Addressing the challenges of climate change and durum wheat production is becoming an important driver for food and nutrition security in the Mediterranean area, where are located the major producing countries . One of the emergent strategies, to cope with durum wheat adaptation, is the exploration and exploitation of the existing genetic variability in landrace populations. In this context, this review aims to highlight the important role of durum wheat landraces as a useful genetic resource to improve the sustainability of Mediterranean agroecosystems, with a focus on adaptation to environmental stresses. We described the most recent molecular techniques and statistical approaches suitable for the identification of beneficial genes/alleles related to the most important traits in landraces and the development of molecular markers for marker-assisted selection. Finally, we outline the state of the art about landraces genetic diversity and signature of selection, already identified from these accessions, for adaptability to the environment. Triticum turgidum L. ssp. durum) (Desf.) is the 10th most cultivated cereal worldwide, with a total production of about 38 million tons (Triticum aestivum), its importance for the countries of the Mediterranean basin is pivotal. DW is considered a staple food as it constitutes the dominant part of the diet for many populations in this area. The main products derived from DW include pasta, cous cous, burghul, and bakery products. Durum wheat-based products have a low glycemic index which makes them healthy products that can be recommended for low-carb diets (Triticum turgidum ssp. dicoccoides) have been at the foundations of food, from the Neolithic period to the Greeks and the Roman Empire, up to the present day (Durum wheat (DW)  Thell (Triticum turgidum L. ssp. dicoccoides) occurred in the Fertile Crescent  about 8000 years BCE (Before Common Era) from limited founder lineages . Until 1950-1955 most of the DW Mediterranean areas were cultivated with DW landraces, local accessions adapted to their place of origin  is a cereal grain evolved from the tetraploid domesticated emmer wheat .) Thell . Domestilineages . Emmer wlineages . Finallyf origin . Howeverf origin . Since tf origin . Afterwaf origin  and effoheat shock, while moderately high maximum temperature (20 to 30°C) for a longer duration is known as chronic heat stress (The climate characteristics of the Mediterranean region have played a significant role in shaping the phenotypic (and the genotypic) configuration of both DW wild relatives and cultivated varieties. This basin is characterized by hot and dry summers, followed by cold and wet winters. Climate change, particularly important in the last decades, points to an increased variability, in which drought events, often coupled with heat waves, can hamper growth and development, eventually affecting crop yield. For example, yield is reduced of about 5% per Celsius degree of increase in temperature, with a gross loss reaching 24% under a growth temperature of 31°C during flowering . Clearlyt stress . In DW, t stress . Heat stt stress . Droughtt stress . Also, tt stress .+/Cl¯ in the soil upper layer, but a variety of ions, mainly K+, Ca2+, Mg2+, and i) cell toxicity of the accumulated ions, which often results in nutrient imbalance and enhanced oxidative stress, and (ii) osmotic stress, due to the extremely low water potential of soil along with a reduction in water assimilation. Therefore, cellular, and metabolic processes involved to counteract salt stress are comparable to those induced by drought , mapped to the long arm of chromosome 2A, responsible for almost 38% of phenotypic variation in low Na accumulation in the mapping population, and this locus, together with closely linked markers, are commonly adopted to select salt tolerant durum genotypes , and events of rising groundwater table can increase the amount of salts in soils. Moreover, anthropogenic activities, such as inappropriate irrigation and drainage practices or irrigation with brackish water, determine salt accumulation in the soil surface or within the solum, causing salinity stress in plants . Soil sa erosion . On the  drought . Interes drought . In fiel drought . Lower lenotypes , which cenotypes . Toleranenotypes . In wheaenotypes . Other cenotypes .Drought, heat, and salt stress, being linked to each other, induce the generation of reactive oxygen species (ROS), including hydrogen peroxide, superoxide, or hydroxyl radicals, which are continuously formed mainly in the cytosol, chloroplasts, and mitochondria . ROS havMany efforts are made by researchers and breeders to constantly look for new sources of genetic variability to improve the elite varieties for adaptation traits, to face climate change. The exploitation of the existing genetic variability, still available in landraces, is one of the best approaches to adopt. According to They are considered a reservoir of useful alleles that can be exploited to broaden the genetic basis of important adaptation traits. Landraces are rich in micronutrients and have high concentrations in total phenol and antioxidant content, as well as in tocols, carotenoids, and lutein . Since tin-situ or ex-situ strategies. The in-situ strategy relies on individual farmers who traditionally cultivate landraces for their production or are sponsored by the government or private companies. The ex-situ conservation is managed by international institutions such as CIMMYT, ICARDA, and USDA or by national projects led by local universities . Graph pangenomes based on 16 public assemblies (Wheat Panache) was developed with the aim to discover genome variation between cultivars and to mine the diversity present in the large and complex wheat genome . HoweverNo pangenome has yet been assembled for durum wheat, although several projects are underway. Indeed, the use of Svevo genome as suitable reference may accelerate the sequencing of new durum cultivars enabling the pangenome construction. As far as is known, at the moment the only reference genome for durum wheat remains Svevo. Given the growing interest in some European landraces Table 2,de novo SNP discovery and genotyping in large genome-size species is the exome sequencing. The workflow involves the fragmentation of high-quality genomic DNA, repair ends, adenylation, adapter ligation, and the selective hybridization of probes for target enrichment. Then two consecutive captures of the hybridization probes ensure high specificity of target region before the sequencing step  is the target RNA sequencing. This technique allows very high precision in the discovery and quantification of genes because it sequences only those of interest. The most important step of target RNA-seq is the design of the specific probes that can be customized to meet the specific needs of each experiment. Since only the genes of interest are sequenced, the coverage can also be very high, allowing to increase the power to assemble low expression transcripts .With the decrease in the NGS cost, sequencing techniques are increasingly used as genotyping tools. However, to afford sequencing in large genome size species, reduced representation sequencing (RRS) approaches can be considered . GenotypNGS technology has also created the basis for the establishment of high-density SNP arrays and the related high-throughput platforms capable of genotyping large numbers of samples .® array of Affymetrix uses photolithographic oligos on an array and the target SNP amplification involves assays with 30-mer probes  and the Axiom technology from Thermo Fisher Scientific  . Technicr probes .In wheat, the first SNP array developed was the 9K Infinium SNP Array by SNP arrays have the advantage of facilitating high-density SNP scanning, have a high call rate, and are also cost effective when there is a need to genotype a high number of markers on many samples. However, a disadvantage, is that the set of SNPs is fixed and cannot be changed; they are also developed in hexaploid wheat, thus the SNPs present in the D genome will be unknown if it is applied in DW.Among the most competitive technologies used for marker assisted breeding, with a medium/low throughput, there are TaqMan , KASP , and rhAmp , widely used in many plant species such as wheat and sugar beet . TaqMan These chemistries have the advantage of being highly reproducible, sensitive, and cost effective; moreover, they can be freely customized both for the number of samples and SNPs that can be analyzed, adapting perfectly to marker assisted selection for crop improvement .Genome wide association study is a powerful tool to study the genetic base of complex traits and detect relationships between phenotypic variations and the associated genetic polymorphisms . The staIn a context of climate change, a technique developed to accelerate breeding procedures and speed up the selection of superior genotypes is genomic selection (GS) . This stIn general, the statistical models developed for GP are based on single-environment assessments. However, in plant breeding the presence of a Genotype x Environment (G x E) interaction may complicate the selection of stable lines. Hence, some genomic prediction models, considering the G x E interaction, help breeder select lines with optimal overall performance across different environments and in a specific target environment. Specifically, a reaction norm model, which is an extension of the random effect Genomic Best Linear Unbiased Predictor (GBLUP) model, was developed by Populus tricocarpa, Beta vulgaris spp. maritima, and Arabidopsis tahaliana.The selective pressure of abiotic stresses often varies in space, causing the evolution of advantageous mutations under their local environment, leading that genotype to have better fitness than the same genotype originating elsewhere. This differentiation process is called local adaptation and is driven by spatially divergent natural selection . To trigMedicago truncatula . Landraces from Ethiopia appeared instead as the more isolated and distant to modern cultivars, while a high admixture level within landrace populations was confirmed .Ppd), the vernalization (Vrn), and the dwarfing (Rht) genes, as well as loci associated with nitrogen use efficiency, plant architecture and grain yield . In addition, genes related to quality such as pasta-making quality and color of semolina and other durum wheat-end products were also divergent between landraces and modern cultivars. In detail, loci for gluten composition , as well as loci involved in the carotenoid pathway (Psy) and polyphenol oxidase reaction (Ppo) were identified in hotspot regions , gene/QTL mapping, and genome-wide association studies (GWAS), supported by the high-throughput genotyping tools and strategies previously described, importantly increased the rate discovery of genes/QTLs regulating biotic, abiotic stress resistance, agronomic and quality traits from landraces. Several studies have been already undertaken so far to identify new genes/traits in landraces useful for breeding . The aptitude of the raw material to be transformed into a high quality end-product mainly depends on grain protein content (GPC) and composition that directly affect wheat’s market price and end-use value  on durum wheat landrace resources paves the way to accelerate the next-generation breeding programs to overcome the gap of knowledge of these underexplored resources and identify advantageous alleles that have been lost in modern varieties.CB: writing original draft. DB and FT: conceptualization and writing. FT, GD, AF: writing, review, and editing. All authors contributed to the article and approved the submitted version.


