全 文 ：Study on Genetic Relationship of Purple Tsai-tai
Germplasms with SSR Markers
Zhuoyi DING*, Zhanbing BAI, Yifei WU, Xiaobo ZHOU
Institute of Vegetable, Hunan Academy of Agricultural Sciences, Changsha 410125, China
Supported by Hunan Provincial Natural Science Foundation of China (2008JJ3035).
*Corresponding author. E-mail: dingzhuoyi@163.com
Received: May 22, 2012 Accepted: August 1, 2012A
Agricultural Science & Technology, 2012, 13(8): 1664-1669
Copyright訫 2012, Information Institute of HAAS. All rights reserved Agricultural Biotechnology
Abstract [Objective] This study aimed to explore the genetic relationship of purple
tsai-tai germplasms using SSR molecular markers. [Method] SSR analysis of 45 pur-
ple tsai-tai samples was conducted with 65 pairs of primers selected from cabbage
primers, and the cluster analysis was carried out. [Result] A total of 23 pairs of
SSR primers were screened; cluster analysis showed that the genetic relationship of
purple tsai-tai germplasms had relatively significantly locality, and the 45 purple tsai-
tai samples can be divided into three groups of Sichuan, Hunan and Hubei; further-
more, the similarity coefficients of 45 purple tsai-tai samples were all greater than
0.5 (ranging from 0.547 0 to 0.910 7), indicating that the closer the genetic relation-
ship among purple tsai-tai samples is, the narrower the genetic basis will be. [Con-
clusion] This study provides a theoretical and technical basis for the identification,
protection and breeding of the resources of local varieties of seaweed sprouts. This
study provided theoretical and technical basis for the identification, protection, breed-
ing and utilization of local purple tsai-tai resource.
Key words Brassica campestris L. ssp. chinensis var. perperea Hort.; SSR; Germplasm;
Genetic relationship
A s a local special vegetable orig-inating in China, purple tsai-tai(Brassica campestris L. ssp.
chinensis var. perperea Hort.), also
known as red tsai- tai, however, has
not attracted much attention in the
resource investigation and conserva -
tion[1]. Especially since the 1980s, many
local purple tsai-tai variety resources
are severely complex due to the signifi-
cant increase of social interaction and
human migration, leading to difficulties
in accurate classification based on the
botanical characters and serious phe-
nomena of synonym, homonym, and
repeated conservation of resources.
Therefore, it has important significance
to develop a molecular marker technol-
ogy to accurately identify the genetic
diversity of purple tsai-tai germplasm
resources for investigation, conserva-
tion and utilization of this unique re-
source in China[2-3].
Compared with a large number of
molecular marker technologies, SSR
marker is more suitable for detecting
large-scale groups as an effective tool
for genetic diversity analysis and ge-
netic linkage map construction due to
its codominance, wide distribution in
the genomes, neutral selection, high
repeatability, abundant polymorphism,
low requirement for DNA quality, rapid
detection by PCR, and extensive ex-
change and use after primer se-
quences are published[4]. SSR markers
should be detected with specific
primers. SSR markers for rape, cab-
bage, Chinese cabbage[5] and other cr-
uciferous crops have already been
studied and a large number of primers
have been developed. Therefore,
primers can be directly selected from
the published sequences for purple
tsai-tai research, which provides tech-
nical support for the selection of SSR
markers in this study.
So far, no research of purple tsai-
tai variety resources has been report-
ed at the molecular level. In this study,
the developed SSR primers of cab-
bage were used for analysis of the ge-
netic diversity of local purple tsai-tai
variety resources collected in the main
producing areas, to provide theoretical
and technical basis for the identifica-
tion, protection, breeding and utiliza-
tion of local purple tsai-tai variety re-
sources.
Materials and Methods
Materials
In this study, 45 local purple tsai-
tai varieties collected by Hunan Veg-
etable Research Institute were used
as experimental materials, which were
shown in Table 1.
