(O ' trnf\ll t"",\,·vt. Ui~Tt\"!N HtJI"'U\A FINAL REPORT 
CO((ABORATIVE PROJECT CIAT-IBPGR 
~ POLYMORPHISM ANO 
DNA FINGERPRINTING OF CASSAVA 
PROJECT INITIATION DATE: MARCH 1990 
PROJECT ENDING DATE: MARCH 1991 
CARRIED OUT IN: BIOTECHNOLOGY RESEARCH UNIT. CIAT , 
" 
'/ 
REPORT PREPARED BY: FERNANDO ANGEL 
DIANA L ARIAS 
WILLlAM M. ROCA 
1461 I 
J 8 FEB. 1991t 
BIOTECHNOLOGY RESEARCH UNIT 
CIAT. Cali. Colombia 
, " 
INTRODUCTION 
When genomic DNAs from two genetically distinct índíviduals are digested with 
a restriction enzyme, electrophoresed, blotted onto a membrane, and probad with a 
radioactively-Iaballed DNA clone, polymorphisms in the hybridization pattems 
sometimes result due to sequence differences batween the individuals. The 
development of restriction fragment length polymorphism (RFLP) technology has 
opened a door to detecting, monitoring and manipulating genetic variation in plants in 
a manner not previously possible (1). One 01 the most ready application 01 this 
technology is in assessing genetic variation in natural populations and exploring the 
evolutionary relationships among plam taxa (2). 
DNA 1ingerpriming techniques are currently being usad as a complement fer 
isozyme characterization and to construct saturatad genetic maps in many CroPS (3-6), 
mainly because it can cover an extended portion of the plant geno me. There are 
several advamages 01 using DNA techniques to measure and monitor genetic variation. 
The ability to score DNA markers is largely unaffected by the envlronment (including 
seasonal fluctuations and geographical origín) and stage of development, problems 
often encountered with morphologícal and even protein markers. In addítion DNA 
techniques can detect variatíon in noncoding as well as in coding reglons 01 the 
genome. 
"DNA Fingerprims' have been reportad in a wide array ot organisms. A major 
advance has been the discovery 01 minlsatellite DNA sequences present in the human 
genome that are hypervarlable in many species ot animals and plants (7, 8). Recently, 
It was tound that wild-type M13 bacteriophage also detect hypervariable minisatellites 
in human, animal and plant DNA (9). 
2 
In this study we usad ONA taehniques to estimate tha leval of ONA variation in 
3 varieties of cassava belonging lo P-IVAG project by using different probes which 
haya been clonad at Ihe BRU. Smallest Cla IIBsm I fragment of the M13 bacteriophage 
including two regions wlth repeats capable to reveal "ONA fingerprints" in sorne 
angiosperms speeies (10) was also usad in this study. 
RESEARCH PROCEDURES 
DNA Extractlon 
Isolation of nuclei from M Col 22 cultivar was earriad out as described previously 
(11). Approximately 10 g of green leaf tissue was quickly frozen in liquid nilrogen and 
grinded to a fine powder in a mortar, then added to 20 mi of a chillad cel! Iysis and 
neutralization buffer (CLAN) composad of 50 mM Tris-HCI pH 7,8, 5 mM MgCI2, 100 
mM KCI, 0,5% PVP-4Q (polyvinyl-pyrrolidone), 10 mM 2-Mercaptoethanol and 0,15% 
Nonidet P-40. The mixture was agitated on a magnetie stirer plate for 3-1 O min al 4°C 
to allow the buffer to thaw, then fíltered through two layers eaeh of cheesecloth and 
miracloth to remove larga debris. The filtrate was centrifugad at 1000 x 9 for 10 min 
at 4°C and the supernatant solution was discardad. The pellet was rasuspendad in 10 
mI of CLAN by sevaral passages through a 10 mI. (2mm diametar tip) plastie pipe!. The 
voluma was brought to 50 mi with preehillad corn salina buffer (CSB) containing 50mM 
tris-HCI pH 7,8, 5mM MgC~, 100 mM KCI and 1mM 2-Marcaptoathanol; and 
centrifuged at 100 xg for 5 min at 4°C. Nuclei ware observad by light mieroscopy. 
Nuclear and genomic ONA isolation proceduras wara similar to that dascribed by 
Dellaporta et al (12). 
