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Li H.,Chinese Academy of Agricultural Sciences | Li H.,Cornell University | Bradbury P.,Robert lley Center For Agriculture And Health | Ersoz E.,Cornell University | And 4 more authors.
PLoS ONE | Year: 2011

Background: Nested association mapping (NAM) is a novel genetic mating design that combines the advantages of linkage analysis and association mapping. This design provides opportunities to study the inheritance of complex traits, but also requires more advanced statistical methods. In this paper, we present the detailed algorithm of a QTL linkage mapping method suitable for genetic populations derived from NAM designs. This method is called joint inclusive composite interval mapping (JICIM). Simulations were designed on the detected QTL in a maize NAM population and an Arabidopsis NAM population so as to evaluate the efficiency of the NAM design and the JICIM method. Principal Findings: Fifty-two QTL were identified in the maize population, explaining 89% of the phenotypic variance of days to silking, and nine QTL were identified in the Arabidopsis population, explaining 83% of the phenotypic variance of flowering time. Simulations indicated that the detection power of these identified QTL was consistently high, especially for large-effect QTL. For rare QTL having significant effects in only one family, the power of correct detection within the 5 cM support interval was around 80% for 1-day effect QTL in the maize population, and for 3-day effect QTL in the Arabidopsis population. For smaller-effect QTL, the power diminished, e.g., it was around 50% for maize QTL with an effect of 0.5 day. When QTL were linked at a distance of 5 cM, the likelihood of mapping them as two distinct QTL was about 70% in the maize population. When the linkage distance was 1 cM, they were more likely mapped as one single QTL at an intermediary position. Conclusions: Because it takes advantage of the large genetic variation among parental lines and the large population size, NAM is a powerful multiple-cross design for complex trait dissection. JICIM is an efficient and specialty method for the joint QTL linkage mapping of genetic populations derived from the NAM design.


Yang Y.,Grape Genetics Research Unit | Jittayasothorn Y.,Grape Genetics Research Unit | Jittayasothorn Y.,U.S. National Institutes of Health | Chronis D.,Robert lley Center For Agriculture And Health | And 3 more authors.
PLoS ONE | Year: 2013

Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four pART27-42 and two pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNA population had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in pART27-42 and pART27-271. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5′ end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.


Hotto A.M.,Boyce Thompson Institute for Plant Research | Schmitz R.J.,Salk Institute for Biological Studies | Fei Z.,Boyce Thompson Institute for Plant Research | Fei Z.,Robert lley Center For Agriculture And Health | And 2 more authors.
G3: Genes, Genomes, Genetics | Year: 2011

Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18-25 nt) involved in posttranscriptional gene silencing, whereas prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected; however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein-coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E, and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 59 end mapping demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast. © 2011 Hotto et al.


Peiffer J.A.,Cornell University | Peiffer J.A.,North Carolina State University | Flint-Garcia S.A.,University of Missouri | De Leon N.,University of Wisconsin - Madison | And 4 more authors.
PLoS ONE | Year: 2013

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.


Park D.H.,Cornell University | Mirabella R.,University of Amsterdam | Bronstein P.A.,Cornell University | Bronstein P.A.,Robert lley Center for Agriculture and Health | And 5 more authors.
Plant Journal | Year: 2010

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gab TGABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mM or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance. © 2010 Blackwell Publishing Ltd.


Cobb J.N.,Cornell University | Cobb J.N.,Robert lley Center For Agriculture And Health | DeClerck G.,Cornell University | Greenberg A.,Cornell University | And 3 more authors.
Theoretical and Applied Genetics | Year: 2013

More accurate and precise phenotyping strategies are necessary to empower high-resolution linkage mapping and genome-wide association studies and for training genomic selection models in plant improvement. Within this framework, the objective of modern phenotyping is to increase the accuracy, precision and throughput of phenotypic estimation at all levels of biological organization while reducing costs and minimizing labor through automation, remote sensing, improved data integration and experimental design. Much like the efforts to optimize genotyping during the 1980s and 1990s, designing effective phenotyping initiatives today requires multi-faceted collaborations between biologists, computer scientists, statisticians and engineers. Robust phenotyping systems are needed to characterize the full suite of genetic factors that contribute to quantitative phenotypic variation across cells, organs and tissues, developmental stages, years, environments, species and research programs. Next-generation phenotyping generates significantly more data than previously and requires novel data management, access and storage systems, increased use of ontologies to facilitate data integration, and new statistical tools for enhancing experimental design and extracting biologically meaningful signal from environmental and experimental noise. To ensure relevance, the implementation of efficient and informative phenotyping experiments also requires familiarity with diverse germplasm resources, population structures, and target populations of environments. Today, phenotyping is quickly emerging as the major operational bottleneck limiting the power of genetic analysis and genomic prediction. The challenge for the next generation of quantitative geneticists and plant breeders is not only to understand the genetic basis of complex trait variation, but also to use that knowledge to efficiently synthesize twenty-first century crop varieties. © 2013 The Author(s).


