G. mangiferae fungi. This species is considered endophytic for citrus plants and is easily isolated from asymptomatic fruits and leaves. G. mangiferae is genetically related and sometimes confused with G. citricarpa which causes Citrus Black Spot (CBS). “Tahiti” acid lime is one of the few species that means to be resistant to this disease because it does not present symptoms. Despite the fact that it is commonly found in citric plants, little is known about the populations of G. mangiferae associated with these plants. Hence, the objective of this work was to gain insights about the genetic diversity of the G. mangiferae populations that colonize “Tahiti” acid limes by sequencing cistron ITS1-5.8S-ITS2. It was verified that “Tahiti” acid lime plants are hosts of G. mangiferae and also of G. citricarpa, without presenting symptoms of CBS. Populations of G. mangiferae present low-to-moderate genetic diversity and show little-to-moderate levels of population differentiation. As gene flow was detected among the studied populations and they share haplotypes, it is possible that all populations, from citrus plants and also from the other known hosts of this fungus, belong to one great panmictic population.Among the citrus plants, “Tahiti” acid lime is known as a host of  Guignardia  encompasses around 330 known species, but some of them with an unknown anamorphic phase [G. mangiferae and also the causal agents of CBS, G. citricarpa, and of fruit rot in guava (Psidium guajava L.), G. psidii. The genus ic phase . Many spMangifera indica) and guava, G. mangiferae was isolated from a wide range of different hosts and was considered endophytic because of the symptomless tissues from which it was isolated. Its hosts include Brazilian tropical plants, such as Apidosperma polineuron, Anacardium giganteum, Myracrodroun urundeuva, Spondias mombin, Bowdichia nítida and Cassia occidentalis [G. mangiferae [ Despite causing foliar and fruit spots in mango  gene cluster, including the internal transcribed spacer (ITS) regions ITS1 and 2, the intergenic spacer IGS, 5.8S rRNA, 18S rRNA, and 26S rRNA genes. This is due to the fact that these multicopy genes are highly conserved within a species but can be quite variable among species. Other commonly used genes include the mitochondrial ATPase subunits, beta tubulin, and elongation factor [Studies about phylogeny and molecular systematic of fungi have utilized ITS rDNA because of the higher number of random copies of this sequence dispersed throughout the genome and their uniformity, which is generally maintained by stabilizing selection . General Ideally, the best way to quantify genetic variation in natural populations should be by the comparison of DNA sequences . HoweverDrosophila at present, they will certainly increase in the future [The introduction of the polymerase chain reaction (PCR), which allows direct sequencing of PCR products and avoids, therefore, their cloning, has changed the situation. Undoubtedly, this has produced a revolutionary change in population genetics. Although population studies at the DNA sequence level are still scarce and primarily carried out in e future .G. mangiferae known hosts as Tahiti acid lime, mango, and guava (Psidium guajava L.), it would be helpful to know and understand these fungi population structure. Despite Tahiti acid lime did not show any disease symptom caused by G. mangiferae, it could be inoculum source for susceptible cultures as mango, and guava. If we consider that citrus, mango and guava plantations are frequently neighbors, G. mangiferae can spread rapidly from one culture to another. So, this disease needs careful observations and monitoring and the knowledge about its population genetic structure would be helpful. Considering that Brazil has larger production areas of G. mangiferae populations, we used the SNP markers found in the ITS1-5.8S-ITS2 region. The objectives of this study were to characterize the population genetics of G. mangiferae from different geographic regions and determine the genetic diversity and population differentiation.Therefore, to obtain valuable information about the genetic structure of The sampling was done in two different geographic areas: in Estiva Gerbi , São Paulo State, and in Itaboraí district , Rio de Janeiro State. In each place, leaves were collected from 24 different acid lime plants in order to obtain one isolate per plant. In the same places, 40 leaves were collected from one plant in order to obtain 24 isolates from the same plant. This sampling, 40 leaves in a same plant, was also done for three different plants in each geographic place.Guignardia isolates from this study were characterized in oatmeal medium according to Baldassari et al. [All i et al. .μL of buffer 1X ; 0.8 μL of MgCl2 5 mM; 0.4 μL of each dNTP 10 mM; 0.3 μL Taq DNA polymerase; 5 pmol of each primer, with 60 ng of genomic DNA and sterile water q.s.p. to 20 μL. DNA was amplified in a Termocycler PTC-100 Programmable Thermal Controller MJ Research, Inc., with 1 initial cycle at 94°C during 2 min, 39 cycles at , and 1 final cycle at 72°C for 5 min. Amplified samples were submitted to electrophoresis in agarose 1.2%, containing ethidium bromide (0.5 μg/mL) and 1 Kb DNA Ladder. The samples were observed under UV light with a GEL DOC 1000-BioRad (data not shown). The DNA fragments obtained were purified and submitted to sequencing PCR with a DYEnamic ET Dye Terminator Kit  according the manufacturer's instructions. The termocycler conditions were the same as those previous described. DNA fragments were precipitated with isopropanol 75%, washed with ethanol 70%, and resuspended with 3 μL of “loading buffer” , heated to a 95°C for 2 min, and then applied to the sequencing gel. Electrophoresis was done in a sequencer ABI Prism 3700 DNA Sequencer . The ITS region of each isolate was submitted to sequencing two times in the two strand ends (Primer Forward + Primer Reverse).DNA from isolates was extracted according the Kuramae-Izioka  protocol ABI Analysis Data Collection and converted in nucleotide sequences with DNA Sequencing Analysis Software Version 3.3. The DNA sequences were then submitted to softwares Phred/Phrap/Consed [The eletropherograms were obtained using the softwarep/Consed  and Sequp/Consed .Genetic distances were calculated between groups of endophytic isolates from the same plant, from different plants and from different geographic origins. These estimates were done in order to evaluate the genetic diversity among the intra- and intergroups according to Nei's equations . The intAverage pairwise differences were estimated from comparisons within a library of the number of sequence differences between a given clone and all other clones  Table 3Table 3. FST values were used to evaluate the genetic diversity within the groups of isolates in relation to the total genetic diversity according to the equation FST = (θT − θW)/θT, where θT is the genetic diversity of all isolates and θW is the diversity within the group of isolates [isolates . Analysiisolates . Populatisolates . Gene flisolates  using thisolates .Guignardia DNA sequences from different species obtained from GenBank (site http://www.ncbi.nlm.nih.gov/). The Guignardia ITS1-5.8S-ITS2 DNA sequences included in each analysis file were G. citricarpa clone 75 (ID:AF346782.1); G. citricarpa (ID: AF346772.1); G. mangiferae voucher ICMP 8336 (ID:AY816311.1); G. mangiferae (ID: AM403717.1); G. laricina (ID:AB041245.1); G. philoprina (ID:AB095507.1); G. philoprina specimen-voucher CBS 447.68 (ID: AF312014); G. aesculi (ID:AB095504.1); G. vaccinii (ID:AB041244.1); G. bidwellii (ID:AB095511.1); G. bidwellii (ID: AB095505); G. bidwellii (ID: AB095509); G. gaultheriae (ID: AB095506.1); G. gaultheriae (ID: AB095506); Phyllosticta pyrolae (ID: AF312010); Phyllosticta pyrolae (ID: AB041242) e Phyllosticta spinarum (ID: AF312009). The aligned sequences were used to verify the genetic relationships among the isolates from same and different plant of Tahiti acid lime from the two places. Dendrograms were built using the Distance method, the grouping algorithm Neighbor Joining  nucleotiPathogenicity tests were performed according Baldassari et al.  using 22Guignardia typical colonies were obtained from all the samples of asymptomatic leaves, in the same and in different plants and geographic origins, in a total of 208 isolates. The samples, from the same and from different plants in the two different regions, were composed of a minimum of 24 isolates and a maximum of 26 isolates  and its ID are showed in Supplementar Data.The obtained sequences were submitted to a quality analysis in order to use only those that showed high quality. All used sequences showed the desired quality up to 20, according to the software Phred/Phrap/Consed. Sequences were edited and ends trimmed. This was done in order to prevent errors during the posterior analysis. All sequences were submitted to GenBank , Itaboraí isolates presented a genetic distance slightly lower (0.036) than the group of isolates from Estiva Gerbi (0.032). The genetic distance between (intergroup) isolates from these two regions was 0.0358. When analyzed according to sampling, isolates from Estiva Gerbi obtained from different plants of “Tahiti” (LC) presented the highest intragroup genetic distance (0.02831), whereas the lowest was presented by isolates from the plant L1 (0.02270) .Itaboraí isolates from one plant (P1) of “Tahiti” presented the highest intragroup genetic distance (0.04210), whereas the lowest was found for isolates from different plants , probably because the same isolate was sampled in different plants.The highest intergroup genetic distance was presented by isolates from different geographic origins . This waThe lowest divergence was presented by groups of isolates from the same geographic origin, Estiva Gerbi, by the group of isolates from “Tahiti” different plants (LC) and those obtained in same plant . These two populations can be considered the ones with the lowest genetic diversity.The diversity indexes show that the highest genetic diversity was found for the groups of isolates from the same plants, Itaboraí/RJ, P1 and P2 . These tG. mangiferae populations was observed at the various hierarchical levels , representing 81.99 percent of the observed genetic diversity. The fixation index among populations within regions was also significant, , representing 15.42% of the observed genetic diversity. The fixation index among the two regions is almost insignificant  representing 2.71 of the observed genetic differentiation. This indicates that there is gene flow between regions.According to the F values, little-to-moderate genetic differentiation of FST = 0.07443, P ≤ 0.005). The lowest was observed for P and P1 . In Estiva Gerbi/SP, the highest differentiation was observed for LC and L3 populations , whereas the lowest for LC and L1 . Between the two regions, L3 and P3 showed the highest genetic differentiation , and L1 and P1 presented the lowest .When differentiation indexes were calculated for groups of isolates according to samples from the same or different plants , it was  Gene flow was also detected among the studied populations . All sam In Estiva Gerbi/SP, the highest level of gene flow between populations was detected for the sample of different plants of LC and the plant L1 (22.6 migrants per generation), and the lowest between the LC population and L3 population (0.44 migrants per generation). The highest levels of gene flow were shown when populations from the two geographic origins were compared .G. mangiferae sequences obtained from GenBank, showing great similarity among them, as demonstrated by the populations obtained from different plants from Estiva Gerbi  was found within the populations. A moderate diversity was found within regions (15.30%) and can possibly be due to the influences of the host over the populations and to the climatic and soil conditions. Low genetic diversity was found between the populations of Estiva Gerbi/SP and Itaboraí/SP, meaning that the same or similar haplotypes were found in all populations, despite the fact that the two geographical areas are distant from each other (around 800 km). The fact that G. mangiferae have several distinct hosts possibly affects the distribution of the diversity within and among populations. We believe that the higher values of genetic differentiation showed by some populations are caused by this. As sampling was done randomly, we probably sampled haplotypes that could be originated from different hosts. Some of the known hosts of G. mangiferae are commonly found near citrus orchards, like eucalyptus , mango (Mangifera indica), guava (Psidium guajava), and native hosts like jaboticaba (Myrciaria cauliflora) and Surinam cherry (Eugenia uniflora). Eucalyptus is also commonly used to protect citrus orchards from winds and to avoid the introduction and dissemination of pathogens to the culture.SNP markers showed that they are useful markers for detecting genetic variation within and between populations of G. mangiferae shared haplotypes and demonstrated gene flow between populations from the same geographic origin and also between the two regions. We believe that this is because the dispersal mechanisms of G. mangiferae allied with the colonization of different hosts. Despite the fact that these mechanisms are not well elucidated for G. mangiferae, we believe that they are very similar to those presented by G. citricarpa. Propagation structures in G. citricarpa are either sexually formed ascospores or asexual pycnidia. The fungal spores generated by mitosis “conidia” formed inside specialized organs “pycnidia” are frequent in G. citricarpa and are found on fruit lesions during the ripening stage, but they are unlikely to function as dispersal units over long distances [G. mangiferae, we believe that these structures are formed without the presence of symptoms in fruits and leaves. Ascospores, whether formed by a homo- or heterothallic process on fallen leaves, are tiny and may disperse over both relatively short and large distances [G. mangiferae were also detected in fallen leaves [G. mangiferae has probably adapted well to conditions during the different seasons, and the generation of dissemination structures is not restricted by some conditions. Different hosts can also permit generation of higher amounts of dissemination structures that can spread over distant regions, facilitating the search for new hosts. Thus, it was demonstrated that populations of G. mangiferae present low-to-moderate genetic diversity, but show little-to-moderate levels of population differentiation. As gene flow was detected among the studied populations and they share haplotypes, it is possible that all populations, from citrus and also from the other known hosts of this fungus, belong to one great panmictic population. This study showed that populations of istances . In G. mistances , whereasistances . Ascospon leaves . BecauseG. citricarpa, a behavior that is also presented by the sour orange (C. aurantium) [G. citricarpa [G. citricarpa, whose extent was unknown at the time.“Tahiti” acid lime plants did not show symptoms of CBS, despite the fact that they permit colonization by rantium) . For “Tatricarpa . These sG. mangiferae is known as a causal agent of fruit and foliar diseases in mango, guava, and banana (Musa sp). Despite the fact that the fungi received names according the host (G. psidium when isolated from symptomatic fruits and leaves in guava and G. musae when isolated from symptoms on banana fruits), recent results showed that all these species probably belong to G. mangiferae [ On the other hand, ngiferae .G. mangiferae, Baayen et al. [P. anacardiacearum) are the same as G. endophyllicola . In addition, Okane et al. [P. capitalensis (teleomorph G. endophyllicola) exists widely as an endophyte in ericaceous plants. The results obtained by Pandey et al. [P. capitalensis has a wide host range as an endophytic fungus. Pandey et al. [P. capitalensis suggests that this fungus may have been described several times as different species, especially since a species name in Phyllosticta is, in many instances, linked to the host. The fact that sometimes the same fungi is identified under different names makes the correct identification of new forms difficult. Despite being identified as n et al.  concludee et al.  demonstry et al.  also supy et al.  and Baayy et al. , concludy et al.  also belG. mangiferae from this work caused symptoms on sweet orange fruits, as shown by pathogenicity tests, reinforces the hypothesis that G. mangiferae is actually endophytic for citrus. The fact that none of the isolates of  Despite the fact that the term “endophyte” is employed for all organisms that inhabit plants, mycologists have come to use the term “fungal endophyte” for fungi that inhabit plants without causing visible disease symptoms, and the term refers only to fungi at the moment of detection without regard for the future status of the interaction .Neotyphodium endophytes in grasses) [ It is known that all fungi invading plant foliage have an asymptomatic period in their life cycle that varies from an imperceptibly short period (like plant pathogens) to a lifetime (like grasses) . Beneficial effects for hosts being colonized by endophytes include increased drought tolerance, deterrence of insect herbivores, protection against nematodes, and resistance against fungal pathogens . The lasG. mangiferae, there is evidence that the guignardic acid that is produced by this species exhibits potent antimicrobial activity [ In the specific case of activity . Some authors hypothesize that there are no neutral interactions, but rather that endophyte-host interactions involve a balance of antagonisms, irrespective of the plant organ infected . There iBotryosphaeriaceae, which are important endophytics for woody plants, are horizontally transmitted between individuals [ Available evidence suggests that ividuals . These aG. mangiferae populations, if populations of different hosts are related one to each other, the range of the presented genetic diversity, and its genetic differentiation. Studies to better evaluate properties like the antifungal substances produced by these fungi, as well as examination of the possibilities of using endophytes for biological control, to replace the actual fungicides are used in Brazilian citriculture. A number of studies suggest that endophytes of woody plants are rather loosely associated with their hosts, with higher correlation between endophyte communities in a specific location, than with a specific host in different locations , 41, 42.This Table contains the Guignardia mangiferae isolates used for this study. ITS1-5.8S-ITS2 DNA region of each isolate was amplified in order to perform the population genetic structure described on this research. The DNA sequence was deposited in GenBank and received an Acession Number presented on Table.Click here for additional data file.


