Haploid Induction In Maize Research Articles

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

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.

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.

Comparative Analysis of the <i>HAP2</i>/<i>GCS1</i>, <i>GEX2</i> Genes Expression in Maize Lines of Saratov Selection

The haploinduction phenomenon have a scientific and practical aspects for development the effective haploid-inducing maize line and diploid homozygous lines creation. The article discusses the relationship between the maize haploid–induction and the violation of the gamete interactions, in particular for GEX2-HAP2/GCS1-mediated adhesion and fusion of gamete membranes in Zarodishevii Marker Saratovskii Purpurnii (ZMSP) maize lines. Using real-time RT-PCR method, it was found that the GEX2, HAP2/GCS1 genes are expressed in sperm and ovules, but the maize haploid-inducing ability does not correlate with their expression. It was shown that the ZMSP haploid-inducing line have 27 SNP, one 9-bp insertion, 2-bp SNP and the corresponding 10 amino acid substitutions with two insertions in the GEX2 protein in comparing with reference B73 line. It was found that HAP2/GCS1, as a conservative protein for membrane fusion has only one amino acid substitution in ZMSP in comparing with B73 maize and Brown Marker maize lines.

Influential factors and transcriptome analyses of immature diploid embryo anthocyanin accumulation in maize

BackgroundAnthocyanins are widely applied as a marker for haploid identification after haploid induction in maize. However, the factors affecting anthocyanin biosynthesis in immature embryos and the genes regulating this process remain unclear.ResultsIn this study, we analyzed the influence of genetic background of the male and female parents, embryo age and light exposure on anthocyanin accumulation in embryos. The results showed that light exposure was the most crucial factor enhancing the pigmentation of immature embryos. The identification accuracy of haploid embryos reached 96.4% after light exposure, but was only 11.0% following dark treatment. The total anthocyanin content was 7-fold higher in immature embryos cultured for 24 h under light conditions compared to embryos cultured in the dark. Transcriptome analysis revealed that the differentially expressed genes between immature embryos cultured for 24 h in dark and light chambers were significantly enriched in the pathways of flavonoid, flavone, flavonol and anthocyanin biosynthesis. Among the genes involved in anthocyanin biosynthesis, five up-regulated genes were identified: F3H, DFR, ANS, F3′H and the MYB transcription factor-encoding gene C1. The expression patterns of 14 selected genes were confirmed using quantitative reverse transcription-polymerase chain reaction.ConclusionLight is the most important factor facilitating anthocyanin accumulation in immature embryos. After 24 h of exposure to light, the expression levels of the structural genes F3H, DFR, ANS, F3′H and transcription factor gene C1 were significantly up-regulated. This study provides new insight into the factors and key genes regulating anthocyanin biosynthesis in immature embryos, and supports improved efficiency of immature haploid embryo selection during doubled haploid breeding of maize.

Variation in anthocyanin pigmentation by R1-navajo gene, development and validation of breeder-friendly markers specific to C1-Inhibitor locus for in-vivo haploid production in maize.

In-vivo maternal haploids serve as the basis of doubled haploid (DH) breeding in maize. R1-navajo (R1-nj) gene governing anthocyanin colouration in the endosperm and embryo is widely used to identify haploid seeds. However, the expression of R1-nj depends on genetic-background of source-germplasm used for deriving DH-lines. Further, presence of C1-Inhibitor (C1-I) gene suppresses the expression of R1-nj, thus makes the selection of haploids difficult. In the present study, 178 subtropically-adapted maize inbreds were crossed with two R1-nj donors 'that do not have haploid induction genes'. Of these, 76.4% inbreds developed purple colour in endosperm, while 23.6% did not show any colouration. In case of scutellum, 62.9% inbreds possessed colour and 37.1% were colourless. The anthocyanin intensity varied greatly, with 19.66% and 42.98% inbreds displayed the least intensity, while 16.85% and 0.84% inbreds showed the highest intensity in endosperm and scutellum, respectively. Two C1-I specific breeder-friendly markers (MGU-CI-InDel8 and MGU-C1-SNP1) covering (i) 8 bp InDel and (ii) A to G SNP, respectively,were developed. MGU-CI-InDel8 and MGU-C1-SNP1 markers predicted presence of C1-I allele with 92.9% and 84.7% effectiveness, respectively. However, when both markers were considered together, they provided 100% effectiveness. These markers of C1-I gene would help in saving valuable resources and time during haploid induction in maize. The information generated here assume great significance in DH breeding of maize.

A reactive oxygen species burst causes haploid induction in maize

Haploid induction (HI) is an important tool in crop breeding. Phospholipase A1 (ZmPLA1)/NOT LIKE DAD (NLD)/MATRILINEAL (MTL) is a key gene controlling HI in maize; however, the underlying molecular mechanism remains unclear. In this study, to dissect why loss of ZmPLA1 function could mediate HI we performed a comprehensive multiple omics analysis of zmpla1 mutant anthers by integrating transcriptome, metabolome, quantitative proteome, and protein modification data. Functional classes of significantly enriched or differentially abundant molecular entities were found to be associated with the oxidative stress response, suggesting that a reactive oxygen species (ROS) burst plays a critical role in HI. In support of this, we further discovered that a simple chemical treatment of pollen with ROS reagents could lead to HI. Moreover, we identified ZmPOD65, which encodes a sperm-specific peroxidase, as a new gene controlling HI. Taken together, our study revealed a likely mechanism of HI, discovered a new gene controlling HI, and created a new method for HI in maize, indicating the importance of ROS balance in maintaining normal reproduction and providing a potential route to accelerate crop 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.

