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Amplified Fragment Length Polymorphism Fingerprinting of 16 Banana Cultivars (Musa cvs.)
Molecular Phylogenetics and Evolution Vol. 17, No. 3, December, pp. 360 –366, 2000 doi:10.1006/mpev.2000.0848, available online at http://www.idealibrary.com on
Amplified Fragment Length Polymorphism Fingerprinting of 16 Banana Cultivars (Musa cvs.) Jin Phang Loh,* Ruth Kiew,† Ohn Set,† Leong Huat Gan,* and Yik-Yuen Gan* ,1 *School of Science, National Institute of Education, Nanyang Technological University, 469 Bukit Timah Road, Singapore 259756; and †Singapore Botanic Gardens, 1 Cluny Road, Singapore 259569 Received October 1, 1999; revised July 26, 2000; published online November 30, 2000
Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits. © 2000 Academic Press
INTRODUCTION The bananas are one of the major subtropical food crops for millions of people worldwide (Grapin et al., 1998). Despite the importance of banana in trade and commerce, little is known of the genetics of its agronomically important traits. Several pathogens seriously threaten banana production and numerous breeding programs have been undertaken to create resistant varieties (Sagi et al., 1998). Breeding programs face extreme difficulty due to the phenomenon of parthenocarpy, female genetic sterility, and structural hybridity (Bhat et al., 1995). The conventional method of classifying Musa cultivars (Simmonds and Shepherd, 1955) is based on 15 morphological traits. Problems associated with variability, environmental factors, and individual bias have made proper identification of banana cultivars difficult. In addition, characterization of cultivars relies heavily on fruit charac1
To whom correspondence should be addressed. Fax: 65-469-8928. E-mail: [email protected]. 1055-7903/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
ters, making it almost impossible to identify sterile plants. Furthermore, there is as yet no genetic basis for this method of classification. DNA fingerprinting techniques, however, will allow for the correct identification of species and cultivars (Kaemmer et al., 1992), as well as being useful in plant patents and for the legal protection of newly bred cultivars in asexual Musa, such as genetically engineered cultivars resistant to diseases. Various laboratory-based techniques have been used to study genetic diversity in banana. These include isozyme analysis (Bhat et al., 1992), restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) (Bhat et al., 1995), inter simple sequence repeat (ISSR) markers (Godwin et al., 1997), and sequence-tagged microsatellite sites (STMS) (Grapin et al., 1998). These techniques have their limitations. Isozymes are generally limited by the relatively low levels of polymorphism detectable and can fail to identify cultivars differing in only a few genes (Jarret and Litz, 1986). RFLP overcame this problem but requires large quantities of relatively pure DNA, and the frequent use of radioisotopes in the detection method makes it technically demanding, laborious, and costly to characterize large numbers of samples. PCR-based RAPD assay overcame many of the technical limitations of RFLPs but has proven sensitive to experimental conditions (Paul et al., 1997). ISSR markers are more reliable than RAPD but, in wheat, they have proven to be less polymorphic and efficient than amplified fragment length polymorphism (AFLP) analysis (Godwin et al., 1997). AFLP is a relatively new PCR-based assay for plant DNA fingerprinting and has been shown to be able to reveal significant levels of DNA polymorphism in plants (Vos et al., 1995). It is a robust and reliable genetic molecular marker assay and the number of polymorphisms detected per reaction is much higher than that revealed by RFLP or RAPD assay. AFLP has been applied successfully to crops such as rice (Mackill et al., 1996), tea (Paul et al., 1997), and wheat (Barrett
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DNA PROFILING ANALYSIS OF BANANA CULTIVARS
TABLE 1 The Banana Genotypes Studied No.
Accession No.
Species/cultivar
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
JP-21 00-10347 101-92-45 146-96-9001 JP-22 53-95-5743 101-92-44 00-10341 53-95-5747 153-95-5740 153-95-5739 JP-23 153-95-5738 33-96-6950 19971030 58-93-271
Cultivar X cv. Blood M. ornata Roxburgh cv. Grand Nain Musa 3 cv. Raja Udang M. coccinea Andr. cv. Dwarf Cavendish cv. Curry cv. Pisang Rastali cv. Pisang Batu cv. Pisang Awak cv. Pisang Assam 2sp 6sp cv. Thousand Fingers
Note. Voucher specimens are kept in the herbarium of the Singapore Botanic Gardens.