Lipotes vexillifer), one of the most critically endangered species in the world, genetic diversity at the major histocompatibility complex (MHC) class II DRB locus was investigated in the baiji. Nine highly divergent DRB alleles were identified in 17 samples, with an average of 28.4 (13.2%) nucleotide difference and 16.7 (23.5%) amino acid difference between alleles. The unexpectedly high levels of DRB allelic diversity in the baiji may partly be attributable to its evolutionary adaptations to the freshwater environment which is regarded to have a higher parasite diversity compared to the marine environment. In addition, balancing selection was found to be the main mechanisms in generating sequence diversity at baiji DRB gene. Considerable sequence variation at the adaptive MHC genes despite of significant loss of neutral genetic variation in baiji genome might suggest that intense selection has overpowered random genetic drift as the main evolutionary forces, which further suggested that the critically endangered or nearly extinct status of the baiji is not an outcome of genetic collapse.To further extend our understanding of the mechanism causing the current nearly extinct status of the baiji ( Lipotes vexillifer), endemic to the Yangtze River of China, has long been recognized as one of the most endangered species in the world The baiji or Yangtze River dolphin  The major histocompatibility complex (MHC) plays a central role in the development of the immune response against pathogens. MHC class I and class II molecules present peptide epitopes to T-cell receptors initiating the adaptive immune response DRB gene was investigated in the baiji. Additionally, we have compared the allelic diversity described in the baiji with those found in other endangered species. Findings from this study will provide basic information for further understanding of how demographic and environmental factors influence MHC variation in the wild animal populations, especially in the endangered species.To determine whether MHC variation has been maintained by natural selection and recombination despite the intense genetic drift implied by the low genetic polymorphism at neutral genetic markers, in this study, highly polymorphic exon 2 of MHC class II DRB unique sequences were identified according to the criteria summarized by Kennedy et al. DRB unique sequences were detected in each individual, indicating that a single-copy DRB locus was amplified from the baiji using this primer sets. These sequences were labeled Live-DRB*01-09 (Accession number: JN202590-JN202598) according to the nomenclature of Klein et al. Live-DRB*01, 02, 03, and 04 were common in the baiji and had high frequency from 11.8% to 29.4%, whereas the frequency of rarer alleles ranged from 2.9% to 8.8% .A total of 233 cloned sequences of 215 bp in length were obtained from the sampled 17 individuals. Nine  to 8.8% . The obsLive-DRB*01 vs. Live-DRB*05) to 57 (Live-DRB*02 vs. Live-DRB*07), whereas amino acid replacements between alleles ranged from 0 (Live-DRB*01 vs. Live-DRB*05) to 30 (Live-DRB*02 vs. Live-DRB*07). Of the 19 PBR (peptide-binding region) sites defined by homology with HLA A total of 72 of 215 (33.49%) nucleotides and 35 of 71 (49.30%) amino acids were variable . The baiDRB locus. First, the rate of nonsynonymous substitutions was significantly higher than the rate of synonymous substitutions when all sites were included in the analysis  and when only PBR sites were included  of the 9 Live-DRB alleles , 0.251 (C), 0.339 (G), 0.184 (T), and the gamma shape parameter α = 0.443. The Bayesian tree and NJ tree were separately reconstructed according to this model (HKY+G). Phylogenetic reconstructions of the baiji and other cetacean DRB sequences showed moderate bootstrap support for a cetacean-specific clade, distinct from the artiodactyl outgroup sequences such as Bibi-DRB and Hiam-DRB sequences .Using the AIC criterion, HKY+G was identified as the most appropriate model for the present equences . Among tDRB alleles were identified in 17 baiji samples, which is much greater than those of some other marine mammal species/populations examined up to date, e.g. only three Mian-DRB alleles in 110 Northern elephant seals (Mirounga angustrirostris) Dele-DRB alleles in 313 beluga Phsi-DRB alleles in 29 Gulf of California harbour porpoises DRB allelic diversity with an average of 13.2% nucleotide (range 1–57) and 23.5% amino acid (range 0–30) differences between sequences, both of which were comparable to or higher than those found in some other endangered species (Oryx leucoryx) Ailuropoda melanoleuca) Bison bison) Mustela lutreola) DQB and MHC-I loci in the baiji. Six MHC-I, eight DQB alleles were separately examined in the same 17 baiji samples examined in this study, most of which were differed more than 20 nucleotides The baiji is now functionally extinct, which would be the first recorded extinction of a well-studied cetacean species to be directly attributable to human influence  species . For exaDRB genetic variation was maintain in the baiji.The unexpectedly high levels of MHC allelic diversity in the baiji may be partly attributable to possibility of severe pathogens and parasites. This prediction was evidenced in humans DRB, DQB, and MHC-I loci. Moreover, several lines of evidences support that balancing selection plays a role in maintaining MHC sequence polymorphism at the DRB, MHC-I and DQB loci. First, the unexpectedly high level of DRB allele variation in the baiji was concentrated in PBR sites with an excess of nonsynonymous substitutions in PBR, which was considered as a primary indication of balancing selection. This inference was further evidenced by the result obtained from comparisons of various models of codon evolution (DRB sequences were highly divergent with up to 26.5% (57/215) and 42.3% (30/71) at the nucleotide and amino acid levels, respectively. The number of pairwise nucleotide differences between alleles ranged from six (Live-I*04 vs. Live-I*06) to 30 (Live-I*05 vs. Live-I*06) and amino acid replacements ranged from 4 (Live-I*03 vs. Live-I*05) to 18 (Live-I*03 vs. Live-I*04). Third, balancing selection was also apparent in the pattern of trans-species polymorphism in phylogenetic reconstructions. Trans-species polymorphism is polymorphism predates speciation events, whereby allelic lineages are passed from species to species and persist over long periods of evolutionary time DRB, DQB and MHC-I loci DRB locus, e.g. Live-DRB*01, 03, 05 clustering with Neph-DRB*06, 02, 09, respectively, etc.On the other hand, it has been suggested that selection may retain MHC diversity even when population bottlenecks lead to the loss of genetic variation elsewhere in the genome, exemplified by an endangered island fox which retained some MHC variation in spite of the virtual lack of neutral variation volution . Models DRB alleles, i.e. Live-DRB*05, 06, 07, 08, 09, were identified only in the two or three baiji samples with frequency ranging from 0.029 to 0.088. This pattern might in line with the mechanism of rare-allele advantage.Besides, two main types of balancing selection were suggested to maintain the unusually high level of MHC polymorphism: heterozygote advantage, rare-allele advantage. The heterozygote advantage hypothesis assumes that heterozygous individuals are favoured as they are able to present a broader array of antigens, and therefore resist a broader array of pathogens than homozygotes DRB locus (s = 0.019) is at least two times higher than for DQB (s = 0.0085) DQ loci was generally insufficient to maintain variation in small populations isolated during hundred or thousand generations. However, on most cases some functional variation was still retained at the DRB locus, consistent with higher selection intensity at this locus. This agrees with theoretical models DRB allelic diversity. Consequently, balancing selection at the MHC must have been intense in the baiji to maintain high levels of variation despite its population size rapidly declined in the past few decades.The maintenance of polymorphism within populations is dependent on the product of selection intensity Live-DRB alleles, and two DNA segments (positions 83–198 and positions 146–189) may have acted as hot spots for recombination. However, the allele Live-DRB*01, 03, 09 were came from one of the hot spots (positions 83–198), and the three alleles were substantially different, suggesting the mutation of alleles might have occurred after recombination. If so, the intragenic recombination must have been very ancient. Thus, intragenic recombination was likely not as major mechanism for the baiji DRB polymorphism.Additionally, intragenic recombination was posed to provide another mechanism that regenerates MHC allelic diversity The significant level of genetic variability at MHC loci, associated with apparent action of balancing selection on this gene, strongly suggested that the baiji, although had a very small population size and dramatically declined in the past decades, still had a strong adaptability and evolutionary potential. In other words, balancing selection might have overpowered genetic drift as the main driving force in shaping the genetic composition of the baiji. The dramatic declining trend or the current nearly extinct status of the baiji, therefore, seems to be dependent mainly on factors outside their genetic makeup. This finding further extends our understanding of the mechanism causing the endangerment or extinction of the baiji. Turvey et al. conducted an analysis of spatial and temporal extinction dynamics in the baiji and found that the population decline of baiji was not associated with any major contraction in geographical range across the middle–lower Yangtze drainage, even in the decade immediately before probable global extinction of this species All the baiji samples used in our study were collected from dead individuals in the wild so that no ehics statement is required. Voucher specimens are preserved at Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University (NNU), China.A total of 17 baiji samples, 3 muscle and 14 skeletal, were collected from stranded or incidentally captured/killed individuals in the Yangtze River. Genomic DNA was extracted by using the DNeasy tissue kit (QIAGEN) for muscle samples and Gene clean for ancient DNA kit (Q. Biogene) for skeletal samples, respectively, following the manufacturer's protocol.DRB gene was amplified with the forward primer DRBex2f2b (5′-CRGTTTAAGAGCGAGTGTC-3′) and reverse primer DRB61a (5′-CCGCTGCACTGTGAAGCT-3′) A 215-bp fragment (exclusive of primer sequences) from exon 2 of the Live-DRB for the baiji Lipotes vexillifer with serial numbers attached.A new allele was identified only when it met the criteria summarized by Kennedy et al. DRB evolution in the baiji, we used two approaches. First, nonsynonymous (dN) and synonymous (dS) substitution rates were calculated for the PBR and non-PBR amino acid positions following the method of Nei and Gojobori dN/dS>1. Six models implemented in this study are: M0 (one-ratio), which assumes the same ω ratio for all codons; M1a , which assumes two categories of sites conserved (ω = 0) and neutral (ω = 1); M2a (positive selection), which adds a third class of sites with ω as free parameter, thus allowing for sites with ω>1; M3 (discrete), which assumes three site classes with the proportions  and ω ratios  estimated from the data; M7 (beta), which assumes ω varies among sites according to a beta distribution with parameters p and q; and M8 (beta and ω), which adds an additional site class to the beta model to account for sites under positive selection In order to test if positive selection has acted on the http://darwin.uvigo.es/rdp/rdp.html) Detection of potential recombinant sequences, identification of likely parental sequences, and localization of possible recombination break points were determined using the Recombination Detection Program (RDP) version 3  and Hippopotamus amphibious (EF017820) were used as outgroups. The best-fit model was selected using the AIC criterion and likelihood scores automatically generated and compared using MODELTEST Phylogenetic analysis was carried out with confirmed alleles using nucleotide sequences of exon 2. The neighbor-joining (NJ) tree was reconstructed from the matrix of distances computed in PAUP 4.0b10


