Abstract
Vaccinium meridionale (section Pyxothamnus), a tetraploid species native to higher-altitude locations in Jamaica, Colombia, and Venezuela, is of interest to Vaccinium breeders for its profuse, concentrated flowering, vigor, and monopodial plant structure, all of which may be useful in breeding for mechanical harvest in blueberry. In this study, tetraploid V. meridionale was successfully hybridized as both female and male with 2x Vaccinium vitis-idaea (section Vitis-idaea, lingonberry). The resultant F1 hybrids with lingonberry were both 3x and 4x, respectively. These hybrids were intermediate in morphology and notably vigorous. Most appear to be evergreen, with small, red-colored fruit. The 4x F1 hybrids displayed good fertility as females in backcrosses to both lingonberry and V. meridionale. Pollen production and quality were evaluated as an indicator of male fertility. Most clones had good pollen shed and high frequencies of well-formed tetrads. The overall fertility suggests that these hybrids, despite being derived from intersectional crosses, might be conventionally used for breeding without substantial difficulty.
This study follows up three previous publications (Ehlenfeldt and Ballington, 2017a; Ehlenfeldt et al., 2018; Ehlenfeldt and Luteyn, 2021). Two of these publications identified the species material used as V. corymbodendron. That material had been tentatively identified as V. corymbodendron (Dunal, 1839) when originally collected and deposited at the U.S. Department of Agriculture Agricultural Research Service (USDA-ARS) National Clonal Germplasm Repository (NCGR, Corvallis, OR). Further taxonomic study by one of the current authors (J.L.L.) has determined the original identification to be erroneous and has determined that the material should be correctly identified as V. meridionale (Swartz, 1788).
Distribution and Habitat
V. meridionale (section Pyxothamnus) is part of a taxonomic complex that includes V. consanguineum Klotzsch (Costa Rica and adjacent Panama), V. floribundum Kunth (Costa Rica to northern Argentina), and probably also V. puberulum Klotzsch (Venezuelan Guyana Highland) (J.L. Luteyn, personal observation).
V. meridionale is known mostly from the Caribbean-facing watershed slopes of northern Venezuela (Coastal Cordillera and Andes), westward into the Andes of northern Colombia where it is quite common at mid-elevations, and then disjunct to the Caribbean island of Jamaica from where the type was described (Swartz, 1788). It is widespread in its range, but only locally common. Its habitat includes isolated populations in high montane cloud forest to sub-páramo thickets, where it is a shrub up to 3.5 m tall. Its elevational range stretches from ≈1000–2800 m. Notably, flowering material has been collected (for herbarium specimens) in nearly every month of the year; mature fruit have been collected in January-February, July-August, and November-December.
V. meridionale is characterized as having small leaves, the blades of which are pinnately nerved and minutely crenate-serrate margined, and by its racemose inflorescences with small, four- to five-merous, white-to-pink, cylindric to ovoid-cylindric, glabrous flowers, with typically as many as 15 to 25 flowers per inflorescence. Its fruit is usually spherical, or more rarely, slightly oblong or flattened, and ≈14 to 20 mm in diameter. Fruit develop as dark reddish, to dark maroon-black, to blue-black berries in which the top of the ovary is prominently convex or dome-shaped (Fig. 1A and B). The fruit is very uniform in size when optimally pollinated. The fruit is relatively thick-skinned, and the interior flesh pulpy. The thick skins are reflective of their high levels of antioxidants (Gaviria et al., 2009). In very ripe fruit, the interior locule surfaces begin to accumulate pigment, suggesting that it might be possible to accentuate flesh pigmentation in the future with well-planned and carefully selected crosses.
Flowers and fruit of Vaccinium meridionale and V. meridionale - Vaccinium vitis-idaea hybrids: (A) Flowers of V. meridionale, (NC 3737); (B) ripe and unripe fruit of V. meridionale (US 2381); (C) Flowers of F1 V. meridionale × V. vitis-idaea (US 1184); and (D) fruit of F1 (US 1184).
