| The sex determination of honey bee depends on its ploidy. In the colony, the queen and worker bee are female and diploid, while drone bee is male and haploid. The complementary sex determination (csd) gene is critical in the sex determination of honey bee, that is, bees with heterozygous csd gene will develop into female, while those with both homozygous, and haploid csd gene will develop into male. The present study analyzed the polymorphism of csd gene in different strains of Apis cerana cerana, multiple subspecies of Apis mellifera, Apis dorsata and Apis florea, as well as an Apis mellifera ligustica strain with high production of royal jelly. The results are as follows:1.Polymorphism Analysis of csd gene in five populations of Chinese honeybee, Apis cerana cerana (Hymenoptera:Apidae)Worker bees collected from Changbaishan (Jilin), Haikou (Hainan), Nanning (Guangxi), Shennongjia (Hubei) and Jingan (Jiangxi) were used as materials; genome DNA was extracted from each sample for PCR amplification of the csd region 3, and PCR products were cloned and 1-3 clones were sequenced, and then polymorphism of csd gene in different geographical populations were analyzed by using the obtained sequences. A total of 131 sequences were obtained representing 69 alleles, and 9 alleles were shared by two populations.The phylogenetic analysis showed that all the haplotypes only form two clade (TypeⅠand TypeⅡ) in the tree. Haplotypes from these five geographical populations are mixed on the phylogenetic tree, not form 5 clades according to different geographical origins. Polymorphism analysis showed that the polymorphism level of typeⅠhaplotypes (π=0.06724) is significant higher than that of typeⅡ(π=0.01477)We further analyzed the polymorphism of TypeⅠhaplotypes in five population. The polymorphisms of Changbaishan (Jilin), Jingan (Jiangxi), Nanning, Haikou and Shennongjia populations are 0.09425,0.09987,0.05096,0.04612,0.03588 in turn. Changbaishan (Jilin) and Jingan (Jiangxi) populations are significantly higher than those of Nanning, Haikou and Shennongjia populations, while polymorphism of csd between Changbaishan (Jilin) and Jingan (Jiangxi) populations, and that among Nanning (Guangxi), Haikou (Hainan) and Shennongjia (Hubei) populations showed no significant difference.We adopted Dxy to measure the population polymorphism of csd gene. The Dxy of five populations is 0.03977～0.09208. Dxy between Changbaishan (Jilin) and Jingan (Jiangxi) populations is maximal (0.09208), while that between Shennongjia (Hubei) and Nanning (Guangxi) populations are minimal (0.03977).An UPGMA tree was constructed based on the Kimura’s 2-parameter genetic distance, the Shennongjia and Nanning populations form a clade at first, then they gathered with Haikou, Changbaishan, Jingan populations in turn.2. Polymorphism analysis of csd gene in six Apis mellifera subspeciesWe analyzed the polymorphism of csd gene in six A. mellifera subspecies, including Apis mellifera anatolica, Apis mellifera caucasica, Apis mellifera carnica, Apis mellifera carpatica, Apis mellifera ssp., Apis mellifera ligustica. Genome DNA was extracted from each sample bees for PCR amplification of the csd region 3; PCR products were cloned; and were sequenced.We obtained 6,10,19,14,28 and 7 haplotypes from A. mellifera subspecies, they are A. m. anatolica, A. m. caucasica, A. m. carnica, A. m. carpatica, A. m. ssp. and A. m. ligustica, respectively. We determined the exons, introns and coding sequences of these haplotypes. Similar to that in A. mellifera, A. cerana and A. dorsata, the coding region of this part also contains an RS domain at the N terminal, a P-rich domain at the C terminal and a hyper variable region between these two domains. The hyper variable region is rich of asparagine (N) and tyrosine (Y), and they form a basic (N)1-4 Y repeats terminated mainly with KK, KQ in each haplotypes.The csd gene has a high level of polymorphism in all the six subspecies. Theπvalue of the A. m. anatolica subspecies is the highest among the six subspecies. It is significantly higher than that of the A. m. ssp. subspecies, but failed rather to be significant compared withπvalues of the other four subspecies. While except for the A. m. anatolica subspecies, there is no significant difference inπvalues among the other five subspecies.The pairwise Fst values between different subspecies are from 0.02570 to 0.23848, of them, Fst value between A. m. anatolica and A. m. carnica is the highest, while that between A. m. carpatica and A. m. caucasica is the lowest. From the Fst values, it indicated that the genetic difference levels between A. m. caucasica and all other five subspecies are very low. The genetic difference between A. m. carpatica and A. m. ligustica, A. m. anatolica and A. m. carpatica are also not significant. Except for these subspecies pairs, the remaining pairs showed significant genetic difference.The kimura’s 2-parameter genetic distance between different subspecies are from 0.04216 to 0.06415. Of them, the genetic distance between A. m. anatolica and A. m. carnica is the largest, while that between A. m. ssp. and A. m. caucasica is the nearest.An UPGMA tree was constructed