With the advances in genetic technology, the pages of the book of human biology and related diseases are turning over and over. Every new page is bringing a different vision into the life of humans not only limited to the scientific area but also to the daily life as well. The material used in genetic methods has found its place in this film with the discovery of a weak acid in the nuclei of white blood cells that we call DNA today. Unraveling the structure of DNA and chromosomes enabled us to find out the causes of many genetic diseases by cytogenetic and molecular genetic techniques. In situ hybridization with DNA probes followed these developments, and genome technology has advanced to the point of high-throughput sequencing nowadays. Throughout this period, the Human Genome Project provided a rapid increase of our insight into the field of human biology. The data obtained from this project allowed us to understand the underlying mechanisms of many genetic diseases and led to great interest among the scientific community.On the other hand, the need for updated information in relation to the use of genetic techniques in the clinical practice of the clinicians increased in parallel with advances in genome technology. The practical use of the aforementioned genetic testing methods and proper interpretation of the generated test results have become a necessity not only for medical geneticists but also for the other specialists. Furthermore, physicians need to know how to order the most appropriate genetic test in the right time for the right indication to prevent under- or overutilization of those tests. That is why this special issue aimed to provide information about genetic techniques and their use in different research areas to improve knowledge, attitudes, and practices of physicians and researchers.In this special issue, we would like to highlight both conventional and novel genetic methods, namely, classical cytogenetics, fluorescence in situ hybridization, PCR, RT-PCR, methylationspecific PCR, pyrosequencing, DNA sequencing, fully automated microarray-based assay, whole genome array CGH, MALDI-TOF mass spectrometry, and Southern blot analysis in a number of clinical conditions such as cancer, periodic fever syndromes, cardiomyopathy, myotonic dystrophy, retinal dystrophies, Wolf-Hirschhorn syndrome, and immunogenetic diseases.Unquestionably those techniques do not comprise all of the methods in genomic studies; however they may give clues to understanding their potential use in clinical practice. Because genetic tests are unique and require robust quality assurance, quality assessment of genetic testing is undoubtedly necessary; therefore a study, which focused on the role of external quality assessments, was also featured in this issue as well. Ozgur CoguluYasemin AlanayGokce A. Toruner


Cervus elaphus) have been an important human resource for millennia, experiencing intensive human influence through habitat alterations, hunting and translocation of animals. In this study we investigate a time series of ancient and contemporary DNA from Norwegian red deer spanning about 7,000 years. Our main aim was to investigate how increasing agricultural land use, hunting pressure and possibly human mediated translocation of animals have affected the genetic diversity on a long-term scale.Red deer (We obtained mtDNA (D-loop) sequences from 73 ancient specimens. These show higher genetic diversity in ancient compared to extant samples, with the highest diversity preceding the onset of agricultural intensification in the Early Iron Age. Using standard diversity indices, Bayesian skyline plot and approximate Bayesian computation, we detected a population reduction which was more prolonged than, but not as severe as, historic documents indicate. There are signs of substantial changes in haplotype frequencies primarily due to loss of haplotypes through genetic drift. There is no indication of human mediated translocations into the Norwegian population. All the Norwegian sequences show a western European origin, from which the Norwegian lineage diverged approximately 15,000 years ago.Our results provide direct insight into the effects of increasing habitat fragmentation and human hunting pressure on genetic diversity and structure of red deer populations. They also shed light on the northward post-glacial colonisation process of red deer in Europe and suggest increased precision in inferring past demographic events when including both ancient and contemporary DNA. Cervus elaphus) are one of the most common and widespread of European ungulates today (Table.4% of thBos taurus) when using primer pair II, a common problem in aDNA studies[None of the negative controls contained red deer DNA, but some samples showed contamination from cattle  were gathered from the extant Norwegian red deer distribution. From these we identified five different haplotypes (NO1 – NO5), identical to five of the ancient samples. The frequency and distribution of the haplotypes differed strongly among the three regions . Pairwise population differentiation was very high between regions and the most geographically distant populations were also the most genetically differentiated  and the combined total genetic diversity of Norwegian deer differed significantly from equilibrium expectations, indicating a recent bottleneck.176 control region sequences , which remains significant even after Bonferroni correction, indicating population expansion. Among the five haplotypes found in the extant population all but NO3 have been found in samples older than any suspected translocation events.Considering only western Norway from where we have most of our ancient samples 70/73) it is clear that both the genetic diversity : 8.15 ×10-8 – 5.23 ×10-7) substitutions per site per year, which is in line with other studies using aDNA datasets[The BSP suggest a relatively stable female effective population size, although with a wide confidence interval, until about 2,000 yr BP, when effective size starts to decrease in a stepwise manner Figure. A Bayesdatasets,67 and tdatasets. NNOR = 7,160  and NEUR = 1.03 mill  and the European and Norwegian populations seemingly diverged from one another tD = 15,280  years ago. There are only weak signs of bottleneck as modal values of NB = 10 , but median value being NB = 22,700, i.e., larger than the NNOR estimate. The larger median value of NB compared to NNOR is likely an effect of assuming an equal population size before and after the bottleneck. The median time with varying population sizes is tBS – tBE = 976 years . All time estimates have relatively flat posterior probability distributions and estimates must be interpreted with caution. The bias (MRB) is low (<1) for NNOR, NEUR and NB effective size estimates and the various time estimates, while moderate for estimated time with possible changes in effective sizes after divergence  . While significant Fu’s Fs indicate a population expansion during the early part of our study period , as indicated by the ABC analysis Table, coincidTwo star shaped patterns are apparent among the western European samples, separated by an A-G transition. One of these centres on NO1, which has been described before, while tOur results indicate that the current genetic diversity of Norwegian red deer can be explained by one post-glacial immigration event followed by a gradual loss of diversity and increasing population sub-division due to heavy exploitation and habitat fragmentation during the last two millennia. They also show that only females from the western European red deer lineage colonised Scandinavia, supporting previous findings that the eastern lineage had a more limited dispersal into Europe,80. The The authors declare that they have no competing interests.JR did the laboratory work, analysed the data and drafted the manuscript. HKS performed the ABC-analyses. JR and AKH picked out the ancient samples. KHR supervised the laboratory work. All authors were involved in the study design, revising of the manuscript and read and approved the final manuscript.Additional file contains details of data collection and some aspects of data analyses. It also contains information on dating of ancient samples as well as an overview of the new sequences provided by this study. The file is in PDF format.Click here for file


Polygonia faunus are supported by genetic data from mitochondrial sequences and eight microsatellite loci. We also investigate the phylogeographic structure of P. faunus and test whether similarities in host-plant use among populations are related to genetic similarity. Neither the nuclear nor the mitochondrial data corroborated subspecies groupings. We found three well defined genetic clusters corresponding to California, Arizona and (New Mexico+Colorado). There was little structuring among the remaining populations, probably due to gene flow across populations. We found no support for the hypothesis that similarities in host use are related to genetic proximity. The results indicate that the species underwent a recent rapid expansion, probably from two glacial refugia in western North America. The mitochondrial haplotype network indicates at least two independent expansion phases into eastern North America. Our results clearly demonstrate that subspecies in P. faunus do not conform to the structuring of genetic variation. More studies on insects and other invertebrates are needed to better understand the scope of this phenomenon. The results of this study will be crucial in designing further experiments to understand the evolution of hostplant utilization in this species.Subspecies are commonly used taxonomic units to formally describe intraspecific geographic variation in morphological traits. However, the concept of subspecies is not clearly defined, and there is little agreement about what they represent in terms of evolutionary units, and whether they can be used as reliably useful units in conservation, evolutionary theory and taxonomy. We here investigate whether the morphologically well-characterized subspecies in the North American butterfly  Indeed, it has been suggested that trinomial names in collections should be maintained since some subspecies may represent true species The concept of species, linked intrinsically to the process of speciation, has been the source of long-standing debate of an intensity matched by few others in biology. Lineages within a species that are presumably under various stages of speciation have been an integral part of this discussion. From a taxonomic perspective, the intraspecific category representing diverging lineages within a species is often the subspecies bona fide subspecies are monophyletic Some authors consider that the majority of WolbachiaWolbachia alone is estimated to have infected up to three-fourths of all insect species Wolbachia, can quickly replace mitochondrial haplotypes in uninfected subspecies. Therefore, relying solely on mitochondrial data to study subspecies in invertebrates is not justified.Mitochondrial DNA is widely used to examine species relationships, and less often for subspecific relationships. The use of mitochondrial DNA in invertebrate taxonomy is complicated by potential