Studies On Genetic Diversity And Construction Of Fingerprinting Of Magnolia Officinalis

Magnolia officinalis is an important medicinal plants distribute in subtropical and northern-tropical of our country, its bark has many medicinal effect. Therefore, the Magnolia officinalis resourse is overuse by humans. Resulted in resources have been seriously damaged, population size have been sharp declined. At present Magnolia officinalis already been a vulnerable species. In order to improve the collection, preservation and make good use of germplasm resources of Magnolia officinalis, to provide technical support for parental set of chromosome in germplasm innovation and for hybrid identification, also In order to enrich the theory Magnolia officinalis genetics, for the choices of Magnolia hybrid breeding parents resources protection of intellectual property, provide a theoretical basis and practical basis for beed improvement and molecular marker-assisted breeding. In this paper, the extraction of Magnolia officinalis polyphenols, excellent germplasm selection, the genetic diversity of Magnolia officinalis from different sources and the construction of fingerprint and et al. were reseached.This study started from the surveys of Magnolia resource in Fujian Province, We selected Magnolia priorities within the Fujian Province based on the content of Magnolia polyphenol and phenotype, and collect some populations from some main producting areas of Magnolia officinalis in China , then we try to do the study begin with the optimize of the RAPD and SRAP marker methods which were used to analysis DNA polymorphism of Magnolia genomic, And use these two DNA markers to systematically study the complex genetic background of germplasm resources of Magnolia officinalis. Therefore, we also studied the genetic diversity and relationships of germplasm resources of Magnolia officinalis, analysised the germplasms'genetic relationship, carried out the molecular identification of germplasm resources, and established an effective DNA fingerprinting which could be used to identificated germplasm resources of Magnolia officinalis, beside that a amplification effect comparison between two markers, a study of the genetic relationship between cultivars in Fujian Province also be carried out.1. Magnolia phenol extraction was experimentally studied through orthogonal design L9(34). The result showed that ethanol concentration is a key factor of extraction, followed by ultrasonic time. The optimal method for extraction was: ethanol concentration 90%, water-curing treatment time 50min, water-curing treatment temperature 60℃, ultrasonic time for 20min2. Seasonal changes have an impact on the content of secondary metabolites during the period of plant growth. The content of phenol in shoot cortex of Magnolia was highest in January. Afterward with the passage of time, the content would decrease at first then increased but in downward trend on September. Finally the total phenol content distribution from tender leaves to old leaves was in Increasing trend until it reached the maximum before the deciduous period.There are some differences of Magnolia content among the different parts of Magnolia but the differences would be minimal among the same organs. Polyphenol content in different organs ranked in the order of root bark ,dry hide ,branch bark, tender bud , seed, fruit, stem wood and needle.With the increase of Magnolia trees age, magnolol and honokiol content trend in both branch bark and stem bark were shown as increasing-decreasing-increasing. while in both middle-aged and young growth plantations, the phenol content would increased with their ages increasing. The value reached the maximum approximately at their ages of 25 but afterward it would be shown as downtrend. However, in hundred years of aged trees, the active component content would show increasing trend in the last stage. The phenol content of Magnolia leaves decreased with age increasing, which reached higher level in their young growth tree leaves. The variation of phenol contents between Magnolia individuals in the same population with the diameter of the similar size were quite large and the difference of phenol contents in various parts between Magnolia individuals were significantly.3. When excellent magnolia provenance was selected, there exited very significant or significant positive correlation with tree height, crown, bark thick, and branches rather than straight degrees, round degree and health degree. Meanwhile, the positive correlation between phenol content of branch bark and traits of trees was very significant or significant.Four main components were extracted, according to the principle in which characteristic root is equal to or greater than 1. Then the principal components were comprehensive evaluated from 24 different provenance. The evaluation result by ranking order was as follow: top front group was zherong,Youxi, Licheng, Fuzhou, which is followed by the Wuping,Yongtai, Shouning,Qingliu.Shaxian,Yongan,Pucheng, campus