Methods
DNA extraction
(1) Tubing: 0.2 g of tender purple
tsai-tai leaves were placed into a 2 ml
test tube, and added with two steel
balls with a diameter of 2 mm and 0.3
ml of 2 × CTAB [2% CTAB (cetyl
trimethyl ammonium bromide),100
mmol/L Tris-HCl (pH 8.0), 20 mmol/L
EDTA and 1.4 mol/L NaCl].
(2) Crushing: 45 test tubes con-
taining leaves were placed on the
crusher for shaking at 28 times /s for 6
min.
(3) Water bath: test tubes con-
taining crushed leaves were placed in
65℃ water bath for 90 min, and gently
reversed several times to fully dissolve
DNA in the extraction buffer.
(4) Test tubes were removed from
the water bath pot, added with equal
volume of chloroform / isoamyl alcohol
(24:1), mixed gently, and centrifuged
at 10 000 r/min for 10 min. The super-
natant was collected and transferred to
10 ml centrifuge tubes. This step was
repeated once when sample in the test
tube was more than 0.2 g.
(5) Two volumes of pre-cooled
-20 ℃ ethanol was added into the
centrifuge tubes, and the white floc
DOI:10.16175/j.cnki.1009-4229.2012.08.037
Agricultural Science & Technology
Vol.13, No.8, 2012 Agricultural Science & Technology
2012
Table 1 Experimental purple tsai-tai germplasms used in this study
No. Code Name Locality No. Code Name Locality
1 S1 Yanzhihong Wuhan City, Hubei
Province
24 S92-10 Hongcaitai Jishou City, Hunan
Province
2 S2 Shiyuehong Wuhan City, Hubei
Province
25 S92-17 Hongcaitai Wuchang County, Hubei
Province
3 S3 Zicaitai Wuhan City, Hubei
Province
26 S92-25 Zicaitai Wuhan City, Hubei
Province
4 S4 Zicaitai Xinyang City, Henan
Province
27 S92-26 Zhonghongcai Mawangdui Town,
Changsha City, Hunan
Province
5 S5 Zicaitai Wuhan City, Hubei
Province
28 S92-27 Hongcaitai Chengdu City, Sichuan
Province
6 S6 Zicaitai Wuhan City, Hubei
Province
29 S92-35 Chunhongcai Shumuling, Changsha
City, Hunan Province
7 S7 Hongcaitai Da’an District, Zigong
City, Sichuan Province
30 S93-3 Zicaitai Wuhan City, Hubei
Province
8 S8 Hongcaitai Huaihua City, Hunan
Province
31 S93-4 Daguzi Wuhan City, Hubei
Province
9 S9 Hongcaitai Yaojiatuo of Changsha
City
32 S93-7 Zicaitai Xianning City, Hubei
Province
10 S15 Hongcaitai Tongling City, Anhui
Province
33 S93-8 Daye hongcaitai Hengyang City, Hunan
Province
11 S19 Changsha chihongcai Lituo Town, Changsha
City, Hunan Province
34 S94-9 Erzaozi Shizhong District,
Leshan City, Sichuan
Province
12 S26 Hongcaitai Yibin City, Sichuan
Province
35 S94-10 Jianyezi
hongyoucaitai
Dabai Town, Longquan
District, Chengdu City,
Sichuan Province
13 S90 Yanjiwei Dong’an Town,
Changsha City, Hunan
Province
36 S94-11 Hongcaitai Yuanling County, Hunan
Province
14 S91-9 Hongcaitai Jingzhou City, Hubei
Province
37 S95-2 Zicaitai Jiujiang City, Jiangxi
Province
15 S402 Zaohongcai Shuiduhe, Changsha
County, Hunan Province
38 S95-3 Zicaitai Jiujiang City, Jiangxi
Province
16 S91-4 Yinhua youcaitai Qianjin Town, Luzhou
City, Sichuan Province
39 S95-11 Hongcaitai Guiyang City, Guizhou
Province
17 S92-2 Hongcaitai Yibin District, Yibin City,
Sichuan Province
40 S98-1 Quanhong Tiaoma Town,
Changsha County,
Hunan Provincev
18 S92-3 Modenghong Caiba Town, Yibin City,
Sichuan Province
41 S98-2 Hongcaitai Liuyang City, Hunan
Province
19 S92-5 Hongcaitai Wanzhou District,
Chongqing Municipality
42 S93-9 Hongcaitai Suizhou City, Hubei
Province
20 S92-6 Xiaoye hongyoucaitai Leshan City, Sichuan
Province
43 S93-15 Hongcaitai Wuhan City, Hubei
Province
21 S92-7 Xiaotaizi Chenghua District,
Chengdu City, Sichuan
Province
44 S92-4 Hongcaitai Changsha City, Hunan
Province
22 S92-8 Hongcaitai Chenzhou City, Hunan
Province
45 S92-16 Hongcaitai Wuhan City, Hubei
Province
23 S92-9 Daye hongyoucai Tangba Town, Qianwei
County, Sichuan
Province
was collected (the mixture was cen-
trifuged to collect precipitate in case no
white floc was obtained). The white
floc or precipitate was dissolved in 0.5
ml of ddH2O for use.
