Transcriptome shock in hybrid intensifies with parental genetic divergence
As described above, the merging of two divergent genomes during hybridization can result in “transcriptome shock”. Many studies reported the altered expression patterns in hybrids. Bell et al.’s study on the intraspecific hybridization of Cirsium found that 70.0% of the studied genes were differentially expressed between the F1 hybrid and at least one of its parents, of which 92.5% were non-additively expressed [20]. Combes et al.’s study on the interspecific hybridization of Coffea canephora × C. eugenioides found that DEGs between hybrids and the parents accounted for ~27% of the studied genes, of which 87.1% presented a non-additive pattern [22]. While for the study of Drosophila melanogaster and D. sechellia, the percent was 96%, of which 84% were non-additively expressed [19]. When it come to our study, ~50% of the genes were differentially expressed between the hybrids and at least one of their parents in either the intra-sectional or the inter-sectional hybridization, and most of them were non-additively expressed in the hybrids (Figure 2). Based on the fragments which are available at NCBI and widely used for phylogenetic analysis (Additional file 1: Table S3), we calculated the genetic distances between the parental species of different studies. Regardless of the intraspecific hybridization of Cirsium, genetic distance between C. canephora and C. eugenioides is 0.025, between D. melanogaster and D. sechellia is 0.048, while between C. chekiangoleosa, C. amplexicaulis and C. azalea are 0.025 and 0.050, respectively. We found there are no linear relationship between the percent of DEGs and the parental genetic distance. A potential reason for this maybe that these works were conducted under different experimental systems. However, in our study, under the same experimental systems, we found that the percent of DEGs between the hybrids and their parents did not increase linearly as genetic distance between the parents become bigger, too. This seems doesn’t meet our expectation that the more divergent the parents are, the greater proportion of genes would be differentially expressed between the offspring and the parents. In fact, Coolon et al. also found that the DEGs did not increase consistently with divergence time, and they speculated that increasing magnitudes of expression differences rather than increasing numbers of genes with divergent expression drive the overall increase in expression differences with divergence time [24]. A potential model may be that, in a definite scope, DEGs between hybrids and their parents would increase with parental genetic distance. However, beyond this scope, new pattern may appear. Our results support this hypothesis. In our study, although the proportion of DEGs decreased to some extant in the inter-sectional hybrid, a greater proportion of DEGs would be non-additively expressed in the inter-sectional hybrid than that in the intra-sectional hybrid. Specifically, more DEGs were transgressively expressed in the inter-sectional hybrid than that in the intra-sectional hybrid. That means the relative proportion of non-additively (especially transgressively) expressed gene within DEGs in hybrids would increase with parental genetic divergence. Correspondingly, the total expression level of genes in the inter-sectional hybrid was more diverge from its parents than that in the intra-sectional hybrid as shown in Additional file 1: Figure S1. These results could serve as important evidence that transcriptome shock in hybrid would intensify with parental genetic divergence.
Relative frequency of cis- and trans-regulatory divergence in different hybrids
According to previous studies, cis- and trans-regulatory divergence have their own ways in affecting gene expression [19]. So, the relative frequency of cis- and trans-regulatory divergence has great influence on the inheritance of gene expression patterns in hybrid [18]. The relative frequency of cis- and trans-regulatory divergence revealed by different studies is always variable. Taking Drosophila for example, McManus et al.’s study on the hybrids of D. melanogaster × D. sechellia found that more genes showed significant evidence of trans- than cis-regulatory divergence [19]. In plants, Combes et al.’s study on Coffea canephora × C. eugenioides and Bell et al.’s study on the intraspecific hybridization of Cirsium, also found more genes were subjected to trans-regulatory divergence [20, 22]. However, when it came to the interspecific hybridization of Arabidopsis thaliana × A. arenosa more genes were significantly influenced by cis- rather than trans- regulatory divergence [21]. Denver et al. speculated that natural selection would eliminate most trans-acting mutations and accumulate cis-regulatory mutations over time [25]. That means the relative frequency of cis- and trans-regulatory changes in hybrids may be related to the divergence time between the parental species. To validate this inference, we calculated the genetic distances of the parental species involved in different studies. According to the nrDNA fragments, the genetic distance between D. melanogaster and D. sechellia is 0.048, between C. canephora and C. eugenioides is 0.025, while between Arabidopsis thaliana and A. arenosa is 0.050. According to these data, cis-regulatory changes tend to be dominant when the parental genetic distance is enough big.
When it came to our study, the cis- and trans-regulatory divergences in different crosses were distinguished using the same method with unified criterions. However, the results were completely different for that the proportions of genes with significant evidence of cis- and trans-regulatory divergence in the intra-sectional cross (C. azalea × C. chekiangoleosa) were 8.09% and 13.34%, respectively, whereas in the inter-sectional cross of C. azalea × C. amplexicaulis were 10.31% and 8.24%, respectively. In other words, trans-regulatory divergence was more prevailing than cis- in the intra-sectional cross, while in the inter-sectional cross was just the opposite. These results indicate that the proportion of genes with significant evidence of cis-regulatory divergence would increase, while with significant evidence of trans-regulatory divergence would decrease with genetic divergence between species. A potential reason for this phenomenon may be that cis-regulatory mutations are more likely to be fixed than trans- under natural selection. This seems to be inconsistent with a neutral model assuming equal probabilities of fixation for cis- and trans-regulatory polymorphisms. In fact, cis-acting mutations in the promoter region may simply alter the transcript levels of gene(s) downstream, whereas a trans-acting mutation in a transcription factor may result in multiple downstream regulatory changes [26]. For selection, it must act more strongly against mutations with pleiotropic effects to operate more efficiently [27]. So trans-regulatory mutations with multiple effects are more likely to be eliminated. Specifically, as Wittkopp et al. [28] speculated, trans-acting mutations may include both highly pleiotropic changes as well as some with limited effects, the former ones are more likely to be eliminated, while the later ones could be accumulated by mutation-selection balance. This may be an important pattern for the evolution of cis- and trans-regulation.
