Instituto de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politécnica de Valencia, 8E CPI, Camino de Vera s/n, 46022 Valencia, Spain.
BMC Genomics. 2010 Nov 12;11:631. doi: 10.1186/1471-2164-11-631.
The EcoTILLING technique allows polymorphisms in target genes of natural populations to be quickly analysed or identified and facilitates the screening of genebank collections for desired traits. We have developed an EcoTILLING platform to exploit Capsicum genetic resources. A perfect example of the utility of this EcoTILLING platform is its application in searching for new virus-resistant alleles in Capsicum genus. Mutations in translation initiation factors (eIF4E, eIF(iso)4E, eIF4G and eIF(iso)4G) break the cycle of several RNA viruses without affecting the plant life cycle, which makes these genes potential targets to screen for resistant germplasm.
We developed and assayed a cDNA-based EcoTILLING platform with 233 cultivated accessions of the genus Capsicum. High variability in the coding sequences of the eIF4E and eIF(iso)4E genes was detected using the cDNA platform. After sequencing, 36 nucleotide changes were detected in the CDS of eIF4E and 26 in eIF(iso)4E. A total of 21 eIF4E haplotypes and 15 eIF(iso)4E haplotypes were identified. To evaluate the functional relevance of this variability, 31 possible eIF4E/eIF(iso)4E combinations were tested against Potato virus Y. The results showed that five new eIF4E variants (pvr2(10), pvr2(11), pvr2(12), pvr2(13) and pvr2(14)) were related to PVY-resistance responses.
EcoTILLING was optimised in different Capsicum species to detect allelic variants of target genes. This work is the first to use cDNA instead of genomic DNA in EcoTILLING. This approach avoids intronic sequence problems and reduces the number of reactions. A high level of polymorphism has been identified for initiation factors, showing the high genetic variability present in our collection and its potential use for other traits, such as genes related to biotic or abiotic stresses, quality or production. Moreover, the new eIF4E and eIF(iso)4E alleles are an excellent collection for searching for new resistance against other RNA viruses.
EcoTILLING 技术可快速分析或鉴定自然种群中目标基因的多态性，并有助于筛选基因库中具有所需特性的基因。我们开发了一种 EcoTILLING 平台来利用辣椒遗传资源。该 EcoTILLING 平台的一个完美示例是它在辣椒属中寻找新的抗病毒等位基因中的应用。翻译起始因子（eIF4E、eIF（iso）4E、eIF4G 和 eIF（iso）4G）中的突变打破了几种 RNA 病毒的循环，而不影响植物生命周期，这使得这些基因成为筛选抗性种质的潜在目标。
我们开发并检测了一个基于 cDNA 的 EcoTILLING 平台，该平台使用了 233 个栽培辣椒属品种。使用 cDNA 平台检测到 eIF4E 和 eIF（iso）4E 基因编码序列中的高变异性。测序后，在 eIF4E 的 CDS 中检测到 36 个核苷酸变化，在 eIF（iso）4E 中检测到 26 个核苷酸变化。总共鉴定出 21 个 eIF4E 单倍型和 15 个 eIF（iso）4E 单倍型。为了评估这种变异性的功能相关性，对 31 种可能的 eIF4E/eIF（iso）4E 组合进行了马铃薯病毒 Y 的测试。结果表明，五个新的 eIF4E 变体（pvr2（10）、pvr2（11）、pvr2（12）、pvr2（13）和 pvr2（14））与 PVY 抗性反应有关。
在不同的辣椒物种中优化了 EcoTILLING 技术，以检测目标基因的等位基因变体。这是首次在 EcoTILLING 中使用 cDNA 而不是基因组 DNA。这种方法避免了内含子序列问题并减少了反应数量。起始因子的多态性水平很高，表明我们的收藏具有很高的遗传多样性，并且可以用于其他特性，例如与生物或非生物胁迫、质量或生产有关的基因。此外，新的 eIF4E 和 eIF（iso）4E 等位基因是搜索其他 RNA 病毒新抗性的极好资源。
