A number of studies have shown that the greater the genetic diversity of parental lines, the greater the heterosis effect. Genetic or phenotypic variation can be estimated by genotype testing on the basis of the observations obtained through prediction (a priori) or the observations and studies (a posteriori). The first method uses data such as the genealogy of a given subject and the information about its geographical origin. The second method is based on the phenotypic observation and studies, as well as on the molecular research. The development of molecular genetics and genotype testing methods at the DNA level has made it possible to rapidly assess the genetic variability regardless of the modifying effect of the environment. The aim of this study was to determine the relationship between the degree of relatedness and the DNA polymorphism  of inbred maize lines and the effect of hybrid-form heterosis. Our analysis demonstrated that the parental components for heterosis crosses can be selected on the basis of the genetic similarity determined using the molecular SSR markers and the Jaccard, Kluczyński, Nei, and Rogers coefficients. Molecular AFLP markers proved less useful for selecting the parental components, but may be used to group lines with incomplete origin data. In the case of the RAPD markers, no clear relationship between genetic distance and the heterosis effect was found in this study. The rapid increase in the global production of maize grain is the result of its versatility, and, hence, high demand. Maize kernels can be used for direct consumption or processed into food products and are also a source of starch and oil. Grain is increasingly used for the production of biofuels. High-quality hybrid varieties play a key role in maize farming. Notably, breeding of hybrid varieties often involves the heterosis effect, which generates tangible economic benefits. Currently, maize heterosis breeding is assisted by genomic selection , 2, 3.Heterosis is a genetic term that describes the beneficial effects of crossbreeding—the vigor of the first generation of hybrids. This phenomenon cannot be preserved across generations and, as the definition implies, occurs only in the first hybrid generation, in which the value of a given feature exceeds that in the best parent. To date, the phenomenon of heterosis has not been fully explained, because its symptoms are too complex to be described by one simple stipulation . Accordicis-regulatory elements in maize , 12.5 μL of genomic DNA, 2 μL of EcoRI and MseI (1.25 units/μL) restriction enzymes and 5.5 μL deionized water. The total volume of the reaction mixture was 25 μL per sample. After adding all the reagents, samples were vortexed and incubated for 3 hours at 37°C and for 15 minutes at 70°C, before being placed on ice. We added 24 μL adapter ligation solution  and 1 μL T-4 DNA ligase (1 units/μL) to the 25 μL mixture containing the digested DNA. All components were mixed on a vortex and incubated for 3 hours at 20°C. After incubation, the samples were diluted 1:10 using TE buffer . The total volume of the diluted samples, after ligation, was 100 μL. At this stage, the samples were frozen at -20°C. In the next stage, preamplification was carried out using 5 μL of the diluted DNA matrix, 40 μL pre-amp primer mix, 3.5 μL 10 × PCR buffer, 1.5 μL MgCl2 (0. 25mM), and 1 μL Taq polymerase (5 U/μL). The samples were centrifuged and subjected to PCR in a T3 BIOMETRA thermal cycler  set to a program of 27 cycles of denaturation at 94°C for 30 s, primer annealing at 56°C for 60 s, and amplification at 72°C for 60 s. Then, dilution was performed as follows: 3 μL of pre-amplification mixture and 147 μL of TE buffer were added to the sterilized tubes. Samples were frozen at -20°C. Selective amplification was carried out in the next step. We prepared a reaction mixture consisting of 5 μL diluted pre-amplified DNA matrix, 5 μL Mix 1  and 10 μL Mix 2 . Fifteen specific primer pairs were used to determine the degree of polymorphism between hybrids and their parental components. The samples were placed in a T3 Biometra thermal cycler  set to 2 cycles of denaturation at 94°C for 1 min, primer annealing at 65°C for 1 min, and amplification at 72°C for 2 minutes; followed by 9 cycles with the amplification temperature being lowered by 1°C per cycle; and 35 cycles of denaturation at 94°C for 1 min, primer annealing at 56°C for 1 min, and amplification at 72°C for 2 min. Once the samples were removed from the thermocycler, they were frozen at -20°C. Electrophoresis was performed on a 5% polyacrylamide gel composed of 52.5 g urea; 40 mL deionized water; 6.25 mL TBE buffer ; 15.5 mL 40% acrylamide:bisacrylamide (19:1); 83.75 μL TEMED; 808 μL APS. Electrophoresis was carried out in TBE 0.5 M buffer for 2.5 hours at 2400 V and 400 mA. After electrophoresis, the gel was stained with silver nitrate.The amplified fragment length polymorphism (AFLP) analysis included nineteen inbred lines and were performed for three biological replicates. Three sets from Invitrogen were employed: the AFLP Analysis System I (cat. no. 10544-013), the AFLP Starter Primer Kit (cat. no. 10483-014), and the AFLP Pre-amp Primer Mix (cat. no. 10792-018). The analysis was carried out in accordance with the instructions provided with the sets. In the first stage, genomic DNA was digested using 2.82, 0.0625 μL BSA, 0.625 μL2 mM dNTP, 0.25 μL primer at 5 pmol/μL, 0.1875 μL Taq polymerase at 5 U/μL, and 0.5 μL extracted DNA at 25 ng/μL. Forty random oligonucleotide primers were used to determine the degree of polymorphism between hybrids and their parental components. The thermal cycler was set to the following program: initial denaturation at 94°C for 1 min; then 10 cycles of denaturation at 94°C for 5 s, primer annealing at 37°C for 30 s, and amplification at 72°C for 30 s; followed by 35 cycles of denaturation at 94°C for 5 s, primer annealing at 37°C for 30 s, and amplification at 72°C for 60 s. After PCR, 1 μL of dye—consisting of 0.25% bromophenol blue, 40% sucrose, and deionized water—was added to each sample. The Taq polymerase used in the mixture was purchased from MBI-Fermentas-ABO, whereas the other reagents were purchased from Sigma-Aldrich . Electrophoresis was carried out for 2 h on a 1.5% TBE-agarose gel stained with 1.0 μL ethidium bromide. Electrophoresis was carried out on TBE 1 × buffer at 100 V and 200 mA. To visualize the results, Molecular Imager Gel Doc XR transilluminator and ImageLab Software  were used.The PCR analysis of nineteen inbred lines was performed for three biological replicates; it was carried out in 12.5 μl of a mixture composed of 9.75 μL deionized water, 0.125 μL 1M Tris HCl with pH 8.3, 1.0 μL 25 mM MgCl2.9We compared the genetic diversity of maize hybrids and their parental components using molecular analysis with the GenStat 18 statistical package. The molecular weights of the generated amplification products were recorded in a binary system, with “1” denoting the presence of the product for a given genotype, and “0” denoting its absence. In order to estimate the genetic similarity (GS) of the examined subjects, the following five coefficients were used: Jaccard , KulczyńThe genetic similarity values were determined for all tested pairs using the five coefficients. We determined the correlation coefficients between the genetic similarity values using the individual coefficients . The difThe genetic similarity coefficients were used to hierarchically group the objects using the average linkage method.The relationship between the degree of relatedness and the genetic similarity values calculated using the five coefficients was determined using Pearson’s linear and Spearman’s rank correlation coefficients.33.1Detailed data on the size of heterosis effects were included in the table presented in Tomkowiak et al. , where t3.2The degree of relatedness between hybrid parental components ranged from 0% to 50% . The gre3.3After converting the PCR product sizes of the generated amplification products to the binary system, we obtained 528 AFLP markers, 234 RAPD markers, and 262 SSR markers. Based on the three types of molecular markers used, we constructed the 3.4The genetic similarity between parental components determined using molecular SSR markers  proved to correlate negatively with the size of the heterosis effect in the hybrids for all the examined yield structure traits other than DCO and NKR . This im4Carefully selected inbred lines with very good combining ability are needed to obtain high-yielding, high-quality maize hybrids. The breeding success of all plant species, not just maize, is determined by access to starting materials with the greatest possible variety. When choosing the starting material, the breeder must remember that, for multigenetic traits (such as yield), the genetic progress achieved by artificial selection depends mainly on the additive genetic variation—i.e., on the heritability and the genetic variability of the selected feature. For the selection to be effective, the desired traits must be present in the starting germplasm because selection can only alter the frequency of genes. Dividing the starting material into heterotic groups results in an increased efficiency and reduced breeding costs. In the light of those facts, assessment of the combining ability of inbred lines and the selection of testers—as well as the correct interpretation of the results—emerge as some of the most important breeding issues .Combining ability is generally assessed using experimental methods, but statistical and genetic parameters are also used . These pThe best combining abilities (SCA) in our study were recorded between the S64417 and S61328 lines, which were the parental components of the Narew hybrid, and the S54555 and S79757 lines, constituting the components of the Popis hybrid. In 2013 and 2014 at Łagiewniki, both hybrids showed the greatest significant heterosis effect for most of the yield structure traits and for the yield itself. Notably, the parental components of those hybrids are not related to each other . The genetic similarity, based on the SSR and the AFLP markers for Jaccard, Kulczyński and Nei measures, between the parental components of the Narew hybrid ranged from 0% to 5%, while for the Popis hybrid this varied from 0% to 14%. It can thus be seen that genetic similarity determined using those markers and measures reflects the degree of relatedness of the parental components.Yield is an important trait and, at the same time, a very complex one. The crop is influenced by environmental and genetic factors, physiological processes, as well as many other causes. Knowing the level of relationship between traits, their mutual correlations, and the relationship between the parental generation and its offspring, can help properly select the lines for crossing, thereby allowing an increase in yield . The anaIn this study, Narew, Popis, and Kozak were the most fertile hybrids. Both the Narew and Popis hybrids showed the greatest significant heterosis effect for most of the yield structure traits in 2013 and 2014 at Łagiewniki. At Smolice in 2013, Narew was also the hybrid with the greatest heterosis effect for LC, DC, NRK, MKC, and WTK, whereas in 2014 Kozak turned out the best hybrid. Parental components of these hybrids may serve as valuable material for heterosis crosses.The first attempts to correlate the estimated genetic similarity between parental forms and the heterosis effect were made as early as 1966 . In receBrassica. In 2007, Liersch and Bartkowiak [Brassica. Tomkowiak et al. [Brassica [Brassica. These authors emphasized that genetic similarity derived from molecular markers is not sufficient to predict the effects of heterosis. Radoev et al. [Many researchers have discussed the relationship between genetic similarity and heterosis. In 2003, Betrán et al.  showed artkowiak  showed aBrassica  came to Brassica  for AbysBrassica  for Brasv et al.  and Kramv et al.  believe v et al.  noticed The lack of unambiguous relationships between the genetic distance determined using RAPD markers and the heterosis effect may be due to the fact that this method has the weakness of generating dominant markers and requires preselection of a primer that gives stable and clear electropherograms. The amplification reaction of RAPD markers was found to be highly sensitive to changes in the concentration and source of Taq polymerase, fluctuations in the amount of plant tissue extract, and the magnesium and potassium cation content. Many authors have also pointed out that the greater the genetic dissimilarity of parental lines, the greater the effect of heterosis and that as a result of crossing genetically differentiated lines with greater frequency, the fertile hybrids are obtained , 60, 61.51 hybrids to create heterosis varieties. This is an important problem for the methodology of plant breeding. It is expected that breeding progress can be obtained through studies of the nuclear genomes and of plasmons and their interdependence and interaction with the environment. Our analysis has demonstrated that the initial selection of parental components for heterosis crossing using molecular markers can be carried out using the SSR technique with the Jaccard, Kluczyński, Nei, and Rogers coefficients, as the high polymorphism of SSR markers allows to distinguish the closely related individuals. Molecular AFLP markers have proved less useful in selecting parental components for heterosis crossing, but they may be used to group the lines with incomplete origin data. All this demonstrates the necessity of using other techniques, such as SSR, to select genotypes for heterosis crosses.We still do not know much about the phenomenon of heterosis and its mechanisms, and can only utilize the vigor of F


