全 文 ：第 16 卷 第 1 期
Vol. 16 No . 1
草地学报
ACTA AGRESTIA SINICA
2008 年 1 月
Jan. 2008
Optimization of the SSR Reaction System for Zoysiagrass
Using Orthogonal Design
GUI Jin-shan, WANG Yun-w en, FAN G Cheng*
( Inst itute of Grassland Science, China Agricultural University, M inist ry of Agricul tu re Key Laboratory of Gras sland
Vegetat ion Restorat ion and Reconst ruct ion, Beijing 100094, Chin a)
Abstract: In order to obtain the optimal SSR-PCR system fo r zo ysiag rass ( Zoy sia j aponica) , an experimental design o f o rthog-
onal diagr am L 16 ( 45 ) was employed to evaluate f ive facto rs ( template DNA, M g2+ , dNTP, T aq DNA po lymerase, and pr imer)
at four different levels. Three randomly selected SSR primers ( RM84, RM220, RM531) w ith resembled T m were used to val-i
date the reaction system. The results indicat e t hat o rthogonal design could be used to optimize the SSR-PCR system; the opt-i
mal system of terminal v olume 20 l consisted of 2 l 10 buffer , 1. 0U Taq DNA po lymerase, 0. 2 mM pr imers, 100ng tem-
plat e DNA , 0. 2 mM dNTP , and 2. 0 mM Mg+ . The opt imal annealing temperatur e w as determined to be 56- 58 by g radient
PCR; t he suitable thermal cycling conditions w ith initia l melt ing at 94 for 4 min, fo llow ed by 35 cycles at 94 for 30 s, 56
- 58 for 40 s, 72 for 60 s; then keep the r eaction mix ture at 4 after a f inal extension st ep of 72 for 10 min. The opt-i
mized system would be effect ive as a solid foundation fo r zoy siagr ass SSR analy sis.
Key words: Zoy sia j aponica Steud. ; SSR markers; Reaction sy stem; O rthogonal design; Optimization
结缕草 SSR反应体系的正交优化
桂金山 , 王赟文, 方程*
(中国农业大学草地研究所,农业部草地植被恢复与重建重点开放实验室, 北京 100094)
摘要: 为了快速获得结缕草( Zoy s ia j aponica ) SSR反应体系, 采用 5 因素(模板 DNA, Mg 2+ , dNTP , T aq 酶和引物) 4 水平正
交设计筛选合适的结缕草 SSR体系, 并通过随机挑选 3 对 T m 相近的引物对该优化体系进行验证。结果表明: 正交设计可应
用于 SSR-PCR反应体系的优化, 20 l最佳 PCR 反应体系中包括 2 l 10 buffer、1. 0U T aq 酶、0. 2 mM 引物、100 ng 模板
DNA、0. 2 mM dNTP、2. 0 mM M g+ ;对结缕草进行梯度退火实验,其最佳退火温度为 56~ 58 ;可行扩增程序是: 94 预变
性 4 min、进行 35 个循环的 94 变性 30 s、56~ 58 退火 40 s, 72 延伸 1 min; 72 延伸 10 min, 4 保存;该最适反应体系的
建立,为今后结缕草 SSR 分析奠定了坚实基础。
关键词: 结缕草; SSR 标记; 反应体系; 正交设计; 优化
中图分类号: Q943 文献标识码: A 文章编号: 1007-0435( 2008) 01-0089-06
Introduction
Zoysiag rass ( Zoy sia j aponica Steud. ) belong s
to Zoy sia Willd. g enus of the g ram inneae fam ily.
It is one o f the most impor tant w arm-season mono-
cotyledonous perennial turfgrass o rig inated in A-
sia[ 1] . It form s a very dense turf by means of slow
g row ing undergr ound rhizomes and aboveg round
sto lons. For centuries, it has been used as turf-
gr ass and forag e in China and other countries in A-
sia. Zoy siagrass has a chromosome number of 40
and is most ly a cross- poll inated, tet raploid spe-
cies
[ 2, 3]
. It is o f good w inter-hardiness and high
temperature tolerance and thus adaptable to a w ide
range of environments
[ 4]
. Zoysiag rass is consider ed
收稿日期: 2007-07-26; 修回日期: 2007-12-12
基金项目:国家自然科学基金项目( 30500354)
作者简介: 桂金山 ( 1982-) , 男,湖南张家界人, 硕士研究生,研究方向为牧草生物技术, E- mail : guijinshan 2008@ 126. com; * 通讯作者
Author for corr espondence, E-mail: Fang chen g@ cau . edu. cn
草地学报 第 16卷
to be a resource eff icient or m inimum maintenance
turfgrass, so the number of progr ams employed for
its improvement has incr eased rapidly
[ 5, 6]
.
