Vigna unguiculata (L.) Walp.) is currently a legume crop of minor importance in Europe but a highly relevant staple crop in Africa and the second most cultivated legume in Mozambique. In Portugal and Mozambique, cowpea’s phenotypic and genetic variation has been maintained locally by farmers in some areas. We used the molecular markers SSR, SilicoDArT and SNP to analyze the genetic diversity and population structure of 97 cowpea accessions, mainly from Portugal (Southern Europe) and Mozambique (Southern Africa). As far as we know, this is the first time that the genetic variation and the relationship between cowpea landraces collected in Portugal with those originated in Mozambique is reported. Despite the shared historical past, the Portuguese landraces did not share a common genetic background with those from Mozambique, and two different gene pools were revealed. Knowledge of the genetic structure of cowpea landraces offers an opportunity for individual selection within landraces adapted to particular eco-physiological conditions and suggests the existence of a valuable gene pool for exploitation in future Portugal-PALOP (Portuguese-speaking African countries) cowpea breeding programs.Cowpea ( Vigna unguiculata (L.) Walp.) is a self-pollinating diploid (2n = 22) species of the Fabaceae family, the third-largest family of plants and the second most important concerning human nutrition [Cowpea  Verdc., widely distributed across Africa [The center of diversity for cultivated cowpea is reported to be in West and Central Africa. The oldest evidence that cowpea existed in West Africa was obtained from carbon dating specimens from the Kimtampo rock shelter in Central Ghana . The cens Africa . Huynh es Africa , using sThe narrow genetic base of improved cowpea varieties can be attributed to its self-pollinating characteristics . HoweverV. unguiculata of Portugal, knowledge is lacking on the Portuguese landraces, which were only partially represented. Characterization and knowledge of the existing levels of diversity within and among these still-cultivated landraces are necessary to establish suitable on-farm conservation strategies and as a first step to introduce these populations in future breeding programs. In Mozambique, despite it being considered a diversity hot-spot for cowpea [In Portugal, there is a detailed morphological description of the landraces grown in the country in the decade of the 1950s, performed by Castillo . Later, r cowpea  and cowpr cowpea ,29 in anV. unguiculata genetic resources. The Portuguese accessions used were collected in farmers’ fields since the decade of the 1980s and were stored at “Banco Português de Germoplasma Vegetal”, with some populations still being cultivated by farmers. There are 199 accessions stored in the Portuguese bank, but for our study, only the most relevant and representative accessions of the different growing areas were chosen. The Mozambican cowpea landraces originated from two gene banks. The first was the Mozambican Germplasm Bank kept at the “Instituto de Investigação Agrária de Moçambique (IIAM)”, the oldest collection in the country. The second source was the newly established “Instituto Superior Politécnico de Manica Gene Bank” in Central Mozambique, which has the highest and most recent collection of cowpea germplasms from Central Mozambique. The accessions used in this study are relevant and distributed through the country. However, they do not yet represent all the existing genetic variability of Mozambican cowpea because collection missions are still ongoing.In this work, we aimed to examine the genetic diversity and structure of Portuguese and Mozambican cowpea landraces, mainly to explore the potential relatedness in existing cowpea landraces in these two Portuguese-speaking countries, where such research has not been conducted yet, and to assess the pertinence of cross-country breeding programs. This knowledge will increase the efficiency in the sustainable conservation and utilization of Portuguese and Mozambican V. unguiculata populations from Portugal and Mozambique, 34 and 52 accessions, respectively, were analyzed using SSR, SilicoDArT and SNP markers. In the SSR analysis, there were additionally 5 accessions from Angola, 3 from Cape Verde, 1 from Spain, 1 from Greece and 1 from Nigeria  Verdc. accessions, from Portugal, Spain and Cape Verde, were used as an out-group. The accession IT-97K-499-35 from Nigeria was also included in the study because it is the reference genome for cowpea.To compare the genetic variation of  Nigeria . For SilTwelve SSR loci amplified a total of 183 alleles, with an average of 15.25 alleles, ranging from 4 (CLM0634) to 27 (VuUGM71), and with an average effective number of alleles (Ne) of 2.61. The Polymorphism Information Content (PIC) ranged from 0.354 (VM5) to 0.892 (VuUGM33), indicating that the used loci displayed a high level of variability and are useful diversity indicators. The locus VuUGM71 displayed the highest values of Ne and He, while the locus CLM0634 displayed the lowest values. The mean observed heterozygosity (Ho) was 0.325 and the mean expected heterozygosity (He) was 0.461 .A total of 61,221 polymorphic SilicoDArT  markers In total, 31,420 SilicoDArT markers were selected for the study. Among these informative markers, around 9% were observed in PIC class 0.45 to 0.5 and 50% in the 0.05 to 0.1 class . The PICA total of 38,889 SNP markers  were ideAll possible SNP types were found in our study . The SNPssp. sesquipedalis) was not particularly separated from the remaining accessions  from Cape Verde was, as expected, isolated in a branch  and the Portuguese commercial variety (Fradel). The accession A14 from Mozambique is also admixed (The Bayesian approach indicated that the most likely number of genetic clusters was K = 2 (ΔK = 2) . Based o admixed .A PCA analysis of the populations explained 28.76% of the variation in the first two axes, 21.01% in the first component and 7.75% in the second component. The Portuguese landraces are on the left side of the graph, while the accessions from Mozambique are on the right side of the graph. The Portuguese landraces CP5650, Lardosa 1A, F. vagem and the commercial variety Fradel did not group with the remaining Portuguese samples. The samples from Angola and Cape Verde are scattered within the Mozambican cloud .Wright’s FST and Slatkin´s RST were used as a measure of the extent of the genetic differentiation among the populations. These parameters are useful differentiation estimators, commonly used to describe population structuring through SSR markers ,31. WhenThe values of ΔK estimated from SilicoDArT markers peaked at K = 2 and K = 4 . At K = V. unguiculata ssp. sesquipedalis , and the commercial variety Fradel (mainly green and yellow)  and three from PALOP landraces . The majority of the Portuguese landraces were pure accessions . Exceptions were the landraces CP5650 , Lardosa 1A (green and yellow), F. vagem, which is  yellow) . The bre yellow) . From AnThere are 58% of Mozambican landraces that are pure, spread across three subpopulations . All the other landraces were admixed. Several landraces from the agroecological region R7, described by Gomes et al.  A71, A7.A PCA analysis of the populations explained 40.6% of the variation in the first two axes, 33.7% in the first component and 6.9% in the second component .V. unguiculata ssp. sesquipedalis) did not group with the remaining Portuguese samples.The Portuguese landraces are on the right side of the graph, while the accessions from Mozambique are on the left side of the graph. The Portuguese landraces CP5650 and Lardosa 1A and the commercial variety Fradel and F. vagem , sampled at the agroecological zone R7, described by Gomes et al. , are thessp. sesquipedalis) and the commercial variety Fradel. With the exception of BPGV 2979, all the other admixed landraces were collected after 2004. Landrace Lardosa 1A stands out as a unique population with no admixture (pink).Considering population structure analyses using SNP markers, the Evanno’s ΔK peaked at K = 3 and K = 5 . At K = Most Mozambican pure accessions clustered in subpopulations that were yellow  and red , 19% and 9.6%, respectively, with all others being admixed .ssp. sesquipedalis) was admixed (Two landraces of Angola were pure (Luanda (yellow) and Malange Claro (red)). The Cape Verde landrace , from the agroecological zone R7, described by Gomes et al. , are groThe PCA analysis using SNP marker data  was veryThe application of different marker systems to the same gene pool stands as an opportunity to compare the performance of those marker types in diversity studies. The difference in results is a consequence of the nature of the markers and the analysis methodology.sesquipedalis) not being discriminated, as expected, by any of the markers. To assess the correlation between the used marker systems, we have applied the Mantel test. Despite low r values, a significant correlation (p < 0.001) was found for the three comparisons  Eco-Regions, where cowpea landraces are still preserved on-farm .Considering Mozambique, Eco-Regions are not yet established. However, in 2019, the second Eco-Regions World Congress within the context of the International Forum on Relevant Territories for Sustainable Food Systems decided to implement an integrated methodology to promote sustainable food systems within the frameworks of Community of Portuguese Language Countries (CPLP) Food Security and Nutrition, the Strategy of the Sustainable Development Goals and the UN Decade of Family Farming . The impTo achieve effective conservation and enhance the use of cowpea germplasm, there is a need for a detailed characterization of the existing diversity. The genetic structure of cowpea populations is highly determined by its mating system, which is characterized by high self-pollination and, so, the cowpea crop is a mixture of a number of distinct homozygous lines .In this work, we present new insights into the diversity of cowpea landraces from Portugal (Europe) and Mozambique (Southern Africa). As far as we know, this is the first time that cowpea landraces collected in Portugal are compared with those originated in Mozambique using SSR, SilicoDArT and SNP markers.FST and RST are useful differentiation estimators, commonly used to describe population structuring ,31. AccoAccording to the three genetic markers used in the study, at least 80% of the Portuguese landraces are pure and they are grouped in a main cluster. Lardosa 1 and Lardosa 1A are landraces grown on-farm in the same region, although only sold at local markets, and have great economic importance for traditional farmers. They are both pure landraces