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. 2016 Feb 4;17(2):203.
doi: 10.3390/ijms17020203.

Use of Repetitive Sequences for Molecular and Cytogenetic Characterization of Avena Species from Portugal

Diana Tomás  1 , Joana Rodrigues  2 , Ana Varela  3 , Maria Manuela Veloso  4   5 , Wanda Viegas  6 , Manuela Silva  7
Affiliations

Use of Repetitive Sequences for Molecular and Cytogenetic Characterization of Avena Species from Portugal

Diana Tomás et al. Int J Mol Sci. .

Abstract

Genomic diversity of Portuguese accessions of Avena species--diploid A. strigosa and hexaploids A. sativa and A. sterilis--was evaluated through molecular and cytological analysis of 45S rDNA, and other repetitive sequences previously studied in cereal species--rye subtelomeric sequence (pSc200) and cereal centromeric sequence (CCS1). Additionally, retrotransposons and microsatellites targeting methodologies--IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism)--were performed. A very high homology was detected for ribosomal internal transcribed sequences (ITS1 and ITS2) between the species analyzed, although nucleolar organizing regions (NOR) fluorescent in situ hybridization (FISH) analysis revealed distinct number of Nor loci between diploid and hexaploid species. Moreover, morphological diversity, evidenced by FISH signals with different sizes, was observed between distinct accessions within each species. pSc200 sequences were for the first time isolated from Avena species but proven to be highly similar in all genotypes analyzed. The use of primers designed for CCS1 unraveled a sequence homologous to the Ty3/gypsy retrotransposon Cereba, that was mapped to centromeric regions of diploid and hexaploid species, being however restricted to the more related A and D haplomes. Retrotransposon-based methodologies disclosed species- and accessions-specific bands essential for the accurate discrimination of all genotypes studied. Centromeric, IRAP and REMAP profiles therefore allowed accurate assessment of inter and intraspecific variability, demonstrating the potential of these molecular markers on future oat breeding programs.

Keywords: Avena Portuguese lines; genomic diversity; molecular markers; repetitive sequences.

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Figures

Figure 1
Figure 1
45S rDNA fluorescent in situ hybridization (green signals) in metaphase cells of diploid A. strigosa. In the landrace from Madeira Island (a) two pairs of nucleolar organizing regions (NORs) with distinct sizes are observed and in the landrace from Elvas; (b) two pairs of NORs with similar sizes are detected.
Figure 2
Figure 2
45S rDNA fluorescent in situ hybridization (green signals) in metaphase cells of Avena hexaploid species. Three pairs of NORs are detected both in A. sativa as in A. sterilis. (a) A. sativa “S. Eulália” presenting NORs with similar sizes; (b) A. sativa from Madeira Island showing one large pair of NORs, one medium size pair and one heteromorphic pair (one medium size and one small); (c) A. sterilis showing one NOR pair with one signal (long line arrow); a second pair composed by two domains with different sizes (short line arrow); and a third one with two domains with similar sizes (arrow head).
Figure 3
Figure 3
PCR amplification products obtained using pSc200 primers. M: molecular marker 1 Kb+; strM—A. strigosa from Madeira Island; strE—A. strigosa from Elvas; satK—A. sativa “Kyto”; satM—A. sativa from Madeira Island; satSE—A. sativa “S. Eulália”; stePt—A. sterilis Pt; Rye—S. cereale.
Figure 4
Figure 4
PCR amplification products obtained using CCS1 primers and correspondent schematic representation. M: molecular marker 1 Kb+; strM—A. strigosa from Madeira Island; strE—A. strigosa from Elvas; satK—A. sativa “Kyto”; satM—A. sativa from Madeira Island; satSE—A. sativa “S. Eulália”; stePt—A. sterilis Pt. Arrowhead indicates the Avena characteristic band that was amplified from all genotypes, isolated and sequenced.
Figure 5
Figure 5
Centromeric sequence fluorescent in situ hybridization (red signals) in metaphase cells. Metaphase cells of A. strigosa (a–c); and A. sterilis (d–f) with DAPI staining (a,d), FISH with centromeric probe (Accession Number KM948610) (b,e) and merge of both fluorescent signals (c,f).
Figure 6
Figure 6
Inter-retrotransposon amplified polymorphism (IRAP) banding profiles obtained with primer for Nikita retrotransposon long terminal repeat (LTR) and correspondent schematic representation. M: molecular marker 1 Kb+; strM—A. strigosa from Madeira Island; strE—A. strigosa from Elvas; satK—A. sativa “Kyto”; satM—A. sativa from Madeira Island; satSE—A. sativa “S. Eulália”; stePt—A. sterilis Pt.
Figure 7
Figure 7
REMAP banding profiles obtained with primer for Nikita retrotransposon LTR and (CA)9G microsatellite anchored primer and correspondent schematic representation. M: molecular marker 1 Kb+; strM—A. strigosa from Madeira Island; strE—A. strigosa from Elvas; satK—A. sativa “Kyto”; satM—A. sativa from Madeira Island; satSE—A. sativa “S. Eulália”; stePt—A. sterilis Pt.
Figure 8
Figure 8
Dendogram based on concatenated centromeric, IRAP and REMAP banding profiles. DICE similarity index [36] and UPGMA clustering mode were used. Cophenetic correlation coefficient of 0.996.
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