ST outlier tests. Candidate genes that may underlie differences in adaptation to specific environments and productive systems and phenotypic traits were detected in potentially selected genomic regions.Genetic characterization of local breeds is essential to preserve their genomic variability, to advance conservation policies and to contribute to their promotion and sustainability. Genomic diversity of twenty European local pig breeds and a small sample of Spanish wild pigs was assessed using high density SNP chips. A total of 992 DNA samples were analyzed with the GeneSeek Genomic Profiler (GGP) 70 K HD porcine genotyping chip. Genotype data was employed to compute genetic diversity, population differentiation and structure, genetic distances, linkage disequilibrium and effective population size. Our results point out several breeds, such as Turopolje, Apulo Calabrese, Casertana, Mora Romagnola and Lithuanian indigenous wattle, having the lowest genetic diversity, supported by low heterozygosity and very small effective population size, demonstrating the need of enhanced conservation strategies. Principal components analysis showed the clustering of the individuals of the same breed, with few breeds being clearly isolated from the rest. Several breeds were partially overlapped, suggesting genetic closeness, which was particularly marked in the case of Iberian and Alentejana breeds. Spanish wild boar was also narrowly related to other western populations, in agreement with recurrent admixture between wild and domestic animals. We also searched across the genome for loci under diversifying selection based on F Pig industry is mainly based on a limited number of cosmopolitan lean breeds which have been extensively used for breeding improvement. These highly selected breeds are raised in intensive production systems, focused on maximizing productivity, and supplying the market with fresh pork. Besides, many local, less performing breeds exist, although some of them are nowadays close to extinction. These traditional breeds are usually associated with local forms of pig husbandry and their meat is used for the production of high-quality and niche products. The Iberian pig, raised in the South-West of the Iberian Peninsula, is probably the most representative local pig breed, although many others are reared in European countries. Common characteristics of these breeds are a good environmental adaptation, rusticity, low muscle mass deposition and high adipogenic potential and, in many cases, superior meat quality traits2.Pork is the most widely consumed meat worldwide3.Conservation of small local breeds is mainly conditioned by their economic relevance but also depends on socio-cultural value, adaptation to local agro-climatic conditions, contribution to the development of local communities and marginal areas, and scientific importance. Efficient utilization of local breeds is needed and for this goal, technological advances, growing knowledge and innovative ideas founded on scientific research must be developed. The genetic characterization of these resources is a preliminary step for the development of conservation programs and to boost local breed promotion, and their sustainable use5 or Casertana7, many other autochthonous breeds have not been analyzed in detail yet and are considered untapped genetic resources. Recently, the diversity at several relevant candidate gene polymorphisms has been evaluated in twenty local European pig breeds8, providing information about the segregation of interesting markers for breeding or traceability10 purposes and giving some first insights into the genetic structure of these populations. Nevertheless, genetic characterization of animal breeds is usually addressed with the analysis of neutral markers like microsatellites or SNPs, as recommended by FAO11. High density SNP panels help to investigate genome wide diversity with a higher resolution. Dense SNP panels can be applied to a variety of genomics studies including inference on population history, structure and admixture12, estimation of effective population size5, QTL mapping strategies13 and whole genome association studies14 and genomic selection15. Also, comparative genomic diversity enables us to explore the degree of genomic variation and linkage disequilibrium (LD) among pig breeds. This as well helps to detect genomic regions that have been subject to selective sweeps in different pig populations16.Despite a few whole genome studies have been carried out for some European local pig breeds, such as the Iberianhttps://treasure.kis.si), a multidisciplinary European Union funded project pointing toward the development of sustainable pork chains in several European local pig breeds.In this study, we analyzed genomic diversity of 20 European local pig breeds: Black Slavonian and Turopolje (Croatia), Basque and Gascon (France), Schwäbisch-Hällisches Schwein (Germany), Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano and Sarda , Lithuanian indigenous wattle and Lithuanian White old type (Lithuania), Alentejana and Bísara , Moravka and Swallow-Bellied Mangalitsa (Serbia), Krškopolje pig (Slovenia) and Iberian and Majorcan Black (Spain). The genotyping information from a high density SNP chip has been employed to assess genomic diversity and structure and to identify selection signatures. This work is framed within the TREASURE project  were successfully genotyped with the GeneSeek ® Genomic Profiler (GGP) 70 K HD Porcine chip . The sample sizes of each analyzed population are included in Table 17. The most widely used Illumina 60 K chip was designed and validated using samples from Berkshire, Duroc, Hampshire, Landrace, Large White, Meishan, Pietrain, Synthetic lines (Large White and Piétrain) and wild boar17. The GeneSeek ® GGP Porcine HD Genomic Profiler used in the present study is an improved version of the latter one, which was designed to include the most informative SNPs from the Porcine60K chip, according to findings in the same major commercial breeds, and that was complemented with new SNPs to improve the coverage of the chromosomes and the telomere regions, to better account for recombination18. In spite of the ascertainment bias implicit in the use of SNP chips, previous results have shown that these tools provide reliable estimates of genomic diversity for comparative studies between European populations, even in local breeds19.In general, the informativity of the SNP chip was moderate in most of the studied breeds, which is not unexpected as these local breeds have not been considered for the design of commercial porcine chipsO) and expected (HE) heterozygosity ranged from 0.195 (Turopolje) to 0.363 (Krškopolje) and from 0.187 (Turopolje) to 0.382 (Sarda), respectively  and 0.303 (±0.054), respectively. These heterozygosity values are considerably lower than those reported previously for European cosmopolitan and Chinese pig breeds23, which ranged from 0.30–0.40 to 0.60–0.70 with an average of about 0.5, and similar to those reported for some local breeds19. The lowest heterozygosity values observed in Turopolje are in agreement with a previous study in which microsatellites were employed to assess the genetic diversity and population structure in eight populations corresponding to Balkan pig breeds24. In addition, Lithuanian indigenous wattle shows the smallest (−0.066) inbreeding coefficient of an individual relative to the subpopulation (FIS) while Apulo Calabrese showed the highest one (0.138) . Negative values indicate random mating among the individuals of the subpopulations but do not necessarily imply lower values of the total inbreeding coefficient which takes into account the accumulated inbreeding along the generations25.Genetic variability parameters for the analysed populations are presented in Table 20. The reason for this could be due to the small effective population size: in some of the breeds, only few founders were left at the beginning of the preservation programs2. This can also be the cause for high level of inbreeding observed in some of these breeds. For instance, the highest FIS values obtained in Apulo Calabrese and Casertana are in agreement with their endangered situation and small census27 and similar values were reported recently in an analysis based on candidate gene polymorphisms8.Lack of selection programs and frequent or recurrent admixture, common to these untapped local pig breeds, should presumably lead to a higher degree of genetic diversity. However, the level of genetic variation in our local breeds is in general lower than that in cosmopolitan pig breedsST) for each breed , indicating that most of the genetic variation occurred within populations rather than between breeds, as previously reported for pig populations29. The value obtained is concordant with previous works28, where the averaged FST value calculated between European breeds was 0.134, ranging from 0.021 to 0.209. The overall FST value obtained in the present work  is considerably lower than the one observed in our previous study (0.27)8 performed with an array of selected SNPs. This difference is expected because in the latter study only 39 causal mutations and polymorphisms in candidate genes were used whereas, in the present work, SNP markers across the whole genome and mostly neutral were used6. In addition to this, the distribution of different genetic diversity parameters estimated for each SNP marker is shown in Supplementary Fig. The level of population differentiation can be quantified using the fixation indexes. The fixation Index  to the maximum value of 0.604   was employed to explore the clustering of individuals of different populations. The first three principal components explained 14.26%, 10.92% and 8.49% of the total variation. PCA allowed the visualization of groups formed by individuals belonging to the same breeds Fig. . Moreove8. Next to the Iberian cluster, Spanish Wild Boar and Majorcan Black pigs are also closely grouped, all composing a nucleus of South-Western European populations. A close relatedness between the domestic breeds and the wild relative is observed. This finding is in agreement with the previously proposed recurrent admixture between wild and domesticated animals in Europe33, which might be especially intense in these local breeds, exploited in free-range systems, favoring a long history of genetic exchange with wild boars. The only breed coming from the Iberian Peninsula and being located far away from the south-western cluster is the Bísara breed, for which the separation from Iberian breed had been previously reported12. This is in agreement with its Celtic origin34. Interestingly  Mangalitsa pigs are located in the middle of the western nucleus, quite far away from Moravka (the other Serbian breed), as already evidenced