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Genetic Diversity of Wild Banana (Musa Balbisiana Colla) in China As Revealed by AFLP Markers

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Genetic Diversity of Wild Banana (Musa Balbisiana Colla) in China As Revealed by AFLP Markers Genet Resour Crop Evol (2007) 54:1125–1132 DOI 10.1007/s10722-006-9004-9 RESEARCH PAPER Genetic diversity of wild banana (Musa balbisiana Colla) in China as revealed by AFLP markers Xiao-Lan Wang Æ Tzen-Yuh Chiang Æ Nicolas Roux Æ Gang Hao Æ Xue-Jun Ge Received: 7 November 2005 / Accepted: 11 July 2006 / Published online: 11 November 2006 Ó Springer Science+Business Media B.V. 2006 Abstract Wild banana Musa balbisiana Colla is 0.3684 at population level, and P = 100%, HT one of the progenitors of cultivated bananas and = 0.3362 and Hsp = 0.5048 at species level. plantains. It is native to Southeast Asia and the Significant genetic differentiation among popula- western Pacific. South China represents the tions was detected based on Hickory’s analysis northern limit of its distribution range. The (27.6%), Shannon’s diversity index (27.0%) and genetic diversity of Musa balbisiana was assessed AMOVA (27.1%). The AFLP results are dis- by the amplified fragment length polymorphism cussed and compared with data obtained by mi- (AFLP) fingerprinting in 15 populations of China. crosatellites method. The estimates of genetic Four primer pairs produced 199 discernible loci. diversity and differentiation between each pair of High levels of genetic diversity were detected, populations computed with microsatellites and with P = 78.5%, HE = 0.241, and Hpop = AFLP markers were not significantly correlated. Conservation strategies for Musa balbisiana in China are proposed. X.-L. Wang Centre for Functional Genomics and Microarray, Keywords AFLP Æ Genetic diversity Æ Guangzhou University, Guangzhou 510405, China Microsatellites Æ Musa balbisiana Æ Wild banana T.-Y. Chiang Department of Life Sciences, Cheng-Kung University, Tainan, Taiwan 701, ROC Introduction N. Roux With an annual production of about 100 million INIBAP, International Network for the Improvement of Banana and Plantain, Parc Scientifique tons, bananas (Musa spp.) are an important crop Agropolis II, 34397 Montpellier Cedex 5, France in the subtropics and tropics. Southeast Asia is the centre of bananas’ domestication. Most of G. Hao edible bananas originated via hybridization be- College of Life Sciences, South China Agricultural University, Guangzhou 510642, China tween Musa balbisiana Colla and M. acuminata Colla. Musa balbisiana is native to Southeast Asia X.-J. Ge (&) and the western Pacific. Populations of South State Key Laboratory of Biocontrol, School of Life China represent the northern most distribution of Sciences, Sun Yat-Sen University, Guangzhou 510275, China the wild banana with a range from tropics e-mail: [email protected] (18°40¢ N) to subtropics (25°30¢ N). 123 1126 Genet Resour Crop Evol (2007) 54:1125–1132 The evaluation and conservation of genetic et al. 1999; Shim and Jørgensen 2000). AFLP has diversity for the progenitors of cultivated plants is been used to detect the genetic diversity of both imperative to guarantee sustainable development. cultivated accessions and wild progenitors (M. Wild banana, M. balbisiana Colla, provides acuminata Colla; Wong et al. 2001). Microsatel- important genetic resources for banana breeding lites (also called short tandem repeats or simple because it has numerous agriculturally advanta- sequence repeats SSRs; Tautz 1989), in turn, geous characters, such as cold- and disease-resis- combine several features of an ultimate genetic tances. Nevertheless, this species has been under marker, owing to their abundance and uniform considerable threats in China in the past decades dispersal in genomes, hypervariability, codomi- due to the destruction of subtropical evergreen nant nature, accessibility for other research lab- broadleaf forests and other human disturbances. oratories (Compbell et al. 2003; Gaudeul et al. Effective conservation of M. balbisiana is urgently 2004). Microsatellites have been widely used to needed to preserve the remaining populations for detect the genetic diversity of plant species. sustaining production of banana. In contrast to the In a molecular breeding study on Musa, Crouch well understood M. acuminata Colla, very few et al. (1999) reported poor correlation between population genetics studies to date have been estimates of genetic similarities derived from dif- carried out on M. balbisiana. Recently, an AFLP ferent types of markers. They suggested that such analysis on 8 accessions of M. balbisiana found data inconsistency stems from differences among high levels of genetic diversity within the species molecular techniques that selectively screened (Ude et al. 2002a, b), which correspond to highly complementary, but not overlapping, regions of diverged morphological characters