The pip, as the most common grapevine archaeological remain, is extensively used to document past viticulture dynamics. This paper uses state of the art morphological analyses to analyse the largest reference collection of modern pips to date, representative of the present-day diversity of the domesticated grapevine from Western Eurasia. We tested for a costructure between the form of the modern pips and the: destination use (table/wine), geographical origins, and populational labels obtained through two molecular approaches. Significant structuring is demonstrated for each of these cofactors and for the first time it is possible to infer properties of varieties without going through the parallel with modern varieties. These results provide a unique tool that can be applied to archaeological pips in order to reconstruct the spatio-temporal dynamics of grape diversity on a large scale and to better understand viticulture history. The models obtained were then used to infer the affiliations with archaeobotanical remains recovered in Mas de Vignoles XIV . The results show a twofold shift between the Late Iron Age and the Middle Ages, from table to wine grape varieties and from eastern to western origins which correlates with previous palaeogenomic results. Vitis vinifera L. subsp. vinifera) is economically one of the most important cultivated fruit species in the world1. Its central economic and cultural role in the Mediterranean Basin goes back beyond the Greco-Roman era2. Modern genetics and archaeobotany concur in locating the origin of domesticated grapevine in the Near East, south of the Caucasus4. Its initial domestication is thought to have occurred during the Neolithic (between 6000 and 3000 BC) but the date is still debated. Chemical analyses of pottery vessels suggest that wine was already produced in the Caucasus area 8000–6000 years ago5. From its Near-Eastern cradle, viticulture spread to most of the Mediterranean and eventually the rest of modern-day Europe, between 3000 BC and 500 CE2. Viticulture could have started in Sardinia and Southern Italy as early as the late 2nd millennium BC6 and in Southern Spain by the beginning of the 1st millennium BC, in connection with the Phoenician influence7.Today, grapevine (Vitis vinifera subsp. sylvestris) to a hermaphroditic reproductive system for most of the varieties, the increase in berry and bunch sizes, the increase in sugar and acid content, and the variation in berry colour and shape9. These changes are so significant that the phenotypic diversity of the domestic grapevine, including its morphological component, is much greater than that of its wild counterpart8. Several thousand varieties can be distinguished10, and are generally classified in two main groups: table (fruits consumed fresh or dried) and wine grapes.Grapevine has been dramatically modified and diversified since its early domestication. The most notable changes concern: the shift from dioecy in wild grapevines ; wild grapes produce roundish pips with short stalks and cultivated varieties produce more elongated pips with longer stalksV. vinifera subspecies in the archaeological record16. Recent quantifications of pip shape that use outline analyses have helped to improve the discrimination towards the identification of individual varieties22.Various pip measurements have been used to distinguish wild and domesticated 23. Table grapes tend to have large berries, sometimes seedless, and have relatively thin skin while wine grapes are smaller, have higher concentrations of sugar, are generally seeded, and have relatively thick skins24.The study of the whole phenotypic diversity in cultivated varieties reveals that two clear types can be discerned: table and wine varieties27 proposed a comprehensive classification of all the known varieties into three major groups or proles. Proles orientalis is composed of table grape varieties, with big berries, typical of the Near and Middle East and regions of the Mediterranean basin. Proles occidentalis gathers wine grape varieties from Central and Western Europe. They are typically more resistant to low temperatures and have smaller, more acidic berries with lower sugar content. The varieties of Proles pontica  have intermediate characteristics between orientalis and occidentalis. They are mostly used for winemaking or for both table/wine purposes.Negrul30, thousands of SNP markers31, or both32. These studies tend to identify a global structure confirming the three major groups described by Negrul25, as well as an additional group of Iberian varieties, and a number of varieties with admixed/intermediate assignment.In recent years, the global diversity of cultivated grapevines has mostly been studied with the use of nuclear microsatellite markers30 were able to identify Negrul’s three major groups, and a second level with two additional groups: “Iberian Peninsula and Maghreb” and “Table grapes from Italy and central Europe”. In Lacombe’s study29, four distinct groups could be recognized using the same markers on a reduced set of varieties, in which closely related genotypes were excluded. In the fourth group, the Iberian cultivars were found to be associated to wine and table varieties from Asia Minor and the Caucasus. Laucou et al.’s findings31, which were based on an array of 18k single nucleotide polymorphisms (SNP), present a similar organization into four main groups, with the Iberian and Negrul’s groups, however, the majority of varieties were admixed.Using 20 microsatellite markers (SSR), Bacilieri et al.V. vinifera, determined by its predominant use by humans and geographic origin, is a result of the long history of grape domestication and of the spread of viticulture. Negrul’s hypothesis proposes that the main groups of cultivars were domesticated from different populations of wild grapevines25. The existence of secondary domestication events with local wild populations as opposed to mere introgression processes in other areas of the Mediterranean is still discussed, and may have helped shaped regional diversity34. The modern diversity of cultivated grapes stems from thousands of years of selection and diffusion through cuttings and seeds combining spontaneous hybridization and somatic variation31.This global structuring of 35, we decided to use seed outline analysis to explore the structure of the diversity of cultivated varieties across the entire Eurasian and Mediterranean area. Our research aims to establish solid foundations on modern material, to further fuel archaeobotanical studies that can provide insights into past grapevine diversity and viticulture history. We used a representative collection of modern grapevine diversity in the form of a photographic pip shape collection, and we tested the reliability of discriminant models based on shape to infer: (i) destination use, (ii) geographical origins, (iii) conformity in the genetic structure found among varieties. Finally, we applied these models on archaeological remains as a first step into drawing finer-grained, morphological-based inferences about viticulture in the past.Following morphometric analyses of grape leaves which demonstrate a weak correlation between leaf morphology and the East/West origin of grape varietiesThis study includes 434 grapevine modern cultivars Table  ESM. TheFor each cultivar, 30 normally developed berries were randomly collected from a single, fully-ripe bunch. The final dataset comprised 12,346 pips.36. We also included genetic assignation31. These two studies and the present one largely used the same set of varieties. However, because these sets were compiled at different time and with different aims, information may be missing, debated or unknown, for any given variety  where large quantities of well-preserved, waterlogged, grape pips dating back to two time-periods  were found in association with other plant remains.38. The site was occupied at different periods between the Neolithic and the early Middle Ages. The first traces of occupation are very sporadic, but from the end of the Bronze Age onwards, the area appears to undergo continuous changes in land use, mainly oriented towards agricultural and craft activities and including few traces of human habitation. During the second century BCE (late Iron Age or Republican period) a large farmhouse was identified nearby; the remains of its northern boundary were uncovered at Mas de Vignoles XIV. This farmhouse was later replaced by two small farms surrounded by areas devoted to agriculture and animal husbandry. The pips from this period were recovered from a well  and a ditch  that was part of a network of ditches delimiting the farming areas.The site is located in the alluvial plain of the river Vistre and was excavated by INRAP39. The presence of beetles associated with stable areas further reinforce the evidence of animal husbandry. Flax and hemp figure among the potentially cultivated plants, other than grapevine. The Vitis pips investigated come from a well  radiocarbon-dated to the Early Middle Ages: Poz-48697: 1200 ± 30 BP .Archaeological remains from the Middle Ages are not abundant, nor well preserved and include very few traces of human habitation; this suggests that people lived further away, probably due to the unfavourable topography and edaphic conditions (depression area with high water table). The archaeological structures found include several ditches, wells, storage pits, animal enclosures and wooden constructions dedicated to farming activities ; the importance of animal husbandry is suggested by the abundance of cattle remains, which is unusual in a region mainly dominated by sheep/goat, but in agreement with local conditions41.Nine pips from Mas de Vignoles XIV previously delivered aDNA results showing that varieties of different origins may have been cultivated during the Late Iron Age and the Middle Ages43. The number of harmonics was chosen to gather 95% of the total harmonic power43, which corresponds to five for both views. In terms of operator error (e.g. while positioning the pip), this is less harmonics, and thus a conservative choice, compared to previous recommendations of six harmonics for both views22. With four coefficients per harmonic, 40 coefficients were obtained and further used as quantitative variables describing the shape. Pip length was derived from outline coordinates. Pip length was shown to be the best predictor of all other lengths measured on pips20 and here helped to analyse form, that is the shape plus size. When compared to manual measurements obtained in a subset of another study20, error was centred and was, on average, below 1% (~ 1/20 mm). The correlation between the berry and the pip size previously shown20 was here tested on a larger dataset using one-tail Wilcoxon rank tests  by the same operator (TP). Outline coordinates  were extracted from these images and two markers (one at each tip of the pips) were used to normalize the position, size, rotation and first point of the outlines by registering them on “Bookstein coordinates”, that are  and  coordinate points. For each view, elliptical Fourier transforms were used to convert the contour geometry into “Fourier coefficients”. Elliptical Fourier transforms are detailed elsewhere44, with the packages Momocs 1.3.243 for everything morphometrics, MASS 7.3-51.645 for linear discriminant analyses, tidyverse 1.2.146 for general data manipulation and visualization, pvclust 2.2-047 for assessing uncertainties in hierarchical clustering and ape 5.048 for unrooted tree representation.Analyses were performed in R 4.0.2First, a principal component analysis was calculated on the full matrix of coefficients to visualize how each level of each cofactor of interest were located in this synthetic morphological space Fig.  