indirect selection on the mitochondrial genome by cytoplasmic endosymbionts such as Wolbachia infection - to investigate whether described subspecies in the well-known butterfly species Polygonia faunus (Green Anglewing) are genetically distinct. Given the problems with using mitochondrial monophyly as the sole criterion to test the validity of subspecies P. c-album - has also shown that differences in hostplant preferences are at least partly genetically determined P. faunus subspecies represent incipient species in intermediate stages of ecological speciation. To answer this, it is paramount to first determine whether these subspecies have a genetic underpinning or if the morphological differences are largely geographic variations expressed due to phenotypic plasticity. The molecular data are also crucial in understanding the population structure and phylogeographic history of the species, thereby gaining a deeper understanding of the evolution of subspecies (or genetic populations) and hostplant preferences. This information also forms the basis for an interesting comparison with the sister species for which similar data have become recently available We here use a holistic approach based on data from three sources - mitochondrial sequences, 8 microsatellite loci and molecular assays to detect Polygonia faunus occurs in woodlands, damp mountain meadows and stream sides, and is often abundant in boreal habitats. The larvae feed on plants from the families Betulaceae, Ericaceae, Grossulariaceae and Salicaceae in the wild, but have also been shown to be able to feed on Urticaceae in the laboratory arcticus feeds on Salix; hylas on Salix and Ribes; smythi on Betula,and rusticus on Rhododendron, although larvae may accept others in the lab P. faunus.To investigate the corroboration between genetic groupings and subspecies in Test whether patterns of hostplant use are correlated with genetic clustering.Wolbachia in P. faunus to that of its sister species P. c-album where Wolbachia is highly prevalent and appears to have had an impact on the diversity of its mitochondrial DNA.Compare the phylogeographic structure and potential effect of ST value was 38.16 (P = 0.00) and pairwise ФST values of population pairs are shown in There were 38 polymorphic sites and 35 unique haplotypes in the 1422 bp COI (cytochrome oxidase subunit I) dataset. Global haplotype diversity was 77.29% (SD ±2.7%); values for each population are given in The statistical parsimony network of haplotypes is depicted in The mismatch distribution curve had a negative slope and did not deviate from that expected under a model of sudden expansion  = 0.21800000). In the Fu’s Fs test of selective neutrality, the simulated Fs value was significantly lesser than the observed value  = 0.00000).ST values are presented in ST values estimated after correcting for the presence of null alleles. ST between subspecies pairs, both with and without ENA correction in the software FreeNA.The number of alleles per locus ranged from four to 22 . Observea priori, the log likelihood peaked at K = 5. The series of analyses with K = 1 to 14 was repeated five times to determine whether the likelihood was consistently highest at K = 5. The best value of K ranged between five and seven. However, all analyses with K = 4 to 7 recovered four well-defined clusters - Arizona, California, Utah and New Mexico+Colorado , Colorado (2), Utah (3), Arizona (1), Washington (2) and Montana (1).11 samples (8%) tested positive for P. faunus. ФST and FST values indicate that most populations and subspecies are differentiated from other populations and subspecies respectively. The haplotype network, phylogeny and the clustering analysis in STRUCTURE all indicate strong structuring of genetic variation in the species, but none of these genetic groupings corresponds to subspecies. Furthermore, we found little evidence for geographic structuring based on hostplant use patterns. We conclude that the morphological and ecological differences among subspecies in P. faunus are either phenotypically plastic traits expressed differentially across populations or local adaptations with genetic underpinnings that evolve rapidly. The use of different hostplants may also be related to differences in availability per se. We note that current knowledge of host use across the range of the species is based mainly on isolated findings, and hence the information available is at a coarse scale. The species is ideally suited for detailed experimental work to understand the factors determining the evolution of female and larval host choice. The results from the current study will prove invaluable in designing such experiments.Although results from the mitochondrial and microsatellite data were not completely congruent, neither dataset corroborated subspecific groupings in P. faunus compared to many other insect species, and typical insect subspecies are circumscribed on the basis of less distinct features . Taxonomists have frequently used highly variable and/or plastic characters to describe subspecies, an excellent example being the use of eyespot (ocelli) number in satyrine butterflies. The number of eyespots has been shown both to evolve rapidly under selection, and to be highly plastic . Another example is given by the ‘faunus’ and ‘arcticus’ morphotypes, which are sympatric at several of the locations sampled for this study  and probably across a broad range of western Canada  diversity in mitochondrial DNA compared to P. c-album. The mitochondrial population structure found in P. faunus was deeper compared to that in its sister species. Results in Wolbachia infection has drastically reduced the diversity and obliterated population structure of the mitochondrial genome in P. c-album. However, the prevalence of Wolbachia in P. faunus is restricted to a small percentage of individuals. It is therefore unlikely that Wolbachia has affected mitochondrial structure in P. faunus to a strong degree.P. c-album, gene diversity (i.e. expected heterozygosity) cannot be compared across the two species. The STRUCTURE analysis in P. c-album indicated that two populations - Morocco and Russia - were genetically distinct, while there was no structure within the European populations. The findings in P. faunus are similar in that there were three distinct genetic clusters, but very limited structure among the remaining populations. Although hostplant use is known to vary across populations in both species, neither study supports the hypothesis that these differences are related to gene flow between populations. Therefore, until there is experimental evidence that supports a genetic basis for the differences in host use, differences among populations should be seen as potentially being plastic responses to varying environmental conditions, chief among which is availability of a particular host species.Since the microsatellite markers were developed for P. faunus. We tested whether subspecies definitions are supported by the genetic data, but find no evidence for this. We instead found that there are three distinct genetic clusters in the species - California, Arizona and New Mexico+Colorado. There was little structuring among the remaining populations, probably due to gene flow across populations. Our results suggest that subspecies in insects need critical re-evaluation, preferably with genetic data, before being considered as important units in biology. However, it is interesting that such phenotypically distinct and geographically separated morphotypes are not supported by genetic data. Further work is needed to understand the evolution of such phenotypes. We found no support for the idea that broad differences in host use are correlated with genetic distance between populations. The results indicate that the species underwent a recent rapid expansion, probably from two glacial refugia in western North America. The haplotype network also indicates at least two independent expansion phases into eastern North America. Results in this study will be crucial in designing further experiments to understand the evolution of hostplant utilization in this species.We have used data from a combination of mitochondrial sequences and eight microsatellite loci to study the population genetics and phylogeography of 137 samples representing 14 populations - British Columbia, Washington, New Mexico, Colorado, Quebec, New Hampshire, Alberta, California, Utah, Montana, Oregon, Arizona, Idaho and Georgia – were collected by us mainly between 2000 and 2007 (A 1450 bp (base pairs) region of the mitochondrial gene - COI - was amplified using two sets of widely used primers LCO-HCO and Jerry-Pat. Primer sequences and PCR protocols are as in ST values. The exact tests of differentiation were each based on a Markov Chain of 100,000 steps. Populations represented by fewer than 5 individuals were not included in the above-mentioned population level analyses. We tested whether the distribution of pairwise nucleotide differences among sequences fit a model of sudden demographic expansion  with ambiguous data for several sequences was excluded from further analyses; the final dataset hence consisted of 1422 bp. Haplotypes were identified and a statistical parsimony network constructed in TCS v1.21 P. c-album sequence (Genbank accession: JN093200) as outgroup, was inferred using maximum likelihood under the GTR+I+G model in the software RaXML v7.2.6 The phylogeny of haplotypes, including a P. c-album  were found to be suitable for use in P. faunus. In this study, the original PCR conditions were modified with a multiplex protocol using the Type-it Microsatellite PCR Kit (QIAGEN). All multiplex PCR reactions were performed according to manufacturer’s guidelines with an annealing temperature of 56°C and 2.0 µl DNA template. Loci Polalb7, 11, 17 & 20 formed one multiplex combination, while Polalb 2, 5, 6 & 8 formed a second. Primer concentrations were adjusted to normalize peak heights during electrophoresis. Polalb10 was incompatible with other loci and hence amplified independently using the protocol described in Eight microsatellite loci developed for ST values were calculated in Arlequin. Statistical significance of pairwise FST values was tested using 100,000 permutations. Hardy-Weinberg equilibrium was tested based on exact tests with a Markov chain of 1,000,000 steps and 100,000 dememorization steps. The software STRUCTURE a priori. In this study, we ran consecutive analyses with increasing values of K from one to 14. Theoretically, one can examine likelihood scores from each run to determine the total number of genetic populations that best explains the data. We imposed the LOCPRIOR model with admixture Allelic variability, global and pairwise population Fwsp (Wolbachia surface protein) gene primers Wolbachia in the P. faunus samples. If the bacterium is present in the cytoplasm of the host tissue from which DNA has been extracted, the primers amplify the wsp (Wolbachia surface protein) gene, which can be visualized electrophoretically. This technique is widely employed to detect the presence of Wolbachia in the host tissue, and our own assay in P. c-album indicates that the species has an almost 100% infection status wsp. 8 µl of product was checked on a 1% agarose gel with ethidium bromide staining. Samples that did not amplify wsp were tested a second time. All PCR reactions included positive and negative controls (ddH2O).PCR-based assays using standard Table S1List of samples used along with the collection localities, Genbank accession numbers, collectors and haplotypes.(XLS)Click here for additional data file.Protocol S1Protocol followed for isolation of 10 microsatellite loci for Polygonia c-album. Six of these loci were used in this study.(DOC)Click here for additional data file.