with the NJ tree. Genetic proximity between Hungarian Mangalitsa and Iberian pigs was reported previously by Herrero-Medrano et al.19. Next, four Italian breeds  are clustered in the middle, together with Moravka and Black Slavonian pigs. The right end of the Italian cluster partially overlaps with Bísara and Krškopolje breeds, which are followed by Schwäbisch-Hällisches Schwein and Lithuanian Indigenous wattle, which is in the right end, close but separated from the other Lithuanian breed.Although the individuals are grouped by breeds, there is some overlapping among them, with a big cluster including many breeds. In the left end of this big cluster, Iberian and Alentejana breeds are completely overlapped, in agreement with their genetic closeness and common breeding history35, which may explain its apparently high genetic diversity, despite several bottlenecks occurred since 2003, leading to a critical situation nowadays. The Schwäbisch-Hällisches Schwein breed was originated by crossing local pigs in Württemberg with Chinese pigs, and later by crossbreeding with pigs imported from England2. The creation of Black Slavonian breed, which was a dominant one on the territory of Croatia up to the mid-20th century, resulted from planned crossing between Mangalitsa, Berkshire, Poland China and Large Black  pig37. On the other hand, several breeds have been subjected to crossbreeding with cosmopolitan European breeds carrying Asiatic introgression, such as Krškopolje, which was likely crossed with German Landrace in times when the breed was cast out. Regarding the two Serbian breeds, Moravka was created as a result of unsystematic crossings of the old pig Šumadinka with Berkshire and possibly with Yorkshire38, while no Asian introgression is known for Swallow-Bellied Mangalitsa. This may explain the separation of both breeds observed in the NJ tree and PCA plots, as well as the proximity of Mangalitsa breed to the south-western populations, which are free of Asiatic introgression.Interestingly, the breeds located in the right end of the PCA plots Fig.  are thos40 and detailed information on LD in domesticated animals is important because it is of high utility for fine mapping of genes41. Usually, a substantial extent of LD has been found in domestic species, which may be due to small effective population size in commercial populations. Our study provides an overview of LD patterns against physical distance in 20 European pig breeds and Iberian Wild Boar.Differences in LD among populations result from the differences in population history and demography2 values in distance bins of 0.05, 0.20 and 5.0 Mb for classes a), b) and c), respectively  ranged from 0.305 (Nero Siciliano) to 0.595 (Mora Romagnola); and at long distances (45–50 Mb) r2 values ranged from 0.028 (Iberian) to 0.089 (Casertana). The persistence and strength of LD varied among breeds. Focusing on the domestic breeds, while the LD of Iberian and Alentejana breeds decreased by the half at 0.15 Mb, showing the highest LD decay, the LD of Mora Romagnola and Turopolje decreased by the half at 1.8 and 1.75 Mb, respectively, showing the highest LD persistence. In addition to this, Fig. 2 < 0.2 at distances lower than 2 Mb except Mora Romagnola and Turopolje, which showed r2 < 0.2 at distances lower than 5 Mb. This high level of long LD extent could point out that these breeds have experienced more unbalanced contributions (bottlenecks) or genetic drift compared with the other ones40. Similar r2 values for all the distances were observed for Iberian and Alentejana breeds, supporting the genetic closeness of these breeds already described above. Wild Boar showed the lowest extent of LD in agreement with previous findings28 and as expected for an outbred and non-admixed population. In a previous work44, European pig breeds showed high levels of significant differences in the extent of LD, in agreement with our results.Overall rces Fig. . As expe2 estimates for short distances (0.05 Mb) were averaged by autosomes in all the breeds (Supplementary Table 2 = 0.728) and SSC10 , respectively.Pairwise rances 0.0 Mb were 2 estimates between all pairs within 50 Mb were computed to estimate Ne across 50 generations  and Mora Romagnola and Turopolje were the breeds with the lowest Ne values . Table e (89.18), and Casertana the lowest value (9.44).Synteny rons Fig. –26 using46 recommended an effective population size of 100 in order to maintain the genetic diversity of a population, which is not accomplished in any of the populations analysed in the present work. Our findings further confirm the need for conservation strategies for many of the studied breeds. The most extreme cases are Casertana, Apulo Calabrese, Turopolje, Mora Romagnola and both Lithuanian pig breeds, for which conservation efforts are currently being undertaken49.MeuwissenIn fact, most local breeds are characterized by having small effective population sizes, which affects their diversity, and leads to high levels of LD and a high proportion of SNPs with fixed alleles. In general, breeds showing the highest levels of LD were those with the lowest effective population size and higher inbreeding .ST index was used in order to detect genomic regions that could be involved in domestication, breed pattern establishment or selective breeding. A genome-wide scan of divergent genomic regions was carried out through the estimation of Wright’s FST at each marker as a measure of genetic differentiation. Candidate regions to diversifying selection were identified as those in the 99th percentile of the empirical distributions of sliding windows , lipid, carbohydrate and protein metabolism , growth and development , cellular homeostasis (ATG5), locomotor behavior  and response to nutrient (BCKDHB) were identified. The region located on SSC8 (100.93–101.74) overlapped in seven breeds  and in Wild Boar and contained genes involved in growth and reproduction. The breeds with the highest number of overlapped regions (with five or more breeds) were Alentejana and Iberian in addition to Wild Boar (12 regions), whereas the breed with the lowest number was Mora Romagnola, in agreement with a specific genetic differentiation in this breed, also highlighted in the PCA analyses.A total of 19 genomic regions overlapped in five or more pig groups (breeds and/or wild boars) on chromosomes SSC1, SSC2, SSC6, SSC7, SSC8 and SSC13 , Gascon (SSC13), Iberian (SSC15), Lithuanian Indigenous wattle (SSC4), Swallow-Bellied Mangalitsa (SSC2) and Mora Romagnola (SSC1). This is in agreement with the large repertoire of functional olfactory receptor genes in pigs, the fast evolution of these genesACOX1 and CPT1A, both involved in fatty acid metabolism are identified. NFKBIA and PPARGC1B which have both been associated with backfat thickness in pigs53 are detected in Krškopolje and Cinta Senese breeds, respectively. DECR1, a positional candidate gene for the first fatness QTL detected in pigs on SSC455, is observed in a potentially selected region in Gascon breed. DLK1, a gene with a fundamental role in muscle growth and fat deposition56, is detected in Majorcan Black. LPIN1, which has been associated with obese pig phenotypes57, is detected in Turopolje breed. Taste receptor genes play a fundamental role in survival through the identification of dietary nutrients or potentially toxic substances, being linked to eating behavior and adaptation to specific geographical locations and diets58 and potentially related to growth and fat deposition59. Among this gene family, the TAS2R16 gene was detected in a selection signature in the Turopolje breed. Genes involved in the endocrine regulation of growth and insulin signalling, are also observed: IRS1 is detected in Casertana; GAL and GALR2 in Basque, and members of the insulin like growth factor binding protein gene family are detected in Black Slavonian. These genes code for signalling molecules that integrate and coordinate numerous biologically key extracellular signals within the cell. Some of them are intermediate of the insulin signalling, with a key role in growth, fatness and energy homeostasis61.Also, candidate genes involved in relevant metabolic functions associated with the adipogenic phenotype of our breeds are identified within the putative selected regions. For instance, in the Basque breed, the genes HEDTC2 and IDE in Alentejana; USP54 in Casertana; CAPN10, RNPEPL1 in Iberian; PSMA6 in Krškopolje, CTSV in Turopolje, or UBE4B, RNF150 and UBE2E1 detected in 5 or more breeds simultaneously  is located very close to PPARD gene (31.22–31.29 cM), related to ear morphology, fat deposition and growth, and detected previously as being located in a differentiated genomic region between European breeds and Wild Boar64.Regarding Wild Boar-specific selection signatures, only three regions were detected. Two of them are located in SSC7, an autosome that has been repeatedly shown to be associated with domestication and behavior-related traits in QTL and GWAS studies64. In these studies, as well as in the present one, genes mapped to these regions can be considered as candidates under selection in pig breeds. Some common biological functions have been detected in different works, such as olfaction, growth or muscle development70. Nevertheless, when comparing different works, variable regions and genes have been observed probably due to differences in the breeds analyzed, statistical methods, SNP density or sample size. Especially, domestic pigs under different evolution and production conditions show different selection signatures and in our case all tested populations are locally produced breeds, which have not been selected for lean meat content, muscularity or enhanced reproduction. Thus, in a differentiation analysis among those breeds, expected signatures may be weaker than those observed in commercial and highly selected genotypes, or more related to domestication and breed standards establishment than actual artificial selection processes.In previous works analyzing different commercial and traditional pig populations, a number of regions showing between-breed signatures of selection has been identifiedThe obtained results were useful for the characterization of the genomic diversity of autochthonous European pig breeds. Results highlighted the genetic closeness among several of these domesticated breeds, and with their wild ancestor, as well as clear differentiation of some other ones and confirm the need of conservation programs to protect their genetic pools. Linkage disequilibrium patterns and extent have been determined at the genome level for a wide repertoire of European traditional breeds, showing potential effects of admixture and inbreeding. Putative signals of selection were detected for regions containing genes involved in growth, muscle