Haploid Induction Rate Research Articles

Genomic estimated selection criteria and parental contributions in parent selection increase genetic gain of maternal haploid inducers in maize.

Parental combinations determined by genomic estimated usefulness and parental contributions of the lines in bridging population can enhance the genetic gain of traits of interest in maternal haploid inducer breeding. Parent selection in crosses aligns well with the quantitative trait performance in the progenies. We herein take advantage of estimated genetic values (EGV) and usefulness criteria (UC) of bi-parental combinations by genomic prediction (GP) to compare the empirical performance of doubled haploid inducer (DHI) progenies of eight elite inducers crosses in a half-diallel. We used parental contribution and discovery of superiors from elite-by-historical bridging populations to enhance genetic gain for long-term selection. In this empirical study, the narrow-sense heritabilities of four traits of interest (Days to flowering, DTF; haploid induction rate, HIR; plant height, PHT; Total primary branch length, PBL) in DHI population were 0.81, 0.71, 0.45 and 0.46, respectively. The genomic estimated EGV_Mid/Mean and EGV/UC_Inferior was significantly correlated with the sample mean of progenies and inferiors in four traits in the breeding and bridging population. EGV/UC_Superior were significantly correlated with the mean of superiors in DTF, PHT, and PBL in breeding and bridging populations. The genomic estimated parent contributions in DH progenies of bridging populations enabled discovery of favorable genome region from historical inducers to improve the genetic gain of HIR for long-term selection.

Efficient induction and rapid identification of haploid grains in tetraploid wheat by editing genes TtMTL and pyramiding anthocyanin markers.

Doubled haploid (DH) technology provides an effective way to generate homozygous genetic and breeding materials over a short period of time. We produced three types of homozygous TtMTL gene-edited mutants (mtl-a, mtl-b, and mtl-ab) by CRISPR/Cas9 in durum wheat. PCR restriction enzymes and sequencing confirmed that the editing efficiency was up to 53.5%. The seed-setting rates of the three types of mutants ranged from 20% to 60%. Abnormal grain phenotypes of kernel, embryo, and both embryo and endosperm abortions were observed in the progenies of the mutants. The average frequency of embryo-less grains was 25.3%. Chromosome counting, guard cell length, and flow cytometry confirmed that the haploid induction rate was in the range of 3%-21% in the cross- and self-pollinated progenies of the mtl mutants (mtl-a and mtl-ab). Furthermore, we co-transformed two vectors, pCRISPR/Cas9-MTL and pBD68-(ZmR + ZmC1), into durum wheat, to pyramide Ttmtl-edited mutations and embryo-specifically expressed anthocyanin markers, and developed a homozygous durum haploid inducer with purple embryo (DHIPE). Using DHIPE as the male parent to be crossed with the wild-type Kronos, the grains with white embryos were identified as haploid, while the grains with purple embryos were diploid. These findings will promote the breeding of new tetraploid wheat varieties.

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.

Development of doubled haploid inducer lines facilitates selection of superior haploid inducers in maize.

Haploid inducers are key components of doubled haploid (DH) technology in maize. Robust agronomic performance and better haploid induction ability of inducers are persistently sought through genetic improvement. We herein developed C1-I inducers enabling large-scale in vivo haploid induction of inducers and discovered superior inducers from the DH progenies. The haploid induction rate (HIR) of C1-I inducers ranged between 5.8% and 12.0%. Overall, the success rate of DH production was 13% on average across the 23 different inducer crosses. The anthesis-silking interval and days to flowering of inducer F1s are significantly correlated with the success rate of DH production (r = -0.48 and 0.47, respectively). Transgressive segregants in DH inducers (DHIs) were found for the traits (days to flowering, HIR, plant height, and total primary branch length). Moreover, the best HIR in DHIs exceeded 23%. Parental genome contributions to DHI progenies ranged between 0.40 and 0.55, respectively, in 25 and 75 percentage quantiles, and the mean and median were 0.48. The allele frequency of the four traits from inducer parents to DHI progenies did not correspond with the phenotypic difference between superior and inferior individuals in the DH populations by genome-wide Fst analysis. This study demonstrated that the recombinant DHIs can be accessed on a large scale and used as materials to facilitate the genetic improvement of maternal haploid inducers by in vivo DH technology.