Echinococcus granulosus sensu lato is a globally prevalent zoonotic parasitic cestode leading to cystic echinococcosis (CE) in both humans and sheep with both medical and financial impacts, whose reduction requires the application of a One Health approach to its control. Regarding the animal health component of this approach, lack of accurate and practical diagnostics in livestock impedes the assessment of disease burden and the implementation and evaluation of control strategies. We use of a Bayesian Latent Class Analysis (LCA) model to estimate ovine CE prevalence in sheep samples from the Río Negro province of Argentina accounting for uncertainty in the diagnostics. We use model outputs to evaluate the performance of a novel recombinant B8/2 antigen B subunit (rEgAgB8/2) indirect enzyme-linked immunosorbent assay (ELISA) for detecting E. granulosus in sheep. Necropsy , western blot (WB) and ELISA diagnostic data were collected from 79 sheep within two Río Negro slaughterhouses, and used to estimate individual infection status (assigned as a latent variable within the model). Using the model outputs, the performance of the novel ELISA at both individual and flock levels was evaluated, respectively, using a receiver operating characteristic (ROC) curve, and simulating a range of sample sizes and prevalence levels within hypothetical flocks. The estimated (mean) prevalence of ovine CE was 27.5% : 13.8%–58.9%) within the sample population. At the individual level, the ELISA had a mean sensitivity and specificity of 55% (95%BCI: 46%–68%) and 68% (95%BCI: 63%–92%), respectively, at an optimal optical density (OD) threshold of 0.378. At the flock level, the ELISA had an 80% probability of correctly classifying infection at an optimal cut-off threshold of 0.496. These results suggest that the novel ELISA could play a useful role as a flock-level diagnostic for CE surveillance in the region, supplementing surveillance activities in the human population and thus strengthening a One Health approach. Importantly, selection of ELISA cut-off threshold values must be tailored according to the epidemiological situation. •Development of a Bayesian LCA model for ovine cystic echinococcosis (CE) in Argentina.•Evaluation of diagnostic performance for a novel rEgAgB8/2 indirect ELISA.•Identification of optimal ELISA thresholds for use as a flock-level surveillance tool.•Improvement of CE surveillance in sheep will help strengthen the One Health approach. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. MGB acknowledges funding from the 10.13039/501100000265Medical Research Council (MRC) Centre for Global Infectious Disease Analysis (MR/R015600/1), jointly funded by the UK MRC and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the 10.13039/501100001713European and Developing Countries Clinical Trials Partnership (EDCTP2) programme supported by the 10.13039/501100000780European Union. The funders had no role in the study design; collection, analysis and interpretation of the data; writing of the report, or in the decision to submit the article for publication.This work received support from the All authors declare no conflicts of interest.


Mitochondrial genome has aseries of characteristics such as simple structure, no recombination, maternalinheritance, stable structure, fast evolution rate, and high copy number. Moreover, it is easy to be sequenced,contains high-resolution phylogenetic information, and exists in a wide rangeof taxa. Therefore, it is widely used in the study of biological phylogeny. Atpresent, phylogenetic studies focus mainly on D-loop region, cytochrome b gene,and protein-coding sequence. Phylogenetic studies using the mitochondrialcomplete sequence are rarely reported in yak. Therefore, the present studyaimed to construct phylogenetic tree using yak mitochondrial complete sequenceand compare the subsequent results with previous findings obtained usingpartial sequences.Complete mitochondrial sequences of five yakpopulations from Qinghai and Xinjiang were obtained. The mitotype diversity ofthe five populations was Xueduo yak (0.992 ± 0.015), Pamir yak (0.990 ± 0.014),Yushu yak (0.963 ± 0.033), Qilian yak (0.948 ± 0.036), and Huanhu yak (0.905 ±0.048), which showed a higher mitotype diversity compared with other breeds fromthe previous reports, including Jiulong yak, Maiwa yak, Zhongdian yak, andTianzhu yak. A total of 78 mitotypes were obtained from 111 individuals. Amongthese, Yushu yak, Huanhu yak, Xueduo yak, and Qilian yak all shared mitotypes,but the Pamir yak did not share mitotypes with these four populations.Phylogenetic analysis showed that yak populations were separable into threedistinct branches. The analysis identified a new phylogenetic branch containingboth wild and domestic yaks. The 155 mitotypes found in 206 individuals weredivided into 3 haplogroups by mitotype clustering. Thehaplogroup was not associated with the geographical distribution of yaks. Theyaks in the same population or the same ecological environment were distributedin different haplogroups. Among the threehaplogroups, haplogroup A and haplogroup B showed a star-shaped distribution ofmitotypes. The central mitotypes were widely distributed and had a highfrequency.Thegenetic diversity of yaks in Qinghai was high. Both domestic and wild yaks clusteredinto three branches.The online version contains supplementary material available at 10.1186/s12864-021-07650-x. Bos grunniens) is a large animal with a compact body, an absence of functioning sweat glands, and a relatively small skin surface area per unit of body weight , are shown in Table Together with the 111 sequences obtained by sequencing, 95 complete mitochondrial genome sequences of yaks that lived in other provinces were downloaded from GenBank  gene sequences [cytb gene is relatively stable in organisms, and its mutation process is slow in mtDNA. Therefore, it is commonly used to reconstruct phylogenetic relationships above the species level. The D-loop region is often used to study the system relationships at the subspecies level because of its rapid variation in mtDNA [cytb gene, D-loop regions, or nuclear genes when evaluating mammalian phylogenetic relationships [As a form of matrilineally inherited genetic material, mtDNA has been widely used to study maternal phylogenetic relationships among mammals , 20. Stuequences . The cytin mtDNA . Completionships –26. The Of the 111 individuals, 78 mitotypes were found, among which Yushu yak, Huanhu yak, Xueduo yak, and Qilian yak all shared some mitotypes each other, but the Pamir share no mitotype with these 4 populations. This might be because the first four populations were geographically close to each other and exchanged genes between their populations, while the Pamir yak was geographically far away from the other four populations, creating geographical isolation. Further, the mitotype specificity of the Pamir yak was caused by the accumulation of mutations during migration from its origin to the Pamir region.Genetic diversity is an integral part of all biological diversity. It is the basis of biological evolution and species differentiation, and is of great significant for population maintenance and reproduction and adaptation to habitat changes. The mitotype diversity and nucleotide diversity are important indicators to measure the degree of genetic variation of the population. The higher the mitotype diversity and nucleotide diversity, the higher the degree of genetic variation of the population. The more the genetic diversity, the more likely it is to adapt to different environments. The mitotype diversity of the five populations was found higher than that of Jiulong yaks, Maiwa yaks, Zhongdian yaks, Tianzhu white yaks, and Huanhu yaks studied by Zhengchao Tu by sequencing the mitochondrial genomes of 111 yaks from 5 populations from Qinghai and Xinjiang . This waPhylogenetic relationship analyses conducted in this study revealed that yak populations were separable into three distinct branches. Compared with the findings of Lai , Guo 2828, and HPrevious studies showed highly differentiated intraspecific genetic branches either from several independent domestication events or from a wild species that diverged in an early stage , 33. AllUsing mitotype clustering, the 155 mitotypes were divided into 3 haplogroups, while Wang  divided In summary, the results revealed that the genetic diversity of yaks in Qinghai was high. Both domestic and wild yaks clustered into three branches. The yaks from Qinghai province, the central producing area of yaks, were selected as the research object, and Pamir yaks from Xinjiang were selected as the comparison group. Blood was collected from yaks not related to each other. The yaks had no sex limitation and were aged between 3 and 8 years. A total of 86 yaks from Qinghai province, including 20 yaks from Yushu , 22 yaks from Qilian , 23 yaks from Xueduo , and 21 yaks from Huanhu , were selected. Further, 25 Pamir yaks from Xinjiang province  were examined. Venous blood samples were collected from all 111 yaks for mtDNA extraction. The collected blood samples were immediately stored in the in-car refrigerator and transported to the laboratory within 24 h. Then, the blood samples were stored at − 80 °C in a refrigerator of the Yak Breeding Engineering Laboratory of Gansu province. The storage number of blood samples was R-5-1-001, and the DNA was extracted within a month later. All procedures involving animals were performed according to the guidelines of the China Council on Animal Care and the Ministry of Agriculture of the People’s Republic of China. The Animal Care and Use Committee of the Lanzhou Institute of Husbandry and Pharmaceutical Sciences Chinese Academy of Agricultural Sciences approved all yak handling procedures (Permit No: SYXK-2014-0002). All blood samples were collected from living yaks, and no yak was sacrificed in this experiment. Before collecting blood samples, the jugular vein of yaks was locally disinfected with alcohol. The blood samples were collected and disinfected with iodine volt to prevent wound infection caused by a needle. In this study, primers designed by Wang  were use2O. The thermocycler settings were as follows: 95 °C for 3 min; 35 cycles of 95 °C for 20 s, 52 °C for 40 s, and 72 °C for 4 min, followed by 72 °C for 10 min. PCR products were detected by 1 % agarose gel electrophoresis. PCR products with appropriate band size were then sent to Xi’an Qinco Biotechnology Co., Ltd  for sequencing.Polymerase chain reaction (PCR) was conducted using a PCR reaction system, with each reaction containing 2 µL of each primer, 25 µL of 2× ProTaq Master Mix, 2 µL of sample DNA, and 19 µL of ddHMAFFT 7.0 was used for sequence alignment , while DAdditional file 1: Table S1. Mitochondrial DNA Haplotype of Qinghai yak and Pamir yak. Table S2. Mitochondrial DNA sequence downloaded from GenBank. Table S3. The haplotype of 206 mitochondrial DNA. Table S4. The accession numbers for mitochondrial DNA.