ESM.To test for a costructure between pip form and cofactors, two approaches were used: hierarchical clustering with robustness assessment, and cross-validation using permutational and balanced linear discriminant analyses. To assess geographical structuration the (putative) countries of origin of cultivars were organized in geographical groups Table  ESM.47 with proportions ranging from r = 0.5 to r = 1.4 with a 0.1 increment and 102 resampling with replacement. The “approximately unbiased p-values” were retained49 and presented in Figs. For hierarchical clustering, the averaged coefficients for each level were used to calculate a distance matrix using correlation as the distance method, on which a hierarchical clustering using average (i.e. UPGMA) was calculated. Topologies obtained were presented as unrooted trees Figs. , C ESM. 2 permutations were used50, and each sampled the minimal group size among all groups. The median value was reported on each node in Fig. This method had the merit of simplicity but did not accounted for: (a) variability within groups, (b) overlapping between groups, and (c) unbalanced groups sizes since coefficients were averaged. Moreover, for archaeobotanical inference, we need predictive models along with their performance assessment. We thus combined this approach with linear discriminant analyses LDA). Class accuracies (i.e. tree leaves) were presented as confusion matrices, and class clustering (i.e. tree nodes) were estimated using classes belonging to a node against all others. The index used is accuracy (the proportion of correctly classified pips), using leave-one-out cross-validation. To cope with unbalanced sample sizes, 10A. Class 50. Across the 102 permutations, we calculated the class accuracies and the proportion of the original group sample size retained, as functions of cut-off value for posterior probability  in distinguishing between pips from wild grapevine individuals and those from domesticated varieties. Further inferences about archaeological pips identified as the domesticated type were then obtained using the 102 balanced models previously presented , geographical origins (LDAGeo), the destination and geographical origins jointly (LDAGeo×Use) and genetical grouping . Inferred proportions of each class are presented with three approaches: no filtering . Similarly, large-sized berries had longer pips than those with medium-sized berries .Our results show that berry size in modern grapes positively correlates with pip size Fig.  ESM: varThe first two components of the PCA obtained from the full matrix of coefficients captured 73.2% of the total variance in form. For the sake of clarity, for each variety only the PC1-PC2 centroid was displayed Fig.  ESM. WitAfter discarding mixed destination varieties, the remaining set included 3,106 pips Table  ESM. The100 = 25%).The first geographical model, based on the putative origin of cultivars according to our bibliography, included all regions except NEW WORLD. For each group, 480 pips were included. The resulting tree Fig.  ESM show100 = 29%—Figs. A second geographical model was restricted to “core” historical regions Fig. . In prac100 = 22%—Figs. With fewer groups, one usually expects better accuracies; since this was not the case, we suspected a latent effect. We thus built a third model including the same core geographical regions, and combined them with their destination use Fig. . Due to 30 and SNP31 data was also echoed in pip form. The first model used SNP4 .The second model used microsatellite data (SSR5) and led to similar results. This model used only 120 pips for each permutation and distinguished two groups EAST_Table and ITACE_Table, and WCEUR_Wine + BALK_Wine + IBER_WT. All nodes presented pvclust values > 85 and cv values > 78% . When applied to archaeological material, LDAstatus classified 81% pips (102/128) and 39% (74/204) pips as the wild morphological type for the Late Iron age (LIA) and the Middle Age (MA) phases. These pips were discarded from further analyses and the remaining sample sizes were thus 26 and 130 for LIA and MA phases. Overall, the different filtering approaches led to congruent results  and geographical origins of V. vinifera genealogies, kinships and, more generally, into the intraspecific structuring of the domesticated grapevine53. Genetic markers are direct, sensitive and accurate proxies, but do not provide clear-cut groups within agrobiodiversity since variety amelioration is the product of a continuous and intertwined history and are rarely performed on the large spatio-temporal scale offered by morphometric studies. The two studies where genetic assignation were used demonstrate a high proportion of admixed varieties31.Analyses of genomic sequences brought direct insights into confirming them. Under certain conditions, for example where a strong population structure and divergent selection are present, phenotypical approaches may be superior to molecular ones for measuring agrobiodiversity54.Pip shape, on the other hand, integrates genotypic, developmental and environmental factors. As a phenotypical trait, it is known to be a much more indirect proxy for measuring and uncovering agrobiodiversity structuring. Because morphology is prone to homoplasy, two identical shapes may not be directly genetically related and may instead reflect a potentially mixed signal of ancestry, similar environmental adaptations, as well as non-adaptive natural processes (i.e. drift). Our results are validated by molecular approaches rather than 20. Several other studies based on phenotypic25 and genetic markers31, have already concluded that grape varieties were structured, above all, according to their use as table or wine.Our results show that grapevine pip diversity is significantly structured by use and, to a lesser extent, by the geographical origins of varieties. Use had the best class accuracies; the pips of wine and table grapevine varieties have different form. This was shown in a previous study, which used a smaller set of varietiesMore importantly, our results also established a significant geographical correlation in the shape of pips. Between the two genetic models, the classifications from SNP4 gave better class accuracies for pips than those of SSR5. The fact that SNP4 was calculated using 10,000 SNP markers scattered along each chromosome, bolsters the findings of our study since the SSR5, only used 20 microsatellite markers. Despite having one class less, the SNP4 dataset may be more representative of pip variability since it was trained using more varieties, and thus more pips in permutations.The large-scale structure in pip shape reflects the same blurred boundaries as those reported by genomic analyses. In genetic analyses, unassigned varieties are mostly attributed to human-assisted movement of cultivars across regions and inter-group breeding. In morphologic analysis, however, additional factors may be involved. i.e. environment and development constraints as well as homoplasy. Nevertheless, the morphology-based geographical tree indicates the clustering of eastern groups and western groups, and Italian varieties clustering with eastern ones. The same pattern is found for the tree combining geography and use, where despite a predominant Use structure, the Italian wine varieties are clustered with eastern ones.Interestingly, incorrect assignations may also reveal meaningful information. Misclassified seeds fall primarily into groups that are closely related in terms of use and geographical origin. For instance, in the Geographical confusion matrix, the eastern Mediterranean group is most frequently misclassified with pips of the “Balkans” groups, and vice-versa. On the same confusion matrix, the “EUR-ITAL” group reflects its intermediate nature between western and eastern varieties. For these reasons, retrieving congruent results using shape alone was far from a foregone conclusion.proles classification proposed by Negrul25 was based on morphological criteria and was later confirmed by genetical studies31. These different approaches are largely congruent and provide evidence that grapevine diversity is not only structured, but that its structuring is related to, and likely a product of, the history and the geography of viticulture.Finally, it is worth noting that the 55, as have the covariation between the berry and the pip size and shape20. With regard to geographical origins, we cannot exclude an indirect link with climatic conditions through crossing of varieties from different origins but we see no reason why a particular geographical origin may directly select a particular pip shape. Berry size likely has a correlation with geographical origin because of the relationship between use and geography. Negrul25 already highlighted the ubiquity of large-berry table varieties in the Near-Middle East, that of small-berry wine varieties in Central-Western Europe, and intermediate varieties in his proles pontica . Overall, it is likely that pip shape was not directly implicated in selection and that the subtle changes in its shape are probably neutral. One can thus reasonably hypothesize that any shape change is therefore caused by genetic drift and/or genetic linkage.Dedicated genotype-to-phenotype association studies could help decipher the mechanisms behind such correlation between the shape of pips and the cofactors of destination use and geographical origins. The destination use is directly related to the phenotypic traits and chiefly those of the berry . Berry trait loci have been reported in other studies21.While