The neuronal coordination of metabolic homeostasis requires the integration of hormonal signals with multiple interrelated central neuronal circuits to produce appropriate levels of food intake, energy expenditure and fuel availability. Ghrelin, a peripherally produced peptide hormone, circulates at high concentrations during nutrient scarcity. Ghrelin promotes food intake, an action lost in ghrelin receptor null mice and also helps maintain fasting blood glucose levels, ensuring an adequate supply of nutrients to the central nervous system. To better understand mechanisms of ghrelin action, we have examined the roles of ghrelin receptor (GHSR) expression in the mouse hindbrain. Notably, selective hindbrain ghrelin receptor expression was not sufficient to restore ghrelin-stimulated food intake. In contrast, the lowered fasting blood glucose levels observed in ghrelin receptor-deficient mice were returned to wild-type levels by selective re-expression of the ghrelin receptor in the hindbrain. Our results demonstrate the distributed nature of the neurons mediating ghrelin action. Ghrelin is a peptide hormone secreted predominantly by a sparsely-distributed group of endocrine cells in the gastrointestinal mucosa The sites of ghrelin action in the coordination of food intake and glucose homeostasis have yet to be definitively identified, but undoubtedly include direct action on one or more of several CNS sites implicated in the control of nutrient intake and blood glucose. For example, GHSRs are expressed within many nuclei comprising the medial basal hypothalamus, hindbrain, and midbrain, including sites already known to mediate food intake and glucose homeostasis In this report, we describe the use of a Phox2b-Cre recombinase-expressing mouse line that permits the selective expression of GHSR in specific hindbrain nuclei implicated in the control of food intake and glucose homeostasis All procedures were conducted in accordance with the UTSW Institutional Animal Care and Use Committee guidelines and those of AAALAC. Furthermore, the UT Southwestern IACUC committee specifically approved the work reported in this current study. Mice were housed in a pathogen-free facility on a 12h light/dark cycle with ad libitum access to food and water unless specified otherwise. Male mice were used for all experiments. GHSR-null mice, which contain a loxP-flanked transcriptional blocking cassette within the endogenous GHSR alleles The extent of GHSR expression resulting from Phox2b-Cre-dependent removal of the transcriptional blocking cassette as compared to that observed in wild-type mice was determined using single-label free-floating ISHH (n = 4), as described previously Six- to eight-week-old male mice were individually housed for 7 days prior to experimentation. Three days prior to experimentation, food was removed and replaced with 2 pellets of mouse chow placed in a petri dish on the cage floor. Mice were acclimated to the handling protocol for 3 days prior to s.c. injection of either saline vehicle or 2 µg/g mouse acyl-ghrelin . At 11 am on the test day, one chow pellet was weighed and placed in a petri dish on the cage floor followed by injection of saline into one half of the cohort and ghrelin into the other. Chow intake was followed for 3 hr. After experimentation, mice were singly housed for 7 days and the experiment repeated. Mice that received ghrelin during the first experiment received saline during the second trial and vice versa.Fed and fasted (18 hr) glucose levels were monitored between 12–3 pm using a OneTouch Ultra glucometer and testing strips . Blood was obtained from tail veins nicked using a disposable razor blade. For the fasting measurements, food was removed just prior to lights off (6 pm) the day preceding measurement of glucose.Mice were injected with acyl-ghrelin, as described above. Mice were subsequently transcardially perfused with formalin, and sectioned brains were stained using anti-c-fos antisera, as detailed previously Brain sections were analyzed with a Zeiss Axioplan light microscope. Data are presented as average +/− SEM. Adobe Photoshop 7.0 was used to adjust only sharpness, brightness, and contrast as well as to combine selected images into plates. Comparisons of food intake following ghrelin or saline injection and of fasting glucose levels were performed using a 1-way ANOVA and Tukey’s post-hoc test.To test the sufficiency of hindbrain ghrelin receptor expression in the mediation of ghrelin-stimulated feeding, we used a mouse model system that is conditionally null for GHSR (GHSR-null). These mice contain a loxP-flanked transcriptional blocking cassette inserted into intronic DNA upstream of the GHSR translational start codon, thus blocking GHSR expression th ventricle or directly into the hindbrain) and peripherally  th ventricle does not induce c-fos in the arcuate nucleus or paraventricular nucleus as it does when administered into the forebrain ventricles or directly into those nuclei, suggesting the presence of partially independent forebrain and hindbrain circuits that respond to ghrelin Multiple studies have suggested that hindbrain neurons exhibit c-fos expression in response to exogenous ghrelin administration – both centrally  expression limited to the hindbrain to determine if such site-selective, hindbrain GHSR expression is sufficient to mediate ghrelin’s actions on food intake and blood glucose. With respect to intake of freely-available food, hindbrain GHSR expression was not sufficient to permit the characteristic orexigenic response to subcutaneous ghrelin administration observed in wild-type animals. With respect to the modulation of glucose homeostasis, hindbrain GHSR expression was sufficient to defend against the exacerbated fasting-induced fall in blood glucose that is otherwise observed in mice with global GHSR deficiency. Interestingly, although subcutaneous ghrelin administration induces c-fos in the hindbrain of wild-type animals, such does not occur when GHSR expression is limited to the hindbrain. Thus, hindbrain c-fos induction seems dispensable for ghrelin-dependent modulation of fasting glucose levels. These data help clarify the relevant sites of ghrelin receptor action in the brain in the modulation of food intake and blood glucose.Several previous studies have implicated both the hypothalamus and the hindbrain as important CNS regions mediating ghrelin’s orexigenic actions. Among the many studies focusing on the hypothalamus, genetic ablation of both neuropeptide Y and agouti-related protein, which are normally co-expressed in a group of GHSR-expressing, arcuate neurons, was shown to completely abolish the acute orexigenic action of intraperitoneally-administered ghrelin While these latter studies indicate that GHSR-containing hindbrain neurons have the capacity to mediate ghrelin-stimulated food intake and that intact vagal signaling may be required for ghrelin’s overall acute effects on food intake, the data here demonstrates that direct sensing of ghrelin by GHSR-expressing hindbrain neurons is not sufficient on its own to mediate these acute orexigenic effects of ghrelin. Conversely, hindbrain ghrelin signaling does contribute to the maintenance of fasting glucose levels, as evidenced by the recapitulation of the usual (e.g. observed in wild-type mice) blood glucose response to an overnight fast by hindbrain-selective GHSR expression.These data complement our prior studies investigating the effects of tyrosine hydroxylase-Cre-driven GHSR expression, in which GHSR expression occurs selectively in catecholaminergic (predominantly dopaminergic) neurons, such as those in the VTA . Notably, and unlike with Phox2b-Cre-driven hindbrain GHSR expression, catecholaminergic GHSR expression was sufficient to partially rescue ghrelin-stimulated acute food intake, while also fully restoring the ability of administered ghrelin and chronic stress to modulate food reward . Also unlike with the hindbrain-selective GHSR expression observed here, fasting blood glucose levels were not rescued by selective GHSR expression in catecholaminergic cells .Regarding the involvement of ghrelin with the control of blood glucose, it most likely is the case that ghrelin’s full effect includes direct actions not only on the hindbrain, but also on other CNS sites and peripheral organs that affect glucose homeostasis. For instance, ghrelin has the capacity to directly bind to GHSRs on pancreatic alpha cells and beta cells, leading to stimulation of glucagon release and inhibition of insulin release, respectively, both of which would tend to raise blood glucose levels


Stalk strength is an important trait in maize (Zea mays L.). Strong stalks reduce lodging and maximize harvestable yield. Studies show rind penetrometer resistance (RPR), or the force required to pierce a stalk rind with a spike, is a valid approximation of strength. We measured RPR across 4,692 recombinant inbreds (RILs) comprising the maize nested association mapping (NAM) panel derived from crosses of diverse inbreds to the inbred, B73. An intermated B73×Mo17 family (IBM) of 196 RILs and a panel of 2,453 diverse inbreds from the North Central Regional Plant Introduction Station (NCRPIS) were also evaluated. We measured RPR in three environments. Family-nested QTL were identified by joint-linkage mapping in the NAM panel. We also performed a genome-wide association study (GWAS) and genomic best linear unbiased prediction (GBLUP) in each panel. Broad sense heritability computed on a line means basis was low for RPR. Only 8 of 26 families had a heritability above 0.20. The NCRPIS diversity panel had a heritability of 0.54. Across NAM and IBM families, 18 family-nested QTL and 141 significant GWAS associations were identified for RPR. Numerous weak associations were also found in the NCRPIS diversity panel. However, few were linked to loci involved in phenylpropanoid and cellulose synthesis or vegetative phase transition. Using an identity-by-state (IBS) relationship matrix estimated from 1.6 million single nucleotide polymorphisms (SNPs) and RPR measures from 20% of the NAM panel, genomic prediction by GBLUP explained 64±2% of variation in the remaining RILs. In the NCRPIS diversity panel, an IBS matrix estimated from 681,257 SNPs and RPR measures from 20% of the panel explained 33±3% of variation in the remaining inbreds. These results indicate the high genetic complexity of stalk strength and the potential for genomic prediction to hasten its improvement. Ostrinia nubilalis H. Diatraea grandiosella D. Gibberella zeaeDiplodia zeaeMaize stalk strength impacts grain yield and silage quality due to its relationship with stalk lodging and stover quality. High stalk strength is important in fields plagued by European corn borer, Dissection of stalk strength into its constituent traits suggests the structural composition of the rind, and not the pith or total girth, is the chief determinant of strength Given the numerous mechanisms mediating stalk strength and the continuous variation observed for the trait, several studies were performed to quantitatively dissect its genetic architecture Previous RPR studies laid a foundation for evaluating stalk strength. But, whole genome sequencing of B73 Stalk strength remains a determinant of harvestable yield and forage quality in the use of maize for ruminant animals. Furthermore, new applications in cellulosic ethanol and biopolymer synthesis have caused a surge of interest in stalk strength related traits such as cell wall composition and biosynthesis In this study, we measured the stalk strength (RPR), days to anthesis (DTA), and primary ear height (EHT) of approximately 200 recombinant inbreds (RILs) from each of 25 families composing the maize nested association mapping panel (NAM) The NAM panel, developed by the Maize Diversity Project, was created as previously described NAM families were also grown in Madison, WI at the Arlington Agricultural Research Station (silt-loam