Prunus avium L.) trees of the German Fruit Genebank were evaluated according to their trueness-to-type. For this purpose, pomological analysis was performed, in which the accessions were assigned totheir historical cultivar names. The pomological identifications were based on several historical reference sources, such as fruit references from historical cherry cultivar and fruit-stone collections, as well as historical pomological literature sources. In addition, the cherry trees were genetically analyzed for cultivar identity using 16 SSR markers. Based on pomological characterization and genetic analysis for the majority of the trees (86%), cultivar authenticity could be confirmed. Most markers were highly discriminating and powerful for cultivar identification. The cherry collection showed a high degree of genetic diversity, with an expected heterozygosity He = 0.67. Generally, high genetic admixture between cultivars of different geographic origin and year of origin was obtained after STRUCTURE analysis, demonstrating the extensive exchange of genetic information between cherry cultivars in the collection over time. However, the phylogenetic tree calculated by DARwin reflected the geographic origin of selected cherry cultivars. After parentage analysis with CERVUS, paternity could not be confirmed for three cultivars, indicating the necessity of further pedigree analysis for these cultivars. The results of our study underlined the general importance of evaluating the authenticity of cultivars in genebank collections based on genetic and pomological characterization.Genebank collections preserve many old cultivars with ancient breeding history. However, often, cultivars with synonymous or incorrect names are maintained in multiple collections. Therefore, pomological and genetic characterization is an essential prerequisite for confirming trueness-to-type of cultivars in gene bank collections. In our study, 1442 single sweet cherry ( Prunus avium L.) is an economically important fruit crop that has been widely cultivated in Europe for hundreds of years. P. avium is usually diploid with 2n = 2x = 16 chromosomes, but sometimes triploid and tetraploid individuals occur naturally in wild cherry [P. avium includes both large-fruited domesticated sweet cherries and small-fruited wild forest so-called mazzard cherries used for timber production. Sweet cherry cultivars are commonly divided into the soft-fleshed heart cherries and the firm-fleshed Bigarreau cherries [Sweet cherry  markers proposed by the European Cooperative Program for Plant Genetic Resources , as stahttps://www.openagrar.de/receive/openagrar_mods_00066782, accessed on 22 March 2021) [A total of 1,442 sweet cherry trees were examined for their cultivar authenticity based on pomological and molecular characterization. By performing the pomological and molecular characterizations twice, on the one hand, ambiguities from the first examination could be clarified and, on the other hand, the pomologists could consider the molecular analyses in their evaluations. According to the workflow for the assessment of cultivar authenticity of cherries, a final determination of cultivar authenticity was made based on phenotypic and genetic data. Molecular analyses of the 1442 trees using the 16 SSR markers and pomological determination resulted in 383 unique genotypes, which were used for the subsequent genetic analyses. The genetic data of the 383 unique sweet cherry genotypes/cultivars were published in Openagrar (dataset available: ch 2021) . With 12ch 2021) .An additional 120 trees, including 55 genotypes, were grouped into their own molecular group, but no reference cultivars were available for the pomological cultivar identification. These were mainly newer international cultivars that were included into the collection either for comparative trials or for breeding work. In addition, old local cultivars, which were pomologically interesting, had partial or no references because not all old cultivars had been described pomologically. For 64 trees, (corresponding to 21 genotypes) a pomological determination was not possible due to lack of fruit quality or quantity, and for 23 trees (corresponding to 15 genotypes), the pomological determination was approved with reservation.The category “no pomological determination” included trees that were not examined because the trees were dying, died, or did not bear fruits during the study period. The category “pomological determination approved with reservation” included the trees bearing, in the years of investigation, insufficient amount of fruits or partially uncharacteristic fruits , which meant that they showed slight differences with fruits of reference trees (in one or more characteristics).In the individual partner collections, 74–92% of the trees were determined as true-to-type. The respective proportion of the categories in the individual collections of the partners is shown in Na = 15.19, ranging from Na = 5 for EMPA003 to Na = 24 for PceGA34  and the highest value for EMPaS06 and PceGA34 (He = 0.87). The allelic richness (Ar) was also high for most SSR markers and ranged between Ar = 3.11 (EMPA003) and Ar = 16.27 (UDP98-412).All 16 SSR markers showed clear polymorphisms and reproducible results, with one or two amplified fragments per genotype. High genetic diversity in the cherry collection was observed, with an average number of different alleles of  PceGA34 . The avePI = 0.18) and ranged from PI = 0.52 (EMPA017) to PI = 0.03 (PceGA34 and EMPaS06), indicating a high discrimination power of the used SSR markers , UDP98-412 (PI = 0.06), PS05C03, BPPCT037 (both PI = 0.07), and EMPaS12 (PI = 0.09). The combination of these six SSR markers were sufficient to discriminate all genotypes in the cherry collection (PI = 0.0). Assuming the existence of related cultivars in the cherry collection, 14 SSR markers were necessary to exclude individuals with the same multilocus genotype that may have been detected by chance.The probability of identity was low for most SSRs (mean  markers . Taking K = 3) (Q > 80%) to their respective genetic cluster, with low genetic admixture with the other clusters. This result suggested a distinct genetic difference between the three genetic clusters obtained in the cherry collection. However, the pairwise Fst values between the genetic clusters were 0.024 (cluster 1 and 2), 0.027 (cluster 2 and 3) to 0.029 (cluster 1 and 3) indicating only slight genetic differentiation between the single clusters [Following STRUCTURE analysis, 383 genotypes were grouped into three genetic clusters (K = 3) . Clusterclusters .Furthermore, a phylogenetic analysis using DARwin was performed to determine the genetic relationship between the single cherry cultivars. Similar to the STRUCTURE output, the dendrogram divided the 383 selected genotypes into three main clusters  and S1.Cluster I was separated into five sub-clusters . Sub.I included mainly cherry cultivars that originated from Canada, such as ‘Sumste’, ’Sunburst’, and ‘Lapins’. Furthermore, single cherry cultivars in these clusters originated from the USA , from Italy  from Czechia  or from France . In contrast, Sub.II and III. included mainly German cultivars originating from the middle eastern and eastern parts of Germany . Particularly, ancient cultivars from Saxony–Anhalt and Brandenburg were part of this branch. Sub.IV and V included cherry cultivars that originated from other European countries, but also German cultivars from the northern, western, and southern parts of Germany .Prunus species accessions  that were used as ECPGR reference genotypes. In Sub.III, cultivars from Switzerland and France were grouped together with German cultivars. Sub.IV, V and VI included mostly German cultivars from different regions and only a few foreign cherry cultivars. Especially in Sub. IV, old varieties of the cherry growing region Altes Land were combined with their breeding products of the Fruit Research Station in Jork, near Hamburg. In contrast, only cultivars of the middle Rhine region were included in Sub.VI.Cluster II was separated into six sub-clusters . Sub.I and II contained cherry cultivars from Great Britain, Hungary, Russia, and the Ukraine, as well as the three wild Cluster III was separated into four sub-clusters. Sub.I and Sub.II included mainly French cultivars  and younger cherry cultivars from Dresden–Pillnitz, Germany , mostly descending from the French variety ‘Souvenir des Charmes’. Sub.III and Sub.IV included 15 cultivars that mainly originated from countries outside Germany, for example ‘Bing’ from USA, ‘Merton Late’ and ‘Merchant’ from Great Britain, or ‘Große Prinzessin’ from The Netherlands. Additionally, several German cherry cultivars, distributed mainly (but not exclusively) in the southern and northern part of Germany, were grouped in these clusters.Parentage analysis was performed for 56 cherry cultivars with known parents and an existing genetic data set of at least one parent in order to verify ancestral information from the literature .For 31 cherry cultivars, both potential parents known from the literature were genetically investigated in this study, therefore genetic data of both parents were available. For these cultivars, the both-parents–progeny trio confidence was calculated using CERVUS. As result, for all cherry cultivars, the parental information was confirmed based on either a strict confidence of 95% (4 cultivars) or a loose confidence of 80% (26 cultivars). Uniquely, for the cherry cultivar ‘Nafrina’, the parents–progeny trio confidence was <80%, indicating that one of the parents could be wrong.For 18 cherry cultivars, only the potential mother was genetically tested in this study. For these cultivars, the mother–progeny pair confidence was calculated using CERVUS. As a result, the putative mother was confirmed for all cherry cultivars. For 14 out of 18 investigated cultivars, confidence over 95% was determined for the presumed mother. Uniquely, for the cultivar ‘Katalin’, the mother–progeny pair confidence was below 80%, so it can be assumed that the presumed mother was erroneous.For seven cultivars, only genetic data of the potential father was available. For these cultivars, the father–progeny pair confidence was calculated using CERVUS. As result, the putative father could be confirmed for six of seven cultivars with a confidence >95%. One exception was the cultivar ‘Swing’, for which ‘Stella’ may not be the true father.The German Fruit Genebank, as a national decentralized genebank network for fruit genetic resources, was established to ensure the effective, long-term conservation and availability of fruit genetic resources for research and breeding. These genebank collections contain mainly German cultivars, including new German selections and cultivars with sociocultural, local, or historical relation to Germany. However, the collections also contain foreign and new cultivars with important pomological traits, especially for breeders. Currently, sweet cherry genetic resources are maintained by eleven partners. In this study, 1442 trees were examined from six collections that had joined the German Fruit Genebank at the time of this study. Several cultivars were present at more than one collection site; these cultivars were sampled multiple times and used for pomological evaluation and SSR genetic analysis in order to identify cultivar authenticity. The aim of combining pomological and molecular determinations was to analyze the trueness-to-type of the cherry cultivars as accurately as possible. Comparison of the results of both determination methods allowed a better identification of potential errors. Especially in the case of cultivars that were difficult to identify, a more accurate assessment of cultivar authenticity could be made combining pomological and molecular determinations than if only one of these methods were used. Beyond the actual comparison of pomology–molecular genetics, the molecular results allowed further investigation into the relationship of the cultivars.The pomological evaluation of cherry cultivars in this study has shown that the determination of the trueness-to-type