3.2.1. Germplasm
A total of 340 samples (Appendix 3.1) which included 301 accessions from Kenya, Uganda and Tanzania, 15 selections from the global minicore set [minicore set being 1% (80) of the global finger millet collection at ICRISAT genebank-India constituted by Upadhyaya et al. (2010)] and 24 checks (elite and blast resistant/susceptible lines from ICRISAT Nairobi breeding program) were used in this study. The 301 accessions represented nine sub-regions viz: eastern Uganda-mid altitude, sub humid with 240-269 length of growing period (LGP in days); western Uganda-mid altitude with 270-299 LGP; northern Uganda-mid altitude with 210-230 LGP; western Kenya – mid altitude sub humid with 240-269 LGP;
Rift Valley Kenya-high altitude low temperature with 120-209 LGP; eastern Kenya-semi-arid mid to low altitude; western Tanzania-mid altitude with 210-239 LGP; northern Tanzania-mid altitude sub humid with 90-149 LGP; and Rukwa region southern Tanzania high altitude with 120-209 LGP. These sub- regions also have differential ethnic representation with occasional overlaps. The 15 minicore accessions
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were selected based on the diversity groups established by Upadhyaya et al. (2010) and were included in order to ascertain if the minicore set adequately captured the total global diversity.
3.2.2. Growing plants
Finger millet seeds were planted in an 8 x 12 well format in plastic trays in soil that was sterilized at 140°C for 30 minutes, and placed in an incubator at 30oC for 24 hours to germinate. The seedlings were then transferred to a greenhouse at the University of Nairobi field station for 2 weeks and were watered regularly.
3.2.3 DNA extraction
Leaf samples of similar size were taken from 10-14 day-old plants from five seedlings in each accession and bulked per accession. The leaf tissue was placed in 12 x 8 well strip tubes with strip caps (Marsh Biomarket, USA) together with two 4 mm stainless steel grinding balls (Spex CertiPrep, USA). To each sample 450 µL of preheated (65°C) extraction buffer (100mM Tris-HCL [pH 8), 1.4 M NaCl, 20 mM EDTA, CTAB [3% w/v], β-mercaptoethanol (0.15% v/v) were added and secured with 8-strip caps (Marsh Biomarket). Grinding of the samples was done in a Spex Certi-prep Inc. Geno/grinder 2000® at 500 strokes/min for 10 min. The samples were then incubated for 30 min at 65°C in a water bath with occasional mixing. DNA extraction was then carried out following the protocol by Mace et al. (2013) with the exclusion of the phenol-chloroform step (de Villiers S, unpublished) where 450 µL of chloroform-isoamylalcohol (24:1) was added to each sample and then inverted twice to mix. The samples were then centrifuge plated at 6200 g for 10 min (Sigma centrifuge model 4K15C with QIAGEN rotor model NR09100: 2 × 1120 g SW). A fixed volume (400 µL) of aqueous layer was transferred to fresh strip tubes (Marsh Biomarket) and 0.7 vol isopropanol (stored at –20°C) added to each sample and inverted once to mix and the centrifuge plated at 6200 g for 15 min. Supernatant from each sample was decanted and the pellet air dried for 30 min. To each sample 200 µL low-salt TE (10 mM Tris, 0.1 mM EDTA [pH 8]) plus 3 µL RNase A (10 mg/mL) were added and incubated at 37°C for 30 min. To each sample 200 µL chloroform-isoamylalcohol (24:1) was added and the sample was inverted twice to mix then centrifuge plated at 4000 g for 15 min. A fixed volume of aqueous layer was transferred to a fresh 96 deep-well plate (Marsh Biomarket). To each sample 315 µL ethanol-acetate solution (30mL EtOH, 1.5mL 3 M NaOAc [pH 5.2]) was added and placed in –20°C for 5 min then centrifuge plated at 4000 g for 15 min. Supernatant was decanted from each sample and the pellets washed with 70% EtOH and centrifuge plated at 4000 g for 5 min then washed again with 70% EtOH. Supernatant from each sample was decanted and air dried for approximately 1 h and the pellet resuspended in 100 µL low-salt TE and stored
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at 4°C. DNA quality and quantity for all samples was determined by agar gel electrophoresis (0.8% w/v) and spectrophotometry (Nano-drop® 1000-Thermo Scientific, USA) then diluted to 10ng/µl in TE buffer (10 mM Tris, 0.1 mM EDTA at PH 8.0).
