For efficient production of doubled haploid (DH) lines in maize, maternal haploid inducer lines with high haploid induction rate (HIR) and good adaptation to the target environments is an important requirement. In this study, we present second-generation Tropically Adapted Inducer Lines (2GTAILs), developed using marker assisted selection (MAS) for qhir1, a QTL with a significant positive effect on HIR from the crosses between elite tropical maize inbreds and first generation Tropically Adapted Inducers Lines (TAILs). Evaluation of 2GTAILs for HIR and agronomic performance in the tropical and subtropical environments indicated superior performance of 2GTAILs over the TAILs for both HIR and agronomic performance, including plant vigor, delayed flowering, grain yield, and resistance to ear rots. One of the new inducers 2GTAIL006 showed an average HIR of 13.1% which is 48.9% higher than the average HIR of the TAILs. Several other 2GTAILs also showed higher HIR compared to the TAILs. While employing MAS for qhir1 QTL, we observed significant influence of the non-inducer parent on the positive effect of qhir1 QTL on HIR. The non-inducer parents that resulted in highest mean HIR in the early generation qhir1+ families also gave rise to highest numbers of candidate inducers, some of which showed transgressive segregation for HIR. The mean HIR of early generation qhir1+ families involving different non-inducer parents can potentially indicate recipient non-inducer parents that can result in progenies with high HIR. Our study also indicated that the HIR associated traits (endosperm abortion rate, embryo abortion rate, and proportion of haploid plants among the inducer plants) can be used to differentiate inducers vs. non-inducers but are not suitable for differentiating inducers with varying levels of haploid induction rates. We propose here an efficient methodology for developing haploid inducer lines combining MAS for qhir1 with HIR associated traits.

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.

Key messageAmong the qhir11 and qhir12 sub-regions of a major QTL qhir1, only qhir11 has significant effect on maternal haploid induction, segregation distortion and kernel abortion.AbstractIn vivo haploid induction in maize can be triggered in high frequencies by pollination with special genetic stocks called haploid inducers. Several genetic studies with segregating populations from non-inducer x inducer crosses identified a major QTL, qhir1, on chromosome 1.04 contributing to in vivo haploid induction. A recent Genome Wide Association Study using 51 inducers and 1482 non-inducers also identified two sub-regions within the qhir1 QTL region, named qhir11 and qhir12; qhir12 was proposed to be mandatory for haploid induction because the haplotype of qhir11 was also present in some non-inducers and putative candidate genes coding for DNA and amino acid binding proteins were identified in the qhir12 region. To characterize the effects of each sub-region of qhir1 on haploid induction rate, F2 recombinants segregating for one of the sub-regions and fixed for the other were identified in a cross between CML269 (non-inducer) and a tropicalized haploid inducer TAIL8. To quantify the haploid induction effects of qhir11 and qhir12, selfed progenies of recombinants between these sub-regions were genotyped. F3 plants homozygous for qhir11 and/or qhir12 were identified, and crossed to a ligueless tester to determine their haploid induction rates. The study revealed that only the qhir11 sub-region has a significant effect on haploid induction ability, besides causing significant segregation distortion and kernel abortion, traits that are strongly associated with maternal haploid induction. The results presented in this study can guide fine mapping efforts of qhir1 and in developing new inducers efficiently using marker assisted selection.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-017-2873-9) contains supplementary material, which is available to authorized users.

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Doubled haploid (DH) technology has become integral to maize breeding programs to expedite inbred line development and increase the efficiency of breeding operations. Unlike many other plant species that use in vitro methods, DH production in maize uses a relatively simple and efficient in vivo haploid induction method. However, it takes two complete crop cycles for DH line generation, one for haploid induction and the other one for chromosome doubling and seed production. Rescuing in vivo induced haploid embryos has the potential to reduce the time for DH line development and improve the efficiency of DH line production. However, the identification of a few haploid embryos (~10%) resulting from an induction cross from the rest of the diploid embryos is a challenge. In this study, we demonstrated that an anthocyanin marker, namely R1-nj, which is integrated into most haploid inducers, can aid in distinguishing haploid and diploid embryos. Further, we tested conditions that enhance R1-nj anthocyanin marker expression in embryos and found that light and sucrose enhance anthocyanin expression, while phosphorous deprivation in the media had no affect. Validating the use of the R1-nj marker for haploid and diploid embryo identification using a gold standard classification based on visual differences among haploids and diploids for characteristics such as seedling vigor, erectness of leaves, tassel fertility, etc., indicated that the R1-nj marker could lead to significantly high false positives, necessitating the use of additional markers for increased accuracy and reliability of haploid embryo identification.