Cultivated crops consist of genetically highly uniform individuals, usually derived from crosses between inbreed lines or highly selected heterozygous plant materials. In this sense, apomictic natural populations with genetically uniform clone-mates and pedigree cultivars of apomictic forage grasses are qualitatively similar, and to some extend comparable to cultivated crop fields with variable levels of heterozygosity and a contrasting type of reproduction involve in the formation of the next generation of seeds. Plant reproductive processes, flowering time, formation of floral organs, pollen viability, etc., are strongly influenced by climatic conditions and can affect seed yields and crop performance (Hampton et al., 2016). Climatic factors govern crop growth and development and are subject to spatial changes in both direction and magnitude, making necessary fine scale analyses to discern the differential spatial responses of crops to climate variability and their impacts on crop yields (Kukal and Irmak, 2018). Therefore, genetically pure sexual crops and apomictic cultivars are expected to have alike responses to variable environmental conditions which might help us understand the short-term effects of ecological and climatic changes on seed production and crop yields, and estimate potential climate-influenced crop yield gain/loss expected from the transfer of apomixis to main crops. Since most apomicts are facultative, in both natural clonal lineages as in commercial forage cultivars, the formation of low proportions of recombinant progeny is expected to increase genetic heterogeneity and inbreeding which may decrease forage productivity. Therefore, at least in forage breeding, a proper reproductive characterization of the selected material using multiple experimental approaches can benefit both breeding programs, by knowing potential rates of hybridization and trait introgression, and field management strategies by estimating proportions of non-maternal offspring expected per generation (Hojsgaard et al., 2016).

Most natural apomicts maintain the pollen function for pseudogamy. For apomictic crops, this means that they could act as pollen donors and introgress adjacent sexual crop stands (van Dijk and van Damme, 2000). To avoid introgression of apomictic variants into sexual ones, apomictic crops would have to be pollen-sterile. However, natural systems show us that the pollen function can be only abandoned with autonomous apomixis, which occurs mostly in plants with or without a weak endosperm formation (see above). Hence, breeding strategies for pollensterile apomictic cultivars may be most useful for forage plants where the major interest of the farmer is in vegetative growth. But for crops plants which are mostly cultivated for their seed yield, the need of pseudogamy for proper endosperm formation requires functional pollen. Breeding strategies aiming at enforced autogamy (e.g., within cleistogamous spikelets or flowers) in crop variants may help to overcome this problem.

Most domesticated crops are highly dependent on use of high inputs (e.g., fertilizers and herbicides), and therefore an escape from cultivation is less likely than an introgression event to a wild relative species (e.g., Arnaud et al., 2003; Uwimana et al., 2012). In contrast, apomictic forage crops are less dependent on inputs and can both introgress a wild relative and escape from cultivation. The same features that make apomictic crops suitable for development of cultivars also make them better invaders, instigating a potential threat to biodiversity and environmental risk. In Latin America, for example, superior Brachiaria grasses for livestock production have been widely adopted covering approximately 25 million hectares1 , and many areas of Brazil have been recorded as invasive (Almeida-Neto et al., 2010; under

1https://ciat.cgiar.org/

the name Urochloa spp. in Zenni and Ziller, 2011). Similar cases are recognized worldwide but little is known about the adverse impacts of such invasions on biodiversity.  A few studied cases show apomictic grasses dominating invaded habitats and displacing native grasslands (Marshall et al., 2012; Dennhardt et al., 2016). Thus, a better understanding surrounding the origin and dynamics of natural apomictic populations, as well as the variation in the expression of residual sexuality and other sources of genetic variation, can help identify and target effective management actions for apomictic crops, which might currently contribute to the sustainable intensification of forage-based systems.

#### CONCLUSION AND OUTLOOK FOR FUTURE STUDIES

Apomixis is a complex, developmental trait expected to have an enormous impact in plant breeding if introduced into main crops, both shortening the time required to develop a new variety and to increase revenues. Currently, apomixis is exploited for the creation of forage cultivars but reports documenting, e.g., lack of genetic homogeneity and genetic erosion in those cultivars are not available. Although eclipsed for a long time, the mechanisms responsible for the rise and dynamics of apomixis in natural plant populations, the potential outcomes of apomixis in natural systems and its role in plant evolution start being disentangled. The knowledge about the genetic and ecological factors governing developmental interactions between meiotic

#### REFERENCES

and apomictic pathways, as well as the population dynamics at local and regional scales can not only help us to decipher the strategies plants use to respond and adapt to the environment, but it also provides valuable information to use on apomictic crop management and production practices.

#### DATA AVAILABILITY

No original datasets were produced for this article.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

Basic research to this manuscript was funded by the German Research Foundation Deutsche Forschungsgemeinschaft (DFG) (projects Ho 4395/4-1, Ho 4395/1-2 to EH and project Ho5462/1-1 to DH).

#### ACKNOWLEDGMENTS

We thank the series editors Emidio Albertini and Fulvio Pupilli for the invitation to present this review article, and reviewers for their valuable comments on the manuscript. We acknowledge support by the Open Access Publication Funds of the Göttingen University.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Hojsgaard and Hörandl. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Boechera Genus as a Resource for Apomixis Research

Vladimir Brukhin1,2 \*, Jaroslaw V. Osadtchiy2 , Ana Marcela Florez-Rueda3 , Dmitry Smetanin3 , Evgeny Bakin1,4, Margarida Sofia Nobre3 and Ueli Grossniklaus3

1 Theodosius Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University, Saint Petersburg, Russia, 2 Department of Plant Embryology and Reproductive Biology, Komarov Botanical Institute RAS, Saint Petersburg, Russia, 3 Department of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zurich, Zurich, Switzerland,

4 Bioinformatics Institute, Saint Petersburg, Russia

The genera Boechera (A. Löve et D. Löve) and Arabidopsis, the latter containing the model plant Arabidopsis thaliana, belong to the same clade within the Brassicaceae family. Boechera is the only among the more than 370 genera in the Brassicaceae where apomixis is well documented. Apomixis refers to the asexual reproduction through seed, and a better understanding of the underlying mechanisms has great potential for applications in agriculture. The Boechera genus currently includes 110 species (of which 38 are reported to be triploid and thus apomictic), which are distributed mostly in the North America. The apomictic lineages of Boechera occur at both the diploid and triploid level and show signs of a hybridogenic origin, resulting in a modification of their chromosome structure, as reflected by alloploidy, aneuploidy, substitutions of homeologous chromosomes, and the presence of aberrant chromosomes. In this review, we discuss the advantages of the Boechera genus to study apomixis, consider its modes of reproduction as well as the inheritance and possible mechanisms controlling apomixis. We also consider population genetic aspects and a possible role of hybridization at the origin of apomixis in Boechera. The molecular tools available to study Boechera, such as transformation techniques, laser capture microdissection, analysis of transcriptomes etc. are also discussed. We survey available genome assemblies of Boechera spp. and point out the challenges to assemble the highly heterozygous genomes of apomictic species. Due to these challenges, we argue for the application of an alternative reference-free method for the comparative analysis of such genomes, provide an overview of genomic sequencing data in the genus Boechera suitable for such analysis, and provide examples of its application.

Keywords: genome assembly, Boechera, apomixis, apomeiosis, diplospory, pseudogamy, genomics, heterozygosity

### GAMETOPHYTIC APOMIXIS AND ITS RELEVANCE TO AGRICULTURE

Apomixis is defined as the asexual reproduction through seeds and results in the formation of genetically uniform progeny (Nogler, 1984a; Asker and Jerling, 1992; Grossniklaus, 2001; Bicknell and Koltunow, 2004; Van Dijk, 2009; Kotani et al., 2014). During sexual reproduction, egg and central cell – the gametes of the reduced female gametophyte (embryo sac) – each get fertilized by one sperm cell to produce the embryo and endosperm, respectively (Dresselhaus et al., 2016).

#### Edited by:

Fulvio Pupilli, Italian National Research Council (CNR), Italy

#### Reviewed by:

Andrea Mazzucato, Università degli Studi della Tuscia, Italy Sònia Garcia, Spanish National Research Council (CSIC), Spain Amal Joseph Johnston, Universität Heidelberg, Germany

> \*Correspondence: Vladimir Brukhin<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science

Received: 29 November 2018 Accepted: 14 March 2019 Published: 02 April 2019

#### Citation:

Brukhin V, Osadtchiy JV, Florez-Rueda AM, Smetanin D, Bakin E, Nobre MS and Grossniklaus U (2019) The Boechera Genus as a Resource for Apomixis Research. Front. Plant Sci. 10:392. doi: 10.3389/fpls.2019.00392

**179**

In contrast, apomictic embryos are not the result of a fusion of male and female gametes but develop clonally from unreduced maternal cell lineages in the ovule (matroclinous inheritance). Characteristic components of gametophytic apomixis are (i) avoidance of meiosis (apomeiosis), (ii) development of the embryo from an unreduced egg cell without fertilization (parthenogenesis), and (iii) formation of functional endosperm either autonomously or by fertilization of the central cell (pseudogamy) (Koltunow, 1993; Grossniklaus, 2001; Koltunow and Grossniklaus, 2003; Hand and Koltunow, 2014).

A drawback of sexual propagation is the segregation of advantageous traits in subsequent generations, such that progeny can lose the advantageous gene combinations of their parents (Spillane et al., 2004; Brukhin, 2017). The study of apomixis has drawn greater interest over the last two decades because of its potential to fix agriculturally valuable characteristics over many generations. The introduction of apomixis into crop plants would allow the long-term fixation of complex genotypes, including those of F1 hybrids often used in agriculture. This would facilitate crop breeding and hybrid seed production and could greatly benefit subsistence farmers by providing them access to high-yielding hybrid crops (Grossniklaus et al., 1998; Spillane et al., 2004; Conner and Ozias-Akins, 2017). Many possible uses of apomixis in agriculture have been proposed and its importance for sustainability and food security has been recognized (Karpechenko, 1935; Jefferson, 1994; Toennissen, 2001; Grossniklaus et al., 2003; Spillane et al., 2004; Conner and Ozias-Akins, 2017). Unfortunately, almost no natural gametophytic apomicts have been found among major crop cultivars and the introgression of apomixis from wild apomictic relatives has so far been unsuccessful (Savidan, 2000).

As an alternative to introgression, genes relevant to apomixis could either be identified in sexual model systems by identifying mutants displaying components of apomixis, or by isolating the relevant genes from an apomictic species (e.g., Grossniklaus, 2001; Pupilli and Barcaccia, 2012; Rodriguez-Leal and Vielle-Calzada, 2012; Barcaccia and Albertini, 2013; Conner and Ozias-Akins, 2017). For the latter approach, an important question is how to choose a convenient apomictic model plant, which will allow the deciphering of the molecular mechanisms underlying the components of apomixis. Three apomictic genera that have been studied in depth, Hieracium, Paspalum and Pennisetum, have large genomes and are polyploid, which is true for the vast majority of currently known apomicts (Asker and Jerling, 1992; Carman, 1997; Hojsgaard et al., 2014). Although these features complicate molecular genetic studies, research in these natural apomicts have greatly contributed to progress in the field (reviewed in e.g., Ortiz et al., 2013; Bicknell et al., 2016; Conner and Ozias-Akins, 2017). In contrast to the natural apomicts mentioned above, Boechera spp. have a relatively small genome (∼170–230 Mb) and Boechera is the only known genus where apomixis is found at the diploid level in the wild (Böcher, 1951; Dobeš et al., 2004b; Sharbel et al., 2005; Voigt-Zielinski et al., 2012). In addition, Boechera spp. are close relatives of the model plant Arabidopsis thaliana, which is very well studied in terms of molecular genetics and functional gene annotation. However, the genomes of apomictic accessions of Boechera are characterized by extremely high heterozygosity, accompanied by alloploidy and aneuploidy that resulted from hybridization events (e.g., Schranz et al., 2005; Koch et al., 2003; Mandáková et al., 2015). This poses challenges to perform a phased assembly and detailed annotation (reviewed in Hirsch and Buell, 2013) of the genomes of apomictic Boechera accessions.

In this review, we will present the particularities of phylogeny, reproduction, and genetics of the Boechera genus, and discuss strategies for assembly and annotation of the genomes of apomictic Boechera accessions.

### TAXONOMY AND HABITATS OF THE MOST IMPORTANT Boechera SPECIES

The genus Boechera comprises mainly North American species of biennial and perennial herbaceous crucifers, characterized by a base chromosome number of n = 7. Previously, these species were included in the genus Arabis L., from which they were excluded based on the difference in the base chromosome number (Löve and Löve, 1976), which is n = 8 in Arabis spp. Molecular genetic data confirmed the difference between the two genera. It was shown that the similarity between them is convergent, representing two evolutionary independent lineages in Brassicaceae (Al-Shehbaz, 2003). Recently, the taxonomy of the genus Boechera has been further developed using molecular markers. Currently, 110 species have been described within the genus, 71 of them are diploid and presumably sexual although diploid apomicts have also been described, and 38 are reported to be apomictic triploids of hybridogenic origin (Windham and Al-Shehbaz, 2006, Windham and Al-Shehbaz, 2007a,b). Thus, Boechera is the fifth largest genus within the Brassicaceae.

Most studies on the reproductive biology of Boechera involve just a small number of species.  A. Löve & D. Löve, and apomicts of a hybridogenic origin previously referred to as Boechera divaricarpa (A. Nelson) A. Löve & D. Löve (Windham and Al-Shehbaz, 2007b). Recently, several studies also used Boechera gunnisoniana (Rollins) W.A. Weber (Ta¸skin et al., 2003, 2004, 2009a; Schmidt et al., 2014; Kirioukhova et al., 2018). The rest of the species were mainly investigated to study particular aspects of apomixis in a geographically large number of species (Aliyu et al., 2010; Corral et al., 2013; Mau et al., 2013, 2015).

