American Journal of Botany: e123–e126. 2011.
AJB Primer Notes & Protocols in the Plant Sciences
CHLOROPLAST MICROSATELLITE MARKERS IN LIRIODENDRON TULIPIFERA (MAGNOLIACEAE) AND CROSS-SPECIES AMPLIFICATION IN L. CHINENSE1 Ai-Hong Yang2,3, Jin-Ju Zhang4, Xiao-Hong Yao2, and Hong-Wen Huang5,6 2Key
Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, Hubei, China; 3Graduate School of the Chinese Academy of Sciences, Beijing 100039, China; 4College of Life Sciences, Jiangxi Normal University, Nanchang 330022, Jiangxi, China; and 5Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, Guangdong, China
• Premise of the study: A set of cpSSR markers were developed for the tree genus Liriodendron L. to investigate population genetic structure and phylogeographic history. • Methods and Results: Primers were designed directly from the chloroplast genome sequences of Liriodendron tulipifera. Among the 55 cpSSR markers tested, 11 polymorphic markers were identified in L. tulipifera. The number of alleles in the population tested ranged from two to five, and the unbiased haploid diversity per locus ranged from 0.074 to 0.644. Eighteen primer pairs generated polymorphic amplification in L. chinense. The number of alleles per locus ranged from two to seven, and the unbiased haploid diversity per locus was from 0.250 to 0.964. • Conclusions: cpSSR markers developed here will be useful for phylogeography and population genetics studies of Liriodendron. Key words: cpSSR; Liriodendron chinense; Liriodendron tulipifera; phylogeography; population genetics.
Trees in the genus Liriodendron L. (Magnoliaceae) were once widely distributed in the Northern Hemisphere and comprised several species (Latham and Ricklefs, 1993). However, extinctions caused by Late Tertiary climate oscillations and the Pleistocene glaciations left only two species: L. tulipifera L. and L. chinense (Hemsl.) Sarg., which display an eastern Northern American and eastern Asian disjunction found in many temperate zone tree genera (Xiang et al., 2000). Liriodendron tulipifera is a fast-growing timber tree that is widespread and common in eastern Northern American broad-leaf forests, while L. chinense is an endangered species that displays similar ecological traits, but occurs in small and isolated populations in southern China and northern Vietnam (Hao et al., 1995). Despite the significant value of L. tulipifera and the endangered status of L. chinense, information on their population genetic structure and geographic variation is limited (Sewell et al., 1996) and could be enhanced with the development of reliable and polymorphic molecular markers. The available chloroplast genome sequences of L. tulipifera (Cai et al., 2006) offer potential sources for the development of chloroplast microsatellite 1 Manuscript received 27 December 2010; revision accepted 12 January 2011.
The authors thank Christopher Dick for helpful discussions and polishing English. This work was partly supported by the KIP Pilot Project of the Chinese Academy of Sciences (KSCX2-EW-Q-16) and the Foundation of the Director of the Wuhan Botanical Garden, Chinese Academy of Sciences (O754581A09). 6 Author for correspondence:
[email protected]. doi:10.3732/ajb.1000532
(cpSSR) markers, which are widely used in population genetic and evolutionary studies of plants (Provan et al., 2001). Here we report on a set of cpSSRs derived from the chloroplast genome of L. tulipifera and evaluate their transferability in L. chinense. METHODS AND RESULTS The cpSSRs were screened from the chloroplast genome sequences of L. tulipifera using WebSat (http://wsmartins.net/websat). The screening criteria were set for detection of mono, di-, and trinucleotide motifs with a minimum of ten, six, and five repeats, respectively. A total of 64 simple cpSSRs were yielded, including one trinucleotide repeat, three dinucleotide repeats, and 60 mononucleotide repeats. Nine cpSSRs contained low GC content in the flanking region and were excluded from primer design. Locus-specific primers were designed for the remaining 55 cpSSRs using the PRIMER 3 web interface program (http://frodo.wi.mit.edu/primer3/). To assess polymorphism, genomic DNA was extracted from young leaves of eight individuals of L. tulipifera using the CTAB method (Doyle and Doyle, 1987). Polymerase chain reactions (PCRs) were performed in a 10-µL reaction solution containing 40 ng genomic DNA, 10 mM Tris–HCl (pH 8.4), 50 mM (NH4)2SO4, 2.0 mM MgCl2, 0.2 mM dNTPs, 0.25 µM each primer, and 0.5 unit Taq polymerase (Fermentas, Vilnius, Lithuania). The amplification protocol used an initial denaturing at 94°C for 4 min, followed by 30 cycles of 45 s at 94°C, 45 s at the appropriate annealing temperature (see Table 1 for details) and 1 min at 72°C, ending with a final extension at 72°C for 8 min. Amplified products were separated on 6% denaturing polyacrylamide gels and visualized by silver staining. A 25-bp DNA ladder was used to identify alleles. Of the 55 cpSSR markers tested, 37 generated successful amplification products, and 11 of these loci were polymorphic. The genetic variability of the 11 polymorphic markers was estimated by genotyping 27 individuals randomly sampled from a seedling population of L. tulipifera (Jurong, Jiangsu, China, 32°06′36″N, 119°13′12″E) introduced from the United States. Population genetic parameters were calculated using GenAlEx 6.1 software
American Journal of Botany: e123–e126, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America
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e124 Table 1.
