Tachycnemis seychellensis, the only species of its genus, is endemic to the four largest granitic islands of the Seychelles archipelago and is reliant on freshwater bodies for reproduction. Its presence in the Seychelles is thought to be the product of a transoceanic dispersal, diverging from the genus Heterixalus, its closest living relative (currently endemic to Madagascar), between approximately 10–35 Ma. A previous study documented substantial intraspecific morphological variation among island populations and also among populations within the largest island (Mahé). To assess intraspecific genetic variation and to infer the closest living relative(s) of T. seychellensis, DNA sequence data were generated for three mitochondrial and four nuclear markers. These data support a sister-group relationship between T. seychellensis and Heterixalus, with the divergence between the two occurring between approximately 11–19 Ma based on cytb p-distances. Low levels of genetic variation were found among major mitochondrial haplotype clades of T. seychellensis (maximum 0.7% p-distance concatenated mtDNA), and samples from each of the islands (except La Digue) comprised multiple mitochondrial haplotype clades. Two nuclear genes (rag1 and tyr) showed no variation, and the other two (rho and pomc) lacked any notable geographic structuring, counter to patterns observed within presumably more vagile Seychelles taxa such as lizards. The low levels of genetic variation and phylogeographic structure support an interpretation that there is a single but morphologically highly variable species of Seychelles treefrog. The contrasting genetic and morphological intraspecific variation may be attributable to relatively recent admixture during low sea-level stands, ecophenotypic plasticity, local adaptation to different environmental conditions, and/or current and previously small population sizes. Low genetic phylogeographic structure but substantial morphological variation is unusual within anurans.The hyperoliid frog  Like alr bodies . The abuts range . The souT. seychellensis from the four islands, including in adult body size and colouration, presence or absence of tubercles on various parts of the body and limbs, presence or absence of grooved digit discs, and several morphometric characters. Four morphometric characters, not dependant on sex, were found to vary significantly between all populations: internarial width, pes length, toe disc length, and length of metatarsal tubercle. An additional 10 male and two female characters varied significantly. Specimens from the more southerly islands of Mahé and Silhouette are morphologically the most similar to each other (T. seychellensis (one marsh- and one stream-associated) only 1 km apart were as morphologically different from each other as they were to T. seychellensis on Silhouette. The populations of T. seychellensis on the more northerly islands (Praslin and La Digue) were morphometrically as distinct from each other as they were from the southern populations. Despite these large morphological differences, T. seychellensis could be challenged by high genetic diversity and/or substantial phylogeographic structure.Using univariate and multivariate analyses, ch other . HoweverTachycnemis seychellensis has a complicated taxonomic history. Since TachycnemisTachycnemis only through bibliographic reference to its single included species  and rho (nuDNA) did so with much support. Paraphyly of Heterixalus with respect to Tachycnemis was also found (though without strong support) by Heterixalus species, Tachycnemis and Heterixalus were sister taxa. Using multiple Heterixalus species in their analyses, Tachycnemis–Heterixalus sister-group relationship. Where trees for individual loci are discordant, coalescence-based methods can be expected to yield more accurate species phylogenies than multilocus concatenation  assess genetic variation within T. seychellensis across its range and test the hypothesis that it is a single, morphologically highly variable species.Here we report phylogenetic analyses of mtDNA and nuDNA data (3228 base pairs (bp)) to (1) test the hypothesised sister-group relationship between 22.1Tachycnemis seychellensis tissue samples  were obtained from 52 voucher specimens from the Seychelles islands of Mahé (15 samples), Silhouette (15 samples), Praslin (15 samples) and La Digue (7 samples) between 1988 and 1991; these correspond to four of the five populations sampled by 2.2T. seychellensis samples using the Qiagen DNeasy™ Tissue Kit. Three mitochondrial gene fragments were sequenced for all samples: cytochrome b (cytb), cytochrome oxidase subunit 1 (cox1) and 16S rRNA (16s). Four nuclear loci were also sequenced: rhodopsin exon 1 (rho), recombination activating gene 1 (rag1), tyronsinase precursor (tyr) and pro-opiomelanocortin (pomc). The rag1 and tyr sequences showed no variation within T. seychellensis, and thus only a subset of individuals from each locality were included in the analyses of the relationships between Tachycnemis and Heterixalus.Genomic DNA was extracted from liver, heart and muscle samples from the 52 2O. Cycling conditions were: denature at 94 °C for 60s; followed by 35  or 40  cycles of denaturing at 94 °C for 30 s, annealing at 48 °C (cox1), 50 °C (16s), 52 °C (cytb), 56 °C (rag1), 60 °C (rho), or 62 °C  for 30 s, and extending at 72 °C for 30 s; and a final extending step of 72 °C for 5 min.Primer information is given in 2.316s were checked for pseudogenes and insertions by searching for stop codons and indels  implemented in BEAST v.1.7.4 .To infer allelic phases from polymorphic sites in the nuDNA, the program PHASE v.2.1  was usedD , tyr, rag1 and rho as four separately evolving loci. Sequence data for Heterixalus spp. were previously published  for all partitions and estimated relative to the mtDNA partition. Two MCMC chains were run for 2 × 108 generations, with trees sampled every 10,000 generations, to ensure convergence was reached the first 5% were discarded as burn-in, although convergence was reached prior to this cut-off. The species-tree Yule-process prior was used with the piecewise linear and constant-root population-size model. Convergence of all parameters was verified using Tracer v.1.5 , 357 bp of rho , and 611 bp of tyr  giving a total sequence length of 3228 bp. Partitioning schemes and nucleotide models used in analyses are presented in We aligned 1500 bp of mtDNA (consisting of 424 variable sites (v.s.), of which 391 were parsimony informative (p.s.)), 760 bp of ∗BEAST dataset by linked loci provided evidence for T. seychellensis being the sister taxon to a monophyletic Heterixalus; a sister-group relationship between H. madagascariensis and H. punctatus; for H. andrakata being the sister taxon to H. tricolor + H. variabilis; and for H. betsileo being the sister taxon to the andrakata–tricolor–variabilis clade (H. rutenbergi and H. luteostriatus) are unresolved , all of which were parsimony-informative (p.s.); cytb 763 bp ; and cox1 786 bp . The dataset was almost complete, with very little missing sequence data across all genes and no genes missing for any individual. No saturation was detected. The rag1 and tyr data were constant in 20 and 19 samples sampled across all populations, respectively. The pomc data consisted of 629 bp  for 50 specimens; and rho 337 bp  for 25 specimens. The sample size for pomc and rho was reduced because of a shorter amplified sequence length of some samples and because of a lack of confidence in the accuracy of the PHASE calling of the small number of variable sites.We aligned three mitochondrial genes for the 52 specimens: The best partitioning strategies and models as determined by PartitionFinder for the mtDNA analyses were the same under AIC and AICc but different under BIC . Thus, tp-distance of 1.5% between any of the seven haplotype groups, and no simple geographic structure is observed in the mtDNA tree  and/or by multiple admixture events possibly during eustatic sea-level fluctuations. The latter is plausible given that all island populations, apart from La Digue, have multiple mtDNA haplotype clades and that nuDNA haplotypes show no clear geographic structuring. The relatively low levels of genetic variation within the Seychelles treefrog are comparable with several other Seychelles taxa such as Drosophila flies  anurans and it is difficult to find any examples in the literature . These explanations could be tested using population-genetic analyses of data from more rapidly evolving nuclear markers.The combination of low levels of genetic diversity (and little phylogeographic structure) within see also . The latcytb p-) distances between populations of T. seychellensis on different islands (see Genetic (ands see  provide ands see , little 5T. seychellensis and a monophyletic Heterixalus. There is little genetic variation within T. seychellensis, even among populations on the four different islands within its range, and the variation is not strongly spatially structured. This is consistent with T. seychellensis is the result of local ecological adaptation and/or small population sizes now and/or in the past, though ecophenotypic plasticity might also be considered.We find support for the sister-group relationship between


Hylaeamys megacephalus and Oecomys aff. roberti, two cricetid rodent species from the mid-Araguaia River in central Brazil, using mitochondrial DNA sequence data. Specifically, we aim to test whether the Araguaia River acts as a barrier to the gene flow of these two species and to assess how ecological traits, such as locomotion habits and habitat preferences, may influence differences in levels of genetic diversity and differentiation. As both species occur in flooded forests, neither showed genetic differences related to river banks. Oecomys aff. roberti showed stronger population structure that appears to be associated with isolation by distance. This arboreal species maintained stable populations in the Araguaia River, while the terrestrial H. megacephalus was more affected by seasonal floods, resulting in a genetic signature of population expansion. Our initial predictions were largely supported by our results given that locomotion habits and habitat preferences of each species appears to have played a role on the genetic structure of these two sympatric rodent species.Although codistributed species are affected by the same abiotic factors, such as rivers and seasonal flooding regimes, ecological traits, such as locomotion habits and habitat preferences, may also influence differences in levels of genetic diversity and differentiation. We examined population genetic structure and diversity of  Genetic differentiation is the result of the interaction of both historical and ongoing evolutionary processes  to compare population genetic structure and diversity of these two cricetid rodent species from the mid-Araguaia River in central Brazil. Specifically, we aim to test whether the Araguaia River acts as a barrier to the gene flow of these two species and to assess how ecological traits, such as locomotion habits and habitat preferences, may influence differences in levels of genetic diversity and differentiation. These two similar-sized species co-occur along the Amazonia-Cerrado ecotone, but they show distinct ecological traits, leading to different predictions regarding levels of genetic diversity and differentiation Table . As bothar Table .Hylaeamys megacephalus and Oecomys aff. roberti were collected from 26 sampling points in the mid-Araguaia River and its surroundings  