Project Number: 3635-31320-008-02
Project Type: Specific Cooperative Agreement

Start Date: Jun 03, 2008
End Date: Sep 30, 2009

Objective:
1. Evaluation of whether the proposed integrative genomics approach is feasible and determination of the associated costs. 2. Determine whether differential gene expression might account for variation in MD incidence observed within and between commercial pure lines. 3. A list of genes and biological pathways associated with response to MDV challenge. 4. Determination of sequence variation within and between lines for ~200 genes that are differentially-expressed with respect to response to Marek¿s disease virus challenge. 5. Determination of MD incidence in two commercial pure lines under controlled conditions.

Approach:
We will evaluate the pure lines for MD incidence under environmentally-controlled conditions. Fifty or more birds from each line will be challenged with MDV at 1 day of age, and monitored for MD incidence and other MD-associated traits up to 8 weeks of age. Our main objective is to determine what percentage of genes exhibit allele-specific expression. First, we produce a list of genes that are differentially-expressed between the two pure lines that vary in MD incidence. Specifically, the two pure lines will be split two groups of ~50 birds each. Half of the birds will be unchallenged controls while the other half will be challenged with virulent MDV at 1 day of age. At 4, 7, 10, and 14 days of age, 6 birds from each line and treatment group will be terminated and the spleens immediately recovered in RNAlater; DNA will be isolated from blood. Total RNA will be extracted using RNAeasy kits (Qiagen), and the quantity and quality evaluated using the Agilent Bioanalyzer 2100 lab-on-a-chip instrument. The RNAs (4 per line/treatment/time point) will be hybridized to the Affymetrix Chicken GeneChip by the Purdue Genomics Core Facility. The data will be analyzed using the Statistical Analysis of Microarrays (SAM) software of Tusher et al. (2001) to identify differentially expressed genes with a 5-10% false discovery rate (FDR). In addition, conservatively significant genes will be subjected to gene ontology (GO) analysis to identify biological pathways and critical nodes of gene action. With GO analysis, every gene on the Affymetrics chip is assigned to a biological process, several features often affect the same process. If the node is real in the pathway, several of the features in the node should also be significant. The number of features in the node is compared to the number significant and a chi-square statistic computed to determine departure from random. In this way, biologically significant genes can be found and pathways verified. To identify SNPs in the identified genes, primers will be designed to amplify 500-1000 bp near the 3¿ end of the transcript. Preference will be given to genes that exhibit high abundance and differential expression between the two lines. We will target ~200 genes and sequence 5 individuals each from the two lines. Finally, we will develop Pyrosequencing assays for ~100 of the SNPs. Material that will be tested will come from the two lines (96 birds), as well as ~64 F1 progeny produced by intermating the two lines. The F1 progeny will be prepared as the pure lines except that we plan to have 8 birds per time point and condition. All samples will be genotyped by testing DNA and we hope to have a significant number of heterozygous individuals. RNA will be converted into cDNA, the relative allele-specific expression determined by Pyrosequencing, and the data analyzed using a paired t-test.