3 
Genomlc Ubrary Constructlon 
Nuclear DNA was dividad into 5 fractions. Each fraction was digestad with one 
01 the following restriction enzymes (Pst 1, Eco RI, Bam HI, Xba 1, Hind 111). Digested 
DNA was ligatad into pUC19 plasmid (13). DH5-alpha bacterial cells were then 
transformad with ligatad plasmíd. Colonies 01 cells containing plasmids with cassava 
inserts were selected basad on X-gal and IPTG screening procedures (14). Individual 
colonies were isolatad and plasmid mini-preps were preparad (15). Insert sizes were 
determinad relative to BstEII-digestad lambda on 1.00/0 agarose gels. 
Restrlctlon dlgests, electrophoresis and Southem analysls 
Three cassava cultivars (M Col 22. M Col 1505, CM507-37) from the P-IVAG 
project were usad in this work. DNA from each cultivar was digestad with the lollowing 
10 enzymes (Bam HI, Eco RI, Hind 111, Pst l. Xba 1, Hae 111, Eco RV, Dra 1, Taq I and 
Hpa 11). Spermidine (4 mM) was added to digests to pro mote complete digestion. 
Electrophoresis of plant DNA (3 ug DNA per lane). Southern blotting and hybridization 
were carried out as describe<! by Southern (16). Whole plasmids including cassava 
inserts batween 0,2 Kb and 3.0 Kb were hexamer labeled with 32p-dATP to high 
specífic activities (1-10 x 108 cpm/ug) (17)and usad as probas on filters 01 cassava 
DNA. Filters were washed at 65°C for 30 min each at 2 x SSC, 1 x SSC and 0,5 x 
SSC (all washes containad 0,1 % SDS) and exposed to X-ray film with intensifier 
screens. 
When M13 bacteriophage was used as proba. DNA Irom each cultivar was 
digested with Hae 111 enzyme. Ten micrograms of digested DNA was electrophorosed 
in 25-cm long 1% agarose gels. JM 105 cells were infected with M13 bacteriophage 
and 500 ug 01 DNA were extracted and purified with polyenthilenglycol as describe<! 
(18). 
4 
Cla IIBsm I fragment (Fig. 1) was eluted Irom the gel and hexamer labalad with 
32p-dATP as aboye. Filters were hybridized in 7% SOS, 1 mM EOTA (pH 8.0), 0.2631'.1 
N~HP04 and 1% bovine serum albumin (Iraction V); and washed twica each for 15 
min. in 2XSSC, 0.1 % SOS at room temperature followad by one 60 min. wash in the 
same solution at 50°C (19). Filters were exposad 10r one day with intensifier screens. 
DNA Sequencing 
Oideoxy method described by Sanger et al., (20) using bacteriophage T7 ONA 
polymerase was used. 
RAPD 
Random amplifiad polymorphism ONA technique recently described (21,22) was 
used. The primer E11 (GAGTCTCAGG) was obtained from OPERON. Amplification 
reactions were performad in volumes of 25 ¡ll containing 10mM Tris-HCI, pH 8.3, 50 
mM KCI, 2mM MgCI2, 0.001% gelatin, 100 ¡lM each 01 dATP, dCTP, dGTP and dTTP, 
0.2¡lM primer, 25 ng 01 genomic ONA and 0,5 unit 01 Taq ONA polymerase. 
Amplification was performed in a thermal cycler programmed for 45 cycles 011 min at 
94°C, 1 min at 36°, 2 min at 72°. Amplilication products were analyzed by 
electrophoresis in 1,5% agarose gels and detected by staining with ethidium bromide. 
RESULTS 
Digestlon with restrlctlon enzymes 
ONA isolated from green leal tissue was digested with ten restriction enzymes. 
The majority 01 the ONA remains high molecular weight when digested with methylation 
- sensitive enzymes (Mspl, Apal, Pst 1) (Figure 2, lanas m, o, p). These enzymes are 
5 
sensitive to cytosine methylation in the S' locatíon (23). Comparative digests with C-
methylation - insensitive enzymes including Eco RI, Eco RV, Taq l. Hinf I shows a 
distribution of 1ragment sizes between 0,2 and 10 Kb (Figure 2, lanes d. f, 1, n). In all 
cases, cassava DNA was cut more effec1ively by C-methylation - ¡nsensitive enzymes 
than was by C-methylation-sensitive enzymes. A comparison of cutting efficiencies 01 
C-methylation-sensitive and insensitive restriction enzymes would suggest that cassava 
DNA is highly methylatad. 
