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23. Molecular characterization of genomes in Musa and its applications - Pillay, M., A. Tenkouano, G. Ude*, R. Ortiz


* Present address: Department of NRSL, Room 1112, HJ Patterson, University of Maryland, College Park, MD 20742, USA

International Institute of Tropical Agriculture (IITA)
Ibadan
Nigeria

International Mailing address:
IITA
c/o Lambourns UK
Carolyn House
26 Dingwall Road
Croydon CR9 3EE
England

Abstract

Many techniques are now available to study the structure of plant genomes. This knowledge is important for the genetic improvement of crop plants. This chapter presents a summary of our current understanding of genomes in bananas (Musa spp.). Four putative genomes, A, B, S and T, are recognized in the genus. Most cultivated bananas are classified into AA, BB, AAA, AAB, and ABB genomic groups. Musa has a relatively small genome size that ranges from 550 to 612 Mbp, that is, 3.5 times bigger than Arabidopsis and comparable in size to that of rice. Effective identification of the genomes is possible by genomic in situ hybridisation. The intensity of cross hybridisation amongst the genomes reflects their degree of sequence homology and corresponds with genetic distances obtained from AFLP data. Methods to identify A and B genome sequences have been demonstrated with RAPD markers. There are significant differences in the nuclear DNA content of the different genomes, with T > S > A > B. AFLP analysis of the Musa genomes clustered accessions according to their genomic groups. But clustering of subspecies in the M. acuminata complex did not correspond to similarities suggested by morphology. Currently, breeding programs in Musa are concerned with only the A and B genomes. The breeding strategy involves crossing triploid (3x) landraces with diploid (2x) accessions that harbour useful genes, to produce tetraploid (4x) hybrids. The 4x hybrids are crossed with improved 2x accessions to produce secondary triploids (3x). Interploidy crosses can generate a range of ploidies. Ploidy selection by flow cytometry and genome determination with molecular tags for the A and B genome have become routine breeding practices at IITA. Molecular breeding is seen as a potential benefit for Musa because of significant barriers to conventional breeding. There is a compelling need for further molecular genetic studies in Musa to widen the knowledge base for the genetic improvement of the crop.

1. INTRODUCTION

A genome is defined as a set of chromosomes corresponding to the haploid set of a species [1]. Several levels of sequence organization can be recognized in plant genomes [2]; the largest is the chromosome and the smallest are regulatory sequences of a few base pairs. Chromosomes make up the nucleus of a cell and are visible at metaphase when they are most condensed and therefore easily observed cytologically. Chromosomes have a special importance in plant breeding since they harbour the genes that constitute genetic linkage groups. Over the years, chromosome studies have shifted from a purely cytogenetic level to the molecular level, ushering in an era of molecular cytogenetics. The development of new techniques for genome analysis, such as restriction fragment length polymorphism (RFLP) [3], random amplified polymorphic DNA (RAPD) [4], amplified fragment length polymorphism (AFLP) [5], and genomic in situ hybridisation (GISH) [6], has made it possible to examine in greater detail the structure of plant genomes. GISH is a technique used to differentiate chromosomes from different species by DNA in situ hybridisation. This technique is effective in distinguishing chromosomes from different plant genomes [7]. The merger of molecular and cytological techniques is providing new insights into chromosome structure and evolution. Molecular cytogenetic research in plants has been concerned with the identification of chromosomes, especially in polyploids, and the physical mapping of genes on individual chromosomes. Learning about the physical organization of genes is a critical element for understanding genome organization and evolution in plants [8]. Genome analysis of crop plants is useful for the integration of physical maps produced by cytogenetic analysis with genetic maps constructed by RFLPs and PCR (polymerase chain reaction)-based markers. The main constituent of the chromosomes, the DNA, plays an important role in assessing genetic diversity in plants and displaying relationships between cultivated plants and their wild progenitors. Knowledge of these relationships is useful for introgression of genes from wild plants into their cultivated relatives.

World production of banana (Musa spp.) is seriously threatened by foliar diseases, nematodes, viruses and insect pests, and many breeding programmes have been set up to create resistant varieties. The use of resistant cultivars is considered the most effective, economical and environmentally friendly approach to controlling diseases and pests [9]. Breeding programs make use of knowledge from several scientific disciplines, including genetics, agronomy, horticulture, crop protection and many others that are an essential part of plant breeding [10]. The exploitation of plant genes and genomes for plant breeding is now becoming better recognized. Although the genus Musa has benefited immensely from such studies, there is scope for greater in depth research into the cytogenetics and molecular genetics of the genus. This chapter provides a summary of our current understanding of the genomes in Musa and their applications in phylogenetic analysis and breeding. Although the cytoplasmic organelles of chloroplasts and mitochondria also contain genomes, they will not be discussed. Similarly, the interested reader will find more information on chromosome structure of Musa elsewhere [11-13]. This review will consider the physical characteristics of the genomes in Musa, methods used for their identification, their value in phylogenetic analysis and finally their role in breeding. However, the evolution and diversity of Musa is reviewed first.

