Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97–99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.Hybrid genotypes have been repeatedly described among natural isolates of  Leishmania promastigotes are able to undergo genetic exchange during their growth and development in the sand fly vector, however, it is still not known if they have a true sexual cycle involving meiosis. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of 44 experimental hybrids generated between different strains of L. major, L. infantum, and L. tropica. In almost every case the number of chromosomes and the allele contributions from each parent matched the inheritance patterns expected under meiosis. Rare instances of hybrid chromosomes that did not fit Mendelian expectations were observed, including gain or loss of somy, and loss of heterozygosity. Strong evidence for a meiotic-like process was also obtained from the genome wide patterns of recombination observed in the offspring generated from backcrosses involving an experimental or natural hybrid, consistent with crossing over occurring between homologous chromosomes during meiosis. The frequency and position of the recombination breakpoints observed on each chromosome allowed us to construct the first physical and genetic maps in Leishmania. The results demonstrate that forward genetic approaches are possible for positional cloning of important genes. Leishmania present a remarkable epidemiologic and clinical diversity, producing a spectrum of human and veterinary diseases ranging from localized, self-limiting cutaneous lesions, to more chronic and destructive mucocutaneous involvement, to disseminating, visceral infection that is fatal in the absence of treatment. Leishmania have a dimorphic life cycle consisting of extracellular promastigotes that multiply within the alimentary tract of the sand fly vector, and intracellular amastigotes that multiply within host mononuclear cells. The diversity of clinical outcomes, as well as reservoir host range and vector species compatibilities, have distinct parasite species associations, with over 20 species associated with human infections. The origins of this diversity, whether by gradual accumulation of mutations through mitotic cell division, and/or by sexual recombination producing admixtures of divergent genomes, remain a matter of considerable debate [Protozoan parasites of the genus e debate .Leishmania strains that have been characterized as hybrids is by now well described. Multi locus genotyping using a variety of techniques identified hybrids between closely related New World species [L. infantum and L. major [L. infantum, L. donovani, and L. tropica [L. tropica strains in particular show high levels of allelic diversity and heterozygosity consistent with full genome-hybridization due to natural outcrossing. Experimentally, we and others have demonstrated that inter- and intraspecific hybrids can be generated in the sand fly vector, formally demonstrating that promastigote stages of Leishmania possess the machinery for genetic exchange [L. major, or with L. major and L. infantum, that in every case appeared to be full genomic hybrids based on their bi-parental inheritance of a limited number of allelic markers distributed across the nuclear genome. The majority of the experimental hybrids were close to diploid, though triploid and tetraploid offspring were also observed. Mating competency was confined to promastigote stages developing in the fly, and both Old and New World vector species could support hybrid formation. Based on the experimental outcrosses performed so far in which only a low frequency of co-infected flies yielded hybrids (2–20%), mating must be considered a non-obligatory part of the parasite life-cycle. Overall, the current debate regarding Leishmania reproductive strategies reflects mainly the mode of genetic exchange, its frequency and impact on population structure, not whether or not it occurs [Hybridization, defined as reproduction between members of genetically distinct populations and producing offspring of mixed ancestry , is comm species –7, betwe species –10, and L. major . Using m tropica –14. Someexchange –18. Usint occurs , 20.Trypanosoma brucei, for which a meiotic process is well supported based on the identification of a haploid parasite stage in the vector [Trypanosoma cruzi [Leishmania [Leishmania, although in vitro cell fusion events were recorded in 1990 for two species, L. infantum and L. tropica [Leishmania [Leishmania [Among kinetoplastid protists, the most well studied mating system is that of e vector , and on e vector , 23. Whie vector  and propma cruzi  and Leisishmania , involvi tropica . Each ofishmania , these oishmania . We haveishmania –32, thenLeishmania, including L. major, L. infantum, and L. tropica, we have used whole genome sequencing to reveal the genome structures, chromosome inheritance patterns, and recombination events present in experimental intra- and interspecies hybrids. The highly predictable somy and allele inheritance patterns, and especially the genome wide recombinations observed in backcrosses involving experimental and natural hybrids, provide strong evidence for a meiotic-like sexual cycle in Leishmania.In the current studies of 3 different species of P. duboscqi and Lu. longipalpis sand flies co-infected with different pairwise combinations of L. major strains originating from across the geographic range of this species [P. duboscqi, a natural vector of L. major transmission, and that had an approximate 2n DNA content, for which assessing parental inheritance is more tractable compared to the polyploid hybrids. The parental and 16 progeny clone sequences were aligned to L. major Friedlin FV1 genome Version 6 (LmjFV1.06)(http://tritrypdb.org) using Novoalign (http://www.novocraft.com/), and yielded an average 60x coverage per sample. The mapped reads were processed to obtain total read depth, reference, and alternate allele frequencies using AGELESS software (http://ageless.sourceforge.net/). The analysis identified 76103 homozygous SNPs in the LmLV39/HYG parent in comparison to LmFV1/SAT, or 0.11 SNPs/Kb, and only a relatively low number (3573) of heterozygous SNPs , and the 0.7% of the chromosomes for which a partial or total loss of heterozygosity (LOH) was inferred (highlighted by blue boxes). The LOH events were visualized using bottle brush plots in which the allele count at each SNP position is displayed. Bottle brush plots of representative chromosomes from hybrids showing either balanced contribution of parental alleles, gain of somy, or complete or partial LOH are shown in L. infantum LLM-320 originating from Spain (LiL/HYG), and the cutaneous L. major strain from Israel (LmFV1/SAT) [Lu. longipalpis sand flies, which is a natural vector of L. infantum, as well as a permissive vector in the laboratory for L. major. All of the progeny clones previously reported, including 5 diploid, 4 triploid, and 1 tetraploid hybrid, and derived from 10 different flies, were subjected to whole genome sequencing to further characterize the hybrid genomes. The LiL/HYG parent had 687923 SNPs, or 21 SNPs/Kb, that were homozygous different from LmFV1/SAT, and 12256 heterozygous SNPs  . The co-ous SNPs . The intous SNPs .Leishmania, in that parent. The extra genome was contributed by LiL/HYG to three of the hybrids, and by LmFV1/SAT to one. The 4n hybrid (LimH10) showed a majority of the chromosomes to be tetrasomic with roughly equal contribution from both the parents. The chromosomes showing somies >4 reflected contributions from the parental non-disomic chromosomes, although unexpected extra contributions from either parent were also observed (e.g. chr 1 & 16). Finally, 7 chromosomes (1.9%) showed partial or total LOH, observed in both the diploid and polyploid hybrids (highlighted in blue boxes).The somy profile of the LiL/HYG parent shows that most chromosomes had somy close to 2, while chromosome 33 was trisomic, and chromosomes 26 and 31 were tetrasomic , Fig 2A.L. tropica is the causative agent of zoonotic and anthroponotic cutaneous leishmaniasis (ACL), which is endemic throughout the Middle East and in some areas of Africa and the Indian sub-continent. Prior population genetic studies have identified discreet geographic regions where L. tropica isolates possess only low levels of heterozygous SNPs, and other regions where they display extensive heterozygosity, consistent with genetic exchange [L. tropica strains has not been demonstrated, so far as we are aware. For the generation of experimental hybrids in L. tropica, we introduced stable drug resistance markers into two different strains, LtMA37/NEO originating from Jordan, and LtL747/HYG from Israel. In comparison to one another, these strains possess 157085 homozygous SNPs, or 4.9 SNPs/Kb, and only 5756 heterozygous SNPs , formally demonstrating the mating competency of members of this species. Ten of the 48 double drug resistant lines recovered from 10 different flies were cloned and tested by PCR to confirm inheritance of both parental antibiotic resistance markers  generated from each of five different hybrid lines were selected for whole-genome sequencing. In each case the two clones presented nearly identical genotypes, and are likely derived from the same hybridization event. All of the hybrids were equivalent to F1 progeny, heterozygous at positions where each of the parents was homozygous for a different nucleotide, resulting in each of the hybrids possessing a high density of 155428–166612 heterozygous SNPs (4.9–5.2 SNPs/Kb) . In the Leishmania species demonstrate that the progeny clones are near full genomic hybrids, with highly predictable somy and allele inheritance patterns that are strongly consistent with a meiotic-like process. Rare instances of chromosomes showing a gain of somy or loss of heterozygosity were also observed.Altogether, the whole genome sequencing of the experimental hybrids generated within and between three different Old World Leishmania we chose a L. major hybrid, 1.16.A1, generated between LmFV1/SAT and LmLV39/HYG with ploidy close to 2n, and that demonstrated robust growth in culture and in flies. The mating studies involved co-infection of P. duboscqi flies with 1.16.A1 and L. major lines stably transfected