DOT Project Number:  90-00-LRTF-409

Fiscal Year:  2004

Award:  $9,000.00

Principal Investigator:  Dr. James Jurgenson, Department of Biology, University of Northern Iowa, James.Jurgenson@uni.edu

Other Project Participants:  Renae Devries, Department of Biology, University of Northern Iowa

Research Report:

ANALYSIS OF GENETIC DIVERSITY OF IOWA'S NATIVE PLANT SPECIES USING THE BECKMAN CEQ 8000 GENETIC ANALYZER

ABSTRACT:
Iowa has lost nearly 99% of its original prairies as a result of agricultural development. Consequently the prairie remnants have suffered from loss of species diversity. Efforts are being made by government agencies and private organizations to maintain genetic diversity of native plant species used in prairie restorations. This is done by producing seed collected from multiple locations. However, little experimental data exists which describes the genetic diversity of seed stocks produced from these collections. In this study amplified fragment length polymorphism (AFLP) was applied to detect the genetic diversity between individuals of Schizachyrium scoparium collected from locations found in northern, central, and southern zones of Iowa. After capillary gel electrophoresis on the Beckman Coulter CEQ 8000 Genetic Analysis Systems, there were 943 potentially parsimony informative characters out of 1170 total characters and produced a cladogram. Parsimony analysis showed clustering of samples within each accession and no noticeable clustering between accessions of the same Ecotype zone. It was concluded that there is genetic diversity between the plants of the three Ecotype zones and a diverse seed mixture can be produced to restore and reconstruct the prairies.

INTRODUCTION:
The tallgrass prairie of Iowa and the rest of the Midwest are completely different from any other type of ecosystem in the world. Even though prairies primarily consist of grasses, it is one of the most diverse natural communities consisting of 200-300 species of grasses and forbes (Chadwick, 1995) (Arbuckle, 1999). Species that inhabit the prairie are adapted to survive life in the open since there is a lack of trees in these areas and other types of cover (Arbuckle, 1999). Another noticeable trait of prairies is the extensive plant root systems. Eighty percent of the biomass of prairie plants is below the surface of the soil. When plants die they leave a large biomass of dead root tissue which provides nutrients to other plants in the area (Ransom, 1998).

Iowa has lost almost 99% of its original prairies as a result of the agricultural development. (Garner, 2002). The untilled remnants are portions of the ancient prairie and may not contain the same diversity of species as the prairie established following the ice age. Many of the symbiotic relationships between microorganisms and host plant species that were established since that time have been disrupted (Risser, 1981). Fragmentation of the prairies has also interrupted the occurrence of wild fires, which many plant species depend on for recycling of organic material and germination of seeds.

Several private and public agencies have been organized to restore and reconstruct the prairies. For instance, the Iowa Ecotype Project (IEP) has been implemented as an “integrated, working model for supplying high-quality, genetically diverse, regionally adapted native seed for prairie restorations in Iowa (Iowa Ecotype, 2004).” IEP has collected seeds from remnant prairies across Iowa and planted these seeds on campus at the University of Northern Iowa, Cedar Falls, Iowa. From this, the IEP can conduct research, develop planting techniques, educate, and increase source identified seed to promote preservation of native vegetation systems. Maintaining diversity is one of the key goals in restoration and reconstruction of the prairies. Inbreeding of native plant species has been a concern as the number of individuals surviving for each species is quite low in remnant populations. This decrease in population size may cause inbreeding to occur, which could cause a loss of heterozygosity and could possibly lead to inbreeding depression thus reducing species hardiness. Increasing the biodiversity can increase the gene pool and introduce new alleles to populations. Increases in genetic heterozygosity may aid in species survival during times of stress such as drought, flood, or pathogenic infections (Sadler, 2001).

In order to determine the genetic diversity of the various species remaining in remnant prairies, an investigation of the genetic diversity of the prairie plants between the three zones of Iowa has been implemented. The three zones were determined by latitude and numbers were assigned to designate the areas of seed collection also known as accessions (Figure 1). A method that has become popular in molecular ecology to study the genetic diversity of various organisms is amplified fragment length polymorphism (AFLP). AFLP is a technique that includes the strategies of restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) in concert to detect selectively amplified fragments (Innan, 1999). This process includes three steps (Figure 2): 1) digestion of the genomic DNA with restriction endonucleases and modification of the fragmented DNA by attachment of synthetic sequences, 2) selective amplification by polymerase chain reaction (PCR), and 3) analysis of fragments by gel electrophoresis. Even when little information is known about a genome, such as its genetic complexity or specific DNA sequences, the AFLP technique can be applied to determine genetic variability successfully (Vos et al., 1995).
AFLP has been used in other studies to detect genetic diversity. For example, a study of genetically resistant germplasm cultivars of Theobroma cacao (the chocolate tree) was conducted by Saunders, Mischke, and Hemeida in 2001. They found that AFLP was a very reliable technique when all the parameters of the AFLP process were followed. It was very efficient for assessing the genetic background of selected cultivars producing a much higher percentage of discriminatory polymorphic bands than other methods such as RFLP or RAPD (random amplified polymorphisms). The position and the size of the DNA fragments when detected by the CEQ 2000XL provided a particular DNA fingerprint that was distinct to that plant. The AFLP process allowed for the detection of the fragments, which could be used to identify one plant from another and establish genetic relationships between plants (Hemeida, 2001).

