Pre- and Postzygotic Barriers Associated with Intergeneric Hybridization between Aronia melanocarpa (Michx.) Elliott x Pyrus communis L. and ×Sorbaronia dippelii (Zabel) CK Schneid. x Pyrus communis

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  • 1 Department of Plant Science and Landscape Architecture, University of Connecticut, Storrs, CT 06269

Intergeneric hybridization between Aronia and Pyrus may provide a pathway for developing novel fruit types with larger, sweeter fruits, while maintaining the high levels of biologically health-promoting compounds present in Aronia fruits. Here we describe a deleterious genetic incompatibility, known as hybrid necrosis or hybrid lethality, that occurs in intergeneric F1 hybrids of Aronia melanocarpa x Pyrus communis and ×Sorbaronia dippelii x Pyrus communis. Pollination experiments revealed that maternal A. melanocarpa and ×S. dippelii pistils are compatible with pollen from P. communis. Controlled pollinations using different mating combinations resulted in varying levels of fruit and seed set. Because every combination produced at least some viable seeds, prezygotic incompatibility does not appear to be present. We attempted to recover putative intergeneric progeny via either in vitro germination or in vitro shoot organogenesis from cotyledons. Progeny of putative hybrids from A. melanocarpa x P. communis only survived for a maximum of 14 days before succumbing to hybrid lethality. Regeneration of ×S. dippelii x P. communis was successful for two seedlings that have been maintained for an extended time in tissue culture. These two seedlings have leaf morphologies intermediate between the two parental genotypes. We also confirmed their hybrid status by using AFLPs and flow cytometry. Putative intergeneric hybrids were grown out ex vitro before showing symptoms of hybrid necrosis and dying after 3 months. Eventually micrografts failed, ultimately showing the same symptoms of hybrid necrosis. These results show that intergeneric hybridization is possible between Aronia and related genera in the Rosaceae, but there are postzygotic barriers to hybridity that can prevent the normal growth and development of the progeny.

Abstract

Intergeneric hybridization between Aronia and Pyrus may provide a pathway for developing novel fruit types with larger, sweeter fruits, while maintaining the high levels of biologically health-promoting compounds present in Aronia fruits. Here we describe a deleterious genetic incompatibility, known as hybrid necrosis or hybrid lethality, that occurs in intergeneric F1 hybrids of Aronia melanocarpa x Pyrus communis and ×Sorbaronia dippelii x Pyrus communis. Pollination experiments revealed that maternal A. melanocarpa and ×S. dippelii pistils are compatible with pollen from P. communis. Controlled pollinations using different mating combinations resulted in varying levels of fruit and seed set. Because every combination produced at least some viable seeds, prezygotic incompatibility does not appear to be present. We attempted to recover putative intergeneric progeny via either in vitro germination or in vitro shoot organogenesis from cotyledons. Progeny of putative hybrids from A. melanocarpa x P. communis only survived for a maximum of 14 days before succumbing to hybrid lethality. Regeneration of ×S. dippelii x P. communis was successful for two seedlings that have been maintained for an extended time in tissue culture. These two seedlings have leaf morphologies intermediate between the two parental genotypes. We also confirmed their hybrid status by using AFLPs and flow cytometry. Putative intergeneric hybrids were grown out ex vitro before showing symptoms of hybrid necrosis and dying after 3 months. Eventually micrografts failed, ultimately showing the same symptoms of hybrid necrosis. These results show that intergeneric hybridization is possible between Aronia and related genera in the Rosaceae, but there are postzygotic barriers to hybridity that can prevent the normal growth and development of the progeny.

Intergeneric hybridization in the Rosaceae, subtribe Malinae (Sun et al., 2018), is an important reproductive mechanism that has facilitated plant speciation and domestication of novel plants (Postman, 2011); however, these wide hybridization events can often be hindered by pre- and postzygotic reproductive barriers. Prezygotic barriers can involve spatial isolation of parental plants, asynchrony in flowering, environmental conditions, and self-incompatibility (De Franceschi et al., 2012; Hiscock and McInnis, 2003; Morgan et al., 2011). Postzygotic barriers include failure of the embryo/seed to fully develop, abiotic and biotic stresses on seed and fruit development (e.g., drought, wind, and animal feeding), and hybrid necrosis (Bomblies and Weigel, 2007; Chen et al., 2016). Hybrid necrosis is the reduced viability of a hybrid, characterized by cell death, tissue necrosis, reduced growth rate and in some cases lethality (Bomblies, 2009).

The molecular mechanisms of hybrid necrosis and lethality are reasonably well understood, and the Bateson-Dobzhansky-Muller (BDM) model which provides a plausible genetic explanation (Chen et al., 2016). The BDM model suggests that as ancestral species diverge, each lineage evolves mutations that are not harmful in their native genome, but interact negatively in a hybrid when two divergent genomes recombine (Bomblies, 2006). Hybrid lethality is induced by these deleterious epistatic interactions, which result in spontaneous activation of plant defense genes, primarily nucleotide-binding site leucine-rich repeat (NB-LRR) type resistance proteins (Bomblies et al., 2007; Deng et al., 2019; Jeuken et al., 2009; Montanari et al., 2016).

Aronia fruits contain high levels of antioxidants and polyphenols that are beneficial for human nutrition (Brand et al., 2017; Wu et al., 2004); however, the fruits are astringent and require processing for improved palatability. Intergeneric hybridization between A. melanocarpa and P. communis may provide a pathway for developing new fruits with desirable traits for both growers and consumers. Fruits of Pyrus and Aronia hybrids may be large and sweet, while still maintaining the high levels of health-promoting compounds present in Aronia fruits. New sources of hybrid germplasm also could be a valuable asset to Pyrus breeding programs and may offer novel traits for disease and pest resistance, improved cultivation, and enhanced fruit quality (Bell and Itai, 2011). The Malinae subtribe has a base chromosome count of 17, and hybridization between species and genera is common (Campbell et al., 2007; Persson Hovmalm et al., 2005; Postman, 2011). Previous reports have confirmed intergeneric hybrids between Aronia x Sorbus (Leonard et al., 2013), Pyrus x Malus (Gonai et al., 2006), and Pyrus x Sorbus (Bell and Itai, 2011; Sax, 1929).

The initial objectives of this study were to create novel sources of pome fruit germplasm via intergeneric hybridization and investigate the mating systems and compatibility involving these wide hybridizations and, later, to document evidence for hybrid lethality. The mating systems associated with the plants chosen as parents are critical for determining the likelihood of hybrid formation. Therefore, the results from this study are essential for making informed breeding decisions involving hybridization among Aronia, Pyrus, and closely related taxa.

Materials and Methods

Plant material and controlled pollinations.

