genetic diversity characterization of cassava cultivars...

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Genetics and Molecular Biology 21,1,105-113 (1998)

Genetic diversity characterization of cassava cultivars (Manihot esculenta Crantz). I) RAPD markers

- _ I Car/os Colombo’, Gerard Second *, Tereza Losada Vale’ and Andre Charrrier

RAPD markers were used to divestigate the genetic diversity of 31 Brazilian cassava clones. The results were compared with the genetic diversityrevealed by botanical descriptors. Both sets of variates revealed identical relationships among the cultivars. Multivariate analysis of genetic similarities placed genotypes destinated for consumption “in nature” in one group, and cultivars useful for flour production in another. Brazil’s abundance of landraces presents a broad dispersion and is consequently an important resource of genetic variability. The botanical descriptors were not able to differentiate thirteen pairs of cultivars compared two-by-two, while only one was not differentiated by RAPD markers. These results showed the power of RAPD markers over botanical descriptors in studying genetic diversity, identifying duplicates, as well as validating, or improving a core collection. The latter is particularly important in this vegetatively propagated crop.

INTRODUCTION

Cassava (Manihot esczileiztii Crantz, Eziphorbin- cem) is one of the world’s most important tropical plants, and is ranked as the fourth source of carbohy- drates in the tropics (FAO, 1995). Unlike many other crops, cassava can be grown with minimal inputs and it is able to produce reasonably well under unfavorable conditions such as low soil fertility, acidic soils or drought. It is a staple crop in various developing nations in Africa, Asia and South America, despite the low attention historically received in research. Cassava is also an industrial crop for starch, flour and animal feed.

Cassava is an outbreeding species originated in the American continent (Rogers, 1972). With highly heterozygous landraces and vegetatively propagated, cassava improvement has been largely limited to mass

Instituto Agronômico (IAC), Av. Barão de Itapura, 1481, Caixa Postal 28, 13001-970 Campinas, SP, Brad. E-mail: ccolombo@ cec.iac.br. Send correspondence to C.C. ORSTOM. 911, Av. Agropolis. 34032-Montpellier, France. ENSAM (Ecole Nationale Supérieure Agronomique de Montpellier). 2, Place Viala. 34060-Montpellier, France.

selection within genotype collections of landraces and F1 segregation progenies (Valle, 1990). An understanding of the genetic structure of this species through molecular markers is important for guiding parental choice in breeding programs and validating a core collection (Hershey et al., 1994). Furthermore, fingerprinting characterization of new varieties will become more and more important, particularly for cultivars used in industrial production, as a result of cultivar protection laws.

Various markers for morphological and agronomic traits are traditionally used for divergence and characterization studies of cassava cultivars (Charrier and Léfèvre, 1987; Pereira et al., 1989; Cury, 1993). Isozyme patterns have also been used as a method to estimate genetic diversity and identification of cassava clones (Zoundjikekpon and Touré, 1985; Hussain and Bushuk, 1987; Ramirez et. al., 1987; Léfèvre, 1989).

Few studies have been published on the use of DNA markers in cassava. The genetic diversity of an in vitro germplasm collection of African cassava clones was evaluated using RFLP (Beeching et al., 1993) and

106 Colombo et nl.

RAPD markers (Marmey et al., 1994). This technology has already been applied to fingerprinting in a wide range of plant species, including rice (Welsh and McClelland, 1990), cocoa (Wilde et al., 1992), papaya (Stiles et al., 1993), apple (Koller et al., 1993), sweet potato (Connolly et al., 1994), and cotton (Multani and Lyon, 1995).

In the present paper we report the use of RAPD markers for cultivar identification, characterization of genetic diversity within a cassava germplasm, and comparison with morphology-based characterization.

MATERIAL A N D METHODS Material

Thirty-one distinct cassava landraces and cultivars originating in different Brazilian regions with different cultivation purposes were used in this study.

Cassava leaf samples from the collection maintained by the Instituto Agronômico at Campinas, São Paulo, Brazil (Table I), were submitted to RAPD analysis at ORSTOM, Montpellier, France.

Morphological data

Morphological data were obtained during regular germplasm characterization in Campinas, Brazil. Nine qualitative and quantitative descriptors recommended by IBPGR and adapted by the Instituto Agronamico Cassava Program were analyzed. The descriptors with their class numbers within parentheses are: color of unexpanded apical leaves (4), color of first fully expanded leaf (4), petiole color (4), width of central lobe (Z), creasing of lobe leaf (3), roughness of film root (Z), storage root film color (Z), color of outer surface of

Table I - Brazilian cassava cultivars studied using RAPD markers and their botanical characteristics.

