American Journal of Plant Sciences, 2013, 4, 2039-2042
http://dx.doi.org/10.4236/ajps.2013.410255 Published Online October 2013 (http://www.scirp.org/journal/ajps)
2039
Search for a Microsatellite Marker Linked with Resistance
Gene to Xanthomonas axonopodis pv. malvacearum in
Brazilian Cotton
Mariana Marangoni1, Larissa Girotto1, Maria Paula Nunes1, Wilson P. Almeida1, Rafael Galbieri2,
Ivan Schuster3, Yeshwant R. Mehta1*
1Instituto Agronômico do Paraná—IAPAR, Londrina, Brazil; 2Instituto Mato-Grossense de Algodão—IMA, Cuiabá, Brazil;
3Coodetec, C.P. 301, Cascavel, Paraná, Brazil.
Received August 1st, 2013; revised September 1st, 2013; accepted October 1st, 2013
Copyright © 2013 Mariana Marangoni et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The cotton cultivar DELTAOPAL is resistant under field as well as under glasshouse conditions to the Brazilian isolates
of Xanthomonas axonopodis pv. malvacearum (Xam). Segregating populations derived from the cross between this
cultivar and one susceptible cv. BRS ITA 90, were utilized to identify molecular marker linked with the resistance gene
to Xam by “Bulk Segregant Analysis (BSA)”. Two hundred and twenty microsatellite (Single Sequence Repeat—SSR)
primers were tested. The amplification products were visualized in polyacrylamide gels stained with silver nitrate. Only
one primer was informative and showed polymorphism between the DNA of the parents and their respective bulks of
homozygous F2 populations contrasting for resistance and susceptibility, and hence was used to analyze DNA of 120 F2
populations. The microsatellite primer yielded one band of 80 bp linked with the resistance locus, which was absent in
the susceptible parent as well as in the bulk of the homozygous susceptible plants of the cross. The segregation ratio as
determined by phenotypic analysis was 3R:1S. It is believed that the microsatellite marker was linked with the resis-
tance locus and hence may offer new perspectives for marker assisted selection against the angular leaf spot disease of
cotton. It is however, felt necessary to repeat the microsatellite analysis and make sure that the primer is tightly linked
with the resistance locus and at the same time verify the genetic distance between the marker and the resistance locus.
Keywords: G o ssypi u m hi rsutum L.; Xanthomonas axonopodis pv. Malvacearum; Genetic Markers; Marker Assisted
Selection
1. Introduction
The angular leaf spot of cotton, also known as “bacterial
blight” and “black arm” of cotton, caused by Xanthomo-
nas axonopodis pv. malvacearum (Xam) is economically
important in several cotton growing areas of Asia, Africa,
USA and the Latin America, including Brazil. The patho-
gen is seed transmitted and can cause appreciable yield
losses. In the USA the estimated annual losses could be
between 0.1% and 2.3% [1]. In Brazil, the disease is re-
ported to cause heavy yield losses under favorable wea-
ther conditions [2]. So far, there is no practical method to
eradicate the pathogen from the seed. Many commercial
cultivars are susceptible and the disease is not controlled
by fungicidal applications. The angular leaf spot disease
however, can be controlled through varietal resistance.
Resistance has been attributed to the combination of
two or more major resistance genes and a modifier com-
plex. Nineteen races of the pathogen currently are recog-
nized in the USA [1,3]. In Brazil, although sources of re-
sistance like cvs. DELTAOPAL, EPAMIG LIÇA and
FIBERMAX 986, are available, breeding for resistance
against this disease is difficult. Conventional breeding
strategies for disease resistance which involve pyramid-
ing of resistance genes are time consuming, expensive
and require artificial inoculations under glasshouse and
field conditions. Invariably, the artificial inoculations with
bacteria give a margin for misinterpretation of the results
because of the inconsistent reactions influenced by un-
successful inoculations especially under field conditions
[4]. Marker assisted selection (MAS) is being advocated
*Corresponding author.

Search for a Microsatellite Marker Linked with Resistance Gene to Xanthomonas axonopodis pv.
malvacearum in Brazilian Cotton
2040
to aid selection of resistant plants in early generations in
some plant species [5-12]. The MAS technique allows us
to identify the presence of resistance gene(s) in segregat-
ing populations of early generations within a few days
and avoids repeated inoculations under field conditions.
It is based on the genotypic reaction and not on the phe-
notypic reaction of the plant. The rapid identification of
segregating plants carrying resistance genes drastically re-
duces the number of segregating plants to be evaluated in
subsequent generations. Besides, the MAS offers com-
plete reliability in identification of plants carrying the re-
sistance gene. For this purpose, however, prior knowl-
edge about the molecular marker linked with the gene of
interest is a prerequisite.
Attempts were made in the present investigation to
identify microsatellite markers tightly linked with the re-
sistance genes in the cv. DELTAOPAL so that they can
be useful in marker assisted breeding programs.
