Are Current Aspergillus sojae Strains Originated from a Native Aflatoxigenic Aspergillus Species Population Also Present in California?

Figures & data

Table 1. Aspergillus genome sequences used in this study.

Species	Assembly	Size (Mb)	Origin	References
A. parasiticus SU-1	GCA_000956085.1	39.47	Uganda	Linz et al. [36]
A. parasiticus CBS117618	GCA_009176385.1	38.39	Argentina	Kjærbølling et al. [37]
A. parasiticus 68-5	GCA_001576805.1	30.14	Georgia, USA	Faustinelli et al. [31]
A. parasiticus E1319	GCA_013145845.1	38.94	Ethiopia	Arias et al. [9]
A. parasiticus E1337	GCA_013145875.1	39.15	Ethiopia	Arias et al. [9]
A. parasiticus E1348	GCA_013146005.1	39.35	Ethiopia	Arias et al. [9]
A. parasiticus E1443	GCA_013146145.1	41.45	Ethiopia	Arias et al. [9]
Aspergillus PWE36	GCA_019419805.1	38.50	California, USA	Hua et al. [6]; this study
A. sojae SMF134	GCA_008274985.1	40.11^a	South Korea	Kim et al. [11]
A. sojae NBRC4239	GCA_000226655.2	39.67	Japan	Sato et al. [15]
A. sojae TK-83	GCA_009687765.1	40.04	Japan	Watarai et al. [29]
A. sojae TK-84	GCA_009687785.1	40.02	Japan	Watarai et al. [29]
A. sojae TK-85	GCA_009687805.1	40.05	Japan	Watarai et al. [29]

^aThe genome has been assembled at the chromosomal level. It contains eight chromosomes, and their GenBank accession numbers are CP035530.1 to CP035524.1.

Table 2. Degrees of sequence identity of PWE36 aflatoxin biosynthesis genes to those of Aspergillus sojae strains and Aspergillus parasiticus isolates.

		A. sojae			A. parasiticus^c
AF genes^a	PWE36	NBRC4239	SMF134	TK strains (3)^b	SU-1	CBS117618	68-5	Ethiopian isolates (4)^b
aflYa/nadA	100.00	99.93	99.93	99.93	97.56	97.64	96.42	95.50, 95.26, 95.50, 96.60
aflX/ordB	100.00	100.00	100.00	100.00	99.25	99.38	97.13	92.24, 92.37, 92.49, 92.24
aflW/moxY	100.00	99.93	99.93	99.93	99.86	99.86	99.17	93.31, 93.25, 93.04, 92.83
aflV/cypX	100.00	100.00	100.00	100.00	99.51	99.64	98.12	94.24, 94.24, 94.30, 94.06
aflK/vbs	100.00	99.95	99.95	99.95	99.45	99.40	97.63	98.89, 98.89, 98.89, 98.69
aflQ/ordA	100.00	100.00	100.00	100.00	99.43	99.49	98.20	97.53, 97.53, 97.69, 99.49
aflP/omtA	100.00	100.00	100.00	100.00	99.26	99.26	96.98	96.37, 96.37, 96.30, 96.17
aflO/omtB	100.00	100.00	100.00	100.00	99.40	99.33	97.68	95.36, 95.36, 95.36, 95.21
aflI/avfA	100.00	100.00	100.00	100.00	98.83	98.60	98.49	91.38, 91.26, 91.38, 90.91
aflL/verB	100.00	100.00	100.00	100.00	99.42	99.55	98.52	93.83, 93.83, 93.64, 94.22
aflG/avnA	100.00	100.00	100.00	100.00	99.63	99.50	98.75	94.05, 93.88, 93.82, 94.24
aflN/verA	100.00	100.00	100.00	100.00	99.35	99.28	98.50	93.27, 93.08, 93.08, 93.27
aflM/ver-1	100.00	100.00	100.00	100.00	99.56	99.67	99.67	96.55, 96.66, 98.44, 96.55
aflE/norA	100.00	100.00	100.00	100.00	98.83	99.14	98.60	98.13, 98.29, 98.13, 98.13
aflJ/estA	100.00	100.00	100.00	100.00	98.10	99.20	96.81	97.50, 97.70, 97.70, 97.50
aflH/adhA	100.00	100.00	100.00	100.00	98.57	98.57	97.97	98.33, 98.45, 98.21, 98.33
aflS/aflJ	100.00	100.00	100.00	100.00	99.38	99.59	98.96	99.04, 99.24, 99.10, 98.97
aflR	100.00	99.93	99.93	99.93	99.40	99.40	97.87	98.14, 98.21, 98.14, 98.14
aflB/hexB	100.00	100.00	100.00	100.00	99.49	99.45	98.62	99.25, 99.20, 99.28, 99.28
aflA/hexA	100.00	100.00	100.00	100.00	99.55	99.77	99.55	99.08, 99.12, 99.14, 99.14
aflD/nor1	100.00	100.00	100.00	100.00	99.39	99.49	99.29	99.80, 99.80, 99.80, 99.80
aflC/pksA	100.00	99.97	99.97	99.97	99.43	99.35	98.45	99.25, 99.36, 99.36, 99.38
aflT	100.00	100.00	100.00	100.00	99.84	99.89	98.37	98.89, 99.00, 99.00 99.31
aflU/cypA	100.00	100.00	100.00	100.00	99.77	99.55	97.45	96.31, 96.42, 99.21, 96.42
aflF/norB	100.00	100.00	100.00	100.00	99.74	99.74	99.13	98.61, 98.61, 98.35, 98.52

