ST), which measures population differentiation, was low for all the studied markers and its mean value was 0.0305. Our analysis also revealed that there has been no inbreeding in the Polish Konik population for the studied group. This monitoring of genetic diversity could be helpful for the Polish Konik breeding and conservation programs and also for making informed decisions in the management of paternal lineages.We investigated the genetic diversity of Polish Konik in sire lines. The Polish Konik horse is a Polish native horse breed of a primitive type and is one of the breeds managed via a conservation program. Due to the small number of breed founders  and paternal lineages with unknown representation, the genetic diversity and structure of the population should be monitored regularly. This is the first characterization of the genetic diversity of Polish Konik paternal lines based on microsatellite markers (STRs). The fixation index  was low (about 3%), the mean inbreeding coefficient (FIT) was low and close to 0, and the mean inbreeding index value (FIS) was negative. The mean expected heterozygosity was established at 0.7046 and was lower than the observed heterozygosity. The power of discrimination and power of exclusion were 99.9999%. The cumulative parentage exclusion probability equaled 99.9269% when one parental genotype was known and 99.9996% with both parents’ genotypic information was available. About 3% of the genetic variation was caused by differences in the breed origin and about 97% was attributed to differences among individuals. Our analysis revealed that there has been no inbreeding in the Polish Konik breed for the studied population. The genetic diversity was high, and its parameters were similar to those calculated for native breeds from other countries reported in the literature. However, due to the small number of breed founders and paternal lineages with unknown representation, the population’s genetic diversity and structure should be monitored regularly. The Polish Konik horse is a primitive native breed included in the genetic resource conservation program in Poland. After World War II, intensive breeding work began, aimed at rebuilding this breed. Now, the whole Polish Konik population is represented by six male founder lines . Individuals representing all six paternal lineages were selected based on their breeding documentation. We performed a fragment analysis with 17 microsatellite markers (STRs) recommended by the International Society for Animal Genetics (ISAG). The genetic diversity and structure within the paternal lineages and the whole of the studied group were investigated. The average allelic richness was 6.497 for the whole studied group. The fixation index (F Equus caballus) is a Polish native horse breed of a primitive type and is one of the breeds managed via a conservation program  a a17] andMicrosatellite markers are commonly used in structure and genetic diversity analyses (within-population and between populations) ,22,23,24We examined the genetic variability and structure of the Polish Konik breed among all six paternal lines. We used blood and hair follicles samples for routine horse parentage testing (via collection of DNA samples from different regions of Poland) and investigated the pedigree of the selected samples. The number of selected samples for each paternal line does not correspond with the paternal line’s size. We could not find any comprehensive reports about general population statistics. We eliminated all samples presenting a doubtful or excluded pedigree. Finally, we tested 196 Polish Konik samples representing the six paternal lineages. The distribution of the individuals among the paternal lineages was as follows: Wicek—48; Myszak—12; Glejt I—35; Goraj—32; Chochlik—36; Liliput—33. These numbers of the individuals as a percentage share of 196 samples are comparable to the percentage share of male lines reported in 2019 by Jaworski et al. .The DNA samples were isolated from 300 μL of peripheral blood using a Wizard Genomic DNA Purification Kit  or Sherlock AX  and from hair follicles using Sherlock AX (A&A Biotechnology). DNA was amplified in a multiplex reaction (17 loci) using an Equine Genotypes Panel 1.1 Kit  with positive and negative DNA controls. The amplification of STRs was performed according to the manufacturer’s protocols using 17 primer pairs labelled with four fluorescent dyes. We analyzed the PCR products in a capillary electrophoresis system using a 3130xl Genetic Analyzer . We conducted products separation using a GeneScan-500 LIZ Size Standard (Applied Biosystems), enabling automated DNA fragment analysis. Then, we processed the received data in GeneMapper 4.0 software (Applied Biosystems). This method was regularly checked and validated through ISAG horse parentage comparison tests and standardized following ISAG recommendations. The DNA profiles were specified using 12 markers from the ISAG core STR panel  and 5 markers from the ISAG additional STR panel .We analyzed the genetic diversity of the six paternal populations via GenAlEx6  and FSTAWe used Structure 2.3.4 software to analyze the genetic structure of the populations by Bayesian clustering methods . Markov o and He) [IS) [1) and both parental genotypes (PE2) and the probability of identity (PID).Due to a lack of differentiation among the paternal lineages and the small amount of six paternal populations, the following analyses were conducted for all samples as a whole population. We conducted population genetic analyses using the pegas package in R  and our  and He) ,32, meanHe) [IS) , and polHe) [IS)  were estHe) [IS) : we calca for all 196 samples was 6.157. We also evaluated Nei’s genetic distances and the highest differentiation was identified between the Liliput and Myszak (DA = 0.120)The results of the analyses of 17 microsatellite loci in six paternal populations, shown in The results for the structure analyses (Structure 2.3.4 software), run to investigate the population structure and eventual admixture patterns among the six paternal populations, are shown in Taking into consideration the analyzed data, ∆K = 2 and ∆K = 3 presented in ST), which measures population differentiation, was low for all studied markers and its mean value was 0.0305. The mean FIT was low and close to 0 (0.0189). The FIS was negative , which suggested that the selected samples in these analyses probably belong to unrelated individuals among the paternal lineages. The mean values were calculated without the X-chromosome linked LEX3 locus.The F-Statistics results of the whole studied population are presented in The population coefficients were calculated and are presented in o and He coefficients (above 0.6) are very high values except HTG4, HTG6, HTG7, and LEX3. The cumulative power of discrimination and the probability of identity were high. Based on the probability of exclusion , we calculated the cumulative parentage exclusion probability knowing one (CPE1) or both (CPE2) parents. CPE1 amounted to 99.9269%, and CPE2 was 99.9996%.The mean expected heterozygosity was 0.7046 and was lower than the observed heterozygosity (0.6519). The power of discrimination and power of exclusion were both close to 1 (99.9999% and 99.9967%). The values of PIC, HFeatures of Polish Konik that are recognized in their conservation program—such as hardiness, natural adaptive instincts, and potential for controlling dense forest invasion of natural wetland areas—have been noticed by environmentalists and scientists in Europe. Polish Konik horses were exported to the Netherlands and then selected to maintain their most favorable phenotypic characteristics. Now, they are called the Dutch Konik horse and—according to the studbook—are descendants of the Polish Konik horse breed . AnotherST level for all samples (the whole studied group) points to the small diversity of the analyzed loci, but the low value of this coefficient is probably due to the nonrandom mode of sample selection. The FST, which measures population differentiation, was low for all studied markers and its mean value was 0.0305, and is caused by breed differences. The FIT values are close to zero, except for LEX3, and indicate that inbreeding depression is not a thread in the present study. The FIS is negative for most loci, and this confirms the low level of inbreeding among the animals selected from the six paternal lineages and random mating within Polish Konik horses. The FIS value is similar to those reported for Thoroughbred [It has been proven that genetic diversity analysis based on Y chromosome variability can be an informative source of data to explain differentiation and the origin of the male founders of many horse breeds ,37. In toughbred  or Czechoughbred  and coulThe Polish Konik population was rebuilt from the small number of surviving individuals after World War II. Our results show a low level of inbreeding, probably due to the well-provided breed management and high genetic diversity of the breed founders. However, a planned investigation of the maternal lines’ genetic diversity should give a broader context for these results. a obtained by those authors was 7.1333 for 53 individuals, in comparison to our Na of 6.157 for 196 samples. Probably, the reason for their higher Na could be related with the genetic variations of other maternal lines or non-Polish origin of samples. The PIC, Ho and He coefficients were also high (above 0.5) for all markers except HTG4 and HTG6 in Gralak et al. [o and He were similar to those reported by Castaneda et al. [Nevertheless, compared to other breeds, the allelic polymorphism was high in the core panel and slightly lower in the additional panel. Allelic polymorphism was high in the core panel (except in HTG4) and rather low in the additional panel (except in CA425 and HMS1). We detected the lowest number of alleles in HTG6, where only three allelic variants were present. The calculated heterozygosity was similar to the value obtained in this locus for the Polish Konik horse breed by Gralak et al.  for 12 mk et al. . The ovea et al.  for Polia et al.  and Renda et al. . In turna et al. . High vaThis study aimed to assess the genetic diversity and population structure of Polish Konik individuals selected by their paternal lines. Our study was conducted on the current STR set (17 loci) recommended by the ISAG and it is the first such research on Polish Konik as previous studies only included 12 STR markers. We performed the analysis with representatives of all paternal lineages, which probably allowed us to include all variants coming from sire lines. Despite the small number of paternal lineages in our research, the genetic diversity parameters were similar to those obtained for other native breeds in other countries reported in the literature. Surprisingly, the inbreeding index indicates that there is no inbreeding in the analyzed Polish Konik population, despite the fact that the population experienced a bottleneck event of genetic diversity several decades ago. Despite the lower parameters for the two loci, overall, the genetic variation of the STR markers was high and similar to that obtained in other research on this breed. However, the breed’s genetic structure should be monitored because the population was derived from a small number of individuals and particular paternal lineages now have an uneven representation. This monitoring could be helpful for the Polish Konik breeding and conservation program and also for making informed decisions in paternal lineages management.


