Genetic Diversity in Banana and Plantains Cultivars from Eastern DRC and Tanzania Using SSR and Morphological Markers, Their Phylogenetic Classification and Principal Components Analyses Genetic Diversity in Banana and Plantains Cultivars from Eastern DRC and Tanzania Using SSR and Morphological Markers, Their Phylogenetic Classification and Principal Components Analyses

Bananas and plantains are edible and vegetatively propagated parthenocarpic species of the genus Musa. They are used as staple food, dessert and cash crop by more than hundred millions of people throughout the world. However, the crop is threatened by several pests and diseases in central and eastern Africa. One way of partly solving this problem is to have diploids which have desirable traits currently lacking in the AAA- Lujugira-Mutika subgroup. The study assessed through 21 microsatellite markers pairs the cladistic closeness of the diploid AA-Mshale accessions with AAA-Lujugira-Mutika with the purpose of inclusion in breeding programmes. Results showed that the eight studied accessions of AA-Mshale were different from each other. AA-Mshale malembo was fairly well established to be among the ancestor of Lujugira-Mutika, suggesting the determinism of its pollen viability and the level of resistance to pests for including in breeding programmes. The use of two pairs of microsatellites per chromosomes linkage group established the existence of alleles ’ deletion, recombination or non-annealing. The closeness among AA-Mshale and AAA-subgroups (Ibota, Gros Michel and Green Red) so far established through other techniques was confirmed. The results recommend the use of microsatellite markers, covering 11 linkage groups for cultivar identification and diver- sity study.


Background
Bananas and plantains are edible and vegetatively propagated parthenocarpic species of genus Musa belonging to the family Musaceae which according to Meng et al. [1] has wild seeded species native to South-East Asia. These seedless edible species are thought to have originated through intra-and interspecies crosses between M. acuminata Colla and M. balbisiana Colla, including some back crosses [2]. These species constitute a staple food, a key commercial crop and a major source of raw materials for both beverage and handicraft industries for hundred millions of people in the world. They include 20% of the population of the United Republic of Tanzania (URT), and its production promotes the country to be the second largest producer after Uganda in east Africa [3][4][5].

Problem statement and justification
The east African highland bananas (EAHBs) are currently threatened by several pests and diseases, which need diploid parents with farmers and other consumers' desirable traits for inclusion in the breeding programme [6]. The edible diploid landrace 'Mshale' (Mchare [7], AA genomic group) of URT was identified to be highly similar to M. acuminata spp. malaccensis cv. 'Pisang lilin'. Research using numerical taxonomy on AAA-EAHB genomic subgroup from Eastern DRC and Tanzania has shown certain level of relationship with 'Mshale malembo', suggesting that it is one of the ancestors [8,9]. These observations were supported by Simmonds [7] and De Langhe et al. [10] but need to be confirmed at a molecular level. Such research has not yet been done and remains dearth for the inclusion of the Tanzania's landrace in breeding programme. Elsewhere, such research using the AFLP technique has been conducted by Ude et al. [11], on phylogenetic origin of AAA-Gros Michel and AAA-Yangambi km 5. The technique has shown that these cultivars have similar ancestors that have contributed to their development. In this respect, M. acuminata spp. malaccensis cv. 'Pisang lilin' was identified as a source of one of their genomes (A). This supported the use of landrace AA 'Paka' from Zanzibar in the improvement of 'Gros Michel' in Jamaïca [7,10].

Hypothesis, technology justification and objective
AFLP technique shows a dominant mode of inheritance and hence constitutes its limiting factor for this study. On the other hand, research using SSR markers has confirmed these preceding findings [12]. Moreover, the fact that the genetic map has 11 linkage groups of Pisang lilin was also reported [13]. Therefore, the determination of identity and confirmation of the contribution of 'Mshale' in the AAA-EAHB using microsatellite markers determined from Pisang lilin could be a useful tool for the regeneration of subgroups escaping genetic erosion due to pests. This would constitute different scientific point of view from the current belief that AAA-EAHB comes from somaclonal variation [14]. The study aimed to establish the cladistic relationship of the banana landrace 'AA-Mshale' in AAA-EAHB which may constitute a way for reconstituting the EAHB through breeding.

Plant materials
Cigar (unfurled) leaf samples from 25 accessions of bananas and plantains ( Table 1) were collected from the existing banana gene bank in the Horticulture Unit of Sokoine University of Agriculture (SUA). The 25 accessions consisted of eight edible diploids (AA), nine, four and two triploids (AAA, AAB, ABB), two tetraploids (AAAA) genomic groups which were determined through numerical morpho-taxonomic classification [15]. Apart from the diploids and triploids AAA-EAHB subgroup, the other subgroups and genomic group were added as control to verify the accuracy of the ancestry. The SUA Musa sp. germplasm was an in situ field conservation located in the plateau zone of Morogoro Urban District of Tanzania [5].

