Thalassia hemprichii, in the Western Indian Ocean (WIO). The aim was to understand genetic population structure and connectivity of T. hemprichii in relation to hydrodynamic features. We genotyped 205 individual seagrass shoots from 11 sites across the WIO, spanning over a distance of ~2,700 km, with twelve microsatellite markers. Seagrass shoots were sampled in Kenya, Tanzania (mainland and Zanzibar), Mozambique, and Madagascar: 4–26°S and 33–48°E. We assessed clonality and visualized genetic diversity and genetic population differentiation. We used Bayesian clustering approaches (TESS) to trace spatial ancestry of populations and used directional migration rates (DivMigrate) to identify sources of gene flow. We identified four genetically differentiated groups: (a) samples from the Zanzibar channel; (b) Mozambique; (c) Madagascar; and (d) the east coast of Zanzibar and Kenya. Significant pairwise population genetic differentiation was found among many sites. Isolation by distance was detected for the estimated magnitude of divergence (DEST), but the three predominant ocean current systems  also determine genetic connectivity and genetic structure. Directional migration rates indicate that Madagascar acts as an important source population. Overall, clonality was moderate to high with large differences among sampling sites, indicating relatively low, but spatially variable sexual reproduction rates. The strongest genetic break was identified for three sites in the Zanzibar channel. Although isolation by distance is present, this study suggests that the three regionally predominant ocean current systems  rather than distance determine genetic connectivity and structure of T. hemprichii in the WIO. If the goal is to maintain genetic connectivity of T. hemprichii within the WIO, conservation planning and implementation of marine protection should be considered at the regional scale—across national borders.This study is the first large‐scale genetic population study of a widespread climax species of seagrass,  Since dispersal of both shoots and fruits of T. hemprichii mainly occurs by passive rafting, we hypothesized that genetic population structure in the region is shaped by the South Equatorial, the North East Madagascar, and the East African Coastal currents. The findings are used to discuss how to improve seagrass management in the WIO region to sustain healthy seagrass populations with high resilience to environmental change.The main aim of the current study was to reveal the genetic population structure, dispersal patterns, and connectivity of the common and important habitat forming seagrass 22.1−1. During the NE monsoon season (October–March), the current is slowed down by the north‐easterly trade winds and as a consequence the current speed is lowered to about 0.5 ms−1 . The climate and pattern (and strength) of currents in the WIO are complex and strongly influenced by the monsoonal circulation Figure . ThroughT. hemprichii. The seagrass meadows are often patchy and where possible samples were collected from different patches. Since a large area was sampled in each site, and T. hemprichii often mixes with other species, there was no site that was fully monospecific. None of the sampling sites were located in conservation areas. At each site, 15–20 individual shoots of T. hemprichii were randomly collected by snorkeling or walking during low tide at a distance of 10–150 m apart to largely avoid sampling of genetically identical clones. Commonly, the distance between samples was 50–100 m. This resulted in the collection of 205 sampling units at 11 locations in Kenya, Tanzania (mainland and Zanzibar), Mozambique, and Madagascar, at pairwise distances between sampling sites of approximately 15–2,700 km , that is clones. As most downstream analyses assume microsatellite loci to be independent from each other  and to be in Hardy–Weinberg equilibrium (HWE), we tested for HWE at each locus and across all loci in each population with Genepop 4.2  need to be removed from the dataset since they cause genotyping errors when one of the allele copies fails to be amplified by the PCR, which leads to missing genotypes and the incorrect assignment of homozygotes. To detect null alleles, we used the software MicroDrop , a measure of the amount of clonal growth, was calculated with the formula (MLG − 1)/(N − 1), which relates the number of MLGs to the number of ramets (N)  in R 2.15.3. We estimated genetic differentiation by calculating pairwise Weir & Cockerham FST among populations  and population differentiation (genetic difference between sampling sites) several calculations were made. Genotypic richness  based on the genetic distance measures of GST  is the relationship of genetic similarity between sites over a geographic distance. The model tests the assumption that the further apart two sites are from each other geographically, the more genetically different they should be. To test for IBD, we correlated the genetic distance matrices with “sea distance” (geographic distance among sampling sites without crossing land). This measure was calculated with the R package 33.1R‐values; Table T. hemprichii samples  across all loci ranged from 1.17 to 1.59, and the observed heterozygosity HO was very low, ranging from 0.014 to 0.09, while expected heterozygosity HE ranged from 0.054 to 0.234  to 7 . There were eleven rare alleles in our dataset (defined as occurring less than three times), which were spread among nine loci. Of these, only three were not previously recorded  and TZF on Zanzibar exhibited signs of a recent expansion .The bottleneck tests indicated that TZC on Zanzibar and ZIS in Mozambique might have experienced a recent population decline  nor G’ST , but sea distance is significantly correlated with Jost's DEST . Although most pairwise FST values are significant, DEST is arguably the most meaningful measure for our dataset given that the observed heterozygosity is very low.“Sea distance” ranged from ~15 to ~2,700 km. The two Madagascan sites are most geographically close, while the samples from Inhaca, Mozambique (ZIN and ZIS), and the Kenyan site (KM) are furthest apart. There was no evidence for a significant correlation between sea distance and the genetic distance measure 4T. hemprichii in the WIO region for regional conservation purposes. The study contributes to the knowledge of population genetic patterns of important species in the region, but also where in fact little is known on population genetic patterns in general. Genotyping of the seagrass T. hemprichii was successful, and we found four distinguished genetic clusters separated geographically on various spatial scales (10s–1000s km). Interestingly, a clear genetic cluster co‐occurs with a local system of eddies between the mainland of Tanzania and Zanzibar. Gene flow was directional and strongest from Madagascar toward the coasts of Kenya and western Zanzibar, spatially coinciding with the NEMC and the “continuing” EACC , as well as by local hydrodynamic patterns (Zanzibar channel). The study, therefore, emphasizes the necessity to consider different spatial scales to understand metapopulation dynamics and identify source populations of a habitat providing plant species.The overall aim of this study was to understand the large‐scale (1000s km) population genetic pattern of the seagrass 4.1T. hemprichii meadows and find a relation between genetic differentiation (DEST) and increasing geographic distance. Nevertheless, large‐scale currents in the area seem to play an important role for the observed genetic differentiation and the four genetic clusters suggested by the TESS analysis conform well with the major oceanographic currents in the WIO. We hypothesized that the northbound SEC, NEMC, and EACC should influence the genetic structure from Madagascar in the South to Tanzania and Kenya in the North, leading to genetic uniformity. Indeed, the sites from the east coast of Zanzibar (TZN and TZC) and Kenya (KM) are not significantly differentiated from each other and show high admixture levels, indicating that they receive propagules from more southern sites. Madagascar also showed a large differentiation compared to the other sampling sites. As predicted based on the directionality of the SEC and NEMC, we found high asymmetric migration from Madagascar to Zanzibar (TZC in particular) and Kenya. The TESS analysis confirms gene flow from the Madagascan cluster  to these two sites, and suggests additional gene flow to one more site on Zanzibar and one in Mozambique  is, however, clearly differentiated from all other sampling sites. The topography of the Zanzibar channel has a strong effect on the bottom current as it creates two gyres systems that turning clockwise and meet in front of the Zanzibar town. The gyres are caused by current systems that move to the north but differ in their velocity between the two revising monsoons. The gyres are stronger in the SE monsoon and are reduced in the NE monsoon, however, the circulation is all year around. In fact, eddies have been recorded in the Zanzibar channel, which could explain the lack of gene flow between these sites and that they are differentiated from all our other sampling sites. Similar patterns of genetic differentiation have also been observed for corals . Only one site in Mozambique (ZIN) showed a dominant asexual reproduction mode. Significantly heterozygote deficits  were observed for all sampled populations, a finding commonly observed in seagrasses in general  compared to the WIO, suggesting that this fish species originated in the EIO (Huyghe & Kochzius, T. hemprichii is in the EIO and Pacific and low genetic diversity may be explained by the fact that our sampling was performed at the edge of its distributional range. Furthermore, a number of studies have shown limited gene flow between the WIO and the EIO and Pacific, while others have shown low genetic diversity in the WIO (reviewed in Ridgway & Sampayo, A previous study on the clown fish 4.3T. hemprichii is to a large extent influenced by predominant currents in the area. Nevertheless, traditional isolation by distance models—where genetic differentiation increases with geographical distance—also seems to play a role at the assessed spatial scales in the WIO, as do local hydrodynamic features. For future conservation planning, the information on genetic clusters and breaks, predominant directions of gene flow and genetic diversity should be used to ensure that connectivity is maintained in the WIO. A regional scale approach to seagrass conservation is recommended, including effective cross‐boundary management.This study is the first large‐scale genetic study of a seagrass in the WIO and results indicate that dispersal of None declared.LMN and MG conceived the idea; LMN secured funding and led the research project; field sampling were conducted by MG, LMN, MEA, SM, AH, and JM; analyses were conducted by MJ; initial analyses were conducted by JL and LMN; SM and MS developed the maps; MJ led the writing and all authors contributed throughout the whole process and agreed on the last version. Click here for additional data file.