The conservation and maintenance of genetic diversity is one of the priorities of the Convention of Biological Diversity and is included in the United Nations (UN’s) Sustainable Development Goals. The evaluation of the genetic variability of a breed is fundamental for its future use in a sustainable way, being indispensable to outline a successful conservation or improvement strategy. Preserving genetic diversity in a population is one of the main objectives for a breed conservation program. Nevertheless, the correct management of genetic diversity is also essential for the adaptation of a population to a new environment, production system or genetic improvement. For the purpose of population monitoring, assessing changes in genetic variability and genetic erosion in animal populations, many methodologies based on pedigree analyses of inbreeding and relationships, and on the probability of genetic origin from different herds, founders and ancestors, have been used. This study presents several genetic diversity indicators in a Portuguese native cattle breed, Mertolenga, assessed by pedigree analysis, and demonstrates the usefulness of these indicators and how they can be used in the genetic management of a breed.The Mertolenga beef cattle, currently with 27,000 breeding females in Portugal, is the largest Portuguese native breed, despite some variation in the breeding stock over the last years. The purpose of this study was to estimate parameters related to the population structure and genetic diversity and to investigate the major factors affecting genetic erosion in the breed, based on the pedigree herdbook information collected since the 1950s, including records on 221,567 animals from 425 herds. The mean generation intervals were 6.4 years for sires and 7.1 years for dams, respectively. The rate of inbreeding per year was 0.183% ± 0.020% and the correspondent effective population size was 38.83. In the reference population , the average inbreeding and relatedness were 8.82% ± 10% and 2.05% ± 1.26%, respectively. The mean relationship among animals from the same and from different herds was 29.25% ± 9.36% and 1.87% ± 1.53%, respectively. The estimates for the effective number of founders, ancestors, founding herds and herds supplying sires were 87.9, 59.4, 21.4 and 73.5, respectively. Although the situation of the Mertolenga breed is not alarming, these results indicate the need to adopt measures to maintain the genetic variability of the population. The Mertolenga beef cattle breed, currently with 27,000 breeding females, is the largest of the fifteen Portuguese native breeds, although only 8000 of them are purebred. The rest are crossbred with international exotic breeds. Recently, this breed experienced a period of strong census decline caused by drastic changes in the livestock sector and unplanned crossbreeding with exotic breeds. With three quite different coat types  digital herdbook database.The Mertolenga breed is a medium sized animal with a red or reddish coat with white patches and white hook-shaped horns. It is known for its remarkable maternal features and adaptability to poor feeding conditions, being traditionally raised in extensive environments, on farms, normally with reduced densities that have, on average, 70 breeding females, but that can range from 5 to 600 cows per herd.In the 1970s, this population was under serious threat of extinction due to land restructuring of the Portuguese livestock and uncontrolled crossbreeding with exotic breeds. However, in 1978, the Mertolenga’s Herdbook (MHB) was created and the population grew/increased during the following decades. Currently, there are around 27,000 breeding females, of which 14,500 cows are registered in the herdbook under the Breeders Association administration, and only 8000 are used for pure breed reproduction.Depending on the Portuguese region, the environmental conditions, the soils and the beginning (and consequently the duration) of the grazing seasons, births are recorded throughout the year but with a peak in the winter–spring period. Some farms use defined mating seasons (3 to 6 months), while others keep the bull in the herd all year long. Reproduction takes place essentially by natural mating, with very limited use of artificial insemination.Breeders usually produce their own replacement females, while bulls are either from their own herd or acquired from other herds. From 2003 onwards, a genetic evaluation by Best Linear Unbiased Prediction (BLUP)–Animal Model methodology was implemented and applied to reproductive and maternal traits . Since 2All the information used in this study was obtained from MHB records and is available through the online database Genpro . These rThe dataset information was used to assess the number of registered animals  and herd size over the years, age distribution of sires and dams, number of offspring per progenitor and several useful demographic and genetic parameters to study the evolution of the genetic variability of the Mertolenga population.Demographic analyses were performed with software specifically developed for this purpose  and werein) was used to assess the degree of pedigree completeness, and calculated as:sn and dn are the number of generations known for the sire and dam respectively, when s and d are known; if s or d are unknown, then sn or dn respectively, assume the value of 1. Base animals were assigned a number of generations known equal to 0.The equivalent number of complete generations known per animal (xF) and additive genetic relationships among pairs of animals (ija) were computed based on the numerator relationship matrix among all animals [xF and ija were used to evaluate the average kinship relationship between animals born on the same, or different farms, and the average animal inbreeding.Individual coefficients of inbreeding .The regression coefficient of individual inbreeding on year of birth was obtained with the General Linear Model (GLM) procedure of SAS® 9.4 , and thiL), which was used to compute the rate of inbreeding per generation (ΔF/g) as ΔF/g = L (ΔF/year). In addition, the effective population size (eN) was then calculated as described by Falconer and MacKay [Generation intervals were calculated for sires and dams of all born calves, and for the 4 paths of selection . The generation intervals for the 4 paths of selection were averaged to obtain a pooled generation interval (d MacKay :(2)Ne=12ef), ancestors (af) and herds (hf) were computed from those genetic contributions as:j stands for founders, ancestors or herds, depending on the situation considered, and kq represents the genetic contribution of a given founder or founding herd, or the marginal contribution of an ancestor [The genetic contributions to the reference population of founder animals, ancestors and founding herds were computed, as described by James , Lacy 1 and Boicancestor .gf), defined as the number of founders that would be expected to produce the same genetic diversity as in the population under study if the founders were equally represented and no loss of alleles occurred [The founder genome equivalents , which corresponds to the mean relationship of each individual with all animals in the pedigree, was computed for each individual, with ENDOG v4.8 [The average relatedness .Annual evolution of the number of registered cows , bulls, calves and herds in the MHB, for the period from 1990 to 2019, is presented in Overall, the total number of breeding females increased until 2014, but has decreased since then, and the number of purebred cows has been gradually decreasing since 2017, on average −280 cows each year.The number of active breeding bulls (with offspring) reached a maximum in 2007  and subsequently decreased to values that have ranged between 189 and 240 bulls depending on the year , followiThe number of pure calves registered annually in the birth section of the MHB reached its highest value in 2007 . Since then, the trend has been to decrease, although with some oscillations, registering since 2010 values between 6300 and 7500 calves per year, which corresponds to an average of 6900 births of purebred Mertolenga animals per year.The maximum number of breeders with Mertolenga females was observed in 2009 (277), but the number of breeders where purebred animals are born has been decreasing since 2007, when the maximum was reached (205 breeders). In 2019, Mertolenga calves were born in only 138 breeders  registered in herdbook.In the last 5 years (2015–2019), the average herd size was 103.5 ± 127.44 registered cows with 27.2 ± 53.5 pure calves per herd/year, with more than 50% of the herds registering less than 10 purebred calves each year and about 40% not registering any Mertolenga calves.The evolution of the number of breeding females registered in the MHB according to each coat variety was slightly different . AlthougThe age distribution of sires and dams of 221,567 calves born between 1978 and 2019 is shown in Most breeding males (53%) are between 4 and 8 years old when their offspring is born . A smallL) , and somewhat later in bulls. These results indicate that, when compared with cows, the use of bulls started at a later age (37.3 ± 9.95 months), but that they were also replaced at a faster rate. Nevertheless, nearly 10% of the offspring was sired by bulls at, or above, 10 years of age, whereas the percentage of calves produced by cows of the same age range was about 24%. In recent years, the percentage of females that calved at 10 or more years of age was about 36% (results not shown). The results obtained for the generation intervals (L)  for calvThe 204,671 calves with a known sire and born between 1978 and 2019 were the offspring of 1629 sires, with an average of 133.7 ± 141.4 calves/sire.The distribution of the number of breeding males according to the number of offspring presented in The degree of pedigree completeness available for the first 3 generations is illustrated in The degree of pedigree completeness was also estimated by the number of generation equivalents, which corresponds to the average number of complete generations known in the pedigree of an individual. The number of generation equivalents increased from nearly 2 for calves born in the 1990s to close to 6 generations for calves born in 2019 a.AR) increased from 1990 to 2009, reaching 2.12% in 2009, and since then has remained between 2.0% and 2.2%.The evolution of inbreeding a indicatAlthough the proportion of animals born from the 3 coat types  is quite distinct over the years, until 2015, the evolution of inbreeding was very similar. However, this evolution was very different in the red coat from 2016, reaching an average inbreeding of more than 13% in 2019, while red spotted and roan animals, on average, are born with less than 9% inbreeding b. In theThe evolution of kinship between animals born in the same herd and between animals born in different herds, regarding inbreeding over 10 years, is illustrated in The average relationship in the reference population and for animals from the same herd was 29.25% ± 9.36% and for animals born in different herds was 1.87% ± 1.53% . The avep < 0.01). From the annual rate of inbreeding and the mean generation interval (7.05 years), the