Physical Locations Of 26 Markers In The Linkage Group 11、13 And 14 Of Genetic Map Of Hevea Brasiliensis

Hevea brasiliensis belongs to the family Euphorbiaceae,and it is a perennial tropical economic tree that produces natural rubber.Many genetic maps of Hevea brasiliensis have been constructed by researchers,most of them were divided into 18 linkage groups,while a few of them were divided into 22、23 or more linkage groups.Therefore,there is a phenomenon that many linkage groups were corresponding to the same chromosome.At the same time,the physical location of the genetic marker on the chromosome is not clear.Molecular cytogenetic map of Hevea brasiliensis is an important tool for its genomics and genetic development.And it can help to reveal the correspondence relationship between the linkage groups and the chromosomes,and the arrangements of genetic markers on the chromosomes and the real physical locations.It can also provide a valuable theoretical basis for gene cloning and marker-assisted breeding of Hevea brasiliensis.In this study,we have selected a total of 26 genetic markers,including 24 RFLPs and 2 SSRs.The genetic markers were selected from LG11,LG 13 and LG 14,which were constructed by Lespinasse et al（2000）.In this experiment,we used fluorescence in situ hybridization and in situ PCR to detect the genetic markers,and combining with the karyotype analysis to construct the molecular cytogenetic maps of LG 11,LG 13 and LG 14.Our results are as follows:First,we used bioinformatics method to find 26 DNA sequences of markers in NCBI.Primer designs were carried out by selecting the DNA sequences of 2500-3500 bp containing the markers.And then,the 26 most specific primers were screened,followed by electrophoresis detection,PCR product sequencing and sequence alignment.Second,the four genetic markers MnSoD,M613,gHbCIR295.PB and gHbCIR636 on the linkage group ll were physically localized by FISH,As a result,they were all located on chromosome 16 of the Hevea brasiliensis Reyan 7-33-97.MnSoD and gHbCIR295.PB were located in the short arm of chromosome 16,and the average percentage distances between marker signals and centromeres were 63.82 and 46.27,respectively.GHbCIR636 and M613 were located in the long arm of chromosome 16,and the average percentage distances between marker signals and centromeres were 28.34 and 65.82,respectively.It shows that there is a corresponding relationship between the linkage group 11 and the chromosome 16.And the cytogenetic map of chromosome 16 was constructed with the markers.Third,the Nine genetic markers gHbCIR671.L2,gHbCIR402,gHbCIR506.L2,gHbCIR31,gHbCIR333,gHbCIR287,gHbCIR360,gHbCIR303 and gHbCIR670.PB were selected from the linkage group 13 for fluorescence in situ hybridization.After karyotype analysis,9 markers were located on chromosome 18 of Hevea brasiliensis Reyan 7-33-97.Among them,gHbCIR671.L2 and gHbCIR31 were located on the short arm of chromosome 18,and the average percentage distances between the marker signals and centromeres were 57.22 and 11.27,respectively.While markers gHbCIR402,gHbCIR506.L2,gHbCIR287,gHbCIR360,gHbCIR333,gHbCIR303 and gHbCIR670.PB were all located on the long arm of chromosome 18,and the average percentage distances between the marker signals and centromeres were 8.39,23.51,29.14,40.18,41.65,60.48 and 60.59,respectively.It shows that there is a corresponding relationship between the linkage group 13 and the chromosome 18.And the cytogenetic map of chromosome 18 was constructed with the markers.Finally,the 1 3 genetic markers were selected from the linkage group 14.The locations of gHbCIR686,gHbCIR425,gHbCIR261,gHbCIR682.L2,gHbCIR631.L2,gHbCIR330,gHbCIR412₄93,gHbCIR602,gHbCIR20.L 1,gHbCIR290,gHbCIR409₄15 and gHbCIR644 were detected by FISH method,and the gHbCIR365.L2 was performed in situ PCR.The results showed that 13 genetic were located on chromosome 12.Among them,5 markers gHbCIR686,gHbCIR365.L2,gHbCIR425,gHbCIR261 and gHbCIR631.L2 were located on the short arm of chromosome 12,and the average percentage distances between the marker signals and centromeres were 32.67,44.56,31.53,29.10 and 11.46,respectively;8 markers gHbCIR682.L2,gHbCIR330,gHbCIR412₄93,gHbCIR602,gHbCIR20.L1,gHbCIR290,gHbCIR409₄15 and gHbCIR644 were located on the long arm of chromosome 12,and the average percentage distances between the marker signals and centromeres were 9.87,45.59,53.82,48.40,53.24,55.24,65.51 and 75.74,respectively.It shows that there is a corresponding relationship between the linkage group14 and the chromosome 12.And the cytogenetic map of chromosome 12 was constructed with the markers.