Preliminary Analysis Of Molecular Response Mechanism Related To Aging In Early Germination Of Brassica Napus Seeds

As one of the main sexual reproduction materials of plants,the aging of seeds will directly reduce the vitality and germination rate.In order to study the molecular response mechanism of aging rape seeds during germination and analyze the regulation mechanism of aging seed components in response to aging during germination,GWAS(genome wide association study,GWAS)was conducted on the five traits(the 12 h relative conductivity(12HRC),the 24 h relative conductivity(24HRC),the relative germination potential(RGP),the relative germination rate(RGR)and the relative germination index(RGI))of the natural population composed of 125 aging(45℃/100%RH/12h)strains in 2019-2020.Quantitative trait locus(QTL)mapping was performed on five traits of the recombinant inbred line population(RILs)composed of 133 aging(45℃/100%RH/12h;45℃/100%RH/24h)lines in 2019-2020 and 2020-2021.Aged Zhongshuang 11 was underwent transcriptome sequencing(RNAsequencing,RNA-seq)at the 12 th,24th and 48 th hours of germination.Combined with GWAS,QTL and RNA seq,candidate genes related to seed aging were screened.The main results are as follows:1.GWAS analysis of correlation between Seed Germination and aging in Brassica napusGWAS results showed that there were 10 SNP significant loci were detected.Among them,the locus associated with 12 HRC was not detected.1 locus associated with 24 HRC was distributed on chromosome A07.T loci associated with RGR were distributed on chromosomes A07 and A08.Two loci associated with RGP were distributed on chromosomes C01 and C07,and Two loci associated with RGI were distributed on chromosomes A01 and C01.The SNP locus with the maximum explanatory phenotypic variation was detected in RGR,with a value of 12.557%.The SNP locus with the minimum explanatory phenotypic variation was detected in RGI,with a value of 8.626%.2.QTL Mapping associated with germination and aging in Brassica napus seedsQTL results showed that there were 94 QTL loci were detected.These QTLs were distributed on18 linkage groups.30 QTLs related to 24 HRC.11 QTLs related to RGR.12 QTLs related to RGP.18 QTLs related to RGI.The LOD values of these QTLs ranged from 2.010 to 7.451,and the phenotypic variation that could be explained was 1.146% to 10.878%.3.RNA-seq analysis related to aging of Brassica napus seeds during germinationA total of 41,788 DEGs(19,584 up-regulated and 22,204 down-regulated)were detected in the three periods,of which 15,317 were detected at 12 h of germination,15,635 were detected at 24 h of germination,and 10,836 were detected at 48 h of germination.GO function showed that these genes were mainly enriched in reactive oxygen species and photosystem,Kyoto Encyclopedia of Genes and Genomes(KEGG)showed that these genes were mainly enriched in carbon metabolism,protein processing in endoplasmic reticulum,amino acid and fatty acid biosynthesis and other metabolisms in the way.4.Combining GWAS,QTL and RNA-seq to screen candidate genes involved in the regulation of seed agingCombined with GWAS,QTL and RNA-seq,a total of 203 candidate genes involved in the regulation of seed aging were screened,involving protein metabolism,sugar metabolism,redox,nucleotide metabolism,fat metabolism and photosynthesis,among them,the genes involved in protein metabolism and glucose metabolism mainly synthesize some osmoregulatory substances such as proline and sugars.The genes involved in redox were mainly antioxidant enzymes(SOD,cat,POD)involved in scavenging oxygen free radicals(ROS);Genes involved in lipid metabolism were mainly involved in the synthesis and decomposition of fatty acids.Different types and different periods of genes work together in response to seed aging.5.Determination of Physiological Indexes in Germination Stage of Aged SeedsExcept for malondialdehyde,the contents of soluble sugar,soluble protein and antioxidant enzymes(SOD,POD,CAT)decreased significantly in aged seeds from 12 h to 24 h.At 12 h of germination,the antioxidant enzyme activities of the aged seeds were significantly increased compared with the control seeds.The analysis showed that the aging caused excessive ROS.In the early stage of germination,the seeds mainly rely on the antioxidant enzymes inside the seeds to scavenge.ROS,maintaining intracellular redox homeostasis.