Vicia faba L.) in Tunisia is largely based on improved varieties of the crop. However, a few farmers continue to produce local cultivars or landraces. The National Gene Bank of Tunisia (NGBT) recently launched a collection project for faba bean landraces, with special focus on the regions of the North West, traditionally devoted to cultivating grain legumes, and where around 80% of the total national faba bean cultivation area is located. The seed phenotypic features of the collected samples were studied, and the genetic diversity and population structure analyzed using simple sequence repeat markers. The genetic constitution of the present samples was compared to that of faba bean samples collected by teams of the International Center for Agricultural Research in the Dry Areas (ICARDA) in the 1970s in the same region, and stored at the ICARDA gene bank. The results of the diversity analysis demonstrate that the recently collected samples and those stored at ICARDA largely overlap, thus demonstrating that over the past 50 years, little genetic change has occurred to the local faba bean populations examined. These findings suggest that farmers serendipitously applied international best practices for in situ conservation of agricultural crops.Cultivation of faba bean ( Vicia faba L., 2n = 2x = 12) is a facultative cross-pollinating species with outcrossing rates varying between 1 and 55% depending on its environment; it belongs to the Fabaceae family, Faboideae subfamily, tribe of Fabeae, and is not interfertile with any other Vicia species [V. faba is unknown, but recent archaeological excavations have allowed, in the Mont-Carmel (Mediterranean Levantine), the discovery of fossilized seeds that are compatible with a wild progenitor of this crop, dating as back as 14,000 ybp [Faba bean  and drought stress occurring in faba bean-growing areas [In Tunisia, faba bean covers more than 70% 59,583 ha) of the total area annually devoted to grain legume crops ,583 ha o. This isng areas . Until tng areas . Some laVicia faba collections has indicated a certain degree of genetic cohesiveness. Using simple sequence repeat (SSR) markers, 16 faba bean accessions, selected from 42 populations collected across eight southern oases arid agro-ecosystems, were analyzed, evidencing genetic cohesiveness among the studied samples [In recent years, thanks to significant achievements in crop breeding, modern high-yielding varieties are widely used in cultivation and have almost completely replaced local populations and landraces . The inc samples ,20,21, tIn the 1960s, major concerns focused on the genetic erosion of biodiversity, eventually leading to fostering of ex situ conservation efforts and the creation of gene banks . As a reVicia faba germplasm, the genetic structure of the samples collected in recent years was compared with those collected by ICARDA in the 1970s. This paper reports on the results of this comparison.In order to better plan an in situ conservation strategy for faba bean and to understand the possible loss of genetic diversity in Tunisian The plant material used in the present study consisted of a collection of 51 Tunisian local faba bean samples . It inclFive seeds of each sample were randomly selected to determine average seed size. The three axial dimensions of seed length (L), width (W), and thickness (T) were measured using a Vernier caliper  with accuracy of 0.05 mm. The geometric mean diameter (Dg) was calculated by using the equation reported by Mohsenin :Dg = (L The sphericity (φ) of faba bean seeds was calculated using the following formula:TM ND-2000  and the quality was verified by separation on 0.8% agarose gel. Equal DNA quantities of the five plants of the sample were then pooled, and all DNA samples were diluted to a standard working concentration of 50 ng/µl by adding ultrapure water .Total genomic DNA was extracted from fresh young leaves—five plants per sample—using the cetyltrimethyl ammonium bromide (CTAB) method, as described by Fulton et al. . DNA conA set of 11 simple sequence repeats (SSRs) markers, retrieved from the literature ,31,32 weHierarchical ascending classification (HAC) clustering analysis based on dissimilarity matrix of morphometric seed data  was performed to evaluate the relationship among the faba samples using XLSTAT statistical software ver. 2016.2 .Na), Shannon’s information index (I), observed Heterozygosity (Ho), expected Heterozygosity (He), Fixation Index (F), and private alleles were calculated using GenAlEx version 6.5 [The genetic indices, number of different alleles  were measured in the samples belonging to both the NGBT and ICARDA faba bean collection. The average mean of the three principal axial dimensions  and Dg of the NGBT and ICAR groups are shown in The values of morphometric seed traits of each samples were used to calculate a dissimilarity matrix based on Euclidean distances. A dendrogram was obtained starting from the matrix, in which three main clusters are evidenced, corresponding to three seed types—large, medium , and small . Each clIn order to evaluate the genetic diversity of 51 faba bean samples, a set of 11 SSR markers was used . A totalAMOVA did not show a molecular diversity among the 29 NGBT faba beans when these were grouped according to the collection sites , suggesting that the NGBT faba bean samples belonged to one genetically cohesive population .According to these results, we investigated the genetic diversity among samples collected in recent years by NGBT and ICAR during the 1970s, in the same Tunisian areas. Similar values for the number of different alleles, Shannon’s information index, Heterozygosity expected, Heterozygosity observed, Diversity index, and Fixation index were found between the two groups, although the ICAR samples appeared slightly more fixed .AMOVA indicated that the NGBT and ICAR groups have no statistical difference, at the molecular level , as mostTo define the genetic relationships among the faba bean samples, the similarity matrix obtained was also used to produce a UPGMA dendrogram . The cluFaba bean is an important crop for sustainable agriculture in marginal areas and advanced agricultural systems, as it plays an important role in soil fertility and nitrogen fixation, being able to grow in diverse climatic and soil conditions. Although faba bean is less consumed in western countries as human food, it is considered one of the main sources of cheap protein and energy for many people in Africa, parts of Asia, and Latin America, where many people cannot afford to buy meat . Seed siThe analysis of morphometric seed traits and molecular markers carried out in this work did not distinguish patterns in the distribution of morphological and genetic variations. All the samples collected by the NGBT teams appeared to belong to a single, genetically cohesive population. Similar results were observed for the faba bean samples obtained by ICARDA and collected from the same areas. When all faba samples were analyzed together, there were no differences between the NGBT and ICARDA groups. In fact, our molecular analyses and seed morphometric variation study demonstrated that the two groups belonged to a single meta population.Seed size is one of the most important morphological traits responsible for yield, and a major target for breeding. Several studies have led to the mapping of QTLs/genes for seed weight/size in soybean , chickpeOur data confirmed that morphometric seed traits were not associated to the markers used. This implies that any selection of lines based on seed traits still retains a variable level of genetic diversity potentially associated to other traits, such as adaptation or resistance, an issue to be taken into account in any faba bean-breeding program based on phenotypic data.The absence of genetic differentiation in different collection sites might depend on many factors. The absence of human selection pressure does not force crop adaptation in a specific direction. The outbreeding habit of faba bean is a second factor; in fact, while the advanced breeding varieties favor inbreeding in search of higher stability, as requested by UPOV standards, the landraces are generally quite allogamous ,48. ReceThe Evanno test yielded three subpopulations within the faba bean collection. In addition, each sample had a coefficient membership lower than 0.50, thus denying that it predominantly belonged to one of these subpopulations. This further supports the fact that the two meta-populations, NGBT and ICARDA, are subsamples of a unique population collected in the same area at different times.The biological significance of the three subpopulations detected with STRUCTURE is unclear, and might depend on the presence of genetic signals that do not parallel clear-cut characteristics. Nevertheless, several authors have reported that hierarchical analyses, such as those based on STRUCTURE or similar software, relay on strict assumptions that might not completely apply to the case studies, thus resulting in incorrect evaluation of the population structure .In conclusion, unintended conservation of ancient faba bean germplasm in Tunisian farms is witnessed, because the farmers cultivating faba bean landraces do not follow seed selection, as well as owing to concurring factors such as the high level of cross-pollination, and the consistent presence of pollinators (due to the limited use or absence of insecticides). This beneficial situation is an empirical application of best practices recommended by research institutions for on-farm conservation of plant genetic resources. In their quest to feed their families and their precious animals, the Tunisian farmers in the small villages mentioned above serendipitously stored and protected their faba bean genetic resources. These findings and implications should be further discussed in the broadest context possible. Future research directions should also be highlighted.


