Buffalograsses: Their Organelle DNA, Chinch Bug Resistance Variation, and Peroxidase
Enzyme Responses to Chinch Bug Injury
By
Osman Gulsen
A DISSERTATION
Presented to the Faculty of
The Graduate College at the University of Nebraska
In Partial Fulfillment of Requirements
For the Degree of Doctor of Philosophy
Major: Agronomy
Under the Supervision of Professors
Robert (Bob) C. Shearman and Kenneth P. Vogel
Lincoln, Nebraska
December, 2004 Buffalograsses: Their Organelle DNA, Chinch Bug Resistance Variation, and Peroxidase
Enzyme Responses to Chinch Bug Injury
Osman Gulsen, Ph.D.
University of Nebraska, 2004
Advisors: Robert (Bob) C. Shearman and Kenneth P. Vogel
Information on genetic diversity and relationship of native buffalograss germplasm is limited and genetic basis of agronomic traits is unknown. The objectives of this research were to determine: 1) the genetic diversity, relationships, and organelle
DNA inheritance based on cpDNA and mtDNA, 2) chinch bug resistance variation in natural buffalograss populations characterized for cpDNA and mtDNA; 3) the degree of correlation between total protein content, basal peroxidase level, chinch bug injury, and ploidy level, and 4) total protein content and peroxidase changes of resistant and susceptible germplasm in response to chinch bugs. Fifty-six, 48,28, and, 6 buffalograsses were evaluated for organelle DNA, chinch bug resistance, correlation analysis, and peroxidase changes, respectively. Six cpDNA and three mtDNA non-coding regions were amplified by polymerase chain reaction, using universal chloroplast and mitochondrial primer pairs. Each amplified fragment was digested with 2 to 6 different restriction enzymes. For the chinch bug study, genotypes were evaluated in replicated trials under greenhouse conditions. Leaf samples were collected for peroxidase changes from infested and control plants at 7, 14,21, and 28 day after exposure (DAB) to chinch bugs. Peroxidase analyses were carried out using native gels stained for anionic peroxidases and enzyme kinetics were measured with a spectrophotometer. Forty-seven of 56 genotypes had identical cpDNA and mtDNA RFLPs and the rest showed only a few polymorphic markers, which suggests a single maternal origin for the four buffalograss
ploidy levels. Based on the use of cpDNA primers amplifying intergenic region between psbC and tmS genes, and restriction enzyme Rae III, cpDNA was determined to be
maternally inherited in buffalograss. The germplasm had considerable diversity for
chinch bug resistance, with approximately 10% of the germplasm having a high
resistance level. There was no significant correlation between chinch bug resistance and
ploidy level or chinch bug resistance and pubescence. Of the genotypes studied, 4 were
highly resistant, 22 were moderately resistant, 19 were moderately susceptible and three
were highly susceptible to chinch bug injury, showing a continuous distribution. Basal
peroxidase expression levels measured in the 28 non-infested plants of resistant and
susceptible buffalograsses did not correlate with chinch bug injury. All six genoptypes
evaluated for chinch bug activity showed an increased level of peroxidase levels in
infested plants, suggesting upregulation in response to chinch bug injury. Relatively low
levels of peroxidase in a highly chinch bug resistant genotype, PX-3-5-1, infers
contribution of other genes to chinch bug resistance. Overall results indicate substantial
genetic variation in buffalograss germplasm that can be used to enhance buffalograss
breeding programs and increase understanding of the chinch bug resistance mechanism. To mother Huriye, my bellowed wife Fatma, and dear sons Askin and Kerem, and
daughter Sumeyra ACKNOWLEDGEMENTS
First and foremost I would like to express my thanks and gratitude to Dr. Robert
C. Shearman and Dr. Kenneth P. Vogel. Under their guide I have been encouraged to explore my potential in plant science. I am especially grateful for their guidance during the apparently endless pursuit of this degree.
I would also like to thank the other members of my guidance committee, Drs.
Tiffany M. Heng-Moss, Donald J. Lee, and P. Stephen Baenziger, who provided inspiration and a friendly environment at UNL. I believe that these are the key issues in career development. Their collaboration greatly helped me to accomplish my expectations at
UNL. I remember that my mother encouraged me to get higher education all the time and for her endless support. I am very thankful and will remember her support forever. My wife, Fatma Funda, deserves special consideration here. Her constant support in all stages of this thesis has been an important part of the pursuit of my PhD degree. I truly appreciate her love, patience, loyalty and assistance.
My special thanks are to members of buffalograss research group for sharing ideas and setting up my experiments at the Department of Entomology: Terrance P.
Riordan, Frederick P. Baxendale, Thomas E. Eickhoff, Hikmet Budak, Wyatt G.
Anderson, and others. Carol Caha, Ismail Dweikat, and Herbert Siqueira backed me up all the times when needed. I also thank to the staff personnel at the Department of
Agronomy and Horticulture, and Entomology.
