Discrimination of the closely related A and D genomes of the hexaploid oat Avena sativa L

Abstract

A satellite DNA sequence, As120a, specific to the A-genome chromosomes in the hexaploid oat, Avena sativa L., was isolated by subcloning a fragment with internal tandem repeats from a plasmid, pAs120, that had been obtained from an Avena strigosa (As genome) genomic library. Southern and in situ hybridization showed that sequences with homology to sequences within pAs120 were dispersed throughout the genome of diploid (A and C genomes), tetraploid (AC genomes), and hexaploid (ACD genomes) Avena species. In contrast, sequences homologous to As120a were found in two A-genome species (A. strigosa and Avena longiglumis) and in the hexaploid A. sativa whereas this sequence was little amplified in the tetraploid Avena murphyi and was absent in the remaining A- and C-genome diploid species. In situ hybridization of pAs120a to hexaploid oat species revealed the distribution of elements of the As120a repeated family over both arms of 14 of 42 chromosomes of this species. By using double in situ hybridization with pAs120a and a C genome-specific probe, three sets of 14 chromosomes were revealed corresponding to the A, C, and D genomes of the hexaploid species. Simultaneous in situ hybridizations with pAs120a and ribosomal probes were used to assign the SAT chromosomes of hexaploid species to their correct genomes. This work reports a sequence able to distinguish between the closely related A and D genomes of hexaploid oats. This sequence offers new opportunities to analyze the relationships of Avena species and to explore the possible evolution of various polyploid oat species.

Figure 1

Southern blot analysis of the DNAs of diploid, tetraploid, and hexaploid oats. The species, in lanes: 1, A. strigosa; 2, A. longiglumis; 3, A. canariensis; 4, A. damascena; 5, A. eriantha; 6, A. murphyi; 7, A. maroccana; 8 and 10, respectively, A. sativa cvs. Prevision and Extra Klock; 9, A. byzantina cv. Kanota; 11, A. sterilis. (a) EtdBr-stained 1.5% agarose gel after separation of DNA samples cut with EcoRV showing similar amounts of plant DNA in each lane. (b) Autoradiogram after hybridization to pAs120 probe.

Figure 2

Nucleotide sequence of a 670-bp fragment cloned in pAs120, which contains a tandem repetitive segment (dark lines) and a interspersed repetitive segment (light lines). The nucleotide sequence data are stored in the European Molecular Biology Laboratory, GenBank, and DNA Data Base in Japan nucleotide sequence databases under the accession no. AJ001922.

Figure 3

Sequence of a 389-bp fragment cloned in pAs120a that includes four closely related monomers. The consensus sequence has been written on the first line. Nucleotides common to the consensus are indicated by hyphens, sequences differences are denoted by the relevant nucleotide, deletions are indicated by dots, and insertions are written under the sequence. The two imperfect inverse repeats are overlined.

Figure 4

Southern blot analysis of the MunI-digested genomic DNA of diploid and polyploid oats. The species, in lanes: 1, A. strigosa; 2, A. damascena; 3, A. canariensis; 4, A. longiglumis; 5, A. eriantha; 6, A. murphyi; 7, A. sativa cv. Extra Klock. (a) EtdBr-stained 1.5% agarose gel of DNA samples. (b) Autoradiogram of a after transfer and hybridization with pAs120a probe. (c) Autoradiogram showing rehybridization of blot in b with pTa71 probe.

Figure 5

FISH of metaphase plates of A. sativa cvs. Extra Klock (a and b) and Prevision (c, d, e, and f). (a) Simultaneous visualization of hybridization sites of digoxigenin-labeled pTa794 (green) and rhodamine-labeled pAs120 (orange). (b) The same cell as in a, shown after in situ hybridization with digoxigenin-labeled pAm1 (green) and rhodamine-labeled pTa71 (red). (c) Double FISH of digoxigenin-labeled pAs120 a (green) and rhodamine-labeled pTa 794 (orange). (d) The same cell as in c, counterstained with 4′,6-diamidino-2-phenylindole. (e) Double FISH of digoxigenin-labeled pAs120a (green) and rhodamine-labeled pTa794 (orange). (f) The same cell as in e, shown after in situ hybridization with rhodamine-labeled pAm1 (orange) and digoxigenin-labeled pTa71 (green). In e, the numbers indicate the A-genome chromosomes. In f, the numbers indicate the A-C and D-C translocated chromosomes.