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.

Chapter 2.

Chapter 3.

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

Buffalograss Germplasm Resistance to Blissus occiduus Hemiptera:

Lygaeidae)

Abstract. 75

Introduction 76

Materials and Methods 78

Results and Discussion 81

Literature Cited 86

Total Protein and Peroxidase Enzyme Changes Among Chinch Bug

Resistance and Susceptible Buffalograsses

Abstract 92

Introduction 94

Materials and Methods 97

Results and Discussion 103

Literature Cited 108

Appendix 120

ii

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

11l

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

iv

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

v

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.

1

(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

2

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

3

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.

4

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

5

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

6

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

7

AMOVA 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.

8

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

9

(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

10

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

11

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.

12

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.

13

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

14

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.

15

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

16

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

17

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

18

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).

19

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

20

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,

21

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.

22

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

23

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

24

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

25

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).

26

Hd Hp

II • IVZ~ I C terminal

I H HI100 amino residues

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

27

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.

28

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.

29

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.

30

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