HORTSCIENCE 53(2):159–166. 2018. https://doi.org/10.21273/HORTSCI12650-17 Deerberries can readily be hybridized in a greenhouse with diploid species in section Cyanococcus, which are close relatives of Hybridization of Cultivated Highbush highbush blueberry (Darrow and Camp, 1945). Most of the hybrids are weak and Blueberry with Vaccinium stamineum: have very low fertility. We have produced hundreds of seedlings by crossing deerberries Phenotype and Fertility of Backcross1 with each of three diploid section Cyanococ- cus species collected from Florida: Vacci- Populations nium darrowii, Vaccinium fuscatum, and Vaccinium elliottii. The F1 hybrids varied in Paul M. Lyrene1 vigor depending on the particular clones of Department of Horticultural Sciences, University of Florida, Gainesville, FL each species used as parents, but almost all have been much weaker than typical high- 32611 bush seedlings. Additional index words. deerberry, wide hybridization, polyploidy, evolution, plant breeding Deerberries have several characteristics that would be useful in highbush blueberry Abstract. Nine F1 hybrids produced by crossing seven tetraploid highbush blueberry cultivars. The plants are very drought tolerant cultivars with three tetraploid Vaccinium stamineum genotypes were backcrossed to an and can produce high berry yields when array of highbush blueberry cultivars to produce 2500 Backcross1 (BC1) seedlings. growing in dry forests. The species is poly- Thirty of the most vigorous BC1 plants were intercrossed in a greenhouse. Fertility of the morphic for berry flesh color. Ripe berries of BC1 plants was studied by examining their pollen at 2503 and by determining the some deerberry plants have purple or bright- number of well-developed seeds per pollinated flower after the crosses. Percent well- red flesh, whereas berries of Vaccinium formed microspores averaged 62.7% for BC1 plants compared with 30.9% for their F1 section Cyanococcus species have green, hybrid parents. Fruit set percentage was high from the BC1 intercrosses, with an average white, or yellow flesh. Freshly harvested ripe of 9.29 plump seeds per pollinated flower. The 2500 BC1 seedlings in the field were highly berries from most deerberry clones, when variable in vigor, but fewer than 10% were as vigorous as the median vigor of highbush 3 mixed with three volumes of water and ’ highbush seedlings. BC1 plants in the field averaged ’1 month later ripening than crushed by hand to liberate flesh and skin highbush 3 highbush seedlings and berries averaged slightly smaller. Berry clusters pigments, produce a liquid that is bright red were very loose compared with those of highbush. Berry flavor was highly variable from to magenta in color. The same procedure plant to plant, but the berries averaged less sweet and lower in acid than highbush using fresh highbush blueberries normally berries. New flavor components not found in highbush seedlings were found in only a few produces a greenish-brown liquid. Only BC1 seedlings. Fresh berries from BC1 plants made bright red juice when crushed in a few highbush clones produce a pink liquid water, whereas berries from most highbush cultivars produced brown to yellow juice/ when their fresh berries are crushed in water. water mixes. Although berry quality in the BC1 population averaged lower than in The corolla tube of deerberry is very short highbush seedlings, some plants had berry quality as high as typical cultivars. Because (Fig. 1). Both anthers and stigma project well V. stamineum is highly drought-tolerant, cultivars bred using V. stamineum introgression beyond the end of the tube. This flower could have improved upland adaptation. structure could make blueberry cultivars easier to pollinate. Carpenter bees (Xylocopa spp.), for example, cut holes in the side of the Vaccinium stamineum (deerberry), a highly berries lack the grit that makes sparkleberries corolla tube of highbush blueberry and rob polymorphic diploid species, is the only spe- (Vaccinium arboreum) nearly inedible, but the nectar without pollinating, but they pollinate cies in Vaccinium section Polycodium (Ashe, skins of deerberries are thicker than those of deerberry flowers legitimately. Deerberry 1931; Baker, 1970; Camp, 1945). Endemic in section Cyanococcus blueberries. flowers are ‘‘open in the bud’’ i.e., the stigma North America, it is found from southwestern Another deerberry characteristic that has is visible in the very small, developing flower Ontario to central Florida and west to eastern discouraged its domestication is berry shat- long before anthesis (Luteyn et al., 1996). Texas, with a few outlying populations in tering. To various degrees, depending on the Deerberry flowers are produced on new central Mexico (Vander Kloet, 1988). In north genotype, berries fall from the plant soon spring growth flushes. Deerberry plants pro- Florida, deerberry plants often produce high after ripening, with the pedicel remaining duce dimorphic branches: sterile vegetative yields of edible berries while growing on attached to the berry. branches and fertile branches with leafy excessively drained, sandy soils in the forest. Deerberry plants can easily be grown bracts, which have a flower in the axil of Cultivated highbush blueberry, which is based from seed, and seedlings grow vigorously in each bract. In some phenotypes, bud size on on hybrids between Vaccinium corymbosum nurseries if the soil is acid and well-drained, dormant plants in late winter differs between and other species in Vaccinium section Cya- but the plants are extremely hard to propagate buds that will give rise to fertile branches nococcus, has become an important world clonally. Neither softwood cuttings nor root and those that will produce purely vege- crop, but deerberry has never been domesti- cuttings are successful, unless cutting wood tative branches (anonymous reviewer). In cated. There are several reasons for this. is taken from very young seedlings. Thus, to Florida, highbush blueberry plants, which Deerberries are large (compared with berries make greenhouse crosses using superior wild produce their flower buds in the fall, usually of native plants of other Vaccinium species), plants from the forest, propagules must be have few flowers the following spring if juicy, and have high Brix, but the skins and taken that contain both roots and stems. This diseases or insects defoliate the plants in sometimes the flesh have bitter components, is easy enough for deerberry plants that have August or September. The same is not true ranging from slightly bitter to almost inedibly a colonial habit, but is difficult for crown- of deerberries. bitter and astringent depending on the popula- forming types that make few sprouts. Deer- In 2010, we started a program to produce tion sampled and the clone sampled within the berries can be grafted onto rootstocks of large populations of tetraploid hybrids be- population. Deerberry seeds are only slightly highbush blueberry or V. arboreum to pro- tween highbush blueberry cultivars and tet- larger than those of highbush blueberry. The duce flowering branches for crossing, but our raploid V. stamineum plants obtained by grafts have not retained vigor for more than colchicine treatment of seeds and seedlings. a few years. The goal was to produce a broad-based Received for publication 25 Oct. 2017. Accepted Uttal (1987) reported a natural hybrid hybrid population that could be used to for publication 18 Dec. 2017. from Virginia between Vaccinium pallidum combine horticulturally desirable features of 1Corresponding author. E-mail: lyrene@ufl.edu. (section Cyanococcus) and V. stamineum. the two taxa and to transfer genetic material

