全 文 ：Received 22 Oct. 2003 Accepted 16 Dec. 2003
Supported by the National Natural Science Foundation of China (30160046) and the Scientific Research Foundation of Chinese Academy of
Tropical Agricultural Sciences (RYKJ9709).
* Author for correspondence. Tel (Fax): +86 (0)28 85412738; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 480-488
AFLP Analysis of Genetic Variability Among Stylosanthes guianensis
Accessions Resistant and Susceptible to the Stylo Anthracnose
JIANG Chang-Shun1, 2, JIA Hu-Sen3, MA Xin-Rong1, ZOU Dong-Mei2, ZHANG Yi-Zheng1*
(1. College of Life Sciences, Sichuan University, Chengdu 610064, China;
2. Tropical Pasture Research Center, Chinese Academy of Tropical Agricultural Sciences, Danzhou 571737, China;
3. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China)
Abstract: Stylosanthes guianensis Swartz, one of the most important tropical forage legumes, is native
to South and Central America and Africa. Anthracnose, caused by the fungus Colletotrichum gloeosporioides
(Penz.) Sacc., is a major constraint to the extensive use of Stylosanthes as tropical forage. Forty-two
accessions of S. guianensis were assessed by amplified fragment length polymorphism (AFLP) for genetic
diversity and for resistance to anthracnose. In AFLP analysis, four selective primer combinations screened
from 96 primer combinations were used to analyze these accessions, which showed a 95.5% level of
polymorphism on average. Genetic similarity from 31.0% to 95.0% among the accessions was calculated
with NTSYS-pc software. The dendrogram was constructed with unweighted pair group method of averages
(UPGMA) based on the AFLP data, and five clusters were defined at 48% genetic simi larity. Principal
coordinates analysis showed that the contribution of the first principal coordinate and the second one to
the genetic variation of 42 accessions is 56.04% and 6.40%, respectively. Two typical strains of C.
gloeosporioides from Stylosanthes in China were used for anthracnose resistance screening. All plant
accessions showed variation in the reaction to two strains, and the correlation of resistance had a value
of 0.904 (P ＜0.01), suggesting the common resistance to the two strains. The resistant accessions were
randomly distributed in the different groups of UPGMA clustering. Also, these results demonstrate that
AFLP analysis is an efficient method for evaluating the genetic diversity among S. guianensis accessions.
Key words: Stylosanthes gu ianensis ; AFLP; genet ic var iat ion; c luster analysis; resistance to
anthracnose
Styl osan th es gui anens is (A es ch ynomenea e,
Papiliondeae), one of the most important tropical fo rage
legumes, is native to South and Central America, and Africa
(Williams et al., 1984). It is used for grazing cattle, raising
livestock, and improving soil fertility in fru it-tree and rub-
ber plantations as cover crops in Australia, South America
and Southern China (Jiang, 1995). The S. guianensis com-
plex has been sub jected to taxonomic stud ies based on
morphological-agronomic traits (Burt et al., 1971), seed pro-
tein patterns (Robinsin and Mergarrity, 1975), isozymes
(Stace, 1982; Vithang and Chakrabority, 1992), rhizobial af-
finities (Date et al., 1979), crossability (Cameron, 1974), and
pollen stainability of hybrids (Hacker et al., 1988). In some
of these studies, different authors treated taxonomic groups
within S. guianensis differently. Mannetje (1984) has rec-
ognized six and seven (Ferreira and Costa; 1984) groups in
S . gu ianensis, res pect ively . Genet ic variat ion in
Stylosan thes genus has been inves tigated with DNA
molecular markers. These techniques have included the use
of random amplified polymorphic DNA (RAPD) (Kemal et
al., 1993a; 1993b; Liu, 1997; Liu and Musial, 1997), restric-
tion fragment length polymophism (RFLP) (Liu and Musial,
1995; Gillies and Abbott, 1996; Liu et a l., 1999), sequence
tagged sites (STS) (Liu et a l., 1996; Liu and Musial, 1997;
Liu et a l., 1999; vander Stappen et a l., 1999a), simple se-
quence repeats (SSR) (vander Stappen et al., 1999b), ribo-
somal DNA internal transcribed spacer (rDNA ITS)(vander
Stappen et al., 1998), ribosomal DNA external transcribed
spacer reg ion (vander Stappen et a l., 2003), and chlo ro-
p last DNA s equence variation (vander Stappen and
Volckaert, 1999; vander Stappen et al ., 1999c; Liu and
Musial, 2001). Few studies have investigated levels of varia-
tion within species. Kemal et al. (1993a) and vander Stappen
et al. (1998; 1999b) examined genetic variation within a col-
lection of S. guianensis with RAPD, rDNA ITS and SSR,
respectively.
