Fermented foods are part of the staple diet in many different countries and populations and contain various probiotic microorganisms and non-digestible prebiotics. Fermentation is the process of breaking down sugars by bacteria and yeast species; it not only enhances food preservation but can also increase the number of beneficial gut bacteria. Regular consumption of fermented foods has been associated with a variety of health benefits , including improved digestion, enhanced immunity, and greater weight loss, suggesting that fermented foods have the potential to help in the design of effective nutritional therapeutic approaches for obesity. In this article, we provide a comprehensive overview of the health effects of fermented foods and the corresponding mechanisms of action in obesity and obesity-related metabolic abnormalities. Obesity is a highly prevalent disease globally and increases the risk for other chronic conditions like type 2 diabetes mellitus, coronary heart disease, osteoarthritis, sleep apnea, and some types of cancer . Anti-obFermentation has been used for centuries to preserve foods and their texture through deactivating spoilage microorganisms ,9. FermeThis paper aims to provide a comprehensive overview of available evidence from recent clinical and experimental investigations on fermented foods and obesity, focusing on the challenges faced, knowledge gaps, and future perspectives, and the potential molecular mechanisms by which fermented foods affect the gut microbiome and immune responses.Fermented foods function as a relatively optimal drug delivery system as they contain a mixture of safe bioactive compounds in sufficient doses to transfer to the target site, thereby facilitating efficacy and long-term compliance ,16,17. TFermented foods also contain live microorganisms or beneficial bioactive compounds that support the symbiosis between the host and the microbiome, resulting in a healthier gut environment ,21. SCFAThe association between the gut microbiome and the risk of obesity has been known for some time ,25,26. DIntestinal permeability is another factor that may mediate some of the associations between the gut microbiome and obesity. The microbiome interacts with inflammatory signal transduction pathways via the gram-negative bacteria membrane lipopolysaccharide (LPS), which binds to CD-14 and toll-like receptor-4 (TLR4) on enterocytes, resulting in the translocation of bacteria through the intestinal barrier and an inflammatory response ,30,31. RThe bioactive compounds in fermented foods can also exert beneficial effects on adipose tissue function via upregulation of the peroxisome proliferator activator receptor γ 2 (PPARγ2) . Excess Understanding the effects of fermented foods on the composition and function of the microbiome can contribute to better nutritional therapies for long-term body weight homeostasis. Since many individuals with obesity can lose weight but cannot maintain it for prolonged periods of time, finding safe and long-term dietary treatments has been of great interest . FermentSCFAs, including acetate, propionate, and butyrate, are the products of fermentation of dietary non-digestible carbohydrates by gut bacteria. They can inhibit lipid synthesis enzymes, reduce pathogen microorganisms, and supply energy for the intestinal epithelium . SCFAs aThere are various types of traditional fermented foods including dairy products, fruit juice, and foods of mixed composition. Although the molecular mechanisms for the observed clinical outcomes linked to consumption of distinct types of fermented food products are too broad to classify in just a few general categories, the major modes of action are summarized in the following sections and are illustrated in Current evidence suggests some lactic acid bacteria, such as Lactobacillus plantarum, have anti-obesity effects; however, their mode of action is not adequately identified. A mixture of L. plantarum species can reduce fat synthesis and storage, the gene expression of adenosine monophosphate-activated protein kinase-α (AMPK-α), fatty acid synthetase (FAS), acetyl CoA carboxylase (ACC), and peroxisome proliferator-activated receptor-γ (PPAR-γ). At the same time, serum concentrations of total and LDL-cholesterol and triglycerides decrease and HDL-cholesterol increase . Among oPumpkin (Cucurbita moschata) seeds have been used for centuries in the Native American medicinal food culture for the reduction of blood glucose, cholesterol, hypertension, and the treatment of intestinal infections and disorders . FermentConcurrent intake of antioxidants and fermentation metabolites can potentially boost metabolic effects. For instance, fermented gallate-containing green tea inhibits adipocyte differentiation without induction of apoptosis and reduces body weight without any change in calorie intake and hepatic cytotoxicity, suggesting that these effects are not dependent on appetite regulation and liver damage pathways . RegardlAppetite control is a key factor in obesity management. Food intake and total energy expenditure are important for long-term body weight homeostasis and can be regulated by multiple physiological pathways . PeripheFermented dairy products slow gastric emptying, so they can influence postprandial responses like those of blood glucose, insulin, and lipids. Sangaard et al. reported a lower gastric emptying effect of fermented milk A38 due to its higher viscosity. A38 intake resulted in a greater increase but also a faster decrease in serum triglyceride concentration in all lipoprotein fractions. The same trajectory pattern (greater peak but faster decrease) was observed for cholecystokinin (CCK) and peptide YY (PYY), gastric inhibitory polypeptide (GIP), and glucagon-like peptide-1 (GLP-1). Increased GIP levels after A38 consumption can be a result of GIP response to dietary intake of fat in conjunction with slower gastric emptying. The increased postprandial CCK levels can be related to the response to the high content of whey protein. Although satiety hormones were significantly elevated, the subjective satiety sensation  did not change . LikewisAnother interesting study evaluated the role of fermented food as a compound delivery system. The researchers investigated whether emulsified lipid Fabuless (Olibra) can reduce dietary energy intake and improve weight management. The findings revealed a non-significant effect of Olibra, separately or with solid food, on postprandial satiety sensation. On the other hand, Olibra plus yogurt was effective in reducing appetite. The anorectic effect of Olibra was enhanced with yogurt consumption by a longer transit time from the mouth to the large intestine . SCFAs are the commonest metabolites of fermented foods, and they have an appetite-suppressing effect ,91. The + T cells, and Th1 cells [Excessive accumulation of white adipose tissue demonstrates a dysregulated pattern of secretion of adipokines associated with abnormal inflammatory cytokine profile and chronic inflammation in obesity ,95. The h1 cells . A chronh1 cells ,98.9 CFU/day) increased the number of immune-regulatory leukocytes like CD4+ T lymphocytes, CD4+ CD25+ Treg cells, and decreased pro-inflammatory leukocytes including CD8+ T lymphocytes, CD11b+ activated leukocytes, and F4/80+ macrophages during weight reduction [LAB strains separated from fermented dairy products have anti-inflammatory and anti-obesity effects. A mixture of LAB strains from Mozzarella di Bufala Campana (1 × 10eduction .8 CFU/day) for 60 days elevates IgA+ cells in the intestine and reduces body weight. The IgA+ cells secrete immunoglobulin A which plays a crucial role in maintaining the efficiency of the epithelial barrier. In addition, increased levels of IL-10 could suppress inflammation in the intestinal epithelium [Supplementation with probiotic yogurt containing L. delbrueckii subsp. bulgaricus Streptococcus thermophilus and probiotic Bifidobacterium animalis , another strain isolated from Chinese fermented foods, has a potential regulatory role in glucose and cholesterol metabolism in obesity and metabolic dysfunction. It reduces serum glucose and total cholesterol concentrations and downregulates pro-inflammatory genes including CD36, Reg3γ, TLR2, and PPAR-α [Sichuan pickles are a traditional food in China that is fermented by lactic, acetic, and ethanol pathways, thereby leading to variable characteristics and chemical features of vegetable pickles. The most abundant metabolites in sichuan are volatile organic products. The concentration of acid, ester, aldehyde, and alkenes depends on the bacterial diversity in the sichuan fermentation process . Lactoba markers . Lactobad PPAR-α . TLR2 ged PPAR-α ,100. TheExercise has been proposed to improve glucose tolerance and body fat-free mass; however, increases oxidative stress damage and pro-inflammatory markers in the muscle. Fermented foods in combination with exercise may exert enhanced beneficial effects on calorie intake and body fat mass as well as immune-related myokines compared to separate administration of fermented food or exercise alone ,85. FermThere is also evidence that fermented foods may have a dose-dependent effect in weight control, glucose and lipid homeostasis, and innate immunity. For example, high-dose fermented mixed grains plus digestive enzymes reduce body weight, adiposity, leptin, keratinocyte chemo-attractant (KC), interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and MCP-1 when compared to low doses .The beneficial role of fermented foods is not limited to the probiotics in these products. The modified proteins in the fermented foods can regulate immune responses through inhibition of inflammatory cytokines  in the liver as well as improved gut microbiota and gene expression patterns of adhesion molecules . Citrus limon) peel is a byproduct that has antioxidant and anti-inflammatory characteristics [Fermentation of food byproducts may have both economical and health-related benefits. Citrus species are rich in ascorbic acid, citric acid, phenolics, and several bioactive compounds. Lemon  is a common probiotic in Korean fermented foods and some pickles and improves oral glucose tolerance and insulin tolerance via increased cellular glucose uptake and decreased protein expression of MCP-1 and IGFBP-3, as well as decreased hepatic mRNA expression of IRS2 and AMPK.Vaccinium angustifolium Ait. (Canadian lowbush blueberry) has been used as a traditional treatment for diabetes in Canada. Fermentation with a natural blueberry flora bacteria called Serratia vaccinii enhances the biological activities of antioxidant compounds in the lowbush blueberry, including phenolic compounds, by modification