Carcass backfat thickness (BFT), carcass lean percentage (CLP) and carcass fat percentage (CFP) are important to the commercial pig industry. Nevertheless, the genetic architecture of BFT, CLP and CFP is still elusive. Here, we performed a genome-wide association study (GWAS) based on specific-locus amplified fragment sequencing (SLAF-seq) to analyze seven fatness-related traits, including five BFTs, CLP, and CFP on 223 four-way crossbred pigs.Sus scrofa chromosomes (SSC), of which 10 SNPs were located in previously reported quantitative trait loci (QTL) regions. On SSC7, two SNPs  for average backfat thickness (ABFT) exceeded 1% and 10% Bonferroni genome-wide significance levels, respectively. These two SNP loci were located within an intron region of the COL21A1 gene, which was a protein-coding gene that played an important role in the porcine backfat deposition by affecting extracellular matrix (ECM) remodeling. In addition, based on the other three significant SNPs on SSC7, five candidate genes, ZNF184, ZNF391, HMGA1, GRM4 and NUDT3 were proposed to influence BFT. On SSC9, two SNPs for backfat thickness at 6–7 ribs (67RBFT) and one SNP for CLP were in the same locus region . These three SNPs were located in the PGM2L1 gene, which encoded a protein that played an indispensable role in glycogen metabolism, glycolysis and gluconeogenesis as a key enzyme. Finally, one significant SNP on SSC14 for CLP was located within the PLBD2 gene, which participated in the lipid catabolic process.A total of 227, 921 highly consistent single nucleotide polymorphisms (SNPs) evenly distributed throughout the genome were used to perform GWAS. Using the mixed linear model (MLM), a total of 20 SNP loci significantly related to these traits were identified on ten A total of two regions on SSC7 and SSC9 and eight potential candidate genes were found for fatness-related traits in pigs. The results of this GWAS based on SLAF-seq will greatly advance our understanding of the genetic architecture of BFT, CLP, and CFP traits. These identified SNP loci and candidate genes might serve as a biological basis for improving the important fatness-related traits of pigs.The online version contains supplementary material available at 10.1186/s12864-022-08827-8. The excessive deposition of fat in pigs leads to low feed efficiency and is not favored by consumers. It is well known that CLP is strongly negatively correlated with BFT , 2. In tDomestic pigs show large phenotypic variation, which is ascribed to approximately 10,000 years of natural and artificial selection . At presCurrently, remarkable advances in fatness-related traits have been made, and many related QTLs and genes have been reported –12. HoweHere, we examined 223 four-way crossbreds with Landrace, Yorkshire, Duroc and Saba pigs as the hybrid parents  raised under the same environmental conditions for BFT, CLP and CFP traits. Subsequently, SLAF-seq was employed to perform GWAS and recover potential alleles controlling these traits. To our knowledge, this was the first SLAF-seq-based GWAS to identify SNP loci and candidate genes linked to porcine economic traits. The results provided a basis for the molecular marker-assisted breeding and improvement of the fatness-related traits in pigs.r > 0, p < 0.001), between five BFT traits and CFP . Five BFT traits and CFP were significantly negatively correlated with CLP . Furthermore, CLP showed the strongest negatively correlated with CFR , while ABFT showed the strongest positive correlations with SBFT, LRBFT and LBFT .Table Oryza sativa indica was used as a control during sequencing. The results showed that the percentage of digestion normally and paired-end mapped reads of control were 90.77% and 95.4%, respectively, indicating that the SLAF-seq process was normal. The density distribution of SLAFs was calculated throughout the pig genome and was shown in Fig. According to the selection principle of the enzyme digestion scheme, two restriction enzymes, RsaI and HaeIII, were selected as enzyme combinations for developing SLAF tags, and the sequence with the length of 314–344 bp was defined as SLAF tags. A total of 1,109.92 million reads were obtained from all individuals. Average Q30 and GC contents were 90.74% and 44.83%, respectively. Similar to the number of expected SLAFs, a total of 1,552,377 SLAF tags  were identified from all individuals with sequencing to an 11.94 average depth. Furthermore, 245,734 SLAFs were identified across the whole genome through genomic mapping, of which 230,239 polymorphic SLAF tags . The density distribution of SNPs was calculated throughout the pig genome and was shown in Fig. P-value calculated by the association study fit the expected ones, which suggested that the population stratification was well-corrected and the association analysis using MLM was reliable. The Q-Q plot of each trait was shown following the Manhattan plot of the corresponding trait  for the traits investigated (Table P = 3.26 × 10–8). Another SNP in proximity to this SNP (rs320451735 for ABFT) exceeded the 10% genome-wide significance level (P = 2.21 × 10–7). Among the detected SNPs, four, three, two, seven, five, six and four SNPs, were significantly associated with SBFT, LRBFT, LBFT, ABFT, 67RBFT, CLP and CFP traits, respectively. These SNPs detected were distributed in ten Sus scrofa chromosomes (SSC), including SSC3, SSC7, SSC8, SSC9, SSC10, SSC11, SSC12, SSC13, SSC14 and SSC15. In this study, 33 genes located within 100 kb upstream and downstream of these significant SNPs were considered potential candidate genes  plots of all traits were drawn. The observed ait Fig. . A totaled Table . The pheZNF184 and ZNF391, while rs1112937671 and SSC7:29,503,670 were located within an intron region of the COL21A1 gene  associated with 67RBFT and another adjacent SNP  related to CLP were located in a region between 8.744 Mb and 8.763 Mb . These three SNPs were located in the PGM2L1 gene.  was searched based on SNP and QTL locations. A total of 20 SNPs were identified in the study, of which 10 SNPs were located in previously reported QTL regions in pigs. The remaining 10 SNPs had not been included in any previously reported QTLs that were associated with BFT, CLP and CFP of pigs. On SSC7, a total of five SNPs significantly associated with BFT were found, which were located in a region from 21.39 to 30.29 Mb . This region was located within 20 previously reported QTLs associated with BFT. Among five significant SNPs, three adjacent SNPs  were located within the 26 BFT-related QTLs that had been previously reported. On SSC9, a total of three adjacent SNPs, including two SNPs associated with 67RBFT and one SNP associated with CLP were located in a region between 8,745 and 8,764 kb , which was located in one formerly acknowledged QTL (from 0.1 to 11.1 Mb) associated with LRBFT and CLP. Besides, one SNP locus (rs344553616) on SSC12 associated with 67RBFT was located within a known QTL region (47.9–59.4 Mb) related to backfat thickness at 10 ribs (10RBFT), another locus (rs319334375) on SSC15 associated with SBFT was located within a formerly reported QTL (57.5–120.1 Mb) related to SBFT. These results were shown in Table STo evaluate whether QTLs associated with seven fatness-related traits in this study replicate any previously known QTLs, the Pig Quantitative Trait Locus (QTL) Database , and 227, 921 highly consistent SNPs were evenly distributed over the entire genome , which was located within 20 previously reported QTLs associated with BFT (Table SZNF184) and zinc finger protein 391 (ZNF391). The result of GO annotation showed that ZNF184 and ZNF391 participated in regulation of transcription, DNA-template and nucleic acid binding. As is known, zinc-finger proteins (ZFPs) represent the largest transcription factor family in mammals [ZNF184 and ZNF391 also are the C2H2-type zinc finger transcriptional factors. According to their structure and function, it was inferred that ZNF184 and ZNF391 might be involved in regulating adipogenesis as C2H2-type transcriptional factors. In addition, another two SNPs  on SSC7 were located within an intron region of collagen type XXI alpha 1 chain (COL21A1). The result of GO annotation showed that the cellular component of COL21A1 was collagen trimer and extracellular matrix (ECM). It has been reported that COL21A1 is VAdomain-containing collagen with a domain structure and which is a part and the smallest of the FACIT family of collagen expressed in various tissues [COL1A1 and COL1A2) influenced porcine fat deposition by affecting ECM remodeling [COL21A1 might play an important role in porcine backfat deposition by affecting ECM remodeling and should be considered a strong candidate gene for porcine BFT traits.The GWAS result showed that four significant SNPs on SSC7 were associated with at least two BFTs. The nearest genes of two SNPs  were zinc finger protein 184 , nudix hydrolase 3 (NUDT3) and high mobility group protein HMG-I (HMGA1), respectively. GO annotation result showed that GRM4 was involved in activation of MAPK activity  gene. GO annotation result showed that PGM2L1 was mainly involved in carbohydrate metabolic process, phosphorylation and glucose-1,6-bisphosphate synthase activity. It is known that PGM2L1, a member of a distinct family of α-phosphohexomutases widely distributed in prokaryotes, is able to produce glucose-1,6-bisphosphate, a crucial metabolic regulator. According to a study, PGM2L1 encodes