The role 01 DNA methylation in eucaryotes and especially plants is poorly 
understood (24). There is evidence that methylation plays a role in gene regulatíon but 
there are many examples 01 a negative correlation between methylation and gene 
expression (25). DNA methylation in cassava must be studiad in detall. 
Clonlng of DNA In pUC19 vector 
Digestad DNA from M Col 22 cultivar 1rom the P-IVAG projec1 was clonad in 
polylinker site of pUC19 plasmid. Five kinds of fragmenta; Pst 1, Bam HI. Xba 1, Eco 
RI and Hind 111 fragmenta, were cloned. After transformation of bacterial cells, the' 
colonies 01 cells containing plaamids with cassava inserts were selectad based on X-
gal hydrolysis by B-galactosidase synthesis in LB medium supplementad with 
ampicillin. White colonies containing recombinant plasmids (Figure 3) were isolatad. 
grown in LB medium and sto red at -20°C in 30% glycerol until required. 
Slze of Inserts 
200 white colonies were grown in LB madium for twelve hours and plasmids 
mini-preps were prepared. Purified plasmids were digested in order to separate the 
insert cloned 1rom the plasmid. The inserts were separated by elec1rophoresis on 1,0% 
agarose gels and their sizes were determinad relative to BstE II-digested lambda. 
6 
Fiva kínd 01 inserts ware obtainad, betwaen 0,2 Kb and 7 Kb (Figure 4), which is the 
optimal size range for detecting polymorphic fragments and to conduct fingerprinting 
analysis. 
Dot blot Hybrldlzatlon 
Genomic clones were preselectad 10r low and repetitiva copy numbar sequences 
by hybridizing with 32P_labellad total genomic DNA. Results 01 this hybridízation are 
shown in Figure 5 where differences in tha hybridization intansity are notable. Strongly 
hibridizing clones were omitted from further screening (clonas 14, 24) and the 
remaining clones were considerad to reprasanl either a single or low copy sequence 
and usad for RFLP studies. 
Southern blot analysis 
The ONA from three cassava lines cultivars M Col 22, M Col 1505 and CM 507-
37, was individually digestad with each of the following restrictíon endonucleasas: Bam 
HI, Ora 1, Eco RI, Eco RV, Hae 111, Hind 111, Hpa 11, Pst 1, Taq 1, Xba 1. Oigestions 
procaadad for over 16 hours lo ansura completion. ONA was depurinated (0,25 M HCI, 
15 mín), denalured (1 ,5 M NaOH - 1',5 M NaCI) and transfarred from Ihe gel onto nylon 
membrana. Whola plasmids including cassava inserts were labalad and hybridizad to 
Iha nylon filters. 
Hind 111, Pst 1, and Xba I probas showed similar ability lo datact polymorphism 
between three cultivars studiad here whereas in Eco RI and Bam HI pro bes this abílity 
was low. When M Col 22 was comparad wilh both M Col 1505 and CM 507-37 
cultivars, the level of polymorphism reached 60% but it was batween 5% and 30% 
when M Col 1505 was comparad with CM 507-37 (Tabla 1). 
7 
• 
Tables 2 to 7 compare the ability of ten different restriction enzymes (seven were six 
cutters and three were four cutters) to detect polymorphism. 
Two enzymas Eco RI and Eco RV displayed high level of polymorphism when 
both Hind 111 and Xba I probas were usad (Tablas 4 and 6). However, when Pst I 
probas were usad Haa 111 restriction enzyme detectad 35% of polymorphism vs 30% 
and 10% displayad by Eco RI and Eco RV respectively (Table 5). 
Ora I and Hpa 11 did nol detect polymorphism between M 0011505 and CM507-
37. Further, polymorphism displayed by these two enzymes between M Col 22 and the 
other two cultivars was very low. Higher polymorphism between M Col 22 and both M 
Col 1505 and CMS07-37 was detected by Eco RV enzyme when Hind 111 probes were 
usad (Table 4). Polymorphism between M Col 1505 and CM507-37 was low in all 
cases. However, Eco RV and Eco RI enzymes also detected higher level of 
polymorphism batween these two cultivars (Table 7). 
Table 1. 
