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

Kelliher, Timothy; Starr, Dakota; Su, Xinxin; Tang, Guozhu; Chen, Zhongying; Carter, Jared; Wittich, P.; Dong, Shiqi; Green, Julie; Burch, Erin; McCuiston, Jamie; Gu, Weining; Sun, Yuejin; Strebe, Tim; Roberts, James K.; Bate, Nic J; Que, Qiudeng

doi:10.1038/s41587-019-0038-x

Cited by 238 publications

(199 citation statements)

References 36 publications

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“…ZmDMP have been linked to pollen-induced production of haploid embryos and other seed 109 defects, which are likely associated with aberrant events at fertilization [17-20] or soon after [21]. 110…”

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confidence: 99%

Highly expressed maize pollen genes display coordinated expression with neighboring transposable elements and contribute to pollen fitness

Warman

Panda

Vejlupkova

et al. 2019

Preprint

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AbstractIn flowering plants, the haploid male gametophyte (pollen) is essential for sperm delivery, double fertilization, and subsequent initiation of seed development. Pollen also undergoes dynamic epigenetic regulation of expression from transposable elements (TEs), but how this process interacts with gene regulation and function is not clearly understood. To identify components of these processes, we quantified transcript levels in four male reproductive stages of maize (tassel primordia, microspores, mature pollen, and isolated sperm cells) via RNA-seq. We found that, in contrast to Arabidopsis TE expression in pollen, TE transcripts in maize accumulate as early as the microspore stage and are also present in sperm cells. Intriguingly, coordinated expression was observed between the most highly expressed protein-coding genes and neighboring TEs, specifically in both mature pollen and sperm cells. To test the hypothesis that such elevated expression correlates with functional relevance, we measured the fitness cost (male-specific transmission defect) of GFP-tagged exon insertion mutations in over 50 genes highly expressed in pollen vegetative cell, sperm cell, or seedling (as a sporophytic control). Insertions in genes highly expressed only in seedling or primarily in sperm cells (with one exception) exhibited no difference from the expected 1:1 transmission ratio. In contrast, insertions in over 20% of vegetative cell genes were associated with significant reductions in fitness, showing a positive correlation of transcript level with non-Mendelian segregation. The gamete expressed2 (gex2) gene was the single sperm cell gene associated with reduced transmission when mutant (<35% for two independent insertions), and also triggered seed defects when crossed as a male, supporting a role for gex2 in double fertilization. Overall, our study demonstrates a developmentally programmed and coordinated transcriptional activation of TEs and genes, and further identifies maize pollen as a model in which transcriptomic data have predictive value for quantitative phenotypes.Author SummaryIn flowering plants, pollen is essential for delivering sperm cells to the egg and central cell for double fertilization, initiating the process of seed development. In plants with abundant pollen like maize, this process can be highly competitive. In an added layer of complexity, growing evidence indicates expression of transposable elements (TEs) is more dynamic in pollen than in other plant tissues. How these elements impact pollen function and gene regulation is not well understood. We used transcriptional profiling to generate a framework for both detailed analysis of TE expression and quantitative assessment of gene function during maize pollen development. TEs are expressed early and persist, many showing coordinate activation with highly-expressed neighboring genes in the pollen vegetative cell and sperm cells. Measuring fitness costs for a set of over 50 mutations indicates a correlation between elevated transcript level and gene function in the vegetative cell. Finally, we establish a role in fertilization for the gamete expressed2 (gex2) gene, identified based on its specific expression in sperm cells. These results highlight maize pollen as a powerful model for investigating the developmental interplay of TEs and genes, as well as for measuring fitness contributions of specific genes.

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confidence: 99%

Highly expressed maize pollen genes display coordinated expression with neighboring transposable elements and contribute to pollen fitness

Warman

Panda

Vejlupkova

et al. 2019

Preprint

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“…Recent studies that utilized the combination of CRISPR/Cas9-mediated gene editing and haploid induction revealed that in vivo haploid induction can enable gene editing in maternal haploids in any background, without mixing the paternal genome (commonly referred to as “HI-edit” or “IMGE”) (Kelliher et al, 2019; Wang et al, 2019). This is promising for future genetic improvement of crops.…”

Section: Resultsmentioning

confidence: 99%

“…Genetic analysis on the QTLs contributing HIR revealed that qhir8 can improve haploid induction efficiency by 3- to 5- fold (Liu et al, 2015), this is a promising result for improving DH breeding efficiency in crop species other than maize. In addition to producing homozygous DH lines, in vivo haploid induction in maize has also been used for gene editing without introducing the genome of the male parent, which is meaningful for genome editing in different genetic backgrounds (Kelliher et al, 2019; Wang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…In vivo haploid induction system has been extended from maize to rice, while both are diploid crop, little is known whether it can be applied to polyploidy plants. Wheat is a very important hexaploidy crop, to extend maize in vivo haploid induction to wheat would create a novel protocol in speeding up wheat breeding and providing a potential method for gene editing in different genetic backgrounds (Kelliher et al, 2019; Wang et al, 2019). Furthermore, it would also give an example for exploring the haploid production and editing system in other polyploidy crops.…”

Section: Introductionmentioning

confidence: 99%

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Extension of thein vivohaploid induction system from maize to wheat