Botanical descriptors

1 2 3 4 5 6 7 8 9

1 SantistajBranca F1117 Ubatuba/SP G GR RV n I ro DB r W 2 PãodeLó F1135 MaresiadSr GV GR RV n 1 ro DB r W 3 CacauI' F1153 Praia Grande/SP GV GR RV n 1 ro DB r W 4 unknown F1162 Peruibe/SP V V R V n w ro DB r W 5 unknown F2023 Jacupiranga/SP GV GR RV n 1 ro DB W W 6 PãodoC6uII F2030 Guapiara/SP GV GR RV b 1 ro DB C Y 7 unknown F3013 SãoSeb.daGrama/SP G R RV n 1 ro LB W C

V V G n w ro DB r Y 18 Canela de Urubu F3039 Franca/SP 19 Unknown E4048 Femandópolis/SP GV GR RG b 1 s L B r W 10 unknown F4072 Araçatuba/SP GV GR RV n 1 ro DB C C 11 Mato Grosso F4113 Banri/ SP GV GR RV b w s L B W W 12 unknown F4130 StaBárbaraDOeste/SP GV G RG n 1 ro DB W Y 13 Vassourinha XII F5075 Ouro Verde/SP VG GR RV b vw ro DB W W 14 VassourinhaXIV F5129 Chavantes/SP GV R RV b vw ro DB W W 15 VassourinhaPta SRTl São Paulo GV GR RV b vw s DB W W 16 BrancadeSC SRT59 Piracicaba/SP VG R RV n 1 s L B W W 17 Santa SRTl20 Ubatuba/SP GV GR RV n 1 ro DB r W 18 Guaxupé SRT454 Guaxupé/MG VG GR RV n 1 ro DB W W 19 CarapéII SRT521 Capela/RS GV G RG b 1 ro DB W W 20 Guaxo SRT1012 Araquari/SC GV GR RV n 1 ro DB W W 21 Taquari SRT1099 Taquari/RS GV G GR n 1 ro DB W W 22 Mico SRT1105 Rio do Sul/SC V R R V n 1 ro DB W W 23 Ciganapreta SRT1116 Cruz das Almas/BA V R R V n 1 ro DB W W 24 Unha SRT1214 São Mateus/ES GV GR RV n 1 ro DB W W 25 Izabel Souza I SRT1229 Paraíba VG GR RV n 1 ro DB W W 26 Unknown SRT1293 Itumbiara/GO GV GR RV n 1 ro DB W W 27 Amarela SRT1333 Coxim/MS GV GR GR b w s D B W C

SRT1337 Jaceara/MS G GR RV n 1 r D B r W 28 PãoXIII 29 Bambu SRT1341 São Francisco/MG GV G GR n 1 ro DB W W 30 Saracura SRT1345 Santa Cruz/RJ GV GR RV b w ro DB r W 31 Apronta a Mesa SRT1351 Rio Grande do Sul GV GR RG b w r D B W W

Botanical descriptors: 1. color of unexpanded apicalleaves; 2. color of first fully expanded leaf; 3. petiole color; 4. width of central lobe; 5. creasing of lobe leaf; 6. roughness of film root; 7. storage root film color; 8. color of outer surface of storage root cortex; 9. storage root pulp color immediately after being opened. G = Green; GV = green-violet; VG = violet-green; V = violet; GR = green-red; R = red; RG = red-green; RV = red-violet; n = narrow; b = broad; 1 = linear; w = winding; vw =very winding; s = smooth; ro = rough; LB = light-brown; DB = dark-brown; W = write; C = cream; r =rose; Y = yellow.

Number Vulgarname Code Origin

RAPD for genetic characterization of cassava 107

storage root cortex (3) and storage root pulp color immediately after being cut (3). The thirty-one landraces and cultivars and their respective descriptors are shown in Table I.