but differ morphologically from each other. The seed of Lardosa 1 has an ovoid form and the hilum is green, and Lardosa 1A usually called “rice bean” and has a globose form and the hilum is brown. Lardosa 1 is grouped in the main Portuguese cluster but Lardosa 1A is not. Physiological studies in Portuguese landraces showed that Lardosa 1 was able to maintain tissue hydration and water use efficiency under the imposed water deficit, which translated into a higher number of grains per plant than in other accessions, indicating adaptation to the Mediterranean climate and conveying the importance of landrace preservation .CP5650 is not a pure landrace (admixed with three subpopulations), is quite distant from all the other Portuguese samples in the three PCAs and is closer to the Mozambican landraces. It should be noted that the cowpea pure landraces are mostly those stored at “Banco Português de Germoplasma Vegetal” (collected before 2000), and also Lardosa 1, Lardosa 1A and Sátão, which are still on-farm cultivated. The CP5650 landrace was only recently collected (2014), which may suggest that it was brought from another geographical region.When analyzing the Mozambique accessions, we observed that, using SilicoDArT or SNP markers, only 50% or 27% are pure landraces. Thus, a high level of admixture was observed for these accessions, as Gomes et al.  previousBriefly, population structure analysis  revealed two different gene pools, corresponding to Portugal (Southern Europe) and Mozambique (Southern Africa). Portuguese landraces did not share a common genetic background with those from Mozambique, confirming previous results. The genetic architecture of cowpea landraces from South Europe and Africa was studied by Huynh et al.  and XionThe present knowledge of the genetic structure of cowpea landraces gives an opportunity for individual selection within landraces adapted to particular eco-physiological conditions. In particular, the assignment of molecular markers for these accessions, potentially linked to abiotic and biotic stress resistance, nutritional richness and a symbiotic capacity for nitrogen fixation, will allow the design of a specific SNP marker panel to be used in molecular-assisted breeding.V. unguiculata accessions of multiple origin were used for genetic diversity and population structure analyses by SSR: 34 from Portugal, 52 from Mozambique, 5 from Angola, 3 from Cape Verde, 1 from Spain, 1 from Greece and 1 from Nigeria , according to the manufacturer´s protocol. DNA quality and concentration were visually checked on 0.8% agarose gel. The DNA concentration was also estimated using a NanoDrop ND2000 spectrophotometer . All accessions were genotyped twice to use as technical replicates.Cowpea genotyping was based on 12 SSR loci, which were developed by Xu et al. , Gupta aThe multiplexed PCR amplification was performed with three SSR loci using the QIAGEN Multiplex PCR Kit in a final volume of 10 µL, according to the manufacturer’s instructions, in a My Cycler Biorad thermocycler.Subsequently, 1.0 µL of the PCR mixture was added to 24 µL formamide and 0.5 µL fragment size standard labeled with WellRED dye D1 . Capillary electrophoresis was performed to separate the PCR products using the CEQ 8000 Genetic Analysis System . Data analysis was performed using the CEQ 8000 Fragment Analysis software, version 9.0, according to the manufacturer´s recommendations . Sizes of fragments were automatically calculated using the CEQ 8000 Genetic Analysis System.The high-throughput DArTseq technology used to genotype cowpea accessions followed the standard procedures  and was Next-generation sequencing technology was implemented using HiSeq2000  to detect SNPs and SilicoDArT markers.GenAlEx 6.503  was usedThe differentiation between the populations was estimated using Wright´s FST and Slatkin´s RST. FST results were interpreted following Del Carpio et al. , where 0Micro checker software v2.2.3  was used4 steps followed by 106 Monte Carlo Markov Chain (MCMC) replicates assuming an admixture model and correlated allele frequencies. K is the probable maximum population number that is assumed to represent and to contribute to the genotypes of sampled individuals. To check the consistency of the results between runs with the same K, eight replicates were run for each assumed K value. The approach suggested by Evanno et al. [The level of genetic stratification among the studied germplasm was assessed using STRUCTURE v.2.3.4 software . The anao et al.  was adopThe SilicoDArT and the SNP data were analyzed using DARTR . MarkersThe SNP and SilicoDArT results were tested for reproducibility (%), call rate (%), Polymorphism Information Content (PIC) and one ratio following Allam et al. .This study allows us to deepen the cowpea genetic diversity knowledge of Portuguese and Mozambique landraces, recently stored in their germplasm banks and also still cultivated on-farm. Considering that on-farm conservation is a relevant strategy to maintain the evolutionary forces within and between components of the agricultural system , it is dThe diversity of cowpea landraces suggests the existence of a valuable gene pool for exploitation in future cross-country breeding programs. A large number of polymorphic markers obtained by SNP and SilicoDArT were identified and are now available as important and useful knowledge for genome studies.


The present study investigated the contributions of environmental microorganisms to the microbial community as well as the volatile compounds in the fermented grains of Sauce-flavor baijiu produced in the Beijing region using high-throughput sequencing combined with sourcetracker analysis, and compared the differences of environmental microorganism and their roles in the production process of Sauce-flavor baijiu from different regions.The results showed that the environmental microorganisms in the tools were the main contributors of the bacterial and fungal communities in fermented grains during heap fermentation and at the beginning of pit fermentation. At the end of pit fermentation, pit mud was the main environmental source of bacterial community in fermented grains, while tools and Daqu were the main environmental sources of fungal community in fermented grains.Environmental microorganisms thrived on the functional microorganisms in the fermented grains of Sauce-flavor baijiu produced in the Beijing region and thus shaped the profiles of volatile compounds. Environmental microorganisms of Sauce-flavor baijiu in the Guizhou province and the Beijing region differed significantly, which is partially responsible for the distinctive characteristics in the microbial community structure of Sauce-flavor baijiu-fermented grains from different regions.The compositions of the microbial community in fermented grains of Sauce-flavor  The main regions producing Sauce-flavor baijiu in China include Moutai and Xishui town in the Guizhou province, Erlang town in the Sichuan province, Beijing, and Tianjin [baijiu produced in these different regions are perceived to exhibit distinctive characteristics [baijiu is traditionally produced by solid-state fermentation in a semi-controlled environment, with its flavor compounds being generated mainly by the diverse and abundant microorganisms during the fermentation process [baijiu produced in the Guizhou province were dominated by Pichia, Saccharomyces, Thermoascus, Wallemia, and Cryptococcus, while those in Beijing were dominated by Issatchenkia, Thermoascus, and Thermomyces [baijiu produced in different regions differed significantly and may be partially responsible for their unique flavor characteristics [Consumers take pleasure in discussing the unique flavor characteristics of fermented foods produced in different geographical areas using similar materials and manufacturing processes. For example, the terroir is often used to describe the regionally distinctive flavor characteristics of wine . Interes Tianjin ,3,4,5. Teristics ,4. Sauce process . Previourmomyces . Overalleristics ,8. Howevbaijiu mainly originate from the Daqu starter used and the specific processing environment [baijiu lasts approximately a year with seven stages, using Daqu as the starter and sorghum as the raw material [baijiu is obtained by distilling the fermented sorghum (fermented grains) [baijiu . The fermented grains are then covered with rice hulls and pit mud and fermented in the pit for 30 d  . Each stfor 30 d . The ferformance . Like San system . The micd grains . Anaerobompounds . These rbaijiu processing environments can differ significantly. The dominant bacterial genera in workshops where Sauce-flavor baijiu was produced in Guizhou province were Sphingobacterium, Enterobacter, and Pantoea [baijiu was produced in the Hebei province were Lactobacillus, Bacillus, and Weissella [baijiu is produced. Environmental factors, such as temperature, air pressure, and light, affect how microorganisms grow and secrete compounds into their surroundings, thereby affecting the diversity of the microbial community [baijiu produced in the Beijing region, and to compare them with those of Sauce-flavor baijiu produced in the Guizhou province.The environmental microorganisms in  Pantoea , while teissella . Howeverommunity ,13. The ommunity . The Beiommunity . The prebaijiu used for pit fermentation were sampled in triplicate. Finally, twenty-seven samples were stored at −80 °C for subsequent analysis.All the samples were collected in May 2021 from a distillery in Beijing, China. The heap and pit fermentation lasted approximately 5 d and 30 d, respectively. The samples in heap fermentation were collected from the upper and lower layers when fermented for 1 d, 3 d and 5 d . At each® soil DNA Kit  according to manufacturer′s instructions. The V3-V4 hypervariable region of bacterial 16S rRNA gene was amplified with primer pair 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTA CHVGGGTWTCTAAT-3′). The internal transcribed spacer 1 and 2 (ITS1 and ITS2) region of fungal 18S rRNA gene was amplified with the primer pair ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS2 (5′- GCTGCGTTCTTCATCGATGC-3′).The extraction, amplification, and sequencing of genomic DNA were conducted according to our previously described method . In brieThe sequencing of PCR amplification was performed on Illumina MiSeq PE300 platform/NovaSeq PE250 platform  with the protocols of Majorbio Bio-Pharm Technology Co., Ltd. . The raw bacterial 16S and fungal 18S rRNA gene sequencing reads were demultiplexed, quality filtered by fastp version 0.20.0, and merged by FLASH version 1.2.7  with the following criteria: (i) the 300 bp reads were truncated at any site receiving an average