全 文 ：园 　艺 　学 　报 　2007, 34 (2) : 311 - 316
Acta Horticulturae Sinica
Rece ived: 2006 - 07 - 08; Accepted: 2006 - 12 - 26
Supported by the Youth Foundation of Shandong Academy of Agricultural Sciences (2005YQ013) , International Science and Technology Co2
operation Projects (2006DFA33130) , and by the Chinese National Program s for H igh Technology and Development (2006AA100108)3 Author for correspondence ( E2mail: qzliu@ sdip1cn)
D evelopm en t of SSR M arkers in Sweet Cherry Using Selec2
tively Am plif ied M icrosa tellite
A I Cheng2xiang1 , YU Xian2mei2 , ZHANG L i2si1 , W E I Hai2rong1 , X IN L i1 , YUAN Ke2jun1 , J IN Song2nan1 ,
SUN Q ing2rong1 , and L IU Q ing2zhong13
(1 Shandong Institu te of Pom ology, Key L abora tory for Fru it B iotechnology B reeding of Shandong, Ta ipian, Shandong 271000,
China; 2 Institu te of Environm ent and Plan t P rotection, Chinese A cadem y of Tropical A gricu ltural Sciences, D anzhou,
Hainan 571737, Ch ina)
Abstract: A total of 100 sequenceswere isolated and cloned by SAM ( Selectively Amp li2
fied M icrosatellite) and another one was obtained from the NCB I and EMBL databases. 82 SSR
sequences were used to design the special p rimers at 77 loci from 69 fragments. Thirty2eight
pairs of special p rimerswere synthesized, matching with 5′anchored degenerate SSR p rimer, to
detect 36 SSR loci. 27 p rimer pairs amp lified clear and robust DNA fragment, of which nine2
teen pairs of SSR p rimers amp lified the corresponding SSR sequences and eight amp lified the
unexpected fragments. 24 polymorphic p rimer pairs were selected from the 27 p rimer pairs by
using the genom ic DNA of 27 sweet cherry germp lasm, and 24 locus2specific SSR markerswere
obtained.
Key words: P runus avium L. ; Germp lasm; SSR markers; Selectively Amp lified M icro2
satellite
甜樱桃 SSR标记的选择性扩增微卫星 ( SAM ) 法筛选
艾呈祥 1 , 余贤美 2 , 张力思 1 , 魏海蓉 1 , 辛　力 1 , 苑克俊 1 , 金松南 1 ,
孙清荣 1 , 刘庆忠 13
(1 山东省果树研究所 , 山东省果树生物技术育种重点实验室 , 山东泰安 271000; 2 中国热带农业科学院环境与植物保
护研究所 , 海南儋州 571737)
摘 　要 : 以甜樱桃 ‘红灯 ’为试材 , 应用选择性扩增微卫星 ( SAM ) 法分离、克隆了 100个 SSR序
列 , 其中 81个非重复 , 可用。加上搜索数据库所获得的 1个 SSR序列 , 一共 82个序列用于特异引物的设
计。仅从 69个序列的 77个基因座设计出特异引物。合成 38对特异引物 , 对其中的 36个基因座进行检测。
其中 19对引物扩增出相应大小的片段 , 另外 8对引物扩增出非预期片段。最后 , 以 27个甜樱桃种质的基
因组 DNA为模板 , 从 27对可扩增出带的引物中 , 筛选出多态性引物 24对 , 获得了 24个甜樱桃基因座特
异性 SSR标记。
关键词 : 甜樱桃 ; 种质 ; SSR标记 ; SAM法
中图分类号 : S 66215　　文献标识码 : A　　文章编号 : 05132353X (2007) 0220311206
Acta Horticulturae Sinica Vol. 34
P runus avium L. is one of the P runus p lants belonging to the fam ily Rosaceae, also called large cherry.
It is the earliest p recocious deciduous fruit tree in North China ( Fang, 1996). The fruit2selection seeds and
bud2selection seeds are most commonly used in sweet cherry ( Sun et al. , 2000). W hile cross breeding of
sweet cherry is just initiated, and the genetic background of sweet cherry is not clear yet. It is necessary to
find out an effective method to analyze the germp lasm s of sweet cherry in an effort to exp lore and utilize these
valuable germp lasm s (W iersma et al. , 2001; Choi et al. , 2002). In recent years, the development of DNA
markers makes it possible to identify sweet cherry cultivars and establish their genetic map (Downey & Iezzo2
ni, 2000; Chen et al. , 2004; W ang et al. , 2005; Cai et al. , 2006). DNA markers are fast and highly effi2
cient, but there should be an accurate analysis standard to increase the results repeatability. Comparatively,
SSR s ( Simp le Sequence Repeats) was a more reliable method to study DNA fingerp rint for its advantages of
high repeatability, rich polymorphism and co2dom inant inheritance ( Yamamoto et al. , 2002).
