Tetraploid Induction And Molecular Basic Of Vegetative Slowly Growth In Tetraploid Populus Spp.(section Tacamahaca)

There are two types of tetraploid plant that the grow faster and the grow slower than vegetative growth diploid.The research on the molecular basis of the formation of polyploid vegetative growth traits is of great significance for promoting the theory and technological progress of plant polyploid breeding.In this paper,full-sib progeny of(P.pseudo-simonii x P.nigra 'Zheyin3#')×(P.× beijingensis)were selected as materials,on the basis of the chromosome doubling technique for inducing adventitious buds of leaves in vitro.The molecular basis of gene expression and the miRNA expression changes in different positions leaves of tetraploid and diploid were researched by using transcriptome sequencing(RNA-seq)and miRNA sequencing.The main results are as follows:(1)We successfully proposed a technical approach of instant discrimination an effective treatment period for inducing somatic chromosome doubling according to the leaves incision callus developmental condition.The induction efficiency of tetraploid of Populus was influenced by the pre-culture time,concentration and duration of colchicine treatment.There was a significant correlation between the incision callus development stage and tetraploid induction efficiency,which could be used as a discriminant basis for chromosome doubling effective period of Populus leaves.There was no significant correlation between tetraploid induction efficiency and genotype in the condition that suitable treatment for chromosome doubling.When the leaves were pre-cultured on the differentiation medium for about 5 d,a few transparent calli appeared at the incision leaves,and when the callus width was less than 1 mm,the effective period for inducing adventitious bud chromosome doubling of most multiple genotypes.At this time,with 30 mg/L colchicine for 3 days,and the induction rate of tetraploid was arrived 5.9 to 12.6%.(2)The studies showed that the Populus tetraploid is the type of growing slowly and dwarf.The leaf shape of tetraploid changed and showed as broad-oval shape.Leaf thickness,leaf vein and stem internodes,xylem cells were significantly larger than diploid,while the high growth rate was significantly less than diploid.For the first 10 leaves from plant tip,the photosynthetic rate of tetraploid was 57.53%?9.50%higher than corresponding leaves of diploid.But with the increase of leaf age,the photosynthesis of tetraploid was decreased by 12.8-48.7%.In addition,the physiological and biochemical indexes such as total protein and MDA content,and POD,CAT activity of tetraploid showed a tendency of decreasing rapidly with the leaf age increase,even significantly lower than the same leaf position of diploid(3)The results showed that the endogenous hormone content and differential expression gene of Populus tetraploid showed regular changes consistent with plant growth with leaf age increased.Compared with diploids,the expression of CPS,KS,KAO,GA3-oxidase,A uxin,Aux/IAA,ARFs,BRI1,which are involved in the regulation of GA3,IAA,and BR synthesis and signal transduction positively related to vegetative growth,were significantly decreased with leaf age increased.However,the related genes expression of endogenous hormone ABA,JA synthesis and signal transduction negatively correlated with vegetative growth,such as ERF,JAZs,PYL,PP2C,SnRKs,ABF,was the trend of increase with the leaf age increasing.Affected by gene regulation,analysis of hormone content found that the contents GA3,IAA,ZT,which promote cell division and stem growth,were decreased with the leaf age increasing in tetraploid,significantly lower than the same leaf position of diploid.In addition,the contents of hormone JA and ABA involved in cell senescence have a tendency to increase with the leaf age increasing.The change of tetraploid hormone content may be one of the important reasons to accelerate senescence and slow down the growth rate of tetraploid leaves.(4)The ability of chloroplast synthesis,light reaction,sucrose and starch synthesis and decomposition showed a tendency of first increasing and then decreasing with leaf age increasing.And the Tetraploid degrades faster than diploid chloroplasts.Compared with diploid,the gene of POR,LHCB3,LHB1B1,ELIP involved in chlorophyll synthesis,two submits protein complexes Psa and Psb gene family in thylakoid membrane,such as PsaF,PsbR,the LHC family gene of LHCA3,LHCB1 involved in photosynthetic reaction,and key genes of SUS,SPS,SS,AMS,BAM,PHS involved in sucrose and starch biosynthesis were down-regulated with the leaf age.However,the genes of PAO1,CLH related to chlorophyll degradation showed a tendency of up-regulation with the leaf age increasing.The determination of important products of gene expression of chlorophyll,sucrose,fructose,glucose content and related SUS,a-AMS,?-AMS activity and the chloroplast ultrastructure were showed that the disintegration of chloroplast was faster,and the photosynthetic reaction and sucrose and starch biosynthesis metabolic ability were lower in tetraploid than in diploid,which were consistent with the trend of gene expression.(5)The miRNAs which related to vegetative growth traits in tetraploid Populus showed dosage effect,in which the expression of miR156,miRNA159,miR167,miRNA390,miR393,miR396,miR399 families were significantly increased with leaf ageing,causing a relatively stronger negative regulation,then the expression of ARF,TIR,GAMA Y,GRF,SPL target genes,which were positively correlated with vegetative growth,were significantly decreased.However,the expression of miR530,miR164,miR172 and miR319 decreased with leaf ageing increasing,causing a relatively weaker negative regulation,then the expression of ERF,NAC,AP2,TCP target genes,which were negatively correlated with vegetative growth,were significantly up-regulation.The miRNA expression showed dose effect may be one of the important mechanisms of vegetative growth slowly in tetraploid Populus.(6)Molecular mechanism of vegetative slowly growth in tetraploid Populus was elucidated.In conclusion,the expression of miRNAs,which were directly related to vegetative growth traits,showed dose effect with leaf aging,in which the miRNAs families expression were significantly increased,causing a relatively stronger negative regulation,then the expression of target genes related to positively correlated with vegetative growth,such as ARFs,GAMYB,were significantly decreased.The content of GA3 and auxin,which promote growth hormones,were significantly lower in tetraploid than diploid.However,the miRNAs expression decreased with leaf ageing,causing a relatively weaker negative regulation,and then the expression of target genes related to negatively correlated with vegetative growth,such as ERF,TCP which were negatively correlated with vegetative growth,were significantly increased.The content of ABA,JA and ET,which regulated senescence,were significantly higher in tetraploid than diploid.As a result,the chloroplast degradation and leaf senescence were accelerated,the photosynthetic rate was decreased,and sugar,starch synthesis and decomposition ability were decreased in tetraploid compared with diploid.Finally,the vegetative growth in tetraploid Populus was slower than diploid.