Crop Science Journal of Natural Resources and Life Sciences Education
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Published online 1 March 2007
Published in Crop Sci 47:767-772 (2007)
© 2007 Crop Science Society of America
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TURFGRASS SCIENCE

Peroxidase Gene Polymorphism in Buffalograss and Other Grasses

O. Gulsen, R. C. Shearman*, T. M. Heng-Moss, N. Mutlu, D. J. Lee and G. Sarath

O. Gulsen, Alata Horticultural Research Institute, Mersin 33740, Turkey; R.C. Shearman and D.J. Lee, Dep. of Agronomy and Horticulture, Univ. of Nebraska, Lincoln, NE 68583; T.M. Heng-Moss, Dep. of Entomology, Univ. of Nebraska, Lincoln, NE 68583; N. Mutlu, West Mediterranean Agricultural Research Institute, Antalya 07110, Turkey; G. Sarath, USDA-ARS, Lincoln, NE 68583

* Corresponding author (rshearman1{at}unl.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Plant peroxidases are a family of related proteins possessing highly conserved domains. Degenerate oligonucleotide primers based on these conserved domains can be used to amplify DNA sequences coding for peroxidases from plants with unsequenced genomes. Polymorphisms in peroxidase genes among buffalograss [Buchloe dactyloides (Nutt.) Engelm.] genotypes and eight other grasses were evaluated, and potential evolutionary relationships were deduced using this approach. Fourteen peroxidase specific primers with alternative forward and reverse primers using 34 rice peroxidase cDNAs were designed based on conserved motifs of this gene family. Targeted polymerase chain reaction (PCR) amplification of genomic DNA from 28 buffalograss, 4 C4, and 4 C3 grass genotypes yielded polymorphisms, differentiating diploids from polyploids within buffalograss and C3 and C4 grass species from each other. A total of 11 peroxidase gene fragments, 7 belonging to buffalograss and 4 to the other grass species, were sequenced. Five of these sequences were clustered with rice (Oryza sativa L.) ascorbate peroxidase known to have chloroplast origin. These results demonstrate that primers targeting the peroxidase gene family can be used to study genotypic diversity and evolutionary relationships on an intraspecific and interspecific basis. The PCR-based peroxidase markers may also have potential for linkage mapping and differential gene expression studies in grasses.

Abbreviations: NCBI, National Center for Biotechnology Information • PCR, polymerase chain reaction • POGP, peroxidase gene polymorphism • SRAP, sequence-related amplified polymorphism • UPGMA, unweighted pair group method arithmetic average.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
PLANT PEROXIDASES belong to a multigene family and play important roles in many stress-related interactions, such as pathogen infection, insect tolerance, salt tolerance, auxin degradation, cell wall lignification, tissue suberization, and plant senescence (Hinman and Lang, 1965; Espelie et al., 1986; Whetten and Sederoff, 1995; Amaya et al., 1999; Chittoor et al., 1999; Heng-Moss et al., 2004; Passardi et al., 2005). Peroxidases are heme-containing proteins that can oxidize compounds in the presence of peroxide (H2O2) or oxygen (O2). They possess three highly conserved motifs: the distal heme-binding domain, the central domain with unknown function, and the proximal heme-binding domain (Hiraga et al., 2001). Yoshida et al. (2003) reviewed the research on the regulatory mechanisms of several peroxidase genes. They reported that tissue wounding, pathogen injury, salt stress, and virus infection were involved in peroxidase gene regulation.

Plants have two classes of peroxidases, intracellular peroxidases, which are related to bacterial peroxidases (class I), and peroxidases targeted to the secretory pathway (class III) (Welinder, 1992). Duroux and Welinder (2003) used 73 class III Arabidopsis thaliana peroxidase genes to study evolutionary relationships among peroxidases in the plant kingdom. Their study indicated that grasses possess some unique peroxidase genes that were not present in dicots. Their findings are not unexpected since monocots and dicots evolved under different abiotic and biotic stresses.

