Accelerating haploid induction rate and haploid validation through marker-assisted selection for qhir1 and qhir8 in maize.

Doubled haploid (DH) technology becomes more routinely applied in maize hybrid breeding. However, some issues in haploid induction and identification persist, requiring resolution to optimize DH production. Our objective was to implement simultaneous marker-assisted selection (MAS) for qhir1 (MTL/ZmPLA1/NLD) and qhir8 (ZmDMP) using TaqMan assay in F2 generation of four BHI306-derived tropical × temperate inducer families. We also aimed to assess their haploid induction rate (HIR) in the F3 generation as a phenotypic response to MAS. We highlighted remarkable increases in HIR of each inducer family. Genotypes carrying qhir1 and qhir8 exhibited 1 - 3-fold higher haploid frequency than those carrying only qhir1. Additionally, the qhir1 marker was employed for verifying putative haploid seedlings at 7 days after planting. Flow cytometric analysis served as the gold standard test to assess the accuracy of the R1-nj and the qhir1 marker. The qhir1 marker showed high accuracy and may be integrated in multiple haploid identifications at early seedling stage succeeding pre-haploid sorting via R1-nj marker.

Haploid identification in maize.

Doubled haploid (DH) line production through in vivo maternal haploid induction is widely adopted in maize breeding programs. The established protocol for DH production includes four steps namely in vivo maternal haploid induction, haploid identification, genome doubling of haploid, and self-fertilization of doubled haploids. Since modern haploid inducers still produce relatively small portion of haploids among undesirable hybrid kernels, haploid identification is typically laborious, costly, and time-consuming, making this step the second foremost in the DH technique. This manuscript reviews numerous methods for haploid identification from different approaches including the innate differences in haploids and diploids, biomarkers integrated in haploid inducers, and automated seed sorting. The phenotypic differentiation, genetic basis, advantages, and limitations of each biomarker system are highlighted. Several approaches of automated seed sorting from different research groups are also discussed regarding the platform or instrument used, sorting time, accuracy, advantages, limitations, and challenges before they go through commercialization. The past haploid selection was focusing on finding the distinguishable marker systems with the key to effectiveness. The current haploid selection is adopting multiple reliable biomarker systems with the key to efficiency while seeking the possibility for automation. Fully automated high-throughput haploid sorting would be promising in near future with the key to robustness with retaining the feasible level of accuracy. The system that can meet between three major constraints (time, workforce, and budget) and the sorting scale would be the best option.

Combining ability of tropical × temperate maize inducers for haploid induction rate, R1-nj seed set, and agronomic traits.

In vivo maternal haploid induction in isolation fields is proposed to bypass the workload and resource constraints existing in haploid induction nurseries. A better understanding of combining ability and gene action conditioning traits related to hybrid inducers is necessary to set the breeding strategy including to what extent parent-based hybrid prediction is feasible. This study aimed to evaluate the following in tropical savanna in the rainy and dry seasons for haploid induction rate (HIR), R1-nj seed set, and agronomic traits: 1) combining ability, line per se, and hybrid performance of three genetic pools; 2) genetic parameters, the modes of gene action, and heterosis; and 3) the relationships of inbred-general combining ability (GCA) and inbred-hybrid performance. Fifty-six diallel crosses derived from eight maize genotypes were evaluated in the rainy season of 2021 and the dry season of 2021/2022. Reciprocal cross effects including the maternal effect barely contributed to the genotypic variance for each trait observed. HIR, R1-nj seed set, flowering dates, and ear position were highly heritable and additive inherited, while ear length showed dominant inheritance. The equal importance of additive and dominance effects was found for yield-related traits. Temperate inducer BHI306 was the best general combiner for the HIR and R1-nj seed set, followed by two tropical inducers, KHI47 and KHI54. The ranges of heterosis were trait-dependent and slightly influenced by the environment, where hybrids in the rainy season consistently had higher heterosis than those in the dry season for each trait observed. Both hybrid groups derived from tropical × tropical and tropical × temperate inducers showed taller plants, larger ear size, and higher seed sets than the corresponding parents. However, their HIRs were still below the standard check of BHI306. The implications of genetic information, combining ability, and inbred-GCA and inbred-hybrid relationships on breeding strategies are discussed.

