45
Genetic Analysis and Identification of RAPD Markers Linked to Northern Corn Leaf Blight Disease
Resistance in a White Maize Population
M.N. Barakat, S.I. Milad A.M. El-Shafei1 and S.A. Khatab1
Biotechnology Laboratory, Crop Science Department, Faculty of Agriculture, Alexandria University, Alexandria, and
1National Research Center, Giza, Egypt
Abstract. Northern corn leaf blight (NCLB) is an important disease occurring in maize producing areas. Understanding the genetic nature of NCLB is regarded as a primer step in maize breeding programs.
This study used the resistant line Sids-63 (Sd-63) and the susceptible line Sids-7 (Sd-7) to develop F1 and F2 generations. P1, P2,F1 and F2 were used to study the genetic nature and to detect RAPD markers associated with the NCLB resistance gene(s) using bulked segregant on analysis (BSA). The genetic studies revealed that the inbred line, Sd- 63, recorded low disease severity (ranged from less than 5% to 10 %) and the inbred line, Sd-7, recorded high disease severity (ranged from 26 % to more than 75 %). F1 severity ranged from less than 5% to 50%. The F2 severity ranged from less than 5% to more than 75 %. The observed ratio (number of plants with high: low disease severity) fitted the Mendelian ratio, 3:1, and suggested the operation of one gene pair in this cross. The degrees of dominance for F1 (h1) and F2 (h2) gave significant negative values (–0.86 and –0.71 respectively). The heritability value for the tested cross was 92.00% and the estimated number of genes was 1.1, indicating that there was a difference in resistance genes between the two inbred lines. Thirty-eight primers were screened to identify two RAPD markers; namely, Pr11180 and Pr11300 linked to NCLB susceptible genotypes. Results from the two RAPD markers fitted the expected Mendalian ratio, 3:1, according to χ2-test .The regression analysis showed that the relationships between the two markers, Pr11180 and Pr11300, and the phenotype of the F2 individuals were significant and they recorded a determination coefficient (r2) = 0.54 and 0.60,for the two markers, respectively.
Introduction
Maize (Zea mays L.) is the third most important cereal crop in the world.
In Africa, maize is increasingly becoming an important non-traditional agricultural export crop. In addition to strong demand as a food crop, the demand for maize is projected to rise with increasing population growth and an expanding need for livestock feed. Production of maize grain is, generally, insufficient relative to the needs of food consumption in many areas. Accordingly, increasing maize production is considered essential for food security in developing countries (CIMMYT, 2002). In Egypt, maize is grown for food, feed, fodder and industrial purposes. Egypt imports, approximately, 35% of its maize need. It is important to develop high-yielding and disease resistant hybrids to meet the country demands.
During the past few decades, a major fungal foliar disease of maize (Zea mays L.) called northern corn leaf blight (NCLB) caused substantial yield losses worldwide. NCLB caused by the fungus Helminthosporium turcicum (Pass) (Leonard, et al., 1989), is one of the most serious diseases occuring on maize throughout the world, particularly in the humid mid-altitude and highland regions of Africa (Ngwira, et al., 1999 and Bigirwa, et al., 1993). Symptoms of NCLB are elliptical leaf lesions, which are at first chlorotic and gray-green later become necrotic and wilted. Lesions may coalesce in susceptible plants and complete destruction of the foliage. Grain yield losses can exceed 50% in susceptible maize cultivar if infection occurs before flowering (Raymundo and Hooker, 1981).
NCLB is mainly controlled by resistant cultivars. The resistance is either qualitative or quantitative. Qualitative resistance is typically race specific and inherited by single genes whereas quantitative resistance is race non–specific and oligogenic or polygenic. The categories qualitative and quantitative refer to the distribution of a trait in a population and not to its effectiveness (Geiger and Heun, 1989).
Molecular markers that are closely linked with target alleles, present a useful tool in plant breeding since they can help to detect the resistant genes of interest without the need of carrying out field disease test. Also, they allow for screening big number of breeding materials at early growth stages and in short time. The Polymerase Chain Reaction (PCR) (Saikl, et al., 1985) offers the potential to lessen the time and expense of molecular mapping. In particular, Randomly Amplified Polymorphic
DNA (RAPDs), involving the use of single short DNA primer to direct amplification of discrete sequences (William, et al., 1990), have shown promise in cereals (Dovidio, et al., 1990; Weining and Langridge, 1991 and Devos and Gale, 1992). Recently, heterotic groups based on yield- specific combining ability data and phylogenetic relationship determined by RAPD markers for 28 tropical open pollinated maize varieties have been reported (Parentoni, et al., 2001).
