Genome expansion in three hybrid sunflower species is associated with retrotransposon proliferation

The origin of new diploid species through inter-specific hybridization may be facilitated by rapid genomic reorganization. There is evidence that this process was involved in the independent origins of three annual sunflower species in the genus Helianthus. The three hybrid taxa, H. anomalus, H. deserticola and H. paradoxus, are products of ancient hybridization events between the same two parental taxa, H. annuus and H. petiolaris [1]. The hybrid species have geographically restricted ranges and occupy habitats that are abiotically extreme relative to other Helianthus species; H. anomalus and H. deserticola are found in desert environments, whereas H. paradoxus is restricted to saline marshes [2]. In addition to several novel karyotypic rearrangements [3], each hybrid taxon has a nuclear genome at least 50% larger than that of either parental species [4]. These genome size differences occur in spite of the fact that the hybrid and parental species are diploids and all possess the same number of chromosomes (n = 17). Because both inter-specific hybridization and abiotic stress have played important roles in the evolutionary history of the hybrid taxa, and because both have been implicated as natural agents of retrotransposon activation and proliferation [5,6], we sought to determine whether the genome size differences associated with hybrid speciation in these sunflowers could be attributable to proliferation of mobile genetic elements in the hybrid taxa. While multiple categories of transposable elements exist in eukaryotic genomes, the class I elements known as long terminal repeat (LTR) retrotransposons most often have been associated with genome size variation in plants [7]. Based on a previously reported Ty3/gypsy-like LTR retrotransposon sequence in Helianthus [8], we developed a PCR probe (887 base pairs from the integrase-domain-encoding region) to use in Southern blot experiments comparing element abundance in the hybrid and parental taxa. The probe was amplified from genomic DNA of the parental species H. annuus. Southern blots revealed a considerably stronger hybridization signal for the hybrid species relative to their parental species (Figure 1), indicating a higher relative abundance of Ty3/ gypsy sequences in the hybrid species’ genomes. This remained the case whether loadings were standardized by genome equivalents, standardized to 1 μg, or standardized to 500 ng without a restriction digest (Figure 1). To gain better quantitative estimates of element copy numbers in these sunflower genomes, we used a quantitative PCR strategy. Examination of 6–10 individuals per species and two different populations of each parental taxon confirmed a stunning increase of Ty3/gypsy sequences in all hybrid taxa (Figure 2), with 5.6 to 23.6-fold increases in copy number in the hybrid species. Statistically significant differences were not observed among different populations of parental species. Assuming a size of 5.2 kb for a Ty3/gypsy element in Helianthus [9] we estimate an additional 1330 Mb, 1162 Mb and 909 Mb of DNA in H. anomalus, H. deserticola and H. paradoxus, respectively, that is attributable to Ty3/gypsy proliferation. These estimates account for ~73%, ~79%, and ~62% of the differences in genome size between the parental species H. annuus (3000 Mb) [10] and H. anomalus, H. deserticola and H. paradoxus, respectively [4]. Retrotransposons represent ancient lineages that often exhibit considerable sub-lineage diversity in plant genomes. So although estimates reported in Figure 2 accurately reflect real differences between the hybrid