Vigna subterrenea L) landraces sourced from different geographical regions  and unknown origin (sourced from United Kingdom) was used to assess genetic diversity, relationship and population structure using DArT SNP markers. The major allele frequency ranged from 0.57 for unknown origin to 0.91 for West Africa region. The total gene diversity (0.482) and Shannon diversity index (0.787) was higher in West African accessions. The genetic distance between pairs of regions varied from 0.002 to 0.028 with higher similarity between Nigeria/Cameroon-West Africa accessions and East-Southern Africa accessions. The analysis of molecular variance (AMOVA) revealed 89% of genetic variation within population, 8% among regions and 3% among population. The genetic relatedness among the collections was evaluated using neighbor joining tree analysis, which grouped all the geographic regions into three major clusters. Three major subgroups of bambara groundnut were identified using the ADMIXTURE model program and confirmed by discriminant analysis of principal components (DAPC). These subgroups were West Africa, Nigeria/Cameroon and unknown origin that gave rise to sub-population one, and Central Africa was sub-population two, while Southern and East Africa were sub-population three. In general, the results of all the different analytical methods used in this study confirmed the existence of high level of diversity among the germplasm used in this study that might be utilized for future genetic improvement of bambara groundnut. The finding also provides new insight on the population structure of African bambara groundnut germplasm which will help in conservation strategy and management of the crop.Understanding the genetic structure and diversity of crops facilitates progress in plant breeding. A collection of 270 bambara groundnut ( Vigna subterranea (L.) Verdc) is an underutilized African legume, grown mostly, by smallholder and subsistence farmers in Sub-Sahara Africa [Vigna subterranea var subterranea (cultivated) and Vigna subterranea var spontanea (wild), which are the two botanical forms both having the same number of chromosomes (2n = 22). It is the third most important legume in terms of consumption and production, after groundnut (Arachis hypogaea) and cowpea (Vigna unguiculata) in semi-arid Africa and plays an important role, especially during times of famine, serving as good source of nutrition and income [Bambara groundnut  conserving the largest germplasm [Bambara groundnut in the genuine wild state was found in North Yola province of Nigeria near Garoua in Cameroon  with mosermplasm  collecteBambara groundnut landraces under low input farming systems have maintained a noticeably amount of genetic diversity  and inveHowever, considering the large genetic resource of the crop globally , only lehttps://au.int/en/member_states/countryprofiles2); while Cameroon and Nigeria, as a possible places of Bambara groundnut domestication, were grouped together. Among the studied accessions, 43.3% were from West Africa, followed by Southern Africa (24.8%), Nigeria/Cameroon (14.4%), Central Africa (10.7%), East Africa (5.6%) and unknown origin (1.1%).A total of 270 accessions of bambara groundnut landraces were used in the study with 262 genotypes obtained from the Gene Bank of International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria and eight genotypes from Crops and Soil Science Research Farm in Bunda, Malawi . The genPstI-TaqI digestion of genomic DNA and ligation of barcoded adapters was done, followed by PCR amplification of adapter-ligated fragments. The library was constructed, according to Kilian et al. [The bambara groundnut leaves at the greenhouse were harvested at seedling stage from three plant of each accession and pooled. The pooled leaves were put inside eppendorf tube with dry ice. The leaf tissue was stored at −80°C, until the tissue was lyophilized, then ground in tubes with stainless steel beads using a plate shaker. DNA extraction was done using Nucleomag Plant Genomic DNA extraction kit, following the manufacturer’s protocol. The quality and quantity of DNA was checked on 0.8% agarose gel. The DArTSeq complexity reduction through n et al. , and wasn et al. . DArTseqThe marker quality was evaluated based on the individual marker related statistics, as suggested by Triticarte Pty Ltd. SNP marker with more than 80% call rate and 95% reproducibility was selected and non-polymorphic markers i.e., having a variance close to 0 with unknown SNP position were removed. The called SNPs were further filtered using TASSEL software , and SNPThe field experiment was conducted in the year 2019 at the Research and Experimental Farm of Jimma University, College of Agriculture and Veterinary Medicine at Ela-Dale. The site is located in the Southwestern part of Ethiopia, in Oromia Regional State, which is 356 km Southwest of Addis Ababa. It is classified as mid-altitude sub humid zone (7° 42 N latitude and 36° 50 E longitude) and has an altitude of 1710 masl. It receives an average annual rainfall of 1250 mm, and has an average maximum temperature of 26.2°C. The average soil pH of the farm is in the range of 5 to 6.0 . The expGenalex ver. 6.502 [Descriptive statistics, phenotypic coefficient of variation, genotypic coefficient of variation and ANOVA were analyzed for the morphological traits using R version 3.4.6 . Major ar. 6.502 , with str. 6.502  was emplPopulation structure, based on minimum cross entropy, was determined using the ADMIXTURE model program in R . The perThe reproducibility and call rate for the mean of the 3343 SNP markers used in the study were 0.99 and 0.85, respectively, showing consistent marker score and high reproducibility , Table 1Genetic diversity was estimated per geographical region of the source of the studied germplasm . The WesThe average Nei genetic distances among the accessions within each population revealed that the Nigeria/Cameroon and West African region had the smallest genetic distance value (0.002) . A similThe AMOVA result showed significant genetic differences within populations . The totThe cumulative percentage contributions of the first three principal components (PCs) to the total variations in the populations was 27.6%, with 14.7%, 9.2% and 3.7% respective contributions to PC1 to PC3 . The WesNeighbor-joining analysis was used to detect the genetic relationship of the bambara groundnut germplasm based on the dissimilarity matrix . The neiThe admixture model-based clustering analysis approach was followed to investigate the population structure of 270 bambara groundnut accessions. The optimum cluster was three (K = 3) based on the model with the lower value of BIC preferred . The thrHighly significant (p < 0.001) genetic variation was observed among all morphological traits in the studied accessions . Among tUnderstanding the genetic diversity and population structure of bambara groundnut landraces originating from various geographical regions of Africa is vital towards its conservation and utilization in developing methods to attain lucrative divergence in the breeding programs of this crop. The genetic diversity in germplasm plays an important role in the proficient investigation of useful alleles existing in landraces and diverse genotypes . The higThe high percentage value of genetic diversity within-population obtained from AMOVA could be as a result of natural adaptation or extensive exchange of seeds among farmers between environments or because of common origin of the population, which might have led to bambara growers using the same seed continuously, without new introductions. In Ghana, seed sources for planting bambara groundnut are from farmer-saved seeds, exchange and market purchase . This isThe cluster dendogram using a neighbor joining tree based on the geographical distribution of accessions showed that most of the bambara groundnut accessions from the same region did not cluster accurately based on their origin or regions. This could be due to low genetic differentiation among the populations, which suggests that the genetic background of bambara groundnut populations does not always correlate with their geographical origin or regions. Although, all the accessions of the same origin did not cluster according to their genetic background but majority of the accessions from the same origin clustered together which provides evidence in the origin and relationship of bambara groundnut landraces. Similar patterns of grouping of landraces according to geographical origin have been reported using morphological markers ,60 and mPopulation structure analysis gives better understanding for genetic diversity and will help towards association mapping studies . It alsoThe differences observed in the morphological traits of the accessions in each cluster/group could be due to the genetic make-up or the influence of environment on the adaptation of the crop or selection of specific traits by farmers. Ntundu et al.  and MoloThe results obtained from the present study indicated that DArT SNP marker is informative and selective and it might be widely used for molecular analysis of bambara groundnut. DArT SNP marker based on molecular characterization of bambara groundnut landraces from different regions revealed that variation exists among the accessions and the pattern of the genetic diversity varied across the five regions. Three main subpopulations of bambara groundnut germplasm was identified. Consequently, this result will contribute substantially to the management, conservation and association mapping and marker assisted selection of bambara groundnut accession for future improvement.S1 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Mesoplodon grayi) from Western Australia and New Zealand found high levels of mtDNA diversity, no geographic structure and stable demographic history. To further investigate phylogeographic and demographic patterns across their range, we generated complete mitochondrial and partial nuclear genomes of 16 of the individuals previously analysed and included additional samples from South Africa (n = 2) and South Australia (n = 4), greatly expanding the spatial range of genomic data for the species. Gray's beaked whales are highly elusive and rarely observed, and our data represents a unique and geographically broad dataset. We find relatively high levels of diversity in the mitochondrial genome, despite an absence of population structure at the mitochondrial and nuclear level. Demographic analyses suggest these whales existed at stable levels over at least the past 1.1 million years, with an approximately twofold increase in female effective population size approximately 250 thousand years ago, coinciding with a period of increased Southern Ocean productivity, sea surface temperature and a potential expansion of suitable habitat. Our results suggest that Gray's beaked whales are likely to be resilient to near-future ecosystem changes, facilitating their conservation. Our study demonstrates the utility of low-effort shotgun sequencing in providing ecological information on highly elusive species.The deep oceans of the Southern Hemisphere are home to several elusive and poorly studied marine megafauna. In the absence of robust observational data for these species, genetic data can aid inferences on population connectivity, demography and ecology. A previous investigation of genetic diversity and population structure in Gray's beaked whale ( However, despite the desire to protect biodiversity in the high seas, the task is a challenge when the ecology of elusive species is still a mystery.The ocean is undergoing rapid ecological change as a result of climate change, ocean acidification and deoxygenation, poor fisheries management, oil and gas extraction, litter and shipping . In an aCurrently, the deep sea is one of the last frontiers of exploration on Earth and there are a great many species for which we know very little, including large cetaceans ,3. DespiMesoplodon traversii) in 2012, Derinayagala's whale (Mesoplodon hotaula) in 2014 and a new species of Baird's beaked whale (Berardius minimus) in 2019 )https://github.com/Mvwestbury/Dstats-topology-test. The significance of these results, i.e. whether |Z| was greater than 3, was determined for each comparison using a one-sample Wilcoxon signed-rank test in R.We computed D-statistics in ANGSD using a random base call approach (-doAbbaBaba 1) and the following parameters: a minimum mapping and quality score of 25 , only consider reads that map to one location uniquely (-uniqueonly 1), calculate genotype likelihoods using GATK algorithm (-GL 2), calculate major and minor alleles using genotype likelihoods (-doMajorMinor 1), skip sites that are triallelic , calculate D using a block size of 1Mb (-blocksize 1 000 000) and specify the Sowerby's beaked whale as the outgroup. We performed a block jackknife approach to test for the significance of the results (Z score) using the Rscript (jackKnife.R) available as part of the ANGSD toolsuite. ANGSD computes every possible combination of individuals, which we filtered based on the sampling localities of the individuals. Scripts on how to filter the ANGSD output to test for population structure can be found at . 