Haploid Induction Rate Research Articles

Genomic prediction of maternal haploid induction rate in maize.

Genomic prediction (GP) might be an efficient way to improve haploid induction rate (HIR) and to reduce the laborious and time-consuming task of phenotypic selection for HIR in maize (Zea mays L.). In this study, we evaluated GP accuracies for HIR and other agronomic traits of importance to inducers by independent and cross-validation. We propose the use of GP for cross prediction and parental selection in the development of new inducer breeding populations. A panel of 159 inducers from Iowa State University (ISU set) was genotyped and phenotyped for HIR and several agronomic traits. The data of an independent set of 53 inducers evaluated by the University of Hohenheim (UOH set) was used for independent validation. The HIR ranged from 0.61 to 20.74% and exhibited high heritability (0.90). High cross-validation prediction accuracy was observed for HIR (r=0.82), whereas for other traits it ranged from 0.36 (self-induction rate) to 0.74 (days to anthesis). Prediction accuracies across different sets were higher when the larger panel (ISU set) was used as a training population (r=0.54). The average HIR of the 12,561 superior predicted progenies (μSP ) ranged from 1.00-18.36% and was closely related to the corresponding midparent genomic estimated breeding value (GEBV). A predicted genetic variance (VG ) of reduced magnitude was observed in the twenty crosses with highest midparent GEBV or μSP for HIR. Our results indicate that although GP is a useful tool for parental selection, decisions about which cross combinations should be pursued need to be based on optimal trade-offs between maximizing both μSP and VG .

Mapping of QTL for kernel abortion caused by in vivo haploid induction in maize (Zea mays L.).

Kernel abortion is common phenomenon in vivo haploid induction and closely linked with haploid induction rate, but little information of kernel abortion is available and its genetic basis still unclear. We used two mapping populations including 186 and 263 F2.3 family lines to analyze the different degree of kernel abortion and identify quantitative trait loci (QTL) responsible for kernel abortion during haploid induction. In total 62 putative QTL, accounting for 3.27–14.70% of the phenotypic variation in kernel abortion traits, were detected across all 10 chromosomes. Ten QTL with over 10% contribution to phenotypic variation were affecting the fifth level of endosperm abortion (EnA5th), endosperm abortion (EnA) and total abortion (TA). Co-localization among kernel abortion traits QTL was observed in both populations and among different kernel abortion types. Five overlaps were indentified in the QTL for kernel abortion traits and HIR traits. Maize chromosome bins 3.01–3.02, 3.04–3.06, 4.05–4.06, 5.03–5.04, 8.06 were QTL hotspots for three or four traits related to the kernel abortion during haploid induction. Total kernel abortion rate (TAR) and HIR showed highly significant positive correlation. These findings may help to reveal haploid induction mechanisms and improve haploid production efficiency.

Generation and characterization of doubled haploid plants in maize

Doubled Haploid (DH) technology has revolutionized the maize hybrid production by developing homozygous inbred lines in 2–3 years which are otherwise produced in 7–8 years using conventional breeding methods. In the present study, Tropically Adapted Inducer Lines (TAIL) hybrid and four maize inbred lines (CM140, LM16, CM123 and I122) were used as target material for haploid production. F1 seeds were obtained from the parental crosses and the Haploid Induction Rate (HIR) obtained from the cross between F1 and inducer line ranges from zero to 9.72 per cent. Haploid seeds were germinated in petri-plates with germination percentage ranging from 89.54 to 93.37 per cent. The germinated seeds were then treated with colchicine solution @0.04% for 12 hours to generate Double Haploid (DH) plants. The survival percentage of the DH plants in the field was 23.37 per cent. The morphological observations of the treated plants conferred only one doubled haploid plant. the doubled haploid plant was confirmed by using cytology and flow cytometry.

