DOI: 10.4197/Met. 23-1.3
37
RAPD Markers for the Detection of Common Smut Resistance Gene(s) in Inbred Lines of Maize (Zea mays L.)
Younis Kh. Hamad; Emadeldin H.Wasfy; Samia A. Farag;
and Aziza K. Darweesh
Department of Plant Pathology, Faculty of Agriculture, Alexandria University, Egypt.
Abstract. This study was conducted to develop the common smut resistance gene(s) associated DNA markers in some maize inbred lines and estimate the genetic diversity among the six maize inbred lines under investigation. Twenty RAPD random primers have been used to determine marker(s) between the resistant and susceptible corn plants. Results indicated that some distinct markers were observed in case of using some primers but with the most of other primers, no markers were observed.
Two markers, in case of primer no. 4 (5- GTCCACACGG 3-), at 280 and 400 bps in case of resistant bulk, were detected. Five markers, in case of primer no.16 (5- TCGGCGATAG 3-), at 620 and 1200 bps in resistant bulk, were detected and also at 330, 400 and 890 bps in susceptible one.
Moreover, two markers, in case of primer no. 19 (5- TTCCGAACCC 3-), one of them at 480 bp in resistant bulk and the second at 900 bp in susceptible one. One marker in inbred line GM 27at 280 bp and the other one in inbred line GM 30 at 400 bp were detected in resistant plants only.
Four cluster groups were identified, however, the first included the inbred line G635, white; the second included two lines GM14 and GM27, white;
the third included two lines GM1002 and GM 1021, yellow; the fourth cluster included only line GM30, white. Accordingly, the shortest genetic distance (highest similarity value) was detected between two white lines GM14 and GM27 (0.55). On the other hand, the highest genetic distance (lowest similarity value) was manifested between two lines; G635, white and GM1002, yellow (0.29).
Keywords: Ustilago maydis, Zea mays, RAPD, resistance genes, inbred lines.
Introduction
The progress of DNA marker technology was tremendous and exciting.
Accordingly, several scientists tackled this important subject in details.
However, the first citation here is about the recent developments in DNA marker technology, together with the concept of marker assisted selection, provide new solutions for selecting and maintaining desirable genotypes (Mohan et al., 1997). In plant breeding programs, DNA markers have been used to measure genetic diversity among lines (Dudley et al., 1991; and Smith and Smith, 1992), which assist in selecting parents (Dudley, 1992), fingerprint lines for legal purposes and use genetic approaches in the seed production process (Smith and Smith, 1989). Advances in molecular biology during the last two decades have provided a new class of genetic markers;
namely, DNA markers. The development of DNA marker technology has enabled breeders to use a Mendelian genetic approach to complement breeding programs. Genetic markers greatly facilitate the selection of parents and assist in the development of new improvement strategies based on marker-assisted breeding (Kumar, 1999). The objectives of the present study were to develop the common smut resistance gene(s) associated DNA markers in some maize inbred lines and estimate the genetic diversity among the six maize inbred lines under investigation.
Materials and Methods
Plant Material
Six pure lines of maize (four white lines, G635, GM14, GM27 and GM30 and two yellow lines, GM1002 and GM1021) were used. Seeds of each line were sown in 25 cm diameter pots (5 seeds/ pot) in sandy clay soil (2:3, w:w). The inoculum (1X106 sporidia /ml) was implemented by a needle stem injection at 4 - 6 – leaf-old plants. The genomic DNA of healthy parts of leaves of young plants, 7 days after galls appearance, were extracted and then tested using PCR technique for both resistant and susceptible young plants of the aforementioned six pure lines.
DNA Extraction
Frozen younig leaves (500 mg) of pure lines of maize were individually ground to a powder in a mortar with liquid nitrogen. The DNA extraction was done using CTAB method (Sagahi-Maroof et al.,
1984). According to Sambrook et al., (1989), it is assumed that DNA at a concentration of 50g/ml has an OD of 1 at 260 nm as follows:
DNA concentration (g/l) = OD260 X 50 dilution factor X 50 g/ml 1000
PCR Amplification
Twenty RAPD primers (Table 1), obtained from Pharmacia Biotech. (Amersham Pharmacia Biotech, UK Limited, England HP79 NA), were tested in this experiment to amplify the template DNA according to Mullis (1993).
