全 文 ：广 西 植 物 Guihaia Jul． 2013，33(4) :465－467 http:/ / journal． gxzw． gxib． cn
DOI:10． 3969 / j． issn． 1000-3142． 2013． 04． 007
田斌，田向楠，许玉兰，等． 木棉的 SSＲ引物开发［J］． 广西植物，2013，33(4) :465－467
Tian B，Tian XN，Xu YL，et al． Developing SSＲ primers in Bombax ceiba (Bombacaceae) ［J］． Guihaia，2013，33(4) :465－467
Developing SSＲ primers in Bombax ceiba (Bombacaceae)
TIAN Bin，TIAN Xiang-Nan，XU Yu-Lan，MA Huan-Cheng*
(Key Laboratory of Biodiversity Conservation in Southwest China，State Forestry
Administration，Southwest Forestry University，Kunming 650224，China )
Abstract:Bombax ceiba is a multipurpose tree species of tropical forests． Nine novel polymorphic microsatellite loci for
B． ceiba were isolated and characterized through the combined biotin capture method． Markers were tested on 32 individ-
uals collected from dry-hot valleys in southwest China． The number of alleles ranged from 2 to 6． The expected and ob-
served heterozygosity values ranged from 0 to 0． 525 and 0． 121 to 0． 561，respectively． One locus showed significant devi-
ation from Hardy-Weinberg Equilibrium． No significant linkage disequilibrium was found． The microsatellite markers
that were characterized in this study were the first to be developed for B． ceiba and would have great potential for differ-
ent genetic studies of wild populations of this species．
Key words:Bombax ceiba;microsatellite markers;genetic diversity
中图分类号:Q943 文献标识码:A 文章编号:1000-3142(2013)04-0465-03
木棉的 SSＲ引物开发
田 斌，田向楠，许玉兰，马焕成*
(西南林业大学 国家林业局 西南地区生物多样性保育重点实验室，昆明 650224 )
摘 要:木棉是分布于热带地区的重要经济树种。通过磁珠富集方法开发了木棉的 SSＲ引物，并在采自于西
南干热河谷的 32个木棉个体中进行了多态性验证。结果表明:这些位点等位基因数量为 2 ～ 6 个，期望杂合
度和观测杂合度范围分别为 0 ～ 0． 525 和 0． 121 ～ 0． 561。仅有一个位点显示 Hardy-Weinberg平衡的偏离。这
些微卫星标记的开发在木棉属中尚属首次，对于木棉的野生种群遗传多样性研究和木棉遗传资源利用上有很
高的潜在价值。
关键词:木棉;微卫星标记;遗传多样性
Bombax ceiba (Bombacaceae)，commonly known as
Cotton Tree，is a multipurpose tree species of tropical for-
ests，providing food，fodder，fiber，and medicine besides
many ecological benefits (Jain et al．，2011)． This lofty
tree species is found in temperate and tropical Asia，Afri-
ca，America and Australia，is an important part of many
tropical dry deciduous forest ecosystems (Li，1987)． To
better understand the genetic diversity，population
structure of B． ceiba，microsatellite loci， useful
codominant markers for population genetics research and
genetic improvement，are needed．
Microsatellites，or tandem simple sequence repeats
(SSＲ) ，have been found in every organism studied so
far since they may be highly polymorphic are useful ge-
netic markers (Tautz ＆ Ｒenz，1984)． To amplify micro-
satellite loci by PCＲ，primers must be developed from
收稿日期:2013-03-01 修回日期:2013-05-03
基金项目:国家自然科学基金(NSFC31260050，31260175) ;国家林业局公益性行业科研专项(201104034) ;云南省高校创新团队(201108)
作者简介:田斌(1983-) ，男，山东兖州人，博士，讲师，主要从事植物遗传育种及群体遗传学方面的研究，(E-mail)tianbinlzu@ gmail． com。
* 通讯作者:马焕成，博士，教授，从事困难地段造林和植被恢复的研究，(E-mail)mahuancheng@ yahoo． com． cn。
the DNA that flanks specific microsatellite repeats． Al-
though microsatellite loci have now been developed for
hundreds of species，these loci have not yet been
isolated from many additional species of interest and re-
main to be developed． This study is the first attempt to
isolate and characterize microsatellite markers for this
