Haploid Induction Research Articles

Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis)

Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis)

Transcriptome Analysis of Potential Regulatory Genes under Chemical Doubling in Maize Haploids

Maize is one of the most successful crops with regard to the utilization of heterosis. The haploid induction technique is one of the fastest methods to obtain pure maize material at the present stage. However, the molecular mechanism of haploid doubling is rarely reported. In this study, we treated B73 and ZNC442 haploid young shoots with colchicine for 0 h, 6.2 h, and 10 h, and analyzed the differentially expressed genes (DEGs). We found that colchicine treatment for 6.2 h and 10 h compared to 0 h resulted in a total of 4868 co-DEGs. GO enrichment analysis and KEGG metabolic pathway analysis found significantly enriched 282 GO terms and 31 significantly pathways, respectively. Additionally, The GO term and KEGG pathway genes of spindle, cytoskeleton, microtubules and nuclear division were selected for analysis, and three candidate genes were screened by taking intersections. Zm00001d033112, Zm00001d010525, and Zm00001d043386 were annotated as kinesin-associated protein 13, kinesin-like protein KIN-10C, and kinesin light-chain LC6, respectively. The real-time fluorescence quantification (RT-PCR) results revealed that Zm00001d033112, Zm00001d010525, and Zm00001d043386 had the same trends as RNA-seq. Interestingly, Zm00001d033112 is homologous gene AT3G20150 in Arabidopsis, which was involved in the regulation of chromosome movement and mitotic spindle assembly. Our study suggests that kinesin genes may play an important role in doubling chromosomes, thus providing valuable information for future studies on the molecular mechanisms of chromosome doubling in maize.

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.

One-step creation of CMS lines using a BoCENH3-based haploid induction system in Brassica crop

Heterosis utilization in a large proportion of crops depends on the use of cytoplasmic male sterility (CMS) tools, requiring the development of homozygous fertile lines and CMS lines1. Although doubled haploid (DH) technology has been developed for several crops to rapidly generate fertile lines2,3, CMS lines are generally created by multiple rounds of backcrossing, which is time consuming and expensive4. Here we describe a method for generating both homozygous fertile and CMS lines through in vivo paternal haploid induction (HI). We generated in-frame deletion and restored frameshift mutants of BoCENH3 in Brassica oleracea using the CRISPR/Cas9 system. The mutants induced paternal haploids by outcrossing. We subsequently generated HI lines with CMS cytoplasm, which enabled the generation of homozygous CMS lines in one step. The BoCENH3-based HI system provides a new DH technology to accelerate breeding in Brassica and other crops.

Cyto-swapping in maize by haploid induction with a cenh3 mutant.

Maize mutants of the centromeric histone H3 (CENP-A/CENH3) gene can form haploids that inherit only chromosomes of the pollinating parent but the cytoplasm from the female parent. We developed CENH3 haploid inducers carrying a dominant anthocyanin colour marker for efficient haploid identification and harbouring cytoplasmic male sterile cytoplasm, a type of cytoplasm that results in male sterility useful for efficient hybrid seed production. The resulting cytoplasmic male sterility cyto-swapping method provides a faster and cheaper way to convert commercial lines to cytoplasmic male sterile compared to conventional trait introgression.

Genome-Wide Identification and Expression Analysis of the DMP and MTL Genes in Sweetpotato (Ipomoea batatas L.).

Sweetpotato (Ipomoea batatas L.) is a strategic crop with both economic and energy value. However, improving sweetpotato varieties through traditional breeding approaches can be a time-consuming and labor-intensive process due to the complex genetic nature of sweetpotato as a hexaploid species (2n = 6x = 90). Double haploid (DH) breeding, based on in vivo haploid induction, provides a new approach for rapid breeding of crops. The success of haploid induction can be achieved by manipulating specific genes. Two of the most critical genes, DMP (DUF679 membrane proteins) and MTL (MATRILINEAL), have been shown to induce haploid production in several species. Here, we identified and characterized DMP and MTL genes in sweetpotato using gene family analysis. In this study, we identified 5 IbDMPs and 25 IbpPLAs. IbDMP5 and IbPLAIIs (IbPLAIIκ, IbPLAIIλ, and IbPLAIIμ) were identified as potential haploid induction (HI) genes in sweetpotato. These results provide valuable information for the identification and potential function of HI genes in sweetpotato and provide ideas for the breeding of DH lines.

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.

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

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

Haploid induction through ovary culture in cucumber

Haploid induction through ovary culture in cucumber

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.

Potential gene editing targets for developing haploid inducer stocks in rice and wheat with high haploid induction frequency.

The online version contains supplementary material available at 10.1007/s13205-023-03857-9.

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.

Chromosome-level changes and genome elimination by manipulation of CENH3 in carrot (Daucus carota).

Hybrid cultivars are valuable in many crop species due to their high yield, uniformity, and other desirable traits. Doubled haploids, which have two identical sets of chromosomes, are valuable for hybrid breeding because they can be produced in one generation, in comparison to the multigenerational process typically used to produce inbred parents for hybrid production. One method to produce haploid plants is manipulation of centromeric histone H3 (CENH3). This method of producing haploids has so far been successful in Arabidopsis, maize (Zea mays), and wheat (Triticum aestivum). Here we describe modification of CENH3 in carrot (Daucus carota) to test for the ability of these modifications to induce uniparental genome elimination, which is the basis for haploid induction. Base editing was used to make cenh3 mutant plants with amino acid substitutions in the region of CENH3 encoding the histone fold domain. These cenh3 mutant plants were then outcrossed with CENH3 wild-type plants. Using PCR-based genotyping assays, we identified two candidates for genome elimination. One candidate was classified as a putative aneuploid plant in which chromosome 7 is in a single copy state. The other candidate was characterized as a putative tetraploid that was likely haploid during its genesis. Our results suggest that this putative tetraploid inherited all of its chromosomes from the CENH3 wild-type parent and that the genome of the cenh3 mutant plant was lost. This study provides evidence that modification of CENH3 in carrot has the potential to induce genome elimination and ploidy changes in carrot.

