Studies On Genetic Diversity And Phylogenetic Relationship Of Nelumbo Nucifera By Srap And Issr Markers

As we all know, N. nucifera is one of the ten traditional flowers and important aquatic cash crops in China, and rich genetic diversity exists among them. In this study, SRAP and ISSR were used to analyze the genetic diversity and phylogenetic relationship of64N. nucifera accessions, which provided the basis for the classification systerm, utilization and genetic improvement of N. nucifera germplasms. The main results were as follows:1. Optimization and establishment of SRAP-PCR reaction system for N. nuciferaN. nucifera Gaertn.’Xin Hong’ leaves were used as the material. By the orthogonal experiment design L16(45), five factors including Mg2+, dNTPs, TaqDNA polymerase, primer and template DNA in SRAP-PCR reaction system were optimized, and the optimization SRAP-PCR reaction system which was suitable for N. nucifera were also established. The optimal SRAP-PCR reaction system was as follows:total volume10μL including2.0mmol·L-1Mg2+,300μmol·L-1dNTPs,0.5U TaqDNA polymerase,4μmol·L-1primer,50ng DNA and10×PCR buffer. The order of each factor in different levels was:TaqDNA polymerase>Mg2+>primer>dNTPs>DNA. The optimal SRAP-PCR reaction system was identified by means of genomic DNA from48N. nucifera varieties And the amplification pattern with clear band and rich polymorphism was obtained. It was concluded that the SRAP-PCR reaction system was steady and reliable.2. Germplasms relationship and genetic diversity of N. nucifera revealed by SRAP markerThe genetic diversity and relationship of64flower lotus germplasms belonging to13populations were analyzed by SRAP marker. The results showed that:(1) Total253bands were detected with21pairs of SRAP primers, and233of253were polymorphic with PPB as92.10%, the Nei’s gene diversity index (H) was0.3941, the Shannon diversity index (1) was0.5741. These indicated a rich genetic diversity.(2) The genetic similarity and the genetic dictance among the varieties were ranged from0.4110-0.8814and0.1262-0.8890 respectively, which indicated that some germplasms existed close genetic relationgship and some germplasms had a relatively lower GS than the others.(3) The clustering results showed that the64germplasms were clustered into two clusters which were N. nucifera Gaertn, the hybrids of N. nucifera Gaertn and N. lutea (Willd). Moreover all of the wild flower lotus accessions were clustered into the N. nucifera Gaertn group, and most of the proliferation flower cultivars were clustered into the double-petalled groups. The hybrids of N. nucifera Gaertn and N. lutea (Willd) can basically been clustered into double-petalled group and few-petalled group, and the double-petalled cultivars could been clustered into big plant type group and medium-small plant type group. The clustering results indicated that morphological characterstics and genetic relationship had a certain degree of correlation.3. Germplasms relationship and genetic diversity of N. nucifera revealed by ISSR markerThe genetic divercity and relationship of64flower lotu germplasms belonging to13populations were analyzed by ISSR marker.The results showed that:(1) Total156bands were detected with12ISSR primers, and127of156were polymorphic with PPB as81.41%, the Nei’s gene diversity index(H) was0.3715, the Shannon diversity index (1) was0.5409. these indicated a rich genetic divercity revealed by ISSR makers.(2) The genetic similarity and the genetic dictance among the germplasms were ranged from0.4259-0.8910, and0.4808-0.9580respectively, which indicated that some germplasms existed close genetic relationgship and some germplasms had a relatively lower GS than the others.(3) The clustering results showed that the64varieties were clustered into two clusters which were N. nucifera Gaertn and the hybrids of N. nucifera Gaertn and N. lutea (Willd), all of the wild flower lotus germplasms clustered into the N. nucifera Gaertn group, most of the proliferation flower cultivars clustered into the double-petalled gtoups. The hybrids of N. nucifera Gaertn and N. lutea (Willd) can basically cluster into double-petalled group and few-petalled group,and the double-petalled accessions can cluster into big plant type group and medium-small plant type group. The clustering results indicated that morphological characterstics and genetic relationship had a certain degree of correlation. This study not only clarified the phylogenetic relationship among the different species and different groups, also provided the scientific basis of the molecular aspects of the lotus classification systerm. 4. Comparision on the results of the diversity of Nelumbo nucifera revealed by SRAP and ISSR markerThe result revealed by two makers were very consistent by comparing the genetic diversity and relationship revealed by SRAP and ISSR makers. The clustering results between the two markers showed that the64varieties could been clustered into two clusters which were N. nucifera Gaertn and the hybrids of N. nucifera Gaertn and N. lutea (Willd). Moveover, all of the wild flower lotus accessions were clustered into the N. nucifera Gaertn group, most of the duplicated-petalled accessions could been clustered into the double-petalled groups. Sino-American Hybrids can basically been clustered into double-petalled group and few-petalled group, and the double-petalled accessions can been clustered into big-plant type group and medium-small plant type group. Correlation analysis was conducted between the two markers, and the result showed that the GS based two markers had a significant correlation (r=0.8560>r0.01, N=2016), which was further verified by the similarity of clustered results Therefore, the two markers can be effectively used for the study of genetice diversity and genetice relationship of lotus resources.