Genetic structure and distinctive features of landraces, such as adaptability to local agro-ecosystems and specific qualitative profiles, can be substantially altered by the massive introduction of allochthonous germplasm. The landrace known as “Cipolla rossa di Acquaviva”  is traditionally cultivated and propagated in a small area of the Apulia region . However, the recent rise of its market value and cultivation area is possibly causing genetic contamination with foreign propagating material. In this work, genotyping-by-sequencing (GBS) was used to characterize genetic variation of seven onion populations commercialized as ARO, as well as one population of the landrace “Montoro” (M), which is phenotypically similar, but originates from another cultivation area and displays different qualitative features. A panel of 5011 SNP markers was used to perform parametric and non-parametric genetic structure analyses, which supported the hypothesis of genetic contamination of germplasm commercialized as ARO with a gene pool including the M landrace. Four ARO populations formed a core genetic group, homogeneous and clearly distinct from the other ARO and M populations. Conversely, the remaining three ARO populations did not display significant differences with the M population. A set of private alleles for the ARO core genetic group was identified, indicating the possibility to trace the ARO landrace by means of a SNP-based molecular barcode. Overall, the results of this study provide a framework for further breeding activities and the traceability of the ARO landrace. Allium cepa L. 2n = 2x = 16) is cultivated on about 5.6 million ha worldwide  and is The landrace known as “Cipolla rossa di Acquaviva”  is cultivated and propagated by local smallholders in the municipality of Acquaviva delle Fonti , an area characterized by freshwater availability and deep well-drained soils. It produces red-colored and flattened bulbs, weighting about 500 g and displaying high solid soluble content and low pungency  14]. Th. Th14]. Genotyping-by-sequencing (GBS) is a reduced-representation sequencing method allowing to perform low-cost and high-throughput genotyping of crops with large and complex genomes ,18,19. MHere we describe the application of GBS on onion samples referable to populations commercialized as ARO and M, aiming to investigate the genetic structure of the ARO landrace and provide a basis for its further genetic improvement and traceability.https://www.doi.org/10.6084/m9.figshare.20301198, accessed on 4 September 2022).Sequencing of a 59-plex GBS library yielded 456 million good barcoded reads. The quality control procedure, including steps to filter variants as well as individuals, resulted in a variant call format (vcf) file containing 5011 SNPs and 53 individuals, which was retained and used for downstream analyses. The filtered vcf file was uploaded and is publicly available at the FigShare repository  was performed as a first non-parametric alternative to study genetic structure . With a In accordance with the results mentioned above, hierarchical clustering highlighted the occurrence of two major clusters, one mainly referable to ARO_K1 individuals, and the other to ARO_K2 and M individuals  . ExceptiST statistics ranged from 0  to 0.113 (ARO5 vs. ARO7)  . Permutapulation . Notablypulation .Population genetic analysis showed a higher number of alleles (Na), the number of effective alleles (Ne), the Shannon’s information index (I) and the observed heterozygosity (Ho) for the ARO_K1 populations, indicating higher genetic diversity. The observed heterozygosity (Ho) was close to the expected heterozygosity (He) for the ARO_K1 populations, thus determining a low fixation index (F), ranging from 0.022 to 0.057. On the contrary, the F values obtained for the ARO_K2 and M populations were considerably higher, ranging from 0.145 to 0.279 .Aiming to provide a tool for product traceability, a search was performed to identify alleles that distinguish the ARO_K1 populations from the M population. In total, 596 private alleles were identified. Private alleles occurred in most cases at low frequency ; howeverThe concept of landrace has been debated for a long time by the scientific community . Zeven  was the The reduction of agronomic performance due to inbreeding , referred to as inbreeding depression, strongly limits genetic gains in outcrossing crop species, including onion ,29,30. Hp ≥ 0.5), suggesting the possibility to implement traceability tests for the ARO landrace. In general, the results of our study indicate the possibility to exploit SNP variation to develop molecular barcodes for onion landrace traceability or protection. This might be facilitated by the availability of user-friendly technologies for high-throughput SNP detection [Genetic traceability of landraces is of great value for the valorization of typical productions, as well as their protection from frauds . Here, tetection ,33,34, tetection .Overall, this study clearly indicates the occurrence of genetic structure in germplasm marketed as ARO, which likely arose from genetic contamination of the original landrace. In addition, it delivers valuable information for breeding activities to be performed on the ARO landrace, and actions to be addressed for its valorization and protection by means of molecular tools.https://www.cipollaramatadimontoro.it/index.php, accessed on 4 September 2022). Based on interviews with smallholders, the population referred to as ARO1 was indicated to certainly descend from germplasm cultivated in the municipality of Acquaviva during the 1980s. Plants were grown under the same environmental conditions at the experimental farm “P. Martucci” of the University of Bari “Aldo Moro” .A collection of 59 individuals was analyzed in this study. Of these, 49 represent seven onion ARO populations, each one collected from a different smallholder operating in the municipality of Acquaviva delle Fonti , and 10 ApeKI restriction enzyme to prepare the DNA library and the Illumina HiSeq 2500 system (paired ends) for sequencing.Young leaves were sampled and stored at −80 °C until use. Leaves were washed three times with the STE buffer   to removTo perform SNP call and generate a variant call format (vcf) file, the UNEAK pipeline  in TASSEParametric analysis of population structure was performed with the software STRUCTURE (v.2.3.4) , consideST estimates among populations and to test the null hypothesis of no significant difference based on 999 permutations. In addition, the same software could compute several diversity indexes , number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Shannon’s information index (I)), and the inbreeding coefficient (F) associated with each population.Genalex 6.5.  was alsoGenalex 6.5. was used to search for private alleles, which only occurred in the set of four ARO populations whose ancestry was mostly referable to the cluster K1 identified by STRUCTURE analysis. In addition, the same software could derive the frequency associated with each private allele.


This Research Topic includes a collection of research papers that highlighted various genetic aspects of indigenous breeds in different countries around the globe. The topic discussed the recent advances, current challenges and latest developments in phenotypic characterization and genetics and genomics of livestock in low input production systems. The research conducted varied from Genome Wide Association Studies (GWAS) and selection signatures to estimation of genetic parameters and heritability to genetic background and genetic diversity of indigenous breeds.Sutera et al., implemented a GWAS based on DEBV to identify genomic regions putatively associated with mastitis using somatic cells count (SCC) as a proxy. Their research, which was carried out in local Valle del Belice sheep, highlighted eight genomic markers associated with somatic cell score that harboured candidate genes related to udder conformation and the immune system. These finding could prove very useful for the fate of this and other sheep breeds in the Mediterranean basin countries as the dairy sheep production there usually relies on well adapted local breeds. Another manuscript implementing GWAS was by Mancin et al. who implemented a single step GWAS to analyse Beef Traits in Local Alpine Breed. Their study revealed the diversity of the pathways and some novel genes impacting a number of beef traits. The discovery of these new pathways and genes shows the importance of genetic research in local breeds which can expand to global livestock populations. Habimana et al. conducted a GWAS to study of growth performance and immune response to Newcastle disease virus of indigenous chicken in Rwanda. Their results showed several genes implicated in body weight and New Castle disease antibody variation.The first manuscript by Verma et al. implemented this approach to discover a panel of 10 reference genes in Indian cattle populations adapted to hot arid normoxia and cold arid hypoxia environments. All together, the previous studies open the door for the discovery of causative genes that could be utilized in selection programs in these local breeds in future.An alternative approach to gene discovery is using a candidate gene approach in which a number of selected genes are selected and genotyped in a population of interest. Serranito et al. was on searching for Selection Signatures Related to Trypanosomosis Tolerance in African Goats. Trypanosomosis is a parasite that greatly impacts various livestock species including camels, cattle, sheep and goats with very little research focusing on it in goats. Therefore, examining selection signatures of innate immunity to it is of vital importance to goat production in Africa. The researchers found 33 selection signatures, 18 of which overlapped previously published research. Such findings are crucial for goat production as they could eventually lead to discovery of genes/allelic combinations of major role on immunity to trypanosome infections in goats. Another attempt to search for selection signatures and genes responsible for trypanotolerence was made by . In this article, the researchers reported several genes potentially involved in tolerance to trypansoma resistance in the Baoulé x Zebu crossbred cattle in Burkina Faso. In addition to the two previous articles, the selection signatures in West African indigenous cattle of Benin was investigated by . In this study, the results unraveled some genes related to production/economic traits in hybrid Beninese cattle and genes related to immunity and feed efficiency in local Somba cattle.Searching for Selection Signatures is another method for discovery of genes impacting traits of interest. Unlike GWAS, it relies on discovery of regions at which nucleotide variations is reduced due to the rise of a new beneficial mutation. The research article by Simčič et al.). In this article, the authors reported medium to high heritabilities for body frame and autochthonous traits and low to medium heritabilities for scored udder traits. These heritabilities are already in use in the prediction of breeding values in Cika cattle which is exemplary for other breeds in other low input systems. The second article was by Hako Touko et al. in which the authors estimated the heritabilities of antibody response to Newcastle disease vaccination impacting local chicken in Cameron. Due to the lack of a large sample size or pedigree data (common to low input systems), the authors used three alternative methods to estimate heritabilities. The first was the breeder’s equation method, the second method was the graphical method and the third was the full-sib/half-sib method. The authors reported low but similar estimates of antibody response to vaccination and a very