Until recently, the Pleistocene relict B. holboellii (Arabis holboellii) was treated in a broad sense as a species with a scattered range (i.e., consisting of several geographically isolated areas due to reasons of historical nature) (Böcher, 1951). However, recent taxonomic studies using molecular markers showed that it is a polyphyletic, artificial taxon, including a number of distinct species (Windham and Al-Shehbaz, 2006; Alexander et al., 2013). At present, B. holboellii is considered in the narrow sense as plants growing in Greenland. It includes sexual and apomictic diploid and triploid forms and the latter ones,

FIGURE 1 | Boechera stricta grown in the greenhouse of the Department of Plant and Microbial Biology of the University of Zurich.

unlike the North American species, appear to be autotriploids (Windham and Al-Shehbaz, 2006).

The continental North American accessions, which previously were included in B. holboellii, are distinguished as series of individual species that form an agamic complex (Stebbins, 1950). The basis of this complex consists of four diploid sexual species, in which, however, the presence of facultative apomixis cannot be excluded: Boechera collinsii (Fernald) A. Löve & D. Löve, Boechera pendulocarpa (A. Nelson) Windham & Al-Shehbaz, Boechera polyantha (Greene) Windham & Al-Shehbaz, and Boechera retrofracta (Graham) A. Löve & D. Löve. The remaining species are triploid apomicts of hybridogenic origin that are morphologically very similar to the parental sexual species: B. consanguinea (retrofracta × fendleri), B. goodrichii (retrofracta × gracilipes), Boechera grahamii (stricta × collinsii), B. pauciflora (sparsiflora × retrofracta), B. pinetorum (rectissima × retrofracta × sparsiflora), Boechera quebecensis (holboellii × stricta), and B. tularensis (retrofracta × rectissima × stricta) (Windham and Al-Shehbaz, 2007a,b). The B. quebecensis is distributed in isolated areas of North-Eastern America, implying the presence of one of its putative parents (Greenlandic B. holboellii s. s.) on the North American continent in the past.

The apomictic B. divaricarpa is probably the most problematic species in the genus from a taxonomic viewpoint. Traditionally, a large diversity of hybrids involving B. stricta as one of the parents (including B. stricta × B. holboellii s. l.) were referred to as B. divaricarpa in many articles on the reproductive biology of the genus Boechera. Such uncareful use of the name could be a potential source of confusion. As Windham and Al-Shehbaz (2007b) state, the correct use of the name B. divaricarpa should be restricted to plants containing genomes of B. stricta and B. sparsiflora. For hybrids of B. stricta × B. collinsii, the name B. grahamii should be used. The hybrids of B. stricta × B. holboellii s. s. should be referred to as B. quebecensis. In cases where the second parent of the hybrid is uncertain, the name "B. divaricarpa" should be avoided and replaced by "B. stricta hybrid."

In terms of prospective models for the study of apomixis, B. gunnisoniana deserves attention. It is a triploid species of presumably hybridogenic origin with diploid sexual species B. oxylobula and B. thompsonii (=B. pallidifolia) as parents (Mateo de Arias, 2015). It is characterized by almost obligate pseudogamous apomixis (Roy, 1995; Ta¸skin et al., 2004; Schmidt et al., 2014), a small plant size, and relatively fast development (approximately 4 months from planting to seed).

Although the vast majority of species of the genus Boechera grows in North America, the occurrence of two putative Boechera species in the Russian Far East has been reported, representing an example of East Asian/North American floristic disjunction. B. falcata (Turcz.) Al-Shebaz from the Russian Far East is closely related to the well-known North American apomicts (Boechera s. s.) based on molecular markers (Al-Shehbaz, 2005; Kiefer et al., 2009; Alexander et al., 2013), and its more detailed study with respect to the potential presence of apomixis is of a great interest. Another species is endemic of the Baikal region and the Russian Far East, Borodinia (=Boechera?) macrophylla (Turcz.) German. Recent molecular genetic studies showed its close relationship with seven Boechera species from the Eastern United States (Al-Shehbaz and German, 2010; Alexander et al., 2013).

### ADVANTAGES OF THE Boechera GENUS FOR THE STUDY OF APOMIXIS

Over the last decade, various species of the genus Boechera have been adopted as a model to study the molecular basis of apomixis, in addition to its well-established role as a study system in evolutionary ecology (reviewed in Rushworth et al., 2011). Among the advantages of Boechera spp. as a model are:

(i) Its close relationship to the model plant A. thaliana (L.) Heynh. (Huang et al., 2016), for which extensive molecular genetic resources are available, whose genome is fully sequenced and very well annotated, and in which many genes required for reproduction are

known, facilitating the search for genes involved in the control of apomixis in Boechera spp.;

### CYTO-EMBRYOLOGICAL STUDIES IN THE GENUS Boechera

The first detailed cyto-embryological studies of apomixis in the genus Boechera were undertaken by the Danish botanist Tyge W. Böcher (1947, 1951, 1954, 1969). He discovered the presence of apomixis in B. holboellii s. l. (referred to by him as Arabis holboellii) in diploid and triploid plants, and described megasporogenesis and microsporogenesis in a number of sexual and apomictic Boechera accessions from Greenland and North America. Particularly remarkable was his description of forms with varying degrees of chromosome synapsis in meiotic prophase. He also noted the presence of plants with different ploidy levels (mainly 2n and 3n, rarely 4n, 5n, and 6n) and aneuploids (2n = 16, 22, 23, and 30), and assumed a hybrid nature for the latter (Böcher, 1954).

Nearly 50 years later, a Dutch-Russian team performed embryological studies of cleared specimens of B. holboellii s. l. (accessions from Greenland and Colorado) using differential interference contrast microscopy (DIC) together with a flow cytometric seed screen (FCSS) analysis (Naumova et al., 2001). The presence of meiotic and apomeiotic events during megasporogenesis was demonstrated (**Figures 2–4**). By screening a large number of cleared ovules, the formation of an unreduced embryo sac through diplospory and parthenogenetic development of the embryo in apomictic Boechera accessions were confirmed. In sexual accessions, embryo sac development follows the Polygonum type (Maheshwari, 1950; **Figures 2**, **4**): the diploid megaspore mother cell (MMC) undergoes meiosis to produce a tetrad of haploid megaspores, three of which degenerate while the functional megaspore undergoes three mitotic divisions to give rise to an eight-nucleate, seven-celled embryo sac comprising an egg cell, two synergids, three antipodal cells, and two polar nuclei that fuse to form the homo-diploid nucleus of the central cell. In the anthers, pollen grains develop from the pollen mother cells (PMCs) that undergo meiosis to produce tetrads of microspores. After their separation, each microspore divides asymmetrically into a large vegetative and a smaller generative cell. After pollination, the vegetative cell germinates producing a pollen tube that transports the sperm cells to the embryo sac, while the generative cell divides once more to form two sperm cells that will later fertilize the egg and central cell, giving rise to a 2n embryo and a 3n endosperm, respectively (**Figures 2**, **4**).

In apomictic Boechera accessions, diplosporous apomeiosis of the Taraxacum type (Crane, 2001) is the most common (Böcher, 1951; Naumova et al., 2001; Schmidt et al., 2014; Mateo de Arias, 2015; Windham et al., 2015; **Figures 3**, **4C**). In meiotic diplospory, the embryo sac originates from an MMC that undergoes an aberrant meiosis without chromosome segregation, resulting in the formation of a dyad of unreduced megaspores. A characteristic feature of the MMC is the lack of callose deposition around the cell (Rodkiewicz, 1970; Nogler, 1984a; Carman et al., 1991). In contrast, callose was observed in the cell wall between the two cells of the dyad (**Figure 3D**). Dyad formation is most commonly observed (Roy, 1995; Ta¸skin et al., 2004; Schmidt et al., 2014); however, rare triads and even tetrads can be found (Schmidt et al., 2014). The chalazal dyad cell undergoes three rounds of mitosis, producing an unreduced eight-nucleate embryo sac that is morphologically similar to the Polygonum type (Crane, 2001; Rojek et al., 2018).

Aposporous apomeiosis was previously thought to be uncommon in Boechera spp., but recent reports described its occurrence in several species. The overwhelming majority of aposporous embryo sacs developed according to the Hieracium type (Crane, 2001). The sexual MMC in this case might degenerate or undergo meiosis as was observed in rare instances in B. microphylla (Carman, 2007; Mateo de Arias, 2015; Carman1 ). In B. retrofracta × stricta hybrids, diplospory of the Antennaria type (Crane, 2001) was also observed rarely (Carman see text footnote 1). In a FCSS, it was also shown that the percentage of mature sexual seeds (in relation to apomictic seeds) in all studied apomicts was significantly lower than the percentage of morphologically normal meiotic tetrads

1https://reeis.usda.gov/web/crisprojectpages/1000552-cytological-andmolecular-characterizations-of-reproduction-in-sexual-and-apomicticboechera-brassicaceae.html

FIGURE 3 | Apomeiotic megasporogenesis in B. holboellii s. l. (A) megaspore mother cell (MMC); (B) diplosporous dyad (Dy); (C) uninucleate diplosporous embryo sac (DES) with remnants of the "megaspore" ("MS"); (D) callose in the cell wall of a diplosporous dyad (B,C – Naumova et al., 2001; A,D – Osadtchiy et al., 2017).

(in relation to apomeiotic events). This implies that meiosis proceeds abnormally in the most cases, resulting in inviable seeds, while apomixis serves as an "escape from sterility" (Mateo de Arias, 2015).

In most apomicts, pollen development is unaffected. In Boechera spp., however, apomeiosis also occurs during microsporogenesis (Böcher, 1951; Ta¸skin et al., 2009a). In triploid apomicts, meiosis I fails as the chromosomes are unable to correctly pair at pachytene. The chromosomes migrate to opposing poles of the PMC and decondense. After cytokinesis the dyad, unlike the meiotic tetrad, is enclosed by a callose wall. Chromosome synapsis with the formation of bi- and trivalents in the metaphase I occurs in B. holboellii s. l., whereas apomeiosis in B. gunnisoniana is completely asynaptic. Investigation of microsporogenesis in apomictic triploid B. holboellii s. l. and B. gunnisoniana showed that in the triploids, the majority of pollen grains are unreduced, formed through apomeiotic dyads (98% in B. holboellii s. l., 90% in B. gunnisoniana), while the rest of the pollen was formed through (partially abnormal) meiosis, resulting in tetrads or triads of microspores (Ta¸skin et al., 2009a), or sometimes even in monads in triploid B. holboellii s. l. (Böcher, 1951). In diploid apomicts, variability in apomeiosis is higher. In different accessions of the B. holboellii complex, pairing and cross-over events can occur normally at pachytene in some accessions, resulting in reduced pollen, or trivalents and even quadrivalents can be formed in others. Some accessions showed mostly diploid pollen formation, while others displayed evidence of haploid and diploid pollen (Kantama et al., 2007; Ta¸skin et al., 2009a; Kirioukhova et al., 2018). In fact, it has been observed that both diploid and triploid apomicts can produce reduced and unreduced pollen in varying proportions (Böcher, 1951; Voigt et al., 2007; Aliyu et al., 2010; Voigt-Zielinski et al., 2012).

In sexually reproducing Boechera spp. double fertilization occurs, while in apomicts only fertilization of the polar nuclei takes place. However, fully autonomous apomixis can also be found in rare cases (Matzk et al., 2000; Naumova et al., 2001), it is more often observed in triploids (at frequencies of up to 15%) than in diploids (1.33% at most) (Aliyu et al., 2010). The formation of embryos with a doubled set of chromosomes as a result of the fusion of unreduced male and female gametes can also occur as a rare event (Naumova et al., 2001). An important conclusion of the FCSS-based study was that, in apomicts, all mature seeds were derived from unreduced female and male gametes (Naumova et al., 2001; **Table 1**).

TABLE 1 | The reproductive modes in B. holboellii s. l. accessions (based on cyto-embryological investigation of ovules and FCSS) (from Naumova et al., 2001).

aUnreduced embryo sac (ES), parthenogenetic embryo, and pseudogamous endosperm development. bUnreduced ES, parthenogenetic embryo, and autonomous endosperm development. cUnreduced ES and double fertilization by unreduced pollen.

In apomicts, endosperm ploidy varies according to the ploidy of the sperm cells, although the most common ratio is a 2 maternal:1paternal (2m:1p) genome ratio. Exceptions, although at lower frequencies, do exist, indicating that there is some degree of flexibility or, at least, that the system is "leaky" (Aliyu et al., 2010).

A large-scale FCSS, covering 16 Boechera spp. revealed a wide variability in reproductive mode within diploid genotypes, ranging from obligate sexual to nearly obligate apomictic. By assessment of the percentage of apomeiosis, sexual and parthenogenetic embryo formation, as well as sexual, pseudogamous, and autonomous endosperm development, it was shown that all investigated facultative apomicts of the same genotype had either a very low (1–3%) or a very high (87–99%) percentage of the apomeiosis, and individuals with intermediate frequencies were not observed. Furthermore, all triploids were found to be obligate apomeiotic. A genotype-specific correlation between apomeiosis on the one hand, and parthenogenesis combined with pseudogamous or autonomous endosperm development on the other hand, showed that frequencies of the latter never exceeded the frequency of apomeiosis (Aliyu et al., 2010). This may indicate a close relationship of their genetic control and a key role of apomeiosis for all subsequent stages of apomictic development.

