Quercus havardii Rydb. We assess the genetic, geographic, and ecological representation of 290 plants from eight ex situ locations, relative to 667 wild individuals from 35 in situ locations. We also leverage a recent dataset of >3000 samples from 11 other threatened plants to directly compare the degree of genetic conservation for species that differ in geographic range size. We found that a majority of Q. havardii genetic diversity is conserved; one of its geographic regions is significantly better conserved than the other; genetic diversity conservation of this widespread species is lower than documented for the 11 rarer taxa; genetic diversity within each garden is strongly correlated to the number of plants and number of source populations; and measures of geographic and ecological conservation  were typically lower than the direct assessment of genetic diversity . This information will inform future seed sampling expeditions to ensure that the intraspecific diversity of threatened plants can be effectively conserved.Although the genetic diversity and structure of in situ populations has been investigated in thousands of studies, the genetic composition of ex situ plant populations has rarely been studied. A better understanding of how much genetic diversity is conserved ex situ, how it is distributed among locations , and what minimum sample sizes are needed is necessary to improve conservation outcomes. Here we address these issues in a threatened desert oak species,  Botanic gardens can provide seed and plant material for ecological restoration, and restoration success can be influenced by genetic diversity , while Hoban, Callicrate, et al. , an uncommon but wide‐ranging shrub species with recalcitrant seeds. We also assess geographic and ecological proxies of genetic diversity conservation. This is, to our knowledge, the first extensive genetic analysis of ex situ collections of a widespread but uncommon species; previous work has mostly focused on highly rare species , and we apply the same molecular analysis techniques  for this widespread species to values recently documented for 11 rarer species.Q. havardii and determine if genetic diversity within a garden is a function of the number of plants.Quantify genetic diversity and structure within each of eight garden populations of Compare the percent of genetic diversity conserved to two non‐genetic measures of ex situ conservation: percent of geographic range and percent of distinct ecological regions from which seed was sampled.To address these major knowledge gaps, we assess patterns of genetic diversity within and among eight botanic gardens , compared to in situ populations  of shinnery oak . Its large seeds  are an important food resource for wildlife. Notably, this species also provides habitat for the lesser prairie chicken (Tympanuchus cupido) and the dunes sagebrush lizard (Sceloporus arenicolus), both listed as Vulnerable on the IUCN Red List and continuing to decline , though detailed observation has not been made for this species.Although uncommon and restricted to a very specific habitat, ight 0.2– m. Its lQ. havardii has a disjunct range . Following best practices to maximize genetic diversity  and SEINET, and suggestions from the International Oak Society. One voucher specimen per population was deposited at each of the following herbaria: MOR, FLD, and NAVA  from 36 populations of georeferenced locations in the summer of 2016 for analyses of in situ genetic diversity. A detailed analysis of in situ genetic structure can be found in Zumwalde et al. . Populat2.4r_d, for each pair of loci  with small modifications Methods . DNA wasi Figure . Four poi Figure . Fragmeni Figure  and INESi Figure  were usei Figure . We did i Figure  and can i Figure , and onl2.52.5.1https://github.com/smhoban/IMLS_Safeguarding), we calculated the percentage of in situ alleles that were present in the ex situ gardens by pooling all individuals held at gardens. We calculated this separately for alleles in categories based on their frequencies as follows: ‘very common’ (>10%), ‘common’ (5%–10%), ‘low frequency’ (1%–5%), and ‘rare alleles’ (<1%), as well as ‘all alleles’. We focused on alleles as the measure of genetic conservation  because they are the aspect of genetic variation on which natural selection can act  between the number of individuals currently held ex situ and the percentage of alleles they conserve, established in this prior study of rare species. If Q. havardii is below the relationship for these 11 rare species, it indicates that less genetic diversity is captured than expected from the number of individuals. The minimum sample size for Q. havardii was also compared to the minimum sample size for the 11 rare species, for which the same resampling procedure was applied.We used an allele accumulation curve  to compare 2.5.3We then calculated the percentage of all in situ alleles present in each botanic garden. We used linear regression models in R to test for a relationship between the number of plants in a garden and the percentage of each category of allele captured. An examination of the residuals suggested that residuals of the linear model are not normally distributed for ‘all alleles’ and ‘low‐frequency alleles’, so we also tested for a relationship between log and square root transformations of the number of plants and the percentage of each category of the allele. Finally, we tested for a relationship between the number of accessions  and the percentage of each category of allele, and the number of populations sourced and each category of the allele. We acknowledge that each garden has various amounts of material from different regions, populations, and maternal lines, which may violate the assumption of linear regression (independence of samples).ST using the R package hierfstat v.0.4.22  overlap with, and the number of ecoregions that ex situ samples are taken from ; 68% of the alleles from the West were captured compared to 93% of the eastern region. The results for the full dataset are similar though all values are lower (Table For the reduced dataset (in which singletons and doubletons are dropped), we found that 79% of the overall species’ alleles are conserved in the 290 ex situ seedlings, with 100% of ‘very common’ and ‘common alleles,’ 94% of ‘low‐frequency alleles’, and 55% of ‘rare alleles’ captured . Considering the ‘reduced’ dataset, if a sampler only wanted to focus on the two regions separately, the minimum sample size for the East alleles is 101, while the minimum sample size for the West is 148. Meanwhile, under the conservative assumption of the full dataset, 481 sampled individuals would be needed overall, 217 for the East, and 284 for the West.Using simulated subsampling of the wild individuals, for the ‘reduced’ dataset, we found that the number of sampled individuals needed to reach a minimum of 95% of the alleles in the ‘all alleles’ category for 3.2Q. havardii was shown to be conserved compared to an expected allele accumulation curve for 11 rare, long‐lived species, including three rare Quercus species  than for 11 rare species (mean of 56). The number of samples needed for Q. havardii for ‘low‐frequency’ alleles was also greater− 73 compared to a mean of 56 for the other 11 species  for each kind of allele. The percentage of ‘all alleles’ conserved showed a strong relationship with the square root of the number of plants, low‐frequency alleles showed a log relationship, and rare alleles showed a linear relationship. There was also a strong relationship between the number of maternal families and the number of populations sampled  when comparing each garden to the West region  than when comparing each garden to the East (FST = 0.0066). Examination of BIC  genetic diversity conservation of this widespread species is lower than for 11 previously studied rarer taxa; (3) genetic diversity within each garden is strongly related to the number of plants in the garden; and (4) the measures of geographic and ecological conservation  were lower than the direct assessment of genetic diversity conservation .Our results provide one of the first studies to quantify genetic conservation value of ex situ plants for a widespread but threatened species, examine genetic diversity and structure among botanic garden populations, and directly compare genetic and ecogeographic conservation. There is a growing need and opportunity for this research due to increasing environmental change and biodiversity loss, heightened conservation mission of botanic gardens  than the rare oak Q. oglethorpensis (94%), which has an estimated extent of occurrence of 130,000 km2 compared to 300,000 km2 estimated for Q. havardii , though Q. oglethorpensis has half as many trees (145) ex situ. However, both species possess similar values for the conservation of ‘low‐frequency alleles’ (94% and 97% respectively). The low percentage conserved for Q. havardii, therefore, relates to the fact that the number of rare alleles will be higher when the range size and census size are larger, as noted in Hoban , Griffith et al. (Leucothrinax morrisii (Arecaceae), Namoff et al. (Sibara filifolia (Brassicaceae) to capture 90% of ‘all alleles,’ depending on the target population. This literature, combined with our results herein and our previous studies . The Global Conservation Consortium for Oaks is one of eight current consortia, and its members work together to prioritize species, populations, and conservation actions (including for Q. havardii) while maintaining diverse living collections across tens of institutions.This guidance regarding minimum sample size and representation across populations is especially vital for species that produce recalcitrant seeds  because they must be held in living collections, requiring significant space. For species that require hundreds of individuals to sufficiently capture wild genetic diversity, and especially for larger specimens such as shrubs or trees, coordinated networks of living collections (metacollections) are required , with some exceptions for EPA Level III ecoregions. This is not entirely unexpected as other work has shown that populations can strongly decline in geographic extent or abundance  diversity may be that geographic analysis counts a location as ‘conserved’ if st Table , there wst Table  that an 4.1An important caveat of this study is that eleven microsatellites are limited in their resolution; the oak genome likely contains 30,000 to 80,000 protein coding genes . It is worthwhile and important to continue collecting additional seeds from additional locations for a larger ex situ metacollection of this species (which could occur across multiple years due to relatively infrequent mast years of large seed production in oaks). The data collected on such future efforts, including sampling across multiple years, will inform future seedsampling expeditions to ensure that intraspecific diversity of threatened plants can be conserved.The authors declare no conflict of interest.Supplementary MaterialClick here for additional data file.


