Salmo trutta) samples to study fine-scale and short-term impacts of different management practices. These had changed from traditional breeding practices, using the same broodstock for several years, to modern breeding practices, including annual broodstock replacement, in the transnational subarctic Pasvik River. Using population genetic structure analyses , four historical genetic clusters (E2001A-D), likely representing family lineages resulting from different crosses, were found in zone E. These groups were characterized by consistently lower genetic diversity, higher within-group relatedness, lower effective population size, and significantly smaller body size than contemporary stocked (E2001E) and wild fish (E2001F). However, even current breeding practices are insufficient to prevent genetic diversity loss and morphological changes as demonstrated by on average smaller body sizes and recent genetic bottleneck signatures in the modern breeding stock compared to wild fish. Conservation management must evaluate breeding protocols for stocking programs and assess if these can preserve remaining natural genetic diversity and morphology in brown trout for long-term preservation of freshwater fauna.Maintaining standing genetic variation is a challenge in human-dominated landscapes. We used genetic  and morphological  measurements of 593 contemporary and historical brown trout ( Hence, standing genetic variation is the basis for responses to anthropogenic pressures and environmental change, and it ensures the long-term survival of species3. Spatial genetic analyses have greatly improved our understanding of intraspecific genetic variation, including phenomena like gene flow and connectivity and/or isolation of populations4. In comparison, temporal changes in genetic variation are less studied, despite their importance for predicting impacts of anthropogenic activities and environmental disturbances on natural populations6. Recent conservative estimates put the global loss of intraspecific genetic variation at 6% since the industrial revolution2. This coincides with estimates that the rate of local population extinction is at least three orders of magnitude higher than species extinctions7.Standing genetic variation is a key factor for the preservation of ecological and evolutionary functions in natural populations, as it is linked to population fitness and adaptive potential6. In fish species, the analysis of temporal genetic changes has attracted interest for studying genetic depletion/replacement and loss of locally adapted populations8. Further, changes in population genetic structure caused by release of translocated broodstocks, stocking, and accidental escapes from fish farms has been in the focus of fishery research8. Other anthropogenic impacts, like overharvesting and the destruction of natural spawning and nursery areas, can lower reproduction resulting in decreasing population sizes and thus, decreased genetic diversity over time4. In addition, hydroelectric dams increase isolation and genetic divergence between river zones4. Negative impacts of anthropogenic infrastructure may have been unforeseen in some cases, but often, such as with construction of hydroelectric dams, they were to some extent foreseen. Moreover, at least in Norway, dam construction projects were often coupled with positivistic stocking programs as mitigation and replacement for lost breeding potentials as part of the concession9.Temporal fluctuations in population size and genetic diversity can cause shifts in allele frequencies that are not completely captured by spatial analyses. Temporal study designs can enhance our understanding of demographic fluctuations, like changes in effective population size and spatial genetic differentiation10. By contrast, modern single-generational supportive breeding, with annual replacement of the parental fish with wild-caught individuals that are used only once for breeding purposes , is expected to have fewer negative impacts because the number of breeders should theoretically be larger and therefore, genetic diversity should be better preserved. Furthermore, the fact that the fish are not as long in captivity should reduce domestication effects and selection for hatchery conditions, which in turn may lead to lower survival and reduced fitness of released fish in the wild12.Different hatchery breeding strategies likely have different impacts on the genetic composition of released fish. For example, rearing repeatedly from a single captive broodstock without replacing individuals, referred to in this paper as traditional multi-generational supportive breeding, can result in inbreeding patterns and genetic drift because the same individuals are crossed and their offspring released to the wild where they can breed with each otherSalmo trutta L. 1758). As shown by4, the natural genetic integrity of the population is threatened by habitat destruction, fragmentation caused by hydropower developments, and the long-term practice of supportive stocking. Different management practices in Norway and Russia have led to distinct genetic diversity patterns in this population. Mainly, river zones situated in Russia are not stocked, whereas Norwegian-Russian zones have been stocked with ~ 5000 offspring of local specimens annually since the 1970s to supplement natural recruitment14. Stocking was introduced because seven hydroelectric dams without fish passes were built between 1932 and 1978. The breeding program was initiated as a requirement for building the last dam . Hypotheses included declining genetic diversity and/or allele frequency changes as well as bottlenecks and reductions in effective population sizes through time due to increasing stocking impacts . Furthermore, we investigated the differential effects of traditional and modern supportive breeding practices, which shifted in the mid-1990s from a multi- to a single-generational  broodstock of local trout specimens. Overall, the aim was to study how different breeding practices have influenced genetic variation and demographics through time in brown trout.4 (DRYAD: https://doi.org/10.5061/dryad.45482t5). For the contemporary samples, we only give the zone designation, as shown in Fig. historical samples, we additionally indicate the collection year as mentioned above. The study was performed in strict accordance with Norwegian legislation. Fish were euthanized by means of cerebral concussion prior to sample collection. A fishing license is required from the fishing right owner. Accordingly, we obtained fishing permissions for the Pasvik watercourse from the County Governor of Finnmark with legal authority through LOV 1992-05-15 nr 47, §13. No ethical permission is required from the Norwegian Animal Research Authority for collection with gill nets and the associated sacrifice of fish . Additional samples were obtained by opportunistic collection of samples from local anglers with permission to fish in the river for personal sustenance.In total, the data set consisted of 593 tissue samples of brown trout from the Pasvik River. From two zones we had time series data including 303 historical samples collected in 2001 and 2007–08 from river zones E and I  loci and PCR conditions, electrophoresis with an Applied Biosystems 3730xl Genetic Analyzer , and allele scoring are detailed in Supplementary Material 25 was used to detect possible genotyping errors, large allele dropouts, and null alleles. Deviations from expected Hardy–Weinberg (HW) proportions were tested with the software GENEPOP 4.7.226 using the Markov chain method with 10,000 dememorization steps, 5000 batches, and 10,000 iterations to estimate exact P-values. The same settings were employed to test for linkage disequilibrium between pairs of loci. Standard summary statistics, including allelic proportions per STR locus for the time series data, were calculated with GenAlEx 6.51b228. ADZE 1.029 was used to estimate allelic richness (AR) and private allelic richness (ARP) corrected for differences in sample sizes when considering the different zones separately (standardized sample size of 10) and at the group level based on genetic clusters, stocking history, and different time periods (standardized sample size of 50). For the second analysis, six major groups were therefore identified: (i) the Russian zones B–C as no supportive breeding occurs there, (ii) E2008/2007 because these samples are from an intermediate time period, (iii) E2001A-E2001D as representative of the traditional breeding practice , (iv) E2001E and E2001F as these represent the modern stock and the stock that most closely resembles wild trout in the river system (we did not separate these two as some admixture occurs between them), (v) zones E–I as these zones are regularly stocked, and (vi) I2007 because these samples represent an intermediate time period.Temporal replicates were treated as separate groups in all analyses unless stated otherwise. MICRO-CHECKER 2.2.330 and it has been shown that detection of genetic clusters in STRUCTURE31 and DAPC32 can be caused by groups of closely related individuals in the data set30. Although we only sampled adult fish 27–70 cm in length, encompassing several age classes, we estimated relatedness based on allele frequencies with the R package RELATED v.1.033 to test whether a higher-than-expected relatedness level is present in any of the groups studied. First, we used the compareestimator function to compare the performance of four different relatedness estimators. The most fitting relatedness estimator based on the highest correlation coefficient (R) was used to estimate relatedness within spatial and temporal groups and to estimate pairwise relatedness within those groups that showed higher-than-expected relatedness.Since stocking is typically performed with offspring from a small subset of individuals, it is expected that stocked fish show closer family relationships. In addition, in salmonid species, the juveniles are often found in shoals/schools of related individuals34 to assess whether recent genetic bottlenecks, linked to breeding practices over the last 20–30 years, could be found. The underlying algorithm assumes that allelic diversity is lost faster than heterozygosity. Hence, it tests for heterozygosity excess compared to expectations at mutation-drift equilibrium35. We tested two mutation models, the infinite-alleles-model (IAM) and the two-phase-model (TPM) following the recommendations by36. The latter encompasses two mutation models, the IAM and the stepwise mutation model (SMM), and different proportions of STRs can display either one of these mutation models. The TPM model was run three times for each population with the percentage of stepwise mutations being 20%, 50%, and 70%, respectively. We used the 1-way Wilcoxon sign-rank test37 and 10,000 iterations to determine significance.We used the program BOTTLENECK 1.2.02versus single-generational), we used the program NeEstimator 2.138. We calculated the effective population size (Ne) with three point estimators , Heterozygote Excess method . In all instances, we used allele frequencies ≥ 0.02 as recommended by38. We obtained confidence intervals with the jackknife method of39. We added the sibship frequency (SF) estimator as a fourth effective population size estimator and used COLONY 2.0.6.542 with default priors, because43 showed through simulations that the most widely used LD method overestimates Ne when sample sizes are considerably smaller than the actual Ne, and that it can be inaccurate when the assumption of random mating is violated.The effective population size may decrease because many individuals from the same parents are released relative to the natural recruitment in the river. To test for differences of effective population size in a spatio-temporal context as well as investigate the effects of different breeding practices  was calculated with the p.adjust function in R version 3.6.047 to minimize Type I and II errors, while controlling FDR by using an ɑ-level of 0.0148.Pairwise population genetic differentiation was estimated with G31 was run with the admixture model with correlated allele frequencies49 for several datasets. An initial run included all samples from the entire river and all time periods. In addition, the two river sections with temporal samples  were separately analysed to check whether additional substructuring could be found and to confirm the initially identified genetic clusters. Using two sets of runs, one with the LocPrior option and one without, K = 1–10 was tested with 40 replicates each. Run parameters further included 1,000,000 MCMC steps and a burn-in period of 100,000. The LocPrior option allows for detection of weak population-genetic structure50. All STRUCTURE analyses were carried out on the CIPRES Portal v3.3 , maximum of means (MaxMeaK), median of medians (MedMedK), and maximum of medians (MaxMedK). Summary bar plots were prepared with the program CLUMPAK55.STRUCTURE 2.3.456. This second set of Bayesian assignment analyses had closely related individuals randomly reduced by ≥ 50% following recommendations by30, whose simulations showed that total purging of related individuals also leads to biases30.As initial tests indicated that highly related individuals were present in some of the identified genetic clusters, we ran a second set of Bayesian assignment analyses with a reduced data set to account for potential false identification of genetic clusters by the presence of closely related specimens in the data set32 in R version 3.6.047. To avoid over-fitting, a cross‐validation function with 100 replicates was used to determine the optimal number of principal components to be retained. Training versus test set proportions were 0.1 and 0.9, respectively.In addition, a Discriminant Analysis of Principal Components (DAPC) was conducted to