of phenolic chemical structure and production of gallic acid [Several plants that have been used for the treatment of diabetes may exhibit stronger effects after fermentation. Although there are few studies about the anti-obesity effects of fermented medicinal plants, the current evidence is promising. lic acid . Adminislic acid . Since olic acid , the reglic acid .Serratia vaccinii contains catechol, chlorogenic, gallic, and protocatechuic acids that have the greatest effect on glucose-6-phosphatase levels and glucose uptake in hepatic  and skeletal muscle cells (C2C12) [The bioavailability of bioactive compounds greatly influences the effectiveness of treatment with medicinal plants, as the phenolic fraction of fermented blueberry juice with  (C2C12) . ConsideThere are several studies indicating that regular consumption of fermented foods modulates intestinal microflora, but not all studies aimed to elaborate the relevant mechanisms(s) of action in subjects with overweight or obesity. Moreover, few studies have been conducted in human subjects for an adequately long period of time to analyze the abundance and diversity of microbiota and mycobiota and their association with gut-brain axis and appetite control.Consumption of fermented foods can increase Prevotella, Bacteroides, Lactobacillus, Leuconostoc spp., and other beneficial microbial populations in the gut. Fermented foods can also affect the gene expression of enzymes that can regulate metabolic pathways of lipid synthesis and blood pressure in the host, for example, kimchi can enhance levels of Acyl-CoA synthetase long-chain family member 1 (ACSL1) through modulation of gut microbiota that inhibits triglyceride synthesis and enhances fatty acid catabolism. In addition, increased mRNA levels of aminopeptidase N (ANPEP) are inversely associated with hypertension, angiogenesis, and inflammation; this may explain the beneficial role of regular intake of kimchi on lipid profile and blood pressure .Muribaculaceae, and decreased abundance of Akkermansiaceae, Coriobacteriaceae, and Erysipelotrichaceae [Bacillus subtilis reduces Firmicutes/Bacteroidetes and Bacteroides/Prevotella ratios in obesity; in fact the Bacteroides/Prevotella ratio shifts towards values observed in lean subjects [Akkermansia and Faecalibaculum, and reduces intestinal permeability and oxidative stress markers, in line with an improved microbiome profile [The microbial community of fermented foods likely determines their anti-obesity characteristics. Administration of a fermented food may exert significant changes in energy metabolism through modulation of the gut microbial population, including increased abundance of ichaceae . Althougsubjects . Inflamm profile .Proteobacteria, Actinobacteria, and Bacteroidetes, and reduces Firmicutes, Clostridiales, Lachnospiraceae, and Akkermansia. The abundance of various genera of the microbiome is distinct and can be associated with the biological pathways affected by different bioactive compounds. The resistant starch content is high in some types of cereals and fermentation may enhance the beneficial effects of a functional food via reduced abundance of endotoxins and pathogens in the intestine, and changes in peripheral blood metabolites [Firmicutes to Bacteroidetes did not change significantly, the abundance of other intestinal bacterial groups such as Akkermansia and Bacteroides and SCFAs levels were augmented [Firmicutes to Bacteroidetes with obesity markers was strong [Some grain-like cereals are rich in dietary fiber, protein, and minerals and have low digestibility . Fermentabolites ,114. In ugmented . The admugmented  and the s strong . Not onls strong .Firmicutes/Bacteroidetes and Bacteroides/Prevotella ratios but can also be related to other bacterial phyla like Lachnospiraceae and Ruminococcaceae, which can be affected by Lactobacillus plantarum HAC01 (HAC01) and L. rhamnosus GG (LGG) intake from fermented foods like kimchi. Despite Lachnospiraceae being a member of the Firmicutes family, a higher abundance ratio has been associated with the anti-obesity effects of kimchi [The mode of anti-obesity action of fermented foods is not only related to f kimchi . Streptococcus genus. Intake of either fermented dairy by Lactobacillus helveticus or Greek yogurt increases abundance of Streptococcus [Another example of altered bacterial phyla in the intestine is the tococcus . Fermenttococcus . Candida spp. and Saccharomyces spp [saccharomyces boulardii, the molecular mechanisms are not well studied, and more research is needed to better understand the underlying biological mechanisms. In addition, it not completely clear whether the health promoting effects of fermented foods originate from the bioactive compounds, such as dairy peptides, or the microbial content of fresh fermented food. The reason behind the beneficial roles of fermented foods through the intestinal microbiome can be related to some unknown molecular mediators that lead to anti-obesity outcomes. Few studies investigated the effect of fermented foods on the intestinal mycobiota as well as microbiota. For example, kefir administration to C57BL/6 mice for 12 weeks markedly augmented not only LAB, but also yces spp . AlthougBacteroidetes:Firmicutes is a common marker to compare gut microbiota in response to lifestyle intervention in individuals with obesity, but there is no fixed value for this ratio that can be used to evaluate the effect of an intervention or differences between groups. A solution to reduce inter-individual variation can be the classification of participants according to the genetic background, dietary history, antibiotic use, etc. [Several factors, including genetic susceptibility and environmental exposure to different chemical, biological, and socio-economic factors, can influence the magnitude of response to any dietary intervention. The baseline characteristics of participants have sometimes been used to distinguish between healthy and unhealthy groups, but this may not be a reliable way of classification of inter-individual variation . The relse, etc. . The richness and diversity of microbial phyla can change in response to ingestion of fermented foods or the combination of fermented foods with polyphenols or micronutrients. Although functional microbiology studies report significant changes after dietary interventions, it is not clear how much of this variation occurs normally and how much can be related to the effect of the administration of a specific bioactive compound or food, because the intestinal microbiome is a dynamic population, and several intrinsic and extrinsic factors play a role to maintain the balance among thousands of groups of microbial species.Few studies have investigated the effect of fermented foods on the gut microbial eukaryote. Some concerns have been raised in this regard: the number of biological specimens, for example, feces, may be not sufficient to analyze mycobiota and targeted metagenomics assays function more efficiently in larger samples. Moreover, there is no stable and core library of fungal communities from the human gut, which makes it difficult to interpret relevant data . AnotherThe interventional studies with fermented foods are highly heterogeneous and there are few randomized controlled trials with a specific fermented food. This makes it difficult to pool data in a systematic manner and draw robust conclusions. Still, the heterogeneity of available clinical trials or animal experiments can be helpful to elucidate molecular mechanisms, map the pros and cons of each type of fermented food, and design better experiments for future research in this area . The length of the experiments is also another factor that may be not sufficient to improve clinical outcomes, such as body weight and body fat mass in a 3-month or 6-month period. The primary outcomes in most studies include weight loss, fat mass, and food intake that depend on multiple factors and confounders that are difficult to control in clinical settings.The amount of food consumed can also affect the findings. There are several doses of diverse types of fermented foods, drinks, or gavages that are not justified well and need more homogenous studies and systematic testing to pool data and make better sense of dose-response relationships.Despite most of the experimental studies in animal models of obesity being methodologically sound, the reproducibility and generalizability of the findings have been considered to a limited extent. The precision, high throughput techniques and availability of biopsy tests and various specimens in animal models are undeniable ; howeverp-value post hoc tests and false positive correction tests [Another methodological concern is the sample size calculation of the experimental and clinical studies, particularly when more than two interventions are compared among more than two groups. The power of the study might be reduced due to low sample size, and multiple test errors should be corrected by the proper statistical tests such as adjusted on tests . It must also be noted that fermented foods are not completely safe. Fermentation of high-protein foods can produce biogenic amines that are hazardous for health. Fermented foods including dry-cured meat, fermented fish, and fermented legumes can produce N-nitroso compounds in the body, which have been linked to cancer . The bioAnother unhealthy compound in fermented foods is their salt content. The amount of salt in some traditional fermented foods, particularly Asian soy paste, and cured meat and seafood, is too high and may contribute to carcinogenesis and other adverse health outcomes ,130. RedRegular consumption of fermented foods can exert beneficial effects on body weight regulation and metabolic function through several mechanisms. Although the level of evidence for some types of fermented foods is still very low and there are considerable knowledge gaps regarding the molecular mechanisms of action of specific fermented foods, the current findings are promising. This may be especially relevant if personalized dietary interventions can be designed to include individual variation and consider multiple factors like genetic, immune, and neurological variation. Compliance to treatment is a key factor to obtain optimal results in intervention studies, and since most fermented foods are integral in the traditional cuisine in most countries, their use as a drug delivery system may increase chances of success in the long-term. Finally, analyzing omics data in addition to clinical outcomes can result in a deeper understanding of the mechanisms of action and the links among metabolism, immune responses, and eating behavior.


