Gene Function Rather than Reproductive Mode Drives the Evolution of RNA Helicases in Sexual and Apomictic Boechera

Abstract

In higher plants, sexual and asexual reproductions through seeds (apomixis) have evolved as alternative strategies. Evolutionary advantages leading to coexistence of both reproductive modes are currently not well understood. It is expected that accumulation of deleterious mutations leads to a rapid elimination of apomictic lineages from populations. In this line, apomixis originated repeatedly, likely from deregulation of the sexual pathway, leading to alterations in the development of reproductive lineages (germlines) in apomicts as compared with sexual plants. This potentially involves mutations in genes controlling reproduction. Increasing evidence suggests that RNA helicases are crucial regulators of germline development. To gain insights into the evolution of 58 members of this diverse gene family in sexual and apomictic plants, we applied target enrichment combined with next-generation sequencing to identify allelic variants from 24 accessions of the genus Boechera, comprising sexual, facultative, and obligate apomicts. Interestingly, allelic variants from apomicts did not show consistently increased mutation frequency. Either sequences were highly conserved in any accession, or allelic variants preferentially harbored mutations in evolutionary less conserved C- and N-terminal domains, or presented high mutation load independent of the reproductive mode. Only for a few genes allelic variants harboring deleterious mutations were only identified in apomicts. To test if high sequence conservation correlates with roles in fundamental cellular or developmental processes, we analyzed Arabidopsis thaliana mutant lines in VASA-LIKE (VASL), and identified pleiotropic defects during ovule and reproductive development. This indicates that also in apomicts mechanisms of selection are in place based on gene function.

Keywords: Boechera; RNA helicases; apomixis; evolution; reproduction; sequence variation.

Fig. 1.

—Sliding window approach to visualize the usage of nucleobases per position in genomic regions of selected RNA helicase genes. For VASL, MEM, and MAA3, the number of different nucleobases comprised in the allelic variants was plotted for all variants of apomictic (blue), facultative apomictic (green), sexual accessions (red), or all accessions (black) with a binning of 12 bases per datapoint. Orange boxes below symbolize exons, blue boxes UTRs, and gray bars noncoding regions. For all genes, a higher sequence variability was observed in noncoding regions as compared with exonic regions or UTRs.

Fig. 2.

—Box and whiskers plot depicting the number of allelic variants per accession for all genes. Boxes indicate the lower and upper quartile of numbers of unique CDS variants. Horizontal lines in each box indicate the medium numbers of variants observed. Accession numbers are given below.

Fig. 3.

—Visualization of mutation density per accession and most detrimental mutations per gene and accession as compared with A. lyrata as reference. (A) Plot of the abundance of synonymous and nonsynonymous SNP per MB of CDS sequences. (B) Visualization of the most deleterious mutation per haplotype and accession with a mutation hierarchy from deleterious to harmless from Nonesense_Mutation leading to a premature stop codon, Frame_Shift_Deletion, Frame_Shift_Insertion, In_Frame_Del, and In_Frame_Ins leading to a deletion or insertion without causing a frameshift, and Nonstop_mutation causing a nonsynonymous SNP. Rows in the plot represent different genes, whereas the 147 allelic variants identified in the study per gene are presented in columns. Boxes between (A) and (B) indicate reproductive mode and ploidy. The plot has been done using the waterfall function from GenVisR (Skidmore et al. 2016).

Fig. 4.

—Plot of mutation density along the CDS of selected RNA helicase genes. For selected RNA helicases, mutations as compared with A. lyrata used as reference were plotted along the CDS in windows of 12 bp. Types of mutations depicted are silent mutations, nonstop mutations (nonsynonymous SNPs), in frame INDELs, nonsense mutations introducing a stop codon, and frameshift INDELs. Depicted are the genes encoding for the conserved proteins EIF4A1 and VASL harboring only silent or nonstop mutations in any accession, MEM and the undescribed helicase AT3G16840 accumulating mutations mainly in the N- and C-terminal regions, and RecQ4A and FAS4 accumulating all types of mutations along the CDS including conserved domains.

Fig. 5.

—Reproductive phenotypes in lines carrying mutant alleles of VASL in A. thaliana. Seed clearing and DIC microscopy was used to study reproductive development in wild-type and lines carrying mutant alleles. (A) FMS in the wild-type. (B–D) Additional or ectopic gametophytic cells in vasl_1/VASL (B, D) and vasl_3/VASL (C). (E) Wild-type like gametophyte after the first mitotic division harboring two nuclei in vasl_3/VASL. (F) Ectopic gametophyte in vasl_1/VASL. (G–I) Slightly protruding (I) or protruding (G, H) gametophytes in vasl_2/vasl_2 (G), vasl_1/VASL (H), and vasl_3/VASL (I), respectively. (ii) Inner integuments; black stars denote FMS; dark gray stars ectopic cells, ectopic, or protruding gametophytes. Scale bars are 20 µm.