The Cactaceae are an exciting and challenging group of plants because of their varied morphology and succulence, their showy flowers, their adaptations to the environment, and their reproductive strategies. The Cactaceae family are New World plants, originating in the North, Central, and South American continents, including the West Indies Islands (in the Caribbean) and the Galapagos Islands. There are four subfamilies: Pereskioideae, Maihuenioideae, Opuntioideae, and Cactoideae. Opuntioideae have a significant diversity of form and habitat and are widely distributed throughout the New World (Griffith andÂ Porter, 2009).
Identification of species within the subfamily Opuntioideae can be difficult, in part because of widespread phenotypic plasticity, interspecific hybridization, polyploidy, and apomixis (clonal seeds and stems), which play important evolutionary roles, particularly in Cylindropuntia and Opuntia (Pinkava, 2002). Opuntia is a large genus that contains many of the pad cacti or prickly pear cacti (Figure 1). Opuntia species have been poorly studied ecologically and taxonomically in the Midwest United States.
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Opuntia fragilisÂ (Nuttall) Haworth was first described by Thomas Nuttall in 1819. He named it Cactus fragilis, and Haworth later moved it to the Opuntia genus: Opuntia fragilis. The specific name 'fragilis' refers to the ease with which the terminal joint is detached, an adaptation for asexual reproduction and dispersal (Ribbens, 2007).
O. fragilisÂ is found further north than any other cactus species in the world, growing in northern Alberta only 4oÂ south of the Arctic Circle (Taylor, 2005). O. fragilis is widely distributed across North America (Figure 2). It occurs from Ontario south to Texas and west to British Columbia, Washington, Oregon, and California (Taylor, 2005). O. fragilisÂ is scattered throughout the upper Midwest, where it is rare in Ontario, Iowa, Illinois, and Michigan and rather uncommon in Minnesota and Wisconsin.
The Flora of North America distribution map of Opuntia fragilis
Figure 2: Distribution of Opuntia fragilis in North America.
Accessed October 21, 2009. Image Source: www.EFloras.org
O. fragilis prefers habitats with exposed bedrocks or sandy and gravelly soils of prairie grasslands and has also been located in areas of light shade at edges of forested coulees and grasslands (Cota, 2002). In prickly pears, each pad, in its first year only, produces areoles with subtending conic leaves. These leaves are usually dropped after a few weeks. The areole also produces leaves modified into spines of two kinds --permanent spines with their bases embedded in cork and small, barbed, easily dislodged glochids. The prickly pears thrive in arid, shallow and well-drained soils, but also on overgrazed sites.
The cladodes are flattened into pads; the fruits are berries, and in many species edible. Opuntias are biologically complex xerophytes. Opuntias flourish in desert-like habitats due to numerous xerophytic adaptations. These include thick, waxy cuticles that reduce the amount of water lost through transpiration and modified leaves, and bud scales in the form of spines and glochids that decrease plant surface areaproducing low transpiration rates to reduce water loss. Malic acid or isocitric acid are accumulated in the vacuoles of photosynthetic cells at night and metabolized to release carbon dioxide during the day. This type of photosynthesis is referred to as Crassulacean acid metabolism or more simply, CAM photosynthesis (Majure and Ervin, 2007).
The highly dispersed taxonomic distribution of CAM photosynthesis, which occurs in 33 families and an estimated 16,000 species of vascular plants, suggests it has arisen on multiple occasions. In families such as the Agavaceae, Cactaceae, and Didiereaceae, almost all species have the capacity for CAM photosynthesis and thus exhibit the presumed apomorphic character-state (Crayn et al., 2004). Cacti have helped evolutionary biologists and ecologists understand CAM photosynthesis (Crassulacean acid metabolism) and succulence (Mihalte et al., 2008). During the night, they open stomata and CO2Â enters the leaf cells where it combines with PEP (phosphoenolpyruvate) to form 4-carbon organic acids (malic and isocitric acids). During the day, their stomata are closed; however they still carry on photosynthesis with the reserve supply of CO2Â that was trapped during the night when the stomata were open and carbon dioxide gas is converted into simple sugars. Thus, there is an increase in water-use efficiency because the stomata open at night; CO2Â is fixed when the air temperatures areÂ lower and the relative humidity is higher, reducing water loss (Hernández-Gonzáles and Villarreal, 2007).
