Abstract
Repetitive DNA including tandem repeats (TRs) is a significant part of most eukaryotic genomes. TRs include rapidly evolving satellite DNA (satDNA) that can be shared by closely related species, their abundance may be associated with evolutionary divergence, and they have been widely used for chromosome karyotyping using fluorescence in situ hybridization (FISH). The recent progress in the development of whole-genome sequencing and bioinformatics tools enables rapid and cost-effective searches for TRs including satDNA that can be converted into molecular cytogenetic markers. In the case of closely related taxa, the genome sequence of one species (donor) can be used as a base for the development of chromosome markers for related species or genomes (target). Here, we present a pipeline for rapid and high-throughput screening for new satDNA TRs in whole-genome sequencing of the donor genome and the development of chromosome markers based on them that can be applied in the target genome. One of the main peculiarities of the developed pipeline is that preliminary estimation of TR abundance using qPCR and ranking found TRs according to their copy number in the target genome; it facilitates the selection of the most prospective (most abundant) TRs that can be converted into cytogenetic markers. Another feature of our pipeline is the probe preparation for FISH using PCR with primers designed on the aligned TR unit sequences and the genomic DNA of a target species as a template that enables amplification of a whole pool of monomers inherent in the chromosomes of the target species. We demonstrate the efficiency of the developed pipeline by the example of FISH probes developed for A, B, and R subgenome chromosomes of hexaploid triticale (BBAARR) based on a bioinformatics analysis of the D genome of Aegilops tauschii (DD) whole-genome sequence. Our pipeline can be used to develop chromosome markers in closely related species for comparative cytogenetics in evolutionary and breeding studies.
Highlights
A large proportion of eukaryotic genomes are represented by various classes of repetitive DNA sequences that are mainly dispersed and arranged in tandem
We present a pipeline for rapid and high-throughput screening of new satellite DNA (satDNA) tandem repeats (TRs) in the whole-genome sequencing data of donor genome and the development of cytogenetic markers based on them that can be applied in the target genome
The novelty of our pipeline is the following: For the first time, we propose to apply a quantitative PCR (qPCR) assay that combines both qualitative and quantitative assessment of a particular TR in related genomes; this facilitates the search for common, highly abundant satDNAs that can be converted into molecular cytogenetic markers
Summary
A large proportion of eukaryotic genomes are represented by various classes of repetitive DNA sequences that are mainly dispersed (mainly transposons and retrotransposons) and arranged in tandem Abundant tandem repeat (TR) DNA sequences, known as satellites, form a significant part of many eukaryotic genomes and are classified as microsatellite, satellite (satDNA), and ribosomal. SatDNA may play a structural role in genome organization and in regulation of gene expression by epigenetic modifications; variations in satellite abundance have been associated with differences and genetic incompatibilities between species. One satDNA family may be present in one species, and different species may share a common set or have species-specific sets. Identifying satDNA in phylogenetically distant species is not possible; inversely, once found in one genome, a given repeat may be found in a closely related genome
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