Probe 
Pst I 
Xba I 
Hind 111 
Eco RI 
Bam HI 
Source of probes compared in their ability to detect polymorphism al 
least wilh one rastriction enzyme. 
M Col 1505/M Col 22 M Col 1505/CM507-37 M Col 22/CM507-37 
60 5 60 
60 20 55 
55 30 55 
40 20 40 
30 5 30 
8 
Table 2. Percent polymorphism with Bam HI Probas. 
Bam HI M Col 1505/M Col 22 M Col 1505/CM507-37 M Col 221CM507-37 
Probes 
Bam HI 30 10 30 
Ora I 
Eco RI 35 10 35 
Eco RV 10 10 
Hind 111 
Hae 111 
Hpall 
Pst I 
Taq I 10 10 
Xbal 
Table 3. Percent polymorphism with Eco RI probes. 
Eco RI M Col 1505/M Col 22 M Col 1505/CM507-37 M Col 22/CM507-37 
Probas 
Bam HI 20 20 
Ora I 
. Eco RI 10 10 
Eco RV 20 10 10 
Hind 111 30 10 30 
Hae 111 20 10 10 
Hpall 
Pst I 30 30 
Taq I 10 10 
Xba I 20 10 10 
9 
Table 4. Percent polymorphism with Hind 111 pro bes. 
Hind 111 M Col 1505/M Col 22 M Col 1505/CM507-37 M Col 221CM507-37 
Probes 
Bam HI 20 20 
Dra I 5 5 
Eco RI 45 20 45 
Eco RV 55 30 50 
Hind 111 5 5 
Hae 111 5 5 
Hpall 5 5 
Pst I 35 5 20 
Taq I 5 5 
Xba I 35 5 20 
Table 5. Percent polymorphism with Pst I probes. 
Pst I M Col 1505/M Col 22 M Col 1505/CM507 -37 M Col 221CM507-37 
Probas 
Bam HI 20 20 
Dra I 10 10 
Eco RI 30 30 
Eco RV 10 10 
Hind III 30 30 
Hae 111 35 30 
Hpall 
Pst I 20 20 
Taq I 
Xba I 10 5 
10 
Tabla 6. Parcant polymorphism with Xba I probas. 
Xba I M Col 1505IM Col 22 M Col 1505/CM507-37 M Col 221CM507-37 
Probas 
Bam HI 20 20 
Ora I 
Eco RI 50 10 45 
Eco RV 45 10 45 
Hind 111 35 5 35 
Haa 111 15 5 10 
Hpall 
Pst I 15 15 
Taq I 5 10 5 
Xba I 15 10 10 
Tabla 7. Pereent polymorphism with five kind of probas. 
Probas M Col 15051M Col 22 M Col 1505/CM507-37 M Col 221CM507-3.7 
Bam HI 22 2 22 
Ora I 3 3 
Eco RI 34 8 34 
Eco RV 29 10 25 
Hind m 20 3 20 
Hae 111 15 3 11 
Hpall 1 1 
Pst I 20 1 17 
Taq I 6 2 6 
Xba I 16 6 8 
11 
Different patterns of polymorphism wera detectad with our genomic clones. A 
Pst I clone, P21, detected polymorphism in M Col 22 when either Eco Al or Hind 111 
restriction enzymes were used (Figure 6), bU1 this clone did not discriminate between 
M Col 1505 and CM507-37. This clone can ba classified as single copy sequence 
because only one or two bands were present, and suggests that polymorphism is due 
to base substitU1ion. M Col 22 cultivar has an additional cleavage site inside both the 
Eco Al and Hind 111 fragments that reduce those fragments in size and produce another 
smaller fragment (Figure 6). 
A Xba I clone X14, detected polymorphism in M Col 22 when the three cassava 
cultivars were digested with Pst I restriction enzyme (Figure 7), but it was not able to 
discriminate between CM 507-35 and M Col 1505 cultivars (Figure 7). Similar to P21 
clone, polymorphism detected by X14 clone is due to an additional Pst I site in M Col 
22. M Col 22 shows an additional cleavage site in the 4,5 Kb fragment of CM507-35 
and M Col 1505, that reduces this fragment in size and produces a 1,0 Kb fragment 
in M Col 22 (Figure 7). 
