The two “berrenda” cattle breeds are important for the conservation of livestock genetic diversity in Spain. They have a great phenotypic and genotypic uniqueness and both of them are important from the cultural and the tourist perspectives. They also contribute to the conservation of the traditional “Dehesa” ecosystem. Both breeds are considered as endangered ones, but their genealogies have never been used for quantifying the risk status of their populations. The aim of this work was to monitor the structure of the “Berrenda en Negro” and the “Berrenda en Colorado” populations, their inbreeding rate and some other parameters that could be useful to prevent losses in their genetic diversity and to conduct and analyze the effect of the conservation programs developed by ANABE Breeders Association and finally, as a tool to implement some selective measures. We found that both “berrenda” cattle breeds retain a huge genetic variability from their founders’ populations, although they have been affected by a shallow depth in their pedigrees; as a consequence, we suggest increasing exchanges of breeding animals among herds, more specifically in the case of the “Berrenda en Negro” breed.Pedigree analyses of two endangered cattle breeds were performed in order to study the structure and the genetic variability in their populations. Pedigree data were analyzed from 12,057 individuals belonging to the “Berrenda en Negro” cattle breed (BN) and 20,389 individuals belonging to the “Berrenda en Colorado” cattle breed (BC) that were born between 1983 and 2020. BN and BC reference populations (RP) were set up by 2300 and 3988 animals, respectively. The generation interval in BN and BC reference populations was equal to 6.50 and 6.92 years, respectively. The pedigree completeness level was 82.76% in BN and 79.57% in BC. The inbreeding rates were 4.5% in BN and 3.4% in BC, respectively. The relationship among animals when they were born in different herds was 1.8% in BN and 5% in BC; these values increased to 8.5% and 7.7%, respectively when comparing animals that were born in the same herd. The effective number of founding herds was 23.9 in BN and 60.9 in BC. Number of ancestors needed to explain 50% of genes pool in the whole population was 50 and 101, in BN and in BC, respectively. The effective population size based on co-ancestries was 92.28 in BN and 169.92 in BC. The genetic variability has been maintained in both populations over time and the results of this study suggest that measures to promote the conservation of the genetic variability in these two breeds would go through for the exchange of breeding animals among farms and for monitoring the genetic contributions before implementing any selective action. Farm Animal Genetic Resources in Spain possess a remarkable variability as a consequence of the country unique geographical location between Africa and Europe. In fact, 46 autochthonous cattle breeds are officially recognized . Among aAlthough the origin of the “berrenda” breeds is considered as ancestral and previous to the Columbian era , the firRegarding their phenotypes , both abBerrenda en Negro and Berrenda en Colorado herds are geographically dispersed in “Dehesa” ecosystems, located in southern and middle Iberian Peninsula  Figure . “DehesaIn addition, and due to their natural character and behavior, these breeds can be easily taught, what makes them to be of special interest among all the others Spanish autochthonous cattle breeds . Oxen ofIn addition, Cañón et al.  have repIn summary, these are two autochthonous cattle breeds of high conservation interest, due to multiple reasons: historical, religious and cultural, linked to local traditional events that are important for the socioeconomic development of the regions where they are reared, mainly linked to tourism activities. They also contribute to the sustainable use and maintenance of the “Dehesa” unique ecosystems, increasing their environmental and economic value by producing meats of differentiated quality, linked to specific type of animals that are reared to be slaughtered (oxen). In addition, as ancestral breeds, they are also of special interest from the genetic point of view. The prospects for the development and conservation of these breeds rely on the ability of consumers and institutions to recognize these values and on the potential of these breeds to face future challenges related to possible changes in consumers’ preferences, to sustainable economy of the territories and to adaptation to climate changes. Market demands could only be met if the implementation of breeding programs developed measures addressed to the prevention of inbreeding. Nowadays, the main objectives of the “berrenda” breeds breeding programs are the conservation of the populations (mainly at in vivo and in situ level) and the maintenance of their genetic variability. Currently, the selective activities carried out by ANABE are focused on not using carriers of phenotypic or genetic disorders, such as Robertsonian chromosomal translocation t1:29, as breeding animals  and alsoMonitoring genetic diversity and monitoring genetic trends in animal populations are the bases for the implementation of any conservation program, as well as a suitable selection of the offspring, in order to reach a sustainable livestock farming . The stoTraditionally, performing pedigree analyses was the first step towards the characterization of the genetic diversity within a population but not many works of this kind related to small populations have been published yet . On the Although the first registrations of animals in BN and BC studbooks took place in 2001, the traditional rearing system under extensive conditions, in combination with the insufficient control of mating by breeders made quite difficult to correctly assign the paternity to the offspring, what negatively affected to the amount of information provided by pedigrees, in the first stages of the breeding program . Since tIn the case of “berrenda” cattle breeds, genetic diversity and paternity of the offspring are annually monitored by analyzing DNA microsatellites, according to FAO recommendations . AlthougWe can find some studies on main Spanish autochthonous cattle breeds based on pedigree data ,42,43,44As a contribution for the development of the conservation programs in both “berrenda” cattle breeds, this work aims to study the current genetic variability and the structure of BN and BC populations based on thorough analyses of their corresponding pedigrees, by quantifying the changes that happened over the time and comparing them to other different autochthonous cattle breeds that have also been traditionally reared in the “Dehesa” ecosystems, following a quite similar farming system.To be more precise, the objective of this work are: (i) characterizing the structure of BN and BC populations by ages and sex and type of herd; (ii) identifying the genetic variability level and the threat of inbreeding in both populations; (iii) identifying the genes origin in both populations. It is also intended to know the possible loss of genetic.This information will be quite useful to establish the most suitable strategies to be implemented in the conservation and selection programs of these valuable genetic resources in risk of extinction.Based on the records from BC and BN breed’ studbooks and after removing all those founders without descendants, the analyses were performed over the BN and BC breeds whole populations.Subsequently, the reference populations were made up of live animals born during the last 7 years (from 2013 to 2020). According to that, BN breed reference population was composed by 2300 animals and BN breed reference population by 3988 individuals. This period has been established, according to the average generation interval from live animals in the whole populations.® for Windows, US, some tables and figures, based on different values: number of animals by sex, number of animals by year of birth, as well as a description of the structure of the populations according to the age of the registered animals in the studbooks that are still alive and the number of registered descendants.For descriptive purposes about the structure of both populations, we have designed on Microsoft Excel 365(i)Generation interval: It is defined as the average age of parents  when its replacement is born ; it is c(ii)Proportion of present ancestors per generation: It was estimated in order to analyze the pedigree completeness level ;(iii)n is equal to the number of generations between the individual and its known ancestor; the individuals without known ancestors were assigned to the base generation [F) and the Average Relationship Coefficient (AR) have been calculated to analyze the inbreeding and the genetic weight of every individual over the whole population. F is defined as the likelihood that an individual carries two identical genes by descent. AR is defined as the average genetic weight of every individual over the whole population and it is equal to the average coancestry coefficient of every individual related to the other members of the population [Pedigree depth: It is calculated by considering the equivalent number of discreet generations equals to neration . The maxpulation ,50.Furthermore, to describe the structure of the studbooks and the integrity of the information than they contain, we analyzed the next parameters in both breeds:∆F) and the Inbreeding after 10 years (10F) and after 50 years (50F) were calculated according to the methodology proposed by Gutiérrez and Goyache [i represents the individual inbreeding and t is the equivalent per complete generation.Moreover, the Inbreeding rate per equivalent generation  and the Effective Population Size after 10 and 50 years (e10N and e50N) were calculated based on the increasing of the individual inbreeding in the animals included in the Reference Population, according to the eN value obtained from the approximation by Gutiérrez et al. [ΔC), following the methodology proposed by Cervantes et al. [In addition, the Effective Population Size Effective number of herds that are rearing sires performing as grandparents, great-grandparents and great-great-grandparents: It was calculated according to Robertson  and it i(ii)ef) that equally contributes and determines the existing genetic diversity in the population: It was calculated according to q1 represents the genetic contribution to the population of founder [Effective number of founders ( founder .(iii)af): It is defined as the number of ancestors, founders or not, that are needed to explain the population whole genetic variability [Effective number of ancestors (iability .(iv)af/ef reveals the possible bottlenecks that could have affected the population under study. It also considers the genetic variability provided by an animal that could not be explained by the contribution of its offspring. It was calculated according to Boichard [Marginal contributions of ancestors Boichard .(v)gf): It can be defined as the number of founders that would lead to a similar genetic diversity in the population under study if the founders were equally represented and with no loss of alleles [Number of Founder Genome Equivalents GCI): It is calculated based on the genetic contributions of all the individuals considered as founders (ip) [Genetic Conservation Index (ers (pi) . Accordi(vii)GD) in the reference population, accounting for loss of diversity due to genetic drift and the unequal contribution of founder was calculated as follows [The amount of genetic diversity  was calculated as follows [The amount of genetic diversity in the reference population accounting for loss of diversity due to the unequal contribution of founders The degree of genetic differentiation among herds, considered as the contribution level of the populations to the whole genetic variability, were estimated using Wright’s F statistics (1978) according to Caballero and Toro  and wereSimilarly, the loss of genetic diversity due to the unequal contributions of founders was estimated by 1 − of 2Nenf .