#### POPULATION GENETIC STUDIES IN THE GENUS Boechera WITH RESPECT TO APOMIXIS

Apomictic and sexual lineages within the genus Boechera can inter-cross (Schranz et al., 2005; Alexander et al., 2013). Distinct evolutionary forces are expected to drive the evolution of lineages that differ in their reproductive modes. In sexual

lineages, recombination increases the probability of elimination of deleterious mutations (Hill-Robertson effect, Hill and Robertson, 1966; Felsenstein, 1976). Apomictic lineages, in contrast, reproduce asexually and do not undergo recombination; thus they cannot recover adaptive alleles once deleterious mutations occur within these alleles (Charlesworth, 2008). Therefore, one expects the accumulation of deleterious alleles, a phenomenon known as Muller's ratchet (Muller, 1964; Charlesworth and Charlesworth, 1997). Recent comparisons of apomictic and sexual lineages in Boechera spp. have supported these population genetic expectations (Lovell et al., 2013, 2014, 2017). Lovell et al. (2017) used apomictic and sexual populations of B. spatifolia and investigated patterns of nucleotide variation across both reproductive modes through whole-genome sequencing. They found an elevated sequence diversity and heterozygosity, together with an increased mutation accumulation, in apomictic populations (Lovell et al., 2017). Likewise, in a larger survey of 37 natural populations of four Boechera spp. (B. stricta, B. retrofracta, B. polyantha, and B. pendulocarpa), microsatellite markers showed the same trend (Lovell et al., 2013): higher levels of heterozygosity were found in apomicts compared to sexuals, independent of the ploidy level of the apomict.

In apomictic lineages, evolution occurs due to both genetic drift and natural selection (Charlesworth and Wright, 2001; Glémin et al., 2006; Brukhin and Baskar, 2019). The lower efficiency of selection expected in apomictic lineages would lead to an increased extinction risk of apomicts through the accumulation of deleterious alleles and an incapacity to adapt to environmental changes (Darlington, 1958; Muller, 1964; Bengtsson, 2009; Brukhin and Baskar, 2019). Contrary to these expectations, the genus Boechera is highly diverse, including several apomictic lineages (Alexander et al., 2013). A likely explanation for the survival of apomictic Boechera lineages is intra- and interspecific gene flow within the genus (Böcher, 1951; Sharbel and Mitchell-Olds, 2001; Dobeš et al., 2004a,b; Schranz et al., 2005; Beck et al., 2012; Lovell et al., 2013, 2017; Schilling et al., 2018). Gene flow mainly occurs from sexuals to apomicts, while apomicts are able to produce reduced pollen that can pollinate sexual lineages and transfer the dominant factor(s) conferring apomixis. Mutual gene flow between apomictic and sexual lineages may allow introgression of adaptive alleles from sexual into apomictic lineages, as posited by Van Dijk et al. (2009) in Taraxacum spp. Moreover, apomixis in Boechera spp. is facultative where different individuals may produce both sexual and apomictic offspring (Schranz et al., 2005; Aliyu et al., 2010). These probable instances of sexuality in apomicts may suffice to purge deleterious mutations and restore the fitness of apomictic lineages, securing their evolutionary survival (Van Dijk et al., 2009).

Differences in the strength of natural selection acting on sexual versus apomictic B. spatifolia populations were described by Lovell et al. (2014). The authors analyzed selection gradients by correlating genotypic trait means with relative fitness measurements, and found a reduction in the strength of adaptive evolution in apomictic relative to sexual lineages. Apomictic lineages experienced relatively less quantitative and molecular genetic differences between populations than sexuals. Also, divergence between apomictic populations was not correlated with environmental variation but, conversely, genomic structure and quantitative traits of sexual lineages were highly correlated with latitude, climatic variables, and elevation (Lovell et al., 2014). A common garden experiment revealed that flowering time was under strong selection in high-altitude sites. This is in agreement with studies in B. stricta (Anderson et al., 2011), which showed flowering time to be under directional selection in a study using recombinant inbred lines subjected to lab and field experiments.

Several studies assessed genetic dynamics and the extent of natural selection in Boechera populations. Earlier work on the population dynamics of the sexual species B. fecunda (Song and Mitchell-Olds, 2007) and B. stricta (Song et al., 2009), using sequence data from several nuclear loci and microsatellites, revealed similar levels of polymorphism and population differentiation in both species, regardless of the marked difference between the widespread B. stricta and the endangered B. fecunda with a reduced range. Similarly, studies comparing the widespread species B. stricta and B. latifolia with the rare species B. crandallii and B. vivariensis did not find strong associations between species size range and within-population genetic diversity (Lovell and McKay, 2015). However, the more widespread species exhibited higher phenotypic plasticity and quantitative trait structure (Qst), while the rare species contained stronger signatures of selection evidenced by higher Qst: Fst ratios, with Fst referring to the fixation index (Lovell and McKay, 2015). Extending the work of Song and Mitchell-Olds (2011) on B. fecunda, Leamy et al. (2014) found regional adaptation through extensive quantitative characterization of populations in Montana (United States) using microsatellite markers. Their analyses of genetic (Fst) and quantitative trait differentiation (Qst) showed evidence for divergent selection acting on water use efficiency and a contribution of the regional environmental conditions to local adaptation. Likewise, Lee and Mitchell-Olds (2011), using microsatellite markers and phenotypic quantitative analyses, demonstrated that water availability was the key environmental variable explaining genetic differentiation between two major genetic groups of B. stricta in Eastern and Western North America. All of these studies relied on microsatellite data and Fst estimations and should be interpreted with caution as microsatellite markers are not ideal for measuring population differentiation (Balloux and Lugon-Moulin, 2002; Putman and Carbone, 2014). Likewise, the use of Fst as a measure of population differentiation has been criticized (Jost, 2008; Meirmans and Hedrick, 2010; Whitlock, 2011; Jakobsson et al., 2013).

Whole-genome sequencing and chromosome painting on the same two major genetic groups of B. stricta investigated by Lee and Mitchell-Olds (2011) identified an inversion in Linkage Group 1 of the B. stricta genome (Lee et al., 2017). Populations carrying the inversion had lower polymorphism in Linkage Group 1, lower Tajima's D, and more linkage disequilibrium than populations without the inversion. Furthermore, the inversion had a strong effect on flowering time in near-isogenic lines under greenhouse conditions. These results showed that this inversion has important ecological impacts on the species and that natural

selection is driving the differentiation of B. stricta populations in North America (Lee et al., 2017).

Hybridization is common between members of the genus Boechera (Böcher, 1951; Sharbel and Mitchell-Olds, 2001; Dobeš et al., 2004a,b; Windham and Al-Shehbaz, 2007a,b). It was reported that hybridization occurs across the whole genus and happened repeatedly and independently (Schranz et al., 2005; Alexander et al., 2013). The earliest molecular evidence supporting hybridization comes from analysis of ITS and chloroplast sequence data, and gene flow between species now known as B. stricta and B. retrofracta was inferred by phylogeographic analyses (Dobeš et al., 2004a,b). It should be noted that conclusions on the hybrid nature of individuals, which are based on a single locus or an organellar genome, may not accurately reflect the history of a clade or population (Doyle, 1992; Maddison et al., 2006). However, Schranz et al. (2005) performed extensive crossing experiments, showing that successful crosses are possible among several members of the genus. This indicates a lack of intrinsic reproductive isolation barriers, and thus the possibility for extensive gene flow among different Boechera species.

Thus far, microsatellite markers have been central in identifying species and putative hybrids in the Bochera genus (Li et al., 2017). Beck et al. (2012) studied Boechera individuals using a set of 13 microsatellites. Hybrids between B. fendleri × B. stricta and B. retrofracta × B. stricta were confirmed using this methodology (Beck et al., 2012). Using similar methods, Lovell et al. (2013) studied 231 individuals from 37 natural populations of four Boechera species (B. stricta, B. retrofracta, B. polyantha, and B. pendulocarpa). They concluded that all triploid individuals were apomictic hybrids. This was not the case for diploid apomictic accessions, which behaved as true species rather than hybrid individuals. Based on these results, it was concluded that hybridization is an indirect correlate of apomixis in the genus Boechera.

With the advent of next generation sequencing technologies, the identification of hybrids is now more refined and precise. By using whole-genome sequencing in B. spatifolia, Lovell et al. (2017) investigated whether apomictic populations had a hybrid origin or not. Analysis of 220 000 haplotype trees across the genome indicated a hybrid origin of the apomictic B. spatifolia accessions. In another study using genotyping-bysequencing methods, Schilling et al. (2018) assessed genomic variation in 79 individuals of eight Boechera species. Admixture analyses allowed to precisely identify hybrid individuals. This study provided evidence of recent and ancient admixture and variation across species.

#### INHERITANCE AND GENETIC ASPECTS OF APOMIXIS IN THE GENUS Boechera

The seminal work of Nogler in the 1970es had shown that apomixis is genetically controlled (summarized in Nogler, 1984a). Subsequent crossing experiments of apomictic individuals as pollen donors with sexual maternal plants showed that apomixis is inherited as a dominant trait in many species (Grossniklaus et al., 2001). Early studies had indicated that apomixis is inherited as a single dominant locus, for instance in Ranunculus auricomus and Panicum maximum, where apomeiosis and parthenogenesis were found to cosegregate (Savidan, 1982; Nogler, 1984b). However, later studies found that different loci control the developmental components of apomixis, i.e., apomeiosis, parthenogenesis, and formation of functional endosperm, in most apomicts. It was also found that the genomic regions conferring apomixis or apomeiosis exhibit suppressed recombination (reviewed in Grossniklaus, 2001; Grossniklaus et al., 2001; Bicknell and Koltunow, 2004; van Dijk and Vijverberg, 2005; Barcaccia and Albertini, 2013; Hand and Koltunow, 2014; Hand et al., 2015; Brukhin, 2017). Apomixis is also frequently associated with hybridization and resulting polyploidy (Koltunow and Grossniklaus, 2003).  Apomixis, as an escape from sterility, has been speculated to be a transitional period in the evolution of neopolyploids, especially when facultative (Hörandl and Hojsgaard, 2012; Hojsgaard et al., 2014). Recent data indicate that apomixis is associated with increased diversity (Hojsgaard et al., 2014), suggesting that apomixis may actually contribute to the establishment of new polyploids (Hojsgaard, 2018) and to the diversification of angiosperms (reviewed in Brukhin and Baskar, 2019).

One of the unique features of apomixis in the genus Boechera is that it can occur at the diploid level. Diploid Boechera apomicts are highly heterozygous hybrids (Beck et al., 2012), and recent cytogenetic and population studies of the sexual and apomictic Boechera spp. have shown that these diploid genomes can be complex. Based on marker and ploidy analysis in diverse Boechera species, the emerging model proposes that, first, genetic factors for apomeiosis would independently arise. Such an individual, apomeiotic in the female side only, would stably generate seeds with a 2C embryo and 5C endosperm by self-pollination with reduced pollen. Other individuals might be apomeiotic on the male side only, generating seeds with a variety of ploidies.  Over time, eventually both femaleand male-apomeiotic individuals would cross, and the resulting seeds with a 2C embryo and 6C endosperm would become stable diploids with unreduced male and female gametes. These diploid apomicts, as they also produce fertile unreduced pollen, are then capable of fertilizing sexual diploids, which could result in triploid apomicts (Lovell et al., 2013).

Metaphase chromosome painting by genomic in situ hybridization demonstrated that all investigated apomictic lineages showed signs of a hybridogenic origin. All were found to be alloploid with a varying number of chromosomes inherited from either B. holboellii s. l. or B. stricta. The structure of their chromosomes was strongly affected by the consequences of

hybridization, resulting in aneuploidy, and the replacement of homeologous chromosomes (Kantama et al., 2007). Therefore, these apomictic Boechera spp. are not univocal diploids, rather they have a polyhaploid origin (Sokolov et al., 2011). It should be noted that these cytogenetic data do not exclude the possible existence of true diploids among Boechera spp., although they cast doubt on it. Inheritance of apomixis-related traits has been proposed to be associated with the heterochromatic chromosomes Het, Het', and Del found in apomictic diploids (Kantama et al., 2007). According to Kantama et al. (2007), all diploid apomictic accessions examined had at least four B. stricta chromosomes, including Het and Del, and the combination of these chromosomes might be important for the manifestation of apomixis.

Recent studies have shown that the Het chromosome is the altered homolog of the first chromosome of B. stricta, which underwent an accumulation of pericentromeric heterochromatin, while the Het' + Del pair is the result of Het breakage followed by a pericentric inversion in the Het' chromosome (Mandáková et al., 2015). According to earlier data, in some lineages Del could have resulted from a translocation fusing the proximal segment of the B. stricta chromosome to the distal segment of the B. holboellii s. l. chromosome (Kantama et al., 2007). However, hybridizing sexual and apomictic Boechera accessions failed to produce apomictic progeny, despite the inheritance of the Het chromosome (Schranz et al., 2006). When crossing sexual B. stricta diploids with apomictic B. divaricarpa allodiploids carrying the Het chromosome, the F1 offspring were triploid and had low fertility but were not apomictic despite carrying the Het chromosome. The F2 population displayed an array of ploidy levels and chromosome numbers, and an equally low fertility. The few F3 individuals seemed to maintain the high ploidy of their parents and fertility increased relative to their F1 and F2 ancestors, but did not reach the levels of the individuals used in the original cross. In any case, there were no apomictic progeny produced. Thus, the genetic control of apomixis in Boechera spp. is not limited to the inheritance of aberrant chromosomes (Schranz et al., 2006).

Chromosomal regions with suppressed recombination around apomixis-related genes, often in a hemizygous state and enriched with repeat sequences and transposons, has been found in many phylogenetically distant apomicts, both dicots, and monocots (Grossniklaus et al., 2001; Ozias-Akins et al., 2003; Van Dijk et al., 2009; Okada et al., 2011; Kotani et al., 2014). It is assumed that such a recombinationally inert region can contain several linked genes with different functions, the synergistic effects of which could lead to apomictic development. However, in many apomicts, the loci controlling the different components of apomixis are in distinct regions of the genome. In Boechera spp., the most likely candidates to carry such loci are the aberrant chromosomes Het, Het', and Del. Taking into account the hybridogenic nature of Boechera apomicts as a mechanism that triggered the emergence and subsequent evolution of such recombinationally inert blocks bearing apomixis-related genes, hybridization of species with incomplete chromosomal homology may have resulted in the formation of nonrecombinant, hemizygous regions from which such blocks evolved (Sharbel et al., 2010).