DNA extraction
The DNA of the 25 accessions ( Table 1) was isolated using DNeasy Plant Mini Kit (Qiagen, USA; www.qiagen.com) following the manufacturer's instructions, quantified in 2% agarose gel (in 0.5 TBE electrophoresis buffer) and stained in 5 μg/ml of ethidium bromide solution.
The DNA quality was checked by ensuring that the 260/280-nm values ranged between 1.4 and 2.2 using spectrophotometer [12]. The PCR was performed using a Gene Amp PCR system 2700 thermocycler (Applied Biosystems). Each reaction was carried out in a total volume of 20 μl, containing 10 ng of genomic DNA, 1. amplifications, temperature cycling was conducted as follows: an initial denaturation step at 95 C for 5 min that was followed by 32 cycles of denaturation at 94 C for 1 min, annealing at each temperature as specified in Table 2 per primer pair for 1 min, and extension (elongation) for 90 s at 72 C. A final extension was carried out at 72 C for 7 min. For gel electrophoresis, a 10-μl aliquot of each amplification reaction was separated at 100 V for 2 h, using 2% agarose gels (0.5Â TBE buffer). Gel images were photographed under UV illumination to check for amplicon size and PCR specificity. Allele sizes were estimated against 2-Log DNA Ladder molecular size standards. All samples were run with three replications starting from DNA extraction to maintain the integrity of the sample.

Data analysis
Alleles (0, 1, 2, …) were scored from 21 SSR marker pairs in the 25 accessions and were used to build the phenetic and cladistic trees. The data were analyzed using Numerical Taxonomy and/or  Table 2. Primer sequences, SSR repeat motif, linkage groups (LG), theoretical annealing temperature ( C) and expected PCR product's size (bp).
Multivariate Analysis System package (NTSYSpc) version 2.1 (Exeter Software, Setauket, USA). The Manhattan method was used to assess similarity among the banana accessions. The genetic similarity matrices were then used to construct the dendrogram with unweighted pair group method with arithmetic mean (UPGMA) algorithms that employed the sequential, agglomerative, hierarchical and nested clustering procedure [16]. The cladistic kinship between accessions was determined based on neighbor joining coefficients using Dice dissimilarity coefficients (matrix using NTSYSpc 2.1. The scattered plot and accuracy of the trees were determined using principal component analysis (PCA) and cophenetic correlation method (from NTSYSpc 2.1). A two-way Mantel statistic test of 500 permutations was performed to get a cophenetic value.

Molecular/genetic relatedness among accessions
The coefficient of dissimilarity varied from 0.28 to 0.66, being <1 or 100% showing no duplication among accessions from the 21 loci covering 11 linkage groups used as shown in Figure 1. Hence, the eight accessions belonging to AA-Mshale group were found to be genetically different. The dendrogram ( Figure 1 The genetic variation causes were allelic deletion or non-annealing and heterozygosis. The mMaCIR168 primer showed allele deletion in cultivars 'Ndyali' and 'Mwanjunjila' (first one and third three after (left) Ladder, Figure 2), and mMaCIR189 showed heterozygosis in cultivars 'Mshale Makyughu', 'Ilalyi' and 'King banana' (first six, nine and second three after ladder) while both primers showed a homozygote allele in cultivar 'Mshale malembo' (the first number six after the ladder). Similarly, alleles' deletion (null alleles) was observed among 19 cultivars for primers mMaCIR117 and mMaCIR174. The alleles' sizes resemble those of Hippolyte et al. [13].
The observed mutation has negatively influenced the principal component analysis (PCA) that resulted in poor fit of the clustering analyses with a cophenetic coefficient of 0.72 from distance matrix and 0.67 from product-moment correlation matrix. Consequently, the variation has spread over the principal component (PC) so that the three first PCs cannot hold the maximum of the variation ( Figure 3) and hence weakened the value of PIC (Polymorphism Information Content).

Cladistic relationship
The cladogram showed three clades which revealed mono-, para-and polyphyly (A, B and C, Figure 4).

Discussion
This clustering from dissimilarity using UPGMA fairly confirms the relationship established by numerical taxonomy between the AA-Mshale malembo and the AAA-Lujugira-Mutika group determined by several authors [2,[7][8][9]. Likewise, the observed alleles' differences among AA-Mshale accessions were in line with the morpho-taxonomic dissimilarity determined previously by the upcited authors. Moreover, the clone sets (Musakala, Nfuuka and Nakitembe) coined subjectively within the AAA-Lujugira-Mutika were linked with the different AA-Mshale accessions following their alleles' closeness [16]. Interestingly, the clustering of AAA-Cavendish, AAA-Gros-Michel, AAA-Ibota, AAB-Plantain and AAB-Silk subgroups as sympatric is similar to results of [11,12,17], while they used other techniques or primers partly covering the 11 linkage groups [13]. This once more established the usefulness and reliability of the alleles from the 11 linkage groups in diversity and cladistic study. The mono-, para-and polyphyletic relationships are in line with those revealed from numerical morpho-taxonomy [7][8][9][10]. The para-and polyphyletic relationship may be explained by the hypothesis of back-crosses developed [2]. The back-crosses theory explains the role of the observed alleles deletion and rearrangement (heterozygosis) in the evolution of AA-Mshale malembo in the AAA-EAHB. These relationships were also similar to results from other microsatellites covering 10 linkage groups [17]. However, there is contrast with the statement of lack of convincing lineage between 'Mutika-Lujugira', 'Red', 'Ibota' and 'Plantain' subgroups, and the diploid M. acuminata accessions. This may be explained by the poor fit of the clustering analysis and the spread of principal components over the variables due to observed mutation.

Conclusion and suggestion
The eight accessions of AA-Mshale were determined at allele level to be different from each other. The contribution of accession AA-Mshale malembo in the ancestry of AAA-Lujugira-Mutika has been ascertained using simple sequence repeat tandem (SSR) markers. This suggests more studies on the parameters like pollen viability, germination and level of resistance to diseases and pests before inclusion in the breeding programme. The SSR markers constitute the best tool for cultivar phylogenetic identification, marker-assisted selection and diversity study.