Studies On Genetic Diversity Of Dioscorea Alata L.

Dioscorea alata L., also known as Chinese yam or water yam; with a short lifecycle and high yield potential, having certain medicinal and nutritional value; In this study, The Agronomic traits of 133 germplasm of Dioscorea alata L collected from Hainan Yunnan Guangdong Guangxi and Fjian in China was registered and analysed; Method of modified CTAB for DNA Extraction from Dioscorea alata L. RAPD and ISSR molecular markers methods were applied to detect the genetic diversity and phylogenetic analysis relationship of 133 germplasms. The main results are as follows:1. Rresearch on classification of agronomic traits.12 agronomic traits of 133 germplasms has been analysed.SAS clustering results demonstrated these yam could be clustered into 6 groups at 1.2 in CEDS.The I group have opposite phyllotaxis and right-hand twist. The II group have left-hand twist. TheⅢ,Ⅳgroups have narrow leaf. The V group have green burgeon. TheⅥgroup have alternate phyllotaxis.2. Extraction of DNA:the improved method of CTAB (100mmol/LTris-HCl PH8.0,1.4mol/LNaCl,20mmol/LEDTAPH8.0,2%CTAB,3%PVP, add2%βME, lug/ml的RNase A in single way) was used to extract the DNA of species in Dioscorea alata L., and extracted with phenol/chloroform/isoamyl alcohol (25:24:1).Because the DNA by the method was suitable for RAPD and ISSR analysis.3. Optimization of RAPD-PCR Reaction System for Dioscorea alata L.and result analyse. RAPD-PCR was performed in a 20μL reaction mixture with 40ng/20μl DNA, 1.0μmol/L random primer,10μL2×Taq PCR MasterMix. The temperature profile used for RAPD-PCR was 94℃for 5 min;followed by 6 cycles of 94℃for 30s,32℃for 90s and 72℃for 90s;and followed by 6 cycles of 94℃for 30s,40-42℃for 90s and 72℃for 90s; and was terminated with a 10 min DNA extension step at 72℃;stored at 4℃.A total of 8 RAPD primers were selected from 100 primers, and 44 amplified sites were detected, of which 42 (95%) were polymorphic. UPGMA clustering results demonstrated that genetic similarity coefficient of 133 materials was ranged from 0.5612 to 0.9762, with the average of 0.687.We can get six genus from all the materials when the similarity coefficient is 0.665.4. Optimization of ISSR-PCR Reaction System for Dioscorea alata L.and result analyse. ISSR-PCR was performed in a 20μL reaction mixture with 60ng/20μl DNA, 1.0μmol/L primer,1U Taq polymerase,1.875mmol/L Mg2+,0.500mmol/L dNTP,.2μL 10×Buffer.The temperature profile used for ISSR-PCR was 94℃for 5 min;followed by 45 cycles of 94℃for 45s,48-52℃for 45s and 72℃for 90s;and was terminated with a 10 min DNA extension step at 72℃;stored at 4℃. A total of 10 ISSR primers were selected from 100 primers, and 91 amplified sites were detected, of which 85 (93%) were polymorphic. UPGMA clustering results demonstrated that genetic similarity coefficient of 133 materials was ranged from0.6223 to0.9315, with the average of 0.674.We can get seven genus from all the materials when the similarity coefficient is 0.665.