Elite, transformable haploid inducers in maize

The introduction of alleles into commercial crop breeding pipelines is both time consuming and costly. Two technologies that are disrupting traditional breeding processes are doubled haploid (DH) breeding and genome editing (GE). Recently, these techniques were combined into a GE trait delivery system called HI-Edit (Haploid Inducer-Edit) [1]. In HI-Edit, the pollen of a haploid inducer line is reprogrammed to deliver GE traits to any variety, obviating recurrent selection. For HI-Edit to operate at scale, an efficient transformable HI line is needed, but most maize varieties are recalcitrant to transformation, and haploid inducers are especially difficult to transform given their aberrant reproductive behaviors. Leveraging marker assisted selection and a three-tiered testing scheme, we report the development of new Iodent and Stiff Stalk maize germplasm that are transformable, have high haploid induction rates, and exhibit a robust, genetically-dominant anthocyanin native trait that may be used for rapid haploid identification. We show that transformation of these elite “HI-Edit” lines is enhanced using the BABYBOOM and WUSCHEL morphogenetic factors. Finally, we evaluate the HI-Edit performance of one of the lines against both Stiff Stalk and non-Stiff Stalk testers. The strategy and results of this study should facilitate the development of commercially scalable HI-Edit systems in diverse crops.

Haploids can be induced in knockout mutants of OsPLA1, but not OsDMP3 or OsDMP6, in rice

Doubled haploid (DH) technology is an important tool in crop breeding because it can significantly accelerate the breeding process. ZmPLA1/MATL/NLD and ZmDMP are two key genes controlling haploid induction (HI) in maize, exhibiting a synergistic effect. However, it is unknown whether knock out of ZmDMP orthologs can stimulate HI in rice. In this study, a ZmPLA1 ortholog (OsPLA1) and two ZmDMP orthologs (OsDMP3 and OsDMP6) were identified in rice. All three genes encode plasma membrane-localized proteins and were highly expressed in mature anthers. Knockout of OsPLA1 in both Minghui 63 and Nipponbare resulted in reduced seed setting rate (SSR) and caused HI. The osdmp3, osdmp6 and the double mutant failed to trigger HI independently, nor increased the haploid induction rate (HIR) when combined with ospla1. Repeated pollinations operations of QX654A with the ospla1 mutant significantly improve SSR, while reducing HIR. RNA-seq profiling of mature ospla1 mutant anthers indicated that a large number of differentially expressed genes (DEGs) were enriched in redox homeostasis and lipid metabolic GO terms, plant hormone signal transduction, and MAPK signaling pathways. These findings provide important insights towards construction of an efficient DH breeding technology and study of the molecular mechanism of HI in rice.

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.

New liguleless (lg2) maize stocks: Genetic resources for leaf architectural and haploid induction rate assessment studies

AbstractLiguleless mutants produce defective ligules and auricles and, consequently, have more upright leaves than their ligulate counterparts, making them useful genetic material for plant architectural studies. Besides, owing to the recessive nature and amenability of the liguleless trait to phenotyping at the seedling stage, liguleless mutants are popularly used for ‘proof‐of‐concept’ demonstration and assessment of haploid induction rate (HIR) of haploid inducer lines (HILs) in maize. The commonly used liguleless testers in maize are of temperate origin and are challenging to use and maintain under tropical/sub‐tropical conditions. In the present study, liguleless lines (V 601, V 602, V 603 and V 604) derived from crosses between agronomically superior locally adapted tropical ligulate lines (V 407 and CM 152) and liguleless donors of temperate origin (PDH‐3 and PDH‐8) were evaluated for different agro‐morphological traits. Liguleless line V 602 was also used as a tester to assess the HIR of haploid inducer line EC937890 (CIM2GTAILP2). The results showed a mean HIR of 12.42% for EC937890, consistent with the HIR reported in other studies, thus demonstrating the efficacy of V 602 as a tester for determining HIR. The agronomically superior liguleless maize lines reported in this study will, therefore, be a valuable resource for leaf architectural studies, assessment of HIR of candidate HILs and maintenance of high HIR in the HILs presently in wide use in the doubled haploid (DH) programmes. Additionally, these genetic stocks carry the liguleless trait in genetic backgrounds with known heterotic affinity with early maturity Indian public maize germplasm and, therefore, can be used directly as parents in hybrid development programmes.

