| For thousands of years,Ginseng(Panax ginseng C.A.Meyer)has been regarded as one of the most precious medical herbs in Chinese traditional medicine and is famed as the "The King of Herbs".Medicinal effects of ginseng have also been confirmed by modern biomedical researches,based on which it has been applied to the treatment of several diseases,including cancer and cardiovascular disease.However,due to its long growth cycle and excessive anthropogenic excavation,the wild ginseng resources are rapidly shrinking and facing depletion.Accordingly,it has been listed in the "First-class National Key Protected Wild Plants".Therefore,the utilization of ginseng mainly depends on cultivated ones.However,there are limited land resources for ginseng cultivation as well as many associated problems in the quality of cultivated ginseng.These factors seriously restrict the sustainable and high-quality development of ginseng industry.The rapid advances of modern biotechnology is expected to help solve the foregoing problems.For example,tissue culture can be used to reproduce rare ginseng germplasm,and genome selection is used to shorten the breeding cycle of ginseng.Notably,the eventual establishment of relevant technologies still requires understanding and resolving relevant fundamental questions,which include(i)what is the regeneration ability(cell totipotency)of ginseng callus by tissue culture?(ii)what is the degree of somatic clonal variation? Both answering foregoing scientific questions and developing modern ginseng breeding technology need high-quality ginseng genome information.Numerous studies have shown that the cell totipotency of plant callus cultured in vitro declines rapidly or even get completely lost after a certain period of subculturing duration(usually within 1-2 years).Although the mechanism underpinning the decline or loss of plant cellular totipotency is not well-characterized,somaclonal variation,including chromosomal instability,is considered to be one of the main causes.Chromosomal instability(CIN)including both numerical and structural chromosomal variations.Numerical chromosome instability refers to the loss and/or gain of whole chromosome(s),while structural chromosome instability includes translocation,inversion and copy number variation of large chromosomal segments.In addition,in the dedifferentiation process of callus induction and subsequent subculturing of callus,the regulation of normal cells will be damaged,which could lead to various molecular level somatic clonal variations including single nucleotide polymorphisms(SNPs)and small fragmental insertion/deletions(Indels).Given current situation and future trend of ginseng research,described above,the objectives of this study are:(1)do the cellular totipotency decline or disappear during the subculture of ginseng callus?(2)are there any somatic clonal variation at chromosome level(CIN)and nucleotide sequence level(SNPs)in the process of ginseng callus culture? if there are,what are their characteristics?(3)is there any correlation between CIN and somatic clonal variation at the nucleotide sequence level?(4)what is the relationship between somaclonal variation(at both CIN and nucleotide sequence level)and cellular totipotency of ginseng callus? As aforementioned,this study also completed a high-quality ginseng genome to facilitate addressing the foregoing questions.The main research contents and respective results completed in this study are briefly described below.First,our study assembled and annotated a high-quality ginseng genome by employing the latest 3rd-generation Nanopore sequencing technology and genome assembly leveraged technology based on Hi-C chromatin capture.The total length of the genomic sequence was 3.36 Gb,contig N50 was 28.71 Mb(99.93% attached to the chromosome),and the total sequencing depth reached 76.74 times of coverage over the ginseng genome sequence,which included 3.07 G bases of annotated repeats and65,913 protein coding genes.Second,our study evaluated the cellular totipotency of ginseng callus,which has been subcultured for 12 consecutive years,and explored their numerical and structural chromosomal variations.It was found that ginseng callus has almost “immortalized totipotency” to regenerate plants,and the chromosome number and structure remain highly stable(no obvious CINs)after 12 years of subculture.Third,this study analyzed the transcriptional expression of potential candidate genes involved in the maintenance of chromosome stability and cellular totipotency in ginseng callus(sampled at the 5th,9th and 12 th year of subculturing).It was found that the ginseng genes homologous to the totipotency-and CIN-related genes identified in the model plant Arabidopsis thaliana and human were stably expressed in the foregoing three ginseng callus samples.Gene ontology(GO)enrichment analysis showed that these genes played multiple important roles in specific biological processes.Finally,we performed whole-genome resequencing and conducted single base-resolution analyses for the genetic variations in the regenerated ginseng plants from long-term cultured calli.Respective genomic characteristics,chromosome distribution,functional category and correlation with expression of the genes were analyzed in detail.It was found that there were somaclonal variations in the form of SNPs in regenerated plants,which harbored the following features:(1)non-random distribution in different chromosomal regions;(2)the rates of non-synonymous mutation was higher than those of synonymous mutation;(3)the mutation rates of genes related to cell totipotency and chromosomal stability were lower than those of the background genes;(4)the expression levels of genes harboring SNPs were higher than those of wild-type genes;(5)genes harboring genetic variants were enriched into genic pathways involved in key biological processes such as cellular component organization,development and reproduction.Overall,based on the successfully assembled high-quality ginseng genome,this study found two unique,and as yet unreported,biological properties of ginseng callus cultured in vitro,namely,long-term cell totipotency and chromosome stability.Our study also provided candidate genes for future exploration about the molecular mechanism underpinning these characteristics.In addition,we also found that the somaclonal variation at the nucleotide sequence level did not affect CIN and cellular totipotency in cultured ginseng callus.Our findings deepened our understanding about the cellular totipotency of plant cultured cells and its relationship with chromosome stability and nucleotide sequence level variation,as well as provided empirical evidence for the feasibility of propagating rare and elite ginseng germplasms through in vitro callus culture.Additionally,the assembled and annotated high-quality ginseng genome lays a foundation for future evolutionary studies of ginseng and for establishing genome-based breeding in this otherwise recalcitrant crop. |
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