a crucial enzyme that is essential for glycogen metabolism, glycolysis, and gluconeogenesis [PGM2L1 should be considered a potential candidate gene for BFT and CLP traits.Furthermore, a significant SNP rs341689410) on SSC7 had located upstream 13.5 kb, downstream 27.6 kb and 27.8 kb of glutamate metabotropic receptor 4 . The result of GO annotation showed that the FAM171A1 gene participated in regulation of cell shape . GO annotation result indicated that the PLBD2 gene participated in lipid catabolic process. Wang et al. found that PLBD2 was predominantly involved in the lipid catabolic process, which was consistent with the favoured selection for fatness in Chinese pigs [PLBD2 gene should be considered a strong candidate gene for porcine CLP trait. Finally, one SNP  related to CFP trait was located in an intron region of KIAA1217. A study found that the KIAA1217 gene showed differences in promoter methylation and mRNA expression in the omental visceral adipose tissue between non-obese and obese individuals [KIAA1217 gene might also be used as a potential candidate gene for the porcine CFP trait.For the traits of CLP and CFP, one significant SNP locus (rs320036825) onSSC10 was located in an intron region of family with sequence similarity 171 member A1  and SSC9  were found to be associated with fatness-related traits. Furthermore, a total of eight candidate genes, including A total of 223 four-way crossbred pigs  used in this study were produced with 7 hybrid boars  and 37 hybrid sows (Yorkshire × (Landrace × Saba), YLS) as the parents. All the animals were reared under the same nutritional and environmental conditions until slaughtered (105.25 ± 15.75 kg) at the pigs and broilers breeding farm in Chuxiong City, Yunnan Province, China. Ear tissue samples were collected from 223 crossbreds.In the study, the studied phenotypes were BFT, CLP and CFP traits. BFT traits included backfat thickness at the shoulder (SBFT), backfat thickness at the last rib (LRBFT), backfat thickness at the last lumbar (LBFT), average backfat thickness (ABFT), and backfat thickness at 6–7 ribs (67RBFT). The SBFT was the backfat thickness at the thickest point over the shoulder. The LRBFT was the backfat thickness at the last rib. The LBFT was the backfat thickness at the last lumbar. The 67RBFT was the backfat thickness between the 6th and 7th ribs. SBFT, LRBFT, LBFT and 67RBFT were measured using the vernier caliper. The ABFT was calculated as the average of three measurements: SBFT, LRBFT, and LBFT. The left side of each carcass was dissected by separating bone, muscle, fat, and skin. Each component was individually weighed and recorded as bone weight (BW), muscle weight (MW), fat weight (FW) and skin weight (SW). CLP and CFP were calculated as follows:The MEANS procedure of SAS  was used to generate descriptive statistics for studied traits. The sample distribution was visualized as a frequency distribution histogram using the R package “ggpubr”. The phenotypic correlation was visualized as a correlation heatmap by the R function “PerformanceAnalytics”.ftp://ftp.ensembl.org/pub/release-102/) to identify expected SLAF yield, avoid repetitive SLAFs, and obtain the relatively uniform distribution of restriction fragments in the genome. As a result, genomic DNA was digested with RsaI and HaeIII restriction enzyme combinations. Meanwhile, to assess the experimental procedure, Oryza sativa indica (http://rapdb.dna.affrc.go.jp/) was used as a control for evaluating the effectiveness of enzyme digestion and paired-end mapped reads. In brief, SLAF library construction and sequencing for each individual was conducted as described previously [Genomic DNA was extracted from ear tissue samples. Total DNA was extracted by the phenol–chloroform extraction method, with concentration and purity measured using the NanodropTM 2000 spectrophotometer  and electrophoresis. A simulated restriction enzyme digestion was carried out on the current pig genome  using BWA software . Local rTotal filtered SNPs  detected from 223 accessions were used for GWAS. GEMMA software  was usedn = 227,921), the threshold p-value for genome-wide 1% and 10% significance were 4.39 × 10–8  and 4.39 × 10–7 , respectively. Considering the complexity of the traits, markers that passed the threshold score or above the threshold − log10(P) were held to be significantly associated with the target trait.where y was the phenotype, X was the genotype, W (fixed effect) was the matrix of population structure calculated by the ADMIXTURE software , and Z wwww.ensembl.org). GO annotation of candidate genes was then performed using Gene Ontology Consortium (http://geneontology.org).Based on the reference –73, the Additional file 1: Figure S1. Frequency distribution histogram for seven fatness-related traits, including five BFTs, CLP and CFP. A SBFT. B LRBFT. C LBFT. D ABFT. E 67RBFT. F CLP. G CFP.Additional file 2: Figure S2. The phenotypic correlation for seven fatness-related traits, including five BFTs, CLP and CFP. The values in the box represented the phenotypic correlation of the traits. Negative values represented negative correlation, and positive values represented positive correlation. *significant at P<0.05, **significant at P<0.01, ***significant at P<0.001. All of the phenotypic correlation coefficients were significant with P < 0.05.Additional file 3: Table S1. Distribution of SLAF tags and polymorphism SLAF tags on Sus Scrofa chromosomes.Additional file 4: Table S2. The information of shared SNPs for fatness-related traits.Additional file 5: Table S3. Comparison of significant SNPs with previously reported QTLs from the pig QTL database and newly significant SNPs for fatness-related traits.Additional file 6: Table S4. The description and GO annotation of the gene with 100 kb upstream and downstream of the significant SNP.


Bemisia tabaci is a threat to agriculture worldwide because of its potential to cause devastating damage to various crops. β-asarone is a bioactive pesticidal chemical originating from Acorus calamus (or “Sweet Flag”) plants, and it displays significant lethal effects against insect pests. In this study, we established a baseline of susceptibility to β-asarone from China and patterns of cross-resistance to other popular insecticides. We found that all the 12 field-collected B. tabaci populations exhibited high susceptibility to β-asarone, and there was no cross-resistance detected for other tested insecticides. We subsequently evaluated the sublethal effects of β-asarone on physiology and biochemistry via LC25 treatment (4.7 mg/L). LC25 of β-asarone resulted in prolonged developmental duration and decreased survival rates in B. tabaci nymphs, pseudopupae, and adults. Significant reductions in oviposition duration, fecundity, and hatchability were also observed. Additionally, the metabolic enzyme activity and expression profiles of selected cytochrome P450 monooxygenase (P450) genes following the LC25 treatment of β-asarone suggest that enhanced detoxification via P450s could be involved in the observed sublethal effects. These findings demonstrate the strong toxicity and significant sublethal effects of β-asarone on B. tabaci and suggest that the induced overexpression of P450 genes could be associated with the response to β-asarone. Bemisia tabaci, the silverleaf whitefly, is a globally distributed, notorious insect pest for agricultural production, and it has been observed to cause significant damage to more than 600 species of plants, mainly by feeding on the phloem of host plants [t plants ,2. Theset plants . For mort plants , but thet plants . Althougt plants ,8,9,10. Acorus calamus (or Sweet Flag) is well-known in Asia for its pesticidal, antifungal, antibacterial, and medicinal properties [Lasioderma serricorne and Tribolium castaneum and exhibit powerful repellent bioactive for T. castaneum [A. calamus essential oil has led to higher larval mortality in diamondback moths and tobacco cutworms [A. calamus seems very promising for developing a safe plant-derived chemical agent for pest management, and it has been reported that β-asarone is the primary component isolated and purified from the essential oil of A. calamus and the main bioactive pesticidal chemical of A. calamus, which shows excellent toxicity to various insect pests [Plant-derived pesticidal chemicals have received much attention in recent years owing to their significantly high safety and low environmental impact. operties ,12,13. Pastaneum . Additiocutworms . A. calact pests ,17. In tct pests ,19,20. Tct pests .1 generation. The sublethal effects of insecticides on insect pests may be complex and diverse owing to differences in insecticide concentration/dose, insect species, treatment settings, or physiological conditions. Various classes of insecticides such as afidopyropen, clothianidin, cyantraniliprole, cycloxaprid, and dinotefuran have previously been shown to display sublethal effects on whiteflies [Nilaparvata lugens [B. tabaci have not been reported so far. In an effort to evaluate the latent capacity of β-asarone as a useful insecticide for populations of B. tabaci, we sought to confirm the compound’s sublethal effects on B. tabaci. More importantly, demonstration of the sublethal effects of this compound for a comprehensive evaluation of β-asarone could contribute to enhancing the efficacy against B. tabaci.Exposure to the sublethal concentrations of these compounds can cause a variety