Amplification of reaction volumes was 25l, each containing 1x PCR buffer with MgCl2 (50mM KCl, mM Tris-HCl (pH=9.0), 2mM MgCl2 and 1% trition x-100), 200M each of dATP, dCTP, dGTP and dTTP, 50PM primer, 50ng template DNA and 1.5l of Taq polymerase.
Reaction mixtures were overload with 15 l mineral oil and exposed to the following conditions: 94C for 3min, followed by 45 cycles of 1 min.
at 94C, 1 min. at 36C, 2 min. at 72C, and a final 7 min. extension at 72C.
Amplification products were visualized with DNA marker on 1.6%
agarose gel with 1x TBE buffer and were detected by staining with an ethidium bromide solution for 30 min. Gels were, then, de-stained in de- ionized water for 10 min. and photographed on Digital camera.
Data Handling and Cluster Analysis
Data were scored for computer analysis on the basis of the presence or absence of the amplified products for each primer. If a product was present in a genotype, it was designated as “1”, if absent as “0” after excluding the irreproducible bands. Pairwise comparisons of genotypes, based on the presence or absence of unique and shared polymorphic products, were used to determine similarity, according to Jaccard (1908).
The similarity coefficients were then used to construct dendrograms, using the un-weighted pair group method with arithmetic averages (UPGMA) employing the SAHN (Sequential, Agglomerative, Hierarchical, and Nested coefficients clustering) from the NTSYS-PC (Numerical Taxonomy and Multivariate Analysis System), version 1.80 (Applied Biostatistics Program) according to Rohlf (1993).
Results and Discussion
Detection of the Common Smut Resistance Gene(s)
In the present investigation, twenty primers of arbitrary nucleotide sequence (Table 1) were used to amplify DNA segments from the two bulks DNA samples as follows. The first bulk (Br, bulk resistant) was constructed using equal amounts of DNA from the ten plants resistant to common smut, for each inbred line, selection was based on phenotypic assessments. The same was done in the second bulk which represents susceptible plants (Bs, bulk susceptible).
Table 1. RAPD primers with the number of amplified products and polymorphic fragments of total resistant and susceptible bulks.
Primers Sequence
5 → 3 No. of amplify.
products (a)
No. of polymorphic products (b)
% of polymorphism (b/a)
1 CCAGCGTATT 0 0 0 %
2 TTGAGACAGG 2 2 100 %
3 ATCTAGGGAC 0 0 0 %
4 GTCCACACGG 17 9 52.9 %
5 TTCCCCCGCT 3 3 100 %
6 CCAGTACTCC 0 0 0 %
7 CAGGCCCTTC 2 2 100 %
8 TGCCGAGCTG 0 0 0 %
9 GACCGCTTGT 4 3 75 %
10 AGGTGACCGT 6 2 33.3 %
11 AGGGGTCTTG 4 3 75 %
12 GGTCCCTGAC 3 2 66.7 %
13 GGGTAACGCC 10 5 50 %
14 GTGATCGCAG 2 2 100 %
15 CAATCGCCGT 8 7 87.5 %
16 TCGGCGATAG 10 10 100 %
17 CAGCACCCAC 13 7 53.8 %
18 TCTGTGCTGG 1 0 0 %
19 TTCCGAACCC 7 6 85.7 %
20 AGCCAGCGAA 4 3 75 %
The number of amplification products produced by each primer (Table 1) varied from as few as one (primer 18, 5 'TCTGTGCTGG 3 ') to as many as 17 (primer 4, 5 'GTCCACACGG 3 '). Four primers (1, 3, 6 and 8) failed to reveal any amplification (no fragments at all). Out of 20 primers tested, 15 revealed polymorphism ranging from 33.3% to 100%
(Table 1).
In all, 96 amplification products were obtained, out of which 66 showed polymorphism, the remaining products were monomorphic across the DNA bulks.
Figures 1 and 2 show that amplification profiles generated by primers, Pr-4, Pr-16 and Pr-19, respectively, in case of the two DNA bulks. Strong polymorphic bands were present in only the total resistant bulk at 280 bp and 400 bp, but absent in the total susceptible bulk (Fig.1).
Fig. 1. An electrophoretic profile showing polymorphism due to using five primers (1-5) to amplify genomic DNA purified from the two bulks (TBr, TBs).
TBr = Total bulk resistant across inbred lines.