2.4.4To assess levels of relatedness between individuals originating from the same geographic location, we used NGSrelate v. 2 . As inpuIn this study, we used the low-coverage genomes to identify the sex of new and previously analysed samples. Our method was as follows: we identified scaffolds in the orca and Sowerby's beak whale assemblies putatively belonging to sex chromosomes using SatsumaSynteny v. 2.01 , default. 3. 3.1. 3.1.1π = 0.00742) in comparison with the other surveyed odontocete species (π = 0.00094–0.00941) .We constructed mitochondrial genomes from all 22 individuals to coverages of between 27.0× and 112.7×, with a mean coverage of 58.3× . Gray's beaked whales exhibit relatively high mitochondrial nucleotide diversity (0.00941) . The hap0.00941) a,b.Figu. 3.1.2a) with an approximate doubling of female effective population size (efN) around approximately 250 thousand years ago (kya). The current estimated median efN is 270 500 whales (95% CI 116 544–1 244 228). Our findings are broadly consistent with those reported by Thompson et al. , South Africa], outgroup], the two South African samples appeared more closely related to each other than they were to any of the individuals from Western Australia. However, we had only two samples from South Africa, and this was not seen when comparing South Africa to any other sampling localities. Therefore, it may be a product of not having enough individuals for this specific comparison.To further investigate the presence of population structure, we used the D-statistics analysis to test for population structure, as this has previously been shown to be more sensitive to small genetic differences among populations  . The results are concordant with previous methods based on the amplification of the SRY gene for a subsample of the samples  . Concord. 4By performing shallow shotgun sequencing of 22 Gray's beaked whale individuals sampled across approximately 13 000 km of the species' range, we were able to successfully recover complete mitochondrial genomes and partial nuclear genomes from all individuals. This increases the amount of genetic data available for this species, increasing the power of evolutionary inference we can make even with few available samples . FurtherWe recovered no discernible geographic population structure in either the mitochondrial or nuclear genome data, suggesting a lack of both maternal and paternal population structure across the study area in this potentially highly mobile marine predator (figures Pagodroma nivea) . Wh. Wh20]. efN approximately 250 kya [a).To explore the genetic diversity of Gray's beaked whales through time, we performed demographic history analysis on the mitochondrial genomes. We uncover relatively stable and high levels of female effective population size over at least the past approximately 1.1 million years, but an approximate doubling of  250 kya  . During this time, sea surface temperature (SST) [c). Moreover, the peak in sea surface temperature 250 kya, shows the ocean being warmer or at least as warm as it is today. A plausible explanation for the increase in efN could be that productivity increased in polar waters at the same time as a shift in temperature, which may have allowed an expansion in suitable habitat at this time. Increases in primary production are likely to have provided more food for whale populations, resulting in increased reproduction with subsequent expansion and divergence of existing lineages.The increase in uctivity . This inuctivity ,62 (figure (SST)  also shoefN approximately 250 kya, the long-term stability of efN of the Gray's beaked whale could reflect that its circumpolar habitat has been relatively stable over the long term. Oceanographic features, such as the Antarctic circumpolar current, subtropical convergence and Antarctic polar front (shown in efN (a).Aside from the doubling in shown in , have aln in Nef a.Our results indicate that Gray's beaked whales have existed as a diverse and stable population across their distribution over the long term, accruing relatively high genetic diversity in comparison with other toothed whales. It is likely that a flexible social system, rich Southern Hemisphere feeding grounds and permanent oceanographic features facilitate gene flow in this species. Given that these whales have never been the focus of whaling—whalers named them ‘scamperdown whales’ due to their elusive behaviour—Gray's beaked whales appear to have been able to maintain ancestral levels of high genetic diversity and abundance.In conclusion, through generating complete mitochondrial genomes, a new low-coverage reference panel of nuclear genomes, and a newly scaffolded Sowerby's beaked whale reference genome, we contribute new and complementary insights into the ecology of a relatively unknown whale. We show that Gray's beaked whales have relatively high levels of maternal genetic diversity, which they have retained over the long term, putatively due to the ability of the species to move across oceans. Such characteristics may facilitate the future survival of this species in a habitat that is undergoing rapid change. Our study shows the ecological information gained from genetic analyses of beached specimens, even with relatively low sequencing effort, and suggests this is a valuable approach for studying highly elusive species where samples are available.Click here for additional data file.


Harnessing heterosis is one of the major approaches to increase rice yield and has made a great contribution to food security. The identification and selection of outstanding parental genotypes especially among male sterile lines is a key step for exploiting heterosis. Two-line hybrid system is based on the discovery and application of photoperiod- and thermo-sensitive genic sensitive male sterile (PTGMS) materials. The development of wide-range of male sterile lines from a common gene pool leads to a narrower genetic diversity, which is vulnerable to biotic and abiotic stress. Hence, it is valuable to ascertain the genetic background of PTGMS lines and to understand their relationships in order to select and design a future breeding strategy.pms3) and temperature sensitive male sterility (tms5) genes. The total gene pool was divided into four major populations as P1 possessing the pms3, P2 possessing tms5, P3 possessing both pms3 and tms5 genes, and P4 containing conventional breeding lines without any male sterility allele. The high genetic purity was revealed by homozygous alleles in all populations. The population admixture, principle components and the phylogenetic analysis revealed the close relations of P2 and P3 with P4. The population differentiation analysis showed that P1 has the highest differentiation coefficient. The lines from P1 were observed as the ancestors of other three populations in a phylogenetic tree, while the lines in P2 and P3 showed a close genetic relation with conventional lines. A core collection of top 10% lines with maximum within and among populations genetic diversity was constructed for future research and breeding efforts.A collection of 118 male sterile rice lines and 13 conventional breeding lines from the major rice growing regions of China was evaluated and screened against the photosensitive (The low genetic diversity and close genetic relationship among PTGMS lines in P2, P3 and P4 populations suggest a selection sweep and they might result from a backcrossing with common ancestors including the pure lines of P1. The core collection from PTGMS panel updated with new diverse germplasm will serve best for further two-line hybrid breeding.The online version contains supplementary material available at 10.1186/s12870-021-03062-x. Oryza sativa L.) is the most important cereal and staple food for more than 50% of global population and 90% of the world’s rice is grown in Asia [indica temperature sensitive genic male sterile line (TGMS) developed in China, which opened a new way of heterosis utilization in rice [Rice  technology is now available for both plant biology research and plant breeding . VariousO. sativa [Currently, single nucleotide polymorphism (SNP)-chip technique is one of the fastest and accurate approaches of plant genotyping as compared to other molecular markers. SNP markers are highly abundant, amendable to high-throughput genotyping, and useful for a number of breeding and genetic applications in crops, including rice . It enab. sativa . The avaIn this study, 131 two-line PTGMS rice lines were clustered on the basis of sterile genes. The clusters were further genotyped by 56 K whole genome rice SNP-chip and the genetic relationship among the materials was analyzed. The core breeding collection of these materials was screened and the selective sweep that occurred during the breeding process was investigated. The results provide important insights into the narrow genetic basis of available PTGMS gene pool and is a reference for the future two-line hybrid breeding and research programs in rice.pms3) and temperature sensitive male sterility (tms5) genes to identify the similarity among them. The total gene pool was divided into four major populations. There were 9.16% (12) lines in the population 1 (P1), possessing the pms3 and denoted as the photosensitive genic male sterile population. A total of 77% (101) lines were in the population 2 (P2) possessing tms5 and denoted as the thermo sensitive genic male sterile population. Only 3.82% (5) lines possessed both pms3 and tms5 genes and were classified as the population 3 (P3). They are denoted as the photo/thermo sensitive genic male sterile population. The other 9.92% (13) lines were the normal breeding lines without any PTGMS gene, classified in the population 4 (P4) and denoted as the conventional breeding population . The genome of all four populations was highly homozygous with 93 to 97% homozygous alleles. The lowest population heterozygosity among alleles was observed in P4 2.66%) followed by P3 3.14%), while the maximum heterozygosity was observed for P1 (6.80%) followed by P2 (3.70%)  genotypes (π = 0.0000339), while the highest genetic diversity (π = 0.0000503) was in P1. The values for genetic diversity among P2 (π = 0.0000348) and P3 (π = 0.0000347) were approximately same  was low, ranging from 7.54 × 10pms3 and tms5 genes in shaping the genetic variation pattern and indicates the genetic similarity of P2 and P3. The linkage disequilibrium (LD) was estimated by r2 for the distance classes of < 1 kb in 30 kb distance around the loci pms3 and tms5. The average r2 value reached the threshold of r2 < 0.5 at the distance of 3.9 kb for markers around the tms5 locus while this value remained above threshold for the pms3 locus  and the conventional lines was not very high (weighted Fst = 0.078), the highest levels (mean weighted Fst > 0.2) of genetic differentiation was revealed by P1 with other populations. It was maximum with P4 (weighted Fst = 0.252) followed by P3 (weighted Fst = 0.178) and P2 (weighted Fst = 0.108)  tree, all the lines could be divided into four clades at the 0.05 genetic distances Fig. . Among tThe results were further supported by the principal component analysis (PCA). The top two principal components PC1 and PC2 explained 19.77 and 7.62% of the total variation, respectively, and divided the germplasm into two major categories. The next two PCs such as PC3 and PC4 explained 