2. EVOLUTION AND DIVERSITY

The genus Musa L. comprises the bananas and plantains of the world. It is generally divided into four sections viz. Eumusa, Rhodochlamys, Callimusa and Australimusa. The section Ingentimusa was created to accommodate the species M. ingens (2n = 14) found in the highlands of Papua New Guinea [14]. Musa spp. is a group of giant herbs that are cultivated in humid and subhumid regions of the world where they serve as staple and cash crops for large fractions of the population. Most cultivated banana and plantain belong to the section Eumusa, and are triploid varieties that evolved from two wild diploid (2n = 2x = 22) seminiferous species, M. acuminata Colla (genomes AA) and M. balbisiana (genomes BB). It is commonly accepted that the diploid ancestors originated in two different regions of the area stretching from Papua New Guinea to the Indian subcontinent, i.e., the tropical Malay region for M. acuminata and the more northern Indian region, characterized by alternating monsoons and droughts, for M. balbisiana [15]. The section Australimusa contains the Fe'i bananas and includes M. textilis, which is of commercial importance for its fibre. The sections Rhodochlamys and Callimusa are only of ornamental interest. Bananas are divided into two main categories: dessert and cooking bananas. Dessert bananas constitute 43% of world production and are usually eaten fresh when ripe, as they are sugary and easily digestible. Cooking bananas make up 57% of world production, are usually starchier when ripe, and are boiled, fried or roasted before being eaten [16].

The evolutionary pathway leading to the emergence of cultivated varieties includes two critical events. Firstly, vegetative parthenocarpy and female sterility appeared in M. acuminata, allowing the production of pulp without seeds, as evidenced by the occurrence of parthenocarpic and seedless diploid M. acuminata [15]. Secondly, crosses within M. acuminata or between M. acuminata and M. balbisiana, coupled with female restitution and haploid fertilization, gave rise to (a) homogenomic hybrids which are essentially AAA dessert and highland bananas, and (b) heterogenomic hybrids comprising the AAB plantains and the ABB cooking bananas.

Plantain and banana were introduced to Africa only about 3000 years ago [17], but a remarkable diversity now exists for both groups in two distinct subregions. The plantains prevail in the humid lowlands of West and Central Africa, while the highland cooking and beer bananas abound in the Great Lakes Zone of the Eastern African highlands. The greatest variability of plantains in the world occurs in West and Central Africa. Therefore, this subregion is considered a secondary centre of plantain diversification. Similarly, Eastern Africa is considered a secondary centre of diversity for the highland bananas of the Musa AAA group.

Most cultivated banana and plantain are highly female sterile, i.e., they produce no seeds and cannot reproduce sexually. Thus only clonal, vegetative propagation is possible, implying that survival in nature and geographical dispersal are not possible without human intervention. Consequently, secondary diversification in areas devoid of wild Musa plants, as is believed to largely be the case in Africa, must be due to somatic mutations of the introduced materials [18]. The cultivars are usually known under a multitude of vernacular names, superimposing linguistic diversity to genetic and ecological diversity of the varieties in any given area [19]. These cultivars are susceptible to black Sigatoka (Mycosphaerella fijiensis), but they remain the most preferred types. It is against this background of complex evolutionary events, genomic and ploidy diversity, as well as human cultural intervention, that Musa breeders attempt to develop new varieties of banana and plantains.

3. GENOMES IN MUSA

Four genomes, A, B, S and T, are known to be present in cultivated bananas. The A, B and S genomes are characteristic of species in the section Eumusa. The A and B genomes were derived from the wild diploid species M. acuminata and M. balbisiana, respectively [20]. The S genome is known to be present only in the diploid M. schizocarpa, while the T genome is characteristic of the section Australimusa (2n = 2x = 20). While the A and B genomes are found in the great majority of cultivars [21], the S and T genomes occur in only a few [22-24]. Classification of edible bananas into genomic groups is based on a system created by Simmonds and Shepherd [21]. This system assigns a score of 1 to 5 for 15 selected morphological features that differentiate M. acuminata from M. balbisiana [21]. The total score determines the relative contribution of these wild species to the constitution of the clone. The system is complicated by polyploidy, and requires chromosome counts of a clone before its genome constitution can be determined. Chromosome counting in Musa is laborious and is complicated by their small size. This limitation is now being overcome by the use of flow cytometry for ploidy determination [25-26]. The classification system described by Simmonds and Shepherd [21] was revised by Silayoi and Chomchalow [27]. The main difference between the two systems is the introduction of BBB clones in the latter system. The existence of pure BBB clones remains questionable. Valmayor et al. [28] endorsed the original classification scheme of Simmonds and Shepherd [21]. Although this system has to a large extent been useful and reliable and agrees with newer molecular methods, it has its limitations. The system is only applicable to diploid, triploid and tetraploid clones that contain the A and B genomes and does not consider cultivars with the S and T genomes. The wide range of genetic variation existing in Musa makes it difficult to study the evolution and taxonomy of the genus by means of morphological, phenological and floral markers [29-30].

Genome composition has played an important role in the classification of bananas. The major genomic groups include diploids (AA, BB, AB) triploids (AAA, AAB, ABB) and tetraploids (AAAA, AAAB, AABB, ABBB). Genomic groups are composed of clones on the basis of morphological characters. Clones that share similar characteristics are considered to have arisen from a single base clone by mutation to form subgroups. Jones [16] and Robinson [29] provide a list of the subgroups and some important clones that constitute each genomic group in Musa. It is worth noting that the bananas that constitute each genomic group can be very different. For example, the AAA group contains the sweet dessert bananas as well as the cooking bananas of the East African highlands. Similarly, the AAB group contains the plantains that are cooked before eating and the Pome subgroup that is used as dessert bananas in some countries. The ABB group appears to be rather homogeneous because all clones are used entirely for cooking.