The objectives of this investigation were to study the nature of genetic resistance to northern corn leaf blight (NCLB) and to identify RAPD markers linked to disease resistance gene(s) in a white population of maize.
Materials and Methods Plant Materials and Disease Evaluation
Genetic analysis and identification of RAPD markers linked to northern corn leaf blight (NCLB) disease resistance were carried out on F1 regeneration (single cross S.C.10W) and segregating F2 population, derived from a cross between two white lines; namely, the resistant line Sids-63 (Sd-63) and the susceptible one Sids-7 (Sd-7) and their parents.
The cross was made during the season of 2005 and was selfed in 2006 to produce the F2 population.
For evaluating against NCLB, the populations of F1 and F2 (259 individual plants) and their parents were planted under field conditions, in the late summer of 2007 at the Experimental Farm Station, Faculty of Agriculture, Alexandria University, Alexandria, Egypt, where environmental conditions allow for a uniform disease infection.
The artificial infection was done to enhance the natural infection, using an isolate of Helminthosporium turcicum cod T-13AS, that was a single spore culture grown in petri-dishes containing potato dextrose agar medium for ten days at 25 ± 2oC. Spore suspensions were prepared by adding sterilized distilled water over fungal growth, which was scraped off, using a sterilized needle. The suspensions were, then, strained through a sterilized cheese-cloth. Spore concentration was adjusted at 2.5
× 103 spore/ml using sterilized distilled water. Plants were inoculated at the three to five leaf stage of growth, in the evening, using a spore suspension. Severity of NCLB, as a percentage of infected leaf area (%
average lesion size), was assessed after flowering growth stage around eight weeks after the inoculation and readings were classified, according to Elliot and Jinkins (1946) as follows:
Genetic Analysis
Frequency distribution values were computed for parental, F1 and F2 populations for NCLB severity percentage under field conditions.
In respect to mode of inheritance, goodness of fit of the observed to the expected ratios of the phenotypic classes, concerning the NCLB severity and infection types, were determined by χ2 analysis, according to Steel and Torrie (1980). Moreover, the minimum number of effective genes, controlling NCLB resistance in the cross, was estimated by the formula of Wright (1968). In this formula,
n = (x¯ P 1 –x¯ P2)2 / 8 (VF2 – VF1) Where:
n = Minimum number of effective genes,
x¯ P 1 a n d x¯ P2 mean of P1 and P2, respectively and VF1 and VF2 variance of F1 and F2, respectively
This formula assumes that there is no linkage, no epistasis, no dominance, all loci have equal effects and all genes controlling resistance are in a single parent of the cross.
Degrees of dominance were calculated, according to the method suggested by Romero and Frey (1973).
Rating scale Leaf area Infected (%)
Resistance level
0.5 <5 Highly resistant (HR)
1.0 6-10 Resistant (R)
2.0 11-25 Moderately resistant (MR) 3.0 26-50 Moderately susceptible (MS)
4.0 51-75 Susceptible (S)
5.0 >75 Highly susceptible (HS)
In this method, the degrees of dominance, symbolized as h1 and h2 for F1 and F2, respectively, were calculated by the two formulae of:
h1 = ( x¯ F1 - x¯ MP) / D , and h2 = 2 ( x¯ F2 - x¯ MP) / D ,
Where:
D = (x¯ hp - x¯ MP), x
¯ F1, x¯ F2 and x¯ hp are the means of F1 , F2 and higher parent, respectively, while, x¯ MP is the mid-parent value.
In addition, the F1 and F2 means were compared with the mid-parent value, using t-test (Steel and Torrie, 1980) to determine whether h1 and h2 values were significantly different from zero.
Heritability, in its broad-sense, was estimated, according to Lush (1949), as follows:
h2 =
p G
V
V × 100
Where:
h2 = broad-sense heritability,
Vp = phenotypic variance of F2 individuals and
VG = Genotypic variance of F2 individuals [VF2 – 1/3 (VF1+VP1+VP2)]
DNA Extraction
Genomic DNA was extracted from fresh leaves of individual F2
plants and their parents, using CTAB (Saghai-Maroof, et al., 1984). RNA was removed from the DNA preparation by adding 10µl of RNAase (10mg/ml) and, then, incubating for 30 min. at 37°C. DNA sample concentration was quantified by using a spectrophotometer (Beckman Du-65).
