Analysis Of High Catechin Tea Plant Germplasm Resources By RAPD And Cloning Of Special Molecular Markers

It has been proved that China is the origin of tea plant(Camellia sinensis(L.)O.Kuntze).There are rich and various tea plant germplasm resources,which provide a good material condition for tea production and breeding.In this study,RAPD was used to analyze a total of 50 high catechin tea plant germplasms from Fuzhou tea germplasm nursery and Zhaoan tea germplasm nursery,which was collected and bred by our laboratory,including genetic diversity analysis,cluster analysis,and molecular identification of 32 tea plant germplasms according to specific amplified and missing bands by 6 random primers.One DNA fragment of Nad1 gene was cloned by analyzing specific bands between five high catechin tea plant germplasms and two low catechin tea plant germplasms,which was associated with catechins synthesis possibly.The results were as follows:1.The traditional CTAB method was improved,and a simple and efficient tea plant genomic DNA extraction and purification was tracked out.After examination,the OD260/OD2g0 value of the extracted and purified DNA ranged from 1.80 to 2.00,and there was almost no impurity.2.A set of best amplified reaction system and amplified procedure for RAPD-PCR in the study were tracked out through optimizing 5 main factors that affected PCR reaction,including the dosage of DNA template and Taq DNA polymerase,and the concentration of primers,dNTPs and Mg2+,and the optimal annealing temperature of primers through temperature gradient test were found.The electrophoresis bands of PCR amplified production were clear and repeatable.3.23 primers selected from 60 random primers were used to amplify a total of 214 loci,among which 183(85.51%)were polymorphic.The average number of DNA locus amplified by each primer was 9.3,the greatest was 13 and the least was 6.7312 DNA bands were amplfied,and the average number of each tea plant germplasm amplified was 146.24.4.Genetic diversity analysis of 50 tea germplasm resources was made by POPGENE v1.32 software.Nei's(1973)genetic diversity H=0.2694,Shannon's Information Index I=0.4088,Nei's(1978)genetic distance between the 50 cultivars ranged from 0.0236(between sample 38 and 39)to 0.6038(between sample 18 and 31).Genetic identity between the 50 tea samples ranged from 0.5467(between sample 18 and 31)to 0.9766(between sample 38 and 39),which explained that genetic diversity of the 50 tea samples was abundant.5.UPGMA cluster analysis of 50 tea plant germplasm resources was made by NTSYSpc v2.10e software.The result showed that 50 germplasms tested could be classified into 2 complex groups and 1 simple group while genetic similarity was 0.74.Sample 31 was classified into group 1;sample 37 to 50 were classified into group 2;other 35 germplasms were classified into group 3.50 germplasms tested could be classified into 4 complex groups and 2 simple groups while genetic similarity was 0.76,sample 40,42 to 50 were classified into group 1;sample 37 to 39,41 were classified into group 2;sample 13 to 24 were classified into group 3;sample 1 to 12,25 to 30,32 to 35 were classified into group 4;sample 31 and 36 were classified into 2 groups independently.6.The molecular identification of the 50 germplasms in different strain was analyzed according to amplification electrophoresis bands status of different primers:"existence" or "nothing",and 32 germplasms could be distinguished,including A1 strain germplasms(including sample 1,2 and 17),A2 strain germplasms(including sample 3 to 9),A3 strain germplasms(including sample 10 and 11),B1 strain germplasms(including sample 12 to 16,and sample 18),DC strain germplasms(including sample 31,38 and 39),BX strain germplasms(including sample 34 and 40),RG strain germplasms(including sample 42 to 46),TTX strain germplasms(including sample 47 and 48)and MLX strain germplasms(including sample 49 and 50).7.One DNA specific fragment was found by analyzing specific bands between five high catechin tea germplasms and two low catechin tea germplasms.The 414bp sequence was identified through recycle,clone and sequence analysis.NCBI homology alignment result showed that the specific sequence shared a high homology in partial base sequence of NADH dehydrogenase subunit 1(Nad1)gene of Couepia guianensis,Licania alba,Licania sprucei,and so on.It showed more than 80%identity with them.Speculating preliminarily that the Nad1 gene of tea plant may be involved in the synthesis of catechin synthesis.