Agaricus bisporus (white and brown) characteristics  and mould/yeast counts, biogenic amine content, fatty acid (FA) and volatile compound (VC) profiles, overall acceptability, and emotions induced for consumers) during a 48 h lactic acid fermentation with Lacticaseibacillus casei No. 210, Lactiplantibacillus plantarum No. 135, Lacticaseibacillus paracasei No. 244, and Pediococcus acidilactici No. 29 strains. Fermented white and brown A. bisporus showed higher LAB count and lower pH, lightness, redness, and yellowness than non-fermented ones. Yeast and fungi counts were similar between non-fermented and fermented samples. All samples contained spermidine  and some of the fermented samples had tyramine . Saturated FA was the highest in non-fermented brown A. bisporus. The highest monounsaturated and polyunsaturated FA contents were found in Lp. plantarum No. 135 fermented white and brown A. bisporus, respectively. For the first time, the VC profile of fermented A. bisporus was analysed. 1-Octen-3-ol content significantly decreased while benzyl alcohol, acetoin, and 2,3-butanediol increased in most fermented samples. Fermented A. bisporus received good acceptability scores. The emotional evaluation showed that the LAB strain and the interaction of the LAB strain and A. bisporus variety were significant on the intensity of emotions “happy” and “sad”, while all analysed factors and their interactions were significant on the intensity of “angry” and “disgusted” (p ≤ 0.05). The findings of this study show the potential of the selected LAB strains and contribute to the increasing body of research on fermented mushrooms.This study aimed to evaluate the changes in  Agaricus bisporus is the most prevalent commercially grown mushroom in the entire world and accounts for 35–45% of the global mushroom market, with Europe and North America being the primary producers [A. bisporus can vary [A. bisporus consists of a high proportion of moisture and is high in protein (11.0–29.1%) and carbohydrate (51.1%) [A. bisporus has a low percentage of fat, it does contain some essential fatty acids including linoleic acid [A. bisporus contains biologically active substances that have been proven to have anticancer, antioxidant, antidiabetic, antibacterial, and anti-inflammatory properties [Edible mushrooms have been regarded as nutritional food, nutraceuticals, pharmaceuticals, and food flavour agents due to their rich chemical composition and health benefits . Agaricuroducers . Due to A. bisporus with a white or brown cap (chestnut mushroom) and mature A. bisporus, so-called Portobello mushrooms, are supplied to the global market [A. bisporus is growing since most customers believe they taste better [A. bisporus is more susceptible to enzymatic browning during storage [A. bisporus lacks a protective cuticle layer but has a high protein, moisture, respiration rate, and enzymatic activity, these mushrooms are difficult to keep stable during storage [A. bisporus, but they all have their limitations [Immature l market . The pope better . However storage . Because storage . Variousitations . As procitations .Lactiplantibacillus plantarum is commonly employed in the food industry considering its ability to enhance antioxidant activity and to improve the flavour of food [Lacticaseibacillus casei and Lacticaseibacillus paracasei are two of the most studied and used probiotic bacteria [Pediococcus acidilactici is a potent producer of bacteriocins as well as a potential probiotic strain, used for the improvement of flavour and texture [Fermentation is one of the most ancient, time- and cost-effective ways of preserving food . It was  of food . Lacticabacteria , while P texture . In cont texture . However texture .A. bisporus fermentation with Lp. plantarum 299v and Lp. plantarum EK3, while the latter strain improved the organoleptic properties of fermented mushrooms. The same LAB strains were used in Jabłońska-Ryś et al. [A. bisporus for LAB isolation. Radzki et al. [Lp. plantarum fermentation of Pleurotus ostreatus mushroom polysaccharide resulted in considerable modifications in its chemical composition.The changes in various mushroom characteristics during fermentation with LAB have been rarely reported. In the study of Jabłońska-Ryś et al. , changesś et al. ’s studieś et al.  used spoi et al.  reportedA. bisporus (white and brown varieties) mushroom characteristics  during the lactic acid fermentation with Lc. casei No. 210, Lp. plantarum No. 135, Lc. paracasei No. 244, and P. acidilactici No. 29 strains.This study aimed to evaluate the changes in A. bisporus mushroom fermentation. Taking into consideration that studies on biogenic amines in mushrooms are scarce and reported data on these compounds significantly differ among species, this study will lead to broader knowledge about the changes in edible mushrooms during lactic acid fermentation.Most of the studies about fermented mushroom composition report changes in the main mushroom parameters without paying attention to the possible formation of undesirable compounds. Despite the fact that LAB has a GRAS status and lactic acid fermentation is an acceptable bio-preservation method for various foods, in this study, we would like to pay attention to the fact that some undesirable compounds can also be formed during this process. The main novelty of this study is based on the complex analysis, which includes both desirable and undesirable changes in the mushroom samples during fermentation. Additionally, as far as we know, No. 210, No. 135, No. 244, and No. 29 strains, previously isolated from spontaneously fermented cereals, were applied for the first time for A. bisporus (white and brown varieties) mushrooms were obtained from the local supermarket in Kaunas (Lithuania). Fresh fruiting bodies were cleaned and boiled in water (200 g mushrooms in 500 mL of water) for 10 min with 6 g of salt. After boiling, mushrooms were cooled to room temperature (22 ± 2 °C) and used for fermentation. Non-fermented boiled mushrooms were analysed as control. Lc. casei No. 210, Lp. plantarum No. 135, Lc. paracasei No. 244, and P. acidilactici No. 29 strains were multiplied in De Man, Rogosa, and Sharpe (MRS) broth culture medium  at 30 °C under anaerobic conditions. Boiled mushrooms (200 g) were inoculated with 5 mL of LAB multiplied in MRS (average cell concentration 9.0 log10 CFU/mL) followed by fermentation for 48 h at 30 ± 2 °C. The principal scheme of the experiment is shown in The LAB strains used in this study were previously isolated from spontaneously fermented cereal and showed a broad spectrum of antimicrobial and antifungal properties . Lc. casBefore measuring the mushrooms’ colour characteristics and pH, samples were homogenized using a blender.The colour coordinates  were assessed using a CIELAB system  .The pH was measured using a pH electrode  .The total titratable acidity  was measured according to ISO 750:1998 standard . TTA was−4 to 10−8 with the same saline were prepared for inoculation. Sterile MRS  agar  of 5 mm thickness was used for bacterial growth in Petri dishes. The dishes were separately seeded with the sample suspension using surface sowing and were incubated under anaerobic conditions at 30 °C for 72 h. All results were expressed in log10 of colony-forming units per gram (log10 CFU/g). The evaluation of the LAB count was performed according to ISO 15214:1998 standard . 10 g of10 CFU/g.Yeasts and fungi count in edible mushroom samples were counted on chloramphenicol agar  after incubation at 25 °C for 5 days . All exp®-5MS capillary column  was used for analysis. The temperature gradient was programmed from a start at 35 °C (5 min hold) to 200 °C (10 °C/min) up to 280 °C (25 °C/min) (5 min hold). The VC was identified according to mass spectra libraries .The VC in edible mushrooms was analysed by gas chromatography–mass spectrometry (GC-MS) according to Bartkiene et al.  with somv/v), and FA methyl esters (FAME) were prepared according to Pérez-Palacios et al. [The extraction of lipids for fatty acids (FA) analysis was performed with chloroform/methanol  was used to separate BA. Ammonium acetate (0.1 mol/L) and acetonitrile were used as the mobile phases at a flow rate of 0.8 mL/min. The sample volume injected was 20 µL and the amines were monitored at 254 nm. The BA were identified based on their retention times in comparison to their corresponding standards.Sample preparation and determination of biogenic amines (BA) in mushroom samples were performed according to the method of Ben-Gigirey et al. , with soThe overall acceptability of the mushroom samples was evaluated by 10 trained judges , according to the International Standards Organization method 8586-1 , using an = 9) ± standard error (SE). Fermentation of mushrooms was performed once by fermenting three parallel samples; all analyses of the parallel samples were performed in triplicate. Results of the overall acceptability and emotions induced for judges by the edible mushrooms were expressed as the mean (n = 10) ± standard error (SE). Results were analysed using the statistical package SPSS for Windows V15.0 . A linear Pearson’s correlation was used to quantify the strength of the relationship between the variables. Results were recognized as statistically significant at p ≤ 0.05.Results of the microbiological and physicochemical analyses were expressed as the mean , but lower a* coordinates , in comparison with brown-variety samples. In all cases (both white and brown varieties of mushroom), fermented samples showed lower L*, a*, and b* coordinates, in comparison with non-fermented ones. Multivariate analysis of variance showed that the variety of mushrooms is a significant factor in samples’ a* coordinates (p = 0.005). However, the LAB used for fermentation and mushroom variety, and the interaction of these factors were not significant on L* and b* coordinates. Colour characteristics and acidity parameters of the non-fermented and fermented mushroom samples are shown in This study’s results confirmed the data published by other authors, which indicate that lactic fermentation influences the colour parameters of mushrooms and reduces the lightness of mushroom fruiting bodies . During Lc. paracasei No. 244 strain . However, the analysed factors (variety of mushrooms and LAB strain used for fermentation) and their interactions were not significant on sample pH. The highest TTA was observed in white mushroom samples fermented with Lc. casei No. 210 and Lc. paracasei No. 244 strains (0.40° N). Despite the fact that a correlation