Determination of DNA concentra-
tion and purity Concentration of the
extracted DNA was determined with
ND-1000 UV Spectrophotometer, and
the extracted DNA was diluted to 60
μg/μl for use.
PCR detection and denaturing
polyacrylamide gel electrophoresis
detection The total PCR reaction vol-
ume was 15 μl, containing 1 × buffer,
125 μmol/L dNTPs, 0.375 U Taq DNA
polymerase, 0.4 μmol/L primers, and
40 ng of DNA. The RAPD amplification
was started with initial denaturation at
94 ℃ for 5 min, followed by 35 cycles
of denaturation at 94 ℃ for 1 min, an-
nealing at 55 ℃ for 1 min, and exten-
sion at 72 ℃ for 90 s; the amplification
was completed by holding the reaction
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2012
Table 2 DNA concentrations determined with spectrophotometer
No. D260/D280 DNA concentration∥ng/μl No. D260/D280 DNA concentration∥ng/μl
1 2.13 753.5 24 2.32 238.2
2 2.10 802.9 25 2.12 886.7
3 2.11 939.8 26 2.18 516.4
4 2.33 423.0 27 2.06 918.3
5 2.23 305.3 28 3.98 153.9
6 2.16 1 136.8 29 2.02 732.2
7 1.7 549.6 30 2.06 703.2
8 2.12 684.7 31 2.07 1 016.2
9 2.12 416.7 32 2.36 496.6
10 2.87 213.6 33 2.29 508.1
11 2.11 747.7 34 2.14 487.9
12 2.19 455.7 35 2.16 894.6
13 2.30 471.7 36 1.92 347.9
14 2.42 302.8 37 2.04 978.7
15 2.20 478.7 38 2.10 217.5
16 2.28 558.0 39 2.21 378.2
17 2.15 700.6 40 1.99 1 202.8
18 2.34 483.5 41 2.12 494.8
19 2.30 534.6 42 2.47 209.0
20 2.24 485.2 43 2.42 400.5
21 2.64 361.3 44 1.81 949.1
22 2.18 447.4 45 2.50 215.6
23 2.11 540.9
Table 3 SSR primers of purple tsai-tai used in this study
Code 5’ sequence 3’ sequence
090326C31 CAAATGTCTCAAGACACATAAACCA CTAAAGCAGCAATTGGGTGTTC
090326C32 ATGCAAGCTTCATGGTGTCA CATCAGCAAAATTTCATTTGTGT
090326C33 GCCTCTACCTGGCTTCAGCA TCATTTGGCGCATACTTCCA
090326C34 CTCGTCTTCTTCACCTACAAC CTGACATCTTTCTCACCCAC
090326C35 CCTCTTTTAATTCAAACAAGAAATCA TTCGGACAATGGCAGTGATA
090326C36 CACCTTATCATCTCTCTATCCC CCTCTGTTTCTCTCCTTGTG
090326C37 CATCCTAATGTTGCTGAGAAAGAGG TATATGAAACCGATGAAGCTCCTTT
090326C38 ACTCAACAACCGAACAAGAAAAACA CGGTAGAGAACAGAGGAAGCCTAAG
090326C39 AACTTAACCGAAACCGAGATAGGTG AATCTCGAAATTCATCGACTTCCTC
090326C40 TTTAACAACAACCGTCACGC CTCCTCCTCCATCAATCTGC
090326C41 AACACTTGCAACTTCATTTTCC CATTGGTTGGTGAATTGACAG
090326C42 TTGAAGTAGTTGGAGTAATTGGAGG CAGCAGCCACAACCTTACG
090326C43 TCGCGACGTTGTTTTGTTC ACCATCTTCCTCGACCCTG
090326C44 TCTTCAGGGTTTCCAACGAC AGGCTCCTTCATTTGATCCC
090326C45 TTCCCAAGCTTGCTGGTACT GAGATTTCCCTCGCTTGATG
090326C46 GCGCCAATTATAAATTTGATTTTC TCCTCCTGAACCTGGTCTTG
090326C47 CAGTGAAGTTCAACCGCAGTA CATGAGTGAACATAAAACAGTGAAA
090326C48 AAAGTCGTGGGAAGTATCGT AGGTGTAAGGATGGTGGTAGT
090327A01 GGATTGCCTGAGTTTATTCTT TCTGGAGTAGATGCTTTGGT
