Transcript profiling of common bean using the Soybean Genome Array: optimizing analysis by masking biased probes

Common bean (Phaseolus vulgaris) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and specificity in common bean cross-species hybridization (CSH) GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV) regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and specificity. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by about 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR analysis of a total of 20 randomly selected genes and purine-ureides pathway genes demonstrated an increased specificity after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The results from this study suggested that transcript profiling in common bean can be done using the soybean GeneChip. However, a significant decrease in sensitivity and specificity can be expected. Problems associated with CSH GeneChip data can be mitigated by masking probes targeting ISV regions. In addition to transcript profiling CSH of the GeneChip in combination with masking probes in the ISV regions can be used for comparative ecological and/or evolutionary genomics studies. Overall design: We hybridized cRNA purified from nodule, leaf, and root of common bean and soybean in, triplicate, to the soybean GeneChip (18 GeneChip hybridizations = 2 species x 3 organs x 3 replicates).普通菜豆（Phaseolus vulgaris）与大豆（Glycine max）同属菜豆族（Phaseoleae），二者具备显著的编码序列同源性。为评估大豆基因芯片（soybean GeneChip）用于普通菜豆转录谱分析的实用性，我们将从普通菜豆和大豆的根瘤、叶片及根系中纯化得到的cRNA以三次生物学重复的方式与大豆基因芯片进行杂交。初始数据分析显示，相较于大豆样本，普通菜豆的跨物种杂交（cross-species hybridization, CSH）基因芯片数据的灵敏度与特异性均有所下降。我们采用了一种对靶向普通菜豆与大豆间种间可变区（inter-species variable, ISV）的推定探针进行屏蔽的方法，并选取了可同时优化灵敏度与特异性的屏蔽信号强度阈值。在对种间可变区完成屏蔽处理后，普通菜豆中鉴定得到的差异表达基因数量提升了约2.8倍，这反映出分析灵敏度的提升。我们对共计20个随机选取的基因以及嘌呤酰脲通路基因开展了定量RT-PCR分析，结果证实屏蔽种间可变区后分析的特异性得到显著提升。此外，我们通过评估每个探针组的屏蔽探针频率，深入解析了普通菜豆与大豆间的序列分化模式。本研究结果表明，普通菜豆的转录谱分析可借助大豆基因芯片完成，但预计其灵敏度与特异性会出现显著下降。通过屏蔽靶向种间可变区的探针，可有效缓解跨物种杂交基因芯片数据存在的相关问题。除转录谱分析外，结合屏蔽种间可变区探针的跨物种杂交策略，还可用于比较生态学和/或进化基因组学研究。 整体实验设计：我们将从普通菜豆和大豆的根瘤、叶片及根系中纯化得到的cRNA以三次生物学重复的方式与大豆基因芯片进行杂交（共计18次基因芯片杂交 = 2个物种 × 3个组织 × 3次生物学重复）。