Genome Diversity, Population Structure and MALDI-TOF MS Profiling of Aspergillus oryzae/flavus Strains from Fermentation and Wild Environments

Genome Diversity, Population Structure and MALDI-TOF MS Profiling of Aspergillus oryzae/flavus Strains from Fermentation and Wild Environments | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome Diversity, Population Structure and MALDI-TOF MS Profiling of Aspergillus oryzae/flavus Strains from Fermentation and Wild Environments Dong-Hyun Kim, Dong-Chan Kim, Donggun Seo, Ki-Tae Kim, Sang-Han Lee, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4865798/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2025 Read the published version in BMC Genomics → Version 1 posted 4 You are reading this latest preprint version Abstract Various strains of Aspergillus oryzae , regarded as a domesticated variant of aflatoxigenic Aspergillus flavus , are utilized in soybean fermentation industry of Korea. This study compared A. oryzae/flavus strains isolated from various environments in Korea, including industrial settings, Meju (brick of dried fermented soybeans), and wild conditions, with globally reported strains using genomic analysis to determine their taxonomic positions and safety. Using population genomics, five distinct groups (A to E) were identified, with all aflatoxigenic Korean strains in Group C and non-aflatoxigenic Korean strains in Groups A, B, and E. Korean strains from Meju and wild conditions are distributed across Groups A and B, and most of the Korean industrial strains form a sub-cluster with Japanese industrial strains in Group A. Comparing secondary metabolite gene cluster mutation pattern, three gene clusters (Aflatoxin, Cyclopiazonic acid and Ditryptophenaline) were revealed as group specific ones. In aflatoxin and cyclopiazonic acid clusters, most of the Group C strains had intact regions compared to other groups strains.Since most of the Group C strains produce aflatoxin and have intact Aflatoxin and Cyclopiazonic acid gene cluster, we considered that this group represent A. flavus . Profiling of MALDI-TOF MS analysis also distinguished Group C from Groups A, B and E by specific proteomic peaks. Among these peaks, those around 12700 to 12900 m/z (Da) are expected to correspond to AflF (nor B), an enzyme involved in Aflatoxin metabolism. These results showed taxonomic positions of Korean strains of A. oryzae/ flavus from various environments and also showed possibility to differentiate between A. oryzae and A. flavus with genome and Maldi-tof analysis. genomics Aspergillus oryzae/flavus complex structure analysis aflatoxin MALDI-TOF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction In Asia, the non-aflatoxigenic filamentous fungal species Aspergillus oryzae plays an indispensable role in the fermentation process of various foods and beverages, including doenjang, soy sauce, and sake [ 1 – 5 ]. This species is considered a domesticated variant of Aspergillus flavus , a species notorious for producing harmful mycotoxins such as aflatoxin and cyclopiazonic acid. [ 3 , 6 ]. A previous study demonstrated that the A. oryzae strain RIB40 is incapable of producing secondary metabolites, aflatoxin, cyclopiazonic acid and produces isomeric metabolites such as aflatrem, miyakamides and ditryptophenaline compared to A. flavus strain NRRL 3357 [ 7 ]. Despite their significant biochemical differences, these two species exhibit a close genomic relationship. Moreover, certain strains of A. flavus exhibited variability in aflatoxin production, influenced by temperature and humidity [ 8 ]. Due to these characteristics, some strains were classified as A. flavus , despite not producing aflatoxin [ 9 ]. Therefore, comparing whole genomes is required to differentiate these closely related species precisely. A previous whole genomic analysis of A. oryzae classified industrial strains used in the Japanese fermentation industry into eight clades [ 5 ]. Further efforts aimed at distinguishing A. oryzae from A. flavus through comparative genomics, with a particular focus on aflatoxin gene cluster and the composition of Carbohydrate-Active enzymes (CAZymes), have not conclusively differentiated the two species [ 10 ]. While extensive genomic studies have been conducted on industrial strains, relatively little attention has been given to non-aflatoxigenic strains derived from traditional meju and wild conditions, which are expected to have fewer events of genetic improvement. In a study on Penicillium roqueforti , traditional strains used in Roquefort cheese production were found to be intermediate between wild and industrial strains. Thus, strains are also presumed to be intermediate between industrial A. oryzae and aflatoxigenic A. flavus [ 11 ]. Therefore, understanding their population structure and aflatoxin production risk at the genomic level is necessary to monitor their distribution. In recent microbial identification and classification study, the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) method has emerged as a powerful tool. For example, Weissella confusa and Weissella cibaria are genetically close and had different roles, with W. confusa contributing to health benefits while W. cibaria having a potential pathogenic role. This relation is similar to relationship between A. oryzae and A. flavus . To distinguish these closely related species, recent studies have developed markers using machine learning to analyze MALDI-TOF MS patterns [ 12 ]. There was also attempt to use MALDI-TOF MS to distinguish A. oryzae/flavus complex, but the study concluded that this method is not suitable for accurate differentiation due to the high overlapping protein profiles of the two species [ 13 ]. Our study aimed to elucidate the distribution of diverse Korean A. oryzae/flavus strains from various environments by comparing their genomes with those of strains from previous studies originated from diverse geographical regions, such as Japan, the United States, and China. Furthermore, variations of mutations within gene clusters associated with mycotoxins and secondary metabolites, related to safety in the food industry, were analyzed. In addition, MALDI-TOF MS for proteomics pattern profiling was conducted to distinguish between clustered groups. Based on the findings, we propose group distribution of A. oryzae and A. flavus , and their specific characteristics. 2. Materials & Methods 2.1. Collection of Genomes of Korean and Global A. oryzae/flavus Strains Korean strains from 4 diverse sources, Korean Industrial non-aflatoxigenic strains (KRI, 5 strains), Korean non-aflatoxigenic strains from meju (KRM, 12 strains), Korean non-aflatoxigenic strains from wild conditions (KRWO, 7 strains), Korean aflatoxigenic strains from wild conditions (KRWF, 15 strains) were collected (Table 1 ). All 39 Korean strains were stored at -80°C. A portion of each stock was cultured on Malt Extract Agar (MEA) at 25°C for seven days for genomic DNA extraction. Table 1 Korean A. oryzae/flavus strains from various condition. KRM are non-aflatoxigenic strains from traditional meju.KRI are non- aflatoxigenic strains used in Korean fermentation Industry. KRWO are non- aflatoxigenic strains from Korean wild condition. KRWF are aflatoxigenic strains from Korean wild condition. Strain name KACC no. Discription Region Aflatoxin norB/cypA omtA MWA1 KRM K46457 Meju, Goisan, omtA A type (Hong et al. (2013) Korea X Type I IA-S-B+ MWA2 K46810 Meju, Sunchang, omtA A type (Hong et al. (2013) Korea X X IA-L-B- MWA3 K46811 Meju, Jeju, omtA A type (Hong et al. (2013) Korea X X IA-L-B- MWB1 K46455 Meju, Gyeongsan, omtA B type (Hong et al. (2013) Korea X Type I IB-L-B- MWB2 K46470 Meju, Yongin, omtA B type (Hong et al. (2013) Korea X Type I IB-L-B- MWB3 K46471 Meju, Incheon, omtA B type (Hong et al. (2013) Korea X X IB-L-B- MWC1 K46456 Meju, Haenam, omtA C type (Hong et al. (2013) Korea X Type I IC-Ao MWC2 K46474 Meju, Damyang omtA C type (Hong et al. (2013) Korea X Type I IC-Ao MWC3 K46469 Meju, Jeju, omtA C type (Hong et al. (2013) Korea X Type I IC-Ao MWX1 K46465 Meju, Buan, no omtA(Hong et al. (2013) Korea X Type I X MWX2 K46466 Meju, Gongju, no omtA(Hong et al. (2013) Korea X Type I X K93210 K93210 Meju, patent strain Korea X Type I IC-Ao KRI1 KRI K46640 Korean industrial strain Korea X Type I IC-Ao KRI2 K46641 Korean industrial strain Korea X Type I IB-L-B- KRI3 K46642 Korean industrial strain Korea X Type I IC-Ao KRI4 K47488 Korean industrial strain Korea X Type I IC-Ao KRI5 K47843 Korean industrial strain Korea X Type I IC-Ao Aor-06 KRWO K46923 Rice straw, Yangyang Korea X Type I ND Aor-17 K46924 Rice straw, Gongju Korea X Type I ND Aor-38 K46909 Soybean, Incheon Korea X X ND SD045 K48145 Peanut, Jinyang Korea X X ND SL041 K46918 Soil, Suwon Korea X Type I ND SL046 K46920 Soybean farm soil, Namhae Korea X Type I ND SL055 K46922 Greentea farm soil, Jeju Korea X X ND Aor-34 KRWF K46927 Rice straw, Yangpyeong Korea O, B Type II IC-L-B+/B- AR018 K46892 Indoor air, Seoul Korea O, B Type II IC-L-B+/B- AR028 K46894 Outdoor air, Suwon Korea O, B/G Type III Out group SD016 K46897 Corn, Hongcheon Korea O, B/G Type III Out group SD022 K46898 Corn, Hongcheon Korea O, B Type II IC-L-B+/B- SD024 K46899 Corn, Hongcheon Korea O, B Type II IC-L-B+/B- SD039 K46902 Peanut, Seosan Korea O, B/G Type II IC-L-B+ SD059 K46903 Peanut, Jangseong Korea O, B Type II IC-L-B+/B- SD061 K46904 Peanut, Danyang Korea O, B/G Type II IC-L-B+/B- SL001 K46928 Soil, Gwangju Korea O, B Type II IC-L-B+ SL005 K46929 Soil, Gwangju Korea O, B Type II IC-L-B+ SL008 K46912 Soil, Deagu Korea O, B Type II IC-L-B+/B- SL015 K46913 Soil, Gwangju Korea O, B/G Type III Out group SL034 K46916 Soil, Suwon Korea O, B/G Type II IC-L-B+/B- SL044 K46919 Garlic farm soil, Namhae Korea O, B/G Type II IC-L-B+/B- The genomic DNA (gDNA) from Korean strains was extracted using DNeasy Plant Mini kit (Qiagen, Hilden, Germany) and its protocol. PCR-free 150-bp paired-end libraries were constructed and sequenced by Macrogen Inc (Seoul) on an Illumina NovaSeq 6000. Raw reads underwent quality control. Trimmomatic (v0.38) was used to remove adapter sequences and low-quality reads [ 14 ] and FastQC was performed to check the read quality ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc ). For the comparative genomic analysis, sequence reads from strains originated from 4 different sources were collected. The sources include Japanese industrial strains (JPI, 17 strains), NCBI non-aflatoxigenic strains (NO, 14 strains), NCBI aflatoxigenic strains (NF, 8 strains), and Afla-guard non-aflatoxigenic strains (AG, 2 strains). All sequence reads were obtained as raw files from the National Center for Biotechnology Information (NCBI) Sequence Reads Archive (Table 2 ). Table 2 Global A. oryzae/flavus strains from NCBI database. JPI are atoxigenic strains used in Japanese industry. Two strains for each clades, 8 clades strains are gathered. TK-24 is not included in any clade. NO are global strains known as atoxigenic. NF are global strains known as toxigenic. AG are strains known as Afla-Guard. Strain name NCBI No. Discription Region Aflatoxin TK-1 JPI DRX154124 Sake, Clade A in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-2 DRX154126 Sake, Clade A in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-20 DRX154161 Sake, Clade B in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-26 DRX154172 Sake, Clade B in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-24 DRX154168 Outer Clade in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-5 DRX154132 Sake, Clade C in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-7 DRX154136 Miso, Clade C in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-59 DRX154211 Soyou, Clade D in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-60 DRX154212 Soyou, Clade D in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-9 DRX154140 Soyou, Clade E in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-13 DRX154148 Soyou, Clade E in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-4 DRX154130 Sake, Clade F in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-11 DRX154144 Miso, Clade F in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-10 DRX154142 Miso, Clade G in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-15 DRX154151 Miso, Clade G in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-12 DRX154146 Soyou, Clade H in Naoki et al. (2019) Japan X, Naoki et al. (2019) TK-14 DRX154149 Soyou, Clade H in Naoki et al. (2019) Japan X, Naoki et al. (2019) 14160 NO SRX013842 Xinyang City, Chacón-Vargas et al. (2021), China X, Chacón-Vargas et al. (2021) BP2-1 SRX6074494 Korean strain in Naoki et al. (2019) Korea X, Naoki et al. (2019) RIB537 SRX147127 Gibbons et al. (2012) America X, Gibbons et al. (2012) RIB949 SRX147131 Gibbons et al. (2012) America X, Gibbons et al. (2012) M2040 * SRX4479827 Biocontroller in Alshannaq et al. (2018) Korea X, Alshannaq et al. (2018) NRRL35739 SRX5329434 Biocontroller in Pennerman et-al. (2019) America X, Pennerman et-al. (2019) SU-16 SRX8635871 Suzhou winery China X, Sun, Liu et al. (2022) WRRL1519 SRX3067799 almond nuts America X, Yin et-al. (2018) 2017 Washington T2 SRX5358295 Biocontrol strain America X, Fountain et al. (2020) A1 SRX8062526 Louisiana State University America X, Fountain et al. (2020) AF36 SRX8062528 Biocontrol strain America X, Fountain et al. (2020) K54A SRX8062531 Louisiana State University America X, Fountain et al. (2020) NRRL30797 (K49) SRX8062530 Biocontrol strain America X, Fountain et al. (2020) VCG1 SRX8062533 Louisiana State University America X, Fountain et al. (2020) 2017 Washington T5 NF SRX5358298 USDA-ARS America O, A9 SRX8062527 Louisiana State University America O, Fountain et al. (2020) CA14 SRX6432310 Pistachio, Hua et al.