6.23 and 3.82% of the total variation, respectively  groups of 28, 5, 23 and 75 genotypes, respectively. Except for the genetically distant five genotypes from P1 grouped as S2 in the structure analysis, a potential widespread genetic introgression from conventional breeding lines of P4 to other populations was observed across K = 2 to K = 4  and male fertile (P4) groups. The high Fst values  were used as criteria for classifying the selective sweeps. There was no selection sweep on chromosome 3, 5, and 8. A total of 1044 candidate genes were found within the sweeps detected on other chromosomes. Some of these genes could be associated with sterility  was studied. A total of 185, 182 and 181 selection sweep signals were observed for pedigree group A, B and C, respectively, in comparison with conventional lines at the Fst threshold of top 5% selective sweep signals , and 11  genotypes were selected from P1 and P2, respectively. Furthermore, one genotype (S239) from P3 and one conventional genotype (Shuhui881) were also included in the core collection.pms3) and temperature sensitive male sterility (tms5) genes could classify the collection of 131 two-line male sterile lines into four major populations in this study. The information about available PTGMS genes and markers will not only help the direct selection of genotypes in hybrid breeding but also for the future research programs. Based on these genetic markers, other germplasm resources could also be evaluated and manipulated to enhance the genetic diversity. In addition, genome-wide marker analysis of various rice populations has demonstrated that shifts in genetic population structures have occurred multiple times in history [Crop breeding programs aim to harness genetic diversity for desirable phenotypes to meet human demands . To achi history . The shi history .Maintenance of genetic purity in inbred lines by minimizing residual heterozygosity (heterogeneity) is important for quality seed production . The thrThere was a low nucleotide diversity (π) in all four populations Fig. , which iTo understand the genetic admixture, the relative kinship coefficients are used as indicators of genetic relationship among the pairs of genotypes. The values of admixture ranged from zero for lack of relation to higher values for stronger relationships. The results from admixture and PCA analyses in this study revealed that the genetic divergence first occurred between the P1 genotypes and their close relatives. The presence of pure lines in each subgroup reinforced the population admixture. The admixture results were reinforced by the phylogenetic analysis and revealed the ancestral relationship among the 131 lines of rice Fig. . As a whpms3 and tms5 genes in shaping the genetic variation pattern, and indicates the genetic similarity of P2 with P3. The results may not totally support the arguments given by [The accumulation of differences in allelic frequencies between completely or partially isolated populations due to evolutionary forces such as selection or genetic drift could be evaluated by population differentiation analysis. The population differentiation was studied by Fst-values. The P1 population was genetically differentiated from other populations. There was no significant difference between population P3 from P4 and the P1 from P2. It suggests a driving force of given by , 33 thatThe genome-wide selection signals related to the male sterility in genotypes was also observed by comparing the male sterile  and male fertile (P4) groups. There are various traits in plants such as plant height, seed color, and stem angle that can be influenced by selective sweep , 34. TheosWAK) protein, its central role in resisting a range of fungal and bacterial diseases [As shown in the Manhattan plot Fig. , the higdiseases .osFBX39), which was reported to contribute in regulation of drought tolerance and regulation of stomatal closure in plants [Two of the selective sweeps on chromosome 2  consisted of 48 and 103 candidate genes, respectively. Amon the candidate genes, we have (i) AP2 domain containing genes which are necessary for flower development, stem cell maintenance, seed development, and abiotic stresses resistance , 39,  based on their breeding history. The ‘Nongkan58S’ with With the identification of genetic similarity and diversity among the PTGMS lines, it was important to design a genetically diverse sample with all possible genetic variation among the population. Selecting a representative sample of all the diversity in the large collection would facilitate the enhanced use of germplasm in the breeding programs . The selpms3) was highly divergent and suggested to be the ancestor of other populations. Several candidate genes were identified in the selective sweeps and could the targets of future studies to unravel their contribution to rice adaptation, genetic differentiation and male sterility.In the present study, the diversity, relationships, selection sweep and breeding inferences were investigated in a collection of 131 rice genotypes including, 118 male sterile lines and 13 conventional breeding lines. The collection was clearly grouped into 4 gene pools according to the presence of the male sterility genes. Based on the within and among populations genetic diversity, a core collection was constructed which will be useful for future breeding efforts. The population containing the photosensitive gene  was conducted in 20 μL containing 3 μL primers, 2 μL 10 × PCR buffer (involving 15 mmol/L MgCl2), 0.3 μL dNTPs (10 mmol/L), 50–100 ng template DNA, 1 U Taq enzyme. The reaction program was pre-denatured at 94 °C for 5 min, 35 cycles with 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C, finally extended for 5 min at 72 °C. Then, the amplified products were electrophoresed on a 6% denatured polyacrylamide gel. On the basis of male sterility genes the germplasm was clustered into four classes as the genotypes carrying “pms3”, “tms5”, both “pms3 and tms5” genes, and the conventional breeding strains without any male sterility allele  ‘pms3’ vs ‘pms3 + tms5’, 3) ‘pms3’ vs Normal, 4) ‘tms5’ vs ‘pms3 + tms5’, 5) ‘tms5’ vs Normal, 6) ‘pms3 + tms5’ vs Normal, and 7)’ pms3,tms5,pms3 + tms5’ vs normal. The results from comparison of normal genotypes to others (PTGMS genes containing) lines were used to identify the candidate genes in selection sweeps and for their GO [To investigate the relationship among genotypes, the population structure was calculated by using all of filtered SNPs in model-based program Structure v2.4.2 . Ten indenotypes  using thenotypes  followedenotypes . PowerMaenotypes  was usedtheir GO  and KEGGtheir GO . FinallyAdditional file 1: Figure 1. Principal component analysis of 131 rice genotypes, with PC1, PC2 PC3 and PC4 classifying the whole germplasm into clusters. Figure 2. The pedigree groups A, B and C obtained from the rice breeding database according to their breeding history, the top one is the ancestor of the other below in the group. The available male sterile gene is indicated in braces with the name of each ancestral genotype. Figure 3. Top 1% selection sweeps on nine chromosomes indicating the genetic differentiation evaluated by Fst values among four populations, the red box in the Chromosome 2 indicated commonly identified and validated top selective sweep. Figure 4. The top enriched gene ontological function revealed Gene Ontology data bases for candidate genes from selective sweep analysis of conventional breeding lines versus pedigree group A (A), B (B) and C (C).Additional file 2: Figure 5. Linkage disequilibrium decay plot, the pairwise LD values (r2) plotted against the physical distance.Additional file 3: Table 1. The list of 118 two-line hybrid environment sensitive genetically male sterile (ESGMS) lines of rice.Additional file 4: Table 2. Chromosome wise values of various genetic diversity parameters for four populations.Additional file 5: Table 3. Top 1% selection sweeps on nine chromosomes indicating the genetic differentiation evaluated by Fst values among four populations.Additional file 6: Table 4. The genome wide selection sweeps indicating the genetic differentiation between the conventional breeding lines and the pedigree groups A, B, and C evaluated by Fst values at the threshold of top 5%.


Olive tree with its main final product, olive oil, is an important element of Mediterranean history, considered the emblematic fruit of a civilization. Despite its wide diffusion and economic and cultural importance, its evolutionary and phylogenetic history is still difficult to clarify. As part of the Mediterranean basin, Algeria was indicated as a secondary diversification center. However, genetic characterization studies from Maghreb area, are currently underrepresented. In this context, we characterized 119 endemic Algerian accessions by using 12 microsatellite markers with the main goal to evaluate the genetic diversity and population structure. In order to provide new insights about the history of olive diversification events in the Central-Western Mediterranean basin, we included and analyzed a sample of 103 Italian accessions from Sicily and, a set of molecular profiles of cultivars from the Central-Western Mediterranean area. The phylogenetic investigation let us to evaluate genetic relationships among Central-Mediterranean basin olive germplasm, highlight new synonymy cases to support the importance of vegetative propagation in the cultivated olive diffusion and consolidate the hypothesis of more recent admixture events occurrence. This work provided new information about Algerian germplasm biodiversity and contributed to clarify olive diversification process. Olea europaea L. sub-sp. europaea var. bot. europaea) is the symbol of the Mediterranean culture and crucial for the Mediterranean diet [Olea sp. in Europe during the Oligocene–Miocene boundary that could be identified as an ancestor of the oleaster (Olea europaea L. sub-sp. europaea var. bot. sylvestris). Phylogenetic analyses have suggested that the most recent common ancestor of the 6 olive subspecies was present during the Late Miocene or Early Pliocene, about 4.0–8.3 million years ago [cuspidata, laperrinei, guanchica, maroccana, cerasiformis, europaea) occurred during the last 3–4 million years, following a series of tectonic and climatic events, thus suggesting a specific evolutionary scenario.The cultivated olive tree, ; some sources also include Islamic Sicily and al-Andalus namely, the Islamic Spain, the name given by the Arabs to the part of the Iberian Peninsula and of Septimania, in the south of France controlled and governed by them. As part of the Mediterranean basin, specifically referred to Maghreb, with a fairly heterogeneous climate between its different regions, Algeria is distinguished by the biological richness of its ecosystems, and it was indicated as a secondary diversification center of several olive subspecies and cultivars . In factAlthough these and other recent studies have provided nuclear and chloroplastic genetic profiles for wild olives and cultivars from the central and southern Mediterranean regions, the Algerian germplasm still remains underrepresented ,10,11,12Efficient and robust molecular markers are increasingly needed for improving varieties, establishing a molecular database for tree identification enabling the spread of common methods for large-scale genetic analysis. At present, Simple Sequence Repeats (SSRs) or microsatellites offer the opportunity to evaluate many potentially polymorphic sites and are the markers of choice for olive DNA fingerprinting and cultivar identification . Microsaeuropaea and laperrinei) in the Eastern and Southern Algeria regions, respectively and characterized them by using 12 microsatellite markers with the main goal to evaluate the genetic diversity and population structure of a wider sample of Algerian olive tree. Furthermore, to provide new insights about the history of olive diversification such as bottleneck, migration and other demography events in the Central-Western Mediterranean basin, we included and analyzed 103 samples of Italian accessions from Sicily considering its peculiar geographic position and, a set of molecular profiles from French, Tunisian, Moroccan, and Spanish cultivars representative of Central-Western