Nature papers on the use of minisatellite probes for DNA fingerprinting of humans . The new technology was soon adopted for many other organisms including plants, and when Hilde Nybom, Kurt Weising and Alec Jeffreys first met at the very First International Conference on DNA Fingerprinting in Berne, Switzerland, in 1990, everybody was enthusiastic about the novel method that allowed us for the first time to discriminate between humans, animals, plants and fungi on the individual level using DNA markers. A newsletter coined “Fingerprint News” was launched, T-shirts were sold, and the proceedings of the Berne conference filled a first book on “DNA fingerprinting: approaches and applications”. Four more conferences were about to follow, one on each continent, and Alec Jeffreys of course was invited to all of them. Since these early days, methodologies have undergone a rapid evolution and diversification. A multitude of techniques have been developed, optimized, and eventually abandoned when novel and more efficient and/or more reliable methods appeared. Despite some overlap between the lifetimes of the different technologies, three phases can be defined that coincide with major technological advances. Whereas the first phase of DNA fingerprinting (“the past”) was dominated by restriction fragment analysis in conjunction with Southern blot hybridization, the advent of the PCR in the late 1980s gave way to the development of PCR-based single- or multi-locus profiling techniques in the second phase. Given that many routine applications of plant DNA fingerprinting still rely on PCR-based markers, we here refer to these methods as “DNA fingerprinting in the present”, and include numerous examples in the present review. The beginning of the third phase actually dates back to 2005, when several novel, highly parallel DNA sequencing strategies were developed that increased the throughput over current Sanger sequencing technology 1000-fold and more. High-speed DNA sequencing was soon also exploited for DNA fingerprinting in plants, either in terms of facilitated marker development, or directly in the sense of “genotyping-by-sequencing”. Whereas these novel approaches are applied at an ever increasing rate also in non-model species, they are still far from routine, and we therefore treat them here as “DNA fingerprinting in the future”.Almost three decades ago Alec Jeffreys published his seminal  Many disciplines in botany are dependent on the ability to differentiate among plant genotypes, and/or to estimate the amount of diversity and relatedness in a set of genotypes. Traditionally, such tasks have been conducted mainly through data on morphological characteristics but these have certain limitations, including insufficient variation among the studied genotypes, subjectivity in the data collection and treatment, and plasticity due to environmentally induced variation. A more neutral and objective tool was offered by molecular markers based on isoenzymes; that is, enzymes that catalyze the same chemical reaction but differ in amino acid sequence and therefore also in the speed taken to travel through an electrophoretic gel. Isoenzymes were introduced into plant science in the early 1960s, and quickly increased in importance throughout the 1970s and 1980s. Co-dominant allozyme data  soon became very popular for studies of, for example, population structure, gene flow, isolation-by-distance (IBD), mating systems and hybridization )While insufficient repeatability of DNA marker profiles can be regarded as a methodological artefact, insufficient germline stability of sequences corresponding to DNA markers can cause “biological artefacts” due to excessively high mutation rates. This problem is most likely to arise with the most sensitive types of markers, such as SSRs. The ability to merge data from different studies, even when developed in different laboratories, is a major asset of this method. The same is true for the other single-locus DNA markers, such as SNPs, SCARs and CAPS, but these are usually only biallelic. Nevertheless, the potential number of SNPs is virtually unlimited, and various SNP-based assay methods have already been developed (see above). In a comparative study on 58 maize inbred lines, SNPs outperformed SSRs both in terms of quality and quantity .Exceptionally high mutation rates and reduced germline stability are often encountered when using retrotransposon-based markers . Thus, sBesides their application for the identification of plant material and for the estimation of similarity and relatedness, DNA markers have been extensively used for the construction of genetic linkage-based genomic maps, with a major aim of identifying markers that are closely linked and therefore co-inherited with genes for specific traits (see "Genetic mapping" below). Dense linkage maps have been constructed for numerous plant species including all major crops using all kinds of markers. For ease of scoring when screening large numbers of progeny, singe-locus biallelic markers such as SNPs are usually preferred for this purpose.Since the humble beginnings in 1988, DNA fingerprinting has become an immensely important instrument for genotype identification in both wild plant species and their cultivated relatives. Plants differ widely in life history traits including reproductive parameters such as propagation method and, for those that propagate by seed, also in breeding system (selfing or cross-pollinated) and in the mode of pollen and seed dispersal. All these factors have profound influence on the amount and partitioning of genetic variability between and within various entities such as cultivars and populations. These differences affect the utilization of DNA markers for fingerprinting individual plants or genotypes.In some cases, DNA-based estimates of similarity among a set of genotypes show a relatively close association with previous morphology-based estimates, but there are also considerable discrepancies in other cases. If the morphological characters are mostly quantitative in nature, correspondence with DNA marker estimates is generally quite high as compared to qualitative characters, which are more likely to reflect only a small number of mutation events. It has also been suggested that molecular data are better at differentiating cultivated genotypes as well as their wild relatives according to origin and pedigree, whereas conventional pomological characterization data are more closely associated with physiological properties .Proper identification of individual genotypes is an important basis for many wild-plant-based studies. As mentioned above, various life history traits affect the amount and partitioning of genetic variation. Inbreeding species are, for example, most useful for forensic applications, since they typically produce suitable-sized patches of genetically identical or almost identical plants . By contCarex curvula, a sedge species found in the European Alps. RAPD analysis of 116 tillers from a small patch (2.0 × 0.4 m) identified a total of 15 multi-locus genotypes. More than half of the sampled tillers proved to belong to a single, large clone estimated to be around 2,000 years old. Invasive species sometimes produce particularly large clones, such as the Japanese knotweed, Fallopia japonica, and the alligator weed, Alternanthera philoxeroides, both of which displayed a single RAPD phenotype in spite of being sampled over very large areas [Eichhornia crassipes were thus shown to consist of at least three different clones according to their RAPD profiles [In other research areas, the variation in plant life history traits can, however, be regarded as a positive factor; a wide range of biological questions can be answered by choosing suitable material and methods. DNA marker analyses have thus been able to estimate genotype age in plant clones, which have often proved to be considerably larger - and therefore often also older - than expected from previous data. For example, Steinger and colleagues  studied ge areas ,106. In profiles .Zostera marina, clonal size proved to be positively correlated with heterozygosity [Gagea spathacea[Information about clonal growth can be very helpful for determination of factors involved in shaping population structure. When a microsatellite-based study was carried out in the marine eelgrass zygosity . Outbreespathacea. All butTaraxacum populations consist of triploid individuals that apparently reproduce through apomixis and therefore are clonal. Such clones can sometimes cover large areas as was demonstrated in an AFLP study [Taraxacum, but that AFLP was more sensitive in detecting also small, mutation-derived differences within each microspecies [Ranunculus carpaticola[DNA fingerprinting has also helped to clarify the reproductive system in species that can produce seeds both sexually and asexually . Many LP study . A compaospecies . By contrpaticola,113. EviTaraxacum clones were recently employed to investigate biodiversity and ecosystem functioning. In one study, five identified Taraxacum clones were used for setting up experimental plots where effects of levels of diversity in both favorable  and unfavorable (mowed lawn) conditions could be quantified [Taraxacum clones [The availability of adequate tools to identify individual genotypes can be immensely useful in plant ecology. Thus, SSR-analysed antified . The genm clones . GenotypPrunus persica, which is self-fertile and self-pollinating to a large extent. In such crops, new cultivars sometimes have DNA fingerprints that are almost identical or at least very similar to those of the seed parent in spite of being derived through sexual recombination. By contrast, variation is sometimes encountered where one expected uniformity. Vegetatively propagated crops are usually still capable of producing sexually derived seeds, and these may germinate and develop into fertile but unnoticed plants in less well-tended fields and orchards. There is therefore an increased risk, especially for older cultivars, that a certain name is being used on several different entities, some of which have originated from seed setting.All plants belonging to a particular cultivar of an asexually propagated crop are expected to share identical DNA fingerprints, except for rare mutations. By contrast, sexually derived cultivars are expected to exhibit non-uniform fingerprint patterns. However, there are crops for which the major breeding method involves selection amongst rather similar seedlings that originate from a very small number of widespread cultivars. This situation can be exemplified by peach, Irrespective of propagation and breeding method, the value of accessions in plant genetic-material collections benefits tremendously from DNA marker-aided identification. This is, however, especially important in vegetatively propagated crops that must be grown in the field or maintained in greenhouses at high costs. Previous reviews ,116 showFor large-scale profiling of, for example, accessions in a genetic resources collection, SSR markers are usually preferred . AlthougTheobroma cacao, and reported an average error rate of only 0.014 for allele drop out and 0.019 for false alleles. Some loci were more error-prone than others, suggesting that putative loci should be evaluated not only for their polymorphism but also for reliability prior to large-scale analyses. Vélez and Ibánez [Zhang and colleagues  checked d Ibánez  checked d Ibánez . InteresThe possibility to merge SSR-derived data from different investigations is often reported as a major asset of this method. This option is, however, dependent on the use of identical SSR loci and suitable standardization procedures. Since absolute allele sizes of the SSR markers often differ when results from different laboratories are compared, a representative reference material with many different alleles should be used at all laboratories involved in the genotyping program, and the material for these standards should be harvested from predetermined plants in one collection only. By comparison with suitable standard alleles, sample alleles can then be defined according to relative number of core repeat units instead of relying only on the absolute fragment length in base pairs.Vitis vinifera, through resequencing of 11 