Paul M. Lyrene

Polyploidy and sexual polyploidization in the genus Vaccinium

Genus Vaccinium,consisting of blueberries, cranberries, lingonberries and many related wild species, includes diploid, tetraploid, and hexaploid species. Most evidence indicates that the tetraploid species are autotetraploids, with non-preferential bivalent chromosome pairing. Although homoploid interspecific crosses usually produce numerous fertile hybrids if the parents are from the same section of the genus, inter sectional crosses at the diploid level normally produce no seedlings, weak seedlings, or seedlings that are very low in fertility. There is a strong but not complete triploid block within Vaccinium. Even with insections, tetraploid × diploid (and the reciprocal) crosses normally give only a few tetraploid and a few triploid hybrids. Hexaploid × diploid crosses within sections are very hard to make, and the few hybrids that have been obtained are pentaploid. The frequency of 2n gametes varies,both among genotypes within species and among species. Vaccinium pollen is normally shed in tetrads, and the frequency of large pollen grains shed in dyads can be used to estimate 2n gamete frequency. Cultivated blueberries occur at both the tetraploid and the hexaploid levels, and there are important genetic resources in the diploids. Unreduced gamete production has permitted transfer of genetic material from the diploid to the tetraploid level and from the diploids and tetraploids to the hexaploid level via triploid hybrids. Intersectional crosses can occasionally produce tetraploid Vaccinium hybrids that appear to behave as amphidiploids and have medium to high fertility. CommercialVaccinium cultivars are normally propagated by cuttings. Intersectional hybridization, chromosome doubling, and asexual propagation could permit the production of novel hybrid combinations with value as ornamentals or in fruit production.

Blueberries and Cranberries

Most blueberry breeding activity is focused on northern highbush, southern highbush and rabbiteye types. The major objectives of blueberry breeders center on high plant vigor, improved disease resistance, flavor, longer storing fruit and expanded harvest dates. Cranberry breeders have concentrated on early maturing fruit, uniform large size, intense color, keeping quality, high productivity, disease resistance and plant vigor. Considerable variability exists in blueberry and cranberry for most of the horticulturally important traits, and while only a limited number of genetic studies have been performed, most inheritance patterns fit quantitative models. Several genes have been identified through molecular, genetic and genomic approaches that are associated with cold hardiness. Wide hybridization is commonly employed in blueberry breeding and southern highbush types were derived primarily by incorporating genes from the diploid species Vaccinium darrowii into the highbush background via unreduced gametes. A wide array of molecular markers has been used in blueberry for fingerprinting and linkage mapping, and a major QTL regulating the chilling requirement in diploids has been identified. Transgenic blueberries have been produced with herbicide resistance and the Bt gene (Bacillus thuringiensis) has been incorporated into cranberry. A large EST library of highbush blueberry has been produced.

Value of various taxa in breeding tetraploid blueberries in Florida

Many species and races of blueberries native to the southeastern United States have been used in the University of Florida breeding program. Highbush blueberry cultivars from a program started in 1908 in the northern United States were used at the start of the Florida program as a source of early ripening, large fruit size and high fruit quality. Vaccinium species native to Florida were used to obtain adaptation to the Florida environment, which is characterized by a long growing season, mild winters interrupted by killing freezes, low number of chill hours, and warm, moist conditions that favor fungal pathogens. The Florida species that have proved most useful in breeding have been southern races of Vaccinium corymbosum, which produce hybrids with high vigor and upright growth habit, and V. darrowi, which produces hybrids that retain their leaves late into the winter and have a very low chilling requirement. Species that have been less useful in breeding are V. elliottii and V. myrsinites. V. arboreum, in Vaccinium section Batodendron, is the only species outside of section Cyanococcus that has been used in breeding Florida blueberries, and has been a good source of vigor and tolerance to soils low in organic matter and high in bicarbonates. Although the gene pool available for improving low-chill highbush blueberries is large and diverse, progress is slowed by the need to improve simultaneously a large number of traits.

