Phocoena phocoena) around the UK by genotyping 591 stranded animals at nine microsatellite loci. The data were integrated with a prior study to map at high resolution the contact zone between two previously identified ecotypes meeting in the northern Bay of Biscay. Clustering and spatial analyses revealed that UK porpoises are derived from two genetic pools with porpoises from the southwestern UK being genetically differentiated, and having larger body sizes compared to those of other UK areas. Southwestern UK porpoises showed admixed ancestry between southern and northern ecotypes with a contact zone extending from the northern Bay of Biscay to the Celtic Sea and Channel. Around the UK, ancestry blends from one genetic group to the other along a southwest--northeast axis, correlating with body size variation, consistent with previously reported morphological differences between the two ecotypes. We also detected isolation by distance among juveniles but not in adults, suggesting that stranded juveniles display reduced intergenerational dispersal. The fine-scale structure of this admixture zone raises the question of how it will respond to future climate change and provides a reference point for further study.Contact zones between ecotypes are windows for understanding how species may react to climate changes. Here, we analysed the fine-scale genetic and morphological variation in harbour porpoises ( HoweverPhocoena phocoena) is one of the smallest and most abundant coastal cetaceans, widely distributed in subpolar to temperate coastal waters of the Northern Hemisphere [P. p. vomerina in the North Pacific Ocean, P. p. phocoena in the North Atlantic Ocean and P. p. relicta in the Black Sea [P. p. meridionalis [et al. [The harbour porpoise . Whidionalis ,16,20, tdionalis . Porpoisdionalis . Given t [et al.  proposed [et al.  from theca 600 years ago), establishing a contact zone on the northern side of the Bay of Biscay, with predominantly northward gene flow [Coalescent-based reconstruction of the evolutionary history of the harbour porpoise populations showed that these three ecotypes in the Atlantic and Black Sea resulted from an initial split between the North Atlantic and Mediterranean porpoises, with the colonization of the Mediterranean Sea during the last Ice Age ,18. Thisene flow ,18,20,31et al. [In this study, we analysed the genetic structure of harbour porpoises around the UK using a dense sampling of 591 stranded animals  archives were provided by P. Jepson  and R. Reid . Data on individual sex, body size, weight, age  and associated temporal, geographical and life-history data were collected following standardized detailed systematic post-mortem examination and sample collection protocols [armap v. 0.9.5 package [b) which correspond to the main distinct maritime areas around the UK and the principal stranding zones used by the United Kingdom Cetacean Strandings Network [Tissue samples collected between 1990 and 2002 for 592 stranded or by-caught porpoises from the United Kingdom Cetacean Strandings Project . This threshold distance was judged as a representative view of the environmental conditions where the porpoises were swimming before dying and stranding.Data on habitat characteristics across the study range with respect to salinity and sea surface temperature were taken from the National Oceanographic Data Centre (NODC) World Ocean Atlas (WOA01) . Bathyme2.3.−1); 1× PCR buffer with 1.5 mM MgCl2 , 0.23 U Amplitaq DNA polymerase (Perkin Elmer), 0.8 mM of each primer and 0.1 mM of each dNTPs except for dCTP which was 0.01 mM. PCR products were labelled during the PCR by direct incorporation of less than 1 µCi 32P-dCTP (0.09 mM). The PCR cycle programme for EV104, EV96 and EV94 was: 1× (95°C for 3 min); 7× ; 25× , 73°C for 1 min) and a final extension (72°C for 15 min). We used the following PCR cycle programme for all other loci: 1× (3 min at 95°C); 35× , 72°C for 10 s) and a final extension (72°C for 15 min). PCR products from 96 individuals at a time were run on 6% denaturing polyacrylamide gels ; visualization was performed by autoradiography using a Fujifilm BAS 2500 phosphor-imager. All genotypes retained for analysis were consistent across two or more genotypings, and all homozygotes were rerun at lower annealing temperature to check for potential allelic dropout after initial analysis for Hardy Weinberg equilibrium on genotypes from the first screen.Genomic DNA was extracted from skin or muscle sample using a standard phenol–chloroform protocol. Individuals were screened at 10 microsatellite loci used previously in harbour porpoises  , allelic richness (Ra) and inbreeding coefficient (FIS) [GENETIX v. 4.05 [FSTAT v. 2.9.3 [b). Per region Ra was computed based on a rarefaction procedure using the minimum sample size available across regions around the UK (n = 13). We conducted permutation tests (105 permutations) in FSTAT to assess potential departures from Hardy–Weinberg (HW) equilibrium for each population. Confidence interval at 95% for the FIS values were calculated using the diveRsity v. 1.9.89 [We estimated the proportion of missing data per locus and region using packages  for the packages . Observent (FIS)  were cal v. 4.05  and FSTAv. 2.9.3 . These s. 1.9.89  package . 1.9.89 .Ra on a grid lattice of 2° where cells included at least two samples. We used a custom R-script to prepare the data, and ADZE 1.0 [Ra based on a standardized minimum sample size of two individuals. We plotted on a map an interpolated surface of Ra calculated using an inverse distance weighted procedure using gstat package for R [We also investigated local patterns of genetic diversity by calculating ADZE 1.0  to calcuge for R .FST [diveRsity [FST comparisons as significant only if two conditions were met: the lower CI > 0, and p < 0.05 following a Bonferroni correction.Levels of differentiation in allelic frequencies between regional groups of porpoises were estimated using pairwise FST  values aiveRsity  package 2.4.2.STRUCTURE v. 2.3.4 [prior assumption that individuals found in the same area are likely to be more closely related to each other than individuals sampled from more distant locations. To implement this, we used the sampling location as a prior information in the Bayesian inference using the Locprior admixture model [We analysed the genetic structure using a Bayesian model-based clustering method implemented in v. 2.3.4 –53. Sincre model .STRUCTURE analyses were conducted by running a series of independent simulations with different numbers of simulated clusters (K), testing all values from 1 to 5. Each run used an admixture model with correlated allele frequencies, 1 × 106 iterations after a burn-in of 1 × 105 iterations. Ten replicates of each run were conducted to test for convergence of the Markov chain Monte Carlos. Results were post-processed using CLUMPAK [K value using the Evanno's method [ CLUMPAK  and custs method , and plob). In addition, to put the local genetic structure into a broader context in the Northeast Atlantic, we combined the UK dataset with data previously obtained from Fontaine et al. [a). To calibrate allele sizes between the two studies, we genotyped 10 samples from the UK and 10 from Fontaine et al. [STRUCTURE analysis on this enlarged dataset considering 25 sampled locations (b) and the 19 zones previously used in Fontaine et al. [We conducted this analysis at a local scale along the UK coasts, considering six zones which correspond to the main maritime areas around the UK b. In adde et al. ,18. The e et al. ,18 for ae et al. ,18 with e et al. ,56 and aocations , includiocations b and thee et al. ,18Figur2.4.3.4 permutations) to interpret the significance of the spatial principal components in the sPCA [adegenet 1.4-2 package [Multivariate analyses of genetic data, such as principal component analysis (PCA), can provide a complementary view to the model-based Bayesian clustering approach ,58, sincthe sPCA . Followithe sPCA . These a package  for R so package .2.4.4.râ statistics analogous to FST/(1 − FST) between demes) is expected to increase with increasing geographical distance [b) between genetic and geographical distance matrices between individuals and evaluated its significance with a Mantel Test  using SPAGEDI 1.4 [et al. [PATHMATRIX [C for improved computational efficiency (available upon request to N. Ray).Patterns of IBD may emerge if dispersal is spatially restricted at the scale of our study . Under tdistance ,62,63. WGEDI 1.4 . InsteadTHMATRIX , implemeWe tested the occurrence of IBD first on all individuals around the UK. Then we tested whether IBD patterns differed among sex and age classes. IBD patterns could indeed differ among sexes and age classes if one of the classes  disperses less than other classes . We tested IBD in adults versus juveniles only, as sample sizes , spatial2.4.5.n = 336) included in the genetic analyses. As two porpoise ecotypes are present in the study area and are known to differ according to their body size [STRUCTURE analysis.Data on body length, age and sex were available for a large subset of the UK individuals . We excluded locus EV104 from further analyses as the proportion of missing data exceeded 10% in some regions  and potential null alleles have been recorded in other studies [He) of the remaining nine loci is shown in The proportion of missing data observed at the 10 microsatellite loci for the UK individuals ranged between 0.5% and 4.9% . All loci but EV104 showed less than 10% missing data in any of the six geographical regions around the UK  as the best solution explaining the genetic structure of the harbour porpoises from the Northeast Atlantic, as suggested by the posterior probability of the data Ln (KP=2|X) = 1 for K = 2 and by the Evanno's method based on the rate of change of this posterior probability . The analyses conducted using the local dataset along the UK coastlines and at a global scale in the Northeast Atlantic by combining the UK dataset together with previous data of Fontaine et al. [a); as pie charts on the map showing the averaged admixture proportion per locality (b); and by colour-coding hybrid individuals which have less than 80% of their genome assigned to either of the two clusters .The individual-based Bayesian clustering analyses of e et al. ,18  are part of this admixed zone between the two ecotypes, as are individuals from the west coasts of UK and the Channel. This admixed background quickly declines in porpoises further east into the Channel and along the UK coasts of the North Sea, as well as northwards along the coasts of Scotland .STRUCTURE using a method that does not rely on any model assumptions. The Global test assessing the significance of positive sPCs showed that the first sPC is significant (p = 0.004) and supports the existence of a global genetic structure such as cline or clusters [p = 0.598). Plotting the individual scores along the first two positive sPCs (a) showed that porpoises from the southwestern region of UK (CWest) depart from the others along the first sPC axis and that the genetic composition of British porpoises gradually changes along a southwest--northeast geographical axis (a). This spatial structure is also well depicted when plotting individual scores for the sPC1 on a map (b).The spatial principal component analysis (sPCA) conducted only on the UK porpoises provides a similar picture of the fine-scale genetic structure along the UK coastline  and confclusters . By contive sPCs a showed cal axis a. This son a map b.Figure3.2.1.We found significant IBD between the 591 porpoises sampled along the UK coasts, indicating that gene flow, and thus individual dispersal, is spatially restricted at that spatial scale . The IBD3.2.2.F2,334 = 261.1, p < 2.2 × 10−16, n = 336). We inspected the geographical variation in the residuals  and observed that porpoises from the southwestern (CWest) area as well as some porpoises from the west area of England displayed significantly larger body size compared to the others . We also observed a strong correlation between individual residuals of body size and individual genetic admixture proportions estimated by STRUCTURE . Combining the genetic ancestry together with the age and sex in the linear model for predicting the body length increased significantly the total variance explained by the linear model up to 67% . This model with genetic ancestry offered a significantly better fit to the data compared to a model where it is not included .As previously reported , we founesiduals a,b and o4.FST comparisons . The admixture proportions quickly decline along a southwest--northeast axis around the UK, blending towards pure individuals from the northern ecotype, with a coincident decrease in body size  in the northern part of the Bay of Biscay may just reflect a temporal snapshot, but could also be