test whether temporal populations within zones E and I are genetically different using the ADEGENET package32 in R version 3.6.047, which is interfaced with the package ADE57. This was done as an additional verification that identified groups in DAPC can also be found without pre-defined groupings.Finally, a Principal Component Analysis (PCA) was carried out in the ADEGENET packageversus wild fish was done by the fishermen, based on characteristic fin damage in stocked fish14. The accuracy of this classification was evaluated by by otolith analysis17 of a large subsample (N = 202), resulting in the same classification as the fin damage method in 97.5% of the cases.For the historical samples in E2001, measures of body size, weight, and a morphology-based classification into stocked and wild fish were available , based on high assignment scores, to break down linkage disequilibrium. In the final population set, linkage disequilibrium was significant (p < 0.05) in only 7/2051 pairwise comparisons after Bonferroni correction. Deviations from Hardy–Weinberg equilibrium were observed in one locus (SsaD157) in two populations (zone I and I2007) after Bonferroni correction.IS estimates. Allelic richness and private allelic richness, as well as allelic proportions per locus were particularly low in the historical genetic clusters, except for E2001E and E2001F, which corresponded to the contemporary populations  in one group (I2007), showing a significant presence of null alleles, and found no signs of large allele dropout at any locus. Furthermore, only one population (zone E) showed potential scoring errors due to stutter at one locus (OMM1152). Hence, all loci were retained for further analysis.4 . Historical samples E2001A–D  by–D Table .4 that non-stocked zones in Russia  do not show signs of demographic bottlenecks while stocked zones G, H, and I showed consistent signs of bottlenecks , but also differences in variances among the groups defined based on genetics (p < 0.0001). Therefore, we used a Welch one-way ANOVA test that does not assume homogeneity of variances in combination with a Games-Howell post hoc test for each variable separately. Prior to the analyses, outliers were removed from the data sets; however, for weight, after the first round of outlier removal, more outliers were found that remained in the analysis. For both length and weight, the ANOVA was significant . For length, the pairwise post hoc tests showed that E2001E and E2001F were significantly different from all other genetic clusters, but not from each other Fig. a. For weOur results revealed that significant temporal changes in genetic structure, effective population size, and allelic composition occurred in the trout population of the Pasvik River during 17 years . Three unique genetic clusters could only be found in historical samples within zone E, likely being the result of traditional breeding practices leading to considerable allele frequency shifts. A fourth genetic cluster became rare with time, although still present in low numbers and as admixed individuals at the end of the 17-year study period. In all these four genetic clusters that either disappeared or declined during the study period, genetic diversity measures and effective population sizes were distinctly lower than in the modern breeding stock and the wild population, which was consistent with indications of recent bottlenecks and considerably lower effective population sizes. Overall, these results suggest a loss of genetic diversity and a homogenization effect as well as isolation by time.62. Potential causes include both lower rates of survival and reproduction and changes in the timing of maturity and spawning introduced by captive-breeding programs67. Shifted spawning timing may also contribute to non-random mating or reproductive isolation by time , resulting in low admixture with wild populations64. If new parents are caught every year for breeding, these effects may be less severe, but they are still present65. For example, in a meta-analysis, relative reproductive success of early-generation hatchery-reared salmonids in the wild was only 50% on average compared to wild fish65. Importantly, genetic effects seem to be present even in F2 generation specimens that have lived their entire lives in natural environments11. Further, mortality rates may be higher in captivity-bred than wild fish because of less efficient foraging behaviour, leading to smaller body sizes and poor physical condition, and more risk-prone or exploratory behaviour that may result in higher predation and angling pressure67. The progeny of hatchery-raised trout may potentially also suffer from fewer resources at egg hatching in the wild, as early juvenile survival is positively correlated to egg size. Egg size typically increases with mother size and may be smaller than in wild fish for two reasons; (1) there may be a hatchery selection towards smaller egg size23, and (2) hatchery fish grow less than wild fish. The latter will also reduce the mothers’ ability to make deep and well-protected nests69. These factors may select particularly against domestic trout that grew up in artificial environments and have reduced fitness in comparison to wild trout11, but also be detrimental to wild fish if the characters are introgressed back to the wild populations.Interbreeding between the historical groups was marginal, except for 2001E and F. Previous studies in salmonids found similarly low introgression levels from domestic fish into the native population8. Although this may partly explain the resilience of the native group with the highest proportion of wild fish (E2001F), it does not explain why domestic fish did not interbreed. Christie et al.10 pointed out that there is a trade-off between the number of stocked fish that should be allowed on the spawning grounds because releasing very few might not help the goal of supportive breeding whereas allowing too many will reduce the effective population size considerably.Introgression between domestic and native trout can be density-dependent, increasing with the relative size of the domestic population70. We can exclude the possibility of food partitioning between juveniles and adults as only adults were collected. Furthermore, as coregonids  are the main prey, constituting > 90% of the diet of adult brown trout in the study system15, we can also exclude the possibility of trophic polymorphism as a mechanism for population sub-structuring.There were no obvious geographical barriers that can explain the strong separation of genetic clusters within zone E. The relaxation of linkage disequilibrium observed when sorting into different groups and the elevated relatedness of individuals in four out of six groups , suggest non-random mating. Ecological, behavioural, historical, and anthropogenic factors may explain genetic sub-structuring of fish populations63. However, size variation within those four groups suggests the presence of multiple generations. Thus, either a low reproduction rate was present, or several age classes were released; however, the latter seemed to have been stopped sometime in the 1980s . In any case, reproductive success, particularly interbreeding with wild trout, was low. Also, stocked fish from groups E2001A–D did not interbreed with each other, suggesting that all released stocks had very low rates of survival and reproductive success.Our results are consistent with these studies, given that the four genetic clusters E2001A–E2001D represent domestic multi-generational broodstocks. The clear genetic separation and low admixture rates of the genetic clusters in combination with the phenotypic classification of being stocked fish support this. Further, the high relatedness and low allelic richness give further credence to this interpretation. Differences in morphological traits, like body size and weight, between domestic and wild fish have also been found to be linked to hatchery-breeding10). The most likely explanation for the four observed genetic clusters is that they correspond to four brood years  with very low reproduction levels in the wild. These traditional breeding practices have also led to significantly smaller body sizes/weight than found in wild fish. Together with the common signatures of recent bottlenecks and low genetic diversity, inbreeding depression was a likely factor in the traditional breeding program.Our results suggest that particularly traditional stocking practices can lead to severe and rapid reductions in effective population sizes and allelic diversity, strong linkage disequilibrium, and increased relatedness in multiple-year breeding programs. Small numbers of breeders plus variation in reproductive success contribute to this pattern. This is consistent with findings by others , which showed higher allelic richness and effective population sizes than the previously stocked groups and which co-exists and interbreeds with wild brown trout in the river. Nevertheless, E2001F, the group that most closely resembled wild brown trout at the time, harboured private alleles that were not found in any modern samples, suggesting that genetic diversity was lost through time. Similarly, bottlenecked modern river zones indicate an ongoing loss of genetic diversity16. Using the Bayesian assignment scores and under the assumption that the Russian non-stocked zones are genetically representative of wild fish, the estimated proportions of wild fish in the contemporary stocked Norwegian-Russian zones equalled 47.8% (zone E), 32.0% (zone F), 13.0% (zone G), 3.9% (zone H), and 8.9% (zone I). This gives an average of 16.2% of wild fish in the stocked zones, but with considerable variation between zones. For example, zone H showed the lowest proportion of wild fish; however, this is the zone in which breeding specimens are caught annually. To preserve natural genetic diversity, breeding practices will need to be refined to increase the number of unrelated breeders and the proportion of wild specimens in the breeding program. Four out of the six zone E populations (E2001B–E) showed largely consistent significant bottlenecks. The main currently surviving wild population in zone E  showed neither elevated relatedness estimates and nor genetic bottlenecks. The other population (E2001E) corresponded to the main contemporary population of the Norwegian-Russian part of the river where stocking and genetic bottlenecks are common4. Several factors may explain these results. The bottlenecked historical populations from E2001B-D could already back then have suffered from considerable population decline and ultimately gone partially extinct, whereas the still occurring contemporary population E2001E may be impacted by stocking practices. Stocking and destruction of spawning habitats occurred also before 2001 and may have led to population declines. The only other surviving genetic cluster E2001F shows partially admixed ancestry with E2001E and this may explain the non-significant bottleneck tests. Effective population sizes in non-stocked Russian zones seem to be comparable to or higher than in stocked Norwegian-Russian zones. Thus, stocking does not appear to increase effective population sizes in any significant way, and in worst case scenarios, decreases them considerably10.One remaining potential problem in this system is that annual catches for the broodstock come from the same zone  and that due to the dams, natural migration is limited or non-existent. This probably results in a situation in which the local fish population encompasses a high proportion of stocked fish, as shown by the current study and others4). Hence, conservation management should carefully assess future breeding protocols to preserve remaining natural genetic diversity in brown trout in the Pasvik River and other rivers where compensatory stockings are implemented. Morphological and molecular screening including the determination of relatedness levels before breeding may be a viable option for quickly implementable conservation measures in this system . However, this cannot replace other efforts like habitat restoration to increase natural reproduction. Alternatives include the abandonment of the breeding program altogether. In many cases this would be very drastic, and potentially detrimental to the fish population and human interest as well if not followed up by other measures. We strongly advise starting by improving conditions for natural reproduction, including increased connectivity by opening fish passages around hydropower installations to facilitate fish migration. Our findings have important implications for stocking program management and indicate that a detailed evaluation will be needed to find the best way forward for stocking practices where such programs exist.In conclusion, our study documents large impacts on the genetic structure and genetic diversity of brown trout populations from annual compensatory stockings following construction of hydropower dams. Four genetic clusters were morphologically identified as stocked fish and showed consistently lower genetic diversity, higher within-group relatedness, lower effective population sizes, and significantly smaller body sizes than the other two genetic clusters, which were genetically more similar to contemporary stocked and wild fish. Over a period of 17 years, three of these genetic clusters within one of the studied river sections went extinct and a fourth cluster distinctly declined, indicating that reproduction success of these groups was low. A change in breeding practices from multi- to single-generational breeding of the locally caught broodstock resulted in the maintenance of higher genetic diversity, but even these modernized breeding practices are not enough to prevent genetic diversity loss when compared to wild fish (see alsoSupplementary Information.


