Physical Localizations Of 25 Markers In The Linkage Group 12,17 And 18 Of Genetic Map Of Hevea Brasiliensis

Hevea brasiliensis is the main source of natural rubber,which is a kind of important tropical crop in our country.Many genetic maps of Hevea brasiliensis have been constructed by different molecular marker techniques,but the relationship between genetic maps and chromosomes is not clear.The integration of genetic maps and karyotypes to construct molecular cytogenetic maps is of great significance for the correct assembly of genomic sequences and sequence splicing.In this study,Hevea brasiliensis 'Reyan 7-33-97' was used as the material.Fluorescence in situ hybridization(FISH)and in situ polymerase chain reaction(in situ PCR)were used to locate 25 genetic markers that from three linkage groups on the genetic map constructed by Lespinasse(2000).In addition,the corresponding relationship between these three linkage groups and nuclear chromosomes was determined.This study will lay a foundation for the construction of a complete molecular cytogenetic map of Hevea brasiliensis,and provid a strong scientific basis for molecular assisted breeding,germplasm resource identification and comparative genomics of Hevea brasiliensis.The results of this study are as follows:(1)25 RFLP markers were selected from the 12th,17th and 18th linkage groups in Hevea brasiliensis genetic map constructed by Lespinasse(2000),and their specific primers were designed and screened.After amplification by PCR,the products detected by gel electrophoresis showed that these specific primers could amplify a clear single band.The products were retrieved,sequenced and compared online.It was found that they belonged to the target sequence.(2)Eight genetic markers were selected from the 12th linkage group and detected by FISH or in situ PCR.The results of karyotype analysis showed that they were all located on chromosome 14 of Hevea brasiliensis ' Reyan 7-33-97',suggesting that the 12th linkage group corresponded to chromosome 14.Among them,the markers gHbCIR387,gHbCIR181,gHbCIR29,gHbCIR513 and gHbCIR80/317 were located on the short arm of chromosome 14,and the percentage distances from signal sites to centromeres were 45.06,38.84,31.86,23.72 and 16.90,respectively.The markers gHbCIR81,gHbCIR145 and gHbCIR164 were located on the long arm of chromosome 14,and the percentage distances from signal sites to centromeres were 2.31,29.27 and 59.77,respectively.The cytogenetic map of chromosome 14 was constructed.(3)Seven genetic markers were selected from 17th linkage group and detected by FISH.The results of karyotype analysis showed that the markers gHbCIR635,gHbCIR366 and gHbCIR339.L2 were all located on the short arm of chromosome 17 of Hevea brasiliensis 'Reyan7-33-97',and the percentage distances from signal sites to centromeres were 30.67,6.23 and 2.83,respectively.In addition,the markers gHbCIR586,gHbCIR526,gHbCIR585 and gHbCIR357 were located on the long arm of chromosome 17,and the percentage distances from signal sites to centromeres were 20.27,42.30,46.79 and 75.65,respectively.Therefore,the 17th linkage group corresponds to chromosome 17 of Hevea brasiliensis 'Reyan7-33-97',and the cytogenetic map of chromosome 17 was constructed.(4)Ten genetic markers were selected from 18th linkage group and detected by in situ PCR or FISH.The results of karyotype analysis showed that the markers gHbCIR266,gHbCIR41/47,gHbCIR227,gHbCIR443,gHbCIR259,gHbCIR101 and gHbCIR618 were all located on the short arm of chromosome 11 of Hevea brasiliensis 'Reyan7-33-97'.The percentage distances from signal sites to centromeres were 70.38,57.87,52.92,44.44,40.36,39.83 and 23.05,respectively.In addition,the markers gHbCIR20,gHbCIR423 and gHbCIR597 were located on the long arm of chromosome 11,and the percentage distances from signal sites to centromeres were 15.58,29.57 and 72.08,respectively.Therefore,the 18th linkage group corresponds to chromosome 11 of Hevea brasiliensis 'Reyan7-33-97',and the cytogenetic map of chromosome 11 was constructed.