with a third antibiotic resistance marker, blasticidin-S deaminase (BSD), and selection for midgut promastigotes that were doubly drug resistant to either SAT + BSD or to HYG + BSD. A series of 4 independent backcross experiments involving 1.16.A1 and LmFV1/BSD resulted in a low frequency of flies yielding hybrids  in patterns consistent with these isolates being naturally occurring full genome hybrids [Lu. longipalpis flies  and performed the analysis as follows: we leveraged 159900 homozygous SNPs in LtMA37/NEO against the reference strain (TritrypDB L590 V.33) and removed 134846 homozygous SNPs that were common with LtL747/HYG. The SNPs at the remaining 23775 markers were homozygous and different between LtMA37/NEO and LtL747/HYG. We created separate Circos plots for the two groups of outcross progeny, and redrew the plots by coloring the SNPs as green if they matched Lt747/HYG, red if they matched LtMA37/NEO, and yellow if they were heterozygous  was originally isolated from a patient with cutaneous leishmaniasis (CL) acquired in the Jordan Valley, and is heterozygous for an allelic replacement of LPG5B on chromosome 18 by a blasticidin S-resistance (BSD) marker [LPG5B; LmLV39/HYG [L. infantum  [L. tropica lines were generated from strains described previously [L. tropica LtMA37 (MHOM/JO/94/MA37) was originally isolated from a patient with CL in Jordan and is heterozygous for either a G418 (LtMA37 /NEO) or a hygromycin B (LtMA-37/HYG) resistance cassette integrated into the 18S rDNA locus on chromosome 27 using the constitutive expression vectors pLEXY neo2.1 or pLEXY hyg2.1, respectively (Jena Bioscience EGE 273 and EGE-272); LtL747/HYG (MHOM/IL/02/LRC-L747) was originally isolated from a patient with CL in Israel and is heterozygous for a hygromycin B resistance cassette, using the pLEXY hyg2.1 vector; LtKub/SAT is derived from a strain isolated from a patient with CL in Syria (MHOM/SY/?/Kub) and is heterozygous for a Nourseothricin resistance cassette (SAT) also integrated into the 18S rDNA locus on chromosome 27 using the pLEXY sat2.1 vector (Jena Bioscience EGE-274); LtRup/HYG and LtRup/NEO are derived from a strain originally isolated from a patient with CL in Afghanistan (MHOM/AF/87/RP) and are heterozygous for either a hygromycin B or G418 resistance cassette using the pLEXY hyg2.1 or pLEXY neo2.1 vectors, respectively. All lines were grown at 26o C in medium M199 as described previously [A summary of the parental lines that were used to generate the experimental hybrids analyzed in this report, including their heterozygous drug resistance loci, is provided in ) marker ; LmSd/BS) marker  and is d) marker  and contLV39/HYG  is derivLV39/HYG  and is hLV39/HYG ; Lm1.16.LV39/HYG  generateLV39/HYG  is derivd MON-1) , isolated MON-1)  interspeeviously : L. tropLutzomia longipalpis and Phlebotomus duboscqi sand flies, collected from field specimens in Brazil and Mali, respectively, were infected by artificial feeding through a chick skin membrane on heparinized mouse blood as previously described [escribed . The mou6 logarithmic phase promastigotes /ml of blood. Midguts were dissected after 7–11 days, and hybrid parasites were recovered by double drug selection as described previously [For the generation of hybrids, the blood was seeded with an equal mixture of the two parental lines containing independent drug resistant markers at 4–8 x10eviously . For theeviously .Total DNA content and inferred ploidy was determined on the different parental lines and hybrid progeny clones (see previous section) using flow cytometry following staining of permeabilized RNAse-treated cells with propidium iodide, as previously described . GenomicDNAs extracted from most of the lines (see previous section) were submitted to whole genome sequencing in Rocky Mountain Laboratory’s Research Technologies Section-Genomics Unit, Division of Intramural Research, NIAID, NIH. DNAs were sheared using the Covaris LE220 for a 200bp insert target size and the fragmented genomic DNA was purified using AMPure XP beads. Libraries were generated with the ThruPLEX DNA-Seq kit (Rubicon Genomics) and quantified using the Kapa SYBR FAST Universal qPCR kit for Illumina sequencing . The libraries were diluted to 2 nM stocks and pooled equally. The 2 nM pooled stock was prepared for sequencing by denaturing and diluting to 11 pM for clustering to a Rapid flow cell. On-board cluster generation and paired-end sequencing was completed on the HiSeq 2500  using a Rapid Paired End cluster kit and 200 cycle sequencing kit. The cluster density averaged 990 k/mm2 per lane resulting in 163 M reads passing filter per lane with 95% > Q30. The samples were sequenced to target 60x coverage, and achieved median coverages across the genome per sample of between 19 and 38x coverage.L. tropica progeny and parental clones which were sequenced in DNA pipelines at Wellcome Sanger Institute (WSI). Differences were that DNA was sheared to a 400bp insert target, and libraries were generated with the NEBNext DNA Library Prep kit (New England BioLabs). These libraries were diluted to 4 nM stocks, and the 4nM pooled stock was diluting to 8 pM for clustering. The cluster density averaged 550 k/mm2 per lane resulting in at least 100 M reads passing filter per lane with 95% > Q30. The raw data was deposited in the European Nucleotide Archive (ENA) in study ERP111791. These samples were sequenced to target 30x coverage, and after removal of duplicates, achieved median coverages across the genome per sample of between 19 and 38x coverage.Similar methods were used for DNA extracted from the 17 intraspecies L. major FV1 Version 6, L. infantum JPCM5 Version 6, L. tropica L590 V. 33) using novoalign software, and using the parameters -F (format) ILMFQ for Illumina 1.3 pipeline, -H enables hard clipping that removes low quality bases from the 5’end, -g 40 for gap opening penalty, -x 6 for gap extend penalty, -R 5 for difference in score between the best alignment and the second best alignment, -r none ignores the reads with similar scores to multiple locations in the genome, -e 1000 for maximum number of alignments for a single read.All samples were subjected to whole genome sequencing using the Illumina Hi-Seq 2500 platform. The 100bp paired end reads were aligned to the closest available reference genome  by dividing the chromosomes into blocks of 5kb and average coverage within each block was calculated. Punctate regions with coverage greater than twice the average coverage for each chromosome or less than half the average coverage for each chromosome were ignored for somy determination. These regions are associated with repeats, gene families and other genomic noise that alter the somy values. The blocks with zero coverage were ignored from further analyses as they indicate noisy/repeat regions filtered in the previous step. The average of block coverages was scaled to the ploidy of the organism to determine the somies.Somies were determined using AGELESS software  is 1% probability of finding a recombination, the calculated probabilities were converted to cM distances by multiplying by 100.We constructed the genetic map based on the recombinations observed in S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 FigPCR was done on DNA extracted from the parental lines LtMA37/NEO and LtL747/HYG, and from 29 hybrids (LtHLM) generated by crossing these two lines in three different experiments . Blank is PCR mix with no template.(TIF)Click here for additional data file.S2 FigA) PCR for SAT, HYG and BSD resistance genes on the parental lines LmFv1/Sat, LmLV39/HYG, 1.16A1 –hybrid generated by these two lines, LmFv1/BSD and two backcross lines, BXLmA and BXLmB generated by crossing LmFv1/BSD and 1.16.A1. B) PCR for HYG and SAT on the parental lines LmFv1/BSD and LmLV39/HYG, and 10 hybrids generated by crossing these two lines (HFL) in two different experiments (A and B). C) PCR for SAT, HYG and BSD in 3 outcross lines generated in two experiments (LmOXA and LmOXB) in which 1.61.A1 hybrid was crossed with LmSd/BSD. D) PCR for HYG and BSD in 7 hybrids generated by crossing LmSd/BSD and LmLV39/HYG. Blank is PCR mix with no template.(PNG)Click here for additional data file.S3 FigA) PCR on DNA extracted from the parental lines LtMA37/NEO and LtKub/SAT, and 13 hybrid clones (LtHKM), or the parental lines LtL747/HYG and LtKub/SAT, and 42 hybrid clones (LtHKL), generated in two experiments (A & B). Hybrids were recovered on selection medium containing SAT and NEO or SAT and HYG. Blank is PCR mix with no template.(PNG)Click here for additional data file.S4 FigPCR for HYG and BSD on DNA extracted from the parental lines LmSd/BSD and LiL/HYG, and 10 hybrids generated by crossing these two lines. Blank is PCR mix with no template.(TIF)Click here for additional data file.S5 FigA) Schematic showing the possible recombinations assuming not more than two crossovers per chromosome. B) Bottle brush plots of representative chromosomes showing the possible inheritance patterns in the 5 hybrid clones generated between the F1 hybrid, 1.16.A1, and either LmFV1/BSD (BC1-2) or LmSd/BSD (OC1-3). SNPs inheritance from LmFV1 or LmSd are shown on the positive x-axis in red, and SNPs inheritance from LmLV39 are shown on the negative x-axis in green. The vertical distance corresponds to the inferred allelic depth, normalized across the entire genome, which was assigned an average somy of 2.(PDF)Click here for additional data file.S6 Fig6 / ml of either LtRupert or LtKubba log phase promastigotes. Flies were dissected days 2 or 8 post-infection and homogenized midguts scored under a hemocytometer for the number of each of 4 different developmental stages of promastigotes. Values shown are individual flies with geometric means +/- 1 s.d., 10 flies / group at each time point.Flies were membrane fed on mouse blood containing with 4x10(TIFF)Click here for additional data file.S7 FigThe read alignments to the genome were translated into somies and depicted as a heatmap, rounded off to the nearest 0.25 value. Overlaid are the parental inheritance values in the format LtKub-SAT/LtL747-HYG or LtKub-SAT/LtMA37-NEO, rounded off to the nearest 0.1 value. Monosomic chromosomes showing a single parent contribution are indicated by blue boxes.(TIF)Click here for additional data file.