and pitfalls (30 L and 60 L) were set in each sampling point. Three sampling periods averaging seven nights (between five and nine nights) were performed in each sampling point  cyt B: initial denaturation at 94°C for 5 min, followed by 39 cycles with denaturation at 94°C for 30 s, annealing at 48°C for 45 s, polymerization at 72°C for 45 s, and a final extension at 72°C for 5 min; (2) D-loop: initial denaturation at 94°C for 2 min, followed by 35 cycles with denaturation at 94°C for 30 s, annealing at 54°C  or 45°C (O. aff. roberti) for 90 s, polymerization at 68°C for 1 min, and a final extension at 72°C for 10 min. Mitochondrial fragments were purified using ExoSap-IT®  and sequenced using an automatic sequencer ABI 3130-XL , with the above-listed primers.DNA was extracted from liver and ear tissue samples preserved in ethanol using the salt-extraction method  from both species were estimated through Bayesian inference (BI) in MrBayes v.3.1.2 .Number of haplotypes and polymorphic sites, haplotype and nucleotide diversity values were estimated with DnaSP v.5 (Librado and Rozas Median-joining (MJ) networks were produced for both markers separately using NETWORK , with the 26 sampling points for each species. K was selected based on the maximum (or plateau) value of fixation index FCT  and on the minimum number of groups with single populations.Spatial analyses of molecular variance were performed for both markers separately using SAMOVA 1.0  and matrix of geographic distances was estimated with the Mantel test, using Isolation by Distance web service v.3.23  and one D-loop haplotype (HHd1) were shared by specimens from both river banks; and one haplotype of each marker (HHc4 and HHd4) was also shared with specimens from the islands  was shared by specimens from both river banks, along with specimens from the islands; one cyt B haplotype (HOc2) and three D-loop haplotypes  were shared by specimens from the eastern river bank and the islands; and one D-loop haplotype (HOd3) was shared by specimens from both river banks  and four (K = 4) groups of populations in both species based on cyt B and D-loop, respectively , and sampling points from Uburu and Tocantins were grouped separately , with the exception of groups SHc1, SHc2, and SHd1 of H. megacephalus and groups SOc2 and SOd3 of O. aff. roberti, which cluster sampling points from both regions , refuting a scenario of isolation by distance; but a positive correlation was found in O. aff. roberti , indicating isolation by distance.We found no correlation between genetic and geographic distances in Fs for the cyt B dataset of H. megacephalus (Table We found no evidence of population expansion in the mismatch distributions of either species Fig. . Consistus Table . All othus Table , therefoAlthough previous studies have shown that the Araguaia River is an important barrier to some small mammals Rocha , we founO. aff. roberti and H. megacephalus, differences in genetic structure and diversity between these two species may be related to biotic factors, like their ecological traits. Differences in overall patterns of genetic diversity at a local scale can be explained by the combination of historical and ongoing processes of differentiation, but also by the ecological traits of each species (Patton et al. Proechimys steerei and P. simonsi, and found that the former, which inhabits the seasonally flooded forest, exhibits high gene flow, while the latter, which occupies upland, unflooded habitats, shows little or no evidence of ongoing gene flow among populations.Because the Araguaia River has not been acting as an effective abiotic barrier to gene flow among populations of Hylaeamys megacephalus is a generalist species with high habitat tolerance (Percequillo et al. H. megacephalus is terrestrial, and we therefore expect population declines during the flooding season, followed by expansions in the dry season when more ground habitat is available. Although an erratic mismatch distribution may indicate a population in equilibrium, significant negative values in neutrality tests, such as shown by the cyt B data from H. megacephalus, as well as star-like genealogies showing very short and very long branch lengths, suggest population expansion (Ray et al. H. megacephalus in the Araguaia River. Additionally, the maintenance of high haplotype diversity of populations that experience demographic declines during the seasonal floods is possible due to high reproductive potential of the species, allowing rapid expansion after the flooding, and to the intermixing of the surviving populations (Matocq et al. O. aff. roberti is more specialized, showing arboreal habits, and preference for flooded forests, thus influencing gene flow among distant populations and resulting in higher genetic differentiation. Although most FST values were not significant, this pattern is corroborated by a significant positive correlation between genetic and geographic distances, suggesting isolation by distance. Additionally, O. aff. roberti is less prone to be affected by the flooding regime in the Araguaia River because of its arboreal habits. Indeed, O. aff. roberti showed nonsignificant values for the neutrality tests and also an erratic mismatch distribution, which indicate stable rather than expanding populations.In contrast, H. megacephalus and O. aff. roberti are well distributed across flooded forests, while other sympatric small mammals, such as the mouse opossum Marmosa murina and the climbing rats Rhipidomys spp., are upland specialists (Patton et al. When comparing several small mammal species within Araguaia River basin, we corroborate previous hypothesis by Moritz et al. , which pH. megacephalus and O. aff. roberti from the mid-Araguaia River. We showed that species with different ecological adaptations have different responses to physical and temporal events, and such events can leave fingerprints on the genetic diversity and differentiation patterns. Although the two rodent species co-occurring in flooded forests are not genetically structured by river bank, the fact that H. megacephalus is terrestrial and O. aff. roberti is arboreal made them more or less prone to local extinction due to the seasonal flooding regime of the Araguaia River. Additionally, differences in habitat tolerance are also important in differentiating populations from distant locations. Further comparative studies will help elucidate in which ways differences in ecological traits may influence the geographic distribution of genetic diversity. The combination of molecular and ecological studies is a powerful tool in understanding evolutionary processes that determine species diversification in the Neotropics.Our initial genetic predictions based on ecological traits were largely supported by the results of both


The analysis of ancient human DNA from South America allows the exploration of pre-Columbian population history through time and to directly test hypotheses about cultural and demographic evolution. The Middle Horizon (650–1100 AD) represents a major transitional period in the Central Andes, which is associated with the development and expansion of ancient Andean empires such as Wari and Tiwanaku. These empires facilitated a series of interregional interactions and socio-political changes, which likely played an important role in shaping the region’s demographic and cultural profiles. We analyzed individuals from three successive pre-Columbian cultures present at the Huaca Pucllana archaeological site in Lima, Peru: Lima , Wari  and Ychsma . We sequenced 34 complete mitochondrial genomes to investigate the potential genetic impact of the Wari Empire in the Central Coast of Peru. The results indicate that genetic diversity shifted only slightly through time, ruling out a complete population discontinuity or replacement driven by the Wari imperialist hegemony, at least in the region around present-day Lima. However, we caution that the very subtle genetic contribution of Wari imperialism at the particular Huaca Pucllana archaeological site might not be representative for the entire Wari territory in the Peruvian Central Coast. The Central Andes in South America are defined by marked geographic contrasts between the Andean highlands and the Pacific coast, with each of the regions being characterized by unique eco-geographic zones. The Central Andes are also closely associated with the history of iconic pre-Columbian South American civilizations such as the Wari, Tiwanaku and Inca –3. The WThe Wari Empire is considered the first imperial state in South America expanding from its political and urban center  located in Ayacucho, Central highland Peru. The empire eventually covered a vast area of the Peruvian territory , 11, 12 Archaeological research has described the nature of the Wari Empire expansion  as a comAncient DNA studies restricted to particular South American regions have clarified micro-evolutionary processes involved in the genetic composition of populations and their interaction with a particular environment –27. HoweThe aim of the study was to contrast the genetic diversity of the successive Lima, Wari and Ychsma cultures in a transect-through-time at the Huaca Pucllana archaeological site. We further analyzed the genetic makeup of Huaca Pucllana cultures in the broader context of South American past and present mtDNA diversity.From a total of 52 individuals collected from the Huaca Pucllana site, we obtained 34 complete mtDNA genome sequences  using hybridization capture with RNA baits in solution and Illumina high-throughput sequencing. The average coverage depth per position was 97.4x : Lima (0.002331), Wari (0.002492) and Ychsma (0.002159). Genetic diversity indices and neutrality tests indicated that individuals of the Ychsma culture had a higher haplotype diversity 0.9810 (p>0.0308) than the two previous occupation phases. Values for Tajima’s D  only suggested a population expansion for individuals associated with the Lima culture (-0.13498), while for Fu’s FS values, neither of the three groups Lima (1.44409), Wari (1.08278) and Ychsma (0.31237) indicated a recent population expansion  belonging to mtDNA haplogroup C1b, two individuals from Ychsma (Ychsma–10722 / Ychsma–10805) belonging to D1, and two individuals from Wari (Wari–10734 / Wari–10765) belonging to C1b. We also observed shared haplotypes between individuals from different cultural layers, e.g. two individuals from Lima and Ychsma (Lima–10811 / Ychsma–10732) belonging to D4/Pre-D1/D1, and three individuals from Wari and Ychsma (Wari–10763 / Ychsma–10709 / Ychsma–10725) belonging to C1b .We evaluated the relationship of cultural groups from Huaca Pucllana with modern and ancient mtDNA diversity in South America based on a genetic distance matrix and visualized as a Multidimensional Scaling Plot (MDS).Individuals from the EIP Lima culture are genetically close to highland Laramate populations from the MH  and LIP We sequenced 34 complete human mitochondrial genomes from the three successive cultural periods Lima, Wari and Ychsma found at Huaca Pucllana. This dataset represents a unique chronological transect in the Peruvian Central Coast.FST values for comparisons between the three groups Lima, Wari and Ychsma indicate no differentiation of sequence haplotypes, meaning that ancient Peruvians from Huaca Pucllana did not undergo dramatic changes in genetic ancestry over approximately ~1000 years. These results suggest population continuity through time, with no major demographic turnovers. Genetic diversity indices do not differ substantially between the three cultural periods and results from neutrality tests (Tajima’s D and Fu’s FS) do not attest for recent expansion in any of the three groups. Overall, samples from the latest Ychsma period are the most diverse, which could be explained by the slightly larger sample size  are present in subsequent LIP Ychsma individuals, while being absent in MH Wari, suggests a potentially exogenous source of the Wari B2b lineages .In addition, half of the MH Wari individuals carried the C1b sub-haplogroup, which is twice as much as the earlier EIP Lima or later LIP Ychsma groups. Since the frequency of haplogroup B2b remains constant during the Wari and Ychsma periods, we suggest that the increase in frequency of haplogroups C1b and B2b during the Middle Horizon might be linked to gene flow from the Andean region. However, we note that haplogroup A2 lineages also increase with the Wari period, but do not seem to become established in the Huaca Pucllana region during Ychsma times, following the collapse of the Wari Empire .We caution that haplogroup composition represents a very broad approach to investigate signals of population structure, in particular because frequencies are subject to genetic drift or stochastic processes, population movements and gene flow . MoreoveNetwork analysis showed that haplotype sharing occurred mostly between individuals from the same archaeological period, and less often between different cultural layers . A shared C1b lineage between Wari and the subsequent Ychsma is very plausible, while the shared Lima–Ychsma haplotypes suggests a local continuity of particular maternal lineages over several centuries, which implies that the Wari incursion was not a replacement but rather brought new lineages to the Huaca Pucllana region .Interestingly, the MDS plot shows an affinity of the Lima population during the EIP  in Peru’s Central Coastal with ancient highland populations rather than other Coastal populations from the same period , suggesting that the population history of the Central Coast is connected with the highlands and did not develop independently unlike Moche and Nasca populations . HoweverIt should be noted that the overall mtDNA haplogroup composition is nonetheless homogenous across cultural periods in Peru Central Coast, even in the light of the expansion of the Wari Empire. To the limits of our resolution, the new results from ancient Huaca Pucllana therefore hint at a very subtle yet negligible genetic contribution of highlands Wari in coastal Peru during the MH.We caution that the situation at Huaca Pucllana in the greater Lima region might not be representative of the overall genetic influx of the Wari Empire in the Central Coast of Peru, supporting the hypothesis that local settlements along the Central Coast were not entirely dominated or affected by Wari imperialism , 24. MorA remarkable observation is that the ancient mtDNA sequences from Huaca Pucllana represent exclusively novel haplotypes  with manAs exemplified by the pre-Columbian expansions of Wari and Tiwanaku and later on the Inca Empire, the Central Andes have always been a dynamic environment with considerable population movements . AnotherThe ~1000-year transect through time of pre-Columbian history at Huaca Pucllana, allowed us to test the impact of the expansion of the highland Wari Empire on the coastal Lima culture, and its legacy in the subsequent Ychsma culture. The results from three archaeological periods indicate that genetic diversity in Huaca Pucllana shifted only slightly through time, ruling out a complete population discontinuity or replacement driven by the Wari imperialist hegemony, at least in the region around present-day Lima.Detailed chronological sampling and high-resolution DNA sequencing efforts are required to refine the analysis of ancient genetic diversity in Peru, but importantly also from modern-day groups from Central Andean and Coastal regions to be able to contrast ancient diversity with present-day populations. Such data would allow the complete contextualization of associated meta-data, testing of demographic scenarios, and exploration of continuity/discontinuity, replacements and bottlenecks on the basis of statistical inferences or simulations. Ultimately, these analyses could elucidate the dynamic of pre-Columbian societies and the dramatic demise of Native American people that coincided with the time of the European arrival , 43.Ethics Statement: All necessary permits were obtained for the described study, which complied with all relevant regulations. Permissions to collect, export and analyze ancient Peruvian specimens from the Huaca Pucllana site were granted by the Ministry of Culture  and the National Museum of Archaeology, Anthropology and History of Peru (MNAAHP) and are available on request .The archaeological site of Huaca Pucllana (12°06’37.01”S–77°01’58.93”W) is located in the Miraflores District of Lima, Peru .Archaeological evidence at the site defined that three cultural layers developed during slightly different times than in the classical chronology for the Central Andes established by Rowe . The Limth and 7th centuries AD, which represents the Lima final phase (500–700 AD) and occupation of the site. Huaca Pucllana is characterized by pyramidal platforms built with sun-dried bricks, or adobes ) . We exclh), nuclear diversity (π), pairwise genetic distances FST, neutrality test Tajima’s D [p.adjust function in R 3.2.3 . Median Joining (MJ) networks [http://www.fluxus-engineering.com) to evaluate genetic relationships among all individuals and to illustrate the extent of haplotype sharing within and among temporal periods and cultures.Haplotype diversity  [https://www.r-project.org/). Since the mtDNA genome data for modern-day populations is heavily biased towards phylogenetic and phylogeographic questions that result in an excess of specific haplogroups, we had to restrict our analyses to mtDNA (HVR–I) sequences compiled from published sources to describe the genetic relationships between ancient samples from Huaca Pucllana and other ancient individuals (n = 352) from 16 populations, as well as present-day South Americans (n = 1090) from 28 populations  plots to visualize genetic similarities and dissimilarities as measured by fixation indices (Slatkin’s FST)  in bi-diulations .fisher.test in R 3.2.3 (http://www.r-project.org) and 10,000 permutations per comparison.We used a Fisher exact test to investigate the variation in sub-haplogroup composition between the three cultural groups. Individuals were assigned to sub-haplogroups A2, B2, B2b, C1b, C1c, C1d, D1, and D4/PreD1/D1. The Fisher extact test was computed using the function S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Populus tremula L.) genotypes were developed on the base on microsatellite (SSR) loci. Out of 33 tested microsatellite loci, 14 were selected due to sustainable PCR amplification and substantial variability in elite clones of aspen aimed for establishment of fast-rotated forest plantations. All eight tested clones had different multilocus genotypes. Among 114 trees from three reference native stands located near the established plantations, 80 haplotypes were identified while some repeated genotypes were attributed to natural clones which appeared as a result of sprouting. The selected set of SSR markers showed reliable individual identification with low probability of appearance of identical aspen genotypes . Case studies demonstrating practical applications of the test system are described including analysis of clonal structure and levels of genetic diversity in three natural aspen stands growing in the regions where plantations made of elite clones were established.Testing systems for molecular identification of micropropagated elite aspen ( This task declares obtaining and maintenance of elite genotypes of forest trees aimed for fast-rotated forest plantations. New highly productive and sustainable to abiotic factors, pests and pathogens cultivars and varieties appear as a result of breeding, selection of mutations, and/or genetic engineering. The clonal propagation of such outstanding individuals ensures fixation of their useful traits both for further breeding experiments and for establishment of targeted forest plantations. In particular, microclonal propagation via efforts –3. in vitro cell culture, individuals may be indiscernible not only individually but also as far as species diagnoses are concerned. Long generation time and ontogenetically late maturation makes the task of genetic passportization of elite clones in woody plants real. The complex multistage process of obtaining, propagation, and introduction of elite cultivars by breeding or genetic engineering increases the probability of various errors.Since morphologically and anatomically different plant clones may look similar, it is essential to reliably identify those including discrimination from each other and from conspecific individuals or representatives of other closely related species. In forestry, the problem of individual identification is especially crucial since the external look of trees is highly dependable on environmental parameters . At partMolecular genetic markers (MGM) proved to be very efficient tools for individual identification. Among different MGM classes microsatellites or simple sequence repeats (SSR) fit best to requirements of testing systems for identification due to their specificity, codominance, selective neutrality, sufficient allelic richness, and heterozygosity caused by high mutation rate. Moreover, due to relative genome conservatism within genera and families of plants, SSR-markers and PCR primers for their amplification can be transferrable from one taxon to another.In several poplar species, the successful DNA fingerprinting and differentiation of clones, cultivars, and varieties were demonstrated by molecular markers including SSR loci –9. Populus tremula L., aimed at the establishment of fast-growing target forest plantations in several regions of Russian Federation and present the results of its application for individual genotypes discrimination, studies of clonal structure in natural aspen stands, and estimation of genetic variability in populations.In this paper we report on the development of SSR-based testing system for molecular genetic identification of elite micropropagated genotypes of aspen,The development of the testing system for the identification of elite genotypes comprised the selection of a specific marker set fitting the requirements of high-resolution discrimination of clones and testing its reliability and identification power on a set of elite genotypes and a sufficient number of representatives of a studied species.Elite aspen and hybrid clones used for development of testing systems for molecular identification have been obtained from micropropagated cell cultures  maintainExperimental aspen clonal plantations derived from these elite genotypes were established in four regions of European part of Russia . Native Prisady. Experimental plot located 1 km westward of settlement Prisady in Serpukhovsky Raion (district) of Moscow oblast' (Russia). Leaves of 52 young or medium-aged (approximately 10–25 y.o.) aspen trees from a natural multiaged stand located in close proximity (1 km) to this plot have been used as a reference population.Voronezh. Seventeen trees were sampled in a stand adjacent to the experimental plot established in Voronezh Oblast. Additional 13 trees were collected along the bank of Voronezh River within the city of Voronezh