The Identification of Bambusa Sp

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The identification of Bamboo using various PCR and Sequencing Techniques

Abstract
Often the incorrect bamboo species is sold to unsuspecting customers at shops. This can have a disastrous effect on their garden. Three separate and unknown Bamboo leaf samples were taken and were required to be distinguished genetically from one another. Using ITS-PCR DNA amplification techniques, the ITS region DNA was amplified and used in PCR-RFLP and RAPD PCR in order to determine the genetic identity of each sample. Sequencing was performed, and results allowed us to distinguish between samples (to a certain extent.)

Introduction
Bamboos are a group of woody perennial green plants (Wikipedia et al. 2006) that are found in many parts of the world. There are 91 genera and about 1,000 species of bamboo (Wikipedia et al. 2006). They are found in diverse climates, from cold mountains to hot tropical regions. Bamboo is a highly desirable plant grown for many reasons in plantations and gardens around the world. Many reasons it is grown are that it is a beautiful ornamental plant with unique properties. Bamboo is also an extremely strong plant that is light; it is used in many building applications for floorboards, and is also often used in furniture making. There are a number of taller growing species that are effective at blocking out the eyes of pepping toms and nosy neighbors.

There are two main forms of bamboo, each form describing the way in which the bamboo itself spreads. These are known as “clumping” (monopodial) and “running” (sympodial) forms. (Wikipedia et al. 2006) Clumping bamboo species tend to spread underground slowly. Running bamboo species are highly variable in their tendency to spread; this is related to both the species and the soil and climate conditions. Some can send out runners several metres a year, while others can stay in the same general area for long periods. If neglected, they can be invasive over time and can cause problems by moving into adjacent areas. The reputation of bamboo as being highly invasive is often exaggerated, and situations where it has taken over large areas is often the result of years of untended or neglected plantings.

Many invasive bamboo species are often sold, unsuspectingly to people, who plant them without realizing this. The result of this is the complete takeover of ones garden. Some species of bamboo can grow at a tremendous rate, some at over 36inches (90cm) a day, providing it is provided with ideal conditions (OneEarth, 2006).

Plant Biosecurity breaches often occur when bamboo plants are imported with incorrect or false labeling, often in an attempt to bring illegal ornamental species in to the country for indoor use. This ‘black market’ operation is a serious threat to native species of plants, and, if a threatening sympodial bamboo species is imported and planted in place of a monopodial (which is preferred, as they do not spread), serious damage to native forests and grasslands can occur (NGIA, 2006). Some of the techniques that can be used to identify to a species level are PCR-ITS, RAPD, and PCR-RFLP. These will be used to identify our unknown samples of bamboo.

Aim
To identify, to a species level, using nucleotide analysis and sequencing techniques, three unknown samples of bamboo.

Materials
For DNA extraction
3 Unknown Bamboo Samples (Leaves)
Mortar and Pestle
Liquid Nitrogen
Quiagen ‘Dneasy’ DNA Extraction Kit
Centrifuge tubes
Pipettes and Tips
Ice and Esky

Quantification of DNA
Well Combs (10uL)
Wells
UV Transilluminator
Agarose
Tris Borate EDTA
Ethidium Bromide
Loading Dye
Centrifuge Tubes
Gel Tank (To run agarose gel electrophoresis)
Pipettes and Tips

For ITS based PCR
5uL of extracted DNA
5x Reaction buffer
MilliQ (Ultra Pure Water)
DNTP’s (dATP, dGTP, dCTP, dTTP)
PCR Machine
MgCl2
Centrifuge Tubes
Pipettes and Tips

For RAPD-PCR
ITS-PCR DNA product
5x Reaction buffer
MilliQ (Ultra Pure Water)
MgCl2
Primers...
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