important tree species． Here we describe the isolation
and evaluation of 9 novel microsatellite loci in B． ceiba，
which will be used in further assessment of the genetic
diversity and germplasm characterization to facilitate
molecular marker-assisted selection and breeding of this
species and its relatives．
1 Materials and Method
Genomic DNA for constructing a microsatellite-en-
riched library was extracted from approximately 30 mg of
silica gel-dried，leaf tissue using the modified cetyltrim-
ethylammonium bromide (CTAB)method (Doyle JJ ＆
Doyle JL，1987)． The selective capture of microsatellite
loci using magnetic beads was used as a methodological
strategy to clone the microsatellite loci． The
microsatellite isolation procedure followed the method of
Hauswaldt ＆ Glenn (2003)． Briefly，about 300 ng ge-
nomic DNA was completely digested with a restriction
enzyme ＲsaI (NEB) and then ligated to SuperSNX
linkers． DNA fragments were enriched for microsatellite
locus by Dynabeads M-280 magnetic streptavidin beads
(Invitrogen，Grand Island，New York，USA)selection
with 5-biotinylated (AG)12，(AT)8，(CG)12，(GT)
12，(ACG)12 and (CCA)8 probes． Captured fragments
were reamplified with adaptor-specific primers by follow-
ing the program:95 ℃ for 2 min，25 cycles of 95 ℃ for
20 s，60 ℃ for 20 s，72 ℃ for 90 s followed by an elon-
gation step of 30 min at 72 ℃． Polymerase chain
reaction (PCＲ)products were linked into the pGEM-T
Easy Vector (Promega，USA) and transformed into
JM109 cells． Positive clones were selected by blue and
white screening and tested by PCＲ using two primers
M13 +/M13 －． PCＲ products (＞ 350 bp) were
sequenced on an ABI 3730xl Genetic Analyzer (Applied
Biosystems)using BigDye Terminator Cycle Sequencing
Kit (Applied Biosystems)． The sequences containing
motifs repeating more than three times were regarded as
microsatellites．
After discarding sequences with few repeat regions
or not suitable for designing primers，a total of 39 clones
were found out of the sequenced 210 sequences to
possess microsatellite motifs using the program SSＲ
Hunter 1． 3． 0 (Qiang Li，Nangjing Agricultural Univer-
sity，Nanjing，China)． These sequences were used the
program Primer Premier version 5． 0 (Clarke ＆ Gorley，
2001)to design primers．
Three individuals from each population were
selected to evaluate the optimal annealing temperature．
A gradient PCＲ procedure was performed using an
initial step of 94 ℃ for 4 min;followed by 35 cycles of
94 ℃ for 40 s，48－65 ℃ for 50 s，and 72 ℃ for 60 s;
and a final extension of 72 ℃ for 10 min． PCＲ was per-
formed in a 25 μL mixture containing 40 ng of genomic
DNA，0． 3 μL dNTPs (10 mmol /L) ，0． 3 μmol /L of
each primer，2 μL of 10 ×PCＲ buffer，and 0． 6 U of Taq
polymerase (TaKaＲa Biotechnology Co．)． In order to
analyze the genetic polymorphism of these isolated mic-
rosatellite loci，32 individuals were selected from dry-hot
valleys in southwest China for genotyping． The microsat-
ellite loci were amplified by PCＲ using the same proce-
dure for the evaluation of the optimal annealing temper-
ature． PCＲ productions were separated using PAGE e-
lectrophoresis using a 50-bp DNA Step Ladder to deter-
mine the allele size．
2 Ｒesults
