Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons

Department of Plant and Environmental Sciences, New Mexico State University, MSC 3Q, Las Cruces, NM 88003, USA.
Journal of Plant Physiology (Impact Factor: 2.56). 12/2011; 168(8):824-30. DOI: 10.1016/j.jplph.2010.10.006
Source: PubMed


MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 (Gossypium barbadense) and Acala 1517-99 (Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.

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Available from: Jinfa Zhang, Aug 15, 2014
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    • "At this time, most miRNAs and target gene research is focused on functional verification through gene over-expression, gene interference, and related methods, but there is paucity in development of miRNA markers and their relationship with phenotypes. Only one report in the literature described miRNA markers- miRNA-AFLP (amplified fragment length polymorphism) [32], but no miRNAs or their target genes have been genetically mapped in cotton. "
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    ABSTRACT: Small RNAs are a group of non-coding RNAs that downregulate gene expression in a sequence-specific manner to control plant growth and development. The objective of the present study was to clone and characterize several small RNAs in cotton. To identify small RNAs that are involved in the development of cotton bolls and fibers, we generated cDNA libraries from cotton bolls at 13 days post-anthesis from two cotton cultivars, Pima Phy 76 (Gossypium bardadense) and Acala 1517–99 (Gossypium hirsutum). Screening of these libraries identified eight small RNAs, seven of which have not been reported in other plant species and appear to be absent in the known sequences of other plant species. Their predicted target genes are known to be involved in cotton fiber development. The cloned small RNAs displayed lower and differential expression in the examined boll developmental stages using RT-PCR and quantitative RT-PCR. The genetic polymorphism of the small RNAs at the DNA level was evaluated by miRNA-amplified fragment length polymorphism (AFLP) analysis using primers designed from the small miRNA genes in combination with AFLP primers. Homologous small RNA gene sequences were further isolated using this homology-based genotyping approach, and potential hairpin structures were identified. The results represent a novel method to isolate small including miRNA genes at the RNA and DNA levels in many plant species where genome sequences are not available or expressed sequence tags are limited.
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