Identification of RAPD Markers for Northern Corn Leaf Blight Resistance in Waxy Corn (Zea mays var. ceratina)
Juthaporn Khampila ,
Piyada Theerakulpisut ,
Kamol Lertrat ,
Weerasak Saksirirat ,
A F2 population from a cross of the waxy
corn inbreds 209W and 241 W was used to determine random amplified polymorphic
DNA (RAPD) markers linked to Northern Corn Leaf Blight (NCLB) resistance
via Bulked Segregant Analysis (BSA). Resistant and susceptible DNA bulks
were constructed using the segregating F2 plants based on phenotypic
reaction against NCLB infection. In total, two hundred and twenty two
decamer primers were used to identify three polymorphic bands observed
between the bulks. The primers OPE02, OPJ18 and OPX04, generated the polymorphic
DNA fragments of approximately 1200, 650 and 500 bp, respectively. These
RAPD bands were present in only in NCLB-resistant bulk and 241W resistant
parent. The study revealed that RAPD markers associated to NCLB resistance
is potentially useful for the identification of genotyped individuals
carrying NCLB resistant traits in breeding programs.
to cite this article:
Juthaporn Khampila , Piyada Theerakulpisut , Kamol Lertrat , Weerasak Saksirirat , Jirawat Sanitchon and Nooduan Muangsan , 2008. Identification of RAPD Markers for Northern Corn Leaf Blight Resistance in Waxy Corn (Zea mays var. ceratina). Asian Journal of Plant Sciences, 7: 18-21.
Exserohilum turcicum (Pass.) K.J. Leonard and E.G. Suggs causes
Northern Corn Leaf Blight (NCLB), a severe and widespread foliar wilt
disease of maize (Zea mays L.) (Leonard et al., 1989). NCLB
causes substantial crop losses in most of the major corn producing areas
throughout the world, particularly in mid-altitude and highland regions
of the tropics and subtropics (Smith and Kinsey, 1993). Symptoms can range
from small cigar-shaped lesions to complete destruction of the foliage.
Grain yield losses can exceed 50% in susceptible maize cultivars if infection
occurs before flowering (Tefferi et al., 1996). To prevent severe
yield losses, the use of resistant cultivars is currently the most effective
and common means of disease control and also very important due to concern
about pesticides and the environment. Breeding for NCLB resistant maize
cultivars by conventional means is considered the most effective and feasible
method to overcome yield losses due to NCLB. However, the conventional
breeding is laborious, time-consuming and dependent on environmental conditions.
The use of molecular markers is an efficient alternative to the tedious
work of phenotype evaluation for NCLB resistance and allows for an efficient
selection of NCLB resistance gene (s). Molecular markers can also accelerate
selection and eliminate the effects of environmental variation during
selection (Malyshev and Kartel, 1997).
Random Amplified Polymorphic DNA (RAPD) analysis has been successfully
used to identify DNA polymorphism linked to many important traits such
as disease resistant genes in sugar pine (Devey et al., 1995),
tobacco mosaic virus resistant gene in tomato (Young et al., 1998),
downy mildew resistant gene in sunflower (Brahm et al., 2000),
angular leaf spot disease resistant gene in common bean (Ferreira et
al., 2000), soybean mosaic virus resistant gene in soybean (Zheng
et al., 2001), powdery mildew resistant gene in grapes (Dalbo
et al., 2001). RAPD is a simple and inexpensive technique compared
to Restriction Fragment Length Polymorphism (RFLP). RAPD markers are also
more-rapidly and more-easily detected than RFLP markers (Welsh and McClelland,
1990; Williams et al., 1990). Michelmore et al. (1991) suggested
an alternative method called Bulked Segregant Analysis (BSA) to find RAPD
markers linked to the trait of interest, such as resistant to NCLB disease.
In the present studies RAPD analysis was used to identify DNA marker (s)
linked to NCLB resistance in waxy corn using bulked segregant analysis
in F2 population.
