Characterization And Genetic Polymorphism Of Toll-like Receptor(TLR) Genes In The Chinese Egret(Egretta Eulophotes)

Genetic diversity is a key parameter that reflects the survival,adaptation and evolution potential of organisms.Its research is helpful to understand the evolutionary processes and the endangered mechanisms of the threatened species for developing effective conservation plans,hence genetic diversity is the core of conservation genetics.TLRs(Toll-like receptors)are an ancient family of innate-immunity genes in animals.Compared to MHC(Major histocompatibility complex)of immunity genes,TLRs are relatively easy to obtain(less pseudogenes and duplications)and have higher levels of genetic diversity.Therefore,TLRs have been increasingly used to estimate adaptive genetic diversity.However,to date,research on TLR genes in ardeid species has not been carried out.The Chinese egret(Egretta eulophotes)is a migratory waterbird,listed as a vulnerable species by IUCN(International Union for Conservation of Nature).In this study,we characterized the TLR genes in the Chinese egret,and then assessed the levels of genetic diversity in this egret,with the aim at understanding its endangered mechanism and facilitating its scientific conservation.The main results are summarized as follows:(1)Based on high-throughput sequencing,we examined and identified the TLR genes in the Chinese egret,and obtained seven TLR genes(TLR1LB,TLR2A,TLR3,TLR4,TLR5,TLR7,and TLR15).All these sequences were consistent with those in jungle fowl(Gallus gallus)using NJ phylogenetic tree analysis.No gene duplication and pseudogenization were found in the TLRs sequences of this egret.Also,no stop codons,frameshift mutations or deletion/insertion were detected in these sequences.(2)Based on the LRR polymorphic regions of TLR genes,we newly developed 9 pairs of primer for amplifying TLR genes(TLR1LB,TLR2A,TLR3,TLR4,TLR5,TLR7 and TLR15)in the vulnerable Chinese egret.This primer set was also confirmed useful for nine other ardeid species(except for TLR15 in yellow bittern Ixobrychus sinensis),including four members in ardeinae(little egret Egretta garzetta,grey heron Ardea cinerea,Chinese pond-heron Ardeola bacchus,cattle egret Bubulcus ibis)and five species in botaurinae(yellow bittern Ixobrychus sinensis,white eared night-heron Gorsachius magnificus,Schrenck’s bittern Ixobrychus eurhythmus,cinnamon bittern Ixobrychus cinnamomeus,black bittern Dupetor flavicollis).(3)Using the above primers,we amplified the LRR regions of seven TLR genes by PCR in the vulnerable Chinese egret(120 individuals from four populations)and the more common widespread species,little egret(20 individuals from two populations).Comparison results showed that the levels of both SNPs polymorphism and genetic diversity of TLRs in the Chinese egret were lower than those in the little egret.We found 26 SNPs(sSNPs:nsSNPs=16:10)and 45 SNPs(sSNPs:nsSNPs=25:20)in the Chinese egret and little egr et,respectively,and the number of non-synonymous SNPs for each TLR in the Chinese egret was less than that of the little egret.Genetic diversity in the Chinese egret was lower compared to the common little egret in China,except for TLR3,TLR5,and TLR7.Our findings are in accordance with previous reports that threatened avian species have lower genetic diversity.(4)Using analysis of molecular variance(AMOVA)and Bayesian clustering analysis(STRUCTURE),we analyzed the genetic differentiation and structure of four natural breeding populations of the Chinese egret in China.Our results suggested that these populations should be classified as a single adaptive unit(AU)for conservation.Although the AMOVA revealed low but significant genetic differentiation(FST=0.011,p=0.043<0.05),STRUCTURE analysis showed that there was no obvious different geographic structure among the four populations.Considering that the gene may exchange among four populations during their long-distance migration,we recommended that these populations had better to be used as an adaptive unit(AU).(5)Using DataMonkey with three methods SLAC,FUBAR and MEME,we tested the selection(ω=dN/dS)and analyzed the positively selected sites in the Chinese egret.Our results indicated that TLR genes(TLR1LB,TLR2A,TLR3 and TLR15)were under purifying selection(ω<1)except for TLR5(ω=48.60>1).No positively selected sites with satisfied any two models(SLAC,FUBAR,and MEME)was detected in the TLR genes,indicating that most mutations in TLRs during the evolutionary process were harmful.In conclusion,this study successfully identified and isolated seven TLRs family members(TLR1LB,TLR2A,TLR3,TLR4,TLR5,TLR7,and TLR15)in the Chinese egret.Based on these gene structures,we developed 9 pairs of primers for amplifying the LRR regions in extracellular,which were successfully amplified in this egret and other nine ardeid species.We analyzed the genetic diversity and population structure in the Chinese egret,and compared its genetic diversity with little egret.The comparison results showed that the genetic diversity of TLRs in the vulnerable Chinese egret was lower than the little egret,indicating that lower genetic diversity might be one of the reasons caused Chinese egret endangered.Furthermore,we found that there was low but significant genetic differentiation and no obvious different geographic structure among the four Chinese egret populations in China,suggesting these populations should be classified as a single adaptive unit(AU)for conservation.