Anatomical tool in the identification of ploids in maize seedlings and potential use in initial stage of double haploides obtainment process

Studies that optimize the haploid technique in the removal of maize lines are necessary. Between the stages that mostly requires attention and it is directly related to the success of the technology is the correctly separation of induced haploids and diploids. Morphological markers are commonly used but have strong influence of the environment, and laboratory methods have been developed and may be more efficient. Thus, the objective was to study the use of the anatomical analysis tool, through the analysis of young maize leaf for use as the indirect markers in the identification of ploidys. The hybrids were crossed with the KEMS haploid inducer. The seeds crossed, were selected according to the R-navajo marker and submitted to two different protocols of chromosome duplication. Plants that survived to the duplication protocols were acclimated in greenhouse and then transferred to the field. After the self-polinization of the DH0 plants, the DH1 seeds were taken to the field, divided into treatments according to the parentals and duplication protocols. At the vegetative stage V4 of the plants, leaf tissue samples were collected to the evaluation of the amount of DNA and identification of ploidys and anatomical analysis. The nuclear DNA review of each sample was performed for the comparison in histograms of the position of G1 peak to the G1 peak of the internal or external reference standard. A high accuracy came to validate an anatomical tool, through the variables studied in this work, as a marker in the differentiation of ploidis in maize plants, and it can be used in selection programs. The anatomy made in some letters is a non-destructible technique and, together with a flow cytometry technique, can be used as an indirect method in haploid cutting programs at the initial stage of the identification of seedlings.


Introduction 53
The success of a breeding program that aims at the production of maize commercial 54 hybrids lies in the fact of obtaining elite lines. Among all the steps, this is considered to be 55 the most time-consuming and costly, and the technology of double haploid emerges as a 56 way of reducing time in obtaining these lines [1].

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The rapid production of homozygotic lines allows a better exploitation of genetic

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The doubling of the chromosome number spontaneously or induced by the application of 70 mitotic agents e.g., colchicine, retrieves the diploid condition and restores fertility [5]. The 71 action mechanism of colchicine involves the irreversible connection to tubulin dimers,

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The analyzes were performed for all characters with the estimation of variance 203 components and the prediction of random effects using the approach of mixed models, the 204 method of restricted maximum likelihood/best linear unbiased prediction (REML/BLUP) [29].

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For this, the following statistical model was used: 218 X, Z and W: incidence matrices for r, and p, respectively.

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The heritability in the average of treatments ( ) and accuracy were estimated, and ℎ 2 220 the significance of the random effects as the genotypic variance among treatments ( ) were

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The genotypic variance was highly significant by the LRT test and likelihood ratio for  (Table 2).  The anatomical differences observed were obtained through anatomical cuts 252 and diaphanization of samples. It was possible to observe a difference in leaf blade as 253 the thickness (Fig 1) and quantity and size of the stomata (Fig 2). In Table 3, it is observed that the treatment 7 presented the highest values of  The importance or contribution of each analyzed variable as a possible marker

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in the separation of the tested plants, demonstrates greater relevance of the thickness 283 of the leaf blade and the stomata, while the thickness of epidermis little contributed to 284 the separation of plants. (Fig 3).

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Maize diploids have the peak G1, located in the region of relative intensity of 299 fluorescence, soon after the mark of 10 2 (Fig 4A). The haploids have lower relative 300 intensity of fluorescence and the peak G1 is located to the left of the mark of 10 2 , i.e., 301 dislocated in the direction of the x axis (Fig 4B). Whereas the triploids and the   altogether, approximately 50% of the contribution of separation (Fig 3).

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The high contribution of leaf blade is due to its constitution. The leaf blade is 388 composed by parenchyma, in which chloroplasts and spaces for CO 2 storage are 389 located, in addition of course, all the other components of the leaf. Therefore its 390 relevance is easily understandable and the importance of variables related to the 391 stomata is also evident, reinforcing what is already described in several academic 392 articles.

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In addition it is possible to suggest that the anatomic variables, as possible 394 markers were efficient on grouping even partially the hybrids (Tables 2 and 3).

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Behavior that reinforces what has been described above, where the traits were efficient 396 anatomical markers for various crops. The treatments that showed higher averages for 397 the analyzed variables were classified as diploids (Table 3), in general with averages 398 exceeding the haploids (Table 2).

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The increase in ploidy level may be the main driving force to facilitate the plants

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The effect "giga" was also related in previous studies with modifications of cell

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The thickness of the leaf blade and the size of the stomata are highly heritable 438 traits in maize.

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The obtained high accuracy validates the anatomical tool through the variables 440 studied in the present work, as a marker in the differentiation of ploidies in maize 441 plants, which may be employed in programs for selection of hybrids.

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The anatomy made in young leaves of maze is a non-destructible technique 443 and in conjunction with the technique of flow cytometry, can be used as indirect method 444 in programs to obtain double haploids, in the initial stage of identification of seedlings.

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The authors declare no conflict of interest.