Genetic Analysis Of A Wl1(Wilting Leaves 1) Mutant In Maize

Maize is an environmentally sensitive crop,and various abiotic stresses brought by climate change seriously affect maize yield.Therefore,the cloning of genes related to maize stress adaptation will contribute to the stability and improvement of maize yield,thereby ensuring food security and the development of animal husbandry.Leaf wilting will seriously affect plant growth and development.However,the molecular mechanism of leaf wilting is rarely reported.In this study,we analyzed a leaf wilting mutant wl1（wilting leaves 1）through phenotype identification and stress treatment.Further positional cloning and analysis of the sequence variation identified a candidate gene named Zm WL1,which encodes anα-L-fucosidase（α-L-fucosidase2）.The main results obtained are as follows:1.wl1 leaves showed obvious wilting.Compared with the wild type,when the wl1mutant grows to the stage of 3 leaves and 1 heart,the leaves are prone to wilting,and the wilting is more serious under stress conditions such as high temperature and drought,so that the plants are dwarf,abnormally developed,and unable to produce seed.It was further found that the development of vascular bundles in the stem of the wl1 mutant was significantly abnormal,and its water transport capacity was significantly weaker than that of the wild type,which easily caused insufficient water supply to the leaves and appeared wilting phenotype,but there were no significant differences in number of vascular bundles,the leaf water loss rate and root growth between wild type and mutant.2.wl1 is sensitive to high temperature,drought and salt stress.The Seedlings at the same growth stage were treated under 40℃.After 24h,the wild type still grew normally,but the wl1 mutant showed severe leaf wilting.Further treatments were carried out under different temperature conditions from 28℃to 40℃,and we found that the leaves of wl1mutants could grow normally below 30℃,but showed temperature sensitivity above 32℃.After 10 days of drought treatment,the leaves of wl1 mutants were wilted and plants were significantly shorter than wild type.After 20 days of drought treatment,the wl1 mutant plants were thin and nearly dead,while the wild type was still surviving.After 2 days of salt treatment,the wl1 mutant leaves appeared obvious wilting,and some of the mutant plants died after 10 days of treatment,but the wild type can still grow normally.The above results indicate that insufficient water transport caused by defects in vascular bundle development makes the wl1 mutant more sensitive to high temperature,drought and salt stress.3.Mapping and cloning of Zm WL1.F_2:3 populations with genetic background of C01and B73 were used to mapping the wl1 leaf wilt phenotype.A single locus controlled the phenotype（3:1,χ² _c=0.1928<χ² 0.05,1=3.84）,the BSR-seq（Bulked segregant RNA-seq）provided the result of preliminarily mapping chr6:114253663-156337858.Using 140isolated mutants and 4023 seeds from F_2:3 group,developing KASP（Kompetitive Allele Specific PCR）tag shrank interval to 400 kb.According to genomic annotation information of B73,there were 7 candidate genes in this region.Seven genes in the wild-type and wl1mutants were re-sequenced,and a 4166bp large fragment of Zm00001d037556 gene in the wl1 mutants was inserted 391bp downstream of the initial codon ATG of the first exon.This gene annotation contains five exons,encoding anα-L-fucosidase,which belongs to the GDSL family.Analysis of RNA-seq data revealed premature termination of transcription at the end of the first exon in wl1.Therefore,Zm00001d037556 gene was identified as a candidate gene and named as Zm WL1.4.Transcriptome analysis of wl1.When wl1 mutant leaves appeared curly under 40℃,the leaves of the wild type and wl1 mutants at the same part and the same leaves under the control condition of 28℃were used for transcriptome sequencing.Data analysis identified1438 differentially expressed genes between wild type and mutant under the control conditions,which mainly enriched in cell wall biogenesis and water loss stress response pathways.Under high temperature treatment,there were 2166 differentially expressed genes identified between wild type and mutant.These genes enriched in pathways include transmembrane transport,hormone response,etc.Therefore,Zm WL1 is likely to participate in these pathways to regulate water transport and stress response.In conclusion,this study isolated a key gene Zm WL1 that controls water transport in maize,which is very important for the normal growth and development of maize and for resisting abiotic stress.The results of this study will provide an important theoretical basis for maize stress resistance molecular breeding and ensuring the normal growth of maize.