DNA extraction

DNA was extracted from fresh and dried leaves at ORSTOM laboratory according to the following procedure: leaves were dried (45-48 C for 24 h in an oven with static aeration) and preserved in silica gel. One hundred milligram of ground tissue was trans- ferred to a 2-ml sterile Eppendorf tube with 1 ml of extraction buffer (0.1 M Tris HC1, pH 8.0; 1.25 M NaC1; 0.02 M EDTA; 4% MATAB (mixed alkyltrimethylam- monium bromide); 1% ß-mercapto-ethanol added just before use). After 90-min incubation at 65 C, the mixture was extracted twice with an equal volume of chloroform/isoamyalcohol (24:l). Fifty pl RNAse (10 mg per ml) was added after the first extraction, and the solution was incubated for 30 min at 37 C. A DNA pellet was obtained after adding 0.8 volume of isopropanol and precipitated by centrifugation (10 min at 10,000 9). After washing in 70% ethanol, vacuum drying and dissolving in 900 pl TE buffer (10 mM Tris HC1, pH 8.0, 1 mM EDTA), 1/10 of the volume of 3 M sodium acetate was added and a second DNA precipitation was done in the same way. Then, it was re-dissolved in 100 p1 buffer TE. DNA quality and concentration were analyzed by electrophoresis in 0.8% agarose gels.

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DNA amplification

PCR was carried out in 25 p1 of a reaction mixture with 10 mM Tris-HC1, pH 8.3,50 mM KC1,1.5 mM MgCl,, 0.001 gelatin, 10 ng template DNA, 0.4 pM primer, 100 pM of each dNTPs, and 0.5 units Taq polymerase (Appligene). DNA amplification was performed in a thermocycler (PTC-100 MJ Research) programmed as follows: 95 C for 4 min, followed by 45 cycles of 1 rnin at 95 C, 1 min at 35 C, 2 min at 72 C, a final stage of 7 min at 72 C, and maintained at 4 C prior to analysis. The amplification products plus 3 pl of buffer (0.5% bromophenol/blue/glycerol: 1:2:1) were electrophoresed on 1.8% agarose gels in l x TBE buffer, stained with ethidium bromide and photographed

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I under W light with Polaroid film.

Data analysis

For analysis of botanical traits, the morphological variables were standardized to O and 1 at a complete disjunctive table. Hence, a novel two-way

matrix (31 cultivars x 29 classes) was created. For PCR amplification products, the bands were scored as presence (1) or absence (O) for each of the 31 cultivars with the 22 primers. Only RAPD bands with good distinctiveness were recorded.

Pairwise comparisons of accessions based on RAPD bands and botanical descriptors were calculated by simple matching (SM) coefficients (Sokal and Michener, 1958). This similarity index was adopted since this model confers equal weight to shared presence and absence bands. Principal coordinate analysis (PCoA) (Gower, 1966) was performed based on these SM coefficients to botanical and RAPD markers, separately.' Calculations were performed using NTSYS-pc software (Rohlf, 1993).

RESULTS

One hundred and ninety primers were tested on four different genotypes. Twelve primers (H13,16, M18, X6, X14, X19, Y3, Y8, Y11, Y19,215,220) did not produce clear bands. The twenty-two primers that produced the clearest bands were chosen for this study. Table II details the RAPD amplifications. The number of total clear bands obtained from each primer varied from 4 to 14, with an average of 9.0 per primer. Seventy-four (37.6%) bands out of 197 were polymorphic. Their molecular sizes ranged from 300 bp to 2000 bp with an average of 900 bp.

To optimize result reproducibility, we exam- ined the influence of template DNA concentration, and the reactions were done several times. Figures 1 and 2 illustrate the patterns of reproducibility using Vassourinha variety (SRT-01). A difference in band intensity was observed under different thermo-cycler settings (not shown).

A simple matching index was used to compare similarities between two-by-two cultivars (Table III). Matching coefficients ranged from 0.47 to 1.00, with an average of 0.65 for RAPD markers. Botanical descriptor values ranged from 0.45 to 1.00, with an average of 0.73. Only two cultivars presented the same genetic fingerprints based on RAPD markers (cultivars 2 and 17). On the other hand, when the cultivars were compared through the botanical descriptors, thirteen pair-wise comparisons obtained the maximum simple matching index (SM = 1).

PCoA of 31 cultivars to RAPD markers and botanical descriptors are represented in Figures 3 and 4. The plan formed by coordinates 1 and 2 explained 25.5% and 38.2% of total variance of RAPD markers (Figure 3) and botanical descriptors (Figure 4),

108 Colombo et nl.

Table II - Total number and polymorphic products detected by RAPD analysis of 31 cassava cultivars. Fragment length is given in pairs of bases (PW.