Autoimmune diseases (ADs) are characterized by a multifactorial etiology, in which genetic and environmental factors are responsible for the loss of immunological tolerance.Although the pathogenic mechanism of ADs is still under investigation, the evidence in favor of a genetic basis for ADs is abundant. The different genetic factors could be associated not only with disease susceptibility but also with specific autoantibodies and disease phenotypes. Knowledge of new aspects of ADs could help us to better understand disease etiology and treatment responses and also contribute to the development of new therapy strategies.The aim of this research topic was to report the most updated views on genetics of autoimmune diseases, with particular attention to organ-specific ones on comorbidities in ADs and their interrelations.This Special Issue put together five original articles, five reviews (four narrative and one systematic) and one very particular case report, summarizing current knowledge on the epidemiology, pathgenesis , clinical long-term experience, clustering and interrelation with other ADs, as well as on the peculiarity of ADs in genetic syndromes. Five very innovative original research studies on genetic basis and predisposition for AD development have been published on this research topic. Cho et al. described two polymorphisms in GPR174 and ITM2A genes on the X chromosome that might be associated with autoimmune thyroid diseases (AITDs) in Korean children [Zou et al. tried to explain that better knowledge of genetic pathomechanisms, using the disease-associated risk gene transcriptional regulation network, could help prevent any therapeutic effect in the ADs . The expThe results of the original study of Almeida et al. suggested a cumulative effect of SNPs at the DHCR7, GC, CYP2R1, and CYP24A1 loci on the susceptibility to type 1 diabetes, due to the roles of these genes in the vitamin D metabolic pathway .In the context of this research topic, there are also five reviews that allowed us to increase our pathogenetic and clinical knowledge based on the wide experience of the authors. Hashimoto’s Thyroiditis and Grave’s Disease in Genetic Syndromes in Pediatric Age by Casto et al. confirmed that AITDs show peculiar phenotypic patterns when they occur in association with some genetic disorders, especially chromosomopathies [The systematic review opathies . Turner,opathies ; Ben-Skoopathies ; Roszkowopathies ; and finopathies .Our Special Issue ends with the description of two siblings, with the same mutations in the AIRE gene associated with two very different phenotypes by Caprino and collaborators . As autoIn conclusion, this research topic provides an important and updated contribution to the subject of autoimmune disease genetics. Several papers highlighted the need for further and prospective studies to clarify certain pathogenetic, clinical, and also therapeutic aspects of ADs.Finally, ADs are confirmed as an active and still growing area of research in pediatric and adult age. Increasing knowledge of genetic aspects and pathogenetic mechanisms will allow to better determine the predisposition to AD development, to diagnose organs and systems involved dysfunction earlier, and to improve treatment using immunogenetic therapy.


Since the 1990s, insurance has been the primary field focused on the social disadvantages of using genetic test results because of the concerns related to adverse selection. Although life insurance is popular in Japan, Japan does not currently have any regulations on the use of genetic information and insurers have largely kept silent for decades. To reveal insurers’ attitudes on the topic, we conducted an anonymous questionnaire survey with 100 insurance company employees and recruited nine interviewees from the survey respondents. We found that genetic discrimination is not generally considered as a topic of human rights. We also found that insurers have uncertain fears and concerns about adverse selection in terms of actuarial fairness but not regarding profits. When it comes to preparing guidelines on the use of genetic information by Japanese insurers, we believe that public dialog and consultation are necessary to gain understanding of the people. Since the 1990s, insurance has been the primary field focused on the social disadvantages of using genetic test results –4 and alBy contrast, while Japan does not currently have any regulations on the use of genetic information for risk assessment purposes, Japanese insurers have explained that they have refrained from using such information for insurance. Under the universal health coverage provided to all citizens since 1961, private Japanese life insurance firms primarily sell death benefit packages to protect the bereaved; these products include whole life insurance, which is sold door to door. Japan has the world’s highest average life insurance value, at about USD 210,000 per household, and 88% of households hold life insurance . Death bAlthough some studies have examined insurers’ views on the use of genetic information , 15, onlIn 2017, some insurers’ policies were revealed to include “taking into account an insured’s heredity” [We questioned the participants to give priority to their concerns on adverse selection. The following concerns were listed in the question, “applicants with the intention of concealing the future illness discovered by a genetic test,” “applicants with the intention of suicide,” “applicants with the intention of murder for insurance claims,” “applicants with the intention of concealing their current physical condition,” “applicants with intention of concealing their current job,” “applicants with the intention of concealing relatives with genetic disorders,” “NA (none of the above),” and the participants were required to rank them as the highest concern, the second highest concern, and the third highest concern. As shown in Fig. Some interviewees were concerned about maintaining actuarial fairness if people with genetic risks were automatically insured. Mr D commented that providing the same coverage to those with higher risks would be unfair as to those with lower risks. Mr I opposed insuring people predisposed to future illnesses predicted by genetic testing while rejecting others based on symptoms of diseases. Only one person (Mr G) commented that insurers should accept people with possible future illnesses revealed by genetic tests to protect human rights.As shown in Fig. This study has some limitations: we cannot generalize the results due to selection bias. Further, we cannot explain why Japanese insurers have not addressed these issues in the past, as our study included only current employees. However, we provide a general overview of Japanese insurers’ attitudes about requesting genetic information from applicants.We found that genetic discrimination is not generally considered as a topic of human rights. We also found that insurers have uncertain fears and concerns about adverse selection in terms of actuarial fairness but not regarding profits. In practice, premium differences based on gender, age, and amount are justified in Japan according to actuarial fairness, while this is prohibited in the EU , 20. It A majority of participants were reluctant to engage the public in open discussions of the issue and would prefer to create internal guidelines for using genetic data. However, we believe that public dialog and consultation are necessary to gain understanding of the people during this process.


European Child and Adolescent Psychiatry on genetic topics reflects this. For example, recent papers focused on patients with a genetic diagnosis (e.g. 22q11.2 deletion syndrome) [European Child and Adolescent Psychiatry editorial pinpointed the integration of genetics into this medical specialty as a major topic within future training of child and adolescent psychiatrists [Genetic information is increasingly part of child and adolescent psychiatry. The number of publications in yndrome) . Furtheryndrome) . A receniatrists , 6.tip of the iceberg. Thus, the expectation was that many more genetic factors underlying ID or mental disorders with and without additional specific clinical symptoms awaited identification, once the technology for a systematic analysis of the genome was available.In the past, genetic testing to confirm or rule out a specific genetic diagnosis was rarely applied in routine clinical child and adolescent psychiatry. If at all, limited genetic testing was offered to patients with intellectual disability (ID) and/or clinical symptoms indicative of a specific underlying genetic syndrome. Examples for the latter include Trisomy 21 (Down syndrome), Rett-syndrome, and fragile (X)-syndrome. It was well understood that these syndromes represent the Over the past ten years, large-scale international science collaborations and major technological advances in the characterization of the human genome facilitated the detailed analysis of the genetic architecture of psychiatric disorders . CurrentThe latter include rare copy number variants  and single base pair changes detected using next-generation sequencing (exome or whole genome sequencing). Genome-wide CNV screening  and/or exome sequencing  are now recommended as part of the diagnostic work-up of patients with ID and/or ASD –11. On adiagnostic odyssey in referrals to potentially multiple clinical specialities trying to make sense of a broad range of symptoms in multiple organs comes to an end [Patients and their parents/caregivers may benefit from receiving a molecular genetic diagnosis in several ways; (i) The o an end . The geno an end . (iv) FiAs child and adolescent psychiatrists, we will be most interested in how a genetic diagnosis underlying ID/ASD might change the psychiatric care of our patients. Particularly for rare genetic disorders, the respective gain in knowledge is currently very limited and oftentimes anecdotal. There are several reasons for this: (i) the discovery of rare, clinically relevant variants with intermediate to strong effect sizes requires very large sample sizes , 8. The The leading example (and currently one of only a few) on how a genetic diagnosis informs psychiatric care, are deletions in the chromosomal region 22q11.2. For more than 30 years, CNVs in this chromosomal region have been the focus of scientific and clinical studies. Deletions in 22q11.2 are a known cause for both ID and ASD . FurtherAs child and adolescent psychiatrists, we will increasingly interact with families of a child diagnosed with a specific genetic disorder and accordingly will be faced with questions in relation to syndrome-specific psychiatric management. It is invaluable to collaborate with self-help groups to come up with appropriate treatments for specific symptoms as for example insomnia (frequent symptom in patients with a genetic disorder). Such treatment schemes may provide guidance even if the empirical evidence is scant. To improve our patient’s care, collecting and sharing information on the psychiatric phenotype and treatment of patients diagnosed with a genetic syndrome is essential.A further aspect requires delineation. Every identified gene/genes relevant to mental disorder sheds light into the disorder’s etiology. Thus, a genetic variant with a strong effect size may entail the elucidation of one or more pathways involved in patients with the same psychiatric diagnosis but without the specific genetic diagnosis. A cross-check of findings obtained in genome-wide association studies within the genetic region of interest may lead to the discovery that common variants (single nucleotide polymorphisms) in the locus of interest contribute to the polygenic architecture of the respective disorder. In conclusion, each genetic finding offers the opportunity to advance our knowledge of how genetic factors can entail the respective phenotype.European Child and Adolescent Psychiatry aims at addressing this need by establishing a specific section for case reports/series of patients with a mental disorder diagnosed with a genetic disorder. The published case reports/series will focus on the mental phenotype of patients with a rare genetic disorder. It needs to include solid data on the mental phenotype based on at least one unpublished patient in combination with a brief review (if available) of all published patients (for details see Genetic case reports/series in instructions for authors (website). We hope that the times are over during which patients were plainly characterized as being hyperactive or as having behavioural abnormalities. We owe it to our patients, their families, and to ourselves to advance this important field by improving the phenotyping of such genetic disorders. There is much to learn. As physicians, we need to incorporate such individualized diagnoses and treatments into our clinical routine.


