Maternal Haploid Induction Research Articles

Doubled Haploid Technology: Opportunities and Challenges for the Rapid Generation of Maize Homozygous Lines.

Maize is used for multiple purposes, including food, feed, and energy production, and since transitioning to hybrid cultivars at around 1930, maize yield has significantly increased. This is largely due to hybrid vigor, which refers to the superior performance of the progeny from two unrelated inbred parents. Consequently, nearly all maize cultivars grown in the United States are hybrids. Hybrid breeding programs comprise two essential components; namely, inbred line development and hybrid production. Traditionally, developing inbred lines takes a long time, requiring six to 10 generations of self-pollination. The doubled haploid (DH) technology, however, accelerates this process, enabling the derivation of fully homozygous lines within two generations. DH technology is applicable in several crop species and has been most successful in maize due to in vivo maternal haploid induction. Here, we review the origins of the DH technology, and discuss advantages and challenges of the technology as well as applications of DH lines.

Doubled Haploid Technology: Generation of Doubled Haploid Maize Lines Using Haploid Inducers.

Doubled haploid (DH) technology allows for the development of completely homozygous lines from heterozygous plants in only two generations. This approach has been widely adopted in maize breeding programs, as it expedites the generation of inbred lines compared to traditional methods. The DH approach is based on the use of maize genotypes that have the ability to induce haploid seeds when used as the pollen parent. The most common method for producing maize haploid plants for the generation of DH lines is in vivo maternal haploid induction. The process involves pollination with a haploid inducer maize line to generate haploid seeds. Then, haploids are screened for and identified (typically via the expression of a particular marker gene), germinated, treated with an exogenous doubling agent to induce genome duplication, and transplanted to the field. Following successful self-pollination, seeds harvested from the ear represent fully homozygous lines. The seed set at this stage, however, is often low, necessitating one or two additional rounds of self-pollination to increase the number of fully homozygous inbred lines. Here, we describe a protocol for the generation of maize DH lines using maternal haploid-inducing maize lines. We outline the steps for setting up the donor material, performing induction crosses, selecting haploids based on two different marker alleles, treating seedlings with colchicine to double the genome, transplanting the treated seedlings to the field, and self-pollinating the treated plants.

Haploid induction: an overview of parental factor manipulation during seed formation.

In plants, in vivo haploid induction has gained increasing attention for its significant potential applications in crop breeding and genetic research. This strategy reduces the chromosome number in progeny after fertilization, enabling the rapid production of homozygous plants through double haploidization, contrasting with traditional inbreeding over successive generations. Haploidy typically initiates at the onset of seed development, with several key genes identified as paternal or maternal factors that play critical roles during meiosis, fertilization, gamete communication, and chromosome integrity maintenance. The insights gained have led to the development of efficient haploid inducer lines. However, the molecular and genetic mechanisms underlying these factors vary considerably, making it challenging to create broadly applicable haploidy induction systems for plants. In this minireview, we summarize recent discoveries and advances in paternal and maternal haploid induction factors, examining their current understanding and functionalities to further develop efficient haploid inducer systems through the application of parental factor manipulation.

Allelic variation and haplotype diversity of Matrilineal (MTL) gene governing in vivo maternal haploid induction in maize

Allelic variation and haplotype diversity of Matrilineal (MTL) gene governing in vivo maternal haploid induction in maize

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.

Prediction of matrilineal specific patatin-like protein governing in-vivo maternal haploid induction in maize using support vector machine and di-peptide composition

