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CCLI Genetics Module

Characterization of Puerto Rican Cassava


Summary
In this research-based laboratory module you will study the Genetic Diversity present in the cassava found in
Puerto Rico. To achieve this we will collect samples from around the island, extract its DNA and use a
molecular technique known as SSR (simple Sequence Repeats) markers for the diversity analysis. SSR markers,
also known as microsatellites, are co-dominant markers that have gained popularity in recent years due to
their ease of use. This lab module will be a multi-week exercise and is aimed at contributing to the .

Introduction to Cassava
Historically, cassava, Manihot esculenta Crantz, has played a unique role on the worlds agricultural stage.
This starchy root is grown in most tropical countries and is a mainstay of some of the most hard-pressed
populations of the world. More than half a billion people living in developing countries depends on its root for
their food and livelihood. The Consultative Group on International Agricultural Research (CGIAR) system has
named cassava the most important food crop in sub-Saharan Africa, where the average consumption exceeds
300 kg per person per year (Mba et al., 2001; Fregene et al., 2001, 2003). By the year 2050, 90% of
humankind will live in developing countries, where agriculture remains the most important economic activity
(Raven et al., 2006). Cassava offers an important opportunity to solve the problem of the calorie
requirements and food security in those areas. Genetically, cassava remains the least understood of any of the
major staple crops (including rice, maize, wheat, and potatoes) that feed mankind. This discrepancy is due to
the heterozygous nature of the crop, its long growing cycle of 9-18 months, its low seed yield per pollination
and the limited funding for research on this crop.
Molecular Markers
In genetics, a molecular marker is a fragment of DNA sequence that is associated to a part of the genome.
Molecular markers are used in molecular biology and biotechnology experiments where they are used to
identify a particular sequence of DNA. As the DNA sequences are very highly specific, they can be identified
with the help of the known molecular markers.
DNA-based molecular markers such as restriction fragment length polymorphisms (RFLP), random amplified
polymorphic DNA (RAPD) and simple sequence repeats (SSR) were used in the development of a cassava
genetic map (Fregene et al., 1997). These molecular markers and others (amplified fragment length
polymorphisms, cDNA RFLPs) have been used to assess the genetic variability of small sets of cassava
germplasm and to establish relationships between cassava and its wild relatives. In a crop where cultivars
have generally been selected and distributed by the farmers themselves and where different cultivars can
often possess the same name (in addition to the same variety bearing more than one name), an objective
method of identification, classification and measurement of genetic diversity is required (Beeching et al.,
1993; Chavarriaga-Aguirre et al., 1998, 1999; Haysom et al., 1994). To achieve this goal, the assessment of the
genetic variability of germplasm collections requires highly polymorphic markers as well as high-throughput
genotyping systems.
SSR markers are particularly attractive to study the genetic variability because they are abundant and widely
distributed in plant genomes. SSR markers also show high levels of polymorphism based on the number of
repeat units found for each locus in any given population, thus making them adaptable to automation
(Morgante & Olivieri, 1993; Fregene et al., 2003).
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CCLI Genetics Module

SSR Markers / Microsatellites


Eukaryote genomes are densely interspersed with simple sequences which consist of stretches of tandemly
repeated nucleotide motifs that can be 1 to 10 nucleotides (but typically 2 to 3 nucleotides). Numbers of the
repeated units vary widely among organisms to as high as 50 copies (figure 2). When such regions are
individually amplified by means of the Polymerase Chain Reaction (PCR), using a pair of flanking unique
oligonucleotides as primers(figure 1), they almost invariably show extensive polymorphism due to site-specific
length variation, which occurs as a consequence of different numbers of repeat units (Morgante and Olivieri,
1993). In eukaryotes one can expect to encounter at least one simple sequence stretch every 10 kb of DNA
sequence. SSR loci are highly variable on account of the number of repeat units found for each locus in any
given population (Morgante and Olivieri, 1993). The high levels of heterozygosity, the PCR-based nature of
these repeat loci and their codominant nature have made SSRs the molecular markers of choice for genetic
mapping and diversity studies (Tautz, 1989; Mba et al., 2001).
Currently, microsatellites have been evaluated for many crop plants, including, sugarbeet, Brassicas, melon,
tomato, pepper, cotton, sorghum, maize, rice, soybean and apple. Furthermore, SSR markers are being
applied to plant mapping projects in many species to saturate the existing genetic maps, for example in wheat,
oat, bean, rice, barley, rye, pearl millet, maize, sorghum, sugarcane, Arabidopsis thaliana, tomato, potato,
hexaploid wheat, soybean, etc (Kumar, 1999).
Figure 1: A diagrammatic representation of the PCR process. 1) Template DNA is denatured by high
temperatures. 2) Primers anneal to template DNA. 3) Thermostable DNA polymerase synthesizes new DNA.
Multiple cycles of the three temperatures result in an exponential increase in the sequence which is amplified
by the PCR process.

