Identification of Oryza Species with the CD Genome Based on RFLP Analysis of Nuclear Ribosomal ITS Sequences

根据核DNA的ITS序列的RFLP分析鉴定稻属CD染色体组物种(英文)

The genus Oryza consists of approximately 24 species with 10 recognized genome types (A, B, C, BC, CD, E, F, G, HJ, HK). The species with the CD genome are endemic to the Central and South America and comprise three species, i.e. O. latifolia Desv., O. alta Swallen and O. grandiglumis (Doell) Prod. Although the three species can be easily distinguished from the species with the other genomes in Oryza by their morphological characteristics, it is very difficult to distinguish them from one another. Recent studies suggested that O. alta and O. grandiglumis should be the same species (O. grandigiumis), whereas O. latifolia retains its taxonomic position unchangeably. In this paper, a total of 77 clones of the nuclear ribosomal internal transcribed spacer (ITS) from 11 samples representing different geographical races were sequenced. Using DNA Strider 1.2 software, the restriction enzyme digestion sites of these clone sequences were analyzed. Based on restriction fragment length polymorphism (RFLP) of ITS sequences, a method to identify the species with CD genome in Oryza was proposed. The method is rapid and convenient and all experimental procedure includes only three steps: (1) to amplify the ITS fragment with the routine primers; (2) to digest PCR products with restriction enzymes Fok I and Dra III or both; (3) to run the digested product on 1% agarose gel and identify the sample based on the restriction profiles.

稻属物种的染色体组类型有10种,其中具CD染色体组的物种有3种(Oryza alta Swallen,O.grandiglumis(Doell)Prod.和O.latifolia Desv.),仅分布在美洲中部和南部。这3个种在形态上和稻属其他染色体组的种比较容易区别,但它们彼此之间鉴别比较困难。近年来的研究表明,O.alta和O.grandiglumis应归并为1个种(O.grandiglumis),而O.latifolia应保持不变。本文基于代表不同分布区11个样品的核糖体转录间隔区(ITS)的77个克隆序列数据,利用DNA Strider1.2软件进行了限制性酶切位点分析,提出了一个鉴别CD染色体组物种的方法。方法的具体步骤是:(1)用通用引物扩增ITS片段;(2)用特异性的限制性内切酶Fok Ⅰ和/或Dra Ⅲ消化PCR扩增产物:(3)用1％的琼脂糖电泳并根据消化产物的多态性特征来鉴别不同物种。基于本文提出的核糖体ITS限制性片段多态性(RLFP)分析,可以快速和可靠地将稻属CD染色体组物种区别开来。