Dominance in self-compatibility between subgenomes of allopolyploid Arabidopsis kamchatica shown by transgenic restoration of self-incompatibility

The evolutionary transition to self-compatibility facilitates polyploid speciation. In Arabidopsis relatives, the self-incompatibility system is characterized by epigenetic dominance modifiers, among which small RNAs suppress the expression of a recessive SCR/SP11 haplogroup. Although the contribution of dominance to polyploid self-compatibility is speculated, little functional evidence has been reported. Here we employ transgenic techniques to the allotetraploid plant A. kamchatica. We find that when the dominant SCR-B is repaired by removing a transposable element insertion, self-incompatibility is restored. This suggests that SCR was responsible for the evolution of self-compatibility. By contrast, the reconstruction of recessive SCR-D cannot restore self-incompatibility. These data indicate that the insertion in SCR-B conferred dominant self-compatibility to A. kamchatica. Dominant self-compatibility supports the prediction that dominant mutations increasing selfing rate can pass through Haldane’s sieve against recessive mutations. The dominance regulation between subgenomes inherited from progenitors contrasts with previous studies on novel epigenetic mutations at polyploidization termed genome shock

Agrobacterium-mediated floral dip transformation of the model polyploid species Arabidopsis kamchatica

Polyploidization has played an important role in the speciation and diversification of plant species. However, genetic analyses of polyploids are challenging because the vast majority of the model species are diploids. The allotetraploid Arabidopsis kamchatica, which originated through the hybridization of the diploid Arabidopsis halleri and Arabidopsis lyrata, is an emerging model system for studying various aspects of polyploidy. However, a transgenic method that allows the insertion of a gene of interest into A. kamchatica is still lacking. In this study, we investigated the early development of pistils in A. kamchatica and confirmed the formation of open pistils in young flower buds (stages 8–9), which is important for allowing Agrobacterium to access female reproductive tissues. We established a simple Agrobacterium-mediated floral dip transformation method to transform a gene of interest into A. kamchatica by dipping A. kamchatica inflorescences bearing many young flower buds into a 5% sucrose solution containing 0.05% Silwet L-77 and Agrobacterium harboring the gene of interest. We showed that a screenable marker comprising fluorescence-accumulating seed technology with green fluorescent protein was useful for screening the transgenic seeds of two accessions of A. kamchatica subsp. kamchatica and an accession of A. kamchatica subsp. kawasakiana

Interspecific crosses between <i>A. halleri</i> and <i>A. kamchatica.</i>

<p>(A, B) Representative incompatible (A) and compatible (B) reactions on <i>A. kamchatica</i> stigmas. Crosses were carried out between <i>A. halleri</i> bearing haplogroup B and <i>A. kamchatica</i> bearing haplogroup B (A), and between <i>A. halleri</i> not bearing haplogroup B and <i>A. kamchatica</i> bearing haplogroup B (B). A bundle of pollen tubes indicates a compatible reaction (yellow arrow). Scale bars = 0.1 mm. (C) Crosses using <i>A. kamchatica</i> from Murodo, Tsurugi-Gozen and Biwako, bearing <i>AkSRK-A</i> and <i>AkSRK-D</i>. The <i>AkSRK-D</i> sequence of Murodo has a 45-bp deletion including one of the 12 conserved cysteine residues (see text). The <i>AkSRK-A</i> sequence of Biwako has a ∼1,700-bp insertion in exon 6 (see text and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s006" target="_blank">Table S1</a>). Note that full-length sequences of <i>AkSRK-D</i> from Tsurugi-Gozen were not confirmed and that crosses with <i>A. halleri</i> bearing haplogroup D were not conducted (indicated by asterisks). (D) Crosses using <i>A. kamchatica</i> from Darling Creek and Potter, bearing <i>AkSRK-B</i> and <i>AkSRK-D.</i> Two <i>A. halleri</i> plants that do not bear <i>SRK-A</i>, <i>SRK-B</i> or <i>SRK-D</i> were also used as pollen donors (indicated as “Other haplogroups”; see Methods). (C, D) Numerators denote crosses where more than 20 pollen tubes penetrated the stigma, indicating compatible reactions. Denominators show the total number of crosses conducted in each combination. NP: not performed.</p

Phylogeny of 76 <i>SRK</i> sequences of <i>A. halleri</i>, <i>A. lyrata</i>, and <i>A. kamchatica</i>.

<p>This phylogeny was obtained by the neighbor-joining method on pairwise proportions of nucleotide divergence. In total, 266 nucleotide positions were used. The evolutionary distances were computed using the Kimura two-parameter method. The tree includes 31 <i>SRK</i> sequences of <i>A. halleri</i> (<i>AhSRK</i>), 40 <i>SRK</i> sequences of <i>A. lyrata</i> (<i>AlSRK</i>) and five <i>SRK</i> sequences of <i>A. kamchatica</i> (<i>AkSRK</i>). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s015" target="_blank">Table S10</a> for the accession numbers of these sequences deposited in GenBank. <i>SRK</i> sequences of <i>A. halleri</i> and <i>A. lyrata</i> are shown in black, and those of <i>A. kamchatica</i> are shown in red.</p

Geographic distribution of <i>S</i>-haplogroups in <i>A. kamchatica</i>.