SSR s has many advantages, but it is not easy to acquire the SSR p rimers. Researchers have developed
SSR p rimers using many methods, such as the classical screening of genom ic library ( Tokuko et al. , 1998) ,
m icrosatellite enrichment (Huang et al. , 1999) , 5′2anchor PCR ( Fisher et al. , 1996) , STMP ( Sequence2
Tagged M icrosatellite Profiling) (Hayden et al. , 2001a) , SAM ( Selectively Amp lified M icrosatellite) (Hay2
den et al. , 2001b) and database blast search (Ram say et al. , 2000). Among them, SAM is a newly2devel2
oped method to develop SSR markers. SSR s have the advantages of high polymorphism of alleles, high recov2
ery of useful SSR, and capable of detecting the polymorphic SSR alleles just with one specific p rimer. Thus,
SAM was used to study SSR markers of sweet cherry in this study so as to p rovide an effective detecting method
to study populational genetic structure of sweet cherry.
1　M a ter ia ls and M ethods
111　Plan t ma ter ia l and D NA isola tion
The 28 cultivars, such as Hongdeng, Sunm i, Sweet heart, Lap ins, were offered by Xingzhuang Taipian,
Sijiazhuang, Dawangzhuang Feicheng, Lüshun Horticulture Field Dalian and Dalian Agricultural Science Insti2
tute. A ll the samp les were conserved in the Taipian Cherry Repository, Shandong Institute of Pomology. Young
leaves of the p lant materials were treated with liquid nitrogen and stored at - 40℃ until being used.
Genom ic DNA was extracted from the young leaves of sweet cherry using Cetyltrimethyl Ammonium B ro2
m ide (CTAB) (A i & L iu, 2006) , and the concentration and purity of DNA samp les were tested.
112　 Isola tion of SSR sequence
SAM segments were isolated from the genom ic DNA of sweet cherry Hongdeng using SAM (Hayden et
al. , 2001b) , after recovery, cloning and sequencing, the fragmentswere analyzed by the software of Sputnik.
SSR sequences were obtained via alignment with other published DNA sequences of sweet cherry blasted from
databases such as NCB I, EM I.
113　SSR pr im ers design
Locus2specific p rimers were designed according to Primer Prem ier 510. The p rimer design parameters
were: length of p rimer (18 - 24 nt) , Tm value ( 51 - 60℃) , GC content ( 45% - 55% ) , rating value of
p rimer ( > 90). The p rimers were synthesized by Beijing SBS Genetech Co. , L td.
114　Selection s of polym orph ic pr im ers
Genom ic DNA of sweet cherry Hongdeng was used to select the polymorphic p rimers. SSR amp lification
parameters: 94℃ 3 m in; 94℃ 45 s, 51℃ 45 s, 72℃ 1 m in, 32 cycles; 72℃ 10 m in. After amp lification,
213
No. 2 A I Cheng2xiang et al. : Development of SSR Markers in Sweet Cherry U sing Selectively Amp lified M icrosatellite
the PCR p roducts were tested by 3% agar2gel electrophoresis and 6% denatured polyacrylam ide gel electro2
phoresis, and then the p rimers p roducing strong bands were selected. Then the polymorphic SSR p rimers were
selected by testing the amp lified efficiency with the genom ic DNA of other 27 sweet cherry cultivars as the tem2
p lates.
2　Results and Ana lysis
211　 Isola tion, clon ing and sequenc ing of SAM segm en ts
Genom ic DNA of sweet cherry Hongdeng was double digested with enzyme M seⅠ and PstⅠ. After being
linked to adap tor, the digested p roducts were amp lified by supp ressed PCR, p re2amp lification and SAM , and
then SAM p roducts were finally isolated after polyacrylam ide gel electrophoresis. The silver2dyed results
showed that there were 18 - 25 legible bands in each gel lane and that the bands size ranged from 80 bp to 650
bp. 100 SAM fragments were random ly selected, cloned and sequenced, 11 of them were identical, and 8 did
not p roduce the desired results, so the remaining 81 segments were used for further study.