genotypes [Citrus[Increased attention has recently been paid to the use of SNP markers for genotype identification in vegetatively propagated cultivars. Advantages of SNPs are their potential abundance and the fact that they do not rely on fragment length variation like SSR, and therefore are easier to standardize across different laboratories and equipment. Numerous high- and low-density SNP arrays have recently been developed for different crops. For example, a set of 48 SNPs was developed in grapevine, enotypes . High-ths [Citrus. Since tSecale cereale[In seed-propagated crops, at least some genetic variation usually persists also within cultivars. This is especially pronounced in highly outcrossing species, thus making DNA-marker-aided cultivar identification considerably more difficult. The situation is further complicated by the fact that each seed production cycle can lead to the introduction of new genetic variation - for example, due to foreign pollen. A considerable influx of new alleles was thus demonstrated after 7 to 13 subsequent regenerations of open-pollinating rye, e cereale.Brassica oleracea, accessions together with first-generation regeneration products from six of these accessions [Even with all the precautions taken in connection with modern gene bank regenerations, changes in allele frequencies can result from just recombination and selection. This was clearly demonstrated in an AFLP analysis of 50 white cabbage, cessions . The genFestulolium cultivars  with 7,680 probes on a microarray [Obviously, a large number of markers are required for proper quantification of genetic changes between generations, and for efficient discrimination among outcrossing, seed-propagated cultivars. Such large numbers are, for example, provided by the DArT technology that proved very useful for distinguishing croarray . In thisTriticum aestivum. Nowadays, SNP markers receive increasing attention also in sexually propagated crops, mainly for the almost inexhaustible number of potential polymorphisms. Genome-specific SNPs have thus been developed from wheat gene intronic regions, and have proven highly useful for cultivar discrimination as well as enabling a quantification of genetic diversity at each of the genomes in this hexaploid crop [Inbreeding crops are usually considered less problematic than outcrossers, since the cultivars are more homogeneous. However, some inbreeding crops still contain intra-varietal variability, especially in the case of primitive cultivars or landraces. Propagation cycles performed in a genebank with such material can cause prominent gene frequency changes due to gene flow and inadvertent selection. In these cases, pure-lining of the accessions may be necessary to avoid loss of diversity, as exemplified by the USDA Soybean Germplasm Collection . In addioid crop .Vitis vinifera, by Franks and colleagues [Spontaneously occurring somatic mutations can give rise to so-called 'sports’. These deviate from the original cultivars in minor but economically important traits such as fruit color in fruit and berry crops, and flower or leaf color in ornamentals. Sports are difficult to distinguish with DNA fingerprinting since the markers usually cover only a minute part of the genome. In addition, chimeras are quite common - that is, mutations that occur in only one of the three meristematic cell layers in the apical meristem that differentiate into the various plant tissues. The existence of chimerism was very elegantly demonstrated in grapevine, lleagues . Althouglleagues , 'Grünerlleagues  and 'Moslleagues  while cllleagues . In thisTy1-copia LTR retrotransposons, a set of 19 bud sports of the apple cultivar 'Fuji’ were investigated with S-SAP [Citrus reticulata, application of eight IRAP primers produced a total of five polymorphic bands whereas RAPD (26 primers), ISSR (16 primers), AFLP (8 primer combinations), S-SAP (9 primer combinations) and SSR (9 primer pairs) revealed, at the most, one (S-SAP) or two (RAPD) polymorphisms [S-SAP analysis has been successful for the genotyping of sports also in other crops, such as apple. Using 15 S-SAP primer combinations, five sports of 'Gala’ and one of 'Braeburn’ could be discriminated, both from each other and from the two original genotypes , whereasth S-SAP . All spoorphisms ,136.Some studies found surprisingly high levels of marker polymorphism within cultivars, such as in olive, where clones have been selected and subsequently multiplied by vegetative propagation for centuries. In one study, 27 putative clones of 'Verdeal-Transmontana’ could be differentiated with ISSR  while evHeritable somaclonal variation - that is, variation among regenerants due to somatic mutations - can be significantly enhanced by some micropropagation techniques. Although often regarded as an undesirable side-effect, these mutations can be valuable in crops that lack sexual reproduction  or have very long generation cycles . In general, axillary branches yield the most stable regenerants, followed by somatic embryogenesis and finally organogenesis. It is, however, impossible to predict whether markers will be able to find any variation in regenerated material, or what methods will prove to be most efficient.Helichrysum italicum, plantlets derived directly from leaves showed the same level of variability as plantlets that had passed through a callus stage [Very few polymorphisms have generally been found in tissue culture regenerants. The extent of DNA marker polymorphism can, however, vary considerably between plant materials - even of the same species - as was shown by comparing the very uniform regenerants of the banana cultivar 'Prata Ana’  with theus stage . Althougus stage .in vitro and in vivo variability [Detailed sequence-based analysis of the molecular events responsible for SCAR marker polymorphism  between somaclones and sexual recombination-derived lines of maize, demonstrated that the same mechanisms apparently determine both iability . Therefoiability  studied Cercidium sp., recovered both from the crime site and from the pick-up truck of a suspect [Polygonum aviculare obtained from germinating seeds found in the wheelhouse of a suspect’s car tire, and from a large number of soil samples taken at the crime site and various reference localities, were analyzed with AFLP [In theory, DNA fingerprints obtained from plant fragments should be able to provide important evidence in crime investigations but success has been limited so far, probably due to problems with isolating DNA of sufficient quality from poorly preserved plant material. SSR markers are often chosen for forensic work since they work comparably well also with heavily degraded DNA. One famous early case, however, involved RAPD analysis of seed pods of the Palo Verde tree,  suspect , while a suspect . In subs suspect . These fith AFLP .Detection of adulterations of food, drink and medicinal products is another area for forensic botany. Licensing arrangements sometimes require that a specified clone, cultivar or landrace is utilized in the manufacturing of food and beverages. Thus, well-defined grapevine clones must be used to receive “appellation d'origine controllée” labelling in France. In one study, musts  from two different grape cultivars could be identified using two SSR markers . In anotOlive oil is also often marketed with PDO labelling. RAPD, ISSR and SSR analysis of Portuguese olive oils allowed the determination of geographic origin of the cultivars on which they had been based . SimilarDendrobium officinale, which is a valuable source of 'Fengdou’ drugs used in traditional Chinese medicine [Medicinal drugs constitute another important product area where adulterants cause major problems. Based on nine SNP sites, all populations except two could be distinguished in DNA isolated from the dried stems of the orchid medicine . The latmedicine  and Sarwmedicine .Cannabis sativa specimens as part of drug enforcement [Cannabis genotypes [A variety of DNA marker methods have been used to demonstrate infringement of Plant Breeder’s Rights, either in court or, in our experience much more common, leading to a settlement outside of court . A relatorcement . In one enotypes . One of enotypes .Discrimination among different genotypes is often only a starting point for the subsequent quantification of genetic variability among these genotypes and analysis of patterns of relatedness and gene flow. The extent of genetic variation in a species and its distribution among and within populations is determined by a large number of factors, such as the breeding system, historical events regarding, for example, habitat availability and immigration, population size, migration between populations and many biotic and abiotic ecological factors. Nybom and Bartish  compiledThe uniparentally inherited plastid genomes behave as a single, haploid character, and the effective population size for plastid markers is therefore only half of that of nuclear  markers. Consequently, population differentiation due to genetic drift occurs much faster for cpDNA markers than for nuclear markers. Because of their relatively high intra-specific variability, chloroplast and mitochondrial micro- and minisatellites are therefore very useful for studying genetic structure at a species-wide scale.Saxifraga oppositifolia[Schinus terebinthifolius[Grevillea mucronulata[Triticum dicoccoides[DNA markers have become a major tool for studying fundamental evolutionary influences of natural selection, mutation, gene flow and genetic drift on wild plant populations. While selection and colonization history is responsible mainly for large-scale structuring of genetic variation, gene flow and genetic drift operate also at a more narrow geographic scale. Among these factors, gene flow especially has received much attention since it is crucial in determining levels of species integrity and subdivision. As already mentioned, breeding system has a profound effect on gene flow and the partitioning of genetic variation between and within populations. The occurrence of IBD between populations has been demonstrated with DNA markers in many different kinds of outcrossing plant species such as, for example, the herb sitifolia, the Brathifolius and the cronulata. IBD hascoccoides. In accococcoides. A correAntirrhinum microphyllum, suggesting a patchy distribution of genetic diversity. This is consistent with the territorial behavior of the main pollinator Rhodanthidium sticticum, short-distance seed dispersal, and a likewise patchy distribution of suitable habitats.In addition to the inherent dispersal capabilities of a species, gene flow is also affected by natural and anthropogenic habitat heterogeneity. Spatial autocorrelation analysis has thus become a valuable tool for studying spatial scale-dependent changes in DNA marker polymorphism within a population or group of closely occurring populations, and the impact of habitat characteristics on the resulting spatial genetic structure (SGS). Several computational methods have been used to calculate autocorrelation coefficients that measure the genetic similarity between individuals that fall within a defined distance class. A positive autocorrelation is frequently encountered over shorter distances, even if there is no overall linear correlation between geographic and genetic distances when calculated across the whole data set. Using RAPD data, Torres and colleagues  found siPulmonaria officinalis exhibited strong differences in allelic and genotypic richness, expected heterozygosity and inbreeding coefficent when analyzed with SSR markers [Thuja occidentalis, showed that SGS could be detected over a six times larger distance (90 m) within peripheral populations compared to within core populations (15 m) [Many plant species comprise both central, so-called core populations as well as more or less peripheral populations. Such populations may experience considerable differences in the magnitude of operating evolutionary and ecological forces. For example, edge and core populations of the herb  markers . Similars (15 m) .Hordeum spontaneum[Autocorrelation analysis has demonstrated IBD also in mainly selfing species but then usually at a very narrow scale, as was shown in the wild barley species pontaneum.Silene latifolia[Hordeum