Prospecting of popcorn inbred lines for nitrogen use efficiency and responsiveness 1

Large amounts of nitrogen (N) fertilizers applied to maize cropping systems support high yields but cause adverse environmental impacts. The development of cultivars with higher N use efficiency is essential to maintain sustainable production. Thus, the present study aimed to select popcorn inbred lines efficient and responsive to N fertilization express - ing high popping expansion. Fifty-one popcorn inbred lines from different origins were evaluated in the field under low and high N availability, arranged in a randomized complete block design with three replications. The two main traits of economic interest in the crop were evaluated: grain yield (GY) and popping expansion (PE). A joint analysis of variance was performed and a scatter plot was generated to classify the inbred lines regarding the response to the N use, focusing on GY. For PE, a Scott-Knott grouping of means was conducted. Within the panel of evaluated popcorn inbred lines, it was possible to identify sources of favorable alleles for nitrogen use efficiency and popcorn expansion, highlighting the inbred lines L205, L206, L217, and L395. These genotypes emerge as potential parents to be included in mating blocks for the development of hybrids and/or breeding populations with high nitrogen use efficiency and popping expansion.


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Submitted on April 28 th , 2021 and accepted on August 16 th , 2023. 1 This work is part of the first author Doctoral Thesis. 2 Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.yure_p-souza@hotmail.com; amaraluenf@gmail.com;tomaz_oft@yahoo.com.br;shahidkhan@aup.edu.pk; julianasaltiresdossantos@yahoo.com.br;samuelkampho@hotmail.com *Corresponding author: yure_p-souza@hotmail.com Nitrogen (N) is the nutrient required in large amounts by plants and is essential for growth and development.In corn, this mineral is of great importance for increasing grain yield (Hirel et al., 2007;Taiz et al., 2017).Thus, N deficiency is, the main abiotic stress that limits the productive potential of the popcorn crop (Zea mays everta).
Popcorn is a special type of corn, which has great popular acceptance, providing economic gains both in the grain production and marketing sector of popcorn (Silva et al., 2019).This crop has been presenting significant commercial demand in the food industry, moving about 10 billion dollars in 2020 (Serna-Saldivar, 2022).
To meet this demand, the cultivation of popcorn has increased in tropical regions, where soils generally have nitrogen availability below that required for the popcorn cultivated species to gain satisfactory yields.Thus, in order to increase agricultural production, farmers become increasingly dependent on nitrogen fertilizers.In 2019, around 107.4 million tons of nitrogen fertilizers were used worldwide.For 2022 it is estimated that 111.6 million tons will be used (FAO, 2019).
Despite the large amount of N fertilizers applied, the nitrogen utilization efficiency of the plants is low about 30 to 40%, which means that much of the applied N is not Yure Pequeno de Souza et al.
used (Ciampitti & Vyn, 2012).Thus, more than 60% of N is lost by leaching, volatilization, runoff, and competition with the soil microbiota, resulting in negative impacts on the environment, as well as increased crop production costs (Sharma & Bali, 2017).
In this scenario, to reduce dependence on nitrogen inputs and maintaining high levels of yields, is essential to ensure sustainable agricultural production for the future.This challenge can be overcome through the development of cultivars nitrogen use efficiency (NUE), which can be obtained by crossing inbred lines adapted to environments with low N availability and, which are responsive to N fertilization, since, the transfer of alleles favorable to descendants depends on their presence in the parent lines (Hallauer et al., 2010).In the aforementioned studies, the number of evaluated inbred lines and their respective genealogies is low.
However, by assessing a larger number of genotypes with diverse genealogical backgrounds, we can increase the likelihood of identifying sources of favorable alleles that contribute to improving NUE and, consequently, fostering the development of superior popcorn hybrids.
Thus, the present study aimed to select popcorn inbred lines efficient and responsive to N fertilization expressing high popping expansion.Köppen (1948) classification, the climate of this municipality is considered tropical humid (Aw), with hot summers and mild winters.During the experimental period, average temperatures of 26.9 °C and precipitation of 291 mm were observed (Figure 1).