Mediterranean area from our internal database. The phylogenetic investigation of 461 genotypes let us to evaluate genetic relationships among Central-Mediterranean basin olive germplasm, highlight new synonymy cases to support the importance of vegetative propagation in the cultivated olive diffusion and consolidate the hypothesis of more recent admixture events occurrence.In this context, we collected 119 endemic Algerian accessions belonging to two different subspecies  dendrogram (p < 0.001) interpreted as a good fit for clustering analysis. The clustering analysis based on Dice coefficient showed two reallydifferent clusters where the subsp. laperrinei was in a single cluster showing a Dice coefficient range between 0 and 0.38. The main cluster included cultivated and putative wild olive genotypes. These genuine olive genotypes did not show a restricted distribution in a single sub-cluster except for WO Kella, Wo Ouled tag, and WO Atounsi but wide genetic variability was however detected among them (Dice coefficient range from 0.48 to 0.7). In the main cluster, six sub-clusters were distinguishable. Two sub-clusters showed a high genetic diversity among olive genotypes even if collected under the same province such in the case of accessions ‘Ballouti’ (Zareza mila) and the accession ‘Messisni ronde’ (Old city mila) 40 km far from each other. Overall, no clear grouping of cultivars in relation to geographical location was observed, except for subsp. laperrinei but clustering seemed mostly related to the fruit end-use  that generated a total variation of 17.18% . The firlaperrinei. The other 41 genotypes (33.6%) were assigned to admixed genotypes.Population structure was investigated using model-based Bayesian clustering applying STRUCTURE analysis without prior information. Olive tree accessions were grouped in two groups of distinct genetic pools (K = 2) and several admixed accessions . AlgeriaAMOVA was calculated between the two genetic groups according to structure analysis results . The obslaperrinei included in a single group. All the other genotypes were included in the second cluster with several differentiated sub-groups. In general, Tunisian germplasm was found in almost all sub clusters.A cluster analysis aimed to evaluate the occurrence of synonymy cases and highlight relationships among the Central-Western olive germplasm, was carried out on a sample of 461 individuals overall. Interestingly, new strict relationships characterized by very high similarity index were brought to light . In thisIn a first sub cluster, a relationship was found between the Sicilian variety ‘Cerasuola’ and the group belonging to the Tunisian variety ‘Zarrazi’ (Dice similarity index: 0.75–0.93) where the accession ‘Sic_45’ corresponded to the authenticated variety ‘Zarrazi’ held in the CREA-OFA collection. In the same sub cluster, the Spanish cultivar ‘Morrut’ clustered with a ‘Chemlali’ group belonging to Tunisian genotypes with a range of similarity beteen 0.7 to 0.82. The accession ‘Sic_22’ was strictly related to the Tunisian accession ‘Rkhami3’ (0.87). Furthermore, the Algerian accessions ‘Ferkani’/’Ferkani Ezzaouia’were really similar to the Tunisian genotypes ‘Chemchali Gafsa’ (0.93).A second sub cluster included 19 Algerian accessions, the French cultivars’ Picholine’, ‘Oliviere’, ‘Picholine di Languedoc’ and ‘Bouteillan’ and the Spanish varieties ‘Arbequina’, ‘Arbosana’, ‘Sabatera’, Joanenca’, and ‘Perafort’ and 8 Sicilian genotypes, three belonging to known cultivars .Another very large sub cluster included most of the Spanish varieties with a wide range of variability. Only 3 Algerian accessions were included in this sub cluster. Relationships were found between Sicilian and Spanish olive germplasm, such as between ‘Pizzutella’ and ‘Ojo de Liebre’ (0.76), ‘Manzanilla de Jaen’ (0.86), and the unknown accession ‘Sic_33’ and ‘Dulzal’ (1).A really interesting sub cluster including 31 Algerian genotypes, 40 Sicilian genotypes, both accessions and known cultivars, and 3 Spanish varieties ‘Manzanillo de Cabra,’ ‘Temprano’ and ‘Gordal Sevillana’. The latter Spanish variety showed strict relationships with the Sicilian varieties ’Pizzo di Corvo’/‘Giarraffa’  and the Algerian genotypes ‘Balbale Fin’, ‘Blilti’, and ‘Grosse du Hamma’ (0.82–1).In a very ‘mosaic’ little subcluster we found Spanish, Algerian, Tunisian, and Sicilian germplasm where the Sicilian cultivar ‘Nocellara del Belice’ showed strict association with Tunisian genotypes. The Algerian accession ‘Gelb al Faroudi’ showed very high similarity with the Sicilian accession ‘Sic_2’ (0.94).Also noteworthy was the substantial genetic uniformity detected between the Sicilian cultivar ‘Sant’agatese’ and the Spanish cultivar ’Cirujal’ (1). The Algerian ‘Messki’ clustered strictly with the Tunisian accession named ‘Meski’ (0.97) and with the Spanish cultivar ‘Fulla de Salze’ (0.85).A well differentiated sub cluster was found including mostly Algerian accessions, 2 Sicilian cultivars ‘Minuta’ (similar to ‘Vaddara’ and ‘Monaca’), ‘Mandanici’ and ‘Cacazzara’. Only one Spanish cultivar (‘Empeltre’) was found in this group. In this group, other strict relationships were also found among Algerian and Tunisian germplasm: ‘Blanquette’/’Rougette’/’Sayali3’/’Chetoui’/’Zeradj’/’ChaibiOntha’/’Tcober’/’Blanquette de Guelma’/’Haouaria’ and ‘Gerboua’ accessions with a variable range of similarity between 0.73 and 1.laperrinei was also detected in a few Spanish cultivars, such as the ancient variety ‘Farga’.Private alleles in the Algerian germplasm were further confirmed by comparison of molecular profiles with the accessions from Tunisia, Sicily, Spain and France  except iThe genetic population structure analysis was conducted on the panel of olive tree accessions excluding putative synonym cases. The Bayesian clustering model implemented in STRUCTURE software indicated K = 2 as the best number of sub-populations  confirmiMolecular variance analysis (AMOVA) was conducted on the two differentiated groups highlighted by STRUCTURE using the same previous described parameters. The observed PhiPT value was highly statistically significant (Fst = 0.274) indicating high differentiation level between the two groups explaining 27% total variance. However, genetic variation within population explained most of the total variance .p = 0.027) confirming results obtained by Diez et al. [Evidence for a bottleneck was evaluated using the genetic differentiated groups individuated by STRUCTURE analysis and here treated as two divergent populations. Three different tests were conducted to detect significant deviations from mutation-drift equilibrium but no significant excess of heterozygosity was detected under one-tailed Wilcoxon rank test. As described above, the two genetic groups did not reflect completely geographic localization of the genotypes here analyzed. In order to evaluate a putative ‘geographic effect’ and infer new hypothesis about diversification process of cultivated olive in the Mediterranean basin, we tried to evaluate bottleneck on dataset including only Spanish and Algerian varieties differentiated by Structure analysis. No evidence of signature for a bottleneck was found in Algerian population while a significant deviation from mutation-drift equilibrium was observed for the sample of 57 Spanish varieties included in the dataset p = 0.07 confirmSicilian varieties such as ‘Giarraffa’, ‘Pizzutella’, and ‘Sant’agatese’ were also included in several offspring combinations. The ‘Giarraffa’ being really genetically similar to the Spanish cultivar ‘Gordal Sevillana’ was included in offspring combinations with several Spanish cultivars and this is an aspect that can mislead the results on the genetic relationships between olive varieties, highlighting the importance to identify potential cases of synonymy for a correct interpretation.However, also ‘Pizzutella’ matched with several Spanish and Algerian varieties. Strict relationships with Spanish germplasm were found also for ‘Sant’agatese’ belonging to the ‘Algerian group’ from the structure analysis. Interestingly the old Spanish variety ‘Farga’ was included in this offspring combination. Several unknown Sicilian accessions showed know Sicilian cultivars as putative parentals, but in a few cases also varieties from other proveniences confirming Sicily as a region really rich in olive trees biodiversity due to its geographical position in the center of the Mediterranean which has favored the passage of many civilizations. The French varieties here considered showed offspring combinations with different provenience genotypes.laperrinei as common ancestral lineage although a low number of samples were available  in order to clarify the diversification process of cultivated olive around the Central-Western Mediterranean basin. Considering the results obtained on the diversity and genetic structure of the Algerian population per se and compared with the germplasm of the Central-Western Mediterranean basin as well as the hypothesis still open on domestication centers, we defined 4 historical scenarios using subsp. vailable . To simpvailable . Since, vailable . We alsolaperrinei. The average number of alleles here observed was higher than those obtained in previous works [laperrinei, one of the first subsp. of the Olea complex that has differentiated following successive phases of aridification of the Saharan region since the Late Miocene [laperrinei and cultivated olive (subsp. europaea var. bot. europaea), while maintaining the interfertility [The first purpose of this work was to improve knowledge on the genetic diversity and population structure of Algerian olive germplasm. For this aim, a set of 12 SSR markers was used for genotyping of 109 cultivars, 8 putative wild  accessions and 2 individuals belonging to subsp. us works ,19. The us works  with thous works ,21. Amon Miocene . Clusterertility ,8,22. Hoertility ,24,25. Alaperrinei) from ITAFV official Algerian collections were analyzed giving new molecular information for further 10 cultivars . In Algeria, the nomenclature of olive cultivars occurs in different languages  and mainly refers to several traits such as the color , surface and size of leaves , the ripening period (Chetoui in winter and Tcober in October) and their origin (Zeboudj and Azeboudj for wild olive). Because of the long history of cultivation and absence of a common criteria for cultivar classification, these indigenous names may have contributed to the confusion in the varieties cataloguing. Cluster analysis highlighted putative synonymies such as the case of the cultivars ‘Aaleh’ , ‘Abani’ and the accession ‘Abani Ezzaouia.’ This result was confirmed also by a previous work [Our results highlighted several groups of synonyms and homonymies, probably due the widest sample ever collected before for Algerian germplasm characterization. In this study, 46 accessions  population. Furthermore, in this model we set up a more recent origin for the Spanish population than the Algerian germplasm. Diez et al.  observedOn the other hand, the STRUCTURE analysis included some Spanish varieties within the ‘Algerian’ group such as the ancient ‘Farga’ variety and the ‘Cirujal’ cultivar genetically very similar to the Sicilian ‘Sant’Agatese’ cultivar. Diez et al.  found thThe large diffusion of the ‘Algerian’ gene pool throughout the Central Mediterranean countries here found corroborates the hypothesis a very early diversification process might have occurred.Olea europaea subsp. laperrinei were collected and georeferenced . The other 76 local accessions (cultivated and WO accessions) were collected in situ both in natural sites and private orchards. The sampling was conducted using different geographical distribution as criteria in the eastern Algeria: 8 accessions from Batna, 1 from Bejaia, 20 accessions, and 1 WO tree from Khenchela, 21 accessions and 5 WO trees from Tassadane Mila, 12 accessions and 2 WO trees from El harrouche Skikda, and 6 accessions from Souk Ahras. A population of 52 Sicilian unknown accessions and a set of 51 reference Sicilian varieties were included in the analysis  olive trees (WO), and 2 accessions of ferenced . Among tanalysis  for the Genomic DNA was extracted from 50 mg finely ground powder of young leaves using a commercial kit . DNA quality and concentration were checked with a NanoDrop 2000 spectrophotometer , diluted to 20 ng/μL and stored at −20 °C until used.2, 2.5 mM dNTPs, 10 μM of forward and reverse primers, and 5 U/μL Taq polymerase, using a thermal cycler . The PCR thermal profile was programmed as follows: a first step at 94 °C for 5 min, 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s. The last step included 7 min of incubation at 72 °C. We also included two reference varieties (Leccino and Frantoio) in PCR amplification to check experimental conditions (data not shown). The GeneScan 500LIZ  was used as internal standard and the SSR migration was carried out using the Applied Biosystems SeqStudio Genetic Analyzer .The following twelve labelled microsatellite markers (SSR) were selected and used for molecular analysis in 4 multiplex combinations: DCA3-6FAM, DCA5-VIC, DCA8-VIC, DCA11-PET, DCA18-6FAM , EMO90-NThe amplified peaks were analyzed using the Gene Mapper v.6.0 software. In order to get a more complete framework of olive relationships in the Central-Western Mediterranean basin, molecular data results from Algerian and Sicilian samples were compared with 154, 74, 10, and 1 molecular profiles of cultivars from Spain, Tunisia, France, and Morocco, respectively, using common markers found in literature ,38,39,40To avoid considering variants of the same accession in the data analyses but also to avoid including families of very closely related individuals that can bias Bayesian clustering analyses , we deciThe number of alleles per locus (A), their frequency (Fa), observed (Ho), and expected heterozygosity (He), polymorphism information content (PIC), were calculated using Cervus v.3.0.7 . The WriA test for evaluation of signature of genetic bottleneck was conducted using Bottleneck v.1.2.02, under the two-phase mutation model (T.P.M.) setting up 70% stepwise mutations and a variance among multiple steps of 12.Relatedness between individuals was evaluated using a parentage analysis computed using Cervus v.3.0.7 software. An approach based on the logarithm of the odds (LOD) score significance was adopted and the following parameters were run: (i) number of offspring: 200,000; (ii) number of candidate parents: 324; (iii) proportion of candidate parents sampled: 0.7; (iv) proportion of loci typed: 0.8218. Default values were adopted for the parameters “proportion of loci mistyped” and “error rate in likelihood calculations”. The relaxed and strict confidence levels were set up to 80% and to 95%, respectively.An Approximate Bayesian Computation (ABC) with DIYABC software v 2.  was usedIn this work we characterized new genetic resources from Algeria which enlarges the olive gene pool. Climate change severely affects the Mediterranean olive growing and new adaptative traits are needed to afford the challenge. A further effort will be needed to evaluate the Algerian germplasm as potentially source of genes encoding adaptative traits for facing olive growing new challenges. The comparison among Algerian olive germplasm and other cultivars and accessions from the Central-Western Mediterranean basin, highlighted new several cases and close relationships among accessions raising a new insight about the importance of vegetative propagation in the olive tree diffusion process along the Mediterranean basin. Demographic analysis corroborated the previous hypothesis of an early differentiation of olive germplasm from Central Mediterranean basin and recent admixture events occurrence confirming the high genetic variability present in the Central Mediterranean area, particularly in the Sicily region. These studies are crucial steps to address the retrieval of genetic resources and begin new pre-breeding activities.


Aldabrachelys gigantea (Aldabra giant tortoise) is one of only two giant tortoise species left in the world and survives as a single wild population of over 100,000 individuals on Aldabra Atoll, Seychelles. Despite this large current population size, the species faces an uncertain future because of its extremely restricted distribution range and high vulnerability to the projected consequences of climate change. Captive‐bred A. gigantea are increasingly used in rewilding programs across the region, where they are introduced to replace extinct giant tortoises in an attempt to functionally resurrect degraded island ecosystems. However, there has been little consideration of the current levels of genetic variation and differentiation within and among the islands on Aldabra. As previous microsatellite studies were inconclusive, we combined low‐coverage and double‐digest restriction‐associated DNA (ddRAD) sequencing to analyze samples from 33 tortoises (11 from each main island). Using 5426 variant sites within the tortoise genome, we detected patterns of within‐island population structure, but no differentiation between the islands. These unexpected results highlight the importance of using genome‐wide genetic markers to capture higher‐resolution genetic structure to inform future management plans, even in a seemingly panmictic population. We show that low‐coverage ddRAD sequencing provides an affordable alternative approach to conservation genomic projects of non‐model species with large genomes. The Aldabra giant tortoise is one of only two giant tortoise species left in the world and survives as a single wild population on Aldabra Atoll, Seychelles. We combined low‐coverage and double‐digest restriction‐associated DNA (ddRAD) sequencing and detected patterns of within‐island population structure, but no differentiation between the islands. These unexpected results highlight the importance of using genome‐wide genetic markers to capture higher‐resolution genetic structure to inform future management plans, even in a seemingly panmictic population. Management efforts often include introductions from these source populations to other suitable locations to lessen the risk of extinction or because the species in question are ecosystem engineers and can be used to restore degraded habitats elsewhere. However, such interventions have important implications for the genetic future of the newly founded population. As only a subset of the individuals in the source population can be moved, genetic diversity is at risk to be lost and artificial population structure may be created in the new populations. Genetic diversity is essential for the adaptive potential of a species, particularly in the face of environmental changes and disease outbreaks Reed, . Hence, One problem with assessing current genetic characteristics of endangered non‐model species is that suitable marker systems, such as simple sets of microsatellites, are often unavailable. Next‐generation sequencing provides promising tools at decreasing costs    Figure . Absolut) Figure . All samDNA extraction was performed with 3 µl of blood (in ethanol) per sample, using the sbeadex™ kit , following the manufacturer's protocol for DNA extraction from nucleated red blood cells. Genomic DNA concentrations were measured with a dsDNA Broad Range Assay kit .2.3in silico restriction digests, performed on the basis of a Chelonoidis abingdonii  genome (NCBI BioProject PRJNA611832), which is the phylogenetically closest available genome for A. gigantea  for the digestion. A single ddRAD‐seq library was prepared by processing the 33 samples following the protocol by Peterson et al.  to 154,599 reads  and heterozygosity <0.5 were retained, the latter to exclude potential paralogs  varied between 2 and 10. The results were analyzed and visualized with CLUMPAK  and each group based on our population structure analyses  was applied to the pairwise FST matrix using the cmdscale function in R .As a measure of population differentiation, weighted pairwise Wickham,  in R . A Tukey's range test  and pop33.1The sequencing effort yielded 23,517,270 raw reads for a total of 33 samples. After adapter removal, quality checking, and demultiplexing, an average of 1,188,685 . The second cluster grouped four of the five GE individuals, six Malabar individuals , one GW individual, and the two remaining P individuals  and 0.008 (range: 0–0.17) among sampling localities Table B. Estima3.3FST estimates calculated among the three Aldabra Islands in the study  were 0, suggesting no evidence for among‐island differentiation. As expected from the PCA and the admixture proportion analysis, the major differentiation was found between M1 and GE (0.06), followed by GE and S&W (0.041) and M1 and M2 (0.039)  = 459, p < 2e−16. All the groups’ mean Watterson's θ values were significantly different from each other at p < .05. Mean π per group was 0.00143–0.00153, with P having the highest and M2 the lowest values  = 75.23, p < 2e−16. Mean π values of all the groups differed from each other, except for GE, which did not differ from M2 or GS&W. While both Wattersons θ and π as well the analyses with the downsampled dataset suggested highest diversity in P, differences in diversity among the other groups were small (but significant) and the relative order of groups differed between analyses.Mean Watterson's t Figure , suggests Figure , again s4A. gigantea population. Our data, although relying on a relatively small sample size (5–11 per island/sampling location), not only revealed the subtle genetic structure of previously bottlenecked populations but also suggested a potentially greater role of passive movement between islands via water in a terrestrial species than previously expected.We used low‐coverage RAD sequencing to investigate the population genetic structure and variation in the endemic A. gigantea) would result in sequencing costs of about 105 USD per sample (2–3× coverage or 6 Gb). Our reduced representation approach could therefore be particularly useful for species with very large genomes.Our study is one of few to focus on a combination of reduced representation sequencing and the genotype likelihood approach to study the population genomics of a non‐commercial and non‐model species. Our case study supports the use of low‐coverage ddRAD sequencing instead of the low‐coverage whole‐genome sequencing . Seawater may play a less important role as a barrier than has been previously assumed . Our study design includes 11 samples (average coverage ~25×) per island , and 5–6 samples (average coverage 11.4–13.7×) per sampling locality/genetic group. The unexpected outcome that there is more population structure than foreseen led to a rather low sample size per genetic group, while the coverage is still within the recommended range. Theoretically one would expect that these recommendations for whole‐genome sequencing would apply for the reduced representation sequencing approach as well, given that the latter is a representative of the former. Our study does indeed show that it is possible to find subtle population structure with this kind of data. Nevertheless, we believe future population genomics studies of Aldabra giant tortoises would highly benefit from more samples per locality and per population as well as more extensive geographical sampling, for example, further western parts of Malabar and small islands in the Aldabra Lagoon.Our study provides the first genomic insight on the wild Aldabra giant tortoise population and the number of tortoises per each main island of the atoll included in this study was limited to 11. Population structure analyses have shown to be robust to extremely low  ; Funding acquisition (supporting); Writing – original draft (lead); Writing – review & editing . Dennis Hansen: Resources ; Supervision (supporting); Writing – review & editing (supporting). Nancy Bunbury: Resources ; Writing – review & editing (supporting). Erik Postma: Conceptualization (supporting); Resources (supporting); Writing – review & editing (supporting). Richard Baxter: Resources ; Writing – review & editing (supporting). Lindsay Turnbull: Resources (supporting); Writing – review & editing (supporting). Arpat Ozgul: Funding acquisition (lead); Supervision (supporting); Writing – review & editing (supporting). Christine Grossen: Conceptualization (lead); Formal analysis (supporting); Funding acquisition (lead); Supervision (lead); Writing – original draft (supporting); Writing – review & editing .Figures S1‐S6Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.