Plasmodium falciparum (PfMSP-142) is the target of an immune response. It has been recognised as one of the promising candidate antigens for a blood-stage malaria vaccine. Genetic structure of PfMSP-142 has been considered to be largely conserved in the P. falciparum population. However, only limited information is currently available. This study aimed to analyse genetic diversity and the effect of natural selection on PfMSP-142 among the Myanmar P. falciparum population and compare them with publicly available PfMSP-142 from global P. falciparum populations.The C-terminal 42 kDa region of merozoite surface protein-1 of P. falciparum clinical isolates collected from Myanmar malaria patients in Upper Myanmar in 2015 were used. The PfMSP-142 region was amplified by polymerase chain reaction, cloned and sequenced. Genetic structure and natural selection of this region were analysed using MEGA4 and DnaSP programs. Polymorphic nature and natural selection in global PfMSP-142 were also investigated.A total of 69 42 were identified in Myanmar isolates of P. falciparum. Myanmar PfMSP-142 displayed genetic diversity. Most polymorphisms were scattered in blocks 16 and 17. Polymorphisms observed in Myanmar PfMSP-142 showed a similar pattern to those of global PfMSP-142; however, they were not identical to each other. Genetic diversity of Myanmar PfMSP-142 was relatively lower than that of PfMSP-142 from different geographical regions. Evidence of natural selection and recombination were found. Comparative analysis of genetic polymorphism and natural selection in the global PfMSP-142 population suggested that this region was not tightly conserved in global PfMSP-142 as previously thought and is under the complicated influence of natural selection and recombination.All three allele types  of PfMSP-142 revealed limited, but non-negligible, genetic diversity by allele types and geographical origins. Complicated natural selection and potential recombination might have occurred in global PfMSP-142. Comprehensive monitoring of genetic diversity for global PfMSP-142 would be needed to better understand the polymorphic nature and evolutionary aspect of PfMSP-142 in the global P. falciparum population. More thought would be necessary for designing a vaccine based on PfMSP-142.Global PfMSP-1The online version of this article (10.1186/s13071-018-3027-x) contains supplementary material, which is available to authorized users. Plasmodium falciparum is the most medically important malaria parasite that is responsible for most malaria-related deaths globally [Malaria is one of the most important health burdens worldwide, especially in tropical and subtropical regions. It results in approximately 216 million clinical cases and about 450,000 deaths annually [42) is further cleaved into 33 kDa (MSP-133) and 19 kDa (MSP-119) fragments when merozoites invades erythrocyte [19 remains on the merozoite surface and enters into erythrocyte whereas the others are released from the merozoite [P. falciparum and P. vivax [19 or MSP-142, is of particular interest since individuals naturally infected with malaria acquire humoral immune responses against this domain [P. falciparum MSP-1 (PfMSP-142) can inhibit the growth of the parasite or invasion of the merozoite into host erythrocytes [42 are associated with protection against P. falciparum infection in endemic settings [19 and PfMSP-142 have shown potential as subunit vaccines in animal models by conferring protection against infections [42 is a promising candidate antigen for blood stage vaccine development.Merozoite surface protein-1 (MSP-1) is a 195 kDa major surface glycoprotein of merozoites. This protein binds to human erythrocytes in a sialic acid-dependent manner. It plays a pivotal role in erythrocyte invasion by merozoites , 3. MSP-erozoite . MSP-1 iP. vivax –10. In p kDa MSP-9 fragmens domain . Antibodhrocytes –19. Antisettings , 21. Botfections , 22–25. P. falciparum antigens, PfMSP-1 also shows polymorphism due to different mechanisms, including single nucleotide polymorphisms (SNPs), insertion/deletion of tandem repeats, and meiotic recombination [42 has been considered to be broadly conserved in different alleles of P. falciparum [42 in small numbers of P. falciparum and P. vivax isolates suggests natural selection may be acting on the area and natural selection is an important factor in the maintenance and generation of genetic polymorphism observed in MSP-142 [42 is a leading candidate for vaccine development, in-depth understanding of the polymorphic nature of this region would be beneficial to design optimised vaccine formulation. In this study, genetic diversity and natural selection of PfMSP-142 in the Myanmar P. falciparum population were analysed. Data from this study were also compared with PfMSP-142 sequences available for P. falciparum isolates from other malaria-endemic countries to gain insights into genetic polymorphism and natural selection of PfMSP-142 in the global population(s).Similar to other bination , 27. PfMbination , 27. Amobination . Therefobination –37. Meanlciparum , 27. How MSP-142 . ConsidePlasmodium falciparum-infected Myanmar malaria patients were used. The patients enrolled in the study attended the public health centres in towns and villages of Naung Cho, Pyin Oo Lwin and Tha Beik Kyin townships in Upper Myanmar in 2015 with clinical symptoms of malaria. Malaria transmission in these areas was heterogeneous and seasonal with most clinical cases reported during the rainy season. Plasmodium falciparum infection was confirmed by Giemsa-stained thick and thin blood smear examination. Finger-prick blood samples were collected from the P. falciparum-infected patients before drug treatment. Plasmodium falciparum infection was further confirmed by polymerase chain reaction (PCR) targeting 18S ribosomal RNA (rRNA) gene [A total of 69 blood samples collected from NA) gene , 40. The42 region flanking nucleotide positions from 3915 to 5163 in 3D7 sequence (XM_001352134)  was used in all PCR steps to minimize the nucleotide misincorporation during PCR amplification. Each PCR product was analysed on 1.2% agarose gel, purified from the gel, and then ligated into T&A cloning vector . Each ligation mixture was transformed into Escherichia coli DH5α competent cells, and positive clones with the appropriate insert were selected by colony PCR with the nested primers. The cloned gene sequences were analysed by automatic nucleotide sequencing. To verify the sequences, at least two clones from each isolate were sequenced in both directions. Some isolates underwent three or four-fold sequence coverage to confirm the existence of rare polymorphisms. The nucleotide and deduced amino acid sequences of PfMSP-142 were analysed using EditSeq and SeqMan in the DNASTAR package . The nucleotide sequences obtained in this study have been deposited in the GenBank database under the accession numbers MF943252-MF943320.Parasite genomic DNA was extracted from the blood filters using a QIAamp Blood Kit . A nested PCR amplified the full-length PfMSP-142 sequences. The number of segregating sites (S), haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and the average number of pairwise nucleotide differences within the population (K) were estimated using the DnaSP ver. 5.10.00 [42 based on a sliding window of 100 bases with a step size of 25 bp. The rates of synonymous (dS) and non-synonymous (dN) substitutions were estimated and were compared with the Z test (P < 0.05) in MEGA4 program [Nucleotide sequence polymorphism analysis was performed on the 69 Myanmar PfMSP-1 5.10.00 . The π v program  with the program  value an program  were ana program . Positiv42 in global P. falciparum isolates was analysed. The PfMSP-142 sequences from Thailand , Philippines , Papua New Guinea , Solomon Islands , Vanuatu , Ghana , and Tanzania  were included in this study to analyse genetic diversity in global PfMSP-142. Nucleotide diversity and the effect of natural selection for global PfMSP-142 were analysed with the same methods described above. Recombination parameter (R), which included the effective population size and probability of recombination between adjacent nucleotides per generation, and minimum number of recombination events (Rm) were analysed by DnaSP ver. 5.10.00 [Population diversity of PfMSP-142 haplotypes, the haplotype network for a total of 499 global PfMSP-142 sequences including 69 Myanmar sequences and the 430 publicly available sequences of global isolates deposited in the GenBank database listed above was constructed using the program NETWORK version 5.0.0.3 with the Median Joining algorithm [To investigate relationships among PfMSP-1lgorithm .42 showed three different allele types  in the Myanmar P. falciparum population. The MAD20 allele type was the most prevalent , followed by K1 , and RO33  allele types. Sequence analysis of MAD20 allele types revealed a total of 21 distinct haplotypes in Myanmar sequences  in block 16 and six amino acid changes  in block 17. Meanwhile, only one amino acid change (I54F) was identified in block 15. These results suggest that blocks 16 and 17 show polymorphic characters in MAD20 allele types of Myanmar PfMSP-142, which might contribute to inner allele diversity. For K1 allele types, a total of six distinct haplotypes were identified in Myanmar PfMSP-142 sequences. Three synonymous and four non-synonymous SNPs were observed in these sequences when compared to K1 (X03371) reference sequence  reference sequence, one tri-morphic (P244H/T) and eight di-morphic amino acid changes  were found in RO33 allele types of 14 Myanmar PfMSP-142 sequences  were novel ones that have not been reported hitherto.Sequence analysis of 69 Myanmar PfMSP-142 were similar to those of global PfMSP-142. However, they differed slightly from each other. MAD20 allele types of Myanmar PfMSP-142 showed a different pattern of amino acid polymorphism compared to those from other geographical regions  were commonly identified in all global PfMSP-142 analysed in this study, although their frequencies were different by country or geographic area. I54F, the only amino acid change identified in block 15, was more prevalent in African (Ghana and Tanzania) PfMSP-142 than that in other origins. Frequencies of F183Y and T245P were relatively high in African PfMSP-142, but they were low or not detected in Asian  or the Pacific  PfMSP-142. D220N was not identified or very rarely identified in Pacific PfMSP-142.Overall patterns of amino acid polymorphisms identified in each allele type  of Myanmar PfMSP-142 from Myanmar and Thailand were not similar  than in Thailand (25.0%).In the case of K1 allele type, only Thailand sequences have been reported so far. Overall amino acid change patterns of K1 allele types of PfMSP-1lar Fig. . E21G, R42 was also analysed .Nucleotide sequence analysis was performed to determine nucleotide diversity and genetic differentiation of Myanmar PfMSP-142 were 0.736 ± 0.109 and 0.00123 ± 0.00041, respectively. The highest π value for RO33 allele type was identified at block 17 (0.00168 ± 0.00108). The dN-dS value was also estimated to examine whether natural selection contributed to the genetic diversity in Myanmar PfMSP-142. The dN-dS values for MAD20 and RO33 allele types were 0.0028 and 0.0015, respectively, implying that positive natural selection might have occurred in these sequences. Meanwhile, dN-dS value for K1 allele type was -0.0012, suggesting that negative natural selection might have affected the allele type, but this value was not significant  for MAD20 allele types, -1.00674 (P > 0.10) for K1 allele types and -1.79616 (P < 0.05) for RO33 allele types. These results suggest that all three allele types of Myanmar PfMSP-142 are under population size expansion and/or purifying selection pressure. Fu and Li’s D and F values for all three allele types of Myanmar PfMSP-142 were also negative  differed by allele types and isolates with different geographical origins  values than those from other geographical origins. Values of π for full-length PfMSP-142 ranged from 0.00168 to 0.00351 in these isolates. However, all isolates showed similar patterns with the highest π values at block 17. Sliding