estimated rate of inbreeding per generation was 1.29%, and the corresponding effective population size was 38.83 , the effective number of founders, ancestors and founding herds were, respectively, 87.9, 59.4 and 21.4 .The cumulative genetic contributions of the most important founders, ancestors and herds  show an Overall, 50% of the genetic pool of the reference population is accounted by the contribution of 56 founders, 41 ancestors and 9 herds , with thgf) to the reference population was 48.7. This estimate indicates that, from the 11,471 founders of the population, if they all were equally represented and no loss of alleles occurred, only 48.7 would produce the genetic diversity currently existent.The estimate of founder genome equivalents —Abóbada Farm, in 1978, recognition of a protected designation of origin (PDO) approved by the European Union in 1994, the inclusion of Mertolenga in the group of breeds at risk and eligible for agri-environment programs that provide financial support in the framework of the European program for conservation of genetic resources, and several breeders’ support activities promoted by Mertolenga Breeders Association (ACBM). All these strategies and activities combined contributed to the resurgence of the Mertolenga breed from a limited base. It currently represents the biggest native beef cattle breed in Portugal, both as a pure breed and mainly as the maternal line in organized crossbreeding programs.Due to the maternal characteristics of the breed and their use as a maternal line, more recently, ACBM started a program to promote F1 females  as a maternal line “Mertolenga F1 Program”, in which the main exotic breeds used in Portugal are collaborating .Currently, there are approximately 27,000 breeding females . Even soIn the beginning of the 21st century, the Mertolenga cattle breed was classified as “non-threatened” according to the criteria used by the European Union (EC Regulation No. 45/2002) to define the animal genetic resources risk status. Recently, in 2015, according to the document of delegated acts for the new Rural Development Regulation adopted by the European Commission, Mertolenga is considered internally to be at risk of extinction, with grade C (lower risk).The different percentages of animals in terms of coat type  have more than 500 sons each and are responsible for more than 11% of the births , which aTo study the genetic variability of a population and the main factors of genetic erosion, it is essential to carry out a broad study over time, taking into account several generations, both in terms of individuals and farms. It is essential to know several indicators and to evaluate them dynamically over the years, such as the size of farms, the age structure of the herd, bottlenecks in terms of excessive use of some breeders and their genetic contribution, the exchange of genetic material between herds, among others.Controlling the rate of inbreeding is usually one of the major targets in conservation and selection programs ,29,30,31In in) increased, on average, by 0.142 ± 0.002 years (results not shown) since 1990 and is presently nearly 6 generations for animals born in 2019. This value is higher than the one for some breeds whose herdbook started at the same time, and close to the values obtained in the Charolais and Limousine breed reported by Bouquet et al. [in above 15 [The complete known generations  estimated for the entire Mertolenga population enrolled in the MHB  is lower than that obtained for the Alentejana breed for animals born between 1968 and 2003 (0.33%/year) [F = 0.183%/year) is inferior to those obtained for the Portuguese breeds Arouquesa [in < 2), naturally, these will have zero or very low levels of inbreeding, which together with individuals born more recently and with more pedigree information, may overestimate the previous estimate of annual rate of inbreeding.The annual rate of inbreeding (Δ3%/year) , and low3%/year) ,49 Spanirouquesa  and Pretrouquesa  and in trouquesa . The anneN = 38.83) is lower than the one recommended (eN = 50) as the minimum number to maintain genetic diversity, both in conservation [The estimate of the effective population size obtained in the Mertolenga breed Ne = .83 is loded Ne =  as the mervation ,61,62,63ervation , and is eN between a local livestock breed, in many cases < 50, and an international breed of worldwide dimension , in some cases with eN > 500, is very uneven. These are distinct realities and also very different possibilities of managing the existing genetic variability. Inbreeding rate per generation should be below 1.0% [The size of the low 1.0% ,63,64,65ef) was 87.9 between 2015 and 2019, and has been decreasing since 2000–2004, when it was 104.82. The slight increase of the ef between 2015 and 2019 compared to the previous period is due to the fact that in the last 5 years, there have been some registered animals in the MHB (side section) without confirmed paternity, as explained above. The effective number of ancestors (af) is currently 59.4 and also shows a decreasing trend during the periods studied. However, these values are not as low as the ones observed in other breeds, including some Portuguese ones, summarized in Of the 11,471 founders who constituted Mertolenga cattle breed, large differences were observed in their genetic contributions to the current population, with five founders contributing to about 19% of the actual genetic pool. The effective number of founders , for the values of the individual inbreeding to remain acceptable without major damage.The pedigree information available in the herdbook is quite complete, but the control of affiliation by DNA analysis should be maintained. It seems that current mating practices to avoid inbreeding work well when used. For long-term maintenance of genetic diversity and dynamics of the breed, the minimization of genetic relationships between candidate breeding animals is the most promising approach, moreover, with the aid of knowledge from molecular genetics.Measures should be taken to avoid further losses in genetic variability in the Mertolenga breed, including the selection of breeding animals with a more extensive representation of ancestors and with a lower relationship with the population, and the rotation of animals among herds, particularly among herds of the same coat type. Restrictions may be applied on BLUP-selection, based on the expected impact of inbreeding on the herd where the animal will be used. Breed management should include in situ or ex situ conservation actions, with conservation nuclei of the different coat types.


Hedgehogs are regularly brought to wildlife shelters. Depending on the area from where animals are accepted, translocation can occur between different regions or populations. In this study, the genetic diversity of wild hedgehog populations was compared with “shelter populations” within central Europe focusing on the western contact zone between both European hedgehog species. Some shelters were hosting both species at the same time, in one this could be shown genetically. Generally, no difference in genetic diversity between shelter individuals and wild populations was found. Two shelters from Innsbruck hosted individuals that probably belong to two subpopulations. This indicates that shelter management-related translocations could facilitate gene flow across a dispersal barrier.Erinaceus europaeus and E. roumanicus. Shelters were mainly hosting one species; in one case, both species were present syntopically. Apart from one exception, the results did not show a higher genetic diversity within shelter populations, indicating that individuals did not originate from a wider geographical area than individuals grouped into one of the wild populations. Two shelters from Innsbruck hosted individuals that belonged to two potential clusters, as indicated in a distance analysis. When such a structure stems from the effects of landscape elements like large rivers, the shelter management-related translocations might lead to homogenization across the dispersal barrier.Hedgehogs are among the most abundant species to be found within wildlife shelters and after successful rehabilitation they are frequently translocated. The effects and potential impact of these translocations on gene flow within wild populations are largely unknown. In this study, different wild hedgehog populations were compared with artificially created “shelter populations”, with regard to their genetic diversity, in order to establish basic data for future inferences on the genetic impact of hedgehog translocations. Observed populations are located within central Europe, including the species  Erinaceus europaeus  and E. roumanicus  are assessed as ‘least concern’ by the IUCN  Red List of Threatened Species [E. europaeus [The two European hedgehog species s Linne, 758 and E Species . Neverthuropaeus ,4,5,6. Iuropaeus . Becauses Linne, 758 and Europaeus ,11, the uropaeus ,13,14. Turopaeus ,16. TherE. europaeus or E. roumanicus) [As likeable and well-known garden species, hedgehogs are often used as flagship or umbrella species in urban nature conservation . Due to this medial presence, and their popularity and presence in gardens, they are often the center of attention when it comes to human-mediated rescue actions. Hedgehogs are frequently translocated and among one of the most abundant species found in wildlife shelters in western Europe . Wildlifmanicus) . Despitemanicus) ,20, the manicus) .E. europaeus and E. roumanicus are not under legal protection by the FFH (Flora-Fauna-Habitat) directive [irective  and are irective . Nature irective .Potential outcomes of uncontrolled translocations may be outbreeding depression, accompanied by the loss of local adaptions, or a reduction in genetic variation and changes in the population genetic composition ,20,24,25E. europaeus and E. roumanicus, which reaches from Poland and the Czech Republic down through Austria to the border between Italy and Slovenia [Because human-mediated translocations can also lead to increased incidents of hybridization between species ,24, a spSlovenia . It is aSlovenia . The degSlovenia ,9,26,27.Slovenia . OngoingE. europaeus and E. roumanicus are known to have overcome the most recent period of repeated glaciations during the Pleistocene era in three different southern refugia. While E. roumanicus survived these glaciations in the Balkan peninsula, E. europaeus was restricted to the regions of the Iberian and Apennine peninsula [E. europaeus and E. roumanicus, and a contact zone separating this divergence between eastern and western Europe [E. europaeus individuals originating from either the Iberian or Apennine peninsula was shown [E. roumanicus, it is suggested that interglacial continental refugia might have led to further genetic differentiation among populations [E. europaeus, E. roumanicus, and E. concolor, as well as intraspecific genetic divergence within E. europaeus, nuclear markers were solely able to resolve the major interspecific splits among the three European Erinaceus species [eninsula ,28,29,30eninsula ,32, are eninsula ,30,33,34n Europe ,30,33,34as shown ,30,33,34ulations . However species ,34. HoweE. europaeus and