The aim of this study was to summarize the experiences of a single medical center for genetic diagnosis and treatment of prenatal patients. This study includes a retrospective data analysis of 2843 prenatally investigated cases using invasive methods during a 6-year period (2013–2019) at a single tertiary care center. Chromosomal abnormalities were detected in 80 out of 1221 amniotic fluid samples;,178 out of 1608 chorionic villus samples, and 1 out of 14 cordocentesis samples. The most common chromosomal abnormality was trisomy 21. At least one mutation was detected in 63 of the 152 molecular tests performed on fetuses.  Clinical procedures such as ultrasounds and genetic tests are able to provide a better clinical follow-up for pregnant women about the possible congenital anomalies or any genetic condition, with proper genetic counseling and testing methodology. Prenatal diagnosis has become widely available in the last decade and an increasing variety of anomalies can be detected via both chromosomal analysis and DNA-based molecular methods. There are two broad types of tests for genetic disorders: screening tests, which evaluate the risk of a fetus for certain genetic disorders and diagnostic tests, which can detect specific genetic disorders actually present in the fetus. Thus, the practice of prenatal screening is an important step for the identification of changes, resulting in the rapid expansion in genomic testing.There is a plethora of genetic disorders that can be diagnosed during pregnancy. These disorders are classified as chromosomal anomalies, monogenic diseases, complex disorders, and teratogenic disorders. Monogenic disorders occur as a result of mutation(s) in a single gene. Although the majority of genetic diseases are caused by single gene mutations, the numerical and structural chromosomal abnormalities are the most evaluated causes of fetal anomalies and congenital genetic disorders [1]. The early and precise diagnosis of such anomalies during pregnancy is the main goal of prenatal diagnostics. The most common aneuploidies observed in prenatal diagnosis during the second trimester are the trisomies of chromosomes 13, 18, or 21, and the gonosomal abnormalities. Trisomy 13, 18, and 21 account for 89% of autosomal chromosomal aneuploidy pregnancies, which can survive to term. However, these patients mostly present with severe phenotypes [2]. Aneuploidies are traditionally detected by full karyotype analysis of cultured cells collected, which has a very high diagnostic accuracy up to 99.8%. Karyotype analyses are, therefore, the reference method for invasive prenatal diagnosis of fetal chromosomal abnormalities. Rapid detection of these aneuploidies following sampling via amniocentesis is also achievable with fluorescence in situ hybridization (FISH) by the utilization of centromere or locus-specific probes. All these assays can be performed via amniotic fluid or chorionic villus sampling (CVS) by an experienced specialized clinician.Çukurova University includes prenatal analysis of CVS, amniocentesis, and cordocentesis in 2843 pregnancy cases. A retrospective survey covering the 6-year period from 2013 to 2019 at the Medical Genetics Department and Adana Genetic Diseases Diagnosis and Treatment Center (AGENTEM) ofThe cytogenetic and molecular data of the 2843 pregnancies were obtained from amniocentesis (AS) (1221), CVS (1608), and cordosentesis (CS) (14). Prenatal cases in which abnormalities were detected were referred to the medical genetics department. Conventional prenatal karyotypic analyses were performed for all of the subjects. All of the patients underwent an invasive prenatal diagnosis both received pre- and posttest counseling from an experienced genetic counselor and/or medical geneticist.The study protocol was approved by the local ethics committee of the study center with a protocol number of 13/68 .The main indications for chromosome analysis were advanced maternal age, increased risk based on maternal serum screening tests, and abnormal ultrasound findings of chromosomal aberrations. Moreover, an affected child or a pedigree analysis revealing a positive familial history are the indicators of molecular testing.Conventional karyotype analysis was performed on cultured biological samples following the European Cytogeneticists Association Guidelines [3]. The chromosomal abnormality determination was performed in two simultaneously cultured flasks, which were made for each patient sample, and at least 20 metaphases were analyzed at the level of 450–550 banding. The FISH analysis was carried out with commercially available probes according to the manufacturers’ protocols. Specific molecular analyses were performed on DNA isolated from samples, based on family history and predefined mutations. A total of 2843 fetal materials were examined and chromosomal analyses were performed in all cases. Fetal material cell culture failure occurred in only 3 cases; these cases were excluded from the statistical data. Chromosomal abnormalities were detected in 80 of 1221 amniotic fluid samples, 178 of 1608 CVS samples, and in 1 of 14 cordocentesis. The most frequent chromosomal abnormality was trisomy 21 (Down syndrome) from both CVS and amniocentesis samples (Table 1). The FISH technique was used to evaluate the microdeletion syndromes via syndrome-specific region probes in 28 pregnancies while 26 of 28 were examined for Di George syndrome, only 1 was analyzed for Prader–Willi syndrome and another 1 for Cri-du-Chat syndrome. A deletion was detected in the Cri-du-Chat FISH test, but the rest were reported as normal. FISH was also used to characterize 131 samples for rapid aneuploidy, of which 94 were determined to be normal. Of the 37 samples with a FISH-identified chromosomal abnormality, 20 were from AS specimens and 17 were from CVS specimens (Table 2). Molecular testing was performed in 152 cases which revealed 41.45% positivity rate (n = 63). The numbers together with the most frequent genes and the mutations detected are listed in Table 3 ). Genetic counseling is defined as a communication process with individuals and their families about the genetic diseases with the aim of risk reducing for the recurrence, mostly through the provision of both prenatal and postnatal diagnosis options and offering the most current therapies together with psychotherapeutic support [4]. Nondirectiveness is the main and basic principle that requires the maintenance of a neutral stance of genetic counselor in order to support and also to respect the patient’s personal values and decisions . A prenatal diagnosis can be either invasive or noninvasive. Invasive prenatal sampling is necessary to apply molecular methods if the results of genetic testing has an impact on clinical decision-making and clinical outcomes such as when the mother is a carrier for X-linked diseases, or if both parents are carriers for an autosomal recessive disorder, as well as when either of the parents is afflicted with an autosomal dominant disease. It is important to not forget that the prenatal genetic testing is an optional strategy that depends on the patient’s needs and decisions. The patients should be informed about what the tests can reveal, before the sampling procedures, as well as after the results are in. The major supporting tool for a medical geneticist is the careful pre- and posttest counseling [6].According to the literature, Down syndrome (trisomy 21) is the most common chromosomal abnormality, with an incidence estimated one in 700–1000 live births. It is considered to be one of the major congenital causes of intellectual disability in the human population. In our study group, numerical chromosomal anomalies were detected in 80 of the AS-diagnosed patients and Down syndrome accounted for 32 (40%) of them. Chromosomal anomalies were found in 178 of the CVS-sampled patients, and Down syndrome was diagnosed in 68 (38.2%) of them. In previous studies, the rate of chromosomal abnormalities had been found lower than that in our study, but reported series were also in accordance with our results as the most common anomaly was the Down syndrome . The higher rates in our study depend on both the sensitivity of screening tests applied to all pregnant women to predict Down syndrome and a well-established clinical algorithm of perinatology and genetic counseling together with clinical follow-ups. The other main reason is the fact that our center and the hospital give a healthcare service as a reference center for prenatal diagnosis and a main hub for genetic counseling, from children to adults. As prenatal screening and diagnostic techniques have become more enhanced and widely available, medical geneticists should expect to provide information and support following a new diagnosis of Down syndrome on a frequent basis. If the fetus is diagnosed with Down syndrome during the prenatal period, the common problems in Down syndrome should be explained to the family clearly, but the decision should be made by the family as to whether to terminate the pregnancy.It has been estimated that a minimum 0.05% of newborns have unbalanced structural chromosomal abnormalities, which encompasses pathological alterations resulting from the breakage or exchange of chromosome material . However, there were no fetuses with structural chromosomal anomalies detected among our case series. All of the chromosomal aberrations we detected were previously known alterations; therefore, the clinical outcomes were predictable and the content of genetic counseling was clear. Uncommon chromosomal abnormalities in prenatal diagnosis require correlations between the cytogenetic aberrations and ultrasonography findings. These rare chromosomal abnormalities are very important for providing easy to understand genetic counseling for the parents [6]. Prior to molecular prenatal testing, genetic counseling should be offered to discuss the information ratio and limitations of prenatal testing along with the consequences of both test results with or without abnormalities (mutations). Hence, a normal prenatal test result cannot absolutely exclude the diagnosis of a genetic disease. Molecular tests were performed as prenatal tests in a 152-person pregnant group. Sixty three of them identified a mutation in the fetus. The most frequent tested genes were spinal muscular atrophy (SMA), cystic fibrosis (CFTR), and phenylketonuria (PAH). All of the families with specific molecular testing had a predetermined history of disease and mutations in the parents had been identified before prenatal testing. The genetic counseling and the family’s understanding of the disease was easier due to familial history in this group.The advances in prenatal diagnosis enabled us to plan the antenatal care and newborn units, as well as the delivery. Together, our data indicates the general acceptance, carrier frequencies, and prenatal testing results in the south-east Mediterranean region of the Turkish population. Moreover, our study describes the first comprehensive frequencies and risks from a large cohort tested from a cytogenetic and molecular genetic perspective in the region. This data can serve as a reference for future screening and genetic counseling. The acceptance of prenatal screening by the efforts of medical geneticists also offers insights into the future potential for whole exome or genome sequencing of all known mutations causing serious diseases of newborns. Thus, preventive medicine is also one of the main aims for all health systems, which includes simultaneous screening of both partners, in conjunction with prescreening genetic counseling. Supplementary MaterialsClick here for additional data file.


