Phylogenetic Analysis Of NBS Gene Familiy In Five Triticeae Species Based On Transcriptome

The Triticeae is an important group of Poaceae.It is a food-fed mixed-use group which not only includes important crops such as wheat,barle,but also a large number of rich forage grasses such as icegrass and new wheatgrass.It contains many excellent disease-resistance genes,providing an extremely rich source for the common wheat(Triticum aestivum L.).Among them,the NBS gene family is a major resistance gene family.This study completed the RNA extraction,sequencing and assembly of five species,and further exploited the bioinformatics method at the transcriptome level to research the NBS gene family.In addition,phylogenetic and evolutionary biology methods were used to systematically analyze the evolutionary patterns of NBS gene families among species.The main results are as follows:1.Transcriptome data of five Triticeae species: We performed next generation sequencing and single-molecule real-time sequencing of Triticum timopheevii,Triticum zhukovskyi,Aegilops umbellulata,Aegilops caudata and Secale Cereal.To obtain high-quality transcripts,full-length transcriptome data were corrected using the "NGS+SMRT" model.The numbers of transcripts of T.timopheevii,T.zhukovskyi,A.umbellulata,A.caudata and S.Cereal were 97653,58207,43573,49870 and 91170 respectively.The lengths of N50 were 1434 and 1742,respectively,1856,1764,1972 bp.2.Homologous search and identification: Homologous search was performed on the protein sequences of five species.The threshold value was set to 1e-50.The results of homologous search in T.timopheevii,T.zhukovskyi,A.umbellulata,A.caudata and S.Cereal were 50,50,96,40,68 and 160 respectively.Representative NBS protein sequences of the embryonic plant were downloaded from the plant resistance genes database,and a phylogenetic tree was rapidly constructed with the candidate NBS protein sequences.No independent branch was found,indicating that the candidate protein sequences obtained can be used as NBS protein sequences for subsequent analysis.3.NBS gene family classification results: We found that only CC-NBS-LRR(CNL)proteins exist in five species,consistent with previous research that TNL mostly not in monocotyledons.According to the presence or absence of the LRR domain in C-terminus,it can be divided into NBS-LRR calss and NBS class.Among them,the NBS-LRR protein sequences were 33,59,27,38,111 and the NBS protein sequences were 17,37,13,30 and 49 respectively.4.Multiple sequence alignment and conserved domain analysis: Multiple alignments of the identified NBS protein sequences with NBS protein sequences in rice were performed.Among the alignment results,there were four conserved motifs which are P-loop,Kinase-2,Kinase-3 and GLPL,indicating that the NBS protein sequences in five species were relatively conserved.5.Phylogenetic analysis: Using the maximum likelihood method to construct the phylogenetic tree of the Marchantia polymorpha as outgroup,these sequences were divided into two distinct branches,indicating that the NBS gene family evolved from two branches.They could be further divided into three groups.Different types of NBS protein sequences in five species were distributed in the three groups and existed in different clades.We inferred that the five species had the same evolutionary pattern.6.Evolutionary pressure analysis: We calculated the synonym substitution rate(Ks),non-synonymous substitution rate(Ka)and the ratio(Ka/Ks)between them for homologous gene pairs.We found that Ks value of paralogous genes were generally higher than orthologous genes,indicating that most of the gene replication events in the five species occured earlier than the species separation time.In addition,the Ka/Ks values of orthologous genes were generally higher than those of paralogous genes,indicating that the orthologous genes were less restricted in their function during evolution and the evolution process was faster.7.Structure analysis of conserved motifs: In the I,II,and III groups,the highest quality 15 CNL protein sequences were selected for analysis of conserved motifs.The results showed that the categories of conserved motifs were different among different groups.Between different sequences,the motif models were not exactly the same.They had differences in the type,number,and order of motifs.It was inferred that the five species evolved through the frequent acquisition and loss of motifs.In addition,the 20 conserved motifs of CNL protein sequences in the five species varied considerably in length from 15 to 50 amino acids,indicating that the NBS gene family members were relatively conserved.