(2012} America O, Hua et al. (2012) E1445 SRX8621731 Ethiopia peanut Ethiopia O, Arias,Mohammed et al. (2020) E1404 SRX8617991 Ethiopia peanut Ethiopia O, Arias,Mohammed et al. (2020) E1402 SRX8617924 Ethiopia peanut Ethiopia O, Arias,Mohammed et al. (2020) MRI19 SRX10945235 Tiger nuts, Schamann et al. (2022) Spain O, Schamann et al. (2022) Tox4 SRX8062532 Louisiana State University America O, Fountain et al. (2020) Afla-Guard AG SRX4479828 University of Georgia America X, Fountain et al. (2020) Yazoo S2 SRX5356519 USDA-ARS(Afla guard) America X, Fountain et al. (2020) * : Non-aflatoxigenic strains from traditional meju; KRM strain. JPI strains, previously categorized into eight clades (Clade A to H), were chosen with two representatives from each clades and an extra strain that did not fit into any clade (TK-24) were selected [ 5 ]. The distinction between the NO and NF strains was not based on the existing nomenclature. Instead, it was determined by previous research studies that examined the aflatoxin production capabilities of these strains [ 3 – 5 , 9 , 15 – 21 ]. 2.2. Single Nucleotide Polymorphisms (SNPs) calling and population genetic analysis For SNP calling, filtered DNA sequencing reads of the 80 individuals were aligned to the reference genome, NRRL 3357, using BWA software v0.7.17 MEM module [ 22 ]. SAM alignment files were sorted and converted to BAM files with SAMtools v1.3 [ 23 ]. Picard was used to delete duplicat reads and create index of reference genome. Next, various programs in GATK packages (HaplotypeCaller for variant calling, CombineGVCFs for merging GVCF files, GenotypeGVCFs for converting GVCF into VCF file, SelectVariants, and VariantFiltration for selecting and filtering SNPs) were used [ 24 ]. The filtering parameters were like this: “QD 80.0 | | MQ 3.0| | MQRankSum < − 12.5| | Read- PosRankSum < − 8.0 | | QUAL < 40.0”. VCF files were converted into fasta files using vcf2fasta to construct a phylogenetic tree ( https://github.com/santiagosnchez/vcf2fasta ). The raw data of Illumina squences were deposited on NCBI Sequence Reads Archive. The tree was drawn using whole-genome-based phylogenetic reconstruction program, SANS serif [ 25 ]. PCA with population-scale SNPs was performed using plink [ 26 ] and GCTA [ 27 ]. A Bayesian population structure assessment was performed using ADMIXTURE v1.3.0 [ 28 ] with block relaxation algorithm, maximum likelihood estimation. Pre-defined genetic clusters were increased from K = 2 to K = 10 (assumed number of ancestral populations). Prior to running ADMIXTURE, SNPs in high linkage disequilibrium (LD) were removed. Specifically, PLINK was used to prune SNPs employing a windowed approach, with an R2 threshold of 0.8. 2.3. Genotypic Analysis of Secondary Metabolites Gene Cluster and Design of Diagnostic Markers Significant phenotypic differences influenced by A. oryzae/flavus domestication are prominently reflected in the mutations of secondary metabolites, with substantial variations in the secretion levels of numerous toxic metabolites. The presence and locations of the aflatoxin gene cluster [ 29 – 31 ], the CPA gene cluster [ 32 ], and the ditryptophenaline gene cluster [ 33 ] in the genome of Aspergillus flavus NRRL 3357 have been elucidated in previous studies. SNPs based on the NRRL 3357 genomic sequence are visualized using the Integrated Genomics Viewer (IGV). This visualization allows the identification of specific mutations or deletions occurring within these gene clusters of each strain. 2.4. MALDI TOF metabolites pattern analysis Mass spectrometry data from MALDI-TOF/MS on KACC strains containing Groups A, B, C and E were analyzed to compare the similarity patterns among groups classified based on genomic analysis results. The group specific mass-spectral loci were explored by comparing presence or absence of mass peak at certain loci and their degree of difference. Each strain underwent individual mass analysis using MicroIDSys® (ASTA Inc.). A sample from each strain is processed by mixing it with a cell lysis solution. After cell lysis, 1.5 µL of the separated supernatant is applied to the µID plate, dried, and then coated with 1.5 µL of CHCA matrix. The raw spectrum data generated by MicroIDSys® ranges from 2,000 to 20,000 daltons (m/z), with an identification cutoff set at ≥ 140 as per the manufacturer’s recommendation. The raw data from MicroIDSys® undergoes normalization to adjust peak height relative to m/z using proprietary software from ASTA Inc. and NQ-Lab. Co.,Ltd. Subsequently, the data is processed with a binning parameter set at 5. An averaging procedure calculates the mean peak heights for each m/z range within the binning. The processed data is then interpreted using a Python-based code, resulting in a combined dataset. The prepared data is analyzed using a machine learning model developed by NQ-Lab., Co., Ltd. Logistic Regression, is the chosen machine learning algorithm tailored with hyperparameters optimized for MALDI-TOF/MS characteristics. The analysis accounts for various factors influencing MALDI-TOF/MS results, such as temporal, physical, and biochemical attributes. To ensure a comprehensive analysis, multiple statistical tests are conducted. The ANOVA test is utilized to examine mean differences between groups, considering the continuity of m/z values. Additionally, the Chi-squared (χ²) test is employed to assess the independence of categories, assuming that the two groups are distinct entities. 3. Results 3.1. Phylogenetic relationship of A. oryzae/flavus strains This comprehensive analysis classified the non-aflatoxigenic and aflatoxigenic A. oryzae/flavus strains into five distinct groups, designated as Groups A to E (Fig. 1 ). Group A predominantly comprises non-aflatoxigenic strains, with a significant presence of KRI and JPI strains. Previous research by Watari et al. (2019) categorized JPI strains into eight clades (Clade A to H), and strains belonging to Clades C to H were integrated into Group A. Notably, most KRI strains were found to cluster within Clade C. Additionally, among the KRM strains, MWC2, MWC3, and M2040 also aligned with Clade C, while K93210 aligned with Clade D. Other KRM and KRWO strains were also classified under Group A, although they did not align with any of the specific clades. Group B primarily includes non-industrial strains such as KRM, KRWO, and NO, apart from a singular KRI strain, KRI2, indicating a diverse genetic background within non-aflatoxigenic populations. Group C encapsulates all aflatoxigenic strains, including KRWF and NF. Intriguingly, this group also contains six non-aflatoxigenic strains from the NO category (2017 Washington-T2, A1, AF36, K54A, NRRL30797, and VCG1), highlighting the complex genetic landscape that does not strictly correlate with aflatoxin production. Group D is characterized by its genetic divergence from Groups A, B, and C, comprising mainly JPI strains that fall into the A and B clades as defined by Watari et al., with the exception of one NO strain, 14160. Group E, distinguished as the most genetically distinct cluster, includes biocontrol strains such as Afla-guard and Yazoo S2, alongside two unique KRM strains (MWA1 and MWA3). 3.2. Genome and Population Structure Analysis of A. oryzae/flavus Principal Component Analysis (PCA) of the A. oryzae/flavus population structure revealed that Group D closely aligns with Groups A and B, demonstrating their closer genetic relationship, while Group E exhibits distinct genetic separateness from all other groups (Fig. 2 A). This distinct positioning of Group E highlights its unique genetic makeup. Admixture analysis further elucidates the genetic diversity and interrelation among the groups. To identify the most optimal K number, we calculated the cross-validation (CV) error values, where a lower CV value indicates a better estimation. This calculation was performed systematically for K values ranging from 1 to 15 [ 34 ]. Using this approach, K = 11 was identified as the most likely number of populations. At K = 11, part of the Group A strains shared population with Group B and D, while part of the Group C strains shared small population with Group E. Phylogenetic network analysis further supports the presence of five main populations and individual population assignment into these populations (Fig. 2 B). 3.3. Secondary Metabolite Gene Cluster Analysis Among the various secondary metabolite gene clusters, three metabolites gene clusters, aflatoxin, CPA and ditryptophenaline gene clusters showed group-specific characteristics. In genome of A. flavus NRRL3357, aflatoxin gene cluster region is located on chromosome 3, 4,940,000 to 5,010,000 (Fig. 3 ). CPA gene cluster region is located on beside the aflatoxin gene cluster, chromosome 3, 5,010,000 to 5,034,262 (Fig. 4 ). The ditryptophenaline gene cluster region is located on chromosome 4, 3,181,000 to 3,195,000 (Fig. 5 ). Group A strains exhibited a shared pattern of deletions and mutations within their gene clusters. The CPA gene cluster pattern depended on the aflatoxin gene cluster pattern. The strain that had a deletion in the aflatoxin gene cluster had a large deletion in the CPA gene cluster, while other strains showed consistent mutation patterns. For the ditryptophenaline cluster, Group A strains shared a similar mutation pattern. Group B showed similar mutation patterns with part of the Group A strains in the aflatoxin and CPA gene clusters, but they had intact ditryptophenaline gene clusters. Most of the Group C strains had intact three gene clusters. Conversely, three aflatoxigenic strains (AR028, SD016, and SL015) exhibited unique mutation patterns in the aflatoxin and CPA gene clusters. This mutation pattern is odd compared to Group A and B mutation patterns. Group D strains presented a distinctive pattern with large deletions in the aflatoxin cluster and complete deletion in the CPA cluster, maintaining an intact Ditryptophenaline gene cluster. In Group E, KRM strains and Afla-guard strains showed distinct features. In contrast to other strains, where the CPA gene cluster pattern depends on the aflatoxin gene cluster's state, KRM strains uniquely exhibited a deleted aflatoxin gene cluster while retaining the CPA gene cluster. Afla-guard strains had a whole deletion in the aflatoxin and CPA gene clusters. However, these Group E strains had an intact ditryptophenaline gene cluster similar to the others. No group-specific characteristics were observed among the groups concerning Aspergillic acid and Aflatrem gene clusters. 3.4. MALDI TOF/MS patterning Comparing patterns of the peaks of Group C and the non-C groups (A, B and E; A. oryzae groups), the two groups were distinctly separated, while non-C groups were not distinctly separated from each other (Supplementary Fig. 1). Group C and the non-C groups exhibited several significantly different loci; 3100–3200 m/z, 6200–6500 m/z, and 12700–12900 m/z showed significant differences (Fig. 6 B). When comparing the non-C groups, no distinct loci were found that significantly differentiated between the groups in overall ranges (Supplementary Fig. 2). Most of the Group C strains had significantly higher peaks in the 6355 to 6385 m/z and 12795 to 12810 m/z ranges, while most of the non-C groups had high peaks at 3145 m/z, 6285 to 6295 m/z, and 12730 m/z. 4. Discussion The phylogenetic analysis and genome structure investigation of A. oryzae/flavus strains from fermentation and wild environment in Korea, compared with globally reported strains, provided significant insights into the genetic diversity and evolutionary pathways of these fungi. The phylogenetic tree, PCA and ADMIXTURE analysis offer a vivid illustration of the genetic distances and relationships among the strains, revealing a broad spectrum of evolutionary divergence within five distinct groups (A to E). In particular, Groups A and B demonstrate closer genetic affiliations, suggesting a shared evolutionary pathway that may be rooted in their non-aflatoxigenic nature and potential adaptations to industrial or natural environments. Conversely, Group E, characterized by its distinct genetic makeup, stands out as the most genetically divergent cluster. This may indicate unique evolutionary pressures or historical genetic isolation that have shaped their current genomic constitution. Group D exhibits an interesting feature since it has a greater genetic distance from Groups A and B than from Group C, yet PCA analysis showed that it aligns more closely with Groups A and B. Considering distribution of Korean strains, industrial strains (KRI), excluding KRI2, clustered within Group A, specifically aligning with JPI Clade C strains. The narrow genetic distances among these industrial strains suggest recent differentiation driven by fermentation functionalities, resulting in their separation as individual strains. Korean Meju-originated strains (KRM) exhibited broader distribution across Groups A, B, and E. Some strains were closely matched to industrial strains, likely due to spore dispersal from industrial environments to traditional fermentation settings. Other Meju strains in Group A, B and E showed distinct genetic positions with industrial strains. Notably, two Meju strains in Group E showed genetic similarity with the biocontrol agent, Afla-guard but differed in aflatoxin and CPA gene clusters, suggesting potential genetic recombination events with wild strains. The distribution of Meju strains(KRM) was similar with that of non-aflatoxigenic strain from wild conditions (KRWO). This supports the nothion that Meju fermentation involves environmental strains