212　Results of Sputn ik ana lysis
The 81 sequenced fragments were analyzed by Sputnik, the results showed that SSR sequence existed in
all the fragments, that most of the fragments contained dinucleotide AG/GA and most of the AG/GA repeats
were (AG) 8 , which accounted for 7912%. There were also (AG) 9 , (AG) 10 , (AG) 11 , (AG) 12 , (AG) 13 ,
(AG) 14 , (AG) 16 and (AG) 21. No. 6922 fragment was sequenced and the SSR length was 38 nt. There were
7 fragments containing more than 10 repeats of AG/GA , which accounted for 814%. Besides the dinucleotide
repeat, there were trinucleotide, tetranucleotide and pentanucleotide repeat in a few fragments. There were e2
ven SSR sequence in the m iddle or double end of few fragments, most of which were simp le SSR. Three of the
fragments contained compound SSR: No. 8521 contained ( GA ) 6 ( GAGAG) 4 , No. 3221 contained ( GA ) 5
(CTT) 4 and No. 4623 contained ( GA ) 5 (CTTG) 3.
213　NCB I, EB I bla st and m icrosa tellite sequence ana lysis
Six sweet2cherry2related gene sequences were obtained by blast in the databases of NCB I and EMBL. Af2
ter Sputnik analysis, it was found that the 28S rRNA gene sequence ( ID No. CG3234545) of Italian sweet
cherry contains a GT repeat: dinucleotide 526: 547 - length 22 scores 10 CACACACACAGCACACACACAC.
It is a discontinuous m icrosatellite, interm itted by a C in the m iddle. It was also used to design the locus2spe2
cific p rimers.
214　Spec if ic pr im ers design
Locus2specific p rimers were designed according to the SSR flanks of 82 DNA fragments by Primer Prem ier
510, among them, 13 fragments were not suitable to design p rimers for resulting in p rimer2dipolymer, harbo2
ring hairp in structure or easy to m ismatch. Finally, locus2specific p rimers were designed according to one or
two flanks of 77 SSR loci existing in the 69 fragments, and then, 38 pairs of p rimers ( numbered SC1, SC2
⋯⋯SC38) were synthesized for polymorphism analysis.
215　Selection of polym orph ic pr im ers
The selected 38 pairs of p rimers were used to amp lify the genom ic DNA of sweet cherry Hongdeng, the
results showed that 19 p rimer pairs ( SC2, SC3, SC6, SC7, SC9, SC10, SC13, SC14, SC16, SC19, SC20,
SC23, SC25, SC26, SC29, SC30, SC32, SC36, SC38) p roduced the desired main bands, and belonged to
functional SSR p rimers; that 8 p rimer pairs ( SC1, SC4, SC11, SC15, SC17, SC22, SC24, SC37) did not
p roduce the desired bands. There m ight be two reasons: one is p rimer design is illogical; the other is sequen2
313
Acta Horticulturae Sinica Vol. 34
cing allows for some errors. The main bands amp lified by these 8 p rimer pairswere not compatible with the de2
sired, but they could be used to test the polymorphism for their recurrence and good legibility.
The selected 27 pairs of p rimers were used to amp lify the genom ic DNA of 27 sweet cherry cultivars, 20
p rimer pairs ( SC1, SC2, SC3, SC4, SC6, SC7, SC9, SC10, SC13, SC16, SC19, SC20, SC22, SC23,
SC26, SC29, SC30, SC32, SC36 and SC37) demonstrated distinct polymorphism: each locus had 2 or more
alleles; SC4 and SC7 had 6 alleles; SC26 had 5 alleles. 4 p rimer pairs ( SC11, SC14, SC17 and SC24) had
only 1 allele, but they demonstrated polymorphism among the studied materials, and p roduced the desired
fragments, so they belonged to polymorphic p rimers. 3 p rimer pairs ( SC15, SC25 and SC38) were regarded
as monomorphic for they p roduced identical band type in most of the samp les and even w ith no p roducts in
few samp les. In conclusion, 24 pairs of SSR p rimers
demonstrated polymorphism among the sweet cherry
cultivars according to 24 SSR loci. The SSR amp lified
p roducts of sweet cherry Hongdeng were tested by
agar2gel electrophoresis ( Fig11 ) and 6% denatured
polyacrylam ide gel electrophoresis ( Fig12 ). From
Fig11 and Fig12, it could be concluded that the latter
had higher resolution p recision than the former and
had the capability to separate the DNA fragments just
having the one2base discrepancy. F ig. 1　SSR prof ile of Hongdeng am plif ied bySSR w ith d ifferen t pr im ers1: Primer SC1; 2: Primer SC2; 3: Primer SC6; 4: Primer SC9;5: Primer SC22; 6: Primer SC26; 7: Primer SC29; M: DNA marker.
