Pellino Marco, Sharbel Timothy F, Mau Martin, Amiteye Samuel, Corral José María
Apomixis Research Group, Leibniz-Institute für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstraße 3, D-06466 Gatersleben, Germany.
BMC Res Notes. 2011 Aug 19;4:303. doi: 10.1186/1756-0500-4-303.
Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates.
Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18), Efalpha1 (Elongation factor 1 alpha), ACT 2 (Actin2), UBQ (polyubiquitin), PEX4 (Peroxisomal ubiquitin conjugating enzyme) and At1g09770.1 (Arabidopsis thaliana cell division cycle 5). Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues) in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed, different optimal combinations were identified in sexual ovules (BoechUBQ and BoechEfα1), in anthers from both reproductive systems (BoechACT2 and BoechEfα1), in apomictic vegetative tissues (BoechEfα1 and BoechACT2) and sexual vegetative tissues (BoechRPS18 and BoechEfα1). NormFinder ranked BoechACT2 as the most stable in the apomictic plant, while BoechRPS18 was the best in the sexual plant. The subgroups analysis identified the best gene for both apomictic and sexual ovules (BoechRPS18), for anthers from both reproductive system (BoechEfα1) and for apomictic and vegetative tissues (BoechACT2 and BoechRPS18 respectively)
From a total of six tested genes, BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ showed the best stability values. We furthermore provide detailed information for the accurate normalization of specific tissue gene expression analyses of apomictic and sexual Boechera.
无融合生殖是植物无性种子生产的一种自然形式,被认为在农业领域具有巨大的生物技术潜力。据推测,性发育途径的去调控可能引发无融合生殖。Boechera属是理解无融合生殖的一个有趣的模型系统,在二倍体水平上既有有性生殖基因型,也有无融合生殖基因型。定量qRT-PCR是验证全基因组基因表达分析最广泛使用的方法,但为了获得可靠的结果,合适的内参基因是必要的。在这项工作中,我们评估了从Boechera的一个454(FLX)衍生cDNA文库中分离出的六个潜在内参基因。来自不同发育阶段的活体显微切割胚珠和花药的RNA,以及无融合生殖和有性生殖的Boechera的营养组织,被用于验证这些候选基因。
基于与拟南芥的同源性,从Boechera的一个454 cDNA文库中选择了六个基因:RPS18(核糖体亚基蛋白18)、Efalpha1(延伸因子1α)、ACT 2(肌动蛋白2)、UBQ(多聚泛素)、PEX4(过氧化物酶体泛素缀合酶)和At1g09770.1(拟南芥细胞分裂周期5)。从17种不同组织中提取总RNA,进行qRT-PCR,并分析原始Ct值以确定引物效率和基因比例。geNorm和normFinder软件分别用于在无融合生殖和有性生殖植物的所有组织以及特定组织组(胚珠、花药和营养组织)中选择最稳定的基因。我们的结果表明,BoechRPS18、BoechEfα1、BoechACT2和BoechUBQ是最稳定的基因。基于geNorm分析,在无融合生殖植物中,BoechRPS18和BoechEfα1或BoechUBQ和BoechEfα1的组合最稳定,而在有性生殖植物中,BoechRPS18和BoechACT2或BoechUBQ和BoechACT2表现最佳。当分析组织样本亚组时,在有性胚珠(BoechUBQ和BoechEfα1)、两种生殖系统的花药(BoechACT2和BoechEfα1)、无融合生殖营养组织(BoechEfα1和BoechACT2)和有性生殖营养组织(BoechRPS18和BoechEfα1)中鉴定出了不同的最佳组合。NormFinder将BoechACT2列为无融合生殖植物中最稳定的基因,而BoechRPS18在有性生殖植物中表现最佳。亚组分析确定了无融合生殖和有性生殖胚珠(BoechRPS18)、两种生殖系统的花药(BoechEfα1)以及无融合生殖和营养组织(分别为BoechACT2和BoechRPS18)的最佳基因。
在总共六个测试基因中,BoechRPS18、BoechEfα1、BoechACT2和BoechUBQ表现出最佳的稳定性值。我们还提供了详细信息,用于准确标准化无融合生殖和有性生殖的Boechera特定组织基因表达分析。