| Tobacco(Nicotiana tabacum L.) is an important annual or perennial economic crop and is also one of the model plants earliest used in molecular biology and genetic engineering. Molecular markers are an important genetic tool for genetic map constructing, gene mapping, genome comparison and genetic diversity. Because of its complex genetic basis, huge genome content, narrow genetic base and low level of polymorphism etc, development and utilization of molecular markers in allotetraploid tobacco are lagging behind, which become a severely problem for researches in tobacco (especially the flue-cured tobacco) genetics and molecular breeding.Developed in the1990s and based on PCR, simple sequence repeats (SSR) is a DNA molecular marker. With significant advantages including evenly distributed in whole genome, highly variable, highly reproducible, generally co-dominant, easy to use and abundant in eukaryotic organisms, SSR is widely used in various fields of life sciences. The large-scale development of SSR molecular markers in tobacco have great significance not only for construction of genetic linkage map, gene loci tagging and map-based cloning, genomic structure and evolution of Nicotiana tabacum L, but also for marker-assisted selection breeding of tobacco.In this study, based on partial genomic data and EST sequences of tobacco, we have developed large-scale tobacco SSR markers and further applied to construction of a genetic map of flue-cured tobacco and mapping of quantitative trait loci underlying six yield-related agronomic traits in flue-cured tobacco(Nicotiana tabacum L.). The main results are summarized as follows:1, Based on the partial genome sequence data (released by Tobacco Genome Initiative: TGI) and the EST sequences data (download from the public data bank, NCBI and TIGR) of Nicotiana tabacum L., we designed5,167simple sequence repeats (SSR) primer pairs. A total of4,886SSR primer pairs including1,365genomic SSR primer pairs (with a prefix of TM: abbreviation of Tobacco Microsatellite) and3,521EST-SSR primer pairs (with a prefix of TME:abbreviation of Tobacco Microsatellite in EST) could obtain the target amplification products, of which676TM markers (49.52%) and216TME markers (6.13%) showed polymorphisms among8cultivated tobaccos (belonging to four different types). The number of alleles and the polymorphic content (PIC) of each TM/TME marker ranged2-6/2-4and0.219-0.781/0.219-0.656with an average of2.88/2.20and0.413/0.298.2, A total of10,005SSR primer pairs (containing two parts:4,886TM/TME primer pairs developed by ours;5,119PT primer pairs published by Bindler et al.(2011)) were initially screened with two map parents, of which590SSR primer pairs showed clear and stable polymorphism. Based on the selection of SSR primer pairs,207DH individuals were analyzed and finally613SSR markers (loci) were detected. By using JionMap version4.0software, a genetic map of flue-tobacco consisting of611SSR loci mapped into24linkage groups was constructed. The map covers a total length of1882.1cM with an average distance of3.08cM between adjacent markers. Each single linkage group of this map comprised of13to58SSR loci and covered28.1cM to120.5cM, with an average distance between markers from1.87cM to5.33cM. All the SSR markers distributed evenly among the linkage groups without clustering of loci. This was the first relatively saturated genetic linkage map of flue-cured tobacco based on SSR markers.3, In this study, we also compared our map constructed based on an intra-type cross (between two flue-cured tobaccos) with the map constructed based on an inter-type cross (between a flue-cured tobacco variety and a specialty variety) by Bindler et al.(2011) through the common SSR markers (PT markers) in the two maps. And the results of collinearity between two tobacco linkage maps suggested that there might be large chromosome structure variations among different tobacco types.4, Based on the newly constructed SSR genetic linkage map of this study, QTL mapping and analyzing of six yield-related agronomic traits in flue-cured tobacco was performed with composited interval mapping (CIM) method using phenotypic data from DH individuals. A total of69QTLs were detected on20linkage groups (excluding LG9,16,21and23). Thirteen QTLs for plant height (PH) and internode length (IL) were detected on ten and nine LGs, respectively; nine QTLs for stem girth (SG) were detected on8LGs; ten QTLs for leaf number (LN) were detected on9LGs and twelve QTLs for width of the largest waist leaf (WWL) and length of the largest waist leaf (LWL) were detected on7and10LGs. Closely linked markers to target trait are substantial base for marker-assisted selection (MAS). In this study, closely linked SSR markers detected for four QTLs (belonging to IL, PH and WWL three traits), together with flanking SSR markers, could be used in MAS for tobacco breeding. |
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