Margaritifera margaritifera). We found multiple paternity in all clutches for which more than two offspring were genotyped, and numbers of sires were extremely high. Thirty‐two sires had contributed to the largest clutch (43 offspring sampled). This study provides the first evidence of multiple paternity in the freshwater pearl mussel, a species that has experienced dramatic declines across Europe. Previous studies on other species of freshwater mussels have detected much lower numbers of sires. Multiple paternity in freshwater pearl mussels may be central for maintaining genetic variability in small and fragmented populations and for their potential to recover after habitat restoration and may also be important in the evolutionary arms race with their fish host with a much shorter generation time.Multiple paternity is an important characteristic of the genetic mating system and common across a wide range of taxa. Multiple paternity can increase within‐population genotypic diversity, allowing selection to act on a wider spectre of genotypes, and potentially increasing effective population size. While the genetic mating system has been studied in many species with active mating behavior, little is known about multiple paternity in sessile species releasing gametes into the water. In freshwater mussels, males release sperm into the water, while eggs are retained and fertilized inside the female (spermcast mating). Mature parasitic glochidia are released into the water and attach to the gills of fish where they are encapsulated until settling in the bottom substrate. We used 15 microsatellite markers to detect multiple paternity in a wild population of the freshwater pearl mussel ( Multiple paternity has primarily been studied in mobile species with distinct mating behavior, such as copulation, and typically internal fertilization , males release sperm, which females concentrate from the water with their filter feeding system. Eggs are fertilized and developed in a pouch on the gills .A total of 52 adult mussels were collected in Slørdal river  on 20 August 2015, without knowledge of sex and reproductive status , but at a time of the year when they were expected to have passed the period of fertilization. The studied population of 2.3Salmo trutta) and salmon , with strong preference for either species in each Norwegian mussel population . In the hatchery, mussels were kept together in a 1 × 1 m tank with circulating permanent water flow (25–35 cm/s). The outgoing water was directed into a tank with fish, ensuring the transport of mussel larvae to a suitable host. Suitable host fishes are trout  in June and 1.14 ± 0.13 mm (range: 0.90–1.53 mm) in September. DNA samples from the 52 collected adult mussels were taken on 22 September 2016 by gently collecting material from the visceral mass with a cotton swab .A minimum of 30 of 52 adult mussels from Slørdal river released larvae (glochidia) in the hatchery, between 5 and 21 September 2015. A total of ca. 4,000 juveniles were produced. Calculating back from the dates larvae were released, eggs were likely fertilized between 8 and 24 August, and thus largely or exclusively before collection of adults in the river. At an age of 9 month (3 June 2016) and 1 year (22 September 2016), 100 juveniles were sampled, respectively, for genetic analysis. Juveniles were randomly sampled and individually stored in ethanol. Sampled juveniles had a length of 0.40 ± 0.04 mm (mean ± 2.4dneasy tissue kits (Qiagen). The mussels were genotyped at 15 loci: MarMa3050, MarMa3621, MarMa4277, MarMa4322, MarMa2671, MarMa4143, MarMa5280 , for each locus and for all loci combined, was calculated in CERVUS. The assumptions of Hardy–Weinberg and linkage disequilibrium were tested on adult genotypes in GENEPOP 4.7 Rousset, . SignifiWe assigned parentage to sampled adults using the likelihood‐based approach in CERVUS 3.0  to determine the critical Delta value (difference in LOD score between first and second most likely candidate pair) for the assignment of parentage. The critical Delta value is affected by the genetic markers, but also by the proportion of candidate parents that had been sampled. We estimated that 18% of the candidate parents had been sampled, based on the assumptions of an even sex ratio among the 52 sampled adults Bauer,  and 10 cWe then performed a maternity analysis with complete sampling of maternal genotypes to assign the remaining offspring to mothers. All parentage analysis in CERVUS was performed with a mistyping rate of 0.01 and a critical Delta value for parentage assignment set for a confidence level of 95%.The sex of adults was inferred from assigned parentage. Adults that were assigned parentage in maternity analysis only, or in both maternity and parental pair analyses, were classified as females. Adults that were assigned parentage in parental pair analysis only were classified as males. This was unambiguous for almost all adults. However, one parental pair consisted of adults that both had additional offspring assigned in maternity analysis only and should thus be classified as females according to the above rules. One adult in that pair (SL61) had only a single offspring that was assigned in maternity analysis only. That offspring had a Delta value close to the critical value in parental pair analysis, and we assigned parentage to both candidate parents. Adult SL61 was then classified as male. Another parental pair consisted of individuals that both had common offspring only. We arbitrarily classified one of them (SL53) as female.2.6We calculated two measures of multiple paternity. MINSIRES , except for locus MarMa4143 (58%). Two offspring were excluded from analysis because genotyping failed. Of the remaining 250 individuals, 138 individuals (55%) were successfully genotyped at all 15 loci, 109 individuals (44%) at 14 loci, two individuals at 13 loci, and one individual at 11 loci.The number of alleles per locus ranged from 1 to 25, with a median of nine . The father was detected among the sampled adults for 13 of the genotyped offspring. Those offspring were assigned to seven males, with each male contributing one to five offspring within a brood. Two males sired offspring with two different mothers. We did not detect any cases of self‐fertilization.A total of 198 offspring were assigned to 20 mothers using CERVUS. The number of offspring assigned to each mother was highly variable  was close to the number of genotyped offspring in all broods Figure . Thus, mWe used simulations to estimate the number of sires in the complete broods of the three females for which most offspring were sampled. The complete brood of female SL51 consisted of approximately 870 juveniles, given that 43 offspring were among a sample of 198 juveniles randomly taken from 4,000 juveniles. We used simulations to estimate the number of sires in the complete brood of 870 offspring, given that 32 sires were detected in a sample of 43 offspring. Those simulations suggested that the number of sires in the complete clutch was most likely approximately 68 and between 44 and 127  that are fertilized by males releasing sperm upstream of the females Bauer, . Given tL. cardium: Ferguson et al., H. cumingii: Bai et al., L. cardium was limited by the size of the studied populations (21–41 adults) (Ferguson et al., H. cumingii were taken from a supposedly large population in a lake (Bai et al., The number of sires was much higher than previously reported for unionid mussels. Between two and four sires were found in broods of other freshwater mussels when 25–29 offspring were sampled (4.2M. margaritifera. Multiple paternity increases the effective population size when compared to monogamous or polygynous mating (Pearse & Anderson, M. margaritifera are small, making them vulnerable to inbreeding and Allee effects (Geist & Kuehn, M. margaritifera can display high genetic variability (Geist & Kuehn, Evidence of extensive multiple paternity offers new perspectives on reproductive potential that can be leveraged to improve conservation of Margaritifera margaritifera is among the most long‐lived invertebrates known, reaching over 200 years in Northern Europe (Lopes‐Lima et al., Not only do our results offer additional evidence of multiple paternity in unionid mussels, our findings illustrate the potential of genetic methods for estimating demographic parameters, including population size. River ecosystems are highly vulnerable to fragmentation, which can prevent completion of bidirectional migratory life cycles like those exhibited by freshwater mussels (Bórquez & Brante, 5Our results show the potential for high levels of multiple paternity in sperm‐casting freshwater mussels. A better understanding of the genetic mating system is critical to the conservation of those species. Future studies need to show whether multiple paternity is widespread in freshwater mussels and how the degree of multiple paternity depends on the size and structure of populations.None declared.S.K. was the project leader, P.J., S.K. and B.M.L. designed and carried out the study. S.W. performed the parentage analysis and wrote the original manuscript. All authors contributed to revisions.https://doi.org/10.5061/dryad.fb2f1v3Adult and juvenile genotypes and R script for simulations of sires in complete clutch are available at the Dryad Digital Repository:  Click here for additional data file.


