scholarly journals Evaluation of the Romosinuano cattle population structure in Mexico using pedigree analysis

2020 ◽  
Vol 33 (1) ◽  
pp. 44-59
Author(s):  
Rafael Núñez-Domínguez ◽  
Ricardo E Martínez-Rocha ◽  
Jorge A Hidalgo-Moreno ◽  
Rodolfo Ramírez-Valverde ◽  
José G García-Muñiz
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Gene Flow ◽  
Population Size ◽  
Effective Population Size ◽  
Genetic Drift ◽  
Pedigree Analysis ◽  
Effective Population ◽  
Genetic Management ◽  
Generation Interval

Background: Romosinuano cattle breed in Mexico has endured isolation and it is necessary to characterize it in order to facilitate sustainable genetic management. Objective: To assess the evolution of the structure and genetic diversity of the Romosinuano breed in Mexico, through pedigree analysis. Methods: Pedigree data was obtained from Asociación Mexicana de Criadores de Ganado Romosinuano y Lechero Tropical (AMCROLET). The ENDOG program (4.8 version) was used to analyze two datasets, one that includes upgrading from F1 animals (UP) and the other with only straight-bred cattle (SP). For both datasets, three reference populations were defined: 1998-2003 (RP1), 2004-2009 (RP2), and 2010-2017 (RP3). The pedigree included 3,432 animals in UP and 1,518 in SP. Demographic parameters were: Generation interval (GI), equivalent number of generations (EG), pedigree completeness index (PCI), and gene flow among herds. Genetic parameters were: Inbreeding (F) and average relatedness (AR) coefficients, effective population size (Nec), effective number of founders and ancestors, and number of founder genome equivalents. Results: The GI varied from 6.10 to 6.54 for UP, and from 6.47 to 7.16 yr for SP. The EG of the UP and SP improved >63% from RP1 to RP3. The PCI increased over time. No nucleus or isolated herds were found. For RP3, F and AR reached 2.08 and 5.12% in the UP, and 2.55 and 5.94% in the SP. For RP3, Nec was 57 in the UP and 45 in the SP. Genetic diversity losses were attributed mainly (>66%) to genetic drift, except for RP3 in the SP (44%). Conclusions: A reduction of the genetic diversity has been occurring after the Romosinuano breed association was established in Mexico, and this is mainly due to random loss of genes.Keywords: effective population size; gene flow; genetic diversity; genetic drift; generation interval; inbreeding; pedigree; population structure; probability of gene origin; Romosinuano cattle. Resumen Antecedentes: La raza bovina Romosinuano ha estado prácticamente aislada en México y requiere ser caracterizada para un manejo genético sostenible. Objetivo: Evaluar la evolución de la estructura y diversidad genética de la raza Romosinuano en México, mediante el análisis del pedigrí. Métodos: Los datos genealógicos provinieron de la Asociación Mexicana de Criadores de Ganado Romosinuano y Lechero Tropical (AMCROLET). Los análisis se realizaron con el programa ENDOG (versión 4.8) para dos bases de datos, una que incluyó animales en cruzamiento absorbente (UP) a partir de F1 y la otra con sólo animales puros (SP). Para ambas bases de datos se definieron tres poblaciones de referencia: 1998-2003 (RP1), 2004- 2009 (RP2), y 2010-2017 (RP3). El pedigrí incluyó 3.432 animales en la UP y 1.518 en la SP. Los parámetros demográficos fueron: intervalo generacional (GI), número