Characteristics of 37 chloroplast microsatellite primers developed in L. tulipifera. Primer sequence (5′–3′)
Locus Lcp1 Lcp2 Lcp3 Lcp4 Lcp5 Lcp6 Lcp7 Lcp10 Lcp12 Lcp13 Lcp15 Lcp16 Lcp17 Lcp18 Lcp19 Lcp21 Lcp22 Lcp23 Lcp24 Lcp25 Lcp26 Lcp28 Lcp30 Lcp31 Lcp33 Lcp35 Lcp37 Lcp39 Lcp43 Lcp44 Lcp46 Lcp47 Lcp48 Lcp49
[Vol. 0
American Journal of Botany
F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R:
TGCCAATCCTGTAAATTGGA GGGTATTGATCCGTTCGATT TCCATGTCAACCAATATCAACA ATCCGACTAGTTCCGGGTTC CCGATCGCTCTTTTGACTTT AGGGTTCTCCTGTGAGTGGA CCAATGGTATGGACGAATCC GGGTAGGGCAATCCAATCTC GAGATTGGATTGCCCTACCC AATGATAGGTTCGCCGGATT AACCACATATGGGCATGGTT ATTTTGCATCAGGCTCCAAT TGTCGACTCAGAATGCATCA GGTAGGATGGGAACAAAGATCA ACTAATTTCGTCGGCTCGAA GTGCTAATATTGGCGTGCTC CCAAGGAAACGAAAGAATCG TGAAACCGGGGACCTAATAA CATGGTCAATCGTTGAATCG CCGCTTGGTCCATTTATTCA GCCTCCATCATCTCTTCCAA CGGTCAATTTCCGGTAGAAG CCGTTCCTGGTGGTATCAAG GAAGCCGAGTAGGTGGATTG CATTTTCCCTTGACCCAATC ATATGGGCCCGTGTGAGTAG AACAACCGGATCCAATCAAA CTTTTGTTGATCAGGCGACA GGTCCACCGCTGAGAATAAA CCGACCTAGAATCCGATGAA TCTCACAACCCGAATTCCTT CCATATCCGCATTTTCTTCG TACGGGTCGGATCCTAGATG GCAAGCAAGATTGGTTGGAT GGGGATACACGACAGAAGGA ACAGAGATGGTGCGATTTGA GCCCTCGGTAACGAGACATA AATCCGGATATTGATACAATTCAT AATCGTGAGGGTTCAAGTCC GTCGGACCCATTTGTGAAAG ACTCCCTCCTACGCGACTTT GAGTAACCGGACTCTCATTCG TCGAACCGTAGACCTTCTCG CACATGGAGCCATCTCGTTA TGATTTCCGCCTATTCTTGTTT TGATTCGCCAAATACATCCA GCCCAATATTTTGAATTGATACG AATGGGTGGCAAAACAAGAG TCTTGATCCTGTTCGTCAAAGA TTTTTGCGCCTATCCAAATC GATAGGCGCAAAAATCCAAG CCCGAAGCAAGAAGAAAGAA CGAAAAAGCCGAGAGATTTG AGAATGACCTCCGGGAAAGT TGAGATTCAGCAATCCCAAA TCTTATGTATCGGGGGTCCA TTGGTGCCTCCTAATTTTGAT TCGCTCGAGAATTGAGACAG GCAATTCCTTCGAAACCTGA ACTTGGTTTCCGACTTGGTG CTGCGGAAAAATAGCTCGAC GAAGTGCGAGAGAAGGGATG CCGATTCACCAGCTCTTCTC CCAGTGACTTGGTCATTTGAA CAAGTATGAACGGCGTTAGAA GGGTCGAAACAAGAGGGAAT GCAGGCTCGTACACATTGAG TGGCCCTCTCTCATTCTCAT
Position
Repeat motif
Expected size (bp)
Ta (°C)
Na
h
trnH-psbA intergenic
(A)10…(A)13…(A)11
242
59
1
–
psbA-trnK intergenic
(A)10
189
59
1
–
matK-trnK intergenic
(A)10
146
59
1
