The Physical Locations Of Ten Markers Loci Of Genetic Map In Cassava

Manihot esculenta Crantz is an important energy plants. At present, many researchers have constructed some cassava molecular genetic maps by using multiple markers, but its genetic maps had not reach saturation. In these genetic maps, some of them had more than18linkage groups, and some of them had just18linkage groups. Except Wang (2012) who came from our group integrated the chromosomes and LG9、LG17、LG18which were reported by Chen in2010, many correspondences between the genetic map and the karyotype were unknown. In the view of this situation, we used fluorescence in situ PCR technology to locate the10markers physically, these markers were selected from two genetic maps of cassava, then we had integrated the karyotype and linkage groups where different markers sites. This research may establish the foundation for the construction of cassava molecular cytogenetic map, and provide the cytogenetic basis for the selection breeding, genetic engineering, germplasm resource identification, comparative genomic science of cassava. The results were as fellows:1. Two markers were selected in the both ends of sixteenth linkage group(LG16) of the genetic map of cassava that reported by Chen in2010, then two pairs of primers NS376-L16and SSRY86-L16were synthesized. A200bp segment could be amplified by NS376-L16, a295bp segment could be amplified by SSRY86-L16in Manihot esculenta Crantz, cv, M. SC6. According to the third(LG3), fourth(LG4), eighth(LG8), fourteenth(LGH) and sixteenth linkage groups(LG16) in the genetic map of cassava that reported by Sraphet in2011, eight markers were chose, and to be synthesized eight pairs of primers:SSRY180-L3, SSRY252-L3, NS368-L8, NS308-L8, SSRY80-L16, NS271-L14, SSRY75-L4and NS356-L4. Two fragments between140-190bp could be amplified by SSRY180-L3in SC6, and a230bp fragment could be amplified by SSRY252-L3. Two fragments between180-220bp were amplified by NS368-L8, and a140bp fragment could be amplified by NS308-L8. A280bp fragment could be amplified by SSRY80-L16, and a220bp fragment could be amplified by NS271-L14. A320bp fragment could be amplified by SSRY75-L4, and two fragments between240-300bp were amplified by NS356-L4.2. The IS-PCR technology system was optimized on the system that founded by Chao Wang (2012). The amplification program for the In situ PCR was as follows: pre-denaturation at94℃for4min,35cycles denaturalization at94℃for30s, annealing at55℃(according to the primers’Tm) for30s, extension at72℃for30s, afinal extension at72℃for5min, save at16℃for10min. The process of after PCR amplification was:0.1×PBS5min at37℃→5%BSA20min at37℃→50μlAnti-DIG-Fluorescein (20μg/ml)1h at37℃→0.1×SSC/Tween202×4min→coversliped with25μl DAPI mixture (using DABCO to diluted DAPI, and makes DAPI final concentration reached2μg/mL)→fluorescence detection.3. The markers NS376and SSRY86from the LG6which was reported by Chen in2010were located on the chromosome of SC6by IS-PCR. The markers NS376and SSRY86were sequentially located on the short arm and long arm of the chromosome4of SC6, the percent distances from centromere to detection site sequentially were31.25and75.86. By the karyotype analysis, we found the marker NS376and SSRY86were located on the same chromosome, but distributed in the different ends, also further revealed the LG16which from Chen (2010) was corresponding to the chromosome4of cassava.4. The markers SSRY180and SSRY252from the LG3which was reported by Sraphet in2011were located on the chromosome of SC6by IS-PCR. The markers SSRY180and SSRY252were sequentially located on the short arm and long arm of the chromosome7of SC6, the percent distances from centromere to detection site sequentially were67.39and30.12. By the karyotype analysis, we found the marker SSRY180and SSRY252were located on the same chromosome, but distributed in the different ends, also further revealed the LG3which from Sraphet (2011) was corresponding to the chromosome7of cassava.5. The markers NS368and NS308from the LG8which was reported by Sraphet in2011were located on the chromosome of SC6by IS-PCR. The markers NS368and NS308were sequentially located on the short arm and long arm of the chromosome5of SC6, the percent distances from centromere to detection site sequentially were83.34and40.46. By the karyotype analysis, we found the marker NS368and NS308were located on the same chromosome, but distributed in the different ends, also further revealed the LG8which from Sraphet (2011) was corresponding to the chromosome5of cassava.6. The markers SSRY80from LG16and NS271from the LG14which were reported by Sraphet in2011were located on the chromosome of SC6by IS-PCR. The SSRY80and NS271were sequentially located on the short arm and long arm of the chromosome13of SC6, the percent distances from centromere to detection site sequentially were69.30and53.44. By the karyotype analysis, we found the marker SSRY80and NS271were located on the same chromosome, but distributed in the different ends, also further revealed the LG16and LG14which from Sraphet (2011) were corresponding to the chromosome13of cassava. And showed they may be integrated to a new linkage group.7. The markers SSRY75and NS356from the LG4which was reported by Sraphet in2011were located on the chromosome of SC6by IS-PCR. The markers SSRY75and NS356 were sequentially located on the short arm and long arm of the chromosome6of SC6, the percent distances from centromere to detection site sequentially were25.70and63.47. By the karyotype analysis, we found the marker SSRY75and NS356were located on the same chromosome, but distributed in the different ends, also further revealed the LG4which from Sraphet (2011) was corresponding to the chromosome6of cassava.