Table 1　Polym orph ic SSR markers ( loc i) and the ir pr im ers
Name of
locus
Primer sequence
(5′→3′)
Repeat
type
Fragment size
( bp) No. alleles
SC1 ATGGTGTGTATGGACATGATGA /CCTCAACCTAAGACACCTTCACT (AG) 8 224 3
SC2 ATTCGGGTCGAACTCCCT/ACGAGCACTAGAGTAACCCTCTC (AG/GA) 8 304 4
SC3 ACCCACAAATCAAGCATATCC /AGCTTCAGCCACCAAGC (AC) 4 TT( TA) 5 175 2
SC4 ATGGAAGGGAAGAGAAATCG/GTCATCTCAGTCAACTTTTCCG (AG) 8 120 6
SC6 TGATACCACCATCCAATCTAGC /TTGCTGGGACATGGTCAG ( GA /AG) 8 195 2
SC7 TGCATGAGAAACTTGTGGC /CCAAGAGCCTGACAAAGC (AG) 8 208 6
SC9 GGACGGACAGAAATGAAGGT/CCTTAACCCACGCAACTCC (AG) 9 286 4
SC10 TGAAGGATGGCTCTGATACC /AATTCATCTACTTCTTCCTCAAGC ( GA) 8 207 3
SC11 CAATTTTAAGCACAGGGATC /CAATGTGAATTGGCCATCAC (AG) 10 225 1
SC13 CACTATTTTATCATGGACGG/CGAATTGAGAGTTCATACTC (AG) 8 195 2
SC14 AATAAGGGAGGAGAGAAAGGGTGC /TCTAGCATTGTCCATCACGTCT (AG/GA) 8 220 1
SC16 GCTAATATCAAATCCCAGCTCTC /TGAAGAAGTATGGCTTCTGTGG (AG) 8 116 2
SC17 ACCACTTTGAGGAACTTGGG/CTGCCTGGAAGAGCAATAAC (AG) 11 214 1
SC19 TGTGCTAATGCCAAAAATACC /ACATGCATTTCAACCCACTC (AG/GA) 8 176 3
SC20 ACGTAAAAAGCCCTCAAACC /TTGCTTACGCGTGGACTAAC (AG/GA) 8 180 2
SC22 CTCCTTGACTTTGAAGTTGC /CTGATCGAGAGTAATAAAG ( GA) 8 264 4
SC23 TTGGAAACGGCCATAACACAAGCC /ATGTGCGAATCTCGGGTCGAT (AG) 14 104 3
SC24 GGAACAACTAGAGAGAACCAAGT/TTGCCTATCCTGCCCCGTATCAC (AG) 9 180 1
SC26 AAGTCAGCAACACCATATGC /CCCACTGTTCATGAGTTTCT (AG) 13 246 5
SC29 TTCTGCGACCTCGAAACCGA /GCTAGGGTTTTCATTTCTAG ( GA) 8 282 3
SC30 CGAGGTTGTTGTTCATCATTAC /GATCTCAAGTCAAAAGGTGTC (CA) 7AA (CA) 5 108 4
SC32 GAACATGATGATTGGCCTC /CCAAACATGACATATGTCCC (AG) 12 172 2
SC36 AAGCTCAATTGGCGTTGCTA /CTTGCCTCGACGGTATGGTA (AG) 8 306 3
SC37 AAGCGGAAAGCACAGGTAG/TTGCTAGCATAGAAAAGAATTGTAG ( GA) 8 220 4
413
No. 2 A I Cheng2xiang et al. : Development of SSR Markers in Sweet Cherry U sing Selectively Amp lified M icrosatellite
F ig. 2　Polym orph ic electrophoresis pa ttern of spec ia l SSR pr im er SC26
M: DNA marker; 1: Zaodaguo; 2: Tieton; 3: B lack tartarian; 4: B ing; 5: Sunm it; 6: Lap ins; 7: Youyi; 8: Sweet Heart;
9: Stella; 10: D ragons Yellow; 11: Zuotengjin ; 12: Hongfeng; 13: Changbahong; 14: Elton; 15: Red Robin; 16: J iahong;
17: Van; 18: Santina; 19: Burlat; 20: Laiyang Short Cherry; 21: O regon; 22: Early Ruby; 23: Jueze; 24: Hongyan;
25: B igarreau Moreau; 26: Rainier; 27: Napoleon.
