| Stemona Lour.is a genus of a small monocotyledonous family Stemonaceae,consists of about 27 species,which distributed in Southeast Asia and Australia.There are 7 species in our country,of which 5 are endemic to China.Fleshy roots of several Stemona Lour.species,also known as ‘Bai Bu',are widely used in the traditional South-East Asian medicine as an antitussive agent and insecticidal remedy.It has certain ornamental value,however,there are some controversies in the relationship between some species.The relationship between them is discussed from the perspective of group in this study.Five chloroplast DNA spacers are used to analyze 25 populations,in addition,one individual of Croomia japonica Miq.(C.jap-FL3)was sequenced as outgroup.In addition,there is no report about the genetic diversity of S.tuberosa.The SSR primers were applied to study the genetic diversity of 12 populations in S.tuberosa.The main results are as follows:1.Study on interspecific relationship of stemona based on chloroplast fragments5 polymorphic chloroplast DNA spacers,trn L-trn F,trn C-ycf6,atp I-atp H,psb Bpsb H and rpl14-rpl36,were screened for the universal primers from the literature,and the interspecific relationships among the species were studied by these molecular markers.The total alignment of atp I-atp H sequence was 649 bp,having 41 variable sites,with 11 parsimony informative sites.The total alignment of psb B-psb H sequence was 581 bp,having 25 variable sites and 12 parsimony informative sites.The total alignment of rpl14-rpl36 sequence was 1152 bp,having 660 variable sites and 57 parsimony informative sites.The total alignment of trn C-ycf6 sequence was 480 bp,having 27 variable sites and 8 parsimony informative sites.The total alignment of trn Ltrn F sequence was 581 bp,having 25 variable sites and 12 parsimony informative sites.The combined alignment of the five cp DNA fragments with a total length of 3,605 bp,with 150 variable sites and 118 parsimony informative sites.Phylogenetic tree and haplotype network showed that the stemona is the monophyletic origin,and the interspecific relationship is clear.Seven species consists of seven clades with various support values.Each species forms its own clade,respectively.Seven species cluster into three major clades(I,II and III).Clade I contains three species,S.japonica,S.sessilifolia and S.mairei.Clade II consists of S.tuberosa and S.kerrii.Clade III is comprised of S.javanica and S.parviflora.The phylogenic studies revealed that S.japonica and S.sessillifolia are sister groups,S.parviflora is the closest relative to S.javanica in this study.It is consistent to the similarities on morphological characteristics.The phylogenic studies support the S.shandongensis as a synonym of S.sessilifolia.The widespread species S.tuberosa formed a highly supported monophyletic group.It possesses a high degree of differentiation and divides into 3 groups with various support values.Samples from Indonesia formed a clade;Samples from the tropics of our country and Fujian composed the another clade.Samples collected from subtropics are clustered together.In order to study the evolution of the Stemona lineages,minimum span network was analyzed based on cp DNA haplotypes.The concatenated cp DNA sequences of 34 accessions differentiate into 26 haplotypes.S.japonica contains 3 haplotypes(H1-H3),S.sessilifolia contains 3 haplotypes(H4-H6),S.parviflora contains 1 haplotypes(H13),S.mairei contains 2 haplotypes(H14-H15)and S.kerrii contains 2 haplotypes(H16-H17),in addition,S.javanica contains 1 haplotype(H12),S.tuberosa contains 14 haplotypes(H7-H11,H18-H26).At the end of the branch in the network,which indicates that the hyplotypes of S.japonica(H1-H3)are derived from H6(S.sessilifolia).Three subgroups are formed in S.tuberosa,which is consistent with the phylogeny tree.2.Genetic diversity of S.tuberosa based on SSR markersWe designed,developed and screened 17 pairs of primers suitable for the study of genetic diversity of S.tuberosa,by dual-suppression-PCR technique.Population genetic analysis was conducted on 123 individuals from 12 populations.A total of 228 alleles were detected,with an average of 13.41 alleles at each locus.The mean of Ho and He were 0.876(0.407-1)and 0.774(0.553-0.942).The average of polymorphism information content PIC was 0.733.The differentiation coefficient FST between the various loci was between 0.003-0.540 and the gene flow Nm was between 0.213-0.638.At the level of population,the mean value of Ho and He was 0.832(0.647-0.939)and 0.560(0.425-0.670),and the Ho of all populations was higher than He,and the average fertility coefficient of the population was-0.495,indicating that the heterozygotes within populations were excess.The PIC of population YUN,ST and GL were 100%,while TN and MTS were 94.12%.The Shannon index I of population is between 0.667 and 1.323,with a mean value of 1.012.The HW balance of TN and MTS populations was found to deviate from the HW equilibrium,and the population of YUN and ST deviated from the HW equilibrium,and the rest of the population conformed to the HW equilibrium,which may be related to the existence of a small population in the S.tuberosa populations.The cluster analysis of principal component analysis(PCA),Structure and NJ cluster analysis support divides 12 S.tuberosa populations into 7 groups.MTS and MTSLJ are clustered into one group,and EMS,HX and YFF are grouped into another group.TN and ST are clustered in a group.YB and YUN groups gather in a group.The results of AMOVA analysis showed that the genetic variation was mainly from the within population,and 7.7% of the genetic variation come among groups,and 40.21% of the genetic variation existed among populations within groups,while 52.09% existed within populations.The distribution pattern of the groups showed that the S.tuberosa have a serious fragmentation of the populations,which resulted in the lack of gene flow among the populations and exist the large differentiation among the populations.The correlation test between geographical distance and genetic distance of the S.tuberosa populations showed that the geographical distance and genetic distance of the hundreds of populations were significantly correlated(A: r = 0.259,p = 0.04).The genetic structure of the population is consistent with the geographical isolation model.There was a negative correlation between He and latitudes,and the regression coefficient was-0.6395.Bottleneck tests showed that YUN and ST populations had a bottleneck effect and may have experienced recent expansion.The excess of heterozygote in other populations is probably caused by founder effect. |
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