Repetitive DNA sequences accelerate molecular cytogenetic research in plants with small chromosomes

Agus Budi Setiawan(1*), Ari Wibowo(2), Chee How Teo(3), Shinji Kikuchi(4), Takato Koba(5)

(1) Laboratory of Genetics and Plant Breeding, Faculty of Agriculture, Universitas Gadjah Mada, Jalan Flora Bulaksumur, Yogyakarta 55281
(2) Indonesian Coffee and Cacao Research Institute, Jalan PB. Sudirman No.90, Jember 68175
(3) Center for Research in Biotechnology for Agriculture, University of Malaya, Kuala Lumpur 50603
(4) Laboratory of Genetics and Plant Breeding, Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271‐8510
(5) Laboratory of Genetics and Plant Breeding, Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271‐8510
(*) Corresponding Author


Repetitive DNA sequences are highly abundant in plant genomes and are favorable probes for chromosome identification in plants. However, it is difficult to conduct studies on the details of metaphase chromosome structures in plants with small chromosomes due to their highly condensed status. Therefore, identification of homologous chromosomes for karyotyping and analyzing chromosome structures is a challenging issue for cytogeneticists without specific probes and precise chromosome stages. In this study, five repetitive DNA probes, i.e., 5S and 45S ribosomal DNAs (rDNAs), melon centromeric sequence (Cmcent), cucumber subtelomeric sequence (Type I), and microsatellite (CT)10 repeats, were used to identify primary constrictions and homologous chromosomes for karyotyping. Four and two loci of 45S rDNA were respectively observed on metaphase and pachytene chromosomes of Abelia × grandiflora. Cmcent was detected on both primary constrictions of melon pachytene and metaphase chromosomes. Furthermore, one pair of 5S rDNA signals were hybridized on melon metaphase chromosomes. Eight and two loci of 45S and 5S rDNA were respectively detected on cucumber chromosomes. Type I and (CT)10 probes were specifically hybridized on subtelomeric and interstitial regions on the chromosomes, respectively. These results suggest that repetitive DNA sequences are versatile probes for chromosome identification in plants with small chromosomes, particularly for karyotyping analyses.


homologous chromosomes; microsatellite repeats; pachytene chromosomes; precise karyotyping; repetitive DNAs

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Biscotti MA, Olmo E, Heslop­Harrison JP. 2015. Repeti­tive DNA in eukaryotic genomes. Chromosome Res 23(3):415–420. doi:10.1007/s10577­015­9499­z.

Cuadrado A, Cardoso M, Jouve N. 2008. Physical or­ganisation of simple sequence repeats (SSRs) in Triticeae: Structural, functional and evolutionary impli­ cations. Cytogenet and Genome Res 120(3­4):210– 219. doi:10.1159/000121069.

Fukui K, Kamisugi Y, Sakai F. 1994. Physical map­ping of 5S rDNA loci by direct­cloned biotinylated probes in barley chromosomes. Genome 37(1):105– 111. doi:10.1139/g94­013.

Ganal M, Hemleben V. 1986. Comparison of the ri­bosomal RNA genes in four closely related Cucurbitaceae. Plant Syst Evol 154(1­2):63–77. doi:10.1007/BF00984868.

Garcia­Mas J, Benjak A, Sanseverino W, Bourgeois M, Mir G, Gonzalez VM, Henaff E, Camara F, Cozzuto L, Lowy E, et al. 2012. The genome of melon (Cu­cumis melo L.). Proc Natl Acad Sci 109(29):11872– 11877. doi:10.1073/pnas.1205415109.

Gerlach W, Bedbrook J. 1979. Cloning and char­acterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res 7(7):1869–1885. doi:10.1093/nar/7.7.1869.

Han YH, Zhang ZH, Liu JH, Lu JY, Huang SW, Jin WW. 2008. Distribution of the tandem repeat sequences and karyotyping in cucumber (Cucumis sativus L.) by fluorescence in situ hybridization. Cytogenet Genome Res 122(1):80–88. doi:10.1159/000151320.

Huang S, Li R, Zhang Z, Li L, Gu X, Fan W, Lucas WJ, Wang X, Xie B, Ni P, et al. 2009. The genome of the cucumber, Cucumis sativus L. Nat Genet 41(12):1275–1281. doi:10.1038/ng.475.

Jiang J. 2019. Fluorescence in situ hybridization in plants: recent developments and future applications. Chro­mosome Res doi:10.1007/s10577­019­09607­z.

Kalendar R, Schulman AH. 2006. IRAP and REMAP for retrotransposon­based genotyping and fingerprinting. Nat Protoc 1(5):2478–2484. doi:10.1038/nprot.2006.377.

Kato S, Ohmido N, Hara M, Kataoka R, Fukui K. 2009. Image analysis of small plant chromosomes by using an improved system, CHIAS IV. Chromosome Sci 12:43–50.

Koo DH, Nam YW, Choi D, Bang JW, De Jong H, Hur Y. 2010. Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA se­quences. Chromosome Res 18(3):325–336.

Kubis SE, Heslop­Harrison JS, Desel C, Schmidt T. 1998. The genomic organisation of non­LTR retrotransposons (LINEs) from three Beta species and five other angiosperms. Plant Mol Biol 36:821.

Kuznetsova MA, Chaban IA, Sheval EV. 2017. Visual­ization of chromosome condensation in plants with large chromosomes. BMC Plant Biology 17(1):1–12. doi:10.1186/s12870­017­1102­7.

Markova M, Vyskot B. 2010. New horizons of ge­nomic in situ hybridization. Cytogenet Genome Res 126(4):368–375. doi:10.1159/000275796.

Mendes S, Moraes AP, Mirkov TE, Pedrosa­Harand A. 2011. Chromosome homeologies and high variation in heterochromatin distribution between Citrus L. and Poncirus Raf. as evidenced by comparative cytoge­netic mapping. Chromosome Res 19(4):521–530.

Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA, et al. 2009. The B73 maize genome: Complexity, diver­sity, and dynamics. Science 326(5956):1112–1115. doi:10.1126/science.1178534.

Schwarzacher T, Heslop­Harrison P. 2000. Practical in situ hyridization. New York: Springer.

Setiawan AB. 2018a. Molecular cytogenetic studies on satellite DNA and retrotransposon in Cucumis species. Ph.D. thesis.

Setiawan AB, Teo CH, Kikuchi S, Sassa H, Kato K, Koba T. 2018b. Cytogenetic variation in Cucumis acces­sions revealed by fluorescence in situ hybridization using ribosomal RNAs genes as the probes. Chromo­ some Sci 21:67–73. doi:10.11352/scr.21.67.

Setiawan AB, Teo CH, Kikuchi S, Sassa H, Koba T. 2018c. An improved method for inducing prometaphase chromosomes in plants. Mol Cytogenet doi:10.1186/s13039­018­0380­6.

Wibowo A, Setiawan AB, Purwantoro A, Kikuchi S, Koba T. 2018. Cytological Variation of rRNA Genes and Subtelomeric Repeat Sequences in Indonesian and Japanese Cucumber Accessions. Chromosome Sci 21:81–87. doi:10.11352/scr.21.81.

Zhang ZT, Yang Sq, Li ZA, Zhang Yx, Wang Yz, Cheng Cy, Li J, Chen Jf, Lou Qf. 2016. Comparative chromosomal localization of 45S and 5S rDNAs and im­ plications for genome evolution in Cucumis. Genome 59(7):449–457.


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