Malus mandshurica, was justified for its excellent tolerance to low phosphorus (low−Pi) stress. To date, however, the genes involved in low−Pi stress tolerance have not yet been unraveled in this species. Currently, the physiological responses of this line for different days to low−Pi stress were characterized, and their roots as well as leaves were used to carry out transcriptome analysis, so as to illuminate the potential molecular pathways and identify the genes involved in low−Pi stress−response. After exposure to low−Pi treatment (32 µmol/L KH2PO4) for 20 day after treatment (DAF) the biomass of shoots was significantly reduced in comparison with that of the stress−free (control), and root architecture diversely changed. For example, the root growth parameters e.g., length, surface area, and total volume somewhat increase in comparison with those of the control. The activity of acid phosphatase (ACP) increased with the low−Pi treatment, whereas the photosynthetic rate and biomass were declining. The activity of antioxidant enzymes, e.g., superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were substantially elevated in response to low−Pi treatment. Many enzyme−related candidate genes e.g., MmCAT1, MmSOD1 and MmPOD21 were up−regulated to low−Pi treatment. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the processes of photosynthesis, plant hormone signal transduction, and MAPK signaling pathway were affected in the low−Pi response. In combination with the physiological characterization, several low−Pi−responsive genes, e.g., PHT, PHO, were identified, and the genes implicated in Pi uptake and transport, such as MmPHT1;5, MmPHO1, MmPAP1, etc., were also obtained since their expression status varied among the exposure times, which probably notifies the candidates involved in low−Pi−responsive tolerance in this line. Interestingly, low−Pi treatment activated the expression of transcription factors including the WRKY family, MYB family, etc. The available evidences will facilitate a better understanding of the roles of this line underlying the high tolerance to low−Pi stress. Additionally, the accessible data are helpful for the use of the apple rootstock M. mandshurica under low−Pi stress.Phosphorus (Pi) is a macronutrient essential for plant growth, development, and reproduction. However, there is not an efficient available amount of Pi that can be absorbed by plants in the soil. Previously, an elite line, MSDZ 109, selected from  Phosphorus (Pi) is an essential macronutrient that is a requisite for the growth, development, and reproduction of plants . AlthougOsSPX1 gene from rice  genes  were up−regulated on the 30 DAF, conversely, SOD [Fe] genes (LOC103431645 and LOC103451457) were down−regulated in comparison with those from the stress−free leaves and roots (MmCAT1 (LOC103445262) gene was up-regulated on the 30th DAF in leaves. A total of 56 differentially expressed POD−related genes were acquired from transcriptome data, including 41 genes of POD, 1 gene of 2−Cys peroxiredoxin BAS1 (PER2 BAS1), 4 genes of peroxiredoxin, 4 genes of L−ascorbate peroxidase (APX), 4 genes of glutathione peroxidase (GPX), and 1 gene of overexpressor of cationic peroxidase (OCP3). The MmPOD6 (LOC103451919) gene was up−regulated on the 30 DAF in leaves and down−regulated in roots. MmPOD3 (LOC103442590), MmPOD4 (LOC103437812), MmPOD11 (LOC103451708), and MmPOD21 (LOC103433277) were up-regulated on the 30 DAF in leaves , manganese SOD (SOD [Mn]), and iron SOD (SOD [Fe]) by Grene . As subjnd roots c. Therefn leaves b. TherefMmPHT1;3 (LOC10344734) and MmPHT1;9 (LOC103428150) were strongly induced on the 30 DAF in comparison with that of the stress−free leaves and roots. MmPHT1;5 (LOC103438596) was strongly up−regulated, a gradually increased tendency was obtained with the progress of stress in roots. MmPHT1;12 (LOC103404046) was up−regulated on the 10 DAF in leaves and on the 30 DAF in roots compared with that of the stress−free. Additionally, low−affinity Pi transporters of MmPHT2;1 (LOC103452033) and MmPHT2;2 (LOC103403064) were weakly up−regulated from exposure to low−Pi treatment on the 30 DAF in both leaves and roots. MmPHT3;5 (LOC103454899) and MmPHT4;8 (LOC103422687) were strongly up−regulated in the roots. Additionally, as subjected to stress, the PHO1 family of MmPHO1;9 (LOC103425833) was strongly down−regulated compared with that of the stress−free, while MmPHO1;3 (LOC103423874) was strongly up−regulated on the 10 DAF. Moreover, 16 acid phosphatase genes were screened, including MmPAP3 (LOC103430610), MmPAP15 (LOC103409175), MmPAP17 (LOC103438263), MmPAP20 (LOC103449530), etc. , respectively a. Exposu0), etc. a. MmPAP1MmWRKY6 (LOC103452669) and MmWRKY9 (LOC103433719) were up−regulated as subjected to low−Pi on the 30 DAF in leaves (MmMYB6 (LOC103401412) and MmMYB53 (LOC103433576) were strongly up−regulated on the 30 DAF in roots. In contrast, MmMYB5 (LOC103441079) and MmMYBR2 (LOC103424517) were down−regulated in comparison with the stress−free roots. However, the mechanism of its regulation remains to be further researched (MmPHR1 (LOC103434216) was down−regulated in comparison with that of the stress−free on the 30 DAF in both leaves and roots, whereas MmPHR2 (LOC103432598) was up−regulated on the 30 DAF in roots. TFs of other families were obtained as they were subjected to low−Pi treatment , MYB, and Cys2/His2 (C2H2), while 44 members of the ERF family, 35 members of the MYB family, and 21 members of the C2H2 family exhibited highly expressed patterns of expression after exposure to low−Pi treatment. Venn diagram analysis of the three datasets suggested that 10 genes and 5 genes overlapped in leaves and roots, respectively a,b. Inten leaves c. MmMYB6searched c. Furthereatment c. TherefLHCA1, LHCA3, LHCA5, LHCA6, LHCA151) and five genes encoding Chla/b binding protein complex II  were down−regulated in LLP20 vs. LLP30  [TaPHR1 (Triticum aestivum) [OsPHR2 (Oryza sativa) [FvPHR1 (Fragaria vesca) [NtMYB12 increased total Pi concentration and increased expression of PHT1 family genes  in tobacco tolerance to low−Pi stress [GmPHT1;1, GmPHT1;4 and GmPHT1;1 [ZmPHR1 can bind to the conserved binding site (P1BS) of ZmGPX−PDE1 and ZmGPX−PDE5 promoters and decompose glycerophosphodiester into glycerol−3−phosphate (G3P), indicating that they are involved in the recovery function of Pi from glycerophosphodiester [MdMYB2 was found to be overexpressed in apples, causing a significant increase in PSI gene expression in response to Pi deficiency [MmMYB5, MmMYB6, MmMYB53, and MmMYBR2 were highly expressed in leaves and roots, indicating that these MYBs have essential roles in various tissues in response to low−Pi treatment of M. mandshurica. Interestingly, our research found that two PHRs were stronger expressed in leaves and roots to low−Pi treatment of M. mandshurica. However, their regulatory roles are unknown in apple rootstocks. We speculate that MmPHR1 and MmPHR2 may be involved in the absorption and transportation of Pi. Additionally, some of available evidences has demonstrated that the PHL (PHR1−LIKE) MYB transcription factors are thus likely candidates as targets of SIZ1, a SUMO E3 ligase, in the coordination of root developmental responses to low−Pi conditions [TFs have been justified to play a vital role in the Pi starvation response via gene expression regulation and signal transmission . The MYBAtMYB2 , GmMYB48[GmMYB48 , AtPHR1 idopsis) , TaPHR1 estivum) , OsPHR2  sativa) , and FvPa vesca) , were rei stress . PreviouGmPHT1;1 . Furtherodiester . Howeverficiency . By analnditions , and thenditions ,66. Malus mandshurica (Maxim.) Kom (M. mandshurica), MSDZ 109, grown in Weining County, Guizhou Province, China  was used as the material, and the seeds were collected during maturity. The seeds were generally stratified at 0−4 °C for 45 days until germination, and then cultured with sterile vegetative soil for 3 months. Selected seedlings with a uniform growth trend were hydroponically experimented on according to the modified Hoagland solution [3 6.0 × 10−3; NH4NO3 0.5 × 10−3; MgSO4 2.0 × 10−3; C10H12N2NaFeO.153 × 10−3; KI 0.5 × 10−5; H3BO3 0.1 × 10−3; MnSO4 0.148 × 10−3; ZnSO4 0.534 × 10−4; NaMoO4 0.103 × 10−5; CuSO4 0.157 × 10−6; COCl2 0.253 × 10−6; Ca(NO3)2 5.759 × 10−3. Before treatment, the MSDZ was pretreated for 3 days with 1/2 of the basic nutrient solution, then treated with low phosphorus . Based on the previous work, 32 µmol/L KH2PO4 (LP) and 2000 µmol/L KH2PO4 (CK) were used as the stress and controls, respectively. The seedlings were cultured in a black plastic culture pot with 2000 mL of complete nutrient solution added to each pot and supported by a punched black plastic plate. A total of 50 seedlings were employed to carry out each treatment with three replications. The cultivation conditions were 400−500 µmol·m−2·s−1 (12 h·d−1), 28 °C day and night, and 60–75% relative humidity. The nutrient solution was renewed every 3 days and adjusted to pH 6.5 ± 0.1 with 0.1 mol·L−1 NaOH or 0.1 mol·L−1 HCl. We rinsed the nutrient pot with deionized water before each change of nutrient solution. The tissues (leaves and roots) of low−Pi treatment  and control  were collected on the 10 DAF, 20 DAF, and 30 DAF of M. mandshurica, with some of them morphologically examined and physiological indicators measured. Others were frozen in liquid nitrogen right away and held at −80 °C until RNA extraction.The apple rootstock of solution . Namely:2O2, which was measured at 240 nm according to the method described by Goth [M. mandshurica, and the extract volume was 1:5−10. The leaves were harvested and homogenized in liquid nitrogen, and enzymes were extracted by extraction buffer, Subsequently, placed on ice for testing after the extraction was centrifuged at 8000× g for 10 min at 4 °C. The products were determined by an auto−microplate reader . All were determined using kits, which were purchased from Suzhou Comin Biotechnology . The experimental operation was carried out in strict accordance with the product