Study On Propagation Habits Of Whitmania Pigra Whitman And Its Genetic Diversity By Molecular Marker

Medicinal leech,including Whitmania pigra Whitman,Hirudo nipponica Whitman and Whitmania acranulata Whitman,has been used in clinical practice for blood circulation promoting and stasis relieving.At present,cardiovascular disease,which has been the first killer to the human health,is expected to be cured by the Chinese Patent Medicine using Leech as major raw materials.It can be carried out and deeply applied in animal feed additives on anti-inflammatory effect.Leech has become indispensable raw materials of Western medicine.and feed industry.With the development of society and research of its related products,leech will in great need.The contradiction between supply and demand appears because of the deteriorating of the wild resources,leading to a worldwide shortage. Leech was included in the list of 1983 IUCN Invertebrate Red Data Book.It is urgent to protect existing leech wildlife resources,so,location,collation and evaluation germplasm resources of Hirudo is becoming an important task.Firstly,we studied the morphological structure of offal and the distribution of skin epidermal cell types of W.pigra.Then,we investigated the physiology,growth, reproductive habit,and residues of pesticide and heavy metals,and also systemically evaluated the inherent quality of different processing drugs and germplasm of W.pigra. Based on the research of the physiology,growth and reproductive habits,we sampled parts of germplasm resources of W.pigra in the distributed places of China and made the four H. nipponica population as reference.We analyzed the genetic diversity,population genetic structure and polymorphism of W.pigra and established a system of classification,which provided a strong theoretical basis for the protection and use of the W.pigra and H. nipponica resource.Secondly,we successfully isolated microsatellite sequence of W.pigra using two methods and develop its SSR markers application in W.pigra.The detail contents and results are listed below: 1.Growth aquaculture habits and quality assessment research(1) Using the methods of paraffin-embedded myocardial specimens and photomicrography,we dissected and analyzed the visceral organ of W.pigra,including of cavity,swallow,esophagus,crop,bowel,rectum,parasomal sac stomach,testicle,seminal vesicle,vasa deferentia duct,ejaculatory ball,prostate gland and skin.As a results,we definited the Digestive System and Reproductive System of W.pigra,we also obtained the knowledge of structure and function of the skin and the organ framework of the digestive and reproductive system,as well as the types and distribution of the mucous cells,which provided information for further study on the living behaviour,reproductive habit,artificial aquaculture environment and species identification of W.pigra.(2) Study the oxygen consumption rate,oxygen demand and suffocation point of W. pigra by the method of respiratory chamber.The result showed that the oxygen consumption rate(OCR) ranged from 0.049 to 0.094mg·g^-1·h^-1,the oxygen consumption volume(OCV) from 0.44 to 0.67mg·p^-1·h^-1,and suffocation point(AP) from 0.9 to 1.51 mg·L^-1,when the average weight of W.Pigra was 10g and the water temperature varied from 15 to 35℃.OCR ranged from 0.044 to 0.058mg·g^-1·h^-1,OCV from 0.19 to 0.77 mg·p^-1·h^-1 and AP from 1.4 to 1.57mg·L^-1,when water temperature was 20℃.We make the conclusion that the OCR will rise as the water temperature increased,but the AP was opposite.The weight was negatively correlated with OCR and positively with OCV.OCR had little variance from day to night.(3) Comparison of the growth of W.pigra in different weight and temperature conditions,and study on the intake habit of W.pigra in 24h.The results show that the appropriate temperature for W.pigra growth is 15～25℃.And with the increase of the weight and the rise of temperature,the intake rate increased.The intake demand reduced with the increase of weight.W.pigra has two intake peaks in 24 hours.(4) Observation the spawning and hatch of W.pigra in different temperature and weight,and determination the best spawning temperature and spawning weight.As a result, we found the most appropriate temperature for W.pigra spawning and incubation is 25℃, and the best spawning weight is 20g.Temperature can influence the weight before and after spawning,but the weight have no obvious effect.When the spawning weight range from 0.1～2.0g,it has the positive correlation with hatch amount,but when the spawning weight exceeds 2.0g,hatch amount will reduce.(5) Analyse the content of heavy metals and pesticide residues for soil and aquaculture water in W.pigra with the help of atomic absorption spectrophotometers,atomic fluorescence spectrophotometers and gas chromatography.The results showed that exception Pb,other indexs including aquaculture base of soil and aquaculture water HCH, DDT and heavy metals residues indicators are consistent with national standards in W. pigra.And we suggested that the animals similar to W.pigra should be separately listed in the national interrelated standards.