Caenorhabditis briggsae, a close relative of the nematode C. elegans, such minor genetic incompatibilities have been identified. One incompatibility between the mitochondrial and nuclear genomes reduces the fitness of some hybrids. To understand the nuclear genetic architecture of this epistatic interaction, we constructed two sets of recombinant inbred lines by hybridizing two genetically diverse wild populations. In such lines, selection is able to eliminate deleterious combinations of alleles derived from the two parental populations. The genotypes of surviving hybrid lines thus reveal favorable allele combinations at loci experiencing selection. Our genotype data from the resulting lines are consistent with the interpretation that the X alleles participate in epistatic interactions with autosomes and the mitochondrial genome. We evaluate this possibility given predictions that mitochondria-X epistasis should be more prevalent than between mitochondria and autosomes. Our empirical identification of inter-genomic linkage disequilibrium supports the body of literature indicating that the accumulation of mito-nuclear genetic incompatibilities might initiate the speciation process through the generation of less-fit inter-population hybrids.Genetic variants that are neutral within, but deleterious between, populations (Dobzhansky-Muller Incompatibilities) are thought to initiate hybrid dysfunction and then to accumulate and complete the speciation process. To identify the types of genetic differences that might initiate speciation, it is useful to study inter-population (intra-species) hybrids that exhibit reduced fitness. In  Biological species comprise genetically related populations of organisms whose inter-population hybrid offspring suffer complete loss of fitness: either hybrid inviability or hybrid sterility . UnderstBut how can genetic differences arise and reproductively isolate two populations without first causing reduced fitness in the original populations? The DMI model addresses this concern by suggesting that neutral genetic differences independently arising in two populations could lead to deleterious epistatic interactions between two (or more) loci only when those differences were combined in inter-population hybrids , 1940. TThe unique relationship between the mitochondrial and nuclear genomes explains how such epistatic interactions exist and how disruption of those interactions at hybridization could be deleterious [reviewed in ]. After As mitochondrial haplotypes (mitotypes) diverge in different populations of a species, then their nuclear genomes might fix different co-evolved alleles that sustain mitochondrial function. These co-evolved population-specific epistatic allele pairs can be disrupted at hybridization. Such a mito-nuclear epistatic interaction thus constitutes a DMI. Mito-nuclear DMI loci separated by hybridization are expected to impact electron transport and to cause hybrid dysfunction. The details of this process have been well detailed by a number of studies in various taxa where disrupting mitochondrial-nuclear (mitonuclear) epistatic interactions by hybridization causes fitness reduction . This grMitochondrial-nuclear DMIs might also help explain Darwin’s corollary to Haldane’s rule : the asyLoci involved in hybrid dysfunction can be identified by genotyping hybrids to identify allele combinations that are selected against due to the reduced fitness they produce. These combinations are then underrepresented in hybrids compared to the Mendelian expectation. Genotypes of hybrids can be assessed for TRD at loci across the genome to map incompatibility loci . To accoIn order to assess the contribution of cytoplasmic alleles to hybrid dysfunction, genetic mapping often also employs reciprocal crosses, with the intent of identifying phenotypes that depend on interactions between maternally inherited cytoplasmic factors  and a hybrid nuclear genome. Cytoplasmic-nuclear hybrids (cybrids), in which the cytoplasm from one population is introgressed into the nuclear background of another, have revealed mito-nuclear epistatic interactions that decrease cybrid fitness in several taxa . Such fiCaenorhabditis briggsae have also revealed mitonuclear epistasis. Like its close relative, C. elegans, it could be considered a non-traditional model for studying speciation genetics, because the presence of a hermaphrodite sex and only rare males means that each wild population is produced predominantly by selfing. Thus, these populations are essentially functionally reproductively isolated from others. However, C. briggsae populations are generally reproductively compatible with other populations when mated in the laboratory  hermaphrodites, as in . Because of the presence of a self-fertile hermaphrodite sex in s, as in . Seven ss, as in . After gs, as in . PurifieLike the AI-RIL, lines were initiated by P0 generation crosses AF16xHK104 and HK104xAF16. Each hybrid line was established in the F1 generation, for each biological replicate of each of the two crosses above, by one sibling-mated hermaphrodite. One additional generation of sibling-mating occurred, followed by ten generations of inbreeding by single hermaphrodite selfing. All of the F2 RIL were cryopreserved using the liquid freezing protocol .Phenol-chloroform extracted and isopropanol-precipitated genomic DNA samples were resuspended and then diluted to 5 ng/μL concentration in 10mM TRIS buffer pH 8.0. Amplification was performed according to  with theC. briggsae, this value is equal to twice the number of replicate lines)  . The nulThirty-nine hybrid lines initiated by reciprocal crosses between AF16 and HK104 (nineteen AF16xHK104 and twenty HK104xAF16) completed the AI-RIL cross design . The mitotypes were all maternally inherited, as expected, with no evidence of heteroplasmy. The fraction of HK104 alleles present at each nuclear marker is shown in Figure To validate AI-RIL genotype results, purified genomic DNA from each F2 RIL was PCR genotyped at a different marker on the X chromosome: cb51757. The fraction of HK104 alleles present at this X locus is shown in Table C. briggsae hybrids. Despite determining genotypes at a limited number of loci across most  nuclear chromosomes, our results are consistent with a number of themes. First, cross direction, and thus possibly mitotype, modulates some TRD patterns. Second, a nuclear chromosome consistently implicated in inter-genomic epistasis was the X chromosome. Below we interpret this result according to theory that suggests the possibility of an increased role of an X chromosome in mito-nuclear interactions. Third, mito-nuclear TRD appears to reveal co-evolution. We discuss these findings in relation to the view that mito-nuclear DMIs play an important role in the process of speciation . We favor the interpretation that differences between the AI-RIL and F2 RIL cross designs account for some observed discrepancies between our datasets. In the AI-RIL cross, recombinant genotypes have opportunity to compete in small populations (reducing the influence of drift), while this did not occur in the F2 RIL cross, where drift might dominate over all but the most severe fitness effects. We previously showed, using different AF16-HK104 F2 RIL and AI-RIL, that this is the case . In thatSecond, we note that the F2 RIL had the fewest biological replicates, which could also influence the ability to detect a significant and consistent signal of TRD. In fact, we find it remarkable, then, that we did observe significant X-chromosome TRD in the HK104xAF16 RIL. This result from F2 RIL, which corroborates the TRD bias in AI-RIL with the same mitotype, raises the possibility that mito-X co-evolution constitutes a strong selective pressure to maintain linkage disequilibrium (LD) between alleles from the same population.C. briggsae AF16-HK104 hybrid lines. Genotypes of AF16-HK104 F2 RIL have shown a slight tendency for inheritance of HK104 alleles over AF16 across the autosomes and X chromosome , the probability of drift resulting in these observations from 20 lines is, conservatively, 0.5ˆ20 = 9.5 × 10-7.Interestingly, alleles on chromosomes III and X  are in perfect LD with the HK104 mitotype and also share a TRD pattern with loci on chromosomes II, IV and V. Using the same probability argument, it would be very unlikely for drift to have caused significant TRD on five chromosomes toward retention of the same allele (HK104), in the case of the AI-RIL with HK104 mitotypes , each mitotype exists in the same (heterozygous) nuclear background. Any subsequent observed differences between reciprocal crosses thus correlate with that singular genetic difference between reciprocal crosses: their mitotypes. The detection of a consistent signal of TRD on the X chromosome in F2 and AI-RIL, and the strong TRD signals observed using multiple biological replicate lines, including across three loci spanning two chromosomes, do support the possibility that a mito-X epistatic interaction exists and possibly signals the co-evolution of alleles within each population.C. briggsae AI-RIL, we observed that mitochondria preferentially segregate with X alleles from the same population. This finding indicates that a separate effect, deleterious epistasis between AF16 and HK104 X alleles, is unlikely to explain the X chromosome TRD , while the autosomal retention of HK104 alleles in the presence of the HK104 mitotype is asymmetric. In this context, it is interesting to note that crosses involving species containing a hermaphrodite sex , as is true in the present study, have been suggested to generate larger effect asymmetries than in dioecious  species  result from genetic incompatibilities between nuclear and parent-of-origin  sources and that symmetrical effects could result from presence of multiple DMIs or from the separation of co-evolved mito-nuclear loci . Althougs Figure  further s Figure , and thas Figure  or symmes Figure . Here, m species .C. briggsae populations, this species is ideal for studying speciation genetics (C. remanei and C. latens (C. briggsae and C. nigoni (C. briggsae.The study of mito-nuclear epistatic interactions segregating within populations offers the opportunity to better understand the genetic basis of incipient speciation . With bogenetics , especiagenetics . Others genetics , that algenetics . Furthergenetics . Althouggenetics . Syntenygenetics . Howevergenetics ; those m. latens  and C. b. nigoni . The preC. briggsae, continue to motivate the exploration of how mito-nuclear epistasis influences fitness and, ultimately, speciation.In future, we will identify the epistatic loci and describe the organismal fitness effects that result in the allele TRD reported here. Improving our understanding of the mechanics of epistasis is important . InitialThe raw agarose gel electrophoresis images supplying the genotype data and the nematode strains or purified genomic DNA samples supporting the conclusions of this manuscript, will be made available by the authors, without undue reservation, to any qualified researcher.RH created the AI-RIL and BM created the F2 RIL. RH and BM isolated and genotyped DNA samples from the RIL, analyzed the data, and conducted statistical analyses. RH and JR wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Camelina sativa, originated from southeastern Europe and southwestern Asia, is being re-embraced as an industrial oilseed crop due to its high seed oil content (36–47%) and high unsaturated fatty acid composition (>90%), which are suitable for jet fuel, biodiesel, high-value lubricants and animal feed. C. sativa’s agronomic advantages include short time to maturation, low water and nutrient requirements, adaptability to adverse environmental conditions and resistance to common pests and pathogens. These characteristics