AFLP has become a very useful technique in determining the genetic diversity of various organisms and has been applied to the tallgrass prairie plant Schizachyrium scoparium (little bluestem) in this study. Schizachyrium scoparium is just one of the many native prairie plant species that is being studied in order to incorporate it into the reconstructed prairies. For example, AFLP studies have been performed on Panicum virgatum (switchgrass) and Coreopsis palmata (prairie coreopsis). Substantial genetic variation was detected between the populations of Panicum virgatum and between Coreopsis palmata populations (Hilker & Jurgenson, 2003).
Schizachyrium scoparium is a native grass of the tallgrass prairie of Iowa and receives its Greek name from its stiff, bunched stems giving it a broom-like appearance (Isaacs, 1992). Because little bluestem can grow in a variety of soils, it can be found all over the United States (Glasscock, 1991). Growing from 1-4 feet tall, the mature, hairless leaves turn a bluish-green or golden-red color when mature and produce fluffy white seeds at the end of the growing season. This bunch grass reproduces only by seeds and usually dominates the area it inhabits. When this perennial sprouts in late spring it grows as several stems with a central, fibrous root system. Little bluestem is often subjected to burning thus releasing its nutrients, which enhances the growth of other prairie plants. At the end of the growing season this grass becomes coarse and is generally not used as forage by wild and domestic animals (Isaacs, 1992).
Little bluestem has many practical uses such as pasture and hay. This species provides nutritional value by supplying seeds for songbirds and upland gamebirds as well as vegetation for livestock, deer, and elk. Ground birds and small mammals rely on the leaf canopy for protection. Because soil erosion is a concern in any agricultural area, especially Iowa, little bluestem is often planted as part of a seed mix in order to help control potential erosion sites (Glasscock, 1991). Thus, little bluestem is a vital part of the prairie ecosystem and maintaining genetically healthy populations of this species is crucial.

METHODS:
Sample Collection:
Little bluestem seed was collected from prairie remnants in the three zones of Iowa (Figure 1). Seed collection was collected by the Iowa Ecotype Project and planted in three zone specific gardens on the campus of the University of Northern Iowa. (Table 1). Vegatative tissue from ~10 plants from each accession was collected from this perennial. Young leaf tissue was ground to a fine powder using liquid nitrogen with a mortar and pestle. The ground frozen tissue was stored at -80C until used for DNA isolation.

DNA Extraction:
DNA was extracted from tissue samples using a modified diatomaceous earth protocol (Boom et al., 1990). Approximately 5-175 mg of powdered tissue was combined with 900 ml of Extraction Buffer (Table 2) containing 0.5% (v/v) b-mercaptoethanol in a 1.5 ml microcentrifuge tube. After incubation for 1 hour in a 60°C waterbath, shaking every 15 minutes, enough Sevag (~500 ml);(Table 2) was added to fill the tube and vortexed to emulsify the mixture. After centrifugation, the aqueous layer was reserved and thoroughly mixed with 875 ml of Adsorption Buffer (Table 2). This mixture was incubated at room temperature for 20-30 minutes with occasional mixing. Then the tube was centrifuged 13,000 RPM at high speed for approximately 30 sEconds and the supernatant was discarded. The pellet was gently resuspended in Guanidine Wash Buffer (Table 2) and centrifuged to remove trace amounts of the supernatant. Once the Wash Buffer was removed the tube was inverted so that the pellet could dry at room temperature for 15 minutes. After suspension in 67 ml of 1X TE buffer (Table 2) the pellet was incubated at 65°C for 30 minutes with occasional mixing. The diatomacious earth was pelleted by centrifugation for one minute. The DNA-containing supernatant was transferred to a new tube. The remaining pellet was suspended in 33 ml of 1x TE buffer and incubated at 70°C for 30 minutes. The supernatant from this step was combined with the first to produce
100 ml of DNA extract.

