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

Abstracts

RAPD markers were used to investigate the genetic diversity of 31 Brazilian cassava clones. The results were compared with the genetic diversity revealed 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.


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 índices 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 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.


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

Carlos Colombo1, Gérard Second 2, Tereza Losada Valle1 and André Charrier 3

1 Instituto Agronômico (IAC), Av. Barão de Itapura,1481, Caixa Postal 28,13001-970 Campinas, SP, Brasil. E-mail: ccolombo@cec.iac.br. Send correspondence to C.C.

2 ORSTOM. 911, Av. Agropolis. 34032-Montpellier, France.

3 ENSAM (Ecole Nationale Supérieure Agronomique de Montpellier). 2, Place Viala. 34060-Montpellier, France.

ABSTRACT

RAPD markers were used to investigate the genetic diversity of 31 Brazilian cassava clones. The results were compared with the genetic diversity revealed 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 esculenta Crantz, Euphorbiaceae) is one of the world’s most important tropical plants, and is ranked as the fourth source of carbohydrates 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 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 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 AND 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.

Number Vulgar name Code Origin Botanical descriptors 1 2 3 4 5 6 7 8 9 1 Santista Branca F1117 Ubatuba/SP G GR RV n l ro DB r W 2 Pão de Ló F1135 Maresias/SP GV GR RV n l ro DB r W 3 Cacau I F1153 Praia Grande/SP GV GR RV n l ro DB r W 4 Unknown F1162 Peruibe/SP V V RV n w ro DB r W 5 Unknown F2023 Jacupiranga/SP GV GR RV n l ro DB W W 6 Pão do Céu II F2030 Guapiara/SP GV GR RV b l ro DB C Y 7 Unknown F3013 São Seb. da Grama/SP G R RV n l ro LB W C 18 Canela de Urubu F3039 Franca/SP V V G n w ro DB r Y 19 Unknown F4048 Fernandópolis/SP GV GR RG b l s LB r W 10 Unknown F4072 Araçatuba/SP GV GR RV n l ro DB C C 11 Mato Grosso F4113 Bariri/SP GV GR RV b w s LB W W 12 Unknown F4130 Sta Bárbara D’Oeste/SP GV G RG n l ro DB W Y 13 Vassourinha XII F5075 Ouro Verde/SP VG GR RV b vw ro DB W W 14 Vassourinha XIV F5129 Chavantes/SP GV R RV b vw ro DB W W 15 Vassourinha Pta SRT1 São Paulo GV GR RV b vw s DB W W 16 Branca de SC SRT59 Piracicaba/SP VG R RV n l s LB W W 17 Santa SRT120 Ubatuba/SP GV GR RV n l ro DB r W 18 Guaxupé SRT454 Guaxupé/MG VG GR RV n l ro DB W W 19 Carapé II SRT521 Capela/RS GV G RG b l ro DB W W 20 Guaxo SRT1012 Araquari/SC GV GR RV n l ro DB W W 21 Taquari SRT1099 Taquari/RS GV G GR n l ro DB W W 22 Mico SRT1105 Rio do Sul/SC V R RV n l ro DB W W 23 Cigana Preta SRT1116 Cruz das Almas/BA V R RV n l ro DB W W 24 Unha SRT1214 São Mateus/ES GV GR RV n l ro DB W W 25 Izabel Souza I SRT1229 Paraíba VG GR RV n l ro DB W W 26 Unknown SRT1293 Itumbiara/GO GV GR RV n l ro DB W W 27 Amarela SRT1333 Coxim/MS GV GR GR b w s DB W C 28 Pão XIII SRT1337 Jaceara/MS G GR RV n l r DB r W 29 Bambu SRT1341 São Francisco/MG GV G GR n l 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 DB W W

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

Botanical descriptors:1. color of unexpanded apical leaves; 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; l = linear; w = winding; vw = very winding; s = smooth; ro = rough; LB = light-brown; DB = dark-brown; W = write; C = cream; r = rose; Y = yellow.

