Identification And Characterization Of Candidate Gene TaCNGC8-A1 And TaGASR7 Associated With Seed Dormancy In Common Wheat (Triticum Aestivum L.)

Pre-harvest sprouting（PHS）in common wheat,as a worldwide problem,leads to a reduction in grain yield and flour quality,brings huge economic losses to farmers and countries,and therefore generates a serious threat to national food security.Identifying the genetic regulators of seed dormancy and accelerating genetic improvement by molecular marker-assisted breeding selection are always the important objectives in wheat breeding for PHS resistance.In our previous study,a major QTL tightly linked to BS00019095＿51 was identified on chromosome 2AL related to seed dormancy using association analysis and linkage analysis.Based on the previous result,we constructed a high-density genetic map on 2AL for fine-mapping using SLAF-Seq and 660K SNP assay,and then identified a cyclic nucleotide gated channel gene TaCNGC8-A1 using Bulked Segregant RNA-Seq.Moreover,we also identified a GA-stimulated regulator TaGASR7 on the 7th homologous chromosomes using BSR-Seq.Therefore,we performed a functional research on them,which would be helpful for deeply understanding the regulatory mechanism of seed dormancy.The main results are as follows:1.The above QTL was fine-mapped in a chromosome region（0.47 Mb）between SLAF0913 and AX3375 using the high-density genetic map,where a cyclic nucleotide gated channel gene（TaCNGC8-A1）contained 8 significantly associated SNPs was identified using BSR-Seq.The CDS of TaCNGC8-A1 was 2,151 bp in length,containing seven exons,and encoded a CNGC protein consisted of 716 amino acids.Four CNGCs were symmetrically combined into a tetramer with a central pore for ion transport.2.The full-length g DNA sequence（6,842 and 6881 bp）of TaCNGC8-A1 was respectively obtained from strongly and weakly dormant materials using CNGC2AL1～CNGC2AL4,and 13 SNPs and 1 39-bp InDel were identified through sequence alignment.Among them,the SNPs in the position of-515,-313,+855,and the InDel in the position of+3579 were developed into three functional markers（CNG2A1～CNG2A3）used for allelic variation analysis.Non-parametric test results showed that two haplotypes（Hap-R and Hap-S）had highy significant difference（p<0.01）in all PHS traits of Jing411/Hongmangchun21 RIL population,and significant difference（p<0.05）in multiple PHS traits of Chinese wheat mini-core collections and natural population.QTL analysis showed that the QTL tightly linked to TaCNGC8-A1 could explain 7.02～16.52%of phenotypic variation across different environments.3.Phylogenetic analysis showed that TaCNGC8-A1 was homologous with Os CNGC6 and At CNGC5～At CNGC9.TaCNGC8-A1 had a higher expression level in the seed growth,maturation and after-ripening processes;its expression level increased gradually in seed growth stage,decreased gradually after entering into the maturation stage,and then increased again in the after-ripening stage;water treatment induced the up-regulated expression of TaCNGC8-A1 with an increasing expression level in the after-ripening process;the expression level in strongly dormant materials was obviously lower than weakly dormant materials,indicating that TaCNGC8-A1 negatively regulated seed dormancy.Furthermore,TaCNGC8-A1 protein localized to the endoplasmic reticulum,suggested it might mediate the response to endoplasmic reticulum stress.4.After subjected to gene editing of,the seed germination index of treatment group in 50μMGA₃was significantly lower than the control group,suggesting its important role in the seed germination regulation of GA.Sequence alignment between strongly dormant materials and weakly dormant materials showed that there existed 3 InDels and9 SNPs in TaGASR7-A1,6 SNPs in TaGASR7-B1,and no variation in TaGASR7-D1.Among them,a long fragment insertion（1360 bp）in the promoter of TaGASR7-A1（-129）resulted in the insertion of 15 cis-acting elements involved in multiple processes including hormone effects and temperature response,and a G-A substitution in the promoter of TaGASR7-B1（-16）led to the deletion of boxE element,which were developed into two functional marker（GS7A1 and GS7B1）used for allelic variation analysis.The results showed that the variation of TaGASR7-B1 had a closer relationship with seed dormancy,and two genotypes had highy significant difference（p<0.01）in most of PHS traits of different population.QTL analysis also showed that TaGASR7-B1was tightly linked a seed dormancy related QTL between Marker3925344 and Marker3862461,which could explain 6.91～12.00%of phenotypic variation.5.TaGASR7-B1 protein with a signal peptide localized to the endoplasmic reticulum,indicated that TaGASR7 might function as a secretory protein.TaGASR7-B1 had a strict tissue-specific expression with a declining expression level in seed growth stage,and was hardly expressed in the maturation and after-ripening stages;water treatment induced the up-regulated expression of TaGASR7-B1 with an increasing expression level in the after-ripening process;the expression level in weakly dormant materials was obviously higher than strongly dormant materials,indicating its significant role in the demise of seed dormancy and seed germination.6.In addition,9 TaGASR family members were identified in a genome-wide scale,and divided into four subgroups with Os GASR and AtGASA protein families by phylogenetic analysis.Among them,TaGASR7 had a close genetic relationship with Os GASR7,AtGASA4,and AtGASA6.All TaGASR members had the conserved GASA domain consisted of 12 cysteines.7.Through the treatment of NO donor,NO scavenger,Ca²⁺chelator,and CNGC blocker,RT-PCR results showed that TaCNGC8-A1 medicated the signal transduction from cyclic nucleotide and Ca²⁺to NO,and NO induced the up-regulated expression of TaGASR7 andα-amylase gene by regulating the biosynthesis and metabolism of ABA and GA,which resulted in the release of seed dormancy.