(viii)TLastly, all the previous parameters were calculated using ENDOG Software (V. 4.8) and they all have been described in detailed by Gutierrez et al. .Berrenda en Negro and Berrenda en Colorado official studbooks started up in 2005, after the registration of 2651 cows and 523 bulls and 1626 cow and 246 bulls in the BC and BN studbooks, respectively.According to birth dates , the annSimilarly, to many other rustic livestock breeds that are extensively reared in the “Dehesa” ecosystem, the reproductive parameters are not quite good. In the case of BN, the average age of parents when their first offspring is born is 5.51 ± 3.68 years for dams and 3.20 ± 1.44 years for sires. In BC, the obtained values have been quite similar: 5.27 ± 3.40 years for dams and 3.56 ± 1.67 for sires. Although there is a huge variation among animals, the average age of parents when their last offspring is born is 8.63 ± 4.50 years for dams and 5.85 ± 2.71 years for sires belonging to BN and in BC are 8.63 ± 4.48 and 6.48 ± 3.00 years for dams and sires, respectively.Moreover, the whole proportion of both BN and BC breeding animals that have offspring when they are more than 10 years old was 12% in sires and 36% in dams; and, 10.4% of dams went on giving birth when they were older than 15 years . Age difThe average number of descendants registered in the studbooks in the whole BN population was 2.96 ± 2.19 calves per dam and 35.30 ± 42.39 calves per sire. In the case of BC, the data are quite similar to BN, presenting 3.13 ± 2.29 registered descendant per dam and 40.61 ± 48.45 registered descendant per sire. Approximately 30% of BN sires have less than 10 descendants and 24% of them have more than 50 offspring. In the case of BC breed, 22% of the sires have less than 10 descendants and 25% of them have more than 50 descendants a. In theD in BN population, responsible of black coat color in BN what could lead to the feasible birth of red or chestnut coat color calves which cannot be registered in the BN studbook nor used as breeding animals [There are some important facts that are limiting the increase in the number of breeding animals registered in both “berrenda” cattle breeds; among them, we will stand out the following ones: (i) 50% of dams are crossbred with meat sires in order to get calves with a higher meat yield; (ii) genetic selection against calves carrying t1:29 chromosome disorder; (iii) the lack of fixation of MC1R locus dominant allele E animals  and (iv)L) considering both the BN and BC whole populations and the BN and BC reference populations are shown in L values were longer than reported by Cañas-Álvarez et al. [L = 3.7 in Avileña, 6.4 in Morucha and 6.2 in Retinta) but shorter than estimates by Cortés et al. [L values are due to Farmers’ bias in favour of the reproductive use of certain animals performing specific external traits. Most popular dams and sires might have been used as breeding animals for long periods of time and their progenies would have had a high influence on the next generation [Estimates of the generation interval  [However, it is necessary to be careful in the control of the mating strategies; on the contrary, there would be a high risk of mating between animals belonging to the same lineage, what would lead to an increase of inbreeding . High L  traits) . Genomic traits) .Generation intervals of the four different pathways were not always similar as there is a certain unbalance among the generation interval values in both “berrenda” breeds. A slightly higher generation interval was observed in the Dam-Offspring pathways than in the Sire-Offspring ones, what could be determined by replacing sires at an earlier age than dams .EqG), what could explain the higher value of this parameter in BN  in comparison to the Dam-Daughter (7.09 ± 0.09) one. According to Gicquel et al. , the exier in BN , despiteL values for dams than for sires) are coincident to those reported by Cañas-Álvarez et al. [These results , despite counting with a high number of registrations in the studbooks . Increase in the number of known generations would provide a higher certainty in the estimations of the population parameters [As a general rule, the evaluation of the population structure, based on relative information, trends to be much more efficient and precise when the number of maximum generations, number of complete generations and number of equivalent generations are high. Usually, during the first steps of every conservation and reproduction program there is an incomplete knowledge of the relationships among the animals that are registered in the studbook, what could lead to an underestimation of the average number of equivalent generations . Considerameters .EqG results are quite similar to those EqG values initially used by Gutiérrez et al. [EqG value was 9.3 in CH and 7.3 in LM, considering the French populations of these two breeds [With low pedigree depths, both in BN and in BC, there will be a trend to overestimate the consanguinity rate and to underestimate the effective population size. Nevertheless, our z et al.  in the Sz et al. . Logicalo breeds . Recentlo breeds  found vaCurrently, and according to Regulation (EU) 2016/1012 , femalesIn small populations as “berrenda” cattle breeds, phenotypic frequencies change by increasing homozygosity; therefore, reducing genetic inbreeding is the first objective in the management of at-risk populations when inbreeding depression occurs. It is also very important in breeding programs to give raise to productive offspring  and negaInb) was 63% in BN and 54% in BC (F) of the animals registered in the BN and BC studbooks was 4.5% and 3.4%, respectively. Low pedigree completeness levels (mostly in the first generations of those pedigrees that apparently are not quite deep) could also lead to an underestimation of the inbreeding levels because descendants from known parents are given an inbreeding value equal to 0, even if they are related to others [Despite not having detected any mating between parents and their offspring, the average percentage of inbred animals (4% in BC . The ave∆F) in “berrenda” cattle breeds present an increase in the consanguinity per equivalent generation (4.02% in BN and 3.01% in BC) as it is shown in ΔF and eN values found for BN and BC exceeded the recommended limits to maintain appropriate levels of genetic variability and to prevent future losses (ΔF < 1% and eN > 50); in any case, BN is in a worse situation than BC. However, we should be aware that pedigree information is still very inconsistent and estimations of ∆F and individual inbreeding coefficients are very sensitive to the quantity and quality of available pedigree information, in the light of the foregoing [The calculated inbreeding rates  Animals with inbreeding rates above 20% were 14.30% and 9.75%, for BN and BC, respectively, point out that a reduction in the inbreeding rate in both “berrenda” cattle breeds should be necessary. The fact that the average inbreeding values in the current generations of both “berrenda” cattle breeds are more than twice as high as the coancestry values, suggests that related animals have been involved in breeders’ mating plans.F) in the BN and BC reference populations were quite similar to those from other local breeds that were reared in extensive farming systems (Dehesa ecosystem) from Portugal  and Spain , despite the fact that these last mentioned breeds showed higher pedigree depths (EqG > 3) than BN and BC breeds.Considering the average inbreeding data from several worldwide cattle breeds reported by Carolino et al. , InbreedF = 1.55) or Bruna del Pirineo breeds (F = 0.25). BN and BC results are worse but in any case, better than those reported in the Pallaresa breed (F = 18.93%) with an effective population size of 4.74 [In any case much higher than estimates from Charolais and Limousin populations located in France, Sweden, Denmark, Italy and Slovakia that were below or close to 1. If we compare these data to others reported by Cañas-Álvarez et al. , regardi of 4.74 . Inbreed of 4.74 , but thiF was 7.4% in the BN reference population and 5.5% in the BC reference population, what confirms that mating within herds is mainly carried out between related animals and as a consequence of this, 9.72% of BC herds and 1.65% of BN herds presented inbreeding rates over 20%  in both “berrenda” cattle breeds  in BN and 5% (0.050 ± 0.062) in BC, increasing to 8.5% in BN and 7.7% in BC when comparing animals born in the same herd. The typical reproductive isolation of BN and BC herds in the “Dehesa” ecosystem, where bulls usually perform as sires in the same herd they have been born and the scarce exchange of animals among herds could explained those results.The average relationship coefficients  and differences between both “berrenda” cattle breeds might also be determined by the different phenotypic  [The tendency to increase inbreeding ( (t1:29) ,15 in boMinimize inbreeding such as the exchanging of breeding animals among herds or the establishment of mating programs, are limited by different reasons: (i) diseases such as tuberculosis or blue tongue are limiting livestock movements among the main geographical areas where the “berrenda” cattle herds are located ; (ii) mindset of some livestock farmers, most of them also owners of bullfighting farms, that are really involved in breeding their own lineages and that are reluctant to share their breeding animals with other farmers; (iii) general problems linked to the implementation of reproductive programs based on artificial insemination, also linked to a lack of frozen semen doses .In view of the problems for implementing rotational or systematic breeding schemes addressed to tackle inbreeding rates by maintaining family groups, ANABE has been providing breeders reports on coancestry rates between males and females at herd level. This plan is flexible and does not restrict the use of any of the sires, allowing breeders themselves to design and program the most appropriate matings, in order to minimize kinship between breeding animals at herd level thanks to a more diverse use of sires .esN) (BN = 147.05 and BC = 190.18); and also, they were estimated for the corresponding individual increasing of inbreeding for a similar contribution of founders (eiN) [BN and BC effective population sizes were estimated for their respective reference populations, based on family size variance (rs (Nei) ,53, beinesN values when considering their reference populations .Considering the imbalances affecting the information about different ancestral pathways provided by the pedigrees of the “berrenda” cattle breeds, metrics as s et al.  could bes et al. . This paaf) lower than the effective number of founders (ef), what would bring to light a decrease in genetic variation derived from bottlenecks increasing the susceptibility to diseases or by reducing its resilience to climate change [Founders’ background in the present populations of the “berrenda” cattle breeds is intended to be preserved. A small part of the genetic diversity might be lost because of the unbalanced contributions of founders and also to the effects of bottlenecks over time that populations could have experienced historically, such as those caused by pyramid selection schemes, also based on the use of artificial insemination ,81. An ee change .ef) and for the effective number of ancestors (af) were also higher  and gf/ef ratio would not suggest any unbalanced contribution of founders that could lead to a loss in genetic diversity; these parameters were slightly higher in BN, what would point out a more intensive use of certain breeding animals in comparison to BC and that could also be determined by a higher average inbreeding or a lower number of individuals under study. On the other hand, gf/af ratio was also close to 1, what would highlight no evidence of genetic drift effects on the loss of genetic variability in these breeds. Rural mechanization process has been blamed as responsible for the bottlenecks suffered by the majority of the European autochthonous cattle breeds. “Berrenda” cattle breeds were also affected by this process and but these two breeds showed a high genetic variability, that apparently had not been affected too much over the years; furthermore, apparently there had not been an excessive unbalanced use of breeding animals.In addition, equivalent number of founder genomes  and Morucha (1.24) breeds [ef/af coefficient values above 2.5 [On the other hand, ) breeds  and Portbérica 1. and Morubove 2.5 .a50N) was 50 in BN and 101 in BC. Similarly, in the reference population, only 36 BN ancestors (6.38% of the population) and 57 BC ancestors (7.01% of the population) were needed to justify 50% of the genetic variability. These results would point out a lower genetic base in BN and also that BN contribution to genetic diversity within the breed was lower than BC contribution. a50N values in BN reference population was lower than those from the seven Spanish meat breeds studied by Cañas-Álvarez [a50N value in BC was very similar to the value observed in the Spanish Avileña Negra Ibérica breed (52) [Some differences were observed in BN and BC between the contributions of founders to the current reference population and the contributions of founders to the total population . The num-Álvarez  and Na50eed (52)  and to teed (52) .Among the five main ancestors that contributed the most to BN and BC reference populations genetic variability, only one female was found in BC . In BN, GCI) [GCI value of 2.80 for the whole BN population and 2.82 for the whole BC population (Alderson’s Genetic Conservation Index (GCI)  was usedpulation .GD *–GD) varied between 0.09% and 0.01%. The 1-GD loss (derived from bottlenecks and genetic drift) in the reference population since the founder population, got a value of 0.4% in BN and 0.1% in BC, respectively. The 1-GD * loss that considers the unequal contribution of founders was equal to 0.5% in BN and equal to 0.2% in BC. The latter might be the main cause of loss of genetic diversity in the “berrenda” cattle breeds.The whole genetic diversity loss  were not relevant in number. This situation might be determined by the fact that 15% of BN and 25% BC breeders were also rearing Bullfighting cattle (“Lidia” cattle) and management