Zhang et al. (2001) surveyed the molecular evolutionary dynamics of 25 multigene families, including peroxidase genes from four grass taxa. Alignment of peroxidases within the grass taxa indicated an average sequence identity of 64.2, ranging from 53.1 to 90.9. The level of sequence identity among peroxidase genes was the second lowest among 25 multigene families. The higher-than-expected peroxidase diversity, compared with the remaining 23 multigene families, indicated an above-average evolutionary force on this gene family. Therefore, comparison of sequences of peroxidase genes could resolve evolutionary relationships in some taxa.

Buffalograss [Buchloe dactyloides (Nutt.) Engelm.] is native to the shortgrass prairie of North America (Shearman et al., 2004). It is a dioecious, low-growing, stoloniferous species that is suitable for turfgrass use (Riordan, 1991). It is also relatively pest free. Current knowledge is limited for the genetic basis of buffalograss agronomic traits and their allelic diversity among genotypes with different ploidy levels. Developing a better understanding of the underlying genetic factors for economically important traits, such as chinch bug (Blissus occiduus Barber) resistance in buffalograsses, would allow breeders to make earlier selections for improved turfgrass performance.

Our study evaluated peroxidase polymorphism among buffalograss genotypes, in particular, and eight other C4 or C3 grasses to determine the evolutionary relationships among grasses based on peroxidase genes. This is the first comprehensive report for which specific peroxidase primers were used to study peroxidase gene polymorphism (POGP) among grass species, with particular emphasis on buffalograss.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
This study used 28 buffalograss genotypes and 8 other grass species (Table 1). The 28 buffalograss genotypes included three cultivars (‘Prestige’, ‘Density’, and ‘378’). The buffalograsses were previously evaluated for chinch bug resistance (Gulsen et al., 2004). The eight other grasses included four warm-season (C4) species (sorghum [Sorghum bicolor Moench], zoysiagrass [Zoysia japonica Steudel], bermudagrass [Cynodon dactylon (L. Pers.)], switchgrass [Panicum virgatum L.]) and four cool-season (C3) species (barley [Hordeum vulgare L.], rice [Oryza sativa L.], wheat [Triticum aestivum L.], rye [Secale cereale L.]) (Table 1). We obtained the 28 buffalograsses and 8 other grasses from plant collections maintained at the University of Nebraska–Lincoln and the USDA Germplasm Repository.


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  		Table 1. Scientific and common names, and ploidy levels of buffalograss and other grass genotypes investigated.

 
Total DNA was extracted from 40 to 50 mg of young, frozen leaf tissue from a single plant genotype by means of a DNA extraction kit, Puregene (Gentra Systems, Minneapolis, MN). DNA concentrations were measured with a fluorometer (Hoefer Scientific Instruments, San Francisco, CA), and 5 ng {upsilon}L–1 DNA templates were made in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0).

Primers were designed based on 34 rice peroxidase cDNA sequences available at the website of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). First, 34 peroxidase cDNA sequences were clustered based on their sequence similarities using CLUSTAL W algorithm nested in Vector NTI Suite Software Version 8.0 (Invitrogen Corp., Carlsbad, CA). Then, forward and reverse primers for each cluster were designed using the Vector NTI multiple sequence alignment module. All primers were anchored at conserved regions using rice peroxidase cDNA sequences. Alternative forward and reverse primers were also designed when additional conserved sequences were found in rice cDNAs. While developing the peroxidase primers, we adjusted primer sizes for the annealing temperatures of forward and reverse primers.

Twenty-two combinations of 14 forward and 16 reverse peroxidase primers were used to target the amplification of peroxidase sequences from genomic DNA of plant genotypes (Table 2). Each 25 µL reaction consisted of 5 pM µL–1 of the primer pairs, 200 µM of each of dNTPs, 2.5 µL of 10X PCR Buffer, 5 µL of Q Solution, 2 mM of MgCl2 as a final concentration, 6 µL ddH2O, 1 unit of Taq polymerase (Qiagen, Valencia, CA), and 25 ng of template DNA. Cycling parameters included one cycle of 2 min at 94°C, 34 cycles of 1 min at 94°C, 1 min at 48 to 57°C, 1 min at 72°C, and for final extension, one cycle of 5 min at 72°C. Polymerase chain reaction (PCR) products were separated on 2.5% agarose gel at 90 V for 4 or 5 h. Bands of amplified DNA were visualized with ethidium bromide staining.


