Characterization of genetic diversity in selected cultivars and identification of a possible molecular marker for drought tolerance in tea Camellia sinensis (L.) O. Kuntze

Abstract

Tea is made from the young leaves of Camellia sinensis, an evergreen, outbreeding tree species. The replacing of seedling with clonal plantations and the use of small numbers of parental plants in breeding programs could cause a decrease in the genetic diversity. The Tea Research Foundation of Central Africa (TRFCA), in Malawi, started tea breeding in 1956. This program has provided new cultivars with increased quality and yield over the first 40 years. However, recently the progress seems to have reached a plateau, suggesting that the maximum potential of the original genetic base has been reached. This study aimed at the evaluation of the genetic diversity within TRFCA cultivars, Malawian seedling accessions and cultivars currently used in China using morphological and microsatellite markers. In the morphological trait analysis, 186 seedling accessions and six TRFCA cultivars were accessed using six morphological traits. The morphological data were scored using two scoring methods, the range scoring method and the mean scoring method. Although the mean scoring method could not discriminate between all the accessions, it is in better agreement with the classification of an expert tea breeder. The six TRFCA cultivars were well distributed among the seedling accessions from Malawi. However, the results of the morphological study were not conclusive since the sample size of the TRFCA cultivars was too small. Microsatellite analysis of 41 TRFCA cultivars, 233 seedling accessions and 22 China origin cultivars revealed only moderate levels of inbreeding. The genetic relationship shows the TRFCA cultivars and the China origin cultivars in their own clusters. The FST values indicated significant differences between the TRFCA and China origin cultivars with all other groups. The allelic richness and the heterozygosity of the TRFCA cultivars are on the low end of the ranges of the groups studied. The TRFCA cultivars may benefit from the introduction of genetically distant tea plants into the breeding program since the level of heterozygosity within this group is relatively low. A second aim of this study is the identification of Random Amplified Polymorphic DNA (RAPD) markers that associate with traits of interest. Primers (17) were selected from previously trait associate plant studies. This selection did not increase the association rate of the primers. The traits that were investigated include Heliopeltis resistance, drought and cold tolerance. One of the tested primers RAPD8, associates with 6 of 8 drought tolerant cultivars. Another RAPD primer previously identified by Mishra and Sen-Mandi (2004) associated with only 4 of 8 drought tolerant cultivars in this study. Neither of the two primers associated with the 8 drought susceptible cultivars. Together these two primers could discriminate all 8 drought tolerant cultivars from the 8 susceptible cultivars and could possibly be used to identify drought tolerant plants. This study contributes to the knowledge of the genetic diversity of tea which may contribute to the management of genetic diversity within tea. In addition a possible trait associated RAPD marker for drought was identified that could be used in the selection for drought tolerant cultivars. AFRIKAANS : Tee word van die jong blare van die Camellia sinensis plant gemaak. Camellia Sinensis is ‘n immergroen boom spesie. Tans word die meeste van die saailing plantasies met klone plantasies vervang wat ‘n negatiewe uitwerking op die genetiese diversiteit van tee het. Tee ontwikkelingsprogramme beïnvloed ook die diversiteit negatief omdat ‘n klein hoeveelheid ouer plante gebruik word. Die Tee Navorsings van Sentrale Afrika (TRFCA) is in Malawi geleë en spesialiseer vanaf 1956 in tee kultivar ontwikkeling. Die kwaliteit en die opbrengs van die vrygestelde kultivars het oor die eerste 40 jaar van die program verbeter. Die vordering in hierdie program is besig om ‘n plato te bereik. ‘n Moontlike verduideliking hiervoor kan wees dat die maksimum kapasitieit van die bestaande genetiese basis bereik is. Die eerste doelwit van hierdie studie is om die genetiese diversitiet van die TRFCA kultivars, geselekteerde saailinge van Malawi en kulticars wat tans in China gekweek word te evalueer deur morfologiese en mikrosateliet merkers te gebruik. In die morfologiese evaluasie is 186 geselekteerde saailinge en ses TRFCA kultivars met behulp van ses morfologiese karaktereineskappe geëvalueer. Die data is geëvalueer deur die gebruik van twee puntestelsels, ’n reeks puntestelsels en ‘n gemiddelde puntestelsel. Alhoewel die gemiddelde puntestelsel nie tussen al die plante kon onderskei nie, het dit ‘n beter groepering ten opsigte van sub-spesies (He was 0.6900) gegee. Die ses TRFCA kultivars is tussen die saailinge van Malawi versprei. Weens die klein hoeveelheid TRFCA kultivars is die gevolgtrekings van die morphologiese evaluasie nie betekenisvol nie. Mikrosateliet evaluasie is gedoen met 41 TRFCA kultivars, 233 geselekteerde saailinge en 22 Chinese kultivars. Gemiddelde vlakke van inteling is opgemerk. In die illustrasie van die genetiese verhouding tussen die plante is die TRFCA kultivars gegroepeer, so ook die Chinese kultivars. Die FST waardes toon betekenisvolle statisitiese verskille tussen TRFCA en die Chinese oorsprong kultivars en al die ander groepe. Die alleliese rykheid van die TRFCA kultivars was aan die onderpunt van die reeks. Hierdie resultate dui daarop dat die TRFCA ontwikkellingsprogram moontlik voordeel kan trek uit die byvoeging van ouer pante wat geneties divers is in die program. Dit mag ‘n positiewe uitwerking hê as gevolg van die ondergemiddelde vlak van die “heterozygosity” in die TRFCA kultivars. ‘n Tweede doelwit van die studie was die identifisering van moontlike “Random Amplified Polymorphic DNA” (RAPD) merkers wat met belangrike kenmerke geassosieer kan word. RAPD merkers (17) wat geïdentifiseer is in vorige kenmerkassosiasiestudies in ander gewassse is geselekteer. Die seleksie van hierdie merkers het nie die assosiasie persentasie verhoog nie. Die kenmerkke wat ondersoek is, was Heliopeltis weerstand, droogte toleransie en koue toleransie. Een van die merkers, RAPD8 assosieer met 6 van die 8 droogte toleransie kultivars. Die RAPD merker wat deur Mishra and Sen-Mandi (2004) geïdentifiseer is, assosieer met 4 van die 8 droogte toleransie kultivars. Beide die merkers assosieer met geen van die droogte sensitiewe kultivars nie. Gesamentlik kon die twee RAPD merkers al 8 van die droogte toleransie kultivars van die sensitiewe kultivars onderskei. Hierdie studie dra by tot die kennis van die genetiese diversitiet van tee wat kan lei to die beter bestuur wan die tee ontwikkelingsprogram. Veder is ‘n moontlike merker vir droogte toleransie geïdentifiseer wat gebruik kan word in die seleksie van moontlike droogte bestande kultivars.