- A genome-wide association study (GWAS) was conducted in a population of 269 maize accessions with 43,737 single nucleotide polymorphisms (SNPs) to identify candidate genes and favorable alleles for controlling Cd accumulation in maize.. - The significant SNP (SYN25051) at position could explained 27.1% of the phenotype variation. - Conclusions: Our findings provide new insights into the genetic control of Cd accumulation and could aid rapid development of maize genotypes with low-Cd accumulation by manipulation of the favorable alleles.. - Cadmium (Cd) is a heavy metal that is highly toxic to all organisms and is one of the major environmental pollut- ants. - Because of a serious concern in wide- spread food safety, research into a better understanding of the mechanisms driving Cd accumulation in crop plants has become increasingly important.. - In plants, many of the transporters for divalent transi- tion metals have Cd uptake activity. - Full list of author information is available at the end of the article. - OsIRT1 increases Cd accumulation [5]. - OsHMA2 is involved in the pref- erential distribution of Zn and Cd at the roots and nodes [11]. - Although the mechanisms of Cd accumulation are widespread and comprehensive in rice and Arabidopsis thaliana , none of homologous genes that have been involved in the natural genetic variation of Cd accumulation in rice and A. - As a result, only a limited amount of natural allelic diversity can be captured in the population, leading to the identified QTLs spanning relatively large genomic regions and making identifica- tion of causal genes more difficult. - In this study, we assessed Cd accumulation in 269 maize genotypes grown at dif- ferent levels of Cd contaminated soils in a greenhouse and under field conditions and conducted GWAS for leaf Cd concentration with the Illumina Infinium maize SNP50K. - Candidate genes and favorable alleles identified in the. - diversity accessions would assist in further revealing genetic control of Cd accumulation in maize.. - Two hundred and sixty nine diverse accessions were evaluated for Cd accumulation of leaves under low-Cd (LSLCd) and middle-Cd (MSLCd) conditions at the seeding stage and under high-Cd condition at the matur- ing stage (HLCd) in 2015 and 2016, respectively. - The results showed that the Cd accumula- tion of leaves in the natural population had a large vari- ation with variation coefficient (CV) over 48%. - In addition, the mean value of leaf Cd concentration in the tropical group was significantly lower than that in the temperate group ( P <. - 3a), indicating a weak relative kinship in the diverse population.. - 300 kb on chromosome 2, and 300–600 kb on the rest of the chromosomes. - The model controlling K and Q + K had similar effects on reducing the false positives, except that LSLCd16 in the Q + K model had lower power than the K model. - Across three soil conditions, 63 SNPs located on 5 of the 10 chromosomes were significantly associated with leaf Cd concentration ( P <. - The association explained approxi- mately 15.9% of the phenotypic variations. - However, no significant associations were detected between SNPs and leaf Cd concentration under the low-Cd condition in 2016 (Additional file 4).. - Forty SNPs in a single region on chromosome 2 were highly associated with leaf Cd concentration (Fig. - Meanwhile, the co-localized peak between SYN bp) and SYN bp) explained an average of 17.5% of the phenotypic variation, based on R 2 values.. - 13.7 at position on Chr2, Table 1 Phenotypic variations for leaf Cd concentration in 269 maize accessions in experiments conducted in 2015 and 2016. - explained 27.1% of the phenotype variation. - QTL mapping for Cd accumulation in IBM Syn10 DH population. - The field results showed that the leaf Cd concentration in B73 (23.3 mg·kg − 1 ) was significantly lower than that in Mo17 (64.1 mg·kg −1. - Leaf Cd concentration ranged from 9.5 to 114.2 mg·kg −1 in the mapping population, but the phenotypic distribution exhibited a transgres- sive segregation (Additional file 6). - The variation in leaf Cd concentration suggested that Cd accumulation was controlled possibly by major-effect QTLs.. - major QTL named qLCd2 (a QTL for leaf Cd concentra- tion on chromosome 2 from GWAS) was identified, spanned a 13.83 Mb region Mb) and ex- plained 41.2% and 38.4% of the phenotypic variation in 2015 and 2016, respectively. - Although LOD values of the other 4 QTLs were high, those QTLs explained only 3.4% to 4.4% of the phenotypic variation for leaf Cd accumulation (Table 2).. - No QTLs in other chromosomal regions were observed by explaining more than 5% of the variation in leaf Cd accu- mulation, suggesting that the qLCd2 is the major genetic locus controlling natural variation in leaf Cd accumulation when maize is grown in Cd contaminated soil.. - In the compara- tively narrow region, GWAS detected a highly significant cluster of 16 SNPs. - To determine the trait- associated loci, all significant SNPs located in the tar- get region were clumped at D’ >. - 2 Analysis of the population structure of 269 maize inbred lines estimated from 5200 SNPs. - b Population structure of the 269 lines from K = 3. - According to the gene functional annotations in the maize B73 genome (RefGen_v2), 8 causal candidate genes were predicted for 12 loci associated with Cd accumulation (Table 3).. - Among these genes in the first LD block,. - 4 Manhattan plots of association analysis between leaf Cd concentration and single-nucleotide polymorphism (SNP) markers in maize. - GWA mapping of leaf Cd concentration under high-Cd field condition at maturing stage of maize in 2015 (c) and 2016 (d). - 5 Logarithm of odds (LOD) score curves for the QTLs for the leaf Cd accumulation of IBMSyn10 DH population in 2015 and 2016. - In the second LD block, SYN33611 and PZE were located 85.30 kb upstream and 81.87 kb downstream of GRMZM2G085939 , respect- ively. - The leaf Cd concentration of individuals with minor frequency alleles (G/G + A/A) at these loci was 28.1 mg higher than those with major frequency alleles (A/A + C/C) (Fig. - 8b, a dramatic upregulation of the GRMZM2G175576 gene was ob- served in response to Cd stress, especially in the stems, exhibiting about a 9.5-fold increase in transcript abun- dance compared to the control. - The relative expression levels of GRMZM2G085939 also significantly increased in the stems and leaves in response to Cd stress. - Furthermore, possible sub-cellular locations of the candidate genes were predicted using ProtComp 9.0.. - The mean leaf Cd concentration in the tem- perate group was nearly double ( P that in the tropical group under high-Cd condition (Fig. - The relative values of Cd concentration in the three subpop- ulations (tropic <. - The low-Cd level in the eight lines of 11GP66-1, T32, 98WV9, Wa138, CIMMYT-1, B047, Y8G and CML282 indicated that these lines could be used for developing new maize varieties with low-Cd accumulation.. - Most previous research on the mechanisms of Cd accumulation has focused on rice and A. - successfully identified a single strong peak of SNPs asso- ciated with leaf Cd accumulation in A. - In this study, we found 63 loci associated with leaf Cd Table 2 QTLs detected for leaf Cd accumulation in maize by composite interval mapping. - c the physical distance (Mb) of the bin makers corresponding to genetic distance. - Noteworthy, we observed a single region on chromosome 2 that contained 40 SNPs highly associated with leaf Cd concentration (Fig. - This QTL could explained over 38% of the phenotypic variation (Table 2), implying that Cd accu- mulation in leaves could be controlled by a major QTL.. - In the comparatively narrow region, GWA detected a highly significant cluster of 16 SNPs on chromo- some 2, which extended to the candidate region to a 300 kb window. - According to a previous study, LD has been observed for the chromosomes that could be involved in the domestication process. - 6 Genome-wide association analysis detected a significant signal associated with leaf Cd concentration in maize. - b The QTL associated with leaf Cd accumulation in maize at chromosome 2. - D ’ values are shown in the squares for values <. - Therefore, the LD might cause overlapping effects and/or co-selection by reorganization, muta- tion, and selection in the evolution process. - Once metal ions are absorbed, trans- location of Cd from the roots