window plot analysis of π also showed that values observed among global PfMSP-142 sequences peaked at blocks 16 and 17, although the overall pattern was slightly distinct throughout sequences . The Rm values for MAD20 allele types of Ghana and Tanzania were 4 and 4, respectively. The values for RO33 allele types in Ghana and Tanzania were 2 and 3, respectively. The values of R (both between adjacent sites and for the entire domain) were also highest in Tanzania , followed by Ghana .To analyse the effect of natural selection on genetic diversity of global PfMSP-1ene Fig. . The mines Table . The hig42 from the Solomon Islands or Vanuatu. Recombination parameters identified in PfMSP-142 were not high, and they differed by geographical population, but a possible recombination event might have occurred in global PfMSP-142.Meanwhile, no recombination event was identified in PfMSP-142 haplotypes from global P. falciparum populations showed a dense network with extremely complex relationships  was a singleton. Haplotype prevalence ranged between 0.2–24.2%. K1 allele type formed a separated group from MAD20 and RO33 allele types. Meanwhile, MAD20 and RO33 clustered into one group with mixed patterns probably due to their high sequence similarities. Interestingly, the rate of a singleton was high in African PfMSP-142, MAD20 and RO33 allele types, which support a high level of genetic diversity in the African PfMSP-142 population.Haplotype network analysis of PfMSP-1ips Fig. . A total42 is the target of immune protection. Therefore, PfMSP-142 has been recognised as a promising candidate antigen for blood stage vaccine development [42 has been considered as genetically stable among P. falciparum isolates [42 in global P. falciparum isolates. In this study, comparative analysis of population structure and natural selection of PfMSP-142 in Myanmar and global P. falciparum isolates was performed.The population structure of malaria parasites plays a vital role in maintaining natural immunity against malaria. Several factors including transmission intensity, parasite recombination rate, antimalarial drug resistance status, and effective malaria control measures can affect the population diversity of parasites. In this respect, population structure analysis of malaria parasites is essential to understand the genetic nature and evolutionary aspect of the parasites, especially for vaccine candidate antigens or drug resistance genes. PfMSP-1elopment , 20–23. isolates , 27. Thi42 displayed limited levels of genetic diversity. However, non-negligible genetic diversity was also found. Thus, it is difficult to regard that PfMSP-142 is genetically well conserved in global P. falciparum population. Genetic diversity of PfMSP-1 has been defined as unique, different allele types: MAD20, K1 and RO33 [42 have been considered to be well conserved in P. falciparum population [42 showed SNPs that were mainly identified in blocks 16 and 17. Overall distribution patterns and frequencies of amino acid changes found in Myanmar and global PfMSP-142 analysed in this study were not identical between or among global PfMSP-142. The implication of these substantial differences in amino acid polymorphisms among global PfMSP-142 by geographical differentiation is currently unclear. Geographical isolation followed by population subdivision may affect genetic differences of PfMSP-142 population in different geographical areas. Major amino acid changes found in global PfMSP-142 were commonly detected among global PfMSP-142, although their frequencies differed among and between populations and only a limited number of PfMSP-142 sequences in each geographical area were analysed in this study. Thus, further examination of PfMSP-142 nucleotide and amino acid variations in a more substantial number of global PfMSP-142 sequences is needed to better understand the polymorphic nature of global PfMSP-142.Myanmar and global PfMSP-1and RO33 , 27. Blopulation , 27, 47.42 than those in Asian and Pacific PfMSP-142, indicating that African PfMSP-142 possessed higher levels of genetic diversities compared to Asian and Pacific PfMSP-142. For both MAD20 and RO33 allele types, the highest π value was identified at block 17 in global PfMSP-142, suggesting that block 17 had the highest genetic variations. Sliding window plot analysis of π across PfMSP-142 revealed similar patterns of nucleotide diversity in global MAD20 and RO33 allele types of PfMSP-142 analysed here, suggesting that global PfMSP-142 might share similar nucleotide diversity across this gene. In the case of K1 allele types, different patterns of π were identified between Myanmar and Thailand PfMSP-142. Nucleotide diversity in Thailand PfMSP-142 was identified only at block 17 while Myanmar PfMSP-142 showed nucleotide diversity at blocks 15, 16 and 17, albeit π values for these regions were not high. Natural selection analysis of global PfMSP-142 suggests that this region is likely to be under natural selection which may maintain or produce genetic diversity in PfMSP-142 population. The dN-dS value for global PfMSP-142 was positive, implying balancing selection might act in this gene. However, dN-dS value for Myanmar K1 allele types was negative (-0.0012), suggesting negative or purifying natural selection in this region. In the sliding window plot analysis of Tajima’s D, overall patterns of Tajima’s D values across full-length PfMSP-142 showed highly complicated patterns that were distinct between and among global PfMSP-142. These results suggested that global PfMSP-142 was under a complicated influence of natural selection, in which either positive natural selection or purifying selection might have occurred in the population depending on allele types and geographical origins. Possible recombination events in global PfMSP-142 were also predicted, although values were not high. Higher values of recombination events were found in African PfMSP-142 than those in PfMSP-142 from other geographical areas, suggesting that African PfMSP-142 might be under more opportunity for inter- or intra-allelic recombination than other geographical PfMSP-142 probably due to high multiclonal infection rate, subsequent cross-fertilisation and recombination in mosquitoes. These results collectively suggest that PfMSP-142 is not highly conserved in global population as known to date and that complicated natural selection acts on this region. Recombination might also contribute to the genetic diversity of global PfMSP-142, albeit recombination parameters were not high.Values of haplotype diversity (Hd) and nucleotide diversity (π) were higher in African PfMSP-142, block 17 (referred to as PfMSP-119) has attracted considerable attention since this region is functionally critical. PfMSP-119 has two EGF-like motifs that play an important role in the attachment of the parasite to specific receptors on the surface of erythrocyte [19 are strongly associated with resistance to both clinical malaria and high levels of parasitemia [19 is conserved mainly between parasite strains with only a few amino acid substitutions [19 might have evolved to evade the human immune response capable of blocking the parasite from invading erythrocytes [19. Therefore, caution is needed when designing polyvalent vaccine constructs targeting PfMSP-119. Comprehensive genetic analysis of global PfMSP-119 is merited to understand the complexity of genetic makeup of this region in the global P. falciparum population.Of three blocks of PfMSP-1throcyte . Antibodasitemia . It has itutions , 27, 47.itutions . SNPs inhrocytes . Polymor42 with global PfMSP-142 revealed that global PfMSP-142 displayed limited levels of genetic diversity in the P. falciparum population. However, results of this study also suggest that global PfMSP-142 is not as genetically conserved as previously thought. Although patterns of major amino acid changes found in global PfMSP-142 were relatively similar, their frequencies differed by allele types and geographical origins. Moreover, some amino acid changes were characteristic only in specific geographical origins. Global PfMSP-142 was under the complicated influence of natural selection, in which either balancing selection or purifying selection might have occurred in the population depending on allelic types and geographical origins. Recombination might have also affected the genetic diversity of the global PfMSP-142 population. These results collectively suggested that more concern would be necessary when designing vaccine based on PfMSP-142 which has been considered to be well conserved in P. falciparum population. This study also warrants continuous monitoring of genetic analysis for PfMSP-142 in global PfMSP-142 to better understand the polymorphic nature and evolutionary aspect of PfMSP-142 in the global P. falciparum population.Comparative analysis of Myanmar PfMSP-1Additional file 1:Figure S1. Sequence analysis for MAD20 allele types of PfMSP-142 in Myanmar Plasmodium falciparum isolates. Regions corresponding to blocks 15, 16 and 17 are marked with different colors. Dots represent identical residues compared to reference sequences. Underlines indicate regions of epidermal growth factor-like (EGF1 and EGF2) domains. Amino acid changes identified in at least one sequence of the corresponding haplotype are marked in red. Cysteine residues in block 17 are shown in blue with an underline. Total numbers of isolates for each haplotype are listed in the right panel. (TIF 3611 kb)Additional file 2:Figure S2. Sequence analysis for K1 allele types of PfMSP-142 in Myanmar Plasmodium falciparum isolates. Regions corresponding blocks 15, 16 and 17 are marked with different colors. Dots represent identical residues compared to reference sequences. Regions of epidermal growth factor-like (EGF1 and EGF2) domains are indicated by underlines. Amino acid changes identified in at least one sequence of the corresponding haplotype are marked in red. Cysteine residues in block 17 are shown in blue with an underline. Total numbers of isolates for each haplotype are listed in the right panel. (TIF 1834 kb)Additional file 3:Figure S3. Sequence analysis for RO33 allele types of PfMSP-142 in Myanmar Plasmodium falciparum isolates. Regions corresponding blocks 15, 16 and 17 are marked with different colors. Dots represent identical residues compared to reference sequences. Regions of epidermal growth factor-like (EGF1 and EGF2) domains are indicated by underlines. Amino acid changes identified in at least one sequence of the corresponding haplotype are marked in red. Cysteine residues in block 17 are shown in blue with an underline. Total numbers of isolates for each haplotype are listed in the right panel. (TIF 1791 kb)


