Chapter 4: Simple sequence repeat (SSR) markers-based genetic diversity and population
4.3 Material and Methods
In early 2008, a total of 202 samples, hereafter referred to as "accessions", were collected from 51 villages, which represented 7 of the 8 rice growing regions in Niger. The collecting trip focused on the Niger River Valley (west and south-west), the Dallol Maouri watercourse valley (south-west), the marshlands and Goulbi of Maradi (central-south), and the Komadougou River Valley (south-east) (Appendix 4.1). In each village, rice varieties were inventoried and a seed sample collected from garrets. Within accessions, seeds were purified visually and then a
"purification-characterization" phenotyping trial was conducted in the 2008 rainy season.
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Morphological features such as ligule length, abundance and distribution of spikelets borne on secondary branches of the panicle, and the attitude of the main axis of the panicle were used to differentiate O. sativa from O. glaberrima (Sano and Sano, 1984). Additionally, plant culm attitude (openness), grain length, awn length and texture were also used to differentiate O. glaberrima from adventive O. barthii Chev. off-types found in the rice samples. Five plants were individually harvested for each accession. The final collection was 370 accessions, but only 321 accessions were used in this study, after the elimination of duplicates. The collection was composed of cultivated rice and 26 wild rice samples, of which 9 were pure O. barthii accessions, collected far from any cultivated rice field; 16 were weedy O. barthii types isolated from rice samples collected with farmers, and one sample was O. longistaminata Chev. et Roehr.
collected in deepwater. Pure and weedy O. barthii types were regrouped together as the samples of O. barthii in this study. Eight varieties such as RAM63 (a floating, photosensitive traditional O. sativa subsp. indica from Mali), IR64 and B6144 (irrigated O. sativa subsp. indica varieties, from the Philippines and Indonesia respectively), Moroberekan and Nipponbare (upland O. sativa subsp. japonica varieties, respectively, from Guinea and Japan), Basmati 307 (an irrigated aromatic O. sativa variety from India), NERICA 7 (an upland interspecific variety from AfricaRice) and CG14 (an upland O. glaberrima from Cote d'Ivoire) were added to the collection as reference varieties.
4.3.2. DNA extraction and genotyping
DNA was extracted by bulking about equal amounts of leaf tissues from 4 plants per accession using the CTAB protocol as described by Rustericci et al. (2000). DNA concentration was by running aliquots of DNA samples on a 1% agarose gel that contained 0.3 µg/mL of ethidium bromide. Twenty six SSR markers, previously used for studying the Generation Challenge Programme rice core collection (CIRAD, Montpellier), were chosen for this study. PCR mix consisted of 1x colorless GoTaq Reaction Buffer (Promega, M7921, Madison, WI, USA), 200 µM of each dNTP (dATP, dGTP, dCGT, dTTP), 0.2 µM of each of the M13-tailed forward and reverse primer, 0.2 µM of a universal M13 fluorescently labelled dye at 700 nm or 800 nm, 1 unit of taq DNA polymerase, and 20 ng template DNA. The 5' end of each forward primer was labelled with a M13 fluorescent dye. The amplification was performed in a T-Gradient thermocycler (Biometra, Goettingen, Germany)
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The PCR products were separated on a 5.5% denaturant polyacrylamide gel on a LiCor IR2 DNA Sequencer. Alleles were called using the Geneprofiler (Version 4.05) software package. Five positive control (reference set) DNA samples (bulked-rice DNA samples of known allele sizes received from CIRAD, Montpellier) and three size markers were loaded on the same gel to serve as size references.
4.3.3. Data analysis
A total of 321 accessions and 8 control varieties were initially genotyped with 26 SSR markers.
Eighteen markers were maintained after dropping the markers with more than 10% missing data.
Fifty seven accessions with identical genotypes were also excluded from the dataset. Thus, the different statistical analyses were performed on the dataset consisting of 264 accessions and 18 SSR markers. Firstly, the software GenAlex (version 6.2) (Peakall and Smouse, 2006), was used to eliminate redundant accessions, with the same genotypic profile for all the markers. Secondly, a genetic dissimilarity matrix was computed based on the simple matching index using the DARwin (Version 5.0.156) software (Perrier et al., 2003).
The distance matrix was then used as input data for constructing a dendrogram using the Neighbour Joining method. Genetic diversity parameters of the collection, such as the number of alleles (Na), heterogeneity (due to heterozygosity and/or bulking of two homozygote genotypes), polymorphic information content (PIC), and gene diversity were calculated using PowerMarker Version 3.25 (Liu and Muse, 2005).
Analysis of Molecular Variance (AMOVA) was used to partition the total genetic variation into among and within population components using Arlequin Version 3.5 (Excoffier and Lischer, 2010). An admixture model-based clustering method was used to infer population structure using the software package STRUCTURE software version 2.3.3 (Pritchard et al., 2000). Allele frequencies were assumed to be correlated and loci were assumed to be unlinked (Falush et al., 2003). The number of populations tested (K) varied from 1 to 12, with ten repetitions each. The procedure was based on a burn-in period of 50,000 iterations and 200,000 iterations of the MCMC. Five independent simulations were performed. The real K value was determined using the method proposed by Evanno et al. (2005), based on the second-order rate of change of likelihood of data between consecutive K values. Accessions with probability of 70% of membership were assigned to the same group while those with lower probability memberships in
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any single group were assigned to an ‘‘admixed’’ group. Finally, O. glaberrima accessions were separated from O. barthii and comparison of related diversity indexes was made.