spontaneum[Highly informative estimations of gene flow can be obtained by genotyping the same plant material using both nuclear and organellar markers. Since the former are biparentally inherited and the latter usually only maternally inherited, the resulting data provide an indication of the relative importance of pollen versus seed migration . This ralatifolia. In selfpontaneum.Beta vulgaris subsp. Maritima, comprising more than a thousand plants from 33 populations along the French coast of the Anglo-Norman gulf, both mitochondrial and nuclear SSRs were applied [While genes can move between populations by seed and/or pollen, colonization of new habitats is dependent on seed only. In coastal plants, seeds often have the potential to disperse over long distances by hydrochory. In a study of wild sea beet,  applied . AnalysiSwietenia humilis in Honduras. Genetic variation was still high in all habitat fragments, but low-frequency alleles were more scarce, thus foreboding future genetic erosion. In another early study on tropical trees, Aldrich and Hamrick [Symphonia globulifera. Seedlings only occurred in primary and remnant forests, but not in pastures. Surprisingly, however, the majority of seedlings in fragmented forests proved to be derived from a few adult trees located in the open pasture land. Thus the genetic bottleneck experienced by the seedlings in remnant forest patches was caused by the reproductive dominance of a few spatially isolated trees in pasture land, in conjunction with unusually high levels of selfing in these trees.Effects of an increased subdivision or fragmentation of natural plant habitats has received much attention lately; dispersal between populations is reduced as well as genetic diversity. Outcrossing species may especially suffer from enforced selfing or biparental inbreeding in fragmented habitats, and lose much of their potential for adaptation to changing environmental conditions. Using SSR markers, White and colleagues  compared Hamrick  reconstrPolylepis multijuga was analyzed with AFLP and shown to contain surprisingly little heterozygosity and to display SGS at short distances, suggesting that most seeds moved only a few meters [Schinus terebinthifolius in western and eastern Florida, respectively, had spread and hybridized in little more than one century [Overall, tree species have been considered as comparatively resilient to fragmentation due to their often highly effective long-distance dispersal mechanisms. Recently, however, the wind-pollinated and wind-dispersed Andean tree w meters . This ty century . Since bQuercus robur and Q. petraea[Euterpe edulis[Abies alba[DNA fingerprinting data are often used to quantify levels of relatedness among genotypes or groups of genotypes, and numerous relatedness estimators have been described and compared. When wild plants are involved, the purpose is often to compare DNA marker-derived estimations of relatedness with current systematic treatment . Other applications include parentage analysis, which is the most direct way to estimate gene flow. SSRs are the most commonly used markers for this purpose but data simulations have shown that multi-locus markers such as AFLP can also be used with high confidence, at least when the dominant alleles occur in frequencies of 0.1 to 0.4 . Using S. petraea. The spape edulis. First, bies alba.Pinus sylvestris, offspring in a progeny test of open-pollinated genotypes in a seed orchard [Access to correctly defined relationships can be very important in plant breeding for the calculation of heritability of specific traits. Various statistical formulae have therefore been developed for determining genetic relationships among individual plants. In a comparison of either purely marker-derived estimations of relationships or combined pedigree and marker-derived estimations, the latter proved to be more informative when analyzing Scots pine,  orchard . Surpris orchard . The molFor cultivated crop plants, estimates of relatedness can provide valuable insights into the domestication process when material originating from different geographic areas is analyzed such as in, for example, Italian olive cultivars . RelatedEstimating the true level of genetic relatedness among cultivars from nuclear DNA marker data is quite difficult, since the obtained information can usually only estimate identity by state  instead of the more desirable identity by descent . One interesting approach towards a true phylogenetic analysis has, however, been achieved within the HiDRAS project . This prDNA marker-based procedures have frequently been applied to assess diversity and relatedness in collections of cultivated plant material - for example, gene banks. Interestingly, the anticipated loss of overall genetic diversity proved to be negligible when studied in 198 Nordic bread wheat landraces and cultivars that were developed during the last 100 years -190.In contrast to animals, many plant groups are characterized by highly variable ploidy levels, often even within the same species. This addition of genomes has certain effects on DNA marker application and data treatment. Moreover, the formation of hybrids by fusion of gametes from two different entities  is also common in plants . While hIris species was investigated using a wide variety of DNA-based methods. First, Iris fulva and I. hexagona were shown to each have a species-specific rDNA profile [I. brevicaulis, and I. nelsonii was shown to have derived from hybridization between all three species [In a series of classical studies, homoploid hybridization among American  profile . Subsequ profile . Diagnos species .Helianthus. RAPD linkage maps were developed for the sympatric and hybridizing species H. petiolaris and H. annuus and subsequently used to analyze the genome of a recently formed hybrid species, H. anomalus, as well as of an artificially generated hybrid [H. annuus and H. debilis, were investigated [Another important set of studies on homoploid hybridization has been undertaken in the sunflower genus, d hybrid ,195. Latd hybrid , and belstigated ,198. Intstigated .Quercus[Eucalyptus from Tasmania [Macaranga[cpDNA-derived information has played a major role in elucidating many cases of homoploid hybridization and subsequent introgression. Studies of multiple taxa in several tree genera have thus shown that chloroplast haplotypes often are closer associated with geographic origin than with species affiliation - for example, in oak trees, Quercus, in EucaTasmania  and in tMacaranga. This inMacaranga.Malus sieversii grows in Kazakhstan and has been suggested as progenitor of cultivated apple, M. domestica, based on morphological, historical and molecular evidence [M. sieversii[M. sieversii, (2) the European wild apple species M. silvestris, and (3) old and modern apple cultivars [M. sylvestris not only had the same common haplotypes as M. domestica, but there was also local sharing of uncommon haplotypes between the two species, suggesting recent inter-specific gene flow. A strong affinity between M. sylvestris and modern apple cultivars was likewise suggested in an SSR analysis of 839 genotypes collected from China to Spain, and representing four wild species as well as cultivated apple [DNA markers are also commonly used for detecting both ancient and ongoing hybridization between crops and their wild relatives. evidence . Nuclearsieversii. The oriultivars . In the ed apple . In thisMagnolia stellata and M. salicifolia together with putative F1 and F2 hybrids and backcrosses. The resulting clusters could be verified also with morphological trait analysis. Simultaneous application of cpDNA SSR markers showed that introgression was heavily asymmetric, with M. salicifolia being the seed parent of almost all hybrids and backcrosses.Two variants of STRUCTURE, InStruct and NewHybrids, were used by Muranishi and colleagues  in a recFestuca and Lolium[Festulolium cultivars and the parental genomes was clearly associated with the type of crossings performed - that is, F1, F2 or backcrosses.In plant breeding, there can be good reasons for analyzing parental contributions in recently developed, experimental hybrids, especially if the breeding process has involved one or several generations of backcrossing. The amount of parental influence could thus be quantified using a microarray analysis with 7,680 probes simultaneously detecting SNPs, indels and methylation differences, in a set of intergeneric hybrids between the commercially important grass genera nd Lolium. The extAstrolepis integerrima, which was recently studied by cpDNA sequencing and AFLP analysis [A. integerrima lineages have been formed through multiple independent hybridizations between A. cochisensis and A. obscura.Polyploidy is very common in the plant kingdom. Although the same marker technologies can be used for genotyping diploid as well as polyploid samples, statistical analyses and interpretations are usually less straightforward when polyploid samples are involved. Many species are allopolyploids and have been derived from their diploid ancestors by hybridization. Moreover, molecular studies of allopolyploid taxa and their putative progenitor taxa have shown that multiple origination is the rule rather than the exception. While frequent gene flow between polyploid lineages and back-crossing to parental taxa can further confound this process, a more easily studied case is offered in apomictic species where the speciation event is more or less frozen in time. One such example is the North American allopolyploid cloak fern, analysis . Six relMusa acuminata) and the other in the B genome . A more easily applied CAPS marker, obtained from PCR amplification of the ITS region followed by restriction with RsaI, has also been applied for this task [Identification of the putative progenitor species of polyploids can be attempted with various types of markers, including the internal transcribed spacer (ITS) region in the nuclear ribosomal RNA gene clusters which was sequenced and analyzed in, for example, polyploid rose cultivars and species . Nair anhis task . More rehis task .Mercurialis annua[Multi-locus based methods such as RAPD and AFLP are sometimes used for studying population genetics in species with different ploidy levels, but problems can arise due to a positive correlation between ploidy level and number of scored bands . In addilis annua. With thFor allopolyploid crops with intermediate levels of similarity among homologous genomes, such as tetraploid potato, SSR primers in general produce a variable number of bands per locus. For example, Fu and colleagues  found a Ranunculus kuepferi[Rosa[In autopolyploids and in allopolyploids with low genomic differentiation, SSR analyses usually produce multiple alleles of a single locus in each genotype, as demonstrated in the autopolyploid and apomictic  kuepferi and in aferi[Rosa-219. To feri[Rosa,220. Usiferi[Rosa. High-quIn crop plants with detailed pedigree information, the so-called microsatellite allele dose and configuration establishment (MADCE) procedure can be used to trace the transmittal of SSR alleles through documented generations of the investigated plant material, and determine the exact allele copy number in the target cultivars . OriginaIn many situations, allele dosage can, however, not be accurately scored with the methods chosen, and much plant material lacks or has only unsatisfactory pedigree information. Specialized programs have therefore been developed for analyzing polyploids with SSR markers, such as, for example, TETRA  and POLYA major drawback with any multi-locus approach is the loss of information about exact levels of heterozygosity and about genome inheritance. In addition, genetic distances between cultivars are exaggerated as compared to distances calculated on the basis of co-dominant data . A methoQuercus robur and Q. petraea. Distribution of markers according to their ability to detect inter-species diversity was clearly L-shaped; apparently only a few markers were located in genomic regions responsible for species differentiation. As expected, these markers were more likely to reside in coding regions than in non-coding regions. In another genome scan based on 88 mapped SSR loci, most loci again showed considerable migration between the analyzed