Breeding strategies for tropical maize targeting in vivo haploid inducers

Abstract: The objective of this study was to compare the selection of plants bred by different pedigree methods using selection among, among and within and only within families. The haploid induction rate of 14 S0:1 and seven S2:3 families, all crossed with the single-cross hybrid GNZ9501, was evaluated. An experimental area of ​​the Department of Biology of the Federal University of Lavras (UFLA), in Lavras, Minas Gerais, in the growing seasons 2012/2013 and 2014/2015, was used for the experiments. In each growing season, one experiment per was carried out, arranged in a complete randomized design, with one and two replications, respectively. Haploid induction was most effective in the families 2 and 6 in both growing seasons. Selection among and within families resulted in higher genetic gains for haploid induction. The results indicated a high genetic variability for haploid induction rate in plants within families.

Genetic dissection of maternal influence on in vivo haploid induction in maize

In vivo haploid induction based on maternal haploid inducers is the first step in deriving completely homozygous maize doubled haploid (DH) lines. Haploid induction rate (HIR) is influenced by both pollen parent inducing haploidy and the maternal source germplasm used in induction crosses. This study was aimed at analyzing the influence of source germplasm on HIR using 671 tropical inbred lines organized in two association mapping panels. These two association mapping panels (AMP1 and AMP2) were crossed to two different Tropically Adapted Inducer Lines (TAILs). For HIR assessment, seeds from induction crosses were planted in the field and ploidy status of each surviving plant was assessed using a gold standard classification based on visual differences between the haploid and diploid plants. The analysis revealed significant variation for HIR and led to identification of several tropical inbred lines that respond very positively to haploid induction. Use of HIR data in a genome wide association study (GWAS) led to identification of twenty-seven and two SNPs that were significantly associated with HIR in AMP1 and AMP2, respectively. Meta-analysis of AMP1 and AMP2 GWAS led to identification of 52 SNPs with significant effect on HIR across both studies. Genome-wide prediction revealed moderate to high prediction accuracy within AMPs using random SNPs. Inclusion of the SNPs detected in GWAS into the prediction model led to improvement in prediction accuracy. Overall, the study revealed that the maternal influence on HIR is controlled by a few moderate and many small effect QTL.

Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system.

The use of CRISPR/LbCpf1 and CRISPR/xCas9 systems in wheat have not yet been reported. In this study, we compared the efficiencies of three CRISPR editing systems (SpCas9, LbCpf1, and xCas9), and three different promoters (OsU6a, TaU3, and TaU6) that drive single-guide (sg)RNA, which were introduced into wheat via Agrobacterium-mediated transformation. The results indicated that TaU3 was a better choice than OsU6a or TaU6. The editing efficiency was higher using two sgRNAs than one sgRNA, and mutants with a large fragment deletion between the two sgRNAs were produced. The LbCpf1 and xCas9 systems could both be used successfully. Two endogenous genes, TaWaxy and TaMTL, were edited with high efficiency by the optimized SpCas9 system, with the highest efficiency (80.5%) being achieved when using TaU3 and two sgRNAs to target TaWaxy. Rates of seed set in the TaMTL-edited T0 transgenic plants were much lower than that of the wild-type. A haploid induction rate of 18.9% was found in the TaMTL-edited T1 plants using the CRISPR/SpCas9 system. Mutants with reverse insertion of the deleted sequences of TaMTL and TaWaxy between the two sgRNAs were identified in the edited T0 plants. In addition, wheat grains lacking embryos or endosperms were observed in the TaMTL-edited T1 generation.

Effect of F1 and F2 generations on genetic variability and working steps of doubled haploid production in maize.