Oxytropis plants are widely distributed in the grasslands in northern China. Some Oxytropis species have been reported to contain the mycotoxin swainsonine, an alkaloid which causes poisoning in livestock, referred to as locoism. Previous studies showed that endophytic fungi (Alternaria oxytropis) symbiotically associate with these Oxytropis species to produce swainsonine. However, the influence of variation within the Oxytropis genus on the fixation or loss of symbiosis and toxicity is poorly understood, as is the influence of environmental factors. Here we used a collection of 17 common Oxytropis species sampled in northern China to assess genetic diversity using genotyping by sequencing which was compared with the levels of the endophyte and swainsonine. Results showed that nine Oxytropis species have detectable A. oxytropis colonisation, and seven Oxytropis species contain sufficient swainsonine to be considered poisonous, whereas the rest may be non-toxic. Species variation rather than the genetic lineage was associated with the fixation or loss of endophyte and swainsonine production, which appears to have resulted from genetic drift. Genotype × Environment (G × E) effects were also found to influence endophyte and swainsonine levels amongst species of the Oxytropis genus. Our study will provide a better understanding about the evolutionary basis of A. oxytropis symbiosis and swainsonine biosynthesis in locoweeds. Oxytropis spp. (Fabaceae) are important perennial herbs that are native to temperate to frigid zones in Europe, Asia and North America +) were converted to ug/mL concentration based on the derivation of a standard curve using commercially obtained swainsonine  that was assessed by FI-FI-ESI-MS.Swainsonine extraction was performed as described by . The samA. oxytropis in plant DNA content (pg/ng). Swainsonine content in each sample was obtained and represented as the percentage of swainsonine in plant dry weight (%). To keep consistent with previous studies, we applied a 0.0001% limit for swainsonine. Any value below this value was considered as not detected [Endophyte levels were calculated and represented as the DNA content of endophyte detected .Oxytropis spp.In order to obtain more accurate plant genetic distance and geographic distance analysis results, after re-dividing all samples into 39 populations according to sampling locations, we used the K2P model in MEGA 7.0 to recalculate the genetic distance matrix. The geographic distance matrix between any two sampling locations was calculated by the latitude and longitude information in Google Earth (V7. 15. 1557). Finally, the “ade4” program in the R package was used to conduct Mental tests to analyse the correlation between the genetic distance and geographical distance of http://data.cma.cn/ (accessed on 20 September 2019)), and Kriging interpolation on the data was performed in ArcGIS 10.2 software based on the coordination of the 39 populations  in the 39 populations using vcftools [Finally, in order to show the relationship between the genetic diversity and the contents of vcftools . Then we


The conservation of genetic diversity, both among and within breeds, is a costly process. Therefore, choices between breeds and animals within breeds are unavoidable, either for conservation in vitro (gene banks) or in vivo . Nowadays, genomic information on breeds and individual animals is the standard for the choices to be made in conservation. Genomics may accurately measure the genetic distances among breeds and the relationships among animals within breeds. Homozygosity at loci and at parts of chromosomes is used to measure inbreeding. In addition, genomics can be used to detect potentially valuable rare alleles and haplotypes, their carriers in these breeds and can facilitate in vivo or in vitro conservations of these genomic regions. Genetic diversity is the set of differences between species, breeds within species, and individuals within breeds present in their DNA or observed in animals as a consequence . GeneticIn the past, these criteria could only be based on the history of breeds and pedigree analysis. The starting-point for these pedigree-based calculations is that each individual receives a random sample of 50 percent of the alleles of its parents in the random process of meiosis. Nowadays, in the era of genomics, DNA-analysis of individuals and parents provides full insight into which alleles of an individual came from its sire and which came from its dam. The measures of genetic relationships and inbreeding based on pedigrees are expectations, while molecular genetic estimates of inbreeding are the particular realisations of such expectations .Inbreeding can be more accurately established with genomics than with pedigree-based measures. Runs of Homozygosity (ROH) play an important role in these measures. Runs of homozygosity are contiguous lengths of homozygous genotypes that are present in an individual due to parents transmitting identical haplotypes to their offspring. Haplotypes are contiguous loci in a very high linkage equilibrium. The length of these ROH may reflect either the diversity in the population or the mating practices, or both. Long ROHs are the result of recent inbreeding, while short ROHs are signs of inbreeding in the past. The accuracy of molecular measures is also higher because pedigree errors are easily detected. Genomics can also be used to detect potentially valuable rare alleles and haplotypes and their carriers in these breeds and promote the conservation of these genomic regions. Nowadays, genomic management is an important tool in breeding programs for livestock animals in large and in small populations .In the conservation of genetic diversity, a lot of attention is given to the conservation of breeds within species. In the genomics era, Sponenberg  defined The conservation of a breed can be realized in vivo: by keeping animals of the breed alive. Numerically, small breeds can still fulfil a sustainable role in society in the utilisation of rural areas . HoweverThe conservation of genetic diversity can also be realized by building cryo-collections of germ plasm. In the past 50 years, in many countries, farm animal gene banks are established that mainly conserved cryo-conserved semen. Gene bank collections might be used to re-establish an extinct breed. Moreover, it can be used to support the live conservation of a small population through the use of semen of sires less related to the animals in the live population . This miIn this Special Issue, you will find the results of recent studies that illustrates that the application of genomic tools in improves the accuracy of decisions to be made in the conservation of animal genetic diversity.


As a result, we demonstrated that import of STAT3 inside mitochondria requires Y705 phosphorylation by Jak, whereas its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. These data were confirmed using mouse embryonic stem cells: although the Y705-mutated STAT3 cannot enter mitochondria, the S727 mutation does not affect import into the organelle and is responsible for STAT3-dependent mitochondrial transcription. Surprisingly, STAT3-dependent increase of mitochondrial transcription appears to be independent from STAT3 binding to STAT3-responsive elements. Finally, loss-of-function experiments, with chemical inhibition of the JAK/STAT3 pathway or genetic ablation of stat3 gene, demonstrated that STAT3 is also required for cell proliferation in the intestine of zebrafish.The STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined  Summary: Mitochondrial import of STAT3 requires Y705 phosphorylation by Jak, whereas STAT3 mitochondrial transcriptional activity and its effect on proliferation depend on the MAPK target S727. Many of the major human malignancies display elevated levels of constitutively activated STAT3 . Most inSTAT3 transcriptional activity is regulated by phosphorylation of two separated residues. When Janus kinases 1/2/3 (JAK1/2/3) phosphorylate its tyrosine 705 (Y705), STAT3 homodimerises, enters the nucleus, binds to STAT3 response elements and triggers transcription of its target genes . On the 2+, with consequences on the mitochondrial Ca2+ levels and on life-death cell-fate decisions. This function is crucial for the maintenance of apoptosis-resistant cells in the tumour niche ; regulation of mitochondrial gene expression; and regulation of the mitochondrial permeability transition pore  and considering the polycistronic nature of mtDNA-transcribed genes that results in stoichiometric mtDNA transcription  of the TeO  analysis of 48 h post fertilisation (hpf) larvae against Pcna protein and, subsequently, we performed in situ hybridisation (ISH) using RNA probes to detect pcna, stat3 and mt_nd2 mRNAs. In pcna mRNA perfectly colocalise, demonstrating that both of these stainings can be used as reliable proliferation markers. Moreover, we demonstrate that Pcna protein expression and mt_nd2 completely colocalise in the TeO at this developmental stage , provided with a nuclear export domain that makes it unable to localise to the nucleus (MLS_mStat3_NES) . This chimeric protein is completely devoid of nuclear functions as assessed by quantitative real time reverse transcription PCR (qRT-PCR) analysis of Socs3, the most direct Stat3 target gene , and efficiently localises only inside the mitochondrion, as revealed by its colocalisation with the mitochondrial marker ATAD3, both in transfected mouse ESCs, and in zebrafish cells .In order to understand whether STAT3 mitochondrial activity regulates mtDNA transcription and promotes proliferation also MLS_mStat3_NES mRNA was injected into zebrafish embryos we could observe that this modified form of Stat3 was unable to induce the expression of its canonical target gene socs3a ; however, we detected, both by ISH and qRT-PCR, a significant increase of mitochondrial transcription at 24 and 48 hpf . Moreover, mitoSTAT3 does not affect the expression of p53  mRNA in zebrafish larvae, demonstrating that MLS_Stat3_NES injection does not induce harmful effects in cellular homeostasis . It is worth noting that, as assayed by pcna analysis, overexpression of MLS_mStat3_NES mRNA also induced a proportional increase of this proliferation marker in the same tissues in which mitochondrial transcription was stimulated, i.e. the PML  mitoSTAT3  have been identified in the mitochondrial D-Loop   and STATitoSTAT3 A,B. It iitoSTAT3 C. This rin vitro studies, to enhance the electron transport chain (ETC) ] with two mutated forms, Y705F and S727A, able to prevent phosphorylation of residues 705 and 727, respectively (in situ hybridisation (FISH) revealed a significant increase of mitochondrial transcription in the PML of the TeO . In conclusion, phosphorylation of both residues is needed for STAT3-mediated increase of mtDNA transcription in the PML. On the other hand, when the mutated isoforms are forcedly targeted to the mitochondrion [by using both the MLS and the