http://narc.gov.np/publicaton/pdf/book/Descriptors%20Sponge%20gourd.pdf
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2008) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36:D480–D484
Kantety RV, La Rota M, Matthews DE, Sorrells ME (2002) Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol 48:501–510
Karmakar P, Munshi AD, Behera TK, Kumar R, Kaur C, Singh BK (2013) Hermaphrodite inbreds with better combining ability improve antioxidant properties in ridge gourd [Luffa acutangula (Roxb.) L.]. Euphytica 191:75–84
Kaur S, Cogan NO, Ye G, Baillie RC, Hand ML, Ling AE, McGearey AK, Kaur J, Hopkins CJ, Todorovic M, Mountford H, Edwards D, Batley J, Burton W, Salisbury P, Gororo N, Marcroft S, Kearney G, Smith KF, Forster JW, Spangenberg GC (2009) Genetic map construction and QTL mapping of resistance to blackleg (Leptosphaeria maculans) disease in Australian canola (Brassica napus L.) cultivars. Theor Appl Genet 120:71–83
Kaur S, Pembleton LW, Cogan NO, Savin KW, Leonforte T, Paull J, Materne M, Forster JW (2012) Transcriptome sequencing of field pea and faba bean for discovery and validation of SSR genetic markers. BMC Genom 13:104
Li YC, Korol AB, Fahima T, Nevo E (2004) Microsatellites within genes: structure, function, and evolution. Mol Biol Evol 21:991–1007
Li Z, Huang S, Liu S, Pan J, Zhang Z, Tao Q, Shi Q, Jia Z, Zhang W, Chen H, Si L, Zhu L, Cai R (2009) Molecular isolation of the M gene suggests that a conserved-residue conversion induces the formation of bisexual flowers in cucumber plants. Genetics 182:1381–1385
Li D, Deng Z, Qin B, Liu X, Men Z (2012) De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.). BMC Genom 13:192
Liewlaksaneeyanawin C, Ritland CE, El-Kassaby YA, Ritland K (2004) Single-copy, species-transferable microsatellite markers developed from loblolly pine ESTs. Theor Appl Genet 109:361–369
Lin MK, Lee YJ, Lough TJ, Phinney BS, Lucas WJ (2009) Analysis of the pumpkin phloem proteome provides insights into angiosperm sieve tube function. Mol Cell Proteomics 8:343–356
Lister R, Gregory BD, Ecker JR (2009) Next is now: new technologies for sequencing of genomes, transcriptomes, and beyond. Curr Opin Plant Biol 12:107–118
Liu Z, Gulya TJ, Seiler GJ, Vick BA, Jan CC (2012) Molecular mapping of the Pl(16) downy mildew resistance gene from HA-R4 to facilitate marker-assisted selection in sunflower. Theor Appl Genet 125:121–131
Martin A, Troadec C, Boualem A, Rajab M, Fernandez R, Morin H, Pitrat M, Dogimont C, Bendahmane A (2009) A transposon-induced epigenetic change leads to sex determination in melon. Nature 461:1135–1138
Nagao T, Tanaka R, Iwase Y, Hanazono H, Okabe H (1991) Studies on the constituents of Luffa acutangula Roxb. I. Structures of acutosides A–G, oleanane-type triterpene saponins isolated from the herb. Chem Pharm Bull (Tokyo) 39:599–606
Ng TB, Chan WY, Yeung HW (1992a) Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from Cucurbitaceae plants. Gen Pharmacol 23:579–590
Ng TB, Wong RN, Yeung HW (1992b) Two proteins with ribosome-inactivating, cytotoxic and abortifacient activities from seeds of Luffa cylindrica Roem (Cucurbitaceae). Biochem Int 27:197–207
Ng YM, Yang Y, Sze KH, Zhang X, Zheng YT, Shaw PC (2011) Structural characterization and anti-HIV-1 activities of arginine/glutamate-rich polypeptide Luffin P1 from the seeds of sponge gourd (Luffa cylindrica). J Struct Biol 174:164–172