Gallus gallus) are among the few remaining ancestors of an extant domesticated livestock species, the domestic chicken, that still occur in the wild. Little is known about genetic diversity, population structure, and demography of wild Red Junglefowl in their natural habitats. Extinction threats from habitat loss or genetic alteration from domestic introgression exacerbate further the conservation status of this progenitor species. In a previous study, we reported extraordinary adaptive genetic variation in the MHC B‐locus in wild Red Junglefowl and no evidence of allelic introgression between wild and domestic chickens was observed. In this study, we characterized spatial genetic variation and population structure in naturally occurring populations of Red Junglefowl in their core distribution range in South Central Vietnam. A sample of 212 Red Junglefowl was obtained from geographically and ecologically diverse habitats across an area of 250 × 350 km. We used amplified fragment‐length polymorphism markers obtained from 431 loci to determine whether genetic diversity and population structure varies. We found that Red Junglefowl are widely distributed but form small and isolated populations. Strong spatial genetic patterns occur at both local and regional scales. At local scale, population stratification can be identified to approximately 5 km. At regional scale, we identified distinct populations of Red Junglefowl in the southern lowlands, northern highlands, and eastern coastal portions of the study area. Both local and long‐distance genetic patterns observed in wild Red Junglefowl may reflect the species’ ground‐dwelling and territorial characteristics, including dispersal barriers imposed by the Annamite Mountain Range. Spatially explicit analyses with neutral genetic markers can be highly informative and here elevates the conservation profile of the wild ancestors of domesticated chickens.Red Junglefowl ( One explanation posits that wild Junglefowl individuals—including adult males, females, juvenile wild chicks, or wild chicks hatched by domestic chickens—do not tolerate captivity  to augment our trapping efforts.Each trapline was approximately 100 m in length and designed to capture territorial Junglefowl. Traplines were made by modifying vegetation at the capture site into a barrier approximately 1 m high Each captured Red Junglefowl was marked with a uniquely numbered aluminum band . At each capture site, we recorded geographical coordinates with a GPS receiver and characterized major vegetation structure. A small blood sample (20–200 μl) was obtained from venipuncture of the brachial vein and stored in a modified lysis buffer   and 20 U AseI (5′‐AT|TAAT‐3′) at 37°C overnight followed by a 20‐min heat inactivation at 65°C. Digested fragments were ligated to double‐stranded oligonucleotide adapters with overhangs complementary to the digested ends with 400 U T4 DNA ligase  overnight at 16°C. Ligated fragments were diluted 1:4 with 10 mmol/L Tris–HCl (pH 8.0) to produce DNA templates suitable for polymerase chain reaction (PCR) amplification.We generated amplified fragment‐length polymorphism (AFLP) fingerprints . Amplifications with lower amplicon concentration  were diluted 1:9 with 10 mmol/L Tris–HCl (pH 8.0).Preselective PCR was performed in 50 μl volumes containing 10 μl diluted ligation mixture with 1× GoTaq Flexi Buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP, 2% deionized formamide, 0.625 U Taq DNA Pol I and 5 pmoles HPLC‐purified primer EcoRI+CAT labeled with 6‐carboxyfluorescein (6‐FAM), and 25 pmoles AseI+GA or with 5 pmoles HPLC‐purified primer EcoRI+GG labeled with 6‐carboxyfluorescein (6‐FAM), and 25 pmoles AseI+GC. Thermocycling conditions consisted of an initial 94°C denature for 1 min followed by 10 cycles of a one‐min annealing touchdown (1°C decrease each cycle) from 65 to 56°C each with a 72°C extension for 2 min. The selective amplification was completed with 18 cycles of 95°C for 50 s, 56°C for 1 min, and 72°C for 2 min. Selectively amplified PCR products were purified over Superfine Sephadex G75 (Sigma) and stored at −80°C. One microlitre of purified product was combined with 13.5 μl deionized formamide and 0.5 μl Geneflo 625 mobility standard  for electrophoresis on an ABI 3730xl DNA Analyzer .Selective PCR amplification was performed in 25 μl volumes containing 5 μl diluted preselective amplification product with 1× GoTaq Flexi Buffer, 2 mmol/L MgClRawGeno package . On the ABI 3730xl device, replicable generation of AFLP fingerprints yielded amplicons with a relative fluorescence intensity (RFI) between 500 and 15,000. Peaks with RFI beyond this range were eliminated from the analysis. Comparing bins created by RawGeno and Dax  helped to evaluate discrepancies in marker assignment. In cases where bins of markers did not match, visual inspection and manual bin reconstruction were performed to correct any errors in marker assignment.Fingerprints of AFLP markers were categorized (binned) by a criterion of size‐homology by a partially automated scoring process procedure. We used 2.3R‐package ecodist . Applications of individual‐based approaches and population‐based approaches in AFLP data have been reviewed and discussed  and population approaches based on allele frequencies (genetic diversity estimates and Bayesian clustering) in our study. At the individual level, we calculated coefficients of similarity Jaccard,  for Red 2.4R‐package ade4  function , proportion of polymorphic sites, and pairwise FST. The significance of the latter was tested with a randomization procedure consisting of 1,000 permutations to create a 95% confidence interval (CI). This procedure tests the null hypothesis of no genetic differentiation among the populations. If the value of FST at the 5% rightmost portion of the CI is less than the observed FST, the null hypothesis is rejected. We used the rarefaction function in the R‐package vegan  iterations, thinning by a factor of 100. A posterior burn‐in of 2,000 iterations was allowed . We ran each model 1,000 times on UW‐Madison’s HT Condor computer cluster and computed the average posterior distribution of the modal ΔK population. We also constructed a dendrogram of all replicates using average linkage of unweighted pair group method with arithmetic mean (UPGMA). The UPGMA topology reflects the mean posterior probability of common genetic cluster memberships observed from the MCMC estimation of ΔK. Importantly it does not depict phylogenetic relationships. A distance metric of 0.00 indicates that two Red Junglefowl were always placed in the same population cluster in 1,000 replicates, whereas a distance of 1.0 indicated that the two Red Junglefowl were never grouped together in any of the replicates. Red Junglefowl that consistently changed their memberships in both global and local clustering models may represent individuals sampled from rare, under‐represented, or cryptic populations.We used the Bayesian clustering method in GENELAND 4.0.4  were parameterized similarly to the models with all samples included . Correlogram models were calculated only at a local scale and had discrete 1 km distance classes up to the maximum sampling distance.Apart from the stochastic nature of the MCMC sampling and the high dimensionality of the dataset, biological phenomena will influence how individuals are apportioned into specific population. First is the relative relationship between our sampling scale and the spatial range of the Red Junglefowl, which we assumed is modulated by home‐range demography and possibly the intrinsic quality of the sampled habitats. Increased habitat connectivity may increase dispersal, thereby preventing genetic differentiation, ultimately reducing the resolution of inferred genetic clusters. Second, as our sample design was opportunistic within sampling sites, some Red Junglefowl may be sampled from cryptic linages or populations that are genetically quite different than the average genetic difference of Red Junglefowl in our sampling pool. If individuals meeting these criteria are sampled at low density, assignment into specific populations will be more difficult to achieve, particularly at the global scale of our sampling design. We addressed the global sampling issue by running additional Bayesian clustering, as well as sPCA ordinations, for each individual sampling site. The additional models for the local sampling sites  were polymorphic, that is, had both the presence and absence of marker bands.A minimum of two and a maximum of five replicate AFLP fingerprints were generated for each individual bird. After marker binning, the two selective primer pairs yielded 431 replicable polymorphic AFLP loci ranging from 50 to 616 bp, of which 90%  separated Red Junglefowl by their sampling locations. Noticeable structure within our two largest and least disturbed sampling sites  was also evident. The second PC  and third PC  supported the observed patterns in PC 1 with CTN and YDN, again, have noticeable within‐site structure. Red Junglefowl sampled in HBA, which is in the northeast foothills of the Annamite Mountain Range, occupied central positions in the PCA plot and then showed some overlap with the Red Junglefowl in CTN and YDN, both of which are further west of the Annamite Mountain Range. This observation implied some low level of genetic exchange among Red Junglefowl across the Annamite (c.f. stepping‐stone model), but it could also be an artifact attributable to both low power and low resolution of PCA model.We observed noticeable patterns of Red Junglefowl in our PCA model Figure a. The fiApplication of the sPCA model at a global scale resulted in high positive eigenvalues and uniformly low negative eigenvalues Figure b. CombinSpatial autocorrelation analyses of cumulative distance classes indicated that Red Junglefowl exhibited genetic correlation at fine scales and were very site specific Appendix B. HBA  was 0.1028 (95% CI −0.0106 to 0.0111), a value nearly identical to that calculated with AMOVA  . Each primary population contained private alleles, which were most abundant in YDN Table . The oveK of nine clusters from the seven study sites . In both the spatial and nonspatial models, the modal number of populations was ΔK = 9  (Appendix The nine genetic clusters include three (or four) distinct clusters for each of three sites west of the Annamite  and five clusters loosely representing HBA Junglefowl and Junglefowl from the other sites . Although cluster membership in this topographically heterogeneous landscape appears stable Figure , the latK of three clusters , the overall FST increased to 0.1974 for the entire sample, ranging up to 0.3169 between individual sites, with the majority of among‐group comparisons greater than 0.1500. This confirmed the strong population structure of Red Junglefowl in South Central Vietnam as already observed by the ordination and Bayesian methods.As commonly done, calculation of ST Table  was bases Figure a, and ats Figure b. EstimaHedrick, , as woul44.1We found large amounts of genetic diversity and strong population genetic structure in wild Red Junglefowl at coarse geographic scales spanning the Annamite Mountain Range of South Central Vietnam. At a local scale, we also found