Applications of GeneChips for Barley Improvement

Abstract

The use of RNA profiling has recently become a powerful tool to examine genome-wide transcript accumulation. The commercial release of the Barley1 Affymetrix GeneChip probe array has provided the resource to conduct RNA profiling of 22,439 barley genes in a single experiment. We have focused primarily on using the Barley1 GeneChip to (1) physically map barley genes to chromosomes; (2) to examine the RNA profiles in barley infected with Fusarium graminearum, and (3) as a proof of concept for targeting markers to genomic regions. In this article, we will describe these applications of the Barley1 GeneChip and discuss some of our results.

Introduction

High-throughput RNA profiling technologies are useful tools for examining the expression of thousands of genes in parallel. Traditionally, gene expression studies have relied on methods and technologies that examine one to a few transcripts at a time. Thus, RNA profiling technology provides a substantial increase in the number of transcripts compared to more classical methods. In 2003, the barley1 Affymetrix GeneChip probe array was fabricated and provided a new resource for barley geneticists to conduct high throughput RNA profiling experiments in barley (Close et al., 2004). This article summarizes the development of the barley1 GeneChip, and applications to barley research and improvement.

The barley1 GeneChip
A USDA-IFAFS grant to a group of U.S. barley geneticists (Andris Kleinhofs, Timothy Close, Roger Wise, Rod Wing and Gary Muehlbauer) provided the funding to develop RNA profiling technology in barley. The genomics company Affymetrix (Santa Clara, CA), which specializes in the development of GeneChip probe arrays, was chosen to develop this resource for the barley research community. The design of the Barley1 GeneChip probe array was based on approximately 350,000 barley expressed sequence tags (ESTs) developed through an effort of barley geneticists in the U.S. (R. Wing, A. Kleinhofs, R. Wise, and T. Close), Scotland (R. Waugh), Japan (K. Sato), Finland (A. Schulman) and Germany (A. Graner). These barley gene sequences were condensed into an exemplary set of sequences for the GeneChip design. The finished product was the Barley1 GeneChip probe array, which represents 22,439 barley genes and thus provides the resource to examine transcript accumulation of all of these genes in parallel (Close et al., 2004).

The 22,439 genes are represented on the Barley1 GeneChip in the form of 22,439 probe sets. These probe sets are comprised of 11 matched and mismatched pairs of 25-mer oligonucleotides. Most of the oligonucleotides were designed from the 3’ end of each exemplar sequence (Close et al., 2004). Hybridization of labeled RNA to each probe set is determined and raw numerical values representing the amount of transcript accumulation are obtained. These values can be examined with a variety of computer programs and statistical packages to address specific questions relating to transcript accumulation.

Each GeneChip experiment results in a tremendous amount of data. To house these data, to conduct data analysis, and to provide a resource for future comparative analysis the MIAME (minimum information about a microarray experiment) compliant BarleyBase (http://barleybase.org/; Shen et al., 2005) database has been established. BarleyBase is an on-line public repository for raw and normalized expression data for Affymetrix GeneChip data. Currently, data from multiple Barley1 GeneChip experiments are housed on this site.

Uses of microarray technology
There are multiple uses of microarray technology including: (1) examining the response to abiotic and biotic stresses; (2) high-throughput gene mapping; (3) determining gene expression patterns associated with malting; (4) identifying tissue-specific gene expression; (5) determining gene expression differences in defined mutant backgrounds; (6) gene cloning; and (7) targeting markers to genomic regions. In this article, we will discuss our work with the Barley1 GeneChip to (1) physically map barley genes to chromosomes; (2) to examine the RNA profiles in barley infected with Fusarium graminearum, and (3) as a proof of concept for targeting markers to genomic regions.

Results and Discussion

High-throughput physical mapping
We developed an approach to utilize the Barley1 GeneChip to physically map large numbers of barley genes to chromosomes. We are using the wheat-barley addition lines to assign barley genes to chromosomes. These disomic chromosome addition lines were developed through wide hybridization between the donor Betzes barley (Hordeum vulgare L.) and the recipient Chinese Spring wheat (Triticum aestivum) (Islam et al., 1981). These genetic stocks contain all 21 wheat chromosome pairs and a single chromosome pair from barley. Wheat-barley disomic addition lines have been developed for six of the seven barley chromosomes including 1(7H), 2(2H), 3(3H), 4(4H), 6(6H) and 7(5H), and ditelosomic addition lines harboring 13 of the 14 barley chromosome arms have been generated (Islam et al., 1981). Our objectives were to use the wheat-barley addition lines in combination with the Barley1 GeneChip to assign barley genes to chromosomes. The basic idea is as follows: transcripts detected in Betzes and the addition lines, but low or no detection in Chinese Spring were derived from Betzes and the barley gene encoding the transcript was assigned to a specific donor barley chromosome.

