Genotypic Difference And Genome-Wide Association Study Of Cadmium Accumulation In Barley

Soil cadmium(Cd)pollution is a major problem for agricultural production globally,which causes harmful effect for crop growth,food quality and human health.Therefore,it is important to understand mechanisms of Cd accumulation in crops and breed new varieties with low Cd accumulation.Barley(Hordeum vulgare)is the fourth important cereal crop in the world,which is widely used for foods,animal feeds and beer material and well adaptable to various environmental conditions.Moreover,barley is well-known for its abundant genetic diversity and elite stress-tolerant gene resources.However,molecular mechanisms of Cd transport and accumulation in barley have not been fully elucidated.In the present study,genome-wide association analysis was used to identify Cd-tolerant gene loci on the basis of assessing Cd accumulation in straws and grains of 100 barley core collection accessions.Meanwhile,we first investigate biological function of HvLCT1 gene.The main results are as follows:1.There were significant genotypic differences of Cd accumulation in 100 barley accessions.With soil culture pot experiment,100 barley accessions were planted in Cd(1.5 mg·kg-1)soils.We determined concentration of Cd and other microelements in grains and straws.The results showed that grain Cd concentration was ranged from 0.03 to 0.41 μg·g-1 DW and straw Cd concentration was ranged from 0.12 to 1.58μg·g-1 DW.It indicated that there was a significant difference of Cd accumulation in orgains among these barley accessions.Futhermore,a significantly positive correlation was found between Cd concentration in the grains and in the straws.Moreover,a significantly positive correlation was also found between Cd concentration and Zn concentration in the grain,between Cd concentration and Zn or Mn concentration in the straw.2.Genome-wide association analysis was performed for Cd concentration in the straws and grains of 100 barley accessions.Barley core collection accessions were genotyped by the RNA-seq technology.We obtained 2.8 Tb sequence data and detected 1,054,371 SNPs and 104,594 InDels.After filtering,279,515 SNPs with high quality were selected for population structure analsyis and genome-wide association mapping.The result showed that 100 barley accessions could be divided into 5 sub-groups.There were 9(P<1×10-4)and 32(P<1×-5)SNPs detected for Cd concentration in the grains and straws.According to rice homologues of Cd transporter genes,we detected HvCAL1,HvLCT1,HvHMA3,HvHMA2 and HvHMAl assoiciated with these SNPs.3.Candidate gene HvLCTl was involved in Cd accumulation in barley grains.Sequence analysis showed that the full-length cDNA of HvLCT1 was 1,942 bp,encoding a peptide with 460 amino acids.The expression of HvLCTl was markedly higher at heading stage than at seedling stage.At heading stage,HvLCT1 was mainly expressed in flag leaf blade and lower leaf blade.HvLCT1-RNAi mutants were generated using Gateway techniques and Agrobacterium-mediated barley immature transgenic systems.We compared Cd accumulation in different tissues between two RNAi lines and the wild-type(WT)plants.There was no significant difference in plant growth and Cd concentration between the RNAi lines and the wild-type at seeding stage.However,the Cd(3 mg·kg-1)soil-culture showed that RNAi plants had higher Cd concentration in the grains,flag leaves and lower leaves than the WT,and also higher Zn concentration in the grains.The result indicated that HvLCTl was involved in Cd and Zn distrubution in the shoot tissues.This study enriches the knowledge of the molecular mechanism of crop Cd accumulation,and provides a theoretical reference for breeding crop cultivars with low Cd accumulation.