of cattle of this kind is usually based on the preservation of their own familiar lineages (“encastes”) ; bullfigST (0.08 in BN and 0.075 in BC), apparently there would not be any subdivision into different subpopulations at herd level; related to FIS (−0.044 in BN and −0.046 in BC), negative values could mean that the average inbreeding rate (F) within a herd could be lower than the average inbreeding rate among individuals, in both “berrenda” breeds, pointing out that average inbreeding rate within a subpopulation would not exceed coancestry between individuals and that matings between related animals would have been avoided [The existing variability between breeds, linked to the possible differences among herds was analyzed according to Wright’s F statistics. So, considering the calculated mean values for F avoided . Accordi avoided , maintai avoided  but it wExpectations regarding the development and conservation of “berrenda” breeds would require that consumers and institutions could acknowledge their values and their potential to face future challenges related to possible changes in consumers’ preferences, to sustainable economy of the territories and to climate change. Production demands could only be met if the implementation of breeding programs developed measures to prevent inbreeding.According to our results, the strategy of minimum coancestrality within herds that is being implemented yet in the “berrenda” cattle breeds, apparently is successfully maintaining their genetic variability throughout generations. The relative isolation that is affecting some BN and BC herds due to the lack of exchanges of breeding animals among herds and to the geographical isolation established by animal health regulations may also be contributing to keep the original genetic variability within herds; but in order to tackle both loss of diversity trends and long-term increase in inbreeding that have been detected, it would be advisable to foster measures for the exchange of breeding animals among farms. Considering that probably, this could not be feasible in all herds, it would be necessary to support the implementation of complementary measures such as artificial insemination or an appropriate control of matings such as planning compensatory matings by minimizing coancestry, prolonging the reproductive use of the breeding animals but limiting the maximum offspring per couple that could lead to a significant reduction of inbreeding in the future .The choice of sires and dams to provide germplasm for in vitro conservation, as well as the selection initiatives to eliminate animals carrying t 1:29 disorder or undesirable alleles of the MC1R gene should be carried out by applying optimal contribution selection approaches, in order to achieve these objectives without increasing individual inbreeding.For all these and future actions, the current information provided by pedigrees of “berrenda” breeds would be sufficient and should be completed in the short term with the information provided by genomic tests that would allow monitoring genetic variability (such as ROH) and identifying genes linked to traits of economic interest, to disease resistance or to climate change.The analysis of the population structure of the two berrenda cattle breeds reared in extensive farming systems (“Dehesa” eco-system) revealed that they are characterized by a lack of precocity, a low number of offspring but the reproductive longevity of dams.According to our results, apparently pedigrees of these two breeds appeared to be not too deep, but they have been improved throughout the last seven years, what highlighted that the paternity monitoring established in their conservation programs was a remarkable success, thanks to the increasing number of animals with known genealogical information.Although it should be noted their rather shallow pedigrees, the average values for inbreeding and relationship coefficient estimated for both “berrenda” cattle breeds would suggest a dangerous and increasing situation of loss in their genetic variability that could be even worse over time if no additional measures were implemented. In order to prevent future losses, highlighting that the situation in BN is even worse than in BC.Currently, both the rate and the level of inbreeding in the “berrenda” breeds are lower than others’ cattle breeds that are reared in the “Dehesa” ecosystem; unfortunately, these trends would show that they would have been suffering some loss of genetic variability what would make necessary the implementation of certain measures addressed to control inbreeding.Our results showed a high presence of founder genotypes that have been maintained in the current “berrenda” cattle populations at herd level. The strategy of minimum coancestrality within herds that has been implemented in the “berrenda” cattle breeds, apparently would be successfully maintaining their genetic variability.The relative isolation that has been affecting some herds as a consequence of the limited exchanges of breeding animals among herds, the lack of implementation of assisted reproductive techniques, such as artificial insemination (IA) and also the geographical isolation established by animal health regulations, might also be contributing to the maintenance of the original genetic variability within herds; but in order to tackle both the loss of diversity trends and the long-term increase in inbreeding, it would be advisable to foster the exchange of breeding animals among farms.In this work, we have analyzed for the first time the genealogy of the two Spanish autochthonous “berrenda” cattle breeds, whose analyses provided very useful information not only to the conservationists dealing with these breeds but also to ANABE breeders. It would be necessary the implementation of suitable institutional financial support that allowed ANABE not only to continue with the adequate management of pedigree information, but also to apply genomic methodologies to achieve the conservation objectives and address future selection actions in the “berrenda” cattle breeds.


We determine the functional form and scaling law of radial artificial land use profiles in 300 European functional urban areas (FUAs). These profiles, starting from a fully artificial surface in the city center, decrease exponentially, the faster the smaller the city. More precisely, the characteristic decrease distance scales like the square root of total population, meaning that the artificial surface of cities is proportional to their population. This also means that the amount of artificial land per capita is independent of city size, and that larger cities are not more or less parsimonious in terms of land use than smaller ones. This obviously influences the relation between population and area: including or not including peripheral areas in the definition of a city has a quite important effect on its artificial area, but not as much on its population size.Cities concentrate residential locations and activities of many people, and also artificial land as a consequence. This yields a main category of definitions of urban areas, which are morphological definitions, based for instance on the contiguity of urban fabric. The artificial area of cities grows with their population, but the relationship between the two quantitities is not very clearMoreover, defining accurately and understanding cities is crucial because they are important elements in a variety of challenges which humanity is facing: they concentrate human activities and the associated pollutions, in a global context of climate change, scarcity of resources and pollution of the environment. They also gather in a reduced space extreme socio-economic inequalities, which makes a dangerous mix. Moreover, they are key nodes for the propagation of epidemics.3 has been dealing with a large variety of observables, including complex social outputs like health, crime or patents. However, it seems important to ground this research in a clear understanding of some fundamental aspects of cities, in particular their physical characteristics and spatial extent. The data necessary for this endeavour is available, at the same time very detailed and covering many urban areas, and urban scaling research needs better tools to account for urban expansion. This is of course not easy, as cities are quite heterogeneous and difficult to model. Here we use the lens of radial (center-periphery) analysis, which is a nice simplifying tool for the study of the internal structure of cities. We apply it to the study of the share of land which is artificial.Research on urban scaling5, we expect a certain scaling and radial behaviour for the artificial land use profiles of European cities. We complement this research using regression methods, and characterize the mathematical form of the radial land use profiles , and their scaling properties. We do not propose an explanatory model, but note that a numerical model reproducing the scaling law studied here is proposed6. The observed scaling behaviour can be called “homothetic scaling”4 instead of the term “isometric scaling” originally used in biology7: it states that large cities present land use profiles which are similar to those of smaller cities when those are dilated proportionally (a zoom in with the right dilation factor), which is the definition of a homothetic transformation.With the results of previous works8 and 30 Chinese urban areas9, the models use at least 3 or 4 parameters, which makes them difficult to compare to the present study.Although quite many works have studied the radial profiles of population density in urban areas, only few works have studied radial profiles of land use. In studies of 40 European cities4 were not uncovered before, despite the amount of literature studying urban radial profiles12 (among others). We note however that the results of this study can be related to the early work of Tobler13 who observed close values of scaling exponents (but not to Nordbeck14).One major specificity of the present work consists indeed in its parsimonious approach, studying hundreds of different cities with very simple mathematical tools, resulting in a very reduced number of model parameters. This may explain why homothetic urban scaling laws15. We combine this dataset with the 1 kmThis work uses the Urban Atlas 2012 dataset developed by the Copernicus land monitoring services, which provides a precise description of land use for the nearly 700 main European cities in 2012, defined at the level of functional urban areas . We study here, in their 2012 version, the 300 urban areas which are available both in the 2006 and 2012 versions of the Urban Atlas. Land use change in those cities between 2006 and 2012 is rather smallysis see , since ts(r) at a given distance r from the city center. Since it is shown to strongly depend on the size of cities5 measured by their total population N, we label it 5 for the radial evolution of these profiles is that they start at 100% in the center, since the city center  is supposed to be on artificial land. They decrease when moving away from the center, that is when the distance r to the center increases and land becomes less artificial. And they should decrease faster for smaller cities. Actually, these profiles scale horizontally4 with the square root of the total population N. This can be written mathematically as We focus here on the study of the artificial share of land 4. However, we would like to clarify with fitting methods how well these laws are followed. In doing so, we try to follow a parsimony principle. We know that more complex models with more parameters have higher performance in terms of the goodness-of-fit, in the sense that the model is closer to the data points. However, the results of the model, which are the fitted parameters, are more difficult to study and understand: the information contained in the model is diluted among the different parameters. This can be related to the problem of overfitting, which is well-known in computer science and machine learning. Physicists usually express this problem using von Neumann’s famous quote about fitting an elephant16. Here we aim as a consequence to use the simplest possible model, with the least possible number of parameters but still a correct description of the dataset.Of course, we do not expect these mathematical properties to be respected exactly: cities have local characteristics which clearly influence these land use profiles and which result in fluctuations around scaling lawsThe exercise to be carried out here can be stated simply. We have 300 artificial land use radial profiles mentclass2pt{minimmentclass2pt{minimN, in order to find their scaling laws. We observe that the results of the non-linear fit are closer to expectations4, and also more pertinent. Indeed, if we consider first the predicted share 4. In order to assess this scaling law more precisely, we subsequently fit the estimated characteristic decrease distances N (using a linear fit of the logarithms of N), followingThe results, which consist in two fitting parameters  variations of land use in peripheral areas, which the non-linear model tends to disregard.18 , in particular for population density. The present analysis suggests to also study non-linear models.This might actually be an important result of this work, which probably also applies to the modelling of population density profiles. Indeed, linear models are often used in the literatureActually, even with the non-linear model, a large part of the cities are predicted to have more than 100% artificial land in their center, which is impossible. Furthermore, we observe that the city hall, which is a building hence artificial, is almost always in a very artificial area, which suggests that the city center is completely artificial. We then try to force this 100% artificial land share in the center entclass1pt{minima4. However, the two models differ in particular in the way they deal with coastal cities, as illustrated by Fig. N, according to the We see that the two fits give similar evolutions of the characteristic size entclass1pt{minimaOn Fig. N, for the two- and one-parameter models respectively. Both models are still compatible with the 1. Furthermore, the characteristic distance Next, we test the two models on continental cities only, and present the results in Table 19, we still consider it in both cases. These two indicators give actually different and complementary insights.Additionally, we study for each individual city and each model two indicators of the goodness of our fits: the Bayesian Information Criterion (BIC), and the usual On one hand, the values of the BIC for all models  decrease on average with city size, indicating that larger cities are better fitted than smaller ones. On the other hand, the values of This suggests that coastal cities are indeed special cases, which do not have a clearly exponential profile due to the land covered by water, and that the one-parameter (SNL) model detects them better. In addition, while this model is clearly more parsimonious, its description of the data is almost on par with the two-parameter model. As it also has the advantage of not overestimating the artificial share of land in the center b called stretching exponent to the SNL model. The SNL model corresponds to b has a surprisingly clear lognormal distribution and seems quite independent of the total population N, as shown on Fig. b often correspond to coastal cities  when averaging over all cities.After having simplified our fitting method to the extreme with the one-parameter model, we can try to make it (reasonably) more complex so as to answer more precise questions regarding the shape of these artificial land profiles. In particular, we wonder whether these profiles show a global tendency to be more convex or concave than the simple exponential form which was very successful so far. In this regard, we use a stretched exponential fit mentclass2pt{minimsee Fig. , whose pIn this work we observe that the radial artificial land use profiles of 300 large European cities have a very clear exponential shape, with fluctuations quite randomly distributed around this exponential form. Moreover, these profiles scale with the square root of total population N. This Eq. , but adapted for the social body of cities: the body mass index of humans is defined as N as We then define the city mass index entclass1pt{minimaIn addition to the fact that coastal cities have low values of the CMI, as discussed before, this map shows a clear national effect: for instance, French and Belgian cities tend to have high values of the CMI, meaning that they are quite extended in terms of artificial land use compared to their total population, while Spanish ones have low values on average. This phenomenon must be studied in more detail in order to understand and improve national planning practices.We note that this work still uses the functional urban area (FUA) as a foundation, in order to compute the total population of each city. However, we observe that there is a lot of heterogeneity between different FUAs from different countries. Fortunately, the total population size does not depend too much on the precise extent of the urban area, because the population density in the periphery is low: most of the urban population is concentrated around the core (see also the discussion in the Supplementary Information.


The ObsQoR-11K score discriminated well between good  and poor recovery . The ObsQoR-11K score showed acceptable internal consistency (Cronbach’s α = 0.78), split-half reliability (0.89), intra-class correlation > 0.4, and no floor or ceiling effect. Of the participants, 100% completed the ObsQoR-11K and median (IQR) time for ObsQoR-11K completion was 81 s (66–97.5 s). ObsQoR-11K is a valid and reliable scoring tool for assessing maternal recovery after elective CS in Korean women.The Obstetric Quality of Recovery (ObsQoR-11) score is a new scoring tool that assesses maternal recovery after cesarean section (CS). We aimed to validate the translated Korean version of ObsQoR-11 (ObsQoR-11K) after elective CS. We validated ObsQoR-11K between March 2021 to August 2021. Validity , reliability , responsiveness, and clinical feasibility (recruitment rate and time for ObsQoR-11K completion) of ObsQoR-11K were evaluated. One hundred and twenty women completed the ObsQoR-11K 24 h after CS, and 24 women repeated it 25 h after CS. We found good convergent validity between the ObsQoR-11K score and the global health numerical rating scale (NRS) (ρ = 0.73 (95% CI 0.64 to 0.81);  Cesarean section (CS) is one of the most frequently performed surgical procedures in obstetrics and gynecology . AdequatRecently, an obstetric-specific recovery scoring tool, the Obstetric Quality of Recovery (ObsQoR-11) score has been formulated and validated in patients undergoing elective CS  and non-http://cris.nih.go.kr, identification number: KCT0005794, date of registration: 20 January 2021; date of last access: 30 September 2021; principal investigator’s name: RyungA Kang) and conducted from March 2021 to August 2021 at Samsung Medical Center, Seoul, Korea. Written informed consent was obtained from all women before surgery, and the study was conducted in accordance with the Declaration of Helsinki. All methods were performed according to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines [This was a prospective observational study. After approval by the Institutional Review Board of Samsung Medical Center, Seoul, Korea (SMC 2020-12-101), this study was registered at the Clinical Research Information Service  who were not involved in clinical care interviewed the participants and asked the questionnaire. Baseline demographic and clinical data  physical status classification) were collected at the time of enrolment. Women were approached while in the postnatal ward 24 h after CS and asked to complete the ObsQoR-11K questionnaire and to choose a point in the 100-mm numeric rating scale (NRS) to evaluate global health status. Women rated each recovery item using an 11-point numerical Likert scale . Global health status was measured using NRS indicated by a 100 mm line . Twenty-four (20%) participants were chosen randomly and asked to repeat the ObsQoR-11K 1 h later  as a measure of test-retest reliability. We also collected data on the duration of surgery, duration of anesthesia, estimated blood loss, duration of post-anesthetic recovery unit (PACU) stay, maximum pain score while in PACU, spinal level at admission and discharge from PACU, pre- or postoperative levels and differences in hemoglobin concentration, and length of hospital stay. All ObsQoR-11K questionnaires were completed under the guidance of the investigator.®; Arrow International, Inc., Reading, PA, USA) at the L2-3 intervertebral space using a midline approach with the loss-of-resistance to air technique and fixed at 10–11 cm into the skin. Spinal tapping was performed at the L3-4 interspace with a 25-gauge Whitacre needle, and 8 mg 0.5% hyperbaric bupivacaine and 20 µg fentanyl were administered. An epidural injection of 10 mL 0.2% ropivacaine was administered thereafter. The success of anesthesia was assessed bilaterally using a cold alcohol swab and pin-prick test, and if the sensory dermatome of T4 was confirmed, surgery was started.All women were instructed to fast for 6 h before surgery and allowed to drink clear fluid until 2 h before surgery. At our institution, a combined spinal-epidural technique using two separate needles with the separate needle technique was a standard clinical anesthesia guideline for elective CS ,15. Upon®, Daewoong Pharmacy, Seoul, Korea) was prescribed twice a day for PODs 2 and 3. Patients with breakthrough pain (NRS > 4/10) were administered 100 mg ketoprofen  intramuscularly up to three times from POD 0 to POD 3. Postoperative nausea or vomiting was treated with 0.3 mg intravenous ramosetron , if needed. Women were encouraged to move 6 h after spinal anesthesia, and the urinary catheter was removed 24 h after surgery.All women received a standardized postoperative supplemental analgesic regimen. Patient-controlled epidural analgesia (PCEA) with 1000 μg fentanyl and 40 mL 0.75% ropivacaine mixed with 210 mL 0.9% saline was initiated in PACU at the first complaint of pain (NRS > 1/10) and programmed to deliver a background infusion of 4 mL/h with 2 mL bolus and a 15-min lockout interval until postoperative day (POD) 2. After the epidural catheter was removed, 100 mg oral aceclofenac  and compared using a two-sample Student’s t-test. When the distribution was not normal, median (interquartile ranges (IQRs)) was presented and the groups were compared using a Mann–Whitney U-test between two independent groups or a Wilcoxon signed-rank test for paired data. Categorical variables are presented as numbers (percentages) and were compared between groups using the chi-square test or Fisher’s exact test, according to their expected counts. Correlations were measured using Spearman’s correlation coefficient (ρ).Validity, reliability, responsiveness, and clinical acceptability of ObsQoR-11K was evaluated based on the following analyses based on previous publications ,9. Firstp-value was < 0.05.Data analysis was performed with SAS version 9.4  or R version 4.0.3 . Differences were considered statistically significant when the From March 2021 to August 2021, 147 women scheduled for elective CS were assessed for eligibility. Of these, 27 women were excluded prior to enrolment because they did not meet the inclusion criteria .A total of 120 women were recruited (recruitment rate: 100%) and completed the ObsQoR-11K questionnaire 24 h after CS (completion rate: 100%). Of these, 24 women (20%) repeated the ObsQoR-11K questionnaire 25 h after CS. Clinical characteristics of the women are presented in Individual item correlation to the global health NRS score is shown in p < 0.001). ObsQoR-11K scores differed significantly between two groups divided according to their global health NRS score; “good”  vs. “poor” postoperative recovery . There was no significant correlation between ObsQoR-11K scores and clinical characteristics , eristics .Inter-item Cronbach’s α was 0.78 and split-half reliability was 0.89. The inter-item correlation matrix for ObsQoR-11K is presented in Inter-item correlations were mostly ρ > 0.15, which indicates good consistency. The test-retest ICC for total ObsQoR-11K scores was 0.76 and ρ > 0.46 (range 0.46–0.92) for all items, suggesting adequate reproducibility and reliability . There wOur study demonstrates that ObsQoR-11K has excellent validity, reliability, clinical acceptability, and feasibility in patients undergoing elective CS. Specifically, ObsQoR-11K showed a strong correlation with global health NRS scores 24 h after CS (ρ = 0.73). Internal consistency, measured by Cronbach’s α and split-half reliability, remained above the recommended levels 0.7–0.9) . These r . These p = 0.02), and a weak positive correlation between parity and ObsQoR-11  in patients who underwent non-elective CS [Our results showed the following characteristics compared with the original version of the ObsQoR-11. First, in our study, no significant correlation was found between the ObsQoR-11K scores and clinical characteristics. In the original version of ObsQoR-11, there was a weak negative correlation between the length of hospital stay and ObsQoR-11  appeared to be very homogeneous for all women enrolled in this study, which may have contributed to the good results of the study. Further evaluation and validation are required in hospitals that serve patients with different characteristics to extend ObsQoR-11K application during the emergency CS or vaginal delivery. Second, ObsQoR-11K was only measured on POD 1 without further serial assessment. As a patient’s mental and physical status undergoes rapid changes during the acute postoperative phase, changes in ObsQoR-11K throughout the postoperative hospital stay may have more profound clinical implications than a single measurement.In conclusion, the Korean version of the ObsQoR-11K has acceptable validity, reliability, and feasibility for assessing the quality of recovery after CS despite cultural differences. It is expected that ObsQoR-11K will be a useful tool for evaluating the quality of recovery of Korean women in the future.


