The Physical Locations Of Ninth,Seventeenth And Eighteenth Linkage Groups Of Genetic Map In Cassava

Manihot esculenta Crantz is one of the most important tuberous crops of the world, and is not only an important food crop, but also an important economic plant. Currently, some of the cassava genetic maps have been constructed by researchers, these genetic maps were not reach saturation, in these genetic maps, some of them had more than18linkage groups, and some of them have just18linkage groups, but the correspondence between the genetic maps and the karyotype was not known. We used fluorescence in situ PCR technology integration molecular genetic map about cassava in this study, and located some markers on the chromosomes of cassava. Can not only determine the accuracy and completeness of the genetic map, but also some genetic maps can be integrated into the karyotype map of cassava. Achieve the target about constructing the molecular cytogenetic maps of cassava. The results were as fellows:1. According to the ninth, seventeenth and eighteenth linkage groups in the linkage of genetic map of cassava that reported by Wang Wenquan in2010, we choosed the marks in the both ends of each linkage groups, then designed and synthesised six pair of primers, the SSRY28-L9and NS198-L9for the ninth linkage group, the SSRY36-L17and EST68-L17for the seventeenth linkage group, and the SSRY161-L18and EST524-L18for the eighteenth linkage group.A190bp segment could be amplified by SSRY28-L9, and four segments of190-490bp were amplified by NS198. A134bp segment could was amplified by SSRY36-L17, four segments which800bp-1500bp were amplified by EST68-L17. A220bp segment could be amplified by SSRY161-L18, and two segments of250bp-270bp were amplified by EST524-L18.2. The IS-PCR technology system was optimized on the base of the technology system that founded by Feng Yaowen (2011). The total volume of reaction solution for in situ PCR was50μl, which contained5×Buffer10μl, MgCl2(25mM)9μl, dATP(10mM)1μl, dGTP(10mM)1μl, dCTP(10mM)1μl, dTTP(3.6mM)1μl, DIG-11-dUTP (20μM)10μl, Taq DNA polymerase(5U/μl)1μl, F-Primer(20μM)5μl, R-Primer(20μM)5μl, ddH2O6μl. The amplification program for the in situ PCR were as follows:predegeneration at95℃for10min;25cycles denaturalization at94℃for1min, annealing at55-60℃(according to the primers'Tm) for1min, extension at72℃for2min; a final extension at72℃for10min. The pretreatment before in situ PCR:0.01M HCl2minâ†'5ug/ml pepsin treated10minâ†'0.5×TBS10minâ†'sterilized water2×5minâ†'70%formamide denatured at70℃for5minâ†'0.1×SSC ice-bath1minâ†'sterilized water ice-bath1minâ†'75%,90%,100%ethanol dehydrated for3min eachâ†'IS-PCR reaction. The process of after PCR amplification was: 0.1x PBS5min at37â†'5%BSA20min at37℃â†'50μl Anti-DIG-Fluorescein (20μg/ml)1h at37℃â†'0.1×SSC/Tween202×4minâ†'50μl PI(1μg/μl)15min at37℃â†'0.1×SSC/Tween20washed2×1minâ†'coversliped with Vectashieldâ†'fluorescence detection.3. The markers SSRY28and NS198in the ninth linkage group were located on the chromosomes of Manihot esculenta Crantz, cv, M. SC6for the first time by IS-PCR. Two signals for SSRY28and one signal for NS198were detected on the different phases of the SC6. According the karyontype analysis, SSRY28was located on the short arm of chromosome3, the percent distances from centromere to detection site was31.67. And SSRY28was located on the long arm of chromosome3, the percent distances from centromere to detection site was48.44. The result indicated SSRY28and NS198were both located on the same chromosome, but distributed in the different ends, also further revealed that the ninth linkage group was corresponding to the chromosome3of cassava.4. The SSRY36marker in the seventeenth linkage group was located on the chromosomes of SC6for the first time by IS-PCR. Only one signal was detected on the different phases of SC6, according the karyontype analysis, SSRY36was located on the short arm of chromosome2, the percent distances from centromere to detection site was60.17. The result indicated that the seventeenth linkage group was corresponding to the chromosome2of cassava.5. The markers SSRY161and EST524in the eighteenth linkage group were located on the chromosomes of SC6for the first time by IS-PCR. There was only one signal for both of them on the different phases of SC6. According the karyontype analysis, SSRY161was located on the short arm of chromosome10, the percent distances from centromere to detection site was65.21. And EST524was located on the long arm of chromosome10, the percent distances from centromere to detection site was45.90. The result indicated that SSRY161and EST524were both located on the same chromosome, but distributed in the different ends, also further revealed that the eighteenth linkage group was corresponding to the chromosome10of cassava.