Cultivars Total Poly- Primers num- mor hic Len th (operon) ber prozucts (PE)

1 2 3 4 S 6 7 8 9 10 11 12 13 14 IS 16 17 IS 19 20 21 22 23 24 25 26 27 28 29 30 31

H6

H7

I1

I8

J9

P O Kl:!

K14

L7

M10

M12

N5

N20

x5

X16 Y11

Y14

Y16

2 4

Z6

z 9

217

Total

10

7

9

7

12

7 11

10

14

12

6

9

9

7

4 10

11

6

10

7

9

10

197

6

2

5

2

5

1 3

6

6

4

2

4

3

3

1 3

2

3

4

3

3

3

74

1380 1100 700 650 600 300

1350 500

1350 1200 650 630 580

1150 900

1600 1100 880 650 500 830

1800 1150 550

2000 1350 1000 750 650 350

1380 980 800 700 670 500 900 830 700 300

1200 1100 1850 1280 1100 800

1200 600 350 950 soo 750 500 780 450 350

1100 1050 800 500 300

1050 1030 1000 950 950 900 700

1250 650 560

1550 950 750

1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 1 0 0 1 1 0 1 1 1 1 1 1 1 1 0 0 0 1 0 0

4 i 1 1 1 1 0 1 1 1 0 1 0 0 1 1 0 1 1 1 1 1 0 1 1 1 0 1 1 1 1 1 1

O ~ O O O O O O O ~ O O O O O O ~ O O O ~ ~ ~ ~ ~ ~ I I ~ I I 1 1 1 0 0 1 1 0 0 0 0 0 1 0 0 1 1 0 1 1 ö 1 ö ö i i õ i ï i ô 1 1 1 1 1 1 1 0 1 1 0 1 0 0 0 1 1 0 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 0 0 0 0 1 0 1 0 0 0 0 1 0 0 1 0 1 0 1 0 0 1 0 1 0 1 0 0 1 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 0 0 I 0 0 0 0 0 1 1 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 1 o o o o o i o o o o i o o i i o o o ~ n ~ n n n n n n n ~ ~ ~ l o o l o l l l l o l l l l l o o o o ö ö õ ï i ï õ i ï i õ õ 0 0 1 0 0 0 0 0 1 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 1 0 0 1 0 0 1 0 1 0 1 0 1 0 1 0 0 0 0 0 0 0 1 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 1 0 1 1 0 1 0 1 1 0 0 1 1 0 1 0 0 0 0 0 1 0 0 1 1 1 0 1 0 1 0 0 0 1 0 1 0 0 0 0 1 1 0 0 1 1 1 1 1 0 0 1 0 0 0 0 1 1 0 0 0 0 1 0 0 0 1 0 0 0 1 1 0 0 0 1 1 0 0 0 0 0 0 0 1 1 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 1 0 0 0 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 1 0 1 1 0 1 0 1 0 0 0 1 1 0 0 1 0 0 1 0 0 0 0 1 0 1 1 1 0 0 1 0 1 1 0 0 1 0 0 1 1 0 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 0 0 0 1 0 0 1 0 0 1 1 0 1 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 1 0 0 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 0 1 1 0 1 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 1 0 0 0 1 0 1 0 0 0 0 1 1 0 0 1 0 0 0 1 0 0 0 0 1 0 1 1 1 0 0 1 1 0 1 0 0 0 0 0 0 1 1 0 1 0 1 0 0 0 1 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 1 0 1 1 0 1 0 0 0 1 0 1 0 1 1 1 0 1 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1 1 1 0 1 1 0 1 0 1 0 0 1 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0 1 1 0 0 1 1 0 1 0 1 0 1 1 0 1 0 0 1 1 1 1 0 1 1 1 0 1 1 0 1 0 0 0 1 0 1 0 1 0 1 1 1 1 0 1 1 0 0 1 1 0 1 0 0 0 0 1 1 1 1 1 0 1 0 1 1 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 1 1 0 1 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 1 0 1 0 1 1 1 1 0 0 0 0 0 0 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 0 0 0 1 1 0 0 0 0 1 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 1 1 0 0 0 0 1 1 1 0 0 1 0 0 1 1 0 1 1 1 1 0 1 0 1 0 1 0 1 1 1 1 1 1 0 0 1 1 0 1 1 1 0 1 0 0 1 0 1 0 1 0 1 0 0 0 1 1 1 1 1 1 0 1 0 0 0 0 O O O ~ ~ O O ~ O O O O O ~ O ~ O ~ ~ ~ I ~ ~ I I ~ ~ ~ ~ ~ ~ 1 1 1 0 1 1 1 O l l l l l l l l l 1 ï 1 i ï ï i i i ï ï ï ï ï 1 0 1 1 1 0 1 1 1 0 0 0 1 1 1 1 0 1 1 0 1 0 0 1 0 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 1 1 1 1 1 1 1 0 0 1 0 1 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 0 0 0 1 0 0 1 1 1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 1 1 1 1 1 1 1 1 0 0 0 0 0 1 0 0 1 1 1 1 1 1 1 0 0 1 1 1 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0