between samples pH and TTA was not established, moderate positive correlations between samples TTA with L* and b* coordinates were found . Comparing the samples’ acidity parameters, the lowest pH was found in samples fermented with the Lc. paracasei No. 244 strain, which, in our previous studies, showed characteristics to ferment a broad spectrum of carbohydrates [Fresh mushrooms’ pH is neutral . The pH hydrates . Additiohydrates . LAB, ye10 CFU/g; yeast and fungi count in non-fermented mushrooms was 1.39 log10 CFU/g) . The LAB count in samples fermented with Lc. paracasei No. 244 and P. acidilactici No. 29 strains was, on average, 6.50 log10 CFU/g. In terms of LAB, in yeast and fungi counts in non-fermented mushrooms, significant differences were not established  . AdditioLp. plantarum [Lactobacillus delbrueckii subsp. bulgaricus, Levilactobacillus brevis, Lc. casei, Lactobacillus helveticus, Lactiplantibacillus pentosus, Streptococcus lactis, Lactococcus lactis, Leuconostoc mesenteroides, and Propionibacterium freudenreichii [Lp. plantarum 299v strain contributed to a rapid reduction of the pH value of mushrooms, and the finished products showed a high number of viable LAB cells.It was reported that many LABs can be applied for mushroom fermentation, including, lantarum ,47,48,49nreichii ,45,46,50nreichii  reported10 CFU/g. However, yeast and mould co-existed with LAB in the process of fermentation [p = 0.004). Venturini et al.  reportedentation . The negentation . Howeverentation . In our Microbial interactions play an important role in the diversity of microbial communities, which plays a key role in selecting functional microbiota and inhibiting the growth of non-desirable microorganisms. However, little is known about microbial interactions during the process of mushroom fermentation, which makes it difficult to control and manage. Although our previous studies showed that the LAB strains used in this experiment possess antifungal properties against some fungi , it can be that fungi that are present in mushroom masses are resistant to these LAB metabolites. Further studies are needed to indicate which fungi are predominant in mushrooms and to select the most appropriate LAB for their inhibition.Lc. casei No. 210 strain and in white variety mushroom samples fermented with Lc. plantarum No. 135 strain, tyramine was formed . The main BA in non-fermented and fermented mushroom samples was spermidine. However, the analysed factors were not significant on spermidine content in mushroom samples. Tryptamine, phenylethylamine, putrescine, cadaverine, and spermine were not found in mushroom samples . Howeverp ≤ 0.001). Positive moderate correlations were found between TTA and tyramine , as well as between TTA and spermidine . The amount of protein and the dry mass of fungal fruiting bodies could vary from 20 to 40% . Our preLp. plantarum 299v, while tyramine was not detected in samples fermented with Lp. plantarum EK3. Finally, this study showed that it is very important to select appropriate LAB strains for the fermentation of edible mushrooms to ensure their safety in the case of BA formation.Certain BA, including tyramine, can be produced by LAB species in different fermented foods . It was A. bisporus mushrooms was linoleic (C18:2), and the highest C18:2 content was established with Lp. plantarum No. 135 strain-fermented brown-variety samples . Additionally, a positive moderate correlation between C18:2 and sample TTA was established . Palmitic acid (C16:0) content in mushroom samples FA profile varied from 11.0 to 13.0% from the total fat content . C16:0 also showed a positive moderate correlation with samples TTA . The highest 9-octadecenoic acid (C18:1) content was found in Lp. plantarum No. 135 strain-fermented white-variety samples . However, with the same strain-fermented brown-variety samples showed the lowest C18:1 content . Between C18:1 content and LAB count in mushrooms, a negative weak correlation was established . Non-fermented brown-variety A. bisporus mushrooms showed the highest palmitoleic (C16:1) and stearic (C18:0) fatty acids content. C18:0 content in samples showed a positive weak correlation with yeast and fungi count . Eicosanoic (C20:0) and docosanoic (C22:0) FAs were found in 4 out of 10 tested mushroom samples, in non-fermented brown-variety mushrooms, in Lc. casei No. 210 strain-fermented white- and brown-variety mushrooms, and in Lp. plantarum No. 135 strain fermented brown variety samples. Multivariate analysis of variance showed that mushroom variety was a significant factor in C22:0 content (p ≤ 0.001); the LAB strain used for fermentation was a significant factor in C16:1, C20:0, and C22:0 content in mushrooms , and as the analysed factors’ interactions were significant on C20:0 and C22:0 content in mushrooms . The fatty acid (FA) content  of the mushroom samples is shown in Lp. plantarum No. 135 strain-fermented white-variety samples . The LAB strain used for fermentation, as well as the analysed factors’ interactions, were significant on MUFA content in mushroom samples . The highest polyunsaturated fatty acid (PUFA) content was found in Lp. plantarum No. 135 strain-fermented brown-variety samples ; however, the analysed factors were not significant on PUFA content in mushrooms. Omega-3 FAs were not found in mushrooms. The highest omega-6 content was observed in brown-variety A. bisporus fermented with the Lp. plantarum No. 135 strain, and the highest omega-9 content was observed in white-variety mushrooms fermented with Lp. plantarum No. 135 strain. Mushroom variety was a significant factor in omega-9 content in samples (p = 0.001). Positive moderate correlations between samples TTA and SFA, PUFA, and omega-6 were established .In comparison to FA profiles of the mushroom samples, the highest saturated fatty acids (SFA) content was found in non-fermented brown variety mushroom samples . However, the analysed factors and their interactions were not significant on SFA content in mushrooms. The highest monounsaturated fatty acid (MUFA) content was found in A. bisporus [A. bisporus lipids profile consists of myristic acid (C14:0) , C16:0 , C16:1 , C18:0 , C18:1n-9 , C18:2n-6 , α-linoleic acid (C18:3 n-3) , C20:0 , and C20:1 n-9 . It was reported that mushroom lipids consist mainly of MUFAs and PUFAs, which are classified as healthy sources of lipids . Althougbisporus . Stojkovbisporus  reportedA. bisporus comprises stearic acid, palmitic, linoleic, caprylic, oleic, erucic, and eicosanoic acids [A. bisporus [A. bisporus’s lipid profile. According to Saiqa et al., the FA profile of ic acids . Additiobisporus . As wellbisporus  reportedA. bisporus from the Netherlands and Portugal showed the highest levels of omega-3 and -9 [A. bisporus lipid profile, linoleic FA is predominant . Therefore, mushrooms are good sources of essential FA omega-6 [However, differences in FA profiles are obtained not just between the species but can also be related to a mushroom’s geographic origin . However3 and -9  and omeg3 and -9 , respect3 and -9  reported omega-6 . However omega-6 . Agaricus sp. can vary [A. bisporus fat content [It should be mentioned that the FA profile in cultivated and wild can vary  and the  content . A. bisporus FA profiles, as well as their changes during fermentation.Additionally, the metabolism of LAB can favour lipid oxidation during fermentation or exert strong antioxidative effects . HydropeLp. plantarum No. 135 strain-fermented white-variety A. bisporus, 1-octen-3-ol was not detected. The LAB strain used for fermentation was a significant factor in 1-octen-3-ol content in mushrooms (p = 0.004). In comparing non-fermented white-variety mushrooms with fermented ones, different tendencies in the benzyl alcohol content were established. In white variety samples fermented with Lc. casei No. 210 and Lc. paracasei No. 244 strains, on average, 1.81 times higher benzyl alcohol content was found. However, in samples fermented with Lp. plantarum No. 135 strain, on average, 27.3% lower benzyl alcohol content was established in comparison with non-fermented samples. White-variety samples fermented with the P. acidilactici No. 29 strain showed similar benzyl alcohol content compared to non-fermented ones. When comparing non-fermented brown-variety samples with fermented ones, samples fermented with the Lc. casei No. 210 strain showed similar benzyl alcohol content compared to non-fermented ones; in samples fermented with the Lp. plantarum No. 135 strain, on average, 37.5% lower benzyl alcohol content was established; samples fermented with Lc. paracasei No. 244 and P. acidilactici No. 29 strains showed, on average, 34.1 and 41.3%, respectively, higher content of benzyl alcohol in comparison with non-fermented samples. The analysed factors and their interactions were not significant in benzyl alcohol content in mushroom samples. Volatile compound profiles of the mushroom samples  are shown in p = 0.001). 2,3-Butanediol was established in the Lc. casei No. 210 strain and in the Lp. plantarum No. 135 strain-fermented brown-variety mushrooms, as well as in Lp. plantarum No. 135 strain- and in with P. acidilactici No. 29 strain-fermented brown variety samples. The analysed factors’ interactions were significant in 2,3-butanediol content in mushrooms (p = 0.027). Benzaldehyde was found in most of the mushroom samples, except in P. acidilactici No. 29 strain-fermented white-variety samples, and the LAB strain used for fermentation as well as the analysed factors’ interactions were significant in benzaldehyde content in mushroom samples . In most of the samples, 3-octanone content was reduced after fermentation, except in P. acidilactici No. 29 strain-fermented brown-variety mushrooms. The LAB strain used for fermentation as well as the analysed factors’ interactions were significant in 3-octanone content in mushrooms (p ≤ 0.001). Ethylhexanol was found in both non-fermented samples as well as in brown-variety mushrooms fermented with Lc. casei No. 210 and Lp. plantarum No. 135 strains. All analysed factors and their interactions were significant in ethylhexanol content in samples (p ≤ 0.001). Benzeneacetaldehyde was identified in most of the mushroom samples, except in Lc. paracasei No. 244-fermented white-variety samples, and mushroom variety as well as the analysed factors’ interactions were significant in benzeneacetaldehyde content in samples . In all the cases, a higher content of nonanal was found in fermented samples, and all analysed factors and their interactions were significant