QTL Mapping And Gene Identification For Cadmium Uptake And Accumulation In Radish (Raphanus Sativus L.)

Cadmium?Cd?is one of the most toxic heavy metals,which not only inhibits the plant growth and development,but also poses a significant threat to human health via the food chain.Radish?Raphanus sativus L.?,an annual or biennial herb of the Brassicaceae family,is an important root vegetable crop with high nutritional and medicinal value in the world,especially in East Asia.Taproot is considered as the major edible organ for radish.Large phenotypic variations in Cd concentration of radish roots and shoots have been observed.Although genetic inheritance of Cd uptake and accumulation has been well studied,the genetic and molecular mechanisms of Cd uptake and accumulation as well as the Cd-responsive genes isolation in radish remain to be elucidated.This study would be conducted to construct a high density genetic linkage map and tag major QTLs for Cd accumulation in radish root and shoot,and isolate a number of Cd-related genes as well as Cd-responsive miRNAs and their targets at the genome-wide level.The main achievements obtained were as follows.?1?Based on two radish advanced inbred lines,a genetic linkage map was constructed using an F2 mapping population derived from a cross between 'Nau-Dysx'?high Cd?and'Nau-Yh'?low Cd?,which showed significant differences in Cd accumulation.The linkage map consisted of 523 marker loci,including 248 SRAP,114 RAPD,76 SSR,47 ISSR,26 RAMP and 12 RGA markers;and had a total length of 1,678.2 cM with a mean distance of 3.4 cM between two markers.Using the CIM apporach,four quantitative trait loci?QTLs?for root Cd accumulation were mapped on LGs 1,4,6,and 9,which accounted for 9.86 to 48.64%of all phenotypic variance?PV?.A major-effect QTL,qRCd9,was identified on LG 9 flanked by NAUrp0011₇54 and EM5me6₂86 markers with a high LOD value of 23.6,which accounted for 48.64%of the total PV in Cd accumulation.qRCd9 showed over-dominance,and the allele controlling root Cd accumulation was coming from'Nau-Dysx'.Two QTLs associated with shoot Cd accumulation were detected on LG1 and 3,and the two QTLs accounted for 46.61%of the total PV.A major QTL qSCd3 with an LOD value of 7.64 was identified on LG3 flanked by EM16gal8₃83 and Nal0A08₄55,which explained 29.53%of the PV.qSCd3 showed over-dominance,and the alleles controlling shoot Cd accumulation was also coming from 'Nau-Dysx'.Moreover,a total of 19 QTLs were identified to be associated with five Cd-related agronomic traits such as RL,SH,RDW,SDW and TDW.These results indicated that the genes located at qRCd9 were responsible for controlling root Cd accumulation in radish.?2?To isolate the differentially-regulated genes involved in Cd stress networks,the CK and Cd400 RNA libraries constructed from radish taproot were sequenced and analyzed using RNA-Seq approach.In all,66,552 contigs and 31,015 unigenes were obtained with an average length of 1241.75bp.Moreover,a total of 2781 significantly differentially-regulated genes consisted of 539 up-and 1565 down-regulated genes responsive to Cd stress were successfully identified,which accounted for 8.97%of all unigenes.GO analysis showed that these differentially-regulated genes could take part into a number of metabolism processes,such as energy metabolism,ion metabolism and binding,stress responses and meal transmembrane transport.Notably,a number of genes,such as PCS1,GST,MT1,MT3,ABC transport proteins and ZIP family proteins were shown to be involved in Cd stress responsive networks,which could take part into some pathways including GSH,metallothioneins and ABC transporter.The expression profiles of a set of Cd-responsive genes such as PCS1,MT1 and IRT2 were validated by qRT-PCR analysis.The full DNA sequence of RsMT1 and RsMT3 were 376bp and 609bp,and the lengths of ORF were 138bp and 201bp with encoding 45 and 66 amino acids,respectively.Both RsMT1 and RsMT3 contained high proportion of Cys.The deduced amino sequence of RsMT1 and RsMT3 shared high homology with BnMTl and BjMT3,respectively.RsMT1 and RsMT3 showed dramatically different expression in different radish organs,and had highest expressions in root.The expreesion patterns of the two genes in taproot were significantly up-regulated with Cd concentration increased and presented steady expressions at 400mg/L.Moreover,the relatively expreesion level of RsMT1 at 100mg/L and 48 h were 3.8 and 3.5 times as that in CK sample;whereas RsMT3 at 150mg/L and 96 h were 4.0 and 3.7 times than in CK sample,respectively.Furthermore,we obtained the promoter sequence of 834bp in 5' UTR region for RsMT1.It contained a few putative cis-acting elements,including