Genetic Diversity And Relationship Of Tea Cultivars In Sichuan Revealed By SSR And ISSR Markers

Southwest of China is the origin of tea plant(Camellia sinensis(L.)O.Kuntze),which has rich of tea germplasms.It provides rich resources for the application of new tea cultivars.SSR(Simple Sequence Repeat) and ISSR(Inter-Simple Sequence Repeat),as newly developed molecular marker techniques,are powerful methods for bio-diversity research.They have been popularized for their rapidity,simplicity and capacity of revealing the genetic diversity in DNA level.Genetic diversity and relationship of 34 tea cultivated species and 3 tea wild species Cultivars including those origins in Sichuan and introduced from Fujian,Taiwan,Zhejiang,Guizhou,Hunan,Guangdong,Anhui and Hainan into Sichuan tea areas,as materials,were revealed by SSR and ISSR Markers.The result as follows:1.The proper SSR-PCR reaction system was determined as follows:25μL reaction system containing 15ng template DNA,12.5ul 2×Master Mix,0.5μmol/L Upstream or downstream primer,9ul ddH₂O.The proper amplification was after 1 cycle initial denaturation at 94℃for 5min,followed by 41 cycles of 1min at 94℃,30s annealing at 52℃or 60℃,and extension of 90s at 72℃,and a final 7min extension at 72℃.The proper ISSR-PCR reaction system was determined as follows:25μL reaction system containing 1×PCR buffer,2.0mmol/L MgCl₂,0.24mmol/L dNTPs,0.6μmol/L primer,1.75U Taq DNA polymerase,20ng template DNA.The proper amplification was after 1 cycle initial denaturation at 94℃for 5min,followed by 41 cycles of 1min at 94℃, 1 min annealing at 52℃or 60℃,and extension of 1min at 72℃,and a final 7min extension at 72℃.2.8 SSR primers selected from 21 SSR primers were used to amplify 93 DNA bands(93%)were polymorphic.The average number of polymorphic DNA bands,the percentage of polymorphic bands(%),PIC amplified by SSR primer were 11.63,92.30,0.91,the average specific bands were 22.38.Each tea cultivar could be amplified 26 to 45 polymorphic DNA bands,with an average of 36.41,the average percentage of polymorphic bands(%) was 83.68.The genetic similarity coefficient between the cultivars varied from 0.490 to 0.880,with an average of 0.699.The SSR results indicated that there was abundant genetic diversity among the tested materials.14 ISSR primers selected from 89 ISSR primers were used to amplify 146 DNA bands(94.81%)were polymorphic.The average number of polymorphic DNA bands,the percentage of polymorphic bands(%),PIC amplified by ISSR primer were 10.43,95.19,0.89,the average specific bands were 13.93.Each tea cultivar could be amplified 37 to76 polymorphic DNA bands,with an average of 57.97,the average percentage of polymorphic bands(%) was 87.63.The genetic similarity coefficient between the cultivars varied from 0.519 to 0.851,with an average of 0.689.The ISSR results indicated that there was abundant genetic diversity among the tested materials. 3.Based on SSR,at similarity coefficient of 0.676,all the tested materials could be clustered into 2 complex groups and 3 simple groups.It was found that there was a certain correlation between the results and the derivation,productive characters of tea cultivars.Most oolong tea cultivars were clustered into a group,some black,green tea were clustered into a group separately too.Based on ISSR,at similarity coefficient of 0.658, all the tested materials could be clustered into 2 complex groups and 1 simple groups.The ISSR results were similar with SSR.4.By the amplication products from genetic DNA of SSR8 and ISSR12 obtained in the study,all the tested materials could be identified.The results indicated that SSR and ISSR were useful tools for molecular identification of tea identification.