MATERIAL AND METHODS
The soil samples were analyzed for pH, organic matter, and availability of P, K, Mg, Ca, and Al (Table 2).Liming was performed with of dolomitic limestone.Subsequently, the planting areas were prepared with mechanized tilling.
The strategy implemented to differentiate the environments regarding the level of N availability was as follows: in the sowing fertilization, the two environments received 32 kg.ha -1 of N, 112 kg.ha -1 of phosphorus (P 2 O 5 ), and 56 kg.ha -1 of potassium (K 2 O), for which the formula NPK (04-14-08) was used.In the high N availability environment (HN), the topdressing provided 118 kg.ha -1 of N, totaling an application of 150 kg.ha -1 of N. In the low N availability environment (LN), the topdressing fertilization was 29 kg.ha -1 of N, which corresponds to 25% of that applied in the high N dosage environment.Urea (46%) was used as the N source.
For both N conditions, the urea application was divided into two equal doses and applied to all inbred lines at the V4 (four fully expanded leaves) and V6 (six fully expanded leaves) growth stages, following the protocols described by Abendroth et al. (2011) and Santos et al. (2017).The experiments were irrigated using sprinklers, including after topdressing fertilization, and weed management was Prospecting of popcorn inbred lines for nitrogen use efficiency and responsiveness  To evaluate the performance of inbred lines, the two main traits of economic interest in the crop were measured: i) grain yield (GY), expressed in kg ha -1 , evaluated around 120 days after sowing, was obtained through the grain production of the plot corrected to 13% moisture and, ii) popping expansion (PE), expressed in mL.g

RESULTS AND DISCUSSION
The genotype (G) effect for the traits GY and PE was significant (p < 0.01), revealing the existence of genetic variability, which is an essential condition for obtaining genetic gains through selection (Table 3).Genetic variability for agronomically important traits has also been reported among popcorn inbred lines under contrasting N levels in other studies (Santos et al., 2017;Almeida et al., 2018).
Significant effects of N level (p < 0.01) and G x N interaction (p < 0.01) were observed for GY (Table 3).The significant effect of N levels demonstrates that the applied N doses were satisfactory in providing differentiation between environments and, consequently, correctly discriminating between efficient and inefficient inbred lines in terms of N use.
The significant G x N interaction for GY (Table 3) suggests that the classification of popcorn inbred lines varied between the two N levels.Therefore, the selection of the most efficient genotypes for N use should be performed specifically for each environment and not based on the overall performance, as the alleles controlling the expression of the trait under N stress are, at least in part, different Prospecting of popcorn inbred lines for nitrogen use efficiency and responsiveness from the alleles controlling the same trait under optimal N supplementation conditions (Fritsche-Neto et al., 2010).
In the low N environment, there was an 18% reduction in the overall average of GY compared to the high N environment (Table 3).According to Bolaños & Edmeades (1996), a yield reduction of 20 to 30% in abiotic stress conditions, compared to non-stress conditions of the same genetic set, is considered indicative of stress.Although the reduction observed in the present study was slightly lower than the recommended range, the results obtained were higher than those reported by Guedes et al. (2015) and Heinz et al. (2019), who observed yield decreases of only 13% and 16%, respectively.
Popping expansion (PE) was not influenced by N levels or the G x N interaction (Table 3), indicating that the selection of superior inbred lines can be based on the average performance in both environments.A similar result was reported by Santos et al. (2017) when evaluating a set of 29 inbred lines of popcorn in environments with contrasting N availability in the soil.
Although popping expansion (PE) shows a pattern of quantitative inheritance, this trait may be little influenced by the environment, as it exhibits high heritability and is influenced by three to five genes with large effects (Robbins & Ashman, 1984;Lu et al., 2003).Babu et al. (2006), mapping quantitative trait loci (QTLs) for PE, identified a total of four QTLs distributed on chromosomes 1, 3, 8, and 10, explaining together 62% of the phenotypic variation.
To perform the selection of individuals under specific environmental conditions, it is initially interesting to understand the magnitude of genetic and environmental influence on the phenotypic variation of the main traits of interest (Falconer & Mackay, 1996).Thus, the coefficient of genotypic determination (Ĥ 2 ) was estimated, which reveals the proportion of phenotypic variance due to genotypic variability among genotype means (Cruz et al., 2014).High estimates of Ĥ 2 (Ĥ 2 > 0.96) were found for both traits in both N levels (Table 3).This result indicates that genetic effects were predominant over residual effects, suggesting promising prospects for genetic gains in the two key economically important popcorn traits through a phenotypic selection of superior individuals in both N availability conditions.: significant at P < 0.01, not significant at the 5% probability level, respectively, in accordance with the F-test.Ĥ 2 : coefficient of genotypic determination; CVg: genotypic coefficient of variation; CV e : envarioment coefficient variation; Î v : Index variation.