and Kidwell, 1998), as well as ornamental plants such as caladium (Loh et al., 1999). The objectives of the present study are (1) to examine the usefulness of AFLPs in differentiating cultivars of banana, (2) to develop molecular markers for the different banana cultivars, and (3) to determine genetic relationships between the cultivars. MATERIALS AND METHODS Plant Materials Fully expanded leaf samples were collected from plants cultivated in the Singapore Botanic Gardens (Table 1). Four samples of unknown origin were also collected and designated cultivar X, Musa3, 2sp, and 6sp. Reference collections and voucher specimens of all cultivars sampled are deposited in the herbarium of Singapore Botanic Gardens. The leaf samples used for AFLP analysis were surface-sterilized first by continuous shaking in 0.75% sodium hypochlorite solution (w/v) with a few drops of Tween 20 for 15 min. Subsequently, the leaves were thoroughly rinsed with autoclaved water, wrapped in aluminium foil, frozen in liquid nitrogen, and stored at ⫺80°C until needed for DNA extraction. DNA Extraction Plant DNA was extracted using the CTAB method according to Reichardt and Rogers (1993). Briefly, leaf tissue was pulverized using liquid nitrogen prior to addition of 4 ml of Solution I (2% w/v CTAB (Sigma), 100 mM Tris–HCl, 20 mM EDTA, 1.4 M NaCl, pH 8.0) per gram of leaf tissue and incubated for 60 min at 65°C. The homogenate was then extracted with an
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equal volume of chloroform/isoamyl alcohol (24:1) and centrifuged at 12,000 rpm for 5 min. The top aqueous phase was recovered and incubated with 1/10 volume of Solution II (10% w/v CTAB, 0.7 M NaCl), prewarmed to 65°C. The aqueous phase was then extracted with 1 volume of chloroform/isoamyl alcohol (24:1) and recovered as before. To the recovered aqueous phase, 1 volume of Solution III (1% w/v CTAB, 50 mM Tris–HCl, 10 mM EDTA, pH 8.0) was added and incubated overnight at 37°C. The mixture was then centrifuged for 5 min at 3500 rpm and the supernatant removed. The DNA pellet was then redissolved in Solution IV (10 mM Tris–HCl, 0.1 mM EDTA, 1 M NaCl, pH 8.0) at 0.5 to 1 ml per gram starting material, followed by ethanol precipitation of the DNA. It was then washed with 70% ethanol, dried, and resuspended in a minimal volume of TE buffer at 0.1 to 0.5 ml per gram starting material. AFLP Analysis The AFLP analysis was carried out according to Vos et al. (1995) with minor modifications. Restriction digests of genomic DNA with EcoRI and MseI were carried out at 37°C for 4 h. Following heat inactivation of the restriction endonucleases, the genomic DNA fragments were ligated to EcoRI and MseI adapters overnight at 15°C to generate template DNA for amplification. PCR was performed in two consecutive reactions. The template DNA generated was first preamplified using AFLP primers, each having one selective nucleotide. The PCR products of the preamplification reaction were then used as template, after dilution fivefold in sterile water, for selective amplification using two AFLP primers, each containing three selective nucleotides. The primer and adapter sequences are shown in Table 2. The EcoRI primers used were not radioactive labeled as in the original protocol. Instead, a modified silver-staining method was used. The final PCR products were run on a 6% denaturing polyacrylamide gel in 1⫻ TBE buffer. Analysis was carried out by silverstaining of the gel (Promega Corp., Madison, WI) and overnight drying before being photographed. Data Analysis For the diversity analysis, bands were scored as present (1) or absent (0) and used as a raw data matrix. A square symmetric matrix of similarity was obtained using Jaccard’s similarity coefficient [a/(n ⫺ d)], where a is the number of fragments in common between two cultivars, n is the total number of fragments scored, and d is the number of fragments absent in both cultivars (Dudley, 1994). Genetic diversity estimates (GDEs) were then calculated as 1 ⫺ Jaccard’s similarity coefficient and used for cluster analysis using the unweighted pair group method using arithmetic averages (UPGMA) technique.
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TABLE 2 Sequence of Primers Used Name/abbreviation GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY
101/ EA⫹ 102/ EA⫺ 103/ MA⫹ 104/ MA⫺ 105/ E-A 107/ E-AAC 108/ E-AAG 109/ E-ACA 110/ E-ACT 111/E-ACC 112/E-ACG 113/ E-AGC 114/ E-AGG 106/ M-C 115/ M-CAA 116/ M-CAC 117/ M-CAG 118/ M-CAT 119/ M-CTA 120/ M-CTC 121/ M-CTG 122/ M-CTT
Enzyme EcoRI EcoRI MseI MseI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI MseI MseI MseI MseI MseI MseI MseI MseI MseI
RESULTS AND DISCUSSION AFLP Profiles of Banana Cultivars The AFLP analysis of eight primer combinations on 16 banana cultivars yielded a total of 555 polymorphic bands and 58 monomorphic bands. The AFLP profiles obtained can be used to distinguish between the different cultivars by their unique banding patterns (Fig. 1). These results compare favorably to those of Bhat et al. (1995), whose RAPD studies with 49 primers yielded 605 polymorphic bands in 57 cultivars and RFLP studies with 19 hybridizations of random genomic clones yielded only 107 polymorphic alleles from the same 57 cultivars, as well as to studies carried out by Grapin et al. (1998), in which nine STMS markers yielded only 72 polymorphic alleles from 59 cultivars of Musa acuminata. The data obtained shows AFLP to be a good method of choice for molecular studies into banana cultivars, by virtue of its ability to pick up more polymorphisms per primer pair, as well as it being a reliable and easily repeatable technique. Cultivar Identification Using AFLP Markers The AFLP analysis of eight primer combinations on 16 banana cultivars was used to identify unique molecular markers specific for each cultivar. Unique markers specific to each cultivar were found for 12 of the 16 cultivars sampled. The number of molecular markers specific for each cultivar ranged from 1 to 52 when the eight primer combinations were used. A total
