C.R.A.-Fruit Tree Research Unit, Via Torrino, 3, 81100, Caserta, Italy.
Mol Biol Rep. 2011 Feb;38(2):1237-49. doi: 10.1007/s11033-010-0223-y. Epub 2010 Jun 25.
English walnut (Juglans regia L.) is the most economically important species from all the 21 species belonging to the genus Juglans and is an important and healthy food as well as base material for timber industry. The aim of this study was to develop a simple technique for specific characterization of English walnut using DNA method. The first and second internal transcribed spacers (ITS1 and ITS2) as well as the intervening 5.8S coding region of the rRNA gene for 18 cultivars of J. regia L. isolated from different geographic origins were characterized. The size of the spacers sequences ranged from 257 to 263 bases for ITS1 and from 217 to 219 bases for ITS2. Variation of GC contents has also been observed and scored as 55-56.7 and 57.1-58.9% for ITS1 and ITS2, respectively. This data exhibited the presence of polymorphism among cultivars. Alignment of the ITS1-5.8S-ITS2 sequences from 18 walnut cultivars showed that there were 244 single nucleotide polymorphisms (SNPs) and 1 short insertion-deletion (indel) at 5' end ITS1. Amplification refractory mutation system strategy was successfully applied to the SNP markers of the ITS1 and ITS2 sequences for the fingerprinting analysis of 17 on 18 walnut cultivars. The prediction of ITS1 and ITS2 RNA secondary structure from each cultivar was improved by detecting key functional elements shared by all sequences in the alignments. Phylogenetic analysis of the ITS1-5.8S-ITS2 region clearly separated the isolated sequences into two clusters. The results showed that ITS1 and ITS2 region could be used to discriminate these walnut cultivars.
欧洲山核桃（Juglans regia L.）是所有 21 个胡桃属物种中经济价值最高的物种，既是一种重要的健康食品，也是木材工业的基础材料。本研究旨在开发一种使用 DNA 方法对欧洲山核桃进行特异性特征描述的简单技术。对来自不同地理起源的 18 个欧洲山核桃品种的 rRNA 基因的第一和第二内部转录间隔区（ITS1 和 ITS2）以及间隔区 5.8S 编码区进行了特征描述。间隔区序列的大小范围为 ITS1 的 257 至 263 个碱基和 ITS2 的 217 至 219 个碱基。还观察到 GC 含量的变化，并分别记录为 55-56.7%和 57.1-58.9%。这些数据显示了品种间的多态性。对 18 个山核桃品种的 ITS1-5.8S-ITS2 序列进行比对，发现有 244 个单核苷酸多态性（SNP）和 1 个 5'端 ITS1 的短插入缺失（indel）。扩增受阻突变系统策略成功应用于 ITS1 和 ITS2 序列的 SNP 标记，用于 17 个山核桃品种的指纹分析。通过检测比对中所有序列共有的关键功能元件，改进了每个品种的 ITS1 和 ITS2 二级结构的预测。ITS1-5.8S-ITS2 区的系统发育分析清楚地将分离的序列分为两个簇。结果表明，ITS1 和 ITS2 区可用于区分这些山核桃品种。
