Indian Academy of Sciences
Molecular phylogeny of the domesticated silkworm, Bombyx mori, based on the sequences of mitochondrial cytochrome b genes AILING LI2,3,4, QIAOLING ZHAO2, SHUNMING TANG2, ZHIFANG ZHANG1,2*, SHENYUAN PAN3 and GUIFANG SHEN1 1
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100 081, China 2 The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212 018, Jiangsu Province, China 3 College of Life Sciences, Xuzhou Normal University, Xuzhou 221 116, China 4 Present address: Shanxi Institute of Education, Xi’an 710 061, China
Abstract Pupae from the Chinese wild mulberry silkworm, Bombyx mandarina, and 11 representative strains of the domesticated silkworm, Bombyx mori were selected for preparation of mitochondrial DNA. The 5′-end fragments of cytochrome b genes (Cytb) were generated by polymerase chain reaction products and sequenced directly. The homologous sequences of the Japanese B. mandarina and three strains of B. mori were from the GenBank database. The sequences of the 16 silkworm strains were analysed with DNASTAR software and a phylogenic tree was constructed using PHYLIP software. The result showed that: (i) The sequence divergence between the strains of B. mori and the Japanese B. mandarina was larger (5.4% ~ 5.8%) compared with that between strains of B. mori and the Chinese B. mandarina (0.8% ~ 1.9%). Analysis of clustering also showed that the sequences of B. mori strains and Chinese B. mandarina clustered into group (B group), while that of Japanese B. mandarina (A group) was outside this cluster. This may be evidence for the hypothesis that B. mori originated from Chinese B. mandarina. (ii) Among 14 strains of B. mori, sequence divergence was small and the most divergence was seen between strains Yanhe-1 and Chuxiong, whose sequences branched off from those of the other B. mori strains on the phylogenetic tree. From this and from historical records, we infer that the strains Yanhe-1 and Chuxiong originated independently from southwest China. [Li A, Zhao Q, Tang S, Zhang Z, Pan S and Shen G 2005 Molecular phylogeny of the domesticated silkworm, Bombyx mori, based on the sequences of mitochondrial cytochrome b genes. J. Genet. 84, 137–142]
Introduction The metazoan mitochondrial genome is a circular, doublestranded DNA molecule, which is known to have small molecular weight, rapid nucleotide mutation and rare recombination, and can be easily handled in a laboratory compared to nuclear DNA (Kim et al. 1998). Because of their structural and evolutional characteristics, mitochon-
*For correspondence. E-mail: [email protected]
drial DNA (mtDNA) sequences have been widely used as molecular markers in the study of molecular evolution in the past several decades (Wolstenholme and Clary 1985; Sankoff et al. 1992; Kim et al. 2000; Yagi et al. 2001). The cytochrome b gene (Cytb) is one of the most important protein-encoding genes on the heavy strand of mtDNA molecule and has been utilised in the studies of molecular evolution and classification of species (Johns and Avise 1998; Tsuchiya et al. 2000; Farias et al. 2001; Sakai et al. 2003). For example, sequence divergence in hymenoptera is associated with adenine
Bombyx mori; Bombyx mandarina; mitochondrial DNA; cytochrome b gene; phylogenetic tree. Journal of Genetics, Vol. 84, No. 2, August 2005
Ailing Li et al. and thymine (A+T) content bases based on comparison of the Cytb gene sequences from Apis mellifera, Drosophila melanogaster, D. yakuba and Ant tetraponera rufoniger (Jermiin and Crozier 1994). Archaeological records and genetics, immunology and biochemistry support the hypothesis that the present silkworm species was domesticated from the ancient wild mulberry silkworm. There are mostly two popular viewpoints on the origin of B. mori and its evolution. One hypothesis is of a multiphyletic origin that B. mori were domesticated independently in different areas and periods based on the studies of philology, archeological records and historic evidences (Jiang 1982). Another is of a monophyletic origin that all geographical strains of B. mori evolved from monovoltine and originated in one area based on the polymorphisms of haemolymph genes and isoenzyme (Yoshitake 1965; Yoshitake 1967; Gamo and Ohtsuka 1980). We analysed the sequence divergence and phylogenetic relationship based on Cytb 5′-end sequences (591 bp) of 14 representative strains of B. mori and two strains of B. mandarina. We discuss here the evolutionary relationship among strains of B. mori and B. mandarina.