Niu SH, Li ZX, Yuan HW, Chen XY, Li Y, Li W (2013) Transcriptome characterisation of Pinus tabuliformis and evolution of genes in the Pinus phylogeny. BMC Genom 14:263
Parra-Gonzalez LB, Aravena-Abarzua GA, Navarro-Navarro CS, Udall J, Maughan J, Peterson LM, Salvo-Garrido HE, Maureira-Butler IJ (2012) Yellow lupin (Lupinus luteus L.) transcriptome sequencing: molecular marker development and comparative studies. BMC Genom 13:425
Partap S, Kumar A, Sharma NK, Jha KK (2012) Luffa cylindrica: an important medicinal plant. J Nat Prod Plant Resour 2:127–134. http://scholarsresearchlibrary.com/JNPPR-vol2-iss1/JNPPR-2012-2-1-127-134.pdf
Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2:225–238
Prakash K, Pandey A, Radhamani J, Bisht IS (2013) Morphological variability in cultivated and wild species of Luffa (Cucurbitaceae) from India. Genet Resour Crop Evol 60:2319–2329
Rajaram V, Nepolean T, Senthilvel S, Varshney RK, Vadez V, Srivastava RK, Shah TM, Supriya A, Kumar S, Ramana KB, Bhanuprakash A, Narasu ML, Riera-Lizarazu O, Hash CT (2013) Pearl millet [Pennisetum glaucum (L.) R. Br.] consensus linkage map constructed using four RIL mapping populations and newly developed EST-SSRs. BMC Genom 14:159
Ramu P, Billot C, Rami JF, Senthilvel S, Upadhyaya HD, Ananda RL, Hash CT (2013) Assessment of genetic diversity in the sorghum reference set using EST-SSR markers. Theor Appl Genet 126:2051–2064
Rohlf F (2002) NTSYS-pc: numerical taxonomy and multivariate analysis system. Version 2.10. Exeter Software, Setauket
Sharopova N, McMullen MD, Schultz L, Schroeder S, Sanchez-Villeda H, Gardiner J, Bergstrom D, Houchins K, Melia-Hancock S, Musket T, Duru N, Polacco M, Edwards K, Ruff T, Register JC, Brouwer C, Thompson R, Velasco R, Chin E, Lee M, Woodman-Clikeman W, Long MJ, Liscum E, Cone K, Davis G Jr, Coe EH (2002) Development and mapping of SSR markers for maize. Plant Mol Biol 48:463–481
Sikdar B, Bhattacharya M, Mukherjee A, Banerjee A, Ghosh E, Ghosh B, Roy SC (2010) Genetic diversity in important members of Cucurbitaceae using isozyme, RAPD and ISSR markers. Biol Plant 54:135–140
Sim SC, Yu JK, Jo YK, Sorrells ME, Jung G (2009) Transferability of cereal EST-SSR markers to ryegrass. Genome 52:431–437
Singh RK, Jena SN, Khan S, Yadav S, Banarjee N, Raghuvanshi S, Bhardwaj V, Dattamajumder SK, Kapur R, Solomon S, Swapna M, Srivastava S, Tyagi AK (2013) Development, cross-species/genera transferability of novel EST-SSR markers and their utility in revealing population structure and genetic diversity in sugarcane. Gene 524:309–329
Thiel T, Michalek W, Varshney R, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet 106:411–422
Torales SL, Rivarola M, Pomponio MF, Gonzalez S, Acuna CV, Fernandez P, Lauenstein DL, Verga AR, Hopp HE, Paniego NB, Poltri SN (2013) De novo assembly and characterization of leaf transcriptome for the development of functional molecular markers of the extremophile multipurpose tree species Prosopis alba. BMC Genom 14:705
Triwitayakorn K, Chatkulkawin P, Kanjanawattanawong S, Sraphet S, Yoocha T, Sangsrakru D, Chanprasert J, Ngamphiw C, Jomchai N, Therawattanasuk K, Tangphatsornruang S (2011) Transcriptome sequencing of Hevea brasiliensis for development of microsatellite markers and construction of a genetic linkage map. DNA Res 18:471–482