AFLP Process:
The extracted DNA was quantitated by electrophoresis on a 0.7% agarose gel containing ethidium bromide with Lambda Hind III digested DNA used as a mass standard. The concentration of the plant DNA samples was determined by measuring the flourescence of the bands of plant DNA compared to Lambda Hind III DNA fragments. Digital images of gels were taken using a COHU high performance CCD camera connected to a Macintosh Quadra 840av and NIH Image capturing program. Kodak 1D software was used to analyze the digital images of the gels. The concentrations of the plant DNA samples were then rEcorded in ng/ml of DNA.

Digestion:
The AFLP process was adapted from Soltis (2002) with minor modifications. DNA sample concentration was adjusted to 100 ng of DNA in 8 ml with water. To each 8 ml sample, 1 ml of 10X OPA (Table 2) was added 1 ml of a restriction enzyme digest mixture (Table 3). The samples were then incubated at 37°C for 1 hour in a Biometra T Gradient Thermocycler. Due to the nature of the two endonucleases (Eco RI and Mse I), a sticky end is left after the palindromic restriction site sequence is identified. A sticky end refers to the single stranded over hang produced by the enzyme once it cuts at the restriction site (Figure 2). When AFLP is being applied to a genome, usually one enzyme is a rare cutter, and one is a frequent cutter. In this case Eco RI cuts less frequently than Mse I. Therefore, it is expected that more fragments will be produced with both ends being cut by Mse I. The only fragments that are applified during this process are those that were cut by the rare cutter/frequent cutter endonucleases (Vos et al., 1995). After digestion, the resultant restriction fragments are modified by addition of synthetic DNA adapters to the ends with DNA ligase.

Ligation:
Preparation of adapters took place by combining Eco RI oligo 1 (5’CTC GTA GAC TGC CTA CC3’) and oligo 2 (5’AAT TGG TAC GCA GTC3’) at 5 mM each. Mse I oligo 3 (5’GAC GAT GAG TCC TGA G3’) and oligo 4 (5’TAC TCA GGA CTC AT3’) were also combined but at 50 mM each. Both oligo mixtures were then heated in the Biometra T Gradient Thermocycler to 94°C and slowly cooled to room temperature. A ligation cocktail (Table 3) was added to the digested samples and incubated overnight at 20°C in the Biometra T Gradient Thermocycler.

Preamplification:
The ligation mixture was diluted with 4 volumes of 1X TE buffer and 5 ml of that was used for the preamplification reaction. A preamplification cocktail was prepared (Table 3) and 46 ml added to the diluted ligation mixture. Amplification was accomplished by using the T Gradient Thermocycler with the following program: 70 °C for 2 minutes, 94°C for 30 sEconds, 56°C for 30 sEconds, 72°C for 2 minutes, return to step two 19 times, then 60°C for 30 minutes, and ending in a 4°C hold.

The primers used for the preamplification select for one base within the sequence next to the restriction site. For instance, the primer (5’ CTC GTA GAC TGC GTA CCA ATT CA3') used on the Eco RI side is complimentary to the adapter and the Eco RI restriction site and has a one base extension, adenine, on the 3’ end. The primer (5’ GAC GAT GAG TCC TGA GTA AC 3’) for the Mse I side of the fragment is complimentary to the adapter and the Mse I restriction site and also has a one base extension, but uses cytosine. These one base extensions help decrease the number of fragments that are amplified, which in turn decreases the number of fragments visualized during capillary gel electrophoresis.

AFLP Amplification:
Once the preamplified DNA was diluted 1:20 with 1X TE buffer, 2.5 ml was combined with an AFLP cocktail (Table 3). Six different primer pairs were used selecting for 3 bases by the restriction site on the end of the fragment (E-ACC/M-CCT, E-ACC/M-CCG, E-AAC/M-CCG, E-AAC/M-CCT, E-AAC/M-CGA, and E-AAC/M-CGC). The primer sequences (Eco RI primer: 5’AGA CTG CGT ACC AAT TCAXX 3’) (Mse I primer: 5’GAT GAG TCC TGA GTA ACXX3”) contain the one base extension just as the preamplication primer as well as a two base extension that is specified by the user. The preamplified DNA was selectively amplified by the following program: 94°C for 2 minutes, 94°C 30 sec, 65°C for 30 sEconds reducing by 1°C per cycle, 72°C for 2 minutes, return to step 2 for 9 more times, 94°C for 30 sEconds, 72°C for 2 minutes, return to step 6 for 35 more times, 60°C for 30 minutes, and 4°C hold.

The AFLP amplification selects for fragments that contain specified bases next to the restriction site sequence. For example, bases that contain 5’ACC3’ on the Eco RI side of the fragment and 5’CCT3’ on the Mse I side of the fragment will be amplified. This step further reduces the number of fragments visualized during capillary gel electrophoresis. The preamplification and AFLP amplification step are performed separately to increase the selectivity of the primers. In the studies by Vos et al., 1995, the amount of background smears was decreased in the fingerprint patterns, and bands were absent when the preamplification step was dismissed. These bands were otherwise present when both the preamplification and the AFLP amplification were performed. It was also found that primers with a three base extension show a decreased amount of base mismatching by the sequence adjacent to the restriction site during PCR in comparison to using four base extension primers. (Vos et al., 1995).

