Identification of in vivo induced maternal haploids in maize using seedling traits

Abstract

In vivo haploid induction in high frequency followed by efficient identification of haploids are important components of deriving completely homozygous doubled haploid (DH) lines in maize. Several genetic marker systems were proposed and/or used for identification of in vivo maternal haploids in maize, such as R1-nj (Navajo), high oil, red root and transgenic markers. In this study, we propose a new method of haploid/diploid identification based on natural differences in seedling traits of haploids and diploids, which can be used in any induction cross independently of the genetic marker systems. Using confirmed haploids and diploids from five different populations, the study established that haploid and diploid seedlings exhibit significant differences for seedling traits, particularly radicle length (RL), coleoptile length (CL), and number of lateral seminal roots (NLSR). In six populations that exhibited complete inhibition of the commonly used R1-nj (Navajo) marker, we could effectively differentiate haploids from diploids by visual inspection of the seedling traits. In the haploid seed fraction identified based on R1-nj marker in ten populations, false positives were reduced several-fold by early identification of haploids at seedling stage using the seedling traits. We propose that seedling traits may be integrated at the haploid identification stage, especially in populations that are not amenable to use of genetic markers, and for improving the efficiency of DH line production by reducing the false positives.

Utilization of Reduced Haploid Vigor for Phenomic Discrimination of Haploid and Diploid Maize Seedlings

“…The selection methods developed here show similar, if not superior, performance compared with traditional R1‐nj or oil‐content‐based haploid selection. The study of Chaikam et al (2017) found similar results in radicle length of haploid seedlings and showed that pairing R1‐nj selection with observations of certain traits such as radicle length, coleoptile length, number of lateral seminal roots, and presence of root hairs reduced the FPR to 9.4%, which was a 3.5‐fold improvement when compared to R1‐nj selection alone. Past experiments reported an average FPR of 25.2 to 40.7% and FNR of 12.7 to 51.7% using R1‐nj selection (Chaikam et al, 2017; Melchinger et al, 2014).…”

“…The study of Chaikam et al (2017) found similar results in radicle length of haploid seedlings and showed that pairing R1‐nj selection with observations of certain traits such as radicle length, coleoptile length, number of lateral seminal roots, and presence of root hairs reduced the FPR to 9.4%, which was a 3.5‐fold improvement when compared to R1‐nj selection alone. Past experiments reported an average FPR of 25.2 to 40.7% and FNR of 12.7 to 51.7% using R1‐nj selection (Chaikam et al, 2017; Melchinger et al, 2014). Moreover, there are some genotypes, such as those that contain the allele C1‐l , that will inhibit the accumulation of anthocyanin, which is responsible for the marker characteristics of R1‐nj (Paz‐Ares et al, 1990).…”

“…Moreover, when haploids represent 20% of the population, the SVM method still represents the best selection method. Compared with standard methods of haploid selection, the SVM selection method improves on both FPR (16% for R1‐nj and 22% for oil content) and FNR (11% for R1‐nj and 13% for oil content) (Chaikam et al, 2017; Melchinger et al, 2014).…”

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

“…Similarly, the relatively small overlap in stomata length of H&DH and C distributions also makes this method promising for detecting C plants. Stomata length could be used in similar situations for identification of C seedlings as proposed for other seedling traits such as radicle length or shoot length (Chaikam et al, ). For example, in the context of DH production, mean stomata length would be determined simply by averaging across all stomata measured per plant and subject to a mixture distribution analysis for each germplasm separately, using the same procedures as described by Melchinger et al () for oil content of maize seeds.…”

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

“…The production of double-haploid lines involves four main steps: in vivo induction of haploidy, identification of possible haploids, chromosome doubling and the self-fertilization of lines obtained for increment of seeds [3]. However, the success of this methodology is still dependent on the use of inductors with high capacity of induction, a precise system of identification and differentiation of haploid and diploid seeds, as well as efficient and reproducible protocols of chromosome doubling [4].…”