Finally, I thank the Department of Agronomy and Horticulture of University of
Nebraska, Lincoln for the financial support for my PhD program here and for giving me opportunities to discover new research areas and myself, and Ministry of Agriculture of Turkey for permitting me PhD study here. Finally I thank Allah for giving me chance to accomplish this program, and hope to use my knowledge I gained here for humanity. TABLE OF CONTENTS
Page
List of Tables iii
List of Figures .iv
Literature Review 1
Chapter 1. Organelle DNA Diversity among Buffalograsses From The
Great Plains Of North America Using CpDNA and MtDNA RFLPs
Abstract 49
Introduction 51
Materials and Methods 54
Results and Discussion 57
Literature Cited 62
Chapter 2. Buffalograss Germplasm Resistance to Blissus occiduus Hemiptera:
Lygaeidae)
Abstract. 75
Introduction 76
Materials and Methods 78
Results and Discussion 81
Literature Cited 86
Chapter 3. Total Protein and Peroxidase Enzyme Changes Among Chinch Bug
Resistance and Susceptible Buffalograsses
Abstract 92
Introduction 94 ii
Materials and Methods 97
Results and Discussion 103
Literature Cited 108
Appendix 120 11l
LIST OF TABLES
Table Page
1.1 Buffalograss genotypes and their ploidy levels, sex expression
and geographic origins used in organelle DNA study 69
1. 2 Primer pairs, corresponding regions, and reannealing temperatures
used in organelle DNA study '" 72
1.3 Chloroplast and mitochondrial DNA primer pairs, restriction enzymes,
and number of restriction fragments scored in each digestion 73
1.4 Pairs of cpDNA and mtDNA primers and PCR product sizes
used in studies of Buchloe, Citrus, and Quercus 74
2.1 Susceptibility of buffalograss genotypes to Blissus occiduus
under greenhouse conditions, and their ploidy level, mean chinch
bug numbers and pubescence 89
3.1 Buffalograss genotypes used in chinch bug evaluation study,
their ploidy levels, and chinch bug resistance 113
Al CpDNA and mtDNA RFLP raw data file 120
A2 Correlations among chinch bug injury, ploidy levels, total protein
content, and basal peroxidase level.. 125
A3 Analysis of variance for plant total protein content. 126
A4 Analysis of variance for peroxidase specific activity changes 127 iv
LIST OF FIGURES
Figure Page
1.1 UPGMA dendogram of 56 buffalograsses and two outgroups 66
1.2 CpDNA restriction fragments of buffalograss accessions and two
outgroups 67
1.3 Verification of maternal inheritance of cpDNA in buffalograsses 68
2.1 Distribution of chinch bug resistance among buffalograss genotypes 88
3.1 Total protein contents of infested and non-infested plants of six
genotypes .115
3.2 Changes in total protein contents among the six infested and
non-infested genotypes 116
3.3 Changes in peroxidase specific activity through 7, 14,21,
and 28 DAE among the six genotypes .117
3.4 Native gels stained for anionic peroxidase activity 14 DAE 118
3.5 Native gels stained for anionic peroxidase activity 28 DAE 119
Al Neighbor-Joining tree based on organelle DNA RFLPs and
produced by Mega software 128
A2 Minimum evolution tree based on organelle DNA RFLPs and
produced by Mega software '" 129
A3 UPGMA tree based on organelle DNA RFLPs and produced
by Mega software .130
A4 Native gel analysis of infested and non-infested plants of six v
genotypes 7 DAB to chinch bugs 131
A5 Native gel analysis of infested and non-infested plants of six
genotypes 21 DAB to chinch bugs 132
A6 Polymorphism for anionic peroxidases among 28 buffalograsses 133 1
LITERATURE REVIEW
Introduction
Buffalograss [Buchloe dactyloides (Nutt.) Engelm.] is a stoloniferous, perennial warm season grass species that is native to the North American Great Plains (Wenger,
1943). It performs well under warmer temperatures. High temperatures tend to increase photosynthetic capacity (Monson et aI., 1983). Although buffalograss performs acceptably under low rainfall and relative humidity, it shows rapid growth when grown under irrigated conditions (Beetle, 1950; Riordan, 1991). Turf type buffalo grasses have been used in home lawns, school grounds, parks, roadsides, cemeteries, golf course fairways and roughs due to the drought resistance and low maintenance requirement (Savage and Jacobson, 1935; Beard, 1973; Riordan, 1991;
Fry, 1995; McCarty, 1995). Its aggressive stoloniferous growth habit and dense sod forming capabilities make it an excellent conservation species (Wenger, 1943;
Pozarnsky, 1983).
It is thought that buffalograss was given its common name by hunters and trappers who observed the large herds of American bison (Bison bison Linnaeus) grazing on the grass (Engelmann, 1859; Bird, 1950). Buffalograss was used to build sod houses by early settlers (Beard, 1973). The first instance of buffalograss being used on lawns can be traced back to as early as 1898 (Beetle, 1950).
Ploidy Diversity
Buffalograsses comprise a polyploid level series of diploids (2n=2x=20), tetraploids (2n=4x=40), and hexaploids (2n=6x=60) (Reeder, 1971). Johnson et aI. 2
(1998 and 2001) used flow cytometry and light microscopy to estimate the DNA content and determine chromosome numbers of buffalograss cultivars, experimental lines, and natural populations. They reported diploids, tetraploids, pentaploids, and hexaploids. Diploids were common in central Mexico, tetraploids occurred in the southern Great Plains of North America, and hexaploids were found throughout the
Great Plains. In general, pentaploids have disadvantages in natural populations due to potential chromosome imbalance in their progeny, and uneven ploidy levels are rare in nature. Johnson and Riordan (2001) reported that 'Tatanka' was comprised from a mixture of pentaploid and hexaploid lines, and has poor seed production and establishment rate characteristics due to chromosome imbalance in the resulting progeny. Probably, low seed production in pentaploids is caused by chromosome imbalance in their progeny. Mixing parents of different ploidy levels in the crossing blocks may result in sterile or genetically unstable progeny, producing undesirable characteristics (Fehr, 1987). These effects could be detrimental for seeded cultivars, but such effects may have no impact on the development of clonal buffalograss cultivars (Burton, 1980). The ploidy levels are difficult to distinguish phenotypically.
Therefore, it is critical for breeders to use ploidy level information to construct appropriate crossing blocks and breeding populations.
The plants with high ploidy levels may have an advantage (Wendel, 2000).
Genes duplicated by polyploidy may retain their original or similar function, undergo diversification in protein function and regulation, or one copy may become silenced through mutational or epigenetic interactions. Changes in polyploidization can influence DNA structure, allowing greater genetic diversity in populations with higher 3 ploidy levels. Hence, genetic modifications coupled with chromosome duplication in polyploids can lead to increased polymorphism, which can be identified by molecular markers such as randomly amplified polymorphic DNA (RAPD), simple-sequence repeats (SSR), and restriction fragment length polymorphism (RFLP). The increased genetic diversity may provide an expanded genetic basis for natural selection, which can explain the larger expansion of hexaploid genotypes.
Taxonomy, Related Species, Origin and Distribution
It is thought that buffalograss in Mexico survived in the ice age as glaciers moved south through north America, then expanded north as temperature warmed
(Webb, 1944; Quinn and Engel, 1986; Shaw et aI., 1987). Rzedowski (1975) and
Stebbins (1987) suggested buffalograss evolved with the five other closely related species in central Mexico in the early to middle Tertiary. Those five closely related species includes Buchlomimus nervatus (Swallen) Reeder, Reeder & Rzedowski;
Cyclostachya stolonifera (Scribn.) Reeder & Reeder; Opizia stolonifera Pres1.;
Pringleochloa stolonifera (Foum.) Scribn., and Soderstromia mexicana (Scribn.)
Morton. Buffalograsses and the five related species are found in the same geographic region in Mexico. They are predominantly dioecious, perennial, and stoloniferous low- growing grasses. The five species are difficult to differentiate from one another and buffalograss based on vegetative characters (Reeder and Reeder, 1963; Reeder and
Rzedowski, 1965). Blue grama, a rhizomatous and relatively distant taxon to buffalograss, when compared to these five species, has the same relative distribution as buffalograss. These seven species are C4 plants with the same variants (Hatters ley and 4
Watson, 1992). They are classified under Family Poaceae, Subfamily Chloridoideae,
Tribe Cynodonteae, and Subtribe Boutelouinae, and are well adapted to arid and semi- arid areas (Renvoize and Clayton, 1992).