de generaciones equivalentes (EG), índice de completitud del pedigrí (PCI), y flujo de genes entre hatos. Los parámetros genéticos fueron: coeficientes de consanguinidad (F) y de relación genética aditiva (AR), tamaño efectivo de la población (Nec), número efectivo de fundadores y ancestros, y número equivalente de genomas fundadores. Resultados: El GI varió de 6,10 a 6,54 para la UP, y de 6,47 a 7,16 años para la SP. El EG de la UP y la SP mejoró >63%, de RP1 a RP3. El PCI aumentó a través de los años, pero más para la SP que para la UP. No se encontraron hatos núcleo o aislados. Para RP3, F y AR alcanzaron 2,08 y 5,12% en la UP, y 2,55 y 5,94% en la SP. Para RP3, Nec fue 57 en la UP y 45 en la SP. Más de 66% de las pérdidas en diversidad genética se debieron a deriva genética, excepto para RP3 en la UP (44%). Conclusiones: una reducción de la diversidad genética ha estado ocurriendo después de que se formó la asociación de criadores de ganado Romosinuano en México, y es debida principalmente a pérdidas aleatorias de genes.Palabras clave: consanguinidad; deriva genética; diversidad genética; estructura poblacional; flujo de genes; ganado Romosinuano; intervalo generacional; pedigrí; probabilidad de origen del gen; tamaño efectivo de población. Resumo Antecedentes: A raça bovina Romosinuano tem estado praticamente isolada no México e precisa ser caracterizada para um manejo genético sustentável. Objetivo: Avaliar a evolução da estrutura e diversidade genética da raça Romosinuano no México, através da análise de pedigree. Métodos: Os dados genealógicos vieram da Asociación Mexicana de Criadores de Ganado Romosinuano y Lechero Tropical (AMCROLET). As análises foram feitas com o programa ENDOG (versão 4.8) para duas bases de dados, uma que incluiu animais em cruzamento absorvente (UP) a partir da F1 e a outra base de dados somente com animais puros (SP). Para ambas bases de dados foram definidas três populações de referência: 1998-2003 (RP1), 2004-2009 (RP2) e 2010-2017 (RP3). O pedigree incluiu 3.432 animais na UP e 1.518 na SP. Os parâmetros demográficos foram: intervalo entre gerações (GI), número de gerações equivalentes (EG), índice de completude do pedigree (PCI), e fluxo de genes entre rebanhos. Os parâmetros genéticos foram: coeficiente de consanguinidade (F) e da relação genética aditiva (AR), tamanho efetivo da população (Nec), número efetivo de fundadores e ancestrais, e número equivalente de genomas fundadores. Resultados: O GI variou de 6,10 a 6,54 para a UP, e de 6,47 a 7,16 anos para a SP. EG da UP e a SP melhorou >63%, de RP1 a RP3. O PCI aumentou ao longo dos anos, mas mais para a SP do que para o UP. Não se encontraram rebanhos núcleo ou isolados. Para RP3, F e AR alcançaram 2,08 e 5,12% na UP, e 2,55 e 5,94% na SP. Para RP3, Nec foi 57 na UP e 45 na SP. Mais de 66% das perdas em diversidade genética foram ocasionadas pela deriva genética, exceto para RP3 no UP (44%). Conclusões: Depois que a associação da raça Romosinuano foi estabelecida no México, tem ocorrido uma redução da diversidade genética, principalmente devido a perdas aleatórias de genes.Palavras-chave: consanguinidade; deriva genética; diversidade genética, estrutura populacional; fluxo de genes; intervalo entre gerações; pedigree; probabilidade de origem do gene; Romosinuano; tamanho efetivo da população.

