A haploid genetics toolbox for Arabidopsis thaliana

Abstract

Genetic analysis in haploids provides unconventional yet powerful advantages not available in diploid organisms. In Arabidopsis thaliana, haploids can be generated through seeds by crossing a wild-type strain to a transgenic strain with altered centromeres. Here we report the development of an improved haploid inducer (HI) strain, SeedGFP-HI, that aids selection of haploid seeds prior to germination. We also show that haploids can be used as a tool to accelerate a variety of genetic analyses, specifically pyramiding multiple mutant combinations, forward mutagenesis screens, scaling down a tetraploid to lower ploidy levels and swapping of nuclear and cytoplasmic genomes. Furthermore, the A. thaliana HI can be used to produce haploids from a related species A. suecica and generate homozygous mutant plants from strong maternal gametophyte lethal alleles, which is not possible via conventional diploid genetics. Taken together, our results demonstrate the utility and power of haploid genetics in A. thaliana.

ARTICLE

Received 23 Jun 2014 | Accepted 21 Sep 2014 | Published 31 Oct 2014

Maruthachalam Ravi1,2,*, Mohan Prem Anand Marimuthu1,3,*, Ek Han Tan1,3,*, Shamoni Maheshwari1,3,*, Isabelle M. Henry1,3, Brenda Marin-Rodriguez1, Guillaume Urtecho1, Jie Tan1, Kristina Thornhill1, Fan Zhu1, Aneesh Panoli1, Venkatesan Sundaresan1, Anne B. Britt1, Luca Comai1,3 & Simon W.L. Chan1,4

Genetic analysis in haploids provides unconventional yet powerful advantages not available in diploid organisms. In Arabidopsis thaliana, haploids can be generated through seeds by crossing a wild-type strain to a transgenic strain with altered centromeres. Here we report the development of an improved haploid inducer (HI) strain, SeedGFP-HI, that aids selection of haploid seeds prior to germination. We also show that haploids can be used as a tool to accelerate a variety of genetic analyses, specically pyramiding multiple mutant combinations, forward mutagenesis screens, scaling down a tetraploid to lower ploidy levels and swapping of nuclear and cytoplasmic genomes. Furthermore, the A. thaliana HI can be used to produce haploids from a related species A. suecica and generate homozygous mutant plants from strong maternal gametophyte lethal alleles, which is not possible via conventional diploid genetics. Taken together, our results demonstrate the utility and power of haploid genetics in A. thaliana.

DOI: 10.1038/ncomms6334

A haploid genetics toolbox for Arabidopsis thaliana

1 Department of Plant Biology, University of California, Davis, California 95616, USA. 2 School of Biology, Indian Institute of Science Education and Research, IISER-Thiruvananthapuram, CET Campus, Thiruvananthapuram 695016, India. 3 Genome Center, University of California, Davis, California 95616, USA.

4 Howard-Hughes Medical Institute and the Gordon and Betty Moore Foundation, University of California, Davis, California 95616, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.R. (email: mailto:[email protected]

Web End [email protected] ).

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Inbred (true-breeding) lines that are homozygous at virtually all loci enable both consistent production of superior hybrid plants as well as facile genetic analysis14. Traditional

inbreeding, however, requires six to eight generations of selng or sib-mating to generate inbred lines. For several crop species, haploid plants can be generated and subsequently doubled to produce true-breeding, entirely homozygous lines in two generations. Currently, the methods for haploid production vary depending on the species1,2. The most widely used method involves in vitro regeneration of plants from haploid cells or tissues, a procedure both tedious and expensive. Arabidopsis thaliana and many crop species have proven to be recalcitrant to in vitro haploid production5,6. In some species, wide crosses712 or intraspecic crosses to lines with specic genetic determinants13,14 result in in vivo haploid embryo because the chromosomes of the haploid inducer (HI) are lost during postzygotic mitotic divisions while the chromosomes of the non-inducer parent are retained15. In a majority of such species, the efcacy of in vivo haploid induction has been hindered by the need for tedious embryo rescue protocols that require in vitro cell culture. We have previously reported a breakthrough in in vivo haploid production: the generation of haploids through seed descent in A. thaliana by simple crossing of a wild-type (WT) strain to a transgenic strain expressing altered forms of CENH3, a centromere-specic histone16. Haploid production using altered CENH3 is particularly attractive and easy to adopt because viable haploid seeds are produced.

Haploid genetics has long provided an invaluable tool for basic genetic research in both prokaryote and eukaryote microbial systems1719 and has recently been extended to mammalian systems through the generation of haploid cell lines20,21. However, although Arabidopsis remains a premiere model system for basic research in higher plants, haploid genetics has been largely inaccessible to the Arabidopsis research community. In this paper, we describe an improved version of the green uorescent protein (GFP)-tailswap haploid induction method, demonstrating that haploid embryos are formed in association with chimeric endosperm and illustrate the utility of haploids in accelerating basic genetic analysis in A. thaliana. Our aim is to spur the utilization of haploid genetics across a wide array of genetic investigations and extend the potential of haploids as a genetic tool in plants.

Specically, we demonstrate that an A. thaliana HI can be used for the following: (1) swapping the cytoplasmic genome of one accession with the nuclear genome of another, (2) generating a series of lower euploids (3 , 2 , 1 ) from a tetraploid (4 )

parent, (3) rapid identication of recessive mutations in the M1 generation of a mutagenized population, (4) pyramiding of multiple mutant combinations and (5) generating plants that are homozygous for gametophyte lethal mutations. In addition, we also show that an A. thaliana HI line can be used to induce haploids from a sister species A. suecica through an interspecic cross, bypassing the need to develop an HI in the related species.