CAAT-box and TATA-box,which could play important roles in plant growth and development and response to abiotic stresses in radish.?3?Using a radish advanced inbred line 'NAU-RG',the RsPCS2 gene was successfully obtained.The full cDNA sequence of RsPCS2 was 1538bp,and it contained a complete open reading frame?ORF?of 1458bp with encoding 485 amino acids.RsPCS2 possessed two significant hydrophilic loops and transmembrance domains,and its deduced amino sequence shared high homology of 92.8%with BnPCS2 gene.RT-PCR and qRT-PCR analysis revealed that sPCS2 had higher expreesion profiles in root and fibrous root than the shoot and leaf,and its expreesion patterns in taproot were significantly up-regulated with the Cd concentration and treatment time increased.The relatively expreesion level of 150mg/L and 24 h were 4.7 and 7.6 times as that in CK sample,respectively.Furthermore,through genomic DNA walking approach,we obtained full promoter sequence of 1383bp in the 5' UTR region of RsPCS2,it contained a number of putative cis-acting elements,such as CAAT-box,TATA-box and TATC-box,which could play critical roles in plant growth and development and response to abiotic stresses in radish.?4?Using a radish advanced inbred line 'NAU-RG',two genes belonging to heavy metal transport protein family were obtained.The full cDNA sequence of RsIRT2 and RsNramp3 were 1038bp and 1527bp,and the lengths of ORF were 1038bp andl527bp with encoding 345 and 508 amino acids,respectively.RsIRT2 and RsNramp3 possessed eight and 11 significant hydrophilic loops and transmembrance domains,respectively,which belonged to classic transmembrance protein.The deduced amino sequence of RsIRT2 and RsNramp3 shared high homology with AtIRT2 and AtNramp3,respectively.qRT-PCR analysis revealed that RsIRT2 was restrictedly expressed under Fe deficiency and/or supplemented with Cd stress.RsIRT2 and RsNramp3 showed dramatically different expression in different radish organs,and their expreesion patterns in taproot showed significantly up-regulated and in-coordinately expreesion profiles with Cd concentration increased and under different Cd treatment time,respectively.The relatively expreesion level of RsIRT2 at 200mg/L and 48 h were 4.6 and 3.6 times as that in CK sample;whereas RsNramp3 at 400mg/L and 24 h were 3.9 and 6.3 times than in CK sample,respectively.?5?Based on Cd-responsive digital gene expression profiling,two heavy metal-related genes belonging to Zinc and Irion protein?ZIP?family were obtained.The full cDNA sequence of RsZIPl and RsZIP4 were 1090bp and 1307bp,and the lengths of ORF were 1038bp and 1527bp with encoding 353 and 420 amino acids,respectively.Both RsZIPl and RsZIP4 possessed eight significant hydrophilic loops and transmembrance domains,and belonged to classic transmembrance protein.The deduced amino sequence of RsZIPl and RsZIP4 shared high homology with ThZIPl and TcZIP4,respectively.RsZIPl and RsZIP4 showed dramatically different expression in different radish organs.The expreesion patterns of the two genes in taproot were significantly up-regulated with the Cd concentration increased and reached the highest expressions at 400mg/L and 150mg/L with 3.9 and 4.2 times than that in CK sample,respectively.Moreover,RsZIPl and RsZIP4 showed relatively highest expressions at 12h and 96h wiith 3.1 and 4.0 times than that in CK sample;and they presented steady expressions after 24 h and 12 h,respectively.?6?To systematically isolate and dissect Cd-responsive miRNAs and their targets at the global level in radish roots,we constructed two small RNA libraries from Cd-free?CK?and Cd-treated?Cd200?roots of radish seedlings.Using Solexa sequencing technology,93 conserved and 16 non-conserved miRNAs representing 26 miRNA families and 28 novel miRNAs representing 22 miRNA families were identified,which showed different expression patterns in six different tissues including root,leave,stem,shoot,flower and bud.A total of 15 known and 8 novel miRNA families were found to be differently regulated under Cd stress.qRT-PCR analysis showed that a set of Cd-responsive miRNAs were differentially-regulated in different Cd treatment time?0,1,6,12,24 and 48 h?.Based on the radish mRNA transcriptome,18 and 71 targets for novel and known miRNA families were identified by degradome sequencing approach,which could take part into in a variety of biological processes,such as energy metabolism,signal transduction,organism development,cell differentiation and biotic and abiotic stresses responses.Notably,a few new target transcripts such as PCS1,ABC transporter protein and Iron transporter-like protein were shown to be involved in plant response to Cd stresses.