2. Material and Methods
Genetic seed material and crosses: Seeds of DELTA-
OPAL as resistant and BRS ITA 90 as susceptible, were
obtained from the germplasm collection of IAPAR and
were multiplied by selfing a typical representative plant
of each cultivar. Crosses were made between the resistant
and susceptible cultivars and F2 seeds were obtained. One
hundred and twenty segregating F2 plants were evaluated
for disease reaction by artificial inoculation under glass-
house conditions. DNA of F2 plants was extracted and
stored for further use. Twenty segregating plants classi-
fied as highly resistant and another 20 classified as high-
ly susceptible were selfed to produce F3 seeds. Four hun-
dred plants derived from the 20 F2:3 families (20 plants of
each F2 plant), were inoculated in the same way as the F2
plants in order to identify the F2 plants homozygous for
resistance and homozygous for susceptibility and the
DNA of only such F2 plants was used for constructing
homozygous “bulks”. Two contrasting “bulks” composed
of equal quantities of DNA of five homozygous F2 plants
for resistance and four homozygous F2 plants for suscep-
tibility were formed and used for “Bulk Segregant Ana-
lysis”—BSA [9]. All populations were conducted in the
glasshouse.
Inoculations and evaluation of disease symptoms:
The inoculation technique, the incubation conditions and
the disease assessment scale were basically the same as
reported earlier [13]. Inoculations were made using the
tooth pick method and an aggressive isolate (N˚ 13403)
of Xam from the culture collection of IAPAR, represent-
ing race 18. Inoculated plants were incubated in a mist
chamber for 24 h, adjusted at 22˚C - 24˚C and about
100% relative humidity, and later were transferred onto
the glasshouse bench. Plants were evaluated for the dis-
ease intensity 20 days after inoculation using a disease
severity scale of 0 - 3 [13], where 0 = resistant (income-
patible reaction) and 1 - 3 = susceptible (compatible re-
action).
DNA extraction: DNA was extracted before inocula-
tion using a protocol as described by Doyle & Doyle [14].
The amount of DNA was quantified by electrophoresis
gels as well as by a DyNa Quant 200 Fluorometer (Phar-
macia). The DNA samples were diluted in 100 L TE
buffer, adjusted to 20 ng·L−1 and stored at −20˚C for
further use. Samples which showed degradation of DNA
in electrophoresis gels were discarded and DNA extrac-
tion was repeated. RNA was eliminated by adding 3 L
of RNAase (10 mg·mL−1).
Bulked segregant analysis and PCR protocols: PCR
reactions were performed in 15 L volumes containing
1.8 L of 1 M MgCl2, 1.5 L of 100 mM/500 mM Tris-
KCl (pH 8.3), 1.5 L of 0.25 mM dNTP, 0.8 L of BSA
1%, 1.5 L of each one of the 0.2 M of primer Forward
and primer Reverse, 3.0 L (30 ng) DNA, 1.0 L (1 unit)
Taq DNA polimerase (Pharmacia, USA), and 3.2 L of
autoclaved distilled water. Negative controls without DNA
were maintained in all the reactions. Two hundred and
twenty microsatellite primers (Invitrogen) were tested
against the DNA of two bulks of F2 populations contras-
ting for resistance and susceptibility and their respective
parents. Amplification was performed in a thermal cycler
(MJ Research, Inc. Watertown, MA, USA), according to
the following program: 94˚C for 4 min followed by 60
cycles of 94˚C for 30 sec, 50˚C for 30 sec, 72˚C for 45
sec, and a final extension at 72˚C for 7 min. Amplifica-
tion products (15 L) were electrophoresed in polyacry-
lamide gels (10%) with TBE running buffer, stained with
silver nitrate, and were scanned into a computer imaging
file using a Kodak EDAS 120 digital camera. After the
identification of the molecular marker, segregation ratio
for the resistance and susceptibility was determined using
DNA of all the F2 plants.
3. Results & Discussion
Analysis of the F2 populations derived from the cross
DELTAOPAL × BRS ITA 90, yielded a segregation ratio
of 3R:1S suggesting the presence of a single dominant
gene for resistance to Xam [3]. Out of the 220 microsa-
tellite primers tested, only one (Primer BNL 2634. Re-
verse, Sequence 5’ to 3’ CCCAGCTGCTTATTGG-TTTC;
Forward, Sequence 5’ to 3’ AACAACATT-
GAAAGTCGGGG), showed polymorphism between the
parents and between the two F2 homozygous bulks con-
trasting for resistance and susceptibility.