of Fujian Agricultural and Forestry University, Pingnan, Mingxi, Ninghua, Zhangping, and at least was Taining, Nanping, Shunchang, Jianyang, Datian, Jianou, Fuan, Guangze.Six principal components extracted by principal component analysis of cumulative contribution rate of greater than or equal to 85% were comprehensively scored from 54 plus tree. The result was shown as follow: the higher scores of plus tree family was the number 11,6,131,146,16,followed by 123,33,49,114, 2. Cluster analysis results were divided into two categories provenance.4. In this paper, the orthogonal design and single factor test design were used to optimize SRAP-PCR amplification system on Magnolia officinalis Rehd.etWils. In four levels of five factors (TaqDNA polymerase, Mg2+, DNA template, dNTPs, and primer, respectively),The results that showed the affected of each factor on the result of PCR was found. The order was dNTPs, TaqDNA polymerase, DNA template, Mg2+ and primer. A most suitable SRAP-PCR system for Magnolia officinalis was established that was total 25μL reaction system containing 1.5 U TaqDNA polymerase, 1.8 mmol·L-1 Mg2+,100 ng DNA template, 0.24 mmol·L-1 dNTPs, and 0.40μmol·L-1primer. The new established SRAP-PCR system of Magnolia officinalis was with good repetition and stability. This optimized system for SRAP marker would become one of the protocols for further research on Magnolia officinalis.5. In order to establish RAPD reaction system suitable for Magnolia officinalis, all factors including annealing temperature, template DNA, Mg2+, dNTP, primers and Taq DNA polymerase were explored. The results showed that the optimal reaction system was 100ng of template DNA, 1.8mM of Mg2+, 0.24mM of dNTP, 0.48μM of primers and 2.0 U Taq polymerase in 25μL total volume. The reaction program was 3 min of pre-denaturation at 94℃, followed by 45 cycles of 45s of denaturation at 94℃, 1 min of annealing at 38℃and 1 min of elongation at 72℃. with a final elongation step for 10 min at 72℃. The new established RAPD-PCR system of Magnolia officinalis was with good repetition and stablility.6. A total of 89 individuals from 28 populations were detected by 19 pairs primers. The results revealed a high level of genetic diversity. At species level, the percentage of polymorphic loci (PPL) was 83%, the effective number of alleles (Ne)was 1.3809, Nei's gene diversity (h) was 0.2342, and Shannon's information index (I) was 0.3638. respectively, At population level, the estimates of PPL =23.62%, Ne=1.1581, H=0.0910 and I=0.1342. The genetic diversity at species level is higher than its at population level. The coefficient of genetic differentiation (Gst) was 0.6101. and the gene differentiation contributes to 61.01% of the total genetic variations between the populations and to 38.99% within the populations. The level of genetic differentiation between populations is higher than that within populations. The total gene flow (Nm) is 0.3195, is low,it shows that genetic drift has become a main cause of the genetic diversity. Conservation and utilization strategies for the species were put forward. 7. The genetic difference and relationship among populations of Magnolia officinalis were revealed at molecular levels on this study, which could be considered as the significant reference bases for the classification and identification and breeding of Magnolia officinalis species .We assessed the genetic diversity within and between populations of this species using RAPD. A total of 89 individuals from 28 populations were detected by 20 RPAD primers. The results revealed a high level of genetic diversity. At species level, the percentage of polymorphic loci (PPL) was 87.36%, the effective number of alleles (Ne)was 1.4552, Nei's gene diversity (h) was 0.2681, and Shannon's information index (I) was 0.4064, respectively. At population level, the estimates of PPL =26.94%, Ne=1.1885, H=0.1071 and I=0.1569. The genetic diversity at species level is higher than its at population level. The coefficient of genetic differentiation (Gst) was 0.6007. and the gene differentiation contributes to 60.07% of the total genetic variations between the populations and to 39.93% within the populations. The level of genetic differentiation between populations is higher than that within populations. It is discovered that coexist inbred and out bred in this species'mating system, and mainly as inbred. The total gene flow (Nm) is 0.3324, is low,it shows that genetic drift has become a main cause of the genetic diversity.8. 