Study On Interspecific Relationships Of Lycoris Herb. And Molecular Markers Of Populations In China

The genus Lycoris(Amaryllidaceae) is comprised of about 20 species,and there are 15 species in China.They widely distribute in many provinces of China,and east China is the diversity center of them.The plants of Lycoris are endowed with medicinal and ornamental values.Alkoilds which are rich in bulbs of Lycoris,have pharmacological effects,for example,galanthamine have been used for the clinical treatment of Alzheimer's disease(AD) in recent years.It is common that different species having the same name or the same species having different names in Lycoris,which cause some confusion in germplasm collection,preservation,and utilization.Since some species are easy to hybridize,there are many natural hybrids.The morphological feature,chromosome number, and karyotype of several species of Lycoris vary in wide range at interspecific and intraspecific level.However,L.radiata(L' Hér.) Herb.as an ornamental and medicinal plant,its genetic diversity has not been studied.It is necessary to identify species with very similar outer characters,to research interspecific relationships of Lycoris and genetic diversity of L.radiata(L' Hér.) Herb.Thus,interspecific relationships and taxonomy of Lycoris were investigated by means of numerical taxonomy which based on the morphology,high-performance liquid chromatography(HPLC),inter-simple sequence repeats(ISSR) markers and cp DNA trn L-F sequences analysis,and genetic diversity of L.radiata were also studied with numerical taxonomy and DNA molecular markers in this paper.The main results were as follows:1.Based on the field observation and the data collected from references,Q cluster analysis for 38 characters of 13 native species and 2 varieties of Lycoris in China and one Narcissus tazetta var.chinensis were conducted.The results of cluster analysis showed that N.tazetta var.chinensis was a monophyletic group and 15 Lycoris species were classified as two groups,group one included L.anhuiensis,L.longituba,L.longituba var.flava,L. chinensis,L.aurea,L.albiflora,L.incarnata,L.squamigera,and L.sprengeri,group two consisted of L.radiata var.pumila,L.radiata,L.rosea,L.straminea,L.houdyshelii,and L. haywardii,which agree with the traditional taxonomic studies.The results supported the viewpoint that hybrid origin of L.squamigera,L.albiflora,L.houdyshelii,L.straminea,L. rosea,and L.haywardii.While L.anhuiensis as an independent species was not supported, it should be recognized as a variety of L.longituba or a hybrid between L.longituba and L. chinensis.The principle component analysis and R cluster analysis revealed that bulb shape and the position relation of the stamen and perianth can be used as the characters of Lycoris for major group division.2.13 native species and 1 variety of Lycoris in China and one N.tazetta var.chinensis were studied with HPLC technique.The result of Q clustering obtained according to the similarity of alkaloids indicated that all Lycoris species were divided into two major groups. The clustering result was generally consistent with that of numerical taxonomy which based on the morphology except for L.haywardii and L.aurea.N.tazetta var.chinensis formed a independent group due to obvious difference of HPLC chromatograms exited between N. tazetta var.chinensis and genus of Lycoris.The differences of HPLC chromatograms also existed between L.aurea and L.chinenesis,L.radiata and L.radiata var.pumila,which indicated there is a far phylogenetic distance between them.3.The cpDNA trnL-F sequence of 17 taxa representing 13 species and 2 varieties of Lycoris and N.tazaetta var.chinensis as one outgroup were determined by using direct sequencing of PCR product,and they were analyzed by means of the software of CLUSTRAL and MEGA.The length of trnL-F of all taxa was 905～1036bp,When the gaps were always treated as missing,there were 14 variable sites,10 were parsim-info ones, and account for 1.13%of the total length.The(G+C) content was 32.9%.At the intergeneric level,4 nucleotides inserteions or deletions and 7 substitutions(6 transversions and 1 transition) occurred.Three substitution sites which located on 22bp, 214bp,and 636bp were used to identify some species of Lycoris.L.incarnata and L. radiata var.pumila had its species-specific SNPs.Maximum-parsimony tree of 17 accessions were constructed based on trn L-F sequence by bootstrap test.N.tazetta var. chinensis was a monophyletic clade,three infrageneric clades of Lycoris were resolved.The trees suggested that L.anhuiensis can not be taken as an independent species,while it may be a variety or a hybrid of L.longituba,and that the maternal parents of L.rosea and L. haywardii are L.sprengeri.The trnL-F sequence of 13 populations of L.radiata,L.radiata var.pumila,L.rosea, and L.haywardii were conducted by using direct sequencing of PCR product.The result showed that minor variation of trnL-F among these taxa occurred and nucleotide substitution was mainly due to transition.There some difference of trnL-F sequences between narrow-leaf type and width-leaf type of L.radiata,while the trnL-F sequences of L.rosea,L.haywardii and Jiangsu,Jiangni population of L.radiata were the same.4.Interspecific relationships of 13 species and 1 variety of Lycoris were evaluated using inter-simple sequence repeat(ISSR) markers,N.tazaetta var.chinensis as outgroup. 