make it an ideal crop for sustainable agricultural systems and regions of marginal land. However, the lack of genetic and genomic resources has slowed the enhancement of this emerging oilseed crop and exploration of its full agronomic and breeding potential. Here, a core of 213 spring C. sativa accessions was collected and genotyped. The genotypic data was used to characterize genetic diversity and population structure to infer how natural selection and plant breeding may have affected the formation and differentiation within the C. sativa natural populations, and how the genetic diversity of this species can be used in future breeding efforts. A total of 6,192 high-quality single nucleotide polymorphisms (SNPs) were identified using genotyping-by-sequencing (GBS) technology. The average polymorphism information content (PIC) value of 0.29 indicate moderate genetic diversity for the C. sativa spring panel evaluated in this report. Population structure and principal coordinates analyses (PCoA) based on SNPs revealed two distinct subpopulations. Sub-population 1 (POP1) contains accessions that mainly originated from Germany while the majority of POP2 accessions (>75%) were collected from Eastern Europe. Analysis of molecular variance (AMOVA) identified 4% variance among and 96% variance within subpopulations, indicating a high gene exchange (or low genetic differentiation) between the two subpopulations. These findings provide important information for future allele/gene identification using genome-wide association studies (GWAS) and marker-assisted selection (MAS) to enhance genetic gain in C. sativa breeding programs.There is a need to explore renewable alternatives  that can produce energy sources to help reduce the reliance on fossil oils. In addition, the consumption of fossil oils adversely affects the environment and human health via the generation of waste water, greenhouse gases, and waste solids.  Camelina sativa (L. Crantz) originated from southeastern Europe and southwestern Asia and is a member of the Brassicaceae (Cruciferae) family, which contains a number of economically important crops such as Brassica napus , Brassica oleracea  and Brassica rapa   can be up to twice that of soybean (18–22%) (C. sativa oil is low in saturated fatty acids (<10%)  . C. satin Turkey , while gn Turkey . C. satiuel crop . The oil(18–22%) . The pros (<10%)  and highs (<10%) . These os (<10%) , low-inps (<10%) , broaders (<10%) , and ress (<10%) , make C.C. sativa accessions. The genome-wide assessments of the genetic landscape of C. sativa germplasm helps facilitate use of accelerated breeding approaches using marker-assisted selection (MAS). Previous works by other groups have yielded a reference genome resource for C. sativa and several relatively small-scale genetic studies. The reference genome  indicates an allohexaploid genome with three ancestral sub-genomes: two sub-genomes with seven chromosomes each derived from an older hybridization event that resulted in an allotetraploid ancestor, and a second hybridization between the tetraploid and a diploid ancestor that resulted in a sub-genome with six chromosomes  (Triticum aestivum L. (winter wheat)  technologies become increasingly cost- and time-effective at the genome-wide level . Among a rapa L. , Ziziphu(jujube) , and Trir wheat) , and morC. sativa accessions assembled from the Canadian germplasm collections in the USDA-ARS National Plant Germplasm System (NPGS) and the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). These accessions are originated from 19 different countries in Europe and Asia. The objectives were to (1) detect and genotype SNPs at a genome-wide scale; (2) characterize the genetic diversity and population structure; and (3) characterize genetic differentiation between and within the subpopulations. This study describes the genetic diversity and population structure in current C. sativa accessions and lays a foundation for future genome-wide association studies (GWAS) or genomic selection (GS) in Camelina breeding programs.In the present study, GBS technology was used to genotype a spring panel of 213 C. sativa accessions, originally collected from different regions of Eurasia  was collected in Costar tubes on dry ice. The tissue tubes were stored in a rack and covered with breathable sealing tape and stored at −80°C until the tissue was lyophilized. C. sativa leaf tissue was ground in tubes with stainless steel beads using a plate shaker. DNA extraction on the lyophilized tissue was done using the Qiagen Plant DNeasy 96 kit following the manufacturer’s protocol. DNA concentration and quality was determined using Quantifluor  and a Synergy H1 plate reader. The PstI restriction enzyme was used for GBS library construction . GBS tags were exported from the database in FASTQ format using the TASSEL TagExportToFastqPlugin and were aligned to the C. sativa genome using BWA MEM   . The VCFHe), is a fundamental measure of genetic diversity in a population, and describes the expected proportion of heterozygous genotypes under Hardy-Weinberg equilibrium  is the frequency of allele I in the population. In addition to GD, MAF, and polymorphism information content (PIC) also indicate genetic properties of SNPs in a population from different aspects. MAF refers to the frequency at which the second most common allele occurs in a given population  based on the rate of change in [LnP(D)] between successive K-values. For the optimal K-value, membership coefficient matrices of five replicates from STRUCTURE were used in CLUMPP (Population structure was estimated using a Bayesian Markov Chain Monte Carlo model (MCMC) implemented in STRUCTURE v2.3.4 . Five ruarvester , using tn CLUMPP  to genern CLUMPP , S2 to cn CLUMPP . Accessin CLUMPP , from whn CLUMPP . The prin CLUMPP .Na), number of effective alleles (Ne), Shannon’s information index (I), observed heterozygosity (Ho) and expected heterozygosity (He) were also calculated using GenAlEx v6.503  in the population varied from 0.1 (142 SNPs) to 0.5  with an average of 0.29, while the PIC values varied from 0.1 (283 SNPs) to 0.4  with an average of 0.24  was plotted against ΔK, which showed a sharp peak at K = 2  showed the highest probability for population clustering and these two subpopulations consisted of 105 and 108 genotypes, respectively  for each of the subpopulations and suggested that there was significant divergence within both of the two subpopulations . An Fst K = 4 . A neighK = 4  was consK = 4 and the results were shown in the The two subpopulations identified in STRUCTURE were then applied in GenAlEx 6.503 to calculate the Analysis of Molecular Variance (AMOVA), Nei’s genetic distance and the genetic diversity indices. The AMOVA, Fst and Nm are provided in Na) and number of effective alleles (Ne) of the two subpopulations were 1.993 and 1.451, respectively  shows higher diversity than pop2 . The percentage of polymorphic loci per population (PPL) ranged from 98.74% (pop2) to 99.82% (pop1) with an average of 99.28%.The grand mean value of different alleles , SUN (33), Poland (POL) (17), and 16 other countries in central Europe and Asia. The origins of 26 accessions were unknown (NA) (C. sativa.To study the genetic diversity within own (NA) . The genCamellia sinensis  and PIC values are both measures of genetic diversity among genotypes in breeding populations, which sheds the light on the evolutionary pressure on the alleles and the mutation rate a locus might have undergone over a time period (He) indicates gene diversity for haploid markers and provides an estimate of the average heterozygosity and genetic distance among individuals in a population (He) and will become closer to GD with more alleles and with increasing evenness of allele frequencies  (Lolium spp. (ryegrass)  . Accordinotypes) , (1) marnotypes) , Lolium yegrass)  and jujuyegrass) . This mayegrass)  and coulyegrass) .K = 2)  gave similar results. The presence of structure in this population meets our expectation for the following reasons. First, according to the pedigree of genotypes   and the K = 2)  indicateenotypes , all theenotypes . Neverthenotypes , resultioduction , and theoduction , therefoultivars , which wC. sativa two subpopulations according to the Fst values obtained from the STRUCTURE , pop1 was slightly higher than pop2, meaning that pop1 was more diverse than pop2 since He depends on both the number of alleles (richness) and the abundance (or evenness) of the alleles in a population. The low genetic diversity and the clusters of two subpopulations were in agreement with a previous population genetics study among a collection of 175 accessions of C. sativa , expected heterozygosity (he), unbiased expected heterozygosity (uhe), and percentage of polymorphism loci (PPL). This knowledge of population structure and genetic diversity of C. sativa accessions will be important for future studies using genomic selection, MAS and GWAS.In this study, high-throughput GBS technology was used to explore genetic diversity and population structure among the current HA-H and NF conceived and designed the study and provided suggestions and comments for the manuscript. NF performed GBS analysis. ZL collected and analyzed the data and wrote the manuscript. ZL, JB, JD, TK, MA, DS, YG, NF, and HA-H revised the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Social processes are associated with depression, particularly understanding and responding to others, deficits in which can manifest as callousness/unemotionality (CU). Thus, CU may reflect some of the genetic risk to depression. Further, this vulnerability likely reflects the neurological substrates of depression, presenting biomarkers to capture genetic vulnerability of depression severity. However, heritability varies within brain regions, so a high-resolution genetic perspective is needed. We developed a toolbox that maps genetic and environmental associations between brain and behavior at high resolution. We used this toolbox to estimate brain areas that are genetically associated with both depressive symptoms and CU in a sample of 258 same-sex twin pairs from the Colorado Longitudinal Twin Study (LTS). We then overlapped the two maps to generate coordinates that allow for tests of downstream effects of genes influencing our clusters. Genetic variance influencing cortical thickness in the right dorsal lateral prefrontal cortex (DLFPC) sulci and gyri, ventral posterior cingulate cortex (PCC), pre-somatic motor cortex (PreSMA), medial precuneus, left occipital-temporal junction (OTJ), parietal–temporal junction (PTJ), ventral somatosensory cortex (vSMA), and medial and lateral precuneus were genetically associated with both depression and CU. Split-half replication found support for both DLPFC clusters. Meta-analytic term search identified “theory of mind”, “inhibit”, and “pain” as likely functions. Gene and transcript mapping/enrichment analyses implicated calcium channels. CU reflects genetic vulnerability to depression that likely involves executive and social functioning in a distributed process across the cortex. This approach works to unify neuroimaging, neuroinformatics, and genetics to discover pathways to psychiatric vulnerability. In particular, disruption in the ability to process social cues is associated with depressive symptomology, and can lead to increased deficits in daily functioning in both patient and subclinical groups3. Depressed individuals’ symptoms relate to specific facets of social behavior, namely, reasoning through others emotions6, fitting under the “understanding mental states” subcategory of the “social dimensions” construct in the US National Institute of Mental Health Research Domain Criteria (RDoC) matrix. Many forms of social response have been associated with depression7, and