E. roumanicus. Additionally, results will be discussed in a phylogeographic context, as the sampling area is assumed to host both refugial lineages of E. europaeus.Only few studies have focused on the effect of animal translocations on gene flow as a whole . We aimeE. europaeus (n = 144) and E. roumanicus (n = 77) by morphological characters. Some samples were already investigated in previous phylogeographical/population genetical assessments [n = 130) and from wild hedgehogs (n = 91). Individuals from one shelter were treated as one population. Corresponding population assignment, as well as the geographical background can be found in A total of 221 samples were used in the present study. Samples were assigned to essments . SamplesIndividuals were either sampled through buccal swabs  or muscle tissue  . All samg. Buccal swab samples were eluted in 20 and 25 µL of elution buffer [DNA isolation followed the methods and procedures described in . For DNAn buffer .E. europaeus and E. roumanicus was conducted through multiplex PCR. In total, 55 different primers were available for this approach, 25 and 30 primer pairs for the species E. europaeus and E. roumanicus, respectively. A list of all primers that have been used for multiplex PCR can be found in the A short sequence repeats genotyping by sequence approach (SSR-GBS), developed by , was usePurification of PCR products in preparation of index PCR was conducted using an inverse magnetic separator. A total 6-μL sample volume was mixed with 4.3 μL of magnetic beads and incubated at room temperature for 5 min. The DNA, bound to magnetic beads, was washed twice with 200 μL of 80% ethanol for 45 s. After discarding the ethanol, the magnetic particles were air-dried for 5 min and DNA was eluted with 17 μL of preheated (65 °C) elution buffer .Index PCR was used to allow for the individual identification of the pooled samples with specific forward and revers primers through specific assignment of indices. On the one hand, the used primers help binding to the before amplified P5/P7 part of the primers used in multiplex PCR. On the other hand, they allow for binding to the flow cell in Illumina Sequencing and label the sample with a unique eight-bp index information, which helps to assign sequenced genotypes to single samples . For index PCR, 1 μL of clean PCR product was mixed with 5 μL of Multimix (QIAGEN), 2 μL of specific P5 forward primer, and 2 μL of specific P7 reverse primer. The following temperature profile was used for the performance of index PCR: 95 °C for 15 min; 10 cycles of 95 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min; with a final extension at 72 °C for 5 min [Following index PCR, samples were pooled and sent to the Biozentrum LMU in Munich, Germany, for sequencing in an Illumina MiSeq machine in both directions/through paired-end sequencing in a sequencing by synthesis approach.The short sequence repeats genotyping by amplicon sequence (SSR-GBAS) method used in this paper follows the pipeline described in ,36, avaip-value below 0.01 for the highest observed expected alignment scores” [Sequences resulting from the Illumina run were supplied in two FASTQ files (read 1 and read 2), containing all sequences per index. The following processing and treatment of the samples was based on a combination of custom-made scripts, as well as third party programs . This in scores” ,38. Mark scores” ,37, as m scores” .Allele definition was largely based on two major steps: (1) Sequence length, as well as (2) the possible occurrence of SNPs in each separate length class. Within each sample, each marker was examined for its most frequent sequence length class. This was done through a custom-made script  and After the initial sequence analysis and exclusion of markers with too much missing data, 41 microsatellite markers were valid to be used see  for popuE. europaeus and E. roumanicus; as well as (3) a separate analysis of shelter and wild populations (on an inter- as well as intraspecific level).Specific analyses were conducted in the following different approaches: (1) Analysis of all 221 hedgehog samples; (2) intraspecific analysis for the two respective species The program STRUCTURE v.2.3.4  was usedTo choose the most likely value for all observed Ks and thereby determine the most likely number of populations (genetic groups) to be found within the underlying set of samples, the “STRUCTURE HARVESTER” ,41 was uThe Excel Add-In GenAlEx  ,45 was uE. roumanicus and E. europaeus was obvious, and intermediate specimens between the species recognized previously were not included in this study. No early generation hybrid was detected in this dataset and no individuals were positioned between the main clusters characterizing the two species , and one for the northern German populations of Berlin and Hamburg) are opposed to one cluster for all E. roumanicus populations , Innsbruck , and Bavaria  form their own clusters. The shelter from Mossautal was assigned to the same cluster as the northern German populations. In E. roumanicus, the analysis of all samples results in one additional cluster for the shelters in Carinthia and the shelter in Graz (shelter “Marilene”), which appears very admixed. In the shelter of Graz, we have some individuals that cluster together with E. europaeus  shows shelters potential to interfere with the two species distribution range. In the thesis of [The syntopic . While isyntopic ,13,14 assyntopic , translosyntopic . The dishesis of , 8 out ohesis of ); howevehesis of ), wherefhesis of . Eventuahesis of .Studies mostly discuss the effect of translocation as the effect on the survival of individuals with lower fitness, which could interfere with natural selection . PotentiE. europaeus and E. roumanicus, hybridization is recognized, but the degree seems to be very low in terms of early generation hybrids [Human-caused augmentation of hybridization pressure due to the translocation of individuals beyond the species range had been considered . For the hybrids ,36,50. LOnly few studies or data can be found about how translocating animals from wildlife shelters affects gene flow . In our E. europaeus and E. roumanicus are sheltered together and are not always differentiated. Our data show that such cases occur, even if in most shelters a genetic effect is not seen. Shelter individuals should therefore be released under the consideration of natural barriers and treated as a species that can express small-scale genetic structure. Future research will investigate this further. Because the main consideration of shelters is animal welfare, also individuals from distant locations can be taken in, but for the individuals tested, this did not apply. Hosting hedgehog individuals in shelters can promote translocation of individuals because it can bring together animals that are isolated by dispersal barriers. In the zone of sympatry, the two different hedgehog species


The narrow genetic base of elite germplasm compromises long-term genetic gain and increases the vulnerability to biotic and abiotic stresses in unpredictable environmental conditions. Therefore, an efficient strategy is required to broaden the genetic base of commercial breeding programs while not compromising short-term variety release. Optimal cross selection aims at identifying the optimal set of crosses that balances the expected genetic value and diversity. We propose to consider genomic selection and optimal cross selection to recurrently improve genetic resources (i.e. pre-breeding), to bridge the improved genetic resources with elites (i.e. bridging), and to manage introductions into the elite breeding population. Optimal cross selection is particularly adapted to jointly identify bridging, introduction and elite crosses to ensure an overall consistency of the genetic base broadening strategy.We compared simulated breeding programs introducing donors with different performance levels, directly or indirectly after bridging. We also evaluated the effect of the training set composition on the success of introductions. We observed that with recurrent introductions of improved donors, it is possible to maintain the genetic diversity and increase mid- and long-term performances with only limited penalty at short-term. Considering a bridging step yielded significantly higher mid- and long-term genetic gain when introducing low performing donors. The results also suggested to consider marker effects estimated with a broad training population including donor by elite and elite by elite progeny to identify bridging, introduction and elite crosses.Results of this study provide guidelines on how to harness polygenic variation present in genetic resources to broaden elite germplasm. Modern breeding has been successful in exploiting crop diversity for genetic improvement. However, current yield increases may not be sufficient in view of rapid human population growth . MoreoveAmong the possible sources of diversity, wild relatives, exotic germplasm accessions and landraces that predate modern breeding exhibit substantial genetic diversity. These ex-situ genetic resources are conserved worldwide in international gene banks and national collections. They provide a promising basis to improve crop productivity, crop resilience to biotic and abiotic stresses and crop nutritional quality , 72. In Different sources of donors can be considered for genetic base broadening. This includes landraces historically cultivated before modern breeding. For instance in maize, open pollinated varieties (OPVs) are landrace populations of heterozygous individuals cultivated before the hybrid maize breeding revolution in the 1950’s , 68. InbWith the availability of cheap high density genotyping, Whittaker et al.  and MeuwIn the context of genetic base broadening, GS is also interesting to fasten and reduce the costs for the evaluation and identification of genetic resources in gene banks , 77. FurIn this study, we propose to take advantage of OCS for selection of bridging, introduction and elite crosses Fig. . Allier The advantage of recurrent introductions in the commercial breeding program after or without bridging depended on the type of donor considered. Donors issued from a panel assembling founders of the breeding pool, referred to as panel donors, showed a large performance gap with the elites they were crossed to. This performance gap increased with advanced breeding generations . Improved donors showed a lower performance gap with elites. Twenty-year old donors showed an intermediate performance gap with elites (− 22 trait units on average over the 60 years period) and five-year old donors showed a reduced performance gap with elites (− 8 trait units on average over the 60 years period).Nobridging_Panel) penalized the breeding population mean performance (μ) at short-term , the short-term penalty was no more significant  did not significantly penalize the short-term mean performance of the breeding population  yielded a penalty in the mid-term compared to not introducing donors  penalized only the short-term performance  also highlighted differences between strategies. The older the donors, the lower the observed increase in frequency of initially rare favorable alleles , the identification of the