Several theories speculate on the mechanisms that control apomixis. Gene mutations have the appeal of the master regulator hypothesis, in which the mutation of a gene upstream of a regulatory cascade would lead to apomeiosis, parthenogenesis, and/or autonomous endosperm development (Koltunow and Grossniklaus, 2003), or the acummulation of mutations in lowrecombining regions for each aspect of apomixis, as evidenced in some of the aforementioned apomictic species. There have been various mutants identified in Arabidopsis that lead to apomeiotic phenotypes (Schmidt et al., 2015).

While no mutants have yet been studied in Boechera, two loci have been identified which correlate with female and male apomeiosis. The APOmixis Linked Locus (APOLLO), which encodes an Asp-Glu-Asp-Asp-His exonuclease, is downregulated in sexual ovules when they enter meiosis and upregulated in apomeiotic ovules (Corral et al., 2013). APOLLO shows biallelic inheritance with "apo-" and "sex-" alleles. These alleles differ in a 20-nucleotide polymorphism in the 50 untranslated region of the exonuclease gene. All tested apomictic Boechera accessions were heterozygous for the APOLLO alleles, having at least one apoallele and one sexallele, while all sexual genotypes are homozygous for sexalleles (Corral et al., 2013). APOLLO's male counterpart is the Unreduced Pollen GRAin Development2 (UPGRADE2) locus, which is exclusively expressed in PMCs of apomictic species. It encodes a chimeric long non-coding RNA (lncRNA) with the potential to form stable secondary structures. UPGRADE2 arose from duplication of UPGRADE1, followed by insertion of a functional gene and subsequent exonization, which made it transcriptionally active (Mau et al., 2013). There is a high correlation between the presence of these apomixis-associated loci and the apomictic mode of reproduction (98.4% for APOLLO, 96% for UPGRADE2 in 275 Boechera accessions from 22 species), although it was also found that, in sexuals, 2.27% had the APOLLO apoallele and 34.48% UPGRADE2. Although APOLLO is thus the most suitable diagnostic indicator of apomixis in different Boechera species and accessions (Mau et al., 2015), its function during reproduction has not yet been elucidated. The independence of APOLLO and UPGRADE2 is consistent with population genetic studies, which showed that male and female apomeiosis are inherited independently, although they usually correlate with each other at the population level (Lovell et al., 2013).

Kliver et al. (2018) found two additional, more distant copies of APOLLO, which may indicate past duplication events. An examination of apo- and sex-alleles of APOLLO indicates that they arose after the separation of the Boechera genus and form two separate clades. Given that B. retrofracta and B. stricta are sexual species, it was not surprising that they carried sexalleles of APOLLO (Kliver et al., 2018). The authors suggest an evolutionary scenario where, after triplication that likely

took place before the separation of the Brassicaceae, one of the APOLLO copies might have acquired a novel function in the common ancestor of Boechera spp., leading to the separation of the apomictic lineages. The Ka/Ks ratio of APOLLO alleles indicates that the branch leading to the apo-alleles is under positive selection (Ka/Ks = 1.4646), which is typical for paralogs that acquired a novel function.

Epigenetic changes in gene regulation have also been proposed to lie at the origin of apomixis. It has been demonstrated that in A. thaliana polyploidization following interspecific hybridization leads to dramatic changes in gene expression (Lee and Chen, 2001), making it a suitable unifier of both the hybridization and gene mutation hypothesis, whereby epialleles rather than mutant alleles would play an initial role in deregulating reproductive genes in space and time (Grimanelli et al., 2001; Grossniklaus, 2001; Spillane et al., 2001; Koltunow and Grossniklaus, 2003). sRNAs have been implicated in epigenetic reprogramming during gametogenesis and post-fertilization events (Martinez and Köhler, 2017), and of the genes involved in the sRNA pathway, AGO9 has been shown to interact with 24-nucleotide sRNAs derived from transposable elements in ovules. It is not clear if the apomeiotic phenotype of ago9 mutants is due to the lack of silencing transposable elements, or a consequence of other sRNAs that interact with AGO9 (Vielle-Calzada et al., 2012). In Boechera, sRNA expression profiling revealed Boechera-specific conserved sRNAs and microsatellite-like RNAs (misRNA), many of which have potential binding sites in exonic regions, the majority of their targets being regulatory factors. The quantitative variation in misRNA target binding was hypothesized to result from microsatellite-length polymorphisms either in their precursors or target genes, which could account for transcriptome-wide shifts in gene regulation between sexuals and apomicts (Amiteye et al., 2013). Such a shift has, in fact, been observed not only in apomictic versus sexual Boechera, but the apomictic ovule has also a significant overrepresentation of transcription factors activity (Sharbel et al., 2010; Schmidt et al., 2014), as well as a significantly different regulation of core cell cycle, sRNA pathway genes (Schmidt et al., 2014), and heterochronic differences in imprinted genes (Sharbel et al., 2010).

Although the genes controlling the components of apomixis in Boechera spp. are not yet identified, the data on the inheritance of apomixis and on apomixis-associated loci provide valuable entry points for further studies. Ultimately, functional studies of candidate genes will be required and the experimental tools required for such analysis need to be further developed.

### MOLECULAR TOOLS FOR THE GENUS Boechera: TRANSFORMATION, LASER CAPTURE MICRODISSECTION, AND TRANSCRIPTOMICS

One of the most useful tools to study molecular mechanisms is transformation for stable transgene expression. Agrobacteriummediated transformation has been the method of choice whenever possible, as any DNA sequence contained between the two tumor-inducing (Ti) borders of the plasmid can efficiently be introduced into a plant genome (Gelvin, 2003). While the "floral dip" method is standard for stable transformation of A. thaliana (Clough and Bent, 1998), most plant species require more elaborate transformation procedures. The most widely used method is co-cultivation of explants with Agrobacterium, which then transform into callus tissue and subsequently undergo organogenesis to regenerate a transformant plant. With this method, even recalcitrant cultivars of various crops can be successfully transformed (reviewed in Altpeter et al., 2016).  Somatic embryos derived from immature cotyledons of the apomict B. divaricarpa (Ta¸skin et al., 2009b) and, as a proof-of-concept, stable transformants of B. gunnisoniana were also generated (Ta¸skin et al., 2003). These advances open up the genus Boechera to the possibilities offered by the study of transgenic lines.

Major advances have also been made in the characterization of transcriptomes in sexual and apomicitic Boechera spp. Microarrays were first used to describe transcriptomes in Boechera spp., followed by various sequence-based approaches, including SuperSAGE using Sanger sequencing (Matsumura et al., 2006). Currently, RNA-sequencing (RNA-seq), based on next generation sequencing technologies, is the method of choice to study transcriptomes (Wang et al., 2009). Continuous methodological improvements now allow high precision and high throughput studies of single cell (Picelli et al., 2014), live (Lovatt et al., 2014), and low input (Schmidt et al., 2012; Florez-Rueda et al., 2016) transcriptomes.

The first transcriptomic studies in Boechera spp. were performed by Sharbel et al. (2009, 2010) using the SuperSAGE technique (Matsumura et al., 2006). They quantified gene expression in manually dissected ovules at the MMC stage of two sexual (B. stricta and B. holboelli) and two apomictic accessions (both B. divaricarpa). Additionally, two cDNA libraries representing apomictic and sexual accessions were sequenced using Roche's 454 technology (Sharbel et al., 2009). These were the first reference transcriptomes for the genus Boechera and formed the basis for future studies (see below). In a second study, Sharbel et al. (2010) quantified gene expression between a single apomict and a single sexual Boechera individual at four different developmental time points but without biological or technical replication. Stage-specific and heterochronic patterns of gene expression were identified (Sharbel et al., 2010). Because these first transcriptomic studies (Sharbel et al., 2009, 2010) used single libraries from single individuals without biological replication, they cannot account

for variation between individuals and lack the statistical power for a robust identification of genes that are differentially expressed in sexuals versus apomicts (Lee et al., 2000; Meyers, 2004; Conesa et al., 2016).

The sRNA fraction of the Boechera transcriptome (Amiteye et al., 2011) was identified by a reanalysis of transcriptome data (Sharbel et al., 2009) and the sequencing of two sRNA libraries (Amiteye et al., 2013). Using a Boechera-specific microarray based on the sexual and apomictic reference transcriptomes (Sharbel et al., 2009), an analysis of copy number variation (CNV) in transcriptionally active regions of 10 sexual and 10 apomictic Boechera accessions was performed (Aliyu et al., 2013). The gene ontology classes found enriched in apomictic CNVs (e.g., pollenpistil interaction), led to the hypothesis that CNV in these gene classes serves to buffer the effects of deleterious mutations.

The first attempt to compare sexual and apomictic development in Boechera spp. at the cellular level was pioneered by Schmidt et al. (2014). While previous studies used whole ovules (Sharbel et al., 2009, 2010), they used laser-assisted microdissection (LAM) to isolate the apomictic initial cell (AIC), nucellus, egg, central, and synergid cells of the triploid apomict B. gunnisoniana (Schmidt et al., 2014). After LAM, cDNA libraries were produced and sequenced using SOLiD technology. A reference transcriptome from pooled floral tissues of B. gunnisoniana was sequenced with Illumina technology (Schmidt et al., 2014). An analyses of gene expression and gene ontology enrichment uncovered the upregulation of spermidine metabolism and patterns of altered expression in the AIC. Comparison to female gametophyte cell-specific transcriptomes of A. thaliana (Wuest et al., 2010) identified regulatory pathways that differ between sexual and apomictic germlines, including hormonal, epigenetic, cell cycle control, and transcriptional regulatory pathways (Schmidt et al., 2014). Likewise, comparison of egg cell-specific transcriptomes of B. gunnisoniana and A. thaliana identified genes expressed only in the apomictic egg cell (Florez-Rueda et al., 2016). Future studies that exploit single-cell transcriptomics by comparing apomictic and sexual Boechera spp. are expected to shed light onto the molecular basis of apomixis in the genus Boechera.

A study of apomictic and sexual Boechera seedlings focused on the response to abiotic and biotic conditions and stressspecific changes that might underlie apomixis (Shah et al., 2016). A relationship between apomixis and environmental conditions is also supported by a phenomenon known as

1Annotated proteins were used for BUSCO benchmarking C, complete; S, complete-single-copy; D, complete-duplicated; F, fragmented; M, missing BUSCO groups were found (Waterhouse et al., 2017).

FIGURE 5 | The B. divaricarpa genome is extremely heterozygous. Distribution of the number of 19-mer occurrences in B. stricta, which has only one peak, indicating a highly homozygous genome, and B. divaricarpa, which has 2 peaks with a pronounced difference in height. Note that the number of occurrences of different K-mers in the first peak (ca. 90) is half of that in the second peak (ca. 180), suggesting that first and second peak represent the heterozygous and homozygous part of the genome, respectively. N50 values are based on the assembly of Illumina paired-end reads (100× coverage) by Platanus. Heterozygosity has an immediate effect on the contiguity of the genome assembly.

"geographical parthenogenesis" (reviewed in Hörandl, 2006). This concept is based on the observation that apomictic lineages have larger distributional ranges than their sexual relatives. In Boechera spp., niche differentiation was found to be driven by ploidy rather than reproductive mode (Mau et al., 2015), indicating low support for geographical parthenogenesis in the genus. Nevertheless, the great variation in ploidy level and reproductive mode and its relationship with niche differentiation make the genus Boechera a good model for the study of plant-environment interactions (Rushworth et al., 2011). From this perspective, the transcriptomes of an obligate triploid apomict and a diploid sexual, both isolated from a droughtprone habitat, were compared. Specific meiotic genes were found to be down-regulated and stress-related transcription factors and chaperons upregulated in apomictic seedlings (Shah et al., 2016), but the relevance of these findings for reproduction is unknown.

#### GENOMIC RESOURCES FOR THE GENUS Boechera AND CHALLENGES TO GENOME ANALYSIS

The advent of next generation sequencing along with progress in bioinformatics tools opened a new chapter in the study of apomixis, allowing the search for apomixis-associated loci and the comparison, genotyping, and phylogenetic analysis of Boechera species and accessions using whole-genome sequencing.

Currently, the genomes of only two Boechera spp. have been assembled and published (**Table 2**). Both B. stricta and B. retrofracta are self-pollinating, diploid sexuals and have largely homozygous genomes, which are straightforward to assemble. Notably, repeats in the genome of B. retrofracta occupy almost 40% of the genome space. Nearly half of them are long terminal repeats (LTRs) (18.27%) (Kliver et al., 2018). In contrast, only 20% of the B. stricta genome are annotated as repeats (Lee et al., 2017, assembly v1.2). The difference in the repeats number correlates with the difference in their genome sizes of the (**Table 2**). In some apomictic species, the apomixis loci are associated with heterochromatin and/or substantial repetitive sequences (Hand and Koltunow, 2014). The chromosomes carrying the LOSS-OF-APOMEIOSIS (LOA) locus in Hieracium praelatum and the APOSPORY-SPECIFIC GENOMIC REGION (ASGR) in Pennisetum squamulatum are characterized by extensive repetitive sequences and transposon-rich regions (Okada et al., 2011). In apomictic Paspalum simplex, the region containing apomixis-related loci has undergone largescale rearrangements due to transposable elements (Calderini et al., 2006). These similarities in repetitive, heterochromatic regions in the genomes of apomicts have led to the hypothesis that these regions might serve as a sink to sequester factors involved in sexual reproduction, triggering apomixis (Grossniklaus, 2001; Koltunow and Grossniklaus, 2003). In line with this idea, some Boechera apomicts have largely heterochromatic chromosomes (Kantama et al., 2007) and some transposon families were found enriched in an apomictic Boechera lineage (Aliyu et al., 2013). Due to extensive hybridization within the Boechera genus, however, the repeat content of the genome might not reflect the mode of reproduction but rather its phylogeographic history.