CCLI Genetics Module


Figure 2: An example of one SSR marker amplification of 3 genotypes. The variable numbers of CCT repeats at
this locus in the 3 genotypes are amplified by a set of primers. A 3rd primer is used to fluorescently label that
amplicons in order to visualize it. In this example Genotype 1 has the same number of repeats for this locus in
both alleles (which leads to one amplicon). Genotype 2 has two different number of repeats for this locus in
both alleles (which leads to two amplicons), but one repeat in common with genotype 1. Genotype 3 has two
different number of repeats for this locus in both alleles (which leads to two amplicons), but one repeat in
common with genotype 2 and another unique repeat (and thus a unique allele).

Cassava in Puerto Rico


There is a cassava collection at the Corozal and Isabela Agriculture Experimental Station in Puerto Rico
consisting of 26 accessions.
Seven of them were introduced to Tropical Agriculture Research Station (TARS) in Puerto Rico in 1994
from the International Center for Tropical Agriculture (CIAT; Cali, Colombia). Those accessions are: CM523,
CM3064, CM3311, CM3380, CM4484, SG804, SM494.
Seventeen accessions have been in the cassava collections of the Agriculture Experimental Stations of
Puerto Rico (EEA, from its acronym in Spanish) for approximately 26 years. Those accessions are: Abuelo,
Brava, Chilena, Cubana, Forastera, Jamaica18, Llanera, PI12900, PI12902, PI12903, Seda, Senon, Serralls,
Tremesiana, Trinidad 14-56, Amarillo and Valencia.
Two accessions were added in 2007 from the International Center for Tropical Agriculture (Cali,
Colombia): 60444 and Mcol2215

CCLI Genetics Module


Member of the laboratory of Dr. Dimuth Siritunga (Department of Biology, UPRM) has utilized
polymorphic SSR markers to assess the allelic diversity within and relationships between the accessions of this
Puerto Rican cassava germplasm. In this study, 36 polymorphic SSR markers were used successfully. Now the
following questions remain to be answered:
Which of this accession is predominantly grown in Puerto Rico? and where?
What of the other accessions are grown in Puerto Rico? and where?
Are there other accessions grown in Puerto Rico (that are not in the collection)? and where?
If there are other accessions grown in Puerto Rico, what are they?
In this exercise we will use SSR markers on DNA that you will extract from unknown cassava (that you
will collect and bring) to assess the diversity of cassava present in Puerto Rico. At the end you would have
participated in an ongoing research project which uses cutting-edge molecular techniques on a very important
food crop of the world. During this exercise you will learn proper sample collection, DNA extraction,
amplification of DNA using Polymerase Chain Reaction of SSR markers using fluorescent primers and
separation of the amplicons using Poly-Acrylamide Gel Electrophoresisc (PAGE).

Lab Exercise #1
Collection of leaf samples
You will be responsible for collection of the cassava samples and it is anticipated that in most cases you
will need to consult their family, friends and neighbors. Use the SAMPLE COLLECTION FORM provided and
follow the handling instructions to ensure data reliability (see below). The following guidelines will be
enforced for the collection of the samples:
Each sampled cassava plant need to be photographed
Leaf to be sampled should be healthy and must come from the third node from the top (young leaves).
Detached the leaf and put it in a zip-lock bag and keep at 40C (as much as possible) until the experiment
Further data to be collected:
o Local name (if available)
o Planting date (if available)
o Exact geographical location of the cassava plant
o Available agronomical data
o Prominent geographical features (rivers, mountains, rocks, construction, etc.)
o Under and over-growth, weeds

