Background and Aims Plant genomes contain many retrotransposons and their derivatives, which are subject to rapid sequence turnover. As non-autonomous retrotransposons do not encode any proteins, they experience reduced selective constraints leading to their diversification into multiple families, usually limited to only few closely related species. In contrast, the non-coding Cassandra terminal repeat retrotransposons in miniature (TRIMs) are widespread in many plants. Their hallmark is a conserved 5S rDNA-derived promoter in their long terminal repeats (LTRs). As sugar beet (Beta vulgaris) has a well-described LTR retrotransposon landscape, we aim to characterise TRIMs in beet and related genomes. Methods We identified Cassandra retrotransposons in the sugar beet reference genome and characterised their structural relationships. Genomic organisation, chromosomal localisation, and distribution of Cassandra-TRIMs across the Amaranthaceae were verified by Southern and fluorescent in situ hybridisation. Key results All 638 Cassandra sequences in the sugar beet genome contain conserved LTRs and thus constitute a single family. Nevertheless, variable internal regions required a subdivision into two Cassandra subfamilies within B. vulgaris. The related Chenopodium quinoa harbours a third subfamily. These subfamilies vary in their distribution within Amaranthaceae genomes, in their insertion times, and the degree of silencing by smallRNAs. Cassandra retrotransposons gave rise to many structural variants, such as solo-LTRs or tandemly-arranged Cassandra retrotransposons. These Cassandra derivates point to an interplay of template switch and recomination processes – mechanisms that likely caused Cassandra’s subfamily formation and diversification. Conclusions We traced the evolution of Cassandra in the Amaranthaceae and detected a considerable variability within the short internal regions, whereas the LTRs are strongly conserved in sequence and length. Presumably, these hallmarks make Cassandra a prime target for unequal recombination, resulting in the observed structural diversity, an example for the impact of LTR-mediated evolutionary mechanisms on the host genome.

The 5S rRNA genes are among the most conserved nucleotide sequences across all species. Similar to the 5S preservation we observe the occurrence of 5S-related non-autonomous retrotransposons, so-called Cassandra. Cassandras harbor highly conserved 5S rDNA-related sequences within their long terminal repeats (LTRs), advantageously providing them with the 5S internal promoter. However, the dynamics of Cassandra retrotransposon evolution in the context of 5S rRNA gene sequence information and structural arrangement are still unclear, especially: 1) do we observe repeated or gradual domestication of the highly conserved 5S promoter by Cassandras and 2) do changes in 5S organization such as in the linked 35S-5S rDNA arrangements impact Cassandra evolution? Here, we show evidence for gradual co-evolution of Cassandra sequences with their corresponding 5S rDNAs. To follow the impact of 5S rDNA variability on Cassandra TEs, we investigate the Asteraceae family where highly variable 5S rDNAs, including 5S promoter shifts and both linked and separated 35S-5S rDNA arrangements have been reported. Cassandras within the Asteraceae mirror 5S rDNA promoter mutations of their host genome, likely as an adaptation to the host's specific 5S transcription factors and hence compensating for evolutionary changes in the 5S rDNA sequence. Changes in the 5S rDNA sequence and in Cassandras seem uncorrelated with linked/separated rDNA arrangements. We place all these observations into the context of angiosperm 5S rDNA-Cassandra evolution, discuss Cassandra's origin hypotheses (single or multiple) and Cassandra's possible impact on rDNA and plant genome organization, giving new insights into the interplay of ribosomal genes and transposable elements.

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