Cloning Of The Major QTL Candidate Gene TpCCX2-4B Underlying Grain Cd Accumulation In Polish Wheat

Wheat is one of the most important crops in the world and the main source of cadmium（Cd）intake of human.Cd pollution in the arable soil of the main wheat-producing areas of China leads to the grain Cd concentration exceeding the standard,which seriously affects the wheat yield and quality.Illustration of Cd accumulation mechanism and breeding of low-Cd cultivars are important for ensuring food security and human health.Polish wheat（Triticum polonicum L.,2n=4x=28,AABB）carries several excellent traits such as long grain,high 1000-kernel weight,high iron and zinc accumulation in grain.It is an important germplasm resource for genetic improvement of common wheat（Triticum aestivum L.,2n=6x=42,AABBDD）.Our previously analysis identified a new major quantitative trait locus（QTL）q Tp Cd-4B underlying the grain Cd accumulation using the recombinant inbred line（RIL）derived from a cross between dwarf Polish wheat（DPW）and tall Polish wheat（TPW）.q Tp Cd-4B is located between 11.43 and 11.91 Mb on chromosome 4B.This region contains13 high-confidence and 7 low-confidence genes.Out of them,only Traes CS4B02G015200（TpCCX2-4B）encodes a metal transproter cation/Ca²⁺exchanger（CCX）.In this study,TpCCX2-4B was further recommended as the candidate gene of q Tp Cd-4B through gene clone,expression pattern analysis,kompetitive allele specific PCR（KASP）marker development and genotyping,subcellular localization,yeast functional complement and overexpression in rice.The main results are as follows:（1）The coding sequence and promoter sequence of TpCCX2-4B are different between DPW and TPW.A total of 14 acting elements are different in the promoter between DPW and TPW,such as MYB transcription factor binding sites and meristem expression cis-acting elements CAT-box.A KASP marker KASP-11433414 was developed.The candidate region of q Tp Cd-4B was further narrowed between the markers AX-110076607 and KASP-11433414,which only includes TpCCX2-4B.According to the genotype of RIL,the grain Cd concentration of TPW-type was significantly higher than that of DPW-type.（2）The genome of common wheat contains 12 CCX genes.Cluster analysis with 48CCX members of Arabidopsis thaliana,rice,wheat,maize,barley,durum wheat and wild emmer wheat indicated that all CCX were divided into three subgroups.CCX of monocotyledon plants including rice,wheat,maize,barley,durum wheat,and wild emmer wheat are close,but distantly related to dicotyledon plant Arabidopsis thaliana.（3）The expression level of TpCCX2-4B in DPW and TPW are different.In root,nodeⅠand flag leaf,the expression of TpCCX2-4B in TPW was significantly higher than DPW;opposite result was found in grains.（4）The subcellular localization results indicated that TpCCX2-4B may be located in the endoplasmic membrane.Expression of TpCCX2-4B significantly decreased the concentrations of Cd and Cu in yeast.Overexpression of TpCCX2-4B^DPW in rice significantly decreased the Cd concentration in the lower nodes,lower stems,lower leaves,nodeⅠand grains;but did not affect the Cd concentration in roots,internodeⅠ,flag leaves,rachises and glumes.Meanwhile,Overexpression of TpCCX2-4B^DPW also significantly reduced the Cd concentration of whole plant,but did not affect root-to-shoot Cd translocation and shoot-to-grain Cd distribution.Taken together,TpCCX2-4B was confirmed as a candidate gene of q Tp Cd-4B.The results provided an important help for revealing the genetic mechanism of wheat grain Cd accumulation and gene functional analysis,also enriched the understanding of wheat metal transporter CCX.