Myc. bovis in an indirect ELISA for the detection of Myc. bovis antibodies in milk samples.The aim of this study was to assess a phage‐displayed MilA protein of milA gene was synthesized and cloned into pCANTAB‐F12 phagemid vector. The expression of the MilA on the phage surface was confirmed by Western blotting. The recombinant phage was used in the development of an indirect ELISA to detect Myc. bovis antibodies in milk samples. There was a significant agreement between the results of phage‐based ELISA and recombinant GST‐MilA ELISA for the detection of Myc. bovis antibodies in milk samples.The desired sequence of Myc. bovis‐associated mastitis.The inexpensive and convenient phage‐based ELISA can be used instead of recombinant protein/peptide ELISA as an initial screening of Myc. bovis is a continuous and serious problem in the dairy industry. Sero‐monitoring of Myc. bovis infection cases are one of the key factors for surveillance of the infections in dairy farms. Despite the existence of some commercially serological assays for Myc. bovis antibodies, they have some limitations regarding their sensitivity and availability. The development of accurate diagnosis tools could contribute to control programmes of Myc. bovis‐associated mastitis in the dairy herds.Mastitis associated with  Myc. bovis is a bacterial pathogen associated with infection in cows of all ages , enzyme‐linked immunosorbent assay (ELISA) and immunofluorescence assays  making it an appropriate antigen for the development of specific serological tests  at position 1156 to 1659 was used. The selected sequence contained 504 nucleotides and was optimized for expression in E. coli. All of the TGA codons in the selected sequence were changed to TGG to match with E. coli expression. The PGEX‐4 T‐1 plasmid construct containing the desired sequence was synthesized . The accuracy of the synthesized sequence was confirmed by Sanger sequencing.To select the proper segment of the protein for expression, the available amino acid sequence (Uniprot accession: A0A454API1) was analysed in BioEdit software based on Kyte and Doolittle scale , SalImilAR primer (5′‐GTCGACATTCTTG AAAATGTTTA‐3′) and ALLTaq™ Master Mix PCR kit . The PCR product was purified and sequenced in both directions to confirm the amplified insert fragment. pCANTABF12 phagemid used in this study was similar to pCANTAB5E but XbaI and SalI cut sites were introduced into multiple cloning sites. The phagemid and milA amplified fragments were digested at 37°C with XbaI and SalI‐HF restriction enzymes in CutSmart buffer . The gel‐purified products were ligated using Invitrogen™ T4 DNA ligase kit (Thermo Fisher Scientific) following the manufacturer's instruction. Ligation product was transfected to electrocompetent E. coli TG1 (Agilent) cells using the electroporation method described by Green and Sambrook  in 2YT broth containing tetracycline. Phage titration was performed by the plaque counting method . Single colony was selected and transferred to 10 ml of 2xYT‐A broth medium. After overnight incubation, 2.5 ml of the medium was added to 500 ml of 2xYT‐A broth medium supplemented with 2% glucose and incubated  until optical density of 600 nm reached the value of 0.6–0.7. At the next step, M13KO7 helper phage with the concentration of 1010 PFU ml−1 was added to the culture medium and incubated for 1 h without shacking, and an additional 1 h with shacking. The culture medium was centrifuged , the supernatant discarded and the pellet resuspended in 500 ml of fresh 2xYT broth medium supplemented with 0.1% glucose, ampicillin (100 μg ml−1), and kanamycin (50 μg ml−1). The culture was incubated overnight at 30°C in a shaker incubator as explained above. After overnight incubation, the culture was centrifuged twice (5000 × g for 20 min) and the supernatant was filtered through a 0.45 μm syringe filter. The resulted bacteriophages were precipitated using 20% polyethylene glycol in 2.5 mol l−1 sodium chloride and incubated at 4°C for at least 2 h. The bacteriophages were precipitated and washed two more times, each time being resuspended in one‐third of the initial volume  and then transferred to PVDF (polyvinylidene difluoride) membrane by Trans‐Blot® Turbo™ RTA Mini PVDF Transfer Kit . After transfer, the membrane was blocked using 10% BSA in PBS for 2 hours at room temperature under rotating. Anti‐E tag antibody (Abcam) in dilution 1:500 and anti‐mouse HRP antibody (Sigma‐Aldrich) in dilution 1:4000 were used as primary and secondary antibodies, respectively, and the membrane was developed using DAB tablet  dissolved in 15 ml of Tris‐buffered saline, pH 7.6 by adding 12 μl of fresh 30% hydrogen peroxide.Myc. bovis in bovine milk samples. Samples were taken from high somatic cell count cows aged 2–10 years without signs of clinical mastitis. Cows belonged to farms with a history of recurrent mastitis and high SCC that did not respond to antimicrobial treatment. The mean SCC value for tested milk samples was 378.04 (±SE = 60.49) × 103 cells ml−1. Positive controls were selected from milk samples that were positive for Myc. bovis culture  was considered the optimal condition. Anti‐bovine IgG‐HRP (Sigma‐Aldrich) as secondary antibody was diluted 1:20,000, 1:40,000, 1:80,000, 1:160,000 and 1:320,000. Optical dilution of secondary antibody was found using optical concentration of phage particle and controls milk.Initially, the optimal concentration of recombinant phage and milk sample (as a primary antibody) was determined by checkerboard titration (with seven dilutions of phage particles and three dilutions of positive and negative milk samples). The highest P/N ratio  was added to each well of 96‐well Nunc MaxiSorp™ flat‐bottom ELISA plate (Thermo Scientific) and incubated overnight at 4°C. After overnight incubation, the plate was washed two times with 0.05% Tween‐20 (v/v) in PBS and then blocked using PBS containing 0.3 mmol l−1 BSA.To assess the phage‐displayed antigen, 50 milk samples with known results for GST‐MilA ELISA and PCRs  Tween‐20). 100 μl of dilution was added to each well and incubated for 2 h at room temperature (23–25°C). The ELISA plate was then washed four times and 100 μl of anti‐bovine HRP antibodies were diluted 1:20,000 in the dilution buffer, added to each well and re‐incubated for 1 h. After washing four times, the wells were developed using TMB  liquid substrate system for ELISA  and incubated at RT for 5–15 min. Then, the reaction was stopped with sulphuric acid solution 2 mol l−1 and the optical density was read at 450 nm wavelength using a microplate reader . Mean OD450 plus two standard deviation of negative milk samples was defined as the threshold value. Samples with optical density greater than threshold value were considered positive  . Furthermore, no reactions were observed to the bacteriophage surface proteins in the tested milk.All samples were previously checked by GST‐MilA ELISA , the inclusion of appropriate positive and negative controls in the test ensures results could be applicable to the external population. The phage‐displayed MilA antigen has some advantages compared to the recombinant MilA protein production. The purification process for recombinant proteins contains complicated steps of affinity purification, protease treatment for removing the fusion tags. It makes the recombinant proteins more expensive, time‐consuming and hard to make in uniform batches , Biovet Inc  and ID screen® Myc. bovis indirect ELISA ). These kits are not yet accessible in some countries. The Sensitivity of the IDvet ELISA and the BIO K302 ELISA for detection of Myc. bovis antibodies in serum sample have been reported, 93.5% and 49.1%, respectively, and the specificity has been shown 98.6% and 89.6%, respectively (Andersson et al., To date, different recombinant antigens have been produced by expression in Myc. bovis‐specific calf sera and was used to optimize the GST‐MilA ELISA. (Wawegama et al., Myc. bovis DNA whilst indirect ELISA detects Myc. bovis antibodies. There was no obligation that either Myc. bovis bacteria or antibodies against Myc. bovis existed in the milk samples at the time of the experiment. Thus, it is our opinion that ELISA may be appropriate for initial screening, followed by qPCR for verifying the active presence of Myc. bovis in the samples.This study showed the sensitivity of phage‐based ELISA was a bit higher than that of GST‐MilA ELISA. It is possible that the correct conformation of peptides on the phage surface leads to better recognition of these peptides in the phage‐based ELISA (Portefaix et al., Candida albicans diagnosis (Quanping et al., C. albicans in ELISA with Sap2 and Hsp90 hybrid phages was higher than in ELISA with Sap2 and Hsp90 recombinant protein (Wang et al., Several studies have revealed the potential use of the phage‐based ELISA to antibody detection. Quanping et al. , reporteMyc. bovis‐associated mastitis. This is supported by the cost‐effectiveness in the production of the phage‐displayed antigen. Indeed, the novel ELISA proposed in this study requires comparison to commercial ELISA kits that have shown high sensitivity and specificity.In conclusion, this study has shown good agreement between the phage‐based ELISA and GST‐MilA ELISA. Due to the advantages of the phage display technology, the phage‐based ELISA rather than the recombinant protein/peptide ELISA could be used as an initial screening of No conflict of interest declared.


