Application of inter-SINE amplified polymorphism (ISAP) markers for genotyping of Cucumis melo accessions and its transferability in Coleus spp


Siti Yuli Meilanda Sormin
Aziz Purwantoro
Agus Budi Setiawan
Chee How Teo


Abstract. Sormin SYM, Purwantoro A, Setiawan AB, Teo CH. 2021. Application of inter-SINE amplified polymorphism (ISAP) markers for genotyping of Cucumis melo accessions and its transferability in Coleus spp. Biodiversitas 22: 2918-2929. An unambiguous characterization of melon (Cucumis melo L.) accessions based on their morphological traits is often laborious and affected by environment when compared with molecular marker genotyping. Short interspersed nuclear elements (SINEs) are highly abundant non-autonomous and non-coding retrotransposons that are widely scattered over all chromosomes of eukaryotes. They can serve as a good molecular marker for routine genotyping in plant breeding and marker-assisted selection. This study aimed to apply inter-SINE amplified polymorphism (ISAP) markers for genotyping of Cucumis melo accessions and its transferability in Coleus spp. Twenty-one C. melo accessions, one C. metuliferus E. Mey. ex Naudin, and 15 accessions of Coleus spp. were used for ISAP marker development. A copy of cucumber-specific long interspersed nuclear element (LINE) and multiple copies of melon-specific SINE were identified and isolated. ISAP primers were designed from the highly conserved region of the SINEs and LINE. The melon and cucumber-specific ISAP markers showed a higher degree of polymorphism (87.5%-100%) than potato ISAP markers (60%-100%) in all the tested melon accessions. The unweighted pair group method with arithmetic average (UPGMA) dendrogram generated from polymorphic ISAP bands clearly distinguish the Cucumis melo accessions from their distantly related wild species C. metuliferus. The transferable nature of Cucumis ISAP marker system was demonstrated in Coleus species, where the marker differentiates the tested accessions.