development, reproduction, metabolism, behavior and sensory perception. Our findings improve the knowledge about the genome biology of European local pig breeds, and provide candidate genes for relevant traits, as well as useful information for future conservation, association or selection approaches.Experts of each country, including personnel from breeding organizations and herd books, selected the animals to be included in the analyses in order to get a representative sampling of each breed. Selection of individuals for sampling was performed by avoiding highly related animals , balancing between sexes and prioritizing adult individuals or at least animals with adult morphology. Blood samples were obtained from 39 to 54 individuals from each one of the 20 local pig breeds included in the study: Black Slavonian and Turopolje (Croatia), Basque and Gascon (France), Schwäbisch-Hällisches Schwein (Germany), Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano and Sarda , Lithuanian indigenous wattle and Lithuanian White old type (Lithuania), Alentejana and Bísara , Moravka and Swallow-Bellied Mangalitsa (Serbia), Krškopolje pig (Slovenia) and Iberian and Majorcan Black (Spain). Besides, seven European wild boars were employed as outgroup. Specialized professionals from each institution that provided animal material obtained blood samples following standard routine monitoring procedures and guidelines, at farm or at slaughter. No procedures with animals were performed that would demand ethical protocols according to Directive 2010/63/EU (2010) and blood samples were obtained as a general breeding procedure or previously collected DNA only reused here. A total of 992 DNA samples were genotyped.71.The genomic DNA was extracted from leukocytes present in 8–15 mL of peripheral blood, collected in Vacutainer tubes containing 10% 0.5 M EDTA  at pH 8.0. The extraction was performed using either a standardized phenol-chloroform, high-salt method or a commercial kitSamples were genotyped with the GeneSeek ® GGP Porcine HD Genomic Profiler v1 , which includes 68,516 SNPs evenly distributed with a median of 25 kb gap spacing and sharing 42,372 markers with Illumina porcine SNP60 chip. The average genotyping call rate was 0.94.72. SNPs with MAF lower than 0.01 or more than 10% missing genotypes were excluded from the analyses in a preliminary filter to inspect the distribution of MAF across the genotyped SNPs. In addition, an individual call rate threshold was set to 95% and ten samples  were removed for further analyses.Genotype quality control (QC) and data filtering were performed using PLINKO) and expected heterozygosity (HE), inbreeding coefficient of an individual (I) relative to the subpopulation (S) (FIS), fixation index (FST) and inbreeding coefficient of an individual (I) relative to the total (T) population (FIT), heterozygosity index based on observed heterozygosity in individuals within breeds (HI) based on expected heterozygosity in subpopulation (HS) and based on expected heterozygosity for overall breeds (HT). Calculations were made with VCFtools software73. Nei’s genetic distances12 were calculated in R74 environment. Pairwise distances were used to build a NJ tree with the nj function belonging to the ape75 library in R. In addition, population structure was inspected trough PCA analyses performed with DISSECT software tool76.A total of 51,219 SNPs mapped on the 18 autosomes on Sus Scrofa 11.1 were used to compute, for the 20 studied breeds, the following indicators of genetic diversity: observed (H−7) and unmapped or mapped in sexual chromosomes were excluded from LD analysis. This QC filtering was carried out independently for each breed and the number of SNP used is showed in Supplementary Table 2 was estimated for all marker pairs less than 50 Mb for each population and autosome independently using PLINK. To plot LD vs physical distance between markers, following Saura et al.4, we divided SNP pairs into three distance classes, (a) 0 to 2 Mb, (b) 2 to 5 Mb and (c) 5 to 50 Mb. Distance bins of 0.05, 0.20 and 5.0 Mb were used for classes (a), (b) and (c), respectively, and average r2 values for each bin were plotted against physical distance. Sample sizes were similar for all the breeds except for Wild Boar. As sample size can have an influence on LD estimation, a correction for sampling size was used to estimate r2 in Wild Boar as follows: (r2 − 1/N) (1 − 1/N)77.Markers with MAF lower than 0.05, with more than a 10% of missing values, significantly deviating from Hardy-Weinberg equilibrium (P < 8.27 × 10e) for each population were computed using the relationship between LD and Ne according to the following equation78:c is the distance between SNPs (Morgans), Ne is the sample size and absence of mutation was assumed  in at least one of the studied breeds were removed. A total of 828 SNPs was extracted from the subset of 51,219 SNPs used to compute genetic diversity parameters. Pairwise Wright’s FST81 were estimated as a measure of genetic differentiation according to the method described in Wilkinson et al.64. For each breed, a total of 21 breed-pairwise comparisons at each SNP were obtained and they were averaged to get overall FST for each SNP per breed. FST values were averaged in sliding windows of 13 SNPs centred at the 7th SNP which determined the genomic location of the window. Regions in the 99th percentile of the empirical distributions of windows per breed were defined as candidate regions to genetic differentiation. Genes were annotated with Biomart tool82.Hardy-Weinberg equilibrium was inspected individually in all breeds. SNPs significantly deviating from Hardy-Weinberg equilibrium (P < 8.27 × 10Supplementary material 1Supplementary material 2Supplementary material 3Supplementary material 4


In France, implementation of genomic evaluations in dairy cattle breeds started in 2009 and this has modified the breeding schemes drastically. In this context, the goal of our study was to understand the impact of genomic selection on the genetic diversity of bulls from three French dairy cattle breeds born between 2005 and 2015  and the factors that are involved.We compared annual genetic gains, inbreeding rates based on runs of homozygosity (ROH) and pedigree data, and mean ROH length within breeds, before and after the implementation of genomic selection.Genomic selection induced an increase in mean annual genetic gains of 50, 71 and 33% for Montbéliarde, Normande and Holstein bulls, respectively, and in parallel, the generation intervals were reduced by a factor of 1.7, 1.9 and 2, respectively. We found no significant change in inbreeding rate for the two national breeds, Montbéliarde and Normande, and a significant increase in inbreeding rate for the Holstein international breed, which is now as high as 0.55% per year based on ROH and 0.49% per year based on pedigree data . The mean ROH length was longer for bulls from the Holstein breed than for those from the other two breeds.With the implementation of genomic selection, the annual genetic gain increased for bulls from the three major French dairy cattle breeds. At the same time, the annual loss of genetic diversity increased for Holstein bulls, possibly because of the massive use of a few elite bulls in this breed, but not for Montbéliarde and Normande bulls. The increase in mean ROH length in Holstein may reflect the occurrence of recent inbreeding. New strategies in breeding schemes, such as female donor stations and embryo transfer, and recent implementation of genomic evaluations in small regional breeds should be studied carefully in order to ensure the sustainability of breeding schemes in the future. In France, genomic evaluations started to be implemented in dairy cattle breeds in 2009. After a brief transition period for the breeding companies to adopt this technology, these new evaluation methods have drastically changed dairy cattle breeding schemes since 2011. Genomic evaluations have allowed breeding companies to increase the number of male candidates for selection and the use of new bulls . In factHowever, since animals with high genomic estimated breeding values (GEBV) can be selected as parents at a very young age, generation intervals are significantly shortened . As a reSuch an increase in inbreeding rate results in a loss of genetic diversity, with lower additive genetic variance leading to a lower response to selection . MoreoveMeasuring genetic diversity is necessary in order to manage it. Typically, genetic diversity is estimated and managed with the help of inbreeding and kinship coefficients based on pedigree data e.g. , 18). Th. Th18]).Falling costs in the field of high-throughput genotyping and sequencing make it possible to obtain more and more genotyping data, and thus to estimate genetic diversity more precisely. These data allow the computation of inbreeding coefficients and genomic relationship matrices (GRM) , 22. TheAnother method to estimate genetic diversity consists in detecting IBD segments, called runs of homozygosity (ROH), by considering that segments of consecutive homozygous SNPs originate from common ancestors , 24. ThuPedigree data are useful to evaluate genetic diversity for individuals that are not genotyped, whereas measures based on molecular data (such as ROH) allow the study of the realized proportion of genome that is autozygous and the pattern of inbreeding along the genome. In spite of their advantage over pedigree-based inbreeding estimates, genomic-based estimates began to be used to manage genetic diversity in selection programs only recently –35.In this context, the goal of our study was to understand the impact of GS on genetic diversity in three French dairy cattle breeds: Montbéliarde and Normande, two national breeds, and Holstein, an international breed. We measured the relative changes in rates of genetic merit and in three different estimates of genetic diversity: (i) pedigree-based inbreeding and kinship, (ii) ROH-based inbreeding coefficients, and (iii) mean length of ROH; and these estimates were taken before and after the implementation of GS in these three breeds in France. Based on our findings, we compare pedigree and ROH-based inbreeding rates within breeds and explore the changes in breeding schemes that could explain the differences observed between national and international breeds.We used three datasets that consisted of all progeny-tested bulls born between 2005 and 2010 and all marketed bulls born between 2012 and 2015 from three French dairy cattle breeds: 1307 Montbéliarde bulls and 1016 Normande bulls , and 4694 Holstein bulls  . Quality control filtering was performed by removing SNPs that were genotyped in less than 95% individuals and SNPs with a minor allele frequency (MAF) lower than 1%. No individual had less than 95% genotyped