| Microsatellite marker, a widely used molecular marker in plant biology and breeding research, is still limited in number for Citrus. This study was to develop a large set of microsatellite markers based on publicly available BAC-end sequences (BESs) and EST sequences, and evaluate their transferability, discriminating capacity of genotypes, mapping ability, performance as a population genetic markers and linkage analysis in Citrus.1. A total of 1025 and 3831 pair primers were successfully designed from the publicly available BES and EST data sets. Our result reveal that 93.92% and 85.06% SSR containing EST and BES sequences, respectively, were suitable for SSR primer modeling. We also found that the SSRs in the Citrus genome tended to skew toward AT rich motifs. Four hundred BES-SSR and 439 EST-SSR primer pairs were assessed to detected PCR amplification rate in the sixteen Citrus and its related genera. The successful PCR amplification rate was 333 (83.25%) for BES-SSR and 368 (83.83%) for EST-SSR primer, respectively.2. Blast search identified 2613(76.22%) EST and 236(27.03%) BES sequences matched with several known proteins. Functional annotation result indicated that 60.12% for EST and 16.38% for BES sequences had similar function with public database. The sum of BES and EST SSR sequences per category did not add up to 100%, as some sequences were classified into more than one category. The GO categorization showed that 40.78% for EST and 13.52% for BES-SSR sequences were homologus to proteins with molecular function. On the other hand 43.82% for EST and 0.57% for BES-SSR sequences were homologus to protein involving with cellular component. Finally,47.17% EST and 11.00% BES-SSR sequences were classified as involvement in biological process.3. Transferability of the BES-SSR and EST-SSR markers was tested using 16 genotypes from Citrus and its relative's genera. Result showed that 76.99% and 65.00% tested markers were transferable across the genera for BES-SSR and EST-SSR markers, respectively. Hexa nucleotides repeats were highly transferable across the genera in both BES-SSR and EST-SSR. Citrus EST-SSR was less polymorphic than the Citrus BES-SSR. We also found that SSR derived from 3'end of EST sequences were superior to those from 5'end of EST sequences. The sequences similarity among the 16 tested genotypes of the EST-SSR primer amplified region was higher than that amplified with BES-SSR primer. Each pair genotypes sequences similarity ranged from 92 to 100 for EST-SSR primer and from 52 to 100 for BES-SSR marker.4. In order to evaluate the utility of EST-SSR and BES-SSR markers for mapping in each putative F1 progeny, we have considered percentage of heterozygous loci, polymorphic heterozygous loci and percentage mappable loci between two genotypes in 169 putative combinations. The higher percentage of heterozygous loci for each Citrus genotype was recorded from BES-SSR marker than that from EST-SSR. We found that Tangerine and Sweet orange were highly heterozygous species; Trifoliate and Sour orange was less and moderate heterozygous species, respectively. In this study combinations including interspecific hybrids such as Grapefruit, Citron, Pummelo, Tangerine and Mandarine gave the higher percentage of heterozygous loci polymorphic which were suitable for comparative genetic maps. In addition, interesting progenies could be obtained from F1 hybrids between Lime and Pummelo, Tangerine and Trifoliate, Lemon and Tangerine, Pummelo and Lemon. 5. BES-SSR and EST-SSR marker techniques have been compared for investigating the genetic diversity among 35 Citrus spp. Our results revealed that each marker technique was capable of detecting genetic variation in Citrus. The highest level of polymorphism was obtained from both type of SSR analysis. The average confusion probability value was lower in BES-SSR than in EST-SSR. The discriminating capacity negatively correlated to the confusion probability, revealing the higher and lower value for the BES-SSR and EST-SSR, respectively. Average limit of discriminating power were very close to the actual discrimination power of each marker. The higher value of effective number of patterns per assay unit (P) was recorded for the BES-SSR (27.48). The values for total number of effective alleles, Assay efficiency index, Effective multiples ratio and Marker index were very similar in both marker system. Phylogenetic analyses of BES-SSR and EST-SSR marker are generally in good agreement with the previous studies. Both marker techniques generated a high degree of similarity in terms of dendrogram topology, though with differences in the positioning of some species.6. Employment of SSR markers in population genetic applications has been limited in Citrus. For the estimation of the utility of BES-SSR and EST-SSR derived primers as population genetic markers in Citrus, we compared the performance of BES-SSR with EST-SSR in characterization population genetic structure for the same set of individuals. The result showed that population genetic parameters generated by both types of SSR were very similar for the same set of individuals from three populations.7. In this study, a total 1291 markers including our developed EST-SSR (524) and BES-SSR (333) markers and publicly available Citrus SSR (434) markers were analyzed on trifoliata orange and red tangerine and their F1 progeny, in order to construct high density linkage map of Citrus. Over all 36.64% of the SSR markers showed putative map ability on trifoliata orange and red tangerine mapping population. Three hundred eighty two loci were found to be segregating among the mapping population in which 66 loci were skewed. A total 313 loci allowed the construction of the frame work map of Poncirus trifoliata and Citrus reticulata. Linkage analysis revealed that among the 313 loci 227 were linked and the rest 86 unlinked loci. Linked loci were distributed among the 15 linkage groups and covering 24293.9 mM map with an average 107.02mM distance per loci. Forty five distorted loci were placed in this frame work map. In addition, most of the distorted loci were placed on the first linkage group.In this work, we developed and characterized a large set of novel SSR markers from the EST and Clementine BES sequences; it will be a valuable resource for the Citrus breeders and useful for mapping and other aspects research in Citrus. So far our knowledge till to date, in this study we used the highest number of molecular markers to developed a frame work map in Citrus.
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