Haploid induction in tropical supersweet corn and ploidy determination at the seedling stage

The objective of this work was to determine the possibility of haploid induction in tropical supersweet corn (Zea mays L. var. saccharata) using a maternal inducer, as well as to identify alternative methods for haploid selection. A single-cross hybrid of field corn and 11 tropical supersweet corn populations were crossed with the haploid inducer. The haploids were pre-selected using the R1-navajo marker and were differentiated into haploids or false positives at the V2-V3 stage, based on the color of the first leaf sheath and on the length of stomata guard cells. The obtained results are indicative of the possibility of inducing maternal haploids in populations of tropical supersweet corn. However, a large number of false-positive haploids were incorrectly selected by the R1-navajo marker. The color of the first leaf sheath was efficient for haploid identification in supersweet corn populations, and the method based on stomata morphometry can be used when there is no color distinction on the first leaf sheath.

Index terms:
Zea mays L. var. saccharata; doubled haploid; haploid induction rate; R1-navajo; stomata

Authorship

Alline Sekiya

Universidade Estadual de Londrina, Departamento de Genética e Biologia Molecular, Rodovia Celso Garcia Cid, Pr 445, Km 380, Campus Universitário, CEP 86057-970 Londrina, PR, Brazil. E-mail: allinesekiya@gmail.com, jessicakpestana@gmail.com, maikon.baptistella@gmail.com, krause.d.matheus@gmail.com, carlosrobertomax@gmail.com, josuemf@uel.brUniversidade Estadual de LondrinaBrazilLondrina, PR, Brazil Universidade Estadual de Londrina, Departamento de Genética e Biologia Molecular, Rodovia Celso Garcia Cid, Pr 445, Km 380, Campus Universitário, CEP 86057-970 Londrina, PR, Brazil. E-mail: allinesekiya@gmail.com, jessicakpestana@gmail.com, maikon.baptistella@gmail.com, krause.d.matheus@gmail.com, carlosrobertomax@gmail.com, josuemf@uel.br

http://orcid.org/0000-0002-5993-8410

Jéssica Kelly Pestana

http://orcid.org/0000-0002-6057-3391

Maikon Guerith Baptistella da Silva

http://orcid.org/0000-0003-1210-1661

Matheus Dalsente Krause

http://orcid.org/0000-0003-2411-9287

Carlos Roberto Maximiano da Silva

http://orcid.org/0000-0002-9134-9704

Josué Maldonado Ferreira ^**Corresponding author: josuemf@uel.br

http://orcid.org/0000-0002-0085-6891

*Corresponding author: josuemf@uel.br

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (3)
Tables (1)

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Figure 1.
Seed classification and mosaic seeds obtained by the process of haploid induction in supersweet corn (Zea mays L. var. saccharata) populations. A, class 1, diploid seed; B, class 2, putative haploid seed; C, class 3, seed without marking; D, parental supersweet corn seed; E, front and rear vision of mosaic seeds; and F, putative haploid seedlings at the V2-V3 stage, showing seedlings with purple first leaf sheath (a), seedlings with green first leaf sheath (b), and albino seedling (c).

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Figure 2.
Metaphasic nuclei obtained from supersweet corn (Zea mays L. var. saccharata) root tips and microscopic images of stomata guard cells of V2-V3-stage seedlings, showing: A1, false positive haploids represented by diploid plants with 2x=20 chromosomes; A2, true haploids represented by plants with x=10 chromosomes; B, F1 diploid plants, originated from class 1 seeds, based on the R1-nj marker; C, parental non-inducer diploid plants; D, putative haploid plants, originated from class 2 seeds by R1-nj and confirmed as diploids by chromosome counting; and E, haploid plants, originated from class 2 seeds by R1-nj and confirmed as haploids by chromosome counting.

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Figure 3.
Averages and mean confidence intervals of stomata guard-cell lengths for 12 populations and four plant categories (DPF1, PDP, HPD, and HPH), with respective comparison of the overall average of each category (lowercase letters above the column) by Tukey’s test, at 5% probability. DPF1, F1 diploids originated from seeds of class 1; PDP, parental non-inducer diploid; HPD, putative haploid confirmed as a diploid by chromosome counting; and HPH, putative haploid confirmed as a haploid by chromosome counting.