^aGene designations are from A. parasiticus SU-1 [13]. The GenBank accession number is AY371490.

^bThe three A. sojae TK strains (83, 84, and 85) have identical nucleotide sequences.

^cCBS117618 was collected from the leaf of an Argentinian wild peanut species (Arachis correntina). SU-1 and 68-5 were isolated from peanut seeds from Uganda and Georgia, USA, respectively. The four A. parasiticus Ethiopian isolates are E1319, E1337, E1348, and E1443 (Table 1).

Table 3. Degrees of sequence identity of PWE36 developmental genes to those of Aspergillus sojae strains and Aspergillus parasiticus isolates.

		A. sojae		A. parasiticus
Genes^a	Gene products	NBRC4239	SMF134	SU-1	CBS117618	68-5
	Conidiation related
fluG	Extracellular developmental signal protein	99.89	99.89	99.10	99.14	98.53
flbA	Developmental regulator	100.00	100.00	99.55	99.71	98.23
flbB	Basic-zipper-type transcription factor	100.00	100.00	99.72	99.72	98.41
flbC	C2H2 conidiation transcription factor	99.92	99.92	99.83	99.92	98.83
flbD	Conidiophore development protein	100.00	100.00	99.79	99.79	99.79
flbE	Conserved hypothetical protein	100.00	100.00	99.55	99.55	98.64
fadA	G-protein complex alpha subunit	99.76	99.76	99.68	99.68	97.90
brlA	C2H2 type conidiation transcription factor	99.92	99.92	99.45	99.45	98.66
hymA	Conidiophore development protein	99.93	99.93	99.93	99.86	98.75
stuA	APSES transcription factor	99.91	99.91	99.67	99.67	99.38
medA	Transcriptional regulator	99.84	99.84	99.79	99.79	98.64
abaA	Transcription factor	100.00	100.00	99.54	99.54	98.63
wetA	Developmental regulatory protein	99.82	99.82	99.59	99.65	98.58
	Sclerotial formation related
sclR	HLH DNA-binding protein	100.00	100.00	98.23	98.34	98.44
veA	Developmental regulator	99.39	99.39	99.66	99.78	98.77
velB	Nucleoside diphosphatase	100.00	100.00	99.36	99.28	97.59
velC	Conserved protein	99.79	99.79	99.16	99.37	96.42
vosA	Developmental regulator	99.90	99.90	99.75	99.85	98.44
nsdC	C2H2 zinc finger protein	100.00	100.00	100.00	99.95	99.75
nsdD	Sexual development factor	100.00	100.00	99.74	99.68	98.41
laeA	Secondary metabolism regulator	99.78	99.78	99.89	100.00	99.66
aswA	C6-type transcription factor	100.00	100.00	99.81	99.81	99.16
sfgA	C6-type transcription factor	99.15	99.15	99.36	99.26	–

^aGenes of A. flavus NRRL33357 were used to BlastN the PWE36 genome sequence. The aligned PWE36 sequences were retrieved and used to BlastN the genome sequences of A. parasiticus and A. sojae. Gene sequences of PWE36 are 100.00%.

