| Huperzzia serrata(Thunb.)Trev,with its name of Qiancengta in China,is one of the traditional medicines.Many alkaloids were isolated from H.serrata,in which the main composition huperzine A(HupA)has been shown to treat Alzheimer’s clinically.However,because of strict requirements on growing environment and long growth cycle(6-15 years),the natural resources of H.serrata is scarce.Many scientists are trying to relieve market demand on HupA in the way of chemical synthesis of HupA and tissue culture of H.serrata.So far,little progress has been made So,wild resources is still the main source of HupA.Our research would be carried out in the next two aspects.Firstly,look for metabolic pathways and functional genes related to the biosynthesis of HupA based on transcriptome sequenceing.Secondly,reveal genetic diversity of H.serrata through developing SSR markers.The results were as follows:(1)Builded cDNA library of H.serrata.Assembly with Trinity software resulted in a total number of 4,128,785 contigs and 111,251 unigenes.We obtained 48989 unigenes in total after annotation.47629(97.2%)unigenes were annotated into Nr;29362(59.9%)unigeges were annotated into GO;there were 28754(58.7%),21507(43.9%),16948(34.6%)and 16782(34.3%)unigenes were annotated into Pfam,Swissprot KEGG and COG respectively.In addition,we found metabolism pathways related to the biosynthesis of HupA,including the lysine biosynthesis and lysine degradation pathways.In lysine degradation pathway,there are two pathways related to the biosynthesis of L-lysine,namely,lysine biosythesis pathway and Biotin metabolism.(2)Many function genes that may participate in the alkaloid biosynthesis were obtained after annotation.When it comes to these unigenes,eight unigenes has been reported to have correlation with biosynthesis of HupA;Thirty-one might participate in the biosynthesis of tropane,piperidine or pyridine alkaloid;there were two unigenes involving in coding enzymes related to lysine metabolism(3)The identification and localization of SSRs from more than 1 KB unigene was performed by MISA.A total of 4395 SSR loci were found in 14913 unigenes.120 SSR markers with di-,tri-,tetranucleotide,pentanucleotide and hexanucleotide repeat motifs were developed and optimized.Of these,10 SSR primer pairs,which produced clear,reproducible polymorphic bands in 177 individuals from five regions were used to detect alleles by Capillary electrophoresis.All SSR primers were polymorphic,the mean of number of Allele(s)was 7.2.(4)Genetic diversity of H.serrata form five geographical regions were analyzed.Among them,Expected heterozygosity(He)ranged from 0.503(SQ)to 0.652(YJ);Shannon information index(I)ranged from 0.845(SQ)to 1.118(YJ);Nei ranged from 0.495(SQ)to 0.641(YJ).The genetic diversity of H.serrata is significantly higher than other endangered species.High genetic diversity indicted that its stronger ability to adapt to the external environment.Therefore,natural selection may not be the main cause of its resources reduced,it may be the result of human interference and unreasonable exploitation.We tested the content of Hup A and HupB in H.serrata using HPLC.The content of HupA from Yunnan province(YJ)(0.1609± 0.0029 mg/g)was significantly higher than other four areas,while,the contents of HupB have no obvious difference.In addition,genetic diversity of H.serrata from YJ is highest(He = 0.652;I = 1.118,Nei = 0.641),therefore,the resources of H.serrata from Yunnan province is significant for its protection(5)STRUCTURE software was used to analysis the relatedness about the 177 H.serrata accessions,the results showed that k=3 is the most likely model for the five population.All accessions could divided into 3 subclusters,each subcluster was consisted of assessions from five regiones respectively.The HJ tree showed that all the populations formed three subclusters,which exhibited the same division pattern as the structure analyses showed.The ANOVA analysis revealed that 88.21%and 11.79%variation accounted for within and among regions,respectively.It indicated that a relatively low level of genetic differentiation(P<0.001)among the five populations.(6)The genetic differentiation among population(Fst)was 0.1093,in this study,there are eight locus deviated from the Hardy-Wein berg,and the heterozygote was excessed,these suggested that there are less inbreeding phenomenon among H.serrata.High gene flow(Nm=2.0379)increased the genetic diversity of H.serrata,meanwhile reduced genetic differentiation among populations,which made H.serrata had stronger adaptability.Then,the fragmental habitat and the continuous decreasing number of H.serrata would lead to the occurrence of inbreeding and the accumulation of harmful mutations,thus,resulting the resoures of H.serrata in confronting with a more serious crisis of extinction.Secondly,the genetic differentiation and genetic structural analysis of five regions of H.serrata.showed sexual reproduction(spore reproduction),maily propagating through wind pollination or other media,was the main factors that affect genetic differentiation of H.serrata. |
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