| Chinese cabbage (B.campestris L. ssp. pekinensis) originated in China, which is the most important leafy vegetable with widely growing cultivation area, many types and rich genetic resources in China. Utilization of male sterile lines is becoming development trend for Chinese cabbage hybrid breeding because of its higher heterosis. Cytoplasmic male sterility (CMS) is an important and valuable genetic phenomena. CMS is useful in Chinese cabbage breeding in which leafy-head is an edible organ. Much effort has been made in recent years to put several CMS types of Chinese cabbage into practice in China, such as Ogu CMS, Pol CMS and CMS96. However, only CMS96 has been used successfully because of its many merits. The male sterile degree and percent of sterile plants in CMS96 are 100%. It is not degenerate by multi-backcrossing generations. It owns normal nectar. It has higher combination ability and so on. In this research, molecular biology, isozymes or protein, botany, transmission electron microscopy (TEM) and scanning electron microscopy techniques are used for 3 sets of 11 Chinese cabbage varieties with the same nuclear and different Pol CMS, Ogu CMS, CMS96, maintainer cytoplasm in order to study male sterile mechanism, especially for the new and valuable CMS96 type. Main conclusions are listed as follows:1. RAPD and AFLP techniques were used in nDNA, 11 Chinese cabbage CMS and maintainer mtDNAs in order to find fragments linked to different CMS types. Cluster analysis by AFLP and RAPD results showed that Ogu CMS and CMS96 had greater genetic similarity.Out of 153 RAPD primers, 20 fragments linked to Ogu CMS and CMS96 could be obtained by OPU04, OPO04, OPAH16 and OPB20 primers. 3 fragments linked to Ogu CMS could be obtained by OPB06 primer. Meanwhile the 1300bp fragments linked to Ogu CMS and CMS96 by OPAH16 primer were converted to SCAR-1300. 900bp fragments linked to all materials by OPAH16 primer were converted to SCAR-900. Out of 64 AFLP primer combinations, 6 fragments linked to CMS96 could be obtained by E-ACC/M-CAT 500, E-ACG/M-CTG 650 and E-ACT/M-CTT 370.6 fragments linked to Ogu CMS could be obtained by E-ACG/M-CAT 540 and E-ACT/M-CAT 610. 22 fragments linked to Ogu CMS and CMS96 could be obtained by 5 primers. 26 fragments linked to Ogu CMS, Pol CMS and CMS96 could be obtained by 5 primers.2. Using homology-based candidate gene method, PCR products could be amplified by 7 primers in 11 Chinese cabbage CMS and maintainer mtDNAs. Results showed that 3 CMS types and their corresponding maintainers in Chinese cabbage could be distinguished by atp6, or/224 and or/222 primers using PCR technique and they also could be distinguished by atp6 and orf222 primer combinations using muti-PCR technique. The mitochondrial chimeric gene in CMS96 was composed by Ogu CMS orfl38 and Nap CMS orf222/nad5c in Brassica napus. PCR products could be amplified by 7 primers in 11 Chinese cabbage CMS and maintainer mtDNAs. Except atp9 and cox I primers, different fragments could be obtained in 11 Chinese cabbage materials by other 5 primers. RT-PCR technique was used for further confirming these fragments linked to CMS types. Then these fragments were cloned and sequenced. After compared in NCBI website, their characteristics were studied. 200bp specific fragments could only be amplified in all Ogu CMS by atp6 primer. Specific fragments could only be amplified in all Pol CMS by orf224 primer. Ogu CMS and CMS96 could be distinguished from others by products of atpA and orf138 primers. Fragments could only be amplified by orf222 primer in Pol CMS and CMS96. But there were 3 differences in Pol CMS and CMS96 by products of orf222 primer. Nucleotide acids of the fragments in Pol CMS and CMS96 were different. The former was 675bp, and the latter was 669bp. ORFs and putative conserved domains were different. 675bp fragments owned ORF224 without putative conserved domain. 669bp fragments owned ORF222 with YMF 19 putative conserved domain. 