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

Arias, Renee

Faustinelli, Paola C.

Lamb, Marshall C.

Mohammed, Abdi

Orner, Valerie A.

Sobolev, Victor S.

English

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University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 
7-23-2020 
Sixteen Draft Genome Sequences Representing the Genetic 
Diversity of Aspergillus flavus and Aspergillus parasiticus 
Colonizing Peanut Seeds in Ethiopia 
Renee Arias 
United States Department of Agriculture, renee.arias@usda.gov 
Abdi Mohammed 
Haramaya University, Dire Dawa 
Valerie A. Orner 
United States Department of Agriculture 
Paola C. Faustinelli 
United States Department of Agriculture 
Marshall C. Lamb 
United States Department of Agriculture, marshall.lamb@usda.gov 
See next page for additional authors 
Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub 
Arias, Renee; Mohammed, Abdi; Orner, Valerie A.; Faustinelli, Paola C.; Lamb, Marshall C.; and Sobolev, 
Victor S., "Sixteen Draft Genome Sequences Representing the Genetic Diversity of Aspergillus flavus and 
Aspergillus parasiticus Colonizing Peanut Seeds in Ethiopia" (2020). Publications from USDA-ARS / UNL 
Faculty. 2330. 
https://digitalcommons.unl.edu/usdaarsfacpub/2330 
This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research 
Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion 
in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of 
Nebraska - Lincoln. 
Authors 
Renee Arias, Abdi Mohammed, Valerie A. Orner, Paola C. Faustinelli, Marshall C. Lamb, and Victor S. 
Sobolev 
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/
usdaarsfacpub/2330 
Sixteen Draft Genome Sequences Representing the Genetic
Diversity of Aspergillus flavus and Aspergillus parasiticus
Colonizing Peanut Seeds in Ethiopia
Renee S. Arias,a Abdi Mohammed,b Valerie A. Orner,a Paola C. Faustinelli,a* Marshall C. Lamb,a Victor S. Soboleva
aU.S. Department of Agriculture, Agricultural Research Service, National Peanut Research Laboratory, Dawson, Georgia, USA
bSchool of Plant Science, College of Agriculture and Environmental Sciences, Haramaya University, Dire Dawa, Ethiopia
ABSTRACT 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 se-
quences that could be targeted by RNA interference to prevent aflatoxin accumula-
tion in peanut seeds.
Aspergillus flavus and Aspergillus parasiticus are commonly found in staple foodgrains such as maize, peanut, and many other crops (1). In seeds, these fungi can
accumulate carcinogenic compounds called aflatoxins (2), as well as the neurotoxin
cyclopiazonic acid (3). Aflatoxin accumulation in seeds can be controlled by plant host
RNA interference-mediated silencing of fungal aflatoxin biosynthesis genes (4, 5); this
requires knowing gene sequences of the invading fungus. A. flavus and A. parasiticus
strains were collected from four peanut-growing districts in Ethiopia, namely, Fedis,
Babile, Darolabu, and Gursum. The fungi were isolated on modified dichloran-rose
bengal (MDRB) agar medium (6), restreaked onto MDRB agar, from which hyphal tips
of single colonies were transferred to slants of Czapek’s medium (7), and identified
following a previously described protocol (8). Cluster analysis of the genetic fingerprints
of the isolates using the 25 insertion/deletion markers within the aflatoxin biosynthesis
pathway and a protocol published by Faustinelli et al. (9) revealed several clades, from
which 16 representative isolates (4 A. parasiticus strains, 5 A. flavus strains, 2 A. flavus L
strains [10], and 5 A. flavus S strains [10]) were chosen for whole-genome sequencing,
as reported in Table 1. DNA was extracted from spores and sclerotia using the DNeasy
plant minikit (Qiagen, Valencia, CA) after growth of the isolates on MDRB agar for 3 days
in the dark at 30°C.
Barcode-indexed sequencing libraries were generated from genomic DNA sheared
on an E220 focused ultrasonicator (Covaris, Woburn, MA) and size selected with a
double-sided solid-phase reversible immobilization (SPRI) protocol with AMPure XP
beads (Beckman Coulter, Brea, CA) using bead-to-sample ratios of 0.6 and 0.73. The
size-selected DNA samples were converted to sequencing libraries using a KAPA
HyperPrep library preparation kit (Kapa Biosystems-Roche, Basel, Switzerland). The
libraries were amplified with six PCR cycles, analyzed with a Bioanalyzer 2100 instru-
ment (Agilent, Santa Clara, CA), quantified by fluorometry with a Qubit instrument (Life
Technologies, Carlsbad, CA), and combined in a pool at equimolar ratios. The pool was
quantified with a KAPA library quantification kit (Kapa Biosystems-Roche) on a Quant-
Studio 5 real-time PCR system (Applied Biosystems, Foster City, CA) and sequenced on
a HiSeq 4000 system (Illumina, San Diego, CA) with paired-end 150-bp reads. Reads
were trimmed for removal of adapters using the Phred quality score in the modified
Mott trimming algorithm for this purpose (average length after trimming was 148.4 nu-
cleotides) and then were de novo assembled with CLC Genomics Workbench version
Citation Arias RS, Mohammed A, Orner VA,
Faustinelli PC, Lamb MC, Sobolev VS. 2020.
Sixteen draft genome sequences representing
the genetic diversity of Aspergillus flavus and
Aspergillus parasiticus colonizing peanut seeds
in Ethiopia. Microbiol Resour Announc
9:e00591-20. https://doi.org/10.1128/MRA
.00591-20.
Editor Christina A. Cuomo, Broad Institute
This is a work of the U.S. Government and is
not subject to copyright protection in the
United States. Foreign copyrights may apply.
Address correspondence to Renee S. Arias,
renee.arias@usda.gov.
* Present address: Paola C. Faustinelli, JLA
International, Albany, Georgia, USA.
Received 11 June 2020
Accepted 1 July 2020
Published 23 July 2020
GENOME SEQUENCES
crossm
Volume 9 Issue 30 e00591-20 mra.asm.org 1
 
