Study On The Genetic Diversity Of Taraxacum Kok-saghyz Rodin Germplasm In Xinjiang

Taraxacum Kok-saghyz Rodin is a kind of perennial herb which can survival at cold area. It is a good alternative source of natural rubber. To develop an alternative source of natural rubber, the United States and European Union are planning to develop Taraxacum Kok-saghyz Rodin as strategic crop. The classification and evaluation for the germplasm resources of Taraxacum Kok-saghyz Rodin is important for the breeding and production of Taraxacum Kok-saghyz Rodin.In this research, we collected181putative Taraxacum Kok-saghyz Rodin samples from10different locations in Xinjiang, China. We identified the samples through phenotypic characters and PCR amplification by using specific primers. The genetic diversity of the63Taraxacum Kok-saghyz Rodin plants had been determined by RAPD-PCR technology.After drying in backlighting ventilation, the seeds of Taraxacum Kok-saghyz Rodin were reserved at the centrifuge tube followed by keeping in4℃refrigerator. The leaves of Taraxacum Kok-saghyz Rodin were used as explants for establishing in vitro regeneration system. The plantlets from the63Taraxacum Kok-saghyz Rodin plants were conserved in vitro separately. The main results that achieved in this research are shown as following:1. Developing the specific primers for identification of Taraxacum Kok-saghyz Rodin through PCR amplificationTo differentiate Taraxacum Kok-saghyz Rodin from Taraxacum mongolicum Hand-Mass, we used software Oligo7.51to design a pair of specific primer designated as TKp2-2L and TKp2-2R that corresponding to an idio-fragment of Taraxacum Kok-saghyz Rodin which GeneBank accession number is HB850815.1. We confirmed the specificity of the primers for Taraxacum Kok-saghyz Rodin through determining the sequencing of the PCR product from the primers. Southern blotting results also confirmed the specificity. The primers can be used to differentiate the Taraxacum Kok-saghyz Rodin from Taraxacum mongolicum Hand-Mass by PCR amplification. We identified63plants of Taraxacum Kok-saghyz Rodin by PCR using the specific primers.2. RAPD analysis of genetic diversity of63plants of Taraxacum Kok-saghyz Rodin germplasm resourcesIn this research, the genetic diversity of62plants of Taraxacum Kok-saghyz Rodin germplasm resources from Xinjiang and1sample from USA determined by using RAPD (Random Amplified Polymorphic DNA). The optimal condition for RAPD amplification has been determined as following: The reactions were performed in a volume of20μl, which containing1.75U Taq DNA polymerase,30ng DNA template,0.25mmol/L dNTPs,0.5μmoL/L random primer,2.0mmol/L Mg2+,2.0μl10×PCR Buffer. The PCR reaction was programmed by1cycle for5min at97℃, followed by45cycles for30s at94℃,30s at37℃, and70s at72℃, and finally, followed by1cycle for10min at72℃.As results showed,225amplified bands were produced by13random primers. The ratio of polymorphic bands reached to99.6%with amplification of224, which illustrated that63plants of Taraxacum Kok-saghyz Rodin germplasm resources had abundant polymorphism and rich genetic resources. According to the diversity of the amplified bands, a dendrogram was constructed to indicate their genetic relationships. When similarity coefficient was0.519,63plants of Taraxacum Kok-saghyz Rodin resource could be separated into3groups which sample from USA belongs to one group separately, samples from Hemu and Kanasi belong to one group, all samples from other locations belong to another group together. When similarity coefficient was0.535, samples from Kashi can set up the fourth group.3. Reservation of Taraxacum Kok-saghyz Rodin germplasm resourcesWe determined the best conditions for the surface sterilization, callus and multiple shoots induction, rooting and transplant by using the seeds and leaves of Taraxacum Kok-saghyz Rodin from Nalati location. The results showed as following:a. The best method of surface sterilization was treated with75%alcohol(30s)+20%(v/v) NaClO(30min)+vernalization(24h) for seeds and with75%alcohol(30s)+20%(v/v) of NaCIO (5min) after drying3days for leaf separately;b. The optimal medium for callus induction from stems and leaves is MS+0.5mg.L-12,4-D or MS+0.1mg.L-12,4-D+0.5mg.L-16-BA, the inductivity was up to100%. The optimal medium for multiple shoots induction is MS+0.3mg.L"1NAA+0.5mg.L-16-BA. The best medium for rooting is1/2MS+15g sucrose+O.5mg.L-1NAA. The best transplanting medium is the vermiculite that should keeping humidity and26℃before transplanting.