to the shoots requires loading of Cd into the xylem from the symplast in the stele. - OsHMA3 has been identified as a firewall by sequestrating Cd into the vacuoles in the roots, keeping the Cd away from the above-ground tissues [8, 22]. - 7 The boxplot of phenotype analysis between the candidate genes for locus associated with leaf Cd concentration in maize and phenotypic difference between minor alleles and major alleles. - a Two combinations of the minor and major between SYN395 and SYN30994 in GRMZM2G175576and GRMZM2G455491region;. - c Two combinations of the minor and major between SYN33611 and PZE-102120786 in GRMZM2G085939region. - d Three combinations of the minor and major among SYN30994, SYN30995 and SYN30993 in GRMZM2G171370 region. - Thus a high expression of such proteins might re- duce Cd accumulation in the plant [24]. - Exposure of plants to Cd in nutrient solution for 12 h caused significant in- crease in the expression levels of GRMZM2G175576 in roots, stems and leaves of B73 (Fig. - We found that the expression of GRMZM2G085939 significantly increased in roots, stems, and leaves in response to Cd stress, suggesting that GRMZM2G085939 might be involved in protection and repair of the cellular proteins.. - Future work will include cloning the genes and illustrating the molecular mechanisms for controlling Cd accumulation in maize plants. - a A heat map illustrating levels of gene expression of the putative genes in nine different tissues from various developmental stages. - Detailed information of the accessions is listed in Additional file 9. - Plants were grown in the green- house for 12 d prior to Cd treatment. - The average air temperatures in the greenhouse were 29 °C/21 °C (day/. - A field trial for GWAS was conducted in the summers of 2015 and 2016 in Deyang city, Sichuan province of China (104°06’N, 31°11′E). - Plants were grown in the contaminated soil with 32.5 mg·kg − 1 of Cd (high-Cd level). - The maintenance of plants in the field followed routine practices of maize during the experiment. - Phenotypic traits were collected from the matured plants of IBMsyn10 DH population in the summers of 2015 and 2016 in Deyang city, Sichuan province of China (104°06’N, 31°. - Determination of Cd concentration and phenotypic data analysis. - The leaves were dried and ground until 95%of the sample could pass through a 1 mm screen. - Subse- quently, the Cd concentration in the solutions was mea- sured by using the inductively coupled plasma-atomic emission spectrometry (ICP-MS) (Nippon-Jarrell-Ash, Tokyo, Japan).. - Analysis of variance (ANOVA) and heritability ( h 2 ) of Cd concentration in leaves were performed using SPSS statistics 21.0. - QTL for leaf Cd accumulation was de- tected using the composite interval mapping method and Model 6 of the Zmapqtl module of QTL Cartog- rapher 1.17 [39]. - The support interval of a QTL was defined as the seg- ment of the chromosome in which the LOD at the peak decreased by half [40].. - Quantile-quantile (QQ) plots for leaf Cd concentration at seeding stage and maturing stage of maize. - SNPs significantly associated with leaf Cd concentration in maize. - Manhattan plots of association analysis for leaf Cd concentration at seeding stage of maize in 2016. - Haplotype analysis of polymorphic SNPs contained in the overlapped region Mb) on chromosome 2. - The frequency distribution of leaf Cd concentration in maize IBMSyn10 double haploid (DH) population. - Details of the candidate genes in the overlapped region and their putative function. - HLCd: The Cd concentration of leaves under the high-Cd condiction at the maturing stage. - LSLCd: Cd concentration of leaves under low-Cd condition at the seeding stage. - MSLCd: The Cd concentration of leaves under the middle-Cd condition at seeding stage. - Genome-wide association study of Fusarium ear rot disease in the USA maize inbred line collection. - Comparative mapping combined with homology-based cloning of the rice genome reveals candidate genes for grain zinc and iron concentration in maize. - Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. - The structure of haplotype blocks in the human genome
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