Shanlin Yu

Identification and Characterization of microRNAs from Peanut (Arachis hypogaea L.) by High-Throughput Sequencing

Background MicroRNAs (miRNAs) are noncoding RNAs of approximately 21 nt that regulate gene expression in plants post-transcriptionally by endonucleolytic cleavage or translational inhibition. miRNAs play essential roles in numerous developmental and physiological processes and many of them are conserved across species. Extensive studies of miRNAs have been done in a few model plants; however, less is known about the diversity of these regulatory RNAs in peanut (Arachis hypogaea L.), one of the most important oilseed crops cultivated worldwide. Results A library of small RNA from peanut was constructed for deep sequencing. In addition to 126 known miRNAs from 33 families, 25 novel peanut miRNAs were identified. The miRNA* sequences of four novel miRNAs were discovered, providing additional evidence for the existence of miRNAs. Twenty of the novel miRNAs were considered to be species-specific because no homolog has been found for other plant species. qRT-PCR was used to analyze the expression of seven miRNAs in different tissues and in seed at different developmental stages and some showed tissue- and/or growth stage-specific expression. Furthermore, potential targets of these putative miRNAs were predicted on the basis of the sequence homology search. Conclusions We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library. This study of the identification and characterization of miRNAs in peanut can initiate further study on peanut miRNA regulation mechanisms, and help toward a greater understanding of the important roles of miRNAs in peanut.

Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR

Quantitative real-time reverse transcription PCR (qRT-PCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on the reference genes have been done with peanut to date. In the present study, 14 potential reference genes in peanut were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms. Expression stability was assessed by qRT-PCR across 32 biological samples, including various tissue types, seed developmental stages, salt and cold treatments. The results showed that the best-ranked references genes differed across the samples. UKN1, UKN2, TUA5 and ACT11 were the most stable across all the tested samples. A combination of ACT11, TUA5, UKN2, PEPKR1 and TIP41 would be appropriate as a reference panel for normalizing gene expression data across the various tissues tested, whereas the combination of TUA5 and UKN1 was the most suitable for seed developmental stages. TUA5 and EF1b exhibited the most stable expression under cold treatment. For salt-treated leaves, TUA5 and UKN2 were the most stably expressed and HDC and UKN1 for salt-treated roots. The relative gene expression level of peanut Cys2/His2-type zinc finger protein gene AhZFP1 was analyzed in order to validate the reference genes selected for this study. These results provide guidelines for the selection of reference genes under different experimental conditions and also a foundation for more accurate and widespread use of qRT-PCR in peanut gene analysis.

Genome-Wide Analysis of Fatty Acid Desaturases in Soybean (Glycine max)

Fatty acid desaturases can introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In the present study, 29 full-length desaturase genes were identified from soybean genome by a thorough annotation exercise. A comprehensive analysis was performed to characterize phylogeny, chromosomal locations, structures, conserved motifs, and expression patterns of those genes. The soybean genes were phylogenetically clustered into nine subfamilies with the Arabidopsis counterparts, FAB2, FAD2, FAD3, FAD5, FAD6, FAD7, FAD8, SLD1, and DES1. Twenty-nine desaturase genes were found to be distributed on at least 15 of the 20 soybean chromosomes. The gene structures and motif compositions were considerably conserved among the subfamilies. The majority of desaturase genes showed specific temporal and spatial expression patterns across different tissues and developmental stages based on microarray data analyses. The study may provide new insights into the origin and evolution of fatty acid biosynthesis pathways in higher plants. Additionally, the characterization of desaturases from soybean will lead to the identification of additional genes for genetic modification of plants to produce nutritionally important fatty acids.

Identification of 30 MYB transcription factor genes and analysis of their expression during abiotic stress in peanut (Arachis hypogaea L.)

The MYB superfamily constitutes one of the most abundant groups of transcription factors and plays central roles in developmental processes and defense responses in plants. In the work described in this article, 30 unique peanut MYB genes that contained full-length cDNA sequences were isolated. The 30 genes were grouped into three categories: one R1R2R3-MYB, nine R2R3-MYBs and 20 MYB-related members. The sequence composition of the R2 and R3 repeats was conserved among the nine peanut R2R3-MYB proteins. Phylogenetic comparison of the members of this superfamily between peanut and Arabidopsis revealed that the putative functions of some peanut MYB proteins were clustered into the Arabidopsis functional groups. Expression analysis during abiotic stress identified a group of MYB genes that responded to at least one stress treatment. This is the first comprehensive study of the MYB gene family in peanut.