CCLI Genetics Module


Lab Exercise #2
Extraction of DNA
Your group will extract DNA from the leaf material your group brought (or provided by the teaching assistant). You will
require less than 2cmX2cm piece of leaf. Please place the rest of the leaf in the original zip-lock bag and return to the
refrigerator for future use.
Since there are 4-5 groups in your lab section, there should be 4-5 DNA samples (Sample A,B,C,D and E) at the end of
this extraction. Each sample should be labeled appropriately.
DNA extraction protocol for Cassava leaves

Step
1

2
3
4
5
6
7
8
9
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Protocol
Take a half piece of leaf, place in 1.5ml tube. Label the tube with
sample #. Add a tip of sand and grind for no more than 3 minutes
with a blue pestle.
Add 500 l of buffer, grind furthermore for 1 minute.
Add 50 l of SDS 20% and mix by inversion.
Incubate at 65C for 8 minutes in a water bath.
Add 250l of cold (-20C) 5M of Potassium Acetate and mix by
inversion.
Incubate on ice for 5 minutes.
Centrifuge at maxim speed (~13000rpm) for 3 minutes. Transfer the
supernatant to new tube. Label the tube with sample #.
Add 500 l cold isopropanol to the supernatant and mix gently by
inversion.
Incubate the samples for 5 min at on ice
Centrifuge 3 min in maxim (~13,000rpm). Discard supernatant.
Add 700l of 70% Ethanol to the pellet and mix gently.
Centrifuge 3 minutes max speed (~13,000rpm). Discard the
supernatant.
Leave the pellet drying for 5 min.
Add 150 l TE (10:1) and 2.25 l of RNAase to the pellet
Incubate for 5 min at 65C in a water bath
Re-label the tube with sample name and place the tube with DNA
at 4OC

Trouble Shooting

Materials and solutions


Extraction buffer: 10 ml de Tris-HCl 1M, 10 ml of 0.5 M EDTA and 10 ml of 5M NaCl and 70ml of distilled water. Mix by gentle
inversion. Final volume is 100 ml. Then autoclave and add 1g of PVP, mix and store at room temperature until use. Preheat the
buffer at 65C before use.
SDS 20%: Weigh 10g of SDS and add 50mL of distilled water, stirred gently (as it can become foamy).
Potassium Acetate 5M: Weigh 122.68g of potassium acetate and add 250 mL of distilled water. Store at -20C.
Isopropanol: Separate 100mL of 100ml of isopropanol y and keep it at -20C.
Ethanol 70%: Final Volume: 100ml, take 70ml of 95% ethanol and add 30mL of distilled water
TE 10:1: Take 2.5ml of 1M Tris-HCl pH8.0 and 500l of 0.5M EDTA and add 247 ml of distilled water.

CCLI Genetics Module


PCR amplification of cassava DNA
You group will now set up an amplifications of 5 cassava samples !!! EACH GROUP WILL WORK WITH 1
SSR-MARKER.
1 SSRY182
2S SSRY4
3 SSRY103
4 SSRY161
5 SSRY82
Your TA will distribute to each group 3 mixed tubes marked Mix1, Mix 2 and Mix3. In addition your TA
will give your group 4 tubes containing different cassava DNA samples (A, B, C, D).
Mix1
MgCl2: 2.5ul
dNTP: 1.0ul
H2O: 4.5 ul

Mix2
Buffer: 2.5ul
Taq: 1.0ul
H2O: 1.5ul

Mix3
Primer M13: 1ul
SSR primer: 1ul each
TE buffer: 2ul

Each student add 8ul of Mix1 + 5ul of Mix2 + 5ul of Mix3 + 7ul of cassava DNA (total volume is 25ul)
The final PCR reaction contains the following: 10 mM Tris-HCl (pH 8.3), 1 Unit of Taq
polymerase, 2 mM MgCl2, 0.2 mM dNTP mix, 0.5 pmoles of the M13 primer and 100 pmoles of
the SSR primers.

Label the PCR tube with your sample name.

SSR Marker #

Sample #

So now your groups PCR tubes should have a name such 1A, 1B, 1C, 1D or 2A, 2B, 2C, 2D or 3A,
3B, 3C, 3D etc. Please note which tubes are your groups.