taxa: sunflower species Helianthus annuus, H. debilis and their inter-specific hybrid [As the availability of DNA-based information increases, more attention is being paid to the genomic patterns of differentiation among plant species. According to the genic view of plant speciation, small “genomic islands” may be responsible for much of the differentiation between taxa through divergent selection or reproductive isolation barriers, while the remainder so-called “porous genome” is more permeable to gene flow ,231. In c hybrid . The genWhen targeting differentiation at a larger taxonomic scale, DNA sequence information of the chloroplast genome and/or nuclear genic regions, is usually preferred over DNA fingerprinting. Proper elucidation of the complex puzzle of plant systematics is, however, often best achieved with a combination of different types of molecular information. In many plant groups, various types of multi-locus or single-locus markers have thus provided important pieces to the puzzle. So far, these marker loci have seldom been placed on a genomic map , anSolanum section Petota and three taxa of Solanum section Lycopersicum[Macaranga[Multi-locus DNA profiling methods such as AFLPs have become the most commonly used DNA fingerprinting tool in plant systematics, mainly in situations where DNA sequencing produces insufficient phylogenetic resolution . In an eopersicum. Ploidy Macaranga. About 3Oryza also comprises of around 22 wild species that have received considerable attention due to their potential importance for rice breeding. Six diploid genomes  and four allotetraploids  have been identified using, among other methods, total genomic DNA hybridization [Oryza species were analyzed with AFLP [Oryza. In a follow-up study based on the sequences of 106 nuclear genes, divergence times and ancestral effective population sizes were also determined [Besides the two species of cultivated rice, the genus dization . In anotith AFLP . Pairwisith AFLP . Comparitermined .Rosa, several different DNA-based methods have been applied for phenetic and phylogenetic analyses, with mostly consistent results - for example SSR [Carolinae, Cinnamomeae and parts of Pimpinellifoliae forming one clade and most of the other seven commonly recognized sections forming the other clade. As for the division into sections, Synstylae appears to be mainly monophyletic and rather closely affiliated with sections Indicae and Rosa[Pimpinellifoliae is apparently polyphyletic, and R. spinosissima should be separated from the other species in this section. In spite of its size  and hybridogenous origination, the mainly European section Caninae  apparently constitutes a well-circumscribed monophyletic group. Another, very large AFLP study was recently conducted on >900 dog rose specimens sampled in a transect across Europe, with more than 200 non-dog rose samples analyzed for comparison [Caninae, Rubigineae and Vestitae. Within the subsections, many species overlapped considerably, and geographic distances often appeared to be at least as important as the conventional taxonomy in explaining similarities between analyzed specimens. Complementary information on phylogeny in Rosa has also been obtained with DNA sequencing. Although sharing some ITS sequence types with species in other sections thereby confirming their hybridogenous origin, the Caninae species also have one unique ITS sequence type which is further evidence of their monophyly [In the large and complex genus mple SSR  and AFLPmple SSR . Two maj and Rosa. Furthermparison . Two linonophyly ,242.Zamia were analyzed with 31 SSR primer pairs [Z. pumila, or as representing three more narrowly circumscribed taxa: Z. erosa, Z. portoricensis and Z. pumila sensu stricto. The SSR analysis showed that Z. erosa is strongly differentiated from the other two species, and thus may represent an independent introduction into Puerto Rico. The data are consistent with an allopatric speciation scenario with Z. portoricensis being the youngest taxon according to Bayesian coalescent analysis and effective population size, and still showing considerable admixture with Z. pumila.Plant systematic studies have occasionally been conducted also using SSR markers, especially when the focus has been on genetic differentiation among closely related taxa. For example, ten Puerto Rican populations of the cycad genus er pairs . These pArachis species from seven sections, including 11 accessions of cultivated peanut, A. hypogaea, were analyzed on the basis of allelic variation at 32 SSR loci [Genetic relationships among 35 SSR loci . A neighFrom the relatively few studies available, it appears that SSR markers do have some potential not only for species delimitation, but also for the reconstruction of genetic relationships among closely related species groups that are only a few million years old. However, SSR markers are usually highly polymorphic and therefore multiallelic within a species. Accordingly, the within-population component of SSR variation is often much higher than the between-population or between-species component . It is tPhylogeography aims to study the spatio-temporal history of a species on the basis of its intra-specific genetic variation . In prinEvolutionary relationships between cpDNA haplotypes are often depicted as networks , which cThere is no clear division between phylogeography on the one hand and traditional population genetics on the other. Accordingly, the use of nuclear SSR markers to study genetic diversity, genetic subdivision and gene flow within and among extant species is sometimes also called “phylogeography” , and theThe majority of plant phylogeographic studies still rely on plastid DNA polymorphisms that can be searched for by either PCR-RFLP, screening of length-variable plastid microsatellites (cpSSRs), or by comparative sequencing of PCR-amplified non-coding DNA . Unique Application areas of marker-based phylogeographic studies are diverse, and include, for example, the analysis of postglacial re-colonization patterns of the Central European landscape by trees and shrubs in the Quaternary ,259, theOne prominent application of molecular markers is the generation of genetic maps which have been established for all major and many minor crops and other plants . Mapping populations usually originate from a cross between two parental lines, which ideally can be distinguished by a large number of polymorphisms that are monitored in the progeny. Particularly convenient mapping populations consist of so-called Recombinant Inbred Lines (RILs), which are generated by selfing single-seed descent from different sibling F Pollard .To identify loci that are very tightly linked with a specific trait, “fine-mapping” is performed by enriching the density of markers in proximity of the responsible genes or, in the best case, markers for the responsible genetic elements themselves. The most commonly applied technique for fine-mapping is Bulked Segregant Analysis (BSA), originally developed by Michelmore and colleagues . In a BSDifferent types of markers can be combined into integrated maps, which become more highly resolved  with each newly added marker. Furthermore, data from different crosses can be integrated in the same map. For example, Wenzl and colleagues  publisheAssociation mapping (AM) aims at linking phenotypes to genotypes, independent of the kinship of the genotypes. The concept of AM has been implemented in humans and model organisms for many years (for example within the human HapMap project that started in 2002), and is now increasingly applied for plant genomes (see the reviews by Abdurakhmonov and Abdukarimov  and SotoAssociation mapping is based on the occurrence of Linkage Disequilibrium (LD) between a particular trait and one or more alleles of a marker locus in a population. In contrast to LM, which refers to the combined inheritance of loci due to their close physical proximity on the same chromosome, LD refers to the non-random occurrence of allele combinations of loci in a population. Thus, the reason for LD can be linkage (and in most cases it is), but also other factors influence LD, such as selection, mutation, mating system, population structure, and so forth, which can result in significant LD even of alleles that are located on different chromosomes . BecauseMore recently, AM-based analyses have been successfully carried out in many crops. In rice, for example, Zhao and colleagues  genotypePicea glauca[Arabidopsis. Nineteen variants were crossed in a random mating scheme, resulting in 527 F4 plants. The RILs originating from the maize NAS recently helped to identify important genes involved in maize kernel composition [Thanks to new, high-density genotyping methods such as the INfinumHD assay that assesses thousands of markers simultaneously, almost all major crops can now be subjected to AM . High-deea glauca, peach [ea glauca, apple [ea glauca and sweeea glauca, and areea glauca presenteea glauca performeposition , resistaposition  and stalposition . In applposition  used an One major aim of genetic linkage analysis in crop plants is marker-assisted breeding (see the review by Jiang ). A partArenaria ciliata and A. norvegica[For genotyping moderate numbers of SNP loci in hundreds to thousands of samples, PCR-based approaches provide a more flexible alternative to microarray-based methods. Besides direct sequencing, three currently popular methods for SNP genotyping of PCR products are high-resolution melting (HRM) analysis, allele-specific PCR (ASP), and the TaqMan assay. In HRM analysis, the PCR product is continuously heated, and the separation of the two DNA strands is monitored in real time . Polymornorvegica.Taq DNA polymerase, which occurs only when the probe has specifically bound to its target site. The ASP and TaqMan™ assays can be assessed in regular quantitative PCR machines while HRM requires specific optics.In ASP, the alternative alleles at a particular polymorphic site are amplified with allele-specific primers that are each labelled with a different fluorochrome. The presence of a particular allele is hence indicated by a diagnostic fluorochrome signal. If primers of different length are used, the ASP products can also be assessed by gel electrophoresis. The TaqMan™ assay dates back to the early 1990s . It invoTaken together, the last two decades have witnessed a prominent increase in the application of various DNA markers for plant DNA fingerprinting. In the beginning, multi-locus dominant markers, especially RFLP, RAPD, AFLP and ISSR, were most popular, but single-locus SSRs and eventually SNP markers rapidly caught up. The chip-based DArT technology is also still used. We believe that traditional multi-locus methods and their various spin-offs will still be employed a decade from now, but mostly for exploratory research that does not necessarily result in published papers. Locus-specific SSR markers will probably remain more popular, due to their co-dominant inheritance, ease of analysis and the fact that new data can easily be added to already existing files. Novel input to DNA fingerprinting was, however, provided by an unforeseen major breakthrough in DNA sequencing technology, which can be envisaged as the starting point for the future of DNA fingerprinting, and which is discussed in the following section.Molecular Ecology 22(11), 2013).The advent of massively parallel high-throughput genomic sequencing about 10 years ago was a breath-taking step forward in the yet short history of molecular genetics and genomics , a waterfowl species endemic to New Zealand. Each read had an average size of 243 bp, adding up to a total of approximately 4.1 Mb. Using appropriate bioinformatic tools, microsatellites were detected in 231 reads. The number of suitable marker loci was, however, reduced to 24 by the necessity to design primers on either side of the microsatellite. Thirteen of the primer pairs displayed polymorphism, and 13 markers were thus generated in a single sequencing run.Quite obviously, random high-throughput sequencing of genomic DNA offers itself as a useful strategy to identify all kinds of repetitive DNA in a genome, including microsatellites. It nevertheless took several years before this potential was realized. In one of the first reports on the