Hybrid incompatibility in Arabidopsis is determined by a multiple-locus genetic network

Abstract

The cross between Arabidopsis thaliana and the closely related species Arabidopsis arenosa results in postzygotic hybrid incompatibility, manifested as seed death. Ecotypes of A. thaliana were tested for their ability to produce live seed when crossed to A. arenosa. The identified genetic variation was used to map quantitative trait loci (QTLs) encoded by the A. thaliana genome that affect the frequency of postzygotic lethality and the phenotypes of surviving seeds. Seven QTLs affecting the A. thaliana component of this hybrid incompatibility were identified by crossing a Columbia × C24 recombinant inbred line population to diploid A. arenosa pollen donors. Additional epistatic loci were identified based on their pairwise interaction with one or several of these QTLs. Epistatic interactions were detected for all seven QTLs. The two largest additive QTLs were subjected to fine-mapping, indicating the action of at least two genes in each. The topology of this network reveals a large set of minor-effect loci from the maternal genome controlling hybrid growth and viability at different developmental stages. Our study establishes a framework that will enable the identification and characterization of genes and pathways in A. thaliana responsible for hybrid lethality in the A. thaliana × A. arenosa interspecific cross.

Figure 1.

Phenotype classification scheme for hybrid seeds and influence of maternal genotype in the A. thaliana × A. arenosa cross. A, Four phenotypic classes were observed in the output from the hybrid crosses: shriveled, green, viviparous, and normal seed. The mature phenotype of these seeds corresponds to defects in major transitions in seed development. B, Visualization of a C24 × A. arenosa 10-DAP silique shows normal and shriveled seeds. The normal seed has an embryo (EM) that fills the seed compartment, whereas the shriveled seed has an embryo arrested in the heart stage. C, The maternal A. thaliana genotype used in the cross to A. arenosa is indicated on the x axis. Bar heights represent mean %NGV values, and se is displayed as black error bars. Significant differences between genotype mean values (P = 0.05) are represented by different letters above the bars (same letter, no significant difference in mean value). Mean numbers are based on data from 15 plants (approximately 1,500 seeds) for each of the Col-0, C24, and F1 crosses, 80 plants (approximately 22,900 seeds) for BC1-Col-0, and 91 plants (approximately 23,300 seeds) for BC1-C24.

Figure 2.

Histograms summarizing the frequency of seed classes in RIL × A. arenosa progeny. The means for the %N, %G, %V, %NGV, and %N/NV (x axes) were calculated from six replicates of each RIL × A. arenosa, and the number of RILs with a given mean value are tallied on the y axis. The means of the entire RIL population, the Col-0 parent, and the C24 parent are indicated by labeled arrows. The distribution of phenotypic performance depends on the trait. For %NGV, and somewhat less for %N, %G, and %V, most RILs fall close to the lower value Col-0 parent. For %N/NV, most lines perform close to the parental midpoint. For all traits, a few lines equal or surpass the mean values of C24.

Figure 3.

Likelihood ratios for QTLs affecting seed set. Composite interval mapping was performed using trait mean values and marker genotypes from 150 A. thaliana RILs crossed to A. arenosa. Seed set was quantified by counting the number of seeds belonging to each of four classes, normal, green, viviparous, and shriveled, and used to calculate five traits: %NGV, %N, %G, %V, and %N/NV. Data were log transformed to improve normality. A line at a likelihood ratio (LR) of 13 is drawn to approximate statistical thresholds calculated by 1,000 permutations. The estimated thresholds for each trait were as follows: %NGV, LR ≥ 12.9; %N, LR ≥ 13.2; %G, LR ≥ 13.3; %V, LR ≥ 13.3; %N/NV, LR ≥ 13.0. All significant QTLs detected by this analysis are marked.

Figure 4.

Loci determining hybrid compatibility interact in a genetic network. The circles depict the A. thaliana genetic map, with intervals represented by circularly arrayed bars colored according to chromosome. A total of 15 epistatic interactions were identified for two of the traits under investigation: %NGV (top circle) and %N/NV (bottom circle). All seven additive QTLs (black outer bars) are involved in at least one epistatic interaction (colored lines). Genetic regions tested for TRD are outlined in gray, with thicker lines and gray fill for those that display TRD (P < 0.05). There are very few interactions shared by early seed traits (%NGV) and maturation/dormancy (%N/NV).

Figure 5.

Fine-mapping of major-effect QTLs reveals multiple loci. A and B, Dissection of the FOE QTL. A, The QTL likelihood plot with FOE localization on chromosome 2 (Fig. 3) is displayed above the map of five NILs with introgressions spanning the FOE QTL. NIL4 to -7 have the Col-0 genomic background, while NIL18 and NIL19 have the C24 background. All contain one introgressed segment, displayed at the bottom, except for NIL7, which contains a C24 introgression on chromosome 4 (middle right). LR, Likelihood ratio. B, Histograms depicting percentage values for four seed traits under investigation (%NGV, %N, %G, and %V). The phenotype was evaluated in NIL F1 × A. arenosa crosses compared with crosses between the background parent (either Col-0 or C24) and A. arenosa (x axis). The mean for each cross is given (y axis) with sd bars. Asterisks denote significant differences in trait means from parental control values based on unpaired Student’s t test. Corresponding P values are given at top right in A. Letters above each NIL denote significant differences in NIL-versus-NIL trait mean comparisons (P < 0.01). The absence of letters above the histogram indicates that the genotypes were not significantly different. All data were derived from three independent biological replicates, and P values have been Bonferroni corrected to reflect multiple tests. C and D, Dissection of the FII QTL. C, The QTL plot displays FII localization on chromosome 3 as well as three NILs with introgressions spanning the FII QTL. All NILs are in the C24 background with one Col-0 introgression. D, Same as B.