Chromosomal Evolution and Apomixis in the Cruciferous Tribe Boechereae

Abstract

The mustard family (Brassicaceae) comprises several dozen monophyletic clades usually ranked as tribes. The tribe Boechereae plays a prominent role in plant research due to the incidence of apomixis and its close relationship to Arabidopsis. This tribe, largely confined to western North America, harbors nine genera and c. 130 species, with >90% of species belonging to the genus Boechera. Hundreds of apomictic diploid and triploid Boechera hybrids have spurred interest in this genus, but the remaining Boechereae genomes remain virtually unstudied. Here we report on comparative genome structure of six genera (Borodinia, Cusickiella, Phoenicaulis, Polyctenium, Nevada, and Sandbergia) and three Boechera species as revealed by comparative chromosome painting (CCP). All analyzed taxa shared the same seven-chromosome genome structure. Comparisons with the sister Halimolobeae tribe (n = 8) showed that the ancestral Boechereae genome (n = 7) was derived from an older n = 8 genome by descending dysploidy followed by the divergence of extant Boechereae taxa. As tribal divergence post-dated the origin of four tribe-specific chromosomes, it is proposed that these chromosomal rearrangements were a key evolutionary innovation underlaying the origin and diversification of the Boechereae in North America. Although most Boechereae genera exhibit genomic conservatism, intra-tribal cladogenesis has occasionally been accompanied by chromosomal rearrangements (particularly inversions). Recently, apomixis was reported in the Boechereae genera Borodinia and Phoenicaulis. Here, we report sexual reproduction in diploid Nevada, diploid Sandbergia, and tetraploid Cusickiella and aposporous apomixis in tetraploids of Polyctenium and Sandbergia. In sum, apomixis is now known to occur in five of the nine Boechereae genera.

Keywords: Cruciferae; North America; apomixis; apospory; autopolyploidy; descending dysploidy; karyotype evolution; speciation.

FIGURE 1

(A) Phylogenetic position of the Boechereae in the Brassicaceae based on Nikolov et al. (2019). In parentheses, the number of genera and species is given based on data from BrassiBase (https://brassibase.cos.uni-heidelberg.de/). (B) Generic relationships within the Boechereae based on Beilstein et al. (2010) (Sandbergia and Yosemitea not shown).

FIGURE 2

Comparative cytogenomic maps of Pennellia micrantha (Halimolobeae), the ancestral Boechereae genome, and 10 analyzed Boechereae species. As subgenomes of the polyploid species/cytotypes have the same structure, only a single (sub)genome is shown for triploids (P. cheiranthoides, S. whitedii) and tetraploids (C. douglasii, P. cheiranthoides, P. fremontii). Color coding of 22 genomic blocks (A to X) reflects their position on the eight ancestral chromosomes (AK1–AK8) in the Ancestral Crucifer Karyotype (Lysak et al., 2016). Blocks split into two segments are labeled as “a” and “b.” Downward-pointing arrows denote the inverse orientation of GBs compared to their position in the ACK represented here by the P. micrantha genome. Black arrows mark the Boechereae-specific inversions which occurred prior to the divergence of the tribe, whereas red arrows denote genus- and species-specific inversions that occurred after the divergence of Boechereae. I^pe: pericentric inversion; I^pa: paracentric inversion; t: whole-arm translocation. All ideograms are drawn to scale, whereby the size of GBs corresponds to the size of homeologous blocks in the Arabidopsis thaliana genome (The Arabidopsis Information Resource, TAIR; http://www.arabidopsis.org). Genome structure of P. cheiranthoides was adopted from Mandáková et al. (2020).

FIGURE 3

The inferred origin of the ancestral Boechereae genome and painted chromosomes of Pennellia micrantha (Hal1–Hal8) and Boechera gracilipes (Boe1–Boe7). The ancestral Boechereae genome (n = 7) originated from a Halimolobeae-like genome (n = 8) through descending dysploidy mediated by an end-to-end translocation accompanied by a centromere inactivaton, two additional reciprocal translocations and a pericentric inversion (see Figure 4 for more details). In the CCP images, different colors correspond to the eight ancestral chromosomes (AK1–AK8) in the Ancestral Crucifer Karyotype (Lysak et al., 2016), whereas capital letters refer to 22 genomic blocks (A to X). A. thaliana BAC clones defining each BAC contig/painting probe in Pennellia and Boechera are listed in Supplementary Tables S2, S3, respectively. Chromosomes were counterstained by DAPI. The fluorescence signals of the painting probes were captured as black and white photographs, and the signals were then pseudocolored to match the eight chromosomes of the ACK. Scale bars, 10 μm.

