Alternaria alternata is an opportunistic human fungal pathogen and a ubiquitous phytopathogen capable of causing diseases to >100 agricultural crops and ornamental plants. To control plant diseases caused by A. alternata, triazole fungicides have been widely used both in open crop and vegetable fields and in indoor growth facilities such as greenhouses. At present, the effect of fungicide use on triazole resistance development in A. alternata populations is not known. Here, we isolated 237 A. alternata strains from nine greenhouses around metropolitan Kunming in Yunnan, southwest China, determined their genotypes using 10 short tandem repeat markers, and quantified their susceptibility to four triazoles . Abundant allelic and genotypic diversities were detected among these A. alternata strains. Significantly, over 17% of the strains were resistant to difenoconazole, and both known and new drug-resistance mutations were found in the triazole target gene cyp51. Our findings of high-level genetic variation of A. alternata in greenhouses coupled with high-frequency fungicide resistance call for greater attention to continued monitoring and to developing alternative plant fungal disease management strategies in greenhouses.IMPORTANCEAlternaria alternata is among the most common fungi in our environments, such as indoor facilities, the soil, and outdoor air. It can cause diseases in >100 crop and ornamental plants. Furthermore, it can cause human infections. However, our understanding of its genetic diversity and antifungal susceptibility is very limited. Indeed, the critical threshold values for resistance have not been defined for most antifungal drugs in this species. Greenhouses are known to have heavy applications of agricultural fungicides. In this study, we analyzed strains of A. alternata from nine greenhouses near metropolitan Kunming in southwestern China. Our study revealed very high genetic diversity and identified strains with high MIC values against two agricultural and two medical triazole antifungals within each of the nine greenhouses. Our study calls for greater attention to this emerging threat to food security and human health. Alternaria is a ubiquitous fungal genus distributed in almost every ecological niche across the globe . Among these categories of fungicides, due to their broad spectrum of activity against different fungal pathogens, DMIs such as triazole fungicides difenoconazole (DIF) and tebuconazole (TEB) have been widely used , boscalid, fluopyram, and isopyrazam (SDHIs) after only 3 to 4 years of consecutive applications of these fungicides  had unimodal but different distribution patterns, suggesting potentially common mechanisms of resistance , random amplified polymorphic DNA (RAPD), and polymorphic microsatellite markers or short tandem repeats (STRs)  –48. Thesribution .Aspergillus fumigatus isolates from nine greenhouses in Jinning county, metropolitan Kunming, Southwest China. A high level of genetic diversity and a high frequency of triazole resistance were observed in the greenhouse populations of As. fumigatus  and geographic region (southwestern China), we are also interested in whether populations of A. alternata from the same greenhouses as As. fumigatus would have similar patterns of genetic diversity and triazole susceptibility. To achieve our objectives, we isolated A. alternata strains from the same soil samples in the same nine greenhouses in Jinning county, Kunming, China. These isolates were genotyped with a panel of 10 short tandem repeat (STR or microsatellite) markers. In addition, we investigated the susceptibility of these A. alternata isolates to four triazoles, two agricultural triazole fungicides, and two medical triazole drugs, to identify the prevalence of triazole resistance among our A. alternata samples. Finally, the DNA sequences of the triazole target gene cyp51 were analyzed to identify potential mutations associated with triazole resistance in our strains.In a recent study, we analyzed umigatus . SimilarA. alternata isolates from the 900 soil samples that we originally used to isolate As. fumigatus in a previous study . To further confirm the species status, we obtained the DNA sequences for nine randomly selected isolates at the following six gene loci: glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase second largest subunit (rpb2), translation elongation factor 1-alpha (tef1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (endoPG), and an anonymous gene region (OPA10-2) as suggested by Woudenberg et al. .We obtained a total of 237 us study . Among tg et al. . The BayA. alternata being haploids. Genotype data based on the 10 STR markers were used to infer genetic diversity and population structure of A. alternata from the nine greenhouses. The results showed that the polymorphic information content (PIC) of each marker was ≥0.5  to 17 (locus c10524) per locus in our sample of 237 isolates . HoweverA. alternata isolates from the nine greenhouses, we found 187 multilocus genotypes (MLGs) based on the 10 STR loci  tree  the relationships among strains from the nine greenhouses, (ii) the distributions of triazole-susceptible and -resistant strains, and (iii) the distributions of azole resistance-associated mutations in the SN) tree . Then, tMSN tree . The resMSN tree . FurtherMSN tree  and C.P = 0.001) (Table S1). We further investigated the extent of genetic differentiation between pairs of greenhouse populations. Our results showed that moderately to highly significant differentiations (P < 0.01) were found between pop.8 and all other eight greenhouse populations (Table S2). The biggest differentiation was found between pop.3 and pop.8 , followed by that between pop.1 and pop.8  (Table S2). However, Mantel test showed no statistically significant correlation between geographical distances and population genetic distances  based on clone-corrected data revealed that 96% of the total genetic variation was found within individual greenhouse populations, and a low (4.5%) but statistically significant genetic differentiation was found among the nine greenhouse populations . STRUCTURE analyses revealed that the optimal number of genetic clusters in the total sample was 2  based on the pairwise mean population haploid genetic distance revealed that the first and second principal coordinate axes explained 56.3% and 30.2% of the total variation, respectively . As show50 values of 183 isolates to DIF, ITR, and TEB were 1 μg/mL, 1 μg/mL, and 8 μg/mL, respectively. However, the lowest MIC to VOR was higher than those of the other three triazoles, and the range of MICs for VOR was from 0.125 to ≥16.00 μg/mL (MIC50 = 4 μg/mL)  and two agricultural triazole fungicides (DIF and TEB) using a serial dilution of drug concentrations from 16.00 μg/mL to 0.0078 μg/mL in a microtiter plate format. The results showed that the MIC values of the 183 isolates to DIF, ITR, and TEB ranged from ≤0.0078 μg/mL to ≥16.00 μg/mL. The MIC4 μg/mL) . Less thA. alternata, we investigated potential cross-resistance among the four triazoles using the Pearson correlation analysis based on their MIC values. Specifically, a statistically significant positive correlation in MIC values between two triazoles would signify evidence for cross-resistance in our A. alternata sample to the two tested triazoles. Our results revealed significant positive correlations between MIC values of DIF and VOR , DIF and ITR , VOR and TEB , and VOR and ITR  (Due to the lack of consensus definition for defining triazole resistance in = 0.016) . TogetheA. alternata were the same as those used for isolating As. fumigatus in our previous study .The soil samples used for isolating us study . The resus study . The conus study . Statistcyp51 gene. Among the 68 SNPs, 56 and 12 were in exons and introns, respectively. Of the 56 SNPs in the exons, 43 were synonymous and 13 were nonsynonymous. Most of the synonymous sites  were observed in fewer than three isolates each, while 30% (13/43) of the synonymous SNPs, including 120C→G, 180C→T, 370C→T, 466C→T, 469C→T, 575C→T, 1226C→T, 1352C→T, 1361G→A, 1364T→C, 1433T→C, 1482G→A, and 1484T→C, were detected in every greenhouse population with overall frequencies of about 24% (53/224), 26% (58/224), 28% (63/224), 35% (79/224), 28% (63/224), 29% (64/224), 17% (38/224), 17% (38/224), 17% (38/224), 67% (150/224), 7% (16/224), 29% (64/224), and 68% (152/224) in the total sample, respectively.A total of 68 single nucleotide polymorphisms (SNPs) and 69 haplotypes were observed among the 224 isolates in which we successfully amplified the The 13 nonsynonymous SNPs resulted in 11 amino acid substitutions, including F76I, Y136F, G182R, N188K, V192I, S237A, A307G, H412Y, E434D, G448S, and G462S . Among tcyp51 gene, the 1,921-bp upstream sequence of cyp51 gene was amplified using primers Ucyp-F/Ucyp-R. An insertion of 6 bp located at position −31 to −36 was observed with a frequency of 66% (148/224) in the total sample. One additional nucleotide insertion at position −339 was detected in 29% (64/224) of the isolates. Interestingly, 63 of 64 isolates with insertion at −339 position simultaneously possessed the 6 bp insertion described above.In order to reveal the polymorphism of promoter region of 2 test) was used