useful genetic diversity to broaden the elite pool is difficult and might dishearten breeders. Consequently, there is a need for global breeding strategies to identify interesting sources of diversity that complement at best the elite germplasm, to improve diversity sources to bridge the performance gap with elites, and to efficiently introduce them into elite germplasm.The identification of diversity sources for polygenic enrichment of the elite pool should account for the complementarity between diversity sources and elites as reviewed in Allier et al. . Allier Different sources of diversity can be considered by commercial breeders. The most original ones, but which show a large performance gap with elites, are landraces In this study, the external breeding program was designed to release every generation several improved lines, later considered as donors for genetic base broadening of the commercial breeding program. The external program started from a broader genetic diversity than the commercial program  and was designed to maintain higher genetic diversity during selection . This was done to mimic in a simple way the outcome of the activity of several companies conducting separate programs and therefore maintaining a global diversity. The external program can also be viewed as a pre-breeding program since it aimed at improving diversity sources to reduce the performance gap with elites while maintaining genomewide diversity . This was done to compensate the increased response to selection due to the higher genetic diversity and ensure that the donors released by the external program underperform the commercial breeding elites. It should be noted that donors outperforming elites might be encountered in practice when considering elite germplasm as source of diversity, but this situation was not considered in this study. In such a situation the direct introduction of donors would be clearly preferable.Our results highlighted a clear beneficial effect of introducing external diversity in the elite program. This benefit increased with increasing performance level of the introduced material from unimproved genetic resources collections (panel donors) to recently improved diversity sources (5-year old donors). This highlights that protection policies that permit a mildly delayed access to improved competitor varieties as diversity sources have a positive impact on long term genetic gain. This also shows that recurrent improvement of diverse and low performing genetic resources such as landraces, i.e. pre-breeding, may be beneficial before introduction in the elite germplasm. More importantly, we show that the approach for introduction should be tuned given the type of external diversity that can be accessed (see next section).When considering recent donors (5-year old), scenarios with introductions after bridging or direct introductions performed similarly. Conversely, for panel and 20-year old donors, introductions after bridging yielded significantly higher mid- and long-term performance compared to direct introductions.For panel donors showing a large performance gap with elites, the direct introductions were not converted into genetic gain. The high inter-family additive variance in this scenario Figure  A reflecFor donors with an intermediate gap level, both direct introductions and bridging brought a higher long-term genetic gain compared to the benchmark. This advantage was higher with bridging. More importantly, bridging reduced to a large extent the short term penalty in genetic gain which was observed for direct introductions. So here again bridging appears as a suitable option to maximize efficiency at different time horizons.These results can be explained by the fact that, when donors (D) were less performing than elites, the fraction of progeny selected in donor by elite bridging families (DE progeny) carried on expectation less than half of donor’s genome . Thus, pWe considered a commercial breeding program with a genetic diversity matching that of an experimental program reported by Allier et al. . BreedinIn scenarios with bridging, we considered by default two distinct bridging and breeding GS models. The prediction of elite (ExE) and introduction (DExE) crosses usefulness and the prediction within crosses were based on a model trained on the breeding progeny of the three corresponding previous generations. Considering a unique genomic selection model trained on both bridging and breeding progeny increased the prediction accuracy within introduction families (DExE) Table . This hiIt is well known that the prediction accuracy is increased for larger TS . At conss Figure . For inss Figure  C. The rs Figure . The opth = 1 for cross selection, for sake of facility. However, we observed that within family prediction accuracies were variable (Table α = 0.7) when selecting bridging crosses in order to reduce the performance gap between donors derived material and elites. When giving less weight to the performance than to the diversity, i.e. α = 0.3, we observed non-significant changes on the short- or long-term performance for scenarios with 5-year and 20-year old donors and a significant increase of long-term performance and novel favorable allele frequency for the scenario with panel donors  or This study highlights a clear beneficial effect of harnessing polygenic variation present in diversity sources to broaden the elite genetic pool, while still achieving significant genetic gain. This interest is all the more important as the level of introduced material is high, which highlights the importance of pre-breeding and the effect of plant protection policies. We show that the strategy for introduction should be tuned given the type of external diversity that can be accessed. This study provides a guideline for reaching an optimized genetic base broadening strategy.We considered 338 Dent maize genotypes from the Amaizing project , 53 as fT, progeny of the three last generations T− 3, T− 4 and T− 5 were considered as potential parents of the next generation. It created overlapping and connected generations as it can be encountered in breeding. We first considered a burn-in period of 20 years with recurrent phenotypic selection from a population of founders. Burn-in created extensive linkage disequilibrium as often observed in elite breeding programs  is the minimal genomewide diversity constraint at time t. The evolution of diversity along time was controlled by the targeted diversity trajectory, i.e. He(t), ∀ t ∈  where t∗ ∈ ℕ∗ is the time horizon when the diversity He(t∗) = He∗ should be reached. For the external and the commercial benchmark without introduction breeding programs, we considered He∗ = 0.10 and He∗ = 0.01 reached after 60 years, respectively. As in Allier et al. . Thus, the UCPC based OCS selected introduction crosses (i.e. DxE if no bridging and DExE if bridging) when necessary to maximize the performance while keeping genomewide diversity constant (Eq. nc| = 20 crosses:V) and the diversity (D), respectively, considering a within family selection intensity of i. α ∈  is the relative weight given to performance compared to diversity. A differential evolution (DE) algorithm was used to find Pareto-optimal solutions of Eq. For the commercial breeding program with introductions, we maintained the genomewide diversity constant after the end of burn-in, i.e. tant Eq. . In casetant Eq. d that mamentclass2pt{minimBridging_Panel, Nobridging_Panel, Bridging_20y, Nobridging_20y, Bridging_5y, Nobridging_5y. We ran 10 independent simulation replicates of the external program that generated donors, the commercial benchmark program without introductions, and the six genetic base broadening scenarios. Note that at a given simulation replicate the commercial breeding program accessed the potential donors released by the corresponding external breeding program simulation replicate.We compared different commercial breeding programs at a constant cost  with recurrent introductions (i) either direct or with a bridging step and (ii) considering three types of potential donors, resulting in the six genetic base broadening scenarios: T ∈  with T = 0 corresponding to the last burn-in generation, we computed the mean true breeding value (TBV) of E progeny μ(T) = mean(TBV(T)) and of the 10 most performing E progeny pj(T) at each QTL j among the 1000 QTLs. We further focused on the QTLs where the favorable allele was rare at the end of burn-in, i.e. pj(0) ≤ 0.05. The results were averaged and standard errors were computed over 10 independent replicates.We followed several indicators in the breeding families , Bridging_20y (Single TS) and Bridging_5y (Single TS). Every generation, we defined the prediction accuracies as the correlation between true breeding values and GEBVs  and 50 introduction (DExE) families of 80 DH progeny. These families were considered as the validation set (VS). We randomly sampled among the 4800 DH progeny different TS of variable sizes and compositions ; Table S2. (Mean performance of the ten best progeny at different generations); Table S3. .Additional file 4:Supplementary Figures contain the supplemental Figure S1. (Evolution of the additive genetic variance intra- and inter-family components in the breeding population); Figure S2. (Summary statistics on the introduction crosses); Figure S3. (Effect of TS composition on intra-family prediction accuracies); Figure S4. (Evolution of the breeding population over generations for two different weightings α).


To assess the conservation status and the genomic variability of Italian chicken breeds, we carried out genome-wide analyses using single nucleotide polymorphism (SNP) molecular markers. These results increase knowledge and can aid with the development of conservation plans for local Italian chicken breeds to safeguard their genetic variability.ROH), was markedly different among the breeds and ranged from 0.121  to 0.607 (Siciliana). In all breeds, short runs of homozygosity (ROH) (<4 Mb in length) were more frequent than long segments. The patterns of genetic differentiation, model-based clustering, and neighbor networks showed that most breeds formed non-overlapping clusters and were clearly separate populations, which indicated the presence of gene flow, especially among breeds that originated from the same geographical area. Four genomic regions were identified as hotspots of autozygosity (islands) among the breeds, where the candidate genes are involved in morphological traits, such as body weight and feed conversion ratio. We conclude that the investigated breeds have conserved authentic genetic patterns, and these results can improve conservation strategies; moreover, the conservation of local breeds may play an important role in the local economy as a source of high-quality products for consumers.The genomic variability of local Italian chicken breeds, which were monitored under a conservation plan, was studied using single nucleotide polymorphisms (SNPs) to understand their genetic diversity and population structure. A total of 582 samples from 23 local breeds and four commercial stocks were genotyped using the Affymetrix 600 K Chicken SNP Array. In general, the levels of genetic diversity, investigated through