Materials and methods Experimental materials
The 11 strains of B. mori used in the study were from the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (SRI) and the Chinese B. mandarina (n = 28) was from the mulberry field in the SRI in April 2003 (table 1). The Taq DNA polymerase was from Invitrogen, the GENECLEAN Kit was from Bio101 Inc.
Preparation of mtDNA
Mitochondrial genomic DNA was extracted from silkworm pupae of the fourth day with the method as described previously (Tamura and Aotsuka 1988; Shen et al. 2000). DNA extracts were ascertained by agarose-gel electrophoresis, respectively. PCR amplification and sequencing
Polymerase chain reaction (PCR) primers for generation of CytB genes were designed as follows based on the sequence of B. mori strain C108 (GenBank accession number: AB070264): primer-H 5299 5′-tat gga cca tta cga tca tca a-3′; primer-L 8917 5′-tgg tac ttt acc tcg tta tcg t-3′, in which the letters H and L refer to the heavy strand and light strand, respectively, and the number refers to the position of the 5′-end base of the primer in the nucleotide sequence of C108. The primers were synthesized by Shanghai Sangon Biological Engineering Technology and Service Company. Using the mtDNA of each silkworm strains as the template, respectively, the PCR amplifications were carried out in a 50 µl volume under the following conditions: denatured for 3 ~ 5 min at 96°C, followed by 35 cycles of 45 s at 94°C, 60 s at 55°C, 90 s at 72°C, finally elongated for 10 min at 72°C. The PCR products were identified by agarose-gel electrophoresis and purified with the GENECLEAN Kit (Ellington 1998). Then the purified products were sequenced directly by TaKaRa Biotechnology (Dalian) Co., Ltd. Sequence divergence and phylogenetic analysis
The partial sequences of CytB were compared using the EditSeq and MegAlign subprograms of DNASTAR software package (Burland 2000). Then the sequences were aligned and saved as *.PHY document by CLUSTALX 1.81 software package (Jeanmougin et al. 1998). The
Brief information on the strains of B. mori and the Chinese B. mandarina sequenced.
Name of strain Yanhe-1 Chuxiong Yanji Deqing-1 Gansu Mysore Yanjinhuang Daisou Japan120 France408-translucent
Bagdad Chinese B. mandarina
Volting and molting V 1 M3 V 2 M4 V 1 M4 V 2 M4 V 1 M3 V 3 M4 V 3 M4 V 2 M4 V 2 M4 V 1 M4 V 1 M4
Cocoon color light yellow gold yellow white white nacarat white gold yellow white white white white light yellow
System or Source
GenBank accession number
Guizhou province, China Yunnan province, China Jilin province, China Zhejiang province, China Gansu province, China India Vietnam Japan Japan Europe Europe Zhenjiang, China
AY496257 AY496252 AY343547 AY496254 AY496255 AY342386 AY496258 AY496253 AY496256 AY342385 AY496251 AY343548
Note: “V1” denotes univoltine, “V2” denotes bivoltine and “V3” denotes multivoltine. “M3” denotes trimoulter and “M4” denotes tetramoulter.
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Molecular phylogeny of the domesticated silkworm phylogenetic tree was constructed by neighbour-joining (NJ) method; Genetic distances were calculated using Kimura 2-parameter method; Statistical support for nodes on the tree was evaluated using bootstrapping (1000 replications) with PHYLIP software package (Felsenstein 2002). The phylogenetic tree was read by TREEVIEW software package (Page 2000).
