Repetitive DNA: A Versatile Tool for Karyotyping in<b><i> Festuca pratensis</i></b> Huds.

Abstract

FISH is a useful method to identify individual chromosomes in a karyotype and to discover their structural changes accompanying genome evolution and speciation. DNA probes for FISH should be chromosome specific and/or exhibit specific patterns of distribution along each chromosome. Such probes are not available in many plants including meadow fescue (Festuca pratensis Huds.), an important forage grass species. In the present study, various DNA repeats identified in Illumina shotgun sequences specific to chromosome 4F of F. pratensis were used as probes for FISH to develop the molecular karyotype of meadow fescue and to reveal a long-range molecular organization of its chromosomes. Five tandem repeats produced specific patterns on individual chromosomes. Their use in combination with probes for rRNA genes enabled the establishment of the molecular karyotype of meadow fescue. Most of the mobile genetic elements were dispersed along all the chromosomes except for the DNA transposon CACTA, which was localized preferentially to telomeric and subtelomeric regions, and a putative LTR element, which was localized to (peri)centromeric regions. Cytogenetic mapping of the 5 tandem repeats in other accessions of meadow fescue showed a highly similar distribution and confirmed the versatility and robustness of these probes.

Comparative analysis of DNA repeats and identification of novel Fesreba centromeric element in fescues and ryegrasses

“…In all fescues and ryegrasses, both probes localized preferentially to centromeric regions of all chromosomes (Figure 7). Whilst the hybridization signals of RT domain were observed almost exclusively in centromeric regions, a probe derived from non-coding LTR region resulted in stronger signals in centromeric and/or pericentromeric regions and weak signals along the chromosomal arms, as previously shown in F. pratensis [34]. Weak signals of LTR part of Fesreba in distal parts of chromosomes indicate the presence of unique LTRs spread over the genome and correspond to higher copy number of LTR non-coding part of Fesreba as compared to its coding sequence.…”

“…The pipeline uses graph-based clustering and is suitable for the analysis of next generation sequencing data to reconstruct and characterize DNA repeats in a particular species, or to compare DNA repeat composition in different genotypes [22,23,48,49]. The pipeline has been used frequently to reconstruct DNA repeats in diversity studies, to create repeat databases for repeat masking [18,45,47] and to identify tandem organized repeats suitable as probes for molecular cytogenetics [34,51,52].…”

“…Southern hybridization with a probe for reverse transcriptase (RT) domain of Fesreba and a probe for its LTR region [34] showed their presence in all fescues and ryegrasses included in this work (Additional file 2: Figure S1). Similar hybridization patterns indicated sequence conservation between Fesreba repetitive DNA elements in these species.…”

“…Centromeric regions in Arabidopsis, Brachypodium, rice and maize, are partly formed from specific satellite DNAs with ~130 bp long units [27][28][29][30], while in other plant species, including cereals, these regions are formed by large blocks of Ty3/gypsy retrotransposons containing chromodomain [28,[31][32][33]. In F. pratensis, a putative LTR element localizing preferentially to centromeric regions was identified [34]. In addition to understanding the molecular organization of chromosome domains, characterization of repetitive parts of nuclear genomes helps to develop cytogenetic markers [20,34,35].…”

“…In F. pratensis, a putative LTR element localizing preferentially to centromeric regions was identified [34]. In addition to understanding the molecular organization of chromosome domains, characterization of repetitive parts of nuclear genomes helps to develop cytogenetic markers [20,34,35]. Repetitive DNA sequences are also used extensively in studies of genetic diversity and to study processes of genome evolution and speciation [36][37][38][39].…”