Recurrent selection in breeding rabbiteye blueberries (Vaccinium asheiReade)

Rabbiteye blueberries are a highly-heterozygous, cross-pollinating fruit species whose genetic improvement began in 1940. Pedigree analysis shows that 15 of the 18 cultivars developed in the United States in the last 40 years trace entirely to 6 plants selected from the wild. The cultivars most recently released are mainly from the second cycle of selection, a fact that implies an intergeneration interval averaging 20 years-long enough to fruit a blueberry seedling 17 times. Although the breeding project has produced cultivars that are far better than the wild plants from which breeding started, it is poorly designed to maximize long term progress. It is proposed that pollen be bulked from 30 new selections from the wild population and used to pollinate 10 to 15 improved cultivars. About 3000 F, plants would be grown from these crosses and recurrent selection with a 5-year generation interval would be initiated. In each cycle, 30 selected seedlings would be polycrossed to produce 3000 seedlings for evaluation.

Unilateral cross compatibility in Vaccinium elliottii × V. arboreum, an intersectional blueberry hybrid

Vaccinium elliottii Chapmn., a diploid blueberry in Vacciniumsection Cyanococcus, was crossed with V. arboreum Marsh, a diploid blueberry insection Batodendron. The goal was toproduce hybrids that could be used toincorporate traits from these species intotetraploid southern highbush cultivars. The crosses were made reciprocally usingtwo clones of each species. A recessiveseedling marker gene for anthocyanin-freecotyledons in the V. elliottii clonesused as female parents permitted thetesting of mentor pollination. For thementor pollinations, V. elliottiiplants that were homozygous for therecessive marker allele were pollinatedwith a small amount of viable V.elliottii pollen carrying the recessivemarker mixed with a large amount of V.arboreum pollen carrying the dominantmarker. Thousands of intersectional hybridseedlings were obtained when V.elliottii was the seed parent, both withand without mentor pollination, but nohybrids were obtained from 2735 flowerspollinated in the reciprocal crosses. Theseeds that gave rise to hybrid embryos weresmaller than normal V. elliottiiseeds, but germinated well. Hybridseedlings grew slowly at first, buteventually produced some vigorous plants inthe field, although many plants remainedmuch smaller than normal seedlings of thetwo parental species. Some of the hybridsproduced numerous flowers during the 6years they were observed in the field, buteven with many diploid, tetraploid, andhexaploid section Cyanococcus blueberryplants growing nearby that could haveprovided pollen for their flowers, thehybrids produced no viable seed.

The Use of Inter-Sectional Hybrids in Blueberry Breeding

The tetraploid species in Vaccinium section Cyanococcus and tetraploid V. uliginosum in section Vaccinium are autotetraploid. The same is probably true of the tetraploid species in other sections of the genus. Chromosome pairing at metaphase I in these species is normally regular and bivalent, but each gamete contains two homologous chromosomes for each of the 12 basic chromosome types (x = 12 in Vaccinium). Thus, F1 hybrids between tetraploid plants from different Vaccinium sections can have regular bivalent chromosome pairing during meiosis and high fertility, even though diploid hybrids involving the same sections are highly sterile. Such fertile tetraploid hybrids are called amphidiploids. Amphidiploidy can give rise to new species; several important crop species are domesticated amphidiploids. The conditions for amphidiploid formation are narrow. The two species that hybridize must be divergent enough to insure faithful homologous bivalent chromosome pairing in the hybrid, where each bivalent consists of chromosomes derived from the same parent species. However, the parent species must be closely enough related to permit formation of vigorous hybrids. The first indication that amphidiploidy could be useful in blueberry breeding was a report by Rousi in 1963 of vigorous, fertile hybrids between tetraploid V. uliginosum (section Vaccinium) and tetraploid V. corymbosum (section Cyanococcus). In Florida, crosses of colchicine-induced tetraploid V. arboreum (section Batodendron) with tetraploid highbush cultivars and with tetraploid Florida V. myrsinites (section Cyanococcus) indicate that Vaccinium sections Batodendron and Cyanococcus also have the right degree of divergence to produce vigorous, fertile tetraploid hybrids. The feasibility of producing other intersectional tetraploid combinations in Vaccinium, and the vigor and fertility of the hybrids, can best be determined by trial and error.

