全 文 ：中国特有种爆杖花的微卫星分子标记开发与评价
*
严丽君1，2，3，张志荣2，李德铢1，2，高连明1＊＊
(1 中国科学院昆明植物研究所东亚植物多样性与生物地理学重点实验室，云南 昆明 650201;2 中国科学院
西南野生生物种质资源库，云南 昆明 650201;3 中国科学院大学，北京 100049)
摘要:爆杖花 (Ｒhododendron spinuliferum)是中国西南地区特有的观赏和药用植物。为了研究爆杖花和碎
米花之间的杂交物种形成过程，本研究利用 FIASCO方法对爆杖花进行微卫星引物开发，从 100对引物中
筛选出 28个微卫星标记，其中 22个为多态。利用爆杖花两个居群共 24个个体对 22个多态性位点进行分
析，结果显示:每个位点具有 2～5个等位基因，平均为 3. 4个，其观测杂合度和期望杂合度分别为 0. 083
～0. 792和 0. 153～0. 744。对筛出的 28个微卫星标记在碎米花的两个自然居群中也做了检测，结果显示:
有 22个微卫星标记成功扩增，其中 20个有多态性;每个多态位点有 2～6个等位基因，平均为 3. 2个，其
观测杂合度和期望杂合度分别为 0. 000～0. 833和 0. 117～0. 736。开发的微卫星标记可用于爆杖花及其近缘
物种的居群遗传学分析和杂交物种形成研究。
关键词:微卫星标记;爆杖花;碎米花;FIASCO;多态性
中图分类号:Q 943 文献标识码:A 文章编号:2095－0845(2014)01－041－06
Isolation and Characterization of Microsatellite Markers
for the Chinese Endemic Species Ｒhododendron
spinuliferum (Ericaceae)
YAN Li-Jun1，2，3，ZHANG Zhi-Ｒong2，LI De-Zhu1，2，GAO Lian-Ming1*
(1 Key Laboratory for Plant Diversity and Biogeography of East Asia，Kunming Institute of Botany，Chinese Academy of Sciences，
Kunming 650201，China;2 Germplasm Bank of Wild Species，Kunming Institute of Botany，Chinese Academy of Sciences，
Kunming 650201，China;3 University of Chinese Academy of Sciences，Beijing 100049，China)
Abstract:Ｒhododendron spinuliferum (Ericaceae)is an ornamental and medicinal plant endemic to southwest Chi-
na． In order to study hybridization between Ｒ. spinuliferum and Ｒ. spiciferum，a FIASCO (Fast Isolation by AFLP of
Sequences Containing Ｒepeats)method was used to develop microsatellite markers in Ｒ. spinuliferum． A total of 28
microsatellite markers were isolated from 100 SSＲ primer pairs，of which 22 were polymorphic． Polymorphism of the
22 polymorphic loci was assessed separately in 24 individuals collected from two wild populations． The number of al-
leles per locus ranged from 2 to 5，with an average of 3. 4，while observed (HO)and expected (HE)heterozygosi-
ties varied from 0. 083 to 0. 792 and from 0. 153 to 0. 744，respectively． The same 28 microsatellite markers were al-
so tested in two wild populations (12 individuals from each)of Ｒ. spiciferum． Twenty two of the markers were suc-
cessfully amplified，of which 20 were polymorphic． Estimates of diversity in two natural populations of Ｒ. spiciferum
based on the 20 polymorphic markers revealed that number of alleles per locus ranged from 2 to 6，with a mean of
3. 2，while observed (HO)and expected (HE)heterozygosities ranged from 0. 000 to 0. 833 and from 0. 117 to
植 物 分 类 与 资 源 学 报 2014，36 (1):41～46
Plant Diversity and Ｒesources DOI:10．7677 /ynzwyj201413019
＊
＊＊
Funding:The Key Program of the Chinese Academy of Sciences (KSCX2-EW-Q-10，KSCX2-EW-Z-2)and Key Ｒesearch Program of the
Chinese Academy of Sciences (KJZD-EW-L07)
Author for correspondence;E-mail:gaolm@ mail． kib． ac． cn
Ｒeceived date:2013－02－20，Accepted date:2013－04－11
作者简介:严丽君 (1988－)女，博士研究生，主要从事杜鹃花属系统发育与物种形成研究。E-mail:yanlijun@ mail． kib． ac． cn
