Organ-specific silencing of duplicated genes in a newly synthesized cotton allotetraploid

Abstract

Most eukaryotes have undergone genome doubling at least once during their evolutionary history. Hybridization followed by genome doubling (allopolyploidization) is a prominent mode of speciation in plants, leading to phenotypic novelty and changes in genome structure and gene expression. Molecular events that take place immediately after polyploid formation can be studied using newly synthesized allopolyploids. Here we studied the extent of gene silencing in a newly created and genomically stable allotetraploid cotton, of genotype AAGG, using an AFLP-cDNA display screen. Over 2000 transcripts were screened and approximately 5% of the duplicated genes in the allotetraploid were inferred to have been silenced or downregulated. Sequencing of 24 AFLP-cDNA fragments revealed genes with a variety of functions. Analysis by RT-PCR showed silencing or a strong expression bias toward one copy for 9 of 13 genes examined. Comparisons of expression patterns among eight organs in the allopolyploid showed that silencing and preferential expression are organ specific. Examination of silencing patterns in two other synthetic polyploids, of genotype AADD, showed that the same gene can be silenced independently in different genotypes. These results provide a detailed portrayal of gene silencing events that can occur following allopolyploidization and suggest epigenetic causal factors.

Figure 1.—

Evolutionary history of the cotton genus (Gossypium), showing the diploids and natural AADD allotetraploid. Genome groups used in this study are shaded. The synthetic allotetraploids were made from AA and GG diploids and from AA and DD diploids.

Figure 2.—

AFLP-cDNA gels. Example fragments of gels run on the ABI 377 with fluorophore-labeled primers are shown. Bands of each class are indicated: (1) a band present in each lane that cannot be scored for the expression status in the polyploid; (2) a band present in all lanes except the A parent, indicating that the maternal G_t copy in the polyploid is expressed; (3) a band present in all lanes except the G parent, indicating that the paternal A_t copy in the polyploid is expressed; (4) a band present in only the A and A + G lanes, suggesting that the A_t copy in the polyploid was silenced; and (5) a band present in only the G and A + G lanes, suggesting that the G_t copy in the polyploid was silenced. Class 4 and 5 bands are highlighted with bullets.

Figure 3.—

Sequencing chromatograms. Shown are sample regions of the chromatograms from RT-PCR products of gene Em2025 (monooxygenase) in the first column and gene Ep550 (a DHHC-type zinc finger protein) in the second column. Bases polymorphic between the A and G parents are marked with boxes.

Figure 4.—

Sequencing chromatograms. Shown are sample regions of the chromatograms from RT-PCR products of gene Em2025 (monooxygenase) from the A and D parents and the two AD allotetraploids. A base polymorphic between the A and D parents is marked with boxes.