Study On Pollenless Traits Of Asiatic Hybrid Lily By SRAP Markers

Due to the serious problem of pollen pollution in lily, cultivating less pollen or pollenless lily varieties has become one of the main direction of lily breeding in the world. In this study, the F1 individuals from the cross of Asiatic hybrid lily ‘H08-03’(♀, pollenless) × ‘Pollyanna’(♂, pollen) were used as materials. Firstly, the fertility inheritance of the materials was discussed; then, the pollenless and pollen pools were mixed respectively according to the BSA method; finally the SRAP molecular markers linkaged with the pollenless trait in Asiatic hybrid lily were screened. The main results are as follows:1. The establishment of SRAP-PCR reaction system on Asiatic hybrid lily. By means of orthogonal intuitive analysis, it showed that the concentration of Mg2+, Taq DNA polymerase, DNA template had great influence on the reaction of PCR. After that, the above 3 factors were analyzed with single-factor design. The results showed that the best reaction system was the combination in 20 μL reaction solution, containing DNA template 50 ng, 1 × PCR Buffer, 2.8 mmol/L Mg2+, 0.5 mmol/L dNTP, 0.7 μmol/L primers, 1 U Taq DNA polymerase and ddH2 O to 20μL. The best amplification procedure was as follows: 94℃ for 5 min; 94℃ for1 min, 35℃ for 1 min, 72℃ for 1.5 min for the 5 recycles; 94℃ for 1 min, 54℃ for 1 min, 72℃ for 1.5 min for 30 recycles, with a finally extension at 72℃ for 8 min.2. Genetic analysis of pollenless trait. By conducting compatibility ?2 test towards the population of pollenless and pollen segregation progenies of ‘H08-03’ × ‘Pollyanna’ and ‘Golden- Horn’ × ‘Pollyanna’. It confirmed that the segregation ratio was in accordance with mendel’s laws of inheritance. In view of the two hybrid groups share the same male parent, it presumably infered the pollenless trait is a quality traits controlled by one recessive gene.3. SRAP markers linkaged with the pollenless trait. With the optimized SRAP-PCR reaction system, 108 pairs of primers were firstly screened with effective amplification out of 272 combinations, accounting for about 40%. Then, 27 primer combinations gave polymorphic amplification patterns, accounting for 25%. After amplificating the 27 primer combinations between the pollenless and pollen gene pools, 3 SRAP markers linkaged with the pollenless trait were acquired, namely EM2/ME3、EM2/ME9 and EM17/ME14. On the basis of testing individual plant in F1 population, the 3 markers were all amplified with differential fragments beween the pollenless and pollen segregation progenies. The length of the differential fragments are respectively about 350bp、200bp、280bp, named as E2M3-350、E2M9-200 and E17M14-280. The study confirmed that the 3 markers can be used for molecular assisted breeding and the early prediction about pollenless germplasm resources of Asiatic hybrid lily.4. Cloning and sequencing of the specific fragment. The E2M3-350, the length of which is 376 bp, was successfully acquired. The results of BLAST showed that there was no similar squence with this squence in the GeneBank.