| Stylosanthes guianensis which belongs to Leguminosae of Stylosanthes Sw is perennial plant.It's grass which has the widely using in tropical area of world and it's the main planting of legume grass in south of China.It uses for grazing,mashing and feeding,orchard intercropping and green manure production.In recent years,molecular biology has been developing.Molecular markers technology which doesn't depend on environment has been developing fastly.Molecular markers are effective measures for rapid breeding.With the development of molecular markers,there are some researches on germplasm resources identification,genetic map and plant classification.However,the researches of sterile gene in S.guianensis in ISSR,SRAP and SCAR are less.In this study,the S.guianensis 1979 was female.S.guianensis cv.Reyan No.5,S.guianensis cv.Reyan No.2,TPRC273 and TPRC90075 were recurrent males.They were called combination A,B,C,D.The experiment materials were their BC1F1 generation plants.With using BSA,we selected the markers which locked to pollen sterile gene in S.guianensis by ISSR.SRAP and SCAR molecular marker technology.The markers were calculated the genetic distance with sterile gene.Then the markers which were successfully confirmed were converted SCAR molecular marker.The study is good for follow-up research in hybrid breeding of S.guianensis.The main conclusions of this study are as follows:(1)This study extracted gene DNA which were sterile and fertile segregated populations of BC1F1 generation by the improved CTAB method.Then sterile and fertile gene pools were set up in the 4 BC1F1 generation.(2)The PCR amplification system of ISSR molecular marker was optimized.The system was 15?L.Among the system,template DNA was 75ng,ISSR primer was 0.66?mol/L,2×EasyTaq PCR SuperMix for PAGE was 7.5?L.(3)There were 33 primers which had polymorphism in sterile pools of 4 BC1F1 generation by 100 ISSR primers.In combination A,9 primers were selected and they were amplified 14 specific bands.By BC1F1 segregated populations confirming,there were 5 specific marks successfully.UBC807-950,UBC834-500 and UBC835-600 were recombinations and recombination rates were 3.33%,3.33%and 13.33%respectively.In combination B,11 primers were selected and they were amplified 15 specific bands.By BC1F1 segregated populations confirming,there were 12 specific marks successfully.UBC808-650,UBC816-600,UBC817-300,UBC817-500,UBC836-250,UBC840-200 and UBC881-220 were recombinations and recombination rates were 23.33%,20.00%,6.67%,13.33%,10.00%,3.33%and 3.33%respectively.In combination C,8 primers were selected and they were amplified 9 specific bands.By BCiFi segregated populations confirming,there were 8 specific marks successfully.UBC811-650 was recombination and recombination rate was 16.67%.In combination D,5 primers were selected and they were amplified 7 specific bands.By BC1F1 segregated populations confirming,there were 4 specific marks successfully.UBC841-700 and UBC853-550 were recombinations and recombination rates were 10.00%and 6.67%.(4)There were 38 primers which had polymorphism in sterile pools of 4 BC1F1 generation by 400 SRAP primers.In combination A,11 primers were selected and they were amplified 12 specific bands.By BCiFi segregated populations confirming,there were 7 specific marks successfully.M1E18-550,M14E20-150 and M14E20-500 were recombinations and recombination rates were 3.33%,10.00%and 6.67%respectively.In combination B,7 primers were selected and they were amplified 7 specific bands.By BC1F1 segregated populations confirming,there were 3 specific marks successfully.M11E8-200 was recombination and recombination rate was 6.67%.In combination C,7 primers were selected and they were amplified 8 specific bands.By BC1F1 segregated populations confirming,there were 4 specific marks successfully.M10E19-400 and M13E12-80 were recombinations and recombination rates were 6.67%and 10.00%.In combination D,13 primers were selected and they were amplified 13 specific bands.By BC1F1 segregated populations confirming,there were 8 specific marks successfully.M2E10-350,M5E10-300,M6E2-550 and M6E7-450 were recombinations and recombination rates were 3.33%,3.33%,6.67%and 6.67%respectively.(5)This study analysised the selected ISSR and SRAP markers and set up relation map which was locked to sterile gene.In combination A,12 markers were locked to sterile gene in S.guianensis.5 ISSR markers were UBC807-950(1.3cM),UBC834-500(1.4cM),UBC835-400(OcM),UBC835-600(8.3cM)and UBC835-700(2.0cM).7 SRAP markers were M1E18-550(1.4cM),M2E18-250(0cM),M4E1-180(0cM),M4E14-200(0cM),M9E16-600(0cM),M14E20-150(6.4cM)and M14E20-500(4.2cM).In combination B,15 markers were locked to sterile gene in S.guianensis.12 ISSR markers were UBC808-350(0cM),UBC816-400(0cM),UBC824-150(0cM),UBC836-400(0cM),UBC843-200(OcM),UBC808-650(14.8cM),UBC816-600(14.3cM),UBC817-300(3.6cM),UBC817-500(9.0cM),UBC836-250(6.3cM),UBC840-200(1.7cM)and UBC881-220(1.7cM).3 SRAP markers were M1E18-300(0cM),M11E8-200(3.2cM)and M14E13-650(OcM).In combination C,11 markers were locked to sterile gene in S.guianensis.8 ISSR markers were UBC807-400(0cM),UBC81 1-200(0cM),UBC820-350(OcM),UBC824-300(0cM),UBC834-200(0cM),UBC846-300(0cM),UBC853-450(OcM)and UBC811-650(11.2cM).3 SRAP markers were M10E19-400(3.5cM),M13E12-600(0cM)and M17E10-500(0cM).In combination D,12 markers were locked to sterile gene in S.guianensis.4 ISSR markers were UBC841-300(OcM),UBC853-400(0cM),UBC841-700(6.8cM)and UBC853-550(4.1cM).8 SRAP markers were M1E2-300(0cM),M4E1-150(OcM),M5E9-300(0cM),M15E16-350(OcM),M2E10-350(1.7cM),M5E10-300(1.4cM),M6E2-550(3.5cM)and M6E7-450(3.0cM).(6)The markers which had been selected were recycled and sequenced in the company.According to sequencing result,20 specific primers of ISSR-SCAR marker were designed.Combination A,B,C,D were designed 3,8,7,2 SCAR marker primers respectively.In SRAP-SCAR,20 specific primers of SRAP-SCAR marker were designed.Combination A,B,C,D were designed 6,3,3,8 SCAR marker primers respectively.Those markers were all comfirmed in their parents and BC1F1 segregated populations and had polymorphism successfully in BC1Fi sterile populations.The SCAR molecular markers which locked to sterile gene in S.guianensis were converted successfully. |
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