<p>(A) Geographic distribution of <i>AkSRK-A</i> (blue), <i>AkSRK-B</i> (green) and <i>AkSRK-C</i> (orange). Black circles: not found. (B) Geographic distribution of <i>AkSRK-D</i> (purple) and <i>AkSRK-E</i> (dark yellow).</p

Nucleotide divergence of <i>AkSRK</i> of five <i>S</i>-haplogroups in <i>A. kamchatica</i> from two parental species.

<p>All estimates were Jukes–Cantor corrected. Bars indicate standard errors. Sequence lengths compared were: 935 bp (<i>AkSRK-A</i>), 3773 bp (<i>AkSRK-B</i>), 1067 bp (<i>AkSRK-C</i>), 555 bp (<i>AkSRK-D</i>) and 550 bp (<i>AkSRK-E</i>). <i>AkSRK-B</i> was 100% identical to <i>AhSRK-B</i> throughout the coding sequence (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen-1002838-g005" target="_blank">Figure 5</a>). Because <i>AkSRK-B</i> belongs to a newly identified <i>S</i>-haplogroup (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#s2" target="_blank">Results</a>), orthologous sequences were not yet reported from <i>A. lyrata</i>, while <i>AhSRK-B</i> from <i>A. halleri</i> was found in this study. Thus, nucleotide divergence from <i>A. halleri</i> only is shown for <i>AkSRK-B</i>. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s007" target="_blank">Table S2</a> for details.</p

The pattern of segregation of <i>AkSRK-A</i> and <i>AkSRK-B</i> in 95 F₂ individuals.

<p>A/B, Amplified by both the <i>AkSRK-A</i>- and the <i>AkSRK-B</i>-specific primers. A/−, Amplified only by the <i>AkSRK-A</i> specific primer. −/B, Amplified only by the <i>AkSRK-B-</i>specific primer. −/−, Amplified by neither the <i>AkSRK-A</i>- nor the <i>AkSRK-B</i>-specific primer. Expected frequencies in each category are shown in parentheses. We confirmed the amplification of <i>AkSRK-D</i> in all 95 F₂ plants and in the F₁ plants.</p

Numbers of examples in which the primary mutations involved in the loss of SI are attributable to male or female components of self-incompatibility.

<p>The pattern is significantly different between wild and cultivated populations (two-tailed Fisher's exact test <i>p</i> = 0.0476; one-tailed <i>p</i> = 0.0238).</p>†<p>Wild populations: <i>Arabidopsis thaliana</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838-Tsuchimatsu1" target="_blank">[41]</a>, <i>A. kamchatica</i> (this study), <i>Capsella rubella</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838-Guo1" target="_blank">[43]</a> and <i>Leavenworthia alabamica</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838-Busch2" target="_blank">[44]</a>. Cultivated populations: <i>Brassica napus</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838-Goring1" target="_blank">[79]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838-Okamoto1" target="_blank">[80]</a>, <i>B. oleracea</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838-Nasrallah4" target="_blank">[111]</a> and <i>B. rapa</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838-Fujimoto3" target="_blank">[112]</a>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s013" target="_blank">Table S8</a> for details of mutations involved in the loss of SI in cultivated <i>Brassica</i> populations.</p

Entire coding sequences of <i>SRK</i> of five <i>S</i>-haplogroups from <i>A. kamchatica</i> and three <i>S</i>-haplogroups from <i>A. halleri</i>.

<p>Thick and thin horizontal bars indicate exons and introns, respectively. Black and white vertical bars indicate single nucleotide polymorphisms found in each haplogroup. Nucleotide sequences were not available for part of the first intron of <i>SRK-A</i> because of repetitive sequences (indicated by gray bars). The gray shaded region in exon 1 was used for generating the phylogenetic tree in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen-1002838-g001" target="_blank">Figure 1</a>.</p

The geographic distribution of <i>S</i>-haplogroups and population structure in <i>A. kamchatica</i>.

<p><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#s2" target="_blank">Results</a> of PCR-based genotyping of <i>SRK</i>, with inference of population structure based on four loci (two homeologous genes each of nuclear <i>WER</i> and <i>CHS</i> genes) for the clustering of <i>K</i> = 2, 3 and 4, by the Bayesian clustering algorithm implemented in InStruct software (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s002" target="_blank">Figure S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s006" target="_blank">Table S1</a>). Numbers below the diagrams correspond to the accessions listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s006" target="_blank">Table S1</a>. Triangles in accession No. 33 (marked with an asterisk) indicate that <i>AkSRK-B</i> and <i>AkSRK-C</i>, as well as <i>AkSRK-D</i>, were found in a single individual. We confirmed these results using the software STRUCTURE instead of InStruct (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s016" target="_blank">Text S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002838#pgen.1002838.s005" target="_blank">Figure S5</a> for details).</p