Investigation Of Intron Length Polymorphisms In Cotton And Conifer And Their Application

With the progress of life science research, molecular markers have been widely used wildspred in genetic mapping, genetic diversity analysis, marker-assisted breeding and so on. Meanwhile, a great amount of EST (expressed sequence tag) data is available because of the development of plant functional genome sequencing projects. Introns are widely distributed in eukaryotic genomes and more variable than coding sequences. Intron length polymorphism (ILP) can be detected most easily among various intronic polymorphisms. Therefore ILP can be developed as molecular markers. ILP can be detected by PCR with specific primers designed on exons flanking the target introns. The key for developing ILP markers is to choose suitable introns. The present study aimed to investigate the feasibility of developing ILP markers in Gossypium and Pinaceae based on putative intron positions predicted according to rice and Arabidosis. Main results are as follows:1. A total of 2,300 PIP markers were developed in loblolly pine (Pinus massoniana).2. Electronic PCR analysis on PIP markers indicated that up to 100% and 88.06% introns predicted by Arabidopsis and rice were correct in Gossypium and Pinus massoniana, respectively, suggesting that intron positions are very conservative.3. Thrity-four pairs of cotton PIP primers were synthesized. PCR analysis with these primers on 16 accessions of cotton and Thespesia populnea showed that 85.29% of the primers amplified products as expected; and the ILP frequency was up to 34.48% among all materials. It can be concluded that intron polymorphism level is high in wild cotton.4. The developed ILP markers were used for cluster analysis of 17 cotton accessions. It was found that dendrogram constructed based on ILP markers was consistent with traditional phylogenetic taxonomy and could distinguish between different genomes, demonstrating that ILP markers are quite suitable for phylogenetic studies of cotton species. 5. Four hundred pairs of loblolly pine PIP primers were synthesized. PCR analysis with these primers on 17 conifer accessions showed that 89.75% of the primers amplified products as expected; and the ILP frequency was up to 64.40% in Pinaceae family, but only 30.90% in Pinus genus.6. The developed ILP markers were used for cluster analysis of 9 conifer accessions. It was found that the ILP markers could strictly distinguish between Pinaceae and other families, and between Pinus and other genera, suggesting that ILP markers can be effectively applied to the studies of cotton classification and evolution.