Background: MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. Results: We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. Conclusions: Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family.

DNA methylation is an epigenetic modification required for transposable element (TE) silencing, genome stability, and genomic imprinting. Although DNA methylation has been intensively studied, the dynamic nature of methylation among different species has just begun to be understood. Here we summarize the recent progress in research on the wide variation of DNA methylation in different plants, organs, tissues, and cells; dynamic changes of methylation are also reported during plant growth and development as well as changes in response to environmental stresses. Overall DNA methylation is quite diverse among species, and it occurs in CG, CHG, and CHH (H = A, C, or T) contexts of genes and TEs in angiosperms. Moderately expressed genes are most likely methylated in gene bodies. Methylation levels decrease significantly just upstream of the transcription start site and around transcription termination sites; its levels in the promoter are inversely correlated with the expression of some genes in plants. Methylation can be altered by different environmental stimuli such as pathogens and abiotic stresses. It is likely that methylation existed in the common eukaryotic ancestor before fungi, plants and animals diverged during evolution. In summary, DNA methylation patterns in angiosperms are complex, dynamic, and an integral part of genome diversity after millions of years of evolution.

Seed protein, oil content and yield are highly correlated agronomically important traits that essentially account for the economic value of soybean. The underlying molecular mechanisms and selection of these correlated seed traits during soybean domestication are, however, less known. Here, we demonstrate that a CCT gene, POWR1, underlies a large-effect protein/oil QTL. A causative TE insertion truncates its CCT domain and substantially increases seed oil content, weight, and yield while decreasing protein content. POWR1 pleiotropically controls these traits likely through regulating seed nutrient transport and lipid metabolism genes. POWR1 is also a domestication gene. We hypothesize that the TE insertion allele is exclusively fixed in cultivated soybean due to selection for larger seeds during domestication, which significantly contributes to shaping soybean with increased yield/seed weight/oil but reduced protein content. This study provides insights into soybean domestication and is significant in improving seed quality and yield in soybean and other crop species.

Soybean [Glycine max(L.) Merr.] was domesticated from wild soybean (G.sojaSieb. and Zucc.) and has been further improved as a dual-use seed crop to provide highly valuable oil and protein for food, feed, and industrial applications. However, the underlying genetic and molecular basis remains less understood. Having combined high-confidence bi-parental linkage mapping with high-resolution association analysis based on 631 whole sequenced genomes, we mapped major soybean protein and oil QTLs on chromosome15 to a sugar transporter gene (GmSWEET39). A two-nucleotide CC deletion truncating C-terminus ofGmSWEET39was strongly associated with high seed oil and low seed protein, suggesting its pleiotropic effect on protein and oil content.GmSWEET39was predominantly expressed in parenchyma and integument of the seed coat, and likely regulates oil and protein accumulation by affecting sugar delivery from maternal seed coat to the filial embryo. We demonstrated thatGmSWEET39has a dual function for both oil and protein improvement and undergoes two different paths of artificial selection. A CC deletion (CC-) haplotype H1 has been intensively selected during domestication and extensively used in soybean improvement worldwide. H1 is fixed in North American soybean cultivars. The protein-favored (CC+) haplotype H3 still undergoes ongoing selection, reflecting its sustainable role for soybean protein improvement. The comprehensive knowledge on the molecular basis underlying the major QTL andGmSWEET39haplotypes associated with soybean improvement would be valuable to design new strategies for soybean seed quality improvement using molecular breeding and biotechnological approaches.

Background: Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship.Results: We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated andBradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed thatCYP93C5(an isoflavone synthase gene) is co-expressed with several genes encoding isoflavonoid-related metabolic enzymes. We then focused on nodulation-induced P450s and found thatCYP728H1was co-expressed with the genes involved in phenylpropanoid metabolism. Similarly,CYP736A34was highly co-expressed with lipoxygenase, lectin andCYP83D1, all of which are involved in root and nodule development.Conclusions: The genome scale analysis of P450s in soybean reveals many unique features of these important enzymes in this crop although the functions of most of them are largely unknown. Gene co-expression analysis proves to be a useful tool to infer the function of uncharacterized genes. Our work presented here could provide important leads toward functional genomics studies of soybean P450s and their regulatory network through the integration of reverse genetics, biochemistry, and metabolic profiling tools. The identification of nodule-specific P450s and their further exploitation may help us to better understand the intriguing process of soybean and rhizobium interaction.

Seed development is programmed by expression of many genes in plants. Seed maturation is an important developmental process to soybean seed quality and yield. DNA methylation is a major epigenetic modification regulating gene expression. However, little is known about the dynamic nature of DNA methylation and its effects on gene expression during plant development. Through whole-genome bisulfite sequencing, we showed that DNA methylation went through dynamic changes during seed maturation. An average of 66% CG, 45% CHG and 9% CHH contexts was methylated in cotyledons. CHH methylation levels in cotyledons changed greatly from 6% at the early stage to 11% at the late stage. Transcribed genes were approximately two-fold more likely to be differentially methylated than non-transcribed genes. We identified 40, 66 and 2136 genes containing differentially methylated regions (DMRs) with negative correlation between their expression and methylation in the CG, CHG and CHH contexts, respectively. The majority of the DMR genes in the CHH context were transcriptionally down-regulated as seeds mature: 99% of them during early maturation were down-regulated, and preferentially associated with DNA replication and cell division. The results provide novel insights into the dynamic nature of DNA methylation and its relationship with gene regulation in seed development.