Somatic Embryogenesis And Preliminary Analysis On Regulatory Network Mediated By MiRNAs In Two Lilies Native To China

Lilies(Lilium spp.)are the famous flowering bulbs all over the word.It has ornamental value,edible and medicine uses.However,there are still many problems in bulb production of our country.For example,the bulb production has not been home-produced,the domestic varieties with independent intellectual property remains limited,and we still have difficulties in germplasm conservation which lead to many precious resources face extinction.Somatic embryogenesis is vital to the germplasm preservation,genetic traits improvement and bulb propagation in Lilium.Current researches focus mainly on selection of plant growth regulation and mediums,but have not involved in mechanism of somatic embryogenesis.Somatic embryos have been obtained in only a few species and could not meet the demands of practical production.miRNA has been demonstrated play a significant role in regulation of large genes network involved in plant somatic embryogenesis.However,the mechanism of somatic embryogenesis regulated by miRNAs remains unclear.Thus,the construction of small RNA library and mining critical miRNAs during Lilium somatic embryogenesis are small but essential step to figure out the regulatory network of miRNA-targets and to elucidate the regulatory roles of miRNAs during somatic embryogenesis in Lilium.The main results in this study are presented as follows:1.Somatic embryos have been successfully induced in Lilium pumilum.DC.Fisch.and Lilium davidii var.unicolor.Additional histological examination and dynamic analysis during Lilium somatic embryogenesis were performed.Results indicated that the combination of 1.0 mg·L-1 picloram and 0.2 mg-L-1 a-naphthaleneacetic acid(NAA)increased the amount of embryogenic callus that formed with a maximum on 100%and 76.7%of all explants which formed 11 and 6 somatic embryos per explant of Lilium pumilum DC.Fisch.and Lilium davidii var.unicolor,respectively.Histological results indicated significant differences between embryogenic cells and non-embryogenic cells in cellular morphology.Embryogenic cells showed large nuclei,dense cytoplasm and concentrated large quantities of starch grains.But non-embryogenic cells had prominent parenchyma cells without an obvious nucleus.Somatic embryos initially formed via exogenous and endogenous origin.In the former,globular embryos developed from superficial cells or sub-superficial cells of embryogenic callus while those derived from an endogenous origin formed from the inner cells of embryogenic callus.There were 3 patterns in the followed developmental stages of endogenous origin.Globular embryos always embedded in inner cells,broke through the inner cells or broke through the superficial cells.The indicators of somatic embryogenesis,including appearance of the protoderm,the bipolar shoot-root development and the suspensor like structure,confirmed the true somatic embryogenesis in Lilium.Changes of starch,sucrose,glucose and fructose contents suggested active carbohydrate metabolism involved in Lilium somatic embryogenesis.2.cDNA libraries of somatic embryos in Lilium pumilum.DC.Fisch.and Lilium davidii var.unicolor have been constructed.RNA sequencing produced a total of 66,422 and 35,450 unigenes in Lilium pumilum.DC.Fisch.and Lilium davidii var.unicolor,respectively.Gene Ontology enrichment indicated that 47,900 and 28,573 unigenes were successfully assigned to GO terms,respectively.Among 42 GO terms involved in somatic embryogenesis,most were distributed among embryo development,terminating in seed dormancy,root development,embryo sac development or seed germination.After analysis of KEGG enrichment,341 and 319 pathways were mapped to KEGG database in Lilium pumilum.DC.Fisch.and Lilium davidii var.unicolor,respectively.Pathways with most unigenes were ribosome,carbon metabolism,biosynthesis of amino acids,starch and sucrose metabolism and plant hormone signal transduction.3.Small RNA libraries of somatic embryos have been constructed in Lilium pumilum.DC.Fisch.and Lilium davidii var.unicolor,respectively.A total of 2,378,760 and 1,727,322 sRNAs were annotated.A length of 24 nt was the most abundant class sRNAs.The highest abundance was observed in cotyledon embryo.After analyzing the results of the alignment against miRbase,a total of 452 known miRNAs belongs to more than 86 miRNA families in Lilium pumlum.DC.Fisch.,and a total of 314 known miRNAs belongs to more than 47 miRNA families in Lilium davidii var.unicolor.Among these miRNAs,miR159 was the largest family and was the most abundant miRNA family.The known miRNA families of both lilies were sorted according to an alignment with their homologs across 24 land plants into the following four categories:highly conserved,moderately conserved,less conserved and species-specific miRNAs.There were 389 and 273 miRNAs that aligned with potential target sequences in Lilium pumilum.DC.Fisch.and Lilium davidii var.unicolor,respectively,and the relationship between miRNA and targets was complicated.In addition,results of GO and KEGG enrichment were different between two lilies.4.In this study,seventeen miRNAs together with two snRNAs,one rRNA and three protein-coding genes were selected as reference candidates,and their expression stability was validated by qRT-PCR among eleven developing somatic embryo cultures in two lilies.Four normalization algorithms,including geNorm,BestKeeper,NormFinder and RefFinder,were also used to evaluate the stability of the reference candidates.For Lilium pumilum DC.Fisch.,lpu-miR159a was the optimal reference gene during SE,followed by lpu-miR408b,while U6 was the least stable reference candidate.For Lilium davidii var.unicolor,FP presented greater stability than did half of the miRNA candidates,but the best reference gene was lda-miR162,followed by lda-miRl59a.Further analysis of the expression level of miR156 and miR529 was used to evaluate the validity of the reference genes in both lilies.In general,miRNAs are superior to common protein-coding genes and snRNAs/rRNAs as reference genes for miRNA expression normalization during Lilium SE,and the most suitable reference miRNA is different between two species in the same Lilium genus.5.Differentially expressed known miRNAs were identified during various stages of somatic embryogenesis.The expression levels of differentially expressed miRNAs and targets were validated using qRT-PCR.Combined with targets annotation,possible function of differentially expressed miRNAs and targets were preliminary discussed.Results suggested complicated expression profiles of 24 miRNAs and 12 targets and some negative relationship between miRNA and targets.For example,in Lilium pumilum DC.Fisch.,miR394a and miR528a may be involved in carbohydrate metabolism by regulated BGA6 and SSG.In Lilium davidii var.unicolor,miR167,miR397and miR408 may participate in cell division of embryogenic cells and synthesis of lignin via rugulating WRKY and LAC.miR395 and miR396 were requested in embryogenic callus induction and cotyledon embryo formation.Analysis on expression profiles of miRNAs and targets is the prerequisite for construction of complicated miRNA-targets network and functional annotation of miRNA during Lilium somatic embryogenesis.In conclusion,focused on somatic embryogenesis of Lilium pumilum DC.Fisch.and Lilium davidii var.unicolor,this study has summarized histological characters and carbohydrate changing regularity based on histological examination and dynamic analysis of carbohydrate.Furthermore,lots of miRNAs and targets related to Lilium somatic embryogenesis have been explored based on high-sequencing technology.Finally,the expression levels of differentially expressed miRNAs and targets have been validated by qRT-PCR.Therefore,this study provides reference value for explanation of plant somatic embryogenesis.