Using high throughput technology to characterize disease response in hexaploid wheat

Elizabeth M Buescher, Purdue University

Abstract

Wheat (Triticum aestivum L.) is vital for human consumption, global economics as well as societal structure. Wheat is found in a wide range of food products as well as forage for livestock. Yield loss in any crop cannot only affect the world food supply, but also economic stability. Plant viruses and pathogens can greatly impact plant health and yield. The Barley- and Cereal yellow dwarf viruses (BYDV and CYDV, respectively), can cause dramatic yield loss, upwards of 50% in some areas. The yellow dwarf viruses (YDVs) have a wide host range and are spread by aphid transmission. YDVs move up the mouth parts of the aphid and into the hindgut. Aphids feeding on the plant phloem with their stylet, deposit the virus. In wheat, there is little natural resistance to a YDV multi-infection. In recent years, an introgression into wheat with a segment of wheatgrass (Thinopyrum intermedium) 7E2 chromosome confers resistance to all YDVs. While the wheatgrass chromosome segment introgression has been mapped to the long arm of 7D, few polymorphic markers have been developed to determine the exact location of the wheatgrass chromosome segment, and thus, YDV resistance. Also, the mode of resistance the wheatgrass introgression has introduced is not well understood. It has been demonstrated in YDV resistant wheat-wheatgrass lines that YDVs are unable to move into the phloem and spread systemically. In other species, both mammalian and plant, it has been demonstrated that small RNAs (sRNAs) are involved in disease response. Likewise, mammalian and plant viruses have developed a set of proteins, called viral suppressors, that counteract the host's response. CYDVs posses such a viral suppressor called P0, which recently has been shown to affect sRNA response machinery. In this thesis, a high throughput approach was taken to examine disease response to YDVs in a YDV resistant wheat-wheatgrass introgression line in two ways: 1.) identify more polymorphic markers to the 7E2 wheatgrass region of the wheat-wheatgrass introgression line, and 2.) identify sRNAs expressed during YDV infection of a YDV resistant wheat-wheatgrass introgression line. To identify polymorphic markers to the wheatgrass region of the wheat-wheatgrass introgression lines, wheat oligonucleotide microarray technology (Affymetrix) was used. Robustified project pursuit (RPP) identified SFPs (single feature polymorphism) candidates by comparing microarray hybridization intensities between the wheat line, Chinese Spring, and the wheat-wheatgrass substitution lines. One hundred fragments were cloned and sequenced and 4 markers were bin mapped to the group 7 wheat chromosomes using Chinese spring deletion lines. Markers specific for the wheatgrass 7E2 chromosome were not identified using the wheat oligonucleotide array because comparison of genotypes would only identify SFPs on the wheat chromosomes. The second part of the thesis was a high throughput examination of sRNA in a YDV resistant wheat-wheatgrass introgression line. A time course study was conducted using three different treatments 1. aphids containing the CYDV-RPV isolate (viruliferous aphids), 2. aphids without any virus (non-viruliferous aphids), and 3. no aphids. Each treatment had three biological replicates. Total RNA was isolated, fractionated, and sRNA libraries were made using the small RNA expression kit (SREK) (Applied Biosystems™) for SOLiD™ (Applied Biosystems™) sequencing. Computational methods developed for wheat SOLiD™ (Applied Biosystems™) sequencing data were developed and identified 5000 unique putative sRNAs in a YDV resistant wheat-wheatgrass introgression line. Of the 5000 unique putative sRNAs, 70 were determined computationally to be conserved microRNAs (miRNAs) and small interfering RNAs (siRNAs) in other plant species. Also, among sRNA between 17 and 28 nucleotides (nts) in length, 76% of the data was 17 nts. Biological validation by time course Northern blotting of 8 putative conserved sRNAs and 4 short (17-18 nt) sRNAs showed no difference in sRNA expression. However, tissue-specific (tissue included root, stem, leaf, spikelet, and flag leaf) Northern blots showed difference in expression of the 12 putative conserved sRNAs. No CYDV-RPV specific viral sRNA (vsRNA) were identified in this data set. However, over 5000 unique sRNAs have yet to be validated. Future directions of this project would be to examine additional unique sRNAs in this data set as well as sRNA expression in a YDV susceptible wheat-wheatgrass introgression line.

Degree

Ph.D.

Advisors

Ohm, Purdue University.

Subject Area

Agronomy

COinS