scholarly journals Population structure of Czech cold-blooded breeds of horses

2011 ◽  
Vol 54 (1) ◽  
pp. 1-9
Author(s):  
L. Vostrý ◽  
Z. Čapková ◽  
J. Přibyl ◽  
B. Hofmanová ◽  
H. Vostrá Vydrová ◽  
...  
Keyword(s):  
Population Structure ◽  
Genetic Variability ◽  
Population Size ◽  
Effective Population Size ◽  
Inbreeding Coefficient ◽  
Effective Population ◽  
The Czech Republic ◽  
Generation Interval ◽  
Average Value ◽  
Inbreeding Coefficients

Abstract. In order to estimate effective population size, generation interval and the development of inbreeding coefficients (Fx) in three original breeds of cold-blooded horses kept in the Czech Republic: Silesian Noriker (SN), Noriker (N) and Czech-Moravian Belgian horse (CMB) all animals of the particular breeds born from 1990 to 2007 were analysed. The average values of generation interval between parents and their offspring were: 8.53 in SN, 8.88 in N and 8.56 in CMB. Average values of effective population size were estimated to be: 86.3 in SN, 162.3 in N and 104.4 in CMB. The average values of inbreeding coefficient were 3.13 % in SN stallions and 3.39 % in SN mares, in the N breed 1.76 % and 1.26 % and in the CMB breed 3.84 % and 3.26 % respectively. Overall averages of Fx were: 3.23 %, 1.51 % and 3.55 % for the breeds SN, N and CMB. The average value of inbreeding coefficient Fx increased by 1.22 % in SN, by 0.35 % in N and by 1.01 % in CMB, respectively. This may lead to a reduction in genetic variability. Reduction in genetic variability could be either controlled in cooperation with corresponding populations of cold-blooded breeds in other European countries or controlled by number of sires used in population

scholarly journals Genetic diversity, population structure, and effective population size in two yellow bat species in south Texas

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10348
Author(s):  
Austin S. Chipps ◽  
Amanda M. Hale ◽  
Sara P. Weaver ◽  
Dean A. Williams
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
North America ◽  
Wind Energy ◽  
Population Size ◽  
Effective Population Size ◽  
Microsatellite Loci ◽  
South Texas ◽  
Population Substructure ◽  
Effective Population

There are increasing concerns regarding bat mortality at wind energy facilities, especially as installed capacity continues to grow. In North America, wind energy development has recently expanded into the Lower Rio Grande Valley in south Texas where bat species had not previously been exposed to wind turbines. Our study sought to characterize genetic diversity, population structure, and effective population size in Dasypterus ega and D. intermedius, two tree-roosting yellow bats native to this region and for which little is known about their population biology and seasonal movements. There was no evidence of population substructure in either species. Genetic diversity at mitochondrial and microsatellite loci was lower in these yellow bat taxa than in previously studied migratory tree bat species in North America, which may be due to the non-migratory nature of these species at our study site, the fact that our study site is located at a geographic range end for both taxa, and possibly weak ascertainment bias at microsatellite loci. Historical effective population size (NEF) was large for both species, while current estimates of Ne had upper 95% confidence limits that encompassed infinity. We found evidence of strong mitochondrial differentiation between the two putative subspecies of D. intermedius (D. i. floridanus and D. i. intermedius) which are sympatric in this region of Texas, yet little differentiation using microsatellite loci. We suggest this pattern is due to secondary contact and hybridization and possibly incomplete lineage sorting at microsatellite loci. We also found evidence of some hybridization between D. ega and D. intermedius in this region of Texas. We recommend that our data serve as a starting point for the long-term genetic monitoring of these species in order to better understand the impacts of wind-related mortality on these populations over time.

Analysis of genetic diversity in four Canadian swine breeds using pedigree data

2010 ◽  
Vol 90 (3) ◽  
pp. 331-340 ◽  
Author(s):  
M G Melka ◽  
F. Schenkel
Keyword(s):  
Genetic Diversity ◽  
Population Size ◽  
Effective Population Size ◽  
Genetic Drift ◽  
Relative Proportion ◽  
Random Genetic Drift ◽  
Pedigree Data ◽  
Effective Population ◽  
Animal Genetic Resources ◽  
Loss Of Genetic Diversity

Conservation of animal genetic resources entails judicious assessment of genetic diversity as a first step. The objective of this study was to analyze the trend of within-breed genetic diversity and identify major causes of loss of genetic diversity in four swine breeds based on pedigree data. Pedigree files from Duroc (DC), Hampshire (HP), Lacombe (LC) and Landrace (LR) containing 480 191, 114 871, 51 397 and 1 080 144 records, respectively, were analyzed. Pedigree completeness, quality and depth were determined. Several parameters derived from the in-depth pedigree analyses were used to measure trends and current levels of genetic diversity. Pedigree completeness indexes of the four breeds were 90.4, 52.7, 89.6 and 96.1%, respectively. The estimated percentage of genetic diversity lost within each breed over the last three decades was approximately 3, 22, 12 and 2%, respectively. The relative proportion of genetic diversity lost due to random genetic drift in DC, HP, LC and LR was 74.5, 63.6, 72.9 and 60.0%, respectively. The estimated current effective population size for DC, HP, LC and LR was 72, 14, 36 and 125, respectively. Therefore, HP and LC have been found to have lost considerable genetic diversity, demanding priority for conservation. Key words: Genetic drift, effective population size

scholarly journals Genetic Resilience of a Once Endangered Species, Tibetan Antelope (Pantholops hodgsonii)