Type
Sequence (5⬘-3⬘)
Adapter ⫹ Adapter ⫺ Adapter ⫹ Adapter ⫺ Primer ⫹1 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹1 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3
CTCGTAGACTGCGTACC AATTGGTACGCAGTCTAC GACGATGAGTCCTGAG TACTCAGGACTCAT GACTGCGTACCAATTCA GACTGCGTACCAATTCAAC GACTGCGTACCAATTCAAG GACTGCGTACCAATTCACA GACTGCGTACCAATTCACT GACTGCGTACCAATTCACC GACTGCGTACCAATTCACG GACTGCGTACCAATTCAGC GACTGCGTACCAATTCAGG GATGAGTCCTGAGTAAC GATGAGTCCTGAGTAACAA GATGAGTCCTGAGTAACAC GATGAGTCCTGAGTAACAG GATGAGTCCTGAGTAACAT GATGAGTCCTGAGTAACTA GATGAGTCCTGAGTAACTC GATGAGTCCTGAGTAACTG GATGAGTCCTGAGTAACTT
of 134 molecular markers were found overall (Table 3). The molecular markers identified could be used to generate specific probes for the different cultivars. Close examination of the AFLP profiles for the banana cultivar Dwarf Cavendish and the unnamed 2sp revealed close similarities. A reinspection of morphological traits showed that plants of these two cultivars were indeed very similar and most closely related to each other. This conclusion was later supported in the UPGMA analysis. AFLP is thus shown to be useful in the identification of clones and related cultivars, as well as for distinguishing between banana cultivars. Genetic Diversity between Cultivars The AFLP data on the 16 banana cultivars from eight selected primer combinations were used for UPGMA cluster analysis (Table 4 and Fig. 2). The outgroup species (M. coccinea and M. ornata) clearly formed distinct clusters separate from the cultivated bananas, which are of hybrid origin between M. acuminata and M. balbisiana. This genetic dissimilarity is to be expected since they are from different sections of the genus: M. coccinea from section Callimusa, M. ornata from section Rhodochlamys, and the cultivated bananas from section Musa. In addition to the two outgroup species, the cultivated bananas fall into two major clusters with Pisang Batu, Rajah Udang, Thousand fingers, cultivar X, and cultivar Curry grouping in one cluster and the rest falling into another cluster which is further subdivided
DNA PROFILING ANALYSIS OF BANANA CULTIVARS
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into three clusters of which the cultivar Blood formed one by itself. The two smaller clusters were formed by Pisang Rastali, Pisang Awak, and Grand Nain in one cluster and Musa 3, 2sp, Dwarf Cavendish, Pisang Assam, and 6sp in the other. If the method used by Simmonds (1966) for classifying germplasm is applied to the cultivars examined in this study, results do not show segregation of cultivars based on their overall hypothetical genetic homologies. For example, those cultivars that Simmonds designated as having AAA genomic constitution (Dwarf Cavendish, Grand Nain, and Raja Udang) fall in separate clusters rather than clustering together, whereas cultivars with three different putative genotypes, e.g., Pisang Rastali (AAB), Pisang Awak (ABB), and Grand Nain (AAA), cluster together (Fig. 2). A close examination of the study by Simmonds (1966) results in several observations. First, Simmond’s method of classifying Musa cultivars does not have a genetic basis due to the difficulty in carrying out conventional breeding, as the cultivated bananas are highly sterile, which means crossing experiments are almost impossible. Neither is the history of the evolution of banana cultivars known, since many are ancient and have been cultivated since prehistoric times. It is therefore not known whether a cultivar recognized by morphological characters has arisen more than once, whether a cultivar in different regions has the same genetic origin, nor what the effect of random genetic drift is over the long period in cultivation. The picture is further complicated as bananas have been transported outside their place of origin since prehistoric times. Current classification based on morphological characters and deduced genotypes based on certain agronomic characters are therefore not reliable. Phenotypic characters, such as sugary or starchy fruits, may not be due to simple euploidy difference (AAA, AAB, or ABB). Different phenotypes could result from allelic differences in single or multiple genes. The difficulty involved in the identification of banana cultivars, which are sterile, therefore highlights the need for a DNA marker system for classification. In the present study, all banana cultivars analyzed originated from the Peninsular Malaysia, where M. acuminata is found as a wild species represented by three subspecies. Using samples collected here may better reflect the true genetic diversity in bananas, as they are closer to their presumed center of origin. Simmonds (1966) found the “AAA” genomic group cultivars
FIG. 1. AFLP profile generated for 16 cultivars of banana using primer pair 28 (E-ACC, M-CAT). Lane 1, cultivar X; lane 2, blood; lane 3, M. ornata; lane 4, Grand Nain; lane 5, Musa 3; lane 6, Raja Udang; lane 7, M. coccinea; lane 8, Dwarf Cavendish; lane 9, curry;
lane 10, Pisang Keling; lane 11, Pisang Batu; lane 12, Pisang Awak; lane 13, Pisang Assam; lane 14, 2sp; lane 15, 6sp; lane 16, Thousand Fingers; lane M, marker. Arrows depict cultivar-specific bands.
AAC* AAG ACA ACC ACG ACT AGC AGG
CAA* CAC CAG CAT CTA CTC CTG CTT
— — — — — — — — —
— 1 1 — 3 4 — 5 14
10 10 6 6 3 6 5 6 52
3 — 2 3 — 1 — 1 10
2 — — 2 — — 2 — 6
1 1 — 1 — — 1 — 4
6 5 — 5 6 4 2 3 31
2 — — 1 1 — — — 4
0.630 — — — — — —
— — — — — — — —
— — — — — — —
— — — — — — — —
Blood
— — — — — — — —
0.678 0.666 — — — — —
M. ornata
— — — — — — — —
0.519 0.468 0.701 — — — —
Grand Nain
— — — — — — — —
0.579 0.525 0.631 0.560 — — —
Musa 3
Note. GDEs represent 1 ⫺ Jaccard’s similarity coefficient.