of cultivars in old collections must necessarily be verified, since numerous accessions were labeled with an incorrect cultivar name. Furthermore, there are numerous synonyms that must be assigned to key names to avoid duplication and to keep the cultivar descriptions in the database correct. A prerequisite for pomological verification is the clear expression of distinctive phenotypic traits that allow the identification of the cultivar by pomologists. Reference cultivars in other collections, stone references, and reference literature are essential for pomological classification. If cultivars are inconsistent in phenotypic characteristics, cultivar authenticity may remain unconfirmed, as was the case with several cultivars in this study. For this reason, genetic analysis with molecular markers is an additional useful tool for evaluating cultivar authenticity ,14,15,16on farm), where information about the cultivars planted decades ago were usually lacking. The cultivars originating from the landscape, more often, could not be assigned to a pomologically-described cultivar due to missing references. These cultivars were given provisional working names . Often,Prunus studies and a future usage of a joint international cherry SSR marker database, we used a set of 16 SSR markers recommended by the ECPGR Prunus working group [PI-values were necessary to discriminate all genotypes in our entire cherry collection  were considerably higher than using SNP markers for sweet cherry cultivar discrimination [PI calculation was probably inaccurate, because it did not consider a possible close relationship between the cherry cultivars [sibPI value, which takes into account the existence of related cultivars in the cherry collection. Using this equation, the number of markers needed for the individual discrimination increased to 14, which still confirmed that the number of markers used in this study was sufficient.Many genetic studies on cherry demonstrated the usefulness of reliable polymorphic SSR markers for determination of the trueness-to-type and the analysis of genetic diversity and structure in genetic resources ,17,18,19ng group ,20. All llection . This reing SSRs ,22 and tmination . Howeverultivars . TherefoIn our study, it was possible to identify 383 unique genotypes in the six investigated sweet cherry collections. Several accessions with the same name from the different collections were confirmed as duplicates, as their SSR profiles were identical. With support of their phenotypic characteristics, about 86% of the investigated trees were assigned to cultivar names with certainty . A similThese ambiguities and mistakes underline the need for detailed analysis. Compared to the other collection institutions involved in the study, at the JKI and the Hessian State Office for Agriculture, a slightly higher proportion of cultivars with “own molecular group but without references for pomological variety identification” was found, probably due to the newer international cultivars in these collections.In our study, for the evaluation of cultivar identity based on genetic data, the threshold for true-to-type was set at >90% allele pattern similarity. This threshold took into account a non-negligible level of PCR artifacts during SSR analysis, which generally occur at a rate of approximately 10% ,28. HoweIn our study, the 383 unique genotypes identified in the sweet cherry collections were investigated regarding genetic diversity and structure. As expected for self-incompatible cultivars, genetic diversity in the collection was high. However, compared to other self-incompatible cultivars, such as apple or pear, the genetic diversity was lower in sweet cherry ,32,33. IAfter STRUCTURE analysis, the 383 genotypes/cultivars were grouped into three genetic clusters . The lowth century originated from random seedling selections in this region, e.g., ‘Schneiders Späte Knorpelkirsche’, ‘Große Germersdorfer’, ‘Dönissens Gelbe’, or ‘Fromms Herzkirsche’. However, for several cultivars, it was not possible to make a definite statement about the geographical origin, as the origin was not always documented.However, these relationships did not document the geographic origin of older cherry cultivars. In our study, we could not find a clear relationship between the geographic distribution of old cherry cultivars and their genetic structure. This reflected the traditional exchange of plant material across geographic regions and the subsequent genetic admixture during the development of newer cultivars over time . ExceptiVitis [Vitis study, much pedigree information was incorrect. The main reason for these errors was thought to be pollen contamination during hand pollination [The parentage analysis in our collection was performed only for cultivars with at least one genetically investigated parent . In our Vitis . In the lination . That cohttps://www.deutsche-genbank-obst.de/, Cherry Network, accessed on 1 December 2022).Incorrect cultivar identification or synonymy in parents’ names is obviously a second involuntary reason that can lead to pedigree errors . This isIn our collection, the confidence level of confirmation was significantly lower for the varieties with two known parents than for those with only one known parent. One reason for this could be erroneous parent assignment due to genotypes containing typing errors. Particularly in collections with a close genetic relationship, as between the investigated sweet cherry genotypes in our study, the assignment of true cultivars can be challenging. Allowing typing errors may improve assignment, but it can impede a clear distinctive parent identification in the collection. Another reason may be more closely related to the analysis itself, since the LOD value for assessing true inheritance is lower when the potential parents carry predominantly common alleles than when they carry more rare alleles. The absence of rare alleles in the cherry collection in our study could explain the low LOD value (confidence level < 95%) for some of the parents studied (data not shown).Therefore, parentage analysis should generally be considered with caution, since these results can be very speculative. Reliable results can only be discovered through extensive genotyping of large collections and additional observations of pomological traits ,38.Prunus avium L.) individual trees, preserved by six different genebank network partners, were investigated . The cultivars within the cherry network of the German Fruit Genebank included (1) German cultivars, including new German selections, (2) cultivars with sociocultural, local or historical relation to Germany, and (3) cultivars with important pomological characteristics, especially for breeders. Some cultivars were present in several collection sites and could be sampled multiple times. The characterization of cultivar identity was based on two evaluation procedures: the pomological evaluation and the molecular analysis.On behalf of the German Federal Ministry of Food and Agriculture, four projects were conducted from 2009 to 2014 and from 2017 to 2020 to characterize the trueness–to-type of the cultivars in the cherry network of the German Fruit Genebank. A total of 1442 sweet cherry .Tree, fruit and stone photos were taken for each cultivar and uploaded to the database of the German National Fruit Genebank .The second evaluation procedure was the molecular DNA fingerprint analysis, which was realized in the years 2012 to 2014 and 2017 to 2019. The microsatellite primers were selected according to the guidelines of the ECPGR  markers : BPPCT03 markers , CPPCT00 markers , EMPA002 markers , EMPaS01 markers , PceGA34 markers , PS05C03 markers , and UDP markers  . Leaf material of the single trees was collected from the different collection sites in 2 mL reaction tubes and dried using silica gel according to a modified protocol by Slotta, et al. . The leaThe second sample set, collected in 2017–2019, was genotyped by Ecogenics GmbH c/o Microsynth AG . Leaf material of each single tree was collected from the different collection sites in a sample bag and was immediately stored on dry ice. Until DNA isolation, the samples were stored at −80 °C. For DNA isolation, the Hotshot method, according to Truett, et al. , was useP. avium F12/1, P. avium ‘Goodnestone Black’, P. avium ‘Napoleon’, P. avium ‘Noble’, P. avium ‘Noir de Meched’, P. incisa E621, P. mahaleb SL64 and P. nipponica F1292 , were used as reference cherry genotypes [Fragment lengths analysis was done on a 3730XL DNA-Analyzer  using the Software GeneMarker V2.6.4 . In addition, the fragments were visually checked for appropriate quality. The eight genotypes enotypes . These rPI) was calculated using GenAlex ver 6.5. In estimating PI, it was assumed that the individuals being compared were unrelated. Because the samples could have contained relatives, we also calculated sibPI, which gave a more conservative estimate of PI.To estimate the number of marker combinations required to distinguish all genotypes, the identity probability , effective number of alleles (Ne), observed heterozygosity (Ho) and expected heterozygosity (He) were calculated for each nuclear SSR loci within the 383 cherry cultivars using the software GENALEX ver. 6.5 [Ar) was calculated with the software ADZE [The mean number of alleles by locus  [Genetic structure within the 383 unique genotypes of the entire cherry collection was analyzed using the software program STRUCTURE. The parameters were 100,000 burn-in periods and 100,000 Markov Chain Monte Carlo repetitions using the admixture model with correlated allele models. To estimate the number of genetic groups in the cherry collection, we ran the program from 2 to 8 with 5 runs for each ARVESTER  was usedK method . The genod [Fst)  using GEThe program DARwin ver.5 was used to estimate the dissimilarity distance matrix of the genetic data of the 383 genotypes considering a bootstrap analysis with 1000 replications . The treCERVUS software version 3.0.7 ,47 was uPomological and genetic characterization of cultivars in genebank collections is an essential prerequisite for confirming the trueness–to-type of cultivars and genebank management. In our study, the trueness-to-type of 86% of cherry trees could be confirmed based on pomological and genetic evaluation. For the remaining unauthenticated accessions, additional pomological studies are needed to clarify open questions on authenticity. In general, a high degree of genetic admixture was found according to geographical location and year of origin, demonstrating extensive exchange of genetic information between old and younger cultivars and between German and foreign cultivars over time. A single parentage could not be confirmed, indicating the need for further pedigree analyses for this cultivar. The results of our study underlined the necessity of assessment of true-to-type cultivars in genebank collections based on genetic and pomological evaluation.https://www.deutsche-genbank-obst.de/). With the determination of cultivar authenticity, the goal of the conservation strategy of the German National Fruit Genebank to secure each variety in at least two different locations can be achieved. Thus, the varieties confirmed as authentic can now be exchanged between the collection partners.The results of the sweet cherry pomological and molecular analyses were forwarded to the DGO partners of the German National Fruit Genebank for revision of their collections and updating of the database (