without artificial starter inoculation, resulting in weak selective pressure. Non-aflatoxigenic strains from Korean wild environments (KRWO) were found in Groups A and B. Group A wild strains exhibited genetic distinctions from JPI strains, suggesting different evolutionary pressures. The lower genetic diversity among these wild strains, compared to industrial strains, may indicate fewer evolutionary changes in natural settings. In contrast, industrial environments likely experienced more frequent selective evolution events. All aflatoxigenic Korean strains (KRWF) were categorized in Group C. Especially strains AR028, SD016, and SL015 had notable genetic characteristics highlighted in previous studies [ 35 ]. PCR analysis patterns of the norB/cypA cluster in these strains were similar to that of A. parasiticus . Genome-wide clustering confirmed their placement within Group C, yet their aflatoxin cluster mutation patterns were notably distinct from NRRL 3357 and other Group C strains. Despite these genomic differences, MALDI-TOF MS results closely matched NRRL 3357 and other Group C strains. Following Aflatoxin mutation patterns, this study reveals that there are points of concordance with previous research as well as advancements in understanding these patterns. Comparing the strains, Afla-guard (NRRL 21882), which had a complete deletion of the aflatoxin gene cluster, and strain 14160, which exhibited deletion starting from the OmtA gene showed the same results. Strains BP2-1, TK-10, TK24, WRRL1519, and NRRL35739 showed deletions beginning from the norA gene. These results are also consistent with previous findings. Additionally, strains 3.042, TK-5, TK-59, and SU-16 were described as having mostly intact gene clusters with partial deletions starting from the AflT gene. Group C strains (A9, CA14, NRRL 3357, VCG1, Washington T5, E1404, E1445) were reported to have partial deletions starting from the CypA gene [ 10 ]. In this study, non-aflatoxigenic strains, including those in Group C, frequently exhibited additional deletions of the cypA gene compared to NRRL 3357. This suggests that the additional deletion in cypA may significantly impact aflatoxin production. This finding also aligns with previous research that classified norB-cypA PCR patterns into type 1 (short read type) and type 2 (long read type), with aflatoxigenic strains being type 2 and non-aflatoxigenic strains being type 1 [ 35 ]. Across Groups A and B, strains with high mutation rates or high deletion rates are mixed and each of them shares a similar mutation pattern. But they don’t share similar mutation patterns with Group C strains. This suggests significant genetic exchange through interbreeding between Groups A and B during domestication, while the two groups appear to be reproductively isolated from the aflatoxigenic group, indicating speciation. Therefore, these strains with high mutation rates or high deletion rates are considered safe since they have less potential for producing mycotoxins and are unlikely to form hybrids with aflatoxigenic strains. Group C mostly consists of aflatoxigenic strains (KRWF and NF), but there are some exceptional non-aflatoxigenic strains (2017 Washington-T2, A1, AF36 and K54A). Among them, AF36 is even known as biocontrol agent to reduce aflatoxin contamination [ 36 ]. This may indicate a complex genetic landscape that does not strictly correlate with aflatoxin production. However, the presence of intact aflatoxin and cyclopiazonic acid gene clusters in these non-aflatoxigenic strains suggests a latent potential for mycotoxin production under certain conditions, raising important considerations for food safety and strain utilization in industrial applications.. The evolutionary direction of Group A, characterized by unique mutations in the ditryptophenaline gene cluster, indicates a potential adaptive response to industrial fermentation processes, possibly due to selective pressures to mitigate negative impacts on food products. Although the toxicity or side effects of ditryptophenaline have not been thoroughly studied, the uniform occurrence of mutations in this cluster among Group A strains implies that such mutations may confer a selective advantage in the context of fermented food production, possibly due to reduced detrimental effects on product quality [ 7 ]. By incorporating MALDI-TOF MS data, we observe significant differentiation, particularly between Group C and the non-C groups (A, B, and E), with unique proteomic features corresponding to their genomic distinctions. Group C exhibited the unique features in the ranges of 3100–3200 m/z, 6200–6500 m/z, and 12700–12900 m/z. A database of various proteins specific for A. flavus and A. oryzae in UniProtKB ( www.uniprot.org ) suggests that these peaks may correspond to significant fungal proteins. For instance, peaks within the 6200 to 6500 m/z range correspond to metabolic enzyme proteins like 3-Hydroxyanthranilate 3,4-Dioxygenase (3HAO) and Short-chain dehydrogenase. Peaks within the 12700 to 12900 m/z range may correspond to proteins such as AflF, also known as norB (primary accession number: A0A7U2MNK6, Mass: 12855 Da). This protein is one of the enzymes included in the aflatoxin gene cluster. No characterized proteins within the 3100 to 3200 m/z range were identified in the database. These specific proteins play crucial roles in cellular metabolism, suggesting their potential as reliable biomarkers for differentiating between groups. Previous attempts to differentiate A. oryzae from A. flavus have been numerous. A previous study showed that targeting the Cyp51A gene could provide differentiation, but the limited strain diversity hindered its broad applicability [ 37 ]. There were also attempts to analyze aflatoxin and cyclopiazonic acid gene clusters to see the difference between two species, and although some differentiation was achieved, it was not definitive [ 3 , 38 ]. There have also been attempts to distinguish the two species through profiling of CAZyme genes and secondary metabolite biosynthesis gene clusters, but the ambiguous similarities between the two species disturbed clear differentiation [ 10 ]. In this study, a comprehensive approach was employed, combining whole-genome SNP-based population structure analysis, detailed secondary metabolite gene cluster variation analysis, and MALDI-TOF MS profiling. Group C formed a distinct cluster in genomic analysis, separating it from other groups. Similarly, MALDI-TOF MS analysis distinguished Group C from other groups, displaying unique peaks. Furthermore, group C strains have comparatively intact gene clusters of aflatoxin and cyclopiazonic acid and produced aflatoxin B. Given these characteristics, Group C aligns with traits traditionally associated with A. flavus . Therefore, this study proposes classifying Group C as A. flavus and the non-C groups as A. oryzae . 5. Conclusion This study provides a comprehensive genomic and proteomic characterization of Korean A. oryzae/flavus complex strains compared to global strains. The analysis highlights the genetic diversity and distinct evolutionary paths of these strains, with particular attention to their safety for food industry applications. Genomic analysis divided the A. oryzae / flavus strains into five groups, with Group C showing relatively intact aflatoxin and cyclopiazonic acid (CPA) gene clusters, suggesting a high potential for producing both metabolites. Furthermore, unique proteomic patterns identified through MALDI-TOF MS provide reliable biomarkers for distinguishing Group C aflatoxigenic strains. Characteristics of group C were well matched with those of A. flavus and the other groups with A. oryzae . These findings establish refined criteria for differentiating A. oryzae from A. flavus , contributing to their safe utilization and better understanding of their genetic dynamics. Abbreviations CPA Cyclopiazonic acid KRI Korean Industrial non-aflatoxigenic strains KRM Korean non-aflatoxigenic strains from meju KRWO Korean non-aflatoxigenic strains from wild conditions KRWF Korean aflatoxigenic strains from wild conditions JPI Japanese Industrial strains NO NCBI non-aflatoxigenic strains NF NCBI aflatoxigenic strains AG Afla-guard non-aflatoxigenic strains. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests Not applicable. Funding This work is supported by the grant from National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea (PJ017286), and the Ministry of Science, ICT (MSIT) of Korea (2021M3H9A1081101). Authors' contributions D.H. Kim wrote the main manuscript text and prepared figures 1-5. S.B. Hong and K.T. Kim provided revisions to the contents of main manuscript. D.C. Kim and D.G. Seo prepared figure 6. S.H. Lee reviewed and corrected the manuscript’s grammar and language. Acknowledgements Not applicable. References Machida M, et al. Genome sequencing and analysis of Aspergillus oryzae. Nature. 2005;438(7071):1157–61. Machida M, Yamada O, Gomi K. Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future. DNA Res. 2008;15(4):173–83. Gibbons JG, et al. The evolutionary imprint of domestication on genome variation and function of the filamentous fungus Aspergillus oryzae. Curr Biol. 2012;22(15):1403–9. Alshannaq AF, et al. Controlling aflatoxin contamination and propagation of Aspergillus flavus by a soy-fermenting Aspergillus oryzae strain. Sci Rep. 2018;8(1):16871. Watarai N, et al. Evolution of Aspergillus oryzae before and after domestication inferred by large-scale comparative genomic analysis. DNA Res. 2019;26(6):465–72. Kjærbølling I, et al. A comparative genomics study of 23 Aspergillus species from section Flavi. Nat Commun. 2020;11(1):1106. Rank C, et al. Comparative chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357). Metabolites. 2012;2(1):39–56. Medina A, Rodriguez A, Magan N. Effect of climate change on Aspergillus flavus and aflatoxin B1 production. Front Microbiol. 2014;5:348. Sun H, et al. Safety evaluation and comparative genomics analysis of the industrial strain Aspergillus flavus SU-16 used for huangjiu brewing. Int J Food Microbiol. 2022;380:109859. Han DM, et al. Comparative pangenome analysis of Aspergillus flavus and Aspergillus oryzae reveals their phylogenetic, genomic, and metabolic homogeneity. Food Microbiol. 2024;119:104435. Dumas E, et al. Independent domestication events in the blue-cheese fungus Penicillium roqueforti. Mol Ecol. 2020;29(14):2639–60. Kim E, et al. Differentiation between Weissella cibaria and Weissella confusa using machine-learning-combined MALDI-TOF MS. Int J Mol Sci. 2023;24(13):11009. Hedayati MT, et al. Discrimination of aspergillus flavus from Aspergillus oryzae by matrix-assisted laser desorption/ionisation time‐of‐flight (MALDI‐TOF) mass spectrometry. Mycoses. 2019;62(12):1182–8. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20. Fountain JC, et al. Draft genome sequences of one Aspergillus parasiticus isolate and nine Aspergillus flavus isolates with varying stress tolerance and aflatoxin production. Microbiol Resource Announcements. 2020;9(37). p. 10.1128/mra . 00478 – 20. Chacón-Vargas K, et al. Comparison of two Aspergillus oryzae genomes from different clades reveals independent evolution of alpha-amylase duplication, variation in secondary metabolism genes, and differences in primary metabolism. Front Microbiol. 2021;12:691296. Pennerman KK, et al. Aspergillus flavus NRRL 35739, a poor biocontrol agent, may have increased relative expression of stress response genes. J Fungi. 2019;5(2):53. Chang PK. Genome-wide nucleotide variation distinguishes Aspergillus flavus from Aspergillus oryzae and helps to reveal origins of atoxigenic A. flavus biocontrol strains. J Appl Microbiol. 2019;127(5):1511–20. Schamann A, Geisen R, Schmidt-Heydt M. Draft genome sequence of an aflatoxin-producing Aspergillus flavus strain isolated from food. Microbiol Resource Announcements. 2022;11(2):e00894–21. Arias RS, et al. Sixteen draft genome sequences representing the genetic diversity of Aspergillus flavus and Aspergillus parasiticus colonizing peanut seeds in Ethiopia. Microbiol Resource Announcements. 2020;9(30). p. 10.1128/mra . 00591 – 20. Hua SST, et al. Characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates from pistachio. Mycotoxin Res. 2012;28:67–75. Li H, Durbin R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics. 2010;26(5):589–95. Li H, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078–9. Van der Auwera GA et al. From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. Current protocols in bioinformatics, 2013. 43(1): p. 11.10. 1-11.10. 33. Rempel A, Wittler R. SANS serif: alignment-free, whole-genome-based phylogenetic reconstruction. Bioinformatics. 2021;37(24):4868–70. Chang CC et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience, 2015. 4(1): p. s13742-015-0047-8. Yang J, et al. GCTA: a tool for genome-wide complex trait analysis. Am J Hum Genet. 2011;88(1):76–82. Alexander DH, Novembre J, Lange K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 2009;19(9):1655–64. Yu J, et al. Clustered pathway genes in aflatoxin biosynthesis. Appl Environ Microbiol. 2004;70(3):1253–62. Amaike S, Keller NP. Aspergillus flavus. Annu Rev Phytopathol. 2011;49:107–33. Caceres I, et al. Aflatoxin biosynthesis and genetic regulation: A review. Toxins. 2020;12(3):150. Chang P-K, Ehrlich KC, Fujii I. Cyclopiazonic acid biosynthesis of Aspergillus flavus and Aspergillus oryzae. Toxins. 2009;1(2):74–99. Kishimoto S, et al. Evaluation of biosynthetic pathway and engineered biosynthesis of alkaloids. Molecules. 2016;21(8):1078. Alexander DH, Lange K. Enhancements to the ADMIXTURE algorithm for individual ancestry estimation. BMC Bioinformatics. 