Recently, cultivars with the S and T genomes have been identified in Papua New Guinea [31,32]. Genomic groups with the S genome include AS, AAS and ABBS, while those with the T genome are AAT, AAAT and ABBT. As discussed by Carreel [24], molecular markers have indicated that M. schizocarpa and one or more species from the section Australimusa played a role in the origin of some cultivars in New Guinea. Confirmation of the involvement of the S and T genomes in some of these cultivars has been provided by genomic in situ hybridisation [33].

4. DNA CONTENT, GENOME SIZE AND BASE COMPOSITION

The nuclear DNA content of many plant species, including Musa, has been estimated by flow cytometry. However, relatively few studies have been conducted in Musa to estimate the DNA content and genome size [34-37]. One of the major shortcomings of these studies is the small sample size representing the different genomic groups. While two of these studies [35,37] involved accessions with only the A and B genomes, only one study included plants with the S and T genomes [36]. The sample size of the latter study is unknown. In addition, there is a large difference in the DNA content estimates obtained by d'Hont et al. [36] compared with those obtained in other studies [35,37], which appear to be more similar. Arumuganathan and Earle [34] provided the first report of the total DNA content and genome size in Musa. A 2C DNA content of 1.81 pg with a genome size of 873 Mbp was reported. The term '2C' represents the DNA content of a diploid nucleus and is represented by the G1 peak in flow cytometric analysis. However, later estimates for both DNA content and genome size [35,37] are significantly lower. The DNA content for AA genome accessions ranges from 1.22 to 1.27 pg [35] or 1.20 to 1.33 pg [37]. Similarly, genome size estimates of Musa range from 534 to 615 Mbp [35] or 560 to 610 Mbp [37]. The estimate of DNA content by Arumuganathan and Earle [34] is similar to those reported for triploid accessions in other studies [35, 37]. This led Lysak et al. [35] to suggest that the 1.81 pg 2C DNA content is probably that of a triploid rather than a diploid accession.

With regard to individual genomes in Musa, the B genome is considered to have the smallest nuclear DNA content, with estimates of 2C values ranging from 1.03 pg to 1.16 pg in different studies [35-37]. Estimates for the average 2C value of the A genome are 1.25 pg [35], 1.11 pg [36] and 1.27 pg [37]. The S and T genomes are reported to have 2C values of 1.18 pg and 1.27 pg DNA, respectively [33]. As indicated earlier, the DNA estimates of d'Hont et al. [33] are significantly lower, and a meaningful comparison of DNA content in the A, B, S and T genomes is not possible. However, the DNA content of the S and T genomes is reported to be greater than that of the A and B genomes [33]. Nevertheless, it is clear that there are significant differences of 12 to 15% in DNA content between the A and B genomes. The expectation is that an accession with two B genomes (ABB) will invariably have less DNA than an accession with one B genome (AAB). However, this is not the case as shown by the results of Lysak et al. [35] and Kamate et al. [37]. The bigger sample size used by Kamate et al. [37] showed a greater number of outliers with respect to genome designation and DNA content. For example, the genome size of Simili Radjah (ABB) was typical of that of a tetraploid. Further, an 11% difference in DNA content was noted within subspecies of the M. acuminata complex. This information, although useful, suggests that great caution has to be exercised in interpreting DNA content data in Musa. The genomic base composition of Musa is estimated to have a median value of 40.8% GC [37].

5. DISCRIMINATION OF GENOMES IN MUSA

Several different methods have been used to identify, either directly or indirectly, the genomes in Musa.

5.1. In situ hybridisation

Osuji et al. [38] were the first to apply molecular cytogenetic techniques for genome identification in Musa. While this study involved accessions with only the A and B genomes and their combinations, a more comprehensive study by d'Hont et al. [33] included accessions with the A, B, S and T genomes and some of their combinations. Both studies used genomic in situ hybridisation to differentiate the chromosomes of the different genomes. In GISH experiments, total genomic DNA from a homogenomic species is used as a probe. In the above studies, genomic DNA from M. acuminata (AA), M. balbisiana (BB), M. schizocarpa (SS) and M. augustigemma (TT) were used as probes to identify chromosomes from the A, B, S and T genomes, respectively. These studies together showed that it was possible to differentiate the chromosomes of the four genomes in banana cultivars and hybrids using fluorochromes. For example, plantains (AAB), were shown to have 22 A genome chromosomes and 11 chromosomes of B genome origin. Similarly, 'Wompa' (AS) had 11 chromosomes each from the A and S genomes [33]. More interesting for practical breeding was the identification of genomes in synthetic hybrids of Musa. In a diploid hybrid (TMP2x) from the cross 'Obino l'Ewai' (AAB) × 'Calcutta 4' (AA), GISH showed that there were 22 A genome chromosomes, strongly suggesting that each parent contributed one set (11 chromosomes) of the A genome. Perhaps more interesting was the observation of 33 A genome and 11 B genome chromosomes in the tetraploid hybrid TMPx 4698-1 from the same cross. This result arose from a normal gamete (A) from 'Calcutta 4' and a 2n = 3x egg (AAB) from 'Obino l'Ewai'. GISH results were also useful in settling cases of disputed genomic constitution. For example the cultivar 'Karoina', considered to be either ATT or AAT from morphological descriptors and molecular markers, was found to be AAT. However there were exceptions. For example, 'Pelipita' (ABB) had 8 A chromosomes and 25 B chromosomes instead of the 11 A and 22 B expected. As discussed by d'Hont et al. [33], 21 A and 12 B chromosomes were identified in two plantains from Cameroon, 'Mbouroukou' and 'Nyombe'.

