全 文 ：广 西 植 物 Guihaia 31(1)：39— 42 2011年 1月
DOI：10．3969／j．issn．1000～3142．201I．O1．009
厚壳 桂新微卫星体分子标记的开发与应用
高丹丹 ～2 3，王峥峰 ，z*，朱 鹏1～2 3，叶万辉 ，2
(1．中国科学院 植物资源保护和可持续利用重点实验室 ，中国科学院 华南植物园，广州 510650；2．广东省数字
植物园重点实验室 ，中国科学院 华南植物园，广州 510650；3．中国科学院 研究生院，北京 100049)
摘 要：生境破碎化是导致全球生物多样性危机的主要原因，即使常见物种也会受到很大影响。厚壳桂
(Cryptocarya chinensis)是我国南亚热带季风常绿阔叶林群落演替顶极物种，由于这一森林植被 的破坏而呈
片断化分布。在以往的研究中，我们利用筛选到的一些微卫星体对其遗传多样性研究后发现其可能以无性生
殖为主。为进一步证实这一发现 ，该文又报道了 11个新微卫星体 ，这些新微卫星体和以往报道的微卫星体将
有助于更好分辨厚壳桂的有性和无性生殖，特别是在空间小尺度(Fine spatial scale)。选取鼎湖山 21个厚壳
桂个体，对这 l1个新微卫星体进行多态性检测。结果表明，这些微卫星体位点所包含 的等位基因数 目为 2～3
个 ，观察杂合度(Observed heterozygosities)和期望杂合度(Expected heterozygosities)分别为 ：0．048-1．000，
0．048~0．535。11个位点中有 8个偏离哈迪一温伯格平衡，FIS在 8个位点表 现为负值 ，并极显著。所有两
两位点在 0．05水平上均表现出连锁不平衡，但这一不平衡在使用 Bonferroni校正(Bonferroni correction)后
不显著。研究同时发现 21个厚壳桂个体中 2O个有相同的多位点基因型(multilocus genotype)。上述遗传多
样性结果符合无性生殖物种的遗传特征，表明厚壳桂以无性生殖为主。
关键词：无性生殖；保育；森林片断化；遗传标记
中图分类号 ：Q943．2 文献标识码：A 文章编号 ：1000—3142(2011)01—0039—04
Isolation and characterization of new set of
microsatellite loci in Cryptocarya chinensis
GAO Dan-Dan1， ，u，WANG Zheng-Feng ， ，
ZHU Peng1，2，u，YE W an-Hui1，
(1．Key Laboratory of Plant Resources Conservation and Sustainable Utili~tion，South C i^na Botanical Garden，
Chinese Academy of Sciences，Guangzhou 510650，China；2．Guangdong Key Laboratory of Digital Botanical
Garden，South China Botanical Garden，ChineseAcademy of Sciences，Guangzhou 510650，China；
3．Graduate School of the Chinese Academy of Sciences，Beijing 100049，China)
Abstract：Habitat fragmentation is the main threat to global biodiversity．Common species could also greatly suffer
from habitat fragmentation．As a result of the destruction of the monsoon evergreen broad—leaved forest in South Chi—
Ha，Cryptocarya chinensis，one of the climax species，is now patchily distributed．Because a few microsatellite loci re—
ported previously might cause low resolution tO detect asexual reproduction in this species，we reported a new set of
microsatellite which wil assist to detect the extensiveness of sexua1 and asexual reproduction at fine spatial scale in
收稿日期：2010-03—1 5 修 回日期 ：2010-09—10
基金项目：国家重点基础研究“973”发展计划项 目(2007CB411600)；国家科技支撑计划项 目(2008BAC39B02) 中国科学院知识创新工程重大项目
(KZCX2一YW-430)；“十一五”国家科技支撑计划项 目(2008BADOB05)[Supported by National Basic Research“973”Program of China(2007CB411600)：the
National Key Technology R ＆ D Program(2008BAC39B02)；Knowledge Innovation Project of the Chinese Academy of Sciences(KZCX2一~_W~-430)：the“E—
leventh Five-Year”National Seienee and Technology Supporting Project(2008BADOB05)]
作者简介：高丹丹(1985一)，女，河南商丘人 ，硕士研究生 ，分子生态学，(E-ma)gaodandan2004@126．corn。
通讯作者(Author for correspondence，E-mail：wzf@scib．ac．cn)
4O 广 西 植 物 31卷
the future．Eleven microsatellite markers from repetitive DNA enriched libraries for C
． chin铆s were devel0Ded．
Twenty—one individuals from Dinghu Mountain in South China were used to characterize their polymorphisHL The
number of alleles of 11 loci ranged from two to three，observed and expected heterozygosities rang ed from 0
． 048 to
1．000，and 0．048 to 0．535，respectively
． Deviations from Hardy-Weinberg equilibrium were detected in 8 loci due to
heterozygote excess(highly significant negative Fis)
． All locus pairs showed significant linkage disequilibrium at the
0．05 significant level，but such significance disappeared after Bonferroni correctioIL 2O of 21 detected individua1s
showed identical muhilocus genotype．Al these results conformed to the genetic characteristic of clonal species。con—
