Microdissection of Haynaldia villosa telosome 6VS and cloning of species-specific DNA sequences

The material T240-6 derived from SC2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution tine of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the "single tube", all the subsequence steps were conducted. After two round of LA (Unker adaptor) - PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600 - 1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with P-32 as probe. The PCR products were purified and ligated into clone vector-pGEM-T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl2. It was estimated that there were more than 17 000 while clones in the library. The size of insert fragments distributed from 100 - 1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa-specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.