On average, large cities in the low-income and lower-middle-income countries had the highest urban population growth, and BUA expansion in the upper-middle-income countries was more than three times that of the high-income countries. Globally, more than 10% of BUAs in 325 large cities showed significant greening (P < 0.05) from 2001 to 2018. In particular, China accounted for 32% of greening BUAs in the 841 large cities, where about 108 million people lived. Our quantitative results provide information for future urban sustainable development, especially for rational urbanization of the developing world.The world has experienced dramatic urbanization in recent decades. However, we still lack information about the characteristics of urbanization in large cities throughout the world. After analyzing 841 large cities with built-up areas (BUAs) of over 100 km Urban development has dramatically increased in recent decades. Analyzing 841 large cities throughout the world for the period from 2001 to 2018, the authors disclosed uneven features of global urbanization in terms of urban expansion, population growth, and greening at different economic levels. By 2050, the global population will reach 9.7 billion, and 68% of the population  will live in urban areas. Undoubtedly, urban sustainable development is highly related to the future of humanity2. However, due to the different levels of socioeconomic development, the process of urbanization is uneven across countries3. Usually, in developed countries, urban expansion is adaptable to the population growth. The residents are served by good public services4 and have access to adequate urban infrastructure, such as water and energy supplies, sanitation5, education, and green space or parks. For many developing countries, the national economic growth and development are inadequate to meet the needs of a growing urban population7. In most cases, these cities lack basic infrastructure, and face overcrowding, pollution, and other urban environmental problems3. At present, urban sustainable development is an increasing concern worldwide, and it has been enshrined in the 2030 Agenda for the UN’s Sustainable Development Goals9. To achieve the goals, it is essential to track the urbanization progress of cities at different development stages in as detailed a way as possible. A large number of studies have focused on multiple aspects of urbanization from the regional scale14 to the global scale17. However, a global perspective that clarifies the urbanization characteristics of large cities around the world is still lacking.According to the latest report from the United Nations (UN), the global population in 2018 was 7.6 billion and the urban population was 4.2 billion18.Our work was mainly motivated by a desire to address the following questions: (1) how have global large cities developed in recent decades in terms of urban expansion, population growth, and urban greenness change? (2) What are the relationships between the urbanization features and the economic levels? To address both questions, we used remote sensing data and economic data for the period 2001−2018 and the gridded population data from 2000 to 2015 to quantify the urbanization characteristics of 841 large cities throughout the world. A quantitative and comparative study of the urbanization of global large cities has not yet been made available, but it is vital to combat poverty and achieve urban sustainable development in the near future5 km2 to 8.0 × 105 km2  per day. During this period, the top 10 countries with the greatest BUA expansion (BUAE) were China , the United States (9%), Nigeria (5.0%), India (3.6%), Indonesia (2.8%), Russia (1.8%), Mexico (1.7%), Malaysia (1.6%), Vietnam (1.5%), and Ghana (1.3%)  Table . Except 19, we merged the BUA pixels adjacent to each other into a united urban patch (see Methods). Our analysis focused on the urban patches larger than 100 km2  around the world. The advantage of defining large cities in this way is that we were able to include more cities with large BUA areas but relatively smaller populations, such as many low-density cities in the United States and other developed countries. It is worth noting that the large cities studied in this paper included clusters of cities; for example, the Yangtze River Delta (YRD) in China is considered one city, but actually includes four cities: Shanghai, Suzhou, Wuxi, and Changzhou. From 2001 to 2018, the number of the large cities increased from 777 to 841  more than $12,375 per capita), upper middle income , lower middle income , and low income  . In contrast, most of the cities in Europe and South America have BUAE of <50 km2  , we found that 80% of the large cities (676 out of 841) experienced population growth. Among them, 22 large cities experienced population growth of more than 2 million  and 1.7 times that in the upper-middle-income countries (3.0 × 105 per city)  of the urban population growth rate (UPGr) to the BUAE rate (BUr) for the 841 large cities (see Methods), and found that the RPB is larger than 1 in 543 large cities (64.5%). In particular, for the 105 large cities with BUAE of over 50 km2, the UPGr in 55% of the large cities (58 out of 105) is faster than the urban expansion rate  Fig. . We calcate Fig. , even th3 per km2, which is ~3.2 times that in the high-income countries (2.0 × 103 per km2)  and Africa (5 large cities) Fig. . The aveTo obtain a rational perspective of global urban greenness change, we assumed that if the greenness of a BUA pixel increased, some new parks, green spaces, and/or green roofs might have been developed in the pixel, or, at a minimum, the street vegetation in the pixel experienced growth. To avoid interference from the vegetation phenology, we chose the annual maximum greenness of the vegetation to represent the best state of urban greening.24 as a greenness indicator and BUA of 2018 as the urban extent, we applied the Mann–Kendall method to evaluate the trend of the annual maximum EVI (EVImax) for each urban pixel of the 841 large cities from 2001 to 2018 (see Methods). Then, we calculated the ratio (Rgreening) of the area of the pixels with a significant increasing EVImax trend (P < 0.05) to the BUA in 2018 of the corresponding large city. Using this method, we evaluated urban greening change since 2001. Most of the pixels with significant greening trends were located in stable BUAs . Finally, all 841 large cities were divided into three levels according to the values of Rgreening  indicated significant greening from 2001 to 2018 Rgreening > 0.1), we found that many greening areas of large cities have a shape like a “fried egg” where the yolk-shaped downtown area has been turning green significantly (P < 0.05); the “egg white” area refers to the area of browning or no significant greening outside the yolk-shaped area, and these areas are usually the suburbs. As one of the ten large cities with the largest greening BUA from 2001 to 2018, the YRD is a typical example of the “fried egg” with a cluster of the yolk-shaped areas of greening BUA   in 2018, and then ranked them into four quarters  of the large cities’ total population lived in cities in Q4; moreover, over 71% (157 out of 211) of these large cities in Q4 were located in high-income countries  among the four classifications    and low-income countries (6.2 × 103 per km2) is also significantly different (P < 0.01)  Fig. , while tty) Fig. . From th01) Fig. . This re01) Fig. , even th01) Fig. , the urb01) Fig. . Hence, max) to represent the best state of urban greening. According to the average EVImax for each city (EVIcity) in 2018, we found that one quarter of the greenness with the highest range of EVIcity, Q4, only encompassed 12% of the total population in the large cities   per capita in 2018 (see Methods) has reached the level of the high-income countries. Thus, we should be cautious in elucidating the impacts of country income levels on urbanization. In addition, in order to provide a global estimate of urbanization, we used 100 km2 as a criterion to screen large cities throughout the world. Consequently, only dozens of the cities in Africa and India were selected, which may not represent the reality of the urbanization process in those countries. In a future study, it will be necessary to adjust the criterion for large cities to get a more comprehensive understanding of the urbanization characteristics in those countries or regions.Overall, we observed that urbanization in the large cities has progressed rapidly in recent decades; moreover, our results indicate there are some economic-related global urbanization characteristics. However, due to the scarcity of the economic data (income levels)9. Therefore, our research using remote sensing data associated with limited demographic and economic data indicates a viable way of synoptically and repeatedly monitoring urbanization process worldwide. Our quantitative results, combined with previous policy studies38 and case studies40, make it possible to assess the unevenness of the urbanization from more perspectives, such as the urban heat island effect41, water security42, and structure25. For the majority of the developing countries, understanding the dramatic uneven urbanization of the past several decades can provide a scientific reference to improve their urban governance43, thereby achieving a virtuous circle among urban expansion, population growth, and urban greening changes. This is vital for achieving urban sustainable development.The systematic monitoring of the global urbanization process, especially in the low-income countries where the data are normally scarce, is essential to achieving the UN’s Sustainable Development Goals19. Any pixel classified as urban and BUA had at least a 30% impervious surface area, including building materials, asphalt, and parking lots. We used the tool “Raster to Polygon” in ArcGIS software to extract the BUA polygon from the raster layer. In this process, only the edge-adjacent (four connectivity) pixels were merged into a united urban patch.The urban and built-up pixels used in this study were extracted from the International Geosphere-Biosphere Programme classification types provided by the collection 6.0 MODIS yearly product known as MCD12Q1. The spatial resolution of the land cover product was 500 m and had a reported 73.6% overall land cover classification accuracy and relatively high accuracy for urban areas2), E2 is less than half of the minimum BUA of the large city (1 km2 < BUAE ≤ 50 km2), and E3 is larger than half of the minimum BUA of the large city (>50 km2). The reason for using 50 km2 as the threshold was that when excluding 12 large cities with BUAE of more than 250 km2, we found that the value of the average urban expansion of the remaining 829 large cities, adding 1 standard deviation to those expansions, was close to 50 km2.In this study, there were three levels of BUAE Fig. : E1 refe2, the second level was between 580 and 2000 km2, and the third level was larger than 2000 km2. For BUAs in 2018, more than 19 large cities had areas over 2000 km2. Excluding those 19 cities, we found that the value of the average BUA of the remaining 822 large cities, adding 1 standard deviation to those