Zingiber zerumbet at a microgeographical scale and compare the results with data for the closely related selfing Z. corallinum and outcrossing Z. nudicarpum. Such studies could disentangle the phylogenetic and sexually reproducing effect on genetic variation of clonal plants, and thus contribute to an improved understanding in the clonally reproducing effects on genetic diversity and population structure.There has always been controversy over whether clonal plants have lower genetic diversity than plants that reproduce sexually. These conflicts could be attributed to the fact that few studies have taken into account the mating system of sexually reproducing plants and their phylogenetic distance. Moreover, most clonal plants in these previous studies regularly produce sexual progeny. Here, we describe a study examining the levels of genetic diversity and differentiation within and between local populations of fully clonal Z. zerumbet was comparable to that of outcrossing Z. nudicarpum and significantly higher than that of selfing Z. corallinum. However, the level of microgeographic genetic diversity of clonal Z. zerumbet is comparable to that of selfing Z. corallinum and even slightly higher than that of outcrossing Z. nudicarpum. The genetic differentiation among local populations of clonal Z. zerumbet was significantly lower than that of selfing Z. corallinum, but higher than that of outcrossing Z. nudicarpum. A stronger spatial genetic structure appeared within local populations of Z. zerumbet compared with selfing Z. corallinum and outcrossing Z. nudicarpum.The results revealed that the level of local population genetic diversity of clonal Our study shows that fully clonal plants are able not only to maintain a high level of within-population genetic diversity like outcrossing plants, but can also maintain a high level of microgeographic genetic diversity like selfing plant species, probably due to the accumulation of somatic mutations and absence of a capacity for sexual reproduction. We suggest that conservation strategies for the genetic diversity of clonal and selfing plant species should be focused on the protection of all habitat types, especially fragments within ecosystems, while maintenance of large populations is a key to enhance the genetic diversity of outcrossing species.The online version contains supplementary material available at 10.1186/s12862-021-01853-2. For most vascular plant species, there are two distinct modes of reproduction: sexual and asexual, through vegetative reproduction and apomixis , 2. For Acacia carneorum contained multiple genets, which can be attributed to occasional sexual recruitment , where ni is the number of samples of genotype i and N is the total number of samples collected for that local population [GST, an indicator of the degree of differentiation between local populations, was calculated using POPGENE. To assess the values of population genetic differentiation (Φ) and the proportion of total variation among and within local populations, the matrix was also subjected to an analysis of molecular variance (AMOVA) implemented in GenAlEx 6.502 [Nm) between local populations was calculated as Nm = 0.5(1 − GST)/GST [The binary matrix was used to calculate the genetic diversity parameters at local population and microgeographic level using POPGENE v. 1.32 . These pgenotype . Two meaples (N) ; (2) Simpulation . GST, anEx 6.502  based onGST)/GST .Z. zerumbet. Mantel tests implemented in GenAlEx were performed to examine whether genetic distances between local populations were related to the corresponding measures of geographic distance.STRUCTURE v. 2.1  was usedr) calculated according to Smouse and Peakall [I, ranging from -1 to 1. To test the statistical significance of the spatial autocorrelation values, a classic two-tailed 95% confidence interval (CI) was generated and bootstrap resampling was performed 999 times.To investigate genetic relatedness of individuals with respect to spatial position within local populations, single population spatial structure analyses were performed to assess the spatial genetic structure of the studied local populations using GenAlEx. To reduce noisy confidence limits in case of uneven sampling, even sample classes were chosen . The aut Peakall  is similAdditional file 1: Fig S1–S5. Fig. S1. Inflorescence of Zingiber zerumbet, Z. nudicarpum and Z. corallinum. Fig. S2. UPGMA dendrogram based on Dice coefficient for individuals in metapopulations of Zingiber corallinum . The figure utilized in this study is from Huang et al. [ Fig. S3. Unrooted Neighbor-Joining trees based on Nei’s genetic distance for individuals in metapopulations of Zingiber corallinum . The figure utilized in this study is from Huang et al. [ Fig. S4. UPGMA dendrogram based on Dice coefficient for individuals in metapopulations of Zingiber nudicarpum . The figure utilized in this study is from Huang et al. [ Fig. S5. Unrooted Neighbor-Joining trees based on Nei’s genetic distance for individuals in metapopulations of Zingiber nudicarpum . The figure utilized in this study is from Huang et al. [g et al. . Fig. S3g et al. . Fig. S4g et al. . Fig. S5g et al. .


Biscogniauxia mediterranea is an endophytic fungus of healthy cork oak trees that turns into a pathogen in trees weaken by environmental stress. Understanding the drivers of B. mediterranea populations diversity and differentiation is expected to allow a better control of cork oak decline and preserve forest sustainability. Endophyte isolates from different cork oak forests were identified as B. mediterranea and their genetic diversity was evaluated using phylogenetic and microsatellite-primed PCR analyses. Genetic diversity and variability of this fungus was correlated with environmental/phytosanitary conditions present in forests/trees from which isolates were collected. High genetic diversity and variability was found in B. mediterranea populations obtained from different forests, suggesting some degree of isolation by distance. Bioclimate was the most significant effect that explained the genetic variability of B. mediterranea, rather than precipitation or temperature intensities alone or disease symptoms. These findings bring new implications for the changing climate to cork oak forests sustainability, cork production and quality.Cork oak is a tree species with ecological importance that contributes to economic and social development in the Mediterranean region. Cork oak decline is a major concern for forest sustainability and has negative impacts on cork oak growth and production. This event has been increasingly reported in the last decades and seems to be related with climate changes.  Quercus suber L.) is an evergreen oak species with high economic, ecological, and social importance in the Mediterranean region2. This species is mainly distributed throughout the Mediterranean Basin, where is well adapted to the climate. Despite that, global climate changes can induce abiotic stress on trees, impacting negatively cork oak growth and productivity4. Indeed, cork oak decline has been increasingly reported in the last decades and seems to be linked to climate changes, which can increase susceptibility to pathogen attack and facilitate infection by opportunistic pathogens6. Biscogniauxia mediterranea is an opportunistic pathogen with an endophytic lifestyle that has been associated with cork oak decline in the Mediterranean region9. This fungus causes charcoal disease that leads to an extensive inner bark and xylem necrosis, often associated with a blackish exudation on the outer bark11. As the appearance of B. mediterranea-related symptoms are mainly associated with weakened trees , the incidence of this disease increased over the last years and was recently reported on young trees13.Cork oak (14. Early studies on B. mediterranea populations used random amplified polymorphic DNA (RAPD), being able to detect a high genetic variability for geographically close populations15. These results indicated a high rate of sexual reproduction and heterothallic mating system displayed by this species. More recently, high genetic variability was reported within a single stromata using microsatellite-primed PCR primers16. Also, B. mediterranea populations from different Mediterranean countries displayed high genetic diversity 17, but studied isolates were not phylogenetic associated with host species or age, geographic position, or charcoal canker expression. However, the analysis of Tunisian populations of B. mediterranea by using microsatellite-primed PCR primers suggested a positive association of their morphological variability and ecological factors, such as altitude, rainfall, and temperature18. Altogether, these findings pointed to the high plasticity and adaptability of this species to the environment17. In this work, we aimed to understand the correlation of B. mediterranea genetic populations with environmental factors and disease symptoms found on cork oak trees from different bioclimates. For increasing the significance of molecular characterization of B. mediterranea populations we used two different typing analyses, a multilocus (6 loci) and a microsatellite (4 primers) approach. This work will increase the current knowledge about drivers of B. mediterranea genetic variability and contribute for the formulation of appropriate disease management.Molecular markers are widely used to study population genetics of phytopathogenic fungiB. mediterranea endophytic isolates from cork oak trees was performed using a multilocus sequence analysis and microsatellite-primed PCR fingerprinting. From all analyzed loci, partial glutamine synthetase (GS) revealed the highest variability with 19.2% of identical sites , followed by actin , translation elongation factor 1-α , β-tubulin 2 , chitin synthase 1  and internal transcribed spacer . As expected, the nucleotide datasets displayed higher percentage of identical sites without outgroups. The corresponding values ranged from 36.9% (GS) to 94.7% (TUB2). The multilocus alignment was in line with this trend .The molecular characterization of B. mediterranea isolates for any factors of interest . The concatenated dataset comprised sequences from 52 B. mediterranea isolates and from the three outgroup species . From a total of 2874 characters, 1775 were constant, 815 parsimony-uninformative and 284 parsimony-informative. Maximum Likelihood (ML) tree produced clades of B. mediterranea isolates with moderate , high  or very high  support values20, but no evident patterns regarding factors of interest were exposed using this approach  from different forests and bioclimates (from the most humid to the driest), also presenting isolates obtained from trees with different disease severity levels. The same was observed for Clade C , which even included an isolate (Bm79) obtained from an olive tree. Moreover, some information can be inferred from the tree. Group III (with moderate ML support) displayed a higher number of isolates from trees with mild symptoms . Clade B (in Group I) only included isolates  from declining trees, while Clade D (Group III) only included isolates  collected from mild diseased trees, though both clades presented isolates from different forests and bioclimates. Overall, isolates from different locations were distributed along the phylogenetic tree, with few of them being placed together. The same distribution pattern was observed regarding isolates from different bioclimates, even though the sequences in Group VII refer to isolates  obtained from two forests (GV and GR) with the same bioclimate (sub-humid). Also, a clear distribution pattern was not observed concerning the presence of exudates on the B. mediterranea host trees. However, subclade A1 only encompassed isolates  obtained from cork oak trees not producing trunk exudates. The opposite was observed for Clade B  and Group VI , where all isolates were recovered from trees producing exudates, even if from dissimilar bioclimatic locations (sub-humid and humid). In line with the low resolution of B. mediterranea isolates by any of the studied factors , the majority of B. mediterranea isolates collected from the same tree were not clustered together. For instance, although pairwise identity within those