1 1 1 0 0 0 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 i i i i i i ï ï ï 1 0 0 1 1 0 0 1 1 0 1 0 1 1 0 1 0 1 1 1 1 1 0 1 0 1 1 0 1 0 1 1 1 0 0 0 1 0 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 1 0 0 0 1 1 0 0 0 0 0 1 0 1 0 0 3 0 0 0 0 1 0 0 0 0 1 1 1 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 1 1 1 1 1 1 1 1 0 0 1 1 1 0 1 0 0 1 1 1 1 1 1 1 1 1 ö B

0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 1 1 1 1 1 0 0 1 0 1 0 0 0 0 0 0 0 1 0 1

0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 O O O O O O O ~ ~ O O ~ O O ~ O O O ~ O O O O I ~ I I ~ ~ I I

0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 \ 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 ¡ ï ï i ï ï i ï 0 0 0 0 1 1 0 0 0 1 0 0 0 0 0 1 0 0 1 1 0 1 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 1 0 1 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 o o o o i o o o o o o o o o o o o 1 o o ~ o 1 o o o o o 1 ~ n 1 1 1 0 0 1 0 0 0 0 0 1 0 1 0 0 1 0 0 0 1 0 1 1 1 ö ~ ~ ~ ö 1 1 0 0 1 0 0 0 0 1 0 1 0 1 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 1 1 0 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 1 1 0 1 1 1 0 0 0 1 1 0 1 0 0 1 0 0 1 0 0 1 0 0 0 0 1 1 1 1 1 0 0 0 0 1 0 0 0 0 1 1 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 1 1 1 1 1 1 0 0 0 1 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0

893 (mean)


Figure 1 - RAPD profile of Vassourinha cultivar (SRT 01) based on different DNA concentrations. Left to right: 1,2,4,8, 16,32, 64 and 128 ng per 25 rJ-1 of reaction mixture, K-14 and N-5 primers.

Figure 2 - Reproducibility of five independent reactions of RAPD amplifications with four different primers in the Vassourinha cultivar (SRT 01): primers 1-8 (A), J-9 (B), K-14 (C) and N-5 (D). The DNA concentration employed was 15 ng per 25 ~1 of reaction mixture.

respectively. These figures show some identities, with various cultivars being grouped identically in the two graphs. Regarding coordinate 1, we can see basically the same cultivars on the left and right of these two graphs, except cultivar 8. In addition, we observed some cultivar identity closeness. Cultivars 1,2,3,10,17 and 28 were grouped together in A, and cultivars 20,21,22, 23 and 24 were grouped together in B. The first presented a simple matching coefficient of 0.8, and the second presented 0.85. On the other hand, cultivars 9, 11, 13, 14, 15, 19, 30 and 31 had a simple matching coefficient of 0.71, and were grouped in C.

Varieties cultivated in large scale for flour production proved to be closely related (simple matching coefficient 0.72 for RAPD markers and 0.87 for

botanical descriptors). Varieties cultivated on the São Paulo State coast, normally destined to "in i z a t ~ m " consumption, also proved to be closely related (simple matching coefficient 0.78 for RAPD markers and 0.91 for botanical descriptors). In both cases, these cultivars presented a small genetic base among themselves.

DISCUSSION

Several parameters are known to affect reproducibility of the RAPD tech- nique. Among them are primer concentration and structure, template quantity, and type of thermo-cycler or polymerase (Kernodle et al., 1993; Penner et al., 1993; Schierwater and Ender, 1993). In our study, we only observed DNA template concentration and reaction repetition. Both the highest and the lowest DNA concentrations used showed the same amplified products, the only difference being the intensity of the bands (Figure 1). However, as a result of practical dispositions, we chose 15 ng/25 pl as the optimum template concentration to optimize scored band selection. The same reproducibility pattern for the Vassourinha cultivar (Figure 2) was observed for other combinations of cultivars and primers. This indicates that the standard PCR conditions allowed uniform amplifications for all primers. Hence, using previously estab- lished amplification conditions, it was pos- sible to obtain a good level of separation for 31 clones studied using a 22-primer system. Due to differences in thermo-cycler

strength, all PCR amplifications utilized the same thermo-cycler.

