**2.2 Data collected**

Data was collected on the following specific parameters:



Grain yield was calculated as follows:

$$fGY = fwt \times \frac{\left(100 - m\right)}{85} \times \frac{10000}{\left(8 \times \phi\right)} \times 0.8 \tag{1}$$

where, GY = grain yield (kg ha−1), fwt = field weight of harvested ears per plot (kg), m = grain moisture content at harvest. 10,000 = land area per hectare (m2 ), Ȣ = land area per plot (0.70 m x 0.4 m), ɸ = number of hills/plot (11) and 0.80 = 80% shelling percentage. Broad-sense heritability (H2 ) was estimated as:

$$H^2 = \sigma\_G^2 / \left(\sigma\_E^2 / re + \sigma\_{GE}^2 / e + \sigma\_G^2\right),\tag{2}$$

Where;

2 σ *<sup>G</sup>* = variation due to genotype, <sup>2</sup> σ *<sup>E</sup>* = variation due to environment, <sup>2</sup> σ *GE* = variation due to genotype by environment interactions, r = number of replications and e = number of environments.

#### **2.3 Source of inoculum and isolation of** *A. flavus*

Aflatoxin contaminated maize samples from Ejura main farms were cut into 3 mm pieces with a sterile scalpel blade, after being surface-sterilized in 1% hypochlorite for 2 minutes, then placed on Potato Dextrose Agar (PDA) and incubated at room temperature for 5 days.

After incubation, colonies of different morphology, shape, and color were observed. A pure culture of each colony was obtained through serial dilution where 1 agar plug containing mycelia was serially diluted into 9 mls of distilled water till a concentration of 1 x 105 was achieved.

One ml of the final dilution was transferred onto water agar (2% agar) and incubated at 31°C in unilluminated growth chamber. Identification slides were prepared by picking spores with isolation needle onto a slide containing a drop of distilled water. *A. flavus* was subsequently identified by observing colony characteristics, conidial morphology as described previously [7, 8].

Isolates that produced large smooth conidial surface and either an average sclerotial diameter > 400 μm or without sclerotia were identified as L-type *A. flavus* using Leica Microscope X 40. Identified isolates were subsequently maintained on potato dextrose agar (PDA) as described by Jha [9]. Maintenance of colonies were done by sub-culturing of the colony onto PDA plates and incubated at room temperature for 5 days.

#### **2.4 Inoculum preparation**

Identified toxigenic isolate was used to prepare the inoculum as described by Windham [10]. The procedure involved multiplication of the isolate on sterile corn cob grit in 500-ml flasks each containing 50 g of grits and 100 ml of sterile distilled water and incubated at 28°C for 3 weeks. Conidia in each flask was washed from the grits using 500 ml of sterile distilled water containing 20 drops of Tween 20 per liter and then filtered through four layers of sterile cheesecloth. The concentrations of conidia was determined with a hemacytometer and adjusted with sterile distilled water to 9 x 107 conidia per ml. Excess inoculum not used immediately was refrigerated at 4°C.

#### **2.5 Inoculation method (wounding)**

The side needle technique described by Scott and Zummo [11] which utilizes an Idico tree-marking gun fitted with a 14-gauge needle was used for inoculations 14 days after mid silk. Ears were inoculated by inserting the needle under the husks on the upper 1/3 of the ear and 3.4 ml of a spore suspension of 9 x 107 conidia/ ml was injected over the kernels. A total of 5 ears per genotype were used for the inoculation study.

#### **2.6 Aflatoxin analysis**

Ears that did not touch the ground were harvested from plots at maturity, approximately 60 days after mid-silk. The cobs were shelled and samples ground using a Romer mill (Romer Industries, Inc., Union, MO) according to manufacturer's instructions. Aflatoxin was extracted using the method described by Sirhan [12] with modifications. Maize samples were homogenized into suspension using a Preethi Mixer Grinder.