ResultsIdentication of haploid seed pre-germination. Among our collection of variant CENH3 strains16,22,23, the previously described GFP-tailswap strain16 is the optimal HI based on its haploid induction efciency and ease of use (see Methods, Fig. 1a, Supplementary Fig. 1). The HI can be used either as a male or a female parent in a cross to generate maternal and paternal haploids, respectively16. This HI line produces 2545% haploids when used as a maternal parent, whereas produces only 5% haploids if used as a paternal parent16. Furthermore, the HI parent produces few viable pollen16,23 and thus we prefer to use

HI as a female parent in crosses to produce haploids. F1 seed from HI WT crosses produce a mixture of hybrid diploids,

aneuploids and haploids in addition to a high frequency of aborted seeds16,24.

The current HI requires all the F1 progeny to be grown to identify haploids demanding more growth space and time. Haploids can be identied by phenotypic, genotypic and cytological methods16,24 (see Methods, Supplementary Fig. 2). A HI that allows the immediate identication of haploid seeds post harvest would allow selective planting of haploid progeny. For example, in Maize, haploid seeds can be selected using R1-nj anthocyanin marker system where hybrid diploid seeds show purple colour in both scutellum (embryo) and aleurone (endosperm) tissues, whereas haploid seeds display purple colouration only in the endosperm and not in the embryo25. Recently, an improved method based on oil content of haploid seeds has also been demonstrated for selection of maize haploids seeds26.

To facilitate selection of haploid seeds in Arabidopsis crosses, we introduced the GFP uorescent marker, expressed under the control of the seed storage protein 2S3 promoter (At2S3:GFP)27 into the HI strain, and named the resulting strain SeedGFP-HI. GFP signal in SeedGFP-HI is visible during late embryo development and persists in the embryo and endosperm27. We expected the diploid and aneuploid F1 seeds from a SeedGFP-HI cross to retain GFP uorescence and haploid seeds to lack any signal in the embryo and endosperm. Examination of well-developed F1 seeds failed to identify seeds completely devoid of uorescence (Supplementary Fig. 3). Instead, we observed a mixture of uniformly uorescent and mottled GFP uorescent seeds (Fig. 1b and Supplementary Fig. 3b and c). In mottled GFP seeds, the GFP uorescence was restricted to the endosperm and the embryo was devoid of GFP signal (Supplementary Fig. 3dj). Next, we scored for GFP uorescence in 35-day-old seedlings germinated from seeds that were either uniformly uorescent (n 50) or mottled (n 193). All the seedlings from the

uniformly uorescent seeds (Supplementary Fig. 3k,n) showed GFP signal throughout the seedling (Supplementary Fig. 3o,r) and the resulting adult plants were hybrid diploids (16%), hybrid aneuploids (32%) and self-pollinated diploids (52%) (Supplementary Table 1). In contrast, the mottled GFP seeds (Supplementary Fig. 3l) gave rise to seedlings devoid of GFP (Supplementary Fig. 3p), in agreement with our embryo dissection experiments (Supplementary Fig. 3i,j). When the seedlings were grown to maturity, 91% of these were haploids and the rest were aneuploids. These results indicate that preselection of mottled GFP seeds followed by selection of seedlings devoid of GFP signal increase the efciency of early haploid selection. In addition, the association of haploid embryos to mottled endosperm poses the interesting question of how genome elimination is coordinated between zygotic tissues.

Exchanging cytoplasmic and nuclear genomes. In Arabidopsis, like in most crop species, the genomes of cytoplasmic organelles are inherited uniparentally from the mother via the egg cell. Despite the importance of maternal cytoplasm in breeding2830, its effect on phenotype is not easy to ascertain because producing different nuclear versus cytoplasmic combinations normally requires hybridization followed by multiple generations of backcrossing. Genome elimination during haploid induction provides a rapid method for producing cytoplasm-swapped genotypes because the maternal cytoplasm is maintained while the entire maternal nuclear HI genome is eliminated16. For example, because HI originated from the Col-0 accession, all paternal haploids derived by crossing any accession to HI inherit

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Schematic for dihaploid production Improved haploid inducer Cytoplasm swap

Col

Haploid inducer (HI) Gamete

Wild type gamete

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&

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Propidium lodide fluorescence (a.u.)

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Figure 1 | Haploid toolbox for ArabidopsisPart I. (a) Cartoon of a (HI) cross depicting uniparental genome elimination. Any genotype of interest can be crossed as a male parent to the HI to induce haploid progeny. A green dot represents weak centromeres from the HI parent, while the black dot represents wild-type centromeres. The HI genome is eliminated after fertilization, producing seeds containing a haploid embryo. Note that haploids inherit the cytoplasm (coloured pink) from the HI parent. (b) SeedGFP-HI crosses produce two types of seeds: seeds with uniform GFP signal that develop as diploids or aneuploids and viable seeds showing mottled GFP uorescence (arrow heads) which predominantly give rise to haploid progeny. Scale bar, 500 mm.

(c) Gel electrophoresis image of PCR assays depicting cytoplasm transfer in haploids after a HI cross. Diploid WT Col-0 (lane 2) and Ler (lane 3) serves as controls for nuclear (top panel) and cytoplasmic (bottom panel) markers to show swapped cytoplasm in the haploids. A Ler haploid in Col-0 cytoplasm (lane 4) and a Col-0 haploid in Ler cytoplasm (lane 5) are shown here. (d) Images of 4 , 3 , 2 and 1 Wa-1 plant ploidy series are shown

with the crossing scheme used for obtaining each of the above plants. (e) Diagram depicting an interspecic uniparental genome elimination cross between A. thaliana HI (2n 10) and the allotetraploid A. suecica (2n 26). Flow cytometric conrmation of ploidy reduction, A. suecica haploid (2n 13)

with half the nuclear content (bottom panel) compared with a A. suecica diploid control (top panel). a.u., arbitrary units.

the Col-0 cytoplasm (Fig. 1c). To demonstrate rapid and convenient swapping of cytoplasms, we generated a HI containing Ler cytoplasm (see Methods) and pollinated this Ler-cytoplasmic HI with Col-0 WT. Marker analysis conrmed that this cross generated Col-0 haploids with Ler cytoplasm (Fig. 1c). This strategy can be employed to generate any desired combination of nuclear and cytoplasmic genome from the hundreds of natural accessions available for A. thaliana in only four generations (Supplementary Fig. 4).

