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. 2013 Sep;25(9):3280-95.
doi: 10.1105/tpc.113.114405. Epub 2013 Sep 30.

The More the Merrier: Recent Hybridization and Polyploidy in Cardamine

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Free PMC article

The More the Merrier: Recent Hybridization and Polyploidy in Cardamine

Terezie Mandáková et al. Plant Cell. .
Free PMC article

Abstract

This article describes the use of cytogenomic and molecular approaches to explore the origin and evolution of Cardamine schulzii, a textbook example of a recent allopolyploid, in its ~110-year history of human-induced hybridization and allopolyploidy in the Swiss Alps. Triploids are typically viewed as bridges between diploids and tetraploids but rarely as parental genomes of high-level hybrids and polyploids. The genome of the triploid semifertile hybrid Cardamine × insueta (2n = 24, RRA) was shown to combine the parental genomes of two diploid (2n = 2x = 16) species, Cardamine amara (AA) and Cardamine rivularis (RR). These parental genomes have remained structurally stable within the triploid genome over the >100 years since its origin. Furthermore, we provide compelling evidence that the alleged recent polyploid C. schulzii is not an autohexaploid derivative of C. × insueta. Instead, at least two hybridization events involving C. × insueta and the hypotetraploid Cardamine pratensis (PPPP, 2n = 4x-2 = 30) have resulted in the origin of the trigenomic hypopentaploid (2n = 5x-2 = 38, PPRRA) and hypohexaploid (2n = 6x-2 = 46, PPPPRA). These data show that the semifertile triploid hybrid can promote a merger of three different genomes and demonstrate how important it is to reexamine the routinely repeated textbook examples using modern techniques.

Figures

Figure 1.
Figure 1.
Reconstructed Comparative Karyotypes in C. amara and C. rivularis (2n = 2x = 16). (A) and (B) DAPI-stained mitotic chromosomes and comparative ideograms of C. amara (A) and C. rivularis (B). The arrow indicates the 2.5-Mb inversion on chromosome CA1, and the open circle shows the ancestral position of the relocated centromere on CR3. (C) Identification of a translocation between chromosomes CA6 and CA8 by CCP of pachytene spreads in C. amara. Arrowheads indicate centromere regions. Bars = 5 µm
Figure 2.
Figure 2.
Genome Structure of the Triploid Hybrid C. × insueta (2n = 3x = 24). (A) GISH revealing eight chromosomes of C. amara (red fluorescence) and 16 chromosomes of C. rivularis (green fluorescence). DAPI-stained chromosome spread shown in the inset. (B) Comparative ideogram of C. × insueta based on comparison with the eight ancestral chromosomes and 24 genomic blocks (A to X) of the ACK (Schranz et al. 2006) and the reconstructed karyotypes of C. amara (chromosomes CA1 to CA8) and C. rivularis (CR1 to CR8). (C) The 2.4-Mb paracentric inversion on the CR8' homolog (blocks O, P, W, and X) in plants from subpopulation #4. Arrows point to the inverted region, and arrowheads indicate centromeres. (D) to (F) GISH revealing chromosomes of C. amara (red) and C. rivularis (green) at pachytene (D), diakinesis (E), and metaphase I (F). Bars = 5 µm
Figure 3.
Figure 3.
Genome Composition of C. × schulzii. (A) GISH in the hypopentaploid (2n = 5x−2 = 38) and hypohexaploid (2n = 6x−2 = 46) accessions. Mitotic chromosome spreads were labeled by gDNA of C. amara (red) and C. rivularis (green) and reprobed by gDNA of C. amara (red) and C. pratensis (green). Arrowheads indicate terminal heterochromatic knobs. (B) Frequency of AK5 and AK8/6 homoeologs and the fusion AK5/8/6 chromosome in genomes of C. pratensis and C. × schulzii. The same chromosomes identified by comparative painting with BAC contig probes for AK5 (green) and AK8/6 (red) homoeologs. Insets and arrowheads show the AK5/8/6 fusion chromosome. Bar = 5 µm. (C) Circos plot showing the collinearity between AK5 and AK8/6 homoeologs in C. amara and C. rivularis and the fusion chromosome AK5/8/6 in the hypotetraploid C. pratensis and in C. × schulzii. Capital letters refer to genomic blocks of the ACK (Schranz et al., 2006), and broken blocks are indexed by small letters.
Figure 4.
Figure 4.
Genome-Specific Variation of Satellite Repeats in Cardamine Accessions. (A) DNA gel blot hybridization showing structural polymorphisms of the Crambo repeats in Cardamine accessions collected at Urnerboden in the course of this study (Kremnica refers to C. pratensis from Slovakia, 2n = 44). (B) DNA gel blot hybridization shows amplification of Crambo repeats in herbarium specimens collected as C. schulzii at the time of its discovery. The absence of signal in the high molecular fraction is due to significant degradation of DNA. (C) to (H) Chromosome localization of Crambo (green fluorescence) and Prasat (red fluorescence) satellites in Cardamine taxa from Urnerboden. (C) C. amara, no hybridization signals. (D) C. rivularis, two interstitial loci of Crambo. (E) C. × insueta, two interstitial loci of Crambo. (F) to (H) Interstitial and/or terminal loci of Crambo (green) and Prasat (red) hybridizing to heterochromatic knobs in the hypotetraploid C. pratensis (F), hypopentaploid C. × schulzii (G), and hypohexaploid C. × schulzii (H). Insets display examples of Crambo-bearing chromosomes. Bar = 5 µm.
Figure 5.
Figure 5.
Ratio of Gene Copy Numbers in Cardamine Accessions from Urnerboden Detected by Pyrosequencing (PyroMark). The red and green bars show the average ratio of four SNP sites between C. amara and C. rivularis/C. pratensis, respectively; error bars indicate the standard deviations based on the C. amara percentage. The last five columns show the analyzed herbarium specimens of C. × insueta (ZT35752 and ZT35751) and C. × schulzii (ZT35755, ZT35754, and ZT35753).
Figure 6.
Figure 6.
Phylogenetic Analyses of cpDNA Sequence Data (rpoB-trnC Intergenic Spacer) of Cardamine Accessions. (A) Maximum likelihood phylogenetic tree, and (B) maximum parsimony network Terminal labels (A) include the accession code and species abbreviation: am, C. amara (green; haplotypes denoted as A1 to A5); riv, C. rivularis (red; haplotypes R1 to R3); prat, C. pratensis (blue; haplotypes P1 to P6); ins, C. × insueta (brown; haplotype I1); sch5x and sch6x, hypopentaploid and hypohexaploid C. × schulzii (brown; haplotype S1). The capital letter U indicates accessions from the Urnerboden site. Values above branches show bootstrap support. Interrupted branches were shortened by half. In the network (B), the circles represent haplotypes (the circle sizes are proportional to haplotype frequencies), the lines indicate mutational steps, and the black dots are unsampled haplotypes.
Figure 7.
Figure 7.
Inferred Parentage of C. × insueta and C. × schulzii.

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