On the other hand. a Bam HI clone named 85. and a Xba I clone X6 detected 
another kind of polymorphism in M Col 22. When those clones were used as probas. 
additiQnal bands were observed in M Col 22 when its hybridization pattern was 
compared to those showed by CM507-37 and M Col 1505 cultivars. The B5 probe 
revealed two additional bands in M Col 22 when the three cultivars were digested with 
Eco AV restriction enzyme (Figure 8. left arrows). The X6 probe showed a similar 
polymorphism to that displayed by B5 probe but only a 0.5 Kb additional fragment was 
detected in M Col 22 when digested with Eco Al enzyme (Figure 9). These results 
suggest the presance of a unique sequence present in M Col 22 but absent in CM507-
37 and M Col 1505. This polymorphism is probably caused by an insertion in M Col 
22. This point was clarified recently when we found a Pst I clone, P12. which 
hybridizes with DNA from M Col 22 but does not with DNA from other cultivars. 
12 
The&e results indicated that P12 insert contains a specific sequence present only 
in sorne cultivars (Fig. 10, Table 8). This fragment may be classified as "single dose 
restriction fragmentO recently described (26). 
DNA sequenclng 
P12 was sequenced partially. Two hundred eighty eight nucleotides were 
sequenced directly in pUC19 by using universal primers. 
109 nucleotides and 179 were sequenced from the universal and reverse primers 
respectively. 
UNIVERSAL SEQUENCING PRIMER 
10 20 30 40 50 
5' CTTCTAGAGC GGACNGTGCT GCCCCTTCTT TCTAGACTTT GATGAAAGTT 
3' GAAGATCTCG CCTGNCACGA CGGGGAAGAA AGATCTGAAA CTACTTTCAA ' 
60 70 80 90 100 
GCTTGCGAAG TGCAATATGA TGATCGTAAA GACCATTTAA TCAAGCGCTG 
CGAACGCTTC ACGTTATACT ACTAGCATTT CTGGTAAATT AGTTCGCGAC 
ATCTAATA3' 
TAGATTAT 5' 
REVERSE SEQUENCING PRIMER 
10 20 30 40 50 
5' TGCAGGGAAA CTTACCTTAC CTTACCTACC TGGCTTGACA AAAGTAAATA 
3' ACGTCCCTTT GAATGGAATG GAATGGATGG ACCGAACTGT TTTCATTTAT 
60 70 80 90 100 
TGTCAAGTTT TGTGCATTCC ATCGAAATAC GTCATGAAAC CAACTCATGT 
ACAGTTCAAA ACACGTAAGG TAGCTTTATG CAGTACTTTG GTTGAGTACA 
110 120 130 140 150 
TGTGCCACTC CAGGACATCA TTGAAATTTA GATCAAGTCT GGAGAGATTA 
ACACGGTGAG GTCCTGTAGT AACTTTAAAT CTAGTTCAGA CCTCTCTAAT 
160 170 
ACTTCGTCGT CAAAGTCAAC AACTCATCA 3' 
TGAAGCAGCA GTTTCAGTTG TTGAGTAGT 5' 
13 
M13 • Cla/Bsm I Probe 
By probing Southem, transfers of endonuclease digested and gel fractionated 
plant ONA axtracts (íncluding the nuclear, chloroplast and mitochondrial components) 
wíth the smallest Cla IIBsm I fragment 01 the M13 bacteriophage, it has been 
demonstrated that it reveals minisatallites bearing endonucleasa fragments in 
gymnosperms and angiosperms. 
We isolated and used as proba this 0,781 Kb fragment (Fig. 1) in order to reveal nONA 
fingerprint" of cassava. The filters were hybridized and washed as describad abova. 
Four cultivars from Colombia (M Col 22, M Col 21, M Col 1505, M Col 638), five 
from Brazil (Br 885, Br 348, Br 12, Br 715, Br 534), two from Venezuela (Ven 331, Ven 
82), two hybrids (CM507-37, CM91-3), one from Argentina (Arg 13) and one from 
Thailandia (Thai-1) were discriminated by this probe (Fig. 11). The origin of each 
cultivar is indicated (Table 8). This probe discriminated between 15 different cultivars 
used here, indicating that M13 is a good probe to carry out nONA fingerprinting" studies 
in cassava. 
Ribosomal gene-specific probes 
Three ribosomal gene-specific probes from soybaan (27), were also used in this 
study. pGmr1, pGmr3 and pXBr1 which include 100%, 50% and 5% 01 a single 
soybean rONA repeat, respectively (Figure 12). 