CRTISO) has emerged as a regulatory step in the carotenoid biosynthesis pathway and could be a good candidate to show how a metabolic pathway gene reflects a species genetic history.Carrot is a vegetable cultivated worldwide for the consumption of its root. Historical data indicate that root colour has been differentially selected over time and according to geographical areas. Root pigmentation depends on the relative proportion of different carotenoids for the white, yellow, orange and red types but only internally for the purple one. The genetic control for root carotenoid content might be partially associated with carotenoid biosynthetic genes. Carotenoid isomerase , and another one mainly from Europe (Western group). The Western and Eastern genetic groups were suggested to be differentially affected by selection: a signature of balancing selection was detected within the first group whereas the second one showed no selection. A focus on orange-rooted carrots revealed that cultivars cultivated in Asia were mainly assigned to the Western group but showed CRTISO haplotypes common to Eastern carrots.In this study, the nucleotide polymorphism and the linkage disequilibrium among the complete CRTISO gene data proved to be complementary to neutral markers in order to bring critical insight in the cultivated carrot history. We confirmed the occurrence of two migration events since domestication. Our results showed a European background in material from Japan and Central Asia. While confirming the introduction of European carrots in Japanese resources, the history of Central Asia material remains unclear.The carotenoid pathway  Selection events along the species or breeding history generally lead to signatures of selection at the molecular level, i.e. local variations of diversity or allele frequencies at genes underlying phenotypic variations or in their surrounding region. The detection of signatures of selection encounters various issues. Firstly, demographic events like population subdivision can modify genetic polymorphism and mimic selective events Daucus carota subsp. sativus), cultivated worldwide, might have lead to such selection signatures. The domestication of this species is thought to have occurred in the Afghanistan region before the 900 s The history of carrot  has emerged as a regulatory step in the carotenoid biosynthesis pathway. CRTISO enzyme catalyses the cis-to-trans isomerisation reactions leading to all trans-lycopene, the substrate for the subsequent lycopene cyclisation to form all trans-α/β-carotene CRTISO as an isomerase was also confirmed by its functional expression in Escherichia coli in which it was able to convert cis-carotenoids to all trans-carotenoids CRTISO mutants, such as ccr2 (Arabidopsis), phs3 (rice) and tangerine (tomato), result in the accumulation of cis-carotenoids in the dark-grown plants Among genes, the carotenoid isomerase , ripe fruit, and the innermost parts of the green fruit CRTISO in plants is presumably to enable carotenoid biosynthesis to occur in the dark and in non-photosynthetic tissues such as the root, case of carrot. By comparison to other crucial steps in the carotenoid pathway, CRTISO may have evolved with fewer constraints due to photoisomerisation, and may have been more prone to be subjected to artificial selection in non-photosynthetic organs, like root. Clotault et al. CRTISO gene has undergone through selection events in cultivated carrot but the polymorphism pattern was observed among partial CRTISO sequence . The particular status of this gene and preliminary results suggest that CRTISO gene could be a good candidate for selection signature research. The analysis of the nucleotide polymorphism and the LD among the complete CRTISO sequence will enable to clarify the selection pattern, depending on the gene structure and in relation with colour types. Moreover, selection effect can spread over genomic regions around the gene by a selective sweep. The study of the intrachromosomic LD will allow testing this hypothesis.During plant evolution, paralogous genes for several carotenoid biosynthesis enzymes have been conserved and subjected to subfunctionalization: the paralogs are differentially expressed in photosynthetic or non-photosynthetic tissues CRTISO, a key gene involved in the carotenoid biosynthesis pathway especially in root organs, should reflect the cultivated carrot history, including selective events during breeding. The present results showed a signature of selection pattern at the CRTISO gene and the critical contribution of CRTISO polymorphism for explaining the cultivated carrot history, especially regarding material from Asia and Central Asia.Our work aimed to know whether polymorphism within Daucus carota subsp. sativus) were sampled . Amplifications were carried out by using a MyCycler thermalcycler . The thermalcycler was programmed as follows: initial denaturation at 94°C for 2 min, 35 cycles at 94°C for 30 s, annealing temperature for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min.PCR reactions were performed in 20 µL volume with 10–50 ng genomic DNA, 1X PCR buffer, 0.2 mM dNTPs, 2 mM MgClThe amplified fragments were analyzed by capillary electrophoresis , and labelled PCR products were automatically sized with Genemapper 3.7 software .5 and a run length of 106, were used for each number of clusters (K) from one to ten. The most likely number of genetic clusters was selected using Evanno’s lnP(P) methods. For each individual, the proportion q of its genome assigned to each cluster and its 95% confidence interval were calculated. An individual was assigned to a cluster if q>0.5. A CA  was also performed by using Genetix 4.05 software Microsatellite data were used to investigate the genetic structure of the sample with STRUCTURE 2.1 software FCT), by using FSTAT 2.9 software Fixation indexes were estimated among the colour groups (Daucus carota subsp. sativus (GenBank accession number DQ192188) in order to obtain the complete CRTISO sequence (exons and introns).Gene-specific primers were designed and synthesized , based o2, 0.2 mM dNTPs, 0.2 µM of both forward and reverse primers, and 1U Taq DNA polymerase . Amplifications were carried out by using a MyCycler thermalcycler . The cycling conditions were: initial denaturation at 94°C for 3 min, 35 cycles at 94°C for 30 s, annealing at 55°C for 45 s, 72°C for 2 min, and a final extension at 72°C for 5 min.PCR reactions were performed in 25 µL volume with 10–50 ng genomic DNA, 1X PCR buffer, 2 mM MgClPCR products were purified with ExoSAP-IT  and sequenced with BigDye® Terminator 3.1 chemistry  in an ABI Prism 3730 sequencer  according to the supplier’s instructions. The primers used to amplify PCR fragments were also used for the sequencing. Eight partial genomic regions were sequenced with forward and reverse primers to discard PCR errors . NucleotThe contiguous sequences of the 39 individuals were aligned with ClustalW software The population genetic parameters were defined by using DnaSP 5.10 software wθ; π; wθ is based on the number of segregating sites while π is based on the pairwise differences between sequences in the population. The analyses of nucleotide polymorphism (wθ) and nucleotide diversity (T)π were based both upon the overall CRTISO sequence and upon a 100 bp window with a step size of 25 bp.The population-level genetic variation was estimated as nucleotide polymorphism θw;  and nuclh and haplotype diversity Hd were then calculated Haplotypes were inferred with DnaSP 5.10 software by including infinite-site violations. The number of haplotypes A median-joining network establishing relationships among haplotypes based on the number of polymorphisms (each insertion/deletion -indel- was considered as a single nucleotide polymorphism -SNP-) was performed with Network 4.61 software 2r because it summarizes both recombination and mutation histories Linkage disequilibrium (LD) is the non-random association of alleles at different loci. The level of LD is influenced by many factors such as population subdivision, selection, genetic linkage, recombination rate CRTISO sequence but also along the linkage group 4 where CRTISO gene and five microsatellite markers were mapped 2r, which was plotted for all pairwise comparisons among SNPs. Given the influence of population subdivision, LD was calculated in each genetic group revealed by population structure analysis.LD was investigated by using TASSEL 3.0 software D test D values reveal an excess of intermediate and low frequency variants, respectively. Tajima’s D test was calculated for the overall CRTISO sequence and upon a 100 bp window with a step size of 25 bp. The statistical significance for the above tests was inferred provided that the observed value was included within the 95% confidence interval of neutral distribution, which was calculated by using 10,000 coalescent simulations in DnaSP by assuming no recombination.Tajima’s D values was also compared to coalescent simulations according to a demographic model. This demographic model aimed at testing the role of genetic bottleneck during domestication and subsequent population subdivision during carrot cultivation. This model included two populations, corresponding to the Eastern and Western genetic groups, assuming constant effective population sizes, EN and WN respectively. At dT generations in the past, these two populations diverged. This divergence was confounded with the end of a genetic bottleneck, started at bT, and characterized by an effective population size bN. Before the bottleneck, the ancestral population size was AN. In domestication modeling studies, the bottleneck intensity d = bT–dT and bN are positively correlated and define the bottleneck severity k = bN/dd value of 100 generations and varied bN in order to test ten k values: 0.2, 0.5, 1, 2, 5, 7, 10, 15, 20 and 50. Other model parameters were sampled from posterior parameter distribution obtained in k value, 5,000 coalescent simulations were made by using ms software D was calculated for each simulation by using Egglib D was inferred provided that the observed value was included within the 95% confidence interval of simulated distribution.The significance of observed Tajima’s All 17 microsatellite loci were polymorphic. A total of 174 alleles were identified with a mean of 10.2 alleles per locus, and a range from two to 19 alleles per locus. Delta K method  suggesteFCT) ranged from 0.003 to 0.142 : these genes are split into 13 exons and 12 introns The CRTISO sequence exhibited a high LD level with an average 2r of 0.61 for the Western genetic group and 0.64 for the Eastern genetic group. LD showed no decay within 4,234 bp  in the Western genetic group.LD was also evaluated along LG4 ISO gene . Indeed,CRTISO genomic sequence, among the whole dataset, the Western versus Eastern genetic group, and the colour groups, is summarized in The observed nucleotide variation of the Among the studied 78 allelic sequences, 15 haplotypes were identified, among which five were found only once (singleton haplotypes). Haplotypes differed by 212 polymorphisms, consisting of 196 nucleotide substitutions and 16 indels. Among the 55 substitutions found in coding regions, 18 were non-synonymous.Tπ = 0.01753). The synonymous diversity value (synπ = 0.02788) was much higher than the non-synonymous one (nonsynπ = 0.00354), yielding a nonsyn/πsynπ ratio of 0.12697. The nucleotide diversity was higher within non-coding regions (non-codingπ = 0.02433) than within coding regions (codingπ = 0.00928)  . High disequence .D statistic detected an excess of intermediate-frequency variants in the whole dataset, which yielded a significant positive D value . High and significant D values were also obtained