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  		Table 2. Forward and reverse peroxidase primers designed using rice (Oryza sativa) peroxidase cDNA sequence.

 
Each band was scored as present (1) or absent (0), and data were analyzed with the Numerical Taxonomy Multivariate Analysis System (NTSYS-pc), version 2.1, software package (Exeter Software, Setauket, NY) (Rohlf, 1993). A similarity matrix was constructed based on Dice's coefficient (Dice, 1945), which considers only one to one matches between two taxa for similarity. The similarity matrix was used to construct a dendogram using the unweighted pair group method arithmetic average (UPGMA) to determine genetic relationships among the germplasm studied.

For sequencing, PCR products were separated on 1% agarose gel at 90 V for 4 h, and bands were excised using sterilized razor blades. The DNA was purified using the QIAquick Gel Extraction Kit (Qiagen) and sequenced at the Genomic Core Facility, University of Nebraska–Lincoln (http://www.biotech.unl.edu). Sequencing was performed using the same forward and reverse primers.

Peroxidase gene sequences from buffalograss and four other grasses (rye, sorghum, bermudagrass, and switchgrass) were aligned and clustered using Vector NTI Suite Software Version 8.0. Default parameters of the software were used to construct phylogenetic relationships among the peroxidase genes based on sequence similarity.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
All primers designed in this study yielded scorable bands except for POX7. Primer sizes ranged from 13 (POX9F) to 24 (POX14R), averaging 18.4 bases. Fifty-two bands were scored in buffalograss, and 64 bands in other grasses. Forty-one out of 52 (79%) bands from buffalograsses and all 64 bands from the other grasses were polymorphic. Band size ranged from 100 to 750 bp, with a mean of 425 bp. Peroxidase gene polymorphism (POGP) bands gave a similarity range between 0.66 and 0.94, averaging 0.80 (Fig. 1 ). Gulsen et al. (2005) reported a slightly lower level of polymorphism, ranging from 0.70 to 0.97 with an average of 0.83, using sequence-related amplified polymorphism (SRAP) markers in the same buffalograss accessions. Therefore, peroxidase gene polymorphism markers appeared to be as useful as SRAP markers in assessing polymorphism in buffalograss.


Figure 1
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  		Figure 1. Unweighted pair group method arithmetic average (UPGMA) dendogram with similarity coefficients for 28 buffalograsses based on analysis of 52 markers as amplified with peroxidase primers is presented here. Chinch bug resistance is identified in the second column as highly resistant (HR), moderately resistant (MR), moderately susceptible (MS), and highly susceptible (HS) (Gulsen et al., 2004).

 
All peroxidase primers except for POX8 gave polymorphisms among the 28 buffalograss genotypes (Fig. 2 ). Genetic similarities among all individuals ranged from 0.66 to 0.94, with a mean similarity of 0.80 (Fig. 1). The UPGMA clustering algorithm grouped the genotypes into meaningful clusters that reflected relationships based on both ploidy level and chinch bug response. The diploid genotypes III-4-9, II-6-6, III-6-6, and DP-47-G grouped together, and the diploid cultivar Density clustered between the four diploids and other genotypes, which possibly reflects its hybrid origin (Fig. 1). The second cluster consisted of buffalograss genotypes with different ploidy levels, which were known to differ in their responses to chinch bug infestation (Gulsen et al., 2004). The resistant (184, 196, PX3-5-1, and Prestige) and highly susceptible (119, 188, and 4A) genotypes grouped in the same branch (Fig. 1). The probability of these seven highly resistant and highly susceptible genotypes or the four diploid genotypes grouping together by chance is quite low. This observation suggests that peroxidase primer sets provide markers that can reveal important genetic associations in buffalograsses.


