HYBRIDIZATION OF TETRAPLOID HIGHBUSH CULTIVARS WITH DIPLOID VACCINIUM ELLIOTTII

By

ELLIOT NORDEN

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

1

© 2017 Elliot Norden

2

To my family and to my friends

3 ACKNOWLEDGMENTS

I thank my parents for their continued support throughout my life. I thank my mentors, Dr. Paul Lyrene and Dr. Jose Chaparro, for their support on my project and expert advice and insights. In addition, I thank both of them for showing me the beauty of plant breeding, and pushing me to complete this degree and be a better person. I express gratitude to my committee members, Dr. Paul Lyrene, Dr. Jose Chaparro and

Dr. Pam Soltis. I thank, Dr. James Olmstead, Werner Collante, and Dr. Kenneth

Quesenberry.

4 TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

ABSTRACT ...... 9

CHAPTER

1 INTRODUCTION ...... 11

2 INTER-SPECIFIC HYBRIDIZATION AND CROSSING BEHAVIOR OF VACCINIUM ELLIOTTII WITH SOUTHERN HIGHBUSH CULTVARS ...... 14

Materials and Methods...... 17 Studies with Vaccinium elliottii ...... 17 Inter-specific hybridization with Vaccinium corymbosum ...... 17 Flower studies ...... 17 Leaf studies ...... 18 Flow cytometry analyses ...... 19 Cytological studies ...... 20 Crossing Studies ...... 21 Intercrosses for F2 generation ...... 21 Backcrosses ...... 22 Berry studies ...... 22 Statistical analysis ...... 23 Results and Discussion...... 23 Studies with Vaccinium elliottii ...... 23 Interspecific hybridization data ...... 23 Putative F1 hybrid selection ...... 24 Morphological Studies of Putative F1 Hybrids (Southern Highbush Cultivars x V. elliottii) ...... 25 Flower characteristics ...... 25 Leaf characteristics ...... 26 Berry characteristics ...... 27 Fertility Studies of Putative F1 Hybrids (Southern Highbush Cultivars x V. elliottii) ...... 28 Cytogenetic Studies ...... 29 Chromosome Counting ...... 31 N-37 ...... 31 N-32 ...... 31 Crossing Data 2016 ...... 31

5 Intercrosses ...... 31 Backcrosses ...... 32 Crossing Data 2017 ...... 33 Intercrosses ...... 33 Backcrosses ...... 33 Conclusions ...... 34

3 PLOIDY LEVEL DETERMINATION IN COLCHICINE-TREATED VACCINIUM ELLIOTII SEEDLINGS AND SUBSEQUENT INTERSPECIFIC HYBRIDIZATION BETWEEN HIGHBUSH BLUEBERRY CULTIVARS AND AUTOTETRAPLOID V. ELLIOTTII ...... 47

Materials and Methods...... 48 Materials ...... 48 Flow Cytometry Analyses ...... 49 Stomata Analyses ...... 51 Pollen Analyses ...... 51 Interspecific Hybridization of Colchicine-Derived Tetraploid Vaccinium elliottii with Tetraploid Highbush Blueberry Cultivars ...... 52 Results and Discussion...... 52 Colchicine Treatment ...... 52 Flow Cytometry Data ...... 53 Stomata and Pollen Size Data ...... 54 Interspecific Hybridization of Colchicine-Derived Autotetraploid Vaccinium elliottii with Tetraploid Highbush Cultivars ...... 55 Conclusions ...... 57

LIST OF REFERENCES ...... 63

BIOGRAPHICAL SKETCH ...... 66

6 LIST OF TABLES

Table page

2-1 Results from 14 tetraploid highbush x Vaccinium elliottii crosses done in the spring of 2014. All highbush flowers were pollinated with pollen bulked from diploid Vaccinium elliottii clonesz...... 37

2-2 Results from the 2016 crosses including: intercrossing of F1 hybrids and b c backcrossing of F1 hybrids to diploid VE (Vaccinium elliottii) and tetraploid VC (Vaccinium corymbosum) varieties...... 38

2-3 Leaf, flower, and berry characteristics of three taxa: V. elliottii (VE), southern highbush (HB) and highbush cultivars x V. elliottii F1 hybrids (HB x VE)...... 39