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 Agronômico 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 (2), creasing of lobe leaf (3), roughness of film root (2), storage root film color (2), color of outer surface of 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 transferred to a 2-ml sterile Eppendorf tube with1 ml of extraction buffer (0.1 M Tris HCl, pH 8.0;1.25 M NaCl;0.02 M EDTA; 4% MATAB (mixed alkyltrimethylammonium bromide);1% b-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 (24:1). Fifty ml 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 adding0.8 volume of isopropanol and precipitated by centrifugation (10 min at10,000 g). After washing in 70% ethanol, vacuum drying and dissolving in 900 ml TE buffer (10 mM Tris HCl, 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 in100 ml buffer TE. DNA quality and concentration were analyzed by electrophoresis in 0.8% agarose gels.

DNA amplification

PCR was carried out in 25 ml of a reaction mixture with10 mM Tris-HCl, pH 8.3, 50 mM KCl,1.5 mM MgCl2,0.001 gelatin,10 ng template DNA,0.4 mM primer,100 mM of each dNTPs, and0.5 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 of1 min 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 ml of buffer (0.5% bromophenol/blue/glycerol:1:2:1) were electrophoresed on1.8% agarose gels in1x TBE buffer, stained with ethidium bromide and photographed under UV light with Polaroid film.

Data analysis

For analysis of botanical traits, the morphological variables were standardized to0 and1 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 (0) 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, I6, M18, X6, X14, X19, Y3, Y8, Y11, Y19, Z15, Z20) 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 to14, with an average of 9.0 per primer. Seventy-four (37.6%) bands out of197 were polymorphic. Their molecular sizes ranged from 300 bp to 2000 bp with an average of 900 bp.

Primers (operon) Total number Poly- morphic products Length (pb) Cultivars 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 H6 10 6 1380 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 1100 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 0 700 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 0 650 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 0 600 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 1 300 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 1 0 1 1 H7 7 2 1350 1 1 1 0 0 1 1 0 0 0 0 0 1 0 0 1 1 0 1 1 0 1 0 0 1 1 0 1 1 1 0 500 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 I1 9 5 1350 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 1200 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 0 650 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 1 630 0 0 0 0 0 1 0 0 0 0 1 0 0 1 1 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 580 1 0 0 1 0 1 1 1 1 0 1 1 1 1 1 0 0 0 0 0 0 0 1 1 1 0 1 1 1 0 0 I8 7 2 1150 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 0 900 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 J9 12 5 1600 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 1100 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 1 880 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 1 650 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 1 500 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 J10 7 1 830 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 K12 11 3 1800 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 1150 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 0 550 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 K14 10 6 2000 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 1350 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 1000 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 1 750 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 1 650 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 0 350 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 L7 14 6 1380 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 1 980 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 0 800 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 0 700 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 1 670 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 0 500 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 M10 12 4 900 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 1 830 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 0 700 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 1 300 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 M12 6 2 1200 0 0 0 1 1 0 0 1 0 0 0 0 0 1 0 1 0 1 0 0 1 0 0 1 1 0 0 0 0 0 0 1100 1 1 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 N5 9 4 1850 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 1280 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 1100 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 1 800 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 N20 9 3 1200 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 0 600 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 0 350 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 X5 7 3 950 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 0 800 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 1 1 750 0 0 0 0 0 0 1 1 1 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 X16 4 1 500 1 1 1 0 0 0 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 Y11 10 3 780 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 1 450 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 0 350 0 0 1 0 1 0 0 1 0 0 0 0 1 0 0 0 0 1 1 1 0 0 0 0 0 1 0 0 0 0 0 Y14 11 2 1100 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 0 1050 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 Y16 6 3 800 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 0 500 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 1 300 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 Z4 10 4 1050 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 1030 0 0 0 0 0 0 0 1 1 0 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 1 1 0 0 1 1 1000 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 950 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 Z6 7 3 950 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 0 900 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 0 700 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0 0 1 0 0 Z9 9 3 1250 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 0 0 1 0 0 1 650 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 0 560 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 Z17 10 3 1550 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 0 950 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 0 750 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 Total 197 74 893 (mean)

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

To optimize result reproducibility, we exam ined the influence of template DNA concentration, and the reactions were done several times. Figures1 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).