different approaches, were lowest in the local chicken breeds compared to those in the commercial stocks. The level of genomic inbreeding, based on runs of homozygosity (F The poultry industry represents an essential and constantly developing branch of agriculture production as an important food provider. Unfortunately, the poultry sector has undergone significant loss in terms of animal genetic resources and the erosion of many genotypes due to replacement by higher-performing commercial hybrids or indiscriminate crossbreeding, related to highly efficient selection programs . Thus, ihttps://www.pollitaliani.it/en/project/)—was developed to evaluate the safeguarding, conservation, and improvement of the Italian poultry genetic resources, represented by many autochthonous breeds that were historically present in the country.In Italy, the interest in the conservation of local poultry breeds began about 20 years ago with regional plans for conservation, followed recently by national involvement . From thDue to the recent advent of high-throughput affordable genotyping techniques, the fine genome-wide analysis of the genetic structure and relationships between chicken populations has become possible . These tThus, in this work, we aimed to provide a high-resolution genetic overview of the genome-wide diversity and population structure of 23 Italian local breeds and four commercial hybrids. Most of the reported breeds are officially recognized by the Italian authorities and by the Food and Agriculture Organization of the United Nations (FAO). Only the Cornuta di Caltanissetta and Valplatani breeds are not yet recognized. We investigated the level of genetic diversity, population structure, admixture patterns, and relationships among these Italian chicken breeds, in order to verify the effectiveness of the conservation programs.Blood samples and animal care were performed in compliance with the European rules (Council Regulation (EC) No. 1/2005 and Council Regulation (EC) No. 1099/2009). The animals identified in this study were intended for slaughter at the end of their production cycle, and samples were collected by official veterinarians who adhered to standard procedures for animals for slaughter.® tubes containing EDTA as an anticoagulant. All the studied breeds were collected from at least three different farms  to obtain a representative sampling of the breeds.A total of 582 samples were collected from 20 to 24 animals  per breed. We sampled 23 different local chicken breeds (Ancona (ANC), Bianca di Saluzzo (BSA), Bionda Piemontese (BPT), Cornuta di Caltanissetta (COR), Livorno Bianca (PLB), Livorno Nera (PLN), Mericanel della Brianza (MER), Modenese (MOD), Mugellese (MUG), Ermellinata di Rovigo (PER), Millefiori di Lonigo (PML), Padovana Argentata (PPA), Polverara Bianca (PPB), Padovana Camosciata (PPC), Padovana Dorata (PPD), Polverara Nera (PPN), Pepoi (PPP), Robusta Lionata (PRL), Robusta Maculata (PRM), Romagnola (ROM), Siciliana (SIC), Valdarnese (VLD), and Valplatani (VLP)). For the four commercial stocks (Broiler Ross 708 (708), Eureka (EUK), Hy-lyne white eggs (HYL), and ISA Brown (ISA)), 9 to 13 animals per breed were included. All of the blood samples (about 2 mL) were collected from ulnar veins and stored in VacutainersDNA extraction and genotyping were performed at Neogen  using a commercial kit and the Affymetrix Axiom 600 K Chicken Genotyping Array, containing 580,961 SNPs. The Gallus_gallus-5.0 chicken assembly was used in this study as the reference genome , with mao) and expected (He) heterozygosity, and genomic inbreeding, which is based on the difference between the observed and expected numbers of homozygous genotypes (FHOM).PLINK 1.9  was alsoK = 2 to 30 [The population structure was investigated by applying the model-based clustering algorithm run in ADMIXTURE from  2 to 30 . The cro 2 to 30 . Pair-wi 2 to 30  and plotape [Phylogenetic relationships among the breeds were analyzed by determining the Reynolds genetic distances using the R studio package ape . Neighboape . A neighape . Graphicape .(--homozig-kb), (2) two missing SNPs and up to one possible heterozygous genotype were allowed in the ROH (--homozyg-window-missing 2 and --homozyg-window-het 1), (3) the minimum number of SNPs that constituted the ROH was set to 100 ([--homozyg-snp 100), (4) the minimum SNP density per ROH was set to one SNP every 100 kb (--homozyg-density 100), and (5) the maximum gap between consecutive homozygous SNPs was 1000 kb ([--homozyg-gap 1000). The total length of the genome covered by ROH was divided by the total chicken autosomal genome length covered by the SNP array  to evaluate the individual genomic inbreeding coefficient using the ROH data (FROH) [Gallus_gallus 5.0 from the Ensembl database (http://www.ensembl.org). To clarify the gene identity, the quantitative trait loci (QTL) database  was interrogated for the presence of QTLs in the ROH islands. To investigate the biological function and the phenotypes that are known to be affected by each annotated gene, a comprehensive search in the available literature was conducted.Assessment of the runs of homozygosity (ROH) was performed for each animal using PLINK 1.9 . To defia (FROH) . Each ROA total of 23 local chicken breeds spread throughout Italy were studied . The finHOMF showed large differences among breeds, ranging from 0.076 ± 0.059 (BSA) to 0.648 ± 0.034 (SIC). All the commercial breeds showed negative values of HOMF , associated with higher He and Ho (The genetic diversity parameters are shown in e and Ho .The MDS plot in K), showed that the best fitting number of populations present in the total sample was K = 25. As shown by the C1 and C2 components of the MDS plot, the inferred breed structure at K = 2 distinguishes PRM and PRL (red), as well as PPA, PPC, and PPD (blue). PLB (green) as well as the COR and SIC breeds were separated from the others (ochre color) at K = 4. At K = 6, the PPP and PER breeds reached their genetic identity, followed by MOD, MER, PLN, and the three commercial breeds  at K = 12. The ROM and ANC breeds exhibited the same genetic structure at K = 16; at this K value, a breed-specific cluster was also observed for VLP. When K increased from 16 to 27, the breeds were progressively assigned to separate clusters: PML, MUG, and VLD breeds at K = 20, and 708 Broiler and BPT at K = 25, whereas BSA never reached a genetic identity; at K = 27 its genetic background is not clear. Notably, EUK, HYL, and ISA shared ancestral genetic components from K = 2 to 27.The population structure inferred by using ADMIXTURE , consideTo provide additional insight into the relationships and patterns of divergence, we constructed a neighbor-net based on Reynolds genetic distances A. ConsisROH for each breed are reported in ROH = 0.607 ± 0.037), whereas the BSA and BTP breeds showed the lowest values . The commercial stocks showed the lowest mean values of inbreeding   . In geneROH results. COR, PPA, SIC, and VLP have a large mean portion of their genome covered in longer ROH >30 Mb.The mean ROH segments per individual ranged from 157.58 (PRM) to 16.22 (ISA). The ROH values were clustered by their physical length into seven categories, and the mean sum of ROH values per breed was evaluated . Breeds TPK1 and LOC107051643—whereas on chromosomes 5, 7, and 8, several annotated genes were found. In addition, located on chromosome (LOC) genes were found inside all of the ROH islands; however, their functions are still unknown . This could be because PER is a dual-purpose chicken, excellent both for egg and meat production. It has been crossed, as has its ancestors, to obtain commercial hybrids. In addition, the PER, PRL, and PRM breeds, as demonstrated by previous studies, shared a proportion of their common Anglo-American derivation [The results, from different analyses, agree with previous studies and with the breeding history of the chicken breeds under investigation ,23. The rivation .ROHF) are increasingly used as an index of inbreeding or a signature of directional selection [ROHF, as observed in the SIC, PPA, and PPP breeds, can be excluded for mating purposes in endangered populations to minimize the loss of genetic diversity and increase the biodiversity [Genome-wide SNPs are a powerful approach for detecting genomic regions with reduced homozygosity. ROH-based F estimates  is the most important gene found on chromosome 2 and is involved in step 1 of the sub-pathway that synthesizes thiamine diphosphate from thiamine. This gene is conserved among several species and seems to be correlated with feed conversion and body weight [ANO (Anoctamin) genes are involved in muscle tissue development and estrogen production [NELL1 (NEL-like protein 1), SLC6A5 (Solute carrier family 6 member 5), and BBOX1 (gamma-butyrobetaine hydroxylase 1), involved in growth factors, embryogenesis events, digestive enzyme activity, and feed efficiency, respectively [FTCD, which plays a main role in feed intake, in accordance with the quantitative trait loci identified [TRMT1L (tRNA Methyltransferase 1-Like) gene is noteworthy as it seems to be involved in chicken adaptation and survival in stressful conditions [After the identification of ROH islands on chromosomes, the presence of genes was observed. Several uncharacterized genes were revealed, reflecting the selection action on uncharacterized regulatory regions or simply the fixation of non-coding DNA by genetic drift due to uncontrolled selection . In conty weight . On chrooduction . Other cectively ,38,39. Tentified . Inside nditions .The TuBAvI project allowed us to evaluate the national poultry biodiversity of the Italian local breeds for the first time through genomic technologies that highlighted the heterogenic conservation of these populations. The results showed the existence of genetic variability and low inbreeding in almost all the breeds. The population structure and genetic distances show a clear separation among the breeds, with some particular clusters related to the region of origin. The genetic background of the commercial stocks is close to that of several breeds from the Veneto region, highlighting the story of their introgression with local breeds.Through the present study, we provided a complete overview of the Italian chicken breeds and contextualized them at a national level. This will promote conservation plans and highlight their role as a genetic reservoir. Consequently, a strategy to increase value for these breeds should be provided in order to guarantee a profit for farmers. This could lead to the development of niche markets, representing the safest long-term strategy for local breed conservation.This work will help with the design of targeted conservation plans. The information obtained represents a useful tool for understanding correct genetic management and supervising conservation activity. In this context, the information from genomic analysis may play a crucial role in the development of mating plans to avoid the negative effects of inbreeding in these breeds. Minor relatedness and low inbreeding are essential for small, local breeds to maintain the native genetic diversity, and good inbreeding management for the progeny is important for preserving biodiversity.