to evaluate the association between the observed mutations and the frequency of isolates in different MIC ranges. The results found that amino acid substitutions of F76I (P = 0) and G462S (P = 0.005) and the 6-bp insertion (P = 0.014) in the upstream region of cyp51 gene were significantly associated with the frequencies of A. alternata isolates distributed in MIC < 0.125 μg/mL, 0.125 μg/mL ≤ MIC ≤ 8 μg/mL, and MIC ≥ 16 μg/mL to DIF. The amino acid substitution of G462S (P = 0) and the 6-bp insertion (P = 0.01) were significantly associated with the frequency of isolates in the same three MIC ranges to TEB. In addition, significant associations were detected between F76I (P = 0), G462S (P = 0), and the 6-bp (P = 0.024) insertion and the frequencies of A. alternata isolates distributed in MIC ≤ 1 μg/mL, 2 μg/mL ≤ MIC ≤ 4 μg/mL, and MIC ≥ 8 μg/mL to VOR. However, for the same MIC ranges to ITR, no statistically significant association was detected in F76I, G462S, or the 6-bp insertion genotype groups.The chi-square goodness-of-fit test  have emerged as the preferred markers for population genetic studies, especially for recently evolved populations. Indeed, studies based on STRs have shown patterns of genetic variation and population structure of Alternaria spp. different from those based on RAPD, AFLP, and RFLP markers  that included geographically diverse samples from Fujian (southeastern China), Henan , Heilongjiang (northeastern China), and Yunnan (southwestern China) provinces based on seven simple sequence repeat (SSR) markers and for an Alternaria tenuissima population (191 isolates) from four broad regions in China   .Alternaria species, including those with or without a known sexual cycle  combining deleterious alleles at different loci from different parents into the same progeny so that such alleles are eliminated more effectively and (ii) combining beneficial alleles from different parents so that the alleles are selected for more effectively , 61. In al cycle , 61, 62.As. fumigatus  was found among the nine greenhouse populations of another fungus, umigatus . Both spA. alternata were found in our total sample of 237 isolates. The number of two genetic clusters was smaller than those reported previously for Alternaria tenuissima and A. alternata based on different population samples  and both live primarily as saprotrophs. As a result, they likely compete for the same nutrients where the competitiveness of one species could have negative consequences on the other. A high genetic diversity in one species could signify that the founding population of the species was diverse, had a high mutation rate, and/or experienced frequent sexual reproduction. In a stable environment, such a population could exert selective pressure against its competitor species where only highly competitive genotypes (thus a low genetic diversity) could be found in the population. To test this hypothesis, the interaction patterns of the strains and genotypes of these two species from these greenhouses need to be determined.Interestingly, though statistically insignificant, a negative correlation was observed in gene diversity between As. fumigatus, A. alternata is an opportunistic fungal pathogen and is the culprit of destructive foliar diseases, such as early blight and brown blotch, in over 100 plants. The additional niche on host plants for A. alternata could provide potentially novel opportunities for selection and evolution to occur for A. alternata that might be absent for As. fumigatus. Specifically, to prevent and control early blight and brown blotch in economic and horticultural crops both within and outside greenhouses, many classes of fungicides have been used. Frequent application of these protectant fungicides could have facilitated the emergence of fungicide resistance in these pathogens on crop plants  could also contribute to the maintenance of genetic diversity.Interestingly, we found negative correlations between gene diversity and MIC values for both ITR and VOR in our greenhouse population of A. alternata is also recognized as a pathogen of immunocompromised patients and those with significant underlying disease conditions . Furthermore, whole-genome sequencing and genome-wide association studies of populations of strains with different MICs could help identify additional mutations outside the cyp51 gene that are associated with triazole susceptibilities in A. alternata  supplemented with 2% manganese chloride tetra hydrate (MnCl2·4H2O) and 50 mg/L chloramphenicol  for nine randomly selected isolates for their species confirmation, based on methods described previously (Genomic DNA was extracted from the mycelia collected from single spore cultures growing on peptone-dextrose agar (PDA) medium using the cetyltrimethylammonium bromide (CTAB) procedure . All isoeviously , 51.Alternaria spp. , 9.5 μL sterile distilled water, 12.5 μL of 2× T5 TSE101 PCR mix (TSINGKE Biotechnology Co. Ltd.), 1.0 μL forward primer, and 1.0 μL reverse primer. The amplification was conducted in an Eppendorf MastercyclerR using the following protocol: an initial denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s, and it ended with a final extension for 5 min at 72°C. The lengths of the obtained amplicons were then determined using an ABI 3730 DNA sequencer (Applied Biosystems), with fragments of different lengths at each locus representing different alleles. The allelic combination at the 10 STR loci represented the multilocus genotype for each strain.A total of 39 primer pairs for STR markers previously developed for ria spp. , 58, 59 lternata , and Alta solani   and polymorphic information content (PIC), were computed using PowerMarker v3.25 software .A. alternata as well as to infer the relationships between genetic and geographical distances (Mantel test) among populations. The same program was employed to calculate population genetic diversity indices over all loci, including observed number of alleles (Na), effective number of alleles (Ne), Shannon’s information statistic (I), allelic frequency, number of private alleles (NPA), Nei’s gene diversity (h), and percentage of polymorphic loci (PPL) of A. alternata in individual greenhouses. Analysis of molecular variance (AMOVA), pairwise population genetic distances, and gene flow were inferred using the GenAlEx 6.5, as well.The STR genotype data were imported into GenAlEx 6.5  to calcuST) and gene flow  were estimated using POPGENE version 1.32  and voriconazole (VOR) are commonly used for treating patients with mycotic infections, including aspergillosis. DIF and TEB were purchased from Shanghai Yuanye Bio-technology Co., Ltd. , at 97% and 99% active ingredient concentrations, respectively. ITR and VOR were purchased from Shanghai Macklin Biochemical Co., Ltd.  at 98% and 99% active ingredient concentrations, respectively. All four triazoles were dissolved in dimethyl sulfoxide, respectively, to obtain stock solution of 1.6 × 10Susceptibilities of 183 randomly selected strains representing all populations were determined using a modified CLSI M38-A2 broth microdilution method . BrieflyTo induce conidial formation for triazole susceptibility testing, all isolates were grown on potato carrot agar for 5 days at 30°C. Approximately 5 mL of sterile 0.85% saline with Tween 20 was placed on top of the sporulating colonies to prepare a suspension by gently probing the colonies with the tip of a transfer pipette. The optical densities of the conidial suspensions at 530 nm were determined and adjusted to between 0.25 and 0.3. Aliquots of 100 μL of each adjusted conidial suspension were added to the medium with serially diluted fungicides in the 96-well plate. The 96-well plates were incubated at 30°C for 72 h before being checked for growth under a microscope.A. alternata resistance for any of the four triazoles. In our study, we examined the germination of conidia and growth of hyphae at each drug concentration for each isolate. We defined the MIC for each drug as the lowest concentration of a fungicide/drug at which conidial germination and hyphal extension of A. alternata strains were completely inhibited as determined using microscopic observations. As shown in Candida strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were used as references.The MIC was defined as the lowest concentration of an antimicrobial agent that causes a specified reduction in visible growth of a microorganism in an agar or broth dilution susceptibility test. At present, there is no recommended cutoff value for defining ATTGGATACCCTGGTCCATGC-3′) and Altcyp51-R (5′-TTAAGACCCGAAATGCGTCG-3′) were used to amplify the cyp51 amino acid-coding gene of A. alternata. Primers Ucyp-F (5′-CAACGGCACATTTGTCAACG-3′) and Ucyp-R (5′-CAGGATAAAGGAGGCGAAGC-3′) were used to amplify the upstream sequence of cyp51 (cyp51 gene were identified by comparing our sequences with the reference sequence of a triazole-susceptible A. alternata strain in GenBank with the accession number MN542658 (cyp51 gene and MICs among isolates for each of the four triazole drugs was assessed using the IBM SPSS statistical software version 25.0.Primers Altcyp51-F (5′-of cyp51 . MutatioMN542658 . The potAll data supporting the conclusions of this study are presented in the article.


