Genetic characterization of banana cultivars - Springer Link

Euphytica 132: 259–268, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

259

Genetic characterization of banana cultivars (Musa spp.) from Brazil using microsatellite markers Silvana Creste1 , Augusto Tulmann Neto2 , Sebastião de Oliveira Silva3 & Antonio Figueira2 1 Departamento

de Genética, Escola Superior de Agricultura ‘Luiz de Queiroz’, Universidade de São Paulo; Av. Pádua Dias, 11 CP 83, Piracicaba, SP, 13400-970, Brazil; 2 Centro de Energia Nuclear na Agricultura, Universidade de São Paulo; Av. Centenário, 303, CP 96, Piracicaba, SP, 13400-970, Brazil; 3 EMBRAPA Mandioca e Fruticultura; R. Embrapa, CP 007; Cruz das Almas, BA, 44380-000, Brazil

Received 6 September 2002; accepted 18 April 2003

Key words: genetic similarity, germplasm, Musa, PCR, ploidy, silver staining, simple sequence repeats

Summary Microsatellite markers were used to characterize 35 banana (Musa spp.) genotypes cultivated in Brazil, including triploid cultivars and tetraploid hybrids. A total of 33 Musa-specific primers were tested, and 11 produced clear, reproducible and discrete bands. The average number of alleles amplified per primer was 6.1, ranging from 4 to 8, with a total of 67 alleles identified. Phenetic analysis based on Jaccard similarity index derived from presence or absence of the alleles agreed with the morphological classification. Bootstrap analysis divided the genotypes into four clusters, according to genomic group and subgroup classification. The first cluster contained the majority of cultivars which have ‘A’ genome alone; while the second contained all triploid cultivars of the subgroup Prata (Pome) and their tetraploid hybrids. The third cluster contained cultivar ‘Maçã’ together with other genotypes considered for breeding purposes as similar to the Silk subgroup. These last two clusters formed a larger group including the majority of genotypes that resulted from hybridization between M. acuminata and M. balbisiana. The microsatellite loci were highly informative, with some pair of primers generating an unique fingerprinting for each genomic group and discriminating a genotype of doubtful classification, although somatic mutants from a subgroup were seldom distinguished from their original clone. Tetraploid hybrids exhibited distortion in the proportion of alleles donated by their triploid female parent. For a few primers, some genotypes exhibited a higher number of alleles than expected from their ploidy level, suggesting the occurrence of duplicated alleles or duplicated chromosomal regions. Abbreviations: AFLP – amplified fragment length polymorphism; bp – base pairs; EMBRAPA – Empresa Brasileira de Pesquisa Agropecuária (Brazilian Agriculture Research Organization); PCR – Polymerase Chain Reaction; RAPD – random amplified polymorphic DNA; RFLP – restriction fragment length polymorphism; SSRs – simple sequence repeats; UPGMA – unweighted pair-grouping with arithmetic average

Introduction Edible bananas (Musa spp.) originated mainly from intra- and interspecific hybridizations between two wild diploid species, M. acuminata Colla (‘A’ genome) and M. balbisiana Colla (‘B’ genome) (Simmonds & Shepherd, 1955). Inter-crossing among species and subspecies has resulted in the appearance of sterility, a trait that was selected for during domest-

ication, together with parthenocarpy and vegetative propagation (Simmonds, 1995). Most current cultivars are triploid, and sometimes diploid or tetraploid. Taxonomically, banana cultivars and hybrids are classified based on ploidy analysis and a set of 15 morphological descriptors into genomic groups, differing for genome constitution and ploidy (Simmonds & Shepherd, 1955). The main genomic groups are AA, AAA, AAB and ABB, although AB, AAAB,