It is often reported in maize experiments conducted in
Yure Pequeno de Souza et al.

The coefficient estimates of experimental variation
(CV e ) obtained under N stress were higher than those observed under conditions of high N availability for GY and PE, as shown in Table 3.This increase in CVe due to N stress is consistent with findings from other studies on corn (Bänziger et al., 1997;Santos et al., 1998;Guedes et al., 2015;Almeida et al., 2018;Torres et al., 2019) reported in the literature.One possible explanation for this difference in CV e is that in environments with a high supply of N, soil deficiencies are corrected through fertilization.However, in environments where this nutrient is not provided, soil fertility issues may arise, leading to greater environmental variation.Additionally, under stress conditions, the average values are generally lower, which can result in a higher estimate of the coefficient of experimental variation (Badu-Apraku et al., 2013).
The variation index (Î v ) is an important parameter as it provides breeders with a meaningful understanding of the trait's status for the breeding program.This index indicates the proportion of genetic effects relative to the experimental error.According to Vencovsky (1987), this estimate should approach or exceed a value of one.For all traits in both nitrogen conditions, we observed Î v values higher than one, indicating that the genetic effects were larger than the experimental error.Specifically, under HN condition, the Iv values were 46% and 19% higher than those obtained under LN condition for GY and PE, respectively (Table 3).These results suggest that despite the higher experimental errors in the low N environment, it is still feasible to successfully select popcorn inbred lines with high agronomic potential for both levels of N availability in the soil.
The identification of genotypes that are adaptable to environments with a lower supply of N fertilizers and show positive responses to potential environmental improvements is a crucial objective of breeding programs focused on developing NUE cultivars.In this regard, a scatter plot was created to classify the inbred lines based on their performance at contrasting levels of soil N availability Prospecting of popcorn inbred lines for nitrogen use efficiency and responsiveness (Figure 2).
Among the analyzed inbred lines, the following genotypes were classified as efficient and responsive (ER) since they exhibited mean grain yield values above the means of environments with low and high nitrogen availability: L261, P2, L292, L216, L328, L272, L217, L206, L205, L381, L204, L511, L270, and L326 (Figure 2).Therefore, these genotypes are noteworthy as potential sources of favorable alleles for improving grain yield under high and low nitrogen conditions.
The inbred lines L263, L509, L291, L293, L562, and L236 showed specific adaptation to the HN environment, that is, they were inefficient and responsive to the use of N since they yielded below the overall average of LN and increased productivity N was applied.On the other hand, it was observed in lines L510, L262, L471, and L395, which showed means whose deviations were positive with respect to the average of the LN environment and negative with respect to the average of the HN environment, thus demonstrating potential adaptative to the stress condition (Figure 2).
It was observed that 51% of the evaluated inbred lines exhibited grain yield (GY) averages below the overall average of both high and low N availability environments.
Consequently, these lines were classified as inefficient and non-responsive (INR) to nitrogen use (Figure 2).Therefore, these genotypes are considered unfavorable for inclusion as parents in breeding programs aimed at improving grain yield under N stress conditions.
We would like to highlight that inbred line P2 was classified as efficient and responsive (ER), while inbred line L80 was classified as inefficient and non-responsive (INR) (Figure 2).This classification aligns with the findings of  Therefore, selecting superior inbred lines for NUE will contribute to obtaining progenies with superior performance.In addition to these results, these authors reported that extrachromosomal genes can influence the expression of NUE in popcorn, as significant reciprocal effects were observed for grain yield.Therefore, the prior evaluation of inbred lines for nitrogen use efficiency allows the breeder to better plan the composition of mating blocks, as superior lines for NUE should be restricted as female parents.
In corn, NUE is usually evaluated considering only grain yield as the main trait.However, in popcorn breeding programs aimed at improving adaptation to low N availability environments, special attention should also be given to popping expansion, as it is the main quality characteristic of the crop.Selecting NUE genotypes associated with high popping expansion (PE), however, is not an easy task.This challenge is well known historically, as grain yield and popping expansion are negatively correlated (Dofing et al., 1991;Daros et al., 2004;Rangel et al., 2011;Cabral et al., 2016).Therefore, popping expansion should be carefully evaluated to select NUE genotypes that simultaneously perform satisfactory for PE.
The selection of inbred lines with high means for popping expansion (PE) is an important criterion to be adopted in popcorn breeding programs, as additive effects are the main components of genetic variation for this trait (Larish & Brewbaker, 1999;Pereira & Amaral Junior, 2001;Scapim et al., 2006).Thus, it is expected that hybrids with high PE can be obtained by crossing superior inbred lines, as in this situation, the F1 mean is equal to the mean of the parents (Lyerly, 1942;Cruz et al., 2014).In this context, for the present study, inbred lines that exhibited PE values equal to or higher than 30 mL.g -1 were considered desirable, as a popcorn cultivar to be released should have a minimum volume expansion of 30 mL.g -1 (Matta & Viana, 2001).
The average performance for popping expansion (PE) of the set of inbred lines, considering the average of HN and LN, is shown in Figure 3.It was observed that the PE means varied from 10.8 (L296) to 33.2 mL.g -1 (L395), forming 11 distinct mean groups, indicating significant genetic variability that can be further explored.Among the inbred lines classified as efficient and responsive (ER) to N use (Figure 3), only L205, L206, and L217 displayed means higher than 30 mL.g -1 for PE.Furthermore, the inbred line L395, despite being non-responsive to N, exhibited adaptation to LN environments.This adaptation led to higher efficiency in N use, associated with a higher mean popping expansion (33.2 mL.g -1 ).
Therefore, these genotypes emerge as promising sources of favorable alleles for both NUE and popping expansion, making them excellent candidates for inclusion as parents in breeding programs aimed at developing superior cultivars for these traits.These findings are consistent with the results reported by Santos et al. (2017), who also suggested the feasibility of selecting inbred lines of popcorn that exhibit efficient N use and high popping expansion simultaneously.
It is worth noting that the aforementioned lines iden-