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Arias et al.
Volume 9 Issue 30 e00591-20 mra.asm.org 2
 
12.0 (Qiagen, Aarhus, Denmark) using default parameters and no minimum contig
length. The average GC content observed across isolates was 47.4%  0.07%, and a
summary of the geographic origins and genome sequencing statistics of the isolates is
shown in Table 1. Assembled contigs of each isolate were converted to BLAST (11)
databases in CLC Genomics Workbench, and then the aflatoxin biosynthesis cluster
(ABC) of A. flavus or A. parasiticus was searched by BLAST analysis within each genome.
In all 16 isolates, the ABC was observed in single contigs; ABC locations were estimated
by alignment to contigs (Table 1).
Data availability. All 16 genomes have been deposited in NCBI GenBank (Table 1).
Raw data are available in the NCBI SRA database (Table 1). Fungal isolates were
obtained from foreign samples and are hosted at the USDA ARS National Peanut
Research Laboratory culture collection; their access requires proper APHIS shipping/
receiving permits (contact renee.arias@usda.gov).
ACKNOWLEDGMENTS
We thank Lutz Froenike for providing detailed information on library construction.
The sequencing was carried out at the UC Davis Genome Center DNA Technol-
ogies and Expression Analysis Core, supported by NIH shared instrumentation grant
1S10OD010786-01; the project was partly funded by grants from the USAID (agree-
ment 58-0210-3-0012) and the USAID-funded Peanut Innovation Laboratory (award
AIDECGA000700001). The overall project was supported by NP301 project 6044-21000-
005-00D. The funders had no role in study design, data collection and interpretation, or
the decision to submit the work for publication.
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Sixteen Draft Genome Sequences Representing the Genetic Diversity of Aspergillus flavus and Aspergillus parasiticus Colonizing Peanut Seeds in Ethiopia

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

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