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.

The Effect of Donors Used in In vivo Maternal Haploid Technique on Haploid Induction Rate

Maize is an important plant grown to obtain grain and silage, and is used in human and animal nutrition. In conventional maize breeding studies, inbred line development studies are carried out for at least 7 years if a single generation is obtained in a year, while it is possible to develop 100% homozygous lines in a short period of 2 years with the in vivo maternal haploid technique. The in vivo maternal haploid technique is widely used in advanced maize breeding programs. The choice of donor or source material to be used for haploid induction depends on the purpose of the breeding program. Generally, breeders use F1 or F2 populations as source material for haploid induction. In this study; 30 F1 genotypes and their F2s were crossed with the inducer line. The putative haploid seed was identified by considering the R1-nj color marker, and the haploid induction rate was determined. The effect of the generations of the donor genotypes on the haploid induction rate was compared by performing an independent sample test, and the haploid induction rate obtained from the F1 donors was found to be higher than the haploid induction rate of the F2 donors. It was determined that there was a change in the haploid induction rate as the genotypes changed within the F1 and F2 donor groups.

Stratified haploid identification system through the R1-nj kernel and reduced seedling vigor in tropical maize germplasm

Abstract. Thawarorit A, Dermail A, Lertrat K, Chankaew S, Suriharn K. 2023. Stratified haploid identification system through the R1-nj kernel and reduced seedling vigor in tropical maize germplasm. Biodiversitas 24: 4262-4268. Haploid identification through the R1-nj marker is commonly applied in in-vivo doubled haploid technology in maize, although misclassification issues often occur. We explored the innate properties of maternal haploids at the seedling stage to verify true haploids and to reduce false positives. There are many questions about whether reduced seedling vigor is effective for haploid verification in tropical maize backgrounds. This study aimed to evaluate the haploid induction rate of Stock6-derived F3 population inducers and to investigate the effectiveness of reduced seedling vigor at the V2/V3stage as an alternative marker to verify putative haploids. Field trials and haploid induction were conducted in the rainy season of 2021 and the dry season of 2021/22 in Khon Kaen, Thailand. Two tropical source germplasm: P789 and S7328, were included as donor females. Haploids were identified through the R1-nj marker and reduced seedling vigor. Significant reduction of haploid frequency up to 2.5% on average between the two identification methods, indicating that a considerable number of false positives could be found using reduced seedling vigor. Inducer population K11 was promising since it performed a stable ability to produce haploids over donors and seasons and had a low misclassification rate. Further breeding strategies and haploid selection schemes are discussed.

Realized genetic gains via recurrent selection in a tropical maize haploid inducer population and optimizing simultaneous selection for the next cycles

AbstractPlant breeders widely use recurrent selection schemes to increase the frequency of favorable alleles for quantitative traits in a population. Although simultaneous selection is complex because it involves several traits combined with selection cycles, the use of selection indexes (SI) is applied to increase the chance of success of the breeding program. Despite many indices are available in the literature, simulations can help breeders to determine which selection index can be better adjusted considering the selection goals, the intensity, and the genetic correlation among traits over breeding cycles. In this context, we estimated the realized genetic gains in a tropical maize haploid inducer population after two cycles of recurrent selection, using external testers and optimizing the simultaneous selection for this breeding population in the long term via stochastic simulations. Furthermore, we proposed a new approach to optimize the initial weights by applying Smith‐Hazel method to maximize the genetic gains for all traits in a balanced way. Overall, the estimated gains in real induction rate were about 63% per cycle, improving the population performance from 0.8% to 2.8%. Moreover, our results confirm that the traditional Smith‐Hazel approach outperformed other methods for long‐term response to selection. Finally, recurrent selection with external testers may be a suitable method to improve the haploid induction rate in tropical maize populations.