cis- and trans-regulatory differences underlying expression divergence between species
McManus et al.’s study on the hybrid of Drosophila showed that the median significant of trans-regulatory divergence was larger than that of cis-regulatory divergence between species, and trans-regulatory divergence correlated more highly with the expression difference between species [19]. Same profile also appeared in the study of Cirsium [20]. Similarly, our results showed that trans-regulatory change contributed more to the expression divergence between C. azalea and the other two species than cis-regulatory change. Correspondingly, the expression differences between C. azalea and the other two species correlated more highly with trans-regulatory changes, too. That means trans-regulatory change plays a larger role than cis-regulatory change in promoting the differentiation of gene expression between species. We also detected the changes of the % cis with the absolute magnitude of total expression divergence between species. As shown in Figure 4B, the relative percent of cis-regulatory divergence decreased with the absolute magnitude of total expression divergence between C. azalea and the other two species. In other words, genes which were more deeply affected by trans-regulatory change would be more divergently expressed between species. This was consistent with the result generated from previous studies [20, 24]. However, as cis-regulatory mutation accumulates over time, its influence on expression divergence increases, too. This could be deduced from the fact that the contribution of cis-regulatory change (% cis) to the expression divergence between C. azalea and C. amplexicaulis at any level was obviously higher than that between C. azalea and C. chekiangoleosa (Figure 4B).
Cis- and trans-regulatory divergence are not mutually exclusive, many genes would be significantly influenced by both cis- and trans-regulatory changes [19, 20, 22, 24, 29]. In our study, 3.62% of the studied genes between C. azalea and C. chekiangoleosa and 3.07% between C. azalea and C. amplexicaulis showed significant evidence of both cis- and trans-regulatory divergence. Interactions between cis- and trans-regulatory divergences can result in quite divergent expression patterns between species. As shown in Figure 4C and 4D, cis- and trans- regulatory changes promoting expression of the same allele (cis + trans) could greatly stimulate the expression divergence between species. Conversely, if two regulatory categories act on the alternate alleles (cis × trans), the divergence of gene expression between species would be relieved to some extent. Specifically, the compensatory effect of cis- and trans-regulatory changes tended to eliminate expression divergence between species. These findings are consistent with the results generated from Coffea [22] and Arabidopsis [21]. According to previous studies, genes significantly influenced by “cis + trans” might be driven by directional selection [16], whereas regulated by “cis × trans” as well as “compensatory” are likely to be driven by stabilizing selection [30]. In our study, the proportion of genes with significant evidence of both cis- and trans-regulatory divergence was lower in the cross of C. azalea × C. amplexicaulis than that in C. azalea × C. chekiangoleosa, mainly because of that fewer genes were affected by “cis × trans” (Figure 3). This may just reflect the evolutionary history of the three Camellia species: C. azalea and C. chekiangoleosa, as two closely related species, have more genes experienced stabilizing selection; while for species from two divergent sections (C. azalea and C. amplexicaulis), fewer genes between them are driven by stabilizing selection.
Contribution of regulatory divergence to gene expression patterns in hybrid
As described above, molecular basis underlying gene-expression difference is the variation of cis- and trans-regulations. Previous studies on Drosophila [19] and yeast [31] showed that cis-regulatory divergence appeared to result in additive inheritance of gene expression more often than trans-regulatory divergence. However, latest studies based on transcriptome analysis reported the opposite result, for that trans-regulatory divergence in these studies accounted for a greater proportion of the regulatory divergence at sites with additive than that with non-additive inheritance patterns [20, 24]. In our study, in the F1 hybrid of C. azalea × C. chekiangoleosa, the median of % trans was significant higher for genes showing additive expression pattern than that showing non-additive expression pattern (Figure 5). However, in the hybrid of C. azalea × C. amplexicaulis, there was no significant difference in the relative percent of cis- and trans-regulatory divergence for neither additively nor non-additively expressed genes. We speculate that the relative contribution of cis- and trans-regulatory divergence (% cis and % trans) to the inheritance of gene expression may depend on the parental genetic divergence. A potential mode is that trans-regulatory divergence is more likely to lead to additive inheritance than cis-regulatory divergence. For hybrids whose parents are closely related species, the relative frequency of trans-regulatory divergence is higher than that of cis-regulatory divergence; however, as genomes between the two parents become more divergent, trans-regulatory mutations are eliminated to some extent and cis-regulatory divergence becomes dominant. This could be used to explain why a higher proportion of genes would be non-additively (especially transgressively) expressed in the F1 hybrid of inter-sectional than that of intra-sectional hybridization.
The interactions between cis- and trans-regulatory divergences can greatly affect gene expression patterns between species. There were studies showed that “cis × trans” regulatory divergence was more common in transgressively expressed genes [19, 29]. However, study on the hybrids of Cirsium found that genes with transgressive expression pattern were mainly regulated by “cis + trans” [20]. In our study, neither “cis × trans” nor “cis + trans” regulation was the major reason leading to the transgressive expression patterns in hybrids (Table 1). Instead, most of the DEGs between the hybrids and their parents followed a “conserved” or “ambiguous” manner. In addition, compared with the intra-sectional hybridization, a greater proportion of DEGs were subjected to “cis only” effect in the inter-sectional hybridization for any expression patterns. So, inheritance of gene expression patterns is more likely to be the result of the comprehensive effects of different regulatory mechanisms, and the change of relative frequency of cis- and trans-regulatory divergence plays an important role in the formation of divergent expression patterns in hybrid.