Seven different cultivars were digested with either Hae 111 or Taq I restriction 
enzymes and hybridized with these three ribosomal probas. When Taql was used 
pGmr3 and pGmr-1 probas displayed some additional bands in M Br 348, M Arg 13, 
M Ven 331, M Ven 82 and M Col 22. However, pattems were very similar (Fig. 13, 
14 
14). No differences were found when Hae 111 digests were hybridized with these 
probes (Fig. 13,14). pXBr1 probe did not detad polymorphism (results not shown). 
RAPD 
Two cultivars M Col 1505 and M Col 22 and one wild Manihot species, M. 
aesculifolia were assayed for RAPO technique using a primer whose content in G-C 
was 60%. Conditions described in research procedures allowed us to discriminate 
among three genotypes (Figure 15) indicating the feasibility of this technique to carry 
out ONA fingerprinting studies. 
. , 
15 
Table 8. 
Cultivar Origin P12 
Brazil 715 South Br + 
Brazil 121 Northeastern Br + 
Brazil534 Northeastern Br + 
Brazil 160 Northeastern Br 
Brazil885 Northeastern Br 
Brazil 191 Northeastern Br 
Brazil348 Amazonas Br 
Brazil502 Amazonas Br 
Brazil 5 NO 
Brazil 12 NO 
Brazil309 NO 
Ecuador 117 Ecuador + 
Ecuador 10 Ecuador + 
Ecuador 166 Ecuador + 
Peru 213 Peru 
Peru 328 Peru 
Venezuela 82 Orinoco 
Venezuela 331 Orinoco 
Argentina 13 Argentina 
Col 21 North + 
Col 22 North + 
Col 1505 NO 
CM 507-37 CIAT hybrid 
Col 638 Llanos + 
Thailandia 1 Thai + 
Thailandia 8 Thai 
Nigeria 5 Nig 
4955-11 Thai-1 x BrS + 
4955-12 Thai-1 x Br5 + 
*Chartaginensis North of Colombia + 
*Grahami Paraguay + 
* Aesculifolia Mexico + 
N.O. Not determined 
* Wild Manihot species 
16 
CONCLUSIONS 
1. Cassava genomic ONA ramainad high in its molacular waight when digested 
with restriction anzymes sensitiva to cytosine methylation in the 5' loestion, 
suggasting that cassava ONA is highly mathylatad. 
2. Pst I and Xba I probas were the best probas to discriminate between M Col 22 
and both CM507-37 and M Col 1505 cultivars. 
3. Hind 111 were the bast probes to discriminate between M Col 1505 and CM507-
37 cultivars. 
4. The best combination probe/enzyme to discriminate between M Col 22, M Col 
1505 and CM507-37 cultivars was Hind IIIIEco RV. 
5. Eco RI and Eco RV restriction enzymes displayed highest polymorphism among 
the three cultivars tested. 
6. Ora 1, Hpa 11 and Taq I restriction anzymas displayed lowast polymorphism 
among the thraa cultivars studied. 
7. The smallest Cla 1/8sm I fragment of the M13 bacteriophage was able to 
discriminate eleven cultivars from different geographical origins indicating that 
this fragment is a good probe to carry out "ONA fingerprinting" studies in 
cassava. 
8. Three ribosomal gene-specific probes from soybean were not good to 
discriminate between different cassava genotypes. 
17 
9. Three different genotypes were easily discriminated by using the RAPD 
technique, suggesting that this technique will be very useful in DNA 
fingerprinting studies of cassava for germplasm characterization. 
10. A fragment callad P12 was only present in fifleen genotypes out of thirty two 
genotypes testad. This fragment could be classified as a "single dose, 
restriction fragment", recently described. 
ACKNOWLEDGMENTS 
We thank to International Board for Plant Genetic Resources IBPGR and its 
Regional Office locatad at CIAT for their financial support to undertake this project to 
a successful point. 
FA was a fellowship from IBPGR (March 1990 - February 1991). 
18 
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restriction fragments. Theor. AppL Genet in press. 
27. Zimmer, EA; Jupe, E.A.; Walbot, V. (1988). Ribosomal gene structure, 
variation and inheritance in Maize and its ancestors. Genetics 120:1125-1136. 