Figure 2
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  		Figure 2. Genomic DNA was amplified by primer pair POX2 and separated at 90 V for 4 to 5 h. Agarose gel (2.5%) images of 28 buffalograss genotypes are as follows: (1) 184, (2) Prestige, (3) 196, (4) PX3-5-1, (5) 240, (6) 193, (7) 209, (8) 170, (9) 83, (10) 203, (11) 47, (12) Density, (13) 174, (14) 45B, (15) 132, (16) 97, (17) 95-55, (18) 87A, (19) 28, (20) 378, (21) 223A, (22) III-4-9, (23) II-6-6, (24) III-6-6, (25) DP-47-G, (26) 4A, (27) 188, and (28) 119; S = standard marker (DNA Ladder VI; Roche Corp., Indianapolis, IN).

 
Correlation analysis was performed to determine association between peroxidase markers and chinch bug resistance. The highest correlation value was r = 0.36, which reflects the relationships revealed by the UPGMA analysis. Chinch bug injury reaction is likely caused by genes at many loci, and some of these genes may regulate peroxidase expression. Heng-Moss et al. (2004) found increased peroxidase activity in Prestige, a highly B. occiduus–resistant buffalograss cultivar, while 378, a highly susceptible cultivar, had decreased or similar peroxidase activity levels in infested and noninfested plants. However, basal peroxidase levels were similar for both cultivars, suggesting that peroxidase level is developmentally regulated, most likely by the contribution of more than one locus. Both B. occiduus (Barber, 1918; Bird and Mitchener, 1950) and buffalograss (Shearman et al., 2004) are native to the North American Great Plains. Given the coevolution of buffalograss and B. occiduus in this region, it is quite likely that the contrasting highly chinch bug resistant and susceptible phenotypes share a common genetic history and that this relationship may be revealed by the peroxidase markers.

Peroxidase primers were also tested in four C3 and four C4 grasses along with the buffalograss cultivar Prestige. All peroxidase primers yielded polymorphic markers (Fig. 3 ). We identified 64 polymorphic markers based on nine primer pairs, and those markers were used to assess relationships among five C4 and four C3 grasses. Genetic similarities among the nine grass species ranged from 0.05 to 0.60. Peroxidase primers designed in this study successfully distinguished C3 and C4 grasses (Fig. 4 ). This result was not surprising because the grass species used in this study evolved under diverse conditions. Genotype groupings corroborated well with previous classification (Gould and Shaw, 1983). Peroxidase genes play roles in resistance to various biotic and abiotic stresses (Grisebach, 1981; Mader and Fussl, 1982; Lagrimini, 1991; Gazaryan and Lagrimini, 1996; Passardi et al., 2005), and peroxidase marker patterns might be relevant to adaptive conditions.


Figure 3
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  		Figure 3. Agarose gel (2.5%) images of five C4 grasses (buffalograss, Buchloe dactyloides; sorghum, Sorghum bicolor; zoysiagrass, Zoysia japonica; switchgrass, Panicum virgatum; and bermudagrass, Cynodon dactylon) and four C3 grasses (barley, Hordeum vulgare; rice, Oryza sativa; wheat, Triticum aestivum; rye, Secale cereale) are presented. Genomic DNA was amplified by primer pairs POX2 and POX8 separated at 90 V for 4 to 5 h as follows: (1) buffalograss, (2) barley, (3) rice, (4) wheat, (5) sorghum, (6) zoysiagrass, (7) bermudagrass, (8) switchgrass, and (9) rye. S = standard marker (DNA Ladder VI, Roche Corp., Indianapolis, IN).

 

Figure 4
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  		Figure 4. Unweighted pair group method arithmetic average (UPGMA) dendogram of five C4 grasses (buffalograss, Buchloe dactyloides; bermudagrass, Cynodon dactylon; switchgrass, Panicum virgatum; sorghum, Sorghum bicolor; and zoysiagrass, Zoysia japonica) and four C3 grasses (barley, Hordeum vulgare; rice, Oryza sativa; rye, Secale cereale; and wheat, Triticum aestivum) based on analysis of 64 markers as amplified with peroxidase primers.