TBs = Total bulk susceptible across inbred lines.
M = Ladder DNA marker.
Accordingly, these markers were completely associated to the resistance gene of common smut. Primer 16 gives strong polymorphic bands in the total resistant bulk at only 620 and 1200 bps (Fig. 2) and in the total susceptible bulk at only 330, 400 and 890 bps (Fig. 2). Also, with primer 19, strong polymorphic bands were observed at only 480 and 900 bps in the total resistant and susceptible bulks, respectively (Fig. 2).
In the present investigation, two markers were associated with resistance to common smut for the primers 4 and 16, whereas, one marker only was associated with resistance to common smut for the primer 19. The number of primers used in the RAPD method should be neither too small, because this could lead to a non-information or biased analysis nor too high, which could result in increased cost. Various numbers of primers have been used in the study of different genotypes of maize that revealed various degrees of polymorphism.
Fig. 2. An electrophoretic profile showing polymorphism due to using five primers (16-20) to amplify genomic DNA purified from the two bulks (TBr, TBs).TBr = Total bulk resistant across inbred lines. TBs = Total bulk susceptible across inbred lines. M = Ladder DNA marker.
Three primers (Pr-4, Pr-16 and Pr-19) were used for screening the bulks containing bulked DNA from only the 10 plants, each from resistant and susceptible inbred line. The PCR amplification of the total resistant and susceptible DNA bulks derived from all six inbred lines, resistant bulk and susceptible bulk for each inbred line using primer 4 are shown in Fig. 3. Reproducible strong polymorphic bands were present only in the resistant bulk at 280 bp and 400 bp for the inbred lines L3 and L4, respectively (Fig. 3), using primer 4 but absent in the susceptible bulk. These results indicated that the Pr-4280 and Pr-4400 bps, used as markers, were associated with the resistance gene to common smut in case of the inbred lines L3 and L4.
Detection of multiple genes for quantitative resistance, using segregating analysis alone, is always a matter of debate, due to the difficulties in differentiating primary genetic effects they affect of environmental factors. (Mc Mullen and Louie, 1991). Therefore, identification and management of durably resistant effective genes for common smut has not been entirely successful (El-Shenawy, 1995). At the same time, bringing more than one gene together into a single genotype by conventional means in many cases is not achievable because screening for one resistance gene interferes with the ability to screen for
another. One promising approach to overcome this problem is RAPD to develop molecular markers, and to dissect the genetic control of quantitative traits (Tanksley, 1993). Therefore, identification of RAPD markers linked with resistance to common smut, allows complex traits to be resolved into simple, tagged, mendelian components (Paterson et al., 1988).
Fig. 3. An electrophoretic profile of bands of DNA after using primer 4. Two bands 280 and 400 bp used as markers and associated with resistance gene to Ustilago maydis (corn smut disease) in L3 and L4 inbred lines, respectively.
Previously, RAPD markers have been successfully used to identify DNA marker(s) linked to many important traits. Poulsen et al. (1995) found RAPD marker linked to leaf rust resistance in barley. Yang et al.
(1997) identified RAPD marker linked to a scab resistance in apple. This marker was converted into SCAR marker and used in turn to screen scab- susceptible and resistant apple cultivars. While, Barakat et al. (2001) identified RAPD marker linked to the leaf rust resistance gene Lr 29 in wheat population. Arnedo-Andres et al. (2002) identified RAPD marker linked to the Pvr 4 locus for resistance to potato virus Y (PVY) in pepper. Recently, Barakat and Imbaby (2005) found RAPD marker linked to yellow rust resistance in wheat.
Genetic Diversity Among Inbred Lines of Maize
The second goal of the present study was to investigate the efficiency of RAPD markers in determining, accurately, the genetic relationship between maize inbred lines under investigation.
Four primers were screened for their ability to amplify the genomic DNA of the inbred lines of maize. The number of DNA fragments amplified ranged from 9 to 21 depending on the primer and the DNA sample with a mean value of 13.5 bands per primer (Table 2). There were no correlations between the number of bands amplified and the degree of polymorphisms. For example, the primers 15 and 17 generated 9 and 21 bands, respectively, of which 100% and 95.2% were polymorphic; on the other hand, 84.6% of the 13 bands amplified by primer 4 were polymorphic (Fig. 4).