In the context of the COVID-19 pandemic, the creation of healthy cities has become an important measure to deal with global public diseases and public health emergencies, and has had a profound impact on the management of municipal solid waste (MSW). This study exploits the Healthy Cities pilot (HCP) program established in 2016 as a natural experiment, and evaluates its impact on MSW management using the difference-in-difference (DID) method. The estimates show that the collection amount and harmless treatment capacity of MSW were increased by 15.66 and 10.75%, respectively, after the cities were established as pilot healthy cities. However, the harmless treatment rate was decreased by 3.544. This conclusion remains valid in a series of robustness tests, including parallel trend test, placebo test, propensity score matching (PSM)-DID, eliminating the interference of other policies, and eliminating the non-randomness of the policy. Mechanism analysis shows that the HCP program increased the collection amount and harmless treatment capacity of MSW by increasing the expenditure on MSW treatment. However, after a city was established as a pilot healthy city, the unsustainable high expenditure of local government on municipal sanitation led to the decrease in the harmless treatment rate of MSW. Moreover, heterogeneity analysis shows that the HCP program had a stronger impact on MSW management in cities with higher administrative levels, more obvious location advantages, and a larger size. Therefore, it is advisable to use the creation of healthy cities as an important tool to gradually improve MSW management, so as to realize the coordinated development of city construction and human health. The continued spread of COVID-19 not only poses a huge challenge to global public health security , but alsTo solve the increasingly serious problem of domestic pollution, the Chinese government proposed the “Healthy China” strategy, trying to build a sound public health system. The Healthy Cities pilot (HCP) program covering 38 pilot citiesThere are two categories of studies related to our work. The first category focuses on the implementation of healthy cities. The second category focuses on how to improve the public health environment of cities. The first category of studies examine the connotation of a healthy city, its metrics, and its impact. The World Health Organization (WHO) defined it in 1994 as people living healthy lives. The goal of creating a healthy city is to make people happier . MetricsThe second category of studies focuses on how to improve the public health environment of cities. Implementing pilot policies is main means of addressing pollution problems in China. First, many studies have focused pilot policies on environmental pollution, and almost all of them reached positive conclusions, that is, the implementation of pilot policies has significantly improved the urban environment. And most of them focus on the control and treatment of industrial pollution sources. For example, Huo et al.  and Qiu To sum up, the existing literature provides many useful leads for examining the impact of the HCP program on MSW management. However, there are still some deficiencies. First, the existing literature on healthy cities , 34 mostThis study provides the following marginal contributions. First, in a theoretical sense, previous studies have paid sufficient attention to the effects of different types of environmental regulations, and the Chinese government has often used pilot policies as a policy vehicle for environmental regulations to study their effects on solving environmental pollution problems, but with mixed environmental praise and criticism. In this paper, we use DID model to assess the impact of HCP program on MSW, which enriches and extends the literature related to the study of policy effects of environmental regulation theory. Second, in terms of research perspective, previous studies have paid sufficient attention to the management of industrial pollution sources , 37, 38,A healthy city is a new model of urban development that focuses on human health in all aspects from urban planning, construction to management, intending to ensure healthy living and work for the general public, and organically combining healthy people, a healthy environment and a healthy society. The selection criteria of healthy cities include “healthy environment,” “healthy society,” “healthy services,” “healthy people,” and “healthy culture” 5 primary indicators, 20 secondary indicators, and 42 tertiary indicators. The specific content of each indicator is shown in Pilot areas have formed a large number of experiences that can be replicated after a period of construction. For example, by promoting the combination of medicine and physical exercise, organizing health lectures, and promoting MSW management, the average life expectancy in the Xicheng District of Beijing reached 84.26 years in 2018, and health literacy increased from 33.4% in 2015 to 36.6% in 2018. Hangzhou has established six special teams for building a healthy society, spreading the health culture, protecting a healthy environment, optimizing health services, cultivating healthy people, and developing the health industry, as shown in The HCP program construction will have an impact on MSW management. First of all, the National Healthy City Evaluation Index System (2018 Edition) takes the harmless domestic waste treatment rate as an important element of the HCP program evaluation index system, which has a certain “Target Leading Effect” and “Binding Effect” on the HCP program. The construction of HCP program will focus on a series of construction activities around the harmless removal and treatment of domestic waste, which in turn will affect MSW management. Secondly, the HCP program is led by the party committee, government-led, and multi-departmental cooperation. As an important symbol of healthy city development, local governments often tend to invest money and technology in the construction of healthy cities in order to ensure the smooth implementation of healthy city work. As an important part of HCP program, MSW management will be influenced by the strong drive of local governments. Finally, under China's political system, the central government directly decides the promotion of local government officials, and the HCP program is implemented by the central government, which has the attribute of “strong leadership,” and local governments are often motivated to build HCP program under the drive of the central government. Therefore, as an important policy of the Healthy China 2030 plan, the HCP program has had an impact on MSW management. The HCP program affects MSW management through the following mechanisms.The HCP program promotes the increase in the amount of MSW collected by increasing related inputs. Guo et al.  believedH1: The HCP program improves MSW management by increasing the inputs.The high expenditure on municipal sanitation may be unsustainable after a city is established as a pilot healthy city, which will lead to a decrease in the harmless treatment rate of MSW. As a typical civic calling card, Healthy Cities are affected by population aging and urban shrinkage in the process of operation and implementation. As a result, local governments will reduce expenditures on public programs, such as the Healthy Cities Initiative , which iH2: During the implementation of the HCP program, the unsustainable high expenditure on municipal sanitation may compromise MSW management.We evaluate the impact of healthy city implementation on MSW management using the DID model. According to Duflo , the folit is the dependent variable which represents the amount of MSW collected (Rem-vol), the harmless treatment capacity of MSW (Tre-cap), and the harmless treatment rate (Tre-rate) of the city i in year t. Hea-Cityit is the core independent variable which is 1 for the year when a city was selected as a pilot healthy city and the subsequent years, and 0 for the other years. Its coefficient β1 reflects the impact of healthy city pilot implementation on municipal sanitation improvement. X is a set of control variables, including financial pressure (fin-pressure), industrial structure (ind-stru), population density (pop-density), level of opening to the outside world (opening), science expenditure (sci-expe), financial development level (fin-deve), and economic development level (lnpergdp). μi is a city fixed effect that controls for the characteristics of a city that do not change over time. νt is a year fixed effect that controls for the macro policy shocks faced by cities. εit is a random disturbance term. This model effectively controls for the characteristic differences between the experimental and control groups and the time variation trend.where Yit), which is measured by the amount of MSW collected (Rem-vol), the harmless treatment capacity of MSW (Tre-cap), and the harmless treatment rate (Tre-rate). The amount of MSW collected is the amount of MSW collected and transported to MSW treatment plants . It is expressed in logarithms in the empirical analysis. The main methods of harmless treatment of MSW are sanitary landfill, high-temperature composting, and incineration by using advanced technology to minimize the environmental harm of MSW. The harmless treatment capacity of MSW refers to the amount of MSW that can be harmlessly treated by waste treatment plants per day (in ton/day). It is expressed in logarithms in the empirical analysis. The harmless treatment rate of MSW is the ratio of the amount of MSW treated harmlessly to the amount generated. However, the amount of MSW generated is often replaced by the amount of MSW collected due to data unavailability. The data are from the China Urban and Rural Construction Statistical Yearbook.The dependent variable is MSW management (Yit). The pilot healthy cities are identified according to the list of the first batch of 38 pilot healthy cities approved by the National Health Commission of China (www.nhc.gov.cn) in 2016.The core independent variable is the pilot healthy cities  are shown in th year of healthy city pilot implementation. The maximum value of k is 4, and the minimum value is −11 during the sample period of this study (2005–2020). For presentation purposes, relative times < −5 are merged into −5. Moreover, it is often necessary to define a base period to avoid multicollinearity when using the event study methodology to test for parallel trends and dynamic effects. The year before healthy city pilot implementation (−1) is selected as the base period. Then, the values of k are −5, −4, ... −2, 0, ...4. The coefficient ofβk represents the difference in MSW management between the experimental and control groups in the kth year of healthy city pilot implementation compared with the year before the implementation.A crucial assumption for the validity of the DID is the parallel trend assumption. Moreover, considering that the effect of the creation of pilot healthy cities on MSW management may be delayed and dynamic, we test for parallel trends and dynamic effects using the event study methodology based on the baseline regression model . The ecoThe results of the tests are shown in p-values are plotted in the same graph, and compared with the coefficients obtained from the baseline regression. The three graphs in p-values for most of the regressions are > 0.1, indicating that the regression results are not significant. This further demonstrates that HCP program implementation significantly increased the collection amount and harmless treatment capacity of MSW, but reduced the harmless treatment rate of MSW. The above conclusions are robust.Another concern with using the DID model to assess the effect of healthy city implementation on MSW management is that the conclusions obtained may be a random phenomenon. To exclude the influence of non-observed factors, this paper uses the method of Li et al.  for refeAlthough the DID model can scientifically identify the average treatment effect of the pilot policy, it may lead to a significant selection effect considering that the establishment of pilot healthy cities may not be completely random. Therefore, PSM-DID is used for robustness testing according to Heckman et al. . GeneralThe steps are as follows: First, financial pressure, industrial structure, population density, level of opening to the outside world, science expenditure, financial development level, and economic development level are used as matching variables. Second, two new data sets are obtained using the two matching methods, for both of which nearest neighbor matching is used. Next, the balance of the two data sets is tested and the data distribution before and after matching is compared. The results of the balance test are shown