Microdissection Of Passiflora Edulis Sims Chromosome And Construction Of Single Chromosome DNA Library

Passiflora edulis Sims is a member of the family Passifloraeae, and one of the most important tropical and subtropical fruit trees. Current studies a re largely centered on cultivation and introduction, fruit processing, evolution and so on. Researches about genetic background of Passion flower are few and the breeding work only can launch through the traditional way with low efficiency. Lack of high-density genetic linkage map isn't favorable to the development of Passion flower molecular breeding. Passion flower single chromosome can be microdissected and microcloned, then build DNA library. Specific markers can be screened in the library, boosting the genetic and physical mapping. The paper using P. edulis seed deals mainly with seed germination, karyotype of P. edulis, microdissection and microcloning the satellite chromosome of P.edulis. A system of constructing integrated DNA library was established for P. edulis satellite chromosome of which specific markers were screened by AFLP. The major results obtained from the study are summarized as follows:1. Effects of soaking time and illumination on the germination of P. edulis seedsStudies were carried out on seed soaking of P.edulis and the results showed that appropriate seed soaking can improve seed vigor and germination. Soaking in H₂O within 3 days could improve seed germination, but more than 3 days reduced seed germination. Soaking for 1 day was the best one. The germination rate of P.edulis seed declined with lengthening of soaking time. The dark environment was advantageous to the germination.2. Effects of various pretreatments on the conformation of P. edulis chromosomesThe results showed that the best treatment was 8-Hydroxyquinoline for 2.5 h. The chromosomes dispersed well, centromeric dots and satellite were clear.3. Karyotype of P. edulisThe results of karyotype of P. edulis indicated that the chromosome number was 18, the 1st and 8th chromosome were submetacentric chromosomes, the others were metacentric chromosomes, and the 4th chromosome had satellite. The karyotype formula was 2n=2x=18=14m(2sat) +4sm.4. Microdissection and microcloning of P. edulis satellite chromosomeP.edulis satellite chromosome was isolated by glass needles, and the chromosomal DNA was amplified by LA-PCR. The PCR products became smear ranging from 210 to 1100 bp. Southern blot revealed that the PCR products were homogeneous with the P. edulis genomic DNA, indicating that DNAs from the satellite chromosome had been successfully amplified. The PCR products were cloned into pUCM-T vector and about 1.17×10⁵ recombinant clones were obtained. The size of the inserted fragments of clones ranged from 300 to 2000 bp.5. Screening specific markers by AFLP in satellite chromosome of P. edullsAFLP analysis was carried out on the chromosomes and there were obvious polymorphism among different chromosomes. Three close fragments of two satellite chromosomes could be amplified steadily by a pair of primers(H+L).