Detailed accession information for the plants used in this research is listed in Table 1. Flowering shoots of Pyrus communis were sent from the US Department of Agriculture–Agricultural Research Service–National Clonal Germplasm Repository in Corvallis, OR, and were used as paternal genotypes in pollinations (Table 1). Container-grown plants were overwintered in a minimally heated hoop greenhouse (8 ± 5 °C) and a dark cooler (4 ± 2 °C) for ≈6 months. To induce flowering, plants were transferred to a heated greenhouse with set points of 22 °C day and 18 °C night. Pollinations were conducted in a pollinator-free greenhouse. Maternal flowers were emasculated when anthers were pink, before anthesis. Yellow anthers were collected from paternal flowers and their pollen was immediately deposited on the maternal stigmas. Pollinations conducted in greenhouses were not covered to prevent unwanted pollinations because insects were excluded. Field pollinations were conducted at the University of Connecticut Research Farm in Storrs, CT, and The Arnold Arboretum in Jamaica Plain, MA. Field pollinations were emasculated before flowers fully opened and were pollinated as described for greenhouse plants. Field pollinations were covered for a period of 2 weeks with dry, waxed paper pollination bags. Control seed was collected from open pollinated maternal plants at the University of Connecticut Aronia germplasm collection. Fruits were harvested at peak maturity and seeds cleaned from the fruits and dried before placement into cool, dark storage (13 ± 2 °C, relative humidity 55% ± 5%).

Table 1.

Germplasm used for controlled pollinations, amplified fragment length polymorphism analysis, and micrografting.

Table 1.

Pollen-tube analysis.

Flowers from a subset of mating combinations were collected 48 h after pollination. Styles were fixed in FPA fixative (70% ethyl alcohol: formaldehyde: propionic acid in a ratio of 80:10:10) and stored at 4 °C until further use. Fixed pistils were softened for 24 h in 8 N NaOH and then rinsed for 4 h in distilled water. Pistils were stained with an aqueous solution of 0.1% aniline blue and 0.1 N K3PO4 for 24 h. Stained pistil samples were covered with a cover slip and softly squashed and observed under a ultraviolet-light microscope (Zeiss Axiophot, Jena, Germany). Pollen compatibility with each pistil was scored by the extent of pollen-tube growth toward the basal end of the style. Mating combinations were determined to be compatible when pollen tubes reached the base of the style. Mating combinations were incompatible if pollen tubes failed to reach the stylar base. Five styles were evaluated for each mating combination.

In vitro seedling regeneration and microshoot rooting.

Mature seeds from putative hybrids and parental genotypes (control group) were used for regeneration. Seeds were surface sterilized for 30 min in 3% (v/v) sodium hypochlorite, rinsed five times in sterile distilled water and then imbibed in sterile distilled water for 48 h at 26 ± 2 °C. Once imbibed, seeds were again surface sterilized in 1% (v/v) sodium hypochlorite for 30 min and rinsed three times in sterile distilled water. Seed coats were aseptically removed in a laminar flow hood with the use of a stereomicroscope.

Seeds with their seedcoats removed were either germinated in vitro, or used as a source of cotyledons for in vitro shoot organogenesis. For in vitro germination, the excised embryo was placed on a petri dish (60 × 16 cm) in direct contact with the medium, which consisted of MS basal medium (Murashige and Skoog, 1962) supplemented with 3% sucrose (w/v) and 0.8% agar (w/v; A1296, Sigma-Aldrich, St. Louis, MO), adjusted to pH 5.8 before autoclaving at 120 °C for 20 min. Cultures were incubated at 26 ± 2 °C under 16-h light photoperiod at ≈50 μmol·m−2·s−1. After 4 weeks, seeds that germinated in vitro had their epicotyl removed and placed on shoot multiplication medium, which consisted of MS basal medium supplemented with 3 µM 6-benzylaminopurine (BAP), 3% sucrose (w/v), 0.8% agar (w/v), and pH of 5.8 before autoclaving at 120 °C for 20 min. Culture environment for epicotyls was as described for in vitro seed germination. Shoot multiplying cultures resulting from plumules were subcultured every 6 weeks.

Attempts to regenerate some of the progeny via shoot organogenesis from cotyledon explants followed the protocol developed by Mahoney et al. (2018). Briefly, the distal two-thirds of the seed were excised and cotyledons were separated and transferred with the abaxial surface in contact with the medium in petri dishes (60 × 16 cm) with 10 mL of the medium. The remaining portions of the seed (embryo axis and radicle) were not discarded, but instead placed into seed germination medium as described previously. For shoot organogenesis, MS basal medium was supplemented with 5 µM 1-naphthaleneacetic acid (NAA) in combination with 10 µM BAP. MS basal medium was supplemented with 3% sucrose (w/v) and 0.8% agar (w/v) with pH 5.8 before autoclaving at 120 °C for 20 min. Cultures were incubated at 26 ± 2 °C in darkness and transferred onto fresh media after 4 weeks. Eight weeks after incubation the quality of calli and number of explants producing shoots were evaluated and recorded.

Microshoots from crosses that were successfully regenerated and multiplied in vitro were rooted ex vitro. Microshoots were dipped in indole-3-butyric acid rooting hormone (Hormidin 1; OHP Inc., Mainland, PA) and rooted in clear plastic salad trays containing a mix of 1 peat: 1 vermiculite. A completely randomized design with three replicates, each containing five cuttings, was used for rooting. Microcuttings were ≈20 mm long, spaced 10 mm apart. Plants were grown at 26 ± 2 °C under 16-h light photoperiod at ≈50 µmol·m−2·s−1 and fertilized once a week with 100 ppm nitrogen (Peters 20–10–20; Scotts, Marysville, OH). After a 21-d rooting period in salad trays, individual rooted microcuttings were transferred to 7.5-cm pots with the same growing mix and grown under the same conditions with a dome lid covering to maintain humidity. Significance of rooting was determined by analysis of variance, and the differences between the rooting percentage were compared by Fisher’s least significance difference test using the R package agricolae (de Mendiburu, 2019).

Phenotypic observations.

Phenotypic observations were collected on the population of plants that successfully rooted. Plants were evaluated every 7 d for 6 weeks following transfer to individual pots. Plants were observed for tissue necrosis, wilting, and shoot elongation.

Micrografting.

Micrografting was used in an attempt to recover and grow out shoots of a putative hybrid (AA15-2) between ×Sorbaronia dippelii (UC123) x Pyrus communis (PC001). Micrografts were made with scions from AA15-2 shoot cultures, cultured as described previously. Scion shoots were micrografted onto rootstocks of Sorbus aucuparia, Pyrus communis, and ×Sorbaronia dippelii. Rootstock shoots were multiplied on the same shoot multiplication medium as were scions. Individual rootstock shoots were transferred to a liquid root initiation medium consisting of MS basal medium supplemented with 3 µM indole-3-butyric acid (IBA). Root initiation cultures were incubated at 26 ± 2 °C under 16-h light photoperiod at ≈50 µmol·m−2·s−1 for 3 weeks. To make the micrografts, individual rootstock shoots were removed from the rooting medium, the top decapitated, and all axillary buds carefully removed. A vertical incision (3 mm) was made centrally at the top of the rootstock. Scion shoot bases were cut at a 45° angle on one side and slipped into the vertical incisions of the rootstocks. Each micrograft was placed into a 50-mL culture vessel with 30 mL of medium consisting of MS basal medium supplemented with 3% sucrose (w/v) and 0.8% agar (w/v) with pH 5.8 before autoclaving at 120 °C for 20 min. Cultures were incubated at 26 ± 2 °C under 16-h light photoperiod at ≈50 µmol·m−2·s−1 for 4 weeks. After the graft union formed and roots developed, micrografts were transferred to salad trays containing 1 peat:1 vermiculite mix. Plants were grown at 26 ± 2 °C under 16-h light photoperiod at ≈50 µmol·m−2·s−1. After a 21-d rooting period in salad trays, individual rooted microcuttings were transferred to 7.5-cm pots with the same growing mix and grown under the same conditions with a dome lid covering to maintain humidity. Micrografts were evaluated every 7 d for 4 weeks.

