Seungho Cho

Transcriptome analysis of the barley-Fusarium graminearum interaction.

Fusarium head blight (FHB) of barley (Hordeum vulgare L.) is caused by Fusarium graminearum. FHB causes yield losses and reduction in grain quality primarily due to the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). To develop an understanding of the barley-F. graminearum interaction, we examined the relationship among the infection process, DON concentration, and host transcript accumulation for 22,439 genes in spikes from the susceptible cv. Morex from 0 to 144 h after F. graminearum and water control inoculation. We detected 467 differentially accumulating barley gene transcripts in the F. graminearum-treated plants compared with the water control-treated plants. Functional annotation of the transcripts revealed a variety of infection-induced host genes encoding defense response proteins, oxidative burst-associated enzymes, and phenylpropanoid pathway enzymes. Of particular interest was the induction of transcripts encoding potential trichothecene catabolic enzymes and transporters, and the induction of the tryptophan biosynthetic and catabolic pathway enzymes. Our results define three stages of E graminearum infection. An early stage, between 0 and 48 h after inoculation (hai), exhibited limited fungal development, low DON accumulation, and little change in the transcript accumulation status. An intermediate stage, between 48 and 96 hai, showed increased fungal development and active infection, higher DON accumulation, and increased transcript accumulation. A majority of the host gene transcripts were detected by 72 hai, suggesting that this is an important timepoint for the barley-F. graminearum interaction. A late stage also identified between 96 and 144 hai, exhibiting development of hyphal mats, high DON accumulation, and a reduction in the number of transcripts observed. Our study provides a baseline and hypothesis-generating dataset in barley during F. graminearum infection and in other grasses during pathogen infection.

Pathotype-specific genetic factors in chickpea (Cicer arietinum L.) for quantitative resistance to ascochyta blight

Ascochyta blight in chickpea (Cicer arietinum L.) is a devastating fungal disease caused by the necrotrophic pathogen, Ascochyta rabiei (Pass.) Lab. To elucidate the genetic mechanism of pathotype-dependent blight resistance in chickpea, F7-derived recombinant inbred lines (RILs) from the intraspecific cross of PI 359075(1) (blight susceptible) × FLIP84-92C(2) (blight resistant) were inoculated with pathotypes I and II of A. rabiei. The pattern of blight resistance in the RIL population varied depending on the pathotype of A. rabiei. Using the same RIL population, an intraspecific genetic linkage map comprising 53 sequence-tagged microsatellite site markers was constructed. A quantitative trait locus (QTL) for resistance to pathotype II of A. rabiei and two QTLs for resistance to pathotype I were identified on linkage group (LG)4A and LG2+6, respectively. A putative single gene designated as Ar19 (or Ar21d) could explain the majority of quantitative resistance to pathotype I. Ar19 (or Ar21d) appeared to be required for resistance to both pathotypes of A. rabiei, and the additional QTL on LG4A conferred resistance to pathotype II of A. rabiei. Further molecular genetic approach is needed to identify individual qualitative blight resistance genes and their interaction for pathotype-dependent blight resistance in chickpea.

Transcriptome analysis of trichothecene-induced gene expression in barley.

Fusarium head blight, caused primarily by Fusarium graminearum, is a major disease problem on barley (Hordeum vulgare L.). Trichothecene mycotoxins produced by the fungus during infection increase the aggressiveness of the fungus and promote infection in wheat (Triticum aestivum L.). Loss-of-function mutations in the TRI5 gene in F. graminearum result in the inability to synthesize trichothecenes and in reduced virulence on wheat. We examined the impact of pathogen-derived trichothecenes on virulence and the transcriptional differences in barley spikes infected with a trichothecene-producing wild-type strain and a loss-of-function tri5 trichothecene nonproducing mutant. Disease severity, fungal biomass, and floret necrosis and bleaching were reduced in spikes inoculated with the tri5 mutant strain compared with the wild-type strain, indicating that the inability to synthesize trichothecenes results in reduced virulence in barley. We detected 63 transcripts that were induced during trichothecene accumulation, including genes encoding putative trichothecene detoxification and transport proteins, ubiquitination-related proteins, programmed cell death-related proteins, transcription factors, and cytochrome P450s. We also detected 414 gene transcripts that were designated as basal defense response genes largely independent of trichothecene accumulation. Our results show that barley exhibits a specific response to trichothecene accumulation that can be separated from the basal defense response. We propose that barley responds to trichothecene accumulation by inducing at least two general responses. One response is the induction of genes encoding trichothecene detoxification and transport activities that may reduce the impact of trichothecenes. The other response is to induce genes encoding proteins associated with ubiquitination and cell death which may promote successful establishment of the disease.