areas, was close to 580 km2. This is why we divided the 841 large cities into three different sizes  was generated from all the acquisitions over a 16-day period.The vegetation index data sets from the MOD13A1 version 6 product were used in this study to match the spatial resolution of the land cover data (500 m). These data sets include two primary vegetation layers: the normalized difference vegetation index (NDVI) and the EVI. Due to the greater sensitivity of the EVI relative to that of the NDVI in the high-biomass regions, our results were based on the use of the EVI. For each pixel, the annual maximum EVI . Using this method, we could exclude those pixels that had changed from rural vegetation to BUA (urban expansion).We used a Mann–Kendall test, which is a nonparametric test, to detect monotonic trends in the time-series data and evaluate the trends of EVImax from 2001 to 2018 for each pixel in a raster file. Then, the Geospatial Data Abstraction Library  was used to mosaic all the raster files (212 files) of the trends into a global raster map of the EVImax trend. Finally, we extracted the EVImax trend for each urban pixel within the urban extent (BUA in 2018) and calculated the ratio (Rgreening) of the area of the pixels with a significant increasing EVImax trend (P < 0.05) to the entire BUA of the corresponding urban extent.In this study, the spatial resolution of the MOD13A1 data was 500 m; thus, we wrote a Python program to calculate the Mann–Kendall trend of EVIThe global country administrative areas were downloaded from the Database of Global Administrative Areas (GADM). The GADM data consisted high-resolution shapefiles at all administrative levels, such as at the country, state, and provincial levels. We used the latest version (v 3.4) in this study.World Urbanization Prospects: The 2018 Revision. The 1 km resolution Gridded Population of the World collection , which was adjusted to the national-level population counts estimated by the UN, was obtained from the Socioeconomic Data and Applications Center. The World Bank classifies economies as low income (<$1025), lower middle income ($1026–$3995), upper middle income , or high income  based on the GNI per capita in 2018. The GDP data of YRD and PRD in 2018 were obtained from the Statistics Bureau of Jiangsu Province and Guangdong Province, respectively.The country population data were obtained from RPB) of the UPGr to the BUr for the 841 large cities. For each large city, the UPGr and BUr were defined as follows:2015 and Pop2000 are the urban populations of a large city in 2015 and 2000, respectively. BUA2018 and BUA2001 are the BUAs of the large city in 2018 and 2001, respectively.In this study, we calculated the ratio (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Enzootic Bovine Leukosis (EBL) caused by the bovine leukemia virus (BLV) has been eradicated in over 20 countries. In contrast, the U.S. and many other nations are experiencing increasing prevalence in the absence of efforts to control transmission. Recent studies have shown that BLV infection in dairy cattle has a greater impact beyond the long-recognized lymphoma development that occurs in <5% of infected cattle. Like other retroviruses, BLV appears to cause multiple immune system disruptions, affecting both cellular and humoral immunity, which are likely responsible for increasingly documented associations with decreased dairy production and decreased productive lifespan. Realization of these economic losses has increased interest in controlling BLV using technology that was unavailable decades ago, when many nations eradicated BLV via traditional antibody testing and slaughter methods. This traditional control is not economically feasible for many nations where the average herd antibody prevalence is rapidly approaching 50%. The ELISA screening of cattle with follow-up testing via qPCR for proviral load helps prioritize the most infectious cattle for segregation or culling. The efficacy of this approach has been demonstrated in at least four herds. Breeding cattle for resistance to BLV disease progression also appears to hold promise, and several laboratories are working on BLV vaccines. There are many research priorities for a wide variety of disciplines, especially including the need to investigate the reports linking BLV and human breast cancer. Enzootic Bovine Leukosis caused by the bovine leukemia virus (BLV) has been eradicated in over 20 countries, while many other nations are experiencing increased prevalence. Recent recognition of BLV’s economic impact has led to increased interest in controlling BLV in our cattle populations. In the last several years, major advancements have improved our understanding of BLV epidemiology, diagnosis and disease control methods, and this review will summarize those advancements and suggest areas for further research.Recent surveys in most nations outside of Europe have reported continuous increases in BLV prevalence in both beef and dairy cattle. Prevalence is generally higher in dairy cattle, with the U.S. Japan, Canada, Brazil, China and Argentina reporting that most herds are infected and are approaching a situation where almost half of their dairy cattle are showing serologic evidence of infection. The following countries and regions around the world are reporting moderate increases in BLV: China , ColumbiMost transmission of BLV is through the provirus-infected lymphocyte. Many modes of BLV transmission are possible, but the most important means of transmission likely varies greatly by geography and agricultural system. For example, transmission via fly bites is thought to be important in some parts of the world but not in other parts. Bloodborne transmission is the most recognized route of transmission, but direct contact may be very important in most all management systems. The importance of milk-borne spread is still unknown, as is the potential role of the free RNA-virus. It is unknown if early exposure as a calf or even in utero may lead to a latent infection which later becomes activated, perhaps at times of stress such as pregnancy, first calving and lactation.The economic loss from BLV-induced lymphoma was previously the only known impact of BLV in cattle. Reportedly occurring in <5% of BLV-infected cattle, lymphomatous tumors remain the top cause of condemnation for U.S. dairy cattle carcasses . Like itBLV-infected dairy cattle may reduce milk production and shorten cow lifespan ,22. The One study  showing BLV-infected cattle were reported to have a shorter lifespan in the herd when compared to their ELISA-negative herd mates ,23,31,32BLV-associated disease and production inefficiencies are thought to be due to a disrupted immune system. A 2015 review by Frie and Coussens  outlinesThe effects of BLV infection on mucosal immunity are not well-characterized despite the importance of mucosal immunity in preventing many cattle diseases. BLV provirus has been detected in a variety of mucosal secretions, including milk, saliva, nasal secretions , vaginalMultiple copies of the BLV provirus can integrate into the host cell genome at multiple sites . QuantitThe correlation between ELISA antibodies and PVL is generally weak, but there is a strong correlation r = 0.855) between BLV PVL and blood lymphocyte counts . Previou between Recently, Hutchinson modeled lymphocyte counts and proviral load over 6-month intervals and measured a small average increase over time . HoweverP = 0.06) 18% greater hazard of being culled compared to their ELISA-negative herdmates. In contrast, cattle with a higher proviral load had an 84% greater hazard of leaving the herd as compared with ELISA-negative cattle (P = 0.01).Compared with dairy cattle, much less is known about the epidemiology of BLV in beef cattle. A US Midwest survey completed by Benitez et al. in 2019 showed that 78% of 28 cow-calf herds had at least one ELISA-positive animal, with 36% ELISA-positive cattle among the 3175 tested . A 24-moAmong breeding beef bulls in the Midwest US, 49% of 39 farms had at least one ELISA-positive bull, and 45% of the 121 bulls tested were ELISA-positive in a recent study . ProviraEarly control efforts were based on culling cattle with high blood lymphocyte counts . More reOne 3000-cow herd in the Midwest U.S. has had great success in screening cows with the ELISA test and measuring PVL on ELISA-positive cows. Cows with the highest PVL were then segregated and prioritized for culling. As of 27 October 2020, only 10 cows with a PVL > 0 remained in a separate pen awaiting culling. Because BLV in the heifers had been controlled via ELISA testing, this large herd is on the verge of eradicating BLV .Genetic diversity within a population is directly linked to that population’s ability to respond to specific pathogens, reflecting the high degree of heterogeneity within genes involved in the innate and adaptive immune system. The major histocompatibility complex (MHC) is a cluster of genes that are responsible for self and non-self-antigen recognition for proper immune function in mammals. While the general structure of the MHC is conserved amongst species, specific exons encoding MHC molecules are highly polymorphic to provide a diversity of adaptive immune responses to emerging pathogens within a population. The Bovine leukocyte antigen (BoLA) in cattle is orthologous to the human MHC or Human Leukocyte Antigen (HLA) complex. Within this cluster of evolutionarily conserved genes, the BoLA Class IIa subregion houses the DR and DQ genes, which have undergone extensive genetic characterization within the context of BLV susceptibility and disease progression ,66,67,68Specifically, DRB3 and DQA1 sequence-based typing (SBT) is becoming a requisite component of BLV cohort studies in order to better understand the propensity of individual disease trajectory. The DRB3 gene has 130 identified alleles to date  and is tPedigree analysis first demonstrated that bovine lymphosarcoma and persistent lymphocytosis track with family lineages, providing the first evidence for genetic influence on susceptibility to BLV . OriginaInitial approaches to BoLA typing involved restriction enzyme digestion and analysis of resulting fragments to identify alleles (RFLP). BoLA-allele identification is now accomplished via different sequencing approaches such as PCR followed by Sanger sequencing. Presently, research groups are utilizing multiplex barcoded primers in order to systematically tag hundreds of animals prior to next generation sequencing and bioinformatic analysis, enabling a wider scope of animals. This approach will enable studies to analyze a much larger number of animals to determine how various BoLA-DRB3 alleles associate with disease state. Due to the high degree of heterogeneity of this genetic locus, SNP-based genotyping is unable to resolve the identity via hybridization probes. This genetic analysis would allow bull studs to determine the BoLA status of their young sires and potentially market BLV-resistance as an economically important trait. Use of this approach as an in-herd selection tool may not be as useful, as cattle do not persist within modern commercial dairy herds long enough to recover an economic benefit. Current studies are under way