Deconstructing a polyploid into a series of lesser euploids. Deconstruction of a polyploid into a series of lower euploids can be achieved easily through HI. Autotetraploids have useful genetic characteristics because they exhibit increased tolerance to DNA damage and salt stress31. However, tetraploidy challenges genetic studies as a result of increased genetic redundancy and complicated tetrasomic inheritance32. We had previously shown that fertile Wa-1 diploids can be created by crossing the natural

autotetraploid accession Wa-1 to HI (ref. 16). However, this diploid Wa-1 contains Col-0 cytoplasm inherited from maternal HI. Here we crossed the synthetic diploid Wa-1 as a pollen parent to natural tetraploid Wa-1 female to generate triploid Wa-1 plants containing Wa-1 cytoplasm. This triploid was subsequently crossed as a female to synthetic diploid Wa-1 male (Col-0 cytoplasm) to produce triploid, aneuploid and diploid progeny33, all with Wa-1 cytoplasm (Supplementary Fig. 5 shows the crossing strategy to maintain Wa-1 cytoplasm in the ploidy series). Crossing the diploid Wa-1 subsequently to HI generated haploid Wa-1 (Fig. 1d). This full ploidy series can be used to study the effects of the different ploidy states in Arabidopsis.

In addition, this strategy can be used to isolate mutant alleles from a polyploid background. Methods such as TILLING (Targeting Induced Local Lesions IN Genomes) can leverage the use a tetraploid population for mutagenesis because genetic redundancy allows for the recovery of greater mutation densities34. Further, dihaploid gametes should buffer against the

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lethal effects of severe alleles that are otherwise impossible to recover through haploid gametes. Thus, TILLING in a tetraploid increases the chance of nding a deleterious mutation in any gene of interest. However, a tetraploid TILLING plant carrying the desired mutant allele must be converted to diploid for easy genetic analysis. In fact, the cenh3-1 allele used to create the HI strain was originally isolated from a tetraploid TILLING population that conventionally required two transitional generations via a triploid bridge to generate a diploid CENH3/ cenh3-1 heterozygote22,33,34. With a HI strategy, we hypothesized that a tetraploid TILLING population can be converted to diploid by a single cross.

To demonstrate this, we identied three new knock-out (KO) alleles (nonsense mutations) in SOG1, a gene encoding a master regulator of DNA damage response35 from the tetraploid TILLING population34 and these heterozygotes were crossed onto HI diploid females. We recovered all three SOG1 KO alleles in the F1 progeny, from which ve individuals were selected for further analysis. Flow cytometry on the selfed F2 progeny of these ve lines conrmed the diploid nature of four progeny, one being aneuploid. Three putative diploids, each corresponding to a different sog1 KO allele, were further characterized via low coverage Illumina sequencing, and all three appeared to have balanced chromosome numbers as expected (Supplementary Fig. 6). While generating mutants in a tetraploid has the disadvantage of adding one generation to the production of diploid mutants, this caveat is largely offset by the benet of having to screen much smaller numbers of progeny, each carrying more mutations than individuals in diploid mutagenized populations34.

Interspecic genome elimination. Developing a HI strain in a new species demands time and effort. Therefore, it would be a signicant advantage if a HI line could be used for uniparental genome elimination not only within its own species but also in crosses to closely related species. This is particularly relevant in a genus such as Brassica, where multiple commercially relevant crop species can be intercrossed to one another. We found that interspecic genome elimination is possible with our HI strategy. As a proof of concept, we crossed the allopolyploid species A. suecica (2 26) as a pollen parent with the A. thaliana GFP-

tailswap HI strain (2 10). Out of 241 viable progeny, two

were identied as A. suecica (2 13) based on their mor

phology and sterile phenotype. This was conrmed by ow cytometry and chromosome dosage analysis36 (Fig. 1e and Supplementary Fig. 7). Thus, an HI strain developed in one species can be successfully employed to generate haploids in a closely related species via interspecic crosses. The pitfalls to this approach are low recovery of hybrid seeds due to interspecies barrier, differences in parental chromosome numbers and endosperm imbalance. Despite these caveats, at least in the case of A. thaliana A. suecica interspecic cross, this method

appears to be straightforward and yields haploids with a frequency of at least 1%.

Pyramiding of multiple mutant loci. Plant breeding and classical genetics often require introgression of selected alleles at multiple loci. Following hybridization, a heterozygous F1 is selfed and the F2 population is screened for the desired homozygous genotype. The cost and complexity of this task increases exponentially with the number of loci according to the expression 1/4n, where n represents the number of loci. For example, two loci result in 1/16 double homozygotes. In a haploid generation, the same frequency is calculated as 1/2n instead. For example, for ve loci, this produces a 32 advantage, and 1 out of 32 haploid progeny is

expected to inherit all 5 loci, as opposed to 1/1024 in the selfed diploid progeny (Fig. 2a). As a proof-of-principle, we crossed a triple heterozygous recessive mutant (YUC1/yuc1;YUC2/ yuc2;YUC8/yuc8) harbouring two independent marker constructs, DR5GFP and an egg cell EC:DsRed marker, both in hemizygous condition (DR5GFP/ ; EC:DsRed/ ) to HI to

generate haploids. Here the ve mutations are not independent because the three YUC genes are on the long arm of chromosome4. In addition, the positions of the two other markers are unknown. We obtained 2 individuals homozygous for all 5 loci out of 113 haploids (1/56.5). This is lower than expected, even if we consider all ve loci to be unlinked (1/32), perhaps because of lower viability of the quintuple mutant or the presence of two mutations on sister chromatids of the same chromosome (unphased mutations) that require a meiotic recombination event for transmission to the same gamete. This experiment suggests that obtaining the same quintuple mutant via traditional diploid crosses would have been challenging and the fact that we were able to obtain two such individuals in a modest screen demonstrates the power of haploid genetics to rapidly generate complex genotypes.