instructions. The photosynthesis rate (Pn) of new fully expanded leaves of M. mandshurica, low−Pi stress on 10 DAF, 20 DAF, and 30 DAF, was quantified using a portable photosynthesis open system  [−2 s−1; leaf temperature 26 ± 2 °C; and relative humidity 60–75%.Following the approach, 15 seedlings were chosen for each treatment, and WinRHIZO root analysis software was used to analyze the root morphological traits after washing with water. The plant height of the above−root sections was measured. The fresh weight of the line was weighed, then dried at 80 °C for 30 min after being heated at 105 °C for 30 min. The dry weight was weighed and statistically assessed. Furthermore, to determine the SOD activity (EC 1.15.1.1) was quantified by monitoring the inhibition of photochemical reduction of nitro−blue tetrazolium (NBT) at 560 nm according to the method described . The CAT by Goth . The POD by Goth . The ACP by Goth . Proline by Goth . In a nuin, USA) . The meaM. mandshurica seedlings with or without low−Pi treatment on the 10 DAF, 20 DAF, and 30 DAF were harvested and ground in liquid nitrogen. The extract volume was 1:5−10, and enzymes were extracted by extraction buffer. Subsequently, they were placed on ice for testing after the extraction was centrifuged at 10,000× g for 10 min. The products were determined by an auto−microplate reader . The content of inorganic phosphorus can be calculated by measuring the light absorption, and then the calculate the total phosphorus content in the leaves. The total phosphorus content of leaves was assayed by a determination kit . The experimental operation was carried out in strict accordance with the product instructions. The total phosphorus content was measured at 660 nm according to the described by the molybdenum blue colorimetry method . BrieflyM. Mandshurica were collected, and the total RNA from leaves and roots at duration treatment was extracted using the TRIZOL kit according to the use method of the manufacturer . The mRNA library was established and sequenced using 36 samples  that were mixed from three biological replicates each. A Nano Drop and an Agilent 2100 bioanalyzer were used to qualify and quantify total RNA . The library was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The final library was amplified with phi29  to make DNA nanoballs (DNBs) that had more than 300 copies of one molecular. DNBs were loaded into the patterned nanoarray and single−end 50−base reads were generated on the BGISEQ500 platform .In total, 36 samples (two conditions (LP and CK) × three stages  × two tissues (leaves and roots) × three biological replicates) of https://en.wikipedia.org/wiki/Hypergeometric distribution (accessed on 6 March 2020)) based on the Hypergeometric test performed GO (http://www.geneontology.org/ (accessed on 24 May 2020)) and KEGG (https://www.kegg.jp/ (accessed on 10 June 2020)) enrichment analysis of annotated distinct expressed genes to obtain insight into the alteration of phenotype. Filter the sequencing data with SOAPnuke (v1.5.2)  and map −∆Ct method. All validations were performed in three biological and technical replicates.To verify the reliability of RNA−seq results, PowerUp SYBR Green Master Mix  was used for qRT−PCR verification. Sixteen differentially expressed genes induced by low−Pi treatment were randomly selected from the treated roots and leaves for experimental validation . Total Rp < 0.1). The Tukey’s test was performed using the SPSS 21.0 statistical software package. All data are represented by at least three repeated means and a standard deviation (SD). All graphs are used in the drawing software Origin 9.0.The parameters were analyzed by variance analysis for statistical testing, and the mean comparison was performed by the Duncan test (MmCAT1, MmSOD1, MmPOD21, MmPHT1;5, MmPHO1, MmPAP1, etc., were also obtained since their expression status varied among the exposure times, which probably notifies the candidates involved in low−Pi−responsive tolerance in this line. Simultaneously, among the TFs, MmWRKY6, MmWRKY9, MmMYB53, and MmPHR2 were up−regulated in the line. (M. mandshurica to low−Pi stress is inextricably linked to its inherent molecular regulatory system. It shows that the response of M. mandshurica to low−Pi stress is a complex procedure and these results provide a broader and better understanding of the Pi response of potential candidate genes. The findings reported herein increase our understanding of the molecular characteristics of M. mandshurica response to low−Pi treatment, which contributes to the identification and application of excellent apple rootstock germplasm with low−Pi environmental adaptation.In conclusion, dramatic morphological, physiological, and transcriptomic changes occurred during exposure to low−Pi stress. The morphological and physiological profiles obviously increased, e.g., total root length, surface area, etc. and the enzymatic activities of CAT, SOD, POD, etc. were in excellent accordance with the enriched pathways of photosynthesis, plant hormone signal transduction and MAPK signaling pathway from the transcriptomic data. Additionally, several enzyme-related candidate genes and low−Pi−responsive genes, e.g., he line. . The tol


These results are consistent with the findings of earlier studies on other genetic features . Therefore, our observations suggest that the genomic changes in the mutants generated by gamma irradiation occurred at the same loci as the mutations in the natural soybean population. These findings are indicative of the existence of mutation hotspots, or the acceleration of genome evolution in response to high doses of radiation. Moreover, this study demonstrated that the integration of GBS and GWAS to investigate a mutant population derived from gamma irradiation is suitable for dissecting the molecular basis of complex traits in soybeans.In this study, we performed a genotyping-by-sequencing analysis and a genome-wide association study of a soybean mutant diversity pool previously constructed by gamma irradiation. A GWAS was conducted to detect significant associations between 37,249 SNPs, 11 agronomic traits, and 6 phytochemical traits. In the merged data set, 66 SNPs on 13 chromosomes were highly associated (FDR  Glycine max L.), which is one of the most important agricultural crops worldwide, is a source of proteins, oils, carbohydrates, lipids, and essential minerals [Soybean .Mutations are sudden genetic changes in the DNA of living cells that are not caused by genetic segregation or genetic recombination. Mutations induced by ionizing radiation range from simple base substitutions to single- and double-strand DNA breaks . Howeverneration . Mutationeration . SpecifiTMS5 of the T98S rice mutant irradiated with gamma radiation can convert cytosine (C) to adenine (A) in cds.71 to identify the mutation and act as a mutation hotspot. In addition, Xiong et al. [Mutation is a common phenomenon in organisms, and all gene sites are not equally mutable. The points that mutate at a higher frequency are often called “mutation hotspots”  and are g et al.  reportedA single nucleotide polymorphism (SNP), which is a genetic variation at a specific nucleotide position of a genomic sequence among individuals, is usually caused by natural mutations and stresses  . AdditioAdvances in next-generation sequencing technologies have aided the development of SNP detection methods. For example, GBS has recently emerged as a promising technique for simultaneously identifying SNPs and genotyping highly diverse species with large genomes . This meA genome-wide association study (GWAS), which is a powerful tool for identifying significant marker–trait associations, involves the detection of causative allelic variations at individual SNP markers related to a natural phenotypic variation. To conduct a GWAS, a large number of markers is required to provide adequate coverage of the entire genome. According to one estimate, tens of thousands of markers are necessary for the soybean genome . Recent In a previous study, we constructed a mutant diversity pool (MDP) from 7000 gamma-irradiated soybean seeds and evaluated the genetic similarity among 208 soybean mutant lines using target region amplification polymorphism (TRAP) markers . In the The GBS library constructed from 192 soybean MDP lines was sequenced using the Illumina HiSeq 2500 platform, which yielded approximately 940 million reads, with a mean quality score of 34.65 . The demFinally, 37,673 SNPs were selected after applying the following filtering parameters: minimum depth = 5, minimum genotype quality = 20, and max-missing = 0.6 . The average depth and average genome-wide transition/transversion ratio after filtering were approximately 22X and 1.6, respectively. The number of SNP transitions ranged from 16 (HK-32) to 12,456 (BS-25), whereas the number of SNP transversions varied from 6 (HK-32) to 7979 (BS-74) . The SNPA UPGMA-based dendrogram was constructed to clarify the genetic relationships among the 192 soybean MDP lines. At a genetic distance of 0.092, the 8 wild-type cultivars and their mutants were divided into approximately 6 major groups . Group 1K values ranging from 2 to 15 and the entire panel of high-quality SNPs. The estimated marginal likelihood was highest for K = 8. Each accession was assigned to one or more groups, depending on whether or not its genotype indicated it was admixed. The results of this analysis were consistent with the dendrogram topology. As denoted by different colors, the main membership composition of the eight wild-type lines in the corresponding groups was as follows: wild-type KAS523-7 in Group 1 was 100% orange, BS in Group 2 was 87% orange, 94Seori in Group 3 was 62% red, PD and KAS360-22 in Group 4 were 92% and 43% red, respectively, HK in Group 5 was 89% blue, and DB and DP in Group 6 were 78% and 100% green, respectively. When the dendrogram and the results of the population structure analysis were considered together, 96% of the 192 mutant lines  were grouped with the