to determine if colostrum from BoLA-DRB3*0902 dams provide additional protection to their young over non-BoLA-DRB3*0902 dams, which may be a useful management strategy for producers. Though selection for this important gene may not resolve the high rate of BLV prevalence in North America, it may be a useful tool for understanding virus-host interaction and BLV’s impact on profitability.BLV is a retrovirus closely related to HTLV-1, whereby definition, it encodes a reverse-transcribed DNA copy of its RNA genome into the host cell that it invades. Once integrated, the proviral genome is propagated via mitosis. Expression of BLV proviral genes occurs through endogenous transcription and translation mechanisms. The BLV genome consists of a common 5′–gag–pro–pol–env–3′ gene cassette encoding the viral capsid, protease and envelope proteins required for viral repackaging. Additionally, the BLV viral genome encodes its own reverse transcriptase (pol) required for creating the DNA version of the virus for integration accomplished via the virally encoded integrase also located within the pol gene. Accessory genes, including the U3 region within the 3’ untranslated region (UTR) and U5 within the 5’ UTR, enhance viral gene transcription via transcription factor binding sites. Two protein coding genes, Tax and Rex, are considered CIS-transcriptional activators that bind to the 3’ LTR and are commonly implicated in viral oncogenesis ,77. UnliThe zoonotic potential of BLV has long been debated, but the understanding of retrovirus biology developed for the AIDS/HIV epidemic has led to more recent findings for the BLV retrovirus . ApproxiEvidence for human infection has not been reported from any investigator. Whole genome sequencing of 51 breast cancer tumors showed no evidence of BLV DNA . A queryPotential routes of transmission between cattle and people are numerous, including direct contact with infected animals and the consumption of unpasteurized or undercooked dairy and beef products, which have been shown to contain the BLV provirus . The detVaccination of cattle for BLV is still being investigated. Unlike some other retroviruses, such as HIV, BLV has a relatively stable genome, and thus, an efficacious vaccine may be possible. However, many previous attempts at developing a vaccine for BLV have been unsuccessful . SeveralGenetically modified vaccine candidates must contend with additional regulatory requirements. Hopefully any developed vaccine will enable vaccinates to be distinguished from cattle with natural infection, and thus, enhance rather than deter any ongoing BLV control efforts.What causes a minority of cattle to develop extremely high proviral load and to what extent is this determined by their genetics or the time at which they become infected (in utero or very early in life via colostrum)?How important are host genetics, whereby certain alleles predispose an animal to a high or low proviral load, antibody titer and/or severity of pathology?Why do first lactation animals seemingly avoid the milk production loss and increased risk of culling that are associated with older ELISA-positive cows?The severe inbreeding of Holstein dairy cattle is such that the effective population size is <50 animals, and they would be considered critically endangered if they were a wild animal species . This raAttribution studies are needed to determine the percentage of new BLV infections that come from direct contact, biting fly transmission, shared hypodermic needles, etc.The role of free BLV RNA in disease transmission should be definitively established.Controlled studies are required to measure the extent to which BLV infection contributes to the causation of the major dairy diseases such as mastitis, lameness, infertility, etc.Longitudinal studies should determine how commonly animals are infected with BLV as calves, but the virus becomes sequestered until the stress of birth and lactation. Genetic methods should be developed to determine if newly recognized infections in cows are usually caused by the same strain found in their dams over two years ago, or the same strains found in their contemporaries.The role of BLV in the induction and pathogenesis of human breast cancer and other human diseases should be definitively established.What role does microRNA play in the pathogenesis of BLV, and can it be used for diagnosis or prognosis?A safe and efficacious BLV vaccine should be developed that allows differentiation between vaccinates and natural infection.The likely return on investment for BLV control and eradication should be determined for test and removal using ELISA and by using ELISA screening with proviral load follow-up testing.E. coli, Campylobacter and Salmonella will become an issue unique to animal agriculture, as it will be much easier to produce a pathogen-free food product coming out of a controlled fermentation or tissue culture process than it will be to produce such a product from a pathogen-abundant farm environment. As the public’s immune systems become increasingly naïve to our common foodborne pathogens, progressively lower pathogen doses will be capable of causing clinical disease.BLV control measures must be targeted to the needs of the future, rather than the needs of the present. A recent Rethinkx analysis of animal agriculture predicts a rapid contraction in the dairy and beef industries as alternative methods of producing protein are developed, e.g., fermentation of specific proteins and tissue culture . They prTo compete with these new food products, the surviving animal industry will need to have adequately solved the previously mentioned issues. Future consumers may not want animal products from herds infected with BLV, which may become recognized as a possible public health hazard and also a cause of animal suffering. If the predicted contraction of herd and cow numbers becomes a reality, many producers will likely forgo controlling BLV if they do not expect their herd to survive for many more years. However, herd managers wanting to reduce the size of their herd may take this as an opportunity for selectively culling BLV-infected cows in the belief that BLV-free herds will be more profitable and appealing to consumers. However, in these uncertain times for the animal industries, many producers may not want to invest funds to eradicate BLV. Some may even decide to wait in the hope that a vaccine is developed that will serve as a useful tool for BLV eradication.Heightened BLV research activity has been driven by BLV’s increasing prevalence in many nations and recent recognition of its impact on milk production, cow lifespan and possible public health implications. The means by which BLV disrupts the cow’s immune system are being described in greater detail than was possible before. It must be determined how newly available BLV diagnostic tools can best be used to control this infection. Many important research questions must be addressed regarding BLV epidemiology, immunology and control. It is likely that the future of the animal industries will see a continued effort to control BLV.


Vitis spp. in this Eastern Mediterranean island has given rise to a plethora of biotypes that have not been adequately characterized, despite their unique attributes and stress tolerance. This ancient germplasm also has an additional value since it survived the phylloxera outbreak; hence, it possesses a large amount of genetic diversity that has been unnoticed. In order to provide useful insights to the lineage of Cypriot vineyards, a two-year-spanning collection of centennial grapevine cultivars mostly regarded to belong to four indigenous variety clusters  was initiated. There were 164 accessions across the broader Commandaria wine zone sampled and characterized using a universal microsatellite primer set. Genetic analysis indicated that considered indigenous Cypriot germplasm has a polyclonal structure with a high level of heterozygosity. Moreover, several lineages or unexplored varieties may exist, since a larger than considered number of discrete genotypes was discovered. Furthermore, it was established that grapevine lineages in Cyprus were shaped across eras via clonal, as well as, sexual propagation. The special attributes of the Cypriot landscape are discussed.Cypriot vineyards are considered as one among the earliest niches of viticulture and a pivotal hub for the domestication and dissemination of grapevine. The millennial presence of  Grapes are the most emblematic deciduous woody vines. The imprint of viticulture to agriculture, society, culture, and even religion rituals has been unparalleled, and it can be stated without any reluctance that viticulture is intertwined with human history throughout eras since antiquity. At present, the total global grape production is 75 million tons and the surface of vineyards spans at around 7.5 million ha (FAO). In terms of revenue, the global wine market alone has perceived a stable expansion over the years and has developed as a multi-billion industry .Vitis vinifera L. or Vitis vinifera subsp. vinifera) have a widespread distribution and can be found across all continents. Nonetheless, Vitis spp. progenitors can be exclusively found at specific areas; V. vinifera subsp. sylvestris (regarded as the Eurasian Vitis wild form) is found in North Africa and Eurasia, while Vitis labrusca is restricted to Eastern North America. The distinction of wild ancestors from cultivated forms can be challenging, since at least in the broader Mediterranean zone, a wide variety of feral forms and hybrids are present. These comprise cultivation escapees, as well as, seed-disseminated weedy types growing in local niches that occur primarily in surroundings areas of vineyards [Taxonomically, grapes belong to the Vitaceae family which consists of 14 genera and about 900 species . Based oV. vinifera subsp. sylvestris is considered to be the wild relative of cultivated grapes (Vitis vinifera subsp. vinifera) and several botanical characteristics are used in order to differentiate the two taxa. One of the most important characters is the flower, as wild grapevines are dioecious, in contrast to virtually all cultivated genotypes that are hermaphrodites [Currently, hrodites . Due to hrodites . Selectihrodites .Increasing indications converge that the domestication of grapevines was disseminated alongside the advent of wine production from the center of domestication placed at the Near East and the Southern Caspian Belt . The difVitis species were introduced as rootstocks, or used for incorporating disease resistant traits to hybrids [Vitis forms, supported by an augmented interest of global wine producers for only a handful of elite varieties [However, there were milestone eras and events that influenced the plethora of genetic grapevine diversity. Early Muslim conquests in the 7th century, and occupation of the primary as well as secondary domestication regions, introduced an extended period of gradual decline of wine production. This in succession could have forced a subsequent extinction of genotypes used for vinification and a turnover towards table grapes production . A more  hybrids . Hence, arieties .Cyprus was on the map of ancient trade routes and, as a