Gene expression non-additivity in immature ears of a heterotic F1 maize hybrid

Non-additive gene regulation has been recently suggested as an important factor promoting phenotypic variation and plasticity. In order to obtain a description of gene expression status at an early stage of ear development in a maize (Zea mays L.) F1 hybrid as relative to its parental inbreds, we compared gene expression profiles in immature ears of elite inbred lines B73 and H99 to one of their F1 hybrids (B73xH99) using cDNA microarray technology. Results show several genes expressed at a significantly different level between both inbred lines and their hybrid. In addition, gene expression non-additivity in the hybrid was detected on a broad scale, consisting of both dominance and over-dominance components, indicating that complex non-additive interactions at the molecular level exist in the developing ear of the studied maize hybrid. Non-additively regulated genes belong to a wide range of molecular functions, indicating that several regulatory and metabolic patterns are possibly affected during ear development in the investigated hybrid. We discuss the possibility that observed gene expression non-additivity in immature ear might be an early molecular manifestation of hybrid vigor, the most exploited factor for maize agronomic improvement. We directly contrasted both B73 and H99 inbred lines vs. their F1 heterotic hybrid transcriptomes in immature ear. Our experimental design consisted of 10 cDNA microarray hybridizations, 5 for each combination of hybrid vs. inbred genotypes, involving 20 separate labeling reactions. Labeling dyes were swapped in two of the five replicates for each combination. In each hybridization, control channel was assigned to the F1 hybrid, and differences in transcriptional levels between B73 and H99 were inferred using the hybrid as a common reference sample in an indirect experimental design. To take in account variability in transcript population among individuals, total RNA coming from different isolations, each collected on multiple individuals, were mixed before poly(A+) RNA purification. All hybridizations on microarray slides were then performed using cDNA independently labeled from the poly(A+) RNA purification product for each genotype. Base-two logarithms of expression ratios were subjected to one-class response significance analysis in SAM v. 2.20 software [Tusher et al. 2001]. For each EST the estimates of additive parameter "a" and dominance parameter "d" (middle-parent heterosis) were obtained as a = (L2 – L1)/2 d = (L1 + L2)/2 (where L1 and L2 are mean base-two logarithms of expression ratios of F1 vs. B73 and F1 vs. H99, respectively). Positive values of "a" indicate expression values bigger in B73 than in H99. The dominance/additivity ratio (d/|a|) was also calculated [Falconer 1989]. Evaluation of statistical significance of parameters was done by calculating standard errors of the estimates "a" and "d" as standard errors of linear functions of the means. Significance testing was done correspondingly, using an F-test for linear contrasts. P-values for the families of tests corresponding to each parameter were subjected to global error analyses using a method based on fitting mixture distribution [Allison et al. 2002], allowing to estimate the false discovery rates (FDR) and false negative rates (FNR). Confidence intervals for d/|a| ratios were obtained by Fieller’s method [Piepho and Emrich 2005], allowing to classify the genes into different dominance type classes.

非加性基因调控（non-additive gene regulation）近来被认为是促进表型变异与表型可塑性的重要因素。为了描述玉米（Zea mays L.）F1杂交种在穗发育早期阶段的基因表达状态（相对于其亲本自交系），我们利用cDNA芯片技术（cDNA microarray），对优良自交系B73、H99及其F1杂交种（B73×H99）的未成熟穗组织进行了基因表达谱比较分析。
结果显示，两个自交系与其杂交种之间存在多个表达水平存在显著差异的基因。此外，该杂交种中广泛存在基因表达的非加性现象，涵盖显性与超显性两种组分，表明本研究中的玉米杂交种在发育的穗组织中，分子层面存在复杂的非加性互作。
受非加性调控的基因涵盖了广泛的分子功能类别，提示本研究的杂交种在穗发育过程中，多种调控与代谢模式可能受到影响。我们讨论了未成熟穗中观测到的基因表达非加性现象，可能是杂种优势（hybrid vigor）的早期分子表征——而杂种优势正是玉米农艺改良中最具应用价值的性状因素。
我们直接比较了B73、H99自交系与其F1杂种优势杂交种的未成熟穗转录组（transcriptome）。本实验设计共包含10次cDNA芯片杂交，每个杂交种-自交基因型组合对应5次杂交，涉及20次独立的标记反应。每个组合的5次重复中，有2次交换了标记荧光染料。在每次杂交实验中，以F1杂交种作为对照通道，采用间接实验设计，以该杂交种作为共同参照样本，推断B73与H99之间的转录水平差异。
为了考虑个体间转录本群体的差异，我们将多次独立提取、且均来自多个个体的总RNA混合后，再进行聚腺苷酸RNA（poly(A+) RNA）纯化。随后，针对每个基因型，从纯化得到的聚腺苷酸RNA中独立标记cDNA，用于芯片杂交实验。
我们将表达比值的以2为底的对数结果，导入SAM v.2.20软件[Tusher et al. 2001]进行单样本响应显著性分析。针对每个表达序列标签（EST），我们计算得到加性参数“a”与显性参数“d”（即中亲优势，middle-parent heterosis）：
a = (L2 – L1)/2 d = (L1 + L2)/2
其中L1与L2分别为F1相对于B73、F1相对于H99的表达比值的以2为底的对数均值。“a”为正值时，表示B73中的表达水平高于H99。我们还计算了显性/加性比值（d/|a|）[Falconer 1989]。
参数的统计学显著性评估通过计算加性参数“a”与显性参数“d”估计值的标准误实现——该标准误由均值的线性函数计算得到。显著性检验采用针对线性对比的F检验。对应每个参数的检验族的P值，通过基于混合分布拟合的方法进行全局误差分析[Allison et al. 2002]，以此估计假发现率（FDR）与假阴性率（FNR）。我们采用Fieller法[Piepho and Emrich 2005]计算d/|a|比值的置信区间，从而将基因划分为不同的显性类型类别。