HORTSCIENCE VOL. 53(2) FEBRUARY 2018 159 in either direction from the F1 hybrids to the Florida. The source plants were 2–4 m tall and crosses produced fewer than 100 seedlings; parent species. Production of the first tetra- were part of a large population of similar plants others produced far more than the 100 ploid hybrids was previously reported, and growing in the understory of a thin stand of seedlings that were saved. One nursery was a description of the fertility and phenotype of native pines (Pinus palustris and Pinus elliot- planted in May 2016, the other in May 2017. the F1 hybrids was given (Lyrene, 2016). The tii). They were selected as a source of seeds Vigor and growth of plants in each nursery purpose of this article is to describe the because they had high yields of large berries wereobservedweeklyuntil1Nov.2017. results of backcrossing these F1 intersectional that were not very bitter or astringent and did On 14 Oct. 2016, 149 d after they had been hybrids to the two parent taxa. not readily shatter. transplanted to the 2016 high-density field To produce the BC1 plants, tetraploid nursery as seedlings 10 cm tall, 2536 BC1 Materials and Methods highbush advanced lines from the Florida seedlings were rated for vigor, along with 351 cultivar-development program were crossed F1 seedlings and 227 seedlings produced by Evaluation of hybrid seedlings in high- with F1 hybrids. Twelve different highbush intercrossing F1 plants (Table 2). One hundred density field nursery. Seeds from five types of advanced lines and 11 different F1 plants and eight seedlings from highbush cultivar · crosses (Table 1) were produced by hand were used. Inbreeding was minimized by highbush cultivar crosses, and 113 from dip- emasculation and controlled pollination in a choosing parent combinations that ensured loid V. stamineum · diploid V. stamineum greenhouse in Jan. and Feb. 2015 and 2016. that every BC1 seedling would have four crosses, which had been transplanted to the The immediate parents of the F1 intersectional distinct genotypes as grandparents. The re- same nursery at the same time, were also hybrids used in these crosses included seven sulting seeds were germinated on peat in a evaluated. Each seedling was given a vigor advanced tetraploid highbush selections from greenhouse under mist in November of the score ranging from 1 to 9. Plants scored in the University of Florida blueberry breeding year the crosses were made. Up to 100 category 1 appeared to be too weak to flower program and three tetraploid V. stamineum seedlings from each cross were transplanted in the following spring. Plants scored 9 were seedlings (Lyrene, 2016). The V. stamineum to trays of peat the following January and equal in vigor to the most vigorous 8% of the tetraploid parents were obtained using February, and the seedlings were trans- highbush seedlings. Category 6 comprised colchicine-treatment of seeds that had been planted to high-density field nurseries at a plants that had the minimum vigor required collected from four plants growing on deep, spacing of 15 cm · 40 cm at the University of in a commercial cultivar. sandy soil in southeastern Alachua County, Florida Plant Science Unit, Citra, FL. Some About 2500 BC1 plants remained in the high-density field nursery in Jan. 2017. Sev- eral hundred of these plants flowered in January, February, or March and ripened fruit in Apr. and May 2017, 1.5 years after the seed was planted to produce them. Many other BC1 plants were too small to flower in 2017. During the ripening season, the plants that had fruit were examined weekly to observe plant and fruit characteristics. Berry size, color, scar, firmness, flavor, flesh color, and cluster looseness were observed for plants that fruited. Evaluation of the fertility of BC1 plants placed in a greenhouse for crossing. On 1 Dec. 2016, from the population of 2536 BC1 seedlings growing in the high-density nurs- ery, 35 of the most vigorous plants were dug, potted into 20 L pots, and placed in a green- house for crossing. Three plants were later discarded because they showed no clear in- dications of being valid hybrids. Fertility of the remaining BC1 seedlings was estimated by examining pollen from each plant and by counting the number of plump seeds pro- duced per pollinated flower after intercross- ing BC1 plants. Pollen from the first flowers Fig. 1. Flowers of diploid Vaccinium stamineum showing long pedicels and stigma and anthers exserted that opened on each BC1 plant was mounted from the corolla tube. in 45% acetic acid on a microscope slide and examined at 250·. Blueberry pollen is shed in tetrads of four microspores. Several hun- Table 1. Number of F1,F2, and backcross seedlings transplanted to high-density field nurseries in May 2016 and May 2017 from crosses made in 2015 and 2016.z Seeds were sown in a greenhouse in dred pollen tetrads were examined from each November of the year the crosses were made and the seedlings were grown in greenhouse trays from plant. Percentage of well-formed microspores January until transplanting. was estimated for each BC1 plant. Percentages were based on individual microspores, not on Number of crosses and number of seedlings perfectly formed tetrads. 2016 nursery 2017 nursery Fertility was also assessed by intercross- y Type of cross Crosses Seedlings Crosses Seedlings ing plants during Jan. and Feb. 2017. In each x F1 (HB · Stam 4X) 10 350 17 860 · cross, the parents were chosen so that every F2 (F1 F1) 6 230 6 600 seedling would have four distinct clones as BC1 (HB · F1) 17 1,480 8 640 BC (F · HB) 12 1,060 19 1,440 grandparents. Twenty-six BC1 clones were 1 1 used as female parents and 13 were used as BC1 (F1 · Stam 4X) 2 48 8 445 zMost crosses produced far more than 100 seedlings, but no more than 100 random seedlings were saved male parents. Clones were selected as males from each cross. if they made numerous flowers and shed y Both parents in all crosses were tetraploid and the seedlings are expected to be tetraploid. pollen well. The BC1 plants used as parents xHB = tetraploid highbush cultivar; Stam 4X = colchicine-induced tetraploid Vaccinium stamineum. had been produced using seven highbush