it is thought that broad deficits in social functioning may be influential in categories of severe depression. In particular, social deficits in theory of mind, the ability to understand others’ thoughts, are related to poor mentalizing/metacognition, or inability to understand the self. Further, theory of mind predicts depression diagnosis above and beyond metacognition in behavioral studies8.As depression follows a normal distribution of risk across the population8), which has been suggested as a marker of internalizing disorder symptom severity10. Although typically examined in the context of externalizing disorders11, CU has also been consistently associated with depression13. This association may arise because, while CU reflects a disregard for others, CU has been related directly to theory of mind, empathy skills, and social processing14, including emotional understanding in social contexts12. Thus, poor social processing/CU may be a mechanism that indexes depression severity15 or sustains depression3. Finally, as CU is related to multiple forms of empathetic behaviors, including cognitive and social-emotional responses, it forms an important first step in exploring the biological overlap between social processes and depression.An inability to understand and respond to others’ emotions due to multiple cognitive deficits may manifest as callousness/unemotionality , thanks to the specificity gained when using high-resolution brain maps. Here, we implement an integrative framework in which we directly map brain areas in which genetic influences on CU and depression overlap. Specifically, the goal of the current study is test whether CU captures some of the genetic vulnerability to depression; and localize the brain areas contributing to this vulnerability. These genetically associated brain areas can then be used with biological and neuro-informatic tools for mapping across different levels within the RDoC matrix, such as RNA expression and biological pathway analyses, to expand understanding of the coinheritance of CU and depression.In contrast to this lack of contextualization and deep phenotyping in genetic research, brain mapping integrates onto other areas of biology/psychology  studies, which employed a voxel-wise spatial resolution, found that MDD was associated with lower brain volume in specific subregions of the rostral anterior cingulate cortex and the dorsolateral and dorsomedial frontal cortex24. And the anterior cingulate cortex shows differential gene expression and differential task activation across the ROI25. Thus, whole-brain approaches that employ a finer degree of spatial resolution are warranted.The largest meta-analysis of neuroanatomical differences in depression to date used region of interest (ROI) measures of cortical thickness. It found that major depressive disorder (MDD) was associated with cortical thinning in the insula, anterior and posterior cingulate, and temporal gyri:26. Additionally, the rostral and dorsal anterior cingulate cortex areas that overlap between CU and depression were also found to distinguish suicidal cases from controls in another VBM study27, giving some evidence to support our hypothesis that CU represents a social severity dimension of depression and that depression and CU symptoms should be jointly studied.With respect to neuroanatomical correlates of CU, decreases in volume of the rostral and dorsal cingulate cortex have been observed, overlapping spatially with regions that have been identified for depressionThe goal of this study is to capture the dimension of depression that overlaps with CU and validate this approach by integrating results from across the imaging genetics literature through one analytical pipeline. Imaging can act as a conduit to understand the mechanisms of genetic effects but also transcriptomics, cognition, and behavior. Mapping genetic association with behavior across the brain enables us to explore multiple levels of biological analysis simultaneously and look for convergence across them to validate theories of behavior.https://github.com/AlexHatoum/Wild-Card-Toolbox). In step 1, we estimated the genetic and environmental association between depression and CU to evaluate the relative importance of shared inherited vulnerability. In step 2, we developed a toolbox that creates genetic and environmental brain maps for each trait. Rather than map standard beta coefficients , our procedure maps effect sizes for genetic and environmental variances , creating brain maps of genetic association between cortical thickness and the two behavioral traits. We estimate areas that represent the genetic vulnerability to CU and depression by overlaying the clusters from the separate depression and CU genetic maps onto one map. Finally, by integrating these brain maps with neuroinformatic tools in step 3, we can begin to characterize likely functions and specific molecular mechanisms of the genes influencing CU and depression, which is impossible in a standard biometrical design. Specifically, in step 3, we used MNI coordinates to align our genetically associated clusters with a meta-analytic database of effects across multiple Functional Magnetic Resonance Imaging (fMRI) and transcriptomic studies. By using the spatial coordinates, we can further investigate our hypothesis about the overlap by comparing our coarser genetically derived phenotypes to results from studies using more specific cognitive functions. Thus, our main analysis is the generation of genetically influenced brain map for depression and CU, and our follow-up analyses explore likely effects of this genetic variance implicated by this map by using high-resolution brain coordinates.Using structural magnetic resonance imaging (MRI) data from 258 young adult twin pairs, we asked, where are the genes influencing the vulnerability to social deficits and depression influencing brain morphology? Do these morphological differences overlap? And, can we map a specific pattern and use this pattern to speculate further on mechanisms? To answer these questions, we used the methodology pictured in Fig. 28. Further, past work has shown there are differences in the genetic variance structure within and between commonly utilized ROIs; thus, measuring genetic variability in ROIs vs. vertices leads to relative differences in genetic variance effects between regions being overestimated29 and more fine-grained metrics, such as voxel or vertex measures, are preferable to ROI approaches for making comparisons across the cortex for individual differences genetics29. Notably for this study, it is these genetic individual differences patterns that are implicated in the mechanisms of psychopathology, requiring high-resolution coordinates to specify accurately. Finally, using high-resolution analysis and Montreal Neurological Institute coordinates (MNI) coordinates allows for integration with functional and transcriptomics literature more broadly.We conduct this analysis in a general population sample to include subsyndromal levels of depression and CU within a large enough sample to find patterns of coheritability between brain and phenotype. We chose high-resolution brain mapping because prior literature in neuroimaging genetics suggests vertex-wise approaches will more appropriately capture the individual differences patterns of genetic effects. In particular, common brain atlases used in anatomical research were derived agnostically to genes influencing individual differences and do not capture the specificity of the architecture of genetic effects on behavioral traits, as has been shown for language-related brain areasM = 28.7, SD = 0.6), recruited from the Colorado Longitudinal Twin Study (LTS). Twin pairs who had completed an ongoing neuroimaging study of neural substrates of executive functions and psychopathology and whose imaging data passed quality control were included. More about the sample can be found in the online methods.Participants were 258 same-sex twin pairs , and 115 dizygotic (DZ), 132 female pairs and 93 male pairs; singletons were used in calculating the mean and variance), aged 28–31 years  = 2400 ms, echo time (TE) = 2.01 ms, flip angle = 8°, field of view (FoV) = 256 mm, and voxel size of 0.8 mm30. We chose this measure because tendencies toward an emotional vulnerability should manifest itself in higher frequency of depression, we wanted to include subsyndromal levels of depression, and this measure has shown reasonable stability across 10 years of longitudinal data31.On the day of the scan, participants completed the Center for Epidemiological Studies-Depression (CESD) scale, a 20-item Likert scale assessing the frequency of past-week depression symptoms13, a 24-item Likert questionnaire with three subscales: callousness , uncaring , and unemotional . We used this scale as a measure of CU because it has been used to define clinical subtypes of conduct disorder in the past13, the ICU total score relates to social and emotional processing12, and, though the factor structure changes in adulthood, the scale retains a high internal consistency and predicts social, emotional, and depressive behaviors in individuals similar in age to our sample13. We conducted all analyses with the ICU total scale, which is more reliable and normally distributed than the subscales, which were all highly intercorrelated For the CESD and ICU, the dependent variable was the average item rating provided that at least 80% of the items were answered, multiplied by the number of items. To improve normality, both scales were square-root transformed . Each vertex and psychopathology measure was residualized on brain mean thickness and sex prior to model estimation.All cortical thickness estimates were processed using a standard Freesurfer pipelineBehavioral genetic ACE models decompose phenotypic variance into three sources: Additive genetic , Common environmental , and non-shared Environmental . Because MZ twins share all their genes, their additive genetic influences correlate 1.0; DZ twins share on average half their genes identical by descent, so their additive genetic influences correlate 0.5. By definition, C effects correlate 1.0 and E effects correlate 0.0 for both types of twins.33, which decomposes the phenotypic covariance between two measures into that explained by genes and environments. The genetic correlation (rG) of the two phenotypes equals (a11 × a12)/√(a112 × (a122 + a22)).To examine the genetic and environmental covariance between the psychopathology measures and brain measures, the standard ACE model for a single variable can be extended to multivariate analyses. To ensure that the estimated component covariance matrices are positive definite, they are expressed as the product of a lower triangular matrix and its transpose Fig. . This isTo examine the etiological overlap between depression and CU, we started by estimating their phenotypic overlap through a partial correlation analyses . We used a series of structural models to show that our association is specific to our measure of depressive symptom frequency and CU, rather than a broad psychiatric vulnerability  used vertex-wise cluster extent p-value correction of values below (0.05) significance at a window of twice the original smoothing kernel (i.e. cluster extent threshold = 20 mm). We chose this procedure partially due to its practicality in integration with genetic estimates and to estimate clusters that were contiguous for follow-up analyses.To determine significant clusters for each disorder, we (1) estimated a chi-square difference test n = 132 pairs; sample 2, n = 126 pairs) and ran the full analyses separately in each sample. In each half of the sample we used a conjunction minimum alpha of 0.05 (ref. 35) and cluster-extent correction of 20 mm to define significant