The number of primers utilized and/or the number of RAPD polymorphism scored bands can determine the informativeness and reliability of the data collected for genomic similarity studies (Bhat and Jarret, 1995). The results presented here closely agree with previous reports in Brassica (Kresovich et al., 1992), papaya (Stiles et al., 1993) and apple (Dunemann et al., 1994). This indicates that approximately 10-30 primers (50-100 RAPD polymorphisms) were adequate to estimate genetic relationships within and/or between species. The advantages of using a large number of loci in estimating genetic distance have been stated, and it was recommended that at least 50 loci be studied (Nei,

110 Colombo et al.

Table III - Simple matching coefficients of similarity from analyses using RAPD product profiles (lower mean table) and botanical descriptors (upper mean table) of 31 cassava cultivars.

Cultivar 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

1. Sanbsta Branca - 093 O93 079 086 072 O72 O70 066 079 059 066 072 06b 066 066 093 086 066 086 072 079 0.79 086 086 086 0.52 072 079 066 2. Pão de Lá 085 - l.& 079 0.93 079 O60 0.79 072 086 066 0.72 0.72 072 O72 066 m 086 072 093 079 079 0'79 093 080 083 059 0.93 079 086 072 3. Cacau I 083 O 8 3 - 079 0.93 079 Ob6 079 O72 086 flhb 072 072 072 O72 066 ,lJ,Q 086 072 0.93 079 079 079 093 086 093 0.59 0.93 079 O8b O72 4. Unknown (F1162) 068 059 066 - 072 OS9 059 O86 OS2 066 059 059 066 Ob6 059 059 O79 072 059 072 066 079 079 O72 072 072 052 079 006 079 066 5.Unknown(F2023) 057 052 057 070 - 079 O72 072 066 086 072 079 0.79 0,79 079 072 093 093 079 loo 086 086 086 093 O b b 086 086 079 0.79 6. Pão do Céu II 077 079 074 065 051 - 059 0.72 006 086 0.66 072 072 0'72 072 052 0.79 072 0.72 079 066 066 066 079 072 0.79 O66 072 066 079 O 7 2 7. Unknown (F3013) 076 073 068 071 062 072 - 066 OS2 072 0.59 Ob6 059 066 052 0.79 Ob6 072 059 072 066 079 07s 072 0.72 O72 O W 072 066 052 052 8. Canela de Urubu 059 056 056 066 060 052 Ob3 - 0.52 072 0.15 072 059 059 0.52 059 079 072 059 072 06b 079 Oï9 072 0.72 O72 045 079 066 066 052 9.Unknown(F4048) 068 061 061 059 057 062 066 068 - 059 079 059 059 O59 072 066 072 059 072 066 059 052 052 066 059 066 066 066 059 O72 0.72

10.Unhown(F4113) 070 072 070 067 061 0.78 O72 055 057 - 059 O72 O6b Obb 066 050 086 0.79 066 086 072 0.72 072 086 079 0 % O66 079 072 072 066 11. Mato Grosso 059 056 059 003 057 060 051 061 071 055 - 052 0.72 072 086 072 Ob6 066 0.66 072 059 OS9 059 072 066 O72 079 059 O59 079 079' 12. Unknown (F4130) 0.59 066 0.59 059 0.72 072 08b 070 086 072 0.72 079 072 079 059 066 0,86 059 072 13. Vassounnha XII 066 Ob1 063 068 060 O62 O73 061 063 067 061 061 - 0.86 086 066 072 O86 072 079 066 072 072 079 086 079 066 072 066 079 0.79 14 Vassourhha XIV 066 ob1 066 063 057 057 063 0bs 071 0.60 073 O63 068 - 086 066 072 072 079 079 072 070 079 O79 072 079 006 065 072 079 079

063 O63 063 056 000 O67 068 O S 4 06b 072 061 -

15. Vassounnha Pta 056 056 056 056 055 060 O61 OM 0.73 Ob0 078 0.73 0.73 0.76 - Ob6 0.72 O72 072 0.79 066 066 066 079 072 079 079 066 066 079 16. Branca de SC O72 Ob7 0.70 077 0.76 O68 OR2 067 067 0.71 057 060 070 067 057 - 066 0.79 059 O 66 079 079 072 079 072 052 066 066 052 17. Santa 087 .L@ 084 057 054 078 072 060 073 0.57 065 0-62 062 057 066 - 086 072 093 079 079 079 093 086 093 0.59 093 079 086 072