090327A02 ATGGCTGTAGAAACACATTGA CTGACAACACGAGCATCTTAC
090327A03 GTTCAGTTCCCAGATTCCTAA TTTCCTCTTCCTTCTCTCTTC
090327A04 CGTCCGTAGCGCTATTTTTCAGA ACGTTGTCGATCGCCCAGTTC
090327A05 GACGCCTCAATTGCTTACTT AGGGAATGAGGATGGGTCTG
mixture at 72 ℃ for 10 min to allow
complete extension of PCR products.
PCR products were detected by using
electrophoresis on 8% polyacrylamide
gel at 160 V for 80 min, stained, ob-
served and photographed.
SSR primer screening and PCR
amplification of experimental mate-
rials Three experimental materials,
S7, S1 and S90, which were respec-
tively collected from Sichuan Province,
Hubei Province and Hunan Province,
were selected as the template for
primer screening; 65 pairs of primers
with more amplified bands were
screened from cabbage primers (of-
fered by Institute of Vegetables and
Flowers, Chinese Academy of Agri-
cultural Sciences) for PCR amplifica-
tion and electrophoresis of various
experimental materials.
Data analysis To ensure the accu-
racy of the experimental results, all
PCR reactions were repeated twice,
and the results showed that stable
bands were amplified in both the two
PCR amplifications. According to the
migration position of the amplified
bands in the same electrophoresis,
results with amplified bands was de-
noted by 1, while that without amplified
band was denoted by 0, to construct a
[1, 0] binary sequence matrix. Subse-
quently, the genetic similarity coeffi-
cients were calculated by using Dice
method of Qualitative in the similarity
analysis module of NTSYS-PC2.1
software, and cluster analysis was
conducted by using UPGMA method
of SAHN in cluster analysis module of
NTSYS-PC2.1 software.
Results and Analysis
Determination of DNA concentra-
tion
One microliter of sample was col-
lected from 0.5 ml of DNA to deter-
mine the DNA concentration under
OD260/OD280 with micro-spectropho-
tometer ND-1000. As can be seen
from Table 2, concentrations of the
extracted DNA were relatively high; to
be specific, DNA concentration of
Sample 28 was the lowest of 153.9
ng/μl, indicating that there might be
DNA fragments; however, DNA con-
centrations of other samples were
about 200 ng/μl; DNA concentrations
of three samples were higher than
1 000 ng/μl, specifically, DNA concen-
tration of Sample 40 was the highest of
1 202.8 ng/μl. OD260/OD280 of various
experimental materials, except Sam-
ple 28, ranged from 1.7 to 2.6.
Amplification results and screening
of different primers
All the 65 pairs of primers ampli-
fied bands, and the number of bands
amplified with each primer ranged
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2012
M, DNA Marker; 1-45, various purple tsai-tai samples.