The mutant matrilineal (mtl) gene encoding patatin-like phospholipase activity is involved in in-vivo maternal haploid induction in maize. Doubling of chromosomes in haploids by colchicine treatment leads to complete fixation of inbreds in just one generation compared to 6–7 generations of selfing. Thus, knowledge of patatin-like proteins in other crops assumes great significance for in-vivo haploid induction. So far, no online tool is available that can classify unknown proteins into patatin-like proteins. Here, we aimed to optimize a machine learning-based algorithm to predict the patatin-like phospholipase activity of unknown proteins. Four different kernels [radial basis function (RBF), sigmoid, polynomial, and linear] were used for building support vector machine (SVM) classifiers using six different sequence-based compositional features (AAC, DPC, GDPC, CTDC, CTDT, and GAAC). A total of 1170 protein sequences including both patatin-like (585 sequences) from various monocots, dicots, and microbes; and non-patatin-like proteins (585 sequences) from different subspecies of Zea mays were analyzed. RBF and polynomial kernels were quite promising in the prediction of patatin-like proteins. Among six sequence-based compositional features, di-peptide composition attained > 90% prediction accuracies using RBF and polynomial kernels. Using mutual information, most explaining dipeptides that contributed the highest to the prediction process were identified. The knowledge generated in this study can be utilized in other crops prior to the initiation of any experiment. The developed SVM model opened a new paradigm for scientists working in in-vivo haploid induction in commercial crops. This is the first report of machine learning of the identification of proteins with patatin-like activity.

In vivo maternal haploid induction system in cotton.

The ghdmp mutant of cotton, generated through the CRISPR system, exhibits a haploid induction rate of 1.06% in F1 progeny as the haploid inducer line.

Genetic basis of maize maternal haploid induction beyond MATRILINEAL and ZmDMP.

In maize, doubled haploid (DH) lines are created in vivo through crosses with maternal haploid inducers. Their induction ability, usually expressed as haploid induction rate (HIR), is known to be under polygenic control. Although two major genes (MTL and ZmDMP) affecting this trait were recently described, many others remain unknown. To identify them, we designed and performed a SNP based (~9007) genome-wide association study using a large and diverse panel of 159 maternal haploid inducers. Our analyses identified a major gene near MTL, which is present in all inducers and necessary to disrupt haploid induction. We also found a significant quantitative trait loci (QTL) on chromosome 10 using a case-control mapping approach, in which 793 noninducers were used as controls. This QTL harbors a kokopelli ortholog, whose role in maternal haploid induction was recently described in Arabidopsis. QTL with smaller effects were identified on six of the ten maize chromosomes, confirming the polygenic nature of this trait. These QTL could be incorporated into inducer breeding programs through marker-assisted selection approaches. Further improving HIR is important to reduce the cost of DH line production.

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.

Loss-of-function of gynoecium-expressed phospholipase pPLAIIγ triggers maternal haploid induction in Arabidopsis.

Production of in planta haploid embryos that inherit chromosomes from only one parent can greatly increase breeding efficiency via quickly generating homozygous plants, called doubled haploid. One of the main players of in planta haploid induction is a pollen-specific phospholipase A, which is able, when mutated, to induce invivo haploid induction in numerous monocots. However, no functional orthologous gene has been identified in dicots plants. Here, we show that loss-of-function of gynoecium-expressed phospholipase AII (pPLAIIγ) triggers maternal haploid plants in Arabidopsis, at an average rate of 1.07%. Reciprocal crosses demonstrate that haploid plants are triggered from the female side and not from the pollen, and the haploid plants carry the maternal genome. Promoter activity of pPLAIIγ shows enriched expression in the funiculus of flower development stages 13 and 18, and pPLAIIγ fused to yellow fluorescent protein reveals a plasma-membrane localization Interestingly, the polar localized PIN1 at the basal plasma membrane of the funiculus was all internalized in pplaIIγ mutants, suggesting that altered PIN1 localization in female organ could play a role in maternal haploid induction.

A simple and highly efficient strategy to induce both paternal and maternal haploids through temperature manipulation.

Haploid production by outcrossing with inducers is one of the key technologies to revolutionize breeding. A promising approach for developing haploid inducers is by manipulating centromere-specific histone H3 (CENH3/CENPA)1. GFP-tailswap, a CENH3-based inducer, induces paternal haploids at around 30% and maternal haploids at around 5% (ref. 2). However, male sterility of GFP-tailswap makes high-demand maternal haploid induction more challenging. Our study describes a simple and highly effective method for improving both directions of haploid production. Lower temperatures dramatically enhance pollen vigour but reduce haploid induction efficiency, while higher temperatures act oppositely. Importantly, the effects of temperatures on pollen vigour and on haploid induction efficiency are independent. These features enable us to easily induce maternal haploids at around 24.8% by using pollen of inducers grown at lower temperatures to pollinate target plants, followed by switching to high temperatures for haploid induction. Moreover, paternal haploid induction can be simplified and enhanced by growing the inducer at higher temperatures pre- and post-pollination. Our findings provide new clues for developing and using CENH3-based haploid inducers in crops.

