Genome Sequence Analysis Of 6VS Chromesome And Identification Of WRKY Transcription Factor Family From Haynaldia Villosa

The wheat 6VS·6AL translocation line selected by Nanjing Agricultural University Cytogenetics Institute carries the broad-spectrum high powdery mildew resistance gene Pm21,and has no adverse effects on the main agronomic traits.In particular,it has a positive effect on thousand kernel weight,spike length,nitrogen utilization,etc and is widely used as a breeding parent by domestic wheat breeding units.According to incomplete statistics in 2018,more than 40 varieties carrying the 6VS·6AL translocation line have been approved and distributed in the main wheat production areas of northwest,southwest,Huanghuai region and the Yangtze River’s middle and lower reaches.These varieties play a vital role in the prevention and control of wheat powdery mildew and the safe production of wheat in China.With the gradual expansion of the application range of the 6VS·6AL translocation line,it is also very urgent to analyze its genome and carry out genetic research and gene mining of other excellent traits.In the early stage of this laboratory,we collaborated with Professor Jaroslav Dolezel of the Czech Republic to sort the 6VS·6AL translocation chromosome with high purity,and used HiSeq method sequencing and Chicago long-range linkage assembly method assembly（Dovetail technology,USA）to obtain the reference genome sequence of this single chromosome（Xing et al.,2018）.This study carried out in-depth sequence analysis and gene annotation on the assembled 6VS·6AL single chromosome genome sequence,developed a batch of 6VS chromosome-specific molecular markers,combining with the 6VS small fragment translocation line created by predecessors,and constructed the fine physical map of 6VS chromosome.At the same time,we have systematically discovered the WRKY transcription factor family members of Haynaldia villosa and systematically identified one of the functions of WRKY1-V which located on 6VS and participated in powdery mildew resistance.The main research results obtained are as follows:1.6VS·6AL single chromosome genome sequence analysisThe 6VS·6AL chromosomes were sorted,sequenced and Chicago assembled,and the length is 574.6 Mb,including 5546 scaffolds.Then compared with IWGSC RefSeq v1.0 database online and removing the 6AL scaffold,3769 scaffolds on the 6VS chromosome which length is 243.39 Mb were obtained.Repeated annotation results show that there are 78.27%（449.7 Mb）repetitive sequences in the 6VS·6AL translocation chromosome genome,with the largest proportion being 67.75%of retrotransposons,and LTR Retroelement accounted for most.Using De novo annotation,homologous annotation and transcriptome annotation methods to predict the genes contained in 6VS,the results showed that 5781 gene sets were counted.The results of collinearity analysis based on high-confidence genes show that the collinearity between 6AL and 6AL in Chinese Spring is very good,and the collinearity between 6VS and 6AS is also good,but the collinearity of the 30 Mb segment is poor.The functional annotation results show that 86.1%of genes can be predicted to have corresponding function by using common five databases as NR,Swiss-prot,KEGG,Pfam and GO comprehensively.2.Construction of 6VS fine physical mapA total of 13 scaffolds with a length larger than 5 Mb were selected from 3769 6VS assembly sequences.The number（length）is arranged in order from the end of the chromosome to the centromere,Sc18QlZ₁327（41.84 Mb）,Sc18Q1Z₁707（22.41 Mb）,Sc18Q1Z₂084（5.45 Mb）,Sc18Q1Z₂155（9.44 Mb）,Sc18Q1Z₂204（6.12 Mb）,Sc18Q1Z₃069（13.2 Mb）,Sc18Q1Z₄548（31.8 Mb）,Sc18Q1Z₅541（5.08 Mb）,Sc18Q1Z₅542（5.01 Mb）,Sc18QlZ₅544（14.69 Mb）.These scaffolds cover 69 percent of the entire 6VS chromosome arm length.Designing primers with the density of each 10 kb,decompose the above scaffold sequence and the corresponding blast line of the Chinese Spring ABD genome,finding the location of the insertion/deletion region obtained,and design a total of 1920 pairs of InDel markers,1089 of which are polymorphism markers.The overall polymorphism rate was 56.7%.Using GISH technology to screen the progeny of radiation-induced mutagenesis translocation lines in the laboratory,14 different types of translocation lines involving 6VS were obtained,including 9 homozygous materials,4 of which were inserted into the translocation line at the top,5 in the middle and 5 hybrid materials,4 of which are inserted into the translocation system at the top and 1 in the middle.Using 1089 molecular markers to carry out the pollen of the chromosomal segment where the foreign fragment contained in the above 6VS structural variant is combined with the distribution of 13 scaffold segments,a fine 6VS physical map consisting of at least 28 chromosomal segments and high-density molecular markers.3.Analysis of WRKY transcription factor family members and the cloning and functional identification of WRKY1-VWRKY transcription factors are widely involved in regulating the stress resistance of plants.Haynaldia villosa has many excellent traits such as resistance to powdery mildew,rust and other wheat diseases,drought tolerance and cold resistance.In order to study WRKY transcription factors in the stress resistance process of Haynaldia villosa,combined with the second-and third-generation transcript sequencing database of Haynaldia villosa,obtaining 14 genes clustered with full-length CDS and 4 non-standard WRKY sequences（WRKY domain contains an incomplete zinc finger motif）through multiple methods such as blast homology search,protein annotation information screening,HMM model screening,and PlantTFDB prediction.The above genes are named according to the sequence similarity to the barley WRKY gene,and are divided into five categories based on WRKY domain and zinc finger structure.Group Ⅰ includes WRKY6,40,41,42,46,61;Group Ⅱa includes WRKY1（which is a homologous gene of HvWRKY1）;Group Ⅱc includes WRKY15;Group Ⅱd includes WRKY8,10 and 58;Group Ⅲ includes WRKY22,28 and 33.Electronic chromosome mapping results show that the 14 WRKY genes are mainly distributed on the second,fourth,fifth,sixth and seventh homologous groups,of which the most distributed on the fifth homologous group,including 8 WRKY genes.Analysis of expression characteristics revealed that multiple WRKY genes have tissue expression specificity and are induced by powdery mildew and induced by salt and drought stress.According to the electronic positioning of chromosomes,WRKY1 is located on the sixth part of the homologous group.The comparison of the above 6VS genomic data shows that WRKY1-V is located on Sc18QlZ₁707 which nears the centromere.It has the highest sequence identity with barley HvWRKY1 gene,reaching 87.15%.Analysis of expression characteristics showed that WRKY1-V was significantly up-regulated after being induced by powdery mildew in the leaves of Haynaldia villosa.Isolation and analysis of its upstream promoter sequence revealed that it contains multiple cis-acting elements related to disease resistance,such as MeJA-mediated,stress-related and light-response related elements.The results of virus induced gene silencing showed that after silencing WRKY1-V gene in the highly resistant powdery mildew 6VS·6AL translocation line,its powdery mildew resistance was partially lost,showing that there were significantly more powdery fungus spores than control group（spores hardly develop）.However,overexpression of WRKY1-V in Yangmai158,which is highly susceptible to powdery mildew,showed that the seven To plants presented a significant increase in powdery mildew resistance.The above results indicate that WRKY1-V plays a positive regulatory role in the process of wheat powdery mildew resistance.