Factors Associated With Attendance for Cardiac Neurodevelopmental Evaluation.

BACKGROUND AND OBJECTIVES: Neurodevelopmental evaluation of toddlers with complex congenital heart disease is recommended but reported frequency is low. Data on barriers to attending neurodevelopmental follow-up are limited. This study aims to estimate the attendance rate for a toddler neurodevelopmental evaluation in a contemporary multicenter cohort and to assess patient and center level factors associated with attending this evaluation. METHODS: This is a retrospective cohort study of children born between September 2017 and September 2018 who underwent cardiopulmonary bypass in their first year of life at a center contributing data to the Cardiac Neurodevelopmental Outcome Collaborative and Pediatric Cardiac Critical Care Consortium clinical registries. The primary outcome was attendance for a neurodevelopmental evaluation between 11 and 30 months of age. Sociodemographic and medical characteristics and center factors specific to neurodevelopmental program design were considered as predictors for attendance. RESULTS: Among 2385 patients eligible from 16 cardiac centers, the attendance rate was 29.0% (692 of 2385), with a range of 7.8% to 54.3% across individual centers. In multivariable logistic regression models, hospital-initiated (versus family-initiated) scheduling for neurodevelopmental evaluation had the largest odds ratio in predicting attendance (odds ratio = 4.24, 95% confidence interval, 2.74-6.55). Other predictors of attendance included antenatal diagnosis, absence of Trisomy 21, higher Society of Thoracic Surgeons-European Association for Cardio-Thoracic Surgery mortality category, longer postoperative length of stay, private insurance, and residing a shorter distance from the hospital. CONCLUSIONS: Attendance rates reflect some improvement but remain low. Changes to program infrastructure and design and minimizing barriers affecting access to care are essential components for improving neurodevelopmental care and outcomes for children with congenital heart disease.

Rapid Whole-Genomic Sequencing and a Targeted Neonatal Gene Panel in Infants With a Suspected Genetic Disorder.

Importance: Genomic testing in infancy guides medical decisions and can improve health outcomes. However, it is unclear whether genomic sequencing or a targeted neonatal gene-sequencing test provides comparable molecular diagnostic yields and times to return of results. Objective: To compare outcomes of genomic sequencing with those of a targeted neonatal gene-sequencing test. Design, Setting, and Participants: The Genomic Medicine for Ill Neonates and Infants (GEMINI) study was a prospective, comparative, multicenter study of 400 hospitalized infants younger than 1 year of age (proband) and their parents, when available, suspected of having a genetic disorder. The study was conducted at 6 US hospitals from June 2019 to November 2021. Exposure: Enrolled participants underwent simultaneous testing with genomic sequencing and a targeted neonatal gene-sequencing test. Each laboratory performed an independent interpretation of variants guided by knowledge of the patient's phenotype and returned results to the clinical care team. Change in clinical management, therapies offered, and redirection of care was provided to families based on genetic findings from either platform. Main Outcomes and Measures: Primary end points were molecular diagnostic yield (participants with ≥1 pathogenic variant or variant of unknown significance), time to return of results, and clinical utility (changes in patient care). Results: A molecular diagnostic variant was identified in 51% of participants (n = 204; 297 variants identified with 134 being novel). Molecular diagnostic yield of genomic sequencing was 49% (95% CI, 44%-54%) vs 27% (95% CI, 23%-32%) with the targeted gene-sequencing test. Genomic sequencing did not report 19 variants found by the targeted neonatal gene-sequencing test; the targeted gene-sequencing test did not report 164 variants identified by genomic sequencing as diagnostic. Variants unidentified by the targeted genomic-sequencing test included structural variants longer than 1 kilobase (25.1%) and genes excluded from the test (24.6%) (McNemar odds ratio, 8.6 [95% CI, 5.4-14.7]). Variant interpretation by laboratories differed by 43%. Median time to return of results was 6.1 days for genomic sequencing and 4.2 days for the targeted genomic-sequencing test; for urgent cases (n = 107) the time was 3.3 days for genomic sequencing and 4.0 days for the targeted gene-sequencing test. Changes in clinical care affected 19% of participants, and 76% of clinicians viewed genomic testing as useful or very useful in clinical decision-making, irrespective of a diagnosis. Conclusions and Relevance: The molecular diagnostic yield for genomic sequencing was higher than a targeted neonatal gene-sequencing test, but the time to return of routine results was slower. Interlaboratory variant interpretation contributes to differences in molecular diagnostic yield and may have important consequences for clinical management.