The final genome annotation of B. stricta and B. retrofracta encompassed about 270 000 genes in both species. The presence of a slightly greater number of predicted transcripts in B. stricta can be explained by lack of gene expression data for B. retrofracta, which resulted in a less complete gene annotation overall, as confirmed by BUSCO benchmarking (**Table 2**).

Assembling the genomes of diploid apomictic Boechera species is difficult because they exhibit high levels of heterozygosity (**Figure 5**), which results from the combination of disparate genomes as consequence of their hybridogenic origin (Beck et al., 2012). For example, the genome heterozygosity rate of B. divaricarpa is around 2.5% as estimated by GenomeScope (Vurture et al., 2017). Because of all the reasons mentioned above, sequencing and de novo assembly of such a plant genome can result in a highly fragmented genome draft. Annotation of the protein coding genes may not always be correct,

TABLE 3 | Publicly available genomic sequencing data1 of Boechera genus suitable for reference-free (k-mer based) analysis.

1 Illumina pair-end libraries only. 2Times of typical Boechera genome size (220Mbases). 3Reproductive mode assignment is based on k-mer profile.

considering that nearly identical genes are notoriously difficult to assemble. Thus, a mosaic sequence can be formed that does not represent any member of the gene family. The high level of fragmentation and mis-assembly could prevent our ability to draw true conclusions about the evolution of apomixis-associated loci and the molecular mechanisms underlying this interesting phenomenon (Claros et al., 2012).

A key challenge is the assembly of the short reads into contiguous sequences (contigs), which then are assembled into chromosome-scale scaffolds. Another complication is the assignment of genetic variants to the correct homeologous chromosome, a process known as haplotyping (Korbel and Lee, 2013).

Only recently, approaches have been developed that are capable to solve the problem of heterozygous genome assembly. Pacific Biosciences long-read sequencing technology and FALCON/FALCON-Unzip algorithms were used to assemble heterozygous genomes including an F1 hybrid of A. thaliana and the widely cultivated Vitis vinifera cv. Cabernet Sauvignon (Chin et al., 2016). Further development of this assembler resulted in FALCON-Phase, a new method that reconstructs contig-length phase blocks using Hi-C short-reads, which is able to produce true diploid assemblies (Kronenberg et al., 2018). Linked-Read sequencing technology (10× Genomics) has recently been successfully employed for a de novo assembly of the heterozygous F1 diploid pepper (Capsicum annuum) hybrid genome (Hulse-Kemp et al., 2018).

Recently, methods for a haplotype-aware, phased assembly of polyploid genomes were developed for cases where the parental species are known (Akama et al., 2014; Kyriakidou et al., 2018). However, speciation in the genus Boechera has a very complex history, where sexual diploids gave rise to multiple apomictic species through hybridization-associated polyploidy, alloploidy, and aneuploidy. This complexity of speciation resulted in the unprecedented genome diversity of the apomictic species in this genus, which was further exaggerated by mutation accumulation (Lovell et al., 2017) and elevated transposon activity in the apomicts (Ferreira de Carvalho et al., 2016). It is thus often not clear what the parental ancestors of polyploid Boechera spp. are. Genomic analysis based on a haploid reference genome might not reflect the reality, especially for apomicts with highly heterozygous diploid/polyploid genomes. In such a situation, many loci might be completely absent in the reference genome because even in the ideal case, it represents only a consensus genome. Bearing in mind also the problems in producing genome assemblies for apomictic species, we would like to outline the potential of an alternative, reference-free approach for comparative genomic analyses of sexual and apomictic species in the genus Boechera.

A reference-free (or, more general, alignment-free) approach to sequence comparison does not rely on alignment and, therefore, it is especially valuable for analyzing genomes of organisms that do not have a reference (Zielezinski et al., 2017). K-mer or word frequency method is one of the most popular alignment-free method for comparative genome analysis, but its successful application can be hindered by insufficient sequencing depth and biases of genome sampling. Illumina paired-end (PE) sequencing of random-primed libraries produce the most suitable data for processing by this approach. **Table 3** provides a selection of next generation sequencing data from Boechera spp. that satisfy the requirements for alignment-free methods.

  The K-mer profile provides an estimate of effective sequence coverage and reflects

the rate of genome heterozygosity, the amount of sequencing errors along with errors of sample preparation and sequencing data processing (Supplementary Note 1 in Vurture et al., 2017).  The K-mer profiles of the apomictic individuals is shaped by a higher level of heterozygosity compared to sexual individuals (Li et al., 2017) and, in case of high sequence coverage, the profile contains two peaks which represent the heterozygous and homozygous part of the genome.

We also used an alignment-free method to investigate genetic relatedness in the B. spatiofolia individuals analyzed by Lovell et al. (2017). **Figure 7** shows genetic variation analysis analogous to the one described by Lovell et al. (2017, **Figure 1B**) but using Mash software for genetic distance estimation (Ondov et al., 2016). In contrast to Lovell et al. (2017) who used A. lyrata as reference for the alignment of B. spatiofolia sequencing reads, this approach is based solely on the data contained in the reads. Nevertheless, the resulting tree is rather similar.

### CONCLUSION

Apomixis produces progeny that is genetically identical to the mother plant, a trait of great agronomical importance. Unfortunately, the molecular mechanisms underlying apomixis are only poorly understood. A better understanding of the genetic networks that control the components of apomixis are crucial for its introduction into crop plants. Apomicts of the genus Boechera represent a convenient model to study apomixis as it also occurs at the diploid level and genomes of Boechera spp. are comparatively small. Despite these advantages, genome assembly and annotation of the apomictic Boechera lineages is complicated due to such phenomena as a high level of heterozygosity of their genomes, which results from chromosome rearrangements, accompanied by alloploidy, aneuploidy, and substitutions of homeologous chromosomes occurring during hybridization events. The use of next generation sequencing and novel bioinformatic approaches should help to overcome these challenges and facilitate generating the first comprehensive genome of an apomictic plant in the near future. Attempts to apply reference-free methods for the assembly and comparative analysis of such genomes are currently underway. A combination of systems biology approaches to analyze RNAseq and genomic data from sexual and apomictic Boechera species, as well as functional approaches in transgenic plants, will facilitate the disentanglement of the genetic control of apomixis at the molecular level. This is prerequisite for the engineering selfsustaining, apomictic hybrids in sexual crop plants.

#### DATA AVAILABILITY

fpls-10-00392 March 30, 2019 Time: 16:29 # 15

All datasets generated for this study are included in the manuscript and/or the supplementary files.

#### AUTHOR CONTRIBUTIONS

VB and UG designed and directed the study, wrote the introduction and conclusion, and advantages of the Boechera genus for the study of apomixis. JO and VB analyzed the systematic position of Boechera and habitats. JO, MSN, and VB carried out cyto-embryological studies in the Boechera

#### REFERENCES

genus. AF-R and UG worked on population genetics of Boechera with respect to apomixis. VB and MSN performed the inheritance and genetic aspects of apomixis in Boechera. MSN and AF-R performed the molecular experiments in Boechera: transformation, laser capture microdissection, and transcriptomics. DS and EB analyzed the available NGS data and genomic resources for Boechera. EB inquired the transcriptomic investigations for analysis of apomictic plants. All authors read and approved the final manuscript.

#### FUNDING

This work was supported by the University of Zurich and grants 16-54-21014 from the Russian Foundation for Basic Research and 1.52.1647.2016 from St. Petersburg State University (to VB), as well as the Marie Curie Action IDP BRIDGES from the European Union and grant IZLRZ3\_163885 from the Swiss National Science Foundation (to UG) through the Scientific & Technological Cooperation Programme Switzerland-Russia (STCPSR). Research in St. Petersburg was carried out within the framework of the state assignment No. AAAA-A18-<PHONE_NUMBER>12-8 to BIN RAS.

by the rust fungus Puccinia monoica in Boechera stricta. PLoS One 8:e75293. doi: 10.1371/journal.pone.0075293

contribute to genomic divergence under asexuality. BMC Genomics 17:884. doi: 10.1186/s12864-016-3234-9

Spring Harb. Symp. Quant. Biol. 77, 17–21. doi: 10.1101/sqb.2012.77. 014894

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer AJ declared a past co-authorship with one of the authors UG to the handling Editor.

Copyright © 2019 Brukhin, Osadtchiy, Florez-Rueda, Smetanin, Bakin, Nobre and Grossniklaus. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Loss-of-Function of a Tomato Receptor-Like Kinase Impairs Male Fertility and Induces Parthenocarpic Fruit Set

Hitomi Takei1,2† , Yoshihito Shinozaki1,2† , Ryoichi Yano1 , Sachiko Kashojiya1 , Michel Hernould3,4, Christian Chevalier3 , Hiroshi Ezura1,5 and Tohru Ariizumi1,5 \*

1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan, 2 Japan Society for the Promotion of Science (JSPS), Kôjimachi, Japan, 3 UMR1332 BFP, Institut National de la Recherche Agronomique (INRA), Villenave-d'Ornon, France, 4 UMR1332 BFP, University of Bordeaux, Bordeaux, France, 5 Tsukuba-Plant Innovation Research Center, University of Tsukuba, Tsukuba, Japan

#### Edited by:

Andrea Mazzucato, Università degli Studi della Tuscia, Italy

#### Reviewed by:

Mariola Plazas, Instituto de Biología Molecular y Celular de Plantas (IBMCP), Spain Giuseppe Leonardo Rotino, Council for Agricultural Research and Economics, Italy

#### \*Correspondence:

Tohru Ariizumi<EMAIL_ADDRESS>†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science

Received: 15 November 2018 Accepted: 18 March 2019 Published: 16 April 2019

#### Citation:

Takei H, Shinozaki Y, Yano R, Kashojiya S, Hernould M, Chevalier C, Ezura H and Ariizumi T (2019) Loss-of-Function of a Tomato Receptor-Like Kinase Impairs Male Fertility and Induces Parthenocarpic Fruit Set. Front. Plant Sci. 10:403. doi: 10.3389/fpls.2019.00403 Parthenocarpy arises when an ovary develops into fruit without pollination/fertilization. The mechanisms involved in genetic parthenocarpy have attracted attention because of their potential application in plant breeding and also for their elucidation of the mechanisms involved in early fruit development. We have isolated and characterized a novel small parthenocarpic fruit and flower (spff) mutant in the tomato (Solanum lycopersicum) cultivar Micro-Tom. This plant showed both vegetative and reproductive phenotypes including dwarfism of floral organs, male sterility, delayed flowering, altered axillary shoot development, and parthenocarpic production of small fruits. Genome-wide single nucleotide polymorphism array analysis coupled with mapping-by-sequencing using next generation sequencing-based high-throughput approaches resulted in the identification of a candidate locus responsible for the spff mutant phenotype. Subsequent linkage analysis and RNA interference-based silencing indicated that these phenotypes were caused by a loss-of-function mutation of a single gene (Solyc04g077010), which encodes a receptor-like protein kinase that was expressed in vascular bundles in young buds. Cytological and transcriptomic analyses suggested that parthenocarpy in the spff mutant was associated with enlarged ovarian cells and with elevated expression of the gibberellin metabolism gene, GA20ox1. Taken together, our results suggest a role for Solyc04g077010 in male organ development and indicate that loss of this receptor-like protein kinase activity could result in parthenocarpy.

Keywords: Solanum lycopersicum, fruit set, male sterility, in situ hybridization, next generation sequencing, gene mapping

**Abbreviations:** Bc1F2, Single backcross and second filial generation; Bc3F2, Backcross three times and second filial generation; Bcip, 5-Bromo-4-chloro-30 -indolyl phosphate; Bsa, Bovine Serum Albumin; F1, First filial generation; F2, Second filial generation; Faa, Formalin-Acetic acid-Alcohol; Ms, Murashige and Skoog medium; Nbt, Nitroblue Tetrazolium; Pbs, Phosphate buffered saline; Rnai, Rna interference; Sgn, Sol Genomics Network; Snp, Single Nucleotide Polymorphism; Spff, Small Parthenocarpic Fruit and Flower; Sspe, Sodium Chloride-Sodium Phosphate-Edta.

## INTRODUCTION

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The flower-to-fruit transition, also known as "fruit set," corresponds to a major developmental shift that transforms an ovary into a fruit (Gillaspy et al., 1993). This genetically programmed process is coordinated by a complex network of signaling pathways that are activated by interacting endogenous and exogenous cues, although the genetic and molecular factors that control the flower-to-fruit transition remain poorly understood (Ariizumi et al., 2013). The development of parthenocarpic fruit has been observed under some conditions; this pollination-independent seedless fruit can arise when fertilization is inefficient, mainly due to male sterility. Some naturally occurring tomato genetic parthenocarpy has been identified, and these parthenocarpic mutants have been designated pat, pat-2, and Pat-k/SlAGL6 (Shinozaki and Ezura, 2016; Klap et al., 2017; Takisawa et al., 2018). The pat mutant is characterized by short anthers, partial male sterility, and the production of small fruits (Mazzucato et al., 1998). The locus of the gene responsible for pat phenotypes was narrowed down to chromosome 3 (Beraldi et al., 2004). In addition, the gene encoding SlGA20ox1, the key enzyme for gibberellin (GA) accumulation in the pollinated tomato ovary, is highly expressed in pat ovaries; this is likely to activate GA metabolism and increase GA levels in the unpollinated ovaries, thus triggering parthenocarpy (Olimpieri et al., 2007). The pat-2 phenotype appears to be caused by a recessive mutation at a single locus on chromosome 4, in a gene encoding a zinc finger homeodomain protein (Nunome, 2016); GA also accumulates at high levels in unpollinated pat-2 ovaries (Fos et al., 2000). Furthermore, it has been shown that fruit set initiation through both pollination-dependent and -independent processes occurs concomitantly with the down-regulation of a family of floral homeotic MADS-box genes, which regulate floral organ identities (Wang et al., 2009; Tang et al., 2015). Indeed, the loss of function of several MADS-box genes can cause tomato parthenocarpy. For instance, the loss of function of tomato MADS-box 29, tomato MADS-box 5, and DEFENCIENS/TOMATO APETALA3/STAMENLESS result in parthenocarpy, together with abnormal stamen differentiation (Pnueli et al., 1994; Ampomah-Dwamena et al., 2002; Mazzucato et al., 2008; Quinet et al., 2014; Okabe et al., 2019). Moreover, parthenocarpy was induced in tomatoes that were genetically transformed in order to inhibit stamen development at an early stage of differentiation via the expression of the BARNASE ribonuclease gene under a stamen-specific promoter (Medina et al., 2013). Although the mechanisms underlying the role of the stamen in parthenocarpy have not yet been fully characterized, it has been hypothesized that stamens could counteract fruit set initiation before pollination in tomato plants, and this may be associated in part with elevated levels of GA (Okabe et al., 2019).

