Abstract
Partial to complete self-incompatibility is normal in most Vaccinium L. (Ericaceae) species. Wild blueberry plants of several Florida provenances and species were self- and cross-pollinated in a greenhouse free of pollinators. Fruit set of V. darrowii Camp (2x), V. corymbosum L. (4x), V. arboreum Marsh (2x), and F1 (V. darrowii × V. corymbosum) hybrids was higher after cross-pollination than after self-pollination. Partial to complete self-incompatibility was present in V. darrowii, V. corymbosum, and their tetraploid F1 hybrids. The three V. arboreum clones tested were fully self-incompatible. Intra- and interpopulation crosses in V. corymbosum, V. darrowii, and V. darrowii × V. corymbosum hybrids were highly successful, and self-pollination reduced all fertility parameters. Advanced selections of V. corymbosum were the most self-compatible clones tested, possibly because self-compatibility has been increased by breeders selecting for reliable fruit set in large fields planted with one or a few clones. One southern highbush selection and two F1 hybrids had fruit set of more than 70% after self-pollination. These plants could be potentially used to breed plants that could be planted in single blocks providing reliable yield.
In the genus Vaccinium L., there are no fundamental sterility barriers between homoploid members of the same phyletic section (subgenus) (Darrow et al., 1949; Meader and Darrow, 1944; Sharpe, 1953; Sharpe and Darrow, 1959). Diploid, tetraploid, and hexaploid species exist within several sections of the genus. In section Cyanococcus L., which includes the cultivated blueberries, partial to complete self-incompatibility and interfertility between homoploid species has allowed formation of interspecific hybrid swarms (Camp, 1942; Vander Kloet 1983, 1988).
Studies of self-pollination and cross-pollination in several Vaccinium species have provided varying results. In most cases, partial to complete self-incompatibility was present, particularly in wild or “unimproved” plants (Bailey, 1938; Coville, 1921; Meader and Darrow, 1944; Merrill, 1936; Merrill and Johnston, 1940; Morrow, 1943; White and Clark, 1939). In cultivated highbush blueberry (tetraploid V. corymbosum L. and its hybrids), cross-pollination usually produced earlier ripening berries, increased berry size, and higher fruit set. Fewer fully developed seeds were found in self-pollinated berries. Partial to complete self-incompatibility, slower pollen tube growth, or collapse of ovules after fertilization could be factors that produced these results (Morrow, 1943).
Meader and Darrow (1944) studied the crossing behavior of several hexaploid rabbiteye (V. virgatum Aiton) varieties. Fruit set was higher after cross-pollination. Berry weight and seed weight per berry were always higher after cross-pollination than after self-pollination. In the same study, several section Cyanococcus species from different ploidy levels were self- and crosspollinated. Tetraploid forms of V. virgatum were found to be self-sterile but gave a fruit set of 1% to 27% when cross-pollinated with tetraploid V. corymbosum. Vaccinium tenellum Aiton (2x) gave a fruit set below 29% when selfed, but when crossed with a different clone of the same species, had over 80% fruit set. Vaccinium darrowii Camp (2x) set 15% when selfed. Vaccinium darrowii, crossed with other diploid species (V. pallidum Aiton and V. elliottii Chapman), gave a fruit set of 71.5% and 62.2%, respectively. Berries formed after pollinating V. darrowii flowers with V. elliottii pollen ripened earlier than those produced by self-pollination of V. darrowii. However, V. myrsinites (4x) produced two-thirds less fruit set when crossed with other tetraploid species than it did when self-pollinated. Fruit set of self-pollinated V. myrsinites Lam. averaged 79%.
Krebs and Hancock (1990) reported that fruit set was significantly greater after crosspollination of V. corymbosum cultivars. They proposed that variable fertility after self- and cross-pollination in cultivated highbush was produced by early-acting inbreeding depression (seed abortion). They proposed that the degree of self-incompatibility depended on the level of zygotic inbreeding, which depended on the parents that were mated. Reduced self-fertility was attributed to homozygosity for sublethal mutations at loci controlling embryo development or to loss of heterotic interactions at these loci (Hokanson and Hancock, 2000; Krebs and Hancock, 1991).
