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. 2017 Oct 30;8(11):298.
doi: 10.3390/genes8110298.

Chromosome Evolution in the Free-Living Flatworms: First Evidence of Intrachromosomal Rearrangements in Karyotype Evolution of Macrostomum Lignano (Platyhelminthes, Macrostomida)

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Chromosome Evolution in the Free-Living Flatworms: First Evidence of Intrachromosomal Rearrangements in Karyotype Evolution of Macrostomum Lignano (Platyhelminthes, Macrostomida)

Kira S Zadesenets et al. Genes (Basel). .
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Abstract

The free-living flatworm Macrostomum lignano is a hidden tetraploid. Its genome was formed by a recent whole genome duplication followed by chromosome fusions. Its karyotype (2n = 8) consists of a pair of large chromosomes (MLI1), which contain regions of all other chromosomes, and three pairs of small metacentric chromosomes. Comparison of MLI1 with metacentrics was performed by painting with microdissected DNA probes and fluorescent in situ hybridization of unique DNA fragments. Regions of MLI1 homologous to small metacentrics appeared to be contiguous. Besides the loss of DNA repeat clusters (pericentromeric and telomeric repeats and the 5S rDNA cluster) from MLI1, the difference between small metacentrics MLI2 and MLI4 and regions homologous to them in MLI1 were revealed. Abnormal karyotypes found in the inbred DV1/10 subline were analyzed, and structurally rearranged chromosomes were described with the painting technique, suggesting the mechanism of their origin. The revealed chromosomal rearrangements generate additional diversity, opening the way toward massive loss of duplicated genes from a duplicated genome. Our findings suggest that the karyotype of M. lignano is in the early stage of genome diploidization after whole genome duplication, and further studies on M. lignano and closely related species can address many questions about karyotype evolution in animals.

Keywords: Macrostomum lignano; flatworms; intrachromosomal rearrangements; karyotype evolution; whole genome duplication.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Scheme of the dissection of the MLI1 chromosome for generation of region-specific DNA probes. Medial part of MLI1 (in yellow color): Mli1med DNA probe; distal parts of MLI1 (in blue): Mli1dist DNA probe. Overlapping regions are shown in grey.
Figure 2
Figure 2
Metaphase plates from the worms of the DV1/10 inbred subline with the “normal” karyotype (2n = 10; four large and six small metacentrics) (A) and with abnormal karyotypes (BI). (B) 2n = 10; four large, five small metacentrics, one small acrocentric chromosome (indicated by the arrow); (C) 2n = 11; four large and seven small metacentrics (additional small chromosomes indicated by the arrow); (D) 2n = 15; four large and nine small metacentrics (additional chromosomes indicated by the arrow); (E) 2n = 12; five large and seven small metacentrics (additional chromosomes indicated by the arrow); (F) 2n = 14; five large and nine small metacentrics (additional chromosomes indicated by the arrow); (G) 2n = 11; four large and seven small metacentrics (additional metacentric indicated by the arrow); (H) 2n = 12; four large and seven small metacentrics and one small acrocentric chromosome (additional metacentric and acrocentric indicated by the arrow); (I) “abnormal” 2n = 8; two large metacentrics, one medium-sized acrocentric chromosome (acrocentric indicated by the arrow) and five small metacentrics. Chromosomes were stained with DAPI (4′,6-diamidino-2-phenylindole) (inverted images are shown). Scale bar: 10 µm.
Figure 3
Figure 3
Painting and fluorescent in situ hybridization (FISH) on chromosomes of metaphase plates. (A) Mli1med (green) and Mli1dist (red) DNA probes; (B) Mli2 (green) and Mli1dist (red) DNA probes. Metaphase chromosomes were counterstained with DAPI (blue). Scale bar: 10 µm.
Figure 4
Figure 4
Scheme of chromosomal regions in the M. lignano chromosomes based on painting patterns of microdissected DNA probes. The regions marked with different colors were painted with different combinations of DNA probes. They were drawn approximately according to the estimation of their size. Microdissected DNA probes derived from the chromosome region provided FISH signal smooth fading in the region borderline. As a result, the position of centromeres in chromosomes MLI2 and MLI3 was not determined precisely in colored bands. For this reason, the centromeres of chromosomes MLI2 and MLI3 were not shown on the scheme.
Figure 5
Figure 5
Localization of the 28S rDNA cluster (green) and the unique DNA fragment R4 (red) in chromosomes of M. lignano. The image with inverted DAPI staining is to the right of the FISH image. Metaphase chromosomes were stained with DAPI (blue). Scale bar: 10 µm.
Figure 6
Figure 6
FISH using Mli2 (Fluorescein, green) and Mli1dist (TAMRA, tetramethylrhodamine, red) on abnormal metaphase plates of M. lignano. (A) Three metaphase plates with the abnormal 2n = 8 chromosome set (two large metacentrics, one unpaired medium-sized acrocentric, and five small metacentrics); (B) abnormal 2n = 10 chromosome set (four large metacentrics, four small metacentrics, and two small acrocentrics). Rearranged chromosomes are marked with arrows. Scale bar: 10 µm.
Figure 7
Figure 7
Localization of rDNA repeats and unique DNA fragments. (A) Clusters of 28S (green) and 5S (red) rDNA; (B) 28S rDNA (green) and unique DNA sequence R1 (red); (C) 28S rDNA (green) and unique DNA sequence R2 (red); (D) 28S rDNA (green) and unique DNA sequence R3 (red); (E) 28S rDNA (green) and unique DNA sequence R4 (red); (F) 28S rDNA (green) and unique DNA sequence R5 (red) in the M. lignano chromosomes. Metaphase chromosomes were stained with DAPI (blue).
Figure 8
Figure 8
Scheme of the localization of the physical markers in the M. lignano chromosomes based on five unique DNA fragments and repeat clusters (5S and 28S rDNA, telomere and pericentromeric DNA repeats).

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