Soil Eukaryotic Microorganism Succession as Affected by Continuous Cropping of Peanut - Pathogenic and Beneficial Fungi were Selected

Peanut is an important oil crop worldwide and shows considerable adaptability but growth and yield are negatively affected by continuous cropping. Soil micro-organisms are efficient bio-indicators of soil quality and plant health and are critical to the sustainability of soil-based ecosystem function and to successful plant growth. In this study, 18S rRNA gene clone library analyses were employed to study the succession progress of soil eukaryotic micro-organisms under continuous peanut cultivation. Eight libraries were constructed for peanut over three continuous cropping cycles and its representative growth stages. Cluster analyses indicated that soil micro-eukaryotic assemblages obtained from the same peanut cropping cycle were similar, regardless of growth period. Six eukaryotic groups were found and fungi predominated in all libraries. The fungal populations showed significant dynamic change and overall diversity increased over time under continuous peanut cropping. The abundance and/or diversity of clones affiliated with Eurotiales, Hypocreales, Glomerales, Orbiliales, Mucorales and Tremellales showed an increasing trend with continuous cropping but clones affiliated with Agaricales, Cantharellales, Pezizales and Pyxidiophorales decreased in abundance and/or diversity over time. The current data, along with data from previous studies, demonstrated that the soil microbial community was affected by continuous cropping, in particular, the pathogenic and beneficial fungi that were positively selected over time, which is commonplace in agro-ecosystems. The trend towards an increase in fungal pathogens and simplification of the beneficial fungal community could be important factors contributing to the decline in peanut growth and yield over many years of continuous cropping.

Dynamic Succession of Soil Bacterial Community during Continuous Cropping of Peanut (Arachis hypogaea L.)

Plant health and soil fertility are affected by plant–microbial interactions in soils. Peanut is an important oil crop worldwide and shows considerable adaptability, but growth and yield are negatively affected by continuous cropping. In this study, 16S rRNA gene clone library analyses were used to study the succession of soil bacterial communities under continuous peanut cultivation. Six libraries were constructed for peanut over three continuous cropping cycles and during its seedling and pod-maturing growth stages. Cluster analyses indicated that soil bacterial assemblages obtained from the same peanut cropping cycle were similar, regardless of growth period. The diversity of bacterial sequences identified in each growth stage library of the three peanut cropping cycles was high and these sequences were affiliated with 21 bacterial groups. Eight phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Planctomycetes, Proteobacteria and Verrucomicrobia were dominant. The related bacterial phylotypes dynamic changed during continuous cropping progress of peanut. This study demonstrated that the bacterial populations especially the beneficial populations were positively selected. The simplification of the beneficial microbial communities such as the phylotypes of Alteromonadales, Burkholderiales, Flavobacteriales, Pseudomonadales, Rhizobiales and Rhodospirillales could be important factors contributing to the decline in peanut yield under continuous cropping. The microbial phylotypes that did not successively changed with continuous cropping, such as populations related to Rhizobiales and Rhodospirillales, could potentially resist stress due to continuous cropping and deserve attention. In addition, some phylotypes, such as Acidobacteriales, Chromatiales and Gemmatimonadales, showed a contrary tendency, their abundance or diversity increased with continuous peanut cropping progress. Some bacterial phylotypes including Acidobacteriales, Burkholderiales, Bdellovibrionales, and so on, also were affected by plant age.

Cloning of Six ERF Family Transcription Factor Genes from Peanut and Analysis of their Expression during Abiotic Stress

Several ethylene-responsive element binding factor (ERF) family proteins have been demonstrated to play important roles in transcriptional regulation of a variety of biological processes, including responses to environmental conditions such as drought, salt, and cold. To date, there have been no reports on this family in peanut. In this study, 40 ESTs in a peanut cDNA library, whose amino acid sequences contained AP2/ ERF domain, have been identified. Of these, full-length sequences of six genes were cloned. Sequence analysis indicated that all six proteins contained only one AP2/ ERF domain and should belong to the ERF family. The six genes were designated Arachis hypogaea ERF 1–6 (AhERF1–6). The expression patterns of AhERF1–6 were analyzed under cold, salt and drought stress. The results indicated that the expression of AhERF4 and AhERF6 were rapidly and substantially enhanced under abiotic stress. The expression of AhERF1 and AhEERF5 were slightly enhanced under certain stress conditions. Some genes were down-regulated when under stress, such as AhEERF3 in leaves under salt stress and AhERF2 in leaves under drought stress. Interestingly, the expression of AhERF3 and AhERF5 exhibited contrary expression patterns in peanut leaves and roots upon PEG treatment. These results suggested that different ERF proteins may have different functions in peanut abiotic stress acclimation.