strong evidence of fine‐scale genetic subdivision at distances as low as 5 km. The average sampling distance of 1.12 km in our study appears to be appropriate for detecting genetically divergent groups of Red Junglefowl, especially as we did not detect IBD at distances greater than 5 km. Here, we discuss the importance of genetic structure and the scale of spatial ranges in wild Red Junglefowl, and the implications of these results for genetic management and conservation.At a broad  scale, the Annamite Mountain Range is likely an impassable barrier limiting long‐distance dispersal of ground‐dwelling organisms, including Red Junglefowl. Although it has been reported that Red Junglefowl occur at elevations up to 1,800 m . We also found strong differentiation between sites in the southern lowlands (CTN) and eastern coastal region . Biogeographical and climatic phenomena may help explain this result beyond a distance‐only interpretation. South Central Vietnam is considered at the convergence of two biogeographic regions, mostly due to the Annamite Mountain Range: the Annamese Mountains Region consists of subtropical drier monsoon habitats in the northwest uplands (including BDP and YDN) and the Cochinchina Region of moist tropical lowlands in the south and an acrid microclimatic region in the eastern coast (including other five sites—HBA shares some Annamese habitats)  phenomenon, where distantly related lineages are incorrectly inferred to be closely related  metapopulation structure of Red Junglefowl in South Central Vietnam. Our current evidence suggests that a fragmentation model does not apply. Overall, we observed well‐defined geographic distributions and inferred some evidence of admixture between sites with close geographic proximity, for example, between the southern lowland sites , the northern highlands , or between the apparent panmictic coastal sites . Long‐distance genetic similarity was rarely observed, and if so it may have resulted from a LBA‐like phenomenon. A classic stepping‐stone model may also not be suitable as we did not find strong correlation between geographic distances and genetic dissimilarity (IBD) at all of our primary sites. Increased sample numbers and additional study of Red Junglefowl demography may resolve this issue. Although we cannot be certain with the current data, we conclude that these observations at least corroborated a classical metapopulation structure in Red Junglefowl. Moreover, a metapopulation structure resulting from fragmentation of a formerly continuous population or a model of completely subdivided populations is not supported either as we observed clear differentiation between inferred genetic clusters, which generally occurred in contiguous and suitable habitats.4.2The MCMC procedure in any Bayesian clustering method—including the GENELAND models employed here—functions by randomly resampling the data as a basis for its inference. When a regular increase or decrease in correlation between genetic variability and geographic distance occurs , it may disrupt the resampling process and generate false‐positives in Bayesian randomization  does not hold at very fine scales, as most living organisms, including Red Junglefowl, are genetically related when they are sampled at close enough distances , we assayed MHC B‐locus haplotype variation. We found 310 of 398 (78%) unique Red Junglefowl haplotypes. Of the 310 unique haplotypes found, none were identified in any commercial  or local heritage chickens (n = 32)  were collected in Vietnam, Laos, and Indonesia”  and the geographic scale sampled , the amount of genetic differentiation observed among wild Red Junglefowl populations in South Central Vietnam (this study) was much greater than in the Okumura et al. study. Indeed, the low levels of Adding adaptive analyses to discussions of wild Red Junglefowl strengthen the appeal of preserving wild Red Junglefowl and other heritage breeds of poultry. For example, conservation of genetic diversity in agriculturally important species is also required for sustainable agriculture. Climate change, newly emergent diseases, increased pressure on land and water resources, and shifting market demands require that [domestic] animal genetic resources are also conserved and used sustainably. Wild Red Junglefowl and heritage poultry breeds certainly experience adaptive selection, acquiring traits allowing them to adapt to differing local environments and cultural conditions, which are unlike any encountered in modern agriculture. The unusually high variation of MHC B‐locus in Red Junglefowl reflects a substantial evolutionary solution to pathogen resistance, a matter of great concern in commercial poultry operations. Similar to the biological conservation of wild species, the potential to augment commercial poultry genetic management is substantial and both wild Junglefowl and heritage poultry breeds should be considered an invaluable genetic reservoir well worth protecting.None declared.HNP and MEB conceived the ideas and wrote the manuscript; HNP conducted the field work with assistance from MEB; HNP and MEB analyzed the data. All authors gave final approval for publication. Click here for additional data file. Click here for additional data file.


The International Institute of Tropical Agriculture maintains the world’s largest collection of cowpea germplasm of over 15,000 accessions. A sub-set of 298 lines from the loosely composed mini core collection of 370 landraces were genotyped based on genotyping by sequencing (GBS). Ward’s minimum variance hierarchical cluster analysis, model-based ancestry analysis and discriminant analysis of principal component (DAPC) were carried out on this sub-set. Three clusters were identified by the different clustering methods. Principal component analysis further supported the three clusters especially when accessions are scattered along the axes of the first two principal components. The first two principal components explained a total of 22.30% of the variation. Cluster one comprises 115 accessions from the largest number of countries and has the highest gene diversity, heterozygosity and polymorphic information content (PIC) values. Cluster two is made up of 102 accessions, 90 percent of which are from West and Central Africa. Analysis of molecular variance shows that the most variation is among accessions and lowest among clusters. No cluster is made exclusively of accessions from a single country. Based on SNP markers, the sub set of cowpea mini core germplasm collection used in this study encompasses the diversity in the crop. More than 75% of the over 15,000 accessions of cultivated cowpea germplasm collected from about 90 countries4 and maintained at the International Institute of Tropical Agriculture (IITA) are from the West Africa sub-region5. Arguably, this supports the assertion that the greatest diversity exists in the sub-region which therefore confirms the area as the crop’s center of origin6. Besides IITA, the United States Department of Agriculture – Genetic Resources Information Network (USDA-GRIN) at Griffin USA7 and University of California, Riverside, USA5 are also conserving collections of about 7,737 and 6,000 cowpea germplasm accessions respectively. Sustained progress in the development of new improved varieties with desirable attributes in any crop depends heavily on the available germplasm, hence the need for their collection and conservation. Such collections are sources of genes needed for enhancing productivity of new, improved varieties in the short, medium and long terms. Due to the often large number of accessions in gene banks, many of them are typically not utilized for crop improvement. To overcome this challenge, the concepts of core8 and mini core collections9 were proposed in which about ten- and one- percent of the total germplasm collections respectively, are selected to be representatives of the crop’s genetic diversity. The smaller number in the core or mini core collections makes characterization and exploitation of the germplasm for crop improvement more realistic10.Cowpea is one of the important grain legume crops in sub-Saharan Africa (SSA) where it contributes to food and nutritional security, fodder for livestock, soil fertility following N fixation as well as income for farmers and food vendors. It is native to SSA where the greatest genetic diversity for this crop exists. While the centre of diversity of cowpea’s wild progenitor is suggested to be in eastern and southern Africa, based on the presence in the sub-region of several primitive, weedy and wild typesVigna which comprised cowpea accessions and some of its wild relatives11. The RFLP markers separated accessions of cultivated cowpea from the wild relatives as well as from the Asiatic Vigna species. Gene derived markers and sequencing were successful in revealing more polymorphisms among Vigna species than within species12. Genetic variation among cowpea breeding lines was evaluated using simple sequence repeats (SSR) markers and a high level of homozygosity detected13. The study also revealed that some recent breeding lines derived from crosses involving several unimproved germplasm lines showed relatively higher levels of genetic diversity. One thousand two hundred  single nucleotide polymorphism (SNP) markers were used to analyse genetic variation among 422 cowpea accessions and the study revealed the existence of two cowpea genepools, one for West Africa and the second for East Africa5. These authors also reported that genetic variation among landraces from outside of Africa was slightly higher than within African landraces.Some studies aimed at understanding the genetic diversity in cowpea using molecular markers have been reported. Restriction fragment length polymorphism (RFLP) markers were used to study genetic variation in the genus 14. The genome-wide molecular marker discovery, highly multiplexed genotyping, flexibility and relatively low cost make GBS an excellent tool in plant genetics and breeding16. GBS is increasingly being used in both genetic diversity analyses of cultivated and wild crop relatives18, development of genetic linkage maps19, genomic selection and genome wide association studies21 GBS was used to study genetic diversity among 768 cowpea germplasm lines and the results indicated that the route of the migration of cowpea germplasm to different parts of the globe could be traced to the two candidate original areas of West and East Africa7.Next-Generation Sequencing (NGS) based genotyping procedures such as Genotyping-by-Sequencing (GBS) represent a high-marker density approach compared with previous technologies such as RFLP, amplified fragment length polymorphism (AFLP) and SSR. GBS is based on reducing genome complexity with restriction enzymes, coupled with multiplex NGS for high-density SNP markers discovery22. On the other hand, it was reported that in grape the core lacked a quarter of alleles found in the whole sativa collections when SSR markers were used23. However, each of the three grape core collections contained almost the whole diversity of the crop when 704 SNP markers were utilized. This suggests that there would be limitations in interpreting results of genetic diversity in grape based on