Studies On EST-SSR Markers And Genetic Transformation Of Sweet Potato (Ipomoea Batatas(L.) Lam.)

Sweet potato (Ipomoea batatas (L.) Lam.) is the seventh most important foodcrop in the world after wheat, rice, maize, potato, barley, and cassava. However, thisspecies has received relatively little genetic research attention.First, the germplasm of sweet potato maintained at CIP (The InternationalPotato Center) needs effective genetic tools to identify and evaluate. In additionthere are many basic questions of sweet potato which are not yet defined, such asthe exact center of origin and domestication of sweet potato; the wild ancestor ofthis species; an alloploid or an autopolyploid of L batatas; the donor of sweet potatoof Oceania.To address above issues, we developed a highly informative and easily scored.set of expressed sequence tags (EST)-simple sequence repeat (SSR) markers. ESTmicrosatellite markers were isolated from the expressed sequence tags of sweetpotato shoot and root. Twenty-five SSR primers were designed and used to screenand test the levels of polymorphism and transferability. Ten out of 25 SSR palmerswere high quality polymorphic markers and have been used for genotyping.Amplification from 9 primer pairs across sweet potato and L trifida indicated thatthe sequences flanking the SSR are not only conserved in sweet potato (6X) but alsoin related species (2X). The nine markers detected 66 polymorphic alleles with anaverage number of 7.33 per locus and allele size from 169 to 393 bp. The coefficientof similarity was ranged from 0.74 to 0.96. The polymorphism of the EST-SSRmarkers was from 0.4230 to 0.8718, with a mean of 0.7145. The actualheterozygosity varies greatly across the 9 loci from 0.30 (IB-S18) to 0.99 (IB-S10)with the mean of 0.71.The EST-derived SSR primers were applied to analyze the diversity of sweetpotato landraces in CIP. One hundred and twenty of landraces from South America (Peru, Ecuador, Venezuela and Colombia), Mesoamerica (Mexico, Nicaragua,Guatemala and Honduras), and Oceania countries (Papua New Guinea, SolomonIslands, Tonga, Fiji and Cook) were analyzed using the 9 EST-SSR primer pairs.The total number of alleles and number of alleles per locus are not evenlydistributed in different geographic regions. The cluster analysis revealed a largegenetic variation in Mesoamerica and Oceania gene pool, far greater than that inPeru-Ecuador. The Guatemala landraces were grouped together with those ofOceania.The results from EST-SSR assay suggest that: I. Sweet potato is a hexaploidyoutbreeding species; 2. I. trifida is most likely one of the diploid progenitors in theorigin of hexaploid I. batatas; 3. Mesoamerica could be origin and domesticatedcenter of sweet potato; 4. Mesoamerica is an alternative explanation for the majororigin of the Oceania sweet potato in stead of Peru-Ecuador; 5. A small part ofaccessions of Oceania associated with Peru-Ecuador germplasm implies thepossibility of prehistory human transfer as" Kumara hypothesis" clamed. Therefore,EST-SSR could be successfully used for sweet potato germplasm conservationmanagement, such as more-direct estimation as functional diversity, genotyping,evolutionary relationships among taxa.Conventional breeding based on sexual hybridization of the sweet potato isdifficult due to its hexaploid genome and self incompacity. The modem geneticrecombination technology shows a promising future for genetic improvement ofsweet potato. The genetic transformation of sweet potato via Agrobacteriurn startedaround 10 years ago but high transformation efficiency is still a major bottleneck.Therefore, it is urgent to develop a completely new transformation protocol forimproving the rate of shoot production, which should be rapid andgenotype-independent.The embryogenesis of an elite cultivar of Africa sweet potato, which was verydifficult to induce embryogenic calli in the transgenic research, was studied. Theinduction conditions of embryogenic calli were optimized for cv. Tanzania. Theresults showed that the rate of embryogenic calli was increased up to 50ï¼…when 1 mg/l 4-fluorophenoxyacetic acid (4-FA) was added in the medium. However thefrequency of plantlets converted from the embryogenic calli were still less than10ï¼….Eight cultivars of China were tested for their transformation efficiency. Anattempt to develop transgenic sweet potato via embryogenesis was made. Producingof recombinant lactoferrin protein has been performed by transformation withAgrobacterium turaefaciens LBA4404, which harbors plasmid pSCL233 containinga human lactoferdn gene (hLFc). Transformation frequency is very different amonggenotypes, from 0ï¼…to 69.35ï¼…. The transgenic events were evidenced by PCR.Finally, we have successfully established a new, rapid and highly effectiveAgrobacterium-mediated transformation method. This strategy was based on denovo (via callus)organogenesis from the petiole with leaf lamina explants of sweetpotato. Stable transgenic sweet potato plants cv. Jewel were obtained in only four tosix weeks after infection with A. turnefaciens hyper-virulent strain EHA105, whichharbors a binary vector pBIN20-D with neomycin phosphotransferase nptâ…¡geneand the dhdps-r1 gene for overproduction of lysine. PCR and Southern analysesconfirmed stable integration of both genes into the sweet potato genome. Theexpression of the nptâ…¡gene was assessed by reverse-transcribed PCR. All thetransgenic lines were survived and had a morphologically normal phenotypeinspected by pot experiment in green house.In conclusion, this organogenesis-based transformation protocol leading to theproduction of transformed plants in 4-6 weeks will facilitate the geneticimprovement of this crop. It will also be very useful for sweet potato varietiesrecalcitrant to regeneration via somatic embryogenesis. Combination with theresistant assay by tissue culture, this efficient process will be useful for producinglarge number of events needed for a transgenic sweet potato improvement.