soil) in the summer of 2009. In WI fields, 3,453 RILs of the NAM panel were randomized and blocked into 10 maturity groups based on previous flowering data At Muskgrave Research Station in Aurora, NY (silt-loam soil) in the summer of 2010, a set of 2,293 NCRPIS inbreds were measured for RPR. The inbreds were planted in single row plots of 12 plants. The same year, 2,453 inbreds from the NCRPIS diversity panel were measured at South Farm Research Station in Columbia, MO (silt-loam soil). Single row plots of 15 plants were grown. B73, and replicates of the 25 alternate NAM parents, were randomly included as checks in the NY and MO field designs. The 2,293 inbreds measured in both environments were used in GWAS and genomic prediction of the NCRPIS diversity panel .To measure stalk strength, a modified Accuforce Cadet digital force gauge  was assembled with a spike and used to manually pierce stalks as previously described RPR measures were collected near the middle of the stalk internode immediately below the primary ear. Measures were taken from three randomly selected plants per plot. This resulted in the collection of 37,548 RPR measures across NAM families in three environments. Measures taken in the IBM family and the NCRPIS inbred diversity panel amounted to 1,735 and 15,399 observations, respectively. These were taken in two environments. To avoid differential nutrient availability and light capture, the end plants of each plot were not measured. Phenotypic data for DTA and EHT were acquired from the same plots as previously described Three molecular marker sets were used for joint-linkage QTL mapping, GWAS, and GBLUP across the NAM and IBM families, as well as linear mixed model GWAS and GBLUP across the NCRPIS inbred diversity panel. In the first marker set, 1,106 markers were genotyped on an Illumina Golden Gate assay across the RIL families to facilitate joint-linkage QTL mapping A second marker set of 1.6 million SNPs reported in the maize HapMapV1 www.panzea.org.A third marker set of 681,257 SNPs from the NCRPIS inbred diversity panel were genotyped using Genotyping-By-Sequencing (GBS) To partition phenotypic variance into genetic and environmental components for RPR, DTA, and EHT, linear mixed modeling was performed using ASReml v3.0 row ∶ AR1col). All environmental effects and residual correlation structures in each environment’s model were tested by backward selection and retained if they met a likelihood ratio significance of p<0.05. BLUPs for each RIL in the NAM and IBM families were then predicted in each environment.For the RIL families, linear mixed models were first separately fit for each environment including a single fixed effect for the grand mean and multiple random effects. Random effects entering the full model included family and RIL nested within family genotypic effects, as well as the environmental effects of the blocks, rows, and columns of each field design. A separate variance component was fit for RIL nested within each of the NAM and IBM families. An additional family term was constructed to accommodate the replicated parental checks. All random effect terms in the model, including the genotypic effects of family and RIL within family, were modeled with independent G structures. However, a correlated R structure was fit among residuals based on a two-dimensional separable first-order autoregressive spatial structure for rows and columns in each environment  line means for these traits . The unbFor each trait, estimates of broad sense heritability on a line means basis across and within RIL families were calculated from the multi-environment models fitting genotypic effects as a random effect, as previously described The denominator of the broad sense heritability estimator, when computed on a line means basis To map genetic associations underlying the estimated heritable variation, the SAS v9.2 statistics package Next, the previously described stepwise joint-linkage mapping procedure After constructing a family-nested QTL model for RPR, DTA, and EHT from the full dataset, each trait’s model was fit to every other trait for estimation of pleiotropic family-nested QTL. Significance of pleiotropy was determined as previously described By modifying family-nested QTL models for RPR, DTA, and EHT constructed from the full dataset, ten additional models, one for each maize chromosome, were built to account for background genetic variation during joint-linkage-assisted GWAS. In the family-nested QTL models, all terms explaining significant trait variation were fit, with the exception of the family term denoting variation between the NAM and IBM families. Family-nested QTL residing on a model’s designated chromosome were also dropped. Residual trait variance after fitting this model was inferred to represent the joint distribution of error and the missing family-nested QTL of the dropped chromosome.For each trait, residuals from each of the ten chromosome’s models were used in joint-linkage-assisted GWAS of the HapMapV1 SNPs Using the Genome Association and Prediction Integrated Tool (GAPIT) We conducted genomic prediction by GBLUP using the package rrBLUP To perform cross-validation, all NAM RILs (not stratified by family), each NAM and IBM family, and the NCRPIS diversity panel were randomly partitioned into five disjoint subsets. Line means from combinations of one , two (10 folds), three (10 folds), and four (5 folds) subsets were used to calibrate models and predict the remaining line mean values. All prediction accuracies were then averaged across folds. This process was repeated 20 times randomly selecting five disjoint subsets each time to estimate prediction accuracy with respect to the number of lines in the model calibration set.For comparison to GBLUP, prediction of RPR BLUP line means by joint-linkage mapping of family-nested QTL was also performed using PROC GLMSelect in the SAS v9.2 statistics package Prediction accuracy was measured as the coefficient of determination obtained by regressing BLUP or BLUE line means from first stage models against predicted line means obtained by GBLUP or the family-nested QTL models of second stage models for each trait upon cross-validation. This differs from traditional estimates of genomic prediction accuracy as the coefficient of determination between genomic estimated and true breeding values. This metric was not calculable given a lack of true breeding values for RPR in RIL families or the NCRPIS inbred diversity panel.Heritable variation between maize lines was identified for RPR, EHT, and DTA within the NAM, and IBM families, as well as the NCRPIS inbred diversity panel . TransgrBroad sense line means heritability estimates for RPR measures in the surveyed germplasm were low . This waAbout 37% of stalk strength variation in the NAM panel was attributed to environmental variation . This waCorrelations between RPR and DTA, were positive among plots across NAM and IBM families , and EHT , were observed across the NAM and IBM BLUP family means.brown midrib mutants involved in lignin biosynthesis of the phenylpropanoid pathway, a putative 4-coumarate-CoA ligase-like gene (AF466202.2_FG012) potentially involved in the same pathway was located near a linkage marker on chromosome ten at about 69.2 cM with a RMIP of 18. Also, a caffeoyl-CoA O-methyltransferase (GRMZM2G077486) of the phenylpropanoid pathway was flanked by linkage markers on chromosome ten at 38.6 and 40.1 cM possessing RMIPs for RPR of 20 and 11, respectively.The resolution afforded by joint-linkage mapping did not provide gene level characterization of RPR associations. In most instances, robust family-nested marker association persisted an interval of one to three cM before dropping below a RMIP of 10. In these intervals, few genes with known involvement in phenylpropanoid or cellulose synthesis pathways were identified. While no robust associations were found near the brittle stalk 2, glossy1–15, and teopod1, 2 were not identified near associations possessing RMIPs for RPR over 10. Similarly, co-localization of QTL mapped in the previous multi-family RPR study and this family-nested QTL study was not substantial Of the 12 known cellulose synthases in the maize genome, the only synthase whose nearest linkage marker possessed a RMIP over 10 was cellulose synthase-9 (GRMZM2G018241). This gene and marker are located on chromosome two at 82.5 cM and possessed a RMIP of 24. Uncharacterized annotations  with predicted cellulose synthase activity by homology and known transcriptional evidence were also identified on chromosome nine near a linkage marker at about 42.8 cM and chromosome ten at about 38.6 cM on the NAM composite map. Both these linkage markers possessed a RMIP of 20, with the latter also flanked by a marker possessing a RMIP of 10. Cloned loci previously identified for vegetative phase transition and other stalk strength related traits, such as the mutants of To further resolve joint-linkage mapped RPR associations, 141 significant associations were identified by joint-linkage-assisted GWAS in the NAM and IBM families . Family-The effect sizes of GWAS RPR associations across the NAM and IBM families were uniformly small and similar in size and distribution to the significant alleles nested within each family during joint-linkage QTL mapping. No RPR effects with an absolute value greater than 0.05±0.02 KgF were observed for GWAS effect estimates. The most robust association identified across the NAM and IBM families possessed a RMIP of 100. This SNP is located on chromosome three at 176,660,475 bp and was flanked by linkage markers that possessed RMIPs of 10 and 13 during joint-linkage mapping. The nearest annotation is 5,139 bp downstream and encodes a transferase (GRMZM2G165192) responsible for transferring acyl groups other than amino-acyl. No annotations within a 1 cM interval surrounding the association were obvious candidates for stalk strength. The second and third most robust associations were both identified on chromosome eight at 163,943,201 bp and 8,415,595 bp. These possessed RMIP of 94 and 73, respectively. Both were also located near regions of the genome neighboring significant markers identified in joint-linkage analysis. The former is located in an interval wherein two linked markers spaced about 1.7 cM apart possessed a combined RMIP of 62. The latter neighbors a linked marker with a RMIP of 14 in joint-linkage mapping. In both instances, no obvious candidates for stalk strength related pathways or developmental processes were apparent. The nearest respective annotations were a glycosyl-transferase (GRMZM2G002023) 4,605 bp downstream and an O-glycosyl hydrolyzing enzyme (AC234160.1_FG003) 3,349 bp downstream.brown midrib, brittle stalk, or teopod loci or near enzymes with known involvement in phenylpropanoid and cellulose synthesis pathways or vegetative phase transition. Comparisons between significant RPR associations identified in the RIL families and those enriched in the NCRPIS inbred diversity panel revealed little discernible commonality.In addition to joint-linkage-assisted GWAS across the NAM and IBM families, sequential single marker GWAS was also performed across the NCRPIS diversity panel regressing BLUP line means for RPR against SNPs. . To accoGiven the highly polygenic architecture of RPR, genomic prediction was performed to determine the ability of all genotyped diversity to explain variation in BLUP line means for RPR, DTA, and EHT across all NAM families possessing a broad sense line means heritability over 0.05 . An IBS Fitting a family-nested QTL model with an included family term, constructed by joint-linkage mapping of calibration sets subsampled from each of the heritable NAM families, wielded lower prediction accuracies than GBLUP . ConstruAn IBS relationship matrix constructed for the RILs of the IBM family was less predictive of variation in BLUP line means for RPR, DTA, and EHT than that obtained across all NAM families