Microscopy Study And Screening Of DNA Linked Fragments In Welsh Onion Male Sterile Lines

Welsh onion (Allium fistulosum L.2n=16) is an important health vegetable and mainly cultivated in East Asian countries. It is a cross-pollinated crop and has obvious heterosis, but its flower organ is small and not convenient emasculation. Therefore, the use of male sterile lines become a best system for the production of Welsh onion hybrids. Welsh onion hybrid seed breeding in China did not begin formally until Zhang et al. found the first male-sterile plant. Numerous studies showed that cytoplasmic male sterility in plants is associated with the mitochondrial genome. Now, there have been few studies on Welsh onion CMS. This study used four pairs Welsh onion maintainer and CMS lines as materials and analyzed in both cellular and molecular levels. The goal is to provide theoretical basis for understanding the CMS mechanism and to accelerate breeding for hybrid seed in Welsh onion. The main content are as followings:1. Cytological studies on male-sterile and maintainer linesAcetocarmine was used to stain anthers, as a result, average length, diameter and stem length of bud were obtained. They are respectively2.32,1.37and3.30mm. Based on this, the process of anther development was observed using paraffin section technology. The results showed that the tapetum of58A1degenerated in advance and therefore affect the meiosis of microspore mother cell; while the tapetum of58A2abnormally thickened in microspore stage and extruded microspores led to abortion.2. Molecular studies on male-sterile and maintainer linesWe proposed a rapid method for extracting Allium DNA based on alkali treatment. It is convenient, fast and suitable for field operation. Analysis of the storage time showed that DNA could be kept for5days at room temperature, at least two months at4℃or-20℃.We amplified eight mitochondrial genes from64A and64B by PCR. The results revealed that amplification products of seven primers have no difference between64A and64B, but one band of2561bp was occured in CMS64A using primer SCS13(L/R). Sequence results showed that37bp is homologous with the coding region of atp9gene and the identity is89%.In this study, mtDNA of nine Welsh onion were analyzed by AFLP method. From the survey of256AFLP primer combinations,65polymorphic bands were obtained. The dendrogran of AFLP showed that genetic relationship of64A and others was very far, it was related to geographical origin. Among them, four bands may be associated with CMS. After cloning, we only obtained k2which was of302bp.145bp homology with A.cepa coxl gene and the remaining157bp was unknown. It is consisted of100bp coxl coding sequence,40bp3’-untranslated region and5bp insertion that was a short repeated sequence. k2of AFLP could distinguish different cytoplasm types. Because of the complex technology, we tried to convert it into one simple SCAR marker. Then two primers were designed and obtained a target band1646bp, these suggested that k2was successfully converted to a SCAR marker, named SCAR1. Verification of other12lines showed SCAR1is stable and can be used to assist breeding.Southern blotting was conducted in nine Welsh onion by16(4X4) combinations of enzyme/probes. There were no hybridization results using probe rrn26. Other combinations showed that differences among the mt genomes of nine Welsh onion were detected. The differences in nad5locus were most obvious and Hind Ⅲ/nad5was suggested as a better technical system for the cytoplasmic classification of Welsh onion with RFLP mtDNA analysis. In addition, specific bands of4-6A and4-6B indicated enzyme sites of BamH I were different.