SSR Primer Selection And Phylogenetic Relationships Based On Issrs For Water Tolerant Bamboo Species

Bamboo is the general name for plants of Bambusoideae belonging to Gramineae,and a very important part of forest resources.There are abundant bamboo resources on the low wetland along the river in south China.They play an important role in land use,adjustment of rural industry structure,and improvement of local ecological environment.However,the genetic relationships among some bamboo species are still in dispute,so that the scientific and effective utilization of these bamboo species are affected.The thesis is composed of the two parts:（1）establishment and optimization of simple sequence repeat-polymerase chain reaction（SSR-PCR）system for water tolerant bamboo species;（2）investigation of genetic diversity and relationships of water tolerant bamboo species based on ISSRs,using the leaves as materials from 11 species of three genera,namely Phyllostachys,Pleioblastus and Indocalamus.Results of the present studies were revealed as follows: 1.SSR-PCR system was established through orthogonal test and optimized through single factor experiment for water tolerant bamboo species,and the SSR primer pairs were selected.The optimal SSR-PCR reaction system was in a volume of 20.0 μL containing DNA（5-10 ng·μL-1）1.1 μL,dNTP（10 mmol·L-1）0.44 μL,forward-and reward-primer（10 μmol·L-1）0.26 μL,respectively,Mg²⁺（25 mmol·L-1）2.0 μL,Taq DNA polymerase（5U·μL-1）0.8 μL,,10 × PCR buffer 2.0 μL and HPLC water 13.14 μL.The amplification PCR program was conducted under the cycling conditions of initial denaturation for 3 min at 94 ℃;44 cycles of denaturation 30 sec at 94 ℃,annealing for 30 sec at 54-56 ℃,extension for 60 sec at 72 ℃;and a final extention for 3 min at 72 ℃.Meanwhile,the results proved the limited cross-species transferability of SSR primers for bamboos.2.The ISSR-PCR system was established and optimized for water tolerant bamboo species.Eleven ISSR primers with clear,polymorphic,and repeatable band patterns were selected.The genetic diversity and phylogenetic relationships among water tolerant bamboo species were investigated based on ISSRs.The high level of genetic diversity among these bamboo species was shown in accordance with their morphological polymorphism.The resultant UPGMA dendrogram based on the Dice similarity index clearly separated all the investigated bamboos into three major groups corresponding to genera Phyllostachys,Pleioblastus and Indocalamus,respectively.The genetic relationships among these three genera revealed in the present study partially supported traditional classification,showing closer relationship of Pleioblastus maculates to genus Phyllostachys,than to genus Indocalamus.The affiliation of species Pleioblastus maculates in taxonomy was also discussed.Preliminary ISSR bands specific to some bamboo species were found,which can serve as diagnostic markers for species identification.The analysis of phylogenetic relationships among bamboo species based on ISSRs have supported and supplemented the traditional taxonomic assessments based on morphological traits.Revelation of genetic diversity and phylogenetic relationships of water tolerant bamboo species will provide theoretical basis and practical guidance for parents selection in cross breeding and plant introduction in the future.