Studies By Genetic Markers And RT-PCR Of CMS In Oilseed Rape (Brassica Napus L.)

Rapeseed is a kind of important oil crop.Heterosis utilization,ultra-high yield,and strong portfolio in rapeseed have arose strong attention from governments and breeder all around the world.At present,most varieties of rapeseed hybrid in China utilize the systerm of CMS.So breeding of new cytoplasmic male-sterile lines in Brassica napus is significant to the production development of rapeseed hybrid in China.In this research,comparative analysis on the floral organ morphology,esterase isozyme,EST-SSR,RAPD and RT-PCR of CMS-related gene in important Brassica napus(Xinl,Ogu,Polima,Shan 2A CMS,maintainer lines, restorer lines and major domestic conventional varieties).It is significant for breeding more high-yield,high-quality hybrid rape by providing theoretical guidance.The main results are as follow:1.It is of great significance for hybrid rapeseed production by observation flower patterns on the period of podding flowering and comparation different male sterile lines with maintainer lines.The stamens of Xinl,Ogu and Shan2A CMS were significantly shorter than the stigma long,filigree not elongation or even slightly longer anther atrophy,no trace of pollen or pollen,show clearly not fertility.Xinl which show significant shrinkage petals,six stamens incomplete or heart of the phenomenon of skin,dry pollen sac hollow,performance for the flower patterns on the ideal of sterile materials.The flower pattern of Pol is similar with the B355,showed hollow-dry,a small amount of pollen,but far less than the pollen of B355.Calyx width of the materials is the only invariable character.2.By means of vertical slab polyacrylamide gel electrophoresid,Esterase Isozyme in leaves of Pol,Shan2A,Ogu and Xin CMS were analyzed.Results showed there are 2 bands of Esterase Isozyme in 4 cytoplasmic male-sterile lines and 1 band in maintainer line.Peroxidase Isozyme pattern differences existed not only between 4 cytoplasmic male-sterile lines and their maintainer line,but also among 4 cytoplasmic male-sterile lines.There are 3 and 5 bands of Peroxidase Isozyme in maintainer and Ogu respectively.4 bands exist in Pol,Shan2A and Xin1.The maintainer missed a band whose moving rate is 0.67.There is a special band of Peroxidase Isozyme in Ogu.But the Peroxidase Isozyme pattern of Pol is identical to Shan2A. 3.35 Brassica napus including 4 sterile lines,3 maintainer lines,10restorer lines and 11 conventional varieties were analyzed by EST-SSR and RAPD markers for the genetic diversity and heterosis.In the result,399 alleles were checked among the 35 varieties,of which 130 alleles accounting for 32.57%showed polymorphic.The range of the 35 varieties'genetic distance was between 0.0280-0.2801 computed by NTSYS(software).On level of genetic distance 0.1401,the varieties were divided into 6 groups.Ogu,Polima and Shan 2A CMS were clustered into the fifth groups.Xinl has further Genetic distance.There were more genetic diversity among sterile lines,restorer lines and conventional varieties than between sterile lines.It was approximately in accordance with their diversity of genetic basis and pedigree sources.4.Six pairs of specific primers are designed in terms of the CMS-related genes orf224, orf222 and orf138 in rapeseed.The buds of the total RNA of Pol,Shan2A,Ogu,Xin CMS and B355 are detected by RT-PCR.The orf138 has obvious polymorphism.Pol and Shan2A are amplified to a 160 bp band,Xinl amplified a 300 bp band,Ogu with a specific about 380 bp band,and B355 with the band of 200 bp.The results showed the polymorphi of CMS-related genes of mitochondria on the RNA level.