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A genetic diversity study of silkworm using cleaved amplified polymorphic sequence (CAPS) markers
Biochemical Systematics and Ecology 34 (2006) 868e874 www.elsevier.com/locate/biochemsyseco
A genetic diversity study of silkworm using cleaved amplified polymorphic sequence (CAPS) markers Jianhua Huang a,b, Muwang Li a,b, Yong Zhang a,b, Wenbin Liu a,b, Minghui Li a,b, Xuexia Miao a, Yongping Huang a,* a
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng Lin Road, Shanghai 200032, PR China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China Received 13 August 2005; accepted 29 June 2006
Abstract The silkworm, Bombyx mori is a beneficial insect of great economic importance in China for its silk production. In this study, we obtained 11 cleaved amplified polymorphic sequence (CAPS) markers and one PCR polymorphism marker from the genes of the silkworm, B. mori. A backcross progeny analysis showed that all these molecular markers were segregated in a Mendelian fashion and that polymorphisms were co-dominant. These markers were used to investigate the genetic diversity among 29 strains of B. mori from China, Japan and Europe. Cluster analysis, based on the genetic similarities calculated from CAPS data, grouped these strains roughly according to their geographical origin. One group contained silkworm strains from Europe and some of the Japanese strains were interspersed into the Chinese groups, whereas other Japanese strains clustered together. Ó 2006 Published by Elsevier Ltd. Keywords: Silkworm; Bombyx mori; CAPS markers; Polymorphism; Genetic diversity; Geographical; Restriction endonuclease
1. Introduction The silkworm, Bombyx mori is of great economic importance in China. About >20 million families in this country raise the silkworm as their economic resource. In addition, the silkworm is one of the best studied lepidopteran insects, including many destructive species for agriculture and forestry, because of the large number of mutants that have been identified (Goldsmith, 1995; Nagaraju and Goldsmith, 2002). This species was domesticated from the wild silkworm, Bombyx mandarina, in China at least 5000 years ago (Xiang, 1995) and then it subsequently spread throughout Asia (e.g. Korea, Japan, and India), and into Europe (France and Italy). At present, silkworm strains have been classified into four main geographical origins: Chinese, Japanese, European and tropical origin (Xia et al., 1998). It is difficult to assume their phylogeny and genetic diversity from their phenotypic traits, though the phenotypic traits of silkworm strains have been noted widely (Tazima, 1964; Doira, 1978; Doria, 1992). In order to better understand the origin and * Corresponding author. Tel.: þ86 21 54924046; fax: þ86 21 54924047. E-mail address: [email protected] (Y. Huang). 0305-1978/$ - see front matter Ó 2006 Published by Elsevier Ltd. doi:10.1016/j.bse.2006.06.011
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the genetic relationships in silkworm, B. mori, it seems ideal to use some kind of valuable markers to study the silkworm strains within different geographical regions. In recent years, DNA-based molecular markers such as random amplified polymorphic DNA (RAPD) (Nagaraja and Nagaraju, 1995; Xia et al., 1998; Lu et al., 2002), amplified fragment length polymorphism (AFLP) (Lu et al., 2001), restriction fragment length polymorphism (RFLP) (Sethuraman et al., 2002), simple sequence repeats (SSR) (Shen et al., 2004; Li et al., 2005) and inter simple sequence repeats (ISSR) (Chatterjee and Mohandas, 2003) have been widely adopted in silkworm genetic diversity studies. In general, these studies provided the positive information to enhance the understanding of silkworm phylogeny. However, until now no attempts have been made to isolate and characterize molecular markers from identified gene sequences, which showed much slower evolution speed than those DNA-based molecular markers. Cleaved amplified polymorphic sequence (CAPS) markers are based on PCR amplification of known genes and cDNA sequences. PCR products are digested by restriction enzymes, generating a simple type of data coded as heterozygote or homozygote (Konieczny and Ausubel, 1993). CAPS markers have been used to study genetic diversity in some species of plants such as Arabidopsis thaliana (Hardtke et al., 1996; Barth et al., 2002), Camellia sinensis (Kaundun and Matsumoto, 2003), and Cryptomeria japonica (Tsumura et al., 1999). In this report, CAPS markers from silkworm gene sequences were developed for determining the phylogeny and genetic diversity of 29 strains of domesticated silkworm strains from different geographical origins. 