Genome-wide Association Studies And Functional Studies Of Candidate Genes For Germination-related Traits Of Low Temperature Tolerance In Maize

Maize(Zea mays L.)originated in tropical and subtropical areas and is naturally sensitive to low-temperature stress,especially during seed germination.As spring maize,seed germination and seedling growth at an early stage are usually subjected to low-temperature stress.Despite the increasing average temperature,further seedling growth also seems to be affected,which may be due to the inability of plants to respond quickly to favorable environmental changes.However,the dynamic changes of maize seed germination at low temperature and the effect of low temperature germination and recovery at normal temperature on seedling growth are still ambiguous.238 maize inbred lines were used to determine the phenotypic traits related to low temperature germination tolerance of maize and establish the evaluation system of low temperature tolerance.Genome wide association studies(GWAS)was carried out for the evaluation index of low temperature tolerance.Low temperature sensitive maize inbred line and low temperature resistant maize inbred line were selected to construct F_2:3families.The F_2:3family extreme pool was constructed for BSA-seq sequencing.Transcriptome analysis of dynamic changes of maize germination at low temperature and compensation mechanism of low temperature germination to normal temperature recovery.Combined with GWAS,linkage analysis and transcriptome analysis,candidate genes were mined.The main results are as follows:1.In this study,18 evaluation indexes of low temperature tolerance germination of 238maize inbred lines were statistically analyzed,of which 12 evaluation indexes of low temperature tolerance had large coefficient of variation.The results of cluster analysis relative decrease of root length is similar to those of comprehensive evaluation cluster analysis of 12low temperature tolerance indexes.The relative decrease of root length ranges from 0.105 to0.936,which conforms to the normal distribution and can distinguish the tolerance of low-temperature germination.Therefore,the relative decrease of root length is used as the evaluation index of low-temperature tolerance.The relative decrease of root length of04Qun0522-1-1 was 0.524,which was low temperature maize resistant maize inbred line(RM).The relative decrease of root length of B283-1 was 0.936,which was low temperature sensitive maize inbred line(SM).2.In this study,the K+Q model was used to set the threshold-log10P=4.61 to score the GWAS of evaluation indexes of low temperature tolerance.A total of 47 SNP loci significantly associated with low temperature tolerance were detected,of which the SNP loci significantly associated with chromosome 8 were the most,and the interpretation range of phenotypic contribution rate was 10.88%-16.31%.It can be used as a candidate interval for subsequent screening and analysis.GWAS analysis showed that there were at least 2evaluation indexes of low temperature tolerance,and a total of 6 SNPs were detected,of which 1 was on chromosome 5 and the other 5 were on chromosome 3.The consistent loci on chromosome 3 were located between PZE-103174386(220,933,020)and PZE-103174950(221,386,301).3.In this study,analyzed the genetic linkage of 574 F_2:3populations constructed by 04Qun 0522-1-1 x B283-1 by BSA-seq method combined with KASP genotyping technology.The results of F_2:3population linkage analysis were consistent with the gene candidate region predicted by GWAS.BSA-seq analysis showed that there were 13 all indexes on chromosome3 between 220.9 Mb and 221.3 Mb.The average value of?all index in the extreme mixed pool of the two offspring was-0.089.There was linkage disequilibrium in the interval.Therefore,the candidate interval of this study is located on chromosome 3 220.9 Mb-221.3Mb.There are 21 candidate genes in the localization interval.4.In order to verify the genes in the waiting area,transcriptome analysis was carried out on two maize inbred lines with different low temperature tolerance.Dynamic changes in seed germination under low-temperature stress in maize.The antioxidant enzyme activity of RM inbred lines was higher than that of SM inbred lines.Transcriptome analysis of dynamic changes in maize germination at low temperature showed that SM inbred lines germinated at low temperature,and Gene Ontology(GO)terms related to photosystem II(GO:0009523,P=2.56E-7)and heat shock protein(GO:0031072,P=8.55E-5)were down regulated from 0to 2 days.Ribosome related genes were down regulated in both maize inbred lines.At 2 to 4 days,the GO terms related to antioxidant activity(GO:0016209)and ribosome(GO:0005840)of two maize inbred lines were up-regulated,and the number and degree of up-regulation of RM were greater than that of SM.The genes related to antioxidant activity are mainly peroxidase(POD)related genes.Vitamin B6(VB6)related GO terms were significantly up-regulated in RM inbred lines from 4 to 6 days.