To test if a scion genotype (AA15-2) that displays symptoms of hybrid necrosis would translocate deleterious compounds to a rootstock and result in hybrid necrosis and lethality of the rootstock, 10 additional micrografts were made with AA15-2 grafted onto S. aucuparia as described above. In this set of micrografts, the axillary buds were not removed from the rootstocks so the rootstock shoot meristems could grow out if they were unaffected by hybrid lethality. Micrografts to test for translocation of lethality were transferred ex vitro and grown out for 4 weeks. Phenotypic observations of the scion and rootstock were collected every 7 d to document any necroses.

Flow cytometry.

Two or three newly emerged leaves (≈50 mg) were thinly sliced with a fresh razor blade in a 55-mm petri dish on a freeze pack with a nuclei suspending solution (Lehrer et al., 2008). The suspending solution was filtered, centrifuged to form a pellet, and resuspended in nuclei staining solution containing propidium iodide. Relative fluorescence of total DNA was measured using a BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA), which quantified 10,000 to 20,000 particles for each sample. Data were analyzed and displayed in histograms by FlowJo Software (Tree Star, Inc., San Carlos, CA) designed to compare standard parental-sample histogram peaks to samples of putative hybrids.

DNA extraction and amplified fragment length polymorphism (AFLP) procedure.

Genomic DNA was extracted following the protocol outlined in Mahoney et al. (2019). The quality and concentration of extracted DNA were determined using a NanoDrop-1000 spectrophotometer (Thermo Scientific, Willington, DE). The AFLP steps, including restriction digestion, adaptor ligation, and preselective and selective amplification reactions, were carried out as outlined in the Applied Biosystems (Foster City, CA) AFLP protocol (Anonymous, 2007). Preselective primers had one selective nucleotide (EcoRI-A + MseI-C). Four primer combinations were used for selective amplification (EcoRI-ACT + MseI-CAC, EcoRI-ACT + MseI-CTA, EcoRI-ACT + MseI-CAT and EcoRI-ACT + MseI-CTG), with fluorescently labeled EcoRI and unlabeled MseI probes. The DNA fragments from selective polymerase chain reaction were visualized by capillary electrophoresis on an ABI3730xl DNA analyzer (Applied Biosystems) with a 500 LIZ size standard. AFLP fragment files were processed into binary matrices with GeneMarker Version 1.95 software (SoftGenetics, State College, PA). Peaks were first binary scored (1 for presence and 0 for absence) using automatic settings followed by visual inspection and manual peak adjustment to ensure accurate scoring. Principal component analysis (PCA) was conducted on the binary matrix with the prcomp function in R (version 3.5.1).

Results

Pollen-tube analysis and seed set.

All outcrossed pollinations had compatible pollen tubes, with pollen tubes reaching the bottom the stylar base, and most of the crosses produced seed (Fig. 1A–D, Table 2). Self-pollinations of P. communis (PC001) resulted in incompatible pollen tubes and no seed set (Table 2). Pollen tubes were not observed in the bottom half of the style of the self-pollinated PC001 pistil (Fig. 1E). The percent fruit set of intergeneric crosses between A. melanocarpa x P. communis ranged from 6% to 38%, with an average of three seeds per fruit. Crosses between ×S. dippelii x P. communis had a relatively low percent fruit set (between 3% and 5%) and an average of 1.5 seeds per fruit. The other controlled crosses between P. communis x ×S. dippelii, and P. communis selfed and outcrossed produced no fruit (Table 2).

Fig. 1.
Fig. 1.

Pollen-tube–pistil analysis used to determine if intergeneric hybrids exhibit a self-incompatibility system. Intergeneric crosses between (A) Aronia melanocarpa (UC023) x Pyrus communis (PC001); (B) ×Sorbaronia dippelii (UC123) x P. communis (PC001); (C) P. communis (PC001) x ×S. dippelii (UC123); (D) P. communis (PC001) x P. communis (PC002); and self-crossed between (E) P. communis (PC001) x P. communis (PC001). Red arrows indicate terminus of pollen-tube development.

Citation: HortScience horts 56, 2; 10.21273/HORTSCI15412-20

Table 2.

Summary of pollen-tube growth, fruit and seed set, and regeneration of progeny from controlled pollinations.

Table 2.

In vitro seed regeneration and microshoot rooting.

The percentage of A. melanocarpa x P. communis seeds germinating ranged from 10% to 100% (Table 2), depending on the parental combination. Of the seeds that successfully germinated, none survived beyond 14 d after in vitro germination began. Parental genotypes did not show symptoms of hybrid necrosis (Fig. 2A), whereas putative hybrid seedlings began showing necrotic lesions between 3 and 14 d after germinating in vitro (Fig. 2B). Hybrid seedlings that had little necrotic lesioning detected on the radicle or cotyledons were observed to exhibit a necrotic plumule, which led to their failure in growth and development (Fig. 2B).

Fig. 2.
Fig. 2.

Regeneration of Aronia-Pyrus putative F1 hybrids and parental controls. (A) Parental genotypes [A. melanocarpa (UC023) and P. communis (PC001)] germinate in vitro and continue growth and development (bar = 5 mm); (B) Aronia-Pyrus putative F1 hybrids showing symptoms of hybrid necrosis 14 d after in vitro germination (bar = 5 mm); (C) shoot organogenesis on paternal genotype [A. melanocarpa (UC023)] cotyledons after 2 months of culture (bar = 5 mm); and (D) necrotic callus development on Aronia-Pyrus putative F1 hybrid cotyledons (bar = 5 mm).

Citation: HortScience horts 56, 2; 10.21273/HORTSCI15412-20

Cotyledons from 50 seeds of A. melanocarpa x P. communis were used to attempt to regenerate progeny via shoot organogenesis. Cotyledons placed on shoot organogenesis medium began to form callus ≈7 d after culture initiation. The control, A. melanocarpa (UC007), produced healthy calli throughout the duration of this experiment. After 8 weeks of culture, A. melanocarpa (UC007) produced an average of seven shoots per explant (Fig. 2C), which was similar to previous results by Mahoney et al. (2018). Cotyledon explants from the A. melanocarpa x P. communis crosses initially formed healthy calli, but at a slightly slower rate than A. melanocarpa (UC007) cotyledon explants. After 7 d in vitro, the Aronia x Pyrus explants began to turn necrotic, and callus development slowed significantly (Fig. 2D). The cotyledons from the intergeneric crosses failed to form shoots (Fig. 2D). As a result, none of the Aronia x Pyrus hybrids was successfully regenerated through in vitro germination or shoot organogenesis.

