Guohua Yin1,5, Sui Sheng T. Hua2, Jiujiang Yu3, Lijing Bu4, Richard T. Sayre5, Joan W. Bennett1
1Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, USA
2Department of Agriculture, ARS, Western Regional Research Center, Albany, California 94710, USA
3Department of Agriculture, Food Quality Laboratory, ARS, Beltsville Agricultural Research Center, Beltsville, Maryland 20705, USA
4Center for Evolutionary & Theoretical Immunology (CETI), Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131, USA
5New Mexico Consortium, Los Alamos, NM 87544, United States
Background: Aflatoxins are fungal secondary metabolites that often contaminate foodstuffs and crops. Aspergillus flavus is the major aflatoxin producing species. Use of non-aflatoxigenic strains of A. flavus to competitively exclude aflatoxin-producing strains has emerged as one of the management practices for reducing aflatoxin contamination, and has led to commercially registered products. The non-aflatoxigenic A. flavus strain WRRL1519 (labelled as M65) was sequenced to define the characteristics of the strain at the genomic and transcriptomic levels.
Methods: Fungal spores were inoculated into potato dextrose broth and cultivated at 28°C for 2 days, with shaking at 200 rpm. Genomic DNA and total RNA extractions were carried out using Qiagen All Prep DNA/RNA/miRNA Universal Kit. Total RNA was extracted from four different samples to achieve all the protein-encoding genes. DNA and RNA-Seq libraries were prepared using Illumina TruSeq Nano DNA LT Library Prep Kit and Illumina TruSeq RNA Library Prep Kit v2, respectively. M65 sequences were de-novo assembled using assembly tool SPAdes (version 3.10.0).
Results: Based on the 17 k-mer statistical analysis, the estimated genome size of M65 (~ 37.97 Mb) is similar to the genome of A. flavus NRRL 3357 (~ 36.89 Mb). The assemblies of M65 consisted of 164 contigs and total length 38.07 Mb with N50 length 534,448 bp. Transcriptome assembly from RNA-Seq reads initially get ~ 38,701 transcripts or transcripts fragments. The G-C content of the genomes was 47.14%. Altogether, 56 secondary metabolite gene clusters were identified using antiSMASH software. Compared with A. flavus AF70, no cyclopiazonic acid gene cluster was found in M65 at nucleotide level similarity search by BLASTN, and the three proteins within the cluster show low identities to M65 (41.85, 47.53, and 39.39% for maoA, dmaT, and cpaA); ten genes (aflT, pksA, nor-1, hexA, hexB, aflR, aflU, adhA, estA, and norA) involved in aflatoxin biosynthesis were deleted. These genes encode for critical enzymes on the aflatoxin pathway and aflatoxins were not detected by HPLC analysis in fungal cultures.
Conclusion: The genomic data demonstrated that in WRRL1519, both aflatoxin and cyclopiazonic gene clusters had deletions. Further analysis of the genomic and transcriptomic libraries defined the metabolic and defense genes of the non-aflatoxigenic strain WRRL1519. The information will provide insight into suitable formulation for field application to achieve the goal of preventing aflatoxin contamination in food crops.
De novo sequencing, re-sequencing, Human seq., RNA seq., metagenomics, etc. , AgriGenomics, livestock genomics, plant genomics