Mapping quantitative trait loci for biomass yield and yield-related traits in lowland switchgrass (Panicum virgatum L.) multiple populations.

Switchgrass can be used as an alternative source for bioenergy production. Many breeding programs focus on the genetic improvement of switchgrass for increasing biomass yield. Quantitative trait loci (QTL) mapping can help to discover marker-trait associations and accelerate the breeding process through marker-assisted selection. To identify significant QTL, this study mapped 7 hybrid populations and one combined of 2 hybrid populations (30-96 F1s) derived from Alamo and Kanlow genotypes. The populations were evaluated for biomass yield, plant height, and crown size in a simulated-sward plot with 2 replications at 2 locations in Tennessee from 2019 to 2021. The populations showed significant genetic variation for the evaluated traits and exhibited transgressive segregation. The 17,251 single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing (GBS) were used to construct a linkage map using a fast algorithm for multiple outbred families. The linkage map spanned 1,941 cM with an average interval of 0.11 cM between SNPs. The QTL analysis was performed on evaluated traits for each and across environments (year and location) that identified 5 QTL for biomass yield (logarithm of the odds, LOD 3.12-4.34), 4 QTL for plant height (LOD 3.01-5.64), and 7 QTL for crown size (LOD 3.0-4.46) (P ≤ 0.05). The major QTL for biomass yield, plant height, and crown size resided on chromosomes 8N, 6N, and 8K explained phenotypic variations of 5.6, 5.1, and 6.6%, respectively. SNPs linked to QTL could be incorporated into marker-assisted breeding to maximize the selection gain in switchgrass breeding.

Insights into the perinatal phenotype of Kabuki syndrome in infants identified by genome-wide sequencing.

Increasing use of unbiased genomic sequencing in critically ill infants can expand understanding of rare diseases such as Kabuki syndrome (KS). Infants diagnosed with KS through genome-wide sequencing performed during the initial hospitalization underwent retrospective review of medical records. Human phenotype ontology terms used in genomic analysis were aggregated and analyzed. Clinicians were surveyed regarding changes in management and other care changes. Fifteen infants met inclusion criteria. KS was not suspected prior to genomic sequencing. Variants were classified as Pathogenic (n = 10) or Likely Pathogenic (n = 5) by American College of Medical Genetics and Genomics Guidelines. Fourteen variants were de novo (KMT2D, n = 12, KDM6A, n = 2). One infant inherited a likely pathogenic variant in KMT2D from an affected father. Frequent findings involved cardiovascular (14/15) and renal (7/15) systems, with palatal defects also identified (6/15). Three infants had non-immune hydrops. No minor anomalies were universally documented; ear anomalies, micrognathia, redundant nuchal skin, and hypoplastic nails were common. Changes in management were reported in 14 infants. Early use of unbiased genome-wide sequencing enabled a molecular diagnosis prior to clinical recognition including infants with atypical or rarely reported features of KS while also expanding the phenotypic spectrum of this rare disorder.

Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3.

Cross bred species such as switchgrass may benefit from advantageous breeding strategies requiring inbred lines. Doubled haploid production methods offer several ways that these lines can be produced that often involve uniparental genome elimination as the rate limiting step. We have used a centromere-mediated genome elimination strategy in which modified CENH3 is expressed to induce the process. Transgenic tetraploid switchgrass lines coexpressed Cas9, a poly-cistronic tRNA-gRNA tandem array containing eight guide RNAs that target two CENH3 genes, and different chimeric versions of CENH3 with alterations to the N-terminal tail region. Genotyping of CENH3 genes in transgenics identified edits including frameshift mutations and deletions in one or both copies of the two CENH3 genes. Flow cytometry of T1 seedlings identified two T0 lines that produced five haploid individuals representing an induction rate of 0.5% and 1.4%. Eight different T0 lines produced aneuploids at rates ranging from 2.1 to 14.6%. A sample of aneuploid lines were sequenced at low coverage and aligned to the reference genome, revealing missing chromosomes and chromosome arms.

Application of growth modeling to assess the impact of hospital-based phthalate exposure on preterm infant growth parameters during the neonatal intensive care unit hospitalization.