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Flowers in the Cactaceae are usually perfect, containing both functional pistils and stamens (Figure 3). Such flowers can either outcross with other individuals or be self-fertile and pollinate themselves (Rebman and Pinkava, 2001). Many prickly pears are apparently self-incompatible (Ribbens and Anderson, 2008). O. fragilis certainly is, at least in Illinois (Ribbens and Anderson, 2008). Asexual reproduction is undoubtedly responsible for the success and spread of this population on the edge of the range of the species (Ribbens and Anderson, 2008). Asexual reproduction is a common occurrence for many Opuntias. The most prevalent type of cloning in this group is vegetative propagation by stem or cladode detachment. The terminal stem segments of some species (e.g., Cylindropuntia leptocaulis (DeCondolle) Knuth, O. fragilis, and O. pubescens Wendland) detach with ease from the parent plant and readily take root, creating clonal individuals (Rebman and Pinkava, 2001).
Figure 3: Opuntia fragilis flower.
Image from website: http://www.geog.ubc.ca/biodiversity/eflora/vasculars.html
The flesh of O. fragilis was used by the Okanagan-Colville Native Americans to treat skin infections and was eaten for its diuretic properties. The stems were used as food by the Okanagan-Colville and Shuswap tribes (Cota, 2002).
It is well known to everyone familiar with handling of cactus cladodes (known in Mexico as nopalitos) or prickly-pear fruit in the kitchen, that when cut, both secrete a characteristic slime. The main constituent of this secreted fluid is polysaccharide mucilage (Cardenas et al., 1997). The mucilage obtained from cactus is commonly described as water-soluble pectin-like polysaccharide (Cardenas et al., 1997). Mucilage production is common to many groups of plants. The mechanism of secretion is apparently the same in different plant species (Trachtenberg and Fahn, 1981). Fresh tissue from cacti presents large amounts of polyphenolics and polysaccharides that co-precipitate with DNA and affect subsequent PCR amplification (Mihalte et al., 2008).
The ability of Cactaceae to retain water under such unfavorable climatic conditions is due in part, at least, to the water-binding capacity of mucilage (Mindt et al., 1975). The carbohydrate composition of mucilages from several Opuntia species has been well established by different chromatographic techniques. From his investigations, Lauterbach (1889) concluded that there are two methods by which the mucilage is formed, one holding true for Opuntia, and the other for the remainder of the cactus groups. In the Opuntia the mucilage arises in a cell containing a small crystal of some oxalate. This crystal seemed to stimulate the growth of the cell. Later the nucleus and crystal might appear suspended on strands of cytoplasm in the midst of the cell and mucilage would then begin to appear in the periphery of the protoplasm (Stewart, 1919).
The genus Opuntia consists of 200 species that are classified based on their morphological and physiological traits (Helson et al, 2007). The use of molecular techniques has proven useful in understanding the genetics of Opuntia. For example, Labra et al. (2003) have suggested that O. ficus-indica should be considered a domesticated form of O. megacantha based on molecular data [chloroplast simple sequence repeat (cpSSR) and amplified fragment length polymorphism (AFLP)], morphological traits and biogeographic distribution.
Butterworth and Wallace (2005) investigated the evolutionary relationships in Pereskia and informal infrageneric groupings. In this study, they isolated total genomic DNA using a modified organelle pellet method suitable for mucilaginous material (Butterworth and Wallace, 2005). With the help of their molecular data they found that Pereskia, generally interpreted as the sister group to the rest of Cactaceae, appears to be paraphyletic. In recent investigations made by Butterworth and Edwards (2008) on pereskia and the earliest divergences in Cactaceae, they present a summary of the two previous molecular (DNA) phylogenetic studies of Pereskia (Butterworth and Wallace 2005; Edwards and others 2005) with their analysis of combined DNA sequence data used in those studies (Butterworth and Edwards, 2008).