MATERIALS AND METHODS
A 217 F2 population from a cross between a highly susceptible
inbred 209W line with a resistant inbred 241W line was grown along with
the parents in the field area of the Department of Plant Science and Agricultural
Resources, Faculty of Agriculture, Khon Kaen University, Thailand during
the normal growing season of maize of the year 2005. The resistant inbred
241W was originally derived from derived from the open-pollinated variety
Sumlee Esan, while the susceptible inbred 209W was originally derived
from a cross between the CK8-F2 and KSC-F2 (Thoungnarin
et al., 2005). The F2 population was screened for NCLB
resistance trait. Severity of NCLB was assessed, based on the percentage
of the total leaf area affected using a slightly modified version of the
standard protocol of Elliot and Jenkins (1946). Parent genotypes were
screened for polymorphism using 222 RAPD primers including 11 primer sets
from Operon technologies, Alameda, CA (Sets A, B, C, E, F, G, H, J, W,
X and Y), UBC244 and UBC9. PCR amplifications were carried out according
to the method described in Williams et al. (1990). For the bulks,
resistant and susceptible bulks were prepared from F2 individuals
by pooling aliquots containing equivalent amounts of total DNA from each
of the fourteen extremely resistant and fourteen extremely susceptible
F2 progenies. DNA was extracted separately from each individual
of the progeny. Genomic DNA of young leaves before inoculation was extracted
by the method of Doyle and Doyle (1987) with minor modifications and adapted
to small tissue quantities (Hormaza, 1999). The PCR reaction volume was
25 μL and contained: 1X PCR buffer, 2.0 mM MgCl2, 300
μM of dNTPs, 0.4 μM of primer, 0.7 U of Tag DNA polymerase
(Promega®, Madison, Wisconsin) and 10 ng of genomic DNA.
DNA amplification was conducted on a programmable thermalcycler (Hybaid®,
USA). Template DNA was initially denatured at 94C for 2 min, followed
by 47 cycles of PCR amplification using the following parameters: 1 min
denaturation at 94C, 1.45 min annealing at 38C and 2 min primer extension
at 72C. A final 7 min incubation at 72C was allowed for completion of
primer extension. PCR products were electrophoretically resolved on 1.5%
agarose gels with 1X TBE (40 mM Tris-borate, 1 mM EDTA) containing 0.5
g mL-1 ethidium bromide and detected on a UV transilluminator.
Bulked segregant analysis: The method involves comparing two pooled
DNA samples from individuals of two extreme phenotypes from a segregating
population. Within each pool or bulk, the individuals are identical for
the trait of interest but are arbitrary for all other traits.
Therefore, the two resultant bulked DNA samples differ genetically only
in the selected region and are seemingly heterozygous and monomorphic
for all other regions. Two DNA pools contrasting for the trait of interest
are analyzed to identify markers that distinguish them. Markers that are
polymorphic between the pools will be genetically linked to the loci determining
the trait used to construct the pools (Michelmore et al., 1991).
Two bulked DNA samples were generated from the F2 segregating
population. One bulk consisted of equal amounts of DNA of fourteen NCLB
resistant F2 plants and the other was similarly formed from
DNA of fourteen F2 plants with susceptible to NCLB following
the procedure giving by Michelmore et al. (1991). PCR reactions
were carried out on the bulks and parental DNA samples using RAPD primers
that were polymorphic between the parents.
RESULTS AND DISCUSSION
Out of 222 arbitrary decamer primers screened for polymorphisms between
209W susceptible and 241W resistant parents, 63 RAPD primers (28.38%)
that gave polymorphic bands between the parent genotypes were identified.
Moreover, 51 oligonucleotide 10-mer primers were not obtained any DNA
bands and 14 RAPD primers gave very weak DNA profiles, while 94 RAPD primers
gave the monomorphic bands when the 222 RAPD primers were used to study
the polymorphism between the parents. The total 1,069 bands were amplified
using the 222 RAPD primers. The number of RAPD fragments that were amplified
ranged from 2 to 11 and the sizes ranged from about 200 to 1,300 bp. Of
these 222 RAPD primers, sixteen primers which produced single, strong
polymorphic bands that were present in only 241W resistant parent, but
absent in 209W susceptible parent, were selected for screening DNA bulks
and their parental DNA.
In RAPD, an often occurring artifact on agarose gels is a primer and
the target sequence not matching 100% would be less so amplification would
be quantitatively less from these loci resulting in faint bands. Extremely
bright RAPD bands observed in the present studies may be the result of
amplification from sequences of high copy number in the genome. The amount
of amplification product of a sequence of high copy number is expected
to be greater compared to that of low copy number which would result in
a very bright band (Malik, 1995). However, a few bright bands split into
sub-bands when ran on relatively high concentration agarose gel (2.5%).