indirect, both in origin and signature, pip shape is informative about variety origins and use. Shape is also often the only exploitable datum on archaeological remains, and these results are therefore of prime interest to help us better understand grapevine agrobiodiversity through both time and space22 and, more recently, to identify domesticated morphotypes that correlate with modern varieties21. The identification of similar modern varieties amongst a diverse subset can be used to make infraspecific conclusions if their properties are compared to those of the already identified varieties51.Archaeobotanical inference is mostly actualistic: insights obtained from modern material generate inferences for archaeological remains. So far, the shape of pips has been used to distinguish between wild and domesticated types22 increased since then56. Here, we moreover directly infer cofactors of interest without the intermediate identification of the most-resembling modern variety. This is an important result because it means that properties of varieties cultivated in the distant past can be inferred directly from the archaeological remains without having to compare them with modern varieties that may not be appropriate counterparts.In our study, we used a more extensive and more representative collection of modern seeds, the largest worldwide, to the best of our knowledge. The pioneer collection used in Terral et al.51. When cut-off thresholds are selected by the “rule of thumb” method, the trade-off between accuracy and sample size is often forgotten. Our results indicated that this approach should be used sparingly and only when classification accuracies were already proven to be better than classification by chance alone. One should also keep in mind that poorly classified pips may actually be “true” intermediate forms, and if filtered out, may accentuate the contrast between or within assemblages. Finally, when using linear discriminant analyses, particularly when groups have significantly unbalanced sample sizes, the use of permutations is preferable for obtaining better estimates of their accuracies50.To increase the robustness of inferences, predictions are sometimes filtered out based on posterior probabilities41. It is an ideal opportunity to combine aDNA approaches with and morphometrics strike force, although not on the same pips.We applied the models trained on modern material to the Mas de Vignoles XIV assemblages whose palaeogenomic information identifying the relationships between ancient grapes and modern diversity had already been obtained16. It can be assumed that this wild type, which is phenotypically different from modern varieties, corresponds to a part of the diversity amongst historically cultivated varieties rather than being gathered wild berries16.Our morphometric analyses of the Mas de Vignoles XIV assemblage demonstrate notable changes in viticulture between the Late Iron Age and Middle Age. The first one was the decreased prevalence of the wild type, from a predominant to a minority proportion. This decrease was previously observed in the study of several other archaeological sites in southern France20. The SSR4 model, that should be considered more robust than the SSR5 as discussed above, similarly exhibits a contrast between these two historical phases: the predominance of Eastern table varieties in the LIA and of Western wine varieties in the MA. According to aDNA results, the three seeds studied from the LIA sample resemble table grapes as well as Eastern and Iberian wine varieties whereas medieval seeds are more similar to Western European wine varieties41. Although the seeds used were not the same as those of our study, they nevertheless corroborate our observations of a geographical shift towards Western wine varieties.Changes were also observed in domesticated pips. The different models agree on a twofold trend “between” the LIA and the MA: a shift from table to wine varieties, and from eastern to western varieties. These changes are probably two facets of the same shift. The pip lengths are also much smaller for the Middle Age assemblages. This direct measure is congruent with the LDA results as well as those of a previous study, which showed that wine varieties have shorter pips than table ones57, archaeological findings have revealed traces of grapevine cultivation on the outskirts of the city of Nîmes, in the close vicinity of Mas de Vignoles XIV, from the second century BC onwards58. Until recently, it was believed that by the late Iron Age the vines cultivated in the South of France were only intended for wine production59. However, while vineyards appeared to be fairly extensive around the city of Nîmes during the early Roman period, wine cellars and wine production equipment were not very widespread in the excavated settlements58. This observation could be consistent with the hypothesis that parts of vineyards were dedicated to the cultivation of table grapes. These new results allow us to imagine the possibility of a viticulture partly destined for table purposes using eastern varieties rather than native or locally domesticated varieties.It is not possible to prove with certainty that the grape seeds found at Mas de Vignoles XIV came from locally grown grapevines, but its rural location may suggest this. While grapes were traded and could be transported over long distances in Roman timesSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.


To date, however, it remains unclear how the selective effects of OA occur within the context of complex genetic interactions underpinning larval development in many of the most vulnerable taxa. Here we evaluated phenotypic and genetic changes during larval development of Pacific oysters (Crassostrea gigas) reared in ambient (~400 µatm) and high (~1600 µatm) pCO2 conditions, both in domesticated and naturalized “wild” oysters from the Pacific Northwest, USA. Using pooled DNA samples, we determined changes in allele frequencies across larval development, from early “D‐stage” larvae to metamorphosed juveniles (spat), in both groups and environments. Domesticated larvae had ~26% fewer loci with changing allele frequencies across developmental stages and <50% as many loci affected by acidified culture conditions, compared to larvae from wild broodstock. Functional enrichment analyses of genetic markers with significant changes in allele frequency revealed that the structure and function of cellular membranes were disproportionately affected by high pCO2 conditions in both groups. These results indicate the potential for a rapid adaptive response of oyster populations to OA conditions; however, underlying genetic changes associated with larval development differ between these wild and domesticated oyster stocks and influence their adaptive responses to OA conditions.The adaptive capacity of marine calcifiers to ocean acidification (OA) is a topic of great interest to evolutionary biologists and ecologists. Previous studies have provided evidence to suggest that larval resilience to high  In this context, it is apparent that estimates of the adaptive capacity of marine invertebrates to environmental stressors, such as OA, require consideration of both the genetic effects of larval development inherent to that species and population, as well as those attributable to external environmental conditions.Another aspect that complicates detection and interpretation of genetic “outliers” in many bivalve species is that larval survival is characteristically very low, with only a small proportion of individuals surviving from fertilization to the postmetamorphic juvenile (spat) stage (type III survivorship). This high larval mortality is reported to be due to high numbers of negative or deleterious alleles (genetic load) present in both selectively bred and wild populations of many marine invertebrates Plough, ; for exapCO2 conditions. This level of consistent difference across both environments was surprising, given that only ~5 generations of controlled breeding separated these two populations and larval traits were not intentional targets of selection in the domesticated oyster stock.A previous set of experiments compared the relative performance of larval Pacific oysters spawned from domesticated stocks to those of their ancestral naturalized “wild” population in the Pacific Northwest USA  and acidified (~1600 pCO2) seawater conditions. We used pooled sequencing of genomic DNA from larvae and spat sampled at 2 and 22 days postfertilization to test three hypotheses comparing these two populations: (1) Relative differences in overall survival through metamorphosis correspond with different amounts of genetic change; (2) larval development in high pCO2 conditions have a greater genetic effect on wild than domesticated larval populations; and (3) genetic changes in both larval populations in each environment are similar in identity (SNPs in common) and function (genes or Gene Ontology). Collectively, these hypotheses were formulated to investigate the adaptive capacity of C. gigas to OA conditions, against a background of genotype‐dependent mortality in these two populations.In this study, we evaluated the extent to which the genetic composition of larval populations, derived from domesticated and wild broodstocks, changed when reared under ambient , Yaquina Bay, Oregon, to conditioning tanks. Broodstock was provided with flow‐through seawater (~2 L min−1) and a mixed algal diet of 50/50 (by cell concentration) Isochrysis galbana (C‐iso) and Chaetoceros gracilis at approximately 20–30,000 cells ml−1. Water temperature was increased from ambient (11°C) to 20°C over the course of two weeks and maintained at this temperature for 25 additional days to facilitate gonad development prior to spawning.The experiment was conducted in 2015 as part of a broader comparison of the phenotypic effects of high o et al. ) and n =2.2For both MBP and wild broodstock groups, 95 single‐pair matings (1♀ × 1♂) were created from available parents. A total of 19 male and 19 female oysters from MBP families were crossed in a semi‐factorial fashion: One male and one female oyster from each family were individually paired with 4–6 individuals (of opposite sex) from other MBP families with a low coefficient of co‐ancestry  and high pCO2  seawater treatment conditions were created by filling two identical 200‐L tanks with seawater  and equilibrating them overnight via vigorous aeration with outside air. High pCO2 treatment water was then aerated for several hours with a gas mixture of CO2‐stripped air and pure CO2 to result in a final pCO2 concentration of ~1600 μatm. This pCO2 level was selected to