of physiological and behavioral alterations, negatively affecting the essential fitness components of arthropods such as prolonging the duration of growth and reducing the fecundity of Fiteflies ,24,25,26a lugens  insect pB. tabaci collected from six provinces of China were monitored for their susceptibility to β-asarone. Researchers found that all of the 12 tested populations exhibited high susceptibility to β-asarone and minimal cross-resistance to other tested chemical agents. The sublethal effects of β-asarone on B. tabaci were then evaluated. Considering that cytochrome P450 monooxygenases (P450s), esterases (ESTs), and glutathione S-transferases (GSTs) in insects are related to tolerance and resistance to most popular chemical agents against B. tabaci [The lethal effects of β-asarone on adults of whitefly were determined, and twelve field-collected populations of . tabaci ,10, to i50 that ranged from 11.78 to 42.80 mg/L  (25-treated group (84.19 ± 4.18%)  A and ovi/female) B, compar± 4.18%) C was low25 groups (25 group significantly increased 1.9-fold compared with that in the CK group. There was no significant difference between the activities of GSTs (elevated 1.3-fold) and esterases (elevated 1.1-fold). Of the twelve CYP candidates, five displayed significantly elevated gene expression in the sublethal treatment group compared with the control: CYP4C64 (increased 2.2-fold), CYP303A1 (increased 2.0-fold), CYP6DZ7 (increased 1.8-fold), CYP6CX3 (increased 1.6-fold), and CYP6CX5 (increased 1.9-fold)  B. tabaci efficiently. β-asarone is the primary bioactive pesticidal chemical of A. calamus [N. lugens, β-asarone achieves a 50% mortality at low concentrations as well as showing clear insecticidal activities against Lasioderma serricorne [B. tabaci adults from 12 field-collected populations from northern China. Furthermore, we investigated the potential patterns of cross-resistance to three popular chemical agents against B. tabaci. Our study indicated that β-asarone showed significant insecticidal activity with no cross-resistance to the three successfully commercialized chemical agents. The above results showed that β-asarone has a powerful lethal effect and offered a useful basis for further study and application of β-asarone against B. tabaci.Plant-derived insecticides are receiving much attention due to their light impact on natural surroundings. Although botanical toxins are considered among the promising alternatives to the application of chemical agents, there are few known to control  calamus ,29,30. Arricorne . In the 25 of β-asarone treatment prolonged the duration of growth and reduced the survival rates of whitefly nymphs, pseudopupae, and adults when compared with the control, and the periods of oviposition, fecundity, and the hatching rate of eggs were also significantly reduced. Similarly, the fecundity of females and the hatching rate of eggs were also significantly decreased in the N. lugens populations treated with sublethal concentrations of β-asarone [Although sublethal doses are not expected to directly kill insect pests, they have been reported to affect some of the most critical functions required for their survival . Pests t-asarone . The subB. tabaci play an essential role in the detoxification response to chemical pesticides [B. tabaci is involved in the processes of detoxification following exposure to various classes of chemical agents. It has been reported that two genes, CYP6CX4 and CYP4G68, are overexpressed in resistant strains of B. tabaci and have a confirmed association with resistance to flupyradifurone and cyantraniliprole [B. tabaci, a series of P450 genes were shown to be associated with different levels of resistance to neonicotinoid insecticides [25, five P450 genes were significantly overexpressed in comparison to the control, suggesting that these P450 genes are involved in the processes triggered in response to β-asarone exposure. In the case of N. lugens, the involvement of several P450 genes in the detoxification metabolism of β-asarone has been confirmed [B. tabaci, resulting in their death. Although the possible mode of action of β-asarone on B. tabaci is not clear yet, the decreased longevity of adults and reduced fecundity display extra functions beyond lethal effects with the application of β-asarone against B. tabaci. Further studies in the lab will be performed to investigate the lethal and sublethal effects of β-asarone on the natural enemies of B. tabaci. Moreover, we plan to carry out RNA-seq and functional studies to study this mechanism further.When insect pests are treated with pesticidal chemicals, detoxifying enzymes are deployed in an effort to mitigate the effects. Former reports have shown that the P450 monooxygenases of sticides ,9,10,34.iliprole ,36. In vcticides ,38. Our onfirmed ,40. We sBemisia tabaci MED-S strain was originally collected from damaged poinsettia (Euphorbia pulcherrima Wild. ex Klotz.) in Beijing, China, in 2009 [B. tabaci were reared on plants of cotton (Gossypium hirsutum L. var. “Shiyuan 321”) under the photoperiod of 16 L: 8D, the temperature of 26 ± 1 °C, and the relative humidity of 55 ± 5%. Adults of the tested B. tabaci no more than seven days old were sampled at random and utilized at about a 1:1 ratio of females and males for all the bioassays conducted for this study.The  in 2009 . Three p in 2009 ,35,42. T in 2009 , and all in 2009 . All theAll the tested chemical agents were according to analytical standards. β-asarone , flupyradifurone , cyantraniliprole , and dimethyl sulfoxide  were purchased from Sigma Aldrich in Shanghai, China. Afidopyropen  was purchased from Dr. Ehrenstorfer in Augsburg, Germany.B. tabaci strains to β-asarone and establish cross-resistance patterns with three other insecticides, bioassays were conducted on adults using an improved feeding method [B. tabaci was counted, and the death rate of each treatment was calculated.To evaluate the susceptibility of g method . The tecB. tabaci shown above, the LC25 value was determined and utilized in the treatment of LC25 as a sublethal treatment strategy on B. tabaci, following the feeding steps shown above. Various fitness components were then recorded, including the duration of growth and the viability of developmental stages in the offspring, the duration of oviposition, the fecundity of females, and the hatching rate of their eggs. Fifty adults of B. tabaci were introduced into the tube with LC25 of β-asarone with two more tubes as replicas, and the tubes were placed in the chamber under the photoperiod of 16 L: 8D, the temperature of 26 ± 1 °C, and the relative humidity of 55 ± 5%. Additionally, another three tubes of B. tabaci adults with no exposure to LC25 of β-asarone were set as controls. After 48 h of feeding, the LC25 of β-asarone treatment and the control groups were ready to be transferred to the plants. Ten whitefly-free plants of cotton were moved into two individual insect-proof cages  with five cotton plants in one of the cages. Plants of cotton in the LC25 cage were treated with β-asarone at the LC25 concentration, while the CK cage was without treatment and set as the group of controls. One hundred adults of B. tabaci that were treated (LC25) by the LC25 concentration were then moved into the LC25 cage for egg laying measurements. One hundred untreated B. tabaci adults were moved into the CK cage. After 24 h of laying eggs, all the plants were moved out of the two cages, and ten leaves were sampled at random from each of the cages. In each of the 10 leaves, 20 eggs were left on each leaf and kept with one leaf clip-cage. All the spots of the eggs on the tested leaves were labeled on the surface with one marker pen, and the cages were under the photoperiod of 16 L: 8D, the temperature of 26 ± 1 °C, and the relative humidity of 55 ± 5%. Newly emerged B. tabaci adults were then moved onto fresh leaves of cotton with clip cages for fecundity measurements that continued until all individuals died, and then the hatchability of eggs was counted and calculated.On the basis of the bioassay conducted with five concentrations of β-asarone on the adults of B. tabaci, 12 candidate P450s, namely CYP6CM1, CYP6DZ4, CYP4G68, CYP6DW2, CYP6CX4, CYP4C64, CYP303A1, CYP6DZ7, CYP6CX3, CYP6CX1v1, CYP6DW3, and CYP6CX5, were chosen, and the gene expressions of the 12 candidate P450s were determined [25, the total RNA was extracted from 100 B. tabaci adults randomly sampled from the treatment population. One hundred of the untreated B. tabaci adults were also randomly sampled and set as the CK group. The gene expression was measured via qPCR using TUB1α and EF-1α for normalizing the data. All the information on primer sequences is displayed in Activities of P450 monooxygenases (P450s), esterases, and glutathione S-transferases (GSTs) were measured based on our previously reported method, with minor modifications . Each sttermined ,38. Afte50 values were evaluated using the PoloPlus software  [B. tabaci treatments were determined via Student’s t-test. The metabolic activity and expression patterns of P450 genes were compared between the two treatment groups using Student’s t-test. All statistical analyses were implemented in the SPSS software  [Differences in mortality between various working concentrations were recorded, an analysis of the bioassay data collected at different working concentrations of the β-asarone was conducted, and LCA, 2002) . TreatmeA, 2011) .