Mutating the maternal haploid inducer gene CsDMP in cucumber produces haploids in planta.

Mutation of DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN in cucumber induces in vivo maternal haploids and suggests prospects for cucurbit breeding.

Engineering synthetic apomixis in different hybrid rice varieties using the Fix strategy

Hybrid rice breeding has significantly increased the yield and stability of rice production. However, due to trait segregation in F2 progeny seeds, the F1 hybrid seeds must be renewed annually. Apomixis is a form of reproduction leading to clonal seeds, which has a revolutionary potential in fixing heterosis and realizing selfretention of hybrid rice seeds. Previously, we successfully engineered synthetic apomixis in rice by combining Mitosis instead of Meiosis (MiMe) with haploid induction (mutation of MATRILINEAL), a strategy known as Fix for Fixation of hybrids. However, only one hybrid rice variety has been tested for the Fix synthetic apomixis system. In this study, we expanded the application of the Fix strategy and achieved synthetic apomixis in multiple hybrid rice varieties. We observed significant variations in seed setting rate across diverse genetic backgrounds and witnessed a remarkable tenfold increase in clonal seed efficiency. Our findings will provide valuable insights into the application of the Fix strategy for synthetic apomixis in hybrid rice.

Use of nurse endosperm for the culture of haploid embryos produced by durum wheat x maize crosses

The use of doubled-haploids in plant breeding programs enables accelerating the release of new varieties adapted to climate change. The durum wheat x maize crosses technique is a method of choice for producing durum wheat haploid plants. The haploid embryos produced by this method develop without albumen and their survival is ensured by post-pollination hormonal treatments. In this study, nine post-pollination treatments with 2,4-Dichlorophenoxyacetic acid (2,4-D), Picloram and Dicamba at the concentrations of 10, 50 and 100 mg.L-1 were applied to 7 durum wheat genotypes. The effects of genotype and post-pollination treatment on durum wheat haploid embryos produced by durum wheat x maize crosses and the use of the endosperm nursing technique for haploid plantlets regeneration were investigated. The haploid induction parameters varied with the durum wheat genotypes as well as the post-pollination treatments. The phenomenon of polyembryony resulting from durum wheat x maize crosses is reported for the first time in this article. The durum wheat genotypes showed different abilities to produce monoembryo and polyembryos. The post-pollination treatments with 2,4-D (10 mg.L-1) and Picloram (10 and 100 mg.L-1) gave a higher embryo formation frequency than the treatments with Dicamba. The embryo conversion to plantlet was greatly improved, especially in recalcitrant genotypes using the durum wheat endosperm as supplemental nourishment in combination with the Gamborg B5 regeneration medium.
 Keywords: Durum wheat; Haploid embryo; Maize; Nurse Endosperm Technique; Polyembryony

CENH3 mediated haploid induction: application and future perspectives in crop plants

CENH3 mediated haploid induction: application and future perspectives in crop plants

Paternal chromosome elimination of inducer triggers induction of double haploids in Brassica napus

A synthetic octoploid rapeseed, Y3380, induces maternal doubled haploids when used as a pollen donor to pollinate plant. However, the mechanism underlying doubled haploid formation remains elusive. We speculated that double haploid induction occurs as the inducer line’s chromosomes pass to the maternal egg cell, and the zygote is formed through fertilization. In the process of zygotic mitosis, the paternal chromosome is specifically eliminated. Part of the paternal gene might have infiltrated the maternal genome through homologous exchange during the elimination process. Then, the zygote haploid genome doubles (early haploid doubling, EH phenomenon), and the doubled zygote continues to develop into a complete embryo, finally forming doubled haploid offspring. To test our hypothesis, in the current study, the octoploid Y3380 line was back bred with the 4122-cp4-EPSPS exogenous gene used as a marker into hexaploid Y3380-cp4-EPSPS as paternal material to pollinate three different maternal materials. The fertilization process of crossing between the inducer line and the maternal parent was observed 48 h after pollination, and the fertilization rate reached 97.92% and 98.72%. After 12 d of pollination, the presence of cp4-EPSPS in the embryo was detected by in situ PCR, and at 13–23 d after pollination, the probability of F1 embryos containing cp4-EPSPS gene was up to 97.27%, but then declined gradually to 0% at 23–33 d. At the same time, the expression of cp4-EPSPS was observed by immunofluorescence in the 3rd to 29th day embryo. As the embryos developed, cp4-EPSPS marker genes were constantly lost, accompanied by embryonic death. After 30 d, the presence of cp4-EPSPS was not detected in surviving embryos. Meanwhile, SNP detection of induced offspring confirmed the existence of double haploids, further indicating that the induction process was caused by the loss of specificity of the paternal chromosome. The tetraploid-induced offspring showed infiltration of the induced line gene loci, with heterozygosity and homozygosity. Results indicated that the induced line chromosomes were eliminated during embryonic development, and the maternal haploid chromosomes were synchronously doubled in the embryo. These findings support our hypothesis and lay a theoretical foundation for further localization or cloning of functional genes involved in double haploid induction in rapeseed.