Agropyron cristatum (L.) Gaertn.] and assess the genetic diversity present in 192 genotypes from 12 tetraploid lines. Bioinformatic analysis identified 45,507 single nucleotide polymorphism (SNP) markers in this outcrossing grass species. The model-based Bayesian analysis revealed four major clusters of the samples assayed. The diversity analysis revealed 15.8% of SNP variation residing among the 12 lines, and 12.1% SNP variation present among four genetic clusters identified by the Bayesian analysis. The principal coordinates analysis and dendrogram were able to distinguish four lines of Asian origin from Canadian cultivars and breeding lines. These results serve as a valuable resource for understanding genetic variability, and will aid in the genetic improvement of this outcrossing polyploid grass species for forage production. These findings illustrate the potential of GBS application in the characterization of non-model polyploid plants with complex genomes.Molecular characterization of unsequenced plant species with complex genomes is now possible by genotyping-by-sequencing (GBS) using recent next generation sequencing technologies. This study represents the first use of GBS application to sample genome-wide variants of crested wheatgrass [ Avena sativa)  is one of the perennial species of the genus Agropyron that comprises 10–15 species in a polyploid series of diploid (2n = 2x = 14), tetraploid (2n = 4x = 28) and hexaploid (2n = 6x = 42) forms with the P genome [Agropyron species are native to temperate-frigid grassland and sandy soils of Eurasia [Crested wheatgrass [CWG;  Eurasia ,17, and  Eurasia . CWG is  Eurasia . It is c Eurasia . This sp Eurasia ,19,20. I Eurasia . It has  Eurasia . In addi Eurasia ,25,26,27 Eurasia . This em Eurasia ,31. An a Eurasia .Agropyron using molecular markers like amplified fragment length polymorphism (AFLP) [Attempts have been made to assess genetic variability within and among the genus m (AFLP)  and simpm (AFLP) ,33,34. Tm (AFLP) ,13 demonA. cristatum; and (3) assess whether the GBS application is useful in the genetic diversity analysis of complex polyploid plants.This study was conducted with the objective to apply GBS in combination with the Universal Network Enabled Analysis Kit (UNEAK)  and the The Miseq run of 192 genotypes from 12 CWG lines  generateThe genetic structure estimated for 192 genotypes from 12 CWG lines without consideration of prior population information in the STRUCTURE  analysisThe principal coordinates analysis (PCoA) revealed that the genetic relationship of 192 genotypes A was notst was also estimated from AMOVA for each line as the weighted variation among individual plants within a line to observe the extent of inbreeding. They were obtained in the range of 0.56 (in line S9491) to 0.64 (in the cultivar Kirk) with mean of 0.60  revealed that most of the SNP variations were present within the lines (84.2%), while much smaller variations reside among lines (15.8%) or among the four Bayesian clusters (12.07%) . Line-sp of 0.60 B. The paThe dendrogram based on AMOVA showed the grouping of the 12 CWG lines into three genetically distinct clusters at the Phi statistic of 0.08 or more A. The deThe optimal numbers of genetic clusters inferred from STRUCTURE analyses with respect to the extent of missing data from M20%, M30%, M40%, and M50% datasets provided 4, 6, 6, and 4 optimal clusters, respectively A. ComparThis study utilized the gd-GBS application, in combination with Haplotag pipeline, for the first time in CWG, to generate a data matrix of 192 genotypes × 45,507 SNP markers, and captured genome-wide genetic variants to evaluate the genetic diversity present in tetraploid CWG. The diversity analysis revealed 15.8% of SNP variation residing among the 12 lines and the model-based Bayesian analysis identified four major clusters of the assayed samples. These research outputs are not only useful for understanding the genetic diversity of CWG and for its breeding, but also are encouraging for molecular characterization of non-model polyploid plants.The revealed patterns of genetic diversity are interesting. First, the model-based Bayesian approach in the STRUCTURE identified four major clusters of the assayed genotypes, while the distance-based approaches like PCoA and UPGMA identified three major clusters; however, the neighbor-joining analysis was in accordance with the result from STRUCTURE analysis. Following the pedigree of the assayed genotypes , we coulOur analysis showed high within-line genetic variation  of assayst, was more in cultivars than most of the breeding lines. Two cultivars “AC-Goliath” and “Kirk” had lower diversity as indicated by higher inbreeding coefficient   B, perhap values) B, higherues) (st B. This gues) , and was eluted in a 1.5 mL Eppendorf tube with Elution Buffer. NanoDrop 8000  was used to measure the quality of the DNA by comparing the 260 and 280 nm absorptions. DNA samples were further quantified through the Quant-iTTM PicoGreen® dsDNA assay kit  and diluted to 60 ng/μL with 1× TE buffer prior to sequencing analysis.For each of the 192 genotypes, DNA was extracted from 0.1 g finely ground tissue following the protocols of NucleoSpinPstI and MspI . Ligation of customized adapters onto the 5′ and 3′ ends of the restriction fragments by T4 ligase was subsequently carried out. Then, the ligation fragments were purified by an AMPure XP kit . Following the purification, Illumina TruSeq HT multiplexing primers were added through PCR amplification. The amplicon fragments were further quantified, concentrated, and pooled to form 4 subgroups of 12 samples each. The samples in the subgroups were pre-selected using a Pippin Prep instrument  for an insert size range of 250–450 bp, before pooling the samples into a library. Each pooled library was diluted to 6 pM, and denatured with 5% of sequencing-ready Illumina PhiX Library Control  that can serve for calibration. Sequencing was completed using an Illumina MiSeq Instrument with paired-ends of 250 bp in length. MiSeq runs generated 384 FASTQ sequence files from 192 genotypes of 12 lines (one forward and one reverse for each of 192 genotypes). All the raw pair-end sequencing data in FASTQ format were deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) with accession number SRP115373 as part of the larger sequencing effort to enhance crested wheatgrass breeding [A genetic diversity-focused GBS (gd-GBS) protocol by Peterson et al.  was usedbreeding . The seqBioinformatic analysis began with sequence (FASTQ) data cleaning, using Trimmomatic version 0.36  to removPstI residue (TGCAG) at the beginning, each FASTQ file of 250 bp was first split into three fragment sets with a custom Perl script fastq184CutandCode-Pst.pl. The first set comprised the first 64 bases with the PstI residual restriction site, and the next two sets each with 59 base portions and an added 5-base PstI residue. The script also provided an arbitrary barcode sequence (CATCAT) at the start of each sequence fragment, since the UNEAK pipeline expects to deconvolute barcoded sequence reads which are not already separated by sample. The three 70-base-long fragments formed, thereafter, were independent, as their relationship was not preserved. Each fragment set was recognized by the UNEAK-GBS pipeline [As the UNEAK-GBS pipeline  only conpipeline , and wasEach fragment set (70 bases long) was analyzed with UNEAK and the Haplotag pipelines , resultiHaplotag was run with the parameters and filtering threshold settings described in the HTinput.txt file, and generated a matrix of samples by SNP loci (online The character by Taxa (CbyT) program supplied by N. Tinker was used to generate a filtered SNP file. In brief, Haplotag generated three separate “HTSNPGenos” files, which were merged before running CbyT. The “minimum presence” value in CbyT was set to 80%, 70%, 60%, and 50% for 20%, 30%, 40%, and 50% missing data, respectively. A SNP-by-sample matrix in the output files was used in further analyses. Additional descriptions of the SNP data matrix and the custom Perl and Shell scripts are available in the online ®. Thereafter, diversity analyses at the individual and line levels were carried out.The diversity analysis was based on 45,507 SNP markers, with 50% or less missing values in 192 genotypes from 12 CWG lines. Data analysis began with calculation of the minor allele frequency and the extent of missing SNP data with Microsoft ExcelThree types of diversity analysis were performed at individual genotype level. First, genetic structure of 192 CWG genotypes was examined using a model-based Bayesian method implemented in the program STRUCTURE version 2.2.3 ,43. Linust values (inbreeding coefficient) for each line [Genetic variation present among the 12 lines was evaluated with AMOVA using Arlequin version 3.5  on 45,50ach line  were genTo estimate the influence of missing SNP data on the genetic diversity analysis, four datasets of 272; 1884; 10,738; and 45,507 SNPs representing 20%, 30%, 40%, and 50% of missing SNPs  were attained for the 192 genotypes, respectively. For each dataset, the among-line variance from AMOVA and the optimal number of genetic clusters from STRUCTURE were obtained and compared among the four datasets of varying percentages of missing data.With the application of GBS, it has been possible to generate 45,507 SNP markers for a diversity analysis of crested wheatgrass. The variation residing among these 12 lines of CWG was found to be 15.8%. Further analysis grouped the assayed samples into four genetic clusters, and revealed the genetic distinctness of two cultivars each from Kazakhstan and Russia, respectively. These results can enhance parental selection for increased genetic variation and improved offspring performance in crested wheatgrass breeding. The findings in this study can also aid in the application of GBS in the characterization of non-model plants with complex genomes.


Echinococcus multilocularis. The metacestodes of E. multilocularis are fluid-filled, asexually proliferating cysts, and they are mainly found in the host's liver in the form of tumor-like growths. In this study, we investigated the genetic variations of E. multilocularis in four mitochondrial (mt) genes, namely, NADH dehydrogenase subunit 5 (nad5), adenosine triphosphate subunit 6 (atp6), cytochrome c oxidase subunit 1 (cox1), and NADH dehydrogenase subunit 1 (nad1). The complete nad5, atp6, cox1, and nad1 genes were amplified separately from each hydatid cyst isolate using polymerase chain reaction (PCR) and then sequenced. Phylogenetic trees were then generated based on the combined mt genes using MrBayes 3.1.2 and PAUP version 4.0b10. The results showed that thirty of 102 voles and two of 49 pikas were infected with E. multilocularis. The genetic variation distances among all E. multilocularis samples were 0.1–0.4%, 0.2–0.4%, 0.1–0.6%, and 0.1–0.4% for nad5, atp6, nad1, and cox1, respectively. Compared to previous studies of the genetic diversity of E. multilocularis based on the cox1 gene, the genetic distances within the same group were 1.3–1.7% (Mongolia strain), 0.6–0.8% (North American strain), 0.3–0.6% (European strain), and 0.1–0.4% (Asian strain). Based on concatenated sequences of the nad5, atp6, cox1, and nad1 genes all haplotypes were divided into two clusters. In conclusion, the genetic diversity of E. multilocularis based on mt genes on a small local area is at low level but between different regions with long distance and different ecological environment each other, the genetic diversity is at relatively high level; genetic variation is higher in the nad1 gene than that in the other three mt genes. The results on a local scale provide basic information for further study of the molecular epidemiology, genetic differences and control of E. multilocularis in Qinghai Province, China.The Qinghai-Tibet Plateau is a highly endemic area of alveolar echinococcosis where a series of intermediate hosts, especially voles and pikas, are infected with  Echinococcus multilocularis is a small cestode that cause the parasitic zoonosis alveolar echinococcosis (AE), which was one of the 17 neglected tropical diseases (NTDs) prioritized by the World Health Organization (WHO) in 2012 , Europe, and Asia , a wide spectrum of mammalian species including small herbivorous, rodents (predominantly) and pikas, and the typical definitive hosts (DH), which are canids and mammalian species, including foxes, wolves, dogs, and cats  and genetic variation in mtDNAs has also been found within E. multilocularis  and M2 , based on four nucleotide substitutions in the nad1 gene , cytochrome b (cytb), and NADH dehydrogenase subunit 2 (nad2)  has an important role in the taxonomy of E. multilocularis in plateau voles (Neodon/ Microtus fuscus) and plateau pikas (Ochotona curzoniae). In addition to the common mt cox1 and nad1 genes, NADH dehydrogenase subunit 5 (nad5) and adenosine triphosphate subunit 6 (atp6) were also included for genetic variation analysis, which provide basic information on molecular epidemiology, genetic variations or differences in E. multilocularis in China.In our study, we chose mtDNAs as molecular markers to assess the diversity of All animals were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People's Republic of China, and the study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (No. LVRIAEC2012-007). In addition, all mice were handled in strict accordance with the animal protection laws of the People's Republic of China .The biohazards, biological select agents, toxins, restricted materials or reagents involved in the research have been carried out according to the “Measures for the prevention and control of environmental