Give the 4 PCR tubes to the TA who will subject all tubes from 4-5 groups to the following
amplification cycle:
Cycle

Temperature (C)

Time

1
2
3
4
5

95
94
55
72
34 cycles since
step 2
72
10

5 min
30 sec
1 min
1 min

6
7

5 min
forever

CCLI Genetics Module


Lab Exercise #3
Electrophoresis of PCR samples

Step
1

Protocol
Measure 50 mL of 1X TAE buffer and pour it into a 250 mL flask. Add to it 0.75g of agarose
powder and mix well gently. Microwave the mixture for upto 1 minute until the agarose powder
is fully dissolved. Be careful when handling the hot flask (use an autoclave glove).
Once the mixture has cooled down (5-15 minutes or so) the instructor will add 10 L of ethidium
bromide (10 ug/mL) and mix gently.
The instructor will now set up the gel electrophoresis tray and gently pour the agarose mixture
onto it. Then put the comb in and let the gel solidify. Let the gel solidify at room temperature.
Once the gel has solidified place it in the appropriate chamber in the correct orientation and add
1X TAE buffer until 1-2 mm above the agarose gel. Now carefully take the comb out.
Aliquot 15L of each PCR amplified sample into a new PCR tube and label it the same way. Add
5L of loading dye each this 15L of PCR sample and mix by gently tapping the bottom of the
PCR tube (if you use PCR buffer that includes loading dye then you do not need to add more
laoding dye here).
Add 20 L (of 15 L if no loading dye is added) of your samples to each well in the gel. On either
side of the 8 sample add 10 L of the molecular weight marker. Note which well your sample is
in.
Loading the gel: Expel the air out of the tip and then submerge the tip into your sample
and take out 20 L. Now enter the buffer in the gel chamber directly on top of the well
you want your sample in. Do not expel the sample until you are sure that the tip is
inserted into a well, and do not insert the tip too far into the well (be careful to avoid
puncturing the bottom of the gel with your pipette tip). When you gently expel the
sample into the well you should be able to see the colored solution settling into the well
and filling it.
After the gel is loaded, your instructor will set the voltage and run the gel for approximately 1
hour. The gel should be viewed immediately following electrophoresis.
View the gel on an ultraviolet light box or use a gel documentation system to photograph
the gel. If the gel is viewed directly (rather that viewing an image of the gel) make sure to
wear appropriate plastic eye protection. Since prolonged exposure to UV light may also
harm the skin, students may wish to wear a plastic face shield and appropriate clothing to
cover exposed skin.

CCLI Genetics Module


Student Name:
Sections:

Sample name:

Questions:
1. What were the approximate base pair sizes of bands you see in each lane of your gel?
Lane
1
2
3
4
5
6
7
8

High weight band

Low weight band

2. For your sample how many of the 5 locus amplified are homozygous and how many are heterozygous?

3. What was the percentage of agarose is in your gel?

4. If you want to see brighter bands in the gel briefly state 2 things you can try next time you do this
experiment:
1.

2.

CCLI Genetics Module


Biol3300: Genetics Course
Department of Biology
University of Puerto Rico Mayaguez

FOR THE TEACHING ASSISTANT


Name of TA: _____________________________
Email of TA: _____________________________
Date Received: __________________________
Lab section #: 3300L-_____

CASSAVA SAMPLE COLLECTION FORM (Fall 2009)

Sample #:_____

FOR THE SIRITUNGA LAB

PERSONAL INFORMATION

Official #:

Your Name:____________________________ ID #:_______________________________


Email: ________________________________ Telephone:__________________________
Major: ________________________________ Level: 1st year / 2nd year / 3rd year / 4th year / other

SAMPLE COLLECTION SITE INFORMATION (if available)


Name of land owner:______________________________

Telephone:_________________________

Barrio/Sector:___________________________________

Municipality:_______________________

Coordinates(GPS)________________________________

Date of Collection:___________________

Is this sample from a commercial farm (yes/no) ?__________


Detailed directions to the site and Address: _____________________________________________________
__________________________________________________________________________________________________
__________________________________________________________________________________________________
____________________
Mark the location of collection site clearly on the map in the reverse side.

SAMPLE INFORMATION:
Approx. height of the plant: _________________________ Approx. age of the plant:________________
Number of nodes in the plant:_______________ Which node (from top) is the sample you collected:_______
What is the local name of this variety: ____________________
Did you place the sample is a zip lock bag immediately:_______ Did you place the sample at 4OC: _____

ADDITIONAL COMMENTS (disease, toxicity, taste, long-lasting, where is this cassava from, other stories about this
cassava etc):

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