A high-yielding micromethod for DNA extraction was established by using only small amounts of a specialized tissue of O. ficus-indica cladodes to be used for genomic characterization. Also, the RAPD analysis of this plant material was performed by eight primers indicating genetic uniformity of the material (Arnholdt-schmitt et al., 2001). However, there are problems associated with extracting DNA from cladodes, leaves or dried roots since they have high levels of polysaccharides, phenolics and other secondary metabolites (Tel-Zur et al., 1999).
Presence of these polysaccharides was indicated by the viscous, glue like texture of all extracts at all stages of DNA isolation (Tel-Zur et al., 1999). The viscous DNA solution was impure and unamplifiable in the polymerase chain reaction (PCR) due to inhibition of Taq polymerase activity (Fang et al., 1992). Though original CTAB method (Doyle and Doyle, 1990; Reichardt and Rogers, 1994) was helpful in successfully extracting and isolating DNA from the cactus species Opuntia ficus-indica and Cereus peruvianus, it was unsuccessful in isolating DNA from certain species of the genera (Tel-Zur et al., 1999).
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In a study made by Mondragón (2001b), he stated that changes such as optimization of sample size, incorporation of insoluble polyvinylpyrrolidone, increased time centrifugation and separation of the acid pill nucleic bath could reduce the problem of the presence of mucilage in the sample, causing gelation of the sample making it difficult to centrifugation and separation of the DNA pellet (Mondragón, 2001b).
To overcome the mucilage problem to an extent and obtain a highly pure DNA, we used roots for extraction of the genetic material. My study examines the genetics of O. fragilis by analyzing ISSR polymorphisms in the Midwest United States. The main objectives of this project are to analyze genetics between and within populations for Illinois, Iowa, Michigan, Minnesota and Wisconsin.
MATERIALS AND METHODS
Forty root samples were harvested from Opuntia fragilis pads that were collected from populations in Midwest (Illinois, Iowa, Michigan, Minnesota, and Wisconsin). Pads were floated in water until they produced roots. These roots were then preserved in Ethanol and stored for future use. Genomic DNA samples isolated from the roots of these O. fragilis pads were prepared by using the CTAB-chloroform protocol (Dr. Alton, personal communication).
The CTAB (Hexadecyltrimethylammoniumbromid) is a cationic detergent that forms a complex with the DNA. The CTAB-DNA complex is then separated from the cellular debris by chloroform. In this step, we can observe two layers: a superior clear aqeous layer containing the DNA and a denser inferior layer containing the chloroform and all other secondary components such as proteins, polysaccharides etc. After centrifugation, cellular debris can usually be observed at the interface. The purification by chloroform can be repeated several times. The topmost aqeous layer is transferred to a clean eppendoff and equal volume of isopropanol is added. This CTAB-chloroform protocol was used to isolate genomic DNA from roots of O. fragilis that would then be subjected to ISSR analysis from the plant.
The isopropanol is poured out leaving behind the precipitate. After the precipitation, the DNA molecule must be washed with ethanol. The DNA is then dissolved and stored in a tris/EDTA (TE) buffer.
Amplification and Sequencing:
Polymerase chain reaction (PCR) was performed for 10 samples in Applied Biosystems Step One plus Real Time PCR system. The Genomic DNA from our samples was amplified using three ISSR primers where ISSR stands for Inter simple sequence repeats. The ISSR primers use microsatellite sequence ending in an extra base to anchor it to specific regions and were suggested by Dr. Jeremy Fant. The ISSR primer sequences are listed below:
Primer 1: AGA GAG AGA GAG AGA GT
Primer 2: ACA CAC ACA CAC ACA CC
Primer 3: ACA CAC ACA CAC ACA CG
Each ISSR reaction was carried out in a total volume of 5ÂµL, containing 5U/ÂµL Master Amp, and 0.5Âµl of Taqman polymerase, 1Âµl of genomic DNA. Initial denaturation was carried out for 10min at 95Â°C, followed by 40 cycles for 1 min at 95Â°C, 1 min at 53.5Â°C for two primers and 1 min at 72Â°C.