Citation: HortScience 57, 4; 10.21273/HORTSCI16285-21
Lingonberry, V. vitis-idaea, is a circumboreal dwarf evergreen shrub of section Vitis-idaea (Moench) W. Koch. (Camp, 1945). Plant stems are semiwoody, with numerous shoots. Leaves are simple, petiolate, evergreen, leathery, and obovate. The leaf upper surface is dark green; the lower surface is pale green and waxy. Plants reproduce by seeds and rhizomes. Flowers are produced singly or in clusters in terminal racemes, with four locules per ovary, four sepals, a bell-shaped corolla and eight stamens with nonspurred anthers (Hall and Shay, 1981).
Utility of V. meridionale for Breeding
Our original interest in V. meridionale was for its high number of flowers per bud, as well as their loose inflorescence structure that might yield hybrids with highbush blueberry amenable to machine harvest (Luby et al., 1991). Plants of V. meridionale also have the potential to develop an upright, tree-like bush structure with a monopodial base. As we recognized that V. meridionale could cross to lingonberry, it suggests that V. meridionale might broaden the lingonberry genepool and allow introgression of traits such as vigor, increased stature, increased flower number, and potentially resistance to root diseases.
In a previous study, 4x V. meridionale was found to produce high numbers of triploids with 2x V. corymbosum (Ehlenfeldt and Ballington, 2017a). Many questions followed about the nature of the crossing behavior of V. meridionale. We also reported that one paradoxical exception to the observed crossing behavior was the production of a highly fertile 4x hybrid of 4x V. meridional with a species of a different section, 2x V. vitis-idaea L. (Ehlenfeldt and Ballington, 2017a, 2017b). In the current study, we examined the crossability in greater detail of 4x V. meridionale with 2x V. vitis-idaea with the goal of determining the ability to recover fertile introgressed hybrids and incorporation of desired characters from V. meridionale.
Materials and Methods
Plant material.
The genotypes of V. meridionale described here were derived from seed collected in Colombia in 1990 by J.L. Luteyn, then a curator at the New York Botanical Garden. Two of these clones are currently held at the USDA-ARS National Clonal Germplasm Repository. Our particular clone of interest, NC 3735, is maintained as CVAC 1146. Among these two clones, NC 3735/CVAC 1146 has a slightly waxy fruit surface and is the more male fertile of the two as judged by pollen shed. NC 3737/CVAC 1148 has darker fruit and in our experience has almost no pollen shed. Ballington and Luteyn also originally had a third genotype NC 3736. US 2381 was a V. meridionale hybrid that was a self of NC 3737 × NC 3736.
‘European Red’, ‘Erntedank’, ‘Sanna’, and ‘Red Sunset’ are commercial lingonberry cultivars. ‘European Red’ originated in 1981 in the United States (Penhallegon, 2006). ‘Erntedank’ is a German selection from wild lingonberry stand (Zillmer, 1985). ‘Sanna’ is a cultivar released in 1987 by the Swedish University of Agricultural Sciences, Balsgard, Sweden (GRIN global, 2021), and ‘Red Sunset’ is an O.P. selection by Hartmann’s Plant Co. (Lakota, MI) from ‘Koralle’ (D. Hartmann, personal communication). All V. vitis-idaea (lingonberry) cultivars were verified by flow cytometry to be diploids 2x = 2n = 24.
Most crosses were done with ‘Red Sunset’ and ‘Sanna’. These cultivars were selected after preliminary experimentation suggested they have the potential to set, retain, and develop fruit even if only a single seed was present. In addition, ‘Red Sunset’ in test crosses gave indications of producing low levels of 2n gametes, as both eggs and pollen.
Among the advanced lingonberry hybrids, US 1184 was derived from a cross of V. meridionale NC 3737 × V. vitis-idaea ‘European Red’. US 1930, US 1933, and US 1993 are selfs of US 1184. US 2389 is a hybrid of ‘Red Sunset’ × US 1930.
Crossing.
Initially, the V. meridionale plants (NC 3735 clones) grew slowly and flowered sparsely. Thus, for our initial crosses, V. meridionale was used as a male with cultivars and selections. To this end, flower clusters were acquired from plants growing at the NCGR and pollen was collected from these flowers in New Jersey. Due to the nature of the flowers (small with narrow corolla openings) only limited amounts of pollen could be collected.
