Discovery Of Genes In Response To Iron Deficiency In Adzuki Bean

Adzuki bean,one of iron uptake strategy?plant,its seed iron content is high as a traditional food for nourishing blood.Iron deficiency in adzuki bean production result in chlorosis and further yield losses.Up to now,the research on mechanism of iron uptake was most in Arabidopsis and tomato,but it was rarely reported in adzuki bean.We obtained several stably genetic mutants of sensitive to iron deficiency and tolerant to iron deficiency by mutagenesis of ⁶⁰Co,EMS and fast neutron tolerance.F₂mapping populations were constructed,which mutants FMY007,FMY009,FMY011andan accession GM309 were crossed with germplasm resources GM684,GM873,CCA026 and a mutant of iron-deficient insensitivity.A total of 35 F₂ populations from 9cross combinations were created.We selected and developed 100 SSR primer pairs uniform distributed on 11 chromosome of adzuki bean.All of the primers were used to screen polymorphism between parents.Nine pairs of core SSR primer were identified for high polymorphism and PCR amplification stability.The nine pairs of core SSR primer had the average of 7.28 alleles per locus and ranged from 2 to 6,The effective amplification frequence from 87.5%to 100.0%,and the average PIC?polymorphism information?of 65.73%.Ten true hybrid populations from GM873×FMY011?FMY011×GM873?FMY011×CCA02?CCA026×FMY009?GM684×FMY007 and GM309×MY076 were identified by mixing-pool.F₂ segregating population with 894 individuals from GM97×FMY007 was used for genetics analysis.Chi-square test showed that segregating ratio of iron-deficient sensitivity to iron-deficient insensitivity in F₂ population coincided with 3:1 by chi-square test.Iron-deficient sensitivity is controlled by single recessive gene.Correlation analysis showed that new leaf chlorosis had significantly positive correlation with the dynamic plant height and leaf apoptosis.Irion-deficient sensitivity gene VaIDS in adzuki bean was mapped on chromosome 5 between SSR markers SSR3 and SSR5 in 470 Kb region at chromosome 5 long arm.Using systematically the geNorm,Norm Finder,BestKeeper,and RefFinder,ELF1B and CYP,SKIP16and PEPKR1 were identified to be optimum reference gene for leaves and stem under iron deficiency stress,respectively.ELF1B/Lectin was best for qRT-PCR analysis of roots and mixing-sample of roots,stems,and leaves under iron deficiency.The transcriptome of wild type Jingnong 6 and mutant FMY007 were sequenced by RNA-Seq under iron deficiency stress.Several new genes in response to iron deficiency,VaWRKY29,VaWRKY25 of WRKY family,VaMYC2 of MYC family,and VaPR-1belonged to PR-1 subfamily were found in adzuki bean by analyzing the transcriptomes.Ten predicted genes in response to iron deficiency were validated by qPCR.In this work,gene discovery was conducted from two strategies,gene mapping and transcriptome analysis.Pathways were predicted.Results of this work will prove theoretical basis for research on mechanism in response to iron deficiency and genetic improvement in adzuki bean.The research will supply populations for mapping and cloning of genes in response to iron deficiency,and will be useful for fine mapping and positional clone of iron uptake genes.The new gene discovery will facilitate to explore pathways of iron deficiency in adzuki bean.It will facilitate iron reinforcement improvement in adzuki bean.