Studies On Molecular Identification And Differentially Expressed Gene Separatation Of Tie Guanyin (Camellia Sinensis) With Varietal Characters Differentiation

Tie Guanyin is a national improved tea cultivar which isoriginally planted in Anxi County, Fujian. It is also a good breedingoriginal material in tea breeding. In recent years, some germplasmcharacters differentiations were found in Tie Guanyin tea treesduring cultivation, especially about the phenotypic characters.Maintaining the variety purity and economic characters of Tie Guanyintea trees were affected by these differentiations. In this research，the morphological variation of Tie Guanyin tea tree was studied, andthe otherness of Tie Guanyin tea trees with different phenotypiccharacter were studied by ISSR technique, and the molecularfingerprint of Tie Guanyin tea tree was established as well. cDNA-AFLP was used to study the gene expression difference between twokinds of Tie Guanyin tea trees with typical phenotypic difference.Themain results were as follows：1. Phenotypic character differentiations among differentTie Guanyin tea trees.Some morphological features of80Tie Guanyin tea trees wereinvestigated. The variability of the posture, bud-leaf color, serratenumber of leaf margin, lateral vein number, leaf thickness, and leaflength and leaf width among these trees were analyzed. The resultsshowed that the posture of the tea tree had the biggest variabilitywhich variable coefficient was48.95%. The second was the bud-leafcolor which variable coefficient was36.83%. Three principalcomponents were picked up by using principal component analysis,which could be used as reference indexes in distinguishing thephenotypic character differentiation of Tie Guanyin tea trees.Genetic similarity analysis and cluster analysis also indicated that the tea trees with higher variety purity had higher geneticsimilarity, and could be clustered in one group.2、The molecular markers differences of different TieGuanyin tea treesThe ISSR amplification reaction system was established andoptimized. The genetic diversity and relationship of thirteen teatrees were investigated by using ISSR. Ninety-one bands weregenerated by PCR using eleven primers, of which45bands werepolymorphic bands, and the polymorphic rate was49.45%. The geneticdiversity index of Nei and Shannon information index of thesethirteen tea germplasms were0.17and0.25, respectively, whichindicated that the genetic diversity level was low among thesesamples. Genetic similarity analysis indicated that the geneticsimilarity coefficient of13tea germplasms ranged from0.687to0.986, with average was0.805. This suggested that the geneticrelationships among these cultivars were close. The thirteen teagermplasms were divided into three groups by cluster analysis, nineTie Guanyin tea trees were clustered in one group, in which the twotrees with higher variety purity were clustered in one sub-group, theother two with variation phenotypic characters were clustered inanother sub-group, but the cluster distance between these two sub-groups was the furthest in the group.3. Molecular fingerprint of Tie Guanyin tea treeFive primers were selected from eleven primers to establish themolecular fingerprint of these thirty tea germplasms, which could beused to identify the samples. The primer UBC-843could be used toidentify the nine Tie Guanyin tea trees from allthe13tea trees, and primer UBC-857could distinguish the two tea trees with highervariety purity from the other trees with characters differentiation.4. The difference of gene expression between two differentphenotyp-es of Tie Guanyin tea treescDNA-AFLP technique was used to identify the two differentphenotype Tie Guanyin tea trees, which one named as Tie Guanyin teatree X and the other named as Tie Guanyin tea tree Y. Total75differently expressed bands were separated by using256pairs of AFLPprimers, of which36bands were higher expressed in Tie Guanyin teatree X, and the other39bands were higher expressed in Tie Guanyintea tree Y. Sixty-three bands were separated from these75bands andbe clone. BlastX comparison results indicated51of these sequenceswere homologous sequences,17sequences of these were related tomaterial synthesis and metabolism,11sequences were related totranscription-regulation,3sequences were related to signaltransduction,3sequences were related to the material delivery, and17sequences were unclear functional.5. qPCR of the differential expressed genes between twoselected Tie Guanyin tea treesTen differential expressed genes were chose to analyze theirexpressed quantity between Tie Guanyin tea tree X and Y by using qPCR.The results showed that there were obvious differences between thesetwo samples. Eight of these ten genes were higher expressed in TieGuanyin tea tree Y, and the relative expressed quantity ranged from1.11to5.56. The expression quantities of the other two genes in TieGuanyin tea tree Y were0.74and0.48. These expression differenceswere probable involved to the difference of bud-leaf color, treeposture, leaf morphology and epiphyllous angle between the two teatrees.