0. 736，respectively． These newly developed microsatellite markers will be used in future studies of hybridization and
the population genetics of Ｒ. spinuliferum and its closely related species．
Key words:Microsatellite markers;Ｒhododendron spinuliferum;Ｒhododendron spiciferum;FIASCO;Polymorphism
Ｒhododendron L． is the largest genus in Ericace-
ae including about 1 025 species，distributed from the
northern temperate zones，throughout tropical south-
eastern Asia to northeastern Australia (Chamberlain
et al．，1996)． There are 571 species in China，of
which 405 are endemic (Fang et al．，2005)． There is
a long horticultural history of Ｒhododendron，and lots
of species in this genus have been used as ornamental
plants (Yang et al．，1999)． Ｒhododendron spinulife-
rum Franch． is one of the important ornamental spe-
cies，which is endemic to southwest China． The
dried stems，leaves and flowers of Ｒ. spinuliferum
can be used as Chinese folk medicine for eliminating
phlegm，diminishing inflammation，relieving cough
and asthma (Chen et al．，1996)．
Natural hybridization has been recognized as an
important factor of speciation and diversification
within Ｒhododendron (Milne et al．，2010)． Numer-
ous instances of natural hybridization have been re-
ported in Ｒhododendron in previous studies (Kron et
al．，1993;Zhang et al．，2007;Milne and Abbott，
2008;Ma et al．，2010;Zha et al．，2010)． Ｒhodo-
dendron × duclouxii is an inferred natural hybrid spe-
cies between Ｒ. spinuliferum and Ｒ. spiciferum
Franch． based on morphological characters (Handel-
Mazzetti，1936)． This had been confirmed by mo-
lecular sequence data recently (Yan et al．，2013)．
To further study the extent of hybridization between
Ｒ. spinuliferum and Ｒ. spiciferum，codominant micro-
satellite markers will be used． In this study，we tried
to isolate and characterize suitable microsatellite
markers from Ｒ. spinuliferum，and test their feasibili-
ty in Ｒ. spiciferum samples．
1 Materials and methods
1. 1 Materials
Leaf samples of Ｒ. spinuliferum used to develop
microsatellite markers were collected from two natu-
ral populations in Yunnan province，China． Total of
24 individuals from two Ｒ. spinuliferum populations
(12 individuals from each)were selected to assess
polymorphisms of the developed microsatellite mark-
ers (Table 1)． The feasibility of the developed mic-
rosatellite markers was also assessed in 24 individu-
als from two Ｒ. spiciferum natural populations (12
individuals from each)(Table 1)． Voucher speci-
mens were deposited in the herbarium of the Kun-
ming Institute of Botany，Chinese Academy of Sci-
ences (KUN)．
1. 2 Methods
1. 2. 1 DNA extraction
Total genomic DNA was extracted from silica-gel-
dried leaves using an improved CTAB (cetyltrimethyl
ammonium bromide)method (Liu and Gao，2011)．
Table 1 Details of materials information used in this study
Taxon Locality Gepgraphic Altitude /m Collection number