160 HORTSCIENCE VOL. 53(2) FEBRUARY 2018 Table 2. Number of seedlings from various crosses in each of nine vigor categories, classified 149 d after V. stamineum. The number of plump seeds per the seedlings were transplanted to the 2016 high-density field nursery. Seedlings were transplanted on pollinated flower was determined. 18 May and evaluated on 14 Oct. 2016. Vigor categoriesz Results 1 2 3 4 5 6 7 8 9 Total type of cross Highbush · Highbush 0 3 19 14 14 11 10 28 9 108 Field nursery evaluations of F1,F2, and y Stam 2X · Stam 2X 1 6 10 24 18 15 16 20 3 113 BC1 seedling populations. Vigor of BC1 · F1 (HB Stam 4X) 195 82 47 16 4 2 3 2 0 351 seedlings on 14 Oct. 2016, after 149 d in · F2 (F1 F1) 102 77 22 18 7 1 0 0 0 227 the 2016 high-density field nursery (Table 2), BC1 (F1 · HB) 305 280 199 140 37 43 33 17 2 1,056 · averaged far lower (median vigor score 2) BC1 (HB F1) 316 458 302 198 97 37 37 34 1 1,480 than highbush seedlings (median score be- BC1 (F1 · Stam 4X) 23 14 9 2 0 0 0 0 0 48 z tween 5 and 6) and V. stamineum seedlings Category 1 = extremely weak; will probably never flower; Category 6 = minimum vigor needed in (median 5) in the same nursery. BC seedling a cultivar; Category 9 = equal in vigor to the most vigorous 8% of highbush x highbush seedlings. 1 yStam 2X = diploid Vaccinium stamineum; Stam 4X = tetraploid V. stamineum; HB = highbush cultivar; vigor was highly variable; 114 of the 2536 BC seedlings received vigor scores of 7, 8, F1 = Highbush cultivar · tetraploid V. stamineum. 1 or 9. Plant vigor varied greatly depending both on the particular clones used in the backcross and on the seedling within the cultivars and nine different F1 hybrids, and of the anther tubules and stigma relative to segregating population of each cross. Some the F1 parents traced back to three different the end of the corolla tube (Luteyn et al., crosses produced only weak BC1 seedlings. V. stamineum tetraploid plants, FL13-869, 1996), presence or absence of anther awns, From other crosses, some BC1 seedlings were FL13-870, and FL13-872 (Lyrene, 2016). and whether the flowers were open in the bud. vigorous enough to be successful cultivars. Mites (Eotetranychus clitus), which in pre- Berry characteristics evaluated in the F1 and The BC1 seedlings averaged higher in vigor vious years had been much more damaging BC1 plants included tendency of ripe berries than the 351 F1 seedlings and the 227 F2 on the F1 hybrids than on plants of either to fall from the plant (shatter), whether the seedlings, both of which had median vigor parent species, were controlled in the green- pedicel remained attached to the berry or scores of 1 (Table 2). F2 seedlings seemed no house using predatory mites (Aphidius cole- stayed on the plant when the berry was more vigorous and no more variable in vigor mani, Koppert Biological Systems; www. picked, presence or absence of waxy bloom than F1 seedlings, even though the F2 plants koppert.com). on the ripe berries, flesh color of mature were obtained by intercrossing unrelated F1 In making the crosses, flowers were berries, and flavor notes. Several berries from plants. In both F1 and F2, leaf color of many emasculated before anthesis and pollinated each seedling were tasted, and a nine-point plants were bronze, rather than green. Most immediately using pollen from other plants in scale was used to rate each of four aspects of F1 plants in the 2014 and 2015 nurseries had the same greenhouse. The number of flowers flavor: sweetness, acidity, bitterness, and other this same leaf color (Lyrene, 2016). Some pollinated and the number of ripe berries flavor components. The first 15 berries that BC1 plants also had bronze-colored leaves, harvested from each female parent were ripened on each BC1 plant were weighed, in but most had green leaves. recorded. At the end of harvest, the seeds three groups of five berries each, and compared By Aug. 2017, 10 months after the orig- were extracted by manually crushing the with the weights of berries picked from high- inal assessment, both average vigor and berries in 500 mL of water in a 4 L plastic bush cultivars and F1 (highbush · tetraploid variability in vigor among plants within the bucket, churning the water vigorously by V. stamineum) plants in the same greenhouse F2 and BC1 crosses had greatly increased in hand, and decanting the skins, pulp, and and with berries harvested from the forest the 2016 field nursery. When the most vig- water. The seeds that remained in the bottom from five diploid V. stamineum plants selected orous F1 and BC1 plants were potted in peat of the bucket were washed several times with because they had large berries. and irrigated with water from which most a small volume of water, and the clean seeds Fertility of BC1 plants obtained by bicarbonates had been removed or were were poured onto paper towels to dry. The backcrossing F1 hybrids to tetraploid V. transplanted to rows irrigated using drip skins, pulp, and water from the first cleaning stamineum. Five BC1 plants produced by tubes with acidified water, many of the hybrid were saved, and the decanting process was backcrossing one F1 hybrid (highbush culti- seedlings became highly vigorous, with ex- repeated several times until few or no addi- var · V. stamineum) to tetraploid V. stam- cellent leaf color. tional good seeds were being recovered. The ineum were dug from the field nursery. Percent Only 20% of the BC1 seedlings in the seeds were dried on a desktop. good pollen was estimated for each BC1 plant high-density nursery flowered and produced The number of plump, well-developed by microscopic examination. Flowers on these fruit after 1 year in the field, compared with seeds that appeared capable of germinating five plants were emasculated and pollinated 60% to 80% of the highbush seedlings in the was estimated for each cross. Where there with pollen from tetraploid V. stamineum same nursery. Many BC1 plants that were were fewer than 200 seeds, all were counted. plants to obtain BC2 seed, and the number of too small to flower in 2017 will probably Where there were more than 200, the total plump seeds per pollinated flower was calcu- flower in 2018. Most BC1 plants in the field seed mass was spread in an oval on a desktop lated when the berries ripened. floweredlaterintheseasonthanhighbush and visually divided into two equal halves, Crosses between tetraploid Vaccinium seedlings in the same nursery, and the mean four equal quarters, or eight equal eighths myrsinites and tetraploid V. stamineum. Vac- date of 50% ripe berries for the BC1 pop- depending on the seed number. The plump cinium myrsinites is an evergreen, rhizomatous ulation was 1monthlaterthanforthe seeds in the smallest division (always 100 or lowbush blueberry species native on fre- highbush seedlings. Although there was more) were counted and an estimate of the quently burned pinelands throughout the state much variation in flowering and ripening total number of seed was calculated. of Florida. It belongs to section Cyanococcus, time among the BC1 seedlings, none flow- Phenotypes of F1 and BC1 plants and fruit and fertile hybrids with highbush cultivars can ered or ripened earlier than seedlings from in the greenhouse. During the period of easily be produced in large numbers. The highbush cultivars such as ‘Jewel’, ‘Emer- flowering and fruiting, various traits for crosses between V. myrsinites and tetraploid ald’, and ‘Farthing’. which highbush and V. stamineum differ V. stamineum were attempted to gain more Most BC1 plants in the field nursery had were examined to see how the BC1 plants information about the genetic distance be- high berry set after open pollination, although compared with the parental taxa and to their tween sections Cyanococcus and Polycodium. one cross produced many seedlings that set F1 parents. These traits included presence or Plants from four clones of V. myrsinites,which fruit poorly and others had one or more absence of pubescence on the leaf petioles had been propagated by stem cuttings from seedlings that flowered but set little fruit. and stems of the new shoots, looseness of the two locations in Alachua County, Florida, Compared with the berries from highbush · flower and berry clusters, position of the tips were pollinated with pollen from tetraploid highbush crosses in the same nursery, berries