Buffalograss adaptation expanded to an area that extends from central Mexico to Canada (Wenger, 1943; Beetle, 1950). Some populations of buffalograsses are found at altitude up to 1825 m (Beetle, 1950). Buffalograss replaces the cool season grasses in the Great Plains when climatic conditions, such as soil moisture and temperature are not optimal for C3 species. Together with blue grama (Bouteloua gracilis (H. B. K.) Laq. Ex Steud), it comprises 90% of the native vegetation on non- sandy soils in the shortgrass prairie (Wenger, 1943).
Janzen (1984) proposed the "foliage is the fruit" hypothesis that suggested large herbivores inadvertently ingested seeds while grazing on buffalograss plants. Large herbivores dispersed viable seeds while migrating. Quinn et al. (1994) reported that buffalograss seeds fed to ruminants can survive 4 to 5 days in the intestine, promoting their plant's dispersal by grazing ruminant species. In addition, passing through an animal and being deposited in its dung increased seedling emergence. Quinn's findings (1994) support Janzen's (1984) theory. This combination of retention time and migratory herbivores during the northward expansion of shortgrass prairie should have enhanced migration of buffalograss northward to Canada from its area of origin in central Mexico. 5
Buffalograss Germplasm
Buffalograss most likely evolved by polyploidization followed by diversification of the extra copies of genes as proposed by Wendel (2000). In addition, buffalograss is cross-pollinated. These two factors would possibly increase buffalograss genetic variation, which may have economic value to mankind (Hawkes et aI., 2000). Genetic variability can be characterized using various techniques including phenotypic, DNA, biochemical, and protein-based markers.
Comprehensive reviews of characterization methods in plants were summarized by Staub and Serquen (1996), Caetano-Anolles (1998) and Chai and Sticklen (1998).
These reviews indicated that a number of methods targeting organelle, nuclear genome markers, and total DNA content could be applied to cultivars or natural populations.
These include phenotypic markers, DNA markers such as RAPD, RFLP, DNA amplification fingerprinting (DAF), amplified fragment length polymorphism (AFLP), chloroplast DNA RFLP (cpDNA RFLP), mitochondrial DNA RFLP (mtDNA RFLP), and biochemical and protein markers. These studies also suggested that phenotypic markers are less expensive than DNA, biochemical, and protein markers, but they have limitations such as low number of markers and environmental modification.
Buffalograss phenotypic traits, such as sex expression (Huff and Wu, 1992), salt tolerance (Wu and Lin, 1994), mealybug resistance (Johnson-Cicalese et al., 1998) and fall dormancy and spring green-up (Kenworthy et aI., 1999), and chinch bug resistance
(Heng-Moss et al, 2002) have been evaluated. Kenworthyet al. (1999) evaluated fall dormancy and spring green-up in 273 natural buffalograss genotypes collected from the
Great Plains of North America and seven cultivars. Significant differences in fall 6 dormancy and spring green-up occurred among accessions. These are important traits of warm season turfgrasses used in transition or northern zones. Therefore, spring
green-up and early fall dormancy variation can be incorporated into breeding programs.
Soil salinity is an escalating problem worldwide (Marcum, 1999). Buffalograss
is adapted to arid and semiarid climates that may associate with saline soils (Hamdy,
1996). Wu and Lin (1994) evaluated diploid, tetraploid, and hexaploid buffalograsses
from natural populations for salt tolerance. They found considerable genetic variation
for salt tolerance and salt exclusion mechanism in buffalograss. In other studies,
buffalograss germplasm was evaluated for mealybug [Tridiscus proboli (Cockerell)]
and chinch bug (Blissus occiduus Barber) resistance (Johnson-Cicalese et al., 1998;
Heng-Moss et al, 2002). USDA (1996 and 2000) studies also indicated that phenotypic
diversity for traits such as color, density, uniformity, quality, and pest resistance is
present in buffalograss germplasm. Based on phenotypic screening, all these studies
suggested potential for cultivar improvement through breeding programs.
Protein-based biochemical markers include isozymes and seed proteins. These
markers are relatively inexpensive and require less effort compared to DNA markers
(Staub and Serquen, 1996). Variation in enzyme or protein mobility is caused by non-
synonymous changes in coding-DNA sequence and post-translational modifications.
Isozymes and protein markers have limited resolving capacity because there are only a
few available detection systems, the systems require high expression levels, and can be
modified by growing conditions.
Harlan and de Wet (1973) suggested that botanical evidence that includes
phenotypic, molecular, biochemical, and protein markers are more reliable than any 7 other evidence such as archaeological or linguistical data. Unlike phenotypic, protein, and biochemical screening, DNA fingerprinting provides more effective genetic information on germplasm variation (Karaca et aI., 2002; Budak et aI., 2004), and includes any markers reflecting changes at the DNA sequence level, such as RFLP and
RAPD markers (Staub and Serquen, 1996). DNA markers can be used for fingerprinting or cultivar identification (Casler et aI., 2003), marker-assisted selection
(Lee, 1996), and phylogeny studies (Yaneshita et aI., 1993).
Molecular markers have several advantages compared to the other botanical evidence (Staub and Serquen, 1996): 1) they are not influenced by the environment, 2) nuclear and organelle genomes can be studied separately using gene specific primers, 3) molecular markers show less pleiotropy, 4) hybrid parentage can be determined since discrete characters inherited from parents can be detected, and 5) an almost indefinite number of markers can be produced by the great range of molecular methods available.
The major advantage of both molecular and biochemical markers is their presumed
selective neutrality, although cases of non-neutrality have been reported, such as the
alcohol dehydrogenase gene in Drosophila melanogaster (Anderson and McDonald,
1983). Selective neutrality increases the probability that marker similarities among
taxa are due to common ancestry rather than to convergence.
The limited molecular studies in buffalograsses have provided some insights on
variation. Huff et aI. (1993) used RAPD markers to detect variation within and among
natural populations from Texas and Mexico. They used seven lO-mer primers to
produce 98 polymorphic bands. Data from their study were used for Analysis of
Molecular Variance (AMOVA). This research was one of the first examples of 8
AMOV A in plant diversity studies. They reported considerable variation within each of the populations, and every individual was genetically different. Similar results were obtained from an isozyme study conducted by Peakall et al. (1995). These findings were different from self-pollinated species in that most individuals are genetically identical or highly similar within a population (Fehr, 1987).