SNPs. After quality control filtering, 39,992, 40,135 and 41,377 SNPs remained in the datasets for Montbéliarde, Normande and Holstein, with on average one SNP every 62.5 ± 66.5 kb, 62.2 ± 66.4 kb and 60.4 ± 63.8 kb, respectively.All the bulls in the datasets were genotyped for 50 K SNPs (Illumina InfiniumIndex Synthèse Unique) is an individual index that combines production, functional and type traits, which are weighted depending on the breed and subsequent breeding goals. The formula to obtain total genetic merit index ISU is built in order to obtain a standard error of the index of 20 points within each breed  and 59%  for Montbéliarde, Normande and Holstein, respectively.Coefficients of variation of pedigree-based inbreeding were equal to 0.33, 0.25 and 0.31 for Montbéliarde, Normande and Holstein, respectively. Coefficients of variation of ROH-based inbreeding were equal to 0.22, 0.18 and 0.21 for Montbéliarde, Normande and Holstein, respectively.The mean difference see Fig. .Fig. 4PaA histogram of the proportion of the total number of offspring per bull for each breed and selection type is available in Additional file The effective number of bulls used was significantly larger between 2005 and 2010 (under PTS) than between 2012 and 2014 (under GS) in Normande and Holstein, but not in Montbéliarde see Fig. . The ratsee Fig. .Fig. 6Efi.e. it is an international breed), whereas Montbéliarde and Normande are French typical breeds managed at the country level . Thus, the results drawn from our study can compare these two situations. To conduct this study, we focused on bulls of these three breeds born between 2005 and 2010 for PTS, and bulls born between 2012 and 2015 for GS.In dairy cattle breeds, GS was expected to increase the annual genetic gain by up to 70 to 108% depending on the scenarios used for the breeding schemes (with different proportions of young genotypes bulls and proven bulls used) , 44 but An increase in annual genetic gain with GS has been observed in most dairy cattle populations. For instance, in the US Holstein cattle breed, the annual genetic gain for milk, fat and protein yield in cows born before or after 2010 has increased by 71, 111 and 81%, respectively, between 2008 and 2014 , 47. ThiWe obtained similar results for Montbéliarde, Normande and Holstein bulls. Their mean annual genetic gains (ΔG) increased by 50, 71 and 33%, respectively, after implementation of GS. At the same time, the bulls’ parental generation intervals were reduced by a factor 1.7, 1.9 and 2 for the Montbéliarde, Normande and Holstein breeds, respectively, suggesting that, similar to the US Holstein situation, most of the extra genetic gain is due to reduced generation intervals. However, there are differences between breeds in terms of increase in annual genetic gain and decrease in generation interval, with the Holstein breed having less increase in gain relative to the reduction in generation interval. This may be due to different formulas used for total merit indices and different breeding goals.Given this global increase in genetic gain for all the breeds studied here along with the decrease in generation interval, one might be concerned about the trend in genetic diversity. Indeed, on the one hand, an accelerated decrease in genetic diversity could be expected due to the decrease in generation interval. On the other hand, genomic evaluations can lead to a more diverse offer in terms of candidates for selection, and thus a moderation of inbreeding rate over time . To studIn all three breeds, the ratio between the effective and actual number of bulls (Ne/N) increased after implementation of GS. This reflects changes in breeding schemes in all three breeds, which should allow for a decrease in inbreeding rate and more generally for a slowdown in the annual loss of genetic diversity. However, we distinguished two contrasting types of behavior of inbreeding based on ROH. First, for the two national breeds, Montbéliarde and Normande, we did not detect any significant change in the annual inbreeding rate (ΔF). Second, for the Holstein international breed there was a significant increase in ΔF, which has increased from 0.080% to 0.55% per year based on ROH, and from 0.088% to 0.49% per year based on pedigree. This result does not align with the fact that all three breeds experienced a significant increase in annual genetic gain, with the lowest increase observed in Holstein while the inbreeding rate in Holstein was highest. This result suggests that changes in breeding schemes reflected by an increase of Ne/N of bulls in Holstein did not allow for a slowdown in the annual loss of genetic diversity.For Holstein, ΔF per generation also increased from 0.39% to 1.36% based on ROH, and from around 0.46% to 1.39% based on pedigree. Thus, the decrease in generation interval did not explain all the increase in ΔF per year for Holstein, since ΔF per generation increased too. The observed inbreeding rate in Holstein is higher than the 1% per generation acceptable rate of inbreeding according to FAO guidelines . Since tWe used a linear regression to estimate the rate of ROH-based inbreeding because it gave better results than a logarithmic regression in terms of residuals of the model  was significantly shorter than that for Normande (1.71 Mb), which in turn was significantly shorter than that for Holstein (1.80 Mb). The length of ROH depends on two main criteria, for given detection parameters and genotyping density: (i) the effective population size and the level of linkage disequilibrium ; and (iiEstimated effective population sizes (Ne) of French dairy cattle breeds were equal to 79 for Montbéliarde, 87 for Normande and 96 for Holstein in 2017 . Based oAfter the introduction of genomic selection, the annual genetic gain increased based on estimated breeding values for bulls of three large French dairy cattle breeds: Montbéliarde, Normande and Holstein. At the same time, the inbreeding rate per year and per generation increased in Holstein bulls, an international breed, but not in Montbéliarde and Normande bulls, two national breeds. Small effective population sizes resulted in highly structured genomes with long haploblocks, particularly in Holstein, as a consequence of the low effective/census number of bulls ratios. A joint increase in genetic gain and conservation of genetic diversity is possible by (i) applying optimal contribution selection; (ii) carrying out on-farm mating plans; and (iii) ensuring that these tools are flexible enough to be used effectively in the field. Our study highlights the fact that changes in breeding schemes can impact genetic diversity, and thus, it is important to continue monitoring genetic diversity and to study the future impact of different breeding schemes strategies on genetic diversity. The recent implementation of genomic evaluations in small regional breeds, for which effective population size can be limited, should be studied carefully in order to ensure sustainability of breeding schemes in the future, in terms of both genetic gain and diversity. The spread of new technologies in breeding schemes, such as female reproductive technologies and embryo transfer that might increase global selection intensity through the dams of bulls, represents a risk in terms of genetic diversity that should be anticipated in the light of our conclusions.Additional file 1: Table S1. Description of the three studied dairy cattle breeds: Montbéliarde, Normande and Holstein.Additional file 2: Table S2. Length of the autosomal genome in three French dairy cattle breeds. Length of autosomal genome on which ROH can be detected corresponds to the length of the autosomal genome covered by SNPs after withholding gaps longer than 150 kb between two SNPs. The proportion of autosomal genome on which ROH can be detected is the length of autosomal genome on which ROH can be detected divided by the total length of the autosomal genome (from Bos taurus assembly 3.1.1).Additional file 3: Table S3. Parental generation intervals. Mean parental generation intervals for bulls born between 2005 and for bulls born between 2010 and 2015 in months and in years, for Montbéliarde, Normande and Holstein.Additional file 4: Figure S1. Square root of standardized residuals for the linear regression of pedigree-based inbreeding depending on birth year (Y5) and selection type (Gen) for Montbéliarde, Normande and Holstein (from left to right). Figure S2. Square root of standardized residuals for the linear regression of pedigree-based inbreeding for the last five generations depending on birth year (Y5) and selection type (Gen) for Montbéliarde, Normande and Holstein (from left to right). Figure S3. Square root of standardized residuals for the linear regression of pedigree-based kinship depending on birth year (Y5) and selection type (Gen) for Montbéliarde, Normande and Holstein (from left to right). Figure S4. Square root of standardized residuals for the linear regression of FROH, inbreeding based on ROH depending on birth year (Y5) and selection type (Gen) for Montbéliarde, Normande and Holstein (from left to right). Figure S5. Square root of standardized residuals for the logarithmic regression of FROH, inbreeding based on ROH depending on birth year (Y5) and selection type (Gen) for Montbéliarde, Normande and Holstein (from left to right) ). Figure S6. Square root of standardized residuals for the linear regression of the mean length of ROH depending on birth year (Y5) and selection type (Gen) for Montbéliarde, Normande and Holstein (from left to right). Figure S7. Square root of standardized residuals for the linear regression of total merit index (ISU) depending on birth year (Y5) and selection type (Gen) for Montbéliarde, Normande and Holstein (from left to right).Additional file 5: Table S4. Pedigree-based inbreeding calculated from the last five generations in percent per year. Table S5. Estimation of the slopes of pedigree-based inbreeding calculated from the last five generations in percent per generation.Additional file 6: Figure S8. Proportion of the total number of offspring per bull for each breed and selection type. Progeny testing selection corresponds to bulls born between 2005 and 2010 and genomic selection to bulls born between 2012 and 2014.Additional file 7: Table S6. Number of bulls and offspring for each birth period in the dataset.Additional file 8: Table S7. ROH categories lengths thresholds.Additional file 9: Figure S9. Confidence intervals of the number of ROH per ROH length category per selection type for Montbéliarde bulls. Figure S10. Confidence intervals of the number of ROH per ROH length category per selection type for Normande bulls. Figure S11. Confidence intervals of the number of ROH per ROH length category per selection type for Holstein bulls.