HORTSCIENCE VOL. 53(2) FEBRUARY 2018 161 on BC1 plants in the field nursery averaged These plants averaged somewhat lower in the corolla tube. In V. stamineum, the tips of smaller, darker, and lower in sugar and acid, percent good pollen than seedlings from the the anther tubes extend well beyond the tip but seedlings were highly variable, both backcrosses to highbush, but the V. stami- of the corolla tube, and the style and stigma within and between crosses. Frequency of neum BC1 plants were barely large enough to extend well beyond the anther tubes (Fig. 1). plants with excessive berry stemminess or flower, and the results were not considered The positions of the anthers and stigmas berry shattering was higher among BC1 conclusive. relative to the tip of the corolla tube in F1 populations, but most plants were free of In the greenhouse crosses of 2017 hybrids was intermediate between the high- these problems. Only a small fraction of the (Table 4), the 26 BC1 · BC1 crosses produced bush and V. stamineum parents, and the BC1 BC1 plants produced berries having exotic an average of 9.29 plump seeds per pollinated plants were intermediate between highbush flavors not expected in highbush seedlings. A flower, which was higher than the highbush · and the F1 (Table 5; Fig. 2). Both the F1 and small fraction of the BC1 plants produced F1 and F1 · highbush crosses, but lower than BC1 populations were quite variable in this some berries having traces of pink in the the 15–30 seeds per pollinated flower that character. Among the 31 BC1 plants mea- flesh, but most plants produced berries with would be expected from highbush · highbush sured, four had stigmas that were 1 mm inside white to greenish-yellow flesh similar to crosses (Lyrene, 1989). Success rates from the corolla tube and two had stigmas 2 mm highbush. Most BC1 plants had flowering similar crosses made in 2015 and 2017 outside the corolla tube. One BC1 plant had racemes with unusually long peduncles and differed considerably, (compare Table 4 with the tips of the anther tubules 3 mm inside the pedicels and berry clusters that were unusu- Lyrene, 2016), but seed set from all crosses corolla tube and four had anther tubules even ally loose. was high enough to allow the highbush and V. with the tip of the corolla tube. Fertility of F1 and BC1 plants in the stamineum gene pools to be combined with Flowers of V. stamineum have anther greenhouse. When the 35 vigorous BC1 little difficulty. awns, prominent curved appendages attached plants that had been moved to the greenhouse Phenotypes of F1 and BC1 plants in the to the pollen sac, whereas the flowers of for use as parents in 2017 began to flower, greenhouse. The plants placed in the green- section Cyanococcus lack these awns. Anther several were discarded because they lacked house received little or no chilling before or awns were present in all but a few BC1 plants, clear signs of V. stamineum introgression after they were dug. Most of the plants still but were much smaller than in V. stamineum. (flowers resembled highbush flowers, lack had some old leaves from the previous A few BC1 plants that lacked anther awns had of pubescence on leaf petioles and new season’s growth when they were dug, but flowers conspicuously open in the bud and growth flushes, and highbush-like leaf color most old leaves abscised within 2 months pubescent stems and petioles, all of which are and venation). after the plants were placed in the green- V. stamineum characters. Petiole pubescence The 33 BC1 seedlings whose pollen was house. Because of the lack of chilling, flush- was present in all BC1 plants and absent from examined had much higher percent good ing of new leaves was slow on some plants. the highbush cultivars. pollen than the 34 F1 plants examined The first flowers opened in late Dec. 2016, The 25 BC1 plants for which berry weight (Table 3). The median percent good pollen and flowering continued through the end of was determined based on berries harvested for the BC1 seedlings was 60%, and 11 of Feb. 2017. Most flower buds on most plants from the greenhouse after hand pollination the plants had percent well-formed pollen opened and produced flowers, but the flower- averaged 1.68 g per berry, compared with similar to what is seen in highbush cultivars. ing period was very long, probably due in part a mean of 2.20 g for the seven highbush No BC1 plants appeared to have less than to the lack of chilling. cultivars with which they were compared 10% good pollen. Pollen was examined for In highbush blueberry flowers, the stigma (Table 6). The 10 F1 plants sampled averaged only five BC1 plants in which tetraploid V. is normally located near the tip of the corolla 1.13 g/berry. Average weight of berries stamineum had been the recurrent parent. tube and the anthers are located well inside harvested from the forest from five V. stam- ineum plants that had been selected for large fruit was 1.00 g. Berries weighed from these Table 3. Number of plants with percent well-formed pollen grains in three hybrid populations. five plants were selected randomly, not be- z y cause they were the largest. Percent good microspores F1 Seedlings BC1 to Highbush BC1 to Stamineum 0–9 2 0 0 Skin color of ripe berries on most of the 26 10–19 6 3 1 hand-pollinated BC1 plants was shiny black, 20–29 9 2 1 but berries on several plants had a medium- 30–39 6 2 0 heavy waxy bloom, similar to that of high- 40–49 3 3 2 bush blueberry cultivars. When polished, the 55–59 5 6 0 berries on all plants had dark black skins, 60–69 1 2 1 indicating normal anthocyanin development. 70–79 0 0 0 None of the BC1 plants from the greenhouse 80–89 0 4 0 showed more than a slight hint of pink in the 90–100 2 11 0 Median score 30 60 45 flesh of the ripe berries, even though one of Mean 30.9 62.7 38.0 the grandparent V. stamineum plants and Standard deviation 21.97 30.29 18.60 several of the parental F1 hybrids had red to purple flesh. When the ripe berries were Backcross1 =BC1. z F1 = Highbush cultivar · tetraploid Vaccinium stamineum. submerged in three volumes of water and yTetraploid plant produced by colchicine treatment. smashed by hand to remove the seeds, the

Table 4. Results of crosses made in the greenhouse in 2017. Type of crossz Number of crosses Total flowers pollinated Plump seeds total number Plump seeds per flower pollinated BC1 · BC1 26 3,410 31,672 9.29 HB · Stam 4X 7 1,415 3,093 2.19 HB · F1 3 428 2,770 6.47 F1 · HB 8 1,111 5,120 4.61 F1 · F1 4 715 131 0.18 F1 · Stam 4X 6 1,184 2,678 2.26 Myrsinites · Stam 4X 4 1,076 134 0.12 z BC1 = (highbush cultivar · tetraploid Vaccinium stamineum) · highbush cultivar; HB = highbush cultivar; Stam 4X = any one of three tetraploid V. stamineum plants; F1 = highbush cultivar · tetraploid V. stamineum; Myrsinites = any one of four clones of the Florida tetraploid, lowbush species, Vaccinium myrsinites.