Muscadinia and Euvitis species.Muscadine berries display enhanced nutraceutical value due to the accumulation of distinctive phytochemical constituents with great potential antioxidant activity. Such nutritional and health merits are not only restricted to muscadine, but muscadine berries accumulate higher amounts of bioactive polyphenolics compared with other grape species. For the genetic study of the antioxidant trait in muscadine, a multi-locus genome-wide association study (GWAS) with 350 muscadine genotypes and 1,283 RNase H2 enzyme-dependent amplicon sequencing (rhAmpSeq) markers was performed. Phenotyping was conducted with several antioxidant-related traits, including total phenolic content (TPC), total flavonoid content (TFC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, and FRAP antioxidant assay in muscadine berry skin. The correlation coefficient analysis revealed that the TPC, and DPPH/FRAP activities were significantly correlated. Through the GWAS analysis, 12 QTNs were identified from the four traits, of which six were pleiotropic QTNs. Two pleiotropic QTNs, chr2_14464718 and chr4_16491374, were commonly identified from the TPC and DPPH/FRAP activities. Co-located genes with the two pleiotropic QTNs were isolated, and two candidate genes were identified with transcriptome analysis. UDP-glycosyltransferase and 4-hydroxy-4-methyl-2-oxoglutarate aldolase were the candidate genes that are positively and negatively correlated to the quantitative property of traits, respectively. These results are the first genetic evidence of the quantitative property of antioxidants in muscadine and provide genetic resources for breeding antioxidant-rich cultivars for both  Muscadinia rotundifolia Michx.) is a native grape of the southeastern United States with great marketing opportunities for juice, wine, and fresh fruit  and Muscadinia (muscadine grapes). While Euvitis has approximately 70 species, only three species, M. rotundifolia, M. munsoniana, and M. popenoei are known in the Muscadinia genus . The DNA for genotyping was extracted from the young leaves of each individual using Qiagen DNeasy Plant Mini Kit .via measuring berry firmness, total soluble sugar (TSS), acidity (TA), and TSS/acid ratio as described previously  markers . Filterik) was identified as 15  . A totalFor transcriptome analysis, muscadine berry samples were collected by three replications from three different genotypes, C5-9-2 (“Ison × Fry”), C6-10-1 (“Southland × Fry”), and Late Fry, at three different berry developmental stages, “Fruit-Set,” “Véraison,” and “Ripe.” These genotypes were selected according to their diversity in TPC, TFC, and antioxidant capacities . The RNAp ≤ 0.05 and r ≥ 0.7 or r ≤ –0.7). The TPC, TFC, DPPH activity, and FRAP activity values during berry development were compared to the TPM expression values of the marker-associated genes at the corresponding developmental stages. The genes that significantly correlated to the antioxidant-related traits were identified and used for further analysis.The Pearson correlation test was used to calculate the correlation between antioxidant contents and expression of marker-associated genes  and FRAP  activities. In contrast, relatively lower, but significant, correlation values of TFC with TPC , DPPH , and FRAP  was observed. It is clear that both TPC and TFC traits contribute to the ultimate antioxidant capacity of muscadine grapes. However, the input of TPC seems to be more significant.To investigate the relationship between the four antioxidant-related phenotypes, we conducted correlation coefficient analysis by Pearson correlation test . It is kEuvitis species . Interestingly, two QTNs, chr2_14464718, and chr4_16491374, were identified as pleiotropic QTNs for these three antioxidant-related traits. In the case of the chr2_14464718 QTN, it was identified with five methods in TPC and six methods in DPPH activity. This indicates that these two QTNs might be highly associated with the antioxidant capacity of muscadine berry skin. For TFC, two associated QTNs were identified, and one of them was a pleiotropic QTN that was also identified in TPC.A total of eight associated QTNs were identified for the TPC trait, of which six among them were pleiotropic QTNs. The TPC and DPPH activity traits shared five of the six pleiotropic QTNs, demonstrating the main contribution of TPC character to the DPPH antioxidant capacity. According to the correlation coefficient analysis, TPC, DPPH activity, and FRAP activity were significant to each other   . To furtactivity . Among tactivity . This inactivity .r ≥ 0.7; p ≤ 0.05). The isoflavone reductase protein is also known to involve the elaboration of flavonoid and isoflavonoid skeletons  by the Pearson correlation test  and 18.92% (chr4_16491374) (Although intensive genetic studies have been conducted with 6491374) . These aWith the two QTNs, the current study identified the positively correlated gene with the antioxidants, UDP-glycosyltransferase, and the negatively correlated gene, 4-hydroxy-4-methyl-2-oxoglutarate aldolase. The genetic studies of antioxidants have identified candidate genes in multiple crops by GWAS analysis. Interestingly, the GWAS results of antioxidants in barley, pearl millet, and common bean identified the UDP-glycosyltransferase gene as the candidate gene for the quantitative property of antioxidants . In applPseudomonas putida, degrades gallic acid to 4-carboxy-4-hydroxy-2-oxoadipate, which is then cleaved by 4-hydroxy-4-methyl-2-oxoglutarate aldolase  can be found below: MP and AD: conceptualization, methodology, investigation, validation, and formal analysis. MP: writing—original draft preparation, software, and visualization. RE, VT, and KS: writing—review and editing. IE-S: conceptualization, methodology, validation, formal analysis, resources, data curation, writing—review and editing, supervision, project administration, and funding acquisition. All authors approved the final manuscript.


Ghnb5, a new recessive genotype of GhNB, was found in 04N-11. Through candidate gene association analysis, SNP 20_15811516_SNV was found to be associated with plant architecture and early maturity in the Xinjiang natural population. The GhNB gene, which is related to early maturity and the plant architecture of cotton, is a branch-type gene of cotton. The 20_15811516_SNV marker, obtained from the Xinjiang natural population, was used for the assisted breeding of machine-picked cotton varieties.With the promotion and popularization of machine cotton-picking, more and more attention has been paid to the selection of early-maturity varieties with compact plant architecture. The type of fruit branch is one of the most important factors affecting plant architecture and early maturity of cotton. Heredity analysis of the cotton fruit branch is beneficial to the breeding of machine-picked cotton. Phenotype analysis showed that the types of fruit branches in cotton are controlled by a single recessive gene. Using an F With the popularization of mechanized cotton harvesting, the selection and breeding of early-maturity varieties with compact plant architecture, concentrated flowering and early maturity are highlighted. The plant architecture of cotton is determined by the fruit branch type, internode length and fruit branch angle. Among them, the fruit branch type is the most influential factor. The study of fruit branch type is helpful in the selection and breeding of machine-harvested cotton. Xinjiang is China’s main cotton planting area, where cotton yield and quality are higher than the national average level. In 2020, Xinjiang’s cotton planting area was 2,501,900 hectares, and the output of cotton was 5.16 million tons . The heaIn recent years, with the continuous improvement in residents’ incomes, the labor cost of cotton production has been increasing. The labor input is the biggest cost in traditional cotton production. The mechanization of planting and harvesting can improve production efficiency, and can reduce the cost of labor and the production cost of cotton. The plant architecture of cotton not only affects the ventilation and light transmission of the population, but also impacts mechanized harvesting . The maiThe type of cotton fruit branch can be categorized as a nulliplex branch, determinate branch or normal branch, according to the number of fruit nodes. Cotton with the nulliplex branch has no obvious fruit branches, with a typical tubular structure; it has mostly early-maturity varieties of plant architecture, and the bolls grow directly in the axils of the main stem . Cotton 2 population or RIL population, is a gene mapping method based on the linkage imbalance principle [BSA, which can locate the major genes of qualitative or quantitative traits based on the Frinciple . Using trinciple , Maize [rinciple , rice [1rinciple , cucumberinciple  and otherinciple . Similarrinciple .GbNB gene was reported to regulate the fruit branch types of cotton in Pima cotton, and the GhNB gene was reported to regulate the fruit branch types of upland cotton [GhNB or GbNB and the flowering period, the effect of the GhNB and GbNB genes on cotton flowering and the molecular markers related to early maturity in cotton were discovered.Previous studies have shown that cotton branches are controlled by a single gene located in the nucleus of the cell. The d cotton . We also04N-11 was a nulliplex-branch mutant of upland cotton, Huaxin 102 was a normal branch variety of upland cotton, and Xinhai 18 was a nulliplex-branch variety of Pima cotton.A total of 248 cotton varieties approved in Xinjiang, including 73 upland cotton early-maturity varieties; 79 medium-maturity varieties; 63 parts of Pima cotton varieties; 11 colored cotton varieties; 20 backbone parents and 2 self-breeding varieties, were collected to construct the Xinjiang natural population  Supplem.The type of cotton fruit branch was divided into nulliplex branch, determinate branch and normal branch according to the boll position. Branches with cotton bolls growing directly in the axils of the main stem are called nulliplex branches; if the cotton has fruit branches but only one fruit node, with bolls clustered at the top of fruit branch, it is a determinate branch; and normal branches have more than one fruit node, and cotton bolls are borne on the top of fruit nodes.2 population. A total of 30 randomly selected plants with nulliplex branches were collected from the population, and DNA was extracted and mixed equally to construct a nulliplex-branch DNA mixing pool. Additionally, 30 randomly selected plants with normal branches were collected from the population, and DNA was extracted and mixed equally to construct a normal-branch DNA mixing pool. Huaxin 102, 04N-11 and two DNA mixing pools were resequenced using an Illumina sequencing platform. The obtained sequencing data were compared with the reference genome of TM-1_ZJU_V2.1 (ftp://ftp.bioinfo.wsu.edu/species/Gossypium_hirsutum/ZJU_G.hirsutum_AD1genome_v2.1) (accessed on 1 May 2022) to identify SNP and InDel mutations between Huaxin 102 and 04N-11. The clean genomic data were generated and deposited in NCBI Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA841236 (accessed on 1 May 2022). According to the SNP markers obtained by sequencing, the index value of each SNP marker in the two DNA mixing pools was calculated as follows: BSA  is a mixed grouping method, which uses a pair of parents with significant differences in traits to construct a separated population