CvX Blood M. ornata Grand Nain Musa 3 Raja Udang M. coccinea Dwarf Cavendish Curry Pisang Rastali Pisang Batu Pisang Awak Pisang Assam 2sp 6sp
CvX
1 — — — — 1 — — 2
— — — — — — — — —
— — 1 — 1 — — — 2
— — — — — — — — —
— — — 1 — — — — 1
— 1 — — — — — 1 2
— — — — — — — —
0.399 0.654 0.688 0.471 0.573 — —
Raja Udang
— — — — — — — —
0.669 0.623 0.655 0.659 0.605 0.658 —
M. coccinea
— — — — — — — —
0.602 0.738 0.638 0.530 0.371 0.557 0.602
Dwarf Cavendish
0.671 — — — — — — —
0.443 0.683 0.706 0.504 0.638 0.348 0.696
Curry
0.450 0.523 — — — — — —
0.470 0.526 0.656 0.391 0.460 0.403 0.669
Pisang Rastali
0.741 0.276 0.581 — — — — —
0.438 0.716 0.734 0.530 0.720 0.405 0.709
Pisang Batu
0.538 0.581 0.237 0.620 — — — —
0.530 0.580 0.716 0.456 0.575 0.532 0.723
Pisang Awak
0.323 0.665 0.448 0.729 0.490 — — —
0.600 0.455 0.651 0.490 0.373 0.579 0.604
Pisang Assam
Mean of the AFLP-Based Pairwise Genetic Diversity Estimates (GDEs) between 16 Cultivars of Banana Using Eight Primer Combinations
TABLE 4
* EcoRI, EcoRI-adapter-based primer; the selective nucleotides added at the 3⬘ end are indicated. * MseI, MseI-adapter-based primer; the selective nucleotides added at the 3⬘ end are indicated.
1 10 19 28 37 46 55 64 Total
0.160 0.664 0.487 0.726 0.527 0.319 — —
0.586 0.489 0.634 0.523 0.399 0.570 0.588
2sp
— — — — — — — — —
0.360 0.704 0.552 0.749 0.526 0.413 0.333 —
0.621 0.563 0.683 0.586 0.474 0.629 0.649
6sp
— — 1 — 1 2 — 1 5
0.588 0.395 0.459 0.429 0.524 0.592 0.570 0.610
0.304 0.651 0.674 0.515 0.576 0.402 0.677
Thousand Fingers
25 18 11 19 15 18 10 18 134
Total number of unique markers per Primer Grand Raja Dwarf Pisang Pisang Pisang Pisang Thousand primer pair EcoRI MseI CvX Blood M. ornata Nain Musa 3 Udang M. coccinea Cavendish Curry Rastali Batu Awak Assam 2sp 6sp Fingers pair
Number of Unique Molecular Marker Bands Specific for Each Cultivar of Banana Detected upon AFLP Analysis Using Eight Primer Combinations
TABLE 3
364 LOH ET AL.
DNA PROFILING ANALYSIS OF BANANA CULTIVARS
365
FIG. 2. UPGMA cluster analysis of AFLP data generated by eight primer combinations for 16 cultivars of banana depicting patterns of genetic diversity.
to be highly diverse morphologically in Peninsular Malaysia. The fact that cultivars given the same name in different regions produce different results suggests that they are not genetically uniform and that there is a need for a comprehensive analysis of genotypes using molecular methods to put the nomenclature on a firm footing. This is because, being clonal in nature and highly sterile due to triploidy, a cultivar should be genetically identical. This study shows that AFLP is a suitable method for this.
CONCLUSION AFLP is shown to be useful in DNA profiling to characterize cultivars and for studying the relationships between banana cultivars. A library of DNA profiles for Musa cultivars needs to be developed. Reassessment of morphological traits and assignment of local names to specific cultivars also need to be reviewed to reflect genetic uniformity of a cultivar. A reevaluation of the current classification of the A and B
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ploidy in M. acuminata using DNA profiles should also be established. ACKNOWLEDGMENTS This research was funded by the Academic Research Fund, National Institute of Education, Nanyang Technological University, Singapore, RP 12/98/GYY. We thank the Director, Singapore Botanic Gardens, for permission to collect the leaf samples.
REFERENCES Barrett, B. A., and Kidwell, K. K. (1998). AFLP-based genetic diversity assessment among wheat cultivars from the Pacific Northwest. Crop Sci. 38: 1261–1271. Bhat, K. V., Bhat, S. R., and Chandel, K. P. S. (1992). Survey of isozyme polymorphism for clonal identification in Musa. II. Peroxidase, superoxide dismutase, shikimate dehydrogenase and malate dehydrogenase. J. Hort. Sci. 67: 737–743. Bhat, K. V., Jarret, R. L., and Rana, R. S. (1995). DNA profiling of banana and plantain cultivars using random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers. Electrophoresis 16: 1736 –1745. Dudley, J. W. (1993). Molecular markers in plant improvement: Manipulation of genes affecting quantitative traits. Crop Sci. 33: 660 – 668. Godwin, I. D., Aitken, E. A. B., and Smith, L. W. (1997). Application of inter simple sequence repeat (ISSR) markers to plant genetics. Electrophoresis 18: 1524 –1528. Grapin, A., Noyer, J. L., Carrel, F., Dambler, D., Baurens, F. C., Lanaud, C., and Lagoda, P. J. L. (1998). Diploid Musa acuminata genetic diversity assayed with sequence-tagged microsatellite sites. Electrophoresis 19: 1374 –1380. Jarret, R. L., and Litz, R. E. (1986). Isozymes as genetic markers in bananas and plantains. Euphytica 35: 539 –549.