ST) between groups. All this implies a low population size and strong bottleneck effects, and indicates a long history of clonal propagation and selection during the evolution of the domesticated gene pool of globe artichoke. Moreover, the comparison between molecular and phenotypic population structures suggests that harvest time, plant architecture , leaf spininess, head morphology  together with the number of heads per plant were the main targets of selection during the evolution of the cultivated germplasm. We emphasize our findings in light of the potential exploitation of this collection for association mapping studies.The knowledge of the organization of the domesticated gene pool of crop species is an essential requirement to understand crop evolution, to rationalize conservation programs, and to support practical decisions in plant breeding. Here, we integrate simple sequence repeat (SSR) analysis and phenotypic characterization to investigate a globe artichoke collection that comprises most of the varieties cultivated worldwide. We show that the cultivated gene pool of globe artichoke includes five distinct genetic groups associated with the major phenotypic typologies: Catanesi , Spinosi, Violetti di Toscana, Romaneschi, and Macau. We observed that 17 and 11% of the molecular and phenotypic variance, respectively, is between these groups, while within groups, strong linkage disequilibrium and heterozygote excess are evident. The divergence between groups for quantitative traits correlates with the average broad-sense heritability within the groups. The phenotypic divergence between groups for both qualitative and quantitative traits is strongly and positively correlated with SSR divergence (F The study of the organization of the gene pool of crops is an essential prerequisite for both theoretical and practical reasons. This provides insight into the evolutionary history of a crop, strengthens our knowledge for the implementation of correct conservation strategies, and rationalizes operational choice in plant breeding.Triticum aestivum  is a perennial open-pollinated crop  TheLater, by analyzing leafy cardoon and globe artichoke accessions using SSR and inter-SSR markers, C. cardunculus var. altilis), and wild cardoon (C. cardunculus var. sylvestris), and showed a clear-cut genetic separation between globe artichoke and the cultivated and wild cardoon. When the analysis was repeated assuming three genetic groups, the authors observed the separation of “Catanesi” from all of the other accessions and that the group of green-headed landraces typical of the Apulia region, Italy (Green Apulian), also formed a well-defined group. Furthermore, similarly to More recently, To the best of our knowledge, no studies are available that compare the molecular and phenotypic population structures for the same collection of globe artichokes. Thus, in this study, we characterized a germplasm collection comprising 110 of the most important globe artichoke varieties cultivated worldwide by conducting a deep phenotypic characterization in a common garden and under field conditions. A total of thirty-five quantitative phenological, morphological, and yield traits and 23 qualitative descriptors from the International Union for the Protection of New Varieties of Plants (UPOV) were considered to describe the plants in the stages of the first, secondary, and tertiary heads. Thus, the phenotypic data were compared with the results of SSR analysis, to investigate the relationships between molecular and phenotypic variability.This study advances our understanding of the evolution of globe artichoke, strengthens the knowledge of the organization of its gene pool, and can help in rationalizing the operational choices when applying association mapping and defining breeding strategies in this species.A total of 110 globe artichoke accessions were analyzed using both SSR and quantitative traits, as listed in Nine clones of “Spinoso sardo” selected by AGRIS based on morphological and production traits were also analyzed .Total genomic DNA was extracted from young leaves (three plants per accession) and characterized using 26 genomic-SSR (gSSR) markers targeting loci distributed through all of the 17 chromosomes of globe artichoke . The marChloroplast-SSR (SR) variation was also investigated using 10 primer pairs designed by ad hoc statistics  clustering algorithm implemented in the Structure software . The dattics ΔK;  with thetics ΔK; .adegenet (find.clusters function was used to detect the number of clusters in the collection adopting the K-means clustering method and determining the most likely number of subpopulations based on the Bayesian Information Criterion (BIC). The number of principal components to be retained was determined using the cross-validation function Xval.dapc, based on the root mean square error.To complement the population structure analysis, discriminant analysis of principal components (DAPC) was used as a multivariate unsupervised method implemented in the R package adegenet . DAPC di2T) was partitioned into two components: among-population (σ2a) and within-population (σ2b). The divergence between populations was quantified as FST = σ2a/σ2T, where σ2T = σ2a + σ2b. The partition of molecular variance was accomplished by the AMOVA implemented in Arlequin v. 3.1.5.2 , RST is based on the stepwise mutation model (SMM), i.e., it also considers the variance of SSR allele size , the observed heterozygosity (HO), and the gene diversity . To determine the coefficient of inbreeding (FIS), total variance (σ2T) was partitioned into three components: among populations (σ2a), among individuals within-populations (σ2b), and within individuals (σ2c). The total FIS was calculated as σ2b/(σ2b + σ2c). Group-specific FIS was also calculated. This coefficient compares the expected and observed heterozygosities and the expected value under the Hardy-Weinberg equilibrium is 0. Thus, FIS > 0 indicates heterozygote deficit compared to the Hardy-Weinberg equilibrium (as a consequence of inbreeding), while FIS < 0 indicates heterozygote excess .The number of alleles per locus  and of expected heterozygosity (HE) is expected, with the former that reduces faster than the latter . As na reduces faster than HE, it is expected that, on average, after a bottleneck HE > HEQ. (p < 0.05) were calculated using the Arlequin software . Individual plants were obtained by vegetative propagation using “Phenotypic data were recorded under field conditions, measuring three plants for each variety (single plant = replicate) in a completely randomized design. A total of twenty-one traits were recorded at the beginning of the first head harvest . Among t2T) was decomposed into two components, within SSR groups (σ2W) and between SSR groups (σ2B), applying Nested Analysis of Variance (NANOVA). For each trait, the variance associated with each component was estimated considering a model with groups, varieties within groups, and replicates as random factors and applying the restricted maximum likelihood (REML) method implemented in the JMP software .The total phenotypic variance , it is assumed that the three replicates of each variety have the same genotype. This means that genetic differences among varieties, both within groups (σ2W) and between groups (σ2B), can be partially due to maternal and non-additive effects (dominance and epistasis), rather than to additive effects. Thus, the sum σ2W + σ2B is equal to the total genetic variance (σ2G) and the ratio between genetic variance (σ2G) and total variance (σ2T) represents the broad-sense heritability (h2B); i.e., h2B = σ2G/σ2T, where σ2T = σ2W + σ2B + σ2E, with σ2E representing the environmental variance.Variance components were first used to calculate the heritability of traits. In globe artichoke, cross-fertilization is promoted by protandry , and wheST = σ2B/(σ2B + 2σ2W)  = (1 + FIS)σ2B/[(1 + FIS)σ2B + 2σ2W]. To calculate the QSTs, we also applied this formula by entering the estimated total FIS (see previous paragraph) and assuming that the FIS of a quantitative trait locus is the same as the FIS of the neutral markers. We presented two QST values calculated as in 2B and σ2W are components of the total genetic variance, as they can include both non-additive and maternal effects. To mark this difference compared to Spitze’s QST and Bonnin’s QST(f), we used the notation QST_B and QST(f)_B, where the subscript “B” means “Broad.”To explore the pattern of phenotypic divergence between genetic groups of globe artichoke varieties, variance components were used to calculate the degree of differentiation for quantitative traits between groups by applying the formula Q + 2σ2W) . The QSTa, HE) and divergence (FqltST_) were calculated as for the SSR markers  were determined. The values of FqltST_ were compared with the FST values obtained for SSRs to flag qualitative traits putatively under selection, using the Arlequin software v. 3.5.1.2 (see below).The qualitative descriptors of the UPOV were statistically treated as molecular markers, considering each trait as a single SSR marker and the states of the trait as different alleles. Thus, for each group, statistics of genetic diversity . To exclude possible selection effects on FST values calculated for the SSR loci, the (putatively) neutral “benchmark” FST was calculated by conducting the outlier tests as implemented in Arelquin v. 3.5.1.2 . This test is similar to that implemented in FDIST computer program (p < 0.05). After obtaining a dataset without markers with signatures of divergent selection, the average FST across loci was assumed as the putatively neutral benchmark. The confidence intervals (C.I.) of the putatively neutral FST were determined by bootstrapping over loci .The levels of molecular and quantitative variations were compared following the rationale of the Fapproach . This is program . We ran e) were detected, giving a mean of 5.85. Gene diversity (HE) per locus varied from 0.231 to 0.822 . A total of 152 alleles were observed: two to 13 alleles per locus  were assigned to their group with qi > 0.80 (K = 3) and then a group that included all the Violetti di Toscana and most green-headed types . The highest frequency (∼30%) of admixed individuals (qi < 0.80) was observed within the VTO-GEA group.Based on these data, from here on, we adopted the label CAT-VPR to designate the nuclear SSR genetic group related to the Catanesi-Violetti di Provenza phenotypic typology; similarly, the labels SPI, VTO-GEA, ROM, and MAC indicate the genetic groups associated with the phenotypic typologies Spinosi, Violetti di Toscana-Green et al., Romaneschi, and Macau, respectively. Overall, at ST) between these five groups was moderate, being 0.170 (ST was slightly lower (0.134).CAT-VPR and SPI showed the highest net-nucleotide distance (0.171), while the lowest distances were observed between MAC and ROM (0.084), VTO-GEA and MAC (0.073), and VTO-GEA and ROM (0.071) . According 0.170  while puST, the average divergence among the genetic groups was 0.148  and the RST calculated by randomizing allele size were not different  to the prevalent genetic groups between 0.489 and 0.644. In two of the four cases, the “mismatches” between the two clustering methods involved varieties that were moved between closely related groups. Indeed, Spinoso di Gonnos was assigned to ROM by Structure, while it was assigned to MAC by DAPC; on the contrary, Testa di Ferro was assigned to MAC by Structure, while it was assigned to ROM by DAPC.The correspondence between the Structure and DAPC classifications is reported in Therefore, both the model-based (Structure) and the unsupervised (DAPC) methods agreed when identifying five groups.a)  . The VTO-GEA group showed the highest HE and was significantly more diverse than SPI and CAT-VPR, which showed the lowest HE values; ROM and MAC were in intermediate positions  that indicated an excess of heterozygotes within the five genetic groups. In all cases except MAC, the group-specific FIS were negative and statistically significant (p < 10–5). The FIS value varied significantly between groups . CAT-VPR, SPI, and ROM showed stronger excess of heterozygotes than MAC and VTO-GEA . The three groups with the highest LD values were also those with the lowest FIS   to 56.0% (CAT-VPR), with an average of 41.1% of pairs of loci in LD  .2 test: p < 0.01). The two most divergent nuclear SSR groups  also show alternative cpSSR alleles: CAT-VPR was monomorphic for allele “I,” while SPI was monomorphic for allele “II”  was polymorphic, with two alleles hereafter named “I” and “II.” The “I” allele was the most frequent (0.67) . Howeverele “II” .p < 10–5). The broad-sense heritability (h2B) within groups varied widely depending