through crossbreeding, backcrossing and other methods. In this study, Huaxin 102 and 04N-11 were used as parents to construct an FP−JSNP is the sequencing depth of the SNP marker genotype in 04N-11, and P−YSNP is the sequencing depth of the SNP marker genotype in Huaxin 102)., the number of days from seeding emergence to flowering period was uniformly converted .GhNB gene in upland cotton and the GbNB gene in island cotton were classified according to resequencing data from the Xinjiang natural population of 248  and 04N-11 (nulliplex branch). Phenotype analysis showed that all F1 plants were normal branch, indicating that the type of branch was a recessive trait controlled by nuclear genes. Then, the F2 population, derived from the self-crossing of the F1 population, were obtained; phenotype analysis showed that the 388 F2 plants contained 278 normal-branch plants and 110 nulliplex-branch plants in 2019, and the 1895 F2 plants contained 1455 normal-branch plants and 440 nulliplex-branch plants in 2020. Chi-square fitness test analysis showed that the separation ratio of normal branch and nulliplex branch was 3:1 in the F2 population  at 10×. The obtained resequencing data were mapped to the reference genome of TM-1_ZJU_V2.1, then to identify SNP and InDel mutations between Huaxin 102 and 04N-11. Through comparative analysis, 815,493 SNPs and InDel variants, with polymorphism between Huaxin 102 and 04N-11, were screened from 730,630 SNP and 84,863 InDel variants. Analysis of the distribution of SNPs and InDels in the whole genome of Huaxin 102 and 04N-11 was performed, and showed aggregation in some genome segments  in the chromosomes of upland cotton was analyzed, and showed that the Δ(SNP-index) values of most of the SNP markers were between −0.3–0.3. Only Δ(SNP-index) values in the 12.16–14.82 Mb range on chromosome D07 exceeded 0.5, which suggests that this region is the candidate region of genes determining the fruit branch type of cotton .GhNB gene at 15.1Mb on the right of the candidate region was reported to control the fruit branch type of cotton [GbNB gene at 15.4Mb on chromosome D07 was reported to control the type of cotton fruit branch in Pima cotton, and the GbNB and GhNB genes were allelic [GhNB or GbNB gene is allelic with the gene determining the fruit branch type of 04N-11, a nulliplex-branch Pima cotton Xinhai 18 (the nulliplex branch was determined by GbNB) and 04N-11 were crossed to observe the progeny phenotype  at the haplotype level, and only one SNP marker, 20_15811516_SNV, was significantly correlated with the flowering period in this linkage interval ) and the marker was significantly correlated with fruit branch type and the flowering period of cotton  associated with early maturity was discovered in the Xinjiang natural population.A candidate interval of 12.16–14.82 Mb on chromosome D07 was obtained in this study. The F


Non-Smoky Glucosyl Transferase 1 (NSGT1), we uncovered extensive genome structural variation (SV) at the Methylesterase (MES) locus. This locus contains four tandemly duplicated Methylesterase genes and genome sequence investigations at the locus identified nine distinct haplotypes. Based on gene expression and results from biparental crosses, functional and non-functional haplotypes for MES were identified. The combination of the non-functional MES haplotype 2 and the non-functional NSGT1 haplotype IV or V in a GWAS panel showed high methyl salicylate levels in ripe fruits, particularly in accessions from Ecuador, demonstrating a strong interaction between these two loci and suggesting an ecological advantage. The genetic variation at the other two known loci, Salicylic Acid Methyl Transferase 1 (SAMT1) and tomato UDP Glycosyl Transferase 5 (SlUGT5), did not explain volatile variation in the red-fruited tomato germplasm, suggesting a minor role in methyl salicylate production in red-fruited tomato. Lastly, we found that most heirloom and modern tomato accessions carried a functional MES and a non-functional NSGT1 haplotype, ensuring acceptable levels of methyl salicylate in fruits. Yet, future selection of the functional NSGT1 allele could potentially improve flavor in the modern germplasm.Methyl salicylate is an important inter- and intra-plant signaling molecule, but is deemed undesirable by humans when it accumulates to high levels in ripe fruits. Balancing the tradeoff between consumer satisfaction and overall plant health is challenging as the mechanisms regulating volatile levels have not yet been fully elucidated. In this study, we investigated the accumulation of methyl salicylate in ripe fruits of tomatoes that belong to the red-fruited clade. We determine the genetic diversity and the interaction of four known loci controlling methyl salicylate levels in ripe fruits. In addition to  Tomato fruit release a wide variety of volatiles and these volatiles are responsible for its unique aroma. One of the important volatiles in tomato flavor is methyl salicylate. Methyl salicylate is important for plant defense, but tomato taste panels routinely associate this volatile with low liking as it features the characteristic odor of wintergreen. Several enzymes involved in the biosynthesis of methyl salicylate have been identified in tomato but the genetic diversity at the gene region has not yet been fully explored. In this study, we investigate the genetic variations at the four known gene regions controlling fruit methyl salicylate levels using a diverse collection ranging from red-fruited wild tomatoes to modern and heirloom varieties. We identified extensive genomic structural variations at two of the four loci. The genetic interaction between two loci determines the volatile levels. We also showed dramatic selection pressures ensued at these loci during evolution and domestication of the tomato. Our study greatly improves the understanding of the genetic basis of methyl salicylate production in tomato fruits and gives breeders far better tools to create agrosystem-specific adapted commercial germplasm. Plants synthesize and emit many volatile organic compounds that play key roles in reproduction, communication, and stress responses. The volatiles emitted from flowers allow plants to attract and guide pollinators to ensure fertilization and reproduction ,2, where−7 to 10−4 of the fresh fruit weight and are responsible for its unique aroma .–35.32–35eostasis . The twonjugates . These dripening ,34. Of tNSGT1 is known for the glycosylation of guaiacol, a smoky volatile that, similarly to methyl salicylate, negatively affects consumer liking. Based on genome structural variations (SV), NSGT1 is represented by five haplotypes that are grouped into functional and non-functional categories based on the presence of a functional enzyme [MES also exhibits SVs [SlSAMT1 and SlUGT5, has also not been explored in red-fruited tomato. The genetic characterization of these loci can provide evolutionary insights and lead to the identification of haplotypes that might be beneficial for crop improvement.l enzyme . Similarbits SVs , but theIn this study, we analyzed a unique and genetically well-characterized tomato collection of fully wild SP, semi-domesticated SLC and ancestral SLL landraces from South and Central America (Varitome Collection) as well as a subset of cultivated accessions that includes heirloom and modern types (SLL_CUL). Collectively, they are referred to as red-fruited tomato in this study. We investigated the genetic diversity at the known genes controlling methyl salicylate levels in tomato fruits and uncovered extensive genome SV underlying the trait in the red-fruited tomato clade. We also show that different selection pressures ensued at these loci during domestication.Quantification of fruit methyl salicylate in 166 accessions from the Varitome Collection and 143 cultivated types showed that the levels ranged from 0.003 nanogram per gram fruit weight per hour (ng/gfw/hr) to 29.360 ng/gfw/hr, representing a 9000-fold difference . Most acMES on chromosome 2 (chr 02), NSGT1 on chr 09, and SAMT1 also on chr 09. For MES, the locus was identified by all three variant types, namely a 4.7 kb deletion at position SL.40ch02:34,464,833, a SNP at SL4.0ch02:34,895,803, and an INDEL at SL4.0ch02:34,943,905 (MES locus contains four Methylesterase genes (Solyc02g065240-80) and the 4.7 kb deletion partially spans the third exon of Solyc02g065260 (SlMES3), whereas the SNP and INDEL mapped 400 kb and 467 kb downstream of the MES locus, respectively. The accessions carrying the 4.7 kb deletion produced significantly higher amounts of methyl salicylate compared to the accessions without the deletion  and an INDEL at SL4.0ch09: 65,401,256 (7.4 kb downstream of NSGT1), explaining 40% of the variation in the population. These overlapping GWAS loci carried three alleles: the reference allele, the alternate (SNP or INDEL) allele, or the 15 kb deletion. Accessions with the deletion at the locus showed significantly higher levels of methyl salicylate compared to accessions without the deletion  produced lower levels of methyl salicylate compared with the alternative allele (GA)  that mapped to approximately 6.5 Mb downstream the gene. For this variant, all accessions with reference allele produced low methyl salicylate levels in fruits, whereas lines with a nucleotide insertion showed significantly higher levels of the volatile   using the whole genome variant information of the Varitome Collection. We identified several GWAS loci associated with fruit methyl salicylate levels  using Si,943,905 . The MESdeletion  and the o fruits . For NSGdeletion . For SAMele (GA) . Similar6.4e-06) .NSGT1 were identified based on the SVs at the locus of which haplotype IV and V were associated with higher methyl salicylate levels [MES, UGT5 and SAMT1 loci with the focus on SVs. For MES, we compared the genomic sequences of the three highest methyl salicylate accumulating accessions  and the three lowest accumulating accessions . The high methyl salicylate producing accessions carried several deletions at MES and multiple mismatches when compared to the Heinz1706 reference genome. On the other hand, the genomic landscape of the low methyl salicylate producing accessions corresponded more closely to the Heinz1706 reference genome  and three missense  mutations in the latter gene. The last haplotype, haplotype 9 consisted of several large deletions at the locus resulting in truncation, internal deletions, and elimination of all four MES genes, and was found only in 13 SLL_CUL accessions . The three genes were found in all accessions and the locus did not show extensive SV except for a single 255 bp deletion  in the promoter of SlSAMT1 (Solyc09g091550) that was found in 27 accessions. The SAMT1 (Solyc09g091550) is known to regulate methyl salicylate levels in tomato fruit [SAMT1 gene Solyc09g091550 carried two synonymous and seven missense mutations compared to the Heinz 1706 reference genome for which one or more were found in 34 accessions. This means that the majority of red-fruited accessions in this study encoded the same SAMT1 protein sequence as Heinz1706. For another SAMT gene, Solyc09g091530, ten synonymous, nine missense, two frameshift, and two nonsense mutations were found compared with the Heinz1706 reference genome. The frameshift and nonsense mutations resulted in null alleles that were found in seven SP, three SLC and one SLL. Lastly, we found six synonymous, seven missense and one frameshift mutation in Solyc09g091540. The frameshift mutation