Association Analysis And Candidate Gene Mining Of Low Temprature Tolerance In Maize From The Germination Stage To Seedling Stage

Low temperature stress from germination to seedling stage is an important abiotic stress that affects the growth and development of spring maize in northern China.Some QTL_S related to the control of low temperature tolerance of maize from germination to seedling stage have been excavated by the previous methods such as preliminary mapping,but the consistent QTL _S obtained by different methods and materials are few and the range is large.Meanwhile,there is more than one main effect QTL,so it is necessary to continue to dig the consistent main effect QTL and determine the candidate genes.In this study,296 excellent inbred lines constitute a natural population of materials at home and abroad,combined with low coverage of heavy sequencing method for genome sequencing,indoors and field respectively for germination to seedling low temperature resistance identification,using genome-wide association analysis method to discover SNPS loci associated with resistance to low temperature,and to determine the consistency site;Then the differentially expressed genes in response to low temperature stress in the seedling stage were analyzed by transcriptome sequencing technology,and the consistent candidate genes co-located with the correlation analysis were mined.At the same time,the candidate genes were verified by q RT-PCR.The purpose of this study was to enrich the genetic theory of low temperature tolerance of maize in cold regions and provide support for the innovation of low temperature tolerance resources and the breeding of new varieties.The specific research contents and results are as follows:1.First of all,296 representative strong maize backbone inbred lines were sel ected as materials,and three interannual field natural low temperature treatment and laboratory temperature control treatment were carried out respectively.In combination with seed emergence rate in the field,relative emergence rate,relative seedling e mergence index,and low temperature stress level,10 low-temperature resistant inbred lines(B144?LX85?XZD270?LX174?LX13?CO228?LX101?LX145?DY7 and SD190)and 4 low-temperature sensitive inbred lines(LH27,Q319,LX120 and LX83)were screened.2.Genotypes of 296 associated populations were analyzed by the low coverage resequencing.The results showed that 24042 high-quality SNPS were screened,which were distributed evenly on10 chromosomes.The number of SNPS distributed on chromosome 1 was the largest(3687),while the number of SNPS distributed on chromosome 10 was the smallest(2976).The mean value of LD between SNP markers on chromosome 9 was the largest(0.122),while that between SNP markers on chromosome 7 was the smallest(0.052).When the number of subgroups was set to 5,296 inbred lines are divided into 5 subgroups.3.When combined with inbred lines 296 field resistance to low temperature properties and genotype data,and in different years of character of germination rate and emergence index as the index,the results show that at the P<1E-4 levels,a total of 7 SNP_S loci were detected significantly to be associated with low temperature resistance,which located in chromosome 5,6,7 and 10,respectively.4.Combined with 296 maize inbred lines of indoor germination and seedling low temperature resistance properties,as well as indoor germination rate and seedling stress level indicators,a total of 23 SNP sites related to indoor germination and seedling tolerance were detected by genome-wide association analysis,including 14 SNP sites related to germination and 9 SNP sites related to seedling.It was found that the SNP site consistent with low temperature tolerance in field and indoor germination to seedling emergence was marker.14070,loca ted on chromosome 5,2205723.5.The selected high-low-temperature resistant maize inbred line B144 and low-temperature highly sensitive maize inbred line Q319 were subjected to low-temperature stress treatment at seedling stage for 12h and 24h respectively,and transcriptome sequencing analysis was conducted with Illumina Hiseq TM 4000 after sampling.The results showed that there were 2,373 DEGs and1,803 DEGs in the two samples and the untreated controls respectively at 12h of low temperature treatment,among which,there were 1,601 differentially expressed genes in B144.When treated at low temperature for 24h,there were 3046 and 2749 differentially expressed genes in each of the 2samples and untreated controls,of which 1831 were differentially expres sed genes in B144.GO annotation was applied to DEGs,and 31 significantly enriched GO annotation classifications were obtained.KEGG annotation showed MAPK signal pathways in plants,plant hormone signal transduction,plant circadian rhythm,styrene acrylic base class biosynthesis,photosynthesis-antenna five metabolic pathways in the two inbred lines were present s significant enrichment,indicating that the two inbred lines in response to the stress of low temperature and signal transduction,adapt to the environment,energy metabolism and secondary metabolic pathway played an important role.6.Based on genome-wide association analysis and transcriptome sequencing,5 differentially expressed genes were detected in the linkage unbalance interval of the consistent locus(marker.14070),namely LOC103633247,LOC100273222,gst2,LOC103629437 and LOC103629384.The annotation function were ubiquitin-60 ribosomal protein,Ras protein ara-3,glutathione s-transferase,Gpi transamide and chloroplast Rubisco large subunit binding protein subunit.7.QRT-PCR was performed on 5 candidate genes related to low temperature tolerance in maize during germination were analyzed.The results showed that the expression patterns of the selected candidate genes were significantly different before and after low temperature stress in high low-temperature resistant inbred line B144 and high low-temperature sensitive maize inbred line Q319.