Camelus dromedarius) in Africa are adapted to arid and the semi-arid environmental conditions, and are valuable for meat, milk and fiber production. On account of the growing demand for camels in this continent, there is a need for knowledge on their phenotypic and genetic diversity. This is fundamental to sustainable herd management and utilization including the design of appropriate breeding and conservation strategies. We reviewed studies on the phenotypic and genetic characterization, breeding objectives, systems of production, productive and reproductive performances, and pathways for the sustainable rearing and use of camels in Africa. The morphological and genetic diversity, productive and reproductive abilities of African camels suggest the existence of genetic variations that can be utilized for breeds/ecotypes’ genetic improvement and conservation. Possible areas of intervention include the establishment of open nucleus and community-based breeding schemes and utilization of modern reproductive technologies for the genetic improvement of milk and meat yields, sustainable management of rangelands, capacity building of the pastoralists and agro-pastoralists, institutional supports, formation of centralized conservation centres and efficient and effective marketing systems.Camels ( Camelus dromedarius), over the years, are renowned for quality milk, meat, and fibre production across the continents  are important domestic animals which are found in arid and semi-arid Africa and the Middle East including western and central Asia (Camelus bactrianus) found mainly in the hot, cold steppes, and Central Asia deserts  are useful in breed discrimination and establishment of phenotypic standards. It is globally important to assess the level of phenotypic variation in camel populations. This paves the way for proper documentation of the available gene pools that will permit the selection of elite animals for the production of superior individuals . Body weCamel bulls appeared to have higher values for size measures, which is an indication of sexual dimorphism . Such diWhen setting a breeding objective, multiple traits are combined irrespective of whether one trait is dominant over others . All ecoIn different African countries, there are different ratings of criteria for the selection of animals for breeding purpose. The choice of camel breeding bull by pastoralists was mostly influenced by performance, conformation, and colour, while hump, temperament and size were less important in Nigeria-Niger Corridor . In OromThe history of camel production systems has highly been on the migratory and extensive side. However, the systems have evolved over the years from a traditional system to a more diverse modern system of production. Camel’s husbandry in different parts of the world can be classified as traditional nomadic management system and transhumant, sedentary, semi-intensive and intensive management system . In AfriCamel milk trait is very important in breeding considering the fact that on average, the milk produced by camels is six times more than that of the indigenous cattle in drylands . Camel mCamel breeds seasonally . ReproduIn terms of breeding, male to female ratio ranged from 1:48–50 (Nigeria), 1:30–50 (Ethiopia), 1:40 (Algeria) to 1:50 (Kenya). Heifer and Bull mean ages at first mating were 3–3.9 and 5.63 years (Nigeria), 3.9–4.7 and 5.5–6.5 years (Ethiopia), 2.96 and 3.6 years (Algeria), 4-5 and 5-6 years (Kenya) and in males only, 5.64–7.4 years . Mean gestation period of 12.1–13 months (Nigeria), 12.8 months (Algeria), 12.5 months (Libya) has been documented. Age at first calving was 4.8–5 years (Nigeria), 5-6 years (Ethiopia), 4.3 years (Algeria), 5.2–6.4 years , 5-6 years (Kenya), 4.9–5.3 years (Niger), 7 years , 6.5 years (Sudan), 5.5 years (Egypt) and 5.04 years (Mauritania) was reported. Calving interval was 23.8 months (Nigeria), 18–31.2 months (Ethiopia), 34 months , 22.32 months (Algeria), 27-28 months (Kenya), 23–28 months (Sudan), 20.8–22.3 months (Egypt) and 12–48 months (Mauritania). The number of calvings in a lifetime was 8.49 calves (Nigeria), 8–11.6 calves (Ethiopia) and 7.8 calves (Egypt). The average birth weight for both female and male calves was 30 kg and 35 kg, respectively (Egypt). The mean herd’s annual fertility was 60% (Nigeria), 56.2% (Algeria) and 44.5% (Sudan), respectively . HoweverGenetic diversity and population structure of camels will assist in sustainable management, selection and improvement of the herd . Comparee) ranged from 0.76 to 0.84 while observed heterozygosity (Ho) was 1.0. The sub-populations showed moderate genetic structure based on fixation index (FST = 0.052)  of 6.5. The expected heterozygosity (H= 0.052) . In fourersified .o and He were 0.52 and 0.63, respectively, indicating the level of genetic diversity. This could have resulted from continuous gene flow with other camel populations occasioned by transhumance along the Nigeria-Niger corridor. However, the molecular genetic data (AMOVA) did not clearly separate the populations into breeds based on coat colour. o per population ranged from 0.44 (Gelleb) to 0.66 (Jigjiga) and the He ranged from 0.70 (Jigjiga) to 0.77 (Gelleb). Similar to the Nigerian camels, genetic analysis of microsatellite data did not show distinct genetic lineages based on geographical locations or designated camel breeds in Ethiopia  depicts maternal inheritance pattern which can be used to show genetic variation between species . The evaIn the recent times, the study of genetic diversity and polymorphisms of genes of economic importance involving the use of bio-makers have evolved with the use of advanced molecular techniques such as single nucleotide polymorphism (SNP). SNPs have been found as proven bio-markers for population-based screening of wide range of QTL in livestock animals . SNP genIn a study to estimate the polymorphism of the tyrosinase (TYR) gene among Maghrebi, Sudani, Somali, and Falahy camel breeds of Egypt, it was observed that The CT heterozygote genotype had the highest frequency of 45%. The C/T SNP of TYR gene was associated with neck length, height at withers and chest girth, teat separation and teat floor distance traits . Abd El-o and local inbreeding differences were observed across the populations from Tunisia, Sudan, and Nigeria. The Ho ranged from 0.194 to 0.327 (Tunisian), 0.293–0.406 (Sudanese) and 0.357 (Nigerian) , g.2126A>G at CSN2 (β-casein), and g.1029T>C at CSN3 (κ-casein) . When thigerian) . Howeverigerian) . Future Mammals inhabiting deserts show remarkable adaptive traits that have evolved repeatedly and independently in different species . There iIn another study, 2a) for TMY, DMY and LP were 0.25, 0.30, and 0.17, respectively. Medium repeatability value (0.19) was obtained for LP, while higher values (0.36 and 0.43) were recorded for TMY and DMY, respectively. The predicted breeding values of the camels for TMY, DMY, and LP were 143.07 kg, 1.5 kg and 113.9 days, respectively  (incorporating indigenous knowledge), involving all relevant stakeholders. The scheme is aimed at initiating systematic breeding at the community level, including an organized animal identification and recording of performance and pedigree data . The breFor industrial use of camels, especially where there are adequate resources, genetic improvement of local breeds/ecotypes utilizing modern reproductive technologies  and embryo transfer (ET) can be exploited. These tools play an effective role in propagating improved breeds .Rangeland is important for the agroecosystem. Rangelands help in the preservation of biodiversity and some of the species of plants that are used for medical purposes. Rangeland policies and implementations with respect to conservation are necessary for sustainable management . It willTo improve livestock production and productivity, there is a need for the capacity building of the herders. Knowledge improvement in the management, health, breeding, proper decision making and problem solving will not only increase production, but the profitability of the camel enterprise . These a. AsadzadAssisting herders in all possible ways will stimulate interests and facilitate increase in camel characterization and production. These include facilitating the conduct of appropriate high quality research targeting the needs of the herders, marketers and consumers; policy and development interventions; legal frameworks, infrastructural supports , and giving incentives to herders in terms of credit facilities and inputs. Such assistance can be made possible by national, regional and international relevant institutions including donor agencies and developmental organizations .in situ (on-farm) and ex situ  conservation. This is to prevent camels from going into extinction. The on-farm (community-based conservation) program should involve the local herders. This is because such bottom-top approach will give the herders sense of belonging, which we make them readily accept and work towards the successful conservation of the camels. According to This involves both The essence of establishing any camel enterprise(s) is to produce for consumption and sales in order to contribute to food security and improve the livelihoods of the increasing rural, peri-urban and urban populations. Therefore, there is a dire need for strong and reliable marketing channels for the sales of live animals and animal products such as milk and meat. In this wise, there should be a synergy in the activities of herders, middlemen, cooperative groups and governments in ensuring that the best quality animals and their products get to the markets. Additionally, there should be price control mechanism to ensure that the herders and other stakeholders within the marketing chain do not run at a loss. This will go a long way in guaranteeing sustainable production of camels which could contribute to the gross domestic products (GDPs) of African countries. Similar submissions have been made on the need for herders, middlemen, the cooperatives and the governments to jointly promote camel meat and milk businesses .Camel populations in Africa have some phenotypic, productive and reproductive attributes including genetic variations, based on the polymorphic nature of the loci reported. However, future genetic studies using robust methods are required to unravel the admixture process between them for proper breeds/ecotypes’ differentiation. Also, there is a need for interventions such as appropriate genetic improvement programs, utilisation of modern reproductive technologies, rangeland conservation, capacity building on sustainable camel production, institutional supports, establishment of centralized conservation stations, and strong marketing strategies. Such interventions are essential for the preservation, utilization and genetic improvement of camels geared towards increased production and productivity including higher profit.


