Single Nucleotide Polymorphisms Of Protein Kinase Genes TaSnRK2.3/4 And Their Association With Agronomic Traits In Wheat

Wheat(Triticum aestivum L.)is the most widely planted crop in the world,and its production is crucial for food security.Drought and extreme temperatures are major stressses limiting the production of wheat.Protein phosphorylation and dephosphorylation regulated by protein kinases and phosphatases,are pivotal biochemical processes in energy metabolism and signal transduction in plants.The sucrose non-fermenting 1-related protein kinase is a kind of Ser/Thr protein kinase.Sn RK2 participates in a variety of signaling pathways,and plays vital roles in abiotic stress tolerance and root development.Mining favorable alles in germplasm resources and developing functional markers are cornerstones for germplasm innovation and molecular breeding in wheat.To facilitate the untilization of TaSn RK2.3 and TaSnRK2.4 in breeding,various experiments including single nucleotide polymorphism analysis,functional marker development,association analysis,and elite haplotype identification were performed.The main results are as follows:(1)The coding regions of TaSnRK2.3s were cloned by Hanxuan 10,and functional markers were developed.The three copies of TaSn RK2.3 were named as TaSnRK2.3-1A,TaSnRK2.3-1B,and TaSnRK2.3-1D based on genomic origins,and their fragment sizes were 2999,2862,and 2817 bp.For TaSnRK2.3-1A,four SNPs were identified in non-exon regions in 32 accessions.Ten nucleotide variations(two 1-bp In Dels and eight SNPs)were detected in exons and introns at TaSnRK2.3-1B,only SNP at 928 bp(C/T)led to an amino acid change(Pro?Ser).There was no polymorphism identified in TaSnRK2.3-1D.Two functional markers(AM1 and AM2)were developed at 1898 and 2905 bp for TaSnRK2.3-1A;two functional markers(BM1 and BM2)were developed at 2153 and 2638 bp for TaSnRK2.3-1B.The four SNPs were recorded as 2.3A-SNP1,2.3A-SNP2;2.3B-SNP1,and 2.3B-SNP2.(2)A natural population with 262 accessions was deployed for association analysis.The genotypes were scaned with the four functional markers,the phenotypic traits were measured under drought-stressed(rain-fed)and well-watered conditions,and the relationships between genotypes and phenotypes were calculated by association analysis.For TaSnRK2.3-1A,three haplotypes(Hap-1A-1,Hap-1A-2,and Hap-1A-3)were identified and all the haplotypes were significantly associated with plant height(PH)and 1000-grain weight(TGW).The two polymorphic sites(2.3A-SNP1 and 2.3A-SNP2)were significantly associated with PH,and both 2.3A-SNP1-C and 2.3A-SNP2-A were regarded as superior sites for reduing PH,and thus their combination Hap-1A-1 might be an elite haplotype in reducing PH and increasing TGW.For TaSnRK2.3-1B,two main haplotypes(Hap-1B-1 and Hap-1B-2)were significantly associated with PH,TGW and stem water-soluble carbohydrates(SWSC).The two polymorphic sites(2.3B-SNP1 and 2.3B-SNP2)were significantly associated with PH,TGW,and SWSC.Both 2.3B-SNP1-C and 2.3B-SNP2-G were regared as superior alleles for increasing TGW and SWSC,and reduing PH,and their combination was Hap-1B-1.(3)Two natural populations,157-landrace and 348-modern cultivar populations were used to reveal the distribution of favored haplotypes in varieties released in different periods and geographical environments in China.From landraces to modern cultivars,the frequencies of the two elite haplotypes Hap-1A-1 and Hap-1B-1 increased across almost all the ten Chinese wheat production zones.In general,the frequencies of Hap-1A-1 and Hap-1B-1 increased over decades.(4)The expression patterns of TaSn RK2.3-1B were further identified in accessions with extreme PH in the natural population.And the higher expression of Hap-1B-1 in peduncle was observed when the fourth node were visible in wheat.(5)Cis-acting elements of TaSnRK2.3-1B promoters were analysed.In the accessions with extreme PH,promoters of TaSnRK2.3-1B haplotypes were cloned,the fragment sizes were 2031 and 2024 bp.There were 14 different sites between Hap-1B-1 and Hap-1B-2.For the promoters of Hap-1B-1 and Hap-1B-2,there were a series of cis-acting elements response to abiotic stresses identified online by Plant CARE.For TaSnRK2.3-1B haplotypes,a cis-acting element as-2-box involved in shoot-specific expression was only detected at Hap-1B-2.(6)The coding regions of TaSnRK2.4s were cloned by Hanxuan 10,functional markers were developed,and association analysis was performed.Fragment sizes of TaSnRK2.4s from A,B,and D genome,were 4257,4457,and 4200 bp.Since TaSnRK2.4s were located on the homologous group of chromosome 3 by a set of Chinese Spring nulli-tetrasomic lines,we named the three genes TaSn RK2.4-3A,TaSnRK2.4-3B,and TaSnRK2.4-3D,respectively.For TaSnRK2.4-3A,only one SNP(A/G)was detected at 2478 bp in 32 accessions,leading to Arg?Cys at the 173 th amino acid and a functional marker 4AM1 was develop accordingly.For TaSnRK2.4-3A,two haplotypes(Hap-3A-1 and Hap-3A-2)were significantly associated with SWSC and TGW.Hap-3A-2 might be a superior allele for increasing TGW and SWSC.From landraces to modern cultivars,the frequencies of Hap-3A-2 increased visibly in most ten Chinese wheat production zones.By association analysis and other methods,elite haplotypes and allelic variations of TaSnRK2.3 and TaSnRK2.4 were identified,which can be used in cross breeding and molecular marker assisted selection,and will benefit the improvement of high yield and abiotic stress tolerance in wheat breeding.