Ｒ. spinuliferum
Kunming，Panlong area，
Shuanglong town
N 25. 10°
E 102. 80°
2070
YLJ-12008，YLJ-12009，YLJ-120010，YLJ-12011，
YLJ-12012，YLJ-12013，YLJ-12014，YLJ-12015，
YLJ-12016，YLJ-12017，YLJ-12023，YLJ-12031
Ｒ. spinuliferum
Chuxiong，Shuangbai
county，Tuodian town
N 24. 68°
E 101. 66°
1775
YLJ-12802，YLJ-12803，YLJ-12804，YLJ-12805，
YLJ-12806，YLJ-12807，YLJ-12808，YLJ-12809，
YLJ-12810，YLJ-12811，YLJ-12813，YLJ-12817
Ｒ. spiciferum
Kunming，Panlong area，
Shuanglong town
N 25. 10°
E 102. 80°
2070
YLJ-12001，YLJ-12018，YLJ-12019，YLJ-12020，
YLJ-12025，YLJ-12027，YLJ-12029，YLJ-12030，
YLJ-12036，YLJ-12037，YLJ-12043，YLJ-12051
Ｒ. spiciferum
Yuxi，Eshan county，
Gaoping town
N 24. 22°
E 102. 32°
1830
YLJ-121089，YLJ-121093，YLJ-121097，YLJ-121111，
YLJ-121120，YLJ-121122，YLJ-121123，YLJ-121124，
YLJ-121126，YLJ-121127，YLJ-121128，YLJ-121130
24 植 物 分 类 与 资 源 学 报 第 36卷
1. 2. 2 Microsatellite loci isolation，PCＲ amplifica-
tion and polymorphism assessment
The microsatellite loci were isolated based on
the FIASCO protocol (Zane et al．，2002)． About
300－ 500 ng genomic DNA was digested with MseI
(New England Biolabs，Beverly，Massachusetts，USA)，
and the digested DNA fragments were ligated to an
MseI AFLP adaptor pair (5-TACTCAGGACTCAT-
3 /5-GACGATGAGTCCTGAG-3)at 37 ℃ for 2 h
with T4 DNA ligase (Fermentas，Burlington，Ontar-
io，Canada)． Total of 5 μL of a diluted digestion-
ligation mixture (1 ∶ 10)was used for amplification
reactions with the adaptor-specific primers MseI-N
(5-GATGAGTCCTGAGTAAN-3) ，with the follow-
ing cycle program:95 ℃ for 3 min，30 cycles of 94 ℃
for 45 s，50 ℃ for 60 s，72 ℃ for 60 s，and a final
extension step of 7 min at 72 ℃ ． The amplified frag-
ments (200－800 bp)were enriched for microsatel-
lite repeats by magnetic bead selection with 5-bioti-
nylated (AC)15 and (AG)15 ． These enriched frag-
ments were amplified again with the MseI-N primers．
The PCＲ products were purified using an EZNA Gel
Extraction Kit (Omega Bio-Tek，Guangzhou，Chi-
na)． The purified PCＲ products with enriched micro-
satellite repeats were ligated into the pGEM-T vector
(Promega，Madison，Wisconsin，USA)and trans-
formed into DH5α cells (TaKaＲa，Dalian，China)．
Identification of recombinant clones was performed in
a blue /white selection assay． Positive clones were
then tested for microsatellite inserts by PCＲ with
(AC)10 /(AG)10 and T7 /Sp6 primers，respectively．
Clones with positive inserts and appropriate size (300
－700 bp)were then sequenced． The sequences of
which contain microsatellite repeats (SSＲs)，and
with sufficient flanking regions were then used for de-
signing locus-specific primers with the program Oligo
6. 0 (Offerman and Ｒychlik，2003)．
The polymorphisms of all microsatellite loci
were then assessed in 24 individuals from two natural
populations (12 individuals from each)of Ｒ． spinu-
liferum (Table 1)． PCＲ reactions were performed in