close to the plantation.Yoshkar-Ola. Young native stand of aspen was the source of trees used as a reference population for an experimental plot located near the city of Yoshkar-Ola, Republic of Mari-El. Leaves from 32 trees were collected.In order to minimize the occasional sampling of individuals having vegetative origin (through sprouting) we collected leaves from trees at a distance not less than 15–20 m from each other. This approach was employed for inclusion into reference samples of predominantly open-pollinated seedlings having maximal genetic diversity. However, based exclusively on external look of the trees and distance among them, sampling of the ramets appearing as a result of sprouting could not be avoided, and subsequent genetic analysis confirmed this.Shoots with leaves were cut off by means of mechanical cutter with an aluminium telescopic mast. Collected shoots with leaves were placed into plastic bags for no more than 6 days at +4°С until processing. Sample preparation included DNA extraction and placement of reserve leaf tissue fragments into labeled zip-bags with silica gel for long-term storage. Outside the period of active vegetation, dormant vegetative buds can be successfully used for DNA extraction. in vitro on a special media (REF) in Petri dishes were used for DNA extraction.Fragments of leaves approximately 350–500 mg taken from explants growingFor trees from native populations, we extracted DNA from 350–500 mg fragments of fresh or 200–300 mg of silica-dried leaf tissues by a modified cethyltrimethylammonium bromide (CTAB) method , 14.Microsatellites, or SSR, represent a class of tandemly repeated DNA sequences with short  motifs differing in copy numbers among individuals due to high mutation rate. Multiple alleles usually found in codominant microsatellite loci create a great variety of unique genotypic combinations which ensures their reliable identification, especially when a sufficient number of loci are employed. Technically, the analysis of SSR polymorphism requires only Polymerase Chain Reaction (PCR) and subsequent electrophoresis (gel or capillary) for fragment analysis. For the development of the testing system we chose the variant of the method that utilizes only basic equipment and simple reagents. This ensured increased reproducibility of the procedures in any PCR laboratory and allowed achieving high cost-effectiveness of the analysis which is crucial for large-scale practical clone identification.http://webofknowledge.com/) and in Molecular Ecology Resources Primer Database (http://tomato.bio.trinity.edu/).Since the substantial number of nuclear SSR loci for poplars was found in the literature and primer databases we decided to select from several publications and test primers that can be used for routine genotype identification based on very simple equipment without using of DNA-analyzers. An initial set of SSR primers for their potential use as elements of the testing system of molecular identification in aspen was a result of search in bibliographical databases  in BioRad Inc. (USA) Dyad Thermo cycler. Microsatellite loci . DNA ofClone identity was determined using multilocus matches analysis for codominant data. Genotype probability (GP), meaning probability of appearance of particular multilocus combination in population, and probability of identity, estimating probability of random matching of two unrelated (PI) or related (PIsib) individuals by particular set of loci, were calculated based on distribution of allele frequencies in population samples. F-statistics for assessment of genetic subdivision among the studied population samples. All the above-mentioned calculations were performed in the add-in for MS Excel, GenAlEx 6.5 [Correspondence of observed genotype distributions for each SSR locus to the expected according Hardy-Weinberg equilibrium was tested by chi-square criterion. Allele number and observed and expected heterozygosities were calculated for each native sample. We employed Wright'sAlEx 6.5 , 18. Populus trichocarpa [ Populus nigra [ in vitro collection stored and propagated in the Pushchino branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences . At this stage, their variability was also tested on 20–24 specimens of wild aspen from Novosibirsk Oblast  and Krasnoyarsk Krai .For initial testing, we selected 33 heterological tri-, tetra-, penta-, and hexanucleotide microsatellite loci from two sets; series ORPM was designed first forchocarpa  and serius nigra . CharactAs a result of the first phase of testing, 24 loci were successfully amplified and nine other loci failed to produce PCR products. Out of 24 loci that produced PCR fragments, 20 loci were shown to be variable with a number of alleles from two to nine, while four loci that have been successfully amplified were monomorphic . After ahttp://dx.doi.org/10.1155/2015/261518. We analyzed up to 8 ramets sampled at different phases of the microclonal propagation. Within clones, genotypes were stable and unambiguously reproduced among ramets independently of the stage of propagation. After exclusion of ramets of the same genet, both elite clones and aspen genotypes from native populations included in the reference samples were 100% different. Probability of appearance of genotypes (GP: genotype probability) among elite clones varied from 2.4 · 10−21 to 1.7 · 10−11, among trees in a reference samples from 4.0 · 10−24 to 5.8 · 10−9. Probability of the occasional coincidence of two unrelated genotypes (PI: probability of identity) varied from 4.8 · 10−10 in Yoshkar-Ola to 4.3 · 10−13 in Prisady. Adjusted to the theoretical probability of descendance of the compared individuals from the same ancestors more conservative estimate (PIsibs) was within the range of 1.0 · 10−4 in Yoshkar-Ola to 9.3 · 10−6 in Voronezh. All values are quite low, so that the theoretical frequency of appearance of repeatable genotypes due to recombination of different gametes in course of seed reproduction was not exceeding about 1 accidentally found identical allele combination out of 10000 comparisons. The relationship of PI and PIsibs from the number of loci used is shown at The obtained multilocus genotypes of eight elite clones and reference genotypes of wild trees from native stands are listed in Table S1 in Supplementary Material available online at  Populus started in late 20th century along with technologies of molecular genetic markers. In 1998, one of the first sets of primers for amplification of dinucleotide SSR loci was designed for American trembling poplar, Populus tremuloides [ P. deltoides, P. nigra, P. x canadensis, and P. maximowiczii, was demonstrated. The authors also postulated the broad spectrum of applications of SSR loci for different purposes including clone identification, analysis of the controlled matings, genome mapping, marker-assisted selection, genetic diversity assays, and support of the programs for conservation and sustainable management of forest genetic resources. Subsequent studies provided eight other dinucleotide SSR loci for Populus tremuloides [ P. nigra [ P. deltoides, P. trichocarpa, P. tremula, P. tremuloides, P. candicans, and P. lasiocarpa. Specific SSR loci primers were designed for Populus euphratica [Development of microsatellite loci for species of the genusP. nigra , 21. Somphratica –25. Latephratica , 27. Populus trichocarpa [Next generation sequencing was the most efficient way of detection of tandem repeats in poplar genome and design on their base transferrable SSR primers as it was demonstrated in case of balsamic poplar,chocarpa , 28. Succhocarpa , 29–31.Microsatellites are also useful for checking of somaclonal diversity within a pool of ramets obtained by microclonal propagation from a single donor tree , for ideSince the very moment of the field sampling of material in wild stands we tried to avoid inclusion of clonal ramets arised as sprouts which is common for aspen. In all studied stands the same sampling scheme was applied keeping at least 15–20 m between the trees. Nevertheless, ramets of the same clone indicating common occurrence of vegetative propagations were found in all of the studied natural populations (Table S1).Prisady. Among the studied 52 trees, we found 41 different haplotypes; one multilocus genotype was found nine times and one was found three times. We concluded that these repeated multilocus combinations resulted from sampling sprouts being ramets of the same clone.Voronezh. Among 17 trees collected at close proximity to the site of the experimental plantation no clonal individuals were found. Thirteen trees collected at the Voronezh River bank were represented by eight genotypes: four of them were in one replicate, three were found to be two ramets of the same clone, and one genotype was found in three copies evidently originated from sprouting of ever existing progenitors. In total, we identified 25 different haplotypes in this reference population sample.Yoshkar-Ola. Thirty-two analyzed trees combined into 13 different haplotypes identified by multilocus matches analysis; one genotype appeared twice, one genotype appeared five times, one genotype appeared seven times, and one genotype appeared nine times. Repeated genotypes evidently corresponded to ramets resulting from sprouting.We concluded that sprouting and high level of clonality are a widespread phenomenon in native aspen stands. In aspen, as well as in many other poplars, vegetative clones are able to occupy large areas. Therefore, for the collection of a sample free of repeated clonal genotypes distances between trees should be increased up to at least 40–50 m. However, more precise estimation of maximal spread of a single clone over stand territory also requires special exploration. P. nigra [ P. alba – P. x canescens on the Iberian Peninsula [Among other applications, nuclear SSR loci were useful for the studies of clonal structure and genetic relationships among clone genotypes in native stands –38 or beP. nigra . ExtensiP. nigra , in the eninsula , and in  Prisady and Voronezh than in Yoshkar-Ola , 10% were among individuals, and 84% were within individuals.The averaged over loci Microsatellites isolated from nuclear as well as chloroplast genomes were employed for the analysis of genetic diversity and genetic structure in various poplar species , 44–60. A substantial number of studies employing microsatellite technique were focused on the detection of hybridization in natural , 61–63 o in vitro collections [The practical task of identity monitoring of commercial clones inlections , 76 was Application of nuclear microsatellite loci for genetic identification and assessment of genetic variability in aspen elite clones and native stands showed high effectiveness of the developed low-cost SSR-based testing system. Reliable authentication of clones ensures genetic monitoring of microclonal propagation and allows revealing clonality in native stands. We demonstrated that the same set of microsatellite loci can be successfully employed for estimation of levels of intra- and interpopulation genetic variability in aspen. Reconstruction of kinship among individual elite clones or genetic relationships of naturally mating populations are perspective tasks that can be realized in the future using the same markers.Suppplementary Table S1 contains a matrix of genotypes of aspen elite clones and trees from three native reference stands by 14 microsatellite loci, probability of appearance of each genotype in a sample and number of ramets of individual clones found in natural populations.