2. Materials and methods 2.1. Animal material Two Chinese silkworm strains, C108 as female and Dazao as male, and their F1 generation, were used to select the CAPS markers. A single pair mating backcross between female C108 F1 (C108 Dazao) was raised to analyze the genetic mode of inheritance of these markers. Both the parental silkworms and the collection of 29 silkworm strains were obtained from the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (CASS), Zhengjiang, Jiangsu Province, China. This collection represents major genetic strains of silkworms in cultures of Chinese, Japanese and European geographical origins. 2.2. Primer design Eleven primer pairs based on known silkworm genes in GenBank (Table 1) were designed with the software Primer Premier 5. All primers were synthesized in Bioasia Company (Shanghai, China). 2.3. DNA isolation and CAPS marker search DNA was isolated from whole silk glands using a slightly modified phenolechloroform extraction (Yasukochi, 1998). The chloroformeisoamyl extraction step was repeated twice in order to purify the DNA. CAPS reactions were modified slightly using the protocol of Konieczny and Ausubel (1993). Each PCR reaction mix (30 ml) for the CAPS marker search used 15 ng DNA in 1 PCR buffer, 0.2 mM dNTP each, 10 pM forward and reverse primers each, and 1 unit Taq DNA polymerase (Dingguo, Beijing, China). Each amplified reaction (3 ml) for Dazao, C108 and their F1 generation was digested with restriction enzymes according to manufacturer’s instructions. Forty different restriction endonucleases (Takara, Tokyo, Japan) were tested, namely, AlwI, AatII, AfaI, AluI, ApaI, BamHI, BclI, BglI, BglII, Cfr13I, DraI, DdeI, EcoRI, EcoRV, FokI, HhaI, HindIII, HaeIII, HincII, KpnI, MboI, MluI, MspI, MscI, NdeI, NsiI, NcoI, NheI, PstI, PvuII, PshBI, PvuI, SalI, ScaI, SacII, SspI, SacI, TaqI, XbaI and XhoI, and the enzyme digestion products were separated on 2% TAE agarose gels for CAPS marker assay. 2.4. Genetic analysis For genetic analysis using CAPS markers, 11 geneeenzyme combinations and one PCR marker were used (Table 2). Each silkworm strain DNA pool for genetic analysis was constructed from the silk glands of 20 individuals.
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Table 1 Primers used to develop CAPS markers and their corresponding gene function with GenBank accession numbers No.
Primer sequence (forward and reverse)
Gene function
Accession number
CAPS01
50 -TCAATCGTATGTCCATCCAGA-30 50 -TCCAACTTACCTACTTTCACCC-30 50 -ATTACTCCAAATCTCCTGTTCAA-30 50 -GTTACTAAAGTCGTTTTCGTGTCTA-30 50 -ACGGTGCTTTGGTATGTGG-30 50 -TCGAACAGGTCGTGATAGAAGT-30 50 -TTGGCAAAGGAGGTCGG-30 50 -TCAAACTGTTCTAATCGGGG-30 50 -AAGAGGGCACTAAGAACAGGG-30 50 -CCAATAACAGGCATCTACGCTA-30 50 -CAACGATGTCGGGAGGAT-30 50 -CTCCAGTGTTTCATTCCAGTCT-30 50 -GCAGTATCATCTCACCCTTCTATC-30 50 -CCTCCAAATCCTCCGCTAT-30 50 -CACTTGATATTTCGCATCCTAA-30 50 -CAGTCTCACAACACGCACG-30 50 -TTTGTCTTTTAGCCTTAGCGA-30 50 -TGGACTGTTTTCACGTGTTTC-30 50 -ATCCTATGGTGCTGTTCGTCA-30 50 -GAGTGTTCGTTGTATTTGCTTTTA-30 50 -TGGACAGTGGCACCTTTCT-30 50 -GGAGCAGCCTCATCTTAACAT-30
Bombyx mori Sui1 (sui1) mRNA
AY426343
Bombyx mori ADP/ATP translocase mRNA
AY227000
Bombyx mori CP8 precursor, mRNA
AY387408
Bombyx mori glucosidase mRNA
AY272037
Bombyx mori pheromone gland-specific acyl-CoA desaturase mRNA Bombyx mori mRNA for ribosome-associated protein P40 Bombyx mori CB10 mRNA for wing disc-specific protein Bombyx mori PRE S42032 embryonal lethal (2)13-1 Bombyx mori cubitus interruptus gene
AF182405
Bombyx mori mRNA for prophenoloxidase
D49370
Bombyx mori mRNA for Boceropsin
AB064496
CAPS02 CAPS03 CAPS04 CAPS05 CAPS06 CAPS07 CAPS08 CAPS09 CAPS10 CAPS11
AB062685 AB062684 wdS00443 AF529422
PCR reaction products (3 ml) were separated on 1% TAE agarose gels for detection, whereas enzyme-digested PCR products were separated on 2% TAE agarose gels. For these CAPS markers, the banding pattern was very stable, and three types of pattern were seen: one band (homozygote), two bands (homozygote) or three bands (heterozygote). Therefore, CAPS markers were scored for banding patterns corresponding to homozygotes or heterozygotes (AA ¼ homozygotes without an enzyme site; BB ¼ homozygotes with an enzyme site for both alleles; AB ¼ heterozygotes). 2.5. Data analysis Popgene v1.32 (Yeh et al., 1997; available at http://www.ualberta.ca/wfyeh/index.htm) was used to apportion diversity among the 29 silkworm strains. The markers were scored as diploid data. The average polymorphic Table 2 Geneeenzyme combinations for 11 CAPS markers and one PCR marker used in silkworm diversity study, their number of banding patterns, observed heterozygosity (Ho), and polymorphic information content (PIC) values Markers
