Studies On Large-scale Development Of Intron Polymorphism Markers Using CDNA/EST Sequences

With the progress made in large-scale plant functional genome sequencing projects, a great amount of EST (expressed sequence tag) data is becoming available. Introns are widely distributed in eukaryotic genomes and are much more variable than coding sequences. Polymorphisms in introns, including intron length polymorphism (ILP) and intron single nucleotide polymorphism (ISNP), can be developed as molecular markers named intron polymorphism (IP). The positions of introns in genes are relatively conservative among species. Therefore, by comparing the EST sequences of a plant with the homologous genes of model plant rice or Arabidopsis, the intron positions (i.e. the joints between adjacent exons) in the ESTs of this plant could be predicted. Thus, by designing a pair of primers on the EST sequence flanking the predicted intron position, a fragment containing the predicted intron could be amplified from that plant and polymorphism in the intron could be detected. In other words, the primers have the potential to develop IP markers. Hence, we call them potential intron polymorphism (PIP) markers. In this study, large-scale development of PIP markers was performed according to the above principle; a PIP database was constructed; ILP markers were developed and applied in cotton; and methods for detecting ISNPs were trialed. Main results are as follows:1. A total of 57,658 PIP markers were developed in 59 plant species.2. A web-based PIP database (http://ibi.zju.edu.cn/pgl/pip) was constructe, providing the following functions: 1) introduction of the procedure and method for developing PIP markers; 2) inquiry and download of PIP markers; 3) online designing of PIP primers on cDNA/EST sequences submitted by users; and 4) comparison of homology between PIP markers from different species.3. Electronic PCR analysis on PIP markers indicated that up to 99.40% and 89.71% introns predicted by Arabidopsis were correct in Medicago truncatula and Populus trichocarpa, suggesting that intron positions are very conservative indeed.4. Comparison analysis on the PIP markers of model plants developed based on themselves showed that the frequencies of ILPs/ISNPs between two subspecies of rice (japonica cultivar Nipponbare and indica cultivar 93-11) and between two ecotypes of Arabidopsis (Columbia and Landsberg) were 17.98%/51.22% and 18.61%/53.18%, respectively, suggesting that the intron polymorphism level is very high in plant and can meet the requirement of practical application.5. Two hundred and twenty pairs of cotton PIP primers were synthesized. PCR analysis with these primers on 19 accessions of cultivated cotton and 25 accessions of wild cotton showed that 92.27% of the primers amplified products as expected; 65.02% amplified single bands; and 94.09% contained introns in their PCR products. The ILP frequency was only 3.18% in cultivated cotton (including G. hirsutum L. and G. barbadense L.), but was up to 47.78% in wild cotton. The PIC values of ILP loci in wild cotton varied 0.061-0.721 with an average of 0.34.6. The ILP markers developed were used for cluster analysis of 25 wild cotton accessions. It was found that ILP markers could strictly distinguish between A, B, C, D, E, F and G genomes and between diploid and allopolyploids, suggesting that ILP markers can be effectively applied to the studies of cotton classification and evolution.7. The feasibility of using TILLING principle to detect ISNP was investigate and technical simplification was made (named simTILLING). The principle of using simTILLING to analyze ISNP markers in segregating populations was proposed.8. The method of detecting SNP by PCR using primers with different lengths was trialed and 6 EST-SNP markers were developed in cotton using this method.