The ×S. dippelii x P. communis crosses (UC123 x PC001 and UC123 x PI61740) formed relatively few seeds, so all of the seeds were used for in vitro germination, not shoot organogenesis (Table 2). All seeds turned necrotic and died soon after germination (Table 2), except for two from UC123 x PC001, which remained green and continued growth. The two seedlings that did germinate in vitro, AA15-2 and AA15-3, had their epicotyls transferred to, and multiplied on, shoot multiplication medium (Fig. 3A). Shoot multiplication cultures have been maintained for more than 4 years by subculturing every other month. There have been no symptoms of hybrid necrosis during this process. Both hybrids produce an adequate number of shoots in tissue culture; however, AA15-3 shoots do not elongate well and produce compact shoot rosettes.

Fig. 3.
Fig. 3.

Putative hybrids (AA15-2 and AA15-3) from ×S. dippelii (UC123) x P. communis (PC001) (A) multiplied in vitro; (B) microcuttings rooted ex vitro (bar = 10mm); (C) microcutting rooting for putative hybrids and parental genotypes. Bars followed by the same letter did not differ significantly according to Fisher’s least significant difference test (P < 0.05).

Citation: HortScience horts 56, 2; 10.21273/HORTSCI15412-20

Putative intergeneric hybrids were successfully rooted ex vitro from microcuttings (Fig. 3B). There was no significant difference in percent rooting (80% to 100%) among AA15-2, UC123, and PC001 (Fig. 3C). AA15-3 had an average rooting of 27%, which was significantly less than the other genotypes (P < 0.0001).

Confirmation of hybrid status.

The two putative hybrids (AA15-2 and AA15-3) were analyzed for their hybrid status based on leaf morphological characteristics, AFLPs, and flow cytometry. The morphological characteristics observed on AA15-2 and AA15-3 suggest that both are intergeneric hybrids. As seen in Fig. 4A, leaves of the two putative hybrids (AA15-2 and AA15-3) and the paternal genotype, P. communis (PC001), tend to be more broadly elliptical, whereas the maternal parent, ×S. dippelii (UC123), has narrowly elliptical leaves. The leaf margins for AA15-2 and PC001 are serrulate, suggesting that this was inherited from the father. The maternal parent (UC123) produces serrate leaf margins, and AA15-3 displays irregular, large serrations at its leaf apices (Fig. 4A).

Fig. 4.
Fig. 4.

Confirmation of putative hybrid status using (A) leaf morphological features (bar = 5 mm); (B) PCA of AFLP marker analysis of putative hybrids and their parental genotypes. Putative hybrids are circled; and (C) flow cytometry showing nuclei relative fluorescence values for the maternal genotype [×S. dippelii (UC123)] and paternal genotype [P. communis (PC001)] and the two putative hybrids (AA15-2 and AA15-3).

Citation: HortScience horts 56, 2; 10.21273/HORTSCI15412-20

AFLP markers were a rapid and efficient tool for confirmation of intergeneric hybrids. Four primer pairs generated 84 total bands, of which 96% were polymorphic among parents and putative hybrids. The PCA partitioned the hybrids between the two parental genotypes, as would be expected (Fig. 4B). We also show that ×S. dippelii (UC123) is an intergeneric hybrid between A. melanocarpa x S. aria, which supports the findings of Leonard et al. (2013).

Flow cytometry provided additional insights into the hybrid status of the two putative intergeneric hybrids. The average relative fluorescence values for PC001, UC123, AA15-2, and AA15-3 were 81,000, 96,000, 85,000, and 75,000, respectively (Fig. 4C). One of the putative hybrids, AA15-2, had a mean fluorescence value that was in-between those of its two parents, UC123 and PC001. AA15-3 produced a mean fluorescence that was less than all other samples, suggesting a loss of genetic material.

Phenotypic observations.

After 21 d of rooting, plants had no symptoms of hybrid necrosis in the rooting trays and were moved to individual pots. AA15-2 started to show symptoms of hybrid necrosis following day 28, with some necrotic lesions occurring at the shoot growing tip. By day 35, both AA15-2 and AA15-3 were alive, but their shoot tips started to become necrotic and growth and development ceased (Fig. 5A). UC123 and PC001 continued to grow vigorously without forming necrotic lesions. In most instances, after 2 months of growing ex vitro, AA15-2 started to show severe symptoms of necrosis and hybrid lethality, which resulted in death (Fig. 5B). AA15-3 showed less severe symptoms than did AA15-2, but was stunted and died after 3 months.

Fig. 5.
Fig. 5.

Phenotypic observations and progression of hybrid necrosis in (A) maternal ×S. dippelii (UC123), paternal P. communis (PC001) and intergeneric hybrid progeny (AA15-2 and AA15-3) over a 3-week period (bar = 20 mm). (B) Intergeneric hybrid AA15-2 at 21 and 63 d (bar = 10 mm).

Citation: HortScience horts 56, 2; 10.21273/HORTSCI15412-20

Micrografting.

Callus formation was visible on micrografts at the graft junction ≈10 d after grafting. Grafted AA15-2 did not survive on any of the rootstocks used in this experiment. The graft union appears to completely fail between 42 to 49 d after grafting resulting in hybrid necrosis and a rapid decline of the scion (Fig. 6A).

Fig. 6.
Fig. 6.

Micrograft between AA15-2 intergeneric hybrid scion [×S. dippelii (UC123) x P. communis (PC001)] and S. aucuparia (UC130) rootstock. (A) Progression of graft union and hybrid necrosis (bar = 10 mm). (B) Growth and development of rootstock axillary bud over a 3-week period (bar = 10 mm).

Citation: HortScience horts 56, 2; 10.21273/HORTSCI15412-20

AA15-2 scions grafted onto S. aucuparia rootstocks with axillary buds left intact for potential grow out, had little effect on the viability of the rootstock. The rootstocks broke bud and continued to grow and develop as the scion developed symptoms of hybrid necrosis 42 d after grafting. By 49 d, scion leaves and stem were exhibiting severe symptoms of tissue necrosis, while the rootstock was growing rapidly without necrosis (Fig. 6B). All 10 replicates developed hybrid necrosis in the scion and normal, vigorous growth and development in the rootstock.

Discussion

Our results demonstrate that postzygotic incompatibilities of F1 intergeneric Malinae hybrids resemble cases previously reported (Bomblies et al., 2007; Montanari et al., 2016). Controlled pollinations provide important insights into intergeneric hybridization among A. melanocarpa, P. communis, and ×S. dippelii. Our study was consistent with others showing that P. communis is self-incompatible (Claessen et al., 2019). Previous studies have suggested that Aronia and closely related intergeneric hybrids are compatible (Connolly, 2014; Leonard, 2011). Compatibility among A. melanocarpa, P. communis, and ×S. dippelii was successful based on stylar pollen-tube germination. Morimoto et al. (2020) found P. communis pollen to be noncompatible with Malus ×domestica pistils, whereas P. pyrifolia was compatible. However, there have been other reports showing hybridization and compatibility between M. ×domestica and P. communis (Fischer et al., 2014). These discrepancies suggest that some genotype-specific mating combinations are more successful than are others. In this study, we observed significant variation in the number of fruits and seeds produced among different intergeneric mating combinations.

The lack of fruit and seed set in outcrossed P. communis maternal plants may be the result of environmental stressors from growing plants in containers and in a greenhouse where temperatures could not always be limited. Similarly, other crosses conducted in the greenhouse exhibited low fruit and seed set which could have been caused by similar events or hybrid barriers during fertilization. Although most of these crosses were conducted in a climate-controlled greenhouse, the temperature during the spring and summer months can be difficult to manage. High temperatures may have been a contributing factor to a reduction in pollen germination, fertilization, and embryo development.