GISH experiments produced broad signals across the centromeric regions of all the chromosomes. Stronger hybridisation signals were observed in some chromosomes than others. The differential hybridisation signals along the length of the chromosome are probably due to the organization of the different classes of repetitive DNA. Our research has shown that the chromosomes of Musa contract differentially during prometaphase and metaphase. The centromeric regions appear to be more highly condensed than the distal regions, suggesting that condensation of chromosomes in Musa begins in the centromeric region. The distal regions lag behind in this process and often appear as lightly stained 'tails'. This may be another reason for the stronger hybridisation in the centromeric regions. Uneven staining patterns are thought to be characteristic of prometaphase chromosomes, especially in plants with small chromosomes [39]. They are caused by differential condensation of the chromatin fibre and are called condensation patterns. Proximal condensed regions are called primary condensations, whereas small interstitial or terminal condensations are termed faint, unstable, or small condensations [39]. Centromeric regions in other species with small chromosomes, such as Arabidopsis, are also known to stain more brightly [40].

The two studies [33,38] reported cross-hybridisation between the genomes. Greater cross-hybridisation existed between the A and B genomes than between the A and S genomes. The least cross-hybridisation occurred between the T genome and the A and B genomes. The intensity of cross-hybridisation is a reflection of the sequence homologies and affinities between the genomes. It also reflects the genetic distances between cultivars representing the different genomes, and corresponds with morphological [41] and molecular markers analyses [24,42,43].

The transfer of useful genes from wild relatives is an important plant breeding strategy, especially in Musa. Effective identification of traits on the chromosomes is essential for understanding the agronomic and horticultural performance of the selected hybrids. GISH could play an important role in identifying gene sequences and alien chromatin in Musa.

5.2. Molecular markers for A and B genome sequences

Identification of PCR markers for detection of A and B genome sequences in Musa was reported by Pillay et al. [26]. Three 10-mer RAPD primers (A17, A18, D10) purchased from OPERON Technologies (Alameda, CA, USA) produced unique banding profiles for the differentiation of M. acuminata (A genome) and M. balbisiana (B genome). Primer A17 amplified two fragments (600 bp, 100 bp) and primer D10 produced one fragment (320 bp) only in M. acuminata. The absence of these fragments in M. balbisiana indicated that they were specific to the A genome of M. acuminata. Similarly, primer A18 produced three fragments (200 bp, 250 bp, 300 bp) that were unique to M. balbisiana and were considered as markers for the B genome. An interesting aspect of the B genome markers was that the B18250 and B18300 fragments were always present in genotypes with at least one B genome while the A18200 fragment was diagnostic for clones with two B genomes. Verification of the informative primers was carried out on a sample of 40 accessions representing landraces and hybrids of different ploidy and genome combinations. The genome composition of the landraces was deduced from morphological descriptors [21], while those of the synthetic hybrids were derived from the genotypes of the parents. PCR assays made it possible to elucidate the genome composition of all the plants. The results were largely in agreement with the genomic designation obtained from phenotypic descriptors. However, there were a few exceptions. The clones 'Monthan Saba' and 'Bluggoe' were previously classified as BBB by Vakili [44] while Valmayor et al. [28] described 'Monthan Saba' as BBB and 'Bluggoe' as ABB. The RAPD marker system showed that both 'Monthan Saba' and 'Bluggoe' are ABB. Similarly, 'Klue Tiparot', originally considered to be a tetraploid from morphological descriptors and reclassified as a triploid from flow cytometry and conventional chromosome analysis [45, 46], has been identified as an ABB clone with the RAPD markers. In later experiments we found that 'Lep Chung Kut', usually classified as BBB [16,47], is an ABB clone. This result puts into question the existence of naturally occurring BBB clones. As shown by Lysak et al. [35], 'Red Dacca' (AAA) clustered with the AAB clones on the basis of nuclear DNA content. The same study also reported that the DNA content of 'Red Dacca' was slightly lower than expected for other AAA clones. The RAPD marker system showed that 'Red Dacca' has an AAB genome constitution.

PCR-RFLP of the internal transcribed spacer regions of the ribosomal RNA genes has also provided markers for the A and B genomes in Musa [48]. A 530 bp fragment unique to the A genome and two fragments of 350 bp and 180 bp specific for the B genome were identified. Interspecific cultivars with both A and B genomes possessed all three fragments. A dosage effect was observed for the B genomes, since the staining intensity of accessions with two B genomes was approximately twice that of accessions with a single B genome.

5.3. Flow cytometry

Flow cytometric analysis of the nuclear DNA content is based on the analysis of the relative fluorescence intensity of nuclei stained with a DNA fluorochrome [49]. Using flow cytometry, d'Hont et al. [36] showed that the sizes of the A, B, S and T genomes are significantly different. The S and T genomes were shown to be larger than the A and B genomes, with the highest value being assigned to the T genome. This information was used as a diagnostic tool to identify the different genomes in Musa. Genome size variation was also used to determine the species involved in interspecific diploid clones as well as the ploidy of banana clones. The validity of using DNA content per se to identify and cluster similar genomes in Musa has to be questioned, especially since the study by Kamate et al. [37] showed that DNA content ranges widely in cultivars having similar genomes. For example, the DNA content for AAB clones ranged from 1.61 pg to 1.79 pg, while those for ABB clones ranged from 1.70 pg to 2.23 pg.

