| The honeybee is an important economic insect and have an unique ability of pollination, which plays an important role in improving the quality of fruit, enhanced pollination progeny seed vitality and resistance to pests and adverse environmental.Nowadays, nine kinds of bees have been recognized, planning an indispensable role in ecological balance and biodiversity. In recent years, with the development of science and technology, micro-satellite has become the second-generation molecular markers,which was widely applied on bee DNA fingerprinting and species identification, disease detection, germplasm conservation and utilization, marker-assisted breeding and pedigree analysis, gene mapping and QTLs analysis, linkage analysis and genetic map.The effective microsatellite marker have been selected in Apis mellifera and used to build genetic map or analyze the genetic diversity. However, the effective microsatellite molecular markers has few report based on the genome of Apis cerana. As published the whole genome sequence of Apis cerana(Korea strains), providing a good opportunity to research the microsatellite. In this study, the genome-wide microsatellite were identified based on the whole genome sequence of A. cerana and the comparison were performed with A. mellifera. The primer used to molecular marker was screened through PCR and polyacrylamide gelelectrophoresis, which were applied to the research of A. cerana diversity. The main result were as follows:1. There were 209991 SSRs through the identification in the genome of A. cerana Korea strains. The number of single nucleotide type SSR > dinucleotide>trinucleotide > tetranucleotide> pentanucleofide> hexanucleotides. Through the comparison between A. cerana and A. mellifera, the total number of SSRs in A.mellifera is larger than that in A. cerana, which is the same sitution in the 5 ’UTR, CDS,and intron. And the SSRs density in gene region in A. mellifera is larger than that in A.cerana, expecting the CDS region. The gene containing the SSR in 5 ’UTR and CDS region were annotated with GO database, finding that the gene of annotated Protein tag and Locomotion contained the SSR in 5 ’UTR in A. cerana, but not found in A.mellifera. In the CDS region gene containing the SSR, comments for the death, and antioxdant translation regulator gene found only in A.cerana. The comments for synapse, synapse part, virion, virion part of the growth and gene exists only in A.mellifera.2. The SSR were identified from the EST data in six tissue of A. cerana and three tissue of A. mellifera. In A. cerana, 24975 SSRs were identified in antennae, 32847 SSRs were identified in brain, 13038 SSRs were identified in hypopharyneal, 47013 SSRs were identified in venom, 71641 SSRs were identified in fatbody, 70043 SSRs were identified in gut. In A. mellifera, 59912 SSRs were identified in antennae, 36977 SSRs were identified in brain, 13241 SSRs were identified in hypopharyneal. The number of SSR identified in EST in A. mellifera is larger than that in A. cerana. The number of five nucleotide and six nucleotide SSR type in A. mellifera, such as AAAAG/CTTTT, ACGAG/CGTCT and AAATAT/ATATTT, is larger than that in A.cerana. Then, the Blast2 go software was used to annotate the gene contained SSR. The function of gene contained the AAAAG/CTTTT and ACGAG/CGTC microsatellite types is consistent between A. cerana and A. mellifera. The genes contained AAATAT/ATATTT microsatellite type were annotated the serine threonine protein kinase in A. mellifera, while those gene were annotated the unknown function protein in A. cerana.3. Through the identification and comparison analysis microsatellites between A.cerana and A. mellifera, 17000 specific primers were designed by QDD software. Then,the product of electronic PCR were used to compare with the homology sequence in A.cerana Thailand and Japan strains. The sequence of insertions and deletions were evaluated and 218 microsatellite primers with polymorphism were selected. By gathering the A. cerana sample of 139 colonies in Chongqing, 5 microsatellite primers with polymorphism were successfully screened through PCR, which may provide the potential molecular marker to research the genetic diversity for Chongqing... |
