Studies On Microtructure And Molecular Phylogenetics Of Nitraria. L In China

The genus of Nitraria.L was a most important constructive species in the desert ecosystem, and possessed a significant eco-efficiency and economical benefits. China was one of distribution and evolution centres of Nitraria.L, and contained abundant germ plasm. It was taken more and more attention because of its ecological and scientific value.The genus of Nitraria.L consists of seven species distributed widely in northwest of China, which including:N.tangutorum Bob, N.roborowskii Kom, N.sibirica Pall, N.sphaerocarpa Maxim, N.praevisa Bobr, N.schoberi, N.pamirica Vas, et al. In this dissertation, the ultra-micro-structure of the leaves and stem, the secondary metabolites and abio-element of the leaves, systematic relationship of the genus Nitraria.L were studied. Based on the full-scale sampling,reasonable experiment design and scientific data analysis, we concluded as follows:1. It was showed that the genus of Nitraria.L was a typical xerophil plant by the ultra-micro-structure observation.The results of the scanning electronic microscope of the leaves shows that there were xerophilly characters in the genus of Nitraria.L and some specialities among the species. The commonness was:the leaves possesses thick keratinization and with waxed epidermal hairs more or less on the face or back. The density of the stoma on the leaves was very obvious, which sunkened in a room shape and rounded with waxed granules. The palisade tissue and the vascular bundle sheath cell was very abundant. The mesophyll cells were fulfilled with white crystal material. The results of the scanning electronic microscope showed that the genus of Nitraria.L was a xerophilly plant with the obvious characters. Such as the lenticel and the trichome, parenchyma were highly developed and the cellular cavity was big in which there were much crystal granule or protonema, which is important to anti-depauperate and supporting capability. The results of the transmission electron microscope of the leaves and the stem of N.tangutorum Bob,N.roborowskii Kom, N.sibirica Pall, N.sphaerocarpa Maxim, N.praevisa Bobr showed that there were much commonness:integrated granum, vacuole inflated and the cell fulfilled with high-density close electric granules and many osmiophilic droplets. The cuticular cell of the stem of Nitraria.L possesses a large number of chloroplast.2. The difference among the secondary metabolites of the genus Nitraria.L was very significant, which was very potential to be exploited and utilized.The Proline, Total Alkaloids and Flavonoids in the leaves of 7 species of Nitrarial.L and Zygophyllum anthoxylon were mensurated. The results showed that the content of proline in genus of Nitraria.L was 4-16 times the content of Zygophyllum anthoxylon. The order of the content of proline of the genus of Nitraria.L was N.tangutorum>N.schober i> N.sphaerocarpa >N.sibirica>N.roborowskii>N.praevisa>N.pamirica. The content of alkaoids in the leaves of genus of Nitraria.L was 4-6 times content of alkaoids of Zygophyllum anthoxylon. The order of the content of alkaoids of the genus of Nitraria.L was N.tangutorum> N.sphaerocarpa>N.schoberi>N.sibirica>N.pamirica>N.praevisa>N.roborowskii. The content of flavonoids in the genus of Nitraria.L was more than other two chemical composition significantly. From the numerical point, the content of flavonoids in N.sphaerocarpa was the most, the second was N.tangutorum, the least was N.praevisa.3. The analysis of the content of abio-elements in the leaves and stems of the N. schoberiThe highest content of the elements on the leaf transect of the N.schoberi was oxygen element, the least was magnesium. The order was O>Na>Ca>P>Cl>Si>Mg. The highest content of the elements on the threadlike material was the oxygen element, the least was magnesium, the order was O>Ca>C>K Na>Si>Mg. the order of the content of the elements in the stem transect parenchymatous cell was O>C>K>Na> Cl>Ca>Si>Mg, in which the K ion and N ion increased significantly.4. The 7 sepcies of gneus Nitraria.L can be clustered into 3 kinds based on AFLP