Liu

Zhong

et al. 2019

Preprint

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3Doubled haploid breeding technology has been one of the most important techniques for 1 4 accelerating crop breeding. In compare to in vivo haploid induction in maize, which is efficient 1 5 and background independent, wheat haploid production by interspecific hybridization pollinated 1 6 with maize is influenced by genetic background and requires rescue of young embryos. Here, we 1 7 analyzed the homologues of maize haploid induction gene MTL/ZmPLA1/NLD in several crop 1 8 species systematically, the homologues are highly conserved in sorghum, millet and wheat etc. 1 9Since wheat is a very important polyploidy crop, as a proof of concept, we demonstrated that the 2 0 in vivo haploid induction method could be extended from diploid maize to hexaploid wheat by 2 1 knocking out the wheat homologues (TaPLAs). Result showed that double knock-out mutation 2 2 could trigger wheat haploid induction at ~ 2%-3%, accompanied by 30% -60% seed setting rate. 3The performance of haploid wheat individual showed shorter plant, narrower leaves and male 2 4 sterile. Our results also revealed that knockout of TaPLA-A and TaPLA-D do not affect pollen 2 5 viability. This study not only confirmed the function of the induction gene and explored a new 2 6approach for haploid production in wheat, but also provided an example that the in vivo haploid 2 7 induction could be applied in more crop species with different ploidy levels. Furthermore, by 2 8 combining with gene editing, it would be a fast and powerful platform for traits improvement in 2 9 polyploidy crops breeding. 3 0

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“…Obviously, an efficient regeneration protocol from calli into plants would eventually allow screening and validating genetically modified plants by advanced allele mining approaches previously used in TILLING projects (Nida et al, 2016). Moreover, new Cas9 delivery systems, which have recently been developed like nanomaterials delivery(Demirer et al, 2019) and Haploid-Inducer Mediated Genome Editing (IMGE)(Wang et al, 2019)(Kelliher et al, 2019) can be utilized to enhance Cas9 delivery system and allow better penetration. On top of that, HDR process is less frequent in somatic cells then NHEJ, therefore it is requiring a procedure that allows increasing HDR over NHEJ pathway e.g.…”

Section: Discussionmentioning

confidence: 99%

RECAS9: Recombining wild species introgression via mitotic gene editing in barley

Lazar

Prusty

Bishara

et al. 2020

Preprint

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Genetic loci underlying variation in traits with agronomic importance or genetic risk factors in human diseases have been identified by linkage analysis and genome-wide association studies.However, narrowing down the mapping to the individual causal genes and variations within these is much more challenging, and so is the ability to break linkage drag between beneficial and unfavourable loci in crop breeding. We developed RECAS9 as a transgene-free approach for precisely targeting recombination events by delivering CRISPR/Cas9 ribonucleotide protein (RNP) complex into heterozygous mitotic cells for the barley (Hordeum vulgare) Heat3.1 locus. A wild species (H. spontaneum) introgression in this region carries the agronomical unfavourable tough rachis phenotype (non-brittle) allele linked with a circadian clock accelerating QTL near GIGANTEA gene. We delivered RNP, which was targeted between two single nucleotide polymorphism (SNPs), to mitotic calli cells by particle bombardment. We estimated recombination events by next generation sequencing (NGS) and droplet digital PCR (ddPCR). While NGS analysis grieved from confounding effects of PCR recombination, ddPCR analysis allowed us to associate RNP treatment on heterozygous individuals with significant increase of homologous directed repair (HDR) between cultivated and wild alleles, with recombination rate ranging between zero to 57%. These results show for the first time in plants a directed and transgene free mitotic recombination driven by Cas9 RNP, and provide a starting point for precise breeding and fine scale mapping of beneficial alleles from crop wild relatives. Amunugama R, Fishel R (2012) Homologous recombination in eukaryotes. Prog. Mol. Biol. Transl. Sci. Elsevier B.V., pp 155-206 Barcelo P, Lazzeri PA (1995) Transformation of cereals by microprojectile bombardment of immature inflorescence and scutellum tissues. Methods Mol Biol. Bloom JS, Boocock J, Treusch S, Sadhu MJ, Day L, Oates-Barker H, Kruglyak L (2019) Rare variants contribute disproportionately to quantitative trait variation in yeast. Elife 8: 8-10 Brachi B, Faure N, Horton M, Flahauw E, Vazquez A, Nordborg M, Bergelson J, Cuguen J, Roux F (2010) Linkage and association mapping of Arabidopsis thaliana flowering time in nature. PLoS Genet 6: 40 Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. Edwards KD, Lynn JR, Gyula P, Nagy F, Millar AJ (2005) Natural allelic variation in the temperature-compensation mechanisms of the Arabidopsis thaliana circadian clock. Genetics 170: 387-400 Fernandes JB, Séguéla-Arnaud M, Larchevêque C, Lloyd AH, Mercier R (2018) Unleashing meiotic crossovers in hybrid plants. Proc Natl Acad Sci U S A 115: 2431-2436 Fernández-Calleja M, Casas AM, Pérez-Torres A, Gracia MP, Igartua E (2019) Rachis brittleness in a hybrid-parent barley (Hordeum vulgare) breeding germplasm with different combinations at the non-brittle...

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

Cited by 238 publications

References 36 publications

Highly expressed maize pollen genes display coordinated expression with neighboring transposable elements and contribute to pollen fitness

Highly expressed maize pollen genes display coordinated expression with neighboring transposable elements and contribute to pollen fitness

Extension of thein vivohaploid induction system from maize to wheat

RECAS9: Recombining wild species introgression via mitotic gene editing in barley

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