Microcloning of a Specific Chromosome in Hot Pepper by Chromosome Microdissection Method

초록

Although hot pepper, Capsicum annuum, is one of the most important vegetable crop species, genomic researches have been meagre. Molecular markers have been utilized not only for crop or domesticated animal breeding programs but also for isolation of specific genes in crop species. The goal of this research was developing protocols for chromosome-specific library construction and providing a specific chromosome rich libraries. The chromosome 12 was isolated under the illumination of inverted microscope and the chromosomal DNA was amplified by DOP-PCR technique. The amplified fragments were either cloned directly into pGEM-easy T vector or subtracted with cDNA expressed in early seedling, or amplified and cloned after amplification with AFLP protocol. Among the colonies obtained (3 × 104), six hundred colonies were selected randomly for characterization, in which approximately 50% of the colonies showed positive signals with genomic probes. There were approximately 20% of the colonies being present more than once in the libraries. Since approximately 30% of the inserted fragments were revealed to be repetitive DNA fragments, approximately 150 colonies might be the low copy sequence fragments which are suitable for RFLP probes in genomic analysis.

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