on the traits considered, from 0.48 (stem diameter) to 0.98 (harvest time)  to 0.77 (VTO-GEA), with ROM (0.60), MAC (0.63), and SPI (0.70) in intermediate positions. These differences between groups were statistically significant based on ANOVA (p < 0.01), while the Tukey-Kramer HD test separated CAT-VPR from the other four groups (p < 0.05).st time) , with anp-values from < 0.01 to < 10–5; ST_B = 0.279), polar/equatorial head-length ratio (QST_B = 0.223), harvest time (QST_B = 0.200), and length of stem (QST_B = 0.183) , diameter of stem (QST_B = 0.011), number of leaves on stem (QST_B = 0.010), and receptacle height (QST_B = 0.014).Based on ANOVA and Tukey-Kramer HD multiple comparison test, 10 out of 21 quantitative traits differed between the five genetic groups (with = 0.183) . Among tp < 10–5) for all the traits examined  and length of the stem , and to a lesser extent, for harvest time . Furthermore, this analysis showed differences in yield performances between the genetic groups, as QST_B were 0.17 and 0.12 for the secondary and tertiary heads, respectively. Among the yield components, QST_B tended to be higher for the average number of heads  than for the average weight of the heads . As expected, when QST_B was calculated considering the observed average negative FIS, the QST values were reduced .At the within-group level, the correlation between HST_B and the average within-groups h2B  and the average heritability within populations (h2B) did not reach statistical significance . Based in QST(f)_B, the traits “plant height” and “head shape” remain statistically significant , i.e., the yield increased with the lateness. Thus, the CAT-VPR yield was 41.7% lower than that of VTO-GEA, the most productive group  varied according to clustering history . Indeed,IS . As the decrease .c, (2.73) and Nei’s diversity index, INei, (0.43), while the lowest INei (0.28) was recorded for CAT-VPR  among the five SSR-derived groups was 0.20. This is close to the estimated value for the SSR markers (FST = 0.17) and quantitative traits (QST_B = 0.18). However, FST_qlt varied widely among the traits  and leaf spininess , followed by bract curvature , spine length  and apex shape .Among the 23 UPOV descriptors applied, 20 were polymorphic throughout the entire collection . Within  CAT-VPR . This coe traits . In fiveing SSRs . The besr = 0.918; n = 10; L95% = 0.685; U95% = 0.981; p = 1.76 × 10–4) (ST_B) and SSR genetic divergence (FST) between the pairs of SSR-derived groups   . This wa= 0.048) .This study shows that the analyzed globe artichoke accessions clustered into two main genetic groups. The first comprises the Catanesi and Violetti di Provenza types and is quite uniform. The second is more complex and includes four additional subgroups related to Spinosi, Green et al., Violetti di Toscana, Romaneschi, and Macau.Based on the phenotypic traits and multivariate analysis, –3 and 10–4, as inferred from genetic data  and the divergence for the quantitative traits (QST_B) and the qualitative traits (FST_qlt).The chloroplast and nuclear genetic polymorphisms as well as the molecular and phenotypic population structures, were associated. This is expected if there are multiple germplasm sources contemporarily differentiated for nuclear, organellar loci, and phenotypic traits. Furthermore, LD was detected within each of the five groups, and the diversity statistics based on SSRs and quantitative and qualitative traits are correlated. These results indicate that the five groups of globe artichoke varieties were genetically relatively isolated, and that the recombination accumulated through crop evolution was overall not sufficiently high (or the time elapsed was too low) to break up the cytonuclear and marker-trait LD. The presence of LD in the whole dataset is also indicated by the correlation between the divergence of the group for SSR markers  or “ovoli” (semi-dormant shoots with a limited root system). Furthermore, our study also shows that some groups have a higher excess of heterozygotes than others and this suggests that different breeding histories and various degrees of historical “clonality” characterize the evolution of the five identified genetic groups.Within the groups, in addition to LD, we observed negative Foduction . Howeverulations . These rE, low h2B) were shorter and produced fewer and smaller heads  than groups with low heterozygotes . This suggests that selection against deleterious mutations might be less effective in groups with high clonality (and high heterozygote excess) than in those with low clonality (with low or null heterozygote excess). Indeed, when there is no recombination, selection against deleterious mutations is less effective, and deleterious mutations have the potential to accumulate   . The accon load) . HoweverST = FST is expected, such that the proportion of total variance allocated among populations  should be equal or similar for both quantitative traits and molecular markers  show different mean values not only because of random demographic processes , but also due to the selection that exerts differential pressure among the populations. On the contrary, the inequality QST < FST suggests uniform selection across populations: populations tend to have the same trait means because the selection has the same direction in all of the populations.Under neutral evolution , Q markers . TherefoST and QST_B obtained among the genetic groups of the globe artichokes identified by the analysis of the population structure. This comparison suggests that some quantitative traits have an “excess” of divergence between groups (QST_B) compared to the putatively neutral markers (FST) (QST_B > FST). For these traits, divergent selection, rather than demographic factors, might be responsible for the phenotypic differences observed between the genetic groups. These traits are harvest time, plant height, head shape, and stem length. Among the qualitative traits, divergent selection can be suggested for leaf spininess, spine length, apex shape, and reflexing of the tip of the outer bracts as they showed an FST_qlt that excedeed the “neutral” FST. In general, it is possible that these traits were under the farmer’s attention when selecting plants for clonal propagation, as they strongly condition both agronomic practice  and consumer preference . However, caution should be exercised when interpreting these data. Indeed, deviations from the Hardy-Weinberg equilibrium might affect the QST–FST comparison. Inbreeding (FIS > 0) can lead to QST < FST under neutrality (instead of QST = FST), which results in low power when detecting divergent selections. On the contrary, when, as in the present case, there is an excess of heterozygotes (FIS < 0), it is possible that under neutrality QST > FST (instead of QST = FST), which leads to a higher risk of false positives  in the sunflower species Helianthus maximilian, which like C. cardunculus var. scolymus belongs to the Asteraceae family, is perennial and is diploid , ceteris paribus, is proportional to h2B , plant architecture , head shape, and spininess (of both leaves and bracts) are suggested as the main targets of selection. The overall covariance between the molecular and phenotypic population structures indicates that genome-wide association mapping strategies must deal with the identified population structure to minimize the risk of false positives.The original contributions presented in this study are included in the article/DR, GA, MR, and EP conceived and designed the experiments. LB, AP, and DS performed the field trial. LB, MR, and GA carried out field data curation. MR carried out DNA extraction. AA and CC performed SSR analysis. DR performed statistical analyzes. DR, EP, and CC wrote the manuscript. All authors contributed to the discussion, revised, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Sudden unexpected death (SUD) is one of the challenging situations encountered in forensic medicine. As a rule, a comprehensive forensic assessment is performed to identify the cause of death in such cases; however, the absence of findings suggestive of a cause, i.e., a negative autopsy, warrants further investigation such as a molecular autopsy. In this review, we aim to highlight the genetic causes of SUD, tools used in a molecular autopsy, and the role of screening in surviving relatives. As per several guidelines, the most preferred samples for DNA extraction are whole blood and fresh frozen tissues. Furthermore, Sanger sequencing and next-generation sequencing are the technologies that are used for genetic analysis; the latter overcomes the former’s drawbacks in terms of cost-effectiveness, time consumption, and the ability to sequence the whole exome. SUD have diverse etiologies; we can generally classify them into cardiac and non-cardiac causes. Regarding cardiac causes, many conditions having an underlying genetic basis are included, such as channelopathies and cardiomyopathies. Regarding non-cardiac causes of SUD, the main etiologies are epilepsy and metabolic disorders. Nevertheless, it has been proposed that there is a genetic overlap between channelopathies, especially long QT syndromes and epilepsy. Additionally, fatty acid oxidation disorders are major metabolic conditions that are caused by certain genetic mutations that can lead to SUD in infancy. Since many SUD causes have an underlying genetic mutation, it is important to understand the genetic variations not only to recognize the cause of death but also to undertake further preventive measures for surviving relatives. In conclusion, a molecular autopsy has a major role in the forensic examination of cases of SUD. Sudden unexpected death (SUD), at any age, is a catastrophic event that results in a significant impact on the grieving family. In the literature, there is considerable variation regarding the definition of the term SUD. According to the International Classification of Diseases, 11th Revision (ICD-11) by the World Health Organization, SUD  is defined as instantaneous death, or a fatal event that is neither traumatic nor explained by a disease, which occurs within 24 hours of symptoms’ onset in an apparently healthy individual . In all SUD is one of the most challenging scenarios encountered by forensic pathologists. It is mandatory to perform an autopsy in all cases of SUD in many jurisdictions. A detailed and comprehensive forensic evaluation should be performed aiming at identifying the cause of death and assisting law enforcement agencies in resolving any suspicions regarding death. Generally, this includes obtaining the medical history, external examination, and gross and microscopic autopsy, followed by advanced forensic investigations such as toxicological analysis, postmortem imaging, and genetic testing -6.In about 65-85% of cases, a macroscopic evaluation is sufficient to determine a conclusive cause of death . CardiacA wide range of conditions may cause SUD with absent or minimal findings at autopsy. For instance, non-cardiac causes include, but are not limited to, epilepsy and metabolic disorders . RegardiSUD, unfortunately, could be the first presentation of an inherited disease . Based oIn this narrative review, we aim to illustrate the importance of molecular autopsy in evaluating SUD cases, highlight postmortem genetic testing in cardiac and non-cardiac-related SUD cases, and elaborate on how this knowledge could aid in SUD prevention in surviving at-risk relatives.Postmortem genetic testing: the molecular autopsyAn appropriate amount of multiple specimens from the decedent’s body at the time of autopsy must be collected in order to proceed with the genetic investigations . Ideally, the preferred samples for genetic analysis are blood in an EDTA tube and fresh frozen tissue from the heart, liver, or spleen -14. TherAccording to the Heart Rhythm Society/European Heart Rhythm Association (HRS/EHRA), in the case of SUD with obscure autopsy findings, performing a comprehensive or targeted four-gene testing for channelopathies may be considered to establish the likely cause of death and subsequently identify at-risk relatives. Targeted genetic testing includes the three genes linked to long QT syndrome (LQTS), namely KCNQ1, KCNH2, and SCN5A, the latter also causing Brugada syndrome (BrS), in addition to the RYR2 gene, which is linked to catecholaminergic polymorphic ventricular tachycardia (CPVT) .Sanger SequencingIn its early years, DNA analysis relied on Sanger sequencing, which represents a first-generation sequencing technology to test for a few defined genes associated with channelopathies . Sanger Next-Generation SequencingTo overcome the limitations of first-generation sequencing technology, a more advanced sequencing technology