clusters. We then overlaid the clusters from (1) the full sample analysis, (2) the analysis in sample 1, and (3) analysis in sample 2. Because the full sample was more conservative than either half, we used the criterion of significant overlap in all three analyses as our standard, i.e., a cluster must have been independently associated below the split-half criteria in both half-samples and by a more conservative threshold with the full sample for us to have high confidence in its effect.To explore the replicability of our approach, we split our sample into halves by families (so that twin pairs would be kept together) by random draw  Neurosynth meta-analytical database of functional activation across over 10,000 fMRI studies19, and the (3) Yeo 7-network parcellation36.Using MNI coordinates, we examined the overlap of our clusters with other sources of data: (1) the Allen brain atlas transcriptomic atlas and genome-wide association study (GWAS) results from the Psychiatric Genomics Consortium depression mega-analyses of 480,359 individuals1 and used Allen brain atlas through Neurosynth gene37 by downloading each gene image, renormalizing it across the cortex with FMRIB Software Library (FSL38) and visualizing its expression. We excluded genes from the major histocompatibility complex, as these associations may be spurious due to long-range linkage disequilibrium, and any genes not obtained through RNA arrays in the Allen brain atlas, leaving us with 100 genes. We put the expression values by each region in one matrix with k-means clustering. We used the elbow method  to account for multiple comparisons.With the Allen brain atlas, we took the list of associated genes from the psychiatric genomics consortium MDD GWAS gene-burden results19. This analysis finds which of our coordinates most overlap with those found in the literature and which terms  are associated with those studies. We then identified which of these terms most commonly appeared across clusters (after filtering out non-specific brain terms). Finally, we overlaid the coordinate of our clusters on the seven resting-state networks from the Yeo parcellation36 to identify to which networks the clusters belonged.With Neurosynth, we entered our clusters from the discovery sample into Neurosynth decoder to obtain “terms” that were most associated with functional activation across studies, as determined by a meta-analytic naïve Bayes classifier across over 10 000 fMRI studiesrG) was 0.40  was not significantly greater than zero . We concluded that the correlation between CU and depressive symptoms was due almost entirely to genetic covariance.We began by estimating the phenotypic, genetic, and environmental overlap between depressive symptom frequency, measured by the CESD, and CU, measured by the ICU. Figure We created a map of areas where cortical thickness genetically correlated with CESD and ICU scores. We then overlaid the clusters from the two maps to discover regions that showed conjunction for genetic prediction.As shown in Fig. 40, though not sufficient to establish a causal relationship. Environmental associations were not consistently in the same direction of effect as the genetic clusters . No other pathways were significant after FDR correction.We used follow-up analyses to gain insight into potential mechanisms involved in this genetic vulnerability. Results of clustering of Psychiatric Genomics Consortium depression-related genes are shown in Supplemental Fig. To identify likely cognitive/behavioral mechanisms reflecting this vulnerability, we conducted a meta-analytic term search using Neurosynth. Supplemental Tables 36. Supplemental Table 41. All but two areas (8 of 10 positively and negatively associated areas) fell in networks with higher-level cognitive functions .We projected our genetic derived clusters onto the Yeo 7-network parcellation, a popular, low-dimensionality parcellation derived from a clustering analysis of resting state data from 1000 participantsBy directly estimating brain areas genetically associated with depression and CU, we found (1) the association between CU and depressive symptoms was entirely genetic in origin. (2) Genetic influences on thicker cortex in right DLFPC sulci, the right PreSMA, left medial and lateral precuneus, OTJ, and TPJ were associated with both traits, and genetic influences on thinner cortex in the right ventral PCC, right medial precuneus, right DLPFC gyrus, and left somatosensory cortex were associated with both traits. (3) Likely molecular pathways are influencing calcium channel depolarization. 4) Likely associated behaviors are “theory of mind”, “inhibit”, and “pain.” (5) Likely neural functions as determined by connectivity are associated with default-mode and higher-level cognitive systems. Figure  Likely a42. Further, our approach allows for expansion of hypotheses in genetic association studies by integrating MRI atlas-based approaches to contextualize the genetic association patterns and implicate molecular pathways and brain functions.We are the first to directly estimate the cortical pattern that represents genetic vulnerability to a psychiatric disorder. Importantly, this approach is not limited to known associations43 and integration with RDoC domains2. Reassuringly, this approach converges on several areas previously associated with depression and social processing literature24, which means past neuroimaging studies of these behaviors may be driven by genetics. However, cortical thickness associations with depression in the temporoparietal and temporo-occipital junctions, key social processing areas, were novel. Finally, we identified likely mechanisms for follow-up analyses using Bayesian meta-analysis, such as theory of mind and inhibition, that are likely targets for behavioral intervention.In this case, we focused on the vulnerability for CU in depression, chosen due to its importance in depression severity44. The association with theory of mind helps validate these results in line with our hypothesis as it implicates a cognitive social process, while the association with inhibition leads us to believe this may be a more top-down mechanism. Spatially, we found clusters specific to the posterior ventral cingulate cortex and DLFPC, which show broad connectivity patterns  between limbic/emotional systems and the association cortex46. Further, almost all clusters were in higher-order cognitive systems.This vulnerability reflects an expanded cognitive network. The association could be explained by both theory of mind (in line with our hypothesis) and inhibition, a trait that we did not hypothesize initially, but that is thought to be related to theory of mind16. Finally, informatic analyses focused on overlap based on spatial coordinates. We did not directly measure the overlap between measures of expression, function, and neuro-anatomy, so we do not know whether these analyses are directly tapping the same underlying variability. Of course, this would be impossible for transcription (in humans) and would require complex multi-mediation patterns for relating functional coordinates and anatomy.There are limitations to our approach. First, the sample is matched on age (at around age 28). While this protects against both linear and non-linear confounding by age, results may not generalize to younger or older ages. Second, we did not examine moderation by sex. However, although sex interaction may be a factor in genetic depressive symptomology, there is still a genetic correlation between males and femalesWe directly mapped genetic vulnerability to CU and depressive symptoms on the brain. We found common genetic variance in CU and depressive symptoms was associated with higher-order cognitive areas and functions. As the genetic vulnerability to psychiatric disorders is discovered, the use of high-resolution cortical methods will be invaluable in contextualizing the patterns of genetic effects.Supplemental Materials


Salmonella serovars, isolated from foods of animal origin.This study aims to study the significance of random amplified polymorphic DNA (RAPD) typing in heterogeneity analysis of Salmonella serovars isolated and identified from different foods of animal origin such as meat, milk, and egg by standard bacteriological methods. DNA isolated from all 10 isolates which are confirmed by biochemical and serotyping methods and then RAPD was performed using the primers OPB 10, primer 1290, NSC I, NSC II, and primer 3. Then, RAPD data were analyzed using the BioNumerics software, Belgium, Germany.Salmonella isolates in combination with other molecular and phenotypic typing techniques. Thus, our results support earlier observation of its significance by different workers on different Salmonella serotypes.RAPD polymerase chain reaction (PCR) using five primers, namely OPB 10, primer 1290, NSC I, NSC II, and primer 3, classified the 10 isolates into 9, 10, 10, 7, and 10 RAPD-PCR types with discriminating powers of 0.1987, 0.423, 0.50889, 0.1842, and 0.2582, respectively. The phylogram constructed with NSC I profile classified isolates based on geographical origin. Primer 1290, NSC II, and primer 3 produced some uniform bands in all isolates indicating their binding ability in conserved genomic region. This study revealed that RAPD profile can be best used for finding out the heterogeneity at molecular level of Salmonella serovars.Repeatability of RAPD-PCR is insufficient to distinguish genetic differences among  Salmonella organisms are most frequently isolated bacterial agents of foodborne outbreaks /1000. The purity was checked as ratio of OD260 and OD280, and the integrity of the purified DNA was assessed by running it in 0.7% agarose gel.DNA isolated from all the 10 biochemically confirmed isolates using HiMedia TM Bacterial Genomic DNA Purification Kit following the manufacturer’s instructions supplied along with the kit with suitable modifications. Briefly, 1.5 ml of overnight broth culture was pelleted by centrifugation at 15,000 rpm for 2 min. The supernatant was discarded, and pellet was resuspended in 180 µl of lysis solution AL. After adding 200 µl lysis solutions CSalmonella isolates as a template with the random primers (Xcelris) mentioned in 2 , 300 µM each nucleotides , and 3.75 units of Taq DNA polymerase  in 50 µl PCR reaction mix. PCR program for NSC I primer included initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation (94°C for 1 min), annealing (25°C for 45 s), and extension (72°C for 1 min). Final extension was carried out at 72°C for 7 min. For primers NSC II, 1290, OPB-10, and primer 5, PCR reaction mix and cycling conditions were same with the exception of their annealing temperature which was adjusted at 29°C, 27°C, 30°C, and 27°C respectively. PCR products were characterized by submarine gel electrophoresis on 0.9% agarose gel. After electrophoresis, the gel was visualized in gel documentation system, and picture was taken. 