Precise Analysis And Gwas Of Vascular Bundle Phenotype In Shank Of Maize

The yield of maize is closely related to the source-sink,and abundant source-sink and efficient flow are the best conditions.Shank,formed by the differentiation and development of the ear,it is the branch stem connecting stem and ear,and the last step from ‘source’ to ‘sink’.The vascular bundle of the shank is an important "flow" organ for transforming maize biological yield to grain yield and its microscopic phenotypic characteristics and genetic analysis are of great significance for promoting the breeding of new varieties with high yield and good quality.In this study,CT images were obtained using the standard process for stem micro-CT data acquisitiontechnology,and its resolution could reach 13.5 um.Moreover,five categories,36-item multi-level phenotypic traits of the shank including cross-section-related,epidermis zone-related,periphery zone-related,inner zone-related,and vascular bundle-related were analyzed through an automatic CT image process pipeline based on the functional zones.Based on two years’ phenotypics data from Beijing and Sanya,best linear unbiased prediction(BLUP)of 36 phenotypic indexes were calculated as the final phenotypic values and genome-wide association analysis was performed to predict candidate genes.The results are as follows:(1)The cross-section of the shank can be divided into nearly round,several other categories,including crescent shape,bell shape,horseshoe shape,and irregular shape.Vascular bundle phenotyping pipeline,vesselparser,was used to realize the high-throughput and automatic analysis of vascular bundle phenotype of maize shank.The frequency distribution of 36 phenotypic traits in the association analysis population showed continuous variation,indicating that the microphenotype of panicle stalk was a typical quantitative trait controlled by multiple genes.Moreover,clustering analysis of 36 phenotypic traits was conducted with hierarchical clustering using the spearman correlation as a distance metric.Group I mainly reflected the distribution properties of vascular bundle,containing 3 phenotypic parameters..Geometric and morphological characters of shank cross-section and functional zones,and total number and area character of vascular bundles were gathered into group II,containing 19 phenotypic parameters.Group Ⅲ was composed of the remaining 14 phenotypic parameters,representing the mean characteristics of vascular bundle shape and size.The systematic and abundant indicators of maize shank vascular bundles provided the phenotypic data basis for the follow-up omics research.(2)We analyzed the phenotypic variation of vascular bundles at the base of the shank between natural population subgroup based on comprehensive phenotypic information.It was found that the number of vascular bundles in the inner zone(IZ＿VB＿A)and the density of vascular bundles in the inner zone(IZ＿A)varied the most among different subgroups.In addition,Duncan’s test showed that the shank cross-section area of NSS subgroup were significantly higher than that of SS,Mixed and TST subgroups,and inner zone area(IZ＿A),inner zone thickness(IZ＿T)of NSS subgroup were much larger.Meanwhile,total number of vascular bundles(VB＿N)and vascular bundles number in the inner zone(IZ＿VB＿N)of NSS subgroup were more than that of SS,Mixed and TST subgroups,and total area of vascular bundles(VB＿A)of NSS subgroup were much higher.On the contrary,vascular bundles density(VB＿D),vascular bundles density in the inner zone(IZ＿VB＿D)and density of vascular bundles in the periphery zone(PZ＿VB＿D)of SS,Mixed and TST subgroups were significantly higher than that of NSS subgroup.(3)The heritability of 36 phenotypic traits of shank and vascular bundles were calculated.36 phenotypic parameters showed different heritability patterns,ranging from0.22 to 0.65.30 items traits had heritability > 0.3 indicating that variability in shank micro-phenotypes was governed in large part by genetic factors.(4)Combined with genome-wide association studies(GWASs),1245 significant single nucleotide polymorphism(SNPs)were identified and 199 specific candidate genes for 30 traits were detected,including the total area of vascular bundles(VB＿A),the total number of vascular bundle(VB＿N),the density of vascular bundles(VB＿D),et al.These candidate genes encode mainly involved in lignin,cellulose synthesis,transcription factors,protein kinases and plant signal transduction and stress response.(5)Pathways enriched by functional enrichment analysis were performed on 199 candidate genes with detailed annotation.A total of 20 KEGG pathways(P-value < 0.05)were enriched,mainly including ‘Biosynthesis of secondary metabolites’,‘Metabolic pathway’,‘ABC transporters’ and ‘Steroid biosynthesis’,et al.In addition,a total of 60 GO terms(P-value < 0.05)were enriched,mainly including ‘glucan metabolic process’,‘regulation of hormone levels’,‘cellular carbohydrate metabolic process’,‘cellular macromolecule metabolic process’ and ‘response to light stimulus’,et al.The results presented here will improve the understanding of phenotypic traits of maize shank,and provide an important phenotypic basis for high-throughput identifying of vascular bundle functional genes of maize shank and promoting the breeding of new varieties with high yield and good quality.