162 HORTSCIENCE VOL. 53(2) FEBRUARY 2018 Table 5. Positions of the stigma and distil end of the anther tubules relative to the distil end of the corolla the same for the BC1 as for the berries of tube for highbush cultivars, tetraploid Vaccinium stamineum,F1 hybrids, and BC1 hybrids. Negative highbush blueberry seedlings. values indicate that the stigma or anthers are inside the corolla tube and positive values indicate they A few ripe berries were tasted from each are exserted. Values are distances in mm.z BC1 plant that was used as a female parent. Genotypes measured (no.) Mean location of stigma Mean location of anthers Bitterness, a characteristic of V. stamineum HBy 15 –0.13 –3.33 berries, was not detected in either the skin or BC 31 0.42 –1.32 1 flesh of more than half of the BC1 plants. F1 18 2.35 0.11 Bitterness was high enough to be objection- Stam 4X 4 10.00 6.00 z able in the berries of only three or four BC1 Within columns, each pair of means differs at P = 0.00001 by Students t test, except for mean positions of plants in the greenhouse, and even these stigma for BC1 and highbush, which differ at P = 0.025. y berries were far less bitter than typical craft HB = highbush cultivars; BC1 = highbush cultivar · F1 or the reciprocal; F1 = highbush cultivar · tetraploid V. stamineum; Stam 4X = tetraploid V. stamineum. beers or red wines. Berry acidity, as judged by tasting the berries, varied from high to low among the BC1 clones, but on average was lower than in highbush berries. Many of the berries sampled did not seem very sweet. Of the 34 BC1 plants evaluated in the greenhouse, 18 had no detectable aromatic flavor compo- nents, 14 had detectable but not high levels, and two were rated as highly aromatic. A desirable feature of V stamineum is the very open flower cluster, resulting from in- florescences in which the pedicels and pe- duncles of the flowering raceme are much longer than those of species in section Cya- nococcus. Long pedicels and peduncles are desirable because the great increase in berry size that has accompanied domestication of the highbush blueberry has resulted in berries that are pressed together in the clusters of some cultivars. Loose clusters make berries easier to harvest mechanically. ‘‘Cluster looseness’’ was rated for the BC1 plants and, with few exceptions, was rated high or very high compared with cultivars such as ‘Emerald’, ‘Jewel’, and ‘Farthing’ (Fig. 3). Results of pollinating BC1 [(highbush · V. stamineum) · V. stamineum] plants with · · Fig. 2. Flowers and leaf of one BC1 hybrid (highbush Vaccinium stamineum) highbush showing pollen from tetraploid V. stamineum. Three exserted style. of the five small BC1 seedlings in which tetraploid V. stamineum was the recurrent parent produced a few flowers in the green-

Table 6. Weight of berries following hand-pollination in the greenhouse for highbush cultivars, F1 hybrid house, and 12 plump seeds resulted when seedlings (highbush cultivar · tetraploid Vaccinium stamineum), BC1 seedlings (F1 · highbush), and these were pollinated with pollen from tetra- for berries harvested from the forest from five heavily laden diploid V. stamineum plants that had been ploid V. stamineum to produce BC2 seed. A selected for high yield and large berries. larger population of BC1 seedlings of this z Highbush cultivars F1 seedlings BC1 seedlings V. stamineum from forest type will be available for crosses in 2018 Number of plants sampledy 510255 (Table 1). Berries weighed per plant 15 15 15 200 Results of V. myrsinites · tetraploid V. Mean berry weight (g) 2.20 ax 1.13 cx 1.68 bx 1.00 stamineum crosses. The 1076 emasculated Standard deviation 0.450 0.341 0.422 0.089 flowers of V. myrsinites that received pollen Standard error of mean 0.170 0.108 0.084 0.040 from tetraploid V. stamineum gave only 134 Backcross1 =BC1. plump seeds, a success rate far lower than z Diploid. All other populations are tetraploid. from the highbush · tetraploid V stamineum yEach plant was a different genotype. x crosses. Because both highbush cultivars and These three means differ at P = 0.05 according to pairwise t tests. V. myrsinites are tetraploids in section Cya- nococcus, the differing results when the two color of the water/juice mixture varied plants in which berries did not shatter were crossed with tetraploid V. stamineum greatly among BC1 plants. In some, the liquid excessively, the pedicels remained attached were unexpected. Seedlings from the V. was dark red to purple, in others pink to red, to many of the ripe berries when picked. myrsinites crosses will be grown to determine and in a few, brown to yellow. When high- When the pedicels were removed by hand, whether they are the intended hybrids and bush blueberries received the same treatment, the scar left on the berries was small for whether they are vigorous enough to flower. a few clones gave a pink to light rose–colored most clones, but was large enough to be liquid, but most produced a liquid that was problematic with a few. Discussion green to yellow to brown. Berry firmness was high in both the high- The tendency of ripe berries to fall easily bush parents and the V. stamineum grandpar- Caution needed in selecting parents for from the plant (shatter) with the pedicel ents of the BC1 plants, and none of the BC1 backcrosses. Putative F1 seedling popula- attached to the berry varied greatly among plants had soft berries. The berries were stored tions from some of the highbush · tetraploid the BC1 plants. In 20% of the genotypes, in paper bags at 5 C for up to 4 weeks as the V. stamineum crosses made during the past 5 shattering was higher than what would be crop was being harvested. Postharvest life was years have contained some plants that were acceptable in a cultivar. On many BC1 notformallyassessed,butseemedtobeabout clearly not the intended hybrids. These plants

HORTSCIENCE VOL. 53(2) FEBRUARY 2018 163 may circumvent problems that make the diploids sterile. Tetraploid intersectional F1 hybrids are expected to have two complete sets of chromosomes from each section, which may allow regular chromosome pair- ing and distribution during meiosis. How- ever, if amphidiploid chromosome pairing explains the increased fertility of the tetra- ploid F1 hybrids compared with diploid hybrids, it is surprising that the BC1 genera- tion is much more fertile than the tetraploid F1 hybrids. BC1 plants would be expected to have three sets of chromosomes from section Cyanococcus and one set from V. stamineum, which should cause meiotic irregularities if highbush chromosomes pair poorly with V. stamineum chromosomes. It is possible that selection against dysfunctional gametes and zygotes when the F1 hybrids were back- crossed to highbush resulted in BC1 popula- tions that had been strongly selected for compatible gene combinations. Berry characteristics. The late ripening of

