Massimo Pindo, PhD Sequencing & Genotyping Platform massimo.pindo@fmach.it Edmund Mach Foundation

FruitBreedomics WorkshopFrom low to high throughput genotyping

for fruit breeding Methods for DNA extraction

April 14th and 15th, 2014, San Michele all’Adige (Italy)

Marker Assisted Breeding: from the greenhouse to the lab

STEP 1Seedling layout

STEP 2Sample collection

STEP 3Run Analysis

Marker Assisted Breeding: from the greenhouse to the lab

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)

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 component.

1. “quick and dirty”

Quick Extract Plant (Epicenter)

Description: QuickExtract™ DNA Extraction Solution can be usedto rapidly and extract PCR-ready genomic DNA fromalmost any sample type using a simple, one-tubeprotocol 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.

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.

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).

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).

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.

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.

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

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.

Next Gen. Sequencing

Whole Genome Seq.Target Reseq.MetagenomicsTranscriptome Seq.

Genotyping &Robotic Platform

DNA & RNA extractionCapillary SequencingMid- to High-plex Genotyping

Next Gen. Sequencing and Protocol Setting

Shot gun & PE gDNA libraryAmplicon LibraryNext Gen. Sequencing Application

Massimo Pindo

Sequencing andGenotyping

Platform

Erika Stefani Daniela Nicolini

Simone Larger

People:

Elisa Banchi Lara Poles