FruitBreedomics WP6 training session

1. Massimo Pindo, PhD Sequencing & Genotyping Platform
massimo.pindo@fmach.it Edmund Mach Foundation
FruitBreedomics Workshop
From low to high throughput genotyping
for fruit breeding
Methods for DNA extraction
April 14th and 15th, 2014, San Michele all’Adige (Italy)

2. Marker Assisted Breeding: from the
greenhouse to the lab
STEP 1
Seedling layout
STEP 2
Sample collection
STEP 3
Run Analysis
Marker Assisted Breeding: from the
greenhouse to the lab

3. PLATEAU A
PLATEAU B
PLATE 1 (A+B)
Sampling for marker-assisted breeding
A young leave (about 1.5 cm) is collected from each sample and put in the
corresponding well of the collection plate kept in ice. Then it is brought to the
lab for freeze-drying, then for DNA extraction.
Cross disposition in greenhouse: the seeds are sewed in 2 plateau correspondent to
the two half of a 96-wellp plate (P: parents of the cross)

4. DNA extraction
MAB usually involves hundreds or thousands of seedlings and common high-
throughput extraction methods, such as columns, are too expensive since DNA
is only screened a small number of times.
1. “quick and dirty”: these products can extract nucleic acid form a small
portion of tissue without any purification of the sample. We tested Quick
Extract Plant (Epicenter) and Extract’n’Amp Plant PCR Kit (Sigma): the latter
one also provide a specific amplification enzyme.
2. “direct PCR”: these products can amplify DNA from few millimetres of crude
tissue. They employ DNA polymerases very robust and tolerant of PCR
inhibitors present in plant material, like polyphenols and polysaccharides.
3. “magnetic particles”: this kit use MagAttract magnetic-particle technology
that combines silica-based DNA purification (the one used with colums) with
the simple handling of magnetic particles.
4. “homemade kit”: the reagent are prepared in the lab and no commercial kit
is needed. It’s called CTAB method from the name of the principal

5. 1. “quick and dirty”
Quick Extract Plant (Epicenter)
Description:
QuickExtract™ DNA Extraction Solution can be used
to rapidly and extract PCR-ready genomic DNA from
almost any sample type using a simple, one-tube
protocol that takes only 3-8 minutes.
Evaluation:
The procedure is easy and fast, and seemed quite reliable, but after a lot that didn’t work
at all the company was not able to provide a new one for long time. The cost is about
0.80 Euro per sample.
Extract’n’Amp Plant PCR Kit (Sigma)
Description:
Extract’n’Amp Plant PCR Kit include a specially formulated hot start PCR ReadyMix for
amplification directly from the extract.
Evaluation:
Procedure is easy and fast, but in different markers we found low reproducibility. The
cost is about 0.60 Euro per sample.

6. 2. “direct PCR”
KAPA3G Plant PCR (Kapa Biosystems)
Description:
KAPA3G Plant PCR (Kapa Biosystems) is based on DNA polymerase engineered for
improved tolerance to common plant-derived PCR inhibitors. Kits permit fast
amplification of plant DNA from crude samples.
Evaluation:
The kit is designed for direct-PCR application, sampling with a 0.5 mm diameter Harris
UnicoreTM (cutter) to get crude sample to amplify directly.
In greenhouse this makes contamination very easy, so we used a “Reducing
contamination protocol” that let us sampling a leaf disc. The cost is about 0.70 euro
per sample.

7. 3. “magnetic particles”
BioSprint 96 DNA Plant Kit (Qiagen)
Description:
BioSprint 96 DNA Plant Kit (Qiagen) use MagAttract magnetic-particle technology of
DNA purification. Needs a specific workstation.
Evaluation:
The protocol is about two hours long (two hours per plate – one at a time) and there is a
lot of plastic waste. The results are quite different from sample to sample and an ad
hoc dilution is needed and it’s not comfortable for MAB when a lot of samples need to
be screened. We didn’t try optimization yet. The cost per sample is about 1.5 euro
(plus the workstation cost).