For both V. meridionale and V. vitis-idaea selections, pollen was extracted from open flowers by manual manipulation, and collected on glassine weighing paper. Pollen was stored for up to a month under refrigerated and desiccated conditions, until used for pollination.
Because of the small, crowded, and somewhat fragile nature of the flowers on many of the parents, no emasculation was performed. All pollinations were made in an insect-free greenhouse, and it was expected that hybrids would be morphologically recognizable. To perform pollinations, a graphite pencil tip was dipped into the collected pollen, and applied to the stigmas of unemasculated flowers. Pollinations were made on mature open flowers.
Ploidy determinations.
Because previous crosses with V. meridionale had produced anomalous triploids, all putative F1 hybrids were assayed for ploidy verification using flow cytometry. For flow cytometry, sampled leaf material (1 cm2/20 to 50 mg) together with leaf material of an internal standard with known DNA content (Zea mays L.) was chopped with a sharp razor blade in 500 mL of extraction buffer (CyStain PI absolute P buffer, catalog number 05-5502; Partec, Münster, Germany) containing RNAse, 0.1% dithiothreitol (DTT), and 1% polyvinylpyrrolidone (ice cold) in a plastic petri dish. After 30 to 60 s of incubation, 2.0 mL staining buffer (CyStain PI absolute P buffer) containing propidium iodide (PI) as fluorescent dye, RNAse, 0.1% DTT, and 1% polyvinylpyrrolidone was added. The sample, containing cell constituents and large tissue remnants of the chopped leaves, was then filtered through a 50-mm mesh nylon filter. After an incubation of at least 30 min at room temperature, the filtered solution with stained nuclei was measured with the flow cytometer [CyFlow ML (Partec) with a green diode laser 50 mW 532 nm (for use with PI); software: Flomax Version 2.4 d (Partec)]. The DNA amount of the unknown samples was calculated by multiplying the DNA amount of the internal standard with the DNA ratio of the relative DNA amount of the unknown sample and the internal standard. DNA amounts were measured and were compared with a set of standards covering a diploid to hexaploid range (2x Vaccinium darrowii ‘Fla 4B’, 4x V. corymbosum cv. Duke, and 6x Vaccinium virgatum cv. Powderblue) to determine basic ploidy levels.
Female fertility.
Cross numbers varied depending on flower availability. Pollinations and fruit set were recorded. Fruit was collected when ripe and measured for fruit size (mm) at the time of seed extraction. Extraction was performed manually under a dissecting microscope, and the seeds were evaluated for number and quality. For our purposes, seeds were classified as: good, good/fair (g/f), fair, fair/poor (f/p), poor, or aborted. “Good” and “fair” described seed that subjectively ranged from those considered fully normal to those somewhat reduced in size and/or development, but nonetheless were judged likely to be capable of germination. “Poor” described seed that displayed reduced size and/or development, often flattened or brown, and judged less likely to be capable of germination. “Aborted seeds” were those that were flat and brown, and generally translucent. Measurements were made of the sizes of aborted seed.
Seeds were germinated on a greenhouse mist bench in a soil mix composed of 50:50, peat:sand mixture. At about a 3 true-leaf stage, seedlings were transplanted to 36-cell flats. All primary hybrids were transferred to 3-L pots in their second season.
Male fertility.
Pollen samples were stained with acetocarmine jelly (75% acetic acid with iron acetate) prepared according to the recipe of Jensen (1962). Pollen samples were assayed for quantity, stainability, and general condition. For general condition, our ratings were as follows: very good = almost all tetrads, good = tetrads and triads, fair = almost exclusively triads, and poor = mostly aborted pollen grains.
Results
Primary crossing.
Working with both V. meridionale and lingonberry (V. vitis-idaea) in New Jersey is a considerable challenge, because neither is well adapted to hot, humid New Jersey summers. Both flower sparingly and sporadically, nonetheless numerous crosses were successful.