Larimichthys polyactis) is a commercially important fish species with high gene flow and its overwintering populations experience heterogeneous environment, suggesting possible population differentiation and adaptive divergence. To delineate patterns of population structure as well as test for signatures of local adaptation, a total of 68,666 quality filtered SNP markers were identified for 80 individuals from four overwintering populations by using restriction site-associated DNA sequencing (RAD-seq). Significant genetic differentiation among overwintering populations from the Central Yellow Sea, the South Yellow Sea and the North East China Sea were detected (Pair-wise FST: 0.00036–0.00390), which were consistent with population division of overwintering groups inferred from traditional ecological approaches. In addition, a total of 126 unique SNPs were detected to be significantly associated with environmental parameters . These candidate SNPs were involved in multiple pathways such as energy metabolism and phagocytosis, suggesting they may play key roles in growth and innate immunity. Our results suggested the existence of hitherto unrecognized cryptic population structure and local adaptation in this high gene flow marine fish and thus gain new insights into the design of management strategies.Exploring factors shaping genetic structure of marine fish is challenging due to fewer barriers to gene flow in the ocean. However, genome-wide sequence data can greatly enhance our ability to delineate previously unidentified population structure as well as potential adaptive divergence. The small yellow croaker ( Clupea harengus) in the Baltic Sea and detected genomic regions associated with temperature and salinity related natural selection. Engraulis encrasicolus) from Atlantic and Mediterranean, which provides comprehensive insights into the understanding of fine-scale genetic structuring in European anchovy and thus improves stock management and conservation actions. Additionally, Anguilla rostrata) and outlier loci detected were found to be significantly associated with environmental variables such as latitude, longitude as well as temperature.Knowledge on the ecological and evolutionary processes influencing biodiversity and dispersal of marine organisms is fundamental to the understanding of their genetic distribution patterns and provides an explicit framework for conservation and management of resource species . PopulatLarimichthys polyactis) is a benthopelagic fish of the family Sciaenidae, widely distributed in northwestern Pacific Ocean, including the Bohai Sea, the Yellow Sea and the East China Sea (FishBase 2014). L. polyactis is a multiple spawner with seasonal asynchronous migrations L. polyacervation .L. polyactis at different spatial scales. However, earlier studies based on microsatellite markers only detected several loci under directional selection in L. polyactis  were used for RAD genotyping.This study focuses on overwintering populations of olyactis , fish spolyactis ; Fig. 1.Eco RI. RAD libraries were constructed using pools of 10–12 individually indexed individuals and DNA fragment of 200–400 bp was isolated and purified. The libraries were enriched by high-fidelity PCR prior to paired-end sequencing from the two lanes of Illumina HiSeq4000 at Novogene in Beijing, resulting in read lengths of 150 bp. Sequence reads from the Illumina runs were sorted according to index sequences (six nucleotides in length and each differed by at least two bases) for individual tracking. To avoid reads with artificial bias, the process_radtags program (Eco RI motif (AATTC). Final read length of the first read (containing the restriction-site) was trimmed to 125 nucleotides to avoid the potential sequencing errors at the tails of the sequences  as the DAPC without prior did not reveal any genetic structure (number of genetic clusters inferred was K = 1). For comparison, principal component analysis (PCA) colored by sampling population was also conducted to illustrate the results of population structure. Moreover, we assessed the distribution of genetic variation across samples by using a Bayesian model-based clustering program ADMIXTURE v1.23 , until we had the optimal number of clusters by plotting the natural probability of four estimators  and the K value was determined where the estimators plateaued as described in K for individual estimator and graphical representations of the results were generated by integrating the CLUMPAK program  to control evolutionary history in the calculation of XTX for individual SNP (using 2 × 105 iterations of the MCMC)  . Three phe MCMC) . Observa package . The envLarimichthys crocea (GenBank assembly accession: GCA_000972845.1), a sister species of L. polyactis  in National Center for Biotechnology Information Search database (NCBI). The scaffolds of L. crocea have been assembled and annotated including both putative and known coding regions, we aim to evaluate the genomic locations of RAD loci and provide annotations for coding regions harboring RAD loci as well as coding sequences within 5 kb of the mapped RAD loci (https://www.ncbi.nlm.nih.gov/genome/gdv/) was used to visualize coding-sequence (CDS) associated RAD loci in the L. crocea genome. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway approach implemented in the Database for Annotation, Visualization and Integrated Discovery (DAVID) web-server v6.8 (https://david.ncifcrf.gov/) was further applied for functional annotation and classification of the candidate genes  as the DAPC without prior did not reveal any genetic structure  and the South Yellow Sea group , although the differentiation between them was to some extent shallow. Taken together, these results indicated three genetically differentiated groups in the overwintering grounds of L. polyactis, including the Central Yellow Sea group (YA), the South Yellow Sea group  and the North East China Sea group (YD).To investigate the extent of population structuring in overwintering populations of 1, K = 1 . However MAXMEAK . A simil MAXMEAK  and all  MAXMEAK , indicatP <0.05, . No diffIn the present study, no significant association between environmental variables was observed based on Pearson’s correlation coefficient. The BAYENV analysis based on the data set of 21,620 SNPs revealed that some SNPs across the genome were associated with the environmental parameters . On the basis of criterion of log10 Bayes factor (BF) greater than 3, we found that 52 SNPs were correlated with variation in temperature, 46 SNPs were correlated with salinity variation and 40 SNPs were found to be correlated with turbidity, respectively . Of all L. crocea genome are provided in L. crocea genome. A hit with a gene was obtained for 80.7% (96 loci) of these 119 RAD loci. Among them, 39 SNPs were located in exonic regions, 44 SNPs were located in intronic regions and 13 SNPs were located within 5 kb of coding sequences (CDS) regions. Based on a standard setting of gene count of 2, the enriched KEGG pathways are summarized in Summaries of alignments and annotations to the L. polyactis and suggesting the existence of hitherto undetected cryptic genetic structure within this fish species. It has remained uncertain whether L. polyactis are genetically structured in the Yellow Sea and the East China Sea  located in the south of Cheju island exhibited genetic similarity with the YB  and these two sampling locations were observed to be clustered together as one group. Therefore, the southern geographical distribution boundary of South Yellow Sea group of L. polyactis might move southwards as compared to previous studies in 1960s. Bearing in mind that understanding population structure is critical for the conservation and management of natural resources  originating from the northward Kuroshio and the Taiwan Warm Current (TWC) intrudes into the central Yellow Sea  Fig. 1)Fig. 1). ine fish . In addiine fish . TherefoClupea harengus, Entosphenus tridentatus, Gadus morhua, Living in water, marine fish have intimate physiological contact with the environment at all times and variations in temperature, salinity and turbidity were widely reported to play important roles in shaping marine fish habitat . TherefoAnguilla anguilla) and American Eel (Anguilla rostrata), which indicate that saccharide metabolism pathways may be among the key biological functions influenced by spatially varying selection in marine realm   that the MHC polymorphism is maintained by pathogen-driven selection  . Teleostex (MHC) . Marine ex (MHC) . In addielection . On the election . TherefoL. polyactis which has not been reported before. In the present study, we found that overwintering populations in the Yellow Sea and the North East China Sea formed three groups, including the Central Yellow Sea group (YA), the South Yellow Sea group (YB and YC) and the North East China Sea group (YD). A number of outlier loci were found to be significantly associated with various environmental factors despite the overall low levels of genome-wide genetic differentiation. We found evidence for adaptation to local temperature, salinity and turbidity in the overwintering grounds, which involved multiple biological processes including energy reserve for growth and innate immune defenses. Moreover, we denote that the patterns of genetic variation in high gene flow marine fish such as L. polyactis may be strongly influenced by ocean fronts as well as ocean currents, which are expected to increase the environmental heterogeneity on small spatial scales. It should be also noted that overwintering samples were collected from the same year in our study, and temporal stability in the genetic structure of L. polyactis will be further assessed in future studies. Nevertheless, our findings suggested that there might be three potential adaptive conservation units (CUs) in the overwintering grounds of L. polyactis and the adaptive markers detected here could be used to identify specific priorities for the future conservation of L. polyactis (L. polyactis and prove useful for elucidating potential population units as well as management implications for other marine migratory fish species.Comprehensive sampling combined with a genome-wide SNP markers in the present study revealed a shallow but significant genetic structure in the overwintering grounds of olyactis . Taken t10.7717/peerj.7242/supp-1Figure S1Click here for additional data file.10.7717/peerj.7242/supp-2Figure S2Click here for additional data file.10.7717/peerj.7242/supp-3Figure S3Click here for additional data file.10.7717/peerj.7242/supp-4Figure S4Click here for additional data file.10.7717/peerj.7242/supp-5Table S1Click here for additional data file.10.7717/peerj.7242/supp-6Table S2Click here for additional data file.