Flowers and fruits are considered to represent sink organs because their development requires high level of nutrients such as sucrose, as a carbon source (Osorio et al., 2014). The vasculature within flowers, fruits, and their pedicels is therefore of major importance because it transports nutrients and water to these organs (Ranci ˇ c et al., 2010 ´ ). XYLEM INTERMIXED WITH PHLOEM1 (XIP1) is one of the proteins with a key role in the organization of vasculature in Arabidopsis (Shiu and Bleecker, 2001). This protein is a leucine-rich repeat receptor-like kinase (RLK) that belongs to a large family with at least 216 members encoded in the Arabidopsis genome. A loss of XIP1 resulted in modification of vascular bundle organization and abnormal lignification of phloem cells, transforming them to xylem cells (Bryan et al., 2012).

To identify key regulators of parthenocarpy, the present study characterized a novel tomato parthenocarpic mutant known as small parthenocarpic fruit and flower (spff ), which was isolated from a population where mutations were introduced via exposure to γ-ray irradiation (Saito et al., 2011). The spff mutant exhibits small flower formation, male sterility, and increased transcription of GA20ox1 in young ovaries. Furthermore, a rapid highthroughput approach followed by functional validation using RNA interference (RNAi) resulted in the identification of a gene encoding a novel RLK protein.

### MATERIALS AND METHODS

#### Plant Material and Growth Conditions

Tomato wild-type (WT) plants, Solanum lycopersicum "Ailsa-Craig" and "Micro-Tom," and spff mutant plants were grown in pots and irrigated daily with Otsuka first and Otsuka second fertilizer solutions under greenhouse conditions in Tsukuba, Japan. The greenhouse was maintained at the ambient temperature and light photoperiod in July and August. WT S. lycopersicum "Micro-Tom" and spff plants for RNA sequence (RNA-seq) analysis, RNAi experiments, and histological analyses were grown in rockwool and irrigated daily with Otsuka first and Otsuka second under vertical farm conditions at 25◦C with a 16/8 h light/dark cycle.

#### Histological Analysis

Histological analysis of flower tissues was processed as described by Hao et al. (2017). Wax-embedded floral buds were cut into 10-µm cross-sections, layered onto glass slides, and dried overnight at 42◦C. The cell size and the number of cell layers were evaluated and the significance of group differences were statistically analyzed using Student t test.

#### Pollen Number and Germination Assay

Pollens were obtained from anthers at the anthesis stage and germinated in 1 mL of pollen germination medium (0.52 M sucrose, 1.6 mM boric Acid, 1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM MgSO4, and 0.01 mM Tris–HCl, pH 7.0). After incubation for 16 h at room temperature, pollen grains were observed under a light microscope. The pollen germination ratio was calculated by dividing the number of germinated pollen (in which the size of the pollen tube is twice or more the diameter of the pollen grain) by the total number of pollen grains; this was defined as the number of pollens observed within one microscopic field. The determinations were made for three replicate biological experiments.

#### High-Density Genetic Mapping

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For genetic mapping by an Infinium assay (Illumina) using the SolCAP single-nucleotide polymorphism (SNP) array1 , an F2 population was derived from a cross between the spff mutant (Micro-Tom background) and WT plants (Ailsa-Craig background). Genomic DNA of 44 F2 plants (43 with spff mutant phenotypes and one with the WT phenotype), together with F1, WT Micro-Tom and parental plants of each genotype, was extracted from fresh leaves using Maxwell 16 DNA purification kits, according to the manufacturer's protocol (Promega). A total of 48 DNA samples were then used for the SolCAP analysis, using the method described by Sim et al. (2012). Of the 7600 markers analyzed, 1956 markers showed polymorphisms that distinguished between Micro-Tom and Ailsa-Craig; these were used for genotyping. SNPs were obtained from the Kazusa Marker Database2 . For the linkage analysis, we examined the genotypes at the position 59,966,064 bp on chromosome 4 with the tomInf4732 SNP marker, with sequences of AAGCTT and AAGATT in Micro-Tom and Ailsa-Craig, respectively. Each genotype was discriminated using the primers listed in **Supplementary Table S1**, followed by restriction digestion with Hind III for 8 h at 37◦C.

### Mapping-By-Sequencing

For further fine mapping based on the mapping-by-sequencing approach (Abe et al., 2012; Garcia et al., 2016), an F2 population was constructed by crossing the spff mutant and WT, in the Micro-Tom background (**Supplementary Figure S1**). Genomic DNA was extracted from fresh leaves of F2 plants that exhibited the spff mutant phenotype, as described above. The same amount of extracted DNA from 20 individual plants was pooled and sequenced by 100 bp paired-end sequencing (HiSeq 2000; Illumina). Mutation or variant information was obtained using the Bowtie2-Samtools-GATK (Genome Analysis Tool Kit) pipeline (Li et al., 2009; McKenna et al., 2010; Langmead and Salzberg, 2012). Briefly, Illumina short reads were aligned onto the tomato genome reference SL2.40 by Bowtie2 version 2.2.13 with default parameters. Mutations or variants including SNPs or insertion-deletions (Indels) were then detected by GATK version 3.5 (McKenna et al., 2010). SNPs and Indels that might cause nonsynonymous amino acid substitution, a premature stop codon, or frameshift were identified using HaplotypeCaller, as described previously (McKenna et al., 2010; Pulungan et al., 2018). Allele frequency datasets were also obtained using GATK. Because the Micro-Tom cultivar is not inbred and relatively many intracultivar variations are present between individuals, we subtracted such intra-cultivar variants from the SNP/Indel datasets using next generation sequencing datasets of several WT Micro-Tom individuals (Pulungan et al., 2018). Candidate genes with a high SNP/Indel index and reliable read numbers (≥10) were then identified. In this analysis, the SNP/Indel index was calculated as the proportion of sequenced reads that included mutant allele SNPs or Indels, in relation to the WT allele.

## Linkage Analysis of the spff Locus

The spff mutant was backcrossed four times with Micro-Tom WT in order to purify the responsible mutation and finally obtain BC4F2 plants (**Supplementary Figure S1**). Linkage analysis was performed using DNA extracted from F2, BC2F2, BC3F2, and BC4F2 populations (**Supplementary Table S1**). Genomic DNA was extracted by DNeasy Miniprep kit (QIAGEN) and amplified by PCR with TaKaRa Ex Taq (TAKARA) and the primer set shown in **Supplementary Table S2**. The PCR products were purified by the Illustra ExoStar kit (GE Healthcare) and then sent to Eurofins Genomics for sequencing.

#### Construction of the RNAi Plasmid

The RNAi construct was designed using Gateway technology (Invitrogen). Total RNA was extracted from WT ovaries using the RNeasy Plant Mini Kit (QIAGEN), followed by the removal of genomic DNA using RNA Clean & Concentrator (ZYMO RESEARCH). cDNAs were then synthesized using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). A 521 bp fragment of the Solyc04g077010 transcript was amplified using the KOD Plus kit (TOYOBO); the cDNA was used as the template, and SlXIPRNAiF1 and SlXIPRNAiR1 were the primers (**Supplementary Table S2**). The amplicon was then cloned into the donor pBI-sense, antisense-GW vector (INPLANTA INNOVATIONS INC., Japan), allowing expression under the control of the constitutive 35S promoter. The resulting plasmid was introduced into WT Micro-Tom by Agrobacteriummediated transformation using A. tumefaciens GV2260 (Sun et al., 2006). Transgenic lines were selected on Murashige and Skoog (MS) agar plates containing kanamycin (100 mg L−1 ).

#### RNA Sequencing

Ovaries were collected from flowers at anthesis, separated into three replicates (15–17 ovaries in each replicate) and ground in liquid nitrogen. Total RNA extraction from the ovaries and subsequent cDNA synthesis were performed as described above. Genome-wide RNA expression levels were analyzed by HiSeq (Illumina) with 100 bp single-end reads. The raw reads were subjected to quality filtering before employing the TopHat2- Cufflinks pipeline to calculate the number of reads and calculate expression levels using the reads per kb of transcript per million mapped reads (RPKM), as described previously (Yano et al., 2018). Comprehensive data were analyzed using multiple t tests (p < 0.05), followed by the Bonferroni correction method, with false discovery rate analysis. Genes with mean RPKM values of ≥1 (three replicates) were considered to be expressed. Genes were considered differentially expressed if the log2 fold ratios were ≥ 1.0 or ≤ −1.0, with false discovery rate adjusted p values (q values) of < 0.05.

### Expression Analysis by Quantitative Reverse Transcription PCR (qRT-PCR) and RT-PCR

For qRT-PCR analysis, the leaves were ground to a fine powder in liquid nitrogen. Total RNA extraction from the samples and subsequent reverse transcription reactions were performed as

1http://solcap.msu.edu

2http://marker.kazusa.or.jp/Tomato/

3https://solgenomics.net/organism/Solanum\_lycopersicum/genome

described above. PCRs were carried out by the CFX96 system (Bio-Rad), using the SYBR Premix Ex Taq kit (TaKaRa) and the appropriate gene-specific primers (**Supplementary Table S2**) according to previously described procedures (Shinozaki et al., 2015). Technical triplicates were performed for each sample, with biological triplicates. The expression levels were calculated using the delta-delta CT method (Pfaffl, 2001), with normalization to the expression of the reference gene, SAND (Expósito-Rodríguez et al., 2008). For RT-PCR analysis, cDNA synthesis was performed as described above and equal amount of cDNA was used as templete to observe level of SPFF mRNA in various tissues.

#### In situ Hybridization

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The riboprobes used to detect spff transcripts were made from a 775 bp fragment amplified from tomato root cDNAs by PCR using the ishF2-ishR1 primer set. The PCR product was used for subsequent PCR using the ishT7F2-ishR1 primer set for sense, and the ishF2-ishT7R1 primer set for antisense, riboprobes; this introduced the T7 RNA polymerase promoter at the 50 and 30 ends, respectively. Labeled riboprobes were synthesized by in vitro transcription in the presence of digoxigenin-UTP (DIG RNA Labeling kit, SP6/T7; Roche) and used for in situ hybridization. The plant tissue processing and in situ RNA hybridization experiments were performed following the protocol described by Sicard et al. (2008). Primer sequences used in this study are shown in **Supplementary Table S2**. For the comparative analysis between WT and spff mutant, both WT and spff mutant samples were mounted on the same glass slides to allow the direct comparison under the same condition.

### RESULTS

#### Identification of the Single Recessive Parthenocarpic spff Mutant

A visual screening of tomato M3 populations obtained after γ-ray irradiation-induced mutagenesis in the genetic background of Micro-Tom, a dwarf and rapid growth variety (Matsukura et al., 2007; Saito et al., 2011), resulted in the isolation of a mutant line (TOMJPG4121) that produced small seedless parthenocarpic fruit (**Figure 1A**). These plants also produced smaller flowers than the WT plant, particularly due to their narrower petals and shorter anthers (**Figure 1B**). We therefore called this line the spff mutant. Although the spff mutant did not produce seeded fruits by practical self-pollination, crossing WT pollen to the spff stigma did result in seeded fruits (**Figure 1A**); these F1 seeds germinated normally, suggesting that spff is male-sterile, with the ovary retaining substantial fertility. Furthermore, all of the resulting six F1 plants exhibited normal flower morphology, with no evidence of parthenocarpic ability, indicating that these mutant phenotypes were recessive. Thirty-three out of 109 F2 progenies obtained through crossing with the WT cultivar Micro-Tom, and 43 out of 186 F2 progenies obtained through crossing with the WT cultivar Ailsa-Craig, exhibited the spff mutant flower morphology and parthenocarpy phenotypes (**Table 1** and **Supplementary Figure S2**). These segregation ratios

FIGURE 1 | Parthenocarpic fruit and small flower production in spff mutant cultivar Micro-Tom. (A) Representative fruit of the spff mutant. Spontaneously obtained parthenocarpic fruit (left) and seeded fruit from manual pollination of the flower with WT pollen (right) are shown. (B) Comparison of WT and spff flowers at anthesis stage. Bars are 1 cm.

corresponded to the expected 3:1 for a single recessive gene (Chi-squared = 1.62 for Micro-Tom and 0.35 for Ailsa-Craig background at p < 0.05 ). These data suggested the presence of a monogenic recessive mutation in the spff line. In the spff and WT cultivar Micro-Tom or Ailsa-Craig F2 populations, anthesis of the first flower was delayed in the plants with the spff phenotype for 19 or 15 days, respectively, as compared to plants with the WT phenotype (**Supplementary Figure S3**); this indicated that the flowering delay trait was tightly associated with the spff flower morphology and parthenocarpy phenotypes.

### Characterization of the Pleiotropic Mutant Phenotypes in spff

For detailed phenotypic characterizations, the spff mutant in the M3 population was backcrossed four times with WT cultivar Micro-Tom pollen to reduce mutagen-induced background mutations (**Supplementary Figure S1**). The resulting BC4F2 plants that exhibited spff phenotypes were analyzed. First, we examined the parthenocarpic phenotype in the spff mutant. The spff yielded obligate parthenocarpic fruit under spontaneous

TABLE 1 | Segregation test of spff mutant traits at F2 progeny of a self-pollinated F1 plant.

