Carelli Maria, Calderini Ornella, Panara Francesco, Porceddu Andrea, Losini Ilaria, Piffanelli Pietro, Arcioni Sergio, Scotti Carla
Consiglio per la Ricerca e la sperimentazione in Agricoltura (C.R.A.) - Centro di ricerca per le Produzioni Foraggere e Lattiero-casearie, Lodi, Italy.
Methods Mol Biol. 2013;1069:101-18. doi: 10.1007/978-1-62703-613-9_9.
Medicago truncatula is one of the model species for legume molecular genetics. In the last decade different types of mutant populations have been created in this species that can be screened by forward and reverse-genetic approaches to identify and functionally characterize genes of interest. TILLING is a reverse-genetic method combining random chemical mutagenesis and a PCR-based screen to identify point mutations in regions of interest. The different steps of the TILLING analysis are described in a mutant collection of ~2,300 M2 individuals for which genomic DNA and M3 seed were obtained. A two-dimensional DNA pooling strategy was adopted to reduce the number of PCR reactions necessary to screen the collection and to unambigously identify the individual M2 plant carrying the mutation. The genotypic and phenotypic analyses of the mutant M3 progeny provide the possibility to study the gene function. In spite of its reduced size, this mutant collection has proved valid in the study of the biosynthetic pathway of a class of secondary metabolites present in the genus Medicago, the triterpenic saponins.
蒺藜苜蓿是豆科植物分子遗传学的模式物种之一。在过去十年中，该物种已创建了不同类型的突变群体，可通过正向和反向遗传学方法进行筛选，以鉴定感兴趣的基因并对其进行功能表征。定向诱导基因组局部突变（TILLING）是一种反向遗传学方法，它结合了随机化学诱变和基于聚合酶链式反应（PCR）的筛选，以鉴定感兴趣区域的点突变。TILLING分析的不同步骤在一个约2300个M2个体的突变体库中进行了描述，该库已获得基因组DNA和M3种子。采用二维DNA混合策略来减少筛选该库所需的PCR反应数量，并明确鉴定携带突变的单个M2植株。对突变体M3后代的基因型和表型分析为研究基因功能提供了可能。尽管该突变体库规模较小，但已证明其在研究苜蓿属中一类次生代谢产物——三萜皂苷的生物合成途径方面是有效的。