260 AABB and ABBB are also possible (Stover & Simmonds, 1987). Highly related clones or cultivars resulting from mutations in a single genotype are allocated to so-called subgroups, characterized by specific morphological and fruit quality attributes (Simmonds, 1973). In many countries (e.g. Ecuador, Costa Rica, Colombia), the production of bananas for export is mainly based on the Cavendish subgroup (group AAA), while a great number of cultivars and landraces (groups AAA, AAB and ABB) are cultivated for local consumption in Africa, Asia and Latin America (Jenny et al., 1999). Brazil has been ranked the third world largest producer of banana. Almost all the production is destined for the local market, which favors fruits from cultivars of the Prata (or Pome) and Silk (Maçã) subgroups (both AAB) (Silva et al., 2001). There is a large number of cultivars planted in Brazil, but their susceptibility to the main diseases (Fusarium wilt, Black Sigatoka, and Yellow Sigatoka) is the major obstacle for banana cultivation (Silva et al., 2001). Despite the high level of sterility in the majority of triploid cultivars, many of them are able to produce seeds when used as the female parent in crosses with diploids, and among the various types of hybrids recovered, with different ploidy levels (diploid, triploid, tetraploid, hyperploid and aneuploids) (Ortiz, 1995), tetraploid hybrids are preferred for breeding. The selection of the best diploids and triploids to be used in hybridization programs to develop improved tetraploid hybrids has been based on agronomic and morphological characteristics (Silva et al., 1998). The occurrence of local names, synonymous and homonymous, plus the high occurrence of somatic mutants for some cultivars has limited the full knowledge of the genetic resources available. Molecular markers have been employed in the characterization and evaluation of genetic diversity in Musa species, including restriction fragment length polymorphism (RFLP, Gawel et al., 1992; Fauré et al., 1994; Carreel et al., 2002); random amplified polymorphic DNA (RAPD, Bhat & Jarret, 1995; Pillay et al., 2000, 2001); amplified fragment length polymorphism (AFLP, Loh et al., 2000; Ude et al., 2002); and microsatellites or simple sequence repeats (SSRs, Lagoda et al., 1998a; Crouch et al., 1998; Kaemmer et al., 1997; Grapin et al., 1998). Of all these techniques, microsatellites have proved to be the best marker for banana typing (Grapin et al., 1998), as they are highly polymorphic, show a co-dominant mode of inheritance, are reproducible and easy to interpret.

DNA microsatellites are polymorphic elements, which are abundant in nuclear genomes and consist of a succession of a small repeat-unit, usually less than four nucleotides long (Wang et al., 1994). These regions are uniformly distributed throughout eukaryotic genomes, generally inserted in regions of single copies (Tautz, 1989). Fragments containing microsatellites can be amplified by the polymerase chain reaction (PCR) using a pair of primers flanking the repeat sequence. The polymorphism between genotypes is due to the variation in the number of repeat units. In Musa, Crouch et al. (1998) have developed specific primers for 24 microsatellite loci, while Lagoda et al. (1998b) have proposed primers for a further 47 loci. The objective of this research was to characterize 35 banana genotypes cultivated in Brazil, including commercial cultivars, landraces and tetraploid hybrids, using these Musa-specific microsatellite loci.

Material and methods Plant material The 35 banana genotypes used in this work included commercial cultivars, landraces and tetraploid hybrids (Table 1), maintained at the Musa germplasm collection of the ‘EMBRAPA Mandioca e Fruticultura’ (Cruz das Almas, BA, Brazil). The cultivars and tetraploid hybrids have been previously morphologically and cytogenetically characterized, and categorized into genomic groups (Silva et al., 1997), while landraces included unclassified materials with localized importance, recognized by a local name. The diploid banana cultivar ‘Lidi’ (AA), a commonly used male parent of tetraploid hybrids in Brazil, was included as an outgroup (Table 1). Primers A total of 33 primer-pairs (referred as primers throughout the text) were tested (Table 2). Twenty seven primers, comprising the complete ‘Ma-series’ developed by Crouch et al. (1998), were purchased from Research Genetics Inc. (Huntsville, AL, USA), and 6 primers (AGMI 24/25, AGMI 93/94, AGMI 121/122, AGMI 157/158, AGMI 67/68, AGMI 161/162) developed by Lagoda et al. (1998b), were synthesized by Life Technologies do Brasil (São Paulo, SP, Brazil).

261 Table 1. The 35 banana genotypes used in the study, with information on genomic constitution, ploidy, subgroup and type (cultivar, mutant, landrace or hybrid) Genotype

Genomic group

Ploidy

Subgroup

Type

Grande Naine Nanica Nanicão PBN Williams

AAA AAA AAA AAA AAA

3x 3x 3x 3x 3x

Cavendish Cavendish Cavendish Cavendish Cavendish

Cultivar Cultivar Cultivar Mutant Cultivar

Gros Michel Bucaneer Ambrosia Calipson

AAA AAAA AAAA AAAA

3x 4x 4x 4x

Gros Michel Gros Michel-derived Gros Michel-derived Gros Michel-derived

Cultivar Hybrid (‘High Gate’ × 2n) Hybrid (‘High Gate’ × 2n) Hybrid (‘High Gate’ × 2n)

Caipira (=Yangambi km 5)

AAA

3x

–

Cultivar

Mysore Thap Maeo

AAB AAB

3x 3x

– –

Cultivar Cultivar

Branca Santa Catarina Enxerto Prata Prata (Cruz das Almas) Prata Ponta Aparada Prata Porte Baixo Prata Santa Maria Prata Jau Prata Anã Pioneira FHIA-01 FHIA-18 SH3640 Pacovan PV42-142 PV42-85 PV42-68

AAB AAB AAB AAB AAB AAB AAB AAB AAB AAAB AAAB AAAB AAAB AAB AAAB AAAB AAAB

3x 3x 3x 3x 3x 3x 3x 3x 3x 4x 4x 4x 4x 3x 4x 4x 4x

Prata Prata Prata Prata Prata Prata Prata ? Prata Prata-derived Prata-derived Prata-derived Prata-derived Prata Prata-derived Prata-derived Prata-derived