quality score of <20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded; reads containing ambiguous characters were also discarded; (ii) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of overlap region is 0.2. Reads that could not be assembled were discarded. (iii) Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted with exact barcode matching and 2 nucleotide mismatch in primer matching. After removing chimeric sequences, the high-quality sequences were clustered into operational taxonomic units (OTUs) with 97% similarity cutoff using UPARSE version 7.11. The taxonomic classification of each 16S rRNA and 18S rRNA gene sequences were analyzed by the RDP Classifier version 2.2  against the SILVA v138 rRNA and greengene database at a confidence level of 70%, respectively .The volatile compounds were quantified according to our previously described method . Brieflybaijiu, and Daqu were set as source. The Spearman rank correlations between volatile compounds and the relative abundance of twenty-three shared genera in fermented grains and environments were calculated using OriginPro2019  and visualized using a heatmap diagram. The statistically significant differences in Shannon and Chao 1 indexes were analyzed using one-way analysis of variance (ANOVA) in OriginPro2019  .All the assays were conducted in triplicate and the results were presented as mean with standard deviations. Principle coordinate analysis (PCoA) was employed to evaluate the ecological distances of different samples on a genus level based on unweighted UniFrac distances. Sourcetracker (v0.9.8)  was used to analyze the source of the microbial communities in fermented grains. Fermented grains-H, fermented grains-P0, and fermented grains-P30 were set as sink, and rice hulls, tools, pit mud, water, tail baijiu, rice hulls, tools and Daqu were significantly higher than those of pit mud and water  in Daqu, tools, and pit mud samples, while Saccharopolyspora, Scopulibacillus, and unclassified_f_Bacillaceae shared subdominant genera (average relative abundance between 10% and 1%) in rice hulls and tools  was further carried out based on the profile of the bacterial community C. The twLactobacillus, 10.89% Kroppenstedtia, 8.62% Virgibacillus, 8.79% Bacillus, 7.27% Oceanobacillus, 2.30% Pediococcus, and 1.80% Staphylococcus in fermented grains during heap fermentation  regions of the fungal rDNA gene in 27 samples were obtained, with a range from 106,734 to 124,340 reads. For the Daqu and environmental samples, the fungal community diversity of tail and Daqu B. The fuand Daqu D. For feand Daqu B; howeveand Daqu D.baijiu, 48 genera were shared with water, 43 genera were shared with pit mud, and 42 genera were shared with rice hulls (baijiu, water, pit mud, tools, and rice hulls, respectively. At the end of pit fermentation, the profile of shared fungal genera between fermentation grains, Daqu, and environmental samples was similar to that during heap fermentation (baijiu, and water as well as pit mud, had relatively higher numbers of shared genera (The fungal taxa at genus level in samples were analyzed B. A totace hulls B. At theentation B. For end genera B.Thermomyces shared predominant genera in Daqu, tail baijiu, water, and pit mud, and Thermoascus shared subdominant genera in rice hulls, tools, water, and tail baijiu (Byssochlamys was predominant in tools while subdominant in other environmental samples. Aspergillus was predominant in rice hulls while subdominant in other environmental samples. Issatchenkia was more abundant in tools but less abundant in tail baijiu (Issatchenkia shared predominant genera and Aspergillus, and Pichia shared subdominant genera among fermented grains samples (Thermomyces and Thermoascus shared predominant genera in fermented grains-H and fermented grains-P30, while there were subdominant genera in fermented grains-P0. Byssochlamys was more abundant in the fermented grains-P0 while less abundant in other fermented grain samples (For Daqu and environmental samples, l baijiu B. Byssocl baijiu B. For fe samples B. Thermo samples B.baijiu were closed to fermented grains-P30 (PCoA was further performed based on the profile of the fungal community D. The reains-P30 D.Issatchenkia, 11.60% Byssochlamys, 6.40% Thermomyces, 5.50% Thermoascus, and 2.00% Pichia in fermented grains during heap fermentation (Issatchenkia and 20.00% Byssochlamys in fermented grains at the beginning of pit fermentation (Issatchenkia, 7.00% Thermoascus, 2.00% Thermomyces, 2.00% Byssochlamys, and 1.00% Aspergillus were derived from tools (Sourcetracker analysis showed that tools was the major contributor of the fungal communities in fermented grains-H and fermented grains-P0 and a partial contributor to the fungal community in fermented grains-P30 B. Tools entation D, and coentation E. At theom tools F.Thermoascus and 1.02% Thermomyces at the beginning of pit fermentation, while contributed 16.19% Thermoascus and 13.53% Thermomyces at the end of pit fermentation (Thermomyces, 6.63% Thermoascus, 2.47% Rasamsonia, and 1.26% Aspergillus in the fungal community of fermented grains-P30 were originated from pit mud (Daqu was another main contributor to the fungal communities in fermented grains-P0 and fermented grains-P30 B. Daqu centation E,F. In a pit mud F.baijiu, the volatile compounds of Daqu and fermented grains were analyzed. A total of 56 volatile compounds were detected in Daqu and fermented grain samples, including 5 acids, 7 alcohols, 3 aldehydes, and 28 esters (To explore the contributions of microorganisms in fermented grains from Daqu and environment to the flavor of 8 esters . The var8 esters .Issatchenkia, as well as Oceanobacillus, were closely related to nine esters, including ethyl 9-hexadecenoate, ethyl iso-allocholate and ethyl acetate.. Byssochlamys exhibited positive correlation with nine esters and hexadecanoic acid. Lactobacillus was closely related to four acids, including acetic acid, isobutyric acid, butyric acid, hexanoic acid, and 18 esters including ethyl caproate, ethyl lactate, ethyl succinate, ethyl dodecanoate (The Spearman correlation coefficients between relative abundance of dominant microorganisms from Daqu and the environment in fermented grains and concentrations of key volatiles were further calculated. Results showed that these microorganisms were important contributors to the formation of volatile compounds . Among tecanoate .baijiu produced in the Beijing region. During the heap fermentation of Sauce-flavor baijiu, the fermented grains are first mixed thoroughly with Daqu and water, and then placed in an open environment for fermentation [baijiu produced in the Beijing region.This study investigated the contribution of microorganisms originating from Daqu and the environment to the microbial community and flavor compounds in the fermented grains of Sauce-flavor entation ,17,18. Aentation ,13. Sourentation . Similarbaijiu produced in the Beijing region, thereby shaping the profiles of those compounds related to flavor. An analysis of the microbial taxonomic profile showed that the relative abundance of Lactobacillus, Oceanobacillus, Issatchenkia, and Byssochlamys was low in Daqu but high in the processing environment (Lactobacillus, which is abundant in the fermented grains of Light-flavor, Strong-flavor, and Sauce-flavor baijiu, is considered to be the main functional genus during the fermentation process of baijiu [Lactobacillus can synthesize lactic acid, acetic acid, ethyl lactate, ethyl acetate, and other compounds related to flavor by producing esterase and lipase, thus providing the fruit flavor and mellow feeling of baijiu [Oceanobacillus can produce enzymes, such as amylase, lipase, and esterase, as well as lactic acid [Byssochlamys can actively participate in the saccharification and fermentation processes by producing pectinase and lipase [Issatchenkia, which is abundant in the fermented grains of Sauce-flavor and Miscellaneous-flavor baijiu as a non-Saccharomyces fungal genus, can produce β-glycosidase, as well as phenethyl alcohol, various acids, and ethyl esters [Lactobacillus, Oceanobacillus, Issatchenkia, and Byssochlamys (The environmental microbiota thrived with the functional microorganisms in the fermented grains of Sauce-flavor ironment A,B. Thesironment A,B, indif baijiu ,20. Lactf baijiu ,20. Oceatic acid ,22. Byssd lipase , which al esters ,25,26,27ochlamys . This inbaijiu differed significantly and were partially responsible for differences in the structure of the microbial community in the fermented grains of Sauce-flavor baijiu produced in different regions. The Guizhou province is the main area producing Sauce-flavor baijiu [baijiu in the Guizhou province showed that the relative abundance of Pichia, Torulaspora, and Wickerhamomyces were high and dominant [Thermomyces, Issatchenkia, Thermoascus, and Byssochlamys on tools were high and dominant (baijiu. As the brewing of batches of Sauce-flavor baijiu is repeated, the dominant microorganisms in the fermented grains with a strong environmental tolerance gradually establish a stable microbial community in that particular environment [baijiu produced in Guizhou province and the Beijing region, and found that Lactobacillus, Virgibacillus, Kroppenstedtia, Oceanobacillus, Bacillus, and Pediococcus were the dominant shared bacterial genera [Pichia, Saccharomyces, Thermoascus, Aspergillus, Byssochlamys, and Monascus being the dominant shared fungal genera [Pichia was more abundant in fermented grains from Guizhou province, while Issatchenkia was more plentiful in fermented grains from the Beijing region. The succession of the microbial community in the fermented grains was strongly influenced by various environmental conditions such as the temperature, oxygen level, and water activity [Pichia, the core non-Saccharomyces fungal genus in the fermented grains of Guizhou province, can produce toxins and organic acids which inhibit the growth of filamentous fungi and bacillus, thus influencing the succession of the microbial community. Pichia can also shape the profiles of flavor compounds in the fermented grains by producing several alcohols, acids, and esters [Pichia in the fermented grains of Guizhou province mainly originated from tools and the indoor environment rather than from the Daqu [Issatchenkia in fermented grains of baijiu from the Beijing region also originated from the tools rather than from the Daqu (baijiu is produced contributed to differences in the fermentation microbiota.The environmental microorganisms in the manufacturing environments of Sauce-flavor r baijiu . The resdominant , while, dominant . This diironment . These eironment ,11,12. Tl genera ,17,18,20l genera ,16,17; hactivity . Differethe Daqu A,B. Overbaijiu from the Beijing region. The results showed that tools were the main contributors of the bacterial and fungal communities in fermented grains during heap fermentation and at the beginning of pit fermentation. Tools contributed 47.20% Issatchenkia, 34.13% Lactobacillus, 11.60% Byssochlamys, 10.89% Kroppenstedtia, 8.62% Virgibacillus, 8.79% Bacillus, 7.27% Oceanobacillus, 6.40% Thermomyces, 5.50% Thermoascus, 2.30% Pediococcus, 2.00% Pichia, and 1.80% Staphylococcus in fermented grains during heap fermentation, and contributed 70.00% Issatchenkia, 23.13% Lactobacillus, 20.00% Byssochlamys, 17.42% Oceanobacillus, 12.13% Kroppenstedtia, 11.88% Virgibacillus, 7.48% Bacillus, and 3.55% Thermoactinomyces in fermented grains at the beginning of pit fermentation. Pit mud contributed 89.30% Lactobacillus, 8.08% Thermomyces, 6.63% Thermoascus, 2.47% Rasamsonia, and 1.26% Aspergillus in fermented grains at the end of pit fermentation, which was the main environmental source of the bacterial community in fermented grains at the end of pit fermentation. Tools and Daqu were the main contributors to the fungal communities in fermented grains at the end of pit fermentation. At the end of pit fermentation, 24.00% Issatchenkia, 7.00% Thermoascus, 2.00% Thermomyces, 2.00% Byssochlamys, 1.00% Aspergillus were came from tools, while 16.19% Thermoascus, and 13.53% Thermomyces were originated from Daqu. The environmental microorganisms on the tools, pit mud, and Daqu shaped the profile of the volatile compounds in the fermented grains. The environmental microorganisms of Sauce-flavor baijiu from Guizhou province and the Beijing region differed significantly, which partially contributed to the distinctive characteristics in the compositions of the microbial community structure in fermented grains.The present study investigated the contributions of environmental microorganisms to the microbial communities and flavor components in fermented grains of Sauce-flavor


The Action Cycle Theory (ACT) is an enactive theory of the perception and a mental imagery system that is comprised of six modules: Schemata, Objects, Actions, Affect, Goals and Others’ Behavior. The evidence supporting these six connected modules is reviewed in light of research on mental imagery vividness. The six modules and their interconnections receive empirical support from a wide range of studies. All six modules of perception and mental imagery are influenced by individual differences in vividness. Real-world applications of ACT show interesting potential to improve human wellbeing in both healthy people and patients. Mental imagery can be applied in creative ways to make new collective goals and actions for change that are necessary to maximize the future prospects of the planet. Since the time of Descartes, perception has been conceived as the reception of sensory information that may or may not lead to any behavioral response or action .Except for the removal of the pineal gland from this system, this ‘S-R’ or theatre model of perception as passive stimulation (S) of an empty organism to produce a response (R) persisted for almost three centuries until Hermann von Helmholtz (1910/1925)  “Yet, the most striking fact is that perceptual experience is far richer than available retinal images; and though neural signaling is slow, it is not usually delayed in time. From these shadowy ghosts in our eyes we see hard solid objects with properties beyond optics. This depends on knowledge of objects, and how they interact, allowing behavior to be appropriate to what is known or assumed, rather than limited to what is being sensed. This is where knowledge comes in, as the past enriches the present, and allows some prediction into the future”.Cognitive Psychology  arguenactive ,66,67. Cenactive ,69. One The association between vivid imagery and covert psychomotor activity has been acknowledged since William Shaw published “The relation of muscular action potentials to imaginal weight-lifting” in 1940 . Shaw haVividness differences are found across tasks and specialist groups with higher or lower than average skill levels, e.g., air traffic controllers, pilots, Olympic athletes , orchestThe ‘mirror neuron’ that is responsive to the observation of motor actions by others was originally identified in the macaque monkey . Then, aEnactive mental imagery is utilized in the development of brain–computer interfaces (BCI), which have brain activity as input and an action carried out by an external device as output. BCI enable humans to interact with the environment without using their muscular system  by exploBlumen and colleagues (2014) examined the behavioral and neural correlates of imagined walking (iW), imagined talking (iT) and imagined walking while talking (iWWT) . Thirty-Since Freud’s famous analysis of Anna O., clinical case studies of the imagery–emotion link remain fascinating but difficult to interpret. The links between imagery and emotion have been said to involve a ‘special relationship’ comprising imagery’s direct influence on emotional systems in the brain to sensory signals . HoweverAgain, one focus has been on visual imagery vividness, which appears to play a role in perceptual decision making following threatening cues. It enhances the planning of actions to protect and to maximize safety, stability and security in light of goals, drives and others’ behavior. The bio-informational theory of emotional imagery of Peter J. Lang  has beenKöchel et al. (2011)  used mulWicken, Keogh and Pearson (2021)  examinedThe powerful causal links between emotion and imagery have been exploited in multiple mental health and neurological interventions and to minimize distress in patients entering hospital for procedures such as surgery. In a controlled trial with patients receiving abdominal surgery, we tested the effects of guided imagery as a preoperative preparation to increase patients’ feelings of coping with surgical stress . Twenty-Mental images are strongly linked to goals, including personal, family and community goals. Images reflect specific goal content and act to maintain goals. A primary function of mental imagery is the selection, rehearsal and planning of goal-directed activity. ACT assumes that mental rehearsal enables the person to guide and improve actions in a way that would be more difficult without it. It is assumed that an action goal includes an internal representation of the external object toward which the action is directed and toward the final state when the object is reached. Conscious mental imagery provides the primary mechanism for goal-directed action planning that needs to be adapted to novel circumstances, whereas unconscious goal-directed action can occur for automatic, habitual actions such as walking or typing.ACT proposes reciprocal causation between imagery, goals, emotion, action, others’ action and schemata. New situations require new images that lead to new goals, and vice versa . VisualiThe different perspectives one uses in imagery of the self can make a difference to future action. There is the visual perspective of oneself from the first-person perspective versus taking an observer’s third-person perspective. One study took place on the eve of the 2004 U.S. presidential election when registered voters in Ohio were instructed to use either the first-person or the third-person perspective to mentally image themselves voting in the election . ImagingEnvironmental Mental Imagery scale to assess how vividly and frequently participants could imagine problems related to the goal of reducing plastic debris floating in the oceans after seeing a video. There were moderately strong correlations between the participants’ environmental imagery vividness and their pro-environmental goals . Emotive environmental imagery can be internalized as mental images to influence individuals’ personal pro-environmental goals and mitigation strategies.The motivational power of mental imagery is strengthened through its close link with emotions. Boomsma, Pahl and Andrade  used an Innovative imagery approaches are available to improve public engagement with climate change . ElsewhePerception is an enactive visuomotor exploration by the eye, hand and body of objects, scenes and others’ actions. As previously noted, the discovery of mirror neurons was a significant step toward an increased understanding of perception . Mirror Mirror neurons fire not only when a person is performing an action but when passively observing a similar action performed by another. Fully consistent with ACT, mirror neurons are designed for prosocial functions of action and emotional understanding or empathy, synchronization and psychological homeostasis. There appears to be a specific kind of type-M schemata for mapping others’ actions, emotions and communications. A meta-analysis of 125 fMRI studies revealed an extensive brain network with mirror-like properties . These rStudies using BOLD fMRI have mapped cortical activation during reaching, observed reaching and imagined reaching in humans . A stronSimilar to mirror regions for observing others’ actions, emotion mirror regions activate neural schemata for particular emotions. The empathic emotion of disgust was noted by Charles Darwin: “Disgust refers to something revolting, primarily in relation to the sense of taste, as actually perceived or vividly imagined; and secondarily to anything which causes a similar feeling, through the sense of smell, touch and even eyesight”  . Humans Multiple examples of applications to action observation treatments (AOT) have been trialed and tested for patients with stroke, Parkinson’s disease, multiple sclerosis, cerebral palsy, orthopedic trauma and postsurgical patients . ParkinsThis review has focused on the visual system. Yet perception and mental imagery are known to be multimodal experiences across multiple senses. The focus has been on psychological research, and other multidisciplinary perspectives on vividness have not been covered . This reThe study of mental imagery has contributed to our basic understanding of vision.The ACT provides a coherent and feasible organization for perception and mental imagery. The six modules and their interconnections receive empirical support from a wide range of studies.All six modules of the perception and mental imagery system are influenced by individual differences in vividness.Real-world applications of ACT show interesting potential to improve human wellbeing in both healthy people and patients.Mental imagery can be applied in creative ways to make new collective goals and actions for change that are necessary to maximize the future prospects of the planet.