In this study, we use advanced growth modeling techniques and the rich biospecimen and data repositories of the NICU Hospital Exposures and Long-Term Health (NICU-HEALTH) study to assess the impact of NICU-based phthalate exposure on extrauterine growth trajectories between birth and NICU discharge. Repeated holdout weighed quantile sum (WQS) regression was used to assess the effect of phthalate mixtures on the latency to first growth spurt and on the rate of first growth spurt. Further, we assessed sex as an effect modifier of the relationship between a phthalate mixture and both outcomes. Nine phthalate metabolites, mono-ethyl phthalate (MEP), mono-benzyl phthalate (MBzP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono-(3-carboxypropyl) phthalate (MCPP), mono-2-ethylhexyl phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) were measured in weekly urine specimens from 101 NICU-HEALTH participants between birth and the first growth spurt. Phthalate levels varied by species but not by infant sex, and decreased over the course of the NICU hospitalization as presented in detail in Stroustrup et al., 2018. There was evidence of nonlinearity when assessing the effect of phthalates on latency to first growth spurt. Above a threshold level, a higher phthalate mixture with dominant contributors MCPP, MBzP, and MEP predicted a shorter latency to the first inflection point, or an earlier growth spurt. A higher phthalate mixture with dominant contributors MECPP, MEHHP, and MEOHP was associated with an increased rate of growth. Results of both models were clearly different for boys and girls, consistent with other studies showing the sexually dimorphic impact of early life phthalate exposure. These results suggest that growth curve modeling facilitates evaluation of discrete periods of rapid growth during the NICU hospitalization and exposure to specific phthalates during the NICU hospitalization may both alter the timing of the first growth spurt and result in more rapid growth in a sexually dimorphic manner.

Environmentally responsive QTL controlling surface wax load in switchgrass.

KEY MESSAGE: Quantitation of leaf surface wax on a population of switchgrass identified three significant QTL present across six environments that contribute to leaf glaucousness and wax composition and that show complex genetic × environmental (G × E) interactions. The C4 perennial grass Panicum virgatum (switchgrass) is a native species of the North American tallgrass prairie. This adaptable plant can be grown on marginal lands and is useful for soil and water conservation, biomass production, and as a forage. Two major switchgrass ecotypes, lowland and upland, differ in a range of desirable traits, and the responsible underlying loci can be localized efficiently in a pseudotestcross design. An outbred four-way cross (4WCR) mapping population of 750 F2 lines was used to examine the genetic basis of differences in leaf surface wax load between two lowland (AP13 and WBC) and two upland (DAC and VS16) tetraploid cultivars. The objective of our experiments was to identify wax compositional variation among the population founders and to map underlying loci responsible for surface wax variation across environments. GCMS analyses of surface wax extracted from 4WCR F0 founders and F1 hybrids reveal higher levels of wax in lowland genotypes and show quantitative differences of ß-diketones, primary alcohols, and other wax constituents. The full mapping population was sampled over two seasons from four field sites with latitudes ranging from 30 to 42 °N, and leaf surface wax was measured. We identified three high-confidence QTL, of which two displayed significant G × E effects. Over 50 candidate genes underlying the QTL regions showed similarity to genes in either Arabidopsis or barley known to function in wax synthesis, modification, regulation, and transport.

The association of prenatal exposure to intensive traffic with early preterm infant neurobehavioral development as reflected by the NICU Network Neurobehavioral Scale (NNNS).

INTRODUCTION: Traffic-related air pollution has been shown to be neurotoxic to the developing fetus and in term-born infants during early childhood. It is unknown whether there is an increased risk of adverse neurobehavioral outcome in preterm infants exposed to higher levels of air pollution during the fetal period. OBJECTIVE: To assess the association between prenatal exposure to traffic-related air pollution on early preterm infant neurobehavior. METHODS: Air pollution exposure was estimated by two methods: density of major roads and density of vehicle-miles traveled (VMT), each at multiple buffering areas around residential addresses. We examined the association between prenatal exposure to traffic-related air pollution and performance on the Neonate Intensive Care Unit (NICU) Network Behavioral Scale (NNNS), a measure of neurobehavioral outcome in infancy for 240 preterm neonates enrolled in the NICU-Hospital Exposures and Long-Term Health cohort. Linear regression analysis was conducted for exposure and individual NNNS subscales. Latent profile analysis (LPA) was applied to classify infants into distinct NNNS phenotypes. Multinomial logistic regression analysis was conducted between exposure and LPA groups. Covariates included gestational age, birth weight z-score, post-menstrual age at NNNS assessment, socioeconomic status, race, delivery type, maternal smoking status, and medical morbidities during the NICU stay. RESULTS: Among all 13 NNNS subscales, hypotonia was significantly associated with VMT (104 vehicle-mile/km2) in 150 m (ß = 0.01, P-value<0.001), 300 m (ß = 0.01, P-value = 0.003), and 500 m (ß = 0.01, P-value = 0.002) buffering areas, as well as with road density in a 500 m buffering area (ß = 0.03, P-value = 0.03). We identified three NNNS phenotypes by LPA. Among them, high density of major roads within 150 m, 300 m, and 500 m buffers of the residential address was significantly associated with the same phenotype (P < 0.05). CONCLUSION: Prenatal exposure to intensive air pollution emitted from major roads may impact early neurodevelopment of preterm infants. Motor development may be particularly sensitive to air pollution-related toxicity.