Landrace Cultivar Cultivar Cultivar Landrace Mutant Landrace Landrace Cultivar Hybrid (‘Prata Anã’ × ‘Lidi’) Hybrid (‘Prata Anã’ × 2n) Hybrid (‘Prata Anã’ × 2n) Hybrid (‘Prata Anã’ × 2n) Cultivar Hybrid (‘Pacovan’ × ‘M53’) Hybrid (‘Pacovan’ × ‘M53’) Hybrid (‘Pacovan’ × ‘M53’)

Ouro da Mata

AAAB

4x

–

Landrace

Maçã Yangambi no. 2 YB42-21

AAB AAB AAAB

3x 3x 4x

Silk – –

Cultivar Cultivar Hybrid (‘Yangambi no. 2’ × 2n)

Terra Lidi

AAB AA

3x 2n

Terra –

Cultivar Cultivar

DNA extraction and PCR conditions Genomic DNA of each genotype was extracted from leaves using the CTAB method proposed by Doyle & Doyle (1990), and quantified by fluorimetry in a DyNA Quant 2000 fluorometer (Amersham Biosciences, Buckinghamshire, UK). All the PCR reac-

tions were performed on a Perkin Elmer 9700 thermocycler (Applied Biosystems, Foster City, CA, USA), in 25 µl reactions, containing 50 ng of genomic DNA; 50 mM KCl; 10 mM Tris-HCl (pH 8.8); 0.1% TritonX; 1.5 or 2.5 mM MgCl2 (as indicated on Table 2); 100 µM of each dNTPs; 0.2 µM of each primer and

262 Table 2. Primers tested in the characterization of 35 Musa genotypes, with original allele length; original annealing temperature (suggested by Research Genetics Inc. for Ma-series); optimized annealing temperatures for touchdown cycle; and description of results of amplification Primers

Expected allele length (bp)z

Suggested Ta ◦ C

Optimized touchdown cycle

Amplification results

Ma-1-2 Ma-1-3 Ma-1-5 Ma-1-6 Ma-1-16 Ma-1-17 Ma-1-18 Ma-1-24 Ma-1-27 Ma-1-31 Ma-1-32 Ma-2–3 Ma-2–4 Ma-2–7 Ma-2–12 Ma-2–23 Ma-3-1 Ma-3-60 Ma-3-68 Ma-3-90 Ma-3-103 Ma-3-127 Ma-3-130 Ma-3-131 Ma-3-132 Ma-3-139 Ma-3-161w AGMI 24/25 AGMI 67/68w AGMI 93/94 AGMI 121/122 AGMI 157/158 AGMI 161/162

142 160 120 139 150 124 150 172 126 133 187 246 175 89 160 250 133 133 244 140 152 152 198 123 160 144 188 248 n.a.y 128 333 143 n.a.y

58 ◦ C 57 ◦ C 56 ◦ C 57 ◦ C 58 ◦ C 57 ◦ C 58 ◦ C 56 ◦ C 56 ◦ C 56 ◦ C 58 ◦ C 57 ◦ C 58 ◦ C 55 ◦ C 58 ◦ C 57 ◦ C 56 ◦ C 56 ◦ C 57 ◦ C 56 ◦ C 56 ◦ C 56 ◦ C 58 ◦ C 56 ◦ C 57 ◦ C 55 ◦ C 58 ◦ C 55 ◦ C 50 ◦ C 55 ◦ C 48 ◦ C 54 ◦ C 50 ◦ C

60–50 ◦ C 60–50 ◦ C 65–55 ◦ C – – 65–55 ◦ C – 65–55 ◦ C 65–55 ◦ C – 65–55 ◦ C – 60–50 ◦ C 60–50 ◦ C – – – – – 60–50 ◦ C 65–55 ◦ C – – – 65–55 ◦ C 65–55 ◦ C 65–55 ◦ C 65–55 ◦ C 60–50 ◦ C 60–50 ◦ C 60–50 ◦ C – –

Failed/Polymorphicx Monomorphic Monomorphic Non-specific Non-specific Polymorphic Non-specific Failed/Polymorphicx Failed/Polymorphicx Non-specific Polymorphic Non-specific Monomorphic Monomorphic Non-specific Non-specific Non-specific Non-specific Non-specific Polymorphic Polymorphic Non-specific Non-specific Non-specific Polymorphic Polymorphic Polymorphic Polymorphic Polymorphic Polymorphic Polymorphic Non-specific Non-specific

z expected length of allele from original clone used to develop primers according to Crouch et al.