Fig.1 SSR amplification results of 45 purple tsai-tai samples
from 1 to 10; averagely each pair of
primers amplified 5 bands, and the two
main bands were clearly visible, indi-
cating that the DNA extraction method
used in this study was feasible.
Specifically, 23 pairs of primers am-
plified more bands with better stability,
which were selected for SSR analysis
of purple tsai-tai resources, as shown
in Table 3.
The selected 23 pairs of primers
were used for PCR amplification and
denaturing polyacrylamide gel elec-
trophoresis of 45 purple tsai-tai sam-
ples, results showed that the ampli-
fied fragments were mostly 200 -
300 bp long, and relatively clear
bands were amplified from all the 45
purple tsai-tai samples. As shown in
Fig.1, amplification results of a single
pair of primers greatly varied, indicat-
ing that purple tsai-tai has relatively
good polymorphism.
Genetic similarity coefficients
Genetic similarity coefficients
among 45 experimental materials
were analyzed by using NTSYS-PC
software, results showed that varia-
tions of the genetic similarity coeffi-
cients among 45 experimental materi-
als ranged from 0.547 0 to 0.910 7,
with an average of 0.718 2. Samples
S92-5 and S92-35, S92-26 and S98-2,
S95-5 and S26, S95-2 and S5, S6 and
S91-4 had relatively great genetic vari-
ations (low similarities) of 0.547 0,
0.552 4, 0.554 5, 0.565 7, and 0.569 0,
respectively; samples S93-7 and S93-
9, S93-7 and S93-15, S93-9 and S93-
4, S93-7 and S93-4, S92-10 and S92-
9 had relatively small genetic varia-
tions (high similarities) of 0.910 7,
0.886 8, 0.877 2, 0.870 4, and 0.866 7,
respectively; to be specific, the simi-
larity coefficient between S93-7 and
S93-9 reached 0.910 7, indicating that
these two sample might be the same
variety. Similarity coefficients among
45 experimental materials were all
greater than 0.5, with an average of
0.718 2, indicating that purple tsai-tai
germplasm resources have relatively
small SSR genetic variations and nar-
row genetic basis.
Cluster analysis
According to the genetic similarity
coefficients, cluster analysis of 45 pur-
ple tsai-tai varieties was conducted
with group average method. As shown
in Fig.2, the 45 purple tsai-tai varieties
could be divided into three groups.
Group Ⅰ included 23 varieties and
could be divided into four sub-groups:
sub-group Ⅰ-1 included S3 (Wuhan
City of Hubei Province), S7 (Zigong
City of Sichuan Province), S92-7
(Chengdu City of Sichuan Province),
S15 (Tongling City of Anhui Province),
S92-2 (Yibin City of Sichuan Pro-
vince), S94-9 (Erzaozi, Leshan City of
Sichuan Province), S92-16 (Wuhan
City of Hubei Province), S94-10
(Jianyezi hongyoucaitai, Longquan
District of Chengdu City), S92-17
(Wuchang County of Hubei Province),
S95-11 (Guiyang City of Guizhou
Province), S402 (Changsha County of
Hunan Province), and S90 (Changsha
City of Hunan Province); sub-group
Ⅰ-2 included S92-26 (Zhonghongcai,
Changsha City of Hunan Province),
Fig.2 Cluster analysis of 45 purple tsai-tai samples
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2012
Responsible editor: Xiaohui FAN Responsible proofreader: Xiaoyan WU
S94-11 (Yuanling County of Hunan
Province), S8 (Huaihua City of Hunan
Province), S92-9 (Daye hongyoucai,
Qianwei County of Sichuan Province),
S92-10 (Jishou City of Hunan Pro-
vince), and S6 (Wuhan City of Hubei
Province); sub-group Ⅰ-3 included S9
(Yaojiatuo of Changsha City), S92-35
(Chunhongcai, Shumuling of Chang-
sha City), and S19 (Chihongcai, Lituo
Town of Changsha City); sub-group
Ⅰ-4 included S92-4 (Changsha City of
Hunan Province) and S92-8 (Chen-
zhou City of Hunan Province). Group