2019 ◽  
Author(s):  
Yue Shi ◽  
Jiarui Chen ◽  
Jianping Su ◽  
Tongzuo Zhang ◽  
Samuel K. Wasser
Keyword(s):  
Genetic Diversity ◽  
Gene Flow ◽  
Population Size ◽  
Effective Population Size ◽  
Genetic Bottleneck ◽  
Migration Routes ◽  
Effective Population ◽  
Human Disturbances ◽  
Population Reduction ◽  
Tibetan Antelope

AbstractPopulation reduction is generally assumed to reduce the population’s genetic diversity and hence its ability to adapt to environmental change. However, if life history traits that promote gene flow buffer populations from such impacts, conservation efforts should aim to maintain those traits in vulnerable species. Tibetan antelope (Pantholops hodgsonii) has experienced population reduction by 95% due to poaching during the 20th century. We hypothesize that opportunities for gene flow provided by their sex-specific migration buffered their genetic diversity from the poaching impacts. We measured the mtDNA (control region, CR) and nuDNA (microsatellites or STRs) diversity, population differentiation, along with the change in effective population size (pre-poaching era vs. post-poaching era) and tested for a genetic bottleneck. Our results showed that Tibetan antelope maintained considerable genetic diversity in both mtDNA CR and STR markers (Hd = 0.9970 and Hobs = 0.8446, respectively), despite a marked reduction in post-poaching effective population size 368.9 (95% CI of 249.3 - 660.6) compared to the pre-poaching average (4.93×103 - 4.17×104). Post-poached populations also had low population structure and showed no evidence of a genetic bottleneck. Pairwise Fst values using CR haplotype frequencies were higher than those using STR allele frequencies, suggesting different degrees of gene flow mediated by females and males. This study suggests that the Tibetan antelope’s sex-specific migration buffered their loss of genetic diversity in the face of severe demographic decline. These findings highlight the importance of recognizing the traits likely to maintain genetic diversity and promoting conservation efforts that allow them to be exercised. For Tibetan antelope, this requires assuring that their migration routes remain unobstructed by growing human disturbances while continuing to enforce anti-poaching law enforcement efforts.

scholarly journals Life histories predict genetic diversity and population structure within three species of Octopus targeted by small-scale fisheries in Northwest Mexico

2017 ◽  
Author(s):  
José F Domínguez-Contreras ◽  
Adrian Munguia-Vega ◽  
Bertha P Ceballos-Vázquez ◽  
Marcial Arellano-Martínez ◽  
Francisco J García-Rodríguez ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Population Size ◽  
Effective Population Size ◽  
Gulf Of California ◽  
Life Histories ◽  
Local Population ◽  
Small Scale ◽  
Effective Population ◽  
Northwest Mexico

The fishery for octopus in Northwest Mexico has increased to over 2,000 tons annually, but to date the specific composition of the catch has been ignored. With at least three main species with varying life histories targeted by artisanal fisheries in the region, lack of information about the distribution of each species and metapopulation size and structure could impede effective fisheries management to avoid overexploitation. Here we tested if different life histories in three species of octopus help to predict observed patterns of genetic diversity, population dynamics, structure and connectivity that could be relevant to the sustainable management of the fishery. We sequenced two mitochondrial genes and genotyped seven nuclear microsatellite loci to identify the distribution of each species in 20 locations from the Gulf of California and the Pacific coast of the Baja California peninsula. We tested four a priori hypothesis derived from population genetic theory based on differences in the fecundity and dispersal potential for each species. We found that the species with low fecundity and without a planktonic larval stage (Octopus bimaculoides) had lower average effective population size and genetic diversity, but higher levels of kinship, population structure, and richness of private alleles, suggesting limited dispersal and high local recruitment. In contrast, two species with higher fecundity and planktonic larvae (O. bimaculatus, O. hubbsorum) showed higher effective population size and genetic diversity, and overall lower kinship and population structure, supporting higher levels of gene flow over a larger geographical scale. Even among the latter, there were differences in the calculated parameters possibly associated with increased connectivity in the species with the longest planktonic larval duration (O. bimaculatus). We consider that O. bimaculoides could be more susceptible to over exploitation of small, isolated populations that could have longer recovery times, and suggest that management should take place within each local population. For the two species with pelagic larvae, management should consider metapopulation structure over larger geographic scales and the directionality and magnitude of larval dispersal between localities driven by ocean currents. The distribution of each species and variations in their reproductive timing should also be considered when establishing marine reserves or seasonal fishing closures.