Elucidation of the human genome has increased understanding of human body responses to drug administration . LikewisThe Coronavirus disease 2019 (COVID-19) pandemic has reinforced the urgent need to study the genetic differences among people with mild symptoms and those with complex responses to the disease . During Examples that illustrate the importance of understanding therapeutic effectiveness responses in target groups include the premature administration of hydroxychloroquine based on affect glycosylation of angiotensin converting enzyme-2, without information on genetic variability  and remdThe reservoirs of genetic material in biobanks in the United Kingdom  and evenBiobanks can hold genetic data for a significant percentage of an entire population . In EstoThere are ethical dilemmas involved with asking a donor to provide unique informed consent. This has, however, been improved with the development of the model of dynamic consent . Ethics Due to high population diversity, Latin America faces the challenge of addressing genetic variability in studies to improve pharmacological responses to therapeutics for diseases. The creation of biobanks, their strengthening, and collaboration among them, would be a fundamental contribution to obtain pharmacogenetic information and efficient therapeutic responses in Latin America.


Durio zibethinus, is a potential agricultural commodity for domestic and international markets. However, its quality is still less competitive or significantly lower to fulfill the export market, compared to a similar one from other countries. This study aimed to determine and analyze the genetic diversity and relationship of the exotic durian (Durio spp.) germplasm originally from Kalimantan, Indonesia, using the rbcL marker.Durian of Indonesia, specifically rbcL region of the durian germplasm has generated 23 variable sites with a transition/transversion (Ti/Tv) bias value of 1.00. However, following the phylogenetic and principal component analyses, this germplasm is separated into four main clades and six groups, respectively. In this case, D. zibethinus was very closely related to D. exleyanus. Meanwhile, D. lowianus and D. excelsus were the farthest. In further analysis, 29 durians were very closely related, and the farthest was shown by Durian Burung (D. acutifolius) and Kalih Haliyang (D. kutejensis) as well as Pampaken Burung Kecil (D. kutejensis) and Durian Burung (D. acutifolius) with a divergence coefficient of 0.011. The Pearson correlation analysis confirms that 20 pairs of individual durians have a strong relation, shown by, e.g., Maharawin Hamak and Durian Burung as well as Mantuala Batu Hayam and Durian Burung Besar.Based on this marker, the durian germplasm has a low genetic diversity (π%=0.24). It may strongly correspond with the variability sites or mutation present in the region. In this case, the While the durian has a low genetic diversity, the phylogenetic analyses revealed that this germplasm originally from Kalimantan, Indonesia, shows unique relationships. These findings may provide a beneficial task in supporting the durian genetic conservation and breeding practices in the future, locally and globally. Durio zibethinus, is a potentially valuable agricultural commodity for domestic and international markets [Durian, specifically  markets . Even it markets . Similar markets . However markets . As a reD. dulcis, D. excelsus, D. grandiflorus, D. graveolens, D. kutejensis, D. lowianus, D. oxleyanus, D. testudinarum, and D. zibethinus. In addition, these durians have other advantages, such as being resistant to diseases, especially patch canker, and having a high tolerance for environmental challenges, such as acid soil [Germplasm collection is a vital component of plant breeding program success or the development of new superior cultivars . Around cid soil . As a reGermplasm characterization is also crucial to aiding the durian plant breeding initiative or generating new superior cultivars . HoweverDurio spp.) germplasm originally from Kalimantan, Indonesia, using the rbcL marker. Following Moura et al. [Oryza sativa [Amorphophallus [Flacourtia inermis [Ilex [This study aimed to determine and analyze the genetic diversity and relationship of the exotic durian  germplasm, excluding an outgroup (Table Bombax ceiba) was obtained from the GenBank database.In this study, we have used a total of eighteen samples of durian  and rbcL-R (5′-GTAAAATCAAGTCCACC RCG-3′) [rbcL) targets were deposited in GenBank with accession numbers of MZ479678–MZ479695  and Geneaid Biotech Ltd., Taiwan GP100). The genetic materials were then quantified using a UV-VIS spectrophotometer  and amplified by a pair of  RCG-3′) . Amplifi RCG-3′) . The amp00. The grbcL sequences of durian were refined manually to a reconstructed consensus using the MEGA-X software [r) analysis, by the criteria of weak (r ≤ 0.35), moderate (r = 0.36–0.67), and strong (r > 0.68), was finally applied to confirm the differences in genetic structure between durian samples [The software . These ssoftware  and Multsoftware . The gensoftware . The physoftware . The intsoftware . The gensoftware . The AMOsoftware . In addisoftware . The Pea samples .rbcL region was successfully amplified. The amplification results show that this region has a size of approximately 650 bp  sample had a different rbcL sequence length, ranging from 527–578 bp  germplasm from Kalimantan, Indonesia, shows unique relationships. This uniqueness lies in the number and composition of durian members in each clade formed. In general, following the maximum likelihood (ML) and neighbor-joining (NJ) methods, this germplasm is grouped into four main clades , which belongs to clade II in ML and clade I in NJ  and three  members only  and Kalih Haliyang (D. kutejensis), and Pampaken Burung Kecil (D. kutejensis) with Durian Burung (D. acutifolius) as well, at a divergence coefficient of 0.011. The Pearson correlation analysis confirms that twenty pairs of individual durians have a strong relation  has a partial rbcL of 250 bp, and Amandita et al. [rbcL sequence of 1428 bp [In this study, the a et al.  about 50 1428 bp .rbcL of durians, in which a conserved region is introduced by polymorphism or other mutational events , and no indels are present. According to Clegg [rbcL substitution rate [Further, a new DNA barcoding motif was discovered in the multiple sequence alignment of the nts Fig. . Based oto Clegg , completion rate .rbcL were highly efficient in Cornaceae, Cupressaceae, Ericaceae, and Graniaceae [rbcL gene evolved more quickly in annual plants, particularly in the Asteridae and Poaceae families, and was dubbed “most morphologically advanced forms” in these families [According to Dong et al. , this geaniaceae . The rbcfamilies .Durio spp.) germplasm originally from Kalimantan, Indonesia, has a low genetic diversity, shown by nucleotide diversity (π%) of 0.24  germplasm from this region has a high diversity. For example, tidal swamp rice (Oryza sativa) shows a genetic diversity of 0.086. According to Teixeira and Huber [Compared to other studies with similar markers used, durian  bias value of 1.00  germplasm from Kalimantan, Indonesia, shows unique relationships, mainly based on the number and composition of durian members in each clade or group formed. In general, following the maximum likelihood (ML) and neighbor-joining (NJ) methods, this germplasm is grouped into four main clades , which belongs to Clade II in ML and Clade I in NJ  and Kalih Haliyang (D. kutejensis), and Pampaken Burung Kecil (D. kutejensis) with Durian Burung (D. acutifolius) as well, at a divergence coefficient of 0.011. Following the Pearson correlation analysis, only 20 pairs of individual durians have a strong relation, for example, Maharawin Hamak and Durian Burung as well as Mantuala Batu Hayam and Durian Burung Besar  germplasm originally from Kalimantan, Indonesia, has a low genetic diversity (π%=0.24). However, following the phylogenetic and principal component analyses, this germplasm is separated into four main clades and six groups, respectively. In this case, D. zibethinus is very closely related to D. exleyanus. Meanwhile, D. lowianus and D. excelsus are the farthest. Individually, 29 durians were very closely related, and the was farthest shown by Durian Burung (D. acutifolius) and Kalih Haliyang (D. kutejensis) as well as also Pampaken Burung Kecil (D. kutejensis) and Durian Burung (D. acutifolius) with a divergence coefficient of 0.011. The Pearson correlation analysis confirms that 20 pairs of individual durians have a strong relation, shown by (e.g.) Maharawin Hamak and Durian Burung, also Mantuala Batu Hayam and Durian Burung Besar. Our results may provide a fundamental paradigm in supporting the durian genetic conservation and breeding practices in the future, locally and globally.Following the