Transcriptome Analysis and Physical Mapping of Barley Genes in Wheat–Barley Chromosome Addition Lines

Wheat–barley chromosome addition lines are useful genetic resources for a variety of studies. In this study, transcript accumulation patterns in Betzes barley, Chinese Spring wheat, and Chinese Spring–Betzes chromosome addition lines were examined with the Barley1 Affymetrix GeneChip probe array. Of the 4014 transcripts detected in Betzes but not in Chinese Spring, 365, 271, 265, 323, 194, and 369 were detected in wheat–barley disomic chromosome addition lines 2(2H), 3(3H), 4(4H), 7(5H), 6(6H), and 1(7H), respectively. Thus, 1787 barley transcripts were detected in a wheat genetic background and, by virtue of the addition line in which they were detected, were physically mapped to barley chromosomes. We validated and extended our approach to physically map barley genes to the long and short arms of chromosome 6(6H). Our physical map data exhibited a high level of synteny with homologous sequences on the wheat and/or rice syntenous chromosomes, indicating that our barley physical maps are robust. Our results show that barley transcript detection in wheat–barley chromosome addition lines is an efficient approach for large-scale physical mapping of genes.

Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress

SummaryLentil is a self-pollinating diploid (2n = 14 chromosomes) annual cool season legume crop that is produced throughout the world and is highly valued as a high protein food. Several abiotic stresses are important to lentil yields world wide and include drought, heat, salt susceptibility and iron deficiency. The biotic stresses are numerous and include: susceptibility to Ascochyta blight, caused by Ascochyta lentis; Anthracnose, caused by Colletotrichum truncatum; Fusarium wilt, caused by Fusarium oxysporum; Sclerotinia white mold, caused by Sclerotinia sclerotiorum; rust, caused by Uromyces fabae; and numerous aphid transmitted viruses. Lentil is also highly susceptible to several species of Orabanche prevalent in the Mediterranean region, for which there does not appear to be much resistance in the germplasm. Plant breeders and geneticists have addressed these stresses by identifying resistant/tolerant germplasm, determining the genetics involved and the genetic map positions of the resistant genes. To this end progress has been made in mapping the lentil genome and several genetic maps are available that eventually will lead to the development of a consensus map for lentil. Marker density has been limited in the published genetic maps and there is a distinct lack of co-dominant markers that would facilitate comparisons of the available genetic maps and efficient identification of markers closely linked to genes of interest. Molecular breeding of lentil for disease resistance genes using marker assisted selection, particularly for resistance to Ascochyta blight and Anthracnose, is underway in Australia and Canada and promising results have been obtained. Comparative genomics and synteny analyses with closely related legumes promises to further advance the knowledge of the lentil genome and provide lentil breeders with additional genes and selectable markers for use in marker assisted selection. Genomic tools such as macro and micro arrays, reverse genetics and genetic transformation are emerging technologies that may eventually be available for use in lentil crop improvement.

Transcriptome Analysis of a Wheat Near-Isogenic Line Pair Carrying Fusarium Head Blight-Resistant and -Susceptible Alleles

Fusarium head blight (FHB), caused primarily by Fusarium graminearum, decreases grain yield and quality in wheat and barley. Disease severity, deoxynivalenol (DON), fungal biomass, and transcript accumulation were examined in a wheat near-isogenic line pair carrying either the resistant or susceptible allele for the chromosome 3BS FHB-resistance quantitative trait locus (Fhb1). Fhb1 restricts spread of disease symptoms but does not provide resistance to initial infection or initial DON accumulation. Wheat exhibits both induction and repression of large sets of gene transcripts during F. graminearum infection. In addition, a difference in the general timing of transcript accumulation in plants carrying either the resistant or susceptible allele at the Fhb1 locus was detected, and 14 wheat gene transcripts were detected that exhibited accumulation differences between the resistant and susceptible alleles. These results indicate that these may be host responses that differentiate the resistant from the susceptible interaction. Comparative analysis of the wheat-F. graminearum and the barley-F. graminearum interactions revealed a large set of conserved transcript accumulation patterns. However, we also detected gene transcripts that were repressed in wheat but not in barley. Based on the disease symptoms, transcript accumulation data, and comparative analysis of the barley and wheat host response to F. graminearum infection, we developed an integrated model for the interactions of wheat and barley with F. graminearum.