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Table 1.
Percentage of seeds classified by the R1-navajo (R1-nj) marker in crossings for haploidy induction between 11 supersweet corn (Zea mays L. var. saccharata) populations and a control, rates of true haploids and false positives, correlation between seeds without R1-nj marking and identified haploids, and factorial analysis of variance for the 12 plant populations and four categories evaluated for stomata guard-cell lengths (μm) and coefficient of variation (CV).

Figure 1. Seed classification and mosaic seeds obtained by the process of haploid induction in supersweet corn (Zea mays L. var. saccharata) populations. A, class 1, diploid seed; B, class 2, putative haploid seed; C, class 3, seed without marking; D, parental supersweet corn seed; E, front and rear vision of mosaic seeds; and F, putative haploid seedlings at the V2-V3 stage, showing seedlings with purple first leaf sheath (a), seedlings with green first leaf sheath (b), and albino seedling (c).

Figure 2. Metaphasic nuclei obtained from supersweet corn (Zea mays L. var. saccharata) root tips and microscopic images of stomata guard cells of V2-V3-stage seedlings, showing: A1, false positive haploids represented by diploid plants with 2x=20 chromosomes; A2, true haploids represented by plants with x=10 chromosomes; B, F1 diploid plants, originated from class 1 seeds, based on the R1-nj marker; C, parental non-inducer diploid plants; D, putative haploid plants, originated from class 2 seeds by R1-nj and confirmed as diploids by chromosome counting; and E, haploid plants, originated from class 2 seeds by R1-nj and confirmed as haploids by chromosome counting.

Figure 3. Averages and mean confidence intervals of stomata guard-cell lengths for 12 populations and four plant categories (DPF1, PDP, HPD, and HPH), with respective comparison of the overall average of each category (lowercase letters above the column) by Tukey’s test, at 5% probability. DPF1, F1 diploids originated from seeds of class 1; PDP, parental non-inducer diploid; HPD, putative haploid confirmed as a diploid by chromosome counting; and HPH, putative haploid confirmed as a haploid by chromosome counting.

Table 1. Percentage of seeds classified by the R1-navajo (R1-nj) marker in crossings for haploidy induction between 11 supersweet corn (Zea mays L. var. saccharata) populations and a control, rates of true haploids and false positives, correlation between seeds without R1-nj marking and identified haploids, and factorial analysis of variance for the 12 plant populations and four categories evaluated for stomata guard-cell lengths (μm) and coefficient of variation (CV).

	Population	Seed total	Seed class by R1-nj (%)⁽¹⁾			Class 2 seeds (%)⁽²⁾
	Population	Seed total	Class 1	Class 2	Class 3	True haploid rate		False positive
	PD1003	7,820	53.8	10.9	35.0	3		97
	PD1010	5,808	41.8	9.1	48.9	2		98
	PD2001	11,579	37.5	5.2	57.3	3		97
	PD2002	7,610	46.3	6.6	47.1	14		86
	PD2003	9,076	41.3	8.7	50.0	9		91
	SD3001	7,518	26.6	1.4	72.0	2		98
	SD3002	4,374	89.0	10.5	0.5	9		91
	SD3004	8,945	86.7	7.2	6.1	16		84
	SD3005	12,970	83.7	11.5	4.8	6		94
	SD3006	16,064	93.0	6.3	0.6	10		90
	SD3007	12,393	58.0	4.2	37.8	5		95
	30F53	14,192	60.8	13.1	26.1	5		95
	Correlation							r
	Class 3 seeds (without marking) x class 2 seeds (putative haploids)							-0.52
	Class 3 seeds (without marking) x true haploid rates							-0.45
Analysis of variance
Source of variation			Populations (P)		Categories (C)	P x C	Error	Average	CV (%)
Degrees of freedom			11		3	32	179	40.5	14.6
Mean square			72.749*		1,930.3**	34.711^ns	34.790

⁽¹⁾
R1-nj seed classes: class 1, diploid; class 2, putative haploid; and class 3, without marking. ⁽²⁾Percentages obtained from 100 class 2 seedlings for each cross. True haploid rates were obtained from haploids identified through first-leaf sheath color, with ploidy level confirmation by chromosome counting. False positives were considered class 2 seeds not identified as haploids by these methods. * and **Significant by the F-test, at 5 and 1% probability, respectively. ^nsNonsignificant.

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Haploid induction in tropical supersweet corn and ploidy determination at the seedling stage

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[1] *Corresponding author: josuemf@uel.br