result, on the crossroads of the westward dissemination of grapevines to Europe. Cyprus and viticulture have been inextricably related since antiquity . RegardlMoreover, a precise number of varieties and clones is not clearly demarcated and there is quite uncertainty regarding the actual number since synonyms and homonyms can occur. Furthermore, ampelographic data are not largely available. Pierre Galet referred that “among the vines of Cyprus, there are (some) fifteen indigenous varieties”: “Xynisteri”, “Mavro”, “Maratheftiko”, “Lefkas”, “Opthalmo”, “Promara”, “Spourtiko”, “Flouriko”, “Yiannoudhi”, “Katomylitiko”, “Kanella”, “Morokanella”, “Michalias”, “Skouro mavro”, “Rodhino rose”, “Rozoudi rose”, and “Maroucho black” . On the Vitis form, it lacks hermaphrodite flowers and is frequently co-planted with other cultivars in order to attain fruit-set and development [One of the most emblematic  dessert wines is Commandaria. Commandaria is an amber-colored traditional wine produced in Cyprus, and it was the first type of wine receiving the controlled appellation of origin certification amid Cypriot wines . One of elopment .terroir are among the most complex and debated issues in viniculture and oenology. The effect of the genotype and the existence of different clones within varieties is often overlooked in studies, hence making it difficult to drive meaningful conclusions (the possible diversity due to genetic background is not considered). Still, clonal selection is extensively practiced in viticulture, signifying that somatic alterations have a substantial unaccounted outcome on berry and wine attributes [Several aspects can affect the outcome of vinification. Among these, primarily the variety (or genotype) and tributes . Since CSince a nationwide molecular characterization of the Cypriot germplasm has not been conducted so far, the size of genetic diversity in Cypriot grapevines must be largely underestimated . Nowadays newly established vineyards are propagated clones of the same genotype; hence, focusing on antique grapevines could in fact be more effective in demarking genetic diversity. The hypothesis when commencing this project was that relic genotypes that are currently considered as a variety (due to the lack of robust ampelographic description and identification) could in fact be landraces (populations), discrete clones, or different varieties that may have a potential in enriching the Cypriot grapevine germplasm. Towards attaining meaningful insights for the Cypriot grapevine landscape, 164 centennial genotypes  were sampled across the Commandaria zone and genotyped using 11 nuclear SSRs in order to reveal their genetic variability. This study is the starting point towards the prominence and safeguarding of local indigenous germplasm, and the highlighting of historical knowledge and heritage concerning the millennia viniculture of Cyprus.A total of 164 centennial grapevine accessions putatively attributed to the four mentioned populations, Maratheftiko, Mavro, Veriko, and Xynisteri , were co2O. DNA concentration and purity were estimated using nanodrop spectrophotometry.For DNA extraction, leaves were excised and kept between humid paper towels on ice, until storage at −80 °C. Leaf tissue (100–200 mg) was weighed in 2-mL rounded Eppendorf tubes, flash frozen in liquid nitrogen and freeze-dried overnight. Two stainless steel balls (3 mm) were added in each Eppendorf, samples were quickly frozen in liquid nitrogen, and tissues were crushed for 30 sec (at full speed) using a mixer mill MM 200 (Retsch). Samples were kept at −80°C until DNA extraction. In preliminary tests, it was established that DNA extraction using standard DNA extraction procedures  proved problematic (for multiplex PCR) due to the highly recalcitrant nature of vine tissues. Hence, before DNA extraction, crushed tissues were pre-washed twice with sorbitol wash buffer  Polyvinylpyrrolidone PVP-40, 0.1% β-mercaptoethanol) to remove interfering metabolites . After tTM, ROXTM, or TAMRATM). This enabled the run of a one-tube, single-reaction nested PCR, as previously described [http://www.vitisdb.it/; Vitis International Variety Catalogue VIVC, http://www.vivc.de/) and pilot single locus PCR reactions, three different panels were selected (TM) was initially tested for multiplexing, it was not finally employed, since it was determined that it interfered with red (ROXTM) and blue (FAMTM) spectra, causing minor peak pull-ups that could result to erroneous scoring.Eleven SSR markers were selected for the analysis of the Cypriot germplasm . Nine ofescribed . Based oselected , in orde2 in a 12 μL final reaction volume. Conditions for the PCR amplification were: 94 °C , followed by 39 cycles at 94/56/72 °C (60 s), and a final extension at 72 °C for 30 min.For PCR reactions, the mix contained 50 ng of template DNA, 10 pmol of a labeled M13 tailed forward primer, 10 pmol of the reverse and 2.5 pmol of the forward primer, 0.2 mM dNTPs, 0.5 U KAPA Taq DNA Polymerase (Kapa Biosystems), and a 2.5 mM final concentration of MgCl2O. One μL of the dilutions was added to 10 μL deionized formamide and 0.2 μL of DNA size standard , before denaturing at 95 °C (5 min). Allele fragments were separated (in three discrete panels) by capillary electrophoresis using an Applied Biosystems 3130® Genetic Analyzer . Two control varieties (Cabernet Sauvignon and Merlot) and four Cypriot genotypes were replicated in every run, to normalize microsatellite allele sizes and associate genotypes across runs and databases. Allele scoring was performed by two researchers independently, and tandem software was utilized to verify/correct bins [Amplification products were verified using a standard 2% agarose electrophoresis and diluted at a 1:40 ratio with dd Hect bins .Microsatellite data curation and formatting was performed via the MS Excel add-in GENALEX v. 6.501 . All genThe same dataset was similarly used to test for linkage disequilibrium and Hardy–Weinberg equilibrium (HWE) in the grapevine accessions. Genetic relationships between individuals (MLGs) were assessed using the dissimilarity distance calculation and visualized as a minimum spanning network (MSN) and a discriminant analysis of principal components (DAPC). All of the above-mentioned statistics/analyses were performed using the Poppr (V. 2.8.5) package  and the A phylogenetic tree was also constructed using the binary template  using the R package polysat . An apprhttp://clumpak.tau.ac.il/).Finally, a Bayesian statistic employing method for estimating genetic kinship was performed using Structure 2.3.4 . The adm−7 to 7.8 × 10−10 across populations.From the broader Commandaria zone of Cyprus 164 vines putatively attributed to four varieties  were genotyped using a primer set of 11 SSR markers. The genotype accumulation curve  indicateAll loci were found polymorphic , and in The Simpson index varied from 0.5 for locus VrZAG112, up to 0.83 for locus VVMD28 (mean 0.73); in general, being proportional to the number of alleles detected among discrete loci. Still, across loci the high percentages of heterozygosity and evenness of alleles, elevated the efficiency of these markers to reveal the genetic polymorphism. Indices indicated the high potential for this marker set to define variability in the Cypriot panel of grapevines.Multi-locus genotype analysis across populations recorded 32 MLGs for Xynisteri, 41 for Mavro, eight for Maratheftiko, and two for Veriko . The mosH = 3.714, Hexp = 0.667), even though many accessions shared a genotype.Even though genotypic abundancy can be primarily inferred from the number of MLGs detected, unequal sample size can cause a partial bias; hence, an estimate of the quantity of genotypes that could be anticipated at the major common sample size established on rarefaction (eMLG) could be more suitable. The expected number of eMLGs was also computed and was found comparable among Xynisteri and Mavro populations 10; . Still, Ia and rbarD); it seems that populations in Cyprus were mostly clonally propagated. Moreover, we explored the probability that loci were under Hardy–Weinberg equilibrium (p < 0.5). This suggests that even though at a reduced rate, sexual propagation has also occurred in the lineage of Cypriot grapevine varieties.In order to determine the mode of reproduction across these centennial varieties, a test of linkage disequilibrium was conducted for clone corrected genotypes. Consequently, disequilibrium indices were calculated  and did not correspond to known varieties/genotypes.Among the 164 Cypriot accessions (registered and putatively assigned to the four clusters), a total of 83 multi-locus genotypes (MLGs) were identified . InteresIn viticulture, it is known that grapevine cultivars frequently consist of discrete clones, sharing mutual phenotypic traits and grouped as a variety cluster . If, howHexp = 0.732). The within cultivar heterozygosity is infrequently reported across studies, but in several cases, it seems that a moderate-to-high proportion of heterozygosity exists. Heterozygosity levels across grapevine clones generally vary from 0.47 for “Tannat” [In genetic studies, when dealing with clonal taxa, analyses can be typically conducted using (or not) clonal correction. Since allele frequencies can be affected by the number of redundant genotypes, clone correction is often advised in order to better depict genetic relationships and kinships, as well as removing potential bias . In the “Tannat”  to 0.87 “Tannat”  cultivar“Tannat” . Despite“Tannat” . In gene“Tannat” ,47,48,49Since Cypriot grapevines have been cultivated for millennia, a high amount of diversity is expected among clones or varieties. Pinot is another characteristic cultivar where several clones are considered, and a great amount of heterogeneity exists. Unfortunately, the lack of precise ampelographic data on Cypriot germplasm complicates the cut-off line that defines the optimal taxonomic status. As a result, the existence of unidentified/misidentified varieties, hybrids, or feral forms considered as a “true-to-type” variety is a possibility. In the current study the predominant MLGs of Xynisteri cluster  differed in one out of 11 loci, hence probably representing different clones of the same variety. This was also the case for the Mavro (MLG 38 and MLG 36) and Maratheftiko (MLG 7 and MLG 8) clusters, which were also identical in 10 out of 11 loci. Conversely, in the study of Hvarleva et al. , genotypThe high amount of heterozygosity within Cypriot-cultivated grapevines could also be the result of a recurrent hybridization and parallel domestication events due to the long cultivation history and the proximity to the hotspot of grapevine domestication. In that direction, in our dataset several tri-allelic profiles across markers and genotypes were observed for markers VVMD32 and VVMD5, indicative of hybridization or chimerism. This phenomenon in clonally propagated grapevines such as monumental cultivars could participate in clonal variation and hamper proper variety identification or pedigree analysis . ZarouriUnfortunately, microsatellite profiles of Cypriot grapevine genotypes  are not available in public databases such as VIVC. Furthermore, partial comparison of the current dataset to the one previously described , revealesylvestris forms as grapevines disseminated to Europe [Nonetheless, the genetic relationships of the current dataset largely correlates to previous studies that included Cypriot genotypes  even thoo Europe . The exto Europe .rbarD values indicated that Cypriot genotypes were also clonally propagated since a considerable disequilibrium among loci was attained signifying conscious selection of elite genotypes during domestication. The clonal propagation of Cypriot genotypes can also be depicted from the network analysis since a limited reticulation can be observed among clusters  reveal that Xynisteri has been broadly used in hybrid crosses in Israel during the 1930s, establishing a series of Hebron varieties used mainly as rootstocks. Nowadays, Xynisteri is largely tested outside the narrow range of Cyprus, at high temperate-arid regions of Australia, highlighting the ready-to-use potential of Cypriot germplasm for grapevine sustainability purposes [The “wild” characteristics of the Cypriot germplasm is also supported by vinification features of these genotypes. Xynisteri (‘Xyno’ is the Greek word for sour) is the predominant white variety and has a sharp taste due to the high level of acids . Other tis forms . In factis forms . Recent is forms ,59 or abis forms . Past pepurposes .In terms of conservation, several projects for the preservation and highlighting of local genetic resources have been directed in grapevine growing countries . As a re