100 bp DNA ladder  and 1 kb DNA ladder  were used as molecular weight markers.RAPD-PCR was performed using genomic DNA of The banding patterns from RAPD were analyzed with BioNumerics Software version 7.5 . The gel images of respective genotypic technique were uploaded as tiff format. The gel image of the genotyping PCR was processed in four steps, namely strip creation, calculation of densitometric curves, normalization of gel image, and detection of bands on the gel. The strip creation step identifies individual lane of the sample and defines the area of it to cut from the main gel. In the densitometric step, the software builds the peaks on the basis of intensity of bands and detects the strength of the positivity of each band. The normalization step helps to save the changes in the edited image and helps to set a reference marker. Finally, in the band calculation step, each of the bands identified on the gel is compared against the reference marker and compared with other bands present on the gel. After processing the gel image, each of the lane data was added to the database of BioNumerics Software version 7.5, in particular, genotyping PCR technique. The data were used to make a comparison of the lanes for the creation of dendrogram by similarity matrix using the Dice coefficient with optimization of 1%. The dendrograms were obtained by means of Unweighted Pair Group Method with Arithmetic Average clustering algorithm.The numerical index of discrimination (D) of each primer was also calculated using the Simpson’s index of diversity  using thth type.Where N is the total number of strains in the sample population, s is the total number of types described, and n is the number of strains belonging to jSalmonella serovars. Keeping this in view, the attempts were made to evaluate the molecular heterogeneity among Salmonella isolates from foods of animal origin using RAPD-PCR. Salmonella enterica isolates obtained from foods of animal origin were subjected to serotyping at CRI, Kasauli, Himachal Pradesh, India. The results of serotyping are presented in Nowadays, advances in molecular biological tools utilizing various typing techniques make it simpler to differentiate isolates of Salmonella species. It was observed that all five primers employed were capable of elucidating polymorphic amplification patterns in all isolates.The application of RAPD analysis -24 basedSalmonella spp. tested  produced by Salmonella Enteritidis isolates. The analysis of fingerprints in the dendrogram displayed that four isolates of Salmonella Lindenburg showed three different types of fingerprints, hence, dividing them into three clonal groups. This indicates the ability of OPB-10 primer to discriminate within the serovar Lindenburg also.RAPD-PCR using primer OPB-10 was successfully able to produce fingerprints of all the 10 isolates of . tested . Some baCR types . A maximet al. [Salmonella Typhimurium. We observed an average genetic similarity between the four isolates of Salmonella Lindenburg (69.3%). The four Salmonella Rough isolates were separated into four different RAPD types with average genetic similarity of 33.9%. The genetic similarity between serotype Salmonella Lindenburg and Salmonella Enteritidis was 33.3% (66.7% average genetic diversity). The genetic similarity between serotype Salmonella Lindenburg and Salmonella Typhimurium was 33.9% (66.1% average genetic diversity). The genetic similarity between serotype Salmonella Enteritidis and Salmonella Typhimurium was 33.3% (66.7% average genetic diversity). The Salmonella isolates from chicken showed an average similarity of 69.3% (30.7% average genetic diversity). Genetic similarity between Salmonella spp. isolated from milk ranged 33.9-71.4% with an average similarity of 52.65% (47.35% average genetic diversity). Average genetic similarity between Salmonella spp. isolated from egg was 33.3% (66.7% average genetic diversity). Average similarity between Salmonella spp. isolated from egg and chicken is 33.3% (66.7% average genetic diversity). The discrimination power of OPB 10 was calculated as 0.1987. Our results were contrary to the reports of Sumithra [Salmonella Typhimurium. As per the results obtained in this study, the discriminative power of OPB-10 primer between the different serovars is very poor.Similarly, Sumithra et al.  reportedSumithra  who repoSalmonella spp. tested . The four Salmonella rough strains were separated into four different RAPD types with an average genetic similarity of 39.8% (60.2% average genetic diversity). The genetic similarity between serotype Salmonella Lindenburg and Salmonella Enteritidis was 50.9% (66.7% average genetic diversity). The level of similarity ranged between 43.2% and 58.6%. The genetic similarity between serotype Salmonella Lindenburg and Salmonella Typhimurium was 33.9% (66.1% average genetic diversity). The level of similarity ranged between 43.2 and 48.3. The genetic similarity between serotype Salmonella Enteritidis and Salmonella Typhimurium was 42.3% (57.7% average genetic diversity).RAPD-PCR using primer 1290 was successfully able to produce fingerprints of all the 10 isolates of . tested . Here alCR types . The anaSalmonella spp. from chicken ranged between 42.3% and 72.7% with an average similarity of 56.5% (43.5% average genetic diversity). Average similarity between Salmonella spp. isolated milk ranged from 43.2 to 58.6%. Average genetic similarity between Salmonella spp. isolated from egg was 21% (79% average genetic diversity) and the similarity between isolates from egg and chicken was 34.65%. The Simpson diversity index of primer 1290 was 0.423.The genetic similarity among Salmonella spp. tested . The level of similarity ranged between 51.7% and 100%. The four Salmonella Rough strains were separated into three different RAPD types with average genetic similarity of 68.25% (31.75% average genetic diversity). The genetic similarity between serotype Salmonella Lindenburg and Salmonella Enteritidis was 65.85% (34.15% average genetic diversity). The level of similarity ranged between 51.7% and 80%. The genetic similarity between serotype Salmonella Lindenburg and Salmonella Typhimurium was 51.7% (48.3% average genetic diversity) and between serotype Salmonella Enteritidis and Salmonella Typhimurium was 36.5% (63.5% average genetic diversity). The discrimination power of NSC I was 0.50889.RAPD-PCR using NSC I also produce fingerprints of all the 10 isolates of . tested . On the CR types . The anaSalmonella spp. from chicken showed a genetic similarity ranged between 51.7% and 100% with an average similarity of 75.85% (24.15% average genetic diversity). Average similarity between Salmonella spp. isolated from milk was 62% (38 % average genetic diversity) and the similarity between the isolates from egg was 36.5% (63.5% average genetic diversity). Average similarity between Salmonella spp. isolated from egg and chicken is 58.25% (41.75% average genetic diversity). This study point toward the power of NSC I primer to some extent group the Salmonella isolates based on their host origin. Sumithra et al. [Salmonella Typhimurium isolates based on geographical origin. The potential ability of NSC I primer to discriminate Salmonella Gallinarum based on their geographical origin was also reported by Habtamu et al. [The genetic similarity among u et al. .Salmonella spp. tested . The four Salmonella Rough strains were separated into four different RAPD types with average genetic similarity of 33.25% (66.75% average genetic diversity). The level of similarity ranged between 23.7% and 42.8%. The genetic similarity between serotype Salmonella Lindenburg and Salmonella Enteritidis was 48.55% (51.45% average genetic diversity). The level of similarity ranged between 34.1% and 63%. The genetic similarity between serotype Salmonella Lindenburg and Salmonella Typhimurium was 34.1% (65.9% average genetic diversity). The genetic similarity between serotype Salmonella Enteritidis and Salmonella Typhimurium was 34.1% (65.9% average genetic diversity).RAPD-PCR using NSC II was also able to produce fingerprints of all the 10 isolates of . tested . On the CR types . The anaSalmonella spp. from chicken ranged between 42.8% and 75%. The average similarity was 61.56% (38.44% average genetic diversity) and for those isolates from milk ranged from 34.1% to 56.3% with an average similarity of 45.2% (54.8% average genetic diversity). Average genetic similarity between Salmonella spp. isolated from egg was 42.8% (57.2% average genetic diversity) and from egg and chicken was 48.55% (51.45% average genetic diversity). The discrimination power of NSC II was 0.1842. A report by Rezk et al. [Salmonella Paratyphi B isolates using NSC II primer when compared to NSC I and NSC III.The genetic similarity among k et al.  showed mSalmonella Gallinarum isolates of poultry origin using NSC II primer when compared to NSC I and 1290. Sumithra [Salmonella Typhimurium. However, on the contrary to previous reports, this study showed that the discriminative power of NSC I primer is better than NSC II for differentiation of different Salmonella serovars. This variation may be due to the difference in reaction mixture such as the amount of magnesium chloride used in the reaction mixture. Also, difference in serotype and origin of the isolates may also be taken into consideration.Similarly, a study by Meenu  showed mSumithra  also repSalmonella spp. tested . The level of similarity ranged from 13.6% to 66.7%. The four Salmonella Rough strains were separated into four different RAPD types with average genetic similarity of 43.75% (56.25% average genetic diversity). The genetic similarity between serotype Salmonella Lindenburg and Salmonella Enteritidis was 21.8% (78.2% average genetic diversity) and between serotype Salmonella Lindenburg and Salmonella Typhimurium was 17.2% (82.8% average genetic diversity). The genetic similarity between serotype Salmonella Enteritidis and Salmonella Typhimurium was 13.6% (86.4% average genetic diversity).RAPD-PCR using primer 3 was also able to produce fingerprints of all the 10 isolates of . tested . On the CR types . The anaSalmonella spp. from chicken showed a genetic similarity ranged between 13.6% and 66.7% with an average similarity of 33.7% (66.3% average genetic diversity) and those milk ranged from 45.8 to 54.8% with an average of 50.15% (49.85% average genetic diversity). Average genetic similarity between Salmonella spp. isolated from egg was 20.8% (79.2% average genetic diversity) and from egg and chicken is 25.9% (74.1% average genetic diversity). The discrimination power of primer 3 was 0.2582.Salmonella serovars. These observations revealed that RAPD profile could be best used for finding out the heterogeneity at the molecular level of Salmonella isolates in combination with other molecular and phenotypic typing techniques. Thus, our results support the earlier observation of its significance by different workers on different Salmonella serotypes [Due to polymorphism inherent in the sequence, as well as, the short length of primers used, the RAPD typing method resulted in a clustering of isolates into highly discriminating genetic trees . Althougerotypes -35.Salmonella isolates, and further, it can be used for the epidemiological studies for the classification of Salmonella isolates based on their geographical origin. However, still, it has demerit due to its non-reproducibility. It once again proved that repeatability of RAPD-PCR is insufficient to distinguish genetic differences among Salmonella serovars.The high discriminative power of NSCI revealed the power of this primer as a potential candidate in RAPD analysis of SSS carried out the research work; AG planned, designed, and supervised the experiment; BJ assisted in planning designing and execution of work. HK and NK assisted in the collection of sample and laboratory work. All authors read and approved the final manuscript.