For doubled haploid (DH) production in maize, F1 generation has been the most frequently used for haploid induction due to facility in the process. However, using F2 generation would be a good alternative to increase genetic variability owing to the additional recombination in meiosis. Our goals were to compare the effect of F1 and F2 generations on DH production in tropical germplasm, evaluating the R1-navajo expression in seeds, the working steps of the methodology, and the genetic variability of the DH lines obtained. Sources germplasm in F1 and F2 generations were crossed with the tropicalized haploid inducer LI-ESALQ. After harvest, for both induction crosses were calculated the haploid induction rate (HIR), diploid seed rate (DSR), and inhibition seed rate (ISR) using the total number of seeds obtained. In order to study the effectiveness of the DH working steps in each generation, the percentage per se and the relative percentage were verified. In addition, SNP markers were obtained for genetic variability studies. Results showed that the values for HIR, ISR, and DSR were 1.23%, 23.48%, and 75.21% for F1 and 1.78%, 15.82%, and 82.38% for F2, respectively. The effectiveness of the DH working step showed the same percentage per se value (0.4%) for F1 and F2, while the relative percentage was 27.2% for F1 and 22.4% for F2. Estimates of population parameters in DH lines from F1 were higher than F2. Furthermore, population structure and kinship analyses showed that one additional generation was not sufficient to create new genotype subgroups. Additionally, the relative efficiency of the response to selection in the F1 was 31.88% higher than F2 due to the number of cycles that are used to obtain the DH. Our results showed that in tropical maize, the use of F1 generation is recommended due to a superior balance between time and genetic variability.

Genome-wide association study to identify genomic regions influencing spontaneous fertility in maize haploids

Efficient production and use of doubled haploid lines can greatly accelerate genetic gains in maize breeding programs. One of the critical steps in standard doubled haploid line production is doubling the haploid genome using toxic and costly mitosis-inhibiting chemicals to achieve fertility in haploids. Alternatively, fertility may be spontaneously restored by natural chromosomal doubling, although generally at a rate too low for practical applications in most germplasm. This is the first large-scale genome-wise association study to analyze spontaneous chromosome doubling in haploids derived from tropical maize inbred lines. Induction crosses between tropicalized haploid inducers and 400 inbred lines were made, and the resulting haploid plants were assessed for haploid male fertility which refers to pollen production and haploid fertility which refers to seed production upon self-fertilization. A small number of genotypes were highly fertile and these fertility traits were highly heritable. Agronomic traits like plant height, ear height and tassel branch number were positively correlated with fertility traits. In contrast, haploid induction rate of the source germplasm and plant aspect were not correlated to fertility traits. Several genomic regions and candidate genes were identified that may control spontaneous fertility restoration. Overall, the study revealed the presence of large variation for both haploid male fertility and haploid fertility which can be potentially exploited for improving the efficiency of doubled haploid derivation in tropical maize germplasm.

The development of homozygous maize lines using an in vivo haploid induction in the Croatian germplasm

The in vivo haploid induction has been widely applied to the maize breeding in recent decades, but it has not been used in the breeding programs in the Republic of Croatia by now. This study's objectives were to examine the haploid induction rates in the Croatian germplasm and to evaluate the properties of the D0 haploids, which are essential for a successful implementation of this method in breeding. The in vivo haploid induction was performed on 11 single-cross hybrids using the Zarodyshevy Marker Krasnodarsky (ZMK) inducer, and colchicine was used as a chromosome doubling agent. Emergence, misclassification rate, colchicine treatment survival, chromosome doubling rate and self-pollination success were examined in the D0 generation. The haploid induction rates ranged from 6.9 to 15.8%, which is consistent with the average induction rates characteristic of the ZMK inducer and the other modern ones. Significant differences were found among the populations of D0 haploids for all tested properties, except for self-pollination success. On average, the misclassification rates were lower, and the seedling survival rates were higher than those reported in other studies, indicating a possibility of a successful application of the doubled haploid method in maize breeding.

Mutation of ZmDMP enhances haploid induction in maize.

