M4 Lesson 6 Plant Molecular Systematics (Lab)

Overview of Plant Molecular Systematics

• Modern Systematic Approaches: While micromorphology and karyotyping are traditional methods for showing species relatedness, the study of plant $DNA$ and $RNA$ provides more convincing evidence of phylogenetic interrelationships.

• Molecular Evolution: Phylogenies are justified through a systematic review of molecular evolution. Molecular data reveal gene-level information that can show correlations among the following types of information to justify the phylogeny of a plant group:     * Macroscopic data.     * Microscopic data.     * Chemotaxonomic information.

• Scope of Molecular Systematics: This field encompasses approaches in which phylogenetic relationships are inferred using information derived from macromolecules of the organisms.

• Types of Molecular Data: Specifically, the data includes:     * $DNA$ sequences.     * $DNA$ restriction sites.     * Allozymes.     * Microsatellites.     * $RAPDs$ (Random Amplified Polymorphic $DNA$).     * $AFLPs$ (Amplified Fragment Length Polymorphisms).

• Exclusion of Chemosystematics: The evaluation of smaller molecules, such as secondary compounds in plants, is generally relegated to the field of chemosystematics and is distinct from molecular systematics.

Acquisition of Molecular Data

• Plant Sample Acquisition: Samples for $DNA$ isolation can be acquired through various means, but consistency in documentation is essential.

• Voucher Specimens: It is vital to collect a proper voucher specimen for every study. This specimen must be properly mounted and accessioned in an accredited herbarium to serve as official documentation for the molecular systematic study.

• Sample Types and Processing:     * Live Samples: These may be collected and immediately subjected to chemical processing (e.g., for allozyme analysis).     * Dried Samples: For many $DNA$ methods, pieces of leaves (containing chloroplast, mitochondrial, and nuclear $DNA$) are removed and immediately dried, typically in a container of silica gel.     * Frozen/Buffer Storage: Alternatively, samples may be frozen or placed in a concentrated extraction buffer. These procedures usually preserve $DNA$ intact.     * Herbarium Samples: Useable $DNA$ can often be successfully isolated from dried herbarium sheets, demonstrating the inherent toughness of the $DNA$ molecule.

DNA Sequence Data

• Significance: Acquiring $DNA$ sequences is considered the most important method for inferring the phylogenetic relationships of life.

• Definition of DNA Sequence Data: This refers to the specific sequence of nucleotides in a particular region of a taxon's $DNA$. The four nucleotides are:     * Adenine ($A$)     * Cytosine ($C$)     * Guanine ($G$)     * Thymine ($T$)

• Comparative Analysis: Relationships are inferred by comparing homologous regions of $DNA$ among the taxa under study to identify characters and character states.

• Sequence Acquisition Process: The first step involves identifying a specific region of $DNA$ to be compared between species. Substantial prior research is required to determine the efficacy of these regions for phylogenetic analysis.

Polymerase Chain Reaction (PCR)

• Isolation and Purification: Once a gene sequence of interest is identified, the sample $DNA$ is isolated and purified via chemical procedures.

• Amplification: The sequence is amplified using the Polymerase Chain Reaction ($PCR$). This technology allows for the rapid replication of thousands or millions of copies of $DNA$. A typical reaction can produce more than $1,000,000$ copies in a few hours.

• Components of a PCR Solution:     1. Isolated and purified sample $DNA$.     2. Multiple copies of primers (short, conserved regions flanking the target sequence).     3. Free nucleotides.     4. $DNA$ polymerase molecules (typically $Taq$ polymerase, which is heat-tolerant).     5. Buffer and salts.

• The PCR Process (Cycle Sequencing):     * Denaturation: The solution is heated. The two strands of the sample $DNA$ separate (denature).     * Priming (Annealing): The temperature is lowered to allow primers to bind to their corresponding complementary conserved regions on the sample $DNA$. Primers are designed not to bind with one another.     * Replication (Extension): Using the sample $DNA$ as a template, free nucleotides attach to the $3' ext{ end}$ of the primer. This reaction is catalyzed by $DNA$ polymerase.     * Repetition: The process of heating (denaturation) and cooling (annealing/extension) is repeated in cycles to exponentially increase the amount of $DNA$.

DNA Sequencing Reaction

• Sequencing Technology: The most common method involves a machine that reads fluorescent dyes with a laser detector. The production of dye-labeled $DNA$ is very similar to the $PCR$ process.

• Reaction Mixture: Replicated $DNA$ is placed in a solution with $DNA$ polymerase, primers, free nucleotides, and a small concentration of dideoxynucleotides ($ddNTPs$).

• Dideoxynucleotides (ddNTPs):     * Types: Dideoxyadenine, dideoxycytosine, dideoxyguanine, and dideoxythymine.     * Function: These compounds resemble the four standard nucleotides but lack a hydroxyl ($-OH$) group.     * Chain Termination: When a $ddNTP$ joins the growing $DNA$ strand, the absence of the hydroxyl group prevents the polymerase from adding further nucleotides, thus terminating the strand.

• Strand Variation: The ratio of $ddNTPs$ to standard nucleotides is kept low so that termination occurs randomly at every possible base position along the gene. This results in millions of newly replicated strands of all different lengths (e.g., primer length plus one base, plus two bases, etc.).

• Electrophoresis and Detection:     * Mechanism: $DNA$ strands are loaded into a capillary or flat gel plate and subjected to an electric current.     * Charge: Higher molecular weight strands migrate slower. Because phosphate components give $DNA$ a net negative charge, molecules migrate toward the positive pole.     * Migration: Migration speed is inversely proportional to the molecular weight of the strand.     * Laser Excitation: Each $ddNTP$ is attached to a different fluorescent dye that emits light at a specific wavelength when excited by a laser.     * Data Output: Light intensity is recorded as a peak. The timing of the migration allows the machine to infer the exact sequence of nucleotides.

Types of DNA Sequence Data in Plants

• Sources of DNA: Plant sequence data comes from three major sources:     1. Nuclear DNA (nDNA): Transmitted via sexual or asexual reproduction (meiosis/mitosis).         * Alcohol Dehydrogenase (Adh): A protein-coding gene used increasingly in systematics.         * Internal Transcribed Spacer (ITS): Located between the $18S$ and $26S$ nuclear ribosomal $DNA$ ($nrDNA$). It contains multiple copies and is valuable for inferring relationships at lower levels (closely related species), though it is also used for higher-level relationships.     2. Chloroplast DNA (cpDNA): Often used to elucidate both lower- and higher-level relationships.         * Structure: Like prokaryotic $DNA$, it is circular. In Nicotiana tabacum (Tobacco), it consists of $155,939$ base pairs.         * Key Regions: Includes a Large Single Copy ($LSC$) region, a Small Single Copy ($SSC$) region, and two Inverted Repeats ($IRA$ and $IRB$).         * Genes: Common genes sequenced include $rbcL$, $atpB$, $matK$, and $ndhF$.     3. Mitochondrial DNA (mtDNA): Along with chloroplasts, these organelles replicate independently of the nucleus.

• Inheritance Patterns:     * Angiosperms: Organelles ($cpDNA$ and $mtDNA$) are usually transmitted maternally (via the egg cell, excluded from sperm).     * Conifers: Interestingly, in conifers, chloroplast $DNA$ is transmitted paternally.