Effect of ploidy level on guard cell length and use of stomata to discard diploids among putative haploids in maize

Abstract

Guard cell (GC) length proved to be efficient in sorting diploids from haploids in doubled haploid development in maize (Zea mays L). It compensates for the weakness of widely used R1-nj marker approach that showed low reliability in haploids identification from tropical genotypes. Guard cell length differs between haploid and diploid plants, and these differences were evaluated in the progeny of three different induction crosses obtained using Krasnodar haploid inducer. Epidermal impressions of the abaxial surface of leaves removed from the second, third, and fourth nodes (from base to apex) were collected and measured using an optical microscope. This was also conducted on the flowering phenotype. Guard cell length varied according to germplasm source, leaf stage, and ploidy level. Mean GC length ranged from 23.67 to 33.82 μm in haploids, and from 36.1 to 41.25 μm in diploids. Based on these differences in GC length at any of the chosen leaf stages (second, third, or fourth), diploid and haploid maize plants were successfully classified. Classification efficiency was found to be more closely related to germplasm source than leaf stage. Comparing GC length according to phenotype (haploid or diploid), GC limits for classification as a diploid plant (threshold points) were estimated and ranged from 29.74 to 34.49 μm, depending on germplasm source. The highest false discovery rate was 2.93% and false negative rate was 15.06%, indicating that classification based on GC length was reliable.

Breeding Maize Maternal Haploid Inducers

“…Haploid plants have narrower, shorter and more erect leaves than diploid plants. Haploid plants also have significantly smaller stomata and guard cell length than crossed, diploid plants [84,85]. Haploidization is also associated with male sterility, and thus the presence or absence of extruded anthers is another indicator of a plant's ploidy level.…”

Identification of haploids and diploids in maize using seedling traits and flow cytometry

“…McNemar 's test was previously adopted by Melchinger et al (2016) to compare test scores, such as R1-Navajo, liguleless, glossy, and resistance to Ultrafox herbicides using results obtained by flow cytometry as the gold standard. Silva et al (2020) also used this test to compare the equivalence between stomatal guard cell length classifications and phenotype. In both studies, it was verified that, in some cases, the test did not indicate associations between the classifications, which is in agreement with our research.…”

“…In these genotypes, the occurrence of genes that inhibit the anthocyanin production pathway, such as C1-I (Chaikam et al 2015), is frequent. Additionally, false positives (Prigge et al 2011, Chaikam et al 2016, Silva et al 2020) and false negatives (Chaikam et al 2016) can be common during visual separation. Thus, obtaining DH inbred lines becomes costly, because numerous diploid seeds move to the chromosomal duplication stage.…”

“…Some studies evaluating alternative haploid selection methods have used the ROC (Receiver Operating Characteristic) curve to establish cut-off points and evaluate the discriminatory capacity of a diagnostic test compared with the standard (gold standard) test. Silva et al (2020) used this analysis when studying the length of stomatal guard cells. In this context, the goal of this study was to determine the optimal T for germination to employ the methodology of seedling traits and verify whether this methodology is efficient in separating haploid and diploid individuals to eliminate the false positives selected by R1-Navajo.…”

Banana leaf anatomy characteristics related to ploidy levels

“…More recently, quantitative anatomy has gained strength in the field of taxonomy and is especially useful for confirming the differences that simple anatomical descriptions are unable to distinguish. This technique has important applications, particularly in the characterization of crop cultivars that have different ploidy levels (Silva et al, 2019, Zeng, Liu, Du, & Kang, 2019. This field is of great interest for understanding the adaptive characteristics resulting from DNA content changes in plants.…”