Selection Of New Carcinogens Cauliflower Germplasm Based On InDel Markers Linked To Major Glucosinolate Genes

199 populations derived from hybrid progeny of broccoli and the double haploid line of cauliflower, A5 and A82, and cauliflower were backcrossed for 3 generations continuely were used as tested materials. And the GSL-ALK and GSL-ELONG genes were detected by the developed marker. The results were as follows:1. Development of the ALK-InDel markerDesigning the primer of GSL-ALK gene at the region of deletion, one primer combination was selected from the primer combinations, which could produced qualified fragments. It was owned the GSL-ALK gene based on the sequencing, naming it was ALK-InDel marker. And the marker was tested with small population of Broccoli and cauliflower.2. Development of the ELONG-InDel markerDesigning the primer of GSL-ALK gene at the region of loss, two primer combinations were selected from the primer combinations. There was large mounts of amplified products, and the non-specific bands was not appear at P12f/P11r primer combination. Therefore, P12f/P11r is the best primer. Based on the sequencing we designed the primers, secondary screening showed that the best primer combination was P11/P15, which could produced qualified fragments, there wasn't non-specific bands and easy to found the difference of the bases. Sequenced the product of PCR displayed that this amplified fragment was owned the GSL-ELONG gene, designated it as ALK-InDel marker. And the marker was tested with small population of broccoli and cauliflower.3. Optimization of PCR reaction system of GSL-ALK geneIn this experiment single factor design with five levels of five factors (annealing temperature, dNTPs, primer, Taq DNA polymerase, concentration of template DNA) was used to optimize the PCR reaction system of GSL-ALK gene. The results showed that a better amplification of GSL-ALK gene was obtained with the reaction system containing dNTPs at 0.2mmol/L, primer at 0.6umol/L, Taq DNA polymerase at 0.1 uL, and DNA at 30ng in the total volume of 20 uL.4. Amplified results of GSL-ALK- gene of backcross populations In this experiment we obtained 56 out of 199 with the genotype of GSL-ALK-, the results showed that the traditional transferring methods also had the high transferring rate. 1281 out of 3889 individuals with the genotype of GSL-ALK-.5. Selection the GSL-ELONG+gene of GSL-ALK determined populations We obtained 4 populations which GSL-ELONG gene expresed the GSL-ELONG+ at the 56 determined populatins, selected 16 single plants at the 4 determined populations. There is 6 single plants expressed ALK-/ ELONG-.6. Selection the GSL-ELONG+gene at the undetermined GSL-ALK populations We selected 3 populations which expressing the GSL-ELONG+ genotype, and selected 22 single plants.7. The effect of different growth stages of plants on extracting DNAThis experiment showed that we can marker the GSL-ALK and GSL-ELONG gene in the whole growth period, and not influenced by natural conditions.