This suggested that they were a mixture of fragments in a small range
of sizes (Amir et al., 2002).
A total of sixteen RAPD primers were used for screening the bulks containing
bulked DNA from fourteen plants each from the resistant and susceptible
F2 plants as described. The sixteen RAPD primers that generated
polymorphic DNA fragments between parents were analyzed and the average
number of bands per primer was again between 4 and 5. Screening of bulks
and parental lines with the primers showed amplification products ranging
in size between 300 to 1,300 bp. The primers OPE02 (5´-GGTGCGGGAA-3´),
OPJ18 (5´-TGGTCGCAGA-3´) and OPX04 (5´-CCGCTACCGA-3´), generated the DNA
fragments of approximately 1200, 650 and 500 bp, respectively (Fig.
1). These RAPD markers were present only in NCLB-resistant bulk and
241W parent, but were missing in NCLB-susceptible bulk and 209W parent
and the markers were reproducible. These three RAPD primers generated
polymorphic fragments that are associated to the NCLB resistance phenotypes.
Random primers used in RAPD analysis usually anneal with multiple sites
in different regions of the genome so that several genetic loci are amplified
and also the markers are inherited as dominant genetic markers. This limits
the application of this marker type, particularly in cases where one would
like to distinguish homozygous from heterozygous genotypes. The PCR amplification
that generates RAPD fragments of interest is sensitive to specific reaction
conditions. Moreover, poor reproducibility can occur in RAPD analysis.
A different population may produce different amplification profiles using
the same primers. Nevertheless, the enormous attraction of RAPD marker
is that the technique is quick, simple, uses small amounts of DNA, sample
throughput can be high and the procedure is automatable (Welsh and McClelland,
1990). There is also no requirement for DNA probes or sequence information
for primer design when one uses RAPDs.
These studies suggested that RAPD technology has great potential in finding
DNA marker (s) for practical breeding programs. Some earlier workers have
demonstrated the use of RAPD in practical plant breeding and DNA marker
assisted back crossing (Stuber, 1995). RAPD markers linked to rust resistance
in barley have been used to carry out successful marker assisted selection
in an F2 population (Poulsen et al., 1995). Speed and
the efficiency of the crop improvement programs can be enhanced significantly
by using marker assisted selection and it also allows consistent progress
in the advancement of selected materials. It is important especially for
those characters which are highly dependent upon the environment for expression
such as drought resistance (Malik, 1995).
||PCR banding patterns generated by primers OPE02, OPJ18
and OPX04 with NCLB resistant parent 241W NCLB susceptible parent
209W, R-bulk the NCLB resistant bulk, S-bulk the NCLB susceptible
bulk and M is 100 bp DNA ladder. Arrows indicate the polymorphic DNA
band corresponding to NCLB resistance
Using a method inspired by BSA, we were able to identify three RAPD markers
associated to NCLB resistance phenotype in waxy corn, Z. mays var.
ceratina. From a total of 1,069 fragments, only three (0.28%) were
linked to the NCLB resistance. The RAPD markers should be useful for marker-assisted
selection. Present results support the idea that BSA can provide fast
detection of molecular markers linked to genes of interest. Traditional
methods of handling breeding populations take very long time for advancement
to a desired stage. Expenditure using conventional means of breeding such
as management and the labor costs of experiments may be higher compared
to using marker assisted selection. It is concluded that RAPD technique
has great potential in plant breeding.
Further work will involve the conversion of the three associated markers,
described in this study to a Sequence Characterized Amplified Region (SCARs)
in order to simplify their use in maize breeding programs. And also the
construction of the RAPD markers combined with simple sequence repeat
(SSR) markers on the genetic map of maize for detecting the Quantitative
Trait Loci (QTLs) linked to NCLB resistance genes.
This research was funded by the University Staff Development Program,
Mahasarakham University, Thailand and the Agricultural Biotechnology Project,
Faculty of Agriculture, Khon Kaen University under the sponsorship of
the Asian Development Bank (ADB) Loan and the Thai Government.
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