5.4. Isozymes and retrotransposons

As shown by Espino and Pimentel [50], isozyme patterns could also be used to distinguish the A and B genomes in Musa. Balint-Kurti et al. [51] showed that hybridisation of the monkey retrotransposon to a HindIII digest of genomic DNA of Musa produced bands that were specific to the A and B genomes. The only exception was the cultivar 'Chakrakeli' (AAB) that did not show the B-specific band. They suggested that 'Chakrakeli' is probably of AAA constitution, which is probably true since Stover and Simmonds [47] listed 'Chakkarakeli' under the AAA group. Due to differences in spelling of the names, a proper identification of the cultivar is necessary to confirm these results. A larger study with a variety of accessions representing different genomic combinations may be needed to determine the ultimate value of the above systems as genome markers.

6. GENOMES AND PHYLOGENETIC ANALYSIS IN MUSA

We have indicated earlier that genomes play an important role in Musa classification. Evolutionary relationships in the genus have not been fully elucidated and are confounded by hybridisation and polyploidy. Knowledge of these natural relationships is essential for future genetic improvement programs in Musa. Many new techniques have become available to study evolutionary and genetic relationships in plants in the last decade. Extensive AFLP (amplified fragment length polymorphism) studies were carried out in IITA laboratories to show genetic relationships among different genomic groups in Musa [42-43]. This section presents a summary of this work.

AFLP analysis of different subspecies of M. acuminata (AA), M. balbisiana (BB) and some of their natural hybrids (AAA, AAB, ABB) showed that the different genomic groups AA, BB, AAB, ABB formed distinct clusters, as expected, which were supported by morphological similarities. The AAA accessions could not be differentiated from the AA clones and appeared as a single entity. It was interesting to find a clear and distant separation of the BB and AA groups, with the ABB clones appearing closer to the BB group. Although the AAB clones occupied a position between the ABB and AA/AAA clusters, it was more distant from the latter group than the ABB group was from the BB cluster. This information suggests that there was a greater divergence of the plantains (AAB) than the cooking bananas (ABB) from the original ancestors. Molecular analysis of the BB genotypes demonstrated, for the first time, the existence of much more variation in the group than could be assessed from morphology. There is wide genetic and morphological variability in M. balbisiana, and further studies are required to determine if subspecies categories could be designated.

Principal co-ordinate analysis of AFLP data showed that the subspecies of the M. acuminata complex formed three broad groups. The most distinct and homogeneous group consisted of 'Calcutta 4' (ssp. burmannicoides) and 'Long Tavoy' (ssp. burmannica). Other studies [52, 53] also reported a close affinity between these subspecies, and recognized them both as ssp. burmannica. The remaining groups were a collection of different subspecies with one subspecies appearing twice in each of the two groups. This information suggests that with the exception of ssp. burmannica, cross-hybridisation has been frequent within the other subspecies of the M. acuminata complex. This work also showed that the dessert bananas 'Gros Michel' and 'Yangambi Km 5' were closely associated with four subspecies - truncata, microcarpa, malaccensis and banksii - suggesting that the origin of dessert bananas could not be assigned to a single subspecies as hypothesized by Simmonds [15]. Using molecular markers, Carreel [24] showed that AA diploid cultivars were derived from ssp. banksii and ssp. errans. She also indicated that dessert cultivars were derived from ssp. malaccensis and ssp. zebrina. Our cluster analysis placed the dessert banana samples closer to ssp. microcarpa. It is hoped that future work will be able to elucidate the exact progenitors of dessert bananas more clearly.

AFLP data were also useful in determining the relationships of the S and T genome accessions to those with A and B genomes. T genome accessions were most distantly related to those with A and B genomes, while the S genome (M. schizocarpa) clustered with almost 40% similarity to the A genome accessions. These results are in agreement with the results from GISH experiments [33].

7. BREEDING STRATEGY AND PROBLEMS

The overall strategy in banana breeding is to incorporate resistance to diseases and pests in the existing cultivars, rather than aiming for genetic materials that are drastically different from the existing cultivars. Hence, IITA's breeding strategy is essentially based on recurrent phenotypic selection coupled with ploidy and genome manipulation [13,54-56].

The initial steps for genetic improvement of plantains or bananas traditionally involve crossing 3x accessions to 2x accessions that are disease-resistant to produce 4x hybrids. The 4x hybrids are both female and male fertile, and this often reduces fruit quality due to the presence of seeds in the pulp, which is overcome by crossing the 4x selections with 2x accessions to produce secondary 3x hybrids. Recurrent diploid breeding to make 2x strains with better resistance or other desirable traits completes this genetic improvement process. Although this process is conceptually straightforward, complex ploidy and genome arrays can arise and complicate selection in all three crossing schemes, as shown in Table 1 for 3x × 2x breeding, Table 2 for 4x × 2x breeding, and Table 3 for recurrent 2x × 2x breeding. The tables show theoretical expectations for ploidy and genomes in non-recombinant gametes.

