Molecular Marker And Its Application In Seedling Stage For Male Sterile Ab Lines Of Marigold(Tagetes Erecta L.)

In this article, marigold male sterile AB lines(provided by Plateau Flower Research Center of Qinghai University) were used as material, and 3 molecular maker methods(RAPD, ISSR, SRAP) were used to screen the male sterile related gene fragments of marigold, then these gene fragments were transformed into stable SCAR markers, and SCAR markers were used to identify the gender of seedlings of marigold AB lines. The main results are as follows:(1) sterile plant DNA of marigold male sterile AB lines 2-2 was used as experimental material, combined with single factor design and L16(45) orthogonal design, we establish and optimized the marigold male sterile lines of RAPD-PCR amplification system. Using 200 RAPD primers to amplify the fertile and sterile gene pool of 2-2 AB lines, we did not screened out the primer which behaved differently in the AB lines.(2) With 20 ISSR primers to amplify the fertile and sterile gene pool of 2-2 AB lines, we screened out 3 ISSR primers that behaved differently in fertile and sterile lines, that is, I1, I7, I15.(3) We used 2-2 sterile plant DNA of marigold male sterile AB lines as experimental material, with single factor design and orthogonal design L16(45), we optimized the 5 parameters which may impact SRAP-PCR reaction system, then the annealing temperature was optimized too. As a result, we got the best SRAP-PCR reaction system and amplification procedure. With the fertile and sterile flower organ DNA of 2-2 AB lines, we conducted SRAP-PCR amplification, and we screened out 4 different primer combinations that can amplify different bands between fertile and sterile lines, and they were me1+em5、me2+em18、me5+em1、me5+em11.(4) Recovery, transformation and sequencing of the specific fragments screened out by above mentioned primers, which included 6 ISSR specific fragments, 4 SRAP specific fragments. By sequencing, the sequences of 5 ISSR specific fragments were obtained, and the sequences of the 4 SRAP specific fragments were obtained too. Based on the gene fragments obtained, 17 pairs of SCAR primers(F1R1-F17R17) were designed. Only one pair of primers(F13R13) could amplify the difference band between fertile and sterile plants, it was absence in sterile, but appeared in fertile. We used F13R13 to test the fertility of 2-2 AB lines, the difference band was absence in four sterile plants when used flower organ as material. When 24 leaf DNA of the 2-2 AB lines were amplified with primers F13R13, the difference band appeared in one of the 24 male sterile plants, and the accuracy was 95.8%, so F13R13 could be used as a marker for detection.