| Researches on genetic diversity of Nelumbo are mainly focused on Chinese lotus with abundant variety resources. About the evaluation of genetic diversity among natural populations of American lotus, Thai lotus and Chinese lotus, few reports was involved. Construction of a genetic linkage map is a powerful research tool for studies on plant genetics and analysis of quantitative trait loci (QTL) underlying traits of economic importance, however, little progress on the genomic constitution of Nelumbo species has been reported. In this studies, genetic diversity and the evolutionary relationship among wild lotus populations in its main distribution area are analyzed. And based on an F1population derived from a cross between Nelumbo nucifera’China Antique’ and Nelumbo lutea’AL1’, a high-density genetic linkage map of lotus has been constructed using SSR and SNP markers which are discovered by restriction site associated DNA sequencing (RAD-Seq). The main results are as follows:1Assessment of genetic diversity among wild lotus populations1.1Wild lotus had a high genetic diversity at species level (I=0.95, He=50and PIC=0.46), and a unique evolutionary history of lotus was likely to be the reason for this. However, a low genetic diversity was revealed at population level. And three possible factors might contribute to the low genetic diversity within populations. Firstly, habitat destruction resulted in the reducing or loss of distribution area and population numbers of wild lotus. Secondly, the natural habitats of wild lotus had become fragmented and isolated, which increased the geographical distance and blocked gene flow between populations. Finally, the specificity of lotus’ breeding system was the main reason for its low genetic diversity.1.2The populations from N. lutea were separated from those from N. nucifera at a similarity coefficient of0.14with the bootstrap of100%, which confirmed that N. lutea and N. nucifera were genetically distinct. And the ranks of genetic distance between lotus populations are AmL&ThL> AmL&ChL> ThL&ChL. The population AmL was related more closely with the population ChL than with the population ThL. 1.3The paper supported that N. nucifera and N. lutea are the two independent species of Nelumbo. According to the fossil evidence, very high similarity of rbcL gene and nuclear DNA ITS sequence, high homology in serological reaction and chromosome analysis, N. nucifera and N. lutea were considered as one species. But on the basis of the facts that N. nucifera and N. lutea were separated by Pacific, and had a certain barrier of reproductive isolation, greater genetic distance, significant genetic variation, divergence in plant traits and the self-imcompatibility of F1plants, the author supported that N. nucifera and N. lutea are two independent species of Nelumbo.1.4Some actions about the protection of lotus population are suggested as follows. Firstly, try to protect the natural habitats of wild lotus from damage. Second, it is imperative to preserve in situ all the populations surveyed to conserve genetic diversity and evolutionary processes. Finally, ex situ conservation could also be applied because of the low genetic diversity within populations. And seeds and rhizomes could be harvested for ex situ conservation.2Construction of a high-density genetic linkage map of lotus2.1A framework map of lotus was constructed. The map for female parent N. nucifera ’China Antique’ was362.52cM with32markers distributed in eight linkage groups. The mean genetic distance between markers was11.33cM and the mean length of linkage group was45.315cM. The map for male parent N. lutea ’AL1’ was495.37cM with137markers distributed in11linkage groups. The mean genetic distance between markers was3.62cM and the average length of linkage group was45.03cM.2.2RAD-seq technology was an effective way to discover rapidly thousands of SNP markers. In line with the reference genomes of N. nucifera’China Antique’, we identified6622potential SNP markers for lotus map construction using high-throughput genotyping of F1population of RAD system.2.3A high-density genetic linkage map of American lotus ’AL1’ was constructed on the basis of a SSR framework map. And the map mainly consisting of RAD markers was563.6cM with1515markers distributed in ten linkage groups. The average distance between markers was0.38cM. There were95to194markers per linkage group, and the genetic distance of each linkage group varied from39.9cM to83.8cM. 2.4RAD was a powerful molecular marker of increasing marker density and extending linkage groups. In this project, compared with the framework map in common with SSR markers, the high-density map mainly consisting of RAD markers contained1378more markers and increased genetic distance of68.23cM in length, and the average distance between markers was decreased by3.24cM.2.5We found that marker segregation distortion was unevenly distributed on linkage groups. There were no markers deviated significantly from Mendelian segregation ratios on LG5, LG7and LG9. Two and three markers of segregation distortion were observed on LG3, LG8and LG10. Many markers on LG1(21), LG2(19), LG4(80) and LG6(127) showed moderate segregation distortion. And obvious clusters of deviated marker loci were investigated on LG4and LG6, which might be explained by the following factors:1) in order to improve the polymorphism of F1population, Nelumbo nucifera ’China Antique’and Nelumbo lutea ’AL1’, diverging strongly in their geographical distributions and important morphological traits, were chosen as parents for hybridization.2) the high level of homozygosity of the female parent Nelumbo nucifera ’China Antique’, which was unexpected, mainly contributed to the considerable segregation distortion in the F1population.3) the number of individuals in F1population was not very large. |
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