–
trnK-rps16 intergenic
(A)16
173
61
1
–
trnK-rps16 intergenic
(T)13
239
60
4
0.595
rps16-trnQ intergenic
(C)14
168
60
1
–
rps16-trnQ intergenic
(T)11
178
60
2
0.433
trnQ-atpF intergenic
(A)20
156
59
2
0.501
atpF-atpH intergenic
(T)11
245
60
1
–
atpH-atpI intergenic
(T)5C (T)10
197
59
2
0.359
rps2- rpoC2 intergenic
(TC)3(T)11
147
59
1
–
rpoC2 gene
(T)12
141
60
1
–
rpoB gene
(T)10
163
59
1
–
rpoB-trnC intergenic
(A)11
246
60
1
–
psbM-trnD intergenic
(C)10
96
59
1
–
psbZ-trnG intergenic
(A)14
200
59
1
–
rps14-psaB intergenic
(A)11
212
59
1
–
ycf3 intron
(A)17
182
60
1
–
rps4-trnT intergenic
(T)10
159
61
2
0.501
trnL-UAA-trnF-GAA intergenic
(A)10
143
60
1
–
trnF-ndhJ intergenic
(T)10
196
60
1
–
trnV-UAC intron
(T)11
182
60
1
–
atpB-rbcL intergenic
(A)10(G)8
244
60
1
–
ycf4-cemA intergenic
(T)10
170
60
1
–
petA-psbJ intergenic
(A)6(G)10…(A)11
224
62
3
0.544
petA-psbJ intergenic
(T)14
198
60
5
0.644
rps18-rpl20 intergenic
(A)10…(T)12
158
61
1
–
clpP intron
(T)12
162
60
1
–
rps8-rpl14 intergenic
(T)16
233
60
1
–
rps3 gene
(T)10
165
60
1
–
rRNA-trnR intergenic
(A)12
141
60
1
–
ndhF-rp132 intergenic
(A)11
181
59
1
–
rp132-trnL–UAG intergenic
(C)12(T)11
174
61
3
0.325
psaC-ndhE intergenic
(T)14
237
59
2
0.074
May 2011] Table 1.
Lcp56 Lcp57
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Continued.
Locus Lcp50
AJB Primer Notes & Protocols—LIRIODENDRON chloroplast microsatellites
F: R: F: R: F: R:
Primer sequence (5′–3′)
Position
Repeat motif
Expected size (bp)
Ta (°C)
Na
h
TACTGACCGGGACAGGAAAA CGGATACAAAAGCGGGATT CACGTGTACCATCCTTCCAA GCCTACGAAAAGATCGCTTG CGCGAGATGGGGATTTTTAT CGCCGTTCATACTTGTTTCA
ndhG-ndhI intergenic
(T)12
138
60
1
–
rp133-rps18 intergenic
(TA)7
174
65
2
0.074
rp132-trnL-UAG intergenic
(AT)6
154
59
2
0.501
F, forward primer; R, reverse primer; Ta, annealing temperature; Na, number of different alleles; h, unbiased haploid diversity determined from 27 individuals. Table 2.
Results of cross-species amplification of 18 polymorphic cpSSR markers in L. chinense.
Locus
GenBank Accession No.