pollution of hazardous chemicals” issued by the state environmental protection administration (No. 27_2005-03-30) and the “Regulations on the management of medical waste” promulgated by the state council of the People's Republic of China (2003-06-16).E. multilocularis were collected from Jiuzhi County, Golog Autonomous Prefecture, Qinghai Province  at an average elevation of 4,100 m above sea level. All samples were collected at random in the field amid a “Rodent control program” that was carried out by the local Center for Animal Disease Control and Prevention. Cystic lesions were collected from two intermediate host species: plateau voles and pikas. They were isolated and placed in 70% (v/v) ethanol before being sent to the laboratory at Lanzhou Veterinary Research Institute (LRVI). The lesions were used for DNA extraction, PCR amplification and sequencing to confirm the parasite species and analyze genetic variations. Additionally, one E. multilocularis metacestode sample from the Xinjiang Uygur Autonomous Region was preserved by our laboratory.The specimens of The cystic lesions were rinsed repeatedly using phosphate buffered saline (PBS) to remove the ethanol, and then, the lesions were disrupted using TissueLysers before DNA extraction. With the use of the TIANamp Genomic DNA Kit , the total DNA was extracted according to the manufacturer's protocols.nad5, atp6, nad1, and cox1 genes , 1 μl DNA template, 1 μl primer F (25 μM), 1 μl primer R (25 μM), and 22 μl ddH2O. The PCR was carried out using a standard 3-step cycle: 94°C, 4 min; 35 cycles of 94°C, 30 s; 50–57°C, 30 s' 72°C, 1–2 min; 72°C, 10 min. Each PCR product purified for sequencing was gel-cut and DNA was recovered through a column (AxyPrep DNA Gel Extraction Kit by AxyGen). The purified products were sent to GENEWIZ, Inc.  to be sequenced using Sanger dideoxy chain termination in an ABI3730 DNA Analyzer.Four pairs of primers were designed for nad5, atp6, nad1, and cox1genes of each isolate were assembled, edited and concatenated into a total sequence using the software package SeqMan  and Clustal W2 . The total sequences (one of the sequences having 100% similarity with the others was chosen) were aligned using BioEdit v7.2.3 and ClustalX 1.83  was used as an out group, because it was considered the sister species of E. multilocularis  version 4.0b10   with the sequences  downloaded from NCBI  and 49 pikas (O. curzoniae) were captured in Jiuzhi County. Of these animals, 32  had cystic lesions in their viscera. From these 32 individuals, 39 cysts were isolated. Twenty-seven cysts were found only in the liver, and the others were found not only in the liver but also in the lungs, the intestinal wall and the muscles , atp6 (516 bp), nad1 (894 bp) and cox1  genes were distributed into 15 haplotypes including one from Xinjiang Uygur Autonomous Region and one from Yushu Tibetan Autonomous Prefecture , 0.2–0.4% (atp6), 0.1–0.6% (nad1), and 0.1–0.4% (cox1). Within the four genes, cox1 had the most mutation sites (with 9 sites), followed by 7 sites in nad5, 2 sites in atp6 and 2 sites in nad1 ; from 120R to 120G ; from 46A to 46V ; from 16K to 16Q ; from 24L to 24V ; from 46E to 46Q ; from 153A to 153V ; from 172V to 172I ; from 443I to 443M ; from 501A to 501P  and from 532F to 532L . There was a heterogeneous substitution in 504 (cox1), from W to R .There were a number of changes in amino acids, with synonymous substitutions in 7 sites, from 460S to 460N , followed by 0.6–0.8% (samples collected from the USA: Alaska and Indiana), 0.3–0.6%  and 0.1–0.4% . The genetic distance within the same group is 0.1–0.3% (Mongolia strain), 0.4% (North American strain), 0.1–0.3% (European strain), and 0.1–0.4% (Asian strain) , belonging to the Asian clade (not shown).The phylogram based on the maximum parsimony method precisely revealed that the 14 haplotypes were divided into two large haplogroups: the JZ03, JZ05-JZ10, and JZ12 haplotypes had a close relationship with each other (classified as C1 haplogroup) and the rest of the haplotypes gathered together (classified as C2 haplogroup) , followed by apt6 , nad5 , and nad1 . According to the results, even though the analysis of the cox1 gene was more meaningful from an evolutionary standpoint, the phylogenetic tree based on only one gene was not accurate. The phylogenetic trees analysis revealed that all 15 haplotypes divided into 3 groups. The 14 samples isolated from Qinghai-Tibet plateau divided into two clades, and the haplotype isolated from Xinjiang province was in a separate group. In C1, the JZ09 haplotype was isolated from the lungs of voles, the JZ12 haplotype was isolated from the intestines of voles, and the remaining haplotypes were found in the liver of voles. In C2, the JZ13 haplotype was isolated from the lungs in voles, the JZ11 haplotype was isolated from the lungs of pikas, and the others were found in the liver of voles. The phylogram indicted that the different intermediate hosts and parasitic sites had no effect on the genetic diversity of E. multilocularis on a local scale. From the results of the genetic divergence within haplotypes based on the cox1 gene, we observed that the European strain has the lowest genetic diversity, and the Asian strain and Mongolia strain have the highest diversity. This also implied that the relationship maybe not important between geographical distances and genetic distances.Until now, a majority of published data on the gene diversity and phylogenetic analysis of the genus nad5, atp6, nad1, and cox1, shows a relatively low genetic variation among the samples from voles and pikas on a local scale. The genetic variation is higher in nad1 than that in the other three mt genes, and the concatenated mt sequences of the cox1, nad1, nad5, and atp6 genes are useful in phylogenetic reconstruction within E. multilocularis isolates.In conclusion, our study, based on the mitochondrial complete coding genes JL, LL, HY, and WJ conceived and designed the experiments. JL, LL, and YF performed the experiments and the data analyses. JL prepared the figures and wrote the manuscript, WJ and HY provided improving paragraphs and BF and XZ provided very constructive suggestions for revisions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors DB.


Ranunculus subrigidus populations distributed throughout the plateau in order to elucidate the relative contribution of geographic distance and environmental dissimilarity to the spatial genetic pattern.Due to the environmental heterogeneity along elevation gradients, alpine ecosystems are ideal study objects for investigating how ecological variables shape the genetic patterns of natural species. The highest region in the world, the Qinghai-Tibetan Plateau, is a hotspot for the studies of evolutionary processes in plants. Many large rivers spring from the plateau, providing abundant habitats for aquatic and amphibious organisms. In the present study, we examined the genetic diversity of 13 R. subrigidus populations.A relatively low level of genetic diversity within populations was found. No spatial genetic structure was suggested by the analyses of molecular variance, Bayesian clustering analysis and Mantel tests. Partial Mantel tests and multiple matrix regression analysis showed a significant influence of the environment on the genetic divergence of the species. Both climatic and water quality variables contribute to the habitat heterogeneity of R. subrigidus and current environmental factors play an important role in the genetic differentiation and local adaptation of aquatic plants in alpine landscapes.Our results suggest that historical processes involving long-distance dispersal and local adaptation may account for the genetic patterns of  By limiting gene flow within species, both spatial distance and environmental heterogeneity influence evolutionary as well as ecological processes , 2. GeneIn alpine ecosystem, physical barriers and a complex topography can significantly reduce the dispersal of organisms , 9. On tMyriophyllum spicatum L. on southeastern edge of the QTP , 51R. suR. subrigidus on the QTP is indicated by the following results. The genetic clusters identified in the present study are not corresponding to the two distant subregions, as suggested by STURCTURE  on the genetic divergence within the species. Long-distance dispersals and historical processes may also contribute to the patterns. The results would further our understanding about how aquatic plants react and adapt to the heterogeneous and harsh environments.R. subrigidus can extend clonally via rhizomes, a genet  may consist of individual shoots (ramets) covering several m2. All samples were therefore collected at an interval of at least 20 m. Fresh leaves were dried in silica gel in the field and frozen at − 20 °C in the laboratory.Field sampling was carried out in the August of 2013 and 2016, respectively. All samples were collected from small water bodies in wetlands and from lakeshores. Idenfication of specimens in the field followed . Water qRanunculus species were used in R. subrigidus [Genomic DNA was extracted using the Novel DNA Plant Kit . Thirteen self-developed EST-SSRs and four feasible SSRs isolated in brigidus . All PCRbrigidus , 1 min apsex) [As the sampling distance of 20 m does not guarantee that the samples do not belong to the same genet (e.g. in case of short-distance propagation by fragments), we carried out an analysis of clone identification using RClone  in R 3.3psex) . Duplicapsex) .Ranunculus subrigidus populations on the QTP were divided into two geographic subregions: the southern and western QTP  and the northeast QTP . The two subregions are separated by the great mountains in the central QTP such as Tanggula Mountains and Nyenchen Tanglha Mountains , which indicated a genet number of no less than five can provide reliable estimation of pairwise FST values. The matrices of genetic distance measured with individual-based and population-based approach were used for the subsequent analysis respectively.The genetic distance was measured based on the individual and population level respectively. Genetic relatedness between individuals was presented by Rousset’s â , using Sâ . The indâ . Consideax 6.501 . Given tstimates . We founM) for direct measurement of gene flow. The effective population size (θ) was also generated in the program. The data were analyzed with Bayesian inference strategy and followed a Brownian motion model. The starting values of both θ and M were generated using FST calculation method. We specified uniform priors for θ with a minimum of 0, maximum of 200, and a delta of 20, and for M with a minimum of 0, maximum of 100, and a delta of 10. We set static heating with four chains , a sampling increment of 100, 50,000 recorded steps, and a burn-in of 50,000.We used the program MIGRATE 3.7.2  to calcu, using the VEGAN package in R [.The pattern of isolation by distance was tested by the Mantel tests with 10,000 permutations based on pairwise genetic similarity index and geographic distance among all populationsage in R .Growing season temperature, extreme low temperate, temperature seasonality and annual precipitation were introduced as climate variables Table . The priR. subrigidus on the QTP. Partial Mantel tests with 10,000 permutations were performed between genetic distance and geographic/environmental distance under the influence of the other factor, using R package VEGAN. MMRR was implemented with 10,000 permutations in R with the MMRR function script [All index of genetic distance and gene flow were suitable for the landscape genetic studies . Becausen script . We alsoAdditional file 1:Table S1 Pairwise individual-based genetic distance (Rousset’s â) between 13 Ranunculus subrigidus populations. Table S2 Pairwise population-based genetic distance (FST) between 13 Ranunculus subrigidus populations. Table S3 Estimates of historical asymmetric migration rates (M)/ number of migrants per generation (Nm) between populations of Ranunculus subrigidus. Table S4 Measures of environmental variables of 13 Ranunculus subrigidus populations. Table S5 Results of dbRDA on the contributions of environmental principal component variables on the genetic pattern.