in Although the HCP program significantly affects domestic pollution, it is undeniable that there are other pilot policies in the same period that may also affect MSW management. For example, the pilot programs of civilized city and low-carbon city  have an i with the year fixed effect νt to the traditional DID model to minimize the non-randomness of the establishment of pilot healthy cities. Specifically, two categories of variables are considered. The first category of variables is natural geographical characteristics (Nature) of the city, such as river density, elevation, and slope. The reason is that natural geographical factors affect vegetation growth, and to a certain extent, the health level of the city , mechanized cleaning rate (Msr), and number of municipal sanitation trucks (San-veh) as the capital input of the local government in MSW collection, and the number of municipal sanitation workers (San-per) as the labor input. As shown by the regression results in China Urban and Rural Construction Statistical Yearbook includes the number of MSW harmless treatment plants (Ghtp), that of landfill plants (Sltp), and that of incineration plants (Inpl). They are used as dependent variables for regression, respectively. As shown by the regression results in The most direct mechanism for healthy city implementation to increase the harmless treatment capacity of MSW is the construction of more waste treatment plants. The As the HCP program continues to be implemented, the harmless treatment rate of MSW has decreased significantly. According to the definition, the harmless treatment rate of MSW is the ratio of the amount of MSW treated harmlessly to the amount collected. The above conclusions of this study indicate that the HCP program increased the amount of MSW collected. Then the decrease in the harmless treatment rate of MSW is due to the fact that the increase in the amount of MSW treated harmlessly (numerator) is smaller than the increase in the amount of MSW collected (denominator). However, as indicated above, the HCP program significantly increased the harmless treatment capacity of MSW. Therefore, the smaller increase in the amount of MSW treated harmlessly is unlikely to be due to poor equipment for MSW harmless treatment, but probably due to the fact that the local government reduced the expenditure on MSW harmless treatment, leading to increased equipment idleness of MSW treatment plants. To test this conjecture, we examine the impact of the HCP program on local government's expenditure on municipal sanitation (Hea-exp). As shown by the regression results in *Hea-City) between the dummy variable (Rank) for city level and the dummy variable (Hea-City) for the HCP policy into the baseline regression, a two-way fixed effects DID regression is performed to examine the difference in the effect of the HCP program on MSW management due to the different administrative levels of cities. The effect of healthy city implementation on MSW management may vary with the administrative level of cities. Accordingly, provincial capitals, municipalities directly under the central government, cities specifically designated in the state plan, and special economic zones are defined as cities with high administrative levels, and other cities are defined as cities with low administrative levels. Cities with high administrative levels are assigned a value of 1, and those with low administrative levels are assigned a value of 0 to generate the Rank variable. By introducing the interaction (Rank*Hea-City) between Location and Hea-City into the baseline regression, a two-way fixed effects DID regression is performed to examine the difference in the effect of the HCP program on MSW management due to the different locations of cities. As shown by the regression results in *Hea-City on MSW collection is significantly positive at the 5% level, indicating that healthy city implementation has a more pronounced effect on increasing MSW collection in eastern cities with obvious location advantages. Likewise, the effect of Location*Hea-City on the harmless treatment capacity of MSW is significantly positive at the 1% level, indicating that healthy city implementation has a more pronounced effect on increasing the harmless treatment capacity of MSW in eastern cities with obvious location advantages. However, the effect of Location*Hea-City on the harmless treatment rate of MSW is negative at the 5% level, indicating that healthy city implementation has a more pronounced negative effect on the harmless treatment rate of MSW in eastern cities with obvious location advantages. The possible reason is that compared with the central and western cities with fewer location advantages, the eastern cities have higher technological levels, better economic foundation, and a more mature concept of healthy living, which provide a good foundation for healthy city pilot implementation, thus allowing a greater improvement in MSW management.The effect of healthy city implementation on MSW management may vary with the location of cities. Accordingly, the sample cities are divided into eastern, central, and western cities by geographic location. Western cities are assigned a value of 1, central cities are assigned a value of 2, and eastern cities are assigned a value of 3 to generate the Location variable. By introducing the interaction (Location*Hea-City) between Scale and Hea-City into the baseline regression, a two-way fixed effects DID regression is performed to examine the difference in the effect of the HCP program on MSW management due to the different sizes of cities. As shown by the regression results in *Hea-City on MSW collection is significantly positive at the 1% level, indicating that healthy city implementation has a more pronounced effect on increasing MSW collection in larger cities. Likewise, the effect of Scale*Hea-City on the harmless treatment capacity of MSW is significantly positive at the 1% level, indicating that healthy city implementation has a more pronounced effect on increasing the harmless treatment capacity of MSW in larger cities. However, the effect of Scale*Hea-City on the harmless treatment rate of MSW is negative at the 1% level, indicating that healthy city implementation has a more pronounced negative effect on the harmless treatment rate of MSW in larger cities. The possible reason is that compared with small cities, large cities have better conditions for MSW treatment, thus allowing a greater improvement in MSW management.The effect of healthy city implementation on MSW management may vary with the size of cities. Accordingly, the sample cities are divided into large and small cities according to the total population. Specifically, cities with a greater than average population are assigned a value of 1, and those with a smaller than average population are assigned a value of 0 to generate the Scale variable. By introducing the interaction  to ensure the sustainability of local financial support for public health improvement. Moreover, efforts should be made to maximize the coordination and linkage of the HCP program with other public health policies. Finally, the HCP program should be implemented according to local conditions. In ordinary cities, cities with fewer location advantages, and small cities, efforts should be made to create an institutional environment that is compatible with the operation of Healthy Cities to promote its effective operation. Cities with high administrative levels, obvious location advantages, and a large size should gain the first-mover advantage and maintain the sustainability of municipal sanitation improvement as pilot healthy cities.This study has two limitations. First, medical waste is an important part of MSW. The collection and harmless treatment capacity of the medical waste directly affect the efficiency of MSW management, especially in the context of the global spread of COVID-19. However, due to the unavailability of medical waste data, this study cannot evaluate the impact of the HCP program on the collection and harmless treatment of medical waste. Second, this study finds that the HCP program has a significant positive impact on MSW management in the short term, but does not assess its impact on people's health—both physical and mental.Based on the existing limitations, future research can consider the following: First, field surveys can be conducted to collect data on the production, collection, and harmless treatment capacity of medical waste in each city. The impact of the creation of healthy cities on the collection and harmless treatment capacity of medical waste can then be evaluated using the DID method based on the survey data. Second, the impact of the HCP program on the healthy growth of adolescents, people's mental health, and the health of the elderly can be evaluated using micro-survey databases to fully understand the direct impact on people.The original contributions presented in the study are included in the article/All authors were involved in the research, writing, proofreading of this paper, and confirmed the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The study investigated the evocation of mental imagery and emotional responses when English–Chinese bilinguals read classic Chinese poems and their English translations to examine (1) the target readers' formation of non-verbal text representations of Chinese poetry and (2) whether different translations affect the target readers' imagery cognition. A total of 20 English–Chinese speaker students enrolled in a Chinese university read a classic Chinese poem in Chinese and its four versions of translation in English. Through questionnaires and interviews, participants rated the visualized words used in the poems for the degree of mental imagery and emotional response evoked based on three indicators of narrative process, salience value, and emotive validity in the theoretical framework of visual grammar. Results showed considerable individual variances in the cognitive differences in forming mental imagery in all versions of the poems and there were also effects of translation strategy. Moreover, visual language information in poetry reading and its translations evoked different emotional responses depending on the use of visual words with cultural features. Our study demonstrates the applicability and accessibility of visual language in describing different readers' mental imagery and the interrelation and interaction between the poetry language system and the emotional, social, and cultural contexts involved in poetry translation. Mental imagery is a cognitive process that involves forming mental representations and images  an argument  work together in poetry translation, as well as how the source poem and the target poem are linked and reformed at the mental imagery level.The poet, the translator, and the target reader are all involved in poetry translation. The perception and interpretation of the imagery vary as the participants' identities alter during the cognitive process of poetry translation. The poet creates imagery in the poem based on real-world objects and the poet's own experiences and emotions. The translator accepts and transforms the imagery by reproducing it in the target language for the target reader see . The traTwenty participants , including senior and post-graduate international students currently studying at a Chinese university, participated in the study. Twelve participants speak English as their native language, and eight are fluent in English (L1). All of them speak Chinese as their second language (L2), and 15 of them have passed Level V of the HSK (Chinese Proficiency Test).A classic Chinese poem and its four versions of translations were used as materials. The title of the poem is “天净沙·秋思” in Chinese. The poem, which contains 28 Chinese characters, was written by Ma Zhiyuan in the Yuan Dynasty (1271–1368). It sketches a picture of a journey at dusk in an autumn field, enumerating the spatial imagery of nine scenes in the landscape of ancient Chinese villages. There are eight noun phrases with the “modifier-head” structure, divided into three groups, each