AFLP markers have previously been used in Aronia because of their high frequency of polymorphisms and reproducibility (Leonard et al., 2013; Mahoney et al., 2019). In addition, Leonard et al. (2013) demonstrated that AFLP markers can be used across Aronia, Pyrus, and other closely related taxa. AFLP marker analysis was used to confirm the hybrid status of two putative intergeneric hybrids, AA15-2 and AA15-3. The results here are the first to demonstrate the hybrid status between ×S. dippelii and P. communis. Flow cytometry analysis was used to confirm the hybrid status of intergeneric hybrids between apple and Japanese pear (Gonai et al., 2006). Flow cytometry conducted in this study showed that AA15-2 had a DNA content that was intermediate between the two parents. AA15-3 had a DNA content that was less than both parents, which may reflect a loss of genetic material. Nunes et al. (2013) found significant reductions in DNA content in interspecific Pennisetum hybrids in comparison with the average DNA content of the parental plants. The reduction in DNA content may act as a mechanism to form genome stability in hybrids (Leitch and Bennett, 2004).

Hybrid necrosis in tobacco has been linked to activation of genes associated with pathogen responses (Masuda et al., 2007). Bomblies et al. (2007) showed that tandem duplication of two R genes in Arabidopsis thaliana was responsible for overexpression of defense-related pathway genes. They also demonstrated that hybrid necrosis in A. thaliana was caused by DM1, an allele of an NB-LRR gene. In our study, hybrids maintained in tissue culture did not display symptoms of hybrid necrosis until removed from the microclimate and rooted ex vitro. In vitro, warmer temperatures and higher humidity may be contributing to a reduction in hybrid necrosis. Higher temperatures have been suggested to interact with the genetic control of plant immune responses (Alcázar and Parker, 2011; Muralidharan et al., 2014; Traw and Bergelson, 2010). Hybrid necrosis-like incompatibilities are dependent on the activation of the salicylic acid stress signaling pathway (Alcázar et al., 2009). Increasing temperatures reduce the salicylic acid pathway, which likely suppress salicylic acid activation (Traw and Bergelson, 2010).

Observing varying degrees of hybrid necrosis is not an unusual biological phenomenon and has been observed in other cases. Montanari et al. (2016) were able to classify seedlings into different phenotypic classes of hybrid necrosis. Similarly, we classified A. melanocarpa × P. communis hybrids into early-stage hybrid necrosis and ×S. dippelii x P. communis hybrids into a late-stage hybrid necrosis. One possible explanation for hybrid necrosis appearing at a later stage is the genetic pedigree of the parental genotypes. Previous studies have documented that S. aria is compatible with A. melanocarpa to form ×S. dippelii and with P. communis to form ×Sorbopyrus spp. (Leonard, 2011; Sax, 1929). It may act as a genetic bridge for intergeneric hybridization between Aronia and Pyrus. Nevertheless, our two intergeneric hybrids between ×S. dippelii and A. melanocarpa did not survive past 3 months after removal from the in vitro environment.

Because Pyrus-Sorbus-Aronia intergeneric hybrids did not survive outside of in vitro culture, we attempted micrografting as a method to alleviate symptoms of hybrid necrosis and lethality. A previous study reported that grafting was required to grow out F1 hybrids between Malus and Pyrus (Fischer et al., 2014). Grafting was necessary for Malus x Pyrus hybrids because the intergeneric hybrid could not produce a root system sufficient to transport water and nutrients. Previous success has been obtained with micrografting Aronia mitschurinii onto Sorbus aucuparia and ×Sorbaronia fallax (Mahoney and Brand, unpublished data) and chip-bud grafting A. mitschurinii onto different Malinae genera (Brand et al., 2018). We did not find that micrografting could reduce hybrid lethality. Instead we observed hybrid necrosis and lethality to occur at a faster rate after grafting. The severity of the micrograft scion succumbing to hybrid lethality may have been accelerated by failure of the graft union. With failure of the graft union, we would expect a rapid reduction of water and nutrients to the scion. In addition, we found that any putative deleterious compounds produced in the scion remain localized there and are not translocated to the rootstock.

In summary, this is the first report of hybrid necrosis in intergeneric hybrids involving A. melanocarpa, ×S. dippelii, and P. communis. In this study, a limited selection of genotypes were used for intergeneric crosses. Future breeding efforts may have greater success if additional genotypes are used in mating combinations. We have shown that, although intergeneric hybridization between these genera is possible, there are postzygotic barriers that result in loss of hybrid offspring. Deleterious epistatic interactions between these divergent lineages follow the BDM model of genetic incompatibility. Transcriptomics and metabolomics could provide further insights into the molecular mechanisms associated with hybrid necrosis in Aronia, Pyrus, and closely related genera. Future studies could attempt chromosome doubling of intergeneric hybrid seedlings, mutagenesis or genome editing of lethal genes to alleviate the symptoms of hybrid necrosis. In addition, exposing the hybrid plants to high temperatures may reduce lethality. Suppression of hybrid necrosis and lethality would allow for further investigation into the molecular function of hybrid necrosis and enable breeders to recover these intergeneric hybrids for crop improvement.

Literature Cited

  • Anonymous. 2007 AFLP plant mapping protocol. Applied Biosystems, Foster City, CA. 2 July 2018. <https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_040959.pdf>

  • Alcázar, R., Garcia, A.V., Parker, J.E. & Reymond, M. 2009 Incremental steps toward incompatibility revealed by Arabidopsis epistatic interactions modulating salicylic acid pathway activation Proc. Natl. Acad. Sci. USA 106 334 339

    • Search Google Scholar
    • Export Citation
  • Alcázar, R. & Parker, J.E. 2011 The impact of temperature on balancing immune responsiveness and growth in Arabidopsis Trends Plant Sci. 16 666 675

    • Search Google Scholar
    • Export Citation
  • Bell, R.L. & Itai, A. 2011 Pyrus, p. 147–177. In: C. Kole (ed.). Wild crop relatives: genomic and breeding resources. Springer, Berlin & Heidelberg

  • Bomblies, K. 2006 Hybrid incompatibility: When opposites attract with a fatal outcome Curr. Biol. 16 R542 R544

  • Bomblies, K. 2009 Too much of a good thing? Hybrid necrosis as a by-product of plant immune system diversification Botany 87 1013 1022

  • Bomblies, K., Lempe, J., Epple, P., Warthmann, N., Lanz, C., Dangl, J.L. & Weigel, D. 2007 Autoimmune response as a mechanism for a Dobzhansky-Muller-Type incompatibility syndrome in plants PLoS Biol. 5 1962 1972

    • Search Google Scholar
    • Export Citation
  • Bomblies, K. & Weigel, D. 2007 Hybrid necrosis: Autoimmunity as a potential gene-flow barrier in plant species Nat. Rev. Genet. 8 382 393

  • Brand, M.H., Connolly, B.A., Levine, L.H., Richards, J.T., Shine, S.M. & Spencer, L.E. 2017 Anthocyanins, total phenolics, ORAC and moisture content of wild and cultivated dark-fruited Aronia species Scientia Hort. 224 332 342