Trillium recurvatum, that has been the focus of a demographic study spanning 26 years at the Meeman Biological Station in Memphis, Tennessee, USA. Using a set of five newly developed simple sequence repeat markers first reported here, we assessed 1) the extent of clonal diversity within the Meeman site, 2) the degree to which genetic diversity varies with stage class  at this site, 3) whether there is spatial genetic structure at the Meeman site, and 4) how measures of genetic diversity and inbreeding at the Meeman site compare to two additional nearby populations. Along with these analyses, we calculated and compared traditional population genetic metrics with information theory-based diversity indices. Although clonal propagation was present, the focal population displayed moderate levels of clonal diversity comprising 81 genets from the 174 individuals sampled. In the focal site, we also found that genetic diversity was highest in the flowering stage class when compared to the non-flowering and juvenile classes. We report that genets exhibited spatial genetic structure in the focal site exhibiting values for the Sp statistic of 0.00199 for linear distance and 0.0271 for log distance. Measures of unbiased gene diversity and the inbreeding coefficient were comparable across the sampled populations. Our results provide complementary genetic data to previous demographic studies in T. recurvatum, and these findings provide data for future studies aimed at integrating the degree of clonality, genetic variation, and population dynamics in this species. Our findings suggest that T. recurvatum at the focal Meeman site displays higher levels of sexual reproduction than were previously suggested, and spatial genetic structure estimates were comparable to other plant species with mixed and outcrossing mating strategies.Combining population genetic studies with demographic surveys in long-lived clonal herbs can yield insight into the population dynamics of clonal plant populations. In this study, we assayed clonal diversity and spatial genetic structure in a population of a long-lived understory herb,  Clonal plants comprise at least 40% of our planet’s flora  and can Studies investigating population dynamics in clonal plant species, e.g., estimating population size, stage structure, and population growth, benefit from knowing the extent of clonality and number of unique genotypes present since these measures can inform mating system and genetic diversity within a population. Estimates of genetic population size  can inform the degree to which a population combines sexual reproduction and clonal growth. The genetic composition of any given population depends on a variety of factors including environmental conditions, breeding strategy, pollination and dispersal biology, and competition, and may fluctuate over time , 12–13. Trillium recurvatum Beck. This species is self-incompatible [Often though, accurate estimates of the genetic population size are difficult in plant species that propagate clonally because genets are not easily distinguished from ramets. Invasive sampling techniques, such as unearthing rhizomes, may be required to determine the extent of clonality; however, genetic markers, when available, can be used in a less destructive manner. Genetic markers can be used to estimate the number of unique genotypes and the extent of clonality, as well as, overall levels of population genetic diversity. The goal for the current study was to understand the extent of clonality and levels of genetic diversity in a focal population of a clonal forest understory herb, mpatible  and has mpatible .T. recurvatum found within the center of the species’ range. The population demographic structure in T. recurvatum reflects clonal growth and rates of transition from young (juvenile) to adult non-flowering to flowering stage class [T. recurvatum as in other Trillium species, ramets can regress from a flowering to non-flowering individual. These ‘backward’ transitions can be affected by biotic factors (e.g. deer herbivory) that cause ramets to regress in stage, have lower fecundity, and have lower probability of producing new ramets [In a study spanning more than 20 years (9 years of data collected), Moore et al.  analysedge class . In T. rw ramets –19.T. recurvatum population has been intensely studied for more than 20 years for demography and population dynamics, little information about the number of genetic individuals, degree of clonality, or spatial structure of genets in the population was known. In this study, we asked four questions: 1) what is the extent of clonality in the Meeman focal population? 2) does genetic variation vary within stage class of the ramet ? 3) what is the spatial genetic structure in the focal population? 4) in general terms, how does the genetic variation and inbreeding in the Meeman focal population compare to nearby T. recurvatum populations? We developed and report here five new simple sequence repeat  markers for T. recurvatum and extensively sampled this population to address these questions.Moore et al.  found thTrillium recurvatum Beck. is a sessile-flowered rhizotomous perennial understory herb with northern limits in southern Wisconsin and Michigan, west to eastern Missouri and Iowa, extending east to Pennsylvania, and south into northern Alabama and Louisiana. The species reproduces both sexually through seed and asexually through rhizomes  and is self-incompatible [T. recurvatum geographic distribution, it is considered rare . In Tennessee, T. recurvatum occurs primarily west of the Cumberland Plateau. In our study area in West Tennessee, plants emerge in mid-to-late February, flower in late March to early April and senesce by the end of April or early May when woody overstory leafburst occurs. Similar to other Trillium species, ants are likely the primary dispersal agent [mpatible . At the al agent . When seal agent .Podophyllum peltatum L. This Trillium site was sampled from a west-facing slope. In addition to the sampling at MBS, we also sampled T. recurvatum from two populations occurring in southwestern Shelby County (SC1 and SC2 populations) roughly 30 Km from the Meeman site in order to provide a comparison for population genetic variation. As much of the land in Shelby County is urban, we chose these sites as they are (to the best of our knowledge) natural, and undisturbed populations of T. recurvatum. Permission to collect leaves was obtained and this study did not involve endangered or protected species.Edward J. Meeman Biological Station (MBS) (owned and operated by the University of Memphis) is a 252 ha site ~ 40 Km north of Memphis, TN (Shelby County), and 3 Km east of the Mississippi River on the third Chickasaw Bluff. The primary soil type is Memphis silt loams composed of loess, which have high erodibility on slopes in this region . The yeaT. recurvatum collected from the Meeman population in early Spring of 2014 were sampled using a standardized transect array consisting of 25 2-by-2 meter (m) quadrats. This five-by-five array  was established in 1990 along a west-facing slope at MBS and is the same array reported in Moore et al. [In the Meeman site, the species tends to grow in both distinct clusters and as dispersed ramets. Leaf samples of e et al. . ColumnsT. recurvatum (two samples pooled from the Meeman site) for short repeats . Enrichment of repeats was critical for developing microsatellites since the genome of T. recurvatum is extremely large for a plant species at ~ 50Gb [de novo into contigs using SeqMan NGen v11 (https://www.dnastar.com/t-nextgen-seqman-ngen.aspx), and microsatellite loci and primer pairs were identified with msatcommander 1.0.3 [Genomic DNA was isolated from a 2 cm portion of leaf tissue obtained from all individual leaves using the SP Omega Biotek Plant Kit. DNA was quantified and quality assayed using a Nanodrop spectrophotometer (Thermo Scientific). Each sample was standardized to 20 ng/μl according to the Nanodrop readings. Five Simple Sequence Repeat (SSRs or microsatellite) markers were utilized for this study: TR-6, TR-7, TR-13, TR-15, and TR-17 , 80mM (NH4)2SO4, and 1.0% Triton X-100), MgCl2 (25mM), dNTPs (each at 20mM), forward primer (5μM), reverse primer (20μM), fluorescently labelled M13 primer (10μM) , Taq DNAfull data set , and 2) in the second, we analysed genetic diversity on a genet-only data set (unique multilocus genotypes). In order to identify the genet-only data set, we used the software program GENODIVE v. 2.0b27 [clonal threshold. This information is used to delineate plants as unique multilocus genotypes (genets vs. ramets) using the stepwise mutation model option and missing data coded as one mutational step. When the genetic distance between two individuals falls below the threshold (set to 2 for our study based upon recommendations by the authors of the programs), the two individuals are assigned to the same genet. For the full data set , we assessed genetic diversity both based on spatial organization in 2 m X 2 m quadrats (up to 25) and by stage class of the sample . Using GENODIVE, we calculated the number of unique multilocus genotypes, effective number of multilocus genotypes based on rarefaction, Simpson’s Diversity, evenness and the Shannon-Weiner index corrected for sample size. Bootstrap tests (1000 replicates) for differences in clonal diversity between pairs of stage classes were also performed in GENODIVE. In GenAlEx v. 6.5 [We sampled 174 plants in the Meeman population; however, some of these samples could represent ramets of the same genet and potentially bias genetic diversity statistics. Following Mandel  and Bent. 2.0b27  to assig. 2.0b27  algorithx v. 6.5 , we calcx v. 6.5 –35) and x v. 6.5 , has demgenet-only data set  consisted of measures of genetic diversity and genetic distance using GenAlEx. Note that from the set of multilocus genotypes, we chose the sample with the least missing data to represent that multilocus genotype. Observed heterozygosity Ho, Nei’s [Genetic analysis of the o, Nei’s  unbiasedpi)2h, where pi is the frequency for each allele observed in the multilocus genotype and h is the number of heterozygous loci. The probability of obtaining n—1 more copies of a particular clonal genotype by chance alone is given by (Pcgen)n—1, where n is the number of times the genotype was observed in the population.A genotype accumulation curve was used to determine the minimum number of loci needed to discriminate between individuals . ThSp statistic which incorporates pairwise kinship coefficients and linear or log distances into the calculations. Estimates of the Sp statistic  were obtained for the Meeman sites using b, the slope of the regression, of Fij on linear and log distance and the mean pairwise kinship coefficient with the formula Sp = -b (1- F1) where F1 is the first distance class following [b were significantly different from zero.A test for genetic isolation-by-distance (IBD) was performed using a Mantel test for matrix correspondence  on all 1ollowing  in SPAGeollowing . Tests ofull data set , we calculated the number of unique multilocus genotypes (G), effective number of multilocus genotypes based on rarefaction (effG), Simpson’s Diversity (D), evenness (E) and the Shannon-Weiner index corrected for sample size (uS-W). We also calculated genotypic richness (R) per site as (G− 1) / (N− 1) where G is the number of genets and N is the number of samples. In GenAlEx, we also calculated Nei's unbiased gene diversity corrected for sample size (uHe). For the genet-only data set (retaining only unique genotypes as in the Meeman population), we calculated observed heterozygosity Ho, Nei’s [ST = (HT- Mean HS) / HT where HT describes the expected heterozygosity if all populations were treated as one, and Mean HS describes the average of the within population expected heterozygosity across populations. We calculated FST among all three sites and between the SC1 and SC2 separately. Genetic distances amongst genets from Meeman and the SC1 and SC2 populations were investigated graphically using Principal Coordinate Analysis (PCoA). For this, a standard genetic distance matrix was constructed based on the nuclear multi-locus genotypes, the resulting matrix was used for the PCoA, and the first two principal coordinates were graphed in 2-dimensional space.As a comparison of genetic diversity measures with the Meeman population, we also sampled 13 and 20 plants from two locations, respectively, in southwestern Shelby County, Tennessee, USA. Even though we attempted to collect in a way in which we maximized unique genetic individuals, we still analyzed the data in the same manner as for the 174 individuals from Meeman. Therefore, for these samples, we also assigned genets (clones) in GENODIVE using the same parameters as above and making genets specific to each population. For the o, Nei’s  unbiasedn—1 