Identification of seed proteins associated with resistance to pre-harvested aflatoxin contamination in peanut (Arachis hypogaea L)

<p>Abstract</p> <p>Background</p> <p>Pre-harvest infection of peanuts by <it>Aspergillus flavus </it>and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. Some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. However, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. Therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism.</p> <p>Results</p> <p>The objective of this study was to identify resistance-associated proteins in response to <it>A. flavus </it>infection under drought stress using two-dimensional electrophoresis with mass spectrometry. To identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar (YJ-1) and a susceptible cultivar (Yueyou 7) under well-watered condition, drought stress, and <it>A. flavus </it>infection with drought stress. A total of 29 spots showed differential expression between resistant and susceptible cultivars in response to <it>A. flavus </it>attack under drought stress. Among these spots, 12 protein spots that consistently exhibited an altered expression were screened by Image Master 5.0 software and successfully identified by MALDI-TOF MS. Five protein spots, including Oso7g0179400, PII protein, CDK1, Oxalate oxidase, SAP domain-containing protein, were uniquely expressed in the resistant cultivar. Six protein spots including low molecular weight heat shock protein precursor, RIO kinase, L-ascorbate peroxidase, iso-Ara h3, 50 S ribosomal protein L22 and putative 30 S ribosomal S9 were significantly up-regulated in the resistant cultivar challenged by <it>A. flavus </it>under drought stress. A significant decrease or down regulation of trypsin inhibitor caused by <it>A. flavus </it>in the resistant cultivar was also observed. In addition, variations in protein expression patterns for resistant and susceptible cultivars were further validated by real time RT-PCR analysis.</p> <p>Conclusion</p> <p>In summary, this study provides new insights into understanding of the molecular mechanism of resistance to pre-harvest aflatoxin contamination in peanut, and will help to develop peanut varieties with resistance to pre-harvested aflatoxin contamination.</p

Utility of EST-derived SSR in cultivated peanut (Arachis hypogaea L.) and Arachis wild species

<p>Abstract</p> <p>Background</p> <p>Lack of sufficient molecular markers hinders current genetic research in peanuts (<it>Arachis hypogaea </it>L.). It is necessary to develop more molecular markers for potential use in peanut genetic research. With the development of peanut EST projects, a vast amount of available EST sequence data has been generated. These data offered an opportunity to identify SSR in ESTs by data mining.</p> <p>Results</p> <p>In this study, we investigated 24,238 ESTs for the identification and development of SSR markers. In total, 881 SSRs were identified from 780 SSR-containing unique ESTs. On an average, one SSR was found per 7.3 kb of EST sequence with tri-nucleotide motifs (63.9%) being the most abundant followed by di- (32.7%), tetra- (1.7%), hexa- (1.0%) and penta-nucleotide (0.7%) repeat types. The top six motifs included AG/TC (27.7%), AAG/TTC (17.4%), AAT/TTA (11.9%), ACC/TGG (7.72%), ACT/TGA (7.26%) and AT/TA (6.3%). Based on the 780 SSR-containing ESTs, a total of 290 primer pairs were successfully designed and used for validation of the amplification and assessment of the polymorphism among 22 genotypes of cultivated peanuts and 16 accessions of wild species. The results showed that 251 primer pairs yielded amplification products, of which 26 and 221 primer pairs exhibited polymorphism among the cultivated and wild species examined, respectively. Two to four alleles were found in cultivated peanuts, while 3–8 alleles presented in wild species. The apparent broad polymorphism was further confirmed by cloning and sequencing of amplified alleles. Sequence analysis of selected amplified alleles revealed that allelic diversity could be attributed mainly to differences in repeat type and length in the microsatellite regions. In addition, a few single base mutations were observed in the microsatellite flanking regions.</p> <p>Conclusion</p> <p>This study gives an insight into the frequency, type and distribution of peanut EST-SSRs and demonstrates successful development of EST-SSR markers in cultivated peanut. These EST-SSR markers could enrich the current resource of molecular markers for the peanut community and would be useful for qualitative and quantitative trait mapping, marker-assisted selection, and genetic diversity studies in cultivated peanut as well as related <it>Arachis </it>species. All of the 251 working primer pairs with names, motifs, repeat types, primer sequences, and alleles tested in cultivated and wild species are listed in Additional File <supplr sid="S1">1</supplr>.</p> <suppl id="S1"> <title> <p>Additional File 1</p> </title> <text> <p><b>List of EST-SSR primers developed from cultivated peanut ESTs</b>. The file contains a table that lists primer names, repeat motifs, primer sequences, allele number and product length for the newly developed EST-SSR markers.</p> </text> <file name="1471-2229-9-35-S1.xls"> <p>Click here for file</p> </file> </suppl

De novo assembly and Characterisation of the Transcriptome during seed development, and generation of genic-SSR markers in Peanut (Arachis hypogaea L.)