in the volatile compound content in mushrooms . Similar tendencies of dodecane content were established, and in all the cases, a higher content of dodecane was found in fermented samples in comparison with non-fermented ones. The LAB strain used for fermentation and the analysed factors’ interactions were significant on dodecane content in mushrooms . Benzo-2,3-pyrrole was found in 3 out of the 10 analysed sample groups, and all analysed factors and their interactions were significant on this volatile compound content in mushrooms . In all the cases, fermentation increased tetradecane content in mushrooms’ volatile compounds profiles, and all analysed factors and their interactions were significant in tetradecane content in samples . In comparison to 2,4-bisphenol content in non-fermented and fermented samples, in most of the white-variety samples, this volatile compound content was increased (except samples fermented with P. acidilactici No. 29 strain). However, in brown-variety samples, after fermentation with Lp. plantarum No. 135 and P. acidilactici No. 29 strains, on average, 2.25 and 1.4 times, respectively, lower 2,4-bisphenol content was found in comparison with non-fermented samples. The LAB strain used for fermentation and the analysed factors’ interactions were significant in 2,4-bisphenol content in mushrooms . Acetoin was found in 3 out of 10 analysed samples and the LAB strain used for fermentation was a significant factor in this volatile compound content in mushrooms  was fermented with Lp. plantarum. Compared to naturally fermented P. eryngii, similar contents of alcohols and higher content of aldehydes, ketones, and esters were found in P. eryngii fermented with Lp. plantarum.The volatile compound profile of  species . During  species . To the g et al. , the ediLc. paracasei and after fermentation of Allomyrina dichotoma larvae with Lactobacillus acidophillus and Lp. plantarum [Eight-carbon compound 1-octen-3-ol is a characteristic volatile of button mushrooms that provides a “mushroom-like, earthy, and buttery” odour and is generated during the enzymatic degradation of linoleic FA . A signilantarum ,84.A. bisporus, whose content significantly decreases after heat treatment [Amino acid-derived benzyl alcohol, with its aromatic, floral, fruity, and sweet odour, is another dominant volatile in white and brown reatment ,81,85. Hreatment .A. bisporus after heat treatment [A. bisporus samples can be related to the LAB carbohydrate metabolism [It was reported that the content of such ketones as 3-octanone , which is generated via enzymatic oxidation of linoleic or linolenic acids, reduces in reatment . Moreovereatment . The pretabolism .A. bisporus species [A. bisporus, Volariella volvacea and Cortinarius odorifer mushrooms and provides floral and cedrol woody notes [Chlorella sp. PR-1 and possesses biocidal activity [The amino acid phenylalanine oxidation metabolites are precursors of benzaldehyde  and benzene acetaldehyde  . Dodecan species . Nonanal species . 2-ethyldy notes ,90,91. 2activity ,93.Lp. plantarum No. 135 strain . The LAB strain used for fermentation and the interactions of the LAB strain used for fermentation and mushroom variety were significant in the intensity of the emotion “happy” induced in judges by mushroom samples . Additionally, between the intensity of the emotion “happy” and volatile compounds 1-octen-3-ol and 3-octanone, positive moderate correlations were found . Additionally, between the intensity of the emotion “happy” and the samples’ pH, a positive weak correlation was established . The overall acceptability and emotions induced for judges by mushroom samples are shown in p ≤ 0.001), and the highest expression of the emotion “sad” was found with Lp. plantarum No. 135 strain-fermented brown-variety mushroom samples and with P. acidilactici No. 29 strain-fermented white-variety mushroom samples . Positive weak and moderate correlations between the intensity of the emotion “sad” and the volatile compounds acetoin and 2,3-butanediol were found . Additionally, negative correlations between the intensity of the emotion “sad” and benzaldehyde; 1-octen-3-ol; 3-octanone; nonanal; dodecane; and tetradecane were established . The LAB strain used for fermentation and the interactions of the LAB strain used for fermentation and mushroom variety were significant in the intensity of the emotion “sad” induced in judges by mushroom samples (p ≤ 0.001), and the lowest expression of the emotion “angry” was found with white-variety mushroom sample groups fermented with Lc. casei No. 210, Lc. paracasei No. 244, and P. acidilactici No. 29 strains. Positive correlations between the expression of the emotion “angry” intensity and the volatile compounds benzaldehyde; benzeneacetaldehyde; nonanal; benzo-2,3-pyrrole; and tetradecane were found . All the analysed factors and their interactions were significant in the intensity of the emotion “angry” induced for judges by mushroom samples phenol), TTA, and tyramine content, significant correlations were established . Additionally, the interaction of the analysed factors was significant in the intensity of the emotion “scared” induced in judges by mushroom samples (p = 0.016). The highest intensity of the emotions “surprised” and “scared” were found with brown-variety mushroom sample groups fermented with p = 0.013; LAB strain used for fermentation p = 0.001; factors interaction p ≤ 0.001), and the highest expression of the emotion “disgusted” was found with white-variety mushroom sample groups fermented with the Lc. paracasei No. 244 strain. Despite that the analysed factors were not significant in the intensity of emotion “contempt” induced in judges by mushroom samples, between the expression of the emotion “contempt” and some volatile compounds , TTA, and tyramine content, significant correlations were established . All the analysed factors and their interactions were significant in the intensity of the emotion “disgusted” induced for judges by mushroom samples . Finally, most of the tested samples’ valences were negative, except for the non-fermented white mushroom variety and Lactiplantibacillus pentosus for 18 days received scores of 7–8 for overall acceptability. In the study of Jabłońska-Ryś et al. [Lp. plantarum 299v and Lp. plantarum EK3 was applied to A. bisporus and the scores of overall acceptability were lower compared to those obtained in our study. The evaluation of food-elicited emotions is an expanding field of interest in the food sector, as the emotional profile can be employed to differentiate foods with identical hedonic scores and plays an important role in estimating consumer purchase intentions [®, of extruded snacks were notably affected when mushrooms Volvariella volvacea and Pleurotus pulmonarius were incorporated into the snack formula.Usually, mushrooms are incorporated into other food products, and their sensory profile and acceptability are evaluated . There aś et al. , 7 days tentions ,96. MushA. bisporus as well as volatile, BA, FA, and elicited emotion profiles. Fermented mushrooms had a darker colour and a higher count of viable LAB, but similar yeast and fungi counts compared to non-fermented ones. The total BA content in fermented A. bisporus was below the maximum level suggested for foods, and in most cases, fermented samples showed a higher content of polyunsaturated FA. The main volatile compounds in mushroom samples were 1-octen-3-ol and benzyl alcohol. The overall acceptability was similar for both non-fermented and fermented mushrooms. The LAB strain used for fermentation and the A. bisporus variety were significant factors in most of the elicited emotion intensities in the judges. This research provided beneficial information that can be further applied to the industrial production of fermented mushrooms. Furthermore, a wider spectrum of LAB for A. bisporus fermentation can be studied in the future to recommend the safest and most acceptable product for the consumer as well as the most appropriate LAB strain for the mushroom fermentation industry.Fermentation with No. 210, No. 135, No. 244, and No. 29 strains induced changes in the physical properties of


Polygonatum cyrtonema Hua is a perennial herbaceous plant of the Polygonatum genus, belonging to the Liliaceae family, with significant medicinal and nutritional value. In China, this species is a traditional medicinal and edible herb with a long history of application and is widely appreciated by the people. However, as the demand for medicinal herbs continues to grow, excessive harvesting has led to the depletion of wild resources and the risk of genetic erosion. In addition, the chaotic cultivation of varieties and the lack of high quality germplasm resources have led to inconsistent quality of medical materials. Therefore, it is urgent to conduct genetic diversity evaluation of this species and establish a sound conservation plan. This study assessed the genetic diversity and population structure of 96 samples collected from seven regions in China using the simple sequence repeat (SSR) molecular marker technology. In this study, a total of 60 alleles (Na) were detected across the 10 polymorphic SSR markers used, with an average of 6.0 alleles generated per locus. The values of polymorphic information content (PIC) values ranged from 0.3396 to 0.8794, with an average value of 0.6430. The average value of the effective number of alleles (Ne) was 2.761, and the average value of the Shannon’s information index (I) was 1.196. The population structure analysis indicates that the Polygonatum cyrtonema Hua germplasm can be classified into three subpopulations  at the molecular level, which corresponds to the previous subgroups identified based on individual plant phenotypic traits. Analysis of Molecular Variance (AMOVA) showed that 74% of the genetic variation was between individuals within populations in different regions. The phylogenetic analysis of the 96 germplasm samples divided them into three main populations. The QY and JD subpopulations are largely clustered together, which could be attributed to their mountainous distribution and the local climate environment. The genetic differentiation coefficient (Fst) value was low at 0.065, indicating relatively low population differentiation. The ratio of the genetic differentiation coefficient (Fst) between the JZ population and the other two populations (QY and JD) is much higher than the ratio between the QY and JD populations. Based on the clustering results and the ratio of the genetic differentiation