16 primers produced 253 discernible DNA fragments,out of which 244 were polymorphic, and the percentage of polymorphic bands(PPB) was 96.44%.The genetic distance between L.houdyshelii and N.tazaetta var.chinensis is farthest(0.8700),while the genetic distance between L.radiata and L.radiata var.pumila is the nearest(0.2104).The DNA differences is obvious between L.chinesis and L.aurea although both of them have similar outer characters.All species of Lycoris were recognized as 2 major groups by UPGMA cluster analysis,which were basically consistent with that of by morphology, karyotype,and RAPD analysis,and also consistent with that of numerical taxonomy which based on the morphology and HPLC chromatograms cluster analysis of Lycoris except for the major group division of L.sprengeri,L.squamigera,and L.incarnata.5.Interspecific relationships of 13 native species and 1 variety of Lycoris in China were discussed by combined the data of numerical taxonomy which based on the morphology,HPLC chromatograms cluster analysis,and ISSR markers,N.tazetta var. chinensis as outgroup.The result indicated(1) N.tazetta var.chinensis was a monophyletic group,which displayed that far relationship between N.tazetta var.chinensis and gnenus of Lycoris.(2) Lycoris was divided into two groups,species in the first group were attributed to Symmanthus subgenus except for L.chinesis and L.aurea,while,species in the second group such as L.radiata,L.radiata var.pumilar were attributed to Lycoris subgenus.(3) Our results confirmed that phylogenetics of 5 primary species of Lycoris in China(L.longituba,L.chinensis,L.aurea,L.sprengeri,and L.radiata var.pwnilar).The status and phylogenetics of hybrid origin species such as L.rosea,L.squamigera,L. haywardii,L.straminea,and L.houdyshelii were supported.However,L.anhuiensis can not be taken as a distinct species,it may be a variety of L.longituba or derived from hybridization between L.longituba and L.chinensis.The status and phylogenetics of L. albiflora and L.incarnata was not resolved in this study.In conclusion,the results obtained by combined analysis were basically accordance with that of by numerical taxonomy which based on the morphology,HPLC chromatograms cluster analysis,and ISSR markers respectively. 6.37 accessions of Lycoris collected from different location in China,including 18 L. radiata,10 L.chinensis,5 L.sprengeri and 4 L.aurea were investigated with the technique of RAPD and ISSR,respectively.The result showed that 229 and 215 DNA bands were amplified with 16 ISSR and 17 RAPD primers respectively,the percentage of polymorphic bands(PPB) in ISSR detection(85.59%) was higher than that in RAPD(69.77 %).The coefficient ranges ofinterspecific and intra-specific GS(genetic similarity) in ISSR and RAPD analysis with the same materials were 0.5459～0.9345(average 0.6836) and 0.6419～1.0000(average 0.7428),respectively.There was significant difference of correlation coefficient(P＜0.01) between ISSR and RAPD(r = 0.8582).The similar molecular cluster results were obtained from RAPD and ISSR analysis,and showed that the genetic diversity of intraspecific was high,and the percentage of polymorphic bands (PPB) of L.radiata was the highest.The coefficient of gene differentiation(Gst) amonginterspecific consists with the result of molecular variance analysis(AMOVA). ISSR and RAPD marker are both efficient methods in revealinginterspecific or intra-specific genetic difference and diversity.7.13 populations ofL.radiata,L.radiata var.pumila,L.rosea,and L.haywardii were studied with numerical taxonomy and cpDNA trnL-F sequence analysis.The result obtained from Q cluster analysis is very similar to that from trnL-F sequence analysis. There were some differences in outer characters,content of galanthimine,and growth and develop habit among populations of L.radiata,and these differences was relation to that variation of trnL-F sequence.The results denoted that L.radiata has abundant genetic diversity.8.Genetic diversity and population genetic structure of L.radiata were examined with the technique of sequence-related amplified polymorphism(SRAP) 218 loci were identified with 10 SRAP primer combinations,out of which 173 were polymorphic and accounted for 79.36%of total genetic diversity at species level.The observed average number of alleles(ha),the effective number of alleles(he),Nei's gene diversity(h), and Shannon's information index(I) at species level were 1.7936,1.4131,0.2415,and 0.3664,respectively.The percentage of polymorphic loci(PPB) was the highest (37.61%) in Guizhou three populations(GZ1,GZ2,GZ3),Sichuan Zhongxian population(SC) was the second(36.70%),the lowest(25.69%) was in Anhui Guangde population(AH1) among populations of L.radiata.A large proportion of genetic variation(96.05%) resided among populations,while only 3.95%resided among samples within populations by Analysis of molecular variance(AMOVA).The result consisted with that of coefficient of gene differentiation(Gst = 0.9748) and Gene flow (Nm = 0.0129).These 24 populations of L.radiata surveyed were classified into two major groups.Group one included 7 populations which came from southwest or northwest of China,except for Jiangsu,Lianyungang population(JS3).Group two included the other 17 populations which distributed in from south to east China.Correlation analysis detected significant correlation between genetic distance and longitude,latitude,annual rainfall, annual average temperature.The coefficients were 0.202,0.248,0.194,and 0.171, respectively.There was no significant correlation between genetic distance and altitude. SRAP marker can detected not only genetic differences among populations,but also among samples within population.The results obtained with molecular markers and numerical taxonomy among populations of L.radiata showed that abundant genetic diversity existed in L.radiata, which can be classified as narrow-leaf type,width-leaf type,and intermediate type.