was found in only three accessions (2 SLC and 1 SLL) and these also carried the predicted null mutations in Solyc09g091530. The haplotype analysis for SAMT1 using the 530 variants at the 21.3 kb locus showed four alleles  with the frameshift mutation in Solyc09g091530 and Solyc09g091540. Haplotype IV was identical to the Heinz1706 reference genome and found in the majority of SLL_CUL. Haplotype I of SlSAMT was the most ancestral haplotype based on the phylogenetic tree constructed from the consensus genomic sequence of the locus and included most of the SP accessions  found in 50 accessions and located 8 kb upstream of the gene. We identified 132 SNPs and 29 small INDELs at the 5.7 kb UGT5 locus compared to the Heinz1706 reference genome. Most of the variants (150) were found in the regulatory regions and only 11 variants  were found in the gene. The frameshift mutation was a likely null allele and was found in 41 accessions. Haplotype analysis using the variants in the 5.7 kb region resulted into four distinct haplotypes . In accessions belonging to haplotype 1 and 3, SlMES3 was well expressed in ripe tomato fruit, whereas the expression of the other MES genes was low or undetectable. Accessions belonging to these haplotypes produced very low amounts of methyl salicylate, ranging from 0.03 to 1.03 ng/gfw/hr  to SLC (41.32%), SLL (44.44%) and SLL_CUL (81.81%)  were under selection in SP but not in cultivated tomato, whereas the opposite was true for MES. For the two loci combined, it appeared that a strong selection for higher levels of methyl salicylate were particularly important to certain SP and SLC accessions from Ecuador and Peru  . Based oand Peru , with a nd NSGT1 . The higMethyl salicylate is an important plant volatile that functions as a signaling molecule within and between plants. Its role in defense responses in leaves is relatively well understood. It serves as an herbivore and pathogen-induced volatile that attracts parasitoids and predators of the herbivores. Plants emit methyl salicylate when attacked by herbivores, sending cues to parasitoids and other predators ,39–41. FMES, along with NSGT1 is a major contributor to fruit methyl salicylate levels in the tomato germplasm. Moreover, the interaction between MES and NSGT1 explained most of the variation in fruit volatile levels in a biparental mapping population [MES haplotype 2 and a non-functional NSGT1 haplotype IV or V resulted in the highest methyl salicylate levels indicating that the interaction of these two loci leads to higher levels of this volatile. Other genes in this pathway, SlSAMT1 and SlUGT5, appeared as minor players in this germplasm. The high or low methyl salicylate level haplogroups of SAMT1 and UGT5 could be explained by their genotype at MES and NSGT1 loci. SAMT1 haplotype II seemed to produce high levels of methyl salicylate. All accessions with SAMT1 haplotype II carried a non-functional MES haplotype (haplotype 2 or 4) which might explain the high volatile levels even though the production of methyl salicylate is dependent on a functional SAMT1. In addition, the 11 accessions carrying a null mutation in Solyc09g091530, whose function in methyl salicylate production is unknown, carried the functional NSGT1 haplotype which could explain the low volatile levels in the fruit. It is likely that there are other fruit-specific glycosyltransferases and methyltransferases with activity on methyl salicylate and salicylic acid respectively, downplaying individual effect of the cloned SlSAMT1 and SlUGT5 [MES and NSGT1) to conclude which was the most likely active or inactive allele at the minor loci.pulation , supportd SlUGT5 ,33,49. AMES and NSGT1 loci have been shaped predominantly by SVs. It is known that SVs greatly affect gene expression and protein functions resulting in variations in phenotype [MES carries several deletions and insertions, spanning the four Methyltransferase genes either partially or completely deleting genes from the locus. These SVs were likely responsible in part for the difference in MES gene expression observed among the haplotypes  in the first exon of SlMES3 found only in accessions in haplotypes 2 and 4. This SNP is predicted to cause an amino acid change from leucine to phenylalanine at position 83, which is one of the salicylic acid binding sites of the protein [SlMES3 is likely functional in addition to SlMES1. For SlMES4, expression levels were associated with the truncation of the protein. Haplotypes 1, 2, 3, 4, and 8 carrying a truncated SlMES4 had low or undetectable expression, whereas the haplotypes 5, 6, and 7 carried full length SlMES4 that showed high expression.The henotype ,50. MES plotypes . SlMES1  protein  and possMES controlling the production of this volatile. Recent evolutionary models proposed by Razifard et al. [MES haplotype distribution. SP from Peru, considered as the most diverse and ancestral, includes accessions with several MES haplotypes , while SP from northern Ecuador carried the non-functional haplotypes (2 and 4). Non-functional MES haplotype and high levels of methyl salicylate may have contributed to their survival in adverse environments. It is also possible that birds or animal dispersers, having preference for minty flavor, contributed to selection and migration, but little is known about this. However, selections imposed by the new environment necessitates adaptation that might have favored the selection of a specific MES haplotype over the other. According to Blanca et al. [MES haplotype 7, with potentially four functional genes, was selected as Peruvian and Ecuadorian SP evolved into SLC in Mesoamerica. MES haplotype 6 evolved from haplotype 7, after a premature stop-codon mutation in MES2. Upon migration back to South America, admixture with Ecuadorian SP resulted in SLC from Ecuador carrying the most diverse MES haplotypes. SLC Peru evolved from admixture with Peruvian SP resulting in at least four MES haplotypes, and together with Ecuadorian SLC spread northwards to evolve into ancestral SLL carrying only haplotype 6 and 7. On the other hand, Razifard et al. [MES haplotype 7 and its derivative haplotype 6 are found in Peruvian and Ecuadorian SP and were selected early as tomato migrated northwards to evolve into SLL. The evolution in MES locus seems to agree more with the model proposed by Razifard et al. [MES locus was maintained in South America whereas migration northwards into Mesoamerican SLC and eventually SLL resulted in the selection of only two MES haplotypes 6 and 7. Thus, the evolution at the MES locus suggests a complex migratory and evolutionary path of tomato.Several events in selection and domestication of cultivated tomatoes have affected the fruit methyl salicylate levels and d et al.  and Bland et al.  can be cplotypes . During a et al. , SP spred et al.  proposedd et al.  as the lMES locus. Moreover, even within each distinct haplotype, we observed wide phenotypic variation  were grown and phenotyped for volatile accumulation as previously described ,28. BaseAll statistical analysis were conducted using R  unless mhttps://www.ncbi.nlm.nih.gov/; SRA: SRP150040, SRP045767, SRP094624, and PRJNA353161). We augmented the original GWAS by Razifard et al. [Raw ILLUMINA read files were downloaded from NCBI . The genomes of these accessions were sequenced using Oxford Nanopore Technology and Illumina short read sequencing and, therefore, were well suited for the identification of SV at each of the loci. For the loci with evidence of SV, MES and NSGT1, SVs were identified in Integrative Genome Viewer (IGV) [MES locus ranged from 15 kb to 35 kb, whereas the size of the NSGT1 locus was as defined previously [SlSAMT1, the locus comprised three tandem duplicated genes without large SVs. To define the haplotypes at the SAMT1 and UGT5 loci, SNPs and small INDELs within the coding region, 3 kb upstream and 1 kb downstream of the termination of transcription were extracted using VCFtools [https://CRAN.R-project.org/package=rnaturalearth) and “ggspatial” . The package “rnaturalearth” uses the public domain map dataset “Natural Earth”  to generate the base layer of the map.To identify the haplotypes at the known genes regulating methyl salicylate levels, we first determined the presence of SVs at or near each by comparing the high-quality genome sequences of 28 Varitome accessions (er (IGV)  on the peviously . For SlSVCFtools . The metVCFtools . VariantVCFtools  using a VCFtools  tool wasMES locus and 10 kb flanking the locus. The consensus nucleotide sequences were generated using IGV and, exported in Geneious Prime v2021.2.2 (https://www.geneious.com/). The sequences were then aligned using the Clustal alignment option. The tree was constructed using Neighbor-Joining method and Tamura-Nei genetic distance model using S. pennellii  as an outgroup. Bootstrap support for the tree was obtained using 100 bootstrap replicates. The tree was then plotted using “ggtree” package in R [A phylogenetic tree was built using Neighbor-Joining method with the genomic sequences of genes at the age in R .ACTIN4 (Solyc04g011500) was used as the control to standardize relative expression levels. The relative expression was calculated using the formula of ΔCq method using a reference gene: R = 2Cq(reference)-Cq(target), where Cq is quantification cycle, reference is ACT4, and targets are MES1, MES2, MES3, or MES4. The expression analysis figure was generated using “ggplot2” package in R [qRT-PCR was performed following a protocol that was used previously . Total Rage in R . The seqhttps://www.ncbi.nlm.nih.gov/; SRA: SRP150040, SRP045767, SRP094624, and PRJNA353161).All the raw sequencing data used in this study are publicly available in NCBI  Alignment of three least methyl salicylate producing accessions against the SL4.0 genome build at the MES locus. Yellow boxes represent gene models.(A) Alignment of three highest methyl salicylate producing accessions against the SL4.0 genome build at the (PDF)Click here for additional data file.S2 FigSlMES1 (B) SlMES2 (C) SlMES3 and (D) SlMES4Alignments of (A) (PDF)Click here for additional data file.S3 FigSlMES2 and SlMES3.The gene model below is a screenshot from SL4.0 genome version of the exact region which is the insertion showing a transposon insertion between (PDF)Click here for additional data file.S4 FigSlMES1, SlMES2, SlMES3 and SlMES4. Yellow boxes represent gene models.Alignment for (PDF)Click here for additional data file.S5 FigSAMT1 haplotypes. (C) Distribution of methyl salicylate in red fruits in different accessions with and without deletion in the SAMT1 locus. (D) Distribution of methyl salicylate between accessions with and without null mutations of Solyc09g091530.(A) Heatmap representing the genotypes of accessions (rows) for the polymorphisms identified (columns). Reference genotypes are represented in blue, alternate in red, heterozygous in yellow and missing data in white. The black rectangular box represents the position of the genes in the locus. (B) Distribution of methyl salicylate in red fruits in different accessions among different (PDF)Click here for additional data file.S6 FigSAMT1 haplotypes. The outer two concentric circles represent the grouping of accessions  and corresponding methyl salicylate levels (MeSA) in the accessions respectively. The numbers on the branches represent the bootstrap values.Different colors of accessions and branches represent different (PDF)Click here for additional data file.S7 FigUGT5 haplotypes. (C) Distribution of methyl salicylate in red fruits in different accessions with and without deletion in the promoter of SlUGT5.