when fit by GBLUP . Cross-vIn addition to RIL families, the accuracy of GBLUP at predicting BLUP line mean variation in RPR, DTA, and EHT was assessed in the NCRPIS inbred diversity panel . This waIn a final assessment of genomic prediction accuracy, BLUE line means of RPR, DTA, and EHT between families and inbreds across environments were predicted by GBLUP . CorrelaIn this study, we inferred the genetic architecture and genomic prediction accuracy of RPR as a proxy for maize stalk strength. In total, over 50,000 plants were measured for RPR across NAM and IBM families, and the NCRPIS diversity panel. These measures were collected in addition to DTA and EHT within three environments for NAM and IBM families, and two environments for the NCRPIS inbred diversity panel.Transgressive segregation for RPR was observed in many NAM and IBM families . This fiDespite the increased genetic variation in RPR among RILs relative to their parental values, estimates of broad sense heritability calculated on a line means basis were low in all families . This coHowever, it is most likely the reduction in heritability resulted from the decreased replication of measures taken on each inbred in this study relative to the former multi-family RPR study Of the heritable RPR variation that was observed in this study, approximately 60±4% was explained by variation between families. With a shared B73 founder, all SNPs differing between families also segregated within at least a fraction of the families depending on an allele’s frequency among founders. Variation between RIL families may then be attributed to mutation and ancestral recombination occurring between the non-B73 progenitor of each family; whereas, variation within families may be attributed largely to recent recombination. Differences in variance within and between families are likely due in part to the reduced functional diversity segregating within a single family relative to across families. But, given the random genetic effects used to model RPR, another reason for increased variance estimates between families, is an increased number of observations available to estimate between family variance. This increase in observations between families relative to within them reduced the error and the shrinkage of random effects modeling between family variance.To resolve family-nested QTL explaining RPR variation, BLUP line means for RPR were used in bootstrapped joint-linkage mapping of the RIL families. After accounting for RPR variation between families, variation in families was mapped to family-nested molecular markers. This identified 78 family-nested markers with a RMIP over 10 . Given tWhile the identified number of family-nested QTL is highly dependent on the mapping resolution and method employed, the number entering into a single model for RPR in our approach was 18±2 before the permutation-based threshold for model entry was reached. Of these family-nested QTL, only about half were significant within a given NAM or IBM family. A single family-nested QTL model explained 81±3% of the variation across RPR BLUP line means. Supporting the presence of repulsion phase linkages of RPR loci in NAM parents, positive and negative allele effects relative to the common parent, B73, were observed within all families. The allele series present in all family-nested QTL may be attributed to the genetic background specificity of the effects and differing linkage patterns among nearby loci whose cumulative effect is explained by the family-nested QTL. Further highlighting the highly polygenic genetic architecture of RPR, all family-nested QTL effects were small and none explained over 2.7% of the BLUP variation in RPR across RILs after accounting for the between family variation. Although covariation was observed for DTA, EHT, and RPR among line and family means, and several loci for these traits co-localized, no significant mutually pleiotropic family-nested QTL were identified. This supports previous studies indicating ear height is a poor proxy for stalk strength during selection After joint-linkage mapping, RPR variation between BLUP line means of the RIL families was further resolved by joint-linkage-assisted GWAS. This analysis revealed the segregation of 141 significant non-nested associations across families, after accounting for variation between families . FurtherIn addition to joint-linkage-assisted GWAS, a sequential single marker linear mixed model GWAS of RPR BLUP line mean variation was performed in the NCRPIS diversity panel . HoweverEven in sizeable populations, such as the NCRPIS diversity panel, only the most common and largest additive effect alleles not severely confounded with population structure will significantly associate with a trait during a single marker mixed model GWAS. If the functional diversity of stalk strength is correlated with fitness and follows the Fisher-Orr model However, it remains to be determined if these studies should focus on candidate genes based on known molecular homology. GWAS associations identified as significant in the NAM and IBM families as well as the top 100 associations with a false discovery rate just over 10% in the NCRPIS diversity panel did not co-localize with genes involved in phenylpropanoid and cellulose synthesis, or vegetative phase transition. This lack of co-localization with genes in pathways known to influence many constituent traits of stalk strength may be due to their conservation in natural variation. This may result as a consequence of negative fitness effects. Nonetheless, given the low heritability of RPR, and high levels of diversity observed, it is likely the numerous anatomical and compositional factors influencing RPR greatly complicate mapping of causal loci. While little pleiotropy was observed between stalk strength and ear height or days to anthesis, interactions among other constituent traits may greatly influence stalk strength, and may do so in a non-additive manner. Future mapping efforts should decompose stalk strength into its constituent traits to reduce latent complexities limiting power and aid mapping of their potentially simpler genetic architectures.Given the high degree of complexity in stalk strength genetic architecture inferred while mapping RPR, genomic prediction was performed using GBLUP to model relatedness based on an IBS relationship matrix. This method revealed substantial variation in BLUP and BLUE line means for RPR, DTA, and EHT was predictable using a fraction of RILs randomly selected from each NAM family . While BAs a comparison to GBLUP, the expected prediction accuracy of family-nested QTL models constructed by joint-linkage mapping subsets of the full data set was also assessed across the BLUP line mean variation for RPR. The variation explained in NAM by family-nested QTL models, 81% , and thaIn the NCRPIS diversity panel, GBLUP was also performed to assess expected prediction accuracies. This was accomplished with an IBS relationship matrix estimated from 681,257 SNPs. While significant prediction accuracies were attained, they remained lower than those observed in the NAM panel. The increased genetic diversity and reduced marker density in the NCRPIS panel relative to the NAM and IBM families may be attributed to this lower prediction accuracy. The potential complexity of the underlying genetic architecture for each trait increases as the diversity in the population increases, the marker density and size of the calibration set limit the degree that GBLUP is both informed of, and able to explain this complexity. Also, given the highly structured nature of the NCRPIS panel and the prevalence of rare alleles, the genetic architecture explaining variation in the surveyed traits may have evolved by differing mechanisms within each subpopulation of the diversity panel. This may introduce genetic background specificity and non-additivity into modeling the global population and is a potential reason for the more limited prediction accuracy of GBLUP in the NCRPIS diversity panel relative to the NAM panel.While the expected RPR prediction accuracy of a given calibration set size is informative, variation in prediction accuracies was present at all the calibration set sizes in both NAM and NCRPIS diversity panels. Variations in accuracy were especially high within the family-nested QTL models in the NAM panel. This suggests the exact set of lines chosen to calibrate a model may have a larger influence on prediction accuracy when family-nested QTL or other marker effects are selected than when a GBLUP model is assumed and all effects are included but shrunken to the mean. In GBLUP, RPR prediction accuracies did not significantly differ if lines included in a calibration set were randomly selected across families, or were stratified by family. Given the large fraction of BLUP line mean variation in RPR explained by between family variation, family stratified sampling should ensure this variation is better modeled. However, a randomly selected 20% of the NAM panel appeared to sufficiently sample most families to accurately capture between family RPR variation. Previous studies have revealed the gains to be had from strategically sampling the phenotypic/genotypic space when developing calibration sets for genomic prediction Stalk strength and its constituent traits will continue to play an important role in the selection of maize varieties maximizing grain harvestability and silage desirability. This study presents a comprehensive look at the genetic architecture of stalk strength and an assessment of its prediction accuracy. Future efforts detailing the genetic architecture of the anatomical, compositional, and phase change traits underlying stalk strength may reveal simpler architectures and better leverage our existing biological knowledge to understand the genetics of stalk strength. Understanding the genetic architecture of these traits and employing crop modeling to realize how they interact to influence stalk strength is a logical path to better inform plant breeding decisions. Breeding efforts in low heritability traits like stalk strength must incorporate a present, but inherently limited, understanding of genetic architecture and an ability to predict breeding values into a unified selection framework. These efforts will improve prediction of a population’s response to frequency-dependent selection on alleles, haplotypes, and genotypes. Given the availability of inexpensive sequence data, this is increasingly becoming an achievable goal in the optimization of crop improvement.Figure S1DTA and EHT transgressive segregation within the RIL families and NCRPIS diversity panel.(TIF)Click here for additional data file.Figure S2RPR, DTA, and EHT correlations within the RIL families and NCRPIS diversity panel.(TIF)Click here for additional data file.Figure S3RPR, DTA, and EHT associations within and across the RIL families and NCRPIS diversity panel.(TIF)Click here for additional data file.Figure S4GBLUP of DTA and EHT BLUP line means across the RIL families and NCRPIS diversity panel.(TIF)Click here for additional data file.Figure S5Family-nested QTL-based prediction of RPR BLUP line means across the RIL families.(TIF)Click here for additional data file.Figure S6GBLUP of RPR, DTA, and EHT BLUE line means across the RIL families and NCRPIS diversity panel.(TIF)Click here for additional data file.Table S1RPR, DTA, and EHT BLUP and BLUE line means across the RIL families.(TXT)Click here for additional data file.Table S2RPR, DTA, and EHT BLUP and BLUE line means across the NCRPIS diversity panel.(TXT)Click here for additional data file.Table S3RPR, DTA, and EHT family-nested QTL associations within the RIL families.(TXT)Click here for additional data file.Table S4RPR joint-linkage-assisted GWAS results across the RIL families.(TXT)Click here for additional data file.Table S5RPR linear mixed model GWAS results across the NCRPIS diversity panel.(TXT)Click here for additional data file.