low estimate of heritability for survival. Nevertheless, the authors reported a significant increase in antibody response when crossing cocks and hens under 1% and 3% selection intensity respectively.A key element of this article collection is research in genetic improvement of local breeds in low input systems. Such improvement is the corner stone of future utilization of these breeds and can help complement their adaptation traits with production traits of interest. The first step to any genetic improvement program is the estimation of genetic parameters of the traits to be improved by the program. Two articles in this article collection discussed genetic parameters estimation in low input systems breeds. The first article discussed the estimation of genetic parameters of type traits in first parity Slovenian Cika cows using a generalized linear model procedure . In the former, the authors analysed the sequence variation in the mtDNA control region of Dromedary Camels. Their results showed that small genetic differences exist between camel types. They reported two haplogroups shared between almost all dromedary camels with one of them being more common in African dromedary camels. In addition, in order to better characterize camel populations, the authors advise future researchers to use WGS or SNP data which is the same advise provided by Piro. In the later article, the authors reported high genetic diversity but absence of population structure in local chickens of Sri Lanka using Microsatellite Markers. In contrast to the findings reported in the previous article, this research showed high genetic diversity in Sri Lanka’s local chicken breeds which translates to a large potential for the development of locally adapted genetically improved chicken breeds. On the other hand, Kebede et al. used Species distribution models (SDMs) and Phenotypic distribution models (PDMs) to phenotypically characterize the relationship between environmental variation and phenotypic differentiation in Ethiopian indigenous chicken. Their results identified three major ecotypes that show phenotypic differentiation in addition to nine major environmental variables conducive to habitat suitability. In another study, Ouhrouch et al. used Whole Genome Sequencing (WGS) data on 87 sheep to study the genetic uniqueness Moroccan sheep belonging to the five major sheep breeds. Their study assessed the genetic diversity, effective population size, population structure and relationships between the breeds. In addition, they were able to find interesting intra-breed selection signatures. Their study showed that the Morocan breeds are not highly genetically differentiated but that they harbor high genetic diversity and effective population size which makes them a suitable reservoir for the development of adaptive traits against the threats of climate change. The study by Babigumira et al. aimed at characterizing the genetic ancestry and inbreeding levels in the local a population of smallholder pigs in Uganda using 422 pigs. The authors used porcine GeneSeek Genomic Profiler (GGP) 50 K SNP Chip to infer the genetic ancestry as well as inbreeding levels. The study showed a relatively low inbreeding in the breed and revealed a genetic background of the breed that is a mix of old British and modern pig ancestries. Pig production, which is managed mostly through low input systems, is one of the main sources of income for African small holder producers.A number of articles in this collection have explored the genetic and phenotypic background of local breeds including (The findings in the previous research articles highlight the importance of preserving local breeds for their notable adaptation to various vector borne diseases. Furthermore, identification of genetic regions/genes can help integrate them from genomes of local lower-production breeds to genomes of other breeds with higher production abilities. Therefore, this article collection will serve as a stepping stone towards the characterization, development and genetic improvement of numerous livestock in low input systems.


Vigna unguiculata (L.) Walp) is an important legume crop with immense potential for nutritional and food security, income generation, and livestock feed in Sub-Saharan Africa. The crop is highly tolerant to heat and drought stresses which makes it an extremely important crop for improving resilience in crop production in the face of climate change. This study was carried out to assess the genetic diversity and population structure of 90 cowpea accessions using single nucleotide polymorphism (SNP) markers. Out of 11,940 SNPs used, 5864 SNPs were polymorphic and maintained for genome diversity analysis. Polymorphic information content (PIC) values ranged from 0.22 to 0.32 with a mean value of 0.27. The model-based Bayesian STRUCTURE analysis classified 90 cowpea accessions into four subpopulations at K = 4, while the distance-based cluster analysis grouped the accessions into three distinct clusters. The analysis of molecular variance (AMOVA) revealed that 59% and 69% of the total molecular variation was attributed to among individual variation for model-based and distance-based populations, respectively, and 18% was attributed to within individual variations. Furthermore, the low heterozygosity among cowpea accessions and the high inbreeding coefficient observed in this study suggests that the accessions reached an acceptable level of homozygosity. This study would serve as a reference for future selection and breeding programs of cowpea with desirable traits and systematic conservation of these plant genetic resources.Cowpea ( Vigna unguiculata (L.) Walp, 2n = 2x = 22) is an important legume crop that belongs to the genus Vigna, family Fabaceae, and order Fabales, with a genome size of 620 million base pairs [Cowpea  ,17, simpThere are sequencing technology-based tools, which is next-generation sequencing (NGS), that have emerged to discover SNP markers, using genotyping-by-sequencing (GBS), which has been reported to be efficient, inexpensive, and fast developing in sequencing plant genomes ,26. In aSNP distribution per chromosome and the gene diversity parameters measured from 90 cowpea accessions are presented in The population structure of the 90 accessions was examined using model- and distance-based structure analyses. At K = 2, the first cluster consisted of 82 genotypes, of which 29% were admixtures while the second cluster contained 8 genotypes in which majority of them (62%) were admixture. At K = 3, the first cluster contained 62% of the cowpea genotypes and 68% were admixtures. The second and the third clusters consisted of 6 and 28 genotypes with 67% admixtures each, respectively . HoweverHowever, the distance-based cluster analysis generated using Nei’s genetic distance using a neighbor-joining algorithm revealed the presence of three distinct clusters in the population represented by the 90 accessions . The cluThe population genetic diversity estimates on 90 accessions were analyzed based on the four subpopulations generated by STRUCTURE and three populations generated by DARwin . SP3 revAMOVA was performed among subpopulations estimated from both model-based and distance-based populations . The resST) estimates among the subpopulations ranged from 0.103 between SP3 and SP4 to 0.239 between SP1 and SP2. The gene flow ranged from 0.8 (between SP1 and SP2) to 2.2 (between SP3 and SP4). The genetic distance among populations ranged from 0.089 between SP3 and SP4 to 0.214 between SP1 and SP2. The genetic identity (GI) ranged from 0.81 between SP1 and SP2 to 0.92 between SP3 and SP4  was observed in this study indicating 67% of the SNP loci used were fixed in the studied accessions. However, a relatively high (FIS = 0.83) was reported by Gbedevi [A moderate fixation Index , while C2 was dominated by accessions with unknown growth habits (44%). C3 was mainly dominated by the prostate (54%) growth habit type. Regarding the wide distribution of cowpea, accession was studied based on growth habit, the prostrate and erect types were more dominant than the semi-erect and climbing types. Growth habit is a morphologically important qualitative trait in cowpea production that highly affects crop yield and tillage method and further defines the shape of the plants and dictates how the plant should be harvested . Plant gST = 0.221). The results indicate high genetic differentiation between four subpopulations based on the standard guideline of Wright [ST) value of 0.423 between two major reported subpopulations of 498 cowpea accessions. However, Sarr [The AMOVA results revealed that the majority of the total molecular variation (59% and 69%) was due to differences among individuals within a population, 22% and 12% of the variation were attributed to the difference among the population, and 18% and 19% was due to variation within individuals. In the model-based approach, the among-population variation was higher than the within individuals variation. The magnitude of variations among and within populations was further quantified by genetic differentiation observed among the populations  gene bank, Pretoria, South Africa. These accessions were collected from different parts of Africa including South Africa, Tanzania, Kenya, and Nigeria. The geographic origin and growth habits of the accessions are presented in The cowpea genotypes were grown in a seed germination chamber at the Biosciences eastern and central Africa International Livestock Research Institute (BecA-ILRI) hub in Nairobi, Kenya for genotyping. Ten-day-old leaf materials were sampled from the seedlings and the leaf samples were frozen in liquid nitrogen and stored at −80 °C for genotyping. DNA extraction was done using a NucleoMag Plant DNA extraction kit from Takara Bio USA, Inc. The genomic DNA extracted was in the range of 50–100 ng/μL. DNA quality and quantity were checked on 0.8% agarose gel. Libraries were constructed according to Killian  using thA total of 90 cowpea accessions were genotyped with 11,940 SNP markers. Monomorphic and SNPs with a minor allele frequency of less than 2% were filtered out and 5864 (49%) SNPs were retained for further analysis. Genotypic data were subjected to analyses of molecular variance (AMOVA) and various measures of genetic diversity within and among inferred subpopulations using GenAlex software version 6.5 . GeneticThe Bayesian genotypic clustering approach of STRUCTURE 2.3.4  was usedThe current study revealed the existence of genetic diversity among and within the cowpea accessions analyzed and showed the effectiveness and reliability of SNP markers. The study revealed that 49% of the selected SNP markers were highly polymorphic and efficiently discriminate the tested cowpea accessions. The low heterozygosity and the high inbreeding coefficients observed among cowpea varieties indicate that the accessions reached an acceptable level of homozygosity. The model-based  and distance-based (UPGM) clustering approaches were used in this study. The model-based analysis revealed the presence of four subpopulations at K = 4 whereas the distance-based cluster analysis classified the cowpea accessions into three distinct clusters. The subpopulations identified exhibited the high level of genetic diversity and were moderately differentiated. These subpopulations could serve as heterotic groups and a relevant source of genes for future breeding and selection of diverse cultivars with different traits. Therefore, the results obtained from this study will be highly valuable for the development of new varieties adapted to diverse environments. Consequently, this study will substantially contribute to the utilization, conservation, and broadening of the use of genetic resources of cowpeas for future improvement.


