Construction Of Single Satellite Chromosome DNA Library Of Zhangzhou Narcissus And Study On Its AFLP Markers

Zhangzhou Narcissus,known as the main variation of French narcissus, is one of perennial bulbous flowers which belong to the Narcissus of Amaryllidaceae. Narcissus tazetta var. chinensis is not only one of the ten Chinese traditional flowers, but also a famed traditional export flower. But recently, the problem of narcissus production in China is more and more serious, single variety, the plant diseases and insect pests, reduce the yield and the quality and so on. So the fundamental research of narcissus breeding is necessary. It can lay a solid foundation of breeding new varieties. For a long time, the sdudy on the origin of chromosomes of Zhangzhou Narcissus is not clear. And the study was limitited on cytology level, less on molecule level. The author holds that the research on three satellite chromosomes will promote the research on the origin of chromosomes of Zhangzhou Narcissus and breeding new varieties.Taking Zhangzhou Narcissus for material, this paper explored the number of satellite chromosomes of Zhangzhou Narcissus, constructed of two single chromosomes (7st) of Zhangzhou Narcissus DNA library by microdissection and microcloning technology, and established molecular markers of the two chromosomes by AFLP molecular markers technology.1. Satellite chromosomes of Zhangzhou NarcissusThe results indicated that Zhangzhou Narcissus was triploid (2n=3x=30), The three chromosomes of the 7st all had satellites but different morphology. One of satellites was too short to identify, when the time of pretreatment to chromosomes was too long.2. Construction of single chromosome DNA library of Zhangzhou NarcissusTwo of Zhangzhou Narcissus satellites chromosomes were isolated by glass needles, and the chromosomal DNAs were amplified by LA?PCR. The PCR products became smear ranging from 200 to 2500bp. Southern blot revealed that the PCR products were homogeneous with the Zhangzhou Narcissus genomic DNA, indicating that DNAs from the satellite chromosome had been successfully amplified. The PCR products were cloned into PMD18-T vector and about 5.0×10~5 recombinant clones and 3.7×10~5 recombinant clones were obtained. The size of the inserted fragments of clones ranged from 300 to 2000 bp by digestion with enzymes of EcoRⅠand HindШ.3. AFLP marker of two chromosomes LA-PCR technology was combined with single enzyme AFLP technology , primer was designed based on LA-PCR adapter sequence. Molecular markers of the two chromosomes were established through denatured polyacrylamide gel electrophoresis and silver staining technique. The result of molecular markers analyse was that the rate of the same bands was 58.8% and obtained preliminary the two satellite chromosomes was not homologous chromosome.