Capsicum pubescens) is a regionally important food crop grown and marketed throughout the mid-highlands of South andCentral America, but little is known about its evolution and the diversity it harbours. An initial scan of genetic diversity and structure across its cultivation range was conducted, the first one using a genomic approach. The RAD-sequencing methodology was applied to a sampling of C. pubescens germplasm consisting of 67 accessions from different American countries, covering its range of distribution/cultivation on the continent. The RAD-seq SNP data obtained clustered the accessions into three major groups, with a high degree of admixture/reticulation among them. Moderate but significant differentiation and geographic structuration were found, depicting a south–north pattern in the distribution of genetic variation. The highest levels of diversity were found among central-western Bolivian individuals, while the lowest was found across Central America-Mexican germplasm. This study provides new genome-wide supported insights into the diversity and differentiation of C. pubescens, as well as a starting point for more efficient use of its genetic variation and germplasm conservation efforts. The findings also contribute to understanding the evolutionary history of C. pubescens, but further investigation is needed to disentangle its origin and diversification under domestication.The locoto chile ( Capsicum, Solanaceae) are famous spices and vegetables consumed worldwide. The genus Capsicum comprises 43 species native to tropical and temperate regions of the Americas, with the centre of diversity in the Andes [C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens. The three economically most important species are C. annuum, C. chinense, and C. frutescens, which are cultivated in many countries around the globe [C. baccatum and C. pubescens, are cultivated and consumed mostly in South and Central America [Chile peppers , holding a greater cultural and economic importance in the Central Andes, especially in Bolivia and Peru [The hot chile ubescens  is betteand Peru ,5,6. It and Peru ,7,8,9, wand Peru . The fruand Peru .C. pubescens in 1799 from plants cultivated in Peru [C. pubescens [Capsicum, C. pubescens forms a morphologically distinct group together with the wild species C. eximium, C. eshbaughii and C. cardenasii [C. pubescens is not an isolated lineage, but a sister species to a small clade formed by C. eximium, C. eshbaughii, and C. cardenasii, all four species conforming the so-called clade Pubescens [Capsicum species would be the wild ancestor to C. pubescens [C. pubescens diverged in the upper Pliocene, earlier than the clade formed by C. eximium, C. eshbaughii, and C. cardenasii, with these three species diversifying from the mid-Pleistocene [Ruiz and Pavon originally described  in Peru , citing  in Peru . It has  in Peru ,14,15, a in Peru , and has in Peru ,17,18. U in Peru . Fruits rdenasii ,17, whicubescens . Those rubescens . Indeed,istocene .C. pubescens is the less exploited among the five domesticated chiles, most likely because of its environmental requirements  and the high fruit fleshiness, which makes them prone to rotting quickly [C. pubescens outside the Americas is very limited, although it has been introduced and grown in Indonesia more than 100 years ago [In addition to its uncertain origin,  quickly . Cultivaears ago . Neverthears ago ,22,23. Tears ago .C. pubescens and the levels of genetic variation and structure it harbours. Previous molecular studies based on a small number of DNA markers, such as amplified fragment length polymorphisms (AFLPs) and simple sequence repeat markers (SSRs), have provided some insight into its variability [C. pubescens in the Americas.Characterising genetic variation within crop species is key in the study of agrobiodiversity, since it may provide a useful framework for the effective use and conservation of that diversity ,26,27. Diability ,29. ThesC. pubescens along its range of distribution/cultivation in the Americas. As a reduced-representation sequencing technique, RAD-seq allows the identification of thousands of SNPs across the genome [C. pubescens? (2) Is the variability of the species genetically structured? (3) How is its genetic variation distributed geographically? and (4) What factors could be influencing the observed genetic variation?This study aimed to lay the groundwork for a genomic perspective of the variability within the locoto chile. To this end, single nucleotide polymorphism (SNPs) markers generated by Restriction-site-Associated DNA Sequencing (RAD-seq)  were usee genome  and has e genome ,32, incle genome ,35,36,37A total of 112,026,584 reads were generated by Illumina sequencing of the RAD-seq libraries. After demultiplexing and filtering of low-quality reads, 103,086,817 were retained. The number of reads per individual ranged from 192,601 to 3,662,664  . Out of The ipyrad pipeline was run separately for datasets used in downstream analyses. For the dataset used in SplitsTree, there were 140,508 prefiltered loci and 44,507 filtered, with 183,569 SNPs and 41,636 unlinked SNPs. For the population inferences dataset, the filtered loci were 101,132 with 345,793 SNPs and 83,813 unlinked SNPs. After filtering out loci/SNPs with more than 80% missing data and a minor allele frequency of less than 0.05, the pruned dataset used consisted of 1462 unlinked biallelic SNPs.C. pubescens, separating the southern from the northern accessions  . The third one clustered samples from Villa Serrano, a central-Bolivian small town situated in mid-elevated highlands (2000 m) about 190 km SW of Santa Cruz de la Sierra. The remaining two groups (among the five recovered) consisted of materials from Peru to Mexico. One of them encompassed samples from Peru to Ecuador, and also a single individual collected in north-western Argentina (# 270). The last group consisted of Central American and Mexican individuals, including two samples of unknown origin .The phylogenetic network showed a clear geographic split in essions) a. While Ten accessions sampled from local markets in La Paz, Bolivia  were located at the centre of the network together with two samples from Cusco, Peru  and one from Salta, Argentina (# 271) a. This sK = 3 genetic clusters, as both the Bayesian information criterion and cross-validation test converged on this number , mostly reflecting the genetic groups identified by the network analysis (K = 3), DAPC supported three geographically structured clusters, with two discriminant functions (dimensions) describing the relationships  and, at K = 5, the remaining samples within G2 were split into individuals from Peru and Ecuador on the one hand and those from central-western Bolivia on the other. Differences with the Admixture results were found at K = 4, where individuals from central-western Bolivia and Villa Serrano were clustered together into a different group from that of the Peruvian-Ecuadorian samples , standard measures of genetic diversity were calculated for the three main clusters of C. pubescens  was the less diverse.Following the best fit model  and low inbreeding (FIS = 0.024) across and within groups of C. pubescens were observed based on Admixture clustering. The DAPC results also converged into this pattern. For K = 3, pairs of clusters were significantly differentiated from one another by pairwise calculations of FST   revealed a significant genetic structure between clusters/populations (= 0.001) . The maj= 0.001) .C. pubescens. The number of RAD-seq reads obtained and filtered, the number of loci/SNPs found and the values of the estimated parameters were consistent with previous reports for population studies in non-model plants e.g., [C. pubescens. Genetically distinct clusters with different levels of genetic diversity could be defined, which correlated with the geographic origin of the accessions. The patterns of genetic diversity and structure observed within C. pubescens would reflect trends in human use and trading of germplasm in the Americas.This is the first study to evaluate genomic variation derived from NGS technologies, specifically Restriction-site-Associated DNA Sequencing (RAD-seq) methodology, for ts e.g., ,39,40 deC. pubescens holds a narrow genetic diversity as a consequence of a founder effect during its domestication [C. pubescens could have low genetic variability due to domestication, the species is known only as a cultigen and not from the wild, so this hypothesis has not been tested empirically. The variability within the species has been partially studied under different approaches such as morphological e.g., [C. pubescens as the domesticated chile having the narrowest genetic diversity, but there is no research undertaken on its variability across its entire cultivation range in the Americas. Even though the current work is not an exhaustive study of C. pubescens genetic variation, since the sampling could be more dense, it represents an initial contribution that stands out from previous work in three main points: the analyses based on genome-wide SNPs, the inclusion of a higher number of accessions to cover a broader cultivation range in America , and (when possible) more detailed information of cultivation and sales locations of the studied samples. For this reason, these results consent to make some comments about what factors could be influencing the observed genetic variation in the locoto chile, as well as its origin and diversification under domestication, and the geographic distribution of diversity.It has been proposed that tication . The speal e.g., ,23,42, pal e.g., ,43,44, aal e.g., ,29. ThesC. pubescens. Two main and clearly differentiated groups corresponded to germplasm from northern Argentina to south-central Bolivia (G1) and from Central America to Mexico (G3), respectively. A third less well-defined group was represented by accessions from central-western Bolivia, Peru to Ecuador (G2). In addition to these three clusters, the Admixture, principal components, and genetic diversity analyses showed that G2 had high levels of genetic admixture and diversity. Within this large cluster, a substructure was recognized as consistent with the phylogenetic network grouping , also indicating a higher admixture and genetic diversity in central-western Bolivian materials. This was in line with the elevated degree of reticulation observed in the network, pointing out considerable levels of gene flow and/or incomplete lineage sorting within G2. Conversely, the G3 showed the lowest levels of genetic diversity and high inbreeding. Taken together, these results revealed that the genetic variation within C. pubescens was structured although moderately differentiated, depicting a south–north pattern in the distribution of genetic diversity and lineage splitting in the species. It is worth noting that variability in the fruit features  was represented in all three groups, with the exception of individuals from La Paz surroundings . A south–north differentiation was also reported by Ibiza et al. [in situ from adult plants and their seeds were afterwards cultivated under controlled conditions, so their particular morphology was corroborated: sparse pubescence , small flowers mostly 5-merous, and the smallest and fleshiest fruits  balanced against the homogenising effect of gene flow and planting of homogeneous cultivars ,49. SincC. pubescens in its putative native range, the South American Central Andes, this work also analysed germplasm from its two latitudinal extremes of cultivation in America: northern Argentina to the south, and central Mexico to the north. The first studies concerning the variation of the species [C. pubescens is recognised as an introduced cultivated species in Argentina [In addition to advancing the study of the genetic variation of  species ,7,51 didrgentina ,53, beinrgentina  , the accessions studied here formed a distinct genetic group (G3) with the lowest levels of genetic diversity and admixture. This would be consistent with records of the introduction of C. pubescens into Central American and Mexican highlands occurring in the 20th century [C. pubescens to Central America would have originated from Peruvian-Ecuadorian sources due to the observed larger affinity. Silvar and García-González [Regarding the northern extreme of the  century ,55,56 ra century . Based oGonzález  also fouGonzález .C. pubescens inferred here following a genome-wide approach represent a useful starting point for understanding the extent and the partitioning of genetic variation across its cultivation range in the Americas. Trends in human use and trading would be key factors that have shaped the patterns observed in this work. The finding of greater genetic diversity in central-western Bolivia and a diversity loss associated with its dispersion towards the north of the continent provides some clues for new studies that seek to unravel the domestication history of the locoto. Of particular value might be an enlargement of the Bolivia to Peru sampling  to provide better coverage of its putative native range. In this context, to better understand the origin and domestication of C. pubescens, its relationship with its wild sister species should be analysed in-depth.Understanding how evolutionary processes  impact crop genetic diversity and characterising the standing genetic variation is key to germplasm conservation and crop improvement efforts. The three (to five) genetic groups of C. pubescens along its cultivation range, a total of 74 accessions from several South–Central American countries were included in the analyses  or the Invisorb® Spin Plant Mini Kit  from leaves dried in silica gel following the manufacturers’ instructions. DNA extracts were purified using the NucleoSpin® gDNA Clean-up kit . DNA was checked for quality by agarose gel electrophoresis and quantified using a Qubit® three Fluorometer .Genomic DNA was isolated using either the DNeasy Plant MiniPhred scores set to 20 and allowing a single mismatch in the barcodes). After demultiplexing reads within each sublibrary based on the individual barcode, six samples were excluded based on low sequencing coverage; additionally, one sample was removed from the dataset after preliminary analysis showed it to be an outlier.RAD-seq libraries preparation followed the protocol of Paun et al.  with modclust_threshold . Clustering threshold (ct) selection approaches aim at determining appropriate ct values to establish homology while avoiding clustering of paralogous RAD-seq loci [Variable SNP loci from all samples were filtered and clustered de novo in ipyrad v0.9.82  using deseq loci . Applicaseq loci ,65,66,67min_sample_locus = 34 , that is 50–51% of samples. Because population genetic analyses are less tolerant of missing data and rare alleles than phylogenetic analysis, additional filtering was performed to obtain the appropriate datasets for the execution of the structure and diversity analyses. First, demultiplexed reads were filtered in ipyrad using the parameter min_sample_locus = 4. The *.vcf file obtained was then processed in VCFtools v.0.1.15 [To produce a dataset for phylogenetic analysis, which can tolerate relatively large amounts of missing data, filtering was conducted using the parameter v.0.1.15 . Only biv.0.1.15 .C. pubescens accessions, an unrooted phylogenetic network was constructed using SplitsTree v.4.17 [To explore the phylogenetic relationships and distances between e v.4.17 . The proe v.4.17  was usede v.4.17  and SpliK = 2–5, 20 pilot runs and 5000 generations, with 5000 initial generations discarded as burn-in) identified zero outliers compared to 74 outliers detected by PCAdapt . As no outliers were common across both approaches, those identified by PCAdapt were considered false positives, therefore the analyses described below were conducted using all loci.The occurrence of outlier/non-neutral loci, interpreted as candidates for loci under selection, was assessed for the pruned dataset  using Bayescan v.2.1  and a PrK-means clustering to identify the optimal number of clusters from K= 1–10, and the appropriate number of clusters was then inferred using Bayesian information criterion (BIC). The cross-validation function Xval.dapc was used to determine the optimal number of PCs to be retained. Second, the genetic structure was also inferred using Admixture v.1.3 [K = 1–10 with 100 replicate runs per K. The most informative value of K was explored using the cross-validation (CV) method and results were plotted using R. Barplots indicating the genetic group assignments were drawn in R. Third, a principal component analysis (PCA) was also conducted in the R package adegenet to visualize samples in two-dimensional genetic space.First, a discriminant analysis of principal components (DAPC)  was implre v.1.3 , as an are v.1.3 , then fure v.1.3 . The anaC. pubescens accessions using the pruned dataset of unlinked SNPs. Total number of alleles (A), observed heterozygosity (HO), and gene diversity  were calculated with the adegenet package v.2.1.3 [R; rarefied to account for population size) and private allelic richness (AP) were estimated in ADZE v.1.0 [IS) was calculated in hierfstat v.0.5–7 [Once genetic structure was inferred, summary population genetic statistics were calculated separately in two subsets based on each clustering approach: (a) for clusters inferred using DAPC, and (b) Admixture clusters with mixed ancestry individuals removed (<70% membership probability) to avoid effects of potential genetic admixture or gene flow effects. Common measures of genetic diversity were calculated for the inferred clusters of  v.2.1.3  in R [75ZE v.1.0 . The inb v.0.5–7  in R.C. pubescens clusters, pairwise values of population differentiation (FST of Weir & Cockerham) [To test for significant genetic differentiation between the inferred ckerham)  were calckerham)  with theckerham)  in R, wi