approximate naturally occurring concentrations of CO2 experienced at hatcheries on the West coast USA during upwelling events  fitted with a sealing lid, and silicone ring seal . No supplemental aeration was supplied to the larval rearing units throughout the experimental period.Ambient (~400 μatm CO−1 (±0.03), respectively.The pH, temperature, and dissolved oxygen of individual culture chambers were monitored daily: Nominal pH and temperature values were measured with an Orion Star A11 pH meter (Thermo‐Fisher USA) with a Ross Ultra pH/ATC triode, calibrated with NIST buffers, and adjusted with a seawater standard . Dissolved oxygen was measured with a YSI 85 meter . The temperature and dissolved oxygen content of culture units was stable throughout larval rearing, with averages (± standard error) of 24.5°C (±0.15) and 8.15 mg L2) solution, and sealed with a metal crimp cap for later analysis. Samples were analyzed at the laboratory of Dr. Burke Hales at Oregon State University, following the procedure outlined by Hales et al. (2), pCO2, and seawater pH, from which Ωarag and Ωcalc values were calculated. These parameters varied somewhat throughout the larval culture period, but remained reasonably within an acceptable range for each treatment. A summary of seawater carbonate chemistry of the experimental treatments is found in Figure Seawater samples for carbonate analysis were also obtained from seawater used to fill the chambers and after 48 h of culture (before each water change) to account for changes in chemistry arising from off‐gassing or respiration. These samples were stored in gas‐tight 350‐ml amber glass bottles, poisoned with 30 µl of saturated mercuric chloride  or high (~1600 μatm) pCO2 seawater. Each treatment level (broodstock × water type) was replicated five times, for a total of 20 culture units. Water was changed every 48 h by sieving larvae on a mesh screen, filling the culture units with fresh seawater equilibrated to treatment pCO2 levels, and re‐stocking the larvae. Antibiotics were added prophylactically in order to reduce bacterial respiration in culture units that would unduly affect seawater carbonate chemistry  at 20,000 cells ml−1 and increasing the ration by 5000 cells ml−1 day−1. The diatom species Chaetoceros gracilis was gradually incorporated into the diet on day 4, starting at 5% (based on cell concentrations) until it accounted for 50% of the algal diet by day 11, where it was maintained for the duration of the experiment. Larval densities were reduced to 10 ml−1 on day 2, 5 ml−1 on day 6, and 1 ml−1 at the pediveliger stage at day 14. Larval density was reduced randomly, with no selection for larval size, and maintained in this fashion to provide equal and optimal environments for growth and survival and to limit respiratory contributions to seawater pCO2 levels. One ambient pCO2 culture unit suffered complete larval mortality on day 10 but all prior data points and samples were retained for analysis. After the appearance of eyed larvae (indicating a readiness to metamorphose), larvae were screened on a 240 μm sieve to retain pediveliger larvae which were subsequently induced to metamorphose by exposure to 1.8E10−4 M epinephrine for 2 h , beginning with C‐iso  were given unique barcodes and pooled in sets of ~32 samples per sequencing lane. Single‐read, 50 base pair (bp), target‐length sequencing was conducted on an Illumina HiSeq2500 platform in the SNP&SEQ Technology Platform at the National Genomics Infrastructure at Uppsala University, Sweden.From each sample, genomic DNA was extracted using a CTAB extraction method with RNAse treatment detailed by  files for each sample were then converted to tab‐delimited files with read counts of each nucleotide at each locus, and the entire dataset was merged. A custom Python script was used to then remove reads with a sequencing depth of <50 or >1000  and has been uploaded, along with custom scripts, at the github page referenced previously. Briefly, de‐duplexed raw reads were truncated to 36 bp in length and quality filtered to include only reads that had <10 bp with phred quality scores <20. The remaining reads were further filtered for adaptor sequence contaminants using BBDuk  and a kTo compare changes in allele frequencies, we interpreted read counts directly from nucleotide frequencies in each pool (replicate) in the output file of SAMBasecaller.pl. All quality filtering and mapping statistics can be found in Supplementary Information Table . All bio2.82.8.1FST values on a SNP‐by‐SNP basis for the 1288 polymorphisms that were retained from quality filtering (see below). Nucleotide diversity was calculated manually with methods adapted from Begun et al.  test. Allele frequencies of each of the five replicates from MBP and wild larval pools were also analyzed with principal components analysis (PCA) using the “prcomp” function in R , and pairwise n et al. . Pairwis2.8.2(A1:A2)∼βStage+βTrt+(βStage×βTrt) where A1 and A2 refer to the read counts of alternative alleles at each locus and βStage and βTrt represent estimates for developmental stage (2 or 22 dpf) and treatment (seawater pCO2), respectively. Initially, these data were modeled with broodstock type (MBP vs wild) as a third fixed effect (and corresponding interactions) but this resulted in an overabundance of two and three‐way interactions that (1) complicated the interpretation of results and (2) effectively over‐fit variation on many loci that were data poor. Analyzing the effects of development and seawater treatment on genetic changes in larval pools of each broodstock group separately also allowed for a more robust detection of significant change arising from the standing genetic variation of each pool. The model for each locus compared read counts of both alleles (A1/A2) in each of five replicates at each of the four levels of the model (stage (2) × treatment (2)). A visualization of the variance among replicates and populations can be found in Figure p‐values were obtained from the type III sum of squares of the GLM and mean allele frequencies of each treatment level were calculated. p‐values were adjusted with a Benjamini‐Hochberg false discovery rate correction (p < 0.05). All data analyses were conducted in R  had reads present and with a starting minor allele frequency (MAF) in at least one broodstock group of >1%. This left 1288 SNPs for statistical modeling. The read count data for each allele at each remaining locus were analyzed with a binomial generalized linear model (GLM) with the formula: 2.8.3n = 334 markers (~25%) were mapped to linkage groups in this way  by comparing genomic mapping locations to markers on a previously published linkage map  which provided putative functions and gene ontology (GO) terms for 204 of the genes predicted in the annotation. Many genes are assigned with multiple, partially overlapping, GO terms resulting in 483 unique GO IDs for this gene set. The entire gene list, and their associated GO terms, was then analyzed with a gene score resampling procedure (GSR) in ErmineJ  but, instead, incorporates p‐values as a continuous measure of significance and evaluates functional over‐representation of specific GO terms from the entire dataset. We conducted GSR analyses for p‐values from each of the main effects (“Stage” and “Trt”) and those in the overlapping category  but, in this case, no functional categories were over‐represented and so the results were not included.Lastly, for functional analyses, genomic regions within 5kb up and downstream of each mapped locus were searched for gene annotations in the reference genome  which is consistent with recent estimates from wild and farmed stocks of C. gigas . Comparisons of genetic diversity and differentiation can be found in Figure By comparing allele frequencies from day 2 samples from ambient conditions , we found that MBP and wild larval groups were moderately differentiated (mean pairwise 3.2pCO2 seawater (p > 0.05). The proportion of fully formed “normal” D‐hinge larvae at day 2, however, was ~13% lower in both groups cultured in high pCO2 seawater ; Durland et al.  for this study were previously reported by Durland et al. . A detaie Figure ; Table Sd et al. ; Table Sd et al. , owing td et al.  and, thu3.3n = 855) exhibited no significant change in allele frequency owing to larval development or seawater treatment in either MBP or wild larval groups. Using type III sums of squares from GLMs on each locus, we classified the remaining ~34% of significantly changed SNPs (n = 433) into the exclusive categories: Stage , Treatment , Stage and Trt (Stage + Trt), or an interaction between these factors (Stage * Trt). The source of genetic changes in each group  was statistically different between groups X2  = 9.53, p < 2.2 × 10−16) indicating that genetic changes were not similarly attributable to each category in MBP and wild larval populations  than MBP larvae  more so in wild (n = 65) than MBP (n = 7) groups but interactive effects between development and treatment (Stage * Trt) were approximately equally abundant in MBP (n = 53) and wild larvae (n = 56). Among all the loci with significantly affected allele frequencies, there were very few that were shared between the groups  across the genome in both MBP and wild larval groups Figure  and whenr Figure . Loci wir Figure . In thesr Figure .3.4pCO2 treatment (Table n = 2–11 for MBP) and marginally significant p‐values (p > 0.05 in wild) and high multi‐functionality (MF) scores (0.217–0.901) warrant caution in interpreting GO categories.Functional enrichment analyses, although low in power with this dataset, successfully identified several GO terms and processes which were over‐represented by genes near mapped loci that were determined to be significantly affected by developmental stage or seawater pCO2 culture (Trt) were much more specific  and populated by more gene assignments  in MBP larvae, two different types of membrane components in wild larvae (GO:0044425 and GO:0016020), and the GO term “integral component of membranes” (GO:0016021) was shared by both MBP and wild groups as a significant function. In contrast to the broad categorization of functions associated with larval development in ambient conditions (Stage), results for high als File .4pCO2/low Ωarag environments.These results contribute to a growing body of literature examining the genetic changes that oysters and other marine invertebrate species undergo during larval development as well as the potential genetic impacts of ocean acidification. Our findings highlight the complexity of the interaction between genetics, environmental stress, and larval development for these taxa. The disparate