<p>Abstract</p> <p>Background</p> <p>The peanut (<it>Arachis hypogaea </it>L.) is an important oilseed crop in tropical and subtropical regions of the world. However, little about the molecular biology of the peanut is currently known. Recently, next-generation sequencing technology, termed RNA-seq, has provided a powerful approach for analysing the transcriptome, and for shedding light on the molecular biology of peanut.</p> <p>Results</p> <p>In this study, we employed RNA-seq to analyse the transcriptomes of the immature seeds of three different peanut varieties with different oil contents. A total of 26.1-27.2 million paired-end reads with lengths of 100 bp were generated from the three varieties and 59,077 unigenes were assembled with N50 of 823 bp. Based on sequence similarity search with known proteins, a total of 40,100 genes were identified. Among these unigenes, only 8,252 unigenes were annotated with 42 gene ontology (GO) functional categories. And 18,028 unigenes mapped to 125 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, 3,919 microsatellite markers were developed in the unigene library, and 160 PCR primers of SSR loci were used for validation of the amplification and the polymorphism.</p> <p>Conclusion</p> <p>We completed a successful global analysis of the peanut transcriptome using RNA-seq, a large number of unigenes were assembled, and almost four thousand SSR primers were developed. These data will facilitate gene discovery and functional genomic studies of the peanut plant. In addition, this study provides insight into the complex transcriptome of the peanut and established a biotechnological platform for future research.</p

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

Peanut gene expression profiling in developing seeds at different reproduction stages during Aspergillus parasiticus infection

<p>Abstract</p> <p>Background</p> <p>Peanut (<it>Arachis hypogaea </it>L.) is an important crop economically and nutritionally, and is one of the most susceptible host crops to colonization of <it>Aspergillus parasiticus </it>and subsequent aflatoxin contamination. Knowledge from molecular genetic studies could help to devise strategies in alleviating this problem; however, few peanut DNA sequences are available in the public database. In order to understand the molecular basis of host resistance to aflatoxin contamination, a large-scale project was conducted to generate expressed sequence tags (ESTs) from developing seeds to identify resistance-related genes involved in defense response against <it>Aspergillus </it>infection and subsequent aflatoxin contamination.</p> <p>Results</p> <p>We constructed six different cDNA libraries derived from developing peanut seeds at three reproduction stages (R5, R6 and R7) from a resistant and a susceptible cultivated peanut genotypes, 'Tifrunner' (susceptible to <it>Aspergillus </it>infection with higher aflatoxin contamination and resistant to TSWV) and 'GT-C20' (resistant to <it>Aspergillus </it>with reduced aflatoxin contamination and susceptible to TSWV). The developing peanut seed tissues were challenged by <it>A. parasiticus </it>and drought stress in the field. A total of 24,192 randomly selected cDNA clones from six libraries were sequenced. After removing vector sequences and quality trimming, 21,777 high-quality EST sequences were generated. Sequence clustering and assembling resulted in 8,689 unique EST sequences with 1,741 tentative consensus EST sequences (TCs) and 6,948 singleton ESTs. Functional classification was performed according to MIPS functional catalogue criteria. The unique EST sequences were divided into twenty-two categories. A similarity search against the non-redundant protein database available from NCBI indicated that 84.78% of total ESTs showed significant similarity to known proteins, of which 165 genes had been previously reported in peanuts. There were differences in overall expression patterns in different libraries and genotypes. A number of sequences were expressed throughout all of the libraries, representing constitutive expressed sequences. In order to identify resistance-related genes with significantly differential expression, a statistical analysis to estimate the relative abundance (<it>R</it>) was used to compare the relative abundance of each gene transcripts in each cDNA library. Thirty six and forty seven unique EST sequences with threshold of <it>R </it>> 4 from libraries of 'GT-C20' and 'Tifrunner', respectively, were selected for examination of temporal gene expression patterns according to EST frequencies. Nine and eight resistance-related genes with significant up-regulation were obtained in 'GT-C20' and 'Tifrunner' libraries, respectively. Among them, three genes were common in both genotypes. Furthermore, a comparison of our EST sequences with other plant sequences in the TIGR Gene Indices libraries showed that the percentage of peanut EST matched to <it>Arabidopsis thaliana</it>, maize (<it>Zea mays</it>), <it>Medicago truncatula</it>, rapeseed (<it>Brassica napus</it>), rice (<it>Oryza sativa</it>), soybean (<it>Glycine max</it>) and wheat (<it>Triticum aestivum</it>) ESTs ranged from 33.84% to 79.46% with the sequence identity ≥ 80%. These results revealed that peanut ESTs are more closely related to legume species than to cereal crops, and more homologous to dicot than to monocot plant species.</p> <p>Conclusion</p> <p>The developed ESTs can be used to discover novel sequences or genes, to identify resistance-related genes and to detect the differences among alleles or markers between these resistant and susceptible peanut genotypes. Additionally, this large collection of cultivated peanut EST sequences will make it possible to construct microarrays for gene expression studies and for further characterization of host resistance mechanisms. It will be a valuable genomic resource for the peanut community. The 21,777 ESTs have been deposited to the NCBI GenBank database with accession numbers <ext-link ext-link-type="gen" ext-link-id="ES702769">ES702769</ext-link> to <ext-link ext-link-type="gen" ext-link-id="ES724546">ES724546</ext-link>.</p