indicative of distinct habitat preferences of porpoises from the admixed zone and the southern ecotype compared to those living further north on the continental shelf. It may also be possible that some local adaptive processes are maintaining these two ecotypes as ecologically and demographically distinct. Testing these hypotheses would require a temporal study of the evolution of this admixed zone and should include a genome-wide perspective of the genetic differentiation to identify molecular evidence of ongoing natural selection.While Iberian porpoises were already recognized as having larger body sizes compared with the northern ecotype , the newet al. [et al. [Interestingly, porpoises from southwestern coasts facing the northern part of the Bay of Biscay displayed slightly lower genetic diversity compared to more northern porpoises . A previous genetic study reported a similar pattern at a larger scale in the Bay of Biscay together with a stronger IBD pattern than in the North Sea . Such r [et al. ,18,31. T [et al. ,62,63.ar estimator, which leads to an imprecise estimate of the slope [D and the variance of intergenerational dispersal distance σ2. While local effective density or effective population size cannot explain the difference, we observed between juvenile and adult porpoises because they are part of the same population, the most biologically sound interpretation is that juveniles have a reduced intergenerational dispersal distance compared to adults. Intuitively, this would be expected if adults show some philopatry and faithfulness to particular breeding areas, as suggested in harbour porpoises, especially in females [The IBD observed around the UK was weak but highly significant, and consistent with patterns observed in other parts of the range . When dehe slope ,63. The he slope ,62,63, t females –71, and 5.The evidence of an admixed contact zone between northern and southern porpoise ecotypes, occurring in the northern Bay of Biscay has been identified recently , but theThe current delimitation of the admixture zone raises the question of what environmental and ecological factors determine the distributions of the ecotypes, extent of the contact zone and whether the distributions are stable or dynamic. Previous work has shown that the structure and distribution of harbour porpoise populations has been influenced by changes in oceanographic conditions which affect food resources ,18. Ther


Total-body CT scanning (TBCT) could improve the initial in-hospital evaluation of severe trauma patients. Indications for TBCT, however, differ between trauma centers, so more insight in how to select patients that could benefit from TBCT is required. The aim of this review was to give an overview of currently used indications for total-body CT in trauma patients and to describe mortality and Injury Severity Scores of patient groups selected for TBCT.A systematic review was performed by searching MEDLINE and Embase databases. Studies evaluating or describing criteria for selection of patients with potentially severe injuries for TBCT during initial trauma care were included. Also, studies comparing total-body CT during the initial assessment of injured patients with conventional imaging and selective CT in specific patient groups were included.Thirty eligible studies were identified. Three studies evaluated indications for TBCT in trauma with divergent methods. Combinations of compromised vital parameters, severe trauma mechanisms and clinical suspicion on severe injuries are often used indications; however, clinical judgement is used as well. Studies describing TBCT indications selected patients in different ways and were difficult to compare regarding mortality and injury severity.Indications for TBCT in trauma show a wide variety in structure and cut-off values for vital parameters and trauma mechanism dimensions. Consensus on indications for TBCT in trauma is lacking.The online version of this article (doi:10.1007/s00068-016-0711-4) contains supplementary material, which is available to authorized users. The work-up of trauma patients by ATLS (advanced trauma life support) guidelines uses a step-up approach for diagnostic imaging. After conventional radiography of the chest and pelvis and focused assessment by sonography (FAST), selective computed tomography can be performed subsequently on indication . OngoingA disadvantage of TBCT scanning is increased radiation exposure for patients that appear to have minor injuries for which selective CT scanning on indication could be sufficient. For the overall group of trauma patients, the proportion of patients receiving a high radiation dose of >20 mSv at the trauma room is increased . For mulJustification for performing a TBCT is only possible in hindsight, when all diagnoses have been confirmed by radiologic imaging, interventions and the clinical course. Moreover, different outcome measures are used to justify TBCT, such as: classification as multiple or severely injured patient by anatomical scoring systems  or certain high-risk profiles for injuries –8. To imFor this systematic review, the preferred reporting items for systematic reviews and meta-analyses (PRISMA) are used as a guideline .Studies evaluating or describing indications for TBCT during initial trauma care were included. Also, studies comparing TBCT during the initial assessment of injured patients with conventional imaging and selective CT in specific patient groups were included. TBCT should at least comprise the following body regions: head, neck, thorax, abdomen and pelvis. For selection of studies, no distinction was made between immediate TBCT and TBCT with preceding conventional radiologic imaging. Reviews, randomized and observational studies describing original data were eligible for inclusion. Study protocols, case reports and editorials were excluded. Literature in a language other than English or German was also excluded.The MEDLINE and Embase library databases were searched for articles published between 1947 and July 2014. The search terms consisted of synonyms of ‘total-body CT’ combined with synonyms and words related to trauma and injury. The full search is presented in Supplementary Appendix 2. The last search was performed in July 2014 and was conducted with the help of a clinical librarian. A cross-reference search was performed on the included articles.Two reviewers independently assessed titles and abstracts of all studies identified by the initial search and excluded irrelevant studies. Second, the full texts of the remaining eligible studies were assessed to determine whether they met the inclusion criteria. Any discrepancies in inclusion were resolved by discussion between the reviewers. In case no consensus was reached, this was solved by a third reviewer. The following data from each included paper were extracted: author, publication year, country, study design, inclusion criteria, sample size, Injury Severity Score (ISS), indications for TBCT, and outcome.The search identified 532 records from the MEDLINE database and 1006 records from the Embase database. 366 duplicates were removed. 30 studies were included for data extraction , high-risk trauma mechanisms (14 studies), clinical suspicion of severe injury (12 studies) and clinical judgement (2 studies). In eight other studies, the decision to perform a TBCT was based only on clinical judgement or suspicion on severe or multiple injuries. Table 5 studiesTable In the appendix, the described TBCT indications after trauma and cut-off values for vital parameters and trauma mechanism dimensions are presented from 30 included articles. These are categorized by vital parameters, clinical suspicious injuries, high-risk trauma mechanism and contraindications. For all the included literature, minor age and isolated penetrating injury were formulated as contraindications for TBCT or indirectly formulated by including only adult patients sustaining blunt trauma.In this systematic review of studies that evaluate or describe indications for TBCT in initial trauma care, we showed similarities and differences of these indications. There is a wide variety of eligibility criteria and outcome measures between studies Table . CombinaDifferences in outcome measures for justification of TBCT in hindsight implicate a lack of consensus toward patient groups that rightfully received a TBCT during their trauma work-up. Anatomical scoring systems with different thresholds for ISS and AIS for body regions are used to justify the performance of TBCT or to select patients who might benefit from TBCT scanning , 8. SeveNot only parameters reflecting severe injury could justify TBCT. Decreased levels of consciousness could be considered an indication on itself since clinical indicators for imaging are unreliable owing to the lack of subjective input from the patient. Routine CT imaging for patients with unreliable physical examination is reported to reveal unsuspected findings in up to 38 %, leading to treatment changes in 19–26 % . FurtherThis review included only three studies for which the main objective was to evaluate indications for TBCT in trauma patients. Studies that described mortality and ISS already chose study eligibility criteria to select patients that might benefit from TBCT. Thereby, the wide variety of eligibility criteria made comparison of mortality and ISS of patient groups selected for TBCT less valuable. Besides limited comparability of methods, there was also a low availability of mortality and ISS for the included studies.An anatomical scoring system such as ISS as indication for TBCT cannot be used in daily practice, because the results are calculated after radiologic imaging is performed. As well as other outcome parameters reflecting severe injury, anatomical scoring systems could only be helpful as an outcome measure for the evaluation of the indication for TBCT and not to define the indication for TBCT.In this overview of TBCT indications, we did not make a distinction between immediate TBCT and TBCT after conventional X-rays and sonography. Future prospective research on the indication for one or both strategies should consider this difference in its design. Furthermore, there was no distinction made regarding different imaging protocols. Contrast enhancement and body position were not described for included studies.Little is known of the predictive value of specific parameters within the sets of indications for severe and multiple injury. However, reduced Glasgow Coma Scale (GCS) after major trauma seems to be a valid indication for TBCT. First, it is reported to independently predict multi-region injury and detection of injury, in general. Second, the unreliability of the physical examination can result in unsuspected findings needing treatment. Decision for a cut-off value for GCS might depend on which goal one pursues: to select multiple and severely injured patients or reduction of missed injuries after major trauma.Future research needs to prospectively determine the positive predictive value of separate TBCT indications for multiple and severely injured patients. Positive predictive values for TBCT indications are useful for determining the proportion of patients that were appropriately selected for TBCT, and the concomitant radiation exposure could, therefore, be accepted. To determine the proportion of the multiple and severely injured patients selected for TBCT, sensitivity of a set of indications has to be calculated. Emphasis on specific diagnostic tests changes when another type of outcome measure is chosen such as reduction of missed injuries.The question remains as to whether we should use fixed sets of indications for TBCT, and, if so, how they should be defined. In the meantime, one should be aware that selection of patients for TBCT by clinical judgement alone could result in relatively low ISS. Independently from which outcome measure is chosen, one should carefully weigh the potential benefits of TBCT to an increased radiation exposure and potential increase of costs. The unsuspected findings and eventual shortening of hospital admission should outweigh the increased radiation exposure to make TBCT beneficial for the less severely injured patients.Indications for TBCT in trauma show a wide variety in formulation and cut-off values for vital parameters and trauma mechanism dimensions. Combinations of compromised vital parameters, severe trauma mechanisms and clinical suspicion on severe injuries are often used. However, clinical judgement on expected severe and multiple injury is used as well. Consensus on outcome measures for justification of TBCT should be obtained to guide further research on the appropriate indications for TBCT in trauma.Supplementary material 1 (DOC 71 kb)Supplementary material 2 (DOC 55 kb)Below is the link to the electronic supplementary material.