Coptis chinensis var. chinensis in recent years. C. chinensis var. brevisepala, the closest wild relative of C. chinensis var. chinensis, has a scattered distribution across southwestern China and is an important wild resource. Genetic diversity is associated with greater evolutionary potential and resilience of species or populations and is important for the breeding and conservation of species. Here, we conducted multiplexed massively parallel sequencing of the plastomes of 227 accessions of wild and cultivated C. chinensis using 111 marker pairs to study patterns of genetic diversity, population structure, and phylogeography among wild and cultivated C. chinensis populations. Wild and cultivated resources diverged approximately 2.83 Mya. The cultivated resources experienced a severe genetic bottleneck and possess highly mixed germplasm. However, high genetic diversity has been retained in the wild resources, and subpopulations in different locations differed in genotype composition. The significant divergence in the genetic diversity of wild and cultivated resources indicates that they require different conservation strategies. Wild resources require in situ conservation strategies aiming to expand population sizes while maintaining levels of genetic diversity; by contrast, germplasm resource nurseries with genotypes of cultivated resources and planned distribution measures are needed for the conservation of cultivated resources to prevent cultivated populations from undergoing severe genetic bottlenecks. The results of this study provide comprehensive insights into the genetic diversity, population structure, and phylogeography of C. chinensis and will facilitate future breeding and conservation efforts.Root rot has been a major problem for cultivated populations of  Deterministic or stochastic forces, such as natural selection and genetic drift, increase genetic diversity at several levels of biological organization  . Higher Plastomes have been extensively used in population diversity analyses and phylogeographic studies because of their small size, low rate of recombination and molecular evolution, and uniparental inheritance pattern . With thCoptis is a perennial herbaceous genus of the Ranunculaceae family that has been used in traditional Chinese medicine for its antibacterial and anti-inflammatory properties for thousands of years , and has been cultivated since the Yuan era (ca. 700 years)  . The hisCoptis chinensis var. brevisepala, the closest wild relative of C. chinensis var. chinensis, exhibits a scattered distribution across southwestern China. It has long been cited as an excellent Huanglian  ] . The areC. chinensis in recent years. Domestication of C. chinensis, as in many cultivated plants, may result in genetic bottlenecks and decrease variation in disease resistance within populations  elucidate the genetic diversity of wild and cultivated C. chinensis and their phylogeographic relationships through the rapid and cost-efficient MPS approach, and (3) propose germplasm conservation strategies for wild and cultivated C. chinensis resources.Here, we used a representative set of plastids and MPS data of members of the genus Coptis to (1) clarify evolutionary relationships within Coptis species were collected for plastid sequencing, and the 227 accessions used for MPS were collected across the entire distribution of Coptis; four plastids were downloaded from GenBank with accession numbers MT773635–MT773638  on an Illumina HiSeq2500 platform (PE 250 sequencing). Procedures for MPS library construction followed the protocol of A total of 23 MT773638 , 3. TotaMT773638 . All thede novo assembly of the high-quality reads with following settings: -F embplant_pt, -R 15 and -K 105  . GetOrgad -K 105 . And Gend -K 105 . Ambiguod -K 105 . If neced -K 105 .1 using sample-specific oligo-tags and primers. Finally, large consensus sequences of different primers and samples were generated using Cotu-Generator.py (https://github.com/YanleiLiu1989/Cotu-master). Procedures of the amplicon data processing followed the protocols of Low-quality sequences and sequences shorter than 200 bp were removed from the PE250 dataset using the NGS QC toolkit v2.3.3 with default settings . The cleCoptis plastids were aligned using the MAFFT v7 online service with the “auto” strategy and manually adjusted using MEGA X, which produced dataset-I  and C. japonica (4.85 Mya) estimated by Divergence time was estimated using BEAST v2.6.6 with the following parameter settings: Relaxed Log Normal clock model, Yule speciation model, and GTR substitution model . The splC. chinensis var. chinensis accessions and 12 C. chinensis var. brevisepala accessions were calculated using DnaSP v5.10 , indels, and Pi of 10 SP v5.10 . The indC. omeiensis accessions were used as outgroups (We composed a supermatrix (dataset-II) using SequenceMatrix v1.7.8 to concatenate 25,197 cpDNA fragments generated by MPS  . For datutgroups .K = 2 to K = 10 with ten runs for each K-value, (2) the initial burn-in period was set to 10,000 with 10,000 MCMCs, (3) the most suitable clusters were determined using the DeltaK method on Structure Harvester2 and aggregated by running the CLUMPP program  the optimal number of clusters was determined by running the K-means clustering algorithm from  program , and (4)de novo assembly of 22 C. chinensis accessions [10 C. chinensis var. chinensis (Ccc) accessions and 12 C. chinensis var. brevisepala (Ccb) accessions] from different provinces in China and one C. deltoidei, as well as four other related species from GenBank. All 27 plastids had a typical quadripartite structure comprising two IR regions  separated by the LSC  and the SSC , and the genome size ranged from 154,156 bp (MT773638) to 154,985 bp (MT773635). Coptis plastids were highly conserved in GC content and the number of genes. The total GC content ranged from 38.1 to 38.2%. In addition, all the sequences contained 80 protein-coding genes, 29 tRNA genes, and four rRNA genes . The region of petA-psbJ contained the greatest number of indels and the highest value of Pi. Overall, the results in the Circos map indicated that the Ccb population was diverse. The marker pairs covered all the variable sites including SNPs and indels were developed for MPS. After the validation step, 111 marker pairs were obtained and used for population analyses.The 22 aligned sequences were 155,489 bp in length. We recovered 926 variable sites, 12 haplotypes, and 334 indels, and the average Pi was 0.00197. The Ccc population had 107 variable sites, and five haplotypes with a diversity (Hd) of 0.822. The Ccb population had 596 variable sites, seven haplotypes, and 222 indels, and Hd was 0.924 . Based oCoptis species in China. The relationships among all taxa were well resolved and fully supported, suggesting that the plastids provided increased resolution for phylogenetic reconstruction . Ccc and Ccb split apart from each other approximately 2.83 Mya (95% HPD: 1.91–3.74 Mya). In addition, diversification of the branches representing different subpopulations within Ccb mostly occurred over a short period ranging from 1.30 to 1.57 Mya; however, the HS and JY subpopulations split recently at approximately 0.49 Mya.A phylogenetic analysis of dataset-I was conducted using IQ-tree methods. The best-fit model was TVM+F+R2 according to the Bayesian information criterion (BIC) . Molecultruction . AccessiLarge consensus sequences of 227 accessions with 111 marker pairs comprised dataset-II, which was 43,354 bp  and had a Pi of 0.00305. Dataset-III was a subset of dataset-II without the four outgroups that was 43,283 bp  and had a Pi of 0.00293 . Pi was P < 0.01), whereas most of the genetic diversity of Ccb existed among populations   that were congruent with their geographic distributions . The gen< 0.001) .K = 2, followed by K = 3 and K = 4  was performed using the 917 SNPs from dataset-III. PCA revealed marked separation between the wild and cultivated subpopulations . Cultivand K = 4 , which iC. omeiensis were used as the outgroup) indicated that wild and cultivated populations formed distinct groups; the clustering of wild accessions was congruent with their geographic distributions; and cultivated populations were highly admixed . Within this monophyletic clade, C. teeta was in the basal position, and a clade formed by C. deltoidea and C. omeiensis was sister to the clade containing C. chinensis var. chinensis and C. chinensis var. brevisepala.in China . Clarifiand rbcL , 2018. PC. chinensis diverged approximately 2.83 Mya. The subpopulations of C. chinensis var. brevisepala showed high diversity, and diversification occurred rapidly from 1.30 to 1.57 Mya, which coincides with the period of increased diversification rates of all members of the Ranunculaceae family approximately 2 Mya  has experienced a severe genetic bottleneck and shows extremely low diversity. Compared with Ccb, the Pi of Ccc (0.00032) was only a quarter of that of Ccb (0.00138). The genetic differentiation (Fst) of wild and cultivated populations also indicated that the cultivated population experienced a genetic bottleneck. Previous studies have indicated that ttleneck . By contttleneck . Thus, mC. chinensis potentially stems from a series of bottlenecks that occurred during its domestication, and this is thought to increase its susceptibility to diseases. Ccc is cultivated over large areas (0.32 million mus) in China, and it is an important resource for many industries and a large segment of the human population. The problems posed by root rot require urgent attention, and the solution to this problem might lie in the effective use of the genetic reservoirs of wild populations.Phylogeographic analysis demonstrated that the wild and cultivated populations were distinct, which is consistent with the results of the network analysis, PCA, and STRUCTURE analysis. The highly mixed germplasm of Ccc with little population differentiation could be roughly divided into two or three genotypes that were not congruent with their geographic distributions. By comparison, the differences in the genotype composition of the Ccb subpopulations were congruent with geographic differences among Ccb subpopulations, and some gene flow occurred among neighboring populations. This medicinal species provides an ideal model for evaluating the genetic and phylogeographic consequences of domestication on wild and cultivated populations. The lack of genetic diversity in cultivated C. chinensis need to be developed. DNA barcoding could be used to facilitate species identification. Most hot spots were identified through comparisons of various sequences in this study, such as ycf1, trnHpsbA, trnK-rps16, and rpl32, which have been widely used as barcodes for discriminating between species or reconstructing phylogenies  that could be used for discrimination (Breeding disease-resistant individuals from wild populations is a long process that requires several steps. First, measures to protect wild resources need to be implemented. Ccc and Ccb are difficult to distinguish morphologically in the non-flowering period, especially in the seedling stage; thus, efficient approaches for distinguishing between wild and cultivated logenies , 2015. Tmination .in situ conservation strategies are needed for Ccb. Smaller populations and endangered species often exhibit lower genetic diversity levels (in situ conservation and preservation of wild habitat, the population size of wild populations needs to be increased while maintaining their high levels of genetic diversity.Genetic diversity is closely associated with the adaptive evolutionary potential and reproductive fitness of populations. The maintenance of genetic diversity is a primary focus of the management of wild populations, especially for endangered species. The genetic diversity of Ccb was high, and the phylogeography of subpopulations was congruent with their geographic distributions. Thus, y levels ; howeverCcc has experienced a severe genetic bottleneck due to its cultivation. Several measures need to be taken to revert this trend. Seed exchange should be more carefully controlled, and wild genotypes should be utilized in the cultivation process. Thus, we suggest that a government-established germplasm resource nursery with several genotypes be initiated to minimize the inbreeding of Ccc. The decrease in diversity could be mitigated if the government or research institutes played greater roles in regulating the distribution of seedlings rather than the private sector. Overall, the information on the plastids and the hotspot regions obtained in this study are important for protecting wild resources and guiding the establishment of cultivated germplasm resource nurseries.OM202488-OM202510.The data presented in this study are deposited in the GenBank, accession numbers: YW and JS did the data analysis and wrote the manuscript. CX and ZZ participated in the experiments. ZZ, PQ, SW, ZX, and MW collected the study materials. QY, LG, and LH conceived and designed the research. All authors read and approved the final manuscript.ZX was employed by Lichuan Jianzhuxi Huanglian Cooperative, China. The remaining 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.


