Analysis Of The Polyploidy Induction,Different Gene Expression Of Dendrobium Officinale And The Development Of Its EST-SSR Markers

Dendrobium officinale Kimura et Migo is epiphytic perennial herb in Orchidaceae.Its medicinal component contains polysaccharides,alkaloids,amino acids,trace elements and other medicinal ingredients,which mainly distributes in Yunnan,Zhejiang,Fujian,Guangdong and Guangxi provinces.With the utilization of D.officinale and its derivative products,the wild resources have been excessively over-exploited and gradually depleted.Cultivars became single due to domestication from wild germplasm.Therefore,massive efforts should be made in the field of protecting wild resources and artificial breeding of new varieties in D.officinale.In this study,polyploid breeding methods were used in induction of D.officinale.Comparing the content of polysaccharide and expression of genes related to the synthesis of polysaccharides in D.officinale between diploid and tetraploid plants.Developing EST-SSR markers for molecular identification of D.officinale gennplasm resources based on transcriptome unigenes.Firstly,colchicine was used to induce protocorms and buds of D.officinal The results revealed that survival rate of protocorms and buds decreased significantly.Survival rate of protocorms were lower than buds in higher concentration treatment.Variation rate were not correlated with treatment time.Variant plants of protocorms were the most in the treatment of 0.6 mg/L colchicine for 30 min,while variant plants of buds concentrated in the treatment of 0.8 mg/L colchicine for 30 min.Protocorms were more sensitive of colchicine than buds.Then,the polyploid of D.officinale from treated materials were accurately identified in terms of morphological features,stomatal guard cell features,chromosome number and nuclear DNA content.Secondly,in order to compare the polysaccharides content of D.officinale between diploid and tetraploid,polysaccharides were extracted by the method of hot water extraction and calculate the content of polysaccharide.The results showed that the content of polysaccharides in tetraploid plants was significantly higher than diploid plants,increasing by 30.34%.Three relevant enzymes genes were selected in the synthesis of polysaccharides:Phosphoenolpyruvate carboxylase gene(PEPC),UDP-glucose Pyrophosphorylase gene(UGP)and Sucrose Phosphate Synthase gene(SPS).From transcriptional level,in order to explain the impact between chromosome doubling and polysaccharide synthesis,different expression of the three genes were quantified in diploid and tetraploid plants.The results showed that PEPC gene in tetraploid D.officinale was 4.006 times of diploid plants;SPS gene was 2.996 times of diploid plants;UGP gene was 2.879 times of diploid plants.The expression of the three genes has shown a rise in tetraploid D.officinale.Finally,EST-SSR is an efficient and convenient method of molecular marker.The research adopted bio-informatics analysis technology for screening 983 EST-SSR based on the transcriptome of D.officinale unigenes.It is found that there are four types units of nucleotide repeats including dinucleotide,trinucleotide,tetranucleotide and pentanucleotide in D.officinale,which the number of trinucleotide up to 84.94%.240 pairs of primers were designed for polymorphic amplification in 18 D.officinale materials.The results showed that 16 EST-SSR markers were successfully developed which may play a role in conservation genetics,phylogeography and genetic breeding of D.officinale.