3.2.4 PCR
The PCR procedure was carried out according to Roux (2009). A 10 µL reaction mix containing ddH2O, Taq buffer (20 mM Tris-HCl (pH 7.6); 100 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.5% (v/v) Triton X- 100; 50% (v/v) glycerol), 2 mM MgCl2, 0.16 mM dNTPs, 0.16 µM of a labeled M13-sequence 0.04 µM forward primer, 0.2 µM reverse primer and 0.2 units of Taq DNA polymerase (SibEnzyme Ltd, Russia) was prepared. In an optical 384 well reaction plate (Applied Biosystems, USA), 7 µL of the reaction mix was added to 30 ng of template DNA and amplified in a PCR machine (Thermocycler-GeneAmp PCR system 9700®, Applied Biosystems, USA). Amplification consisted of initial denaturation of the template DNA at 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, annealed at 59°C for 1 min, with first extension at 72°C for 2 min and final extension at 72°C for 20 min. To verify amplification, PCR products were electrophoresed on a 2% agarose gel. The amplified DNA was visualized under UV light after staining with GelRed® (Biotium, USA). The DNA samples described above for each accession were subsequently subjected to SSR genotyping using the best 23 markers selected from a reference microsatellite kit of 82 markers which were evaluated across ten finger millet cultivars at ICRISAT-Nairobi molecular lab (de Villiers S, unpublished) to determine their amplification efficiency, polymorphism and ability to discern genetic diversity in finger millet. All the forward primers contained an M13-tag (5'-CACGACGTTGTAAAACGAC-3ʹ) on the 5' end that was fluorescently labeled to allow detection of amplification products (Sheulke, 2000). Depending on the efficiency of amplification, 1.5-3.5µL of three different amplification products were co-loaded together with a size standard that ranged from 50-500 bp (GeneScanTM -500 LIZ® Applied Biosystems) and Hi-DiTM-Formamide (Applied Biosystems) and amplified fragments separated by capillary electrophoresis using an ABI Prism® 3730 Genetic analyser (Applied Biosystems) (Kuomi et al., 2004). Gene Mapper 4.0 (Applied Biosystems) was used to score allele sizes in base pairs.
3.2.5 Phenotypic characterization
A total of 420 finger millet accessions (301 genotyped from Kenya, Uganda and Tanzania genotyped as above, plus an additional 39 accessions from the three countries, 80 global minicore accessions and five checks) were phenotyped at Kiboko (a dry lowland location 960 meters above sea level, 2°20’S 37° 45’E)
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in eastern Kenya. The five checks were ‘Kahulunge’-farmer preferred in Tanzania,
‘Nakuru FM1’- released in Kenya for cool high altitudes, ‘Seremi 2’-released in Kenya and Uganda for mid-altitudes, ‘KNE 479’-blast susceptible check, and ‘KNE 814’-blast resistant. The materials were planted in an augmented design in single row plots of 4 m length with inter-row spacing of 0.40 m. The trial was arranged in twenty blocks of twenty six plots each with all check cultivar replicated once in each block. Seed was manually drilled in furrows 2.5-3 cm deep and plants were thinned to one plant per hill at intervals of 0.10 m two weeks after emergence. Standard fertilizer rates were applied. Qualitative data (plant colour, growth habit, ear shape, ear size, and grain colour) were collected according to morphological descriptors for finger millet (IBPGR2, 1985) (Table 3.2-1) from five randomly selected plants in each plot.
Table 3.2-1. Description and scoring for phenotypic traits
Trait Description/scoring
Plant colour At flowering (0-tan; 1-pigmented)
Growth habit Tillering attitude 40 days after sowing (3-decumbent; 5-erect; 7- prostrate)
Panicle shape Shape of panicle at dough stage (1-droopy; 2-open; 3-semi- compact; 4-compact; 5-fisted)
Glume covering Proportion of grain covered by glume at maturity (3-exposed; 5- intermediate; 7-enclosed
Grain colour Post-harvest (1-white; 2-light brown; 3-copper brown; 4-purple brown; 5-others)