2-4 Comparative analysis of DNA content of etiolated tissue versus Non-etiolated tissue from V. elliottii (VE), highbush (HB) and highbush x V. elliottii F1 hybrids using flow cytometry...... 40

2-5 Genome size estimation by flow cytometry of VC (Vaccinium corymbosum) x VE (Vaccinium elliottii) F1 hybrids utilizing Vigna radiata as a diploid standard with a genome size of 1.06 pg. Pollen size of VE selections was also used as a predictor of ploidy level. Pollen shed and % fully formed tetrads were used as indicators of fertility...... 41

2-6 Results from the 2017 crosses, including: Intercrosses of F1 hybrids to obtain F2 populations and backcrosses to VCb (Vaccinium corymbosum) varieties...... 43

2-7 Comparative success of Highbush x Highbush, Vaccinium elliottii x Vaccinium elliottii, and Highbush x Vaccinium elliottii crosses 2014z...... 43

3-1 Population one, genome size estimation by flow cytometry of colchicine-treated V. elliottii clones utilizing Vigna radiata as a diploid standard with a genome size of 1.06 pg. Stomata size and pollen size were also used as predictors of ploidy level...... 58

3-2 Population two, genome size estimation by flow cytometry of colchicine-treated V. elliottii clones utilizing Vigna radiata as a diploid standard with a genome size of 1.06 pg. Stomata size and pollen size were also used as predictors of ploidy level...... 60

3-3 Results of crosses between southern highbush cultivars and tetraploid colchicine-treated V. elliottii during winter and spring 2017...... 62

7 LIST OF FIGURES

Figure page

2-1 Principal component analysis that explains 76.5% of the variation between flower morphology features from V. elliottii, F1 hybrids and highbush flowers...... 44

2-2 Principal component analysis that explains 100% of the variation in leaf length and width from V. elliottii, F1 hybrids and highbush leaves...... 44

2-3 Principal component analysis and K-means clustering that explains 60% of the variation, with K=3, for fruit traits from V. elliottii, F1 hybrids and highbush blueberries...... 45

2-4 Typical flowers of diploid V. elliottii, F1 hybrids and a tetraploid highbush cultivar...... 46

2-5 Meiotic analysis at metaphase I of F1 (Highbush x V. elliottii) hybrids. A) Tetraploid N-37, metaphase I, 24II. B) Triploid N-32, Metaphase I, 12II+12I. 1000x...... 46

3-1 Pictures showing flower size variation between autotetraploid and diploid V. elliottii...... 62

8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

HYBRIDIZATION OF TETRAPLOID HIGHBUSH CULTIVARS WITH DIPLOID VACCINIUM ELLIOTTII

By

Elliot Norden

December 2017

Chair: Jose Chaparro Major: Horticultural Sciences

In February 2014, ten plants, generated from wild diploid V. elliottii cuttings taken from southwest Alabama, were used to pollinate 19 different southern highbush selections. Some crosses between tetraploid southern highbush selections and diploid

V. elliottii produced tetraploid hybrids because of unreduced gametes from V. elliottii. In total, 78 plump seeds were generated from 1677 pollinations. Fifty-five of the 78 seeds germinated and were evaluated for hybridity. Twenty-eight of the fifty-five plants were selected as putative hybrids for crossing studies. Crosses made in 2016, before determining ploidy and hybridity, gave low numbers of plump seeds per pollinated flower (PPF). Using principal component analyses for morphological floral, leaf and berry features allowed for the identification of hybrids from this population. Using crossing behavior, pollen fertility and flow cytometry as a predictor of ploidy aided in distinguishing triploids from tetraploids. Crosses made in 2017, after determining ploidy and hybridity, resulted in a large number of plump seeds per pollinated flower. Eight tetraploid V. elliottii plants were generated from colchicine treatment of seedlings.

Stomata size was an accurate and efficient indicator of chromosome doubled V. elliottii plants. Flow cytometry, as a predictor of ploidy, confirmed findings from stomata data

9 and aided in identifying chimeras. Crosses between colchicine derived tetraploid V. elliottii plants and southern highbush selections resulted in low plump seeds per pollinated flower. This was attributed to chimeric plants producing diploid and tetraploid flowers, and some diploid flowers being used for crosses with tetraploid highbush selections.

