INDIANA
PURDUE UNIVERSITY
Departments of Agronomy, Entomology, and Botany and Plant Pathology, and the USDA-ARS, Purdue University, West Lafayette, IN 47907, USA.
J.M. Anderson, S.E. Cambron, C. Crane, S.B.
Goodwin, A. Johnson, J.A. Nemacheck, S. Scofield, B. Schemerhorn,
R.H. Shukle, and C.E. Williams (USDA-ARS); H.W. Ohm, L. Kong,
H.C. Sharma, X. Shen, and J. Uphaus (Department of Agronomy);
G Buechley, D. Huber, G. Shaner, and J.R. Xu (Department of Botany
and Plant Pathology); and J. Stuart (Department of Entomology).
Indiana farmers harvested 178,138 ha (440,000 acres) of wheat in 2004, up 2 percent from 2003. According to the USDA National Agricultural Statistics Service, wheat yield in Indiana averaged 4,170 kg/ha (62 bu/acre) in 2004, down 6 % from the average yield in 2003. Seeding of wheat was completed generally on a timely schedule, but wet soil conditions were common especially early in the seeding season, autumn 2003. The winter was mild or there was snow cover during cold periods, resulting in little winter kill. The spring growing season was cool and wet through May, resulting in loss of nitrogen through denitrification, and accentuating effects of soil compaction from wet soil conditions at seeding in the autumn, and a resulting shallow root system especially in low-lying areas. Ample soil moisture and generally cool conditions prevailed through June, resulting in higher than typical test weight and yield of late maturing cultivars.
Two new SRWWs, licensed cultivars, INW0411 and INW0412, were released and seed is being increased. INW0411, tested as P97397E1-11-2-4-1-14, has low incidence and moderate type II resistance to FHB, is awnless and early like cultivar Patterson, but 4 inches shorter than Patterson, has resistance to leaf rust, powdery mildew, Soil borne mosaic virus, nodorum glume blotch, Septoria leaf blotch, is susceptible to Hessian fly biotype L; and has very good soft wheat milling and baking qualities. INW0412, tested as P981359C1-4-2-1-8, is early like Patterson, typically 1 to 2 inches taller than Patterson but has stronger straw, is awned, has high test weight and large kernels; has low incidence and moderate type-II resistance to FHB, nodorum glume blotch, Septoria leaf blotch, leaf rust, powdery mildew, WSBMV, is susceptible to Hessian fly biotype L, and has acceptable soft wheat milling and baking qualities.
Fusarium head blight, not expected to be serious due to cool conditions through mid June, developed rapidly during grain fill, and causing 5 % to 20 % estimated yield losses throughout the state. Septoria leaf blotch and nodorum blotch were present throughout Indiana and were moderately severe in the southern part of the state on susceptible cultivars. Powdery mildew was present in some areas, but not severe. Leaf rust was moderately severe on susceptible cultivars. Stripe rust occurred in some areas of the state, but was not severe.
Functional analysis of genes required in disease resistance pathway of wheat (Scofield, Amanda Brandt, and Lauren Grieg). A virus-induced gene silencing (VIGS) system has been developed for the rapid analysis of gene function in hexaploid wheat. In VIGS, viruses carrying sequences derived from plant cDNAs activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus contains transcribed plant sequence, homologous host mRNAs are also targeted for destruction, resulting in silencing the expression of the targeted gene. As the silencing mechanism is homology-dependent, it should be able to silence expression of the closely related gene present in polyploid plants. The VIGS system is based on barley stripe mosaic virus (BSMV). This virus has a tripartite single-stranded RNA genome composed of the a, b and g RNAs. Short fragments of cDNAs (150-300 bp) of wheat genes to be silenced are cloned into a DNA plasmid encoding the g RNA. In vitro transcribed viral RNAs are then prepared from this plasmid and those encoding the a and b genomes. These transcripts are mixed together and used to mechanically inoculate the first and second leaves of wheat seedlings. Quantitative PCR measurements of the expression of targeted genes in the third leaf (uninfected) indicate that significant suppression occurs within 5 days of viral inoculation and persists until at least 21 days after inoculation.
We are particularly interested in using VIGS to identify genes that are essential in various disease resistance pathways. Preliminary studies probing the Lr21 leaf rust resistance pathway, in collaboration with Bikram Gill and Li Huang of Kansas State University, demonstrate that silencing Lr21 results in conversion of resistance to susceptibility while infection with control viral constructs has no effect. We are beginning to screen for novel plant genes that encode essential functions in this resistance pathway.