Figure 1 - RAPD profile of Vassourinha cultivar (SRT01) based on different DNA concentrations. Left to right:1, 2, 4, 8,16, 32, 64 and128 ng per 25 ml 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 (SRT01): primers I-8 (A), J-9 (B), K-14 (C) and N-5 (D). The DNA concentration employed was15 ng per 25 ml of reaction mixture.

A simple matching index was used to compare similarities between two-by-two cultivars (Table III). Matching coefficients ranged from0.47 to1.00, with an average of0.65 for RAPD markers. Botanical descriptor values ranged from0.45 to1.00, with an average of0.73. Only two cultivars presented the same genetic fingerprints based on RAPD markers (cultivars 2 and17). 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).

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. Santista Branca - 0.93 0.93 0.79 0.86 0.72 0.72 0.79 0.66 0.79 0.59 0.66 0.72 0.66 0.66 0.66 0.93 0.86 0.66 0.86 0.72 0.79 0.79 0.86 0.86 0.86 0.52 1.00 0.72 0.79 0.66 2. Pão de Ló 0.85 - 1.00 0.79 0.93 0.79 0.66 0.79 0.72 0.86 0.66 0.72 0.72 0.72 0.72 0.66 1.00 0.86 0.72 0.93 0.79 0.79 0.79 0.93 0.86 0.93 0.59 0.93 0.79 0.86 0.72 3. Cacau I 0.83 0.83 - 0.79 0.93 0.79 0.66 0.79 0.72 0.86 0.66 0.72 0.72 0.72 0.72 0.66 1.00 0.86 0.72 0.93 0.79 0.79 0.79 0.93 0.86 0.93 0.59 0.93 0.79 0.86 0.72 4. Unknown (F1162) 0.68 0.59 0.66 - 0.72 0.59 0.59 0.86 0.52 0.66 0.59 0.59 0.66 0.66 0.59 0.59 0.79 0.72 0.59 0.72 0.66 0.79 0.79 0.72 0.72 0.72 0.52 0.79 0.66 0.79 0.66 5. Unknown (F2023) 0.57 0.52 0.57 0.70 - 0.79 0.72 0.72 0.66 0.86 0.72 0.79 0.79 0.79 0.79 0.72 0.93 0.93 0.79 1.00 0.86 0.86 0.86 1.00 0.93 1.00 0.66 0.86 0.86 0.79 0.79 6. Pão do Céu II 0.77 0.79 0.74 0.65 0.51 - 0.59 0.72 0.66 0.86 0.66 0.72 0.72 0.72 0.72 0.52 0.79 0.72 0.72 0.79 0.66 0.66 0.66 0.79 0.72 0.79 0.66 0.72 0.66 0.79 0.72 7. Unknown (F3013) 0.76 0.73 0.68 0.71 0.62 0.72 - 0.66 0.52 0.72 0.59 0.66 0.59 0.66 0.52 0.79 0.66 0.72 0.59 0.72 0.66 0.79 0.79 0.72 0.72 0.72 0.52 0.72 0.66 0.52 0.52 8. Canela de