    • Search Google Scholar
    • Export Citation
  • Brand, M.H., Lubell, J., Mahoney, J. & Wojtyna, N. 2018 Performance of Aronia mitschurinii ‘Viking’ grafted onto Pyrinae tree rootstocks HortScience 53 S387 S388

    • Search Google Scholar
    • Export Citation
  • Campbell, C.S., Evans, R.C., Morgan, D.R., Dickinson, T.A. & Arsenault, M.P. 2007 Phylogeny of subtribe Pyrinae (formerly the Maloideae, Rosaceae): Limited resolution of a complex evolutionary history Plant Syst. Evol. 266 119 145

    • Search Google Scholar
    • Export Citation
  • Chen, C., Zhiguo, E. & Lin, H.-X. 2016 Evolution and molecular control of hybrid incompatibility in plants Front. Plant. Sci. 7 1208

  • Claessen, H., Keulemans, W., Van de Poel, B. & 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

    • Search Google Scholar
    • Export Citation
  • Connolly, B.A. 2014 Collection, Description, Taxonomic Relationships, Fruit Biochemistry, and Utilization of Aronia melanocarpa, A. arbutifolia, A. prunifolia, and A. mitschurinii. Doctoral Dissertations. University of Connecticut, Storrs, CT

  • De Franceschi, P., Dondini, L. & Sanzol, J. 2012 Molecular bases and evolutionary dynamics of self-incompatibility in the Pyrinae (Rosaceae) J. Expt. Bot. 63 4015 4032

    • Search Google Scholar
    • Export Citation
  • Deng, J., Fang, L., Zhu, X., Zhou, B. & Zhang, T. 2019 A CC-NBS-LRR gene induces hybrid lethality in cotton J. Expt. Bot. 70 5145 5156

  • Fischer, T.C., Malnoy, M., Hofmann, T., Schwab, W., Palmieri, L., Wehrens, R., Schuch, L.A., Müller, M., Schimmelpfeng, H., Velasco, R. & Martens, S. 2014 F1 hybrid of cultivated apple (Malus × domestica) and European pear (Pyrus communis) with fertile F2 offspring Mol. Breed. 34 817 828

    • Search Google Scholar
    • Export Citation
  • Gonai, T., Manabe, T., Inoue, E., Hayashi, M., Yamamoto, T., Hayashi, T., Sakuma, F. & Kasumi, M. 2006 Overcoming hybrid lethality in a cross between Japanese pear and apple using gamma irradiation and confirmation of hybrid status using flow cytometry and SSR markers Scientia Hort. 109 43 47

    • Search Google Scholar
    • Export Citation
  • Hiscock, S.J. & McInnis, S.M. 2003 The diversity of self-incompatibility systems in flowering plants Plant Biol. 5 23 32

  • Jeuken, M.J.W., Zhang, N.W., McHale, L.K., Pelgrom, K., den Boer, E., Lindhout, P., Michelmore, R.W., Visser, R.G.F. & Niks, R.E. 2009 Rin4 causes hybrid necrosis and race-specific resistance in an interspecific lettuce hybrid Plant Cell 21 3368 3378

    • Search Google Scholar
    • Export Citation
  • Lehrer, J.M., Brand, M.H. & Lubell, J.D. 2008 Induction of tetraploidy in meristematically active seeds of Japanese barberry (Berberis thunbergii var. atropurpurea) through exposure to colchicine and oryzalin Scientia Hort. 119 67 71

    • Search Google Scholar
    • Export Citation
  • Leitch, I.J. & Bennett, M.D. 2004 Genome downsizing in polyploid plants Biol. J. Linn. Soc. Lond. 82 651 663

  • Leonard, P.J. 2011 Aronia mitschurinii: Solving a Horticultural Enigma. Master’s Thesis. University of Connecticut, Storrs, CT

  • Leonard, P.J., Brand, M.H., Connolly, B.A. & Obae, S.G. 2013 Investigation of the origin of Aronia mitschurinii using amplified fragment length polymorphism analysis HortScience 48 520 524

    • Search Google Scholar
    • Export Citation
  • Mahoney, J.D., Apicella, P.V. & Brand, M.H. 2018 Adventitious shoot regeneration from in vitro leaves of Aronia mitschurinii and cotyledons of closely related Pyrinae taxa Scientia Hort. 237 135 141

    • Search Google Scholar
    • Export Citation
  • Mahoney, J.D., Hau, T.M., Connolly, B.A. & Brand, M.H. 2019 Sexual and apomictic seed reproduction in Aronia species with different ploidy levels HortScience 54 642 646

    • Search Google Scholar
    • Export Citation
  • Masuda, Y., Yamada, T., Kuboyama, T. & Marubashi, W. 2007 Identification and characterization of genes involved in hybrid lethality in hybrid tobacco cells (Nicotiana suaveolens × N. tabacum) using suppression subtractive hybridization Plant Cell Rep. 26 1595 1604

    • Search Google Scholar
    • Export Citation
  • de Mendiburu, F. 2019 agricolae: Statistical Procedures for Agricultural Research. R package version 1.3-1. <https://CRAN.R-project.org/package=agricolae>

  • Montanari, S., Brewer, L., Lamberts, R., Velasco, R., Malnoy, M., Perchepied, L., Guérif, P., Durel, C.E., Bus, V.G.M., Gardiner, S.E. & Changé, D. 2016 Genome mapping of postzygotic hybrid necrosis in an interspecific pear population Hort. Res. 3 15064

    • Search Google Scholar
    • Export Citation
  • Morgan, E.R., Timmerman-Vaughan, G.M., Conner, A.J., Griffin, W.B. & Pickering, R. 2011 Plant interspecific hybridization: Outcomes and issues at the intersection of species Plant Breed. Rev. 34 161 220 John Wiley & Sons, Inc., Hoboken, NJ

    • Search Google Scholar
    • Export Citation
  • Morimoto, T., Inaoka, M., Banno, K. & Itai, A. 2020 Genetic mapping of a locus controlling the intergeneric hybridization barrier between apple and pear Tree Genet. Genomes 16 5

    • Search Google Scholar
    • Export Citation
  • Muralidharan, S., Box, M.S., Sedivy, E.L., Wigge, P.A., Weigel, D. & Rowan, B.A. 2014 Different mechanisms for Arabidopsis thaliana hybrid necrosis cases inferred from temperature responses Plant Biol. 16 1033 1041

    • Search Google Scholar
    • Export Citation
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bio assays with tobacco tissue cultures Physiol. Plant. 15 473 497

  • Nunes, J.D., Azevedo, A.L.S., Pereira, A.V., Paula, C.M.P., Campos, J.M.S., Lédo, F.J.S. & Santos, V.B. 2013 DNA elimination in embryogenic development of Pennisetum glaucum x Pennisetum purpureum (Poaceae) hybrids Genet. Mol. Res. 12 4817 4826

    • Search Google Scholar
    • Export Citation
  • Persson Hovmalm, H., Jeppsson, N., Bartish, I.V. & Nybom, H. 2005 RAPD analysis of diploid and tetraploid populations of Aronia points to different reproductive strategies within the genus: RAPD analysis of populations of Aronia Hereditas 141 301 312

    • Search Google Scholar
    • Export Citation
  • Postman, J.D. 2011 Intergeneric hybrids in Pyrinae (=Maloideae) subtribe of Pyreae in family Rosaceae at USDA Genebank Acta Hort. 918 937 943