(A) Heatmap representing the genotypes of accessions (rows) for the polymorphisms identified (columns). Reference genotypes are represented in blue, alternate in red, heterozygous in yellow and missing data in white. The black rectangular box represents the position of the gene in the locus. (B) Distribution of methyl salicylate in red fruits in different accessions among different (PDF)Click here for additional data file.S8 FigUGT5 haplotypes. The outer two concentric circles represent the grouping of accessions  and corresponding methyl salicylate levels (MeSA) in the accessions respectively. The numbers on the branches represent the bootstrap values.Different colors of accessions and branches represent different (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableMES haplotype information is provided in the column besides the accession names. The annotation information is provided along each variant. 0|0 represents reference allele, 1|1 represents alternate allele, and 1|0 represents heterozygous allele. The yellow highlighted variants are the variants discussed in the text.The (XLSX)Click here for additional data file.S5 TableSAMT1 haplotype information is provided in the column besides the accession names. The annotation information is provided along each variant. 0|0 represents reference allele, 1|1 represents alternate allele, and 1|0 represents heterozygous allele. The yellow highlighted variants are the variants discussed in the text.The (XLSX)Click here for additional data file.S6 TableUGT5 haplotype information is provided in the column besides the accession names. The annotation information is provided along each variant. 0|0 represents reference allele, 1|1 represents alternate allele, and 1|0 represents heterozygous allele. The yellow highlighted variants are the variants discussed in the text.The (XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 TableThe data are presented in different worksheets.(XLSX)Click here for additional data file. 23 Mar 2023Dear Dr van der Knaap,Thank you very much for submitting your Research Article entitled 'Structural variation underlies functional diversity at methyl salicylate loci in tomato' to PLOS Genetics.The manuscript was fully evaluated at the editorial level and by two independent peer reviewers. We apologize for the lengthy amount of time that this paper was in the review process - we had a difficult time securing the necessary reviews.  The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript. Neither of the reviewers had major concerns or large suggestions for additional work that are necessary.  However, both of the reviewers make a number of suggestions regarding the presentation or interpretation of the results that could be quite helpful towards improving the manuscript. We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.In addition we ask that you:1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.archive. 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SpringerAcademic EditorPLOS GeneticsLi-Jia QuSection EditorPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: A manuscript entitled “Structural variation underlies functional diversity at methyl salicylate loci in tomato” investigates how the allelic variation of four genetic loci shape the quantitative variation of methyl salicylate in tomato fruit and how this variation might have evolved during evolution and domestication. The manuscript contains a large amount of novel interesting information, that can prove useful for both fundamental understanding of metabolic variation in tomato fruit as well as for applied proposes, such as breeding for specific chemical composition of tomato fruit. The quality of the study and the presentation of the results is high, but there are a few points that I’d like to see addressed.Line 80 “While the levels of methyl salicylate are generally low in red-fruited tomato including heirlooms, some produce intermediate levels of the volatile in ripe fruit, suggesting that selections for yield and disease resistance were at the expense of better flavor, a common concern among consumers” – selection for yield and resistance is indeed often blamed for the poor flavor of many modern tomato cultivars. However, I don’t immediately see how varying levels of MeSa suggest that “selections for yield and disease resistance were at the expense of better flavor”. The data present in literature suggests that the ratio between relatively high and low MeSA tomatoes is roughly 50/50 and fruit type independent. Wouldn’t this suggest that the MeSA fruit phenotype is rather neutral for both flavor and tomato plant well-being? Besides, a lower MeSA content would be rather beneficial since a higher content of this volatile often correlates with off-flavor.Besides that, the importance of MeSA as a selection factor for flavour is questionable. The maximum concentration in the accession collection reported here, which represent a wide genetic diversity, is about 30 ppb whereas the odor threshold reported for MeSA is about 40 ppb  and this is water meaning that in a fruit matrix it would be even higher. The observation that MeSA has often been associated with off-flavor comes from the correlation and regression analyses, which do not necessarily prove the causal effect. In this particular case the off-flavor could be more likely caused by e.g. guaiacol, which often highly correlates to MeSA (mostly due to NSGT), has a comparable concentration range, but a much lower odor threshold.Of the four loci studied only NSGT and MES seem to explain certain amount of variation in MeSA. Line 332 states that “Overall, we could not show functional association of SAMT1 and UGT5” - Nevertheless there's a significant association of MeSA and these two loci in the GWAS. Does this mean SAMT1 and UGT5 are not the genes underlying the variation in these loci?Besides that, accessions with identical MES/NSGT haplotypes show quite different MeSA contents. For instance, in table S3 accession TS-275 - low MeSA - has exactly the same HPT combination as the top producer TS209. Thus, what % variation these do these two loci actually explain? Does this mean there's something else? And why it doesn’t come out of the GWAS?The discussion on the evolutionary development of the loci is very interesting, but, in my opinion, the authors have put too much weight on MeSA as a driving force of selection for these loci. The effect of the NSGT locus is certainly not restricted to MeSA only, but it also glycosylates other compounds. The same might hold for MES that might act on other ester substrates, which, as recent studies showed, are present in fruits of wild tomato species. They can also demethylate some non-volatile secondary metabolites that have not been analysed in the tomato material studied here.The picture of both structural alterations and expression levels is kind of scattered throughout the manuscript which makes it difficult to grasp. Can these somehow be summarized  in e.g. a table or a figure so that a reader could in one glance see what likely to cause a certain MeSA chemotype in different haplotypes, e.g. is that a deleterious mutation in a certain gene(s) or expression or both?Reviewer #2: Structural variation underlies functional diversity at methyl salicylate loci in tomato.This is a nice manuscript that takes advantage of the VARITOME collection that has been genotyped and phenotyped by this consortium of partners with the aim to define the origins and evolution of tomato and eventually use of this knowledge in breeding.In this MS authors focus their attention in the different loci contributing to one of the VOCs that are produced by tomato fruits and generally associated negatively with good tomato flavor MeSA and the authors speculate about a hypothetical agroecological role it could have in the tomato evolution from the center of origin in South and Meso Americahttps://doi.org/10.1093/plphys/kiac509 ). NSGT molecular genetics also in Alonge et al (2020) Cell. 2020 Jul 9; 182(1): 145–161.e23. doi: 10.1016/j.cell.2020.05.021The key genes regulating MeSA and their diversity in the tomato clade has been described in a few seminal papers that counted in some with the participation of the authors of this paper. Namely, the key role of the MES cluster and the NSGT have been described in detail from evolution to biochemistry .  and some of the RT PCR results don’t reveal anything substantially new that was not revealed in the PP paper.Also de authors describe in the text many details that can be directly obtained from figures or unnecessary.Authors should cleary indicate what data were generated and original for this ms ad which already reported and indicate were to find the original data as required by PlosGenetics.To discuss about the “functional” diversity in relation to a putative eco/evolutionary advantage is highly speculative as it is based on single measurements at the ripe stage of fruit (from probably/growing under standard in greenhouse conditions) and the role of MeSA in ripe fuir in relation to these aspects has still to be substantiated with more results. MeSA is known to respond to evironmental cues and the authors know and reported in the PP. This is MeSA-centric ripe fruit-centric paper and when talking about interaction with biotic and abiotic conditions, it misses to include in the analysis SA  measurements of MeSA in ripe fruit samples so it should not sound like these fruit MeSA related loci can explain ecological adaptations that may depend on plant SA levels for instance more than in red fruit MeSA.Authors take for granted that the triglycosilated form of MeJA produced by NSGT is an end irreversible product and this appears to be the case in tomato but is it possible that some accessions of SP or SLC may have a specific glycosidases that could make this last step reversible by cleaving the triglyco20sid form of MeSA?Please give details on how VOCs were determined. If is by cutting the fruits and flow and trap from tomato wedges as in the PP papers is it possible that MeSA production changes during wounding, release and capture and theexpression of corresponding biosynthetic and metabolism genes. If so then could it be that the MES steady state levels obtained by RNA extraction of ripe fruit are missing a possible induction produced in the wounded tomato wedges during MeSA capture and also does not capture the eventual activataion of the MES, NSPGT etc in response to the different environmental conditions that the accessions were experiencing during their evolution.In summary the story build on the evolution of the traits and the underlying haplotypes is interesting and well constructed ad of interest. Not sure if adds much in comparison to the PP paper about clues for flavou breeding. Technically is ok but I found it highly speculative for the ecological story build around it.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesNo: See comment above.Reviewer #2: **********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  29 Apr 2023PGENETICS-D-22-01442R1 Structural variation underlies functional diversity at methyl salicylate loci in tomato Dear Dr van der Knaap, We are pleased to inform you that your manuscript entitled "Structural variation underlies functional diversity at methyl salicylate loci in tomato" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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