Urubu 0.59 0.56 0.56 0.66 0.60 0.52 0.63 - 0.52 0.72 0.45 0.72 0.59 0.59 0.52 0.59 0.79 0.72 0.59 0.72 0.66 0.79 0.79 0.72 0.72 0.72 0.45 0.79 0.66 0.66 0.52 9. Unknown (F4048) 0.68 0.61 0.61 0.59 0.57 0.62 0.66 0.68 - 0.59 0.79 0.59 0.59 0.59 0.72 0.66 0.72 0.59 0.72 0.66 0.59 0.52 0.52 0.66 0.59 0.66 0.66 0.66 0.59 0.72 0.72 10. Unknown (F4113) 0.70 0.72 0.70 0.67 0.61 0.78 0.72 0.55 0.57 - 0.59 0.72 0.66 0.66 0.66 0.59 0.86 0.79 0.66 0.86 0.72 0.72 0.72 0.86 0.79 0.86 0.66 0.79 0.72 0.72 0.66 11. Mato Grosso 0.59 0.56 0.59 0.63 0.57 0.60 0.51 0.61 0.71 0.55 - 0.52 0.72 0.72 0.86 0.72 0.66 0.66 0.66 0.72 0.59 0.59 0.59 0.72 0.66 0.72 0.79 0.59 0.59 0.79 0.79 12. Unknown (F4130) 0.63 0.63 0.63 0.56 0.60 0.67 0.68 0.54 0.66 0.72 0.61 - 0.59 0.66 0.59 0.59 0.72 0.72 0.86 0.79 0.86 0.72 0.72 0.79 0.72 0.79 0.59 0.66 0.86 0.59 0.72 13. Vassourinha XII 0.66 0.61 0.63 0.68 0.60 0.62 0.73 0.61 0.63 0.67 0.61 0.61 - 0.86 0.86 0.66 0.72 0.86 0.72 0.79 0.66 0.72 0.72 0.79 0.86 0.79 0.66 0.72 0.66 0.79 0.79 14. Vassourinha XIV 0.66 0.61 0.66 0.63 0.57 0.57 0.63 0.68 0.71 0.60 0.73 0.63 0.68 - 0.86 0.66 0.72 0.72 0.79 0.79 0.72 0.79 0.79 0.79 0.72 0.79 0.66 0.66 0.72 0.79 0.79 15. Vassourinha Pta 0.56 0.56 0.56 0.56 0.55 0.60 0.61 0.63 0.73 0.60 0.78 0.73 0.73 0.76 - 0.66 0.72 0.72 0.72 0.79 0.66 0.66 0.66 0.79 0.72 0.79 0.79 0.66 0.66 0.79 0.79 16. Branca de SC 0.72 0.67 0.70 0.77 0.76 0.68 0.82 0.67 0.67 0.71 0.57 0.60 0.70 0.67 0.57 - 0.66 0.79 0.59 0.72 0.66 0.79 0.79 0.72 0.79 0.72 0.52 0.66 0.66 0.52 0.52 17. Santa 0.87 1.00 0.84 0.57 0.54 0.78 0.72 0.55 0.60 0.73 0.57 0.65 0.62 0.62 0.57 0.66 - 0.86 0.72 0.93 0.79 0.79 0.79 0.93 0.86 0.93 0.59 0.93 0.79 0.86 0.72 18. Guaxupé 0.61 0.63 0.66 0.68 0.72 0.57 0.63 0.68 0.61 0.60 0.59 0.63 0.59 0.61 0.59 0.70 0.62 - 0.72 0.93 0.79 0.86 0.86 0.93 1.00 0.93 0.59 0.86 0.79 0.72 0.72 19. Carapé II 0.66 0.61 0.63 0.63 0.67 0.70 0.68 0.56 0.71 0.70 0.59 0.59 0.68 0.56 0.59 0.74 0.60 0.61 - 0.79 0.86 0.72 0.72 0.79 0.72 0.79 0.66 0.66 