coefficient (Fst), it can be inferred that the genetic relationship between the QY and JD subpopulations is closer, with a certain degree of genetic differentiation from the JZ subpopulation. This study supports the conservation of germplasm resources of Polygonatum cyrtonema Hua in China and provides new parental material for germplasm genetic improvement and breeding programs. Polygonatum in China [Polygonatum. The Polygonatum cyrtonema Hua (P. cyrtonema) is widely distributed in the Yangtze River basin, and Anhui Province is one of the main production areas, of which Qingyang County is the Geo-Authentic Product Area.There are more than 30 species of in China , which iP. cyrtonema is distributed in Qimen, Shitai, Yixian, Xiuning, Shexian, Tongling, and Jinzhai, among others. These areas are all located in the southern and western mountainous regions of Anhui Province. Currently, the highest quality P. cyrtonema is produced in Jiuhua Mountain and its surrounding areas in Qingyang County, southern Anhui. The plant, with a medicinal history in China spanning over 2000 years, is recognized as one of the source plants for the medicinal herb rhizoma polygonati specified in the Pharmacopoeia of the People’s Republic of China [According to the "Flora of Anhui"  the P. cof China . With itof China , immune of China , blood lof China , memory of China , anti-tuof China , and antof China . Moreoveof China .P. cyrtonema has gradually become scarce, posing a risk of genetic erosion. Therefore, it is essential to evaluate the genetic diversity and kinship among populations to provide valuable references for the establishment of conservation programs.With the increased demand for the medicinal herbs of rhizoma polygonati and the investigation of its edible value, the related industry has seen rapidly increased growth . RelatedP. cyrtonema are closely associated with the geographical areas [P. cyrtonema at the molecular level to validate the previous research on phenotypic traits and establish a correlation. The objective is to establish a solid foundation for the conservation of resources, breeding programs, and industrial development of this species.Wild germplasm resources of the species are mainly found in the Dabie Mountain system in western Anhui, and the Jiuhua Mountain system and the Huangshan Mountain system in southern Anhui. The variations in plant traits of al areas . Throughal areas . The phePolygonatum, and since then, molecular biology techniques such as RAPD [P. cyrtonema, a study has confirmed the viability of employing transcriptome data from this species for the development of SSR markers [As a result of gene interactions, multiple genotypes interact in a similar manner, giving rise to similar phenotypic phenomena , 16. Som as RAPD , SRAP [3 as RAPD , SCoT [3 as RAPD , ISSR [3 as RAPD , 40 and  as RAPD , 42, whi markers .Simple Sequence Repeats (SSR), also known as Microsatellites, are a class of short sequence repeat units that exist widely in the genome. SSRs exhibit high variability and genetic polymorphism in the genome. They are analyzed by PCR amplification of the SSR region in the target sequence and subsequent analysis of the size differences in the amplified products. SSR are favored for their advantages of co-dominant inheritance, abundance in the genome, high polymorphism, high repeatability among closely related species, and transferability . SSR marP. cyrtonema germplasm resources. The objectives are to elucidate the kinship and population genetic structure of germplasm resources from different sources and evaluate the diversity of germplasm resources more effectively. To be more specific, based on previous studies on traits, this research aims to provide genetic background information and novel materials for breeding high-quality germplasm resources with stable agronomic traits, high yield, and strong stress resistance. In summary, this study provides fundamental data for the conservation and utilization of the genetic resources of this species, offering valuable references for future resource management, conservation, and molecular marker-assisted breeding.Therefore, in this study, the SSR molecular marker technology was selected for optimizing the analysis of genetic diversity in P. cyrtonema. The original plant was identified by Prof. Nianjun Yu and Prof. Helin Liu of Anhui University of Traditional Chinese Medicine as P. cyrtonema. More than 10 individuals were collected from each area . Fresh young leaves were collected, dried with silica gel, and stored in an ultra-low temperature refrigerator at -70°C for backup. A voucher specimen of this plant was stored in the Center of Herbarium, Anhui University of Chinese Medicine, Hefei, China . The sample collection information is shown in  method, commonly used for extracting plant genomic DNA, yields high-quality and high-purity DNA suitable for molecular biology research, molecular marker analysis, gene cloning, and other applications . The met-1), 5 μL of MIX enzyme(2×M5 HiPer plus Taq HiFi PCR mix (with blue dye)), and ddH2O added to achieve a total system volume of 10 μL. The amplification program included an initial denaturation step at 96°C for 3 minutes, followed by denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 1 minute. This was repeated for a total of 20 cycles. After the cycling, there was a final extension step at 60°C for 10 minutes, followed by storage at 12°C. The amplified product was stored in a -20°C refrigerator.Based on the relevant studies, 50 pairs of primers were screened in this paper , 54–58, TM is a highly integrated and fully automated capillary electrophoresis system used for the analysis and quantification of biological molecules such as DNA, RNA, and proteins. It utilizes microfluidic technology and proprietary analysis software to measure the size, concentration, and quality of target molecules rapidly and accurately in the sample. The instrument is equipped with a proprietary analysis software called ProsizeTM2.0, which allows for real-time monitoring and analysis of the electrophoretic data from the samples. It can generate high-quality electrophoresis profiles, quality scores, and quantitative data, and provides visual representation of the data. Therefore, in this study, the Fragment AnalyzerTM automatic capillary electrophoresis system was used for sequencing, and the ProsizeTM2.0 software was applied to score the polymorphic DNA bands in the electrophoresis profiles [The Fragment Analyzerprofiles .P. cyrtonema, and the primer details are shown in , major allele frequency (MAF), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) were calculated using Power Marker software , 61, andshown in .Genetic diversity was assessed using the allelic binary data set obtained from the ten polymorphic SSR markers. GenAlEx V.6.5 is a commonly used genetic data analysis software that provides a range of analysis tools and statistical methods for handling various types of genetic data, including microsatellite (SSR) markers, nucleotide sequence data, SNP data, and more. The Shannon’s information index (I) and the number of effective alleles(He) were determined with GenAlEx V.6.5 software . PowerMahttp://taylor0.biology.ucla.edu/structureHarvester/) website [NTSYS-PC 2.1 is a software used for phylogenetic analysis and classification studies based on genetic marker data. In this study, the NJ phylogenetic tree was constructed using this software. The genetic structure of 96 germplasm was then analysed using STRUCTURE software  based on website . The bes website , 71.P. cyrtonema.A total of 10 pairs of SSR primers with good polymorphism were screened from 50 pairs of primers, and the detection information is shown in . Based oP. cyrtonema from seven regions were initially divided into three populations , and the data of genetic diversity analysis by SSR amplification are shown in  and mean expected heterozygosity (He) of the 96 germplasm were 0.458 and 0.570, respectively. The mean observed heterozygosity (Ho) was 0.428, 0.66, and 0.481 for the JZ, QY, and JD populations, respectively, and the mean expected heterozygosity (He) was 0.568, 0.574, and 0.568, respectively. The QY population had the highest observed heterozygosity and expected heterozygosity, and all three populations had lower observed heterozygosity than expected heterozygosity, indicating low intra-population gene exchange.Genetic differentiation between population and principal coordinate analysis. Analysis of Molecular Variance (AMOVA) showed a highly significant difference (P<0.001), the results of AMOVA analysis are shown in  and ΔK maximum, setting the population size K as 2 to 10. The results showed that ΔK was maximum when K = 3  were identified (The Neighbor-Joining (NJ) method is a clustering method based on a distance matrix used for constructing phylogenetic trees and inferring evolutionary relationships among species. It has the advantages of being simple, intuitive, computationally efficient, and widely applicable. In this study, an NJ evolutionary tree of 96 entified . ClusterThe present study employed fluorescence-labeled capillary electrophoresis technique for detection and analysis, which offers high sensitivity, high resolution, flexibility, and wide applicability. This technique allows for multi-color labeling, multi-channel analysis, and real-time monitoring , 74. In Fluorescence labeled capillary electrophoresis technology does not require gel making and silver staining, which saves time and effort and realizes automated data processing and reduces human errors. The size of amplified fragments can be accurately calculated, and data from different batches of reactions can be processed uniformly. High-throughput detection of samples and high-throughput detection of loci can be performed. Comparative studies on the detection of gene mutation sites and SSR sites have also demonstrated that capillary electrophoresis technique is superior , 78. In P. cyrtonema and closely related species. And 10 primer pairs with high polymorphism were screened from the collected primers. The 10 primer pairs produced major allele frequencies(MAF) ranging from 0.1823 to 0.7083 with a mean value of 0.4370 on the 96 germplasm polymorphisms studied. A genetic differentiation analysis study on six species of Polygonatum based on 49 SSR markers found that the major allele frequencies (MAF) ranged from 0.40 to 0.98, with a mean value of 0.77 [In this study, we collected a total of 50 primer pairs from the previously published studies on  of 0.77 . This