Analysis of retrotransposon abundance, diversity and distribution in holocentric Eleocharis (Cyperaceae) genomes

Abstract

Background and aims: Long terminal repeat-retrotransposons (LTR-RTs) comprise a large portion of plant genomes, with massive repeat blocks distributed across the chromosomes. Eleocharis species have holocentric chromosomes, and show a positive correlation between chromosome numbers and the amount of nuclear DNA. To evaluate the role of LTR-RTs in karyotype diversity in members of Eleocharis (subgenus Eleocharis), the occurrence and location of different members of the Copia and Gypsy superfamilies were compared, covering interspecific variations in ploidy levels (considering chromosome numbers), DNA C-values and chromosomal arrangements.

Methods: The DNA C-value was estimated by flow cytometry. Genomes of Eleocharis elegans and E. geniculata were partially sequenced using Illumina MiSeq assemblies, which were a source for searching for conserved proteins of LTR-RTs. POL domains were used for recognition, comparing families and for probe production, considering different families of Copia and Gypsy superfamilies. Probes were obtained by PCR and used in fluorescence in situ hybridization (FISH) against chromosomes of seven Eleocharis species.

Key results: A positive correlation between ploidy levels and the amount of nuclear DNA was observed, but with significant variations between samples with the same ploidy levels, associated with repetitive DNA fractions. LTR-RTs were abundant in E. elegans and E. geniculata genomes, with a predominance of Copia Sirevirus and Gypsy Athila/Tat clades. FISH using LTR-RT probes exhibited scattered and clustered signals, but with differences in the chromosomal locations of Copia and Gypsy. The diversity in LTR-RT locations suggests that there is no typical chromosomal distribution pattern for retrotransposons in holocentric chromosomes, except the CRM family with signals distributed along chromatids.

Conclusions: These data indicate independent fates for each LTR-RT family, including accumulation between and within chromosomes and genomes. Differential activity and small changes in LTR-RTs suggest a secondary role in nuclear DNA variation, when compared with ploidy changes.

Fig. 1.

Correlation between chromosome number, ploidy levels and estimated amount of DNA for nine samples belonging to seven species of Eleocharis. (A) Comparison between 2C values (total DNA) and the probable monoploid complement (Cx). Note that although the increase in DNA content accompanied the increase in the ploidy level, significant variations were observed between species with the same chromosome number, as well as in the dysploid species E. maculosa (2n = 6) and E. niederleinii (2n = 42). (B) Correlation between the chromosome number and DNA C-value, based on the monoploid complement (Cx), showing R² = 0.712

Fig. 2.

Relative distribution (%) of repetitive DNA (Classes 1 and 2) in the genomes of E. elegans and E. geniculata, based on the assembly of low coverage reads after Illumina sequencing. Note in (A) that LTR-RTs (Class 1) were more abundant in the data set than Class 2 elements and integrated viruses. (B) Relative distribution (%) of Copia and Gypsy superfamilies in the genomes of E. elegans and E. geniculata. (C) Observe that Oryco, SIRE and Retrofit of Sirevirus (Copia) and Athila/Tat families (Gypsy) are predominant in these two data sets.

Fig. 3.

Genetic diversity graph obtained after alignment of reverse transcriptase conserved sequences with ClustalW and Muscle, elaborated with the FigTree tool. Reverse transcriptase sequences were grouped and well delimited according to the phylogeny proposed by Llorens et al. (2009), i.e. Sirevirus (Oryco, SIRE and Retrofit) and Tork of Copia, and Chromovirus (Del, Reina and CRM) and Athila/Tat of Gypsy

Fig. 4.

In situ hybridization using Copia LTR-RT family probes against holocentric chromosomes of diploid and polyploid Eleocharis species. Images appear organized according to Sirevirus (Oryco and SIRE) and Tork clades. Chromosomes were stained with DAPI (blue) and probes labelled with biotin-11-dUTP and detected with avidin–FITC conjugate (green). The Oryco probe hybridized to several small clusters in E. maculosa 2n = 6 and 2n = 10 (A and B, respectively). Differences were observed between E. geniculata (C), E. elegans (D) and E. niederleinii (E), especially in the number of hybridized chromosomes that have intense signals. In E. niederleinii (E), for example, the four large chromosomes (associated with dysploidy) exhibited contrasts in FISH signal accumulation. The SIRE probe showed small clustered signals in E. maculosa with 2n = 10 (F), but in E. elegans (G), E. sellowiana (H) and E. niederleinii (I), the FISH studies reveal a predominance of scattered signals. Note that in E. niederleinii the four large chromosomes showed stronger homogeneous signals than seen in the others. The FISH using the Tork probe exhibited signals predominantly scattered, with differential signal accumulation between chromosomes and karyotypes. Small clustered signals were also observed. E. maculosa (J, L and M), E. geniculata (K), E. elegans (N) and E. niederleinii (O). Scale bar = 10 μm

Fig. 5.

In situ hybridization using Gypsy LTR-RT family probes against holocentric chromosomes of diploid and polyploid Eleocharis species. Images are organized according to Chromovirus (Del and CRM) and Athila/Tat clades. Chromosomes were stained with DAPI (blue) and probes labelled with Cy3-11-dUTP (red). The Del probe hybridized in a dispersed manner along chromosomes, but in E. maculosa 2n = 6 and 2n = 10 (A and B) and E. elegans (C), signals appeared homogeneously in all chromosomes. In contrast, E. geniculata (D) and E. montana (E) exhibited stronger signals on half of the chromosomes. The CRM probe showed a slightly larger diversity in FISH location, with hybridization signals varying within chromosomes, such as in E. maculosa with 2n = 6 and 2n = 10, (F) and (G), respectively, as well as within the karyotypes in E. geniculata (H). In E. montana (I) and E. niederleinii (J), part of the chromosomes exhibited stronger FISH signals. Note also that, in E. niederleinii (J), the four larger chromosomes derived from dysploidy were the ones that accumulated more FISH signals with the CRM probe. The Athila/Tat probe was the one that exhibited the greatest diversity of FISH signal distribution among the Gypsy probes. Eleocharis maculosa with 2n = 6 and 2n = 10 (K and L, respectively), and E. sellowiana (M) exhibited scattered and clustered signals. In E. geniculata, with 2n = 20 (N), four chromosomes accumulated signals along chromatids, while the remaining FISH signals were finer and homogeneously dispersed. Eleocharis montana (O) exhibited numerous clustered signals, in well-delimited blocks or dots in all chromosomes. In E. niederleinii (P), Athila/Tat signals predominate in two of the four dysploid chromosomes, and signals with less intense brightness appeared in 16 other chromosomes with intermediate size. In the remaining chromosomes, signals were inconspicuously dispersed. Scale bar = 10 μm.