isolates was high, isolates from a single tree, such as Bm60, Bm61, Bm63 and Bm67 (pairwise identity of 95.4%) and Bm46, Bm56 and Bm58 (99.6%), were found to cluster better with isolates obtained from other forests and/or were placed apart in the phylogenetic tree. Similarly, isolates collected on olive trees (Bm79 and Bm80) were distantly placed from each other, in separate lineages.Phylogenetic trees of each single locus were not able to individually resolve B. mediterranea isolates generated different banding patterns. Primer (GTG)5 generated 15 bands (from 0.25 to 1.5 kb), (CAG)5 generated 17 bands , (ACAC)5 generated 17 bands (from 0.25 to 1 kb) and M13 generated 26 bands (from 0.15 to 1.5 kb). The monomorphic band was removed and a binary dataset with the remaining (74) band positions was used for molecular analysis. Using this approach, the genetic diversity of B. mediterranea varied within different factors of interest/populations (Table Na) varied from 1.40 to 1.97 and the number of effective alleles (Ne) from 1.45 to 1.57, which were found considering ‘forest’ and ‘exudates’ factors, respectively. These results were corroborated by the genetic diversity found within populations , Nei’s gene diversity (h = 0.33) and percentage of polymorphic loci (PPL = 98.7), while ‘forest’ factor shown the lowest genetic diversity . In addition to ‘exudates’, other disease-related factors also revealed high genetic diversity levels for total populations, in particular when considering ‘cankers’ and ‘disease severity levels’ factors  and AL (humid) were the forests with the lowest diversity. Isolates collected from healthy trees (low disease severity level) revealed the lowest genetic diversity and declining trees were associated with more diverse B. mediterranea isolates. This result is in line with the highest genetic diversity found among B. mediterranea isolates collected from trees producing trunk exudates. However, the opposite was found for trees with trunk cankers, in which higher genetic diversity was associated with isolates from trees without this symptom. Regarding defoliation, the isolates obtained from trees with very accentuated damage were also less genetically diverse, while those from trees with moderate and light damages presented higher genetic diversity.Microsatellite-primed PCR fingerprinting of 68 Gst coefficient (Table Gst = 0.239), followed by ‘defoliation’ (Gst = 0.151), and ‘bioclimate’ (Gst = 0.097) populations. The lowest genetic differentiation was found in ‘cankers’ (Gst = 0.046), ‘disease severity levels’ (Gst = 0.045) and ‘exudates’ (Gst = 0.032) populations. As expected, gene flow (Nm) values were opposite to Gst, being higher among populations with lower genetic differentiation and vice-versa , followed by ‘bioclimate’ (6%), ‘cankers’ (4%) and ‘exudates’ (3%), all at p < 0.001 . For further understanding the relative contribution of forest location, environmental, and disease-related factors in driving the genetic diversity of B. mediterranea populations, a redundancy analysis was performed , being all the others  not significant. The combination of all significant variables explained 9.602% (p = 0.001) of B. mediterranea genetic variance. Most of this variation is due to the ‘bioclimate’ and ‘forest location’ factors that together contribute to 8.831% of variation (p = 0.001), in contrast with the shared contribution of ‘exudates’ and ‘cankers’ variables of only 2.722% of variation (p = 0.001). Indeed, when excluding all other variables, ‘exudates’ and ‘cankers’ do not present a significant contribution for B. mediterranea genetic diversity (p > 0.05). The production of exudates showed higher correlation with ‘bioclimate’ (0.188%) than with ‘forest location’ (0.081%). The opposite was found for the presence of cankers, in which no correlation was found with ‘bioclimate’, but a 0.943% correlation was found for ‘forest location’ variable. The index of association for the estimation of linkage disequilibrium revealed that LI and GV forests, as well as forests from the humid, sub-humid and semi-arid bioclimates was significantly different from 0, which indicates linkage disequilibrium  did not revealed clusters of cork oak isolates, according to the factors of interest . The evaluated isolates comprised those obtained from cork oak trees, thriving in different forests, bioclimates, and displaying distinct charcoal disease severity levels and symptoms. This experimental approach is expected to provide new information about which factors/variables contribute the most to this species diversity and variability. Other isolates obtained from olive trees were also analyzed to evaluate host-specificity. As expected, all studied B. mediterranea isolates were phylogenetically apart from other Biscogniauxia species included in this study as outgroups, revealing the genetic divergence of B. mediterranea species. In general, individual  and concatenated  phylogenetic analyses did not correlate B. mediterranea isolates with any of the studied environmental/disease variables. Also, after determining the microsatellite polymorphic patterns, B. mediterranea isolates did not clustered according to the factors of interest. These results suggest a high genetic diversity and variability of B. mediterranea populations, which is concordant with other reports18, even when isolates come from the same stroma16. Furthermore, the inability to resolve B. mediterranea isolates obtained from cork oak and those obtained from olive tree reinforces current knowledge about the high adaptability of this fungus to different hosts, as suggested by17.In this study, the genetic diversity of endophytic B. mediterranea isolates was mostly explained by ‘forest location’ (6.9%) and ‘bioclimate’ (4.6%). Indeed, when these factors were combined, they explained 8.8% of B. mediterranea genetic variation. Considering ‘forest location’, B. mediterranea populations demonstrated the lowest genetic diversity within population, while also revealing the highest genetic differentiation and lowest gene flow among populations. These results suggest some isolation by distance of B. mediterranea communities, which agrees with the significant correlation (Mantel test) found between the B. mediterranea genetic diversity and forest geographic location of cork oak forests from which they were obtained. This contrasts with previous studies, where B. mediterranea intraspecific polymorphism and genetic diversity were not associated with geographic position of host trees17. However, the contribution of geographic isolation in B. mediterranea genetic differentiation was suggested for cork oak populations in Tunisia18. The high genetic variability of B. mediterranea has been related with the heterothallic mating system of this species15 and sexual reproduction with the production of a high number of variable ascospores21. Our results suggest that global B. mediterranea population displays linkage disequilibrium and a clonal genetic structure. Despite that, populations from the majority of forests  demonstrated to have random mating with frequent sexual reproduction. B. mediterranea ascospores are primarily dispersed by wind after the occurrence of precipitation22, although insects could also play a role for their spreading in short- and long- distances, depending on their bioecology22. Our results suggest low migration rates between geographically distant B. mediterranea populations, indicating short-distance dispersal. The clonal structure of the populations from the majority of bioclimates and the finding that PG-ER and AL forests presented the lowest genetic diversity among all other sampled forests reinforces this suggestion. In contrast with other sampled forests, both are characterized by a high density of mixed forest trees and low anthropogenic interference, which may restrain spore dispersal to and from distant locations. Indeed, canopy architecture and the use of mixed tree species have been reported as a management strategy to reduce spore dispersal of pathogenic fungi25. In addition, high genetic differentiation among forest populations can be a result of random events but there is enough gene flow to refute the effects of genetic drift26. While a significant variation was found among forest populations, high variability of B. mediterranea was also described within populations. This result concurs with the high intraspecific genetic variability found within populations described by Henriques et al.27, indicating an adaptation of B. mediterranea to the environment and ensuring species long-term survival.The genetic variability of B. mediterranea populations occurred in response to ‘bioclimate’, although precipitation and temperature alone were not significantly correlated with B. mediterranea variability, as revealed by variation partitioning redundancy analysis. In contrast, other studies indicated a positive correlation between B. mediterranea genetic diversity with temperature and rainfall18. The ability of B. mediterranea to develop in a wide range of temperatures27, associated with the significant variability of this fungus in different bioclimates may represent a problem for charcoal cork oak disease management. This will be further challenged by the effect of combined bioclimate and charcoal symptoms (exudates in cork oak trunk) in increasing B. mediterranea variability. Indeed, in Tunisia, the correlation between bioclimate and charcoal disease development has been suggested7. In any case, the variability promoted by disease symptoms (exudates and cankers presence) was better explained when taking into consideration the forest location, suggesting that other factors characteristic of each forest  are contributing to B. mediterranea variability. Moreover, B. mediterranea isolates collected from declining trees or trees with charcoal symptoms, like trunk exudates, presented a higher genetic diversity than those collected from healthier trees.Besides ‘forest location’ factor, a significant variation on Quercus suber populations and their associated microbiota are vulnerable to different bioclimates30 and will be affected by the predicted climate changes31. Therefore, as cork oak forests currently displaying a moist bioclimate become more arid, they will be increasingly affected by environmental stressors. Taking all together, the results obtained in this work support the previous suggestions that B. mediterranea isolates have facilitated adaptation. Our findings reinforce the previous knowledge of B. mediterranea opportunistic behavior and reveal the importance of bioclimate as a source of B. mediterranea variability, exacerbating the implications of a changing climate on cork oak forests sustainability which will affect cork production and quality.Several reports suggested that Biscogniauxia mediterranea isolates were obtained from cork oak twigs as endophytes32. Twigs were sampled, during 2017, from eight Portuguese cork oak forests with different locations and bioclimate classifications , humid (AL and LI), sub-humid (GV and GR) and semi-arid (HC-MA and HC-CT)]. If existent, trees with different disease severity levels were collected from each location. Evaluated disease symptoms included defoliation , as well as canopy and trunk damages   medium and obtaining pure cultures through re-plating outgrowing fungi into fresh PDA medium30. DNA of pure cultures was extracted using Quick-DNA Fungal/Bacterial Miniprep Kit  and B. mediterranea isolates were identified using universal primer pairs ITS1-F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)35. A total of 74 B. mediterranea isolates obtained from cork oak were distinguished and identified by sequencing (Table Olea europaea twigs (Bm7836) and olives (Bm79 and Bm8037), collected from cvs. Cobrançosa (Bm78 and Bm80) and Madural (Bm79). These O. europaea-derived isolates were included in this study to evaluate host-specificity in B. mediterranea. All methods complied with relevant institutional, national, and international guidelines and legislation.ons Fig. . Bioclime) Table . DiseaseITS), translation elongation factor 1-α (TEF), partial glutamine synthetase (GS), actin (ACT), chitin synthase 1 (CHS) and β-tubulin 2 (TUB2). PCR amplifications were performed according to each pair of