Sporophila maximiliani) is an endangered South American bird that has suffered from trafficking and the destruction of its natural habitat. In contrast, there are over 180,000 Great-billed Seed-finches legally raised in captivity in Brazil. The interest as a pet for Great-billed Seed-finches is due to their exceptional ability to sing. In the present research, the unknown genetic structure of the Great-billed Seed-finch captive population was investigated by quantitative analysis of 6,226 pedigree records. Additionally, 7,671 phenotypic records were available to estimate genetic parameters such as heritability and evolvability of a song-related trait of these birds for competitions. The captive Great-billed Seed-Finch population faces many of the problems commonly encountered in domestic animal populations such as a high level of inbreeding , pedigree bottlenecks, unbalanced contribution of breeding animals and structuring . Despite this, most genetic diversity remains preserved within aviaries. The high generation interval (5.74 years) found for this population should help to prevent a rapid increase in inbreeding and genetic drift. These results should serve as strong motivation and support for urgent actions to manage the genetic diversity of captive Great-billed Seed-Finches. From the viewpoint of genetic improvement for singing time in tournaments (STT), this trait presents sufficient variability to allow response to artificial selection given the heritability of 18.7% and evolvability of 2,447%. In contrast, inbreeding and high generation interval appear to be the most considerable barriers that may limit the genetic gain for STT. Widespread adoption of techniques such as optimal genetic contribution selection and implementation of routine genetic diversity monitoring via pedigree analysis and molecular tools can be crucial both in terms of breeding and conservation of genetic diversity of captive Great-billed Seed-Finches.The Great-billed Seed-finch ( Sporophila maximiliani) is a South American bird, occurring mainly in flooded areas and marshy borders in the Brazilian Cerrado biome −Cij, where Cij is the average pairwise coancestry between birds of aviaries i and j, including all Ni×Nj pairs, and Cii and Cjj are the average pairwise coancestries within aviaries i and j, respectively. A distance matrix (10 × 10) was constructed, which has 0 on the diagonals and Dij on the off-diagonals. A cluster analysis was applied to the distance matrix using the unweighted pair group method with arithmetic mean and a dendrogram was produced.Within the reference population, the birds of ten of the most popular commercial aviaries in Brazil were identified. For these aviaries was calculated the Nei's minimum genetic distance  as Dij =The pedigree analyses were performed using the PEDIG  and RELAad libitum access to water, food, and a bathtub. Almost all birds participating in the competitions had a female with which they were mated. The mating of Great-billed Seed-finches is a crucial point in stimulating male-male competition. In the reproductive period, these birds become extremely territorial and aggressive [Fiber tournaments are very popular for several bird species in Brazil, each with its own rules. For Great-billed Seed-finches, the competitions were held in large gyms with good lighting and ventilation . The birgressive . The femThe trait analyzed in the present study was defined as singing time in tournaments (STT), measured in seconds. At around 12:00 pm, an electronic record of the total time of each bird sang within 15 minutes was recorded. Each technician positioned about 1.5 m in front of a bird's cage used a timer provided by the event organizer for the registration of the STT. The winning bird of an event was the one with the highest STT record.Only birds whose pedigree were known as well as their hatch date were considered in this study. Contemporary groups were defined by birds that participated in the same tournament, i.e., in the same city, place, year, month, and day. Records of birds in contemporary groups with fewer than three animals were eliminated, as well as data exceeding 3.5 standard deviations above or below the mean of the trait within the respective contemporary group. Thus, after quality control of data, 7,549 records of 602 birds were effectively used to estimate genetic parameters. The pedigree file of birds with phenotypic records comprised 2,138 animals. A summary of the data set is shown in σ2 are variances the additive genetic (a), permanent environmental (c), breeder (b), and residual (e) effects, respectively; A is the numerator of the relationship matrix among all birds, and I is the identity matrix. The variance components were obtained under a single trait repeatability animal model [t generations with evolvability IA and strength of selection βμ was computed as described in [IAβμ)t. The parameter βμ was considered equal to 1, which means that a 1% change in the trait produces a 1% change in fitness.The mixed model adopted to estimate the genetic parameters of STT included the fixed effects of contemporary group (as defined above), the age of the bird at the date of competition (linear and quadratic effects) and the random effects of animal , breeder and residual. The (co)variance structure follows:al model  using Gial model . The prial model . Narrow-al model ,43. The ribed in :  were male and 3,688 (59.24%) female. The ratio of males to females in reproduction was approximately 1 to 1.8. This number reflected the adoption of polygamy as a mating practice. The average number of progeny was 5.95 and 3.28, while the maximum was 185 and 40 per father and mother, respectively. The average number of hatchlings per clutch was 1.47 (standard deviation of 0.59). Approximately 79% of hatches occurred between November and March, with 19.7% in January.As previously reported, the reference population was defined based on the generation interval  to refleApproximately 97% of the total Great-billed Seed-Finches analyzed in the reference population had known parents . The meaMost birds analyzed were inbred (70.47%). The average inbreeding for the population was 8.26% and reached 11.73% for inbred birds. Some individuals presented an extremely high inbreeding coefficient, above 40%. In the last decade, there has been an increase of 0.57%/year in the average inbreeding of the population . It is iFIS value was greater than zero (FIT) was higher than the expected level of inbreeding under random mating (FST). As an illustration of this scenario, the estimate of effective population size based on coancestry (55.60) was more than double the effective size based on inbreeding rate (19.10). A small effective population size associated with a high level of inbreeding may lead to increased loss of genetic diversity in the next generations.The han zero , indicatfe is dependent on both the number of founders and the balance of their expected contributions to the gene pool. When founders contribute to the reference population more equally, the effective number of founders is higher. In the present population, fe represented only 5% of the total number of founders  supports the loss of genetic diversity due to genetic drift. Approximately 4% of the genetic diversity of the current population was lost, most of which was due to genetic drift.The parameter founders . In thisNeCi and NeFi allows to estimate the equivalent number of subpopulations (S), which was 2.91 in the present research. Therefore, the current population of captive Great-billed Seed-Finches is subdivided. The dendrogram based on Nei's minimum genetic distance among ten popular and representative Brazilian commercial aviaries  made it possible to identify subpopulations of captive Great-billed Seed-Finches  approximately 1.7 years [Petroica australis rakiura) 4 years [The average generation interval found for the Great-billed captive Seed-finches was relatively high compared with other species of birds such as canary Lizard (.7 years  and Stew 4 years . DiffereTaeniopygia guttata), those authors estimated an average inbreeding of 4.30% while using an SNP-based technique obtained an estimate of 6.40% (3.6 to 10.20%). With caution in any comparison, the estimate obtained in the present study was within limits observed for other bird populations with an old history of domestication, free-living or even endangered species such as song sparrows (Melospiza melodia) 7.60% [Passer domesticus) 12.9% [Serinus canaria) 15.83% [Notiomystis cincta) 8.00% [Petroica australis rakiura) approximately 7.00% in the last year evaluated by the author [FIT) was more than twice the inbreeding expected under random mating (FST). Despite the negative aspects related to effective size and inbreeding depression of restricted matings within subpopulations, the fragmentation into some isolated groups can be beneficial for preserving the genetic diversity of the population. In the long term, as different allelic variants will be fixed in each group, they become genetic reservoirs of variation [The population of Great-billed Seed-finches captives exhibited a high level of inbreeding mean of 8.26%) and an alarming trend of increase in the last decade. The cumulative aspect of inbreeding in captive populations is worrisome since, in a few generations, it can lead to fitness impairment . Inbreeda) 7.60% , house ss) 12.9% , Lizard ) 15.83% , New Zeaa) 8.00%  and Stewe author . There i.26% and fe and fg were very different from the total number of founders indicating an unbalanced contribution of founders to the current population. The value of fg was much lower than that of fe, revealing a potential loss of founding alleles in the current population of captive