Direct phenotypic characterization of recessive mutations. Chemical mutagenesis and insertion element mutants are heterozygous in their rst generation34,37 and lesions of interest are often recessive and are therefore not visible in the phenotype of the mutagenized M1 population. Therefore, forward genetic screens require the production of M2 generations to allow recessive mutations to homozygose at the predicted 1/4 frequency. Functional genomics approaches such as forward and reverse genetic screens can be combined with haploid induction for faster analysis of non-lethal mutations. To demonstrate the principle of instantaneous screens, we combined mutagenesis of pollen followed by haploid induction to phenotypically screen for mutants in the M1 haploid generation. Specically, pollen from Ler gl1 plants was irradiated with varying doses of g rays (100 Gy and 200 Gy) to induce double-stranded DNA breaks. The mutagenized pollen was used to pollinate the HI. Out of 240 haploids obtained (100 Gy irradiation), we obtained three phenotypically distinct haploid M1 mutants (Supplementary Fig. 8). The M1_01 (Fig. 2b) mutant has mottled leaves, which phenocopies mutant phenotypes of VAR1, VAR2 and IMM. Analysis of mapped Illumina whole genome reads from M1_01 revealed a 2 bp indel in exon 4 of VAR2 (At2g30950) but not in Ler gl1 haploid control. This lesion was further conrmed by targeted Sanger sequencing (Fig. 2b). This indel causes a frame shift, resulting in a nonfunctional truncated VAR2 protein that is a likely candidate for causing the variegated phenotype. We envision using haploid induction in this manner to signicantly reduce the time and resources often associated with forward and reverse genetic screens. However, the disadvantage of using a haploid M1 screen is that mutations that need to be propagated in a heterozygous state, such as embryo lethal mutations and haploinsufcient gene mutations, cannot be obtained using this screen.

Production of gametophyte lethal homozygous mutants. Maternal and paternal gametophyte lethal mutations cannot be transmitted either through the female or male lineage, respectively38,39. It is therefore impossible to ascertain the loss-of-function phenotype of these genes on the sporophyte, except in the rare transmissions of weak alleles38. Because sex-specic lethal alleles can be transmitted through the unaffected sex, we hypothesize that haploid plants produced from the unaffected sex should inherit the mutant allele and permit the recovery of a

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Mutation stacking

Rapid M1 mutation screen

WT haploid

-irradiated pollen

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mut1 mut2 mut3 mut4 mut5

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Expect 1/32 vs 1/1024

Figure 2 | Haploid toolbox for ArabidopsisPart II. (a) Cartoon comparing the generation of a quintuple mutant from a quintuple heterozygous parent by haploid genetics and conventional genetics. The haploid genetic scheme (left) has a 32 advantage over the conventional diploid genetic scheme (right).

(b) Schematic diagram showing the rapid recovery of phenotypic mutations in the M1 haploid generation using g-irradiated pollen. Images of the M1_01 haploid mutant and its doubled haploid progeny with variegated leaf phenotype are shown. The candidate mutation that confers the M1_01 phenotype is a 2 bp deletion in exon 4 of VAR2 (At2g30950) but not in WT conrmed by Sanger sequencing as shown on the chromatogram. (c) Cartoon depicting the recovery of a homozygous female gametophyte lethal mutation. DME, a gene essential to the female gametophyte cannot be recovered through regular self-fertilization of diploid plants (left). dme-2 male gametes from DME/dme-2 can successfully fertilize HI female gamete to give rise to haploid dme-2 plants, which upon spontaneous chromosome doubling generate diploid dme-2/dme-2 plants (right panel).

mutant sporophyte. To test this hypothesis, we crossed maternal gametophytic lethal DME/dme-2 heterozygote38 as the male to HI. Out of 37 haploids, we obtained 19 WT DME haploids and 18 dme-2 mutant haploids (Supplementary Fig. 9). The 1:1 segregating ratio indicates that it is indeed possible to propagate maternal gametophyte lethal alleles as paternal haploid plants. The dme-2 mutant haploids were phenotypically similar to WT haploid siblings except for one sibling (Supplementary Fig. 9), indicating that DME is not essential in adults. However, sporadic developmental abnormalities have been reported for homozygous mutant plants derived from heterozygous plants carrying the dme-1 weak allele, via transmission of the mutant allele through the female lineage38. These plants randomly produced individual owers with reduced or increased oral organs mostly petals and sepals apart from improperly fused carpels, petaloid anthers, fused stamen laments and so on38. In the case of the dme-1 weak allele, the abnormal oral phenotype was sporadically observed across random owers in an inorescence. When we produced haploid mutants carrying the dme-2 strong allele that show no female transmission, these abnormalities were conned to a single plant (Supplementary Fig. 9b,c) while all other mutant haploid plants (n 17) looked WT (Supplementary Fig. 9a). Furthermore, oral

abnormalities were observed almost uniformly across all the owers in that single plant. This observation suggests that the

developmental phenotype of this stronger allele of dme-2 is incompletely penetrant and manifests phenotypically only in certain mutant siblings.

Similarly, mea-1 (ref. 39) haploids could also be obtained (Supplementary Fig. 9). Surprisingly, both dme-2 and mea-1 haploids were partially fertile and produced diploid progeny that were homozygous recessive for their respective KO allele (Fig. 2c). The molecular basis for this unexpected result is currently being investigated. These results demonstrate the utility of this tool in determining the sporophytic phenotype of gametophyte lethal mutations and the importance of their characterization to understand gene function.

