| In this test,we constructed the genetic linkage map by using Masson pine megagametophyte based on SSR markers.The population number was 160 and the primer pairs was 235.The results were as follows:1,We found that the SDS method was more suited than CTAB method in the DNA extraction.2,The best reaction system for SSR was established . The amplification was in a10μL volum,15ng DNA templates,0.1μM primer,0.3mMof each dNTPs,2.0mM Mg2+,1.0Utaq.The best PCR procedure:94℃,4 min;94℃,30s;Tm-5℃,15s;72℃,30s;40cycles;72℃extended 30min;4℃conservation.The PCR products were tested on 8% PAGE,displayed by Ag dyed method.Through the ways above,the experiment had been approved good results.3,Tested by using 160 megagametophytes,97 separated locis were gained by using 30 pairs of screening primers,the average loci/primer was 1.62.4,The linkage map was constructed by using the software FsLinkageMap2.0.Based on the 86 loci,the genetic linkage map of 74 loci was constructed, they were distributed evenly into 11 linkage groups.The total map distance was 927.91cM,the biggest map distance was 359.89cM including 18 markers while the smallest map distance was 20.44cM including 2 markers;the largest gap was 29.2 cM between two markers and the smallest gap was 0 cM with the average was 12.54cM.It was covered 36.5% of the length of the genome,which was estimated to be as long as 2541.21cM. |