7.1 Ploidy selection

Ploidy estimation is classically carried out by chromosome counting [57,58], but this procedure may not be suitable for large-scale screening of breeding materials. Several alternative methods have been investigated, such as those based on stomata size and density [59] or chloroplast density in stomata guard cells [60], but they are not foolproof in Musa. We have therefore adopted a rapid method for ploidy determination that uses flow cytometry [26,61]. This method is precise and non-destructive, requiring a small amount of fresh leaf tissue, and can be used to analyse large populations of cells in one sample, making it easier to detect mixoploidy.

One practical application of this tool is the determination of the frequency distribution of ploidy status in crosses, which in turn provides insights into breeding behaviour of parents and helps to address some theoretical issues regarding breeding methodology. For example, breeding triploid hybrids could theoretically be carried out through 4x × 2x or 2x × 4x crosses. A third possibility is via 2x × 2x crosses with unilateral sexual polyploidisation in one 2x parent.

To determine which of the first two methods offered the best prospects for producing triploids, we used flow cytometry to analyse large segregating breeding populations grown at IITA. The data showed that 4x × 2x crosses produced predominantly triploid progeny (>90%) while 2x × 4x crosses produced less than 3% triploid progeny. In contrast, the frequency of diploid progeny was about 3% in 4x × 2x crosses and > 95% in 2x × 4x crosses. Secondary hybrids of higher ploidy (tetraploids and pentaploids) were also recorded in both 4x × 2x and 2x × 4x crosses, although at a very low frequency. Hence, ploidy polymorphism in this population is attributed to the complex microsporogenesis in the 4x parent. Diploid progenies from 2x × 4x crosses may have resulted from double reduction in the 4x male parent or a differential survival rate of unbalanced pollen (i.e. 1x gametes) compared with 2x gametes in the 4x pollen donor. This result suggests that it is microsporogenesis that determines the ploidy of progeny from inter-ploidy crosses in Musa. Therefore, the choice of male parents is very critical in the genetic improvement of this species.

7.2. Genome selection

There is wide consensus about the attributes conferred by A or B genomes in interspecific natural or artificial hybrids of M. acuminata and M. balbisiana. Hence it is accepted that edibility of mature fruits arose from mutations causing parthenocarpy and female sterility in diploid M. acuminata [15]. It is also commonly accepted that hardiness to drought is contributed by the B genome, since M. balbisiana clones thrive in areas experiencing pronounced dry seasons alternating with monsoons. Also attributed to the B genome are fruit characteristics such as starchiness and acid taste, causing AAB plantains to be starchier but less sweet and less palatable when raw than AAA dessert bananas [15].

This implies that deterministic breeding strategies aimed at high-yielding parthenocarpic varieties of the different utilization classes must incorporate some element of genome substitution. This breeding strategy has been successfully used to add or replace chromosomes by heterologues in species with fully differentiated genomes, notoriously in wheat. Because differentiated genomes behave as separate groups during meiosis and fertilization, it is relatively easy to manipulate chromosomes, compared to situations where the genomes are only incompletely differentiated as is the case for Musa.

Regardless of the complexity of the task facing the breeder, being able to tag genomes is a prerequisite for genome substitution. This underscores the importance of the discovery of molecular markers specific for the A and B genomes [26, 48]. Unlike previous methods of ascribing genome composition [62], these molecular markers can be used at any developmental stage of the plant, do not rely on subjective scoring of morphological traits, and provide an objective means for genome classification in Musa. Clearly, the robustness of these markers depends on (a) their even distribution across chromosomes within the tagged genomes; (b) their location on genetically or structurally conserved regions of the genomes they are associated with; and (c) the non-involvement of such regions in chromosome translocations during meiotic recombination. Notwithstanding this, we have used molecular markers, in conjunction with ploidy analysis, to determine the genome composition of all germplasm accessions (ca. 400) and selected hybrids (ca. 150) available at IITA. This in turn is now being used to design informed crosses with the highest probability of generating breeding lines with putative plantain, dessert or cooking banana characteristics.

8. CONCLUSIONS AND FUTURE PROSPECTS

Genomic research in Musa is still in an early stage. The research efforts and funding spent on other major food crops in the world are much greater than that spent on Musa, which is at the bottom end of the scale. Very few laboratories are devoted to full-time genomic research in Musa, yet genetic information is a prerequisite for developing scientific breeding strategies. Most of the genetic knowledge of Musa has been gained over the last decade [63]. This crop, once considered intractable to genetic improvement, has witnessed the development of improved germplasm through conventional cross-breeding [64]. However due to significant barriers inherent in conventional breeding of bananas, molecular breeding is seen as a potential benefit for the crop [65]. This highlights the need for further molecular genetic studies in Musa.

A wide disparity exists in the DNA content estimates in Musa. Clearly there is a need for further in depth studies in this regard. A greater number of accessions representing different sections, subspecies and genomic combinations in Musa may make a rewarding study, and provide useful knowledge of the evolutionary aspects of this complex genus. Comparisons of DNA contents between studies would be easier if researchers selected the same standard cultivar and used similar fluorochromes.