2011;12:1–6. Hong S-B, et al. The proportion of non-aflatoxigenic strains of the Aspergillus flavus/oryzae complex from meju by analyses of the aflatoxin biosynthetic genes. J Microbiol. 2013;51:766–72. Doster MA, Cotty PJ, Michailides TJ. Evaluation of the atoxigenic Aspergillus flavus strain AF36 in pistachio orchards. Plant Dis. 2014;98(7):948–56. Nargesi S, et al. Differentiation of Aspergillus flavus from Aspergillus oryzae targeting the cyp51A gene. Pathogens. 2021;10(10):1279. Toyotome T, et al. Comparative genome analysis of Aspergillus flavus clinically isolated in Japan. DNA Res. 2019;26(1):95–103. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure.docx SupplementaryTable.xlsx Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 09 Aug, 2024 Editor assigned by journal 08 Aug, 2024 Submission checks completed at journal 08 Aug, 2024 First submitted to journal 06 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4865798","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":337979242,"identity":"c68acd8f-9518-4253-b21c-772a56103d94","order_by":0,"name":"Dong-Hyun Kim","email":"","orcid":"","institution":"Korean Agricultural Culture Collection, National Institute of Agricultural Sciences, RDA","correspondingAuthor":false,"prefix":"","firstName":"Dong-Hyun","middleName":"","lastName":"Kim","suffix":""},{"id":337979243,"identity":"134af90e-c5a0-4ca2-aa96-ffddc88fc4dd","order_by":1,"name":"Dong-Chan Kim","email":"","orcid":"","institution":"NQ-Lab, Inc","correspondingAuthor":false,"prefix":"","firstName":"Dong-Chan","middleName":"","lastName":"Kim","suffix":""},{"id":337979244,"identity":"11402960-3572-4670-ad6a-03c6c2a71619","order_by":2,"name":"Donggun Seo","email":"","orcid":"","institution":"NQ-Lab, Inc","correspondingAuthor":false,"prefix":"","firstName":"Donggun","middleName":"","lastName":"Seo","suffix":""},{"id":337979245,"identity":"2754318e-474f-4f88-8d20-a05c007d35a3","order_by":3,"name":"Ki-Tae Kim","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"prefix":"","firstName":"Ki-Tae","middleName":"","lastName":"Kim","suffix":""},{"id":337979246,"identity":"cb9ef084-12d1-43f7-b990-70487aa8a0d0","order_by":4,"name":"Sang-Han Lee","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Sang-Han","middleName":"","lastName":"Lee","suffix":""},{"id":337979247,"identity":"53fd63f8-51ef-409f-95be-534df1c7cdc4","order_by":5,"name":"Seung-Beom Hong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBAC9gYwZWMA4R4gQgsPRFEa6VoOk6JFIvfwa96288b80ocPfmA4c48YLXlp1rxtt80k+9KSJRhuFBPWYi+RY2YM1GJjcIbHQILhQwIxtoC1nANq4f/8g1gtxo952w6YAW1hAzqMGC08b8wY55xLNpbsYTOzSDhDjBb2HOMPb8rsDPt5mB/f+HCMCC1AwCbByAZlEqeBgYH5A8MfIpWOglEwCkbByAQATPA2MGemvIQAAAAASUVORK5CYII=","orcid":"","institution":"Korean Agricultural Culture Collection, National Institute of Agricultural Sciences, RDA","correspondingAuthor":true,"prefix":"","firstName":"Seung-Beom","middleName":"","lastName":"Hong","suffix":""}],"badges":[],"createdAt":"2024-08-06 05:51:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4865798/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4865798/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-11596-9","type":"published","date":"2025-04-30T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63962225,"identity":"dcba5a42-cc56-4607-bce3-639997a8fdae","added_by":"auto","created_at":"2024-09-04 09:01:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":189716,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of 80 \u003cem\u003eA. oryzae/flavus\u003c/em\u003e complex strains from eight different sources, categorized into five distinct groups (A-E). Color coding denotes the different sources: KRI (Korean Industrial non-aflatoxigenic strains, Blue), KRM (Korean non-aflatoxigenic strains from meju, Cyan), KRWO (Korean non-aflatoxigenic strains from wild conditions, Green), KRWF (Korean aflatoxigenic strains from wild conditions, Red), JPI (Japanese industrial strains, Purple), NO (NCBI non-aflatoxigenic strains, Pale green), NF (NCBI aflatoxigenic strains, Orange), and AG (Afla-guard non-aflatoxigenic strains, Grey). Branch lengths represent genetic distances.\u003c/p\u003e","description":"","filename":"OnlineFigure18.png","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/984a9ebf4a61b279b749022b.png"},{"id":63962227,"identity":"58433967-7ab1-4fae-a0bd-b8b9dbd7be69","added_by":"auto","created_at":"2024-09-04 09:01:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":233204,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Principal component analysis (PCA) plot of 80 \u003cem\u003eA. oryzae/flavus\u003c/em\u003e strains. Each dot represents an individual strain, with colors corresponding to the groups identified in the phylogenetic analysis: KRI (Blue), KRM (Cyan), KRWO (Green), KRWF (Red), JPI (Purple), NO (Pale green), NF (Orange), and AG (Grey). The first two principal components (PC1 and PC2) capture the majority of the genetic variance among the strains, highlighting the closer genetic relationships between certain groups and the distinctiveness of others. (B) Admixture plot depicting the estimated population structure and genetic admixture of A. flavus and A. oryzae strains across varying numbers of ancestral populations (K values). Each vertical bar represents an individual strain, partitioned into colored segments that reflect the strain's estimated proportion of membership in each of the K-inferred genetic clusters.\u003c/p\u003e","description":"","filename":"OnlineFigure26.png","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/295fca339683f37756a096e9.png"},{"id":63962226,"identity":"7892c098-857a-4b5a-aafe-aaca6f022660","added_by":"auto","created_at":"2024-09-04 09:01:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":464644,"visible":true,"origin":"","legend":"\u003cp\u003eAflatoxin biosynthesis gene cluster genotype comparison. Grey bars indicate genomic matches to the reference genome (NRRL 3357), showing no mutations. Cyan bars depict homozygous mutations differing from the reference. Blue bars mark heterozygous mutations. Absent bars signal deletions, where genomic regions are missing.\u003c/p\u003e","description":"","filename":"OnlineFigure37.png","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/f39c7c3394cc319730198842.png"},{"id":63964156,"identity":"4ad4e8b4-a9bb-4ff8-ba61-0d4251bb5cf7","added_by":"auto","created_at":"2024-09-04 09:17:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":342218,"visible":true,"origin":"","legend":"\u003cp\u003eCyclopiazonic acid biosynthesis gene cluster genotype comparison. Grey bars indicate genomic matches to the reference genome (NRRL 3357), showing no mutations. Cyan bars depict homozygous mutations differing from the reference. Blue bars mark heterozygous mutations. Absent bars signal deletions, where genomic regions are missing.\u003c/p\u003e","description":"","filename":"OnlineFigure43.png","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/2e272f3e9dd6bd8d9ce897d5.png"},{"id":63962229,"identity":"c092f0e7-c8fe-4e47-9d0a-1625f2f8edde","added_by":"auto","created_at":"2024-09-04 09:01:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":408146,"visible":true,"origin":"","legend":"\u003cp\u003eDitryptophenaline biosynthesis gene cluster genotype comparison. Grey bars indicate genomic matches to the reference genome (NRRL 3357), showing no mutations. Cyan bars depict homozygous mutations differing from the reference. Blue bars mark heterozygous mutations. Absent bars signal deletions, where genomic regions are missing.\u003c/p\u003e","description":"","filename":"OnlineFigure53.png","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/51016da2988cbcd4659628ad.png"},{"id":63962950,"identity":"764ffe40-74e2-4bdf-bad2-58ba980a4ae0","added_by":"auto","created_at":"2024-09-04 09:09:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":325718,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Average intensity of Group C and Non-C group strains in 2100 to 16000 m/z. (B) The matrix of peak intensity of mass-spectral loci among KACC strains. The mass-spectral loci according to the difference (p \u0026lt; 0.001) between Group C and non-C groups are listed on the right side of the matrix.\u003c/p\u003e","description":"","filename":"OnlineFigure62.png","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/3502a8dc4a64ff85b46f83b7.png"},{"id":81988054,"identity":"d5e28bb8-dc86-4026-b5ba-21e9c9a49805","added_by":"auto","created_at":"2025-05-05 16:07:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2992238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/10c55189-f11a-47f3-a02c-1c866173355b.pdf"},{"id":63962948,"identity":"42ea759d-a078-478c-bc1f-a093d39564be","added_by":"auto","created_at":"2024-09-04 09:09:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4336214,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/538df73017e4991b8d805646.docx"},{"id":63962945,"identity":"041b9b10-848c-4409-89e2-314662c1aec4","added_by":"auto","created_at":"2024-09-04 09:09:42","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23203,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4865798/v1/2a34543d125a1d695ebe7275.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome Diversity, Population Structure and MALDI-TOF MS Profiling of Aspergillus oryzae/flavus Strains from Fermentation and Wild Environments","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn Asia, the non-aflatoxigenic filamentous fungal species \u003cem\u003eAspergillus oryzae\u003c/em\u003e plays an indispensable role in the fermentation process of various foods and beverages, including doenjang, soy sauce, and sake [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This species is considered a domesticated variant of \u003cem\u003eAspergillus flavus\u003c/em\u003e, a species notorious for producing harmful mycotoxins such as aflatoxin and cyclopiazonic acid. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA previous study demonstrated that the \u003cem\u003eA. oryzae\u003c/em\u003e strain RIB40 is incapable of producing secondary metabolites, aflatoxin, cyclopiazonic acid and produces isomeric metabolites such as aflatrem, miyakamides and ditryptophenaline compared to \u003cem\u003eA. flavus\u003c/em\u003e strain NRRL 3357 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite their significant biochemical differences, these two species exhibit a close genomic relationship. Moreover, certain strains of \u003cem\u003eA. flavus\u003c/em\u003e exhibited variability in aflatoxin production, influenced by temperature and humidity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Due to these characteristics, some strains were classified as \u003cem\u003eA. flavus\u003c/em\u003e, despite not producing aflatoxin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, comparing whole genomes is required to differentiate these closely related species precisely.\u003c/p\u003e \u003cp\u003eA previous whole genomic analysis of \u003cem\u003eA. oryzae\u003c/em\u003e classified industrial strains used in the Japanese fermentation industry into eight clades [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Further efforts aimed at distinguishing \u003cem\u003eA. oryzae\u003c/em\u003e from \u003cem\u003eA. flavus\u003c/em\u003e through comparative genomics, with a particular focus on aflatoxin gene cluster and the composition of Carbohydrate-Active enzymes (CAZymes), have not conclusively differentiated the two species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile extensive genomic studies have been conducted on industrial strains, relatively little attention has been given to non-aflatoxigenic strains derived from traditional meju and wild conditions, which are expected to have fewer events of genetic improvement. In a study on \u003cem\u003ePenicillium roqueforti\u003c/em\u003e, traditional strains used in Roquefort cheese production were found to be intermediate between wild and industrial strains. Thus, strains are also presumed to be intermediate between industrial \u003cem\u003eA. oryzae\u003c/em\u003e and aflatoxigenic \u003cem\u003eA. flavus\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, understanding their population structure and aflatoxin production risk at the genomic level is necessary to monitor their distribution.\u003c/p\u003e \u003cp\u003eIn recent microbial identification and classification study, the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) method has emerged as a powerful tool. For example, \u003cem\u003eWeissella confusa\u003c/em\u003e and \u003cem\u003eWeissella cibaria\u003c/em\u003e are genetically close and had different roles, with \u003cem\u003eW. confusa\u003c/em\u003e contributing to health benefits while \u003cem\u003eW. cibaria\u003c/em\u003e having a potential pathogenic role. This relation is similar to relationship between \u003cem\u003eA. oryzae\u003c/em\u003e and \u003cem\u003eA. flavus\u003c/em\u003e. To distinguish these closely related species, recent studies have developed markers using machine learning to analyze MALDI-TOF MS patterns [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. There was also attempt to use MALDI-TOF MS to distinguish \u003cem\u003eA. oryzae/flavus\u003c/em\u003e complex, but the study concluded that this method is not suitable for accurate differentiation due to the high overlapping protein profiles of the two species [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study aimed to elucidate the distribution of diverse Korean \u003cem\u003eA. oryzae/flavus\u003c/em\u003e strains from various environments by comparing their genomes with those of strains from previous studies originated from diverse geographical regions, such as Japan, the United States, and China.