Preparation Of The Peripheral Blood Cells Chromosome Slides And Mapping Of Polymorphic Microsatellite Loci On Chromosomes In Chinese Alligator

The Chinese alligator(Alligator sinensis)is an endangered freshwater crocodilian species endemic to China.This species is listed as critically endangered by IUCN.With the efforts of scientific personnel,the number of Chinese alligator population in the reserve is increasing.However,there are few studies on the structure and function of its chromosomes and the mapping of molecular markers.The morphological structures of the chromosomes possess unique information of species.Molecular markers have also been applied to many biological fields for their unique advantages.Our research includes two parts.The first part is the preparation of metaphase chromosome specimens from peripheral blood cells of Chinese alligator.Based on the materials,there are four main methods to prepare the metaphase chromosome specimens.We chose the method of utilizing peripheral blood to produce metaphase chromosome specimens because this method can minimize the damage to the research object and produce more mitotic phases.It provides a practical method for the future research of chromosomes of Chinese alligator.Moreover,in the second part,we mapped the polymorphic microsatellite loci on the chromosomes of Chinese alligator.Microsatellites are a class of highly polymorphic genetic markers.Mapping of the microsatellite loci is a method that can contribute to the selection of genetic markers with no significant linkage and the construction of the precise genetic linkage map of Chinese alligator.All blood samples of Chinese alligator used in this research were drawn from Changxing Yinjiabian Chinese alligator Nature Reserve.We utilized peripheral blood to produce metaphase chromosome specimens.On this basis,Giemsa staining of chromosomes was performed by trypsinase treatment.Using the ImageJ software,we measured and analyzed the five pairs of large chromosomes of Chinese alligator.Based on the well-splitting slides,the fluorescence in situ hybridization method was used to locate the microsatellite loci on the Chinese alligator chromosomes.The main findings are as follows:(1)Chromosome slides were prepared from peripheral blood cells of Chinese alligator,and 200 metaphase chromosome slides with well-dispersed and complete chromosomes were obtained.(2)The arm ratio and relative length data of five pairs of large chromosomes of Chinese alligator were obtained.It was found that all of them belonged to metacentric chromosomes.(3)By fluorescence in situ hybridization,the microsatellite locus CXA-138 of Chinese alligator was located on a pair of homologous chromosomes.In this study,a large number of metaphase chromosome slides were prepared using peripheral blood cells,and mapping of the microsatellite loci on the Chinese alligator chromosomes were achieved successfully using fluorescence in situ hybridization.It lays the foundation for the research on chromosomes and gene mapping of the Chinese alligator.