firming extensive elonal growth in C．chinensis
．
Key words：asexual reproduction；conservation；forest fragmentation；genetic marker
Habitat fragmentation is the main threat to
global biodiversity．Alike to rare and endemic spe—
cies，common species could also greatly suffer from
habitat fragmentation (Hooftman et a1．，2003；
Jump＆ Pefiuelas，2006)．As a successional climax
species，Cryptocarya chinensis(Lauranceae)is the
main component of typical monsoon evergreen
broad-leaved forest in lower subtropical China
(Peng，1996)．As a result of the destruction of the
monsoon evergreen broad—leaved forest，it is now
patchily distributed and an ideal system to study
the influence of forest fragmentation on the popu—
lation genetics of forest species， Surprisingly，our
previous study revealed unexpected clonal growth
in C．chinensis in fragmented forest(W ang et a1．。
2007，2008)．Because only eight previous microsat—
ellite 1oci might cause low resolution to detect sex—
ual growth in this species，we reported here new
set of microsatellite which will assist to detect the
extensiveness of sexual and asexual growth in C．
chinensis at fine spatial scale in the future．
C．chinensis is a forest—dwelling species and
occupies the second or third level of the tree layer．
Its hermaphroditic flowers form paniculate inflo—
rescences and are pollinated by insects．Seeds of C．
chinensis are dispersed by vertebrates(e．g．birds
and small mammals)and gravity(Wang et a1．，
2003)．
Genomic DNA was extracted from one dry leaf
tissue by using CTAB method(Doyle，1991)．Ap-
proximately 250 ng of the total genomic DNA was
digest ed by a restriction enzyme M seI(NEB)and
the resulting fragments ligated with M seI adaptor
(5 一TACTCAGGACTCAT一3 ／5 一GACGATGAG—
TCCTGAG-3 )with T4 ligase(NEB)overnight at
1 6 ℃ ．The digestion-ligation mixture was subse—
quently diluted 10 times，and 2 L was used for
PCR amplification using adaptor—specific primers
(5 一GATGAGTCCTGAGTAAN一3 ，i．e．MseI—N)．
PCR products hybridized to a 5 biotin—labeled oli—
gonucleotide probe(GA )15．Subsequent probe—
bound DNA fragments were enriched for GA re—
peats using streptavidin—coated，magnetic beads
(NEB)．Enriched fragments were recovered with
PCR amplification using M seI-N as primer．PCR
products were then ligated into the pGEM—T plas—
mid vector(Promega)，and transformed into the
Escherichia coli DH5a competent cells(Takara)．
The PCR—based method described by Lunt et a1．
(1 9 9 9)was used to screen the recombinant clones．
Identified positive clones were sequenced by United
Gene Holdings，LTD(Shanghai，China)with M 13R
or M 1 3F as primer． Primers were designed using
OLIGO 6．5 4 software(MBI)for the sequences con—
tain microsatellite repeats．
Polymorphisms of these mieosatellite loci were
assessed by 2 1 C．chinensis individuals。newly col—
lected from Dinghu M ountain，Guangdong Prov—
inee，China．PCR amplification were performed in
10 L reaction mixtures，consisting of approxi—
mately 5 ng of template DNA，5O mmol／L KC1，2O
mmol／L Tris—HC1(pH 8．0)，1。5 mmol／L MgC12，
0．5／~mol／L of each primer，0．2 mmol／L of each
dNTP，and 1U of Taq DNA polymerase(Takara)．
The reaction mixture was sub ected to PCR ampli—
fication in a PTC-100(MJ)using a PCR program，4
min at 95。，followed by 35 cycles of 94℃ for 3O s，
5 2—6 O ℃ (depending on locus)annealing tempera—
高丹丹等：厚壳桂新微卫星体分子标记的开发与应用 41
ture for 30 s。and 72℃ for 45 s，followed by 10 min