 
Eleven buffalograsses and four C3 or C4 markers were sequenced. A search of the GenBank databases revealed that 11 markers were indeed unique peroxidase genes with significant matches to peroxidase cDNAs in rice. The remaining four markers were not peroxidase genes and were likely due to evolutionary relationships between peroxidase genes (Table 3). The sequences generated in our study have been deposited in the NCBI website (http://www.ncbi.nlm.nih.gov/), and GenBank numbers were assigned (Table 3).


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  		Table 3. Gene identification number, plant source of the gene, E-value of similarity, and homologous sequence.

 
The CLUSTAL W algorithm nested in Vector NTI classified 11 peroxidase genes into two different groups (Fig. 5 ). The seven buffalograss peroxidase genes were grouped in the upper branch, while the remaining four peroxidase genes were placed in the lower branch. These data suggest that the buffalograss peroxidases are more closely related to each other. Cluster analysis was performed on the 34 rice peroxidase genes used to design primers and the 11 peroxidase genes sequenced in this study, using CLUSTAL W algorithm nested in Vector NTI Suite Software Version 8.0 (Fig. 6 ). Genes AY728139, AY751439, AY751444, and AY751445 sequenced in this study were clustered with AB114856, D45423, AB050724, and AB053297. These four genes represent different rice ascorbate peroxidase genes (http://www.ncbi.nlm.nih.gov). The AY751447 gene was clustered with a different L-ascorbate peroxidase gene, CA762991. Ascorbate peroxidases catalyze reduction of H2O2 in the presence of ascorbate (Foyer and Halliwell, 1977) and play a critical protective role in plants (Davletova et al., 2005; Tsai et al., 2005). The six other peroxidase genes sequenced in this study were clustered with the 16 rice peroxidase genes that had unknown functions.


Figure 5
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  		Figure 5. Phylogenetic relationships among the peroxidase genes sequenced in this study were constructed with Vector NTI Suite Software version 8.0 (Invitrogen Corp., Carlsbad, CA), using CLUSTAL W module. The upper branch includes buffalograss peroxidase genes, while the lower branch includes peroxidase genes from the other grasses studied.

 

Figure 6
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  		Figure 6. Phylogenetic relationships between the rice peroxidase genes used to design primers and peroxidase genes sequenced in this study. The tree was constructed with CLUSTAL W algorithm nested in Vector NTI Suite Software Version 8.0 (Invitrogen Corp., Carlsbad, CA). The rice peroxidase cDNA sequences were obtained from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/).

 
Peroxidase markers might be used to define relationships among plant genotypes from different geographic locations. Grass species from different locations are exposed to varying biotic or abiotic stresses that could enhance developmental variations in peroxidases based on varying selection pressure. Therefore, peroxidase marker diversity and relationships likely reflect exposure to stress factors. There are approximately 100 peroxidase genes in a single plant species that vary in sequence and function. The peroxidase primers designed in this study may be helpful in identifying peroxidase genes in grasses, and studying gene expression profiles in the presence of abiotic or biotic stresses.

Yoshida et al. (2003) indicated that more information was needed regarding peroxidase gene expression. Hiraga et al. (2001) reported that as more peroxidase genes are identified, microarray technology may have a greater potential to improve our understanding of peroxidase gene expression due to conserved sequences among genes.

In this study we used PCR-based peroxidase markers to assess polymorphism within buffalograss and among grass species. The C3 and C4 grasses differed from each other as expected, and diploid buffalograss genotypes differed from polyploid buffalograsses using these primers. This source of polymorphism could potentially be used in genetic mapping. Eleven peroxidase marker fragments (seven buffalograss and four other grass species) were sequenced. The five peroxidase genes were clustered with ascorbate peroxidase. These primers may have potential to improve our understanding of stress-related resistance and evolutionary relationships among peroxidase genes from C3 and C4 grasses, which were known to have evolved in different geographic and adaptive conditions. The targeted gene family approach provides advantages over the use of random or anonymous loci to study evolution, mapping and diversity. The targeted loci reveal polymorphism in genes that have a characterized biological role in the plant. This approach may be applied to the study of other gene families that carry conserved motifs similar to the peroxidases.


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 NOTES
ABSTRACT
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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Received for publication July 31, 2006.


   REFERENCES
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