V. meridionale - 2x V. vitis-idaea hybrids were arrived at in a somewhat circuitous manner. The original clone of this type, V. meridionale × V. vitis-idaea ‘European Red’, was a rare 4x offspring among a set of 4x V. meridionale × 2x section Cyanococcus crosses that were all shown to be triploids. Original tabulations have been lost, but the original hybrid arose from a small set of pollinations done as 4x V. meridionale NC 3737 × V. vitis-idaea ‘European Red’. This original hybrid, US 1184, was a slow-growing plant that took several years to initiate flowering. When it ultimately flowered, there were neither V. meridionale nor V. vitis-idaea flowers or pollen available for test crosses. It was, therefore tested for self-fertility, and the few flowers present were self-pollinated. From these self-pollinations, ≈20 offspring were produced. Soon after this initial selfing, US 1184 perished. US 1184 morphologically appeared intermediate to the two parents, but most notably produced spherical fruit that ripened to a deep red color, rather than the black or blue-black that might be expected (Fig. 1C and D). Considerable variation existed among the US 1184 self-progeny. Although all resembled US 1184, there were considerable differences in vigor and propensity to flower. Ultimately, three S1 genotypes stood out as being vigorous, prolific, and fertile. These were US 1930, US 1933, and US 1993. US 1930 was uncontestably the best of these. These S1 clones were evaluated by flow cytometry and all found to be 4x. All shed pollen well, and pollen was regular and fertile (see “Male fertility of hybrids,” below).
As research progressed, we recognized the need to repeat the original cross of 4x V. meridionale × 2x V. vitis-idaea (lingonberry) and also test its reciprocal. In this endeavor, no 4x lingonberry was available, so repeat success, if any, in these crosses was expected to be dependent on the functioning of 2n gametes. Because the presence of 2n gametes in lingonberry is undetermined, a selection of lingonberry cultivars was evaluated as both males and females. As previously noted, after several years of evaluations, we came to rely on ‘Red Sunset’ and ‘Sanna’ for vigor, profuse flowering, and fruit retention, and particularly on ‘Red Sunset’ for indications it gave of 2n gamete production.
Using V. meridionale US 2381 as a female, 91 pollinations were made: 75 with ‘Red Sunset’, none of which produced any seed, and 16 with ‘Sanna’, which ultimately produced four triploid hybrids (Table 1). Using US 2381 as a male, 53 pollinations were made: 21 with ‘Red Sunset’, which resulted in two tetraploid hybrids, and 32 pollinations with ‘Sanna’ that failed to produce any seed (Table 1). Thus, from these interspecific crosses, we currently hold six F1 hybrids: two tetraploids in lingonberry cytoplasm from ‘Red Sunset’, and four triploids in V. meridionale cytoplasm from crosses with ‘Sanna’. In the first case, V. meridionale succeeded as a male with 2n eggs from ‘Red Sunset’. In the latter case, V. meridionale, as a female, succeeded in crosses when pollinated with 1n pollen (by contrast, the original US 1184 hybrid resulted from functioning of 2n pollen). Across the two successful combinations, crosses with ‘Red Sunset’ (as female) set 0.3 seed/fruit (s/f), and crosses with ‘Sanna’ as the male set 1.6 s/f. The low frequency of these results prevents us from drawing any specific conclusions about triploidy vs. tetraploidy as it relates to cross directionality.
Hybridizations of Vaccinium vitis-idaea with Vaccinium meridionale, and with V. meridionale - V. vitis-idaea hybrids.
Morphological appearance of 4x V. meridionale-V. vitis-idaea hybrids.
V. meridionale has a relatively small, elliptical, uniformly flat leaf with fine lobe-like scalloping along the edges with a matte surface, and no waxiness.
Lingonberry has elongate-obovate leaves, indented (or nearly indented) tips (visible in Fig. 2), moderate texture and a significant midvein crease; margins are minutely serrate and moderately revolute.
Leaves of Vaccinium meridionale, S1 hybrid, US 1930 (F1 selfed), and Vaccinium vitis-idaea.