Diploid V. darrowii has been extensively used in breeding blueberries, but most crosses have involved only one V. darrowii clone, Fla. 4B. Vaccinium darrowii in Florida is quite variable. Lyrene (1986) described three types of V. darrowii in Florida: 1) the Florida panhandle race, a petit form with highly glaucous leaves, which matches Camp's original description of the species (see Camp, 1945); 2) the Ocala Forest race, a tall form with shiny green leaves (glaucous on new growth flushes); and 3) the Istokpoga race, found at the southern end of the species range in the Florida peninsula, a highly variable population with short and tall plants. Introgression from a diploid highbush species (V. fuscatum) was clearly occurring in the Florida peninsula. The use of a wider range of V. darrowii accessions in breeding would provide beneficial diversity in the cultivated gene pool.
Vaccinium arboreum (sparkleberry) is a diploid species that is abundant and widespread in the southeastern United States. Because it is in section Batodendron Marsh. and does not readily make fertile hybrids with cultivated blueberries, which are in section Cyanococcus, it has not been much used in breeding. Recently, genes from V. arboreum have been shown to be useful in breeding (Brooks and Lyrene, 1995; Lyrene, unpublished data), and more information on the pollination biology of this species is needed. The purpose of this research was to determine levels of self-compatibility within the populations of several Vaccinium species and to search for plants with high self-compatibility that could be used in breeding self-compatible southern highbush cultivars.
Materials and Methods
Self- and cross-pollination experiments.
Self- and cross-pollination were conducted using plants of V. darrowii (2x), V. arboreum (2x), V. fuscatum (2x), V. corymbosum (4x), and F1 (V. darrowii × V. corymbosum) hybrids. The diploids, V. darrowii, V. fuscatum, and V. arboreum clones, were propagated from the wild. Vaccinium darrowii clones came from the vicinity of Sumatra and Wilma in the Florida panhandle and from the Florida peninsula in the vicinity of Lake Istokpoga. Vaccinium fuscatum clones (a Florida diploid highbush taxon resembling V. corymbosum) were propagated from plants growing in a disturbed wetland near Davenport in the central Florida peninsula. Softwood cuttings from the selected wild plants were rooted in a 1:1 mixture of sphagnum peat and perlite. Rooted cuttings were transplanted to 4-L nursery containers filled with sphagnum peat and perlite (1:1). The plants were maintained in containers in a greenhouse at the University of Florida, Gainesville, FL.
Vaccinium arboreum plants were grown from open-pollinated seeds collected in Clay County in northeast Florida. The seedlings were transplanted to a high-density nursery at the University of Florida Plant Science Unit in Citra, FL, in May. In November, 15 plants were dug with a root ball of soil and fitted to 8-L nursery containers using sphagnum peat. The containers were placed in a greenhouse in Gainesville, FL.
Tetraploid southern highbush selections from the Florida breeding program were selected from a field nursery, potted in 8-L nursery containers of sphagnum peat, and placed in a greenhouse in Gainesville, FL. For simplicity, these tetraploid products of the breeding program will be designated V. corymbosum, although other species are included in their genetic background. Tetraploid Vaccinium darrowii × V. corymbosum hybrids were selected from crosses made at the University of Florida. The F1 hybrids were grown in a field nursery for 8 months and then selected and transplanted to 8-L nursery containers of sphagnum peat and maintained in a greenhouse in Gainesville, FL. Self- and cross-pollination experiments for southern highbush selections, F1 hybrids (V. darrowii × V. corymbosum), V. darrowii, V. fuscatum, and V. arboreum clones were made. The results from the individual crosses of each type were averaged and compared.
In February of each year, the highbush plants and F1 hybrids were chilled for more than 1000 h in a walk-in cooler at 4 °C with no light, after which they were placed in a greenhouse. The V. arboreum, V. darrowii, and V. fuscatum clones, which are evergreen and have no chilling requirement, were not chilled. Each V. darrowii plant was divided into two sections to compare self- and crosspollination as described by Morrow (1943). Flowers that had opened previously were removed. Flowers were emasculated before pollination. Pollinations were made in a greenhouse free of pollinators. For crosspollination, new flowers were pollinated with pollen from a different clone of the same species with the exception of V. fuscatum, which was cross-pollinated with V. arboreum pollen. For self-pollination, new flowers were pollinated with pollen from the same plant. Pollen was collected by tapping the male flower on the thumbnail. The stigmas of emasculated flowers were touched by the thumbnail. For each test, ≈100 to 250 flowers were self-pollinated and 100 to 250 flowers were cross-pollinated.
