| Foxtail millet is one of the major cereal crops in northern China.In recent years,with the adjustment of the industrial structure of foxtail millet and the emphasis on dry farming,the planting area of foxtail millet has gradually increased,and the incidence of downy mildew of foxtail millet has gradually developed into one of the most serious diseases in foxtail millet production,so that seriously reduce the yield and quality of foxtail millet.Screening and breeding resistant cultivars are effective ways to improve the resistance of foxtail millet to downy mildew.However,no gene has been cloned for downy mildew of foxtail millet.This study was to investigate the resistance of 18 foxtail millet varieties and identify the resistance germplasm resources by investigating the incidence rate in the field.Meanwhile,the DNA mixed pool was constructed by using the F2 population obtained from the cross between the reiatant varity G1 and the high susceptible cultivar Jingu 21 SR,and the resistance genes were identified by BSA-Seq.Then,the laccase gene family of foxtail millet was selected from the candidate genes for bioinformatics analysis.The main research results are as follows:1.Based on the disease classification standard established by our previous research,the resistance of18 foxtail millet varieties to downy mildew was evaluated.18 foxtail millet varieties were divided into five grades: high resistance,resistance,medium resistance,sensitivity and high sensitivity;Yugu 1,G1 and Fen Xuan 3 are high resistance varieties;Jingu 35,Jinfen 52,Qinhuang 2,changnong35,Jingu 42 and Jingu 29 are resistance varieties;Jingu 20,Jingu 21 d,Changsheng 08,Jingu 54 and Jingu 57 are medium resistance varieties;Jingu 21 and Jingu 40 are susceptible varieties;dabaigu and Jingu 21 SR are high sensitivity varieties.2.Using G1(male parent)and Jingu 21SR(female parent)as materials,and F2 population including144 single plants,20 highly resistant and highly sensitive extreme isolated single plants were selected from the isolated population,and a mixed pool was constructed,respectively.BSA-Seq sequencing was used to analyze and screen the genes of resistance to downy mildew.Six resistance candidate genes(Seita.8g241700,Seita.8g241900,Seita.8g211800,Seita.8g202900,Seita.8g199500,Seita.8g225400)were identified.The functional annotation were GUB-WAK protein kinase gene,laccase gene,leucine rich repeat protein gene and Arabidopsis broad spectrum resistance protein RPW8.Subsequently,q RT-PCR was used to analyze the expression level of the candidate genes.3.Previous GWAS association analysis and this study identified that laccase gene was involved in the resistance of foxtail millet to downy mildew,we used bioinformatics to analyze the physical and chemical properties,chromosome location,phylogenetic tree,gene structure,protein motif and gene expression of 28 laccase family related to foxtail millet resistance.The 28 genes were found on chromosome 3,5,7,8 and 9,and the family members were divided into three subfamilies by phylogenetic tree analysis,28 laccase genes all contain exon and intron structure,protein sequence analysis shows that there are differences in the number and type of 28 laccase motifs,gene expression analysis shows that laccase genes are differentially expressed in 3,5 and 7 leave stages of susceptible foxtail millet varieties infected with Sclerospora graminicola.It is suggested that some laccase genes play an important role for foxtail millet response to S.graminicola.In this study,through the evaluation of the disease resistance of 18 foxtail millet varieties,the germplasm resources of resistance to downy mildew were screened;the candidate genes of resistance to downy mildew were screened by BSA-Seq method,and the candidate genes for resistance to downy mildew were verified by q RT-PCR;bioinformatics analysis of the laccase gene family related to resistance to downy mildew provides further theoretical basis for the mapping,cloning and functional verification of foxtail millet resistance to downy mildew genes. |
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