corresponding wild-type line.The population structure of the 192 soybean MDP lines, based on the genotypes acquired in this study, was analyzed using fastSTRUCTURE. Specifically, the population structure was assessed using The data for five qualitative  and six quantitative  agronomic traits, as well as six phytochemical traits  for the 192 soybean MDP lines were obtained from earlier studies ,41.p < 0.05) with the following four agronomic traits: DF (33 SNPs), FC (16 SNPs), NN (6 SNPs), and SCC (11 SNPs) (p = 2.20 × 10−10). One SMTA was detected on chromosomes 1 (p = 8.22 × 10−5), 7 (p = 0.00011), 11 (p = 3.58 × 10−6), 12 (p = 7.24 × 10−5), 15 (p = 2.50 × 10−5), and 20 (p = 1.52 × 10−5). Two SMTAs were detected on chromosomes 2 (p = 8.55 × 10−6) and 13 (p = 4.30 × 10−5), whereas three were detected on chromosome 19 (p = 5.62 × 10−6) (p < 0.05) with FC. Fourteen major associations were detected on chromosome 13 (p = 1.02 × 10−10). One SMTA was detected on chromosomes 5 (p = 1.64 × 10−5) and 12 (p = 2.51 × 10−5) (p = 2.37 × 10−7). One SMTA was detected on chromosome 9 (p = 1.40 × 10−5). The analysis also confirmed the presence of major SMTAs on chromosome 19  (p = 2.43 × 10−6), whereas three were detected on chromosome 20 (p = 5.92 × 10−6). We confirmed the presence of major SMTAs on chromosome 8  (p ≥ 0.05)  . The ass × 10−6) a. A tota × 10−5) b. The as × 10−7) c. Our an × 10−7) d. In con ≥ 0.05) . Althoug ≥ 0.05) .Glyma.06g198100, Glyma.06g204600, Glyma.06g205600 (RGP3 and RGP), Glyma.06g205900 (GAUT11), Glyma.06g208300 (TET11), and Glyma.06g211600. All of the SNPs were located in genic regions, including four and three nonsynonymous and synonymous SNPs, respectively. The locus most highly associated with DF, Chr06_19,461,588  in Glyma.06g205600, was revealed to contain a nonsynonymous SNP. Specifically, compared with the wild-type sequence, T was replaced by C, which resulted in an amino acid change from Val (GUA) to Ala (GCA). For FC, the 13 detected SNPs were located in 13 different genes, of which the following 12 were on chromosome 13: Glyma.13g070400, Glyma.13g070800, Glyma.13g070900 , Glyma.13g071400 (ATHSP22.0), Glyma.13g072000 (SHT), Glyma.13g072600, Glyma.13g073400 (MYB33 and ATMYB33), Glyma.13g073500, Glyma.13g076300, Glyma.13g076800 (EIN3 and AtEIN3), Glyma.13g078500, and Glyma.13g078800  in Glyma.13g073400, contained a nonsynonymous SNP; the C-to-T change caused the amino acid to change from Ser (UCG) to Leu (UUG). The analysis of the SNPs in the candidate genes for NN indicated that all six SNPs were in genic regions . For SCC, nine candidate genes were identified, among which the following five included six major SNPs and were detected on chromosome 8: Glyma.08g124900 (ZKT), Glyma.08g126500, Glyma.08g136600, Glyma.08g247500, and Glyma.08g249910 (RGP2 and ATRGP2). These SNPs were mainly located in genic regions .To identify candidate genes for DF, FC, NN, and SCC, the genes that were highly associated with SNPs  were ana3g078800 . The locIn this study, we identified SNPs in 192 soybean MDP lines using GBS data. Approximately 980 million reads were obtained, with 30.6 (Gm01) to 49.6 (Gm13 and 18) SNPs per Mb (average of 38.5) . In an eA previous examination of genetic diversity on the basis of 16 TRAP markers divided 208 soybean mutants into four groups . Additiop < 0.05) with DF (33 SNPs), FC (16 SNPs), NN (6 SNPs), and SCC (11 SNPs). We confirmed the existence of a major SMTA for DF on chromosome 6  (p = 7.08 × 10−8) on chromosome 6. Several studies of natural soybean populations [p = 1.02 × 10−10) (p = 2.37 × 10−7) (p = 5.89 × 10−36) on the same chromosome determined by Fang et al. [p = 1.16 × 10−7) (p = 1.20 × 10−17) on this chromosome identified during an earlier GWAS analysis of a natural population [p < 0.05 in seven agronomic and six phytochemical traits  at extremely low doses under natural conditions [TMS5 may be a mutation hotspot in the thermosensitive genic male sterile rice line T98S (induced by 300 Gy gamma irradiation). On the basis of transcriptome sequencing data, Xiong et al. [dm1–dm4). Although all four of these mutants were derived from the winter cultivar Jing411, two were the result of a 250 Gy gamma irradiation (dm1 and dm2) and two were generated following a 1–1.5% EMS treatment (dm3 and dm4). In the current study, we compared the wild-type and mutant lines to detect the presence of 66 SNPs highly associated with specific traits in the mutants. Of the 20 SNPs associated with DF , 19 were detected in all 15 PD mutants and in 39–49 of the 53 DP mutants. Similar results were obtained for the SNPs associated with FC, NN, and SCC. More specifically, all 5 BS mutants had 12 SNPs associated with FC; 29–50 of 60 DB mutants had 14 SNPs associated with FC; 12–15 of 15 PD mutants had 7 SNPs associated with FC; 7–13 of 53 DP mutants had 14 SNPs associated with FC; 1–2 of 4 94Seori mutants had 6 SNPs associated with FC; 25–29 of the DB mutants had 5 SNPs associated with NN; 6–7 of the PD mutants had 5 SNPs associated with NN; 34–35 of the DP mutants had 2 SNPs associated with NN; all BS mutants had 1 SNP associated with SCC; 2–10 of the DB mutants had 6 SNPs associated with SCC; and 1–6 of the DP mutants had 3 SNPs associated with SCC. However, in the HK mutants, the genomic regions corresponding to the 66 SNPs were relatively unaffected by the gamma irradiation, with only 4–5 of 45 mutants with 3 SNPs associated with FC, and only 1 mutant with 1 SNP associated with SCC. Accordingly, our findings may reflect the existence of mutation hotspots. These mutated loci might exhibit accelerated genome evolution in response to high gamma-ray doses. Indeed, all of the M1 seeds of the MDP lines were irradiated with 250 Gy gamma rays using the 60Co gamma irradiator when the soybean MDP was constructed [In this study, we identified candidate genes and validated our GBS approach. A genome-wide association analysis was performed for 11 agronomic traits  and 6 phytochemical traits . A total of 66 SNPs on 13 chromosomes were highly associated FDR × 10−10) a. In a Gulations ,31,32,45× 10−10) b. This r with DF  SNPs, FC× 10−10) . We also × 10−7) c. This Sg et al.  in a pre × 10−7) d, which pulation . Anotherpulation  also detl traits . There a with DF  SNPs, FCnditions ,52. Manynditions  suggeste−10 a. Instructed .Glyma.06g205600  on chromosome 6 was identified as a candidate gene for DF and was predicted to be highly correlated with the flowering time (AtRGP2); the flowering time of the transgenic lines was 1.5- to 2-times longer than that of the wild-type control. Moreover, the transgenic plants were stunted, with a rosette-like growth pattern, and their source leaves exhibited severe chlorosis, increased photoassimilate retention, and starch accumulation, which resulted in increased fresh and dry leaf weights. Thus, we speculate that the protein encoded by the candidate gene Glyma.06g205600 modulates the flowering time because it adversely affects the pollen structure. Ambawat et al. [Glyma.13g073400  associated with FC encodes MYB33. In Arabidopsis, AtMYB33 influences responses to hormones [In the present study, ing time . The prot et al. ,58. Anthhormones , as wellGenome-wide association studies are useful for identifying genetic loci related to traits of interest . The det12 generation were phenotypically assessed for the following 11 agronomic traits: growth type (GT), flower color (FC), seed coat color (SCC), seed hilum color (SHC), and stem anthocyanin (SA) ; and days of flowering (DF), maturity days (MD), seed index (SI), plant height (PH), node number (NN), and ramification number (RN)   used in this study were derived from 2 soybean landraces (KAS523-7 and KAS360-22) and 6 representative Korean soybean cultivars  from an earlier study . The 192m et al. ). The fom et al. ).Genomic DNA was extracted from the leaves of each mutant line using the DNeasy 96 Plant kit . After quantifying the extracted DNA using the NanoDrop ND-1000 spectrophotometer , the DNA concentrations were adjusted to 50–100 ng/µL  for the GBS analysis.ApeKI . Bar-coded adapters were then ligated to the digested fragments. The 192 barcode sequences (4–8 nucleotides long) used for tagging the samples are listed in A GBS approach was used for SNP genotyping. Specifically, a GBS library was prepared by digesting the DNA of individual soybean plants with K = 2… 15. This program has been widely used to calculate posterior inference on the basis of the Bayesian framework [Genetic relationships were investigated by constructing a UPGMA-based dendrogram using TASSEL software  . The popramework . A phyloTo ensure sufficient evaluation of the genetic diversity of the 192 soybean MDP lines, 17 phenotypic traits, comprising 5 qualitative traits , 6 quantitative traits , and 6 phytochemical traits  were included in the GWAS analysis. After filtering SNPs with <10% missing data, 37,249 SNPs were selected and used for the GWAS, which was performed using a compressed mixed linear model . All anaIn summary, we previously constructed an MDP core collection that reflects the diversity among soybean lines in terms of agronomic traits. We focused on identifying loci that were identical to the genetic loci in existing natural populations. We revealed substantial variations in 17 soybean agronomic traits, including 6 phytochemical traits, and demonstrated the utility of GWAS for detecting genetic factors underlying important agronomic traits. The genetic basis of these traits was then dissected in an association analysis using 37,249 SNPs obtained by GBS. A total of 66 SNPs on 13 chromosomes were determined to be highly associated with the examined traits. All of the major SNP markers for four agronomic traits  were detected at the same genomic loci in the mutants and the natural populations. The soybean MDP core collection is a useful resource for future research on soybean genetic diversity, as well as soybean mutation breeding. Furthermore, the SNPs described herein may be applicable as new markers in future soybean studies.


Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management. Gambierdiscus, identified for the first time as G. toxicus in the Gambier Archipelago (French Polynesia) PbTx-3) or fluorescent (Bodipy®-PbTx2) compounds [Gambierdiscus matrix, several parameters were evaluated before the quantification of G. polynesiensis toxic samples.The RBA is based on the competition for site 5 of VGSCs between CTXs and a brevetoxin (PbTx) labelled with radioactive , methanol (MeOH), or dimethylsulfoxide (DMSO), can be used by up to 40% without inducing any unspecific effect .G. polynesiensis TB92 sample extract at 4 °C for 90 min versus 37 °C for 30 min  obtained under the two incubation conditions did not show any significant difference between the two tested temperatures and incubation durations. In most RBA-based studies, the binding reaction usually occurs at a temperature of 4 °C from 60 to 120 min for the detection of both VGSC activators, i.e., CTXs and PbTxs [G. polynesiensis, so this protocol was further applied in the following experiments. Of note, McCall, et al. [The effective concentration at 50% , four strains that were previously found to be negative by CBA-N2a (data not shown) were extracted using the same chemical protocol  and testell mL−1 . This va studies ,88.The Pacific ciguatoxin CTX3C showed a response typical of the mode of action of VGSC activators, illustrated by a sigmoidal dose–response curve with a negative slope .80, EC50, and EC20 values (n = 7) with coefficients of variation (CV) ranging from 7.6 to 14.5 % with good repeatability  of CTX3C was three-fold higher than those previously reported using the fRBA [20 and EC80 were comparable to several previous studies . However, despite the different origins of CTX3C (ILM versus Wako), incubation conditions, and labelling of PbTx-2 or PbTx-3, our results were consistent with rRBA results that were recently published by Díaz-Asencio, et al. [80, EC50, and EC20 values of CTX3C (after their conversion into ng ml−1 unit)  . In our q cell−1 , which aG. polynesiensis, competitive binding curves with a negative slope were obtained below the MCE from the least toxic to the most toxic sample, i.e., RG92-b and NHA4-b, respectively , RIK7 (n = 12), RAI1 (n = 8), and NHA4 (n = 7), regardless of the ages of the cultures and culture conditions  ATPase pump blocker ouabain (O), together with the sodium-channel activator veratridine (V), which induces permanent activation of the VGSCs without cytotoxicity (non-destructive OV treatment) [80 and EC50 values of CTX3C were estimated at 0.63 ± 0.05 and 1.5 ± 0.23 pg mL−1 (n = 6) with CVs of 8.2 and 15.6%, respectively, showing overall good repeatability (As CTXs have no direct cytotoxic effect on neuroblastoma (N2a) cells, their detection requires the addition of the sodium/potassium (Naeatment) . When CTeatment) . This speatment) . The EC8tability , as in V80 and EC50 values were consistent with those previously reported for CTX3C in some studies [Gambierdiscus matrix were two-fold higher than previous studies  for all 30 samples, from the lowest RG92-a and RG92-b to the highest NHA4-g toxic samples . In thisnditions ,115.50 values ranged from 0.15 ± 0.04 to 2.28 ± 0.54 cell mL−1, with CV varying from 12 to 30%, except for the NHA4-d sample, for which a CV of 38% was observed , RIK7 (n = 12), RAI1 (n = 8), and NHA4 (n = 7), regardless of culture ages and culturing conditions (−1), followed by those of RIK7 and RAI1, i.e., 2.29 ± 0.86 versus 2.96 ± 0.83 pg CTX3C eq cell−1, with NHA4 displaying the highest mean value of 5.69 ± 2.08 pg CTX3C eq cell−1  and analyzing their fragmentation pathways. The Multiple Reaction Monitoring (MRM) used in this study is a sensitive and specific technique that can selectively quantify compounds within a complex matrix. Its principle resides in (i) the selection of a charged ion corresponding to the molecule of interest (parent or precursor ion), (ii) the fragmentation of this parent ion to produce a range of daughter ions, and (iii) the selection of one or more of these daughter ions for quantification. Using LC-MS/MS, the LOD and LOQ values for CTX3C were determined with the ordinary least-squares regression data method [−1 (n = 3) according to method 2, as described in Sibat, et al. [The LC-MS/MS is a chemical method for detecting, identifying and quantifying molecules of interest by measuring their mass-to-charge ratio  that has been previously separated by liquid chromatography in a sample. The ionic abundance is represented by a chromatographic peak with a retention time and a peak area . RegardiG. polynesiensis samples was mainly composed of CTX3B and CTX3C, ranging from 56 to 91% of the total CTX content, with CTX3B as the dominant congener compared to CTX3C, as previously highlighted by Longo et al. [The CTX profile of all o et al. . Nonethe−1 with CV ranging from 0% to 14%, showing good repeatability (−1), followed by RIK7 (3.67 ± 1.38 pg CTX3C eq cell−1) and RAI1 (4.17 ± 1.08 pg CTX3C eq cell−1), with NHA4 strain displaying the highest concentration (7.04 ± 0.53 pg CTX3C eq cell−1). These results confirmed the hierarchy relative to CTX-production among these strains, as previously outlined by Longo et al. [The CTX contents of the 30 samples varied by 8.17-fold from the lowest to the highest value, i.e., from 1.01 ± 0.14 to 8.25 ± 0.45 pg CTX3C eq celltability . When cotability , signifio et al. ,23. NeveG. polynesiensis as the most toxic Gambierdiscus species described to date, compared to toxicity levels in the range of femtograms reported in other Gambierdiscus species [G. polynesiensis, as also evidenced in other Gambierdiscus species [With CTX levels ranging from 0.68 ± 0.03 to 9.72 ± 1.64 pg per cell estimated by the three methods, our results confirm  species . Our dat species .−1) was also more sensitive than the colorimetric ELISA , the immunosensor and immunoassay  [Regarding the sensitivity of the three methods used, based on the LOD and LOQ values for CTX3C, the fRBA appeared to be from 1.8 to 2.9 times more sensitive than LC-MS/MS . Conversctively) ,112.Gambierdiscus matrix, the difference between the three methods was less important than for the pure toxin CTX3C . These differences in repeatability could be explained, in part, by the fundamental differences related to the principles of these three methods .G. polynesiensis between the three methods, there was a significant difference among the three assays (p < 0.05), although variations in toxicity among samples led to similar means when grouped together across assays , generating n = 90 values of CTX contents. The distribution of all CTX contents obtained with fRBA and LC-MS/MS were similar and differed from that obtained with CBA-N2a (r2 = 0.841) found between the fRBA and LC-MS/MS , but this is likely due to the limited sample size (n = 3 samples). Conversely, significant differences in toxicity estimates were observed between CBA-N2a and fRBA for RIK7, RAI1, and NHA4, while no difference was observed between fRBA and LC-MS/MS for any of the three strains. The CBA-N2a also yielded significant differences in toxicity estimates compared to LC-MS/MS for strains RIK7 and RAI1, as was also found by Longo, et al. [r2 values). In other words, high toxicity samples measure high on all assays, while low toxicity samples measure low , fRBA can detect moderate to high CTX levels (≥100 fg CTX eq cell−1) and is considered as a complementary detection method to CBA-N2a. However, its high requirements regarding the amount of standard material, i.e., 50 ng per experiment for fRBA versus 60 pg and 29 ng for CBA-N2a and LC-MS/MS, respectively, potentially limits the use of fRBA to laboratories with large amounts of CTX standards, as is already the case with rRBA. Similarly, for the lowest toxic G. polynesiensis sample, the highest cell biomass required for one experiment was 20,000 cells by fRBA, versus 100 and 500 cells for CBA-N2a and LC-MS/MS, respectively. The great advantage of both in vitro methods is their high-throughput format allowing for (i) the qualitative screening (presence or absence of composite CTX-like activity) of 120 samples at a time, tested at a single concentration), and (ii) the quantification of CTX contents in 15 positive samples at a time, if tested at a range of eight distinct concentrations.Our results showed the great sensitivity and repeatability of fRBA, CBA-N2a and LC-MS/MS when applied to the detection of CTXs in In algal-based ciguatera risk-monitoring programs, an efficient approach would be to carry out a qualitative screening using CBA-N2a and/or fRBA to quickly discriminate between negative samples and those showing from low to high ciguatoxicity. This approach allows for a rationalization in testing efforts by focusing only on CTX-positive samples in quantitative analyses. With an intermediate analysis time of 23 h for accurate quantification and only 20 min for qualitative analysis, LC-MS/MS is still more specific than fRBA and CBA-N2a because it is able to identify all known analogs of CTXs. However, its main limitation is that it is not able to detect unknown CTX analogs, whereas the composite activity of both known and unknown CTX analogs can be detected by fRBA and CBA-N2a.G. polynesiensis , G. toxicus  and G. pacificus (TKR9) originating from different islands and/or archipelagos of French Polynesia   and dimethylsulfoxide (DMSO) HPLC grade .Chemicals used for extraction, toxins and samples resuspension and dilution were methanol (MeOH), ethanol (EtOH), and dichloromethane (CHG. polynesiensis TB92 strain following purification by high-pressure liquid chromatography (HPLC), as detailed in Chinain, et al. [−1, 20 ng mL−1, and 2 µg mL−1 for fRBA, CBA-N2a and LC-MS/MS, respectively. All toxins were stored at −20 °C. Gilson Microman positive-displacement pipettes  were used to transfer all the solvent solutions used in the assay for greater accuracy.Ciguatoxin CTX3C sourced from the Institut Louis Malardé (ILM) bank of CTX standards and was obtained from , et al. . This toG. polynesiensis, TKR9 G. pacificus and HPT3, RIK31, and TKU5 G. toxicus strains were obtained under routine culturing conditions, as described in [G. polynesiensis strains , samples were cultured under six different conditions, three conditions using nitrate as N source at three distinct pH values , and the three others using urea  as described in Longo et al. [−1, as assessed by LC-MS/MS . Each sample was then stored at −20 °C until further extraction for toxicity screening.Prior to cell harvest, the total cell yield was determined by automated counting using a Multisizer IIITM particle-counter . As described in Longo, et al. , cultureGambierdiscus cells were extracted by adding 20 mL of MeOH directly into the tubes containing each freeze-dried cell sample. Subsequently, tubes were vortexed for 1 min and then placed in an ultrasound bath for 1 h 30. Following a centrifugation step at 2800× g for 10 min, the resulting supernatant was recovered in a 250 mL flask. This extraction step was repeated once in MeOH and once in aqueous methanol (MeOH:H2O 50/50). The three supernatants were further pooled and dried under vacuum using a rotary evaporator , and the resulting crude cellular extract was resuspended in 4 mL of pure MeOH. Half of this solution (2 mL MeOH) was further partitioned between 6 mL of CH2Cl2 and 2 × 3 mL of aqueous MeOH (MeOH: H2O 60/40) according to the procedure previously described in Chinain, et al. [−1 for fRBA, 10,000 