Boops boops throughout most of its widespread distribution. Our results identified the existence of three clades, one comprising specimens from the Azores and eastern Atlantic/Mediterranean, another with individuals from the Canary Islands, Madeira and Cape Verde archipelagos, and the third with samples from Mauritania only. One of the branches of the northern subtropical gyre (Azores Current) that drifts towards the Gulf of Cádiz promotes a closer connection between the Azores, southern Portugal and the Mediterranean B. boops populations. The Almería-Oran Front, widely recognised as an oceanographic barrier for many organisms to cross the Atlantic-Mediterranean divide, does not seem to affect the dispersal of this benthopelagic species. The southward movement of the Cape Verde Frontal Zone during the winter, combined with the relatively short duration of the pelagic larval stage of B. boops, may be potential factors for preventing the connectivity between the Atlantic oceanic archipelagos and Mauritania shaping the genetic signature of this species.Marine species exhibiting wide distributional ranges are frequently subdivided into discrete genetic units over limited spatial scales. This is often due to specific life-history traits or oceanographic barriers that prevent gene flow. Fine-scale sampling studies revealed distinct phylogeographic patterns in the northeastern Atlantic and the Mediterranean, ranging from panmixia to noticeable population genetic structure. Here, we used mitochondrial sequence data to analyse connectivity in the bogue  Nonetheless, an increasing number of studies reported significant population diversification over limited spatial scales in species exhibiting large geographic ranges due to various processes2. Among those, life-history traits associated with limited dispersal abilities, such as determined by short pelagic larval durations (PLD)3 or oceanographic barriers that avert gene flow5, may play an essential role in shaping the genetic structure of the species. Studies performing meta-analyses and fine-scale sampling revealed the existence of distinct phylogeographic patterns in the northeastern Atlantic and Mediterranean. These patterns range from fish species displaying noticeable population structure6 to others exhibiting panmixia throughout their geographic ranges7. Historical events and oceanographic patterns generate species-specific responses8.The existence of population genetic structure in the marine realm is often considered paradoxical given the apparent absence of physical barriersBoops boops  (family Sparidae) is a gregarious, benthopelagic fish species inhabiting depths between 0 and 350 m9. This coastal species is broadly distributed in the Mediterranean and eastern Atlantic (from Norway to Angola), including the oceanic archipelagos at these latitudes10. It represents a vital fishery resource, particularly in the eastern Mediterranean11, inhabiting diverse habitats, including rocky substrates, sandy bare seabeds, seaweeds and seagrass meadows12. Boops boops have a PLD of 16 to 18 days and exhibit a vertical pattern of larval assemblages: smaller larvae occur at the surface while larger individuals are more frequently found at the bottom13. During the summer, groups of adult individuals approach shallow coastal waters, while in winter migrate to waters deeper than 100 m14. Until now, studies on the bogue have focused on biological aspects17 or on the species' impact on fish community assemblages and aquaculture19. Defining the spatial structure of intraspecific genetic diversity is paramount for fisheries stock assessment20 and understanding how oceanographic features affect population connectivity.The bogue B. boops throughout most of its geographic range in the eastern Atlantic, from the Bay of Biscay to Mauritania, including the archipelagos of Madeira, Azores, Canary Islands and Cape Verde .In the present study, we used the mitochondrial control region (D-loop) to infer genetic connectivity in rde Fig. . The speB. boops tissue (n = 335) were obtained from several locations across the distributional range of the species in the Atlantic and Mediterranean: Bouharoun, Algeria (ARG); Azores, Portugal (AZO); Bay of Biscay, Spain (BIS); Cádiz, Spain (CAD); Canary Islands, Spain (CAN); Mindelo, Cape Verde (CPV); Brač Island, Croatia (CRO); Galícia, Spain (GAL); Cesenatico, Italy (ITA); Lampedusa, Italy (LAM); Tyre and Tripoli, Lebanon (LEB); Lisbon and Tróia, Portugal (LIS); Madeira Island, Portugal (MAD); Nouadhbou, Mauritania (MAU); Murcia, Spain (MUR); Ria Formosa, Portugal (ALG); Sicily, Italy (SIC); Bizerte and Kélibia, Tunisia (TUN)  Fig. . Specime22. The PCR protocol was performed in a 20 μl total reaction volume with 10 μl of REDExtract-N-ampl PCR mix (Sigma-Aldrich), 0.8 μl of each primer (10 μM), 4.4 μl of Sigma water and 4 μl of template DNA. PCR conditions were the following: initial denaturation at 94 °C for 2 min., followed by 35 cycles  and a final extension at 72 °C for 10 min. The same primers were used for the sequencing reaction, and the PCR products were purified and sequenced in STABVIDA (http://www.stabvida.net/). Chromatograms were edited with Codon Code Aligner , and sequences were aligned with Clustal X 2.123. All sequences were deposited in GenBank (Accession numbers ON605268-ON605602).Total genomic DNA was extracted with the REDExtract-N-Amp Kit (Sigma-Aldrich) following the manufacturer's instructions. The mitochondrial control region (D-loop) was amplified in a Bio-Rad Mycycler thermal cycler, using the primers L-pro1 and H-DL124 packages haplotypes25 and pegas26 were used in RStudio27 to estimate standard descriptive measures of genetic diversity in B. boops, including the number of haplotypes and private haplotypes, haplotype .The R32 (https://github.com/jessicawleigh/popart-current.git) was used to build a haplotype network based on the Minimum Spanning algorithm34. To further visualize relationships between haplotypes at population level, we used the software Network v. 10.2.0.034 to build a Median Joining network (https://www.fluxus-engineering.com/sharenet.htm). Pairwise nucleotide distances  within and between B. boops clusters defined by AMOVA were computed in MEGA735. We used the pheatmap function of the R package with the same name36 (https://cran.r-project.org/web/packages/pheatmap/index.html) to obtain a heatmap, which is a graphical representation of the individuals in a matrix to visualize the similarity and dissimilarity among individuals with colors, and simultaneously generate a dendogram to better visualise the clusters.The PopART software37 (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html) to analyse putative cryptic diversity within B. boops. ABGD is based on identifying the barcode gap, i.e., a larger divergence among individuals of different species than among conspecific individuals. We selected the Jukes-Cantor model, the X-value of 1.5 (minimum relative barcoding gap width), and prior intraspecific divergences ranging from Pmin = 0.001 to Pmax = 0.1 to run the barcode gap analysis.We used ABGD (Automatic Barcode