(1998) and Lagoda et al. (1998b). y n.a. = not available. x failed/polymorphic is equivalent to lack of amplification for some genotypes. w 2.5 mM MgCl concentration, all the others 1.5 mM MgCl . 2 2

1.5 units of Taq polymerase (Life Technologies do Brasil). Each primer was initially screened for amplification of a specific product from banana genomic DNA using the originally suggested annealing temperature (Table 2), followed by testing a 65 to 55 ◦ C touchdown PCR program, comprised of an initial step of 3 min at 94 ◦ C, followed by 9 cycles of 40 s at 94 ◦ C; 40 s at annealing temperature decreasing from

65 to 56 ◦ C by 1 ◦ C every cycle, and 60 s at 72 ◦ C, followed by 30 identical cycles, except for the annealing temperature at 55 ◦ C for 40 s. For those primers that exhibited no product with these conditions, a 60 to 50 ◦ C touchdown program was tested (Table 2).

263 Electrophoresis and polymorphism detection Sequencing gels (6% polyacrylamide, 8 M urea) were run under standard conditions and the products were visualized by silver staining according to the procedure described by Creste et al. (2001). Data analysis Amplified fragments were scored for presence or absence in all the 35 genotypes. The genetic similarity between all genotypes was calculated according to the Jaccard coefficient. Relationships among genotypes were evaluated with a phenetic cluster analysis, using the unweighted pair-grouping with arithmetic average (UPGMA) clustering, and plotted in a phenogram using NTSYS-PC version 2.0j (Exeter Software, Setauket, NY, USA). Bootstrap analysis was performed using the WinBoot program (Yap & Nelson, 1996), with 1000 repetitive samplings of the microsatellite data to compute bootstrap P values. Heterozygote frequency was calculated for each loci based on the frequency of individuals with two or more alleles.

Results A total of 33 primers were tested to amplify specific products in PCR reactions of 35 banana genotypes. When no amplification occurred or products were nonspecific, attempts to optimize the PCR were carried out by altering the annealing temperatures; by applying a touchdown program; or by adjusting the concentration of MgCl2 (Table 2). Of the 33 primers tested, 11 (33.3%) amplified products resulting in discrete, repeatable polymorphic bands; four (12.1%) produced monomorphic bands; 15 (45.4%) amplified unspecific products, even after changing amplification conditions; and three (9.1%) did not amplify products in some genotypes (Table 2). To ensure that the occurrence of null alleles was not a failure of reaction, the assays were repeated several times. Primers producing putative null alleles were not further considered for analysis. From the 11 primers that amplified polymorphic products, 67 alleles were detected, with a mean of 6.1 alleles per locus (Table 3). The highest degree of polymorphism, with 8 alleles detected, was observed for primers Ma-1-17 and Ma-3-90, and the lowest (4 alleles) with primers Ma-3-132 and AGMI 121/122. The

Table 3. Microsatellite primers which produced clear and distinct amplification products used in this study Primer

Variation in allele size (bp)

Number of alleles

Heterozygote frequency

Ma-1-17 Ma-1-32 Ma-3-90 Ma-3-103 Ma-3-132 Ma-3-139 Ma-3-161 AGMI 24/25 AGMI 67/68 AGMI 93/94 AGMI 121/122 Total Overall Mean

104–138 243–277 129–169 126–160 150–186 114–141 103–141 250–310 186–220 129–143 293–350

8 5 8 6 4 6 7 7 7 5 4 67 6.1

1.00 0.63 0.87 1.00 1.00 0.97 0.97 0.87 0.87 0.87 0.83 0.90

mean heterozygote frequency was 90%, ranging from 63 to 100% for each microsatellite locus (Table 3). The number of alleles per genotype ranged from one to two for the diploid; one to five for triploids; and one to six for tetraploids. Most genotypes exhibited two (40% of cultivars) or three (35%) alleles for each locus, but four primers detected more alleles than expected, considering the basic ploidy level of the genotypes. Primer Ma-1-17 detected 4 alleles in the triploids from subgroup Cavendish, and genotypes ‘Gros Michel’ and ‘Maçã’, as well as 5 alleles in the tetraploids ‘Bucaneer’, ‘Ambrosia’, and ‘Calipson’. Similarly, primer Ma-3-139 amplified 4 alleles from some triploids of the Prata subgroup (‘Enxerto’; ‘Prata’; ‘Prata Porte Baixo’; ‘Prata Cruz das Almas’; ‘Prata Santa Maria’; ‘Prata Ponta Aparada’; and ‘Prata Anã’), and genotype ‘Maçã’, while primer Ma-3-161 detected 5 alleles in ‘Thap Maeo’ and ‘FHIA-18’, and 6 alleles in ‘FHIA-01’. Primer AGMI 93/94 revealed 4 alleles in the triploid ‘Branca Santa Catarina’and 5 alleles in the tetraploid ‘SH3640’. Primer Ma-3-103 detected two duplicated loci (Figure 1A), identifying two alleles (130 and 150 bp) present only in genotypes with ‘B’ genome (except for genotypes ‘FHIA-18’ and ‘Terra’), as well as two alleles (140 and 160 bp) present only in genotypes that possess the M. acuminata genome alone (except for ‘Terra’). The AGMI 24/25 primer was highly informative (Figure 1B), able to detect the ploidy level of 29 genotypes (83%) and to generate a unique finger-