Episyrphus balteatus in northern China. Our work reveals how E. balteatus migrate northward during spring–summer and exhibits return (long-range) migration during autumn. The extensive genetic mixing and high genetic diversity of E. balteatus populations underscore its adaptive capacity to environmental disturbances, for example, climate change. Pollen markers and molecular gut analysis further illuminate how E. balteatus visits min. 1012 flowering plant species (39 orders) over space and time. By thus delineating E. balteatus transregional movements and pollination networks, we advance our understanding of its migration ecology and facilitate the design of targeted strategies to conserve and enhance its ecosystem services.Worldwide, hoverflies (Syrphidae: Diptera) provide crucial ecosystem services such as pollination and biological pest control. Although many hoverfly species exhibit migratory behavior, the spatiotemporal facets of these movement dynamics, and their ecosystem services implications are poorly understood. In this study, we use long-term (16-year) trapping records, trajectory analysis, and intrinsic  markers to describe migration patterns of the hoverfly  Migration plays a key role in the evolution and life history of many organisms, with insects being the most abundant, speciose, and economically important group of terrestrial migrants . Across Hoverflies (Diptera: Syrphidae) are a speciose family of beneficial insects – deemed to be the second most important pollinators after bees . The larIn recent years, several new technologies have helped to uncover insects’ seasonal migration patterns and population genetic structure. Stable isotope analysis, molecular genetics, tethered flight mill assays, insect radar, and aerial trapping have all yielded insights into the migration behavior of hoverflies . AttemptEpisyrphus balteatus (DeGeer)  , a commo(DeGeer) . Owing t(DeGeer) .E. balteatus in China. More specifically, we conducted long-term (16-year) searchlight trapping on Beihuang (BH), a small isolated island with sparse vegetation in the center of the Bohai Strait, to clarify whether E. balteatus engages in long-range migration and to describe the ensuing migration patterns. Second, we deployed backward trajectory analysis and stable isotope analysis to infer the E. balteatus migration routes and source areas. Third, we employed a population genetics approach to compare the genetic makeup and demographic history of migrant and field-collected individuals throughout China. Fourth, we described E. balteatus host–plant associations by identifying the pollen grains attached to hoverfly bodies. Lastly, we paired molecular gut content analysis with high-throughput sequencing (HTS) to investigate the spatiotemporal distribution of its (flower) host plants across a broad geographic range. As such, our work characterized E. balteatus migration behavior and captured its broader ecological relevance, for example, in terms of flower visitation networks, pollination, or natural biological control services.In northern China, numerous insect species annually undertake (two-way) migration across the Bohai Strait, influenced by the East Asian monsoon cycle . In thisE. balteatus larvae on Beihuang (BH) island. Yet, night-time trapping consistently yielded E. balteatus adults from late April to October   , with pe7.9 days .E. balteatus migrants on BH was identified through backward trajectory analysis using the HYSPLIT model. For ‘mass migration’ events over 2003–2018    . By compradients , seasona< 0.001) . IsotopeE. balteatus genetic diversity and population structure using one mitochondrial DNA gene (Cytb) and two nuclear DNA genes . Upon analysis of 670 field-collected specimens and 133 light-trapped individuals (representing a respective 16 and 2 populations), high haplotype diversity and low nucleotide diversity was recorded (π) from 0.000110 to 0.00536 , respectively. Conversely, the concatenated nuclear gene possesses improbably high haplotype diversity and low nucleotide diversity. Up to 145 haplotypes were detected among 260 individuals with Hd ranging from 0.810 to 1  and π ranging from 0.00392 to 0.01384 .To gain genetic evidence of its regional migration, we described recorded . Based uCytb and 33–88% for nuclear genes  and unimodal distribution for both markers . It should be noted that most plant hosts were identified from one single hoverfly, with only few adults carrying pollen from different plant species.Morphological characteristics and barcode markers were used to identify pollen species attached to migrating entified . Out of E. balteatus bodies equally exhibited seasonal variation. Specifically, 29 pollen species were identified from spring–summer migrants, with Taraxacum mongolicum, Ailanthus altissima, Amorpha fruticose, and Chenopodium giganteum the most common plant hosts . Conversely, 17 pollen species were identified from autumn migrants, with Artemisia L., Chrysanthemum zawadskii and Ambrosia trifida accounting for a 82.2% total carrier rate  and April (17%). Also, the identity of pollen grains that adhered to ier rate .Citrus L., Sedum japonicum, or Euonymus myrianthus endemic to central and southern China hinted at migration origins in south-central China. Conversely, during autumn, the presence of C. zawadskii mirrored potential migration origins in northeastern China  that were consumed by E. balteatus adults.Under laboratory conditions, a polymerase chain reaction PCR-based assay was developed to identify the plant species that are consumed by E. balteatus guts for up to 9-day postfeeding, the maximum digestion time in our experiments. Moreover, qPCR methods further yielded standard curves to quantify the degradation of the plant DNA through digestion, and a negative exponential equation to compute the time to reach a 50% detection probability  in bability . DetectiE. balteatus migrants on BH and 436 field-collected individuals at 19 different locations in China were elucidated. MiSeq paired-end sequencing of the ITS2 gene yielded 3,348,773 raw reads from 616 samples. After quality filtering and reference-based chimera removal, a total of 2,952,448 sequences remained, ranging from 100 to 28,582 sequences per sample. These valid reads were clustered into Operational Taxonomic Units (OTUs) for further analysis (Asteraceae (84), Poaceae (37), Apiaceae (21), and Fabaceae (21) (count at genus level) the dominant families accounting for 38.0% of all plant taxa  or Monocotyledons . Woody plant hosts were more common than herbaceous ones  ,  diet profiles of 180 light-trapped analysis . Accordiant taxa . Further 0.0001) . These rApiaceae, Poaceae, Asteraceae, Brassicaceae, Fabaceae, Ranunculaceae, and one genera Brassica observed in all of the populations across different geographical sites and sampling times , we further assessed how sampling time did affect the primary (plant) foraging resource. The results showed that the overall plant community composition exhibited important within-year variability, as reflected by the host-associated community separation visualized in the principal coordinates analysis (PCA) profile and the significance test based on analysis of similarities , Flueggea and Maclura became more common pollen hosts; during autumn (September–October), Ajania and Artemisia was the most represented species  dispersal patterns of the widely distributed Helicoverpa armigera and natural enemies such as the dragonfly Pantala flavescens  journey over sea. Further field observations or manipulative assays  wind currents for their migratory movements . In our  species . On the E. balteatus over hundreds of kilometers. Specifically, we showed how E. balteatus migrates between agriculture-dominated regions of Northern China and Northeast China. Hoverflies thus disperse northwards in prevailing southerly winds during spring–summer , followed by a return migration in prevailing northerly winds during autumn (August to October). Similar achievements have been made for other insects in the study area  are added. Hence, follow-up studies are needed to generate finer-resolution migration patterns. Nonetheless, considering how migrant hoverflies involve in the transport of energy, nutrients and biomass, pest regulation, and pollen transfer at a macroscale  conservation or pest management interventions . Howeverudy area  but alsoudy area . Yet, wecroscale , our worCytb and concatenated nuclear gene, all our population genomics analyses of field-caught E. balteatus from different geographical locations and BH migrants uncovered high haplotype diversity and low nucleotide diversity; a distinguishing feature of migratory species. Meanwhile, the lack of a pronounced genetic differentiation and genetic structure has been recorded for other hoverfly populations  while conventional molecular markers proved to be effective for E. balteatus genetic analyses, NGS can yield a more in-depth characterization of genetic structure and evolutionary history.Population genetics studies are routinely used to assess adaptive capacity of different organisms including insects, but can also characterize migratory movements through an ecoevolutionary lens . Here, aulations  and refl species . Elevatebundance . NeverthCitrus spp.) were routinely associated with spring–summer migrants. Meanwhile, pollen from C. zawadskii  was present in autumn migrants. As several E. balteatus individuals transported pollen grains over long‐distance flights, they likely assume a prime role in transcontinental pollination and (insect-mediated) gene flow between geographically disjunct plant populations. Moreover, our work also identified the main  foraging resources for E. balteatus. For example, spring–summer migrants relied extensively upon T. mongolicum, A. altissima, A. fruticosa, or C. giganteum, while C. zawadskii and A. trifida were frequently observed in autumn migrants. As these species may contribute to E. balteatus population build-up, these plant resources deserve further attention in nutritional ecology studies.Upon flower visitation, pollen is regularly deposited on insects’ bodies and carried during flight . Pollen E. balteatus guts. For a  set of plants, this method permitted detecting DNA in larval guts for up to 9-day postfeeding and comparing (plant- or herbivore-specific) consumption rates through qPCR. With the advent of the NGS approaches, molecular gut content analysis has become an exceptionally powerful tool – allowing high-throughput analysis of samples to simultaneously reveal multiple trophic linkages  between hoverflies and their host plants. Future work can use these new methods to assess flower visitation networks, define the coevolutionary processes between hoverflies and their host plants, and to ultimately design strategies to bolster hoverflies’ ecosystem service delivery. Aside from confirming the role of E. balteatus as a key pollinator of wildflowers  at BH island . With a size of approx. 