known as next-generation sequencing (NGS), has been developed. Over recent years, genetic alterations associated with SUD have been increasingly identified. Being very fast and cost-effective for large-scale genetic analysis, NGS allows us to perform a broader targeted gene panel testing, or a more comprehensive genomic study, whole exome sequencing (WES) . WES resAlthough WES is a fascinating technology, it is challenging to assess and identify the variants’ pathogenicity due to the massive number of variants it detects. A large number of these variants are of undetermined significance (VUS). This results in a huge dilemma in the current genetic investigations, as there is insufficient evidence regarding their pathogenicity. If such variants are misinterpreted, it will lead to inaccurate diagnosis, unnecessary treatment, and a profound psychological impact on affected individuals . To overThe Use of miRNAsSince its discovery in the biomedical field in 1993, microRNA (miRNA) analysis has attracted the interest of several researchers. However, the forensic application of miRNA analysis has only been proposed in the last 10 years and is currently receiving great attention. Body fluid identification has been the main goal of the forensic use of miRNA analysis in order to provide confirming universal analysis of unidentified biological stains received from crime scenes or evidence objects. However, there are other forensic uses for miRNA profiling that have demonstrated value but are mostly unexplored and warrant more research, including the identification of organ tissue and the estimation of donor age . AccordiPostmortem genetic testing for the evaluation of SUD is recommended in standard guidelines. However, it is not routinely performed, perhaps due to the unavailability of specialist personnel, the lack of equipment in many forensic institutions, and the high cost associated with the procedure . SurprisGenetic findings in certain specific conditionsThe etiologies of SUD in young individuals are extremely diverse. However, in autopsy cases with obscure findings, genetic cardiac diseases are the most probable etiology . These gIn this section of the review, the causes of SUD are further discussed and explained.Postmortem Genetic Testing in Cardiac-Related CausesCardiovascular diseases are responsible for about 90% of all sudden death cases in most developed countries . It inclDewar et al. conducted a study to investigate the genetic etiologies in obscure autopsy SUD cases using molecular autopsy . The stuChannelopathies are disorders caused by genetic mutations in the genes encoding for cardiac ion channels or the proteins associated with these channels. In cardiac channelopathies, there are no anatomic abnormalities in the heart; instead, there are electrical abnormalities such as ventricular fibrillation or polymorphic ventricular tachycardia leading to sudden death ,24. CardLong QT syndrome (LQTS): LQTS is a term used to describe the inherited ion channel disorder and it does not include the acquired causes of QT prolongation such as heart diseases, drugs, hypokalemia, or stroke, where the term “acquired LQTS” is used. Prolonged repolarization is the hallmark of this syndrome. It can be diagnosed by measuring the heart-rate-corrected QT interval (QTc) of more than or equal to 480 ms or by Schwartz criteria, which include clinical history, family history, and 12-lead ECG ,24.There are 17 different genes for LQTS. A mutation in any of these genes will lead to the development of LQTS. The most frequently identified genotypes are LQTS1, LQTS2, and LQTS3, which are caused by a loss of function mutation of KCNQ1, KCNH2, and a gain of function mutation of SCN5A, respectively. LQTS has an autosomal dominant mode of inheritance ,24. EachA study done by Tester et al. aimed to investigate LQTS in 49 cases of SUD in the young age group . The autMarcondes et al. conducted postmortem genetic testing for LQTS in 128 SUD cases . Their pBrugada syndrome (BrS): BrS is prevalent in approximately one in every 10,000 individuals, most commonly in South Asian countries, and it appears to have a male predominance . The di: CPVT occurs most frequently in the pediatric age group of 4-12 years and male sex. It is triggered by exercise, especially swimming, presenting as syncope or cardiac arrest. It is less common than LQTS; however, it is more severe as the postmortem findings of CPVT are nearly as common as LQTS in the autopsy of sudden arrhythmic death syndrome (SADS) cases. The most frequently detected pathogenic mutations are RYR2 and CASQ2. CPVT does not have any findings in resting ECG. After the exclusion of structural cardiac disease, CPVT is diagnosed by inducing premature ventricular contractions (PVCs) in the presence of heart rates of more than 100 bmp during a stress ECG test. The PVCs may convert to polymorphic ventricular tachycardia and occasionally to the classic pathognomonic sign of CPVT, which is bidirectional ventricular tachycardia [Catecholaminergic polymorphic ventricular tachycardia (CPVT)hycardia .Lahrouchi et al. investigated 302 cases of SADS . The stuShort QT syndrome (SQTS): SQTS is much rarer than other channelopathies. It is diagnosed by a QTc of 340 ms or less, or a QTc of 360 or less plus one or more of the following additional features: the presence of a gain of function mutation in the genes encoding for potassium channels including KCNQ1, KCNH2, KCNJ2; secondly, the presence of SQTS in the family or SCD in young family members; thirdly, surviving a cardiac arrest without any structural abnormalities in the heart .: in cardiomyopathies, HCM is one of the most frequent autosomal dominant genetic disorders. It is a major cause of SCD in youths and athletes. It is identified by the ventricular septum being asymmetrically hypertrophied, ventricular wall thickening, increased heart weight, and narrowed ventricular cavity. There are two types of pathogenic mutations implicated with HCM: familial and sporadic.Hypertrophic cardiomyopathy (HCM)Hereditary cardiomyopathy is the third most common cause of SCD, accounting for 5.9-6.2% of all SCDs . BecauseMarey et al. assessed 35 patients who died of cardiac arrest and were suspected of having cardiomyopathy on the basis of autopsy or clinical evidence . They diGaertner-Rommel et al. reported a case of a 19-year-old with SCD who was further investigated using a combination of forensic and molecular autopsy techniques. In FHL1 and MYBPC3, which are genes found in cardiac muscles, the molecular autopsy revealed two (possibly) harmful genetic variations. The MYBPC3 variant only demonstrated partial penetrance. The FHL1 mutation revealed a de novo type of mutation. In muscle samples, low levels of FHL1 mRNA and no FHL1 protein were found. Indicating nonsense-mediated mRNA decay and/or damaging of the truncated protein revealing a possible disease mechanism in victims of SCD .Arrhythmogenic cardiomyopathy (ACM) and arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D):ARVC is a poorly recognized and frequently misdiagnosed disorder of the right ventricle (RV) marked by the substitution of the myocardium into fibroadipose tissue. It is estimated that ARVC affects one out of every 5000 individuals, accounting for nearly 20% of SCDs in individuals below the age of 35 years . AccordiSato et al. investigated mutations in DSP, DSG2, and PKP2 in 15 cases of sudden death in which the cause of death was unknown at autopsy . DSP mutLikewise, in one recent case report published by Leone et al., a molecular autopsy was performed on a 13-year-old boy who suddenly died after physical exertion following cardiopulmonary resuscitation . A samplPostmortem Genetic Testing in Non-Cardiac-Related CausesEpilepsy: SUDEP is one of the main causes of the increase in mortality of epileptic patients and can account for up to 18% of deaths in this population ,36. It iThe exact mechanism of SUDEP is unknown; however, many theories have been proposed to explain the underlying mechanism of SUDEP, mostly pertaining to dysfunction in the respiratory, cardiac, and nervous systems ,36.2 levels. Moreover, this can be worsened by airway obstruction that can occur during sleep in the prone position, which was found to be common in SUDEP cases. In addition, normal arousal response to the increased CO2 level might be blunted by the suppressed autonomic nervous system. Several studies indicate the significance of respiratory suppression mediated by the seizure activity in the brain in SUDEP cases [Suppression of the autonomic nervous system control over respiration during seizure episodes can lead to severe hypoxia and increased blood COEP cases .The cardiac conduction system may also be affected by seizure episodes; it can either lead to shortening or prolonging of the QT interval, which is a known pathophysiological mechanism of sudden unexpected death in general, such as in autosomal dominant LQTS . It has Many epileptic syndromes have underlying genetic mutations with increased risk of SUDEP,e.g., Dravet syndrome with a genetic variant in SCN1A, early infantile encephalopathy with a genetic variant of SCN8A ,36, and Metabolic disorders:SUDI is a broader phenomenon referring to all sudden unexpected deaths in which the age is between birth and up to 12 months, including sudden infant death syndrome (SIDS) cases . SIDS reMetabolic disorders are a group of inherited disorders that can lead to SUDI. The majority of these disorders have an autosomal recessive inheritance fashion, and they are generally asymptomatic in early life. Deficiency in the N-acetylglutamate synthase enzyme can occur when there is a mutation in the NAGS variant leading to hyperammonemia. Fatty acid oxidation disorders are major metabolic disorders that are classified into several types, including deficiency of the following enzymes: very long-chain acyl-CoA dehydrogenase, 3-hydroxy-3-methylglutaryl-CoA lyase, and carnitine palmitoyl transferase II; caused by mutations of these genetic variants ACADVL, HMGCL, and CPT2, respectively . The defOutcomes of postmortem genetic testingMost of the genetic variations that underlie SUD owing to cardiomyopathies or channelopathies are autosomal dominant and have a 50% chance of inheritance. As a result, understanding the genetic variations implicated in SUD cases is critical, not only to know the cause of death in the deceased but also to decide whether genetic screening for surviving relatives should be undertaken .A full clinical history of the deceased should be obtained, including the assessment of the type and level of activities before death and any symptoms that preceded the event. After this, a detailed autopsy including postmortem genetic testing should be performed . GeneticIn a study by Marcondes et al., 24 families underwent screening after an occurrence of SUD in their families . The resEthical and financial implications of postmortem genetic testingIndications of postmortem genetic testing include scientific research purposes, request of the decedent’s family, or, in many countries, as part of judicial investigations into death, especially in young victims ,41. ChilIn medical practice, informed consent, which is the act of voluntary agreement between the patient and doctor regarding medical care, is a crucial part before any action is performed and is typically obtained from the competent patient . ConsentConfidential information regarding individuals must not be disclosed without their authorization even after an individual’s death. However, given the familial nature of genetic information, sharing positive results with close family members may help in preventing serious harm in living individuals who might carry a similar genetic risk . In suchThe cost of postmortem genetic testing has been frequently reported as a significant barrier. Currently, most third-party payers and insurance companies do not cover the costs of postmortem genetic testing, and hence family members who are willing to do the test are responsible for paying out of their pocket . It is eCounseling families affected by sudden death in the young about the results of the molecular autopsy is undoubtedly challenging. In general, the focus of counseling is to explain the findings to the relatives in a way they can understand the scenario, identify other at-risk relatives, and provide referrals for follow-up as needed . In someA molecular autopsy is an essential tool for the forensic evaluation of SUD cases requiring medico-legal death investigation; it helps to reach a genetic diagnosis when routine autopsies are unclear, with the ultimate purpose of determining the cause of death besides collaterally leading to the genetic screening of the deceased individual’s family.