\u003c/p\u003e \u003cp\u003eFurthermore, variations of mutations within gene clusters associated with mycotoxins and secondary metabolites, related to safety in the food industry, were analyzed. In addition, MALDI-TOF MS for proteomics pattern profiling was conducted to distinguish between clustered groups. Based on the findings, we propose group distribution of \u003cem\u003eA. oryzae\u003c/em\u003e and \u003cem\u003eA. flavus\u003c/em\u003e, and their specific characteristics.\u003c/p\u003e"},{"header":"2. Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Collection of Genomes of Korean and Global \u003cem\u003eA. oryzae/flavus\u003c/em\u003e Strains\u003c/h2\u003e \u003cp\u003eKorean strains from 4 diverse sources, Korean Industrial non-aflatoxigenic strains (KRI, 5 strains), Korean non-aflatoxigenic strains from meju (KRM, 12 strains), Korean non-aflatoxigenic strains from wild conditions (KRWO, 7 strains), Korean aflatoxigenic strains from wild conditions (KRWF, 15 strains) were collected (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All 39 Korean strains were stored at -80\u0026deg;C. A portion of each stock was cultured on Malt Extract Agar (MEA) at 25\u0026deg;C for seven days for genomic DNA extraction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKorean A. oryzae/flavus strains from various condition. KRM are non-aflatoxigenic strains from traditional meju.KRI are non- aflatoxigenic strains used in Korean fermentation Industry. KRWO are non- aflatoxigenic strains from Korean wild condition. KRWF are aflatoxigenic strains from Korean wild condition.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKACC no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiscription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRegion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAflatoxin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003enorB/cypA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eomtA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003eKRM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46457\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Goisan, omtA A type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIA-S-B+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Sunchang, omtA A type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIA-L-B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Jeju, omtA A type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIA-L-B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Gyeongsan, omtA B type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIB-L-B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Yongin, omtA B type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIB-L-B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Incheon, omtA B type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIB-L-B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46456\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Haenam, omtA C type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-Ao\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Damyang omtA C type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-Ao\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Jeju, omtA C type (Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-Ao\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWX1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Buan, no omtA(Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWX2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46466\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, Gongju, no omtA(Hong et al. (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK93210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK93210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeju, patent strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-Ao\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKRI1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eKRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKorean industrial strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-Ao\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKRI2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKorean industrial strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIB-L-B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKRI3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKorean industrial strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-Ao\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKRI4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK47488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKorean industrial strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-Ao\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKRI5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK47843\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKorean industrial strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-Ao\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAor-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003eKRWO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46923\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRice straw, Yangyang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAor-17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRice straw, Gongju\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAor-38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46909\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoybean, Incheon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK48145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeanut, Jinyang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46918\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoil, Suwon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoybean farm soil, Namhae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGreentea farm soil, Jeju\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAor-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"14\" rowspan=\"15\"\u003e \u003cp\u003eKRWF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46927\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRice straw, Yangpyeong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAR018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46892\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIndoor air, Seoul\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAR028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46894\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOutdoor air, Suwon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOut group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCorn, Hongcheon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOut group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46898\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCorn, Hongcheon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46899\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCorn, Hongcheon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeanut, Seosan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46903\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeanut, Jangseong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46904\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeanut, Danyang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoil, Gwangju\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46929\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoil, Gwangju\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoil, Deagu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoil, Gwangju\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOut group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46916\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoil, Suwon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK46919\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGarlic farm soil, Namhae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, B/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eType II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIC-L-B+/B-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe genomic DNA (gDNA) from Korean strains was extracted using DNeasy Plant Mini kit (Qiagen, Hilden, Germany) and its protocol. PCR-free 150-bp paired-end libraries were constructed and sequenced by Macrogen Inc (Seoul) on an Illumina NovaSeq 6000. Raw reads underwent quality control. Trimmomatic (v0.38) was used to remove adapter sequences and low-quality reads [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and FastQC was performed to check the read quality (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the comparative genomic analysis, sequence reads from strains originated from 4 different sources were collected. The sources include Japanese industrial strains (JPI, 17 strains), NCBI non-aflatoxigenic strains (NO, 14 strains), NCBI aflatoxigenic strains (NF, 8 strains), and Afla-guard non-aflatoxigenic strains (AG, 2 strains). All sequence reads were obtained as raw files from the National Center for Biotechnology Information (NCBI) Sequence Reads Archive (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGlobal A. oryzae/flavus strains from NCBI database. JPI are atoxigenic strains used in Japanese industry. Two strains for each clades, 8 clades strains are gathered. TK-24 is not included in any clade. NO are global strains known as atoxigenic. NF are global strains known as toxigenic. AG are strains known as Afla-Guard.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNCBI No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiscription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRegion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAflatoxin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"16\" rowspan=\"17\"\u003e \u003cp\u003eJPI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSake, Clade A in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSake, Clade A in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSake, Clade B in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSake, Clade B in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOuter Clade in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSake, Clade C in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMiso, Clade C in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoyou, Clade D in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoyou, Clade D in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoyou, Clade E in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoyou, Clade E in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSake, Clade F in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMiso, Clade F in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMiso, Clade G in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMiso, Clade G in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoyou, Clade H in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK-14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDRX154149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoyou, Clade H in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"13\" rowspan=\"14\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX013842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eXinyang City, Chac\u0026oacute;n-Vargas et al. (2021),\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Chac\u0026oacute;n-Vargas et al. (2021)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX6074494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKorean strain in Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Naoki et al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRIB537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX147127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGibbons et al. (2012)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Gibbons et al. (2012)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRIB949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX147131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGibbons et al. (2012)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Gibbons et al. (2012)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM2040 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX4479827\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiocontroller in Alshannaq et al. (2018)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Alshannaq et al. (2018)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNRRL35739\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX5329434\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiocontroller in Pennerman et-al. (2019)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Pennerman et-al. (2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSU-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8635871\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSuzhou winery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Sun, Liu et al. (2022)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWRRL1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX3067799\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ealmond nuts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Yin et-al. (2018)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2017 Washington T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX5358295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiocontrol strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8062526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLouisiana State University\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAF36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8062528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiocontrol strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK54A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8062531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLouisiana State University\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNRRL30797 (K49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8062530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiocontrol strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVCG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8062533\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLouisiana State University\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2017 Washington T5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003eNF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX5358298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUSDA-ARS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8062527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLouisiana State University\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCA14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX6432310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePistachio, Hua et al.