Although GISH provides direct and reliable information on the identification of genomes in Musa, it is not robust enough to detect intergenomic rearrangements, substitutions and other small changes. GISH may not be useful in distinguishing chromosomes that share a high degree of DNA homology, as has been reported for the A and B genomes of Musa, and this could pose a problem in detecting small chromosomal exchanges between different genomes. GISH combined with other techniques has been more useful for identifying smaller regions of individual chromosomes. Chromosome banding and GISH have been the most popular techniques for identifying alien chromatin in plants with large chromosomes [7, 66]. While GISH identifies the parental origin of the chromosomes, banding methods are used to identify individual chromosomes. However, chromosome banding has been difficult in plants with small chromosomes such as in Musa. To overcome this limitation, GISH is now being combined with various molecular markers such as RFLP, and repeated and low-copy sequences, to identify specific regions on individual chromosomes. Many laboratories are now developing BAC (bacterial artificial chromosome) libraries. Song et al. [67] showed, using fluorescence in situ hybridisation (FISH), that it was possible to isolate a set of BAC clones specific to each of the 12 potato chromosomes. It was also shown that BACs could be used as chromosome-specific cytogenetic DNA markers (CSCDM) for potato chromosome identification. These advances in molecular cytogenetics open up new avenues for the development of Musa CSCDM, and could lead to the establishment of karyotypes in the genus.

Although GISH experiments suggest that there is little differentiation between the A and B genomes, our laboratory has identified both RAPD and PCR-RFLP markers for these genomes. The RAPD markers have been converted to SCARS (sequence characterized amplified regions) to test the validity of these markers. FISH experiments with these markers would locate them precisely on the chromosome. In our research, we observed that the RAPD markers identified 'Pelipita' as an ABB clone implying that it should have a full complement of 11 A chromosomes. However, GISH showed that 'Pelipita' has only 8 A chromosomes [33]. This shows that although the RAPD markers are useful for identifying A and B genome sequences, it cannot be assumed that full genome complements are present.

Although phylogenetic relationships using AFLP in Musa are being addressed, there is still a need for further research in this area using a greater number of accessions. It is also important to have correct identification of the accessions under study to confirm the congruence of results obtained from DNA analysis with those from morphological data. In this way discrepant results could be settled. In this regard, there ought to be greater interaction between banana taxonomists and molecular geneticists. One of the problems researchers are faced with in this area is the acquisition of plant material for evolutionary studies, especially from the areas of origin of Musa.

Musa breeding is complicated by a number of factors [13,54,68]. The development and use of new tools such as flow cytometry for ploidy selection, and molecular markers for genome identification, has widened our knowledge of the behaviour of genomes in Musa crosses. The value of the S and T genomes in Musa breeding has not been studied in detail. More effort should be directed at examining these genomes, which could harbour useful resistance genes for Musa improvement.

Genomic research in Musa has progressed in phases from the initial determination of chromosome numbers to the study of genome relationships in artificially produced hybrids. Identification of the chromosomes that constitute the A, B, S and T genomes using fluorescence microscopical techniques has been accomplished. Major landmarks such as the NOR locus and the smaller 5S ribosomal DNA sites have been located. Researchers are now poised to embark on the next phase of dissecting the fine structure of the Musa genomes.

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[48] NWAKANMA, D. C., et al., PCR-RFLP of the ribosomal DNA internal transcribed spacers (ITS) provides markers for the A and B genomes in Musa L., 4th Int. Symp. Mol. Cell. Biol. Banana., September 2002, Leuven, Belgium (2002).

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Table 1 Theoretical expectations for ploidy and genome segregation in gametes and zygotes during crosses between triploid female and diploid male parents, assuming chromosome segregation. Gametes are shown in brackets

Chromosome segregation in triploid female parent

Chromosome segregation in diploid male parent

AA Parent

BB Parent

AB Parent

Normal

Abnormal

Normal

Abnormal

Normal

Abnormal



[A]

[O]

[AA]

[B]

[O]

[BB]

[A]

[B]

[O]

[AB]

AAA Parent












Normal

[A]

AA

AO

AAA

AB

AO

ABB

AA

AB

AO

AAB

[AA]

AAA

AAO

AAAA

AAB

AAO

AABB

AAA

AAB

AAO

AAAB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[AAA]

AAAA

AAAO

AAAAA

AAAB

AAAO

AAABB

AAAA

AAAB

AAAO

AAAAB

AAB Parent












Normal

[A]

AA

AO

AAA

AB

AO

ABB

AA

AB

AO

AAB

[AB]

AAB

ABO

AAAB

ABB

ABO

ABBB

AAB

ABB

ABO

AABB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[B]

AB

BO

AAB

BB

BO

BBB

AB

BB

BO

ABB

[AA]

AAA

AAO

AAAA

AAB

AAO

AABB

AAA

AAB

AAO

AAAB

[AAB]

AAAB

AABO

AAAAB

AABB

AABO

AABBB

AAAB

AABB

AABO

AAABB

ABB Parent












Normal

[B]

AB

BO

AAB

BB

BO

BBB

AB

BB

BO

ABB

[AB]

AAB

ABO

AAAB

ABB

ABO

ABBB

AAB

ABB

ABO

AABB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[A]

AA

AO

AAA

AB

AO

ABB

AA

AB

AO

AAB

[BB]

ABB

BBO

AABB

BBB

BBO

BBBB

ABB

BBB

BBO

ABBB

[ABB]

AABB

ABBO

AAABB

ABBB

ABBO

ABBBB

AABB

ABBB

ABBO

AABBB

NB: The number of letters A and B designate the respective frequency of A and B genomes, the sum of which gives the ploidy level. The letter O indicates the lack of A or B genomes (aneuploids).

