Chromosomal characterization of the three subgenomes in the polyploids of Hordeum murinum L.: new insight into the evolution of this complex

Abstract

Hordeum murinum L. is a species complex composed of related taxa, including the subspecies glaucum, murinum and leporinum. However, the phylogenetic relationships between the different taxa and their cytotypes, and the origin of the polyploid forms, remain points of controversy. The present work reports a comparative karyotype analysis of seven accessions of the H. murinum complex representing all subspecies and cytotypes. The karyotypes were determined by examining the distribution of the repetitive Triticeae DNA sequences pTa71, pTa794, pSc119.2, pAs1 and pHch950, the simple sequence repeats (SSRs) (AG)10, (AAC)5, (AAG)5, (ACT)5, (ATC)5, and (CCCTAAA)3 via in situ hybridization. The chromosomes of the three subgenomes involved in the polyploids were identified. All tetraploids of all subspecies shared the same two subgenomes (thus suggesting them to in fact belong to the same taxon), the result of hybridization between two diploid ancestors. One of the subgenomes present in all tetraploids of all subspecies was found to be very similar (though not identical) to the chromosome complement of the diploid glaucum. The hexaploid form of leporinum came about through a cross between a tetraploid and a third diploid form. Exclusively bivalent associations among homologous chromosomes were observed when analyzing pollen mother cells of tetraploid taxa. In conclusion, the present results identify all the individual chromosomes within the H. murinum complex, reveal its genome structure and phylogeny, and explain the appearance of the different cytotypes. Three cryptic species are proposed according to ploidy level that may deserve full taxonomic recognition.

Figure 1. In situ hybridization with probes pTa71 (45S rDNA), pTa794 (5S rDNA), pAs1, (AG)₁₀, (AAC)₅, (AAG)₅, (ACT)₅, (ATC)₅, and the telomeric (Tel.) probe (CCCTAAA)₃, in three metaphases (panels a–f, g–h and i–j respectively) of the diploid H. murinum accession BCC2002.

Each panel shows merged images to facilitate the visualization of the in situ signals with respect to DAPI (blue) staining (a). In a, the arrows point to the two pairs of satellited chromosomes; in h, the arrows and arrowheads point to 5S rDNA and 45S rDNA loci respectively. In i and j, the arrows point to the pair of chromosomes carrying interstitial telomeric repeats and both ribosomal sequences. (k) Karyotypes showing one chromosome of each homologous group chosen from the metaphases shown in g–h (top and two bottom rows) and a–f (the remaining rows). Scale bar = 10 µm.

Figure 2. In situ hybridization in accessions GRA1144 (a–b), GRA2735 (c–d) and BCC2007 (e–h) illustrating the patterns obtained with probes pHch950, pTa71, pTa794, pSc119.2, pAs1, (AAG)₅, (AAC)₅, (AG)₁₀ and the telomeric (Tel.) probe (CCCTAAAG)₃ in tetraploid H. murinum taxa.

In a, the arrows point to (AG)₁₀ signals in five pairs of chromosomes. One pair carrying weak signals is shown in the insets. In b and e, the arrows point to pSc119.2 signals. The arrowheads in f point to the 14 chromosomes labeled with pHch950. In g, the arrows and arrowheads point to minor pTa794 and pTa71 sites respectively. In h, the arrows and arrowheads point to interstitial telomeric repeats and stronger, derived pTa71 signals (see g) respectively. The satellited chromosome pair belonging to subgenome Xu carrying pTa71, pTa794 and interstitial telomeric sequences is amplified in g′ and h′. Scale bar = 10 µm.

Figure 3. (a) (AAG)₅ karyotypes from each tetraploid genotype studied showing one chromosome of the seven homologous groups in the Xu and Xv subgenomes. Chromosomes of each karyotype were chosen from the same metaphase. Fig. 2c and e show those of GRA2735 and BCC2007. Note that the in situ patterns for each accession - even those belonging to different H. murinum subspecies - are very similar to one another. (b) Karyotypes of accession GRA2894, providing a representative sample of the chromosomal distribution of different probes in tetraploids of H. murinum. Note that signals from probes pTa71, pTa794 and pSc119.2 were drawn over the DAPI-stained chromosomes. Asterisks indicate the polymorphic sites (presence/absence) observed among the tetraploid accessions (Table 2).

Figure 4. In situ hybridization with probes pHch950, pTa71 (45S rDNA), pTa794 (5S rDNA), pSc119.2, (AAG)₅, (AAC)₅, and the telomeric (Tel.) probe (CCCTAAA)₃, in three metaphases cells (panels a–b, c–d and e–g, respectively) of the hexaploid H. murinum accession GRA1183.

Each panel shows merged images to facilitate the visualization of the signals with respect to the blue DAPI staining. In a, the arrows point to the 14 chromosomes revealed with pHch950. In c, the arrows and arrowheads point to the 5S rDNA and 45S rDNA minor loci respectively. In d, the arrows point to interstitial telomeric repeats (enlarged in the insets). In e, the arrowheads indicate pTa71 signals derived from previous hybridizations. (g) The four chromosome pairs carrying pSc119.2 signals were chosen from the metaphase shown in e–f. Scale bar = 10 µm.

Figure 5. Karyotypes of hexaploid H. murinum accession GRA1183 probed with pTa71, pTa794, (AAG)₅, pAs1, (AG)₁₀ and pSc119.2, showing one chromosome of the seven homologous groups of the Xu, Xv and Xw subgenomes.

Chromosomes of each karyotype were chosen from the same metaphase (that shown for [AAG]₅ in Fig. 4b). Note that the pTa71, pTa794 and pSc119.2 signals were drawn over the DAPI-stained chromosomes. The orange signals in the pAs1 karyotype are the stronger red signals observed in the (AG)₁₀ karyotype.

Figure 6. Photomicrographs showing meiotic metaphase I chromosome spreads of pollen mother cells from the tetraploid accessions BCC2007 (a–d) and GRA2735 (e–f) probed with multiple repeat DNA in sequential hybridizations.

The chromosome identities present as bivalents Xu-Xu and Xv-Xv are indicated in white and yellow letters, respectively. In a, the arrows point the seven bivalents revealed with pHch950 (some of them in the inset). In c, the arrows point the bivalents carrying pSc119.2 signals. In d, the arrows and arrowheads point to the (AG)₁₀ and 45S rDNA signals respectively. In f, the arrowheads point to the pSc119.2 signals. Scale bar = 10 µm.