Sources of clinically significant neonatal intensive care unit phthalate exposure.

In the United States each year, more than 300,000 infants are admitted to neonatal intensive care units (NICU) where they are exposed to a chemical-intensive hospital environment during a developmentally vulnerable period. Although multiple studies have demonstrated elevated phthalate biomarkers in NICU patients, specific sources of NICU-based phthalate exposure have not been identified.In this study, premature newborns with birth weight <1500 g were recruited to participate in a prospective environmental health cohort during the NICU hospitalization. Exposure to specific NICU equipment was recorded daily during the NICU hospitalization. One hundred forty-nine urine specimens from 71 infants were analyzed for phthalate metabolites using high-performance liquid chromatography/tandem mass spectrometry.In initial analyses, exposure to medical equipment was directly related to phthalate levels, with DEHP biomarkers 95-132% higher for infants exposed to specific medical equipment types compared to those without that equipment exposure (p < 0.001-0.023). This association was mirrored for clinically relevant phthalate mixtures whether composed of DEHP metabolites or not (p = 0.002-0.007). In models accounting for concurrent equipment use, exposure to respiratory support was associated with DEHP biomarkers 50-136% higher in exposed compared to unexposed infants (p = 0.007-0.036). Phthalate mixtures clinically relevant to neurobehavioral development were significantly associated with non-invasive respiratory support (p = 0.008-0.026). Feeding supplies and intravenous lines were not significantly associated with clinically important phthalate mixtures.Respiratory support equipment may be a significant and clinically relevant NICU source of phthalate exposure. Although manufacturers have altered feeding and intravenous supplies to reduce DEHP exposure, other sources of exposure to common and clinically impactful phthalates persist in the NICU.

Cohort profile: the Neonatal Intensive Care Unit Hospital Exposures and Long-Term Health (NICU-HEALTH) cohort, a prospective preterm birth cohort in New York City.

PURPOSE: The Neonatal Intensive Care Unit Hospital Exposures and Long-Term Health (NICU-HEALTH) longitudinal preterm birth cohort studies the impact of the NICU exposome on early-life development. NICU-HEALTH collects multiple biospecimens, complex observational and survey data and comprehensive multisystem outcome assessments to allow measurement of the impact of modifiable environmental exposures during the preterm period on neurodevelopmental, pulmonary and growth outcomes. PARTICIPANTS: Moderately preterm infants without genetic or congenital anomalies and their mothers are recruited from an urban academic medical centre level IV NICU in New York City, New York, USA. Recruitment began in 2011 and continues through multiple enrolment phases to the present with goal enrolment of 400 infants. Follow-up includes daily data collection throughout the NICU stay and six follow-up visits in the first 2 years. Study retention is 77% to date, with the oldest patients turning age 8 in 2019. FINDINGS TO DATE: NICU-HEALTH has already contributed significantly to our understanding of phthalate exposure in the NICU. Phase I produced the first evidence of the clinical impact of phthalate exposure in the NICU population. Further study identified specific sources of exposure to clinically relevant phthalate mixtures in the NICU. FUTURE PLANS: Follow-up from age 3 to 12 is co-ordinated through integration with the Environmental Influences on Child Health Outcomes (ECHO) programme. The NICU-HEALTH cohort will generate a wealth of biomarker, clinical and outcome data from which future studies of the impact of early-life chemical and non-chemical environmental exposures can benefit. Findings from study of this cohort and other collaborating environmental health cohorts will likely translate into improvements in the hospital environment for infant development. TRIAL REGISTRATION NUMBERS: This observational cohort is registered with ClinicalTrials.gov (NCT01420029 and NCT01963065).