Gap Discovery)No experiments on live vertebrates were performed in this study.B. boops individuals collected from 18 locations throughout the Western Atlantic and Mediterranean distribution. Haplotype diversities were high, ranging between 0.924 (in samples from the Canary Islands) and 1.000 in several locations of the Atlantic and Mediterranean . Nucleotide diversities varied between 0.013 (in samples from the Canary Islands) and 0.042 (in samples from Lisbon)   of the several groups tried (results not shown). The Φst-based values  the Mediterranean and eastern Atlantic, including the Azores (hereafter designated as ATL/MED), 2) Canary Islands, Madeira and Cape Verde archipelagos (hereafter designated as ILS) and (3) Mauritania (hereafter designated as MAU) is supported by the highest highly significant Φ Canary IB. boops coinciding with the AMOVA results.Both haplotype network building algorithms, Minimum Spanning Fig.  and MediB. boops, one comprising specimens from the Azores and eastern Atlantic/Mediterranean, another with individuals from the eastern Atlantic archipelagos , and the third with samples from Mauritania , in which the strong swimming abilities of the larvae seem to promote connectivity among populations43. Also, it has been proposed that the Portuguese coast could be considered a sort of "Mediterranean appendix, rather than a biogeographic bridge"44, which is supported by our results.Identifying processes generating population differentiation within a species is crucial to understanding its evolutionary dynamics. The interplay between life-history traits  and oceanographic features often plays an important role in the dispersal range of a species45. If the dispersal of B. boops in this area depended on the adults' movement, we would expect a closer relationship between Cape Verde and Mauritanian populations. Instead, the genetic analyses revealed that specimens from Cape Verde, Madeira and Canary Islands grouped into a cluster different from the one including the Mauritania samples  than between Atlantic/Mediterranean vs. Mauritania (0.075 ± 0.012). These results further support the low levels of connectivity between the eastern Atlantic islands and the Mauritanian population.From June to October, the Cape Verde Frontal Zone migrates eastward from Guinea-Bissau and Cape Verde waters to the Cape Blanc headland (Mauritania), while during the rest of the year it moves southwardes Figs.  and 3. B8. For instance, the sand-smelt (Atherina presbyter) populations from the Azores show a closer relationship with the eastern Atlantic archipelagos (Canary Islands and Madeira) and are markedly distinct from specimens of the Mediterranean and southern European Atlantic shores47. This pattern may have resulted from the colonisation of the Azores by the Canary Islands and Madeira acting as stepping stones and glacial refugia for fish coming from West Africa47. Boops boops haplotypes from southern Portugal and the Mediterranean are closely related to samples from the Azores. Yet, it is impossible to quantitatively infer the direction of the dispersal because there are few shared haplotypes and none with the Azores. Nonetheless, a West–East direction is expected due to a branch of the northern subtropical gyre (Azores Current) that drifts towards the Gulf of Cádiz. This current promotes the displacement of larvae and adults from the Azores to southern Portugal and into the Mediterranean, which is consistent with our pattern  combined with the relatively short PLD of this species averts the genetic admixture between populations from Mauritania and the neighbour eastern Atlantic archipelagos. The gregarious nature of the adults that remain in shallow waters during the summer to feed in the highly productive marine system of the Mauritanian upwelling, further prevents connectivity with nearby oceanic islands' populations. The observed genetic discontinuities do not conform to an isolation-by-distance model but instead reflect the role of specific life-history traits and local oceanographic features.Identifying the processes that generate population differentiation in a species with a broad distribution is pivotal to understand its evolutionary dynamics. Here we show the existence of three clades within the bogue Supplementary Information.


Since the basic biochemical mechanisms of photosynthesis are remarkably conserved among plant species, genetic modification approaches have so far been the main route to improve the photosynthetic performance of crops. Yet, phenotypic variation observed in wild species and between varieties of crop species implies there is standing natural genetic variation for photosynthesis, offering a largely unexplored resource to use for breeding crops with improved photosynthesis and higher yields. The reason this has not yet been explored is that the variation probably involves thousands of genes, each contributing only a little to photosynthesis, making them hard to identify without proper phenotyping and genetic tools. This is changing, though, and increasingly studies report on quantitative trait loci for photosynthetic phenotypes. So far, hardly any of these quantitative trait loci have been used in marker assisted breeding or genomic selection approaches to improve crop photosynthesis and yield, and hardly ever have the underlying causal genes been identified. We propose to take the genetics of photosynthesis to a higher level, and identify the genes and alleles nature has used for millions of years to tune photosynthesis to be in line with local environmental conditions. We will need to determine the physiological function of the genes and alleles, and design novel strategies to use this knowledge to improve crop photosynthesis through conventional plant breeding, based on readily available crop plant germplasm. In this work, we present and discuss the genetic methods needed to reveal natural genetic variation, and elaborate on how to apply this to improve crop photosynthesis. Natural genetic variation is an undervalued resource in the study of photosynthetic functioning. Quantitative and molecular genetics can be used to exploit natural genetic variation for improving photosynthetic performance of crops. Approximately 1.5 billion years ago a cyanobacterium was engulfed by a phagotrophic eukaryote . This un3, the different forms of C4, and CAM plants  underlying phenotypic differences in photosynthetic processes ; Fig. 1.To reveal genetic variation for a given phenotype, it is essential to correlate phenotypic variation to causal genotypic variation. For decades this has been done by genetic linkage mapping approaches . A linka2 populations, or it could be ‘immortal’ populations of recombinant inbred lines (RILs), near-isogenic line (NILs), back-cross inbred lines (BILs), or doubled haploid (DH) lines  doubling of the haploid genome , but in an agricultural context it will be important that the improvement can be seen to act at the level of the canopy or field. With the global human population increasing and the climate changing rapidly, the need for agriculture to constantly push the limits of crop production is likely to increase.While there is plenty of natural and induced genetic variation for photosynthetic functioning, the question arises whether photosynthetic performance can actually be improved by breeding, using either conventional or novel plant breeding techniques. The term ‘improving’ is very broad, and depending on the perspective it may have different meanings . In this3 species, the current efficiency of intercepted radiation is such that 1.5% of the full spectrum of solar energy per unit area is converted into biomass  light energy interception by the canopy, (ii) the conversion of that absorbed light energy into the chemical energy of biomass, and (iii) the harvest index  dispenses with the costs of the above ground architecture necessary to support and position its leaves so they can function well as light absorbers for photosynthesis and at the same time be supplied with water and be able to export assimilates. This also implies that understanding the optimization of photosynthesis is not a matter that can be understood by analysing just leaf-level photosynthesis, but needs to be understood at the whole plant or, as is the case in agriculture, the canopy level.If energy conversion in