Doubled haploid (DH) breeding based on in vivo haploid induction has led to a new approach for maize breeding1. All modern haploid inducers used in DH breeding are derived from the haploid inducer line Stock6. Two key quantitative trait loci, qhir1 and qhir8, lead to high-frequency haploid induction2. Mutation of the gene MTL/ZmPLA1/NLD in qhir1 could generate a ~2% haploid induction rate (HIR)3-5; nevertheless, this mutation is insufficient for modern haploid inducers whose average HIR is ~10%6. Therefore, cloning of the gene underlying qhir8 is important for illuminating the genetic basis of haploid induction. Here, we present the discovery that mutation of a non-Stock6-originating gene in qhir8, namely, ZmDMP, enhances and triggers haploid induction. ZmDMP was identified by map-based cloning and further verified by CRISPR-Cas9-mediated knockout experiments. A single-nucleotide change in ZmDMP leads to a 2-3-fold increase in the HIR. ZmDMP knockout triggered haploid induction with a HIR of 0.1-0.3% and exhibited a greater ability to increase the HIR by 5-6-fold in the presence of mtl/zmpla1/nld. ZmDMP was highly expressed during the late stage of pollen development and localized to the plasma membrane. These findings provide important approaches for studying the molecular mechanism of haploid induction and improving DH breeding efficiency in maize.

Production of Double Haploid Plants Using In Vivo Haploid Techniques in Corn

This research was conducted at the breeding station of the Turkish breeding company Agromar A.S in the city of Bursa in Turkey during the 2013 and 2014 growing seasons. Used from within the same heterotic group crossings, 7 donor materials were obtained during the 2012 winter season in the greenhouse. The inducer line RWK-76xRWS, provided by University of Hohenheim, Germany, was used for generating haploid seeds. The donor and inducer crossing was performed during the 2013 summer season. The haploid selection and chromosome doubling were performed during the 2014 summer season. Seven donors were used for haploid induction which name are DNR1, DNR2, DNR3, DNR4, DNR5, DNR6, DNR7 respectively, from each donor different amount of ear crosses were performed (DNR1:16 ears, DNR2:10 ears, DNR3:10 ears, DNR4:12 ears, DNR5:11 ears, DNR6:13 ears, DNR7:11 ears). According to the present study, the average induction rate found ranged from 7.1 to 12.8%, and the average seedling survival rate in the greenhouse after colchicine application ranged from 57.9 to 77.6%. After transplanting to the field, 78.3-92.6% of these plants survived. As a result of this research, the chromosome doubling rate ranged from 22.5 to 48.3% depending on the donor material. These result indicates that maternal haploid selection visually is easy. Haploid induction rate (HIR) changes from donor to donor, its mean genotype and environment is effective for HIR. Average chromosome doubling rate is lower than other researchers’ results, it is also effected by genotype and chromosome doubling methods. Zea mays; Haploid line; Double haploid line; In vivo induction

Marker-Assisted Breeding of Improved Maternal Haploid Inducers in Maize for the Tropical/Subtropical Regions

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.

Genome-Wide Association Study of Haploid Male Fertility in Maize (Zea Mays L.).

Large-scale application of the doubled haploid (DH) technology by in vivo haploid induction has greatly improved the efficiency of maize breeding. While the haploid induction rate and the efficiency of identifying haploid plants have greatly improved in recent years, the low efficiency of doubling of haploid plants has remained and currently presents the main limitation to maize DH line production. In this study, we aimed to assess the available genetic variation for haploid male fertility (HMF), i.e., the production of fertile pollen on haploid plants, and to investigate the underlying genetic architecture. To this end, a diversity panel of 481 maize inbred lines was crossed with “Mo17” and “Zheng58,” the F1 hybrids subjected to haploid induction, and resulting haploid plants assessed for male fertility in two environments. Across both genetic backgrounds, we observed a large variation of HMF ranging from zero to ~60%, with a mean of 18%, and a heritability of 0.65. HMF was higher in the “Mo17” than in the “Zheng58” background and the correlation between both genetic backgrounds was 0.68. Genome-wide association mapping identified only few putative QTL that jointly explained 22.5% of the phenotypic variance. With the exception of one association explaining 11.77% of the phenotypic variance, all other putative QTL were of minor importance. A genome-wide prediction approach further corroborated the quantitative nature of HMF in maize. Analysis of the 14 significantly associated SNPs revealed several candidate genes. Collectively, our results illustrate the large variation of HMF that can be exploited for maize DH breeding. Owing to the apparent genetic complexity of this trait, this might best be achieved by rapid recurrent phenotypic selection coupled with marker-assisted selection for individual QTL.