Broad spectrum developmental role of Brachypodium AUX1.

Targeted cellular auxin distribution is required for morphogenesis and adaptive responses of plant organs. In Arabidopsis thaliana (Arabidopsis), this involves the prototypical auxin influx facilitator AUX1 and its LIKE-AUX1 (LAX) homologs, which act partially redundantly in various developmental processes. Interestingly, AUX1 and its homologs are not strictly essential for the Arabidopsis life cycle. Indeed, aux1 lax1 lax2 lax3 quadruple knock-outs are mostly viable and fertile, and strong phenotypes are only observed at low penetrance. Here we investigated the Brachypodium distachyon (Brachypodium) AUX1 homolog BdAUX1 by genetic, cell biological and physiological analyses. We report that BdAUX1 is essential for Brachypodium development. Bdaux1 loss-of-function mutants are dwarfs with aberrant flower development, and consequently infertile. Moreover, they display a counter-intuitive root phenotype. Although Bdaux1 roots are agravitropic as expected, in contrast to Arabidopsis aux1 mutants they are dramatically longer than wild type roots because of exaggerated cell elongation. Interestingly, this correlates with higher free auxin content in Bdaux1 roots. Consistently, their cell wall characteristics and transcriptome signature largely phenocopy other Brachypodium mutants with increased root auxin content. Our results imply fundamentally different wiring of auxin transport in Brachypodium roots and reveal an essential role of BdAUX1 in a broad spectrum of developmental processes, suggesting a central role for AUX1 in pooideae.

Neonatal intensive care unit phthalate exposure and preterm infant neurobehavioral performance.

Every year in the United States, more than 300,000 infants are admitted to neonatal intensive care units (NICU) where they are exposed to a chemical-intensive hospital environment during a developmentally vulnerable period. The neurodevelopmental impact of environmental exposure to phthalates during the NICU stay is unknown. As phthalate exposure during the third trimester developmental window has been implicated in neurobehavioral deficits in term-born children that are strikingly similar to a phenotype of neurobehavioral morbidity common among children born premature, the role of early-life phthalate exposure on the neurodevelopmental trajectory of premature infants may be clinically important. In this study, premature newborns with birth weight <1500g were recruited to participate in a prospective environmental health cohort study, NICU-HEALTH (Hospital Exposures and Long-Term Health), part of the DINE (Developmental Impact of NICU Exposures) cohort of the ECHO (Environmental influences on Child Health Outcomes) program. Seventy-six percent of eligible infants enrolled in the study. Sixty-four of 81 infants survived and are included in this analysis. 164 urine specimens were analyzed for phthalate metabolites using high-performance liquid chromatography/tandem mass spectrometry. The NICU Network Neurobehavioral Scale (NNNS) was performed prior to NICU discharge. Linear and weighted quantile sum regression quantified associations between phthalate biomarkers and NNNS performance, and between phthalate biomarkers and intensity of medical intervention. The sum of di(2-ethylhexyl) phthalate metabolites (∑DEHP) was associated with improved performance on the Attention and Regulation scales. Specific mixtures of phthalate biomarkers were also associated with improved NNNS performance. More intense medical intervention was associated with higher ∑DEHP exposure. NICU-based exposure to phthalates mixtures was associated with improved attention and social response. This suggests that the impact of phthalate exposure on neurodevelopment may follow a non-linear trajectory, perhaps accelerating the development of certain neural networks. The long-term neurodevelopmental impact of NICU-based phthalate exposure needs to be evaluated.

Sequencing and functional validation of the JGI Brachypodium distachyon T-DNA collection.

Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21 165 T-DNA lines, 15 569 of which were produced in this study. This increased the number of unique insertion sites in the T-DNA collection by 21 078, bringing the overall total to 26 112. Thirty-seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500 bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T-DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T-DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/.

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits.

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution.

Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum.

The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.

Brachypodium distachyon genotypes vary in resistance to Rhizoctonia solani AG8.