(6) Determine the moisture,alcohol extract,total ash,acid-insoluble ash,antiplatelet aggregation enzyme of the wild and the artificial aquaculture W.pigra using the method referred in the book (2005 edition).Mineral elements including iron,zinc,copper,chrome,lead,cadmium and Hg etc.are identified by the method of ICP-MS(Inductive Coupling Plasma Launching).In the results,in the parts of water there is no obvious difference between wild and artifical processing drugs(P＜0.05). The talcum powder bums have the highest content of total ash,which has obvious difference with other processing drugs(P＜0.05).Acid-insoluble ash:The talcum powder bums＞lives exposes to the sun＞the liquor product;alcohol extract:The talcum powder burnsz＞the liquor product＞to live exposes to the sun;antiplatelet aggregation enzyme:Lives exposes to the sun＞the liquor product＞the talcum powder to burn; Apart from artificial aquaculture W.pigra moisture and antithrombin higher than which in the wild,but have no significant difference(P＜0.05),the rest were lower than in the wild. The activity of antiplatelet aggregation enzyme in domestic W.pigra is higher than those in wild ones and has no significant difference(P＜0.05).But total ash,acid-insoluble ash and alcohol extract axe less than the wild.(7) Analysis the moisture,alcohol extract,total ash,acid-insoluble ash,antiplatelet aggregation enzyme of concocting wild and the artificial aquaculture W.pigra using the methods consulted with the (2005 edition),and analysis the Xanthine and Hypoxanthine by HPLC.The results showed that the moisture,alcohol extract,total ash,acid-insoluble ash,antiplatelet aggregation enzyme,Xanthine and Hypoxanthine of artificial aquaculture population are better than other population.2.Analysis of the genetic diversity of W.pigra germplasm resources in China using Molecular marker technology(1) 225 samples from 15 germplasm resources distributed in 8 provinces were used to analyze genetics deiversity.Based on analysis of RAPD technology,the results showed that at the level of species the polymorphic loci percentage(PPL) is 99.66%,Nei'S gene diversity index(He) is 0.3599.At the population level,PPL is 30.30%～56.06%,averaging at 44.14%,in which the highest population is in Maanshan,followed by the Liyang population,Nanjing cultivated,the lowest of population.Genetic differentiation factor(G_ST) within groups is 0.5487,population gene flow(Nm) is 0.4028.Data shows that inter-population has more diversity,and intra-population has a certain extent variation. Cluster analysis revealed that when the similar coefficient is 0.80,15 materials can be divided into eight groups,in which Guangzhou,Guilin,Dali and Maanshan four population have lower similar to most of the other population.(2) Analysed genetic diversity of W.pigra using SSR molecule marker technology. The results showed that at the species level PPL is of 99.56%,He is 0.3490.At the population level PPL is in 25.00%～53.95%,averaging at 37.73%,highest is the Guilin population,followed by Guangzhou,Nanjing cultivated the lowest population.G_ST is to 0.5730,and Nm of 0.3727.The results also showed that intm-population has much differentiation,and the inter-population has a certain level of genetic differentiation.Cluster analysis showed that as the coefficient is 0.78,15 population of W.pigra were divided into five groups.(3) SRAP marker is a new technology.In this paper,we analysed the SRAP marker applicability in research of the genetic diversity of W.pigra.The results showed that at the species level,PPL is 99.02%,He is 0.3687.While at the population level PPL is within 31.69%～58.86%,averaging at 50.59%.the highest population is in Le'an,followed by Liyang,Nanjing cultivated is the lowest population.G_ST is to 0.4699,Nm is 0.5641.Cluster analysis revealed that as coefficient of 0.82,15 population were divided into 6 groups.By comparing SRAP,RAPD,ISSR data,the new technology can be used in W.pigra genetic structure study.(4) To understand variation type within species,we tested the ribosomal ITS base sequences and analysed the characteristics of them.We obtained rDNA ITS complete sequences from 14 samples of 14 main W.pigra and H.nipponica population.The length of ITS was 857～863bp.The base frequence has more variation.ITS has more conservative.It constructed the phylogenetic tree using Neighbor-adjacent method,showing that there are at least 3 variations in W.pigra.Combined with RAFD,ISSR,and SRAP molecular markers data,it was suggested that 14 population should be divided into four grades to be protected and utilized.3.Microsatellite exploiture of W.pigra The main strategies of microsatellites development were reviewed in the paper. Analysis and evaluation of several representative methods are helpful to develop Micro-satellite method.Two micro-satellites development strategies for W.pigra were provided based on the existing methods.(1) First method:Because of the strong affinity of biotin for streptavidin,the 5' end biotin of microsatellite probe was combined with magnesphere paramagnetic particles,and then combinations hybridization with the digested W.pigra DNA fragments both ends of them connected with special adaptor.Elution of the other DNA fragments,the micro-satellite library was established.The adaptors as PCR primers clone the products direct,and screening the results by technology of bacteria PCR amplification in the end.(2) Second method:Connected the special adaptors to digested genomic DNA fragments,then make it as a template.The microsatellite library was established by inhibition PCR with mergers primer.Then analyze results of clone sequence after two screening by bacteria PCR.The results show that the two methods can quickly and efficiently be used to develop micro-satellite method.(3) Finally,combining the isolated micro-satellites sequences from genome DNA,we analysis the characteristics of W.pigra.As a result,we found the dinueleotide repeat type in W.pigra SSR has absolute advantage,frequency nearly reached 93%.Dinucleotide repeat motif(AC)_n is up to 53.33%.In addition,14 pairs of SSR primers were designed,11 pairs of which can be used to effectively detect the genetic diversity of W.pigra.