Fig. 3. Immature berries from a BC1 hybrid showing long pedicels and peduncle. BC1 seedlings in the field nursery compared with highbush seedlings was not surprising. V. stamineum ripens in north Florida during were more vigorous than the intersectional the BC1 plants placed in the greenhouse July, whereas southern highbush cultivars hybrids and had no obvious V. stamineum produced large numbers of flowers for plants ripen from mid-April to mid-May. characteristics. Most resembled highbush · of their size and age. The most vigorous BC1 The berries of Vaccinium stamineum par- highbush seedlings. They were not used in plants in the field nursery also flowered ent FL13-872 had purple flesh, and in most F1 data sets or in crosses. Seedling populations prolifically. hybrids obtained by crossing it with highbush from difficult crosses, in which hundreds of The flowers of Vaccinium section Cyano- cultivars, flesh color ranged from pink to flowers must be pollinated to produce a few coccus species are pollinated in the forest by purple (Lyrene, 2016). Berry flesh in almost hybrids, often contain a few plants from self- sonicating bees (Cane et al., 1985). The all BC1 plants, both in the field and in the pollination, pollination from other unin- anthers are located well inside the corolla greenhouse, was white, with tints of yellow, tended sources, or from seeds splashed from tube, and even though the stigma normally green, or brown, which is typical of highbush one pot to another. A few extremely vigorous extends to the outer edge of the corolla tube, berries. When fully ripe, berries of a few BC1 seedlings have appeared in our F1 highbush · nectar, and pollen are not readily available to seedlings had pink-white to pink flesh. A few tetraploid V. stamineum populations that visiting honeybees. Large blueberry planta- very-mature highbush berries from the same were neither typical F1 hybrids nor typical tions in Florida and Georgia often have nursery had pink streaks in the flesh, but pink highbush seedlings. These were interesting reduced fruit set because honeybees placed flesh was much more common and more because of their high vigor and unusual in the fields to pollinate blueberries abandon strongly expressed in BC1 seedlings. It is phenotypes, but were not studied further. the fields to work in flowers of native plants hoped that the BC1 · BC1 crosses will pro- Phenotypes of hybrid plants: vigor. Vigor such as red maple (Acer rubrum) and titi duce some seedlings with red pigments in the of F1 and BC1 seedlings resulting from (Cyrilla racemiflora) in the surrounding for- flesh. hybridization between Vaccinium section ests and swamps. The problem is exacerbated Many BC1 seedlings from the greenhouse Cyanococcus species and Florida V. stami- when carpenter bees (Xylocopa sp.) cut slits produced bright red juice/water mixes when neum depended greatly on the particular at the base of the corolla tube, after which fresh, undamaged berries were smashed in clones chosen as parents. This was true of honeybees rob nectar from slits rather than water to remove the seeds (3 volumes of diploid seedlings from crosses between dip- visiting the corolla aperture. Both the stigmas water per 1 volume of berries). Most high- loid V. stamineum and diploid Cyanococcus and the anthers of V. stamineum are exserted bush clones produce a brown-to-green water/ species (V. darrowii, V. elliottii, and V. well beyond the end of the corolla tube and juice mix with similar treatment. A few fuscatum) and in the tetraploid intersectional offer much easier access to a variety of highbush clones give a pink-to-red water/ crosses and backcrosses reported here. Some potential pollinators. F1 and BC1 populations juice mix, but none of the hundreds tested in hybrid populations have been extremely were highly variable in flower structure. In this study produce the deep red obtained from weak. Some tetraploid crosses produced general, F1 plants had flowers intermediate most BC1 seedlings. Purees made with a food many seedlings with vigor approaching that between the two parents. BC1 populations blender from fresh BC1 berries would be of highbush cultivars. Because the tetraploid remained highly variable with respect to the visually much more attractive than those F1 hybrids that were used to make backcrosses relative positions of the stigma, the anthers, made from fresh highbush blueberries. The were extremely variable, it was not surprising and the end of the corolla tube, indicating that berries of BC1 seedlings either had more red that vigor varied greatly among BC1 seedling it should be rather easy to select for flowers pigments in the skin than highbush berries, or populations derived from different F1 plants. It that give easier access to honeybees and other their pigments were more readily released was more surprising to find that crosses in- pollinating insects. when the fresh, undamaged berries were volving the diploid species gave seedling Fertility of hybrids. It is not clear why crushed. After highbush blueberries are populations ranging from barely viable to tetraploid hybrids between highbush culti- cooked or frozen, dark purple pigments that medium vigorous, depending on the particular vars and tetraploid V. stamineum were so are released from the skin make brightly plants chosen to make the crosses. much more fertile than diploid hybrids be- colored jellies and pies. Flowers and flowering. Vaccinium stam- tween diploid V. stamineum and diploid The most problematic aspect of berry ineum plants of the type used in these crosses section Cyanococcus species, such as V. flavor among the BC1 clones was low sugar. are prolific flower producers, in the forest, in darrowii, V. elliottiii, and V. fuscatum. Ge- Low sugar may have been due in part to the field nurseries, and as potted plants. Many of netic redundancy provided by tetraploidy fact that the plants in the greenhouse had

164 HORTSCIENCE VOL. 53(2) FEBRUARY 2018 limited foliage while the berries were de- attached stems, and flavor were highly vari- There is no easy way to move genes from the veloping and maturing. The plants had not able in BC1 populations, and some seedlings tetraploid to the diploid level, and a breeder been chilled before the crosses were made, were near cultivar standards in all respects. would not want to breed deerberries at the and the berries tended to develop faster than Allard (1960) and Stebbins (1950) hy- tetraploid level if only a small amount of V. the new leaves. The old leaves had mostly pothesized that characteristics of the re- stamineum diversity could be made tetra- abscised or were senescent by the time the current parent are rapidly recovered in ploid. Breeding tetraploid deerberry cultivars berries were ripe. Berry sweetness improved backcrosses involving wide hybrids because while avoiding a genetic bottleneck by using in the last-harvested berries as the new leaves gametic and zygotic selection in partially a large number of tetraploid V. stamineum matured. Many of the BC1 plants in the fertile wide hybrids favors gametes and plants in the foundation crosses might be the high-density field nursery also seemed low zygotes that most resemble those of the best approach. in sugar compared with highbush seedlings parent species, in which harmonious gene Phylogenetic distances separating in the same nursery. Some BC1 seedlings, combinations have been selected over count- Vaccinium sections Cyanococcus (highbush both in the greenhouse and in the field, had less generations. blueberry), Polycodium (V. stamineum), and berries that tasted as good as those of blue- In the field nursery, highbush · tetraploid Batodendron (V. arboreum). These are the berry cultivars such as Emerald, Jewel, and V. stamineum F1 hybrids were clearly distinct three Vaccinium sections represented by spe- Farthing. from highbush seedlings, even before they cies native in Florida. Little is known