Department of Crop Plant Biology, University of Pisa, Pisa, Italy.
Theor Appl Genet. 2011 Apr;122(6):1039-49. doi: 10.1007/s00122-010-1509-0. Epub 2010 Dec 24.
With the aim to study variability in genes involved in ecological adaptations, we have analysed sequence polymorphisms of eight unique genes putatively involved in drought response by isolation and analysis of allelic sequences in eight inbred lines of sunflower of different origin and phenotypic characters and showing different drought response in terms of leaf relative water content (RWC). First, gene sequences were amplified by PCR on genomic DNA from a highly inbred line and their products were directly sequenced. In the absence of single nucleotide polymorphisms, the gene was considered as unique. Then, the same PCR reaction was performed on genomic DNAs of eight inbred lines to isolate allelic variants to be compared. The eight selected genes encode a dehydrin, a heat shock protein, a non-specific lipid transfer protein, a z-carotene desaturase, a drought-responsive-element-binding protein, a NAC-domain transcription regulator, an auxin-binding protein, and an ABA responsive-C5 protein. Nucleotide diversity per synonymous and non-synonymous sites was calculated for each gene sequence. The π (a)/π (s) ratio range was usually very low, indicating strong purifying selection, though with locus-to-locus differences. As far as non-coding regions, the intron showed a larger variability than the other regions only in the case of the dehydrin gene. In the other genes tested, in which one or more introns occur, variability in the introns was similar or even lower than in the other regions. On the contrary, 3'-UTRs were usually more variable than the coding regions. Linkage disequilibrium in the selected genes decayed on average within 1,000 bp, with large variation among genes. A pairwise comparison between genetic distances calculated on the eight genes and the difference in RWC showed a significant correlation in the first phases of drought stress. The results are discussed in relation to the function of analysed genes, i.e. involved in gene regulation and signal transduction, or encoding enzymes and defence proteins.
为了研究参与生态适应的基因的变异性，我们通过分离和分析来自不同起源和表型特征的 8 个向日葵自交系的等位序列，分析了 8 个假定参与干旱响应的独特基因的序列多态性，这些自交系在叶片相对含水量（RWC）方面表现出不同的干旱响应。首先，我们使用来自高度自交系的基因组 DNA 通过 PCR 扩增基因序列，然后直接对其产物进行测序。如果没有单核苷酸多态性，我们就认为该基因是独特的。然后，我们在 8 个自交系的基因组 DNA 上进行相同的 PCR 反应，以分离等位变体进行比较。这 8 个选定的基因编码一个脱水素、一个热休克蛋白、一个非特异性脂质转移蛋白、一个 z-胡萝卜素去饱和酶、一个干旱响应元件结合蛋白、一个 NAC 结构域转录调节剂、一个生长素结合蛋白和一个 ABA 响应 C5 蛋白。为每个基因序列计算了同义和非同义位点的核苷酸多样性。每个基因序列的π(a)/π(s)比值范围通常非常低，表明存在强烈的纯化选择，尽管存在位置间的差异。就非编码区域而言，只有在脱水素基因中，内含子的变异比其他区域更大。在测试的其他基因中，其中一个或多个内含子发生，内含子的变异性与其他区域相似甚至更低。相反，3'-UTR 通常比编码区域更具变异性。所选基因中的连锁不平衡平均在 1000bp 内衰减，基因间差异很大。在干旱胁迫的早期阶段，根据 8 个基因计算的遗传距离与 RWC 之间的差异进行的成对比较显示出显著的相关性。这些结果与所分析基因的功能有关，即参与基因调控和信号转导，或编码酶和防御蛋白。