264

Figure 1. Amplification products generated by primers Ma-3-103 (A); AGMI 24/25 (B) and Ma-3-90 (C). Key: m = 123 base pair ladder marker; 2 = ‘Williams’; 3 = ‘Grande Naine’; 4 = ‘Nanica’; 5 = ‘Nanicão’; 6 = ‘PBN’; 7 = ‘Ouro da Mata’; 8 = ‘Branca Santa Catarina’; 9 = ‘Gros Michel’; 10 = ‘Bucaneer’; 11 = ‘Ambrosia’; 12 = ‘Calipson’; 13 = ‘Mysore’; 14 = ‘Thap Maeo’; 15 = ‘Prata Jau’; 16 = ‘Prata’; 17 = ‘Prata (Cruz das Almas)’; 18 = ‘Prata Santa Maria’; 19 = ‘Prata Ponta Aparada’; 20 = ‘Enxerto’; 21 = ‘Prata Anã’; 22 = ‘Pioneira’; 23 = ‘FHIA-01’; 24 = ‘FHIA-18’; 25 = ‘SH3640’; 26 = ‘Pacovan’; 27 = ‘PV42-142’; 28 = ‘PV42-85’; 29 = ‘PV42-68’; 30 = ‘Caipira’; 31 = ‘Maçã’; 32 = ‘Yangambi no. 2’; 33 = ‘YB42–21’; 34 = ‘Terra’; 35 = ‘Prata Porte Baixo’; 36 = ‘Lidi’; c = control. The arrows indicate the marker for genome ‘B’.

printing for all the genomic groups studied. For this primer, ‘Prata Jau’ exhibited two B alleles of distinct size, while ‘Terra’ presented a B allele slightly larger. Bootstrap analysis divided the genotypes into four significantly different clusters, considering significant bootstrap P values above 80% (Figure 2). The first cluster contained the majority of cultivars which have ‘A’ genome alone (P = 81.7%). Group II (Figure 2) contained all triploid cultivars of the subgroup Prata and their tetraploid hybrids (P = 82.9%). The third group (P = 99.5%) contained cultivar ‘Maçã’ from the Silk subgroup, plus the triploid ‘Yangambi no. 2’ and the tetraploid hybrid ‘YB 42-21’, all considered for breeding purposes as similar to Silk (Silva et al., 2001). These last two clusters formed a larger group (P = 66.5%) including the majority of genotypes resultant of hybridization between M. acuminata and

M. balbisiana. Cultivars ‘Mysore’ and ‘Thap Maeo’ formed another robust cluster (group IV; P = 100%), more distantly from the major interspecific cluster. Cultivars ‘Terra’ and ‘Prata Jau’ were outliers from the major groups (Figure 2). The triploid cultivar ‘Caipira’ and the diploid ‘Lidi’ exhibited the lowest similarity with all the other genotypes and were not grouped. Cultivars of the Cavendish subgroup exhibited 100% similarity and could not be distinguished with the primers used. The same was observed for cultivars of the Prata subgroup, except for cultivar ‘Branca Santa Catarina’ and ‘Pacovan’, which could be discriminated with primer Ma-3-161 and AGMI 121/122, respectively. The cultivar ‘Thap Maeo’ is a variant of the ‘Mysore’, but it was possible to be distinguished from the other with the primer Ma-3-161.

265

Figure 2. Phenogram demonstrating the genetic relationships among banana cultivars based on microsatellite markers, derived from Jaccard coefficient of similarity. Bootstrap P values are given at the corresponding node for each cluster.

Tetraploid hybrids sharing the same female parent clustered together, e.g. hybrids ‘PV42-142’, ‘PV4268’ and ‘PV42-85’ derived from the ‘Pacovan’ cultivar were highly similar, as were the tetraploids ‘FHIA01’, ‘FHIA-18’, ‘SH3640’ and ‘Pioneira’ derived from the cultivar ‘Prata Anã’ (Figure 2). The hybrids ‘Ambrosia’ and ‘Calipson’, derived from ‘High Gate’ cultivar (a dwarf mutant from ‘Gros Michel’ cultivar) showed 100% similarity, but the hybrid ‘Bucaneer’ was discriminated from these two hybrids by primer Ma-3-90 (Figure 1C). Discussion A small proportion of the primers tested (33.3%; 11 in 33) amplified scorable polymorphic products from the 35 banana genotypes, while the other primers (54.5%) amplified either unspecific products or failed to amplify products in some genotypes (Table 2). The lack of amplification of products in some genotypes has been a common observation in Musa (Crouch et al., 1998, 1999a, b) and it may reflect divergences in the sequences flanking the microsatellite loci, leading to the production of possible null alleles, or totally restricting amplification.