(2012}\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, Hua et al. (2012)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE1445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8621731\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthiopia peanut\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEthiopia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, Arias,Mohammed et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE1404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8617991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthiopia peanut\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEthiopia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, Arias,Mohammed et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE1402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8617924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthiopia peanut\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEthiopia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, Arias,Mohammed et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRI19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX10945235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTiger nuts, Schamann et al. (2022)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSpain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, Schamann et al. (2022)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTox4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX8062532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLouisiana State University\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAfla-Guard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX4479828\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUniversity of Georgia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYazoo S2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRX5356519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUSDA-ARS(Afla guard)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmerica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX, Fountain et al. (2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e* : Non-aflatoxigenic strains from traditional meju; KRM strain.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eJPI strains, previously categorized into eight clades (Clade A to H), were chosen with two representatives from each clades and an extra strain that did not fit into any clade (TK-24) were selected [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe distinction between the NO and NF strains was not based on the existing nomenclature. Instead, it was determined by previous research studies that examined the aflatoxin production capabilities of these strains [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Single Nucleotide Polymorphisms (SNPs) calling and population genetic analysis\u003c/h2\u003e \u003cp\u003eFor SNP calling, filtered DNA sequencing reads of the 80 individuals were aligned to the reference genome, NRRL 3357, using BWA software v0.7.17 MEM module [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. SAM alignment files were sorted and converted to BAM files with SAMtools v1.3 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Picard was used to delete duplicat reads and create index of reference genome. Next, various programs in GATK packages (HaplotypeCaller for variant calling, CombineGVCFs for merging GVCF files, GenotypeGVCFs for converting GVCF into VCF file, SelectVariants, and VariantFiltration for selecting and filtering SNPs) were used [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The filtering parameters were like this: \u0026ldquo;QD\u0026thinsp;\u0026lt;\u0026thinsp;2.0 | | FS\u0026thinsp;\u0026gt;\u0026thinsp;80.0 | | MQ\u0026thinsp;\u0026lt;\u0026thinsp;20.0| | SOR\u0026thinsp;\u0026gt;\u0026thinsp;3.0| | MQRankSum\u0026thinsp;\u0026lt;\u0026thinsp;\u0026minus;\u0026thinsp;12.5| | Read- PosRankSum\u0026thinsp;\u0026lt;\u0026thinsp;\u0026minus;\u0026thinsp;8.0 | | QUAL\u0026thinsp;\u0026lt;\u0026thinsp;40.0\u0026rdquo;. VCF files were converted into fasta files using vcf2fasta to construct a phylogenetic tree (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/santiagosnchez/vcf2fasta\u003c/span\u003e\u003cspan address=\"https://github.com/santiagosnchez/vcf2fasta\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The raw data of Illumina squences were deposited on NCBI Sequence Reads Archive.\u003c/p\u003e \u003cp\u003eThe tree was drawn using whole-genome-based phylogenetic reconstruction program, SANS serif [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. PCA with population-scale SNPs was performed using plink [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and GCTA [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A Bayesian population structure assessment was performed using ADMIXTURE v1.3.0 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] with block relaxation algorithm, maximum likelihood estimation. Pre-defined genetic clusters were increased from K\u0026thinsp;=\u0026thinsp;2 to K\u0026thinsp;=\u0026thinsp;10 (assumed number of ancestral populations). Prior to running ADMIXTURE, SNPs in high linkage disequilibrium (LD) were removed. Specifically, PLINK was used to prune SNPs employing a windowed approach, with an R2 threshold of 0.8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Genotypic Analysis of Secondary Metabolites Gene Cluster and Design of Diagnostic Markers\u003c/h2\u003e \u003cp\u003eSignificant phenotypic differences influenced by \u003cem\u003eA. oryzae/flavus\u003c/em\u003e domestication are prominently reflected in the mutations of secondary metabolites, with substantial variations in the secretion levels of numerous toxic metabolites. The presence and locations of the aflatoxin gene cluster [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], the CPA gene cluster [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and the ditryptophenaline gene cluster [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] in the genome of \u003cem\u003eAspergillus flavus\u003c/em\u003e NRRL 3357 have been elucidated in previous studies. SNPs based on the NRRL 3357 genomic sequence are visualized using the Integrated Genomics Viewer (IGV). This visualization allows the identification of specific mutations or deletions occurring within these gene clusters of each strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. MALDI TOF metabolites pattern analysis\u003c/h2\u003e \u003cp\u003eMass spectrometry data from MALDI-TOF/MS on KACC strains containing Groups A, B, C and E were analyzed to compare the similarity patterns among groups classified based on genomic analysis results. The group specific mass-spectral loci were explored by comparing presence or absence of mass peak at certain loci and their degree of difference.\u003c/p\u003e \u003cp\u003eEach strain underwent individual mass analysis using MicroIDSys\u0026reg; (ASTA Inc.). A sample from each strain is processed by mixing it with a cell lysis solution. After cell lysis, 1.5 \u0026micro;L of the separated supernatant is applied to the \u0026micro;ID plate, dried, and then coated with 1.5 \u0026micro;L of CHCA matrix. The raw spectrum data generated by MicroIDSys\u0026reg; ranges from 2,000 to 20,000 daltons (m/z), with an identification cutoff set at \u0026ge;\u0026thinsp;140 as per the manufacturer\u0026rsquo;s recommendation.\u003c/p\u003e \u003cp\u003eThe raw data from MicroIDSys\u0026reg; undergoes normalization to adjust peak height relative to m/z using proprietary software from ASTA Inc. and NQ-Lab. Co.,Ltd. Subsequently, the data is processed with a binning parameter set at 5. An averaging procedure calculates the mean peak heights for each m/z range within the binning. The processed data is then interpreted using a Python-based code, resulting in a combined dataset.\u003c/p\u003e \u003cp\u003eThe prepared data is analyzed using a machine learning model developed by NQ-Lab., Co., Ltd. Logistic Regression, is the chosen machine learning algorithm tailored with hyperparameters optimized for MALDI-TOF/MS characteristics. The analysis accounts for various factors influencing MALDI-TOF/MS results, such as temporal, physical, and biochemical attributes. To ensure a comprehensive analysis, multiple statistical tests are conducted. The ANOVA test is utilized to examine mean differences between groups, considering the continuity of m/z values. Additionally, the Chi-squared (χ\u0026sup2;) test is employed to assess the independence of categories, assuming that the two groups are distinct entities.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Phylogenetic relationship of \u003cem\u003eA. oryzae/flavus\u003c/em\u003e strains\u003c/h2\u003e \u003cp\u003eThis comprehensive analysis classified the non-aflatoxigenic and aflatoxigenic \u003cem\u003eA. oryzae/flavus\u003c/em\u003e strains into five distinct groups, designated as Groups A to E (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGroup A predominantly comprises non-aflatoxigenic strains, with a significant presence of KRI and JPI strains. Previous research by Watari et al. (2019) categorized JPI strains into eight clades (Clade A to H), and strains belonging to Clades C to H were integrated into Group A. Notably, most KRI strains were found to cluster within Clade C. Additionally, among the KRM strains, MWC2, MWC3, and M2040 also aligned with Clade C, while K93210 aligned with Clade D. Other KRM and KRWO strains were also classified under Group A, although they did not align with any of the specific clades. Group B primarily includes non-industrial strains such as KRM, KRWO, and NO, apart from a singular KRI strain, KRI2, indicating a diverse genetic background within non-aflatoxigenic populations.\u003c/p\u003e \u003cp\u003eGroup C encapsulates all aflatoxigenic strains, including KRWF and NF. Intriguingly, this group also contains six non-aflatoxigenic strains from the NO category (2017 Washington-T2, A1, AF36, K54A, NRRL30797, and VCG1), highlighting the complex genetic landscape that does not strictly correlate with aflatoxin production.\u003c/p\u003e \u003cp\u003eGroup D is characterized by its genetic divergence from Groups A, B, and C, comprising mainly JPI strains that fall into the A and B clades as defined by Watari et al., with the exception of one NO strain, 14160.\u003c/p\u003e \u003cp\u003eGroup E, distinguished as the most genetically distinct cluster, includes biocontrol strains such as Afla-guard and Yazoo S2, alongside two unique KRM strains (MWA1 and MWA3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Genome and Population Structure Analysis of \u003cem\u003eA. oryzae/flavus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003ePrincipal Component Analysis (PCA) of the \u003cem\u003eA. oryzae/flavus\u003c/em\u003e population structure revealed that Group D closely aligns with Groups A and B, demonstrating their closer genetic relationship, while Group E exhibits distinct genetic separateness from all other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This distinct positioning of Group E highlights its unique genetic makeup.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdmixture analysis further elucidates the genetic diversity and interrelation among the groups. To identify the most optimal K number, we calculated the cross-validation (CV) error values, where a lower CV value indicates a better estimation. This calculation was performed systematically for K values ranging from 1 to 15 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Using this approach, K\u0026thinsp;=\u0026thinsp;11 was identified as the most likely number of populations. At K\u0026thinsp;=\u0026thinsp;11, part of the Group A strains shared population with Group B and D, while part of the Group C strains shared small population with Group E. Phylogenetic network analysis further supports the presence of five main populations and individual population assignment into these populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Secondary Metabolite Gene Cluster Analysis\u003c/h2\u003e \u003cp\u003eAmong the various secondary metabolite gene clusters, three metabolites gene clusters, aflatoxin, CPA and ditryptophenaline gene clusters showed group-specific characteristics. In genome of \u003cem\u003eA. flavus\u003c/em\u003e NRRL3357, aflatoxin gene cluster region is located on chromosome 3, 4,940,000 to 5,010,000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). CPA gene cluster region is located on beside the aflatoxin gene cluster, chromosome 3, 5,010,000 to 5,034,262 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The ditryptophenaline gene cluster region is located on chromosome 4, 3,181,000 to 3,195,000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGroup A strains exhibited a shared pattern of deletions and mutations within their gene clusters. The CPA gene cluster pattern depended on the aflatoxin gene cluster pattern. The strain that had a deletion in the aflatoxin gene cluster had a large deletion in the CPA gene cluster, while other strains showed consistent mutation patterns. For the ditryptophenaline cluster, Group A strains shared a similar mutation pattern.