The Sheltered Genetic Load Linked to the S Locus in Plants: New Insights From Theoretical and Empirical Approaches in Sporophytic Self-Incompatibility

Inbreeding depression and mating systems evolution are closely linked, because the purging of deleterious mutations and the fitness of individuals may depend on outcrossing vs. selfing rates. Further, the accumulation of deleterious mutations may vary among genomic regions, especially for genes closely linked to loci under balancing selection. Sporophytic self-incompatibility (SSI) is a common genetic mechanism in angiosperm that enables hermaphrodite plants to avoid selfing and promote outcrossing. The SSI phenotype is determined by the S locus and may depend on dominance relationships among alleles. Since most individuals are heterozygous at the S locus and recombination is suppressed in the S-locus region, it has been suggested that deleterious mutations could accumulate at genes linked to the S locus, generating a “sheltered load.” In this article, we first theoretically investigate the conditions generating sheltered load in SSI. We show that deleterious mutations can accumulate in linkage with specific S alleles, and particularly if those S alleles are dominant. Second, we looked for the presence of sheltered load in Arabidopsis halleri using CO₂ gas treatment to overcome self-incompatibility. By examining the segregation of S alleles and measuring the relative fitness of progeny, we found significant sheltered load associated with the most dominant S allele (S15) of three S alleles tested. This sheltered load seems to be expressed at several stages of the life cycle and to have a larger effect than genomic inbreeding depression.THE main genetic mechanism causing inbreeding depression is believed to be the expression of recessive mildly deleterious mutations in inbred individuals (Charlesworth and Charlesworth 1999). These deleterious mutations are generally supposed to be distributed throughout the genome. However, some genomic regions where loci under balancing selection are present may be more inclined than others to accumulate deleterious mutations and could lead to the formation of what is generally called a “sheltered load” (Uyenoyama 1997; van Oosterhout 2009). The sheltered load has been suggested as a potential reason why MHC genes, mating-type systems in fungi, and self-incompatibility systems in plants generally show longer terminal branches in their genealogies than expected (Richman 2000). Despite its potential importance, the extent of the sheltered load is still largely unknown.Homomorphic self-incompatibility is widely distributed among angiosperm families (de Nettancourt 2001; Igic et al. 2008). Self-incompatibility (SI) is controlled by genes under strong balancing selection. SI prevents self-fertilization and promotes outcrossing by the presence of a gamete recognition system involving proteins expressed in both the pollen and the pistil. The proteins controlling the recognition system are generally encoded by genes located in a single genomic region, the S locus. Each plant in a self-incompatible population expresses an S specificity and is unable to mate with other plants expressing the same specificity. In species with gametophytic self-incompatibility (GSI), the S specificity is controlled by interactions between protein expressed in the pollen''s haploid genome, the male gametophyte, and the pistil''s diploid genome. In species with sporophytic self-incompatibility (SSI), S specificity is controlled by interactions between gene products of the diploid sporophyte expressed on the pollen coat and those on the stigmatic surface. In this mating system, three reasons may facilitate the accumulation of recessive deleterious mutations in this region, namely a sheltered load (Uyenoyama 1997). First, high heterozygote frequencies are expected in populations at the S locus but also at other linked loci in the S-locus genomic region (Kamau et al. 2007). Second, negative frequency-dependent selection, a form of balancing selection, is the main selective force acting on the S locus and on linked genes (Castric and Vekemans 2004). Third, the recombination rate is low in the S-locus region (Casselman et al. 2000; Charlesworth et al. 2003). Such a sheltered load may have important evolutionary consequences for SI evolution: it can slow down the rate of emergence of new S alleles (Uyenoyama 2003), considerably extend the conditions for the persistence of GSI (Porcher and Lande 2005), and, finally, substantially increase the inbreeding depression in a small population (Glémin et al. 2001), which can have large consequences for endangered species and the viability of their populations.The magnitude of the sheltered load should depend on the size of the genomic region in which heterozygosity is enforced because of linkage to the S locus and also on the number of genes affecting fitness in that region. From an analysis of recombination rates in the S-locus genomic region in Arabidopsis lyrata, a species with SSI, Kawabe et al. (2006) suggested that the number of genes in the S-genomic region is probably not high enough for a large sheltered load to have an impact on fitness compared to the overall genomic load. Dowd et al. (2000) indeed found only 13 genes near the S locus in Petunia inflata. However, two studies have demonstrated the existence of transmission ratio distortion of some S alleles in A. lyrata (Bechsgaard et al. 2004; Leppala et al. 2008). The authors proposed that this could be indirect evidence of the existence of a sheltered load. To the best of our knowledge, the existence of sheltered load in SI species was specifically demonstrated so far only in Solanum carolinense, a species with GSI: Stone (2004) crossed individuals sharing alleles at the S locus, using bud pollination to overcome self-incompatibility. By looking at seed number and genotype of the progeny, a sheltered load linked to only two of seven S alleles investigated was detected. Direct evidence and estimations of the extent of the sheltered load are thus lacking.In SSI, complex dominance interactions among S alleles are usually observed [Ipomoea trifida (Kowyama et al. 1994), Brassica campestris (Hatakeyama et al. 1998), A. lyrata (Mable et al. 2003), and A. halleri (Llaurens et al. 2008a)]. The effect of these dominance interactions on the occurrence of a sheltered genetic load has not been investigated either theoretically or empirically, but may potentially be large. Indeed, recessive S alleles are expected to be more often homozygous in natural populations than dominant alleles (Schierup et al. 1997), and so may rapidly purge strongly deleterious recessive mutations, and thus should limit the sheltering effect. The sheltered load could thus differ depending on the dominance levels of the associated S alleles.In this study, we first investigated the theoretical conditions for the accumulation of a sheltered load in a SSI system, using stochastic simulations. Then, we empirically tested the existence and strength of an S-linked sheltered load in relation to dominance levels in SSI. We focused on A. halleri, a member of the Brassicaceae family. In this family, the S-locus region includes two major genes: SCR (also called SP-11), encoding a cysteine-rich protein of the pollen envelope, and SRK, encoding a receptor kinase located across the membrane of the papilla cells. High heterozygote frequencies at the S locus have been found in several species like B. insularis (Glémin et al. 2005) or A. lyrata (Schierup et al. 2006). The SRK and SCR genes are tightly linked, since they are located close to each other, and recombination suppression in the S-locus region has been suggested in several studies: in Brassica (Casselman et al. 2000) and in A. lyrata (Kamau and Charlesworth 2005; Kawabe et al. 2006). The conditions thus may be suitable for the existence of sheltered genetic load in A. halleri. We performed controlled pollinations in A. halleri to specifically measure the magnitude of the potential sheltered load of three S alleles with different dominance levels: a dominant, an intermediate, and a recessive allele. To evaluate the effect of the sheltered load on these crosses, we looked at the number of seeds produced, as well as at the development and the genotype at the S locus of the progeny.