use of next-generation DNA sequencing for microsatellite marker development, Abdelkrim and colleagues  used a 4Fusarium circinatum), an insect (the wasp Sirex noctilio) and a nematode (Deladenus siridicola). Two methods, ISSR-PCR and “fast isolation by AFLP of sequences containing repeats” (FIASCO), were used to enrich the template DNA for microsatellites prior to sequencing. Altogether, 1.2 to 1.7 Mb of DNA were sequenced, and 873 potentially amplifiable microsatellites were identified with sufficient flanking sequence available for primer design. A set of 28 SSR-flanking primer pairs were developed for Fusarium circinatum. Of these, 19 yielded single fragments in the expected size range, and 13 produced polymorphic amplicons from a set of fungal isolates. The authors also generated a traditional library from F. circinatum DNA enriched for microsatellites by the ISSR method. Sanger sequencing of 100 clones from this library yielded only eight potentially amplifiable microsatellites.Santana and colleagues  also useAgain using a 454 platform, Allentoft and colleagues  searchedBioTechniques and indicated for the first time that high-throughput sequencing has a strong potential to isolate SSR markers from non-model organisms where no genomic information exists. The novel methods save a lot of time, and are also cost-efficient as compared to traditional enrichment cloning. For example, Csencsics and colleagues [Typha minima within 6 weeks. In their study, 307 di-, tri- and tetranucleotide repeats were found in a total of 76,692 sequence reads. One hundred loci were selected for primer design, 30 primer pairs were tested and yielded 17 polymorphic markers.The above three papers were published in the same issue of lleagues  spent apTypha minima[Amaranthus tuberculatus[Vigna radiata[The relatively long read-lengths provided by 454 sequencing facilitate the identification of enough flanking sequence on either side of the SSR for the design of PCR primers. 454 technology was therefore initially preferred over other next-generation sequencing approaches for generating SSR markers. In plants, 454 sequencing was first applied to microsatellite isolation in ha minima, Amaranterculatus and Vigna radiata. Many moa radiata). More ra radiata,307, espa radiata showed iGiven that sequencing methodology is still developing at a rapid pace, a further decrease of costs may be expected. The large amount of sequence data obtained in even a small-scale experiment allows a top-down selection of the most promising candidates . Enrichment for microsatellite motifs prior to sequencing is sometimes advocated ,308, butAnser albifrons. Whatever technology is used for sequencing, it is essential that appropriate bioinformatic tools and hardware are available to cope with the huge numbers of sequences that have to be screened.Concerning the read-length of high-throughput sequencing platforms, a major breakthrough was recently reached by Pacific Bioscience, whose SMRT cells produce read-lengths of up to 15 kb. Sequence quality is, however, relatively low. It was therefore suggested to additionally produce reads with another sequencing platform  to correct the long PacBio reads in a hybrid sequencing approach . AnotherAmaranthus tuberculatus along with a contig representing an almost complete chloroplast genome, mitochondrial DNA fragments, transposable elements and numerous nuclear genes of interest [Dyckia marnier-lapostollei (Bromeliaceae) chloroplast genome from only 59,624 pyrosequencing reads. A total of 34 chloroplast microsatellites (cpSSRs) with a minimum number of 10 A´s or T´s were also found, and flanking primers were constructed that produced highly polymorphic amplification products in various Dyckia species.An additional benefit of the use of next-generation sequencing for SSR marker development is the large amount of random nuclear and organellar sequence data generated as a “by-catch” of microsatellite identification. For example, 382 SSRs were sampled in the course of 454 sequencing of interest . Krapp ainterest  assembleThe radically increased throughput in sequencing capacity at 50 to 100,000 times lower costs per base as compared with traditional Sanger sequencing also had a large impact on DNA fingerprinting technology itself. High-throughput sequencing opened completely new and unprecedently fast avenues for genotyping (up to the highest possible resolution) the resequencing of the entire genomes of populations of plants . WhSeveral strategies were developed that aim to enrich particular portions of the genome prior to sequencing. One obvious approach is to sequence only the transcriptome that is available in ESTs or cDNA libraries (for details see "Transcriptome sequencing: RNAseq and related approaches" below). Other techniques rely on the prior amplification of certain genomic subsets using PCR with selected primers , or enrDepending on its recognition sequence, any given restriction enzyme targets only a small proportion of the entire genome. Restriction digestion is therefore the first (and sometimes the only) step of reducing genomic complexity in REAS. As the restriction sites in genomes of closely related genotypes are usually shared, orthologous genetic regions are sequenced throughout the different genotypes. By choosing an appropriate (pair of) restriction enzyme(s), the extent of reduction of genome representation can be tailored experimentally. For example, a four-base cutter would  cut every 256 bp, and a six-base cutter every 4,096 bp. In order to focus the sequencing on the non-repetitive, transcriptionally active parts of the genome, most applications make use of methylation-sensitive enzymes that digest only non-methylated sites. Although different epigenetic regulation and accompanying methylation patterns might occur in the different genotypes of interest and hence there is the risk of sequencing and comparing different genetic regions in different genotypes, the advantages outweigh this risk.One version of the many different REAS techniques is summarized in Figure The primary data comprise the sequence information of the region flanking the restriction site to which the adapters were ligated. These restriction site-associated DNA sequences are referred to as “tags”, or “RAD tags”. Sequencing can be performed either by single end- or by paired-end sequencing. The latter is usually more cost efficient and facilitates mapping of the sequenced region to known related genomes ,328. By Provided that the genome under investigation is not too large, and an adequate sequence coverage is obtained, each particular RAD tag is resequenced many times. The data obtained from an REAS experiment can therefore be exploited for identifying huge numbers of SNP markers ,330. REACynara cardunculus, Scaglione and colleagues [Brassica napus. Yang and colleagues [Lupinus angustifolius. Finally, Pfender and colleagues [Lolium perenne and found several major QTLs for rust resistance.Numerous applications of REAS have recently been described in plants (see the review by Poland and Rife ). For exlleagues  discoverlleagues  sequencelleagues  used RADlleagues  used RADde novo assembly or annotation, the protein sequence as well as its variants are directly revealed. These polymorphisms represent the ideal markers for gene identification in a mapping approach, as the gene itself and not a co-segregating, anonymous genomic region is marked. (2) The method reduces the complexity of a genome to its transcriptionally active parts and thus allows the comparison of very large genomes. (3) RNAseq simultaneously allows both the determination of a genotype, and the assessment of quantitative gene expression, which permits the analysis of expression QTLs. This concept was described by Harper and colleagues [Brassica napus to correlate differences in gene sequences and gene expression on the one hand with trait variation on the other. The downside of the RNAseq approach is that, in order to be able to sequence a transcript, it must be expressed under the given circumstances - that is, in the investigated tissue - and at the time point of RNA isolation. Another problem is the uneven distribution of the transcript species, which either requires very deep sequencing, or normalization prior to sequencing. This problem especially concerns rare transcripts, such as those coding for transcription factors or other regulatory elements.Another widely used application of high-throughput sequencing for the discovery of polymorphisms is the sequencing of cDNA in an approach coined RNAseq (sequencing of transcribed regions). RNAseq is basically EST sequencing without prior cloning and on high-throughput sequencing machines. The technique and its applications in plants were recently reviewed by Martin and colleagues , and an lleagues  as “assoH. bulbosum into barley [RNAseq has also been used in BSA. For example, Trick and colleagues  fine-mapo barley .Molecular Ecology .Where do we go from here? Most certainly, the full technological capabilities are still far from being attained, and “next-next-generation” single-molecule sequencing is currently replacing some of the pioneering methods such as 454 and SOLiD, which are already on the brink of extinction. Further improvements and innovations of sequencing methodology can be expected, and will soon allow for cost-effective genotyping-by-sequencing of any non-model plant species. Even whole-genome sequencing might become feasible for certain applications, such as the analyses of somaclonals or mutation screening. However, obtaining complete genomic sequence data for all individuals included in a study would be unnecessary in most situations, and only inflate the costs. The tremendously huge data sets generated by next-generation sequencing need to be handled and stored, and bioinformatics has already become a real challenge. An important question will therefore have to be asked at the onset of each study - that is, how to allocate the finite number of resources between (1) sequence coverage , (2) sampling density , and (3) the fraction of the genome that is subjected to sequencing . We enviAFLP: Amplified fragment length polymorphism; AM: Association mapping; ASP: Allele-specific PCR; bp: Basepairs; BSA: Bulked Segregant Analysis; CAPS: Cleaved amplified polymorphic sequences; cDNA: copied DNA; cM: centiMorgan; cpDNA: chloroplast DNA; cpSSR: chloroplast simple sequence repeat; CRoPS: Complexity Reduction of Polymorphic Sequences; DAMD: Direct amplification of minisatellite DNA; DArT: Diversity Arrays Technology; EST: Expressed sequence tag; FIASCO: Fast isolation by AFLP of sequences containing repeats; GS: Genomic selection; GWS: Genome-wide selection; HRM: High-resolution melting; IBD: Isolation-by-distance; IRAP: Inter-retrotransposon amplified polymorphism; ISSR: Inter-simple sequence repeat; ITS: Internal transcribed spacer; LD: Linkage Disequilibrium; LM: Linkage mapping; LTR: Long terminal repeat; MAC-PR: Microsatellite DNA allele counting - peak ratios; MADCE: Microsatellite allele dose and configuration establishment; MAS: Marker-assisted selection; mtDNA: Mitochondrial DNA; NAS: Nested association mapping; PCR: Polymerase chain reaction; PDO: Protected denomination of origin; QTL: Quantitative trait loci; RAD: Restriction Site Associated DNA; RADseq: Restriction Site Associated DNA Sequencing; RAPD: Random amplified polymorphic DNA; rDNA: Ribosomal DNA; REAS: Restriction Enzyme Anchored Sequencing; REMAP: Retrotransposon-microsatellite amplified polymorphism; RFLP: Restriction fragment length polymorphism; RGAP: Resistance gene-analog polymorphism; RIL: Recombinant Inbred Line; RNAseq: Sequencing of transcribed regions; SAMPL: Selective amplification of polymorphic microsatellite loci; SCAR: Sequence characterized amplified region; SGS: Spatial genetic structure; SNP: Single nucleotide polymorphism; SRAP: Sequence-related amplified polymorphism; S-SAP: Sequence-specific amplification polymorphism; SSR: Simple sequence repeat; STR: Synthetic tandem repeat; TRAP: Target region amplification polymorphism; UTR: Untranslated region; WGS: Whole genome sequencing.The authors declare that they have no competing interests.HN, KW and BR participated in the writing of the manuscript. All authors read and approved the final manuscript.