The spff mutant phenotypes were evident as small flower and parthenocarpic fruit formation.

achi-squared test (p < 0.05). ns, not significant.

production, and this was not observed in WT plants (**Figure 2A**). Compared to the pollinated WT fruits, the spff parthenocarpic fruits were smaller and lighter (**Figures 2B–E**). For cytological characterization of parthenocarpy at the early developmental stage, we prepared cross-sections of the ovaries at anthesis and examined the number of cell layers and cell size within the pericarp (**Figure 3**). The spff mutant cells were significantly larger than the WT cells, by approximately 1.3-fold (WT = 202 ± 17 µm2 , spff = 272 ± 11 µm2 ), and spff had fewer

cell layers. This suggested that spff parthenocarpy was associated with cell enlargement, rather than active cell division.

Further, the smaller flowers produced in spff reflected the presence of smaller constitutive tissues, including the petals, style, and anthers; the clearly defective anther may explain the malesterility of this mutant (**Figures 4A–H**). To evaluate the male fertility of spff, cross-sections of the WT and spff anthers at the anthesis stage were compared. The oval-shaped WT anther locules included pollen grains that showed a germination rate

of approximately 60 ± 5% (**Figures 4D,I,K,L**). In contrast, the spff anther locules were shrunken and contained very few pollen grains, which were unable to germinate (**Figures 4H,J–L**); this indicated that the spff mutant was fully male sterile. In addition, histological observations of the spff and WT ovaries at the bud length 4 mm indicated the presence of equivalent internal structures, except for their size (**Figures 4M–O**), consistent with the fact that the spff retained substantial female fertility (**Table 1**).

We also found that spff affected plant architecture, with an altered pattern of axillary shoot development (**Supplementary Figures S4A–C**). The lateral branches of spff showed increased sympodial growth, in which vegetative and inflorescence stems were more actively developed from the individual first axillary buds, leading to a bushy plant morphology. These data characterizing the phenotypes of spff indicated that the mutation conferred pleiotropic effects on both reproductive and vegetative morphology in tomato plants.

We next compared yield potential between WT and spff mutant. Since spff mutant showed significant growth delay compared to WT leading to late fruit production (**Figures 5A,B**), which made it difficult to conduct comparative yield quantification, WT and spff mutant plants were grown in a greenhouse for 112 and 173 days, respectively, until they nearly reached vegetative growth maturation, determining the yield of ripe red fruits as well as the total number of fruits per plant. The yield (total weight) of ripe red fruit in spff mutant was reduced to 28 % of WT albeit longer growth period and higher number of fruits per plant, suggesting less impact of its potential for improving yield (**Figures 5C–F**).

### Identification of the Gene Associated With the spff Phenotype

The spff mutation was mapped using an F2 population obtained by crossing spff mutants (Micro-Tom background) with WT plants (Ailsa-Craig background) by an Illumina SNP Infinium analysis with the SolCAP array (Sim et al., 2012). We generated in total of 186 F2 plants consisting of 143 plants showing WT phenotypes and of 43 plants showing spff mutant phenotypes (**Table 1**). Genotyping of 43 plants with the spff mutant phenotype using 1956 SNP markers pointed to a 2.6 Mbp region flanked by two SNP markers (solcap\_snp\_sl\_36809 and solcap\_snp\_sl\_3746) on chromosome 4. Based on the tomato genome release SL2.40, these two markers flanked locations at positions 57,939,715 bp and 60,553,996 bp, respectively (**Figure 6A** and **Supplementary Figure S5**). No other possible candidate loci were detected, consistent with the fact that the spff phenotypes reflected a monogenic recessive mutation (**Table 1**). According to the Kazusa Marker Database3 (based on SL2.40), this candidate region included 267 protein-coding genes. The tomInf4732 SNP, which discriminated between Ailsa-Craig and Micro-Tom alleles within the candidate region using primer set F4-R4 (**Supplementary Table S2**), was used to further genotype 73 F2 plants. These included 43 plants with spff phenotypes and 30 with WT phenotypes, allowing us to narrow down the region of interest to 2.0 Mbp, which included 205 genes.

We next employed mapping-by-sequencing (Abe et al., 2012; Garcia et al., 2016) of an F2 population derived by crossing spff with WT in a Micro-Tom background. DNA from 20 individual mutant phenotype F2 plants was sequenced by Illumina HiSeq, and cleaned reads were mapped onto the cultivar Micro-Tom reference genome; polymorphisms were substituted against the cultivar Heinz reference genome version SL2.40 (Kobayashi et al., 2014). The Bowtie2-Samtools-GATK pipeline identified and calculated the frequencies of potential spff -specific SNPs and Indels. This analysis identified 77 mutant homozygous SNPs and Indels within the region narrowed down by SNP Infinium analysis (**Supplementary Table S3**). These 77 mutations were present in the coding regions of 46 genes, which were considered to represent candidate genes for

structure of Solyc04g077010. Chromosome 4 is indicated by the green bar. The gray boxes indicate the untranslated regions, orange boxes indicate the exons, separated by the introns. (B) Nucleotide and amino acid sequences of the WT and the spff mutant around the mutation point. The upper black letters indicate nucleotides, and the lower red letters indicate putative amino acids. The 2 bp deletion indicated by hyphens introduces a premature stop codon in the first exon in the spff mutant. (C) The putative amino acid length and domain structure of the RLK encoded by Solyc04g077010, in WT and the spff mutant. In B, C ∗ indicate the stop codon.

the spff phenotype (**Supplementary Table S4**). Five of these candidates (Solyc04g076020, Solyc04g076100, Solyc04g076250, Solyc04g076320, and Solyc04g077010) were chosen for further linkage analysis. These were selected because of their relatively high expression levels in flowers and fruits, according to tomato eFP browser (Winter et al., 2007; The Tomato Genome Consortium, 2012), and because of the predicted impact of the mutation on the encoded protein. Their linkages with the spff phenotypes were analyzed using marker-based approaches at F2 and backcrossed populations listed in **Supplementary Table S1** with the five primer sets shown in **Supplementary Table S2**. The F18-R18 marker for a 2 bp deletion in the Solyc04g077010 gene (**Figure 6B**), which encodes an RLK, showed perfect segregation with the spff phenotypes. All of the 83 mutant-phenotype plants, and none of the 80 non-parthenocarpic plants, were homozygous for this mutation; the non-parthenocarpic plants were either heterozygous or azygous for this mutation, while four other mutations were not perfectly linked with the spff phenotypes (**Supplementary Table S5**). We realized that the gene model of Solyc04g077010 in the tomato gene annotation ITAG2.3/SL2.40 differed from the latest ITAG3.2/SL3.04 , in which Solyc04g077010 consists of two exons spanning 2871 bp and encoding 957 amino acids. The mutation identified in the present study was located in the first exon and led to a frame shift, which

introduced a premature stop codon at position 494 and therefore generated a truncated protein composed of 493 amino acids (**Figure 6C**). RLK proteins are structurally characterized by three conserved domains: a receptor domain containing a varying number of leucine-rich repeats; a transmembrane domain; and a kinase domain that transduces the downstream signal via autophosphorylation (Shiu and Bleecker, 2001). The RLK protein encoded by Solyc04g077010 harbors a single transmembrane domain between amino acids 505 and 524. This suggested that the mutation would cause a loss-of-function of this protein, thus resulting in the spff mutant phenotypes.

To confirm this, RNAi was used to reduce Solyc04g077010 expression. The RNAi vector targeted the first exon of this gene, which encoded a highly specific receptor domain that was confirmed to be unlikely conserved in other tomato genes encoding RLK proteins by the BLAST search. The RNAi vector was introduced into Micro-Tom plants and three transgenic lines were obtained; these showed significantly reduced mRNA expression of the target protein (**Figure 7A**). These three independent transgenic plants showed resemblance to spff phenotypes such as producing small flowers and fruits with parthenocarpy (**Figures 7B–H**). Further, those RNAi showed complete male sterility, while pollination of WT pollen gave rise to mature viable seeds as observed in spff mutant. These

4https://solgenomics.net/organism/Solanum\_lycopersicum/genome

analyses demonstrated that the spff phenotypes resulted from a loss-of-function of this RLK protein-encoding gene.

### Vasculature-Specific Expression of SPFF Gene in Flower Receptacle

The in silico expression profile obtained by RNA-seq and RT-PCR analyses (Winter et al., 2007; The Tomato Genome Consortium, 2012) revealed that Solyc04g077010 was expressed in various plant organs, including roots, leaves, buds, and flowers (**Supplementary Figures S7A**, **S8**). Previously published transcriptome data (Ezura et al., 2017) indicated that this gene was expressed in floral organs both before and after anthesis, and transcripts were observed in individual floral organs including the ovary/pistil, anther, petal, and sepal, with the highest expression observed in the ovary/pistil at 1 day before anthesis (**Supplementary Figure S7B**). Interestingly, a spatiotemporal analysis of the transcriptome of developing tomato fruits (Fernandez-Pozo et al., 2017; Shinozaki et al., 2018b) revealed vasculature-specific expression of Solyc04g077010 in the fruit pericarp throughout development (**Supplementary Figure S7C**). Consistent with this, predominant expression of this gene was also found in fruit internal tissues, columella and placenta (**Supplementary Figure S7D**), with a high abundance in thick vascular bundles.

To unravel the spatio-temporal expression pattern of Solyc04g077010 during flower development, in situ mRNA hybridization was performed in WT floral buds at different stages of development. In the early developing 1.1 mm bud, the transcript signal was exclusively observed in the vasculature tissues of the receptacle (**Figures 8A,D**). As development proceeded, the SPFF transcripts were also detected in the vasculature of the pedicel (2.9 mm bud) (**Figures 8B,E**), and in the vasculature of the columella tissue (4.5 mm bud) (**Figures 8C,F**). We also observed reduced SPFF transcripts in receptacle and leaves of spff compared to WT (**Supplementary Figure S6A**), indicating that the spff mutation influences both transcript abundance and protein function.

#### Solyc04g077010 Mutation May Affect Hormonal Regulation at the Transcriptional Level

To obtain insights into the molecular mechanisms underlying parthenocarpy in the spff mutant plant, the ovarian transcriptome at the anthesis stage, corresponding to flowerto-fruit transition, was compared to that of WT plants. Our RNA-seq analysis identified a total of 25 differentially expressed genes; 13 of these were significantly up-regulated in spff plants (log2 fold-change > 1) and 12 were significantly down-regulated (log2 fold-change < −1) (q values < 0.05 for the comparison with WT, **Supplementary Table S6**). Notably, the up-regulated genes in the spff ovary included SlGA20ox1 (Solyc03g006880), which encodes a key GA biosynthetic enzyme that is induced by pollination and is also highly expressed during parthenocarpy in the pat mutant (Olimpieri et al., 2007; Serrani et al., 2007b). In spff, the expression level of SlGA20ox1 was > 10-fold of that observed in the WT plant. This result suggested that GA

buds. Distribution of Solyc04g077010 expression was detected by an antisense probe (A–C) and a low background signal was detected by a control sense probe (D–F) in 1.1 mm (A,D), 2.9 mm (B,E), and 4.5 mm (C,F) buds. Black arrows indicate signals detected in vasculature bundles and their distribution in the receptacle, pedicel, and/or columella. Bars are 100 µm.

is involved in the parthenocarpic early transition from flower to fruit exhibited by the spff mutant. To gain further insights into this, we compared our differentially expressed genes with previously published transcriptomic data obtained from GAtreated and -untreated unfertilized ovaries (Tang et al., 2015). One of our 13 up-regulated genes (SlGA20ox1) and three of our 12 down-regulated genes [Solyc02g078150 (Plant-specific domain TIGR01615 family protein), Solyc12g094620 (catalase), and Solyc05g005150 (F-box/Kelch repeat-containing F-box family protein)] were found in the list of genes that were up- and down-regulated by GA treatment, respectively.

#### Flower Receptacle Development Is Not Likely to Be Affected in the spff Mutant

A database BLASTP search showed that the protein encoded by XYLEM INTERMIXTED WITH PHLOEM1 (XIP1) is the closest homolog to tomato SPFF, with 63% amino acid identity (E-value 0, score 1153 bits, and 77% positives) with the Arabidopsis counterpart (GenBank accession no. BAC42540.1). Arabidopsis xip1 loss-of-function mutants showed excessive

anthocyanin accumulation in the leaves and severe defects in plant growth, while fertility was not affected (Bryan et al., 2012). Here, the spff mutant did not show excessive anthocyanin accumulation in the leaves and showed severe male sterility (**Figure 4** and **Supplementary Figure S4D**). Nevertheless, the fact that the xip1 mutants altered plant vascular development, represented by intermixed xylem with phloem, suggests a similar function for the SPFF protein, whose expression was indeed localized to vasculature in the fruit and inflorescence tissues (**Figure 8** and **Supplementary Figure S7C**). To unravel this, we compared xylem-phloem distribution patterns between WT and spff mutant receptacles. Cross-sections of receptacle were stained with Safranin O and Astra blue to visualize lignified (seen as red) and unlignified (seen as blue) tissues. **Supplementary Figure S9** shows that the stained receptacle cross-sections did not reveal significant xylem-phloem intermixing in the spff mutant.

### DISCUSSION

#### The Gene Associated With the spff Phenotype Encodes a Putative RLK Involved in Flower and Fruit Development

This study aimed to identify and characterize the gene underlying a newly isolated tomato mutant, named spff, which showed parthenocarpy and floral organ dwarfism as its major phenotypes (**Figures 1**–**4**). A high-throughput approach combining highdensity genetic mapping (**Supplementary Figure S5**) and mapping-by-sequencing, followed by conventional genetic linkage analysis (**Supplementary Tables S3**–**S5**), allowed the rapid identification of a potential causal mutation in a gene located on chromosome 4, Solyc04g077010 (**Figure 6**). This gene encodes a potential RLK that appeared to be mainly expressed in the receptacle of young floral buds (**Figure 8** and **Supplementary Figure S6**). A 2 bp deletion mutation was identified, which introduced a premature stop codon that leads to the production of a truncated RLK protein (**Figure 6**) as well as to reduced transcript abundance (**Supplementary Figure S6**). Using RNAi approach, we confirmed that the spff phenotypes could be reproduced by silencing Solyc04g077010 (**Figure 7**), and thus concluded that this is the causative gene for the spff mutant.