JIANG Chang-Shun et al.: AFLP Analysis of Genetic Variability Among Stylosanthes guianensis Accessions Resistant and
Susceptible to the Stylo Anthracnose 481
This study aims to (1) describe the distribution of genetic
variability within a group of accessions representing the ge-
netic diversity available in S . guianensis germplasm; (2) as-
sess their resistance or susceptibility to anthracnose in order
to determine if resistance is general or specific; and (3) help in
identifying accessions that could be selected for improve-
ment programs and in studies of host-pathogen interaction.
1 Materials and Methods
1.1 Plant materials
Forty-two acces sions of Stylosan thes guianensis
Swartz were used for anthracnose resistance screening and
genetic analysis. Of these, 25 access ions were from the
Genetic Resource Unit of the International Center for Tropi-
cal Agriculture (CIAT), because these accessions have been
identified for anthracnose resistance with South American
isolates of Colletotrichum gloeosporioides (Penz.) Sacc.
(Kelemu et al., 1996). The others were from Chinese Acad-
emy of Tropical Agricultural Sciences (CATAS), Institute
of Guangxi Animal Sciences (IGAS) of China, respectively.
All thes e accessions were either landraces or improved
clones classified as ELITE in relation to agronomic traits,
such as resistance to biotic o r abiotic s tresses and yield.
The accessions are coded as follows: the landraces are
coded by collection numbers of CIAT, Australia, and Florida
state o f USA. The others are the improved access ions of
CATAS, IGAS, respectively (Table 1).
1.2 Resistance screening
Stylosanthes anthracnose resistance was conducted on
th e fo rt y - two ac ce s s ion s . Two s t r ains o f C .
gloeosporioides, named CATAS292 (Biotype A) and
CATAS100 (Biotype B) from stylo in China, previously char-
acterized as virulent (Yi, 2003), were employed in this study.
For short-term storage of up to 1 year, fungal disks (8 mm
diameter) were removed from 4- to 6-d old potato dextrose
agar (PDA) cultures of monocondial isolates and maintained
in s crew-cap tubes of s terile distilled water at room tem-
perature (25 ℃). Isolates were reactivated by placing pieces
of disks on plates with fresh PDA and incubating at 28 ℃
for 6 d before inoculation. The Stylosanthes plants for test-
ing were grown from seeds in sterile so il. Ten plants per
access ion and per fungal strain were tes ted in the green-
hous e at 28/19 ℃ (day/nigh t ), under a 12 h daylight
pho toperiod, and 80% relative humidity, at CATAS,
Danzhou, China. The 6-week-old plants were inoculated as
described by Kelemu et a l. (1996). Diseas e severity was
determined at 10 d after inoculation by visual estimation of
leaf tissue necrosis based on a Horsfall-Barratt (1945) rat-
ing scale where 0-9 means zero, 1%-3%, 4%-6%, 7%-12%,
13%-25%, 26%-50%, 51%-75%, 76%-87%, 88%-94%,
95%-100% disease symptom, respectively. Reactions with
a mean disease rate of less than or equal to 1.5 were scored
as resistant (R), and all other reactions were rated as sus-
ceptible (S).
Table 1 Stylosanthes guianensis accessions used in the analysis
No. Accessions Origin Source No. Accessions Origin Source
1 CIAT13 Mexico CIAT 22 USF873014 USA IGAS
2 CIAT15 Bolivia CIAT 23 USF873015 USA IGAS
3 CIAT1283 Brazil CIAT 24 USF8730161 USA IGAS
4 CIAT1297 Brazil CIAT 25 USF8730162 USA IGAS
5 CIAT1500 Venezuela CIAT 26 USF873017 USA IGAS
6 CIAT1507 Venezuela CIAT 27 Cook Bolivia CATAS
7 CIAT1534 Venezuela CIAT 28 White cook China CATAS
8 CIAT1850 Venezuela CIAT 29 CPI18750A Australia CATAS
9 CIAT1890 Venezuela CIAT 30 CIAT1044 Colombia CIAT
10 CIAT1927 Venezuela CIAT 31 S. guianensis cv. 907 China IGAS
11 CIAT1959 Venezuela CIAT 32 CIAT184 Colombia CIAT
12 CIAT2031 Brazil CIAT 33 Reyan 2 China CATAS
13 CIAT2160 Brazil CIAT 34 Reyan 5 China CATAS
14 CIAT2222 Brazil CIAT 35 Reyan 7 China CATAS
15 CIAT2312 Colombia CIAT 36 Reyan 10 China CATAS
16 CIAT2340 Colombia CIAT 37 CIAT136 Colombia CIAT
17 CIAT2950 Brazil CIAT 38 L7-1 China CATAS
18 CIAT11363 Colombia CIAT 39 L7-2 China CATAS
19 CIAT11369 Colombia CIAT 40 L3 China CATAS
20 CIAT11370 Colombia CIAT 41 L8 China CATAS
21 CIAT11371 Colombia CIAT 42 S. guianensis cv. Graham Australia IGAS
CATAS, Chinese Academy of Tropical Agricultural Sciences; CIAT, International Center for Tropical Agriculture; IGAS, Institute of Guangxi
Animal Sciences.