28. Appels, A.; Honeycutt, A.L (1986). rDNA: evolution over a billion years. pp. 81-
135. In: DNA Systematics, Edited by S.K. Dutta. CRG Press, Boca Raton, Fla. 
22 
6529 Finl 
6508 M,tl! I 
6493 G. 
6468 H!lIEII~\ 
6431 agn \ \\ 6425F.P'~ 
6405 Pvul--
6293 Gdill_ JaeZ' 
Polycloolng Ille __ 
6036 Gdl1l 
6001 Ah.U. NBt1':::::::: 
5914 Av"U __ 
5625 A.a' __ 
5716 D/lIII,,-,,--
5643 BanlJ .... " 
5613 Naa'. Cfr' ~ 
6882 elsl 
~$UI 6899 \ Sgll'6935 ,\ I 7000 Xmn' 357 ~==~ u ~ 
Bacterlophage 
M13mp18/ M13mp19 
(7.25 kb) 
4000 
1000 
Sn.B'1266 
s.m"
746 l 
2000 
<4---J 
Figure 1. Restriction map of bacteriophage M13 vector. Smallest ela IIBsm I 
fragment from 1746 to 2527 was alutad from tha gel and used as proba. 
23 
Figure 2. 
abcdetgh 
• 
ONA from cassava laaves cleaved with difterent restriction enzymes. 
Molecular weights are in Kilobases. 
a: 
b: 
c: 
d: 
e: 
f: 
g: 
h: 
undigested 
Hae 111 
Hind 111 
Eco RI 
Lambda-BstE 11 
Eco RV 
891 11 
Ora 1 
i: Lambda - Pst I 
j: Barn HI 
k: Bam HI 
1: Taq I 
m: Msp l· 
n: Hinf 1 
o: Apa 1-
p: Pst 1* 
• 5'-C-methylation sensitive restriction enzymes. 
24 
Figure 3. Selection of recombinant plasmids based on X-gal and IPTG screaning 
procedures. White colonias (Bgal-) containing racombinant plasmids 
were ¡solated and its ínsart characterizad. 
25 
Figure 4. 
kb 
5,0 ..... 
2.7 -
0,7 ...... 
0,2-
_ pUC19 
Electrophoresis on 1% agarose gel 01 inserts afier digestion. Size 01 
inserts were between 0,2 Kb and 7 Kb. Molecularweights are expressed 
in Kilobases. 
26 
Figure 5. 
, ?~ < • 
4r 
... 
., 
" 
'2- 2., ,q 
• • 
<l • 
.. 
I ~ 
," 7-
• • ,., ,.. n " ? 
• r 
~. 
• • • 
• ... J ..... . 
Dot blot hybridization of recombinant plasmids with 32P-labelled genomic 
DNA. Ten nanograms of each plasmid were usad in this experiment. 
Single and low copy sequences were selected according to hybridization 
intensity. 
27 
Figure 6. 
Bam H l l Ora llEco RIIEcoRVIHae IlIlHindlll1 Hpa IIIPst 1I Taq 1 I Xba 1 
kb 
10- '" 4,5 , 
..... 
ttt 
ABe 
-
...-
-
"P-
• 5IL IId.4Sl 
..... 
-
Variation at DNA level detected with proba P21. Southem blots were 
axposed by 2 days at -70°C with intensifier screens. A"" M Col 22; B = 
M Col 1505; C '" CM507-37. Restriction endonucleases Eco RI and Hind 
111 detectad DNA variation in M Col 22. Restriction endonucleases used 
to digast genomic DNAs are shown (Iisted aboye). 
28 
Figure 7. 
).,EcoRI,EcoRV,Haelll Taql ,Xbal ,)... 
kb 
kb 
-...--4,5 
~ , ' . 
t t t 
ABe 
Variation at DNA level detected with probe X14. A = CM507-37; B"" M 
Col 1505; C '" M Col 22. Restriction endonuclease Pst I detected DNA 
variation in M Col 22 cultivar. 
29 
Figure 8. 
... 
_ "t .••• 
- l;' 
t t t 
ABe 
'. 
-
tdentification of additional bands in M Col 22 (Iaft arrows) with proba B5 
when DNA was digested with Eco RV enzyme. A = CM507-37; 8 = M 
Col 1505; C = M Col 22. 
30 
Figure 9. 
kb IAIECORlliílHaellll",:q,I,I,Xb~1 lA 
4,3- .:: ~ ." !!'. 
