In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed "progressively partial endoreplication" (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical "horseshoe" pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.

Biotechnological Centre Faculty of Agriculture University of South Bohemia in České Budějovice Czech Republic

Centre of the Region Haná for Biotechnological and Agricultural Research Institute of Experimental Botany Olomouc Czech Republic

Department of Ecosystem Biology Faculty of Science University of South Bohemia in České Budějovice Czech Republic

Department of Experimental Plant Biology Faculty of Science Charles University Prague Czech Republic Prague Botanical Garden Prague Czech Republic

Institute of Botany the Czech Academy of Sciences Průhonice Czech Republic Department of Botany Faculty of Science Charles University Prague Czech Republic

Institute of Botany the Czech Academy of Sciences Průhonice Czech Republic Department of Botany Faculty of Science Charles University Prague Czech Republic Biotechnological Centre Faculty of Agriculture University of South Bohemia in České Budějovice Czech Republic

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Barow M, Jovtchev G. 2007. Endopolyploidy in plants and its analysis by flow cytometry In: Doležel J, Greilhuber J, Suda J, editors. Flow cytometry with plant cells. Analysis of genes, chromosomes and genomes. Weinheim (Germany: ): Wiley-VCH; p. 349–372.

Bass HW, et al. 2014. A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development. J Exp Bot. 65:2747–2756. PubMed

Begum R, Alam SS, Menzel G, Schmidt T. 2009. Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh. Ann Bot. 104:863–872. PubMed PMC

Bory S, et al. 2008. Natural polyploidy in Vanilla planifolia (Orchidaceae). Genome 51:816–826. PubMed

Bracht JR, et al. 2013. Genomes on the edge: programmed genome instability in ciliates. Cell 152:406–416. PubMed PMC

Capesius I, Nagl W. 1978. Molecular and cytological characteristics of nuclear DNA and chromatin for Angiosperm systematics: DNA diversification in the evolution of four orchids. Plant Syst Evol. 129:143–166.

Chevalier C, et al. 2011. Elucidating the functional role of endoreduplication in tomato fruit development. Ann Bot. 107:1159–1169. PubMed PMC

D’Amato F. 1952. New evidence on endopolyploidy in differentiated plant tissues. Caryologia 4:121–144.

D’Amato F. 1989. Polyploidy in cell differentiation. Caryologia 42:183–211.

D'emerico S, Galasso I, Pignone D, Scrugli A. 2001. Localization of rDNA loci by fluorescent in situ hybridization in some wild orchids from Italy (Orchidaceae). Caryologia 51:31–36.

De Veylder L, Larkin JC, Schnittger A. 2011. Molecular control and function of endoreplication in development and physiology. Trends Plant Sci. 16:624–634. PubMed

Del Priore L, Pigozzi MI. 2014. Histone modifications related to chromosome silencing and elimination during male meiosis in Bengalese finch. Chromosoma 123:293–302. PubMed

Doležel J, Bartoš J, Voglmayr H, Greilhuber J. 2003. Nuclear DNA content and genome size of trout and human. Cytometry 51:127–128. PubMed

Doležel J, et al. 1998. Plant genome size estimation by flow cytometry: inter-laboratory comparison. Ann Bot. 82:17–26.

Doležel J., et al. 2012. Chromosomes in the flow to simplify genome analysis. Funct Integr Genomics. 12:397–416. PubMed PMC

Doležel J., et al. 2014. Advances in plant chromosome genomics. Biotechnol Adv. 32:122–136. PubMed

Doležel J, Greilhuber J, Suda J. 2007. Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc. 2:2233–2244. PubMed

Doležel J, Sgorbati S, Lucretti S. 1992. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Plantarum. 85:625–631.

Goday C, Esteban MR. 2001. Chromosome elimination in sciarid flies. BioEssays 23:242–250. PubMed

Johnston JS, Schoener M, McMahon DP. 2013. DNA underreplication in the majority of nuclei in the Drosophila melanogaster thorax: evidence from Suur and flow cytometry. J Mol Biol Res. 3:47–54.

Joubes J, Chevalier C. 2000. Endoreduplication in higher plants. Plant Mol Biol. 43:735–745. PubMed

Kelly LJ, et al. 2015. Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size. New Phytol. 208:596–607. PubMed PMC

Klemme S, et al. 2013. High-copy sequences reveal distinct evolution of the rye B chromosome. New Phytol. 199:550–558. PubMed

Kondorosi E, Kondorosi A. 2004. Endoreduplication and activation of the anaphase-promoting complex during symbiotic cell development. FEBS Lett. 567:152–157. PubMed

Lan T, Albert VA. 2011. Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid. BMC Plant Biol. 11:126.. PubMed PMC

Lee YI, Chang FC, Chung MC. 2011. Chromosome affinities in interspecific hybrids reflect phylogenetic distances among lady's slipper orchids (Paphiopedilum). Ann Bot. 108:113–121. PubMed PMC

Macas J., et al. 2015. In depth characterization of repetitive DNA in 23 plant genomes reveals sources of genome size variation in the legume tribe Fabeae. PLoS One 10:e0143424.. PubMed PMC

Macas J, Neumann P, Navrátilová A. 2007. Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula. BMC Genomics 8:427.. PubMed PMC

Muller F., et al. 1982. Nucleotide sequence of satellite DNA contained in the eliminated genome of Ascaris lumbricoides. Nucleic Acids Res. 10:7493–7510. PubMed PMC

Nagl W. 1972. Evidence of DNA amplification in orchid Cymbidium in vitro. Cytobios 5:154 145.

