New tools to screen wild peanut species for aflatoxin accumulation and genetic fingerprinting.

BACKGROUND: Aflatoxin contamination in peanut seeds is still a serious problem for the industry and human health. No stable aflatoxin resistant cultivars have yet been produced, and given the narrow genetic background of cultivated peanuts, wild species became an important source of genetic diversity. Wild peanut seeds, however, are not abundant, thus, an effective method of screening for aflatoxin accumulation using minimal seeds is highly desirable. In addition, keeping record of genetic fingerprinting of each accession would be very useful for breeding programs and for the identification of accessions within germplasm collections. RESULTS: In this study, we report a method of screening for aflatoxin accumulation that is applicable to the small-size seeds of wild peanuts, increases the reliability by testing seed viability, and records the genetic fingerprinting of the samples. Aflatoxin levels observed among 20 wild peanut species varied from zero to 19000 ng.g-1 and 155 ng.g-1 of aflatoxin B1 and B2, respectively. We report the screening of 373 molecular markers, including 288 novel SSRs, tested on 20 wild peanut species. Multivariate analysis by Neighbor-Joining, Principal Component Analysis and 3D-Principal Coordinate Analysis using 134 (36 %) transferable markers, in general grouped the samples according to their reported genomes. The best 88 markers, those with high fluorescence, good scorability and transferability, are reported with BLAST results. High quality markers (total 98) that discriminated genomes are reported. A high quality marker with UPIC score 16 (16 out of 20 species discriminated) had significant hits on BLAST2GO to a pentatricopeptide-repeat protein, another marker with score 5 had hits on UDP-D-apiose synthase, and a third one with score 12 had BLASTn hits on La-RP 1B protein. Together, these three markers discriminated all 20 species tested. CONCLUSIONS: This study provides a reliable method to screen wild species of peanut for aflatoxin resistance using minimal seeds. In addition we report 288 new SSRs for peanut, and a cost-effective combination of markers sufficient to discriminate all 20 species tested. These tools can be used for the systematic search of aflatoxin resistant germplasm keeping record of the genetic fingerprinting of the accessions tested for breeding purpose.

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds.

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance. Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes (stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins. We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

Draft genome sequence data of Nothopassalora personata, peanut foliar pathogen from Argentina.

Late leaf spot (LLS) caused by the Ascomycete Nothopassalora personata (N.p.) (Syn. Cercosporidium personatum) is the main foliar disease of peanuts in Argentina and in peanut producing areas of the world, causing up to 70% yield losses. The extremely slow growth of this fungus in culture, that takes around one month to form a 1 cm colony (0.45 mm/day), and the lack of adequate young tissues from where to extract nucleic acids, have hindered genetic studies of this pathogen. Here, we report the first genome sequence of a N. personata isolate from South America, as well as genetic variants on its conserved genes, and the complete sequence of its mating-type locus MAT1-2 idiomorph. The N. personata isolate IPAVE 0302 was obtained from peanut leaves in Córdoba, Argentina. The whole genome sequencing of IPAVE 0302 was performed as paired end 150 bp NovaSeq 6000 and de novo assembled. Clean reads were mapped to the reference genome for this species NRRL 64463 and the genetic variants on highly conserved genes and throughout the genome were analyzed. Sequencing data were submitted to NCBI GenBank Bioproject PRJNA948451, accession number SRR23957761. Additional Fasta files are available from Harvard Dataverse (https://doi.org/10.7910/DVN/9AGPMG and https://doi.org/10.7910/DVN/YDO3V6). The data reported here will be the basis for the analysis of genetic diversity of the LLS pathogen of peanut in Argentina, information that is critical to make decisions on management strategies.

First draft genome and transcriptome of Cercosporidium personatum, causal agent of late leaf spot disease of peanut.