Great-billed Seed-Finches. A similar situation was found by [Porphyrio hochstetteri) population in New Zealand, where the value of fg was almost half of the fe. Those authors demonstrated how the unbalanced representation of founders, the high level of inbreeding (8.9%), and the small population size of Takahe contributed to a 7.5% loss of the original heterozygosity. Also, the fa/fe ratio in the current captive Great-billed Seed-Finches population has evidenced the presence of genetic bottlenecks due to the unbalanced use of birds as breeding animals. The main consequence of pedigree bottlenecks is the loss of genetic diversity, which was about 4% in the present population. Genetic drift was the most important cause of loss of genetic diversity, accounting for about 64% of the total. According to [The values of found by  for the rding to , the selrding to  explaineFIS assumes a positive value. In the case of captive Great-billed Seed-Finches, this has been verified either by the common practice of mating between relatives, intensive use of some birds as breeding animals, or genetic isolation of some aviaries. The dendrogram based on genetic distances between ten of the most popular commercial aviaries (NeFi) and coancestry (NeCi) rates. The NeFi is more appropriate for cases where the objective is to assess inbreeding depression, which depends on the inbreeding coefficient [Under non-random mating conditions, coancestry is usually lower than inbreeding, and thus, aviaries  supportefficient . Howeverfficient ,59. Despfficient , see alsSporophila angolensis.For the first time, a trait related to singing and territorialism of Great-billed Seed-Finches was defined and evaluated, which was called STT. As stated earlier, Great-billed Seed-Finches are territorial birds , which dThe posterior mean of the heritability estimates for STT was of low magnitude 0.187), but with a relatively wide highest posterior density interval (0.099 to 0.290). Thus, it is possible to state that the heritability for STT is statistically different from zero and that this trait can respond to artificial selection. The estimate found here is within limits observed by  employin7, but wiBased on the evolvability estimate obtained here, it was possible to predict a 2.45% change for STT per generation under unit selection. Thus, in about four generations, it would be possible to change by about 10% of the STT. Therefore, as stated by , a stronThe repeatability of STT was low but statistically different from zero, as verified by the highest density interval obtained (0.277 to 0.371). Thus, it is expected that many birds with a high STT value in one event will not perform as well in another event and vice versa. It is a common sense of breeders that birds that exhibit stable performance between different competitions are relatively rare. The repeatability estimate for STT was within the range reported in , betweenFinally, it is worth making some additional considerations. Great-billed Seed-Finches have been the subject of concern by researchers, entities, national and international organizations. As stated by  and reinThe captive Great-billed Seed-Finches population faces many of the problems commonly encountered in domestic animal populations such as a high level of inbreeding, pedigree bottlenecks, unbalanced contribution of breeding animals and structuring. Despite this, most genetic diversity remains preserved within aviaries. The high generation interval found for this population should help to prevent a rapid increase in inbreeding and genetic drift. These results should serve as strong motivation and support for urgent actions to manage the genetic diversity of captive Great-billed Seed-Finches. Greater genetic exchange among aviaries and mating of unrelated individuals are simple measures that can be recommended. From the viewpoint of genetic improvement for singing and territorialism, defined here as STT, this trait presents sufficient variability to allow response to artificial selection. In contrast, inbreeding and high generation interval appear to be the most considerable barriers that may limit the genetic gain for STT in captive Great-billed Seed-Finches. Widespread adoption of techniques such as optimal genetic contribution selection and implementation of routine genetic diversity monitoring via pedigree analysis and molecular tools can be crucial both in terms of breeding and conservation of genetic diversity of captive Great-billed Seed-Finches.S1 Data(ZIP)Click here for additional data file.S2 Data(ZIP)Click here for additional data file.


The influence of species life history traits and historical demography on contemporary connectivity is still poorly understood. However, these factors partly determine the evolutionary responses of species to anthropogenic landscape alterations. Genetic connectivity and its evolutionary outcomes depend on a variety of spatially dependent evolutionary processes, such as population structure, local adaptation, genetic admixture, and speciation. Over the last years, population genomic studies have been interrogating these processes with increasing resolution, revealing a large diversity of species responses to spatially structured landscapes. In parallel, multispecies meta‐analyses usually based on low‐genome coverage data have provided fundamental insights into the ecological determinants of genetic connectivity, such as the influence of key life history traits on population structure. However, comparative studies still lack a thorough integration of macro‐ and micro‐evolutionary scales to fully realize their potential. Here, I present how a comparative genomics framework may provide a deeper understanding of evolutionary process connectivity. This framework relies on coupling the inference of long‐term demographic and selective history with an assessment of the contemporary consequences of genetic connectivity. Standardizing this approach across several species occupying the same landscape should help understand how spatial environmental heterogeneity has shaped the diversity of historical and contemporary connectivity patterns in different taxa with contrasted life history traits. I will argue that a reasonable amount of genome sequence data can be sufficient to resolve and connect complex macro‐ and micro‐evolutionary histories. Ultimately, implementing this framework in varied taxonomic groups is expected to improve scientific guidelines for conservation and management policies. For instance, Aguillon et al.  used a pNe) and the per‐generation migration rate (m) on gene flow (Nem). Thus, the effect of time on allele frequency changes can be represented on a genetic drift intensity scale, and evolutionary independence can be assessed from estimated migration rates  identifying selected loci in population genomic data, but also for (b) the fundamental interest of understanding the demographic history of populations per se  at linked neutral markers, on a chromosomal scale that depends on recombination , irrespective to the relative amounts of genetic load in the donor and recipient populations , that is, in the absence of associative overdominance. In this case, a positive correlation between introgression and recombination can be obtained if the donor population has a lower longer‐term Ne  than the recipient population  and mechanisms of action  of mutations involved in fitness may be undistinguishable based on the sign of the correlation between recombination and introgressed ancestry alone. However, the recent findings of such correlations in several systems tend to support the polygenic nature of the underlying mechanisms  choosing similar sampling locations for all species to compare the effect of homogeneous geographic distances across taxa, and using the same molecular strategy with the same sample size for all sites and species. The repeatability and traceability of bioinformatics pipelines and statistical approaches are also crucial, given the need to execute in parallel numerous steps to move from raw sequence reads to evolutionary parameter inference in each species. The use of workflow management tools  and containers  is a good way to achieve exchangeability of analysis software within a collaborative project. The phylogenetic scale is also a matter of concerns for standardization, since overly distant taxa may pose technical difficulties due to strong differences in their genomic architectures .Finally, different species with contrasted life history traits are likely to display a wide diversity of genetic connectivity patterns. These need to be classified to communicate the results more efficiently and more clearly to managers. The use of reference study systems has been proposed to make progress toward standardized decision‐making for a closely related issue, the delineation of species using genomic data Galtier, . The ide6How different species with contrasted ecological traits experience connectedness within a similar landscape? To what extent and why do they differ? Linking micro‐ and macro‐evolutionary scales is probably the crux to understanding the diversity of genetic divergence and connectivity patterns and in particular its relationships to species biology and ecology  between two daughter populations in terms of molecular divergence thus depends on the amount of ancestral polymorphism and the rate of lineage sorting during divergence  could be associated to increased chances of colonizing isolated habitat patches, maintaining diversified metapopulations in fragmented landscapes (Cahill et al., 7This review on the connectivity of spatially based evolutionary processes may be found surprising in its lack of consideration of the spatial dimension of the studied processes. This is by no means the sign of a lack of interest in the issue nor an attempt to devaluate spatially explicit approaches to identify environmental features and factors that affect connectedness across landscapes. This frustrating gap mainly reflects a limitation inherent to comparative studies, imposed by the need for a compromise between the extents of spatial and genome sampling resolution. This review deliberately took the path of an approach based on a limited number of genomes sampled in a few populations thought to be representative of the targeted processes. The main motivation for this choice comes from the wish to better connect micro‐ and macro‐evolutionary scales in connectivity research. However, recent developments in the analysis of genome‐wide genealogies combined with increasing sequencing capacities make it possible to foresee in the near future alternative approaches based on a random sampling of individuals through space (Bradburd & Ralph, Connectivity disruptions caused by human activities cover a wide range of effects from habitat fragmentation to increased genetic exchanges between geographically isolated lineages within species. Depending on the context, the eco‐evolutionary aspects of connectedness that are the most relevant to conservation may impose different measures of connectivity. This may benefit from a reference‐based classification process, in an attempt to attend the conditions necessary for continuing evolution (Frankel & Soulé, None declared.