Nagl W. 1977. Nuclear structures during the cell cycle In: Rost TI, Gifford EM, editors. Mechanisms and control of cell division. Stroudsburg (US: ): Dowden Hutchinson and Ross, Inc; p. 147–193.

Nordman J, Li S, Eng T, Macalpine D, Orr-Weaver TL. 2011. Developmental control of the DNA replication and transcription programs. Genome Res. 21:175–181. PubMed PMC

Novák P, et al. 2014. Genome-wide analysis of repeat diversity across the family Musaceae. PLoS One 9:e98918.. PubMed PMC

Novák P, Neumann P, Macas J. 2010. Graph-based clustering and characterization of repetitive sequences in next-generation sequencing data. BMC Bioinformatics 11:378.. PubMed PMC

Novák P, Neumann P, Pech J, Steinhaisl J, Macas J. 2013. RepeatExplorer: a Galaxy-based web server for genome-wide characterization of eukaryotic repetitive elements from next-generation sequence reads. Bioinformatics 29:792–793. PubMed

Piednoël M, et al. 2012. Next-generation sequencing reveals the impact of repetitive DNA across phylogenetically closely related genomes of Orobanchaceae. Mol Biol Evol. 29:3601–3611. PubMed PMC

Pigozzi MI, Solari AJ. 1998. Germ cell restriction and regular transmission of an accessory chromosome that mimics a sex body in the zebra finch, Taeniopygia guttata. Chromosome Res. 6:105–113. PubMed

Pigozzi MI, Solari AJ. 2005. The germ-line-restricted chromosome in the zebra finch: recombination in females and elimination in males. Chromosoma 114:403–409. PubMed

Rudkin GT. 1969. Non-replicating DNA in Drosophila. Genetics 61:227–238. PubMed

Sabelli PA, Larkins BA. 2009. The contribution of cell cycle regulation to endosperm development. Sex Plant Reprod. 22:207–219. PubMed

Sanchez L. 2014. Sex-determining mechanisms in insects based on imprinting and elimination of chromosomes. Sex Dev. 8:83–103. PubMed

Schweizer D, Nagl W. 1976. Heterochromatin diversity in Cymbidium and its relationship to differential DNA replication. Exp Cell Res. 98:411–423. PubMed

Šimková H, et al. 2008. Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley. BMC Genomics 9:294.. PubMed PMC

Smith JJ, Antonacci F, Eichler EE, Amemiya CT. 2009. Programmed loss of millions of base pairs from a vertebrate genome. Proc Natl Acad Sci U S A. 106:11212–11217. PubMed PMC

Smith JJ, Baker C, Eichler EE, Amemiya CT. 2012. Genetic consequences of programmed genome rearrangement. Curr Biol. 22:1524–1529. PubMed PMC

Sonnhammer ELL, Durbin R. 1995. A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene 167:GC1–G10. PubMed

Suda J. 2004. An employment of flow cytometry into plant biosystematics. PhD thesis, MS., Depon. Prague: Dept. of Botany, Faculty of Science Charles University.

Suda J, Kron P, Husband BC, Trávníček P. 2007. Flow cytometry and ploidy: Applications in plant systematics, ecology and evolutionary biology In: Doležel J, Greilhuber J, Suda J, editors. Flow cytometry with plant cells. Analysis of genes, chromosomes and genomes. Weinheim (Germany: ): Wiley-VCH; p. 103–130.

Tobler H, Etter A, Muller F. 1992. Chromatin diminution in nematode development. Trends Genet. 8:427–432. PubMed

Trávníček P, et al. 2011. Remarkable coexistence of multiple cytotypes of the fragrant orchid (Gymnadenia conopsea agg.): evidence from flow cytometry. Ann Bot. 107:77–87. PubMed PMC

Trávníček P, et al. 2015. Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication. Cytometry A. 87A:958–966. PubMed

Wang J, et al. 2012. Silencing of germline-expressed genes by DNA elimination in somatic cells. Dev Cell. 23:1072–1080. PubMed PMC

Wang J, Davis RE. 2014. Programmed DNA elimination in multicellular organisms. Curr Opin Genet Dev. 27:26–34. PubMed PMC

Wilkins TA, Rajasekaran K, Anderson DM. 2000. Cotton biotechnology. Crit Rev Plant Sci. 19:511–550.

Yarosh W, Spradling AC. 2014. Incomplete replication generates somatic DNA alterations within Drosophila polytene salivary gland cells. Genes Dev. 28:1840–1855. PubMed PMC

Integrating genome-wide traits and multi-loci phylogeny to investigate orchid evolution-A case study on Pleurothallidinae

Reference standards for flow cytometric estimation of absolute nuclear DNA content in plants

Integrative Study of Genotypic and Phenotypic Diversity in the Eurasian Orchid Genus Neotinea