The basic/helix-loop-helix (bHLH) proteins constitute a superfamily of transcription factors that are known to play a range of regulatory roles in eukaryotes. Over the past few decades, many bHLH family genes have been well-characterized in model plants, such as Arabidopsis, rice and tomato. However, the bHLH protein family in peanuts has not yet been systematically identified and characterized. Here, 132 and 129 bHLH proteins were identified from two wild ancestral diploid subgenomes of cultivated tetraploid peanuts, Arachis duranensis (AA) and Arachis ipaensis (BB), respectively. Phylogenetic analysis indicated that these bHLHs could be classified into 19 subfamilies. Distribution mapping results showed that peanut bHLH genes were randomly and unevenly distributed within the 10 AA chromosomes and 10 BB chromosomes. In addition, 120 bHLH gene pairs between the AA-subgenome and BB-subgenome were found to be orthologous and 101 of these pairs were highly syntenic in AA and BB chromosomes. Furthermore, we confirmed that 184 bHLH genes expressed in different tissues, 22 of which exhibited tissue-specific expression. Meanwhile, we identified 61 bHLH genes that may be potentially involved in peanut-specific subterranean. Our comprehensive genomic analysis provides a foundation for future functional dissection and understanding of the regulatory mechanisms of bHLH transcription factors in peanuts.

Background: Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with more than 200 nucleotides in length, which play vital roles in a wide range of biological processes. Powdery mildew disease (PM) has become a major threat to the production of melon. To investigate the potential roles of lncRNAs in resisting to PM in melon, it is necessary to identify lncRNAs and uncover their molecular functions. In this study, we compared the lncRNAs between a resistant and a susceptible melon in response to PM infection. Results: It is reported that 11,612 lncRNAs were discovered, which were distributed across all 12 melon chromosomes, and > 85% were from intergenic regions. The melon lncRNAs have shorter transcript lengths and fewer exon numbers than protein-coding genes. In addition, a total of 407 and 611 lncRNAs were found to be differentially expressed after PM infection in PM-susceptible and PM-resistant melons, respectively. Furthermore, 1232 putative targets of differently expressed lncRNAs (DELs) were discovered and gene ontology enrichment (GO) analysis showed that these target genes were mainly enriched in stress-related terms. Consequently, co-expression patterns between LNC_018800 and CmWRKY21, LNC_018062 and MELO3C015771 (glutathione reductase coding gene), LNC_014937 and CmMLO5 were confirmed by qRT-PCR. Moreover, we also identified 24 lncRNAs that act as microRNA (miRNA) precursors, 43 lncRNAs as potential targets of 22 miRNA families and 13 lncRNAs as endogenous target mimics (eTMs) for 11 miRNAs. Conclusion: This study shows the first characterization of lncRNAs involved in PM resistance in melon and provides a starting point for further investigation into the functions and regulatory mechanisms of lncRNAs in the resistance to PM.

As a typical geocarpic plant, peanut embryogenesis and pod development are complex processes involving many gene regulatory pathways and controlled by appropriate hormone level. MicroRNAs (miRNAs) are small non-coding RNAs that play indispensable roles in post-transcriptional gene regulation. Recently, identification and characterization of peanut miRNAs has been described. However, whether miRNAs participate in the regulation of peanut embryogenesis and pod development has yet to be explored. In this study, small RNA and degradome libraries from peanut early pod of different developmental stages were constructed and sequenced. A total of 70 known and 24 novel miRNA families were discovered. Among them, 16 miRNA families were legume-specific and 12 families were peanut-specific. 30 known and 10 novel miRNA families were differentially expressed during pod development. In addition, 115 target genes were identified for 47 miRNA families by degradome sequencing. Several new targets that might be specific to peanut were found and further validated by RNA ligase-mediated rapid amplification of 5′ cDNA ends (RLM 5′-RACE). Furthermore, we performed profiling analysis of intact and total transcripts of several target genes, demonstrating that SPL (miR156/157), NAC (miR164), PPRP (miR167 and miR1088), AP2 (miR172) and GRF (miR396) are actively modulated during early pod development, respectively. Large numbers of miRNAs and their related target genes were identified through deep sequencing. These findings provided new information on miRNA-mediated regulatory pathways in peanut pod, which will contribute to the comprehensive understanding of the molecular mechanisms that governing peanut embryo and early pod development.