DiscussionWe envision that the haploid genetic methods described in this study can in principle, be extended to any plant species for which a successful haploid production method exists. In vivo haploids are better suited for realizing the potential of haploids in basic genetics studies, especially in the case of methods such as swapping of nuclear and cytoplasmic genomes and for inducing interspecic uniparental genome elimination described here. Further, in a majority of crop species, in vitro production of haploids is genotype dependent6,40 and cannot be exploited to its full advantage if multiple genotypes/strains are involved in the

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genetic experiments. Uniparental genome elimination leading to in vivo haploid production has been documented by plant breeders mostly from studies of interspecic hybridization experiments from several plant species712. In maize, an intraspecic in vivo haploid production system using Stock 6 genotype13 derivatives is one of the commercially exploited haploid production system used for the development of hybrid maize cultivars41. However, the genetics behind both intraspecic and interspecic uniparental genome elimination are too poorly understood to engineer an in vivo haploid inducing system in any desired species. Recently, it has been shown in barley interspecic crosses (Hordeum vulgare H. bulbosum) that elimination of H.

bulbosum genome is associated with a selective loss of CENH3 from H. bulbosum centromeres, resulting in the production of haploids from H. vulgare42. This nding is encouraging because it links CENH3 function with genome elimination in an unrelated system. Engineering an in vivo haploid induction system through targeted manipulation of CENH3, as demonstrated in Arabidopsis16, could be successful in other plant species. A strategy for developing a CENH3-based haploid induction system in a plant species of interest are elaborated in a review43. Interestingly, a QTL mapping approach to identify the genetic loci behind haploid induction in Maize did not identify CENH3 loci, suggesting a different genetic mechanism in this case44. A better understanding of the maize uniparental genome elimination system and the CENH3-based haploid production system will pave a way towards employing in vivo-generated haploids for basic genetic studies besides their routine use for hybrid breeding in a wide variety of plant species.

An insight that hints at the crucial role of the endosperm in haploid seed production came from our work with the seedGFPHI strain and this should be considered as HI systems are developed in other species. The endosperm results from a double fertilization event and its development is sensitive to deviations from the 2:1 ratio of maternal/paternal genomes, as well as to specic epigenetic states of certain imprinted genes45. Haploid seed could be, in principle, associated with uniform triploid, uniform haploid or mottled endosperm. Failure of nding triploid endospermhaploid embryo seed indicates coordination between the fates of HI chromosomes in the zygotes. Further, the absence of haploid endospermhaploid embryo seed is most likely dependent on the necessity to maintain this ratio of parental genome dosage in the endosperm and implies that a failure to do so might underlie the large proportion of aborted seeds in an HI cross. We hypothesize that partial loss of mutant parent chromosomes in the endosperm might be responsible for the mottled GFP uorescence seen in the endosperm of haploid seeds. This hypothesis is the subject of ongoing investigations.

We have demonstrated novel facets of haploid genetics that will signicantly reduce cost and time in comparison to currently employed approaches. In some cases, such as in the generation of homozygous recessives from sex-specic lethals, haploid induction has enabled the phenotypic characterization of a developmental stage that would otherwise be unattainable. Characterization of sex-lethal transmission after haploidization should add useful information on the function and regulation of these genes. These ndings add to our previous work in which we demonstrated that haploids can be used to rapidly generate mapping populations46, chromosome substitution lines47, parental lines for reverse breeding47 and for engineering clonal reproduction through seeds48. Finally, like in haploid yeast, it is possible to measure meiotic crossover and gene conversion using Arabidopsis haploids49 and to study meiotic recombination events in the absence of a homologous partner during haploid meiosis50. Taken together, these methods constitute a novel and complementary genetic haploid toolbox for the Arabidopsis community.

Methods

Plant materials and growth conditions. Plants were grown in Sunshine Professional Peat-Lite Mix 4 (SunGro Horticulture) in a controlled environment growth room at 203 with a 16 h/8 h light/dark photoperiod. The WT diploid(2n 2 10) accessions used in this study are Col-0 and Ler. DME/dme-2 and

MEA/mea-1 plants are in Ler background. The yuc1 (At4g32540) mutant is a SALK transfer DNA (SALK T-DNA) insertion mutant (Salk_106293), yuc2 (At4g13260) mutant is Salk_030199 and the yuc8 (At4g28720) mutant is a dSpm insertion from the SLAT collection. Ler gl1 pollen was used in M1 mutant screen for easy phenotypic identication of haploid plants. The tetraploid (2n 4 20) Arabi

dopsis accession Wa-1 (CS6885) was obtained from the Arabidopsis Biological Resource Center (ABRC), Ohio State University. The tetraploid TILLING population used to identify the SOG1 lesion is of Col-0 accession and the seeds were obtained from UC Davis TILLING facility. A. suecica #1 (2n 26), the allote

traploid used for the interspecic cross with A. thaliana HI was a strain described in the study by Hanfstingl et al.51

Using and maintaining GFP-tailswap as a HI. The HI used is homozygous for the cenh3-1 mutation as well as the GFP-tailswap transgene. GFP-tailswap/GFP-tails-wap cenh3-1/cenh3-1 plants (referred to as HI) that we have bred for haploid induction can be easily distinguished from WT plants by their bushy and stunted phenotype (Supplementary Fig. 1a,b).