Preparation of samples for fragment detection:
The AFLP amplified DNA was diluted 1:10 in water and 2 ml was transferred to a well in a sample plate along with a 23 ml of a mixture containing formamide (23 ml) and a 600 base pair fluorescently labeled Size Standard (0.2 ml). A drop of mineral oil was added to the top of each sample. Another tray was filled with 1.1 X ACE Separation Buffer that corresponded with the filled wells in the sample plate.

Fragment Analysis:
The samples were run on the Beckman Coulter CEQ 8000 Genetic Analysis System and analyzed with the AFLP analysis software. The CEQ computer software analyzes the fragments by detecting the length in comparison to a 600 base pair size standard, and assigns a “1” if the fragment is present or a “0” if the fragment is absent. Data were then transferred to McClade 3.06 for formatting and generation of the Nexus file required by the phylogenetic analysis software PAUP (Swofford, 2002). Parsimony analysis was performed using PAUP with 1000 heuristic searches, each with random input order to produce the shortest tree.

RESULTS:
Six AFLP primer pair combinations ((E-ACC/M-CCT, E-ACC/M-CCG, E-AAC/M-CCG, E-AAC/M-CCT, E-AAC/M-CGA, and E-AAC/M-CGC ) gave a total of 1170 AFLP bands for 93 individual plants of little bluestem distributed between 10 accessions (Table 1). From the 1170 characters analyzed, 26 characters were invariable, 202 variable characters were parsimony uninformative, and 942 variable characters were parsimony informative according to the PAUP software.(Table IV) PAUP constructed a tree with the length of 9157. Some primer pairs produced more fragments in comparison to others. For instance the AFLP primer pair E-AAC, M-CGC yielded 287 characters whereas E-AAC, M-CGA produced only 107. The primer pair E-AAC, M-CCT produced 277 AFLP bands.
Individuals in an accession tended to group together with a few exceptions on the cladogram produced (Figure 3). There were three distinct clusters produced, two minor and one major. One of the minor clusters consisted mainly of accession 146 and the other consisting of mainly of a few central Iowa accessions (204, 232, 207) and a few northern and southern. The major cluster contained samples from all of the accessions, but only one sample from 146. The distances ranged from approximately 40-200 changes in the major grouping and 20-120 changes in the minor groupings. Even though the samples within the accessions clustered together on the cladogram, the accessions from the northern, central, and southern regions did not group together.

DISCUSSION:
The cladogram produced showed that the clustering of individuals within accessions indicates genetic diversity between accessions. The PAUP software detected 942 characters as being potentially parsimony informative from the total of 1170. This indicates that almost 80% of the characters showed a distinct difference between the samples from the accessions. This high percentage shows high diversity between the accessions. The cluster 1 consisting of the accession 146 shows a distinct difference from the rest of the accessions and could be a result from high percentage of discriminatory characters (Figure 3). A few members of the accession 110 is grouped by 146. Genetic diversity still prevails since the branch has 9 differences from its neighboring branch.
The samples from all other accessions in cluster 2 group together with most giving approximately 5-13% difference between neighboring samples. From this it can be concluded that samples within an accession are genetically similar, but are genetically dissimilar to others. Because there is little grouping between plants from the same zones there is an indication that even plants from the same latitude are genetically diverse. This is particularly noticable when comparing the accession 146 to the other northern plants since it is grouped completely separate from the other accessions. Even though there is grouping of the plants within each accession, the accessions themselves are intermixed with accessions from all three zones. This indicates that there are many genetic similarties, but since the total number of discriminatory characters was 942 out of 1170, they are still genetically diverse. This study has provided information that the plants from the three zones are genetically diverse.
High genetic diversity between prairie plants from the three Ecotype zones has also been seen in AFLP studies performed on Panicum virgatum and Coreopsis palmata. As stated before, most of the genetic variation observed was between the populations for both species (Hilker and Jurgenson, 2003). Analysis of little bluestem has shown similar clustering of plants within an accession and less between the populations from the three ecotype zones.
It can be concluded that there is a large degree of AFLP diversity between the accessions of little bluestem. Knowledge of this diversity can help those in the efforts to restore and reconstruct prairies by producing a seed mix that contains seed from the various ecotypes. This genetically diverse seed mix can aid the restoration and reconstruction of the prairies by incorporating a variety of alleles that can enhance the survival of the plants during environmental changes.

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