Brassica oleracea var. italica) is one of the most important and nutritious vegetables widely cultivated in China. In the recent four decades, several improved varieties were bred and developed by Chinese breeders. However, the efforts for improvement of broccoli are hindered by limited information of genetic diversity and genetic relatedness contained within the available germplasms. This study evaluated the genetic diversity, genetic relationship, population structure, and fingerprinting of 372 accessions of broccoli representing most of the variability of broccoli in China. Millions of SNPs were identified by whole-genome sequencing of 23 representative broccoli genotypes. Through several stringent selection criteria, a total of 1,167 SNPs were selected to characterize genetic diversity and population structure. Of these markers, 1,067 SNPs were genotyped by target sequencing (GBTS), and 100 SNPs were genotyped by kompetitive allele specific PCR (KASP) assay. The average polymorphism information content (PIC) and expected heterozygosity (gene diversity) values were 0.33 and 0.42, respectively. Diversity analysis revealed the prevalence of low to moderate genetic diversity in the broccoli accessions indicating a narrow genetic base. Phylogenetic and principal component analyses revealed that the 372 accessions could be clustered into two main groups but with weak groupings. STRUCTURE analysis also suggested the presence of two subpopulations with weak genetic structure. Analysis of molecular variance (AMOVA) identified 13% variance among populations and 87% within populations revealing very low population differentiation, which could be attributed to massive gene flow and the reproductive biology of the crop. Based on high resolving power, a set of 28 KASP markers was chosen for DNA fingerprinting of the broccoli accessions for seed authentication and varietal identification. To the best of our knowledge, this is the first comprehensive study to measure diversity and population structure of a large collection of broccoli in China and also the first application of GBTS and KASP techniques in genetic characterization of broccoli. This work broadens the understanding of diversity, phylogeny, and population structure of a large collection of broccoli, which may enhance future breeding efforts to achieve higher productivity.Broccoli ( Brassica oleracea var. italica) is an economically important vegetable widely grown in many countries and is gaining popularity as a human diet due to its rich nutritional value as well as presence of anti-cancer glucosinolates in its florets  has established the “distinctness, uniformity, and stability” (DUS) testing for new varieties before registration . In thisThe molecular markers can assess the genetic diversity of crop species based on the variation of DNA that arises from substitution, insertion, and/or deletion in the chromosomes. Over the past three decades, several different DNA marker technologies, including amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), and multiple nucleotide polymorphisms (MNPs) have been widely applied in DNA fingerprinting, genetic diversity, population structure analysis, and marker-assisted breeding . SSRs ar1). GBTS is mainly divided into multiplex PCR-based target sequencing and probe-in-solution-based target sequencing. Nowadays, this platform contains several main technologies, such as AmpliSeq  to identify genome-wide SNPs through whole-genome sequencing (WGS) of a set of diverse genotypes of broccoli; (ii) to develop a GBTS platform suitable for genotyping broccoli accessions; (iii) to develop a KASP platform suitable for fingerprinting of broccoli accessions for varietal identification; (iv) to decipher the genetic diversity, genetic relationship, and population structure of 372 broccoli accessions.Based on our previous study on phylogenetic relationship, a set of 23 diverse genotypes were selected for WGS because of their genetic distinctness. Among the 23 diverse broccoli genotypes, 10 were double haploid (DH) lines, and the others were advanced-generation inbred lines. These materials  are reprTo analyze the phylogenetic relationship, genetic variability, population structure, and also to carry out the fingerprinting for varietal identification, a set of 372 accessions were used. Most of these accessions (367) were provided by 11 broccoli breeding groups in China, and five hybrids (widely planted in China) were from the Sakata Seed Corporation of Japan. The 372 broccoli accessions comprised of 102 breeding lines , 248 landraces, and 22 commercial hybrids, could nearly cover all agro-ecological zones of China, where broccoli is widely cultivated.Genomic DNA of 23 broccoli genotypes was extracted from young leaf tissues by a modified cetyl trimethyl ammonium bromide (CTAB) method . The DNAThe original image data generated by the Illumina sequencing machine were converted into sequence data via base calling (Illumina pipeline CASAVA v1.8.2), and the raw reads were then subjected to quality control (QC) procedure and adapter trimming to remove the unusable reads. The available draft genome sequence of HDEM broccoli with a genome size of 630 Mb  was downAfter SNPs were called for 23 broccoli genotypes, the quality of each SNP was assessed based on minor allele frequency (MAF) and polymorphic information content (PIC) using PowerMarker v3.25 software . SeveralThe GBTS library construction consisted of two rounds of PCR. In the first round, the target SNPs in plant DNA samples were amplified and captured using a multiplexed PCR panel. In the second round, a unique barcode was added to the captured product for each DNA sample. First, the multiplexed PCR was performed in 30-μl reactions including 20 ng of DNA template, 10 μl of GenoPlexs 3 × T Master Mix . The PCR conditions were as follows: 95°C for 5 min, then 16 cycles of 95°C for 30 s and 60°C for 4 min, and an extension at 72°C for 5 min. The PCR products were purified by magnetic bead suspension and 75% alcohol. Second, PCR was conducted in 30-μl reactions consisting of 11 μl of purified PCR product from the first round, 10 μl of GenoPlexs 3 × T Master Mix, and 1 μl of sequencing connector with barcode sequence. The PCR conditions were as follows: 95°C for 5 min, then nine cycles of 95°C for 30 s, 60°C for 20 s, and 72°C for 30 s, and an extension at 72°C for 5 min. The second round of PCR products was purified with 100 μl of 75% alcohol and 23 μl of Tris-HCl buffer . Thereafter, the constructed library was sequenced using Illumina HiSeq X Ten platform.To better characterize the 372 broccoli accessions, the genotyping data were further filtrated as follows: (1) removing the SNPs having missing data points of >20%, (2) removing SNPs with the MAF values of <0.05, and (3) removing the SNPs with the PIC values of <0.2.The available SNPs were filtered based on several criteria to choose the best loci for KASP marker development. The filtration of SNPs was carried out by removing (1) SNPs having missing data points of >20%, (2) SNPs with the MAF values of <0.05, (3) other variations located within a distance of 50 bp upstream and downstream of the target SNPs, and (4) SNPs with the PIC values of <0.2. Among the SNPs amenable to KASP assay development, SNPs were further selected for designing of KASP primers by (1) removing the primer sequence with GC content lower than 0.3, (2) based on the loci present within the exonic, intergenic, and upstream/downstream of the functional genes, and (3) loci selected based on physical position, which are evenly distributed across the chromosomes.2 in an optimized buffer solution, which is universal to every KASP genotyping assay. The genomic DNA was extracted from 100 mg of young leaf tissue from each broccoli accession by a modified cetyl trimethyl ammonium bromide (CTAB) method  for visualization of allelic discrimination of each genotype.For the selected KASP loci, primers were designed, and genotyping of the 372 broccoli accessions was carried out. Basically, the KASP assay contains three components: KASP assay mix, KASP master mix, and template DNA. The KASP assay mix (72X) contains two different, allele-specific, competing forward primers with unique tail sequences at the 5′ end  and one common reverse primer. KASP master mix (2X) contains FAM and HEX-specific FRET (fluorescence resonance energy transfer) cassette, ROX passive reference dye, KASP Taq DNA polymerase , dNTPs, and MgCl) method . The DNAThe allelic data of the 372 broccoli accessions genotyped by GBTS and KASP panel were integrated. All the SNP markers were analyzed, and various genetic diversity summary statistics including observed heterozygosity (Ho), expected heterozygosity (He) also called gene diversity (GD), minor allele frequency (MAF), and polymorphic information content (PIC) were measured using the PowerMarker software v3.25 . The genK value from 1 to 10 was performed using 10 iterations for each K. For each run, a burn-in period of 50,000 was used to minimize the effect of the starting configuration, which was followed by an additional 100,000 iterations using a model with admixture (genotype might have mixed ancestry) and correlated allele frequencies. The likelihood of optimal K value was calculated, and the K value corresponding to the highest likelihood was interpreted as the number of subpopulations in the samples. The most probable K value was determined from the uppermost level of population structure, detected using an ad hoc statistic ΔK based on the rate of change in the