Banding pattern
Ho
PIC
CAPS01/MboI CAPS02/SacII CAPS03/HhaI CAPS04/XhoI CAPS05/SspI CAPS06/BamHI CAPS07/BclI CAPS08/MscI CAPS09/BclI CAPS09/MluI CAPS10/NsiI CAPS11/PCR
3 3 3 3 3 3 3 3 3 3 3 2
0.2143 0.2759 0.2069 0.1379 0.4643 0.2759 0.4483 0.8621 0.2759 0.4483 0.8667 0.3103
0.6024 0.4588 0.4418 0.5047 0.6595 0.4588 0.6887 0.6853 0.6531 0.6912 0.6870 0.6927
Total: 35
Mean: 0.3938
Mean: 0.6020
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informationP content (PIC) P was P calculated for each CAPS marker according to the equation from Smith et al. (1997), PIC ¼ 1 ni¼1 p2i ni¼1 nj¼iþ1 2p2i p2j , where p is the relative frequency of the jth pattern for CAPS marker i. Pairwise genetic P similarities were P calculated for each marker system using the similarity coefficient of HS ¼ ð1=kÞ ks¼1 HSs ¼ ð1=kÞ ks¼1 ½1 q2s ð1 qs Þ2 (Nei and Li, 1979), where k is the total number of loci, and qs is the frequency of one of the two alleles at the sth diallelic locus. Associations among the 29 strains were analyzed using cluster analyses from the CAPS raw data, applying the unweighed pair-group method with arithmetic averages (UPGMA) based on genetic similarities’ estimates (Sneath and Sokal, 1973). 3. Results 3.1. CAPS markers and their characterization The primer pairs designed from 11 cloned genes with known function (Table 1) were used to amplify fragments of DNA from Dazao, C108 and their F1 generation. Forty kinds of restriction endonucleases were employed to find nucleotide polymorphisms. The enzyme site appearing only once in the predicted amplified sequences were selected, which facilitated band identification and analysis. As a result, we found 11 CAPS and one PCR polymorphism marker between Dazao and C108 silkworm strains in these 11 silkworm genes (Table 2). In addition, two enzyme sites in the gene sequence CAPS09 were found to be polymorphic, which we considered as two CAPS markers. In order to test the inheritance pattern of these CAPS markers, we examined 190 BC1 offspring (obtained using an F1 male, from a single pair mating of a C108 female with a Dazao male, to mate a C108 female). In general, the alleles segregated in a Mendelian fashion at all informative loci and were consistent with the parental genotypes. The segregation patterns for the 12 polymorphic markers did not differ significantly from 1:1 (Chi square, p > 0.05, all tests) (Table 3). These results confirmed that the selected CAPS markers behaved as simple Mendelian markers and therefore could be used to determine variation in genetic diversity among different silkworm strains. 3.2. Genetic diversity of the silkworm determined by CAPS markers After these markers were amplified among the 29 silkworm strains (four strains from Europe, five strains from Japan and the others from China), each pair of primers produced a single PCR product except the PCR polymorphism marker. Each fragment-enzyme combination that was polymorphic in the 29 silkworm strains had only two alleles, corresponding to the presence or absence of a single restriction site (Fig. 1A). The results for PCR polymorphism were shown in Fig. 1B. Table 3 Segregation of polymorphic alleles in 190 progenies from the backcross of female C108 (C108 Dazao) Markers
Parent genotypes C108 (C108 Dazao)
Genotypic frequencies
Chi square value
Significance* ( p ¼ 0.05)
CAPS01/MboI CAPS02/SacII CAPS03/HhaI CAPS04/XhoI CAPS05/SspI CAPS06/BamHI CAPS07/BclI CAPS08/MscI CAPS09/BclI CAPS09/MluI CAPS10/NsiI CAPS11/PCRa
BB (AA BB) BB (AA BB) BB (AA BB) AA (BB AA) BB (AA BB) AA (BB AA) BB (AA BB) BB (AA BB) AA (BB AA) AA (BB AA) AA (BB AA) AA (BB AA)
BB/AB 102/88 BB/AB 105/85 BB/AB 99/91 AA/AB 90/100 BB/AB 93/97 AA/AB 84/106 BB/AB 97/93 BB/AB 97/93 AA/AB 88/102 AA/AB 89/101 AA/AB 93/97 AA/AB 98/92
1.03 2.11 0.34 0.51 0.08 2.46 0.08 0.08 1.03 0.76 0.08 0.19
NS NS NS NS NS NS NS NS NS NS NS NS
Homozygote for the absence of the site (AA), homozygote for a polymorphic restriction site (BB). *NS: not significant. a AA represents as the longer homozygote, BB represents as the shorter one.