SSR. In addition, many of the marker systems are constrained by high costs required for large-scale analyses24. The allele specific SNP markers have limitations as they require upfront investments including time and cost for discovery of informative SNPs and assay development. However, the high throughput genome complexity reducing procedures such as GBS have advantages over the conventional and allele specific SNP markers as they enable simultaneous discovery and genotyping of genome wide SNP markers within a shorter time and at lower cost.Despite their usefulness in studying genetic diversity in crop species, different types of molecular markers are not equally informative. For example, SSR markers were found to be more informative in the classification of some wild rice lines when compared to SNP even though a higher number of the latter marker type was detectedet al.25 reported that molecular markers detected significant gene flow in the United Kingdom’s Beta maritima populations whereas isozymes analyses suggested very little gene flow. The authors explained this observation as evidence of selection for traits associated with isozymes. A study of closely related Avena barbata and A. hirtula and the analysis of multi-locus dynamics through several generations support that inbreeding, even when coupled with tight linkage does not necessarily hold the genotypes together26. However, if the adaptive properties are due to associated loci, they behave as a virtually indivisible unity with the genetic markers analysed. The development and maintenance of plant’s adaptiveness within populations are favoured by self-pollination which also enhances the development of spatial differentiation27. It is advisable, therefore, that when making germplasm collections for conservation purposes, efforts should focus not only on alleles that contribute to adaptiveness but also multi-locus combinations of alleles that interact with one another in an epistatic favourable way26. In the current study, we carried out GBS on 298 of the cowpea mini core lines to understand the underlying genetic diversity and population structure among the cowpea germplasm assemblage maintained at IITA.As much as molecular markers can help in understanding the extent of genetic diversity in crop germplasm and its management, caution need be exercised in applying results obtained from molecular marker-based diversity studies especially as they pertain to germplasm conservation efforts. This is because in the process of evolution, adaptiveness plays a major role in the survival of individuals in populations and it is not easy to determine if plant selection acts on markers directly or on associated loci responsible for adaptive properties. Raybould The 298 lines used in this study were collected from 50 countries and they make up most about 81%) of the cowpea mini core  from discriminant analysis of principal components (DAPC) to determine the appropriate number of sub-populations show rapid decline from K = 1 to K = 3 and from 1 to 3 respectively Fig. , indicatThe distribution of the tested cowpea mini core lines based on hierarchical clustering dendrogram Fig. , the modMembership clustering for DAPC ranged from 91 to 107. Group one is made up of 100 accessions with most accessions from Nigeria. Group two has 107 accessions with majority of them from Nigeria and Niger while group three has 91 accessions with most of the accessions coming from India and Egypt. Group membership from DAPC is to a large extent in agreement with the hierarchical dendrogram clustering. Group one has 99 accessions in agreement with the hierarchical dendrogram out of the 100 members assigned by the DAPC while group two has 100 accessions in agreement with hierarchical dendrogram out of the 107 members assigned to the group. The third group with 91 accessions has 81 accessions in agreement with the hierarchical dendrogram.Principal component analysis (PCA) was performed and it further supported the groupings of the samples based on the membership assignment from DAPC Fig. . The firDiversity indices statistics for the three observed groups show that, irrespective of clustering method used, group one shows the highest polymorphic information content, number of effective alleles, expected and observed heterozygosity Table . HoweverAnalysis of molecular variance was also carried out on the identified groups on the hierarchical dendrogram and DAPC. The results of analysis are identical irrespective of the clustering method used for the structure analysis. The observed variance partitioned among the three groups is 16 percent, 66 percent of the variance partitioned among individual accessions and 18 percent within the accessions Table .Table 3AST = 0.16, F’ST = 0.24), indicating that the groups are relatively genetically distinct. The FIS and FIT values are 0.78 and 0.82 respectively indicating that the cowpea lines making up the groups are inbred lines.There is a moderate amount of differentiation between the three groups  the domesticated form was derived from a narrow selection of the wild germplasm and 2) chloroplast gene flow between wild and cultivated types has been very limited. In addition, the authors detected no homoplasy in tested cultivated and wild relatives following construction of a hierarchical tree. Genetic diversity was reported to be low among cowpea populations collected from Benin Republic30, Ghana31 and Sudan32. In explaining what could be responsible for the narrow genetic base for cowpea Coulibaly et al.1 suggested that a single domestication event may have occurred between cultivated and the wild progenitor and this concurs with one of the conclusions reached by Vaillancourt and Weeden29.The world’s largest cowpea germplasm collection maintained at the International Institute of Tropical Agriculture (IITA) headquarters in Ibadan Nigeria has more than 15,000 landraces and over 2,000 wild relatives. These accessions have served and continue to serve as sources of genes for desirable traits contributing to the successes being recorded in variety development especially in sub-Saharan Africa. In this study involving a sub-set of the mini core collection, a low number of robust SNP markers was observed which can be attributed to the non-availability of reference SNP discovery pipeline (when the study was performed) or the acknowledged narrow genetic base of cowpeaIS mean value of 0.75 obtained across samples indicated that most accessions are inbred. Xiong et al.7 also reported low heterozygosity among the 768 cowpea accessions used in their diversity study. These observations are to be expected since cowpea is a highly self-pollinated crop. Depending on the genotype and environment the extent of outcrossing in cowpea is low and could range from less than 0.15 to up to 1.58%33. However, He = 0.4344 in a population comprising 105 cowpea accessions from Kenya, Niger, Nigeria and China has recently been reported34.The relatively high Fet al.7 that reported a diverse set of 768 cowpea germplasm maintained at United States Department of Agriculture – Germplasm Resources Information Network (USDA-GRIN) formed three well defined groups and Qin et al.35 who also found three clusters among 369 accessions belonging to the USDA’s core collection. It can therefore be stated, based on the results of the present study, that the set of mini core lines we have used represent a significant amount of the diversity present in the world cowpea collection maintained at IITA. Hence, the test lines represent a broad picture of the crop’s genetic diversity. It is also worth noting that the 369 USDA cowpea core collections came from 47 countries whereas the 298 mini core accessions we used are obtained from 50 countries.The sampled mini core accessions of the world cowpea collection maintained at IITA and used in the present study are distributed into three main clusters on the dendrogram generated following hierarchical cluster analysis. These three clusters are also identified by DAPC and ADMIXTURE. The distribution of the germplasm accessions into three main clusters in this study agrees with the findings by Xiong et al.34 who found that two of the four clusters from their study were made up of accessions from Niger and Nigeria and the other made of accessions from Kenya. The identification of three clusters (gene pools) each from America (North America and Latin America), the three regions of Africa  and Central West Asia, Europe and Oceania were reported7. The composition of the three clusters identified in our study does not agree with these observations when sources of their origin are considered. Rather, our results align more with those of Huhyn et al.5 in their study in which they identified three gene pools among 422 accessions that included 46 wild cowpea relatives.Two of the three groups formed in this study have most of their members collected from different parts of Africa thus agreeing with results by Chen 36. Many authors have submitted that the center of diversity for cultivated cowpea is the West and Central African sub-region37 from where it moved to the other parts of the world - Asia, North and South America and Europe38.The presence of accessions from West and Central Africa especially Nigeria in each of the three groups is further evidence that the sub-region is a centre of diversity for cultivated cowpea4. However, many of the accessions from West and Central Africa constitute the majority of members in group two while most members of group three are from East and Southern Africa. Clustering of accessions according to countries of origin varies across crop species. For example, in African rice (Oryza glabberima), a self-pollinated crop like cowpea clustering of genotypes was more according to country of origin than to agro-ecology39 whereas in lentil only a weak correlation was observed between geographic origin and genetic relationships among tested landraces40. Cowpea lines from Niger and Nigeria clustered together and separated from those collected from Kenya and China34. Clustering together of genotypes from Niger and Nigeria was attributed to their coming from similar agro-ecologies34. In their study Xiong et al.7 reported no clustering of cowpea genotypes according to country of origin. However, geographic variation in germplasm distribution is nearly always impossible to separate from ecologically determined variation hence the recognition of ‘ecogeographic factors’ in plants’ genetic diversity41. It is reasonable to believe that the cowpea germplasm lines have remained in West and Central Africa sub-region over a long period after having been domesticated there. The plants have become adapted to the agro-ecologies prevalent in the sub-region rather than countries especially since a common farming system of intercropping is mostly practised in the different countries. Also, the high level of self-pollination, as in cowpea, may have resulted in the rapid fixation of alleles and accumulation of mutant genes in tomato42. It is also worth noting that movement of seeds among farmers in the West and Central Africa sub-region is not limited by national boundaries as farmers in neighbouring communities exchange seeds freely with one another39 and farmer-to-farmer