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Use of chromosome walking in discovery of single-nucleotide polymorphism in noncoding regions of a candidateactin gene inPinus radiata

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Abstract

Untranslated regions (UTRs) of eukaryotic mRNAs play crucial roles in post-transcriptional regulation of gene expression via the modulation of nucleocytoplasmic mRNA transport, translation efficiency, subcellular localization, and message stability. Single-nucleotide polymorphisms (SNPs) in UTRs of a candidate gene may also change the post-transcriptional regulation of a gene or function by nucleotide mutation. For species that have not been entirely sequenced genomically, new methods need to be devised to discover SNPs in noncoding regions of candidate genes. In this study, based on the expressed sequence tag (EST) ofPinus radiata (Monterey pine), we obtained all the sequences of UTRs of theactin gene by using a chromosome walking method. We also detected all the SNPs in and around the coding region of theactin gene. In this way, the full genomic sequence (2154 bp) of theactin gene was identified, including the 5’UTR, introns, the coding sequence, and the 3’UTR. PCR amplification and DNA fragment sequencing from 200 unrelatedP. radiata trees revealed a total of 21 SNPs in theactin gene, of which 3 were located in the 5’UTR, 3 in the introns, 10 in the coding sequence, and 5 in the 3’UTR. We show that chromosome walking can be used for obtaining the sequence of UTRs, and then, based on this sequence, to discover SNPs in the noncoding regions of candidate genes from this species without an entire genomic sequence.

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Authors and Affiliations

  1. Key Laboratory for Genetics and Breeding of Forest Trees and Ornamental Plants, Ministry of Education, National Engineering Laboratory for Forest Tree Breeding, Beijing Forestry University, Beijing, China

    W. Li & H. Li

  2. College of Forestry, South China Agriculture University, 510642, Guangzhou, China

    X-Y Chen

  3. CSIRO Plant Industry, Canberra, Australia

    W. Li & H. Wu

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  1. W. Li
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  2. H. Li
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  3. H. Wu
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  4. X-Y Chen
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Correspondence to X-Y Chen.

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Li, W., Li, H., Wu, H. et al. Use of chromosome walking in discovery of single-nucleotide polymorphism in noncoding regions of a candidateactin gene inPinus radiata . J Appl Genet 51, 275–281 (2010). https://doi.org/10.1007/BF03208856

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  • DOI: https://doi.org/10.1007/BF03208856

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