Objective Isolation and identification of resistance gene analogs (RGAs) of Lagenaria siceraria would lay the foundation for a further cloning of disease resistance genes and marker-assisted selection (MAS) of resistance breeding.
Method According to the conserved domains of nucleotide binding site and leucine rich repeat (NBS-LRR) type of disease-resistance genes in most known plants, degenerate primers were designed and synthesized to isolate resistance gene analogs from genomic DNA of bottle gourd resistant variety " Dazihu", with length variation, conservative domain, homology alignment and phylogenetics analyzed by various bioinformatics softwares.
Result and conclusion Twenty three RGAs were obtained and named as HNB1 - HNB23, and the GenBank accession numbers were KJ908192 - KJ908214.Sequences analyses and alignment results indicated that the full-length of RGAs varied from 242 nt to 261 nt, and the deduced amino acids sequences contained typical conserved domains of NBS R genes, such as P-loop and Kinase-2a.Eighteen sequences had continuous open reading frames (ORFs).These RGAs showed a great homologous differences with the similarity ranging from 41. 5% to 98.8%, and the amino acid sequence similarity varied from 21.5% to 100.0%.At the nucleotide level, the sequence identity of 23 RGAs ranged from 40% to 100% with the cloned NBS R genes from other plants, especially cucumber, wax gourd, luffa and calabash.The result of clustering analyses showed that all RGAs were divided into 5 groups.These RGAs were ranked into non-TIR-NBS-LRR type by homology and evolution analyses, which was consistent with the classification result based on multiple alignment of deduced amino acid sequences.
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