20 μL volumes containing 50－100 ng genomic DNA，
0. 6 μM of each primer，7. 5 μL 2× Taq PCＲ Mas-
terMix (containing 0. 1 U Taq polymerase /μL，0. 5
mM dNTP each，20 mM Tris-HCl (pH= 8. 3) ，100
mM KCl，3 mM MgCl2 (Tiangen，Beijin，China)．
The PCＲ amplifications were conducted under the
following conditions:95 ℃ for 3 min followed by 32
cycles at 94 ℃ for 30 s，an annealing temperature op-
timized specifically for each primer pair(Table 2)for
45 s，72℃ for 60 s，and a final extension step at 72℃
for 7 min． The amplified fragments were separated on
8% polyacrylamide denaturing gels with a 20 bp mo-
lecular size standard ladder (Fermentas，Burlington，
Ontario，Canada)and visualized by silver staining．
The polymorphic information content was calculated
by PIC Calculator． Standard genetic diversity param-
eters and deviations from the Hardy-Weinberg equi-
librium were estimated in GENEPOP version 4. 0. 10
(Ｒousset，2008)for all polymorphic loci． Estimation
for linkage disequilibrium between pairs of loci was
performed also in GENEPOP version 4. 0. 10．
2 Ｒesults and discussion
A total of 294 clones with positive inserts and
appropriate size were sequenced． Among these se-
quences，217 (73. 8%) sequences were found to
contain microsatellite repeats (SSＲs)，and 133 of
these sequences with sufficient flanking regions were
suitable for designing locus-specific primers． Final-
ly，total of 100 primer sets were designed for develo-
ping microsatellite loci．
The evaluation criteria for the amplification suc-
cess rate of the loci followed Gao et al． (2012)． Of
the 100 primer pairs tested，28 loci were successful-
ly amplified，of which 22 showed polymorphisms，
and six were monomorphic (Table 2)． Sequences of
these primers were deposited in GenBank under the
accession numbers KC155596 to KC155623． For these
polymorphic primers，the number of alleles per locus
(A)ranged from 2 to 5，with an average of 3. 4，and
the values for the observed (HO) and expected
(HE)heterozygosities ranged from 0. 083 to 0. 792
and from 0. 153 to 0. 744，respectively． Five of the
341期 YAN Li-Jun et al．:Isolation and Characterization of Microsatellite Markers for the Chinese Endemic …
22 polymorphic microsatellite loci deviated from Har-
dy-Weinberg equilibrium (P ＜ 0. 01)(Table 3) ，
likely due to the presence of null alleles or few test-
ed samples included． There was no significant geno-
typic linkage disequilibrium (LD)between any pair
of loci at P＜0. 001．
Table 2 Characteristics of 28 microsatellite loci developed in Ｒ． spinuliferum
Locus Primer sequence (5－3) Ｒepeat motif Size range /bp Ta /℃
GenBank
accession No．
Polymorphic microsatellites
Ｒh003*
F:TCTTCGTCTCCCTCTATCTTT
Ｒ:AACACACACAGACCTCAAATC
(TC)8 152－176 58 KC155596
Ｒh005*
F:ATCATTGCTTCTTTTTCCCT
Ｒ:TCCACCCTCTGTCTCACTCT
(AG)12 164－182 55 KC155597
Ｒh008*
F:TTGGAGTGAGAACAGAGAGG
Ｒ:TAATAGGCAGCATCTCCCAT
(AG)14 202－234 55 KC155598
Ｒh009*