The Bardigiano horse is a native Italian breed bred for living in rural areas, traditionally used in agriculture. The breed counts about 3000 horses, and it is nowadays mainly used for recreational purposes. The relatively small size and the closed status of the breed raise the issue of monitoring genetic diversity. We therefore characterized the breed’s genetic diversity based on molecular data. We showed a critical reduction of genetic variability mainly driven by past bottlenecks. We also highlighted homozygous genomic regions that might be the outcome of directional selection in recent years, in line with the conversion of Bardigiano horses from agricultural to riding purposes.e) was equal to 39 horses, and it showed a decline over time. The average inbreeding based on runs of homozygosity (ROH) was equal to 0.17 (SD = 0.03). The majority of the ROH were relatively short (91% were ≤2 Mbp long), highlighting the occurrence of older inbreeding, rather than a more recent occurrence. A total of eight ROH islands, shared among more than 70% of the Bardigiano horses, were found. Four of them mapped to known quantitative trait loci related to morphological traits  and disease susceptibility. This study provided the first genome-wide scan of genetic diversity and selection signatures in an Italian native horse breed.Horses are nowadays mainly used for sport and leisure activities, and several local breeds, traditionally used in agriculture, have been exposed to a dramatic loss in population size and genetic diversity. The loss of genetic diversity negatively impacts individual fitness and reduces the potential long-term survivability of a breed. Recent advances in molecular biology and bioinformatics have allowed researchers to explore biodiversity one step further. This study aimed to evaluate the loss of genetic variability and identify genomic regions under selection pressure in the Bardigiano breed based on GGP Equine70k SNP data. The effective population size based on Linkage Disequilibrium (N The Bardigiano is an Italian autochthonous horse breed whose origin can be traced back to the age of the Roman Empire in the Belgian Gaul province . The breROH) [ROH is able to capture variation due to Mendelian sampling and linkage during gamete formation [ROH does not rely on pedigree quality, which may be a limiting factor in inbreeding estimation based on genealogical data [Recent techniques in genomics permit present researchers to gain more insights into genetic diversity, population history and selection signatures compared to pedigree-based methods ,7. SeverROH) ,19,20,21cal data ,23. Overcal data ,24,25. Scal data ,31,32,33cal data ,31,32,34ROH, and (iii) evaluate the presence of potential signs of selection based on ROH islands.The detailed aims of this study were to (i) investigate genomic effective population size throughout generations, (ii) calculate within-breed genomic diversity based on Fn, where n is the number of generations separating the individual from each of its known ancestors [®  SNP chip, containing 65,157 SNPs separated on average by 40 kb. The SNP physical positions were remapped from the former reference genome EquCab2 to EquCab3 [Equus caballus autosome (ECA) chromosomes were retrieved and used in this study. The quality control (QC) was performed in PLINK v1.9 [p < 10−6 and minor allele frequencies (MAF) <0.01. Horses with a call rate lower than 0.90 were excluded.The study included genotype data from 89 Bardigiano horses . The horses submitted to genotyping were carefully selected to represent the most of genetic variability from the pedigree information [ncestors ) lower tncestors ; (iii) n EquCab3 , as desc EquCab3 . Only SNe) were estimated with the SNeP v.1.1 software [e slope analysis (NeS) [e changes occurring throughout generations. The NeS analysis identifies subtle changes in the inferred Ne curve not visually explicit in the Ne plot. The slope of each segment linking pairs of neighboring Ne estimates was first calculated and then normalized using the median of the two most proximal past Ne slope values.Past and present effective population sizes   to look ROH), average length of ROHs (LROH) and the average ROH number (SROH) were summarized according to each ROH length category. The genomic inbreeding coefficients (FROH) were calculated following the method described in [ROHL being the length of ROHs in each individual and AUTOL being the length of the autosomal genome, which was set to 2276 Gb, based on the genome length covered by SNPs. Based on the hypothesis that the length of ROH reflects the chronological time points when inbreeding happened [The ROH segments were detected using the DetectRUNS  package ribed in :(1)FROH=happened , the genhappened ,45, and happened , we estiPutative ROH islands were determined based on overlapping homozygous regions within more than 70% of the horses. The EqCab3 genomic coordinates of these regions were used to retrieve candidate gene lists and annotations from the Biomart web interface in Ensembl  and fromAll horses except one passed the genotype data quality control , and 58,047 SNPs were retrieved. The average genotype call rate was 0.99 and the average pedigree depth based on complete generation equivalent of the selected horses was 6.28 (SD = 0.79). The horses descended from 54 stallions with an average of 1.65 offspring each (SD = 0.82) and 76 mares with 1–2 offspring each. In total, 80% of the horses were born between 2011 and 2019, whereas 18 horses were born between 2001 and 2010.e calculation, we only included animals born between 2011 and 2019, which numbered 70 horses. The Ne declined over time, and was estimated to be ~39 horses one generation ago. Instead, the estimated Ne 20 generations ago was around 195 horses . The majority of ROHs were shorter than 2 Mbp, with 69.5% being shorter than 1 Mbp and 21.4% being between 1 and 2 Mbp length . The proThe number of ROHs and length of ROH segments varied across chromosomes, as shown in ROH equal to 0.17 ± 0.03 (SD). To differentiate ancient and recent inbreeding, we calculated FROH based upon five ROH length classes, as presented in ROH calculated per length below or equal to 2 Mbp. For the ROH length class above 8 Mbp, four horses did not have ROHs and thus their inbreeding resulted in being equal to 0. The degree of inbreeding based on the longest ROH class showed an average inbreeding equal to 0.02, reflecting inbreeding events which happened <8 generations ago.The inbreeding, measured as the proportion of the genome covered by ROH, resulted in an average FThe inbreeding calculated per chromosome showed large variation among chromosomes both in terms of average values and the distribution of inbreeding level per individual . The higA total of five chromosomes  contained ROH islands that were shared by more than 70% of individuals in the total sample. Three ROH islands were detected on ECA3, two on ECA10, and the remainder were equally distributed among ECA11, ECA15 and ECA19 . The thrThe evaluation of genetic diversity with traditional methods based on pedigree analyses have been widely used to manage animals’ genetic resources. In small livestock populations, where financial support is generally a limiting factor, pedigree-based methods are still commonly adopted due to their cost–effectiveness ratio ,58,59,60e), computed using genealogical data, could be biased if pedigree depth and quality are low [e in livestock populations [e in the Bardigiano horses based on LD information was approximately 39 horses in the last generation. This value of Ne is lower than that calculated in the Persian Arabian Horses (Ne = 113) [e = 48.1) and in the Rhenish German Draught Horse (Ne = 46.1) [e estimate based on pedigree data , yet comparable to, that found by molecular data analyses [e estimate obtained from pedigree can be considered a proper approximation of molecular based Ne in the case of Bardigiano horses. The estimation of Ne in the last 20 generations showed a steady decrease in effective population size, in line with the known history of the breed. The NeS analysis highlighted two sharp reductions in Ne in the Bardigiano breed, approximately ten and six generations ago. Since the average generation interval in this breed is 8.74 years [e about 10 generations ago. The Bardigiano studbook was founded in 1977 with the aim to recover the breed, just after the second sharp reduction in Ne found in this study. This second sharp reduction in Ne might highlight the loss of genetic diversity due to the selection of breeding candidates during the initial steps of the breed recovery. In contrast, the general increase and stabilization of the Ne from the 4th generation onwards might reflect the breeding policies applied by the Bardigiano studbook to conserve the breed.Effective population size  , in the  = 46.1) . Interesbreeding ,66 in thanalyses . This ev74 years , we hypoROH ranged from 17.0%, considering all ROH regardless of the size, to 2.0%, considering only the longest class (ROH > 8Mbp). This last result highlights the occurrence of older inbreeding, rather than inbreeding which happened in the last few generations. Nevertheless, the average FROH in the Bardigiano was higher compared to other small-sized horse populations. For example, in the Croatian Posavje horse breed, the FROH was equal to 8.59%; in the Bosnian mountain horse (originating from Bosnia and Herzegovina) this value reached a mean of 13.21%; and in the Austrian Noriker horse it ranged between 8.00% and 13.00%, depending on coat color strains [ROH to those in the Bardigiano horses were found in the Polish Koniks breed (mean = 16.00%), which is a primitive breed closely related to the extinct wild Tarpan [The extent and frequency of ROH have been widely used to infer ancestry at the individual and breed level . Long RO strains ,34. Simid Tarpan , and in d Tarpan . In contd Tarpan . As exped Tarpan ,70, the LCORL) gene [LCORL gene is a transcription factor that has been associated with human height [LCORL was identified in a screen for loci under selection [NCAPG, has been implicated in prenatal growth [LASP1) gene is located. The LASP1 gene mediates cell migration and survival, and its expression is induced by insulin-like growth factor (IGF1) [In general, the ROHs caused by inbreeding tend to be distributed unevenly over the genome, as we have found in the Bardigiano horse, with a different distribution of numbers and sizes of ROH for each chromosome . HoweverRL) gene . The LCOn height ,74,75. Ielection , and thel growth  as well l growth . The othr (IGF1) . HoweverMC1R). The MC1R encoded by the Extension (E) locus controls, together with the peptide antagonist agouti-signaling-protein (ASIP), the amounts of melanin pigments in mammals [In addition, this latter region of ECA11, together with two ROH islands on ECA3 between 35.48 and 37.59 Mbp, overlapped with known QTLs for equine insect bite hypersensitivity (IBH). IBH is a pruritic skin allergy caused primarily by biting midges, Culicoides spp . This sk mammals . We, thee based on LD and ROHs detected in this study. We identified a decrease in the LD-based Ne, when calculated over the last 20 generations, in line with the known history of the breed. The ROH analysis provided additional information to further optimize the breeding and management decisions needed to ensure the long term survival of the breed. Inbreeding levels, especially those based on short ROH segments, were moderate to high, suggesting the presence of past bottlenecks in the genome of Bardigiano horses. Conversely, the lowest percentage of ROH resided in the longest ROH classes, showing a small reduction in genetic variability during the last generations. Eight ROH islands were found in the breed; these islands were shared among more than 70% of the Bardigiano horses analyzed in this study. Four of them overlapped with known QTLs associated with conformation traits and disease susceptibility. Genes related with conformation traits, coat color and disease susceptibility appear to be targets of directional selection in the Bardigiano breed. This study has outlined the first genome-wide map of selection signatures in an Italian native horse breed, and provides material for further functional studies on the biological mechanisms of complex traits in horses.The genetic diversity of the Bardigiano breed was analyzed via N