Agarose electrophoresis in 1X TAE buffer was used to visualize the size of bands expected. Product lengths of 3 samples were analyzed on a 1.5% agarose gel and visualized using 0.5 Âµg/ml Ethidium Bromide stain.
I was not able to successfully perform PCR analysis on these samples. I believe this is due to high polysaccharide compositions in the samples. Subsequent electrophoretic analysis showed a distinct band of high-molecular-weight DNA and a smear of RNA (Figure 4). Isolation of good quality DNA from O. fragilis is complicated by the presence of large amounts of polysaccharide-based mucilage in its roots. Also, the other possibilities of not getting DNA bands on the gel could be insufficient quantity or concentration of DNA loaded on the gel or the DNA was degraded or the DNA was electrophoresed off the gel and so electrophoresing the gel for less time might have been helpful.
Figure 4: Photo of gel loaded with 6 DNA samples.
The main objective of this project was to deal with the genetics of Opuntia fragilis to analyze the ISSR polymorphisms in the Midwest United States. This was not achieved due to possibility of the contaminating polysaccharides present in the genomic DNA extracted from the roots of O. fragilis.
For many years the role of mucilage in succulents was thought to be in water conservation (Evans, 1932). Sutton et al. (1981) suggest that mucilage serves as a carbohydrate reserve in addition to glucan. The biochemistry for this potential reserve role is presently unknown (Holthe and Szarek, 1985). Despite the improvements in the extraction of DNA, numerous protocols are limited to specific plant groups (Cota et al., 2006). It is true that extraction of DNA from cacti can be more difficult than from other plants because of their low yields of DNA. Cacti, among other succulents, may often contain large amounts of polysaccharide-based mucilage which can bind water in extraction buffers, causing difficulty in pipetting and centrifugation (Griffith and Porter, 2003). They also contain high amounts of secondary metabolites which form insoluble complexes with nucleic acids during extraction. (Guillemaut and Marechal, 1992).
Studies of the molecular biology of Opuntia fragilis are limited. The inclusion of molecular data will be helpful to clarify classification within the Opuntia genus.The monophyly of the tribe Cactaceae was tested using rpl16 intron sequence variation data which helped resolve the intergeneric relationships in cactaceae and also the monophyly in previously proposed cacteae genera was assessed using this molecular data (Butterworth et al., 2001). In a study conducted by Dr. Wang et al. (1998) molecular analysis of RAPD marker patterns demonstrated the usefulness of molecular markers in the classification of Opuntia accessions. Their experiments also indicated the feasibility of a comprehensive effort to determine the relationships among Opuntia species using molecular markers (Wang et al., 1998). High levels of polymorphism in Galapagos prickly pear (Opuntia) cactus species were observed by developing 16 microsatellite markers and studying the population genetic structure of Galapagos prickly pear (Opuntia) cactus species (Helsen, 2007). Amplified fragment length data can be used to answer a wide range of genetic questions (Mueller and Wolfenbarger, 1999). With the help of amplified fragment length technique, even small amounts of genomicÂ DNA can be used to produceÂ DNAÂ "fingerprints" that are highly specific to particularÂ species. AFLPÂ can also be used to determineÂ genetic variationÂ across different populations (Chial, 2008). Thus, Amplified fragment length technique generates the results in the form of large number of fragments which gives us an estimate of variation across the entire genome. These large numbers of fragments thus gives a good general picture of the level of genetic variation of the O. fragilis.
However, DNA isolation in my study was extremely difficult because of the presence of acidic polysaccharides and polyphenols. Therefore, I conclude that the interferences with the DNA in my samples are due to the Opuntia polysaccharide compositions and not a contaminating artifact. Further improvement of theÂ extraction methodology will allow investigators to isolate good quality DNA from roots of O. fragilis and analyze the ISSR polymorphisms among species. DNA sequence-based phylogeneticÂ estimations may indicate that a genome wideÂ approach may yield more useful variation for the genusÂ Opuntia.