Progress and Opportunities of Doubled Haploid Production

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In addition, replacement of the N-terminal tail of A. Attempts by N-terminal tail editing have been performed in diverse species [ 49 ], and HI has been successfully achieved in maize [ 48 ], tomato and rice [ 50 ].

Progress and Opportunities of Doubled Haploid Production

Although the HI rates are relatively low 0. These results imply that modification of the HFD may create a haploid inducer. This strategy was proven to be feasible by recent studies from two labs [ 37 , 38 ]. Karimi-Ashtiyani et al. Meanwhile, Kuppu et al. They first identified conserved amino acids from the HFDs of A. Unlike the tailswap-CENH3 haploid inducer, all of these point-mutant plants were viable and fully fertile when self-pollinated, without any obvious phenotypic effects [ 37 , 38 ]. Studies in Arabidopsis have also shown functional conservation across a broad evolutionary distance, as the exogenous CENH3s from a wide range of species can target A.

A consistent phenomenon in the centromere-mediated HI is that the haploid plants contain only WT chromosomes. Recently, a centromere-size model provided a plausible explanation for the elimination of chromosomes with defective CENH3 [ 56 ]. Briefly, defective CENH3 is expected to have a lower efficiency in recruiting some key kinetochore proteins, leading to the smaller centromere sizes in outcrosses.

These smaller centromeres have less accurate metaphase plate alignment, causing inefficient recruitment of centromeric factors and leading to a high level of stochastic chromosome loss.

Progress and Opportunities of Doubled Haploid Production - Semantic Scholar

The findings that tailswap- and point-mutant CENH3s lead to reduced centromere loading [ 37 , 47 ] provide indirect evidence to support the above assumption. Furthermore, when an inducer is self-pollinated, all centromeres are on an equal defective level and thus are competitively retained. Thus, the centromere-size model can also explain the observation that homozygous tailswap-CENH3 and other inducer lines are self-fertile and do not produce haploids.

In contrast, small centromeres that can expand to match the average size of other centromeres will be retained in the progeny, producing aneuploid or diploid individuals. This centromere-size model is derived from the concept that each centromere has a similar size within a species [ 57 , 58 ]. Zhang and Dawe [ 57 ] examined ten species of grass and observed similar immunoassay signal intensities from each centromere that suggested uniform centromere size within a species.

Studies through chromatin immunoprecipitation followed by sequencing ChIP-seq using an anti-CENH3 antibody confirmed the uniform size of centromeres within a species [ 59 , 60 , 61 , 62 , 63 ]. However, electron microscopy and immunoassay observation analyses revealed that centromere sizes across species are different and correlate with genome size and total centromere volume [ 57 , 64 ]. Therefore, the similar size of maize and oat centromeres observed in oat-maize addition lines indicates that the alien maize centromeres have adopted a similar size to oat centromeres.

Thus, the retention of the maize chromosomes in oat-maize addition lines can be attributed to expansions in centromere size allowing them to match the average size of the other centromeres, whereas failure of the smaller centromeres from one parent to adopt the required size may cause genome elimination in a hybrid. In fact, during wide hybridizations between a large-genome species such as oat, barley and wheat and a small-genome species such as maize, pearl millet, adlay millet, perennial rye grass or sorghum , chromosomes from the small-genome parent are often eliminated in early embryogenesis [ 65 , 66 , 67 ].

Thus, these findings not only support the centromere-size model but also suggest that the centromere-size model can explain the HI caused by wide hybridization. According to the centromere-size model, this lack of HI suggests that all the centromeres should have a uniform size in those hybrid plants. A reasonable explanation is that WT CENH3 may competitively load to all centromeres, thus generating a uniform centromere size and producing diploid progenies. In contrast, if the WT and mutant CENH3s were constrained to load into the corresponding WT- and mutant-derived centromeres, it was expected to see haploid progeny, because the centromeres loaded with mutant CENH3 would be smaller, and they would be eliminated, producing haploid or aneuploid offspring.

In oat-maize addition lines, the additional maize centromeres are consistently incorporated by oat CENH3 [ 68 ] and adopt a similar size to the oat centromeres [ 59 ]. In addition, HTR12 A. All these findings support our hypothesis.

Progress and Opportunities of Doubled Haploid Production

The model of centromere-mediated chromosome elimination. WT CENH3 may competitively load to all centromeres, thus generating a uniform centromere size and producing diploid progenies. In contrast, haploid inducer-derived centromeres may load with defective CENH3, generating smaller or defective centromeres, and they would be eliminated, producing haploid or aneuploid offspring. These defective mutant CENH3s continuously target the inducer-derived centromeres and produce smaller centromeres due to their inefficient centromere loading procedure.

These smaller centromeres are noncompetitive and are later eliminated when encountering WT centromeres.


However, mutant CENH3 loading must occur in a short time frame early in the zygotic mitoses. Alternatively, the WT CENH3 would competitively load into some or all of the mutant-derived centromeres, producing normal large-sized centromeres and generating normal diploid or aneuploid plants. Clearly, our revised model is consistent with and based on the centromere-size model, but it also presents a plausible explanation for both HI incapability despite co-ocurrence of WT and mutant CENH3s [ 32 , 37 ] and CENH3 loading in the centromeres of alien species in stable hybrids [ 68 , 69 ].

This hypothesis is testable through further centromere loading assays involving WT and mutant CENH3s during the early stage of zygotic development. In maize, the line Stock6 has been widely used to generate haploids for inbred line production.