values ranged from 0.0056 to 3.5E-63. Thus, the statistical probability that sampled plants with the same multilocus genotype were different genetic individuals was low and below the 0.05 threshold for all 81 genotypes. Evidence for null alleles, large allele dropout, and stuttering was assessed and the potential for null alleles or stuttering was suggested at Locus 6 and Locus 17; however, the results were not consistent across the populations . The genAmong the three stage classes , the flo2 = 0.0006, p = 0.06; R2 = 0.0015, p = 0.22; R2 = 0.0004, p = 0.33; R2 = 0.003, p = 0.28). However, the results of the spatial genetic structure analyses as assessed by the Sp statistics estimated values of 0.00199 for linear distance and 0.0271 for log distance, and permutation tests showed that the kinship coefficient (0.192) and regression slopes b, which were negative (-0.00248 for linear and -0.0338 for log distance), were significantly different from zero in all cases.In order to represent clonal diversity graphically, full data sets  demonstrated lower values in the focal Meeman population as compared to the other Shelby County populations surveyed as measured by the effective number of multilocus genotypes, Simpson’s Diversity, Evenness, the Shannon-Weiner Index, and gene diversity . The two southwestern Shelby County populations also mostly overlapped but showed some differentiation along PC1, and FST between these two sites was similarly low: FST 0.041 (p < 0.05).Diversity statistics for the iversity . Note, tiversity . The PCoT. recurvatum primarily reproduced vegetatively as only 11% of stems originated from seed. The population genetic data we present here demonstrates that more sexual reproduction is occurring than was previously thought. Indeed, our finding that T. recurvatum harbors moderate levels of genetic diversity is in agreement with population genetic studies in other clonal species  and a mean unbiased gene diversity of 0.54. Moreover, the inbreeding coefficient, as measured here, was positive which is in contrast to other findings in highly clonal species where values are often highly negative . Moore es e.g., ; 43). Mo. MoT. res  and could contribute to the distribution of plants along the hillside. The potential for soil composition and the physical characteristics of the hillside to play a role in population density and structure has previously been noted for this site [In the Meeman site, measures of clonal diversity varied across the quadrats as some genets were found in only one portion of the sampling area whereas others were more widespread. For example, genet/clone 1 is present in five quadrats spread across the study site, and 52 genets are present only once throughout the site. This finding may be related to the high erodability (providing mobility of ramets during periods of rain) of the loess soil (Memphis silt loams) in which these plants grow at the Meeman site . The rhihis site , and as Trillium species; [T. recurvatum. Some studies have predicted that genetic variation of genets may be linked to levels of fitness (as measured by reproductive output) [T. recurvatum are difficult to mark in this site, however, future research assessing the reproductive output of genets and levels of genetic variation could yield insight into these hypotheses. Finally, when compared to the other stage classes, the flowering stage generally comprises the fewest numbers of individuals across years [We found the flowering stage class to have significantly higher levels of genetic variation when information theory-based calculations were employed whereas traditional population genetic measures demonstrated no detectable differences. The finding that Shannon-Wiener based-measures are more likely to detect differences among means has recently been noted with regard to crop-wild gene flow literature –35 and rspecies; ) to thosspecies; ). Explan output) . Ramets ss years ; we hypoSp statistics (which represents the rate of decrease in pairwise kinship with distance) demonstrated significant spatial genetic structure. Our estimate of the Sp statistic based upon the logarithm distance slope (Sp = 0.0271) is comparable with the predominately outcrossing Trillium species, T. grandiflorum (Sp = 0.02494) and can be placed in context with other plant species falling between the mixed mating and outcrossing means of species [Sp values for gravity dispersed seeds. The clonal nature of T. recurvatum likely adds to the structuring of genetic variation. The related clonal species Paris quadrifolia also demonstrated strong spatial genetic structure of genets [We found no evidence for a correlation between spatial distance of stage classes and genetic distances as might be expected, for example, if seed dispersal was restricted and seeds were dispersed in clusters. However, spatial genetic structure analyses of genets as assessed by  species . The pat species . The degSp = 0.024 and canT. recurvatum in Shelby County, the Meeman site displayed comparable levels of genetic variation and inbreeding . The among population structure when all populations were considered was very low, though significantly different from zero. The sampled sites in Shelby County, fall within the central distribution of the range for T. recurvatum and this species is relatively common with likely additional populations intervening and connecting our populations. At the northern and southern edges of the range, the species is considered rare and of concern; additional genetic studies of those edge populations would be valuable to understanding the population dynamics of this clonal species.When compared to two other nearby populations of T. recurvatum at the Meeman site. The prevailing view based upon field-based surveys indicated that the majority of recruitment was occurring via asexual production. Our findings of moderate levels of genetic variation demonstrate sexual reproduction is relatively common, and population structure measures indicate that a substantial amount of gene flow, e.g., via seed or pollen, occurs amongst populations. We surveyed sites that were approximately 30 km apart, and a sampling of natural populations in the intervening areas would be beneficial for understanding gene flow at a finer scale. Our nuclear makers suggest a high level of gene flow; however, the use of plastid markers would also be beneficial in this system to assess the relative contribution of seed vs. pollen mediated dispersal for T. recurvatum. As mentioned previously, highly clonal populations often display negative values of F; and our finding here of positive F coefficients for all three sites studied perhaps indicates biparental inbreeding in the Meeman sight (given the self-incompatibility in the species).Our results provide novel insights into mating system in T. recurvatum at the focal Meeman site displays higher levels of sexual reproduction than were previously suggested [T. recurvatum, especially at the ranges of the species where it is considered rare, would be of value to understanding the population dynamics of this species. Our findings also suggest that other ecological genetic studies may benefit from analyzing data in an information theory framework; for example, in the related species, T. maculatum, where no evidence for genetic diversity differences among stage classes was reported [T. recurvatum.In closing, we report that uggested . We alsouggested  who callreported , a reanaS1 FigBox plot describing the genotypic resolution of microsatellites in the data set.(TIF)Click here for additional data file.S1 TablePrimer sequences and PCR reaction conditions for the newly developed microsatellite markers.(XLSX)Click here for additional data file.S2 TableResults of MICRO-CHECKER.(XLSX)Click here for additional data file.S3 TableThis table provides number of different alleles, number of effective alleles, observed heterozygosity, expected heterozygosity, and unbiased (corrected) expected heterozygosity per population for each locus per population and for each quadrat surveyed.(XLSX)Click here for additional data file.S4 TableThis table provides coordinates of each sampled plant (81 genets) given within quadrats.(XLSX)Click here for additional data file.S5 TableThis table provides the raw data for (XLSX)Click here for additional data file.S6 Tablemicrosatellite data.This table provides the genotype scores for the (XLSX)Click here for additional data file.


The origin of native and locally developed Russian cattle breeds is linked to the historical, social, cultural, and climatic features of the diverse geographical regions of Russia. In the present study, we investigated the population structure of nine Russian cattle breeds and their relations to the cattle breeds from around the world to elucidate their origin. Genotyping of single nucleotide polymorphisms (SNPs) in Bestuzhev (n = 26), Russian Black-and-White (n = 21), Kalmyk (n = 14), Kholmogor (n = 25), Kostromsky (n = 20), Red Gorbatov (n = 23), Suksun (n = 20), Yakut (n = 25), and Yaroslavl cattle breeds (n = 21) was done using the Bovine SNP50 BeadChip. SNP profiles from an additional 70 breeds were included in the analysis as references.O = 0.337–0.363) except for Yakut (Ho = 0.279). The inbreeding coefficients FIS ranged from -0.028 for Kalmyk to 0.036 for Russian Black-and-White and were comparable to those of the European breeds. The nine studied Russian breeds exhibited taurine ancestry along the C1 axis of the multidimensional scaling (MDS)-plot, but Yakut was clearly separated from the European taurine breeds on the C2 axis. Neighbor-Net and admixture analyses, discriminated three groups among the studied Russian breeds. Yakut and Kalmyk were assigned to a separate group because of their Turano-Mongolian origin. Russian Black-and-White, Kostromsky and Suksun showed transboundary European ancestry, which originated from the Holstein, Brown Swiss, and Danish Red breeds, respectively. The lowest level of introgression of transboundary breeds was recorded for the Kholmogor, Yaroslavl, Red Gorbatov and Bestuzhev breeds, which can be considered as an authentic genetic resource.The observed heterozygosity levels were quite similar in eight of the nine studied breeds  contains supplementary material, which is available to authorized users. Livestock breeding is an important sector of agriculture in Russia and is inextricably linked with the historical, social, cultural, and climatic features of the regions of this country. The beginning of cattle breeding and spreading throughout Russia is associated with the relocation of ancient Slavic tribes during the sixth century . The hisMany studies have been undertaken to clarify the demographic history of cattle breeds in the European, Asian, and North and South American countries, as well as in Africa –14, but Here, a genome-wide comparative study of nine native and locally developed Russian cattle breeds was performed with the aim to characterize their genetic architecture and clarify the contribution of the worldwide breeds to their origin.A total of 195 samples (sperm or blood) collected from nine locally derived Russian cattle breeds, namely, Bestuzhev , Russian Black-and-White , Kalmyk , Kholmogor , Kostromsky , Red Gorbatov , Suksun , Yakut , and Yaroslavl , were analysed. Blood samples from eight of the nine breeds (excluding Russian Black-and-White) were collected from purebred herds during the 2004–2016 period, whereas Russian Black-and-White was represented by semen samples  of bulls born in the 1970–1980s. Blood samples were collected during routine veterinary procedures. Sperm samples were provided by the artificial insemination (AI) stations according to specific scientific collaboration agreements. Figure Genomic DNA was extracted using Nexttec columns  and following the instructions of the manufacturer. DNA concentration was estimated by measuring the absorbance at 260 nm, and DNA quality was determined by separation on agarose gels. Whole-genome SNP genotyping was performed using the Bovine SNP50 BeadChip .The genotypes of 746 individuals representing 70 breeds distributed around the world were included in the dataset  were excluded from further analyses. LD filtering was performed by removing SNPs with the squared correlation coefficient between the two alleles of the two SNPs (r2) higher than 0.5 within 50-SNP sliding windows with each window overlapping by 5 SNPs (–indep-pairwise 50 5 0.5). SNPs located on sex chromosomes or with unknown map positions were also removed. Individuals for which < 90% of SNPs were genotyped (–mind 0.1) were removed. A Hardy–Weinberg equilibrium test was not performed for comparisons with the worldwide breeds because too many SNPs would have been excluded due to the Wahlund effect [To assess the accuracy and efficiency of SNP genotyping, the GenCall (GC) and GenTrain (GT) scores were used. A cut-off