re of 0.77 , 82. Ada of 0.77 .Polygonatum sibiricum Red. germplasm materials. The average number of alleles (Na) was 1.9843, the average effective allele number (Ne) was 1.5278, and the polymorphic information content (PIC) was 0.46 [Polygonatum. In contrast, a related study using only P. cyrtonema as the study material reported that the number of alleles (Na) ranged from 2–11 with a mean value of 5.916, and the polymorphism information content (PIC) ranged from 0.232–0.851 with a mean value of 0.585 [P. cyrtonema germplasm from similar geographic regions, different primer types, and the amount of germplasm we used. And the results of Chen (He = 0.619 Ho = 0.458) [In this study, the number of alleles detected per core primer pair was 3.000–8.000, the average number of alleles (Na) and effective alleles (Ne) were 6.000, 2.761, respectively, and the polymorphic information content (PIC) ranged from 0.3396–0.8794 with a mean value of 0.6430. Some scholars used 12 primer pairs for SSR polymorphic locus analysis of 32 wild was 0.46 . The smawas 0.46  were lowof 0.585 , which iof 0.585 , and theof 0.585  was sign= 0.458)  were cloThe higher values of QY allele (Na), effective allele (Ne) and Shannon’s Information Index (I) in the three populations  indicate that the QY population has the highest genetic diversity and more complex genetic background. It may be caused by the fact that Qingyang is the main cultivation area with many exotic germplasm cultivars. The expected heterozygosity (He) was higher than the observed heterozygosity (Ho) in all three populations, suggesting a low intra-population gene exchange. Low levels of gene flow can lead to increased genetic drift, resulting in the loss of beneficial genes, accumulation of harmful genes, and reduced genetic diversity . AdditioP. cyrtonema [The differences in these correlation parameters among the three populations were generally not significant, with two populations, QY and JD, being closer to each other and relatively more different from the JZ population. The genetic differentiation coefficients Fst and ΦST indicated that there was some degree of genetic differentiation between the QY, JD populations and the JZ population, while no significant genetic differentiation was produced between the QY and JD populations. This is consistent with the analysis of molecular variation (AMOVA) showing the lowest percentage of genetic variation among populations (7%). The richness of genetic diversity in a population has significant impacts on its ability to adapt to environmental changes, resistance to diseases, population stability, and genetic health –90. The yrtonema . Whereasyrtonema , 58. In yrtonema , 58.The genetic structure analysis showed that the 96 germplasm could be divided into three subgroups, and the results were the same as the clustering results of the diversity study of morphological traits. However, there was a small amount of admixture in the three gene pools, indicating that there was some gene exchange between different populations and that these materials had the same or similar parents. The NJ cluster analysis did not show clear boundaries between germplasm from different geographical regions. However, the QY and JD populations in southern Anhui are relatively distantly related to the JZ population in western Anhui, and there are obvious geographical features. It suggests that geographic isolation and ecological differences due to different geographic distributions may be important factors contributing to this result. The genetic grouping was closely related to the mountain range, and the different origins of this species were genetically distant and did not cluster into one group. This indicates that intraspecific genetic diversity is related to geography, and these studies are consistent with the results of the present study. Dabie Mountain has a high altitude, relatively dry climate, relatively monotonous vegetation types, and is relatively barren. On the other hand, the southern Anhui mountainous region features complex topography, a more humid climate, diverse vegetation types, and soils that have good water retention capacity and fertility. Among the top eight ecological factors that have the most significant impact on the growth of this species, include rainfall, vegetation type, and soil type. These environmental differences may be important factors leading to a certain degree of genetic differentiation between the populations in southern Anhui  and western Anhui (JZ).P. cyrtonema. This may be one of the important reasons for the abundant genetic diversity and high medicinal quality of the QY population. In addition, the conversion of many wild resources into cultivation, the continuous use of rhizomes for asexual propagation and excessive fertilization may all be responsible for the differences in plant morphology.The QY and JD populations belong to the same mountainous area in southern Anhui, and the genetic differentiation between them is low, but there are obvious differences in plant morphology. This could be attributed to the fact that the QY population belongs to the Jiuhua Mountain range, while the JD population belongs to the Huangshan Mountain range, which exhibit variances in vegetation and ecological environments. Both Mount Jiuhua and Mount Huangshan are influenced by the monsoon climate, and they share many similarities in terms of their ecosystems and climatic environments. This could potentially lead to lower genetic differentiation between the two populations. Mount Jiuhua belongs to a subtropical humid climate and is primarily characterized by subtropical deciduous broad-leaved forests and mixed forests. On the other hand, Mount Huangshan belongs to a temperate humid climate, and its ecosystem includes subtropical evergreen broad-leaved forests and coniferous forests. Based on previous studies on ecological factors, the more abundant rainfall on Mount Jiuhua may be the main factor leading to the differences in plant traits between the two populations. The unique geographical landforms, climatic conditions, and vegetation types of Mounts Jiuhua create a shaded, cool, and humid environment. This environment aligns with the shade-loving and moisture-loving growth habits of P. cyrtonema population has a high genetic diversity. The population exhibits high genetic diversity, with most of the variation occurring within the population. The QY population has the highest genetic diversity and has great potential for use in resource conservation and breeding material selection. The genetic structure of populations tends to correlate with environmental factors such as geographical distribution and climatic conditions. The limited number of SSR primers restricts adequate coverage of the target species’ genome, thus compromising the analysis of diversity and accuracy of genetic analysis to some extent. Although the number of markers used in this study was small, but 10 pairs of SSR primers with highly polymorphic loci and abundant genetic variability in 96 germplasm were suitable for the study of genetic diversity of P. cyrtonema, which can still provide a reference for germplasm selection and resource conservation. In future studies, whole genome sequencing, population dynamics analysis, screening for adaptive genes and studying the interaction between environment and genetics will be of great importance for this species.Our research study confirms that the Chinese S1 Appendix(DOCX)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Global climate change poses a grave threat to biodiversity and underscores the importance of identifying the genes and corresponding environmental factors involved in the adaptation of tree species for the purposes of conservation and forestry. This holds particularly true for spruce species, given their pivotal role as key constituents of the montane, boreal, and sub-alpine forests in the Northern Hemisphere.Picea crassifolia. Our comprehensive analysis employing ADMIXTURE, principal component analysis (PCA) and phylogenetic methodologies showed that the species has a complex population structure with obvious differentiation among populations in different regions. Concurrently, our investigations into isolation by distance (IBD), isolation by environment (IBE), and niche differentiation among populations collectively suggests that local adaptations are driven by environmental heterogeneity. By integrating population genomics and environmental data using redundancy analysis (RDA), we identified a set of climate-associated single-nucleotide polymorphisms (SNPs) and showed that environmental isolation had a more significant impact than geographic isolation in promoting genetic differentiation. We also found that the candidate genes associated with altitude, temperature seasonality (Bio4) and precipitation in the wettest month (Bio13) may be useful for forest tree breeding.Here, we used transcriptomes, species occurrence records, and environmental data to investigate the spatial genetic distribution of and the climate-associated genetic variation in Our findings deepen our understanding of how species respond to climate change and highlight the importance of integrating genomic and environmental data in untangling local adaptations.The online version contains supplementary material available at 10.1186/s12870-023-04539-7. In recent years, there has been increased interest in the effects of natural selection on local populations, particularly in the context of climate change and its potential impacts on biodiversity –3. AcrosPicea is a prime example of a dominant tree species found mainly in the northern temperate forest ecosystems of Asia, North America, and Europe [Conventional ways for studying local adaptation, such as reciprocal transplant or common garden experiments –20, posed Europe , 33.Picea crassifolia, an endemic, ecologically important tree species in China, to investigate its local adaptation and potential response to climate change. P. crassifolia is a highly-valued afforestation tree in northwest China due to its superior wood quality, serving as a source of wood for bridges, furniture and papermaking. P. crassifolia thrives at elevations ranging from 1,750 to 3,100 m, inhabiting mountainous areas characterized by overcast and semi-overcast slopes, as well as wet valley. While past studies have focused on the speciation between P. crassifolia and closely-related species, little is known about the species' adaptation to climate change and how its distribution may change in the future [In this