The Application Of ISSR Markers In Plant Genetic Analysis

ISSR (Inter Simple Sequence Repeat) molecular marker is a technique, which involves the use of microsatellite sequences as primers in a PCR (Polymerase Chain Reaction) to generate multilocus markers. No sequencing is required to design the primers and amplification of DNA segments present at an amplifiable distance between two identical microsatellite repeat regions oriented in opposite direction. It is a simple and quick method that combines most of the advantages of AFLP (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeat) and RAPD (Randomly Amplified Polymorphism DNA). ISSR markers are highly polymorphic and useful in studies of plant, especially genetic relationships and genetic diversity. In order to discuss the value of the application of ISSR in plant studies, we applied ISSR analysis to 59 Phalaenopsis, 22 Canarium and 5 Hibiscus. The main results are as follows:1. Improve the methods of extracting genome DNA for the three plants respectively. We used the method of FastPrep for Phalaenopsis, which make it convinent and rapid. In order to solve the problem of polyphenol and amylase, some improvements were applied.2. ISSR-PCR reaction system for Phalaenopsis was optimized through each component. ISSR-PCR reaction system for Canarium was optimized through orthogonal test. We can see that both of them are useful, but orthogonal test is better. Based on the reaction system of Phalaenopsis and Canarium and some adjustments, reaction system for Hibiscus is obtained. ISSR-PCR reaction system for plant has commonality.3. The result shows that we can get enough ISSR markers for genetic analysis and the PPB is high, so ISSR technical is suitable for the study of Phalaenopsis, Canarium and Hibiscus. Moreover, we obtained many specific bands, so ISSR represents a useful method for the identification of variety.4. Using 14 primers in the ISSR analysis of 59 Phalaenopsis, we gained 192 bands, of which 165 bands are polymorphic, and the average percentage of polymorphic bands is 86%. The range of genetic distance is 0.035~0.386, revealing that most cultivars have small genetic distance, but the substantial genetic divergence between few cultivars from abroad. All 59 cultivars could be distinguished by ISSR markers and 35 cultivar-specific ISSR bands were obtained for 20 of the 59 Phalaenopsis cultivars. The result of UPGMA clustering analysis was generally consistent with color, but there were some exceptions between clustering result and flower colors which may result from hybridization between cultivars.5. Using 8 primers in the ISSR analysis of 22 Canarium, we gained 164 bands, of which 127 bands are polymorphic, and the average percentage of polymorphic bands is 77.4%. The range of genetic distance for 21 Canarium species is 0.048~0.138, revealing that their genetic diversity is not high and there is no substantial genetic divergence between species. Otherwise, all 21 species could be distinguished by ISSR markers and 11 species-specific ISSR bands were obtained for 8 of the 21 Canarium species.6. Using 5 primers in the ISSR analysis of 5 Hibiscus, we gained 66 bands, of which 48 bands are polymorphic, and the average percentage of polymorphic bands is 72.7%. From the Dendrogram of genetic distance, we can see that H. schizopetalus has a high genetic similarity with the four varities of H.rosa-sinensis, which supports the view of“H. schizopetalus should be a varity of H.rosa-sinensis”. Meanwhile, ISSR-PCR analysis revealed that the amplified fragments respectively unique to H.schizopetalus and H.rosa-sinensis L.‘Carminatus’showed additivity in H.rosa-sinensis L.‘Scarlet’. The molecular evidence indicated that H.rosa-sinensisL.‘Scarlet’was a hybrid origin.