Mytilus galloprovincialis) from 18 sites along the eastern Adriatic Sea revealed little or no spatial variation. In contrast, seascape genetics analysis revealed a pronounced locus‐specific gradient in allelic and genotypic frequencies across the study region. At a sixth locus, MGE7, the frequencies of two alleles, MGE7243 and MGE7249, were strongly associated, negatively and positively, respectively, with a single environmental variable – minimum salinity (minSAL). The frequency of the MGE7243/243 homozygous genotype was strongly negatively associated with minSAL, whereas the frequencies of the MGE7246/249 and the MGE7249/249 genotypes were strongly positively correlated with minSAL. Interpretation of these pronounced gradients is confounded by the fact that minSAL and another environmental variable, maximum sea surface temperature (maxSST), are highly correlated (R = −.911) and are therefore not necessarily acting independently. BLAST searches of the MGE7 locus against M. galloprovincialis whole genome shotgun sequence returned an alignment with contig mg10_S01094 (accession UYJE01010330.1) and 7 predicted M. galloprovincialis proteins VDI82194.1 ‐ VDI82200.1. Conserved domain searches revealed a similar structure to the transcriptional regulator Msx2‐interacting protein. The BLASTp search also returned significant alignments to Msx2‐interacting proteins in Mytilus coruscus, Crassostrea virginica, and Haliotis rubra. The existence of the MGE7 gradient highlights the role that environmental variation may play in retarding gene flow among wild M. galloprovincialis populations, and also how the success of collection of young mussels (spat) from one site and their transfer to another site (the farm) may be influenced by a single factor such as minSAL or maxSST on a localized scale.Population genetic analysis of variation at five neutral microsatellite loci for Mediterranean mussels  from 18 sites along the eastern Adriatic Sea, using six microsatellite loci. Population genetic analysis of variation at five neutral microsatellite loci revealed little or no spatial variation. In contrast, seascape genetic analysis revealed a pronounced association between allelic and genotypic frequencies at a sixth locus, MGE7, and the environmental variable minimum salinity, highlighting the role that environmental variation may play in retarding gene flow among wild M. galloprovincialis populations.We investigated the genetic variation of the economically and ecologically important species the Mediterranean mussel ( Three cyclonic gyres, corresponding to northern, central, and southern basins, operate with varying strength through the year and significant variation between years may form barriers to larval dispersal and therefore lead to genetic discontinuities Figure . The AdrM. galloprovincialis, along the eastern Adriatic coast.The Mediterranean Sea is predicted to be particularly strongly influenced by global warming trends, with increasing occurrences of marine heat waves and temperature‐related mass mortality events  along the eastern coastline of the Adriatic Sea, a region spanning four countries  and a variety of coastal environments . Individuals were genotyped using seven microsatellite loci described in three published papers as follows: Mgμ1 .Different versions of the data set have been used for analyses Table . This isTo assess whether sample sizes were sufficiently large to characterize the allelic variation of each population, the rarefaction procedure in the software package PopGenKit  and Linkage Disequilibrium (LD) were performed using Genepop 4.7.0 Rousset, . DepartuNe, was estimated using the linkage disequilibrium method of Hill , and the fixation index (FIS) were calculated using GenAlEx 6.503  and private allelic richness (PAR) were calculated using HP‐RARE 1.1 , observed and expected heterozygosities , together with delta values  that reflect the full range of variability. Three geospatial variables were also employed, latitude (Lat), longitude (Long), and the total coastal distance . TotalCD was calculated following Wei et al.  as the t2.7To test for independence correlations between pairs of all twelve variables  were calculated and relationships visualized using principal component analysis using Statistica 7.1 (StatSoft). We removed variables so that |R| < 0.85 for all pairs of variables to avoid excess collinearity  which is based on the complete data set of five neutral loci, (2) LinFST6  which includes the locus that is putatively under selection, (3) LinFSTMGE7 which was based only on the MGE7 locus only, (4) f(MGE7243) ‐ the site‐specific frequency of allele MGE7243, and (5) f(MGE7243/243) ‐ the site‐specific frequency of the homozygous genotype. Estimates of linearized FST (LinFST) were calculated using GenAlEx 6.503  was used to describe the relationship between a genetic response variable and site‐specific environmental variation. GLZ analysis was carried out for five separate metrics: (1) LinGLZ with a normal distribution and a Log‐link function was employed to identify the best‐fit model based on the Akaike information criterion (AIC). For each of the five genetic indices, the top‐ranked model  and a set of best‐fitting models were used to evaluate which of the individual or combined predictor variables were most important in explaining variation in the genetic index. The number of models selected for the set of best‐fitting models was chosen by inspecting the plot of AIC values against the number of models and finding a point of inflection in the plot. This inflection is the point at which successive models add only a small and relatively constant amount to the AIC and are therefore not additionally informative . The importance of each independent variable in explaining genetic structure was evaluated by calculating the percentage of best‐fitting models containing that independent variable Figure .2.9R2). DISTLM analysis was carried out on the 6‐locus data set .To test the effect of the six independent environmental/geospatial variables  on site‐specific genetic variation, a distance‐based linear model was employed  and also for the heterozygote genotype MGE7243/X where X is any other allele.We tested MGE7 alleles and genotypes for correlation with environmental variables that were important in explaining population‐specific genetic variation in the GLZ and DISTLM analyses. We tested correlations for all alleles and genotypes with frequencies greater than 0.05 . The percentage of each locus scored per population was broadly consistent between the two service providers.Six of seven microsatellite loci were amplified satisfactorily with 85.1%–98.7% of individuals scored. Locus Mgμ1 showed stuttering and multiple peaks for a proportion of samples, with 44.1% of individuals able to be reliably scored overall and the percentage scored per population ranging between 28.0% and 64.7%. Because results of analyses with and without this unreliable locus were similar, we have only reported results not including this locus. There was no relationship between percentage scored per population and population latitude  and the six locus data set (including MGE7).The outlier analysis did not detect any significant deviations in the The application of Microchecker identified an excess of homozygote genotypes across all 18 populations for four of the six loci . For the remaining two loci, MGE3 and MGE5, only one and three populations, respectively, had excess homozygotes. All 18 populations showed significant departures from HWE expectations with four individual loci being out of HWE at every population. Loci MGE3 and MGE5 met expectations at 15 and 11 populations, respectively. There was no evidence of linkage disequilibrium for any pair of loci, either at population level or across all populations.Ne, were large . This is reflected in the high inbreeding coefficient, FIS, for which values ranged from 0.267 to 0.453 across all populations and was 0.365 overall  had lower FIS, 0.052 and 0.115, respectively, compared to the loci that did have excess homozygotes at every population .Average allelic richness, All Table . The indG‐tests, was <1 × 10−20 across all loci, less than 1 × 10−5 for individual loci MT282, MGE4, MGE7, and MT203, 0.021 for MGE5 and 0.187 for MGE3. The probability that genotypes from each population were drawn from the same distribution for six loci was 4.4 × 10−7 across all loci, less than 1 × 10−5 for MGE7, 0.018 for MT282 and not significant for the remaining loci.The probability that alleles from each population were drawn from the same distribution for six loci, calculated by exact R or FIS between farmed and wild populations . Regional allele frequencies are illustrated in Figure 243, showed an increasing southward trend, with average frequency 46.5% in the North (range 39.1%–54.4%), 51.4% in the Central region (range 45.8%–59.5%), and 66.9% in the South (range 54.9%–77.0%)   but not for the five neutral loci . The FST index, which in the hierarchical AMOVA is an index of variation explained between regions and populations to total variation, was significant for all six loci  and also for the five neutral loci .AMOVA Table  revealedFRT values among regions were statistically significant (p ≤ .012) whereas for the five locus data set only the North‐Central comparison was significant  but weaker than the corresponding correlation from the Structure program. For the five neutral loci, only one cluster was evident and when K = 2, the correlation between cluster 2 membership and latitude was not significant .AWclust analyses Figure  of the s3.4Based on PCA and correlation analysis with a critical cut‐off value of |R| > 0.85, we removed six of 12 environmental and geospatial variables from the data set Figure . Subsequ3.5FST5, LinFST6, LinFSTMGE7, f(MGE7243), and f(MGE7243/243) , p = .071 for all loci including MGE7 (LinFST6), p = .023 for MGE7 only (LinFSTMGE7), p < .0001 for both allele frequency (f(MGE7243)) and genotype frequency (f(MGE7243/243)). The variable minSAL was the most important of all predictor variables in