A Comparison between Inbred and Hybrid Maize Haploid Inducers.

The effectiveness of haploid induction systems is regarded not only for high haploid induction rate (HIR) but also resource savings. Isolation fields are proposed for hybrid induction. However, efficient haploid production depends on inducer traits such as high HIR, abundant pollen production, and tall plants. Seven hybrid inducers and their respective parents were evaluated over three years for HIR, seeds set in cross-pollinations, plant and ear height, tassel size, and tassel branching. Mid-parent heterosis was estimated to quantify how much inducer traits improve in hybrids in comparison to their parents. Heterosis benefits hybrid inducers for plant height, ear height, and tassel size. Two hybrid inducers, BH201/LH82-Ped126 and BH201/LH82-Ped128, are promising for haploid induction in isolation fields. Hybrid inducers offer convenience and resource-effectiveness for haploid induction by means of improving plant vigor without compromising HIR.

Seasonal Variation of Tropical Savanna Altered Agronomic Adaptation of Stock-6-Derived Inducer Lines.

Tropicalization is one of the major objectives in breeding haploid inducers to address the poor adaptation of temperate haploid inducers in doubled haploid production in tropical maize. Gaining a better understanding of weather profiles in targeted agroecology is important. This study aimed to investigate the seasonal variation of tropical savanna climate and its impact on agronomic traits and haploid induction rate (HIR) of Stock-6-derived haploid inducer lines. A total of 14 haploid inducers were evaluated across two typical growing seasons between 2020 and 2021. Weather data were collected on daily minimum and maximum temperatures, relative humidity, precipitation, and solar radiation whereas phenotypic data were recorded on plant phenology, tassel attributes, plant stature, ear components, inducer seed rate (ISR), and HIR. The effects of season, genotype, and genotype by season were significant for all traits except season factor on ISR. Seasonal variation existed where the dry season was more suitable for haploid induction and inducer maintenance, as haploid inducers revealed better agronomic performance and seed set, delayed flowering dates, and higher HIR. Since the crossover performance of haploid inducers over seasons was detected, further implications on genotype selection in each season are discussed.

Selection Gain of Maize Haploid Inducers for the Tropical Savanna Environments.

Lacking elite haploid inducers performing high haploid induction rate (HIR) and agronomic performance is one of fundamental factors hindering the rapid adoption of doubled haploid technology in maize hybrid breeding, especially under tropical savanna climate. Breeding haploid inducers for specific agro-ecology, thus, is indispensable yet challenging. We used temperate inducer Stock6 as genetic source for haploid induction ability and eight tropical maize genotypes as principal donors for agronomic adaptation. Three cycles of modified ear-to-row with 5% intra-family selection were applied in a population set of 78 putative haploid inducer families emphasized on agronomic performance, R1-nj anthocyanin intensity, and inducer seed set. Genetic gains, variance components, and heritability on given traits were estimated. Hierarchical clustering based on five selection criteria was performed to investigate the phenotypic diversity of putative families. Cycle effect was predominant for all observed traits. Realized genetic gain was positive for HIR (0.40% per cycle) and inducer seed set (30.10% or 47.30 seeds per ear per cycle). In this study, we reported the first haploid inducers for regions under tropical savanna climate. Three inducer families, KHI-42, KHI-54, and KHI-64, were promising as they possessed HIR about 7.8% or 14 haploid seeds per tester ear and inducer seed rate about 95.0% or 208 inducer seeds per ear. The breeding method was effective for enhancing the seed set and the expression of R1-nj anthocyanin marker of inducers, yet it showed a low effectiveness to improve haploid induction rate. Introgression of temperate inducer Stock6 into tropical gene pool followed by phenotypic selections through modified ear-to-row selection on inducer seed set and R1-nj marker did not compromise the agronomic traits of tropical inducer families. Implications and further strategies for optimizing genetic gain on HIR are discussed.