The RAPD markers, produced by four primers, were used to construct a similarity matrix (Table 3). Simple matching coefficient, ranging from 0.29 to 0.55. (Table 3) indicates the genetic similarity estimates of the 15 pairwise comparisons among the maize inbred lines, based on 50 polymorphic bands (Table 2).
Table 2. Number of amplification and polymorphic products, using four primers in inbred lines of maize, L1 to L6.
Polymorphism (b/a) No. of
polymorphics (b) No. of
amplifications (a) Sequence
5 → 3
Primer number
84.6%
11 13
GTCCACACGG 4
90.9%
10 11
GGGTAACGCC 13
100%
9 9
CAATCGCCGT 15
95.2%
20 21
CAGCACCCAC 17
Fig. 4. An electrophoretic profile showing RAPD polymorphism in maize inbred lines. M = Marker; Inbred line L1 = G635 W;Inbred line L2 = GM14W; Inbred line L3 = GM27W; Inbred line L4 = GM30W; Inbred line L5 = GM1002Y; Inbred line L6 = GM1021Y.
11
Table 3. Simple matching coefficient of similarity matrix for 6 maize inbred lines determined by RAPD analysis using four different primers, 4, 13, 15 and 17.
G635 W GM14W GM27W GM30W GM1002Y GM1021Y
G635W 1
GM14W 0.49 1
GM27W 0.46 0.55 1
GM30W 0.33 0.33 0.4 1
GM1002Y 0.29 0.39 0.32 0.38 1
GM1021Y 0.43 0.44 0.46 0.47 0.53 1
Figure 5 represents the clustering of maize inbred lines generated by UPGMA analysis of the six maize inbred lines. The results of characterization analysis revealed a high diversity between the inbred lines. Four clusters could be observed. The first cluster included only line G635W, while the second one included line GM14W and line GM27W.
The third cluster included line GM1002Y and line GM1021Y. The last cluster included only line GM30W. It can be seen from this figure that the shortest genetic distances (the highest similarity value, 0.55) was found between lines GM14W and GM27W, whereas, the highest genetic distance (the lowest similarity value, 0.29), was observed between lines G635W and GM1002Y.
Fig. 5. Dendrogram of genetic distances constructed according to RAPD data and using UPGMA method of clustering, shows DNA similarity between maize inbred lines.
In conclusion, these results must be regarded as preliminary studies because of the small size of analyzed samples, the low number of used primers and the low number of generated bands. Nevertheless, they are encouraging. Moreover, they give information on the level of genetic polymorphism existing among these inbred lines and brings new prospective for the use of such markers in a breeding program for improving disease resistance in maize such as resistance to common smut.
The PCR amplification that generates RAPD fragments of interest is very sensitive to specific reaction conditions. Moreover, poor reproducibility can occur in RAPD analysis (Karp et al., 1997). In order to increase the specificity of the reaction and to simplify the use of markers linked to common smut resistance in maize breeding programs, the markers will be transformed into sequence characterized amplified region (SCAR) in future study. Converting RAPD markers to SCAR markers have been reported to facilitate screening of genotypes for a particular trait as they are identified as distinct single bands in agarose gel. Moreover, in some reports this type of markers can be used to differentiate heterozygotes from homozygotes (Paran and Michelmore, 1993). Future work will facilitate the improvement of horizontal resistance to common smut without the drawbacks of pathological tests and will involve the mapping of the molecular markers on the genetic map of maize for detecting the QTLs linked to common smut resistance genes, which will significantly increase their usefulness.
These results indicated that RAPD technique could be successfully applied to field crops. Genetic distance of inbred lines and predication of maize single-cross performance using RAPD markers have been reported (Lanza et al., 1997; Shieh and Thseng, 2002; Garcia et al., 2004).
Acknowledgment
The authors wish to express their gratitude to Prof. Dr. Mohamed N. Barakat, Professor of Crop Science Department, Biotechnology lab., Faculty of Agriculture, Alexandria University, for his help, guidance, and valuable ideas concerning this study.