669bp fragments in CMS96 were 100% homological to orf222/nad5c in Brassica Nap CMS. Using orf138 and orf222 primer combinations, 3 specific fragments could be amplified in CMS96 types.By muti-PCR technique using atp6 and orf222 primer combinations, a new and rapid technique for distinguishing 3 CMS types and maintainers in Chinese cabbage was founded with only one PCR reaction. In this method, 800bp fragments could be named as difference of maintainer. 1500bp and 2300bp fragments were specific for Pol CMS type which were produced by atp6 and orf222 primer combinations. 200bp fragments were specific for Ogu CMS type. 700bp fragments were specific for CMS96 type.3. Isoelectric focusing (IEF) electrophoresis technique was used in 2 sets of 8 Chinese cabbage CMS types and maintainers for EST, ACP, POD and total protein analysis in flowers, different buds, leaves in order to study differences between CMS and maintainer. Results showed POD, ACP isozyme and total protein between CMS and maintainer were different except EST isozyme. For EST isozyme, there was no difference between 8 Chinese cabbage materials in flowers, buds and leaves. EST bands in all materials were about 10～11.For POD isozyme, the amounts of POD isozyme in 3 CMS types and maintainers were different. C bands in 3 CMS types were more active than those of maintainer. Then C bands of Ogu CMS and CMS96 were more active than those of Pol CMS. Meanwhile, C bands of all CMS types in the length of buds more than 0.45cm were more active than those of buds less than 0.45cm. There were significant differences of POD isozyme in 2 CMS96 materials. In No. 2 set, B bands in CMS96 were significantly active than those of CMS96 in No. 3 set, and they were more active than those of other materials in same set. CMS96 in No. 2 set had A bands which were absent in other materials, but existed in No. 3 set. In No. 3 set, the amounts of POD isozyme were similar.For ACP isozyme, the amounts of ACP isozyme in 3 CMS types and maintainers were similar, but flowers of Ogu CMS had active A and B bands which differed from others. The length of buds more than 0.45cm had active A and B bands in Ogu CMS and CMS96 which differed from others. The length of buds more than 0.45cm in maintainers had active C and D bands which differed from others. For the length of buds less than 0.45cm, there was no difference among all the materials, and the amounts of ACP isozyme were 5～6. For total protein, the length of buds from 0.35 to 0.55cm in maintainers had active A bands which differed from others. The amounts of total protein were similar in other materials.4. Using transmission electron microscopy and scanning electron microscopy techniques, anther and pollen developmemt, surface of anther and pollen grains were studied in order to compare differences between CMS with maintainer. Preliminary results showed that the main sterile stage and characteristics of CMS96 occurred at the early pollen mother cell (PMC) stage. Middle layer and tapetal cells were separate early. PMC could normally perform meiosis, but there were many larger gaps between them. PMC showed abnormal shapes. CMS96 could form pollen grains with pollen vein and structural exine, but the compositions inner the pollen grains were empty. The abnormal pollen grains adhered to the inner anther without dispersing.5. To enlarge the usage of CMS96 in Chinese cabbage breeding, CMS96 lines and CMS96 hybrids were compared with maintainer and maintainer hybrids in flowery organ and mainly biological characteristics. It proved that CMS96 was stable in male-sterility. CMS96 was not degenerate by muti-backcrossing generations. And CMS96 had higher combination ability with 100% crossing rate. It further proved that CMS96 and its hybrids had potential and wide value. 31 stable CMS96 lines in Chinese cabbage were obtained by muti-backcrossing generations with different maintainers. 4 CMS96 hybrids 03-10, 04-2, 05-4 and 06-5 were propagated. The procedure of producing CMS96 hybrids seeds was drafted. Sowing rate of CMS96 and father line was 3 to 1. CMS96 is the best CMS type in Chinese cabbage now.
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