2.5 kmistances . High-alistances . This liE. balteatus migrants that were trapped on BH island during spring–summer (April to July) and autumn (August to October). First, for trajectory simulation, we arbitrarily selected dates during 2003–2018 in which more than 40 E. balteatus individuals were caught . A total of 76 ‘mass migration events’ were thus identified, including 42 and 34 in the spring and autumn migration period, respectively  simulation model in MeteoInfo software (version 1.3.3)  , the BH cordings , simulatD) has been successfully applied  are efficiently used as endogenous markers. For migratory insects including hoverflies, the hydrogen isotope deuterium , Middle-Lower Yangtze Plain , Northern Region , and Northeastern Region   . Each saE. balteatus specimens were removed with dissection scissors and subsequently sent to the Stable Isotope Mass Spectrometry Facility, Chinese Academy of Forestry  for hydrogen isotope (δD) measurements as per 2H:1H) of the combusted wings was measured using a Flash EA 1112 HT Elemental Analyzer  and Isotope Ratio and Mass Spectrometer . Calculations were done using the formula δ2H‰ = (Rsample/Rstandard −1) × 1000 in which R is the abundance ratio of heavy isotope to light isotope, namely 2H/1H. The laboratory error was estimated to be ±2 ‰. Results are expressed in typical delta (δD) notation, in units of per mil (‰), and the relative standard of δ2H was the Vienna Standard Mean Ocean Water (VSMOW).As wing tissue is not part of the active metabolism after adult eclosion, it is generally used for isotope analyses . AccordiE. balteatus genetic diversity and population structure using one mitochondrial gene and two nuclear genes.Population genetics studies are routinely used to assess adaptive capacity of different organisms including insects; the resulting data equally reveal migratory movements within an ecoevolutionary perspective . In thisE. balteatus adults were collected. These included 133 long-distance migrants that were caught in BH searchlight traps, and 537 individuals collected using sweep-net sampling at 16 sites ] and two nuclear genes  were chosen as molecular markers. Primers were used as previously described  gene eLife. Your revised article has been evaluated by Meredith Schuman (Senior Editor) and Youngsung Joo (Reviewing Editor).Thank you for resubmitting your work entitled "Windborne migration amplifies insect-mediated pollination services" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below.Essential revisions:Please address these three points in your revision and response letter. The individual reviews are provided for your information and will give details to guide your response to the essential revisions, but you do not need to provide a point-by-point response to the reviews – only to the essential revisions.1) Please elaborate on figures 8 and 9 in the results. Currently, even experienced insect migration researchers are still having some difficulty with these figures, so any extra clarification you can provide regarding the take-home message of these figures and their relevance to the story would be helpful. Alternatively, these figures could be moved to supplementary material and briefly summarized in the text.2) Describe the geographical and biological background of Beihuang Island in the introduction, as the Materials and methods have been moved to the end of the paper.Reviewer #1 (Recommendations for the authors):I would like to make one more comment before the acceptance. It is necessary for the introduction to describe the geographical and biological background of Beihuang Island as the Materials and methods have been moved to the end of the paper.Reviewer #2 (Recommendations for the authors):The authors have done a fine job in implementing some of the changes that I suggested – most importantly, I was concerned about the long trajectory durations and the assumption that nocturnal flight was the norm, and so I am pleased that they have made important changes in the manuscript with respect to these points.In general, the paper is written to a high standard throughout, and contains some extremely interesting data. The study represents an exceptionally detailed and thorough examination of an insect migration system, and is very worthy of publication.I still feel  that perhaps too much material is presented, as some sections  are not explained clearly enough for me – but this is perhaps something for the editorial board to make a decision on. Essential revisions:Please respond only to these essential revisions, which are determined from the consultative review process, in your response letter and your revision. In doing so you may refer to relevant comments from the individual reviews.1) None of the reviewers are convinced that hoverflies migrate at night on purpose, and suggest that other factors may contribute to their behavior at night. According to Reviewer 2 (major comment 2) and Reviewer 3 (comment ii), the nocturnal migration of the hoverfly can possibly be explained by the geographical location of the trap (an island): specifically, the hoverflies likely start to migrate during the day but, at the end of the day, find themselves over water and so must keep flying until they reach land (the island). Please address this concern in your revision.We agree with the reviewers’ perspective and have now removed reference to a “dual migration strategy” from the Abstract section. Furthermore, we have expanded our discussion on this topic -according to reviewers’ comments- in lines 312-328.2) As a result of the species' diurnal activity, a flight lasting up to 24 hours is highly unlikely. A flight lasting no more than 12 hours is a more likely maximum duration, with flights typically lasting no longer than eight hours (please see Reviewer 2 major comment 3). Please address this concern and revise your trajectory analyses accordingly.We are grateful for these valuable comments. We have now revised our trajectory analyses and recalculated backward trajectories every hour from 10:00 to 22:00, by setting the max flight duration to 12 h.3) Many interesting outcomes are described, but insufficient explanations are provided . The data should be discussed in more detail. A large part of the present manuscript was devoted to the methods section, despite the fact that the manuscript was already quite long. The main text of eLife consists of about 5000 characters, excluding the methods part, so there is ample space for a detailed description of the results and their discussion. All three reviewers provide suggestions for how to focus the text, and for which aspects need more presentation in the manuscript. Please substantially revise the manuscript along these lines and explain these revisions in your response letter. In doing so it is not necessary to follow the specific recommendations of all reviewers, but you must convincingly address the general concern that the results are not sufficiently presented and discussed.We wish to thank all reviewers for these comments. As suggested, we have now carefully addressed all concerns and adapted our manuscript accordingly. Aside from addressing the specific recommendations of individual reviewers, we have also attempted to improve the overall presentation (and discussion) of our key findings.4) Along the same lines, the figures, especially Figures 4-8, should be revised substantially in terms of data presentation. These present too much information that cannot be distilled into solid take-home messages, and many of the subfigures are auxiliary. Furthermore, the data from different regions are not seasonally aligned, so they cannot be compared with the data from BH. Please substantially revise the presentation of the data in the figures and explain in your response letter how this has been revised to make each figure coherent, and support rigorous conclusions. Please see suggestions especially from Reviewers 1 and 3. Again, in doing so, you do not need to respond to each individual reviewer comment.As suggested, we have reworked the Results -and specifically Figures 4-8. We have also strengthened the overall presentation of the data e.g., by also amending other figures. Below, we provide more detailed information on how specific elements of the Results section were revised.Reviewer #1 (Recommendations for the authors):FormatThe main text of the article should usually be structured and ordered as follows: Introduction; Results; Discussion; Materials and methods (or Methods); Acknowledgements; References; Figures with the corresponding legend below each one; and Tables.We have now changed our article structure as suggested.Figure 1.Migration fluctuations were quite strong each year. Is there any explanation for this? Further, the authors suggest that this fluctuation is related to outbreaks of aphids, but no data or references are provided. Please provide further details.In the initial draft of our manuscript, we discussed the  determinants of the (strong) inter-annual fluctuations in migration patterns in the second paragraph of the Discussion section. However, in order to clarify this further to future readers, we now discuss this topic in further detail in lines 291-311.Figure 2.According to the literature, hydrogen isotope data should be interpreted in relation to elevation. Therefore, I believe it is necessary