QUANTIFYING THE GENETIC DIVERGENCE AMONG MAIZE HYBRIDS USING WARD-MLM STRATEGY

ABSTRACT – The aim of this study was to estimate the genetic divergence of twelve maize hybrids grown in Cerrado-Pantanal ecotone, based on nine agronomic traits and using the Ward-MLM procedure. The experiment was installed at Universidade Estadual de Mato Grosso do Sul, Aquidauana Unit, in a randomized blocks design with four replications. The following traits were measured: plant height, ear insertion height, ear diameter, ear length, stem diameter, number of kernels per rows, number of rows per ear, weight of hundred grains and grain yield. The significance of the nine traits was analyzed by F test at 5% probability. Data were simultaneously analyzed using the Ward-MLM to compose the hybrids groups through the cluster. There was genetic variability among the maize hybrids assessed. Hybrids with high heterotic effect can be obtained by crossing individuals of the group II with group III. Key words: dissimilarity, hybrid development, genetic breeding, Zea mays L. QUANTIFICACAO DA DIVERGENCIA GENETICA ENTRE HIBRIDOS DE MILHO USANDO O PROCEDIMENTO WARD-MLM RESUMO – O objetivo deste trabalho foi estimar a divergencia genetica usando o procedimento Ward-MLM em 20 hibridos de milho cultivados no ecotono Cerrado-Pantanal baseada em nove caracteres agronomicos. O experimento foi instalado na Universidade Estadual de Mato Grosso do Sul, Unidade de Aquidauana Unit, em um delineamento de blocos casualizados com quatro repeticoes. Mensuraram-se os seguintes caracteres: altura de plantas; altura de insercao da primeira espiga; diâmetro da espiga; diâmetro do colmo; numero de graos por fileira; numero de fileiras por espiga; peso de 100 graos; e produtividade. A significância dos nove caracteres foi analisada pelo teste F a 5% de probabilidade. Os dados foram analisados simultaneamente usando o procedimento Ward-MLM para compor os grupos de hibridos por meio da analise de agrupamento. Existe variabilidade genetica entre os hibridos de milho avaliados. Hibridos com alto efeito heterotico podem ser obtidos do cruzamento ente individuos contidos no grupo I com os do grupo II. Palavras-chave: dissimilaridade, desenvolvimento de hibridos, melhoramento genetico, Zea mays L.

Knowing the genetic divergence in available germplasms is fundamental for the optimal designing of breeding programs, and its efficiency can be increased if superior crossings are pre-established (Rigon et al., 2015).At last five decades, a big number of maize (Zea mays L.) hybrids have been developed from genotypes with a restricted genetic base.This causes the risk of loss of genetic divergence and restricts the possibility of crossing between genetically divergent genotypes.Knowledge of the genetic relationships among breeding materials can help to prevent the great risk of increasing uniformity in the elite germplasm, and can also ensure long-term selection gains (Oliveira et al. 2004).
The adequate choice of genotypes as sources of lines extraction can to determine the success or economic return of a breeding program to develop maize hybrids.The trends are in order to use F2 populations with genetic variability or double hybrids with yield potential originated from crosses of single hybrids, synthetic elite lineages and even commercial hybrids, as it the inappropriate choice of population will result in loss of time and resources destined for breeding (Smith et al. 1997;Gutierrez et al. 2003;Franco et al., 2005;Ortiz et al, 2008;Oliveira et al., 2010;Rotili et al. 2012;Simon et al. 2012;Gomes Júnior et al. 2014).
Front to numerous genitors frequently used in the maize breeding programs, hundreds of new lineages are generated each year.Thus, the difficulty arises in relation to combination capacity studies for determination of heterotic groups, which are highly correlated with genetic divergence between the genitors.The determination of genetic dissimilarity among genitors contributes in the extraction of endogamic lines with considerable specific combination ability and thus allows exploiting the heterosis phenomenon in hybrid crosses, increasing the chances to obtain segregating generations.These estimates are of great use in breeding programs and also in the choice of genitors for mapping of genes (Rigon et al., 2015).
In a population genetic divergence study, quantitative morphological descriptors are subjected to multivariate biometric techniques, allowing unifying multiple information of a trait set.The modified location model (MLM), proposed by Franco et al. (1998), is a new strategy to quantify the variability using quantitative and qualitative traits simultaneously.MLM has two stages.In the first, Ward clustering method (Ward, 1963) defines the groups using the Gower dissimilarity matrix (Gower, 1971).In the second stage, the vector average of the quantitative trait is estimated by MLM procedure, for each subpopulation, regardless of the qualitative traits values.
In this context, the aim of this study was to estimate the genetic divergence with Ward-MLM procedure in twelve maize hybrids cultivated in the Cerrado-Pantanal ecotone based on nine agronomic traits.