Kaemmer, D., Afza, R., Weising, K., Kahl, G., and Novak, F. J. (1992). Oligonucleotide and amplification fingerprinting of wild species and cultivars of banana (Musa spp.). Bio/technology 10: 1030 –1035. Loh, J. P., Kiew, R., Kee, A., Gan, L. H., and Gan, Y. Y. (1999) Amplified fragment length polymorphism (AFLP) provides molecular markers for the identification of Caladium bicolor cultivars. Annals Bot. 84: 155–161. Mackill, D. J., Zhang, Z., Redon˜a, E. D., and Colowit, P. M. (1996). Level of polymorphism and genetic mapping of AFLP markers in rice. Genome 39: 969 –977. Paul, S., Wachira, F. N., Powell, W., and Waugh, R. 1997. Diversity and genetic differentiation among populations of Indian and Kenyan tea (Camellia sinensis (L.) O. Kuntze) revealed by AFLP markers. Theor. Appl. Genet. 94: 255–263. Reichardt, M. J., and Rogers, S. J. (1993). Plant DNA isolation using CTAB. In “Current Protocols in Molecular Biology” (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, Eds.), Supplement 22, Wiley, New York. Sa´gi, L., May, G. D., Remy, S., and Swennen, R. (1998). Recent developments in biotechnological research on bananas (Musa spp.). Biotech. Genet. Engineer. Rev. 15: 313–327. Simmonds, N. W. (1956). Botanical results of the banana collecting expedition, 1954 –1955. Kew Bull.: 463– 489. Simmonds, N. W. (1966). “Bananas,” Longman, London, UK. Simmonds, N. W., and Shephard, K. (1955). The taxonomy and origin of the cultivated banana. J. Linn. Soc. Bot. 55: 302–312. Simmonds, N. W. (1995). Bananas. In “Evolution of Crop Plants” (J. Smartt and N. W. Simmonds, Eds), pp. 370 –375. Longman, Essex, UK. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kupier, M., and Zabeau, M. (1995). AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res. 23: 4407– 4414.
Amplified Fragment Length Polymorphism Fingerprinting of 16 Banana Cultivars (Musa cvs.) Jin Phang Loh,* Ruth Kiew,† Ohn Set,† Leong Huat Gan,* and Yik-Yuen Gan* ,1 *School of Science, National Institute of Education, Nanyang Technological University, 469 Bukit Timah Road, Singapore 259756; and †Singapore Botanic Gardens, 1 Cluny Road, Singapore 259569 Received October 1, 1999; revised July 26, 2000; published online November 30, 2000
Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits. © 2000 Academic Press
INTRODUCTION The bananas are one of the major subtropical food crops for millions of people worldwide (Grapin et al., 1998). Despite the importance of banana in trade and commerce, little is known of the genetics of its agronomically important traits. Several pathogens seriously threaten banana production and numerous breeding programs have been undertaken to create resistant varieties (Sagi et al., 1998). Breeding programs face extreme difficulty due to the phenomenon of parthenocarpy, female genetic sterility, and structural hybridity (Bhat et al., 1995). The conventional method of classifying Musa cultivars (Simmonds and Shepherd, 1955) is based on 15 morphological traits. Problems associated with variability, environmental factors, and individual bias have made proper identification of banana cultivars difficult. In addition, characterization of cultivars relies heavily on fruit charac1
To whom correspondence should be addressed. Fax: 65-469-8928. E-mail: [email protected]. 1055-7903/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
ters, making it almost impossible to identify sterile plants. Furthermore, there is as yet no genetic basis for this method of classification. DNA fingerprinting techniques, however, will allow for the correct identification of species and cultivars (Kaemmer et al., 1992), as well as being useful in plant patents and for the legal protection of newly bred cultivars in asexual Musa, such as genetically engineered cultivars resistant to diseases. Various laboratory-based techniques have been used to study genetic diversity in banana. These include isozyme analysis (Bhat et al., 1992), restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) (Bhat et al., 1995), inter simple sequence repeat (ISSR) markers (Godwin et al., 1997), and sequence-tagged microsatellite sites (STMS) (Grapin et al., 1998). These techniques have their limitations. Isozymes are generally limited by the relatively low levels of polymorphism detectable and can fail to identify cultivars differing in only a few genes (Jarret and Litz, 1986). RFLP overcame this problem but requires large quantities of relatively pure DNA, and the frequent use of radioisotopes in the detection method makes it technically demanding, laborious, and costly to characterize large numbers of samples. PCR-based RAPD assay overcame many of the technical limitations of RFLPs but has proven sensitive to experimental conditions (Paul et al., 1997). ISSR markers are more reliable than RAPD but, in wheat, they have proven to be less polymorphic and efficient than amplified fragment length polymorphism (AFLP) analysis (Godwin et al., 1997). AFLP is a relatively new PCR-based assay for plant DNA fingerprinting and has been shown to be able to reveal significant levels of DNA polymorphism in plants (Vos et al., 1995). It is a robust and reliable genetic molecular marker assay and the number of polymorphisms detected per reaction is much higher than that revealed by RFLP or RAPD assay. AFLP has been applied successfully to crops such as rice (Mackill et al., 1996), tea (Paul et al., 1997), and wheat (Barrett
360
DNA PROFILING ANALYSIS OF BANANA CULTIVARS
TABLE 1 The Banana Genotypes Studied No.
Accession No.