composed of three visual words. On the surface, there appears to be no link between the nine scenes, but the context of the poem reveals that all of the scenes are utilized to portray a profound and emotional love for one's birthplace. The four versions of English translations are titled “Autumn”, “Tune: Tian Jin Sha”, “Tune to “Sand and Sky”-Autumn Thoughts”, and “Autumn Thoughts” by translators Weng Xianliang, a Chinese translator Weng, , Wayne SAll participants were first familiarized with the concept of visual grammar and its three critical indicators, “narrative structure”, “salience value”, and “emotive validity” in the theoretical framework. Each participant was then given five copies of the poem, one original Chinese version and its four versions of English translations. Participants were asked to read the materials and filled a questionnaire regarding the observed visual images presented by the words used in the materials. An experimenter then conducted an interview with each participant to collect explanations and reasons for the participant's answers to the questionnaire and the cognitive process behind it. According to the previous empirical study on mental imagery in literary translation and visual grammar  poem  and its four versions of English translations , 15 participants identified 12 separate images in the source poem, TT2, and TT3. Sixteen reported 13 images in TT1, involving one visual word, “the far bank” which does not exist in the source poem. For TT4, sixteen participants identified the same 12 images as ST, but four participants identified more than 12 images. These four participants explained that “returning crows” and “at dusk” formed two separate images in their mental imagery and the same as “a narrow bridge” and “below the bridge.”over a sparkling stream, and on the far bank, a pretty little village” (TT1), and “A few houses hidden past a narrow bridge, and below the bridge a quiet creek running” (TT4). All participants suggested that these words evoked imagery of pastoral life in their minds. Verbs can relate to others, such as Line 3, “west wind moaning, horse groaning” (TT1), and “A lean horse comes plodding” (TT4), which formed imagery of a very burdened, tired horse.However, single visual pictures must be connected to disclose the poem's idea and produce an emotional and aesthetic experience on the reader's part. For example, the same linguistic construct  was used to communicate three images in the first three lines of the source verse, which might be connected to make more significant pictures. Participants were asked to combine those images in each line and report whether or not they could “see” the pictures or determine the representational meaning of imagery superimposition. As can be seen in Recognition of imagery superimposition in Line 1, Line 2, and Line 3 in the source poem and its four versions of English translations, respectively, can be seen in The current study selected the Chinese verb “下”, which means “come” or “go down” in English, as the critical visual word to examine participants' narrative structure of imagery. Both a verbal . Most participants could feel the nostalgia and the sadness in the poem, but it was still hard for the five of them, who knew less about Chinese poetry, to construct a complete picture in their minds, such as the sunset scenery in autumn. Nine participants found calmness, positioning themselves for the poem's second line, which describes the peaceful life through three images: bridge, stream, and homes. Participants also expressed their emotional responses to each salient element from As seen in The evocation of mental imagery and emotions can assist in understanding and appreciating poetry in different cultural backgrounds. The recognition that target readers access both verbal and non-verbal imagery when reading poems is essential in selecting translation strategies. These strategies can exploit non-verbal aspects  of comprehension with target readers whose vocabulary, linguistic, and cultural knowledge of the poem's native language is rather limited. From a psychological point of view, imagery transformation is the process of selecting and matching the imagery to languages. In addition to the multiple influences of the translator's individual variables, such as knowledge, experience, and cognitive style, the conversion is also subject to external factors, such as social, cultural, and thinking traditions. Compared with the aesthetic imagery presented in the original poem, what is restored and reproduced by the translator may be the same, similar, or even wholly different “imagery.”The findings of the current study showed that the cognitive transformation of imagery contains considerable individual variances. Therefore, the ultimate goal of poetry translation is to achieve imagery-to-imagery equivalence, which refers to the fact that the imagery created has the same semantic and aesthetic implications in the translated versions as in the original language, based on the commonality of thinking and psychological identity. Although different cultures have distinct languages and ways of thinking, there are similarities in understanding the same thing and the translator's style of thinking, processing, and aesthetic experience. As a result, the “meaning” and “imagery” can be connected in the original as in translated languages. At this point, the translator will utilize more image-based reasoning to extract pictures of particular items in the original language, as well as the visual implications that go along with them. Translators will also search the translation system for objective items that fit the meaning and emotions so that they can link the two.For the imagery in translation works to have the same or similar emotional effect like the one in the original, “image for image” strategy can also be used. This strategy refers to searching for different imagery in the target culture to replace the original one with the translator's imagination. Due to external environmental factors, the materials or information available for people's imagination might be different. That is to say, the equivalent image in the translated context cannot be found to represent the “image” in the original poem to a perfect level. However, since human beings have a high degree of consistency in their understanding of objective things in nature, the content of thinking (the “meaning” in imagery) will stay the same or comparable even if the output of thinking (language) changes in imagery reduction Zoltan, .Furthermore, our results suggest that extracting the translated picture with the same visual impact from memory is not always possible. To be “faithful” to the original poem or let the target readers understand the exotic flavor, the translator needs to retain or highlight the imagery of the original work. The semantic or formal equivalence between the “original image” and the “translated image” may be obtained through this process, and the translator must make the meaning of the image explicit by adding notes or supplementary explanations to convey the original imagery's meaning and emotion. Finally, sometimes it is necessary to discard the image to reach the meaning, which means that the translator can ignore images developed in the original poem entirely and only look for the visual word with a similar meaning or effect. This strategy prevents the tautologies of adding long explanations or notes for imagery words in the original poem. This strategy also releases the translator from the burden of looking for expressions in the target language to restore and reproduce the original imagery. The translator may choose to abandon the imagery words in the transformation process based on his aesthetic judgment.Our findings suggest that the theoretical framework of visual narrative provides a new perspective for analyzing poetry translation. First, we can comprehensively and systematically compare different visual narratives between the original and translated poems. Second, we can explain in more depth how visual imagery regulates readers' emotional engagement and attracts their interests by selecting visual words with almost the same imagery effect and providing theoretical guidance and practical strategies for poetry translation. The concepts of “narrative process,” “salience value,” and “emotive validity” help examine the readers' representational imagery from reading both the source poem and the translated poems. It can be found that there are interpersonal differences in recognizing imagery through visual words and between parallel poems involved in translation because meanings arise in social contexts and personal interaction, which are variables of cultural backgrounds .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Creole sheep represent a strategic genetic resource for populations living in marginal areas under financial restrictions on the American continent. Six Colombian sheep breeds  and four hair  were genotyped using the Illumina Ovine SNP50 BeadChip. Data was also included from international 44 breeds from International Sheep Genomics Consortium (ISGC) and from data published in previous a previous work on the Caribbean and African breeds. Although geographically separated, wool  and hair types  presented little genetic differentiation (FST 0.05) at a global level but several groups of animals separated suggesting local clustering due to geographical isolation. The OPCP underwent a recent crossing with Mexican Pelibuey, explaining its differentiation. Findings in this work such as the proximity to West African Djallonké (WAD) and Barbados Black Belly (BBB), suggest different introductions of African type animals from the Caribbean region on a pre-existing genetic basis formed by animals deriving from the first importations coming from Europe in colonial times. As expected, Colombian wool breeds showed, in particular in Admixture software results, a greater genomic component in common with European breeds and in particular with Iberian ones (Churra). This study provides a basis for future research into the genetic diversity within and between the Colombian sheep breeds analysed, and scientific data for policy decisions on Farm Animal Genetic Resources (FAnGR). Previous studies have described several populations using morphology, microsatellites, and mitochondrial DNA markers [In the American continent, Creole sheep represent a strategic genetic resource for populations living in marginal areas with scarce economic resources ,2,3. The markers ,14. Amon markers . his ter markers . The objA total of 237 samples were collected from the six breeds previously described [® DNA Mini Kit . DNA quantity and quality were evaluated by spectrophotometry using ColibriTM  and agarose gel electrophoresis using a standard protocol to evaluate sample degradation. Additionally, six samples of Ecuadorian Creole sheep were included as representatives from the nearest country in the region.1. Genotyping and dataset preparation.Animals were ~50% of both sexes, excluding OPCP, where all samples were from males. A total of 10 mL of blood from each animal were collected in BD vacuum tubes  with k3-EDTA. Genomic DNA was extracted from 237 blood samples using the QIAamp® Assay, including 54,241 SNPs . Using the PLINK v1.9b5.2 program [The genotyping was performed using an Illumina Ovine SNP50 BeadChip Infinium program , the raw program  represen program  permitte program , data weSeparately the process with the same parameters was repeated for only the Colombian population resulting in a dataset of 42,119 SNPs and 237 samples (4097 variants removed due to missing genotype only).o and He) was estimated for each breed using the ‘--het’ and ‘--family’ commands in PLINK [SNPRelate’ R package [Observed and expected heterozygosity  was estimated for each animal using PLINK and the following criteria were used: the minimum length was set to 1 Mb (--homozyg-kb), one missing SNPs and up to one possible heterozygous genotype were allowed in the ROH (--homozyg-window-missing 1 and --homozyg-window-het 1), the minimum number of consecutive SNPs included in an ROH was set to 30 (--homozyg-snp 30), the minimum SNP density per ROH was set to one SNP every 100 kb (--homozyg-density 100), and the maximum gap between consecutive homozygous SNPs was 500 kb (--homozyg-gap 500). The inbreeding coefficient based on ROH (FROH) for each animal was calculated as follows: FROH = LROH/LAUT where LROH is the total length of all ROH in the genome of an individual and LAUT is the specified length of the autosomal genome covered by SNPs on the chip (2452.06 Mb).’ [The pairwise genetic relationships were estimated using a matrix of genome-wide identity-by-state (IBS) genetic distances calculated by PLINK . After, ’ , to expl’ .50 2 0.1” PLINK [cv” flag, a 20-fold cross-validation procedure was performed for a range of k . Cross-validation error per K plots are reported in After a quality control filter, LD pruning was performed on the quality filtered data  using the “--indep 1” PLINK  command 1” PLINK  was used1” PLINK , which uplotSelection” option , while for OPCP, and OPCW, values between 0.36 and 0.35 were found. Similar results have been observed for BCL and NCL sheep breeds (o) varied between 0.37 in OPCP. At the same time, it was OPCS for 0.36 and 0.35 for OPCE, and for wool sheep (NCL and BCL) indicated values between 0.31 and 0.35, respectively. These results show that the (Ho) values were consistently lower than (He), except for OPCP. Average MAF values , while the hair Creoles from the Caribbean  and wool Creoles (BCL and NCL) indicated a low value of FST (0.01 and 0.05). At a global level, the OPCP showed low values of FST with all Brazilian hair breeds (0.11) and Barbados Black Belly from the Caribbean (0.08). The same happens with OPCE, OPCS, OPCW, and the wool BCL. The NCL breed generally showed low FST values compared with other wool-type American breeds, and the closest relationship was with the Brazilian Wool Creole (BSC) (0.05). WAD, from the Atlantic coast of Africa, showed a lower value with OCPE, OCPS, and OCPW (<0.1). Creole hair sheep from Colombia showed a low FST (<0.16) with some African breeds such as the African White Dorper (AWD), African Dorper (ADPR), Egyptian Barki (EBI), Norduz (NDZ), Qezel (QEZ), Ronderib Afrikaner (RAD), and Ethiopian Menz (EMZ). In contrast, MacArthur Merino (MCM) and Namaqua Afrikaner (NQA) breeds show an FST > 0.27 with all the Colombian Creole breeds.Pairwise F results , calculaIn the MDS analysis , as wellA Neighbor-Net network constructed using 42,294 SNPs generally grouped all the populations according to their geographic origin . ColombiThe Admixture in The haplotype exchange analysis between populations  is basedThese results align with what was found in the ADMIXTURE analysis  when it The MDS graph constructed using the prime due dimensions for the sole six Colombian populations  shows a E differentiated from the OPCW, OPCS, BCL, and NCL. At the same time, the OPCP remains heterogenous but differentiated from the rest across all the tested K. At a higher value of K, the populations were progressively assigned to separate groups but with no homogeneous classification of every single individual. NCL and BCL breeds have a common genomic background, even if the NCL has a lower degree of admixture than the BCL. Concerning the hair-type creole sheep, both a high degree of admixture and an intense subdivision into sub-groups within each breed entity are appreciable at K 10 and 14. Curiously, although the OPCW is geographically separate and phenotypically different from the wool creole, they share a common genomic component with NCL and BCL, confirming the previous analysis.The population structure for the six Colombian sheep breed sheep was inferred separately by ADMIXTURE , consideUsing a K-NN of 30, all samples showed significant interconnection in a cluster where OPCP is at one extreme, connected to a central group constituted by OPCS, OPCW, and OPCE. NCL and BCL formed a divergent group directly connected to OPCE and OPCW individuals at the other extreme. Interestingly, some OPCE and OPCS samples remained isolated in independent groups even at this level.South American sheep breeds, called creoles, are typically animal descendants from the introductions in the colonial age. Still, this term is generally used to define animals that are in a certain way autochthonous of the South American continent.For decades, Colombian creole hair sheep  have been misnamed by foreign names, while the scientific community has erroneously denoted them as Ethiopian Sudan . MeanwhiThe diversity parameters of the six breeds distributed throughout the national territory  indicatee values (0.30 and 0.35) lower than those found in Colombian Creoles. Comparing Colombian Creole sheep with African breeds, for example, the Namaqua Afrikaner breeds (He = 0.22) and other European breeds such as the Merino (He = 0.321) [e was lower than those found in this study. This difference seems to be related to the recent crossbreeding experienced by Colombian breeds. Although European breeds show higher heterozygosity values, Deniskova et al. [e for the Baikal Fine-fleeced breed (0.39). Similarly, Mastrangelo et al. [e values for Sicilian breeds range between 0.37 and 0.39.On the other hand, Spangler et al.  in Creol= 0.321) , He was a et al. , using to et al.  reportedIS values were found for OPCS, OPCW, NCL, and BCL  in seven Brazilian sheep breeds (five hair types and two wool types), slightly higher values than the OPCS, OPCW, NCL, and BCL breeds.In the Valle del Cauca nature reserve, where these animals were sampled, directed matings and a certain degree of management and selection are carried out, including ram interchange, which would probably explain the small inbreeding in these populations. Vivas et al.  indicateThe population structure for OPCP was the most divergent among the Colombian populations, observed in all the statistical tests. Caicedo  reportedOn the other hand, the Colombian hair types OPCE, OPCS, and OPCW, although phenotypically different ,50, seemHowever, the arrival of the first hair sheep in the country is unclear. Probably some specimens arrived at the time of the slave trade from the African continent ,2. The pST < 0.014). Similar results were obtained by Revelo et al. [Another important finding was that wool  and hair types , although geographically separated, presented very little genetic differentiation  or camuro  are used. In the global comparison, both the DMS , while in 1970, 25 Persian breed specimens were introduced to the Huila region near Cali . These a the DMS  and Admi the DMS  analysesThe WAD breed deserves an additional discussion as, being a West African genetic type, it would represent the possible introductions of animals during the late colonial era. Spangler  proved sIn all the statistics, genetic divisions were detected that separate the European, African and Brazilian sheep from the Creole sheep of Colombia. Similar results were reported by Paim et al. , Kijas eThe extant scientific information on south American sheep breeds is limited because they are usually reared in marginal and low input systems in a region where cattle is the most important genetics resource. However, sheep are strategically important for many people in South America for their adaptive capabilities to the different agroclimatic conditions. Promoting their recognition and characterizing their production ability and genetic uniqueness is necessary. In this scenario, the knowledge of the population structure and the genetic diversity of the Colombian Creole sheep becomes essential. In this work, we analyzed all sheep breeds in Colombia, including the Wayuu breed (OPCW). The results show that the genetic basis of Colombian sheep is of European origin, as seen with mitochondrial DNA. Subsequently, various introductions of animals of African origin followed through different geographical routes and at different historical moments.


Since the early days of DNA sequencing using the Sanger dideoxy synthesis  and MaxaApplication in Evolutionary Novelties and Diversities: Medicine, Agriculture, and Conservation, to yield more insightful intellectual chemicals.”As an old science, evolutionary biology occupies a central position in the biological sciences. Most evolutionary models and theories were developed early with the establishment of evolutionary biology and population genetics. Until recently, however, questions about the genetic basis of evolutionary novelty and biodiversity could be answered directly through in-depth analysis of sequences and molecular biology technologies. Our knowledge of biodiversity and novelty is growing rapidly and enormously in species of academic, social, and economic importance. In light of this critical bridging role of sequencing data and biotechnology between questions and applications, we have launched a Frontiers Research Topic: “etc. In this Research Topic, young genes were identified and analyzed in a plant species, Olea europaea subsp. Cuspidate, a widespread close relative of olive. Notably, a burst (19.5%) of gene transposition events was detected in the common ancestor of olive subspecies, suggesting the importance of the emergence of new genes in the evolution of olive species.Broadly speaking, the Research Topic aims to promote practical applications of genetic markers in medicine, agriculture, and conservation and exploration with evolutionary novelties and/or diversity. The novel genetic elements of a species have long attracted special interest. Among these markers, evolutionary young genes are known to play important roles in phenotypic novelties with medical importance , adaptivnuclear genetic diversity and structure of wild populations of Anastrepha ludens were analyzed with microsatellite markers. Gálvez-Reyes et al. examined nine microsatellite loci and answered interesting questions about population diversity, structure, gene flow, and population size of the Mexican fruit fly, Anastrepha ludens, an important pest that causes widespread damage to a range of fruit crops in Mexico. In addition, Sun et al. conducted a landscape genetic study and linked genetic differentiation to bioclimatic factors for the parasitoid Stethynium empoasca. Guan et al. produced a genome assembly for Luehdorfia taibai, an endangered butterfly endemic to the Qinling Moutains in China with extremely small populations, which showcases the whole-genome as a powerful and complete biomarker to study conservation biology. Ke et al. conducted temporal sampling and network analysis on Plutella xylostella and uncovered rapid population change and dynamic migration patterns in overwintering regions of this cosmopolitan pest. Guo et al. conducted a mendelian randomization study based on single-nucleotide polymorphisms (SNPs) to analyze the causal relationship between vitamin D and uterine fibroids, demonstrating the applications of genomic variants in addressing questions of cause and effect with medical considerations. Mamat et al. performed population genetics for Tolai hares, Lepus tolai, in Xinjiang, China, using genome-wide SNPs from SLAF-seq and mitochondrial markers. Mu et al. performed transcriptome analysis to understand the mechanisms underlying the adaptation of plateau pika, Ochotona curzoniae, on the Qinghai-Tibet Plateau. Tian and Ma proposed to use natural hybrids in horticulture to accelerate the breeding of woody ornamental plant. Although the specific questions are different, these studies demonstrate the power of genetic, genomic, or transcriptomic data at species or population level in address questions involving biodiversity and evolution.In addition to evolutionarily young genes, other biomarkers, such as microsatellites, are also extensively explored for conservation genetics. In this Research Topic, the In summary, this Research Topic has gained updated knowledge input from multidiscipline scientists who studied genetic markers considering the importance of novelty and diversity. Future fine-scale studies and multi-omics data from more diverse species and geographical populations would enrich our understanding of the evolutionary significance of genetic novelty and biodiversity on our planet.


