| Genetic diversity of 20 Apis cerana populations from 18 provinces, cities and municipalities in China were established to DNA GenBank of Apis cerana in China and evaluated with morphology markers, microsatellite markers and mtDNA sequences firstly and roundly. Genetic variability within populations, population structure and the relationship of them were estimated to provide academic reference for farther protection and utilize of Apis cerana in China. The main results were summarized as follows:1.The 6 main indexes of external morphology such as the length and width of the anterior wing, the cubital vein indexes, the length of proboscis and the length of terga 3+4 were detected. There were significant differences (P <0.05 and P <0.01) among 6 characteristics of 20 populations in China. Tibet bee and Diqing bee had differences in one or more characters in length of bee right forewing, width of bee right forewing, area of the anterior wing from other populations, indicating better collecting ability; there were no difference in length of proboscis among Conghua bee, Tonglu bee, Xishuangbanna bee, A-ba bee, Xingcheng bee and Changbaishan bee, and their values were higher than other ones; Changbaishan bee and Tibet bee had the longest length of terga 3+4, which indicated that they had the best honey storing capability. The research result showed that significant difference (P <0.01) existed in the length, width and area of the anterior wing ,the length of proboscis and the length of terga 3+4; there was no difference between the cubital vein indexes and other characters(P>0.05), which indicated that cubital vein indexes was an independent morphological features. The principal component analysis (PCA)of the 6 main indexes of external morphological and 8 ecological features of 20 populations showed that the difference among morphological features of 20 populations were performed by 7 principal components included both morphological features and ecological features, and contribution rate of the two was 89.386%. Not only induviduals'morphological features but also ecological features should be considered in principal component analysis.2. 21 microsatellite markers suited for Apis cerana were selected from 56 microsatellite markers used in Apis mellifera to be used to analyze genetic diversity of 20 Apis cerana populations. 502 alleles were found, and the average value was 24.1430. The average value of gene heterozygosity (He) of 21 microsatellite loci was 0.8689±0.0525, The PIC was 0.8564±0.0603. All 21 microsatellite loci in this study showed high levels of polymorphism. The average value of gene heterozygosity (He) of all populations was 0.8378±0.0136, showed rich genetic diversity and higher selected potentiality. The number of populations deviated from Hardy-Weinberg equilibrium per locus ranged from 6 to 20. The average value of Fst was 42.3% (P <0.001), and all loci were contributed significantly (P <0.001) to this differentiation. There was significant genetic differentiation among 20 populations. Reynolds'distance values varied between 0.020 (Diqing bee-Wuding bee pair) and 1.085 (Beijing bee-Nanchang bee pair). The Nm value was ranged from 0.128 (between Nanchang bee and Beijing bee) to 12.376 (between Diqing bee and Wuding bee). The Nm value between Changbai bee, Beijing bee and the others were low, so they maybe separate populations from the others.3. Seqence of mtDNA tRNAleu-COⅡo f 20 Apis cerana populations were sequenced and analyzed. The result showed the average ratio of A,C,G,T in the sequence of mtDNA COⅡwas 38.2%,10.0%,4.6%,47.1%, respectively. High A+T was contained in the sequence demonstrating the bias of base composition. The average ratio of A,C,G,T of noncoding area was 47.5%,8.3%,5.0%,39.2%. Similarly, High A+T showed the bias of base composition. There were 16 polymorphic sites represent 6.18 % of total analyzed sites in COⅡ, no insertion/deletion were found in this region, the ratio of transition and transversion in this study was 0.07.There were 17 polymorphic sites represent 17.53% of total analyzed sites in noncoding area, contained 2 insertion/deletion, and the ratio of transition and transversion in this study was 0.25. Transitions were only found in Huangshan bee, Hainan bee and Feixian bee. The variability measure of noncoding area of Apis cerana populations was more than COⅡ. 4. 18 haplotypes were found in COⅡsequence,among which 5 haplotypes were shared among some populations, 13 haplotypes were unique for one population. H2 appeared the most times which was the main haplotype of 20 populations. 