The UK and Ireland have many native pony breeds with historical and cultural importance as well as being a source of uncharacterized genetic diversity. However, there is a lack of comprehensive research investigating their genetic diversity and phylogenetic interrelationships. Many studies contain a limited number of pony breeds or small sample sizes for these breeds. This may result in erroneous grouping of pony breeds that otherwise have intricate interrelationships with each other and are not evaluated correctly when placed as a token subset of a larger dataset. This is the first study that specifically investigates the genetic diversity within and between British and Irish native pony breeds using large sample numbers from locations of their native origin.This study used a panel of microsatellite markers and sequence analysis of the mitochondrial control region to analyze the genetic diversity within and between 11 pony breeds from Britain and Ireland. A large dataset was collected , and previously published data were used to place the British and Irish ponies in a global context.The native ponies of Britain and Ireland were found to have had a complex history, and the interrelationships between the breeds were revealed. Overall, high levels of genetic diversity were maintained in native breeds, although some reduction was evident in small or isolated populations . Unusual mitochondrial diversity distribution patterns were apparent for the Carneddau and Dartmoor, although among breeds and global haplogroups there was a high degree of haplotype sharing evident, well‐represented within British and Irish ponies. Ancestral maternal diversity was maintained by most populations, particularly the Fells and Welsh ponies, which exhibited rare and ancient lineages. The maternal and paternal histories of the breeds are distinct, with male‐biased crossings between native breeds, and other shared influences, likely Arabs and Thoroughbreds, are apparent. The data generated herein provide valuable information to guide and implement the conservation of increasingly rare native genetic resources. This study is the first to specifically investigate the genetic diversity within and between British and Irish native pony breeds using large sample numbers from locations of their native origin and used a panel of microsatellite markers and sequence analysis of the mitochondrial control region to analyse the genetic diversity within and between 11 pony breeds from Britain and Ireland. The native ponies of Britain and Ireland were found to have had a complex history, and the interrelationships between the breeds were revealed. Overall, high levels of genetic diversity were present, including maintenance of rare ancient matrilines in the Welsh and Fell breeds. The study provides valuable information to guide and implement the conservation of increasingly rare native genetic resources. Worldwide, pony populations that have historically undergone population reduction but have been subject to carefully managed conservation programs have shown success in improving their genetic viability. For example, following a 10‐year breeding management and conservation plan, the previously endangered Pottoka breed now displays high genetic diversity .SSR genotyping of samples was performed for 17 SSR loci using commercially available kits and protocols as described previously  from Hardy–Weinberg expectations using the exact test in Arlequin 3.5 ] between successive values of K) was calculated using STRUCTURE Harvester v0.6.7 (http://taylor0.biology.ucla.edu/struct_harvest/) as per Evanno, Regnaut, and Goudet  clustering of samples was conducted via the STRUCTURE v2.2.3 software  (1972) using Populations 1.2.31 . This matrix was visualized by drawing an unrooted neighbor‐joining phylogenetic tree with MEGA 5.1  and visualized by drawing a NeighborNet in SplitsTree 4.13.1  were calculated using Arlequin 3.5  calculating the average number of allelic size differences between populations.Population summary statistics, pairwise Slatkin, . The RSIn order to account for difference in sample size, allelic richness (AR) was calculated for each locus and population based on a minimum sample size of 21 individuals using FSTAT v2.9.3.2 Goudet, .2.4*.phy) format.UK samples were sequenced for a 606bp mtDNA fragment of the mitochondrial control region (MTCR) as described previously . In line with other studies, hypervariable nucleotide positions 15,585, 15,597, and 15,650 were removed (downweighted to 0) and nucleotide positions 15,604, 15,659, and 15,737 were downweighted (from 10 to 5). Nucleotide positions refer to that of the complete horse reference sequenced by Xu and Arnason  and observed (HO) heterozygosity values of 0.721 and 0.659, respectively. The New Forest pony displayed the highest overall diversity, with the highest AR value (6.83) and HO (0.731) and low FIS . The Section C had the lowest AR (5.61) and the highest FIS . The Shetland showed the lowest HO at 0.680. The Section A also had low HO (0.687) and relatively high FIS (0.033), but had the second highest AR and MNA .Summary statistics for microsatellite (SSR) data analysis are presented in Table 3.1.2p < .001), with 6.94% of the total variation existing between populations. Variation within individuals was 91.51%.Molecular analysis of variation demonstrated highly significant genetic structure within the dataset   Nei,  between s) Nei, . As alsos) Nei,  and 4. TNm) was highest between the Section C and Section D, while the Highland shared the highest Nm with the Fell , with 14% of the total variation found between populations. Table p < .001) from the other Welsh populations. The Section B group was not found to be significantly differentiated from the Irish Connemara pony, with a very low value for the average pairwise differences and correspondingly high effective number of migrants (Nm) between the two populations. The Section B and the Dartmoor ponies also appear to share maternal lineages and were not significantly divergent from each other. The Carneddau were significantly differentiated from every other population analyzed (p < .001). The Shetland pony was also highly divergent, though demonstrated closer maternal links with the Fell breed than with any other population. The Fell pony breed shared maternal lineages with the Welsh Sections A, C, and D.Significant genetic structure was apparent , but after the J‐K haplogroup split. It is thus designated here as belonging to haplogroup J. This cluster was comprised solely of Welsh ponies and is not represented in any other British or Irish pony breeds. The G haplogroup contains a large number of Shetland individuals, as well as Welsh ponies  and a single New Forest, but no other breeds. Haplogroup N consists of predominantly Highland and New Forest. Haplogroup B is limited to (predominantly) Connemara, as well as two Fells and a New Forest.Certain breeds are particularly noteworthy due to their diversity patterns across the haplogroups Figure . HoweverThe New Forest is the most diverse group; it has animals represented within all haplogroups except the ancestral J node that is restricted to the Welsh populations. Many breeds frequently shared haplotypes with other breeds within each haplogroup. However, the New Forest had one or more breed‐specific haplotypes within most haplogroups, including clusters of multiple animals. However, there was a distinct lack of intermediate nodes connecting these unique clusters; that is, the New Forest ponies were poorly represented within, or missing from, the nearest “shared” haplotype node. In contrast, the Section A showed few breed‐specific haplotypes yet also had representative animals within most haplogroups . However, most examples of shared haplotypes for this breed occurred in the more ancestral nodes for each haplogroup.4The results revealed lower autosomal genetic differentiation according to breed (8%) compared to previously reported data of other horse breeds worldwide, in which 10%–17% of the genetic diversity could be partitioned this way  is placed between these nodes within the network. Therefore, Welsh ponies appear to be established from an ancient ancestral population, with some rare matrilines maintained within the population to this day. The presence of this node suggests the divergence of the ancestral stock during the periglacial period and that today's British ponies may represent a relic of the expansion of animals following the retreat of the last glacial period rather than more recent introductions in the historical period. Jansen et al.  suggesteThe Shetland pony showed evidence of more restricted diversity for mitochondrial analyses than other breeds. It is highly diverged from other pony breeds of Britain and Ireland for both autosomal and mitochondrial analyses and has been found to cluster with Nordic breeds, indicating a predominantly Scandinavian origin or influence . This phenomenon has been seen in other species such as goats, chickens, and wild sheep . The admixture shown for the SSR STRUCTURE plots, the lack of breed‐specific clustering in the individual N‐J tree, and the low divergence also confirms the mixed heritage of the New Forest ponies for the autosomal data. Prystupa, Juras, et al.  , et al.  and Van , et al.  found si, et al.  similarlEdwards, .Fell ponies displayed very high maternal diversity, with a broad distribution of haplogroups, and moderately high nuclear diversity. Fell ponies, like many other native breeds, suffered population declines following the increased use of motor vehicles and mechanized farming in the early 1900s. Despite this, traditional shepherding of the fell uplands continued to some extent, as did limited keeping of free‐roaming herds on the mountains . The Fell breed suffers from foal immunodeficiency syndrome (FIS), a fatal autosomal recessive disease. The breed society currently recommends the practice of breeding FIS clear ponies with carriers and testing the offspring for FIS status in order to avoid genetic degradation . It is important that this practice is adhered to (rather than avoiding breeding carriers entirely) in order to maintain the extensive maternal diversity within the Fell breed.The Fell ponies showed the closest autosomal relationship with the Welsh Ponies and Cobs (particularly Section C), the Connemara, and the Highland. The Fell and the Welsh Sections share a strong phenotypic trait of a notably fast trotting ability. For both populations, trotting races were a particularly popular activity in the 1800s and stallions were recorded to have been shared between the breeds Lewis, . The FelK = 9, indicating strong sharing of alleles between these breeds. Interestingly, while Petersen et al. , cosupervised the student, and proof‐read and refined the manuscript. Mina Davies Morel provided historical context and interpretation of the data. Charly Morgan supported all laboratory analyses. Deborah Nash devised the study, wrote grant‐funding applications, supervised the student, and assisted with writing the manuscript.