0.86 0.72 0.86 20. Guaxo 0.70 0.70 0.70 0.74 0.71 0.73 0.72 0.62 0.57 0.78 0.55 0.60 0.67 0.60 0.50 0.80 0.68 0.70 0.72 - 0.86 0.86 0.86 1.00 0.93 1.00 0.66 0.86 0.86 0.79 0.79 21. Taquari 0.68 0.63 0.66 0.66 0.62 0.62 0.66 0.51 0.63 0.65 0.56 0.66 0.61 0.59 0.56 0.67 0.62 0.68 0.63 0.57 - 0.79 0.79 0.86 0.79 0.86 0.66 0.72 1.00 0.66 0.72 22. Mico 0.70 0.70 0.67 0.74 0.73 0.71 0.77 0.62 0.60 0.78 0.62 0.62 0.70 0.62 0.60 0.85 0.68 0.65 0.72 0.83 0.62 - 1.00 0.86 0.86 0.86 0.52 0.79 0.79 0.66 0.66 23. Cigana Preta 0.70 0.70 0.62 0.67 0.68 0.68 0.77 0.57 0.55 0.76 0.55 0.70 0.62 0.57 0.57 0.73 0.68 0.67 0.57 0.76 0.70 0.76 - 0.86 0.86 0.86 0.52 0.79 0.79 0.66 0.66 24. Unha 0.70 0.65 0.60 0.72 0.66 0.63 0.77 0.67 0.65 0.71 0.50 0.70 0.62 0.62 0.60 0.73 0.63 0.67 0.62 0.68 0.67 0.76 0.78 - 0.93 1.00 0.66 0.86 0.86 0.79 0.79 25. Izabel Souza I 0.72 0.72 0.72 0.67 0.61 0.76 0.74 0.55 0.65 0.71 0.60 0.79 0.62 0.55 0.60 0.73 0.71 0.65 0.62 0.66 0.77 0.71 0.78 0.73 - 0.93 0.59 0.86 0.79 0.72 0.72 26. Unknown (SRT1293) 0.62 0.70 0.62 0.62 0.61 0.61 0.65 0.70 0.65 0.61 0.65 0.60 0.65 0.60 0.65 0.63 0.68 0.67 0.67 0.68 0.60 0.66 0.63 0.61 0.61 - 0.66 0.86 0.86 0.79 0.79 27. Amarela 0.65 0.65 0.62 0.62 0.56 0.66 0.62 0.62 0.62 0.78 0.65 0.70 0.65 0.67 0.72 0.59 0.66 0.60 0.60 0.66 0.52 0.63 0.66 0.66 0.59 0.66 - 0.52 0.66 0.72 0.79 28. Pão XIII 0.78 0.83 0.68 0.59 0.48 0.79 0.80 0.61 0.63 0.72 0.54 0.63 0.61 0.61 0.59 0.65 0.82 0.56 0.59 0.70 0.56 0.70 0.74 0.70 0.72 0.62 0.67 - 0.72 0.79 0.66 29. Bambu 0.70 0.62 0.62 0.74 0.68 0.73 0.72 0.65 0.67 0.63 0.65 0.60 0.65 0.65 0.65 0.80 0.61 0.70 0.77 0.73 0.65 0.78 0.63 0.66 0.63 0.61 0.61 0.65 - 0.66 0.72 30. Saracura 0.62 0.70 0.67 0.57 0.51 0.73 0.72 0.60 0.70 0.68 0.62 0.60 0.60 0.55 0.65 0.63 0.68 0.60 0.57 0.63 0.60 0.68 0.63 0.61 0.68 0.71 0.61 0.74 0.61 - 0.86 31. Apronta a Mesa 0.70 0.74 0.65 0.60 0.51 0.71 0.67 0.57 0.70 0.66 0.60 0.67 0.67 0.70 0.67 0.63 0.73 0.62 0.65 0.61 0.60 0.66 0.56 0.66 0.61 0.66 0.73 0.72 0.66 0.68 -