Scurria viridula and Scurria zebrina, that occur in parapatry and whose poleward and equatorward range edges intersect in the 30–35°S SEP biogeographic transition zone. We sampled 118 specimens sourced from nine sites from Tocopilla (22°S) to Chiloé (41°S) including one site where both species overlap and analyzed over 8000 biallelic single nucleotide polymorphisms. We found evidence of hybridization between these species in the contact zone and found significant but contrasting population structures for both species. Our results indicate that the genetic structure in S. viridula, which is currently expanding its range poleward, follows a simple isolation by distance model with no traces of natural selection (no evidence of outlier loci). In contrast, S. zebrina, which has  its equatorward range edge at the transition zone, displayed a pronounced genetic break approximately at 32–34°S, along a region of marked environmental heterogeneity in association with a semi‐permanent coastal upwelling regime. For S. zebrina we also found 43 outlier loci associated with this genetic break, with a significant proportion of them clustering in a single linkage group. This marked difference in the presence of outlier loci between species suggests that they could be responding differently to local environmental challenges found at their overlapping geographic range edges, thus providing important new insights about genomic changes around biogeographic transition zones in sister species and the forces that shape genetic diversity in intertidal marine species.The study of sister species that occur in parapatry around biogeographic transition zones can help understand the evolutionary processes that underlie the changes in species composition across biogeographic transition zones. The South Eastern Pacific (SEP) coast is a highly productive coastal system that exhibits a broad biogeographic transition zone around 30–35°S. Here, we present a comparative genome‐wide analysis of the sister species  Here, we report a novel comparative population genetics study of two parapatric sister marine limpets endemic to the South Eastern Pacific and whose poleward and equatorward range edges intersect in the 30–35°S SEP biogeographic transition zone. We used RADseq to survey thousands of SNPs along with the distribution of both species, including one locality where they overlap. Our results provide evidence of hybridization between species and contrasting patterns of genetic structure between species. These species appear to be responding differently to local environmental challenges found at their overlapping geographic range edges, thus providing important new insights about genomic changes around biogeographic transition zones in sister species and the forces that shape genetic diversity in intertidal marine species. It is a heterogeneous seascape characterized by an intense and spatially structured coastal upwelling circulation  belongs to the most ancestral group of living Gastropods, the order Patellogastropoda  data, all endemic to the SEP  and Scurria zebrina . These sister species diverged approximately 15 My ago  and Huentelauquén (31.6°S), and S. zebrina were collected between Huentelauquén and Chiloé (41.8°S)  of the Chilean government.2.3DNA was extracted using the GenJet Genomic DNA purification kit  following the manufacturer's protocol. Genomic DNA was shipped to Floragenex  where libraries were prepared using the restriction enzyme SphI, fragment size selection of 300–500 bp, and individual 10 bp barcodes for each sample. Two libraries were constructed and sequenced using Illumina 4000 technology, 101 bp single end.Sequences were quality‐filtered and demultiplexed using the process_radtags module of STACKs vers. 2.53  using bowtie2  with alpha = 10. We also used the program NewHybrids v1.1  and individuals of S. zebrina from the southernmost sampling site (CH) were assigned as pure individuals for each species, respectively. We used the R package ParallelNewhybrids  using the “poppr.amova” function from the poppr R package  passed population filter constraints. Of these 2188 were variable. For the dataset including S. zebrina samples only, 12,784 loci  passed population filter constraints. Of these 4424 were variable.Stacks assembled a total of 393,581 RAD‐tag derived loci. For the dataset including samples from both species, 8966 loci  passed population filter constraints . Of these 5288 were variable. For the dataset including oH) and expected (eH) heterozygosity, FIS and genetic diversity (π) per sampling location are presented in Table S. viridula: min =0.0004, max =0.00043, S. zebrina: min =0.0008, max =0.00087) but was slightly higher in the northern sampled localities in both species. Genetic diversity values in S. zebrina were on average two times higher than in S. viridula.Summary statistics including observed  fell in between the northern population group of S. zebrina samples and S. viridula samples group when plotting PC1 against PC2. Also, four samples classified as S. viridula in the field clustered with S. zebrina samples from the northern localities (HU and TA) , and two S. zebrina samples from HU as backcrosses (S. zebrina × F1). All assignments had a posterior probability = 1.0.PCA results when considering the full dataset (both species) separated samples into three major groups. PC1 clearly discriminated between samples from both species, while PC2 separated two groups of populations within a Figure . PC3 fura Figure . Interes) Figure . LEA res3.3S. viridula, the results of the PCA separated individual genotypes into three clear groups , 2% was explained by differences between samples within populations (FIS = 0.020), and the rest of the variation was explained by variation within samples. For S. zebrina, results from AMOVA showed that 5.7% of genetic variation was explained by differences among populations  and 3.6% of the variation was explained by differences among samples within populations (FIS = 0.037).The AMOVA results showed that for 3.4S. viridula . For S. zebrina, the correlation between genetic and geographic distance matrices was significant but weaker . A visual inspection of the correlation between genetic and geographic distance matrices clearly shows that for S. viridula genetic distance between populations increases linearly with geographic distance. For S. zebrina, this relationship is not linear  with a genetic break at 32–34°S. Below, we discuss these results in the geographic context where these species are found. We also discuss the perspectives and novel opportunities this system offers to study hybridization and to better understand the role of evolutionary forces shaping genetic diversity in intertidal marine species  from the remaining sites to the south (29–31°S). This genetic transition coincides approximately with the 30°S biogeographic break. We note, however, that for S. viridula, the genetic composition of samples between 29 and 31°S is homogeneous and differentiation further north follows neutral expectations and isolation by distance. Our sampling design indicates that a genetic break in this species, if present, lies somewhere between 25 and 29°S. Yet, the clear pattern of isolation by distance and the lack of selection footprints suggests that genetic differentiation for S. viridula is rather gradual and could be explained simply by the limited dispersal potential in these organisms  is that intrinsic pre‐ or post‐zygotic genetic incompatibilities are in fact responsible for this genetic differentiation pattern but are coupled with exogenous barriers associated with ecological selection  displays clear footprints of divergent selection, including 43 outlier loci. We also found that a large proportion of these outliers was concentrated in one of the linkage groups, a pattern that is consistent with the idea of divergent selection operating over a narrow genomic region  could have adapted to the heterogeneous environmental conditions of this transition zone, which would explain its high densities, high genetic diversities, and natural selection footprints. We note, however, that it is also possible that these highly divergent regions could be shaped by differential introgression resulting from the hybridization of these two lineages and complex demographic histories  were highly differentiated from other populations further south, away from this transition zone and this differentiation involves several loci that appear to be under divergent selection. Indeed, the phenotypic response of range edge populations at Talcaruca for S. zebrina, were significantly different from populations inside the range  have shown that The authors declare no conflict of interests.Pablo Saenz‐Agudelo: Conceptualization (lead); Data curation ; Formal analysis (lead); Funding acquisition (lead); Investigation ; Methodology ; Project administration (lead); Resources (lead); Validation ; Visualization ; Writing – original draft (lead); Writing – review & editing . Livia Peluso: Investigation ; Methodology ; Resources ; Writing – original draft (supporting); Writing – review & editing . Roberto Nespolo: Conceptualization ; Formal analysis ; Investigation ; Writing – original draft (supporting); Writing – review & editing . Bernardo Broitman: Conceptualization ; Data curation ; Investigation ; Methodology ; Writing – original draft (supporting); Writing – review & editing . Pilar A. Haye: Investigation ; Resources ; Writing – original draft (supporting); Writing – review & editing . Marco Lardies: Conceptualization ; Formal analysis ; Investigation ; Methodology ; Writing – original draft (supporting); Writing – review & editing .