Budak et al. (2004) evaluated the 53 buffalograss genotypes, including cultivars and experimental lines, using sequence related amplified polymorphism (SRAP) markers. All genotypes in their study could be distinguished from each other. They identified a core collection of 41 buffalograss accessions by eliminating individuals through the use of the principal component analysis (peA). A core collection of genotypes that includes most of the gene diversity in a given population can assist plant breeders in reducing the numbers of genotypes needed to maintain a core collection, while saving cost.
Gulsen et al. (unpublished) studied 52 natural buffalograss populations along with four cultivars using SRAP markers. In their study, all genotypes were distinguished from each other with varying similarity values. Their results may have been anticipated because buffalograss is cross-pollinated. These results were consistent with two previously conducted studies by Huff et al. (1993) and Budak et al. (2004).
Gulsen et al. also found that higher ploidy levels were positively correlated with a
higher number of markers, which supports Wendel's hypothesis (2000). Natural buffalograss populations were less overlapped than cultivars and experimental lines
used in Budak et al. (2004). This can be expected because some genotypes used in their study were repeatedly used as parents to develop many of the cultivars. 9
Therefore, a high number of cultivars and experimental lines may have a common ancestry, resulting more overlaps among their germplasm.
The nuclear genome studies of buffalograsses described previously suggested considerable diversity among buffalograss germplasm accession. However, organelle
DNA variation was not known. DNA markers from chloroplast and mitochondria organelles may extend information on diversity and relationships among genotypes because organelle genomes of most grass species are maternally inherited (Corriveau and Coleman, 1988).
Organelle Genome Analysis
CpDNA and mtDNA, also called organelle DNAs, are particularly well suited for diversity and evolution studies because of the uniparental mode of inheritance of the organelle genomes. Of 235 angiosperm species evaluated by Corriveau and Coleman
(1988), 192 showed maternal inheritance for cpDNA, the rest of the species showed paternal or biparental inheritance. A number of reviews have discussed the value of the chloroplast genome for inferring relationships in plants (Curtis and Clegg, 1984;
Palmer, 1985, 1987; Palmer and Stein, 1986; Zurawski and Clegg, 1987; Palmer et aI.,
1988).
CpDNAs have been studied at the intra-and inter-species levels in numerous plant species: tomato (Lycopersicum spp.) (Palmer and Zamir, 1982); pearl millet
(Pennisetum americum) (Clegg et al., 1984); bromegrass (Bromus spp.) (pillay and
Hilu, 1990); various turfgrasses (Yaneshita et al., 1993); grass family (poaceae) (Davis and Soreng, 1993); switchgrass (Panicum virgatum) (Hultquist et al., 1997); kiwi 10
(Actinidia spp.) (Cipriani et aI., 1998); Abies (Parducci and Szmidt, 1999); and Citrus spp. (Gulsen and Roose, 2001). These studies indicate that organelle DNA studies may
successfully identify maternal lineages and inter-species organelle DNA variation is higher than intra-species variation. These studies used different procedures that vary in
cost and efficiency.
Three methods have been used to study variation in cpDNA and mtDNA
RFLPs. The first method was used in a considerable number of plant species during the
1980s and involves digestion of DNA with restriction enzymes, size separation of
fragments on agarose gel, transfer of fragments to membranes, and hybridization with
probes to detect specific cpDNA sequences (Lee et aI., 1988). The second procedure is
the same as the first, with the exception that that no probe-hybridization is required.
Instead, after isolation of cpDNA from the nuclear and mitochondrial DNA, and
digestion with restriction enzymes, restriction fragments are separated on gel and
detected with one of the available DNA detection procedures (Pillay and Hilu, 1990).
Both approaches are laborious, and require relatively large amounts of DNA. Since
cpDNA has a lower mutation rate than any other cell genomes (Wolfe et aI., 1987),
'universal' primers anchored within coding sequences can amplify non-coding regions
of the cpDNA across species (Taberlet et aI., 1991). Universal primers for amplification
of specific cpDNA sequences can overcome the limitations of the two previously
mentioned procedures. As a result, PCR-based cpDNA and mtDNA RFLPs emerged,
and involves PCR amplification of cpDNA and mtDNA, digestion of PCR products
with endonucleases, separation of fragments by electrophoresis and detection of
digested PCR products. Either specifically extracted cpDNAs and mtDNAs or total 11
DNA extractions can be readily used via cpDNA and mtDNA gene specific primers that may amplify cpDNA and mtDNA from a great range of plants (Demesure et aI.,
1995).
Gepts (1993) suggested that cpDNA and mtDNA have different evolutionary dynamics. MtDNA has high levels of rearrangements, low rates of point mutations, and the presence of foreign sequences such as viral sequences. Lilly and Havey, (2001) reported that mtDNA consisted of a considerable amount of repetitive DNA sequences.
This makes mtDNA difficult to establish phylogenetic relationships based on mtDNA
RFLPs. On the other hand, the gene order and genome size of cpDNA is highly conserved, but its nucleotide substitution rate is higher than mtDNA (Olmstead and
Palmer, 1994). Therefore, cpDNA RFLPs are more commonly used in plants. CpDNA
RFLP, thus, is expected to be more efficient in studying buffalograss evolutionary relationships and cytoplasmic diversity.
Variation in cpDNA and mtDNA RFLP occurs due to insertions, deletions, or base changes, usually within non-coding cpDNA sites. Caetano-Anolles (1998) suggested that the RFLP markers generated from polymorphic organelle DNAs of grass species have provided consistent evidence for accurate identification and clarification of phylogenetic relationships. For example, cpDNA polymorphism distinguished
lowland and upland ecotypes among switchgrasses (Hultquist et aI., 1996). CpDNA
variation was also associated with ploidy levels. Tetraploid switchgrass cultivars or
experimental strains had either the upland or lowland cytotypes, while only the upland
ecotypes had 6 pg DNA content in their nucleus. Cytotype classification is important in
switchgrasses because to date, no successful crosses between lowland and upland 12 ecotypes have been reported. CpDNA RFLPs also distinguished warm and cool season turfgrasses (Yaneshita et aI., 1993).
CpDNA RFLP variations were studied in diploid and polyploid species of
Bromus subgenera Festucaria and Ceratochloa (Pillay and Hilu, 1990). No variation was detected among the hexaploids and octaploid Ceratochloa spp. studied. Similarly, polyploid species of subgenus Festucaria, except for B. aulaticus, were identical in cpDNA restriction sites for the same cpDNA analyzed. However, diploid species of subgenus Festucaria had varying degrees of variation. This suggests either different organelle DNA origin or restriction site gain/loss mutation in cpDNA for some diploids studied. This study also suggests that cpDNA RFLPs may help diversity and identifying evolutionary relationships among buffalograsses with varying ploidy levels.
