Allele-specific, bidirectional silencing of an alcohol dehydrogenase gene in different organs of interspecific diploid cotton hybrids

Abstract

Interspecific hybridization is a common and important process that generates phenotypic novelty and can lead to hybrid speciation as well as to changes in gene expression. Using two different interspecific cotton (Gossypium) diploid hybrids, we show organ-specific, bidirectional allelic silencing at a heterozygous Adh locus, with alternate alleles being silenced in leaves and many floral organs, respectively. These results show that developmental regulation of gene expression is changed immediately upon hybridization between diploid species, possibly due to epigenetic factors or regulatory mismatch.

Figure 1.

(A) Diagram showing synthesis of two different interspecific hybrids. (B and C) SSCP-cDNA gels showing transcript levels of alcohol dehydrogenase A (AdhA) alleles in the F₁ hybrids of G. trilobum × G. gossypioides (B) and G. raimondii × G. gossypioides (C). D6, D8, and D5 correspond to the species used in the crosses (see A) and hence reveal gel migration positions of the alleles derived from each parent. (D) Agarose gels showing AdhA RT-PCR products from parental species. Reactions were performed with (+) or without (−) reverse transcriptase. Lanes are numbered in pairs as follows: 1, D5 bract; 2, D5 stamen; 3, D5 stigma and style; 4, D5 ovule; 5, D6 leaf; 6, D8 stamen (repeat); 7, D8 petal; 8, D8 stamen; and 9, D8 stigma and style. “m” indicates size marker. Plant materials: G. gossypioides (accession HC8065), G. raimondii (Wendel laboratory stock), and G. trilobum (accession D8-1) were crossed as described in the text and as shown in A. The same perennial G. raimondii and G. gossypioides individuals used to make the hybrids and a plant from the accession of G. trilobum were used for RNA extractions. Two progeny plants derived from the same cross were used as biological replicates for each of the diploid hybrids. All plants were grown under the same conditions in the greenhouse at Iowa State University. Methods: RNA was extracted from the plants (flowers were collected between 10:00 am and noon on the day of flower opening), the quality was checked by electrophoresis on agarose gels, and the concentration was estimated with a spectrophotometer as previously described (Adams et al. 2003). The RNA was treated with DNase to remove residual DNA and then it was reverse transcribed also as previously described (Adams et al. 2003). PCRs were performed with cDNA templates, using primers that amplify both alleles, as described in Adams et al. (2003). Primer sequences were 5′-GGTGAGGGTGTGAATGATTTG-3′ and 5′-GTACATATCCCACAGCTAATGG-3′. RT-PCR products were separated by SSCP-cDNA (Cronn and Adams 2003); gels included 2% urea and were run at 4°. Bands were quantified from dried gels by phosphorimaging using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). RT-PCR products showing one allele on the SSCP gels were directly sequenced to verify the presence of only one allele.