872
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Fig. 1. (A) CAPS markers used in the 29 silkworm strains, which was designated as AA, BB and AB corresponding to the presence or absence of a single restriction site. Homozygote for the absence of the site (AA), homozygote for a polymorphic restriction site (BB) and a heterozygote state (AB) with only one of the two alleles being cut. (B) PCR polymorphism marker results, AA represents the longer homozygote gene sequence, BB represents the shorter homozygote gene sequence, while AB means heterozygotes.
The genetic analysis was based on 35 banding patterns derived from 11 geneeenzyme combinations and one PCR marker. PIC (polymorphic information content) values ranged from 0.4418 to 0.6927 with an average of 0.6020. The observed heterozygosity ranged from 0.1379 to 0.8667 with an average of 0.3938, suggesting a high degree of variation in these loci (Table 2). Using these data from the 11 CAPS and one PCR markers, genetic similarity between all pairs of lines ranged from 0.3722 to 0.9444. In the UPGMA dendrogram, one group contained silkworm strains all from Europe, while some of the Japanese strains were interspersed into the Chinese groups and other Japanese strains clustered together (Fig. 2). The monovoltine strains and bivoltine strains were also distinguished well in these groups. The distinctness of the different groups was possibly due to their geographical origin. 4. Discussion Differences in the distribution of polymorphic alleles among silkworm strains have already been reported for fibroin (Ueda et al., 1984), PTTH (Shimada et al., 1994) and diapause hormone genes (Pinyarat et al., 1995).
Fig. 2. UPGMA clustering of silkworm, Bombyx mori ecotypes based on genetic similarity calculated from these CAPS markers. Chinese strains, C; Japanese strains, -; Europe strains, :. The monovoltine and bivoltine strains have been mentioned in this dendrogram.
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PCR-RFLP analysis has also been identified for silkworm mutations in DNA base pairs that control the presence or absence of the restriction enzyme (Yasukochi, 1999). It is because of the natural selection that allows some genes to mutate freely in some strains while the others retain the original DNA sequences to tackle with the changes of environments (Andolfatto, 2001). The insertion or deletion of gene fragments and the nucleotide mutation indicate the basic characteristics of an organism. The analysis of the difference of these genes will offer the information of the closely related strains. In the present study, we developed CAPS markers from 11 silkworm gene sequences between Dazao and C108. Using these markers, we further analyzed 29 silkworm strains with different geographical distribution. The silkworm strains could be clustered into different groups according to the geographic areas initially observed. Our results also indicated that the Chinese monovoltine strains seem to be differentiated from B. mandarina M. in the earliest year, while the appearance of bivoltine strains is after the monovoltine strains during the evolution, which agrees with the previous conclusions (Xia et al., 1998; Lu et al., 2001). Our conclusion also accorded with the hypothesis that the Chinese silkworm monovoltine strains are the only original center (Yoshitake, 1967; Jiang, 1982; Jiang and Xiang, 1986) (Fig. 2). The voltinism seems to be related to the silkworm’s geographical origin. In summary, our data suggest that CAPS marker is highly reproducible, rapid, and the data obtained are relatively easy to interpret and score. Now a web-based program for generating CAPS markers has been established (Michaels and Amasino, 1998). The most important and preponderant factor is that CAPS markers are derived from known gene sequences, which will not only help us to finish the measurement of genetic diversity, but also lead to the direct mapping of these genes. We draw a conclusion that CAPS markers for diversity analysis in the silkworm, B. mori, will be more efficiency and will group these strains more ideally if the selected CAPS markers can ascertain an even representation of evolution related gene sequences. Acknowledgments The authors sincerely thank Dr. S.C. Levings, Prof. M.R. Goldsmith and Prof. X.X. Zhu for their comments on earlier drafts of this manuscript. This research was sponsored by grants from National Basic Research project (973) (2005CB121000) and NSFC (30471310). References Andolfatto, P., 2001. Adaptive hitchhiking effects on genome variability. Curr. Opin. Genet. Dev. 11, 635e641. Barth, S., Melchinger, A.E., Lubberstedt, T., 2002. Genetic diversity in Arabidopsis thaliana L. 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A genetic diversity study of silkworm using cleaved amplified polymorphic sequence (CAPS) markers Jianhua Huang a,b, Muwang Li a,b, Yong Zhang a,b, Wenbin Liu a,b, Minghui Li a,b, Xuexia Miao a, Yongping Huang a,* a