PCR Amplification
Thirty-eight primers were used in the present investigation to amplify the templated DNA. Each amplification reaction was performed in a 25-µl vol., containing 50 ng of genomic DNA, 1x PCR buffer Mg
Cl2 (60 mM KCl, 10mM Tris- HCl (pH 9.0), 2mM MgCl2 and 1% Triton x-100), 200 mM each of dATP, dCTP, dGTP and dTTP (promega), 50 pM primer, 50ng template DNA and 1.5 µ of Taq DNA polymerase.
Amplifications were carried out in an MJ Research PTC-100 thermal cycler with amplification conditions, adopted from Williams, et al.
(1990): DNA denaturation at 94°C for three minutes and 45 cycles of melting at 94°C for one min., annealing at 36°C for one min. and extending at 72°C for two min. This was followed by a seven-min. final extension step at 72°C, then, the reactions were kept at 10°C. RAPD fragments were size-fractionated in a 2% agarose gel in 0.5 × TBE buffer, with a 1-kb ladder molecular weight marker. Gels were stained in ethidium bromide solution and, then, photographed.
Bulked Segregant Analysis
Bulked-segregant analysis (BSA) was used, in conjunction with RAPD analysis, (Michelmore, et al., 1991) to find markers linked to genes of interest. Resistant and susceptible bulks were prepared from F2
individuals by pooling aliquots, containing equivalent amounts of total DNA, approximately, 50 ng/µl from each of fourteen susceptible and fourteen resistant F2 plants selected, based on phenotypic assessments.
RAPD primers were, then, screened on the parents and the two bulk DNA samples, from which some primer combinations revealed bands that were polymorphic, not only between parental genotypes, but also between the pair of the bulk DNA. Based on the evaluations of DNA bulks, individual F2 plants were analyzed with cosegregating primers to confirm RAPD marker linkage to the NCLB resistance genes.
Data Analysis
Goodness of fit to a 3:1 ratios was calculated for RAPD marker by Chi-square test. The association between RAPD markers and resistance to NCLB trait was assessed with a simple regression analysis, using PROC REG in SAS version 9.1 software packages (SAS Institute, Cary, NC, 2007). Magnitude of the marker associated phenotypic effect was described by the coefficient of determination, R2, which represented the fraction of variance explained by the polymorphism of the marker.
Results and Discussion Genetic Analysis
The qualitative analysis of the resistance to NCLB was carried out, according to the response of the tested inbred lines, F1 and F2 populations against Helminthosporium turcicum pathogen at the adult stage, under field conditions, using the single spore culture isolate, T-13AS.
The frequency distributions of NCLB severity for the two inbred lines under investigation, F1 and F2 were classified and presented in Table 1. The results revealed that the inbred line, Sd-63, was the most resistant to NCLB and exhibited low disease severity, which ranged from less than 5% to 10%. On the other hand, the inbred line, Sd-7, expressed high susceptibility to NCLB, with a disease severity ranged from 26% to more than 75%.
The disease severity of F1 plants ranged from less than 5% to 50%.
This result indicated that low disease severity was partially dominant over high disease severity.
The F2 disease severity frequency distribution of the F2 population ranged from less than 5% to more than 75%. Furthermore, the number of plants with low: high NCLB severity was 183:76. This observed ratio fitted the theoretical expected ratio, 3:1, and suggested the operation of one gene pair in this cross.
To study the genetic behavior of white maize resistance to NCLB, the two inbred lines, F1 and F2 populations were tested in the adult stage under field conditions. Population means and variances of the inbred lines, F1 and F2 were used to estimate the degrees of dominance for F1
(h1) and F2 (h2), heritability in broad-sense and the number of functioning genes. The F1 mean value in the cross was 10.8%, and significantly lower than its respective mid-parent value, indicating the presence of complete dominance for low disease severity (partial resistance) .The F2 mean was 25.9%. Such mean was significantly different from its expected value based on P1, P2 and F1, revealing the presence of epistasis (Table 1). The obtained result, also, revealed that the estimated values of degree of dominance for the F1(h1) and for the F2(h2) were –0.86 and –0.71, respectively. The significant negative values of h1 and h2 suggested that the average degree of dominance was close to complete dominance in the
F1 and supported the F1 result. This indicates the importance of dominance gene action in the inheritance of NCLB resistance in this cross. Similar results were reported by Luo, et al. (2005) in wheat resistance to stripe rust. Their results indicated that the resistance to stripe rust was controlled by a single dominant gene. In the present investigation, the heritability value for the tested cross was 92%
indicating that the NCLB resistance is a heritable character and the effect of environment on the expression of this trait was small in respect to genetic effect .
