Tissue culture is an important experimental technique widely used for plant transformation and can induce somaclonal variation that is shown to be associated with genetic and epigenetic changes. However, the molecular basis of somaclonal variation and plant cell response to tissue culture has yet to be fully understood. In this study, we investigated gene expression, DNA methylation, and small RNA changes in regenerated lines (RL) compared with the wild-type progenitor plants (WT) of rice cv. Hitomebore. Using microarray, we identified many genes that were differentially expressed in the shoot-tip tissue and showed that TEs were generally activated in RL. Methylation Sensitive Amplification Polymorphism (MSAP) analysis of 5′CCGG sites combined with bisulfite sequencing detected a generally reduced DNA methylation in the RL lines. Small RNA sequencing analysis detected widespread changes in small RNA accumulation between RL and WT. In particular, repeat and TE-associated 24-nt size class of small RNAs, the inducer of RNA-directed DNA methylation, was in general down-regulated in RL, consistent with reduced CHG and CHH methylation at some of the differentially methylated TE loci. A large number of differentially expressed miRNAs were identified in RL and WT lines, including known and novel miRNAs. The expression of some of these miRNAs exhibited inverse correlation with the predicted target genes, suggesting a regulatory function. The RL plants looked similar to WT plants under normal conditions but showed significant phenotypic alterations under abiotic stress conditions. The widespread changes in DNA methylation, small RNA accumulation and gene expression in regenerated plants supports the role of epigenetic changes in tissue culture-induced somaclonal variation.

Polyploidization is a driving force in plant evolution. Chromosomal variation often occurs at early generations following polyploid formation due to meiotic pairing irregularity that may compromise segregation fidelity and cause homoeologous exchange (HE). The trends of chromosomal variation and especially factors affecting HE remain to be fully deciphered. Here, by whole-genome resequencing, we performed nuanced analyses of patterns of chromosomal number variation and explored genomic features that affect HE in two early generations of a synthetic rice segmental allotetraploid. We found a wide occurrence of whole-chromosome aneuploidy and, to a lesser extent, also large segment gains/losses in both generations (S2 and S4) of the tetraploids. However, while the number of chromosome gains was similar between S2 and S4, that of losses in S4 was lower than in S2. HEs were abundant across all chromosomes in both generations and showed variable correlations with different genomic features at chromosomal and/or local scales. Contents of genes and transposable elements (TEs) were positively and negatively correlated with HE frequencies, respectively. By dissecting TEs into different classes, retrotransposons were found to be negatively correlated with HE frequency to a stronger extent than DNA transposons, whereas miniature terminal inverted elements (MITEs) showed a strong positive correlation. Local HE frequencies in the tetraploids and homologous recombination (HR) rates in diploids within 1 Mb sliding windows were significantly correlated with each other and showed similar overall distribution profiles. Nonetheless, non-concordant trends between HE and HR rates were found at distal regions in some chromosomes. At local scale, both shared and polymorphic retrotransposons between parents were negatively correlated with HE frequency; in contrast, both shared and polymorphic MITEs showed positive correlations with HE frequency. Our results shed new light on the patterns of chromosomal number variation and reveal genomic features influencing HE frequency in early generations following plant polyploidization.

Chloroplasts and mitochondria, descendants of ancient prokaryotes via endosymbiosis, occupy a pivotal position in plant growth and development due to their intricate connections with the nuclear genome. Genes encoded by the nuclear genome but relocated to or being functional within these organelles are commonly referred as organelle-targeting nuclear genes (ONGs). These genes are essential for maintaining cytonuclear coordination, thereby determining the stability of the life cycle. While molecular function and cytonuclear coordination of some endosymbiosis-derived ONGs (E-ONGs) have been extensively studied, the evolutionary history and characteristics from a more widespread range of non-endosymbiosis-derived ONGs (NE-ONGs) remain largely enigmatic. In this study, we focused on 12 representative species within the Poaceae family to systematically identify NE-ONGs and investigated their evolutionary history and functional significance on a phylogenetic timescale. Upon aligning these 12 species’ evolutionary histories, we observed the following phenomena: (i) an exploration of NE-ONGs between the BOP and PACMAD clades unveiled dynamic compositions, potentially influencing their photosynthetic divergence; (ii) the majority of the abundant species-specific NE-ONGs exist in a single-copy status, and functional enrichment analysis further underscored their specialized roles, which could be crucial for species adaptation; and (iii) comparative analyses between plasmid- and mitochondria-related NE-ONGs (pNE-ONGs and mNE-ONGs) revealed a prevalence of pNE-ONGs, indicating tighter control for chloroplast function in Poaceae. In summary, this study offers novel insights into the cytonuclear co-evolutionary dynamics in Poaceae speciation and draws attention to crop improvement by using NE-ONGs.