Analysis Of Rice Mutant WGS And RNA-seq Induced By Maize DNA

Exogenous DNA directly introduced into rice embryo cell which is a new way of molecular breeding method and a convenient method for creating new rice germplasm and new germplasm.To study the mutant molecular mechanism of exogenous DNA directly into rice embryo cell,we use the genome sequencing and transcriptome sequencing,to explore how exogenous DNA acts on the receptor genome and the receptor genome variation,reveal the inherent law of variation,and provide a theoretical basis of the breeding method for rice molecular marker assisted breeding,the discovery of new genes and provide theoretical data.By using the method of germ cells in situ into laboratory(embryo conversion technology),the maize genome DNA into rice(Nipponbare)embryo cells,obtained in plant height,tiller number,panicle type,root traits,genetic traits have changed significantly,compared with the control strain RMIM92.In this study,the whole genome sequencing technique was used to carry out high-throughput sequencing of the mutant strain RMIM92,The results showed that: 1,the mutation of RMIM92 genome were detected in 258 DNA fragments from corn,these corn fragment length between 8bp-205 bp,2,the insertion of maize fragments into the genome of the recipient genome is random,and when integrated into the rice genome,most of the rice sequences are replaced by rice sequences of different lengths.3,The maize sequences were inserted in 103 rice genes,Among them,43 genes are missense mutations,3 genes are only splice site mutations,33 genes are missense mutations and splice site mutations,12 genes are missense and frameshift mutations,1 gene is splice site mutations and frameshift mutations.6 genes are simultaneously missense mutations,splice site mutations and frameshift mutations.4,analysis of maize sequences into(replace)rice base site found that the highest frequency of the side inserted in the base sequence maize between AnA/AnA(10.4%),the lowest frequency in the base insertion between CnG/GnC(3.1%).5,Analysis of SNP mutation types,revealed that the frequency of conversion(52.69%)was greater than the frequency of the occurrence of the transition(47.31%).6,analysis of four kinds of nucleotide mutation frequency,found that: C(26.84%)>G(26.73%)>A(23.58%)>T(22.84%),C and G had higher frequency of nucleotide mutation and mutation frequency is basically the same,while A and T mutation frequency and mutation frequency relative to the minimum consistent.7,The base of mutation pro side statistics found that: ANG(8.20%)> ANC(7.64%)> ANT(7.49%)> GNC(7.15%)> CNT(6.73%)> ANA(6.62%)> TNT(6.36%)> TNC(6.21%)> CNA(6.17%)> GNT(5.91%)> CNC(5.72%)> CNG(5.68%)> GNA(5.64%)> GNG(5.04%)> TNA(4.82%)> TNG(4.63%),showed that ANG type had the highest frequency and lowest frequency of TNG.8,the analysis of continuous mutation bases: single nucleotide mutation(94.12%)> continuous double mutation(5.49%)> three consecutive mutations(0.28%)> continuous mutation(0.10%),indicating the mutation mainly by single base mutation.Study of the mutant RMIM92's flag leaf and internode at flowering period,to explore the 103 ricegenes that inserted into maize sequences,Compared with CK1,The results show that: 1,In mutant sword leaf and stem section 16 up-regulated genes expressed,the highest increase rate of gene is OS01G0115750 in the sword leaves 5.6 fold increase in OS06G0568400 in the stem increased 9.2 times.2,In sword leaf and stem section variants in the expression of 12 genes were down regulated OS01G0123900 down 3.14 times in the flag leaf,OS04G0344100 down 10.3 times in the stem section,cut the highest rate of.3,there are one genes OS01G0124000 expressed in the stem of the leaves,which are expressed in the stem node.There are two genes that are not expressed in flag leaf,OS01G0152500 and OS04G0344100,respectively.4,there are 3 genes that are not expressed in flag leaf and stem node,OS02G0614966,OS04G0165200 and OS12G0554000 respectively.5,There are 8 genes up-regulated in flag leaves and down expressed in stem nodes 6,There were 5 genes expressed up-regulated in the stem node and down regulated g in flag leaves.The 1-6 results showed that exogenous DNA into rice genome directly affects the expression of the rice genes can cause gene expression,the gene expression level decreased,leading to gene silencing and activation of gene silencing.7,To further analyze the relationship between the mutation and phenotype of mutation,the known gene expression levels were analyzed,the results show that compared with CK1 control,plant height,number of grains per spike,heading pleiotropic gene OS07G026120,to control the width of grain weight of main gene OS02G0244100,gene controlling tiller angle upregulation of OS06G0704300 in flag leaf and stem section;grain length and grain weight of OS03G0407400 gene and expression of gene OS09G0529300 in controlling tiller angle of flag leaf and stem section cut;dwarf gene OS05G0333200,dwarfing gene OS06G0127800 few tillers in the flag leaf was up-regulated,down regulated expression in stem section;plant height,tiller and spikelet fertility gene OS01G0751600,control of blade erect and dwarf gene OS03G0227700 expression in the flag leaf,up-regulated in stem section.Mutations in the known functional genes and expression levels change may be related to phenotypic changes that corresponding to the variant.In short,germplasm introduction method is an effective biological mutation technique can make the exogenous DNA fragment inserted into the recipient genome,and cause the receptor mutation;study found mutations,mutation,mutation pro side bases results provided important theoretical data for the further study of functional genes,molecular marker assisted breeding and precise mutation breeding for new varieties and create rice gene expression regulation in the analysis and study of molecular genetic approaches have very important significance.