study, we focused on e future , 35.P. crassifolia, we conducted a transcriptome analysis on 82 individuals derived from 16 populations of this species across Gansu Province, Qinghai Province, and their surrounding areas in China. The aims of this study were (i) to examine genetic differentiation and population structure in P. crassifolia, (ii) to assess the respective influence of environmental and geographic factors on inter-population genetic differentiation while identifying key environmental variables that drive local adaptation, and (iii) to quantify the future potential distribution of this species under projected future climate scenarios.To investigate the spatial pattern of genetic variation and the underlying genetic mechanisms of local adaptations in P. crassifolia was acquired through the NCBI database (NCBI BioProject Accession ID PRJNA846694). Additionally, we obtained the same data for a P. wilsonii individual from NCBI (NCBI BioProject Accession ID PRJNA401149) to serve as an outgroup. Each population consisted of a range of 3–10 individuals, and detailed information regarding RNA extraction, library construction and sequencing protocols can be found in the works of Feng et al. (2023) and Ru et al. (2018). The locations of the 16 populations are visually represented in Fig. Primary raw RNA-seq data encompassing 82 individuals from 16 populations of Picea abies [http://broadinstitute.github.io/picard/) and were excluded from downstream analysis. Indels were detected and realigned using the “RealignerTargetCreator” and “IndelRealigner” tools from GenomeAnalysisTK v3.8 [The paired-end raw reads were filtered and trimmed with fastp v0.20.0 . This inea abies  using thea abies  with defsTK v3.8 .For variant calling, we utilized the “mpileup” command from SAMtools v0.1.19 , incorpohttp://www.iqtree.org), employing the Akaike information criterion (AIC). GTR + F + R3 was identified as the best-fitting model. Subsequently a maximum likelihood (ML) tree of 82 individuals was constructed using IQ-TREE v2.0.3 [P. wilsonii was set as an outgroup based on SNP data set.The best nucleotide substitution model was determined using ModelFinder  and implE v2.0.3  with 1, To identify genetic clusters, a model-based genetic clustering analysis was conducted using ADMIXTURE v1.3.0 . The SNPπ) in non-overlapping 100 bp windows. Pairwise genetic differentiation (FST) per locus was calculated using VCFtools v0.1.14 [Using VCFtools v0.1.14 , we calc v0.1.14  to estimP. crassifolia, we collected 56 environmental variables . All 55 environmental variables (averaged over the period from 1970–2000) and the topographic variable  were downloaded from Worldclim v2.1 (https://www.worldclim.org) at a spatial resolution of 30 arc-seconds. Species occurrences data were obtained from the Chinese Virtual Herbarium , the Global Biodiversity Information Facility  and field surveys. To avoid overfitting the niche models due to spatial autocorrelation of records [To investigate the local adaptation of  records , we rare records  in ArcGIr) among 56 environmental variables from 85 localities and eliminating one variable from each pair where |r|> 0.7. Consequently, 6 final environmental variables were selected, including Bio3 , Bio4 , Bio14 (precipitation in the driest month), Bio15 , Wind8 (wind speed in August) and Alt .Multicollinearity was avoided by examining the Pearson correlation coefficients (P. crassifolia. A niche comparison was conducted using the niche overlap tool in ENMTools v1.4.4 [D and standardized Hellinger distance (I). Both D and I had values ranging from 0 (no overlap) to 1 (complete overlap). The niche identity test was used to determine whether ecological niche modeling habitat suitability scores generated by the pairs of the three groups demonstrated statistically significant divergence from each other. The null distribution was generated by repeating the randomization procedure 100 times and comparing observed niche overlap values with the null distribution using a one-tailed test.Next, the maximum entropy approach was implemented in MAXENT v3.4.4  to deters v1.4.4  to quanthttps://www.worldclim.org) database at a resolution of 30 arc-seconds.Further, ecological niche modeling was utilized to predict potential distributions for all individuals both today and in the future . To predict future distribution, future climate layers from the SSP-126 and SSP-585 scenarios under the Sixth Phase of the Coupled Model Intercomparison Project (CMIP6) were downloaded from the WorldClim v2.1  were selected and analyzed.r) among the 56 environmental variables from our 16 sampling sites, eliminating one variable in each pair where |r|> 0.7. To calculate pairwise geographic distances, we utilized the geographic coordinates of the sampling sites and employed the Geosphere v1.5–16 R package .To illustrate the influence of IBD and IBE on genetic composition, we calculated the relationship between environmental distance, geographic distance and genetic distance. To address multicollinearity, we assessed the pairwise Pearson correlation coefficients (https://github.com/vegandevs/vegan).To calculate environmental distance (Euclidean distance) among the 16 populations, we employed the retained environmental variables and utilized the “dist” function in R software. We use Arlequin v3.5  to calcuGiven that the genetic variation explained by environmental variables was greater than that explained by geographic variables (see results), only SNPs associated with environmental variables were identified. An RDA was conducted to identify candidate SNPs involved in local adaptations responding to the multivariate environment , 51. PriArabidopsis thaliana protein database using DIAMOND v0.9.14 [–5. Gene ontology (GO) enrichment analyses were conducted with the ClusterProfiler v4.2.2 R package [p-values, and GO terms with corrected p-values ≤ 0.05 were considered significantly enriched.To determine the function of candidate loci, we searched for homologous genes in the non-redundant  v0.9.14  with an  package . BenjamiP. abies was used as the reference for variant calling, and 120,855 high-quality SNPs were obtained after strict filtering of the initial variant loci.The total raw data comprising 82 individuals amounted to 677.06 Gb, with an average of 8.26 Gb per individual. After filtering the raw data, 645.44 Gb of clean data was obtained. The reference transcriptome of P. crassifolia in Gansu and Qinghai provinces  for the overall population and for each group, we also determined the number of SNPs exclusive to each group, the shared SNPs, and the FST values between groups. The π value at the species level was 0.003364, while the π of the three groups ranged from 0.003658 to 0.003843, with the highest in the QH group and the lowest in the GS group (Table Pinus densata (π = 0.0028), Pinus yunnanensis (π = 0.0024), Picea purpurea (π = 0.00392) and Picea wilsoni (π = 0.00392) [FST values ranged from 0.013994 (QL vs. GS) to 0.023407 (QL vs. QH)  , 55. AmoP. crassifolia. A total of 85 occurrence records for P. crassifolia were obtained using our rigorous methods, with 20 records belonged to the QH group, 26 to the GS group, and 39 to the QL group  generated by Maxent runs were below the critical values for null distributions concerning group pairs, indicating high niche differentiation in group pairs , which predicts a more pronounced warming trend, the potentially suitable habitat for P. crassifolia faces even greater threats  exerted a stronger influence on genetic structure than did geographic distance  . Likewise, when controlling for geographic variables , environmental variables significantly influenced genetic variation, explained 3.41% of genetic variation alone . The combined effect of confounded geographic and environmental variables accounted for 0.86% of the genetic variation. The total genetic variation explained by the model incorporating both geographic and environmental variables (model gen. ~ geo. + env.) was 6.60%. Remarkably, environmental variables  explained a higher proportion of genetic variation compared to geographic variables . In summary, both geographic and environmental variables significantly influenced the genetic differentiation of the P. crassifolia population. However, the impact of environmental variables was more pronounced than that of geographic variables.To investigate the effects of IBE on genetic structure, we selected five environmental variables  for further analyses. Subsequently, we used the partial Mantel test to examine the relationship between geographic/environmental distance and genetic distance. The results revealed a significant relationship between genetic distance and both geographic distance and environmental distance. Notably, environmental distance (01) Fig. . We furtp ≤ 0.001) between the SNP matrix and environmental variables along the first three axes. Genetic variation was explained by RDA axes 1, 2, and 3 at rates of 28.3%, 23.7%, and 19.9%, respectively. RDA axis 1 divided 16 P. crassifolia populations into the QH group and the QL + GS group, and RDA axis 2 separated the QL group from the QL + GS group , we extracted SNPs loaded on the axis tails.In our analysis, SNP loadings were calculated from the first three significant constrained axes , and the histograms of the loadings on each RDA axis appeared to follow a relatively normal distribution  among the three P. crassifolia groups, though the cluster π values were notably higher than the species-level value , and GO analysis showed that the most representative terms were related to GTP binding, rRNA processing, GTPase activity, and proton-transporting ATP synthase activity , and enrichment analysis showed that Golgi apparatus, glycosyltransferase activity, GTPase activity and protein transport were the most representative terms , the greatest Annual Precipitation (Bio12), and the smallest Temperature Annual Range (Bio7) , relative to the current situation  of Picea crassifolia. Additional file 2. Cross-validation error plot of each K (K = 2 to 10). Additional file 3. The significant environmental and geographic variables retained by the initial step-forward selection method. Additional file 4. Histograms of SNPs loaded on the first three significant RDA axes.Additional file 5. Environmental associated loci (EALs) identified using RDA. Additional file 6. GO enrichment for 73 candidate genes for the QL group. Additional file 7. GO enrichment for 686 candidate genes for the QH group.