In vivo maternal haploid induction based on genome editing of DMP in Brassica oleracea.

In vivo maternal haploid induction based on genome editing of DMP in Brassica oleracea.

Overexpression of Modified CENH3 in Maize Stock6-Derived Inducer Lines Can Effectively Improve Maternal Haploid Induction Rates.

Maize (Zea mays) doubled haploid (DH) breeding is a technology that can efficiently generate inbred lines with homozygous genetic backgrounds. Haploids are usually produced through in vivo induction by haploid inducer lines in maize. Currently, two approaches are usually used to develop maize haploid inducer lines. One is through the conventional breeding improvement based on the Stock6 germplasm, and this strategy is extensively used to induce maternal haploids in commercial maize DH breeding. Another strategy, newly developed but less utilized so far, is by genetic manipulation of the Centromeric Histone3 (CENH3) in regular lines. However, whether both approaches can be combined to develop the haploid inducer line with higher maternal haploid induction rate (HIR) has not been reported. In this study, we manipulated the Stock6-derived inducer lines by overexpressing maize CENH3 fused with different fluorescent protein tags and found that the engineered Stock6-derived lines showed an obvious increase in the maternal HIR. Intriguingly, this above strategy could be further improved by substituting a tail-altered CENH3 for the full-length CENH3 in the tagged expression cassette, resulting in a maternal HIR up to 16.3% that was increased by ~6.1% than Stock6-derived lines control. These results suggested that integration of two in vivo haploid induction methods could rapidly and effectively improve the maternal HIRs of maize Stock6-derived inducer lines, and provided a potentially feasible solution for further optimizing the process of commercial maize DH breeding.

Loss-of-function alleles of ZmPLD3 cause haploid induction in maize

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR–Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

Haploid Embryos: Being Like Mommy or Like Daddy?

In planta haploid embryo induction is a powerful plant breeding tool, but is limited to very few crops. Two recent studies by Wang et al. and Lv et al. report seed-based haploid systems that produce paternal haploid embryos by engineering CENTROMERIC HISTONE H3 (CENH3). Together with recent translation of maize maternal haploid induction ability into wheat and rice, this extends our collection of haploid inducer lines.

Maternal haploid induction in African marigold (Tagetes erecta L.) through in vitro culture of un-fertilized ovules

A protocol for production of haploids from two heterozygous male sterile lines of Tagetes erecta using un-pollinated ovules as explant was standardized. Various factors affecting the in vitro gynogenic response viz., growth regulators, ovule developmental stage, basal media, sucrose concentration, photoperiod and cold shock duration were assessed in Tagetes erecta. Direct induction of parthenogenic embryos occurred in cultivar ‘Local Orange’ when the un-pollinated ovules were cultured in EMS (MS medium enriched with coconut water, AgNO3, PVP etc.) medium supplemented with 2 mg l−1 BAP and 0.5 mg l−1 NAA or 2 mg l−1 BAP along with 2 mg l−1 2,4-D. The developmental stage of the flower buds was vital in embryo induction from the excised ovules. The ovules collected from half-open flower buds were more responsive to gynogenesis as compared to ovules collected from un-open and fully open flower buds. EMS basal medium was found best for gynogenesis over the other commercially available basal media. Cold pre-treatment of flower buds at 4 oC had no stimulatory effect and negatively affected the gynogenesis. The dark culture condition was imperative for direct parthenogenic embryo induction than 16 h light duration. The ploidy levels of 17 regenerants were determined by cytological studies, revealed 47.06% as diploids, 41.18% as haploids and 11.76% as mixoploids. These results were reconfirmed with flow cytometry analysis. The determination of ploidy level by counting the number of chloroplasts in stomatal guard cells of marigold was also established for rapid screening of haploids. The resultant haploids were successfully diploidised and are being utilized in the hybrid breeding programme at our institute. The standardized protocol for doubled haploids (DHs) production by un-pollinated ovule culture paved the way for Tagetes F1 hybrid breeding. This is the first successful report of in vitro gynogenesis in Tagetes spp for production of doubled haploids (DHs). The protocol for obtaining high frequency parthenogenic embryo induction from unpollinated ovules was standardized and validated for rapid production of homozygous lines.