photosynthesis is so important, then why has this not been maximized by natural selection and evolution? The answer to this is complex, but it is likely that photosynthesis has been optimized rather than maximized. Photosynthesis is clearly an important process for plants that brings with it the benefits of fixed carbon and energy, but it also brings with it costs; reconciling these costs with the benefits of the process in different environments results in different optimal combinations of photosynthetic properties. In a rather crude way this can be seen in the morphology of parasitic plants in which photosynthesis is no longer used. The parasitic orchid 3 photosynthesis a maximum theoretical CO2 assimilation rate of 55 μmol m−2 s−1 at an irradiance 2000 μmol m−2 s−1. This compares favourably with the highest measured rate of C3 photosynthesis   view of photosynthesis and focusing on leaves, there are numerous parameters that can be used to define photosynthesis. One of the most important of these is the maximum rate of photosynthesis as this defines a boundary to the effectiveness of the process as a source of carbon and a means for storing energy. The maximum rate can be that of a leaf, a genotype, a species, a type of plant, etc. Surprisingly, we know of no thorough theoretical analysis of the maximum leaf-level photosynthetic rate since that of esis see  of abou m−2 s−1 , and the m−2 s−1 . In addi m−2 s−1 , light-lis) e.g. , and resis) e.g. . Photosyutrition . So the Chylismia claviformis, occurring in the dry semi-deserts of the southwestern part of North America. This species has one of the highest CO2 assimilation rates reported for C3 plants, exceeding 60 μmol m−2 s−1  short. Such conditions occur, but are rare. Think about semi-deserts, with generally sparse vegetation, but occasionally sufficient rainfall to allow short-lived, abundant plant growth. Some of the species with the highest photosynthetic rates are found at such sites, such as the winter annual of 42 °C . These eTo place this into perspective, in natural environments the fittest genotypes are selected, where fittest is defined as the ability to reproduce best . Even thWhile natural systems may rarely select for plants with maximum photosynthesis due to various environmental and genetic constraints, these constraints may be removed for crop species, as agricultural systems are fundamentally different from most natural environments. Environmental conditions impairing production are actively minimized by watering, fertilizing, weeding, and pest management of crops. Trade-offs that may exist in a natural condition, favouring optimal over maximal photosynthesis, may be irrelevant in agriculture. Since natural selection for optimal photosynthesis has resulted in genetic variation for photosynthetic traits in many species, such could be exploited to improve photosynthetic performance of crops in agriculture. Plant breeding can facilitate gene flow to interconnect the optima of fitness landscapes, and allow the best photosynthetic performance to be selected in a way that would be difficult to achieve in nature. Studies into historically released cultivars show that in some crops increases in photosynthetic rate have been made, but in other crops there is no sign of such increase . So, pla3 to C4 photosynthesis  values. Examples of quantitative traits are flowering time, plant biomass, and the efficiency of photosynthesis. The identification of genes underlying quantitative traits can be resolved by genetic mapping . In ordeBi- and multiparental populations  are veryGWAS make use of diversity panels, rather than bi- or multiparent populations, which means a GWAS population, or diversity panel, typically represents much more genetic diversity and a higher density of recombination events between genomic regions than present in bi- or multiparental populations. The genomic region in which no recombination is found, when comparing all accessions in the diversity panel, is often referred to as being in linkage disequilibrium (LD)  above these thresholds are most likely to be valid QTLs that warrant further follow-up study. However, one should realize that QTLs that do not reach the significance threshold may still be valid, and QTLs that exceed the threshold may be false , because of which one may focus on a likely candidate and miss the actual causal gene, and thus the opportunity to shed further light on the biology underlying the target phenotype  underlying a QTL, a common mistake is to assume that the causal gene is the one closest to the marker with the highest genotype–phenotype association. Such is generally not the case, and proper gene validation is needed using a limited set of candidate genes. In the case of QTL mapping in bi- or multiparental mapping approaches, QTLs are often mapped to a large genomic region containing hundreds of genes. Resolving these will need one or more rounds of fine-mapping, using the generation of additional segregating populations, to reduce the QTL region by means of additional recombinations e.g. . Especiahenotype . As furtwww.arabidopsis.info or abrc.osu.edu), or by CRISPR–Cas-mediated gene editing. As genotypes within a species can vary substantially for photosynthetic traits (FWA gene, controlling flowering time in Arabidopsis (When the number of candidate genes has been narrowed down to about 10, either upon fine-mapping in the biparental population, or upon LD analysis in GWAS, the gene validation can start. As a first step, and as a first step only, a mutant analysis of the remaining candidate genes is often the preferred next step. If a mutant with a loss-of-function allele produces a phenotype consistent with the function of the hypothetical wild-type gene giving rise to the phenotype used in the QTL analysis, then that mutant may point to the causal gene. Knock-out lines may be obtained via available stock centres, such as the T-DNA insertion lines for Arabidopsis (c traits , it is i1s with wild-type or mutant plants will be similar for both allelic variants, while it will be different if the target gene is indeed the one for which allelic variation underlies the QTL. A case in which this has been successfully used was presented by YS1 in photosynthetic response to an increase in irradiance.Once there are only a few candidate genes remaining, meaning genes for which there is allelic variation between genotypes that may explain the phenotypic differences between the genotypes, and which have a function in line with the studied trait, the final step of the gene validation can start. In general two approaches are used to confirm the identity of the causal gene underlying a QTL. Of those two approaches transgenic complementation is most often used. This involves transforming the allelic variants into a loss-of-function mutant background, to recreate the phenotypic difference or variation initially used to identify the QTL e.g. . This reOnce the causal gene is convincingly identified, the different alleles can be used to study the physiological role of the gene  and learAll of the methods described above focus on the exploitation of genetic variation in nuclear genomes, thus ignoring the genetic contribution from organellar genomes. Photosynthesis, however, is predominantly associated with processes occurring in the chloroplasts. The chloroplast holds roughly 70 protein-coding genes, most of which are essential for photosynthetic performance . Also, mConsequently, when studying genetic variation for photosynthetic traits, not only the nuclear genome, but also the organellar genomes and the nuclear–organellar interaction should be considered. This variation can be exploited for breeding purposes using recurrent backcrossing or the construction of cybrids, which are genotypes with novel combinations of nuclear and organellar genomes (In this review we have described how to identify natural genetic variation for photosynthetic traits, and perform validation of the genes and alleles involved. Photosynthesis is not an easy trait to study: it is highly polygenic and phenotypically highly responsive to environmental conditions (Breeding for elite cultivars has largely ignored improvements in photosynthesis. While promising advances in improved photosynthesis may be achieved by genetic modification (