Gracilaria chilensis colonised the Chilean coast from New Zealand. Combining archaeological observations with phylogeographic data provided evidence that exchanges between New Zealand and Chile have occurred at least before the Holocene, likely at the end of the Last Glacial Maximum (LGM) and we suggest that migration probably occurred via rafting. Furthermore, the remarkably low microsatellite diversity found in the Chilean populations compared to those in New Zealand is consistent with a recent genetic bottleneck as a result of over-exploitation of natural populations and/or the process of domestication. Therefore, the aquaculture of this seaweed, based essentially on clonal propagation, is occurring from genetically depressed populations and may be driving the species to an extinction vortex in Chile.The history of a domesticated marine macroalga is studied using archaeological, phylogeographic and population genetic tools. Phylogeographic and population genetic analyses demonstrated that the cultivated red alga  Humans have always exploited species of plants and animals. Although archaeological evidence has shown the natural environment was altered by hunter-gatherers, the major impact of human activities on biodiversity is considered to have begun when humans started the domestication process, over 13,000 years ago, at the beginning of the Holocene period Domestication of marine species appears very recent compared to terrestrial examples. The earliest tentative record was dated around 600y BP but the vast majority of marine species were domesticated in recent decades Gracilaria chilensis Bird, McLachlan et Oliveira and analyse the historical interaction between humans and seaweeds. Archaeological evidence suggests that this species was used for food and/or medicine by the first inhabitants of Monte Verde in Southern Chile, about 14,600 years before present G. chilensis were extensively harvested in Chile for agar production, until they collapsed in the late 1980s because of overexploitation G. chilensis is by far the most important seaweed crop cultivated in Chile G. chilensis displays a disjoint, trans-oceanic distribution range Gracilaria in Australia and New Zealand Here we consider the case of the red alga G. chilensis on both sides of the Pacific Ocean to better understand the state of the species genetic resources in Chile, a region where the species has faced long-term extensive exploitation and recent domestication. We first investigate the history of the disjoint distribution through a phylogeographic approach. In particular, we test the hypothesis of a range extension from New Zealand through trans-Pacific dispersal. We also investigate potential human impacts on genetic diversity using population genetic analyses in an attempt to resolve the recent history of this domesticated species.In this study we retrace the evolutionary history of Gracilaria chilensis was extracted in Chile. For the eight populations from New Zealand, collections were made under a Special Permit to NIWA from the New Zealand Ministry of Primary Industries. GPS coordinates of all locations are available in the Nineteen populations were sampled in New Zealand and in Chile covering the disjoint range distribution of the species : 187 tetrbcL, plastid) and compared with two sequences of G. chilensis from Chile already deposited in GenBank: AY049396 from Coquimbo rbcL amplifications were carried out following the procedures described by rbcL (GENBANK N° HQ998840 to HQ998848) were obtained with an ABI 3100 Sequencer . Sequences were edited using CHROMAS DNA extraction followed To minimise the rate of genotyping errors, a second and third round of PCR and electrophoresis were performed for individuals with dubious multilocus genotypes . Because of the very high amplification failure rate  of the locus Grc-AC/CT23 FIS was calculated over all loci according to http://cran.r-project.org/). We applied sample size intervals of one unit, varying from one to a maximum of 410 individuals for Chile and a maximum of 187 individuals for New Zealand.For nuclear microsatellites loci, expected heterozygosity (He), observed heterozygocity (Ho), allele number and the number of private alleles were calculated in each population using FSTAT v 2.9.3 A median-joining network of ITS2 ribotypes was constructed for both the New Zealand and Chilean data sets using NETWORK v 4.5 p). We ran STRUCTURE 50 times at K = 5 according to the highest Ln(p) in the previous set of runs, and combined the result of all these runs with the programme CLUMMP post hoc analyses. POPULATION GRAPH was implemented on the web (http://dyerlab.bio.vcu.edu/wiki/index.php/) using the population genetic dataset. A test for distinct clustering of New Zealand and Chilean populations was conducted a posteriori using the methods outlined by Dyer and Nason To compare population structure in New Zealand and Chilean populations using microsatellites, we employed two different methods. First we used a Bayesian, model-based genetic admixture analysis implemented in STRUCTURE 2 statistic were estimated and significant departure from mutation-drift equilibrium was tested by 10,000 bootstrap replicates using DnaSP version 5.10.01 To detect bottlenecks, we used the graphical method developed by Luikart et al rbcL from Chile , New Zealand  and Chatham Island  displayed the same haplotype as G. chilensis sequences from Chile deposited in GenBank Over the whole dataset , nuclear microsatellite loci displayed moderate to high numbers of alleles, ranging from three to 22 for the loci 6C7 and 7D3, respectively. For the 201 ITS2 sequences, characterised by 24 polymorphic sites and four indels, 21 ribotypes were identified. The most genetically diverse population was found in Chatham Island . ObserveWe found strong evidence for genetic divergence between New Zealand and Chilean populations supported by both model based clustering and graph theory analyses of microsatellite data . First, We found evidence of a bottleneck in Chile where rare alleles  were 10 times less common than in New Zealand . New Zea2 values G. chilensis. Expansion dates were estimated at 360,000  years for the West coast of New Zealand, 100,000  years for Chatham Island and 19,000  years for Chile based on ITS2 data.Signatures of demographic expansion were detected in three regions  by some of our estimators. A significant signature of expansion was observed by Fu’s Fs rbcL and ITS2 data confirmed that specimens of G. chilensis found in New Zealand and Chile are conspecific G. chilensis, originally described in Chile G. chilensis arrived in Chile as a result of recent trans-oceanic long-distance dispersal from New Zealand. Firstly, G. chilensis is the unique representative species of Gracilaria on the Chilean coast  supports the hypothesis of a recent co-ancestry. In Chile, the predominant ribotype r1 is likely to be the ancestral allele since it occupied the centre of the network from which private ribotypes were connected by only one mutational step. In agreement with other phylogeographic studies  Papenf. Gracilaria sp., reported as undergoing rapid range expansion, is the formation of extensive mats in muddy bays by vegetative fragmentation G. chilensis to survive for long periods out of the water, enabling transportation and dispersal. Transport and exchanges of this alga are active between coastal communities of fishermen and have contributed to a very recent expansion in the range of distribution of this species in Chile Successful establishment and expansion of G. chilensis has been documented to have deeply affected the distribution range of the species and the genetic structure of populations G. chilensis, remains an open question that could be further addressed with more powerful markers.Modern anthropogenic transport of living The signature of bottleneck was observed with the typical shift of the microsatellite allele frequency distribution and could explain the strikingly low genetic diversity in Chile when compared to New Zealand. Because this distortion is transient and likely to be detectable for only a few dozen generations G. chilensis cultivated in Chile, with a modification of life history traits G. chilensis, the erosion of genetic diversity along the Chilean coast may have seriously limited the capacity of individuals to adapt to changing environments and/or to respond to further human selective pressure for aquaculture needs. In conclusion, this study emphasises how the conjunction of recent bottleneck with the predominance of clonal propagation seems to be driving this species into an extinction vortex in Chile.Domestication is a process that usually lasts up to several millennia as a result of a mostly unconscious human selection (at least in the early stages), leading to complex evolutionary trajectories associated with changing selective pressures across the history of human utilisation/cultivation Figure S1Comparison of allele accumulation curves between Chilean and New Zealand (including Chatham Island) samples.(PDF)Click here for additional data file.Table S1Sampling locations and diversity measures for microsatellites (5 loci) and ITS2 sequences in G. chilensis. N, number of individuals per sampling locations used for microsatellite genotyping; (Ho) observed heterozygosity, (He) expected heterozygosity, Ae, multi-locus estimates of expected allelic richness based on the smallest sample size ; Pa: number of private alleles; Weir and Cockerham’s FIS estimate ; n, number of individuals used for sequencing; nR, number of ribotypes; Re, expected haplotype richness based on the smallest sample size (5 ribotypes); Ribotypes: details of the different ribotypes observed in the populations, private ribotypes are in bold. Mean ± SEM are indicated for all New Zealand samples  and all Chilean samples .(PDF)Click here for additional data file.