N-glycan profile, and disulfide linkage were investigated. Biological attributes were evaluated by target/receptor binding analysis and in vitro/ex vivo cell-based assays, which are linked to mechanisms of action. As a result, LBAL had the identical amino acid sequence, similar post-translational modifications and N-/C-terminal variants, and comparable primary, secondary, and tertiary structures and disulfide linkage profile. However, some differences in N-glycan profiles were observed. Biological activities, including tumor necrosis factor (TNF) binding, TNF-neutralization, apoptosis, Fc receptor binding, and complement-dependent cytotoxicity, were largely consistent. Despite a slightly lower antibody-dependent cellular cytotoxicity activity in LBAL, this difference was not significant under physiological conditions. As indicated, this extensive analytical characterization and functional comparison assessment showed that LBAL was similar to Humira®, with minor differences of no clinical relevance. Taken together, our comparative assessment of physicochemical and biological attributes demonstrated that LBAL is structurally and functionally very similar to Humira®, supporting the biosimilarity of clinical efficacy and safety.LBAL was developed as an adalimumab (Humira®) biosimilar using Chinese hamster ovary cell lines. Comparable quality, safety, and efficacy between a biosimilar and its reference product should be ensured for regulatory approval. Here, we present the results of a comprehensive physicochemical and biological characterization between LBAL and Humira®. As physicochemical attributes, primary and higher-order structure,  A biosimilar is a biological product with high similarity in quality, potency, efficacy, and safety to a pre-approved reference product (RP)  was first approved by the FDA in 2002 and by the EMA in 2003, respectively (Derbyshire LBAL is another Humira® biosimilar developed and manufactured by LG Chem using Chinese hamster ovary (CHO) cells. During the LBAL development periods, the analytical  quality attributes relevant to the MOA or with the potential to impact efficacy, potency, and safety were selected through the risk assessment according to the regulatory guidelines US FDA  and A-MaHere, we present a comprehensive analytical characterization of the physicochemical and biological features of LBAL and Humira® to prove the biosimilarity. These similarity assessment results successfully show that LBAL is highly similar to Humira® at both the structural and functional levels, underpinning the biosimilarity of the following non-clinical and clinical trials.More than 20 lots of Humira® were procured from a pharmacy in Korea. Multiple batches of LBAL were produced by LG Chem (Korea). All chemical reagents, cell culture reagents, and the Biacore™ T200 instrument and its reagents were purchased from Sigma-Aldrich , Gibco , and GE Healthcare , respectively, unless otherwise noted. The recombinant proteins were purchased from R&D Systems , Sino Biological , Quidel , and NIBSC .N-linked glycans, and the disulfides were reduced with dithiothreitol.The molecular masses of intact samples were measured using a quadrupole time-of-flight mass spectrometer  coupled with an Acquity ultra-performance liquid chromatography (UPLC) instrument (Waters). The spectra data were deconvoluted by the BiopharmaLynx™ software (Waters). For molecular mass analysis of reduced and deglycosylated samples, the samples were pretreated with PNGase F  to remove Samples were denatured, then either reduced or non-reduced and alkylated with iodoacetic acid. The peptides digested by trypsin or Lys-C  were separated by UPLC. Eluting peaks were detected by UV (214 nm), and Q-TOF MS was used to determine the molecular mass. Disulfide bonds were analyzed by non-reduced tryptic peptide map analysis. The disulfide-linked peptides were identified by liquid chromatography-mass spectrometry (LC-MS) analysis.N-glycans released by PNGase F treatment were labeled with 2-aminobenzoic acid , followed by hydrophilic interaction liquid chromatography (HILIC) purification. The 2-AA labeled N-glycans were subsequently separated by UPLC-fluorescence detection (FLD). The N-glycan structure of each peak was determined by mass spectrometry (MS) analysis. The MS/MS spectrum was obtained using the GlycoWorkbench software (developed by Alessio Ceroni).The −1. The melting temperature (mT) was measured by differential scanning calorimetry (DSC). The thermogram was obtained using the DSC calorimeter .A Chirascan™ Plus spectrophotometer  was used to obtain circular dichroism (CD) spectra. The absorption difference between left- and right-handed circularly polarized light in the far-UV (195–250 nm) and near-UV regions (250–320 nm) was measured. The measured CD signals were converted to mean residue ellipticity (MRE) using the CDNN software. Fourier transform infrared (FTIR) spectroscopy was performed using the Bruker Tensor™ II FTIR instrument equipped with an AquaSpec™ spectrophotometer. FTIR spectra were obtained in the range from 1800 to 1400 cmka, kd) and binding affinity (KD) for sTNF-α and FcγRI binding were determined from sensorgrams by 1:1 binding fit-model analysis using BIAevaluation™ software. Binding affinity for other FcRs was determined from sensorgrams by the steady-state response model.Amine-coupling was used to immobilize the target protein on a CM5 chip in a Biacore™ T200 instrument. For the sTNF-α binding assay, the fragment crystallizable (Fc) capture Ab was immobilized on the chip surface. The sample and TNF-α were sequentially injected, and the chip was regenerated. For Fc receptor (FcR)-binding, His-tagged FcγRI and FcγRIIb were captured on the chip surface via pre-immobilized anti-His capture Ab. FcγRIIa (R/H), FcγRIIIa (V/F), FcγRIIIb, and neonatal Fc receptor (FcRn) were directly immobilized on the chip. Serially diluted samples were injected, and an appropriate regeneration solution was used. The kinetics , followed by treatment with the fluorogenic substrate . Fluorescence intensity was detected by a microplate reader. For C1q binding, the TNF-α-coated microplate was blocked with gelatin. Samples and human C1q were sequentially treated, followed by anti-C1q-HRP Ab . ABTS substrate  was added, and absorbance was measured by a microplate reader at 415 nm.Samples were pre-incubated with TNF-α  and actinomycin D , and then the mixtures were treated to L-929 cells . The cell viability was measured by the MTS assay . The absorbance (490 − 650 nm) was measured by a microplate reader. The percentage of neutralization was calculated from the dose − response curve.Samples were incubated with the tmTNF-CHO cells in the presence of rabbit complement serum  at 37°C for 2 h to induce cell lysis. The cell viability was analyzed by the MTS assay, and the percentage of cytotoxicity was calculated from the dose–response curve.The ADCC reporter assay kit (Promega) was used. tmTNF-CHO target cells were treated with samples and subsequently co-incubated with Jurkat reporter effector cells at 37°C for 6 h. Bio-Glo reagent was added, and the luminescent signal was measured on a luminometer . PBMCs were isolated from healthy donors under the guidance of the institutional review board. For ADCC using PBMCs, tmTNF-CHO cells were treated with samples and PBMCs. After incubation, the luminescent cytotoxicity indicative of dead-cell protease activity was measured by the CytoTox-Glo® cytotoxicity assay (Promega). The relative activity was calculated from the dose–response curve.Apoptosis was analyzed using a MEBCYTO® apoptosis kit . Samples were incubated with Jurkat-tmTNFα   regression model using the parallel line analysis (PLA) software . The quality range (QR) for the similarity assessment is provisionally established as the mean ± 3 × standard deviation (SD) of RPs. The equivalence testing (α = 0.05) by the Minitab 18 software was applied to sTNF-α binding and neutralization. If the 90% confidence interval (CI) of the mean difference is within the equivalence acceptance criteria (EAC) defined by 1.5×SD of the Humira® lots, the analytical similarity is acceptable -, Fc-, and Fab-/Fc-related biological activities. A list of quality attributes, analytical methods used for similarity assessment, and summary results are described in The examined quality attributes included the primary structure, N-linked glycans with either zero, one, or two terminal galactose residues  were observed as the prevalent species, consistent with the glycan map results  were mainly observed. Afucosylated (Afucose) complex-type (lacking core fucose) and high mannose (HM)-type N-glycans were found at low abundance in both Humira® and LBAL. LBAL had relatively similar levels of %galactosylation but slightly higher levels of afucosylation and sialylation, with lower levels of the HM-type than Humira® (Supplementary Table S1). Therefore, the impact of these differences in N-glycan profiles on biological activity needs to be demonstrated in relation to the biological and functional attributes.The profiles of  species C. NeutraMultiple biophysical methods were used to analyze the higher-order structure of LBAL and Humira®. The secondary structure was investigated by far-UV CD and FTIR spectroscopy, the tertiary structure by near-UV CD spectroscopy, and the thermal stability by DSC  and CH3  domains , one sTNF-α neutralization, and one apoptosis.Results of the relative sTNF-α-binding affinity measured by SPR exhibited statistically equivalent activity between LBAL and Humira®. The 90% CI for the difference of the means between LBAL and Humira®  was within the EAC of Humira® (±17.3%), demonstrating that LBAL and Humira® are highly similar in sTNF-α-binding activity A. The abn = 3), method variability, and the high sensitivity of SPR. Collectively, LBAL showed very similar Fc-related biological activities to Humira®. These findings are further detailed in Supplementary Table S3.The Fc region of adalimumab interacts with a variety of FcγRs expressed on immune cells or soluble C1q in complement systems as well as FcRn on epithelial/endothelial cells, contributing to modulation of diverse immune responses, besides affecting its serum half-life/pharmacokinetics  were within the QR of Humira® (16.0–24.4%).The CDC activity was measured by the cell viability (MTS) assay using tmTNFα-CHO target cells in the presence of soluble C1q. The CDC activity of LBAL (94–107%) resembled that of Humira®, within the QR of Humira® (78–109%) A. Next, ng PBMCs C, which in vivo is known to be attenuated by serum/IgG  and similar N-terminal variants , C-terminal Lys variants, and post-translational modification profiles, including Asn deamidation and Met oxidation. Full amino acid sequencing and N-/C-terminal sequencing using trypsin/Lys-C peptide mapping combined with LC-MS/MS further confirmed 100% sequence coverage (214 amino acids for LC and 451 amino acids for HC) (data not shown). The free sulfhydryl contents were comparable by the Measure-iT™ thiol assay (data not shown).A biosimilar should have an identical amino acid sequence to an RP as a prerequisite requirement EMA . We haveThe secondary and tertiary structures of a protein are essential for their action as the three-dimensional structural motifs define the protein’s functionality. Therefore, the comparison of higher-order structures is a critical element in the similarity assessment of biosimilar products. Disulfide linkage by LC-MS, spectroscopic, and calorimetric approaches, including far-UV CD, near-UV CD, FTIR, and DSC, confirmed the highly similar secondary and tertiary structure between LBAL and Humira®.N-glycosylation profiles are regarded as a critical quality attribute among physicochemical quality attributes. N-glycan profiles were analyzed by HILIC-UPLC/MS. We found that neutral biantennary N-glycans were dominant species, and Afucose- and HM-types were minor species in LBAL and Humira®. The level of galactosylation (%) was similar, albeit there were small differences in the relative abundances of afucosylation, HM, and sialylation. LBAL contained slightly higher levels of %[Afucose] (approximately 2% difference) and %[sialylation] (<1% difference), and lower levels of %[HM] (about 6% difference) (Supplementary Table S1). We showed that LBAL and Humira® have similar binding activities for C1q, FcγRs, and FcRn and CDC, but not ADCC. The difference in %[HM] content was deemed clinically irrelevant, considering similar pharmacokinetic profiles in non-clinical/clinical studies (data not shown), which were consistent with the comparable FcRn-binding activity between LBAL and Humira®. It has been reported that the sum of %[Afucose + HM] content correlates with an increase in both FcγRIIIa-binding and ADCC activities , and mannose-binding receptors  were evaluated by a similarity assessment. As Fc-related biological attributes, the binding to FcγRs , FcγRIIb, FcγRIIIa (V/F), and FcγRIIIb), C1q, and FcRn were investigated. Furthermore, as Fab- and Fc-related biological attributes, the effector functions, such as CDC and ADCC, were determined to demonstrate the biosimilarity of LBAL to Humira®. As mentioned in the justification for the difference in %[Afucose + HM] contents, the ADCC potency is likely to be similar between LBAL and Humira® under physiological conditions. Therefore, the observed differences in the most sensitive ADCC reporter assays under the absence of human serum or IgG were expected to be clinically irrelevant, which was supported by clinical studies (data not shown).N-glycan profiles, and disulfide linkage as structural/physicochemical quality attributes, in addition to the Fab-, Fc-, and Fab-/Fc-mediated biological activity as biological/functional quality attributes. Some minor differences in quality attributes were detected. However, these differences have been fully examined and justified as clinically irrelevant. The findings presented here are considered a foundation to support the overall similarity in non-clinical and clinical studies of LBAL.In summary, the analytical similarity between LBAL and its RP Humira® has been comprehensively demonstrated in compliance with the appropriate regulatory guidance. The similarity studies addressed the primary and higher-order structures,


Samples were then scanned , and three-dimensional (3D) images reconstructed. The relative volume of gaps (VG%) and voids (VV%) present on each material was calculated. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey’s HSD test (p < 0.05). Mean VG% for BC, MTA, and BIO groups were, respectively, 0.513%, 1.128%, 1.460%, with BC presenting statistically (p < 0.05) fewer gaps formation than the other groups. Mean VV% were, respectively, 0.018%, 0.037%, and 0.065%. The was no statistical difference regarding VV%. There were no gap-free and void-free samples. BC group had the lowest VG% among the groups with a significant statistical difference (p < 0.05).This study aimed at evaluating volumetrically gaps and voids of calcium-silicate based materials of different generations and handling properties  in simulated furcal perforations in an ex vivo setup by microcomputed tomography (Micro-CT) analysis. Thirty-six extracted human mandibular molars with sound furcation areas were selected. Standardized perforations were created in the furcation area of the pulp chamber using #4 diamond burs. The specimens were randomly assigned to three groups (BC, MTA and BIO;  Furcal perforations are overall considered inconvenient accidents for clinicians. They happen due to several reasons, including extensive caries, resorptions, or iatrogenic factors  These inMTA was first introduced in the 90s  and sincThe evaluation of calcium silicate-based materials in furcal perforation repair is still limited since valuable alternatives for this clinical condition are mostly based on unpredictable case reports. Those investigations included unclear features that may have affected the outcome of perforation repair other than the investigated materials themselves increasing the need for studies under controlled conditions. Regarding this, microcomputed tomography (Micro-CT) analysis is an alternative method to assess the physical behavior of biomaterials , providiConsidering the lack of studies evaluating gaps and voids in cases of furcal perforation, and the advantages of Micro-CT analysis and its ability to complement conventional tests for endodontic materials, the present study aimed at evaluating the volume of gaps and voids of calcium silicate-based materials with different handling and mixing features in first mandibular molars with furcal perforations.The local institutional review board  approved the use of freshly extracted human teeth in the present study under registration no. 201903881. Due to the absence of specific studies using the Micro-CT analysis to evaluate gaps and voids in furcal perforations filled by different materials, a pilot study was performed by the same operator (C.C.T.), a specialist with 5 years of experience, who was also responsible for the experimental procedure and evaluation. Based on the data from the pilot study regarding percentage values of gaps and voids, the sample size calculation revealed that a minimum of eight teeth per group would be appropriate to show a 5% difference in the volume percentage of gaps and voids with a power of 90%.n = 12 per group) freshly extracted human first mandibular molars with sound furcation area were selected based on general dimensions and a similar pulp chamber height of at least 2 mm and less than 3 m. By evaluating teeth under a dental microscope  at 10× magnification, teeth presenting fractures, cracks, perforations, caries, resorption and previous root canal treatment were excluded. Calculus bone and soft tissue were removed with Gracey curettes. To avoid bacteria proliferation, teeth were stored in 0.1% thymol solution at 4 °C prior to the study enrollment.Thirty-six  to mimic the alveolar bone and periodontal ligament, avoiding the extrusion of the materials during their placement  of #4 diamond burs and the depth of the perforation ranged from 2 to 3 mm according to the dentin-cementum thickness in the furcation area. All the procedures were performed (by C.C.T.) under a dental microscope  at 10× magnification. Prior to the calcium silicate-based materials placement, all cavities were carefully rinsed with saline solution, dried with paper points , and filled with cotton pellets. Teeth were then placed individually in a 24-well plate  with silicone impression material ;MTA—ProRoot MTA , andBIO—Biodentine .Specimens were randomly assigned into three groups as follows:Both MTA and BIO were mixed according to the instructions provided by the manufacturer. For the BC group, no mixing was needed. Cavities were incrementally filled with the perforation repair materials to their extension by the same single operator in order of one by one. Each increment was gently compacted by using a hand plugger . For the MTA group, the material was also carried by the microapical placement (MAP) system, as specified by the manufacturer. As calcium silicate materials need moisture to set, teeth were stored at 37 °C and relative humidity for one week, and afterward subjected to Micro-CT scanning.The same operator (C.C.T.) who performed the pilot study and the experimental procedure also evaluated the samples. To guarantee a standardization, and an accurate assessment of the data, a calibration of the density ranges of each material, dentinal walls and empty spaces (gaps and voids) was performed by analyzing successive Micro-CT images of each tooth. In addition, the Intraclass Correlation Coefficient (ICC) was calculated  to assess the intra-rater reliability during the analysis for both the pilot and final study. The intra-rater reliability score was 1.0, an excellent reproducibility.https://www.paraview.org/, accessed on 4 March 2022)  to analyze the volume of gaps  and voids  in cavities simulating furcal perforation. Samples were scanned at 125 kV and 80 μA using a voxel size of 21 (μm), a 1-mm thick Aluminium filter, a rotation step of 0.6 (deg), and an exposure time of 2630 s. Prior to each scan, a flat field correction was performed. Digital data were reconstructed using NRecon 1.7.1.0 software . Reconstructed images were first visualized by Paraview   and then3) of the calcium-silicate based materials within the cavities (Vm), gaps (Vg) and voids (Vv) were obtained by using the 3D  analysis tool of the CT-An software . The region of interest (ROI) was selected in an area over the furcal perforation depth by using the “top” and “bottom” tools. Once a suitable ROI was selected, the proper grayscale threshold was established to distinguish the root dentin wall from the cavity, comprehending the repair material itself, gaps, and voids  of gaps and voids were compared between the groups by using one-way analysis of variance (ANOVA) and Tukey’s HSD as post-hoc