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004482
1.3 DNA extraction
Seeds were germinated on moist filter paper in Petri dishes
at room temperature. Total plant DNA was extracted from 6-
d-old seedlings according to the following protocol modi-
fied from the method described by Liu and Musial (1997).
Ten seedlings of each accession were collected and ground
into powder in liquid nitrogen. The powder was then trans-
ferred into a 1.5 mL eppendorf tube and extracted in 600 mL
extraction buffer (2% SDS; 100 mmol/L Tris-HCl, pH 8.0;
100 mmol/L NaCl; 50 mmol/L EDTA, pH 8.0), shaken and
incubated at 65 ℃ for 30 min. Next, proteins were removed
by addition of 600 mL of phenol:chloroform:isoamyl alco-
hol (25:24:1) followed by precipitation with isopropanol
(80%), then stored overnight at -20 ℃. Samples were cen-
trifuged at 13 000 r/min and the supernatant was removed.
The pellet was washed with 1 mL of 70% ethanol, dried for
30 min, and then redissolved in 100 mL 1× TE buffer (10
mmol/L Tris-HCl, pH 8.0; 1 mmol/L EDTA, pH 8.0). DNA
was treated with RNase for 30 min at 37 ℃, and visualized
on an agarose gel (0.8%). The DNA was quantified with an
ultrav iolet spect rophotometer, and stored at -20 ℃ un til
use.
1.4 AFLP analysis
Sequences of the primers and adapters were synthesized
by Saibeisen (SBS) Genetech Co. Ltd. (Beijing, China). Taq
DNA polymerase and dNTPs were purchased from Sino-
American Bio technology Co. AFLP was perfo rmed as
des cribed by Vos et a l. (1995), using 300 ng of genomic
DNA. Each DNA was digested with 1.0 U EcoRⅠ and 1.0 U
MseⅠ (New England BioLabs, Inc.). The digested DNA
was ligated to specific EcoRⅠ and MseⅠ adapters (Table
2) with T4 DNA ligase (TaKaRa Biotechnology Co. Ltd.,
Dalian, China). The pre-amplification was carried out using
universal primers, M00 and E00 (Table 2). For selective PCR,
special primer combinations were employed, and 5 mL of
the dilu tion (1:50) from the firs t PCR was used as DNA
template. The successful reaction products were assessed
by visualizing the smear pattern on a 2.0% agarose gel, and
subsequently an aliquot o f 3 mL of reaction product was
mixed with 3 mL of formamide dye, denatured for 3 min at 95
℃, and chilled on ice immediately. The reaction products
were then size-fractioned on 6% denaturing polyacrylamide
gel on a DNA sequencing apparatus. Electrophoresis was
carried out for 1.5-2 h in 1×TBE at 40 V/cm and 50 ℃. At
the end of the electrophoresis period, gel was stained with
0.1% silver nit rate (Bas sam et al ., 1991). The resu lting
banding pattern was analyzed manually.
1.5 Data analysis
AFLP markers were generated by using four selected
primer combinations . Each clear marker was treated as a
separated character and scored as either present (1) or ab-
sent (0) across the forty-two accessions, and recorded in a
binary data matrix. Genetic similarities between pairs of ac-
cessions were estimated with the Nei and Li (1979) formula,
Gs = 2Nxy / (Nx + Ny), where Gs is the measure of genetic
similarity coefficient between the xth and yth accession,
Nxy is the number of bands shared by x and y, Nx and Ny are
the total number of bands in x and y, respectively. The
resultant similarity matrix was input into both an unweighted
pair group method of averages (UPGMA) cluster analysis
and a principal coordinates analysis (PCO). All these analy-
ses were performed with NTSYS-pc software (Version 2.0)
(Rohlf, 1997). The similarity matrix was calcu lated with
SIMQUAL, the PCO was performed us ing EIGEN
procedures, and UPGMA cluster analysis was done with
SAHN clustering. Dendrograms were displayed graphically
with TREE plot option. Performing a PCO on the data can
be cons idered as a check of the clusters fo rmed by the
cluster analysis.
