| Genus Nelumbo, belongs to Nelumbonaceae family, comprise of two species namely Nelumbo nucifera Gaertn.and Nelumbo lutea (Willd.) Pers. As the only one aquatic plant of the top ten famous Chinese traditional flowers, lotus (Nelumbo nucifera) owns high ornamental and economic values because of its long cultivation history, unique cultural and religious significance, ornamental and edible uses. During its long history of cultivation with more than two thousands years, abundant variations arose. Meanwhile, Nelumbo germplasm of different geographical origins are genetically differentiated which is probably due to extended periods of geographical isolation.In recent years, lots of researches of genetic diversity and cultivars classification of genus Nelumbo on morphological characters, cytology, isozyme and DNA molecular markers were performed, but it was poorly known about genetic relationships and genetic diversity of Nelumbo germplasm of different origins or different eco-types. In addition, few researches on pollen morphology were made, and few analyses were using different types of genetic markers study on genetic diversity of Nelumbo germplasm.In this study, genetic diversity and genetic divergence of Nelumbo germplasm of different origins were analysed using RAPD,1SSR molecular markers and pollen morphological markers in order to provide a basis for germplasm or cultivars identification, conservation, classification and breeding.The main results were obtained as follows:1. Research of 87 Nelumbo germplasm using RAPD and ISSR analysis(1) RAPD markers and ISSR markers were used to investigate the genetic diversity and genetic relationships among different germplasm of 87 Nelumbo germplasm (including 70 Chinese ornamental cultivars,7 wild Thai genotypes,2 Nelumbo lutea genotypes and 8 hybrids of Nelumbo nucifera and Nelumbo lutea).11 selected RAPD primers yielded a total of 111 amplified bands, of which 107 were polymorphic (96.4%), and the avenue value of Jaccard’s similarity coefficient was 0.49.13 selected ISSR primers yielded a total of 205 fragments and 187 of them (91.2%) were polymorphic, and the avenue value of Jaccard’s similarity coefficient was 0.77. The Nei’s genetic diversity index and the Shannon’s information index were RAPD (0.17,0.28) and ISSR (0.20,0.33) respectively, both suggesting that the relatively high level of genetic diversity existing in those 87 Nelumbo accessions. (2) Clustering analysis using both RAPD and ISSR data revealed that the clustering pattern of Nelumbo germplasm was tightly linked with their geographical origins. The combined dendrogram was constructed based on RAPD and ISSR data showd N. lutea and N. nucifera germplasm were clustered separately which indicated significant genetically differentiated occurs. Morever, the Thai wild genotypes and Chinese genotypes showed genetic divergence. And then the germplasm of different origins was suggested as a categorical standard added into the current lotus cultivars classification system.(3) Molecular markers could help the morphlogical character markers for identifying lotus cultivars more easily and precisely. However, low correlation between molecular cluster and morphlogical character classification, only the data on plant size was consistent with the molecular dendrogram to some extent.The reasons for the genetic divergence in those Nelumbo germplasm of different origins were possibly as follows. Firstly, geographical isolation and lack of gene exchange. Secondly, geographical location appears to have affected genetic diversity due to adaptation of the lotus plants to the different environments. Therefore, in the future breeding program, the breeding materials should be introduced from different regions, and parental lines from diverse regions should be utilized on purpose so as to make the most of the genetic diversity reserved in the different germplsm.2. Research of 31 Nelumbo germplasm using pollen morphology analysis(1) Quantitative and qualitative analysis were assessed on pollen morphology of 31 Nelumbo germplasm (including 10 Chinese ornamental cultivars,1 Nelumbo lutea genotypes,6 Sino-American hybrids,5 Thai genotypes, and 9 Sino-Thai hybrids), which presented: all pollen morphology is subglobose and the shape of equator view is oval; there have two types of polar view shape,3-labed-rounder type and suborbicular type, most accessions are 3-labed-rounded type; all accessions have 3 colpus, besides several accessions also have 6 colpus. According to the NPC system classification, most accessions belong to N3P4C3 type. The pollen grain size varied in different Nelumbo germplasm which ranged from 52.19 x 55.90μm to 41.93 x 43.98 um (polar axis length x equator axis length).(2) There have a lot differences in the colpus characters, namely:shape of colpus, length, width and depth of colpus, arrange pattern in colpus are all showed differences in varied extent. These microscopic differences indicated genetic diversity existing in those 31 Nelumbo accessions. Meanwhile the style of lirae has two types, namely most accessions are showed cerebroid lirae, minority accessions are rugulate lirae type. The shapes of lumina, the width and depression of lirae are also varies in different germplasm indicated genetic divergence existed.(3) In principal component analysis, the first 7 principal components, from which the cumulative contribution reached 89.0936%, corresponded to the 7 characters, i.e. pollen size, L/P (length of colpus/length of polar axis), style of pollen, shape of colpus, character of lirae, width of lirae and shape of pollen.(4) By means of Q cluster analysis, the Nelumbo accessions used in this study were categorized into two main groups at first. The main differences between the two groups were mainly reflected in pollen size, length of colpus, style of pollen, width and depth of colpus. The main characters of the two groups obtained by Q cluster were tightly linked with the first 3 principal components by principal component analysis. However, compare with the molecular markers cluster, the pollen morphology cluster didn’t show Chinese germplasm were clustered separately with either N. lutea genotypes or Thai germplasm, but still show differences in the cluster analysis. The reasons for the cluster divergence using molecular markers and pollen morphological markers were possibly as follows, firstly, there have some differences of Nelumbo accessions used in the two experiments. Secondly, the genomes dectected by the two kinds of genetic markers are different. Thirdly, data analysis methods used in the two kinds of genetic markers are different.The obvious differences of pollen morphology of those 31 Nelumbo germplasm, especially in the characters of colpus and lirae would well reflected genetic diversity and genetic divergence. This study considered that pollen morphological markers could provide valuable basis for genetic relationship analysis in Nelumbo germplasm but not suitable to apply directly in Nelumbo germplasm classification.Therefore, combined analysis using different types of genetic markers would supply a more comprehensive understanding of genetic diversity in the current Nelumbo germplasm. |
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