Citation: HortScience 57, 4; 10.21273/HORTSCI16285-21
Hybrids with V. meridionale were most often recognizable by V. meridionale’s influence. The morphology of US 1184 appeared in most obvious ways to be intermediate (Fig. 1C and D). Among the second set of F1 hybrids of V. meridionale – V. vitis-idaea, all were fairly distinct in their morphology, having leaves that are relatively small (3 cm × 1.5 cm), pointed, and lance-shaped to obovate (Fig. 2). Leaves under ideal conditions were dark green, lightly textured in a pattern reminiscent of lingonberry, very slightly serrate, and possessing slightly revolute edges. Leaves were very regular in size, and in the greenhouse, shoots developed in an indeterminate growth pattern, rather than distinct flushes. At this point, few of the F1s have flowered. However, among the S1s, the flowers were intermediate, but more similar to those of V. meridionale, than to those of V. vitis-idaea, which possesses relatively open-throated flowers (Fig. 1C). The hybrids generally had elongate, oval-shaped flowers with a slightly constricted throat. Flowering occurred in branched clusters, and flowering appeared to be indeterminate. Ripe fruit of these hybrids was a medium red, similar to lingonberry (Fig. 1C). S1 and F1 hybrids appeared to be vegetatively more vigorous than either parent under greenhouse conditions (Fig. 3). Beyond vigor, one of the notable characteristics of the F1s, S1s, and backcrosses was their non-rhizomatous nature. Unlike the rhizomatous nature of lingonberry, S1 genotypes branched freely at their bases, but no recognizable rhizomatous growth was observed.
Plants of S1 hybrid, US 1930 (Vaccinium meridionale ×Vaccinium vitis-idaea) (left) and ‘Red Sunset’ V. vitis-idaea (right).
Citation: HortScience 57, 4; 10.21273/HORTSCI16285-21
Male fertility of hybrids.
In 2021, only a limited number of these selections had flowered (Table 2). What was seen in these evaluations is that the five F1-type genotypes (F1s and S1s) all exhibited well-formed tetrads as well as triads with well-formed subunits. US 1930 shed well and functioned well as a male in crosses with ‘Red Sunset’. US 2389 successfully self-pollinated (Table 1). The two backcross hybrids similarly both exhibited good pollen shed and produced sufficient levels of well-formed tetrads and triads that suggested well-functioning pollen (Fig. 4). Although the number of genotypes evaluated was small, these genotypes serve as a proof-of-concept that male fertility can exist in both F1 and BC1 generations.
Male fertility of Vaccinium meridionale - Vaccinium vitis-idaea hybrid germplasm.
Pollen images of F1 Vaccinium meridionale × Vaccinium vitis-idaea (US 1184), F1 V. meridionale × V. vitis-idaea (US 2513-A), S1 US 1930 (= US 1184 selfed), and BC1 US 2389 (= V. vitisidaea ‘Red Sunset’ × US 1930) (×200).
Citation: HortScience 57, 4; 10.21273/HORTSCI16285-21
BC1-type crosses to lingonberry.
Thus far, the 4x S1 genotypes, US 1930 and US 1933 have been used most extensively in further crossing with 2x ‘Red Sunset’ and 2x ‘Sanna’. Similar to the F1 crosses, these crosses had a low level of success due to ploidy differences, but over several years, multiple hybrids (based on morphology) have been produced: US 1930 × ‘Red Sunset’ (11 hybrids: 7@4x, 5@3x); ‘Red Sunset’ × US 1930 (1 hybrid@4x); US 1930 × ‘Sanna’ (3 hybrids@4x); US 1933 × ‘Red Sunset’ (1 hybrid@4x); US 1930 × ‘Erntedank’ (1 hybrid@4x), and ‘Red Sunset’ × US 1933 (1 hybrid@4x) (Table 1). It is worth noting that among these crosses, both 1n and 2n pollen from both ‘Sanna’ and ‘Red Sunset’ succeeded in producing hybrids, but as females only 2n eggs from ‘Red Sunset’ resulted in hybrid production. Again, it should be noted that these crosses had a very low number of seeds per fruit (range 0.5 to 1.8).