10 CHAPTER 1 INTRODUCTION

Blueberry cultivation began in 1908, when Frederick Coville, a chief botanist for the United States Department of Agriculture, realized the potential for domesticating the wild blueberry. In 1911, Coville began making crosses using wild species from New

Hampshire as parents (Coville, 1937). Selections from these crosses led to several cultivars (Moore, 1965). Over the next 50 years, blueberry production expanded slowly but steadily. After 1950, the expansion of blueberry production was much more rapid, with the release of new cultivars adapted to new growing regions.

In 1949, the University of Florida Blueberry Breeding program began, under

Professor Ralph Sharpe, with the goal of creating a blueberry that would be harvestable early while still retaining a high quality (Lyrene, 1997). Until this time, blueberry production in Florida was limited to plantings of wild rabbiteye selections (Vaccinium virgatum) dug from the swamps of west Florida (Lyrene and Sherman, 1984). These plants flowered late, ripened late and were highly variable in quality and productivity

(Lyrene and Sherman, 1984). There was a need for early-ripening cultivars that were well adapted to Florida’s climate and disease pressure.

Utilizing cultivars and advanced selections from northern breeding programs,

Sharpe began his attempts to develop highbush varieties adapted to Florida’s climate

(Lyrene, 1997). Due to lack of chill hours, the highbush varieties bred by Coville and his successors in New Jersey and Michigan suffered from lack of chilling when planted in

Florida, and this adversely affected flowering and productivity. Native blueberry species were seen as a source of genes to overcome these problems (Sharpe and Darrow,

1959).

11 Many species of Vaccinium are native to the southeastern United States (Camp,

1945). Three native species were exploited to develop the southern highbush blueberries: Rabbiteye (Vaccinium virgatum (6x)), Darrow’s blueberry (Vaccinium darrowii (2x)), and shiny blueberry (Vaccinium myrsinites (4x)). Selections from crosses between high-chill northern highbush cultivars and these species gave cultivars with reduced chilling requirements (Sharpe, 1953).

Vaccinium elliottii, a wild diploid species native to the southeast, was seen as source of genes for early ripening, upright-growth habit, disease resistance, and high quality fruit (Lyrene, 1997). V. elliottii was considered to have low potential for crossing with highbush cultivars because it is diploid and cultivars are tetraploid. There exists a strong triploid block in the genus Vaccinium (Lyrene and Sherman, 1984). The production of unreduced gametes and the triploid block in the genus Vaccinium facilitate the production of tetraploid hybrids from tetraploid x diploid crosses (Sharpe and

Darrow, 1959).

Multiple attempts to introduce traits from V. elliottii into cultivated germplasm have been made (Lyrene and Sherman, 1983; Dweikat and Lyrene, 1991; Ballington et al., 1996; Wenslaff and Lyrene, 2001). These crosses have led to the development of cultivars like ‘Snowchaser’ and ‘Kestrel’. However, V. elliottii was used only sparingly as a parent because highbush cultivar hybrids with V. elliottii were often short-lived, had disease problems, and produced small dark fruits.

Large populations of wild V. elliottii occur in north Florida and in Alabama.

Because V. elliottii has not been extensively used in breeding and because the two

12 Florida cultivars having V. elliottii in their pedigrees ripen unusually early and have berries with excellent flavor, the following studies were undertaken.

13 CHAPTER 2 INTER-SPECIFIC HYBRIDIZATION AND CROSSING BEHAVIOR OF VACCINIUM ELLIOTTII WITH SOUTHERN HIGHBUSH CULTVARS

The cultivated blueberries belong to the family Ericaceae, subfamily:

Vaccinioideae, genus: Vaccinium. Worldwide Vaccinium is estimated to include anywhere from 150-450 species, distributed among 30 different sections. Cyanococcus is the largest section, containing about 20 species, including the cultivated blueberry,

Vaccinium corymbosum (highbush blueberry), Vaccinium elliottii (Elliott’s blueberry),

Vaccinium darrowii (Darrow’s blueberry), and Vaccinium virgatum (Rabbiteye blueberry, previously named Vaccinium ashei) (Hancock et al., 2008). These Vaccinium species are endemic to eastern North America, which is thought to be the center of origin for section Cyanococcus. Other Vaccinium species are distributed across the world from the Americas to Asia. Vaccinium species within section Cyanococcus include diploids

(2n=2x=24), tetraploids (2n=4x=48) and hexaploids (2n=6x=72) (Camp, 1945). Many of these species have been harvested from the wild for hundreds of years, but it has only been in the last century that major breeding efforts have been taken (Lyrene, 1998).