Species/cultivar
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
JP-21 00-10347 101-92-45 146-96-9001 JP-22 53-95-5743 101-92-44 00-10341 53-95-5747 153-95-5740 153-95-5739 JP-23 153-95-5738 33-96-6950 19971030 58-93-271
Cultivar X cv. Blood M. ornata Roxburgh cv. Grand Nain Musa 3 cv. Raja Udang M. coccinea Andr. cv. Dwarf Cavendish cv. Curry cv. Pisang Rastali cv. Pisang Batu cv. Pisang Awak cv. Pisang Assam 2sp 6sp cv. Thousand Fingers
Note. Voucher specimens are kept in the herbarium of the Singapore Botanic Gardens.
and Kidwell, 1998), as well as ornamental plants such as caladium (Loh et al., 1999). The objectives of the present study are (1) to examine the usefulness of AFLPs in differentiating cultivars of banana, (2) to develop molecular markers for the different banana cultivars, and (3) to determine genetic relationships between the cultivars. MATERIALS AND METHODS Plant Materials Fully expanded leaf samples were collected from plants cultivated in the Singapore Botanic Gardens (Table 1). Four samples of unknown origin were also collected and designated cultivar X, Musa3, 2sp, and 6sp. Reference collections and voucher specimens of all cultivars sampled are deposited in the herbarium of Singapore Botanic Gardens. The leaf samples used for AFLP analysis were surface-sterilized first by continuous shaking in 0.75% sodium hypochlorite solution (w/v) with a few drops of Tween 20 for 15 min. Subsequently, the leaves were thoroughly rinsed with autoclaved water, wrapped in aluminium foil, frozen in liquid nitrogen, and stored at ⫺80°C until needed for DNA extraction. DNA Extraction Plant DNA was extracted using the CTAB method according to Reichardt and Rogers (1993). Briefly, leaf tissue was pulverized using liquid nitrogen prior to addition of 4 ml of Solution I (2% w/v CTAB (Sigma), 100 mM Tris–HCl, 20 mM EDTA, 1.4 M NaCl, pH 8.0) per gram of leaf tissue and incubated for 60 min at 65°C. The homogenate was then extracted with an
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equal volume of chloroform/isoamyl alcohol (24:1) and centrifuged at 12,000 rpm for 5 min. The top aqueous phase was recovered and incubated with 1/10 volume of Solution II (10% w/v CTAB, 0.7 M NaCl), prewarmed to 65°C. The aqueous phase was then extracted with 1 volume of chloroform/isoamyl alcohol (24:1) and recovered as before. To the recovered aqueous phase, 1 volume of Solution III (1% w/v CTAB, 50 mM Tris–HCl, 10 mM EDTA, pH 8.0) was added and incubated overnight at 37°C. The mixture was then centrifuged for 5 min at 3500 rpm and the supernatant removed. The DNA pellet was then redissolved in Solution IV (10 mM Tris–HCl, 0.1 mM EDTA, 1 M NaCl, pH 8.0) at 0.5 to 1 ml per gram starting material, followed by ethanol precipitation of the DNA. It was then washed with 70% ethanol, dried, and resuspended in a minimal volume of TE buffer at 0.1 to 0.5 ml per gram starting material. AFLP Analysis The AFLP analysis was carried out according to Vos et al. (1995) with minor modifications. Restriction digests of genomic DNA with EcoRI and MseI were carried out at 37°C for 4 h. Following heat inactivation of the restriction endonucleases, the genomic DNA fragments were ligated to EcoRI and MseI adapters overnight at 15°C to generate template DNA for amplification. PCR was performed in two consecutive reactions. The template DNA generated was first preamplified using AFLP primers, each having one selective nucleotide. The PCR products of the preamplification reaction were then used as template, after dilution fivefold in sterile water, for selective amplification using two AFLP primers, each containing three selective nucleotides. The primer and adapter sequences are shown in Table 2. The EcoRI primers used were not radioactive labeled as in the original protocol. Instead, a modified silver-staining method was used. The final PCR products were run on a 6% denaturing polyacrylamide gel in 1⫻ TBE buffer. Analysis was carried out by silverstaining of the gel (Promega Corp., Madison, WI) and overnight drying before being photographed. Data Analysis For the diversity analysis, bands were scored as present (1) or absent (0) and used as a raw data matrix. A square symmetric matrix of similarity was obtained using Jaccard’s similarity coefficient [a/(n ⫺ d)], where a is the number of fragments in common between two cultivars, n is the total number of fragments scored, and d is the number of fragments absent in both cultivars (Dudley, 1994). Genetic diversity estimates (GDEs) were then calculated as 1 ⫺ Jaccard’s similarity coefficient and used for cluster analysis using the unweighted pair group method using arithmetic averages (UPGMA) technique.