Berries were harvested when fully ripe. Berry weight and number of seeds per berry were determined for the first 20 berries ripe from each cross. Seeds were classified as plump and round (large seeds) and shriveled and shrunk (small seeds). Seeds from additional berries were removed using a food blender. Seeds were washed free of pulp and skins with water, dried on a benchtop at room temperature, and stored in coin envelopes at 5 °C. Seeds were planted in Nov. 2007 on the top layer of 4-L nursery containers of sphagnum peat. The containers were kept in a greenhouse with intermittent mist for 2 to 3 months until germination was complete. Seedlings from each cross were counted.
First-pollination to first-ripening interval was calculated from the first day of pollination to the first day of berry ripening. Average pollination-to-ripening interval was calculated from the mean day of pollination to the mean day of harvesting. Fruit set percentage, berry weight, number of seeds per berry, number of plump seeds per pollinated flower, pollination-to-ripening interval, average pollination-to-ripening interval, and number of seedlings per pollinated flower were determined for self- and cross-pollinated flowers. An index of self-incompatibility (ISI) was calculated by dividing the average number of plump seeds per pollinated flower obtained after self-pollination by the same following cross-pollination. A score of 0 to 0.2 was classified as self-incompatible, a score of 0.2 to 1 as incompletely compatible, and a score of 1 as self-compatible (Zapaya and Arroyo, 1978). Means for fruit set percentage were separated using the χ2 “test of independence” with a significance level of 5%. Means for berry weight, number of seeds per berry, number of plump seeds per pollinated flower (PPF), number of seedlings per pollinated flower (SPF), pollination-to-ripening interval, and average pollination-to-ripening interval for the different treatments were separated using least square means by Tukey's test with a significance level of 5%. Data analysis was performed using the GLM and FREQ procedures of SAS (Statistical Analysis System Version 9.1; SAS Institute, Cary, NC). In the χ2 tests for fruit set, each pollinated flower was considered an observation with two possible responses: set or nonset. The null hypothesis of the χ2 “test of independence” was that the ratio of set to nonset was the same for the two crosses being compared.
Results
Fruit set (percentage).
Fruit set for V. darrowii was less than 30% after self-pollination and more than 70% after crosspollination (Table 1). For V. arboreum, fruit set was 0% after self-pollination and ranged from 3.3% to 54.2% after cross-pollination (V. arboreum × V. arboreum) (data not shown). For V. corymbosum selections, fruit set after self-pollination averaged 39.4% (range for five crosses, 9.8% to 74.1%). Fruit set of V. corymbosum after cross-pollination averaged 82.8% (range for 74 crosses, 37.1% to 94.3%, data not shown).
Results of self- and cross-pollination in Florida Vaccinium species measured by fruit set (%), first-pollination to first-ripening interval (pol-ripe interval), and average pollination to ripening (pol-ripe interval average).
Fruit set for self-pollination of 17 F1 (V. darrowii × V. corymbosum) hybrids ranged from 0% to 90.3%. Two hybrid plants had a selfed fruit set over 80% (data not shown). Fruit set after cross-pollination of F1 (V. darrowii × V. corymbosum) hybrids was 59.8% for one cross and 98.2% for a second. Two of the three V. fuscatum clones tested had fruit set over 50% after self-pollination. Fruit set after self-pollination did not differ significantly between V. fuscatum and southern highbush selections (Table 1). Mean fruit set of V. darrowii, V. corymbosum, and their F1 hybrids after self-pollination was lower than after cross-pollination (P < 0.05) (Table 1).
Pollination-to-ripening interval.
First-pollination to first-ripening intervals and average pollination-to-ripening intervals did not differ significantly for self- and cross-pollination in either V. darrowii or V. fuscatum (Table 1). In southern highbush selections and in F1 (V. darrowii × V. corymbosum) hybrids, first-pollination to first-ripening interval was longer after self-pollination than after cross-pollination (P < 0.05). For southern highbush selections, average pollination-to-ripening was also significantly longer after self-pollination (Table 1).