Benzothiadiazole (BTH) is a commercial chemical elicitor that can induce an innate immune response in grapevines and improve the phenolic components and color quality of grapes and corresponding products. The literature on the influence of BTH on the accumulation and metabolism of phenols from grapes is extensive. However, many unknown bio-mechanisms involved have been poorly investigated, which opens a gateway for pioneering research that needs to be done in this field. To this purpose, this review aims to analyze and explore the gaps in current research so that subsequent studies may be geared towards them. Grape is a valuable economic crop from which various products such as raisins, table grapes, grape juice, and wine  can be obtained. Grape quality is greatly influenced by secondary metabolites such as phenolic compounds ,2,3,4. ABotrytis cinerea), powdery mildew (Erysiphe necator), and downy mildew (Plasmopara viticola) impair the healthy development of grapevines and the quality of their berries . Th. Th84]. p-coumaroyl and acetylated derivatives.In Merlot variety, studies showed that anthocyanin concentrations increased in BTH-treated grapes ,52,59,61Regarding the Syrah variety, BTH treatments increased the anthocyanin concentration of grapes and corresponding wines, with the total of 3-monoglucosides increasing up to 27% and 10%, except for cyanidin-3-glucoside, in the treated grapes. For the acetylated compounds, BTH increased the contents of petunidin-3-acetyl-glucoside, cyanidin-3-acetyl- glucoside, and peonidin-3-acetyl-glucoside in treated grapes and increased the concentration of cyanidin-3-acetylglucoside and peonidin-3-acetyl-glucoside in the resultant wines. In addition, the concentration of coumarates (peonidin-3-coumaryl-glucoside and cyanidin-3-coumaryl-glucoside) increased in wines produced from the grapes treated with BTH .For the Monastrell variety, some authors found that the anthocyanins content of BTH-treated grapes at harvest was high ,33,44, bFlavonols are a group of the most studied phenolic compounds for their antioxidant capacity and biological activities , as wellThe total content and pattern of flavonols are affected to some extent by BTH . When exGómez-Plaza et al.  detectedIn BTH-treated Monastrell grapes, the flavonol content was higher than the control throughout the ripening phase. The greatest differences were observed in control grapes a week before harvest . ContrarNonetheless, in two years of investigation, the results were not entirely consistent. Similarly, Ruiz-García et al.  noticed There are two kinds of tannins in wine: condensed tannins from grapes and hydrolyzable tannins from oak barrels. In this review, more focus is placed on condensed tannins from grapes. In general, condensed tannins are also referred to as proanthocyanidins. The application of BTH can increase the proanthocyanidin content of Merlot grapes by up to 36% and reduce the incidence and consequences of gray mold . Ruiz-GaIn addition, the previous study found that BTH treatment showed partial differences in promoting proanthocyanidin synthesis in grapes. Gil-Muñoz et al.  reportedFurthermore, significant differences were observed in the terminal and extension units of skin tannins in the control and BTH treatments. In particular, only (+)-catechin  showed significant variations, while the content of (+)-catechin, (−)-epicatechin, and epicatechin-3-o-gallate  increased in the control samples compared to the BTH treatments. In seeds, the content of (+)-catechin and (−)-epicatechin increased in BTH treatments . In the cis- and trans-resveratrol in the skin of Merlot grapes with the increase in trans-resveratrol being significantly higher than that of cis-resveratrol [trans-resveratrol concentration in Syrah grapes [−1 BTH, a significantly higher amount of stilbenes was found compared to control grapes during incubation [In recent years, multiple pre-harvest BTH treatments have been shown to promote the buildup of veratrol . Similarh grapes  and stilh grapes . This mah grapes . The devcubation . Based on the report by Beckers and Spoel , the incAltogether, BTH can affect the buildup of phenolic compounds in several ways: directly  and indirectly . It has been shown that the effects of BTH treatments on grape phenolic content strongly depend on the variety ,88, climRegarding BTH treatment time, Boss et al.  reportedIn wines made from BTH-treated grapes, the increase in anthocyanin content is not usually observed . Due to The soluble form of phenolics is mainly located in the vacuoles of plant cells, which may be in free or conjugated form, while the insoluble phenolics are mainly found in the cell wall matrix ,93. The p-coumaroyl-CoA, which are biogenetically derived from phenylpropanoids and acetate pathways [p-coumaryl-CoA are transformed into phenols by stilbene synthase (STS) and chalcone synthase (CHS) through the formation of an aromatic ring (by adding three more carbon groups consisting of two C atoms) [The synthesis of phenolic compounds originates from the branching of phenylpropanoids  44,62,9,962,95. C atoms) . ThroughC atoms) . 2O2, and other reactive oxygen species (ROS) in the cell wall. ROS can cause rapid cross-linking or interlacing of cell wall phenolic compounds. Wang et al. [2O2. Cellular hypersensitive defense responses, including activation of defense genes and induction of defense compounds  in plants during oxidative burst could be attributed to the accumulation of H2O2 in plant cells [2O2 production in cultured V. vinifera cells, triggering the expression of defense-related genes or phytoalexins  synthesis [Presently, there are more studies on pre-harvest BTH treatment to enhance phenolic accumulation in grapes but fewer studies on the effects of BTH treatment on the phenolic metabolism of grapes. Based on the mechanism of SAR pathway, BTH  can stimulate the expression of protein defense genes  associated with pathogenesis via the protein non-expressor gene 1 (NPR1), which is also associated with pathogenesis ,98,99. Eg et al.  also fount cells . Using eynthesis ,106,107.However, BTH can reduce the levels of primary metabolites such as amino acids and soluble sugars in treated grapes ,22. In pThe increase of anthocyanins and stilbenes content in BTH-treated grapes could also be due to BTH induction of PAL . Iriti eAnthocyanins can be affected by BTH treatments ,111. GómStudies on pre- or post-harvest BTH treatment have been conducted not so much in terms of secondary metabolic mechanisms in grapes as in various plants. In one study, post-harvest BTH treatment resulted in a higher anthocyanin content in strawberries and increased the activities of metabolic enzymes such as PAL, 4-coumarate/coenzyme A ligase (4-CL), cinnamate-4-hydroxylase (C4H), dihydroflavonol 4-reductase (DFR), glucose-6-phosphate dehydrogenase (G6PDH), tyrosine ammonia-lyase (TAL), and shikimate dehydrogenase (SKDH) . This suOverall, the influence of BTH on grapes has been reported in the literature, particularly on grape color, phenolics, variety, and metabolism . Based oBTH treatment is a good strategy to improve the synthesis of grape phenolic compounds. However, BTH treatments do not always produce consistent results. The effects of BTH treatment rely on several factors, including variety, climate, environmental conditions, the timing of BTH treatments, and maturity. This review found that BTH can improve phenolic compounds and color quality by directly affecting secondary metabolism or altering primary metabolism. Although some encouraging progress has been made, the complete process is not yet clearly understood. The influence of BTH on phenolic compounds and their synthesis is still unclear. In particular, the metabolic and physiological changes resulting from the inducer are still unknown and require further studies. In the future, a clear understanding of the mechanisms of BTH at the cellular, molecular, and genetic levels would help overcome these problems. In addition, focusing on the influence of climate change, variety, and environmental conditions on BTH-induced phenols accumulation in grapes can help producers use BTH scientifically, effectively, and safely. Therefore, the comprehensive and in-depth analysis of the regulatory mechanism of phenols enhancement by BTH is of great importance for improving our knowledge of the regulatory control of phenol synthesis in grapes.