scholarly journals The paradox of retained genetic diversity of Hippocampus guttulatus in the face of demographic decline

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rupert Stacy ◽  
Jorge Palma ◽  
Miguel Correia ◽  
Anthony B. Wilson ◽  
José Pedro Andrade ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Gene Flow ◽  
Population Size ◽  
Effective Population Size ◽  
Population Decline ◽  
Genetic Bottleneck ◽  
Effective Population ◽  
Evolutionary Potential ◽  
Hippocampus Guttulatus ◽  
Ria Formosa

AbstractGenetic diversity is the raw foundation for evolutionary potential. When genetic diversity is significantly reduced, the risk of extinction is heightened considerably. The long-snouted seahorse (Hippocampus guttulatus) is one of two seahorse species occurring in the North-East Atlantic. The population living in the Ria Formosa (South Portugal) declined dramatically between 2001 and 2008, prompting fears of greatly reduced genetic diversity and reduced effective population size, hallmarks of a genetic bottleneck. This study tests these hypotheses using samples from eight microsatellite loci taken from 2001 and 2013, on either side of the 2008 decline. The data suggest that the population has not lost its genetic diversity, and a genetic bottleneck was not detectable. However, overall relatedness increased between 2001 to 2013, leading to questions of future inbreeding. The effective population size has seemingly increased close to the threshold necessary for the population to retain its evolutionary potential, but whether these results have been affected by sample size is not clear. Several explanations are discussed for these unexpected results, such as gene flow, local decline due to dispersal to other areas of the Ria Formosa, and the potential that the duration of the demographic decline too short to record changes in the genetic diversity. Given the results presented here and recent evidence of a second population decline, the precise estimation of both gene flow and effective population size via more extensive genetic screening will be critical to effective population management.

scholarly journals Differentiation with drift: a spatio-temporal genetic analysis of Galápagos mockingbird populations ( Mimus spp.)

2010 ◽  
Vol 365 (1543) ◽  
pp. 1127-1138 ◽  
Author(s):  
Paquita E. A. Hoeck ◽  
Jennifer L. Bollmer ◽  
Patricia G. Parker ◽  
Lukas F. Keller
Keyword(s):  
Genetic Diversity ◽  
Gene Flow ◽  
Population Size ◽  
Genetic Drift ◽  
Bird Species ◽  
Effective Population ◽  
Island Size ◽  
Island Populations ◽  
Population Sizes ◽  
Spatio Temporal

Small and isolated island populations provide ideal systems to study the effects of limited population size, genetic drift and gene flow on genetic diversity. We assessed genetic diversity within and differentiation among 19 mockingbird populations on 15 Galápagos islands, covering all four endemic species, using 16 microsatellite loci. We tested for signs of drift and gene flow, and used historic specimens to assess genetic change over the last century and to estimate effective population sizes. Within-population genetic diversity and effective population sizes varied substantially among island populations and correlated strongly with island size, suggesting that island size serves as a good predictor for effective population size. Genetic differentiation among populations was pronounced and increased with geographical distance. A century of genetic drift did not change genetic diversity on an archipelago-wide scale, but genetic drift led to loss of genetic diversity in small populations, especially in one of the two remaining populations of the endangered Floreana mockingbird. Unlike in other Galápagos bird species such as the Darwin's finches, gene flow among mockingbird populations was low. The clear pattern of genetically distinct populations reflects the effects of genetic drift and suggests that Galápagos mockingbirds are evolving in relative isolation.

scholarly journals Genetic characterization of the endangered and endemic anchialine squat lobster Munidopsis polymorpha from Lanzarote (Canary Islands): management implications