across geo- the genome. Therefore, integration of genetic graphical regions (Sotto and Rabara 2000). With estimates from different molecular techniques was its north limit in its distribution range and variable proposed to provide a clearer picture of Musa environmental conditions, it is necessary to esti- genetic relationship and generate highly accurate mate the level of genetic diversity in natural estimates of genetic similarity in germplasm populations of M. balbisiana in China. analysis (Crouch et al. 1999; Wong et al. 2001). In Various molecular markers, especially differ- order to obtain a better understanding of the ent PCR-based molecular markers including population structure in M. balbisiana Colla, the AFLP, RAPD, microsatellites, have been fre- simultaneous use of AFLP and microsatellites will quently used for assessing genetic diversity and be very informative. phylogenetic relationship in wild banana and In this study, we analysed the genetic diversity cultivation accessions (i.e., Grapin et al. 1998; and population structuring in wild banana, M. Loh et al. 2000; Wong et al. 2001; Carreel et al. balbisiana Colla, from 15 different populations in 2002; Ude et al. 2002a, b; Creste et al. 2003; China based on AFLP fingerprints. The results Nwakanma et al. 2003). Of fingerprinting tech- were compared to the data of a previous study niques, amplified fragment length polymorphism from the same material produced by microsatel- (AFLP) and microsatellite are among the most lites (Ge et al. 2005). We intended to draw rec- informative. The two PCR-based marker systems ommendations for conservation purposes based differ in principle, and there are different on the comparative analysis of genetic diversity strengths and limitations. The AFLP technique is with different markers. based on the selective amplification of restriction fragments obtained from the digestion of total genomic DNA. Given their dominant and biall- Materials and methods elic nature, AFLP markers have been increasingly applied to various plants, mainly owing to the Sampling capabilities of detecting a very high number of polymorphisms in a single assay, good repeat- Musa balbisiana Colla (2n = 22, Horry et al. ability and reasonable coverage of the genome 1997) has a wide range in China, with its distri- (Vos et al. 1995; Cervera et al. 1998; Vuylsteke bution centred in Guangdong Province. In this 123 Genet Resour Crop Evol (2007) 54:1125–1132 1127 study, a total of 218 individuals from 15 popula- the sequencing system (Li-Cor 4300L; Li-Cor tions of M. balbisiana were analysed. Populations Inc., Lincoln, Nebraska, NE, USA) using elec- were collected from all the provinces in China trophoresis. The 50–700 size standard (Li-Cor, within the species range, one population each Lincoln, NE, USA) was run with the samples to from Fujian, Hainan, and Yunnan, 10 populations estimate the size of fragments. AFLP patterns of Guangdong, and two populations of Guangxi were visualized and recorded by SAGAMX3.1 (Fig. 1, Table 1). About 12–15 individuals were software (Li-Cor, Lincoln, NE, USA). The bands analysed per population. Young, healthy leaves were scored as either present (1) or absent (0) were collected and dried in silica gel. DNA was across all loci. extracted using the modified CTAB method (Murray and Thompson 1980). Data analysis AFLP analysis The computer program POPGENE 1.31 (Yeh et al. 1999) was used to provide information on AFLP analysis was carried out following Vos the percentage of polymorphic loci (P), Nei’s et al. (1995) with modifications of the labelling of expected heterozygosity fromP Hardy–Weinberg 2 the EcoRI-primers. Near infrared (NIR) fluores- assumption (HE =1– pi P) (Nei 1973) and cence technology was used for imaging labelled Shannon’s diversity (Ho =– pi log2pi), where DNA bands. The EcoRI+3 primers were labelled pi is the frequency of a given AFLP fragments. Ho with IRD 700 and IRD 800 fluorescence dyes was calculated at two levels: the average diversity (Li-Cor, Lincoln, NE, USA). After digestion with within populations (Hpop), and the total diversity EcoRl and MseI, adaptors were ligated on both (Hsp). Then the proportion of diversity among ends of genomic fragments and a two-step populations was estimated as (Hsp – Hpop)/Hsp. selective amplification was performed. We chose An analysis of molecular variance (AMOVA) four selective primer pairs: M-CAG/E-ACA, was performed using Arlequin 2.000 (Schneider M-CAG/E-AAC, M-CTA/E-ACG, M-CTC/E- et al. 2000). The hierarchical analysis was con- ACA. Amplification was conducted on a PTC 200 ducted at two levels: (1) among populations; and Peltier Thermal Cycler (MJ Research). PCR (2) within populations. A simplified estimate of products were mixed with loading buffer and FST of Wright (1951) was obtained by AMOVA. loaded on 7% polyacrylamide gels after heat As an alternative to AMOVA, population struc- denaturation.
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