Mining Of Genes In Pre-Harvest Sprouting (PHS) Resistance Control And Their Molecular Genetic Mechanism In Wheat

Pre-harvest sprouting（PHS）is a complicated agronomic trait that is controlled by a series of genes with a profound genetic and molecular biological basis,has a negative impact on grain yield and quality,and is closely related to seed dormancy and germination.Wheats with higher dormancy level ensure their survival and reproduction in the severe environment.However,long dormancy is insufficient for efficient modern industrial production.As a result,domestication has shortened the dormancy period of wild species via artificial intervention.Although a high germination rate speeds up the process of brewing,bread manufacturing,and feed production,it also increases the risk of PHS.In recent years,wheat PHS has become increasingly of concern,and numerous quantitative trait loci（QTL）controlling wheat PHS have been identified.However,these QTL are still distributed among the wheat genomes confused and widely.In addition,due to the complexity of the wheat genome（16 Gbps）,the basic research on wheat PHS still lags,only a few genes in the PHS regulation have been identified at the whole genome level,seriously restricting the mining and cultivation of related excellent germplasm resources and impacting the national seed safety.Therefore,it is important for the depth resolution of the wheat PHS molecular genetic mechanism and the mining of excellent variation loci and alleles by using the large-scale wheat accessions with substantial genetic diversity by genome wide association studies（GWAS）.This study also provides a reference value for the custom cultivation of wheat varieties with different PHS resistance in various regions and fields by molecularly assisted breeding,as well as theoretical supports for PHS problem improvement and agricultural economic production and efficiency enhancement.In view of this,our study first systematically summarized and analyzed the relevant studies on wheat PHS.In addition,the association between wheat PHS-related traits and the wheat SNP660K chip at the genome-wide level was established and three important loci selected to mine the causal genes for further mechanistic analysis.The following are the main research findings obtained in this study:1.Meta-analysis of PHS related-QTL in wheat.A search for‘PHS-controlled-QTL mapping’articles published in the last two decades（2001–2020）was conducted and 188valid QTL from 40 articles were extracted.Then,meta-analysis using information from these QTL was performed,and 66 meta-QTL（MQTL）were obtained.Moreover,28 wheat PHS regulatory genes and their corresponding alleles were also marked on the map.Through comparison,21 known genes were found to co-locate with 11 MQTL,and 15 known genes were distributed around the 10 MQTL intervals.2.Identification of wheat PHS-controlling QTL at the genome-wide level.The genetic architecture of germination rate,sheath length,root length,percentage of visible sprouted kernels（PVSK）in Yangling,Shaanxi province and Qingdao,Shandong province were explored using 760 worldwide wheat accessions with rich natural variations.The association between these traits and wheat SNP660K chips was established using the mixed linear model（MLM）approach.A total of 4634 single nucleotide polymorphisms（SNP）sites were mapped at the genome-wide level,spanning 131 genomic regions.In addition,the genes controlling seed dormancy,germination,and PHS in Arabidopsis,rice,maize,and barley were systematically summarized,and 330 homologous wheat genes by sequence alignment were obtained.Subsequently,through comparison,28 known wheat PHS-controlling genes were found to co-locate with 23 loci and 61 wheat homologs involved in dormancy and germination in other plant species co-located with 51 loci.The remaining 61 loci are novelly harboring 2,380 potential candidate genes.3.The"TaSn RK2.1/TaABI3-TaPI4K"module regulates wheat PHS.The causal gene corresponding to Qphs2A.8＿nwafu was mined and named TaPI4K-2A.The expression of TaPI4K-2A decreased during the germination stage and increased during the maturation stage,and have a strong response to ABA,co-expressed with two key regulators in ABA signaling,TaABI3,and TaSn RK2.1.A series of molecular biology experiments proved that,the expression of TaPI4K-2A was specifically induced by TaABI3 at the transcriptional level;TaPI4K-2A specifically interacts with TaSn RK2.1 and phosphorylated by TaSn RK2.1 at the post-transcriptional level.TaPI4K-OE lines showed strong PHS-resistance,while TaPI4K-RNAi lines were PHS-susceptible,demonstrating that TaPI4K-2A is a powerful downstream performer of ABA signaling for PHS regulation.Natural variations in TaPI4K-2A confer wheat different PHS resistance via the"TaSn RK2.1/TaABI3-TaPI4K"molecular module.The gene-based association（GBA）analysis and resequencing data showed that,one deletion mutation（In Del-1359）and two SNP sites（SNP-1216,SNP-1217）in the promoter of TaPI4K-2A resulted in one ABRE element deletion and core sequence of the G-box element alteration;two SNP sites（SNP512,SNP718）in the coding region resulted in two missense mutation.The five sites are closely linked,and classified this gene into two major haplotypes,TaPI4K-2A^Hap1 and TaPI4K-2A^Hap2.The evolutionary analysis showed that TaPI4K-2A^Hap1 is favorable allele to resistant PHS.Subsequent molecular experiments revealed that variations in TaPI4K-2A^Hap2 promoter reduced its up-regulation by TaABI3.SNP718 in TaPI4K^Hap2resulted in the loss of its autophosphorylation activity and poor protein stability,which weakened the binding affinity of TaPI4K^Hap2 to TaSn RK2.1 and attenuated its ability in PHS resistance.4.The"TaABI3-TaATG8"molecular module regulates wheat PHS.Combined RNA＿Seq data with importance of variation site analysis,the causal gene corresponding to Qphs2A.7＿nwafu was mined and named as TaATG8-2A.The GBA analysis indicates that the ABRE element insertion in the promoter of TaATG8-2A is strongly associated with PHS.A series molecular assays showed that TaATG8-2A was upregulated by TaABI3 TO response to ABA.In transgenic lines,TaATG8-2A expression was proportional to PHS resistance.The above results indicate that TaATG8-2 conferred wheat PHS resistance via the"TaABI3-TaATG8"module.5.The"TaMKK6-TaMAPK6-TaABI4"molecular module regulates wheat PHS.The TaMAPK6-7D was identified as the causal gene for Qphs7D.1＿nwafu.The expression analysis revealed a co-expression relationship between TaMKK6,TaABI4 and TaMAPK6-7D.The interaction between TaMKK6,TaABI4 and TaMAPK6-7D was further determined by molecular assays.The TaMKK6-TaMAPK6-TaABI4 molecular module was initially proposed for wheat PHS regulation.Taken together,this study comprehensively and systematically analyzed the molecular genetic mechanism of wheat PHS regulation.First,abtained 66 MQTL through Meta analysis of the identified PHS related QTL.Then 760 representative wheat accessions with rich genetic diversity and characteristics of different ages were selected for large-scale analysis of PHS and seed germination phenotype,with the help of GWAS tools,multiple PHS-controlling QTL were identified on the genome level.Three key wheat PHS regulatory genes（TaPI4K-2A,TaATG8-2A,and TaMAPK6-7D）have been identified,and their functional mechanisms have been extensively elucidated.The remarkable findings fully exploit the excellent existing wheat germplasms and genetic resources and provide theoretical support for the improvement of wheat and also other gramineous crops like rice and barley,etc.