about Pleasant tertiary flavor components (in flowered. In the BC1 populations, most seed- when, where, and how the three sections addition to sugar and acid) are conspicuous lings were clearly different from highbush, originated, diverged genetically, and be- in the berries of some highbush blueberry but a few would not have been conspicuous came widely established in Florida. Which cultivars when these are grown in environ- had they been mixed with seedlings from is more closely related to highbush blue- ments that favor complex flavor develop- highbush crosses. Only the combination of berry: V. stamineum or V. arboreum? Evi- ment. In the V. stamineum populations from flower, fruit, leaf, and stem characteristics dence from berry anthocyanins suggests that which our parent plants came, the berries, showed that these plants were valid BC1 V. arboreum is closer (Ballinger et al., 1982). despite some bitterness, have a unique flavor hybrids. One additional backcross to high- The anthocyanins in sparkleberry are simi- component that most people consider pleas- bush cultivars would probably produce many lar to those in blueberry, whereas those ant, or at least interesting. Failure to find BC1 seedlings that could scarcely be distinguished in deerberry resemble those in cranberry seedlings in either the greenhouse or the field by phenotype from seedlings having no (Vaccinium macrocarpon, section Oxycoccus; whose berries had intense, pleasant flavors Polycodium genes. Because a breeder would Ballinger et al., 1982). not found in highbush blueberry was disap- be selecting for berry and plant characteris- Darwin (1859) listed three ways to esti- pointing. Some plants gave hints of exotic tics desired in cultivars, most of which re- mate the phylogenetic distance between two flavors, and these flavors could probably be semble the highbush phenotype, the return to taxa using crossing experiments: how easy is intensified by recurrent selection. Bitter fla- highbush-like plants would be fast. Undoubt- it to make the cross?; how vigorous or weak vors, which can be objectionable in V. stam- edly, many V. stamineum chromosome seg- are the hybrids?; and how fertile are the ineum, were absent or greatly reduced in BC1 ments would persist in the BC2 seedlings that hybrids when intercrossed or backcrossed to berries. most resembled highbush cultivars. These the parent species? Darwin wrote before Outlook for moving useful characteristics introgressed genes from V. stamineum would chromosomes and polyploidy were discov- from V. stamineum into highbush-type give a richer, more-variable gene pool, and ered and before the triploid block was recog- cultivars. Hybridization with V. stamineum could lead to interesting phenotypes when nized. His method of estimating genetic could increase drought tolerance and upland BC1 or BC2 seedlings are intercrossed. distance based on the ease of hybridizing adaptation in blueberry cultivars. Highbush Prospects for domesticating the taxa works in Vaccinium only with homo- blueberry is a ‘‘swamp’’ species. In the wild, deerberry. The ability of deerberry to pro- ploid crosses because the triploid block it is normally found on well-drained, acid duce high yields of large berries while grow- imposes a superficial secondary barrier to soils that are high in organic matter and have ing on droughty soils has been noted hybridization that is not directly related to a dependable moisture supply. In the south- (Ballington et al., 1984; Ballington, 1995). genetic consanguinity. eastern United States, V. stamineum thrives Deerberries are extremely variable, both Diploid species in section Cyanococcus, on excessively drained, coarse, white sand, in within locations and between locations, and such as V. darrowii, V. elliottii, and diploid V. forested communities of drought-tolerant genes are probably available within the spe- fuscatum, can readily be crossed as female plant species. The basis for drought tolerance cies for solving the major problems that have parent with both V. stamineum and V. arbor- in V. stamineum has not been studied. Three- so far prevented its domestication. These eum, producing hundreds if not thousands of year-old plants dug from our high-density include difficult vegetative propagation, bit- seedlings (P.M. Lyrene, unpublished data). field nurseries do not have large, deep roots as ter or astringent flavor components, and the V. stamineum and V. arboreum seem to differ V. arboreum plants dug from the same tendency for ripe berries to shatter from the little in the ease with which such diploid nursery. Drought tolerance could be a major bush. Both shattering and berry bitterness hybrids can be obtained. Despite substantial benefit from introgression of V. stamineum vary greatly among plants in wild popula- attempts during several crossing seasons, genes, but we have not grown BC1 seedlings tions of V. stamineum. If time and effort equal hybrids using diploid Cyanococcus species in an environment that would test drought to what was spent domesticating the high- as the pollen parent and V. stamineum or V. tolerance. bush blueberry were devoted to V. stami- arboreum as the seed parent have not been Obtaining commercial cultivars following neum, deerberry could probably become an obtained. hybridization of highbush cultivars with V. important new crop. Highbush cultivars have been easier to stamineum will require that most of the de- Whether introgressing genes from high- cross with tetraploid V. stamineum than with sirable features of the highbush cultivars be bush cultivars would be necessary, or even tetraploid V. arboreum. In part, this is be- quickly recovered by backcrossing, whereas useful, in domesticating the deerberry, is cause tetraploid V. stamineum flowers shed selected useful traits from V. stamineum are uncertain. Essential traits such as nonshatter- much more pollen than tetraploid V. arbor- retained. BC1 populations showed rapid ing berries, easy vegetative propagation by eum flowers under our greenhouse condi- progress back toward a highbush phenotype. stem cuttings, and freedom from bitter flavor tions. With patience, large numbers of In both field and greenhouse (Table 6), components are almost universal in highbush tetraploid hybrids can be obtained from both berries on BC1 plants averaged only slightly blueberries. The fact that highbush blue- types of crosses if fertile tetraploid plants of smaller than berries from highbush seedlings berries are tetraploid and the great genetic V. arboreum and V. stamineum are available. and cultivars. Plant architecture of most diversity in V. stamineum is at the diploid At both the diploid and tetraploid level, hybrids was similar to that of highbush level complicates the use of highbush culti- hybrids between Cyanococcus species and V. cultivars. Berry color, scar, freedom from vars in the domestication of the deerberry. arboreum have been more vigorous than

