A VISUAL AID FOR STATISTICIANS AND MOLECULAR BIOLOGISTS WORKING WITH MICROARRAY EXPERIMENTS

The use of microarrays to measure the expression of large numbers of genes simultaneously is increasing in agriculture research. Statisticians are expected to help biologists analyze these large data sets to identify biologically important genes that are differentially regulated in the samples under investigation. However, molecular biologists are often unfamiliar with the statistical methods used to analyze microarrays. Presented here are methods developed to graphically represent microarray data and various types of errors commonly associated with microarrays to help visualize sources of error. Two case studies were used. In case study one, genes differentially regulated when two corn lines, one resistant and one sensitive, were treated with Aspergillus flavus isolate NRRL 3357 or left untreated were investigated. Analyses and images showing 3 types of variation are shown. Genes were ranked according to fold change and re-ranked after adjusting for potential sources of error. In case two, cotton genes differentially regulated in 1-day-old fiber compared to whole ovules or older fibers were investigated. Data and sources of error were imaged as described for case one and genes with significant changes in gene expression were identified

Identification of Maize Genes Associated with Host Plant Resistance or Susceptibility to Aspergillus flavus Infection and Aflatoxin Accumulation

infection and aflatoxin accumulation. inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04∶86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E. infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation

Primer sequences of candidate genes and the house keeping gene GAPDH in qRT-PCR study.

<p>Primer sequences of candidate genes and the house keeping gene GAPDH in qRT-PCR study.</p

2D plot showing gene expression levels over a period of 21 days after the fungal inoculation.

<p>These genes are highly expressed in Va35 than in Mp313E.</p

Kernel aflatoxin levels in the four maize inbred lines used for the DNA microarray analysis.

<p>Kernel aflatoxin levels in the four maize inbred lines used for the DNA microarray analysis.</p

Genes differentially expressed in the resistant maize inbred line Mp313E and the susceptible maize inbred line Va35 verified by qRT-PCR method.

*<p>p value <0.05, **p value <0.001, ***p value <0.0001. The significance levels were determined by a paired t-test for the qRT-PCR data. ^a AW017563 chromosome and bin information is not available.</p

Plot showing the grouping of Mp313E and Va35 samples in the qRT-PCR study by principal component analysis.

<p>Notice the Mp313E and Va35 samples were grouped into two distinct groups. It indicated the criteria used for candidate gene selection from the microarray data were effective in reflecting host plant specific responses to the fungal infection.</p

Plot of PCA analysis on the 50 candidate genes differentially expressed in Mp313E versus Va35.

<p>Genes highly expressed in Mp313E were presented in the area with positive values of y axis coordinates, whereas genes located in the area with negative values of y axis coordinates were those expressed more inVa35.</p

2D plot showing gene expression levels over a period of 21 days after the fungal inoculation.

<p>These genes are highly expressed in Mp313E than in Va35.</p