We examined transcript accumulation in seedling tissues of Betzes barley, Chinese Spring wheat and wheat-barley chromosome addition lines carrying barley chromosome 2H, 3H, 4H, 5H, 6H, or 7H. By examining only those transcripts that were detected in Betzes and one or more of the addition lines, we identified 482, 331, 352, 392, 246 and 421 transcripts in the addition lines carrying barley chromosome 2H, 3H, 4H, 5H, 6H and 7H, respectively. Based on these results, we assigned 2,224 genes to barley chromosomes. Our results were validated through extensive genomic PCR and by in silico comparisons to the wheat and rice genomes. We found that our physical map positions were highly syntenic with the wheat and rice genomes and that our genomic PCR results were consistent with our GeneChip interpretations. We also examined transcript accumulation in ditelosomic addition lines carrying the long and short arm of chromosome 6H and assigned 139 and 105 genes to chromosome 6HL and 6HS, respectively. The chromosome 6H ditelosomic addition line results validated the location of 244 out of the 246 genes assigned to chromosome 6H. Therefore, we have substantially increased the number of genetic markers for use in marker-assisted selection, map-based cloning and for scaffolds for full-genome sequencing. Our results show that this is an efficient method to physically map barley genes to chromosomes.

Fusarium head blight of barley
Fusarium head blight (FHB) of barley is caused by F. graminearum and related Fusarium species. FHB is a major disease problem for barley growers in the United States and in the barley growing regions of the world (Parry et al., 1995). Trichothecenes mycotoxins, such as deoxynivalenol (DON) are produced by the fungus during infection and accumulate in the harvested grain grain. Barley grain containing measurable levels of DON results in reduced malting quality. Therefore, our goals are to understand the interaction between barley and F. graminearum with the intent to identify genes that provide resistance to FHB. Our approach is to use the Barley1 GeneChip to gain an understanding of the interaction between barley and F. graminearum during infection and to use the gene expression data to direct marker development for FHB resistant QTL-containing regions of the genome.

Transcript accumulation in Morex during Fusarium graminearum infection
Four replications of spikes from the FHB susceptible barley cultivar Morex at 1, 2, 3, 4, and 6 days after F. graminearum and water inoculation and a fifth replication at 1 and 3 days after F. graminearum and water inoculation were sampled for RNA isolation. RNA profiles were examined at these treatment/timepoints using the Barley1 GeneChip. Three hundred and fifty seven transcripts were differentially expressed between F. graminearum-and mock (water) inoculated barley spikes at one or more time points. The differentially accumulating transcripts were placed into two subgroups. One subgroup of 182 transcripts was identified based on the presence versus absence test of transcripts between F. graminearum and mock-inoculated spikes and referred to as qualititatively-induced during infection. The other subgroup of 175 transcripts was identified as significantly induced between F. graminearum- and mock-inoculated barley spikes and referred to as quantitatively-induced during infection. The transcript accumulation from all detected genes was greater in the F. graminearum-treated plants, there were no transcripts that were down regulated in this experiment. These transcript accumulation patterns were validated via RNA gel blot analysis

Examination of the transcript accumulation profiles resulted in the following three major observations. (1) There are three major stages of disease progression: an early stage between 0-2 days after inoculation (dai), an intermediate stage between 2-4 dai; and a late stage between 4-6 dai. (2) Most of the induced genes were identified at 3 dai during the intermediate stage, indicating that this is an important host response timepoint. (3) We observed upregulation of the tryptophan biosynthetic pathway. This observation demonstrates a specific biochemical host response to infection. These observations provide the theoretical basis for a better understanding of the plant response to infection.