Carollia castanea) throughout much of its range. Information on population genetic structure can inform management strategies for this widespread frugivorous bat, and effective protection of C. castanea will also benefit the more than 20 mutualistic plant species of which this bat is the primary seed disperser. To facilitate understanding of population genetic structure in this species, fourteen novel microsatellite markers were developed using restriction-site-associated DNA libraries and Illumina sequencing and tested on 28 individuals from 13 locations in Costa Rica. These are the first microsatellite markers developed for C. castanea. All loci were polymorphic, with number of alleles ranging from 2–11 and average observed heterozygosity of 0.631. Markers were also cross-amplified in three additional frugivorous bat species threatened by habitat loss and fragmentation: Sowell’s short-tailed bat (Carollia sowelli), Seba’s short-tailed bat (Carollia perspicillata), and the Jamaican fruit bat (Artibeus jamaicensis), and 10, 11, and 8 were polymorphic, respectively.Rapid anthropogenic land use change threatens the primary habitat of the Chestnut short-tailed bat ( Carollia castanea) is a frugivorous bat which inhabits tropical forests from Honduras to Bolivia, and is the primary seed disperser of many pioneer plant species found in regenerating forests , Seba’s short-tailed bat (Carollia perspicillata), andthe Jamaican fruit bat (Artibeus jamaicensis). These novel markers can be used to quantify population genetic structure, identify populations that have become genetically isolated due to habitat loss and fragmentation, and evaluate correlations between genetic diversity, gene flow, and land use in fragmented agricultural landscapes. Future studies can also use these markers to increase understanding of mating and dispersal strategies in C. castanea; preliminary evidence of female-biased dispersal has been found in this species using mitochondrial DNA markers . Data was de-multiplexed and quality-cleaned using Stacks V.1.21 , 2X Qiagen Multiplex PCR Master Mix, 0.5X Q solution, and 0.10 µL of each 10 µM primer solution. Cycling conditions consisted of a 15 min initial denaturation at 95 °C, followed by 15 cycles of a touchdown protocol of 94 °C for 30s; 63 °C for 90 s; 72 °C for 60 s, and then 20 additional cycles of 94 °C for 30 s; 57 °C for 90 s; 72 °C for 60 s.All sequences were screened using strict criteria to select only perfect microsatellites with di- or tetranucleotide motifs, at least five repeats, and low alignment scores with known transposable elements. From the 656 candidate loci identified with these criteria we selected 32 high-quality loci to test for amplification. Unlabeled forward and reverse primers for these loci were synthesized through Applied Biosystems. Primers were diluted to a 10uM solution containing both forward and reverse primers, and tested on eight C. castanea individuals. To obtain estimates of population genetic parameters representative of the study area, we selected these individuals from thirteen different patches across the study area: the same eight remnant forest patches from the previous step, plus an additional five patches . Fourteen of the twenty loci were identified as definitively polymorphic. These loci were multiplexed into two reactions  and test patches . Cycling patches . All locNA), observed heterozygosity (HO), expected heterozygosity (HE), and tests for departures from Hardy-Weinberg equilibrium (PHWE) were calculated in GenAlEx 6.5    , and all(±0.227) . These lthan 0.7 .K = 1 had the largest log likelihood (closest to zero) of all tested values. This indicated that the most likely number of genetic groups was one (K = 1), with a posterior probability the first run of ln Pr (X∕K) = 0.49. These results are in line with the findings from C. castanea populations in the same region. However, it is important to note that since the primary purpose of the present study is to develop new microsatellite markers, only a very small number of individuals were sampled (n = 20). It is possible that analyzing additional individuals and individuals from more isolated forest patches could reveal significant genetic structure at the scale of the study area.For all three independent replicate runs in STRUCTURE, C. castanea and enable tests of whether levels of genetic diversity and gene flow in isolated populations are correlated with land use change and habitat loss and fragmentation. Understanding the impact of these processes on C. castanea is especially important since this bat is known to disperse at least 20 species of Neotropical plants . In addition, allele frequencies reported in C. perspicillata and directly developed for A. jamaicensis, these additional markers will add resolution and power to future studies of genetic patterns in these species.Our analyses were also successful in determining the utility of these microsatellite markers in the three co-distributed frugivorous bat species. Ten loci amplified and were polymorphic for  species . Eleven ymorphic . Rates o10.7717/peerj.2465/supp-1Figure S1Carollia castanea. For all charts allele size is listed on the y-axis.Distribution of raw allele size for each of the fourteen microsatellite loci, across all 20 individuals of Click here for additional data file.


Phragmites australis is a subcosmopolitan species typical of wetlands being studied in Europe for its disappearance from natural stands, a phenomenon called reed die-back syndrome (RDBS). Although it is conjectured that low genetic variability contributes to RDBS, this aspect remains neglected to this day. Using a molecular fingerprinting approach and a sequence analysis of the trnT-trnL/rbcL-psaI regions of cpDNA, this study aimed to compare the genetic structure of stable vs. RDBS-affected P. australis stands from five wetlands of central Italy. Beforehand, in order to characterize the health condition of reed populations, the occurrence of the main macromorphological descriptors for RDBS was considered on 40 reed stands. Soil samples were also collected to examine the total content of heavy metals. The current study analyzed cpDNA in 19 samples and AFLP profiles in 381 samples to investigate the genetic structure of Phragmites populations. Based on the multinomial-Dirichlet model, an analysis of candidate loci under selective pressure was also performed. The relationships among AFLP data, RDBS descriptors and chemicals were evaluated with the use of Linear Mixed Models. The analysis of the cpDNA shows the occurrence of the haplotypes M (the most widespread), and K here recorded for the first time in Italy. Three new haplotypes were also described. The DNA fingerprinting analysis has produced a total of 322 loci (98% polymorphic) and shows the medium-to-high amount of genetic diversity. The significant genetic differentiation among wetlands (Fst = 0.337) suggests either low gene flow or small effective population size. Moreover, the low amount of outlier loci , seems to indicate the scarce occurrence of selective pressure upon the reed’s genome. Genetic diversity increased in relationship to the decrease in diameter and of flowering buds of the reed, two of the trends associated with the die-back. The current study rejects the hypothesis that genetic diversity massively contributed to RDBS. Moreover, significant relationships between genetic diversity and the total concentration of some heavy metals  were highlighted, indicating possible genotoxic effects on P. australis. The current study represents a fact-finding background useful for the conservation of common reed. Phragmites australis (Cav.) Steud. (Common reed ‘die-back’ syndrome (hereafter RDBS) refers to the non-reversible, spontaneous retreat or disappearance of mature stands of the common reed, ) Steud. . RDBS ap) Steud. , but its) Steud.  has high) Steud. , 2014. T) Steud. . Althoug) Steud. . Specifi) Steud. .P. australis populations affected by die-back  could exceed the toxicity threshold in the aerial portion of this species  To evaluate the frequencies of outlier loci and their relation to demographic and morphological traits indicators of RDBS; and (iii) To evaluate the possible genotoxic effect on the populations due to heavy metals detected in the rhizosphere.The current study had the objectives of testing the three hypotheses presented above: (i) To compare the genetic structure of vigorous stands of Figure 1): Fucecchio Marshland, at Le Morette ; Chiusi lake ; Trasimeno lake ; Colfiorito marsh  and Vico lake . The five sites differ in their pedo-morphological characteristics, passing from lowland marshlands (‘Fu’) or lakes (‘Ch’ and ‘Tr’) to the Apennine mountain marshland (‘Co’) or a volcanic basin (‘Vi’), and surface areas, with the smallest site (‘Fu’) covering 1.02 km2 to the largest wetland (‘Tr’) .The study area included five wetlands of central Italy (2 plots (four flooded and four emerged) were randomly selected, for a total of 40 plots sampled. Macromorphological traits accounting for RDBS  were available according to the methods of The five areas had been formerly investigated in order to understand the RDBS process and diagnostic traits, by way of, stratified random sampling including two ecologically distinct conditions: (1) ‘flooded stands,’ submerged by water also in the driest season (August–September) and (2) ‘emerged stands,’ more or less long-flooded during the year, but not so during the driest season .For the molecular analysis, each selected plot was considered as the center of a circle with a radius of 10 m in which foliar tissue samples were randomly collected from at least 10 individuals spaced about 5 m apart. Sampling was carried out at the end of the vegetative season, corresponding to the reaching of the maximum standing crop of Each leaf tissue sample was first dried on silica-gel and ground in a mortar with sterile sand. The DNA was extracted by using the 2x cetyltrimethylammonium bromide (CTAB) protocol . The quaP. australis were edited in BioEdit v7.0 , 32 Phragmites s.l. sequences (see Supplementary Table Hakonechloa macra (NC 025235) and Monachather paradoxus (KF169836-KF169834). A total of 53 sequences were grouped on the basis of a previous parsimony network analysis . A cut-off value, fixed at 5% of the maximum profile showed in the chromatograms, was decided upon after the analysis of replicate samples, taking into account only those bands present in all the replicates.Fst coefficients, as done here by the Bayesian method, the AFLP loci under selection could be quantified  was then calculated directly from the measure of population differentiation (Fst) as: Nm = (1-Fst)/4Fst /4Fst . As sugg-1 and a pressure of 2.5 bars, to improve the instrument’s detection limits which ranged from 0.1 to 1.5 mg⋅kg-1. Commercially produced  standard solutions (1000 mg⋅L-1) in nitric acid were used to prepare the appropriate elemental calibration standards.The concentrations of heavy metal pollutants were determined for Cd, Pb, Zn, Cr, Ni, Cu. These heavy metals were considered due to their high environmental significance . The tot®, 65%) and 2 ml of ultrapure solution