Cloning and functional analysis of three diacylglycerol acyltransferase genes from peanut (Arachis hypogaea L.).

Diacylglycerol acyltransferase (DGAT) catalyzes the final and only committed acylation step in the synthesis of triacylglycerols. In this study, three novel AhDGATs genes were identified and isolated from peanut. Quantitative real-time RT-PCR analysis indicated that the AhDGAT1-2 transcript was more abundant in roots, seeds, and cotyledons, whereas the transcript abundances of AhDGAT1-1 and AhDGAT3-3 were higher in flowers than in the other tissues examined. During seed development, transcript levels of AhDGAT1-1 remained relatively low during the initial developmental stage but increased gradually during later stages, peaking at 50 days after pegging (DAP). Levels of AhDGAT1-2 transcripts were higher at 10 and 60 DAPs and much lower during other stages, whereas AhDGAT3-3 showed higher expression levels at 20 and 50 DAPs. In addition, AhDGAT transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. The activity of the three AhDGAT genes was confirmed by heterologous expression in a Saccharomyces cerevisiae TAG-deficient quadruple mutant. The recombinant yeasts restored lipid body formation and TAG biosynthesis, and preferentially incorporated unsaturated C18 fatty acids into lipids. The present study provides significant information useful in modifying the oil deposition of peanut through molecular breeding.

Transcriptome identification of the resistance-associated genes (RAGs) to Aspergillus flavus infection in pre-harvested peanut (Arachis hypogaea)

Pre-harvest aflatoxin contamination caused by Aspergillus favus is a major concern in peanut. However, little is known about the resistance mechanism, so the incorporation of resistance into cultivars with commercially-acceptable genetic background has been slowed. To identify resistance-associated genes potentially underlying the resistance mechanism, we compared transcriptome profiles in resistant and susceptible peanut genotypes under three different treatments: well watered, drought stress and both A. flavus and drought stress using a customised NimbleGen microarray representing 36158 unigenes. Results showed that the profile of differentially expressed genes (DEGs) displayed a similar pattern of distribution among the functional classes between resistant and susceptible peanuts in response to drought stress. Under A. flavus infection with drought stress, a total of 490 unigenes involved in 26 pathways were differentially expressed in the resistant genotype YJ1 uniquely responding to A. flavus infection, in which 96 DEGs were related to eight pathways: oxidation reduction, proteolysis metabolism, coenzyme A biosynthesis, defence response, signalling, oligopeptide transport, transmembrane transport and carbohydrate biosynthesis/metabolism. Pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that eight networks were significantly associated with resistance to A. flavus infection in resistant genotype YJ1 compared with susceptible Yueyou7. To validate microarray analysis, 15 genes were randomly selected for real-time RT-PCR analysis. The results provided in this study may enhance our understanding of the pre-harvest peanut-A. flavus interaction and facilitate to develop aflatoxin resistant peanut lines in future breeding programs.

Erratum to: Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of peanut (Arachis hypogaea L.)

The cultivated peanut is important oil crop and salt stress seriously influences its development and yield. Tolerant varieties produced using transgenic techniques can effectively increase peanut plantation area and enhance its yields. However, little is known about how gene expression is regulated by salt stress in peanut. In this study, we screened genes regulated by salt stress in peanut roots using microarray technique. In total, 4828 up-regulated and 3752 down-regulated probe sets were successfully identified in peanut roots subjected to 3 and 48 h of salt stress. Data analysis revealed that different response groups existed between the up and down-regulated probe sets. The main up-regulated biological processes involved in salt stress responses included transcription regulation, stress response, and metabolism and biosynthetic processes. The main down-regulated biological processes included transport processes, photosynthesis and development. The Kyoto encyclopedia of genes and genomes pathway analysis indicated that metabolic pathway, biosynthesis of unsaturated fatty acids and plant–pathogen interaction, were mainly up-regulated in peanut under salt stress. However, photosynthesis and phenylalanine metabolism were mainly down-regulated during salt stress. The function of some probe sets in salt stress regulation was not clarified (e.g., protein functioning in cell cycle regulation and xylem development). Many of the genes we identified lacked functional annotations and their roles in response to salt stress are yet to be elucidated. These results identified some candidate genes as potential markers and showed an overview of the transcription map, which may yield some useful insights into salt-mediated signal transduction pathways in peanut.