HORTSCIENCE VOL. 53(2) FEBRUARY 2018 165 hybrids between the same Cyanococcus spe- Vaccinium species into 30 sections. Vacci- quality characterization of 11 Vaccinium spe- cies and V. stamineum. The most vigorous F1 nium sections vary greatly in how closely cies. J. Amer. Soc. Hort. Sci. 109:684–689. hybrids from V. darrowii · V. arboreum and they are related to each other. As a conse- Ballinger, W.E., E.P. Maness, and J.R. Ballington. from diploid V. fuscatum · V. arboreum were quence, the results reported here for Cyano- 1982. Anthocyanins in ripe fruit of the sparkle- easy to maintain in field plantings, and some coccus crosses with sections Polycodium and berry, Vaccinium arboreum Marsh. Can. J. Plant Sci. 62:683–687. plants began heavy annual flowering after Batodendron may have limited value in Brooks, S.J. and P.M. Lyrene. 1998. Derivatives of several years in the field. By contrast, most predicting what will happen when other in- Vaccinium arboreum · Vaccinium section diploid hybrids between section Cyanococcus tersectional crosses are attempted. The first Cyanococcus. II. Fertility and fertility param- species and V. stamineum have died before intersectional Vaccinium cross made on a eters. J. Amer. Soc. Hort. Sci. 123:997–1003. flowering. Tetraploid interspecific hybrids large scale and studied in great detail was Camp, W.H. 1945. The North American blue- have been stronger than the diploids, but made by Rousi (1963) in Finland between berries with notes on other groups of Vaccinia- highbush hybrids with V. stamineum have still highbush cultivars and Vaccinium uliginosum ceae. Brittonia 5:203–275. been weaker than those with V. arboreum. (Vaccinium section Vaccinium). Thousands Cane, J.H., G.C. Eickwort, F.R. Wesley, and J. Spielholz. 1985. Pollination ecology of Vacci- With respect to fertility of F1 hybrids, V. of F1 and backcross seedlings were grown, arboreum and V. stamineum crosses differed and the cold-hardy cultivar Aron was se- nium stamineum (Ericaceae: Vaccinioideae). Amer. J. Bot. 72:135–142. little. Diploid Cyanococcus hybrids with both lected. ‘Aron’ was recommended for hobby Darrow, G.M. and W.H. Camp. 1945. Vaccinium V. arboreum and V. stamineum were highly cultivation in southern Finland, but was not hybrids and the development of new horticul- sterile, except that V darrowii · V. arboreum considered sufficiently productive for com- tural material. Bull. Torrey Bot. Club 72:1–21. F1 hybrids produced a few viable unreduced mercial blueberry production (Rousi, 1963; Darwin, C. 1859. On the origin of species. John gametes, and a few seedlings were obtained Hiirsalmi, 1989). Murray, Kent, London, UK. after F1 flowers were pollinated with pollen Tsuda et al. (2013) produced fertile hybrids Hiirsalmi, H.M. 1989. Research into Vaccinium from tetraploid highbush cultivars (Brooks by crossing colchicine-induced tetraploid cultivation in Finland. Acta Hort. 241:175– and Lyrene, 1998). Diploid V. darrowii · V. Vaccinium bracteatum (section Bracteata), 184. stamineum hybrids have so far been too weak which is native in Japan, with the highbush Luteyn, J.L., W.S. Judd, S.P. Vander Kloet, L.J. to permit backcrosses to tetraploids. cultivar ‘Spartan’. The hybrids were expected Dorr, G.D. Wallace, K.A. Kron, P.S. Stevens, and S.E. Clemants. 1996. Ericaceae of the At the tetraploid level, most F1 hybrids of to be useful in breeding cultivars with higher southeastern United States. Castanea 61:101– both types are partially fertile, with much sugar content, abundant phytochemicals, ex- 144. variation in fertility among individual F1 tensive environmental adaptability, and late Lyrene, P.M. 1989. Pollen source influences fruit- plants. Average percent well-formed pollen flowering and fruit maturity. ing of ‘Sharpblue’ blueberry. J. Amer. Soc. grains averaged 39.7% for 86 highbush · Blueberry breeders have been interested Hort. Sci. 114:995–999. tetraploid V. arboreum F1 seedlings (Lyrene, in crossing highbush cultivars with bilberry, Lyrene, P.M. 2013. Fertility and other characteris- 2013) and 30.9% for 33 highbush · tetraploid Vaccinium myrtillus (section Myrtillus), tics of F1 and Backcross1 progeny from an V. stamineum F seedlings. In seedling pop- a diploid, low-growing, rhizomatous species intersectional blueberry cross ((highbush culti- 1 · · ulations produced by backcrossing a diverse native in the British Isles and in parts of var Vaccinium arboreum) highbush culti- population of F hybrids of each type to northern Europe (Ritchie, 1956). Bilberries var). HortScience 48:146–149. 1 Lyrene, P.M. 2016. Phenotype and fertility of are valued for their small, acid berries which highbush cultivars, percent good pollen av- intersectional hybrids between tetraploid high- eraged 70.3% for V. arboreum backcrosses have purple flesh and high levels of anthocy- bush blueberry and colchicine-treated Vacci- and 62.7% for V. stamineum. Similarities anin in both skin and flesh. Large quantities nium stamineum. HortScience 51:15–22. between the two types of crosses were more of bilberries are gathered annually from the Ritchie, J.C. 1956. Vaccinium myrtillus L. J. Ecol. striking than the differences. In both cases, forest and sold in European markets. Bilberry 44:291–299. the most fertile tetraploid F1 hybrids were hybrids have been produced that combine Rousi, A. 1963. Hybridization between Vaccinium fertile enough to allow easy production of genes from other Vaccinium sections, but so uliginosum and cultivated blueberry. Ann. Agr. Fenn. 2:12–18. thousands of BC1 seedlings. In both cases, the far these crosses have not led to cultivars. Stebbins, G.L. 1950. Variation and Evolution in resulting BC1 hybrids averaged much higher in fertility than their F parents. Literature Cited Plants. Columbia Univ. Press, New York, NY. 1 Tsuda, H., H. Kunitake, M. Yamasaki, H. Komatsu, Taken together, the results of these Allard, R.W. 1960. Principles of Plant Breeding. and K. Yoshioika. 2013. Production of inter- crosses and backcrosses support the conclu- Wiley, New York, NY. sectional hybrids between colchicine induced- sion suggested by previous anthocyanin data: Ashe, W.W. 1931. Polycodium. J. Elisha Mitchell tetraploid shashanbo (V. bracteatum)and V. stamineum is not as closely related to Sci. Soc. 46:196–213. highbush blueberry ‘Spartan’. J. Amer. highbush blueberry as is V. arboreum. Both Baker, P.C. 1970. A systematic study of the genus Soc. Hort. Sci. 138:317–324. F1 and BC1 seedlings in blueberry hybrids Vaccinium L. subgenus Polycodium (Raf.) Uttal, L.J. 1987. The genus Vaccinium L. in with V. stamineum tend to be weaker than Sleumer in the southeastern United States. Virginia. Castanea 52:231–255. blueberry hybrids with V. arboreum. The wide- Univ. North Carolina at Chapel Hill, NC, PhD Vander Kloet, S.P. 1988. The genus Vaccinium spread leaf bronzing in most highbush · V. Diss. in North America. Publ. 1828. Agriculture stamineum F hybrids is not seen in highbush · Ballington, J.R. 1995. The deerberry (Vaccinium Canada. 1 stamineum L. Vaccinium section Polycodium Vander Kloet, S.P. and T.A. Dickinson. 2009. A V. arboreum hybrids. (Raf.) Sleumer): A potential new small fruit subgeneric classification of the genus Vacci- Implications for further Vaccinium crop, p. 21–28. In: R.E. Gough and R.F. Korcak nium and the metamorphosis of V. section intersectional crosses. Interest in using (eds.). Blueberries: A century of research. Bracteata Nakai: More terrestrial and less intersectional crosses in breeding Vaccinium Haworth Press, Inc., New York, NY. epiphytic in habit, more continental and less cultivars is increasing. Vander Kloet and Ballington, J.R., W.E. Ballinger, W.H. Swallow, insular in distribution. J. Plant Res. 122:253– Dickinson (2009) divided the world’s 400 G.J. Galletta, and L.J. Kushman. 1984. Fruit 268.

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