of hydrogen peroxide  were added to 0.200 g from each dried sediment sample and digested in a Mars microwave oven (working at a power of 1000 W). Microwave digestion consisted of two steps: 130°C (200 psi) for 1 min, and 180°C (300 psi) for 10 min. The mixture was cooled, filtered , and diluted with ultrapure water (18.2 MΩ) to 50 ml. The heavy metal concentrations were measured based on two replicates of the overall analytical procedure for each sample of sediment examined. Differences among the chemical sediment composition of the five studied sites have been tested adopting an ANOVA. A Shapiro–Wilk test was performed in order to test the normality of Residuals , relationships with heavy metals and the ecological status were analyzed through a linear mixed-effects model considering the genetic diversity as response variable, the metals and the status (flooded/emerged) as main factors  and the sites as random factor. The use of mixed models and random effects allowed to take in account the spatial correlation among the observation from the same site and obtain more general and robust results . The nor“r2glmm” . AccordiAll the analyses were performed in the R environment ; the linTable 2; it ranged from 0.001 for the “North America native group” and “P. japonicus related group,” to 0.002 for the “P. australis s.l. group” and “HapI/U group.” The HapI/U groups were well differentiated, with an equal genetic distance between the groups (0.005). In terms of their genetic distance, the North American lineage was equally distant from all the other groups (0.005). The haplotype network analysis  showed that the haplotype M was observed in all the accessions from ‘Fu’ and partially for ‘Ch’ and ‘Tr.’ In addition to haplotype M, the common reed from ‘Ch’ hosted accessions with sequences similar to haplotype K (CH2.1 and CH2.2). The multi-alignment of sequences showed that HapK and the accessions CH2.1 and CH2.2 were characterized by a small nucleotide subunit (32 bp long), which was repeated twice, from position 182 to 278 of region trnT-trnL , that differed from the haplotype M only by a single insertion at position 855 of the alignment , 148 for the combination hex_EcoRI-CTA/MseI-ATG and 174 for that of fam_EcoRI-TAC/MseI-ATG. The percentage of polymorphic loci (PPL) ranged from a maximum of 66.9 (Fu09) down to a minimum of 28.8 (Tr01). Within each population, the average percentage of polymorphic loci varied from 52.3 for ‘Figure 3), representing just 1.5% of the all analyzed loci. The highest level of high-differentiated loci found was for ‘Ch’ whereas the lowest was for ‘Co.’ Three stands apparently lacked any outlier loci .The BayeScan analysis identified five outlier loci that had a posterior probability greater than 0.76 . No significant differences were found among the five wetlands for the values of AGD_out . The lowest AGD value was observed for ‘Vi’ for the all loci, whereas the ‘Co’ showed the AGD lowest value for the outlier loci . However, the population from ‘Table 3). Among all the hypothetical groupings, the partition of the sites based on their provenance with Ch2 and Ch6 + Tr11 isolated from each other, accounted for the highest percentage of among-group variation (9.51%). This indicates that each site was a plausible genetic center of propagation and that Ch2, Ch6, Tr11 were different haplotypes within the respective populations (Table 4). The genetic differentiation among the five populations was relatively high (Fst = 0.337) and the estimated effective number of migrants was less than one (Nm = 0.497), suggesting restricted gene flow or low effective population size. Instead, the genetic differentiation among groups within populations, decreased significantly (Fst = 0.095), and the Nm was 2.371, suggesting increased gene flow within each population, and is expected to retain genetic connectivity . Each lake had different levels of genetic organization: ‘Fu,’ ‘Tr,’ and ‘Vi’s distinguished the flooded and emerged stands . By contrast, the flooded stands from the ‘Co’ separated into two different terminal clades and were merged with the emerged accessions . The samples Ch02 and Ch06 were the most divergent among the accessions of ‘Ch.’ Two emerged samples, Ch11 and Ch12, were clustered with the Ch01, whereas the remaining flooded samples of Ch04 and Ch05 were clustered with Ch03 . Statistical support for the genetic distances are reported in Supplementary Table The AMOVA showed that the greatest amount of variation occurred at the intra-sampling site level (66.28%), rather than among the sampling sites . The concentration levels showed in the present study, were compatible with the common ranges of heavy metals in Italian soils . For Zn a strong interaction with the ecological status was found (p = 0.0063). The same relationships were observed between AGD_neut vs. Zn and AGD_neut vs. Cu . Also in this case, for Zn a strong interaction with the ecological status was found (p = 0.0057).The relationships between metals and components of genetic diversity were statistically assessed. Zn and Cu were significantly associated with AGD_all . An overall positive relationship was observed between Cu and the two indices , and a positive relationship was observed between Cr and AGD_neut .No significant relationships between heavy metal concentration and AGD_out was found. The trends of the relationships between heavy metals and the two indices of genetic diversity AGD_all/AGD_neut were complex. An overall negative relationship was found between Zn and the two indices even if this metal showed a different trend according to the ecological status, with a strong negative relationship with the emerged status and a slight positive relationship with the flooded status  and AGD_neut (figure not shown) with culm diameter (p = 0.0472 and p = 0.0421) and flowering percentage (p = 0.0204 and p = 0.0172). For the latter trait the log transformation was applied in order to obtain the normality of residuals. No relationships were observed between macromorphological traits and the AGD_out.As for the relationships between macromorphological traits and the genetic diversity indices, the analysis showed negative relationships between AGD_all  populations, the most represented haplotype is M, with a lot of its variants  Clayton [=P. australis susp. chrysanthus (Mabille) Soják]; this taxon is rather rare in Italy, reportedly at present only in some northern regions , the occurrence of diverse haplotypes of common reed from Chiusi and Trasimeno Lakes, may be deducted. Indeed the sampling sites CH2 (attributable to the haplotype K) and CH6 (attributable to the haplotype CHTR) for ‘Ch,’ and the sampling site TR11 (ascribable to the haplotype CHTR) for ‘Tr’ diverged from each other within each population. Moreover, ‘Ch’ hosted three different haplotypes , and had the highest mean level of genetic diversity . However, despite the remarkable variability observed in terms of haplotype distribution, the AMOVA shows that only the 9.51% of the total variation among populations (33.7%) could be explained through the subdivision by geographical location and the haplotypes occurrence (Table 4).The present study provides the first estimates for an analysis of genetic structure of natural common reed populations as characterized by their differing health status. Much like the other Italian populations of ustralis , 2008, australis . This reustralis . Indeed,ustralis  or by thustralis . A firstl scales . In workvariants . In the variants . The M1  regions  and Sard regions . The obsFst should be carefully interpreted. Nevertheless, Fst may give reasonable estimates of Nm when some factors are controlled, for instance a reduced spatial scale and a large number of loci analyzed. Moreover, one of the approaches to indirectly estimate the rate of migration consists in resorting the Nm after the exclusion of outlier loci from the total analyzed , thus indicating a similar per-lake fingerprinting profile, likely related to the local wetland ecology of the site. These results are in line with the observations of P. australis adaptation to changes in local environmental factors and, in this present study, it could be considered as proxy indicators of the selective pressure in populations under RDBS. In the current study, the low occurrence of outliers  compared to neutral loci, suggests there is negligible evidence for divergent selection. In addition, there was no find any significant correlations between the frequency of outlier loci and ecology or macro-morphological indicators of RDBS. In support of the low effect of genetic drift among the populations, the AMOVA showed that most of the genetic variation occurs within the populations (66.28%) rather than between populations. The observed levels of molecular variance are typical for perennial, long-lived, outcrossing plant , two of the trends associated with the die-back syndrome (Table 5) as short and long-term objectives for sediment remediation policies based on Sediment Effect Concentrations (SECs). The consensus I values were close to the “non-effect” of SQG and correspond to the averages between Lowest Effect Levels and Threshold Effect Level (Co’ and ‘Vi’) to moderately (anthropogenic) polluted . In particular the mean values for Cr and Ni were above the consensus II value for the ‘Ch,’ ‘Tr,’ ‘Co,’ and ‘Fu’ sites. The mean values for Cd and Cu were in the range between the consensus I and II value to all the sites. The mean values for Zn and Pb were below the consensus I value for ‘Ch,’ ‘Vi,’ ‘Tr,’ and ‘Co.’ Considering the overall mean concentrations of the heavy metals the ‘Fu’ wetland was the most polluted followed by ‘Ch,’ ‘Tr,’ ‘Co,’ and ‘Vi.’ Many in vivo and in vitro studies demonstrated that a high concentration of chemicals could produce the phenomena of DNA random-breaking, mutations in the target sequences of the restriction enzymes  in the common reed plant can increase with that of the soil or substrate . It must be pointed out that these all five haplotypes were found both in vigorous and in RDBS affected stands, indicating that this syndrome does not seem related with a specific haplotype composition of the population. In addition to the complex distribution of haplotypes, moderate to high levels of genetic diversity were observed. The first hypothesis that low genetic diversity contributes to reed die-back is therefore not supported. On the contrary, the genetic diversity is positively related with morphological trends that typify RDBS. The scarce occurrence of outlier loci and the absence of correlations between frequency of outlier loci and macro-morphological indicators of RDBS suggest no evidence for a selective pressure in populations under die-back, rejecting thus the second hypothesis. The internal genetic diversity of AC, LL, and LR designed the work. AC, LL, MC, VF, DG, and LR collected the samples. AC, LL, RS, and DC acquired and analyzed the data. AC drafted the manuscript. LL, DC, MC, FF, BF, DG, RV, DV, RS, and LR critically revised the article. All the authors approved the version of the manuscript to be published.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.