explaining genetic variation: it was included in the top‐ranked model for all genetic indices except LinFST5 and had a significant all effects test for all genetic indices except LinFSTMGE7. It was included in an increasing percentage of the best‐fitting models as the genetic index focused on the MGE7243 allele  and f(MGE7243/243)). Although minSST and minCHL‐a were included in the top‐ranked model (together with minSAL) for f(MGE7243/243), the three lowest ranked models of all possible models included only minSST and/or minCHL‐a.GLZ analyses were performed on five genetic response indices ‐ Lin7, fMGE723, and f(3.6p = .0016), Lat (p = .0434))  to 0.432 (six variables). The variable minSAL was included in all six of the six best‐fit models, indicating that it is the single variable with greatest power to explain variation in the genetic data set.Marginal analyses revealed that two of the six variables explained significant variation in the 6‐locus data set (minSAL ) Table . Model b)) Table . This mo3.7243, was negatively correlated with minSAL . This was partially counterbalanced by the positive correlation of MGE7249 . The second most common allele, MGE7246, was not correlated with minSAL  , while there was no correlation between the heterozygous genotype, MGE7243/246 and minSAL. Both MGE7246/249  and MGE7249/249  were positively correlated with minSAL , the expected frequency was 0.13 (n = 100.6 mussels expected). The ratio of observed to expected for this genotype (0.07) was very low, even in a background of heterozygote deficiencies. The ratios of observed to expected for the three other heterozygous genotypes with expected frequency >0.05, MGE7243/246, MGE7240/243, and MGE7246/249, were 0.37, 0.50, and 0.87, respectively.The MGE7243/243) but neither was significantly correlated with any MGE7 allele or genotype.The variables minSST and minCHL‐a were included with minSAL in the top‐ranked model for the GLZ analysis based on the genetic response index f were not statistically significant and minSAL was not included in the top‐ranked model, the measures that included the MGE7 locus (LinFST6 and LinFSTMGE7) were strongly associated with the geospatial variable Lat and, to a lesser extent, minSAL. The genetic indices based solely on the frequency of the allele MGE7243 and on the homozygous genotype MGE7243/243 were strongly associated minSAL, and the models were statistically highly significant (p < .0001). The geospatial variable Lat was not statistically significant in these multivariate models, this result being consistent with the stronger single variable correlation of MGE7243 allele frequency with minSAL  than with Lat . The association between the MGE7243 allele and minSAL extended to the homozygous MGE7243/243 genotype, but not to the heterozygous MGE7243/X genotype. Analysis of regional subsets of the data  and analysis with or without individual sites  revealed that the overall relationship is robust and not dependent on the inclusion or exclusion of specific sites (results not shown). Variations in allele frequencies that coincide with changes in an environmental variable or geographic cline may arise from chance effects and historic events . The MGE7243 allele appears to confer an advantage in low‐salinity environments only when alleles MGE7246 and MGE7249 are not also present as the heterozygote combination, while MGE7249 may be advantageous in high‐salinity environments either as a homozygote or heterozygote. This is supported by the low frequency of the MGE7243/249 genotype which was lower than expected even in a background of heterozygote deficiency.The potential role of selection in generating a gradient of genetic variation is supported by the seascape analyses that revealed a strong association between the frequency of allele MGE7M. galloprovincialis whole genome shotgun contigs returned an alignment with contig mg10_S01094 (accession UYJE01010330.1) and a BLASTp search found 7 predicted proteins VDI82194.1 ‐ VDI82200.1. The 7 coding sequences span nucleotides 66,989–122,497 of UYJE01010330.1 with VDI82194.1 and VDI82195.1 covering the entire range. A conserved domain search retrieved 4 RNA recognition motif (RRM) superfamily domains (cl17169) close to the N‐terminal and a SPOC SHARP domain (cd21543) close to the C‐terminal. This is similar to the structure of the SHARP protein  which has 3 N‐terminal RRM domains and a C‐terminal SPOC SHARP domain ; data curation (lead); formal analysis ; funding acquisition ; investigation ; methodology ; resources (supporting); software ; validation ; visualization ; writing – original draft ; writing – review and editing (lead). Marina Piria: Conceptualization ; funding acquisition ; methodology ; project administration (supporting); resources (lead); writing – review and editing (supporting). Ana Gavrilović: Resources (supporting); writing – review and editing (supporting). Mirna Mrkonjić Fuka: Resources (supporting); writing – review and editing (supporting). Lidija Svečnjak: Resources (supporting); writing – review and editing (supporting). Slađana Nikolić: Resources (supporting); writing – review and editing (supporting). Rigers Bakiu: Resources (supporting); writing – review and editing (supporting). Gardner Jonathan: Conceptualization ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration (lead); resources (lead); software ; supervision (lead); validation ; visualization ; writing – original draft ; writing – review and editing (lead).Data S1. Supporting Information.Click here for additional data file.


Triticum species spread in cultivation in Sicily and neighboring regions over the centuries, which led to the establishment of a large genetic diversity. Many ancient varieties were widely cultivated until the beginning of the last century before being replaced by modern varieties. Recently, they have been reintroduced in cultivation in Sicily. Here, the genetic diversity of 115 and 11 accessions from Sicily and Calabria, respectively, belonging to Triticum species was evaluated using a high-density SNP array. Einkorn, emmer, and spelta wheat genotypes were used as outgroups for species and subspecies; five modern varieties of durum and bread wheat were used as references. A principal coordinates analysis (PCoA) and an unweighted pair group method with arithmetic mean (UPGMA) showed four distinct groups among Triticum species and T. turgidum subspecies. The population structure analysis distinguished five gene pools, among which three appeared private to the T. aestivum, T. turgidum subsp. Turgidum, and ‘Timilia’ group. The principal component analysis (PCA) displayed a bio-morphological trait relationship of a subset (110) of ancient wheat varieties and their wide variability within the T. turgidum subsp. durum subgroups. A discriminant analysis of principal components (DAPC) and phylogenetic analyses applied to the four durum wheat subgroups revealed that the improved varieties harbored a different gene pool compared to the most ancient varieties. The ‘Russello’ and ‘Russello Ibleo’ groups were distinguished; both displayed higher genetic variability compared to the ‘Timilia’ group accessions. This research represents a comprehensive approach to fingerprinting the old wheat Sicilian germplasm, which is useful in avoiding commercial fraud and sustaining the cultivation of landraces and ancient varieties.Several  Wheat is one of the key crops to ensure that food and nutritional needs are provided for an ever-growing human population in the future. This is attributable to its ability to adapt to a wide range of climates worldwide. Wheat and its derivatives are foreseen to provide about 20% per capita consumption of protein and calories for an estimated global population of 9.7 billion people in 2050  by contrTriticum aestivum L.) comprises roughly 90% to 95% of the total wheat production worldwide, whereas durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn.) represents just 5–7%. However, durum wheat is a staple food in Mediterranean countries due to its adaptation to hot dry conditions and is useful for producing pasta, couscous, bulgur, and bread [Wheat represents the second most important cereal grain after maize and is the most widely grown crop in the world, with a cultivated area of approximately 219 million hectares and a production of about 760 million tons . Bread ond bread . Italy iPoaceae and the tribe Triticeae. Einkorn wheat  is the earliest diploid species of cultivated wheat. Tetraploid wheat  includes emmer (T. turgidum ssp. dicoccum), which was widely cultivated before bread wheat [T. turgidum ssp. durum), which was cultivated mainly in the Mediterranean basin [(T. aestivum L.) is an allohexaploid  that arose from the interspecific hybridization of T. turgidum ssp. dicoccum (AABB)) and Aegilops tauschii Coss.  originated from a North African population created by Nazzareno Strampelli in 1915.T. turgidum subsp. turgidum) landrace group or so-called ‘Bufale’ are distinguished by color, all showing a mealy (softer) fracture of the kernel, a trait closer to bread than durum wheat [Rivet wheat  was reported in Sicily at the beginning of the 19th century [‘Farro Lungo’  at Caltagirone . Over the last century, different populations of the same variety have been distinguished. Currently, the ESS contains more than 200 accessions of different This study aimed to assess the morphogenetic diversity of the ESS germplasm collection using a high-density SNP array. In the analysis, durum and bread wheat landraces from the Calabria region were included that could also be conveniently preserved and exploited in future breeding programs. Interestingly, the genetic diversity of the EES collection was compared to the improved varieties of both old and modern wheat using the same SNP array while also defining their population structure. Moreover, the inter- and intrapopulation genetic variations in the historical Sicilian durum wheat varieties were also estimated. This research laid the foundation for expanding the genetic variability of cultivated wheat through the use of these