Berry weight.
Mean berry weight of V. darrowii and F1 (V. darrowii × V. corymbosum) hybrids was lower after self-pollination than after cross-pollination (P < 0.05). Mean berry weight of southern highbush selections and V. fuscatum clones was higher after self-pollination than after cross-pollination (P < 0.05) (Table 2).
Results of self- and cross-pollination in Florida Vaccinium species measured by mean berry weight (g), number of large seeds per berry, number of small seeds per berry, total number of seeds per berry, number of plump seeds per pollinated flower (PPF), number of seedlings per pollinated flower (SPF), and index of self-incompatibility (ISI).
Seeds and seedlings per flower.
Number of PPF after self-pollination of V. darrowii ranged from 0.06 to 3.32, depending on the clone that was selfed. SPF ranged from 0.02 to 0.32. Cross-pollinated V. darrowii gave PPF ranging from 3.39 to 37.96 and SPF ranging from 1.23 to 9.83 (data from individual crosses not shown). For PPF and SPF, self-pollination of V. darrowii gave lower values than cross-pollination (P < 0.05; Table 2). SPF of V. darrowii after cross-pollination was ≈20 times higher than after self-pollination (P < 0.05). The average number of seeds per berry was lower when V. darrowii clones were self-pollinated compared with crosspollination (P < 0.05). Few plants were produced from self-pollinated V. darrowii seeds. The ISI was 0.08 for V. darrowii, classified as self-incompatible.
After cross-pollination of V. arboreum, PPF ranged from 0.46 to 6.78 and SPF ranged from 0.10 to 1.80 compared with zero values for self-pollinated V. arboreum (data from individual crosses not shown). Self-pollinated V. arboreum flowers abscised a few weeks after pollination. The three V. arboreum clones tested appeared to be completely self-incompatible with an ISI score of 0.
In southern highbush selections, PPF for self-pollination ranged from 0.52 to 8.13 compared with 3.08 to 11.50 for crosspollination (data from individual crosses not shown). Number of large seeds per berry and total number of seeds per berry were lower for self-pollination than for cross-pollination (P < 0.05) (Table 2). Cross-pollinated berries had fewer small seeds compared with self-pollinated berries (P < 0.05). The ISI score for the southern highbush selections was 0.38, classified as incompletely compatible.
In V. darrowii × V. corymbosum hybrids, PPF ranged from 0.00 to 12.22 for self-pollination and from 8.69 to 21.90 for crosspollination (data from individual crosses not shown). Two F1 hybrid plants gave a PPF higher than 7.00 when self-pollinated. PPF and seeds per berry after self-pollination were lower than after cross-pollination (P < 0.05). The ISI score for the F1 hybrids was 0.22, classified as incompletely compatible (Table 2). SPF for the V. darrowii × V. corymbosum hybrids and for V. fuscatum were not significantly different between cross- and self-pollination experiments (P < 0.05) (Table 2). The ISI score for V. fuscatum was 0.46, classified as incompletely compatible.
Discussion
Coville's (1921, 1937) first attempts to self-pollinate highbush blueberry cultivars failed. Self-pollinated plants ripened few berries, and those that ripened contained few seed. In cross-pollinations among different plants of the same species, he obtained many berries with numerous seeds. Krebs and Hancock (1991) attributed the reduced fertility of highbush blueberry after self-pollination to postzygotic events that aborted most selfed seeds. In our study, fruit set of V. darrowii, V.corymbosum, V. arboreum, and F1 (V. darrowii × V. arboreum) hybrids after cross-pollination was higher than after self-pollination. Differences in fruit set between self- and cross-pollination were similar to those reported by Coville (1921), but the degree of self-incompatibility varied among the taxa.
Although the experiments were not specifically designed to compare the length of the fruit-development period among taxa, it is clear that V. arboreum, a fall-ripening species, has a very long fruit development period; that southern highbush selections, which have been selected for early ripening, have a short fruit-development period; and V. darrowii, the Florida native species used in breeding to impart southern adaptation to highbush cultivars, is intermediate.
