Function Mechanism Analysis And Candidate Gene Screening For Soybean Necrotic Root Gene

Root necrosis is a harmful trait that severely affects the yield and quality of plants.Most of the root necrosis occur due to biotic stress.However,a necrotic root(nr)mutant has been identified in soybean(Glycine max),which is a genetic mutation that causes necrotic root spontaneously without any biotic stress.This phenotype is consistent with the phenotype of soybean roots after inoculation with the fungus.Therefore,the nr mutant is a lesion mimic mutant which the lesion manifests in the roots.The nr root exhibit an increased tolerance to Phytophthora sojae.Therefore,the study for nr is important for exploring the defense response to pathogens in soybean.In this study,nr mutant is investigated using cytology observation,transcriptomics and molecular genetics,and the main findings were as follows:(1)The phenotype and cytology observation of nr mutant.The nr causes browning and necrosis root during growth.While pure lethality in the field,a few nr mutant can grow to pods when grown in a greenhouse.However,the nr growth is worse than wild type,and nr has less pods and leaves comparing to wild type.We further characterized the period of browning and cell death by of root tissues sections observation.And it was found that browning and cell death appeared in the cortex in 4 days growth post light(6 days after germination).(2)Transcriptome data analysis of T329＿WT(Nr Nr)and T329＿nr(nr nr).We sequenced root transcriptome data of T329＿WT and T329＿nr at three time points,before browning(2dpl),early browning(4dpl)and total browning(6dpl).We found that PPO,encoding the key enzyme in melanogenesis process,was substantially up-regulated in nr mutants.So we suspected that browning roots might be associated with melanin deposition.Comparing to WT,ROS raised in nr which may stimulated a hypersensitive response leading to cell death.In nr,defense responses genes are up-regulated,such as JA synthesis and signal transduction and glyceollin biosynthesis.This may be the reason why nr root exhibit an increased tolerance to Phytophthora sojae.(3)Map-based cloning for nr gene.The progeny derived from the cross XD 0632(Nr Nr)×T328(nr nr)was used as the locus population for map-abased cloning.The nr was located between molecular markers M7 and M9 on chromosome 18.(4)Screening of candidate genes.With reference to Williams 82,approximately 290 kb in candidate region,containing 20 genes.Comparison of sequences between the wild type(T322)and mutant(T328＿nr,T329＿nr and T332＿nr),there are no mutant loci in candidate region.The mutations in the nr may be structural variants that could not be revealed in resequencing results.Gm Sam is the only differentially expressed gene in the interval and its expressed lower in the mutant than WT.the variation is We speculate that the structural variation is occurred near Gm Sam,which caused the Gm Sam expressed at a lower level in the mutant than in the wild type,ultimately leading to necrotic roots in the mutant.So,Gm Sam may be a candidate gene for nr.