ACKNOWLEDGMENTS, FINANCIAL SUPPORT, AND FULL DISCLOSURE
We thank the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) that financed part of the study.This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001.There is no conflict of interest in the conduct and publication of the work.
The selection of parents in a breeding program aiming at the development of hybrids NUE is usually carried out by evaluating the combining ability of their parental inbred lines, via diallel or top crosses.However, prior to this stage, the selecting inbred lines by performance per se in environments with and without N restriction, can be an effective strategy to increase the chances of obtaining superior hybrids in the N use.Santos et al. (2020) observed that the hybrids classified as efficient and responsive to N use came from the crosses in which at least one of the parents demonstrated efficiency and responsiveness to N use based on their performance per se.Nevertheless, in popcorn there are still few studies that address this theme, mainly in field conditions, where the cultivation environment can best be represented, especially considering the site of adaptation to which one intends to develop a breeding program.Among these studies, Santos et al. (2017) conducted research to identify inbred lines of popcorn with high N use efficiency (NUE) in a field setting.They evaluated a panel of 29 inbred lines originating from three different genealogies and ultimately selected 13 lines that demonstrated efficiency and responsiveness to N fertilization.Similarly, Almeida et al. (2018) aimed to select popcorn inbred lines with enhanced NUE.They evaluated 19 inbred lines from two genealogies and identified a specific genotype that exhibited superior NUE.