seed movement has been recognised as a means of disseminating seeds of newly improved varieties.The germplasm lines making up the bulk of IITA’s cowpea mini core did not cluster according to countries of origin despite that geographical location along with agronomic and botanical descriptors were used to define the core collection35. Low levels of precipitation in the areas of sub-Saharan Africa (SSA) where cowpea is mostly found may partly contribute to the relatively lower genetic diversity in lines from the region as compared to those from other parts of the world where more precipitation occurs. For example, an appreciable level of genetic diversity has been reported among yard-long-bean  found mostly in the moist Asian countries especially China and India43. The yard-long-bean evolved in Asia from the cultivated cowpea introduced originally from the savannahs of SSA where they are not found. The greater precipitation and lower sunlight in Asia was suggested as being responsible for the evolution of the yard-long-bean from cowpea5. A relatively higher genetic diversity has been reported in tomatoes from the western Galapagos islands and this was attributed to the higher precipitation in the area44. It has also been suggested that plant species richness is correlated with annual precipitation as observed in the Neotropics45.The genetic diversity within cluster two of which 92 percent of members are from West and Central Africa is lowest of the three clusters. Total genetic variation among cowpea accessions from outside of Africa was slightly greater than in the African landraces7.In this study, members of the cluster with the most number of accessions (115 accessions) are from 27 countries 16 of which are from outside of Africa and 11 African countries. This also showed the highest level of genetic variation among the three clusters. The higher genetic variation observed among accessions in this most cosmopolitan cluster may be the result of the high number of countries from where the members are collected. Most genetic variance in the USDA cowpea world collection was found within, instead of, among geographic regions and within instead of among countries34. There is therefore, in cowpea higher genetic divergence among individual accessions which could not be explained by the groups. However, in African rice molecular variance was reported to be highest within than among groups when based on structure and cluster analysis and the diversity could not be explained by ecology39.The analysis of molecular variance (AMOVA) showed that highest variation was present among accessions followed by within accessions and smallest among clusters. Similarly, the highest amount of variation was found among individuals, followed by within individuals and least among populations7. It can be suggested that many parental lines used for making crosses in the USA while generating breeding lines may be of West African origin. Whit46 suggested that cowpea came to the USA through slaves who may have brought them along from West Africa. We also observed that some USA lines are in close proximity with accessions from Egypt and India. This may justify the findings that some of the USA lines came through other sources than from only West Africa5. It had been suggested that cowpea germplasm was also dispersed from sub-Saharan Africa to Europe and elsewhere through Egypt in North Africa47.Accessions from the USA were distributed among different clusters but the most number were found in cluster one where they are found to be closely associated with accessions from West Africa thus suggesting that relatively high similarities exist between USA and West African lines. The observed 86% similarity between American breeding lines and accessions from West Africa agree with this observation49. Available records show that the work carried out at the Agricultural Research Station, Bihar, India in which crosses were made to generate segregating populations from where early maturing lines with economically desirable traits were selected may probably be one of the earliest in the history of cowpea variety development50. Although most cowpea is produced and consumed in Nigeria, only limited improvement activities were carried out in the country until 1960s. Widespread dissemination of improved varieties to farmers in the West Africa sub-region is even more recent and can be traced to the establishment in early 1980s of the Semi-Arid Food Grain Research and Development (SAFGRAD) Program with support from the USAID.The bulk of cowpea accessions maintained in the Genetic Resources Center at IITA (>71%) are from Africa and would therefore be mostly landraces. These lines would to a very large extent represent the diversity present in the indigenous cultivated cowpea. The history of cowpea variety development in Africa is recent. Early reports on genetic studies in cowpea are from the USA and date back to early 1900s. Inheritance of several seed related traits in cowpea were the earliest reported studies from the USAThis diversity study was carried out using genotyping by sequencing on a sub-set of the mini core from the world’s largest assemblage of cowpea germplasm collections and the results further confirm West and Central Africa as center of origin for cowpea. Further, the constitution of the mini core collections was successful in encompassing the diversity that may be present in the cowpea gene bank maintained at IITA as three groups were identified which is in concurrence with previous diversity studies in cowpea. It can be inferred from the study therefore that SNP markers are effective in placing cowpea germplasm lines in appropriate clusters based on relationships at molecular level and with the current accumulation of genomic tools in cowpea the crop promises to benefit from the deployment of these tools especially in the development of new varieties that should meet the immediate and future challenges facing its productivity.51 with some modifications. DNA concentrations of samples were normalized and working solutions contained between 10–100 ng/µl. Ninety-five samples were placed in a 96 well plate. As a DNA quality measure, 5 µl of each of 10 samples in a plate were digested using EcoRI restriction enzyme and run on a 1% w/v agarose gel along with the λ HindIII size standards.Seeds of the 370 accessions making up the cowpea mini-core collections were obtained from the IITA’s Genetic Resources Center, sown in pots containing 5.0 kg top soil and placed in the screenhouse. DNA was extracted from a newly expanded young trifoliate leaf of emerged seedlings of each line using SDS method14. A total of about four 96-plex GBS libraries were constructed and sequenced on the Illumina HiSeq 2000. Due to unavailability of reference genome (as at the time of the sequencing), SNP variants were discovered using the UNEAK pipeline18 (http://www.maizegenetics.net/gbs-bioinformatics) as implemented in TASSEL Version: 5.052 . In summary, reads were trimmed to a 64-bp length and unreliable markers filtered out using a network filter, designed to detect and eliminate markers showing unexpected relationships with others that could be a result of paralogous sequences and/or sequencing errors18. A total of 121,910 SNPs was called out of the UNEAK pipeline. The called SNPs were further filtered using TASSEL software52. SNPs with more than 20% missing data were removed as were those with minor allele frequency (MAF) below 0.05. Non-polymorphic markers i.e., having a variance close to 0 were identified (only six markers were discarded based on this criterion). SNPs resulting from sequence paralogy were further removed, resulting in a final set of 2,276 SNPs.Genome reduction for sequencing was achieved by digesting genomic DNA with the restriction enzyme, ApeKI, which recognizes a degenerate 5 bp sequence , and sequencing was performed following the standard procedure53, power marker54 and PLINK55.Diversity analysis was carried out using the filtered 2,276 SNPs. Genetic diversity parameters which include minor allele frequency (MAF), polymorphic information content (PIC), the effective number of alleles (Ne), expected heterozygosity (He), observed heterozygosity (Ho) and inbreeding coefficient (Fis) were established using GenAlex version 6.4156 and 3) assumption-free discriminant analysis of principal components (DAPC)57. For the hierarchical clustering, the pairwise genetic distance  matrix was calculated among all individuals using PLINK55. A Ward’s minimum variance hierarchical cluster dendrogram was then built from the IBS matrix using the Analyses of Phylogenetics and Evolution (ape) package58 implemented in R59.Population structure was characterized using three complementary approaches: 1) distance-based hierarchical clustering analysis; 2) a model-based maximum likelihood estimation of ancestral sub-populations using ADMIXTURE56. For the analysis, the number of subpopulations, K, varied from 2 to 10. According to ADMIXTURE’s cross-validation procedure a good value of K will exhibit a low cross-validation error compared to other K values55. Hence, the most appropriate K value that is useful and better describes the data and also has good correspondence with the clustering pattern obtained by the hierarchical tree, was selected after considering the 10-fold cross-validations.In the second approach, population structure and accession ancestry were determined using the ADMIXTURE method which assumes linkage equilibrium among loci and Hardy-Weinberg equilibrium within ancestral populationsST) and standardized FST  of the observed sub-clusters were assessed using analysis of molecular variance (AMOVA) implemented in GenAlex 6.41 and the genetic diversity parameters of each observed cluster determined. Genetic diversity parameters were also calculated for each of the identified sub-clusters.To complement the results from ADMIXTURE, we carried out DAPC using the R package ‘adegenet’. The optimal number of clusters from DAPC was inferred using k-means analysis by varying possible number of clusters from two to 40 using a Bayesian Information Criterion (BIC) to assess the best supported model. The number of clusters at which BIC decreases were considered to determine the most likely K value. Following the information based on hierarchical clustering and ADMIXTURE analysis the most appropriate K expected to provide useful summaries of the data was selected. DAPC clustering was thereafter performed on the clusters identified using the first 70 principal components. The membership probabilities of each individual for the different groups were obtained from DAPC and the results of DAPC analysis, ADMIXTURE, and the hierarchical tree were compared. As sub-clusters were observed within the mini core samples, the fixation index (FSupppementary Table 1Supplementary Table 2