Results Nucleotide sequence analysis
By sequencing the PCR products, the 12 5′-end sequences of CytB (591 bp) were obtained, including that of the Chinese B. mandarina and those of 11 strains of B. mori (table 1). The sequences have been submitted to the GenBank database and their accession numbers are listed in table 1. The other 4 homologous sequences were obtained from the GenBank database, including that of the Japanese B. mandarina (NC_003395), C108 (AB070264), Aojuku (AB083339) and Backokjam (NC_002355). All
of these 16 sequences were analysed for sequence divergence and phylogenetic relationship. The 5′-end sequences of CytB (591 bp) started from the initiation codon “ata” and no termination codon appeared. The percentage of A+T content was higher than that of G+C (table 2). Multiple alignments of the sequences (figure 1) revealed that neither deletions nor insertions occurred. But there were total 53 base substitutions with 39 (73.58%) occurring at the third positions of codons and 44 of which (83.02%) were transitions. Most transitions (77.27%) were T-C transitions (table 2). In the 53 substitutions, 41 (77.36%) didn’t change the encoded amino acids, that is, most substitutions were synonymous mutations. The significance test showed that the difference of the average rate of base substitution between the strains of B. mori and the Japanese B. mandarina was significant (χ 2df=1 = 4.97, P < 0.05), whereas there was no significant difference between the strains of B. mori and the Chinese B. mandarina (χ 2df=1 = 0.36, P > 0.05).
Table 2. Nucleotide average content of the stains of B. mori and frequency distribution of nucleotide substitutions of CytB segment sequences (591 bp). First position of codon
Second position of codon
Third position of codon
34 (77.27%) 10 (22.73%)
A-T A-C G-T
1 0 1
2 0 1
1 3 0
4 (44.45%) 3 (33.33%) 2 (22.22%)
A+T (content) G+C (content)
140 (71.07%) 57 (28.93%)
126 (63.96%) 71 (36.04%)
170 (86.29%) 27 (13.71%)
436 (73.77%) 155 (26.23%)
Table 3. Percentage of the nucleotide difference (%) (above the diagonal) and the Kimura 2-parameter distances (below the diagonal) of CytB segment sequences. Name of strain 1 2 3 4 5 6 7 8 9
Daisou Yanji Yanjinhuang Gansu Deqing-1 Chuxiong Yanhe-1 Chinese B. mandarina Japanese B. mandarina
— 0.0017 0.0034 0.0017 0.0068 0.0102 0.0137 0.0085 0.0470
0.2 — 0.0017 0.0000 0.0051 0.0085 0.0119 0.0068 0.0452
0.3 0.2 — 0.0017 0.0068 0.0102 0.0137 0.0085 0.0470
0.3 0.2 0.3 — 0.0051 0.0085 0.0119 0.0068 0.0452
0.3 0.2 0.3 0.3 — 0.0034 0.0171 0.0119 0.0505
1.0 0.8 1.0 1.0 0.7 — 0.0171 0.0119 0.0505
1.2 1.0 1.2 1.2 1.2 1.2 — 0.0188 0.0471
1.0 0.8 1.0 1.0 1.0 1.9 1.9 — 0.0381
5.6 5.4 5.6 5.6 5.6 5.8 5.6 5.2 —
Note: The sequences of 7 strains of B. mori, including Aojuku, Japan120, Mysore, France408-translucent, Backokjam, C108 and Yanji, are identical, so only that of Yanji is listed. The sequences of Deqing-1 and Bagdad are identical, so only that of Deqing-1 is listed. Journal of Genetics, Vol. 84, No. 2, August 2005
Ailing Li et al.
Figure 1. Multiple alignment of CytB segment sequences. Note: The sequences of 7 strains of B. mori, including Aojuku, Japan120, Mysore, France408-translucent, Backokjam, C108 and Yanji, are identical, so only that of Yanji is listed. The mark “-” denotes the identical base compared with the sequence of Yanji.
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Molecular phylogeny of the domesticated silkworm Sequence divergence
The sequence comparison (table 3) showed that the divergence between the strains of B. mori and the Japanese B. mandarina (5.4% ~ 5.8%) was larger than that between the strains of B. mori and the Chinese B. mandarina (0.8% ~ 1.9%). The sequences of the 7 strains of B. mori including Aojuku, Japan120, Mysore, France408-translucent, Backokjam, C108 and Yanji, were identical. The sequences of the other 5 strains of B. mori including Deqing-1, Bagdad, Daisou, Gansu and Yanjinhuang showed one base difference (0.2%) compared to the other 7 sequences, respectively. The sequences of Deqing-1 and Bagdad were also identical. The sequence of Yanhe-1 showed the maximum divergence compared with that of the other strains of B. mori (1.0% ~ 1.2%), and that of Chuxiong was the second (0.7% ~ 1.2%) strand to show divergence. Phylogenetic relationship
The phylogenetic tree consisted of two major groups. The sequence of the Japanese B. mandarina branched off independently (A group). And the sequences of the strains of B. mori and the Chinese B. mandarina formed another group (B group), which consisted of three subgroups: the first one contained the sequences of 12 strains of B. mori (B-1 subgroup), the second one included that of the other 2 strains of B. mori Yanhe-1 and Chuxiong (B-2 subgroup), and the third one was that of the Chinese B. mandarina (B-3 subgroup) (figure 2).