The potent ial o f biotechno logy applied to turf-
g rass improvement has been recognized in recent
year s
[ 7, 8]
. Mapping of simple sequence repeat
( SSR) markers w ere established for zoysiagrass [ 9] .
It exhibits r epeat units of 1-6 bp in leng th. SSRs
occur ubiquitously and abundant ly in eukar yot ic
genomes
[ 10, 11]
. T heir successful use fo r g enetic a-
nalysis in a number o f plant species reveals that
they ar e highly polymorphic, somat ically stable
and inherited in a co-dominant Mendelian manner.
The abundance and amount of informat ion derived
from such markers, tog ether w ith easy ident ifica-
t ion, make them a valuable too l for genet ic divers-i
ty est imat ion, g enetic linkage const ruct ion, phy s-i
cal mapping , and var ietal ident ificat ion[ 12~ 14 ] . In
practice, how ever, many at tempts to perform PCR
failed to yield meaning ful results, so opt imal PCR
condit ions ar e very important . Here w e report the
ef f icient opt imizat ion o f SSR-PCR sy stem for zo y-
siag rass using or thogonal design[ 15] . T his novel
opt imizat ion sy stem allow s a mo re pr ecise and d-i
r ect w ay to obtain the specif ic optimal r eact ion sy s-
tem w ithout the need of burdensome w or ks.
Materials and methods
Plant materials and Reagents
T hr ee lines o f g enus Zoy sia ( Haiqing1, Ha-i
qing2 and Yesheng zoysiagrass) w er e used in this
study. Among them Haiqing1 and Haiqing2 are
cult ivars of Z. j aponica. var. pallida Nakai ex
Honds, but Yesheng w as a w ild type of Z. j apon-
ica in nat ive countryside of Jiaozhou, Shandong
province. All o f them were under open pollinat ion
env ironment . Seeds w er e al low ed to germinate and
the seedling s w er e g row n in greenhouse in China
Ag ricultural University up to the appearance of
three leaves. T he three leaves each of 10 plants per
bulked sample w ere collected and analy zed. The
primers sequences used in the exper iment w ere
seed do rmancy QTL o f r ice obtained from pr ofes-
so r Gu[ 16, 17] ( RM 84, RM 220, RM 531) and pr ofes-
so r Wan[ 18] ( RM 11) , and synthesized by AuGCT
Biotechnolo gy Synthesis Lab. All of the T aq DNA
polymer ase, 10 buf fer, dNTP, and M g2+ were
pur chased from T IANGEN Biotech ( Beijing) CO. ,
LTD.
DNA preparation
DNA was isolated from fresh leaves of 18-day-
o ld zoy siagrass seedlings using the CTA B method
described by M urray et al
[ 19]
w ith a slight modif-i
cat ion. T he ex t racted DNA was quant if ied by an
ult raviolet spect rophotometer ( U traspec 3100, Bio-
chr om Ltd. ) and its intactness w as analy zed by
1% agarose gel elect rophor esis and diluted to
w orking concentration of 25 ng/ l, stored at 4 . The
results are show n in the table 1 and fig. 1.
Table 1 Optical density and DNA concentration of experiment materials tested by Utraspec 3100
Zoysiagrasses
st rain
Opt ical dens ity( nm )
260 280 320
OD 260 /OD 280
Concent rat ion
g/ m l
YS 0. 463 0. 221 0. 002 2. 10 1152
YS 0. 438 0. 214 0. 006 2. 08 1080
H I 0. 544 0. 269 - 0. 005 2. 00 1372
H I 0. 508 0. 253 - 0. 005 1. 99 1282
H
1
0. 473 0. 220 0. 000 2. 15 1184
H
1
0. 511 0. 235 - 0. 004 2. 15 1288
H
2
0. 136 0. 057 - 0. 008 2. 20 362
H
2
0. 101 0. 042 - 0. 007 2. 20 272
Note: YS: Yesheng; H I: H aiqin g1; H 1: H aiqing2, H 2: Haiqing2
SSR-PCR amplification
The PCR r eact ion system for Z. j ap onica
w ith total 20 l reaction solut ion w as perfo rmed on
a Biometra T-Gr adient Thermoblock pro grammed
for an init ial melting temperature at 94 fo r 4
m in, fo llow ed by 35 cycles at 94 fo r 30 s, 56
90
第 1期 桂金山等:结缕草 SSR 反应体系的正交优化
fo r 40 s, 72 fo r 60 s. A f inal ex tension step at
72 for 10 min w as per formed af ter the 35 cycles.