No previous self-pollination studies have been reported in V. arboreum. In our study, complete self-incompatibility was observed in the three clones of V. arboreum that were tested. Self-pollinated V. arboreum flowers abscised a few weeks after pollination. In addition, several V. arboreum clones that flowered in a greenhouse in Gainesville, FL, without access to pollination set no berries. Under similar conditions, some clones of V. corymbosum, V. darrowii, V. fuscatum, and F1 (V. darrowii × V. fuscatum) natural hybrids set a few berries (unpublished data).
The 17 F1 (V. darrowii × V. corymbosum) hybrids that were self-pollinated in these experiments were produced from V. darrowii (2x) × V. corymbosum (4x) crosses. Approximately 95% of these hybrids were believed to be tetraploids and had high pollen viability determined by staining the pollen with acetocarmine (Chavez and Lyrene, 2009). The results of cross- and self-pollination of the F1 hybrids were similar to those for the southern highbush selections and V. darrowii clones. One southern highbush clone and two F1 hybrid plants had fruit set over 70% after self-pollination. The higher self-compatibility of highbush selections is attributed to breeding and selection for plants that set fruit reliably in large clonal plots. High-chill northern highbush blueberry cultivars, which are grown from North Carolina to Michigan, and in the Pacific Northwest, vary in self-compatibility. Those that have become most popular with growers such as ‘Duke’ and ‘Bluecrop’ are highly productive in large fields planted to single clones. Cultivars with low self-compatibility tend to give inconsistent yields from year to year (Lyrene, personal communication).
Fruit set, first-pollination to first-ripening interval, average pollination-to-ripening interval, berry weight, number of large seeds per berry, number of small seeds per berry, total number of seeds per berry, and PPF were different for cross- and self-pollination of V. corymbosum selections (Table 2) and indicated varying levels of self-incompatibility. Similar results were described by Vander Kloet and Lyrene (1987), who found strong self-incompatibility in wild V. corymbosum plants. They found that intra- and interpopulation crosses in V. corymbosum were equally fertile, but self-pollination resulted in reductions in all fertility parameters. Morrow (1943) observed that after self-pollination in V. darrowii, pollen tube growth is slower, ovules may collapse after fertilization, and the number of fully developed seeds is reduced. In another study, V. darrowii gave low fruit set when selfed (15%) but high fruit set when cross-pollinated with other diploid species, V. elliottii and V. pallidum (62.2% and 71.5%, respectively) (Meader and Darrow, 1944).
In our study, cross-pollination increased berry size for V. darrowii and F1 hybrids but not for southern highbush selections or V. fuscatum clones. This result is surprising because most previous studies in blueberry have shown a positive correlation between berry size and berry seed content. Phenotypic variation between the plants used as female parents may be the reason for this difference. Morrow (1943) reported that cross-pollination increased berry size of three northern highbush cultivars: Scammel, Weymouth, and Dixi. Meader and Darrow (1944) studied 10 rabbiteye (V. virgatum) varieties. Without exception, berries were larger after cross-pollination than after self-pollination. In the same study, V. tenellum and V. darrowii cross-pollinated flowers resulted in larger berries than self-pollinated flowers. All fertility parameters were reduced for self-pollination, including a lower number of seeds per berry that contributed to a smaller berry size.
Average berry weight, number of seeds per berry, and PPF of V. darrowii and F1 (V. darrowii × V. corymbosum) hybrids were higher when these species were cross-pollinated rather than self-pollinated. Reduction in total number of seeds per berry may have been the result of reduced pollen germination or pollen tube growth, both prezygotic events. Reduction in PPF and SPF may have been the result of embryo abortion after self-pollination, which is a postzygotic event. Both pre- and postzygotic events were believed to be involved with the self-incompatibility reaction. Few plants, with abnormal morphology, were produced from seeds of V. darrowii, V. fuscatum, and V. darrowii × V. corymbosum hybrids after self-pollination (Table 2). Similar results were obtained by Coville (1937). He found that self-pollination of highbush cultivars produced few berries. Most seeds were abnormal and lacked embryos, and few useful plants were obtained from self-pollination. Krebs and Hancock (1990) obtained few plants after self-pollination of V. corymbosum. They attributed the low seedling numbers after self-pollination to increased homozygosity of deleterious alleles at loci that are critical to embryo vigor and survival.
