Cultivars used in molecular characterization using SSR markers.
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 diversity study.
- AA-Mshale malembo
- SSR markers
Bananas and plantains are edible and vegetatively propagated parthenocarpic species of genus
1.2. 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 . The edible diploid landrace ‘Mshale’ (Mchare , AA genomic group) of URT was identified to be highly similar to
1.3. 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 . Moreover, the fact that the genetic map has 11 linkage groups of Pisang lilin was also reported . 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 . 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.
2. Materials and methods
2.1. 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 . 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
|No||Name of cultivars||Genomic group||Subgroup, clone set|
|04||Ngego I||AAB||French Plantain|
|05||Ngego Halisi||AAB||French Plantain|
|09||Bukoba||AAA||EAHB-Musakala, cooking type|
|10||Embwailuma||AAA||EAHB-Nakitembe, cooking type|
|11||Mwanjunjila||AAA||EAHB-Nfuuka, cooking type|
|12||Muhowe||AAA||EAHB-Nfuuka, beer type|
|13||Kimalindi fupi||AAA||Dwarf Cavendish|
|15||Yangambi km 5||AAA||Ibotabota (or ‘Ibota’ in short)|
|21||King banana||AA||Wild diploid|
2.2. 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 . 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, 1.2 mM MgCl2, 10 mM dNTPs, 0.2 μM of each primer, 1.25 U of Taq polymerase and 10x Go Taq flex buffer (New England Biolabs, Inc.). Twenty-one SSR primer pairs (Table 2) distributed across the 11 linkage groups were used. This SSR primer selection was done among established linkage groups covering banana genome . During 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 (0.5× 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.
|SSR||Motif||LG||Forward primer (F)||°C||bp|
|Reverse primer (R)|
2.3. 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 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 . 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.
3. Results and discussion
3.1.1. 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) established two main clusters (A and B). In the first cluster (A), AAA-Lujugira-Mutika accessions (‘Bukoba’/Musakala, ‘Muhowe’/Beer (Mbidde) and ‘Embwailuma’/Nakitembe) were clustered with the seven accessions of AA-Mshale (‘Ndyali’, ‘Mshale makyughu’, ‘Ilalyi’, ‘Mshale malembo’, ‘Nshonwa mshale’, ‘Ijihu’ and ‘Huti’). They included the tie of AAAA-FHIA (17 and 23) accessions with ‘Yangambi km 5’ (AAA-Ibota), ‘Mzungu mwekundu’ (AAA-Green-red) and ‘Green bell’ (AAA-Cavendish). Whereas in the second cluster (B), six heterogenomic accessions named ‘Kisukari’ (AAB-Silk), ‘Ngego I’, ‘Ngego Halisi’ and ‘Mzuzu’ (AAB-French Plantain), ‘Unyoya’ and ‘Bokoboko’ (ABB) were tied to three homogenomic accessions (AAA) ‘Jamaica’ (Gros Michel), ‘Kimalindi fupi’ (Dwarf-Cavendish) and Mwanjunjila (EAHB having a yellow male bud). The accession ‘King banana’ (AA) was an outline.
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. .
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).
3.1.2. Cladistic relationship
The cladogram showed three clades which revealed mono-, para- and polyphyly (A, B and C, Figure 4). The eight AA-Mshale accessions were subdivided into two clades. The first clade (A) was a monophyletic group composed of eight accessions in which six belonged to AA-Mshale genomic group (‘Ndyali’, ‘Mshale malembo’, ‘Ijihu’, ‘Nshonwa mshale’, ‘Huti’ and ‘King banana’) and two of triploid (‘Green bell’ (AAA-Cavendish) and ‘Mzungu mwekundu’ (AAA-Green-red).
The second clade (B, Figure 4) that encompassed AAA-EAHB accessions was subdivided into two subclades (B1 and B2) and formed paraphyletic group with the first clade. The first subclade (B1) was made of three accessions, ‘Mzuzu’, ‘Bukoba’ and ‘Yangambi km 5’, that belonged to AAB-French Plantain, AAA-EAHB-Musakala and AAA-Ibota, respectively. Whereas, in the second subclade (B2), the AAA-EAHB accessions ‘Muhowe’ and ‘Embwailuma’ shared the ancestry with AA-Mshale (Mshale makyughu and Ilalyi) and AAAA-FHIA (17 and 23). The last clade (C) had ‘Kimalindi fupi’ (AAA-Cavendish), ‘Mwanjunjila’ (AAA-EAHB) and Jamaica (AAA-Gros Michel) sharing a common ancestry with AAB-Silk (Kisukari), AAB-French plantain (Ngego Halisi and Ngego I) and ABB (Bokoboko and Unyoya). The clade (C) established a polyphyly with the two first clade (A and B) that had AA genomic group accessions. Whereas, in reference to accession ‘Jamaica’, there was a paraphyly between the clades B and C.
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 . 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 . 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 . 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 . However, there is contrast with the statement of lack of convincing lineage between ‘Mutika-Lujugira’, ‘Red’, ‘Ibota’ and ‘Plantain’ subgroups, and the diploid
4. 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.
The authors would like to thank the Canadian International Development Agency (CIDA) (through Bioscience in East and Central Africa Network/NEPAD) for funding this research.
Conflict of interest
The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this chapter.