DNA fingerprinting

1. Malawian Groundnut Variety Identification
Using DNA Fingerprinting
Sep/2010
Santie de Villiers,Vincent Njunge,Moses Siambi,Emmanuel Monyo
ICRISAT-Nairobi and ICRISAT-Lilongwe
For legumes, such as groundnuts, it is
often difficult to differentiate the seeds
from various varieties and to be sure that
seeds are genetically“true-to-type”.
Conventionally, seed samples are drawn
from large lots and planted so that the
morphological features can be compared
against a set of descriptors,but this takes
time and is expensive.
The identification of molecular markers
that can be used for variety identification
through DNA fingerprinting is not only
quicker but also cheaper.Any plant tissue
(seed, leaf, root) can be used at any stage
of growth and it is not dependent on the
particular cropping season.
Identification of fingerprinting markers
• Nuclear seed from breeders and/or genebank accessions of all
cultivated varieties (six released varieties and 13 local ones) were sent
for analysis in Nairobi at the BecA-ILRI hub
• The seeds were planted and DNA isolated from seedlings
• The isolated DNA from all the varieties was analyzed by genotyping
with 20 publicly available GCP SSR markers (http://gcpcr.grinfo.net/
index.php?app=datasets&inc=search_simple) and results visualized
using an ABI3730 sequencer.
• From the 20 markers used, three were identified that can distinguish all
19 varieties. They are:
- TCF12
- TC7H11
- 5D5
• Training of Malawi seed system unit staff in sampling, DNA extraction
and PCR technology that can be done in-house.
DNA fingerprinting process
• Seed inspectors or breeders collect samples from seed lots and/
or farmer fields according to an internationally approved sampling
protocol
• Samples are sent for DNA fingerprinting to an appropriate facility such
as ICRISAT-India, BecA-Nairobi, the Malawi Seed Systems Unit or an
outsourced company/laboratory)
• DNA extraction and genotyping with the three fingerprinting markers
• Comparison of test samples with DNA from each documented variety
• Confirmation of sample identity and purity
• Results sent back to SSU for certification
Applications
• Assist in seed certification through :
confirmation of varietal identity
seed purity testing/confirmation
• Tracking of disseminated varieties in farmers’fields to adoption of
released varieties and a measure of the impact from the release of new
varieties.
An example of a groundnut DNA fingerprint displayed on an agarose gel.
The known fingerprints are on the left (no’s 1 to 6).The unknown samples
on the right are compared with these known fingerprints – a matter of
matching the bars. In this example, the column with the arrow is clearly
the same as known variety no.3,which is the fingerprint for variety Baka/
ICGV-12991.
With funding from Irish Aid in 2010, ICRISAT has identified a set of three DNA markers for groundnut that can
distinguishallthelocalandimprovedgroundnutvarietiesthatarecultivatedinMalawi.Thisgeneticfingerprinting
technique will assist both breeders and seed regulatory agencies in ensuring that seed quality standards are
maintained in the Malawi Seed Alliance and through maintenance breeding.