Xiaoyuan Chi

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.

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 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.

Genomic and Transcriptomic Analysis Identified Gene Clusters and Candidate Genes for Oil Content in Peanut (Arachis hypogaea L.)

Peanut (Arachis hypogaea), a major source of vegetable oil in many Asian countries, has become an integral part of human diet globally due to its high nutritional properties and option to consume in different forms. In order to meet the demand of vegetable oil, many peanut breeding programs of China have intensified their efforts in increasing oil content in newly bred varieties for reducing the import of edible oils in China. In this context, transcriptome sequencing data generated on 49 peanut cultivars were analyzed to identify candidate genes and develop molecular markers for seed oil content across multiple environments. Transcriptome analysis identified 5458 differentially expressed genes (DEGs) including 2243 positive DEGs and 3215 negative DEGs involved in oil synthesis process. Genome-wide association study identified 48 significant insertion/deletion (InDel) markers associated with seed oil content across five environments. A comparative genomics and transcriptomics analysis detected a total of 147 common gene clusters located in 17 chromosomes. Interestingly, an InDel cluster associated with seed oil content on A03 chromosome was detected in three different environments. Candidate genes identified on A03 form a haplotype, in which variable alleles were found to be different in oil content in an independent population. This locus is important for understanding the genetic control of peanut oil content and may be useful for marker-assisted selection in peanut breeding programs.