log probability of data between successive K values  was computed using GenAlEx v6.502  to estimTwenty-three diverse broccoli genotypes were used for WGS, and paired-end sequencing libraries were generated for each sample. The libraries were of good quality according to sizing profiles generated by high-sensitivity lab chip kit , and the fragment size of the libraries ranged from 450 to 750 bp. Sequencing of these libraries in the Illumina HiSeq2500 instrument yielded 13.1 to 18.7 Gb of raw data per sample . A totalThe number of reads per sample ranged from 87.7 to 124.9 million . The HisThe putative SNPs identified through the re-sequencing of 23 broccoli genotypes were further filtered for the GBTS and KASP panel developments, respectively.For the GBTS panel, 210 MNPs consisting of 1,332 SNPs were selected, with an average of 6.3 SNPs within each MNP. The utility of these SNPs in deciphering the genetic relationship and diversity of 372 broccoli accessions was assessed by calculating the missing data, MAF, and PIC values. As a result, 1,067 (80.1%) SNPs were retained for further analysis . These SFor the KASP panel, a total of 13,621 SNPs with the PIC values between 0.2 and 0.5 were retained after filtration. Of these, only 8,768 (64.4%) SNPs could be successfully designed as KASP markers. From this set, 2,515 SNPs were from exonic , intronic, intergenic regions, and from the upstream or downstream of the functional genes. Based on their functional role in regulating important agronomic traits and also based on the physical position and uniform genomic distribution, finally, 500 SNPs were selected for KASP marker development. Of the targeted 500 KASP markers, only 347 (69.4%) could be genotyped successfully with high-quality genotype clusters , while cKey descriptive statistics for measuring the utility and informativeness of SNP markers were assessed by analyzing the genetic diversity of the 372 broccoli accessions . Of the The pairwise genetic distance matrix ranged from 0.0004 to 0.6418 with a mean of 0.3894 , indicatBased on the genotyping data of 1,167 SNP markers in 372 broccoli accessions , a phyloK) of the accessions was estimated by setting the number of clusters (K) from 1 to 10 with 10 replications for each K. The average logarithm of the probability of likelihood [LnP(D)] and standard deviations for different number of sub-populations (K = 1 to 10) are presented in K) was selected by comparing the logarithmized probabilities of the LnP(D) and ΔK data  was plotted against ΔK, which showed a sharp peak at K = 2  was 0.126, supporting low population differentiations. The population differentiation based on AMOVA revealed that only 13% (p < 0.001) of the total variation was found among the subpopulations, while the rest (87%) was within the populations  was carried out using GenAlEx v6.502. The ulations . In addiulations . Nei’s sthe rest 7% was wiThe subpopulation I  shows higher diversity than the subpopulation II  . The per2. This barcode can be scanned to obtain the information used for creating the 2D barcode. To build a rapid and cost-effective way of varietal identification, we selected 28 KASP markers from the genotyping database for the fingerprinting of every accession , 3. ThesIn the current study, Illumina platform was chosen to undertake the whole-genome sequencing of 23 diverse broccoli genotypes and discover genome-wide SNPs. The Illumina platform was preferred for WGS due to its throughput, cost effectiveness, and indexing capabilities. The WGS data were generated with a high coverage (approximately 28×), and a total of 346.18 Gb of raw data for 23 broccoli genotypes were generated. Sequencing with high coverage gives confidence for SNP calling as low coverage sequencing will make SNP calling difficult with high genotyping errors . The rawBrassica species , and the alignment percentage ranged from 98.1 to 98.98 with an average of 98.65%. The high mapping percentage signifies high quality of the sample reads. Among the SNPs, transition SNPs were more frequent than the transversions, which is a common phenomenon in  species . This ha species  and sugg species . The ave species . The gen species . The intIn this study, a total of 1,067 markers were selected for the GBTS panel development. This GBTS panel with larger numbers of markers can effectively be used for genetic diversity and population structure analysis. In maize, a series of high-quality GBTS panels, including 20 K, 10 K, 5 K, and 1 K SNP loci were developed, making it an affordable genotyping platform for maize marker-assisted breeding . Based oFor the fingerprinting of the broccoli lines, few KASP markers will be more cost effective and flexible. KASP assay has been widely used by plant breeders for genetic diversity study, genetic mapping, and genetic purity test in several major crops including pigeon pea , chickpeA total of 372 broccoli accessions represents a wide variability and comprised of several improved lines from China, elite breeding lines, parents of commercial hybrids, landraces, and a few commercial hybrids. All these accessions were genotyped by 1,167 SNPs developed from the GBTS and KASP panels. The PIC values and expected heterozygosity  are two parameters in measuring genetic diversity in any population. In the present study, the PIC values of the SNPs ranged from 0.18 to 0.37 with a mean value of 0.33. In a previous study by The MAF value of the SNPs ranged from 0.11 to 0.50, with a mean of 0.33. MAF threshold dramatically affects population structure inference, and inference of population structure is sensitive to MAF . SNPs wiThe pairwise genetic distance matrix ranged from 0.0004 to 0.6418 with a mean of 0.3894. Most of the accessions were found considerably distant, though a few accessions, mostly improved cultivars, were found genetically similar indicating the accessions having a common breeding history. Maximum genetic distance of 0.6418 was found between the broccoli accession ZAAS-8 and KR8. Interestingly, the genetic distance between several broccoli accessions belonging to different breeding groups was reported to be as low as 0.0004 (M31 and KR18). This could be due to the massive exchange of materials between different broccoli breeding groups in China resulting in the development of genetically similar broccoli cultivars. Recently, 1 hybrids were grouped in cluster II and were placed closely with several accessions such as M14, T19, and KR 32. As mentioned earlier, broccoli was introduced into China from Japan four decades back  lines are widely used for the development of commercial FIn this context, DNA fingerprinting could act as an insurance for the breeders in safeguarding the elite varieties and germplasm. DNA fingerprinting have advantages in identifying varieties and are free from environmental impact, compared with customary field inspection . Molecul1 hybrids. For hybrid purity, three to five KASPs will be adequate to ascertain the genetic purity. The markers can be selected from the 100 KASPs according to the parental genotypes of a specific hybrid of broccoli.Furthermore, the KASP markers developed in our study can be utilized in genetic purity of the broccoli FThis study provides comprehensive information about the genetic diversity of broccoli accessions in China based on a large population of 372 broccoli accessions. Genome-wide SNPs were discovered by WGS of 23 diverse broccoli genotypes, and the SNPs were converted into GBTS and KASP panels for genotyping of a large set of broccoli accessions. Evaluation of genetic diversity demonstrated that low to moderate genetic diversity prevails in broccoli cultivars widespread in China, and few accessions are in close relationship. This indicated the prevalence of a narrow genetic base among broccoli accessions and the need to broaden the gene pool by adding diverse genotypes into the breeding program. Phylogenetic and PCA analyses based on 1,167 SNPs revealed the presence of two groups but did not show strong groupings. The STRUCTURE results also suggested the presence of two subpopulations with weak genetic structures. The population differentiation was found to be low indicating extensive gene flow. A set of 28 KASP markers was chosen for DNA fingerprints of the broccoli accessions for varietal identification. The SNP genotype of each broccoli accession was used to generate a 2D barcode containing the fingerprinting information of a large collection (372) of broccoli accessions in China. The KASP markers developed in our study could also be used for seed authenticity and purity evaluation of broccoli cultivars. To our knowledge, this is the first study to measure diversity and population structure of a large collection of broccoli in China and also the first application of GBTS and KASP techniques in broccoli for genetic studies. The information generated in the present study will assist in the selection of suitable genotypes for breeding in developing new cultivars as well as physiological and molecular studies in broccoli.https://www.ncbi.nlm.nih.gov/, PRJNA681704.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: HG designed the experiments, supervised the study, and revised the manuscript. YS and RS analyzed the experimental data and wrote the manuscript. JW and ZZ collected the accessions and selected the core germplasms for re-sequencing. HY, XS, and SL designed the KASP markers and performed the research. All authors discussed the results and contributed to the final manuscript.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.


