Construction and Analysis of Suppression Subtractive cDNA Library from Fertile Disk Floret Buds of Zinnia elegans

Abstract

In order to isolate genes related to anther development and understand molecular basis of male sterility, cDNA library of Zinnia elegans was constructed using suppression subtractive hybridization (SSH) approach. 672 different expressed clones were selected from the fertile disk floret buds. PCR results showed that cDNA inserts were ranged from 100 to 750 bp. 303 positive clones screened by dot-blot hybridization were sequenced. 273 out of 303 sequenced clones produced readable sequences; these sequences represent 87 non-repetitive sequences. The homology alignment showed that 76 expressed sequence tags (ESTs) had functional annotations in GenBank, the other 11 ESTs without any homology to the known gene. In addition, 87 ESTs were divided into 17 groups according to MIPS of Arabidopsis thaliana database. Sequence data from the cDNA library have been deposited with the GenBank under the accession numbers GT067016-GT067085. As an important result in this study, 7 genes related to anther development were isolated. Results from semi-quantitative RT-PCR showed 6 genes were expressed only in disk florets of fertile plants compared with that of male sterile plants. These ESTs obtained provide important clues for further isolation and identification of fertility-related genes in Z. elegans.

Keywords

• Zinnia elegans
• suppression subtractive hybridization (SSH)
• male sterility
• anther development
• expressed sequence tags (ESTs)