Microarrays for global expression constructed with a low redundancy set of 27,500 sequenced cDNAs representing an array of developmental stages and physiological conditions of the soybean plant

Abstract

Background: Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays.

Results: We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines.

Conclusions: Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype.

Figure 1

Steps in the construction and documentation of cDNA microarrays using a low redundancy soybean 'unigene' set of 27,513 cDNA clones. See Methods for details. (a) NSF Plant Genome Program project "A Functional Genomics Program for Soybean" (NSF DBI #9872565); (b) Soybean Public EST project [5]; (c) Washington University Genome Center, St. Louis, MO; (d) Center for Computational Genomics and Bioinformatics, University of Minnesota, Minneapolis, MN [20]; (e) Genome Systems, St. Louis, MO, until its closure; (f) Keck Center for Comparative and Functional Genomics, University of Illinois, Urbana, IL [21] (g) Soybean Functional Genomics, Department of Crop Sciences, University of Illinois, Urbana, IL [30]; (h) databases maintained by the National Center for Biotechnology Information, Bethesda, MD [22].

Figure 2

Gene discovery is increased by selection of weakly expressed cDNAs clones from a cDNA library made from immature cotyledons. (A) Phosphorimager pattern: A high density membrane containing 18,432 double spotted colonies from the Gm-c1007 cDNA library made from immature cotyledons was hybridized with ³³P-labeled cDNAs transcribed from mRNAs isolated from immature cotyledons. (B) Graphical representation of the new cDNAs selected by the normalization process using filter hybridization. Circles represent the 931 total sequences obtained from the non-normalized source cDNA library Gm-c1007 versus the 1799 sequences of Gm-r1030 that were selected as weakly expressed sequences from the filter hybridization experiments shown in part (A). The intersection of the circles represent sequences common to both sets. H = number of sequences with hits in the databases; N = number of sequences that did not have a hit in the databases; and T = total number of sequences.

Figure 3

The scatter plots of the log values of expression data from two duplicate microarray slides before (left) and after flagging and normalization (right). RNAs were extracted from seed coats of the 50–75 mg per seed fresh weight range by standard methods [13]. Replicate 1 was hybridized with Cy5 labeled RNA from seed coats of the T/T genotype and Cy3 labeled RNA from seed coats of the isogenic t*/t* mutant line. Replicate 2 is a dye swap experiment in which the mRNA from the T/T genotype is labeled with Cy3 and the isogenic t*/t* line is labeled with Cy5. The lines in each graph indicate expression either two-fold higher or two-fold lower than equivalent levels of expression. The dots encircled by the box represent repeats of flavonoid 3' hydroxylase cDNAs on the array that are overexpressed in the RNA samples from seed coats of the T/T genotype.

Figure 4

One of the scatter plots of the log values of expression data from microarray slides hybridized with Cy3 labeled RNA from leaves and Cy5 labeled RNA from roots. Many cDNAs have differential expression above or below the two-fold level as indicated by the lines.