Research on the neural basis of speech-reading implicates a network of auditory language regions involving inferior frontal cortex, premotor cortex and sites along superior temporal cortex. In audiovisual speech studies, neural activity is consistently reported in posterior superior temporal Sulcus (pSTS) and this site has been implicated in multimodal integration. Traditionally, multisensory interactions are considered high-level processing that engages heteromodal association cortices (such as STS). Recent work, however, challenges this notion and suggests that multisensory interactions may occur in low-level unimodal sensory cortices. While previous audiovisual speech studies demonstrate that high-level multisensory interactions occur in pSTS, what remains unclear is how early in the processing hierarchy these multisensory interactions may occur. The goal of the present fMRI experiment is to investigate how visual speech can influence activity in auditory cortex above and beyond its response to auditory speech. In an audiovisual speech experiment, subjects were presented with auditory speech with and without congruent visual input. Holding the auditory stimulus constant across the experiment, we investigated how the addition of visual speech influences activity in auditory cortex. We demonstrate that congruent visual speech increases the activity in auditory cortex. In daily conversations, speech is not only heard but it is also seen – auditory speech is typically accompanied by congruent visual speech. Visual cues provide powerful information and aid audition when speech occurs in noisy environments ,2. IndivSeveral neuroimaging studies have examined the neural basis of visual speech perception. Lip-reading, without auditory input, activates a network of auditory and language regions such as portions of auditory cortex in the superior temporal gyrus, posterior superior temporal sulcus, inferior frontal gyrus, and premotor cortex -10. StudWhile the literature clearly indicates that auditory and visual speech interact at higher levels of cortical processing , what is less clear is how low in the cortical hierarchy multisensory interactions may occur. Several lines of evidence suggest that audio-visual speech interaction may occur at the earliest functional-anatomic stages of cortical processing. For example, research in non-human species has demonstrated multisensory interactions within primary auditory cortex -19. In tThe present study addresses whether the influence of visual speech extends into core regions of auditory cortex. Although previous studies of silent lip-reading have found activation in lower-level auditory cortical regions ,6,22 oneTwenty participants  between 18 and 36 years of age were recruited from the University of California, Irvine (UCI) community and received monetary compensation for their time. The volunteers were right-handed, native English speakers with normal or corrected-to-normal vision, normal hearing, no known history of neurological disease, and no other contraindications for MRI as assessed by self-report. Two subjects were omitted from data analysis due to poor image quality. Written informed consent was obtained from each participant prior to participation in the study in accordance with guidelines from UCI Institutional Review Board, which approved this study.In a block design experiment, participants were presented with one of four syllables over headphones . The auditory syllables were paired with matching visual speech, which were videos of mouths articulating the syllables , or the auditory syllables were presented with a still face (Auditory-Speech Only Condition). Although we assume that any differences in auditory cortex between the audiovisual speech and audio-speech only conditions can be attributed to AV speech integration, it is a possibility that visual motion generally could drive the effect. However, this would not undermine the claim that visual information modulates the auditory system; rather, it would simply generalize it to include non-speech-specific dynamic information as a modulator.Stimuli section for details) followed by a 2.7s silent period during which subjects made a button press response to indicate which syllable was presented. In each fMRI session, there were 8 Auditory-Speech Only blocks (2 blocks of each syllable) and 8 Audiovisual blocks. In addition, two rest trials (scanner noise presented with a still face) 15s in length were randomly interspersed throughout each session. Presentation order of the blocks was randomized for each subject with respect to speech sound category  and stimulus condition .A single block was 15s in length and this was created by concatenating 8 videos with syllables of a single type  for a total duration of 12.3s  while audio was recorded on a separate microphone and digitized at 44.1 kHz. Audio information was also captured by the built-in microphone on the digital video camcorder. Audio and video files were synced manually by aligning the auditory waveform with the built-in audio recording. Thus, the natural timing of audiovisual speech information was preserved in all stimuli.During recording, a talker produced approximately 20 samples of each syllable using natural timing and intonation, and pausing briefly between each sample over the course of a single continuous session. A set of 12 tokens were chosen for each syllable based on informal evaluation of loudness, clarity and quality of the audio recording. For each token, a 46-frame (1.533s) video was extracted from the continuous recording with the visual speech information centered in time. The corresponding auditory information was extracted and synced as described above (mean auditory syllable length = ~ 400ms). The fundamental frequency (f0) was estimated for each auditory stimulus and all stimuli were then normalized to the overall mean f0  and matched for root-mean-square power.Stimulus blocks were created by concatenating eight videos from within a speech sound category. A single token was never repeated within a block and the order of tokens within a block was chosen pseudorandomly such that each token appeared an equal number of times across all blocks. For the Auditory-Speech Only condition, the audio was extracted and presented with a 12.3s clip of a still frame of the talker’s face at rest.The experiment started with a short exposure session to familiarize subjects with the task and learn the mapping between syllable and button box. Subjects were scanned during the exposure session to ensure they could comfortably hear the stimuli through the scanner noise, and to acclimatize them to the fMRI situation. Following 9 experimental sessions, the study ended with a localizer scan which consisted of 10 cycles of amplitude modulated broadband noise (8 Hz) alternating with rest (scanner noise) in 15s intervals. All stimuli were presented with MR compatible headset and stimulus delivery and timing were controlled using Cogent software (http://www.vislab.ucl.ac.uk/cogent_2000.php) implemented in Matlab 6 .MR images were obtained in a Philips Achieva 3T  fitted with an 8 channel RF receiver head coil, at the high field scanning facility at the University of California, Irvine. Images during the experimental sessions were collected using Fast Echo EPI . After the functional scans, a high resolution anatomical image was acquired with an MPRAGE pulse sequence in axial plane .We utilized both a standard whole brain group analysis to replicate previous studies and an individual subject, ROI-based approach to allow us more power in assessing our specific hypothesis.http://afni.nimh.nih.gov/afni). First, motion correction was performed by creating a mean image from all of the volumes in the experiment and then realigning all volumes to that mean image using a 6-parameter rigid-body model [Data preprocessing and analyses were performed using AFNI software . Second-level analysis was then performed on the linear contrasts of the parameter estimates from each participant, treating participants as a random effect and voxel-wise t-tests were performed. Group analysis was thresholded at q<0.05 using the false discovery rate correction.First level analysis was performed on the time course of each voxel’s BOLD response for each subject using AFNI software . RegressIn each subject, auditory cortex voxels of interest were functionally identified using the localizer session. In addition to selecting voxels in auditory cortex using a functional definition, an additional analysis was performed on voxels selected using an anatomically defined ROI. First, we used a cytoarchitectonic probability map (included in the AFNI software package) to create a new auditory cortex mask that only included cytoarchitectonic areas Te1.0, Te1.1 and Te1.2 which covers Heschl’s gyrus . This maIn group analysis, a contrast of the Audiovisual Condition compared with the Auditory-Speech Only condition (AV>A) yielded activation in left posterior superior temporal sulcus (pSTS), left middle temporal gyrus and right superior temporal gyrus . We also found significant activation in several other regions such as bilateral inferior and middle occipital cortices, bilateral anterior cingulate, left insula and left superior frontal gyrus. The first ROI analysis was performed on the voxels extracted from auditory cortex using the functional localizer scan. We performed a repeated measures ANOVA treating condition and hemisphere as factors. The ANOVA revealed a significant main effect of Condition, F=6.993, p=0.02. There was greater activity in the Audiovisual condition compared to the Auditory-Speech Only condition  bilaterally to a greater extent than auditory speech alone. Auditory regions lower in the cortical hierarchy in the supratemporal plane did not show the same effect in this whole brain analysis. However, the response of these areas was examined more closely using a functionally defined ROI approach in individual subjects, which revealed that visual speech does modulate activity in lower cortical stages of the auditory processing hierarchy.A number of previous studies have reported effects of visual stimulation in auditory cortex including studies of lipreading  ,27, elecThe present study sought to circumvent these ambiguities in interpretation by using a low-level functional localizer to define ROIs in and immediately surrounding Heschl’s gyrus  and by assessing whether the addition of visual speech to auditory speech further boosts activation levels within the ROIs (thus minimizing the possibility of auditory imagery driving the effects). As such, our results suggest that auditory and visual interactions occur not only in high-level language-related regions such as pSTS, as has been documented repeatedly, but that interactions can occur lower in the cortical processing stream. This finding is consistent with work in both nonhuman primates and other species suggesting that there are direct anatomical connections between primary cortices and that multisensory responses can be observed in unimodal regions ,34.There may be several routes for visual speech to exert influence on auditory processing in and around the auditory core. First, there could be inputs from multisensory regions such as pSTS, with higher level processing influencing low level processing. Second, activity in the auditory cortex could be directly modulated by input from visual cortex. For example, in non-human primates, anatomical connections between primary auditory cortex and primary visual cortex have been demonstrated  and in hIt seems clear that multisensory interactions occur at multiple levels in the processing hierarchy ,36,40. O