patterns of genetic change in domesticated and wild oyster stocks also provide an interesting insight into the potentially broad network of physiological functions that are crucial to successful larval development as well as the more specific set of functions that are affected by high 4.1Larval development was the single most impactful driver of genetic change in both MBP and wild larvae  and genotypic distortions in spat, relative to parental genotypes Plough, . It is dTraditional theories predict that purging of genetic load is favored by periods of inbreeding  on larval pools from wild than MBP broodstock, supporting our second hypothesis. The genetic effects of seawater treatment on larval survival also interacted extensively with larval developmental processes, in both compounding and contrasting ways. In wild larvae, ~1/3 of all SNPs with MAFs affected by high pCO2 seawater were also additively impacted by developmental stage , which also points to the possibility of adaptation to hatchery conditions or reduced genetic load in these selected lines.Similar to the genetic effects of larval development, we found that high pCO2 stress and larval development, there was also an abundance of loci with alleles which exhibited interactive effects between these two processes  reared in low and high pCO2 seawater with no significant difference in survival. In the current study, wild larvae had ~230% more SNPs with altered allele frequencies in response to seawater treatment, suggesting genetic trade‐offs were markedly greater in this population than in larvae from domesticated oyster stocks.It is interesting to note that we detected genetic changes in high r et al. , who obsThe extent to which selection pressures and physiological trade‐offs assist or constrain the long‐term adaptive capacity of marine organisms that inhabit variable environments, such as oysters, remains one of the pressing questions for conservation biology in the era of climate change  started with MAF values of <5% in both groups, indicating that small changes in low variance SNPs were not overtly obscured by our analytical methods. Overall, we find insufficient evidence to conclude that differences in genetic diversity adequately explain the disparate genetic changes associated with larval development in MBP and wild populations.The most unexpected finding from this study was not that there was a quantitative difference in genetic changes between MBP and wild larvae but that these changes were largely unique to each group and category of effect Table . This reAnother possible way to explain the disparate genetic changes that we observe is that pooled DNA samples inaccurately represent the allele frequency of a population when individuals within the sampled pool are unequal in size . This technical artifact may have arisen in day 22 samples where spat were significantly larger than pediveliger larvae. MBP groups had more and larger spat than wild larval populations Figure  and, thuIn addition to differences in genetic diversity between the populations or variance arising from a poolseq approach, it could also be proposed that linkage disequilibrium decay may account for the stark contrasts in the observed genetic changes. With a short‐read RADseq approach, one of the analytical assumptions is that polymorphisms (SNPs) are associated with, or “linked” to, causal genetic variants uniformly across populations. In two distantly related populations, the stochastic processes of meiotic recombination and drift may reduce the frequency of this association, known as linkage disequilibrium decay Baird, . For theWith the inability to directly tie observations of disparate genetic changes to a single causal mechanism, we must allow for the possibility that several of these factors contributed independently to these findings. Even so, we are unable to validate the first part of our third hypothesis—that patterns of genetic change in each group share a similar identity. Determining the biological cause of these dissimilar genetic identities would be valuable in understanding adaptive responses as well as the challenges in using molecular genetic tools to identify consensus effects in populations of marine invertebrates.4.4Although the genetic changes we found were largely specific to each group, one remaining explanation to account for this disparity is that different genetic vulnerabilities were detected which shared a similar functional role. We found that for larval development under ambient conditions, no specific gene ontology categories were uniquely over‐represented with compelling statistical strength Table . Given tpCO2 culture conditions appear to be significantly over‐represented by GO categories concerning aspects of membrane function and structure in both larval groups  effects on allele frequencies at numerous loci, genetic marker‐based approaches for predicting breeding values for larval traits, such as fitness in high None declared.Fig S1‐S6Click here for additional data file.Table S1‐S10Click here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Scientific Reportshttps://doi.org/10.1038/s41598-021-92314-9, published online 17 June 2021Correction to: The original version of this Article contained an error, as the paper has erroneously stated that there were no mtDNA studies conducted on Moors previously. As a result, Reference 9 was omitted and is listed below,Leg. Med. (Tokyo)17, 539–546, https://doi.org/10.1016/j.legalmed.2015.05.007 (2015).Ranasinghe, R., Tennekoon, K. H., Karunanayake, E. H., Lembring, M. & Allen, M. A study of genetic polymorphisms in mitochondrial DNA hypervariable regions I and II of the five major ethnic groups and Vedda population in Sri Lanka. Consequently, in the Introduction,6, Y chromosomal7 and mitochondrial markers8, none has utilized those present on the X chromosome.”“Although a few previous studies that had been conducted to understand the genetic substructure and underlying heterogeneity of Sri Lankan ethnicities using autosomalnow reads:6, Y chromosomal7 and mitochondrial markers8,9, none has utilized those present on the X chromosome.”“Although a few previous studies that had been conducted to understand the genetic substructure and underlying heterogeneity of Sri Lankan ethnicities using autosomalIn the Results and discussion section, under the subheading “Population differentiation among Sri Lankan ethnicities”,8.”“Although there are no mtDNA studies conducted on Moors, research on the other three ethnic groups have reported the existence of a very fine genetic structure with a more closer genetic relationship among Sinhalese and Sri Lankan Tamils, in comparison to that among Sinhalese and Indian Tamilsnow reads:8. In addition, haplotype sharing between Sinhalese and Moors has also been demonstrated9.”“Despite that the number of mt DNA studies conducted on Sri Lankan ethnicities are very limited, the available reports closely agree with our observations and indicated a very fine genetic structure with a more closer genetic relationship among Sinhalese and Sri Lankan Tamils, in comparison to that among Sinhalese and Indian TamilsAll the subsequent references have been renumbered.The original Article has been corrected.


The role of regulatory light chains (RLCs) in cardiac muscle function has been elucidated progressively over the past decade. The RLCs are among the earliest expressed markers during cardiogenesis and persist through adulthood. Failing hearts have shown reduced RLC phosphorylation levels and that restoring baseline levels of RLC phosphorylation is necessary for generating optimal force of muscle contraction. The signalling mechanisms triggering changes in RLC phosphorylation levels during disease progression remain elusive. Uncovering this information may provide insights for better management of heart failure patients. Given the cardiac chamber-specific expression of RLC isoforms, ventricular RLCs have facilitated the identification of mature ventricular cardiomyocytes, opening up possibilities of regenerative medicine. This review consolidates the standing of RLCs in cardiac development and disease and highlights knowledge gaps and potential therapeutic advancements in targeting RLCs. Myosin regulatory light chains (RLCs) play a primary role in striated muscle contraction by regulating the movement of myosin head molecules for cross-bridge formation. The phosphorylation of RLC residues promotes the movement of myosin heads towards the actin filaments. A notable feature of RLCs is that they can be disengaged and reconstituted at their designated locations on the myosin molecule in vitro . Such spThe RLCs of myosin are ~20 kDa proteins present in striated muscles with isoform variability between muscle fibre types. The RLC isoforms have similar structures, but amino acid sequences are only evolutionarily conserved to some extent a, sugges2+ or Mg2+ [2+ ions under the relaxed state of skeletal muscles and is increasingly occupied by Ca2+ ions as its concentration increases in the event of muscle contraction [−1) on the time-scale of contraction (~50 ms for twitch contractions) [The RLC is positioned on the myosin heavy chain (MHC) molecule (myosin molecule) which ends with a globular head b. It is  or Mg2+ ,7,8,9. Ttraction . The sigactions) ,12. Thisactions) ,14. The actions) . Asparagactions) . Howeveractions) . Thus, iThe heart is the first organ to be developed during embryogenesis. The precursor of the heart is the primitive heart tube, from which atria, ventricles, outflow track and inflow track form via major morphological events, namely, cardiac looping and chamber septation . Myosin Even though the sequence of developmental milestones in cardiogenesis remains largely similar in vertebrates, the expression dynamics of RLCs are variable among different species ,28,29,30In situ hybridisation experiments have shown that MLC-2a gene is clearly expressed in the early stages (E8) of murine embryogenesis ,37. By EUnlike in humans and mice, zebrafish express only one type of MLC-2 gene throughout the heart . The losJust as in the murine and zebrafish models, the MLC-2v gene is detected in very early stages (in the anterior part of the cardiac primordia) of avian cardiogenesis and is restricted to the ventricular chambers during development. Again, disruption to MLC-2v gene expression results in abnormal cardiac looping and sarcomeric organisation. The literature seems to suggest there is only one type of MLC-2 gene expressed in birds . From thWe have learned that the ablation of ventricular or atrial MLC-2 results in embryonic lethality, emphasising RLCs’ crucial role in cardiac development. However, loss-of-function