061 O63 066 068 0.72 057 063 061 0.60 059 O63 059 061 059 0.70 062 - 0.72 093 079 086 086 093 066 061 063 063 0.67 070 068 0 5 b 071 070 059 059 068 056 059 074 060 061 - 079 086 077. 072 079 072 070 070 070 0.74 O71 073 072 Ob2 057 078 055 060 067 O60 050 O R O 06R 070 072 086 OR6 086 19p 093

0.6R 063 066 066 Ob2 062 066 051 063 065 056 O b 6 061 O59 056 067 062 068 063 057 - 079 079 086 079 070 070 067 0.75 073 071 077 062 O60 078 062 062 070 062 060 O85 068 065 0.72 083 062 - loo 086 086 086 O52 079 079 066 066 0.70 070 062 067 068 068 077 057 055 070 055 070 062 057 057 073 008 067 OS7 076 070 076 - O86 086 086 052 0.79 079 0.66 066 070 0.05 060 072 066 O63 077 067 065 071 050 070 062 062 060 073 003 067 062 068 067 0.76 078 - 093 UQ 066 086 086 079 079

20. Guaxo 21. Taquari 22. Mico 23. Cigana Preta 24. Unha

-

15. IzabeI Souza I 072 on 072 0.67 061 0.76 0.74 055 065 071 o60 079 062 055 ofin 073 071 065 0.62 066 077 071 0.78 a73 - 093 059 086 0.79 072 1172 26. Unknown(SRT1293) 062 070 062 062 061 061 065 070 065 061 065 O60 065 O60 Ob5 063 068 Ob7 067 068 060 066 063 061 061 - 066 O86 486 0.79 079 27 A m a d a 065 065 062 062 056 066 0.62 062 O62 078 065 070 065 0.67 072 059 066 0.60 060 066 052 O63 066 066 059 O66 - 052 066 072 a79 28 Päoxm 078 083 068 OS9 048 079 O80 001 063 O72 054 063 061 061 059 065 O 8 2 056 OS9 070 056 070 074 070 O72 062 067 - 072 079 066 29 Bambu 0.70 062 062 0.74 068 O 7 3 0.72 flb5 067 0.63 065 060 065 065 065 O80 061 070 077 0.73 065 078 0.63 Ob6 OM 061 0.bl 0.65 - 066 O72 30. Saracura 062 070 067 057 051 073 072 060 070 068 062 060 0.bO 055 065 063 068 060 057 Ob3 060 068 063 o b 1 068 0.71 0.bl 074 0.61 - 086 31 A p o n t a a Mesa 070 074 065 O60 O51 071 067 057 070 066 060 0b7 067 0.70 067 063 073 062 O 6 5 O61 060 066 0.56 066 Ob1 066 073 072 060 068 -

1978). Therefore, the closeness among cultivars discussed in this analysis, based on 74 genetic markers, seems to be relatively 0.8 accurate.

Similarity among all pairwise E 0.4

cultivars obtained using the botanical 8 descriptors presented thirteen pairwise + comparisons with complete similarity (SM = 8 -0, 1). The same comparisons using RAPD markers resulted in only one complete .g -0.8 similarity (Table II). Using 29 randomly selected RAPD markers in this comparison

that presented complete similarity. Principal coordinate 1 (13.5%) produced only one pair (cultivars 2 and 17) -0.8 -0.4 O 0.4 0.8 1.2

Therefore, we observed that the botanical 'lass used to were not capable to differentiate thirteen

Figure 3 -Principal coordinate analysis (PCoA) of thirty-one cassava cultivars based on genetic distances calculated with 74 RAPD markers. A, B, C - Genotype groups with similar botanical characteristics.

the

pairwise cukvars that were separated by RAPD markers. In order to check the results of cultivars with identical botanical characteristics, detailed observations of field collection were made. We noticed differences in relation to other characteristics that are not regularly obtained by botanical description, except for cultivars 2 and 17. These cultivars were very similar, and could possibly be the same genotype. These results

illustrated the power of RAPD markers over botanical descriptors in studying the genetic diversity of cassava collections and identifying duplicates.