  • Sax, K. 1929 Chromosome behavior in Sorbopyrus and Sorbaronia Proc. Natl. Acad. Sci. USA 15 844 845

  • Sun, J., Shi, S., Li, J., Yu, J., Wang, L., Yang, X., Guo, L. & Zhou, S. 2018 Phylogeny of Maleae (Rosaceae) based on multiple chloroplast regions: Implications to genera circumscription BioMed Res. Intl. 2018 1 10

    • Search Google Scholar
    • Export Citation
  • Traw, M.B. & Bergelson, J. 2010 Plant immune system incompatibility and the distribution of enemies in natural hybrid zones Curr. Opin. Plant Biol. 13 466 471

    • Search Google Scholar
    • Export Citation
  • Wu, X., Gu, L., Prior, R.L. & McKay, S. 2004 Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity J. Agr. Food Chem. 52 7846 7856

    • Search Google Scholar
    • Export Citation

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Contributor Notes

This research was partially supported by The Arnold Arboretum Deland Award and US Department of Agriculture (USDA) Multistate Hatch NC007 Plant Germplasm and Information Management and Utilization.

We gratefully acknowledge the USDA–Agricultural Research Service–National Plant Germplasm System and The Arnold Arboretum of Harvard University for providing germplasm and resources for this study.

M.H.B. is the corresponding author. E-mail: mark.brand@uconn.edu.

  • View in gallery

    Pollen-tube–pistil analysis used to determine if intergeneric hybrids exhibit a self-incompatibility system. Intergeneric crosses between (A) Aronia melanocarpa (UC023) x Pyrus communis (PC001); (B) ×Sorbaronia dippelii (UC123) x P. communis (PC001); (C) P. communis (PC001) x ×S. dippelii (UC123); (D) P. communis (PC001) x P. communis (PC002); and self-crossed between (E) P. communis (PC001) x P. communis (PC001). Red arrows indicate terminus of pollen-tube development.

  • View in gallery

    Regeneration of Aronia-Pyrus putative F1 hybrids and parental controls. (A) Parental genotypes [A. melanocarpa (UC023) and P. communis (PC001)] germinate in vitro and continue growth and development (bar = 5 mm); (B) Aronia-Pyrus putative F1 hybrids showing symptoms of hybrid necrosis 14 d after in vitro germination (bar = 5 mm); (C) shoot organogenesis on paternal genotype [A. melanocarpa (UC023)] cotyledons after 2 months of culture (bar = 5 mm); and (D) necrotic callus development on Aronia-Pyrus putative F1 hybrid cotyledons (bar = 5 mm).

  • View in gallery

    Putative hybrids (AA15-2 and AA15-3) from ×S. dippelii (UC123) x P. communis (PC001) (A) multiplied in vitro; (B) microcuttings rooted ex vitro (bar = 10mm); (C) microcutting rooting for putative hybrids and parental genotypes. Bars followed by the same letter did not differ significantly according to Fisher’s least significant difference test (P < 0.05).

  • View in gallery

    Confirmation of putative hybrid status using (A) leaf morphological features (bar = 5 mm); (B) PCA of AFLP marker analysis of putative hybrids and their parental genotypes. Putative hybrids are circled; and (C) flow cytometry showing nuclei relative fluorescence values for the maternal genotype [×S. dippelii (UC123)] and paternal genotype [P. communis (PC001)] and the two putative hybrids (AA15-2 and AA15-3).

  • View in gallery

    Phenotypic observations and progression of hybrid necrosis in (A) maternal ×S. dippelii (UC123), paternal P. communis (PC001) and intergeneric hybrid progeny (AA15-2 and AA15-3) over a 3-week period (bar = 20 mm). (B) Intergeneric hybrid AA15-2 at 21 and 63 d (bar = 10 mm).

  • View in gallery

    Micrograft between AA15-2 intergeneric hybrid scion [×S. dippelii (UC123) x P. communis (PC001)] and S. aucuparia (UC130) rootstock. (A) Progression of graft union and hybrid necrosis (bar = 10 mm). (B) Growth and development of rootstock axillary bud over a 3-week period (bar = 10 mm).

  • Anonymous. 2007 AFLP plant mapping protocol. Applied Biosystems, Foster City, CA. 2 July 2018. <https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_040959.pdf>

  • Alcázar, R., Garcia, A.V., Parker, J.E. & Reymond, M. 2009 Incremental steps toward incompatibility revealed by Arabidopsis epistatic interactions modulating salicylic acid pathway activation Proc. Natl. Acad. Sci. USA 106 334 339

    • Search Google Scholar
    • Export Citation
  • Alcázar, R. & Parker, J.E. 2011 The impact of temperature on balancing immune responsiveness and growth in Arabidopsis Trends Plant Sci. 16 666 675

    • Search Google Scholar
    • Export Citation
  • Bell, R.L. & Itai, A. 2011 Pyrus, p. 147–177. In: C. Kole (ed.). Wild crop relatives: genomic and breeding resources. Springer, Berlin & Heidelberg

  • Bomblies, K. 2006 Hybrid incompatibility: When opposites attract with a fatal outcome Curr. Biol. 16 R542 R544

  • Bomblies, K. 2009 Too much of a good thing? Hybrid necrosis as a by-product of plant immune system diversification Botany 87 1013 1022

  • Bomblies, K., Lempe, J., Epple, P., Warthmann, N., Lanz, C., Dangl, J.L. & Weigel, D. 2007 Autoimmune response as a mechanism for a Dobzhansky-Muller-Type incompatibility syndrome in plants PLoS Biol. 5 1962 1972

    • Search Google Scholar
    • Export Citation
  • Bomblies, K. & Weigel, D. 2007 Hybrid necrosis: Autoimmunity as a potential gene-flow barrier in plant species Nat. Rev. Genet. 8 382 393

  • Brand, M.H., Connolly, B.A., Levine, L.H., Richards, J.T., Shine, S.M. & Spencer, L.E. 2017 Anthocyanins, total phenolics, ORAC and moisture content of wild and cultivated dark-fruited Aronia species Scientia Hort. 224 332 342

    • Search Google Scholar
    • Export Citation
  • Brand, M.H., Lubell, J., Mahoney, J. & Wojtyna, N. 2018 Performance of Aronia mitschurinii ‘Viking’ grafted onto Pyrinae tree rootstocks HortScience 53 S387 S388

    • Search Google Scholar
    • Export Citation
  • Campbell, C.S., Evans, R.C., Morgan, D.R., Dickinson, T.A. & Arsenault, M.P. 2007 Phylogeny of subtribe Pyrinae (formerly the Maloideae, Rosaceae): Limited resolution of a complex evolutionary history Plant Syst. Evol. 266 119 145

    • Search Google Scholar
    • Export Citation
  • Chen, C., Zhiguo, E. & Lin, H.-X. 2016 Evolution and molecular control of hybrid incompatibility in plants Front. Plant. Sci. 7 1208

  • Claessen, H., Keulemans, W., Van de Poel, B. & 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