In recent years, neuroimaging techniques such as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) have played a significant role in elucidating the neural underpinnings of posttraumatic stress disorder (PTSD). However, a detailed understanding of the neural regions implicated in the disorder remains incomplete because of considerable variability in findings across studies. The aim of this meta-analysis was to identify consistent patterns of neural activity across neuroimaging study designs in PTSD to improve understanding of the neurocircuitry of PTSD.We conducted a literature search for PET and fMRI studies of PTSD that were published before February 2011. The article search resulted in 79 functional neuroimaging PTSD studies. Data from 26 PTSD peer-reviewed neuroimaging articles reporting results from 342 adult patients and 342 adult controls were included. Peak activation coordinates from selected articles were used to generate activation likelihood estimate maps separately for symptom provocation and cognitive-emotional studies of PTSD. A separate meta-analysis examined the coupling between ventromedial prefrontal cortex and amygdala activity in patients.Results demonstrated that the regions most consistently hyperactivated in PTSD patients included mid- and dorsal anterior cingulate cortex, and when ROI studies were included, bilateral amygdala. By contrast, widespread hypoactivity was observed in PTSD including the ventromedial prefrontal cortex and the inferior frontal gyrus. Furthermore, decreased ventromedial prefrontal cortex activity was associated with increased amygdala activity.These results provide evidence for a neurocircuitry model of PTSD that emphasizes alteration in neural networks important for salience detection and emotion regulation. In the aftermath of highly distressing and shocking events such as combat, genocide, and rape, a subset of individuals develop posttraumatic stress disorder (PTSD), which is characterized by distressing memories of the event, physiological hyperarousal, and impairment in daily functioning. With the growing interest in PTSD due in part to its high prevalence among veterans of the Iraq and Afghanistan wars, there is an urgency to understand the neural pathogenesis of the disorder. Neuroimaging studies have been conducted to examine brain regions involved in PTSD -26. BaseThe aforementioned brain regions, which play a key role in nonhuman animal fear conditioning , likely However, fear conditioning models are limited in their ability to explain the full range of human experience and emotion. Fear conditioning can occur outside of conscious awareness, yet conscious processes such as voluntary and effortful avoidance of thoughts and memories of the trauma play a vital role in the development and maintenance of the disorder . This haIn the present study, we performed a quantitative meta-analysis of neuroimaging studies in PTSD using activation likelihood estimation (ALE). This method calculates the probability that a given voxel is activated consistently across studies rather than a single study  and therUsing keywords “PTSD,” “neuroimaging,” “fMRI,” and “PET,” a literature search in PubMed and Published International Literature on Traumatic Stress (PILOTS) was conducted for PET and fMRI studies of adult PTSD that were published before February 2011. The article search resulted in 79 functional neuroimaging studies. Included studies contrasted a traumatic or negative emotional condition with a resting baseline, positive condition, or neutral condition, conducted between-group analyses using subtraction methodology, and reported between-group peak activation coordinates in standard space. For relevant articles that did not report whole-brain results, the authors were contacted to request activation coordinates ,10. CaseFor each of the articles listed in Table In studies that included two levels of control groups  or PTSD patients , only foci from one of the between-group comparisons were used . Coordinates reported in MNI space were converted to Talairach space using the Lancaster transform [Coordinate-based random-effects meta-analyses were conducted using GingerALE software version 2.1  to assess for above chance clustering between experiments using a threshold at false discovery rate (FDR) corrected P < 0.05 and a less conservative cluster-extent of 40 mm3  was used.To explore the hypothesis that activity in the ventromedial prefrontal cortex and the amygdala was inversely related, we first identified whole-brain studies that reported increased ventromedial prefrontal cortex activity in controls relative to PTSD patients (which would suggest that this region was hypoactive in PTSD) and also reported regions of increased activity in PTSD relative to controls. Six studies were identified that met these criteria ,12,21-23Separate meta-analyses were run to examine the neural activity across and within symptom provocation and cognitive-emotional tasks in PTSD. Because of the variability in naming conventions of medial prefrontal cortex regions across different studies, activated regions are listed in the text and tables both by their structure specific name  and a general name signifying their contribution to a broader, less defined area .The regions that were hyper- and hypoactive when studies were collapsed across task type  in PTSD relative to control subjects are reported in Table Notably, there were several regions of hypoactivation in PTSD relative to controls including the medial frontal gyrus . The whole-brain voxel-wise analysis revealed hyperactivity in supplementary motor area. Bilateral amygdala and medial frontal gyrus  activity was observed when ROI studies were included . The analysis showed that when the ventromedial prefrontal cortex was hypoactivated, greater amygdala activation was observed in PTSD, supporting the hypothesis that activity in the ventromedial prefrontal cortex and amygdala are inversely related. Other regions that showed increased activity included the right middle and inferior temporal gyrus, left superior temporal gyrus, bilateral precuneus, and right putamen Table .The present study used quantitative meta-analysis to examine the pathophysiology of PTSD. The results confirmed involvement of a subset of regions implicated in fear-circuitry models of PTSD, including robust hyperactivity in the dorsal anterior cingulate cortex, hypoactivity in the ventromedial prefrontal cortex in PTSD, and an inverse relationship between activity in the ventromedial prefrontal cortex and amygdala. However, additional regions were found to be hyper- and hypoactive in PTSD, suggesting that a broader view of the neural circuitry of PTSD should be considered. Collapsing across symptom provocation and cognitive-emotional studies, the whole-brain voxel-wise analysis revealed hyperactivation of the mid/dorsal anterior cingulate cortex, supplementary motor area, and superior temporal gyrus in PTSD. These regions have been previously shown to be part of a putative ‘salience network’ that processes autonomic, interoceptive, homeostatic, and cognitive information of personal relevance ,43. UltiThe dorsal anterior cingulate cortex is a key node in the salience network. Earlier conceptualizations of the region suggested that its role was primarily in “cold” cognitive processes, in contrast to the ventral aspects of the anterior cingulate cortex that were thought to be involved in affective processing . HoweverInterestingly, the present results highlight the contribution of the mid-cingulate in PTSD, adding to the growing evidence that this region plays an important role in this disorder -51 and mWith the addition of ROI analyses, amygdala activity was observed for cognitive-emotional tasks but not symptom provocation tasks, suggesting that the type of task employed within a study influences amygdala activity in PTSD. There is emerging recognition that the amygdala may play a more general role in processing ambiguous and salient stimuli in the environment -55, of wIn the present study, widespread hypoactivity in prefrontal cortex in PTSD was observed, including both medial and lateral regions. Notably, hypoactivity in the ventromedial prefrontal cortex was present in both symptom provocation and cognitive-emotional study designs. To examine the relationship between the ventromedial prefrontal cortex and amygdala, we performed a meta-analysis that identified regions of hyperactivity within a subset of studies that showed a decrease in ventromedial prefrontal cortex activity in PTSD patients. We reasoned that under conditions of diminished ventromedial prefrontal cortex activity, which may signify reduced top-down governance of interconnected regions, we would observe greater amygdala activity. The results showed that when the ventromedial prefrontal cortex was hypoactive, the amygdala, putamen, and temporal cortex were hyperactivated. These results support the notion that a consequence of hypoactivity of the ventromedial prefrontal cortex may be greater responsivity of the amygdala in the face of negative information. Although the direction of this effect cannot be determined conclusively because the neural connections between the amygdala and ventromedial prefrontal cortex are bidirectional, there is a well-established literature showing the involvement of ventromedial prefrontal cortex in regulatory control across species . It is iImportantly, additional prefrontal cortex regions such as the inferior frontal gyrus were hypoactivated in PTSD. This finding is notable as previous work has implicated the role of inferior frontal gyrus in emotion regulation, including inhibition from emotional distraction  and emotP < 0.05, cluster-extent = 24 mm3). Future studies could isolate resting-state networks as a more powerful and robust method towards understanding the functional connections between nodes of the salience network in PTSD. Interestingly, a recent resting-state study in PTSD revealed greater connectivity between the amygdala and insula in patients with PTSD than trauma-exposed controls [A constraint of the current study is the availability of studies that met our criteria for inclusion into the analyses. Although the literature search started with 79 studies, the exclusion of studies that did not include stereotaxic coordinates likely reduced our power to detect less robust activations. Although the number of foci included in this study is more than the minimum recommended for a meta-analysis, it remains an open question whether a larger sample will reveal additional networks central to the PTSD diagnosis. For example, amygdala activity was observed in the PTSD group only when considering ROI analyses or using a less stringent spatial extent. Therefore, the limited number of studies available for the meta-analysis may have had an impact on the ability to detect amygdala activity within the whole-brain analysis. Activity in another key node within the salience network, the anterior insula, was observed in the PTSD group using a less stringent cluster threshold , supplementary motor area, and superior temporal gyrus.Furthermore, regulatory control regions include two primary networks that appear to be dysfunctional in PTSD, including ventromedial prefrontal cortex control over the amygdala and lateral prefrontal regions putatively involved in modification of thought and inhibition of distracting emotions. This model is consistent with the findings that therapies designed to both extinguish fear responses and promote emotion regulation through challenging negative cognitions are helpful for the treatment of PTSD.The author(s) declare that they have no competing interests.Ms. Amanda Mikedis conducted a literature review, performed data analysis, and assisted in writing the Methods section. Dr. Jasmeet Hayes and Dr. Scott Hayes helped with the literature review and data analysis, and wrote the paper. All authors read and approved the final manuscript.


