There is growing evidence to suggest that epigenetic tags, especially DNA methylation, are critical regulators of fruit ripening. To examine whether this is the case in sweet pepper (Capsicum annuum) we conducted experiments at the transcriptional, epigenetic, and physiological levels. McrBC PCR, bisulfite sequencing, and real-time PCR demonstrated that DNA hypomethylation occurred in the upstream region of the transcription start site of some genes related to pepper ripening at the turning stage, which may be attributed to up-regulation of CaDML2-like and down-regulation of CaMET1-like1, CaMET1-like2, CaCMT2-like, and CaCMT4-like. Silencing of CaMET1-like1 by virus-induced gene silencing led to DNA hypomethylation, increased content of soluble solids, and accumulation of carotenoids in the fruit, which was accompanied by changes in expression of genes involved in capsanthin/capsorubin biosynthesis, cell wall degradation, and phytohormone metabolism and signaling. Endogenous ABA increased during fruit ripening, whereas endogenous IAA showed an opposite trend. No ethylene signal was detected during ripening. DNA hypomethylation repressed the expression of auxin and gibberellin biosynthesis genes as well as cytokinin degradation genes, but induced the expression of ABA biosynthesis genes. In mature-green pericarp, exogenous ABA induced expression of CaDML2-like but repressed that of CaCMT4-like. IAA treatment promoted the transcription of CaMET1-like1 and CaCMT3-like. Ethephon significantly up-regulated the expression of CaDML2-like. Treatment with GA₃ and 6-BA showed indistinct effects on DNA methylation at the transcriptional level. On the basis of the results, a model is proposed that suggests a high likelihood of a role for DNA methylation in the regulation of ripening in the non-climacteric pepper fruit.

Elongated tomato fruit shape is the result of the action of the fruit shape genes possibly in coordination with the phytohormone auxin. To investigate the possible link between auxin and the fruit shape genes, a series of auxin (2,4-D) treatments were performed on the wild-type and the fruit shape near-isogenic lines (NILs) in Solanum pimpinellifolium accession LA1589 background. Morphological and histological analyses indicated that auxin application approximately 3 weeks before anthesis led to elongated pear-shaped ovaries and fruits, which was mainly attributed to the increase of ovary/fruit proximal end caused by the increase of both cell number and cell size. Fruit shape changes caused by SUN, OVATE and fs8.1 were primarily due to the alterations of cell number along different growth axes. Particularly, SUN caused elongation by extending cell number along the entire proximal-distal axis, whereas OVATE caused fruit elongation in the proximal area, which was most similar to the effect of auxin on ovary shape. Expression analysis of flower buds at different stages in fruit shape NILs indicated that SUN had a stronger impact on the transcriptome than OVATE and fs8.1. The sun NIL differentially expressed genes were enriched in several biological processes, such as lipid metabolism, ion transmembrane and actin cytoskeleton organization. Additionally, SUN also shifted the expression of the auxin-related genes, including those involved in auxin biosynthesis, homeostasis, signal transduction and polar transport, indicating that SUN may regulate ovary/fruit shape through modifying the expression of auxin-related genes very early during the formation of the ovary in the developing flower.

There are many agronomic traits of pepper (Capsicum L.) with abundant phenotypes that can benefit pepper growth. Using specific-locus amplified fragment sequencing (SLAF-seq), a genome-wide association study (GWAS) of 36 agronomic traits was carried out for 287 representative pepper accessions. To ensure the accuracy and reliability of the GWAS results, we analyzed the genetic diversity, distribution of labels (SLAF tags and single nucleotide polymorphisms (SNPs)) and population differentiation and determined the optimal statistical model. In our study, 1487 SNPs were highly significantly associated with 26 agronomic traits, and 2126 candidate genes were detected in the 100-kb region up- and down-stream near these SNPs. Furthermore, 13 major association peaks were identified for 11 key agronomic traits. Then we examined the correlations among the 36 agronomic traits and analyzed SNP distribution and found 37 SNP polymerization regions (total size: 264.69 Mbp) that could be selected areas in pepper breeding. We found that the stronger the correlation between the two traits, the greater the possibility of them being in more than one polymerization region, suggesting that they may be linked or that one pleiotropic gene controls them. These results provide a theoretical foundation for future multi-trait pyramid breeding of pepper. Finally, we found that the GWAS signals were highly consistent with those from the nuclear restorer-of-fertility (Rf) gene for cytoplasmic male sterility (CMS), verifying their reliability. We further identified Capana06g002967 and Capana06g002969 as Rf candidate genes by functional annotation and expression analysis, which provided a reference for the study of cytoplasmic male sterility in Capsicum.

Cytoplasmic male sterility (CMS), which is controlled by mitochondrial genes, is an important trait for commercial hybrid seed production. So far, genes controlling this trait are still not clear in pepper. In this study, complete mitochondrial genomes were sequenced and assembled for the CMS line 138A and its maintainer line 138B. The genome size of 138A is 504,210 bp, which is 8618 bp shorter than that of 138B. Meanwhile, more than 214 and 215 open reading frames longer than 100 amino acids (aas) were identified in 138A and 138B, respectively. Mitochondrial genome structure of 138A was quite different from that of 138B, indicating the existence of recombination and rearrangement events. Based on the mitochondrial genome sequence and structure variations, mitochondrion of 138A and FS4401, a Korean origin CMS line, may have inherited from a common female ancestor, but their CMS traits did originate separately. Candidate gene selection was performed according to the published characteristics of the CMS genes, including the presence SNPs and InDels, located in unique regions, their chimeric structure, co-transcription, and transmembrane domain. A total of 35 ORFs were considered as potential candidate genes and 14 of these were selected, with orf300a and 0rf314a as strong candidates. A new marker, orf300a, was developed which did co-segregate with the CMS trait.