Some triploid and tetraploid genotypes presented more alleles than expected for four microsatellite loci. Crouch et al. (1999a) had also detected an unexpected higher number of alleles amplified from the diploid genotype ‘Calcutta 4’ (AA), the triploid plantain ‘Obino l’Ewai’ (AAB) and their progeny, and proposed that a high frequency of duplicated alleles or duplicated chromosomal regions may occur in genomes A and B of Musa. There were some problems to assign the exact length of some alleles. For example, the specific allele for ‘B’ genome generated by primer AGMI 24/25 was reported by Kaemmer et al. (1997) as 254 bp long, but in our study it was estimated as being 290 bp. The size of some other alleles were also different to what had been expected (Lagoda et al., 1998b; Crouch et al., 1998). It has been suggested by Testolin et al. (2000) that the possible causes for this discrepancy may include the denaturation conditions during electrophoresis; the type of molecular weight marker (ladder) used as the standard; and differences in the base pair composition of the microsatellites. The clustering analysis (Figure 2) based on microsatellite polymorphism is in agreement with characterization based on morphological descriptors. The

266 primers used here were able to separate cultivars containing the M. acuminata genome alone, from interspecific hybrids of M. acuminata and M. balbisiana, and to arrange the genotypes into groups and subgroups. A high degree of polymorphism within the genomic groups was detected, possibly reflecting the high degree of variability, which exists within the M. acuminata complex of subspecies (Jenny et al., 1999; Carreel et al., 2002). However, the occurrence of polymorphism was substantially reduced, or absent, within the subgroups. No polymorphism was detected within the Cavendish subgroup. Cultivars from the Cavendish subgroup presented 100% similarity based on the microsatellite primers used and could not be distinguished, although they present some differences in morphology. The same was observed for the genotypes from subgroup Prata, except for a few genotypes (‘Branca Santa Catarina’ and ‘Pacovan’) that could be distinguished with specific primers (Ma-3-161 and AGMI 121/122). The most cultivated bananas in Brazil are from Prata subgroup, and there are innumerous unclassified landraces with localized importance, recognized by a local name associated with the Prata subgroup (e.g. ‘Prata Jau’). Here, we have shown that most of the landraces evaluated (e.g. ‘Prata Santa Maria’, ‘Prata Ponta Aparada’) are highly similar or identical to the cultivar ‘Prata’. However, the landrace ‘Prata Jau’, with doubtful classification, did not cluster with the other ‘Prata’ cultivars, despite the clear name relationship with subgroup Prata. ‘Prata Jau’ might be classified in ABB group since it exhibited two distinct B alleles with primer AGMI 24/25. Thus, it is possible that other landraces, with specific morphological and/or fruit quality attributes similar to Prata, but unrelated, might occur in Brazil, with potential use in breeding. Cultivars ‘Enxerto’ and ‘Prata Anã’ are duplicates, and therefore synonymous (Silva et al., 1997) and appeared to be identical based on all the markers tested. The microsatellites here used showed to be suitable for Musa classification into group and subgroups, and to identify synonymous and homonymous cultivars/landraces. The division of Musa species into subgroups is based on common origin or mutations in a cultivar, although these mutations may have important effects for commercialization (Dantas et al., 1997). Such mutations are mainly reflected in changes in the color of leaves and the pseudostem as well as changes in the size and vigor of the plants, while maintaining fruit quality (Jenny et al., 1999). Large variations in mor-

phological characteristics do not necessarily reflect the same degree of genetic variation (Gawel et al., 1992). According to Jenny et al. (1999), the variability within each subgroup is mainly dependent on the genotype and the frequency with which each clone is multiplied and planted. High levels of genetic similarity are expected between cultivars from the same subgroup because they share a common origin. The observation of monomorphic band profiles among cultivars of the same subgroup reflects the maintenance of allele composition during successive cycles of vegetative propagation. The use of SSRs to discriminate between somatic mutants and the clone from which the mutants originated have been studied in other vegetatively propagated fruits. Sánchez-Escribano et al. (1999) employed eight SSR markers to discriminate between 43 cultivars of table grapes, but could not detect somatic mutants, while Testolin et al. (2000) used 26 SSR markers to investigate 50 peach clones and were able to detect only a few somatic mutants. In Musa species, superior tetraploid hybrids (AAAB) have been generated through hybridization of triploid landraces (AAB) with diploid genotypes (AA) (Silva et al., 2001). Under the hypothesis that the full female genotype is represented in the tetraploid hybrid offspring of the crosses, recombination might only occur for the diploid parent. In this study, some hybrids presented distortions in the proportion of alleles donated by the female parent. For instance, the hybrids ‘SH3640’, ‘FHIA-18’ and ‘Pioneira’ had one allele less than their female parent ‘Prata Anã’, as detected by primers Ma-1-17, Ma-3-103 and Ma-3-139 respectively, while hybrid ‘PV42-68’ presented only one allele for primer AGMI 121/122 although the female parent (‘Pacovan’) exhibited three alleles. These data support the observation of Crouch et al. (1998) that gametic recombination is occurring in the female parent. The co-dominant nature of SSR markers allows one to estimate the allelic relationships among the genotypes. However, in this study, the band pattern observed did not allow the definition of the allelic relationships between genotypes belonging to distinct subgroups. This effect occurred because of errors in scoring microsatellite polymorphism in polyploid heterozygous species, in which each allele is interpreted as a unique character without considering the effect of gene dosage (Provan et al., 1996). For example, in two triploid cultivars containing the same two alleles in a specific locus, the first cultivar being duplex for the first allele (A1 A1 A2 ) and the second, duplex for the

