The Transcriptome Sequencing Of Three Avian Species And Evolution Of NADH Gene Family

Recent studies have shown that mitochondria play a crucial role in the energy production through the process of oxidative phosphorylation complexes(OXPHOS).Complex I(NADH: ubiquinone oxidoreductase),is the largest and first enzyme of the OXPHOS system,including both nuclear-and mitochondrial-encoded proteins.To date,numerous cases of adaptive evolution of OXPHOS have been reported in vertebrates,such as fishes,reptiles and mammals.However,patterns of natural selection and phylogenetic implications of complex I remain unclear in aves.We performed transcriptome sequencing of three Passerida aves and combined with the public released data,then discussed the following scientific problems:(1)the transcripts' characteristics of P.montanus,L.lutea,and G.canorus;(2)comparing the evolutionary rate of mitochondria with nuclear-encoded genes from NADH gene family;(3)the evolutionary rate analyses for aves,mammals,amphibians and reptiles(4)the phylogenetic relationships of Passerida.The main findings are as follows:1.High-throughput sequencing and de novo assemblyIn this study,we used the Trizol to extracting total RNA from P.montanus,L.lutea and G.canorus,and then constructed c DNA libraries.Transcriptome sequencing was performed with the pair-end sequencing at the Illumina Hi SeqTM 2500 sequencing platform.After the assembly of raw reads and the analysis of quality control,it is found that the number of raw reads of L.lutea was the least.there is 9,115,852,062 and the number of base sequences of the raw reads is 60,530,122 bp;After removing the low quality fragments,there is 58,616,456 clean reads,accounting for 96.84%;P.montanus has the largest number of raw reads(10,534,487,628),and its base number is 69,904,416 bp;after removing low-mass fragments,the number of base sequences of raw reads is 9,465,945,091 bp,accounting for 89.86%;the assembled unigenes number of P.montanus is the highest,which is 237,822 with 145,747 kb.The length of N50 is 751 bp.The average length of unigenes is 612 bp.The collected unigenes from L.lutea is the least.There are 198,193,which is 142,511 kb.The length of N50 is 739 bp.The average length of unigenes is 604 bp.2.Functional annotationThe whole transcripts were annotated in seven frequently used databases,i.e.,the NR,NT,Swiss-prot,GO,KOG,Interpro and KEGG databases.Among them,the three birds' transcriptome data has 4487 unigenes in P.montanus with the most annotated information in the seven commonly used databases,and the minimum is 2282 for the L.lutea.In the 24 annotated categories of KOG,the unigenes annotated ingeneral function prediction genes(R)and signaling mechanisms(T)were the most.Compared with the KEGG pathway database it reveals that 6,600,6,405,and 6,488 unigenes in the transcriptomes of P.montanus,L.lutea,and G.canorus,respectively,are successfully annotated and mapped to different metabolic pathways.The unigenes in the three birds' transcriptome data accounts for the majority of metabolic processes.During the unigenes' protein sequences categorize as GO databases,the GO analysis distributions of the three transcriptomes' unigenes are highly similar.Based on the results of preliminary SSR analysis,it is found that the total number of P.montanus transcriptome data is 48,460,including 42,736 SSR sequences,while the number of SSRs in transcriptome of L.lutea are 57,198,including 44,460 SSR sequence.In the SSR analysis of the three birds' transcriptomes,the single-base tandem repeats account for the most in the tandem repeats.3.The analysis of evolutionary rate(1)The analysis of evolutionary rate for avesIn this study,the NADH gene Family of Taeniopygia guttata was used as a template,and the Basic Local Alignment Search Tool was used for the extraction of the birds' NADH gene Family.After screening,a total of 48 genes from 65 species of birds were used for further analysis.Based on the Datamonkey's SLAC,FEL,REL models and the site model of the PAML software,positive selection sites were screened.At the same time,the branch-site model was used to determine the information between loci of specific branch.Secondly,comparing the evolutionary rate of mitochondrial coding subunits with its nuclear encoding genes,we used the paired sample T-test from IBM SPSS Statistics v.23 software to evaluate the significant difference between groups.In addition,Tree SAAP software was used to determine the physicochemical properties of the amino acid at the protein level and the positive selection sites screened by the above two methods at least.Based on the results of SLAC,REL,FEL and site model,it was found that some genes have undergone possibly potential positive selection,including three mitochondrial genes(MT-ND2,MT-ND4,MT-ND5)and three nuclear genes(NDUFS1,NDUFS7,NDUFV2).Through the analysis of changes in physical and chemical properties,we found that 72.92% of the amino acid sites have undergone a strong change.In our study,nu genes of NADH gene Family have the fastest evolutionary rate,while it was the slowest for nu core genes.By analyzing the average d N/d S(?),we found that there are extremely significant differences between the mt genes and the nu core genes;the nu non-core genes were different from the mt genes encoding the core members.By comparing the evolutionary rate of the accelerated gene with the reference gene,the average ? value of the nu non-core genes is greater than that of the accelerated genes.The average ? is the smallest for the reference genes.In addition,the ? value of the NDUFV3 was the largest(0.68),while the ? value of the RPS7 was the smallest(5e-09).(2)Comparison of evolutionary rates of birds,mammals,amphibians and reptilesBy comparing the average d N / d S(?)of mt genes,the results showed that birds were significantly different from that of mammals,amphibians and reptiles.It is worth noting that based on the results of SLAC,REL,FEL and site models,we found that there is potential positive selection for MT-ND5 in birds,mammals,amphibians and reptiles.However,NDUFA9,NDUFA10,NDUFB9 and NDUFS5 of birds,mammals and amphibians may have undergone potential positive selection for nu non-core genes.The NDUFV3 is the largest value of 0.56.4.Phylogenetic analysis of the encoding complex I genes for birdsIn this study,we constructed MP,ML and BI phylogenetic trees based on seven mitochondrial genes and seven nu core genes,and we found that they have similar topological structures.The main difference in the topology is the phylogenetic position of Pseudopodoces humilis.Our results provide a new insight into the molecular evolution of birds' complex I.In this study,the high-throughput sequencing and the released data were used to study the evolution of the birds' NADH gene family.At the same time,we compared the NADH gene family,accelerated genes and reference genes for birds,mammals,amphibians and reptiles.In addition,we constructed a phylogenetic tree using NADH gene family to investigate the current problems within the phylogenetic relationship of Passerida.The results lay a solid foundation for studying molecular evolution of the NADH family in the future.