FIGURE 4

The origin of Boechereae-specific chromosomes. Parsimoniously reconstructed origin of the ancestral Boechereae chromosomes Boe1, Boe2 (A), Boe3, and Boe5 (B) from the ACK/Halimolobeae-like chromosomes Hal1–Hal3, Hal5, and Hal8. Chromosome images show painting of the Boe5 homeolog at pachytene in Boechera gracilipes including the K-L/M-N region with the inactive AK5 paleocentromere (magnified inset). Downward-pointing arrows refer to GBs inverted compared to their orientation in the ACK/Halimolobeae genome. Capital letters mark the ancestral GBs of the ACK (Lysak et al., 2016). Green flash-like symbols indicate chromosome breakpoints; red arrows indicate the lost of Hal5 (AK5) centromere on Boe5. Scale bar, 10 μm.

FIGURE 5

Quadrivalents in Polyctenium fremontii (2n = 4x = 28). The 28 chromosomes form seven quadrivalents during the first meiotic division. The quadrivalent of Boe3 homeologs was identified by CCP of pachytene chromosomes with BAC contigs specific for genomic blocks F, G, W, and X (Supplementary Table S3). Chromosomes were counterstained by DAPI. Scale bars, 10 μm.

FIGURE 6

Species- and genus-specific chromosomal rearrangements in diploid Boechereae revealed by CCP. Red downward-pointing arrows denote inverse orientation of GBs compared to their position in the ancestral Boechereae genome (Figure 3). I^pe: pericentric inversion; I^pa: paracentric inversion; t: whole-arm translocation. Chromosomes were identified by CCP with A. thaliana BAC contigs labeled by biotin-dUTP (red flourescence), digoxigenin-dUTP (green) and Cy3-dUTP (yellow). A. thaliana BAC clones defining each BAC contig/painting probe are listed in Supplementary Tables S4, S5, S7, S8. Chromosomes were counterstained by DAPI. Scale bars, 10 μm.

FIGURE 7

Sequence comparison of the AK5 centromeric region on homeologous chromosomes in Arabidopsis thaliana, A. lyrata and Boechera stricta. (A) The functional centromere on chromosome 3 and 5 in A. thaliana and A. lyrata, respectively, is located between genomic blocks K-L and M-N, between At3g32980 and At3g33530. The 13-kb region corresponding to the inactive AK5 centromere is located between genes Bostr.0556s0638 and Bostr.0556s0640 on chromosome Boe5. (B) A dot-plot self-comparison of the 13-kb region on Boe5 showing the absence of tandem repeat arrays. (C) Annotation of the B. stricta scaffold 556 including the 13-kb region and regions 20 kb upstream and downstream. Transposable elements and their remnants do not exhibit a higher abundance within the 13-kb region.

FIGURE 8

Frequencies of dyads vs. tetrads, sexual vs. aposporous gametophytes, and parietal cells or tissues (≥1 parietal cell) vs. parietal tissues (>1 parietal cell). Numbers next to bars represent observations contributing to each variable pair.

FIGURE 9

Megasporogenesis and sexual and aposporous gametophyte formation in P. fremontii (A,K–N) and sexual C. douglasii (B,D,F,G), N. holmgrenii (C), and S. perplexa (E,H–J). (A) Archesporial cell (AC) at the budding integument stage. (B) Mitotic division of an AC yielding a proximal MMC and a distal parietal cell (P). (C,D) Anticlinal and paraclinal divisions, respectively, of a P to yield a two-celled parietal tissue. (E,F) Dyads (D) with one and two Ps, respectively. (G) Tetrad showing the functional megaspore (FM) and three degenerating megaspores (DM). Also shown is a parietal tissue consisting of three Ps that formed from two paraclinal divisions of the original P. (H) Two-nucleate sexual gametophyte (G2) showing a central vacuole (v), two of three DM, and a P. (I) Four-nucleate sexual gametophyte (G4) with three nuclei visible. (J) Eight-nucleate sexual gametophyte (G8) showing egg apparatus formation at the micropylar end of the gametophyte. (K) 1-nucleate aposporous gametophyte (AG1) from a nucellar cell at the MMC stage. (L) AG1 from a parietal cell (P-AG1) at the tetrad stage. (M) AG1 from a nucellar cell at the late tetrad stage showing functional megaspore degeneration (DFM), DMs, degenerating nucellar cells, and a degenerating parietal cell (DP). (N) Two-nucleate aposporous gametophyte (AG2) at the late tetrad stage showing a DFM, DMs, degenerating nucellar cells, a degenerating parietal cell (DP), and degenerating epidermal cells. Scale bars, 20 μm.