primers and target region  was used to eliminate poorly aligned positions and divergent regions, allowing smaller final blocks38. Geneious version 2010.4.8.5 was then used to concatenate alignments. Some isolates were not sequenced with enough quality in some targeted loci and were excluded from individual alignments before concatenation. DNA sequences from closely related taxa—Biscogniauxia atropunctata, B. nummularia and Xylaria hypoxylon40—were used as outgroups and were retrieved from GenBank , unless stated otherwise. For each isolate and molecular marker, forward and reverse sequences were trimmed (0.05 error probability limit), assembled and consensus sequences were created. Consensus sequences obtained in this study were deposited in GenBank 41 to find best-fit partition schemes of each loci42. Bayesian inference (BI) trees were computed using Markov chain Monte Carlo (MCMC) algorithm in MrBayes version 3.2.743. Models (lset) and prior probability distributions (prset) were set according to PartitionFinder2 results. Two independent runs were performed with one million generations and four chains in each run. The two runs were converged with a burnin of 25% and tree with posterior probabilities was generated. Maximum Likelihood (ML) trees were generated using W-IQ-TREE 44, a web server for IQ-TREE45. Best-fit model was computed using ModelFinder version 1.4.246, with an edge-linked partition model47. Branch support analysis was performed using 1000 ultrafast bootstrap replicates48 and minimum correlation coefficient of 0.99. Phylogenetics trees were visualized in FigTree version 1.4.449. ML bootstrap (BS) and BI posterior probability (PP) values and topologies obtained by both phylogenetic inference methods were compared using TreeGraph 2 version 2.0.4050. This allowed the distinction of clades with reliable support value20 or groups of sequences showing limited support. Additional data from environmental/disease factors of interest were added using the vector image editor Inkscape version 0.9251.Phylogenetic trees for each individual locus were generated using sequences from 74 B. mediterranea isolates was also evaluated by using microsatellite-primed PCR (MSP-PCR) with four sets of primers, (GTG)5, (CAG)5, (ACAC)5 and M13 18. PCR reactions were prepared in 10 µl volume using 0.2 U/µl of NZYTaq II 2× Green Master Mix , 1 µM of each primer and 1 µl of DNA. The amplifications with (GTG)5 were performed using the following PCR program: initial denaturation at 94 °C, for 4 min; 35 cycles of 45 s at 94 °C, 45 s at 56 °C and 30 s at 72 °C; and final elongation at 72 °C for 10 min. Amplifications with (CAG)5, (ACAC)5 and M13 primers comprised the following PCR program: initial denaturation at 94 °C, for 2 min; 40 cycles of 30 s at 93 °C, 60 s at 53 °C and 30 s at 72 °C; and final elongation at 72 °C for 10 min. Amplifications with each primer were performed in duplicate for reproducibility. PCR products were run on a 1.5% (w/v) agarose gel, stained with Green Safe Premium . The visualization and image acquisition were performed using an UV transilluminator . DNA fingerprinting gel images were analyzed using GelAnalyzer version 19.1  and each band was scored as 0 (absence) or 1 (presence).The molecular diversity of 68 B. mediterranea isolates was evaluated by grouping samples into populations, based on their host origin, such as climate parameters (including bioclimate), forest provenience, or disease-related parameters . Frequency- and distance-based genetic diversity of each population was evaluated using GenAlEX version 6.51b252. Frequency-based genetic diversity was evaluated considering the number of different alleles (Na), number of effective alleles (Ne), Shannon’s Information index (I), diversity [h = 1 − (p2 + q2)] and percentage of polymorphic loci (PPL). Distance-based genetic diversity was accessed using different analyses: Principal Coordinates Analysis (PCoA) to find patterns within dataset; Nei’s pairwise genetic distance to calculate genetic distance between populations; Analysis of Molecular Variance (AMOVA) to calculate hierarchical partitioning of genetic variation within and among populations (999 permutations); and Mantel test to calculate statistical correlation between genetic diversity and geographic distance (999 permutations). F-statistics to analyze diversity and genetic differentiation among populations were calculated in POPGENE version 1.3253. Given the reduced sample size, B. mediterranea isolates collected from olive tree  were included only in phylogenetic and PCoA analyses.Molecular diversity of B. mediterranea composition to environmental  and disease variables , by making use of the R version 4.0.254. Analyses were performed using the package vegan version 2.5-755, except when stated otherwise. Spatial trend was included in RDA using a trend surface analysis. Latitude–longitude data was transformed into flat Cartesian coordinates using geoXY of SoDA package (version 1.0-6.1)56. To compute polynomials of degree 3, poly of STATS package (version 4.0.2) was used. Spatial and environmental variables were analyzed separately on a first approach. RDA was performed using rda, while anova.cca was used to perform Monte Carlo permutation test (1000 permutations) and test significance of global model. Forward selection of explanatory variables was performed using ordistep with 999 permutations, using spatial and environmental variables to find most parsimonious RDA. Then, another RDA was performed, as described before, for selected variables. The contribution to variation of those variables was performed using RsquareAdj and Monte Carlo permutation test (1000 permutations) to determine significance. For explaining the variation on B. mediterranea composition, variation partitioning was performed for the best model, using the most explanatory variables , and making use of varpart. Partial-RDA was performed to evaluate the influence of conditional variables obtained from variation partition. Statistical significance was performed as described for RDA. Spatial variable was referred as ‘forest location’ variable in sections related with this analysis. Linkage disequilibrium of B. mediterranea populations was evaluated by the index of association (IA) and the unbiased index of association (d), in which clonal populations have results significantly different from 0 and sexual populations do not have statistical significant IA and d61. Analysis was performed using popsub to specify population and ia with 999 permutations for calculation using poppr package (version 2.9.0)62.Redundancy analysis (RDA) was used to explore responses of Supplementary Information.


Vespa velutina nigrithorax) is an invasive species in South Korea with negative economic, ecological, and public health impacts. We investigated genetic and morphological variation in the species populations on Mt. Jiri, the tallest mountain in South Korea. We hypothesized that a high-altitude would be negatively correlated with the genetic diversity of the hornet population, and hornet wing morphology would change with an increase in altitude. Our results showed that the genetic diversity of yellow-legged hornets did not decrease as altitude increased. Regardless of the altitude, the inbreeding coefficient was high at the newly colonized sites. A single genetic population occurred in the mountainous areas examined and gradually expanded its range. Wing morphology, especially shape, did not change with an increase in altitude or decrease in temperature. Although snow cover and cool temperatures at high altitudes could limit nest-building activities, they did not prevent the extension of the range of the species. Therefore, the yellow-legged hornet cannot be controlled naturally by climate or topography; combined approaches, including chemical control, nest removal, and bait-trapping techniques should be implemented.The yellow-legged hornet ( Vespa velutina nigrithorax), native to China, was introduced in South Korea in 20031 and it has rapidly colonized a large part of the country2. The spread of V. velutina nigrithorax has had negative economic, ecological, and public health effects5. However, approaches to control the species are still lacking and need to be studied continuously6.The yellow-legged hornet  across seven loci and 39 yellow-legged hornet samples was 2.846 (2.49–3.286); the overall number of effective alleles (NE) was 2.444 (2.188–2.896); overall observed heterozygosity (HO) was 0.718 (0.524–0.857); overall expected heterozygosity (HE) was 0.667 (0.468–0.619); overall Shannon’s information index (I) was 0.920 (0.758–1.069); overall inbreeding coefficient relative to the population was 0.292 (− 0.529 to 0.121); and overall molecular diversity indices  was 0.639 (0.562–0.743). In addition, overall inbreeding coefficient to the population was − 0.292  based on geographical distances among yellow-legged hornets from the 13 sampling sites  among the hornets from the 13 sampling sites  than wing size in G10. There were no significant differences in wing size among the other groups.Centroid size, which is a proxy for wing size, was significantly different , F = 2.304, p < 0.0001) and CV2  were significantly different among yellow-legged hornets from the 13 groups. Hornets in groups G3, G5, G8, and G9 were distinct  from all the other groups based on CV1 , hornets of groups at seven sites  were separated from those of other groups  results revealed two major morphological variations among the hornets from 13 sampling sites based on the two major axes CV1 and CV2 Fig. a, b. CV1p < 0.05) from that in G8. Wing shape in G10 was significantly distant (p < 0.05) from that in G11 and wing shape in G13 was significantly distant (p < 0.05) from those in G7 and G8.The morphological distance is expressed based on Procrustes distance from CVA Table . Unlike 2. In the present study, adult hornets were collected at altitudes as high as 1100 m a.s.l. in mountainous areas. In Italy, adult hornets have been found at altitudes of 1200 m a.s.l, however, most nests of the yellow-legged hornet have been reported within 700 m a. s. l.15. The yellow-legged hornet can be observed at such high altitudes because its colony foraging radius is greater than 700 m16. Furthermore, in autumn, when foraging activity is the greatest, workers are found at sites relatively far from their nests17. Although snow cover and cool temperatures at high altitudes could limit hornet nest-building activities, Mt. Jiri, the highest mountain in South Korea, has not prevented the extension of the range of the yellow-legged hornet.The yellow-legged hornet is expanding its range in mountainous areas, regardless of altitude18. The genetic bottlenecks were not drastic enough to limit the expansion of the ranges of the yellow-legged hornet into mountainous areas. In addition, the hornets are likely to experience temporary genetic bottlenecks at the local level when expanding their colonies annually. In South Korea, there is a debate on whether the yellow-legged hornet invaded from a single or several sites19. In our study, although many hornets and nests were observed at relatively lower altitudes, their genetic structure did not show altitude-specific patterns. The yellow-legged hornet on Mt. Jiri appeared as a single population. The single genetic population is spreading at a slightly slower rate than in other countries, without being restricted by mountainous areas as geographical barriers.In this study, yellow-legged hornet genetic diversity did not decrease significantly with an increase in elevation. Yellow-legged hornet only exhibited a high inbreeding coefficient at a specific site. The high level of inbreeding is consistent with the genetic bottleneck experienced by hornets following their introduction in South Korea20. A decrease in temperature due to an increase in altitude does not seem to be sufficient to alter wing shape. However, our study might be limited due to insufficient number of samples at high altitudes. We conducted permutation tests to resolve this limitation. Nevertheless, no altitude-specific pattern of morphological variation was observed. Our results demonstrated genetic and morphological adaptations specific to altitude in the hornets of Mt. Jiri; however, an