Bibliographic Information

The original Stock6 inducer line was created by Coe in with HI rates of 2. Several quantitative trait loci QTLs associated with HI have been identified, indicating that the HI in Stock6 is controlled by multiple genes [ 75 , 76 , 77 , 78 , 79 ]. Among those candidate QTLs, qhir1 , which is located in bin 1. Researchers from Syngenta conducted the first trial to isolate the HI gene from the qhir1 region [ 80 ]. After fine mapping, they narrowed the QTL to a 0. This 4-bp insertion in the fourth exon leads to a frame shift, causing 20 altered amino acids and a premature transcription termination that truncates the protein by 29 amino acids.

After functional verification gene knockdown and knockout , they verified that MTL was responsible for HI. Notably, large-scale sequence comparisons revealed that the 4-bp insertion was a distinct feature restricted to only the inducer lines, confirming its critical role in HI [ 81 ]. In addition, the absence of such a 4-bp insertion from the ancestral variety, teosinte, suggested that this mutation occurred after maize domestication [ 81 ].

Another interesting finding is that some gene-editing and knockdown events increased HI rates, suggesting that it is possible to create a high-HI-rate inducer by modifying MTL. MTL encodes a patatin-like phospholipase and is expressed specifically in maize pollen [ 80 , 81 , 82 ]. Phospholipase alterations are associated with delayed pollen germination and pollen tube growth [ 83 ], which explain the pleiotropic phenotypes accompanying with maize HI capability [ 73 , 80 ]. However, the mechanisms of HI that contribute to single fertilization [ 84 ] or postzygotic genome elimination [ 85 , 86 ] remain unclear.

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A subcellular localization assay revealed that the MTL protein targets the sperm cell plasma membrane in both maize and Arabidopsis. In contrast, the truncated protein from the inducer line PK6 was absent from the plasma membrane [ 80 ]. Further in silico analysis of the MTL protein demonstrated that the absence of a lipid anchor site from the truncated protein C-terminal may contribute to its mislocalization. Collectively, these results indicate that membrane integrity or mechanisms involving signaling precursor triggered by defective MTL might be responsible for HI capacity [ 82 ]. Recently, Li et al. The proportion of viable inducer pollen is lower than that of inbred lines, suggesting that defective pollen development may be attributed to HI. After single-nucleus sequencing of mature pollen, they demonstrated that chromosome fragmentation that begins around pollen mitosis may be the cue causing pollen abortion and HI.

Given that the haploid is an abortive kernel with a haploid embryo, only those that initiate fragmentation after the 2nd mitosis can generate haploid nonaborted kernels [ 87 ]. However, detailed embryo development studies still need to be performed, because the possibility of single fertilization fragmented sperm not fusing with the egg cannot be excluded [ 87 ].

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However, the results of Li et al. As HI is potentially caused by pollen sperm fragmentation after the 2nd mitosis, increasing the frequency of chromosome fragmentation initiated after the 2nd mitosis could potentially improve the HI rate. Alternatively, decreasing early chromosome fragmentation, before the 2nd mitosis, would be an efficient strategy to improve kernel viability. The CENH3 function is highly conserved across eukaryotes. Moreover, the centromere is composed of multiple proteins, which means that other key centromere proteins, in addition to CENH3, are also expected to be potential targets for HI inducer development.

To date, in addition to the model plant Arabidopsis , haploids have been successfully induced in maize [ 48 ] using the tailswap strategy.

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However, it is perplexing that the CENH3 tailswap plants in maize did not induce haploids as frequently as in Arabidopsis [ 32 ]. Due to the enigmatic structure of centromeres [ 88 ], functional divergence of CENH3s in other plants compared with the dicot model plant Arabidopsis is possible. It therefore implied that the HI ability might be correlated with the plant fertility. In fact, the plant with the highest HI rate was also the plant with the strongest knockdown of CENH3 in maize [ 48 ] supporting the above hypothesis.

Therefore, it will be interesting to explore the correlation of inducer sterility and HI rate. Moreover, the usage of WT parent is another factor that should be considered since the HI rates can be nearly two-fold change when different WT lines were crossed with the same inducer in Arabidopsis [ 32 ]. Theoretically, given that the HI rated are still low using the tailswap strategy in crops, the methods of HFD modification, CENH3 replacement and genetic editing other kinetochore components provide alternative choices for the development of inducers with high HI rates.

Importantly, point mutations can be achieved by nontransgenic chemical mutagenesis, and then, the results can be directly used in crop breeding. The centromere-size model provides a basis to explain HI resulting from both wide hybridization and CENH3-mediated systems. Based on the centromere-size model, crosses between inducer and larger-centromere lines should produce haploids with high efficiency. Thus, this model will allow us to predict HI capacity before crossing experiments, which will greatly improve the application of HI to crop breeding.

The HI capacity of maize Stock6 is caused by at least seven potential genes or QTLs [ 75 , 79 ], indicating that a potentially enhanced HI inducer could be created by combining multiple HI-related genes. In addition, alteration of the MTL gene renders it possible to extend this tool to other species, at least in Poaceae [ 80 , 90 ], because of the high-level conservation of MTL orthologs Fig. Its successful application in rice is very encouraging [ 91 ]. However, the presence of numerous co-orthologs [ 82 ] and non-pollen-specific expression in dicots [ 92 ] Fig.

Thus, a careful function s assessment of orthologs will be required before experimental trials in other species. Phylogenetic analysis of MTL gene orthologs in plant. The orthologs which showed expression pattern in reproductive organ, were highlighted by asterisk. Copy numbers of MTL orthologs were indicated in the brackets. Notably, the CENH3-mediated inducer can induce haploids as both female and male.