3　D iscussion s
M icrosatellites were traditionally isolated by constructing genom ic library and then hybridized with SSR ra2
dioactive2isotope2labeled or D IG2labeled p robes, it is not only a waste of manpower and money, but also low
efficient, and not easy to obtain positive clones (only 1% - 3% of possibility) ( Tokuko et al. , 1998; Hay2
den et al. , 2001b). W hile the possibility of obtaining positive clones increased to 50% by m icrosatellite en2
richment. In this study, SSR sequenceswere isolated efficiently using SAM , and 100 DNA fragmentswere iso2
lated and cloned totally. After sequencing, all the fragments were confirmed to harbor SSR sequences, inclu2
ding the 8 fragments p roducing non2ideal sequence results. The actual possibility of positive clones was 89%
excep t 11 identical fragments.
Generally, functional SSR p rimers were those which can amp lify desired fragments, if the size of amp li2
fied fragments was m ismatched, or the p roducts were dispersed and even nothing, the p rimers were regards as
non2functional SSR p rimers. Fragments amp lified by non2functional SSR p rimers were usually monomorphic
(RÊder et al. , 1998). In this study, 24 p rimer pairs were obtained, and among them, 19 pairs p roduced the
desired bands, so they belonged to functional SSR p rimers. The fragments amp lified by p rimer SC1, SC11,
SC17, SC22, SC37 and so on, were not the desired, while they demonstrated polymorphism and their electro2
phoresis pattern has the typ ical characters of SSR markers, so they should not be classified into non2functional
SSR p rimers. Itm ight resulted from the amp lification p rocess, where the desired fragments of gene locus failed
to amp lify, while the homologous fragments of nonhomologous gene locus were amp lified. However, SSR se2
quences existed in these fragments, so they could be used as locus2specific SSR markers.
SAM is a newly2developed method to study SSR markers, it could p roduce SSR fingerp rint of multilocus
and has a high recovery of useful SSR s. B read wheat was used to clone 20 SAM sequences using SAM to de2
velop locus2specific SSR markers. 20 pairs of SSR p rimers were designed totally, the results showed that 12
pairs of p rimers p roduced the desired fragments, and among the 12 p rimer pairs, 6 pairs demonstrated allele
variation. The possibility of obtaining polymorphic SSR markers was 30% according to the sequenced frag2
ments (Hayden et al. , 2001a). In this study, 100 SAM sequences were isolated and cloned using SAM , and
the SSR p rimers were designed according to 77 loci, of which, 38 p rimer pairs were used for the following
study and 19 pairs p roduced the desired fragments, 8 pairs failed to p roduce the desired fragments. 24 out of
38 pairs demonstrated polymorphism and were used as locus2specific markers. The possibility of obtaining
polymorphic SSR markers in this study was 63% according to the sequenced fragments. The development of
SSR markers will simp lify the evaluation of sweet cherry fam ily, the study of gene flow and populational varia2
513
Acta Horticulturae Sinica Vol. 34
tion, establishment of DNA fingerp rint and genetic map, acquiring of purpose2gene2linked SSR markers of
sweet cherry and molecular2assistant breeding.
References
A i Cheng2xiang, L iu Q ing2zhong. 2006. Rap id isolation of sweet cherry DNA. Deciduous Fruits, (2) : 4 - 5. ( in Chinese)
艾呈祥 , 刘庆忠. 2006. 甜樱桃 DNA的快速提取法. 落叶果树 , (2) : 4 - 5.
Cai Yu2liang, L i Shan, Cao Dong2wei, Q ian Zeng2qiang, Zhao Gui2fang, Han M ing2yu. 2006. U se of amp lified DNA sequences for the genetic a2
nalysis of the cherry germp lasm. Acta Horticulturae Sinica, 33 (2) : 249 - 254. ( in Chinese)
蔡宇良 , 李　珊 , 曹东伟 , 钱增强 , 赵桂仿 , 韩明玉. 2006. 利用 DNA扩增片段序列对樱桃种质资源的遗传分析. 园艺学报 , 33
(2) : 249 - 254.