Casacuberta, J.M., Santiago, N., 2003. Plant LTR-retrotransposons and MITEs: Control of transposition and impact on the evolution of plant genes and genomes. Gene 311, 1–11.
Diekmann, K., Seibt, K.M., Muders, K., Wenke, T., Junghans, H., Schmidt, T., Dehmer, K.J., 2017. Diversity studies in genetic resources of Solanum spp. (section Petota) by comparative application of ISAP markers. Genet. Resour. Crop Evol. 64, 1937–1953.
Endl, J., Achigan-Dako, E.G., Pandey, A.K., Monforte, A.J., Pico, B., Schaefer, H., 2018. Repeated domestication of melon (Cucumis melo) in Africa and Asia and a new close relative from India. Am. J. Bot. 105, 1662–1671.
Flavell, A.J., Knox, M.R., Pearce, S.R., Ellis, T.H.N., 1998. Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis. Plant J. 16, 643–650.
Garcia-Mas, J., Oliver, M., Gómez-Paniagua, H., de Vicente, M.C., 2000. Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. TAG Theor. Appl. Genet. 101, 860–864.
Ghebretinsae, A.G., Thulin, M., Barber, J.C., 2007. Relationships of cucumbers and melons unraveled: molecular phylogenetics of Cucumis and related genera (Benincaseae, Cucurbitaceae). Am. J. Bot. 94, 1256–1266.
Gonzalo, M.J., Oliver, M., Garcia-Mas, J., Monfort, A., Dolcet-Sanjuan, R., Katzir, N., Arús, P., Monforte, A.J., 2005. Simple-sequence repeat markers used in merging linkage maps of melon (Cucumis melo L.). Theor. Appl. Genet. 110, 802–811.
Hall, T.A., 1999. BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT, 41, 95-98. Nucleic Acids Symp. Ser. 41, 95–98.
Kalendar, R., Amenov, A., Daniyarov, A., 2019. Use of retrotransposon-derived genetic markers to analyse genomic variability in plants. Funct. Plant Biol. 46, 15–29.
Kalendar, R., Grob, T., Regina, M., Suoniemi, A., Schulman, A., 1999. IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. TAG Theor. Appl. Genet. 98, 704–711.
Kalendar, R., Khassenov, B., Ramankulov, Y., Samuilova, O., Ivanov, K.I., 2017. FastPCR: An in silico tool for fast primer and probe design and advanced sequence analysis. Genomics 109, 312–319.
Kalendar, R., Schulman, A.H., 2014. Molecular Plant Taxonomy, Methods in Molecular Biology. Humana Press, Totowa, NJ.
Kalendar, R., Schulman, A.H., 2006. IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nat. Protoc. 1, 2478–2484.
Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 33, 1870–1874.
Leigh, F., Kalendar, R., Lea, V., Lee, D., Donini, P., Schulman, A.H., 2003. Comparison of the utility of barley retrotransposon families for genetic analysis by molecular marker techniques. Mol. Genet. Genomics 269, 464–474.
Lou, Q., Chen, J., 2007. Ty1-copia retrotransposon-based SSAP marker development and its potential in the genetic study of cucurbits. Genome 50, 802–810.
Mirani, A.A., Teo, C.H., Markhand, G.S., Abul-Soad, A.A., Harikrishna, J.A., 2020. Detection of somaclonal variations in tissue cultured date palm (Phoenix dactylifera L.) using transposable element-based markers. Plant Cell. Tissue Organ Cult. 141, 119–130.
Mliki, A., Staub, J.E., Zhangyong, S., Ghorbel, A., 2001. Genetic diversity in melon (Cucumis melo L.): An evaluation of African germplasm. Genet. Resour. Crop Evol. 48, 587–597.
Monforte, A.J., Garcia-Mas, J., Arús, P., 2003. Genetic variability in melon based on microsatellite variation. Plant Breed. 122, 153–157.
Nagaki, K., Tanaka, K., Yamaji, N., Kobayashi, H., Murata, M., 2015. Sunflower centromeres consist of a centromere-specific LINE and a chromosome-specific tandem repeat. Front. Plant Sci. 6, 1–12.
Pitrat, M., 2008. Melon, in: Prohens, J., Nuez, F. (Eds.), Handbook of Crop Breeding. Vol. I: Vegetables. Springer, New York, pp. 283–315.
Renner, S.S., Schaefer, H., Kocyan, A., 2007. Phylogenetics of Cucumis (Cucurbitaceae): Cucumber (C. sativus) belongs in an Asian/Australian clade far from melon (C. melo). BMC Evol. Biol. 7, 58.
Rohlf, F.J., 2009. NTSYSpc. Numerical taxonomy and multivariate analysis: version 2.2. Exeter Software Setauket, New York.
Schmidt, T., 1999. LINEs, SINEs and repetitive DNA: Non-LTR retrotransposons in plant genomes. Plant Mol. Biol. 40, 903–910.
Schulman, A.H., Flavell, A.J., Paux, E., Ellis, T.H.N., 2012. The Application of LTR Retrotransposons as Molecular Markers in Plants, in: Mobile Genetics Element: Protocols and Genomic Applications, Method in Molecular Biology. pp. 115–153.
Sebastian, P., Schaefer, H., Telford, I.R.H., Renner, S.S., 2010. Cucumber (Cucumis sativus) and melon (C. melo) have numerous wild relatives in Asia and Australia, and the sister species of melon is from Australia. Proc. Natl. Acad. Sci. U. S. A. 107, 14269–73.
Seibt, K.M., Wenke, T., Muders, K., Truberg, B., Schmidt, T., 2016. Short interspersed nuclear elements (SINEs) are abundant in Solanaceae and have a family-specific impact on gene structure and genome organization. Plant J. 86, 268–285.
Seibt, K.M., Wenke, T., Wollrab, C., Junghans, H., Muders, K., Dehmer, K.J., Diekmann, K., Schmidt, T., 2012. Development and application of SINE-based markers for genotyping of potato varieties. Theor. Appl. Genet. 125, 185–196.
Setiawan, A.B., Teo, C.H., Kikuchi, S., Sassa, H., Kato, K., Koba, T., 2020a Centromeres of Cucumis melo L. comprise Cmcent and two novel repeats, CmSat162 and CmSat189. PLoS One 15, e0227578.
Setiawan, A.B., Teo, C.H., Kikuchi, S., Sassa, H., Kato, K., Koba, T., 2020b. Chromosomal Locations of a Non-LTR Retrotransposon, Menolird18, in Cucumis melo and Cucumis sativus, and Its Implication on Genome Evolution of Cucumis Species. Cytogenet. Genome Res. 160, 554–564.
Teo, C.H., Tan, S.H., Ho, C.L., Faridah, Q.Z., Othman, Y.R., Heslop-Harrison, J.S., Kalendar, R., Schulman, A.H., 2005. Genome constitution and classification using retrotransposon-based markers in the orphan crop banana. J. Plant Biol. 48, 96–105., T., Seibt, K.M., Döbel, T., Muders, K., Schmidt, T., 2015. Plant Genotyping, Methods in Molecular Biology. Springer New York, New York, NY.
Zhu, H., Guo, L., Song, P., Luan, F., Hu, J., Sun, X., Yang, L., 2016. Development of genome-wide SSR markers in melon with their cross-species transferability analysis and utilization in genetic diversity study. Mol. Breed. 36.

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