Cultivated varieties have come from rabbiteye (V. virgatum), lowbush (V. angustifolium), and both northern and southern highbush (V. corymbosum). The primary gene pool for breeding consists of cultivars formed from these species. Wild species within section

Cyanococcus make up the secondary gene pool, and species outside of section

Cyanococcus comprise the tertiary gene pool (Lyrene and Ballington, 1986).

Chilling requirements and cold tolerance restrict cultivation of V. corymbosum.

Northern, intermediate and southern blueberry varieties were bred to extend the area over which blueberries could be cultivated (Hancock, 2004). Northern and intermediate varieties were poorly adapted for growth in the Southeast. They were unable to handle

14 the disease pressure, and mild winters prevented the accumulation of chill hours needed to break dormancy. In 1949, the University of Florida began a breeding program to create cultivars adapted to cultivation in north and central Florida. Wild blueberry selections would be used to overcome climactic adaptation and disease resistance

(Sharpe and Sherman, 1971; Lyrene, 1997).

Introgression of genes from wild blueberry species has been attempted since the beginning of blueberry breeding programs. Homoploid and heteroploid crosses have been attempted with different species from section Cyanococcus. Inter-specific homoploid crosses tend to be successful, producing fertile F1 hybrids with few sterility barriers. Heteroploid crosses are more variable in success, but can give rise to fertile F1 hybrids (Camp, 1942; Darrow and Camp, 1945).

Rabbiteye blueberries (V. virgatum) were the first cultivated blueberries in

Florida, and consisted of large plantings of seedling selections from the wild. Eventually, improved rabbiteye cultivars were bred using a few elite clones selected from the wild.

However, rabbiteye blueberries (V. virgatum) are very late ripening compared to highbush blueberries (V. corymbosum), and rabbiteye growers miss the early part of the market window, when the prices are highest (Lyrene, 1997). Eventually, interest and production of cultivated rabbiteye blueberries dwindled in the Southeast, as new inter- specific hybrids led to the production of early-ripening highbush cultivars based on V. corymbosum. Diploid V. darrowii and V. elliottii were selected as primary genetic sources for adaption of highbush varieties to the Florida climate (Lyrene and Sherman

1984).

15 Southern highbush blueberries were a result of successful hybridization between northern highbush cultivars and V. darrowii. 1,600 pollinations resulted in 31 fertile hybrids (Sharpe and Darrow, 1959). V. darrowii produces unreduced gametes at a high rate, which facilitated the heteroploid cross (Ortiz et al., 1992). The genus Vaccinium also possesses a strong triploid block, a postzygotic barrier, which aided in the selection of fertile hybrids. These hybrids, combined with northern highbush cultivars, became the primary gene pool for phenotypic recurrent selection in breeding for Florida adapted blueberries.

V. elliottii is an early-ripening diploid species endemic to north-central Florida, west to Texas, and north to Virginia (Camp, 1945). Its upright form and very low chill requirement made it an ideal genetic source for the improvement of low chill varieties in

Florida. In the late 1970’s and early 1980’s, Lyrene and Sherman (1983) made major efforts to incorporate genes from V. elliottii into the southern highbush germplasm. F1 hybrids showed potential; they were vigorous, highly fertile, flowered and ripened early in the season. However, after 15 years of backcrossing, V. elliottii was mostly abandoned as a parent due to small fruit size, soft berries and a lack of wax on the blueberry giving it a black appearance (Lyrene and Sherman, 1983).

This study seeks to evaluate an F1 population of inter-specific hybrids between

Vaccinium elliottii and southern highbush cultivars, re-evaluate V. elliottii’s potential as a parent using new wild germplasm collected from southwest Alabama, and create a segregating F2 generation.

