Study On Phylogenetic Relationship Of Picea Mongolica And Its Relative Species Based On Fluorescence In Situ Hybridization

Picea trees are important species of forest in China and the world which have inestimable ecological value and economic value.The study of its karyotypes,system classifications and evolutionary relationships of origins has crucial importance in biology and forestry respects.In this study,seven kinds of Picea natural forest seeds were used as materials,On the basis of conventional chromosome production methods,we optimized the spruce metaphase chromosome preparation technology and establish a perfect section preparation system;18S rDNA and 25 S rDNA gene fragments were cloned using the homologous sequence method,after sequence analysis,they were identified as probes of dual-color fluorescent in situ hybridization,and made gene mapping in the metaphase chromosomes of seven species of spruce.It provides a more effective method for the identification of the homologous chromosomes,analysis of genetic relationship and phylogenetic relationships of various spruces.The main findings were as follows:1.To optimize the Flame-drying protocol after enzyme digestion of cell wall,getting the most ideal pretreatment condition was that 0.1% colchicine(containing 1% dimethylsulfoxide)was immersed was dark immersed at 115 rpm at 23°C for 6.5 h;Enzymolysis condition was that the apical roots were dissociated at 37°C for 70-75 minutes in the enzyme mixed solution(2% cellulase + 1% pectinase solution),and root tip metaphase chromosomes with good dispersibility and good morphology were obtained,which improved the efficiency and quality of the production.2.The 18 S rDNA and 25 S rDNA gene fragments were amplified by PCR using genomic DNA of P.Mongolica as a template,and after sequencing,the respective sequence sizes were 1472 bp and 364 bp.The BLAST analysis showed that the two cloned sequences shared 99% sequence homology with some of the plant genes published in GenBank,confirming their applicability as FISH probes.3.The 18 S rDNA and 25 S rDNA were mapped to 8 signal sites on the chromosomes of P.Mongolica and P.meyeri and the four pairs of homologous chromosomes of the two spruce were identified,6 signal sites on the chromosomes of the other five species of spruce and the three pairs of homologous chromosomes of them were identified respectivly.It provides the evidence for the identification of homologous chromosomes and karyotype analysis.4.Based on the results of rDNA-FISH signals of seven species of spruce,and the Mendelian inheritance laws were used to preliminarily determine that P.mongolica and P.meyeri were clustered into one and their genetic relationship was close.The other five species of spruce were clustered into one.