Discussion Relative relationship between B. mori and B. mandarina
The domestication history of B. mori is about 5000 years
Figure 2. NJ tree based on CytB segment sequences. Note: The sequence of Samia cynthia ricina was used as outgroup; Numbers at the nodes indicate bootstrap values (1000 replicates). The sequences of 7 strains of B. mori, including Aojuku, Japan120, Mysore, France408-translucent, Backokjam, C108 and Yanji, are identical, so only that of Yanji is listed; The sequences of Deqing-1 and Bagdad are identical, so only that of Deqing-1 is listed.
old (SRI 1987; Zhu 1992). It was suggested that B. mori was separated from the Japanese B. mandarina about 7.1 million years ago based on the comparison of nad 5 sequence of C108 with that of the Japanese B. mandarina (Yukuhiro et al. 2002). The two results show that the relative relationship between the B. mori and the Japanese B. mandarina is a little remote. Our results from sequence divergence, significance test of base substitution rate and phylogenetic analysis also suggested that the relative relationship between the B. mori and the Chinese B. mandarina is closer than that between the B. mori and the Japanese B. mandarina. It provides the evidence for the hypothesis that B. mori originated from Chinese B. mandarina (Yoshitake 1965; Jiang 1982) at the molecular level. Evolution analysis of B. mori strains
The sequences of 14 strains of B. mori formed two major subgroups (B-1 and B-2 subgroups) on the phylogenetic tree. In the following analysis, we will treat them separately. According to history recordation, sericicultural technology spread to Korea in the 10th century B.C., to Japan in the 2nd century A.D., to India in the 4th ~ 5th century A.D. and to Arabia in the 6th century A.D. (Jiang 1982). The 12 strains of B. mori (B-1 subgroup) are widely geographically distributed, not only in East and North China, but also in Europe, India, Japan etc. and the sequence divergence among them is small. It is interesting that the sequences of Yanhe-1 and Chuxiong shaped clearly a subgroup (B-2 subgroup) on the phylogenetic tree. We deduced the domestication of the strains of B. mori Yanhe-1 and Chuxiong as an independent event. The reasons are as the follows: (i) Yanhe-1 and Chuxiong are geographic strains collected from the countryside of Guizhou and Yunnan provinces during the 80s ~ 90s of the 20th century, respectively. Yunnan and Guizhou provinces were not entirely ruled by central government until Yuan Dynasty (A.D. 1271) (Xn et al. 1985), so the areas had little communication with other parts of China and the probability of introduced strains of B. mori was small at that time; (ii) As described in the last paragraph, strains of B. mori were spread to other countries around 1500 ~ 2000 years ago, but only a small sequence divergence (0 ~ 0.2%) occurred. Yanhe-1 and Chuxiong showed a large sequence divergence with other strains of B. mori (0.7% ~ 1.2%), while the domestication history of B. mori is only about 5000 years, so Yanhe-1 and Chuxiong were not likely introduced from other areas; (iii) On the basis of literature recorded, there were two minority tribes in southwest China. One was “Bulangmi” and the other was “Ersu”. They reared silkworm mostly for harvesting the pupae as food, while the aim for introduction of strains of B. mori is filature generally. So Yanhe-1 and Chuxiong were not likely introduced from
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Ailing Li et al. other areas. Taken together, the strains of B. mori Yanhe1 and Chuxiong originated independently from Guizhou and Yunnan provinces of the southwest China. It provided the evidence for the hypothesis of a multiphyletic origin at the molecular level. Our results not only supported that the evolution of B. mori, but also reflected some interesting events. More experimental data and more extensive study will provide better a understanding of this issue. Acknowledgements This work was supported by National Natural Science Foundation Grant of China (No: 3 0 2 7 1 0 0 7 ) .
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Received 23 February 2004; in revised form 23 August 2004
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