In conclusion, some level of self-fertility was seen in some plants of every taxon we tested except V. arboreum. One southern highbush selection from the breeding program and two F1 V. corymbosum × V. darrowii tetraploid hybrids had fruit set of more than 70% after self-pollination. This indicated the potential for developing low-chill highbush cultivars that would give reliable fruit set when planted in solid blocks containing a single clone.
Literature Cited
Bailey, J.S. 1938 The pollination of the cultivated blueberry Proc. Amer. Soc. Hort. Sci. 35 71 72
Brooks, S.J. & Lyrene, P.M. 1995 Characteristics of sparkleberry × blueberry hybrids Proc. Fla. State Hort. Soc. 108 337 339
Camp, W.H. 1942 On the structure of populations in the fenus Vaccinium Brittonia 4 189 204
Camp, W.H. 1945 The North American blueberries with notes on other groups of Vacciniaceae Brittonia 5 203 275
Chavez, D.J. & Lyrene, P.M. 2009 Interspecific crosses and backcrosses between diploid Vaccinium darrowii and tetraploid Southern Highbush blueberry J. Amer. Soc. Hort. Sci. 134 273 280
Coville, F.V. 1921 Directions for blueberry culture USDA Bulletin 974
Coville, F.V. 1937 Improving the wild blueberry USDA Yearbook 559 574
Darrow, G.M., Dermen, H. & Scott, D.H. 1949 A tetraploid blueberry from a cross of diploid and hexaploid species J. Hered. 40 304 306
Hokanson, K. & Hancock, J. 2000 Early-acting inbreeding depression in three species of Vaccinium (Ericaceae) Sex. Plant Reprod. 13 145 150
Krebs, S.L. & Hancock, J.F. 1990 Early-acting inbreeding depression and reproductive success in the highbush blueberry, Vaccinium corymbosum L Theor. Appl. Genet. 79 825 832
Krebs, S.L. & Hancock, J.F. 1991 Embryonic genetic load in the highbush blueberry, Vaccinium corymbosum (Ericaceae) Amer. J. Bot. 78 1427 1437
Lyrene, P.M. 1986 Variation within Vaccinium darrowi blueberry in Florida HortScience 21 512 514
Meader, E.M. & Darrow, G.M. 1944 Pollination of the rabbiteye blueberry and related species Amer. Soc. Hort. Sci. 45 267 274
Merrill, T.A. 1936 Pollination of the highbush blueberry Michigan Agriculture Experimental Station of Technology Bulletin 151
Merrill, T.A. & Johnston, S. 1940 Further observations on the pollination of the highbush blueberry Proc. Amer. Soc. Hort. Sci. 37 617 619
Morrow, E.B. 1943 Some effects of cross-pollination experiments versus self-pollination in cultivated blueberries Proc. Amer. Soc. Hort. Sci. 42 469 472
Sharpe, R.H. 1953 Horticultural development of Florida blueberries Proc. Fla. State Hort. Soc. 66 188 190
Sharpe, R.H. & Darrow, G.M. 1959 Breeding blueberries for the Florida climate Proc. Fla. State Hort. Soc. 71 308 311
Vander Kloet, S.P. 1983 The taxonomy of Vaccinium Cyanococcus: A summation Can. J. Bot. 61 256 266
Vander Kloet, S.P. 1988 The genus Vaccinium in North America. Publ. 1828 Research Branch, Agriculture Canada Ottawa, Canada
Vander Kloet, S.P. & Lyrene, P.M. 1987 Self-incompatibility in diploid, tetraploid, and hexaploid Vaccinium corymbosum Can. J. Bot. 65 660 665
White, E. & Clark, J.H. 1939 Some results of self-pollination of the highbush blueberry at Whitesbog, New Jersey Proc. Amer. Soc. Hort Sci. 36 305 309
Zapaya, T.R. & Arroyo, M.T.K. 1978 Plant reproductive ecology of a secondary deciduous tropical forest in Venezuela Biotropica 10 221 230