Extending the depth of field (DOF) is especially essential in thick and 3D sample imaging. However, it's difficult to achieve both large DOF and high resolution in a zoom microscope. Currently, the use of optical sectioning to expand DOF still has the problem of inconstant magnification. Here, we develop an extended the depth of field (EDOF) and zoom microscope, which can realize EDOF with constant magnification and high resolution. Besides, the proposed microscope can achieve optical axial scanning at different NA and magnifications in real time without any mechanical movement. The proposed varifocal lens is employed to realize optical axial scanning, zooming, and keeping constant magnification when extending the DOF. Experimental results show that the proposed microscope can realize a continuous optical zoom of 10–40×, NA from 0.14 to 0.54, and the DOF of microscope can be extended to 1.2 mm. Generally speaking, obtaining the transverse and axial resolution resolution is accompanied by a loss of horizontal resolution. Therefore, it's difficult to achieve both large DOF and high resolution at various magnifications in a zoom microscope.In order to accurately obtain all the information of the observed object, extending the depth of field (DOF) and zooming have always been the research hotspots in the field of microscopy. The DOF of the microscope is determined by the correlation between the NA and magnification3, pupil filter4 and wavefront coding6, etc. And in the practical applications, the most common method is the optical sectioning techniques, which collects the information of the continuous focal plane by scanning or zooming to reconstruct the object. The current microscopic techniques, such as wide-field microscopy7, confocal microscopy8, two-photon microscopy9 and light sheet microscopy10, can realize 3D imaging by this way. There are many ways to obtain optical slices. Traditional axial scanning method realizes axial focus scanning by changing the distance of the lens group in the microscope or moving the position of the stage12. Although easy to operate, mechanical disturbance may affect the stability of the microscope system and even cause the loss of observation objects. In order to break through the limitations of mechanical scanning, new zoom elements without mechanical movement are used to solve this problem. The multi-focal microlens array (MLA) can get information of different focal planes by switching the optical power14, which shows high spatial resolution and dynamic speed. However, crosstalk between different zones within lenslet and adjacent lenslets may affect the image quality. Besides, the aperture of the microlens array is relatively small, which limits its application in the microscope with high NA. The varifocal lens is a good alternative for not only axial scanning but optical zooming. The varifocal lens, including the electrowetting lens20, liquid crystal lens23, tunable acoustic gradient (TAG)24 and polymer lens26, can vary its focal length by changing the curvature or index of lens. The varifocal lenses have good performance in zoom speed and continuity, and can eliminate some negative effects of mechanical scanning. However, the problem of inconstant magnification still exists in in axial scanning. The issue of inconstant magnification directly affects the accuracy of image reconstruction. At present, there are some methods to correct this problem. For example, setting an electrically adjustable lens to the aperture stop of the microscope objective27 or using an adaptive lens with multiple adjustable surfaces28 can achieve it. Though inconstant magnification can be corrected, the small aperture and low optical power of the electrowetting lens is not suitable for microscopes with a high NA and large zoom range. In addition, it is difficult to use external components in the zoom microscope with limited working distance. At present, there is no integrated microscope that can achieve extension of DOF at different magnifications and correct the problem of inconstant magnification.Numerous methods were proposed to extend the DOF of the microscope, including aperture codingIn this paper, we develop an EDOF and zoom microscope, which can realize EDOF with constant magnification and high resolution. Besides, the proposed microscope can realize optical axial scanning at different NA and magnifications in real time without any mechanical movement. As the core part, the EDOF objective includes four varifocal lenses, which are specially designed and fabricated to realize zoom, optical axial scanning, and keeping constant magnification. At present, there is no microscope with the same function and our microscope objective has many possibilities to be adapted to other microscopy.The proposed microscope can achieve a continuous optical zoom of 10–40× and NA from 0.14 to 0.54. In addition, the DOF of microscope can be extended to 1.2 mm.Schematic illustration of the proposed microscope is shown in Fig. As the key part of the proposed microscope, the EDOF and zoom microscope objective consists of several glass lenses and four varifocal lenses. Simplified schematic diagram of the microscope objective is shown in Fig. In order to make full use of the zoom capability of the varifocal lens, we adopt a splicing design of the front and rear groups in the optical path. The image plane of the front group coincides with the object plane of the back group, and the image space NA of the front group matches the object space NA of the rear group. In this way, we can use the front group to increase the NA of the objective, while the rear group is used to expand the zoom ratio. In addition, both the front and rear groups have zoom capability, so they can work together to achieve continuous optical zoom by controlling the curvature of the four varifocal lenses. In addition, the change of the magnification The transverse magnification of the microscope objective  can be simplified to the Eq. .6\documeThe imaging quality of the proposed PDMS lens is tested as shown in Fig. −1–32 m−1. In addition, when the optical power is small, the correlation between the optical power of the PDMS lens and the injected liquid volume is close to a linear relationship. The capture images of “e” specimen under different liquid volume are shown in Fig. Focusing characteristics of the proposed PDMS lens are shown in Fig. z + 0 mm to z + 1 mm. Additionally, as shown in Fig. z + 0 mm to z + 1 mm, and keep the magnification constant.In order to achieve high resolution, the EDOF objective is optimized at different magnifications and different focal distances. A 2/3’’ CMOS is selected as the image sensor. The largest diagonal field-of-view (FOV) of the EDOF microscope objective is 0.55 mm (V) in the object space. In addition, polychromatic wavelengths of 486 nm, 587 nm and 656 nm are set in the lens model. By adjusting the curvature of the four varifocal lenses we can get the zoom range of the objective from 10× to 40×. The corresponding relationship between optical power of proposed varifocal lens and the movement (z) of the focal plane at different magnifications (M) are shown in Fig. The corresponding diffraction MTF at different magnifications (M) and focal distance (z) are shown in Fig. The fabricated EDOF and zoom microscope is shown in Fig. In the first experiment, we tested the actual imaging quality of the proposed EDOF microscope in a large DOF at different magnifications. The depth of imaged sample was up to approximately 0.32 mm, which was measured by manually translating the sample mounted on the micrometer stage. In the experiment we recorded 4 object plane images with different axial depths for each magnification, and the experimental results are demonstrated in Fig. 30 to image the whipworm slices at 20×. The depth of the imaged whipworm slices is up to approximately 1.2 mm, which is measured by manually translating the sample mounted on the micrometer stage. In the experiment we recorded almost 15 process images with different axial depths for each magnification, and the experimental results are demonstrated in Fig. We further conducted another experiment to verify if the proposed microscope can maintain the same magnification when expanding the DOF. In the experiment, we respectively used the proposed microscope and the conventional microscopeComparing the two synthetic EDOF images shown Fig. In this work, we develop an extended the depth of field (EDOF) and zoom microscope, which can realize extension of the DOF with constant magnification and high resolution. Besides, the proposed microscope can achieve optical axial scanning at different NA and magnifications in real time without any mechanical movement. As the core element of microscope, the proposed varifocal lens is employed to realize optical axial scanning, zoom and keeping constant magnification. In addition, experimental results show that the proposed EDOF microscope can achieve a continuous optical zoom in 10–40×, and the DOF can be extended to 1.2 mm with constant magnification. Besides, the objective has a higher degree of freedom to be combined with various microscopy.Supplementary Information.