A weight of 2 g of slurry was weighed into a 15 ml centrifuge tube and toppedup with a 4 ml of 60:40 (v/v) methanol:acetronitrile solution, and vortexed for

3mins. 1.32 g of anhydrous MgSO4 and 0.2 g of NaCl were added to the mixture, and vortexed for additional 1 min. The tube was centrifuged for 5 min at 4000 rpm and the upper organic layer filtered through a 0.45 μm nylon syringe prior to injection. A volume of 100 μl of the filtered extract was injected into the HPLC.

A Cecil-Adept Binary Pump HPLC coupled with Shimadzu 10 AxL fluorescence detector (Ex: 360 nm, Em: 440 nm) with Phenomenex Hyper Clone BDS C18 Column (150 x 4.60 mm, 5 μm) was used for analysis. The mobile phase used was methanol: water (40:60, v/v) at a flow rate of 1 ml/min with column temperature maintained at 40°C. To 1 liter of mobile phase were added 119 mg of potassium bromide and 350 μl of 4 M nitric acid (required for postcolumn electrochemical derivatisation with Kobra Cell, R-Biopharm Rhone). Aflatoxin Mix (G1, G2, B1, B2) standards (ng/g) were prepared from Supelco® aflatoxin standard of 2.6 ng/μL in methanol. Concentration of B1 and G1 were 0.5, 1, 2, 8, 16 ng /g per 100 μl injection of each standard.

Concentration of B2 and G2 were 0.15, 0.3, 0.6, 2.4, 4.8 ng/g per 100 μl injection of each standard. Limit of Detection and Limit of quantification of total aflatoxin were established at 0.5 ng/g and 1 ng/g respectively. The unit (ng/g is equivalent to ppb). Aflatoxin concentration was estimated as:

$$\mathbf{n} \mathbf{g} / \mathbf{g} = \mathbf{A} \times (\mathbf{T} / \mathbf{I}) \times (\mathbf{1} / \mathbf{W}) \tag{3}$$

where A = ng of aflatoxin as eluate injected, T = final test solution eluate volume (μl), I = volume eluate injected into LC (μl), W = mass (g) of commodity represented by final extract.

#### *2.6.1 Validation of HPLC method*

Recovery studies were conducted to check for precision and accuracy. Blank samples were spiked at 5 (five) replicated maize samples at 13 ng/g, 26 ng/g and 104 μg/g with recoveries 91 ± 1.75%, 98 ± 1.33% and 102 ± 1.87% respectively. Blanks that were run periodically contained no detectable amount of target analyte. Trueness was further validated using a certified reference material (TR-A1000) from Triology laboratory, USA. The value obtained, 20.17 ± 1.14 μg/kg from ten replicates was within the recommended range of the certified value of 21.0 ± 2.9ug/kg. Coefficient of variation was less than 15% for replicates.

#### **2.7 Statistical analysis**

Analysis of Variance (ANOVA) was performed on plot means for grain yield and all other agronomic traits for each environment and across environments using PROC GLM procedure of SAS software, version 9.4 [13]. Data on aflatoxin contamination was transformed as Ln (y + 1) where y is the aflatoxin level whilst Ln is Log base e.

This transformation was done to reduce the heterogeneity of variance of contamination levels. Genotype or entry means were adjusted for block effects and analyzed according to lattice design [14]. Each environment was defined as season x location x *A. flavus* inoculation treatment. Effects of environment were considered as random while genotypes were classified as fixed effects. Additionally, genetic correlations between aflatoxin accumulation and selected agronomic traits were performed using the meta menus program implemented in SAS to examine the relationships among the traits and also predict strategies to enhance their improvement.

#### *2.7.1 Clustering analysis using agro-morphological traits*

Classification of genotypes was based on significant agro- morphological traits. The significant traits were standardized and used to generate Euclidean genetic distance co-efficient whiles Ward's minimum variance method implemented in SAS software version 9.4 [13] was used for the clustering.