2012 ◽  
Vol 69 (6) ◽  
pp. 1030-1037 ◽  
Author(s):  
Patricia Cabezas ◽  
Fernando Alda ◽  
Enrique Macpherson ◽  
Annie Machordom
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Population Size ◽  
Effective Population Size ◽  
Canary Islands ◽  
Genetic Characterization ◽  
Effective Population ◽  
Management Implications ◽  
Squat Lobster

Abstract Cabezas, P., Alda, F., Macpherson, E., and Machordom, A. 2012. Genetic characterization of the endangered and endemic anchialine squat lobster Munidopsis polymorpha from Lanzarote (Canary Islands): management implications. – ICES Journal of Marine Science, 69: 1030–1037. Anchialine species show restricted geographic ranges, high habitat specificity, and small population sizes. These factors make them particularly vulnerable to human activities, yet little is known about their ecology and evolutionary history. Munidopsis polymorpha is a decapod endemic to an anchialine cave system of the Corona lava tube in Lanzarote (Canary Islands). The present study, the first genetic survey conducted on this largely unknown species, was designed to characterize its genetic diversity, population structure and recent demographic history, using sequence data for the cytochrome oxidase I gene and eight microsatellites. A single haplotype was identified in the mitochondrial dataset. Nuclear genetic diversity was also low (average = 4.375 ± 1.685). No significant genetic structure was detected between sampling sites and years, either by analysis of molecular variance (FST = 0.006, p = 0.110) or Bayesian clustering analysis (K = 1), indicating this species should be treated as a single management unit. Neither did we find evidence for a recent bottleneck event, and estimates of effective population size were extremely low (∼50). The lack of population structure, low genetic diversity and extremely low effective population size reinforce the high degree of isolation and endemicity of this species, and, consequently, the need to implement appropriate management actions.

scholarly journals Genetic characteristics and diversity of Korean Jeju Black cattle by whole genome SNP markers

2020 ◽  
Author(s):  
Mohammad Zahangir Alam ◽  
Hyo Jung Son ◽  
Yun Mi Lee ◽  
Lauren Hulsman Hanna ◽  
David Riley ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Population Size ◽  
Effective Population Size ◽  
Genetic Improvement ◽  
Effective Population ◽  
Model Based Clustering ◽  
Cattle Breeds ◽  
Model Based ◽  
Jeju Black Cattle

Abstract Background Conservation and genetic improvement of cattle breeds requires to know the information about genetic diversity and population structure of animals. This study investigated the genetic diversity and population structure among the three breeds raised in Korean peninsula. Three popular breed found in Korea , i.e. Jeju Black, Hanwoo, Holstein with other six breeds such as Angus, Hereford, Brown Wagyu, Black Wagyu, Brahman and Nellore was examined in this study. Genetic diversity within the cattle breeds was analyzed using the popular measures of genetic diversity namely minor allele frequency (MAF), observed and expected heterozygosity (H O and H E ), inbreeding coefficient (F IS ) and past effective population size (N E ). Molecular variance and population structure were performed among the nine cattle breeds using model-based clustering (ADMIXTURE) analysis. Genetic distances between breed pairs were evaluated using Nei’s genetic distance (D A ) and with Weir and Cockerham’s F ST . Results This study revealed that Jeju Black cattle had lowest level of heterozygocity (HE = 0.21) among the studied taurine cattle breeds ranging from 0.25 to 0.30, and low MAF of 0.16, while other breeds have MAF ranging 0.11~0.21. The level of inbreeding was -0.076 in case of Jeju Black as compared to other breed (-0.018 ~ -0.118). PCA analysis and neighbor-joining (NJ) tree showed a clear separation of Jeju Black cattle from other local and exotic cattle breeds. Model-based clustering also revealed a distinct pattern of admixture of Jeju Black cattle having no clustering with other studied populations. The F ST value between Jeju Black cattle and Hanwoo was 0.106, which was lowest across the breeds ranging from 0.161 to 0.274, indicating some degree of genetic closeness of Jeju Black cattle with Hanwoo. The N E of Jeju Black cattle was 38, whereas Hanwoo was 209 in the most recent 13 generation ago. Conclusion This study indicates an alarming trend of reducing effective population size in Jeju Black cattle. Thus, a sustainable breeding policy should be implemented to increase the population of Jeju Black cattle for the genetic improvement and future conservation.

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