Both sets of variables (RAPD markers and botanical descriptors) were able to show the relationship among cultivars with some common characteristics. Groups A and B (Figures 3 and 4) show

t ' " ' " ' . ' ' 1G

7


16 1

O 0.4 0.8 1.2 -0.8

-0.8 -0.4 Principal coodinate I (23.2%)

Figure 4 - Principal coordinate analysis (PCoA) of tlurty-one cassava cultivars based on genetic distances calculated with 9 cassava botanical descriptors. A, B, C - Genotype groups with similar botanical characteristics.

these relationships. We observed in group A the closeness of cultivars 1,2,3,6,10,-5 and 28, all destined to "in natura" consumption, except cultivar 25. On the other hand, a second group of cultivars including 16/19, 20,21,22,23 and 24 is very useful for flour production, except cultivar 20.

Another group, 4,5,7,9,11,13,14,15,26,27,30 and 31, represents landraces from all over the country. Despite the little agronomic information available for these genotypes, smaller values of similarity were found between some of these two-by-two cassava accessions based on botanical traits as well as RAPD markers. Consequently, a broad dispersion of these cultivars is observed in Figures 3 and 4. This indicates a very large varietal divergence. Normally utilized as family subsistence crop, they represent an answer to different levels of farmer's exigency. These cultivars were probably maintained in isolation by small farmers. They probably underwent moderate selection pressures which may explain their important varietal diversity. Therefore, these cassava landrace accessions can represent an important resource of genetic variability. Cultivar 8 is a very interesting genotype that most botanical descriptors note for its strong red color. This cultivar was isolated in both multivariate analyses.

Regarding cassava's genetic diversity of different traits, several authors were able to reveal relationships among cassava cultivars based on botanical descriptors (Cordeiro et al., 1995 and Cury, 1993) and isoenzyme markers (Zoundjikekpon and Touré, 1985; Hussain and Bushuk,1987; Ramirez et al.,1987; Lefèvre, 1989). A preliminary study of some African cassava cultivars concluded that RAPD markers were better in understanding genetic diversity of cassava clones

(Marmey et al., 1994). Although the number of genotypes utilized in this study was reduced, RAPD markers and botanical descriptors revealed an interesting genetic cultivar structure. This result is very important in guiding parental choice in breeding programs, because more segregation will occur between more divergent parents crossed. Moreover, parental choice can be guided to obtain an efficiently segregated population and derive a genetic map useful in marker- assisted selection. RAPD is particularly important to validate or improve a core collection in this vegetatively propagated crop. In relation to both sets of variables, RAPD can be an interesting and complementary tool for identification and characterization of cassava varieties. According to Bayley (1983), who identified environmental stability and experimental reproducibility as basic criteria for cultivar identification, RAPD markers have advantages over botanical descriptors because they are not influenced by environmental conditions. In addition, this method is rapid, genetically neutral and simple. By selecting only strength amplified DNA frag- ments as informational bands, we can establish different fingerprint patterns among closely related cassava cultivars. Such a study of genetic diversity of cassava species based on RAPD markers is now in progress.

ACKNOWLEDGMENTS

The authors gratefully acknowledge CAPES for financial support and fellowships conceded to C. Colombo. The authors are also grateful to Dr. C. Pommer for critically reading the manuscript.

Publication supported by FAPESP.

RESUMO

Marcadores moleculares do tipo RAPD foram utilizados para estudar a diversidade genética de 31 clones de mandiocas brasileiras. Os resultados foram comparados com a diversidade genética fornecida por descritores botânicos. As relações de parentesco obtidas foram semelhantes para os dois tipos de marcadores utilizados. A análise multivariada baseada nos indices de similaridade genética entre os cultivares deste estudo permitiu o grupamento de genótipos mais indicados para o consumo "in natura", assim como daqueles genótipos destinados à produção de farinha num outro grupo. A maioria das raças locais, procedentes de várias regiões do país, apresentaram ampla dispersão, revelando serem uma importante fonte de variabilidade genética para a espécie. Os descritores botânicos foram incapazes de diferenciar treze pares de cultivares comparados dois-a-dois, enquanto que, através dos marcadores RAPD, apenas um par não foi possível diferenciar. Os resultados deste estudo

112 Colombo et al.

mostram o poder da técnica RAPD em relação aos descritores botânicos para estudos de diversidade genética de mandiocas, assim como para a identificação de duplicatas e para a formação de ”core collections”, que são particularmente importantes nesta espécie de propagação Vegetativa.

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(Received June 26,1997)

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