studies in animal models have impeded the interrogation of RLCs in adult hearts. Instead, molecular and structural studies on RLCs have revealed that phosphorylation of RLC residues is required for proper cardiac muscle contractility in normal and diseased hearts. The RLC is positioned at the IQ motif (lever arm) of the myosin heavy chain molecules . In cani2+ binding properties. Mutations at close proximity to the phosphorylation sites, A13T, F18L and P95A, decrease muscle energetics  [In humans, there are a number of RLC missense mutations  that are associated with cardiomyopathies ,56,57,58ctivity) . These dctivity) ,61. Likectivity) . Structuctivity) . Cardiacctivity) . Indeed,One mechanism by which RLCs regulate the movement of the myosin head is via phosphorylation. The phosphorylation of RLCs at their serine residues induces negative charges at the myosin head region. As a result myosin heads are repulsed from the positively charged myosin filament backbone region and move towards the actin filaments (shown by electron microscopic studies), facilitating cross-bridge formation for muscle contraction ,65,66. T2+ sensitivity and the rate of cross-bridge kinetics across various species [Studies have shown that the baseline phosphorylation levels of ventricular RLCs are about 0.3–0.5 mol Pi/mol RLC  for optimal cardiac function . The RLC species ,74,75,76 species ,77,78.Structural studies have shown that the phosphorylation of RLCs reduces interfilament spacing (distance between actin and myosin filaments) . This th2+ binding to RLC [RLC phosphorylation levels are significantly reduced in heart failure patients ,83,84. Ag to RLC . Thus, pThe mechanisms regulating RLC phosphorylation remain elusive. There are conflicting reports on RLC phosphorylation being initiated by β-adrenergic stimulation or inhibition ,92. None2+/calmodulin-dependent kinase and is activated during activation of muscle contraction, namely when cystolic Ca2+ ion concentration is high [2+/calmodulin-dependent autoregulatory segments, enabling the binding of RLC to MLCK [2+/calmodulin-dependent, are there other kinases involved in maintaining phosphorylation levels in diseased hearts with compromised contraction?cMLCK is postulated to be a Ca is high . As the  to MLCK . As card to MLCK  until a  to MLCK . The fol2+ sensitisation, muscle contraction, apoptosis) [Studies have shown that other kinases are able to phosphorylate RLCs. Zipper-interacting protein kinase (ZIPK) is ubiquitously expressed in the heart. The RLC is a substrate of ZIPK, as identified by unbiased substrate search . ZIPK phoptosis) ,100. Othoptosis) . Cardiacoptosis) . CaM-depoptosis) . Thus, rIncreasing RLC phosphorylation may be a promising approach towards improving the cardiac function of diseased hearts and rescuing the functional consequences of RLC mutations. Many studies have reported disparate changes of RLC phosphorylation with heart failure progression in humans or animal models. Possible reasons, including biological disparities , should be delved into. Finally, elucidating the complex enzymatic pathways in normal and diseased hearts would open up novel therapeutic avenues based on RLC phosphorylation.The detection and quantification of RLC phosphorylation extracted from cell or tissue samples can be challenging due to the low abundance of phosphoproteins . VariousAt the moment, there are no imaging modalities to directly visualise RLCs in the sarcomere. Immunohistochemistry (IHC) provides the spatial distribution of RLCs in cells or tissues, electron microscopy enables the movement of myosin heads and actomyosin interactions to be visualised and low-angle X-ray diffraction provides dynamic data on contractile protein motions . TechniqBiochemical protein exchange experiment is one of the most powerful experiments involving RLCs because muscle cell contractile function can be monitored before and after the exchange. This technique enables native RLCs in permeabilised muscle fibres to be exchanged with recombinant RLCs with various chemical treatments, primarily involving trifluoperazine . The excThere are useful tools such as in situ hybridisation to locate the longitudinal expression patterns and loss-of-function studies via genetic knockout using oligonucleotides or genetic manipulation to study the role of RLCs in cardiogenesis. As the RLC ventricular isoform is restricted to the ventricles until adulthood, it has the potential to serve as a genetic locus (promoter) for driving ventricle-specific gene expression, as well as a robust marker for ventricular cardiomyocyte selection and enrichment (flow cytometry) and detection (immunohistochemistry) ,114. StuThe selection of pure ventricular cardiomyocytes from hiPSC differentiated cells is useful for improving regenerative-medicine-related treatments such as cardiomyocyte cell patches and cell delivery techniques. Even though RLCs are one of the earliest markers that arise in cardiac development, they are used as a late-stage detection marker in iPSC-derived cardiomyocytes, as these are developmentally less mature than human hearts ,120. ThuTaken together, the findings related to RLCs are derived from in vitro or in situ conditions, thus far. It will be useful to derive methods to study their activities in an in vivo setting for translation to therapeutic purposes.The structure and functional roles of the RLC have been studied for the past five decades , but dis


The identification of pathogenic variants in genes associated with chronic kidney disease can provide patients and nephrologists with actionable information to guide diagnoses and therapeutic plans. However, many nephrologists do not use genetic testing despite costs decreasing over time and more widespread availability.We conducted a survey to uncover the perceptions of general adult nephrologists about the utility of and barriers to genetic testing in clinical practice.The online survey was administered to board-certified nephrologists  in the United States.We analyzed demographic characteristics of the survey respondents and their responses in the context of their use of genetic testing in routine clinical practice.A total of 149 nephrologists completed the survey, with 72% (107 of 149) reporting genetic test use in their practice. On average, tests were ordered for 3.8% of their patient population. Thirty-five percent of responses from nephrologists without a history of genetic test use ranked perceived barriers as “extremely significant” compared with 23% of responses from those who had previously used genetic tests. However, both users and nonusers of genetic tests indicated high cost  and poor availability or lack of ease  of genetic testing as the most significant perceived barriers to implementation.The survey used in this study was not previously validated; additionally, because of the relatively small number of responses, there might have been a selection bias among the responders.Although most nephrologists reported using genetic tests in clinical practice, high costs and poor availability or the lack of ease of use were perceived as the most important barriers to routine adoption. These observations indicate that educational programs that cover a range of topics, from genetics of chronic kidney disease to selection of the test, may help mitigate these barriers and enhance the use of genetic testing in nephrology practice. The identification of mutations in genes associated with chronic kidney disease (CKD) can provide patients and nephrologists with actionable guidance for diagnostics and therapeutic plans. However, many nephrologists do not use genetic testing, despite decreasing costs and widespread availability. Therefore, we conducted a survey to uncover the perceptions of board-certified general adult nephrologists about the utility of, and barriers to genetic testing in clinical practice. Although most nephrologists reported using genetic tests, the costs, availability and ease of use were perceived as the most important barriers to routine adoption. Educational programs covering a range of topics from genetics of CKD to test selection may help mitigate these barriers and enhance the use of genetic testing in nephrology practice.Chronic kidney disease (CKD) is a significant public health challenge, with few curative therapies and a prevalence of ∼15% in the United States.Over 30% of patients with CKD report a family history.The management of genetic and acquired forms of CKD can differ, and determining the underlying cause of CKD using traditional diagnostic tools can be challenging.Historically, the use of genetic testing in patients with CKD was limited by the high costs of DNA sequencing. However, advances in next-generation sequencing have enabled the development of several lower-cost clinical platforms offering simultaneous testing of multiple CKD genes.We developed a 29-question online survey based on previous market research surveys and patient focus groups conducted by Natera, Inc. The survey contained multiple-choice, ranking, and fill-in-the-blank questions . For queThe survey was hosted by SurveyHealthcareGlobus, which used a technology suite with a proprietary healthcare professional panel to identify and administer the survey to potential respondents. The survey was distributed to 10,054 nephrologists in the United States via email . An analysis of questions involving the ranking of barriers and resources was performed by the quantification of the responses using the 5 (significant barrier or extremely important) categories.Descriptive statistical analyses were performed to summarize demographics and the frequency of the survey responses. Comparisons of test ordering habits based on levels of education were analyzed using the Kruskal-Wallis test . However, 60 responses were excluded because of ineligibility (n = 50), incompletion of the survey (n = 9), or invalid responses (n = 1). In a final cohort of 149 respondents (effective response rate of 1.5%), general nephrologists represented 35 states (including the District of Columbia), with the largest proportion of 38.9% (58 of 149) located in the southern region, consistent with the breakdown of US board-certified nephrologists in 2020, as reported by the American Board of Internal Medicine .9 Sixty-Among the 149 respondents  comprising the final cohort, 107 (72%) reported ordering ≥1 genetic test, on average, per month (designated as "users"), whereas 42 (28%) reported ordering an average of 0 genetic tests per month (designated as "nonusers"); Iit is possible that “nonusers” also ordered genetic tests, but at a rate of <1 per month. The median number of genetic tests reported by “users” with