Description of the study area

The evaluation of gains in the cycles of selection was done in plots at the Cinta Larga Technological Innovation and Research Center, in Mixquiahuala, State of Hidalgo (20° 11’ N, 99° 14’ W, 2100 masl). The crop was sown on March 28, 2019 in the spring-summer planting cycle, under rainfed conditions. The field evaluation was carried out in the state of Hidalgo and the laboratory activities described were performed at the Colegio de Postgraduados, Campus Montecillo, State of Mexico.

Maize germplasm evaluated

The genetic material evaluated in this study consisted of six breeding cycles (C₀ to C₅) of a population with white endosperm and five cycles (C₀ to C₄) of a population with yellow endosperm. The genetically broad based maize populations of white and yellow endosperm were developed including 45 and 55 collections of diverse germplasm, including commercial hybrids, improved varieties and native populations collected in the Mexican Highlands. The different components included in the formation of the base populations were recombined in isolated fields for two consecutive cycles in the Ejido Santa Teresa Tiloxtoc, in Valle de Bravo, State of Mexico (19° 13’ N, 100° 107’ W, 1740 masl), planting each component in two rows, each one 10 m long, which were used as females, alternating with one row as male which included a mechanical balanced composite of all the materials included as females.

After two cycles of recombination for their homogenization, the method for the intrapopulational selection of S₁ families began in both populations. Each cycle was completed in two years. Year one: in each white or yellow grain population, 400 plants were self-pollinated to generate S₁ families of each population. During the growing cycle and until harvest, plants with undesirable characters were eliminated. Year two: seeds from the best 60-70 S₁ selected families were inoculated with head smut (S. reilianum) teliospores, as described by ^{Quezada-Salinas et al. (2013}) and were planted ear-to-row in the field. Out of the total S₁ families under evaluation, a selection pressure of 33% was applied and selected S₁ families from each population were recombined. The best plants of each family selected, with desirable characters, healthy, with no symptoms of head smut and common rust (P. sorghi) were manually recombined with a pollen mixture. A balanced, mechanical mixture of seeds of the recombined families formed the next cycle (C₁). This sequence was repeated to obtain the six cycles of the white endosperm population and the five of the yellow endosperm population evaluated in this study.

A completely randomized block design with three replications was used in the evaluation trials of the different cycles of selection, in 3m long rows, 80 cm between rows, 16 cm between plants and 44 plants per row to adjust to 22 and at a density of 80 000 plants ha^-1. Each row constituted a plot. Soil preparation and crop management were carried as done at the Cinta Larga Technological Innovation and Research Center, in Mixquiahuala, State of Hidalgo. Furrow irrigation was applied after planting, followed by once or twice monthly depending on rainfalls. Weed control done with four applications of herbicide (potassium salt of 3,6-dichloro-2-methoxybenzoic acid and 6-Chloro-N2-ethyl-N4-isopropyl-1,3,5,triazine-2,4 diamine). There was no fertilizer application or pest and disease control.

Trials of artificial inoculation in white and yellow endosperm maize populations

To evaluate genetic gains by cycle of improvement of head smut resistance, two replications were included with seeds inoculated with S. reilianum teliospores were included. The inoculum was collected in September 2018 from infected maize plants from the previous planting at the Cinta Larga field, in Mixquiahuala, Hidalgo. The inoculum was collected from infected tassels and ears dried for seven days in a greenhouse, collecting the teliospores after cleaning with a 117 µm sieve and storing them at 20 ± 2 °C for their later use. Seeds from each cycle of both populations were treated using a suspension of 1x10⁶ teliospores mL^-1, immersed in white glue (Polyvinyl acetate = Resistol 85^®) diluted to 20% with water (v/v) and drying them for 2 h at 35 ± 2 °C. Smut incidence was evaluated in the six cycles of the white endosperm population and the five cycles of the yellow endosperm population. The purpose of this trial was to evaluate the incidence of head smut with artificial inoculation and to evaluate the efficiency of white glue used to stick the teliospores to the seeds. The same conditions, design and management were applied as in the yield trial.

To evaluate the differences and gains obtained between the different cycles of selection, the following variables were determined:

1) Days to male flowering (anthesis): Registered as the number of days between the day of planting until 50% of plants began sheding pollen. 2) Days to female flowering (emission of silks): Recorded as the number of days between date of planting to 50% silking. 3) Plant height (cm): height between the basis of the stalk and the insertion of the tassel. 4) Ear height (cm) was measured from the base of the stalk to the basis of the main ear. 5) Grain moisture (%): determined by plot at harvest time, taking 500 g of seeds from the central part of five good ears. It was measured using a Dickey John miniGAC plus moisture tester. For the analysis, grain yield was adjusted to 15% moisture and transformed to t ha^-1. 6) Rust severity: Data were taken at flowering stage using an arbitrary scale of 1 to 5 where 1: No damage (foliar tissue 100% healthy), 2: few lesions (90% healthy), 3: regular (80% healthy), 4: bad (60 - 70% healthy) and 5: very bad (0 - 60% healthy). 7) Head smut incidence: The response to infection was determined a few days after the emission of both inflorescences. Incidence was determined as a percentage (Num. of diseased plants divided by the total number of diseased plants per plot x 100). The variables studied were determined following ^{IBPGR (1991)} and ^{Edmeades et al. (2000}).

Statistical analysis

All data were analyzed using the software SAS (Statistical Analysis System, version 9.4) (^{SAS Institute Inc., 2013}), using the procedure PROG GLM and PROG REG to obtain the linear regression. An analysis of variance was used along with a DMS comparison of means with α ≥ 0.05.

Yield trial in white and yellow populations

Due to low germination problems, cycle two (C₂) of the populations was not included. Table 1 shows the measurements for the different traits described. For the variables of plant and ear height and head smut incidence, there were no significant differences based on the DMS (0.05) in both populations. For the trait of days to male and female flowering, significant differences were observed between cycles of selection in both populations. For the yellow endosperm cycles, the variables of rust severity and grain yield showed significant differences but not in the white endosperm population. In the variables that did not present a statistically significant response, the advanced selection cycles express an improvement in comparison with the initial cycles. To better appreciate the behavior of the traits evaluated in the different cycles of both populations, data were analyzed by linear regression to get the average gain cycle^-1 (Table 2). For the ear height, an increase of 3.5 and 4.3 cm cycle^-1 was observed, and for ear height there was also an increase of 1.7 and 2.2 cm cycle^-1 in the white and yellow endosperm populations, respectively. For days to tassel, a reduction of -1.51 and -1.6 day cycle^-1 was observed, and for days to silking -1.8 and -2.2 days cycle^-1 was recorded in both populations, indicating that, as the selection cycles in the populations advanced, populations tend to be earlier in flowering. A reduction of -0.2 and -0.6 cycle^-1 was found for common rust severity (Figure 1). For head smut incidence, a reduction of -0.9 % cycle^-1 was found in the population of white endosperm and -1.2 % cycle^-1 in the yellow endosperm population (Figure 1). For grain yield, there was an increase of 0.3 and 0.6 t ha^-1 cycle-¹ for the white and f yellow populations, respectively (Figure 1).