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TABLE 2 Sequence of Primers Used Name/abbreviation GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY GYY
101/ EA⫹ 102/ EA⫺ 103/ MA⫹ 104/ MA⫺ 105/ E-A 107/ E-AAC 108/ E-AAG 109/ E-ACA 110/ E-ACT 111/E-ACC 112/E-ACG 113/ E-AGC 114/ E-AGG 106/ M-C 115/ M-CAA 116/ M-CAC 117/ M-CAG 118/ M-CAT 119/ M-CTA 120/ M-CTC 121/ M-CTG 122/ M-CTT
Enzyme EcoRI EcoRI MseI MseI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI MseI MseI MseI MseI MseI MseI MseI MseI MseI
RESULTS AND DISCUSSION AFLP Profiles of Banana Cultivars The AFLP analysis of eight primer combinations on 16 banana cultivars yielded a total of 555 polymorphic bands and 58 monomorphic bands. The AFLP profiles obtained can be used to distinguish between the different cultivars by their unique banding patterns (Fig. 1). These results compare favorably to those of Bhat et al. (1995), whose RAPD studies with 49 primers yielded 605 polymorphic bands in 57 cultivars and RFLP studies with 19 hybridizations of random genomic clones yielded only 107 polymorphic alleles from the same 57 cultivars, as well as to studies carried out by Grapin et al. (1998), in which nine STMS markers yielded only 72 polymorphic alleles from 59 cultivars of Musa acuminata. The data obtained shows AFLP to be a good method of choice for molecular studies into banana cultivars, by virtue of its ability to pick up more polymorphisms per primer pair, as well as it being a reliable and easily repeatable technique. Cultivar Identification Using AFLP Markers The AFLP analysis of eight primer combinations on 16 banana cultivars was used to identify unique molecular markers specific for each cultivar. Unique markers specific to each cultivar were found for 12 of the 16 cultivars sampled. The number of molecular markers specific for each cultivar ranged from 1 to 52 when the eight primer combinations were used. A total
Type
Sequence (5⬘-3⬘)
Adapter ⫹ Adapter ⫺ Adapter ⫹ Adapter ⫺ Primer ⫹1 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹1 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3 Primer ⫹3
CTCGTAGACTGCGTACC AATTGGTACGCAGTCTAC GACGATGAGTCCTGAG TACTCAGGACTCAT GACTGCGTACCAATTCA GACTGCGTACCAATTCAAC GACTGCGTACCAATTCAAG GACTGCGTACCAATTCACA GACTGCGTACCAATTCACT GACTGCGTACCAATTCACC GACTGCGTACCAATTCACG GACTGCGTACCAATTCAGC GACTGCGTACCAATTCAGG GATGAGTCCTGAGTAAC GATGAGTCCTGAGTAACAA GATGAGTCCTGAGTAACAC GATGAGTCCTGAGTAACAG GATGAGTCCTGAGTAACAT GATGAGTCCTGAGTAACTA GATGAGTCCTGAGTAACTC GATGAGTCCTGAGTAACTG GATGAGTCCTGAGTAACTT
of 134 molecular markers were found overall (Table 3). The molecular markers identified could be used to generate specific probes for the different cultivars. Close examination of the AFLP profiles for the banana cultivar Dwarf Cavendish and the unnamed 2sp revealed close similarities. A reinspection of morphological traits showed that plants of these two cultivars were indeed very similar and most closely related to each other. This conclusion was later supported in the UPGMA analysis. AFLP is thus shown to be useful in the identification of clones and related cultivars, as well as for distinguishing between banana cultivars. Genetic Diversity between Cultivars The AFLP data on the 16 banana cultivars from eight selected primer combinations were used for UPGMA cluster analysis (Table 4 and Fig. 2). The outgroup species (M. coccinea and M. ornata) clearly formed distinct clusters separate from the cultivated bananas, which are of hybrid origin between M. acuminata and M. balbisiana. This genetic dissimilarity is to be expected since they are from different sections of the genus: M. coccinea from section Callimusa, M. ornata from section Rhodochlamys, and the cultivated bananas from section Musa. In addition to the two outgroup species, the cultivated bananas fall into two major clusters with Pisang Batu, Rajah Udang, Thousand fingers, cultivar X, and cultivar Curry grouping in one cluster and the rest falling into another cluster which is further subdivided
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into three clusters of which the cultivar Blood formed one by itself. The two smaller clusters were formed by Pisang Rastali, Pisang Awak, and Grand Nain in one cluster and Musa 3, 2sp, Dwarf Cavendish, Pisang Assam, and 6sp in the other. If the method used by Simmonds (1966) for classifying germplasm is applied to the cultivars examined in this study, results do not show segregation of cultivars based on their overall hypothetical genetic homologies. For example, those cultivars that Simmonds designated as having AAA genomic constitution (Dwarf Cavendish, Grand Nain, and Raja Udang) fall in separate clusters rather than clustering together, whereas cultivars with three different putative genotypes, e.g., Pisang Rastali (AAB), Pisang Awak (ABB), and Grand Nain (AAA), cluster together (Fig. 2). A close examination of the study by Simmonds (1966) results in several observations. First, Simmond’s method of classifying Musa cultivars does not have a genetic basis due to the difficulty in carrying out conventional breeding, as the cultivated bananas are highly sterile, which means crossing experiments are almost impossible. Neither is the history of the evolution of banana cultivars known, since many are ancient and have been cultivated since prehistoric times. It is therefore not known whether a cultivar recognized by morphological characters has arisen more than once, whether a cultivar in different regions has the same genetic origin, nor what the effect of random genetic drift is over the long period in cultivation. The picture is further complicated as bananas have been transported outside their place of origin since prehistoric times. Current classification based on morphological characters and deduced genotypes based on certain agronomic characters are therefore not reliable. Phenotypic characters, such as sugary or starchy fruits, may not be due to simple euploidy difference (AAA, AAB, or ABB). Different phenotypes could result from allelic differences in single or multiple genes. The difficulty involved in the identification of banana cultivars, which are sterile, therefore highlights the need for a DNA marker system for classification. In the present study, all banana cultivars analyzed originated from the Peninsular Malaysia, where M. acuminata is found as a wild species represented by three subspecies. Using samples collected here may better reflect the true genetic diversity in bananas, as they are closer to their presumed center of origin. Simmonds (1966) found the “AAA” genomic group cultivars
FIG. 1. AFLP profile generated for 16 cultivars of banana using primer pair 28 (E-ACC, M-CAT). Lane 1, cultivar X; lane 2, blood; lane 3, M. ornata; lane 4, Grand Nain; lane 5, Musa 3; lane 6, Raja Udang; lane 7, M. coccinea; lane 8, Dwarf Cavendish; lane 9, curry;
lane 10, Pisang Keling; lane 11, Pisang Batu; lane 12, Pisang Awak; lane 13, Pisang Assam; lane 14, 2sp; lane 15, 6sp; lane 16, Thousand Fingers; lane M, marker. Arrows depict cultivar-specific bands.