Columbus (1999) indicated that some species of Bouteloua were more closely related to other genera than to congeners and speculated that buffalograss should be reclassified as Bouteloua dactyloides (Nutt.) J. T. based on only one region of cpDNA sequence and the nuclear ribosomal internal transcribed spacer region. Conclusions based on a single chloroplast region could be misleading because a single gene may have differential conservation compared to other genes. Instead, polymorphism based on several non-coding organelle DNA segments may better help define relationships among related species because of less conservation in intergenic regions (Taberlet et aI., 1991).
To date, there has been no study defining buffalograss organelle DNAs.
CpDNA and mtDNA RFLPs as a uniparentally inherited organelle marker could help to define relationships among buffalograsses that have five different ploidy levels. 13
Particularly, extended ploidy variation among buffalograsses raises the question on origin and evolution of buffalograsses. Cytoplasmic incompatibility can likewise be a factor in directing breeding programs. There is no report on cytoplasmic incompatibility in buffalograsses from crossing studies. CpDNA and mtDNA RFLP variations could be related to cytoplasmic incompatibility. Organelle genome studies may contribute in clarification of genetic origin and cytoplasmic incompatibility-related questions in buffalograss.
Organelle genomes are taking more attention in plant improvement through plant transformation. Gene escape from cultivated transgenic plants to wild or weedy
species is an important consideration in public health and environmental protection.
The reason is to prevent formation of super weeds and possible adverse impact(s) on
other organisms such as insects, birds, and other animals. Most gene introduction
strategies introduce exogenous genes to crop species by inserting the transgene into the
nuclear genome (Stewart et aI., 2003). Daniel et aI. (1998) has proposed chloroplast
engineering as a mode of transgene containment because maternal transmission of the
organelle genome limits transgene escape to the seed. To date, although only a few
species have proven to be amenable to placement of the transgene in an organelle
genome, a considerable number of studies target organelle genome, particularly
chloroplast genome (Danielle et aI., 1998). Corriveau and Coleman (1988) reported
that the mode of organelle genome inheritance could be maternal, paternal, or
biparental as mentioned above, and therefore determining the mode inheritance of
organelle genome is a prerequisite to cultivar improvement through plant chloroplast
transformation. 14
Mode of organelle genome inheritance can be determined by examining progenies from parents that are polymorphic for the organelle genome as in Pring et al.
(1982) and Lee et al. (1988), or pollen compounds as in Corriveau and Coleman (1988).
Corriveau and Coleman (1988) examined whether pollens from various plant species contained plastid compounds to determine mode of chloroplast inheritance, and suggested those species that do not contain chloroplast compounds have maternal chloroplast inheritance. Lee et al. (1988) examined progenies using chloroplast markers and found both paternal and maternal chloroplast markers, suggesting that chloroplast inheritance in alfalfa (Medicago sativa L.) is biparental. Pring et al. (1982) indicated progenies from inter-specific crosses had maternal cpDNA markers and suggested maternal inheritance of cpDNA in sorghum (Sorghum vulgare). Organelle
DNA inheritance in buffalograss can also determined by examining progenies when a polymorphic marker is identified.
Statistical Methods for Phylogeny Analysis
Phylogeny analyses based on phenotypic, protein, biochemical, and DNA
markers are used to estimate degree of diversity and relationships, and understand evolution of plant species or particular genes families, and manage germplasm
organization. There are three phylogeny methods: 1) distance, 2) parsimony, and 3) maximum likelihood methods (MLM). Each has a different algorithm, a specific set of operations to construct a tree, and is implemented through various computer packages
used in phylogeny studies. Although some distance methods such as neighbor joining and unweighted group method arithmetic average (upGMA) do not have any 15 optimality criteria, parsimony and MLM methods have optimality criteria for deciding which, among a set of trees, is best. Each one has advantages and disadvantages.
In choice of methods, the number of samples is an important criteria, because computation time differ considerably among the three methods. Parsimony methods take hours when high numbers of genotypes (>50) are used, this computation time is usually seconds in distance methods. It was found that type of genotypes is also important in selecting an appropriate method for phylogeny analyses (Lucinda, 1997).
When hybrids and non-hybrid taxa were analyzed together, distance and parsimony methods gave different groupings. The parsimony method tends to cluster known
hybrids in a separate group rather than with or close to one of the parents that
contributed to the genome of these hybrid taxa. In conclusion, Lucinda (1997)
suggested that if identification of ancestral genotypes among a set of potential parental
genotypes is the goal, pairwise distances are more useful than parsimony method. The
second possible disadvantage of the parsimony method is this method minimizes
differences among genotypes studied. Organelle genomes have lower diversity than the
nuclear genome. Thus, few marker differences detected among genotypes may not be
seen in dendograms produced with parsimony method. The distance procedure might
be useful where there is a low level of polymorphism among target germplasm. In the
genus Buchloe, single species, Buchloe dactyloides, occurs and the level of organelle
DNA variation is expected to be low compared to interspecies diversity. Therefore, the
distance procedure is more appropriate to construct a phylogeny tree based on organelle
genome in buffalograsses. 16
In contrast with phylogenetic methods that analyze character data directly, distance methods use similarity or distance values that summarize character data as pairwise comparisons either between taxa or characters. A similarity matrix is needed before constructing the phylogenetic tree.
In distance methods, pairwise distances using binary data are calculated by one of several methods, including simple matching, Nei and Li's coefficient (Nei and Li,
1979), and Dice's coefficient (Dice, 1945). These distance measures differ in what similarities are scored as matches between two taxa. The first two consider both 1-1
(character present in both taxa) and 0-0 (character absent in both taxa) matches as similarity between two taxa, but Dice's coefficient includes only 1-1 matches as evidence of similarity. Distance measures that ignore 0-0 matches are preferable for most molecular marker data (Mumm and Dudley, 1994), because 0-0 matches may not necessarily show similarity. After a similarity matrix is produced, the phylogenetic tree is constructed, using one of several methods such as single linkage, UPGMA, and average linkage. Mumm and Dudley (1994) compared the methods listed above and found that the results of UPGMA were more consistent with pedigree information on
27 maize inbreds. This method is also recommended by Sneath and Sokal (1973) and by Romesburg (1984).
PCA and Principal Coordinate Analysis (PCoA) were recently reviewed by
Mohammadi and Prasanna (2003). PCA and PCoA can be utilized to derive a 2- or 3- dimensional scatter plot of individuals, such that the geometrical distances among individuals in plot reflect the genetic distances among them with minimal distortion.