Ⅱ included only five varieties: S98-2
(Liuyang City of Hunan Province),
S95-3 (Jiujiang City of Jiangxi Pro-
vince), S91-4 (Yinhua youcaitai, Qian-
jin Town of Luzhou City), S93-8
(Hengyang City of Hunan Province),
and S95-2 (Jiujiang City of Jiangxi
Province). Group Ⅲ included 17 vari-
eties and could be divided into three
sub-groups: sub-group Ⅲ-1 included
S5 (Wuhan City), S2 (Shiyuehong,
Wuhan City), S91-9 (Jingzhou City of
Hubei Province), and S93 -3 (Wuhan
City); sub-group Ⅲ-2 included S93-4
(Wuhan City), S93-9 (Suizhou City of
Hubei Province), S93-7 (Xianning City
of Hubei Province), S93-15 (Wuhan
City), S1 (Yanzhihong, Wuhan City),
S4 (Xinyang City of Henan Province),
S92-25 (Wuhan City), and S92-27
(Chengdu City of Sichuan Province);
sub-groupⅢ-3 included S98-1 (Quan-
hong, Tiaoma Town of Changsha
County), S92-5 (Wanzhou District of
Chongqing Municipality), S26 (Yibin
City of Sichuan Province), S92-3
(Modenghong, Yibin City of Sichuan
Province), and S92-6 (Xiaoye hongy-
oucaitai, Leshan City of Sichuan
Province).
The three groups have significant
locality. Specifically, group Ⅰ covers
most varieties, which are mainly Hu-
nan and Sichuan varieties, sub-groups
Ⅰ-1 andⅠ-2 are mainly composed of
Sichuan varieties, while sub-groups
Ⅰ-3 andⅠ-4 are mainly composed of
Hunan varieties, suggesting that
Sichuan and Hunan purple tsai-tai va-
rieties have relatively close phyloge-
netic relationship and abundant genet-
ic basis; however, group Ⅲ is mainly
composed of Wuhan varieties, with
close phylogenetic relationship, indi-
cating that Wuhan purple tsai-tai vari-
eties have no abundant resources.
Discussions
Rapid extraction of purple tsai-tai
DNA
Acquiring high-quality genome
DNA is the basis of molecular biology
experiments. Although the technolo-
gies for DNA extraction from plant tis-
sues are relatively mature, large-scale
DNA extraction with traditional CTAB
and SDS methods is time-consuming
and labor-consuming, while DNA ex-
traction using kits leads to low quantity
and high cost.
In this study, the total DNA ex-
traction method has fewer steps, less
reagents, low cost, and relatively easy
operation, which is unlikely to cause
cross-contamination and ensures the
accuracy of PCR analysis. During the
extraction process, the collected
leaves were quickly transferred to 2 ml
test tube and added with 0.3 ml of 2 ×
CTAB buffer to effectively control the
browning, and the remaining opera-
tions require gentle movements and
avoid excessive vibration, to prevent
DNA fragmentation and degradation;
the centrifugation speed should be
controlled within 13 000 r/min, other-
wise it will be difficult for extracted
DNA dissolving in water or TE. In order
to prevent oxidation, the proportion of
β-mercaptoethanol in extraction buffer
can be appropriately increased to 2％
CV/V and the amount of PVP can be
enhanced, which can effectively ex-
clude the interference of polyphenols
to prevent browning. UV spectropho-
tometer and SSR-PCR detection
showed that the extracted DNA has
large quantity and good quality.
Genetic diversity analysis of purple
tsai-tai
All markers should be able to
evenly cover the entire genome for
genetic diversity analysis of germ-
plasm resources. The 23 pairs of SSR
primers adopted in this study were of-
fered by Institute of Vegetables and
Flowers, Chinese Academy of Agri-
cultural Sciences, while the specific
distribution of these primers in the
chromosomes is unclear and requires
for further improvement. However, the
results were accurate, and the cluster
analysis well revealed the phyloge-
netic relationship of the experimental
materials.