Botrytis cinerea is the most harmful postharvest disease of table grapes. Among the strategies that can be envisaged for its control, the use of coatings based on natural products is particularly promising. The study presented herein focuses on the assessment of the antagonistic capacity of two Streptomyces species and their culture filtrates against B. cinerea. Firstly, the secondary metabolites were characterized by gas chromatography–mass spectrometry, with N1-(4-hydroxybutyl)-N3-methylguanidine acetate and 2R,3S-9-guanine acetate as the main compounds produced by S. lavendofoliae DSM 40217; and cyclo(leucyloprolyl) and cyclo as the most abundant chemical species for S. rochei DSM 41729. Subsequently, the capacity of S. lavendofoliae DSM 40217 and S. rochei DSM 41729 to inhibit the growth of the pathogen was tested in dual culture plate assays, finding 85–90% inhibition. In agar dilution tests, their culture filtrates resulted in effective concentration values (EC90) in the 246–3013 μg·mL−1 range. Upon the formation of conjugate complexes with chitosan oligomers (COS) to improve solubility and bioavailability, a synergistic behavior was observed, resulting in lower EC90 values, ranging from 201 to 953 μg·mL−1. Ex situ tests carried out on ‘Timpson’ and ‘Red Globe’ table grapes using the conjugate complexes as coatings were found to maintain the turgor of the grapes and delay the appearance of the pathogen by 10−15 days at concentrations in the 750−1000 µg·mL−1 range. Hence, the conjugate complexes of COS and the selected Streptomyces spp. culture filtrates may be put forward as promising protection treatments for the sustainable control of gray mold. Table grapes are considered a product with functional characteristics since they offer a wealth of health benefits due to their high nutrient and antioxidant contents , which aPostharvest diseases of table grapes in favorable conditions—and particularly in developing countries—can cause losses in total production of up to 55% . HoweverBotrytis spp., is the most harmful postharvest disease in, given that it can grow and spread even at very low temperatures (0.5 °C). Infections frequently occur in the field, where they remain dormant until ripening  = 1.81 × 10−3 M0.93 pyrazine−1,4-dione; hydrazones; butyrolactone; deoxyspergualin; cyclo(leucyloprolyl); cyclo; and oxazinoquinoxalines. In the in vitro mycelial growth inhibition tests, the presence of these chemical species resulted in EC90 values in the 246–3013 μg·mL−1 range, which were substantially improved (EC90 = 201–953 μg·mL−1) upon the formation of conjugate complexes with COS. Ex situ bioassays showed that the COS–S. rochei DSM 41729 secondary metabolites conjugate complex at a concentration of 750 μg·mL−1 fully inhibited the growth of B. cinerea on cv. ‘Timpson’ and cv. ‘Red Globe’ grapes for 15 days. Regarding the clusters treated with COS–S. lavendofoliae DSM 40217 secondary metabolites conjugate complex at 1000 μg·mL−1, full protection was observed for cv. ‘Timpson’ berries, while an incidence of ca. 20% was registered in cv. ‘Red Globe’ grapes after 15 days. These promising results support that coatings based on secondary metabolites of S. rochei DSM 41729 and S. lavendofoliae DSM 40217 can be a promising strategy to control B. cinerea, preserve the quality, and improve the shelf life of table grapes.Gas chromatography–mass spectrometry analyses evidenced that


Acropora tenuis, and its associated endosymbiotic algae along the entire expanse of the Great Barrier Reef (GBR). We use SNPs derived from genome‐wide sequencing to characterize the cnidarian coral host and organelles from zooxanthellate endosymbionts (genus Cladocopium). We discover three distinct and sympatric genetic clusters of coral hosts, whose distributions appear associated with latitude and inshore–offshore reef position. Demographic modelling suggests that the divergence history of the three distinct host taxa ranges from 0.5 to 1.5 million years ago, preceding the GBR's formation, and has been characterized by low‐to‐moderate ongoing inter‐taxon gene flow, consistent with occasional hybridization and introgression typifying coral evolution. Despite this differentiation in the cnidarian host, A. tenuis taxa share a common symbiont pool, dominated by the genus Cladocopium (Clade C). Cladocopium plastid diversity is not strongly associated with host identity but varies with reef location relative to shore: inshore colonies contain lower symbiont diversity on average but have greater differences between colonies as compared with symbiont communities from offshore colonies. Spatial genetic patterns of symbiont communities could reflect local selective pressures maintaining coral holobiont differentiation across an inshore–offshore environmental gradient. The strong influence of environment (but not host identity) on symbiont community composition supports the notion that symbiont community composition responds to habitat and may assist in the adaptation of corals to future environmental change.Genomic studies are uncovering extensive cryptic diversity within reef‐building corals, suggesting that evolutionarily and ecologically relevant diversity is highly underestimated in the very organisms that structure coral reefs. Furthermore, endosymbiotic algae within coral host species can confer adaptive responses to environmental stress and may represent additional axes of coral genetic variation that are not constrained by taxonomic divergence of the cnidarian host. Here, we examine genetic variation in a common and widespread, reef‐building coral,  Rather, dynamic tensions between divergence and homogenization shape genetic diversity among taxa that interbreed occasionally in spatially and (or) temporally variable manners . Cryptic differentiation in corals often coincides with environmental gradients, including depth  to environmental stress  and the wider tropical Indo‐Pacific, with broadcast spawning reproduction. Previous microsatellite surveys of A. tenuis  in the cnidarian hosts and SNPs from symbiont plastids. We aimed to: (1) determine whether A. tenuis resolved into two or more cryptic taxa as evidenced by distinct genetic clusters; (2) document spatial extents of such cryptic taxa and their associations with latitude and shelf position; (3) test for gene flow among cryptic taxa using demographic modelling; and (4) characterize patterns of symbiont diversity within and among coral colonies with respect to latitude, shelf position and cryptic cnidarian host taxon identity. Our data suggest that at least three discrete taxa comprise what is currently recognized as A. tenuis on the GBR. We demonstrate that diversity in the cnidarian host and its symbionts are costructured by similar environmental processes but with much stronger genetic‐environment associations for symbionts than coral hosts. Additionally, we find that structuring of symbiont variation is independent of genetic identity of cnidarian hosts, suggesting that these discrete but cryptic cnidarian taxa share the same, environmentally determined symbiont pool. This study provides the most spatially comprehensive joint survey of genomic variation in cnidarian hosts and their symbiont partners for any coral species on the GBR to date.Here, we characterize 22.1A. tenuis despite recent evidence that this species on the GBR may be different from the A. tenuis type specimen from Fiji and the appropriate GBR species name may be Acropora kenti . Libraries for 698 individual colonies and four technical replicates were sequenced on an Illumina HiSeq HT (125 bp paired end reads) to approximately 5× coverage.Details for all methods including function calls and settings can be found in the Supplemental Information in Appendix 2.2FastQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc) and bbtools .ANGSD v0.921  and combined them with two previously published samples . To extract UCE and exon sequences from whole‐genome data , we extracted read alignments at the ITS1 region and obtained consensus sequences using bcftools v1.3  effect , as these individuals exhibit low admixture and therefore are most characteristic of their genetic cluster . We calculated pairwise geographic distances from latitude and longitude using the Vicinity formula for ellipsoids that accounts for the Earth's curvature and tested for isolation by distance (IBD) between reefs using Mantel tests with 1000 permutations in the R package vegan v2.5‐7  a divergence model (div), which represents strict isolation with no gene flow between clusters; two isolation‐with‐migration (IM) models with (ii) symmetric and (iii) asymmetric gene flow; and (iv) a secondary contact (SC) model, which entails strict isolation followed by SC with gene flow. To balance sample sizes and improve computational efficiency, we subsampled individuals from clusters 1A (n = 15) and 1B (n = 13) to match the low total number of individuals in Cluster 2 (n = 14) and prioritized individuals with the highest depth of coverage. From Cluster 1A, we used three groups of individuals (each group composed of 15 samples) sampled in the northern , central  and southern  GBR to represent the large spatial coverage due to detected IBD within this cluster. For each cluster and each group, we selected individuals that were from the same or nearby reef(s). Using these subsets of individuals, we then generated seven different joint‐SFS representing seven comparisons between three Cluster 1A datasets  and Cluster 1B (north) and Cluster 2 (south). Our pairwise comparisons were thus made between sympatric corals , and allopatric corals . All the folded joint‐SFS were generated using ANGSD v0.934 following the same settings as described for single population SFS. For each of the seven joint‐SFS, we estimated the likelihood of our four different models and estimated their parameters following the optimization of Portik et al.  to partition the effects of latitude and shelf position on coral host genotypes. We fit the model:Y, was a matrix of scaled inferred individual allele frequencies ranging from 0 to 1: 0, a homozygote for the major allele; 0.5, a heterozygote; and 1, a homozygote for the minor allele. Y comprised 448 coral samples and 14,789 SNP loci. The response matrix was conditioned on PC scores of genomic variation .Our data on symbionts include only short reads obtained from shotgun sequencing of the coral colony, and therefore, typical approaches based on matching longer sequence reads to reference databases were not feasible. Rather, we draw upon strategies for examining allelic variation in pooled sequence data and focus on the community of symbionts within each coral as a pooled sample. We began analyses of symbiont variation by testing whether there were compositional differences of Cladocopium plastid genome  to (i) measure symbiont community diversity within each coral colony, and (ii) characterize the distribution of symbiont diversity among different coral taxa or across environmental gradients.Our novel approach of using plastid genome allele frequencies to characterize symbiont variation is analogous to that well established in pool‐seq studies, whereby the goal is to estimate the composition of allele frequencies in a pooled sample and not determine individual genotypes or haplotypes  for the number of symbiont alleles and an RDA. Our GLM of