test. Statistical analysis was performed using Microsoft Excel . The level of significance adopted was 5%.Micro-CT analysis exposed a clear overview of tooth dentin, calcium silicate-based materials, and unfilled areas at assigned grey values. All samples showed both gaps and voids after Micro-CT analysis .p < 0.05) and no significant statistical difference was found regarding VV%  percentage of gaps (VG%) and voids (VV%) through Micro-CT analysis.Over the years, tooth extraction was the treatment of choice in cases involving the furcation area. However, the furcal perforation repair is the best option in terms of dental structure preservation. The evaluation of the furcal perforation healing is still limited once valuable alternatives for this clinical condition are frequently based on unreliable clinical reports, increasing the need for more studies under controlled conditions  Even witHigh-resolution Micro-CT analysis is a highly accurate method which has been used for the assessment of 3D microstructures in ex vivo models . Micro-CUnfilled areas should be prevented for a better prognosis since they act as a pathway for bacteria and their toxins through the perforation cavity . For thiWilling to assess the filling capacity of calcium-silicate based materials with different generations, and handling/mixture characteristics, a hand-mixed material (Pro-Root MTA), a mechanically mixed one (Biodentine) and a ready-to-use putty (Endosequence BC RRM-Fast Set Condensable Putty) were selected. When the distribution of unfilled areas was evaluated, all the specimens presented both unfilled areas between the material and the surrounding dentin (gaps) and within the material (voids).p > 0.05), BC showed fewer gaps (VG%) formation than the other groups (p < 0.05), as shown in Based on our results, all samples showed a minor number of empty spaces represented by gaps and voids, with means varying from 0.513%, 1.128% to 1.460% for gaps, and from 0.018%, 0.037% to 0.065% for voids on BC, MTA, and BIO, respectively. Even though gaps and voids seem to be consistent structural features, while voids (VV%) were equally found among the groups  and their widths were standardized to the diameter of the burs (1.40 mm). The final VG% and VV% were calculated, considering the total amount of the materials (Vm) to overcome these limitations.As a promising technique, the Micro-CT analysis used in this study provided a great understanding of the volumetric measurements of gaps and voids because of its highly accurate features, since two dimensional techniques cannot be suitable to measure a three-dimensional structure ,33. To tNone of tested materials were gaps and voids free. The Endosequence BC putty had the lowest volume of gaps among the three tested materials.


Evaluating population genetic background and genomic selection signatures can provide important insights into the uniqueness and the genetic relationship among breeds. In this study, we analyzed the genetic diversity, population structure, and phylogenetic relationships between Huaxi cattle and its ancestors. The results showed that Huaxi cattle has formed its own unique genetic structure features, which could be clearly distinguished from its ancestors and similar populations. Moreover, some genes associated with important economic traits, including growth and development, reproduction, milk quality, and immune response, were identified by scanning potentially selected genomic regions, which may play an essential role in the excellent environmental adaptability and growth performance of Huaxi cattle. Our study will contribute to the genetic evaluation and rational breeding strategy, and give an extensive reference for understanding the genetic basis of economically important traits in Huaxi cattle.KCNK1, PDLIM5, CPXM2, CAPN14, MIR2285D, MYOF, PKDCC, FOXN3, and EHD3) related to ion binding, muscle growth and differentiation, and immunity were detected by both methods. Our study sheds light on the unique genetic feature and phylogenetic relationship of Huaxi cattle, provides a basis for the genetic mechanism analysis of important economic traits, and guides further intensive breeding improvement of Huaxi cattle.Huaxi cattle, a specialized beef cattle breed in China, has the characteristics of fast growth, high slaughter rate, and net meat rate, good reproductive performance, strong stress resistance, and wide adaptability. In this study, we evaluated the genetic diversity, population structure, and genetic relationships of Huaxi cattle and its ancestor populations at the genome-wide level, as well as detecting the selection signatures of Huaxi cattle. Principal component analysis (PCA) and phylogenetic analysis revealed that Huaxi cattle were obviously separated from other cattle populations. The admixture analysis showed that Huaxi cattle has distinct genetic structures among all populations at K = 4. It can be concluded that Huaxi cattle has formed its own unique genetic features. Using integrated haplotype score (iHS) and composite likelihood ratio (CLR) methods, we identified 143 and 199 potentially selected genes in Huaxi cattle, respectively, among which nine selected genes ( Cattle are vital agricultural economy species worldwide, providing meat, milk, and labor for humans. China has abundant cattle genetic resources, with rough feeding tolerance, excellent meat quality, and strong adaptability . For a lMYH10, RLF, ARHGAP31, SQOR, and TBCB, were identified as associated with the growth and development traits [RLF gene had a significant effect on the average daily gain from birth to yearling by GWAS of 3996 Huaxi cattle [PLAG1 gene as the candidate gene for knuckle, biceps, and shank [TMEM236 for fat color, SORL1 and TRDN for meat color, S100A10 and AP2S1 for marble score, KCTD16 and LOC506594 for longissimus muscle area, as well as DHX15 and BRINP3 for shear force [FASN gene at 51.3 Mb on BTA19 could explain 6.49% and 10.04% of the genetic variances for C14:1 cis-9 and C14:0, respectively, and the ELOVL5 gene was identified in 25.1 Mb regions on BTA23 explaining 1.5% of the genetic variance for C14:1 cis-9 [Huaxi cattle, formerly known as “China Simmental beef cattle”, is a new specialized beef cattle breed in China, which consists of 24/32 Northern America beef Simmental cattle, 5/32 of dual-purpose Simmental cattle from Germany and Austria, 1/32 Charolais cattle, 1/32 Sanhe cattle, and 1/32 Mongolian cattle . The brend shank , and Chand shank . MoreoveGHSR related to body weight, TG associated with meat fat and marbling, and CANCNA2D1 related to cattle flesh color, the dressing percentage and backfat thickness were screened [Genomic selection technology has been widely used in Huaxi cattle breeding. Multiple strategies have been explored, including the additive dominance model , parallescreened .Linkage disequilibrium (LD) is the non-random association of alleles at different loci. The LD patterns provide relevant information about past demographic events, which reflect the population history of natural and artificial selection . CrossbrMAF), observed heterozygosity (Ho), expected heterozygosity (He), and inbreeding coefficient (FROH). Then, the population structure was elucidated by principal component analysis (PCA), neighbor-joining tree (NJ tree), population admixture, and (Nei) genetic distance analysis. Furthermore, the genomic selection signatures associated with important traits in the Huaxi cattle were determined based on the integrated haplotype score (iHS) and composite likelihood ratio (CLR) methods. Our study will help to evaluate the genetic background and plan the long-term breeding strategy of Huaxi cattle.Nowadays, the intercrossing and trait fixation of Huaxi cattle has reached the fifth generation. A comprehensive assessment of its genetic diversity, population structure, and genomic selection signatures are necessary for uncovering genetic divergence among Huaxi cattle and other breeds. In this study, Illumina BovineHD SNP array was used to genotype nine populations, including Huaxi cattle, its ancestors, and similar populations. Then, the genetic diversity of nine populations was estimated by average minor allele frequency (n = 55), its maternal ancestors including Mongolian cattle  and Sanhe cattle , and its paternal ancestors including Charolais , Fleevicht cattle , American Simmental cattle , and Canadian Simmental cattle , as well as two similar populations, Australian Simmental beef cattle  and dual-purpose Montbeliard cattle from France . Sample details were presented in To understand the genetic background and genomic selection signatures of Huaxi cattle, we collected 228 individuals from Huaxi cattle ; (4) individual with more than 10% missing genotypes; (5) closely related individuals were removed . In addition, only SNPs located on autosomal chromosomes were used for subsequent analysis [Genomic DNA was extracted from the blood samples of all cattle using the TIANamp Blood DNA Kit . DNA was qualified when the DNA concentration was above 50 ng/uL and the OD260/280 ratio value ranged from 1.7 to 2.1. Genotyping was performed using the Illumina BovineHD SNP array that contained 777,962 SNPs. Single nucleotide polymorphisms (SNPs) were scanned using the iSCAN platform, and genotype calling was conducted using GenomeStudio software. Quality control of the raw data was performed using PLINK v1.9 software [analysis .MAF), observed heterozygosity (Ho), and expected heterozygosity (He) were estimated with PLINK v1.9. In addition, we calculated the inbreeding coefficient based on ROH (FROH) by the total length of individual ROH divided by the length of the autosomal genome with a sliding window approach using the default settings of “-homozyg” command [2) between paired SNPs within 500 kb [In order to assess the genetic diversity of nine diverse cattle populations, the average minor allele frequency  in STRUCTURE 2.3.4. Each process was performed