22 haplotypes were found in noncoding aera,among which 7 haplotypes were shared among some populations, 15 haplotypes were unique for one population. H1 concluded the most individuals, was the main haplotype to test the population of honeybee. The result showed that, Wuding bee and fengxian bee had the most haplotypes in the noncoding area of COⅡ, and all were 4; Changbaishan bee had the fewest, 1 and 2 respectively. The distribution of all haplotypes among the populations was disequilibrium and the diversity of haplotypes in COⅡwas ranged from 0 to 0.778, the average diversity of haplotypes was 0.621±0.038; The diversity of haplotypes in noncoding area was ranged from 0 to 0.778.The average diversity of haplotypes was 0.699±0.035. The average number of nucleotide divergence (K) and average nucleotide diversity (Pi) of COⅡwere 0.939 and 0.851%, respectively. The average number of nucleotide divergence (K) and average nucleotide diversity (Pi) of noncoding area were 1.003 and 1.034%, respectively. Inter-population Nucleotide Divergence (Dxy) of COⅡin 20 Apis cerana populations was ranged from 0%～0.965%, Inter-population Nucleotide Divergence (Dxy) of noncoding area in 20 Apis cerana populations was ranged from 0%～2.062%. The results indicated that the genetic diversity of 20 Apis cerana populations was very abundant.Inter-population Net Nucleotide Divergence (Da) in COⅡin 20 Apis cerana populations was ranged from -0.007%～0.781%, Inter-population Net Nucleotide Divergence (Da) in COⅡin 20 Apis cerana populations was ranged from -0.037%～1.489%. Fst value of mtDNA COⅡand noncoding area of 20 Apis cerana populations was 0.4978 and 0.4332 respectively, which indicated the genetic variation was extremely significant within populations (P < 0.001). There was significant divergence among 20 Apis cerana populations.5. The phylogenetic relationship among 20 Apis cerana populations in China was analyzed with 21 microsatellite loci. The 6 populations from Eastern China fell together in the first place by NJ phylogenetic tree and Sturcture procedure, and the reason may be that Apis cerana from Eastern China had formed the same genetic characters. nshui bee, Hainan bee, Wuding bee), related to their complex genetic basis. Populations from Conghua, Nanning and Fengxian, populations from Tibet and Xishuangbanna had similar genetic basis, and the results suggested that they were affected by the same phylogenetic origin. The genetic diversity of Diqing bee was low, it maybe a separate population, and had great far relationship with Tibet bee. Huangshan bee had two different ecological groups, and had more genetic exchanges. Analysis of haplotypes of mtDNA COⅡand noncoding area among 20 populations estimated with molecular phylogenetic trees and network and molecular phylogenetic trees based on Kimura-2 parameters indicated that there was a complex origin for Apis cerena cerena populations, which was validated by molecular phylogenetic trees of the haplotypes of mtDNA COⅡand noncoding area of Apis cerena both from GENBANK and our research. Analysis of polymorphism and genetic relationship among 20 Apis cerena cerena populations estimated with 21 microsatellite loci and mtDNA sequences indicated that the genetic diversity were occurred as a result of certain geographic populations. Genetic diversity was only difference among different populations, rather than differentiation of the subspecies. So more reasonable measures should be made according to the charaters of different geographical populations to protect and make use of the Apis cerena resource.6. Analysis of genetic distance and geographical distance among 20 Apis cerena cerena populations were estimated with 21 microsatellite loci and mtDNA. The result suggested that the equation Fst/ (1-Fst) = 0.209 +0.084ln (d) and the result from Mantel's test (P=0.055) did not provide enough support for a significant correlation between the genetic and geographical pair wise distances. There were no significant correlations between the genetic diversity of mtDNA D-loop and sequence of noncoding area and the distributing of these populations. The results concluded that the geographical distributing maybe not the determinant influence on the genetic structure of Apis cerena cerena populations during the course of their developed history.
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