molecular marking technology. The systematic relationship among the 7 species of genus of Nitraria.L and 1 Zygophyllum anthoxylon (as the outgroup) were perfectly analyzed by using Flu AFLP with 8 primer pairs, in which a large number of effective polymorphic markers were obtained. The study generated clear and credible 6245 AFLP bands totally, of which 1327(21.24%)bands were polymorphic. Based on DNA bands, the similarity coefficient were calculated and their average value was 0.57, ranging from 0.31 to 0.74,When all of the polymorphic bands were analyzed by cluster analysis. The results showed that the N.tangutorum and the N.roborowskii was clustered in one kind and the N.sphaerocarpa and the N.pamirica were clustered farsidly in the phylogenetics tree which shows the sib-relationship was far.5. The phylogenetic tree of the genus of Nitraria.L were constructed based on the psbA-trn H,trn S-trn G and ITS sequence.The psbA-trn H,trn S-trn G and ITS sequence were selected after strict prophase test to be the basic sequence for analyzing the phylogenetics relationship among the genus of Nitraria.L, and the trn S-trn G and ITS sequence were also tested in outgroup Zygophyllum anthoxylon. The pair length of the psbA-trn H was 559bp,34 variation site (5.8%) were found, of which 19 site (3.3%) were information site. And the genetic distance ranged from 0.0025 to 0.021, the distance between genus of Nitraria.L was 0.032～0.0409. The pair length of the trn S-trn G intron was about 832bp, which ranged from 725 to 902bp on length, consist of 48 variation site (5.7%) and 31 information sites个(3.72%)。The genetics distance between species counted from these sequences ranged from 0.0012～0.0217 and the distance to the outgroup was 0.0235～0.0389. The pair length of ITS Pland P4 intron was about 557bp, consist of 76 variation site (13.6%) and 47 (8.4%) information site, the genetic distance based on these sequences was 0.0102～0.1482, and the distance between the outgroup was 0.0295～0.2357. The combined sequence of three sequences used above consists 1729 pairs and 95 (5.3%) variation site and 67 (3.7%) information site and was used to calculating the genetic distance, which ranged from 0.0011～0.0373 and the distance between the outgroup was 0.014～0.1977. 6. The phylogenetic tree based on the sequence of psbA-trn H, trn S-trn G and ITS sequence.Four phylogenetic tree were construced based on psbA-trn H,trn S-trn G,ITS P1-P4 intron and combining sequence, the maximum parsimony (MP) and neighbor-joining(NJ) analysis methods respectively. It can be concluded from the calculation result that the phylogenetic tree reflects the evolutionary law of the species of genus Nitraria.L was coincident, although there were some differences among the topological structure based on different sequence respectively.Seven species of genus Nitraria.L could be concluded into three subdivsions from the MP tree and NJ tree constructed on psbA-trn H sequence, one subdivision consist of N.praevisa,N.roborowskii and N.tangutorum, one consists of N.schoberi, N.sibirica and N.pamirica, the third one consist of N.sphaerocarpa only.It could be inferred from the MP tree and NJ tree constructed on trn S-trn G sequence that N.schoberi and N.sphaerocarpa and N.sibirica and N.praevisa located at the basal site, while N.roborowskii and N.tangutorum take a close position, and little different from the position on the phylogenetic tree.It could be inferred from the phylogentic tree of the genus Nitraria.L based on the ITS sequence that seven species should be clustered into three subdivisions, one included N.schoberi, N.pamirica, N.sibirica, one included N.tangutorum, N.roborowskii and N.praevisa, and N.sphaerocarpa separately.In the phylogenetic tree based on the combined sequence, the N.sibirica, N.pamirica and N.schoberi were very close between each other, and N.tangutorum, N.praevisa and N.roborowskii were very close between each other, while N.sphaerocarpa far from other species. This result of the clustering took some similitude to the clustering results of the AFLP phylogenetic tree, in which N.pamirica separated from other species.