\u003c/p\u003e \u003cp\u003eGroup B showed similar mutation patterns with part of the Group A strains in the aflatoxin and CPA gene clusters, but they had intact ditryptophenaline gene clusters.\u003c/p\u003e \u003cp\u003eMost of the Group C strains had intact three gene clusters. Conversely, three aflatoxigenic strains (AR028, SD016, and SL015) exhibited unique mutation patterns in the aflatoxin and CPA gene clusters. This mutation pattern is odd compared to Group A and B mutation patterns.\u003c/p\u003e \u003cp\u003eGroup D strains presented a distinctive pattern with large deletions in the aflatoxin cluster and complete deletion in the CPA cluster, maintaining an intact Ditryptophenaline gene cluster.\u003c/p\u003e \u003cp\u003eIn Group E, KRM strains and Afla-guard strains showed distinct features. In contrast to other strains, where the CPA gene cluster pattern depends on the aflatoxin gene cluster's state, KRM strains uniquely exhibited a deleted aflatoxin gene cluster while retaining the CPA gene cluster. Afla-guard strains had a whole deletion in the aflatoxin and CPA gene clusters. However, these Group E strains had an intact ditryptophenaline gene cluster similar to the others. No group-specific characteristics were observed among the groups concerning Aspergillic acid and Aflatrem gene clusters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4. MALDI TOF/MS patterning\u003c/h2\u003e \u003cp\u003eComparing patterns of the peaks of Group C and the non-C groups (A, B and E; A. oryzae groups), the two groups were distinctly separated, while non-C groups were not distinctly separated from each other (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eGroup C and the non-C groups exhibited several significantly different loci; 3100\u0026ndash;3200 m/z, 6200\u0026ndash;6500 m/z, and 12700\u0026ndash;12900 m/z showed significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). When comparing the non-C groups, no distinct loci were found that significantly differentiated between the groups in overall ranges (Supplementary Fig.\u0026nbsp;2). Most of the Group C strains had significantly higher peaks in the 6355 to 6385 m/z and 12795 to 12810 m/z ranges, while most of the non-C groups had high peaks at 3145 m/z, 6285 to 6295 m/z, and 12730 m/z.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe phylogenetic analysis and genome structure investigation of \u003cem\u003eA. oryzae/flavus\u003c/em\u003e strains from fermentation and wild environment in Korea, compared with globally reported strains, provided significant insights into the genetic diversity and evolutionary pathways of these fungi. The phylogenetic tree, PCA and ADMIXTURE analysis offer a vivid illustration of the genetic distances and relationships among the strains, revealing a broad spectrum of evolutionary divergence within five distinct groups (A to E). In particular, Groups A and B demonstrate closer genetic affiliations, suggesting a shared evolutionary pathway that may be rooted in their non-aflatoxigenic nature and potential adaptations to industrial or natural environments. Conversely, Group E, characterized by its distinct genetic makeup, stands out as the most genetically divergent cluster. This may indicate unique evolutionary pressures or historical genetic isolation that have shaped their current genomic constitution. Group D exhibits an interesting feature since it has a greater genetic distance from Groups A and B than from Group C, yet PCA analysis showed that it aligns more closely with Groups A and B.\u003c/p\u003e \u003cp\u003eConsidering distribution of Korean strains, industrial strains (KRI), excluding KRI2, clustered within Group A, specifically aligning with JPI Clade C strains. The narrow genetic distances among these industrial strains suggest recent differentiation driven by fermentation functionalities, resulting in their separation as individual strains.\u003c/p\u003e \u003cp\u003eKorean Meju-originated strains (KRM) exhibited broader distribution across Groups A, B, and E. Some strains were closely matched to industrial strains, likely due to spore dispersal from industrial environments to traditional fermentation settings. Other Meju strains in Group A, B and E showed distinct genetic positions with industrial strains. Notably, two Meju strains in Group E showed genetic similarity with the biocontrol agent, Afla-guard but differed in aflatoxin and CPA gene clusters, suggesting potential genetic recombination events with wild strains. The distribution of Meju strains(KRM) was similar with that of non-aflatoxigenic strain from wild conditions (KRWO). This supports the nothion that Meju fermentation involves environmental strains without artificial starter inoculation, resulting in weak selective pressure.\u003c/p\u003e \u003cp\u003eNon-aflatoxigenic strains from Korean wild environments (KRWO) were found in Groups A and B. Group A wild strains exhibited genetic distinctions from JPI strains, suggesting different evolutionary pressures. The lower genetic diversity among these wild strains, compared to industrial strains, may indicate fewer evolutionary changes in natural settings. In contrast, industrial environments likely experienced more frequent selective evolution events.\u003c/p\u003e \u003cp\u003eAll aflatoxigenic Korean strains (KRWF) were categorized in Group C. Especially strains AR028, SD016, and SL015 had notable genetic characteristics highlighted in previous studies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. PCR analysis patterns of the norB/cypA cluster in these strains were similar to that of \u003cem\u003eA. parasiticus\u003c/em\u003e. Genome-wide clustering confirmed their placement within Group C, yet their aflatoxin cluster mutation patterns were notably distinct from NRRL 3357 and other Group C strains. Despite these genomic differences, MALDI-TOF MS results closely matched NRRL 3357 and other Group C strains.\u003c/p\u003e \u003cp\u003eFollowing Aflatoxin mutation patterns, this study reveals that there are points of concordance with previous research as well as advancements in understanding these patterns. Comparing the strains, Afla-guard (NRRL 21882), which had a complete deletion of the aflatoxin gene cluster, and strain 14160, which exhibited deletion starting from the OmtA gene showed the same results. Strains BP2-1, TK-10, TK24, WRRL1519, and NRRL35739 showed deletions beginning from the norA gene. These results are also consistent with previous findings.\u003c/p\u003e \u003cp\u003eAdditionally, strains 3.042, TK-5, TK-59, and SU-16 were described as having mostly intact gene clusters with partial deletions starting from the AflT gene. Group C strains (A9, CA14, NRRL 3357, VCG1, Washington T5, E1404, E1445) were reported to have partial deletions starting from the CypA gene [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this study, non-aflatoxigenic strains, including those in Group C, frequently exhibited additional deletions of the cypA gene compared to NRRL 3357. This suggests that the additional deletion in cypA may significantly impact aflatoxin production. This finding also aligns with previous research that classified norB-cypA PCR patterns into type 1 (short read type) and type 2 (long read type), with aflatoxigenic strains being type 2 and non-aflatoxigenic strains being type 1 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcross Groups A and B, strains with high mutation rates or high deletion rates are mixed and each of them shares a similar mutation pattern. But they don\u0026rsquo;t share similar mutation patterns with Group C strains. This suggests significant genetic exchange through interbreeding between Groups A and B during domestication, while the two groups appear to be reproductively isolated from the aflatoxigenic group, indicating speciation. Therefore, these strains with high mutation rates or high deletion rates are considered safe since they have less potential for producing mycotoxins and are unlikely to form hybrids with aflatoxigenic strains.\u003c/p\u003e \u003cp\u003eGroup C mostly consists of aflatoxigenic strains (KRWF and NF), but there are some exceptional non-aflatoxigenic strains (2017 Washington-T2, A1, AF36 and K54A). Among them, AF36 is even known as biocontrol agent to reduce aflatoxin contamination [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This may indicate a complex genetic landscape that does not strictly correlate with aflatoxin production. However, the presence of intact aflatoxin and cyclopiazonic acid gene clusters in these non-aflatoxigenic strains suggests a latent potential for mycotoxin production under certain conditions, raising important considerations for food safety and strain utilization in industrial applications..\u003c/p\u003e \u003cp\u003eThe evolutionary direction of Group A, characterized by unique mutations in the ditryptophenaline gene cluster, indicates a potential adaptive response to industrial fermentation processes, possibly due to selective pressures to mitigate negative impacts on food products. Although the toxicity or side effects of ditryptophenaline have not been thoroughly studied, the uniform occurrence of mutations in this cluster among Group A strains implies that such mutations may confer a selective advantage in the context of fermented food production, possibly due to reduced detrimental effects on product quality [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy incorporating MALDI-TOF MS data, we observe significant differentiation, particularly between Group C and the non-C groups (A, B, and E), with unique proteomic features corresponding to their genomic distinctions. Group C exhibited the unique features in the ranges of 3100\u0026ndash;3200 m/z, 6200\u0026ndash;6500 m/z, and 12700\u0026ndash;12900 m/z. A database of various proteins specific for \u003cem\u003eA. flavus\u003c/em\u003e and \u003cem\u003eA. oryzae\u003c/em\u003e in UniProtKB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.uniprot.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) suggests that these peaks may correspond to significant fungal proteins. For instance, peaks within the 6200 to 6500 m/z range correspond to metabolic enzyme proteins like 3-Hydroxyanthranilate 3,4-Dioxygenase (3HAO) and Short-chain dehydrogenase. Peaks within the 12700 to 12900 m/z range may correspond to proteins such as AflF, also known as norB (primary accession number: A0A7U2MNK6, Mass: 12855 Da). This protein is one of the enzymes included in the aflatoxin gene cluster. No characterized proteins within the 3100 to 3200 m/z range were identified in the database. These specific proteins play crucial roles in cellular metabolism, suggesting their potential as reliable biomarkers for differentiating between groups.\u003c/p\u003e \u003cp\u003ePrevious attempts to differentiate \u003cem\u003eA. oryzae\u003c/em\u003e from \u003cem\u003eA. flavus\u003c/em\u003e have been numerous. A previous study showed that targeting the Cyp51A gene could provide differentiation, but the limited strain diversity hindered its broad applicability [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. There were also attempts to analyze aflatoxin and cyclopiazonic acid gene clusters to see the difference between two species, and although some differentiation was achieved, it was not definitive [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. There have also been attempts to distinguish the two species through profiling of CAZyme genes and secondary metabolite biosynthesis gene clusters, but the ambiguous similarities between the two species disturbed clear differentiation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, a comprehensive approach was employed, combining whole-genome SNP-based population structure analysis, detailed secondary metabolite gene cluster variation analysis, and MALDI-TOF MS profiling.\u003c/p\u003e \u003cp\u003eGroup C formed a distinct cluster in genomic analysis, separating it from other groups. Similarly, MALDI-TOF MS analysis distinguished Group C from other groups, displaying unique peaks. Furthermore, group C strains have comparatively intact gene clusters of aflatoxin and cyclopiazonic acid and produced aflatoxin B. Given these characteristics, Group C aligns with traits traditionally associated with \u003cem\u003eA. flavus\u003c/em\u003e. Therefore, this study proposes classifying Group C as \u003cem\u003eA. flavus\u003c/em\u003e and the non-C groups as \u003cem\u003eA. oryzae\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study provides a comprehensive genomic and proteomic characterization of Korean \u003cem\u003eA. oryzae/flavus\u003c/em\u003e complex strains compared to global strains. The analysis highlights the genetic diversity and distinct evolutionary paths of these strains, with particular attention to their safety for food industry applications. Genomic analysis divided the \u003cem\u003eA. oryzae\u003c/em\u003e/\u003cem\u003eflavus\u003c/em\u003e strains into five groups, with Group C showing relatively intact aflatoxin and cyclopiazonic acid (CPA) gene clusters, suggesting a high potential for producing both metabolites. Furthermore, unique proteomic patterns identified through MALDI-TOF MS provide reliable biomarkers for distinguishing Group C aflatoxigenic strains. Characteristics of group C were well matched with those of \u003cem\u003eA. flavus\u003c/em\u003e and the other groups with \u003cem\u003eA. oryzae\u003c/em\u003e. These findings establish refined criteria for differentiating \u003cem\u003eA. oryzae\u003c/em\u003e from \u003cem\u003eA. flavus\u003c/em\u003e, contributing to their safe utilization and better understanding of their genetic dynamics.