8. 4. “homemade kit”
CTAB method
Description:
CTAB method is based on modified Doyle and Doyle (1990) protocol. Ref: Doyle and
Doyle (1990) Isolation of plant DNA form fresh tissue.
Evaluation:
After some optimization, we gain good quality extracted DNA with half young leaf.
Analysis of SSR markers gave good results even with multiplex. The use of CTAB
instead of a kit permits the use of any enzyme for PCR amplification because there is
no need to use the kit’s one. The protocol takes about three hours for two 96-wells
plates. This is the extraction method we are using for MAB. The cost per sample is
the lowest, about 0.15 Euro (include only extraction).

9. CONCLUSION
KAPA 3G Plant PCR and Extract’n’Amp Plant PCR Kit: the unpurified nucleic acid from
these extractions can give problems for plants like apple, which are rich in
polyphenols and polysaccharides.
With 3G Plant PCR we managed to amplify markers in multiplex and found the major
difficulties with some SCAR markers.
Even with Extract’n’Amp it was possible to multiplex SSRs but we found inconsistencies
with some samples and markers.
CTAB permits a quite homogenous yield and clean result.
BioSprint 96 DNA Plant Kit didn’t meet our requirements in terms of time and throughput
and yield that was very different to sample to sample.
We found that KAPA 3G Plant PCR is a valuable, cheap and fast extraction method
expecially for short fragments analysis.
CTAB is a robust and cheap. The protocol is longer than KAPA but suitable for long-
storage needs and reliable even for longest fragments.

10. 3G Plant PCR Optimisation
Results:
• by gel electrophoresis: good resolution
• by capillary electrophoresis: clear and easy to optimize for
DATA analysis using the genemapper software.

11. 3G Plant PCR Optimisation
Results:
• by gel electrophoresis: low resolution
• by capillary electrophoresis: good resolution specific
alleles are evaluable but there are some aspecific
products

12. CONCLUSION
We found that KAPA 3G Plant PCR is a valuable, cheap and fast extraction method
expecially for short fragments analysis.
With the proposed protocol, we also reduced time and cost of the MAS analysis: there
are no step of samples dilution from the extraction to the capillary electrophoresis,
and the reduced volume of the reagents permits to perform more reactions without
increased costs (about 0.70 Euro per sample).
We used this protocol for MAS in 2013 and 2014. The main problems encountered were
associated with poor quality “quick-and-dirty” extractions; however, following
optimization less than 10% of the seedlings had been re-sampled.
CTAB is a classic low cost home made extraction method, in this 96-wells plate version
permits a quite high throughput (four plate/day per person) and a homogenous yield
(e.i. 150-200 ng/µL for younger leaves) and a cleaner result.
Plus, while “quick-and-dirty” give the chance to conserve the samples only for a short
time because they are thought for an immediate use, in this case is possible to stock
the plates for longer period if it’s needed. Using hazardous reagents, the availability
of a fume hood is necessary.

13. Next Gen. Sequencing
Whole Genome Seq.
Target Reseq.
Metagenomics
Transcriptome Seq.
Next Gen. Sequencing
Whole Genome Seq.
Target Reseq.
Metagenomics
Transcriptome Seq.
Genotyping &
Robotic Platform
DNA & RNA extraction
Capillary Sequencing
Mid- to High-plex Genotyping
Genotyping &
Robotic Platform
DNA & RNA extraction
Capillary Sequencing
Mid- to High-plex Genotyping
Next Gen. Sequencing and
Protocol Setting
Shot gun & PE gDNA library
Amplicon Library
Next Gen. Sequencing
Application
Next Gen. Sequencing and
Protocol Setting
Shot gun & PE gDNA library
Amplicon Library
Next Gen. Sequencing
Application
Massimo Pindo
Sequencing and
Genotyping Platform
Erika Stefani Daniela Nicolini
Simone Larger
People:
Elisa Banchi Lara Poles