The development of early and personalized diagnostic protocols is considered the most promising avenue to decrease mortality from cancer and improve outcome. The emerging microfluidic-based analyzing platforms hold high promises to fulfill high-throughput and high-precision screening with reduced equipment cost and low analysis time, as compared to traditional bulky counterparts in bench-top laboratories. This article overviewed the potential applications of microfluidic technologies for detection and monitoring of cancer through nucleic acid and protein biomarker analysis. The implications of the technologies in cancer cytology that can provide functional personalized diagnosis were highlighted. Finally, the future niches for using microfluidic-based systems in tumor screening were briefly discussed. Although much progress has been made in the diagnosis and treatment of malignancy, cancer is still the most common cause of death worldwide. Most patients with cancer have symptoms and distant metastases when diagnosed, which makes it more difficult to successfully treat the disease. Therefore, accurate prediction and diagnosis at early time are the most critical issues in cancers. Recently, significant efforts have been put into finding informative cancer biomarkers that can contribute to the establishment of cancer diagnosis . The bioTypically, microfluidic technologies enable the actuation of fluids and manipulation of bioparticles  at the microscale . Fluid fThis article presented a current overview about the potential application and perspectives of microfluidic technology in clinical oncology. Notably, due to the wide range and rapid proliferation of the subject matter, the paper was not intended to provide a comprehensive review but to provide the reader with a practical appreciation on the use of microfluidic devices in clinical cancer research.Genetic changes in cancer cells which lead to altered gene expression patterns can be utilized as biomarkers for detection and diagnosis of cancer . Gene exThe development of a high-speed, high-throughput DNA sequencing method represented the most attractive challenge for clinical molecular diagnostics, especially mutation detection and genetic screening. Capillary array electrophoresis (CAE) appears to be a prime candidate to be employed among various DNA sequencing methods . Since cThe use of microchips as a miniaturized polymerase chain reaction (PCR) platform realizes an autonomous lab-on-a-chip system, which can reduce the overall analysis cost as well as preventing the potential for sample cross contamination, and importantly, provide the more accurate and high-throughput means of cancer gene research . GeneralAlthough gene analysis has opened up a few applications in cancer research, discrepant changes in gene expression might not be reflected accurately to the level of protein expression or practical function . Protein4 theoretical plates at 460 V/cm. Total analysis time including immunoassay was nearly half an hour for a single sample. Further improvements in throughput could be aquired by increasing more sample reservoirs or parallel channels on the chip [Integrated CAE microchips have potential to address needs in high-throughput assays of proteins, in particular, capturing and quantifying cancer biomarkers. A polymethyl methacrylate (PMMA) microchip CAE system was fabricated with integrated on-chip fluorescence derivatization function . The devthe chip .For various solid malignancies, the routine diagnostic methods rely on qualitative tissue immunohistochemistry (IHC). IHC allows the identification of proteins of interest and provides information on protein localization and tissue morphology. However, it is now apparent that panels of protein cancer biomarkers, as opposed to single biomarkers, will be necessary for reliable cancer detection and monitoring . UnfortuOver the past century, mass spectrometry (MS) has evolved into a primary analytical tool in the proteomics area because of its sensitivity and accuracy. Advances in MS have been achieved by combining with several techniques, including gas and liquid chromatography, CE, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) ,31. The The research of cancer cytology is the most straightforward way for screening and monitoring precancer and cancer lesions . BioanalThe accurate separation of tumor cell subpopulations is a preliminary but often tedious step for cancer cell biology. Currently, the most widely used methods of cell sorting are fluorescence-activated cell sorting (FACS) and magnetically-activated cell sorting (MACS) -38. The Significant progress has so far been made toward developing microfabricated FACS (μFACS), enabling high screening throughput and high purity enrichment. Optical force was firstly developed in a μFACS system to sort fluorescent Hela cells . The syset al reported a three-dimensional (3D) microfluidic platform for rapid isolation and detection of cancer cells from a large sample volume [Alternatively, many other types of microfluidic-based sorting devices have also been proposed lately. Microsystem based MACS technologies (μMACS) allows high-throughput sorting of target cancer cells from the clinical samples based on target-specific affinity markers ,48. Liene volume . With the volume . DEP hase volume . In thise volume . The deve volume . The chiThe heterogeneity of cancer cells which can dramatically influence cell fate and biological responses has led to increased interest in individual cell studies. A number of single cell analysis approaches have been established, among which flow cytometry and fluorescence microscopy are most commonly employed . These mIntact single-cell analysis Cancer cell populations represent intrinsically variable and stochastic systems characterized by high levels of spatiotemporal complexity . Howeveret al reported a multi-channel parallel μFCM that was based on analog detection combined with parallel microfluidics [Rapid analysis and characterization of primary cancer cells using microfluidic technology has aroused many interest for clinical research of oncology. At present, microfluidic flow cytometry (μFCM) has offered promising avenues that leverages the multiparameter and high-speed measurements owing to their established nature. For instance, McKenna fluidics . The mulfluidics ,57. Thisfluidics .Alternatively, an innovative design was developed using a miniaturized diagnostic magnetic resonance (DMR) system for multiplexed, quantitative and rapid analysis of cancer cells . With thCell lysate analysis Cell Lysis is a crucial step upon which the subsequent analysis of extracted intracellular components depends. Mechanical cell lysis system was fabricated in early stage by using a microfluidic filter with nano-barbs . DespiteStochasticity and complexity in gene expression, protein or metabolite levels contribute to cell-cell variations. Ideally, the relationship between the cellular heterogeneity and cell behavior, which is key to understanding the development, progression, and treatment of cancers should be elucidated at the single-cell level . SubstanMicrofluidic devices provide numerous advantages over the traditional macroscale methods and slowly emerge as new platforms in a wide range of researches in clinical oncology. At molecular level, the analysis methods of gene biomarkers including gene mutations, cancer-related methylation, and other alterations were firstly introduced which included microfluidic chip based CAE and PCR. Protein biomarker analysis were then reviewed which mainly focused on the interface of microfluidics with IHC, ELISA, and MS. At cellular level, different microfluidic based methods were extensively employed which include cell sorting, cell culture and unique manipulation on isolated single cancer cells. These were, thus, particularly attractive for the clinical and diagnostic laboratories, allowing rapid analysis of only small amounts of patient derived cells. Moreover, the reconstruction of cancer microenvironment on chip provided new vistas for integrated cancer cytomics in clinical diagnostics and drug screening routines.Regardless of the advancement in the field of micofluidic system, this system remains in its infancy and still has several disadvantages, such as (1) the complicated fabrication processes, for example, the use of valves and pumps to fabricate large arrays of microchannels often require experimental setups and equipment not readily available in most labs; (2) the suitable interfaces for fluid transfer into, within, and out of the microfluidic chip may not be entirely satisfactory especially for portable and field-use systems; (3) the commonly used PMDS for fabricating the microfluidic device present with hydrophobicity and propensity for protein absorption, which may disturb the bioassay results; (4) electrokinetics methods driving fluid flow in microchannels suffer from the limitations of buffers incompatibility, electrolytic bubbles formation, evaporation of solvent and electrophoretic demixing due to different electrophoretic mobility; (5) the use of microfluidic systems often requires expertise which is not the part of training for the life science professionals.Several of the issues described above has raised a recurring theme throughout extensive applications of microfluidics that device integration for true on-chip functionality without requiring cumbersome and costly external ancillary equipment such as power supplies, capillary pumps, lasers, and mass spectrometers can be practically used and more widely adopted in a variety of laboratory, or commercial settings. These tests afforded by microfluidic-based lab-on-a-chip devices can be administered at a patient’s locale and even by the patient himself, offering not only convenience but significantly more rapid diagnosis than conventional lab-based testing. In the long term, a detection system can be envisioned that consists of an inexpensive, single-use, disposable cassette in conjunction with a portable analyzer. Specially for cancer detection, the microfluidic system will provide a means for cancer typing and staging, defining the clear boundaries of tumor mass, monitoring disease progression and recurrence, assessing the effectiveness of therapies, and detecting drug resistance. A more immediate and achievable near-term goal is perhaps the seamlessly interfacing of microfluidic devices with conventional benchtop laboratory processes through modular “plug and play” platforms . MoreoveAlthough the outlook of microfluidic technology is promising, the journey towards this lofty goal will be an extremely exciting and challenging one. There is a pressing need for a genuine collaborative and interdisciplinary effort, spanning from fundamental academic research to the commercial pathway towards translational biomedical technology.LOC: Lab-on-a-Chip; μTAS: micro-total analysis systems; CAE: Capillary array electrophoresis; PCR: Polymerase chain reaction; IHC: Immunohistochemistry; MS: Mass spectrometry; ESI: Electrospray ionization; MALDI: Matrix-assisted laser desorption ionization; FACS: Fluorescence-activated cell sorting; MACS: Magnetically-activated cell sorting; DACS: Dielectrophoresis (DEP)-activated cell sorter; FADS: Fluorescence-activated droplet sorter; μFCM: Microfluidic flow cytometry; WGA: Whole genome amplification.Both authors declare that they have no competing interests.Both LY and QW participated in the design of the manuscript and drafted the manuscript. Both authors read and approved the final manuscript.