Table 2 Theoretical expectations for ploidy and genome segregation in gametes and zygotes during crosses between tetraploid female and diploid male parents, assuming chromosome segregation. Gametes are shown in brackets

Chromosome segregation in tetraploid female parent

Chromosome segregation in diploid male parent

AA Parent

BB Parent

AB Parent

Normal

Abnormal

Normal

Abnormal

Normal

Abnormal

AAAA Parent


[A]

[O]

[AA]

[B]

[O]

[BB]

[A]

[B]

[O]

[AB]

Normal

[AA]

AAA

AAO

AAAA

AAB

AAO

AABB

AAA

AAB

AAO

AAAB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[A]

AA

AO

AAA

AB

AO

ABB

AA

AB

AO

AAB

[AAA]

AAAA

AAAO

AAAAA

AAAB

AAAO

AAABB

AAAA

AAAB

AAAO

AAAAB

[AAAA]

AAAAA

AAAAO

AAAAAA

AAAAB

AAAAO

AAAABB

AAAAA

AAAAB

AAAAO

AAAAAB

AAAB Parent












Normal

[AA]

AAA

AAO

AAAA

AAB

AAO

AABB

AAA

AAB

AAO

AAAB

[AB]

AAB

ABO

AAAB

ABB

ABO

ABBB

AAB

ABB

ABO

AABB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[A]

AA

AO

AAA

AB

AO

ABB

AA

AB

AO

AAB

[B]

AB

BO

AAB

BB

BO

BBB

AB

BB

BO

ABB

[AAA]

AAAA

AAAO

AAAAA

AAAB

AAAO

AAABB

AAAA

AAAB

AAAO

AAAAB

[AAB]

AAAB

AABO

AAAAB

AABB

AABO

AABBB

AAAB

AABB

AABO

AAABB

[AAAB]

AAAAB

AAABO

AAAAAB

AAABB

AAABO

AAABBB

AAAAB

AAABB

AAABO

AAAABB

AABB Parent












Normal

[AB]

AAB

ABO

AAAB

ABB

ABO

ABBB

AAB

ABB

ABO

AABB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[AA]

AAA

AAO

AAAA

AAB

AAO

AABB

AAA

AAB

AAO

AAAB

[BB]

ABB

BBO

AABB

BBB

BBO

BBBB

ABB

BBB

BBO

ABBB

[AAB]

AAAB

AABO

AAAAB

AABB

AABO

AABBB

AAAB

AABB

AABO

AAABB

[ABB]

AABB

ABBO

AAABB

ABBB

ABBO

ABBBB

AABB

ABBB

ABBO

AABBB

[AABB]

AAABB

AABBO

AAAABB

AABB

AABBO

AABBBB

AAABB

AABBB

AABBO

AAABBB

ABBB Parent












Normal

[AB]

AAB

ABO

AAAB

ABB

ABO

ABBB

AAB

ABB

ABO

AABB

[BB]

ABB

BBO

AABB

BBB

BBO

BBBB

ABB

BBB

BBO

ABBB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[A]

AA

AO

AAA

AB

AO

ABB

AA

AB

AO

AAB

[B]

AB

BO

AAB

BB

BO

BBB

AB

BB

BO

ABB

[ABB]

AABB

ABBO

AAABB

ABBB

ABBO

ABBBB

AABB

ABBB

ABBO

AABBB

[BBB]

ABBB

BBBO

AABBB

BBBB

BBBO

BBBBB

ABBB

BBBB

BBBO

ABBBB

[ABBB]

AABBB

ABBBO

AAABBB

ABBBB

ABBBO

ABBBBB

AABBB

ABBBB

ABBBO

AABBBB

The number of letters A and B designate the respective frequency of A and B genomes; their sum is the ploidy level. The letter O indicates the lack of A or B genomes (aneuploids).

Table 3 Theoretical expectations for ploidy and genome segregation in gametes and zygotes during crosses between diploid female and diploid male parents, assuming chromosome segregation. Gametes are shown in brackets

Chromosome segregation in diploid male parent

Chromosome segregation in diploid female parent

AA Parent

BB Parent

AB Parent

Normal

Abnormal

Normal

Abnormal

Normal

Abnormal

[A]

[O]

[AA]

[B]

[O]

[BB]

[A]

[B]

[O]

[AB]

AA Parent












Normal

[A]

AA

AO

AAA

AB

AO

ABB

AA

AB

AO

AAB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[AA]

AAA

AAO

AAAA

AAB

AAO

AABB

AAA

AAB

AAO

AAAB

BB Parent












Normal

[B]

AB

BO

AAB

BB

BO

BBB

AB

BB

BO

ABB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[BB]

ABB

BBO

AABB

BBB

BBO

BBBB

ABB

BBB

BBO

ABBB

AB Parent












Normal

[A]

AA

AO

AAA

AB

AO

ABB

AA

AB

AO

AAB

[B]

AB

BO

AAB

BB

BO

BBB

AB

BB

BO

ABB

Abnormal

[O]

AO

OO

AAO

BO

OO

BBO

AO

BO

OO

ABO

[AB]

AAB

ABO

AAAB

ABB

ABO

ABBB

AAB

ABB

ABO

AABB

NB: The number of letters A and B designate the respective frequency of A and B genomes, their sum is the ploidy level. The letter O indicates the lack of A or B genomes (aneuploids).


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