Search for a Microsatellite Marker Linked with Resistance Gene to <i>Xanthomonas axonopodis</i> pv. <i>malvacearum</i> in Brazilian Cotton

doi:10.4236/ajps.2013.410255

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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.

Email: *[email protected]

Received August 1st, 2013; revised September 1st, 2013; accepted October 1st, 2013

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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