Genetic Diversity And Relationships Researches And ITS Sequences Analysis In Magnolia Officinalis

Magnolia officinalis belongs to Magnolia genus Magnoliaceae family, an endangered and deciduous plant to China. It was listed as state Endangered Plants II. It is an original species, has special classification status, high medicinal and ecological value, which has been attracted much attention. However, with the increase of demanding, people slinkingly digged wild M. officinalis resources, which led to the number and size of their wild populations constantly becoming small. Their habitats became fragmentated. Habitat fragmentation blocked gene exchanging among natural populations, and the survival situation is becoming more and more anxious. So the research of genetic diversity and relationships in M. officinalis populations is imminent.This paper studied the genetic diversity and relationships of M. officinalis populations with ISSR molecular marker and explored the geographical genetic differentiation. So as to point out the endangered cause of M. officinalis from the molecular level and put forward corresponding protection strategies. At the same time, we also studied base variability of ITS sequence in 11 major producing areas of M. officinalis, which provide molecular identification of authentic producing areas and introduction between different producing areas. The main results are as follows:(1) The results of extraction methods in M. officinalis genomic DNAExtracted genomic DNA of M. officinalis with different methods, SDS-CTAB was the best method, particularly the introduction of high concentrations of NaAc and the corresponding freezing can improve the purity of genomic DNA. At the same time, with quartz sand and 4% PVP solution grinding leaves instead of liquid nitrogen can also achieved better effects.(2) The results of ISSR primer selection and optimizationWe confirmed the parameters that impacted ISSR-PCR reaction by orthogonal test. The amplification system was as follows: primer concentration 0.3μmol?L-1, Taq enzyme 0.04 U?μl-1, DNA concentration 4 ng?μl-1, Mg2 + and dNTP concentrations were 1.5 and 0.2 mmol?L-1, respectively. The amplification procedure: pre-denaturation at 94℃for 5 min, then followed by 40 cycles, denaturation at 94℃for 30 s, annealing at 50℃~ 60℃(annealing temperature set with different primers) for 45 s, DNA extension at 72℃for 90 s, then 72℃for 8 min, terminate the reaction at 4℃. Finally, we successfully screened out 21 ISSR primers for the research of genetic diversity in M. officinalis with the above system.(3) The results of ITS sequence analysis in different producing areas ITS analysis in the 11 producing areas of M. officinalis showed that the length of ITS sequence was 593 ~ 600bp (without considering Gap, ITS1 and ITS2 sequences length were 214 ~ 217bp and 215 ~ 219bp, respectively, 5.8S rDNA coding region was extremely conservative with fragment length of 164bp). The whole sequence had 33 variable sites (all occurred in ITS1 and ITS2 sequences, Gap as missing treatment). The base frequency and content also showed some changes (GC contents were 54.42% ~ 55.35% and 59.82% ~ 60.95% in ITS1 and ITS2 sequence, 5.8S was 46.95%). Genetic distance and cluster analysis showed some relationship between partial M. officinalis producing areas, which reflected the introduction and cultivation status in our country. There was no causal link between ITS sequence variation sites and changes of leaf shape in different producing areas.(4) The results of genetic diversity and relationships in M. officinalis populationsThe results of amplification in 666 individuals of 28 M. officinalis populations showed that the 12 ISSR primers can stably amplify in all individuals. With a total of 137 bands amplified, 114 was polymorphic and the percentage of polymorphic loci (PPB) ranging 75.00% - 91.67%. The genetic diversity of M. officinalis natural populations was higher at the species level (PPB = 83.21%, H = 0.342), while relatively lower at population level (PPB = 49.76%, H = 0.194), which may be the results of special historical formation. The genetic diversity index showed differences between the 28 natural populations. However, there was no relationship between the genetic diversity among populations and altitude, longitude and latitude changes (Pearson correlation test, P > 0.05). There was a certain degree of differentiation between the 28 populations and mainly existed within populations (GST = 0.4278). The results of AMOVA (Analysis of molecular variance) coincided with that. It also indicated that there was significant genetic differentiation within and among the populations (AMOVA analysis, P <0.001). The 28 M. officinalis natural populations were obviously divided into 3 groups based on NJ (Neighbour-joining) clustering. The results consistent with genetic structure analysis and principal coordinate analysis (PCoA) (2Dim and 3Dim). Mantel test based on the natural logarithm of geographical distance and genetic distance showed that genetic differentiation of M. officinalis natural populations didn't accord with the IBD (Isolation By Distance) model (R = - 0.0054, P = 0.5120).