of 0.5 for both the GC and GT scores was applied to determine the valid genotypes for each SNP . SNP quad effect .R), observed (HO) and unbiased estimate of the expected heterozygosity (HE) [FIS).Within-breed genetic variability was evaluated using the R package ‘diveRsity’  to calcuity (HE) , and inbFST value [FST values [FST values were also calculated with the R package ‘diveRsity’ [To characterize the genetic differences between the Russian breeds, we calculated the overall ST value  using thST value . Breed dT values  and multveRsity’ . MDS anaveRsity’ .FST) and inbreeding [FST values [To reduce the bias that is caused by longest genetic distances  was inferred based on the results of linear regression analysis performed on Ne estimates ranging from 10 to 60 generations ago. A “Ne changing ratio” (NeC) analysis was used as a proxy of the speed in Ne changes in the 13 most recent generations. The slope of each segment that links a pair of neighbouring Ne estimates was calculated and normalized using the median of the 13 most recent Ne estimates.Trends in historical effective population size (Ne) were estimated from linkage disequilibrium (LD) as implemented in eP v1.11 . DefaulteP v1.11 ), and theP v1.11 . FollowieP v1.11 , the curR version 3.3.2 was used as an instrument for creating input files .R and HO values were similar in eight of the nine breeds analysed (AR ranged from 1.918 to 1.958 and HO from 0.337 to 0.366), except for the Yakut breed, for which a significantly lower level of variability was observed (AR = 1.780 and Ho = 0.279). Six of the nine breeds analysed had a significant (95% CI) excess of heterozygotes, while in the three remaining breeds  a deficiency in heterozygotes was identified. The largest excess in heterozygote was detected in Kholmogor (FIS = − 0.028), while the Russian Black-and-White breed was characterized by the largest deficiency in heterozygotes (FIS = 0.036) (Table 0 = 63 (Red Gorbatov) to Ne0 = 161 (Bestuzhev).After quality control and filtering, 35,874 SNPs remained for further analyses. The summary statistics for genetic diversity are in Table (Bos taurus) and indicine cattle (Bos indicus). Most of the studied Russian breeds  formed a cluster that overlapped with the breeds from Northern Europe and Great Britain as well as those from Southern Europe . The Yakut breed exhibited taurine ancestry along the C1 axis, but was clearly separated from the European taurine breeds on the C2 axis.Multidimensional scaling revealed the clear differentiation of breeds from the European part of Russia Fig. a. ComponBos taurus turano-mongolicus). Among the remaining breeds, the Red cattle breeds clustered together at the opposite end, and among these the Red Gorbatov breed was the most divergent. The Russian Black-and-White breed was integrated in the Red breeds’ cluster between the Suksun and Bestuzhev breeds, which indicates the presence of possible common ancestry. The intermediate positions between the Turano-Mongolian breeds and the Red cattle breeds were occupied by branches comprising the native Yaroslavl and Kholmogor breeds, and the Kostromsky breed on the opposite end. Neighbor-Net of Eurasian breeds  of most of the linear regression models ranged from 5 to 10, whereas Kalmyk and Yakut had values of 21 and 3, respectively (Table r2 > 0.99).Most of the breeds showed a slow decline in the effective population size over time Fig. . The Ne FST value of 0.0982 was obtained for the Russian breeds, which suggests that 9.8% of the variability was due to between-breed differentiation and the remaining 90.2% was due to allelic variations within breeds. The level of genetic variability  was highest in the Bestuzhev, Russian Black-and-White, Kalmyk, and Suksun breeds, which indicates the participation of several genetically distinct breeds in their development. The level of genetic variability observed in eight Russian breeds, excluding Yakut, was similar to that in other taurine breeds [E = 0.278; AR = 1.780; Ne0 = 64) was recorded for the Yakut breed of Siberia, which could be due to the extremely small size of the population  to 0.036 (Black-and-White). Our observations were in agreement with Gautier et al. [FIS values for 28 of the 29 European breeds in their study (FIS ranging from − 0.040 to 0.027) based on 44,706 SNPs. Five of the studied Russian breeds had FIS values close to zero (ranging from − 0.006 to 0.007), while four breeds showed relatively higher deviations. The Kholmogor, Kostromsky and Suksun breeds were characterized by an excess in heterozygotes (FIS ranging from − 0.013 to − 0.028). For the Kostromsky and Suksun breeds, the negative FIS values were probably due to recent crossbreeding with Brown Swiss (for Kostromsky), and Danish Red and Red Holstein cattle (for Suksun). For the Kholmogor breed, the excess in heterozygotes may be associated with breeding schemes that use bulls of various genetic origins. We observed a deficiency in heterozygotes (FIS = 0.036) in Russian Black-and-White cattle, which was probably due to the greater use of a limited number of bulls and higher selection pressures for a limited number of productive traits compared to other breeds.Understanding the genetic diversity and population structure of local cattle breeds is necessary for their genetic improvement and the development of effective conservation programs as well as for understanding their capacity to survive under certain environmental conditions , 55. Nume breeds , 58. TheBy applying a variety of statistical estimations to whole-genome SNP data, we unravelled the genetic structure within Russian cattle breeds. Furthermore, we assessed the distinctiveness of Russian cattle in a scenario including world-wide cattle. The results of the admixture, MDS-plot, and Neighbour-Net analyses were consistent regarding the genetic relationship and population structure patterns in the Russian breeds analysed in this study Figs. a, 3, 4.We identified three major groups within the studied Russian breeds, based on their historical origin and degree of conservation of ancestral genomic components. In contrast, based on results of a principal component analysis, Yurchenko et al.  grouped FST ranging from 12.9 to 19.0%) and from other Eurasian breeds (FST ranging from 13.6 to 27.5%)   and Gray Ukrainian breeds (FST = 3.3%)  in the pairwise comparison  . It is kFST = 7.7%) and Pirenaica (FST = 8.1%)  and the other breeds. The Red Gorbatov breed has its own branch within the British-Northern European cluster in the Eurasian Neighbor-Net tree Fig. b and clu0) showed that several breeds approached a Ne0 of ~ 50 . The third group comprised the Kholmogor, Yaroslavl, Red Gorbatov, and Bestuzhev breeds, which are those that have been the least influenced by introgression with non-Russian breeds, and thus they represent the authentic genetic resources. Such results provide a significant contribution to our understanding of the origin and modern genetic composition of the Russian cattle breeds, and provide the basis for developing more accurate programs for their conservation and genetic improvement.Additional file 1: Table S1. Short description of the local Russian breeds under study. This table provides information about the Russian cattle breeds used in this study, including the year of the breeds’ official recognition, dynamics of their population size, the productivity type, and a short description of the breeds’ history.Additional file 2: Table S2. Description of the reference samples included in the analyses. This table provides information about the reference samples used in this study including the sample size, country of origin, and the references where the genotypic data were previously published.Additional file 3: Figure S1. CV-error according to the K-number. This graph shows the CV-error for the number of clusters (K) from 2 to 40.Additional file 4: Figure S2. Trend in slope changes of historical effective population size (Ne). This graph shows the trend in slope changes of the historical effective population size (Ne) for the period starting approximately 70 generations ago. For the full definition of breeds, see Table S1 [see Additional file Additional file 5: Table S3. Pairwise Wright fixation index (FST). This table provides the values of Wright’s fixation index (FST) at pairwise comparisons between the nine Russian cattle breeds and worldwide reference breeds. For the full definition of breeds, see Tables S1 and S2 [see Additional file


Signal transducer and activator of transcription 3 (STAT3) plays a critical role in promoting the proliferation and survival of tumor cells. As a ubiquitously-expressed transcription factor, STAT3 has commonly been considered an “undruggable” target for therapy; thus, much research has focused on targeting upstream pathways to reduce the expression or phosphorylation/activation of STAT3 in tumor cells. Recently, however, novel approaches have been developed to directly inhibit STAT3 in human cancers, in the hope of reducing the survival and proliferation of tumor cells. Several of these agents are nucleic acid-based, including the antisense molecule AZD9150, CpG-coupled STAT3 siRNA, G-quartet oligodeoxynucleotides (GQ-ODNs), and STAT3 decoys. While the AZD9150 and CpG-STAT3 siRNA interfere with STAT3 expression, STAT3 decoys and GQ-ODNs target constitutively activated STAT3 and modulate its ability to bind to target genes. Both STAT3 decoy and AZD9150 have advanced to clinical testing in humans. Here we will review the current understanding of the structures, mechanisms, and potential clinical utilities of the nucleic acid-based STAT3 inhibitors. PTPR genes .,84.83,84GQ-ODNs have been proposed as a class of unique, anti-cancer STAT3 inhibitors which directly destabilize the homodimerization of STAT3, and thus interfere with its DNA binding activity ,90. CompPreliminary in vitro assays demonstrated inhibition of IL-6-induced STAT3 activation in Hep2G cells incubated with the PEI-GQ-ODN T40214 (90% inhibition at 50 ng/mL). Preincubation at the same concentration inhibited the expression of anti-apoptotic mediators, with complete blockage of Bcl-XL mRNA upregulation and 50% blockage of Mcl-1 mRNA upregulation .Treatment with GQ-ODN has demonstrated inhibition of STAT3 and tumor growth in in vivo models of head and neck squamous cell carcinoma (HNSCC) , breast 50 for STAT1 over STAT3 [GQ-ODN selectivity for STAT3 is a potential consideration, given that a negative regulator of cell growth, STAT1 shares 50% sequence similarity with STAT3. Computational docking models indicate a two- to four-fold greater ICer STAT3 , and 3D er STAT3 . Howeverfos gene. We derived the decoy by systematically shortening the double stranded oligonucleotide to determine the smallest formulation that retained binding activity to STAT3 on gel shift assays. fos genes, demonstrating nearly perfect conservation across these species. Subsequent studies identified optimal binding of STAT3 to a variant sequence of hSIE, specifically the sequence 5′-CATTTCCCGTAAATC-3′  response element termed human serum-inducible element (hSIE) in the promoter region of the c-We synthesized the double-stranded STAT3 binding sequence B, the STThe inhibition of cell growth resulting from treatment with STAT3 decoy was associated with induction of apoptosis and downregulation of the STAT3 target gene encoding Bcl-XL. Further investigation  revealedFusion of the STAT3 decoy with CpG has enabled targeting of the decoy to TLR9-expressing leukemia and lymphoma cells ,107. TreA major limitation of the first generation STAT3 decoy was the necessity for intratumoral injection. In preclinical studies, systemic delivery of STAT3 decoy failed to inhibit xenograft tumor growth , presumaThe therapeutic value of treatment with the STAT3 decoy, as with other STAT3 inhibitors, may be best realized in combination with other anticancer agents. In this regard, STAT3 decoy treatment has been shown to enhance the sensitivity of head and neck xenograft tumors to cisplatin ,104. SimHyperactivation of STAT3 in malignant cells and tumor-associated immune cells promotes cancer survival and growth through expression of anti-apoptotic and proliferative genes, as well as cytokines that generate an immunosuppressive tumor microenvironment. Understanding the role of STAT3 in cancer has led to promising strides in the development of specific nucleotide inhibitors . These aCurrently, there are four classes of drugs that directly inhibit STAT3: SH2 domain inhibitors; DNA-binding domain inhibitors ; N-terminal domain inhibitors; and STAT3 antisense and siRNA . As a whThe future of STAT3 inhibition is likely to be in combination therapy with currently existing or newly developed drugs. Blocking STAT3 could result in synergistic anti-tumor effects in combination with inhibition of EGFR or other tumorigenic dysregulated transcription factors ,118. STA