Solanum lycopersicum var. lycopersicum (SLL), S. lycopersicum var. cerasiforme (SLC), and S. pimpinellifolium (SP) that were genotyped using 7,720 SNPs. Additionally, we explored the allelic frequency of six loci affecting fruit weight and shape to infer patterns of selection.Domestication modifies the genomic variation of species. Quantifying this variation provides insights into the domestication process, facilitates the management of resources used by breeders and germplasm centers, and enables the design of experiments to associate traits with genes. We described and analyzed the genetic diversity of 1,008 tomato accessions including Our results revealed a pattern of variation that strongly supported a two-step domestication process, occasional hybridization in the wild, and differentiation through human selection. These interpretations were consistent with the observed allele frequencies for the six loci affecting fruit weight and shape. Fruit weight was strongly selected in SLC in the Andean region of Ecuador and Northern Peru prior to the domestication of tomato in Mesoamerica. Alleles affecting fruit shape were differentially selected among SLL genetic subgroups. Our results also clarified the biological status of SLC. True SLC was phylogenetically positioned between SP and SLL and its fruit morphology was diverse. SLC and “cherry tomato” are not synonymous terms. The morphologically-based term “cherry tomato” included some SLC, contemporary varieties, as well as many admixtures between SP and SLL. Contemporary SLL showed a moderate increase in nucleotide diversity, when compared with vintage groups.This study presents a broad and detailed representation of the genomic variation in tomato. Tomato domestication seems to have followed a two step-process; a first domestication in South America and a second step in Mesoamerica. The distribution of fruit weight and shape alleles supports that domestication of SLC occurred in the Andean region. Our results also clarify the biological status of SLC as true phylogenetic group within tomato. We detect Ecuadorian and Peruvian accessions that may represent a pool of unexplored variation that could be of interest for crop improvement.The online version of this article (doi:10.1186/s12864-015-1444-1) contains supplementary material, which is available to authorized users. The domestication process of crop plants led to dramatic phenotypic changes in many traits that result from changes in the genetic makeup of the wild species ancestors ,2. The aSolanum lycopersicum L.) (SL) is a member of the family Solanaceae, genus Solanum L., section Lycopersicon [S. pimpinellifolium L. (SP) is thought to be the closest wild ancestor to cultivated tomato [Cultivated tomato  Spooner, G.J. Anderson & R.K. Jansen (SLC) and S. l. var. lycopersicum (SLL). SLC is native to the Andean region encompassing Ecuador and Peru, but it is also found in the subtropical areas all over the world [he world . SLC grohe world . SLC thrhe world ,11,12. Ahe world , the AndMany details of tomato domestication remain debated, especially regarding the role of SLC in this process. The South American SLC native to the Ecuadorian and Peruvian Andes has been proposed to be an evolutionary intermediate between SP and cultivated SLL ,9,14 or,Extensive breeding efforts have modified tomato over the last 100 years. Breeding goals were focused on improving SLL for disease resistance, adaptation to diverse production areas, yield and uniformity. These efforts resulted in the introduction of many introgressions from SP and more distant tomato relatives , leadingFAS, SUN, OVATE and LC genes [The traits that most likely have been selected during the domestication of tomato were fruit weight and, to a lesser extent, shape. In recent years, several genes affecting these traits have been identified -29. As tLC genes . What isThe aim of this study was to better delineate the evolutionary history of tomato including its domestication. By using a dataset with over 7,000 SNPs and 1,008 accessions of SP, SLC and SLL we aim to compare and contrast the genome-wide molecular diversity of populations spanning the entire red-fruited clade. Additionally, the allele frequency of six fruit weight and shape genes have been measured in order to elucidate the domestication process.Solanum lycopersicum var. lycopersicum , S. l. var. cerasiforme , S. pimpinellifolium , Solanum galapagense S.C.Darwin & Peralta , Solanum neorickii D.M.Spooner, G.J.Anderson & R.K.Jansen , Solanum chmielewski  D.M.Spooner, G.J.Anderson & R.K.Jansen , crosses between S. lycopersicum and S. pimpinellifolium , and one hybrid between S. l. lycopersicum and S. pennellii. The hybrids were included to determine the ability of detecting heterozygous SNPs with the genotyping platform.We analyzed 1,008 tomato accessions from the species representing the red-fruited clade of tomato . The SolCAP SNP discovery work-flow was described [http://solgenomics.net/), the SNP annotation file is available (http://solcap.msu.edu/tomato_genotype_data.shtml), and sequences are available through the Sequence Read Archive (SRA) at the National Center for Biotechnology Information .All samples were genotyped using the Tomato Infinium Array  developed by the United States Department of Agriculture (USDA) funded SolCAP project  and diluted to 50 ng/μl in TE buffer . Genotyping was performed using 250 ng of DNA per accession following the manufacturer’s recommendations. The intensity data were analyzed in GenomeStudio version 1.7.4 . The automated cluster algorithm generated from the SolCAP project was used to obtain initial SNP calls. Visual inspection was used to assess the default clustering of each SNP, and calls were modified when the default clustering of a SNP was not clearly defined.There are three methods for SNP calling for the Illumina Infinium array: relative to the reference , the design  or the TOP strand (a designation based on the polymorphism itself and its flanking sequence). To merge data sets from three different laboratories that had used different SNP calling methods, we developed a Python script to facilitate detection, reorientation and merging of the data such that all SNPs are called relative to the design strand (the script is available upon request to J. Blanca).The accessions were genotyped with 7,720 SNPs . A three level classification scheme, based on a series of hierarchical PCAs, was used to define genetic groups within species and genetic subgroups within genetic groups. PCAs were performed with the smartPCA application included in the Eigensoft 3.0 package [Principal Component Analyses (PCA) were used to explore the patterns of genomic variation in the entire collection without considering the  package ,35. ThisPairwise genetic distances were computed among accessions within each group at each level of the hierarchical classification. Kosman and Leonard’s distance method  was usedWhen an accession was genotyped more than once and both genotypes were inconsistent  all data for the accession was removed from the analysis  to check the reproducibility between genotyping datasets coming from different laboratories. For the genetic analyses, unless stated to the contrary, only one randomly chosen genotype representative of the uniquely named accessions was used.e) and observed (Ho) heterozygosity were calculated using custom scripts for each hierarchy of the genetic classification. Differentiation among genetic subgroups was explored by calculating differentiation index Dest [st using Arlequin v. 3.5.1.3 [est and Fst was assessed after 1,000 permutations.For polymorphic loci with a major allele frequency lower than 0.95 (P95), the expected  [Allelic richness and private allelic richness  were estimated using the rarefaction method implemented in the software ADZE . LD was  (LOESS)  implemen (LOESS) . Rarefachttp://www.worldclim.org) at 30 arc-seconds resolution (approx. 1 km). A PCA was carried out with all the climatic information and the resulting scores were used to obtain the pairwise climatic distances based on a Euclidean metric. Significance of the correlations between distance matrices was assessed with a Mantel test based on 1,000 permutations implemented by the PyCogent Python library [kde2d function in the R ‘MASS’ package [Correlations between genetic, geographic and climatic distances were analyzed to infer patterns of isolation by distance or the effect of ecological conditions on the genetic structure. Pairwise genetic distances between accessions were computed using Kosman and Leonard’s distance method . Pairwis library . A densi package .S. galapagense, S. neorickii and S. chmielewski.A phylogenetic tree was built with SNAPP  to infer as well as the fruit weight loci (fw2.2 and fw3.2) were genotyped (Table lc (locule number) and fas (fasciated) control the number of locules, an important feature affecting fruit weight as well as shape.The gene lc is hypothesized to be an ortholog of WUSCHEL which is required to maintain stem cell identity [fas mutation affects a YABBY2 transcription factor which encodes a member of the family regulating organ polarity [sun [ovate [fw2.2 negatively controls cell division and encodes a member of the Cell Number Regulator (CNR) family [fw3.2 encodes an ortholog of KLUH, a P450 enzyme which increases weight through increased cell number in pericarp and septum tissues [Six markers that distinguish wild type and causal derived alleles of the fruit shape loci identity . The faspolarity ,51. Two ity [sun  and ovatn [ovate , positiv) family ,52. fw3. tissues .Table 1sun, were genotyped by amplification using standard PCR following previously published methods [fas) or after restriction enzyme-digestion  by electrophoresis on 3% TBE  agarose gels. The sun duplication was scored as an RFLP using standard Southern blotting and hybridization protocols [All markers, except  methods . PCR prorotocols .e = 0.21), followed by SLC (He = 0.17) and SLL (He = 0.12)  to He = 0.15 (Peruvian SP)  and sub-clusters within each cluster , additional PCAs were conducted in a hierarchical fashion with the accessions belonging to the same species Figure A. These P) Table .Figure 2e = 0.19 and SLC Peru He = 0.18, Table For SLC, the first two PCs explained 16.0% of the total variance and showed a clustering based on geography  compared with the Peruvian and Ecuadorian SLC (He = 0.188-0.177)  separated five main genetic groups: vintage, fresh, processing 1, processing 2 and SLL 1  (data not shown). The distance among repeated samples of the uniquely named accessions was very low indicating a high consistency among genotyping experiments.To determine the consistency of the structure obtained by PCA analyses, we compared the distribution of genetic distances within the following hierarchy levels: species, genetic group, genetic subgroup and samples of the same uniquely named accession  accession clustered very close to the Ecuadorian SP, a grouping which was also found in the PCA , as well as for genetic and geographic distances   than in SP, whereas the genetic versus geographic was similar . When considering the SLC accessions together, a low correlation between genetic and climatic  as well as genetic and geographic distances  were observed.We noted that most clusters in SP and SLC corresponded to the location of where the accessions were collected. Therefore, we sought to evaluate the significance of this finding by calculating the correlation between genetic, climatic and geographic distances Table . The hige) and observed heterozygosity (Ho) decreased in the succession from SP to SLC and SLL (Table e = 0.19 and 0.18) compared to SP Ecuador and SP Montane. In contrast with the high diversity of the Ecuadorian and Northern Peruvian SLC, the other SLC subgroups exhibited low diversity, similar to that found in vintage SLL. For SLL a similarly low level of observed heterozygosity was typical for most subgroups. However, when combining the contemporary SLL subgroups (processing and fresh), slightly higher levels of diversity were found when compared to SLL vintage (He = 0.12 vs. 0.09), a situation that is likely due to the effect of introgression during breeding and differentiation into distinct market classes . The lc mutation was also common in SLL vintage and SLL fresh accessions whereas the derived allele was not found in the SLL processing types. The derived allele of fw2.2 was nearly fixed in all SLL groups. For fw3.2, the derived allele was found in SLC Ecuador and SLC Peru albeit at lower frequency compared to lc and fw2.2. Fixation of the derived allele did not occur in the SLL vintage but instead became nearly complete in the contemporary SLL accessions. The derived alleles of fas and ovate were most likely to have arisen in the Ecuadorian or Peruvian SLC accessions and were maintained at low frequency in the remaining SLC accessions. Of the SLL vintage, 20 and 30% carried the derived alleles of ovate and fas, respectively. In other SLL groups, the derived allele for ovate and fas were found at low frequency in this dataset. However, the derived allele of ovate is quite common among Italian vintage cultivars where 38 to 47% of the accessions carry the mutation [Sun is present at low frequency in SLL vintage, fresh and processing whereas the allele has neither been detected in Ecuadorian and Peruvian SLC nor in the Mesoamerican accessions.Several genes involved in the transition from small and round to large and variably shaped tomato have been cloned in recent years. In all cases, the nucleotide polymorphism that is associated with the change in fruit appearance is known. We wanted to investigate when and where the derived alleles of the six fruit shape and weight loci arose and how they migrated through populations in the evolution of tomato. For all fruit morphology loci, the derived allele was at very low frequency or not found in the SP accessions Figure . The derKey questions regarding the evolutionary history of cultivated tomato include where and when the crop was domesticated and the position of SLC in this evolutionary process. In this study, we interrogated a selection of 2,313 SNPs from the SolCAP array in nearly 1,000 unique accessions comprised of SLL, SLC and the red-fruited wild relative SP. By combining accessions with robust passport data we were able to test hypotheses about the origin of cultivated tomato. Our results support the two-step domestication hypothesis proposed by Blanca et al. , and areLycopersicon section. Our data also supports the view that the Northern Ecuadorian SP is the closest ancestor of SLC. Northern Ecuadorian SLC was likely to have originated from Ecuadorian SP, yet its high genetic diversity and its reticulate structure in the phylogenetic network suggests a complex history. The position of SG within SP contrasts with a recent study [et al. and the present one, are based on a few number of accessions and Koenig et al. lacked SP accessions from Ecuador.The phylogenetic tree Figure , neighbont study  in whichThe data suggest two possible scenarios for the origin of SLC. Ecuadorian SLC features twice the level of the genetic diversity as Ecuadorian SP Table , thus itThe Mesoamerican SLL vintage subgroup appeared to be the most ancestral SLL according to the phylogenetic trees and the network. This SLL genetic subgroup was closely related to SLC Peru 2 in the phylogenetic network and tree. Thus, our data clearly support that SLC evolved into Mesoamerican SLL. According to the analyses with this dataset, all other SLL are monophyletic and all SLL groups originated from the SLL Mesoamerican accessions.lc, fw3.2 and fw2.2 are already prevalent in the ancestral SLC accessions from Northern Peru and across Ecuador  (Additional file Contemporary tomatoes are the result of introgressing genes from wild species into SLL starting before 1920 . The PCAlc, fas, fw2.2 and fw3.2. At the same time there are numerous resistances that have been bred into germplasm, with any given accession having multiple introgressed alleles that are missing from the vintage class, hence increased genetic diversity instead of phenotypic diversity in the contemporary class.The distribution of the fruit weight and shape alleles is skewed in processing and in fresh market classes of SLL, suggesting high selection pressures of shape loci for each market class. In contrast, the distribution of these alleles is more varied in the vintage group which is why this class is characteristically variable in shape and weight  while coS. pimpinellifolium. The data are an excellent resource for evolutionary biologists and plant breeders. Our analysis support a two-step domestication as proposed by Blanca et al. [This work represents an effort to show a comprehensive view of genomic variation in tomato and closely related species. We have analyzed and classified 1,008 tomato accessions, including the complete set of its closest wild relative, a et al. ; a firstAll supporting data are included as supplementary files.