Positive and negative correlations were detected among damage categories and between sampling times. For example, sap suckers on leaves in June were positively correlated with sap suckers on leaves (P<0.001) but negatively correlated with skeletonizer damage (P<0.01) in August. The seven forms of leaf damage were used as a proxy for seven functional groups of insect species. Significant variation in insect association occurred among the hybrid offspring, including transgressive segregation of susceptibility to damage. NMDS analyses revealed significant variation and modest broad-sense heritability in insect community structure among genets. QTL analyses identified 14 genomic regions across 9 linkage groups that correlated with insect association. We used three genomics tools to test for putative mechanisms underlying the QTL. First, shikimate-phenylpropanoid pathway genes co-located to 9 of the 13 QTL tested, consistent with the role of phenolic glycosides as defensive compounds. Second, two insect association QTL corresponded to genomic hotspots for leaf trait QTL as identified in previous studies, indicating that, in addition to biochemical attributes, leaf morphology may influence insect preference. Third, network analyses identified categories of gene models over-represented in QTL for certain damage types, providing direction for future functional studies. These results provide insight into the genetic components involved in insect community structure in a fast-growing forest tree.Identifying genetic sequences underlying insect associations on forest trees will improve the understanding of community genetics on a broad scale. We tested for genomic regions associated with insects in hybrid poplar using quantitative trait loci (QTL) analyses conducted on data from a common garden experiment. The F Populus systems, plant genotype has been shown to predict arthropod community structure Describing the genetic mechanisms underlying species interactions is a central aim of community genetics. This relatively new field of study expands the principles of population genetics to the associated species and larger ecosystem Populus system, the role of condensed tannins has been well documented as influencing nutrient cycling and possibly community structure Eucalyptus globulesStudies to identify possible mechanisms underlying community interactions often focus on major resistance genes or biochemical products of known function. In the natural a priori understanding of causal traits. Using such a quantitative genetic approach to identify loci significantly correlated with community structure may reveal genetic mechanisms other than those predicted by secondary compounds or defensive traits. Additional insight into the genetic basis of community interactions may come by combining QTL and genomic data for the host species The mechanisms influencing the community structure may be identified through quantitative genetic analyses, which do not rely on Populus species  frequently act as keystone species within their community, and are associated with a large number of insect, vertebrate, and fungal species Populus host species and the diversity of the associated insect community have shed light on the complexities of community genetics Populus fremontii, P. angustifolia and their hybrids, significant correlations were identified between the genetic similarity of individual trees, the chemical properties of their leaves, and the structure of the insect communities on each individual Salix have revealed genomic regions  associated with insect damage in willow Populus.Forest trees provide a powerful system to examine the relationship between host genotype and phytophagous insects due to the long-lived nature of the host organism, relative frequency of insect occurrence on trees, and ease of assessing insect abundance. P. trichocarpa to examine the genetic variance in insect community structure on hybrid poplar. Conducting a QTL study on a poplar pedigree provided the ability to investigate possible genetic mechanisms using the vast genomic resources available. The genomic sequence for Populus trichocarpa, a model tree Populus species have been the focus of transcriptomic studies In this study we combined traditional quantitative trait loci experiments with the genomic data available for 2 pedigree of P. trichocarpa x P. deltoides (Populus systems.We examined the correlation between genomic variation and insect association using quantitative trait loci (QTL) analysis. As the insects are difficult to detect, but the damage they cause can be assessed at any time, variation in insect association was classified into seven damage types , and quantified through visual inspection of leaf damage in an Feltoides . After ieltoides , typicalPopulus trichocarpa (black cottonwood) and P. deltoides (eastern cottonwood) are widely distributed and can occur sympatrically P. trichocarpa (sect. Tacamahaca) and P. deltoides (sect. Aigeiros) are most easily distinguished by leaf size and shape, with the former displaying lanceolate leaves and the latter deltoid to cordate leaf bases Populus have been repeatedly chosen for hybrid analyses into the genetic basis of complex phenotypic traits In North America, 2 hybrid pedigree of Populus trichocarpa x P. deltoides, consisting of the maternal grandparent P. trichocarpa, 93-968, crossed with the paternal grandparent P. deltoides, ILL-129, to produce hybrid F1 progeny  1 parents (53-242 and 53-246) and 184 of the F2 progeny. One pure P. deltoides genet was analyzed to assess the variation in the paternal species, but the paternal grandparent was not available for assessment. In the spring of 2000, three replicates (ramets) of each genet were planted in a fully replicated randomized block design as part of an ongoing short rotation coppice experiment. The field work was undertaken at a closed Forest Research, Forestry Commission UK nursery site at Headley, Hampshire, U.K. , with their permission, as previously described Family 331 is a F progeny . Two of 2 progeny were genotyped for 91 microsatellite markers, and 165 F2 progeny were genotyped for 92 AFLP markers. The linkage map resolved 21 Linkage Groups defining a total map distance of 1,453.1 cM, with markers spaced an average of 8 cM across the map. Linkage maps in this F2 family were described elsewhere P. trichocarpa genome (v. 2.0). For this, the forward and reverse primer sequences for each microsatellite locus were aligned to the genomic sequence using BLASTN with a word length of 4. Alignments were conducted using linear interpolation over each marker pair via the tools provided on www.phytozome.net/poplar (accessed 3 May 2010), and revealed some putative inversions between the linkage map and physical sequences, consistent with observations in other pedigrees The genetic linkage map for the Family 331 pedigree was kindly provided by G. Tuskan (pers. comm.). The map was constructed from microsatellite  and fully-informative amplified fragment length polymorphism (AFLP) markers using JoinMap v. 3.0 Interactions between insects and trees were quantified as different categories of leaf damage. Plants were scored for damage in June and August 2009. Each individual ramet score was the average of 30 leaves chosen at random from each plant. Damage was scored in 7 categories. Percentage of leaf area lost was scored for chewer, skeletonizer, miner and gall damage. The number of leaf rollers was counted, and the presence or absence of sap suckers on the leaf or stem (distinct categories) was scored. Measurements were made by three observers with the aid of printed guides depicting a scale of leaf damage (in percentages), photos of damage types for each class, and images of common phytophagous insect species. Prior to scoring and intermittently while in the field, the three observers scored the same tree and compared mean damage scores to maintain standardized measures while assessing the common garden. Finally, the observers changed starting locations for the August assessment in order to minimize the potential of influencing block effects by scoring the same plants twice.ijY is the phenotype of the ith genet in the jth block, αi is the genet  effect, βj is the block effect, and ε is the residual error. As there was no replication within blocks, this was a fully cross-factored model with no nesting. In addition, due to mortality of ramets in the field site, replication was reduced from the initial design so that approximately 30% of genets were represented by three replicates, 34% by two replicates, and 36% by a single replicate. Those traits found to have a significant block effect  were treated to a block correction by adding the difference of the block mean and the grand mean to each ramet score within each block. These block-corrected values were then treated to the same GLM to gain corrected estimates of within and among genet variance. Broad-sense heritability (H2) was then estimated for each trait as:B) and error mean squares (MSE) from the GLM (B – MSE)/r, where r is the number of replicates) and Due to the skewed nature of the data collected, raw measures were either arcsine transformed (for percentage measures) or square root transformed data (for count measures) prior to analysis. Transformed data were treated to the general linear model (GLM):In order to assess phenotypic correlations between different categories of insects, Pearson’s correlation coefficient was calculated over genet means for all pairs of damage categories and scoring month. Block-corrected data were used when appropriate. The correlation coefficients and 2-tailed measures of significance were calculated in SPSS v. 17.0 .k = 2 axes, and variation among genets was examined using a distance matrix-based analysis of variance based on the linear model described for the univariate analyses above. Permutations of raw data values were used to estimate pseudo-F statistics for the terms in the linear model. The analysis provided estimates of variance (mean squares) that were then used to estimate the broad-sense heritability of community structure following the manner described for individual damage levels. All analyses were conducted using the multiMDS and adonis functions provided in the R package vegan .To assess variation in insect community structure across genets, a non-metric multidimensional scaling (NMDS) ordination procedure was applied. Specifically, we were interested in quantifying differences in the composition of the insect community associated with each tree, rather than differences in each damage category. The NMDS procedure reduces multivariate  data to a smaller number of orthogonal ordination axes, and has been repeatedly applied to similar questions 2 progeny in this study. Significance was determined through 1000 randomizations of all markers along a linkage group, with permutation results used to estimate the critical value (F-ratio) for each trait-chromosome combination a priori value for likelihood ratio or LOD scores P. deltoides) and maternal (P. trichocarpa) effects were calculated following 0, pure parental species) was heterozygous at each locus so that the effects of four alleles are considered in each estimate. This approach is justified by the high level of heterozygosity revealed by resequencing of multiple Populus genotypes The mean scores for each genet were treated to a quantitative trait loci (QTL) analysis. QTL were identified using Grid-QTL Populus genome were plotted in order to identify co-location of insect association QTL and possible biochemical defences. Second, a test for co-location between insect association and leaf trait QTL was conducted. A total of 105 QTL for leaf traits identified in Family 331 were collected from studies in the UK nathaniel.street@plantphys.umu.se).We then undertook a three-step approach to identify potential mechanisms underlying each QTL. First, the physical location of shikimate-phenylpropanoid pathway genes  using regional distance ratios defined by the two anchored microsatellite markers closest to each QTL. This allowed us to extract an approximate bp location of each QTL and 95% confidence interval in the genomic sequence. All gene models, whether well annotated or not, within each confidence interval were identified and extracted using the BioMart tool at www.phytozome.net/poplar (accessed October 2010). Gene ontology categories for three independent classifications  were extracted for each gene model P. trichocarpa genome as a whole. Tests were conducted using singular enrichment analysis (SEA), with adjustments for multiple tests made using Benjamini-Hochberg false discovery rate (FDR) for p = 0.05. All analyses were conducted in agriGO v. 1.2, a web-based analysis service that provides gene model GO classification and background level analyses Third, a bioinformatics analysis of functional categories of genes in QTL regions was used to identify genes or genetic pathways for further analysis. We aligned the position and 95% confidence interval of each significant QTL with the P. trichocarpa parent and F1 progeny displayed lower damage levels than many F2 genets . Thus, the distribution of damage levels in the F2 progeny is consistent with transgressive segregation of susceptibility to insect damage.Significant variation in levels of damage was observed for the majority of insect categories scored among the full-sib genets, indicating that the insect community is associated with poplar genets in a non-random manner. Damage levels varied across guilds, and the majority of trees showed little damage in several categories . In both2 genets . While tr = 0.213, d.f.  = 181, P = 0.004), sap suckers on leaves , and skeletonizers . Damage by skeletonizers in June was positively correlated with August levels of chewer damage  and leaf roller presence . Similarly, the presence of leaf galls in June was positively correlated with the occurrence of leaf rollers in August . Skeletonizer damage in August was negatively correlated with the presence of sap suckers on leaves in June , indicating these species may avoid previously damaged leaves or be deterred by ant mutualists (which were observed but not quantified during the season). Patterns of damage in August revealed a negative correlation between the damage by skeletonizers and both the number of leaf miners  and the presence of sap suckers on leaves , indicating that skeletonizers may deter other phytophagous insects, or that the insect community may be stratified across the tree canopy.Correlations between damage levels varied among insect categories and sampling times. Nine pairs of damage types were significantly correlated . Three dF186, 175 = 1.476, P = 0.001). The variance among genets corresponded to a broad-sense heritability of H2 = 0.137. These values indicate genetic factors influence community structure as a whole, not just individual insect guild host choice, in hybrid poplar.Examination of insect community structure using NMDS  revealedA total of 14 QTL were identified for seven of the season-trait combinations assessed , Fig. S1For the August data, 11 QTL were identified for four damage categories: chewer, skeletonizer, leaf miner, and sap suckers on leaves . Individχ2 = 12.96, d.f. = 12, P = 0.371).A three-step approach using genomic resources was taken to identify possible mechanisms or candidate genes for future study. First, we identified QTL containing shikimate-phenylpropanoid pathway genes within the 95% confidence interval. These genes represent potential biochemical defence pathways that may serve as selective agents against phytophagous insects. The number of shikimate-phenylpropanoid genes within a QTL ranged from 0 to 4 . Given tSecond, a catalogue of QTL for eight leaf traits from three previous studies identified a total of 106 QTL . The hot2 pedigree, which resulted in relatively large confidence intervals on some linkage groups. Analysis of the distribution of gene ontology (GO) classifications for each gene model revealed a non-random pattern of gene function within insect categories. All but two damage category/GO class combinations displayed at least nine over-represented GO categories, with a maximum of 114 in the chewer QTL for August damage  within individual QTL regions . The larPopulus fremontii, P. angustifolia and naturally occurring hybrids SalixOur assessment revealed significant variation in insect association among hybrid poplar genotypes in both early and late summer. Levels of leaf damage were correlated among time points: Genets damaged by chewers, skeletonizers or sap suckers on leaves in June were more likely to display higher levels of the same damage in August. This pattern was likely due in part to genetic basis of insect preference when choosing host plants, consistent with the heritable patterns of insect richness on 2 progeny indicates genotype explained 13% of the variation in community structure among trees, a smaller percentage than was estimated for tri-trophic interactions among P. angustifolia and back-cross hybrids  and soil microbial composition among P.angustifolia individuals . These genomic regions are spread across nine linkage groups, indicating the insect community responds to wide ranges of genetic variation on host plants. Given the modest number of F2 progeny assessed for each trait (fewer than 200), the number of QTL identified likely underestimated the true number involved in these plant-insect interactions (biased towards QTL of large effect), yet overestimated the effect size of each QTL due to the Beavis effect Salix (willow), another member of the Salicaceae. In Salix, the number and position of QTL varied by site, with differences attributed in part to the composition of the local insect community Populus, QTL for different damage categories only co-located in one instance, with QTL for chewer and leaf miner damage in August occurring on LG XVII. The 95% confidence intervals for these QTL occupy a large portion of the linkage group, and the region is not robustly aligned to the physical map, but two shikimate-phenylpropanoid pathway genes, chorismate mutase (CM) and phenylalanine ammonia-lyase (PAL), are adjacent. Both genes are involved in the PAL-dependent phenolic glycoside pathway Despite the economic and ecological importance of this genus, this is the first study to report QTL for insect association in Insect levels 2 cross are native to North America, where natural hybridization occurs but is infrequent Oenothera biennis showed that environmental variation drove species richness across diverse habitats, but that plant genotype explained a greater portion of the variance within microhabitats P. trichocarpa or P. deltoides in their native range. Such a caveat should not nullify this study, for three reasons. First, as the first attempt to identify the quantitative variation underlying community genetics in a Populus, this study provides a proof of concept. We found significant heritability of insect association, although the level may be higher in the species’ native ranges. Given our ability to identify QTL for association with non-native insect species, it is likely that the same number or even more significant QTL would be identified for co-evolving insect species within the trees’ native ranges. Further, the poplar pedigree examined here has a wealth of genetic and genomic tools available, and has been the focus of numerous physiological studies in varying environments P. deltoides germplasm) are regularly used as stock in plantations across Europe, meaning these findings may transfer directly to ongoing studies and biomass breeding purposes. Second, examining native insects on a non-native host may provide insight into the genetic basis of novel species interactions. By examining the genetic basis of host choice in non-native tree species, these findings may better reflect the mechanism of host switching or invasion rather than the mechanism of adaptation in a natural stand. Repeating this study in different environment will be necessary to determine the robustness of these QTL, in particular any genotype by environment interactions that may influence the extended phenotype of these trees. Third, many of the F2 trees displayed community structure or individual insect association levels transgressive to those on the F1 parents and pure species. These observations are consistent with studies showing natural Populus hybrid zones to be a center of biodiversity The study site used for this work presents a significant caveat for the interpretation and transfer of these findings. The poplar genotypes presented novel host plants to the native insect community of southern England as the two parental species of the FP. trichocarpa to search for possible mechanisms underlying each QTL identified. Our three-step approach provides insight for future studies based on candidate gene sequence and functional analyses. First, nine of the 13 QTL examined against the P. trichocarpa physical map contained at least one of the shikimate-phenylpropanoid pathway genes described by P. tremuloides, providing evidence of varying susceptibility among phytophagous insects, a possible mechanism of defense activity Populus, and members have been shown to be highly upregulated in response to wounding, indicating these genes may serve a defensive role We harnessed the genomic resources available for F2 pedigree. Alternatively, the genomic hotspots containing both classes of QTL may be due to genetic linkage of independent causative genes or the pleiotropic effects of a single locus.Second, we examined the co-location of insect association and leaf morphology QTL to ask whether morphological and not strictly biochemical factors may affect insect host choice. Two QTL for insect association co-located to genomic hot-spots for leaf morphology. Both of these genomic regions also contain one gene involved in the shikimate-phenylpropanoid pathway, but the feeding guilds involved may respond to leaf morphology or phenology. A third QTL hotspot contained only leaf morphology QTL, with a QTL for leaf miner association in August adjacent (LG VIIIa). Insect association may reflect leaf morphology or biochemistry other than defensive compounds. Total leaf nitrogen content  and leaf toughness may influence the level and diversity of phytophagous insects on a tree ++ influx relating to stomatal movements Populus.Finally, we tested for the over-representation of gene ontology categories within the QTL regions identified for each damage type. These inferences would be improved by increasing the density of the linkage map used in the analyses, which would be expected to reduce the confidence intervals of significant QTL. Nonetheless, similar GO classes were observed to be over-represented in QTL for the different damage types, indicating similar functional components may underlie the genetic basis of insect association. For instance, bioinformatics analyses identified gene models related to extracellular glutamate-gated ion channel activity to be overrepresented in QTL regions related to chewer abundance. These glutamate receptors (GLRs) are a class of ligand-gated, non-selective cation channels found in animals and plants P. trichocarpa, we identified possible mechanisms underlying the QTL, including shikimate-phenylpropanoid pathway genes, genomic hot-spots for leaf morphology, and over-represented gene ontology categories. Together, these genes help to narrow the list of candidate genes for future functional and sequence-based studies aiming to identify the mechanisms of community structure in this ecologically and economically important forest tree.By quantifying the direct interaction between phytophagous insects and hybrid poplar in a common garden experiment, we identified 14 QTL for insect association, revealing genomic regions putatively involved in the genetic components of community structure. Multivariate analyses revealed the community structure to vary among genotypes, consistent with a moderately heritable community trait (broad-sense heritability  =  0.13). Using the genomic resources available for Table S1Pearson’s correlation coefficients among leaf damage categories assessed in early (June) and late (August) summer in hybrid poplar(PDF)Click here for additional data file.Table S2Leaf morphology QTL from three previous studies of hybrid poplar used for co-location analyses.(PDF)Click here for additional data file.Table S3Complete list of GO classifications over-represented in QTL for insect association identified in hybrid poplar.(PDF)Click here for additional data file.Figure S1Test statistics across each chromosome, calculated as F - ratios, were used to identify significant QTL for each month/damage category. Each horizontal axis represents the length of one linkage group from the F2 linkage map. The vertical axis indicates the magnitude of the test statistic. Solid lines represent the observed F-ratio, and dashed lines represent the 5% critical value calculated from 1000 chromosome-wide permutations.(PDF)Click here for additional data file.Figure S2QTL for insect damage (red) co-locate to QTL hotspots (gold) for leaf traits (green) on two linkage groups in a hybrid poplar pedigree . (A) The presence of sap suckers (primarily aphids) on leaves co-locates with QTL for leaf size on LG XII. (B) The frequency of skeletonizers early in the summer co-locates with QTL for leaf size and growth rate on LG XIV.(TIFF)Click here for additional data file.