at 72℃ ．PCR products were then resolved on 6
denaturing polyaerylamide gels and visualized by
silver staining．
Observed heterozygosity(HO)，the unbiased
expected heterozygosity(HE )and fixation index
(FIs)were calculated using GDA 1．1(Lewis
Zaykin，200 1)． Deviations from Hardy—W einberg
equilibrium (H WE)for each locus and genotypic
linkage disequilibrium(LD)between all pairs of loci
were tested using FSTAT 2．9．3(Goudet，1995)．
The number of alleles per 1Oeus varied from 2
to 3，observed and expected heterozygosities ranged
from 0．048 to 1．000，and 0．048 to 0．535，respec—
tively(Table 1)． Deviations from H WE were de-
tected in 8 loci due to highly significant negative
Ffs． All locus pairs showed significant LD (P<
O．05)without Bonferroni correction．
Table 1 Details of mierosatelite loci in Cryptocarya chinensis including locus name，forward and reverse
primer sequences，repeat motif，annealing temperature(Ta)，numbers of aleles(A)，observed／expected
heterozygosities(HO／HE)，fixation index(Fls)by Weir& Cockerham s(1984)，significant
value of HWE(P—valueof HW E)，GenBank accession number
T ⋯ ． Primer sequence L。。“。 (5
一
3 ) Repeat (r
℃
a
) A HO HE FB p-va1ue GenBa
．
nk
of HW E acces
，
sion
n um Der
256 F：GTTCCCAAGAAACCGATAGAA
R：GTAGTAGTGAATGGCGTAG
185 F：CCAAATGGCAATATGCAA
R：CTAACACTATCCACGCATCTC
225～2 F：GGGTTTGTTTGGGCGGTGAG
R：GAAGGTGGGCCATCGCATCAG
155 F：CTGTTTATTCATTCATAAGGG
R：CCAGTTGCTGCAAATCCAA
2 1 Z F：GAACATGCGGAATGAG
R：GTGTCTCCAACTGCTCGTTCA
1 04 F：GGGTCACCATCCATTGCATCT
R：GGACGCATGTCGAATCTATCT
170 F：TGCGCTTCTCTATCTT
R：CTCACTTCTGCCATCAATCTC
353 F：GCTTCTCTGCCTACTATGCCT
R：CCATTTCACTGCTCCAACCAT
268 F：GGAAATATGGGAAATAGTCCT
R：GCGCAGACGTGCTAAC
1 72 F：GGCCATGAATGCAATAA
R：TCTGCAACGAAACCCTAGAAG
174 F：GGGCGAATCTTATATT
R：GGACTTGGTAACTGGCTAACA
(AG)20 58 2 0．048 0．048 0．000 0．911 GU117095
(CT)21 52 3 1．000 0．535 —0．909 0．000 GU117096
(GA)16 60 2 0．048 0．048 0．000 0．911 GU117097
(TC)20 54 3 1．000 0．535 —0．909 0．000 GU117098
(CT)22 56 2 0．952 0．511 —0．905 0．000 GU117099
(TC)22(AC)2 60 2 0．952 0．511 —0．905** 0．000 ’GU117100
(TC)19
(TC)16
52 3 1．000 0．535 —0．909 0．000 GU117102
60 2 0．048 0．048 0．000 0．911 GU117103
T10GT(TC)19 60 2 0．952 0．511 —0．905 * 0．000 GU117104
(AG)6AT(AG)4 52 2 0．952 0．511 —0．905 * 0．000 GU117105
Complex 52 3 1．000 0．535 —0．909 0．000 GU117106
： T23C(CTT)4CCTT(CCT)8(CT)1 6； P< O．O1 after Bonferroni corection
Unlike its congener Cryptocarya concinna，which
distributes in the same Dinghu Mountain，showed no
clonal growth by using microsatellite markers(Wang et
a1．，2009)，C chinensis showed unexpected elonal growth
in our previous study(Wang et al。，2007，2008)．The
new set of microsatelite confirmed this．A1thou【gh no
linkage disequilibrium occurred between any locus pairs
after Bonferroni corection，eight of total eleven 1oci
showed highly significant negative F,s value．Genotypic
diversity of G／N(where G was number of observed mul—
tilocus genotypes，N was number of sample size)was
0．048，much lower than previous reported G／N value in
clonal species(see references in Wang et a1．2008)．In
fact，total twenty individuals showed the same identical
mutiloci genotype．Strong negative F values(meaning
excess of heterozygote)，high occurrence of identical gen—
otypes(G／N values)confirmed growth mode of Crypto—
carya chinensis is predominantly clonal(Balloux et nZ．，
2003；Halkett et a1．，2005)．
42 广 西 植 物 3l卷
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