extension of these results may be dangerous.Yellow-legged hornets that expand their ranges across or into mountainous areas may have no morphological adaptations. Although intraspecific minor variability in wing morphology may exist in the population investigated in the present study, wing morphology was not correlated with altitude. Additionally, yellow-legged hornets prefer cooler highlands and mountains in their original habitatsVespa simillima4. However, V. simillima, which is less aggressive than the yellow-legged hornet, could have been forced out of the mountainous area, and the hornet occupied its place mountainous areas in South Korea4. Typically, nest removal and bait-trapping are used to control the yellow-legged hornet. However, eradicating and controlling hornets that occupy large mountainous areas using such techniques is challenging and impractical21. Therefore, chemical control techniques, including the use of insect growth regulators that have not been considered so far, could be adopted in mountainous areas, which is a core habitat of the yellow-legged hornet in South Korea.In conclusion, South Korea does not have a climate or topography that can naturally prevent the spread of wasps. In the early stages of the invasion of the yellow-legged hornet, the spread and establishment of the hornet in mountainous habitats was hampered by competition with an ecologically similar hornet, We collected yellow-legged hornets using traps at Mt. Jiri National Park in September 2019 when the colony was at its maximum size and the production of new queens resulted in high demand for hornet workers. At a lower altitude (approximately 200–800 m) and a higher altitude (approximately 800–1200 m), a total of 20 and 3 hornet nests were identified, respectively. Sugared water with acetic acid and 95% ethanol was used as the trap solution in 2 L plastic containers. The traps were monitored every two weeks. The collected hornets were preserved in 95% ethanol for use in later molecular and morphological analyses. Same individuals were used in both genetic and morphological analysis, for tracking response of individual units in two types of variations. Traps were placed in 30 sites and hornets were captured from 13 sites Fig. , Table 322 for the yellow-legged hornet . We performed amplifications using the procedures for each locus detailed in22. Seq-Studio Genetic Analyzer  was used to visualize the amplicons, and GeneMapper version 6.0 was used to evaluate the dataset for genotype errors and the presence of null alleles.We collected muscle samples from the hind legs of each worker for genomic DNA extraction. DNeasy Blood and Tissue kits  were used to extract genomic DNA according to the manufacturer’s protocol. We measured the concentrations and quality of genomic DNA using a NanoDrop 2000 spectrophotometer , and the concentrations of genomic DNA were diluted between 10 and 20 ng/µL range. Seven polymorphic microsatellite loci were amplified using primers developed by23 was used to confirm deviations in Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium of seven microsatellite loci. Null alleles, significant deviations from HWE, or evidence of linkage disequilibrium were not observed in any of the seven loci. In addition, seven loci could sufficiently separate 39 samples of yellow-legged hornets , effective number of alleles (NE), observed heterozygosity (HO), expected heterozygosity (HE), Shannon’s information index (I), molecular diversity (h), and inbreeding coefficient relative to the sub-population (FIS) of each population. The UPGMA based on Euclidean distances was used to construct a dendrogram based on Nei’s genetic distances obtained using GenAlEx version 6.5, using PAST 326. Paired population differentiation (FST) was performed using Arlequin version 3.5. The linearized FST value (FST/[1 − FST]) with geographic distance was used in the Mantel test (number of permutations: 999) to find evidence of genetic isolation by distance from the hornet using the ‘vegan’ package in R27.The MS Excel add-in, GenAlEx version 6.528. STRUCTURE is an admixture model, which allows us to confirm whether ancestors in population k have passed a portion of their genetic material to individual i. Simulations  were performed in each analysis after an initial burn-in of 100,000 simulations. We used the ΔK method29 in STRUCTURE Harvester30, which supported the estimation of the best‐identified k value. The range of one to 14 possible clusters with three independent runs each was employed in STRUCTURE Harvester. In the STRUCTURE harvest analysis results, the best-supported K value among hornets across 13 populations was identified as 7  of each canonical variate (CV) in CVA. Variation in wing shape among the hornets from 13 sites was visualized in a wireframe graph along the first two (CV1 and CV2) axes. We also identified the values and significant distances of the Procrustes from CVA to explore the morphological similarity between groups. Significant morphological distances were obtained using CVA after 10,000 permutation rounds. One-way ANOVA was used to test significant differences in centroid size, CV1, and CV2, among the hornets using GraphPad Prism version 7.0 for Windows . When significant differences were found in the ANOVA test, Tukey’s post hoc tests were performed. All differences were considered significant at p < 0.05.Landmark-based geometric morphometrics were used to analyze the wing shapes of the hornets. TpsDig


Verticillium dahliae is a fungal pathogen that causes Verticillium wilt on many economically important crops including lettuce. We assessed the dynamics of changes in the V. dahliae genome under selection in a long-term field experiment.Plant pathogens and their hosts undergo adaptive changes in managed agricultural ecosystems, by overcoming host resistance, but the underlying genetic adaptations are difficult to determine in natural settings. Verticillium dahliae race 1 strain (VdLs.16) was introduced. A derivative 145-strain population was collected over a 6-year period from this field in which a seggregating population of lettuce derived from Vr1/vr1 parents were evaluated. We de novo sequenced the parental genome of VdLs.16 strain and resequenced the derivative strains to analyze the genetic variations that accumulate over time in the field cropped with lettuce. Population genomics analyses identified 2769 single-nucleotide polymorphisms (SNPs) and 750 insertion/deletions (In-Dels) in the 145 isolates compared with the parental genome. Sequence divergence was identified in the coding sequence regions of 378 genes and in the putative promoter regions of 604 genes. Five-hundred and nine SNPs/In-Dels were identified as fixed. The SNPs and In-Dels were significantly enriched in the transposon-rich, gene-sparse regions, and in those genes with functional roles in signaling and transcriptional regulation.In this study, a field was fumigated before the V. dahliae evolves at a whole genome scale to accumulate SNPs/In-Dels nonrandomly in hypervariable regions that encode components of signal transduction and transcriptional regulation.Under the managed ecosystem continuously cropped to lettuce, the local adaptation of The online version contains supplementary material available at 10.1186/s12915-021-01061-w. Evolution of pathogens occurs by local adaptation in both natural and agricultural ecosystems . In the Plasmopara viticola [Zymoseptoria tritici populations collected from warm environments showed faster growth than the populations collected from cold environments [A diversity of factors in agricultural ecosystems also play important roles in local adaptation . For insviticola . Similarviticola . For insronments , 8. Therronments .vs resistance gene) theory [Rlm1 and Stb4 into Brassica napus cultivars resulted in Leptosphaeria maculans avirulence genes evolving in agricultural systems. However, in most cases, the local adaptation of pathogens is largely determined by genes other than a member of the gene-for-gene interactions governing plant pathogen-host interactions. Examples include Rhynchosporium commune [Zymoseptoria tritici [Parastagonospora nodorum [Rhynchosporium commune, regulate functions related to biotic and abiotic stress responses, in contrast to the prevailing view that a small number of gene-for-gene interactions governing plant pathogen evolution [Aspergillus niger [Cochliobolus carbonum [Magnaporthe oryzae [M. oryzae [Alternaria alternata [Adaptations of pathogens in the agricultural ecosystem may also include those involving effector genes which are under strong positive selection that may be highly detrimental or beneficial for the pathogen . To a la) theory . For ins tritici , and Par nodorum . The arm nodorum . For insvolution . As an evolution . TEs serus niger , Cochliocarbonum , and Mage oryzae . Such ele oryzae –21. TE-m. oryzae  or loss lternata .Verticillium dahliae is an asexually reproducing, soilborne fungus that causes vascular wilt in over 200 plant species, including many economically important crops [V. dahliae is capable of infecting epidermal cells for limited proliferation [Verticillium dahliae produces an exoproteome that plays important roles during colonization and proliferation in the vascular system, disease establishment, and symptom development [nt crops –26. The nt crops , 28. Thent crops . Even whferation , 32. Verelopment , 33, 34.Vr1 is the resistance locus against the avirulence gene Ave1 of race 1 in V. dahliae [Vr1/vr1 parents. The results offered no evidence for altered mating-type, virulence loci, or phenotypes in this managed agricultural ecosystem [V. dahliae in fields continuously planted with resistant lettuce within a managed agricultural ecosystem, is unknown.Lettuce  dahliae . In a prcosystem . Howevercosystem –39 in thV. dahliae VdLs.16 strain and 145 isolates derived from this strain that were collected over 6 years in a lettuce disease nursery [V. dahliae exploring genome-wide single-nucleotide polymorphisms (SNPs), insertions-deletions (In-Dels), and other trackable genetic variations; and (3) to identify genes under selective pressure and ongoing genetic fixation in V. dahliae in a managed agricultural ecosystem.The overall aim of this study was to investigate the occurrence of genome-wide variations in a field population of  nursery . To accoAve1 and Av2 that characterize race 1 and 2, respectively) [V. dahliae genomes. Nearly 30% of the TEs (1.15 Mb) were of the LTR/Gypsy type  and VdLs.17  , 41. Thectively) , was assV. dahliae, a derivative VdLs.16 population was collected from symptomatic roots samples from the field that evaluated a resistant (carrying resistance gene Vr1 against Ave1 of race 1 strain) × susceptible (vr1 genotype) segregating population of lettuce during 2010–2015 [Ave1), non-defoliating phenotype, and mating-type 2 (MAT1-2)  and insertions-deletions (In-Dels) were compared by mapping the high coverage and depth of sequenced reads to the VdLs.16 reference genome. In total, only 3519 genetic variations were called from the VdLs.16 population, including 2769 SNPs with four main mutation types  and 750 In-Dels   and changes in the predicted gene promoter regions (800 bp of 5′ sequence) in response to local adaptation. There were 386 nonsynonymous SNPs and 52 In-Dels in the coding sequences  , kinase activity , regulation of macromolecule metabolic processes  (vs 0.97/10 kb) and increased transposon sequences (2.99/10 kb vs 1.08/10 kb), but fewer genes (2.99/10 kb vs 1.93/10 kb) in the RIV sequences . The distribution of genetic variation present in the VdLs.16 genome was dispersed into 20 regions of increased variation (RIVs) in windows containing more than 30 SNPs and In-Dels Fig. . While tV. dahliae genome.Analysis of the RIVs along with the 100-kb flanking sequences showed significantly enriched LTR/Gypsy type of TEs Fig. . BecauseP < 0.05), including DNA-binding transcription factor activity , regulation of biological process , and regulation of cellular process   and the variation co-present in more than 143 strains (two variations), 509 variants  were identified as fixed genetic variations in the VdLs.16 population  that ensured high mapping depth  that consist of few coding genes and numerous transposons may also