Doubled haploids in maize: Development, deployment, and challenges

AbstractHaploids are naturally produced in maize (Zea mays L.) at different rates and can also be induced through different methods. Haploids are used to develop doubled haploids (DHs), which have many potential uses. The development of DH lines in maize involves haploid induction, haploid identification, chromosome doubling, and field sowing for self‐pollination of D0 plants. Different potential methods are used for haploid induction, in‐vivo maternal haploid induction being the most prevalent. Haploid induction is highly reliant on the unambiguous identification of haploids among a mixture of different ploidies. Haploid identification is facilitated by visual morphological markers, chromosome counting, flow cytometry, molecular markers, and many other approaches. Chromosome doubling may be achieved by spontaneous doubling or by induction with different antimicrotubular treatments. Among the potential uses of DH lines are the development of inbred lines, genomic selection (GS), quantitative trait loci (QTL) mapping, and unlocking new genetic variations. Although DH technology can potentially accelerate maize breeding, it still faces challenges at each step of DH line development. This article aims to highlight the importance, procedural steps, potential opportunities, and key challenges in DH line development in maize.

A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis

Doubled haploid technology using inducer lines carrying mutations in ZmPLA1/MTL/NLD and ZmDMP1-4 has revolutionized traditional maize breeding. ZmPLA1/MTL/NLD is conserved in monocots and has been used to extend the system from maize to other monocots5-7, but no functional orthologue has been identified in dicots, while ZmDMP-like genes exist in both monocots and dicots4,8,9. Here, we report that loss-of-function mutations in the Arabidopsis thaliana ZmDMP-like genes AtDMP8 and AtDMP9 induce maternal haploids, with an average haploid induction rate of 2.1 ± 1.1%. In addition, to facilitate haploid seed identification in dicots, we established an efficient FAST-Red fluorescent marker-based haploid identification system that enables the identification of haploid seeds with >90% accuracy. These results show that mutations in DMP genes also trigger haploid induction in dicots. The conserved expression patterns and amino acid sequences of ZmDMP-like genes in dicots suggest that DMP mutations could be used to develop in vivo haploid induction systems in dicots.

Development of doubled haploid maize lines by using in vivo haploid technique

The doubled haploid technology is now an integral component of modern maize breeding programs. In this study, the maternal haploid induction (gynogenesis) method was used to derive Doubled-Haploid (DH) lines from elite maize germplasm adapted to Turkey. Temperate haploid inducers (RWS, RWK-76, RWS x RWK-76 and WS14) were used as pollinators, and a set of 30 single-crossses (in FAO 650-700 maturity groups) were used as source materials. Putative haploid seeds were selected based on expression of R1-nj anthocyanin color marker. Highest haploid induction rate (20.42%) was recorded by using RWK-76 as inducer line, and the lowest haploid induction rate (17.75%) was obtained through WS14. Putative haploid seeds were germinated and seedlings were treated with 0.06% colchicine + 0.5% dimethylsulfoxide solution. Following transfer of seedlings into the field, 2178 D0 plants were obtained out of a total of 3012 treated haploids. Live plants were from 89% of 2178 seedlings which are planted to the field. Fertile plants were formed 57% of live plants. Inbreeding was succeeded in 31.23% of fertile plants and only 7.8% of inbreeding plants were able to produce seeds. Consequently, 27 doubled haploid lines were developed.

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 .