Results of multiple pollination in blueberry (Vaccinium elliottii Chapm.)

Blueberry (Vaccinium section Cyanococcus, family Ericaceae) is a shrub that produces multiple-seeded berries in which only a fraction of the ovules develop into viable seeds. A recessive yellowleaf marker gene was used to evaluate the efectiveness of a single pollination versus multiple pollinations in producing seeds in Vaccinium elliottii Chapm. A multiple-pollination experiment was conducted to see if the first pollen applied or the second produced more progeny, and to see if multiple pollinations increased the number of seedlings produced. Flowers of diploid yellowleaf Vaccinium elliottii were pollinated from one to four times at daily intervals. Pollen from redleaf and yellowleaf plants was used to produce two types of seedlings that could be distinguished visually. Generally, percent berry set, seeds per berry, and seed germination were not increased by multiple pollination. After multiple pollination, the pollen applied first always produced the most seedlings. Prior pollination greatly reduced seed set from subsequent pollination, but subsequent pollination caused little or no decrease in the number of seeds set by the first pollination.

Use of Vaccinium octoploids to facilitate 4x-6x gene transfers

The use of an 8x Vaccinium produced through doubling a tetraploid hybrid with colchicine was studied as a means of facilitating gene exchange between V. corymbosum L. and V. asheiReade. Analysis included meiotic observations and crossability studies of the 8x plant, as well as attempting to develop 6x breeding lines. Meiotic analysis revealed the presence of micronuclei in Telphase II products and polyspory in sporads. In the crossability studies five 4x highbush plants were pollinated with 8x o2 4x pollen. Data gathered included: % fruit set, average weight/fruit, average number of seeds/fruit, average number of seedlings/pollination, and number of 6x seedlings. Octoploid pollinations were significantly lower than 4x pollinations in all parameters. One 6x plant was produced from the 4x-8x crosses but was found to be mitotically unstable, having somatic cells with chromosome numbers ranging from 48 to 168.

Assessing genetic diversity of wild southeastern North American Vaccinium species using microsatellite markers

Wild species representatives from Northwestern, Central and Southern Florida, and neighboring U.S. states were collected in multiple United States Department of Agriculture (USDA) exploration expeditions and are being preserved at the USDA, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon. Germplasm from these southeastern regions of North America is particularly vulnerable to loss in the wild due to encroachment of human development in key habitats and biotic and abiotic stresses from climate change. Fourteen simple sequence repeats (SSRs), previously developed from the highbush blueberry (Vaccinium corymbosum) cultivar ‘Bluecrop’, were used to estimate genetic diversity and genetic differentiation of 67 diploid individuals from three species, including 19 V. elliottii, 12 V. fuscatum, and 35 V. darrowii accessions collected throughout the species’ ranges. Results from our analyses indicated that the samples from each species could be reliably resolved using genetic distance measures with ordination and neighbor joining approaches. In addition, we estimated admixture among these species by using Bayesian assignment tests, and were able to identify a mis-labeled accession of V. darrowii ‘Johnblue’, two mis-classified accessions (CVAC 735.001 and CVAC 1223.001), and four accessions of previously undescribed hybrid origin (CVAC 734.001, CVAC 1721.001, CVAC 1741.001, and Florida 4B CVAC 1790). Allele composition at the 14 SSRs confirmed that Florida 4B CVAC 1790, the donor of low chilling for the southern highbush blueberry, was the critical parent of US 74. Genetic diversity assessment and identification of these wild accessions are crucial for optimal germplasm management and expand opportunities to utilize natural variation in breeding programs.