As an indicator of educational opportunity, social scientists have studied intergenerational mobility—the degree to which children’s attainment depends on that of their parents—and how it varies across place or time. We combine this research with behavior genetics to show that societal variation in mobility is rooted in family advantages that siblings share over and above genetic transmission. In societies with high intergenerational mobility, less variance in educational attainment is attributable to the shared sibling environment. Variance due to genetic factors is largely constant, but its share as a part of total variance, heritability, rises with mobility. Our results suggest that environmental differences underlie variation in intergenerational mobility, and that there is no tension between egalitarian policies and the realization of individual genetic potential. Intergenerational education mobility—how strongly educational attainment persists from parent to child—is commonly used to indicate societies’ degree of openness or equality of opportunity , 2. A li5In other words, it matters not only whether education is inherited, but also how . One appors (e2) . While sIn h2 B tells uh2   compiledGenetic variance components are from a metaanalysis by Branigan et al. . The h2 Twin studies make some strong model assumptions such as no genetic parental assortative mating, no gene–environment interaction, and that different types of twins share environmental influences effectively to the same extent. In our comparative study design, we mainly need to assume that potential model violations do not correlate with mobility patterns.The GDIM spans cohorts born from the 1940s through the 1980s; genetic variance components span cohorts born throughout the century. We match each twin estimate to the GDIM based on country, gender, and the closest year of birth. Of the 34 estimates available, we exclude 2 cohorts that lack overlap in the GDIM data, and a further 3 that are from local or nonrepresentative samples in the United States. This leaves a total of 26 data points. The data contain a few negative variance estimates, which we set to zero.https://osf.io/c549j/. There we also show that our results are robust to analytical decisions taken, including the choice of parent, whether to smooth over cohorts, whether to exclude nonrepresentative US samples, whether to recode negative variance estimates, and how to account for clustering. Moreover, we confirm that our results are not driven by any one country, by repeating analyses excluding each.Data and code to replicate all findings can be found at


Cytauxzoon felis is a tick-transmitted apicomplexan that causes cytauxzoonosis in domestic cats (Felis catus). Even with intensive care, the mortality rate of acute cytauxzoonosis approaches 40% in domestic cats, while bobcats (Lynx rufus), the natural intermediate host of C. felis, remain clinically asymptomatic. However, multiple reports of domestic cats surviving acute disease without any treatment exist. One hypothesis for survival of these cats is infection with unique C. felis genotypes of lower pathogenicity. Prior studies have identified genetically distinct C. felis isolates containing polymorphisms within internal transcribed spacer regions (ITS) of the rRNA operon. However, these polymorphisms do not correlate with the clinical outcome of cytauxzoonosis, and so additional genetic markers are needed to test this hypothesis. We selected C. felis apical membrane antigen-1 (ama1) as a potential genetic marker of differential pathogenicity. AMA1 is a vaccine candidate for relatives of C. felis within Plasmodium spp.; however its historically high level of genetic polymorphism has resulted in escape from vaccine-induced immunity. While such diversity has hindered vaccine development, the expected polymorphism within the ama1 gene may be useful to evaluate population genetics.C. felis ama1 gene was PCR-amplified from 84 domestic cats and 9 bobcats and demonstrated 99.9% sequence identity across all samples. A single nucleotide polymorphism (SNP) was identified in domestic cats and bobcats with evidence for co-infection with both genotypes identified in two domestic cats. The prevalence of the two genotypes varied with geographical distribution in domestic cats. Nucleotide diversity (π) and haplotype diversity (H) were calculated for C. felis ama1 and ama1 of related apicomplexans to assess genetic diversity. Based on these values (π = 0.00067 and H = 0.457), the diversity of the C. felis ama1 gene region analyzed is considerably lower than what is documented in related apicomplexans.A 677 bp sequence of the C. felis ama1 gene is highly conserved. While lack of genetic diversity limits utility of C. felis AMA1 as a genetic marker for clinical outcome, it supports further investigation as a vaccine candidate for cytauxzoonosis.In surprising contrast to related apicomplexans, our results support that the sequence of the  Felis catus) caused by the tick-transmitted apicomplexan Cytauxzoon felis. A recent study found an overall prevalence rate for C. felis of 6.2% in three U.S. states, with over 15% in Arkansas alone, demonstrating relevance of infection to veterinarians [Lynx rufus) is the natural host for C. felis and unlike domestic cats, infected animals are typically asymptomatic [C. felis with the use of a combinatorial selamectin (6.0 mg/kg) plus sarolaner (1.0 mg/kg) topical product  as well as with 10% imidacloprid/4.5% flumethrin collars  [C. felis infection in 18 cats with and without treatment [C. felis infection. Similar sporadic reports in other areas exist [C. felis genotypes of different pathogenicity.Cytauxzoonosis is a life-threatening infectious disease of domestic cats  , 4. For , Bayer) . A studyC. felis populations geographically, and suggested a diverse population structure for C. felis; however, ITS polymorphisms were not associated with pathogenicity and clinical outcome. Additional genetic markers will be required to fully address questions of population genetics and, importantly, the possibility that particular C. felis genotypes are linked to clinical outcome in domestic cats [Studies to date attempting to link specific genotypes to clinical outcome have focused on identifying polymorphisms within the first and second internal transcribed spacer regions of the rRNA operon ITS1 and ITS2) –11. Thes and ITS2tic cats , 10, 12.ama1) gene. AMA1 is a surface membrane protein unique to apicomplexan pathogens that allows for efficient invasion of host cells. Blockade of AMA1 via specific antibodies has been associated with inhibition of host cell invasion for several apicomplexans including Plasmodium spp. [Babesia spp. [Toxoplasma gondii [Theileria parva [Plasmodium spp. [Babesia spp. [C. felis population genetics, we investigated the ama1 gene of C. felis for sequence variation and association with clinical outcome of infection in domestic cats, as well as geographical origin of infection in bobcats and domestic cats.One candidate for identification of polymorphisms that may be associated with clinical outcome is the apical membrane antigen-1 . A single nucleotide polymorphism (SNP) coding for a synonymous substitution (cytosine or thymine) at position 612 was identified  in domestic cats with single-genotype infections. In Arkansas, the sequence from all 53 domestic cats contained a thymine at position 612, whereas in Georgia the presence of cytosine at this position was more common  and haplotype diversity (H). Nucleotide diversity (π) is the average number of nucleotide differences per site in pairwise comparisons, and haplotype diversity (H) is the probability that two randomly chosen haplotypes will be different. The values of π and H for the C. felis ama1 gene are considerably lower than those of related apicomplexans. While such measurements depend upon factors related to the population sampled, based upon available gene sequences in GenBank, the C. felis ama1 gene region analyzed has a nucleotide diversity 15–30-fold lower than closely related parasites  driven immune responses present in more diverse human populations. This possibility is supported by work showing that the bobcat populations in the southeastern USA, where all of our C. felis samples were obtained, cluster into one subclade of the continental bobcat population when analyzed by either mitochondrial DNA (mtDNA) or microsatellite data [AMA1 has been a major focus of vaccine development against human malarial parasites; however, successful vaccine development has been hampered by the presence of extensive polymorphism, with over 200  in Mali . The laclciparum . There aite data .P. falciparum, which it should be noted still maintain significantly greater levels of genetic diversity in AMA1 [ama1 gene in infected populations of domestic cats and bobcats. Two lines of evidence suggest that this is not the case. First, in the comparison of nucleotide diversity between related apicomplexans, all sample sets, save that for P. falciparum, had significantly fewer samples, yet showed dramatically higher diversity suggesting sample size is not limiting our ability to detect diversity. Next, in previous work using the ITS regions we successfully characterized population structure in 88 samples from Arkansas and Georgia [C. felis ama1 gene makes this a poor marker for exploring correlations between parasite genetics and clinical outcomes. Thus far, neither the now widely used rRNA internal transcribed spacer regions nor the ama1 gene reported here have been adequate for fine-scale population genetic studies, nor for rigorous testing of genetic correlates of clinical outcome, highlighting the need for better molecular markers for the study of population genetics within C. felis.Another possibility is that the observed lack of genetic polymorphism is due to a relatively recent population bottleneck, as has been postulated for some populations of  in AMA1 . A final Georgia . WhateveC. felis ama1 gene is uniquely conserved compared to other related apicomplexans. While limiting its utility as a genetic marker for clinical outcome, the lack of polymorphism in C. felis  AMA1 suggests that it may be an effective target for vaccine development for cytauxzoonosis.Our results support that the sequence of n = 53), Georgia (n = 30) and Florida (n = 1). EDTA whole blood samples from 9 bobcats originating from Arkansas (n = 2), Georgia (n = 1), Florida (n = 5) and California (n = 1) were obtained from the United States Department of Agriculture and the Southeastern Cooperative Wildlife Disease Study. Genomic DNA (gDNA) was purified from 200 µl of whole blood using the QIAmp DNA Blood Mini Kit . To confirm C. felis infection status and assess for the presence of inhibitors, real-time polymerase chain reaction (PCR) for the C. felis 18S rRNA gene and for the feline reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed on purified gDNA samples using previously published methods [Ethylenediaminetetraacetic acid (EDTA) anticoagulated whole blood samples from 84 domestic cats with acute cytauxzoonosis were obtained from diagnostic laboratories and private veterinary hospitals in Arkansas , 1× concentration of PCR Buffer II , 1.25 U of Taq Polymerase, 5 µl of gDNA template, 1.5 mM MgCl2, and 200 µM of each dNTP. Thermal cycling parameters included an initial denaturation at 95 °C for 5 min, followed by 36 amplification cycles . PCR products were visualized by gel electrophoresis, purified using a QIAquick® PCR Purification Kit , and sequenced bi-directionally via Sanger sequencing.Using a homology based PCR approach, degenerate primers for C. felis ama1 gene region from 84 domestic cats and 9 bobcats were performed using ClustalW software [PCR amplification and sequencing primers were derived from the amplicon sequence produced by the degenerate primers and designed with Geneious v9.1. PCR was performed on all samples using above described parameters and 25 pmol of each primer (5′-ACA CAT TAT TAT CGT ATG CCA-3′ and 5′-ACT AAA GTT ATT ATT CCT TAA CTC C-3′). PCR products were purified and sequenced bi-directionally to yield a 677 bp region for analysis. Histograms from all samples were analyzed to confirm presence or absence of nucleotide substitutions, insertions and/or deletions. Nucleotide and amino acid alignments of the software .Theileria annulata, Babesia bovis, Babesia bigemina, Plasmodium falciparum and Plasmodium vivax was constructed using Geneious v10.2 and MUSCLE v 3.8.425. Based upon this alignment, homologous nucleotide regions were determined from nucleotide alignments of ama1 genes from each of the species and submitted to DnaSP for analysis.Two measures of genetic diversity, nucleotide diversity (π)  and hapl


