Physiological Analysis And Candidate Gene Mapping Of Waterlogging-sensitive Mutant γ2214 In Maize

Maize is very sensitive to waterlogging stress,which has caused great losses to spring maize production in southern China.To explore the key genes of maize waterlogging tolerance,analyze its function and molecular mechanism,accumulate new biological knowledge for revealing the molecular mechanism of maize waterlogging tolerance,and provide new genetic resources and theoretical guidance for the genetic improvement of waterlogging tolerance.Our research group constructed a maize mutant library and screened out the waterlogging sensitive mutantγ2214.In this study,γ2214was used as the material in order to explore the target gene of the mutant which cause the waterlogging sensitive phenotype.The main results of the study are as follows:（1）Waterlogging stress causes premature senescence of the first leaf of the mutant.The chlorophyll content of the mutant and wild-type was analyzed,and the results showed that after waterlogging treatment,the total chlorophyll content of the mutantγ2214decreased by 82.03%,chlorophyll a and chlorophyll b decreased by 81.94%and 82.6%,respectively.Trypan blue staining showed that the coloring area of the mutant leaves was larger and darker after waterlogging stress.q RT-PCR analysis showed that the expression of NYE1 and SAG12 were significantly up-regulated in the mutants after waterlogging stress compared with the wild-type.（2）Waterlogging stress disrupted the balance of active oxygen species in the first leaf of mutant.After waterlogging stress,the contents of H₂O₂ and O₂^-in the first leaf increased by 24.38%and 32.2%in the mutant compared with the control group,respectively;The activities of catalase,peroxidase,ascorbate peroxidase,superoxide dismutase and glutathione reductase in the antioxidant enzyme system were also 36.57%,34%,18.3%,54%and 29.51%higher than those in the control group,respectively.（3）The cell homeostasis is disequilibrium in mutant under waterlogging stress.After waterlogging stress,the content of malondialdehyde was increased by 40%in the first leaf of the mutant compared with the wild-type,which reflects the degree of membrane lipid peroxidation.While the content of soluble sugar was 67.95%lower in mutant than that of the wild-type.The content of proline decreased by 31%in mutant compared with wild-type after waterlogging.（4）The mutant is sensitive to ethylene.The first leaf of the mutantγ2214 exhibited premature senescence after spraying exogenous ethylene,which was consistent with the traits after waterlogging stress,while the wild-type did not exhibit phenotype.The content of ACC in the first leaf of mutantγ2214 and wild-type was measured under normal condition,results showed that the content of ACC in the first leaf of mutantγ2214（3.56ng/g）was significantly higher than that of wild-type（2.88 ng/g）,In vitro leaf culture experiments also showed that compared with wild-type,γ2214 exhibited premature senescence under 100μL/L exogenous ethylene treatment.（5）Fine mapping of target gene ofγ2214 mutant.F₁ was obtained by crossing theγ2214 mutant with the inbred line Qi205,and the F₂generation population was constructed by self-crossing F₁.A total of 173 F₂ plants were used to verify the isolation ratio.It was found that the ratio of waterlogging normal type and waterlogging sensitive type was 130:43,which was consistent with the segregation ratio of 3:1（X²<3.84）,indicating that theγ2214 mutant was a recessive mutation of a single gene controlled.In F₂ generation,40 waterlogging normal type DNA and 35 waterlogging sensitive type DNA were used to form two mixed pools.Meanwhile,two parent mutantγ2214 and inbred line B73 were used to construct the mixed pool.The mutant gene was preliminarily located in the region of 85.93-178.97 Mb on chromosome 4 by BSA technique.Subsequently,20 pairs of SNP markers and 16 pairs of In Del molecular markers were designed for further fine mapping,and the interval was shifted to the right and narrowed to within the 1.04Mb interval of 180.06-181.1Mb on chromosome 4,which contained 29genes in total.The above results show thatγ2214 is a waterlogging sensitive mutant,which exhibited the phenotype of yellowing and cell death of the first leaf after 120 hours of waterlogging treatment in V2 stage.This phenotype may be due to the high content of ethylene synthesis precursor ACC in the mutant leading to high content of ethylene,which induces senescence of first leaf.And the accompanying changes include the increased MDA content,decreased soluble sugar and proline content,increased H₂O₂ and O₂^-content,and enhanced antioxidant enzyme activity.Genetic analysis revealed that the mutation was caused by a recessive mutation of a single gene.Finally,BSA analysis combined with fine mapping narrowed the gene range to 180.06-181.1 Mb on chromosome 4.