Brule-Babel and Fowler (1988) stated that low heritability estimates are generally associated with large experimental errors and narrow crosses, while intermediate to high heritability estimates are associated with wider crosses. This idea corresponds with the results of our study, because the parents were quite different in responding to NCLB resistance, while applying the Wright’s formula to detect the number of genes controlling resistance was small and amounted to 1.1, indicating that there was a difference in resistance genes between the two inbred lines. Several assumptions for the validity of this equation were not fulfilled, therefore it is anticipated that the number of genes obtained would be less than the expected one. Milus and Line (1986) and Sofalian, et al. (2006), when they studied the genetic control of cold tolerance in wheat, stated that the genes controlling quantitative traits could be linked and could, therefore, segregate as a group or effective factor. If this was true, the formula would have estimated the number of effective factors and the number of individual genes would have been greater.
RAPD Markers Analysis
Out of 38 arbitrary primers (Table 2) screened for polymorphisms between the two tested inbred lines, Sd-63 resistant and Sd-7 susceptible parents, 27 RAPD primers (71.1%), that gave polymorphic bands suitable to differentiate between the two parents were identified. The total 201 bands were amplified, using 38 RAPD primers, produced an average of five bands per primer. The number of RAPD fragments, that were amplified, ranged from one to four and the sizes ranged from about 130 to 1200 bp. Of these twenty seven RAPD primers, Pr11 primer (5' CAATCGCCGT 3'), which produced two strong polymorphic bands at 180 and 300 bp, that were present in only the susceptible parent (Sd-7)
Table 1. Leaf blight severity classes, Chi - square analysis of F2 population generated from the cross, Sd-63 X Sd-7, degree of dominance, heritability in its broad sense (%) and number of genes. Leaf blight severity Means
Variance Phenotypes Expected ratio 2 χ
Degree of Dominance
Heritability % No. of genes
Genotype
No. of tested plants HR R MR MS S HS R : S R : S h1 h2 (Sd-63) 52 40 12 5 48.5 (Sd-7) 55 15 30 10 66.5 34.3 –0.86 –0.71 92 1.1 F1 45 15 10 10 5 10.8 12.9 F2 259 43 40 100 62 11 3 25.9 431.4 183: 76 3 : 1 2.6ns
(Fig. 1) for screening DNA bulks and their parental DNA. The primer, Pr11, generated the two polymorphic fragments at 180 and 300 bp, which were present only in NCLB-susceptible bulk and Sd-7 parent and were missing in NCLB-resistant bulk and Sd-63 parent (Fig.1). These RAPD markers (Pr180 bp and Pr300 bp) were regarded as candidate markers linked to NCLB susceptible gene in maize.
Table 2. Number of amplifications and polymorphic products of thirty-eight primers used to screen the two inbred lines (Sd-63 and Sd-7).
Primer
Nucleotide sequence
(5' 3')
No. of amplification
products
No. of polymorphic
products
Pr1 CAGGCCCTTC 3 1
Pr2 TGCCGAGCTG 3 0
Pr3 AGTCAGCCAC 4 1
Pr4 AATCGGGCTG 7 3
Pr5 AGGGGTCTTG 4 0
Pr6 GGTCCCTGAC 6 2
Pr7 GAAACGGGTG 6 2
Pr8 GTGACGTAGG 4 3
Pr9 GGGTAACGCC 4 3
Pr10 GTGATCGCAG 5 2
Pr11 CAATCGCCGT 5 3
Pr12 TCGGCGATAG 4 1
Pr13 CAGCACCCAC 12 3
Pr14 TCTGTGCTGG 5 1
Pr15 TTCCGAACCC 3 1
Pr16 AGCCAGCGAA 6 0
Pr17 GACCGCTTGT 3 0
Pr18 AGGTGACCGT 5 2
Pr19 CAAACGTCGG 8 3
Pr20 GTTGCGATCC 13 2
UBC321 ATCTAGGGAC 0 0
UBC475 CCAGCGTATT 2 1
UBC532 TTGAGACAGC 6 3
OPA02 TGCCGAGCTG 6 4
Table 1. Contd.
