Crested Ibis Mitochondrial Genome And Population Genetics Based On Microsatellite Markers

The Crested Ibis(Nipponia nippon),endemic to East Asia,is listed in the IUCN Red List of Threatened Species(EN)and among Level-? Key State-Protected Animals in China.Ever since its rediscovery in 1981,there are many aspects involved in its research,but only a few have dealt with its genetics and genomics.There has been controversy over the taxonomic placement of the Crested Ibis;it was once placed under the order Ciconiiformes,but is now under the order Pelecaniformes.In addition,genetic diversity of a population is fundamental to its long-term survival.A good insight into such information would be important to a successful management of endangered species.However,to date,little is known about the genetic background of wild,re-introduced and captive populations in Shaanxi Province.To gain an insight into the taxomonic status of the Crested Ibis and the genetic diversity of its populations in Shaanxi Province,two major tasks were carried outin the present study.Frirstly,its complete mitochondrial genome has been assembled and characterized by high-throughput sequencing and bioinformatic technology.And Bayesian phylogenetic analysis was conducted to ascertain its taxonomic placement based upon mitochondrial protein-coding genes.Secondly,a novel panel of microsatellite markers was developed,and was utilized to reveal the genetic diversity and structure of wild,re-introduced and captive populations in Shaanxi Province.The results would provide scientific reference for the establishment of effective conservation and management strategies.The major findings of the present study are summarized as follows:(1)The mitochondrial genome of the Crested Ibis is 16,800 bp in length,and harbors 37 genes(incl.13 protein-coding genes/PCGs,22 tRNA and 2 rRNA genes)and a non-coding control region.The A+T content(53.79%)is higher than G+C content(46.21%).PCGs are initiated with four types of start codons(ATA?ATC?ATG>G),with GTG as the start codon for COX1.Besides,there are five types of stop codons(AGA?AGG?TAA?TAG&T),and two PCGs(COX3&ND4)are terminated with the incomplete stop codon T.As found in many other birds and reptiles,the insertion of a single C base is also present at the location of 174th base in the gene ND3.All 22 tRNAs have the typical cloverleaf-like secondary structure.A tandem repeat region consisting of nine‘CAAACAACAAA'motifs is also present near the 3' terminal of the control region.Phylogenetic tree based on the Bayesian analysis of 13 mitochondrial PCGs of 49 bird species added support to the inclusion of Nipponia nippon within the family Threskiorothidae,but failed to support the current taxonomic framework of the order Pelecaniformes.Furthermore,its relationship to the order Suliformes may also need further investigation.(2)Primers were designed for a total of 53 trinucleotide microsatellite loci,and a panel of ten polymorphic loci were characterized from 39 amplifiable primer pairs.A test of the ten loci against 215 DNA samples identified a total of 30 alleles.(3)The ten polymorphic loci were utilized to reveal the population genetics of N.nippon.The mean expected(He)and observed(Ho)heterozygosities are 0.531 and 0.359 at the population level,respectively.Population YX1 possesses the highest He(0.519)and Ho(0.412).Among the re-introduced populations,populations TC and NS1 have the same He(0.474),and that of population QY1 is 0.396.As for the captive populations,the He values of populations ZZ,YX2,QY2 and NS2 are 0.429?0.413?0.389and 0.355,respectively.Hence,the overall genetic diversity is low,and displays the pattern of‘Wild populations>Re-introduced populations>Captive populations'.(4)AMOVA analysis revealed that the majority of genetic variability(83.33%)occurred within populations of the Crested Ibis.The Genetic differentiation coefficient(FST=0.123)also suggests moderate among-population genetic differentiation.Cluster analysis based on principal coordinates analysis(PCoA)revealed no obvious genetic distribution pattern among the eight populations.(5)The expected heterozygosities(He)are higher than the observed heterzygosities(Ho)in all eight populations,with an overall Fis of 0.396(P<0.001),suggesting the occurrence of inbreeding.Measures should be taken to minimize the inbreeding in any future conservation and restoration plans.