OsMATL mutation induces haploid seed formation in indica rice.

Intraspecific haploid induction in maize (Zea mays) is triggered by a native frameshift mutation in MATRILINEAL (MATL), which encodes a pollen-specific phospholipase. To develop a haploid inducer in rice (Oryza sativa), we generated an allelic series in the putative ZmMATL orthologue, OsMATL, and found that knockout mutations led to a reduced seed set and a 2-6% haploid induction rate. This demonstrates MATL functional conservation and represents a major advance for rice breeding.

New insight into the mechanism of heterofertilization during maize haploid induction

To study the influence of both pollen competition ability (PCA) and haploid induction rate (HIR) on heterofertilization, we used maize haploid inducers with different HIRs as male parents. For these experiments, maize plants underwent dual pollination, first by the inducers and then by self-pollination at different time intervals. We found that PCA strongly influenced heterofertilization, as measured by the resulting ratio of hybrid kernels to selfed kernels. When log10 (hybrid kernel/selfed kernel) was close to 0, i.e., when pollen from the different parents arrived at the embryo sac nearly simultaneously, the heterofertilization rate (HFR) was high. Haploid inducers with a high HIR, such as CAU5 and CAUB73, led to not only more haploids and embryo aborted kernels but also more heterofertilized kernels, as compared with a non-inducer or with a haploid inducer with a low HIR. We thus conclude that PCA is an important factor that affects HFR through polytubey. In addition, during maize haploid induction, abnormal fertilization could also contribute to heterofertilization, which may be due to fertilization recovery of kernels that otherwise would have been aborted at early stage.

Dissection of a major QTL qhir1 conferring maternal haploid induction ability in maize

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.In 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.

MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction.

Sexual reproduction in flowering plants involves double fertilization, the union of two sperm from pollen with two sex cells in the female embryo sac. Modern plant breeders increasingly seek to circumvent this process to produce doubled haploid individuals, which derive from the chromosome-doubled cells of the haploid gametophyte. Doubled haploid production fixes recombinant haploid genomes in inbred lines, shaving years off the breeding process. Costly, genotype-dependent tissue culture methods are used in many crops, while seed-based in vivo doubled haploid systems are rare in nature and difficult to manage in breeding programmes. The multi-billion-dollar maize hybrid seed business, however, is supported by industrial doubled haploid pipelines using intraspecific crosses to in vivo haploid inducer males derived from Stock 6, first reported in 1959 (ref. 5), followed by colchicine treatment. Despite decades of use, the mode of action remains controversial. Here we establish, through fine mapping, genome sequencing, genetic complementation, and gene editing, that haploid induction in maize (Zea mays) is triggered by a frame-shift mutation in MATRILINEAL (MTL), a pollen-specific phospholipase, and that novel edits in MTL lead to a 6.7% haploid induction rate (the percentage of haploid progeny versus total progeny). Wild-type MTL protein localizes exclusively to sperm cytoplasm, and pollen RNA-sequence profiling identifies a suite of pollen-specific genes overexpressed during haploid induction, some of which may mediate the formation of haploid seed. These findings highlight the importance of male gamete cytoplasmic components to reproductive success and male genome transmittance. Given the conservation of MTL in the cereals, this discovery may enable development of in vivo haploid induction systems to accelerate breeding in crop plants.