Agave angustifolia var. pacifica, an agave used for bacanora  production in northwestern Mexico. We sampled 34 wild and cultivated sites and used over eleven thousand genome-wide SNPs. We found shallow genetic structure among wild samples, although we detected differentiation between coastal and inland sites. Surprisingly, no differentiation was found between cultivated and wild populations. Moreover, we detected moderate inbreeding (FIS ~ 0.13) and similar levels of genomic diversity in wild and cultivated agaves. Nevertheless, the cultivated plants had almost no private alleles and presented evidence of clonality. The overall low genetic structure in A. angustifolia var. pacifica is apparently the result of high dispersibility promoted by pollinators and the possibility of clonal reproduction. Incipient cultivation history and reliance on wild seeds and plants are probably responsible for the observed patterns of high genetic connectivity and considerable diversity in cultivated samples. Due to the recent increase in demand for agave-based beverages, many wild agave populations have experienced rapid decline and fragmentation, whereas cultivated plants are now managed at monocultural plantations, in some cases involving clonal propagation. We examined the relative effect of migration, genetic drift, natural selection and human activities on the genetic repertoire of  Plant populations are not arbitrary assemblages of genotypes but are structured in space and time . The majMigration among plant populations is affected by life history traits , pollinators and seed dispersal mechanisms, and by the continuity of geographical distribution ,6,7. SomAnother important factor affecting plant population genetic structure is spatial distribution ,15. PlaneN) and connectivity among plant populations comes from direct extraction and lumbering [Genetic structure in plants is also affected by human use and cultivation ,22. Usuaumbering ,23. On tumbering . Artificumbering . Otherwiumbering ,27. GiveAgave L. (Asparagaceae) may be excellent models to investigate the relative importance of natural and/or anthropogenetic causes that affect population structure and diversity in plants. Agave is a species rich genus of perennial monocots found primarily in the arid regions of Mexico and the southwestern United States [Agaves are keystone species that disproportionately affect their ecological communities relative to their abundance by producing a large inflorescence with copious amounts of nectar and pollen [Agave has a long history, and nowadays it has become the primary use of agave [Agave L. are used for beverage production [In this regard, species of d States ,29. Agavd pollen ,31,32. Od pollen . The prooduction ; yet, thoduction . Neverthoduction  with difoduction . In the oduction . This troduction . Agave spp. utilized in Mexico is Agave angustifolia. A. angustifolia is also the species with the widest geographic distribution within the genera; it is found from Sonora and Tamaulipas in Mexico to Costa Rica [A. angustifolia is a diploid, apparently self-incompatible [Leptonycteris bats, birds and bees [A. angustifolia is sometimes considered to comprise several varieties [A. angustifolia var. pacifica, which is the focus of the present study. One of the most important sta Rica . A. angumpatible ,40 specimpatible . It is pand bees . Becausearieties . In nortAgave angustifolia has a rich ethnobotanical history [A. angustifolia is the most important agave species used for mescal elaboration in Oaxaca, the state with the highest percentage (over 90%) of mescal production, and other places [A. angustifolia—“Espadín” [A. angustifolia is used for production of bacanora [A. angustifolia var. pacifica is now often carried out in monocultural extensive areas, the majority of plantations are small, with important reliance on wild seeds and plants.  history ,42. Currr places ,43. The Espadín” . In Sonobacanora , a less A. tequilana was drastically reduced [The increase in mezcal production, and in particular, the accelerated extraction of wild individuals, constitutes an important environmental problem . This is reduced ,47, whic reduced . The ava reduced ,50. On t reduced ,52,53. A reduced . Given tA. angustifolia var. pacifica, an agave used for bacanora  production in the Sonora state in northwestern Mexico. We analyzed 96 individuals from wild and cultivated sites of A. angustifolia var. pacifica, genotyping them with over 11,000 SNP markers . After filtering and adapters removal, the final data set resulted in an average of 2,630,790 high quality reads per sample . From this data, 638,704 variants were called using the de novo pipeline. After filtering, our final data set consisted of 95 A. angustifolia individuals and 11,619 SNPs with 2.8% of the data missing. Average sequencing depth for each individual and missingness on per site and per individual level are presented in The genotyping by sequencing (GBS) on 96 A. angustifolia showed shallow genetic differentiation, with the exception of some cultivated sites and one wild site . Similar results were found using Nei’s D index, as cultivated plants had negligible differentiation from their wild counterparts (0.006). We further carried out differentiation analyses at site level for cultivated and wild individuals and only for the wild sites (FST ranged from 0.1 to 0.57). One of these sites corresponded to a plantation that had in vitro propagated plants  was highly differentiated . When these coastal sites were excluded, the differentiation was low and homogenously distributed, ranging from FST −0.01 to 0.01 , anothe to 0.01 . A. angustifolia, we used an ADMIXTURE analysis. When all samples (cultivated and wild) were used, the cross-validation error estimates showed that model fit was optimized at K = 4  that presented higher differentiation. The cross-validation criterion performed with  plateau , but theK = 2  but non-significant (p = 0.89) local spatial structures  samples of two coastal sites from low elevation near Navojoa city; (ii) a group including three coastal sites of low elevation near San Carlos; and (iii) an inland group comprising the rest of the sites.At K = 2 A, we foute group B. At K =analysis C. Spatiaructures , indicatructures , corroboFST) between wild sampling sites increased with geographical distance ; nevertheless, the relationship was not linear  for all samples was 0.22 (SD 0.13). The mean oH among 53 wild agave individuals was lower  than the expected heterozygosity , indicating a deficit of heterozygous individuals. Almost identical results were obtained for the cultivated samples . The difference between expected and observed heterozygosity was significant, even after Bonferroni correction . Individual based multilocus heterozygosity (MLH) was similar to the oH for both wild and cultivated plants  for both wild and cultivated agaves. In wild individuals, f ranged from almost zero to as high as 0.37  differences in MLH and isF between inland and coastal sites, with coastal sites presenting lower diversity and higher inbreeding  , indicatWhen the number of private alleles was compared between managed and wild plants, we found that wild individuals harbored a higher number of alleles that were not shared with cultivated plants , 299 vs. 12.A. angustifolia var. pacifica from northwestern Mexico. Agave angustifolia is a species with a wide distribution and a long history of human use that recently has escalated up to intensive management and heavy pressure on wild populations (Molina Freaner pers. observ.). Nevertheless, probably due to a combination of factors, including aspects of the species biology , pollination biology , the incipient cultivation history and particularities of bacanora production , we found low genetic structure, and homogeneously distributed levels of diversity in both wild and cultivated individuals. As for the wild samples, we found low but detectable spatial population genomic structure, apparently driven by the ecological characteristics of the habitat.We analyzed patterns of genetic diversity and population structure of wild and cultivated A. angustifolia. We found that wild populations of A. angustifolia, with a relatively continuous distribution in dry, mid-elevation areas throughout northwestern Mexico [One of the aims of this study was to understand how natural forces, life history traits and cultivation practices may affect patterns of genetic structure of n Mexico , do not n Mexico . A. angustifolia var. pacifica, shallow genetic structure is probably the result of high genetic connectivity, which homogenizes populations and reduces the effects of genetic drift and selection. Species of nectar-feeding bats in the genus Leptonycteris have been proposed as important pollinators of agaves in general, and of A. angustifolia in particular [Overall, genetic structure is created by limited migration among populations, genetic drift and different selection regimes occurring across populations . In A. articular ,55, Agavrticular ,50,52. Irticular ,57.Another important life history trait that may contribute to the observed lack of differentiation is the possibility of clonal reproduction and, as a consequence, enhanced longevity of genets ,42,58. IAlthough we did not detect population genetic structure using traditional methods, we did find interesting spatial clustering of the samples. Thus, we detected that low elevation coastal sites (with an elevation of less than 50 m) are differentiated from the inland genetic cluster. Moreover, those sites were also differentiated from each other, a fact that may also explain non- linearity in Mantel tests. Coastal regions have unique environmental characteristics, such as high salinity, greater availability of atmospheric moisture, wind strength and tidal influence, therefore constituting distinct landscapes with unique abiotic and biotic composition ,62. We tA. tequilana, present considerable genetic differentiation from its wild relatives and among cultivars [A. angustifolia var. pacifica is relatively recent and therefore, there may have been little time for differentiation.The genetic differentiation between wild and cultivated agaves has been the focus of extensive research ,49, withultivars ,64. Basiultivars . We founA. angustifolia var. pacifica. Our findings expand the knowledge of genetic differentiation and diversification processes in agaves across coastal and inland areas of northwestern Mexico. Our results are concordant with previous suggestions ,58 that The most widespread and concerning consequence of plant management and domestication is the erosion of genetic diversity ,66. DuriA. tequilana, A. salmiana and A. americana) presenting low levels of diversity [A. angustifolia in northwestern Mexico have not lost genomic diversity, nor did they present increased inbreeding in comparison to their wild counterparts. These findings are consistent with other studies reporting that traditional management of agave that involves gathering of wild specimens and seeds, instead of clonal propagation, allows the persistence of high genetic diversity in cultivated agaves [FST > 0.3) of some cultivated sites and individuals, particularly at some of the intensively managed plantations. Unfortunately, we were not able to determine the original geographic source of these samples, as probably they were moved from locations outside the sampling area. We suggest that future studies should focus on analysis of A. angustifolia throughout its whole distribution area and to establish a standard set of markers that would be useful to trace the origin of the cultivated individuals and to compare their diversity levels. The case of agave is probably more complicated, with some cultivated varieties  alleles (299 vs. 12), which represent an important gene pool of standing genetic variation not found in the cultivated plants used for bacanora production. This genetic resource could be relevant, for example, in future crop improvement programs. These differences indicate that the cultivated plants do not include the total genomic diversity of f = 0.13 for both wild and cultivated plants) were observed, and these occurred consistently among sites. Interestingly, in the Agave genus, relatively high levels of inbreeding (FIS) have also been reported in other studies [A. maximiliana , in A. 21–0.32; ). Agave schottii apparently do not experience strong negative selection and, as a consequence, inbred individuals continue to make up a substantial proportion of the population. Nevertheless, this hypothesis has not been tested in A. angustifolia, and further research focusing on optimal outcrossing distances and effects of inbreeding depression in this species would be helpful in elucidating how moderate levels of