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Studies on Intron Length Polymorphism and EST Single Nucleotide Polymorphism in Brassica

Author: ZhangGeng
Tutor: WuWeiRen
School: Zhejiang University
Course: Crop Science
Keywords: Intron Brassica Phylogenetics EST SNP
CLC: S184
Type: Master's thesis
Year: 2009
Downloads: 39
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Abstract


For the organisms whose genomes have not been completely sequenced, the EST (expressed sequence tag) sequences are the most abundant resources in the public databases. According to the homology of genes and the conservation of intron positions, the intron positions in any species can be predicted by comparing its EST sequences with the genome sequences of model plants. Introns as well as ESTs contain rich polymorphisms. Therefore, they can be developed as molecular markers for the use of genetic studies and plant breeding.In order to detect potential intron polymorphisms (PIPs), it is necessary to amplify introns using primers designed on flanking exons. In this study, by comparing the EST sequences of Brassica napus and B. rapa with the Arabidopsis thaliana genome sequence, 5264 pairs of PIP primers were designed. 200 of the primer pairs were randomly selected to examine the correctness of the intron prediction and intron length polymorphisms (ILPs), study the intron length variation in Brassica genus, and construct the phylogenetic tree of Brassica based on ILP markers, by experimentally analyzing 29 Brassica accessions with Raphanus sativus and Sinapis arvensis as outgroup. The results indicated that: (1) 95% of the PIP markers could obtain good PCR products in B. napus and B.rapa, and the bands appeared to contain introns according to their sizes, suggesting that the prediction of intron positions was reliable; (2) Intron length variation occurred frequently during the process of new species forming via large-scale chromosomal rearrangement, but the frequency of intron length variation decreased after the genome of new species became stabilized; (3) The phylogenetic tree constructed based the ILP data could clearly classify the accessions according to their genome types, suggesting that ILP markers are very suitable for phylogenetic studies, especially for that among different genome types.In this study, SNP and InDel mining was also performed from Brassica EST data by making use of the redundancy of EST sequences; 31788 EST-SNPs were obtained and their variation was analyzed. The results indicated that: (1) The redundancy of EST sequences should be large enough for the EST-SNP mining, otherwise efficiency of finding EST-SNPs would be very low; (2) The main types of EST-SNP in Brassica are C/T and A/G, which account for about half of the total number; (3) The Brassica ESTs contain abundant SNPs, which could be developed as molecular markers for constructing high-density genetic map and for marker-assistant breeding.

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CLC: > Agricultural Sciences > Agriculture as the foundation of science > Agriculture and Biology > Agricultural botany
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