Identification And Analysis Of Cold-responsive Genes At Germination And Seedling Stages In Maize

Maize is an important food crop.In recent years,its sown area and yield have ranked first among the three major food crops in China.However,maize originated in tropical region and is a thermophilic crop.Low temperature stress during germination and seedling stages will affect seed germination and the growth and development of seedling roots,stems,and leaves,and affect grain filling and the final yield and quality of maize.Therefore,it is of great significance to dig out the cold stress-related candidate genes in maize.In this study,we evaluated the cold tolerance of different maize inbred lines by investigating low temperature phenotypic traits at germination and seedling stages under cold stress conditions.We identified cold-tolerant germplasms at germination and seedling stages based on phenotypic statistics,identified significant SNPs associated with cold-tolerant phenotypes at germination and seedling stages by GWAS,explored key candidate genes for cold response with the help of RNA-Seq and 3’Ribo-Seq technologies.The main results are as follows:1.We used germination index and vitality index as the evaluation indicators of cold tolerance,and identified 274 maize inbred lines for cold tolerance at germination stage.The germination ability of different materials showed a certain difference under cold conditions.The generalized heritability both germination index and vitality index were high（H²=0.83）,indicating that genetic factors had a greater influence on phenotypic variation.2.We performed GWAS on germination index and vitality index.At the threshold of P=10^-5,BLUP results detected a total of 55 SNPs that were significantly associated with germination index,which could explain 8.38%-14.83%of the phenotypic variation,significant markers are located within 30 candidate segments;BLUP results detected a total of 155 SNPs that were significantly associated with vitality index,which could explain8.06%-14.64%of the phenotypic variation,and significant markers were located within 27candidate segments.30 SNPs with P values less than 0.0001 in at least two replicates of germination index and vitality index were distributed in 8 candidate segments and mapped to 8 candidate genes by defining the gene closest to the extremely significant marker in each segment as the candidate gene.3.By investigating the wilting length of the second leaf of maize seedlings treated at4℃for 72 h,using the wilting ratio as an index,we evaluated the cold tolerance of 292maize inbred lines at the seedling stage,found the cold tolerance of different inbred lines was different after 4℃.The generalized heritability of the wilting ratio was high（H²=0.75）,indicating that the variation was mainly caused by genetic factors.4.We performed GWAS on the wilting ratio at seedling stage.At the threshold of P=10^-5,BLUP results detected a total of 144 SNPs that were significantly associated with leaf wilting ratio,which could explain 7.41%-12.17%of the phenotypic variation.Significant markers were located within 15 candidate segments.The 14 significant SNPs all had high P values(-log₁₀P≥4.5)in at least 2 replicates were distributed in 4 significant segments,and mapped to 4 candidate genes.5.Taking cold-tolerant inbred line CML285 and cold-sensitive inbred line CIMBL20as materials,we performed sequencing analysis at the transcriptional and translational levels on maize seedlings treated at 4℃for 0 h and 72 h.We identified 12,349 and 716DEGs at transcriptional and translational levels in cold-tolerant material,respectively,identified 9,077 and 671 DEGs at transcriptional and translational levels in cold-sensitive material,respectively.We compared the DEGs identified in the two materials and found that 3,039 and 125 genes were up-regulated in both materials at the transcriptional and translational levels,respectively,2,017 and 37 genes were down-regulated in both materials,the identification of these genes indicated that the two materials had commonalities in response to cold stress.In addition,we also identified a number of genes that responded differently in the two materials.For example,at the transcriptional level,209 genes were up-regulated in cold-tolerant material and down-regulated in cold-sensitive material,246genes were down-regulated in cold-tolerant material but up-regulated in cold-sensitive material.The genes that respond differently in cold sensitive materials may be related to their differences for cold tolerance.6.In order to analyze the difference in low temperature response between cold-tolerant and cold-sensitive materials,we studied the low-temperature response of 31photosynthesis-related genes and 66 reported genes involved in low-temperature response in two materials.For photosynthesis genes,we found that after treatment at 4℃,8 and 3genes were down-regulated in cold-tolerant and cold-sensitive materials,respectively.Among them,7 genes were only down-regulated in cold-tolerant material,and the number of down-regulated genes in cold-tolerant materials was higher than cold-sensitive material.For the reported cold-responsive genes,we found that there were 35 and 30 DEGs at the transcriptional level in cold-tolerant and cold-sensitive materials after treatment at 4℃,respectively.Among them,7 genes were specifically up-regulated in cold-tolerant material.These genes that are specifically up-regulated in cold-tolerant material may cause differences in cold-tolerance among different cold-sensitive materials.7.Genome-wide association study for cold tolerance trait in seedling stage located 17candidate genes in significant segments on chromosomes 1 and 4.We analyzed the expression of these candidate genes at different omics levels,we found that the genes Zm00001d030256 and Zm00001d053313 were differentially expressed at the transcriptional level.Zm00001d030256 was up-regulated in cold-tolerant material,but not differentially expressed in cold-sensitive material.Zm00001d053313 was up-regulated in cold-tolerant material and down-regulated in cold-sensitive material.At the translational level,the two genes were not differentially expressed,but the expression patterns were inconsistent between cold-tolerant and cold-sensitive materials.In conclusion,the genes Zm00001d053313 and Zm00001d030256 co-localized in GWAS and different omics sequencing technologies are key candidate genes for cold tolerance trait at the seedling stage in Maize.