Diverse retrotransposon families and an AT-rich satellite DNA revealed in giant genomes of Fritillaria lilies

Abstract

Background and aims: The genus Fritillaria (Liliaceae) comprises species with extremely large genomes (1C = 30 000-127 000 Mb) and a bicontinental distribution. Most North American species (subgenus Liliorhiza) differ from Eurasian Fritillaria species by their distinct phylogenetic position and increased amounts of heterochromatin. This study examined the contribution of major repetitive elements to the genome obesity found in Fritillaria and identified repeats contributing to the heterochromatin arrays in Liliorhiza species.

Methods: Two Fritillaria species of similar genome size were selected for detailed analysis, one from each phylogeographical clade: F. affinis (1C = 45·6 pg, North America) and F. imperialis (1C = 43·0 pg, Eurasia). Fosmid libraries were constructed from their genomic DNAs and used for identification, sequence characterization, quantification and chromosome localization of clones containing highly repeated sequences.

Key results and conclusions: Repeats corresponding to 6·7 and 4·7 % of the F. affinis and F. imperialis genome, respectively, were identified. Chromoviruses and the Tat lineage of Ty3/gypsy group long terminal repeat retrotransposons were identified as the predominant components of the highly repeated fractions in the F. affinis and F. imperialis genomes, respectively. In addition, a heterogeneous, extremely AT-rich satellite repeat was isolated from F. affinis. The FriSAT1 repeat localized in heterochromatic bands makes up approx. 26 % of the F. affinis genome and substantial genomic fractions in several other Liliorhiza species. However, no evidence of a relationship between heterochromatin content and genome size variation was observed. Also, this study was unable to reveal any predominant repeats which tracked the increasing/decreasing trends of genome size evolution in Fritillaria. Instead, the giant Fritillaria genomes seem to be composed of many diversified families of transposable elements. We hypothesize that the genome obesity may be partly determined by the failure of removal mechanisms to counterbalance effectively the retrotransposon amplification.

Fig. 1.

Phylogenetic relationships of selected species according to Rønsted et al. (2005) with their genome size and dot-blot hybridizations of total DNAs from these Fritillaria species using F. affinis and F. imperialis genomic DNAs as a probe. Genome size data for Lilium martagon taken from Zonneveld et al. (2005), otherwise C-values are estimated from this study or correspond to data published by Leitch et al. (2007), as indicated. Cross-hybridization signals of genomic DNAs were related to hybridization intensity of the species used as a probe (100 %).

Fig. 2.

Proportion of different classes of repetitive elements identified in 140 kb of DNA sequenced from four fosmid clones of F. affinis (A) and F. imperialis (B).

Fig. 3.

Neighbour-joining tree of Ty3/gypsy-type retrotransposons inferred from alignment of reverse transcriptase protein domains. The elements identified in F. affinis and F. imperialis are highlighted. Classification of lineages is according to Llorens et al. (2008).

Fig. 4.

Identification and sequence characterization of the FriSAT1 satellite repeat in species of subgenus Liliorhiza. (A) MboI digestion of genomic DNA of various Fritillaria species (M = marker). ‘Relic’ band corresponding to the FriSAT1 repeat encircled. (B) A dot-plot sequence analysis of the DOP-PCR-amplified c1063 clone against itself.

Fig. 5.

Proportion of dispersed repetitive elements estimated using quantitative dot-blot hybridization in 11 Fritillaria species and in Lilium martagon.

Fig. 6.

FISH localization of the FriSAT1 satellite repeat in three species from subgenus Liliorhiza: (A) F. affinis, (B) F. pudica and (C) F. camschatcensis. On DAPI-stained chromosomes (left), heterochromatin domains and bands are visible as intensely stained, dark regions; hybridization sites of the FriSAT1 repeat are shown as violet signals (right). Arrowheads indicate heterochromatin bands (A) and whole chromosomes without the FriSAT1 repeat (B). Scale bar = 10 µm.

Fig. 7.

FISH localization of repetitive DNAs in Fritillaria affinis (A–D) and F. imperialis (E, F). Chromosomes of F. affinis stained with DAPI (A), probed with Ty3/gypsy repeat Fragy86 (green hybridization signal; B) and reprobed with FriSAT1 satellite repeat (purple hybridization signal; C). Images of (B) and (C) are overlaid in (D). DAPI-stained chromosomes of F. imperialis (E) probed with the Ty1/copia repeat Frico2 LTR retrotransposons (F; red hybridization signal). Arrows indicate strong bands of hybridization on two acrocentric chromosomes. Scale bars = 10 µm.