Determination Of DNA Content Of Mulberry,and Preliminary Exploration Of Gene Expression Differences Of Mulberry Autopolyploids

Mulberryis one of the woody plants which are of significant economic and ecological value which have abundant germplasm resources and complex ploidy level.The basic scientific data of mulberry are still short of C-value.Polyploid breeding is an crucial approach to increase utilized value.Therefore,analysing the gene expression changes after polyploidization has profound significance.In this study,optimization of the method for determiningchromosome ploidy and nuclear DNA content of mulberryby flow cytometry（FCM）was operated,and chromosome ploidy and nuclear DNA content of mulberry varieties was determined.Meanwhile,we obtained expression level per cell of the genes based on number of cells in RNA-Seq samples and expression level of exogenous internal standard RNAs by RNA-Seq.Therefore,we preliminarily comparedthe genes expression differences between mulberry diploid and autopolyploids.The main contents and conclusions of this study are as follows:1.Optimization of the method for determiningchromosome ploidy and nuclear DNA content of mulberryby FCMThe results indicated that the following steps constitute a suitable sample preparation method for determiningchromosome ploidy and nuclear DNA content of mulberry:taking0.2 g young leaves as material,adding MgSO₄ dissociation buffer,chopping the leaves quickly on chilled plain glass with double-edged razor blade,transferring the sample into a petri dish and keeping static for 3⁵ min,and sieving the sample into 1.5 mL centrifugation tube using300 mesh cell strainer.The resultant single nucleus suspension（500μL）was added with PI solution and RNase A solution,stained for 30 minutes under dark condition at 4℃,sieved with 300 mesh cell strainer,and finally sent to testing on flow cytometer.We preliminary established a suitable method for determining chromosome ploidy and nuclear DNA content ofmulberry by FCM which couldachieve optimalresult that includes less cellulardebris,clear main peak and less miscellaneous peaks,etc.2.Determining nuclear DNA contentof mulberry varietiesQindou 8 was used as internal standard,we determined the nuclear DNA content and C-valueof 22 mulberry varieties included part of varietiescommonly used for industry and Xinjiang black mulbrerry by FCM.The rage of C-value of mulberry varieties determined in this study which involved different mulberry species and ploidy levels is 0.281⁰.402 pg.3.Analysing differences of gene expression between mulberry diploid and autopolyploids by RNA-SeqIn this study,4 DNA fragments of E.coli was in vitro transcribed to obtain exogenous internal standard RNAs which was added into RNA-Seq sample of mulberry.Basing on copy number of internal standard RNAs and cell number of RNA-Seq sample,we preliminarily established the method for comparing the gene expression differences between different ploidy mulberry according to gene expression level normalized by the expression level of internal standard RNAs.This study determined nuclear DNA content of mulberry varieties which enrichs the cytogenetical data.In addition,the nuclear DNA content and optimized method for determining chromosome ploidy and nuclear DNA content of mulberry provide reference and theoretical basis for fundamental study,polyploid breeding,utilization of germplasm resources of mulberry.Moreover,this study preliminarily established the method for analysing the gene expression changes per cell after polyploidization by RNA-seq,which has an important meaning for exploring physiological and molecular mechanism of the character changes after polyploidization.