to present elevation information for each place and interpret it accordingly.We agree with reviewer #1 that elevation does affect the hydrogen isotope ratio. However, when sampling is carried out over a relative narrow elevational range, its impact on isotope ratio is rather small. As all our sampling was conducted at low elevation (<1000 masl), we have opted not to include elevational data in Figure 2A.Figure 3.The neutrality test indicates that PE and SH can be isolated from other populations. However, the authors did not interpret the details of the neutrality test. In addition, although the authors provided regional information, Figure 3D is still difficult to comprehend for those who are not Chinese. It would be easier to understand if each circle was positioned on a map of China. Lastly, I think Figures 3A and B should be included in the supporting information.We agree with the reviewer’s assessment. We have opted to maintain Figures 3B and D in the main body of the manuscript, while transferring Figures 3A and C to the Supplementary Information.Figure 4.It is well known that hoverflies are generalist pollinators. Therefore, the results showing that hoverflies carry various pollen grains are not considered significant. It would be useful to include a graph showing whether the seasonal migration pattern, which is the take-home message of this study, is well expressed in plants carried by migrants. Particularly, the author discussed that it is possible to estimate whether the hoverfly has passed through a specific ecological zone based on plants that only inhabit a particular geographical area. However, no data or references are provided for L501 – 506. Please provide further details.We are grateful to reviewer #1 for this excellent suggestion. As recommended, we have now added a graph showing the extent to which seasonal migration patterns are mirrored in hoverfly pollen loads. We equally extended our discussion on these findings and analysis in lines 300-500.Figure 5,6,7,8.Since each region has a different sampling period, the data appear to be incomparable. For instance, the seasons surveyed in the CD region appear to be longer than those in other regions. I am not sure if this interpretation is correct since the vegetation structure can change according to the season. Therefore, I recommend focusing on the data from BH. It would be helpful if you could provide a correlation between the phenology and spatial distribution of migrants collected from BH (or CD?) as they change by season.As suggested, we have carried out further detailed analyses of the data from BH.Reviewer #3 (Recommendations for the authors):Figure 1A, the circular histogram of annual catches looks very strange, I think it should be better to show in simple histogram with cartesian coordinates.We are grateful to reviewer #3 for this valuable suggestion. As indicated above, we have now converted the rose diagram to a simple histogram.Figure 2B could be removed into the Supplementary, and the sites in Figure 2A also don't need be labeled.As this work aimed to analyze the migrants collected at BH, we believe that it is important that keep Figure 2A in the main body of the manuscript. Doing so should improve the overall flow of the paper and further accentuate the significance of our work.Figure 3 is very complicated, I couldn't catch the main idea of this figure. I am not expert on molecular or genetics studies. For me, I am very interesting on the geneflow, and I think Figure 3B and 3D are the most important. I really hope the paper can give more explanation, why the pattern from mitochondrial and nuclear was different? Why the general migration trend towards the Yangtze basin?As indicated above, we have moved many data  into the Supplementary Information section. We are hopeful that the (multi-panel) figure has thus been simplified. We further emphasize that other studies have also reported (similarly) different patterns for mitochondrial vs. nuclear analyses; we clarify this for future readers in a revised Discussion section (see lines 362-397).Figure 4 and 5 could be merged together. Firstly, I don't think it is necessary to show all pollen pictures of 46 species. Secondly, map in Figure 5A and 2A is very similar, is it necessary showed it again and again? Third, Figure 5B did not showed very important information and might be better to move it to the Supplementary.We are hesitant to merge Figure 4 and 5, as they represent markedly different findings . To help future readers with the interpretation of both figures, we have (slightly) amended Figure legends. Meanwhile, as per reviewer #3’s suggestion, we have moved Figure 5A -B to the Supplementary Information.Figure 6-8, only a very short paragraph described these three large figures. I think each figure should be simpler, and need more description and more explanation.As suggested, we have now made substantial revisions to both figures. We also opted to incorporate more information on both figures in the Results  and Discussion sections .The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below.Essential revisions:Please address these three points in your revision and response letter. The individual reviews are provided for your information and will give details to guide your response to the essential revisions, but you do not need to provide a point-by-point response to the reviews – only to the essential revisions.1) Please elaborate on figures 8 and 9 in the results. Currently, even experienced insect migration researchers are still having some difficulty with these figures, so any extra clarification you can provide regarding the take-home message of these figures and their relevance to the story would be helpful. Alternatively, these figures could be moved to supplementary material and briefly summarized in the text.We agree with the reviewers’ perspective. To avoid confusion among future readers, we have now transferred Figures 8 and 9 to the Supplementary Information.2) Describe the geographical and biological background of Beihuang Island in the introduction, as the Materials and methods have been moved to the end of the paper.We are grateful for this valuable comment. We now add this information in the revised draft of our revised manuscript (lines 90-91).


Hordeum vulgare L.) cultivars, which corresponded to 120 years of this crop’s breeding in Poland. The analysis was based on 11,655 DArTseq-derived SNPs evenly distributed across seven barley chromosomes. Five groups were assigned in the studied cultivars according to the period of their breeding. A decrease in observed heterozygosity within the groups was noted along with the progress in breeding, with a simultaneous increase in the inbreeding coefficient value. As a result of breeding, some of the unique allelic variation present in old cultivars was lost, but crosses with foreign materials also provided new alleles to the barley gene pool. It is important to mention that the above changes affected different chromosomes to varying degrees. The internal variability of the cultivars ranged from 0.011 to 0.236. Internal uniformity was lowest among the oldest cultivars, although some highly homogeneous ones were found among them. This is probably an effect of genetic drift or selection during their multiplications and regenerations in the period from breeding to the time of analysis. The population genetic structure of the studied group of cultivars appears to be quite complex. It was shown that their genetic makeup consists of as many as eleven distinct gene pools. The analysis also showed traces of directed selection on chromosomes 3H and 5H. Detailed data analysis confirmed the presence of duplicates for 11 cultivars. The performed research will allow both improvement of the management of barley genetic resources in the gene bank and the reuse of this rich and forgotten variability in breeding programs and research.This study was undertaken to investigate the diversity and population structure of 83 spring barley ( The International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA) in 2001 defined cultivar as “a plant grouping, within a single botanical taxon of the lowest known rank, defined by the reproducible expression of its distinguishing and other genetic characteristics” [The advances in plant breeding achieved in the 20th century have had a tremendous impact on the agricultural landscape and have contributed to improving global food security through significant increases in crop productivity . A milesBarley breeding has focused on improving yield and biotic and abiotic stress tolerance. Malting quality is also important due to barley’s use in the brewing industry. Various traditional breeding methods have been employed, i.e., selection , haploid and doubled haploid production, mutation, single-seed descent (SSD), compound crosses, backcrossing, interspecific and intergeneric crosses. Male sterile-facilitated recurrent selection (MSFRS) and diallel selective mating system (DSMS) have also been used, which facilitate breakage of existing linkage blocks and expansion of the gene pool by providing large amounts of genetic diversity into barley cultivars . Due to Using molecular biology tools in breeding has significantly facilitated and accelerated the identification of genotypes that determine a specific and desired phenotype. The molecular characterization of preserved collections performed in gene banks helps in the preliminary identification of germplasm potentially applicable to current breeding programs. This is crucial, especially where there is a fragmented structure of the breeding companies producing cultivars for the local market, which usually do not have the financial resources and laboratory infrastructure to perform their own rapid screening of gene bank collections to identify components for crossbreeding. However, it is essential to provide open access to genetic data.The beginning of spring barley breeding on Polish territory dates back to the end of the 19th century. Beginning in 1870, breeding stations and companies were established in the partitioned Polish territory. Initially, breeding was