F:GGTAGCCACACTGTTGAAAT
Ｒ:CTTCCCCTCCATCTTGTTCT
(AG)8 216－230 54 KC155599
Ｒh017*
F:TTTGGCTCATCGCTTTTAGT
Ｒ:GAGAGCATCCAAGTCCCTAT
(TC)10 151－175 54 KC155600
Ｒh020*
F:GCATCTCAAGAACACAATA
Ｒ:TCAAGAAGGTCCTCCCAGTC
(AG)9 109－143 51 KC155601
Ｒh031*
F:GAGGAGAGAAAAGGACAAG
Ｒ:AGTCTTCTTCCTTACCAACG
(AG)14 231－237 49 KC155603
Ｒh032*
F:GGGCAAACATTCATACATAA
Ｒ:AGGCAGGCAGGCACCAGAAG
(TC)16 296－308 59 KC155604
Ｒh034*
F:CAAAAAACACACCGCAGACG
Ｒ:TGATGGGTGGATGGATAAT
(AG)9 193－203 52 KC155605
Ｒh037
F:CCTGGGCAAGAGAGAAAACT
Ｒ:ACAGCGATGGCGATTTGAAC
(AG)8…(AG)11 279－287 55 KC155606
Ｒh039*
F:TCCTAATCCCTCCATCTCCC
Ｒ:GCCGTTCCATACAGTACCAA
(TC)10 156－168 57 KC155607
Ｒh041*
F:CGATTGCCATTTGCCACTACCT
Ｒ:CCACAACTCCGCTGCTACTG
(TC)7 148－178 55 KC155608
Ｒh043*
F:AGTTCCCCAAATCTCTTCTC
Ｒ:TCATTTTCTTTTCTCTGCCT
(AG)23 149－175 53 KC155610
Ｒh054
F:TGTAGCAAACCCATCTCACC
Ｒ:TCACCTGGGCATAACTAATC
(TC)8 261－275 58 KC155611
Ｒh058*
F:GATATGGACTCCGACAAGGT
Ｒ:GGCGAGATCGTGGAGAAAAT
(TC)9 174－180 58 KC155612
Ｒh060*
F:AAGAGATTGGAAGGGTTGAT
Ｒ:TCATAGTGTGGCAAAACGAC
(AG)7…(AG)7 166－172 54 KC155613
Ｒh063
F:TGACGACATGGGACTTTAGA
Ｒ:ACCCTTTCTTCATCTTCCAG
(TC)20 164－172 52 KC155614
Ｒh065
F:TAAAAAAATGGGGCTAAAGT
Ｒ:GACATTGACGCAGCCGAACC
(AG)16 261－283 50 KC155616
Ｒh072*
F:GCTCTACCCTTATCATTTTA
Ｒ:AAGACGGACGAAACACATC
(TC)25 169－181 57 KC155617
Ｒh076*
F:ATACACCACCATTCATACGC
Ｒ:TAGAGAGTGGGGTTGATTAG
(AG)17G(AG)6…(AG)8 258－310 20 KC155618
Ｒh078*
F:CAATGATGTGAAAGCCCTGG
Ｒ:AGGATTCCAATTAGTAAACG
(TG)8 284－300 50 KC155619
44 植 物 分 类 与 资 源 学 报 第 36卷
Table 2 continued
Locus Primer sequence (5－3) Ｒepeat motif Size range /bp Ta /℃
GenBank
accession No．
Ｒh086*
F:ATCACCCAAGCAATAGTCTG
Ｒ:ATTTTCCACACGATACAGGC
(TC)9… (TG)8 269－281 57 KC155620
Monomorphic microsatellites
Ｒh023*
F:CTACCATCAACATCACACTG
Ｒ:AGTAAAAAGAGAAGGGGAGT
(TC)8 131 52 KC155602
Ｒh042*
F:CACAAGTGTTCCAAGATTCG
Ｒ:GACGGGAGTTATCGGTGAAG
(TC)7C(TC)9 165 55 KC155609
Ｒh064*
F:GATGGTAGTTTCAACGCAAG
Ｒ:ACTCCTTTCTTTTCTCACCT
(AG)9 193 52 KC155615
Ｒh087
F:AGAATAGAAGGTTGAAGGGT
Ｒ:AAGGCTTGAATGAGGTTGAT
(TC)13 217 52 KC155621
Ｒh096*
F:CCCTCCTCTCTCAACAAAAG
Ｒ:TCAGAGTTGTTCGGTGTGTG
(TC)10 157 54 KC155622
Ｒh098
F:AAACCCCATTACAGTAGATT
Ｒ:ACTGGACCCTTGAAACCTAAC
(AG)9 189 50 KC155623
Note:Ta，PCＲ annealing temperature;* ，successful amplification in Ｒ. spiciferum
Table 3 Ｒesults of the polymorphic microsatellite loci evaluated in two wild populations (12 individuals from each)
of Ｒ. spinuliferum and Ｒ. spicifeum respectively
Locus
Ｒ. spinuliferum
NA HO HE PHW
Ｒ. spicifeum
NA HO HE PHW
Ｒh003 4. 000 0. 739 0. 616 0. 113 3. 000 0. 833 0. 542 0. 005*
Ｒh005 4. 000 0. 375 0. 318 1. 000 2. 000 0. 250 0. 278 0. 502
Ｒh008 4. 000 0. 417 0. 357 1. 000 4. 000 0. 667 0. 587 0. 142
Ｒh009 2. 000 0. 083 0. 153 0. 128 2. 000 0. 545 0. 496 1. 000
Ｒh017 5. 000 0. 750 0. 724 0. 254 2. 000 0. 500 0. 469 1. 000
Ｒh020 4. 000 0. 792 0. 744 0. 090 2. 000 0. 542 0. 430 0. 355
Ｒh023 2. 000 0. 042 0. 117 0. 063