cell mL−1 for CBA-N2a, and 100,000 cell mL−1 for LC-MS/MS., et al. , to sepaThe fluorescent receptor binding assay (fRBA) developed in this study was based on previously published protocols ,143,149,®-PbTx-2) was provided by the University of North Carolina at Wilmington (UNCW) [−1). Stock and intermediate (dissolved in assay buffer) solutions were covered with aluminum foil to protect from light. Synaptosomes were prepared following the protocol described by Dechraoui, et al. [−1 of proteins and stored at −80 °C for use in subsequent assays.The fRBA assay buffer was composed of 50 mM HEPES , 130 mM choline chloride , 5.4 mM potassium chloride , 1.7 mM magnesium sulfate , 5.5 mM glucose , 6.1 mM ethylene glycol . For 1 L of assay buffer, 200 μL of protease inhibitor cocktail was added . Then, the buffer was brought to pH 7.4 with Trizma base  and stored at 4 °C. For each experiment, a complete assay buffer was made containing 100 mg of serum bovine albumin  and one drop of Tween-20 detergent  added to 100 mL, and mixed at room temperature for 15–20 min. The fluorescent brevetoxin (BODIPYn (UNCW)  and diss, et al.  from fro, et al. . Then, t®-PbTx-2 mixture was prepared containing 230 µL of complete assay buffer and 50 µL of 10 nM Bodipy®-PbTx-2 per well and a synaptosome mixture containing 150 µL of complete assay buffer and 50 µL of 1 mg mL−1 of synaptosomes per well. These two mixtures were stored at 4 °C and protected from light, particularly for the mix containing the Bodipy fluorochrome. An eight-point serial 1:2 dilution of CTX3C and each Gambierdiscus dry extract were prepared (v = 100 µL per concentration) using a U-bottom 96-well microtiter and 20 µL of each concentration of pure toxin or Gambierdiscus extract were directly added in wells. All reagents were added to 96-deepwell microplates  in the following order: 280 µL of Bodipy®-PbTx-2 mix , 20 µL of CTX3C or Gambierdiscus extracts and 200 µL of synaptosomes mix  to reach a final volume of 500 µL. To avoid early binding of one of the competitors towards the sodium channel receptors, synaptosomes were added at the end and all reagents were homogenized using multichannel pipettes. Then, the 96-deepwell microplates were sealed with 96-well plate film and incubated in the dark. Before filtration, 250 µL of complete assay buffer was added to each well of a Pall Acroprep™ Advance 350 1 µm glass fiber plates (96 well format) previously placed on a Pall® multi-well plate manifold vacuum apparatus and then aspirated through the filter under vacuum. After a drying step [n = 36) were subtracted from each well to account for any background fluorescence. The mean RFU value measured from the control wells (n = 8 in each experiment) was composed of Bodipy®-PbTx-2 + synaptosomes + assay buffer only and served as total binding reference (100%). Net fluorescence data were fitted to a sigmoidal dose-response curve (variable slope) based on the four-parameter logistic model (4PL) using GraphPad Prism v9.1.0 software . From the sigmoidal curves, the effective concentrations inducing 80% and 50% of binding were determined as EC80 and EC50 for CTX3C, respectively, and expressed in pg mL−1. The EC50 of Gambierdiscus samples were expressed in cell mL−1.First, a Bodipying step , 2 × 240To evaluate if the presence of solvent may interfere in the response of the fRBA toward CTXs, eight-point serial 1:2 dilutions of EtOH, MeOH, and DMSO were tested. As described in G. polynesiensis TB92 strain was used with final concentrations ranging from 0.02 to 3 µg mL−1 of dry extracts corresponding to 16 to 2000 cell mL−1, with each concentration tested in duplicate wells for four independent experiments.In the initial protocols ,143,149,Gambierdiscus dry extract (MCE) that does not induce unspecific binding was established using G. pacificus (TKR9) and G. toxicus  previously found negative by CBA-N2a (data not shown). These strains were extracted using the same chemical protocol [−1 for HPT3, RIK31, TKR9 and TKU5, respectively, corresponding to 500 to 64,000 cell mL−1 for HPT3, RIK31, and TKU5, and to 1000 to 128,000 cell mL−1 for TKR9, and incubated for 30 min at 37 °C.The maximum concentration of protocol . Eight-pGambierdiscus extracts were estimated using the following Equations (1) and (2):The limit of detection (LOD) and the limit of quantification (LOQ) of fRBA applied to −1.Both the LOD and LOQ were expressed in fg CTX3C eq cellGambierdiscus sample. This was estimated by comparing the EC50 value of CTX3C with the EC50 value of each tested Gambierdiscus sample and was further calculated using the following Equation (3): The CTX content is the concentration of CTXs in each −1.The CTX content was expressed in pg CTX3C equivalents (eq) cellG. polynesiensis samples were selected from the study of Longo, et al. [−1 as evaluated by LC-MS/MS from crude extracts , the culture medium was replaced by 200 µL of 2% fetal bovine serum RPMI-1640 supplemented medium containing a non-destructive treatment of ouabain-veratridine (OV+) at a final concentration of 90/9 µM for all the internal wells. These treated N2a cells in OV+ conditions were exposed to a serial dilution 1:2 of eight concentrations of CTX3C or DSFs of G. polynesiensis samples. For the dry extracts of Gambierdiscus samples, the maximum concentration of dry extract (MCE) that does not induce unspecific mortalities in N2a cells was established at 9500 pg µL−1 corresponding to 1904 cell mL−1 [−1 and from 0.02 to 2.97 and from 0.18 to 23.81 cell mL−1, from the most toxic to the least potent sample, respectively. Each concentration was tested in triplicate wells per plate. Following an overnight incubation period, the viability of N2a cells was assessed using the MTT assay [50 values  in cell mL−1 using Prism v9.1.0 software . The LOD and LOQ values of CBA-N2a and toxin content of Gambierdiscus samples were calculated using Equations (1)–(3). Fifteen samples were tested at the same time in parallel with CTX3C in three independent assays on different days, and the same was carried out for the 15 remaining samples. At the end, each sample was tested in n = 3 independent experiments and CTX3C in n = 2 × 3 = 6 independent experiments.The CBA-N2a experiments were conducted following the procedure described previously . The mouell mL−1 . Final cTT assay . The absG. polynesiensis for the P-CTX group based on the protocol method 2 by Sibat, et al. [In this study, one LC-MS/MS acquisition method was used to determine the toxin profile of the strains of , et al. . A brief® electrospray ionization source (ESI). The instrument control, data processing, and analysis were conducted using Analyst software 1.7.2 .Experiments were performed using a UHPLC system  coupled to a hybrid triple quadrupole-linear ion-trap API4000 QTRAP mass spectrometer  equipped with a TurboV−1, the injection volume was 5 μL, and the column temperature 40 °C. The elution gradient was as follows: 78% B to 88% B from 0 to 10 min, hold at 88% B for 4 min, decrease from 88% to 78% in 1 min and hold for 5 min at 78% B. The optimized ESI+ parameters were set as follows: curtain gas at 25 psi, ion spray at 5500 V, turbo gas temperature at 300 °C, gas 1 and 2 were set at 40 and 60 psi, respectively, and an entrance potential at 10 V.A linear gradient using water as eluent A and MeOH as eluent B, with both eluents containing 2 mM ammonium formate and 50 mM formic acid, was run on a Zorbax Eclipse Plus C18 column, 50 × 2.1 mm, 1.8 μm, 95 Å . The flow rate was 0.4 mL min−1 in triplicates in three independent experiments (n = 3). In addition, a mix of P-CTX standards (ILM) were injected in each experiment to obtain a reference for the retention times. The MCE of Gambierdiscus matrix was established at 100,000 cell mL−1 and G. polynesiensis samples were tested at a concentration of 20,000 cell mL−1 in three independent experiments.Mass spectrometric detection was performed in positive ionization mode using the scheduled Multiple Reaction Monitoring (MRM) algorithm . This alGambierdiscus sample were calculated against the CTX3C calibration curve and expressed in the equivalent CTX3C. The sum of the estimated concentrations of all CTX analogs allowed for an estimation of the total CTX content (expressed in pg CTX3C eq cell−1). All 30 Gambierdiscus samples were analyzed in three independent experiments on different days (n = 3).Quantitation of the different P-CTX analogs was performed using CTX3C (ILM) as a reference standard. Assuming an equal molar response and applying the same LOD and LOQ, the estimated concentrations of each CTX analog in 50 values generated at different temperatures and incubation times were analyzed using a paired t-test . To compare strain differences using individual assays, one-way ANOVAs were conducted with Tukey’s multiple comparisons posthoc analysis (GraphPad Prism v.7.05).To compare fRBA incubation conditions, EC50 values and CTX contents obtained from three independent experiments on different days of each sample tested by each assay.Descriptive statistics were performed to calculate the mean ± standard deviation (SD) of ECThe coefficient of variation (CV) is defined as the ratio of the standard deviation to the mean and calculated according to the following Equation (4):The CV  is expressed in percentages (%) and is a statistical measure of the dispersion of data points around the mean, used to express the precision and repeatability of an assay.r2) (GraphPad Prism v.7.05). To assess if the assays yielded significantly different estimates of toxicity, repeated measures ANOVAs were performed on individual sample with Tukey’s multiple comparisons posthoc analysis (GraphPad Prism v.7.05). Comparisons were considered significant if a p-value of less than 0.05 was obtained.The distribution of CTX content for each assay was graphed using the histogram analysis in GraphPad Prism v.7.05. To determine the degree to which the different assays were correlated with each other, Pearson’s correlation analyses were performed to assess the correlation coefficient  the lack of a reference detection method duly validated at an international level, (ii) the lack of or poor availability of commercial or certified CTX standards, as well as biological reference materials (positive and negative samples), and (iii) the absence of clearly defined regulatory thresholds ,151. At