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.

PCoA of 31 cultivars to RAPD markers and botanical descriptors are represented in Figures 3 and 4. The plan formed by coordinates1 and 2 explained 25.5% and 38.2% of total variance of RAPD markers (Figure 3) and botanical descriptors (Figure 4), respectively. These figures show some identities, with various cultivars being grouped identically in the two graphs. Regarding coordinate1, 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. Cultivars1, 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 of0.71, and were grouped in C.


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.


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

Varieties cultivated in large scale for flour production proved to be closely related (simple matching coefficient0.72 for RAPD markers and0.87 for botanical descriptors). Varieties cultivated on the São Paulo State coast, normally destined to" in natura" consumption, also proved to be closely related (simple matching coefficient0.78 for RAPD markers and0.91 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 technique. 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 (Figure1). However, as a result of practical dispositions, we chose15 ng/25 ml 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 established amplification conditions, it was possible 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 approximately10-30 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,1978). Therefore, the closeness among cultivars discussed in this analysis, based on 74 genetic markers, seems to be relatively accurate.

Similarity among all pairwise cultivars obtained using the botanical descriptors presented thirteen pairwise comparisons with complete similarity (SM =1). The same comparisons using RAPD markers resulted in only one complete similarity (Table II). Using 29 randomly selected RAPD markers in this comparison produced only one pair (cultivars 2 and17) that presented complete similarity. Therefore, we observed that the botanical class traits used to discriminate the cultivars were not capable to differentiate thirteen pairwise cultivars 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 and17. 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 these relationships. We observed in group A the closeness of cultivars1, 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 including16,19, 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; Léfèvre,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 markerassisted 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 fragments 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 índices 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 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.

REFERENCES

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Beeching, J.R., Marmey, P., Gavalda, M.C., Noirot, M., Hayson, H.R., Hughes, M.A. and Charrier, A. (1993). An assessment of genetic diversity within a collection of cassava (Manihot esculenta Crantz) germplasm using molecular markers. Ann. Bot. 72: 515-520.

Bhat, K.V. and Jarret, R.L. (1995). Random amplified polymorphic DNA and genetic diversity in Indian Musa germplasm. Genet. Resour. Crop Evol. 42:107-118.

Charrier, A. and Léfèvre, F. (1987). La diversité génétique du manioc: son origine, son évaluation et son utilisation. In: La Mosaique Africaine du Manioc et son Contrôle (Fauquet, C. and Fargette, D., eds.). Proceedings of the International Seminar, Yamoussoukro,1987. Institut Français de Recherche Scientific pour le Développement en Coopération (IFRDC), Côte d’Ivoire, pp. 71-81.

Connolly, A.G., Godwin, I.D., Cooper, M. and DeLacy, I.H. (1994). Interpretation of randomly amplified polymorphic DNA marker data for fingerprinting sweet potato (Ipomoea batatas L.) genotypes. Theor. Appl. Genet. 88: 332-336.

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Hershey, C., Iglesias, C., Iwanaga, M. and Tohme, J. (1994). Definition of a core collection for cassava. In: International Network for Cassava Genetic Resources. Report of the the First Meeting of the International Network for Cassava Genetic Resources. CIAT, Cali, Colombia,18-23 August1992. International Crop Network Series No.10. International Plant Genetic Resources Institute, Rome, Italy.

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Rogers, D.J. (1972). Some further considerations on the origin of M. esculenta. Trop. Root Tuber Crops Newslett. 6: 4-10.

Rohlf, F.J. (1993). NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System. Version1.80. Exeter Software, Setauket, N.Y.

Schierwater, B. and Ender, A. (1993). Different thermostable DNA polymerases may amplify different RAPD products. Nucleic Acids Res. 21: 4647-4648.

Sokal, R. and Michener, C.D. (1958). A statistical method for evaluating systematic relationships. Univ. Kans. Sci. Bull. 38:1409-1438.

Stiles, J.I., Lemme, C., Sondur, S., Morshidi, M.B. and Manshardt, R. (1993). Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars. Theor. Appl. Genet. 85: 697-701.

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(Received June 26,1997)