Epinephelus marginatus, within Malta’s Fisheries Management Zone for the purpose of localised conservation planning. Epinephelus marginatus is a long-lived, sedentary, reef-associated protogynous hermaphrodite with high commercial and recreational value that is at risk of extinction throughout its global distribution. Based on global trends, population substructuring and gaps in local knowledge this has led to an increased interest in evaluation of local stock. Assessment of Maltese demography was based on historical and contemporary catch landings data whilst genetic population structure and regional connectivity patterns were evaluated by examining 175 individuals collected within the central Mediterranean region between 2002 and 2009 using 14 nuclear microsatellite loci. Demographic stock assessment of Maltese E. marginatus’ revealed a 99% decline in catch landings between 1947 and 2009 within the Fisheries Management Zone. A contemporary modest mean size was observed, 3 ± 3 kg, where approximately 17% of the population was juvenile, 68% female/sex-changing and 15% were male with a male-to-female sex ratio of 1:5. Genetic analysis describes the overall population of E. marginatus’ within the Fisheries Management Zone as decreasing in size (ƟH = 2.2), which has gone through a significant size reduction in the past (M = 0.41) and consequently shows signs of moderate inbreeding  with an estimated effective population size of 130 individuals. Results of spatially explicit Bayesian genetic cluster analysis detected two geographically distinct subpopulations within Malta’s Fisheries Management Zone and that they are connected to a larger network of E. marginatus’ within the Sicily Channel. Results suggest conservation management should be designed to reflect E. marginatus’ within Malta’s Fisheries Management Zone as two management units.The objective of this study is to describe the genetic population structure and demographic history of the endangered marine fish,  Genetic analysis is a method of assessment used as a complimentary appraisal to demography for defining population structure, geographic boundaries (i.e. breeding stock) and vulnerability (i.e. genetic diversity) of the subject species in question.Molecular conservation genetics seeks to manage biological threats by assessing genetic composition and implementing management strategies aimed at maintaining or restoring natural dynamics in order to protect unique species and their genetic diversity. The integration of population distribution mapping, identification of extrinsic environmental factors and population genetic theory play a significant role in the qualitative and quantitative assessment of species status and determination of sustainable conservation strategies. When evaluating marine species, stock assessment is traditionally the first evaluation of population status and trends. A decline in the number of observed individuals, catch size, landings data (kg) or an imbalance in population demography  for future colonisation . UnderstEpinephelus marginatus, within Malta’s Fisheries Management Zone (FMZ), a marine area 23,600 km2 that extends 25 nautical miles from the islands shorelines , Brazil2, E. Algeria) found that the Maltese dusky grouper mean body size was modest; however it was in accordance with the body size of individuals from the neighbouring populations of S. Italy and E. Algeria in the central Mediterranean region suggesting that this may be a regional trait [et al. [A comparison of the weight-length relationships of Maltese dusky groupers to discrete global populations (listed in descending order of size (g/TLcm): Senegal, W. Algeria, Brazilal trait ,120–125. [et al. , the numO = 0.691–0.795; HE = 0.695–0.759) were comparable to those in a similar Mediterranean study  using the same technique [IS = 0.10, P < 0.001) when compared to these nine neighbouring Mediterranean populations  where fNe) individuals. However, as Jamieson and Allendorf [Ne/Ncensus ratio of 0.10 [Ne/Nc ratio for dusky groupers is currently unresolved, for the gag grouper (Mycteroperca microlepis) which has similar biology and generation time it is estimated to be on the order of 0.01 [Within the management zone, measurement of adult effective population size was low, estimated to be around 130 individuals with fewer than a dozen effective breeders per reproductive cycle. In order for a species to retain its evolutionary potential, to avoid localised extinction and survive deterministic and stochastic events, it is recommended that immediate conservation action be put in place in Malta to restore natural dynamics. As a general guideline, in order to ensure short-term survival and minimise inbreeding depression within populations, Franklin  proposedllendorf  point ou of 0.10 , where h of 0.01 , where aApproximately 90% of dusky groupers sampled within the FMZ were identified as resident individuals with evidence of localised genetic clustering found between the island complex and Hurd Bank, located 12 nm offshore. Genetic homogenisation and consistent gene flow within the archipelago, based on observed surface current direction during spawning season , could bet al. [et al. [et al. [et al. [Regional patterns of genetic structure observed were similar to results seen by Schunter et al.  and de I [et al.  that fou [et al.  or de In [et al. , detecteM. microlepis) with an estimated regional Ne of 16,500 [Generally, in order to minimise loss of genetic diversity, between one and 10 migrants per generation is considered effective at maintaining within population heterozygosity and preventing long-term deleterious loss of polymorphisms . For newf 16,500 . AlthougNe [Ne) initially display a relative reduction in genetic and allelic variation and an increase in LD caused by genetic drift. This is often followed by population expansion and an increase in allelic frequencies, but not allelic richness  which leads to divergence from progenitor populations in absence of selection and immigration. Based on life history traits and environmental needs of dusky grouper settlers within shallow seagrass beds, the existence of an open water population at 40–50 m depth is somewhat surprising. Due to genetic divergence from nearby demes, juvenile stage migration related to intraspecific interaction, ontogenetic changes or seasonal micro-migration does not appear to be a plausible explanation [E. marginatus restocking programme [Influences of geographic and genetic isolation of Hurd Bank leading to significant genetic partitioning and differentiation within the region may be attributed to the founder effect. The founder effect is a type of bottleneck caused by a selective sweep or colonisation event by a few individuals. Whilst several founder models have been proposed –136, HurNe . Small elanation . Investirogramme ,138 thatrogramme . TherefoEpinephelus striatus) that have been heavily targeted by fishermen are not known to re-establish once they have been fished out [Effects of geographic productivity and environment on dusky grouper size and population density have been documented from several protected areas throughout the Western Mediterranean Sea, including Ustica Island Marine Reserve, IT established in 1986 ,140–151.shed out –156. Altshed out , and theshed out ,141. TheRecommendations are that the local population may benefit from a combination of a network of biotic targeted MPAs aimed at increasing biomass and a ban on spearfishing–at minimum—corresponding to the local spawning months, between May and August, which would help to mitigate further skewing of the male-to-female sex ratio, encourage repopulation and provide an environment able to sustain this repopulation. A minimum catch size limit is not supported because the most important demographic parameter to protect in this protogynous hermaphroditic species to achieve population stability are larger size females at a flexible transitional life stage.Monitoring recommendations include an annual fish based visual census, utilising the dusky grouper as an indicator species, especially where new MPAs or Marine Reserves have been established to examine the trends and evaluate the effectiveness of different levels of protection on the ecosystem as well as to elucidate shifts in biological landscapes caused by a climate change. In well managed MPAs, results of the visual census can been seen in as little as three years, whilst reaching carrying capacity for the dusky grouper is on the order of decades. Complimentary or in absence of a visual census, it is recommended that a molecular genetic based study be conducted every 14 years, the low minimum population doubling time for the dusky grouper. As a goal, the minimum viable effective population size should not be allowed to fall below 50 individuals in order to ensure short-term survival, with emphasis on preventing deviation from historical levels of the local male-to-female sex ratio and increasing allelic richness in order to retain long-term evolutionary potential.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.