The oldest among the S1 – V. vitis-idaea offspring, US 2389 (= ‘Red Sunset’ × US 1930), is essentially a BC1 hybrid to lingonberry. US 2389 displayed both fruit set and fruit development upon selfing and upon backcrosses (BC2) to ‘Red Sunset’ (Fig. 5). It also showed excellent pollen shed (Table 2, Fig. 4).
(A) Flowers of S1 Vaccinium meridionale × Vaccinium vitis-idaea (US 1930); (B) fruit of S1 (US 1930); (C) flowers of BC1 (US 2389 = US 1930 × ‘Red Sunset’); and (D) Fruit of BC1 (US 2389).
Citation: HortScience 57, 4; 10.21273/HORTSCI16285-21
Backcrosses, S1 × V. meridionale.
These crosses were easily accomplished and the offspring possessed a hybrid appearance. The most successful among these backcrosses (US 1930 × V. meridionale, US 2381) yielded 5.6 hybrids per pollination (Table 1). By comparison, the best cross among the S1 × lingonberry combinations, US 1930 × ‘Red Sunset’, yielded only 0.069 hybrids per pollination. The most notable morphological feature of these offspring was a pronounced rosy tinge to young growth, which was not seen in the other hybrids generated. The results from these crosses suggested the success that might be possible in backcrosses with 4x lingonberry, if such clones were available. These hybrids may be useful parental material, being three-fourths V. meridionale introgressed with lingonberry.
Discussion and Conclusions
Vaccinium meridionale represents a remarkable bridging species that is just beginning to demonstrate its potential to allow germplasm movement among Vaccinium species. The primary cross in this study, V. meridionale × V. vitis-idaea is an intersectional cross between sections Pyxothamnus and Vitis-idaea. Moreover, it is a cross between a species recognizable as blueberry and lingonberry. This study complements similar reports of crosses between V. meridionale and V. corymbosum (Ehlenfeldt and Luteyn, 2021), and crosses of V. meridionale and 4x Vaccinium macrocarpon (Ehlenfeldt et al., in preparation).
Several salient points can be made regarding the crosses reported here:
Despite being intersectional crosses, it was possible to generate hybrids with 4x V. meridionale with relative ease, and hybrids were generated with more than one crop-parent genotype. Under our conditions, in most cases, hybrids are more vigorous than either parent, especially with respect to growth and stature, and at least subjectively, they appear to have better root rot resistance than lingonberry. The vigor of these hybrids would seem to make them significant contenders for improving lingonberry.
The crosses of 4x V. meridionale × 2x lingonberry (and the reciprocals) generated relatively low numbers of hybrids, but this is not unexpected from 4x - 2x crosses. Success of such crosses depends on either the functioning of 2n gametes (producing tetraploids) (Hanneman and Peloquin, 1968) or other exceptional fertilization events (producing triploids). Similar crosses done within section Cyanococcus have had equally low or lower success rates (Dweikat and Lyrene, 1988; Vorsa and Ballington, 1991). In our previous work with V. meridionale (Ehlenfeldt and Ballington, 2017a), high levels of triploids were produced from 4x × 2x crosses, and some of our success here might be reflective of V. meridionale’s propensity to produce triploid offspring.
One of the significant limitations in the crosses with lingonberry, is the lack of 4x germplasm. We currently have six verified tetraploid lingonberry seedlings from colchicine-treated seed, but they will not be of any practical use until mature. In addition, these seedlings are from unselected populations. No other domestically available 4x lingonberry germplasm has been identified, although it has been reported to occur naturally in Belarus (Morozov, 2007). In the meantime, ‘Red Sunset’ lingonberry produces at least limited levels of 2n gametes (yielding 4x hybrids). ‘Sanna’ lingonberry also has been a successful and interesting parent, producing F1 triploids and both triploids and tetraploids in BC1 type crosses.
Notably, our hybrids, the 4x particularly, were highly fertile. Fertility of F1 allotetraploids might be expected because every chromosome is expected to have a homologous pairing partner (Clausen and Goodspeed, 1925). Furthermore, recent findings of high levels of synteny among Vaccinium spp. genomes suggest little differentiation in general among Vaccinium genomes (although V. vitis-idaea is not among those published genomes) (Qi et al., 2021, Wu et al., 2021). High levels of synteny suggest that ongoing recombination and fertility may be possible.
