The Diversity of Sequence and Chromosomal Distribution of New Transposable Element-Related Segments in the Rye Genome Revealed by FISH and Lineage Annotation

Abstract

Transposable elements (TEs) in plant genomes exhibit a great variety of structure, sequence content and copy number, making them important drivers for species diversity and genome evolution. Even though a genome-wide statistic summary of TEs in rye has been obtained using high-throughput DNA sequencing technology, the accurate diversity of TEs in rye, as well as their chromosomal distribution and evolution, remains elusive due to the repetitive sequence assembling problems and the high dynamic and nested nature of TEs. In this study, using genomic plasmid library construction combined with dot-blot hybridization and fluorescence in situ hybridization (FISH) analysis, we successfully isolated 70 unique FISH-positive TE-related sequences including 47 rye genome specific ones: 30 showed homology or partial homology with previously FISH characterized sequences and 40 have not been characterized. Among the 70 sequences, 48 sequences carried Ty3/gypsy-derived segments, 7 sequences carried Ty1/copia-derived segments and 15 sequences carried segments homologous with multiple TE families. 26 TE lineages were found in the 70 sequences, and among these lineages, Wilma was found in sequences dispersed in all chromosome regions except telomeric positions; Abiba was found in sequences predominantly located at pericentromeric and centromeric positions; Wis, Carmilla, and Inga were found in sequences displaying signals dispersed from distal regions toward pericentromeric positions; except DNA transposon lineages, all the other lineages were found in sequences displaying signals dispersed from proximal regions toward distal regions. A high percentage (21.4%) of chimeric sequences were identified in this study and their high abundance in rye genome suggested that new TEs might form through recombination and nested transposition. Our results also gave proofs that diverse TE lineages were arranged at centromeric and pericentromeric positions in rye, and lineages like Abiba might play a role in their structural organization and function. All these results might help in understanding the diversity and evolution of TEs in rye, as well as their driving forces in rye genome organization and evolution.

Keywords: Secale cereale; TE lineages; fluoresence in situ hybridization; nested transposition; variation.

FIGURE 1

Fluorescence in situ hybridization (FISH) mapping of rye species-specific sequences on metaphase chromosomes. Chromosomes were counterstained with DAPI (blue signals), rye species-specific sequences were labeled with Texas Red (red signals), and rye chromosomes were distinguished by pSc 119.2 (green signals). (A–C) The signal distribution of HK5-38 on chromosomes of allohexaploid triticale (AABBRR, 2n = 42), Secale cereale L. var. King II and “Chinese Spring” wheat (AABBDD, 2n = 42). (D–F) The signal distribution of HK11-4 on chromosomes of allohexaploid triticale, Secale cereale L. var. King II and “Chinese Spring” wheat. (G–I) The signal distribution of HK16-18 on chromosomes of allohexaploid triticale, Secale cereale L. var. King II and “Chinese Spring” wheat. (J–L) The signal distribution of HK15-13 on chromosomes of allohexaploid triticale, Secale cereale L. var. King II and “Chinese Spring” wheat. The signal of each rye species-specific sequence (red signals) was typically displayed by the enlarged 1R chromosomes placed in the inset, with pSc 119.2 (green signals) removed. Bars = 10 μm.

FIGURE 2

Fluorescence in situ hybridization mapping of sequences common to wheat and rye. Metaphase chromosomes were counterstained with DAPI (blue signals), sequences common to wheat and rye were labeled with Texas Red (red signals), and rye chromosomes were distinguished by pSc 119.2 (green signals). (A–C) The signal distribution of HK5-64 on chromosomes of allohexaploid triticale, Secale cereale L. var. King II and “Chinese Spring” wheat (AABBDD, 2n = 42). (D–F) The signal distribution of HK1-68 on chromosomes of allohexaploid triticale, Secale cereale L. var. King II and “Chinese Spring” wheat (AABBDD, 2n = 42). (G–I) The signal distribution of HK15-21 on chromosomes of allohexaploid triticale, Secale cereale L. var. King II and “Chinese Spring” wheat (AABBDD, 2n = 42). The signal of each sequence hybridized with both wheat and rye chromosomes (red signals) was typically displayed by the enlarged 1B and 1R chromosomes placed in the inset, with pSc 119.2 (green signals) removed. Bars = 10 μm.

FIGURE 3

Fluorescence in situ hybridization mapping of centromere-specific sequences on allohexaploid triticale metaphase chromosomes. Chromosomes were counterstained with DAPI (blue signals), and rye chromosomes were labeled and distinguished by pSc 119.2 (green signals). (A) The signal distribution of HK15-13. (B) The signal distribution of HK1-71. (C) The signal distribution of HK5-64. (D) The signal distribution of HK12-3. (E) The signal distribution of HK11-15. (F) The signal distribution of HK1-62. Bars = 10 μm.

FIGURE 4

Immuno-colocalization of centromeric sequences and CENH3 at the rye nuclei interphase stage. Nuclei were counterstained with DAPI (blue signals), and CENH3 binding was detected by the secondary antibody anti-rabbit coupled to fluorescein isothiocyanate (FITC; green signals). The centromeric sequences probes were labeled with Texas Red (red signals). (A) Co-localization of HK15-13 and CENH3. (B) Co-localization of HK1-71 and CENH3. (C) Co-localization of HK5-64 and CENH3. (D) Co-localization of HK12-3 and CENH. (E) Co-localization of HK11-15 and CENH3. (F) Co-localization of HK1-62 and CENH3. Bars = 10 μm.

FIGURE 5

Number of sequences homologous with different TE lineages. (A) Venn diagram showing number of sequences homologous with different TE class, the numbers in overlapped regions representing the number of sequences homologous with multiple TE class. (B) Pie chart showing numbers of sequences homologous with different TE lineages, six unknown lineages were included. (C) Pie chart showing numbers of non-chimeric sequences homologous with different TE lineages, one unknown lineage was included. (D) Pie chart showing numbers of chimeric sequences homologous with different TE lineages, six unknown lineage were included.