Supporting research on making linkage maps of marker, RFLP and RAPD loci in genome of sugi, Cryptomeria japonica D.Don

日本柳杉是日本古代重要的林业树种。单倍体染色体数目为11条，具有相似的表型特征，但有两条染色体具有随体或次生收缩。本项目首先研究了在有或不存在致死因子的情况下，标记基因座之间重组值的估计方法，包括同工酶、RFLP和RAPD基因座。根据观察到的分离比与预期分离比的偏离程度，分别检验了致死基因座的存在。模型的适合性通过LOD评分进行评估。将该方法应用于同工酶分离，发现了分别与Lap、G6P、Dia-3连锁的致死基因。在熊本与冲之山杂交的三代后代中，将128个RFLP、33个RAPD、2个同工酶和1个矮秆等164个基因座放入13个连锁群的连锁图谱中，总图谱距离为1190 cM。在13个连锁群中，有8个连锁群的座位分离比例偏离。在6个连锁群中检测到一个或两个致死基因，发现有致死基因的图谱距离比不含致死基因的图谱距离拉长。通过对单倍体大配子体植物的分析，绘制了175个随机扩增多态性DNA基因座的图谱。共14个连锁群，总长2,212.3 cM，用冷处理和荧光原位杂交方法鉴定单倍体染色体。对方法进行了改进，用激光切割后回收了染色体片段。

Sugi, Cryptomeria japonica D.Don has been a important forestry species in Japan sice old times. It has haploid chromosome number of 11 with similar phenotypic features except to two chromosomes which have a satelite or secondary constriction.In this project, firstly, studies were made on the methods for estimatin of recombination values between marker loci including isozyme, RFLP and RAPD loci with or without the presence of a lethal factor. Existence of a lethal locus was examined by the magnitude of deviation of the observed segregation ratio from expected one for each locus separately. The fitness of the model involving a lethal factor were evaluated by LOD scores. This method was applied for isozyme segregation and it was found a lethal gene linked to Lap, G6p, Dia-3 respectively.In a three generation progeny derived from a cross of Kumotooshi and Okinoyama sugi, 164 loci including 128 RFLP,33 RAPD,2 isozyme and 1 dwarf were put in a linkage map with 13 linkage groups with total map distance of 1,190 cM.Out of 13 linkage groups, there were 8 linkage groups which had clustered loci with deviated segregation ratios. One or two lethal genes were detected in 6 linkage groups, and it was found that with the lethal genes, map distances were elongated than those estimated by free of lethal gene. Another map in which 175 RAPD loci were placed, was made by analysis of haploid megagametophytes. It had 14 linkage groups with 2,212.3 cM in total.Discrimination of haploid chromosome was tried by clod treatment and fluorescence in situ hybridization. It was tried to improve the methods and chromosome fragments were recovered after cutting with laser beam.