267 second allele (A1 A2 A2 ), both appeared identical by the SSR markers, when in reality they are only 66% similar. Discrimination between these two cultivars is only possible using methods that allow the estimation of allele dosage, such as quantitative PCR. Even though the genetic similarity revealed by SSR markers in polyploid heterozygous species has a limited level of correspondence with real genetic similarity, the occurrence of multiple alleles and/or duplicated chromosomal regions makes them a powerful tool for fingerprinting and discrimination between different genotypes. In our research, a good example of the usefulness of SSR markers is the primer AGMI 24/25, which generated a unique pattern of bands for each of the genomic groups studied, determined the ploidy level of 29 (83%) genotypes and also discriminated the ‘Prata Jau’ cultivar, which had no relationship with cultivars of the Prata subgroup. In conclusion, the 11 microsatellite primers here described were able to discriminate most of the banana cultivars investigated. In some cases, the primers were able to discriminate clones from their respective somatic mutants; to detect erroneously classified cultivars; and to identify duplicates, as was the case for the Prata subgroup.

Acknowledgements We acknowledge financial support from FAPESP (97/10969-4) and CNPq (fellowship to SC, ATN, SOS).

References Bhat, K.V. & R.L. Jarret, 1995. Random amplified polymorphic DNA and genetic diversity in Indian Musa germplasm. Genet Res Crop Evol 42: 107–118. Carreel, F., D.G. Leon, P.J.L. Lagoda, C. Lanaud, C. Jenny, J.P. Horry & H.T. du Montcel, 2002. Ascertaining maternal and paternal lineage within Musa by chloroplast and mitochondrial DNA RFLP analyses. Genome 45: 679–692. Creste, S., A. Tulmann-Neto & A. Figueira, 2001. Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining. Plant Mol Biol Rep 19: 299–306. Crouch, H.K., J.H. Crouch, R.L. Jarret, P.B. Cregan & R. Ortiz, 1998. Segregation at microsatellite loci in haploid and diploid gametes of Musa. Crop Sci 38: 211–217. Crouch, J.H., H.K. Crouch, A. Tenkouano & R. Ortiz, 1999a. VNTR-based diversity analysis of 2x and 4x full-sib Musa hybrids. Eletron J Biotechn 2: 99–108. Crouch, J.H., H.K. Crouch, H. Constandt, A. Van Gysel, P. Breyne, M.V. Montagu, R.L. Jarret & R. Ortiz, 1999b. Comparison

