Analysis Of Drought Tolerance Related Regulatory Networks By Transcriptome Sequencing In Maize

Maize?Zea mays L.?is one of the most important crops in the world.As the climate change and decreasing of water resource,drought has become one of the most serious abiotic stresses affecting maize production.It is an effective strategy to reduce the yield loss caused by drought with improving the drought tolerance of maize and breeding drought tolerant maize lines or hybrids.In our previous study,two maize inbred lines with contrasting drought tolerance,H082183?drought tolerant?and Lv28?drought sensitive?,were identified.Moreover,these two lines showed significant different tassel size and leaf angle.Thus,a F_2:3 population were constructed by the cross of these two lines and the QTL controlling tassel size and leaf angle were identified.Furthermore,to explore the drought-related regulatory networks mediated by miRNAs,a miRNome analysis of the leaves and roots of H082183 and Lv28 under different water treatments were conducted using deep sequencing.And the drought-related miRNA-mRNA modules were validated by gene expression analysis and degradome sequencing.In addition,the transcriptome of 10 maize inbred lines under different water conditions were analyzed by RNA sequencing to identify the drought responsive genes.Gene co-expression networks of the drought responsive genes were established.The main results in the present study are as following:1.High-density quantitative trait locus mapping of leaf angle and tassel size in maize.In the present study,an F_2:3 population,including 213 families,was developed using the cross of Lv28 by H082183,the latter having more erect leaves and small tassels.The population and parents were genotyped by Maize56K SNP array.A modified hidden Markov model?HMM?was used for bin map and linkage map construction.The linkage map contained 1965 bins,and covered 1495.79 cM.The QTL analysis identified six leaf angle,which could explain 55.08%of the phenotypic variance.Seven tassel branch number QTL explained 60.76%of the phenotypic variance.And ten tassel dry weight QTL explained 61.26%of the phenotypic variance.Interestingly,the significant SNPs associated with tassel branch number and the selection region in qTBN8 were consistent at 5'non-coding region of GRMZM5G873917.And the nucleotide diversity analysis revealed that the 5'regulatory region of GRMZM5G873917 was under strong selection.2.Genome-wide identification and comparative analysis of drought-related microRNAs in two maize inbred lines.Two maize inbred lines,H082183?drought tolerant line?and Lv28?drought sensitive line?,were planted in the field and treated with drought and well-watered treatments.The expression of miRNAs in leaves and roots under different water conditions were analyzed by small RNA sequencing.The expression of miRNA-target genes were obtained from mRNA sequencing,and the target sequences were validated by degradome sequencing.In H082183,the miRNA-mRNA pairs of miR164-MYB and miR164-NAC showed responsive to drought in leaves,and miR159-MYB,miR390-LRR and miR398-SBP were responsive to drought in roots.In Lv28,miR160-ARF uniquely responded to drought in leaves.However,the miR156-SPL were responded to drought in both leaves and roots of Lv28,but in different regulation patterns.The gene function analysis showed the putative genes regulated by miR164-MYB and miR164-NAC in leaves of H082183 were related to stress response,but the genes regulated by miR156-SPL and miR160-ARF were enriched in photosynthesis and development functions.In addition,the expression of miR164-MYB and miR164-NAC were regulated by ABA.3.Identification of drought-related genes and co-expression networks in 10 maize inbred lines by RNA sequencing.Ten maize inbred lines were planted and treated with different water conditions.A total of 7278 drought responsive genes were identified by differential expression analysis between different water treatments.Furthermore,the gene co-expression networks showed numerous re-wiring and de-wiring between drought and well-watered conditions,which were established by WGCNA.Under drought treatment,there were five co-expression modules had significant correlation with leaf relative water content.Finally,eight transcription factor genes and eleven protein kinase genes were consider as drought tolerance-related key genes,whose expression showed highly correlated with leaf relative water content.