Ｒh031 4. 000 0. 333 0. 510 0. 011 4. 000 0. 500 0. 688 0. 148
Ｒh032 3. 000 0. 565 0. 638 0. 336 4. 000 0. 333 0. 641 0. 001*
Ｒh034 3. 000 0. 250 0. 227 1. 000 4. 000 0. 708 0. 736 0. 023
Ｒh037 4. 000 0. 227 0. 714 0. 000*
Ｒh039 4. 000 0. 667 0. 643 0. 565 3. 000 0. 542 0. 594 0. 477
Ｒh041 4. 000 0. 583 0. 513 1. 000 3. 000 0. 500 0. 492 0. 301
Ｒh043 2. 000 0. 458 0. 430 1. 000 3. 000 0. 250 0. 624 0. 000*
Ｒh054 2. 000 0. 174 0. 476 0. 002*
Ｒh058 4. 000 0. 417 0. 506 0. 091 4. 000 0. 792 0. 711 0. 104
Ｒh060 3. 000 0. 250 0. 473 0. 001* 3. 000 0. 333 0. 645 0. 008*
Ｒh063 3. 000 0. 625 0. 555 1. 000
Ｒh065 3. 000 0. 708 0. 661 0. 670
Ｒh072 3. 000 0. 208 0. 659 0. 000* 5. 000 0. 708 0. 728 0. 523
Ｒh076 4. 000 0. 375 0. 704 0. 000* 6. 000 0. 333 0. 538 0. 016
Ｒh078 3. 000 0. 333 0. 351 0. 157 3. 000 0. 333 0. 586 0. 015
Ｒh086 3. 000 0. 208 0. 320 0. 056 2. 000 0. 000 0. 287 0. 000*
Ｒh096 2. 000 0. 208 0. 492 0. 006*
Note:NA，number of alleles revealed;HO，observed heterozygosity;HE，expected heterozygosity;* ，polymorphic microsatellite loci deviating
from Hardy-Weinberg equilibrium (P＜0. 01)．
541期 YAN Li-Jun et al．:Isolation and Characterization of Microsatellite Markers for the Chinese Endemic …
The 28 microsatellite markers were also tested
in Ｒ. spiciferum using the same PCＲ conditions as in
Ｒ. spinuliferum． Of the 28 loci tested，22 SSＲ mark-
ers were amplified successfully，of which 20 loci
showed polymorphisms and two loci were monomor-
phic (Ｒh042 and Ｒh064)in Ｒ. spiciferum (Table 2
＆ 3)． The two monomorphic microsatellite markers
(Ｒh023 and Ｒh096)in Ｒ. spinuliferum showed pol-
ymorphism in Ｒ. spiciferum (Table 2 ＆ 3)． For the
20 polymorphic markers of Ｒ. spiciferum，the number
of alleles per locus ranged from 2 to 6，with a mean
of 3. 2． The observed (HO)and expected (HE)het-
erozygosity ranged from 0. 000 to 0. 833 and from
0. 117 to 0. 736，respectively． Six of the 20 polymor-
phic microsatellite loci deviated from the Hardy-
Weinberg equilibrium (P＜0. 01)(Table 3)．
In summary，of the 28 microsatellite markers
firstly developed in Ｒ. spinuliferum，most worked in
Ｒ. spiciferum (79%)． Thus，these codominant mic-
rosatellite markers developed in this study will be
very useful to investigate the hybrid speciation sce-
nario between Ｒ. spinuliferum and Ｒ. spiciferum，and
also be useful to assess the genetic diversity and
population structure of Ｒ. spinuliferum and other
closely related species．
Acknowledgements:We are grateful to JB Yang，J Yang，
HT Li，WB Yuan and CY Lin for their help in lab work and
data analysis． We also thank Michael Mller from Ｒoyal Bo-
tanic Garden Edinburgh for improving English of the MS． La-
boratory work was performed at the Laboratory of Molecular
Biology at the Germplasm Bank of Wild Species，Kunming
Institute of Botany，Chinese Academy of Sciences．
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64 植 物 分 类 与 资 源 学 报 第 36卷