Pisum sativum L.). To examine the powdery mildew pathogen’s morphological, molecular, and genetic diversity, we collected samples of powdery mildew-affected pea crops from ten distinct locations in the Nilgiris district of Tamil Nadu, India. The pathogen Erysiphe pisi was identified morphologically based on anamorphic characters. Molecular identification of E. pisi isolates was befitted by targeting the internal transcribed spacer (ITS) region of rDNA and specific primers of powdery mildew fungi. The genetic variation between ten different E. pisi isolates collected from topographically distinct mountainous areas was studied using random amplified polymorphic (RAPD). Based on its morphological characteristics, the powdery mildew fungus presented high similarities to E. pisi. Molecular characterization of the ITS rDNA of E. pisi produced 650 bp nucleotides, PMITS  primers produced 700 bp nucleotides, and an Erysiphe specific ITS primer pair amplified and synthesized 560 bp nucleotides. According to the findings, the collected E. pisi strains exhibited a low level of genetic diversity and only a slight differential in virulence on the host. In the study, E. pisi isolates from Anumapuram, Emerald Valley, Indira Nagar, and Thuneri showed a greater disease incidence in the natural field conditions and shared the same genetic lineage with other isolates in UPGMA hierarchical cluster analysis based on RAPD markers. There was no evidence of a link between the occurrence of the disease and these grouped populations.Powdery mildew is an omnipresent disease that reduces the yield and quality of pea crops ( According to the Legume Taxonomy Working Group, the family Leguminosae  is the third most diverse and socio-economically influential flowering family. It is a single monophyletic group that includes highly proteinaceous beans, legumes, and peas. There are over 800 genera and 23,000 species in this group, and they have been climatically resilient, cosmopolitan in distribution, and cultivated worldwide . Along wPisum fulvum Sibeth. & Sm. and Pisum sativum L. are crucial cultivated species for fixing atmospheric nitrogen in the soil through a symbiotic relationship with Rhizobium bacteria. Due to its high protein content, the pea crop is an ideal animal feed and a suitable meat substitute for human nutrition. Mendel’s discovery of inheritance laws used the P. sativum (2n = 2x = 14) as a model species, providing the framework for modern plant breeding, cytology, and genetics [P. sativum is powdery mildew, which causes economic losses on a global scale [P. fulvum is a new source of resistance in cultivated peas against powdery mildew and bruchid pest. Also, an intensive breeding program is needed for the wild species, P. sativum sub sp. elatius (Bieb.) Aschers. & Graebn., to develop resistant pea genotypes [er1, er2, and the single dominant gene, Er3, are being extensively exploited using linked markers in breeding programs worldwide [Pea  is a widely domesticated winter crop of the Leguminosae family consumed as a pulse and a green vegetable. al scale . The maiorldwide . As a reErysiphe pisi DC , a filamentous fungus responsible for 25–70% of yield losses in pea cultivation [Erysiphe pisi var. pisi DC. Furthermore, species such as Erysiphe trifolii Grev. or Erysiphe baeumleri U. Braun and S. Takam., in addition to E. pisi, may affect peas in specific environments [E. pisi is naturally an obligate biotrophic fungus, only minimal information about its genotypic diversity, epidemiology, or the importance of population dynamics is available in the Nilgiris District of Tamil Nadu.Pea powdery mildew is an airborne fungal disease caused by tivation . Recentlronments ,9. Howevronments . It is aronments . Powderyronments . The mycronments ; since EIn pulse crops, rapid and accurate pathogen identification or disease diagnosis is essential for providing a suitable recommendation for sustainable disease management. Conventionally, powdery mildew species are identified based on the morphological characteristics of the anamorphic conidia and teleomorphic chasmothecia over the infected plant surface . Due to E. pisi have been concerned and demonstrated the applicability of molecular markers for diagnosing and analysing E. pisi diversity. The internal transcribed spacer region (ITS) of fungi has been sequenced routinely. It has been confirmed to assist in developing molecular systematics at the species level and even within a species [E. pisi from pea fields in 10 different places in the Nilgiris district of Tamil Nadu were molecularly characterized using the ITS region and the RAPD marker used to reveal its genetic diversity. Based on the aspects mentioned above, the purpose of this study is to determine the genetic diversity of pea powdery mildew fungus (Erysiphe sp.) in the Nilgiris as well as to correlate any differences between isolates in terms of physiological, virulence, and regional variability by employing both specific PCR amplification and RAPD marker analysis.Molecular markers are becoming increasingly relevant for studying taxonomic and evolutionary relationships among various fungi . These m species . In addi species . Among t species ,19. In tWe collected pea powdery mildew diseased leaves from ten growing villages in the Nilgiris ecosystem to study the phenotypic and genotypic characteristics of the pea powdery mildew pathogen. Then, the anamorphic propagules of isolates, including mycelium, conidiophores, and conidia, were separated from the infected leaves under aseptic conditions. These isolates were designated based on the regions, Anumapuram (P-ANU1), Emerald Valley (P-EMV1), Indira Nagar (P-IDN1), Thuneri (P-TNI1), Iduhatty (P-IHY1), Kenthorai (P-KTR1), Bigatti (P-BGI1), Palada (P-PLD1), Coonoor (P-CNR1), and Kotagiri (P-KGI1), for further studies . The colE. pisi isolates were established at a genus level using ITS-1 and ITS-4, and the 5.8S ribosomal DNA (rDNA) region of the genus was amplified using ITS-1 and ITS-4 universal forward and reverse primers [E. pisi isolates. The PCR amplification was performed as per the protocols cited [Erysiphe-specific PCR products with the comparison of 1 kb marker already loaded on the gel along with the samples. primers . Then, t primers  and EryF primers  were usels cited  using anE. pisi  separately. Using a thermocycler  amplification was performed under the following cycles: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 36 °C for 1 min, extension at 72 °C for 2 min, and final elongation at 72 °C for 5 min. The amplified PCR products were subjected to a 1.5% agarose to separate the amplified products by primer sequence. Subsequently, gels were then individually illuminated with a UV transilluminator and photographed using the BioRad gel documentation systemTwenty random amplified polymorphic DNA (RAPD) oligo-primers  were used to determine the genetic variability among the ten isolates of E. pisi . The 20 E. pisi populations.After repeating the RAPD-PCR experiment three times, only the consistently randomly amplified bands that emerged were evaluated. We started with the most considerable fragment and counted the banding patterns of each RAPD primer, moving down to the smallest fragment. Each band present or absent in each isolate was assigned a 1 or 0. A similarity matrix analogue was created to disclose genetic relationships using a statistical tool called Plymouth Routines in Multivariate Ecological Research (PRIMER v7) by Quest Research Limited, New Zealand. This multivariate analysis was produced by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). It evaluated the similarities, dissimilarities, and distances using hierarchical cluster analysis (HCA) with the Bray–Curtis coefficient. Non-metric multidimensional scaling (NMDS) was used to scale unorganized E. pisi isolates was studied based on its polymorphism. We quantified the total number of bands, the number of monomorphic and polymorphic bands, the number and frequency of bands in each primer, and the ratio of polymorphic to monomorphic bands. In addition, we applied the developed formula to calculate Polymorphic Information Content (PIC).The efficacy of primers in determining the genetic variability of Erysiphales is a taxonomic order of ascomycetous fungi that cause powdery mildew disease with a nearly global distribution. This order consists of 18 genera and about 900 species [Erysiphales is the most common parasitic fungus and affects over 10,000 plant species, including many vital to the economy [Erysiphe is the largest, compared to additional teleomorphic genera, and accounts for about 50% of powdery mildew genera. Powdery mildew species develop within plant tissue and are generally epiphytic, forming symptomatic white mycelial mats on almost every plant organ [Erysiphe spp. have already been conducted in various geographical areas. Most often, abundantly produced asexual spores are spread by the wind and cause the infection. Occasionally, a sexual recombination process develops more virulent strains for widespread adaptability to varied habitats [E. pisi, is a severe and recurring disease. According to Bahadur et al., 2008 [E. pisi fungal parasite penetrates chloroplast-deficient epidermal cells through haustoria. Furthermore, initial mycelial colonization starts on basal mesophyll cells and then produces superficial mycelia over the plant surface, which interrupts the photosynthetic activity, and a severe outbreak can alter total biomass, plant height, pods per plant, seed yield, and node formation, resulting in crop death and production losses of up to 50% [ species . Erysiph economy . Erysiphnt organ . Numerouhabitats . Due to l., 2008 , pea powp to 50% . In Indip to 50% .E. pisi were studied for characteristics such as colour, shape, and size. The observations showed that the fungal hyphae were superficial, ectophytic, thin-walled, septate, and branched over the leaf surface, anchored with the support of absorptive haustoria. Conidiophores are cylindrical, with three to four cells, and produce chains of hyaline, single, thin-walled, immature ellipsoid-ovoid to mature dolliform  conidia  revealed an amplicon of 700 and 560 bp, respectively  based on the Bray–Curtis similarity index, which accurately distinguished individuals of E. pisi . This scE. pisi . As per E. pisi, leading us to conclude that the pathogen has evolved the ability to adapt to both the macro- and micro-environments to which it is exposed during pea cultivation. The RAPD primers revealed the genetic variation among E. pisi isolates through the amount of polymorphism. In addition, dealing with an obligate parasite such as E. pisi challenges the selection procedure for developing powdery mildew resistance. To overcome these challenges, most RAPD markers studied were linked to finding powdery mildew resistance genes, which may play a prominent role in recognizing resistance loci and pyramiding resistance genes in various pea cultivars [In most genetic and taxonomic studies of different fungi, nucleic acid-based molecular markers, such as Random Amplified Polymorphic DNA (RAPD), have been utilized to characterize the pathogen’s diversity in the study area. The ability of a pathogen to adapt to its environment is reflected in its genetic diversity. As a result, the degree of pathogenic variation would contribute to determining the degree of adaptability. Our research discovered a substantial genetic variability in ultivars .E. pisi genetic variation occurs within a specific region in a particular period, further validating its use in marker-assisted selection [In the present research, RAPD primers produced many polymorphic bands, indicating pairwise genetic distances in the genomic region targeted by the primers possessed a high level of variation. P-ANU1, P-EMV1, P-IDN1, and P-TNI1 isolates were collected from geographically nearby locations with a greater incidence of disease in the field and belonged to the same genetic lineage as other isolates. There was no indication of a link between the disease occurrence and these populations in the region. According to the study’s findings, the RAPD marker is more useful for defining small pathogenic clade or closely related clade with sequence similarities. As a result, the RAPD marker can be used to determine how much election .Over many generations, powdery mildew isolates subjected to ongoing evolutionary pressures, either with efficient vertical resistance genes or fungicides, might demonstrate considerable adaptability. In this study, it was evident that low-level genetic variability among the pathogens, favourable climatic conditions for pathogen infection, and selective host parasitism on peas in the region contributed to a wide range of potential influences for the rapid spread of pathogenic species. This work explored some powdery mildew-infested biogeographical regions that could provide valuable scientific insight to promote the resistant cultivars of those remaining locations. Additionally, this is the first investigation into the fungal diversity in the Nilgiris biosphere region. Furthermore, collecting additional powdery mildew samplings of peas from wider regions is desirable for better marker-assisted selection in future work.formae specialis/strains. Understanding the degree and spread of genetic variation among pathogen populations assists in defining its ecology and designing efficient standard precautions against infection. Understanding evolutionary dynamics brings us closer to predicting disease population increases in agricultural habitats. This knowledge is beneficial for plant pathologists and breeders seeking to develop appropriate management tactics.Pea is the most widely cultivated pulse crop in the Nilgiris region of India. Diseases such as powdery mildew, however, have dampened its productivity. Powdery mildew, the most frequent field pea disease in the Nilgiris district, can be identified and characterized as a result of this research, which will aid in the detection and characterization of the disease. The use of polymerase chain reaction (PCR) for molecular investigations helps clarify the identification at the species level as well as the genetic diversity between isolates. Identifying appropriate and long-lasting control strategies is a massive obstacle to sustainable agricultural research in pea. Also, to maintain the lifespan of recently developed resistant cultivars in the region, it is crucial to understand the genetic variation of local fungal populations and the mechanisms that contribute to distinct