Genetic Approaches to Enhance Multiple Stress Tolerance in Maize.

The multiple-stress effects on plant physiology and gene expression are being intensively studied lately, primarily in model plants such as Arabidopsis, where the effects of six stressors have simultaneously been documented. In maize, double and triple stress responses are obtaining more attention, such as simultaneous drought and heat or heavy metal exposure, or drought in combination with insect and fungal infestation. To keep up with these challenges, maize natural variation and genetic engineering are exploited. On one hand, quantitative trait loci (QTL) associated with multiple-stress tolerance are being identified by molecular breeding and genome-wide association studies (GWAS), which then could be utilized for future breeding programs of more resilient maize varieties. On the other hand, transgenic approaches in maize have already resulted in the creation of many commercial double or triple stress resistant varieties, predominantly weed-tolerant/insect-resistant and, additionally, also drought-resistant varieties. It is expected that first generation gene-editing techniques, as well as recently developed base and prime editing applications, in combination with the routine haploid induction in maize, will pave the way to pyramiding more stress tolerant alleles in elite lines/varieties on time.

MYO, a Candidate Gene for Haploid Induction in Maize Causes Male Sterility.

Doubled haploid technology is highly successful in maize breeding programs and is contingent on the ability of maize inducers to efficiently produce haploids. Knowledge of the genes involved in haploid induction is important for not only developing better maize inducers, but also to create inducers in other crops. The main quantitative trait loci involved in maize haploid induction are qhir1 and qhir8. The gene underlying qhir1 has been discovered and validated by independent research groups. Prior to initiation of this study, the gene associated with qhir8 had yet to be recognized. Therefore, this research focused on characterizing positional candidate genes underlying qhir8. Pursuing this goal, a strong candidate for qhir8, GRMZM2G435294 (MYO), was silenced by RNAi. Analysis of crosses with these heterozygous RNAi-transgenic lines for haploid induction rate revealed that the silencing of MYO significantly enhanced haploid induction rate by an average of 0.6% in the presence of qhir1. Recently, GRMZM2G465053 (ZmDMP) was identified by map-based gene isolation and shown to be responsible for qhir8. While our results suggest that MYO may contribute to haploid induction rate, results were inconsistent and only showing minor increases in haploid induction rate compared to ZmDMP. Instead, reciprocal crosses clearly revealed that the silencing of MYO causes male sterility.

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.

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.

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.

One-step genome editing of elite crop germplasm during haploid induction.

Genome editing using CRISPR-Cas9 works efficiently in plant cells1, but delivery of genome-editing machinery into the vast majority of crop varieties is not possible using established methods2. We co-opted the aberrant reproductive process of haploid induction (HI)3-6 to induce edits in nascent seeds of diverse monocot and dicot species. Our method, named HI-Edit, enables direct genomic modification of commercial crop varieties. HI-Edit was tested in field and sweet corn using a native haploid-inducer line4 and extended to dicots using an engineered CENH3 HI system7. We also recovered edited wheat embryos using Cas9 delivered by maize pollen. Our data indicate that a transient hybrid state precedes uniparental chromosome elimination in maize HI. Edited haploid plants lack both the haploid-inducer parental DNA and the editing machinery. Therefore, edited plants could be used in trait testing and directly integrated into commercial variety development.

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.

Hetero-fertilization together with failed egg–sperm cell fusion supports single fertilization involved in in vivo haploid induction in maize

In vivo doubled-haploid technology is widely applied in commercial maize breeding programs because of its time-saving and cost-reducing features. The production of maize haploids primarily depends on the use of Stock6-derived haploid inducer lines. Although the gene underlying haploid induction, MTL/ZmPLA1/NLD, was cloned recently, the mechanism of haploid induction is still unknown. Hetero-fertilization can occur via a single fertilization, which provides a means to investigate single-fertilization events by studying the hetero-fertilization phenomenon. In this study, we found that the hetero-fertilization rate increased significantly when female maize lines were first individually crossed with pollen from the inducer CAU5 in dual-pollination experiments 4 h before a second pollination with common lines. We also examined embryogenesis during haploid induction by confocal laser-scanning microscopy and observed single-fertilized ovules, indicating that single fertilization occurred during haploid induction. We therefore postulate that both single fertilization and chromosome elimination contribute to haploid induction in maize. We also propose a scheme for the formation of hetero-fertilized and haploid kernels. Our results provide an efficient approach to identify hetero-fertilized kernels for research on interactions between embryo and endosperm.

Single nucleus sequencing reveals spermatid chromosome fragmentation as a possible cause of maize haploid induction

Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize. However, the underlying mechanism of haploid induction (HI) is unclear. We develop a method to isolate three nuclei from a pollen grain and four microspores from a tetrad for whole-genome sequencing. A high rate of aneuploidy is observed at the three-nucleus stage (6/22 pollens) rather than at the tetrad stage (1/72 microspores) in one HI line CAU5. Frequent aneuploidy is also observed in another two inducer lines, but not in two regular lines, which implies that HI may be associated with pollen aneuploidy. We further sequence the individual embryos and endosperms of 88 maize kernels crossing between regular and inducer lines. Genome-wide elimination of the CAU5-derived chromosome is identified in eight of 81 embryos. Together, these results suggest that continuous chromosome fragmentation occurring post meiosis in the gametophyte may cause haploidy of the embryo.

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.