CYP11A1, a gene belonging to the family 11 of cytochrome P450, encodes a crucial steroidogenic enzyme that catalyzes the initial step in the production of all classes of steroids. Many studies show that CYP11A1 plays a role in ovary function. However, the role of CYP11A1 in goose reproductive cycle remains largely unknown.CYP11A1 cDNA of Zhedong goose was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA consisted of a 96-base pair (bp) 5′untranslated region (UTR), a 179-bp 3′UTR and a 1509-bp open reading frame. The open reading frame encodes a putative 503 amino acid protein that shares high homology with CYP11A1 of other birds. The amino acid sequence possesses conserved domains of the P450 superfamily, which include the steroid-binding domain and the heme-binding region. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed CYP11A1 mRNA was expressed ubiquitously in every Zhedong goose tissue analyzed, including the heart, liver, glandular stomach, lung, spleen, kidney, intestinum tenue, intestinum crassum, cerebrum, cerebellum, muscle, oviduct, pituitary, hypothalamus and ovary.. The relatively low levels of CYP11A1 mRNA were detected in pituitary, ovary and oviduct tissues at ovulation when compared with levels at oviposition. Interestingly, higher expression was observed in ovary and oviduct tissues during brooding. Lastly, higher mRNA expression of Yangzhou geese was detected during the ovulation period than that of Zhedong geese.In this study, full-length Our findings reveal the sequence characterization and expression patterns of the CYP11A1 gene during the goose reproductive cycle, which may provides correlative evidence that CYP11A1 expression is important in reproduction activity. CYP11A1 gene) plays a major role in the regulation of steroidogenesis by mediating the conversion of cholesterol to pregnenolone [CYP11A1 has been the focus of many studies. Recently, more and more evidence shows that CYP11A1 plays a role in ovary function. In fowl, many investigations have found that CYP11A1 expression changes dynamically in developing follicles [CYP11A1 was principally driven by multiple trans-acting factors, like Sp-1 [Cytochrome P450 side chain cleavage  is a commercially important food source that is widely cultivated in China. It is an ideal animal model for characterization of fowl reproduction because of obvious reproductive stages and strong broodiness [CYP11A1 as an important candidate gene through transcriptome profiling of ovaries from laying and brooding geese [CYP11A1 in the goose reproductive cycle, we cloned Zhedong goose (Anas cygnoides) CYP11A1 and characterized its spatiotemporal expression patterns by Real-time qPCR. We also investigated the differences in CYP11A1 expression in high egg production breed and low egg production breed(Zhedong goose). These data may facilitate a better understanding of expression patterns of the CYP11A1 gene during the reproductive cycle in goose.The goose  and the Standards for the Administration of Experimental Practices .CYP11A1 expression patterns in different tissues. The Zhedong geese were anesthetized with sodium pentobarbital and the heart, liver, glandular stomach, lung, spleen, kidney, intestinum tenue, intestinum crissum, cerebrum, cerebellum, muscle, oviduct(infundibulum), pituitary, hypothalamus and ovary(stroma) were removed and immediately frozen in liquid nitrogen before storage at−80 °C for RNA isolation. Tissue samples from the oviduct(infundibulum), pituitary gland, hypothalamus, and ovary(stroma) were obtained from twelve sacrificed 380-day-old adult geese, including three laying Zhedong geese with an egg in the oviduct , three laying Zhedong geese without an egg in the oviduct , three brooding Zhedong geese, and three laying Yangzhou geese with an egg in the oviduct (ovulation). The tissue samples were obtained promptly, as described above, to characterize developmental expression patterns.One hundred female Zhedong geese and Yangzhou geese were selected from the breeding farm of Jiangsu Lihua Animal Husbandry Co. Ltd ), which laid about thirty and seventy eggs, respectively. They were both housed semi-enclosed building and raised the same conditions according to the farm’s standard practice. During the experiment, geese were fed ad libitum with rice grain supplemented with green grass or water plants whenever possible. The feed was provided during the daytime when the geese were released to an open area. The geese were exposed to natural light and ambient temperature throughout this study. The five 120-day-old young (prelaying) Zhedong female geese were sacrificed to investigate CYP11A1 gene [CYP11A1 were amplified via rapid amplification of cDNA ends (RACE) using the SMART RACE cDNA amplification protocol  and the 3′-Full RACE Kit , respectively. RACE primers .Total RNA was extracted from collected tissue samples using TRIzol reagent according to the manufacturer’s instruction . The RNA was resuspended in RNase-free water, and the concentration and purity were determined using a NanoDrop Spectrophotometer . After purification, total RNA (2 μg) was reverse transcribed using M-MLV reverse transcriptase  according to the manufacturer’s protocol. BLAST analysis of one unigene revealed that it was highly similar to the chicken 1A1 gene . Primershttp://au.expasy.org/tools/), respectively. Homology analyses were carried out using Clustal W (http://www.ebi.ac.uk/Tools/msa/). The phylogenetic analyses and statistical neighbor-joining bootstrap tests of the phylogenies were performed using the MEGA package (version 6.0) and the bootstrap method was used to indicate confidence values for tree nodes based on 1000 replicates. The conserved domain analysis was predicted by SMART (http://smart.embl-heidelberg.de/).The Zhedong goose CYP11A1 cDNA and the deduced amino acid sequences were analyzed using DNAssist (version 2.2) and the Expasy search program  and performed on an ABI two-step RT-PCR system  with diluted first-strand cDNA. qPCR programs for CYP11A1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were: 1 cycle of 95 °C for 5 min,40 cycles of 95 °C for 10 s, 60 °C for 34 s data collection, followed by 1 cycle for melting curve analysis. All cDNA synthesis reactions were carried out using 100 ng of total RNA per reaction and assayed in three to four technical replicates for each set of biological samples. The same methods were used to determine the Zhedong CYP11A1 mRNA expression profile during the reproductive cycle. The GAPDH gene served as an internal reference gene and the hypothalamus tissue from prelaying Zhedong geese served as a calibrator tissue for differential expression analyses. To analyze the difference of expression patterns between different egg production goose breeds(Zhedong geese and Yangzhou geese), the mean ΔCt value of the hypothalamus tissue of Zhedong geese within each group was used as the calibrator.To study Statistical analyses were performed using t-tests independent of group and SPSS version 13.0 software.CYP11A1, RT-PCR and RACE were carried out. The CYP11A1 cDNA was only found to be 1784 nucleotides in length, which included a 96-nucleotide 5′ UTR, a 179-nucleotide 3′ UTR, and a 1509-nucleotide open reading frame putatively encoding a single 503 amino acid protein. The other transcript variant did not been found among the tissues tested in Zhedong goose.To obtain the full-length cDNA of goose To evaluate the relationship between goose CYP11A1 and that of other birds, sequence alignment and phylogenetic analysis were carried out. Alignment of the amino acid residues of goose CYP11A1 with those of other birds forms is shown in Fig. Phylogenetic analysis of CYP11A1 resulted in clear segregation into three groups, one branch containing mammalian CYP11A1, one branch of birds CYP11A1, another branch of fish CYP11A1Fig. . Goose CCYP11A1 was ubiquitously expressed in fifteen tissues tested, but the expression levels were distinctly different . We observed different CYP11A1 expression profiles in the different tissues at different times of the reproductive cycle  and Yangzhou geese (high egg production) in ovulation periods . The expression of CYP11A was significantly higher in the examined reproductive tissues of Yangzhou geese in the ovulation period in pituitary and ovary tissues than that of Zhedong geese (Fig. Additionally, to explore the differences in ese Fig. .Fig. 5CoCYP11A1 were cloned from Chinese goose ovaries. The deduced amino acid sequence of CYP11A1 shows high identity with that of other birds and contains some structural domains typical of the cytochrome P450 superfamily, specifically a steroid-binding domain and a heme-binding domain [In this study, cDNAs encoding g domain . Both doAs expected on the basis of the predicted goose CYP11A1 amino acid sequence, phylogenetic analysis clearly clustered goose CYP11A1 with other Phasianidae forms and showed that birds CYP11A1 appear to be significantly diverged from mammalian forms and fish forms.CYP11A1 mRNA was expressed ubiquitously in every tissue analyzed except heart and muscle, albeit to different degrees. As expected, CYP11A1 mRNA was easily detected in reproductive tissues and endocrine tissues. These results are in agreement with recent molecular studies aimed at determining the temporal expression of CYP11A1 in mammals [CYP11A1 was previously well documented in tissues such as the adrenals, gonads, placenta [CYP11A1 to be expressed in various nonendocrine tissues, including the lung, spleen, stomach, liver, brain, gut and kidney. During the last few years, CYP11A1 has been shown to be expressed in various tissues that are not involved in steroid synthesis. Sigel A et al. have elaborated CYP11A1 own ubiquitous roles [ mammals , 20. Theplacenta , 22, panplacenta , skin [2placenta , gut [25placenta  and certplacenta , where iplacenta , 28. We us roles .CYP11A1 transcript abundance drastically changed in ovary, oviduct, and pituitary tissues during the different stages of the reproductive cycle. As expected, CYP11A1 mRNA levels were relatively low at ovulation compared with oviposition. CYP11A1 catalyzes the conversion of cholesterol to pregnenolone in the first step of steroid biosynthesis [CYP11A1 mRNA level. The higher CYP11A1 expression was observed in ovary and oviduct tissues during broodiness, which is in concordance with previous studies that showed that gonadal steroid levels are maintained at a high level in broody birds [CYP11A1 expression was high in pituitary than the oviduct during prelaying time. In contrast, the expression of gene was high in ovary/oviduct during oviposition, ovulation and brooding, but low in hypothalamus/pituitary (Fig. CYP11A1 mRNA expression.The qPCR analysis revealed that ynthesis . It has ynthesis , 31 whicdy birds . Interesary Fig. . There mCYP11A1 expression in Yangzhou geese than in Zhedong geese during the ovulation period. Yangzhou geese are known for high egg production, while Zhedong geese show low egg production. Yangzhou geese might need to release more pregnenolone to ovulate, so the observed higher CYP11A1 expression levels may be required to ensure sufficient hormone secretion.We also observed higher CYP11A1, and analyze its expression during the goose reproductive cycle. The relatively low levels of CYP11A1 mRNA were detected at ovulation when compared with levels at oviposition. The higher mRNA expression was investigated during the ovulation period of Yangzhou geese than that of Zhedong geese. Our findings provides correlative evidence that CYP11A1 expression is important in reproduction activity.In summary, we present the molecular cloning and characterization of the goose