Liu Z.,Huazhong Agricultural University | Liu Z.,Cold Spring Harbor Laboratory | Kumari S.,Cold Spring Harbor Laboratory | Zhang L.,Cold Spring Harbor Laboratory | And 3 more authors.
PLoS ONE | Year: 2012

Waterlogging of plants leads to low oxygen levels (hypoxia) in the roots and causes a metabolic switch from aerobic respiration to anaerobic fermentation that results in rapid changes in gene transcription and protein synthesis. Our research seeks to characterize the microRNA-mediated gene regulatory networks associated with short-term waterlogging. MicroRNAs (miRNAs) are small non-coding RNAs that regulate many genes involved in growth, development and various biotic and abiotic stress responses. To characterize the involvement of miRNAs and their targets in response to short-term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the 24 candidate mature miRNA signatures (22 known and 2 novel mature miRNAs, representing 66 miRNA loci) and their 92 predicted targets in three inbred Zea mays lines (waterlogging tolerant Hz32, mid-tolerant B73, and sensitive Mo17). Based on our studies, miR159, miR164, miR167, miR393, miR408 and miR528, which are mainly involved in root development and stress responses, were found to be key regulators in the post-transcriptional regulatory mechanisms under short-term waterlogging conditions in three inbred lines. Further, computational approaches were used to predict the stress and development related cis-regulatory elements on the promoters of these miRNAs; and a probable miRNA-mediated gene regulatory network in response to short-term waterlogging stress was constructed. The differential expression patterns of miRNAs and their targets in these three inbred lines suggest that the miRNAs are active participants in the signal transduction at the early stage of hypoxia conditions via a gene regulatory network; and crosstalk occurs between different biochemical pathways.


Zwickl D.J.,University of Arizona | Stein J.C.,Cold Spring Harbor Laboratory | Wing R.A.,University of Arizona | Ware D.,Cold Spring Harbor Laboratory | And 2 more authors.
Systematic Biology | Year: 2014

We describe new methods for characterizing gene tree discordance in phylogenomic data sets, which screen for deviations from neutral expectations, summarize variation in statistical support among gene trees, and allowcomparison of the patterns of discordance induced by various analysis choices. Using an exceptionally complete set of genome sequences for the short armof chromosome 3 in Oryza (rice) species,we applied these methods to identify the causes and consequences of differing patterns of discordance in the sets of gene trees inferred using a panel of 20 distinct analysis pipelines.We found that discordance patterns were strongly affected by aspects of data selection, alignment, and alignment masking. Unusual patterns of discordance evident when using certain pipelines were reduced or eliminated by using alternative pipelines, suggesting that theywere the product of methodological biases rather than evolutionary processes. In some cases, once such biaseswere eliminated, evolutionary processes such as introgression could be implicated.Additionally, patterns of gene tree discordance had significant downstream impacts on species tree inference. For example, inference from supermatrices was positivelymisleading when pipelines that led to biased gene treeswere used. Several resultsmay generalize to other data sets: we found that gene tree and species tree inference gave more reasonable results when intron sequence was included during sequence alignment and tree inference, the alignment software PRANK was used, and detectable "block-shift" alignment artifacts were removed. We discuss our findings in the context of well-established relationships in Oryza and continuing controversies regarding the domestication history of O. sativa. © The Author(s) 2014.


Kumari S.,Cold Spring Harbor Laboratory | Ware D.,Cold Spring Harbor Laboratory | Ware D.,Robert lley Center For Agriculture And Health
PLoS ONE | Year: 2013

Transcription initiation, essential to gene expression regulation, involves recruitment of basal transcription factors to the core promoter elements (CPEs). The distribution of currently known CPEs across plant genomes is largely unknown. This is the first large scale genome-wide report on the computational prediction of CPEs across eight plant genomes to help better understand the transcription initiation complex assembly. The distribution of thirteen known CPEs across four monocots (Brachypodium distachyon, Oryza sativa ssp. japonica, Sorghum bicolor, Zea mays) and four dicots (Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Glycine max) reveals the structural organization of the core promoter in relation to the TATA-box as well as with respect to other CPEs. The distribution of known CPE motifs with respect to transcription start site (TSS) exhibited positional conservation within monocots and dicots with slight differences across all eight genomes. Further, a more refined subset of annotated genes based on orthologs of the model monocot (O. sativa ssp. japonica) and dicot (A. thaliana) genomes supported the positional distribution of these thirteen known CPEs. DNA free energy profiles provided evidence that the structural properties of promoter regions are distinctly different from that of the non-regulatory genome sequence. It also showed that monocot core promoters have lower DNA free energy than dicot core promoters. The comparison of monocot and dicot promoter sequences highlights both the similarities and differences in the core promoter architecture irrespective of the species-specific nucleotide bias. This study will be useful for future work related to genome annotation projects and can inspire research efforts aimed to better understand regulatory mechanisms of transcription.


Bouis H.E.,International Food Policy Research Institute | Welch R.M.,Robert lley Center For Agriculture And Health
Crop Science | Year: 2010

Minerals and vitamins in food staples eaten widely by the poor may be increased either through conventional plant breeding or through use of transgenic techniques, a process known as biofortification. HarvestPlus seeks to develop and distribute cultivars of food staples (rice [Oryza sativa L.], wheat [Triticum aestivum L.], maize [Zea mays L.], cassava [Manihot esculenta Crantz], pearl millet [Pennisetum americanum Leeke], beans [Phaseolus vulgaris L.], sweet potato [Ipomoea batatas L.]) that are high in Fe, Zn, and provitamin A through an interdisciplinary global alliance of scientific institutions and implementing agencies in developing and developed countries. Biofortified crops offer a rural-based intervention that, by design, initially reaches these more remote populations, which comprise a majority of the undernourished in many countries, and then penetrates to urban populations as production surpluses are marketed. Thus, biofortification complements fortification and supplementation programs, which work best in centralized urban areas and then reach into rural areas with good infrastructure. Initial investments in agricultural research at a central location can generate high recurrent benefits at low cost as adapted biofortified cultivars become widely available in countries across time at low recurrent costs. Overall, three things must happen for biofortification to be successful. First, the breeding must be successful—high nutrient density must be combined with high yields and high profitability. Second, efficacy must be demonstrated—the micronutrient status of human subjects must be shown to improve when consuming the biofortified cultivars as normally eaten. Third, the biofortified crops must be adopted by farmers and consumed by those suffering from micronutrient malnutrition in significant numbers. © Crop Science Society of America.

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