| Cornel(Cornus officinalis Sieb.et Zucc.),alias dogwood,dogwood meat,Zaopi and so on,is a plant which belongs to the Cornus and Cornaceae.It is an expensive traditional Chinese medicines and has a long history of cultivation in our country,which has been recorded in "Shen Nong's Herbal Classic ","Collective Notes to the Canon of Materia medica "," Wu Pu Materia Medica ",Herbal Classics" and other classics.Its flavor is sour,astringent,tepid and has the effect on tonifying liver and kidney and astringent essence as it falls into the liver and kidney.In recent years,it is also used in landscape and lots of people love it because of its unique ornamental characteristics of flowers,fruit,leaves and tree pose.Cornel plant is mainly distributed in Henan,Zhejiang,Shaanxi and other provinces in China,and it is also distributed in Sichuan,Anhui,Shandong.For a long time Cornel is mostly in the semi-wild state,and because of cross-pollination and differences in the growth of the environmental conditions,which results into a large variation within species,mixed breed,uneven in quality,big quality difference.In this research 63 samples of Cornel leaves from different areas were conducted by statistical analysis of morphological index and comparing the morphological differences between the different areas of the blade.48 samples of Cornel fruit materials from Xixia,Neixiang were determined of the polysaccharides content by phenol-sulfuric acid method and analyzing the content of polysaccharide in the different superior strains,which provided a reference test for the breeding of new varieties of Cornel.In this study,100 samples of Cornel from different areas as materials were conducted by PCR sequencing to obtain ITS and chloroplast gene sequences,and to compare the differences between groups in different areas of Cornel in molecular level by analysis of ITS and chloroplast gene sequence of using bioinformatics software.At the time to explore the affiliation between different Cornel groups and the differential problem of ITS sequences,which in order to understand the genetic relationship and germplasm genetic background of different areas from the DNA level,and which provide a theoretical basis on Classification conservation and utilization of Cornel resources.The main findings are as follows:(1)In this study,the statistical analysis of 63 leaves Cornel showed: The larger coefficient of variation of Cornel leaves,the richer diversity;there are obvious differences between the main producing areas: Henan(Funiu Mountain)Cornel leaves is small and the average leaf length is 7.91 cm,which is less than that of 8.86 Northwest areas and 8.48 in South Africa.The variation coefficient of Cornel leaves in Northwest is as small as 11.05.The average width of the blade in Henan is 4.35 cm,also less than the Northwest(4.60cm)and South Africa(4.72cm),and Blade amplitude of the in the Northwest is minimum.(2)In this study,the measuring of 48 Cornel fruit polysaccharide content by the phenol sulfuric acid method shows: the variation coefficient of polysaccharide content is larger,and there are a a significant difference between different superior variation strains of Cornel.(3)This study of Cornel ITS,trn C-F,trn Q-rps16 and trn L-trn F sequence cloning,amplification,purification,sequencing have improved the method of DNA extraction of Cornel,and established the optimal expansion of PCR increase system and procedures.(4)The ITS sequence length of Cornel ranged from 658 to 668bp(without regard Gap),and wherein the ITS1 sequence length is 238-247 bp,ITS2 sequence length of 263-267 bp.The length of 5.8S r DNA is very conservative.By comparison,the reference sample of 5.8S r DNA length is 153-155 bp,which starts at 263 rd bases,ends at 424 rd bases.The nucleotide content of Cornel showed differences among the sample ITS1 and ITS2.The GC content of ITS 1 sequence is 62.6% ~ 72.5%,and that of ITS 2 sequence is 58% ~ 67%.The GC content of 5.8S r DNA in code area is 51.9% ~ 56.1%.The G + C content of each area are high,and the G + C content of G ITSl and ITS2 region was significantly higher than that of 5.8S.150 variation sites were detected from the 675 bp in total sites of all 100 ITS,which accounted for 22.2% of the total sites.The 80 parsimony informative sites accounted for 11.85% of the total site.Wherein 87 variable sites were determined in ITS1 region accounting for 34.8% ITS1 sequence;The 64 parsimony informative sites accounted for 25.6% of the total site.Whereas 56 variable sites were determined in ITS2 region accounting for 20.81% of its total length.The 18 parsimony informative sites accounted for 6.7% of the total site.While 5.8Sr DNA coding region was more conservative,only eight variable sites accounted for 5.1% of its total length,no parsimony informative sites.(5)Diversity of ITS sequences groups haplotype(h)and nucleotide diversity(?)of all 13 provinces were tested,and results showed that: Cornel germplasm showed a high level of genetic diversity(h = 0.735,? = 0.01027)and a greater difference(h = 0.000 ~ 1.000,? = 0.000 ~ 0.02238)on the level of nucleotide polymorphism among different groups.Overall the level of genetic diversity of Zhejiang(ZJ)group(h = 0.853,? = 0.02238)is the highest among the Cornel groups.(6)In this experiment,genetic distance within groups and genetic distance between groups were conducted to show that: the average distance between groups of the candidate ITS fragment is greater than that within populations,and the degree of overlap is less.Therefore,ITS has the plant barcode potential between Cornel groups in different producing areas.(7)In this experiment,basis on ITS sequences,Construction of phylogenetic tree by maximum parsimony(Maximum parsimony,MP)and neighbor-joining(NJ)analysis showed that: Different areas introducted Cornel frequently and genetic relationship between different groups were significantly different.Comprehensive identification rate was only 1.4%.(8)The test results showed that: The range of genetic distance between groups is between 0.000 to 0.052,with an average of 0.018.Wherein between Zhejiang(ZJ)groups and Chongqing(CQ-FJ)groups,the genetic distance in Zhejiang(ZJ)and Hunan were furthest with highest degree of differentiation;and Hunan(HN-GY)groups and Chongqing(CQ-FJ)groups were minimum with a relatively low degree of differentiation.(9)In the results of this experiment showed:The length of Cornel chloroplast trn Q-rps16 gene fragment was 1512 bp with the G + C content of 51.8%;The length of Cornel chloroplast trn C-F gene length was 874 bp with G + C content of 48%;The length of chloroplast trn L-F gene length was 385 bp with G + C content of 52%.Chloroplast gene sequences were almost identical between each sample,which indicating chloroplast genome was rather conservative. |
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