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Summary Searching for amino acid or nucleic acid sequences unique to one organism may be challenging depending on size of the available datasets. K-mer elimination by cross-reference (KEC) allows users to quickly and easily find unique sequences by providing target and non-target sequences. Due to its speed, it can be used for datasets of genomic size and can be run on desktop or laptop computers with modest specifications. Availability and implementation KEC is freely available for non-commercial purposes. Source code and executable binary files compiled for Linux, Mac and Windows can be downloaded from https://github.com/berybox/KEC. Supplementary information Supplementary data are available at Bioinformatics online.

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.

With more than 160-fold variation, Orchidaceae are currently the most diverse angiosperm family with respect to the amount of nuclear DNA. This study provides first genome size estimates for approximately 50% of species currently recognized in subfamily Apostasioideae, which is sister to the other four orchid subfamilies. The estimated 1C-values range from 0.38pg in Apostasia nuda to 5.96pg in Neuwiedia zollingeri var. javanica, a nearly 16-fold range. The two genera show non-overlapping genome sizes, with those in Apostasia being distinctly smaller than those in Neuwiedia. In fact, most Apostasia spp. are at the lower end of the range of orchid C-values. Observed discontinuities in DNA amounts in genera most probably reflect interspecific variation in ploidy. In addition to ploidy heterogeneity in N.zollingeri var. javanica, intraspecific variation in genome size (up to 17.7%) was also detected in some species; this can be plausibly related to the incidence of different geographical variants or unrecognized taxonomic heterogeneity. The AT content varied from 62.6 to 66.0%, which is in the upper range recorded for angiosperms. The genome size data obtained in this study fill a major phylogenetic gap in Orchidaceae and show that (very) small genomes prevail in subfamily Apostasioideae.

†Background and Aims One of the prerequisites for polyploid research in natural systems is knowledge of thegeographical distribution of cytotypes. Here inter- and intrapopulational ploidy diversity was examined in theGymnadenia conopsea aggregate in central Europe and potential explanations and evolutionary consequencesof the observed spatial patterns investigated.†Methods DAPI flow cytometry supplemented by confirmatory chromosome counts was used to determineploidy in 3581 samples of the G. conopsea aggregate from 43 populations. The fine-scale spatial pattern of cyto-type distribution (intra- and interploidy associations) was analysed with univariate and bivariate K-functions.†Key Results Gymnadeniatissues undergo a progressively partial endoreplication, which accounts for about 60 %and 75 % of the total genome in G. conopsea and G. densiflora, respectively. Flow cytometric profiles are there-fore species-specific and can be used as a marker for rapid and reliable species recognition. Two majority (4x,8x)and three minority (6x,10x,12x) cytotypes were found, often in mixed-ploidy populations (harbouring up to allfive different ploidy levels). The scarcity of the minority cytotypes (about 2.7 %) suggests the existence of strongpre- or postzygotic mating barriers. Spatial structure was observed in plots of populations with the highest cyto-type variation, including clumping of individuals of the same ploidy and negative association between tetra- andoctoploids.†Conclusions The remarkable ploidy coexistence in the G. conopsea aggregate has reshaped our perception ofintrapopulational ploidy diversity under natural conditions. This system offers unique opportunities for studyingprocesses governing the formation and establishment of polyploids and assessing the evolutionary significance ofthe various pre- and postzygotic mating barriers that maintain this ploidy mixture.Key words: Coexistence, contact zone, cytotype mixture, flow cytometry, Gymnadenia conopsea, hybridization,mating barriers, polyploidy, progressively partial endoreplication, spatial distribution, sympatry.