AAC* AAG ACA ACC ACG ACT AGC AGG
CAA* CAC CAG CAT CTA CTC CTG CTT
— — — — — — — — —
— 1 1 — 3 4 — 5 14
10 10 6 6 3 6 5 6 52
3 — 2 3 — 1 — 1 10
2 — — 2 — — 2 — 6
1 1 — 1 — — 1 — 4
6 5 — 5 6 4 2 3 31
2 — — 1 1 — — — 4
0.630 — — — — — —
— — — — — — — —
— — — — — — —
— — — — — — — —
Blood
— — — — — — — —
0.678 0.666 — — — — —
M. ornata
— — — — — — — —
0.519 0.468 0.701 — — — —
Grand Nain
— — — — — — — —
0.579 0.525 0.631 0.560 — — —
Musa 3
Note. GDEs represent 1 ⫺ Jaccard’s similarity coefficient.
CvX Blood M. ornata Grand Nain Musa 3 Raja Udang M. coccinea Dwarf Cavendish Curry Pisang Rastali Pisang Batu Pisang Awak Pisang Assam 2sp 6sp
CvX
1 — — — — 1 — — 2
— — — — — — — — —
— — 1 — 1 — — — 2
— — — — — — — — —
— — — 1 — — — — 1
— 1 — — — — — 1 2
— — — — — — — —
0.399 0.654 0.688 0.471 0.573 — —
Raja Udang
— — — — — — — —
0.669 0.623 0.655 0.659 0.605 0.658 —
M. coccinea
— — — — — — — —
0.602 0.738 0.638 0.530 0.371 0.557 0.602
Dwarf Cavendish
0.671 — — — — — — —
0.443 0.683 0.706 0.504 0.638 0.348 0.696
Curry
0.450 0.523 — — — — — —
0.470 0.526 0.656 0.391 0.460 0.403 0.669
Pisang Rastali
0.741 0.276 0.581 — — — — —
0.438 0.716 0.734 0.530 0.720 0.405 0.709
Pisang Batu
0.538 0.581 0.237 0.620 — — — —
0.530 0.580 0.716 0.456 0.575 0.532 0.723
Pisang Awak
0.323 0.665 0.448 0.729 0.490 — — —
0.600 0.455 0.651 0.490 0.373 0.579 0.604
Pisang Assam
Mean of the AFLP-Based Pairwise Genetic Diversity Estimates (GDEs) between 16 Cultivars of Banana Using Eight Primer Combinations
TABLE 4
* EcoRI, EcoRI-adapter-based primer; the selective nucleotides added at the 3⬘ end are indicated. * MseI, MseI-adapter-based primer; the selective nucleotides added at the 3⬘ end are indicated.
1 10 19 28 37 46 55 64 Total
0.160 0.664 0.487 0.726 0.527 0.319 — —
0.586 0.489 0.634 0.523 0.399 0.570 0.588
2sp
— — — — — — — — —
0.360 0.704 0.552 0.749 0.526 0.413 0.333 —
0.621 0.563 0.683 0.586 0.474 0.629 0.649
6sp
— — 1 — 1 2 — 1 5
0.588 0.395 0.459 0.429 0.524 0.592 0.570 0.610
0.304 0.651 0.674 0.515 0.576 0.402 0.677
Thousand Fingers
25 18 11 19 15 18 10 18 134
Total number of unique markers per Primer Grand Raja Dwarf Pisang Pisang Pisang Pisang Thousand primer pair EcoRI MseI CvX Blood M. ornata Nain Musa 3 Udang M. coccinea Cavendish Curry Rastali Batu Awak Assam 2sp 6sp Fingers pair
Number of Unique Molecular Marker Bands Specific for Each Cultivar of Banana Detected upon AFLP Analysis Using Eight Primer Combinations
TABLE 3
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FIG. 2. UPGMA cluster analysis of AFLP data generated by eight primer combinations for 16 cultivars of banana depicting patterns of genetic diversity.
to be highly diverse morphologically in Peninsular Malaysia. The fact that cultivars given the same name in different regions produce different results suggests that they are not genetically uniform and that there is a need for a comprehensive analysis of genotypes using molecular methods to put the nomenclature on a firm footing. This is because, being clonal in nature and highly sterile due to triploidy, a cultivar should be genetically identical. This study shows that AFLP is a suitable method for this.
CONCLUSION AFLP is shown to be useful in DNA profiling to characterize cultivars and for studying the relationships between banana cultivars. A library of DNA profiles for Musa cultivars needs to be developed. Reassessment of morphological traits and assignment of local names to specific cultivars also need to be reviewed to reflect genetic uniformity of a cultivar. A reevaluation of the current classification of the A and B
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ploidy in M. acuminata using DNA profiles should also be established. ACKNOWLEDGMENTS This research was funded by the Academic Research Fund, National Institute of Education, Nanyang Technological University, Singapore, RP 12/98/GYY. We thank the Director, Singapore Botanic Gardens, for permission to collect the leaf samples.
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