Fig. 1. RAPD fragments, produced by primer Pr11.
M: Molecular weight.
P1and P2 parents, Sd-63 and Sd-7, respectively.
Br: bulk resistance; Bs, bulk susceptible. Primer
Nucleotide sequence
(5' 3')
No. of amplification
products
No. of polymorphic
products
OPA06 GGTCCCTGAC 0 0
OPA07 GAAACGGGTG 4 0
OPB08 GTCCACACGG 9 4
OPB09 TGGGGGACTC 0 0
OPB13 TTCCCCCGCT 4 1
OPC04 CCGCATCTAC 6 1
OPC15 GACGGATCAG 7 3
OPE20 AACGGTGACC 7 4
OPF15 CCAGTACTCC 0 0
OPH13 GACGCCACAC 8 1
OPJ04 CCGAACACGG 8 2
OPJ10 AAGCCCGAGG 6 0
OPU06 ACCTTTGCGG 10 0
OPZ03 CAGCACCGCA 8 1
These polymorphic markers, Pr11180 bp and Pr11300 bp, were further used to check their linkage to the NCLB susceptible gene, using a segregating F2 population, derived from the cross between the resistant parent Sd-63 and the susceptible parent,Sd-7. When analyzing the individual plants of F2 population, the Pr11180bp and Pr11300 bp fragments were amplified in the DNA obtained only F2 susceptible ones. The PCR amplification of resistant parent (Sd-63), susceptible parent (Sd-7), resistant bulk, susceptible bulk, five F2 resistant and five F2 susceptible individuals, using primer, Pr11 are shown in Fig. 2.
Fig. 2. RAPD fragments, produced by primer Pr11.
M: Molecular weight.
PI and P2 parentsSd-63 and Sd-7, respectively.
Br, bulk resistance; Bs, bulk susceptible.
F2 individuals in the cross, Sd-63 X Sd-7.
R: resistant; S: susceptible.
For the RAPD markers Pr11180 bp and Pr11300 bp, 76 of 259 (29.3%) individuals, in the F2 population, exhibited the amplified polymorphic fragments (180 and 300 bp), while , the remaining did not (Fig. 2). The ratio fitted the expected Mendalian ratio, 3:1 (χ2 = 2.6, P < 0.01).
To check for potential co-segregation of DNA fragments and NCLB resistant phenotypes, simple regression analysis was carried out in order
to confirm an association between the Pr11180 bp and the Pr11300 bp markers and the resistance to NCLB in all 259 F2 progenies.
The results showed that the regression analysis for the relationship between the two markers, Pr11180 bp and Pr11300 bp, and the phenotypes of F2 individuals were significant and they recorded r2 = 0.54 and 0.60, respectively. This indicates that the two markers were linked with the NCLB susceptible gene.
Bulked segregant analysis (BSA) (Michelmore, et al., 1991), combined with several types of molecular marker, have been extensively used to find markers linked to disease resistance genes in a number of species (Reiter, et al., 1992; William, et al., 2003 and Barakat and Imbaby, 2005). In several previous studies, RAPD combined with BSA have been successfully used to identify DNA marker(s) linked to many important traits. For example, Poulsen, et al. (1995) found RAPD marker (OPU022700) linked to leaf rust resistance in barley. The OPU022700
marker was shown to be useful in the identification of the individual F2
plants originally misclassified as having susceptible infection types.
Motawei, et al. (2001) reported the presence of RAPD marker (Pr7700) linked to the leaf rust resistance gene Lr29 in wheat. Moreover, Motawei, et al. (2003) found two RAPD markers (Pr8450 and Pr18700) linked to stripe rust resistance gene in wheat.
Based on the results of the present study, it could be stated that the dominance gene action is responsible for the inheritance of NCLB resistance in this maize cross, and because of high broad sense heritability, selection for NCLB resistance should be effectively practiced in breeding programs using this cross. In addition, the results indicated that RAPD markers, combined with bulked segregant analysis, could be used to identify molecular markers linked to NCLB resistance gene in maize. Once these markers are identified, they may facilitate genetic improvement of NCLB resistance in these programs, as a selection tool in early generation.
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