HI plants are predominantly male sterile during early to mid stages of the inorescence growth, however, as the inorescence matures distal owers show increasing fertility and are more likely to self-pollinate. This is an advantageous feature for crossing and maintenance of HI genotype. First, male sterility in the early inorescence allows pollination for haploid induction to be carried out without the need for tedious emasculation. Successful haploid induction has been achieved even in opened owers (23 days after ower opening) as long as they have a receptive stigma. Even if accidental self-pollination occurs while crossing, the contaminant selfed progeny can be distinguished from haploids, hybrid diploids and aneuploids by the characteristic phenotype of HI plants as shown in Supplementary Fig. 1a. Second, viable seeds collected from self-pollinated siliques from later stages of inorescence can be used to raise a uniform population of HI plants (Supplementary Fig. 1b), without the need to segregate out homozygous HI plants from a heterozygous parent. About 95% of such selfed seeds give rise to diploid HI plants that can be phenotypically distinguished from the 5% aneuploid siblings (Supplementary Fig. 1b).

HI pistils elongate normally after successful pollination with viable pollen, which suggests that our HI has good female fertility. Because of the very low male fertility, self-pollination is minimal except in later stage owers and thus unpollinated pistils fail to elongate. This property of HI can be used to distinguish a pollinated pistil from an unpollinated one while crossing. This is especially important when bulk pollen (collected by vacuum method) is randomly dusted onto open owers.

Even though siliques develop normally after successful crossing, about 7095% of the developing seeds (depending on the Arabidopsis accession) within the silique abort during embryogenesis such that only 520% of viable seeds are typically recovered from a cross. Among the viable progeny, 4060% are haploid with the remaining being either diploid or aneuploid. On an average, we recover B13 haploid progeny per silique after crossing to a WT parent. Hence, we recommend crossing at least twice as many owers as the required number of haploids.

All HI lines used in these experiments were identied from a segregating population of a selfed CENH3/cenh3-1 GFP-tailswap/GFP-tailswap individual by phenotype (Supplementary Figs 1 and 2) and conrmed by PCR genotyping using a derived Cleaved Amplied Polymorphic Sequence (dCAPS) assay. Oligosused for genotyping are cenh3-1_XbaI_Fwd: 50-AGAATTTTAGGTTTTTTATT

TCGATTTTGTAACCCTAGATTTCGAATCTGAAATTTCTA-30 and cenh3-1_XbaI_Rev: 50-GCCTCTCCTTGTCGGGGTCTTCA-30. The WT 212 bp CENH3 PCR product is cleaved by XbaI to produce a band at 148 bp but the mutant cenh3-1 allele remains uncut.

Recovery of haploid and dihaploid progeny. To maximize the recovery of haploid progeny from HI crosses, we recommend germinating F1 (HI WT) seeds

on MS (Murashige and Skoog) agar plates. We nd that the late germinating seedlings (as late as 23 weeks after other siblings from the same cross) are enriched with haploid progeny. The chance of recovering the slowly germinating haploid progeny is maximized under the optimal conditions provided by MS agar.

Haploid plants can be phenotypically distinguished from hybrid diploid, aneuploid and selfed siblings (Supplementary Fig. 2a) as early as 10 days post germination16. Yet another way to identify haploids is to screen for the presence of GFP uorescence at centromeres by observing nuclei in different tissues (Supplementary Fig. 2b,c). Root tips from germinating seedlings can be used for early screening or ower parts such as petals, pistil or anthers can be used at later stages for scoring. Since haploids always originate from a parent devoid of centromeric GFP protein, plants without centromeric GFP protein are haploids. On the other hand, selfed HI progeny, hybrid diploid and aneuploid siblings display GFP uorescence at their centromeres.

Alternatively, haploid plants can be identied by PCR-based genotyping because they are homozygous for the WT CENH3 allele, whereas diploid and

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6334 ARTICLE

aneuploid siblings are heterozygous for the cenh3-1 allele contributed by the HI parent. In addition, the haploid plants can also be PCR tested for absence of the GFP-tailswap construct. For details on the primers and methodology for PCR-based genotyping, please refer to an earlier publication24. Final conrmation of haploids can be done by counting the number of chromosomes either in mitosis or meiosis (Supplementary Fig. 2f,g).

For propagation and genetic analysis sterile haploids must be converted to fertile diploid plants called doubled haploids (DH). The traditional method to convert a haploid to a DH is to treat the plants with the microtubule depolymerizing drug colchicine. Although the latter works well, an eco-friendly way to produce DHs is to collect those spontaneous seeds from haploid plants produced as a result of spontaneous mitotic and/or meiotic chromosome doubling16. To achieve maximum harvest of spontaneous DH seeds, we recommend growing the haploid plants under optimal conditions, such as feeding with full strength MS nutrient solution, to achieve healthy growth. Under such conditions, the haploids tend to produce more owers than WT diploid as a response to sterility. Production of more owers increases the probability of spontaneous meiotic and/or mitotic doubling and the chances of obtaining DH seed. A single viable double haploid seed/plant is sufcient to propagate the genotype of that haploid plant. In practice, depending upon the accession, a single haploid typically produces 50 to 5,000 spontaneous DH seeds.

Construction of seed marker HI line. The binary vector pFP91 harbouring At2S3::GFP27 and introduced into Agrobacterium tumefaciens strain GV3101, which was used to transform a heterozygous CENH3/cenh3-1 GFP-tailswap/GFP-tailswap plant by oral dip. Transformed GFP-positive T1 seeds harbouring At2S3::GFP construct were identied by visualizing the seeds using a uorescence stereomicroscope. GFP-positive seeds were selected and grown to identify CENH3/ cenh3-1 GFP-tailswap/GFP-tailswap T1 individuals. T2 seeds that show 100% inheritance of the At2S3::GFP transgene were selected and selfed to ensured the stable expression of the endosperm marker in the T3 generation. A T3 plant of the genotype cenh3-1/cenh3-1 GFP-tailswap/GFP-tailswap At2S3::GFP/At2S3::GFP was designated as SeedGFP-HI. The F1 seeds from SeedGFP-HI Col-0 WT were broadly

categorized into two groups based on the nature of GFP uorescence:seeds with uniform GFP uorescence and those with mottled GFP uorescence (Fig. 1b and Supplementary Fig. 3). Sorted seeds from each category were surface sterilized and sown on MS agar plates for germination. All plated seeds were labelled and imaged for GFP uorescence using a uorescence dissection stereomicroscope. After 3 days of cold treatment, the plates were transferred to the growth room. Germinating seedlings (35 days post germination) were imaged for GFP uorescence and transferred to soil. The progeny were scored for ploidy as described above.

