Small RNAs Contribute Significantly To The Phenotypic Variation In The Distant Reciprocal Tomato Hybrids

Different phenotypes between the reciprocal hybrids and transgression in the hybrids had been reported in the distant hybridization. Many mechanisms underlying those focused on relevant theoretical models, for example, the dominance model and overdominant model, heterologous cytoplasms have not been received widespread attention in distant hybrids. In this study, the distant hybridization between Solanum lycopersicum cv. Micro-Tom and S. pimpinellifolium line WVa700 were performed. Different phenotypes between the reciprocal hybrids were observed, AFLP (amplified fragment length polymorphism) and MSAP (methylation-sensitive amplification polymorphism) were used to explore the difference on genetic changes, the patterns and the levels of DNA methylation. The difference of small RNAs between the reciprocal hybrids were systematically analyzed by using high-throughput sequencing of small RNAs to explore the effect of small RNAs in heterologous cytoplasm on the phenotypes of the distant reciprocal hybrids. This study revealed the regulation of small RNAs in heterologous cytoplasm on the phenotypes of the distant reciprocal hybrids, providing a way to clarify the molecular mechanism of different phenotypes between the reciprocal hybrids in the distant hybridization. The main results were summarized as follows:1. In the reciprocal hybrids of Solanum lycopersicum and S. pimpinellifolium, significantly different phenotypes between Micro-Tom×WVa700 and WVa700xMicro-Tom, including fruit shape index, single fruit weight and plant height, were found. Compared with parents, Micro-TomxWVa700 had bigger leaf area and crown width and smaller fruit shape index, WVa700xMicro-Tom had longer leaf length and smaller fruit shape index.2. The genetic alterations were analyzed by AFLP, the result showed that 68 fragments (4.3%) displayed alterations in Micro-Tom×WVa700, while 16 fragments (1.01%) in WVa700xMicro-Tom, the significant differences were showed (P<0.01). Therefore, the differences of genetic alterations between the reciprocal hybrids of tomatoes were significant.3. The analysis of the pattern and level of DNA methylation by MSAP showed that the proportion of changes of methylation pattern in WVa700×Micro-Tom was significantly higher than Micro-TomxWVa700 (x2=14.098, P<0.01). Therefore, the differences of changes of methylation pattern between the reciprocal hybrids of tomatoes were significant. Moreover, Micro-Tom×WVa700 showed significantly higher levels of the externally hemi-methylation (1.44 and 1.46) and significantly lower levels of the internally fully-methylation (0.73 and 0.69) than that of the parents (P<0.05). Similarly, WVa700×Micro-Tom displayed dramatically higher levels of the externally hemi-methylation (1.41 and 1.43) and dramatically lower levels the internally fully-methylation (0.65 and 0.61) and the levels of DNA methylation (0.88 and 0.84) than that of the parents (P<0.05). Thus, there were significantly different levels of the externally hemi-methylation, the internally fully-methylation and DNA methylation between F1 hybrids and the parents.4. Using high-throughput sequencing to analyze the differences of small RNAs and their target genes between the reciprocal hybrids, the result showed that the expression levels of 76 known miRNAs were highly variable between Micro-Tom×WVa700 and WVa700×Micro-Tom, and 3 miRNAs (miR172e-3p, miR482a, miR5081) dramatically differently expressed in F1 hybrids and the parents. Target predictions of the 76 differently expressed miRNAs in the reciprocal hybrids resulted in 410 target genes. Gene ontology (GO) annotation displayed that those 410 target genes are those associated with lots of proteins and involved in many metabolic processes. Differentially expressed miRNAs negatively regulated their target genes to contribute to the different phenotypes between the reciprocal hybrids and transgression in the hybrids during distant hybridization. Moreover, the changes of the amount of unique sRNAs during the process of distant hybridization were compared. The result showed that the quantity of sRNAs was decreased and some sRNAs even disappeared. The buffering capacities of sRNAs in Micro-Tom cytoplasm were stronger than that of WVa700 cytoplasm.