The Detection Of STS Markers In Populus And Study Of SCAR Markers On Anti-black Spot Disease

Carrying out the detection of STS markers by PCR-SSCP in Populus and study of SCAR makers on anti-black spot disease of 187 F1 clones, which are the progenies of Populus deltoides Bartr. "Lux"(l-69/55)and P.euramericana cv.(I-45).The optimization of conditions affecting the results of PCR-SSCP analysis was also discussed. The main results as following:1.In this paper, comparing and analyzing the experimental results under various controlled conditions such as the concentration and cross-linker of PAGE, the presence of glycerol in gels, the voltage and temperature of the gel during electrophoresis. Results showed that the concentration of PAGE, the voltage and temperature of the gel during electrophoresis were the important factors which effected the results of PCR-SSCP analysis. Each fragment size needed its own optimal running conditions such as suitable concentration of PAGE, stable-low temperature and voltage during electrophoresis.2.Out of 29 STS primers, three primers(Win3 , P164 and P770) showed polymorphism by PCR-SSCP analysis. Sequencing the homozygotes and heterozygotes (the parents and two progenies) and then analyzing the sequences with clustal W and blastn. The results showed:The lengths of four sequences of Win3 marker were: 45,255bp; 69,254bp; 116,255bp; 117,256bp respectively. The analysis results of Clustal W showed: there were differences between the 148th (116 pluged a T and 117 pluged a GX the 163rd (117 pluged a T) and the 168th (A transformed G) base. Through searching nucleotide acid databases by Blastn, the sequences of Win3 marker had high homologous with Populus x generosa isolate H11 -11 serine proteinase inhibitor (win3.12) gene, Populus trichocarpa wound responsive gwin3 gene and Populus nigra Win3 marker sequence.The lengths of four sequences of P164 marker were all 164bp. The analysis results of Clustal W and Blastn showed: there were differences between the 124th (A transformed G)base and no homologous sequences existed in NCBI nucleotide acid databases.The lengths of fours sequences of P770 marker were: 69,641bp; 45,643bp; 10,650bp; 08,651bp. The analysis results of Clustal W and Blastn showed: there were obvious differences among them and No homologous sequences existed in NCBI nucleotide acid databases.3.TwoRAPD markers: OPAI17-1550 and OPAI13-900 markers that linked to anti-black-spot disease gene locus, were successfully converted into SCAR markers named SAI17-land SAI13-2 respectively. The examinations results of PCR products by 1% gel electrophoresis showed: SAI17-1 and SAI13-2 were validated in the resistant, susceptible pools and F1 population. The SAO17-1 had amplification products with templates of the positive OPAI17-1550 F1 population and resistant pool and did not have amplification products with templates of the negative OPAI17-1550 F1 population and susceptible pool. The SAI13-2 amplified two bands with templates of the positive OPAI13-900 F1 population and resistant pool and amplified one band with templates of the negative OPAI13-900 F1 population and susceptible pool.Labelled the target fragment with Dig and hybrided with the PCR products of primers OPAI13 and OPAI17. The results of Southern hybridization showed; the sequences among positive clones were homologous, there were several homologous sequences in genome respectively.