reduce the functional gene divergence and probably facilitate purges of mutations introduced by transposable elements. Therefore, our studies suggested that as an asexual pathogen, V. dahliae also significantly accumulates genetic variations in an agricultural ecosystem, and these are linked with transposable elements.TEs with high mutagenic potential serve as an active site of evolution in several pathogens , 17 and ariation , 44, whiariation , as some dahliae  and Neura crassa . Further dahliae . TherefoV. dahliae populations challenged in the managed agricultural ecosystem were significantly enriched in the regulatory factors, especially the transcription factors and protein kinases  regions in the genome relative to the core genomic sequence [V. dahliae, with greater numbers in VdLs.16  were defined as genes under selection pressure  nonrandomly in hypervariable regions associated with transposon enrichment in the managed agricultural ecosystems. The increased variation of genes encoding products obviously involved in signaling and transcriptional regulation may play important roles in local adaptation in V. dahliae in managed agricultural ecosystems.In conclusion, our study showed that the strictly asexual pathogen V. dahliae race 1 isolate, VdLs.16 [V. dahliae infestation was fumigated using methyl bromide (67%) and chloropicrin (33%) mixture at 361 kg/ha in 2009 before nursery establishment. Following fumigation, the absence of residual V. dahliae inoculum in the fumigated field was confirmed from soil assays [vr1) were first grown in a greenhouse in germination trays filled with sterilized potting mix in the spring and fall seasons of 2009 and 2010. The 14-day-old seedlings were inoculated in trays with 3 mL of 1 × 107 conidia/mL suspension of isolate VdLs.16  per plug and repeated twice within a week. Following the last inoculation, seedlings were grown for two additional weeks in the greenhouse and moved outside the greenhouse for acclimatization for 10 days before transplanting. Inoculated seedlings were transplanted into the field at a spacing of approximately 28 cm within and between two seed lines on each bed. Plants were grown to maturity and incorporated into the soil by disking. This process of planting inoculated lettuce seedlings, growing the crop to maturity, and incorporating the residue elevated the soil inoculum levels to > 100 microsclerotia g−1 soil. This high inoculum density was maintained throughout the study years 2010–2015 by planting susceptible lines each season interspersed with the plants with race 1 resistance.All isolates used in this study were obtained from a Verticillium wilt disease nursery at the USDA Research Station in Salinas, California. The disease nursery was established with the  VdLs.16 . Brieflyl assays . LettuceVr1) and other cultivars of the iceberg, romaine, and leaf type lettuce were screened in approximately 45% of the test plots in the nursery. Plant introductions with resistance to race 1 or partial resistance to race 2 isolates in a greenhouse were planted in another 5% of test plots. The remainder of the plots were occupied by susceptible germplasm. Planting patterns were completely randomized each year.Between 2010 and 2015, an early and intermediate generation of families and inbred lines derived from a cross between La Brillante  root tissue on semi-selective NP-10 medium  enrichedus study . The purTM Fugnal/Bacterial Midiprep Kit  according to the manufacturer’s protocol.From the cultures of actively growing isolates on PDA, two to three 3-mm-diameter plugs were transferred into 50 mL potato dextrose broth (PDB) and grown for 1 week on laboratory benches at room temperature (23 ± 2 °C). The fungal mycelium was harvested after 1 week, washed, lyophilized, and processed for total genomic DNA extraction using FastDNA Kit . Total DNA was quantified, diluted, and used for downstream processing. The high-quality genomic DNA was extracted using the Quick-DNAIsolate VdLs.16 collected from a commercial lettuce field in Watsonville, CA, in 1996, was used for de novo genome sequencing. The library construction, sequencing, reads filtering, and assembly procedure of the VdLs.16 reference genome was performed as described in our previous study . For reshttp://www.phi-base.org) [Protein-coding genes in the VdLs.16 genome, and the gapless reference genome of VdLs.17 (GenBank assembly accession: GCA_000952015.1) and JR2 GCA_000400815.2) [0815.2 4. The secase.org) . The PKsase.org) . The trahttp://www.repeatmasker.org). The output files were summarized using a custom Perl script.Transposable elements were identified by RepeatMasker  and RepeatProteinMask   . SNPs weTo identify In-Dels within the sequenced isolates, Illumina clean reads for the VdLs.16 population were aligned to the VdLs.16 reference assembly using a BWA program (-o 1 -e 63 -i 90 -L -k 2 -l 31 -t 4 -q 10) . In-DelsAve1 in the VdLs.16-derived population was determined by mapping the reads of the VdLs.16 progeny population to the Ave1 gene with 1-kb flanking sequence, and the depth of each base was calculated and standardized by the base with highest depth (set as 100%).The sequence divergence of To determine the accuracy of genetic variations in the resequenced whole-genome data, 24 genetic variations in individual strains were randomly selected for PCR amplification using primers designed to produce a 400–600-bp amplicon  suspension was spread on the PDA medium and cultured for three additional days. By 1 week, the plates with lettuce seedlings were covered by V. dahliae growth and served as the induced strain. The induced strains were collected at 36 h and 60 h, respectively. The uninduced VdLs.16 strain was used as the control. The AxyPreP Multisource Total RNA Miniprep Kit  was used to isolate total RNA, and first-strand cDNA was synthesized using a TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix Kit according to the manufacturer’s instructions . Reverse transcription-quantitative PCR (RT-qPCR) was performed under the following conditions: an initial 95 °C denaturation step for 5 min, followed by denaturation for 30 s at 95 °C, annealing for 30 s at 60 °C and extension for 30 s at 72 °C for 40 cycles. The V. dahliae elongation factor 1α (EF-1α) was used as the endogenous reference. Relative transcripts levels of genes were determined using the 2−∆∆CT method [t tests were performed to determine statistical significance at P < 0.01 to make pairwise comparison of treatments.Sterile lettuce seedlings were grown in tissue culture dishes with roseite as the substrate in liquid Murashige and Skoog medium at 25 °C in a greenhouse under an alternating 16-h light and 8-h dark regime. VdLs.16 strain was cultured on potato dextrose agar (PDA) medium and then was transferred to liquid Cazepk medium and incubated for 4 days at 25 °C. A 50 μL conidial  SNPs, In-Dels, and fixed variations in the genomic sequences of the VdLs.16 population. (B) Numbers of genes with genetic variations in their coding sequences and gene flanking sequences. (C) Venn diagram characterization of the numbers of genes with fixed genetic variations in gene coding and flanking sequences. Supplementary Figure S2. Identification of the genetic variation at the Ave1 locus of Verticillium dahliae. The reads of the VdLs.16 progeny population were mapped to the Ave1 gene with 1-kb flanking sequence, and the depth of each base was calculated and standardized by the base with highest depth (set as 100). The red square box represents the region of the Ave1 gene. Supplementary Figure S3. Population genomics comparison between the regions showing enriched variation in the VdLs.16 genome. (A) Genetic variations in regions compared to the VdLs.16 population. (B) The density of genetic variations and genome annotation contents in the regions of the VdLs.16 genome with increased genetic variation (RIVs). The density of variations was calculated by the average in 10 kb windows by the total number of variations in the full genome sequence. Supplementary Figure S4. Genes encoded in the regions of increased genetic variation within the VdLs.16 genome. The significant GO catalogs of genes encoded in the regions of increased genetic variation were selected by the Pearson Chi-Square test (P < 0.05). Supplementary Figure S5. Distribution of LTR/Gypsy transposons and gene enrichment in the regions of increased genetic variations in strain VdLs.16 of Verticillium dahliae. (A) Enrichment of the LTR/Gypsy transposons in the regions of increased variation. The enrichment of the LTR/Gypsy transposons was assessed by the number of LTR/Gypsy transposons per sequence length of 10 kb. The density of coding genes and genes under selection was assessed (B) within the regions and (C), within the regions plus 100-kb flanking sequence. Supplementary Figure S6. Distribution of the fixed genetic variations in the VdLs.16 population of Verticillium dahliae. The orange color represents the strains with the genetic variations compared with the VdLs.16 reference genome. Supplementary Figure S7. Annotation of the genes from strain VdLs.16 of Verticillium dahliae under selection in starch and sucrose metabolic pathways. The pink box represents the genes under selection mapped to the pathway by KEGG database annotation. Supplementary Figure S8. Expression levels of genes with fixed genetic variations under induction by lettuce. (A) and (B) The PDA plates with one-week-old lettuce seedlings were inoculated with a 50 μL conidial (5 × 106 conidia/mL) from the VdLs.16 strain. The strain was harvested at 36 h and 60 h after co-cultivation with lettuce seedlings. RT-qPCR was performed to determine the expression levels of randomly selected genes (encoding transcription factors and protein kinases) with fixed genetic variations, relative to V. dahliae elongation factor 1-α (EF-1α). Error bars represent standard errors and asterisks represent statistical significance at P < 0.01 (**) and P < 0.001 (***), respectively, based on unpaired Student’s t-tests. Supplementary Figure S9. PCR detection of the accuracy of genetic variations determined by resequencing. (A) 24 genetic variations determined in individual strain were selected randomly for PCR detection. (B) Determined the genetic variation by sequence alignment of sequencing of PCR amplicon with the refine genome sequence. Red triangle represents the genetic variation (C) Gel of PCR products amplified from 24 individual samples.Additional file 2: Supplementary Table S1. Clean data of VdLs.16 genome sequenced by PacBio RS II and Illumina technologies. Supplementary Table S2. Prediction of the transposons in the sequenced genomes. Supplementary Table S3. The CAZymes in VdLs.16 genome. Supplementary Table S4. The CAZymes sub-families in VdLs.16 genome. Supplementary Table S5. The CAZymes in VdLs.16 genome. Supplementary Table S6. The secreted CAZymes sub-families in VdLs.16 genome. Supplementary Table S7. The protein kinase subfamilies VdLs.16 genome. Supplementary Table S8. The PHI homologs in VdLs.16 genome. Supplementary Table S9. The transcription factors in the VdLs.16 genome. Supplementary Table S10. Information of VdLs16 population collected from disease nursey. Supplementary Table S11. Sequencing data of the VdLs.16 population. Supplementary Table S12. Coverage and depth of the resequencing VdLs.16 population. Supplementary Table S13. Determination of the genotype of VdLs.16 population by gene markers. Supplementary Table S14. Statistics of the SNP variations in VdLs.16 population. Supplementary Table S15. Statistics of the insertion-deletion variations in VdLs.16 population. Supplementary Table S16. Identification of the genetic variations in VdLs.16 population. Supplementary Table S17. Functional annotation of the genetic variations in VdLs.16 population. Supplementary Table S18. Information of the genetic variation regions under selective in VdLs.16 genome. Supplementary Table S19. Statistics of the genetic variations density between genome and genetic variation regions. Supplementary Table S20. Statistics of genes under selection in the regions of increased genetic variation. Supplementary Table S21. Pathway annotation by KEGG database with Verticillium dahliae model. Supplementary Table S22. Primers used in this study.