et al. 2014). This lack of homology is due to the age
of the transposons, which mutate over time. In
Spirodela, the relatively few LTRs (264) had an
average age of 4.3 million years, while the average
in Brachypodium and rice was found to be 1.8 and
0.7 million years, respectively. In the later analysis
of the 9509 genome, TEs were annotated by
homology to other known TEs, and by mapping
22–24nt siRNAs known to regulate them through
methylation. This showed that the genome is 25%
TEs, with a Gypsy/Copia ratio of 1.5. In accordance with the age of the LTRs, the Spirodela
genome was found to be purging them through
illegitimate recombination resulting in the highest
ratio of deactivated solo to intact LTRs seen in any
plant genome.
After the Spirodela 7498 genome was published, the draft genome of Lemna minor 5500
was published due to its importance in ecotoxicological studies (Van Hoeck et al. 2015). While
Lemna minor strains vary in genome size from
323 to 760 Mb strain 5500 is 481 Mb in size and
only has 14% more annotated genes than Spirodela polyrhiza 7498 (Table 8.1). Compared to
Spirodela 94.5% of the difference in genome size
is due to repeats. These repeats make up 61% of
the genome and 36% of the genome is TEs,
mainly retrotransposons, which is slightly higher
than Spirodela. The count of LTRs increased
*10-fold to 210,531. There was a final category
of unclassified repeats that made up 21% of the
genome. In strain, 7498 DNA-based transposons
were difficult to annotate based on their old age
and low homology, and in strain, 9509 the
annotation relied on siRNAs. Therefore, the
unclassified repeats may include many ancient
unannotated transposons.
The relative lack of TEs in Spirodela brought
attention to the RNA directed DNA methylation
(RdDM) pathway. This is a mechanism of
silencing transposons through siRNAs where
Pol IV creates a ssRNA transcript and RDR2
makes it a dsRNA (Matzke et al. 2015). Then,
DCL3 cleaves it into 24nt het-siRNAs (heterochromatic) that are loaded onto AGO4, which
binds to DRM2 and RDM1 proteins that
methylate the 5’ end of cytosine in GC, CHG,
and CHH sequences. To finish the process a
collection of proteins in a histone-modifying
complex converts the methylated TE sequence to
silenced heterochromatin. This pathway is highly
conserved across all land plants, with the notable
outlier of the Norway Spruce, which has relatively few 24nt het-siRNAs, mainly localized to
reproductive organs (Matzke et al. 2015).
In Spirodela polyrhiza, it was noticed that 24nt
sRNAs were rare, comprising 7.3% of the small
RNAs in strain LT5a and 1% in strain 7498
(Fourounjian et al. 2019). While the 9509 genome
had the lowest DNA methylation rate of any plant
sequenced at 9%, the TEs had an average methylation rate of 20% (Michael et al. 2017). Furthermore, older TEs were annotated based on the
mapping of 22–24nt siRNAs, suggesting that they
were expressed and active. The Spirodela genome
also revealed a low number of old TEs suggesting
that it has been very successful at halting their
proliferation (Wang et al. 2014; Michael et al.
2017). Taken together it looks like the RdDM
pathway is working with little to no 24nt
het-siRNAs. This could be similar to the results
seen in Norway spruce where 24nt het-siRNAs are
localized to flowers, which are very rare in Spirodela, or perhaps other mechanisms may be at
play. The mystery of how the Lemnaceae, particularly Spirodela, regulate their TEs is an exciting
field of research that is still currently unfolding.
Acknowledgements Thank you to Dr. Alex Harkess for
reviewing this chapter to confirm its accurate, but not
complete description of the RdDM pathway.
References
Biscotti MA, Olmo E, Heslop-Harrison JS (2015) Repetitive DNA in eukaryotic genomes. Chromosom
Res 23:415–420. https://doi.org/10.1007/s10577-0159499-z
Cao HX, Vu GTH, Wang W et al (2016) The map-based
genome sequence of Spirodela polyrhiza aligned with
its chromosomes, a reference for karyotype evolution.