The treatment of an acute left main coronary artery occlusion still poses a challenge. In this case report we present a 50-year-old patient with an acute occlusion of the left main artery. After a successful angioplasty without “stenting” due to the complexity of the stenosis the patient underwent a successful bypass surgery. We discuss the therapeutic options of acute left main occlusion regarding medical, interventional and surgical options. The medical treatment of the unprotected left main coronary artery has high mortality.th postoperative day due to septic shock. A 50-year old male patient was admitted to our hospital in cardiogenic shock with ST-elevation myocardial infarction (STEMI). The cardiovascular risk factors were dyslipidemia, hypertension, and smoking. The coronary angiography -3 presen-3 The initial attempt of the percutaneous coronary intervention (PCI) documented frequent serious comorbidities and high event rates. In contrast the additional stenting of the unprotected left main coronary artery results in better prognosis. Since the appearance of drug eluting stents (DES) with reduction in restenosis rates, recent studies compared PCI with DES versus bypass surgery. In this connection PCI shows non-significant results with lower 6-month4 and 12-month mortality and myocardial infarction rates.5 Acute occlusion of the left main coronary artery is rare and generally fatal. The mechanism is mostly acute thrombosis and the clinical presentation shows an extensive infarction usually with cardiogenic shock. Bypass surgery shows a survival benefit compared to medical therapy, and is the current standard of therapy for patients with unprotected left main coronary artery occlusion.4 and 12-month target vessel revascularization rates,5 due to instent restenosis. To our knowledge, our case report represents one of the first time a patient with an acute left main coronary artery occlusion within STEMI, who received a successful PCI for the myocardial revascularization. In this case the PCI is used as a bridge over to CABG. The fact that the patient died the 11th postoperative day due to non cardiac death, signifies basically the acute resolution. Patients with renal dysfunction, prior bypass surgery, advanced age, and severe heart failure are at highest risk. As mentioned our patient presented a highly reduced left ventricular ejection fraction. We may advise PCI as an alternative for patients who are ineligible for bypass surgery, or for emergency myocardial infarction as a bridge over to CABG in selected patients with acute left main occlusion like in our case report. Nevertheless, there also exists data with significantly higher 6-month


Close to half of the human genome is derived from transposable elements (TEs), and some TE families continue to generate new insertions through RNA-mediated mechanisms. Due to its mutagenic potential, such retrotransposition is normally suppressed by epigenetic and post-transcriptional mechanisms. However, the epigenetic and regulatory disruptions commonly observed in cancers may allow for TE activation, and a few examples have been reported in lung and colon cancer previously.To systematically evaluate the frequency of such events across different tumor types and assess their impact in human cancers, we developed Tea , a computational pipeline to detect TE insertions at single nucleotide level and extract their mechanistic signatures. We applied Tea to the high-coverage (>30×) tumor and matched normal genome pairs from 43 cancer patients across five tumor types as well as three healthy individuals.We identified 194 high-confidence somatic TE insertions , most of which were generated through endonuclease-mediated retrotransposition mechanism. The novel L1 and Alu insertions were all found in the epithelial cancers , and none were detected in the examined blood or brain tumor samples. The somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, and disrupt the expression of the targeted genes. To further illustrate the distinct genomic distribution of the somatic TE landing sites, we compared their placement with the 7,449 non-reference polymorphic TE insertions that we have identified from 44 normal genomes. The TE landing sites are strongly biased towards genomic regions that exhibit cancer-specific decrease in DNA methylation.Our analysis illustrates the functional impact of somatic TE insertions and suggests resulting positive selection toward tumorigenesis.


Prevention of infections by vaccination remains a compelling goal to improve public health. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion – a term coined for non-inflammatory antibody shielding of internal body surfaces, mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA), produced by local plasma cells (PCs) stimulated by antigens that target the mucose. SIgA was early shown to be complexed with an epithelial glycoprotein – the secretory component (SC). A common SC-dependent transport mechanism for pIgA and pentameric IgM was then proposed, implying that membrane SC acts as a receptor, now usually called the polymeric Ig receptor (pIgR). From the basolateral surface, pIg-pIgR complexes are taken up by endocytosis and then extruded into the lumen after apical cleavage of the receptor – bound SC having stabilizing and innate functions in the secretory antibodies. Mice deficient for pIgR show that this is the only receptor responsible for epithelial export of IgA and IgM. These knockout mice show a variety of defects in their mucosal defense and changes in their intestinal microbiota. In the gut, induction of B-cells occurs in gut-associated lymphoid tissue, particularly the Peyer’s patches and isolated lymphoid follicles, but also in mesenteric lymph nodes. PC differentiation is accomplished in the lamina propria to which the activated memory/effector B-cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue but by different homing receptors. This compartmentalization is a challenge for mucosal vaccination, as are the mechanisms used by the mucosal immune system to discriminate between commensal symbionts , pathobionts, and overt pathogens (elimination). The existence of an external antibody system was proposed by Alexandre Besredka at the Pasteur Institute, Paris, when he in 1919 showed that rabbits, after oral immunization with killed Shigella, were protected against fatal dysentery irrespective of the serum antibody titer . These a+ PCs, and their immediate precursors (plasmablasts) are ∼20 times as numerous as IgG+ PCs.A molecular basis for this field emerged when it was shown that saliva contains immunoglobulin (Ig) molecules . Conclus+ PCs produce mainly dimers and perhaps some larger polymers  rather than monomers (+ (and IgM+) PCs and SC-expressing serous-type of secretory epithelial cells , has indicated that almost 90% of them are variably involved in production of polymers  hinge region. The same is true for oral bacteria, especially some strains of S. sanguinis (previously S. sanguis) and S. mitior (previously S. mitis) but also Porphyromonas/Prevotella (previously Bacteroides) and Capnocytophaga species which are involved in periodontal disease  from Porphyromonas gingivalis , Bacteroides fragilis, and Escherichia coli is carried mainly by in the IgA2 isotype  of the distal ileum and other parts of GALT, such as the numerous isolated lymphoid follicles (ILFs) and the appendix . From thIt is not always well delineated which MALT structures are most important for induction of SIgA responses subsequently expressed at a specific secretory effector site, but there is convincing evidence both in animals and humans that activated B-cells migrate from GALT to the upper aerodigestive tract and lactating mammary glands –76. Howe+ cells, as mentioned previously. Although some studies have concluded that the predominance of IgA2+ plasmablasts/PCs in the distal intestine (particularly in the colon) can be explained by in situ switching to this subclass in the lamina propria  and CCR7  cells which are specialized for sampling and transport of especially particulate antigens like bacteria .+IgM+) which pass into a network formed by antigen-capturing follicular dendritic cells (FDCs). The origin of FDCs remains obscure, but both their development and the clustering that allows follicle formation depend on lymphotoxin (LT) signaling  and a “light zone” (dominated by centrocytes) where cells proceed to memory B-cell or terminal plasmablast/PC differentiation Figure . In huma+ T cells, which have been presented with processed antigen by MHC class II-expressing interdigitating DCs  antibody which binds circulating antigen with low affinity. Although still somewhat unclear, it seems that some of these cells use their complement receptors to carry opsonized antigen or immune complexes into the follicles to become deposited on FDCs . AntigenIt remains elusive which factors govern the GC decision for the selected high-affinity memory/effector B cells to go into terminal differentiation to become plasmablasts/PCs Figure ; but it FH) cells (FH cells to secrete IgA-switch factors such as transforming growth factor (TGF)-β, and the local availability of the vitamin A metabolite retinoic acid, are probably important for the IgA-inducing capacity of a particular MALT structure  cells  which inH) cells . The captructure , 71.Several components of the innate immune system, in addition to complement, may be involved in the GC activity, such as the cytokines A Proliferation-Inducing Ligand (APRIL) and B-cell-Activating Factor of the TNF Family (BAFF). Under certain conditions, IgA differentiation driven by gut bacteria may even bypass the usual sIgM (or sIgD)-BCR and T cell requirement  – that i+ PCs (+ B1 (CD5+) cells, which can give rise to polyreactive  SIgA antibodies, particularly directed against polysaccharide antigens from commensal bacteria as a result of T cell-independent responses (H) genes as a sign of selection  antigens are directly mitogenic for B cells – including sugars, lipid structures, and certain nucleic acids. Type 2 T cell-independent (TI-2) antigens, on the other hand, are not by themselves mitogenic but cause extensive cross-lining of BCR by repeating epitopes; their B-cell activation is exerted through synergy with soluble factors  and interaction with various types of accessory cells. There are notable species differences in the mechanisms involved; for instance, while LPS acts as a TI-1 antigen on mouse B cells, it acts as a TI-2 antigen on human B cells. Interestingly, microbial polysaccharides may also function as T cell-dependent antigens and exert profound homeostatic effects on the immune system by stimulating CD4dulation .It is well established that the gut microbiota is critically required for activation of GALT with normal intestinal PC development , althoug+ plasmablasts/PCs which accumulated in the lamina propria and produced both “natural” polyreactive (or cross-reactive) and specific IgA. Individual bacterial species were shown to differ, however, with regard to the maximal amount of total IgA induced and the fraction that could be shown to be specific for antigens of the colonizer , which become a major gut colonizer of the distal ileum of mice after weaning. Colonization of formerly germ-free weanlings resulted in a transient GC reaction in GALT and seeding of the lamina propria with IgA+ plasmablasts, providing an SIgA level comprising 50–70% of that seen in conventional mice; but notably, only about 1% of this IgA showed specificity for the SFB  inhibited activation of innate response markers such as oxidative burst and NFκB, thereby inducing crucial modulation of immune homeostasis in the gut as well as microbial antigenic drift J exons of Ig H and Ig light (L) chains. This process of SHM may be enhanced when antigen recall drives B cell to re-enter the dark zone for a new GC cycle Figure , or to ebacteria .+ B cells, the CD27-expressing subset was indeed considered the best memory candidate with signs of a prolonged AID impact during multiple immune responses, most likely involving MALT (−IgA+ B cells which seemingly were completely T cell-independent and showed molecular similarities to IgA+ B cells in colonic mucosa. This finding was deemed to support the notion that IgA2-producing PCs in the distal human gut develop in the lamina propria by direct CSR from IgM to IgA2 in a T cell-independent manner (With regard to circulating IgAing MALT . Howevert manner . As allu+ B cells (Although mucosal IgA responses to commensal bacteria may be multi-centered, of low affinity and diverse, oral immunization of mice with a cholera toxin-adjuvanted novel antigen was recently shown to result in a strongly oligoclonal response of affinity-matured IgA B cells . Interes B cells .The difficult issue of affinity maturation of mucosal B cells and how the mucosal immune system can distinguish between the indigenous microbiota and overt exogenous pathogens has been discussed in several recent articles –177. TheIn vivo coating of bacteria with IgA present in external secretions can be directly demonstrated by immunostaining (Figure g Figure A; and alg Figure , it is cg Figure , 51.Salmonella typhimurium infection it was found that bacteria coated with such “natural” IgA showed reduced shedding and less liability to horizontal spreading by the fecal-oral route (In human feces, some 40% of the anaerobic bacteria are normally coated with IgA  and thisal route .In mice, the IgA coating of gut bacteria has been shown to be unrelated to the total amount of SIgA exported to the intestinal lumen, suggesting that a specific reaction is involved . Also, o+ subset (Altogether, SIgA does not seem to cause clearance of commensal bacteria, but controls in various ways their colonization and inhibits the penetration of agents that could potentially cause hypersensitivity reactions or infection . In the + subset .V. cholera and enterotoxigenic E. coli seems to depend largely on SIgA, and so does herd protection against horizontal fecal-oral spread of enteric pathogens under naïve or immunized conditions – with a substantial innate impact both on cross-reactivity and memory.Secretory immunoglobulin A constitutes the largest humoral immune system of the body and performs antigen exclusion at mucosal surfaces and neutralizes virus and endotoxin within epithelial cells without causing tissue damage , 199. ThLike natural infections, live attenuated vaccines or adequate combinations of non-replicating vaccines and mucosal adjuvants administered by the oral route to target GALT, give rise not only to SIgA antibodies but also to longstanding serum IgG and IgA responses . HoweverNevertheless, relatively few mucosal vaccines have been approved for human use, and more basic work is needed in vaccine and adjuvant design, including particulate or live-vectored combinations . It is aThe 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.