underexplored southern Italian landraces.Triticum species, which are widespread in Southern Italy , was investigated by using a high-throughput genotyping system based on a wheat 90k SNP array. After filtering, 20,899 high-quality SNPs out of 81,587 SNPs were retained . All the durum wheat accessions and the more distant emmer wheat (T. turgidum ssp. dicoccum) landrace ‘Garfagnana’ and variety ‘Padre Pio’ comprised the fourth-largest cluster. In the latter, it was possible to distinguish several durum wheat subclusters (The first cluster included the ossella’ . The secclusters A.T. turgidum subp. turanicum) gathered within this last subcluster maintained their distinctness except for the “Farro lungo 03” accession. The most mixed subcluster, which encompassed 84 Sicilian durum wheat accessions based on gene bank information, was originally classified into four groups . The 14 durum-T samples and ‘Francesa_01′ accessions that clustered showed narrow genetic distances. Eleven durum-RI accessions were distinguished from the durum-R group except for ‘Sicilia Lutri’, Russello03, and Sicilia01. Four landraces from Sicily  were also included in this subcluster. Finally, the last subcluster included 11 durum-R group, Ruscia01, and 17 durum wheat landraces  were grouped together; the older improved varieties  were clustered with the older varieties from North Africa . The turanicum wheat . Instead, a low differentiation (<0.17) was detectable in the other tetraploid subspecies (rivet wheat and ‘Timilia’ and ‘Russello Ibleo’ durum groups), with the lowest value (0.025) highlighted between the pair T. turgidum subsp. durum and the Russello group (R). Among the four durum wheat groups previously described, a low Fst (≤0.07) was evident.Overall, moderate pairwise fixation index (Fst) values among the wheat species and subspecies were recorded . The higturanicum wheat accessions, were gathered in a unique cluster. The population structure of the 126 Triticum accessions was inferred using fastSTRUCTURE, which did not account for linkage disequilibrium (LD) between genetic markers (K) was between 4 and 6 when using the algorithm for multiple choices chooseK.py, with K = 6 as the model used to explain the structure in the dataset (K = 5 (K = 5). The inferred population membership coefficients are shown in K5), in agreement with the phylogenetic , while the rivet wheat accessions were in K1  of the wheat germplasm under study. Sixteen accessions were not included in the analysis based on the twenty-four International Union for the Protection of New Varieties of Plants (UPOV) descriptors  because T. turgidum subsp. turanicum accessions clustered together with ‘Turanicum_PI283795′, an accession from Afghanistan included in the ESS collection since 2012, and ‘Giustalisa’, a Sicilian landrace of durum wheat.The clustering pattern of the morphological traits was calculated based on the Euclidean distance . All of durum-R group were clustered together with two durum wheat accessions . The remaining accessions of the durum-R group were distributed in other clusters together with the durum wheat accessions belonging to the RI and T groups. A slight coloration of the ear characterized the RI and R groups. All of the T group accessions showed a pyramidal ear shape.Another cluster included all seven Calabrian rivet wheat accessions together with BufalaSB02, while the other Sicilian rivet wheat accessions were distributed in different clusters. Six accessions included in the T. turgidum subsp. turanicum (top-left quadrant) and T. aestivum (top-right quadrant), with var3, var5, and var 6 (2 = 0.60)  was included in the bottom-left quadrant. Of the traits, var7 and var10 were the most influential components  of the standardized data was applied to display the wheat morphological trait relationships and their application in germplasm characterization . The firnd var 6  showing  = 0.60) . Likewismponents . OverallA Pearson correlation matrix showed that variables 3, 4, and 5 (glaucosity traits) were highly correlated in a positive manner, as were var 9 and var10 (ear and plant length) . These wT. turgidum subsp. durum (84 accessions) were then evaluated first through an evaluation of their main genetic parameters.The genetic background of the samples that belonged only to eH, oH, AR, and Fis values were calculated  to 0.17 (old durum varieties), as well as a low oH that ranged from 0.15 (old durum varieties) to 0.20 (RI). Allelic richness (AR) was largely uniform in all four groups, with the highest value recorded by the old durum varieties  and ranged from −0.037 (T. turgidum subsp. durum-R) to −0.301 (T. turgidum subsp. durum-RI), which highlighted a slight heterozygous excess.The lculated , which clculated  and morplculated . A low Harieties . The FisThe genetic relationships among the Sicilian durum wheat accessions and the population structure were analyzed using the NJ and DAPC analyses, respectively. Minimum spanning networks (MSNs) were also computed for the same subpanel. The Bayesian information criterion (BIC) method allowed six clusters as the optimal number of groups . MembersFinally, the two accessions ‘Castiglione GL03′ and ‘Castiglione PUB01′ together with the old varieties ‘Gioia’ and ‘Niuru’ were included in pool 4, while the accession ‘Francesa01′ was included in pool 2, which was in agreement with the UPGMA cluster analysis. In contrast, the accession ‘Francesa02′ was assigned to pool 3 together with the old durum wheat varieties .The neighbor-joining (NJ) tree, which included only the accessions belonging to T. turgidum subsp. durum, highlighted a large cluster that gathered all improved durum wheat varieties and the landrace ‘Vera’ A. The redurum group (dark green) widespread in the network and admixed mainly with durum-R samples, which showed a close relationship (durum-RI (blue) and durum-T (deep pink) appeared in distinct nodes and had a greater diversity than the other groups (The minimum spanning network (MSN) confirmed the NJ tree results B, with ationship B. In conr groups B.durum-R and durum-RI groups had been introgressed in the remaining durum landraces/varieties. In contrast, the durum-T group accessions’ gene pool was shown to remain underutilized by breeding.The multidimensional scaling (MDS) plots  illustraTriticum landraces cumulated across the centuries and reached a maximum at the beginning of the previous century when breeders began the distribution of new improved varieties [The presence of wheat in the Mediterranean basin dates to 7000 BP (before the present) ; later, arieties . This wiarieties , althougarieties .From 1929 until 1948, De Cillis headed the “Experimental Sicilian Station for wheat crop” (ESS) at Caltagirone (province of Catania in Sicily), which collected information on the morphological, physiological, and technological traits of Sicilian wheat landraces/varieties. The first description/classification based on the Percival method  placed sMany studies were carried out to characterize and identify wheat species accessions based on morphological characterization according to UPOV traits ,54,55, wThe development of the first molecular marker classes furnished novel tools for avoiding misclassification due to the environment. Among others, the simple sequence repeats (SSR) represent more informative molecular markers that are largely detectable in plant genomes. The genetic diversity of several wheat germplasm collections has been evaluated and distinguished species, subspecies, and genotypes ,56,57,58T. monococcum (EES collection), and many subspecies related to T. turgidum  were included. Our results confirmed the ability of SNP technology to easily distinguish Triticum species and subspecies. In addition, we were able to classify 84 durum wheat accessions  for genetic distances and structures in order to assess their genetic relationships. Moreover, three groups/populations related to ‘Timilia’, ‘Russello’, and ‘Russello Ibleo’ were also included, thereby also defining the intrapopulation genetic variability, as recently reported by Taranto et al. [Several studies assessed the genetic diversity of the Sicilian wheat germplasm described by De Cillis using both SSR  and SNP o et al. .T. aestivum subsp. spelta and four ancient bread wheat landraces collected in Calabria were also included.A study of 56,342 hexaploid accessions from CIMMYT and ICARDA germplasm banks showed that a large portion of genetic variability of hexaploid wheat varieties has not been sampled in breeding selection for modern varieties . In our T. turgidum subsp. turanicum and, more distant, the two emmer wheat varieties: ‘Garfagnana’  and the variety ‘Padre Pio’. This result did not agree with Ganugi et al. [turanicum, dicoccum, and durum accessions.The distinctness between the 14 rivet wheat accessions and the other three tetraploid subspecies, as previously reported ,62, was T. turgidum subsp. turanicum), better known under the brand Kamut, has recently drawn a large commercial interest due to its high protein content and valuable nutritional qualities [turanicum wheat. It was reported that they are related to durum wheat [turanicum and durum wheat varieties [Khorasan wheat  value was close to 1 for the more recent durum wheat varieties (0.130), while the negative values recorded for the T, RI, and R groups highlighted a slight heterozygous excess. Both the ‘Timilia’ and ‘Russello’ accessions belonged to the two ancient wheat varieties, which were widely cultivated in Sicily during the 18th and 19th centuries, respectively. Thereafter, the cultivation of these two accessions spread to many Sicilian areas. The accessions belonging to the RI (‘Russello Ibleo’) group remained confined to their area of origin (Monti Iblei) and appeared to be distinguished by ‘Russello’ accessions. Recently, intra- and interpopulation genetic variations of two large collections of ‘Russello’ and ‘Timilia’ were explored by Taranto et al. [durum-R samples, while durum-RI (blue) and durum-T (deep pink) appeared in distinct nodes with greater diversity than other groups. These