Mapping barley genes to chromosome arms by transcript profiling of wheat-barley ditelosomic chromosome addition lines

Wheat-barley disomic and ditelosomic chromosome addition lines have been used as genetic tools for a range of applications since their development in the 1980s. In the present study, we used the Affymetrix Barley1 GeneChip for comparative transcript analysis of the barley cultivar Betzes, the wheat cultivar Chinese Spring, and Chinese Spring - Betzes ditelosomic chromosome addition lines to physically map barley genes to their respective chromosome arm locations. We mapped 1257 barley genes to chromosome arms 1HS, 2HS, 2HL, 3HS, 3HL, 4HS, 4HL, 5HS, 5HL, 7HS, and 7HL based on their transcript levels in the ditelosomic addition lines. The number of genes assigned to individual chromosome arms ranged from 24 to 197. We validated the physical locations of the genes through comparison with our previous chromosome-based physical mapping, comparative in silico mapping with rice and wheat, and single feature polymorphism (SFP) analysis. We found our physical mapping of barley genes to chromosome arms to be consistent with our previous physical mapping to whole chromosomes. In silico comparative mapping of barley genes assigned to chromosome arms revealed that the average genomic synteny to wheat and rice chromosome arms was 63.2% and 65.5%, respectively. In the 1257 mapped genes, we identified SFPs in 924 genes between the appropriate ditelosomic line and Chinese Spring that supported physical map placements. We also identified a single small rearrangement event between rice chromosome 9 and barley chromosome 4H that accounts for the loss of synteny for several genes.

The genetics of barley low-tillering mutants: low number of tillers-1 (lnt1).

Barley (Hordeum vulgare L.) carrying recessive mutations at the Low number of tillers1 (Lnt1) gene does not develop secondary tillers and only develops one to four tillers by maturity. Double mutant analysis determined that the lnt1 mutant was epistatic to five of the six low and high tillering mutants tested. Double mutants of lnt1 and the low tillering mutant intermedium-b (int-b) resulted in a uniculm plant, indicating a synergistic interaction and that Lnt and Int-b function in separate tillering pathways. RNA profiling identified 70 transcripts with either increased or decreased abundance in the lnt1 mutant compared to wild-type. One gene with reduced transcript levels in the lnt1 mutant was the BELL-like homeodomain transcription factor JuBel2. The JuBel2 allele in the lnt1.a mutant contained a frameshift mutation that eliminated most of the predicted polypeptide, indicating that the Lnt1 gene encodes JuBel2. Previous studies with the low-tillering mutant absent lower laterals (als) showed that the tillering phenotypes and genetic interactions of als and lnt1 with other tillering mutants were very similar. However, the transcriptomes were very different; many transcripts annotated as stress and defense response exhibited increased abundance in the als mutant. This difference suggests a functional separation between Als and Lnt1 in the genetic control of tillering.

The genetics of barley low-tillering mutants: absent lower laterals (als)

Barley (Hordeumvulgare L.) carrying the recessive mutation absent lower laterals (als) exhibits few tillers and irregular inflorescence development. To gain an increased understanding of the genetic control of tillering in barley, we conducted morphological, genetic, and transcriptome analysis of the als mutant. Axillary buds for primary tillers, but not for secondary tillers, developed in als plants. Double mutant combinations of als with one low-tillering and four high-tillering mutants resulted in a tillering phenotype similar to als, indicating that als was epistatic to these tillering genes. However, double mutant combinations of als with another low-tillering mutant, intermedium-b, reduced tiller numbers, indicating there were at least two genetic pathways regulating tillering in barley. Next, we used simple sequence repeat markers to map the Als gene on the long arm of barley chromosome 3H, Bin 11. Finally, the Affymetrix Barley1 GeneChip was used to identify differentially accumulated transcripts in als compared to wild-type. Forty percent of the transcripts with twofold or greater accumulation in als tissues corresponded to stress and defense response genes. This finding suggested that a tillering pathway may modulate the stress response.

Single-feature polymorphism discovery by computing probe affinity shape powers

BackgroundSingle-feature polymorphism (SFP) discovery is a rapid and cost-effective approach to identify DNA polymorphisms. However, high false positive rates and/or low sensitivity are prevalent in previously described SFP detection methods. This work presents a new computing method for SFP discovery.ResultsThe probe affinity differences and affinity shape powers formed by the neighboring probes in each probe set were computed into SFP weight scores. This method was validated by known sequence information and was comprehensively compared with previously-reported methods using the same datasets. A web application using this algorithm has been implemented for SFP detection. Using this method, we identified 364 SFPs in a barley near-isogenic line pair carrying either the wild type or the mutant uniculm2 (cul2) allele. Most of the SFP polymorphisms were identified on chromosome 6H in the vicinity of the Cul2 locus.ConclusionThis SFP discovery method exhibits better performance in specificity and sensitivity over previously-reported methods. It can be used for other organisms for which GeneChip technology is available. The web-based tool will facilitate SFP discovery. The 364 SFPs discovered in a barley near-isogenic line pair provide a set of genetic markers for fine mapping and future map-based cloning of the Cul2 locus.