Physical Locations Of 15 Markers In The Genetic Map From Hevea Brasiliensis

In the study hereby,the fluorescence in situ hybridization(FISH)has been used to locate the 15 markers from two linkage groups of the Hevea brasiliensis genetic map constructed by Lespinasse et al.,and two linkage groups have gotten integrated with rubber tree karyotype diagram.It not only lays the foundation for the construction of molecular cytogenetic map of the rubber tree,but also provides scientific bases for rubber tree molecular assisted breeding,identification of germplasm resources,comparative genomics,comparative genomics,etc.The main results of this study are as follows.First,special primers have been set for the 15 RFLP markers selected from the two linkage groups of Hevea brasiliensis genetic map constructed by Lespinasse,et al.Among them,6 markers(ghbCIR 540,ghbCIR229,ghbCIR327,ghbCIR580,ghbCIR282 and ghbCIR393)were chosen from the linkage group 2,and the left 9 markers from linkage group 3(gHbCIR 148?gHbCIR 319?gHbCIR 359?gHbCIR 439?gHbCIR 478?gHbCIR 497?gHbCIR 524?gHbCIR 536 and gHbCIR 659).The 40 pairs of primers were designed from these marker sequences and among which after screening the specificity of 15 pairs has been verified very well.Using Reyan7-33-97 genomic DNA as PCR template,each of the 15 pairs of specific primers was able to amplify a clear single DNA band between 800bp and 1400bp.The recovery,sequencing and comparison of the single bands showed PCR-amplified sequences were for the purpose of sequence.Second,The purified PCR products of 6 RFLP markers from linkage group 2 were fluorescently labelled as FISH probes.After FISH and karyotype analyzing,the 6 genetic markers were located in chromosome 6 of Hevea brasiliensis Reyan 7-33-97.The marker gHbCIR 229,gHbCIR 282,gHbCIR 393 and gHbCIR 580 were located on the long arm of the chromosome 6,the percent distances form signal to centromere were 41.46,48.48,45.65 and 51.02,respectively.The marker gHbCIR 540 and gHbCIR 327 were located on the short arm of the chromosome 6,the percent spacing form signal to centromere were 61.90 and 20.0,respectively.These results indicated that the linkage group 2 corresponded to the chromosome 6.Last,The purified PCR products of 9 RFLP markers from linkage group 3 were fluorescently labelled as FISH probes.After FISH and karyotype analyzing,the 9 genetic markers were located in chromosome 7 of Hevea brasiliensis Reyan 7-33-97.The marker gHbCIR 148,gHbCIR 359,gHbCIR 439 and gHbCIR 659 were located on the long arm of Hevea brasiliensis chromosome 7,the percent distances form signal to centromere were 78.26,60.78,58.18 and 20.59,respectively.The marker gHbCIR 319,gHbCIR 478,gHbCIR 497,gHbCIR 524 and gHbCIR 536 were located on the short arm of the chromosome 7,the percent spacing form signal to centromere were 8.33,26.09,45.45,52.94 and 10.71,respectively.These results indicated that the linkage group 3 corresponded to the chromosome 7.