within non-coding regions  varied from 0.01105 to 0.02071, with an average value of 0.01614. The white-, orange- and yellow-rooted carrots showed globally higher nucleotide diversity than the red and purple ones  and D (haplotypes 3 and 15) corresponded only to four individuals, with white or orange roots. Three out of four individuals belonged to the Western genetic group, whereas the two clusters were overall closer to the cluster B. The clusters C and D could constitute intermediate clusters.CRTISO gene showed that cultivated carrot was divided into two genetic groups: one from Asia (Eastern genetic group), and another one mainly from Europe (Western genetic group). This subdivision is consistent with previous studies about microsatellite markers or genes CRTISO was especially marked by the genetic subdivision between the Western and Eastern genetic groups.In the present study, both results from microsatellites and CRTISO gene. This was consistent with results obtained by Populus tremulaNo decay of linkage disequilibrium was detected within 4,234 bp for CRTISO gene was mapped on the linkage group 4 near the centromeric region CRTISO gene, the Tajima’s D value varied a lot along the sequence, varying from significant to non significant values, especially in the whole dataset and the Western genetic group. The partial sequence analyzed in By considering population differentiation obtained here, LD remained high in the Western and Eastern genetic groups, suggesting the population subdivision could not explain the absence of LD decay. However, k ≥2 to k ≥15, depending on the considered groups) during carrot domestication and a subsequent population subdivision between the Eastern and Western groups, confirmed the significance of observed D values. However, it should be noted that for an intense bottleneck hypothesis (k <2), the significance disappeared. Bottleneck intensity usually found for domestication ranges from k = 0.2 . Several models of selection maintaining diversity were suggested: heterozygote advantage at a locus, frequency-dependent selection, temporally or spatially heterogeneous selection One explanation for the potential balancing selection observed in the Western genetic group would be the selection for some colour types. A signature of balancing selection was found at CRTISO polymorphism in yellow and orange carrots as evidence for signature of selection. Indeed, the differentiation between colour groups was considerably influenced by the subdivision between the Western and Eastern genetic groups. White carrots were mostly in the Western genetic group, four among six. Otherwise, all red and purple carrots  were assigned to the Eastern genetic group. The yellow group was half constituted by Western and Eastern individuals concordantly for microsatellite and CRTISO data, while the orange group was composed of individuals mainly assigned to the Western genetic group, with some individuals cultivated in Asia showing CRTISO haplotypes common to Eastern carrots. Therefore, these two colour groups are probably the most made of the two genetic groups. The respective effect of population subdivision, which could mimic balancing selection, and real balancing selection in these two colour types should be investigated in the future.However, caution is needed while interpreting the CRTISO gene might lead to a selective sweep around this gene CRTISO genomic region could be developed thanks to carrot genome sequencing, and therefore could allow improving the study of selection events in this species.Selection footprint and high LD extent detected among CRTISO polymorphism analyses allowed testing the hypotheses about cultivated carrot history. Microsatellite markers gave information about the entire genome and were therefore very suitable to understand gene pool origin. The incongruity between the CRTISO haplotype network and genetic structure given by microsatellite analysis informed about genetic admixture.Neutral loci and CRTISO sequence data. By comparison, the clustering of yellow cultivars half into the Western group and half into the Eastern group, whether through microsatellite or CRTISO sequence analyses, suggested that the yellow type had experienced two separate migration events, following domestication.Literature considers the purple and yellow carrots as the two original colour types CRTISO data. This is congruent with the breeding of this colour type. Indeed some European cultivars were exported and adapted for cultivation to Japan in the 1900 s. They were used in breeding programs and crossed with Asian carrots CRTISO data. It remains therefore unclear if Central Asia cultivars came from European material crossed locally with Eastern material, or they originated from an Eastern migration from Japan.According to their assignment probability and large confidence interval estimated by STRUCTURE analysis, orange carrots sampled from Japan and Central Asia were assigned to the Western genetic group but with some admixture between Western and Eastern carrots whereas they belonged clearly to the Asian cluster depending on CRTISO polymorphism analyses were shown to be complementary in order to recount the breeding history of cultivated carrot. Indeed, human activities  could affect allele diversity, which resulted in the distinction of two genetic groups and large diversity at the CRTISO gene. These results may suggest that human preferences for carrot root colour have varied depending on periods and geographical areas. It brings new insights about the history of orange carrots. The orange-rooted carrots spread from Europe to other continents including Asia. Therefore the breeding of this carrot form was adapted to different markets. In spite of nucleotide polymorphism and the distinction of several haplotype clusters within the orange carrots, corresponding individuals belonged mainly to the Western genetic group based on microsatellite data. This would confirm that the introduction of European carrots in Asian breeding programs is relatively recent, as genetic background remains common to both plant materials. The knowledge of the breeding history of this species, based both on neutral and functional loci, allows a better characterisation of plant material, and offers substantial insight to secure, manage and exploit carrot genetic resources while maximising genetic diversity.Neutral loci and Figure S1Plots of (A) Delta K and (B) the log likelihood, from the STRUCTURE analysis.(TIF)Click here for additional data file.Figure S2Genetic structure of 39 carrot cultivars based on a correspondence analysis (CA) on microsatellite data. Squares and circles represent respectively individuals from the Western genetic group and individuals from the Eastern genetic group according to STRUCTURE results.(TIF)Click here for additional data file.Figure S3Significance of Tajima’s D according to the genetic bottleneck severity k, for (A) the total sequence, (B) the coding sequence and (C) the non-coding sequence. Ten values of bottleneck severity from 0.2 (most severe) to 50 (least severe) were tested. The significance was displayed for the whole dataset (black line), Western group (blue line), Eastern group (green line), white group (grey line), yellow group (yellow line), orange group (orange line), red group (red line) and purple group (purple line). The significance is shown as P-values for two-tailed Tajima’s test, with a logarithmic scale (y-axis). P-values were calculated by comparing the location of observed Tajima’s D with the simulated dataset obtained by the demographic model. In each plot, the areas with P<0.001, P<0.01 and P<0.05 are represented with red, orange and yellow backgrounds, respectively.(TIF)Click here for additional data file.Table S1Characteristics of 17 carrot microsatellite markers .(XLSX)Click here for additional data file.


The ability of species to adapt to different environments resides in their genetic diversity. This diversity, most commonly manifested as Single Nucleotide Polymorphisms (SNPs), can provide clues to the adaptive processes and population histories that have played a role in the species’ evolution. A number of different techniques for identifying SNPs have been developed, all having their limitations.Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variations in natural or mutant populations. Ecotilling, a variant of TILLING  technique, allows high-throughput analyses of natural genetic diversity in plants , particuJatropha curcas L. is a perennial, monoecious shrub of the Euphorbiaceae family, native to America but distributed widely in the tropical and subtropical areas [J. curcas can grow well under unfavourable climatic and soil conditions [J. curcas has attracted a great deal of attention worldwide, regarding its potential as a new energy plant. The seeds of J. curcas contain 30-45% oil [J. curcas is an interesting model species, since it has a relatively small genome (2C DNA content of 0.850 ± 0.006 pg or C DNA content of 0.416 × 109 bp) [al areas . Wild ornditions . J. curcJatropha for wider ecological adaptation, disease resistance and novel seed quality, the use of germplasm from different group and regions is necessary. Understanding the population structure of the alternative bioenergy plant Jatropha curcas is challenging due to limited genetic variability and information on phylogenetic relationships between accessions and related species. The development of cultivars of Jatropha curcas by conventional breeding will profit largely from biotechnological support .However, to achieve specific breeding goals in J. curcas remains limited and little genomic research has been done so far [J. curcas is not well-developed and only few molecular markers exist that could be used to clearly distinguish world wide accessions. Therefore, a resource database of SNPs in J. curcas would provide researchers with a tool for answering questions concerning population structure or adaptation and allow comparison of this species with related species.The knowledge about e so far . In factJatropha species. ISSRs (Inter Simple Sequence Repeats) also were considered as a tool in selecting germplasm for breeding purposes.The identification of novel SNPs that account for natural variation was used to study genetic diversity and the relationships between and within in vitro germplasm collection of 1300 accessions from 12 countries was established. This collection will serve different purposes: a) conserve valuable genetic resources, b) survey genetic variation, and c) serve as starting material for genetic improvement with different breeding goals.An Jatropha curcas and related species. Four different pooling strategies were used to identify homozygous and heterozygous SNP variations. In fact, variation was analyzed both within a single tree (heterozygous) as well as between individual trees and a reference samples. Due to the reported low variations between Jatropha accessions [Ecotilling was applied to 12 different genes of interest related to stress tolerance, toxin and oil metabolism. 50 ISSR primers were used to assess the genetic diversity of cessions ,9 and laJatropha accessions from different regions and related species, a dendrogram was produced using NJ analysis of NeiÂ´s genetic distance for 5 ISSR markers. The dendrogram is divided into two groups, one containing all Jatropha accessions and the other containing the related species. The main Jatropha curcas cluster is divided into two subclusters, one containing samples from Kenya and the other containing the remaining Jatropha accessions. The data showed clear variations not only among individuals but also between different regions.To elucidate genetic relationship among Jatropha, compared to RAPD, SSR and AFLP. Ecotilling is a low cost, high-throughput reverse genetic method for haplotyping and SNPs discovery.The level of differentiation observed was based on the geographic distribution pattern, i.e. it was higher in the centre of origin. ISSR analysis yielded highly reproducible patterns with 5/50 primers.Ecotilling was found to be more efficient for large-scale studies of genetic variation in