Short tandem repeats (STRs) are attractive to genetic applications like forensic, anthropological and population genetics studies. The Huaxia Platinum System was specifically developed to allow co-amplification and detection of all markers in the expanded CODIS core loci and the Chinese National Database. In this study, in continuation to our previous validation study, 568 unrelated individuals were firstly genotyped to investigate the effectiveness of this novel assay in 3 main ethnic groups of China . The combined power of discrimination (CPD) were 0.9999999999999999999999999992, 0.999999999999999999999999992, 0.999999999999999999999999998, respectively, and the combined power of exclusion (CPE) were 0.9999999999, 0.999999995, 0.999999998, respectively. Next, genetic relationships along administrative and ethnic divisions were analyzed using pairwise genetic distances, multidimensional scaling (MDS), principal component analysis (PCA) and phylogenetic analysis. The Han ethnicity showed a high genetic homogeneity all across China, and significant genetic differences existed between Han groups and some minority groups, most prominently for the Tibetans, the Uyghurs, the Kazakhs, the Miaos, the Zhuangs and the Dais. Aforementioned results suggested that the Huaxia Platinum System is polymorphic and informative, which provides an efficient tool not only for human forensics, but also for population genetics studies. STR genotyping has now been applied in various aspects of human identification in forensic investigations for nearly 30 years3. Recently, to minimize adventitious matches and to facilitate data sharing, the Combined DNA Index System (CODIS) was upgraded from 13 loci to 20 loci (i.e. the expanded CODIS)4, while a parallel process occurred in China where 20 loci are required for uploading DNA profiles to the Chinese National Database (CND), the world’s biggest DNA databases6. However, 6 non-overlapped loci exist between the expanded CODIS and the CND7. The Huaxia Platinum System  is a 25-locus, six-dye, multiplex that allows co-amplification and fluorescent detection of the 23 autosomal loci  including all the recommended core loci in the expanded CODIS and the CND as well as Amelogenin and Y-InDel (rs2032678) for gender determination8. Our previous validation study demonstrated that this novel assay is robust, sensitive, specific and reliable, and forensic parameters is polymorphic and informative in three main ethnic groups of China, Sichuan Han, Xinjiang Uygur and Tibet Tibetan7.Short tandem repeats (STRs) are DNA fragments with a variable number of tandemly repeated short (2–6 bp) sequence motifs, such as (GATA)n10. The Han population, accounting for 92% of the total population in China, is widely distributed in mainland territory of China. The Yi people are a typical ethnic minority in China and the largest ethnic minority group in Sichuan Liangshan Yi Autonomous Prefecture. Most of them live in mountainous regions and often carve out their existence on the sides of steep mountain slopes. The Tibetan is one of the oldest peoples in China and South Asia and Tibetan population mainly reside throughout the Qinghai-Tibetan Plateau for hundreds of generations, and has genetic adaptations of distinct combinations of phenotype in high-altitude (>4000 m). With economic growth and traffic development, some Tibetans began to migrate to plain areas. Sichuan is home to a large community of Tibetans, with 30,000 permanent Tibetan residents and up to 200,000 Tibetan floating population.China, the world’s most populous country and the world’s second-largest state by land area, emerged as one of the world’s earliest civilizations in the fertile basin of the Yellow River in the North China Plain. The nation officially recognizes 56 distinct ethnic groups, widely disseminated in 34 administrative regions, and the population substructure is further complicated11, the present study characterizes the genetic diversity of the Huaxia Platinum System in 3 main ethnic groups of China . Additionally, genetic data of our present investigated individuals and other previously studied 56 Chinese populations44 were used to investigate genetic relationships along administrative and ethnic divisions.In continuation to our previous studies11, in the present study Sichuan Yi  and Tibetan people  were analyzed.Hainan, separated by the Qiongzhou Strait from the Leizhou Peninsula of Guangdong, is the smallest and southernmost province of China. The population density of Hainan is low compared to most Chinese coastal provinces. This study was carried out to provide the first batch of 23 STRs data of Hainan Han population  using the Huaxia Platinum System. Meanwhile, we continue to evaluate the forensic efficiency of this novel assay for application in 2 main ethnic groups in Sichuan. Sichuan consists of two geographically distinct parts: the eastern part is mostly within the fertile Sichuan basin and the western part consists of the numerous mountain range. Han Chinese, the majority of the province’s population, mainly reside in the eastern portion, while significant minorities of Yi and Tibetan people reside in the western portion that are impacted by inclement weather and natural disasters. Sichuan Han have been investigated in our previous studiesNo significant deviation from Hardy-Weinberg disequilibrium was observed Table  and no sTM DNA ID system 20 A kit and AGCU EX22 kit were estimated separately and listed in Supplementary Table A total of 246 alleles were observed in Hainan Hans with corresponding allele frequencies ranging from 0.0026 to 0.5207. The PD, PE, TPI, Ho, He varied from 0.8028 to 0.9840, 0.2290 to 0.8411, 1.0966 to 6.4333, 0.5412 to 0.9227 and 0.6233 to 0.9233, respectively. The combined power of discrimination (CPD), combined power of exclusion (CPE) are 0.9999999999999999999999999992 and 0.9999999999. For Sichuan Yi population, a total of 245 alleles were identified with corresponding allele frequencies varied from 0.0028 to 0.5057. The values of PD, PE, TPI, Ho, He spanned from 0.7927 to 0.9794, 0.3060 to 0.7475, 1.2899 to 4.0455, 0.6158 to 0.8757 and 0.6327 to 0.9111, respectively. The CPD, CPE are 0.999999999999999999999999998 and 0.999999998. For Sichuan Tibetan population, a total of 231 alleles were detected with corresponding allele frequency varied from 0.0025 to 0.5808. The PD, PE, TPI, Ho, He spanned from 0.7677 to 0.9841, 0.2227 to 0.7333, 1.0815 to 3.8269, 0.5404 to 0.8687 and 0.5878 to 0.9207, respectively. The values of CPD and CPE are 0.999999999999999999999999992 and 0.999999995. Meanwhile, the CPD and CPE based on the loci covered by the PowerPlex 21 System, Goldenst and corresponding p values showed that statistically significant differences were observed between Hainan Han and Yunnan Miao at 11 loci, between Yunnan Vietnamese at 6 loci, and between Xinjiang Uyghur at 4 loci after Bonferroni adjustment (p < 0.00004) . The Locus-by-Locus FPrincipal component analysis among the 59 groups was carried out been prepared on the basis of allele frequency distributions of 19 STR loci. As displayed in Fig. st = 0.0940, Rst = 0.1103 and Rst = 0.1442, respectively), while Guangzhou Han is the most closely related to Hainan Han (Rst = 0.0107), Shanghai Han is the most closely related to Sichuan Yi (Rst = 0.0116), and Yunnan Bai is the closest to Sichuan Tibetan (Rst = 0.0199).To further explore the genetic diversity and phylogenetic characteristic among Chinese populations, Nei’s standard genetic distances were calculated among 59 Chinese populations and presented in Supplementary Table Furthermore, evolutionary relationships among 59 Chinese populations were inferred from MDS  and Han populations. The results of genetic population substructure pattern can happen for the reasons of large-scale population migration, ethnic intermarriage, random mating and gene flow among different ethnicities or one nationality from distinct geographic regions.Human blood samples were collected upon approval of the Ethics Committee at the Institute of Forensic Medicine, Sichuan University. Written informed consent was obtained from each participant. All the methods were carried out in accordance with the approved guidelines of Institute of Forensic Medicine, Sichuan University. This study was approved by the Ethics Committee of Sichuan University .568 peripheral blood samples were collected from 193 unrelated Han Chinese recruited from Hainan Province, 177 unrelated Yi Chinese recruited from Sichuan Liangshan Yi Autonomous Prefecture and 198 Tibetan Chinese recruited from Sichuan Province.Human genomic DNA was extracted using the Purelink Genomic DNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The quantity of the DNA template was determined using Quantifiler Human DNA Quantification Kit (Thermo Fisher Scientific) on a 7500 Real-time PCR System (Thermo Fisher Scientific). DNA samples were then normalized to 1.0 ng/μl and stored at −20 °C until amplification.PCR amplification was performed with 27 PCR cycles in a ProFlex PCR System (Thermo Fisher Scientific) following the manufacturer’s protocol. Amplification products were separated and detected on an Applied Biosystems 3500 Genetic Analyzers using POP-4 polymer and 36 cm capillary array according to the manufacturer’s recommendations. Allele allocation was carried out with GeneMapper ID-X v.1.4 software (Thermo Fisher Scientific) using the allelic ladder and the set of bins and panels provided by the manufacturer.49. Forensic parameters were estimated by calculating power of discrimination (PD), power of exclusion (PE) and typical paternity index (TPI) using modified PowerStats V12 spreadsheet (Promega)50.To evaluate the forensic efficiency of this novel STR system for application in 3 main ethnic groups of China, genotype data of 568 unrelated individuals including 193 Han, 177 Yi and 198 Tibetan were analyzed. Population indices including allele frequency, heterozygosity, Hardy–Weinberg equilibrium (HWE) and the possible presence of linkage disequilibrium (LD) among loci pairs were obtained using Arlequin software v3.5.2.244 was conducted using Locus-by-Locus comparisons (Fst) based on 19 overlapping STR loci  following Slatkin’s linearized Fst51. The pairwise Fst’s can be used as short-term genetic distances between populations, with the application of a slight transformation to linearize the distance with population divergence time. The detailed information and abbreviations of aforementioned populations are shown in Supplementary Table 52 and multidimensional scaling analysis (MDS) was conducted in SPSS software . Unbiased estimate of Nei’s standard pairwise genetic distance was calculated using the Phylip3.695 package. A neighbor-joining phylogenetic tree was delineated in the Molecular Evolutionary Genetics Analysis 7.0 (MEGA 7.0) software53.Furthermore, to further investigate the phylogenetic relationships among Chinese populations, a comprehensive population comparison among 59 groups2O were used as positive and negative controls respectively for each batch of amplification and genotyping. All experiments were conducted at the Forensic Genetics Laboratory of Institute of Forensic Medicine, Sichuan University, which is an accredited laboratory (ISO 17025), in accordance with quality control measures. Additionally, the laboratory has been accredited by the China National Accreditation Service for Conformity Assessment (CNAS).Control DNA 007 (Thermo Fisher Scientific) and ddHSupplementary Figure S1 and Supplementary Tables S1-12