Otoba parvifolia/glycycarpa (Myristicaceae), Clarisia biflora, Poulsenia armata, Ficus insipida , and Jacaratia digitata (Caricaceae) across the western Amazon Basin. Plastid DNA  and nuclear ribosomal internal transcribed spacers  were sequenced to assess genetic diversity, genetic differentiation, population genetic structure, and demographic patterns. Overall genetic diversity for both markers varied among species, with higher values in populations of shade‐tolerant species than in pioneer species. Spatial analysis of molecular variance (SAMOVA) identified three genetically differentiated groups for the plastid marker for each species, but the areas of genetic differentiation were not concordant among species. Fewer SAMOVA groups were found for ITS, with no detectable genetic differentiation among populations in pioneers. The lack of spatially congruent phylogeographic breaks across species suggests no common biogeographic history of these Amazonian tree species. The idiosyncratic phylogeographic patterns of species could be due instead to species‐specific responses to geological and climatic changes. Population genetic patterns were similar among species with similar biological features, indicating that the ecological characteristics of species impact large‐scale phylogeography.Various historical processes have been put forth as drivers of patterns in the spatial distribution of Amazonian trees and their population genetic variation. We tested whether five widespread tree species show congruent phylogeographic breaks and similar patterns of demographic expansion, which could be related to proposed Pleistocene refugia or the presence of geological arches in western Amazonia. We sampled  Given the independent nature of the sampling schemes and different spatial scales of previous studies , Clarisia biflora Ruiz & Pav., and Poulsenia armata (Miq.) Standl. (both Moraceae) are shade‐tolerant species characteristic of primary forest, while Ficus insipida Willd. (Moraceae) and Jacaratia digitata (Poepp. & Endl.) Solms (Caricaceae) are light‐demanding pioneer species that tend to grow in secondary forest. Pollen of these species is dispersed by different insects, and seeds are dispersed by animals (Table O. parvifolia/glycycarpa (Markgr.) A.H. Gentry that was not consistently distinguished morphologically from O. glycycarpa (Ducke) W. Rodrigues & T.S. Jaramillo. These two species were also not distinguished genetically using trnH‐psbA sequences . Here, they are treated as one group of samples (O. parvifolia/glycycarpa) because both genetic evidence and morphological evidence suggest that they are the same species. The phylogeographies of four species are characterized in this study for the first time, while the phylogeographic patterns of F. insipida were previously published by Honorio Coronado et al. . For each individual collected, leaf samples were dried and stored in silica gel. All individuals were photographed in the field, and at least one herbarium voucher was collected from each population to allow subsequent verification of identification. These vouchers were collected by E. Honorio, K. Dexter, and A. Monteagudo and deposited at LOJA in Ecuador, HOXA and MOL in Peru, and UAP and USZ in Bolivia.Samples were collected in regions where these species occur in western Amazonia, giving priority to sites where at least three were previously reported. We sampled 34 different sites in Ecuador, Peru, and Bolivia Figure , with 222 north of FA vs. 130,554 km2 south of FA), and the mean geographic distance between sampled populations did not differ significantly between the two regions . We therefore chose the FA to divide the sampled populations into two regional groups for some of the downstream analyses. The area north of the FA is generally termed the northwest Amazon (NWA) and is thought to have experienced a stable climate through Pleistocene climatic oscillations , a geological feature that divides the Amazonia into a northern and southern foreland basin  of the nuclear ribosomal DNA was amplified and sequenced using the ITS1 and ITS4 primers  and nucleotide substitutions, indels , and inversions were visually checked against the original electropherograms. The alignment was created manually in Mesquite v. 2.74 . Genetic diversity was computed independently for each species using the trnH‐psbA and ITS sequences by estimating the probability that two individuals carry distinct haplotypes or ribotypes (h) and the mean genetic distance between haplotypes or ribotypes of two distinct individuals (v) averaged over populations (hS and vS) and over all population pairs (hT and vT) in Permut CpSSR 2.0  that makes use only of haplotype or ribotype frequency and NST (=1−vS/vT), for which sequence similarities between the haplotypes or ribotypes are taken into account. The presence of phylogeographic signal was assessed by testing whether NST was significantly higher than GST based on 10,000 permutations in Permut CpSSR 2.0 . Genetic structure using pairwise nucleotide differences was further examined by analysis of molecular variance in Arlequin v. 3.5  was performed for each species for 2.6trnH‐psbA and ITS sequences for all populations. We used the neutrality test, Fu's Fs , and demographic analyses were estimated for each geographic region by independently analyzing populations located in the NWA and SWA regions. The regional analyses were only performed for trnH‐psbA sequences, because of data availability.The number of haplotypes/ribotypes and private haplotypes/ribotypes, overall genetic diversity , slightly lower in F. insipida (0.71), and much lower in J. digitata (0.57) , much lower in F. insipida (0.14), while measures were not available for J. digitata because it showed no intrapopulation genetic variation for ITS  was widely distributed in all populations of southern Peru and Bolivia. In this species, populations in Ecuador and northern Peru hosted the most divergent haplotypes  present in populations of southern Peru. Jacaratia digitata and F. insipida displayed closely related ribotypes, which were widely distributed in all populations  and P. armata (96%) showed significant genetic differentiation among populations  of 0.68 for F. insipida, 0.97 for J. digitata, 0.46 for C. biflora, 0.92 for P. armata, and 0.61 for O. parvifolia/glycycarpa. For ITS, F. insipida and J. digitata showed no geographic genetic structure  and two groups for P. armata (ΦCT = 0.92).Genetic differentiation among populations for the es Table . In the ns Table . The SAMs Figure a, with v3.3trnH‐psbA and ITS sequences were not significant in most species (p > 0.02; Table C. biflora and marginally for F. insipida, both for ITS. A unimodal mismatch distribution was only observed in C. biflora for trnH‐psbA and ITS. However, based on the HRI statistics, the recent demographic expansion model was consistently accepted for all species and markers, except for O. parvifolia/glycycarpa (p > 0.05; Table Neutrality tests for the s p > 0.0; Table 23.4trnH‐psbA, NWA and SWA regions had haplotypes private to each region in all species, but other haplotypes were present in both regions such as H10 in O. parvifolia/glycycarpa, H4 in C. biflora, and H1–H3 in F. insipida (Figure C. biflora and P. armata, while diversity in NWA was much greater than in SWA in J. digitata and slightly greater in O. parvifolia/glycycarpa and F. insipida (Table J. digitata in the SWA region. Within each regional group, recent expansion was consistently accepted for the SWA region for all species and for C. biflora, P. armata, and F. insipida in NWA (Table For a Figure a. Overal44.1Inga species by Dexter et al. (Inga species in Dexter et al. (Across five Amazonian rain forest tree species, we did not find evidence for shared phylogeographic break that could have been congruent with either geological paleoarches or putative Pleistocene refugia. Of the various geological paleoarches in the Amazon (Motamayor et al., r et al. , but it r et al.  showed tRecent work suggests stability of forest across northwestern Amazonia at the Last Glacial Maximum (Anhuf et al., F. insipida and F. adhatodifolia; da Costa et al., For each of the five tree species in our study, we found significant genetic differentiation among populations for the plastid marker, with clear geographic breaks between more genetically homogeneous sets of populations, but these breaks are not geographically coincident. Genetic breaks found in western Amazonia could represent areas of secondary contact of previously differentiated lineages put in contact by subsequent dispersal events. Moreover, incomplete lineage sorting and introgression after hybridization with close relatives could also increase the genetic distance between lineages in different regions (e.g., 4.2J. digitata, and to a lesser degree for O. parvifolia/glycycarpa and F. insipida. This regional pattern is consistent with the postglacial rain forest expansion reported on the southern margin of the Amazon rain forest in Bolivia, based on pollen and charcoal records (Burbridge, Mayle, & Killeen, A regional signal of low overall genetic diversity in SWA for the plastid marker was observed in J. digitata and F. insipida compared to the shade‐tolerant species. Lowe et al. (trnH‐psbA here. Moreover, we highlight the presence of a dominant and widespread plastid haplotype in SWA for our two pioneer species. This pattern was also found in other pioneer species such as Ceiba pentandra across Amazonia (Dick, Bermingham, Lemes, & Gribel, Cordia alliodora across the Neotropics (Rymer, Dick, Vendramin, Buonamici, & Boshier, Our results showed lower overall genetic diversity in e et al.  suggestee et al.  in that e et al.  and trnHJ. digitata and F. insipida is dispersed by sphingid moths (Bawa, Bullock, Perry, Coville, & Grayum, Pithecellobium elegans; Chase, Moller, Kesseli, & Bawa, F. insipida have been reported to travel over distances of up to 14 km in the Neotropics (Nason, Herre, & Hamrick, C. pentandra (Dick et al., C. alliodora (Rymer et al., Schizolobium parahyba (Turchetto‐Zolet et al., Symphonia globulifera (Dick & Heuertz, The two pioneer species also showed low genetic diversity and no genetic differentiation among populations for ITS compared to shade‐tolerant species. The lack of genetic diversity for ITS may reflect the slow rate of evolution of this region (Dick, Abdul‐Salim, & Bermingham, 5en masse, but rather each individual lineage did so idiosyncratically. The one signature of genetic concordance that we uncovered is that of recent, rapid population expansion in southwestern Amazonia, which is consistent with paleoecological evidence for the expansion of the southern fringe of the Amazonian rain forest since the Last Glacial Maximum (Burbridge et al., This comparative phylogeographic study of trees in western Amazonia demonstrates that genetic breaks are not congruent among species. We found no evidence consistent with the effects of Pleistocene refugia or paleoarches. These results imply that Amazon forest trees did not evolve, disperse, and successfully occupy the Amazon environment The lack of congruent patterns of genetic structure is consistent with studies that suggest a key role for idiosyncratic historical dispersal in shaping the biogeography of Amazonian tree species (e.g., Dexter et al., In sum, our results suggest that idiosyncratic dispersal and ecological differences among species together played key roles in shaping patterns of genetic diversity in neotropical rain forest tree species. This would explain why this comparative phylogeographic study, and most prior studies of individual species (e.g., Dick et al., The authors have no conflict of interest to declare.E.H. and R.T.P. conceived the idea; E.H. and K.D. collected the samples; E.H. and M.H. carried out the DNA laboratory work; E.H. performed statistical analyses; E.H., R.T.P., and K.D. wrote the paper; and M.H and O.P. contributed to the writing. All authors read and approved the final manuscript.