Fo llow ing the thermocycling, the react ion m ix tur e
w as automatically held at 4 until 1. 5% agarose
gel elect rophoresis w as r un w ith 1T AE buf fer at
100V for 1h and v isualized w ith Bio Imaging Sy s-
tem ( Bio-Rad Laborato ries) after ethidium bromide
( EB) staining.
Confirmation of the factors of PCR and orthogonal
diagram design
Although the basic pr inciples of PCR tech-
nique are so simple, many at tempts performing
PCR in pr act ice failed to yield meaningful results.
Based on the published papers, many people at-
tempted to opt imize PCR sy stem w ith single factor
experiment design. In fact it becomes burdened
w ith some disadvantag es. In or der to conf irm the
f ive main factors of PCR, w e used L16 ( 4
5
) orthog-
onal diagram as exper imental design, each treat-
ment w ith tw o replicat ions. The PCR react ion lev-
els of factors and o rthogonal diag ram design are
shown in table 2 and table 3, r espect ively.
Fig. 1 Results of template DNA electrophoresis
Note: 1- 3: YS ; 4- 6: HI; 7- 9: H 1; 10- 12: H 2; each
sample hole w as loaded w ith 5l DNA solut ion and 1l 6 loadin g
buf fer
Table 2 Factors and levels of SSR-PCR system
Levels
Factor s
T aq E U Mg+ mM Template DNA ng dNT P mM Primer M
1 0. 5 1. 5 25 0. 10 0. 2
2 1. 0 2. 0 50 0. 15 0. 3
3 1. 5 2. 5 75 0. 20 0. 4
4 2. 0 3. 0 100 0. 25 0. 5
Table 3 Orthogonal design[ L16 ( 45 ) ] for Zoysiagrasses SSR-PCR system
T reatment No. T aq E U Mg+ mM Template DNA ng dNT P mM Primer M
1 0. 5 1. 5 25 0. 10 0. 2
2 0. 5 2. 0 50 0. 15 0. 3
3 0. 5 2. 5 75 0. 20 0. 4
4 0. 5 3. 0 100 0. 25 0. 5
5 1. 0 1. 5 50 0. 20 0. 5
6 1. 0 2. 0 25 0. 25 0. 4
7 1. 0 2. 5 100 0. 10 0. 3
8 1. 0 3. 0 75 0. 15 0. 2
9 1. 5 1. 5 75 0. 25 0. 3
10 1. 5 2. 0 100 0. 20 0. 2
11 1. 5 2. 5 25 0. 15 0. 5
12 1. 5 3. 0 50 0. 10 0. 4
13 2. 0 1. 5 100 0. 15 0. 4
14 2. 0 2. 0 75 0. 10 0. 5
15 2. 0 2. 5 50 0. 25 0. 2
16 2. 0 3. 0 25 0. 20 0. 3
Annealing temperature selection
Based on the optimal PCR sy stem, the annea-
l ing temperatures o f the primers w ere selected u-
sing a Biometra T-Gradient T hermoblock pro-
g rammed for gradient PCR. Set ting 53 as the
mid-po sit ion temperature o f the block and gradient
at 12 , then 12 g radient test reactions w ere pro-
duced by the PCR appar atus f rom 49. 0 lef t side
to 59. 0 on right side of the block. The product
w as detected by 1. 5% agarose gel elect rophoresis.
Test of the optimal PCR system
T hr ee SSR primers ( RM 84, RM 220, RM 531)
w ere random ly cho sen according to their Tm resem-
91
草地学报 第 16卷
blance. DNA sample of three zoysiag rass lines
w ere used as template DNA. Primers and tem-
plates w ere amplified in PCR apparatus in order to
test the opt imized SSR-PCR system.