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng Lin Road, Shanghai 200032, PR China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China Received 13 August 2005; accepted 29 June 2006
Abstract The silkworm, Bombyx mori is a beneficial insect of great economic importance in China for its silk production. In this study, we obtained 11 cleaved amplified polymorphic sequence (CAPS) markers and one PCR polymorphism marker from the genes of the silkworm, B. mori. A backcross progeny analysis showed that all these molecular markers were segregated in a Mendelian fashion and that polymorphisms were co-dominant. These markers were used to investigate the genetic diversity among 29 strains of B. mori from China, Japan and Europe. Cluster analysis, based on the genetic similarities calculated from CAPS data, grouped these strains roughly according to their geographical origin. One group contained silkworm strains from Europe and some of the Japanese strains were interspersed into the Chinese groups, whereas other Japanese strains clustered together. Ó 2006 Published by Elsevier Ltd. Keywords: Silkworm; Bombyx mori; CAPS markers; Polymorphism; Genetic diversity; Geographical; Restriction endonuclease
1. Introduction The silkworm, Bombyx mori is of great economic importance in China. About >20 million families in this country raise the silkworm as their economic resource. In addition, the silkworm is one of the best studied lepidopteran insects, including many destructive species for agriculture and forestry, because of the large number of mutants that have been identified (Goldsmith, 1995; Nagaraju and Goldsmith, 2002). This species was domesticated from the wild silkworm, Bombyx mandarina, in China at least 5000 years ago (Xiang, 1995) and then it subsequently spread throughout Asia (e.g. Korea, Japan, and India), and into Europe (France and Italy). At present, silkworm strains have been classified into four main geographical origins: Chinese, Japanese, European and tropical origin (Xia et al., 1998). It is difficult to assume their phylogeny and genetic diversity from their phenotypic traits, though the phenotypic traits of silkworm strains have been noted widely (Tazima, 1964; Doira, 1978; Doria, 1992). In order to better understand the origin and * Corresponding author. Tel.: þ86 21 54924046; fax: þ86 21 54924047. E-mail address: [email protected] (Y. Huang). 0305-1978/$ - see front matter Ó 2006 Published by Elsevier Ltd. doi:10.1016/j.bse.2006.06.011
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the genetic relationships in silkworm, B. mori, it seems ideal to use some kind of valuable markers to study the silkworm strains within different geographical regions. In recent years, DNA-based molecular markers such as random amplified polymorphic DNA (RAPD) (Nagaraja and Nagaraju, 1995; Xia et al., 1998; Lu et al., 2002), amplified fragment length polymorphism (AFLP) (Lu et al., 2001), restriction fragment length polymorphism (RFLP) (Sethuraman et al., 2002), simple sequence repeats (SSR) (Shen et al., 2004; Li et al., 2005) and inter simple sequence repeats (ISSR) (Chatterjee and Mohandas, 2003) have been widely adopted in silkworm genetic diversity studies. In general, these studies provided the positive information to enhance the understanding of silkworm phylogeny. However, until now no attempts have been made to isolate and characterize molecular markers from identified gene sequences, which showed much slower evolution speed than those DNA-based molecular markers. Cleaved amplified polymorphic sequence (CAPS) markers are based on PCR amplification of known genes and cDNA sequences. PCR products are digested by restriction enzymes, generating a simple type of data coded as heterozygote or homozygote (Konieczny and Ausubel, 1993). CAPS markers have been used to study genetic diversity in some species of plants such as Arabidopsis thaliana (Hardtke et al., 1996; Barth et al., 2002), Camellia sinensis (Kaundun and Matsumoto, 2003), and Cryptomeria japonica (Tsumura et al., 1999). In this report, CAPS markers from silkworm gene sequences were developed for determining the phylogeny and genetic diversity of 29 strains of domesticated silkworm strains from different geographical origins. 