TM 19 amplification system and genetic background of Kham Tibetan remain uncharacterized. Thus, we genotyped 19 forensic genetic markers in 11,402 Kham Tibetans to gain insight into the genetic diversity of Chinese high-altitude adaptive population. Highly discriminating and polymorphic forensic measures were observed, which indicated that this new-developed DNATyper 19 PCR amplification is suitable for routine forensic identification purposes and Chinese national DNA database establishment. Pairwise genetic distances among the comprehensive population comparisons suggested that this high-altitude adaptive Kham Tibetan has genetically closer relationships with lowlanders of Tibeto-Burman-speaking populations . Genetic substructure analyses via phylogenetic reconstruction, principal component analysis, and multidimensional scaling analysis in both nationwide and worldwide contexts suggested that the genetic proximity exists along the linguistic, ethnic, and continental geographical boundary. Further studies with whole-genome sequencing of modern or archaic Kham Tibetans would be useful in reconstructing the Tibetan population history.Tibetans residing in the high-altitude inhospitable environment have undergone significant natural selection of their genetic architecture. Recently, highly mutational autosomal short tandem repeats were widely used not only in the anthropology and population genetics to investigate the genetic structure and relationships, but also in the medical genetics to explore the pathogenesis of multiple genetic diseases and in the forensic science to identify individual and parentage relatedness. However, genetic variants and forensic efficiency of DNATyper This mo (InDel) . STR mut (InDel) . Human p (InDel) . TishkofTM 19 kit, was developed and validated by the Institute of Forensic Science in the Ministry of Public Security , which can co-amplify 18 autosomal STRs and one sex-determination marker of Amelogenin focused on Chinese populations.In forensic science, multiplex STRs genotyping by the fluorescent labeled PCR amplification combined with the CE approach is recognized as the current gold standard in the personal identification, kinship testing and missing person’s identification . Since sThe Tibetan Plateau is generally considered to have been covered by the ice sheet during the last glacial maximum. Until recently, there has been no consensus view about when colonization began, how Tibetans got there and how they occupied and adapted this cold, arid, hypobaric, and hypoxic environment. Archeological evidence from the Heimahe, Jiangxigou sites suggested that the gradual expansion of foragers’ occupation of Tibet began from 40–25 thousand years ago (kya) . AbundanTM 19 PCR amplification system and explored the detailed genetic variants, genetic diversity and forensic efficiency of STRs in the Kham Tibetans in this study. Furthermore, we performed two comprehensive population comparisons (nationwide population relationship investigation among 64 groups and worldwide genetic affinity exploration among 53 groups) to dissect the genetic differentiation between the Kham Tibetans and reference populations and simultaneously provide some new insights for patterns of global or local population substructure based on autosomal genetic variability.Thus, we conducted and reported the first large-scale autosomal STRs study in this unique high-altitude adaptive Tibetan population based on a new-generation DNATyperThis project and corresponding protocol were considered and approved by the Ethics Committee of the Institute of Forensic Medicine, West China School of Basic Science and Forensic Medicine, Sichuan University . Our participants are needed to be the indigenous Tibetans and no intermarriage or long-distance migration at least three generations. Our subjects have signed written informed consent and analyzed anonymously. A total of 11,402 unrelated healthy individuals  were collected from the east of Tibet Kham Tibetan autonomous region (Chengdu country), Aba and Muli city in Sichuan province. To insure the included the donors which meet the aforesaid requirement, we followed the following criteria: (1) both parents and grandparents being Tibetans; (2) the language used first is Tibeto-Burman language; (3) all participants residing in the same village or owning the same family names are need to check with relative relationships with previous included subjects to avoid included close relatives; (4) in the past three generations, there is no documented ancestors from other ethnic groups. Besides, to avoid the potential included close relatives, we employed a large sample size to dilute the sample collection bias. Blood samples are collected using FTA cards or cotton swab. All datasets generated and analyzed for this study are included in the TM 19 PCR amplification system on a GeneAmp PCR System 9700 Thermal Cycler  on the basis of the manufacture’s instruction. We employed the following PCR amplification conditions: decomposition at 72°C for 20 min and denaturation at 95°C for 11 min, and then amplification for 26 cycles of denaturation for 30 s at 94°C, anneal for 2 min at 59°C and extension for 1 min at 72°C, following a final extension at 60°C for 60 min, and holding at 25°C. PCR products are mixed with the deionized Formamide and Typer500, and then isolated using the capillary electrophoresis on an ABI 3500 XL Genetic Analyzer . Electrophoresis results were visualized and checked using the GeneMapper ID-X Software v1.5 .Nineteen forensic genetic markers labeled with multi-fluorescent dyes  were amplified simultaneously using the DNATyper1  and expected heterozygosity (He), were carried out using the Arlequin version 3.5.2.21 . Online 1  was used1  and positive control (9947A) were genotyped along with each batch of samples.This study was in accordance with the recommendations of scientific standards for studies in forensic genetics proposed and advocated by the International Society for Forensic Genetics (ISFG) . The expTM 19 amplification system (Supplementary Table p < 0.05/18 = 0.0028). Simultaneously, pairwise Linkage Disequilibrium (LD) among 153 locus pairs was conducted, and we identified 34 pairs existing linkage or associated inheritance in the Kham Tibetan . This reference population panel comprises of 43 geographically diverse Han Chinese populations, four Uyghur populations, two Manchu, Hui, Yi and Tibetan populations and one Kazakh, Bai, Vietnamese, Miao, Zhuang, Hani and Xibe , which is in accordance with the observed patterns of population genetic relationship in the PCA Figure .We finally carried out phylogenetic relationship reconstructions on the basis of the neighbor-joining (N-J) algorithm. A N-J tree based on the pairwise Reynolds’ genetic distance among 64 Chinese populations  should be accurately obtained and understood to evaluate the forensic efficiency and paternity probability. Ho, He, and PIC values observed in this study indicated that the 18 autosomal STRs are high diversity and polymorphic in the Tibet Kham Tibetan. The overall forensic efficiency values of the combined power of discrimination (CPD) and the combined probability of exclusion (CPE) are 0.99999999999999999999974 and 0.999999931, respectively. This new PCR amplification system is more polymorphic and informative compared with the forensic effectiveness of 21 non-Combined DNA Index System (CODIS) autosomal STRs included in the AGCU 21+1 system, which CPD and CPE are, respectively, 0.9999999999999999999 and 0. 999997 in the Liangshan Tibetan and 0.9999999999999999993 and 0.999999 in the Liangshan Yi . Simultaectively . Moreoveectively . Thus, n∗ method . Finally, genetic substructure analyses in the nationwide and worldwide context suggested that the genetic proximity exists along with linguistic, ethnic, and continental geographical boundary. Additional studies with whole-genome sequencing of modern or archaic Kham Tibetans would help in reconstructing Tibetan population history.In summary, we presented the first batch population data of large sample size  to comprehensively evaluate the genetic diversity and forensic efficiency of DNATyperThis study was carried out according to the Declaration of Helsinki and the recommendations of “Ethical Committee of Sichuan University, China” with written informed consent from all subjects. Our protocol was approved by the “Ethical Committee of Sichuan University” .XZ and GH wrote the manuscript. MW, JL, PC, BG, SW, and ZL collected the samples and extracted DNA. GH, MW, XZ, and JL helped to conduct the statistical analysis. ZW revised the manuscript. YH designed this study. All authors agreed to the submission of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Olea europaea L.) is a major fruit crop in the Mediterranean Basin. Ex-situ olive management is essential to ensure optimal use of genetic resources in breeding programs. The Worldwide Olive Germplasm Bank of Córdoba (WOGBC), Spain, and Marrakech (WOGBM), Morocco, are currently the largest existing olive germplasm collections. Characterization, identification, comparison and authentication of all accessions in both collections could thus provide useful information for managing olive germplasm for its preservation, exchange within the scientific community and use in breeding programs. Here we applied 20 microsatellite markers (SSR) and 11 endocarp morphological traits to discriminate and authenticate 1091 olive accessions belonging to WOGBM and WOGBC . Of all the analyzed accessions, 672 distinct SSR profiles considered as unique genotypes were identified, but only 130 were present in both collections. Combining SSR markers and endocarp traits led to the identification of 535 cultivars (126 in common) and 120 authenticated cultivars. No significant differences were observed between collections regarding the allelic richness and diversity index. We concluded that the genetic diversity level was stable despite marked contrasts in varietal composition between collections, which could be explained by their different collection establishment conditions. This highlights the extent of cultivar variability within WOGBs. Moreover, we detected 192 mislabeling errors, 72 of which were found in WOGBM. A total of 228 genotypes as molecular variants of 74 cultivars, 79 synonyms and 39 homonyms as new cases were identified. Both collections were combined to define the nested core collections of 55, 121 and 150 sample sizes proposed for further studies. This study was a preliminary step towards managing and mining the genetic diversity in both collections while developing collaborations between olive research teams to conduct association mapping studies by exchanging and phenotyping accessions in contrasted environmental sites.Olive ( Olea europaea, ssp. europaea, var. europaea Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Dear Editor,The manuscript entitled: Characterization of Worldwide Olive Germplasm Banks of Marrakech (Morocco) andCórdoba (Spain): towards management and use of olive germplasm in breeding programs, describe worldwide olive germplasm using morphological and molecular approaches. The article can suitable for olive breeder and useful for extended genetic diversity, which is a main role in plant breeding. the manuscript could be consider for publication in PLOS ONE, but before that require some parts revision as following of:- The manuscript need to be minor revised for grammatical using native speaker English language because some parts hard understands for readers.-please prepare high resolution of figures, because the quality is low and some parts no clear in details.-in Figure 2 and Fig S3, 2-D distribution (PCO) how does it and which of traits ? The first two PCO accounting 12.34%in total. This is very, when you are established PCO for morphological traits/markers because of different characters for example environmental conditions (Morocco & Spain) you should get high genetic diversity. Unless, some of genotypes have a clone and so express as the same function. This is also expected when we are used molecular data  with limited primers . These cause that's use markers not comprehensively covered the olive genomes. The authors need to be declared this event and explained.