New Phytol 209:354–363. https://doi.org/10.1111/
nph.13592
Ernst E, Martienssen R (2016) Status of the Lemna gibba
7742a and Lemna minor 8627 genomes. Duckweed
Forum, 12
8 Repetitive Sequences: Impacts and Uses in the Spirodela Genome
89
of the transposons, which mutate over time. In
Spirodela, the relatively few LTRs (264) had an
average age of 4.3 million years, while the average
in Brachypodium and rice was found to be 1.8 and
0.7 million years, respectively. In the later analysis
of the 9509 genome, TEs were annotated by
homology to other known TEs, and by mapping
22–24nt siRNAs known to regulate them through
methylation. This showed that the genome is 25%
TEs, with a Gypsy/Copia ratio of 1.5. In accordance with the age of the LTRs, the Spirodela
genome was found to be purging them through
illegitimate recombination resulting in the highest
ratio of deactivated solo to intact LTRs seen in any
plant genome.
After the Spirodela 7498 genome was published, the draft genome of Lemna minor 5500
was published due to its importance in ecotoxicological studies (Van Hoeck et al. 2015). While
Lemna minor strains vary in genome size from
323 to 760 Mb strain 5500 is 481 Mb in size and
only has 14% more annotated genes than Spirodela polyrhiza 7498 (Table 8.1). Compared to
Spirodela 94.5% of the difference in genome size
is due to repeats. These repeats make up 61% of
the genome and 36% of the genome is TEs,
mainly retrotransposons, which is slightly higher
than Spirodela. The count of LTRs increased
*10-fold to 210,531. There was a final category
of unclassified repeats that made up 21% of the
genome. In strain, 7498 DNA-based transposons
were difficult to annotate based on their old age
and low homology, and in strain, 9509 the
annotation relied on siRNAs. Therefore, the
unclassified repeats may include many ancient
unannotated transposons.
The relative lack of TEs in Spirodela brought
attention to the RNA directed DNA methylation
(RdDM) pathway. This is a mechanism of
silencing transposons through siRNAs where
Pol IV creates a ssRNA transcript and RDR2
makes it a dsRNA (Matzke et al. 2015). Then,
DCL3 cleaves it into 24nt het-siRNAs (heterochromatic) that are loaded onto AGO4, which
binds to DRM2 and RDM1 proteins that
methylate the 5’ end of cytosine in GC, CHG,
and CHH sequences. To finish the process a
collection of proteins in a histone-modifying
complex converts the methylated TE sequence to
silenced heterochromatin. This pathway is highly
conserved across all land plants, with the notable
outlier of the Norway Spruce, which has relatively few 24nt het-siRNAs, mainly localized to
reproductive organs (Matzke et al. 2015).
In Spirodela polyrhiza, it was noticed that 24nt
sRNAs were rare, comprising 7.3% of the small
RNAs in strain LT5a and 1% in strain 7498
(Fourounjian et al. 2019). While the 9509 genome
had the lowest DNA methylation rate of any plant
sequenced at 9%, the TEs had an average methylation rate of 20% (Michael et al. 2017). Furthermore, older TEs were annotated based on the
mapping of 22–24nt siRNAs, suggesting that they
were expressed and active. The Spirodela genome
also revealed a low number of old TEs suggesting
that it has been very successful at halting their
proliferation (Wang et al. 2014; Michael et al.
2017). Taken together it looks like the RdDM
pathway is working with little to no 24nt
het-siRNAs. This could be similar to the results
seen in Norway spruce where 24nt het-siRNAs are
localized to flowers, which are very rare in Spirodela, or perhaps other mechanisms may be at
play. The mystery of how the Lemnaceae, particularly Spirodela, regulate their TEs is an exciting
field of research that is still currently unfolding.
Acknowledgements Thank you to Dr. Alex Harkess for
reviewing this chapter to confirm its accurate, but not
complete description of the RdDM pathway.
References
Biscotti MA, Olmo E, Heslop-Harrison JS (2015) Repetitive DNA in eukaryotic genomes. Chromosom
Res 23:415–420. https://doi.org/10.1007/s10577-0159499-z
Cao HX, Vu GTH, Wang W et al (2016) The map-based
genome sequence of Spirodela polyrhiza aligned with
its chromosomes, a reference for karyotype evolution.
New Phytol 209:354–363. https://doi.org/10.1111/
nph.13592
Ernst E, Martienssen R (2016) Status of the Lemna gibba
7742a and Lemna minor 8627 genomes. Duckweed
Forum, 12
8 Repetitive Sequences: Impacts and Uses in the Spirodela Genome
89