Mapping algorithms and gene expression (Charles Crane). A polynomial-time genetic mapping algorithm was completed and tested extensively on simulated data and on real data from wheat and honeybee. Programs were also written to analyze type and frequency of repeated sequence motifs from one to 300 bases long in database-derived genomic sequence from rice, Arabidopsis, eight fungal genera, and in EST sequence from 55 genera of seed plants, including Triticum, Aegilops, Hordeum, Secale, Leymus, Avena, Oryza, Pennisetum, Saccharum, Sorghum, and Zea, among the grasses. These repeats could be simple or compound, having distinct repeated submotifs over a short span of sequence. Grasses have the most GC-rich genomes among seed plants, and this is reflected in the relative frequencies of GC-rich and AT-rich motifs. However, seed plants also vary widely in motif frequencies of similar AT content, and as might be expected, the most similar motif distributions occur in the most closely related species. Additional programs were written to relate gene expression measures between microarrays and "GeneCalling", a type of fluorescent AFLP based on cDNA obtained at different timepoints after inoculation of wheat with Mycosphaerella graminicola. Although problems with adaptor sequence apparently remain unresolved, the low correlation of these methods in this study falls in line with similar discrepancies in other gene-expression studies.
Transcriptome analysis of the Hessian fly midgut (O. Mittapalli and R.H. Shukle). We have undertaken an EST project on the larval Hessian fly midgut. Analysis of data sets from 1st instars and 2nd instars indicates a high proportion of full-length or near full-length cDNA clones and annotation of the assembled sequences has identified Hessian fly genes with digestion, protein metabolism, cellular communication/signal transduction, bioenergetics, detoxification/antioxidant defense, and immune responses. The annotated EST set we are developing will provide a useful resource for microarray and functional genomic studies with the Hessian fly to reveal knowledge about gene expression in the midgut at critical times in compatible and incompatible interactions with wheat. Despite its importance as a pest of wheat, knowledge of the Hessian fly and its interactions with wheat at the molecular level is limited. This research will contribute toward understanding the ability of the insect to respond to resistant wheat and the experimental dissection of the Hessian fly wheat interaction. A manuscript from this work has been accepted for publication in Insect Molecular Biology in 2005. A second manuscript on expression profiling of two Cytochrome P450s expressed in the larval Hessian fly midgut during compatible and incompatible interactions with wheat has been submitted to Insect Biochemistry and Molecular Biology.
Population history of the Hessian fly (A.J. Johnson, B.J. Schemerhorn, and RH Shukle). We have sequenced the mitochondrial 12S rRNA gene in Hessian fly populations from North America, the Mediterranean basin, and Southwest Asia. The complete complement of 12S sequences in the Hessian fly populations was subjected to a phylogenetic reconstruction. This analysis supported one of the haplotypes from the Middle East as most ancestral and revealed trends among relationships for the remaining haplotypes. To provide for a more robust phylogenetic reconstruction and analysis of population history, we are currently sequencing an 829-bp intron in a Hessian fly ortholog of the Drosophila melanogaster white gene for inclusion with the 12S sequences. Results to date suggest the nuclear sequence will increase the confidence values assessed by bootstrap resampling for groupings in phylogenetic trees. A manuscript from this work was published in 2004 in The Annals of the Entomological Society of America.
Hessian fly resistance genes (N. Sardesai, S. Subramanyam, M.P. Giovanini, and C.E. Williams). A new gene that confers resistance to biotype L of the Hessian fly, H32, was identified in the Synthetic parent of the ITMI mapping population. We scored 114 NILs from this population for resistance, and the phenotypic data were added to the marker data on the internet for mapping. The gene is located on chromosome 3DL and is flanked by an SSR that is 3.9 cM and an RFLP 3.8 cM from the gene. Linkage was confirmed by testing for cosegregation of the SSR with resistance on the 114 lines, and the map position was confirmed by using the SSR with nested deletion lines.
New wheat genes responding to feeding by first-instar Hessian fly larvae were identified. Both compatible and incompatible interactions were studied. The genes up-regulated in incompatible interactions are agglutinin isolectin (Hfr-3; 3,000-fold up-regulation) and flavone 3-hydroxylase. Genes up-regulated in compatible interactions are GST, connective tissue growth factor and sorbitol transporter. The gene down-regulated incompatible interactions is a lipid transfer protein.