the number of symbiont alleles was used to assess how symbiont diversity varied over latitude, shelf position and coral host. We determined the number of unique alleles per plastid locus for each sampled coral colony:NA was the number of alleles at a locus, counts were the number of mapped reads at a locus, latitude and shore reflect colony position, host cluster was based on PCAs of the host, and locus and sample were fit as random effects to allow a unique intercept for each plastid locus and each coral colony, respectively. All variables were scaled and normalized. The GLM was fit using the lme4 v1.1‐27.1 R package . After the initial filtering, each sample retained a mean of 3,181,264 reads . Alignment of the filtered sequences to the reference genome resulted in an average 94% mapping rate (SD = 11%) with ~5% of samples having a mapping rate <73%. The baited regions  had a mean coverage of 2.01 reads , with read depth ≥3 for 25% of SNPs. The final high‐quality data set resulted in 463 individuals and 296,667 loci falling within baited and nonbaited genomic regions Table , where mn = 1), Fitzroy Island (n = 2), Kelso Reef (n = 2), Orpheus Island (n = 1), Seagull Reef (n = 1), Bugatti Reef (n = 2), Pine Island (n = 1) and Reef 20‐344 (n = 1). After filtering, reducing MLGs to a single representative colony, and removing technical replicates, we retained 60,733 high‐confidence loci falling within baited and nonbaited genomic regions  below a threshold of 0.17, the upper 95% confidence limit among technical replicates. We identified 11 putative clones as evidenced by shared MLG from eight reefs based on these criteria: Tydeman Reef  in s Figure  with 1.50 Figure .A. tenuis reference sequence  clade containing the e Figure .ITS1 host consensus sequences for additional phylogenetics analysis, we were left with n = 46, n = 26 and, n = 8, for genetic Clusters 1A, 1B and 2, respectively. All ITS1 consensus sequences from our study were grouped in a single monophyletic clade with the A. tenuis sequence AF538489  9 Figure . An addiA. tenuis populations  from our data set combined with five previously published A. tenuis individuals shared between both studies did not resolve the three major genomic clusters  and may have been able to leverage extra discriminatory power among groups as compared to a reduced data set (n = 365). Regardless, microsatellites both here , genome‐wide average FST values between Cluster 2 and either Cluster 1A (0.196) or Cluster 1B (FST = 0.212) were substantively greater than between Cluster 1A and 1B (FST = 0.018). Site‐specific FST values varied across the genome  at 42 SNPs relative to Cluster 1A and at 59 SNPs relative to Cluster 1B, whereas comparisons between Clusters 1A and 1B did not indicate any fixed sites (FST range: 0–0.77). Within cluster differentiation was globally weak (mean weighted FST = 0.02) and varied between 0.01 and 0.06 among all pairwise comparisons. We found a significant association between genetic differentiation and reef distance within Cluster 1 , but associations were not significant within Cluster 1B and Cluster 2 with fewer sampled populations  were similar among Clusters 1A (mean = 0.024) and 1B (mean = 0.023), but lower for Cluster 2  model with models involving gene flow and fitted them to 2D joint‐SFS involving seven cluster pair combinations as described in the methods . The effect of latitude  and shore position  were both significant but small , after partialling out the effect of host genetic cluster. The effect size for spatial variables may be downwardly biased as Clusters 1B and 2 were geographically restricted; therefore, the constrained ordination was driven by samples belonging to the host genetic Cluster 1A, for which we had a large, and well distributed, number of samples. The effects of latitude and shore position on genomic variation were largely uncorrelated, as shown by the essentially perpendicular directions of effect in RDA1/RDA2 dimension space, where RDA1 accounted for 43% of the constrained variation  and RDA2 accounted for 30% of the explainable variation .The first two PC axes of the partial RDA examining spatial distribution of genotypes across the GBR described 4.7% of the total variation in individual allele frequencies Figure . The ful3.5ITS2 reference sequences recovered only matches to Clade C (genus Cladocopium) (data from 312 colonies retained following filtering). The C40c reference matched 100% of samples, C72k in 0.64% of samples, and C72d in 0.32% of samples, with 4–275 reads mapped (mean = 17.7 per colony) to the ITS2 locus. Having verified that symbionts were Cladocopium, we focussed on plastid sequence variation to describe symbiont diversity within and between coral colonies. Mapping short reads to plastids provided substantial allelic diversity and coverage (data from 223 colonies retained following filtering), with 19 loci (SNPs and indels) from two contigs (contig 1 and contig 7), with 2–17 alleles per locus. The diversity of symbiont plastid alleles within each coral colony varied from a minimum of 1, to a maximum of 8, alleles per locus, including SNPs  and indels. The average median read depth at a plastid locus was 86.74 reads, with a range of 26–775 median reads.Mapping coral host short reads to a set of Symbiodiniaceae 3.6F1,190 = 12.84, p < 0.001), with increasing alleles per locus with increasing latitude . There was also a significant effect of shelf position . Relative to inshore reefs, offshore reefs had more alleles per locus , whereas midshelf reefs did not significantly differ from inshore reefs . The effect of the host genetic cluster  did not significantly predict the number of alleles per colony. Not surprisingly, there was a significant effect of read counts on the number of symbiont alleles per coral colony  and an increase in alleles per locus with increasing read counts . In summary, after accounting for variation in read counts, symbiont plastid allele diversity exhibited clinal variation with latitude, and lower diversity in inshore and midshelf reefs.Spatial patterns of symbiont plastic diversity showed strong associations with geographic features. The GLM identified a significant effect of latitude , which explained 1.4% of variation in symbiont allele composition. The effect of shelf position was also significant  and explained 18.3% of the variation in symbiont allele composition. There was a nonsignificant effect of the host genetic cluster , which explained 1.2% of the variation in symbiont allele composition. The effect of latitude was almost completely perpendicular to the effect of the offshore factor, indicating that the effects of latitude and offshore position had largely uncorrelated effects on symbiont allele composition on the first two RDA dimensions. Only the first dimension (RDA1) was significant , accounting for 86% of the explained variation in symbiont allelic composition. Read counts accounted for 6% of variation in symbiont allele composition.For the partial RDA examining how symbiont plastid allele composition was structured Figure , we obse4A. tenuis, considering both spatial genomic diversity for the cnidarian host and associated symbiont communities across the entire GBR. We found that the cnidarian host comprised at least three well differentiated but sympatric taxa, with strong evidence for gene flow among them. Our data are consistent with growing evidence that many morphologically defined coral species comprise multiple cryptic co‐occurring taxa that are connected over evolutionary time by low‐to‐moderate levels of ongoing gene flow. Despite this differentiation in the cnidarian host, all identified A. tenuis taxa shared a common symbiont pool, dominated by the genus, Cladacopium. Rather than segregated by cnidarian host taxa, symbiont allelic variation was largely structured by shelf position, supporting the idea that broadcast spawning corals possess locally acquired and environmentally filtered endosymbiont communities. Habitat matching of coral symbionts might therefore provide an important mechanism for rapid adaptive change in corals.Here, we present a uniquely rich data set for a common coral species, 4.1Acropora tenuis is common on the GBR and throughout the Indo‐Pacific, and among the first corals to be characterized in population genomic investigations  leverage genotype compositions  reported between s Figure . In all s Figure . Few fix4.3A. tenuis, we found that both cnidarian host genotypes and symbiont allelic variation were structured with respect to latitude and shelf position. For the cnidarian host, latitude and shelf position together explained a very small but significant proportion of variation (0.77%) in individual allele frequencies. This signal was largely driven by Cluster 1A for which we had the most widely distributed samples . All coral colonies contained the C40c strain, and <1% contained C72k and C72d. Yet, by recovering plastid sequences from colony‐level shotgun sequencing, we could further characterize allelic variation and test the effects of the environment on symbiont structure. Our novel method of describing symbiont diversity based on plastid derived alleles appears to yield more information than strain identity based on ITS2 sequences. Because genomic sequencing is typically obtained from samples of the coral holobiont, our approach demonstrates the great potential of using cocaptured symbiont genome sequences to study variation variations in these endosymbiotic bacteria alongside their cnidarian hosts.In contrast, genetic differentiation by latitude and shelf position was substantial for symbionts, explaining 19.7% of variation in compositional turnover Figure . ITS2 seCladocopium plastids varied by shelf position, and to a lesser extent by latitude, both in terms of alpha  and beta  diversity. These distinct aspects of spatial diversity were evidenced in the GLM results that focussed on spatial patterns of within‐colony diversity. Colonies from northern and further offshore sites had more plastid alleles per locus, compared with colonies from southern, inshore and midshelf locations. Our RDA of symbiont allelic composition showed substantial turnover along latitudinal and shelf gradients , greater environmental variance might promote variation among inshore locations (i.e. increased beta diversity).Our observations that e et al. . In theiA. tenuis at the scale of the GBR. However, symbiont strains can exhibit significant turnover across very short physical distances, for example, between leeward and windward sides of coral islands  that investigators try to build explicit links between new and previously published DNA sequence‐based data sets. Throughout this investigation, we have endeavoured to place our new results in the context of previous work on s Figure , and cons Figure .A. tenuis samples with those from a low‐coverage whole‐genome study by Cooke et al. .Genetic data: Raw sequence reads are deposited in the SRA (BioProject PRJNA849642) and individual genotype data are available on Data Dryad (48096232EVA‐2022‐007‐OA.R1). Sample metadata can be found in GEOME , and the research addresses a priority concern, in this case the conservation of corals. Benefits from this research accrue from the sharing of our data and results on public databases as described above.Appendix S1Click here for additional data file.