16 Materials and Methods

Studies with Vaccinium elliottii

Inter-specific hybridization with Vaccinium corymbosum

During August 2013, softwood cuttings were taken from 80 Vaccinium elliottii seedlings, from an 8-km stretch of Perone Creek, in southwest Alabama by Dr. Paul

Lyrene. Cuttings were rooted in a greenhouse under mist using peat and perlite mix

(1:1). Four ramets of each plant were obtained from these cuttings. Ramets were grown in pots of pure peat in a greenhouse until March 2014. In early February, they began to flower. Concurrently, nineteen southern highbush selections that had been placed in a cooler to accumulate chilling were taken out and placed in the bee-proof greenhouse.

Flowers on the southern highbush selections were emasculated before opening, and roughly 200 flowers per plant were pollinated with pollen from ten of the original eighty

Vaccinium elliottii selections (Lyrene, 2015). The seeds were extracted, dried on paper towels, and then placed in paper envelopes for storage. The berries from all of these crosses yielded only 78 plump seeds (Table 2-1). In November 2014, the seeds were sown on the surface of Canadian peat under mist. Fifty-five of the 78 total seeds germinated. In January 2015, the seedlings were transplanted to a tray of peat. They were grown in a greenhouse until June 2015, when they were transplanted into a nursery plot at the University of Florida’s Plant Science Unit, in Citra, FL.

Flower studies

On December 1, 2015, the 55 putative F1 hybrids were evaluated based on phenotypic characteristics (Table2-2). In total, 38 plants whose phenotypes were intermediate between the parents were dug and transplanted into pots with pure peat, and placed in a cooler at 4°C to accumulate chilling. On January 1, 2016, the plants

17 were taken from the cooler and moved to a bee proof greenhouse for flowering. Plants began to flower in late January 2016. The first two flowers that opened on each plant were used for pollen analysis. Open flowers were harvested and the pollen was shed into a drop of 45% acetic acid on a microscope slide for evaluation. Pollen was observed at 250x magnification using a microscope (Leitz Wetzlar Model 664012,

Wetzlar, Germany). Pollen shed, pollen size, and percent fully formed pollen tetrads were recorded (Table 2-5). A fully formed tetrad consisted of 4 enlarged spores combined to form a full tetrad. It should be noted that percent fully-formed tetrads is likely to be much lower than percent potentially viable microspores, since some sporads that were not full tetrads had one, two, or three well-formed microspores that were potentially functional.

The next ten flowers that opened on each plant were used for floral morphology measurements. Open flowers were collected in the morning from each plant and placed in separately labeled petri dishes. They were dissected and analyzed the same day using a razorblade and tweezers (Table 2-3). Floral measurements taken consisted of corolla length, corolla diameter, stigma position vs. corolla length, and style length. All measurements were taken using Fisher Scientific™ Traceable™ Digital Carbon Fiber

Calipers.

Leaf studies

In August 2017, parental and F1 hybrid plants that were growing outside were selected for leaf morphology studies. The F1 hybrids used for this report were from the original 2014 interspecific crosses. One random fully expanded leaf was collected from ten different V. elliottii and V. corymbosum genotypes, and two fully expanded leaves were collected from ten different F1 hybrid genotypes for analysis (Table2-3). Length

18 and width were recorded for each leaf using Fisher Scientific™ Traceable™ Digital

Carbon Fiber Calipers.

Flow cytometry analyses

Etiolated tissue was generated by placing 5 stem cuttings from each F1 hybrid in

8L plastic buckets with 1 L of tap water for estimation of genome size. The buckets were placed under 100 L paper bags to exclude light, and the water was replaced with fresh water every week until soft new etiolated growth could be harvested. The stems ranged in length from 30 to 75 cm and in basal diameter from 0.5 to 2 cm. They were tied into bundles of 5 to keep them upright. The covered tissue was kept in a climate controlled room at 22 °C ± 1 °C. After 5 weeks, the etiolated tissue was harvested periodically until tissue had been collected from all genotypes. Tissue was collected in the early morning and placed between wet napkins until it was processed. Tissue was processed the same day it was harvested.