Chen Xiao2liu, Chen Xue2sheng, Shu Huai2rui, Xu Heng. 2004. RAPD anlaysis of 15 cherry cultivars. Journal of Fruit Science, 21 (6) : 556 -
559. ( in Chinese)
陈晓流 , 陈学森 , 束怀瑞 , 许　衡. 2004. 15个樱桃品种的 RAPD分析. 果树学报 , 21 (6) : 556 - 559.
Choi C, Tao R, Andersen R L. 2002. Identification of self2incompatibility alleles controlled pollination. Euphytica, 123: 9 - 20.
Downey S L, Iezzoni A F. 2000. Polymorphic DNA markers in black cherry ( Prunus serotina) are identified using sequences from sweet cherry,
peach, and sour cherry. J. Am. Soc. Hort. Sci. , 125: 76 - 80.
Fang Zhong2da. 1996. Plant pathology research. Beijing: China Agricultural Press. ( in Chinese)
方中达. 1996. 植病研究方法. 北京 : 农业出版社.
Fisher P J, Gardner R C, R ichardson T E. 1996. Single locusm icrosatellites isolated using 5′anchored PCR. Nucleic Acids Research, 24 (21) :
4369 - 4371.
Hayden M J, Sharp P J. 2001a. Sequence2tagged m icrosatellite p rofiling ( STMP) : a rap id technique for develop ing SSR markers. Nucleic Acids
Research, 29 (8) : e43.
Hayden M J, Sharp P J. 2001b. Tagged development of informative m icrosatellite ( SSR) markers. Nucleic Acids Research, 29 (8) : e44.
Huang H B, Song Y Q, H seiM, Zahorchak R, Chiu J, Teuscher C, Sm ith E J. 1999. Development and characterization of genetic mapp ing re2
sources for the Turkey (M eieagris gallopavo) . The Journal of Heredity, 90 (1) : 240 - 242.
Ram say L, Macaulay M, degli Ivanissevich S, MacLean K, Cardle L, Fuller J, Edwards K J, Tuvesson S, Morgante M, Massari A, Maestri E,
Marm iroli N, Sjakste T, GanalM, PowellW , W augh R. 2000. A simp le sequence repeat2based linkage map of barley. Genetics, 156: 1997
- 2005.
RÊder SMarion, V ictor Korzum, KatjaW endehake, Jens Plaschke, Marie2Helene Tixier, Philippe Leroy, Martin W Ganal. 1998. A m icrosatellite
map of wheat. Genetics, 149: 2007 - 2023.
Sun Q ing2rong, Sun Hong2yan, Shi Yin2p ing, Yang J ian2m ing. 2000. Study of inducing shoot regeneration from matured cotyledons of Laiyang
dwarf cherry. Journal of Shandong Agricultural University (Natural Science) , 31 (3) : 291 - 293. ( in Chinese)
孙清荣 , 孙洪雁 , 石荫坪 , 杨建明. 2000. 莱阳矮樱桃成熟胚子叶不定梢诱导研究. 山东农业大学学报 (自然科学版 ) , 31 (3) : 291
- 293.
Tokuko U jino, Takayuki Kawahara, Yoshihiko Tsumura, Teruyoshi Nagam itsu, H iroshi Yoshimaru, W ickneswari Ratnam. 1998. Development
and polymorphism of simp le sequence repeat DNA markers for Shorea curtisii and other dip terocarpaceae species. Heredity, 81: 422 - 428.
W ang Cai2hong, Tian Yi2ke, Zhao J ing, Dai Hong2yi, W ang Dong. 2005. RAPD analysis of the genetic differences among several cherry varieties.
Acta Bot. Boreal. 2Occident. Sin. , 25 (12) : 2431 - 2435. ( in Chinese)
王彩虹 , 田义轲 , 赵　静 , 戴洪义 , 王　东. 2005. 樱桃品种资源间遗传差异的 RAPD分析. 西北植物学报 , 25 ( 12) : 2431 -
2435.
W iersma P A, W u Z, Zhou L, Hamp son C, Kappel F. 2001. Identification of new self2incompatibility alleles in sweet cherry ( P runus avium L. )
and clarification of incompatibility group s by PCR and sequencing analysis. Theor. App l. Genet. , 102: 700 - 708.
Yamamoto T, Kimura T, Sawamura Y, Manabe T, Kotobuki K, Hayashi T. 2002. Simp le sequence repeat for genetic analysis in pear. Euphyti2
ca, 124: 129 - 137.
613