Thrombophilia, commonly manifested as venous thromboembolism (VTE), is a worldwide concern but little is known on its genetic epidemiology in many parts of the globe particularly in the developing countries. Here we employed TaqMan genotyping and pyrosequencing to evaluate the prevalence of known common nucleotide polymorphisms associated with thrombophilia in a Somali population in the Puntland region of Somalia. We also employed next generation sequencing (NGS) to investigate other genetic variants in a Somali patient with deep venous thrombosis (DVT). As expected, we found no existence of factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) in the Somali population. The G allele of ABO [261G/delG] polymorphism (rs8176719) was found at a frequency of 29%, similar to that observed in other African populations. We found the lowest so far reported frequency of MTHFR C677T (rs1801133) polymorphism in the Somali population . A novel and deleterious single nucleotide variation in exon 11 of coagulation factor V (c.1631A>G) causing Gln544Arg exchange in factor V was identified in a 29 years old Somali female with DVT. The same patient was heterozygous to VKORC1 Asp36Tyr polymorphism (rs61742245) that predisposes to warfarin resistance. In conclusion, this study shows that common hereditary factors for thromboembolism found in Caucasians are either less frequent or absent in the Somali population—similar to the situation in other Africans. NGS is possibly a better choice to detect genetic risk variants for thrombosis in this ethnic group. Venous thrombosis, usually evolving from thrombophilia and commonly manifested as deep venous thrombosis and pulmonary embolism (VTE), is a major health concern worldwide. There are known strong genetic risk factors for VTE. These involve deficiencies in the innate anticoagulants protein C, protein S and anti-thrombin—typically with low frequencies in the general population due to their severity —but alsoThe two most evaluated genetic risk factors for VTE are factor V Leiden  and prothrombin G20210A (rs1799963), each increasing thrombotic risk by threefold to fivefold , and methylenetetrahydrofolate reductase (MTHFR) C677T in the Somali population. These SNPs (excepting MTHFR C677T) were found to provide a predictive power for venous thrombosis in Caucasians .All genotyping and NGS analysis were carried out at the division of Clinical Chemistry, University Hospital in Linköping, Sweden.Using Catch-All Sample Collection swabs , buccal cells were collected from unrelated medical students and university staff (n = 101) at the East Africa University in Bosaso city of the Puntland region of northern Somalia. Extraction and purification of DNA was performed with the PureLink Genomic DNA Kits using the standard protocol supplied by the manufacturer . TaqMan SNP assays for genotyping factor V Leiden (rs6025), prothrombin G20210A (rs1799963) and MTHFR C677T (rs1801133) were purchased from Life Technologies. TaqMan genotyping was performed in a 25 µl reaction with TaqMan Genotyping Master Mix on a Stepone Plus instrument following assay protocols provided by the manufacturer (Life Technologies).http://genome.ucsc.edu/cgi-bin/hgPcr) and on 2% agarose gel electrophoresis.A new pyrosequencing  method wA novel c.1631A>G heterozygous mutation in exon 11 (Gln544Arg) of coagulation factor V found in a patient was verified by pyrosequencing using the following PCR and sequencing primers: B-5′-ACTTGGGGTCATCACGTTTC-3′ (forward biotinylated PCR primer), 5′-TCCCTATTGCTTGCTTTTGTCA-3′ (reverse PCR primer), and 5′-GCAGCAGACATCGAA-3′ (sequencing primer). The sequence-to-analyse was CRGCAGGCTGTGTTTGCTGTGTTTGAT where “R” represents the variable position (A/G). All pyrosequencing reactions were run on a PyroMark Q24 instrument using standard reagents and protocols provided by the instrument manufacturer . Allele frequency was calculated by gene counting.GRCh37/hg 19).DNA of a thrombosis patient was isolated from EDTA-blood using EZ1 BioRobot (Qiagen). Targeted NGS using HaloPlex Target Enrichment reagents for library preparation  was applied to sequence coding sequences of 15 loci involved in haemostasis (Table https://www.ncbi.nlm.nih.gov/snp/) and is to be publicly available in Build B151 under the sequence identifier number ss2137510516.A novel SNV, c.1631A>G in coagulation factor V, was submitted to the dbSNP database   was low in the Somali population. We identified only three individuals heterozygous to this SNP corresponding to a T allele frequency of 0.015 (1.5%). This indeed represents the lowest T allele frequency of MTHFR C677T SNP found so far in any population when compared with data reported in the 1000 Genomes Project Phase 3 (801133 waWe used a newly developed pyrosequencing method to genotype the O blood group discriminating [−/G] polymorphism (rs8176719) in exon 6 of the ABO locus. This INDEL polymorphism at codon 261 [261G/delG] is associated with VTE risk , 23 and We used NGS to explore genetic variants that might be associated with thrombosis in a patient with DVT. Using a panel comprising 15 loci involved in haemostasis Table  suggestiAlthough VTE is a complex disease, with lifestyle and environmental factors as important elements, the genetic component remains significant. Prevalence of common genetic risk factors for VTE has been investigated in numerous ethnic populations. Vast majorities of these studies were performed in developed western countries, while there is still little data, or no data at all, reported from many developing nations. Somalia is a country that is recovering from more than a quarter century of unrest and refugee crisis. Infectious diseases and emergency medicine consume whatever available of the overstretched and scarce health care resources in the country and thrombosis is not on the public agenda. Since collapse of the central government in 1991 there has been very little medical research conducted in the country and there is no national registry for diseases such as thrombosis. Incidences of thrombosis in Somalia are therefore unknown. Although some research has been performed on Somali refugees living in the west, the genetic panorama of inherited disorders is virtually unexplored in the Somali population. In the current study, we studied the frequency of common genetic markers for thrombosis in the Somali population using a limited number of samples from 101 unrelated volunteers and one thrombosis patient. First, we found that the two most common VTE genetic risk factors in Caucasians, factor V Leiden and prothrombin G20210A, are non-existent, or at least very rare, in the Somali population, supporting the Neolithic farming origin of these SNPs in the Middle East . Ethnic The G allele frequency of ABO [261G/delG], a third common VTE risk factor, was found to be similar to that found in other African countries (29%) and lower than in Eurasians. Strikingly, a study performed in 1987 by the Somali Red Crescent Society and the Finnish Red Cross Blood Transfusion Service who analysed 1,026 blood samples of Somalis from the entire country, found that the phenotype O blood group had a frequency of 58% , which pIn the Somali population a low T allele frequency of MTHFR C677T polymorphism was observed, indeed lower than in any other world population studied up to now. In addition to its reported role in cardiovascular diseases, MTHFR 677T allele is associated with neural tube defects and pregnancy loss . RemarkaOn the whole, our study shows that the most known common genetic risk factors for VTE are either absent or less frequent in the general Somali population when compared with other ethnic populations. However, this study also shows that there may be other genetic risk factors for VTE in the Somali population as we now report for the unique factor V c.1631A>G (Gln544Arg) heterozygote mutation that we found in a young Somali female patient with DVT. Further studies on this mutation will be necessary before its role in VTE can be confirmed. The Gln544 residue is located in a highly conserved region of factor V that is close to the cleavage site of protein C (534–535). Glutamine is a neutral polar amino acid whereas arginine is positively charged and it is therefore not inconceivable that its transition to arginine may affect factor V inactivation and cause protein C resistance.One limitation of this study is that we genotyped samples collected from one region (Puntland) in Somalia. However, ethnic Somalis were found to be genetically highly homogenous regardless regional or clan affiliation , 30 and Finally, it is our opinion that NGS is the most appropriate option available when investigating genetic bases of VTE in non-Caucasian patients as these individuals usually do not carry the most characterised VTE genetic risk factors (i.e. factor V Leiden and prothrombin G20210A) often included in the standard laboratory analysis for thrombophilia investigation in many countries. With NGS being the leading diagnostic tool for genetic diseases, it is now possible to detect inherited traits associated with both VTE as well as drug response (pharmacogenetic), which enables a more personalised treatment. In the current study, for instance, NGS enabled simultaneous detection of Gln544Arg as well as VKORC1 Asp36Tyr mutations in the same patient. The latter mutation causes warfarin resistance , a drug In conclusion, this study shows that common genetic predictors used to assess risk for VTE in Caucasians are less relevant in the Somali population. Instead we believe that NGS is a more appropriate diagnostic tool for discovery of hereditary thrombosis markers in individuals of non-Caucasian origin.