dominated by cultivars selected from elite materials imported from abroad, landraces and ecotypes. Barley breeding in Wielkopolska, which at that time was part of the Prussian partitioning, was carried out by Hildebrand, Kirhoff and Stiegler. Their cultivars were widely grown on Polish lands and used in further breeding work. At the beginning of the 20th century, Polish breeders started to work on components and cultivars from Moravia (now the Czech Republic) . As earlHere, emphasis was placed on investigating changes in the gene pool of the Polish spring barley cultivars collection during 120 years of breeding based on the analysis of DArTseq-derived SNPs. For five breeding periods, both the size of gene pools, their structure and internal level of diversity were assessed. Traces of targeted breeding were examined along the chromosomes. The level of heterogeneity within the studied cultivars was also estimated. The analysis presented here also provided an opportunity to verify and identify duplicates in the germplasm collection. The DArTseq results also enabled the core collection to be selected. Therefore, the results will improve the efficiency of collection management and its use in research and breeding.Sequencing yielded over 75,000 SNP loci, from which loci with low reproducibility (RepAvg ≤ 0.95), low call rate , and low minor allele frequency (MAF < 0.01) were removed. As a result, approximately 85% of the loci obtained were removed, and loci, 11,655 in number, that met all quality parameters were used for analysis. The distribution of loci on chromosomes before and after filtering was also checked. Filtering did not affect the uniform distribution on chromosomes, which ranged from 10 to 15%. However, the proportion of loci with unknown chromosomal location decreased by 2% compared to the raw data. The highest number of loci analyzed was located on chromosome 2H (1695) and the lowest on 1H (1150). On average, 1 bp per 470 Kbp in the genome was analyzed. Loci distribution along each chromosome showed a similar pattern, i.e., the number of studied loci was higher at the ends of chromosomes and decreased towards centromere .The frequencies of transitions  and transversions (other variants) among SNPs were 60.2% and 39.8%, respectively. On chromosome 3H there were significantly less purine transitions and significantly more pyrimidine transitions compared to the other chromosomes. Among the transversions, their significantly decreased frequency was observed on 1H (A > C) and 3H (A < T), and a simultaneously increased frequency on 7H (C > A) .The polymorphism information content (PIC) ranged from 0.02 to 0.49 with mean 0.19 and median 0.13. Over 40% of loci had PIC below 0.1 b. BetweeThe coefficients of variation such as observed heterozygosity (uHo), expected heterozygosity (uHe) and fixation factor (F) were calculated for the studied material. The mean values of these were 0.058, 0.197 and 0.706, respectively. The mean uHo for 3H, 5H and 6H (~0.06) was significantly higher than for the other chromosomes (~0.05) . The meaThe analysis of diversity coefficients  in groups of cultivars, assigned based on the period of breeding, showed the presence of significant differences . HeterozAnalysis of the diversity coefficients in relation to the period of breeding and chromosome showed that the pattern of changes in the level of uHo was in most cases consistent with the main pattern . The divBecause DArTseq analysis was conducted on pooled samples, where each cultivar was represented by eight seedlings, it was possible to estimate the level of intrinsic heterogeneity of the cultivars studied. Barley is a self-pollinating species; therefore, heterozygous loci are due to the presence of different genotypes in the sample. Thus, it can be assumed that the heterozygosity observed reflects the heterogeneity of the cultivar. Within 83 tested cultivars Ho ranged from 0.011 (‘Kazimierski’) to 0.236 (‘Cesarski Stieglera’) . In the The number of unique alleles was also compared among the groups . As a thFrom the perspective of individual chromosomes, the greatest magnitude of change was in chromosome 5H . During An analysis of genetic distance showed that the lowest distance occurred between the two accessions representing the “Klimek” cultivar, and the highest between ‘Mazowiecki’ and ‘Stratus’ . Low dis, ‘Mobek’ and ‘Gwarek’) exhibit greater similarity to the group of recent cultivars than to cultivars bred in the same period. Among the most recent cultivars, those bred in Poland display a link to historical domestic materials. Foreign cultivars, on the other hand, show some distinctness. Polish cultivar ‘Podarek’ is the most genetically similar to foreign cultivars, especially to ‘Alianz’ and ‘RGT Planet’. The 3D plot also clearly shows the distinctiveness of the five accessions. Among them, the outermost, i.e., ‘Klimek’ and ‘Mazowiecki’, are multi-row. The other two are ‘Polo’ and ‘Start’. Both are two-row, like the rest of the tested cultivars, but they originated from crosses of foreign cultivars.Principal coordinate analysis (PCoA) performed for 83 spring barley cultivars indicated that the first three axes account for 32.69%, i.e., 13.81%, 10.69% and 8.19% of the variation, respectively . GraphicAnalysis of Molecular Variance (AMOVA) performed for 83 spring barley cultivars assigned to five breeding periods showed that most of the variation occurred within the groups (91%), and only 9% was inter-group variation.The admixture model in the STRUCTURE software  was implGenomic regions involved in differentiation of cultivars bred before 1945 and after 2000 were revealed by plotting FST values for all loci with known locations in the genome . RegionsIn the studied set of 83 spring barley cultivars, as many as 31 accessions had passport data indicating that they appeared to be duplicates or even triplicates of cultivars. These accessions were submitted to the gene bank in different years. One of the aims of this study was to verify whether these accessions were indeed duplicates. For final verification, an identity by descent (IBD) analysis was performed  and its An advanced maximization strategy through a modified heuristic algorithm (A*), which is complete and optimal, i.e., it finds a path if only one exists, and the shortest path, was used to identify the minimum group of cultivars representing the full diversity. Out of the studied 83 cultivars, a set of 50 that should form the core collection was extracted. The cultivars are marked in Described in this paper, the analysis of 83 spring barley cultivars representing 120 years of Polish breeding is the next step towards a molecular characterization of the collection conserved at NCPGR using high-resolution and genome-wide genotyping via the DArTseq method. This is a direct continuation of the study by Dziurdziak et al. ,26 in whThe analyzed loci, relatively uniform, represented all barley chromosomes, and their proportion and density was consistent with previous results obtained by DArTseq for barley . At the The distribution of the analyzed loci along chromosomes, i.e., their high frequency in the distal parts of chromosomes and low or complete absence in the centromeric and pericentromeric regions, was also observed in previous studies on barley, durum wheat, and soybean ,25,37,38The analysis showed the presence of all possible SNP types in the studied cultivar set. The number of transition-type SNPs was 1.5 times higher than the transversion-type. An excess of transversions was also observed in previous studies involving NGS technology for cowpea, wheat, rice, barley, and common bean, among others ,43,44,45Polymorphism of the examined loci, determined by the PIC coefficient, was slightly lower in the cultivars than in the landraces previously studied . HoweverFor thousands of years, since their domestication, crops have been cultivated as populations with a complex genetic structure. Selection occurred on farms either as a result of human efforts or as a result of pressure from local ecogeographic conditions. This resulted in a differentiation between populations and the formation of landraces ,50. The Looking ove4 120 years of barley breeding in Poland it is clearly visible that the average variability within old cultivars is almost three times higher than in the group of modern cultivars, which are very uniform. Breeding-related selection is even more pronounced when the results obtained here are compared with the previous ones for landraces. Even the most internally differentiated cultivar, i.e., ‘Cesarski Sieglera’ (Ho = 0.236), is almost twice as less heterogeneous than the Polish landrace PL503844 (0.422) . Thus, iBased on the results obtained, no loss of genetic diversity was observed between the oldest and the newest cultivars studied. However, a detailed analysis of changes in allele frequency clearly indicated genetic erosion. In the course of breeding, about 600 alleles were lost from the gene pool of barley cultivars over the years. They have been preserved only thanks to the activity of the gene bank. Gradually, during breeding, ’old’ unique alleles were driven out from Polish cultivars and replaced by new allelic variation. As many as 11% of the 11,655 loci examined have completely different alleles in the group of the oldest and the newest cultivars. On the basis of the few pedigree data, we can state that alleles representing the native gene pool from landraces occurring in Poland and the Czech Republic were almost completely suppressed in breeding programs. This result also indicates that researchers should be very cautious about the results of the analysis of genetic diversity in the context of changes over time.Genome-wide DArTseq analysis provided an opportunity to evaluate changes in the genetic structure of spring barley cultivars bred in Poland. Both PCoA and STRUCTURE showed the merging of consecutive groups of gene pools. Breeding in Poland follows European trends, so it may be assumed that changes in population structure reflect a breeding focus on increasing yield and, in recent years, also on increasing resistance to pathogens. A gradient of