Figure 1. Alignment of portions of pksA/aflC sequences (A); aflR sequences (B) from Aspergillus parasiticus isolates, PWE36, and Aspergillus sojae strains. SNPs in blue that yield pre-termination codons in pksA/aflC (TAA) and aflR (TGA) genes, respectively, are yellow highlighted. Numbers correspond to nucleotide positions in A. sojae gene sequences.

Figure 2. Schematic representations of deletions in the CPA gene clusters of Aspergillus parasiticus isolates, PWE36, and Aspergillus sojae strains. The CPA gene cluster of Aspergillus flavus AF36 (16.8 kb) was used as the alignment template. The comparisons on the lower panel were drawn to scale. Blank space indicates deleted portions. Grey lines are nonhomologous regions of replacement and the 112 bp deletions. Thick red lines are highly homologous sequences.

Figure 3. Schematic representation of homologous regions in genome sequences of Aspergillus sojae strains, PWE36, and Aspergillus parasiticus isolates. The genome sequence of A. sojae SMF134 that had been assembled at the chromosome level was used as the reference; it was concatenated in chromosome order (from I to VIII). Colored blocks are locally collinear blocks (LCBs), which indicate homologous regions between two genomes. The LCB weight was set at around 550 except for the SMF134/TK-84 pair, of which the lowest score was 24,283. Regions that were split and translocated in a compared genome are shown as inverted segments at the bottom.

Figure 4. (A) Total SNP counts from paired genome sequence comparisons among PWE36, Aspergillus sojae strains, and Aspergillus parasiticus isolates; (B) Phylogenetic tree inferred from concatenated sequences of total SNPs by the Neighbor-Joining method with 100 bootstrap iterations. The number of total SNP count for each sequence was 618,048. The estimated divergence time (1.1 mya) between A. parasiticus SU-1 and A. sojae NRBC4239 was derived from the reference divergence time scale (3.8 mya) between Aspergillus flavus NRRL3357 and Aspergillus oryzae RIB40 [38].

Linz JE, Wee J, Roze LV. Aspergillus parasiticus SU-1 genome sequence, predicted chromosome structure, and comparative gene expression under aflatoxin-inducing conditions: evidence that differential expression contributes to species phenotype. Eukaryot Cell. 2014;13(8):1113–1123.

 PubMedGoogle Scholar

Kjærbølling I, Vesth T, Frisvad JC, et al. A comparative genomics study of 23 Aspergillus species from section flavi. Nat Commun. 2020;11(1):1106.

 PubMed Web of Science ®Google Scholar

Faustinelli PC, Wang XM, Palencia ER, et al. Genome sequences of eight Aspergillus flavus spp. and one A. parasiticus sp., isolated from peanut seeds in Georgia. Genome Announc. 2016;4(2):e00278–e00216.

 PubMedGoogle Scholar

Arias RS, Orner VA, Martinez-Castillo J, et al. Aspergillus section flavi, need for a robust taxonomy. Microbiol Resour Announc. 2021;10(48):e0078421.

 PubMed Web of Science ®Google Scholar

Hua SST, Parfitt DE, Sarreal SBL, et al. First report of an atypical new Aspergillus parasiticus isolates with nucleotide insertion in aflR gene resembling to A. sojae. Mycotoxin Res. 2018;34(2):151–157.

 PubMed Web of Science ®Google Scholar

Kim KU, Kim KM, Choi YH, et al. Whole genome analysis of Aspergillus sojae SMF 134 supports its merits as a starter for soybean fermentation. J Microbiol. 2019;57(10):874–883.

 PubMed Web of Science ®Google Scholar

Sato A, Oshima K, Noguchi H, et al. Draft genome sequencing and comparative analysis of Aspergillus sojae NBRC4239. DNA Res. 2011;18(3):165–176.

 PubMed Web of Science ®Google Scholar

Watarai N, Yamamoto N, Sawada K, et al. Evolution of Aspergillus oryzae before and after domestication inferred by large-scale comparative genomic analysis. DNA Res. 2019;26(6):465–472.

 PubMed Web of Science ®Google Scholar

Yu J, Chang P-K, Ehrlich KC, et al. Clustered pathway genes in aflatoxin biosynthesis. Appl Environ Microbiol. 2004;70(3):1253–1262.

 PubMed Web of Science ®Google Scholar

Steenwyk JL, Shen XX, Lind AL, et al. A robust phylogenomic time tree for biotechnologically and medically important fungi in the genera Aspergillus and Penicillium. mBio. 2019;10(4):e00925–19.

 PubMed Web of Science ®Google Scholar