O. glaberrima and O. sativa (RAM3/Jin23B//Jin23B///YuetaiB). The proportion of O. glaberrima genome (PGG) in the ILs ranged from 0.3% to 36.7%, with an average value of 12.32% which is close to the theoretically expected proportion. A total of 250 polymorphic alleles were amplified by 21 AFLP primer combinations with an average of 12 alleles per primer. Population structure analysis revealed that the IL population can be divided into four genetically distinct subpopulations. Both principal component analysis and neighbor-joining tree analysis showed that ILs with a higher PGG displayed greater genetic diversity. Canonical discriminant analysis identified six phenotypic traits  as the main discriminatory traits among the ILs and between the subpopulations and showed significant phenotypic distances between subpopulations. The effects of PGG on phenotypic traits in the ILs were estimated using a linear admixed model, which showed a significant positive effect on grain yield per plant (0.286±0.117), plant height (0.418 ± 0.132), panicle length (0.663 ± 0.107), and spikelet number per panicle (0.339 ± 0.128), and a significant negative effect on filled grain percentage (-0.267 ± 0.123) and days to flowering (-0.324 ± 0.075). We found that an intermediate range (10% − 20%) of PGG was more effective for producing ILs with favorable integrated agronomic traits. Our results confirm that construction of IL population carrying O. glaberrima genomic fragments could be an effective approach to increase the genetic diversity of O. sativa genome and an appropriate level of PGG could facilitate pyramiding more favorable genes for developing more adaptive and productive rice.The introduction of closely related species genomic fragments is an effective way to enrich genetic diversity and creates new germplasms in crops. Here, we studied the genetic diversity of an introgression line (IL) population composed of 106 ILs derived from an interspecific tetra cross between  Oryza sativa and Oryza glaberrima were independently domesticated in Southern Asia and Western Africa, respectively, from different wild species progenitors [O. sativa is now cultivated globally, whereas O. glaberrima is largely restricted to West Africa. However, O. glaberrima varieties offer distinct advantages such as weed competitiveness, high light use efficiency, drought tolerance, and pest and disease resistance [O. glaberrima has the ability to grow in a wide range of challenging ecosystems such as rain-fed hilly areas, deep-water conditions, and in coastal mangrove areas [The cultivated rice species genitors . The higsistance , 6, 7, 8ve areas , 10. ThuO. sativa genome is a promising approach for breeding new inbred rice varieties. Introgression lines (IL) and chromosome segment substitution lines (CSSL) have been developed for the genetic improvement of rice via transfer of favorable traits from O. glaberrima into O. sativa [O. sativa and O. glaberrima at the African Rice Center. The NERICA varieties are high-yielding, drought and pest resistant, and are adapted to the local conditions of West Africa [O. glaberrima genomic fragments into drought susceptible O. sativa backgrounds can enhance grain yield under drought stress [O. glaberrima genes [The introduction of genes from evolutionarily distant germplasms into the . sativa , 14, 15.t Africa , 16. Furt stress . Quantitma genes . Therefoma genes , 20.O. glaberrima) cytoplasm has been developed to increase the cytoplasmic genetic diversity of three-line hybrid rice; a new dominant restorer gene was identified from O. glaberrima in introgression restorer lines [O. sativa and introgression restorer lines carrying O. glaberrima genes has been reported to result in a positive interspecific heterosis for grain yield [Recently, further utilization of ILs carrying African genes is considered a promising approach in breeding hybrid rice varieties. A new type of CMS rice with African rice (er lines . The devin yield .Oryza rufipogen etc.) and Asian rice (Oryza sativa) [Oryza glaberrima and Oryza sativa is limited. The effect of O. glaberrima introgression segments on the phenotypic traits of the IL population has not been reported.Information of population structure and genetic diversity of the germplasm is very important for both genetic researches and breeding projects , 24. Man sativa) , 26, 27.Oryza sativa, it is still inadequate for O. glaberrima [Echinochloa spp. [Calotropis procera in the West Africa region [Oryza glumaepatula wild rice populations in Costa Rica [A variety of molecular markers can be used to evaluate the genetic diversity of populations, such as simple sequence repeats (SSRs) and amplified fragment length polymorphisms (AFLPs). SSR markers are codominant, generally locus specific, and randomly dispersed throughout the plant genome but need completed genome sequence information to determine their exactly location on the genome , 31. Altaberrima . AFLPs, aberrima  independaberrima . Therefoloa spp. . Sixteena region . Eight Asta Rica . In thisO. glaberrima genomic fragments (PGGs) in the ILs; (2) to analyze the genetic diversity and phenotypic variation of ILs with different PGGs; (3) to evaluate the impact of PGG on phenotypic variation of agronomic traits of the ILs.The objectives of this investigation were to (1) estimate the proportion of O. glaberrima [O. sativa varieties and maintainer lines for indica CMS lines widely used for hybrid rice production in China [A set of 131 ILs were derived from an interspecific tetra cross RAM3/Jin23B//Jin23B///YuetaiB. Data of 106 ILs was obtained. RAM3 is a genetically representative accession of aberrima , while Jin China .The ILs were grown at Huazhong Agricultural University , Wuhan, Hubei province of China, in the summer season of 2012. A randomized complete block design with three replications was used and each plot contained 24 plants in two rows with 15 × 20 cm spacing. Eight phenotypic traits were evaluated: days to flowering (DF); plant height (PH); panicle length (PL); panicle number per plant (PN); spikelet number per panicle (SN); filled grain number per panicle (FGN); thousand grain weight (TGW); and yield per plant (YP). FGP was calculated as follows:Three grams of fresh leaves were ground in liquid nitrogen using a pestle and mortar. Total genomic DNA extraction was performed using the cetyltrimethy lammonium bromide method (CTAB) as described by Adedze et al. (2015) with minor modifications . The DNAAFLPs were detected using a method similar to that described by Vos et al. . The AFLAFLPs were used to assess genetic diversity by determining the number of alleles, the number of polymorphic bands, the proportion of polymorphism (%), Nei’s genetic diversity index, and the Shannon diversity information index. POPGENE 3.2 software was used to calculate Nei’s genetic diversity index and the Shannon diversity information index. Two hundred and fifty five AFLP alleles which showed dominance (present) in all three parents but recessiveness (absent) in some of the ILs were selected to estimate the frequency of variable loci (FVL) as follows:K) ranging from 1 to 10 with the admixture model, a burn-in period length of 5000, a run length of 50,000, and 10 replications for each K value. Both LnP(D) and Evannos’s ΔK methods were used to estimate the number of subpopulations. The run of the estimated numbers of subpopulations showing the maximum likelihood was used to assign genotypes with membership probability ≥ 0.65 to subpopulations. Genotypes with membership probability ≤ 0.65 were assigned to an admixed group. A principal component analysis (PCA) was carried out using the Eigen program of NTSYS-pc 2.1e based on the DIST (average taxonomic distance) coefficient. Genetic and phenotypic differentiations in the IL population were estimated by constructing a neighbor-joining (NJ) tree in MEGA 5.0. The SAS software  was used for statistical analyses of the phenotypic data. The phenotypic differences among ILs and subpopulations  were assessed via the canonical discriminant analysis using the CANDISC procedure [The STRUCTURE software program was used to infer the structure of the IL population . Ten indrocedure . Canonicrocedure .O. glaberrima fragments in the genome (PGG) of each IL was estimated with a Bayesian method according to the admixture model described by Pritchard et al. [K = 2, referring to the genetic sources of O. glaberrima and O. sativa; a burn-in period length of 10,000 and a run length of 100,000 were performed. The probability of membership for the cluster including RAM3, from the run with lowest log likelihood value (-4593.1), was used to estimate the PGG of each IL. The plot of the STRUCTURE results was generated by the DISTRUCT program.The proportion of d et al.  and Gouyd et al. . PGG wasb2I), d is the vector of random effects within different pedigrees ~N, I is the identity matrix, e is the vector of residual error of the model, and z1 and z2 are incidence matrices. A likelihood ratio test was performed to test for the fixed effect of the proportion of the genome arising from O. glaberrima.The effect of PGG on phenotypic traits was assessed via the following mixed model:To avoid systemic errors due to different dimensions, a standardizing treatment was performed by a zero-mean normalization before the data were used for the mixed model. All these models were computed using SPSS 19 software .Twenty-one primer combinations showing rich molecular diversity were selected to produce the AFLP profiles for the 106 ILs evaluated in this study. A total of 533 alleles were identified. The number of bands produced by the primer combinations ranged from 18 (Eatc/Mcag) to 39 (Ega/Mcaa). The number of polymorphic bands per primer combination varied from 5 (Ega/Mcta) to 24 (Eaag/Mtag), with an average of 12 bands per combination . The proO. glaberrima fragments in the genome for every IL and identify the population structure  and Evannos’s Δs K = 4; . With ths K = 4; . Net nucA principal component analysis (PCA) based on marker genotypes was performed to determine the consistency of the differentiation of the populations defined by the cluster analysis . The anaO. glaberrima fragments.An