35 samples from 13 different geographical populations located different areas in Fujian province were studied by SRAP and RAPD markers. The results indicated that 19 pairs of SRAP and 20 RAPD primers amplified 219 and 202 polymorphic loci respectively. The percentage of on species level was 73% and 77.39%, it indicated that there were abundant genetic diversity on species level.in each population there was 61.4 and 61.6 polymorphic loci were detected and the percentage of polymorphic loci was 20.46% and 23.58%. Genetic differentiation between and within 13 geographical populations of Magnolia officinalis was estimated by genetic differentiation coefficient(Gst)which showed that 63.25%,60.98%genetic variance was among populations revealed by SRAP and RAPD markers, genetic variance existed between populations is higher than its existed within population. The genetic diversity Comparing with percentage o polymorphic loci, Nei's gene diversity index and Shannon's information index,the genetic diversity from JY, PC and MX was the highest, SC ,LC and FSG was lower, but the others have similar genetic diversity.We consider that it should bring the three highest genetic diversity populations into the precedence conservation system. UPGMA cluster analysis indicated that except No.3, 4, and 5 population have different clustering result in two kind of markers, No.1 and 2 get together, and the other 8 populations get together basically, No.1 and 2, No.6 to No.13 are two groups which have similar genetic diversity. When genetic distance is 0.17, the 13 populations can be divided into three groups and mostly populations have a similar compartmentalize in SRAP and RAPD markers.9. The SRAP and RAPD fingerprinting of 89 Magnolia officinalis germplasm were constructed. It was of much importance using minimum primers to obtain the maximum identification ability. Two independent ways, including unique SRAP markers and combination of the band patterns, can identify the varieties. The result showed SRAP were very effective in identifying Magnolia officinalis germplasm. The results show that the presence or absence of 12 unique markers obtained from SRAP primers made it possible to identify 12 Magnolia officinalis germplasms, The amplified result of any of two primer pairs between these 5 pairs of primers (Me5?Em2,Me6?Em2,Me1?Em7,Me1?Em5,Me1?Em8) can be as a fingerprint of 89 Magnolia officinalis germplasms, We can descry that SRAP marker is a efficient technology. RAPD was very effective in identifying Magnolia officinalis germplasm as well. There are 15 Magnolia officinalis germplasms can be indentified by 15 unique markers. Choose S60,S69,S91,S1412,S1422 and S330 six primers with good polymorphism to construct RAPD fingerprint of 89 Magnolia officinalis germplasms, These 11 pairs of primer include S60 and S69,S60 and S91,S60 and S1412,S60 and S330,S60 and S1422,S69 and S91,S69 and S1412,S69 and S1422,S91 and S1412,S1412 and S1422,S1412 and S330 can identify every germplasms, and can be used to construct fingerprint of 89 Magnolia officinalis germplasms.10. Totally 249 polymorphic loci were generated with 19 pairs SRAP primers. The mean the effective number of alleles, the mean Nei′s gene diversity (h) and the mean Shannon′s information index (I) of the tested germplasm were1.3881,0.2367,0.3656, respectively. Totally 228 polymorphic loci were generated with 20 RPAD primers. the mean ne,h and I, three index is 1.4368,0.2575,0.3912 respectively ,These suggests that the genetic diversity of germplasm was high and the genetic base was wide. The cluster analysis based on markers revealed that the Magnolia officinalis were clustered into three groups, in which the third group could be further classified into two sub-groups. The dendrogram showed clear genetic relationships among the tested Magnolia officinalis germplasms, although the dendrograms based on SRAP and RAPD markers were not all the same, some germplasms were clustered into different groups, it phenomena may be relative to the characteristics of the two markers. The investigation suggested that would be a better choice, and a long breeding program would be better to use both RAPD and SRAP.11. To compare amplification effect of the two kinds of SRAP and RAPD markers, the results of PCR amplification of the two kinds of markers were compared, the result show that the percentage of polymorphic loci of two kinds of molecular markers is 83% and 87.36% respectively, reveal that the genetic diversity of Magnolia officinalis is high. SRAP markers'index were slightly higher than the RAPD markers'by comparing the total number of loci results from the amplification, the average number of loci and the average number of polymorphic loci of the two markers. However, detection of genetic diversity between the two markers still have a larger similarity, Coefficient of genetic differentiation indicated that the gene differentiation contributes to 60% of the total genetic variations between the populations and to 39% within the populations. Detection effect is similar. By comparing the genetic distance, it shows that RAPD marker's scope of genetic distance is more widely than SRAP marker. Therefore, genetic information reflected by RAPD markers is more than the SRAP markers, And a higher genetic diversity among germplasm. This may be because RAPD markers are distribute throughout the genome, revealing the diversity of the entire genome, while the region of SRAP marker amplified is an open reading frame (ORF).