Preliminary Mapping Of Cadmium Enrichment Genes In Brassica Napus

With the development of industrialization and urbanization of human society,heavy metal pollution continues to increase.The area of cadmium contaminated soil in China has reached 20 million hectares.Cadmium pollution has a significant impact on crop growth and food security.Rape,as a potential species for soil cadmium5 pollution remediation,has been reported many times,and rape has the advantages of large biomass,easy harvesting,strong climate adaptability,and the oil squeezed from rapeseed planted in the polluted area can be used in power plants or biodiesel to avoid waste.Therefore,the remediation of cadmium contaminated soil by rape is an efficient,economic and environment-friendly way of remediation of heavy metal pollution,and has broad development prospects.The study of cadmium enrichment genes in Brassica napus is of great significance to speed up the breeding of cadmium enriched varieties and explain the mechanism of cadmium enrichment in Brassica napus from the molecular level.It also provides a theoretical basis for the remediation of Cd contaminated soil by Brassica napus.Parental materials for this study were obtained from a cadmium enriched parent P₁（18M08485）and a low absorbing parent P₂（Zheyou 28-2）previously screened in142 Rapeseed cultivars or lines（conventional Rapeseed）with rich genetic backgrounds.F₁ was obtained from a two parent cross,F₂ was derived from an F₁ inbred cross,QTL mapping was performed on the F₂population using BSA-seq,while cadmium enrichment related genes were screened by transcriptome analysis of both parents using RNA-seq.The findings are as follows:（1）Through the statistics of phenotypic data at seedling stage and the determination of cadmium content in aboveground and underground at maturity,it was found that the cadmium content in aboveground part of cadmium enriched parent P₁was higher than that in underground part,and the transfer coefficient was 153.75%,while the cadmium content in aboveground part of cadmium low absorption parent P₂was less than that in underground part,and the transfer coefficient was 48.72%.The distribution of cadmium content in both aboveground and underground parts is generally normal distribution,and there is a positive correlation between aboveground cadmium content and underground cadmium content,and the correlation coefficient is0.523.（2）Nine candidate intervals were obtained on five chromosomes by BSA-seq.In this experiment,44 groups of KASP primers were developed in the candidate interval.Three QTLs were by using shoot cadmium content and underground cadmium content as phenotypic indexes:FJ-DS-C3 a QTL locus with shoot cadmium content as an index,LOD value of 4.6897 and contribution rate of 28.41%;The LOD values of QTL loci FJ-DX-C3 and FJ-DX-A9 with underground cadmium content as index are 21.4115and 3.9489 respectively,the contribution rates is 45.94%and 9.17%,respectively.（3）Through GO enrichment analysis of genes in the candidate interval obtained by BSA-seq,61 genes related to cadmium enrichment were screened,including 20genes on chromosome C3,5 genes on chromosome A9,7 genes on chromosome C2,23 genes on chromosome C5 and 11 genes on chromosome C8.These genes were enriched in GO:0046873:metal ion transmembrane transporter activity,GO:0030001:metal ion transport GO:0042546:cell wall biosynthesis and GO:0042743:hydrogen peroxide metabolism,involving the key mechanisms of heavy metal detoxification in plants,such as cell wall deposition,heavy metal transfer and antioxidant defense mechanism.（4）A total of 5427 up-regulated genes and 4908 down-regulated genes were detected by RNA-seq.Go enrichment and KEGG enrichment of differential genes were analyzed.The up-regulated genes are mainly enriched in physiological and biochemical pathways such as metal transporters,cell wall biosynthesis,immune response and thiometabolism.The down-regulated genes are mainly enriched in light response,light and function and chloroplast function.Physiological processes such as ion binding and cell redox balance are closely related to plants’resistance to external heavy metal stress.The up regulation of the expression of related genes is conducive to the normal growth of plants,which proves that the cadmium enriched parent P₁ has made a positive response to external cadmium stress,and the genes involved in these processes play an important role in the normal growth and metabolism of cells in the process of light reaction and hydrogen peroxide metabolism,The down-regulation of its expression showed that cadmium treatment had a significant inhibitory effect on the normal metabolism of cadmium low absorption parent P₂.（5）Using BSA-seq and RNA-seq data of two parents after cadmium stress treatment,19 differentially expressed genes were found in the QTL locus FJ-DS（X）-C3,including 7 up-regulated genes and 12 down-regulated genes;17 differentially expressed genes were found in the QTL locus FJ-DX-A9,including 3 up-regulated genes and 14 down-regulated genes.We further detected them by QRT PCR,and the results also confirmed the reliability of RNA SEQ analysis results.These 36 genes need more attention in the later experiments.This study further uses qRT-pcr detection,and the results also verify the reliability of RNA-seq analysis results.These 36 genes need more attention in later experiments.