2. Materials and methods 2.1. Animal material Two Chinese silkworm strains, C108 as female and Dazao as male, and their F1 generation, were used to select the CAPS markers. A single pair mating backcross between female C108 F1 (C108 Dazao) was raised to analyze the genetic mode of inheritance of these markers. Both the parental silkworms and the collection of 29 silkworm strains were obtained from the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (CASS), Zhengjiang, Jiangsu Province, China. This collection represents major genetic strains of silkworms in cultures of Chinese, Japanese and European geographical origins. 2.2. Primer design Eleven primer pairs based on known silkworm genes in GenBank (Table 1) were designed with the software Primer Premier 5. All primers were synthesized in Bioasia Company (Shanghai, China). 2.3. DNA isolation and CAPS marker search DNA was isolated from whole silk glands using a slightly modified phenolechloroform extraction (Yasukochi, 1998). The chloroformeisoamyl extraction step was repeated twice in order to purify the DNA. CAPS reactions were modified slightly using the protocol of Konieczny and Ausubel (1993). Each PCR reaction mix (30 ml) for the CAPS marker search used 15 ng DNA in 1 PCR buffer, 0.2 mM dNTP each, 10 pM forward and reverse primers each, and 1 unit Taq DNA polymerase (Dingguo, Beijing, China). Each amplified reaction (3 ml) for Dazao, C108 and their F1 generation was digested with restriction enzymes according to manufacturer’s instructions. Forty different restriction endonucleases (Takara, Tokyo, Japan) were tested, namely, AlwI, AatII, AfaI, AluI, ApaI, BamHI, BclI, BglI, BglII, Cfr13I, DraI, DdeI, EcoRI, EcoRV, FokI, HhaI, HindIII, HaeIII, HincII, KpnI, MboI, MluI, MspI, MscI, NdeI, NsiI, NcoI, NheI, PstI, PvuII, PshBI, PvuI, SalI, ScaI, SacII, SspI, SacI, TaqI, XbaI and XhoI, and the enzyme digestion products were separated on 2% TAE agarose gels for CAPS marker assay. 2.4. Genetic analysis For genetic analysis using CAPS markers, 11 geneeenzyme combinations and one PCR marker were used (Table 2). Each silkworm strain DNA pool for genetic analysis was constructed from the silk glands of 20 individuals.
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Table 1 Primers used to develop CAPS markers and their corresponding gene function with GenBank accession numbers No.
Primer sequence (forward and reverse)
Gene function
Accession number
CAPS01
50 -TCAATCGTATGTCCATCCAGA-30 50 -TCCAACTTACCTACTTTCACCC-30 50 -ATTACTCCAAATCTCCTGTTCAA-30 50 -GTTACTAAAGTCGTTTTCGTGTCTA-30 50 -ACGGTGCTTTGGTATGTGG-30 50 -TCGAACAGGTCGTGATAGAAGT-30 50 -TTGGCAAAGGAGGTCGG-30 50 -TCAAACTGTTCTAATCGGGG-30 50 -AAGAGGGCACTAAGAACAGGG-30 50 -CCAATAACAGGCATCTACGCTA-30 50 -CAACGATGTCGGGAGGAT-30 50 -CTCCAGTGTTTCATTCCAGTCT-30 50 -GCAGTATCATCTCACCCTTCTATC-30 50 -CCTCCAAATCCTCCGCTAT-30 50 -CACTTGATATTTCGCATCCTAA-30 50 -CAGTCTCACAACACGCACG-30 50 -TTTGTCTTTTAGCCTTAGCGA-30 50 -TGGACTGTTTTCACGTGTTTC-30 50 -ATCCTATGGTGCTGTTCGTCA-30 50 -GAGTGTTCGTTGTATTTGCTTTTA-30 50 -TGGACAGTGGCACCTTTCT-30 50 -GGAGCAGCCTCATCTTAACAT-30
Bombyx mori Sui1 (sui1) mRNA
AY426343
Bombyx mori ADP/ATP translocase mRNA
AY227000
Bombyx mori CP8 precursor, mRNA
AY387408
Bombyx mori glucosidase mRNA
AY272037
Bombyx mori pheromone gland-specific acyl-CoA desaturase mRNA Bombyx mori mRNA for ribosome-associated protein P40 Bombyx mori CB10 mRNA for wing disc-specific protein Bombyx mori PRE S42032 embryonal lethal (2)13-1 Bombyx mori cubitus interruptus gene
AF182405
Bombyx mori mRNA for prophenoloxidase
D49370
Bombyx mori mRNA for Boceropsin
AB064496
CAPS02 CAPS03 CAPS04 CAPS05 CAPS06 CAPS07 CAPS08 CAPS09 CAPS10 CAPS11
AB062685 AB062684 wdS00443 AF529422