IS and intra-breed structure show that there is some heterogeneity and structure within the breed. However, inter-breed structure results underline that this breed is differentiated from other Creole breeds, because crossbreeding with other breeds was not detected; therefore, we must take advantage of this valuable genetic resource, and ensure its conservation and selection.An individual from a population presents a series of characteristics that differ from the rest and that increase as the kinship relationships are lower; this leads to the fact that two populations that stop exchanging genetic material through mating eventually come to present characters common to all members of each, but different between the two. This was what happened with the animal populations brought to America more than 500 years ago from the Iberian Peninsula in the colonization period, resulting in Creole populations that inhabit the harshest environments of our immense geography. The Creole goat “Chusca Lojana” has adapted to live in the dry forest region of Southern Ecuador, where environmental conditions are warm-dry, with sparse vegetation and a rather irregular topography. In the present study, the intra-breed genetic diversity of this goat is analyzed as well as its genetic relationships with other breeds. Significant FIS value of 0.11. Between-breed genetic diversity among 43 goat populations  showed the following values: FIS = 0.087, FIT = 0.176, and FST = 0.098. Regarding the analysis of the population structure, the results showed that the Creole Chusca Lojana goat population is homogeneous and no genetic separation was observed between the different biotypes (FST = 0.0073). In conclusion, the Chusca Lojana goat has a high genetic diversity, without exhibiting a genetic substructure. Therefore, it should be considered as a distinct population because crossbreeding with other breeds was not detected.The largest population of goats (62%) in Ecuador is in the dry forest region in the south of the country. A Creole goat, named “Chusca Lojana”, has adapted to the dry forest region where environmental conditions are warm-dry, with sparse vegetation. Knowledge of the genetic information of the Creole goat is important to determine intra-racial diversity, the degree of genetic distance among other breeds of goats, and the possible substructure of the population, which is valuable for the conservation of such a species’ genetic resources. A total of 145 samples of the Creole goat was taken from the four biotypes previously identified. Genetic analyses were performed using 38 microsatellites recommended for studies of goat genetic diversity (FAO-ISAG). The results of within-breed genetic diversity showed a mean number of alleles per locus (MNA) of 8, an effective number of alleles (Ae) of 4.3, an expected heterozygosity (He) of 0.71, an observed heterozygosity (Ho) of 0.63, polymorphic information content (PIC) of 0.67, and an F Biodiversity and abiotic factors are responsible for maintaining the balance and stability of ecosystems. According to , biodiveIn the last two decades, however, such genetic heritage has been jeopardized and more than one million species have become endangered ,3. RegarGoat farming is of great significance for the economy of rural populations worldwide, especially in developing countries due to the rusticity of goats ,8. GoatsHair samples were obtained from 145 Chusca Lojana goats of four different biotypes, the same ones that differ from each other in the size and shape of the ears  and in the absence of horns (“muco” biotype). Sampled individuals belonged to different herds and locations and kinship was minimized, so genetic variability of sampling was ensured. Samples were collected in paper envelopes with individual information and stored at room temperature until genetic analyses were performed at the Laboratory of Applied Molecular Genetics of the University of Cordoba, Spain. Sampling was carried out by qualified veterinarians during routine technical assistance, so the approval of an animal ethics and welfare committee for biological sampling collection was not required. The genomic DNA of the hair follicle samples was extracted according to the methodology described by .BM1258, BM1329, BM1818, BM6506, BM6526, BM8125, CSRD247, CSRM60, CSSM66, ETH010, ETH225, HAUT27, HSC, ILSTS008, ILSTS011, ILSTS019, ILSTS030, ILSTS087, INRA005, INRA006, INRA023, INRA063, INRA172, MAF065, MAF209, McM527, MM12, OarFCB011, OarFCB020, OarFCB048, OarFCB304, SPS115, SRCRSP05, SRCRSP08, SRCRSP23, SRCRSP24, TGLA053, and TGLA122. After amplification by the polymerase chain reaction (PCR) technique, size separation of the amplified fragments was carried out by polyacrylamide gel electrophoresis in an ABI 3130XL automatic sequencer by means of Genescan® 400HD ROX Size Standard . Fragment analysis and allelic typing were performed using Genescan Analysis® 3.1.2  and Genotyper® 2.5.2 software , respectively  for genetic diversity studies in goat species. The microsatellites included were ectively .IS fixation index values with a 95% confidence interval were calculated with the GENETIX v software 4.05 [The MICROSATELLITE TOOLKIT software for Excel  was usedare 4.05 . The Harare 4.05 , which aare 4.05  and Bonfhttps://biogoat.jimdo.com/razas-breeds/).To obtain the genetic differentiation, structure, and distance information data of the Chusca Lojana goat was compared with data of other 42 goat populations worldwide, which included some native Creole breeds from Spain, Europe, America, and Africa . Data onIT, FST, and FIS [Wright’s F statistics, namely F and FIS , were ca and FIS . A Facto and FIS  was dete and FIS . With th and FIS  to graphSA) were calculated [The genetic distances between individuals . After Bonferroni correction, only the INRA023 and SRCRSP24 markers were unstable compared with the Hardy–Weinberg equilibrium of the population. Only the INRA005 and SPS115 markers showed a homozygous defect, but their FIS values were not significant. Half of the microsatellites (n = 19) showed a significant excess of homozygosity, and the other microsatellites (n = 19) displayed FIS values that were not significantly different from 0.Shown in IS with a 95% confidence interval and 1000 randomizations was significant (FIS = 0.1099 (0.08773–0.12549)), which indicates that the population could deviate from the Hardy–Weinberg equilibrium.The mean number of alleles (MNA) is an indicator of the genetic variability within populations. The MNA is 8.26 in the Chusca Lojana goat, and with an average Ae value of 4.25. We assessed the genetic diversity within a population by calculating the proportion of heterozygous individuals or heterozygosity. In ST = 0.0073).To make a preliminary assessment of the population’s homogeneity, a tree diagram of the genetic distances between individuals was designed , in whicA more complex analysis of the population structure was also carried out using STRUCTURE v. 2.1. This software  allowed IS = 0.0871 (0.065–0.116), FIT = 0.176 (0.154–0.205), and FST = 0.0978 (0.089–0.108).The genetic differentiation among the 43 populations of goats included in the present study was high, with the following statistical values for F: FThe results of the Correspondence Factor Analysis  show thaST values between pairs of goat populations are shown in The Reynolds’ genetic distance data and the FIn the graphic representation of Reynolds’ genetic distances in a network dendrogram , it can Shown graphically in The characterization process is the first step of the zoogenetic resources conservation programs. So, studies of this nature are generally recommended to save locally adapted breeds from extinction .The high genetic diversity of the Chusca Lojana breed is demonstrated by the mean number of alleles (MNA = 8.3) and the effective number of alleles (Ae = 4.3). These values are similar to those obtained for the Spanish goat of the USA  and higher than those of the Creole goats of Argentina, Bolivia, Brazil, Colombia, Cuba, Peru, and Venezuela . It is noteworthy that the Galapagos Creole goat displayed lower MNA and Ae values , and the Creole Goat of Paraguay showed higher MNA and Ae values  [IS value of the Chusca Lojana breed was significant (0.1098) and higher than the mean of the corresponding values reported for the other American goat populations [IS value could be due to non-random mating within the breed. Significant inbreeding levels and deviations from the Hardy–Weinberg equilibrium can be shown in a short time if no measures are taken.The Chusca Lojana breed exhibited higher expected (He = 0.71) and observed heterozygosity values (Ho = 0.63) than the mean of the corresponding values for the other American Creole goat populations . The only exception was the Paraguayan Creole, which showed higher He and Ho values . The genulations ,27, beinIS of the 57 Asian breeds was lower (0.073) and the FIT = 0.191 was similar to the corresponding values in the present study.Interestingly, the Chusca Lojana breed showed higher genetic diversity  than reported for 57 native goat breeds in Asia  . HoweverST) of the Chusca Lojana breed and the other 42 Creole and transboundary breed populations was lower (FST = 0.098) than the values reported for 24 Creole and three cross-border breeds in America , for the Creole breeds of Asia (FST = 0.127) [ST = 0.112) [ST of the Chusca Lojana breed was higher than the values obtained for goats from Northern Europe and Central-Eastern Mediterranean (0.07) [The genetic differentiation  , and for= 0.112) . On the n (0.07) , West Asn (0.07) , and Afrn (0.07) .By graphic representation of Reynolds’ genetic distances, it could be demonstrated that results in the present study are in accordance with the data previously reported , in whicFinally, both the work carried out by  and the The Chusca Lojana goat breed exhibits a high genetic diversity, with a heterogeneous structure inside the population; but, the existence of four different biotypes empirically admitted inside the breed was not supported by our genetic results. The Chusca Lojana breed belongs to the genetic group of the American Creoles, descendants of the of the Iberian populations imported by the Spanish colonizers five centuries ago. No genetic introgressions from the most important international breeds  were detected in the Chusca Lojana breed.The Chusca Lojana breed is a valuable zoogenetic resource, well adapted to its difficult environment; its conservation and selection should be encouraged, and actions should be taken to avoid future genetic erosion.