Results
Orthogonal experimental results
According to o rthogonal design diagram
show ed in table 3, the amplified product w as run
w ith 1. 5% agarose gel elect rophoresis and 1
TAE buf fer at 100V for 1h and visualized w ith Bio
Imaging System ( Bio-Rad Laboratories ) af ter
ethidium bromide ( EB) staining. The results show
significant dif ference in f igur e 2 due to the dif ferent
concentrat ion combinations of the f ive react ion fac-
tors ( T aq DNA polymerase, primers, template
DNA, dNTP and M g
+
) . Amplificat ion w as detec-
ted fo r al l t reatments w ith an except ion of t reat-
ment 1. Although the amplif icat ion lanes w ere de-
tected, but only t reatment 3, 10, 15 had strong
product amplificat ion. In or der to obtain signif-i
cant pr oduct amplificat ion, t reatment 3, 10, 15
w ere used to retest , but only t reatment 10 alw ays
received stable amplif icat ion among them. So, w e
cho se t reatment 10 ( 1. 5 U Taq DNA polymer ase,
0. 2 mM pr imer s, 100 ng template DNA, 0. 2 mM
dNTP and 2. 0 mM M g
+
) as the opt imal sy stem
for zoy siagrass amplif ication.
Fig. 2 Amplification results of each treatment based on the orthogonal design diagram (Table 3)
Note: M : Mar ker; 1~ 16: Treatment 1~ 16 w ith tw o repeats ( see tab le 3) ; Prim er: RM11; each sample hole w as
loaded w ith 5 l PCR r eact ion s olut ion and 1 l 6 loading buf fer; same as th e follow s
PCR anneal ing temperature selection
In many situations, changes in annealing tem-
perature are effect ive w ays to reach the special am-
plif ication o f specif ic materials [ 20] . As show n in
figure 3, spur ious PCR products could be signif-i
cant ly reduced by raising the annealing tempera-
ture. Therefore, it is useful to set up a series o f
test react ions o f the g radient annealing tempera-
tures so that the temper ature yielding the desired
ratio of appropriate products to spurious products
can be selected. As the annealing temperature in-
creases and approaches to the Tm of the pr imer s,
the yield o f the desired product usual ly falls . How-
ever, in the experiment , annealing the primer
RM 11 ( Tm= 58. 0) at 59. 0 high temper ature a-l
so resulted in considerable amplificat ion, possibly
due to the pr imer having a high G + C content
( GC% = 61. 1) . According ly, the 57. 9 annea-
ling temperature w ould be recommended as opt imal
temperature of this primer.
Fig. 3 Results of gradient annealing temperature
in the SSR system
Note: Th e annealin g temperatur e 1- 12 represents 47. 0 , 47.
3 , 48. 1 , 49. 4 , 50. 8 , 52. 3 , 53. 7 , 55. 1 , 56. 6 ,
57. 9 , 58. 7 , and 59. 0 , respectively
Test results of the optimal PCR system
In opt im izing PCR condit ions, the three prim-
ers ( RM 84, RM 220, RM531) combined w ith the
92
第 1期 桂金山等:结缕草 SSR 反应体系的正交优化
template DNA of the thr ee zoy siagrasses lines w er e
used to test the reliability of the optimal sy stem.
According to f igure 4, the thr ee primers yielded
the desired amplificat ion, but t reatment 7 w as an
exception. Based on the previous experiences, w e
only altered the T aq DNA po lymerase fr om 1. 5 U
to 1U in the vo lume of 20 l react ion system, and
then all the t reatments yielded the stable and de-
sired amplif ication. The result is show n in the f ig-
ure 5.