Ta
Allele sizes (bp)
Na
h
Lcp4 Lcp5 Lcp6 Lcp7 Lcp12 Lcp15 Lcp19 Lcp21 Lcp23 Lcp24 Lcp26 Lcp31 Lcp33 Lcp37 Lcp39 Lcp47 Lcp48 Lcp49
HQ824812 HQ824813 HQ824814 HQ824815 HQ824817 HQ824819 HQ824820 HQ824821 HQ824822 HQ824823 HQ824824 HQ824825 HQ824826 HQ824828 HQ824829 HQ824830 HQ824831 HQ824832
60 60 60 60 60 60 60 61 61 61 61 60 62 60 60 60 60 60
170, 171 243, 255, 256, 257, 258, 259, 260 166, 167 176, 178, 179, 180 271, 272, 273 150, 154, 156, 157, 158, 161 95, 96 198, 199, 200 179, 180 159, 160 197, 198, 199, 200 171, 172 219, 222, 223, 224, 225 154, 155 159, 160 180, 181 170, 171, 173, 174, 175 233, 234, 235, 236, 237
2 7 2 4 3 6 2 3 2 2 4 2 5 2 2 2 5 5
0.250 0.964 0.250 0.750 0.607 0.893 0.250 0.714 0.429 0.250 0.821 0.250 0.893 0.250 0.250 0.250 0.786 0.857
Ta, annealing temperature; Na, number of different alleles; h, unbiased diversity per locus (Peakall and Smouse, 2006). The number of alleles per locus ranged from two to five, with an average value of 2.636. The unbiased haploid diversity per locus was from 0.074 to 0.644, with an average of 0.414 (Table 1). The same set of 55 cpSSR markers were tested on L. chinense with eight individuals randomly collected from natural populations in China. Eighteen out of 55 primer pairs were polymorphic. The number of alleles per locus ranged from two to seven, and the unbiased haploid diversity per locus was from 0.250 to 0.964 (Table 2). To verify whether the PCR products of the cross-species amplification were the target fragments, PCR products were purified using E.Z.N.A Gel Extraction Kit (OmegaBiotek, Lilburn, Georgia, USA), and then the purified fragments were ligated into the pMD 18-T plasmid vector (Takara, Dalian, Liaoning, China), followed by transformation into E. coli H5α competent cells (Invitrogen, Shanghai, China). Recombinant clones were screened using M13 primers, and positive clones were sequenced with ABI BigDyeTM Terminators Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) in an ABI 3730xl automated sequencer (Applied Biosystems). The GenBank accession numbers of the polymorphic loci are shown in Table 2. The results revealed that all the polymorphic cpSSR loci in L. chinense were highly homologous to the corresponding sequences in L. tulipifera, which confirms the high conservation of the chloroplast genome in Liriodendron.
CONCLUSIONS This set of highly polymorphic cpSSR markers (Table 2) should be useful for further studies of the population genetic structure of Liriodendron species. Because the markers were evaluated on an introduced population of L. tulipifera, we expect that higher levels of variation will be found in populations
of equal size sampled in their native range. In combination with previously available nuclear SSR markers (Xu et al., 2006; Yao et al., 2008), the polymorphic cpSSR markers developed in this study will enhance our capability to integrate both nuclear and cytoplasmic SSR markers for further investigation on parental and maternal patterns of gene flow, and ultimately to provide a better understanding of the phylogeographic history of Liriodendron. LITERATURE CITED Cai, Z., C. Penaflor, J. V. Kuehl, J. Leebens-Mack, J. E. Carlson, C. W. dePamphilis, J. L. Boore, and R. K. Jansen. 2006. Complete plastid genome sequences of Drimys, Liriodendron, and Piper: Implications for the phylogenetic relationships of magnoliids. BMC Evolutionary Biology 6: 77. Doyle, J. J., and J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–15. Hao, R.-M., S.-A. He, S.-J. Tang, and S.-P. Wu. 1995. Geographical distribution of Liriodendron chinense in China and its significance. Journal of Plant Resources & Environment 4: 1–6 (in Chinese with English abstract). Latham, R. E., and R. E. Ricklefs. 1993. Continental comparisons of temperate-zone tree species diversity. In R. E. Ricklefs and D. Schluter [eds.], Species diversity in ecological communities, 294– 314. University of Chicago Press, Chicago, Illinois. Peakall, R., and P. E. Smouse. 2006. GENALEX 6: Genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6: 288–295.
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Provan, J., W. Powell, and P. M. Hollingsworth. 2001. Chloroplast microsatellites: New tools for studies in plant ecology and evolution. Trends in Ecology & Evolution 16: 142–147. Sewell, M. M., C. R. Parks, and M. W. Chase. 1996. Intraspecific chloroplast DNA variation and biogeography of North American Liriodendron L. (Magnoliaceae). Evolution; International Journal of Organic Evolution 50: 1147–1154. Xiang, Q. Y., D. E. Soltis, P. S. Soltis, S. R. Manchester, and D. J. Crawford. 2000. Timing the Eastern Asian-Eastern North American
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floristic disjunction: Molecular clock corroborates paleontological estimates. Molecular Phylogenetics and Evolution 15: 462–472. Xu, M., H. Li, and B. Zhang. 2006. Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera. Molecular Ecology Notes 6: 728–730. Yao, X., J. Zhang, Q. Ye, and H. Huang. 2008. Characterization of 14 novel microsatellite loci in the endangered Liriodendron chinense (Magnoliaceae) and cross-species amplification in closely related taxa. Conservation Genetics 9: 483–485.
