Development Of PIP And SSR Markers And Their Applied Research In Nicotiana

It is an urgent to distinguish major tobacco cultivars from the molecular level because oftheir relatively low polymorphism and morphologically similar. In this paper, usingbioinformatics approaches，a total of1,223,537accessions of N. tabacum genomic sequencesand171,570EST sequences of four Nicotiana species (N. tabacum, N. sylvestris, N.benthamiana and N. langsdorffi×N. sanderae) were downloaded from the TGI andPlantGDB databases for designing PIP and SSR markers. Combined using this two markers toanalysis genetic diversity in64Nicotiana germplasms and construct a DNA fingerprinting of9major tobacco cultivars, we draw the conclusion as follows:1. A total of12,388PIP and76,848SSR markers were designed and uploaded to aweb-accessible database (http://yancao.sdau.edu.cn/tgb/).2. E-PCR analysis showed that PIP and SSR rarely overlapped and were stronglycomplementary in the tobacco genome. The density was3.07PIP and1.72SSR markers per10kb of the known sequences.3. For real PCR, a total of153and166alleles were detected by22PIP and22SSRmarkers in64Nicotiana accessions. SSR produced higher PIC (polymorphism informationcontent) values and identified more alleles than PIP, whereas PIP could identify largernumbers of rare alleles.4. Mantel testing demonstrated a high correlation coefficient (r=0.949, P <0.001)between PIP and SSR. The UPGMA dendrogram created from the combined PIP and SSRmarkers was clearer and more reliable than the individual PIP or SSR dendrograms. Itsuggested that PIP and SSR can make up the deficiency of molecular markers not only intobacco but also in other plants.5. A DNA fingerprinting was constructed of9major tobacco cultivars using SSR.21SSR primer pairs with high polymorphisms and good repeatability were screened out from62candidates as core primers to construct the fingerprinting database. A total of125alleles weredetected by21SSR markers in9accessions with an average of6alleles per locus. Thenumber of alleles ranged from2to12. The PIC value ranged from0.37to0.86with anaverage of0.67. There were9primers amplified specific amplifications in5accessions. Inaddition,9major cultivars could be completely identified using4primers. Therefore, it’s a promising prospect of application in purity identification of major tobacco cultivars byfingerprinting.