H3-H6-H9-H15 linkage block (L. Kong, H.W. Ohm, S.E. Cambron, and C.E. Williams). A sequence characterized amplified region (SCAR) marker, SOPO05909 , was developed from a RAPD marker linked to gene H9. Linkage of the SCAR marker to H9 was confirmed in two different F2 populations. Linkage analysis identified H9 as located near the STS marker, STS-Pm3, and eight microsatellite markers, all previously mapped to the short arm of chromosome 1A. Thus, H9 and closely linked genes H3, H6, and H15 are located on chromosome 1AS, contrary to their previously reported location on chromosome 5A. The locus Xbarc263 was 1.2 cM distal to H9 and H9 was 1.7 cM proximal to loci Xcfa2153, Xpsp2999, and Xgwm136. Xgwm136, Xcfa2153, and SOPO05909 were shown to be specific to H9 and not diagnostic to several other Hessian fly-resistance genes.
Mapping (S.B. Goodwin laboratory). Testing of segregating populations to map QTL for resistance to Septoria tritici blotch continued in a collaborative project with Dr. Hugh Wallwork at the South Australian Research and Development Institute, Adelaide. Preliminary analyses identified possible QTL on chromosomes 3A, 3D, 4A, and 7D. Detection of the QTL on 3A and 3D varied depending on the isolate used for inoculations, indicating some possible isolate specificity. Other testing identified a possibly new major gene for resistance in an Australian wheat cultivar and mapping of that gene continues.
Real-time PCR to estimate fungal biomass was tested as an approach to discriminate resistant and susceptible lines of wheat to Septoria tritici blotch. However, fungal biomass remained low even in highly susceptible plants until near the time of symptom expression, so this method may not save much time over traditional phenotypic testing and scoring of symptoms by eye. A surprising result was that fungal biomass was detected in resistant plants even 27 days after inoculation, so a resistant plant apparently does not kill the fungus. The RT-PCR approach is now being used to test whether QTL based on fungal biomass estimations are the same as those identified by phenotypic scoring.
Analyses of plant gene expression during the resistance response identified 10-50-fold increases of particular gene products occurring within a few hours after inoculation of resistant plants with the pathogen. These responses began before penetration of the host by the fungus. Additional work identified numerous genes that are expressed late during the resistance response, but only in resistant plants. Expression of these genes was much higher, from 200 to more than 1,000-fold higher, than water-inoculated controls at the same time points. Therefore, both early and late responses probably contribute to the resistance response of wheat to Septoria tritici blotch. Experiments with the Affymetrix Barley GeneChip Array identified numerous genes that probably are involved in the non-host resistance response of barley to the Septoria tritici blotch pathogen of wheat. These responses mainly involved cell wall strengthening and were different from R-gene responses seen against the closely related barley pathogen S. passerinii or in susceptible interactions.
Fungal genetics (S.B. Goodwin laboratory). A bioinformatics analysis of fungal EST sequences identified approximately 100 microsatellite-containing loci in the Septoria tritici blotch pathogen. Primers were designed for each locus and tested for polymorphism on a selection of field isolates plus isolates of the related fungus S. passerinii, the cause of speckled leaf blotch of barley. Approximately half of the primer pairs worked well and were polymorphic, and 23 of these were placed on the genetic map of the Septoria tritici blotch pathogen. These markers should be very useful for future studies of fungal genetics and population variability.
For more information see the Goodwin lab web site at: http://www.btny.purdue.edu/USDA-ARS/Goodwin_lab/Goodwin_Lab.html.
Resistance (H. Wiangjun and J.M. Anderson). Previous data has resulted in three possible models for the mechanism of intermediate wheatgrass (Th. intermedium)-derived CYDV resistance (Wiangjun and Anderson 2003, Phytopath 94:1102-1106). This resistance has been given the gene name of Bdv3. Further study has shown that while CYDV replication can occur in nonvascular and vascular tissue in resistant and susceptible wheat lines cellular analyses have demonstrated that the most likely mechanism is an inhibition of long-distance transport. CYDV can spread from the infected companion cells into adjacent sieve elements but its movement beyond this point is blocked. These analyses have also shown that the feeding behavior of the aphid is affected in the resistant line. The aphid deposits virus more often in non-vascular cells in the resistant line compare with the susceptible line suggesting that the aphid needs to probe more before it finds the vascular tissue.