Sheep milk (SM) is an alternate dairy source, which despite many similarities, has both compositional and structural differences in lipids compared to cow milk (CM). Studies are yet to examine the apparent digestibility of SM lipids, relative to CM, and the potential impact on the plasma lipidome.To determine the response of the circulatory lipidome to equal volume servings of SM and CM, in females who avoid dairy products.n = 30; 24.4 ± 1.1 years) drank SM or CM  following an overnight fast. Blood samples were collected at fasting and at regular intervals over 4 h after milk consumption. The plasma lipidome was analyzed by LC-MS and fatty acids were quantified by GC-FID.In a double-blinded, randomized, cross-over trial, self-described dairy avoiding females (p = 0.003), well before any postprandial lipemic response. This was despite greater quantities provided by SM. However, there were notable differences in the postprandial fatty acid response, with SM leading to an increase in short- and medium-chain fatty acids (MCFAs)  and several long-chain fatty acids (LCFAs) . This corresponded to a greater postprandial response for medium chain triglycerides (MCTs) C10:0, including TG(10:0/14:0/18:1), TG(16:0/10:0/12:0), and TG(16:0/10:0/14:0) (interaction time × milk p < 0.05).The overall postprandial triglyceride (TG) response was similar between SM and CM. TG concentrations were comparable at fasting for both groups, however they were higher after CM consumption at 30 min (interaction milk × time Despite a higher fat content, SM ingestion resulted in a greater circulating abundance of MCTs, without increasing total postprandial triglyceride response, when compared to CM. The greater abundance and postprandial appearance of MCTs may provide advantageous metabolic responses in children and adults.https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=375324.U1111-1209-7768;  Avoidance of dairy products is evident globally , 2, wher2 response in dairy-avoiding females, as reported previously (sn-1/3 positions (48.3–67.3 vs. 12.8–47.1%) of triglycerides (TGs) in SM compared to CM (in vitro model (in vitro study such as the specificity of human digestive enzymes and the impact of protein in fat metabolism.Sheep (ovine) milk (SM), is an alternate ruminant milk which maintains many of the key nutritional features of CM. However, there are differences between the two milk types: for example SM proteins are more digestible than CM proteins, and provides a greater quantity of branched-chain amino acids (BCAAs) with a corresponding elevation of the postprandial circulating BCAA response . Despiteeviously . Compareeviously –19. Addied to CM , 21. Thero model , 22. TheThe aim of this study was therefore to compare, on a “portion-for portion” basis, the appearance of circulating fatty acids following ingestion of SM or CM, and further to compare the appearance of other lipid species  in circulation. Spray dried powdered milk was selected as a shelf stable milk suitable for both SM and CM. To improve the generalizability of the results, reconstituted SM powder from two different producers and commercially available reconstituted CM powder were used as the test drinks in this study.We hypothesized that consistent with the compositional differences, concentrations of circulating SCFAs and MCFAs would be greater following the ingestion of SM compared to CM.The double-blinded, cross over randomized controlled trial was conducted at the Liggins Institute, The University of Auckland between July and November 2018. The primary outcome of the study has been reported by Milan et al. . Seconda2 were recruited using digital and printed advertisements. Two subjects withdrew prior to the completion of the study and were excluded from further analyses. All participants self-reported dairy avoidance. Subjects with known dairy allergy, current or history of gastrointestinal, cardiovascular, or metabolic disease, consuming medications expected to interfere with normal digestive and metabolic processes like proton pump inhibitors, antibiotics, or prebiotics (3 months prior to the study) were not eligible. Participants with pre-existing cardiovascular diseases or self-reported alcohol intake >28 units per week were also excluded.A total of 32 healthy young women aged 20–40 years with BMI 18–28 kg/mwww.randomizer.org. Both participants and investigators were blinded to the treatment identity and allocation was implemented through sealed envelopes. One day prior to the visits, subjects were advised to avoid vigorous physical exercise, dairy and fiber rich food, and were provided with a standardized low fat, low dietary fiber dinner after which they were to remain fasted from 10.00 p.m. Upon arrival the following morning, fasting blood samples were collected. Subjects then consumed 650 mL of milk within 10 min. Following milk ingestion, blood samples were collected every 30 min until 2 h and hourly thereafter until 4 h.Eligible participants were randomized to consume 650 mL of either SM or CM test drinks, which were made from reconstituted whole milk powders, on two occasions at least 1 week apart. Milk test drinks were prepared from milk powder the evening prior to the visit using heated (30°C) filtered water, shaken vigorously, then stored over night at 4°C. Pre-weighed portions of sheep or cow milk powder  were reconstituted in 585 mL water to provide a total solid content equivalent to the respective lipid SM or CM. The reconstituted milk was served chilled. Randomization sequences were computer generated using Venous blood samples were collected in EDTA containing vacutainers , and plasma was removed after centrifugation at 2,000×g for 15 min at 4 °C and frozen at -80°C prior to analyses.Plasma cholesterol, triglycerides, high-density lipoprotein-cholesterol and low-density lipoprotein-cholesterol levels at baseline were measured using a Cobas c311 clinical chemistry analyzer .Whole SM powder was provided by Blue River Dairy Ltd. (batch no. F2125/HC08) and Spring Sheep Milk Co. (batch no. MAN: NOV17-JAN18) and these were blended 1:1 prior to weighing and reconstitution for both consumption and all subsequent milk analysis. Whole CM powder was commercially sourced from NZMP . Milk powders were stored at -20°C prior to use.The volume of 650 mL was selected to align with previous studies investigating milk digestion dynamics , to provThe proximate compositions per 100 mL of the sheep and cow milk test drinks are listed in sn-1 or sn-2 positions are more highly abundant in SM, and the same is true for DGs containing C4:0, C8:0, C:10, and C12:0  and unsaturated FAs such as C18:1 t9, C18:1 t11, and C18:2n6 were also higher in SM than CM. In contrast, C14:1 c9 (myristoleic acid), C16:0 , C16:1 c9 , and C18:3n3 (α-linolenic acid) were found in higher proportions in CM.In general, sheep milk contained higher concentrations of all fatty acids than cow milk, which is not surprising given the higher fat content of sheep milk . In addiiso-octane (>99%) were purchased from VWR Chemicals BDH . 2-Propanol (>99%), hexane (>99%), sodium methoxide (>95%) and glacial acetic acid (>90%) were purchased from Sigma-Aldrich .Sodium acetate trihydrate  was purchased from Merck KGaA . Sulphosalicylic acid (>99%), chloroform (>99%), methanol (>99%), d4, propionic acid-d3, iso-butyric acid-d7, butyric acid-d7 and 13C-labeled isovaleric acid), and internal standard of C23:0 methyl ester were purchased from Supelco . Internal standard for lipidomics (16:0 d31–18:1-PE) was purchased from Avanti .The Carboxen/PDMS solid-phase microextraction fibers, the internal standard mix (containing acetic acid-FAs (C2:0–C24:0) in milk samples were analyzed as follows: Cow or sheep milk powders  were reconstituted in 90 mL MilliQ water. Reconstituted milk (2.0 mL) was mixed with 2-propanol (4.0 mL), and hexane (3.0 mL) was then added. After shaking vigorously, the hexane layer was transferred to a new tube and evaporated to dryness. The residue was re-suspended in hexane (4.0 mL). Sodium methoxide in methanol  was added and the sample was vortexed for 5 s, repeating 4–5 times over 10 min. Glacial acetic acid (5 μL) was added to neutralize the sodium methoxide. The sample was dried by adding anhydrous calcium chloride to ~1 cm depth, and was then incubated at room temperature for 60 min. The top layer was collected and transferred to 1.5 mL GC vials and stored at 4 °C until analysis.Fatty acid analysis was performed using a Shimadzu GC2030 gas chromatograph (GC) equipped with a flame ionization detector (FID). Fatty acids were separated on a Restek RTX 2,330 column . The column oven was held at 75 °C for 5 min and was then increased to 175 °C at a rate of 15 °C/min and held for 17 min. Thereafter the temperature was increased to 250 °C at a rate of 5 °C/min and held for 6 min. The carrier gas was hydrogen with a linear velocity of 50 cm/s. The injection volume was 1 μL, with a split ratio of 60:1. The injector temperature was 260 °C and the detector temperature was 265 °C.FAs in milk samples were reported as mg/mL and as a percentage of total FAs after peak areas were corrected for detector response using theoretical FID response factors. The equations for generating the response and conversion factors to quantify individual fatty acids from the FAMEs were obtained from American Oil Chemists' Society .3:MeOH = 1:1, v/v) and stored at -20°C for 60 min. MilliQ water (400 μL) was added, and the samples were centrifuged at 13,663×g for 10 min at 4 °C. The lower, organic layer (300 μL) was transferred to a centrifuge tube and evaporated to dryness under a stream of nitrogen at room temperature. Lipid residues were reconstituted in 300 μL of modified Folch solution  and centrifuged . Supernatant (20 μL) from each sample were pooled to form a quality control (QC). The remaining supernatant was used for lipid liquid chromatography-mass spectrometry (LC-MS) analysis. LC-MS analysis of lipids, raw data processing, lipid annotation and lipid identification were performed using a previously published method  was mixed with 800 μL of pre-chilled extraction solvent (CHCld method . Lipidom2 (ddMS2) scans were collected in profile data acquisition mode. For full scan mode, a mass resolution setting of 35,000 was set to record a mass range of m/z 200–2,000 with a maximum trap fill time of 250 ms. In ddMS2, MS2 measurements are activated when a set peak intensity threshold is achieved. For ddMS2 scan mode, the same mass resolution setting was maintained with a maximum trap fill time of 120 ms. The isolation window of selected MS1 scans was ±1.5 m/z with a normalized collision energy of 30 eV. Samples were run in both positive and negative ionization modes separately. Positive ion mode parameters were as follows: spray voltage, 4.0 kV; capillary temperature, 275 °C; capillary voltage, 90 V; tube lens 120 V. Negative ion mode parameters were as follows: spray voltage, -2.5 kV; capillary temperature, 275 °C; capillary voltage, -90 V; tube lens, -100 V. The nitrogen source gas desolvation settings were the same for both modes (arbitrary units): sheath gas, 40; auxiliary gas, 10; sweep gas, 5. The Xcalibur software package provided by the manufacturer was used to create these settings.Both full and data dependent MS2 (ddMS2). Fragmentation data on 4 samples in total per ionization mode (positive and negative) were used for identification of lipid ions/classes.The sequence of runs comprised blanks (containing no milk or plasma extract), QCs and samples in that order. To verify and maintain data quality, the QC sample was injected once every 10 samples. Retention time, signal intensity, and mass error of the internal standard were constantly monitored during the runs. For every 10 samples, one sample was selected at random for data dependent MSTM was based on comparison of the ddMS2 data obtained from select samples with in silico calculations/database. To ensure correct identification of lipid species by the LipidSearchTM software, one lipid species from each class was manually identified according to published method  was mixed with 20% sulphosalicylic acid (100 μL) and 100 μL internal standard mix. Samples were frozen overnight and were then centrifuged at 22,000×g for 45 min at 4 °C. Fatty acids were extracted from 300 μL of the supernatant using a Carboxen/PDMS solid-phase microextraction fiber for 5 min at room temperature. The fiber was then thermally desorbed at 280 °C for 5 min.m/z were monitored and used for quantitation.SCFAs and MCFAs were analyzed by gas chromatography-mass spectrometry (GC-MS) using a Shimadzu QP2010 instrument running