Figure 1 shows the banding pattern obtained in BSA
with one microsatellite primer. The microsatellite primer
yielded one band of 80 bp linked with the resistance lo-

Search for a Microsatellite Marker Linked with Resistance Gene to Xanthomonas axonopodis pv.
malvacearum in Brazilian Cotton
2041
cus which was absent in the susceptible parent as well as
in the bulk of the homozygous susceptible plants. The se-
gregation ratio as determined by phenotypic analysis was
1:2:1 (Table 1). It can be observed that all the F2 plants
which composed the susceptible bulk have the same al-
lele as the susceptible progenitor. The plants which com-
posed the resistant bulk have the same allele as the resis-
tant progenitor, in homozygous or in heterozygous form.
Since the plants which composed the bulks were selected
by analyzing their respective progenies as being homo-
zygous, the heterozygous genotype for the marker may
be resultant of the recombination. It is believed that the
microsatellite marker was linked with the resistance lo-
cus and hence may offer new perspectives for marker
assisted selection against angular leaf spot disease of cot-
ton in Brazil.
The cv. DELTAOPAL is resistant to the populations
of Xam occurring in Brazil, including the race 18. So far,
there is no report of angular leaf spot symptoms on this
cultivar under the diversified cotton growing areas of
Brazil. Dominance for resistance in this cultivar was
complete. The number of F2 individuals used in con-
structing two contrasting bulks was small. According to
Michelmore et al. [9], loci not segregating in the popula-
tion, whether linked or not, will not distinguish the bulks.
The search for molecular markers tightly linked with
the resistance gene is a random process. We tested 220
SSR primers which represent only a very small portion of
the cotton genome. Since the resistance to R. areola is
governed by one gene, only one locus of the whole ge-
nome would have this gene. SSR primers which do not
amplify regions near this locus would always be segre-
gating irrespective of the resistance indicating that there
is no genetic linkage. Only the primers which amplify re-
gions near the locus which has the resistance gene would
segregate with it and would indicate the existence of ge-
netic linkage. It is for this reason that several SSR prim-
ers encompassing different regions of the genome need
to be tested to increase the chances of identifying primers
tightly linked with the resistance gene.
Further work is necessary to verify whether the mi-
crosatellite marker identified in the present investigation
is tightly linked with the resistance locus in cv. DEL-
TAOPAL. It would also be interesting to verify whether
the microsatellite marker is linked with the resistance
gene(s) in other cotton cultivars with different genetic
background including cultivars of G. barbadense. Other
than marker-assisted selection in breeding populations,
resistant lines or accessions from germplasm collection
can also be screened once a molecular marker tightly link-
ed with the resistance gene is identified. Molecular mar-
ker has an additional advantage in the sense that the
80 bp
20 bp
Figure 1. Bulk segregant analysis showing banding pattern obtain in polyacrylamide gel with microsatellite primer BNL 2643.
Lane A = resistant bulk; B = resistant parent (DELTAOPAL); C = susceptible bulk; D = susceptible parent (ITA 90). Lanes
E-J = F2 homozygous resistant; Lanes G-I = heterozygous resistant individuals. Lane K-N = F2 homozygous su sceptible and
O = susceptible parent.
Table 1. Segregation of disease reactions for the leaf area infected by Xanthomonas axonopodis pv. malvacearum (Xam),
among parental cotton cultivars and their segregating populations of the cross DELTAOPAL X BRS ITA 90.
Number of individuals in each category of infection*
Observed Expected
Parents and progeny Leaf reaction** Total No. of
indivíduals
Theoretical
ratio (R:S) R S R S

2 Prob.
%
DELTAOPAL R 23 1:0 23 0 23 0
BRS ITA 90 S 18 0:1 0 18 0 18
F1 R 20 1:0 20 0 20 0
F2 Segreg. 127 3:1 95 32 95.25 31.75 0.002 94.91
*Inoculations were performed in the glasshouse, on 25 days old plants, using an aggressive isolate of Xam and disease reactions were observed 20 days after
oculation; **R = Resistant; S = Susceptible; Segreg = Segregating. Source: Zandoná et al. (2006). in

Search for a Microsatellite Marker Linked with Resistance Gene to Xanthomonas axonopodis pv.
malvacearum in Brazilian Cotton
2042
expression of the marker is not masked by epistatic in-
teractions that occur between resistance genes, permitting
thereby, pyramiding of resistance genes in agronomically
desirable cultivars [5,15].
4. Conclusion
Out of 220 SSR primers screened, only one primer show-
ed polymorphism between resistant and susceptible par-
ents and their bulks contrasting for resistance and sus-
ceptibility for angular leaf spot of cotton. The results
indicate that the marker is linked with the resistance gene
and could be used in breeding programs aimed at marker
assisted selection in Brazil. However, further research is
needed to verify if the marker is tightly linked with the
resistance loci and the distance between the marker and
the resistance locus.
5. Acknowledgements
The present research was conducted under the financial
support of IMA, MT, Brazil. Thanks are also due to Lu-
celeine P. Lopes, Carla Zandoná, and Priscila Alves, for
the technical support.
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