Brachypodium distachyon (L.)P.Beauv. (Bd) has previously been developed as a pathosystem model for the wheat root rot pathogen Rhizoctonia solani Kühn anastomosis group 8 (AG8). Here we explore variation in resistance to R. solani AG8 in Bd, to determine whether genomic tools could be used to find Bd genes involved in the grass defence response, with the aim of using this information for the improvement of Rhizoctonia root rot resistance in wheat. We looked for variation in resistance to R. solani AG8 in a diverse Bd natural accession collection and in Bd T-DNA insertion lines selected based on putative mechanisms reported for tagged genes. All lines were susceptible to the pathogen. Repeatable and significant variation in resistance was measured in both groups, with greater variation in resistance found across the natural accessions than in the T-DNA lines. The widest and most repeatable variation in resistance was between lines Koz-3 and BdTR 13a. The ratio of R. solani AG8-inoculated to uninoculated root length for line Koz-3 was 33% greater than the same ratio for line BdTR 13a. The increased resistance of Koz-3 was associated with nodal root initiation in response to the pathogen. A negative correlation between seedling vigour and resistance was observed, but found not to be the sole source of variation in resistance to R. solani AG8. The only T-DNA line with significantly greater resistance to R. solani AG8 than the reference line had an insertion in a putative galactosyltransferase gene; however, this result needs further confirmation. Genetic resistance to Rhizoctonia root rot is not available in wheat cultivars and only a few instances of quantitative resistance to the pathogen have been described within close relatives of wheat. Brachypodium distachyon offers potential for further investigation to find genes associated with quantitative resistance and mechanisms of tolerance to R. solani AG8.

Brachypodium as an emerging model for cereal-pathogen interactions.

BACKGROUND: Cereal diseases cause tens of billions of dollars of losses annually and have devastating humanitarian consequences in the developing world. Increased understanding of the molecular basis of cereal host-pathogen interactions should facilitate development of novel resistance strategies. However, achieving this in most cereals can be challenging due to large and complex genomes, long generation times and large plant size, as well as quarantine and intellectual property issues that may constrain the development and use of community resources. Brachypodium distachyon (brachypodium) with its small, diploid and sequenced genome, short generation time, high transformability and rapidly expanding community resources is emerging as a tractable cereal model. SCOPE: Recent research reviewed here has demonstrated that brachypodium is either susceptible or partially susceptible to many of the major cereal pathogens. Thus, the study of brachypodium-pathogen interactions appears to hold great potential to improve understanding of cereal disease resistance, and to guide approaches to enhance this resistance. This paper reviews brachypodium experimental pathosystems for the study of fungal, bacterial and viral cereal pathogens; the current status of the use of brachypodium for functional analysis of cereal disease resistance; and comparative genomic approaches undertaken using brachypodium to assist characterization of cereal resistance genes. Additionally, it explores future prospects for brachypodium as a model to study cereal-pathogen interactions. CONCLUSIONS: The study of brachypodium-pathogen interactions appears to be a productive strategy for understanding mechanisms of disease resistance in cereal species. Knowledge obtained from this model interaction has strong potential to be exploited for crop improvement.

Brachypodium distachyon.

The small grass Brachypodium distachyon has attributes that make it an excellent model for the development and improvement of cereal crops and bioenergy feedstocks. To realize the potential of this system, many tools have been developed (e.g., the complete genome sequence, a large collection of natural accessions, a high density genetic map, BAC libraries, EST sequences, microarrays, etc.). In this chapter, we describe a high-efficiency transformation system, an essential tool for a modern model system. Our method utilizes the natural ability of Agrobacterium tumefaciens to transfer a well-defined region of DNA from its tumor-inducing (Ti) plasmid DNA into the genome of a host plant cell. Immature embryos dissected out of developing B. distachyon seeds generate an embryogenic callus that serves as the source material for transformation and regeneration of transgenic plants. Embryogenic callus is cocultivated with A. tumefaciens carrying a recombinant plasmid containing the desired transformation sequence. Following cocultivation, callus is transferred to selective media to identify and amplify the transgenic tissue. After 2-5 weeks on selection media, transgenic callus is moved onto regeneration media for 2-4 weeks until plantlets emerge. Plantlets are grown in tissue culture until they develop roots and are transplanted into soil. Transgenic plants can be transferred to soil 6-10 weeks after cocultivation. Using this method with hygromycin selection, transformation efficiencies average 42 %, and it is routinely observed that 50-75 % of cocultivated calluses produce transgenic plants. The time from dissecting out embryos to having the first transgenic plants in soil is 14-18 weeks, and the time to harvesting transgenic seeds is 20-31 weeks.

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines.

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.