... PI :¡ 
2,0- -
.1 
••• 
--- ---
-0,5-
-
.. _-
t t t 
ABe 
Identification of an additional band (O,5Kb) in M Col 22 with proba X6 
whan DNA was digested with Eco RI anzyme. A = CM507·37; B = M Col 
1505; C = M Col 22. 
31 
AH(:[)~!'GlltJ 
• 
- - . -
•. 11 ..... 
t:..-o 11'1 
"'" 1 
Figure 1 O. Identification of a specific sequence with probe P12. A = M Col 21; B = 
M Bras 191; C = M Sras 534; O = CM507·37; E = M Col 22; F = M Col 
1505; G = M Grahami; H = M. Chartaginensis; 1 = M. Peru 328; J = M. 
Ecuador 117. Hybridization was detected in six of ten cultivars testad 
with any of the three restriction endonucleases. Restriction 
endonucleases used to digest genomic ONAs are shown (listad below). 
32 
a bcd e f g h i j k. 1. Il (1 
Figure 11. Fingerprinting of different cassava cultivars digested with Hae 111 and 
hybridized with smallest Cla 1/8sm I frag ment 01 the M13 bacteriophage. 
a: M Ven 331; b: M Ven 82; e: M Br885; 
d: M Br 348; e: M Br 12; 1: M Col 1505; 
g: CM91-3; h: CM507-37 i: M Col 638; 
j: M Co121; k: M Col 22; 1: M Br 534; 
m: M Br715; n: M Arg 13; o: M Thai 1 
33 
1&S 11$ uS IGS 'IS ITS nS IGS t.S iTS uS IGS 
.. I _ti xa , 'L~_UI"·. __ t"'~ .~ 1m1'lR5lt~_=é;& 13 .... ~
I 
O 
.XfM 
"G.. , 
...... 
1-
~ 
I 
10 
• CQdin{j Rt;¡lon 
n 
E'l'".07_7 /7Z<27ZZZ1 
I I 
1$ ~O 
KilOblJ'U Pau$ 
- Notlt:!::!lng Rr\jl;m 
I 
2~ 
I 
30 
Figure 12. A typical array 01 plant ribosomal DNA. Taken trom Zimmer el al. (27). 
This figure depicts Ihe general features 01 the sequences that code lar 
the large cytoplasmic ribosomal ANAs 01 higher plants. Aibosomal 
repeat units 01 8-12 kbp axist in the genoma in tandem arrays; each 
repeat consists 01 coding and noncoding regions (28). Indicated on the 
figure are diagnostic restriction endonuciease cleavage sites (X= Xba 1; 
B= Bam HI; E= Eco Al). Tha Bam HI site in the 268 coding region in 
plant ribosomal gene arrays is not susceptible to cleavage in al! arrays. 
Abbreviations on the figure denote the following: 188 = 18S rRNA coding 
region; IT8= internal transcribed spacer; 268= 268 rANA coding region; 
IGS= intergenic spacer. The position 01 the cloned probes (pxBrl. 
pGmr3. pGmrl) used lor sequential probing is shown below the repeat 
unit. 
34 
• 
, 
t 
• 
-
abrdcfg hl 
.. ., 
• 
-. 
I 
' ... 
i k I m n 
Figure 13. Hybridization with pGmr-1 probe 01 different cassava cultivars digested 
with aither Hae 111 (lanas a-g) or Taq I (Ianes han). 
a,h = M Br 348 
b,í = M Br 885 
c,j = M Arg 13 
d,k = M Ven 331 
e,1 = M Ven 82 
f,m = M Ec 10 
g,n = M Col 22 
35 
• 
t 
J 
, ... 
Figura 14. Hybridization with pGmr-3 proba 01 diffarant cassava cultivars digastad 
wíth aither Taq I (Ianes a-g) or Haa 111 (lanas h-n). 
a,h = M Sr 348 
b,í = M Sr 885 
c,j = M Arg 13 
d,k = M Van 331 
a,l = M Van 82 
l.m = M Ec 10 
g.n = M COl 22 
36 
a b e 
Figure 15. Amplification 01 cassava DNA. DNA was amplified from thrae different 
genotypes using a primer of arbítrary nucleotide sequence. Amplification 
products were resolved by electrophoresis in a 1,5% agarose gel which 
was stained with ethidium bromide and photographed. 
a: M Col 1505; 
b: M Col 22; 
e: M. aesculifolia 
37