| Several markers linked to cytoplasmic fertility loci and restore genes were obtained with sterile line 209A and maintainer line and several cultivars as materials in welsh onion. The feasibility of their application in marker assisted selection was also discussed in this paper. The details were as follows:1. Markers linked to cytoplasmic fertility lociCpDNA and mtDNA from the sterile line 209A and maintainer line 209B were analyzed by RAPD technique with 220 random primers. Five fragments linked to cytoplasmic fertility loci, S132800, S38960, S72300, S73I100 and S2002400, were identified and only S72300 linked to fertile loci. The recombination rates between linkage fragment and cytoplasmic loci were 0, 7.5%, 0, 4.2%, and 0 respectively. However, only S132800 could be used to identify N and S cytoplasm in some cultivars and S2002400 could be used in all tested cultivars. Progeny test indicated the two linkage fragments could be stably inhereted, which was useful in marker assisted selection.Polymorphic bands S132800and S2002400were recovered, purified and then inserted into pGEM-T-easy vector, and transferred into E coli. DH5 a . According to the sequences, three pairs of 22-mer specific primers, SCS13L/SCS13R, SCS200L/SCS200R1 and SCS200L/SCS200R2 were designed. SCS13 could be used to identify N and S cytoplasm, and its optimal annealing temperature was 59. Unfortunately SCS200 could not reproduce the polymorphic S2002400. Furthermore, the frequencies of N and S cytoplasm were analyzed by SCSI3 and S2002400 in different cultivars, such as Zhangqiu dacong, Longyao dacong, Laiwujituicong, Hanguo dacong and Dagonghei. A procedure of marker assisted sterile line and maintainer line selection was also described in this paper.First, DNA fragment linked to cytoplasmic loci, assisted selection could be used to estimate primary plant number for selecting sterile and maintainer lines. Second, 20% to 80% descrease of crosses and selfcrosses for identifying a maintainer genotype could beachieved by the fragment linkage-assisted selection comparing to traditional selection method. Third, 1-2 breeding cycles could be reduced comparing to conventional breeding procedure. So this method could improve breeding efficiency and save labor, time, and cost if it's used in practice.MtDNA of sterile line and maintainer line were digested by EcoR I and Hind ni and electrophoresized on 0.8% agrose. Southern blot was conducted with 32P labeled SI32soo probe. The result showed several positive signals and polymorphism between the two lines.2. Markers linked to restore genesBased on the segregation ratio of 24 cross combinations, a hypothesis was proposed that male sterility was controlled by interaction between the cytoplasm and two main nuclear genes and polygenic system. Then a method to tag restore gene by BSA analysis was established with unknown genotypes and without restore line. Two pairs of bulked sterile and fertile pools were obtained in the populations with segregation ratio 1:3. Four RAPD markers, S21590, 569^, S8561g, 81022^2 linked to restore gene MSI were developed with recombination rates of 14.9%, 12.6%, 9.2%, and 8.0% respectively. Another marker, S3271061, linked with MS2 with a recombination rate of 6.7% was laso identified. Three polymorphic bands, S85618, S3271061 andS1022482, which showed lower recombination, were isolated and cloned into pGEM-T-easy vector and sequenced. Three pairs of SCAR primers were designed based on their sequences and SCR85L/SCR85R could not reproduce polymorphism between sterile and fertile plants. SCR327 combining with SCR1022 could identify individuals' genotype in cultivars Zhangqiu dacong, Longyao dacong, Riben 3, Laiwujituicong, et al.AFLP analysis and silver staining system of welsh onion genome were established., It revealed polymorphisms between sterile and fertile pools in welsh onion. However their linkage relations with restore genes were not established yet.Finally, the feasibility of applying the markers linked to main restore genes in marker assis...
|
PDF Full Text Request