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 [15]. Apart from the diploids and triploids AAA-EAHB subgroup, the other subgroups and genomic group were added as


Table 1. Cultivars used in molecular characterization using SSR markers.

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].

#### 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 [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, 1.2 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 [13]. During


Genetic Diversity in Banana and Plantains Cultivars from Eastern DRC and Tanzania Using SSR and Morphological… 63 http://dx.doi.org/10.5772/intechopen.79922


Table 2. Primer sequences, SSR repeat motif, linkage groups (LG), theoretical annealing temperature ( ˜C) and expected PCR product's size (bp).

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.

#### 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 [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.
