In order to find out a new pathway for utilizing heterosis of Zinnia elegans and accelerate breeding process, the mechanism of anther development of a male sterile line was explored. Backcross, sibmating, selfing of fertile plants and testcross with inbred lines were analyzed and identified in the field, and cytology was observed. Recessive nucleus male sterile line AH209AB capable of being a maintainer was obtained by successive backcrosses with male sterile plants and fertile F1 plants as male parents. Cytological and anatomical studies indicated that: (1) The wall of normal anther was constituted of four layers of cells such as epidermis, powder chamber wall, middle level and tapetum cells. The process in meiosis of pollen mother cell in Zinnia elegans was normal and cytoplasm divided simultanously. Mature pollen grain was tricellular type. (2) The petal of male sterile plant degraded as a thread-like structure, the stamens were villiform in appearance and no pollens were formed. The result showed that the anther of male sterile plant no longer proceed to differentiate spore mother cell and the pollen sac after the formation of the tissue of sporogenous cells, there was no evident boundary between tapetum cell, middle lamella and inner wall of PMC, tapetal cells did not develop from the very beginning. So the abortion type was completely structural male sterility. The male sterile line belongs to non-sporange male sterile type and is of great use in F1 seeds production.
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.
PANG Rui-huaMA Guang-yingLOU Xue-yuanBAO Man-zhuYE Yao-mei
