Molecular Genetic Analysis And Management Strategies Of Black Stork In Captivity

The black stork（Ciconia nigra）is a large and migratory wading bird with a wide distribution.Black stork is an important endangered species,and only 1000 individuals are estimated to exist in China.At present,a stable artificial breeding population has been established in China’s zoos,which has become the living gene bank of this species.In order to better manage the captive black stork population,the genetic diversity of the black stork population was studied usingmitochondrial D-Loop region and microsatellites.Male and female black storks are identical in appearance,thus it is difficult to distinguish their sex by morphology.This study used molecular biology methods to identify the sex of black storks.A pair of primers,CHD-1F/CHD-1R of the CHD gene,and two pairs of primers,AWS05/NRD4 and SINT-F/SINT-R of the EE0.6 gene were used to identify their sex.The identification results of the two groups of primers were the same.The results showed that the sex ratio of the population was 1:1.When we amplified the D-Loop region of the black stork population,we found that the population had nuclear gene copying,and the length of the copied fragment was between 3400 and 4200 bp.This insertion is a neutral mutation and has no adverse effects.We analyzed the sequence of the mitochondrial D-Loop region of the black storks,and obtained 10 haplotypes and 23 polymorphic sites.The haplotype diversity（Hd）was 0.768,and the nucleotide diversity（π）was 0.00744.The average number of nucleotide difference（k）was 4.504.The results of the neutrality test showed that the population had an insignificant pressure of negative selection.In the analysis of the haplotypes,these 6 haplotypes only had one sample of the 10 haplotypes.In order to effectively manage genetic diversity,we should pay special attention to these 6 haplotypes,otherwise it is easy to lose genetic diversity.Using the haplotype to calculate the genetic distance and construct a phylogenetic tree,the genetic distance between the 10 haplotypes was 0.0033-0.0168,and the overall genetic distance was 0.0101.The ML tree and NJ tree were constructed for the haplotypes.The results of the two tree constructions were similar.The 10 haplotypes were divided into two branches,and the two branches were divided into several small lineages.Regarding the attribution of Hap₂,the NJ tree and the MJ tree were different,and the confidence of the two classification methods was low,so we separated Hap₂ alone.In this study,30 pairs of primers with good amplification and polymorphic microsatellite loci were selected from 29 loci.A total of 18 primers were successfully amplified,1 of which（Cc01）was difficult to genotype.10 loci namely Cc05,Cc06,WSμ13,WSμ14,Cbo102,Cbo121,Cbo133,Cbo151,Cbo168 and Ah208 were monomorphic,3 loci namely Cc03,Cc37 and Cc42 showed only 2 alleles each,and four loci namely Cc02,Cc04,Cc07,Cc10 were all highly polymorphic,with 4 to 10 alleles per locus.Polymorphic information content（PIC）ranged from 0.497 to 0.784,and all loci were at Hardy-Weinberg equilibrium（P=0.088～0.601）.The four loci are applicable to population genetic analysis and genetic management of the black stork.We constructed NJ trees using these six loci,and divided the population into three branches.The distribution of individuals within the branches was uneven,indicating that the genetic relationship of the population was relatively close.These results suggest that the success rate of cross-amplification of microsatellite is associated with the closeness of species;the closer the species,the higher the success rate.However,polymorphism is not mostlyrelated to the species closeness,but it is determined by evolutionary history of species.Finally,based on the genetic polymorphism of the population,we have made some recommendations for their genetic management.