variation and gradual targeted shifts were also observed in earlier studies on barley ,57,58.Mlg, a powdery mildew resistance gene in the gene-dense pericentromeric region [Thanks to the knowledge of the barley genome sequence and the mapping of DArTseq data to it, it was possible to determine the chromosomal localization of the analyzed loci. This allowed observation not only of the changes in genetic diversity in time, but also to what extent this affected individual chromosomes. In general, for most chromosomes there was the same pattern of change over time, i.e., a decrease in observed heterozygosity and an increase in inbreeding along with breeding progress. Comparison of the polymorphism level of loci along chromosomes in cultivars representing extreme breeding periods allowed detection of regions showing a lack of variation. These regions did not change during 120 years of breeding and were located in the centromeric and pericentromeric fragments of chromosomes 1H, 2H, 4H and 7H. Interestingly, in landraces of spring barley, such “empty” regions were observed at 1H, 2H and 4H  and, in c region ,60, whilc region ,64,65,66Rph2 [FST analysis enabled identification of regions in which, during breeding, different alleles were fixed compared to the oldest cultivars. These regions occurred mainly on 5H. Their presence in the pericentromeric region was also found in modern European cultivars . The fixRph2 ,67. The Rph2 . Wild-tyRph2 . DeletioRph2 . It was Rph2 . AccordiRph2  study, VRph2 ,74. The Rph2 ,76,77,78DArTseq analysis will also enable improved management of the germ plasm collection. On the one hand, verification or identification of duplicate accessions was performed, and on the other, a core collection was selected. In a group of 74 cultivars stored in the Polish gene bank, for 15 cultivars, there were two or even three separate accessions.Duplicates in gene banks arise when, by mistake, a cultivar or other type of accession becomes added to a collection multiple times . Here, dImproved barley collection management will also be provided by the selected core collection. The idea behind the establishment of core collections is to facilitate scientists and breeders in using the genetic resources stored in germplasm collections . This alFrom the spring barley collection held at the National Center for Plant Genetic Resources (NCPGR), 74 accessions classified as advanced/improved cultivars were selected and analyzed. In addition, nine cultivars that are currently cultivated and have not yet been accessioned into the gene bank collection were included in the analysis .For each investigated cultivar, information about the period and place of its breeding and the time of its entry and presence in the official register of cultivated varieties, maintained by the Research Centre for Cultivar Testing (RCCT), was collected. Data for historical cultivars were obtained from Arseniuk et al.  and for Seeds, were obtained from long term storage of NCPGR or directly from breeding stations, were sown in a greenhouse in a substrate dedicated to planting seeds. From eight, random seedlings in the second leaf stage, the middle part of the second leaf about 10 mm long was collected. A modified CTAB protocol ,82 was uDArTseq results in a form of a table containing codominant single nucleotide polymorphisms (SNPs) were transformed into a binary matrix. Each locus was recorded as two lines where homozygotes were denoted as 1/1 or 0/0 and heterozygotes as 1/0. In the first step the array was filtered according to reproducibility (RepAvg ≥ 0.95), call rate , and the minor allele frequency (MAF > 0.01).Further preliminary analysis included determination of the proportion of polymorphic loci and calculation of polymorphic information content (PIC), observed (Ho) and expected heterozygosity (He), and inbreeding coefficient (F) according to the formulas published in Dziurdziak et al. .The distribution of the investigated loci on the chromosomes and PIC, Ho and F along the chromosomes were also assessed using the sliding window method with 500 kb intervals at 250 positions for each chromosome.10) was performed [Values of variation coefficients were calculated for groups of cultivars using a formula excluding the effect of sample size. Analysis of variance ANOVA and Tukey’s post hoc test were used to compare the degree of variation. The level of allelic richness (AR) was assessed based on rarefaction method. Analysis of molecular variance AMOVA was also performed. The Wright’s FST parameter was used to estimate genome wide group differentiation, and to increase plot resolution transformation by rising FST to the 10th power  was performed. Moreover, the identity by descent (IBD) was estimated for all pairwise comparisons among the accessions. Duplicates were defined as having IBD > 0.95 among accessions.The final step of the analysis was to perform clustering based on the Bayesian model to analyze the genetic structure of examined accessions. In order to obtain the most probable value of K, a search was conducted in the range from 1 to 16 with six independent repetitions per K for cultivars analysis, whereas analysis of the compiled cultivars and preexisting landraces results was performed for K up to 11 with six independent runs/K. A LINUX cluster hosted by the Interdisciplinary Centre for Mathematical and Computational Modelling at the Warsaw University was used to run the analysis of batch files. The number of clusters was determined based on the posteriori data probability for a given K and ΔK  and the A core collection was extracted using the advanced M strategy implemented through a modified heuristic algorithm (A*).The above mentioned analyses were performed using Microsoft Excel 2016, XLSTAT Ecology , GenAlEx 6.501 , HP-RAREThis study showed that the gene pool structure of spring barley cultivars has changed significantly during 120 years of breeding in Poland. Many alleles have been displaced and replaced by new ones. These changes were associated with breeding priority evolution over time. Traces of directed selection are particularly visible on chromosomes 3H and 5H. The genetic uniformity of the cultivars increased with the progress of breeding. In contrast, the low variation within some of the old cultivars is the result of selection that probably occurred before they were obtained by the gene bank. A side effect of the analysis was the identification and verification of duplicates and the establishment of a core collection and thus DArTseq analysis will contribute to more efficient management of the barley collection in the gene bank. Analysis of changes in the level of genetic diversity over time may not reflect changes in genetic structure, so its results should be treated with caution.


Brassica napus is an important oil source. Its narrow gene pool can be widened by interspecific hybridization with the Brassicaceae species. One of the agronomically important traits, that can be transferred through the hybridization, is the resistance to blackleg, a dangerous disease mainly caused by Leptosphaeria maculans. Hybrid individuals can be analyzed with various molecular markers, including Simple Sequence Repeats (SSR). We investigated the genetic similarity of 32 Brassicaceae hybrids and 19 parental components using SSR markers to reveal their genetic relationship. Furthermore, we compared the field resistance to blackleg of the interspecific progenies. The tested set of 15 SSR markers proved to be useful in revealing the genetic distances in the Brassicaceae hybrids and species. However, genetic similarity of the studied hybrids could not be correlated with the level of field resistance to L. maculans. Moreover, our studies confirmed the usefulness of the Brassicaceae hybrids in terms of blackleg management. Brassica contains three diploid species, i.e., B. rapa (AA genome), B. nigra (BB genome), and B. oleracea (CC genome), and allotetraploid species obtained as a result of natural interspecific crosses, namely B. napus (AACC), B. juncea (AABB), and B. carinata (BBCC). Another representative of the Brassicaceae family is Sinapis alba, a yellow mustard plant closely related to Brassica, well known for possessing many potentially useful traits [Brassicaceae is a family of high agroeconomic importance comprising fodder, oilseed plants, vegetables, ornamental species, as well as plants of medical and scientific importance. Furthermore, ecological, morphological, and genetic diversity of this family makes it a perfect model for relationship and evolution studies . The genl traits .Brassica napus (rapeseed) is one of the most important oil crops, accounting for over 12% of worldwide oil production (USDA). Due to a relatively short history of cultivation and use of conventional breeding methods, rapeseed displays limited genetic diversity [B. napus gene pool. One of the most effective approaches to solve this problem is interspecific hybridization [B. napus germplasm and to enable the transfer of genome fragments carrying desirable traits, which could further improve the cultivar’s characteristics. The sexual incompatibility and differences in the genome sizes of parental components may result in hybridization failure [L. maculans, is a fungal disease which can cause significant yield losses [iversity ,4; theredization . Crossin failure . Barrier failure . The Depd losses . The reld losses . The bred losses . It reliVarious molecular marker systems such as RFLP, SSR, and RAPD can be used to determine the genetic distance of the Brassicaceae species . Simple We are aiming to gain insight into the genetic relationship between hybrids with different parental components, which are diverse in terms of resistance to blackleg. Therefore, the objectives of this research are to determine the genetic similarity of hybrid and parental genotypes from the Brassicaceae family and to evaluate the usefulness of the chosen SSR markers for genetic diversity analysis.9 and F10 generation and 19 parental genotypes were used as research material (1 generation were developed at the Department of Genetics and Plant Breeding (Poznań University of Life Sciences), with