Porcula salvania) is an endangered species with a small population inhabiting the tall sub-Himalayan grasslands of Assam, India. A conservation breeding program of pygmy hog from six founders has produced a multi-generational captive population destined for reintroduction into the wild. However, the impact of conservation breeding on its genetic diversity remained undocumented. Here, we evaluate temporal genetic changes in 39 pygmy hogs from eight consecutive generations of a captive population using genome-wide SNPs, mitochondrial genomes, and MHC sequences, and explore the relationship between genetic diversity and reproductive success. We find that pygmy hog harbors a very low genome-wide heterozygosity (H) compared to other members of the Suidae family. However, within the captive population we find excess heterozygosity and a significant increase in H from the wild-caught founders to the individuals in subsequent generations due to the selective pairing strategy. The MHC and mitochondrial nucleotide diversities were lower in captive generations compared to the founders with a high prevalence of low-frequency MHC haplotypes and more unique mitochondrial genomes. Further, even though no signs of genetic inbreeding were observed from the estimates of individual inbreeding coefficient F and between individuals (FIS) in each generation, the kinship coefficient showed a slightly increasing trend in the recent generations, due to a relatively smaller non-random sample size compared to the entire captive population. Surprisingly, male pygmy hogs that had higher heterozygosity also showed lower breeding success. We briefly discuss the implications of our findings in the context of breeding management and recommend steps to minimize the genetic effects of long-term captive breeding.Long-term captive populations often accumulate genetic changes that are detrimental to their survival in the wild. Periodic genetic evaluation of captive populations is thus necessary to identify deleterious changes and minimize their impact through planned breeding. Pygmy hog ( Captive breeding has become a major conservation tool for safeguarding critically-endangered species from extinction and restoring declining populations in the wild. The primary goal of such programs is to establish a demographically-secure and genetically-healthy population through planned breeding, which will make them self-sustaining in the wild post-release . HoweverThe increased availability of genome-wide data of several non-model organisms over the last decade due to reduced cost of sequencing technology has enabled the study of genetic diversity patterns at a genomic scale, giving critical insights about their evolutionary processes. For example, the underlying mechanism of inbreeding depression in a small and isolated population of grey wolves in Isle Royale, Michigan, USA could be unravelled through genome-wide analysis of their heterozygosities . SimilarIn addition to genome-wide diversity, studies on local diversity at markers of functional significance are increasingly gaining importance since quantitative difference of some ecologically important traits is better explained by the genetic variation at functional markers . Some ofSuidae family and the sole representative of the genus Porcula. Its small population size, and a very limited habitat range in the grasslands south of the Himalayan foothills puts it at high risk of extinction. A captive breeding program for pygmy hog was initiated by the Pygmy hog Conservation Breeding Program (PHCP) as a part of a species recovery program after its population plummeted to only a few hundred individuals (∼150) in the wild. The program, which started in 1996 with only six wild-caught hogs from Manas National Park (MNP), Assam, has been successful in achieving its demographic goal by increasing the captive stock to a sizable number that can insure the species from extinction due to any stochastic events. With the inclusion of one rescued male in 2001 and three additional wild hogs to the captive stock in 2013 from MNP for genetic enrichment, a total of 683 individuals have been bred, comprising seven generations by 2020. The successful breeding followed by reintroduction of 130 pygmy hogs between 2008 and 2020 into their previous ranges have resulted in an increase in their numbers and range size, which prompted IUCN to downgrade the status of pygmy hog to endangered (EN) from the critically endangered (CR) category in 2019. Nevertheless, in a long-term captive rearing program, the possibility of fitness impairment due to unrecognized biases in mate-pair selection and adaptation to captivity may impact the future breeding success and compromise the reintroduction program. Therefore, the objectives of the current study are (1) to examine the genetic variability in a captive population of pygmy hogs using SNPs, mitochondrial, and MHC markers and (2) to test if the changes in genetic variability had any impact on reproductive success in the focal population. The outcomes of the present study will be helpful in devising effective management plans for breeding and reintroduction.The pygmy hog is the smallest member of the n = 39) representing all the eight generations based on their availability in the veterinary facility of PHCP. The generation and pedigree inbreeding coefficient of each individual were obtained from SPARKS v 1.66  in Assam, India. The captive population, which has been bred up to eight generations till February 2020, is generated by breeding mate pairs based on lowest kinship and highest mate suitability index values as calculated by genealogy record keeping tool SPARKS v 1.66 and PMx. All samples were collected in accordance with the ethical and legal regulations of India and as per Wildlife Protection Act 1972. The program was approved by the Institutional Animal Care and Use Committee and Internal Ethical committee of the Centre for Cellular and Molecular Biology vide No. IEM/CCMB 07/2020. We opportunistically collected tissue or blood samples (S v 1.66 . The sams groups . In SPARWe collected various life-history traits like lifetime breeding success (LBS), annual breeding success (ABS), juvenile survival (JS), neonatal mortality (NM), and age at first breeding for individuals which were paired for breeding in the dataset. While LBS and ABS were used as determinants of reproductive success in both males and females, JS and NM were considered only for females as piglet survival into adulthood is reliant on and is an indicator of maternal fitness . LBS wasBlood and tissue of piglet ear notch were collected from live animals and stored in EDTA vacutainer and 100% ethanol solution respectively. From dead animals, soft tissues of the kidney, brain, and heart were stored in 100% ethanol. All samples were stored at −20 °C until further use. The DNA was extracted using standard phenol-chloroform method . DNA purSus scrofa chromosome 7 that harbors the SLA-DRB1 gene. The PCR reaction yielded a product of 242 bp (excluding primers) in the pygmy hog. The amplicons were cloned in a blunt-ended cloning vector  and transformed to E. coli cells of DH5α strain according to The DNA samples were used to amplify the exon 2 region of MHC class IIB gene in the pygmy hog. The primers , designed at the intron-exon junctions of exon2, correspond to 7896 and 8195 nucleotide positions on the in silico using DDRADSEQTOOLS (In order to select a suitable pair of restriction enzymes for Restriction site-Associated DNA sequencing (RADseq), we simulated the DNA fragmentation process SEQTOOLS . We seleSEQTOOLS  containiA total of 36 samples out of 39 passed the required purity and concentration for the preparation of high-throughput sequencing libraries. Two micrograms of genomic DNA from each sample was as digested with 20 units of EcoRV-HF and SspI-HF at 37 °C. The digestion was carried out sequentially with two hours of incubation for each enzyme, followed by a heat inactivation step at 65 °C for 10 min. For library preparation, a minimum of one microgram of double digested DNA was used as starting DNA concentration for each sample. We adapted the ezRAD protocol from https://sapac.support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html). The raw paired-end fastq files were processed using the Stacks v2.54 pipeline  and FIS were calculated using the populations program of Stacks. The H for each individual was generated by considering only the number of heterozygous sites among all the genotyped sites within a given individual. Since the calculation of individual heterozygosity is independent of the number of shared SNPs across samples, we did not employ any filtering of missing sites. The SNPs were exported into a VCF format from Stacks and further analyzed using VCFtools  were calculated from complete mitochondrial genomes using DnaSP v.6. The change in diversity over time was estimated by comparing the genetic variability from founders to the current breeding population. Additionally, the same indices were calculated separately for 1,150 bp of mitochondrial CytB gene and 440 bp of control region with those of Sus scrofa.Number of unique haplotypes (h), haplotype diversity (Hd) and nucleotide diversity  and various fitness measures. Fitness traits like lifetime breeding success (LBS), annual breeding success (ABS), neonatal mortality (NM), and juvenile survival (JS) were considered as response variables. They were defined as functions of genetic variables in coordination with some non-genetic explanatory variables on the basis of a biologically plausible hypothesis that best explained the response variables. For example, in LBS models, we fitted the number of times an individual has been paired for breeding (no. of breeding seasons) as a covariate to account for the unequal breeding opportunity given to individuals in the captive breeding set up. To fit the discrete data of LBS, poisson distribution with the identity link function was used. To fit the continuous data of ABS, JS and NM, a Gaussian distribution with identity link function was used. A separate analysis was carried out for each genetic parameter. A null model (only intercept) was also tested for each response variable to check for any bias in the model. We modelled the association studies separately for males and females. To rule out the presence of any confounding effects between neutral and functional markers, we tested the association between genome-wide kinship coefficient of individual pairs with their MHC allelic distance. All the models were carried out using “glm” function of lme4 package in R .The models were ranked based on Akaike information criteria for small sample size (AICc). Each model was supported with ΔAICc and the cumulative AICc weight (ranging from 0 to 1). The model with the lowest ΔAICc score was considered as the best-fit model for a given response parameter. The AICc was implemented using the R package AICcmodavg .We obtained a total of 431.67 gigabases of raw data from a single sequencing run containing 36 samples. Upon demultiplexing, an average of 52.76 million (SD 11.04M) 250 bp paired-end reads were assigned to each sample. About 3.28 million (SD 1.89M) RAD-tags containing the EcoRV and SspI overhangs were extracted from each sample. A final catalog of 417,278 unique RAD-loci (stacks) were assembled from 36 samples with a coverage of 9x . An average of 399.74 sites (SE 0.13) were genotyped per locus with a mean coverage of 8.26 samples per locus. A total of 273,122 polymorphic sites were obtained with an average of 62,373.63 SNPs per sample .H) estimates for 36 individuals ranged from 0.0002 ± 0.00006 to 0.00083 ± 0.00001 (HO ± SE); the lowest was recorded in founder male PH159 and the highest value was estimated in a fourth-generation male, PH294  relative to the founders  exhibited negative values for all individuals except PH159 . The negbreeding . Howeverbreeding . The higbreeding .We obtained a total of 68 MHC reads that contained 35 distinct alleles from 38 pygmy hog individuals. The distinct alleles were translated into 30 unique amino acid sequences of 80 residues each, without any stop codons. Each individual carried either one, two, three, or four of these alleles which points at a classical double copy of SLA-DRB1 MHC class II gene, amplified either from homozygous or heterozygous loci. Out of 35 alleles, 31 were not shared between any two individuals. Five haplotypes were unique to the wild individuals and three were shared between wild and captive-bred pygmy hogs.