PCR reaction products (3 ml) were separated on 1% TAE agarose gels for detection, whereas enzyme-digested PCR products were separated on 2% TAE agarose gels. For these CAPS markers, the banding pattern was very stable, and three types of pattern were seen: one band (homozygote), two bands (homozygote) or three bands (heterozygote). Therefore, CAPS markers were scored for banding patterns corresponding to homozygotes or heterozygotes (AA ¼ homozygotes without an enzyme site; BB ¼ homozygotes with an enzyme site for both alleles; AB ¼ heterozygotes). 2.5. Data analysis Popgene v1.32 (Yeh et al., 1997; available at http://www.ualberta.ca/wfyeh/index.htm) was used to apportion diversity among the 29 silkworm strains. The markers were scored as diploid data. The average polymorphic Table 2 Geneeenzyme combinations for 11 CAPS markers and one PCR marker used in silkworm diversity study, their number of banding patterns, observed heterozygosity (Ho), and polymorphic information content (PIC) values Markers
Banding pattern
Ho
PIC
CAPS01/MboI CAPS02/SacII CAPS03/HhaI CAPS04/XhoI CAPS05/SspI CAPS06/BamHI CAPS07/BclI CAPS08/MscI CAPS09/BclI CAPS09/MluI CAPS10/NsiI CAPS11/PCR
3 3 3 3 3 3 3 3 3 3 3 2
0.2143 0.2759 0.2069 0.1379 0.4643 0.2759 0.4483 0.8621 0.2759 0.4483 0.8667 0.3103
0.6024 0.4588 0.4418 0.5047 0.6595 0.4588 0.6887 0.6853 0.6531 0.6912 0.6870 0.6927
Total: 35
Mean: 0.3938
Mean: 0.6020
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informationP content (PIC) P was P calculated for each CAPS marker according to the equation from Smith et al. (1997), PIC ¼ 1 ni¼1 p2i ni¼1 nj¼iþ1 2p2i p2j , where p is the relative frequency of the jth pattern for CAPS marker i. Pairwise genetic P similarities were P calculated for each marker system using the similarity coefficient of HS ¼ ð1=kÞ ks¼1 HSs ¼ ð1=kÞ ks¼1 ½1 q2s ð1 qs Þ2 (Nei and Li, 1979), where k is the total number of loci, and qs is the frequency of one of the two alleles at the sth diallelic locus. Associations among the 29 strains were analyzed using cluster analyses from the CAPS raw data, applying the unweighed pair-group method with arithmetic averages (UPGMA) based on genetic similarities’ estimates (Sneath and Sokal, 1973). 3. Results 3.1. CAPS markers and their characterization The primer pairs designed from 11 cloned genes with known function (Table 1) were used to amplify fragments of DNA from Dazao, C108 and their F1 generation. Forty kinds of restriction endonucleases were employed to find nucleotide polymorphisms. The enzyme site appearing only once in the predicted amplified sequences were selected, which facilitated band identification and analysis. As a result, we found 11 CAPS and one PCR polymorphism marker between Dazao and C108 silkworm strains in these 11 silkworm genes (Table 2). In addition, two enzyme sites in the gene sequence CAPS09 were found to be polymorphic, which we considered as two CAPS markers. In order to test the inheritance pattern of these CAPS markers, we examined 190 BC1 offspring (obtained using an F1 male, from a single pair mating of a C108 female with a Dazao male, to mate a C108 female). In general, the alleles segregated in a Mendelian fashion at all informative loci and were consistent with the parental genotypes. The segregation patterns for the 12 polymorphic markers did not differ significantly from 1:1 (Chi square, p > 0.05, all tests) (Table 3). These results confirmed that the selected CAPS markers behaved as simple Mendelian markers and therefore could be used to determine variation in genetic diversity among different silkworm strains. 3.2. Genetic diversity of the silkworm determined by CAPS markers After these markers were amplified among the 29 silkworm strains (four strains from Europe, five strains from Japan and the others from China), each pair of primers produced a single PCR product except the PCR polymorphism marker. Each fragment-enzyme combination that was polymorphic in the 29 silkworm strains had only two alleles, corresponding to the presence or absence of a single restriction site (Fig. 1A). The results for PCR polymorphism were shown in Fig. 1B. Table 3 Segregation of polymorphic alleles in 190 progenies from the backcross of female C108 (C108 Dazao) Markers
Parent genotypes C108 (C108 Dazao)
Genotypic frequencies
Chi square value
Significance* ( p ¼ 0.05)
CAPS01/MboI CAPS02/SacII CAPS03/HhaI CAPS04/XhoI CAPS05/SspI CAPS06/BamHI CAPS07/BclI CAPS08/MscI CAPS09/BclI CAPS09/MluI CAPS10/NsiI CAPS11/PCRa