  • Bayley, D.C. (1983). Isozymic variation and plant breeders’ rights. In: Isozymes in Plant Genetics and Breeding (Tanksley, S.D. and Orton, T.J., eds.). Part A. Elsevier, Amsterdam, pp. 425-441.
  • Beeching, J.R., Marmey, P., Gavalda, M.C., Noirot, M., Hayson, H.R., Hughes, M.A. and Charrier, A. (1993). An assessment of genetic diversity within a collection of cassava (Manihot esculenta Crantz) germplasm using molecular markers. Ann. Bot. 72: 515-520.
  • Bhat, K.V. and Jarret, R.L. (1995). Random amplified polymorphic DNA and genetic diversity in Indian Musa germplasm. Genet. Resour. Crop Evol. 42:107-118.
  • Charrier, A and Léfèvre, F. (1987). La diversité génétique du manioc: son origine, son évaluation et son utilisation. In: La Mosaique Africaine du Manioc et son Contrôle (Fauquet, C. and Fargette, D., eds.). Proceedings of the International Seminar, Yamoussoukro,1987. Institut Français de Recherche Scientific pour le Développement en Coopération (IFRDC), Côte d’Ivoire, pp. 71-81.
  • Connolly, A.G., Godwin, I.D., Cooper, M and DeLacy, I.H (1994). Interpretation of randomly amplified polymorphic DNA marker data for fingerprinting sweet potato (Ipomoea batatas L.) genotypes. Theor. Appl. Genet. 88: 332-336.
  • Cury, R. (1993). Dinâmica evolutiva e caracterização de germoplasma de mandioca (Manihot esculenta Crantz) na agricultura autóctone do Sul do Estado de São Paulo. Master’s thesis, ESALQ-USP, Piracicaba, SP.
  • Dunemann, F., Kahnau, R. and Schmidt, H. (1994). Genetic relationships in Malus evaluated by RAPD fingerprinting of cultivars and wild species. Plant Breed.113:150-159.
  • F.A.O. (1995). Annuaire de la Production1994. Vol. 48. Division de la Statistique, FAO, Rome, Italie.
  • Gower, J.C. (1966). Some distance properties of latent root and vector methods used in multivariate analysis. Biometrika 53: 325-338.
  • Hershey, C., Iglesias, C., Iwanaga, M. and Tohme, J. (1994). Definition of a core collection for cassava. In: International Network for Cassava Genetic Resources Report of the the First Meeting of the International Network for Cassava Genetic Resources CIAT, Cali, Colombia,18-23 August1992. International Crop Network Series No.10. International Plant Genetic Resources Institute, Rome, Italy.
  • Hussain, A. and Bushuk, W (1987). Identification of cassava (Manihot esculenta Crantz) cultivars by electrophoretic patterns of esterase isozymes. Seed Sci. Technol15:19-22.
  • Kernodle, S.P., Cannon, R.E and Scandalios, J.G. (1993). Concentration of primer and template qualitatively affects products in random-amplified polymorphic DNA PCR. Biotechniques14: 362-364.
  • Kresovich, S., Williams, J.G.K., McFerson, J.R., Routman, E.J. and Schaal, B.A. (1992). Characterization of genetic identities and relationships of Brassica oleracea L. via a random amplified polymorphic DNA assay. Theor. Appl. Genet. 85:190-196.
  • Léfèvre, F. (1989). Ressources génétiques et amelioration du manihot, Manihot esculenta Crantz, en Afrique. Doctoral thèsis. ORSTOM ed. coll TDM nEJ 57, Paris.
  • Marmey, P., Beeching, J.R., Hamon, S. and Charrier, A (1994). Evaluation of cassava (Manihot esculenta Crantz) germplasm collections using RAPD markers. Euphytica 74: 203-209.
  • Multani, D.S. and Lyon, B.R. (1995). Genetic fingerprinting of Australian cotton cultivars with RAPD markers. Genome 38:1005-1008.
  • Nei, M. (1978). Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89: 583-590.
  • Penner, G.A., Bush, A., Wise, R., Kim, W., Domier, L., Kasha, K., Laroche, A., Scoles, G., Molnar, S.J. and Fedak, G. (1993). Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories. PCR Meth. Appl. 2: 341-345.
  • Pereira, A.V., Vencovsky, R. and Cruz, C.D. (1989). Divergência genética em germoplasma elite de mandioca. Rev. Bras. Mand. 8: 77-95.
  • Ramirez, H., Hussain, A., Roca, W. and Bushuk, W. (1987). Isozyme electrophoregrams of sixteen enzymes in five tissues of cassava (Manihot esculenta Crantz) varieties. Euphytica 36: 39-48.
  • Rogers, D.J. (1972). Some further considerations on the origin of M. esculenta Trop. Root Tuber Crops Newslett. 6: 4-10.
  • Rohlf, F.J (1993). NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System Version1.80. Exeter Software, Setauket, N.Y.
  • Schierwater, B. and Ender, A. (1993). Different thermostable DNA polymerases may amplify different RAPD products. Nucleic Acids Res 21: 4647-4648.
  • Sokal, R. and Michener, C.D (1958). A statistical method for evaluating systematic relationships. Univ. Kans. Sci. Bull. 38:1409-1438.
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  • Publication Dates

    • Publication in this collection
      06 Jan 1999
    • Date of issue
      Mar 1998

    History

    • Received
      26 June 1997
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