Germplasm enhancement of maize: a look into haploid induction and chromosomal doubling of haploids from temperate‐adapted tropical sources

AbstractThe allelic diversity (AD) project of the Germplasm Enhancement of Maize (GEM) programme utilized the double haploid (DH) breeding method to expedite development and release of lines derived from 300 exotic maize races. Using 18 races in this study, differential effects on haploid induction rates (HIRs) and doubling rates (DRs) by the recurrent parents PHB47 and PHZ51, the elevation that the race is traditionally grown at, and by the race itself were addressed in this study. Races from the AD project were grouped by elevation of their origin, high, middle or low altitude. Six races per elevation were randomly selected and backcrossed using both recurrent parents to generate 36 populations. Ten replications were randomized in a complete randomized design for two growing seasons. The recurrent parent effect was significant, with PHB47 having a higher HIR than PHZ51. Effect of elevation was significant with higher HIR associated with low‐elevation origin, and race also proved to be significant. Effects of elevation, recurrent parent and race were not significant for DR.

Development and Validation of Red Root Marker‐Based Haploid Inducers in Maize

One of the critical limitations for the in vivo production of doubled haploid (DH) lines in maize (Zea mays L.) is the inability to effectively identify haploids in a significant proportion of induction crosses due to the possibility of complete or partial inhibition of the currently used R1‐nj (Navajo) color marker. In this study, we demonstrate that the R1‐nj marker could result in a high proportion of false positives among the haploids identified, besides being ineffective in germplasm with natural anthocyanin expression in pericarp tissue. To address these limitations, we developed haploid inducer lines with triple anthocyanin color markers, including the expression of anthocyanin coloration in the seedling roots and leaf sheaths, in addition to the Navajo marker on the seed. Although these inducers show acceptable haploid induction rates ranging from 8.6 to 10.2%, they exhibited relatively poor agronomic performance compared with tropicalized haploid inducers within tropical environments. The addition of the red root marker more accurately identified haploids among the germinating seedlings, including four tropical inbred lines and eight breeding populations that showed complete inhibition of R1‐nj. We also demonstrate that the red root marker can be used for haploid identification in germplasm with natural anthocyanin expression in the pericarp. A survey of 546 tropical inbreds and 244 landraces showed that anthocyanin accumulation in the roots of germinating seedlings is very rare compared with anthocyanin accumulation in the seed and leaf sheath tissues. As a result, the red root marker can serve as a highly complementary marker to R1‐nj to enable effective identification of haploids within a wide range of tropical maize germplasm.

In Vivo Haploid Induction in Maize: Comparison of Different Testing Regimes for Measuring Haploid Induction Rates

The doubled‐haploid (DH) technology, based on in vivo induction of haploid seeds by pollen from inducer genotypes, has become routine in maize (Zea mays L.) breeding. De novo and maintenance breeding of inducers requires measuring the haploid induction rate (HIR) of numerous plants. We compared five testing regimes in combination with three inducers to find the best method for estimating HIR by discrimination of haploid (H) and crossing (C) seeds or plants with low false discovery rates (FDRs) and false negative rates (FNRs) in two experiments. Three testing regimes were based on tester genotypes LGT, GLT, and UFT equipped with the liguleless (lg2), glossy (gl), and herbicide‐resistance (uf) gene, respectively. One testing regime used classification of H and C seeds in crosses with a high‐oil tester (HOT). Embryo coloration as a result of the R1‐nj marker gene was used as another testing regime. Flow cytometry analysis or phenotyping of adult plants served as gold standard classification (GSC). With HOT, H and C seeds were not clearly distinguishable because of considerable overlapping of their oil content (OC) distribution. Estimates of HIR from LGT and UFT generally agreed well with HIR from GSC and showed acceptable FDR and FNR. The HIR determined with GLT showed a high Matthew correlation with the GSC but lower estimates as a result of higher FNR. The HIR estimated with the R1‐nj marker deviated considerably from HIR of GSC and had high FDR and FNR. Thus, we recommend LGT for determining HIR, but testing regimes UFT and HOT should be revisited.