| Verticillium wilt(Verticillium dahliae)is an important disease causing loss of cotton yield and quality Previous research progress on cotton resistance to Verticillium Wilt always focused on the protein-coding gene’s resistance mechanism.Then,we evaluated the non-coding RNAs at whole genome level and also focused on coding proteins faimiliy RLPs to identify and analyze long-non-coding RNAs involved in resistance to Verticillium dahliae and receptor-like protein family which was closely related to Verticillium wilt resistance.In addition,we obtained the Verticillium genome by third generation sequencing,thus finally revealed the interaction of cotton and Verticillium in the view of Verticillium genome variation.The main results were as follows:1 Firstly,we described a comprehensive disease-responding IncRNA profiles in defense against a cotton fungal disease Verticillium dahliae.We further revealed the conserved and specific characters of disease responding process between two cotton species,Gossypium barbadense and G.hirsutum.Conservatively for two cotton species,we found the expression dominance of induced IncRNAs in the Dt subgenome,indicating a biased induction pattern in the co-existing subgenomes of allotetraploid cotton.Comparative analysis of IncRNA expression and their proposed functions in resistant line Gossypium barbadense cv.’7124’ versus susceptible line G.hirsutum cv.’YZ1’ revealed their distinct disease response mechanisms.Species-specific(LS)IncRNAs containing more SNPs displayed a fiercer inducing level post infection than the species-conserved(core)IncRNAs.Gene Ontology enrichment of LS IncRNAs and core IncRNAs indicates distinct roles in the process of biotic stimulus.Further functional analysis showed that two core IncRNAs GhlncNAT-ANX2-and GhlncNAT-RLP7-silenced seedlings displayed an enhanced resistance towards V dahliae and Botrytis cinerea,possibly associated with the increased expression of LOX1 and LOX2.This study represents the first characterization of IncRNAs involved in resistance to fungal disease and provides new clues to elucidate cotton disease response mechanism.2.Among studies of cotton against Verticillium dahliae,the only site that was confirmed to be completely resistant to Verticillium dahliae was Vel,which belongs to the receptor-like protein.However,there is no Vel in cotton,so we hope to find the key genes controlling Verticillium wilt by screening the whole genome of cotton.Using validated methods by Arabidopsis,we screened four genomes of Gossypium barbadense,G.hirusutum,G.raimondii and G.arboreum and identified 100 to 300 RLPs from the above genomes.In addition,we also compared the different characteristics of the RLP family between two tetraploid cotton genomes,such as evolution rate,genomic distribution and tissue expression preferences.The results showed most of RLPs were under purifying selection.In G.barbadense,more RLPs located in the At subgenome.For RLPs in G.hirusutum,most are from the Dt subgenome;RLPs from G.barbadense have higher tissue specific level in root and stem.Finally,we identified the candidate RLPs of cotton Verticillium wilt by VIGS method and found that RLP 13 positively regulates cotton resistance,which will provide a powerful candidate gene for cotton Verticillium resistance breeding.3.To understanding the interaction of cotton and Verticillium Wilt better,we assemblied genomes of two distinct field isolates,Defoliating isolate V991 and Non-defoliating isolate lcd3-2 based on single-molecule real-time sequencing developed by Pacific Biosciences(PacBio).Illumima sequenced short reads were used to polish assembled genome.Finally we produced two near-complete high quality genomes.Main results as following:We finally produced defoliating isolate V991 genome(35.bMb)and Non-defoliating isolate lcd3-2 genome(34.5Mb).The contig N50 respectively reached 3.8Mb and 3.5Mb;By ab initio,homology and transcriptome prediction,we predicted 10941 protein-coding genes of V991 and 10971 proteins of lcd3-2.Comparative genomics analysis allows systematic characterization of genomic structural variations,presence/absence variations between two isolates,core genes and strain-specific genes.The results showed striking divergence on gene feature and function between core genes and strain-specific genes.The occurrence of PAV incidents will lead to the new formation of strain-specific genes,which might contribute to strain-specific virulence and pathogenicity.Additional epigenetic information of DNA methylation showed a biased distribution of 6mA and 4mC in different isolates.Intriguingly,we found 4mC is highly associated with active genes.Thus the low distribution of 4mC for strain-specific genes might lead to their lower expression level compared with core genes.These results will provide novel insight into the genomic evolution basis of V dahliae and facilitate the understanding of fungal virulence divergence. |