with 10,000 burn-in cycles, followed by 10,000 replications under admixture and correlated allele frequency models [To ascertain the genetic relationship among Huaxi cattle and other breeds, we first pruned the marker set by excluding related SNP in a window of 50 SNP, sliding the window by 5 SNP, and r2 threshold of 0.2. A total of 63,510 SNPs were obtained after LD filtered to conduct the principal component analysis (PCA). Pairwise genome-wide identity-by-state (IBS) distances were estimated using PLINK v1.9, and the PCA graph was visualized through the ggplot2 R package. Then, we calculated the genetic distance matrix between pair-wise individuals. PHYLIP v3.69 was used to construct the neighbor-joining phylogenetic tree . The phyy models .https://faculty.washington.edu/browning/beagle/beagle.html, accessed on 10 October 2021) with default settings was used to impute missing alleles and infer the haplotype phase for all individuals of Huaxi cattle [https://cme.h-its.org/exelixis/web/software/sweed/, accessed on 10 October 2021) to calculate the CLR values for sites every 20 kb across each chromosome of the genome [To detect the genomic regions under selection in the breeding process of Huaxi cattle, two analysis methods including the integrated haplotype score (iHS) and composite likelihood ratio (CLR) test were applied for selection signature analysis. The iHS estimates the relative decay of extended haplotype homozygosity (EHH) of the ancestral and derived core allele ,38. The i cattle . The iHSi cattle ,41. We ai cattle . For thee genome . To defie genome ,45. We uhttps://www.animalgenome.org/cgi-bin/QTLdb/BT/index, accessed on 10 October 2021) to detect whether the selected genes have been reported to be associated with important functional traits of cattle.Genes within the candidate regions were retrieved from the University of California Santa Cruz (UCSC) genome browser using the bovine UMD3.1 reference genome. To further investigate the potential biological function of selected genes in selected regions, we performed Gene Ontology (GO) function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis by the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 . AdditioMAF), average observed heterozygosity (Ho), average expected heterozygosity (He), and inbreeding coefficient based on ROH (FROH) to assess polymorphism of nine cattle populations. As shown in MAF of the Huaxi cattle population was 0.256, which was between Montbeliard cattle and Canadian Simmental cattle. Among all groups in this study, the Ho of Huaxi cattle was 0.375, which was between Mongolian cattle and Sanhe cattle. The He of Huaxi cattle was 0.34, which was between Canadian Simmental cattle and Montbeliard cattle. Overall, the value of He was considerably lower than the value of Ho in each population, except for Mongolian Cattle, a native breed of China. The FROH value of Huaxi cattle was 0.053, which was lower than that of the three commercial beef Simmental cattle groups from American (0.15), Canadian (0.094), and Australian (0.071), and approximate to that of Mongolian cattle (0.049), Sanhe cattle (0.052), and Fleevicht cattle (0.053). Moreover, Charolais has the maximum values of each parameter of genetic diversity and the minimum value of inbreeding coefficient when compared with other cattle populations, while American Simmental cattle were the opposite.After quality control, all samples showed high genotyping call rates  and low inter-individual relatedness . Finally, a total of 592,920 autosomal SNPs of 228 individuals were obtained for subsequent analysis. We calculated the average minor allele frequency  levels in each population . To measTo investigate the cluster patterns among the animals analyzed, principal component analysis (PCA) was conducted . In the Then, a neighbor-joining tree was constructed using the pairwise individual genetic distance matrix. Consistent with the PCA result, phylogenetic analysis showed that the individuals from the Huaxi cattle group were roughly clustered together and formed an independent branch, presenting high genetic consistency . MoreoveNext, to determine admixture degree in the nine cattle populations, STRUCTURE v2.3.4 software was used to perform the population admixture analysis. The hypothetical ancestral groups ranged from K = 2 to 5 . At k = To explore the genetic variation at the population level, we further evaluated Nei’s genetic distance among the nine populations. The results showed that the Nei’s distance between the nine populations ranged from 0.0775 to 0.2103 . Among tKCNK1, PDLIM5, CPXM2, CAPN14, MIR2285D, MYOF, PKDCC, FOXN3, and EHD3) in fifteen overlapping genomic regions covered 1.5 Mb were jointly detected by both methods.In this study, the genomic regions under the recent selection of the Huaxi cattle were determined by two methods, iHS and CLR. The genome-wide distribution of |iHS| values for 100 k non-overlapping windows on autosomes is depicted in p-value < 0.05 were observed. Among them, genes were significantly enriched in metabolism-related and immune-related biological functions, such as interleukin-1-mediated signaling pathway , B-1a B cell differentiation , negative regulation of interleukin-6 production , pantothenate metabolic process , proteolysis . In addition, we obtained five significant enriched pathways, including ubiquitin mediated proteolysis , PI3K-Akt signaling pathway , pantothenate and CoA biosynthesis , nicotine addiction , and pathways in cancer , which were related to immune, metabolism, and signal transduction.We further performed the GO function annotation and KEGG pathway analysis on the selected genes. As shown in Huaxi cattle is a new specialized beef cattle breed that has been bred by Chinese breeders for 43 years. Due to its fast growth rate, rough feeding resistance, wide adaptability, and good economic effect, Huaxi cattle is now widely distributed in many provinces of China, such as Inner Mongolia, Jilin, Henan, Hubei, Yunnan, and Xinjiang, greatly promoting the increase in beef cattle industry and farmers’ economic income in China. In this study, to better understand the characteristics of Huaxi cattle, we comprehensively evaluated the genetic diversity, population structure, admixture, and phylogenetic relationship by comparing Huaxi cattle with its ancestors and similar populations.Genetic diversity is the basis for species evolution to adapt to the environment and an important reference index to evaluate the status of germplasm resources . Our anaPCA and NJ tree analyses were further conducted to reveal the genetic relationship and genetic differentiation among Huaxi cattle and other breeds. The results showed that the individuals from Huaxi cattle gathered clearly and obviously separated from other cattle breeds. STRUCTURE analysis also demonstrated that Huaxi cattle has formed various genetic backgrounds compared to other breeds. Furthermore, we calculated the genetic distance among different cattle herds at the population level. We found that the genetic relationship between Huaxi cattle and beef Simmental cattle was slightly close, and far from Mongolian cattle, Sanhe cattle, and Charolais cattle, which was consistent with the results of the phylogenetic tree. Overall, after the initial population formation and subsequent artificial selection in the breeding, the Huaxi cattle population has been genetically stable over time and developed into a unique breed.Natural selection or intensively artificial selection may have left selection footprint on the genomes of individuals within the population . IdentifTBC1D5, CDK6, RXRA, LCORL, and CAPN2 genes were involved in growth and development [ZNF280B, CA10, LAP3, POLB, HELB, IRAK3, and ANO5 genes were associated with carcass and beef quality [KDR, a receptor for vascular endothelial growth factor, has been reported to be associated with susceptibility to bovine respiratory disease [CAPN14 and EHD3 are associated with calcium ion binding [CPXM2 regulates the early differentiation of connective tissues and has been recognized to be a gene associated with fetal growth restriction [MYOF is a member of the Ferlin family involved in the recycling of insulin-like growth factor (IGF) receptor, which plays major roles in controlling somatic growth and participate in skeletal muscle growth and differentiation [PDLIM5, a cytoskeleton-related protein that tethers protein kinases to the Z-disk in striated muscles, plays a key role in the proliferation and differentiation of the skeletal muscle, nervous, myocardium, and tumor [PKDCC gene is a putative protein kinase that has been reported to be involved in embryonic development and plays an essential role in bone development of the human and mouse [KCNK1 belongs to the family of background K channel [FOXN3 was identified as a selected candidate gene, which has been reported to be involved in cell proliferation, apoptosis, and pathogenesis in cancer, and functions as a tumor suppressor [An important goal of this study was to identify candidate genes involved in the domestication and artificial selection of Huaxi cattle. Through gene retrieval, we found that some genes were related to important functional traits in the potential selected regions. For example, elopment ,65,66,67 quality ,69. By e disease . In addi binding ,72, and triction ,74. MYOFntiation . It has ntiation . PDLIM5,nd tumor . The PKDnd mouse ,79. KCNKppressor . AccordiIn this study, we confirmed that Huaxi cattle has formed its own unique genetic structure feature, and we identified numerous candidate genes that have potential effects on important phenotypic traits such as growth and development, meat quality, and immune response. Our findings will conduce to a comprehensive understanding of the unique genetic features and phylogenetic relationships in Huaxi cattle, and provide a new perspective on the genetic mechanisms controlling specific phenotypes under selection.