Three main genetic pools were identified within this collection , and the commercial varieties were located across the pools. The ploidy level was the main driver of pool identification, followed by a robust representation of accessions from Phureja and Andigenum cultivar groups based on former taxonomic classifications. We also found divergent heterozygosity values within genetic groups, with greater diversity in genetic groups with tetraploids  than in diploid accessions (CCC_Group_A: 0.14). We subsequently generated one mini-core collection size of 3 percent (39 entries) and three further core collections sizes of 10, 15, and 20 percent  from the total samples genotyped. As our results indicated that genetic diversity was similar across the sampled core collection sizes compared to the main collection, we selected the smallest core collection size of 10 percent. We expect this 10 percent core collection to be an optimal tool for discovering and evaluating functional diversity in the genebank to advance potato breeding and agricultural-related studies. This study also lays the foundations for continued CCC curation by evaluating duplicity and admixing between accessions, completing the digitalization of data, and ploidy determination using chloroplast count. The highly diverse Colombian Central Collection (CCC) of cultivated potatoes is the most important source of genetic variation for breeding and the agricultural development of this staple crop in Colombia. Potato is the primary source of income for more than 100.000 farming families in Colombia. However, biotic and abiotic challenges limit crop production. Furthermore, climate change, food security, and malnutrition constraints call for adaptive crop development to be urgently addressed. The clonal CCC of potatoes contains 1,255 accessions ― an extensive collection size that limits its optimal assessment and use. Our study evaluated different collection sizes from the whole clonal collection to define the best core collection that captures the total genetic diversity of this unique collection, to support a characterization more cost-effectively. Initially, we genotyped 1,141 accessions from the clonal collection and 20 breeding lines using 3,586 genome-wide polymorphic markers to study CCC’s genetic diversity. The analysis of molecular variance confirmed the CCC’s diversity with a significant population structure (Phi=0.359;  Solanum tuberosum L.) is a species with great diversity in the primary gene pool. As domestication took place, several cultivated species co-evolved, from the most ancient cultivated potato, S. stenotomum, to the modern and most globally commercialized potato , from western Venezuela to the south in southern Bolivia and northern Argentina, except for the Chilotanum group landraces found in the lowlands of the central and south-central area of Chile . The laner yield . This exin situ and ex situ. The in situ conservation depends on some isolated indigenous communities and smallholder farmers who still conserve traditional landraces  and universities such as Universidad Nacional de Colombia, among others, using three different systems: seeds, field, and in vitro. Farmers and private companies also try to conserve and promote the commercialization of native potatoes. The Colombian Central Collection (CCC) of potato was started in the 1950s as the basis for a genetic platform for potato research and breeding initiative promoted by several Colombian plant scientists, agronomists, and farmers, supported by the Agriculture Minister of Colombia, and guided by Dr. Jack Hawkes . Similarly, we assembled a core and mini-core collection using the Average Accession to Nearest Entry (A-NE) method and validation recommended by in vitro copies taken from the clonal CCC of potatoes genebank managed by AGROSAVIA and 20 commercial varieties and advanced breeding lines obtained from different sources. This CCC contains mainly landraces and a small proportion of released varieties. Based on S. tuberosum group Andigenum, and few accessions to S. tuberosum group Chilotanum. However, the classification of the material in the potato collection follows S. phureja, 681 S. tuberosum subsp. andigenum, 67 S. tuberosum subsp. tuberosum, one (1) S. rybinii (older classification), while for 269 accessions we currently do not have the information because it is in the curation process and digitalization of data from hard copy records. Based on the original classification, this study named the accessions Andigena, Phureja, and Tuberosum types. The geographic origin of the accessions is diverse; we have records for 802 accessions, from which 670 are from Colombia, 95 from other Latin American countries, 26 from Europe, and 11 from the United States , following the manufacturer’s technical recommendations. Then, we genotyped each accession with the Illumina single nucleotide polymorphism (SNP) Infinium Potato Array version 8303 . Clustercall package  a Principal Component Analysis (PCA) calculated using the R-Adegenet package  in the R-packages dartR  a Bayesian model implemented in the software STRUCTURE 2.3  without  package . For the package  using th package . The NbC package  was usedes dartR  and Poppes dartR , respectWe predicted the ploidy level of each accession using the proportion of simplex and triplex genotype scores  across all SNPs per accession, according to We used the Core Hunter 3 program  to definWe assessed the quality of the A-NE method for core collection selection by calculating and comparing the average criterion distance for A-NE and two additional distance-based metrics evaluated by To validate that the genetic diversity representation was kept across collections and select the most suitable core collection for breeding and research, we compared the average observed heterozygosity (Ho) measurements from the core and mini-core collections to the average in the whole CCC. Additionally, a PCA was carried out for each collection to show the distribution of the samples selected in each proposed core collection compared to the whole CCC. Finally, for the 3 percent mini-core collection identified using Core Hunter, we replaced some selected entries to match and contain all accessions within the 10 percent core collection. For this, we identified the entries that differed between the 3 percent mini-core and 10 percent core collections from the initial Core Hunter entry selection. Then, using the MRD matrix, we calculated the difference between the genetic distance of each entry identified in the 3 percent mini-core and the 10 percent entries to identify the 10 percent entry with the minor genetic distance difference. Finally, the 3 percent entries were replaced with the corresponding 10 percent core entry identified, which were instead selected for 19 accessions.We validated the effectiveness of the accessions in the core and mini-core collections to represent the whole spectrum of phenotypic variation for three different traits: i) Average Tuber Weight (ATW) in grams, ii) Number of Tubers per Plant (NTP), and iii) Total Tuber Yield (TTY) in kg/plant. For this we used the phenotypic data of 846 genotyped accessions  for which preliminary field evaluations had been conducted for ATW, NTP, and TTY. Data were collected between 2013 and 2015. Accession data collected across these years depends on the accessions evaluated during each field campaign. Due to financial and operational capacity limitations, we prioritized accessions and traits to be evaluated by year. Andigenum and later the Phureja accessions were evaluated in three consecutive years. The evaluations were conducted in a highland area of Zipaquirá municipality, in the department of Cundinamarca. Field data for each year was collected from an average of 20 clones planted in one tier per accession, using a complete randomized design for the entire CCC in the field. Thus, each year’s data represents a repetition. We used the mean values from all-year evaluations per trait and accession for the analyzes. We expected that, independently of the size, a good core collection would show a similar phenotypic distribution as the one for the 846 accessions. Moreover, we analyzed to see if the identified entries for the different core and mini-core collections represented the phenotypic diversity of all accessions by comparing their average criterium distance for A-NE, E-NE, and E-E metrics. The Gower distance  between p-value=0.001) and three  genetic groups detected in the CCC. The PCA also showed similar results  and CCC_Group_B with mostly tetraploid (2n=4x=48) accessions. The second analysis conducted using Nbclust detected three genetic groups, the same diploid cluster (CCC_Group_A) previously identified, while the tetraploid cluster was separated into two new genetic groups (CCC_Group_B1 and CCC_Group_B2). As shown in S. rybinni, and for 39.9 percent (n=99) we have no information. There has been no digitalization of hard copy records of the taxonomic classification assigned to these accessions when the CCC material was acquired. Therefore, this documentation is currently in the process of curation. The CCC_Group_B1 had 640 accessions, 96.7 percent (n=619) tetraploid accessions, and 3.2 percent (n=21) with unknown ploidy. Taxonomically, most of the accessions, 81.4 percent (n=521), were Andigenum, and a lower proportion Tuberosum, 2.7 percent (n=17), and Phureja, 0.3 percent (n=2). The remaining 15.6 percent (n=100) are currently undergoing taxonomic classification. Finally, the CCC_Group_B2 genetic group had 100 percent (n=273) tetraploid accessions. At the taxonomic level, 56 percent (n=153) corresponded to Andigenum, 16.8 percent (n=46) Tuberosum, 1.5 percent (n=4) Phureja, and 25.6 percent (n=70) are pending taxonomic classification. Finally, the accessions evaluated in this study that are released varieties or breeding program germplasm ― some of which are part of the CCC ― were evenly located across genetic groups. Accordingly, some were diploids and other tetraploids.In the latest taxonomic classification, the most traditional and modern cultivated potatoes are classified within a single potato species. However, the Hawkes taxonomy originally assigned to the CCC aligns with a clear genetic differentiation inherent to the germplasm. Thus, for this paper, we designated the accessions as Phureja, Andigenum, and Tuberosum types. The population structure analysis conducted using STRUCTURE discriminated the CCC into two genetic groups, the CCC_Group_A with mainly diploid . The CCC_Group_A shows lower genetic differentiation with CCC_Group_B1 than with