Comprehensive Evaluation Of Low-Cd Rice And Molecular Marker-assisted Development Of A Doubled Haploid Population By Anther Culture

In the past 20 years,a lot of research has been carried out to uncover the mechanism of cadmium?Cd?absorption and accumulation in rice,and low-Cd accumulating rice materials have been developed by physical/chemical mutation induction and CRISPR/Cas9-based genome editing.The present research is a follow-up study on mutant lines of OsLCT1 and OsNRAMP5 generated by the CRISPR/Cas9 system in our laboratory.Two mutants,lct1x1+1?a frameshift mutation caused by 1bp insertion of the 1st exon of OsLCT1?and nramp5x7+1?a frameshift mutation caused by 1bp insertion of the 7th exon of OsNRAMP5?were tested in paddy fields and pot experiments,together with a wild-type control variety XiuShui134 to evaluate the accumulation of Cd in brown rice and yield performance.Meanwhile,a segregating population was developed by crossing of the nramp5x7+1 mutant line and a conventional japonica rice line A703 for developing and testing of a functional ACT-PCR marker for the nramp5x7+1 mutation.Genotyped F₂ plants were subjected to anther culture using both heterozygous and homozygous F₂ plants.A doubled haploid?DH?population was developed and further genotyped and subjected to hydroponic and field experiment.Through the analysis of physiological and agronomic traits,the effects of low-Cd accumulation mutant gene?nramp5x7+1?on Cd and manganese?Mn?accumulation and plant growth under different genetic backgrounds were studied.The main findings are as follows:1.Multi-location tests of low-Cd mutant lines.The Cd content of mutants brown rice decreased by different degrees in pot and field experiments,and there was still a certain gap between the yield with the control.In the pot experiment,when the external Cd was added to 1 mg/kg,the Cd content in the brown rice of Xiushui134 was similar to that in the lct1x1+1 mutant,and both exceeded the national safety standard?0.20mg/kg?;while the Cd content in the mutant nramp5x7+1 was only 0.06 mg/kg.When exogenous Cd was added to 3 mg/kg,the Cd content of the control XiuShui134 and lct1x1+1 mutant brown rice increased to about 1.0 mg/kg.While,the nramp5x7+1mutant was 0.16 mg/kg within the standard.In the field experiment with Cd pollution of about 0.8 mg/kg,the Cd content in the lct1x1+1 and nramp5x7+1 mutants was increased to 0.15 and 0.06 mg/kg,respectively,which met the standards.Except for the Mn,the content of other metal elements in the nramp5x7+1 mutant was not was significantly lower than those of the control varieties.Based on these results,the lct1x1+1 mutant line is suitable for planting in fields with low Cd pollution?⁰.8 mg/kg?,while the nramp5x7+1 mutant can be grown in higher Cd-contaminated soil?<3mg/kg?for low Cd rice.Yield evaluation showed that the yields of lct1x1+1 and nramp5x7+1 mutants in Jiaxing and Quzhou were lower than that of XiuShui134 by15.8%²9.7%,and there was a large gap compared with the control variety.2.Development of functional molecular markers for the nramp5x7+1 mutation.After the cross between the nramp5x7+1 mutant and the elite japonica rice line A703,F₂ population contained genetically separating plants,namely wild type,heterozygous,and homozygous mutant plants.A functional molecular marker was developed using ACT-PCR?the annealing at critical temperature-PCR?methods.In practice,two pairs of primer were designed to match wild-type or homozygous mutants,respectively.Wild-type or mutant genotype plants can be successfully amplified by wild-type?mutant?specific primers at critical annealing temperatures,as well as the heterozygous mutation plants.This method uses two pairs of primers to amplify a single plant genomic DNA separately and mixes the PCR products for electrophoresis to genotype according the band size and presence or absence.A total of 396 F₂ plants were divided into 206 heterozygous mutants,87 homozygous mutants,and 103 wild-type plants by this method.3.Development of a DH population from a cross of the nramp5x7+1 with a wild-type line.Heterozygous and homozygous F₂ plants with the nramp5x7+1 mutation were selected for anther culture via anther inoculation,callus induction,differentiation,and rooting.A total of 404 and 393 regenerated plantlets were produced from heterozygous and homozygous F₂ plants,respectively.After spontaneous chromosome doubling,a total of 99 heterozygous and 98 homozygous DH plants were obtained,respectively.The DH plants from heterozygous F₂ plants consisted of 48 wild-type and51 nramp5x7+1 plants.These DH lines laid the material foundation for further research on the mutation effect of nramp5x7+1 and breed selection.4.Cd tolerance test of wild-type and nramp5x7+1 DH lines.In hydroponic experiments with different Cd/Mn concentrations,the nramp5x7+1 DH lines grew weaker than the wild-type under low Mn supply,and its growth was also affected to some extent with the treatment of 5?M Cd;When the Mn supply was restored to normal levels?7?M?with or without the addition of Cd,the content of Mn in shoots was still very low,only 13.1 mg/kg to 31.7 mg/kg,but it was not affected by Cd stress.The trends of Mn and Cd contents in roots or aboveground of wild-type and nramp5x7+1DH lines were similar to their parents.In the 5?M Cd treatment test,the nramp5x7+1DH lines were less affected,and its dry biomass weight reduction was less than 20%;while the wild-type showed a significant Cd suppression effect,and the dry biomass weight reduction was 25.9%³9.4%.5.Agronomic traits of nramp5x7+1 DH lines.The agronomic traits such as plant height,effective panicle number,1000-grain weight,seeding rate were investigated for the mutant and the wild type DH lines in field experiment.It was observed that most nramp5x7+1 DH lines had the plant height of 75⁹5 cm and 12 to 15 panicles per plant;The seeding rate ranged from 70%to 85%;nramp5x7+1 DH lines had 1000-grain weight slightly higher than its nramp5x7+1 parent line.In summary,the main agronomic traits of the nramp5x7+1 DH lines were between the parents,and there are some nramp5x7+1 DH lines had agronomic traits better than their parent nramp5x7+1.The DH lines with the excellent agronomic traits could be selected as genetic material for breeding low-Cd accumulation rice varieties.