results agreed with those previously reported by Taranto et al. [The genetic analysis was then intensified and limited to the o et al. . In agreo et al. .Triticum germplasm collection maintained at ESS highlighted the importance of preserving this precious reservoir of genetic resources that have developed and evolved over the centuries in the Mediterranean environment.Southern European crops after the 2000s have been subjected to high temperatures in summer, early drought, and very hot temperatures at heading, exacerbating the risk of yield loss . Van de T. aestivum L. (19 accessions), and T. aestivum subsp. spelta L. Thell. (one); the cultivated einkorn T. monococcum L. subsp. monococcum (two); four tetraploid subspecies of T. turgidum (104) consisting of 14 rivet wheat accessions ; four T. turgidum subsp. turanicum accessions; two T. turgidum subsp. dicoccum accessions; 75 Sicilian landraces and nine improved varieties of durum wheat . A principal component analysis (PCA) was also performed using the R package FactoMiner [p < 0.05) was also calculated among the morpho- and quality-related traits using the R/Hmisc package . A scatter plot with the correlation coefficients and their significance was developed using the PerformanceAnalytics package in R .Samples of wheat grains were analyzed for 24 main morphological traits . To easictoMiner . Finally®ND-1000 .Genomic DNA was extracted from fresh leaves of young seedlings of each variety. The GenElute Plant Genomic DNA Miniprep Kit  was used. DNA quantity and quality were checked via both electrophoresis (1% agarose gel) and a NanoDropTwo hundred nanograms of genomic DNA were used as template for each reaction in the wheat 90k SNP array  for genoT. turgidum subsp durum (84) collected. The distance-based dendrogram with Nei’s genetic distance [Fst) [Fst values was developed using R/gplots .Phylogenetic and PCoA analyses were performed to estimate the overall relationship among varieties while considering the entire wheat germplasm investigated (126 genotypes) and the samples only belonging to distance , unweighdistance  and R/addistance , respectdistance . The Wrice [Fst)  was compsp durum  collecteK) was computed for the entire wheat dataset using an admixture model performed through fastSTRUCTURE [K) by evaluating the model complexity that maximized the marginal likelihood and model components used to explain structures in the data. The prediction error for each K was also computed using the cross-validation (--cv) function to extract the optimum K. A Bayesian analysis was also carried out for the T. turgidum subsp durum subset (84 samples) using a discriminant analysis of principal components (DAPC) implemented in R/adegenet. The Bayesian information criterion (BIC) method [K-means clustering. The analyses were performed independent of the membership group.The number of genetic pools (TRUCTURE  using thTRUCTURE  and the ) method  was usedT. turgidum subsp durum. The main genetic parameters ) were evaluated using the R/OutFLANK package [T. turgidum subsp durum, T. turgidum subsp durum-R (Russello), T. turgidum subsp durum-RI (Russello Ibleo), and T. turgidum subsp durum-T (Timilia), a minimum spanning network (MSN) was also developed in R/poppr [The filtering procedure and LD (linkage disequilibrium) pruning (r2 = 0.20) resulted in datasets of high-quality SNP markers that were used to evaluate in detail the genetic diversity of samples belonging to  package . A pairw package  was calc R/poppr .Triticum germplasm. The relationships among old Southern Italian landraces, populations, and old and modern varieties were confirmed. We developed a definitive panel of 126 accessions that were mainly preserved at the ‘Experimental Sicilian Station (EES) for Granicoltura’, including the collection derived from the Nazareno Strampelli research activity and described by De Cillis, among which many landraces and old varieties were cultivated in Sicily and Southern Italy since the beginning of the 20th century. They represent an important reservoir of genes/alleles that can be used in durum and bread wheat breeding to counteract the effects of climate change. The adopted germplasm collection and SNP array allowed us to distinguish not only the Triticum species (as was expected), but also many T. turgidum subspecies. Interestingly, we were able to distinguish the subspecies dicoccum and turgidum from most cultivated durum. However, the subspecies turanicum, including the commercial cultivar Khorasan, was not distinguishable. In addition, two Sicilian old landraces/populations, namely ‘Timilia’ and ‘Russello’ and its variant ‘Russello Ibleo’, were characterized among the 84 durum wheat accessions (landraces and old and modern varieties). The old Sicilian landraces/populations were clearly clustered and distinguished from the remaining durum wheat accessions, including the ‘Russello Ibleo’ variant. The present research confirmed the importance of the broad genetic base available not only in the Sicilian landraces, but also among the old varieties cultivated in Southern Italy over the last century and recently re-utilized for their high-quality traits. The interpopulation genetic diversity observed for ‘Timilia’ and ‘Russello’, together with the synonyms and misclassifications of some old wheat varieties, should be taken into account when registering old varieties in the National Registers of Conservation Varieties. By so doing, it will be possible to avoid commercial fraud and contribute to sustaining the profits of farmers that cultivate landraces and ancient varieties.In conclusion, SNPs represented powerful tools for exploring genetic diversity in


Ctenocephalides felis and Pulex irritans can parasitize various animals closely related to humans and are of high veterinary significance.Fleas are the most economically significant blood-feeding ectoparasites worldwide. cox1 and cox2, the population genetics and genetic variation of C. felis and P. irritans in China were further investigated.In this study, 82 samples were collected from 7 provinces of China. Through studying the nuclear genes ITS1 and EF-1α and two different mitochondrial genes C. felis and P. irritans ranged from 0 to 3.9%. The interspecific variance in the EF-1α, cox1, and cox2 sequences was 8.2–18.3%, while the ITS1 sequence was 50.1–52.2%. High genetic diversity was observed in both C. felis and P. irritans, and the nucleotide diversity of cox1 was higher than that of cox2. Moderate gene flow was detected in the C. felis and P. irritans populations. Both species possessed many haplotypes, but the haplotype distribution was uneven. Fu's Fs and Tajima's D tests showed that C. felis and P. irritans experienced a bottleneck effect in Guangxi Zhuang Autonomous Region and Henan province. Evolutionary analysis suggested that C. felis may have two geographical lineages in China, while no multiple lineages of P.irritans were found.The intraspecies differences between C. felis and P. irritans populations. Both species possessed many haplotypes, but the distribution of haplotypes varied among the provinces. Fu’s Fs and Tajima’s D tests indicated that both species had experienced a bottleneck effect in Guangxi and Henan provinces. Evolutionary analysis suggested that C. felis may have two geographical lineages in China, while no multiple lineages of P.irritans were found. This study will help better understand fleas' population genetics and evolutionary biology.Using sequence comparison and the construction of phylogenetic trees, we found a moderate amount of gene flow in the  Ctenocephalides felis and the human flea Pulex irritans are characterized by various hosts, including humans, carnivores, rodents, and ungulates [C. felis and P. irritans are closely related to human and animal life and are, therefore, of high veterinary significance [Fleas are the most economically significant blood-feeding ectoparasites worldwide. They account for 50% of skin diseases in cats and dogs at veterinary clinics , and > $ngulates , 9 and eificance , 10. Undificance , 12.cox1 and cox2, are frequently used to evaluate genetic diversity and identify cryptic species and the population structure of invertebrates [C. felis and P. irritans [With the development of genetic technologies, which complement, to some extent, the limitations of traditional morphology, molecular biology methods have been widely used in taxonomy, systematics, and population genetics, including those of fleas , 14. Thetebrates , 21–23. tebrates . Hornok irritans .C. felis has so far been investigated in Africa [c oxidase subunit I (cox1) and II (cox2) to study the genetic lineages of the C. felis population in Malaysia and revealed two main lineages, with Malaysian haplotypes closely related to tropical haplotypes [The genetic diversity of n Africa , the USAn Africa , 27, Eurn Africa , Asia [2n Africa , 29, andn Africa , 30, 31.plotypes .P. irritans from two populations in Spain and Argentina, using the internal transcribed spacer (ITS)1 and ITS2 and partial cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) genes. The study found a considerable degree of molecular differentiation between the two populations and found two clear geographic genetic lineages, indicating the presence of two cryptic species [In 2019, Zurita et al. studied the classification, origin, evolution, and phylogeny of  species .C. felis and P. irritans. In this study, two nDNA markers, ITS1 and EF-1α, were combined with two different mtDNA markers, cox1 and cox2, to (i) provide detailed information that expands the knowledge of the genetic diversity of C. felis and P. irritans in various provinces of China; (ii) provide a basis for the re-evaluation of the population structure and genetic differentiation of C. felis and P. irritans in China; and (iii) infer phylogenetic relationships from the geographical distribution of different isolates.Fleas have previously been studied in parts of China , 32, but® SV Genomic DNA Purification System; Promega Madison, WI, USA) from the manufacturer. The sample codes, host, locality, and