Correlations Between Alnus Host Species And Associated Endosymbiotic Frankia Populations From Nodules

Total 58 leaf and nodule samples of Alnus nepalensis were collected naturally occurring at the Laifeng Mountains, Gaoligong Mountains, Jizu Mountains, Cang Mountains and Wuliang Mountains in Yunnan Province. And total 21 leaf and nodule samples of A.sibirica, A.tinctoria and A. mandshurica were collected naturally occurring at the Changbai Mountains, Jilin province of Northeast China. The genomic DNAs of plants and Frankia strains were extracted by improved CTAB method.The RAPD-PCR reaction mixtures and program were refined. Then, the polymorphism of plant DNA was analyzed on optimum condition by RAPD-PCR. Finally, the stable results amplified with 6 primers were analyzed. The DNA molecular UPGMA dendrograms were established by NTSYS software, and Nei's gene diversity and Shannon's information index among the host plants were computed by POPGENE software. The genetic diversity of genomic DNA of Frankia strains was analyzed by rep-PCR and nifD-nif K IGS PCR-RFLP. Then the DNA molecular UPGMA dendrograms of Frankia strains were established.Combined the results of the genetic analysis of corresponding endosymbiotic Frankia strains from nodules with it of hosts, the relationship between Alnus and Frankia in symbiosis was discussed preliminarily. The conclusions were as follows:It was optimum that PCR amplification was performed in 20μL reaction mixtures containing 10ng template DNA approximately, 2μL of 10×PCR buffer, 4mmol·L-1Mg2 +, 0.2 mmol·L -1 dNTP mix, 1μmol·L-1 primer, 2.5u Taq Polymerase. The optimum reaction program included an initial denaturation at 95℃for 5min was followed by 45 cycles, and each cycle comprised of denaturation at 94℃for 30s, annealing at 33℃for 1min and extension at 72℃for 90s. This was followed by an extension time of 10 min at 72℃at the end of the run.The dendrogram showed that A. nepalensis located in Yunnan were divided into six groups on the similarity level of 68 percent. The groupsⅠandⅣalmost spreaded in all five areas, and the other groupsⅡ,Ⅲ,Ⅴ,Ⅵwere peculiarly owned by Gaoligong Mountains, Laifeng Mountains, Wuliang Mountains, Jizu Mountains and Cang Mountains, respectively. Furthermore, every area had the main group, which was also peculiar at Gaoligong Mountains, Wuliang Mountains, Jizu Mountains and Cang Mountains. The Shannon's information indexs of different populations of A. nepalensis were various. The indexs of populations of Jizu Mountains Gaoligong Mountains, Wuliang Mountains, Laifeng Mountains, and Cang Mountains were 0.3929, 0.3489, 0.3472, 0.3247 and 0.3220, respectively. The result of genetic diversity indicated that A. nepalensis occurring in Yunnan Province possessed of an abundant genetic diversity. The genetic diversity of A. nepalensis at Jizu Mountains was the highest, which followed by the populations of Gaoligong Mountains, Wuliang Mountains, Laifeng Mountains, and Cang Mountains orderly. The Shannon's information index of A. nepalensis was 0.3254 along the southern slope, and it was 0.3031 along the northern slope at Wuliang Mountains.In other words, the genetic diversity of A. nepalensis was richer along the southern slope than the northern slope at Wuliang Mountains. And it was positively related to the altitude at the range of 1300 to 2400 meters at Gaoligong Mountains. It was concluded that there was close relationship between the genetic structure of hosts and their geographic distribution. The genetic diversity of A. nepalensis characterized regionality. The distribution of various genetic groups of A. nepalensis is related to altitude. The degree of genetic variation was positively related to the environmental stress.Compared the results of analyzing A. nepalensis naturally occurring at the Gaoligong Mountains in Yunnan Province with A.sibirica, A.tinctoria and A. mandshurica naturally occurring at the Changbai Mountains, Jilin province of Northeast China, it has been found that the species A. nepalensis was likely more primitive than A.sibirica, A.tinctoria and A. mandshurica, A. mandshurica was the most evolutionary species, and the relation of A.sibirica and A.tinctoria is closer. This conclusion is coincident with Chen zhiduan's research.Samples located at Gaoligong and Changbai Mountains were chosen as the representations of the center of origin and of divergence for genus Alnus. Preliminary study of Alnus-Frankia symbiosis demonstrated that the genetic distance of hosts and it of corresponding symbionts Frankia were nearly consistent. It also can be said that the genetic proximity of hosts and the corresponding symbiotic Frankia was similar. And the genetic diversity of populations of hosts and it of Frankia also indicated consistency. It also can be understood that if the host population shows the highest level of genetic diversity, the corresponding Frankia symbionts may also show the highest level of genetic diversity. In this paper, it can be embodied by this result that hosts of A. nepalensis and their symbiotic Frankia populations had the highest genetic diversity, hosts of A. sibirica and A.tinctoria and their symbiotic Frankia populations were intermediate, and hosts of A. mandshurica and their symbiotic Frankia populations were the lowest. In other words, the degree of genetic diversity of the Frankia populations was closely related to the evolutionary ages of their host plants.According to the rep-PCR analysis of Frankia populations from nodules of three species of Alnus from Changbai Mountains and of A. nepalensis from Jizu and Wuliang Mountains, different clusters of hosts and Frankia associated were matched up, and the symbiotic frequency and correlation coefficient were calculated. It was found that the selectivity of host-frankia was shown on genetic level, and it was related to certain environmental factors such as altitude.Various genetic groups of Frankia had various symbiotic faculty. Some could associate with diverse groups of hosts, another could only associate with limited groups of hosts. Every symbiosis possessed a main model(as host group-frankia group).It was suggested that co-evolution of the host and the microsymbiont existed, and the evolutionary pattern may have some similarity. The further genetic matching may be functional in establishment of this symbiotic association. And it was more likely that the common progenitor of genus Alnus entered into an association with Frankia. A. nepalensis-Frankia in Southwest China may be ancient.