BB (AA BB) BB (AA BB) BB (AA BB) AA (BB AA) BB (AA BB) AA (BB AA) BB (AA BB) BB (AA BB) AA (BB AA) AA (BB AA) AA (BB AA) AA (BB AA)
BB/AB 102/88 BB/AB 105/85 BB/AB 99/91 AA/AB 90/100 BB/AB 93/97 AA/AB 84/106 BB/AB 97/93 BB/AB 97/93 AA/AB 88/102 AA/AB 89/101 AA/AB 93/97 AA/AB 98/92
1.03 2.11 0.34 0.51 0.08 2.46 0.08 0.08 1.03 0.76 0.08 0.19
NS NS NS NS NS NS NS NS NS NS NS NS
Homozygote for the absence of the site (AA), homozygote for a polymorphic restriction site (BB). *NS: not significant. a AA represents as the longer homozygote, BB represents as the shorter one.
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Fig. 1. (A) CAPS markers used in the 29 silkworm strains, which was designated as AA, BB and AB corresponding to the presence or absence of a single restriction site. Homozygote for the absence of the site (AA), homozygote for a polymorphic restriction site (BB) and a heterozygote state (AB) with only one of the two alleles being cut. (B) PCR polymorphism marker results, AA represents the longer homozygote gene sequence, BB represents the shorter homozygote gene sequence, while AB means heterozygotes.
The genetic analysis was based on 35 banding patterns derived from 11 geneeenzyme combinations and one PCR marker. PIC (polymorphic information content) values ranged from 0.4418 to 0.6927 with an average of 0.6020. The observed heterozygosity ranged from 0.1379 to 0.8667 with an average of 0.3938, suggesting a high degree of variation in these loci (Table 2). Using these data from the 11 CAPS and one PCR markers, genetic similarity between all pairs of lines ranged from 0.3722 to 0.9444. In the UPGMA dendrogram, one group contained silkworm strains all from Europe, while some of the Japanese strains were interspersed into the Chinese groups and other Japanese strains clustered together (Fig. 2). The monovoltine strains and bivoltine strains were also distinguished well in these groups. The distinctness of the different groups was possibly due to their geographical origin. 4. Discussion Differences in the distribution of polymorphic alleles among silkworm strains have already been reported for fibroin (Ueda et al., 1984), PTTH (Shimada et al., 1994) and diapause hormone genes (Pinyarat et al., 1995).
Fig. 2. UPGMA clustering of silkworm, Bombyx mori ecotypes based on genetic similarity calculated from these CAPS markers. Chinese strains, C; Japanese strains, -; Europe strains, :. The monovoltine and bivoltine strains have been mentioned in this dendrogram.
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PCR-RFLP analysis has also been identified for silkworm mutations in DNA base pairs that control the presence or absence of the restriction enzyme (Yasukochi, 1999). It is because of the natural selection that allows some genes to mutate freely in some strains while the others retain the original DNA sequences to tackle with the changes of environments (Andolfatto, 2001). The insertion or deletion of gene fragments and the nucleotide mutation indicate the basic characteristics of an organism. The analysis of the difference of these genes will offer the information of the closely related strains. In the present study, we developed CAPS markers from 11 silkworm gene sequences between Dazao and C108. Using these markers, we further analyzed 29 silkworm strains with different geographical distribution. The silkworm strains could be clustered into different groups according to the geographic areas initially observed. Our results also indicated that the Chinese monovoltine strains seem to be differentiated from B. mandarina M. in the earliest year, while the appearance of bivoltine strains is after the monovoltine strains during the evolution, which agrees with the previous conclusions (Xia et al., 1998; Lu et al., 2001). Our conclusion also accorded with the hypothesis that the Chinese silkworm monovoltine strains are the only original center (Yoshitake, 1967; Jiang, 1982; Jiang and Xiang, 1986) (Fig. 2). The voltinism seems to be related to the silkworm’s geographical origin. In summary, our data suggest that CAPS marker is highly reproducible, rapid, and the data obtained are relatively easy to interpret and score. Now a web-based program for generating CAPS markers has been established (Michaels and Amasino, 1998). 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