GenBank accession numbers are listed in Table A total of 82 fleas were obtained from different regions of China Fig. . The flecox1 was amplified using LCO1490 (5ʹ-GGTCAACAAATCATAAAGATATTGG-3ʹ) [cox2 was amplified using F-Leu (5ʹ-TCTAATATGGCAGATTAGTGC-3ʹ) and R-Lys (5ʹ-GAGACCAGTACTTGCTTT CAGTCATC-3ʹ) [ITS1 was amplified with the primers Sen-ITS1 (5ʹ-GTACACACCGCCCGTGCGTACT-3ʹ) and Rev-ITS1 (5ʹ-GCT GCGTTCTTCATCGACCC-3ʹ) . The cycTTGG-3ʹ)  and Cff-TTGG-3ʹ) . The cycCATC-3ʹ) . The cyc2O. All PCRs were run on a thermocycler . The results were validated by electrophoresis on 1% agarose gels. PCR products were bi-directionally sequenced by Sangon Biotech Co., Ltd. . BLAST analysis was performed on the sequencing results of four genes of all samples to further determine the species of flea collected.All PCR reactions (25 μl) contained 1 µl of DNA sample, 0.5 μl of each primer, 12.5 μl of 2 × Premix EmeraldAmp Max HS PCR Master Mix , and 10.5 μl of ddHcox1, and cox2 sequences were compared using Clustal X 1.83 software and then manually cut. MegAlign software was used to analyze the sequence differences among four genes [cox1, cox2, and concatenated sequences, DnaSP 5.0 was used to calculate Tajima's D [C. felis in six regions and P. irritans in two regions. DnaSP 5.0 and PopART 1.7 were used to create a statistically parsimonious network to infer haplotype relationships [All  ITS1, EF-1α, ur genes . The Dnaur genes . DnaSP wur genes . For thejima's D  and Fu’sjima's D  statistiionships .cox1 + cox2 of C. felis and P. irritans from China were compared and analyzed. For the EF-1α sequence, Megabothris calcarifer was used as the outgroup, and for cox1 + cox2, Ceratophyllus wui was used as the outgroup.The EF-1α sequence and the tandem sequence of cox1 and cox2 were compared using Gblock 0.91 [Clustal X 1.83 was used to align the sequences of these four genes separately , and theock 0.91 . Using Mock 0.91 . RelativC. felis and 23 P. irritans) from seven provinces in China  were amplified and uploaded to GenBank.A total of 82 samples , while Guangdong and Shanxi provinces had the lowest Fst (− 0.05614). The Fst value of population pairs in Guangdong and Shanxi provinces was not significantly different, and the Fst values of population pairs in Hunan and Guangdong provinces and Hunan and Shanxi provinces were moderate, while the Fst values of population pairs in other regions were significantly different. Among the six sampling regions, the gene flow was the largest in Guangdong and Hunan provinces (1.66) and the smallest in Shanxi and Guangdong provinces (− 9.41). Low levels of gene flow were found between Guangxi Zhuang Autonomous Region and Hunan province, Guangxi Zhuang Autonomous Region and Guangdong province, Jiangsu province and Guangxi Zhuang Autonomous Region, Shanxi and Guangdong provinces, and Shanxi province and Guangxi Zhuang Autonomous Region. Medium levels of gene flow were found between Guangxi Zhuang Autonomous Region and Henan province, Guangdong and Jiangsu provinces, Hunan and Jiangsu provinces, and Shanxi and Jiangsu provinces. High gene flow was found between the other regions. For P. irritans, the level of genetic differentiation between Henan province and Sichuan province was high (Fst = 0.37433), and the level of gene flow was intermediate (Nm = 0.84).According to the known classification criteria, Fst > 0.25, is defined as great differentiation; 0.15 < Fst < 0.25 is defined as moderate differentiation, and Fst < 0.15 is defined as a trivial difference . AccordiC. felis, the Fu’s F neutrality test of cox1, cox2, and the serial sequence were positive, except for the cox2 sequence in Guangxi Zhuang Autonomous Region, and cox1, cox2, and the serial sequence in Jiangsu province. In Tajima's D neutrality test, cox1 from Jiangsu province, cox2 from Guangdong province, and cox1, cox2, and their serial sequences from Hunan province were all positive, while cox2 from Guangxi Zhuang Autonomous Region had no Tajima’s D neutrality test value, and the rest were negative. There were no statistically significant differences except for cox1 in Henan province and cox1 and the serial sequence in Guangxi Zhuang Autonomous Region. The significance of the neutrality test in Guangxi Zhuang Autonomous Region is a sign of population balance. cox2 in Henan province and cox1 and the tandem sequence in Guangxi Zhuang Autonomous Region were positive, indicating that these two regions' populations shrank dramatically because of equilibrium selection and bottleneck effects , 8 haplotypes for cox2 (B1-8), and 21 haplotypes for cox1 + cox2 (AB1-21) of C. felis. The cox1 haplotype A1 (n = 21) was the most common, followed by A4 (n = 9), and the least common types of haplotypes were A6, A7, A8, A9, A10, A11, A12, A13, A15, A16, and A17. In cox2, the most common haplotype was B4 (n = 16), followed by B1 (n = 14), and the least common haplotypes were B6 and B8. In the cox1 + cox2 series, haplotype AB1 (n = 14) was the most common, followed by AB5 (n = 9), and the least common haplotypes were AB4, AB6, AB8, AB9, AB12, AB13, AB14, AB15, AB16, AB17, AB18, AB19, AB20, and AB21.The haplotype distribution of P. irritans, there were 17 haplotypes for cox1 (C1-17), 13 haplotypes for cox2 (D1-13), and 20 haplotypes for cox1 + cox2 (CD1-20). In cox1, haplotype C1 (n = 7) was the most common, while in cox2, haplotype D1 (n = 7) was the most common, followed by haplotype D2 (n = 4) and haplotype D5 (n = 2). In the cox1 + cox2 series, haplotype CD1 (n = 4) was the most common, and all the other haplotypes had one sample.For C. felis and P. irritans was constructed  were distributed in a star shape around the A1 haplotype, which contained 21 individuals, including 4 from Hunan province, 5 from Guangdong province, 5 from Jiangsu province, and 7 from Shanxi province. Henan province had the largest haplotypes, with ten haplotypes , while Guangxi Zhuang Autonomous Region had the fewest haplotypes (A3 and A4). The A4 haplotype was found only in Guangxi Zhuang Autonomous Region, the A6-A13 haplotypes were found only in Henan province, the A14 and A15 haplotypes were found only in Hunan province, the A16 haplotype was found only in Jiangsu province, and the A17 haplotype was found only in Shanxi province  were fewer and less widely distributed  formed a star shape with the C1 haplotype as the center. C1 was the only haplotype that appeared in both Sichuan and Henan provinces. The C2-12 haplotypes were found only in Henan province, and the C13-17 haplotypes were found only in Sichuan province , the D2 and D3 haplotypes were found only in Sichuan province, and the D4-13 haplotypes were found only in Henan province, except D1 was found in both places  and many rare haplotypes , indicating that the genetic differentiation of C. felis and P. irritan was greater. Ctenocephalides felis and P. irritan had moderate gene flow , which may result from restricted gene flow between different regions. China is a vast country with complex terrain. Flea transmission between regions may be limited despite well-developed road transportation and frequent human exchanges, resulting in a low distribution of flea haplotypes and limited gene flow.es Figs. , Table 4Malaysia , 29–31, ustralia . Howeverribution . At the C. felis and P. irritan in this study were compared with previous results. Among them, C. felis from Vietnam and Philippines matched their cox1 gene with haplotype A4 from Guangxi Zhuang Autonomous Region in this study [cox2 gene from Malaysian C. felis studied by Azrizal-Wahid et al. [P. irritans, haplotypes C1 and C10 matched haplotypes H6 and H4 from Spain, respectively [C. felis.To further understand the population outcome and genetic differentiation of fleas, is study . The hapd et al.  was consectively . Other hcox1 + cox2 sequences showed that C. felis could be divided into two different branches, and C. felis from Guangxi Zhuang Autonomous Region had undergone genetic differentiation. Our study identified two clades of C. felis from China through the cox1 and cox2 genes of mtDNA, indicating two geographical lineages. Only one branch in the evolutionary tree was constructed by EF-1α and cox1 + cox2 sequences. There were two geographical lineages of P.irritans in Spain and Argentina in previous studies, with two possible hidden species [P.irritans were found, which may be due to the lack of hidden species of P.irritans in China or the limitation of sample size.Phylogenetic analysis based on  species . In our C. felis populations were geographically isolated, and high levels of genetic diversity and moderate levels of gene flow indicated that C. felis and P.irritans had undergone genetic differentiation and lineages recombination. C. felis may have two genetic lineages, and the Guangxi Zhuang Autonomous Region has undergone genetic differentiation. In contrast, no multiple lineages have been found in human fleas. These results complement previous studies and better understand Chinese fleas population genetics and evolutionary biology.Our study showed that C. felis samples and 23 P. irritans samples from seven provinces in China were analyzed for population genetic variation and structure using the nuclear genes ITS1 and EF-1α and the mitochondrial genes cox1 and cox2. The results showed that C. felis populations were geographically isolated. The high level of genetic diversity and a moderate level of gene flow indicated that the populations of C. felis and P. irritans had undergone genetic differentiation and lineage recombination. The results of the neutral test showed that C. felis and P. irritans in Guangxi Zhuang Autonomous Region and Henan province had experienced equilibrium selection and bottleneck effects. Phylogenetic results indicate that there might be two genetic lineages in C. felis, and genetic differentiation has occurred in Guangxi Zhuang Autonomous Region, while no multiple lineages were found in P. irritans. These results contribute to a better understanding of fleas' population genetics and evolutionary biology and provide a theoretical basis for further studies.In this study, 59