Aggregations of individuals in such a plot will reveal sets of genetically similar 17 individuals (Melchinger, 1993; Crossa et aI., 1993; Karp et aI., 1997; Warburton and
Crossa, 2000; Warburton et aI., 2002). Based on numbers of aggregations, number of genotypes can be reduced to establish core collections as in buffalograsses (Budak et aI., 2004). Reducing number of genotypes preserved in germplasm is important to save cost and increase efficiency of germplasm management.
Cophenetic correlations are used to test for the goodness of fit of a clustering to a set of data. COPH module to calculate cophenetic correlations is nested within
NTSYS-pc version 2.1 software package (Exeter Software, Setauket, N.Y.) (Rohlf,
1993). COPH module calculates the correlation between the similarity matrix produced to make a dendogram and ultrametric distances among taxa. First, the software takes a hierarchical system of clusters and produces a symmetrical matrix of "cophenetic"
(ultrametric) similarity or dissimilarity values. Simply speaking, it shows how well a dendogram represents a similarity or dissimilarity matrix. A discussion of the properties of cophenetic values is given in Rohlf and Sokal (1981). After construction
of a dendogram for buffalograss germplasm, goodness of fit of buffalograss clusters can
be tested by using COPH module.
Plant Resistance to Insects
Genetic resistance to insects offers a great deal of benefits for plants and is a
major component of Integrated Pest Management (IPM) (Panda and Khush, 1995).
Resistance reduces the use of chemical pesticides, and can lessen environmental
damage and save producer's money. Tolerant plants manage to survive under high
insect pressure and show little or no damage. Observations of plant resistance have 18
been documented for many years, and include wheat cultivars (Triticum spp.) to the
Hessian fly [Mayetiola destructor (Say)], the apple cultivar (Malus communis D. C.)
Winter Majetin against the wooly apple aphid, Eriosma lanigerum (Hausman) (Lindley,
1831), and grape rootstocks (Vitis vinifera L.) against the grape phylloxera [Phylloxera
vitifoliae (Fitch)], sorghum to the grasshopper Melanoplus spp. (Panda and Khush,
1995) and cotton Gossypium spp. against the leafhopper (Empoaska spp.) (Parnell,
1935). Numerous plant cultivars with insect resistance have been developed by
breeders in many plant species and they play an important role in plant cultivation for
crop species such as wheat (Triticum aestivum L.), (Souza et al. 1997a, b; Lage et aI.,
2004; Martin and Harvey 1995, 1997); soybean (Glycine spp.) (Hill et aI., 2004;
Onstad, 2001); maize (Zea mays L.), (Malvar et al., 2004); tall fescue (Festuca
arundinacea Schreber) (Bughara et al., 2003); cassava (Manihot esculenta Crantz) (Riis
et aI., 2003); rice (Oryza sativa L.) (Reay-Jones et aI., 2003); tomato (Lycopersicum
esculentum Mill. L.) (Liu and Trumble, 1997); and barley (Hordeum spp.) (Porter and
Momhingweg,2004).
Biotic and abiotic factors determine plant resistance to insects (Panda and
Khush, 1995; Onstad, 2001). Biotic factors include the plant, insect traits, and other
living-organisms such as fungi, virus, and mycoplasm, while abiotic factors are caused
by physical factors such as light, nutrients, pH, and temperature. Plant factors are plant
genotype, age, size, height, and density. In example, increased plant density may
positively affect insect infestation, whereas older age of plants may prevent insect
infestation, hence increasing plant resistance. Insect factors are sex, age, and
preconditioning. The infection of plants by pathogens can alter the fitness of host plant 19 in a positive or negative way (Hammond and Hardy, 1988). For example, barley yellow dwarf virus caused an increased performance of the aphid Rhopalosiphum padi
(Gildow, 1980), while Nephotettix virescens virus significantly reduced plant hopper damage on rice (Lee et a1., 1984). Therefore, plant, insect, and other living-organisms can significantly modify plant resistance to insects, and variation in these factors should be considered in evaluation studies for plant resistance to insects.
Abiotic factors are the microenvironment in the host plant, soil pH, temperature, light intensity, relative humidity, fertilizers, pesticides, and air pollutants. For example, the sorghum plants grown below pH 5.4 showed increased fall armyworm injury when compared to plants grown greater >pH 6.0 (Garder and Duncan, 1982). Abiotic factors may also have direct effect on insect behavior (Stephanou et a1., 1983) or host plant response (Dahms and Painter, 1940). Therefore, each biotic and abiotic treatment should be uniform if possible before studying host plant resistance to insects.
Germplasm resources are critical for developing improved insect resistant genotypes provided that variation for insect resistance occurs in germplasm (Martin and
Harvey 1995, 1997; Souza et a1. 1997a,b; Liu and Trumble, 1997; Onstad, 2001;
Bughara et a1., 2003; Riis et al., 2003; Reay-Jones et a1., 2003; Hill et a1., 2004; Lage et
al., 2004; Malvar et al., 2004; Porter and Momhingweg, 2004). When resistant
genotypes are detected in germplasm, they are incorporated into breeding programs,
which offers effective insect contro1. Chemical pesticides, on the other hand, still are
major components of commercial plant cultivation where germplasm variation is not
sufficient for selection. However, excessive use of chemicals for particular insect
control in agriculture has caused numerous 'ecocatastrophe' (Metcalf, 1986). 20
Therefore, genetic plant resistance, in combination with other IPM components such as mechanical removal, irrigation, biological control, fertilization, and chemical application, have cost and environmental benefits.
Host plant resistance with transgenes may provide an efficient control against insect pest. Transgenic plants carry insect toxin proteins from various organisms
(Panda and Khush, 1995). In a transgenic approach, the genes from various organisms that are the same species or distant taxa provide plant resistance to insect. By 1998, more than 40 different genes conferring insect resistance had been incorporated into crops (Schuler et al., 1998). Bacillus thuringiensis (Bt) toxin genes have been used in several important crops (Schuler et al., 1998). The genes encodes toxin protein and provides control against lepidopteran insects. Other transgenes such as lectins, proteinase inhibitors, and amylase inhibitors from various organisms are under investigation. Peroxidase genes have recently been used in cotton transformation against chilling injury (Payton et al., 2001) and tomato to compare growth effects
(Yoshida et al., 2003). There is no report of using of peroxidase transgenes against insect pests. Transferring peroxidase genes into plants could advantageous because their effect is to increase tolerance of plants to an insect by eliminating toxic compounds. In contrast, Bt toxin genes direct the plant to make large quantity of protein that have toxic effects on target insects, but could cause environmental concern in the future.
Identification of insect resistance mechanism in buffalograsses is a basic step to plant improvement that can direct traditional breeding and genetic engineering so that 21 desirable genes can be selected for or manipulated into the commercial plant cultivars.
Insect resistance mechanism may vary among the genotypes (Panda and Khush, 1995).