NJ tree based on the AFLP markers showed clusters similar to that of the PCA and STRUCTURE analysis . The genSignificant variation for eight agronomic traits was observed among the ILs , indicatAn NJ tree  was perfc1 = 0.836) and between the ILs and the second canonical variate (rc2 = 0.504) indicate that this canonical variates can explain the phenotypic differentiation of this IL population. The first canonical discriminant function  explained 84.3% of the variance and was dominated by large coefficients from the plant phenotypic traits—yield per plant, panicle number per plant and days to flowering. Canonical variate 2 accounted for 12.3% of the variation and was dominated by large coefficients from this traits—plant height, filled grain percentage, panicle length and panicle number per plant. The two canonical variates together accounted for 96.5% of the variation among ILs.The canonical coefficients showing the contribution of each measured trait to the total variation are presented in x and y axis to visualize the subpopulation clusters (D2. The greatest phenotypic distance was observed between SP1 and SP2 (D2 = 19.96) followed by SP2 and AD (D2 = 8.96), SP1 and SP3 (D2 = 7.12), SP1 and AD (D2 = 5.32), SP2 and SP3 (D2 = 5.20), and SP3 and AD (D2 = 1.31). All pairwise distances between the subpopulation clusters were significant.The centroid values of the first two canonical discriminant functions for the accessions were plotted as the clusters . The extAgronomic performance in ILs was further analyzed with respect to population structure based on AFLP markers. Significantly differences were found between the means of subpopulations for all the traits . Plant hComparison of the genotypic and phenotypic DIST distance matrixes in the ILs revealed both similarities and differences . GeneticGenotypic and phenotypic DIST distances varied between ILs within different subpopulations. For ILs of SP1 and SP2, genotypic and phenotypic distances were relatively low, < 0.30 and < 1.50, respectively, while for most ILs of SP3, they were relatively high, > 0.45 and 1.00 − 3.50, respectively.Larger genetic distances were present in ILs from different subpopulations. The average genotypic and phenotypic distances between SP1 and SP2 were 0.459 and 1.621, respectively. The average genotypic and phenotypic distances between ILs from SP1 and SP3 were 0.480 and 2.732 respectively; those between SP2 and SP3 were 0.490 and 1.385, respectively.Exceptionally high genotypic and phenotypic genetic distances were found between ILs with high PGGs . The topr = 0.275**), spikelet number per panicle (r = 0.415**), panicle length (r = 0.583**), and negative correlations were found for thousand grain weight (r = -0.264**), filled grain percentage (r = -0.503**), days to flowering (r = -0.707**). No significant correlation was found for plant height or effective tiller number.The relationship between the PGG in ILs and eight phenotypic traits was examined using Pearson’s correlation analysis . SignifiThe effect of different levels of PGG on phenotypic traits was analyzed by the mixed model using the frequency of variable loci (FVL) as the co-variate . PGG hadO. glaberrima genomic fragments might be more favorable for improving agronomic traits.To visualize the PGG effects to phenotypic traits, the performance of 8 phenotypic traits in four groups of ILs with different amounts of PGG  are presented in O. glaberrima genomic fragments in the ILs. The average PGG (12.32%) of the ILs estimated by Bayesian method is close to the theoretically expected introgression level of 12.5%, while the frequency of introgression alleles showed an average of 5.53%. Although PGG values resulted from the two methods are different, they are significant correlated   , S3 Fig.O. glaberrima genomic fragments into the O. sativa genome.ILs incorporating African rice genomic fragments have been developed and studied for different purposes, such as identification and screening on yield related traits of ILs under biotic and abiotic stress, etc. , 19, 45.O. glaberrima fragments into O. sativa genome background is an effective way to increase the genetic diversity of rice.In this study, four subpopulations were detected with STRUCTURE in a population consists of 106 ILs based on 250 polymorphic AFLP sites, and greater genetic variation was found in subpopulations containing ILs with higher GGP. PCA and NJ phylogenetic tree analysis were constructed based on molecular markers to analyze the genetic differentiation, both results showed the ILs with lower PGGs (< 10%) were mostly assigned into two clusters while the ILs with high PGGs (10% − 40%) were distributed discretely, indicating higher PGG might lead to greater variation between the genotypes. There was a significant correlation between PGG level and the proportion of variable sites, with a correlation coefficient of 0.725 (P < 0.01). These results indicate that the introduction of It is important to exploit the genetic diversity found in rice genetic resources which can be classified according to the phenotypic variability of agronomic traits . In thisThe yield related traits were found to be significantly different between pairs of subpopulations carry different PGGs as revealed by the t tests . The ILsO. glaberrima genes have been reported to develop partial interspecific hybrid rice express positive yield heterosis [O. glaberrima introgression effectively increase the genetic diversity would encourage and facilitate the use of interspecific hybridization for high yielding and more adaptive rice breeding programs.Genetic variation provides the potential for a species to adapt to adverse environmental changes . Geneticeterosis . Our resO. sativa and O. glaberrima [Grain yield has been reported to increase by more than 20% in the progeny of crosses between aberrima . In thisO. glaberrima genomic fragments into O. sativa allows the importation of genes controlling adaptive traits such as drought tolerance and biotic resistance [African rice has many favorable genes that could potentially be used to improve the agronomic traits of Asian rice, although it is inferior to Asian rice in yield related traits such as panicle size and plant type , 50. Mansistance , thus, tsistance , 20. TheO. sativa and O. glaberrima F1 hybrids are sterile due to the sexual barrier between the two species. Many genes responsible for this hybrid sterility have been identified, such as S1, S3, S18, S19, S20, S21, S29(t), S36(t), S37(t), S38(t), and S39(t) [O. glaberrima controlling interspecific sterility might persist in ILs with higher PGG levels.Most d S39(t) , 56, 57.O. glaberrima and O. sativa, was detected in the ILs. We included the FVL in the mixed model to estimate the random effects of genetic variation as a co-variate to avoid its interference with our analysis of PGG effects. Significant correlations were found between FVL and PGG and between FVL and phenotypic traits, such as filled grain percentage and days to flowering , which were distinct from both lowering . This anO. glaberrima fragments is an effective way to broaden the genetic diversity and improve phenotypic traits, it is crucial to determine the optimal level of PGG for efficient development of elite ILs. Here, we found that a PGG level of 10% − 20% appeared to be most favorable; thus, the interspecific tetra cross with an average PGG of 12.5% was considered suitable for producing ILs for genetic improvement of yield related traits in O. sativa. The results of this study demonstrated that moderate introgression levels (10% − 20%) from the O. glaberrima genome can facilitate both the incorporation of beneficial genes and elimination of interspecific sterility genes to achieve high grain yield.While the introgression of Oryza glaberrima and Oryza sativa was divided into four distinct subpopulations by STRUCTURE analysis. AFLP marker genotypes and morphological traits were analyzed to evaluate the PGG levels and its effects on genetic diversity and phenotypic variation in the ILs. An estimated population mean of PGG of 12.32% was found in the IL population, consistent with to the theoretical ratio of a random sample population. PCA and NJ tree analysis revealed greater genetic diversity and larger phenotypic variations in ILs with higher PGG levels. Canonical discriminant analysis showed great phenotypic distance between subpopulations, and six agronomic traits  were the main discriminatory characters among the ILs and between the subpopulations. PGG had positive effects on yield per plant, plant height, panicle length, spikelet number per panicle, and negative effects on filled grain percentage, and days to flowering. Additionally, we found an intermediate range (10% − 20%) of PGG was favorable for the incorporation of more beneficial genes and the elimination of harmful genes from Oryza glaberrima. Overall, our results could facilitate the further exploitation of O. glaberrima germplasm to breed more productive and adaptive inbred and hybrid rice varieties.An IL population derived from an interspecific cross between S1 FigPC1, PC2 and PC3 refer to the first, second and third principal components, respectively. The numbers in parentheses refer to the proportion of the variance explained by the corresponding axes. Most ILs (47/48) with lower PGGs (< 10%) were clustered into two groups, Cluster I (37), Cluster II (10), while Cluster III contained 12 ILs with intermediate PGG (10% − 30%). The remaining 46 ILs with intermediate or higher PGG (10% − 40%) were distributed discretely.(TIF)Click here for additional data file.S2 FigMost ILs (43/48) with lower PGG (< 10%) were assigned into two clusters on the phenotypic NJ tree. There were two smaller clusters. One contained ILs (9/10) with PGGs in the range 0% − 20% and another contained 10 ILs with PGGs in the range 10% − 30%. The remaining 38 ILs with PGGs (10% − 40%) were dispersed to different branches.(TIF)Click here for additional data file.S3 Fig(a) PGG in the ILs estimated by using introgression alleles. (b) Comparison of PGG estimated by Bayesian method vs. PGG estimated by using introgression alleles in ILs.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.