Transcript accumulation in contrasting alleles at the Chromosome 3H DON accumulation QTL
To identify potential genes that are involved with FHB resistance and markers that are linked to a DON accumulation resistant QTL, we examined transcript accumulation in a barley near-isogenic line (NIL) pair carrying resistant and susceptible alleles at the DON resistant chromosome 3H (BIN 6) QTL. The DON resistant QTL was identified in the Fredrickson/Stander recombinant inbred line population (Smith et al., 2004). An NIL pair carrying resistant and susceptible alleles at the chromosome 3 DON QTL was provided by Kevin Smith (University of Minnesota).

We used the Barley1 GeneChip to examine transcript accumulation in plants carrying the resistant and susceptible alleles at the chromosome 3H DON QTL at 48 and 96 hours after inoculation. We identified seven genes that are differentially expressed in the lines containing the differing alleles at the barley chromosome 3H QTL. These transcript accumulation differences were due solely to genotype not the treatment. No genes were identified that exhibited differential transcript accumulation between the contrasting alleles due to F. graminearum infection.

Based on the allelic differences in the NIL pairs carrying the resistant and susceptible alleles, some of the 7 differentially expressed genes may map to the chromosome 3H QTL region. We mapped two of the seven genes on the Fredrickson/Stander mapping population in the chromosome 3H DON accumulation QTL region. Our results show that the Barley1 GeneChip can be used to identify allelic differences that can be converted into genetic markers that target specific regions of the genome.

Impact of trichothecene accumulation on barley gene expression
F. graminearum infection of barley results in the fungus synthesizing trichothecene mycotoxins. These mycotoxins are a major detriment to grain quality, especially for grain intended for use as malt. In wheat, the ability of F. graminearum to synthesize trichothecenes increases the virulence of the fungus (Proctor et al., 1995). Loss-of-function mutations in the Tri5 gene, the first committed step in the trichothecene biosynthetic pathway, results in the inability of the fungus to synthesize trichothecenes and a reduction of virulence on wheat. To determine the host response to trichothecene accumulation in barley, we examined the transcript accumulation profiles in Morex barley inoculated with a wildtype strain of F. graminearum, the Tri5 mutant and water at 48 and 96 hours after inoculation.

Examination of the transcript accumulation data revealed three classes of genes that respond differentially to trichothecene biosynthesis. We identified 37 genes that were only expressed in barley during Tri5 mutant infection (no trichothecene accumulation), and 96 genes that were only expressed during wildtype infection (trichothecene accumulation). We also identified 27 genes that are statistically significantly upregulated in wildtype-infected plants versus Tri5 mutant infected plants. These results show that there are genes that are specifically upregulated and downregulated during trichothecene accumulation. Further analysis and annotation of the genes is ongoing.

Acknowledgements

Support for this research was from grants to GJM and DFG from U.S. Barley Genome Project, and grants to GJM from the U.S. Wheat and Barley Scab Initiative and the USDA-IFAFS.

"This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture."

References

Close, T. J., S.I. Wanamaker, R.A. Caldo, S.M. Turner, D.A. Ashlock, J.A. Dickerson, R.A. Wing, G.J. Muehlbauer, A. Kleinhofs and R.P. Wise. 2004. A new resource for cereal genomics: 22K barley GeneChip comes of age. Plant Physiol. 134: 960-968.

Islam, A. K. M. R., K. W. Shepherd, and D. H. B. Sparrow. 1981. Isolation and characterization of euplasmic wheat-barley chromosome addition lines. Heredity 46: 16 l-174.

Parry, W.D., P. Jenkinson, and L. McLeod. 1995. Fusarium ear blight (scab) in small grain cereals – a review. Plant Pathol. 44:207-238.

Proctor, R.H., T.M. Hohn, S.P. McCormick. 1995. Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol. Plant-Microbe Interact. 8: 593-601.

Shen, L., J. Gong, R.A. Caldo, D. Nettleton, D. Cook, R.P. Wise and J.A. Dickerson. 2005. BarleyBase - an expression profiling database for plant genomics. Nucl. Acids Res. 33 (Database issue): D614-D618.

Smith, K.P., C.K. Evans, R. Dill-Macky, C. Gustus, W. Xie and Y. Dong. 2004. Host genetic effect on deoxynivalenol accumulation in Fusarium head blight of barley. Phytopath. 94:766-771.

Gary J. Muehlbauer (1), David F. Garvin (2), Kevin Smith (1), Jayanand Boddu (1) and Seungho Cho (1)
(1) Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108;
(2) Plant Science Research Unit, United States Department of Agriculture-Agricultural Research Service, St. Paul, MN 55108

Presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005