Dryolimnas [cuvieri] aldabranus) is the last surviving flightless bird in the western Indian Ocean, and the only living flightless subspecies within Dryolimnas cuvieri, which is otherwise volant across its extant range. Such a difference in flight capacity among populations of a single species is unusual, and could be due to rapid evolution of flight loss, or greater evolutionary divergence than can readily be detected by traditional taxonomic approaches. Here we used genetic and morphological analyses to investigate evolutionary trajectories of living and extinct Dryolimnas cuvieri subspecies. Our data places D. [c.] aldabranus among the most rapid documented avian flight loss cases . However, the unusual intraspecific variability in flight capacity within D. cuvieri is best explained by levels of genetic divergence, which exceed those documented between other volant taxa versus flightless close relatives, all of which have full species status. Our results also support consideration of Dryolimnas [cuvieri] aldabranus as sufficiently evolutionary distinct from D. c. cuvieri to warrant management as an evolutionary significant unit. Trait variability among closely related lineages should be considered when assessing conservation status, particularly for traits known to influence vulnerability to extinction (e.g. flightlessness).Flight loss has evolved independently in numerous island bird lineages worldwide, and particularly in rails . The Aldabra white-throated rail ( Organisms living in island environments frequently undergo remarkable evolutionary changes –4. One sThe avian family with the highest incidence of flight loss worldwide is the Rallidae , with over 25% of the extant rail species being flightless . The famca. 1500 years BP). However, it is estimated that such documented cases are greatly outnumbered by undocumented human-induced rail extinctions, which may total 2000 species in the Pacific islands alone  aldabranus), which occurs only on Aldabra Atoll in the southern Seychelles. Historically, D. cuvieri occurred on all four islands of the Aldabra group–Aldabra, Assumption , endemic to Assumption  aldabranus and the poorly volant D. c. abbotti from the volant lineage of Madagascar. Genetic variation among populations of D. [c.] aldabranus is used to refine our understanding of important dispersal events in the biogeographic history of this lineage. We also show how differentiation among D. [c.] aldabranus subpopulations can be used to inform effective management of this unique bird, the last survivor among 12–17 flightless avian lineages that once occupied the Western Indian Ocean region before human arrival  aldabranus occurs in an area of ca. 37.2 km2, on the raised atoll of Aldabra , Polymnie (1.9 km2) and Malabar (25.9 km2). A UNESCO World Heritage Site since 1982, Aldabra has been managed entirely for research and conservation since 1979 with only a very small resident human population.The total population of D. [c.] aldabranus originates from a successful reintroduction of 18 rails from Malabar in 1999 [Dryolimnas [c.] aldabranus also occurred until very recently on the smaller islet of Île aux Cèdres, and was reportedly more morphologically distinct (leg and bill size) from D. [c.] aldabranus on other islands than was D. c. abbotti [D. [c.] aldabranus subpopulations on Grande Terre and Picard were extirpated  following the introduction of feral cats  aldabranus samples , four D. c. abbotti , and nine D. c. cuvieri from different locations in Madagascar . The samples include individuals from all D. [c.] aldabranus subpopulations, except the extinct Grande Terre subpopulation, for which no museum specimens exist. Specimens from the extinct Picard subpopulation were available from museum skins. The 19 contemporary D. [c.] aldabranus blood samples were collected on Aldabra  in two periods (years 2000 [Île aux Cèdres] and 2011–2014). We used only historical D. c. cuvieri samples after attempts to obtain contemporary samples were unsuccessful .Thirty-eight samples , represehttp://www.ncbi.nlm.nih.gov/tools/primer-blast/; FinchTV 1.4 (Geospiza), BioEdit 7.2.0  aldabranus and D. c. abbotti subspecies, respectively) are indeed most closely related to the volant white-throated rail of Madagascar . Although phylogenetic relationships are congruent with existing taxonomy in this respect, levels of genetic divergence are somewhat incongruous, with substantial genetic divergence within Dryolimnas cuvieri; the highest absolute sequence divergence of 2.1% between flightless D. [c.] aldabranus and volant D. c. cuvieri in Cytb, and 1.7% divergence between D. [c.] aldabranus and poorly volant D. c. abbotti, exceeds the minimum absolute divergence of 1.2% in Cytb encountered between other flightless and volant rail taxa, all of which have full species status [Dryolimnas subspecies derived from the concatenated (593 bp) mtDNA dataset are noteworthy in three cases  D. c. abbotti and D. [c.] aldabranus; and 3) D. [c.] aldabranus populations native to the southern islands of Aldabra  and D. [c.] aldabranus populations native to northern islands of Aldabra .Tree topology is highly concordant between Bayesian and ML analyses, between Bayesian analyses with different tree priors, and among replicate Bayesian analyses with the same tree prior. Bayesian analyses converged, with date estimates for supported (PP≥0.95) ingroup nodes varying by a maximum of 2.6% (1600 years) between replicate chains. Based on Bayesian analyses of 10 million generations, divergence time estimates show consistent variation depending on the tree prior used . We therefore selected Yule and Coalescent-Inversegamma tree priors for our final two Bayesian analyses (each was run for 30 million generations), thereby obtaining longer chains for the two tree priors that cover the full range of divergence estimates based on findings from shorter chains. Our Bayesian analyses  confirm s status . Mean nuee cases  between:ca. 0.07–0.13 Myr ago. The Assumption population (D. c. abbotti) forms a monophyletic group within the species Dryolimnas cuvieri . Tree topology is consistent with a lack of monophyly for all other subspecies; e.g., D. [c.] aldabranus populations from Malabar, Malabar* and Polymnie , p = 0.19; Aldabra, p = 0.17; and Madagascar, p = 0.18). Therefore, signal in our CR and Cytb data neither provides significant support for nor against the monophyly of these populations–both scenarios remain plausible.Our relaxed clock analysis suggests that the divergence of Aldabra and Assumption populations from those on Madagascar occurred D. cuvieri within the Aldabra group. Dryolimnas c. cuvieri of Madagascar is intermediate between two groups of D. [c.] aldabranus on each side of the network. This pattern in the concatenated mtDNA network . Loss of flight must have evolved rapidly, in less than 130,000 years based on our estimations, which concords with inferences made from subfossils  aldabranus and volant D. c. cuvieri, and 1.7% between D. [c.] aldabranus and poorly volant D. c. abbotti. species . Our resbfossils . This plbfossils ,13,15,41et al. [D. [c.] aldabranus was already flightless by 100,000 ybp, as a fossil D. [c.] aldabranus tarsometatarsus from this period  measures within the size range of the present flightless population of D. [c.] aldabranus. Flightlessness may result from variations in development of several physical traits [D. [c.] aldabranus  aldabranus than D. c. abbotti and D. c. cuvieri was found in both sexes .The rapid evolutionary change associated with such cases of flight loss, despite low genetic divergence, is generally believed to be driven by selection rather than genetic drift, as maintaining such traits as energetically costly flight muscles ,58 is pret al. ,59 propol traits , such asanus see ), increaanus see ,61. Chanl length . Our find Abbott ) and Bend Abbott , but notd Abbott . Bill sid Abbott  and the y Benson , might bPhalacrocorax harrisi), a series of candidate function-altering genetic variants was found that likely contributed to the evolution of flightlessness  aldabranus and D. c. cuvieri are still extant, a genome-wide study should provide further insights into the adaptive evolution of flightlessness.Morphological changes are frequently due to selection on a limited number of loci. In the flightless Galapagos cormorant  lagoon islet, closest to Grande Terre (distance: 253m) and separated from Malabar by a ca. 15m wide, deep channel (ca. 15m depth), and between Picard and Polymnie  are conD. [c.] aldabranus. Our understanding of extant genetic diversity would have been greatly impoverished without access to extinct genetic diversity archived in museum specimens.Our study provides a good example of the value of museum collections in understanding biogeographic and evolutionary history, and in informing conservation management of closely related extant species. Genetic and morphological data from museum specimens of extinct rail populations were essential to outline the evolutionary pathway of populations and identify appropriate conservation recommendations for Dryolimnas on Aldabra and Madagascar, species boundaries have long been debated as it is argued that the populations must have been isolated for considerable time for flightlessness of the Aldabra population to have evolved. The surprisingly high genetic divergence and marked morphological differences of the Aldabra and Assumption subspecies from those of Madagascar, warrant the management, protection and assessment of the remaining Aldabra population as distinct from the Madagascar population. The small population size of D. [c.] aldabranus and its history of local extirpation, combined with the fact that it has evolved flightlessness and is consequently more vulnerable, increases the need for appropriate conservation management.Phylogenetic data, combined with data on morphology and behaviour, is a useful basis upon which to assess whether a population is sufficiently evolutionarily distinct from others to be treated as a separate conservation management unit. Despite morphological similarities between Dryolimnas cuvieri is currently Red-Listed as ‘Least Concern’  aldabranus much more vulnerable to extinction. A Red List status that actually applies to a widely distributed, volant and less threatened subspecies is inappropriate and could compromise conservation management  aldabranus should at least be treated as a subspecies Vulnerable to extinction, based on IUCN criteria B and D2  is now extinct. Nonetheless, it is possible that a few individuals are still present and, until this possibility is ruled out, translocation of individuals of Native North Aldabra origin to Île aux Cèdres (or Grande Terre) should be avoided. Performed prematurely, such a translocation risks extinguishing Native South Aldabra rail genetic diversity through hybridisation;Efforts to reinforce D. [c.] aldabranus subpopulations on Picard, Grande Terre and possibly also on Île aux Cèdres. Cats could easily colonise Aldabra's other islands from Grande Terre, so it is important to eradicate cats as soon as is logistically feasible on this large and remote island.It is likely that the introduction of cats caused the extirpation of the original D. [c.] aldabranus, although the effects may be limited (but not absent) as this species has been reported to be able to defend itself against, and even kill, rats [see Rats may also compromise breeding success of rats see . NeverthD. [c.] aldabranus should be considered to other islands in the Aldabra group  and Western Indian Ocean preferably only when rat- and cat-free. Translocated groups should contain individuals from both Polymnie and Malabar.Translocation of Dryolimnas, and on its colonisation history, with important implications for conservation management. The flightless D. [c.] aldabranus is clearly on a separate evolutionary trajectory from the volant D. c. cuvieri. Its evolutionary uniqueness, based on genetic and morphological divergence, warrants treating D. [c.] aldabranus as an independent conservation management unit.As the last extant flightless bird in the Western Indian Ocean, the Aldabra white-throated rail has unique conservation significance. Our research sheds new light both on the phylogeny and evolution of flightlessness in S1 Appendix(DOC)Click here for additional data file.S2 Appendix(DOC)Click here for additional data file.S3 Appendix(1) Phylogenetic tree from (DOC)Click here for additional data file.S4 Appendix(DOCX)Click here for additional data file.S5 Appendix(DOCX)Click here for additional data file.