Specifically, female fertility has been demonstrated by success in selfing, BC1-type backcrosses to both species parents, and S1 sibbing (data not shown). The most advanced hybrid, US 2389 exhibited relatively low seed set in self-pollinations; however, even these low levels of fertility stand as a proof-of-concept result for V. vitis-idaea – V. meridionale introgression. The generation of additional hybrids and subsequent inbreeding might be expected to stabilize and improve self-fertility.
Similarly, the 4x hybrids as F1, S1, and BC1 all exhibit an observable degree of male fertility. Among these hybrids, all are producing good levels of well-formed tetrads.
Crosses and hybrids of somewhat similar nature have been documented previously. Ritchie (1955a, 1955b) identified natural hybrids of V. vitis-idaea and Vaccinium myrtillus that he named Vaccinium intermedium and conducted crossability studies with the same. Christ (1977) made crosses of V. vitis-idaea and V. macrocarpon. Zeldin and McCown (1997) created hybrids between lingonberry and Vaccinium reticulatum (‘Ōhelo berry) and conducted further crosses. However, a critical difference between those previous hybrids and the current ones are the resultant ploidy levels. The earlier crosses occurred between diploids and resulted in hybrids with limited or no fertility. The truest antecedent to the current crosses are those of Morozov (2007) in Belarus, who generated fertile 4x hybrids between 4x Vaccinium uliginosum (bog bilberry) and 4x accessions of V. vitis-idaea. Morozov’s (2007) findings are analogous to the current findings in that the 4x hybrids possessed viable fertility and successfully backcrossed to a range of 4x Vaccinium material. Morozov (2007) envisioned the improvement of lingonberry by introgression of other Vaccinium germplasm.
Much remains to be understood about intersectional hybrids in Vaccinium. However, given these results, and the results of several other studies (Ehlenfeldt and Polashock, 2014; Lyrene, 2011, 2016; Morozov, 2007), intersectional crossing barriers appear to be modest at best. The hybridization of Vaccinium padifolium (Ehlenfeldt and Polashock, 2014) would have seemed to be an extraordinarily difficult one, based on modern molecular-based taxonomic determinations that listed sect. Hemimyrtillus species among the furthest outliers from section Cyanococcus (Powell and Kron, 2002), yet these hybrids were made with only moderate difficulty, and the resultant tetraploid hybrids were highly fertile. There seems to be no doubt that diploid intersectional hybrids present fertility issues, but if hybrids can be made at the tetraploid level, allotetraploid fertility and apparent tetraploid buffering almost surely guarantee some level of success will exist in the backcrossing of intersectional hybrids to tetraploid cultivated germplasm. To be sure, in wide crosses, even after seed is produced, not all genetic combinations are fully viable. After germination, seedlings may manifest morphology ranging from viable, to sub-lethal, to lethal. Among sub-lethal types we see, there are those that display reddish pigmentation and develop slowly, others that are deeply pigmented and barely grow beyond the cotyledonary stage, and still others that stagnate and die within a brief period. Occasionally sub-lethal genotypes transition from stagnation to relatively normal growth. Of true value, though, are the plants that display relatively normal growth and a vigorous genotype.
Like Morozov, we believe V. vitis-idaea may benefit significantly in the short-run from introgression of new germplasm. The accessible genepool of lingonberry is small. V. meridionale has significantly expanded the pool of usable germplasm. Many temperate growers have experimented with lingonberry, only to find it too susceptible to root disease to be a viable option. In limited observations, our F1 hybrids have shown greater tolerance to root rot than pure lingonberry material. An allotetraploid lingonberry with appropriate amounts of germplasm introgressed from other Vaccinium species might open new options in lingonberry or lingonberry-like fruit production.
Based on our studies, we feel that V. meridionale will play a critical role in the future in producing wide hybrids, as it appears to be a very accepting parent. We believe we are on the brink of gaining access to a wide array of tertiary Vaccinium germplasm that will benefit both conventional and molecular aspects of Vaccinium breeding.
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