OBJECTIVE: Two main fungal leaf spot diseases occur in peanut, namely early leaf spot (ELS) and late leaf spot (LLS), these cause a yearly average of $44 million losses. Limited genetic information, 3534 bp of sequencing, exists about the causal agent of LLS, Cercosporidium personatum (syn. Nothopassalora personata, syn. Phaeoisariopsis personata). The extremely slow growth of this fungus, approximately 1 cm colony in 6 months, and challenges in nucleic acid extractions have hindered research on LLS. Our goal in this work is to provide a reference genome for research on this pathogen. RESULTS: Whole genome and transcriptome sequencing of the LLS fungus were obtained. A total of 233,542,110 reads of the genome were de novo assembled resulting in 1061 scaffolds, and estimated genome size 27,597,787 bp. RNA sequencing resulted in 11,848,198 reads that were de novo assembled into 13,343 contigs. Genome annotation resulted in 10,703 putative genes. BUSCO analysis of the genome and annotation resulted in 91.1% and 89.5% completeness, respectively. Phylogenetic dendrograms for 5442 bp and 4401 bp of RNA Polymerase II largest and second largest subunits, and for 5474 bp of the ribosomal RNA cistron of C. personatum are presented in relation to closely related fungi.

First draft genome of Thecaphora frezii, causal agent of peanut smut disease.

OBJECTIVES: The fungal pathogen Thecaphora frezii Carranza & Lindquist causes peanut smut, a severe disease currently endemic in Argentina. To study the ecology of T. frezii and to understand the mechanisms of smut resistance in peanut plants, it is crucial to know the genetics of this pathogen. The objective of this work was to isolate the pathogen and generate the first draft genome of T. frezii that will be the basis for analyzing its potential genetic diversity and its interaction with peanut cultivars. Our research group is working to identify peanut germplasm with smut resistance and to understand the genetics of the pathogen. Knowing the genome of T. frezii will help analyze potential variants of this pathogen and contribute to develop enhanced peanut germplasm with broader and long-lasting resistance. DATA DESCRIPTION: Thecaphora frezii isolate IPAVE 0401 (here referred as T.f.B7) was obtained from a single hyphal-tip culture, its DNA was sequenced using Pacific Biosciences Sequel II (PacBio) and Illumina NovaSeq6000 (Nova). Data from both sequencing platforms were combined and the de novo assembling estimated a 29.3 Mb genome size. Completeness of the genome examined using Benchmarking Universal Single-Copy Orthologs (BUSCO) showed the assembly had 84.6% of the 758 genes in fungi_odb10.

Transformation of Major Peanut (Arachis hypogaea) Stilbenoid Phytoalexins Caused by Selected Microorganisms.

The peanut plant accumulates defensive stilbenoid phytoalexins in response to the presence of soil fungi, which in turn produce phytoalexin-detoxifying enzymes for successfully invading the plant host. Aspergillus spp. are opportunistic pathogens that invade peanut seeds; most common fungal species often produce highly carcinogenic aflatoxins. The purpose of the present research was to evaluate the in vitro dynamics of peanut phytoalexin transformation/detoxification by important fungal species. This work revealed that in feeding experiments, Aspergillus spp. from section Flavi were capable of degrading the major peanut phytoalexin, arachidin-3, into its hydroxylated homolog, arachidin-1, and a benzenoid, SB-1. However, Aspergillus niger from section Nigri as well as other fungal and bacterial species tested, which are not known to be involved in the infection of the peanut plant, were incapable of changing the structure of arachidin-3. The results of feeding experiments with arachidin-1 and resveratrol are also reported. The research provided new knowledge on the dynamics of peanut stilbenoid transformations by essential fungi. These findings may contribute to the elucidation of the phytoalexin detoxification mechanism involved in the infection of peanut by important toxigenic Aspergillus spp.

Genotyping tools and resources to assess peanut germplasm: smut-resistant landraces as a case study.

Peanut smut caused by Thecaphora frezii is a severe fungal disease currently endemic to Argentina and Brazil. The identification of smut resistant germplasm is crucial in view of the potential risk of a global spread. In a recent study, we reported new sources of smut resistance and demonstrated its introgression into elite peanut cultivars. Here, we revisited one of these sources (line I0322) to verify its presence in the U.S. peanut germplasm collection and to identify single nucleotide polymorphisms (SNPs) potentially associated with resistance. Five accessions of Arachis hypogaea subsp. fastigiata from the U.S. peanut collection, along with the resistant source and derived inbred lines were genotyped with a 48K SNP peanut array. A recently developed SNP genotyping platform called RNase H2 enzyme-based amplification (rhAmp) was further applied to validate selected SNPs in a larger number of individuals per accession. More than 14,000 SNPs and nine rhAmp assays confirmed the presence of a germplasm in the U.S. peanut collection that is 98.6% identical (P < 0.01, bootstrap t-test) to the resistant line I0322. We report this germplasm with accompanying genetic information, genotyping data, and diagnostic SNP markers.