a history of ordering was 2 per month (range: 1-100). The average proportion of patients for whom genetic tests were ordered was 3.8% (SD 7.4%). Eighty-three percent (n = 89) of the respondents indicated that they tested ≤5% of their patients, whereas 49% (n = 52) indicated that they tested <1% of their patient population A.Figure P < 0.001)  indicated “extensive”, 72% (n = 108) indicated “limited”, and 7% (n = 10) indicated no prior education in genetics. Comparison of the reported numbers of tests ordered per month, when stratified by each respondent’s level of education in genetics, revealed that nephrologists with “extensive” education in genetics ordered, on average, more genetic tests (20.2 ± 27.7) than those with “limited” (3.1 ± 6.1) or no education  B.The surveyed nephrologists were asked to identify clinical scenarios in which they thought that genetic testing held the most value among the provided categories: (i) all patients with CKD, (ii) patients with CKD of unknown etiology, (iii) patients with specific clinical diagnoses, or (iv) pediatric patients. The majority, regardless of their history of genetic testing use, indicated that genetic testing was the most valuable for patients with specific clinical diagnoses  and for the evaluation of patients with CKD of unknown etiology  on a scale of 1 (least significant) to 5 (most significant) (n = 730 responses). The largest proportion of responses from nonusers were ranked as “5” , whereas the highest proportion of responses from users  scored barriers as “4” on the 5-point scale A. RegardThe nephrologists also ranked the importance of 4 resources that have been proposed to overcome the barriers to genetic testing on a scale of 1 (not important) to 5 (extremely important) (n = 596 responses). All provided resources were considered extremely important, with 46% (273 of 596) of the total responses falling into the “5” category . FurtherGenetic testing has transformed diagnostic, prognostic, and therapeutic approaches to many inherited disorders, including CKD. However, the extent of adoption of genomic medicine practices, including genetic testing, in nephrology remains low.,,COL4A3, COL4A4, or COL4A5 genes, and steroid-resistant nephrotic syndrome, which is associated with variants in over 40 genes.APOL1, which are present in ∼10% of the African American population.Our study revealed that most nephrologists who responded to our survey had a history of using genetic testing (72%) and that they appreciated its value in the management of inherited forms of CKD. The highest perceived utility was observed for CKD of unknown etiology and for cystic and glomerular diseases. Such results suggest that general nephrologists understand the value of genetic testing, especially in clinical contexts that are likely to yield a genetic diagnosis. Therefore, it is expected that an enhanced understanding of relevant genetic factors that contribute to kidney function loss  will foster broader adoption of genetic testing by general nephrologists. For example, genetic testing has been shown to uncover inherited causes in up to 20% of cases of CKD with unknown etiologyOur findings also revealed that 28% of the nephrologists had not used genetic tests in their practice. This value might have grossly underestimated the actual proportion of nonusers of genetic tests, given the low response rate to our survey and the possibility that genetic test users might have been more likely to respond to this survey. These findings are consistent with those of a recent study of attitudes toward the use of genetic testing among nephrology practitioners in Australia.Similar to our study, Jayasinghe et alOur study also identified the lack of ease of the genetic test ordering process as a barrier among nephrologists (by both users and nonusers of genetic tests). The inability to determine the appropriate genetic test was also recently identified as a major barrier to the use of genetic tests by nephrologists.Among the nephrologist characteristics that we stratified for in our analysis (including US region and institution type), genetics education was the only characteristic that yielded significant differences in ordering patterns . DespiteOur survey revealed high interest in educational resources spanning multiple topics, from the genetics underlying CKD to insurance implications. Two recent studies similarly indicated that nephrologists identified discomfort in speaking with patients about the resultsThe current study’s major limitation is that it remains unknown how well our survey responses represent the entire surveyed group of nephrologists. Because of the small sample size and low response rate, it is possible that the survey responses were biased. Users of genetic tests might have been more motivated to respond to the survey, resulting in strongly overweighted sampling toward frequent users of genetic tests. Such a bias may explain the relatively high proportion of patients for whom genetic testing was ordered among all patients seen by individual nephrologists. Despite the potential for bias, this study provides valuable information related to the perceptions about genetic testing among nephrologists with or without a history of genetic test use. Another concern is the accuracy of the answers provided; this is because this survey was not administered directly by research staff and relied on the respondents’ opinions. For example, some responses provided for the number of genetic tests ordered  seem excessively high. Finally, because of the relatively small number of responses, this study was underpowered to allow stratification based on specific responses and subsequent mediation analyses.In conclusion, our survey sheds light on the clinical utility of, advantages of, and perceived barriers to using genetic tests among general nephrologists. These observations may support the development of educational programs that cater for increased appreciation of using genetic tests, which may help with patient management and improved formal practical guidance for the use of genetic testing in nephrology practice.


Vitis vinifera L.), besides being one of the most extensively cultivated fruit trees in the world, is also a fascinating subject for evolutionary studies. The domestication process started in the Near East and the varieties obtained were successively spread and cultivated in different areas. Whether the domestication occurred only once, or whether successive domestication events occurred independently, is a highly debated mystery. Moreover, introgression events, breeding and intense trade in the Mediterranean basin have followed, in the last thousands of years, obfuscating the genetic relationships. Although a succession of studies has been carried out to explore grapevine origin and different evolution models are proposed, an overview of the topic remains pending. We review here the findings obtained in the main phylogenetic and genomic studies proposed in the last two decades, to clarify the fundamental questions regarding where, when and how many times grapevine domestication took place. Finally, we argue that the realization of the pan-genome of grapes could be a useful resource to discover and track the changes which have occurred in the genomes and to improve our understanding about the domestication.Domestication is a process of selection driven by humans, transforming wild progenitors into domesticated crops. The grapevine ( Vitis vinifera L. is one of the most economically important fruit crops in the world. It is used in the global wine industry, covering approximately 7.5 million hectares in 2019 and producing more than 67 million tons of grapes [V. vinifera subsp. sylvestris, and the domesticated one, V. vinifera subsp. vinifera (or sativa). Subspecies sylvestris is considered the progenitor of the subspecies vinifera and, phenotypically, the two subspecies differ in some traits relating to the morphology of flower, seed and leaf, to berry and bunch size , while those of the vinifera are monoicous (hermaphrodite flowers) [sylvestris was neglected for several years, but recent studies have evidenced a great morphological variability and a presumed subdivision in different variants [vinifera, can grow in a wide range of habitats with wide adaptability to different soils, including forested wetlands, along seasonal rivers in closed forests and sand dune shrublands [nch size  and sugaflowers) . A recenflowers) . The subsylvestris and vinifera), based only on fossil seeds, is tricky and the morphological change of grape pips is indicative of consolidated winemaking practice but not informative about the beginning of domestication [The grapevine domestication process occurred in the Near East, in some place along a large area that ranges between Central Asia and the Black Sea . An areatication .vinifera is cultivated in Europe, Asia, Northern America, Southern America, Northern Africa, South Africa, New Zealand and Australia. The domesticated form includes a huge number of varieties, with more than 10,000 cultivars believed to exist in the world [Proles based on the phenotype. Individuals from Central Asia and Caspian Sea were included in the Proles orientalis, the ones from Central and Western Europe in Proles occidentalis, the ones from Eastern Europe, Georgia and Turkey in Proles pontica. P. occidentalis individuals have small berries, high acidity and scarce sugar content. P. orientalis individuals have opposite traits, while P. pontica individuals show intermediate traits, such as scarce acidity and discrete sugar level.The current dispersal area of wild subspecies ranges from Western Europe to Western Himalaya, in areas 900–1000 m above sea level [he world , compliche world  classifiOver the years, in terms of domestication, the necessity to move from a detailed to an overall view arose. For this reason, initially, the researchers included a pool of samples coming from a limited single area of interest, then they involved an increasing number of samples coming from different wine-growing areas, covering large geographic regions ,14,15. TThe study of domestication is a research area that does not only concern how plants were modified by humans in the past but allows us to understand how to design ideal crops for more sustainable agriculture . This asGenerally, domestication occurred in areas of high biodiversity and some authors have proposed that over 160 taxonomic families and 2500 species could have been involved ,22. PlanIf compared with the evolution of species, several changes may have been collected in the domesticated form throughout time, forming an independent monophyletic group. However, unlike