π ± SD). The pattern of π across generations showed a remarkable drop in generation 2 and 4, followed by a rise in generation 5 and 6 . Tajima’s D analysis of MHC haplotypes showed significantly negative D values in generations 2, 4, and 6, suggesting an accumulation of novel mutations . The outcome of McDonald–Kreitman test between pygmy hog and wild boar showed an α and neutrality index (NI) values of −1.556 and 2.556 respectively, suggesting the non-synonymous to synonymous variations within populations are higher than the variations that exist between populations. However, these values were not statistically significant.The study population harbored a high MHC diversity with a haplotype diversity of 0.828 ± 0.047 (Hd ± SD) and nucleotide diversity of 0.074 ± 0.019 .We assembled 22 complete and 14 partial mitochondrial genomes (mitogenomes) from the randomly fragmented reads of ezRADseq. The complete circular mitogenomes had an average length of 16,652.5 bp (SD 91.5 bp) and contained all the 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. The partial mitogenomes had an average length of 12,086 bp  and contained scaffolded gaps filled with ambiguous bases (N). The average base coverage in the assembled mitogenomes was 159.5x . For an accurate estimation of genetic variability in pygmy hogs, we sampled individuals with complete mitochondrial genomes (π) of the entire captive population was 0.00022 ± 0.00005 (π ± SD). There was a reduction in π in captive-born pygmy hogs compared to the founders (π was observed in generation 4 (0.00007 ± 0.00003), consistent with the MHC results.We found eight distinct haplotypes from 22 complete mitogenomes, of which, three were present in the founders while the remaining five were unique to the captive-bred individuals. Of the three founder-specific mitogenomes, two were shared by PH1, PH11 and PH21, and, PH159 and PH41 respectively. In spite of the shared mitochondrial haplotypes, the genomic pairwise kinship coefficient of founders ranged from −5.93E−05 to 0.055046, showing low relatedness between its members. Of the 13 variable sites detected in the mitogenomes, 10 were parsimony-informative. Notably, the three singleton mutations were unique to only captive-bred individuals. The mean nucleotide diversity , MHC allelic p distance, and the number of MHC alleles per individual. In order to get an unbiased estimate of the correlation scores, we checked if the effects of H and MHC diversity on fitness were mutually exclusive. Therefore, we tested if MHC p distance was associated with H in the captive population. We found no correlation between these two diversity indices  and MHC allelic p-distance in candidate pairs to check any signature of inbreeding in the population. We found a negative correlation between Φ and p-distance which was not significant .To check the genetic diversity on lifetime breeding success (LBS), we used an additive model. We included the number of times an individual was paired for mating (mBrseason & fBrseason) as a covariate in the interactive models to account for the higher number of offspring produced by any individual due to more breeding opportunities. We found a strong positive association between the number of times an individual mated and lifetime breeding success both in males and females. However, in the interactive model for males, In case of MHC, both its measures of diversity  displayed negative correlation with LBS in male and female pygmy hogs ; Fig. 3AH) showed a significant negative correlation with ABS in males  as opposed to non-significant negative correlation in females  in pygmy hog  with breeding success to test if the apparent gain in genetic diversity in the captive-bred hogs had any reproductive fitness advantage. We found H was negatively correlated with both lifetime breeding success (LBS) and annual breeding success (ABS) in males. In females, H established a non-significant HFC with LBS and ABS. The result was unexpected as similar findings were rarely reported in animal systems. One such rare example was in a reintroduced population of Arabian oryx, which was derived from a captive breeding program after it was hunted to extinction in the wild (H and breeding success in males, hint at the possibility of locally adapted sub-populations of pygmy hogs in the grassland range from which the founders were picked.We tested the associations of genome-wide heterozygosity (the wild . The autH the MHC analysis relied on much fewer variable sites, the power of the HFC models to resolve a significant relationship, if any, could be improved with a larger sample set.For MHC diversity, we found no significant correlations with any measures of fitness, both in male and female pygmy hogs  and 3B. Genetic management of captive populations has gained importance over the last few decades and supplementing captive populations with wild individuals has been a recurrent management theme for highly endangered species , with thThe observation of negative correlation between heterozygosity and lifetime breeding success suggests that conservation breeding protocols employed to achieve higher genetic diversity in the captive population could be overlooking the possibility of outbreeding depression. In order to avoid this, some modifications in the existing protocol of mate pair selection could be explored. Furthermore, studies on the genetic structure in wild population could be undertaken to ascertain the presence of locally adapted sub-populations that might impact the efficiency of breeding programs.10.7717/peerj.12212/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12212/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12212/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12212/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.12212/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.12212/supp-6Supplemental Information 6Click here for additional data file.


Triplophysa tenuis is an important indigenous fish in the Xinjiang Tarim River. In this study, we collected 120 T. tenuis individuals from 8 T. tenuis populations in the Tarim River. Through genotyping-by-sequencing (GBS), a total of 582,678,756 clean reads were generated for all the genotypes, and after quality filtering, 595,379 SNPs were obtained for the population genetic analyses. Multiple genetic parameters showed that the 8 T. tenuis populations had high genetic diversity. Phylogenetic tree analysis indicated that all T. tenuis individuals were divided into five branches, the individuals from the north of Tarim River were grouped into cluster 1 (SF and WS) and cluster 3 , while the AETS, WLWT and LF individuals from the south of Tarim River were clustered into cluster 2. The result was consistent with the admixture analysis, which supported that the 8 T. tenuis populations were clustered into three subgroups. Furthermore, the pairwise FST values and genetic distance indicated that there was a large genetic differentiation between WS and other T. tenuis populations. Collectively, this study provides valuable genome-wide data for the conservation of natural T. tenuis populations in the Tarim River. Triplophysa tenuis, which belongs to Triplophysa, Nemacheilidae, and Cypriniformes, inhabits the backwater of rivers over sandy or muddy bottoms. It is recognized as an important indigenous fish with certain economic value in southern Xinjiang Tarim River, such as Kaidu River, Aksu River  is a special group in the Qinghai-Tibet Plateau, which has strong adaptability to the highland environment. In recent years, affected by natural and anthropic factors, the Tarim River has experienced runoff curtailment and river desiccation, accompanied by river salinization, biodiversity decrease, and ecosystem service loss  investigate fine-scale genetic variations of T. tenuis populations by the GBS database; 2) provide genomic evidence on the population structure of T. tenuis by a phylogenetic tree, admixture analysis, and principal component analysis; 3) determine the genetic diversity of 8 T. tenuis populations by detecting the genetic parameters, as well as explore the population genetic differentiation of 8 T. tenuis populations by the pairwise FST and genetic distance. This study is essential to the germplasm resource conservation of T. tenuis in Xinjiang Tarim River, as well as provides a valuable reference for genome-assisted breeding of T. tenuis.Large-scale genotyping at the whole genome level is becoming more and more important for population genetic studies . Herein,T. tenuis samples were collected from the branches of the Tarim River, including the Krakech River , the Yurungkash River , the Yarkant River , the Kashgar River , the Toxkan River , the Muzart River  in 2021   method, the distance matrix was calculated by treebest software (To explore the population structure of (v1.3.0)  was usedsoftware  was usedsoftware . The gctsoftware  was usedsoftware .FST) between populations was calculated using the R package genepop , Hardy-Weinberg (He), observed heterozygosity (Ho), polymorphism information content (PIC), effective number of alleles (Ne), observed number of alleles (Na), nucleotide diversity (Pi). The vcftools software was used to calculate these genetic indicators , and nine independent runs for each K value were conducted  to 0.2224 (WLWT with WS), with an overall FST value (0.1126), suggesting a moderate genetic differentiation among these T. tenuis populations. Notably, we found that the FST values ranged from 0.1777 (WS with SF) to 0.2224 (WS with WLWT), indicating that there was a large genetic differentiation between WS population and the other T. tenuis populations. Moreover, the genetic distance between pairs of the 8 T. tenuis populations ranged from 0.0010 (KZE with TKX) to 0.2515 (WLWT with WS). Likewise, there was a large genetic distance value between WS population and the other 7 T. tenuis populations, indicating an obvious genetic differentiation. By these analyses, we observed that there was a large genetic differentiation between WS and WLWT, a low genetic differentiation between KZE and TKX.The genetic differentiation among the distance . The FHW-P index showed the relationship between allele frequency and genotype frequency. We found that HW-P values ranged from 0.8495 (WS) to 0.8777 (WLWT), indicating that all populations reached genetic equilibrium. The He index represents the expected value of heterozygosity based on Hardy Weinberg equilibrium, which depends only on allele frequency. The result showed that He values ranged from 0.1298 (WLWT) to 0.1450 (WS), with an average of 0.1359. The Ho index indicates the proportion of heterozygotes observed in a population. We observed that Ho values ranged from 0.1198 (WLWT) to 0.1307 (SF), with an average of 0.1245. The PIC is an indicator of polymorphism level. The result showed that PIC values ranged from 0.1085 (WLWT) to 0.1214 (WS), with an average of 0.1139. Additionally, Ne, Na and Pi indices are the basic parameters of genetic diversity, the result indicated that the Ne values ranged from 1.2011 (WLWT) to 1.2268 (SF) with an average of 1.2100, and the Na values ranged from 1.5584 (WLWT) to 1.6172 (WS) with an average of 1.5931. Moreover, the Pi values ranged from 0.1348 (WLWT) to 0.1508 (WS), with an average of 0.1411.The genetic parameters are shown in Triplophysa fishes can well adapt to these severe natural conditions  in the north of Tarim River, and three sampling points  in the south of Tarim River. Through population genetic structure analysis, we found that the individuals from the south of Tarim River were clustered together, the individuals from the north of Tarim River were divided into two subgroups according to the geographical isolation.The harsh environment, characterized by high altitude, cold habitat and poor nutrition in the Qinghai-Tibet Plateau, has brought severe challenges to local species . Neverthnditions . Given taptation . In rece decline . To inveFST values indicated that there was a moderate gene differentiation among 8 T. tenuis populations, indicating that the genetic exchange among these T. tenuis populations was very restricted. Likewise, very large pairwise FST values were also estimated among T. yarkandensis populations in the Tarim River . Importantly, coupled with the fragmentation of the Tarim River, it may increase the possibility of outbreeding decline.Tarim River is the largest inland river in China, formed by the confluence of 114 rivers of 9 major water systems . The KraT. tenuis within the Tarim River ecosystem, which is essential to develop the population management unit. Further, we envision that artificial propagation and releasing are conducted to restore the T. tenuis population in the Tarim River ecosystem. The genome-wide molecular markers can be available to establish a parent genetic information database and select an appropriate population for proliferation and release, which can not only be conducive to the restoration, but also avoid the genetic bottleneck effect and prevent genetic drift. Meanwhile, to maintain genetic diversity and avoid depletion of genetic resources, the genetic consequences of releasing the T. tenuis populations into the wild need to be evaluated.In conclusion, our study represented an important step in better understanding the genetic diversity and differentiation of