CCC_Group_B2 genetic groups , while the tetraploid groups are more similar . Thus, the CCC_Group_B1 has an intermedium differentiation level between CCC_Group_A and CCC_Group_B2 genetic groups. The results show high genetic variation in the CCC germplasm, confirmed by a higher significant variation within genetic groups [62.95 percent for genetic structure with two groups (K2) and 64.08 percent for three groups (K3)] than between groups (37.04 percent for K2 and 35.91 percent for K3). Based on divergent heterozygosity values (Ho) for the genetic structure of two (CCC_Group_A: 0.14 ± 0.04 and CCC_Group_B: 0.42 ± 0.08) and three genetic groups , the genetic variation within the CCC_Group_B, B2, and B1 groups was greater than that observed in CCC_Group_A group (The genetic differentiation is significant between the CCC_Group_A and the CCC_Group_B genetic groups (F_A group 2C.x=24), 27 tetraploids (2n=4x=48), and two triploids (2n=3x=36). As expected, the Solanum genus’ primary number of chromosomes (12) was duplicated 2−4 times according to each accession’s ploidy level . The chloroplast count in guard cells could not predict triploid accessions. Most diploid accessions  had 7−8 chloroplasts/guard cell average, and a few  were in a grey area between 9−10 chloroplasts/guard cell average. The tetraploids were more consistent with 12−14 chloroplast/guard cells for 96 percent of accessions (26/27) and one in a grey area of 10 chloroplasts/guard cells.We used a reference sample of 112 accessions to confirm the accuracy of predicting the ploidy level of the CCC accessions using the proportion of simplex and triplex SNP marker scores per sample, according to p-value < 0.001), as shown in When using the proportion of SNP genotype scores as an indirect method for assessing ploidy, this analysis showed an average of 4.8 percent ±9.04 simplex and triplex SNP genotype scores for diploid accessions and a 27.1 percent ±1.53 for tetraploid accessions. Moreover, the ANOVA confirmed significant differences between the average for diploid and tetraploid samples , 15 (CCC_15), and 20 percent (CCC_20) of the total number of samples used in this study , plus one mini-core collection with 3 percent (CCC_3) of the original size (39 entries). The accuracy of the A-NE algorithm used to identify diverse core collections was confirmed. Lower values of average A-NE distance were observed for the core and mini-core collections identified using the A-NE. In contrast, the same entries in the different core collections had an average distance near the optimum maximum values for the E-E or E-NE distance criteria Figure 4Finally, we used the available phenotypic data of three agronomical traits  for 846 genotyped accessions of the CCC to validate the representation of phenotypic variation in the core and mini-core collections. The validation strategy demonstrated that the 846 genotyped and phenotyped accessions had a similar distribution range of values for central and dispersion statistics in the whole collection (CCC_100) and across the different core (CCC_20-10) and mini-core (CCC_3) collections for the three different traits Table 3.Moreover, we compared the behavior of three distance-based metrics used for core collection identification to validate ― using phenotypic data ― the quality of the core and mini-core collections identified with SNP markers and the A-NE metric Figure 7S. tuberosum subsp. andigenum, 18 are S. phureja, 17 are S. tuberosum subsp. tuberosum, and 12 are currently undergoing curation. This 10 percent core collection contains 70 accessions from ten collection sites in Colombia, the larger representation from Nariño (22), Boyacá (14), and Cundinamarca (14) departments, followed by Cauca (6), Antioquia (5), Valle del Cauca (5), and one accession from Caldas, Quindío, Santander, and Tolima. Moreover, the core collection has accessions from Peru (7), Bolivia (3), Ecuador (2), Mexico (2), the USA (2), the Netherlands (2), and Germany (2). Thirty-nine of these accessions are of unknown origin. Of the 39 accessions in the mini-core collection (3 percent), 25 are S. tuberosum subsp. andigenum, six are S. phureja, two are S. tuberosum subsp. tuberosum, and six have yet to be identified and are in the process of curation. Regarding their origins, the mini-core collection has 22 accessions from Colombia collected from seven departments: Nariño, Cundinamarca, Boyacá, Antioquia, Cauca, Quindío, and Valle del Cauca. The mini-core collection has four accessions from other countries, two from Peru, one from Mexico, and one from the Netherlands. Finally, the mini-core collection has 13 accessions of unknown origin.The 10 percent core and 3 percent mini-core collections contain accessions from different taxonomic groups and geographic origins (Our study genotyped 90.9 percent (1141/1255) of the clonal collection of the CCC of potatoes and a representation of released varieties. These genomic data allowed us to study the population structure and genetic diversity, determine ploidy, and define core and mini-core collections representing the genetic diversity of the whole CCC. Below we discuss our study results and how we can use them to curate and improve the Colombian potato collection in agreement with the Global Conservation Strategy for potato .Solanum tuberosum L.). According to our previous study that used 809 accessions  collection in Peru, with around 4,500 landrace accessions . Geneticpotatoes . NotablyIn different population structure and genetic diversity studies of genebank collections of traditional cultivars and breeding programs germplasm of potato, the material has been discriminated following ploidy, taxonomy, and characteristics of breeding selection . In geneThis study used the frequency of simplex and triplex scores of all SNPs in each accession as a ploidy level predictor, following In this study, the frequency of simplex and triplex SNP scores identified in the reference set of samples varied compared to Heterozygosity values estimated for potato samples genotyped with the Infinium Potato Array have also been related to ploidy levels. A reference set of samples with ploidy information generated by other indirect (chloroplast count per guard cell or flow cytometry) and/or direct (chromosome count) methods would better define the threshold separation in ploidy assessment. In our case, we compared data from SNP scores, the number of chloroplast/guard cells, and chromosome counts. The threshold separation for the number of chloroplast/guard cells (6−8 for diploids and 12−14 for tetraploids) was consistent with previous reports . AlthougWe proposed the 10 percent subset as the core collection because it already captured the representation of the total diversity of the CCC. The reduced but highly diverse core collection has a more manageable and versatile number of accessions for trait discovery, evaluations, and use in Colombia’s potato breeding and crop improvement. The representation of phenotypic diversity for three agronomic traits  within the core and mini-core collections confirmed their potential use. In general, the evaluation of the CCC for sources of new alleles for different traits has taken several years because of the size of this collection. Now, faster screening can be conducted, and the mini-core collection, which is even smaller in size, represents an alternative when evaluating the core collection is not feasible.As expected, after eliminating redundancy in the collection, the genetic diversity measured using the Ho average increased in the core and mini-core collections. Similar results have been reported for recently developed core collections of potato . The PCAThe selected 10 percent core collection size was within the range of 5−20 percent core collection size reported and corresponded with the most recommended size . We willS. tuberosum subsp. andigenum. The assignment to genetic groups, ploidy, morphology, and historical records should be considered when reassigning taxonomic classification. Some diploid accessions can be Andigenum, and some tetraploid accessions Phureja. In potatoes, the germplasm ploidy can be increased due to crosses involving 2n gametes (polyploidization) or reduced by pickle pollination (haploidization). Phureja accessions are mainly diploids, but triploids and tetraploids can be found in a low proportion  into a single species is well supported, a suite of morphological descriptors, length of plant cycle, photoperiod adaptation, and cytoplasm type support their differentiation , biological status , and ethnobotanical classification (Chauchas and Guatas). Regarding the different taxonomic classifications, it is important to maintain the information of Hawkes’ classification, which facilitates the management and use of germplasm in the conservation and breeding programs. Even though the classification based on ntiation . If thisin vitro and field collection to verify the identity of germplasm. This study successfully genotyped 90.9 percent of the clonal collection of the CCC of potatoes and a representation of released varieties. This result is an outstanding achievement considering this collection’s size  and its importance for the region. Moreover, we are interested that this collection become in a regional reference. Therefore, we would recommend aligning the CCC conservation strategy with the ten actions suggested for improving the global-scale conservation of potato resources, as proposed by several institutions and organizations . Some ofThe genotyping data generated in this study can also be compared with other collections, such as CIP’s collection, genotyped using the same technology. This comparison could serve to identify signatures of selection, materials with potential use in breeding, duplicity, genetic gaps, homologate accessions between the collections, and assess the extent to which CCC diversity is representative of the world’s largest collection of native potatoes. This paper is also available in Spanish to continue the discussion of this topic within Colombia and more broadly in the region (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/RY, JB-C, IC-S, and PR-H conceived the study. JB-C, ZL-P, and PR-H organized the CCC databases and designed the analysis. ZL-P and IC-S collected leaves from selected accessions. JB-C and IC-S worked in the wet lab. JB-C and PR-H performed the analysis, including descriptive graphics. NM-C led the manuscript writing and performed part of the analyses. All authors contributed to the article and approved the submitted version.