Hyphaene compressa) is a perennial economic plant primarily growing in Kenya’s Arid and Semi-Arid Lands (ASALs). It is heavily relied upon for food, animal feed, construction materials and medicine, making it an ideal plant for resource sustainability. However, the limited information on its genetic resources has hindered its breeding and conservation studies. This study used the genotyping by sequencing approach to identify Single Nucleotide Polymorphisms. These SNPs were further used to assess the genetic diversity and population structure of 96 H. compressa accessions from Coastal, Northern and Eastern ASAL regions of Kenya using two approaches; reference-based and de novo-based assemblies. STRUCTURE analysis grouped the sampled accessions into two genetic clusters (Cluster 1 and Cluster 2). Cluster 1 included accessions from the Northern region, whereas Cluster 2 included all accessions from Eastern and Coastal regions. Accessions from Kwale  had mixed ancestry from both Cluster 1 and Cluster 2. These STRUCTURE findings were further supported by principal components analysis, discriminant analysis of principal components and phylogenetic analysis. Analysis of molecular variance indicated greater genetic variation within populations (92.7%) than among populations (7.3%). An overall FST of 0.074 was observed, signifying moderate genetic differentiation among populations. The results of this study will provide information useful in breeding, marker-assisted selection and conservation management of H. compressa.Doum palm ( Hyphaene compressa H. Wendl., also known as the East African doum palm, is a member of the Arecaceae family. It is integral in the agroforestry system of coastal and riverine parts of Africa  that are able to acquire in depth data on parts of the genome and are as effective but less costly compared to whole genome sequencing . GBS is H. compressa remains unknown despite its economic and subsistence role in Africa’s ASALs. There were no H. compressa or other palms in the genus Hyphaene with assembled genomes at the time of this study. Due to the scanty genetic information coupled with the pressure already demonstrated on this palm, there is a need to decipher its genetics. This study assumes that the different accessions of H. compressa growing in Kenya are diverse. The present study aimed to identify genome-wide SNPs, assess the level of genetic diversity, determine the population structure and estimate gene flow between H. compressa accessions collected from four ASAL regions of Kenya using GBS approach. The data obtained from this study will be an important genomic resource that will be used to inform the conservation, management, breeding and propagation of H. compressa.Thus far, the genetic diversity of H. compressa accessions collected between February and August of 2018 were used in this study. These accessions were collected from different ASALs of Kenya; Eastern (Tharaka Nithi County), Northern (Turkana County), Coastal  as shown in A total of 96 H. compressa leaves using DNeasy® Plant Mini Kit . The purity and quantity of the DNA were determined using Qubit Fluorometer (Invitrogen) or microplate reader , while a 1% agarose gel was used to confirm its integrity. Commercial GBS sequencing was done at Beijing Genomics Institute . A total of 96 samples; Tharaka (27), Turkana (21), Tana River (20) and Kwale (28), passed the sample quality check (QC) and proceeded to library preparation.Genomic DNA was isolated from Library preparation was done following the method previously described by de_novo and reference-based approaches. In the de_novo assembly, ipyrad version 0.9.74  genome  using the parameters ‘mem–t4 –k32 –M’ (The data was processed using the n 0.9.74  was used) genome . A confa) genome  since H.–k32 –M’ . SNP fil–k32 –M’  with theThe quality of the filtered VCF files were assessed using the R package tidyverse . The reaK (K = 1–10) were run with three replications using a burn-in of 100, 000 generations and 100, 000 Markov Chain Monte Carlo (MCMC) iterations. The software STRUCTURE HARVESTER web Version 0.6.94  was constructed using the R software package SNPrelate  was also used to evaluate the population structure of on 2.1.3  in R. ToNPrelate  to deterHo), Expected heterozygosity (He) fixation index (FST), inbreeding coefficient (FIS), Analysis of Molecular Variance (AMOVA) and pairwise FST values of the population were determined using Arlequin version 3.5.2.2  was used to improve the MCMC searches. One gene flow model was designed with direct migration from Tharaka to Tana River and to Kwale. The drainage of the Tana basin was used to design this model. Turkana accessions were excluded from this model since structure analysis and PCA demonstrated little historical gene flow. To judge whether the runs converged on good answers, the histograms and the effective population sizes were checked.H. compressa accessions yielded an average of 2.4 million reads per sample. The de_novo-based assembly using ipyrad software resulted in 3,941 raw loci. After filtering, a total of 2,096 SNPs with a mean depth of 35.7  were retained using the de_novo based assembly. On the other hand, reference-based assembly using date palm as a reference genome resulted in 3.4 million loci. After filtering, 23,146 biallelic SNPs with a mean depth of 3.5  were obtained using the reference-based assembly.Paired-end sequencing of 96 de_novo based assembly had higher depths than the reference-based assembly, as shown by the individual sequencing depth and the mean depth. The proportion of missing data per accession was low for both the de_novo based and reference-based assemblies, with a maximum of 0.04 and 0.4, respectively. These VCF quality statistics, including the mean depth, observed heterozygosity, depth per individual and missing data per individual, are presented for both the de_novo based assembly  transition SNPs and 813 (38.8%) transversion SNPs with the following types: A↔G type , C↔T type , A↔C type , A↔T type , C↔G type , G↔T type . There were 16,598 (70.9%) transition SNPs and 6,818 (29.1%) transversion SNPs with the following types: A↔G type , C↔T type , A↔C type , A↔T type , C↔G type , and G↔T type  in the reference based assembly. The A↔G and C↔T transition SNPs had the highest counts for both assemblies  than cluster 2 (He = 0.23). However, similar expected heterozygosity values were observed in the reference-based assembly for the two clusters (He = 0.30). The genetic variation among populations in Cluster 1 was higher (de_novo FST = 0.68 and reference-based FST = 0.17) than Cluster 2 (de_novo FST = 0.3 and Reference-based FST = 0.06). A total of seven accessions from Kwale had admixed ancestry between Cluster 1 and Cluster 2 using the reference based assembly  or gene diversity, observed heterozygosity (Ho), FIS and FST. The genetic variation among the H. compressa populations was moderate . The observed heterozygosity was higher than the expected heterozygosity in all the populations . Kwale had the highest polymorphic sites  followed by Turkana  as shown in He = 0.23, Polymorphic sites = 8,370) of all the sampled regions  and 0.105 (Turkana and Tana River). High pairwise FST was recorded among Turkana and Tana River samples , followed by Kwale to Tharaka (102.7), Tana River to Kwale (63.1), Tana River to Tharaka (59.9), Tharaka to Kwale (50.3) and Kwale to Tana River (57.7). These results indicate that the gene flow along the Tana basin was mostly asymmetrical  and diversity studies to aid future germplasm conservation.This study is the first to report the use of SNPs through the GBS approach to characterize de_novo-based approaches) were used to infer the population structure and genetic diversity of H. compressa. In the reference-based assembly, Phoenix dactylifera was used as a reference genome. This confamilial genome was used because H. compressa had no assembled genome at the time of this study. In the absence of a reference genome of the same species (conspecific) or genus (congeneric), a confamilial reference genome can be used to provide similar estimates of diversity (de_novo-based assembly (2096). The reference-based assembly has also been previously demonstrated to outperform de_novo assembly in determining the number of SNPs in olive cultivars (de_novo assemblies. GBS of H. compressa accessions showed considerable SNP variations with transition SNPs (purine-purine or pyrimidine-pyrimidine) being the most frequent mutations. This high frequency in transition SNPs has also been observed in many plants such as Capsicum annuum, Vigna unguiculata, Elaeis guineensis, and Camelia sativa  were pooled into cluster 1. Cluster 2 had the highest number of genotypes that included accessions collected along the Tana basin sites (Tharaka Nithi and Tana River) and Kwale. In addition, an admixture of accessions was reported for accessions from Kwale that showed mixed ancestry from Tharaka Nithi and Tana River. This admixture may be due to genetic exchange between Kwale, Tharaka Nithi and Tana river. These accessions also exhibited reciprocal monophyly based on phylogenetic analysis. This corroborates the phenotypic diversity studies on H. compressa where accessions from Kwale exhibited the highest phenotypic diversity compared to other regions  is an informative method for measuring population differentiation among populations  than cluster 2 . On the other hand, cluster two had higher levels of heterozygosity (He) compared to cluster 1. This indicates high diversity in accessions along the Tana basin. The high heterozygosity and lower genetic differentiation between H. compressa accessions along the Tana Basin (cluster 2) might be due to genetic exchange arising from gene flow. Although Tharaka Nithi is found approximately 163 and 391 miles away from Tana River and Kwale respectively, gene flow between these three counties seems high. This could be due to the flow of the River Tana  within population diversity than among population diversity (7.3%).The negative us plant , a condius plant . Dioecy us plant . High geH. compressa provides useful information for future selection and appropriate conservation strategies. High priority should be given to the conservation of all populations with high genetic diversity. The conservation of H. compressa must consider the two identified clusters to ensure that the high diversity within populations is retained. This can be achieved by maximum collection and ex situ conservation of germplasm especially for cluster 2 which had the most diversity.The understanding of the genetic diversity and population structure within H. compressa using the GBS approach. Hyphaene compressa in Kenyan ASALs is delineated into two gene pools. Cluster 1 comprising accessions in the north of Kenya while cluster 2 comprising accessions found along the River Tana basin. Further, accessions from the Tana basin are more diverse than those found in the northern part of Kenya. In addition, the results indicate that H. compressa accessions are interconnected with high gene flow and moderate genetic differentiation, evidenced by high within-population variation than among population variation. The high within population diversity can be harnessed for future breeding and improvement programs for various adaptive traits in H. compressa.This study was able to show the genetic diversity and population structure of