Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars.
Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars.
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DOI：
10.1371/journal.pone.0072998
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Figueiredo A
中科院分区：
文献类型：
--
作者：
Monteiro F;Sebastiana M;Pais MS;Figueiredo A
栽培葡萄藤（葡萄属vinifera L.）在许多国家，由于其对霜霉病病原体葡萄霜霉病的高度易感性，经济受到损害。最近的研究已经确定了一组与抗性相关的基因，这些基因可能用于追踪霜霉病的感染。这些抗性基因的表达的定量需要使用在所研究的系统中具有稳定表达的参考基因对qPCR数据进行归一化。在这项研究中，评估了一组11个基因（VATP 16、60 S、UQCC、SMD 3、EF 1 α、UBQ、SAND、GAPDH、ACT、PsaB、PTB 2），以在两种葡萄基因型和葡萄疫霉菌之间相互作用的最初几个小时（6、12、18和24 hpi）内识别参考基因。使用两种分析来选择参考基因：在接种葡萄生疫霉后0小时、6小时、12小时、18小时和24小时直接比较敏感的Trincadeira和抗性的Regent葡萄品种（V. vinifera）（基因型效应）;以及在接种时间过程中比较每个基因型与模拟接种样品（生物胁迫效应）。使用了三种统计方法，GeNorm，Normalization和BestKeeper，可以将UBQ，EF 1 α和GAPDH鉴定为基因型效应最稳定的基因。在生物胁迫效应中，感病品种Trincadeira的EF 1 α、SAND和SMD 3最稳定，抗病品种Regent的EF 1 α、GAPDH和UBQ最稳定。此外，在接种时间过程中，对两个数据集分析了编码枯草杆菌蛋白酶样蛋白的三种防御相关转录物的表达。综上所述，我们的研究结果为参考基因的选择提供了指导，以更准确和更广泛地使用qPCR来研究不同葡萄藤品种和葡萄疫霉之间的第一个小时的相互作用。
The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (VATP16, 60 S, UQCC, SMD3, EF1α, UBQ, SAND, GAPDH, ACT, PsaB, PTB2) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two V. vinifera genotypes and P. viticola. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, V. vinifera cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with P. viticola (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify UBQ, EF1α and GAPDH as the most stable genes for the genotype effect. For the biotic stress effect, EF1α, SAND and SMD3 were the most constant for the susceptible cultivar Trincadeira and EF1α, GAPDH, UBQ for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, CYP and PR10, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and P. viticola.
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