Peanut zygotes typically divide a few times to form a pre-embryo before further embryonic development halts under normal day/night photoperiods. Ovary elongation, however, continuesforming a downward growing peg-like structure. When the peg is buried in the soil, embryo development resumes in the darkness. The embryo-located region (ER) of the peg begins to enlarge and form a pod, while the basal region (BR) of the peg has a distinct fate. The molecular mechanisms governing these unique embryo development processes are unknown. In this study, histological analysis demonstrated that from 4 days after pollination to 3 days after soil penetration, the peanut pre-embryo remained morphologically similar. By 9 days after soil penetration, the embryo had changed to a globular embryo. Transcriptome analysis revealed differentially expressed genes in the ER and BR before and after peg soil penetration. In addition to light signaling and plant hormone metabolism genes, we identified differentially expressed genes in the ER that contribute to embryo development and pod formation processes, including MADS-box transcription factors, xyloglucan endotransglucosylase/hydrolase protein, cellulose synthase, homeobox-leucine zipper (HD-Zip) protein family genes, amino acid permease, and seed growth and embryo morphogenesis regulators (DA1, TCP3, and YABBY). A large number of genes were found to be differentially expressed in the ER and BR across three developmental peg stages. Exact changes in gene expression were also identified in the ER during early embryo and pod development. This information provides an expanded knowledgebase for understanding the mechanisms of early peanut pod formation.

Early peanut pod development is an important process of peanut reproductive development. Modes of DNA methylation during early peanut pod development are still unclear, possibly because its allotetraploid genome may cause difficulty for the methylome analysis. To investigate the functions of the dynamic DNA methylation during the early development of the peanut pod, global methylome and gene expression analyses were carried out by Illumina high throughput sequencing. A novel mapping strategy of reads was developed and used for methylome and gene expression analysis. Differentially methylated genes, such as nodulin, cell number regulator-like protein, and senescence-associated genes, were identified during the early developmental stages of the peanut pod. The expression levels of gibberellin-related genes changed during this period of pod development. From the stage one (S1) gynophore to the stage two (S2) gynophore, the expression levels of two key methyltransferase genes, DRM2 and MET1, were up-regulated, which may lead to global DNA methylation changes between these two stages. The differentially methylated and expressed genes identified in the S1, S2, and stage 3 (S3) gynophore are involved in different biological processes such as stem cell fate determination, response to red, blue, and UV light, post-embryonic morphogenesis, and auxin biosynthesis. The expression levels of many genes were co-related by their DNA methylation levels. In addition, our results showed that the abundance of some 24-nucleotide siRNAs and miRNAs were positively associated with DNA methylation levels of their target loci in peanut pods. A novel mapping strategy of reads was described and verified in this study. Our results suggest that the methylated modes of the S1, S2, and S3 gynophore are different. The methylation changes that were identified during early peanut pod development provide useful information for understanding the roles of epigenetic regulation in peanut pod development.

The WRKY proteins constitute a large family of transcription factors that have been known to play a wide range of regulatory roles in multiple biological processes. Over the past few years, many reports have focused on analysis of evolution and biological function of WRKY genes at the whole genome level in different plant species. However, little information is known about WRKY genes in melon (Cucumis melo L.). In the present study, a total of 56 putative WRKY genes were identified in melon, which were randomly distributed on their respective chromosomes. A multiple sequence alignment and phylogenetic analysis using melon, cucumber and watermelon predicted WRKY domains indicated that melon WRKY proteins could be classified into three main groups (I-III). Our analysis indicated that no recent duplication events of WRKY genes were detected in melon, and strong purifying selection was observed among the 85 orthologous pairs of Cucurbitaceae species. Expression profiles of CmWRKY derived from RNA-seq data and quantitative RT-PCR (qRT-PCR) analyses showed distinct expression patterns in various tissues, and the expression of 16 CmWRKY were altered following powdery mildew infection in melon. Besides, we also found that a total of 24 WRKY genes were co-expressed with 11 VQ family genes in melon. Our comparative genomic analysis provides a foundation for future functional dissection and understanding the evolution of WRKY genes in cucurbitaceae species, and will promote powdery mildew resistance study in melon.