In addition, the similarity coeffi-
cients among various experimental
materials were all greater than 0.5, in-
dicating that these samples have rela-
tively close phylogenetic relationship,
which to some extent suggests that
current purple tsai-tai germplasms
have small genetic variation and nar-
row genetic basis. Therefore, hy-
bridization with distantly related sub-
species such as Chinese cabbage and
pak choi should be carried out in fur-
ther breeding to expand the genetic
basis of purple tsai-tai.
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灵芝群体交配基因型分析
陈裕新 1，2，3，夏志兰 1，2，刘 鹏 1，2，康信聪 1，2，洪亚辉 4，刘东波 1，2* （1.湖南农业大学园艺园林学院，湖南长沙 410128；2.国家中医药管理局亚健康干
预技术实验室，湖南长沙 410128；3.湖南金叶众望科技股份有限公司，湖南临湘 414300；4.湖南农业大学生物技术学院，湖南长沙 410128）
摘 要 [目的] 研究灵芝群体交配基因型，并与酯酶同工酶试验相比较，探讨它们在群体亲缘关系分析上的异同点。[方法] 采用 OWE-SOJ技术
对 24个灵芝菌株的单孢分离菌株进行标准交配型鉴定，及群体间交配基因型的测定，并结合酯酶同工酶试验对群体间的亲缘关系进行分析。[结
果]鉴定出 7大类交配基因型，并发现 A因子含有 4个等位基因，B因子含有 4个等位基因，及一个特殊的 A混合基因，四类交配型出现了一定程
度的偏分离。酯酶同工酶试验检测出了 28条不同酯酶酶谱带，在相似系数为 0.732 14时，样品被分为 9大类。比较交配基因型分析与酯酶同工
酶试验结果，发现两者具有很高的相似性。[结论] 交配基因型测定可以作为分析菌株间差异和鉴定品种的一种重要补充手段。
关键词 酯酶同工酶；OWE-SOJ技术；交配型；菌落形态
基金项目 国家科技支撑计划“中药产业区域发展及特色产品研究开发”(2006BIA06A20)；科技部科技人员服务企业行动计划“珍稀食用菌系列
产品开发”(2009GJD20012)。。
作者简介 陈裕新（1987-），男，广西来宾人，硕士，从事微生物工程方面的研究, E-mail: hotdogchenyuxin@163.com。*通讯作者，副教授，硕士生
导师，博士，研究方向为中药资源与开发，E-mail: chinasaga@163.com。
收稿日期 2012-06-08 修回日期 2012-07-30
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Responsible editor: Jianli YIN Responsible proofreader: Xiaoyan WU
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SSR分子标记技术研究紫菜薹资源的亲缘关系
丁茁荑 *，白占兵，吴艺飞，周晓波 （湖南省农科院蔬菜研究所，湖南长沙 410125）
摘 要 [目的] 利用 SSR分子标记技术研究紫菜薹资源的亲缘关系。[方法]用从大白菜引物中筛选出的 65对引物，对收集到的 45份紫菜薹地
方资源进行 SSR分析，并对结果进行聚类分析。[结果]试验共筛选出 23对 SSR引物；聚类分析表明，紫菜薹亲缘关系表现出较为明显的地域性，
大致可分为四川、湖南、湖北 3个类群；同时，45份资源的相似系数都大于 0.5（介于 0.547 0～0.910 7之间），表明紫菜薹各品种间亲缘关系较近，
遗传基础较为狭窄。[结论]该研究为紫菜薹地方品种资源的鉴定、保护以及育种利用提供了理论和技术依据。
关键词 紫菜薹；红菜薹；SSR；种质；亲缘
基金项目 湖南省自然科学基金项目“SSR分子标记研究紫菜薹的分类与亲缘关系”（2008JJ3035）。
作者简介 丁茁荑（1966-），男，湖南桃江人，副研究员，在读博士，从事菜薹遗传育种研究，E-mail : dingzhuoyi@163.com。*通讯作者。
致 谢 中国农科院蔬菜花卉所王晓武研究员为本项工作的 SSR引物筛选给予了大力支持和帮助，特此致谢！
收稿日期 2012-05-22 修回日期 2012-08-01
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