    • Search Google Scholar
    • Export Citation
  • Connolly, B.A. 2014 Collection, Description, Taxonomic Relationships, Fruit Biochemistry, and Utilization of Aronia melanocarpa, A. arbutifolia, A. prunifolia, and A. mitschurinii. Doctoral Dissertations. University of Connecticut, Storrs, CT

  • De Franceschi, P., Dondini, L. & Sanzol, J. 2012 Molecular bases and evolutionary dynamics of self-incompatibility in the Pyrinae (Rosaceae) J. Expt. Bot. 63 4015 4032

    • Search Google Scholar
    • Export Citation
  • Deng, J., Fang, L., Zhu, X., Zhou, B. & Zhang, T. 2019 A CC-NBS-LRR gene induces hybrid lethality in cotton J. Expt. Bot. 70 5145 5156

  • Fischer, T.C., Malnoy, M., Hofmann, T., Schwab, W., Palmieri, L., Wehrens, R., Schuch, L.A., Müller, M., Schimmelpfeng, H., Velasco, R. & Martens, S. 2014 F1 hybrid of cultivated apple (Malus × domestica) and European pear (Pyrus communis) with fertile F2 offspring Mol. Breed. 34 817 828

    • Search Google Scholar
    • Export Citation
  • Gonai, T., Manabe, T., Inoue, E., Hayashi, M., Yamamoto, T., Hayashi, T., Sakuma, F. & Kasumi, M. 2006 Overcoming hybrid lethality in a cross between Japanese pear and apple using gamma irradiation and confirmation of hybrid status using flow cytometry and SSR markers Scientia Hort. 109 43 47

    • Search Google Scholar
    • Export Citation
  • Hiscock, S.J. & McInnis, S.M. 2003 The diversity of self-incompatibility systems in flowering plants Plant Biol. 5 23 32

  • Jeuken, M.J.W., Zhang, N.W., McHale, L.K., Pelgrom, K., den Boer, E., Lindhout, P., Michelmore, R.W., Visser, R.G.F. & Niks, R.E. 2009 Rin4 causes hybrid necrosis and race-specific resistance in an interspecific lettuce hybrid Plant Cell 21 3368 3378

    • Search Google Scholar
    • Export Citation
  • Lehrer, J.M., Brand, M.H. & Lubell, J.D. 2008 Induction of tetraploidy in meristematically active seeds of Japanese barberry (Berberis thunbergii var. atropurpurea) through exposure to colchicine and oryzalin Scientia Hort. 119 67 71

    • Search Google Scholar
    • Export Citation
  • Leitch, I.J. & Bennett, M.D. 2004 Genome downsizing in polyploid plants Biol. J. Linn. Soc. Lond. 82 651 663

  • Leonard, P.J. 2011 Aronia mitschurinii: Solving a Horticultural Enigma. Master’s Thesis. University of Connecticut, Storrs, CT

  • Leonard, P.J., Brand, M.H., Connolly, B.A. & Obae, S.G. 2013 Investigation of the origin of Aronia mitschurinii using amplified fragment length polymorphism analysis HortScience 48 520 524

    • Search Google Scholar
    • Export Citation
  • Mahoney, J.D., Apicella, P.V. & Brand, M.H. 2018 Adventitious shoot regeneration from in vitro leaves of Aronia mitschurinii and cotyledons of closely related Pyrinae taxa Scientia Hort. 237 135 141

    • Search Google Scholar
    • Export Citation
  • Mahoney, J.D., Hau, T.M., Connolly, B.A. & Brand, M.H. 2019 Sexual and apomictic seed reproduction in Aronia species with different ploidy levels HortScience 54 642 646

    • Search Google Scholar
    • Export Citation
  • Masuda, Y., Yamada, T., Kuboyama, T. & Marubashi, W. 2007 Identification and characterization of genes involved in hybrid lethality in hybrid tobacco cells (Nicotiana suaveolens × N. tabacum) using suppression subtractive hybridization Plant Cell Rep. 26 1595 1604

    • Search Google Scholar
    • Export Citation
  • de Mendiburu, F. 2019 agricolae: Statistical Procedures for Agricultural Research. R package version 1.3-1. <https://CRAN.R-project.org/package=agricolae>

  • Montanari, S., Brewer, L., Lamberts, R., Velasco, R., Malnoy, M., Perchepied, L., Guérif, P., Durel, C.E., Bus, V.G.M., Gardiner, S.E. & Changé, D. 2016 Genome mapping of postzygotic hybrid necrosis in an interspecific pear population Hort. Res. 3 15064

    • Search Google Scholar
    • Export Citation
  • Morgan, E.R., Timmerman-Vaughan, G.M., Conner, A.J., Griffin, W.B. & Pickering, R. 2011 Plant interspecific hybridization: Outcomes and issues at the intersection of species Plant Breed. Rev. 34 161 220 John Wiley & Sons, Inc., Hoboken, NJ

    • Search Google Scholar
    • Export Citation
  • Morimoto, T., Inaoka, M., Banno, K. & Itai, A. 2020 Genetic mapping of a locus controlling the intergeneric hybridization barrier between apple and pear Tree Genet. Genomes 16 5

    • Search Google Scholar
    • Export Citation
  • Muralidharan, S., Box, M.S., Sedivy, E.L., Wigge, P.A., Weigel, D. & Rowan, B.A. 2014 Different mechanisms for Arabidopsis thaliana hybrid necrosis cases inferred from temperature responses Plant Biol. 16 1033 1041

    • Search Google Scholar
    • Export Citation
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bio assays with tobacco tissue cultures Physiol. Plant. 15 473 497

  • Nunes, J.D., Azevedo, A.L.S., Pereira, A.V., Paula, C.M.P., Campos, J.M.S., Lédo, F.J.S. & Santos, V.B. 2013 DNA elimination in embryogenic development of Pennisetum glaucum x Pennisetum purpureum (Poaceae) hybrids Genet. Mol. Res. 12 4817 4826

    • Search Google Scholar
    • Export Citation
  • Persson Hovmalm, H., Jeppsson, N., Bartish, I.V. & Nybom, H. 2005 RAPD analysis of diploid and tetraploid populations of Aronia points to different reproductive strategies within the genus: RAPD analysis of populations of Aronia Hereditas 141 301 312

    • Search Google Scholar
    • Export Citation
  • Postman, J.D. 2011 Intergeneric hybrids in Pyrinae (=Maloideae) subtribe of Pyreae in family Rosaceae at USDA Genebank Acta Hort. 918 937 943

  • Sax, K. 1929 Chromosome behavior in Sorbopyrus and Sorbaronia Proc. Natl. Acad. Sci. USA 15 844 845

  • Sun, J., Shi, S., Li, J., Yu, J., Wang, L., Yang, X., Guo, L. & Zhou, S. 2018 Phylogeny of Maleae (Rosaceae) based on multiple chloroplast regions: Implications to genera circumscription BioMed Res. Intl. 2018 1 10

    • Search Google Scholar
    • Export Citation
  • Traw, M.B. & Bergelson, J. 2010 Plant immune system incompatibility and the distribution of enemies in natural hybrid zones Curr. Opin. Plant Biol. 13 466 471

    • Search Google Scholar
    • Export Citation
  • Wu, X., Gu, L., Prior, R.L. & McKay, S. 2004 Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity J. Agr. Food Chem. 52 7846 7856

    • Search Google Scholar
    • Export Citation
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