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

Vijay Chaikam,Prasanna M Boddupalli,Luis Antonio Lopez,Leocadio Martinez,Juan Burgueño

doi:10.1007/s10681-017-1968-3

Vijay Chaikam, Prasanna M Boddupalli + Show 3 more

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https://doi.org/10.1007/s10681-017-1968-3

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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.

Highlights

Doubled haploid (DH) technology has become an invaluable tool in maize breeding programs to produce177 Page 2 of 9 homozygous lines from heterozygous populations within just two cycles compared to 6–8 cycles through conventional inbreeding
One of the critical requirements for large scale production of DH lines in maize is efficient and accurate identification of a small proportion of haploids from the diploids among the seeds obtained through induction crosses, before subjecting the selected haploids to chromosomal doubling treatment (Chaikam et al 2016)
coleoptile length (CL) and radicle length (RL) increased with increasing time of germination, while the number of lateral seminal roots (NLSR) remained same across time points (Online Resource 1)

Summary

Introduction

Doubled haploid (DH) technology has become an invaluable tool in maize breeding programs to produce177 Page 2 of 9 homozygous lines from heterozygous populations within just two cycles compared to 6–8 cycles through conventional inbreeding. One of the critical requirements for large scale production of DH lines in maize is efficient and accurate identification of a small proportion of haploids from the diploids among the seeds obtained through induction crosses, before subjecting the selected haploids to chromosomal doubling treatment (Chaikam et al 2016). All of the currently used maternal haploid inducers in maize are equipped with an anthocyanin marker for haploid identification conditioned by R1-nj (Navajo) gene (Melchinger et al 2015) When such R1-nj based inducers are crossed as male parent with the source germplasm/population (from which DH lines are desired) as the female parent, the resultant seeds will predominantly have diploids marked with anthocyanin coloration on both endosperm and embryo whereas the maternal haploids have anthocyanin coloration only on the endosperm (Nanda and Chase 1966; Greenblatt and Bock 1967). Navajo based haploid versus diploid classification can result in high false positives (Rober et al 2005; Prigge et al 2011; Melchinger et al 2014; Chaikam et al 2016) and false negatives (Rober et al 2005; Chaikam et al 2016), and is not useful when the source germplasm has either color inhibitor C1-I or natural anthocyanin coloration (purple/red) on the pericarp/endosperm as is the case in many maize landraces (Chaikam et al 2016)

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