Tyagi S, Sharma P, Siddiqui SA, Khandelwal RC (2010) Combining ability for yield and fruit quality in Luffa. Int J Veg Sci 16:267–277
Varshney RK, Graner A, Sorrells ME (2005) Genic microsatellite markers in plants: features and applications. Trends Biotechnol 23:48–55
Wang H, Ng TB (2002) Luffangulin, a novel ribosome inactivating peptide from ridge gourd (Luffa acutangula) seeds. Life Sci 70:899–906
Wang Z, Fang B, Chen J, Zhang X, Luo Z, Huang L, Chen X, Li Y (2010) De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas). BMC Genom 11:726
Wang Z, Li J, Luo Z, Huang L, Chen X, Fang B, Li Y, Chen J, Zhang X (2011) Characterization and development of EST-derived SSR markers in cultivated sweetpotato (Ipomoea batatas). BMC Plant Biol 11:139
Wang S, Wang X, He Q, Liu X, Xu W, Li L, Gao J, Wang F (2012) Transcriptome analysis of the roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish. Plant Cell Rep 31:1437–1447
Wei W, Qi X, Wang L, Zhang Y, Hua W, Li D, Lv H, Zhang X (2011) Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers. BMC Genom 12:451
Wu HB, Wang B, Chen Y, Liu YG, Chen L (2013) Characterization and fine mapping of the rice premature senescence mutant ospse1. Theor Appl Genet 126:1897–1907
Xia J, Xiang C (2008) Analysis of genetic diversity in Luffa via morphological and RAPD markers. China Veg 10:21–25
Xu P, Wu X, Luo J, Wang B, Liu Y, Ehlers JD, Wang S, Lu Z, Li G (2011) Partial sequencing of the bottle gourd genome reveals markers useful for phylogenetic analysis and breeding. BMC Genom 12:467
Yadav S, Tomar AK, Yadav RN, Yadav S (2013) Screening of antifungal proteins from plants of Cucurbitaceae family against Fusarium oxysporum: potential as biofungicides. Int Res J Environ Sci 2(6):91–96. http://www.isca.in/IJENS/Archive/v2/i6/15.ISCA-IRJEvS-2013-109.pdf
Yan Y, Lin X, Zhang Y, Wang L, Wu K, Huang S (2005) Isolation of peanut genes encoding arachins and conglutins by expressed sequence tags. Plant Sci 169:439–445
Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:W293–W297
Yeh FC, Yang R, Boyle T (1999) POPGENE. Version 1.31. Microsoft Windows-based freeware for population genetic analysis. University of Alberta, Edmonton
Yeung HW, Li WW, Ng TB (1991) Isolation of a ribosome-inactivating and abortifacient protein from seeds of Luffa acutangula. Int J Pept Protein Res 38:15–19
Yi G, Lee JM, Lee S, Choi D, Kim BD (2006) Exploitation of pepper EST-SSRs and an SSR-based linkage map. Theor Appl Genet 114:113–130
Zhang J, Wu K, Zeng S, Teixeira DSJ, Zhao X, Tian CE, Xia H, Duan J (2013) Transcriptome analysis of Cymbidium sinense and its application to the identification of genes associated with floral development. BMC Genom 14:279
Acknowledgments
Our sincere gratitude goes to Xi Chen for assistance with manuscript editing. This research was funded by the President Foundation of Guangdong Province, China (No. 201107) and Science and Technology Program of Guangzhou of China (The breeding and introduction of new species of ridged Luffa characterized by heat-tolerance, high quality and fusarium wilt resistance).
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Wu, HB., Gong, H., Liu, P. et al. Large-scale development of EST-SSR markers in sponge gourd via transcriptome sequencing. Mol Breeding 34, 1903–1915 (2014). https://doi.org/10.1007/s11032-014-0148-6
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DOI: https://doi.org/10.1007/s11032-014-0148-6