The Study On Homology-based Candidate Gene Method In R Gene Cloning

A further study on the homology-based candidate gene method for cloning resistance gene was done by scanning genomic DNA in maize near-isogenic lines (NIL) (OH43, OH43HI,, OH43 Ht2;B37, B37Ht,, B37Ht2) with the degenerate oligonucleotide primers designed from sequence motifs conserved in the type of NBS-LRR resistance gene. The results are as follows;1. All kind of oligonucleotides (16 sense primers, 8 antisenset primers, 128 different primer combinations in all) were designed from the conserved motifs GLPL and CFLY (distance between them is approximately 210bp) which existed only in the type of NBS-LRR resistance gene and used as primers for scanning the genomic DNA. No specific products (which is approximately 210bp in length and obtained from genomic DNA containing Ht gene) was obtained though amplified products of approximately 500-lOOObp appeared in six different primer combinations. The results indicate that conserved motif GLPL and CFLY does not exist in Ht gene, in other words, Ht gene does not belong to the type of NBS-LRR resistance gene.2. Oligonucleotides based on sequence motifs GGVGKTT and GLPLAL 1 (distance between them is about SOObp) conserved in tabacoo N and arabidosisi RPS2 were used as PCR primers for scanning genomic DNA. The amplifiedfragments of approximately 500bp were obtained in two different primercombinations, but were not considered as Ht gene's analog for they also appeared in the genomic DNA without Ht gene.-33-3. the blindness of homology-based candidate gene method for cloning resistance gene originated from diversity and unpredictability of resistance genes.