Chromosome Karyotype Analysis And Microseparation Microdissection And Microcloning Of Single Chromosome In Sunflower

Sunflower (Helianthus annuus L.) is one of the important oil crops. Its karyotype analysis and microdissection, microclone of the single chromosome are favorable for establishing high-density genetic map and cloning its key genes. In this paper, the triple hybrid and single cross hybrid of"Inner Mongolia Hibrid oil sunflower 3"was used, the karyotype was analysed, the single chromosome was isolated and amplificated, and the single chromosome DNA library was constructed. The major results as follows:1. The root tip was treated with 0.05% colchicine, 0.002mol.L-1 8-hydroxyquino- line and 4℃low tempreture, the results showed that the best treatment was 8-hydroxyquinoline 5h and colchicine 4.5h, the chromosome dispersed well, and centromeric dots were clear.2. The results of karyotype anylysis indicated that the total numbers of the triple hybrid and single cross hybrid chromosomes were 34, and each had a pair of satellite chrosome. The triple hybrid chrosomes were composed of 4 submetaxentric chromosome and 30 metacentric chromosomes. The relative length ranged from 4.092% to 7.729%, and the karyotype formula was 2n=2x=34=30m+4sm(2sat). The single cross hybrid chrosomes were composed of 34 metacentric chromosomes. The relative length ranged from3.661% to 8.128%, and the karyotype formula is 2n=2x=34=34m (2sat).3. The satellite chromosome of triple hybrid and single cross hybrid sunflower were isolated by glass needles, and the chromosomal DNA was amplified by LA?PCR. The PCR products were a smear which ranged from 250 to 1500bp. The result of southern blot revealed that the PCR products were homogeneous with the sunflower genomic DNA, so DNAs from the satellite chromosome had been successfully amplified.4. The PCR products of satellite chromosome in triple hybrid and single cross hybrid sunflower were cloned into pGEM?T?Easy vector, and their DNA library was constructed. 2.26×105 recombinant clones were obtained from the triple hybrid sunflower. They were through PCR examination and restriction enzyme digestion, and the results demonstrated that the inserted fragments were approximately from 200-700bp, the average size was 535bp. 2.57×105 recombinant clones were obtained from the single cross hybrid. They were through PCR examination and restriction enzyme digestion, and the results showed that the inserted fragments were approximately from 200-500bp, the average size was 480bp.