ABSTRACT

The present study was undertaken to develop a simple, robust, and reliable polymerase chain reaction-based molecular marker that can identify a critical nonsense mutation in a receptor like protein (RLP) gene of tomato. The Solyc04g014400 locus was found to code for a RLP with leucine-rich repeats, a trans-membrane domain, and characteristic three-dimensional structure. In silico analysis revealed the gene to be Pseudomonas-responsive. The gene was previously reported to contain a single nucleotide polymorphism (SNP) in the second exon, which converts the Trp 76 codon (TGG) to a premature stop (TAG) codon. Hence, a tetra-primer amplification refractory mutation system (T-ARMS) assay was developed to detect this functional SNP in 20 tomato genotypes in a co-dominant manner. Authenticity of the allele-specific amplicons was also tested, where the primers were observed to be discriminatory for the allelic variants. Suitability of the developed assay for determining heterozygosity in developed F₁ hybrids was also confirmed on the basis of the results. Thus, the developed T-ARMS marker will be useful for functional characterisation of this gene in the light of its Pseudomonas-responsiveness.

摘要

本研究旨在开发一种简单、稳健和可靠的基于聚合酶链反应的分子标记，可以识别番茄受体样蛋白 (RLP) 基因中的关键无义突变。发现 Solyc04g014400基因座编码具有富含亮氨酸重复、跨膜结构域和特征性三维结构的 RLP。计算机分析显示该基因是假单胞菌属-反应灵敏。此前报道该基因在第二个外显子中包含单核苷酸多态性 (SNP)，它将色氨酸 76 密码子 (TGG) 转换为过早终止 (TAG) 密码子。因此，开发了一种四引物扩增耐药突变系统（T-ARMS）测定法，以共显性方式检测 20 种番茄基因型中的这种功能性 SNP。还测试了等位基因特异性扩增子的真实性，其中观察到引物区分等位基因变体。在结果的基础上还证实了所开发的测定在开发的F _{1杂种中杂合性的适用性。}因此，根据其假单胞菌反应性，开发的 T-ARMS 标记将可用于该基因的功能表征。