Gene expression analysis (B. Balaji and J.M. Anderson). Over 200 genes whose expression changes in YDV resistant and susceptible lines after YDV infection were identified using Suppressive Subtractive Hybridization (SSH) Quantitative Real time-PCR (Q-RT-PCR) has been used to verify that these are differentially expression. To facilitate these studies it was necessary to choose the appropriate endogenous control genes as internal references in Q-RT-PCR. Two classes of genes were found that could be used as controls for genes that accumulate high levels of transcripts with abundant messages (28S and 18S rRNA) and low amount of transcripts (GAPDH, rbcL). These studies also showed that ubiquitin and rbcS were not useful endogenous control genes as their expression was not constant in our resistant and susceptible lines with or without virus infection.
Wheatgrass molecular markers (Platteter, Mullen, Francki, and J.M. Anderson). Utilizing the data derived from the NSF funded wheat ESTs bin-mapping project as a resource those ESTs which had microsatellite sequences within them were examined to identify dominant or codominant wheatgrass/wheat SSR marketers for integrating biotic or abiotic resistance genes into wheat. Using addition and substitution lines containing Lophopyrum elongatum chromosomes several SSR markers were found for each Lophopyrum chromosome except 4E. The proportion of polymorphic markers was greater when the primer containing regions flanking the SSR were highly conserved between two species such as wheat, barley, rice, and oat. Additional work has identified more wheat ESTs that produced SSR markers for the L. elongatum 1E, 2E, and 7E chromosomes.
Wheat-Thinopyrum mosaic chromosomes (L. Ayala, N. Thompson, and J.M. Anderson). The PCR markers described above and other PCR markers were used to increase the resolution of the RFLP-based map from Th. intermedium/wheat M4 recombinant lines (Crasta et al. 2000, Genome 43:698-706) we added PCR-derived markers. Fluorescent in situ hybridization using GISH and repetitive (FISH) DNA probes also were used to physically analyses the lines. The F2 progeny of two M4 lines crossed to Chinese Spring were examined with the PCR markers for the presence of Th. intermedium segregating fragments. These data showed that significantly more recombination had occurred in these lines then previously thought, given the analysis of these chromosomes in the M4 generation because in most of the F2 recombinant lines tested, the chromosome 7D/7E appeared to be a mosaic of wheat and Th. intermedium chromatin sections. These observation contrasts with most of the literature which suggests that recombination between homeologous chromosomes from different genomes have very low rates of recombination. Our data suggest that introgressing the many agronomically useful traits in Th. intermedium or other related wheatgrass species into wheat may be much more feasible then previous thought.
7E translocations (H.C. Sharma, X. Shen, and H.W. Ohm). The process of shortening the 7E chromosome segment in P961341 and KS24-2-11 continued. P961341 is a translocation with yellow dwarf viruses resistance from Th. intermedium and KS24-2-11 is a translocation with FHB resistance from Lophopyrum (Thinopyrum) ponticum. The F4 populations from 'P961341/ph1ph1' and 'KS24-2-11/ph1ph1' crosses were characterized to isolate potential recombinants.
Alien addition lines of A. cristatum
in wheat, received from J. Jahier, France, were tested for winter
hardiness in Indiana during the 2003-04 wheat season. No winter
hardiness was found in these lines.
Tika Adhikari, postdoctoral associate with Steve Goodwin, accepted a position as assistant professor in the Department of Plant Pathology at North Dakota State University, where he will continue to work on cereal diseases. Jessy Gilsinger completed M.S. degree requirements under the guidance of Herb Ohm and is studying for a Ph.D. at North Carolina State University in soybean genetics/breeding under the guidance of Dr. Joseph Burton. Ligia Ayala, postdoctoral associate with Joe Anderson, accepted a postdoctoral position at CSIRO in Canberra Australia with Phil Larkin where she will continue to work on YDV and other cereal diseases. Boovaraghan Balaji, postdoctoral associate with Joe Anderson, accepted a postdoctoral position in the Plant Pathology Department at the University of Missouri examining differential gene expression following virus infection in Nicotiana. Nicole Thompson, postdoctoral associate with Joe Anderson, accepted a postdoctoral position at Adelaide University working on Mundulla Yellows, a disease affecting eucalyptus.