in selected ion monitoring (SIM) mode on a Supelco Nukol column (30 m × 0.25 mm ID × 0.25 μm film thickness). The column oven was held at 80 °C for 1 min and was then increased to 200 °C at a rate of 75 °C/min and held there for 7 min. The carrier gas was helium with a linear velocity of 40 cm/s. The injection was thermally desorbing the fiber with a split ratio of 20:1, and the injector temperature was 280 °C. Ions were detected in SIM mode with an ion source temperature of 200 °C. The detector voltage was initially 1.3 kV, and was increased to 1.6 kV after 3.52 min. The 3 solution (2.0 mL), and heated at 80 °C for 30 min. The samples were then cooled again, mixed with iso-octane (2.0 mL) for 1 min, then mixed with a saturated NaCl solution (5 mL) and finally centrifuged at 1,500×g for 5 min at room temperature. The iso-octane layer was transferred to glass vials and the remaining aqueous phase was extracted again using a further 1.0 mL of iso-octane. The combined iso-octane phases were evaporated to dryness at 40 °C under a stream of nitrogen. The residue was reconstituted in iso-octane (100 μL).LCFAs in plasma were analyzed as follows: Plasma (800 μL) was mixed with 1.0 mL internal standard (C23:0 methyl ester) and 8.0 mL chloroform:methanol 1:1 and was shaken for 10 min. Samples were then centrifuged at 2,000×g for 5 min at room temperature and the supernatant transferred to new tubes. Chloroform (4.0 mL) and MilliQ water (2.4 mL) were added, and samples were centrifuged at 2,000×g for 5 min at room temperature after which the upper aqueous layer was discarded. The solvent was evaporated from the chloroform phase at 40 °C under a stream of nitrogen. Methanolic NaOH solution  was added, and the samples were heated at 80 °C for 10 min. The samples were then cooled, mixed with methanolic BFLCFAs were analyzed using the same equipment and column as the FA analysis in milk samples described in section 2.5.2. The column oven was held at 175 °C for 20 min and was then increased to 220 °C at a rate of 10 °C/min and held for 12 min. Thereafter the temperature was increased to 260 °C at a rate of 20 °C/min, and finally to 275 °C at a rate of 1 °C/min. The carrier gas was hydrogen with a linear velocity of 50 cm/s. The injection volume was 1 μL, with a split ratio of 30:1. The injector temperature was 300 °C and the detector temperature was 300 °C.FA in plasma are reported as μg/mL. Quantitation of SCFAs and MCFAs was based on a 5-point calibration curve using the calibration curve function of the GCMS Solutions software . The concentration in μg/mL plasma was calculated directly by GCMS Solutions and exported to MS-Excel for data analysis. LCFAs were quantitated after peak areas were corrected for detector response using theoretical FID response factors. The equations for generating the response and conversion factors to quantify individual fatty acids from the FAMEs were obtained from American Oil Chemists' Society . The corrected areas were multiplied by the internal standard (IS) ratio (IS concentration/IS area) to give μg FAME/sample. This value was divided by the sample volume (0.8 mL) to give μg/mL, this was then converted to μg FA/mL plasma by the ratio of the molecular weights of the fatty acid/FAME.−1)   with a pt-test with the null hypothesis that there is no difference between the test drink treatments. The null hypothesis was rejected, and the difference between treatments statistically significant, if P < α and t > tcrit. All outcomes with multiple factors were analyzed by repeated factor generalized linear models with milk and time compared within-subject and adjusted for multiple comparisons using a Sidak Holm adjustment. The Huynh-Feldt correction was used where Mauchly's sphericity test failed. Alpha was set at P < 0.05 for all tests.Single factor comparisons, such as iAUC, were made using Student's paired Heat maps were created using R software version 2.15.2  with gpl2) completed the study. All subjects had lipid profile values within a healthy range , the overall post-meal response was similar between the milks, despite the difference in total fat intake.Postprandial lipemic responses, or total blood triglyceride responses, were different between SM and CM treatments  was greater than in the test drinks themselves. In contrast, the number of TG species in plasma was only about 10% of the species found in the test drinks (37 in plasma compared to 362 in the test drinks).p < 0.05 main time effect), except for TG (18:0/18:1/18:2) (p > 0.05). In general, saturated TGs showed the greatest increase relative to the baseline SM concentrations, followed by the monosaturated TGs, while the polyunsaturated TGs changed the least. Changes in three species containing C10:0  were greater after SM ingestion than CM, while 10 species with LCFAs [e.g., TG(15:0/16:0/16:0), TG(16:0/14:0/14:0), TG(16:0/14:0/16:0), TG(16:0/16:0/16), and TG(18:0/16:0/16:0), interaction time × milk p < 0.05; p < 0.05; p < 0.01; The relative abundance of each lipid is presented as the log fold % change relative to the SM baseline concentrations to illustrate the pattern of response across all lipid species over time. As most differences dependent on milk type were in the meal-derived lipids, TGs, DGs, MGs, and ZyE are presented in p = 0.043; p = 0.038; p > 0.05; Of the other lipid classes, only two were significantly different in circulation following ingestion of the milks. SphM(d34:1) was more abundant 1 h after SM ingestion  and FAs with low abundance, e.g., C5:0 and C7:0 (p > 0.05).Most measured fatty acids (FAs) were responsive to milk ingestion . These fatty acids also showed a greater iAUC with SM than CM, aside from C20:0  and is likely due to the relative low abundance of this FA, combined with differences observed at baseline, which impacts the iAUC calculations. Iso C5:0, anteiso C15:0, and iso C16:0 were more abundant in circulation after SM than CM, although this was not dependent on time  was greater after CM digestion compared to SM. Of these, only C14:1 c9 showed a similarly higher iAUC with CM  have been demonstrated for ingested quantities of 15 vs. 30 g of fat, but this relationship does not continue for intakes >30 g . Howeversn-1/-3 positions are more abundant in SM compared to CM according to our previous in vitro work (sn-) of fatty acids in the TG backbone, the postprandial plasma response demonstrated greater abundances of C10:0-containing TGs, including TG(10:0/14:0/18:1), TG(16:0/10:0/12:0), and TG(16:0/10:0/14:0), after SM ingestion. However, many other differences in FAs between milk types were not evident in the TG fraction. For the SCFAs and MCFAs, this may be reflective of the rapid absorption and subsequent mitochondrial β-oxidation in the liver  . SM conttro work . Whilst he liver , 35. Thihe liver , 37. It he liver .sn-1/-2 position are more abundant in SM (CM has a greater abundance of some LCFAs (C14:0–C18:0), and there were some notable differences in the LCFA species between the milks . It has nt in SM . In genent in SM . This want in SM  and ~120nt in SM . howeverThe lipidomic analysis performed in this study also included other major classes of lipid species, including PLs and SphM, which have important signaling and regulatory roles, particularly within cellular membranes . Milk PLTrans fatty acids (TFAs) are a class of unsaturated fatty acids in which one or more double bonds are present in the so-called trans configuration. The most common origin of TFAs in the Western diet is industrially generated (predominately C18:1t9), produced with the partial hydrogenation of vegetable oils (t11 (trans-vaccenic acid) being the most abundant in milk (t11 (trans-vaccenic acid) was found in the SM (p < 0.01) concentration at 240 min   concentration at 240 min  is a term referring to a family containing many different FAs with 18 carbons and two conjugated double bonds . The mose system . In the n the SM . This in 240 min  and 1.89 240 min . WhetherOur study included a small cohort of healthy women aged 20–40 years, and the results may not be generalizable to men, people of a different age or those with different metabolic health status. The subjects were also those who self-reported dairy avoidance, with the possibility their digestion may be altered relative to the wider community. Analysis was performed on a fixed volume of milk, made from reconstituted powder. The benefit of MCFAs provided by SM needs to be further studied. Given the inherent differences in lipid composition, the sheep milk in this study resulted in a greater quantity of lipids ingested. To fully elucidate the differences in lipid digestibility, metabolism and lipidomic response, studies are required that compare equal quantities of total lipids. However, such a study is not reflective of how foods are consumed .This study provides a highly comprehensive determination of the lipidome with 415 species detected in SM and CM, and 131 species in blood plasma. The importance of lipidomic analysis, beyond the insights that are gained with measurement of FA species alone is detailed data on the relative distribution of FAs into the differing biological active lipid compartments including TGs, PLs and SphM. As highlighted in this study, there is extensive SM metabolism, with the composition of the ingested beverage only partially relating to the circulating SM lipidome.t11, c9, t11-CLA, and C20:0) compared to CM. These differences are not only a result of compositional differences between SM and CM, but also attributed to the inherent differences in FA positioning and the lipid digestibility. The greater uptake of MCFAs may make SM a potential fast energy source for consumers seeking CM alternatives, especially for those with higher energy requirements such as infants or athletes. In addition, the greater availability and uptake of potentially biologically beneficial lipids including C18:1 t11 and c9, t11-CLA with SM warrant further investigation into the role of SM in human health.In conclusion, the ingestion of SM and CM results in different circulatory lipidome responses when compared “portion-for-portion.” Although providing a ~1.6-fold higher fat content, SM does not result in a greater circulating increase of total postprandial triglyceride compared to CM. SM results in a greater circulating increase in three TG species containing C10:0, SCFAs and MCFAs  and several long-chain fatty acids (LCFAs) (i.e., C18:1 The original contributions presented in the study are included in the article/The study involving human participants was reviewed and approved by Central Health and Disability Ethics Committees, Ministry of Health, Ethics Department, 133 Molesworth Street Wellington 6145 New Zealand. The patients/participants provided their written informed consent to participate in this study.FT conducted research, analyzed data, performed statistical analyses, and wrote the first draft of the paper. LS designed research, managed the project, performed statistical analyses, and wrote the paper. AM designed and conducted research, performed statistical analyses, and wrote the paper. ASu analyzed lipids in milk and blood samples. MA analyzed fatty acids in milk and blood samples. ASh conducted research, analyzed data, and performed statistical analyses. DC-S designed the research and reviewed the paper. LD designed research, reviewed the paper, and had primary responsibility for the final content. All authors approved the final version of the manuscript before submission.


Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne pathogen capable of causing severe, invasive illness (listeriosis). Three serotypes, 1/2a, 1/2b, and 4b, are leading contributors to human listeriosis, with 4b including the major hypervirulent clones. The multiplex PCR scheme developed by Doumith and collaborators employs primers targeting specific lineages  or serotypes . The Doumith scheme (DS) is extensively employed for molecular serotyping of L. monocytogenes due to its high accuracy, relative ease, and affordability. However, for certain strains, the DS serotype designations are in conflict with those relying on antibody-based schemes or whole-genome sequence (WGS) analysis. In the current study, all 27 tested serotype 4b strains with sequence type 782 (ST782) within the hypervirulent clonal complex 2 (CC2) were designated 1/2b/3b using the DS. These strains lacked the serotype 4b-specific gene LMOf2365_1900, while retaining LMOf2365_2059, which, together with prs, yields the DS 1/2b/3b profile. Furthermore, 15 serotype 1/2a strains of four STs, mostly from water, were designated 1/2b/3b using the DS. These strains lacked the lmo0737 cassette but harbored genomic islands with LMOf2365_2059, thus yielding the DS 1/2b/3b profile. Lastly, we investigated a novel, dual 1/2a-1/2b profile obtained using the DS with 21 serotype 1/2a strains of four STs harboring both the lmo0737 cassette and genomic islands with LMOf2365_2059. The findings suggest that for certain strains and clones of L. monocytogenes the DS designations should be viewed with caution and complemented with alternative tools, e.g., traditional serotyping or WGS analysis.IMPORTANCEListeria monocytogenes is a foodborne pathogen responsible for severe illness (listeriosis), especially in pregnant women and their fetuses, immunocompromised individuals, and the elderly. Three serotypes, 1/2a, 1/2b, and 4b, account for most human listeriosis, with certain serotype 4b clonal complexes (CCs) overrepresented in human disease. Serotyping remains extensively employed in Listeria epidemiologic investigations, and a multiplex PCR-based serotyping scheme is widely used. However, the PCR gene targets can be lost or gained via horizontal gene transfer, leading to novel PCR profiles without known serotype designations or to incorrect serotype assignments. Thus, an entire serotype 4b clone of the hypervirulent CC2 would be misidentified as serotype 1/2b, and several strains of serotype 1/2a would be identified as serotype 1/2b. Such challenges are especially common in novel clones from underexplored habitats, e.g., wildlife and surface water. The findings suggest caution in application of molecular serotyping, while highlighting Listeria’s diversity and potential for horizontal gene transfer. Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne bacterial pathogen notorious for its persistence in food-processing facilities. It is the causative agent of human listeriosis, typically via the consumption of ready-to-eat (RTE) processed foods  1, 2, 4, and 6 , with most cases of human listeriosis involving lineages I and II. Serotypes 1/2b, 3b and most strains of serotype 4b are members of lineage I, while serotypes 1/2a, 1/2c, 3a, and 3c belong to lineage II , found in all L. monocytogenes strains, as well as genes associated with specific lineages and serotypes (lmo0737 (lineage II); LMOf2365_2059 ; LMOf2365_1900 ; and lmo0118, harbored by the lineage II serotypes 1/2c and 3c (https://www.ncbi.nlm.nih.gov/pmc/) as of 21 September 2022. The recent emergence of whole-genome sequencing (WGS) has allowed the in silico serotyping of L. monocytogenes strains based on core genome multilocus sequence typing (cgMLST) allele clustering  harbors lmo0118, thus yielding the DS profile 1/2c/3c , which is shared by all serotypes, serotypes 1/2b and 4b share the lineage I-specific target LMOf2365_2059 (ORF2819), while 4b strains harbor an additional serotype 4b-specific gene, LMOf2365_1900 (ORF2110)  than that of the surrounding genes (39.2%) in the reference serotype 4b strain F2365 , its GC content suggests that it was acquired by serotype 4b strains via HGT and subsequently eliminated from ST782. In silico analysis of 11 additional ST782 strains from the Listeria database at the Centers for Disease Control and Prevention (CDC), including 9 human clinical strains and 1 each from food and water, revealed that all lacked LMOf2365_1900 and would thus yield the serotype 1/2b/3b designation using the DS .ST782 belongs to the hypervirulent CC2 (serotype 4b); the ST782 strains that we examined were derived from human listeriosis and surface water . All tesORF2110) . Howeverr origin . LMOf236in F2365 . Thus, en = 14) strains of ST1055 (n = 3), ST1082 (n = 1), ST1492 (n = 3), and ST1494 (n = 7) yielded the serotype 1/2b/3b designations using the DS  remaining, while the flanking genes remained intact  having GC values similar to the L. monocytogenes chromosomal average of approximately 38%  was much lower  and F2365 .Several  or lineage I, e.g., strains F2365 and PNUSAL002394 (both of serotype 4b) or INV971911 (serotype 1/2b)  .L. monocytogenes database at the CDC and the Institut Pasteur’s BIGSdb (https://bigsdb.pasteur.fr/listeria/) for additional strains with the same STs. No additional ST1492 strains could be identified, suggesting that this novel ST remains uncommon. However, we identified two additional strains of ST1055 (one each from fresh produce and blood), as well as a blood-derived strain of ST1494 and 28 strains of ST1082, mostly from food and environmental swabs (Table S1). The additional ST1055 and ST1494 strains (Table S1) lacked lmo0737, while harboring LMOf2365_2059, suggesting that this trait may be a clonal feature of these two STs. On the other hand, in silico screening of the additional ST1082 strains indicated that they would yield the 1/2a/3a DS designation (Table S1). Thus, the findings for the ST1082 strain in our panel  provided strong evidence that the dual 1/2a-1/2b profile observed in ST1503 was clone specific. All other ST1503 strains identified through interrogation of the CDC and Pasteur Institute databases are serotype 1/2a but would yield the 1/2a-1/2b profile using the DS (Table S1). These strains were relatively recently isolated from human clinical samples  as well as from the environment (soil) (Table S1). On the other hand, analysis of the additional strains of ST912, ST124, and ST1365 from the CDC database revealed that they would yield the serotype 1/2a/3a designation using the DS (Table S1). Thus, the dual 1/2a-1/2b profile for the ST912, ST1365, and ST124 strains in our panel  may be uLMOf2365_2059 into serotype 1/2a genomes appears to be recent, with the oldest strain being from 2014, this gene appears to have disseminated to multiple, unrelated serotype 1/2a STs from clinical, water, soil, and wildlife sources  or surface water. It is conceivable that L. monocytogenes community composition and environmental conditions in these previously unexplored habitats may promote such HGT events or confer fitness advantages to the resulting strains.It is noteworthy that while the movement of LMOf2365_2059 genomic region revealed a hypervariable mobile genetic element identified previously (orfA) , a S-adenosylmethionine (SAM)-dependent methyltransferase, and LMOf2365_2059 . However, they also harbored several additional genes, namely, a glyoxalase family protein and two resolvase/integrase bin proteins  . The flat not 4b . Analysiproteins . Overallproteins , highligLMOf2365_1900, LMOf2365_2059, and the lmo0734-lmo0739 cassette has large impacts on L. monocytogenes isolate classifications, it is also important to consider the functional outcomes of the gain or loss of these genes. For instance, the lmo0734-lmo0739 cassette is essential for d-allose metabolism in lineage II strains, and strains of lineage II can grow with d-allose as the sole carbon source, while strains of lineages I or III lack this capacity , was implicated in growth at low pH, high osmolarity, and high temperatures  and, as discussed above, is on a diverse genomic island harboring a multitude of genes, including a SAM-dependent methyltransferase and a glyoxalase family protein  remain undersurveilled for L. monocytogenes. As such environments become more extensively sampled, we may identify more strains with novel DS profiles or with profiles that may disagree with the actual serotype of the strains. Findings from a recent study of numerous  L. monocytogenes strains from the Salinas River watershed  confirm this expectation. For instance, strains of CC842 , ST2135, and ST2137 harbor prs and the serotype 4b-specific LMOf2365_1900; they would yield a novel profile in the DS but belong to a lineage II clade based on WGS phylogeny  in urban and suburban regions of the southeastern United States, as previously described  were used for L. monocytogenes serotyping. This agglutination-based method was performed according to the manufacturer’s instructions. For ELISA, strains were passaged three times on BHI plates containing 0.3% agar and grown at 30°C to enrich for flagellum-producing cells. For O-antigen assessment, cultures were grown in BHI broth overnight with shaking (100 to 120 rpm) at 37°C, while for H-antigen assessment, they were grown in BHI broth overnight statically at 30°C. The cultures were all centrifuged  for 5 min. The cell pellets for the O-antigen preparations were resuspended in equal volumes of 0.2% NaCl, autoclaved for 15 min at 121°C, centrifuged again, and resuspended in 0.2% NaCl to an A600 value of 0.1 to 0.3. The cell pellets for the H-antigen preparations were resuspended in equal volumes of 4% (wt/vol) formaldehyde in 0.2% NaCl, incubated at room temperature for 30 min, centrifuged, and resuspended in 0.2% NaCl to an A600 value of 0.1 to 0.2. Cell suspensions (70 μL) were pipetted into ELISA well strips  and dried overnight at 40°C in an oven. ELISA was performed using the Denka-Seika Listeria antiserum kit (part number 294616) and interpreted as described , ORF2110 (LMOf2365_1900), and prs (lmo0199)  and investigation of their chromosomal locations were completed using the Basic Local Alignment Search Tool (BLAST) and Genome Browser functions of the PathoSystems Resource Integration Center (PATRIC) Bioinformatics Resource Center  designations using BIGSdb- Pasteur . Sequence Center .Accession numbers for the WGS data for the strains used in this study are listed in