-One of the important issues in the evaluation and characterization of Plant Germplasm (PG) is their use in plant breeding programs. So knowledge of gene diversity and genetic variation is very important. The authors need to be clarified of it based on Nei & Li .This is procedure could better helps to identified all the best diversity for selection of genotypes in breeding programs.- In Figure 3, dendrogram based on UPGMA using Dice coefficient similarity. Why used dice index for clustering? The authors require to analysis Mantel test and so find the best correlation coefficient among  so we could described our results based on all the best. For stability and consistency analysis of the cluster, bootstrap analysis could better results combined with STRACTURE.- In Figure 4 the patterns of morphological showed different variation, but we do not see in PCO- analysis! 12.34% is a high!- Determining the genetic structure of populations is becoming an increasingly important aspect of genetic studies. One of the most frequently used methods is the calculation of F-statistics using an Analysis of Molecular Variance (AMOVA).Linking AMOVA and K-means cluster could better find results in survey of germplasm and is one of significant approaches in plant breeding. Please see AMOVA-Based Clustering of Population Genetic Data .Reviewer #2: REFEREE COMMENTSThe manuscript "Characterization of Worldwide Olive Germplasm Banks of Marrakech (Morocco) and Córdoba (Spain): towards management and use of olive germplasm in breeding programs" by Bakkali et al.is a very interesting paper which aims to discriminate and authenticate 1091 olive accessions belonging to Worldwide Olive Germplasm Bank of Córdoba (WOGBC), Spain, and Marrakech (WOGBM), Morocco, applying 20 microsatellite markers (SSR) and 11 endocarp morphological traits, providing useful information for managing olive germplasm for its preservation, exchange and use in breeding programs.I recommend publishing this paper with minor revision.I found very few points to improve, as follows:Figure are not of good quality, but probably it is a problem of the transferred copy.Page 2, Line 46: I doubt of these data; please, check itPage 3, Line 99: On this subject, there are more recent papers worthy to be mentioned:for SSR analysishttps://doi.org/10.7717/peerj.5260di Rienzo V. et al., 2018. Genetic flow among olive populations within the Mediterranean basin. PeerJ 6:e5260 Boucheffa S et al., 2017. The coexistence of oleaster and traditional varieties affects genetic diversity and population structure in Algerian olive (Olea europaea) germplasm. (doi: 10.1007/s10722-016-0365-4).for SNP analysisD’Agostino, N. D. et al., 2018. GBS-derived SNP catalogue unveiled wide genetic variability and geographical relationships of Italian olive cultivars. Scientific Reports., 1–13. doi:10.1038/s41598-018-34207-y.Taranto et al., 2018. SNP diversity in an olive germplasm collection. Acta Horticulturae 1199, pp. 27-32. doi: 10.17660/ActaHortic.2018.1199.5Page 23 Line 917: Table 4 (space)Page 13 Line 485 On this subject, there are more recent papers worthy to be mentioned:di Rienzo et al., 2018. The preservation and characterization of Apulian olive germplasm biodiversity. Acta Hortic. 1199, 1–6. doi:10.17660/ActaHortic.2018.1199.Page 15 line 555Boucheffa S. et al., 2019. Diversity Assessment of Algerian Wild and Cultivated Olives (Olea europeae L.) by Molecular, Morphological, and Chemical Traits. Eur. J. Lipid Sci. Technol.121, 1800302 DOI: 10.1002/ejlt.201800302.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  23 Sep 2019Response to reviewersReviewer #1: Dear Editor,The manuscript entitled: Characterization of Worldwide Olive Germplasm Banks of Marrakech (Morocco) and Córdoba (Spain): towards management and use of olive germplasm in breeding programs, describe worldwide olive germplasm using morphological and molecular approaches. The article can suitable for olive breeder and useful for extended genetic diversity, which is a main role in plant breeding. the manuscript could be consider for publication in PLOS ONE, but before that require some parts revision as following of:- The manuscript need to be minor revised for grammatical using native speaker English language because some parts hard understands for readers.The article has been reviewed by a professional scientific English mother-tongue reviser (David Manley).-please prepare high resolution of figures, because the quality is low and some parts no clear in details.The quality of the figures has been reviewed by using the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool implemented in PlosOne website-in Figure 2 and Fig S3, 2-D distribution (PCO) how does it and which of traits ? The first two PCO accounting 12.34% in total. This is very, when you are established PCO for morphological traits/markers because of different characters for example environmental conditions (Morocco & Spain) you should get high genetic diversity. Unless, some of genotypes have a clone and so express as the same function. This is also expected when we are used molecular data  with limited primers . These cause that's use markers not comprehensively covered the olive genomes. The authors need to be declared this event and explained.For the PCoA in Figure 2, we used only SSR data. Due to the high genetic diversity within both collections and to high number of alleles par locus , the total variation is described by the N-1 axis, where N is the number of alleles. In the figure below, more than 100 axes are presented with the inertia proportion exceeding 0.1%. The first two axes explained the highest variation, i.e. 12.34%. This pattern is mostly observed in studies based on SSR . -One of the important issues in the evaluation and characterization of Plant Germplasm (PG) is their use in plant breeding programs. So knowledge of gene diversity and genetic variation is very important. The authors need to be clarified of it based on Nei & Li .This is procedure could better helps to identified all the best diversity for selection of genotypes in breeding programs.We agree on the fact that knowledge of gene diversity and genetic variation is very important, especially in breeding programs. We examined the organization of genetic diversity using two approaches, the first one was the model-based clustering method implemented in Structure  and the second one was PCoA based on simple matching coefficient . We did not use clustering analysis based on Nei & Li . As additional information to the reviewer, we used the DAPC approach implemented in Adegenet package in R to classify the 672 olive genotypes in different groups and then we constructed one dendrogram based on NJ clustering and Fst for each pairwise group of genotypes (see below for details). We obtained similar pattern as Structure and PCoA analyses.- In Figure 3, dendrogram based on UPGMA using Dice coefficient similarity. Why used dice index for clustering? The authors require to analysis Mantel test and so find the best correlation coefficient among  so we could described our results based on all the best. For stability and consistency analysis of the cluster, bootstrap analysis could better results combined with STRUCTURE.For the dendrogram in Figure 3, we used the UPGMA method and Dice similarity index to classify the set of 74 different cultivars showing molecular variants. For each of these cultivars, their molecular variants are distinguished by only one to four dissimilar alleles. Our aim here was not to analyze the genetic relationships between all of the 672 olive genotypes identified in both collections, but we focused on the analysis of molecular variants within each of the 74 cultivars with presumed somatic mutations. We examined the genetic relationships between the 672 olive genotypes using the Structure and PCoA approaches described in Figures 1 and 2. Furthermore, we proposed additional analyses based on the clustering approach described above. - In Figure 4 the patterns of morphological showed different variation, but we do not see in PCO- analysis! 12.34% is a high!We choose to remove the figure 4 from our manuscript as it doesn’t show clearly the similarity in the endocarp traits between synonymous cases of Picholine marocaine  and Frantoio  cultivars. The PCoA was performed based only on SSR markers.- Determining the genetic structure of populations is becoming an increasingly important aspect of genetic studies. One of the most frequently used methods is the calculation of F-statistics using an Analysis of Molecular Variance (AMOVA). Linking AMOVA and K-means cluster could better find results in survey of germplasm and is one of significant approaches in plant breeding. Please see AMOVA-Based Clustering of Population Genetic Data .Upon the reviewer’s request, we performed an additional analysis to identify the number of clusters using the K-means clustering approach, as implemented in the adegenet package in the R environment. The most probable group was detected at 14 (figure a below). Based on these 14 groups, Pairwise Fst differentiation was calculated and ploted using NJ method (see figure b below). The 14 identified groups were classified based on pairwise Fst into three major clusters  with a high bootstrap. such genetic pattern is similar to Bayesian model approach  and PCoA analysis.Reviewer #2: REFEREE COMMENTSThe manuscript "Characterization of Worldwide Olive Germplasm Banks of Marrakech (Morocco) and Córdoba (Spain): towards management and use of olive germplasm in breeding programs" by Bakkali et al. is a very interesting paper which aims to discriminate and authenticate 1091 olive accessions belonging to Worldwide Olive Germplasm Bank of Córdoba (WOGBC), Spain, and Marrakech (WOGBM), Morocco, applying 20 microsatellite markers (SSR) and 11 endocarp morphological traits, providing useful information for managing olive germplasm for its preservation, exchange and use in breeding programs.I recommend publishing this paper with minor revision.I found very few points to improve, as follows:Figure are not of good quality, but probably it is a problem of the transferred copy.OK, the quality of the figures has been reviewedPage 2, Line 46: I doubt of these data; please, check itThe data were updated based on IOOC and FAO statistics (most recent data from 2018)Page 3, Line 99: On this subject, there are more recent papers worthy to be mentioned:for SSR analysis https://doi.org/10.7717/peerj.5260di Rienzo V. et al., 2018.. PeerJ 6:e5260 Boucheffa S et al., 2017. The coexistence of oleaster and traditional varieties affects genetic diversity and population structure in Algerian olive (Olea europaea) germplasm. (doi: 10.1007/s10722-016-0365-4).for SNP analysisD’Agostino, N. D. et al., 2018. GBS-derived SNP catalogue unveiled wide genetic variability and geographical relationships of Italian olive cultivars. Scientific Reports., 1–13. doi:10.1038/s41598-018-34207-y.Taranto et al., 2018. SNP diversity in an olive germplasm collection. Acta Horticulturae 1199, pp. 27-32. doi: 10.17660/ActaHortic.2018.1199.5These references were included in the final version of the manuscript.Page 23 Line 917: Table 4 (space)This comment has been taken into consideration in the final versionPage 15 line 555Boucheffa S. et al., 2019. Diversity Assessment of Algerian Wild and Cultivated Olives (Olea europeae L.) by Molecular, Morphological, and Chemical Traits. Eur. J. Lipid Sci. Technol.121, 1800302 DOI: 10.1002/ejlt.201800302.This reference has been Taken into consideration in the final versión.AttachmentResponse to Reviewers.docSubmitted filename: Click here for additional data file. 27 Sep 2019Characterization of Worldwide Olive Germplasm Banks of Marrakech (Morocco) and Córdoba (Spain): towards management and use of olive germplasm in breeding programsPONE-D-19-19157R1Dear Dr. KHADARI,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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