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the linoleic acid metabolism and phenylpropane biosynthesis pathways of RM inbred lines were significantly enriched earlier than SM inbred lines.Low temperature germination of maize is a complex regulatory mechanism.5.The two maize inbred lines germinated at 13?for 4 days,compared with 1.5 days at25?,the total antioxidant capacity and POD activity of low-temperature germination decreased,and the total antioxidant capacity of SM decreased more.Antioxidant enzyme activity:the POD activity of SM inbred line decreased significantly and the superoxide dismutase(SOD)activity increased not significantly,while the POD activity of RM inbred line decreased not significantly and the SOD activity increased significantly.Transcriptome analysis showed that the antioxidant activity related genes of the two inbred lines were down-regulated,but the down-regulation degree of RM was less than that of SM,and the abscisic acid response related genes were up-regulated,but the up-regulation degree of RM was less than that of SM.The two maize inbred lines germinated at 13?for 4 days and recovered at 25?for 2 days.Compared with 25?for 4 days,the total antioxidant capacity of RM inbred lines increased significantly,while the total antioxidant capacity of SM inbred lines decreased significantly.The lipid peroxidation of SM inbred lines was higher than that of RM inbred lines.The SOD activity of RM inbred lines increased significantly and that of SM inbred lines decreased significantly.The results showed that the recovery ability of antioxidant activity of SM inbred line was weaker than that of RM inbred line after low temperature stress.Further transcriptome analysis revealed that seed germination under low-temperature stress caused the down-regulation of photosynthesis-related GO terms in two lines.However,the downregulation of RM inbred lines was less than that of SM inbred lines.Moreover,the SM line displayed down-regulation of ribosome and SOD related genes,whereas genes involved in SOD and vitamin B6 were up-regulated in the RM line.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis revealed that photosynthesis and antioxidant metabolism-related pathways played essential roles in response to low-temperature stress during seed germination.6.Combined with transcriptome analysis,12 candidate genes have transcriptional expression in this transcriptional analysis,of which Zm00001d044300 and Zm00001d044301have great differences in transcription level.Five low temperature extremely sensitive inbred lines,five low temperature resistant inbred lines and F_2:3extreme cell were used to verify that only Zm00001d044301 was differentially expressed in extreme materials.Therefore,Zm00001d044301 was used as a candidate gene in this study.Zm00001d044301 encodes PP2C protein,homologous comparison of protein sequences shows that ZmPP2C gene is very conserved in maize,rice and Arabidopsis.Phylogenetic analysis showed that the genetic relationship between maize and rice was closer.Subcellular localization showed that ZmPP2C protein was localized in the cytoplasm.7.Wmv12-ZmPP2C overexpression vector was constructed and transformed into Arabidopsis.The low temperature germination rate of the overexpressed strain was faster than that of the wild type,showing low temperature resistance.The results showed that the expression of At PP2C in PP2COE plants at 8?was higher than that in wild type.PP2C downstream gene At Sn RK2.2/2.3/2.6,the PP2COE lines at 8?for 9 days were higher than those of the wild type,and the overexpression lines at 8?for 15 days were lower than those of the wild type.The phenotype of MU mutant of Zmpp2c was analyzed.The results showed that the germination percentage,seedling dry weight,root length and seedling length of Zmpp2c MU mutant were significantly lower than B73 under low temperature stress.When germinated at 13?,the expression of ZmPP2C in B73 was higher than that of Zmpp2c MU mutant,and the total antioxidant capacity of B73 was significantly higher than that of Zmpp2c MU mutant.