Spike trains with temporal correlations have been related to cognitive and sensory processing , and extIt is known that excitatory and inhibitory activities are typically balanced , and maiIn this study, we use a Leaky-Integrate-And-Fire neuron with inputs that model a canonical disynaptic feedforward-inhibitory circuit Figure . We showOur findings provide insight in the role of inhibitory STDP, in maintaining detailed balance and enhancing transmission efficacy in disynaptic neural circuits.


At a species level, occurrence of TCs is significantly higher in transport than in collection phloem. TCs are absent from release phloem, but occur within post-sieve element unloading pathways and particularly at interfaces between generations of developing Angiosperm seeds. Experimental accessibility of seed TCs has provided opportunities to investigate their inductive signaling, regulation of ingrowth wall formation and membrane transport function. This review uses this information base to explore current knowledge of phloem transport function and inductive signaling for phloem-associated TCs. The functional role of collection phloem and seed TCs is supported by definitive evidence, but no such information is available for stem node TCs that present an almost intractable experimental challenge. There is an emerging understanding of inductive signals and signaling pathways responsible for initiating trans-differentiation to a TC morphology in developing seeds. However, scant information is available to comment on a potential role for inductive signals  that induce seed TCs, in regulating induction of phloem-associated TCs. Biotic phloem invaders have been used as a model to speculate on involvement of these signals.Transfer cells (TCs) are ubiquitous throughout the plant kingdom. Their unique ingrowth wall labyrinths, supporting a plasma membrane enriched in transporter proteins, provides these cells with an enhanced membrane transport capacity for resources. In certain plant species, TCs have been shown to function to facilitate phloem loading and/or unloading at cellular sites of intense resource exchange between symplasmic/apoplasmic compartments. Within the phloem, the key cellular locations of TCs are leaf minor veins of collection phloem and stem nodes of transport phloem. In these locations, companion and phloem parenchyma cells  Transfer cells (TCs) are located at “bottlenecks” for membrane transport of nutrients within plants. Their unique wall architecture and abundance of transporter proteins provides these cells with an enhanced membrane transport capacity. They occur at interfaces between plants and their environment  and at symplasmic/apoplasmic interfaces in loading/unloading of vascular pathways and exchange between them; in developing reproductive organs; in secretory organs  and in association with biotic symbionts (nitrogen-fixing bacteria and mycorrhiza) and intruders  . These are comprised of sieve elements (SEs). At maturity, SEs contain a parietal cytoplasm and are joined end-to-end and interconnected through sieve pores in their shared transverse walls to form STs extending from source leaves to heterotrophic sink organs. In metaphloem, each SE is symplasmically coupled with, and metabolically dependent upon, a cytoplasmically-enriched companion cell (CC) to form a sieve element/companion cell (SE/CC) complex. For protophloem, SEs may, or may not, be accompanied by CCs. In all cases, SEs or SE/CC complexes are embedded in a matrix of phloem parenchyma cells.−2 ST cross-sectional area s−1 and 500 L m−2 ST cross-sectional area s−1, respectively  and developmental and defense molecules form the cargo transported over long distances through STs , this brief analysis of phloem transport identifies a short list of candidate sites along the source-to-sink phloem path where cell types could be modified to a TC morphology. In the collection phloem, phloem parenchyma cells and CCs of apoplasmic loaders could be modified to a TC morphology to enhance nutrient release to, and uptake from, phloem apoplasms, respectively Figure . Retrievtrans-differentiate from non-vascular cells.With the evolutionary advancement of vascularization, TCs are found located in close proximity to sieve and xylem elements throughout the vascular highway. As anticipated (see Section Key Apoplasmic Steps in the Phloem Transport Pathway), TCs are found in the collection phloem of leaf minor veins and the transport phloem of the major vein network of leaves, petioles, stems, rhizomes and roots. For the release phloem, they are located within post-SE unloading pathways at apoplasmic/symplasmic interfaces, for example, in developing seeds  with the development of the megaphyllous habit . However, in  Winter, .Tradescantia virginiana—Gunning et al., Triticum aestivum—Bushby and O'Brien, Pisum sativum—Newcomb and Peterson, Nodal xylem parenchyma TCs may have either a reticulate morphology with their ingrowth walls polarized to a xylem element  TCs occur at a number of locations during embryogenesis and in the pre-storage and storage phases of seed development . These TCs form in one or both generations with their reticulate ingrowth walls polarized to the interface. An analysis of 18 genera  supporting the spore-bearing capsule, there are extensive symplasmic pathways to, and from, TCs bordering the gametophyte/sporophyte interface  or solely adjacent  to SEs . The high light growth conditions evinced a 50 percent increase in plasma membrane surface areas of collection phloem SE/CC complexes attributable to a more extensive development of wall ingrowths in their CC/TCs. The increased plasma membrane surface area of CC/TCs was accompanied by a comparable relative increase in rates of phloem loading  biosynthetic mutants and, in particular, vte2. Raised under cold conditions, the vte2 mutant exhibits a biochemical phenotype of excessive carbohydrate accumulation in source, but not sink, leaves consistent with attenuation of phloem loading and export as verified by 14C-photoassimilate export rates from excised leaves  and sites of emergance of lateral roots  of developing kernels. The resulting shrunken kernels are attributed to an attenuated resource delivery to the endosperm through reduced wall ingrowth formation in the TC layer  do TCs perform at host/biotroph interfaces. For leafhoppers, development of wall ingrowths in host transport phloem CCs could be a defense response to limit loss of transport phloem content to the surrounding stem apoplasm and hence the invading leafhoppers , the regulatory mechanisms directing assembly of their ingrowth walls are poorly understood  induced to form wall ingrowths on their outer periclinal walls  synthase 2 (VfACS2), demonstrated that a rapid burst in ethylene biosynthesis coincided with adaxial epidermal cells becoming competent to form wall ingrowth papillae  are rapidly induced  localized to basal endosperm cells, has been accounted for by a lowered glucose signal, sensed through a hexose kinase-dependent pathway, driving a positive regulation of auxin biosynthesis and hence auxin levels in these cells  within or proximal to the phloem comes from studies of nematode giant cell development. For instance, a spike in auxin concentration occurs as early as 18 h post-infection in giant cell progenitor cells which then declines from 42 h post-infection to being absent at 10 days post-infection  maxima functioning as a developmental signal initiating Pisum sativum, CC/TCs differentiate concurrently with the minor vein network  are an universal requirement for TC formation, this may account for why only 20 to 30 percent of Angiosperm species form TCs in their collection phloem  or symbiotic (nitrogen-fixing bacteria), involves suppression of host defenses coupled with a localized re-programming of host cell development  and re-programming development during establishment of a host plant/biotrophic relationship. Thus, a strong burst in extracellular ROS production by host cell NADPH oxidases and peroxidases accompanies root knot nematode invasion at 12 to 24 h post invasion before declining to lower levels during giant cell formation  migrates through cell walls of adjoining vascular parenchyma cells . Arabidopsis phloem parenchyma TCs are being used to address these knowledge gaps for collection phloem TCs. However, the peculiarities of ingrowth wall development in the less common phloem parenchyma TCs  indicates caution will be needed in extrapolating conclusions to inductive mechanisms for CC/TCs. There is an even greater dearth of information about regulatory mechanisms controlling induction and intracellular targeting of membrane transporters in developing phloem TCs. Arguably the root-knot nematode system affords the best opportunity to study inductive signaling of TCs associated with the phloem . Transcriptomic analyses are identifying cohorts of TC specific genes putatively responsible for organizing and constructing their ingrowth walls. At the mechanistic level for constructing ingrowth walls , the crucial process to enhance plasma membrane surface area and thus membrane transport capability, is to determine the signal(s) defining loci for wall ingrowth deposition.While there is an emerging body of evidence demonstrating a positive relationship between wall ingrowth amplification of plasma membrane surface areas and resource transport rates of phloem loading and unloading, the functional contribution of transporters targeted to these membranes remains essentially untested . Similarly, current knowledge of inductive signals initiating trans-differentiation, the potential exists for development of this functionally important phloem cell type in strategic locations for resource flow in species where TCs are absent. A combination of broad transcriptomic analyses, directed molecular analysis and mechanistic cell biology studies will be needed to achieve such an outcome.Since all plant species have the genomic capacity for TC The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.