Using the stylo resistance data categorized as resistant
(R) o r suscept ible (S) as described previously, a correla-
tion index was calcu lated to determine the independence
Table 2 List of primers and adapters used in the AFLP analysis
Primer/adapters Sequences
MseⅠ adapter 5-GACGATGAGTCCTGAG-3
3-TACTCAGGACTCAT-5
M00 (universal primer) 5-GATGAGTCCTGAGTAA-3
MseⅠ + 1 primers M11, M12, M13, M14 M00 + A, M00 + T, M00 + C, M00 + G
MseⅠ + 3 primers M1, M2, M3, M4, M5, M6, M15, M00 + CTG, M00 + CTA, M00 + TCG, M00 + ATT,
M16, M17, M18, M19, M20 M00 + GAT, M00 + GTA, M00 + ACC, M00 + CAA,
M00 + CAC, M00 + CAT, M00 + CTC, M00 + CAG
EcoRⅠ adapter 5-CTCGTAGACTGCGTACC-3
3-CATCTGACGCATGGTTAA-5
E00 (universal primer) 5-GACTGCGTACCAATTC-3
EcoRⅠ + 2 primers E6, E7 E00 + AA, E00 + AC
EcoRⅠ + 3 primers E1, E2, E8, E9 E00 + AGG, E00 + ACC, E00 + AGC, E00 + ACG
JIANG Chang-Shun et al.: AFLP Analysis of Genetic Variability Among Stylosanthes guianensis Accessions Resistant and
Susceptible to the Stylo Anthracnose 483
or corr elat ion o f res is ta nce to t he d iffer en t C .
gloeosporioides strains.
2 Results
2.1 Resistance screening
Variation in the reaction of S. guianensis accessions to
C. gloeosporioides st rains was observed for all p lant
materials: 30.9% of all acces sions were resistant to
CATAS292, 26.2% to CATAS100, 26.2% to both strains,
and 69.0% sus cept ible to bo th st rains . The accessions,
CIAT1534 and CIAT1890, were resistant to CATAS292,
while susceptible to CATAS100 (Table 3). The correlation
of res istance to strains CATAS292 and CATAS100 had a
value of 0.904 (P < 0.01), suggest ing the common res is-
tance to the two strains.
2.2 Primer combination screening
Initially, six accessions: Reyan 2, Reyan 5, Reyan 7, S .
guianensis cv. 907, Reyan 10, and S. guianensis cv. Graham,
were chosen to test the variation of primer combinations.
With these accessions, the po lymorphism rates and the
total number of peaks with 96 primer combinat ions were
evaluated (Tab le 4). The most useful primer combinations
were considered to be those having the highest polymor-
phism rate that also generated a reasonable number of clearly
detectable total fragments. Using the results of the evalua-
tion of 96 primer combinations based on six accessions of
S. guianensis, eleven selected primer combinations were
further screened with 42 representative accessions. The
mos t-po lymophic p rimer combinat ions were E2/M17,
E2/M19, E8/M20 and E9/M17 (data not shown), which were
selected for the subsequent analysis.
2.3 AFLP profile
AFLP analysis was performed with four selective primer
combinations (E2/M17, E2/M19, E8/M20 and E9/M17) on
42 access ions of S. guianensis. The number of amplifica-
tion bands varied between 50 and 60 for each primer
combination. The size of the fragments ranged from 34 to
501 bp, with a reproducible banding pattern between 67 to
331 bp. The primer combination E2/M19 (Fig.1) generated
the highest percentage of po lymorph ic bands . A total of
225 clear bands were obtained with four s elected primer
combinations. Of these, 215 bands were polymorphic, show-
ing a 95.5% level of polymorphism on average.
2.4 Relationship between resistance/susceptibility and
AFLP fragments
Specific fragments o f 310 bp and 120 bp, which were
present in eleven resistant accessions and the susceptib le,
respectively , were ident ified with primer combination
E2/M17. A 385 bp specific fragment, which was present in
res istan t accession S. guianensis cv . 907 and absent in
CIAT184, was cloned. The accession S. guianensis cv. 907
was bred from CIAT184 mutated from cobalt treatment
(Liang et al., 1998).