speciation in which we can delimit the split between two species at the end of the process, the domestication process can potentially be considered an infinite process. At any time, the human-caused reunion between crops and its wild relatives can trigger the formation of gene flow with the consequent development of introgression events, while an increase of divergent traits can determine reproductive isolation between different forms so that they are no longer able to produce fertile progeny. Moreover, the domestication can revert at any time through a feralisation process in which the crops return to the wild environment.Usually, crops show evidence of genetic bottlenecks that remain well imprinted during initial domestication, but the intensity can vary from crop to crop and additional bottlenecks or gene flow may occur confounding the reconstruction of the domestication process . This caWhether the grapevine was domesticated only once, or whether some varieties were domesticated independently, is a mystery hotly debated and different scenarios are proposed . The maii) from Mount Ararat to Greece through Mesopotamia and Egypt or through Anatolia; (ii) from Greece to Magna Graecia , France (Marseille) and Spain; (iii) from France to the north of Europe [Based on the anthropological condition, historical and ampelographic evidence, additional grapevine domestication centres in Europe were hypothesised . A seconf Europe ,31 , IMIDRA and ICVV repositories (Spain), JKI repository (Germany) [In the last few years, new arrays holding tens of thousands of SNP loci have become available, increasing the capacity to investigate the genetic variability hidden in the genomes. Marrano et al.  array inu et al.  array, cGermany)  and someGermany) ,65,66,67Germany) ,63,66. MGermany) , the secGermany) , while tGermany) . PaschouGermany)  demonstrGermany) ,44) and Although plastid genomes tell one side of the evolutionary history due to the uniparental inheritance, molecular markers are very helpful for exploring the origin of several plants . PrelimiIn phylogeographic studies, findings about origin, conservation and diversity of grapevine are obtained analysing the haplotypes distributed in wild populations collected from different geographical areas. The Caucasus, harbouring the highest haplotype diversity observed in plastid genome, was proposed as the centre of origin of the species, while the Iberian and Italian peninsulas are the result of refugial persistence and accumulation of variation over several ice ages . High leVitis taxa [V. vinifera and other grapes. Some authors have proposed the origin of an ancient Eurasian clade [V. jacquemontii R. Parker could be the sister species of V. vinifera [Unfortunately, the exploration of plastid DNA has only partially resolved the evolutionary origin and domestication of grapevine, probably due to the low number of loci analysed or the slow mutation rate of grape plastomes . Moreovetis taxa ,82,83,84tis taxa . Today, tis taxa ,87. For an clade ,85 and Vvinifera . The ideDomestication is defined as a process by which a species becomes adapted to human use by selecting desirable traits occurring over generations. In this way, one or more populations are separated from their natural ecological context and placed under anthropogenic-driven pressures . General−9 per nucleotide per year previously observed in the Brassicaceae family [−9 mutations per nucleotide per year in grapevine. Although these studies agree with the recent theories that propose a long pre-domestication phase for the crops [Demographic inference methods are available today to analyse the population size changes overtime and genome-wide resequencing methods are applied to infer the population structure and demographic history . Zhou ete family . Mutatioe family ,98. Thuse family  have proe family , Liang ee family  have deche crops ,100, somhe crops . The incVitis genus [Hybridisation is a natural process generally attributed to taxa range changes but in certain circumstances can be imputed to human activity. In recent years, molecular studies have proved that interspecific hybridisation and introgression are central processes in the evolution of is genus ,102,103.is genus . On the sylvestris and vinifera have proven reproductively compatible [sylvestris and vinifera. Zecca et al. [sylvestris. D’Onofrio [Some authors have questioned the existence of a really wild grapevine suggesting that wild plants observed in nature can be feral ,105,106.mpatible ,108. Altmpatible  have shoa et al. , using pa et al. , analysi’Onofrio  showed t’Onofrio , analysisylvestris plants with a significant reduction in gene diversity and heterozygosity of populations in respect to vinifera, is causing a serious demographic decline of wild grapevine [Generally, in genetic studies a preliminary selection based on morphological and ecological traits is conducted to exclude feral in successive analysis thus the real introgression in nature might be particularly intense and involve far more than 10–15% of the samples. Today little is known about the effect of introgression, but the continuing extinction of rapevine ,108,111.rapevine ,114,115.rapevine ,62,116, The increasing accessibility to genomic data and the new tools of evolutionary genomics developed recently are improving the ability to trace the process of admixture. For example, different approaches that integrate population genomics and phylogenetic methods could be used to co-estimate both splits between subspecies and migration events. The TreeMix model , developsylvestris seeds are spherical with a small beak, while the vinifera seeds are pyriform-shaped with a well-developed beak [A limit of the research aimed to investigate the origin of grapevine domestication is, undoubtedly, the interpretation of the past through the study of modern cultivars. An important contribution in dealing with this issue comes from archaeological remains. So far, the most common method used to study the diversity of archaeological pips has been based on morphometric analysis. Wild and domesticated grapevine seeds can be distinguished based on seed shape, contributing greatly to connecting past to present diversity ,122,123.ped beak ,125,126.ped beak , and an ped beak ,131,132 ped beak . Secondlped beak .sylvestris to vinifera in Western Europe, as hypothesised by Arroyo-García et al. [The next-generation sequencing techniques provided a significant boost to aDNA studies. Indeed, short read length makes this new technology ideal for aDNA research, guaranteeing reliable results . Wales ea et al.  and Mylea et al. . AlthougThe first study on nuclear aDNA was published in 2019 by Ramos-Madrigal et al. . The autA further step forward towards understanding the grapevine domestication process has been taken by Bouby et al. , when geAll genetic variations, that ranging from SNPs to large genomic structural variants (SVs) such as duplications, inversions and transpositions, are the result of selection through time. The origin of SVs in plants is relatively poorly understood as are the mechanisms that govern the gene gain and loss, even though many important agronomic traits may be determined by these changes , such asIn the future, comprehensive evolutionary studies need to resequence an extensive number of genomes including wild relatives of crops. Unfortunately, a part of genetic diversity included in the wild grapevine lineages may be lost due to climate changes that occurred in the past or the anthropogenic pressures in the recent times . AccessiVitis genus. Recent studies propose to produce a super-pan-genome for each crop, using at least one de novo assembly from each species [Vitis genus counts around 60–70 taxa widespread throughout Eurasia and Northern America and several of them are inter-fertile [Generally, the pan-genome refers to a full complement of genes of a species, but in grapes the concept could be expanded to the clade that includes the wild species of  species . In this-fertile ,146. Wil-fertile ,150,151.-fertile . We highsylvestris to vinifera aided by human activity have contributed to the domestication processes. Wild populations have had enough time to adapt to local environments and they harbour desirable traits useful for the improvement of the varieties. On the other hand, gene flow from vinifera to sylvestirs has been widely detected and it can have a significant impact on the conservation of wild populations. On the basis of data reported, the introgression is far from uncommon, thus, we propose that a detailed screening of gene flow which occurred between sylvestris and vinifera germplasms should be conducted urgently.Over the last two decades, an increasing number of studies aimed to examine the genetic resources of grapevine and to investigate its ancestries. In this review, we have discussed the main findings focused on answering important issues regarding the origin of the grapevine. Several studies proposed that the main domestication occurred in the southern regions of Caucasus, but recent insight suggests that a primo-domestication centre could be extended to Central Asian countries. In these areas, the grapevine has suffered from a history of protracted cultivation, thus, the pre-domestication phase may have begun earlier than previously thought. Even if these results fit well with the protracted model of crop domestication proposed by Allaby et al. , successToday the grapevine domestication process is a challenge which is far from being completely solved and, though some aspects have found a preliminary answer, some issues remain pending: (1) routes of migration and secondary domestication centres should be verified by specific tests; (2) phylogeography of wild grapevine populations is understood but little is known about time and mode of diffusion in the Mediterranean basin; (3) new tools of evolutionary genomics should be applied to distinguish between domestication and diversification centres.We argue that the realisation of a pan-genome of grapes could be a useful resource to track the change of genes which have occurred during different phases of domestication. In the future, whole-genome resequencing analysis will make it possible to explore a large portion of the variabilities in grapevine and some of the abovementioned pending issues are expected to be solved. However, we are also aware that only by increasing the sampling of landraces and wild populations distributed in remote regions and particularly in Central Asian countries, will it be possible to increase the chance to obtain a complete picture of genetic relationships and go back to the roots of the domestication process.