Carthamus tinctorius L.) is a multipurpose crop of dry land yielding very high quality of edible oil. Present study was aimed to investigate the genetic diversity and population structure of 131 safflower accessions originating from 28 different countries using 13 iPBS-retrotransposon markers. A total of 295 iPBS bands were observed among which 275 (93.22%) were found polymorphic. Mean Polymorphism information content (0.48) and diversity parameters including mean effective number of alleles (1.33), mean Shannon’s information index (0.33), overall gene diversity (0.19), Fstatistic (0.21), and inbreeding coefficient (1.00) reflected the presence of sufficient amount of genetic diversity in the studied plant materials. Analysis of molecular variance (AMOVA) showed that more than 40% of genetic variation was derived from populations. Model-based structure, principal coordinate analysis (PCoA) and unweighted pair-group method with arithmetic means (UPGMA) algorithms clustered the 131 safflower accessions into four main populations A, B, C, D and an unclassified population, with no meaningful geographical origin. Most diverse accessions originated from Asian countries including Afghanistan, Pakistan, China, Turkey, and India. Four accessions, Turkey3, Afghanistan4, Afghanistan2, and Pakistan24 were found most genetically distant and might be recommended as a candidate parents for breeding purposes. The findings of this study are most probably supported by the seven similarity centers hypothesis of safflower. This is a first study to explore the genetic diversity and population structure in safflower accessions using the iPBS-retrotransposon markers. The information provided in this work will therefore be helpful for scientists interested in safflower breeding.Safflower ( Carthamus tinctorius L.) belongs to the Compositae family, it is self-pollinated and has a haploid genome size of about 1.4 GB and 2n = 24 chromosomes [Carthamus spp. were found at sites in Syria about 7500 BC ago [Safflower , Plant Genetic Resources Institute Pakistan, and from the Turkish Central Research Institute for Field Crops . A total2 and 7.6 μL distilled water. Reactions were performed in the sequence of denaturation at 95 oC for 3 min, subsequently followed by 30 denaturation cycles at 95 oC for 15 sec, annealing temperature 50–65 oC for one minute depending upon the primer, and a final extension for five minute at 72 oC [Seventy iPBS-retrotransposon primers were initially screened using eight randomly selected accessions of safflower for PCR amplifications . Out of at 72 oC . The ampStrong, clear, and unambiguous bands were selected for scoring. iPBS-retrotransposon markers are dominantly inherited markers and were therefore scored using the binary system: 0 or 1, respectively, for the absence and presence of specific bands with respect to 100 bp+ DNA ladder . For indThirteen most polymorphic iPBS-retrotransposon primers produced a total of 295 clear and strong scorable bands with an average of 22.69 bands per primer across 131 safflower accessions. Out of the 295 scorable bands, 275 (93.22%) were polymorphic with an average of 19.77 bands per primer . The higTo clearly understand the broader picture of genetic diversity, pairwise genetic distance among 131 safflower accessions was measured with the Jaccard coefficient. The mean Jaccard genetic distance across the evaluated accessions was 0.288. The highest genetic distance (0.51) was observed between Turkey3 and Afghanistan4 accessions. Similarly, lowest genetic distance (0.05) was present between Afghanistan4 and Afghanistan5 accessions. Genetic distance was also calculated across the populations and mean genetic distance for population A (0.22), population B (0.24), population C (0.24), population D (0.31), and unclassified population (0.28).Analysis of molecular variance (AMOVA) was carried out considering two main population strata: the model based structure and the country of origin of the accessions . AMOVA rIn accordance with the observed most suitable goodness of fit (K = 4), the Bayesian clustering model implemented in STRUCTURE software divided the evaluated safflower accessions into four main populations; 31 accessions  in the population A (black), 22 accessions  in the population B (red), 33 accessions  in the population C (blue), 27 accessions  in the population D (pink) . EighteeTo the best of our knowledge, the present investigation represents the first attempt to elucidate the genetic diversity and population structure of safflower accessions at DNA level using iPBS-retrotransposons. It was observed that retrotransposons are abundant and widely distributed throughout plant genome  and hugePolymorphism information content (PIC) is a widely used metric of the usefulness of molecular markers . The PICTo know the genetic diversity more clearly, diversity metrics like; overall gene diversity (0.24), Fst (0.21), and Fis (1) were also computed. The Fst (a measure of genetic differentiation) obtained in this work was comparable with the findings of Ambreen et al.  as they The evaluation of pairwise genetic distance showed a mean of 0.288, with the highest genetic distance between accessions Turkey3 and Afghanistan4, followed by Afghanistan2 and Pakistan24 with respective distance values of 0.51 and 0.49. Greater similarity was found between Afghanistan4 and Afghanistan5 accessions showing least genetic distance of 0.05. One understandable reason behind the presence of maximum genetic similarity might be due to their origin from the common parents. To explore the genetic diversity more clearly, genetic distances were also calculated at the population level and mean maximum genetic distance was reflected by the population D and minimum was resulted by population A. Within populations, Turkey16 and China9 reflected maximum genetic distance and minimum was present between Afghanistan4 and Afghanistan5 accessions belonging to population A. Within population B, maximum genetic distance was observed between accessions Iraq1 and Jordan4, while minimum genetic distance was shown between accessions Jordan4 and Jordan5. Argentina1 and Iran8 were the two most distinct accessions reflecting maximum genetic distance in the population C and Australia1 and Turkey6 were found two most genetically similar accession of population C representing minimum genetic distance. Within population D, Turkey3 and Iran9 were most diverse accessions and Kazakhstan1 and Pakistan14 were two genetically distinct accessions belonging to unclassified population. Germplasm containing desirable plant traits can be usefully integrated in breeding programs to develop superior cultivars [The analysis of molecular variance (AMOVA) was used to determine the pattern of the partition of the total gene diversity among and within populations, and the countries of origin . AMOVA sThe model-based structure application proved more robust and informative in previous investigations –59. StruOn continents basis, population A clustered a total of 7 and 24 accessions belonging to American and Asian continents respectively. In population B, 3, 11 and 8 accessions originated from Africa, Asia and Europe, respectively. Population C comprised accessions from America (2), Asia (29), Europe (1), and Oceania (1). In population D most of the accessions originated from Asia (23), while a few accessions came from Africa (3) and Europe (1). The unclassified population contained genotypes mostly from Asia (11), while the other few came from Africa (2) and Europe 5) accessions also made divergence from above four populations by making their separate group. Clearly, the clustering based on molecular markers did not discriminate the origins of the safflower accessions evaluated in this work, which was also confirmed by the AMOVA inferences. Accessions from different countries clustered together, implying that kinship was more determinant for the population structure than the geographical provenance. In addition to sharing common parentage, similarities of accessions in same group during clustering might also be due to convergent evolution and selection . It can  accessioIn 1969, Knowles recognized the existence of seven safflower similarity centers across the world. Overall, the centers of similarity were represented by several accessions in this study. However, the molecular marker data used in this study did not provide much support to the above Knowles’s hypothesis on the similarity centers. Indeed accessions belonging to different similarity centers were clustered together. This lack of importance of similarity centers in defining molecular-based populations was reported in scientific literature . In popuThe investigation of genetic relationships between the 131 accessions using UPGMA clustering algorithm resulted in a clustering pattern comparable with the model-based algorithm with a few exceptions as two and five population B accessions clustered with population D and population C, respectively, and UPGMA discrepantly clustered the accession Iran8 (population C) in population A . Since tPrincipal coordinate analysis (PCoA) greatly supported the structure based clustering of 131 safflower accessions using 13 iPBS-retrotransposon primers . The fouA good level of genetic diversity was identified among 131 safflower accessions. The importance of genetic populations on the genetic structure was significant, but its magnitude was lesser than the importance the variations of individuals within genetic populations. The provenance of the samples showed no effects on the genetic structures in the 131 accessions. Our results most probably obey the seven similarity centers hypothesis of safflower but still there is need to conduct further research works to confirm these similarity centers at the molecular level. Generally, safflower accessions from Asian countries like Afghanistan, Pakistan, China, Turkey, and India were found diverse. Specifically, among 131 safflower germplasm, accessions Turkey3, Afghanistan4, Afghanistan2, and Pakistan24 were found most diverse at molecular level and might be recommended as a candidate parents for future safflower breeding programs.