There are three categories of insect resistance in plants. The first, antixenosis affects behavior of that insect population to a particular plant genotype. The resistant plant genotype avoids colonization by an insect population by having physical barriers or biochemical factors. This adverse interaction reduces either the number of insects or sometimes causes starvation of the insects (Painter, 1968). Schultz (1988) speculated that all green plants contain one or more chemicals that function as a repellents, antifeedants, and/or toxins to at least some insects during some part(s) of their life cycle. Physical barriers could be glandular as in Solanum spp. and Lycopersicum spp. or non-glandular trichomes, composition of leaf structure, number and thickness of leaves, epicuticular waxes, tissue toughness (Brettell, 1980; Southwood, 1986).
Another factor is color, which has been shown to influence insect behavior. i.e. red apple fruit is preferred by codling moth, yellow onion leaves are preferred by onion fly, red cabbage is deterrent to the cabbage aphid (Singh and Ellis, 1993). Wisser (1986) suggested that biochemical factors that are constitutively present in leaves are repellents and deterrents. For example, lignification of plant leaves driven by plant genetic factors as a response to biotic or abiotic stress may affect probing of piercing-sucking insects (Whetten et aI., 1998). Antixenosis can be determined by using free-choice experiments.
The second, antibiosis, also is an insect reaction to a plant genotype that has an adverse effect on insect fecundity, development time, size and/or survival (panda and
Khush, 1995). There are alternative names for antibiosis: vertical gene resistance, 22 monogenic resistance, and single gene resistance (van der Plank, 1963). It sometimes is difficult to distinguish antibiosis from antixenosis as an insect's response may be the same or similar. It may significantly reduce an insect population compared to antixenosis and tolerance (Kennedy et al., 1987). In antibiosis, natural enemies of insect pests can be a concern, since it may reduce population size significantly (panda and Khush, 1995). On the other hand, reduction in prey populations may change both predator behavior to other prey and reduce predator populations in nature. Kennedy et al. (1987) suggested that antibiotic effects on insects may range from mild to lethal.
Potential toxins produced by a plant may include nicotine, rotenone, pyrethrum, and dimboa (Norris, 1986). Antibiosis can be evaluated through no-choice experiment in which insects are forced to feed on only one plant genotype.
The third, tolerance, also called horizontal or polygenic or quantitative resistance, is a plant response to an insect pest, opposite to the other two categories of resistance (Robinson, 1980; Panda and Khush, 1995). Insect biology, fecundity, size, weight, and population size are not adversely affected. Therefore tolerance does not have a major negative impact on the insect pest, predator population or non-target organisms. Since pest density is not negatively affected, a cost of maintaining an insect population on infested plants is expected. This cost is met by several different pathways: increasing photosynthetic capacity, changing sink-source relationships, and resource reallocation. Plant tolerance can be measured by evaluating control and infested plants of susceptible and resistant plants together. 23
Insect pests of Buffalograss
Buffalograsses are known as relatively pest free species. However, a few important pests occur in buffalograsses. Those arthropods of concern include white grubs [Phyllophaga crinita (Burmeister)]; grasshoppers, leafhoppers; mound-building prairie ants; the buffalograss webworm, Surattha indentella (Kearfott); the rodesgrass mealybug, Antomima gramimis (Maskell); eriophyid mite, (Eriophyes slykhuisi (Hall); and two grass feeding mealybugs, Tridiscus sporoboli (Cockerell) and Trionymus sp.
(Rainhard, 1940; Wenger, 1943; Chada and Wood, 1960; Sorenson and Thompson,
1979; Crocker et al., 1984; Baxendale et al., 1994). Several beneficial arthropods have also been reported, including ants, big-eyed bugs, ground beetles, rove beetles, spiders, and numerous hymenopterous parasitoids (Heng-Moss et aI., 1998).
The chinch bug, Blissus occiduus Barber, has recently emerged as an important insect pest of buffalograss (Baxendale et al., 1999). Current distribution of B. occiduus includes California, Colorado, Montana, Nebraska, and New Mexico in the United
States, and Alberta, British Columbia, Manitoba, and Saskatchewan in Canada (Bird and Mitchener, 1950; Slater, 1964; Baxendale et al., 1999).
Chinch bugs spend most of their time in the crown area of the buffalograss plant, which limits many control options. Therefore, there are few effective management options available for controlling chinch bugs in buffalograss (Baxendale et aI., 1999). The development of resistant buffalograss cultivars offers an attractive approach for managing this pest. Heng-Moss et al. (2002) found variation among 11 buffalograss cultivars for chinch bug resistance. Based on chinch bug injury, 'Prestige'
(or NE91-118), 'Tatanka', 'Bonnie Brae', and 'Cody' were highly to moderately 24 resistant. These four buffalograss cultivars exhibited minimal damage, even though all were heavily infested with chinch bugs in greenhouse experiments. Field experiments confirmed that 'Prestige' had high levels of resistance, while 'Cody' and 'Tatanka' were moderately resistant, and the buffalograss cultivar, '378', was highly susceptible to chinch bugs. This study indicated that there was variation in buffalograss cultivars.
However, variation in natural populations is unknown, and additional germplasm may strength our understanding of buffalograss resistance mechanism to chinch bugs as well as improving buffalograss cultivars.
The categories of chinch bug resistance in buffalograsses was investigated by
Heng-Moss et a1. (2003). Tolerance studies showed that 'Prestige', 'Cody', and
'Tatanka' were moderate to highly tolerant to chinch bug based on damage rating and plant height, whereas 'Bonnie Brae' was moderate-to highly susceptible, which was consistent with their previous study (Heng-Moss et al., 2002). They also found that
'Prestige' had antixenosis to chinch bug, while 'Cody' and 'Tatanka' showed no antixenosis. Antibiosis studies indicated no significant differences in chinch bug fecundity, nymphal development, or survival among the resistant and susceptible buffalograsses.
Recently, the susceptibility of 18 warm and cool season grass species including turfgrasses, crops, and weeds were evaluated in no-choice studies (Eickhoff et a1.,
2004). In these studies, damage ratings ranged from high susceptibility to non- susceptibility. These studies also found that B. occiduus produced offspring on 15 of the 18 turfgrass, crop, and weed species evaluated. These results suggest that chinch 25 bugs may find host(s) and survive under the absence of one or a few of the hosts identified above.
Another insect pest of buffalo grasses is mealybugs (Baxendale et aI., 1994).