Material and Methods
The experiment was installed at Universidade Estadual de Mato Grosso do Sul, Aquidauana Unit Experimental design used was randomized blocks with four replications.The area was divided into four blocks with a total of seventy-four plots, each with 15.75 m² (3.15 x 5.0 m), spaced two meters spacing between blocks.The treatments consisted of 20 maize hybrids (Table 1), which it has shown satisfactory agronomic performance in the study region (Torres et al., 2013;Teodoro et al., 2014).
In the preparation of the experimental area, it was performed application of glyphosate herbicide for desiccation.After drying and complete death of the plants, the furrows were opened using a seeder.
Seeding was done manually under no-tillage, on 2012-02, ten days after desiccation, which were distributed six seeds per meter in the rows, spaced 0.45 m.With about fifteen days after plants emergence, we carried thinning keeping four plants per linear meter for establishment of 88,889 plants ha -1 .
Fertilization at sowing time consisted of 300 kg ha -1 , at formulation 4-20-20.In topdressing, urea was used as nitrogen source, applying 100 kg ha -1 on the surface when the plants showed five to eight fully and number of rows per ear (NRE).
Harvest and threshing of maize ears were performed manually in three central rows of five meters length, according to the cycle of each hybrid.
The weight of hundred grains (WHG) was determined by manual counting, weighing and correction of moisture to 13%.Grain yield (YIE) was estimated by extrapolation of harvested production in 1 ha, correcting by plant stand and for 13% wet basis.
The significance of the nine agronomic traits was analyzed by F test (ANOVA), at 5% probability.Data were analyzed simultaneously, using Ward-MLM for composing the hybrids groups through the cluster.For the Ward cluster method, the distance matrix was provided by Gower's algorithm (Gower, 1971).The definition of the ideal number

Results and Discussion
There was significant differences (p<0.01) between genotypes for all evaluated traits, allowing to infer about the existence of genetic variability in the population (  It can be observed in Table 4 that the groups I and II were the most similar between them.Such groups, by presenting the same pattern of similarity, are not recommended for use in breeding programs by hybridization, so that genetic variability is not restricted, in order to derail the gains to be obtained by selection.His occurs because genetically related parents tend to share many genes or alleles in common, and when two of these parents are crossed, there is little stimulus, attributed low level of allelic heterozygosity at the crossing (Cruz et al., 2014).
The longest distance was found between the groups II and III.This high divergence, at first, allows recommending the crossing between these pairs, in order to maximize heterosis in progenies and increase the possibility of segregating in advanced generations due to different numbers of loci in which the dominance effects are evident (Cruz et al., 2014).
In addition, to consider the grain yield extremely important trait for selecting superior hybrids in breeding programs, individuals from group II and III presented the highest means.Thus, it may be possible to generate hybrids with high heterotic effect due to different numbers of loci in which the dominance effects are evident.
In the analysis based on the canonical variables (CAN), we found that the first two variables accounted for 100% of the variance.Thus, the two-dimensional representation is the most suitable for representing the data set.The Ward-MLM procedure was employed to quantify the genetic variability in research on maize (Gutierrez et al. 2003;Franco et al., 2005;Ortiz et al, 2008), turnip-feed (Padilha et al, 2005), tomato (Goncalves et al., 2009), common bean (Cabral et al., 2010), chili/pepper (Sudre et al., 2010) and banana (Pestana et al., 2011).These authors observed that the first two CAN explained the variability between the groups higher than 80% and the two-dimensional graph was suitable for viewing the relationship between the groups.
By the graphical analysis of the first two CAN, we observed a move away from group III from other groups formed by Ward-MLM procedure and its greater distancing with the hybrids from the group II (Figure 1).These results reinforce the hypothesis of obtaining hybrids with high grain yield by crossing between individuals from group II and III.

Conclusions
There is genetic variability of the evaluated traits in hybrids adapted to ecotone Cerrado/Pantanal region.
Ward-MLM statistical procedure is a useful tool for detecting genetic divergence and group hybrids, using agronomic traits.
F2 populations with high genetic variability or double hybrids with high heterotic effect may be obtained bye crossing of individuals from group II with group III.
III from other groups formed by Ward-MLM procedure and its greater distancing with the hybrids from the group II (Figure 1).These results reinforce the hypothesis of obtaining hybrids with high grain yield by crossing between individuals from group II and III.

(
UEMS/UUA), in the municipality of Aquidauana-MS, located in the Cerrado/Pantanal ecotone, comprising the coordinates 20º27'S and 55º40'W, with an average elevation of 170 m.The soil was classified as Ultisol sandy loam texture.The region climate, according to the classification described by Köppen-Geiger, is Aw (Savanna Tropical), with cumulative rainfall over the experiment of 450 mm and maximum and minimum temperatures of 33 and 19ºC, respectively.
of groups was performed according to the pseudo F and pseudo t² criteria.Difference among groups, correlation between the variables and canonical variable (CAN) were graphically evaluated.The distance for the traits distribution proposed byFranco et al. (1998) was used for determining the dissimilarity among the formed groups.All analyzes were carried using the SAS statistical software(SAS   INSTITUTE, 1999).

FIGURE 1 .FIGURE 1 .
FIGURE 1. Graph of the first two canonical variables (CAN 1 and CAN 2) for the three groups of maize hybrids formed by Ward-MLM procedure.

TABLE 1 .
Commercial Name, business and genetic class of 20 maize hybrids gowing in Aquidauana,

TABLE 2 .
Summary of the analysis of variance for the traits evaluated in 20 maize hybrids grown in ecotone Significant at 1% probability by F test; SV: sources of variation; CV: coefficient of variation; DF: degrees of freedom; EL: ear length; ED: ear diameter; NRE: number of rows per ear; NKR: number of kernels per rows; SD: stem diameter; EIH: ear insertion height; PH: plant height; WHG: weight of hundred grains; YIE: yield grain. *

TABLE 3 .
Groups, number of hybrids per group, hybrids and mean of each trait evaluated in 20 maize hybrids grown in ecotone Cerrado/Pantanal.