GISH, PCR-RFLP and RFLP analysis of intergeneric hybrids combining for Ascocenda and Phalaenopsis

• Main Authors: Win-Lin Liu, 劉文粦
• Other Authors: Yu-Chung Chiang, Ph. D
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/25455442480587729275

碩士 === 國立屏東科技大學 === 生物科技研究所 === 97 === The Phalaenopsis is one of potential and high economic crop in Taiwan. The genetic inheritance of intergeneric hybrids combining Ascocenda John De Biase "Blue" (♀) and Phalaenopsis Chih Shang's Stripes (♂) (F1) were detected by genomic in situ hybridization (GISH), PCR-restriction fragment length polymorphism (PCR-RFLP) and restriction fragment length polymorphism (RFLP). The intergeneric hybrids (F1 plants) can be confirmed that from Ascocenda John De Biase "Blue" (♀) and Phalaenopsis Chih Shang's Stripes (♂) by GISH. No recombination can be found between two species. Twenty-seven randomly selected for following DNA anaysis. Both internal trandscribed spacer (ITS) DNA and external transcribed spacer (ETS) of nuclear ribosomal DNA (nrDNA) were separately analyzed by PCR-RFLP analysis. It is not in agreement between ITS and ETS analysis. In ITS analysis, those hybrids showed biparental patterns, however, they only showed the DNA pattern of maternal parent, Ascocenda John De Biase "Blue"(♀), in ETS analysis. Furthermore, the trnL intron of chloroplast genome (cpDNA) was also analysis by RCR-RFLP. The result shows that cpDNA is maternal inheritance in the intergeneric hybrids. For further realizing aforementioned incongruence of genetic inheritance between ITS and ETS by PCR-RFLP, RFLP analysis was used. The result shows that ETS region is also biparental inheritance same as that of ITS region. It indicates that the bias of PCR amplification for ETS region is happened in all hybids. After sequencing of ETS region for both parents, the bias of PCR amplification resulting of two-base mismatch between the primer designed in ETS region and the DNA template of Phalaenopsis Chih Shang's Stripes (♂) is suggested. In addition, seven hybrids show one unique DNA pattern of ETS by RFLP analysis. Inspection of the morphology of these seven hybrids, they show leaves than others. In final, GISH, PCR-RFLP and RFLP can be used for detecting the genetic inheritance of intergeneric hybrids. However, it should be kept from the primer competition in PCR amplification for mixed genome when we use PCR-RFLP analysis.