The spontaneous return of fertility after bone marrow transplantation or heterotopic grafting of cryopreserved ovarian cortical tissue has surprised many, and a possible link with stem cells has been proposed. We have reviewed the available literature on ovarian stem cells in adult mammalian ovaries and presented a model that proposes that the ovary harbors two distinct populations of stem cells, namely, pluripotent, quiescent, very small embryonic-like stem cells (VSELs), and slightly larger “progenitor” ovarian germ stem cells (OGSCs). Besides compromising the somatic niche, oncotherapy destroys OGSCs since, like tumor cells, they are actively dividing; however VSELs persist since they are relatively quiescent. BMT or transplanted ovarian cortical tissue may help rejuvenate the ovarian niche, which possibly supports differentiation of persisting VSELs resulting in neo-oogenesis and follicular development responsible for successful pregnancies. Postnatal oogenesis in mammalian ovary from VSELs may be exploited for fertility restoration in cancer survivors including those who were earlier deprived of gametes and/or gonadal tissue cryopreservation options. Fetal pig skin stem cells [in vitro have also been shown to generate oocyte/follicle-like structures. Daley [Stem cells hold tremendous potential and promise for regenerative medicine and have raised the hope of the public for a cure for several diseases. Reproductive biologists and infertile couples are further excited by the concept of deriving “synthetic gametes” from pluripotent stem cells, but one wonders whether generation of “synthetic gametes” is more of science fiction or a realistic option for healthy babies in the future. Hübner et al.  were theem cells  and rat em cells  cultureds. Daley  summarizs. Daley . Althougs. Daley . The othAnother fast expanding area is the presence of stem cells in adult mammalian ovaries. The mammalian ovary harbors stem cells and possibly undergoes postnatal oogenesis during reproductive life rather than being endowed with a finite pool of primordial follicles at birth. Johnson et al.  provideddark spermatogonial stem cells (SSCs), which have cytoplasmic OCT-4B. OCT-4 expression is lost as the testicular germ cells undergo further differentiation and meiosis. Similar stem cell biology also exists in the adult mammalian ovary, which will be explained in the subsequent sections. Unlike testis, OCT-4 continues to be expressed in growing follicles, since it is a maternally inherited gene but this will not be elaborated further as it is beyond the scope of this paper.One of the markers to identify stem cells is OCT-4 (Pou5f1), an octamer binding nuclear transcription factor. It is normally used to define pluripotent state of a stem cell and is well studied in embryonic (ES) and carcinoma stem cells. It is also a germline-specific maternally expressed factor . During μm were cultured for more than 15 months and on transplantation in busulfan-treated mice resulted in live-births demonstrating postnatal oogenesis. Pacchiarotti et al. [μm) sized cells in the ovarian surface epithelium, medium (20–30 μm) and big (50–60 μm) oocytes in the follicles. Ploidy analysis based on flow cytometry showed that 70% of these cells were tetraploid oocytes and 30% were diploid stem cells. Gong et al. [in vitro to embryonic-like state. They have also reported that a small subgroup of the dissociated cells from adult ovary  is immunoreactive for both OCT-4 and NANOG (pluripotent marker). Reverse transcription-PCR (RT-PCR) results also demonstrate the presence of transcripts for both Oct-4 and Nanog in adult ovarian tissue. Mitotically active germ cells expressing mouse VASA homolog (MVH) and synaptonemal complex protein 3 (SCP3) were reported in the adult mouse ovarian surface epithelium (OSE) by Johnson et al. . Niikurai et al.  have demg et al.  derived μm in the presence of a medium with phenol red (estrogenic stimuli). Virant-Klun and her group [in vitro. We have recently shown the presence of VSELs in ovaries which can be easily isolated by gentle scraping of OSE in adult rabbit, sheep, monkey, and perimenopausal women. These stem cells spontaneously differentiate into oocyte-like structures and parthenotes in vitro [Studies on human ovarian stem cells are relatively few in number because of scarcity of the ovarian tissue for research. Bukovsky et al.  were theer group –29 identin vitro  in agreein vitro , 29, 31.The VSELs are probably the PGCs persisting into adulthood as suggested by others as well , 33. RatAt this juncture, it becomes crucial to comprehend and consolidate the various published studies so that a strong and clear concept emerges. dark SSCs in the testis, OGSCs in the ovaries also have a relatively dark nucleus after Hematoxylin and Eosin (H & E) staining. This possibly reflects simple stem cell biology in vivo wherein the open euchromatin of pluripotent VSELs possibly gets compacted, appears dark, and undergoes remodeling and reprogramming for differentiation into a particular lineage. During “nuclear reprogramming” a dramatic change in facultative heterochromatin occurs [dark SSCs in testis and OGSCs in ovary, similar to that reported during ES cell differentiation [dark SSCs appear dark.Ovary harbors two distinct populations of stem cells, namely, VSELs and OGSCs . VSELs an occurs . Cells wntiation . Thus, bin vitro. The differentiating oocyte undergoes meiosis and exhibits various germ cell markers, formation of Balbiani body-like structures, and characteristic cytoplasmic streaming in vitro (unpublished data). Similar views have been recently put forth by other groups as well [in vivo occurs in the tunica albuginea region of the ovary and may be involved in primordial follicle assembly. During three-week culture of the scraped OSE cells, the stem cells give rise to oocyte-like structures whereas the epithelial cells undergo epithelial-mesenchymal transition (EMT) to give rise to somatic granulosa-like cells . The gra as well , 42. BukMenopause implies exhausted ovarian follicle reserve and may be age related or induced prematurely by gonadotoxic insults including oncotherapy in the case of cancer survivors. But several groups have shown the presence of stem cells in the OSE of postmenopausal ovary –30 and iWe studied the presence of VSELs and OGSCs in chemo-sterilized mouse ovaries. We have observed that the quiescent VSELs persist and are resistant to therapy whereas the rapidly dividing OGSCs and mature follicles are lost resulting in premature ovarian failure (unpublished results). Similar resistance of VSELs has recently been demonstrated in mouse bone marrow after whole body irradiation .It becomes pertinent to refer to two published studies here. Firstly Lee et al.  could reThe current available options offered to female cancer patients for fertility preservation include gonadal shielding, cryopreservation of egg/embryo, and/or ovarian cortical tissue prior to oncotherapy. The eggs or embryos are utilized to achieve parenthood by standard assisted reproductive techniques when required whereas the cryopreserved ovarian tissue fragments are transplanted at either orthotopic or heterotopic site to serve as a source of gametes . To dateFertility restoration in these cases could be because of (i) restoration of lost germ stem cells or (ii) improved functionality of compromised niche in the atrophied ovary that is now able to support oogenesis and follicular assembly. Veitia et al.  providedResearch efforts must be intensified to identify the actual factors that are essential to restore functionality of the gonadal niche. Similar regenerative signals exist in young and aged male blood  which cain vitro culture of OSE to generate autologous oocytes or maturation of primordial follicles in vitro may be overcome. This approach will open up new and novel, non-invasive avenues for fertility restoration, offer new means to treat female infertility, and delay menopause. Moreover, even patients who were deprived of fertility preservation options prior to oncotherapy stand to benefit by advances in this field.This paper consolidates the published literature and discusses it in the context of the existence of two distinct stem cell populations in the ovary in an effort to bring more clarity in the field of adult mammalian oogenesis. It also discusses the possibility of restoring fertility by reconstructing the ovarian somatic niche. If true, various epigenetic and genetic concerns associated with long-term culture and differentiation of embryonic stem cells to make “synthetic gametes” or