References
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ـلا لئلاد مادختسا محفتلا رطفل ةمواقملا تانيج ديدحتل RAPD
داعلا ي ف ي ةيماشلا ةرذلل ةيقنلا تلالاسلا
دمح نوع للهاريخ سنوي
، و
،ىفصو نيسح نيدلادامع
و جرف معنملا دبع ةيماس
، و شيورد لماك ةزيزع
تابنلا ضارما مسق
، ةعارزلا ةيلك
، ةعماج لإا ةيردنكس
، رصم
لختسملا ص . ـلا تارابتخا تيرجأ مادختساب RAPD
داـب02 ئ يئاوـشع
لـــيلد دـــيدحتل أ
و أ يـــب ةــــقرفتلل رـــثك ةـــلباقلاو ةــــمواقملا ةرذـــلا تاـــتابن
ةباصلإل . اـ نم ، ـيقن تلالاـس تـس سـلع تاـساردلا لذـه تـيرجأ ـيح
يـــــــهو اـــــــ يب ةـــــــعبرأ ،G635
و ،GM14
و
، GM27
و ـــــــنثاوGM30
ا
هو ارفص ي
GM 1002
، و
GM 1021
، و لئلادـلا ـم ددـع رـ ظ دق ـم
ئداوبلا ضعب
، ر ظ دقف ،ةح او لئلاد ا مظعم م ر ظي ملو يـنثا
مــقر ئداــبلا ــم لئلادــلا لذــه ــم 4
(5- GTCCACACGG 3-)
دــنع
bp 280
دــنعو
400 bp
ةــمواقملا ةــلاح يــف
، ةدــع ترــ ظو لــئلاد
ــم
ئداــبلا مــقر 61
(5' TCGGCGATAG 3')
دــنع ترــ ظف
620 bp
،
1200 bp
مواـقملا ةـلاح يـف دـنعو
330 bp
، و
400 bp
، و
890 bp
يـف
ةـــــلاح يـــــلد رـــــ ظ لذـــــك ،ةباـــــصلإل لـــــباقلا لا
مـــــقر ئداـــــبلا ـــــم 61
(5' TTCCGAACCC 3')
ر ظ يح يلد
ل دنع دحاو
480 bp
ةلاح يف
دــنعو مواــقملا
900 bp
ةــلاح يــف لــباقلا
ةباــصلإل . رــ ظ لذــك يــلد
لا
ـم مبـس اـمك دـنع TBr
280 bp
، و
400 bp
، رـ ظ دـقف
marker
يـف
مـقر ةللاـسلا
(GM 27)
دـنع
280 bp
، مـقر ةللاـسلا يـفو 4
(GM 30)
دـنع
400 bp
و ، طـقف ةــمواقملا تاــتابنلا يـف ــلذ ةباــصلإل ةــلباقلا ود
.
تــطعأ لذــك ئاــتن ةيئاوــشع ئداوــب 4
إ ــم ةــيباجي ةرذــلا تاــبنDNA
لـــــمعل ـــــلذو
cluster analysis
تلالاـــــسلا لذـــــه ةـــــبارق ةـــــجرد ةـــــفرعمل
ا ــــ عبل
، ـــــلا رــــطفل ةــــمواقملاو ةــــيبرتلا مارــــب يــــف ا مادختــــسلا ــــلذو
U. maydis
ةللاـسلا اـ نم سـلووا تلمـش تاعومجم ةعبرأ تر ظ دقو ،
اــــــــ يبلا
G 635
اهدــــــــحو
، يواــــــــ يبلا يتللاــــــــسلا تلمــــــــش ةــــــــيناثلاو
GM 14
، و
GM 27
، يوارفــصلا يتللاــسلا ةــثلاثلا تلمــشو
M 1002
و
GM 1021
ةعبارلا ةعومجملا تلمش امنيب اـ يبلا ةللاـسلا
GM 30
اهدحو ةظحلام كمي .
ا ئاـتنلا لذه م رـصقأ
ةـينيج ةفاـسم (
سـلعأ
باـــشت ةـــميق )
اـــ يبلا ةللاـــسلا يـــب تـــناك
GM 14
اـــ يبلا ةللاـــسلاو
GM 27
( )2500 ةــينيج ةفاــسم لوــطأ اــمنيب (
باــشت ةــميق لــقأ )
تــناك
ا يبلا ةللاسلا يب
G 635
ارفصلا ةللاسلاو
GM 1002
( .)2501
ةيحاتفملا تاملكلا :
سدييام وجلايتسوي رطف –
ةيماشلا ةرذلا –
لعافت
- RAPD
ةمواقملا تانيجلا –
ةيقنلا تلالاسلا .