nuclear export signal (NES)], the S727A mutation prevented mitoSTAT3-mediated activation of mt_nd2 gene expression, whereas the STAT3-Y705F mutated isoform retained its mitochondrial transcriptional activity . This finding implies that Y705 is not directly involved in mitochondrial transcription.It is known that JAK1/2/3-mediated phosphorylation on Y705 controls the nuclear activity of STAT3 and also ensures the stability of STAT3 monomers in the cytoplasm . On the in (ETC)  as well in (ETC) . To veriectively . Interes the TeO A,B. Incr the TeO C,D. Nota−/−Stat3 ESCs transiently transfected. Although transient transfection of STAT3-Y705F resulted in its nuclear and sporadic mitochondrial localisation, transfected mitoSTAT3-Y705F localised exclusively to mitochondria .As phosphorylation of S727 is needed for mitoSTAT3-driven mtDNA transcription, we tested whether this post-translational modification is also required for the increased expression of the proliferation marker Pcna in the PML. The expression of the proliferation marker Pcna in the PML of 48 hpf embryos injected with NES mRNA A, indicain vivo the involvement of the MAPK pathway in phosphorylation of S727, and its downstream effects for mitoSTAT3-driven cell proliferation, we decided to use the MEK kinases inhibitor PD98059 , we administered this compound to zebrafish embryos. Interestingly, WT larvae treated from 24 to 48 hpf with PD98059 displayed a significant reduction of mt_nd2 and pcna transcript levels, suggesting that MEK inhibition reduced the expression of mitochondrial genes and the proliferation marker pcna  in which the S727 is substituted with aspartic acid. This modification has been already classified as phosphomimetic . In addition, proliferation-related activity was found to be significantly reduced in the PML of 72-hpf AG490-treated larvae, as assayed by ISH using anti-pcna probe   A. The acon (dpf) ; (2) STAon (dpf) ; (3) Staon (dpf) . The admon (dpf) A,B. Moreon (dpf) C,D and ron (dpf) E,F. TakeStat3 constructs, indicate that STAT3 regulates mitochondrial transcription and cell proliferation. We decided to genetically support our observations. First, we used the zebrafish ia23stat3 mutant   reporter line characterised in stat3 gene locus confirmed that stat3 had been successfully mutagenised in exons 14, 22 and 23  , which int lines , or to aSPR/Cas9 . We eval2 and 23 A″. Notabgulation B and sta mutants , was not mutants C. Hence,of Stat3 D-G.Fig.stat3 knockout displayed a significant and clear reduction of both mt_nd2 and pcna transcripts . Moreover, in order to evaluate the volume of mitochondria, we crossed stat3 mutants with the Tg(CoxVIII-mls:EGFP) transgenic line that expresses a mitochondria-localised form of enhanced GFP. No clear change in the total mitochondria volume was present in the intestine of −/−stat3 larvae compared with +/+stat3 sibling larvae . Together with previous results, this highlights that mitoSTAT3 is only acting as a regulator of mitochondrial transcription, without impacting on mitochondria biogenesis or homeostasis. Notably, ∼70% of −/−stat3 larvae displayed severe defects in the development of intestinal epithelium , our data strongly support, in vivo, a direct link between mitoSTAT3 activity and mitochondrial transcription. This is consistent with the mitochondrial transcriptional role of mitoSTAT3 found in vitro in murine ESCs, as previously reported . Surprisingly, this inhibitor does not have any effect on mitochondrial transcription. It is known from literature that other kinases, such as CDK8  that induces dimerisation and translocation to the nucleus, and at serine 727 (S727), the function of which has been reported to have unclear effects on STAT3 nuclear transcriptional activity . On the + uptake . Here, w as CDK8 , TBK-1  STAT3. Together with the fact that mitochondrial STAT3 has been identified as a contributor to RAS-dependent cellular transformation , we supp4 and 5 g NaHCO3 for 1 l) and kept in a 12 h light/12 h dark (LD) cycle. All experimental procedures complied with European Legislation for the Protection of Animals used for Scientific Purposes (Directive 2010/63/EU) and were approved by the Animal Experiments Committee of the University of Padua and the Italian Ministry of Health (23-2015-PR).Zebrafish were staged and fed as described by ia23stat3 mutants and ia28Tg(7xStat3-Hsv.Ul23:EGFP) transgenic zebrafish are described in Tg(CoxVIII-mls:EGFP) transgenic zebrafish line is described in ®  for qRT-PCR analysis.The following chemical compounds were used: AG490 ; PD98059 ; PD0325901 ; and Balapiravir . Before drug administration, a hole was made in the chorion of 8 hpf embryos, whereas 24 hpf embryos were dechorionated. All drugs were dissolved in DMSO and stored in small aliquots at −20°C. We performed 100 µM AG490 treatment from 24 to 48 hpf or from 24 to 72 hpf. We administered 60 µM AG490 in 3-6 dpf treatments, and 12.5 µM PD98059 or 10 μM PD325901 (same treatment as PD98059) treatments were administered from 24 to 48 hpf. Balapiravir solution (50 µM) was administered from 8 to 48 hpf. After treatments, embryos were anaesthetised and fixed in either 4% paraformaldehyde (PFA)  in PBS for ISH, FISH and IHC or in TRI Reagentstat3 gene was produced as described in stat3 gene were designed with CHOPCHOP software https://chopchop.cbu.uib.no/ and provided by Synthego.sgRNA against exon 14 of the stat3 genes were injected into one-cell-stage eggs. Subsequently, 48 hpf injected larvae were collected for DNA and RNA extraction and for imaging. Sequences of sgRNAs and of primers used for genotyping are listed in Tables S1 and S2, respectively.Cas9 protein  and sgRNAs against antisense of exons 14, 22 and 23 of mStat3, mStat3_Y705F and mStat3_S727A coding sequences were obtained from pCEP4-Stat3-WT, pCEP4-Stat3-Y705F and pCEP4-Stat3-S727A plasmids  and sub-cloned into a pCS2+ backbone using the In-Fusion® HD Cloning Kit (Clontech). MLS_mStat3_NES CDS, containing the murine Stat3 cDNA flanked by an MLS and an NES, was subcloned into a pCS2+ plasmid from a 70_pPB-CAG+MLS+Stat3+NES-pA-pgk-hph-2-2 plasmid by digestion with XbaI and BamHI. Mutated forms of MLS_mStat3_NES mRNA were obtained from pCS2+MLS_mStat3_NES by site-directed mutagenesis using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs); primers are indicated in Table S3.in vitro transcribed using the mMESSAGE mMACHINE® SP6 Transcription Kit (Thermo Fisher Scientific) and purified using the RNA Clean and Concentrator kit (Zymo Research). A mix containing mRNA , Danieau injection Buffer and Phenol Red injection dye, was injected into one-cell-stage embryos.mRNAs were ® EvaGreen® qPCR Mix Plus  following the manufacturer's protocol. The cycling parameters were: 95°C for 14 min, followed by 45 cycles at 95°C for 15 s, 60°C for 35 s and 72°C for 25 s. Threshold cycles (Ct) and dissociation curves were generated automatically by Rotor-Gene Q series software. Sequences of specific primers used in this work for qRT-PCR and RT-PCR are listed in Table S4. Primers were designed using the software Primer 3 (https://primer3.ut.ee/). Sample Ct values were normalised with Ct values from zebrafish gapdh and results were obtained following the method described in For expression analysis, total RNA was extracted from pools of 15 7 dpf larvae or 35 48 hpf embryos with TRIzol reagent . mRNA was treated with RQ1 RNase-Free DNase  and then used for cDNA synthesis with Superscript III Reverse Transcriptase  according to the manufacturer's protocol. qPCR was performed in triplicate with EvaGreen method using a Rotor-gene Q (Qiagen) and the 5× HOT FIREPolImmunoblotting was performed as previously described in 2O2 solution added immediately before use). The samples were then postfixed with 1% osmium tetroxide plus potassium ferrocyanide 1% in 0.1 M sodium cacodylate buffer for 1 h at 4°C. After three water washes, samples were dehydrated in a graded ethanol series and embedded in an epoxy resin (Sigma-Aldrich). Ultrathin sections (60-70 nm) were obtained with an Ultrotome V (LKB) ultramicrotome, counterstained with uranyl acetate and lead citrate and viewed with a Tecnai G2 (FEI) transmission electron microscope operating at 100 kV. Images were captured with a Veleta (Olympus Soft Imaging System) digital camera.Cells were fixed in a 24-well plate with 4% PFA in PBS (pH 7.4) for 30 min at room temperature (RT). After fixation cells were washed five times with PBS (5 min each), blocked and permeabilised with 5% normal goat serum and 0.1% saponin in PBS for 30 min, and then incubated with primary antibody anti-STAT3 mouse monoclonal  overnight at 4°C in PBS 5% normal goat serum and 0.05% saponin. After five washes with PBS (5 min each), cells were incubated with HRP-conjugated Fab fragments of the secondary antibody for 2 h at RT. After five washes, cells were incubated in the DAB solution  or HOECHST 33342  where specified. Images were acquired using a Leica SP2 confocal microscope equipped with a CCD camera.ESCs were grown and transfected as previously described by in situ hybridisation (WISH) on zebrafish embryos was performed as previously described (stat3 probe was obtained by PCR amplification from embryo cDNA using stat3_probe-fw (5′-TGCCACCAACATCCTAGTGT-3′) and stat3_probe-rv (5′-GCTTGTTTGCACTTTTGACTGA-3′) primers. The mt_nd2 probe was obtained by PCR amplification from embryo cDNA using mt_nd2-fw (5′-GCAGTAGAAGCCACCACAAA-3′) and mt_nd2-rv (5′-GGAATGCCGCGGATGTTATA-3′) primers. The pcna probe was obtained as described by sox9b probe was obtained as described by Whole mount RNA escribed . It is wCombined IHC and WISH were carried out as follows. IHC was performed as described in pcna, stat3 and mt_nd2. Alkaline phosphatase-mediated Fast Blue staining was performed by incubating samples in 0.25 mg/ml Fast Blue BB , 0.25 mg/ml Naphtol-AS-MX-phosphate , 100 mM Tris HCl (pH 8.2), 50 mM MgCl2, 100 mM NaCl and 0.1% Tween 20. After glycerol-based mounting, green (from IHC) and far-red (from WISH) emissions were acquired using a Leica SP5 confocal system. Signals were analysed by Volocity 6.0 software (Perkin Elmer) and images organised by Adobe Photoshop v. 21.1.1.After IHC, samples were rinsed in 1× PBS and subjected to WISH, according to Larvae were anaesthetised and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. After that, samples were dehydrated, embedded in epoxy resin, and prepared according to standard protocols by the Trasmission Electron Microscopy facility at the Department of Biology, University of Padova .t-test. The P-values are indicated with the following symbols: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. For quantitative analysis, the sample size for each experiment was calculated assuming a confidence level of 95% (z-score 1.96), a standard deviation of 0.5 and a confidence interval (margin of error) of 5%.Statistical analysis was performed with GraphPad Prism V6.0. Data are presented as mean±s.e.m. and statistical analysis was determined using the unpaired two-tailed Student's