Development of Specific Molecular and Phenotypic Marker-Based Haploid Inducers in Rice

Doubled haploid (DH) technology is an efficient strategy for producing completely homozygous lines for breeding programs. Mutations in the MATRILINEAL (MTL) phospholipase trigger intraspecific haploid induction in cereals. Although an in vivo haploid induction system based on OsMTL-edited plants has been established in rice (Oryza sativa), DH technology is still limited by other factors, such as haploid identification, which is one of the essential steps required for DH technology. In the study, we addressed this technical challenge by integrating specific molecular and phenotypic markers into rice haploid inducers. We first generated large fragment insertion or deletion mutations within the OsMTL gene and designed a pair of primers flanking the mutational sites to be used as the specific and universal molecular markers between wild-type and Osmtl plants. Next, we screened for hairy leaf as a single dominant trait and integrated it into specific molecular marker-based haploid inducers using the cross and self-cross method. When crossing cytoplasmic male sterile lines with these haploid inducers, we utilized the specific InDel marker and hairy leaf phenotypic marker to identify putative haploids (or double haploids). These putative haploids were further confirmed through ploidy and phenotypic analysis, demonstrating the high efficiency of haploid identification using these markers. The haploid induction rate (HIR) of the developed specific molecular and phenotypic marker-based haploid inducers ranged from 3.7% to 12.5%. We have achieved successful integration of distinct molecular and phenotypic markers into rice haploid inducers. Our advanced marker-based system has significantly enhanced the accuracy of haploid identification, thereby expediting the adoption of DH technology in rice breeding.

Protocol optimization and assessment of genotypic response for inbred line development through doubled haploid production in maize

BackgroundDoubled haploid technology offers the fastest route of inbred line development by rapidly fixing the desirable combinations in a single year. However, the differential response of haploid induction to genetic background of maternal lines accompanied with low induction rate and high mortality rate due to artificial chromosomal doubling of haploid seedlings creates hindrance in doubled haploid production on a commercial scale under tropical conditions. To speed up the hybrid breeding programme in sub-tropical maize, efforts are reported here to optimize the protocol for efficient production of fixed lines using haploid inducers. The second-generation haploid inducers i.e. CIM2GTAILs obtained from CIMMYT, Mexico were used for haploid induction in 13 F1s of diverse backgrounds. For standardization of chromosomal doubling protocol, various concentrations of colchicine and two seedling growth stages were used to determine the extent of chromosomal doubling and survival rate of doubled haploid plants.ResultsA high mean haploid induction rate is obtained from CIM2GTAIL P2 (10%) as compared to CIM2GTAIL P1 (7.46%). Out of four treatments, CIMMYT reported protocol of chromosome doubling in tropical maize comprising combination of 0.07% colchicine and 0.1% DMSO at V2 stage is highly effective for acquiring doubled haploid plants in sub-tropical adapted maize with high survival rate of 52.7%. However, increasing the colchicine concentration from 0.07 to 0.1% led to high mortality rate.ConclusionAccording to the findings, the haploid induction rate, survival rate and overall success rate varied depending upon the genotype of the inducer and the source population along with the concentrations of chemical used. The optimized protocol developed using CIMMYT haploid inducer CIM2GTAIL P2 for efficient doubled haploid production will not only fasten the breeding programme but will also reduce the production cost of doubled haploid with great efficiency in sub-tropical maize.

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.

DNA-free genome editing for ZmPLA1 gene via targeting immature embryos in tropical maize

ABSTRACT Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal (ZmPLA1) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger’s sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.

Production of double haploid watermelon via maternal haploid induction.

Production of double haploid watermelon via maternal haploid induction.

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.

Development of homozygous maize lines differing in oil and zein content using in-vivo maternal haploid technique

This study was carried out in order to develop homozygous lines that differ in grain quality from the local maize population. Twelve different local maize landraces were used as donor materials in the study. These populations were subjected to induction crossing under greenhouse conditions in September 2020 with the ADAIL-I inducer line. In September 2021, a total of twelve haploid lines were grown in greenhouse conditions. Some plant traits and some grain quality characteristics were examined. Zein protein fractions were also analyzed with SDS-PAGE analysis. The haploid induction rates (HIR) of donor materials ranged from 6.08% to 11.71%. The average HIR value of the ADAIL-I inducer line was determined as 8.20%. The average value of plant height of developed lines varied between 123 cm and 250 cm; first ear height between 54 cm and 120 cm; stem diameter between 0.7 cm and 1.2 cm; crude oil content between 2.39%, and 7.54%; oleic acid content between 15.34% and %30.98; linoleic acid content between 50.4% and 67.8%; protein content between 6.75% and 13.74%; and zein content between 4.58%, and 5.04%. Some the homozygous lines carry the desired protein bands in terms of zein fractions.

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.