Cloning,Development Of Fuctional Marker And Associaton Mapping Of Key Gene TaSPP2 Related To Sucrose Metabolism In Wheat

Drought is an important abiotic stress factor affecting wheat production in arid and semi-arid regions of C hina.Water soluble carbohydrates(WSC)as osmotic regulation substances not only alleviate physiological damage of plant caused by drought stress,but also deliver the important carbon source to compensate grain-filling.Sucrose is the main component of WSC remobilization and storage,which plays an critical role in carbohydrate metabolism.Sucrose-6-phosphoric acid hydrolase catalyzes the last step of the synthesis of sucrose.Therefore,it is important to explore the different alleles of the gene and develop specific molecular markers,which are important to cultivate high-yield wheat varieties.In order to well-understand the spatiotemporal physiological characteristics and genetic variations on sucrose accumulation and remobilization of the post-anthesis regulated by drought,a natural population composed of 196 winter wheat varieties(lines)from different geographic origins was used to determine the phe notypes of different organs of the sucrose content and its accumulation,remobilization and contribution rate to grain filling at the different growth period,of which the phenotypic variation,correlation,and cluster analysis were performed under the drought stress(DS)and well-watered(WW)conditions.The genome specific primers were designed to clone TaSPP2 gene and to develope molecular markers.Combining the gene TaSPP2 marker with 2982 SNP marker on 3A,3B,5D and 6A chromosomes,four SSR markers o f TaSPS?gene,and one SSR marker of Ta1-FEH genes were utilized to analyze the genetic diversity and population structure of the natural populations.It was further to explore the candidate markers tightly associated with the phenotypes of sucrose accumula tion and remobilization by the strategy of association mapping.The findings would provide important theoretical basis for drought-resistant molecular markers assisted selective breeding.The results are as follows:1.All traits had higher variation coefficients(13.57~80.60%/DS and 20.90~72.55%/WW)and phenotype diversity indices(0.42~0.89/DS and 0.45~0.87/WW),but lower heritabilities(0.31~0.64/DS and 0.29~0.65/WW).The factors,such as water,growth stage and organ,played predominant roles in a ffecting the phenotypic variations.Drought stress significantly promoted the sucrose accumulation and remobilization to grain of different organs.Sucrose content at mid-grain filling stage was significantly higher than those at flowering and mature stage.All of phenotypic values of peduncle and the penultimate internodes seemed remarkably higher than those of other organs.Sucrose remobilization rates and contribution rates at the pre-anthesis were significantly higher than sucrose remobilization rates and contribution rates at the post-anthesis.There were significantly positive correlations between sucrose contents at flowering and mid-grain filling stage and its remobilization rate and contribution rate to grain filling.Based on the characters related to sucrose accumulation and remobilization of peduncle and penultimate internodes,the population was clustered into four categories,and eleven varieties(lines)with higher phenotypic values were selected under two water conditions.2.The genome specific primers were designed to clone the TaSPP2 gene from hexaploid wheat.Based on different hexaploid wheat varieties TaSPP2 gene sequence alignment analysis,it was found that the gene sequences contained eight exons regions and seven introns regions.3.The allele-specific PCR primers F2/R2 were developed based on the SNP at the position 1,460 bp in TaSPP2 gene.The marker was used to genotype polymorphisms in the intergression lines(ILs)backcrossed by Jinmai47 and Xifeng20,and was located on chromosome 5D,which genetic distance was 22.22 cM from the right marker Xwmc357.4.The genetic diversity of the natural population was analyzed by 2988 polymorphic markers.A total of 5976 allelic variants were detected with average two alleles for each site.The frequency of alleles of polymorphic markers differed from 0.487 to 0.978.The diversity index of each marker site ranged from 0.031 to 0.489.The polymorphic information content(PIC)of each marker site varied from 0.040 to 0.460.The heterozygosity of nucleic acid at each marker site altered from 0.041 to 0.497.By UPGMA clustering and population structure analysis,the natural population was divided into four groups,in which the same geographical origin of wheat germplasms were classified to same the group.5.By the association mapping,145 polymorphic markers were significantly associated with related traits to sucrose accumulation and remobilization in wheat(P <0.0001).Among them,79 markers were significantly correlated with sucrose contents,and 76 markers were greatly associated with sucrose remobilization and contribution rates.In addition,10 markers were detected with pleiotropic effect.O f these,the SNP marker AX-9470607 was greatly associated with sucrose contents at mature stage and sucrose remobilization rates at the pre-anthesis in flag leaves.Both AX-95194715 and AX-94494956 were significantly associated with sucrose contents at flowering and contribution rate to grain filling at the pre-anthesis in the third leaf from top and the peduncle internode.The marker AX-95037003 was remarkably associated with sucrose contents at flowering and contribution rate to grain filling at the post-anthesis in the peduncle internode.The marker AX-94438075 was significantly associated with sucrose conte nts at flowering and contribution rate to grain filling at the post-anthesis in the third internode from top.The marker AX-94974281 was greatly associated contribution rate to grain filling at the pre-anthesis and post-anthesis in the peduncle internode.The marker AX-94719090 was significantly associated remobilization rates at the pre-anthesis in flag leaves and sucrose contents at flowering in the penultimate internode.The marker AX-94637777 was significantly associated sucrose contents at mature stage in the peduncle internode and third internode from top.The marker AX-94539031 was greatly associated remobilization rates and contribution rate to grain filling at the post-anthesis in the third leaf from top and remobilization rates at the pre-anthesis in the third internode from top.The marker AX-95000434 was significantly associated remobilization rates at the pre-anthesis in the third internode from top under drought stress condition and remobilization rates at the pre-anthesis in the penultimate internode under well-watered condition.