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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. Brazils 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 worlds 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 Agronmico (IAC), Av. Baro 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 Suprieure Agronomique de Montpellier). 2, Place Viala. 34060-Montpellier, France.

selection within genotype collections of landraces and F1 segregation progenies (Valle, 1990). An under- standing 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, finger- printing 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 Lfvre, 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 Tour, 1985; Hussain and Bushuk, 1987; Ramirez et. al., 1987; Lfvre, 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 Agronmico at Campinas, So 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 PodeL 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 PodoC6uII F2030 Guapiara/SP GV GR RV b 1 ro DB C Y 7 unknown F3013 SoSeb.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 Femandpolis/SP GV GR RG b 1 s L B r W 10 unknown F4072 Araatuba/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 StaBrbaraDOeste/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 So 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 Guaxup SRT454 Guaxup/MG VG GR RV n 1 ro DB W W 19 CarapII 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 So Mateus/ES GV GR RV n 1 ro DB W W 25 Izabel Souza I SRT1229 Paraba 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 PoXIII 29 Bambu SRT1341 So 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% -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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