Genetic variation between various plant species determines differences in their physio-chemical makeup and ultimately in their hyperspectral emissivity signatures. The hyperspectral emissivity signatures, on the one hand, account for the subtle physio-chemical changes in the vegetation, but on the other hand, highlight the problem of high dimensionality. The aim of this paper is to investigate the performance of genetic algorithms coupled with the spectral angle mapper (SAM) to identify a meaningful subset of wavebands sensitive enough to discriminate thirteen broadleaved vegetation species from the laboratory measured hyperspectral emissivities. The performance was evaluated using an overall classification accuracy and Jeffries Matusita distance. For the multiple plant species, the targeted bands based on genetic algorithms resulted in a high overall classification accuracy (90%). Concentrating on the pairwise comparison results, the selected wavebands based on genetic algorithms resulted in higher Jeffries Matusita (J-M) distances than randomly selected wavebands did. This study concludes that targeted wavebands from leaf emissivity spectra are able to discriminate vegetation species. A geh other)  and the  [et al.  discrimii.e., cell walls and cuticular membranes), which can act as a fingerprint for discriminating vegetation [Till recently, vegetation spectra in the mid to thermal infrared (2.5–14 μm) was perceived as a line without any spectral features . Howevergetation –29. The 2.2.1.The dataset of leaf samples used in this study was the same as used in the . The lea2.2.2) gas was used to continuously purge the spectrometer from water vapor and carbon dioxide. A mid-band mercury-cadmium-tellurium (MCT) detector cooled with liquid nitrogen was used to measure the DHR spectrum of the adaxial (upper) surface of the leaf samples between 2.5 and 14 μm (−1. Thirty five (35) leaves were measured per species, thus 455 leaves were measured in total. Each leaf measurement was referenced against a calibration measurement of gold plate  with a high reflectance (approximately 96%). One thousand  scans were averaged to produce each leaf spectrum. The spectra between 6 to 8 μm were noisy (due to water absorption) and were excluded from the analysis. The DHR spectra were converted to emissivity using Kirchhoff's law (Emissivity = 1 − R) [A Bruker VERTEX 70 FTIR spectrometer  was used to acquire the Directional Hemispherical Reflectance (DHR) spectrum of each leaf. Nitrogen  –32. For = 1 − R) ,33.2.3.i.e., undergo the mechanism of crossover and mutation) a second generation that consists of hybridized offspring of the first generation. Of this second generation, again the solutions with the highest fitness are selected to reproduce a third generation, and so on, until the improvement in fitness between subsequent generations levels off to a pre-set threshold. Parameters that have to be selected before starting the algorithm are the chromosome size , the population size , the fraction of a generation that is selected to be the “parents” for the new generation, and when to stop the algorithm. The reproduction operators, objective function, and selection mechanism are summarized in the next subsection, while the detailed practical implementation (step by step procedure) can be found in Goldberg [Genetic algorithms, introduced for the first time by Holland , are a pGoldberg . The gen2.3.1.For problem solving, the selected chromosomes directly undergo crossover and mutation. In the crossover operation the two selected parent chromosomes merge and produce offspring (new chromosomes) that share the properties of both parents. A single point crossover was used in this study, where two parent chromosomes split into four segments (two segments per parent). Then the exchange of gene segments produces two offspring from every two parents. In mutation, a single gene  in the offspring chromosome is randomly altered and as a result the characteristics of the offspring differ from the parental chromosome combination.2.3.2.i.e., target and reference) by calculating the angle between them in an n-dimensional space. To calculate the fitness function, half of the spectra of each species (17 spectra per species) were used for training purposes, and the remaining half for validation purposes. For each species, the average spectrum of training dataset was used as a reference spectrum.An objective function is required to assign a value to each chromosome. The associated value of each chromosome is an indication how well it fits the solution it represents. The spectral angle mapper (SAM) nearest neighbour classifier was used to evaluate the fitness values  of the chromosome population during the process of evolution. The SAM determines the spectral similarity between two spectra , the parent chromosomes were selected to reproduce offspring using random (roulette wheel) selection. The chromosomes with higher fitness values have a higher chance of being selected for reproduction and to generate a new chromosome.On the basis of fitness value  = 2.The initial parameters were configured as follows: Population size = 1,000, maximum number of generations = 500, crossover probability = 1, probability of mutation = 0.01, elite count  was set to 85% [In order to define the number of genes in a chromosome for maintaining high classification accuracy, the genetic algorithms were run with different gene numbers per chromosome. The minimum threshold for class separability , crossover, mutation and elite count processes.2.4.The performance of the genetic algorithms in separating the species was assessed by using the Jeffries Matusita (J-M) distance . The J-MThe average J-M distance between each species pair selected using the genetic algorithm's selected bands were compared with the average J-M distance derived from the randomly selected bands. The significance of difference in the J-M distances between the genetic algorithm based bands and randomly selected bands was tested using a t-test.3.3.1.The results  compare 3.2.Illustrating the process of evolution, The overall classification accuracy using the winning chromosome genes are illustrated in The genetic algorithm was run 40 times to check consistency. The wining chromosomes along with classification accuracies (based on the SAM) are reported in The eight waveband regions (where selected genes were grouped) correspond to the spectral wavebands positions of the Mid-wave infrared Airborne Spectrographic Imager (MASI600) and the Thermal infrared Airborne Spectrographic Imager (TASI600). The MASI600 and TASI600 are pushbroom hyperspectral sensors operating in the mid-wave infrared (3–5 μm) and thermal infrared (8–11.5 μm), having 64 continuous spectral bands. These sensors can acquire data at a maximum altitude of 3,048 m (above sea level). The spatial resolution varies between 1 m and 3.5 m  with a spatial coverage of 600 pixels. The first four waveband regions  correspond to the wavebands of MASI600 and the last three regions  lay within the spectral range of TASI600.3.3.p ≤ 0.05).The Jeffries Matusita (J-M) distances between different species pairs calculated using the bands selected by the genetic algorithm, were compared with the randomly selected bands. The five selected bands  were used to calculate the J-M distance between each species. The average J-M distance values of genetic algorithm based selected bands were higher than the value of randomly selected bands. The result of the t-test  confirmset al. [post-hoc Tuckey HSD test. The spectral features  were then manually selected. In this study, the genetic algorithms selected the bands, further improving the classification accuracy.The classification accuracy based on genetic algorithms selected bands was higher than results obtained by Ullah et al. . They us4.This study tested the applicability of genetic algorithms for the selection of bands from the mid and thermal infrared emissivity spectra to discern thirteen vegetation species. The visible to shortwave infrared domain have been widely used for discriminating vegetation species, but mid to thermal infrared emissivity spectra have received little attention. The outcome of the study  demonstrThe improved classification accuracy of the bands selected by genetic algorithms compared to the randomly selected bands could be attributed to the fact that genetic algorithms provide several possible solutions, evaluate them on the basis of an objective function and pick the best one for the next generation.2 bond of methylene in leaf surface waxes [The validity of the combination of genetic algorithm based selected bands used for the spectral discrimination of vegetation species in the mid to thermal infrared emissivity spectra may be attributed to the spectral positioning of the selected bands. The emissivity spectra of the different plant species contain unique features due to the variation in physio-chemical composition of the superficial epidermal layer of the plant leaves. The emissivity signature of plant leaves is dominated by a feature associated with major classes of cellulous of the epidermis –28,35–38ce waxes –43. The ce waxes . Differece waxes ,41. The ce waxes ,46. The ce waxes .Discriminating vegetation species using laboratory measured emissivity spectra is prerequisite for the future vegetation mapping campaigns from air-borne and space-borne data. However, there are a number of problems associated with extending this work to field level. The calibration of remotely sensed signals in the MIR (around 3 μm) is complicated by the difficulty associated with the overlap of reflected and emitted energy in the MIR. Other problems associated with field condition are the distance between target and sensor, spectral and spatial resolution, atmospheric condition, and seasonal changes. The cavity effect of plant leaves causes blackbody emittance in the TIR and reduces spectral contrast in the signal. The cavity effect problem is noticeable in small and needle leaved species and also in species with funnel-like leaf arrangements . One cou5.This study has demonstrated the potential of genetic algorithms as band selectors using high resolution mid to thermal infrared emissivity spectra to differentiate between vegetation species at laboratory level. It is concluded that the bands selected by genetic algorithms are more useful for discriminating vegetation species than randomly selected bands are, when using laboratory emissivity spectra. The genetic algorithm based selected bands were actually found to have potential for floristic mapping. Bands selected with genetic algorithms may correspond to physiochemical characteristics of vegetation leaves (as seen in the previous studies) as leaves of different species possess unique surface materials. The genetic algorithm based selected bands help to understand the section of the electromagnetic spectrum that has a high potential for discriminating vegetation types, which may be useful when designing new sensors for vegetation studies. The outcome of this study is that the genetic algorithm band selection procedure can differentiate between plant species using laboratory measured thermal emission spectra. It would be very interesting to extend this work to the field and at airborne level with the advancement of hyperspectral thermal infrared sensors.