Molecular biological and evolutionary analysis of hybrid weakness in Capsicum annuum x Capsicum chinense

概要

Hybrid weakness, a type of postzygotic isolation in reproductive isolation, is defined as abnormal growth in F1 plants derived from crosses between two normal parents. This phenomenon often prevents plant breeding programs due to difficulty in obtaining offspring. Moreover, it is thought that hybrid weakness contributes to speciation. Therefore, the study of hybrid weakness is important in academic and industrial fields. However, the mechanism of hybrid weakness is not yet fully understood, even though it has been extensively studied in recent years.

This study aimed to investigate the mechanism of hybrid weakness in the cross of Capsicum annuum (chile or sweet pepper) × Capsicum chinense (bonnet pepper). The hybrid weakness is caused by two complementary dominant genes, and it is reported that C. annuum can possess the aaBB genotype while C. chinense and C. frutescens can possess the AAbb genotype (Inai et al. 1993, Yazawa et al. 1989). Despite these previous studies, however, the research on Capsicum hybrid weakness had not been advanced any further. In Chapter 1, I investigated the morphological and physiological characters of Capsicum F1 hybrids showing hybrid weakness. In Chapter 2, I investigated when hybrid weakness was induced in the plant growth phase. In Chapter 3, I focused on the changing time course after the induction of hybrid weakness. In Chapters 4 and 5, I studied about the relationship between hybrid weakness and speciation in Capsicum.

Chapter 1. Characterization of Capsicum hybrid weakness
I characterized morphologically and physiologically hybrid weakness in Capsicum. F1 hybrids did not show weaker growth than their parents after 20 days after germination (DAG), but at 40 DAG, the hybrid weakness phenotype was evidenced by almost complete arrest of new leaf formation, delayed increase in plant height, and reduced upper internode length. The shoot apical meristem (SAM) of F1 hybrids exhibited delayed development and an abnormal structure characterized by a flat shape and the presence of fuzzy cell layers on the surface. These abnormal SAMs of F1 plants may lead to dwarfism. Dead cells and accumulation of H2O2, one of the reactive oxygen species, were visually detected in leaves of F1 hybrids, and cell death was considered to be programmed, as it was accompanied by internucleosomal fragmentation of DNA. The expression of immunity marker genes PR1 and PR2 was upregulated in leaves of F1 hybrids. These results suggest that a hypersensitive response-like reaction is involved in Capsicum hybrid weakness.

Chapter 2. Abnormalities in juvenile-to-adult transition in Capsicum hybrid weakness
I surveyed morphological traits that are reported to change during juvenile-to-adult (JA) transition in vegetable phase in several plant species using the first to 9th leaves at 60 DAG. Length / width ratio, and presence or absence of trichome in the leaves didn’t almost change by leaf position in Capsicum. Petiole / leaf blade ratio decreased as leaf position increased. The decrease of petiole / leaf blade ratio reached plateau at 5th, 6th, and 7th leaves position in respectively C. annuum, C. chinense, and F1 hybrids. The number of lateral veins increased as leaf position increased in both parents and F1 hybrids, and those in F1 hybrids were less than those in both parents. In toluidine blue staining, the color was pink until 5th leaves, and it was blue from 7th leaves in both parents, but the color didn’t change in leaves of F1 hybrids. The number of stomata increased as leaf position increased, and rapidly increased from 6th leaves at both parents. However, the increase kept moderate in F1 hybrids, and the number of stomata in F1 hybrids was less than that of both parents from 6th leaves. Based on these morphological surveys, I estimated that juvenile phase was until 5th leaves, and adult phase was from 7th leaves in both parents. On the other hand, the JA transition was delayed or not progressed in F1 hybrids. The phenotype of hybrid weakness appeared when the 5th leaves almost fully expanded. Therefore, hybrid weakness is suggested to be induced during JA phase transition.

Chapter 3. The time course of temperature-dependent Capsicum hybrid weakness in gene- expressional, physiological and morphological changes
I showed that Capsicum hybrid weakness was suppressed at 30 and 35°C, and was induced at 15, 20, and 25°C. Moreover, I investigated the time course of hybrid weakness in cell death, metabolite content, and gene expression in leaves of plants transferred to 20°C after growing at 30°C for 21 days. The expression of pathogen defense-related genes was upregulated 1 day after transfer to 20°C (DAT). Cell death was detected at 7 DAT, plant growth had almost stopped since 14 DAT, and sugars were accumulated at 42 DAT in hybrid plants. The study revealed that some sugar transporter genes, which had been upregulated since 7 DAT, were involved in sugar accumulation in Capsicum hybrid weakness. Thus, these results demonstrated that gene expression changes occur first, followed by physiological and morphological changes after induction of hybrid weakness. The responses observed in this study in Capsicum hybrid weakness are likely to be owed to plant defense responses-like reactions.

Chapter 4. Phylogenetic analysis of Capsicum based on rDNA-ITS region
The rDNA-ITS sequences and morphological traits of domesticated and wild Capsicum species were examined. The accessions of C. annuum, C. chinense and C. frutescens were closely related according to morphology characters because no trait can distinguish one another. The phylogenetic tree based on rDNA-ITS formed the C. annuum clade, the C. chinense and C. frutescens clade, the C. baccatum clade, and the C. pubescens clade. C. annuum, C. chinense and C. frutescens, which may have A or B allele for hybrid weakness, were also closely related in the phylogenetic tree; especially, C. chinense and C. frutescens were genetically very close.
C. annuum accessions having b allele belonged to one clade in the C. annuum clade. It was suggested that Capsicum hybrid weakness could be involved in speciation of C. annuum.

Chapter 5. Phylogeographic analysis of Capsicum hybrid weakness
I surveyed whether 63 C. annuum accessions had B or b allele for hybrid weakness by test crossing with C. chinense having A allele. Out of the 63 C. annuum accessions, five accessions had B allele; three accessions were native to Latin America, and two accessions were native to Asia. I calculated the percentage of geographic distribution of the B carriers of C. annuum by Latin America, Europe, Africa, and Asia, based on the results of the present and previous studies (Yazawa et al. 1989). The percentage of B carriers was 42% in Japan, 13 % in Asia excluding Japan, 8% in Latin America, and 0% in Europe and Africa. Additionally, the 48 accessions of
C. annuum from various countries were subjected to SSR analysis. The number of the clade with high percentages of B carriers was two in UPGMA tree, and was one in NJ tree. In the graph by principal coordinate analysis, most B carriers were localized in a single group although the group also included b carriers. I presumed that the B allele was acquired in some C. annuum lines in Latin America, and both B-carrying and b-carrying lines were introduced to the world during the Age of Discovery.

I discussed the mechanism of Capsicum hybrid weakness based on the results of my research. Capsicum hybrid weakness was triggered by beginning JA transition, suggesting that a factor expressed during JA transition may affect the causal genes of hybrid weakness. Then, the interaction of two causal genes induced auto-immune responses accompanied with reaction oxygen species, cell death, and upregulation of genes of PR proteins and sugar transporters. Causal genes have not yet identified, but I guessed that one or two of the causal genes are the leucine-rich repeat receptor-like protein kinase genes as reported in hybrid weakness in other species, because auto-immune response occurs in Capsicum hybrid weakness. Moreover, I presumed that activation of sugar transporters by auto-immune responses may prevent sugar translocation, which plays an important role in JA transition. That may be why F1 hybrids cannot completely transit to adult phase in vegetable phase. Besides, I suggested the possibility that Capsicum hybrid weakness is involved in speciation of C. annuum, C. chinense and C. frutescens.