inbreeding and low population structure can be maintained [Explaining moderate levels of inbreeding coupled with the considerable polymorphism that have been found in many plants has not been an easy task . One intintained ,82. A. angustifolia’s level of genetic diversity in a broader context of Agavoideae species, and it is unclear what levels of diversity and inbreeding should be expected. These challenges result from the general lack of studies that cover the whole distribution range of this or other agave species and a myriad of different molecular markers that have been used over the last 50 years [The major concern of the current increase of mezcal and related beverage production is that in the eagerness for satisfying ever-growing demand, there could be an unprecedent negative impact on natural habitat and on wild agave populations ,83. Curr50 years ,49.Nevertheless, we discovered an alarming trend in some cultivated samples, particularly collected from the intensively managed plantations. Although at each plantation we sampled distant individuals, we found several samples with a high relatedness coefficient (over 0.5), indicating clonal propagation. These findings are in contrast with the fact that almost all wild individuals and samples from the traditionally managed plantations were genetically unrelated. Clonal propagation has several advantages for cultivation; for example, it is easier and faster than propagation from seeds, and it also ensures that favorable genotypes and mutations are passed on to the next generation . NeverthBased on our findings, we argue that there is an urgent need to conduct systematic research focused on (i) delineating distribution limits of agave species used for spirits production; (ii) determining conservation and management units within each species; (iii) evaluating the impact of wild plant extraction by humans on species distribution and numbers; (iv) developing a set of genomic markers that would allow reproducibility and comparison among studies. Moreover, ancient and historical genetic data, such as those from specimens stored in herbarium collections and remains from archaeological sites, can add a temporal dimension to conservation of agaves by providing baseline levels of diversity .A. angustifolia collected from 34 sites in the state of Sonora, Mexico in October 2021 . The choice of enzymes was based on a previous standardization for de novo assembled using ipyrad v. 0.9.79 [https://ipyrad.readthedocs.io/, accessed on 20 February 2022). As no published genome of any Agave species is yet available, we used a de novo assembly strategy. We set the level of sequence similarity for clustering at 90% after comparing the number of recovered single nucleotide polymorphisms (SNPs) using values between 85–95% (see p ≤ 0.05 after Bonferonni correction). Finally, to ameliorate the confounding effects of linkage disequilibrium (LD), we eliminated markers within the specified distance from one another using --thin argument (--thin 250) as implemented in VCFtools. The final VCF file was then produced for all downstream analyses.The raw data were filtered by removing adapters and low-quality bases using TRIMMOMATIC . For the. 0.9.79  with par–95% see . The finStAMPP package [a priori grouping was inferred using the method implemented in ADMIXTURE v.1.23 [K), with ten replicates for each K value. The support for different values of K was assessed according to the likelihood distribution , as well as by visual inspection of the co-ancestry values for each individual. We also used the R package SNPRelate to perform a principal component analysis (PCA) of A. angustifolia individuals based on the genetic covariance matrix calculated from the genotypes [A. angustifolia samples: (i) including both wild and cultivated plants, and (ii) including only wild individuals. Several complementary approaches were used to test for patterns of genetic structure between wild and cultivated agave plants and among sites. We estimated genetic distance between wild and cultivated samples, and among all pairs of sites using the  package . ClusterE v.1.23 ,97. Admienotypes . We ran A. angustifolia, we used the spatially explicit clustering program TESS3R [K = 1–10 possible genetic groups with 20 replicates of each K and kept the most supported model  within each of the 20 replicates. Map locations were colored by the resulted dominant ancestry cluster, with transparency reflecting the percent ancestry of that cluster and the largest value assigned as opaque. To explore the role of geography in the population genetic structure of m TESS3R , which dTESS3R, we conducted a spatial principal components analysis (sPCA) [adegenet [While classic PCA is appropriate for detecting obvious genetic structure , it doess (sPCA) ,102. Thiadegenet . Tests fadegenet  were madade4 package to estimate the correlation between the genetic distance, geographical and altitudinal distance matrices [p value correspond to the proportion of times the randomized regression coefficient is equal or greater than the observed one. We calculated the pairwise genetic distance matrices among sites using FST and pairwise geographical distance matrices using the Geographic Distance Calculator [Finally, we employed a Mantel test using the matrices . The teslculator . eH) and observed heterozygosity (oH) were calculated for the full dataset, and separately for each management type using the R packages adegenet and hierfstat [Rhh and inbreedR [FIS statistics. Following Marshall et al. [f) of zero as ‘none’, below 0.125 as ‘low’, 0.125 ≤ f < 0.25 as ‘moderate’, and f ≥ 0.25 as ‘high’. Wilcox tests were then used to test for significant differences in the diversity indices between management types, sites and populations identified with spatial analysis in TESS3R using the R package stats [poppr package [Expected heterozygosity  from northwestern Mexico using SNP markers. We focused on wild and cultivated sites composed by individuals of the morphologically predominant morphotypes. Nevertheless, even this criterion did not help us in sampling morphologically similar but genetically different plants at one wild site (CamW) and several cultivated sites. Unfortunately, due to the considerable gap in agave phylogenetic and population genetic studies, we could not trace the geographic original source of these genotypes. Besides these outlier cultivated samples, we did not detect genetic differentiation between wild and cultivated individuals, neither did we find loss of genetic diversity in plants under management, suggesting that seeds of wild individuals are readily used for cultivation. Interestingly, the data analysis of wild samples revealed a partition of the samples into three groups—two coastal groups and one inland cluster. Nevertheless, being aware of our experimental limitations, at this moment we are unable to distinguish between local adaptation and neutral divergence; we therefore may only hypothesize reasons for such a separation.The current study aimed to dissect the genetic architecture of bacanora agave (


Perilla crop is highly regarded in South Korea, both as a health food and traditional food. However, there is still a lack of Perilla SSR primer sets (PSPSs) for studying genetic variation among accessions of cultivated and weedy types of Perilla crop (CWTPC) from South Korea. In this study, 30 PSPSs were newly developed based on transcriptome contigs in P. frutescens, and 17 of these PSPSs were used to study the genetic diversity, phylogenetic relationships and structure population among 90 accessions of the CWTPC collected from South Korea. A total of 100 alleles were detected from selected 17 PSPSs, with an average of 5.9 alleles per locus. The gene diversity (GD) ranged from 0.164 to 0.831, with an average of 0.549. The average GD values from the cultivated var. frutescens, weedy var. frutescens, and weedy var. crispa, were 0.331, 0.588, and 0.389 respectively. In addition, most variance shown by Perilla SSR markers was within a population (73%). An analysis of the population structure and phylogenetic relationships showed that the genetic relationship among accessions of the weedy var. frutescens and weedy var. crispa is closer than that for the accessions of the cultivated var. frutescens. Based on association analysis between 17 PSPSs and three seed traits in 90 Perilla accessions, we detected 11 PSPSs that together were associated with the seed size and seed hardness traits. Therefore, the newly developed PSPSs will be useful for analyzing genetic variation among accessions of the CWTPC, association mapping, and selection of important morphological traits in Perilla crop breeding programs.The  Perilla frutescens (L.) Britt. is an annual self-pollinating crop of the Labiatae family. The Perilla crop has been widely cultivated for a long time, mainly in East Asia, and is also widely distributed in Southeast Asia. In East Asia, the Perilla crop is divided into two varieties (or cultivated types) according to its morphological characteristics and availability as follows: cultivated var. frutescens, which is used as an oil, condiment, and vegetable  crop; and cultivated var. crispa, which is used as a Chinese herbal medicine and vegetable  crop [frutescens, var. crispa) of the Perilla crop are extensively cultivated and are used mainly in South Korea and Japan. In particular, var. frutescens is extensively cultivated and used mainly in South Korea, while var. crispa is extensively cultivated and used mainly in Japan [Perilla crop is believed to be China, because China has a long cultivation history, high genetic diversity, and many resources distributed in the country [Perilla crop in East Asia have not yet been definitively identified, many weedy plants of the Perilla crop are widely distributed in China and South Korea [Perilla crop have their weedy types, which grow naturally and are usually found in habitats such as wastelands, roadsides, stream or river sides, and the land around farmers’ houses and fields.ds) crop ,2,3,4,5.in Japan ,7. Howev country ,9,10. Alth Korea ,11. In pth Korea , Nitta ath Korea , and NitPerilla crop (CWTPC). The two cultivated types (var. frutescens and var. crispa) of the Perilla crop can be distinguished based on seed size, color of flower, color of leaf and stem, plant fragrance, and other morphological characteristics such as seed dormancy, seed hardness, pubescence degree, plant height, and leaf shape [frutescens and var. crispa) of the Perilla crop have not been clearly distinguished because of the presence of intermediate types [Perilla crop breeding programs.In East Asia, many researchers have conducted studies to distinguish between the accessions of cultivated and weedy types of the af shape . Howeverte types . The accte types . Additiote types ,17,18,19Perilla crop, molecular marker technology has recently provided more comprehensive information on the genetic variation and phylogenetic relationships between and within accessions of the CWTPC. Molecular markers such as randomly amplified polymorphic DNA (RAPD) [Perilla crop. Among them, SSR markers are considered to be the preferred choices of markers for genetic studies because of their codominance, polymorphism, high repeatability, and rich genetic information [Perilla SSR primers [Perilla SSR primer sets (PSPSs) developed for analyzing the genetic variation and phylogenetic relationships of the CWTPC is still insufficient.In the A (RAPD) ,12, amplA (RAPD) , and simA (RAPD) ,20,21,22ormation . In prev primers ,25,26 ha primers ,28,29. HPerilla species [Perilla crop, Tong et al. (2015) [frutescens (PF98095) of the Perilla crop using transcriptome sequencing, conducted via RNA sequencing. In addition, Sa et al.  [Perilla SSR loci, which were classified as dinucleotide SSRs, trinucleotide SSRs, and tetranucleotide SSRs based on the number of repeating units. In addition, using this information, novel PSPSs were developed in the Perilla crop [Recently, RNA-sequencing has presented a powerful tool for discovery of new genes, and the development of eSSR primers or microsatellite sites for non-model crops ,31. This species ,26,32. I. (2015)  were the8, 2019) ,26 deteclla crop ,26.Perilla crop and performed genetic diversity, phylogenetic relationship and association mapping analysis on the CWTPC collected in South Korea, using the newly developed PSPSs. The current research results are expected to provide useful information for future genetic research, such as understanding the differentiation process of the CWTPC and genetic and breeding studies of Perilla germplasm accessions.Therefore, in this study, we developed novel PSPSs in the Perilla species , ambiguous banding patterns (31), and poor or no amplification banding patterns (85) (data not shown). Among the 30 PSPSs, 17 PSPSs presented clear band patterns that differentiate the Perilla species. Therefore, in our study, to understand the genetic variation among accessions of the CWTPC collected from South Korea using the newly developed PSPSs, we performed genetic diversity and phylogenetic relationship analysis among the 90 accessions of the CWTPC . In doing so, we made use of 17 PSPSs from among the newly developed 30 PSPSs  of the 90 accessions of the CWTPC with the 17 new PSPSs, the average number of alleles was 3.3, 4.9, and 3.1 from the cultivated var. frutescens, weedy var. frutescens, and weedy var. crispa, respectively. The average MAF values were 0.749, 0.545, and 0.722 from the cultivated var. frutescens, weedy var. frutescens, and weedy var. crispa, respectively. The average GD values were 0.331 and 0.588 in the cultivated and weedy types of var. frutescens, and 0.389 in the weedy var. crispa. The average PIC values were 0.305 and 0.536 in the cultivated and weedy types of var. frutescens and 0.346 in the weedy var. crispa , the 90 accessions of the CWTPC were clustered into six major groups with 46.6% genetic similarity . Group Itionship .frutescens showed a large size of 2 mm or more and soft seeds, whereas all accessions of the two weedy types of var. frutescens and var. crispa showed a small size of less than 2 mm and hard seeds. For SCC, the 30 accessions of cultivated var. frutescens showed white, gray, brown, and dark brown. Among them, three accessions were white, two accessions were gray, 13 accessions were brown, and 12 accessions were dark brown. Meanwhile, the 30 accessions of weedy var. frutescens showed brown and dark brown, of which 10 accessions were brown, and the remaining 20 accessions were dark brown. The 30 accessions of weedy var. crispa showed brown and dark brown, of which 7 were brown, 22 accessions were dark brown, and the remaining one accession had mixed brown and dark brown seeds , seed hardness (SH), and seed coat color (SCC). As shown in wn seeds .p ≤ 0.01 , were used to confirm significant marker–trait associations (SMTAs) using TASSEL 3.0 software. From the results, we detected 11 PSPSs  that were together associated with the SS and SH traits using GLM at a significance level of p ≤ 0.01 . Additiop ≤ 0.01 .Perilla crop is still insufficient. That is, the classification of the CWTPC is not clear because of a lack of PSPSs for analyzing the genetic variation of the Perilla crop and its related weedy types. Therefore, the new PSPSs developed in the current study provide information for further determination of the genetic variation among accessions of the CWTPC, and such information may be useful in understanding the genetic differentiation and phylogenetic relationships of accessions of the CWTPC collected from South Korea. In particular, in recent years the cultivated var. frutescens of the Perilla crop has become one of the most important economic crops in South Korea because of an increase in oil consumption, along with its use as a leaf vegetable. Therefore, genetic diversity analysis using the newly developed PSPSs on the Perilla germplasm resources collected in South Korea will provide good information for the selection of useful germplasm resources, the conservation of germplasm resources of the CWTPC, the differentiation study of the CWTPC, and the development of the Perilla cultivars (or varieties) in future studies.Understanding the genetic variation of plant genetic resources in crop species is necessary for the conservation of biodiversity and the development of new cultivars of crop species. In recent years, PSPSs have been widely used to identify genetic diversity and phylogenetic relationships, as well as for analysis of population structure and association mapping among accessions of the CWTPC ,22,27,29Perilla crop, Sa et al. (2018) [Perilla crop, such as the value of 9.2 detected by Sa et al. (2013) [frutescens, 30 accessions of weedy var. frutescens, 30 accessions of weedy var. crispa) of the CWTPC was analyzed using the 17 PSPSs. The number of these accessions of the CWTPC is the most used for diversity analysis research in South Korea so far. Therefore, the results of this study obtained using the new PSPSs are expected to provide useful information for understanding the genetic diversity and phylogenetic relationship of the accessions of the CWTPC in South Korea. As a result, the average GD values of the accessions of cultivated and weedy types of var. frutescens and weedy var. crispa from South Korea were 0.331, 0.588, and 0.389, respectively  [As already mentioned in the Introduction and Materials and Methods sections, in the case of the . 2018)  reported. (2018) . Therefo. (2018) . Therefo. (2013) , the 9.8. (2013) , and the. (2013) ; but it . (2013) , 3.3 det. (2013) , and 5.8. (2013) . The ave. (2013) ,35, Park. (2013) , and Kim. (2013)  and slig. (2013) . General. (2013) ,39. Furtectively . From th018  repoi (2003)  using AFPerilla crop, seed characteristics, especially SS, SH, SCC, and SD showed great differences between the CWTPC [frutescens showed a large seed size of 2 mm or more, whereas the two weedy types of var. frutescens and var. crispa showed a small seed size of less than 2 mm. For SH, most accessions of cultivated var. frutescens showed soft seed characteristics, whereas all accessions of the two weedy types of var. frutescens and var. crispa showed hard seed characteristics. For SCC, cultivated var. frutescens showed various seed coat colors such as white, gray, brown, and dark brown, whereas the two weedy types of var. frutescens and var. crispa showed only brown and dark brown seed coat colors. However, there was no significant difference between the two weedy types of var. frutescens and var. crispa of the Perilla crop  [Perilla crop have been found in East Asia, these accessions of the two weedy types of the Perilla crop are thought to have an important place in the origin of the two cultivated types of the Perilla crop. Therefore, the novel PSPSs developed in this study were expected to provide useful information for the evaluation of genetic variation in accessions of the CWTPC collected from South Korea.In crop species, seed characteristics are one of the useful traits that distinguish cultivated type from wild or weedy types. For example, SS, SH, seed dispersal, and seed dormancy (SD) differ dramatically between cultivated grain crops and their wild ancestors ,41,42,43he CWTPC ,22. In olla crop . In addiion rate ,22. In pion rate . Therefoi (2003) ,45. TherΔK proposed by Evanno et al. (2005) [ΔK = 2  . Based o [ΔK = 2 a, and bene group b. This ranalysis  also shohe CWTPC c. Thereffrutescens and the weedy var. crispa. Although most accessions of the two weedy types of var. frutescens and var. crispa did not form different groups, all accessions of cultivated var. frutescens were clearly different from the two weedy types of var. frutescens and var. crispa. These results indicate that, as with morphological characteristics such as seed traits  [frutescens could be an escaped form from cultivation, or otherwise likely derived from hybrids between accessions of the CWTPC. Although there is still no accurate report on the natural hybridization rate in the Perilla crop and its weedy varieties, recently Lim et al.  [frutescens and weedy var. crispa.On the other hand, in the analysis of the UPGMA dendrogram for accessions of the CWTPC , the cluaits see , SSR maraits see , the six. 2002)  and Lee 002  and 9, 2021) ,18 and Ket al. 20, 2021 [1frutescens and var. crispa were well distinguished from each other in the UPGMA dendrogram, but some accessions were poorly differentiated . The present study revealed that genetic variation occurred mainly within the population rather than among the three populations, as shown by the population structure and UPGMA dendrogram analyses . In particular, many accessions of the CWTPC were found throughout South Korea [Perilla germplasm accessions and novel PSPSs compared with previous reports by Lee et al. (2002) [Perilla crop.Today the var. th Korea ,6,11,28.. (2002) ,21, Lee . (2002) , and Ma Perilla crop  were used for the selection of 200 newly developed PSPSs  were selected from the published germplasm on the website of the Korean RDA-Genebank of the Republic of Korea . The 90 accessions of the CWTPC used in this study were selected to represent, as much as possible, all regions of South Korea, and the collection information is shown in Perilla accession was extracted from the young leaves at the seedling stage based on a modified Doyle’s CTAB method [In the preliminary experiment of our study, four accessions of the ed PSPSs . In addiB method .frutescens (PF98095) RNA-seq data using Trinity software (http://TrinityRNASeq.sourceforge.net) (accessed on 1 January 2021). As previously reported by Sa et al.  [http://pgrc.ipk-gatersleben.de/misa) (accessed on 1 January 2021) was used to search for new Perilla SSR or microsatellite sites in the assembled transcriptome sequences of the accession of cultivated var. fruteseces (PF98095) of the Perilla crop. In accordance with the Perilla SSR flanking sequences of PF98095, new PSPSs were designed using PRIMER 3 software. In previous studies, Sa et al.  [Perilla SSR loci (100%), which were classified as dinucleotide SSRs , trinucleotide SSRs , and tetranucleotide SSRs  based on the number of repeating units. Therefore, in this study, we designed 200 PSPSs from the 9910 dinucleotide SSRs based on the number of di-repeat units. The SSR amplification method for accessions of the CWTPC has been described in a previous study by Sa et al.  [To develop PSPSs based on the previous published results of Tong et al. (2015) , we perf8, 2019)  and Kim 8, 2019)  detected8, 2019) . After p8, 2019) .K statistic was based on the rate of change of the logarithmic probability of the data between K values [http://taylor0.biology.ucla.edu/structHarvester/, accessed on 1 December 2021) based on the STRUCTURE results. Association mapping analysis was performed for the 17 novel PSPSs and three seed characteristics  of the 90 accessions of the CWTPC to confirm SMTAs using TASSEL 3.0 software [DNA fragments amplified by PSPSs were scored as present (1) or absent (0). The number of alleles, allele frequency, GD, MAF, and PIC for the 17 PSPSs was calculated with the PowerMarker 3.25 software program . GeneticK values , which wsoftware , which wsoftware  and Parksoftware .Perilla crop is highly regarded as a health food and traditional food in South Korea owing to the use of its seeds and leaves. However, PSPSs for studying genetic variation among accessions of the CWTPC from South Korea are still lacking. In this study, we developed 30 PSPSs (dinucleotide SSRs) using transcriptome sequencing by RNA-sequencing and used 17 of these PSPSs to analyze the genetic variation and phylogenetic relationships between and within 90 accessions of the CWTPC collected from South Korea. The number of these accessions of the CWTPC is the largest number used so far for diversity analysis research in South Korea. Therefore, the results of this study obtained using the new PSPSs are expected to provide a useful information for understanding the genetic diversity and phylogenetic relationship of the accessions of the CWTPC in South Korea. In our study, 100 alleles in total were detected from the 90 accessions of the CWTPC with an average of 5.9 alleles per locus, ranging from 130 to 315 bp. The average GD values of the accessions of cultivated and weedy types of var. frutescens and weedy var. crispa from South Korea were 0.331, 0.588, and 0.389, respectively. The results showed that the accessions of weedy var. frutescens had the highest genetic variation compared with the accessions of cultivated var. frutescens and weedy var. crispa. In the Perilla crop, seed characteristics, especially SS, SH, and SCC, showed great differences between the CWTPC. Of the 17 PSPSs used in the analysis, 11 PSPSs  were together associated with the SS and SH traits. According to analysis of the seed characteristics, population structure and phylogenetic relationship, although most accessions of the two weedy types of var. frutescens and var. crispa did not form different groups, all accessions of cultivated var. frutescens were clearly different from the two weedy types of var. frutescens and var. crispa. In this study, most variance shown by the Perilla SSR markers was within a population (73%) so that the genetic variation occurred mainly within the population of the CWTPC. Therefore, the newly developed PSPSs will be useful for analyzing genetic variation among accessions of the CWTPC, association and QTL mapping, and selection of important morphological traits in Perilla crop breeding programs.The