based on the Kimura’s 2-parameter genetic distance, the A. m. caucasica and A. m. ssp form a clade at first, and then A. m. carnica, A. m. carpatica, A. m. ligustica and A. m. anatolica gathered with them in turn.3.Polymorphism analysis of csd gene in Apis floreaA. florea samples were collected from Wuming County, Guangxi Province, China. Genome DNA was extracted from each sample for PCR amplification of the csd region 3; PCR products were cloned; and sequenced.From the 60 A. florea workers, we obtained a total of 54 sequences representing 37 alleles. A genealogy tree was constructed based on these alleles, all the alleles fall into three clades, they are typeⅠ, typeⅡand typeⅢ. Both typeⅡand typeⅢalleles showed extremely low diversity, while theπvalue of typeⅠwas more than 10 times higher than those of typeⅡand typeⅢ. We speculate the typeⅡand typeⅢalleles might be from other genes. Therefore, further analysis we used only 12 typeⅠalleles since they are most likely the actual csd gene.We compared the nucleotide diversity (π) of csd coding region among four different Apis species, including A. florae, A. dorsata, A. cerana, A. mellifera. Theπvalue of A. florea csd gene is significantly higher than those of the other three species.We determined the exons, introns and coding regions of type I haplotypes. Similar to A. mellifera, A. cerana and A. dorsata, the coding region of this part also contains an RS domain at the N terminal and a P-rich domain at the C terminal, with a hypervariable region between these two domains. The hyper variable region is rich of asparagine (N) and tyrosine (Y), and they form a basic (N)1-4Y repeats in each allele, and about half of the alleles have another (KHYN)1-4 repeats following the (N)1-4Y repeats. The (N)1-4Y and (KHYN)1-4 repeats are two important motifs found in the hypervariable region.We analyzed the relationship between the number of nonsynonymous changes per synonymous site (dN) and the syonnymous changes per synonymous site (dS). For most of the pairs, dN is higher than dS. The average dN/dS ratio of all allele pairs is 1.28. Moreover, for newly diverged alleles, the regression line is above the dN/dS=1 ratio, whereas for anciently diverged alleles, and the regression line drops below dN/dS=1. These results indicate that nonsynonymous mutation are selectively favored in young alleles.4. Polymorphism analysis of csd gene in Apis dorsataA. dorsata samples were collected from Chongzuo county, Guangxi province, and Qiongzhong county, Hainan province, China. Genome DNA was extracted from each sample for PCR amplification of the csd region 3, PCR products were cloned and sequenced.We obtained 34 and 24 different alleles from region 3 of the csd gene in Guangxi and Hainan populations, respectively. There ais a total of 52 haplotypes,6 haplotypes were shared by both Guangxi and Hainan populations. The nucleotide diversity (π) values of csd in Guangxi and Hainan populations were 0.04889±0.00404 and 0.03832±0.00541, respectively, without significant difference between these two populationsME phylogenic tree showed csd alleles do not form two branches reflecting the Guangxi and Hainan samples. Rather, the two groups are well mixed among each other.We then constructed another tree using ME method, using all the available csd data for A. dorsata, including these obtained in the current study, and that from Cho et al (2006) and Hasselmann et al (2008). Again, it shows very little or no clustering according to geographical origins of each csd allele.The Fst distance between the Hainan and Guangxi A. dorsata populations was 5.32%, that is indicating a moderate level of genetic differentiation. There is also a high gene flow value (Nm=6.73) between these two populations, suggesting frequent exchange of genetic material between these two populations in history. All these results demonstrated that the genetic differentiation between these two populations was very low.5. Polymorphism analysis of csd gene in Apis mellifera ligustica with high production of royal jellyAnalysis of csd gene polymorphism was carried out for artificial breeding honeybee with high yield royal jelly, and Apis mellifera with low yield royal jelly, csd gene polymorphism has been observed. Samples were collected from Jianshan of Zhejiang Province, China. Genome DNA was extracted from each sample for PCR amplification of the csd region 3, PCR products were cloned and sequenced.The nucleotide diversity (π) values of csd in Apis mellifera and Apis mellifera ligustica with high production of royal jelly populations were 0.04380±0.00575 and 0.05785±0.00492 respectively. Z test showed that there is a significant difference between theπvalues of these two populations.When all the alleles from these two populations were used to construct a phylogenic tree by using the ME method, csd alleles do not form two branches reflecting the two samples. Rather, they are well mixed among each otherThe Fst distance between Apis mellifera and Apis mellifera ligustica with high production of royal jelly populations was 3.69%, indicating a weak genetic differentiation between these two strains. |
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