| Magnolia sinostellata(Magnoliaceae)is a rare and endangered species which is endemic to subtropical China,the species is of high scientific significance and ornamental value.However,M.sinostellata has been listed as an "endangered" species due to its extremely narrow distribution,small natural population,low fertilization rate,and anthropogenic disturbance.In order to explore the germplasm genetic status and the endangered mechanism of M.sinostellata,we employed RAD-seq technology for SNP discovery,and used the screened SNPs to study the population genetics of the six wild populations by analyzing of the genetic diversity,genetic structure and population differentiation.The study will provide an important reference for the protection of germplasm resources and rational utilization.At the same time,the Next Generation Sequencing approach was used to study the complete chloroplast(cp)genome of M.sinostellata.The phylogenetic tree of Magnoliaceae was reconstructed,and a total of 34 chloroplast SSR primers were developed,providing the theoretical basis for future research.The main results are as follows:1.Total 394.5Gb clean data were obtained from the six populations of M.sinostellata using the RAD-seq technology,with an average of 8.4 Gb per sample,and sequencing depth of more than 3 times.Bioinformatics analysis yielded 49,066,178 RAD-Tag,a total of 267,168 representative SNP sites for subsequent population genetic analysis.2.Genetic diversity of six populations was evaluated by the representative SNPs.The results showed that both the species(He=0.27312,Pi=0.32323)and population levels(He,0.07391-0.1585;Pi,0.09986-0.19583)of M.sinostellata is low,which may be caused by three reasons:(1)The reproductive system obstacles of itself,such as self-infertility,anomalies during microcpore mother cell meiosis,etc.;(2)Individuals reduction in population and insufficient sampling caused by habitat fragmentation and human poaching;(3)The low seed germination rate and the special sprouting proliferation mode in the wild.3.Based on STRUCTURE analysis,PCA analysis,the six populations of M.sinostellata can be divided into four genetic groups.ML phylogenetic analysis recovered six wellcircumscribed lineages corresponding to the six sampled populations.Two geographically close related populations(NTS and QJD)form sister relationship on the tree,indicating they are closely related genetically.4.The Fst value analysis showed that the genetic differentiation among the populations of M.sinostellata was at a moderate level(Fst,0.05962-0.44565),and there was significant differentiation between the BGJ population and other populations.AMOVA analysis showed that the genetic variation among populations of M.sinostellata was 19.28%(P<0.001),most of the genetic variation occurred within population(80.72%).When we performed AMOVA analysis according to the genetic group divided by STRUCTURE,the genetic variation among groups was 17.1%,among populations within group was only 4.13%,while most of the genetic variation(78.77%)occurred within population.It indicates that there has been a certain extent of differentiation among populations of M.sinostellata,but the variation within population contributes a relatively greater contribution to the overall variation.5.The complete chloroplast genome of M.sinostellata is 160,076 bp,and exhibits a typical quadripartite structure comprising a pair of inverted repeat regions(IR),a large single copy region(LSC),and a small single copy region(SSC).The whole cp genome of M.sinostellata encodes 113 unique genes,including 79 protein-coding genes,30 transfer RNA(t RNA)genes and four ribosomal RNA(r RNA)genes.Finally,34 pairs of chloroplast SSR primers were developed.The phylogenetic tree based on the chloroplast genome sequence and protein coding sequence shows that the systemic position of Liriodendron is clear,and the internal relationship of Magnolia is not well resolved,especially the relationship of the subgenus Yulania and subgenus Gynopodium. |
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