of PCR-based molecular marker analyses of Musa breeding populations. Mol Breeding 5: 233–244. Dantas, J.L.L., K. Shepherd, S.O. Silva & W.S. Soares-Filho, 1997. Classificação botânica, origem, evolução e distribuição geográfica. In: E.J. Alves (Ed.), A Cultura da Banana: aspectos técnicos, socioeconômicos e agroindustriais, pp. 27–34. Brasília, Embrapa. Doyle, J.J. & J.L. Doyle, 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13–15. Fauré, S., J.L. Noyer, F. Carreel, J.P. Horry, F. Bakry & C. Lanaud, 1994. Maternal inheritance of chloroplast genome and paternal inheritance of mitochondrial genome in bananas (Musa acuminata). Curr Genet 25: 265–269. Gawel, N.J., R.L. Jarret & A.P. Whittemore, 1992. Restriction fragment length polymorphism (RFLP)-based phylogenetic analysis of Musa. Theor Appl Genet 84: 286–290. Grapin, A., J.L. Noyer, F. Carreel, D. Dambier, F.-C. Baurens, C. Lanaud & P.J.L. Lagoda, 1998. Diploid Musa acuminata genetic diversity assayed with sequence-tagged microsatellite sites. Electrophoresis 19: 1374–1380. Jenny, C., F. Carreel, K. Tomekpe, X. Perrier, C. Dubois, J.P. Horry & H.T. Montcel, 1999. Les bananiers. In: P. Hamon, M. Seguin, X. Perrier & J.C. Glaszman (Eds.), Centre de Coopération Internationale en Recherche Agronomique pour le Développement. Diversité Génétique des Plantes Tropicales, pp. 113–139. Montpellier, CIRAD. Kaemmer, D., D. Fisher, R.L. Jarret, F.-C. Baurens, A. Grapin, D. Dambier, J.L. Noyer, C. Lanaud, G. Khal & P.J.L. Lagoda, 1997. Molecular breeding in the genus Musa: a strong case for STMS marker technology. Euphytica 96: 49–63. Lagoda, P.J.L., D. Dambier, A. Grapin, F.-C. Baurens, C. Lanaud & J.L. Noyer, 1998a. Nonradioactive sequence-tagged microsatellite site analyses: A method transferable to the tropics. Electrophoresis 19: 152–157. Lagoda, P.J.L., J.L. Noyer, D. Dambier, F.-C. Baurens, A. Grapin & C. Lanaud, 1998b. Sequence tagged microsatellite site (STMS) markers in the Musaceae. Mol Ecol 7: 657–666. Loh, J.P., R. Kiew, O. Set, L.H. Gan & Y.Y. Gan, 2000. Amplified fragment length polymorphism fingerprinting of 16 bananas cultivars (Musa cvs.). Mol Phylogenet Evol 17: 360–366. Ortiz, R., 1995. Musa genetics. In: S. Gowen (Ed.), Bananas and Plantains, pp. 84–109, Chapman & Hall, London. Pillay, M., D.C. Nwakanma & A. Tenkouano, 2000. Identification of RAPD markers linked to A and B genome sequences in Musa L. Genome 43: 763–767. Pillay M., E. Ogundiwin, D.C. Nwakanma, G. Ude & A. Tenkouano, 2001. Analysis of genetic diversity and relationships in East African banana germplasm. Theor Appl Genet 102: 965–970. Provan, J., W. Powell & R. Waugh, 1996. Microsatellite analysis of relationships within cultivated potato (Solanum tuberosum). Theor Appl Genet 92: 1078–1084. Sánchez-Escribano, E.M., J.P. Martin, J. Carreño & J.L. Cenis, 1999. Use of sequence-tagged microsatellite site markers for characterizing table grape cultivars. Genome 42: 87–93. Silva, S.O., E.J. Alves, K. Shepherd & J.L.L. Dantas, 1997. Cultivares. In: E.J. Alves (Ed.), A Cultura da Banana: aspectos técnicos, socioeconômicos e agroindustriais, pp. 85–105. Brasília, Embrapa. Silva, S.O., A.P. Matos & E.J. Alves, 1998. Melhoramento genético da bananeira. Pesqui Agropecuária Brasileira 33: 693–703. Silva, S.O, M.T. Souza-Junior, E.J. Alves, J.R.S. Silveira & M.B. Lima, 2001. Banana breeding program at Embrapa. Crop Breed Appl Biotechn 1: 399–436.

268 Simmonds, N.W. & K. Shepherd. 1955. The taxonomy and origins of the cultivated bananas. Bot J Linn Soc 55: 302–312. Simmonds, N.W., 1973. Los plátanos: técnicas agrícolas y producciones tropicales. Editorial Blume, Barcelona, Spain. Simmonds, N.W., 1995. Bananas. In: J. Smartt & N.W. Simmonds (Eds.), Evolution of Crop Plants, pp. 370–375. Longman, Essex, UK. Stover, R.H. & N.W. Simmonds, 1987. Bananas. 3rd Edn. Longman Scientific & Technical. New York, USA. Tautz, D., 1989. Hypervariability of simple sequences as a general source of polymorphic markers. Nucl Acids Res 17: 6463–6471. Testolin, R., T. Marrazzo, G. Cipriani, R. Quarta, I. Verde, M.T. Dettori, M. Pancaldi & S. Sansavini, 2000. Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars. Genome 43: 512–520.

Ude, G., M. Pillay, D. Nwakanma & A. Tenkouano, 2002. Genetic diversity in Musa acuminata Colla and Musa balbisiana Colla and some of their natural hybrids using AFLP markers. Theor Appl Genet 104: 1246–1252. Wang, Z., J.L. Weber, G. Zhong & S.D. Tanksley, 1994. Survey of plant short tandem repeats. Theor Appl Genet 88: 1–6. Yap, I.V. & R.J. Nelson, 1996. WinBoot: A Program for Performing Bootstrap Analysis of Binary Data to Determine the Confidence Limits of UPGMA-based Dendrograms. Manila: International Rice Research Institute – IRRI, 22 pp. (Discussion paper series, 14).