A total of 19 out of the 39 primers pairs
successfully amplified the target regions and 9 of them
showed polymorphic banding patterns(Table 1)． The
characteristics of the nine novel polymorphic
microsatellite loci for B． ceiba are listed in Table 1 and
their corresponding sequences are deposited in Genbank
under the accession numbers from KC471333 to
KC471341． The number of alleles per locus (Na) ，ob-
served heterozygosity (Ho)and expected heterozygosity
(He)were calculated using software GenALEx 6． 4
(Peakall ＆ Smouse，2006) ;nine primer pairs were suc-
cessfully amplified， and all of them showed
664 广 西 植 物 33 卷
polymorphism． The observed and expected heterozygosi-
ties varied from 0 to 0． 525 and 0． 121 to 0． 561，re-
spectively (Table 1)．
Table 1 Characteristics of nine polymorphic microsatellite markers from Bombax ceiba
Locus Primer sequence (5→3) Ｒepeat motif Size range (bp)
Ta
(℃)
GenBank
Accession No．
Na Ho He PHW
BC01 F:CCATCAGCGAGGACATTG
Ｒ:CATAAGGCATCGGCATAG
(GGA)4AGTGA(TGG)5 370－382 53 KC471333 4 0． 121 0． 459 0． 372
BC02 F:AAGGCTACTACAACTTGACTG
Ｒ:CTTTGCTCTATTCCCACA
(ATG)3A(TGG)3 167－170 52 KC471334 2 0． 000 0． 169 0． 521
BC03 F:GATGCTTCTCCTGCTTTA
Ｒ:AATGTGGGCTTATTGTCT
(GAC)6 334－340 50 KC471335 3 0． 172 0． 205 0． 339
BC04 F:ATTTGAATCTTGCGTGTTAC
Ｒ:TTCCTCACCCTCCTCTTT
(TG)6(TGCG)3(TG)4 321－335 52 KC471336 6 0． 467 0． 561 0． 441
BC05 F:TGGTGGTAAAGCAAGGATCG
Ｒ:TGCACTGAGTGACCATGACA
(TG)12 103－117 59 KC471337 5 0． 525 0． 533 0． 213
BC06 F:CTTGAGAGCTCCGCTTGAAC
Ｒ:AACGGGAATGGGAAAAGTG
(CCA)5 167－173 52 KC471338 3 0． 210 0． 3210． 002*
BC07 F:AAAGCCAACATGATCGGAGAT
Ｒ:TGGCAGTTTCGGCATACA
(GTG)9 92－107 59 KC471339 5 0． 456 0． 452 0． 078
BC08 F:AACGGGAATGGGAAAAGTG
Ｒ:CTTGAGAGCTCCGCTTGAAC
(GGT)6 136－148 50 KC471340 4 0． 328 0． 532 0． 568
BC09 F:AGCTTTACAAGCCTCATG
Ｒ:AAGGTATCTATTCCAGCA
(GT)9 290－298 50 KC471341 5 0． 378 0． 541 0． 662
Ta:annealing temperature (℃) ;Na:number of alleles;Ho:observed heterozygosity;He:expected heterozygosity;PHW from exact tests for Hardy-Weinberg equilibrium(*
P＜0． 01)．
Hardy-Weinberg equilibrium (HWE)and linkage dise-
quilibrium were calculated using AＲLEQUIN 3． 5 (Ex-
coffier ＆ Lischer，2010)． After Bonferroni adjustments，
one locus，BC06，was found to be deviated from HWE
(P＜0． 01) (Table 1) ，and no significant evidence of
linkage disequilibrium was observed．
3 Conclusions
The number of loci scored，degree of polymorphism
of each locus and sample size are of paramount impor-
tance for the statistical power of microsatellites to be ef-
fective (Zane et al．，2002)． In this study，9 novel poly-
morphic microsatellite markers were characterized out of
a total of 210 clones sequenced． These primers are the
first to be developed for B． ceiba and have great
potential for use in different genetic studies of wild and
cultivated populations of this species． These markers
will be used to gain a better understanding of various
evolutionary questions including population genetic di-
versity and differentiation，population demography，and
gene flow of B． ceiba are also expected to be useful for
construction of linkage maps and marker assisted selec-
tions of this useful species．
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7644 期 田斌等:木棉的 SSＲ引物开发