Table 3 Disease reaction and AFLP cluster of Stylosanthes guianensis accessions
Disease reaction Disease reaction
Accession Cluster
CATAS292 CATAS100
Accession Cluster
CATAS292 CATAS100
CIAT13 E S S USF873014 D5 S S
CIAT15 C S S USF873015 D5 S S
CIAT1283 D5 R R USF8730161 D5 S S
CIAT1297 D5 R R USF8730162 D5 S S
CIAT1500 D1 R R USF873017 D5 S S
CIAT1507 D1 R R Cook D5 S S
CIAT1534 D5 R S White Cook D5 S S
CIAT1850 D5 S S CPI18750A D5 R R
CIAT1890 D5 R S CIAT1044 D5 R R
CIAT1927 D1 S S S. guianensis cv. 907 D4 R R
CIAT1959 D5 S S CIAT184 D4 S S
CIAT2031 D5 R R Reyan 2 E S S
CIAT2160 D5 R R Reyan 5 D1 S S
CIAT2222 D5 R R Reyan 7 D4 S S
CIAT2312 B S S Reyan 10 D3 S S
CIAT2340 D5 S S CIAT136 D4 S S
CIAT2950 A R R L7-1 D4 S S
CIAT11363 D5 S S L7-2 D4 S S
CIAT11369 D5 S S L3 D3 S S
CIAT11370 D5 S S L8 D4 S S
CIAT11371 D5 S S S. guianensis cv. Graham D2 S S
Plants with a disease reaction ≤ 1.5 were grouped as resistance (R), and those with a score >1.5 were classified as susceptible (S).


Acta Botanica Sinica 植物学报 Vol.46 No.4 2004486
0.45) (Kemal et al., 1993a), and it was no significance differ-
ence with SSR (0.1-0.7) (vander Stappen et al., 1999b). In
addition, our experiments were repeated twice and the re-
sults were highly reproducible. Therefore, AFLP can be
used as an ideal method for germplasm identification and
genetic diversity studies of S. guianensis.
3.2 Genetic diversity in S. guianensis and its application
From our experiments, both polymorphism and genetic
diversity are high among S. guianensis accessions. Upon
UPMGA cluster analysis, 42 accessions were divided into
five groups, and the resistant accessions were randomly
distributed in the different groups (Fig.2). And it was con-
firmed with PCO analysis. It is possible that the anth ra-
cnose resistance of S. guianensis is polygenic and addi-
tively inherited. In efforts to improve S. guianensis for re-
sistance to anthracnose, we suggest that the accessions
CIAT2950, S. guianensis cv. 907, CIAT1283, CPI18750A,
CIAT2160, and CIAT1044 be used as parents, because they
have shown the high resistance and genetic variation. We
also recommend us ing the access ions CIAT1534 and
CIAT1890 to be tested as host different ials with a wide
range of C. gloeosporioides strains , because they are re-
sist to one strain and highly susceptible to the other. The
accession CIAT2312 belonging to cluster B might be used
as a host to test the pathogen icity of C. gloeosporioides
strains , because it is h ighly sus cept ible to both typical
strains.
3.3 Identi fication of AFLP fragments linked to res is-
tance
The use of the AFLP technology to map resistance loci,
and to clone the resistant gene(s), may help us better un-
derstand the genetic basis of resistance against pathogens.
In this study, we have isolated the specific fragment of 310
bp, which is present in eleven resistant access ions and
abs ent in the sus ceptible ones. With a cross between a
highly resistant parent and susceptible one, we could test
whether the fragment of 310 bp is associated with resistance.
If it is confirmed, further work is to clone including the
resistant gene(s). In addition, we have also cloned the spe-
cific fragment of 385 bp from S. guianensis cv. 907 (Liang et
al., 1998), which was bred from CIAT184 mutated from co-
balt t reatment. This fragment is possibly associated with
resis tance, because it is abs ent in s usceptib le acces sion
CIAT184 and this finding is in agreement with our previous
RAPD analysis (unpublis hed data). Studies on the func-
tion of the fragment are being undertaken.
Acknowledgements: We acknowledge Dr. Daniel Debouck
of Genetic Res ource Unit of the International Center for
Tropical Agriculture, Prof. LIANG Ying-Chai of Institute of
Guangxi Animal Science, and Mr. HE Hua-Xian of Tropical
Pasture Research Center of Chinese Academy of Tropical
Agricultural Sciences for their kind providing Stylosanthes
guianensis seeds. We also thank Ms. CHAI Bi-Yun for her
assistance in greenhouse.
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(Managing editor: ZHAO Li-Hui)