Swapping of cytoplasm between Ler and Col-0 accessions. To generate the HI with Ler cytoplasm (Supplementary Fig. 4), a CENH3/cenh3-1 GFP-tailswap/GFP-tailswap plant (Col-0) was used as a male parent and crossed to Ler. Among the resulting F1, CENH3/cenh3-1 GFP-tailswap/ progeny was self-pollinated and the

F2 segregant with cenh3-1/cenh3-1 GFP-tailswap with Ler cytoplasm marked by characteristic phenotype of HI (Supplementary Fig. 1a) was crossed to Col-0 pollen. The resulting F1 seeds were germinated on soil and 30% (n 56) of the

established plants were phenotypically identied as haploid. To distinguish Col-0 and Ler cytoplasm, a natural single nucleotide polymorphism in the chloroplast gene Atpt69346 was used in CAPS assay using the following oligos. CP717: 50-GTCATTTACCCTGTTAGTCCG-30 and CP718: 50-GAAATACAAGACAGC

CAATCC-30. The PCR amplied product was then digested with the HinfI restriction enzyme and resolved on a 3.0% agarose gel (Fig. 1c). The nuclear genome of Col-0 and Ler ecotype was differentiated using microsatellite marker nga8 located on chromosome 4. The microsatellite locus nga8 was PCR amplied using oligo combination CP662: 50-TGGCTTTCGTTTATAAACATCC-30 and

CP663: 50-GAGGGCAAATCTTTATTTCGG-30 and the resulting products were resolved on a 3% agarose gel (Fig. 1c).

Ploidy reduction of the Wa-1 tetraploid. Ploidy reduction of the natural tetraploid Wa-1 was achieved by a two-step sequential genome elimination procedure as described above. A natural single nucleotide polymorphism between the Col-0 and Wa-1 chloroplast gene Atpt69346, which utilizes the same dCAPS assay16 as described above was used to distinguish Col-0 and Wa-1 cytoplasm. The PCR amplied product is digested with the HinfI restriction enzyme, which cleaves the Wa-1 fragment twice and the Col-0 fragment once (Supplementary Fig. 5).

Diploidization of SOG1 mutation. To identify new KO alleles for the SOG1 gene, we screened a TILLING population derived from 528 tetraploid ethyl methane-sulfonate-mutagenized plants34. We identied three nonsense (KO) and 21 missense mutations in SOG1. Eight tetraploid plants representing each of the three sog1 KO alleles were genotyped by targeted Sanger sequencing of the SOG1 locus using following primers SOG_F1 50-CTTTCACTGCTAGGTTGGGGT-30,

SOG_R1 50-CTGTTGTGGCTGCTGGTAGA-30 and SOGseq 50-GATAATTCTG CTTTGTGTAG-30. No plant was found to be homozygous for any of the three sog1 alleles. A heterozygous plant representing each mutation was crossed as pollen donor onto HI plants. Approximately 100 HI owers were crossed for each line; the

deposition of large quantities of pollen onto each ower was conrmed using a dissecting microscope. The HI plants were then bagged in a glycine bag to prevent contamination by diploids present in the same chamber. Viable seeds were rare (3 9 viable seeds/100 owers crossed), reecting the generally poor seed set obtained when crossing tetraploid Col-0 onto diploid Col-0 (ref. 45). The F1 seeds were sown on MS plates and the resulting seedlings were transplanted to soil and genotyped again by targeted Sanger sequencing. Plants carrying the mutation (heterozygotes) were identied and allowed to self-pollinate. To determine the frequency of diploids produced from HI Tetraploid TILLING mutant crosses,

ploidy analysis was done by ow cytometry on a bulk sample of 50 F2 seeds collected from each of the 5 F1 plants.

Generation of A. suecica haploids. To produce A. suecica haploids, interspecic crosses were performed by pollinating A. thaliana HI (2n 10) with pollen col

lected from A. suecica (2n 26) plants. The resulting F1 seeds were germinated on

MS media and of the 576 germinated seedlings that were transplanted to the soil media only 241 survived. Only a small fraction of the 241 plants appeared to be phenotypically interspecic hybrids while the majority (B160) of the plants resulted from self-pollination of the HI. Flow cytometry and chromosome dosage analysis36 using Illumina sequencing (Supplementary Fig. 7) were used to conrm the haploid karyotype of the two A. suecica (2n 13) plants.

Multiple mutant analysis. The YUC1/yuc1; YUC2/yuc2; YUC8/yuc8 triple heterozygous plants harbouring independent insertions of the DR5GFP and EC::DsRED transgene were genotyped by locus-specic PCRs detailed below. The haploids obtained after crossing this quintuple hetero/hemizygous mutant to the HI were PCR genotyped to identify homozygotes at all loci using the following set of oligos: The genotype for YUC1 locus (At4g32540) was identied using 50-CCT