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCyclopiazonic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKRI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKorean Industrial non-aflatoxigenic strains\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKRM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKorean non-aflatoxigenic strains from meju\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKRWO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKorean non-aflatoxigenic strains from wild conditions\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKRWF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKorean aflatoxigenic strains from wild conditions\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eJapanese Industrial strains\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNCBI non-aflatoxigenic strains\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNCBI aflatoxigenic strains\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAfla-guard non-aflatoxigenic strains.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Consent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work is supported by the grant from National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea (PJ017286), and the Ministry of Science, ICT (MSIT) of Korea (2021M3H9A1081101).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eD.H. Kim wrote the main manuscript text and prepared figures 1-5. S.B. Hong and K.T. Kim provided revisions to the contents of main manuscript. D.C. Kim and D.G. Seo prepared figure 6. S.H. Lee reviewed and corrected the manuscript\u0026rsquo;s grammar and language.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMachida M, et al. Genome sequencing and analysis of Aspergillus oryzae. Nature. 2005;438(7071):1157\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachida M, Yamada O, Gomi K. Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future. DNA Res. 2008;15(4):173\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGibbons JG, et al. The evolutionary imprint of domestication on genome variation and function of the filamentous fungus Aspergillus oryzae. Curr Biol. 2012;22(15):1403\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlshannaq AF, et al. Controlling aflatoxin contamination and propagation of Aspergillus flavus by a soy-fermenting Aspergillus oryzae strain. Sci Rep. 2018;8(1):16871.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatarai N, et al. Evolution of Aspergillus oryzae before and after domestication inferred by large-scale comparative genomic analysis. DNA Res. 2019;26(6):465\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKj\u0026aelig;rb\u0026oslash;lling I, et al. A comparative genomics study of 23 Aspergillus species from section Flavi. Nat Commun. 2020;11(1):1106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRank C, et al. Comparative chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357). Metabolites. 2012;2(1):39\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedina A, Rodriguez A, Magan N. Effect of climate change on Aspergillus flavus and aflatoxin B1 production. Front Microbiol. 2014;5:348.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun H, et al. Safety evaluation and comparative genomics analysis of the industrial strain Aspergillus flavus SU-16 used for huangjiu brewing. Int J Food Microbiol. 2022;380:109859.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan DM, et al. Comparative pangenome analysis of Aspergillus flavus and Aspergillus oryzae reveals their phylogenetic, genomic, and metabolic homogeneity. Food Microbiol. 2024;119:104435.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDumas E, et al. Independent domestication events in the blue-cheese fungus Penicillium roqueforti. Mol Ecol. 2020;29(14):2639\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim E, et al. Differentiation between Weissella cibaria and Weissella confusa using machine-learning-combined MALDI-TOF MS. Int J Mol Sci. 2023;24(13):11009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHedayati MT, et al. Discrimination of aspergillus flavus from Aspergillus oryzae by matrix-assisted laser desorption/ionisation time‐of‐flight (MALDI‐TOF) mass spectrometry. Mycoses. 2019;62(12):1182\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFountain JC, et al. Draft genome sequences of one Aspergillus parasiticus isolate and nine Aspergillus flavus isolates with varying stress tolerance and aflatoxin production. Microbiol Resource Announcements. 2020;9(37). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ep. 10.1128/mra\u003c/span\u003e\u003cspan address=\"p. 10.1128/mra\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 00478\u0026thinsp;\u0026ndash;\u0026thinsp;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChac\u0026oacute;n-Vargas K, et al. Comparison of two Aspergillus oryzae genomes from different clades reveals independent evolution of alpha-amylase duplication, variation in secondary metabolism genes, and differences in primary metabolism. Front Microbiol. 2021;12:691296.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePennerman KK, et al. Aspergillus flavus NRRL 35739, a poor biocontrol agent, may have increased relative expression of stress response genes. J Fungi. 2019;5(2):53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang PK. Genome-wide nucleotide variation distinguishes Aspergillus flavus from Aspergillus oryzae and helps to reveal origins of atoxigenic A. flavus biocontrol strains. J Appl Microbiol. 2019;127(5):1511\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchamann A, Geisen R, Schmidt-Heydt M. Draft genome sequence of an aflatoxin-producing Aspergillus flavus strain isolated from food. Microbiol Resource Announcements. 2022;11(2):e00894\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArias RS, et al. Sixteen draft genome sequences representing the genetic diversity of Aspergillus flavus and Aspergillus parasiticus colonizing peanut seeds in Ethiopia. Microbiol Resource Announcements. 2020;9(30). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ep. 10.1128/mra\u003c/span\u003e\u003cspan address=\"p. 10.1128/mra\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 00591\u0026thinsp;\u0026ndash;\u0026thinsp;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua SST, et al. Characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates from pistachio. Mycotoxin Res. 2012;28:67\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Durbin R. Fast and accurate long-read alignment with Burrows\u0026ndash;Wheeler transform. Bioinformatics. 2010;26(5):589\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan der Auwera GA et al. \u003cem\u003eFrom FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline.\u003c/em\u003e Current protocols in bioinformatics, 2013. 43(1): p. 11.10. 1-11.10. 33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRempel A, Wittler R. SANS serif: alignment-free, whole-genome-based phylogenetic reconstruction. Bioinformatics. 2021;37(24):4868\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang CC et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience, 2015. 4(1): p. s13742-015-0047-8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, et al. GCTA: a tool for genome-wide complex trait analysis. Am J Hum Genet. 2011;88(1):76\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexander DH, Novembre J, Lange K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 2009;19(9):1655\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu J, et al. Clustered pathway genes in aflatoxin biosynthesis. Appl Environ Microbiol. 2004;70(3):1253\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmaike S, Keller NP. Aspergillus flavus. Annu Rev Phytopathol. 2011;49:107\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaceres I, et al. Aflatoxin biosynthesis and genetic regulation: A review. Toxins. 2020;12(3):150.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang P-K, Ehrlich KC, Fujii I. Cyclopiazonic acid biosynthesis of Aspergillus flavus and Aspergillus oryzae. Toxins. 2009;1(2):74\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKishimoto S, et al. Evaluation of biosynthetic pathway and engineered biosynthesis of alkaloids. Molecules. 2016;21(8):1078.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexander DH, Lange K. Enhancements to the ADMIXTURE algorithm for individual ancestry estimation. BMC Bioinformatics. 2011;12:1\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong S-B, et al. The proportion of non-aflatoxigenic strains of the Aspergillus flavus/oryzae complex from meju by analyses of the aflatoxin biosynthetic genes. J Microbiol. 2013;51:766\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoster MA, Cotty PJ, Michailides TJ. Evaluation of the atoxigenic Aspergillus flavus strain AF36 in pistachio orchards. Plant Dis. 2014;98(7):948\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNargesi S, et al. Differentiation of Aspergillus flavus from Aspergillus oryzae targeting the cyp51A gene. Pathogens. 2021;10(10):1279.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyotome T, et al. Comparative genome analysis of Aspergillus flavus clinically isolated in Japan. DNA Res. 2019;26(1):95\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"genomics, Aspergillus oryzae/flavus complex, structure analysis, aflatoxin, MALDI-TOF","lastPublishedDoi":"10.21203/rs.3.rs-4865798/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4865798/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVarious strains of \u003cem\u003eAspergillus oryzae\u003c/em\u003e, regarded as a domesticated variant of aflatoxigenic \u003cem\u003eAspergillus flavus\u003c/em\u003e, are utilized in soybean fermentation industry of Korea. This study compared \u003cem\u003eA. oryzae/flavus\u003c/em\u003e strains isolated from various environments in Korea, including industrial settings, Meju (brick of dried fermented soybeans), and wild conditions, with globally reported strains using genomic analysis to determine their taxonomic positions and safety. Using population genomics, five distinct groups (A to E) were identified, with all aflatoxigenic Korean strains in Group C and non-aflatoxigenic Korean strains in Groups A, B, and E. Korean strains from Meju and wild conditions are distributed across Groups A and B, and most of the Korean industrial strains form a sub-cluster with Japanese industrial strains in Group A. Comparing secondary metabolite gene cluster mutation pattern, three gene clusters (Aflatoxin, Cyclopiazonic acid and Ditryptophenaline) were revealed as group specific ones. In aflatoxin and cyclopiazonic acid clusters, most of the Group C strains had intact regions compared to other groups strains.Since most of the Group C strains produce aflatoxin and have intact Aflatoxin and Cyclopiazonic acid gene cluster, we considered that this group represent \u003cem\u003eA. flavus\u003c/em\u003e. Profiling of MALDI-TOF MS analysis also distinguished Group C from Groups A, B and E by specific proteomic peaks. Among these peaks, those around 12700 to 12900 m/z (Da) are expected to correspond to AflF (nor B), an enzyme involved in Aflatoxin metabolism. These results showed taxonomic positions of Korean strains of \u003cem\u003eA. oryzae/ flavus\u003c/em\u003e from various environments and also showed possibility to differentiate between \u003cem\u003eA. oryzae\u003c/em\u003e and \u003cem\u003eA. flavus\u003c/em\u003e with genome and Maldi-tof analysis.\u003c/p\u003e","manuscriptTitle":"Genome Diversity, Population Structure and MALDI-TOF MS Profiling of Aspergillus oryzae/flavus Strains from Fermentation and Wild Environments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-04 09:01:37","doi":"10.21203/rs.3.rs-4865798/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-09T05:58:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-09T02:28:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-09T02:28:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-08-06T05:48:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b8afa27b-1158-4811-ad9d-96e69a523a84","owner":[],"postedDate":"September 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T16:04:51+00:00","versionOfRecord":{"articleIdentity":"rs-4865798","link":"https://doi.org/10.1186/s12864-025-11596-9","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-04-30 15:57:12","publishedOnDateReadable":"April 30th, 2025"},"versionCreatedAt":"2024-09-04 09:01:37","video":"","vorDoi":"10.1186/s12864-025-11596-9","vorDoiUrl":"https://doi.org/10.1186/s12864-025-11596-9","workflowStages":[]},"version":"v1","identity":"rs-4865798","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4865798","identity":"rs-4865798","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}