| Saline-alkali stress has seriously restricted food production worldwide,which has become a growing threat to food security.Foxtail millet has been regarded as a model crop for exploring mechanisms under stress.Considering its extreme abilities to adapt to adverse ecologies,it is of great importance to reveal the mechanism of plant response to saline-alkali.However,due to the continuous deterioration of saline-alkali soil and the reduction of cultivated land,the growth and yield of the foxtail millet have been severely affected.Mixed saline-alkali stress is different from simple salt stress.With salt containing a large number of alkaline salts,the damage of mixed saline-alkali stress to the plant is much larger than salt stress.So far,there have been many reports on the research of foxtail millet stress,while the mining and utilization of genes under mixed saline-alkali stress are obviously backward..Therefore,it’s of great significance to study the saline-alkali resistance and excavate saline-alkali stress response gene for breeding saline-alkali tolerant foxtail millet varieties,which will improve the utilization rate of saline-alkali land and ensure the foxtail millet stability production in China.A total of 360 natural populations of foxtail millet obtained 3.82 × 106 high quality SNPs through resequencing technology.The whole genome association analysis was used to analyze the fresh weight above ground,dry weight above ground,plant height,leaf length,chlorophyll content,survival rate and other related data of the treatment and control group.In addition,the transcriptome sequencing of saline-alkali tolerant foxtail millet varieties B103 and B241,and salt sensitive foxtail millet varieties B323 and B355 at bud stage and two leaf stage were carried out under saline-alkali stress and control conditions.The differential genes in the two categories with different saline-alkali tolerance were screened and analyzed.With the help of genome-wide association analysis and haplotype technology,we hope to find new genes of saline-alkali tolerance in foxtail millet to improve millet varieties.The main results are as follows:(1)Statistical analysis was performed on the six saline-alkali tolerance related traits of foxtail millet seedling stages under treatment compared with control and single treatment data.The results showed that the higher the comprehensive evaluation value D was,the stronger the saline-alkali tolerance of foxtail millet.The foxtail millet could be divided into five grades of saline-alkali damage,in which B103 and B241 were saline-alkali tolerant varieties,and B323 and B355 were saline-alkali sensitive varieties.It proved that the natural population of foxtail millet used in this study showed the diversity of individual differences in response to salt-alkali stress.(2)According to the analysis of population structure(Q),the natural population of foxtail millet selected in this study could be divided into 9 subgroups according to their planting areas,which were distributed in different climatic environments in China.The phylogenetic(NJ)tree showed that there were distant genetic relationships among individuals in the population,which was a good population for genome-wide association research.(3)The mixed linear model(MLM)of TASSLE 5.0 software was used to predict the-log10(P)of significant loci.When-log10(P)was higher than the threshold of 6,it could be judged that there was a significant association between the SNP site and the phenotype related to saline-alkali response.A total of69 SNPs were detected,which were significantly associated with saline-alkali tolerance in foxtail millet;and 121 genes were found to have haplotype difference effect in LD block.Among them,25 genes were annotated into GO term and KEGG pathways related to saline-alkali stress in foxtail millet.(4)The transcriptome sequencing of saline-alkali tolerant foxtail millet varieties B103,B241 and saline-alkali sensitive foxtail millet varieties B323,B355 under saline-alkali stress and control conditions was carried out.The differences of cross transcriptome expression between saline-alkali sensitive and saline-alkali tolerant foxtail millet varieties were analyzed.The common genes in bud stage(Group C)and two leaf one heart stage(Group D)were counted respectively;the common genes in control group and treatment group of saline-alkali tolerant varieties B103 and B241 were counted in bud stage(Group E)and two leaf one heart stage(Group F),respectively.A total of 9152 unique differentially expressed genes(DEGs)were detected in C,D,E and F combinations,most of which were enriched in GO functions related to abiotic stress such as stimulation,stress response and ion transmembrane transport;KEGG pathway enrichment analysis showed that DEGs were mainly involved in MAPK cascade signal transduction,plant pathogen interaction and endoplasmic reticulum protein processing.(5)Cluster analysis of differential genes expression in B103,B241,B323 and B355 foxtail millet varieties between C and D groups showed that saline-alkali sensitive and saline-alkali tolerant millet varieties were clustered into one group,respectively,and both groups were divided into seven cluster groups;GO and KEGG analysises of cluster groups in C and D groups showed that most of the genes in the largest cluster were annotated to GO term and pathway related to saline-alkali stress.(6)In C group and D group,the frequencies of variable splicing events corresponding to intron retention(RI),exon skip(SE),5’terminal variable splicing(Alt5’ SS),3’terminal variable splicing(Alt3’ SS)and exon mutual exclusion(MXE)were approximately the same,and SE was the main significant variable splicing event.Most of the variable splicing genes had only one splicing form and there were few genes with five kinds of variable splicing forms at the same time.(7)In this study,25 genes related to saline-alkali stress were obtained through genome-wide association and haplotype analysis,and 10 candidate genes related to saline-alkali tolerance were screened by transcriptome analysis of two types of foxtail millet varieties with different saline-alkali tolerance. |
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