| Miscanthus is one of the most potential herb cellulose energy plants.In recent years,the genetics and genomics research of Miscanthus and the germplasm utilization have become hot fields in international biological research.Miscanthus originated in South East Asia and China is rich in genetically diversified wild Miscanthus germplasm resources.Miscanthus has unique biological properties,including herbaceous perennial,fast growth,high efficiency of water and nitrogen utilization rate,high photosynthetic efficiency and biological yield,strong root system,superb biotic and abiotic stress tolerances,low production cost,wide range of application,easy storage and transportation,benefit of soil and water conservation,environment friendly.These characteristics make it stand out in the numerous energy plants and become the most development potential resource of the cellulosic energy crops in the world.Thus Miscanthus possesses important values for scientific research.M.lutarioriparia is an endemic species in C hina,with characteristics of widely distributed,resistant to cold and drought,high photosynthetic efficiency.The genome of M.lutarioriparia is large and complicated,about 2500 Mb,and equivalent to the size of the human genome and maize genome.Genomic research,physical map construction,gene cloning and molecular biology are developed on the basis of the BAC library.The innovation and improvement of modern biological technology and methods greatly improved the efficiency and quality of BAC library construction for complex plant genomes.The genomic BAC library construction and library scree ning technology has been quite mature.Construction of a BAC library and physical map based gene cloning techniques has been widely used in genomic study.The BAC library of the M.lutarioriparia in China was successfully constructed through the procedure of vector preparation,high quality large DNA fragments obtaining,determination of optimal conditions for partial digestion,twice successive selections of large fragment,connection and transformation.The experimental results show that high yield DNA wa s obtained using young leaf with less pollution,and all of the sizes of the fragments were bigger than 700 kb.The optimum digestion condition for each 1/2plug is digesting with 3U Hind III enzyme 10 min at 37℃.The highest efficiency was achieved under the ratio of the weight of large fragment DNA and vector 10:1.In this experiment,the BAC library consists of 130560 clones.The clones were stored in 340 pieces of 384-well bacteria culture plate in the form of monoclones,and the superpool was also constructed.One hundred clones were selected randomly for testing.The results show that the sizes of insert fragments were almost ranged from 90 to 120 kb and the average size was 106 kb.The insert fragment sizes showed good uniformity,and the rate of empty clone was 1%.The BAC library covers 5.6 times of M.lutarioriparia genome.Five randomly selected clones still can be inherited steadily after about 100 generations cultivation,each clone were consistent between every generation with no loss or mutation,indicating good stability of the BAC clone.Library coverage,insert size and stability can respond to the needs of physical map construction and gene cloning in the follow-up experiments.This is the first time to construct the BAC library with deep coverage for M.lutarioriparia,the unique Miscanthus species of cellulosic biomass energy plants in China.The library can provide an effective platform for genome research of Miscanthus,and play an important role in exceptional gene cloning and genomics research of M.lutarioriparia. |