The Solyc04g077010 homolog in Arabidopsis, xip1, was reported to be involved in vascular bundle differentiation (Bryan et al., 2012). The xip1 mutant shows aberrant xylemlike cells within the phloem in inflorescence stems. Although Solyc04g077010 appeared to be expressed in close vicinity to the vascular bundle (**Figure 8** and **Supplementary Figures S6**, **S7**), xylem-like cells were not present within the phloem (**Supplementary Figure S9**). Moreover, fertility was not affected in Arabidopsis xip1 mutant plants, where the inflorescence stems are shorter than those of the Col-0 accession plants, and the cotyledons and rosette leaves show a purple color, indicative of anthocyanin accumulation. Since these phenotypes were not observed in the present spff mutant (**Figures 1**, **4** and **Supplementary Figure S4**), Solyc04g077010 does not seem to be a functionally conserved ortholog of XIP1. It is more likely to be a novel gene that has possibly acquired a specific function in tomato, although further analyses are needed to confirm this functional dissimilarity with the Arabidopsis XIP1 gene.

### Hypothesis for How the spff Mutant Induces Parthenocarpy

Parthenocarpy can mimic the molecular mechanisms underlying pollination-dependent ovary growth (Li et al., 2014). Fruit set initiation and parthenocarpy are regulated by complex hormone networks. Molecular genetic studies of many mutants/genotypes and transcriptome analyses of early fruit development have suggested that parthenocarpy is in part induced through a hierarchical scheme of temporal regulation by multiple hormones, initiated by the accumulation of auxin; this induces intense cell division, with the subsequent induction of GA metabolism triggering active cell expansion (Martí et al., 2007; Serrani et al., 2007a, 2008). Thus, GA should act as the downstream signal and cell expansion most likely plays a crucial role for fruit set initiation in tomato (Serrani et al., 2008; Shinozaki et al., 2015). The present study revealed that the spff mutant exhibited higher levels of GA20ox1 than WT plants (**Supplementary Table S6**); this is one of the key factors involved in GA biosynthesis in tomato ovaries (Olimpieri et al., 2007; Serrani et al., 2007b). Further, three GA-down regulated genes (Solyc02g078150, Solyc12g094620, and Solyc05g005150) were found in the list of differentially expressed genes identified by the RNA-seq analysis in the unfertilized ovary of spff mutant (**Supplementary Table S6**). In addition, the small parthenocarpic fruits produced by the spff mutant were characterized by enlarged cells, rather than an increased number of cell layers in the ovary pericarp, most likely due to a lack of intense cell division (**Figure 3**). This was consistent with the characteristics of parthenocarpic fruit induced by increased GA sensitivity (Martí et al., 2007). In contrast, auxin-induced parthenocarpy is associated with intensive cell division in the pericarp, resulting in an increased number of cell layers (Wang et al., 2009). The spff mutant also showed reduced pollen fertility (**Figures 4I–L**), which could reflect an increased GA response (Livne et al., 2015). These results suggest that the RLK encoded by Solyc04g077010 functions to repress the GA response in reproductive organs, and that spff parthenocarpy may result in part from an increased GA response.

Additionally, the association of parthenocarpy with early male organ developmental abnormality has been observed in tomato plants. Mutations or genetic suppressions of MADSbox genes, which inhibit functional stamen development by causing homeotic conversions, can induce parthenocarpy (Pnueli et al., 1994; Ampomah-Dwamena et al., 2002; Mazzucato et al., 2008; Quinet et al., 2014; Okabe et al., 2019). Furthermore, the over-accumulation of BARNASE mRNA under a stamen-specific promoter triggers early anther ablation and parthenocarpy (Medina et al., 2013), while loss of function of SEXUAL STERILITY/HYDRA results in complete male sterility and parthenocarpy (Hao et al., 2017; Rojas-Gracia et al., 2017). Recently, a

tap3 mutant has also been described in which stamens are converted into a carpelloid structure and GA over-accumulates in unfertilized ovaries, most likely due to the overexpression of GA metabolism genes such as GA20ox1 (Okabe et al., 2019). Taken together with the fact that the spff mutant shows male sterility and GA20ox1 is highly expressed in the unfertilized ovary of the spff mutant (**Supplementary Table S6**), it is possible that parthenocarpy in the spff mutant involves increased levels of GA20ox1 transcripts through the association with male sterility. Since our transcriptome analysis revealed no differential expression of MADS-box genes between WT and spff mutants (**Supplementary Table S6**), and no homeotic conversion phenotypes were observed in the spff mutant (**Figures 1**, **4**), the association of floral homeotic genes with the Solyc04g077010 gene, and the mechanisms involved in GA20ox1 gene regulation, require further elucidation.

The in situ mRNA analysis showed that Solyc04g077010 was strongly expressed in vascular bundle cells of the floral receptacle and pedicel (**Figure 8**). Vascular systems in inflorescence stems are important for nutrient and signal transportation during developmental events in the reproductive organs (Ranci ˇ c´ et al., 2010). We therefore hypothesize that the RLK encoded by Solyc04g077010 may be involved in the transportation of molecular substances essential for normal floral organ development, and that loss-of-function mutations of this gene may lead to the disruption of integrity of such a system, which may then cause anther abortion. Since we identified little cytological evidence for structural differences between the vascular bundles observed in WT and spff mutant plants (**Supplementary Figure S9**), future studies are required to investigate this possibility in more detail.

Although the role of RLK family proteins in the regulation of fruit development has yet to be fully delineated, a celltype specific transcriptome study of tomato ovaries showed that several genes encoding RLKs were enriched in the cluster that is mainly expressed in the funiculus of the developing seed. These included a homolog of the Arabidopsis HAESA gene, which is involved in specifying seed abscission zones, suggesting that the tomato homolog may possess a similar function (Pattison et al., 2015). Furthermore, silencing of an invertase inhibitor gene in the SlINVINH1-RNAi line, causing increased cell wall invertase activity, was associated with an overall reduction in the transcription of RLK family members in young ovaries, suggesting that RLK may play a role in sensing the modification of cell wall components, thereby regulating downstream gene expression (Ru et al., 2017). Elucidation of RLK activities, including the identification of ligands and kinase domain target proteins, would provide valuable insights into the involvement of RLK proteins in the regulation of fruit development.

#### CONCLUSION

In conclusion, this study identified a novel tomato mutant showing parthenocarpy and this was caused by the loss of function in the gene encoding a receptor kinase gene designated as SPFF. The parthenocarpic variety potentially shows improved fruit productivity due to increased fruit set efficiency (Shinozaki et al., 2018a), although the spff produced delayed growth, smaller mature fruits and reduced yield compared to WT (**Figure 5**). Such unfavorable traits render this mutant less attractive for breeding application, but it would be interesting to identify hypomorphic (weaker) alleles of spff carrying less detrimental phenotypes through screening from TILLING populations or genome editing approaches (Okabe et al., 2011; Shimatani et al., 2017), and investigate their potentials for impact on breeding application.

### AUTHOR CONTRIBUTIONS

YS and TA contributed to the mutant screening. HT, YS, RY, and TA contributed to genetic mapping and transcriptomic analysis. HT and YS performed phenotypic characterizations of mutant plants. SK and HT contributed to expression analysis. HT, MH, and CC contributed to histological analysis and in situ hybridization assays. HT, YS, MH, CC, HE, and TA wrote the manuscript. All authors reviewed and approved the final manuscript.

### FUNDING

This work was supported by JSPS KAKENHI, grant no. 15KK0273, Program to Disseminate Tenure Tracking System, and JSPS bilateral program to TA, Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, Japan (Grant No. 26013A) to HE, a grant from the Japan Society for the Promotion of Science to YS (16J00582), HT (18J20505), and SK (18J00528).

### ACKNOWLEDGMENTS

We thank all members in our lab for critical comments on this research. Seeds of Micro-Tom WT (TOMJPF00001), Ailsa-Craig WT (TOMJPF00004), and spff (TOMJPG4121) were obtained from the National BioResource Project (NBRP) of the Japan Agency for Medical Research and Development (AMED). This research was partly supported by the "Sustainable Food Security Research Project" in the form of an operational grant from the National University Corporation.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00403/ full#supplementary-material

### REFERENCES

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Micro-Tom of tomato. J. Plant Growth Regul. 26, 211–221. doi: 10.1007/s00344- 007-9014-7

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Takei, Shinozaki, Yano, Kashojiya, Hernould, Chevalier, Ezura and Ariizumi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Finding a Compatible Partner: Self-Incompatibility in European Pear (*Pyrus communis*); Molecular Control, Genetic Determination, and Impact on Fertilization and Fruit Set

#### *Hanne Claessen1 , Wannes Keulemans1 , Bram Van de Poel2 and Nico De Storme1 \**

*1 Laboratory for Plant Genetics and Crop Improvement, Division of Crop Biotechnics, Department of Biosystems, KU Leuven, Leuven, Belgium, 2 Laboratory for Molecular Plant Hormone Physiology, Division of Crop Biotechnics, Department of Biosystems, KU Leuven, Leuven, Belgium*

#### *Edited by:*

*Emidio Albertini, University of Perugia, Italy*

#### *Reviewed by:*

*Irene Serrano, University of Göttingen, Germany Luigi Russi, University of Perugia, Italy Celia M. Cantin, Fundacion Agencia Aragonesa para la Investigacion y el Desarrollo, Spain*

*\*Correspondence:*

*Nico De Storme<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science*

*Received: 15 November 2018 Accepted: 18 March 2019 Published: 16 April 2019*

#### *Citation:*

*Claessen H, Keulemans W, Van de Poel B and De Storme N (2019) Finding a Compatible Partner: Self-Incompatibility in European Pear (Pyrus communis); Molecular Control, Genetic Determination, and Impact on Fertilization and Fruit Set. Front. Plant Sci. 10:407. doi: 10.3389/fpls.2019.00407*

*Pyrus* species display a gametophytic self-incompatibility (GSI) system that actively prevents fertilization by self-pollen. The GSI mechanism in *Pyrus* is genetically controlled by a single locus, i.e., the S-locus, which includes at least two polymorphic and strongly linked S-determinant genes: a pistil-expressed *S-RNase* gene and a number of pollen-expressed *SFBB* genes (S-locus F-Box Brothers). Both the molecular basis of the SI mechanism and its functional expression have been widely studied in many Rosaceae fruit tree species with a particular focus on the characterization of the elusive *SFBB* genes and S-RNase alleles of economically important cultivars. Here, we discuss recent advances in the understanding of GSI in *Pyrus* and provide new insights into the mechanisms of GSI breakdown leading to self-fertilization and fruit set. Molecular analysis of S-genes in several self-compatible *Pyrus* cultivars has revealed mutations in both pistil- or pollen-specific parts that cause breakdown of self-incompatibility. This has significantly contributed to our understanding of the molecular and genetic mechanisms that underpin self-incompatibility. Moreover, the existence and development of self-compatible mutants open new perspectives for pear production and breeding. In this framework, possible consequences of self-fertilization on fruit set, development, and quality in pear are also reviewed.

#### Keywords: *Pyrus communis*, gametophytic self-incompatibility, fertilization, fruit set, S-RNase, SFBB

## INTRODUCTION

Self-incompatibility (SI) refers to all genetic mechanisms in flowering plants that prevent self-fertilization through the recognition and rejection of self-pollen by the style of a flower (DeNettancourt, 1977). SI is generally classified into two types: heteromorphic and homomorphic SI. The heteromorphic SI system includes distyly and tristyly and inhibits self-fertilization through the production of more than one morphological flower type. In contrast, homomorphic SI inhibits self-fertilization through genetic or biochemical mechanisms that operate regardless of flower morphology (Charlesworth, 2010; Orević et al., 2014). There are two main types of homomorphic SI: gametophytic selfincompatibility (GSI) and sporophytic self-incompatibility (SSI) (Kao and Huang, 1994) (**Figure 1**). In GSI, the genotype of the haploid pollen itself (gametophyte) determines its incompatibility type, while in SSI, the genotype of the diploid parental plant (sporophyte) that acts as the pollen donor determines the incompatibility type (Hiscock and Tabah, 2003). GSI is considered the most prevalent SI system in the plant kingdom and occurs in Solanaceae, Rosaceae, and Plantaginaceae (Franklin-Tong and Franklin, 2003a).

European pear (*Pyrus communis*) exhibits an RNase-based gametophytic self-incompatibility system (Sassa et al., 1992). This system is genetically controlled by a single locus, named the S-locus, which includes at least two polymorphic genes that are tightly linked: a pistil-expressed gene and one or several pollen-expressed genes (Entani et al., 2003; Kao and Tsukamoto, 2004). The pistil S-gene encodes an S-RNase, which is highly expressed in the style and catalyzes degradation of RNA (Sassa et al., 1992, 1996; Zhou et al., 2016). In *Pyrus* species, the pollen S-determinant is proposed to consist of multiple *F-box* genes, called *SFBBs* (S-locus F-Box Brothers) (Sassa et al., 2007). Across all *Pyrus communis* genotypes, there exists great variability in the S-locus haplotype, i.e., as reflected by the allelic variability in both the pollen- and pistil-expressed genes. For fertilization to take place, the S-haplotype of the pollen grain must differ from the two S-haplotypes of the diploid pistil, otherwise the growth of the pollen tube is arrested after it has migrated through about one-third of the length of the style (DeNettancourt, 1977; Franklin-Tong and Franklin, 2003b). The GSI mechanism therefore inhibits specific hybridizations between *Pyrus communis* genotypes that carry the same S-haplotypes.