Dylan Bale, A Nuffield student in my lab summer 2010.

1. www.aber.ac.uk/wpsi
Summer 2010
Nuffield Science Bursary Project
Dylan Bale Ysgol Gyfun Ystalyfera
Investigating quality traits in the bio-energy
crop Miscanthus using molecular techniques
Introduction
Miscanthus is a C4 perennial grass native to tropical climates. It has very efficient photosynthesis, growing at
high speeds, which makes it ideal as a bio-energy crop. It can be used either as a biomass crop (burnt dry) or as a
biofuel by fermenting to ethanol. Miscanthus also has the advantage it can tolerate cold temperatures and so is
not limited to the tropics. Important traits for any bioenergy crop include high yield, high quality, and low input
requirements.
Another quality of biomass crop is the heating value; this is determined by factors such as moisture content, oxygen concentration, ash concentration
and lignin mass. Lastly, entophytic bacteria (symbiotes living within a plant, often a bacteria or a fungus) are thought to have a positive effect on
the acceleration of seedling emergence and therefore can form bigger plants , by the feeding of essential vitamins to the plant, and by modifing
metabolism and nitrogen fixation. Endophytes have been shown to be present in Miscanthus, although their role is not yet known. It is possible
that Nitrogen fixing bacterial endophytes might contribute to the low nutrient requirement of this crop.
Method Results
Primers were extracted from the dry matter material of the Miscanthus in order to investigate
DNA markers for later comparison in the BAC (Bacteria Artificial Chromosomes) libraries to
The CAD primer: The F5H primer:
indicate positive quality traits. The DNA Bands shown in this gene specific The DNA bands found in this gene specific
The following five stages were carried out: primer were in the positions 1H, 8F and 10 primer were found in the wells 1C, 4C and
1. DNA extraction: (using Qiagen DNeasy Plant Mini Kit Protocol) H. Bands shown here are somewhat faint 12E. The DNA bands here are not as faint as
but manageable to read. the CAD primer, therefore easily read.
2. Superpool DNA screening - PCR set up
3. - agarose gel set up
4. - gel imaging
5. - band selection by masking
Full details can be found in the accompanying report
(copies can be obtained by e-mailing wpsi@aber.ac.uk).
Discussion
Errors inPCR-based screening can be common. For example, incorrect pipetting of the
super pool DNA or the evaporation of the combination of the PCR mastermix , loading
dye and superpool DNA in the PCR machine. Care needs to be taken over this and cross-
contamination. Variations in annealing temperatures for the development of bands can be
also difficult to achieve best results.
Conclusion
The super pool initially used with the primers (CAD and F5H) during PCR and electrophoresis
will be involved in later experiments in order to isolate specific bands and positions, to
determine quality traits in the bio energy crop Miscanthus. This will then enable the plants
to be more efficient as a bio energy crop and give the best possible quality of biofuel or
biomass in the future.
With thanks to
• The Nuffield Foundation for funding, • The Institute of Biology, • Dr Debra Croft (Centre for Widening
and Bryan Hatton (Co-ordinator in Environmental and Rural Sciences Participation and Social Inclusion) and
Wales) (AberystwythUniversity) – especially the Wales Summer University team
Dr Kerrie Farrar and team (Aberystwyth University)