Mealybugs have been collected from buffalograss stands throughout Nebraska
(Baxendale et aI., 1994), Texas, and Arizona (Johnson-Cicalese et aI., 1998). Johnson-
Cicalese et aI. (1998) found dramatic differences among buffalograss selections for mealybug resistance. Of the 62 buffalograss genotypes evaluated, 'Prairie' and '609' were highly resistant and the rest of the genotypes were moderately susceptible. They also found that pubescence was positively correlated with mealybug injury. It has been speculated that pubescence provides a holding point for the mealybug. There is no report on whether buffalograss pubescence has a similar impact on chinch bugs. Heng-
Moss et aI., (2003) found the same levels of pubescence in resistant 'Prestige' and susceptible '378', suggesting no association of pubescence and chinch bug resistance.
The two studies mentioned above (Johnson-Cicalese et al., 1998; Heng-Moss et aI.,
2002) revealed considerable variation in buffalograss resistance to these two important insect pests of buffalograss and the potential for buffalograss cultivar improvement against these two insect pests in breeding programs. However, due to extensive ploidy variation among buffalograss germplasm, characterization of additional germplasm would provide an opportunity for efficient breeding programs.
Peroxidases
Toxic molecules such as superoxide and hydroxide radicals can be found in cells due to the presence of oxygen. These are by products of aerobic respiration and 26 increases by the presence of abiotic stress factors. These toxic molecules are eliminated by a number of enzymes present inside the cell. Superoxide, for example, is destroyed by superoxide dismutase. The degradation, however, produces more hydrogen peroxide (H202), which is, in turn, destroyed by peroxidase enzymes, a class of enzymes in animal, plant, and microorganism. Thus, peroxidases are oxidoreductases, which use H202 as an electron acceptor for catalyzing different oxidative reactions (Mavelli et aI., 1982). The overall reaction is as follows:
Donor + H202 < = > oxidized donor + 2 H20
Some peroxidases require the presence of certain molecules for enzymatic activity.
These molecules are called cofactors. In peroxidases, the bound cofactor for its enzymatic activity is heme. The additional information on peroxidase is available at: http://www.chem.admu.edu.phl-nina/rosby/intro.htm.
Higher plants have a large number of peroxidase isozymes, which are encoded by multigene families (Hiraga et al., 2001). Plant peroxidases (POX) have three highly
conserved domains, the distal heme binding domain, the central domain of unknown
function, and the proximal heme-bindind domain (I, n, and ill in figure below)
(modified from Hiraga et aI., 2001).
Hd Hp II IVZ~ I C terminal I •H HI 100 amino residues 27
Although a high level of conservation at the amino acid level was reported by Hiraga et al. (2001), its DNA conservation has not been reported. If these three highly conserved amino acid regions are also conserved at DNA level, this may allow us to design primers based on genomic DNA or cDNA sequences from related species and study their diversity, relationship, differential response in the presence of a stress factor, and evolution of peroxidase gene family.
The procedures to study peroxidase enzymes via enzyme kinetics are well established (Hiraga et al., 2001). Peroxidases generally react to compounds containing a hydroxyl group(s) attached to an aromatic ring. For example, guaiacol (o-methoxy phenol) is commonly used as a substrate for the measurement of peroxidase activity.
Dehydrogenative oxidation of guaiacol by peroxidase results in the formation of phenoxy radicals, and the subsequent coupling of unstable radicals leads to the non- enzymatic polymerization of monomers.
One possible insect-resistance mechanism is the formation of the radicals and peroxides in tissues of resistant and susceptible genotypes upon pest-induced wounding. Hildebrand et al. (1986) indicated that resistant genotypes may detoxify higher levels of radicals and peroxides, while susceptible plants fail to detoxify the same level of those toxic chemicals. In addition to removal of H202, the reported functions of peroxidases in plants include biosynthesis and degradation of lignin in cell walls (Grisebach, 1981; Mader and Fussl, 1982; Lagrimini, 1991), auxin catabolism
(Gazaryan and Lagrimini, 1996), defense responses to wounding (Dowd and Lagrimini,
1997) and defense against pathogen and insect attack (Ye et al., 1990; Dowd and
Lagrimini, 1997). Although information on the functions and structures of peroxidase 28 enzymes is available, very little is known about the signal transduction for inducing
expression of the peroxidase genes. In general, ethylene and jasmonic acid are the
main compounds for signal transduction in many insect-infested plants of resistant and
susceptible genotypes (Argandona et aI., 2001; Sasaki et aI., 2002; Yoshida et aI.,
2003). The roles of these compounds need to be investigated for buffalograss chinch
bug resistance
Plant protein profiles and expressions of plant oxidative enzymes are modified
in response to insect feeding (Green and Ryan, 1972; Hildereband et aI., 1986; Felton et
aI., 1994a and b; Miller et aI., 1994; Rafi et aI., 1996; Stout et aI., 1999; Hiraga et aI.,
2000; Chaman et aI., 2001; Heng-Moss et aI., 2004). Miller et ai. (1994), Rafi et ai.
(1996), and Jerez (1998) have reported changes in the protein profiles of resistant plants
after insect feeding. Hildebrand et ai. (1986) found increased peroxidase activity in
resistant soybean plants after exposure to mites. Tomato plants expressed higher levels
of peroxidases in response to both pathogens and insects (Stout et aI., 1999). Increased
levels of peroxidases were observed in both the cytoplasm and the cell wall of barley
infested with aphids (Chaman et aI., 2001). These studies suggested that peroxidase
enzymes are involved in many plant species resistance to insects.
Heng-Moss et ai. (2004) found increased levels of peroxidase activity in
infested plants of a highly resistant buffalograss, 'Prestige' while a decrease in a highly
susceptible genotype, '378'. This study suggests that peroxidase enzymes may play
role(s) in chinch bug resistance in buffalograsses. Since multiple genes are expected in
tolerance mechanism as mentioned earlier, roles of other genes remain unclear. 29
Peroxidase enzymes may serve as markers for early selection of resistant genotypes in breeding programs. Since there are a number of peroxidase enzymes from a gene family comprising multiple functionally and structurally diverse peroxidases, particular type(s) of peroxidases responsible for chinch bug resistance should be determined. In order to further study role(s) of peroxidase enzymes, broader based germplasm characterized for chinch bug resistance is required to detect the overall contribution of peroxidases and potential of other genes. 30
GOALS OF THE STUDY
The objectives of this research were to determine: 1) genetic diversity and relationships based on cpDNA and mtDNA, and cpDNA inheritance based on molecular markers; 2) chinch bug resistance variation in natural buffalograss populations characterized for cpDNA and mtDNA; 3) the degree of correlation among total protein content, basal peroxidase level, chinch bug injury, and ploidy level among the non-infested plants of resistant and susceptible buffalograsses, and 4) total protein content and peroxidase changes of resistant and susceptible germplasm in response to chinch bugs. 31
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