Fig. 4 Amplification results from the best treatment
( Figure 2 No. 10) system
Note: 1 - 3: Primer RM84; 4- 6: Primer RM220; 7 - 9:
Prim er RM 531; 1, 4, 7: T emplate YS; 2, 5, 8: T em plate HI; 3,
6, 9: Template H
Fig. 5 Amplification results of treatment 10 ( Table 3)
with a small adjustment
Discussion
In this study, the orthogonal experimental de-
sign w as employed and proved reliable in the analy-
sis of opt im izing SSR-PCR sy stem . T o our know-l
edge, this is the f ir st report o f opt imizing SSR-
PCR system for zoysiag rass using orthogonal de-
sign. T he study concluded that the opt imal reac-
t ion system consisted of a total volume of 20 l
w ith 2 l 10 buf fer, 1. 0 U Taq DNA po lymer-
ase, 0. 2 mM primers, 100 ng template DNA, 0. 2
mM dN TP and 2. 0 mM M g+ . The suitable ther-
mal cycling conditions were init ial melt ing at 94
fo r 4 min, followed by 35 cycles at 94 for 30 s,
56- 58 fo r 40 s, 72 for 60 s. A f inal ex tension
step at 72 for 10 min w as perfo rmed and then the
react ion m ix tur e w as automat ically held at 4 .
T he quality and r eproducibility o f PCR prod-
ucts are markedly affected by several parame-
ter s
[ 20]
. In part icular, the quality of DNA tem-
plate, Taq DNA polymerase, the M g+ concentra-
t ion, the dN TP concentrat ion, and the specific
primer sequences gr eat ly influence the y ield of the
PCR-generated pr oducts.
In practice, the GC% and potent ial for sec-
ondary structure dif fer for each target sequence be-
cause the ability of the primers to interact w ith
mult iple sequences in the template and w ith each
other v aries fo r each primer pair. So, in order to
opt imize the condition, it is useful to set up a se-
ries of test react ions of the g radient annealing tem-
peratures so that the opt imal annealing tempera-
tur e can be selected direct ly.
PCR as a technolog y is a fundamental tool fo r
molecular biolog y analysis. Due to it s simplicity,
sensit ivity, specificity, and r eliability[ 21] , PCR is
an integ ral part in every molecular laboratory.
How ever, a uniform r eaction condition is not avai-l
able, so optim izing the system is a common w ork
to do. T here are many opt imizing tact ics for SSR-
PCR system, such as sing le facto r design and com-
pletely blo cks design. How ever, they are signif-i
cant ly t ime and energy consuming and can no t de-
tect the interaction among the f iv e main factors.
M oreover, they do not ensure the opt imal comb-i
nat ion of PCR sy stem . This novel optimizing o r-
thogonal experimental design allow s hitt ing the
specif ic opt imal react ion system dir ect ly w ithout
the requir ement of many treatments like the com-
plete block design.
Acknowledgments
We thank Pr of. Michael D. Casler ( U . S.
Dairy Fo rage Research Center ) for his valuable
comments on the manuscript. We also thank Sisi
L iu for her cr it ical reading o f the manuscript and
the Eng lish correction.
(下转 102页)
93
草地学报 第 16卷
stylo santhes under cold st ress, but it w as not af fected under heat st ress. RuBPcase and FBPase act ivit ies
in both legumes decr eased under both heat and cold str ess w ith low er activit ies in alfalfa and higher act iv-i
t ies in Guyana sty losanthes under heat str ess than cold st ress, respectively.
3N assimilation in response to temperature stress
Act ivit ies of nit rate r eductase ( N R) , nit rite reductase ( NiR) , g lutamine synthase ( GS) , and gluta-
mate synthetase ( GOGA T) w er e great ly decr eased in leaves o f alfalfa under heat st ress, w hile they w ere
litt le af fected under co ld st ress. On the contrary, they w ere gr eat ly decreased in Guyana sty losanthes un-
der cold st ress but lit t le af fected under heat st ress. T emperature st resses induced the accumulat ion of
NO-3-N and NH +4-N in leaves o f both legumes, and higher levels w ere accumulated under heat str ess in
comparison to cold st ress. T he decreased activ it ies o f NR and NiR caused by temperatur e st resses resulted
in the accumulat ion o f NO3--N and NH4+-N in leaves.
In summary, alfalfa exhibited tolerant to co ld st ress but sensit ive to heat str ess, w hile Guyana
stylo santhes w as tolerant to heat but sensit iv e to cold str ess. T emperature str esses resulted in the injury of
photosyntem and therefo re inhibited pho tophosphory lation, w hich led to the lim its of AT P supply in
leaves, decreased photosynthesis, and N assim ilat ion.
Key words: Alfalfa; Guyana stylosanthes; Nit rate reductase; Pho to synthesis, AT P synthase; Sucro se
synthase
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(上接 93页)
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