Sixteen Draft Genome Sequences Representing the Genetic Diversity of Aspergillus flavus and Aspergillus parasiticus Colonizing Peanut Seeds in Ethiopia.

Draft genomes of 16 isolates of Aspergillus flavus Link and Aspergillus parasiticus Speare, identified as the predominant genotypes colonizing peanuts in four farming regions in Ethiopia, are reported. These data will allow mining for sequences that could be targeted by RNA interference to prevent aflatoxin accumulation in peanut seeds.

Draft genome assembly of Passalora sequoiae a needle blight pathogen on Leyland cypress.

OBJECTIVE: Passalora sequoiae (family Mycosphaerellaceae) causes a twig blight on Leyland cypress that requires numerous fungicide applications annually to minimize economic losses for ornamental plant nursery and Christmas tree producers. The objective was to generate a high-quality draft assembly of the genome of P. sequoiae as a resource for primer development to investigate genotype diversity. DATA DESCRIPTION: We report here the genome sequence of P. sequoiae 9LC2 that was isolated from Leyland cypress 'Leighton Green' in 2017 in southern Mississippi, USA. The draft genome was obtained using Pacific Biosciences (PacBio) SMRT and Illumina HiSeq 2500 sequencing. Illumina reads were mapped to PacBio assembled contigs to determine base call consistency. Based on a total of 44 contigs with 722 kilobase (kb) average length (range 9.4 kb to 3.4 Mb), the whole genome size was estimated at 31,768,716 bp. Mapping of Illumina reads to PacBio contigs resulted in a 1000 × coverage and were used to confirm accuracy of the consensus sequences.

Analysis of small RNA populations generated in peanut leaves after exogenous application of dsRNA and dsDNA targeting aflatoxin synthesis genes.

Previously, we have shown that RNA interference (RNAi) can prevent aflatoxin accumulation in transformed peanuts. To explore aflatoxin control by exogenous delivery of double-strand RNA (dsRNA) it is necessary to understand the generation of small RNA (sRNA) populations. We sequenced 12 duplicate sRNA libraries of in-vitro-grown peanut plants, 24 and 48 h after exogenous application of five gene fragments (RNAi-5x) related to aflatoxin biosynthesis in Aspergillus flavus. RNAi-5x was applied either as double-stranded RNA (dsRNA) or RNAi plasmid DNA (dsDNA). Small interfering RNAs (siRNAs) derived from RNAi-5x were significantly more abundant at 48 h than at 24 h, and the majority mapped to the fragment of aflatoxin efflux-pump gene. RNAi-5x-specific siRNAs were significantly, three to fivefold, more abundant in dsDNA than dsRNA treatments. Further examination of known micro RNAs related to disease-resistance, showed significant down-regulation of miR399 and up-regulation of miR482 in leaves treated with dsDNA compared to the control. These results show that sRNA sequencing is useful to compare exogenous RNAi delivery methods on peanut plants, and to analyze the efficacy of molecular constructs to generate siRNAs against specific gene targets. This work lays the foundation for non-transgenic delivery of RNAi in controlling aflatoxins in peanut.

Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut.

Aflatoxin contamination is a major constraint in food production worldwide. In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumulation of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B1 than the controls. The objective was to determine the putative involvement of the small RNA populations in aflatoxin reduction. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. We furthermore found two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins.

Draft Genome Sequence of Cercospora arachidicola, Causal Agent of Early Leaf Spot in Peanuts.

Cercospora arachidicola, causal agent of early leaf spot, is an economically important peanut pathogen. Lack of genetic information about this fungus prevents understanding the role that potentially diverse genotypes may have in peanut breeding programs. Here, we report for the first time a draft genome sequence of C. arachidicola.