Identification of haploid maize by flow cytometry, morphological and molecular markers

Abstract

The development of homozygous breeding lines in maize may be accelerated through the use of haploids. Thus, the obtaining and prior identification of haploids generated by the haploid inducer lines is an important factor. The purpose of this study was to identify haploids by flow cytometry and to correlate the nuclear DNA content to the morphological and morphometric traits of the seeds that gave rise to them. In addition, molecular markers were used to confirm the androgenetic nature of the haploid. The seeds obtained were derived from the cross between the inbred line W23 and the commercial hybrid P30F90. Among these seeds, a group was selected, putative haploids, whose embryo was white and the pericarp purplish. This group, consisting of 330 seeds, was characterized based on seed morphology, seed morphometry and nuclear DNA content. Flow cytometry analyses identified four haploids, and all of them were small size plants and had brittle leaves. The weight, length, thickness and width of the haploid seeds were very variable indicating that morphometric traits do not constitute reliable data for visual selection of haploid seeds. Based on results, the inbred line W23 induced haploid maize even under tropical conditions. Microsatellite molecular markers (SSR) proved to be efficient, confirming the androgenetic trait of the haploids.

Verification and characterization of chromosome duplication in haploid maize

“…Flow cytometry is advantageous in experiments involving chromosome duplication (Choe et al, 2012;Dang et al, 2012;Battistelli et al, 2013;Couto et al, 2013), as the sample is not degraded because only a piece of the leaf is sufficient for the analyses and little time is required for cytometry measurements (Loureiro and Santos, 2004). In this study, we analyzed all doubled plants in less than one week.…”

“…Belicuas et al (2007) and Couto et al (2013) used microsatellite markers to detect androgenetic haploids in maize and observed only one band in the haploid individual. Battistelli et al (2013) used SSR to confirm whether the plants that presented peaks for haploid cells in the histograms generated by flow cytometry were truly haploids.…”

“…Because flow cytometry can be used to directly evaluate DNA content and indirectly determine the ploidy level, this technique allows for the analyses of haploid induction as well as the chromosome duplication (Dang et al, 2012;Battistelli et al, 2013;Couto et al, 2013).…”

“…Because they are codominant and stable, these markers allow for the separation of homozygous and heterozygous individuals, enabling the study of genetic inheritance and gene introgression (Belicuas et al, 2007, Li et al, 2009Battistelli et al, 2013;Couto et al, 2013).…”

Early diagnosis of ploidy status in doubled haploid production of maize by stomata length and flow cytometry measurements

“…Meanwhile, a high‐throughput platform is available which is capable of analysing 1,000 samples per day (Erich Pollähne GmbH, ; Saaten‐Union Biotec GmbH, ). In maize, flow cytometry has been used for discriminating H and C plants (Belicuas et al, ; Couto, Davide, Bustamante, Pinho, & Silva, ; Wu, Ren, Li, & Chen, ) and for monitoring chromosome doubling to compare different treatment protocols (Couto et al, ). However, these studies neither determined the association between ploidy classification of D 0 plants as H or DH by flow cytometry and classification by phenotyping of plants in the field nor evaluated the effectiveness of flow cytometry for discriminating H from C plants in early growth stages.…”