INRA023 and CSRD247 with 16 alleles, and the smallest polymorphism was noted for thelocus D5S2 with 8 alleles. The level of observed heterozygosity was in the range OarFCB20 locus showed thehighest level of genetic variability. Excess of heterozygosity was observedat three loci; MAF065, McM042, and OarFCB20.The highest genetic distance was observed betweenKazakh Arkhar-Merino and Edilbay-1, whereas the genetic distance betweenEdilbay-1 and Edilbay-2 is the smallest using Nei's standard geneticdistance. The Edilbay-1 sheep breed possesses the largest genetic diversityamong these five populations.A total of 75 individuals from five sheep populations in Kazakhstanwere investigated based on 12 STR  markers in order to study their geneticstructure and phylogenetic relationship based on genetic distances. Thesesheep had a high level of genetic diversity. In total, 163 alleles werefound in all the populations using 12 microsatellite loci. The meannumber of alleles, effective number of alleles, and polymorphism information content (PIC) values per loci were13.4, 5.9, and 0.78, respectively. Comparing the allelic diversity betweenthe populations, the highest genetic diversity was observed in the Edilbay-1 sheep breed ( Among them, thesheep breeds which first appeared in Kazakhstan by origin and history areEdilbay, Kazakh Arkhar-Merino, Kazakh Finewool, and Kazakh fat-tailedcoarse wool. These breeds are well adapted to the various climaticconditions in Kazakhstan. Various breeds of sheep in one way or another aredifferent in terms of biological efficiency. This study aims atinvestigating the genetic diversity of local sheep breeds. The animalsstudied belong to purebred and farms' own selection and tribal cards. TheKazakh Arkhar-Merino sheep breed was investigated on Kumtekey breedingfarm, where the best sheep of this breed are kept. The Kazakh Arkhar-Merinois based on the interspecific hybridization of wild Arkhar rams with fine wool ewes of Novo-Caucasian Merino, Précoce, and Rambouillet breeds that occurred during 1934–1950. R-Kurty farm is also a breeding farm where the highlyproductive animals of the Kazakh Finewool breed are concentrated. Edilbaysheep breed is bred in Birlik breeding centre, where the purebred andhighly productive sheep of this breed are kept. The Kazakh fat-tailedcoarse wool sheep breed is the product of popular selection which haslasted many years. They are widely bred in many sheep farms all overKazakhstan. However, highly productive animals typical of this breed areconcentrated in Kabyl-Nur farm. Thus, four sheep breeds selected by usfor molecular genetic studies are, firstly, the most common sheep breeds inKazakhstan, which are well adapted to different climatic conditions ofbreeding and housing. Secondly, these breeds differ from each other in theirorigin and method of breeding, and thus they are excellent subjects forcomparative molecular genetics research. Thirdly, the specification ofbreeding farms from which sheep were studied is extremely important, sincewe use the results of the molecular genetics study of these breeds in thesefarms to improve breeding work and speed up the selection process in orderto create a highly productive breeding core in a short period of time. Sinceresearch started in 2010, the molecular database has grown, and thecharacterization of genetic diversity in farm animals has become particularlypertinent .Today, the FAO and the ISAG–FAO Advisory Group on Animal Genetic Diversityrecommend specific sets of STR  loci for genetic analyses, such as for horse,cattle, and pig breeds. Of the different types of molecular markers, STRs are suitable for studying genetic diversity because oftheir abundance – the large amount of allelic variation at each locus ishighly polymorphic, their distribution throughout the genome is random, and inheritance is codominant . In addition, STRs are able to generate information for theplanning of crossings and further selection of genotypes in genetic breedingprograms . The objective of thecurrent research was to investigate 12 STR loci based on genetic diversityin sheep herds as well as the differentiation and relationship among the number of alleles and genetic links between Kazakh sheep breeds.2Blood samples were taken from five sheep populations: two of them wereEdilbay-1, though of two various Edilbay-1 sheep herds, and threeothers were different sheep breeds in the categories Kazakh Finewool, KazakhArkhar-Merino, and Kazakh fat-tailed coarse wool. The 15 animals were chosenrandomly from each population. Genomic DNA was extracted using a commercialkit .Both the quality and concentration of DNA were verified by spectrophotometricand agarose gel electrophoresis. In this study, 12 STR primers were used, andall of them are recommended by the International Society of Animal Genetics. Amplification was carried out using a Tetrad 2thermal cycler (Bio-Rad). Polymerase chain reaction (PCR) products were attached in the ABI310 Genetic Analyser, andGeneMapper software was used to determine fragment size. The number ofalleles, effective number of alleles, polymorphism information content (PIC)values, observed and expected heterozygosities, Wright's 3INRA023 and CSRD247, whereas D5S2 showed thelowest number , and the average numberof alleles was 13.4 per locus.All examined markers were polymorphic for all the populations examined. Intotal, 161 alleles were found in five native Kazakh sheep breeds based on12 STR loci (Table 1). The highest number of alleles was 16 for markersThe effective number of alleles for each marker varied between 3.2 and 9.1,with a mean value of 5.9. The PIC values ranged from 0.65 to 0.88, with amean of 0.78. Observed heterozygosity values varied from 0.52 to 0.89, withan average of 0.73, while the expected heterozygosity values ranged from 0.69to 0.89 with a mean value of 0.81 (Table 1). Our study demonstrated a highgenetic polymorphism in the investigated sheep breeds. These estimates arehigher than those reported for other sheep breeds .The average number of alleles, the effective number of alleles, and the expected and observed heterozygosity for each breed are shown in Table 2. The averagenumber of alleles for the Edilbay-1 breed is 8.33. This analysis showed thatthere was no significant differentiation among the groups from the followingpopulations: Kazakh Arkhar-Merino (7.08), Kazakh Finewool (7.91), Edilbay-2(7.58), and Kazakh fat-tailed coarse wool (7.41). With the exception of INRA006, while the lowestMcM527. The maximum and minimum INRA006 and OarFCB20 respectively. The highest The neighbour joining tree for all samples was constructed using pairwisepopulation matrix of Nei's genetic distances in order to represent therelationships between the sheep breeds . Edilbay-1 and Edilbay-2were initially classified as sub-clusters, which were further clustered intoKazakh fat-tailed coarse wool, whereas Kazakh Arkhar-Merino and Kazakh Finewool sheep breeds were grouped around the same node. Moreover, in thefactorial correspondence analysis, the distinction of three clusters isillustrated by three axes showing variances of 37.93 %, 24.35 %, and22.49 %, respectively .4INRA023 and CSRD247, with a large number of alleles. According to Ozerov et al. (2008),the Edilbay sheep breed had the highest genetic diversity among the populationsstudied. Similarly, our analysis of the data showed that the greatestgenetic diversity was detected in the Edilbay-1 sheep breed, with a mean value ofalleles of In this study, 12 STR (microsatellite) markers were used to evaluate geneticdiversity in five populations. The analysis revealed no significantdifferences in the main genetic characteristics of the interbred population:number of alleles, effective number of alleles, and expected and observedheterozygosities. A high level of genetic diversity was observed in lociPIC value for the five populations was equal to 0.78, and allthe markers were higher than 0.5, which means all the investigated STRloci were highly informative. This value was higher than that reported forChinese sheep . Also, PIC values showed thatthe most informative markers were OarFCB20 and INRA023. However, PIC values were lowerthan heterozygosities. According to Botstein et al. (1980), PIC must bealways less than the expected heterozygosity. Aside from KazakhArkhar-Merino, in all examined populations, the average PIC value had asimilar ranking. Consequently, these markers are suitable for studying thegenetic diversity in Kazakh sheep breeds.The overall average of MAF065, McM042, and OarFCB20.The fixation index was estimated on a per locus basis, and due to negativeassortative mating, an excess of heterozygosity was found at markersMAF065 and McM042, in all loci the level of observed heterozygosity was lowerthan expected heterozygosity; therefore there were more homozygous individuals in the investigated flocks. As well as the mean expectedheterozygosity in each population being higher than that of theheterozygotes, our results showed that heterozygotic deficiency was noted inall populations. Compared to the average heterozygosity value in thepopulation, the observed heterozygotes fluctuated from In addition, to estimate the genetic variability of the studied sheepbreeds, we calculated the expected and observed heterozygosity values.Except for Previously, a study was conducted by Ozerov et al. (2008) on four sheepbreeds of Kazakhstan  using 20 microsatellite loci. As a result, it wasdetermined that all studied sheep breeds showed a high level of polymorphismin all 20 microsatellite loci and were in a state of genetic equilibrium according to the Hardy–Weinberg ratio. However, in our studies of the Kazakh Arkhar-Merinosheep breed, there were significant differences between the expected andobserved heterozygosity  between the five populations of sheepwere calculated using 12 STR loci (Table 3), which varied from 0.469 to 0.217. Theresults of genetic distance of the present study were lower than thefindings of Bai et al. (2015), who reported that the genetic distanceranged from 0.21 to 0.62 for Chinese indigenous sheep breeds, which washigher than that of Egyptian sheep breeds . The valuesof genetic distances showed that the investigated sheep populations arecharacterized as having a high range of variability of the allele pool,presence or absence of certain alleles, and differences in frequency ofoccurrence alleles. The largest genetic distance was observed between KazakhArkhar-Merino and Edilbay-1 (0.469). These two breeds were absolutely differentfrom each other originally: by phenotype, according to the different branchesof stock breeding and geographical places. Kazakh Arkhar-Merino is a meat-woolly breed with fine wool. Kazakh Arkhar-Merino is well adapted to breeding athigh altitudes, and these sheep differ favourably from other breeds inconditions of mountain pasture. In contrast, the Edilbay-1 sheep breedis classified as the coarse wool sheep from the meat-fatty category. They arewell adapted to the severe desert and semi-desert conditions of Kazakhstan.Also, Kazakh Arkhar-Merino is geographically most distant from Edilbay-1. Incomparison with other populations, a closer relationship was found betweenKazakh Arkhar-Merino and Kazakh Finewool (0.25), which could be attributedto the low geographical distance between these two populations. The same resulthas been found between Kermani and Lori-Bakhtiari Pakistani sheep (0.25),which both breed in Iran . Both of them(Kazakh Arkhar-Merino and Kazakh Finewool) refer to the meat and wool typesof sheep breeds. Kazakh fat-tailed coarse wool and Edilbay-2 had less similarity to the Kazakh Finewool than Edilbay-1.Further, to assess the genetic relationships among the population, aphylogenetic tree was constructed using the neighbour joining method based on Nei's genetic distance. The results ofthe phylogenetic tree indicated that the Edilbay-1 and Edilbay-2 sheep breedswere clustered into Kazakh fat-tailed coarse wool, which are coarse woolbreeds and have a common origin. However, Kazakh Finewool and KazakhArkhar-Merino were grouped in the same node, both being fine wool sheep,as these breeds have the same ancestral background. Likewise, these twobreeds were categorized together on the same branch of the phylogenetic treein the previous study .In addition, factorial correspondence analysis demonstrated that KazakhArkhar-Merino and Kazakh Finewool were isolated from the otherstudied populations due to the high level of differentiation and no sharing of alleles. Edilbay-2 is an admixture of both Edilbay-1 and Kazakhfat-tailed coarse wool. According to the FCA findings, this is connected withhistorical origin of these Kazakh sheep breeds.5In this study, within and among herds, the genetic diversity of five Kazakhsheep populations was assessed using 12 microsatellite markers. Based on ourresults, all five populations examined show high genetic diversity through a high effective number of alleles, a large mean number of alleles, high PIC values, and 12 completely polymorphic tested microsatellites. Moderatedifferentiation was found between Kazakh Arkhar-Merino and Edilbay-1,whereas differentiation between Edilbay-1 and Edilbay-2 was lower. Therefore,the evaluation of the results of Nei's genetic distance, neighbour joining, and FCA agreed with the historical origin of animals. As our study showed, although10 years have passed since the research by Ozerov et al. (2008), it hasbeen discovered that genetic diversity remains in sheep breeds other than Kazakh Arkhar-Merino. The main reasons for this fact are as follows.Scientists and livestock breeders have always been working on theimprovement of these breeds. The advantages of this research also includethe ability of the breeds to transmit all useful economic traits to theiroffspring. Breeding work is carried out mainly along the lines using severalherds. Of course, not only the best males, but also elite queens, areselected to replenish livestock. Lines of sheep are created according tosome outstanding qualities – precocity, weight, size of fat tail, and qualityof wool. The work consists mainly of mating animals with distant degrees ofkinship in the line. In addition, based on the data obtained, it is possibleto recommend the Kazakh Arkhar-Merino breed for the selection againsthomozygous individuals. Further, the results achieved on STR loci are proposed to beused to control and conserve the genetic diversity of native sheep breeds.