Studies On 1575A Of Cytoplasmic Male Sterile Lines In Brassica Napus L.

Cytoplasmic male sterility (CMS) is a main pollination control system for oilseed rape hybrids production in the application of heterosis. At present, only a few kinds of CMS lines can be used in oilseed rape breeding, and the use of a single cytoplasm for a long time hinders the development of rape hybrids. Therefore exploiting and establishing new CMS lines are of importance to oilseed rape hybrids production. The Brassica napus 1575A is a new CMS line developed by the oilseed rape breeding team of Northwest Agriculture and Forestry University, whose sterility is very stable by test cross experiments for years. Almost all kinds of Brassica napus can be used as 1575A's maintainer line, but it's hard to find the restorer line. This feature is similar to ogu CMS. While the most obvious difference between the two CMS lines is that seedlings of 1575A will grow well and have normal nectaries instead of becoming yellow when encountering low temperature. In order to carry on preliminary study and further identify 1575A in the future, a series of studies such as morphological observation, determination of restoring and maintaining relationship and molecular identification were conducted. The main results were described as follows:1. Petals of 1575A are quite smooth with wide and large surface. The stamen degenerates, and the pistil appears externally. In short, flowers of 1575A are quite different from CMS lines ogu CMS and nap CMS. Different maintainer lines have different maintaining effect, and the effect mainly has four types. The first kind is the stamen degenerates completely; the second one is that petals are big and round and stamens are short hidden in the petals; the third one compared to the second one is that petals are narrow and long; finally the flower is close to normal ones, but no pollens. In addition, heart leaves won't become yellow when encountering low temperature, and the nectary grows well with bottle green color compared to ogu CMS.2.41 species of Brassica napus were hybridized with 1575A and Shaan 3A(Belong to shan 2A),respectively. The test-cross results showed that 41 materials were all 1575A's maintainer lines, in which 14 materials could restore sterility of Shaan 3A, and 27 materials could be used as the maintainer line. This point indicated that 1575A and Shaan 3A were different in restoring and maintaining relationship, not belonging to Shaan 2A series. In addition, a restorer line of 1575A was found after hybridizing with 168 test-cross materials (including Brassica napus, Brassica juncea, Brassica related species) in 2010.3. Primers were designed according to the reported fertility-related gene orf224, orf138 and orf222 and the mtDNA amplification of 4 CMS lines (including 1575A, Shaan 2A, ogu CMS and nap CMS) of Brassica napus were carried out by PCR. It proved that mtDNA of 1575A contained homologous sequences of both genes orf138 and orf222, and the specific fragment and the sequence of gene orf138 (Accession numbers:AB055435) and orf222(Acce- ssion numbers:DQ872162) reached 100% in homology.4. Gene orf138 and orf222 were both cloned in Brassica napus 1575A by semi-quantita- tive RT-PCR. The result showed that there was no difference in gene expression of orf138 between 1575A and ogu CMS; gene expression of orf222 in 1575A was lower than that in nap CMS, suggesting that fertility of 1575A was mainly affected by gene orf138, and might be regulated by gene orf222. In addition, gene orf138 and orf222's expression in 1575A and ogu CMS were detected during different developmental stages, and the quantitative analysis showed that there was no difference in gene orf138's expression in ogu CMS during different stages, while in 1575A the expression was lower in etiolated seedlings than that in green seedlings and buds. It suggested that sterility of ogu CMS had nothing to do with the phenomenon of chlorosis, and restoring of 1575A's iron deficiency chlorosis might be associated with nap CMS cytoplasm's infiltration.