the use of in vitro cultures. Next, selected combinations were self-pollinated multiple times in order to obtain stable hybrid lines.A total of 32 various Brassicaceae hybrids of Fmaterial . InterspB. rapa using the ISSR-suppression-PCR method. Preliminary screening was performed in order to assess their usefulness in the present study. Genomic DNA was extracted from young seedling leaves of the studied individuals using the Genomic Mini AX Plant kit  according to the manufacturer’s protocol. PCR was performed in a total volume of 12.5 µL , 2 × 0.5 µL primers, 4.25 µL H2O, and 1 µL DNA template) under the following conditions: initial denaturation at 94 °C for 5 min, thirty-five cycles of amplification , followed by a final extension step at 72 °C for 7 min. Primer sequences and annealing temperatures are presented in 15 SSR markers were selected to characterize the genetic background of the research material. The markers were chosen according to the literature data . This seElectrophoresis was performed on agarose gel stained with Midori Green Advance , 5 µL per 100 mL of TBE buffer. All image data obtained from the electrophoresis gels were examined in the same way: for each marker, the presence or absence of a band of particular size was scored as ‘1’ or ‘0’, respectively. Next, a binary data matrix was created which was further analyzed with Peak Scanner Software v1.0 .ijp denotes frequency of the jth allele for i-th marker among a total of k alleles [The polymorphic information content (PIC) was calculated for each marker using the formula: alleles ,22.GS) was estimated for each pair of genotypes on the basis of Nei and Li [ABN denotes the number of bands in genotypes A and B, AN and BN denote the number of bands in A and B, respectively. The similarity matrix was used to construct a dendrogram using the unweighted pair group method with arithmetic mean (UPGMA) to determine genetic relationships among the genotypes studied. The principal component analysis (PCA) was calculated on the basis of the similarity matrix. All the analyses were conducted using the GenStat 18.2 edition  statistical software package. The analysis of molecular variance (AMOVA) was made using GenAlEx 6.5 [STF) was calculated using the formula:TH denotes the probability that two alleles drawn at random from the entire group differ in state and SH denotes the probability that two alleles drawn at random from a subgroup differ in state. Groups for AMOVA, presented in the B. napus × S. alba hybrids were grouped together with S. alba. Four B. napus cultivars were added to each group.Genetic similarity , and July, BBCH 70-89 (term II). Phoma leaf spotting (term I) was evaluated according to the scale from 0 to 4, where 0 was no visible disease symptoms and 4 was numerous (over 10) leaf spots per plant [All hybrid combinations have been studied in terms of resistance to phoma leaf spotting/blackleg in field conditions. The assessment was carried out in testing fields at the Poznań University of Life Sciences experimental station Dłoń, located in Wielkopolska Voivodeship. The soil and weather conditions were typical for this region of Poland, and no fungicides or pesticides were used on the testing field. The agricultural practices were optimal for the local ecological conditions. The experiment was set up in a completely randomized block design with five replications; the size of a single plot was 10 mer plant . The blaer plant . ObtaineThe set of 15 primer pairs allowed for the detection of 2 monomorphic and 98 polymorphic alleles . The aveB. napus cv. Zhongshuang9 × B. rapa ssp. pekinensis 08 006169 (34) and B. napus cv. Zhongshuang9 × B. rapa ssp. pekinensis 08 006169 (51), whereas the lowest genetic similarity (0.22) was found for B. carinata (7) and B. fruticulosa PI 649097 (18). The mean value of genetic similarity was 0.63. The SSR marker data were used to group cultivars by the UPGMA method. The relationships between genotypes are presented in the form of a dendrogram (B. rapa ssp. chinensis (COBORU)), which had less than a 0.5 similarity with other genotypes; Cluster II comprised only one individual, genotype 18 (B. fruticulosa PI 649097); Cluster III comprised only one individual, genotype 43 (B. napus cv. Lisek × B. fruticulosa—PI649099); Cluster IV comprised genotypes 42, 44, 47, and 50 ; Cluster V, 14, 38, 39, 40, and 48 , and B. rapa ssp. pekinensis 08 006169); Cluster VI comprised only one individual, genotype 49 ; Cluster VII comprised genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 ; Cluster VIII comprised two genotypes, 19 and 20 , while the ninth cluster contained the remaining 26 genotypes  was found between genotypes ndrogram , in whicenotypes .STF = 0.059; p = 0.011) between the genotypes among the groups presented in The significant differentiation , although those genotypes are still considered as moderately resistant. Examples of lesions observed on hybrid combinations are presented in The performed analysis allowed to distinguish the genotypes with the highest resistance level to blackleg . SixteenThe assessment of diversity between species is important for the management of germplasm resources and for the curation of genetic databases. As the phenotypic assessments partially relay on environmental conditions, they do not allow for a clear discrimination of related species. Thus, in this study, genotypic analysis using SSR markers was performed for the unbiased determination of genetic diversity.B. rapa  and were developed using the ISSR-suppression-PCR method by Tamura et al., [Brassica species has been suggested by the aforementioned authors. The Brassicaceae family consists of approximately 3000 species [S. alba (S genome), and B. fruticulosa (F genome), although conserved regions of gene content and gene order are present among the family [Molecular DNA markers are important tools for genetic similarity studies. SSR markers are especially valuable, as they enable multi-allelic detection and can be applied using various laboratory systems . The marAA, 2n =  and were species  with dive family . This atPolymorphism Information Content (PIC) is an indicator that allows to evaluate the discriminatory ability of molecular markers and to study the genetic diversity . The PICB. rapa ssp. chinensis (COBORU) shows weak connection to their progeny or other genotypes form the same species. Furthermore, the distinctiveness of this genotype was confirmed with the PCA method. The weaker-than-expected association between species can be explained by a different origin  or outbreeding [The UPGMA allowed for the distinction of nine groups, based on genetic similarity. Generally, the applied method permitted the assessment of the genetic distance of the studied hybrids and their parents, but not all of the results are in line with the predictions. For example, breeding .B. rapa and B. carinata and their hybrid progeny. The rest of the genotypes were generally more scattered around the diagram. However, attention should be drawn to the short distance revealed for two pairs of genotypes: B. napus cv. Lisek × B. fruticulosa PI649099 and B. napus cv. Californium × S. alba cv. Bamberka, and B. napus MS8 line × B. rapa ssp. pekinensis 08 006169 and B. napus cv. Jet Neuf × B. oleracea var. alboglabra. These hybrids’ male parental components present entirely different genomic structures, however their genetic similarity can be explained by the unequal inheritance of the B. napus genome during hybridization. It should also be emphasized that the markers used in this study derived from B. rapa, which possess A genome, which might have an impact on the obtained PCR products.The PCA analysis was conducted to confirm the complicated structure of the studied individuals, and the results confirmed a close relationship for Brassica and their wild relatives. The genetic similarity of the studied genotypes varied from 0.22 to 0.97. The extensive range of the similarity coefficient values show that the Brassicaceae germplasm collection reflects a diverse and varied population. These results are in line with the findings of Kumari et al. , as wellL. maculans and incorporating their germplasm into the B. napus gene pool. All individuals that had B. carinata, B. fruticulosa, and S. alba as one of the interspecific cross components showed the lowest infestation level. This indicates that particular attention should be paid to these parental species, as they may hold valuable resistance genes that could help to control the disease. This is especially important considering the previously reported resistance breakdowns [The level of field resistance to blackleg varied between the studied genotypes. We managed to select sixteen combinations with the lowest pathogen infestation, which might be especially valuable in future studies focusing on finding a durable resistance to eakdowns . The afoeakdowns ,37,38, wHybrid individuals with the lowest blackleg infestation could be found in five out of nine groups distinguished with UPGMA and were spread evenly on the PCA diagram. This indicates that the genetic similarity of the studied hybrid genotypes is not correlated with their level of field resistance. On the other hand, it might be simply explained by the fact that applied molecular markers are not linked to the regions of the genome containing the resistance genes.L. maculans resistance outside the B. napus gene pool.In conclusion, the tested SSR markers proved to be useful in revealing the genetic distances in Brassicaceae hybrids and species. The ability to properly characterize and organize the genetic resources is key to the effective conservation of accessions. More precise and quick determination of the relationship of genotypes and the amount of variation within or among accessions in a collection can be accomplished by using molecular diagnostic techniques. Other than successfully maintaining the collections, genetic markers are invaluable for crop improvement and plant breeding programs. Moreover, our studies confirmed the usefulness of the Brassicaceae hybrids in terms of blackleg management and the importance of searching new sources of