GAAGCCAAGTAGGCACGTT-30 and 50-CGTTCATGTGTTGCCAAGGGAGA TAC-30 for the WT allele, followed by 50-CCTGAAGCCAAGTAGGCACGTT-30 and 50-GGCAATCAGCTGTTGCCCGTCTCACTGGTG-30 for T-DNA insertion. The WT YUC2 locus (At4g13260) was identied using 50-CGTCCAATACCTT GAGTCTTACGC-30 and 50-CTGCATACAATCCGCTTTCGC-30 and the T-DNA insertion was identied using 50-GGCAATCAGCTGTTGCCCGTCTCACTG GTG-30 and 50-CTGCATACAATCCGCTTTCGC-30 oligo pairs. Similarly for YUC8 (At4g28720), the WT allele was identied using 50-CTAGTGCTCAAC CGTCACAAACCCC-30 and 50-AACGTTGATTTACCCATTACTTCCCTCGG-30 while the T-DNA allele was identied using the 50-TACGAATAAGAGCGTCCATTTTAGAGGA-30 and 50-GAACTGACGCTTCGTCGGGTAC-30 oligo pairs. The presence of EC::DsRed transgene was identied by PCR using CP1118: 50-GTCATCACCGAGTTCATGCGCT-30 and CP1119: 50-ACGCCGAT

GAACTTCACCTTGTA-30. Lastly, DR5::GFP was identied using oligo combinations CP844: 50-ACAACAGCCACAACGTCTATATC-30 and CP845: 50-GGTGTTCTGCTGGTAGTGGTC-30. The genomic location of the T-DNAs encoding the uorescent DsRed and GFP markers was not determined.

Haploid mutant M1 screen. Bulked pollen from Ler gl1 was collected using the vacuum collection method and exposed to 0 Gy (no irradiation), 100 Gy and200 Gy of g irradiation at the Center For Health and the Environment Facility in

UC Davis. Irradiated pollen from the different treatments was used to pollinate HI plants. We observed very poor seed set from siliques pollinated with 200 Gy irradiated pollen, suggesting that this dose might be lethal to the male gametophyte. These crosses were not analyzed further. The 100 Gy pollen gave good seed set and the typical mixture of haploid, diploid and aneuploid progeny was recovered (Supplementary Fig. 8). M1 haploids were scored based on their trichome-less Ler gl1 phenotype of the male parent. Out of 240 haploids obtained from the 100 Gy cross, we identied three Ler gl1 haploids exhibiting visible morphological mutant phenotypes (Supplementary Fig. 8). Spontaneously produced DH seeds were collected from these plants and the DH mutants were recovered (Fig. 2b). DNA from WT Ler gl1 and M1_01 were puried using DNA Phytopure Kit (GE) and sequencing libraries were prepared using the standard NEB Next DNA Library Prep with NEXTFlex-96 Adapters from BIOO Scientic and sequenced on Illumina HiSeq 2000. After mapping the reads to the Col-0 TAIR10 reference genome, a 2 bp indel in exon four of VAR2 (At2g30950) was identied in M1_01. To conrm the indel polymorphism, we designed primers VAR2_Fwd: 50-AGTTGATGAAACTGAGTGTTTGAGACTGT-30 and VAR2_-

Rev: 50-GCCAATAGTTTCTTTCTCGAGAAGCACT-30, and PCR amplied the fragment from WT and M1_01 for Sanger resequencing.

Generation of homozygotes from gametophyte lethal mutants. To generate dme-2 (At1g02580) and mea-1 (At5g04560) haploid mutants, heterozygous DME/ dme-2 and MEA/mea-1 plants were crossed as pollen parent to HI plants. The resulting F1 seeds were directly sown on soil and the haploids were identied as described. All the haploids were PCR genotyped to identify the DME WT allele using CP1261: 50-AGTAACTGATGTGTCCAAACCAGCTCC-30 and CP1262: 50-

CTGCATTGTAAAACCACCATGGGTT-30 oligo combinations, whereas haploids were PCR genotyped to identify the dme-2 mutant allele using CP1262 and a left border-specic oligo LB: GACGTGAATGTAGACACGTC-30.

In the case of MEDEA, the WT allele was identied using oligo combinations

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6334

50-GCGTAGCAGTTAGGTCTTGCTG-30 and 50-GTTTGACCCGTCAGGAC TCTC-30, and the mea-1 allele using 50-GCGTAGCAGTTAGGTCTTGCTG-30 and 50-CGTTCCGTTTTCGTTTTTTACC-30.

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Acknowledgements

We would like to also acknowledge the previous members of the Chan lab: Patrick Dunn, Joel Valencia, Joseph Ramahi, Daniel Melters, Pak Kwong, Maryam Sanei, Martijn Heddes and all the undergraduates who were members of the Chan lab when the experiments were being performed. This work was funded by the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation through grant GBMF3068 (to L.C.), the NSF Plant Genome Grant DBI-0924025 and the Ofce of Science (BER), US Department of Energy (to L.C.), Gordon and Betty Moore Foundation through Grant GBMF 2550.03 to the Life Sciences Research Foundation (to S.M.) and the NSF DMB award #1158443 (to A.B.B.). S.W.L.C. was a Howard Hughes Medical InstituteGordon and Betty Moore Foundation Investigator. M.R. is supported by the Ramalingaswami Fellowship awarded by Department of Biotechnology (DBT), Government of India.

Author contributions

S.W.L.C. and M.R. outlined the paper. M.R., M.P.A.M., K.T. and J.T. made the SeedGFPHI line. I.M.H. generated the diploid Wa-1 with Wa-1 cytoplasm. M.P.A.M. did the cytoplasm swapping experiment. A.B.B. and F.Z. performed sog1 ploidy reduction. S.M. made the A. suecica haploids. M.R. and B.M.R. isolated the quintuple loci haploid. E.H.T and G.U. did the M1 haploid mutagenesis screen. M.R. and B.-M.R. made haploid gametophyte lethal mutants. A.P. and V.S. contributed materials. M.R., M.P.A.M., E.H.T., S.M. and L.C. drafted the manuscript. M.R., M.P.A.M., E.H.T., S.M., I.M.H., A.B.B. and L.C. participated in data interpretation and edited the manuscript. All authors read and approved the nal manuscript.

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How to cite this article: Ravi, M. et al. A haploid genetics toolbox for Arabidopsis thaliana. Nat. Commun. 5:5334 doi: 10.1038/ncomms6334 (2014).

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