Figure 1 :
Figure 1: Rainfall (mm), maximum, average, and minimum temperature (°C) observed during the experimental period in the agricultural year 2018/2019 in Campos dos Goytacazes, Rio de Janeiro, Brazil.Sowing was carried out on December 4th, 2018, and the harvest on April 5th, 2019.Source: National Institute of Meteorology (INMET).
-1 , obtained by the relation between the volume of expanded popcorn and the mass of 30 g, using the average of two samples per plot.The classification of genotypes regarding their efficiency and responsiveness to N use was based on the deviations of the mean grain yield of each inbred line from the overall mean of each environment.These deviation values were then plotted on a scatter plot, with the X-axis representing the deviations under high N levels (responsiveness in N use) and the Y-axis representing the deviations under low N availability (efficiency in N use).Based on this classification, the lines were grouped into four categories: efficient and non-responsive (ENR), efficient and responsive (ER), inefficient and non-responsive (INR), and inefficient and responsive (IR).Data were tested for normality of residuals (Shapiro-Wilk test) and no transformation was needed.Individual analysis of variance was performed for each N level separately to verify the homogeneity of residual variances (O'Neill and Mathews test), followed by joint analysis of variance, considering genotype and environment (N level) as fixed effects.The genetic parameters were estimated using the mean squares of the individual analysis of variance.The coefficient of genotypic determination (Ĥ 2 ) was estimated using the following expression: Ĥ 2 =(MSg-MSe)/MSg, in which MSg: corresponds to the mean square of the genotype; MSe: corresponds to the mean square of the error; genotypic coefficient of variation (CVg), obtained by and µ correspond to replicate and general average, respectively; environment coefficient variation (CV e ), obtained by 100  e QMr CV x µ;and Index variation (Î v ), estimated by Î v =CV g /CV e .For the PE trait, means were clustered by theScott & Knott (1974) algorithm at 5% probability and represented on a circular bar chart.The statistical analyses were executed using the GENES(Cruz, 2013) computer program.

Figure 2 :
Figure 2: Classification of 51 popcorn inbred lines for efficiency and responsiveness to the N use.ENR: efficient and non-responsive; ER: efficient and responsive; INR: inefficient and non-responsive; and IR: inefficient and responsive.
Santos et al. (2017) who evaluated 29 popcorn lines in a field study with two contrasting N levels.Similarly, Khan et al. (2020), evaluating the same inbred lines (P2 and L80) and the F1 hybrid resulting from their cross, in experiments with different doses of nitrogen

Figure 3 :
Figure 3: Means clustering of popping expansion (mL.g -1 ) of popcorn inbred lines evaluated.*Barsfollowed by the same letters belong to the same group according to Scott-Knott test at 5% probability.
tified as superior for NUE and popping expansion were derived from the testcross hybrid IAC-125 (L205, L206, and L217) and the open-pollinated variety SAM (L395), which are cultivars known for their good agronomic performance.These cultivars likely contributed favorable alleles for grain yield and popping expansion in the process of developing their respective inbred lines.Analyzing the genealogical context along with the performance of the lines regarding N use and popping expansion, different strategies of gene recombination can be adopted to develop hybrids and/or breeding populations that simultaneously express the two main economically important traits for the crop, GY, and PE.In this scenario, crossing L395, which showed merit for both PE and GY in LN, and belongs to a distinct gene pool from the other elite lines (L205, L206, and L217), holds high potential Prospecting of popcorn inbred lines for nitrogen use efficiency and responsiveness for heterosis exploitation and, consequently, generating superior hybrids in terms of NUE and popping expansion.CONCLUSIONSWithin the panel of evaluated popcorn inbred lines, it was possible to identify sources of favorable alleles for nitrogen use efficiency and popcorn expansion, highlighting the inbred lines L205, L206, L217, and L395.These genotypes emerge as potential parents to be included in mating blocks for the development of hybrids and/or breeding populations with high nitrogen use efficiency and popping expansion.

Table 1 :
List of popcorn inbred lines evaluated with their respective genealogies, number of inbred lines per genealogy (N° lines), country of origin, and climate adaptation

Table 2 :
Chemical attributes of the soil in the experimental area in Campos dos Goytacazes-RJ, at layers of 0-10 and 10-20 cm

Table 3 :
Summary of the analysis of joint variance for grain yield (GY) and popping expansion (PE) evaluated in 51 popcorn inbred lines under conditions of high and low nitrogen availability in the soil **, ns