Tissue from plants that were not etiolated was collected for comparative analyses

(Table 2-4). To obtain this tissue, these plants were pruned heavily, removed from the crossing greenhouse, and placed outside. When the plants began to flush out, the tender new growth was harvested and placed between wet napkins until it was processed. Tissue from all F1 hybrids was collected and prepared the same as etiolated tissue.

Flow cytometry analysis was done using the BD Accuri™ C6 instrument at the

University of Florida’s ICBR (Interdisciplinary Center for Biotechnology Research),

Gainesville, FL. Tissue was prepared using the Sysmex CyStain® PI Absolute P kit.

Prior to tissue preparation, RNAseA stock solution and staining solution were prepared.

RNAseA stock solution was prepared by adding 1.5 ml H2O to 1 tube of RNAseA. The

19 stock solution was stored at -20°C. The staining solution was prepared by combining 20 ml staining buffer, 120 μL propidium iodide and 60 μL RNAseA stock solution. The staining solution was stored at 4°C in bottles wrapped in aluminum foil to exclude light until used. Five hundred mg of etiolated tissue was harvested from each plant. Within 8 hours, the tissue was chopped for 60 seconds in 500 μL of nuclei-extraction buffer on a

7.5 cm petri dish using a microtome blade. The solution was filtered through 50μm mesh into a 5ml glass tube. 2 ml of staining solution was added to each 5 ml tube, and the tube was immediately placed on ice in the dark to incubate until being analyzed.

The equipment settings were held constant for all samples. Each sample ran

10,000 events at a medium flow rate of 35 μl/min and a maximum fluorescence emission threshold of 1,000 on FL2-H. The Gate was set according to each sample.

Samples were vortexed before being analyzed. To determine the ploidy of each sample, a Vaccinium elliottii nuclei solution was prepared and used as an internal diploid standard. The diploid standard was run first and then the unknown samples were run individually. Since a tetraploid was expected to have double the DNA content of a diploid, and the diploid RFI peak was known, any peak from an unknown sample that had a an RFI peak twice as big as the diploid standard was considered a potential tetraploid (Table 2-5).

Cytological studies

To confirm the ploidy estimation by flow cytometry and phenotypic measurements, anther squashes were prepared from flower buds that had been gathered in February 2017 at about the time meiosis was expected to be occurring and stored in a fixative solution consisting of 3 parts ethanol (95%): 1 part glacial acetic acid.

The tissue was collected and placed in the fixative solution and stored at 4°C until

20 processed. The samples were processed under a dissecting microscope with 40x magnification. Flower buds were prepared for squashing by dissecting leftover pedicles, removing individual buds from the cluster and only keeping the actively dividing immature buds. Individual flower buds were digested by immersion in a solution of 30% viscozyme® L and citric acid (Sigma-Aldrich, St. Louis, USA) with a ratio of 3:7 v/v, for

20 minutes, then washed in 70% ethanol for 30 seconds, and transferred to 45% glacial acetic acid for 1 minute. The remaining tissue was used to prepare cell squashes by placing the tissue in an 8-μl drop of 45% glacial acetic acid on a microscope slide, covering it with a cover slip, and applying homogeneous pressure using the thumb until the tissue was completely squashed. The wooden end of a dissecting needle was used to further flatten the tissue by tapping gently on the cover slip. The cover slip was sealed to the microscope slide by applying clear nail polish around the edges of the cover slip to inhibit evaporation.

Prepared cell squashes were visualized using a phase contrast microscope

(Leitz Wetzlar Model 664012, Wetzlar, Germany). Squashes were first observed at 250x lens to locate isolated cells showing chromosomes, then moved up to 400x lens to obtain coordinate location so they could be observed at 1000x using an oil immersion lens with phase contrast. Pictures of individual cells were taken using a Moticam 1000

1.3M pixel camera (Motic, Richmond, British Columbia, Canada).

Crossing Studies

Intercrosses for F2 generation

In January 2016, 38 putative F1 hybrid plants were moved from the cooler to a bee-proof greenhouse for crossing. These plants were simultaneously used for the flower studies and crossing studies in 2016. The results from the flower studies were

21 used to decide which putative hybrids would be intercrossed. Plants with a fully formed tetrad percentage greater than 15% were intercrossed (Table 2-2). Unopened flowers on plants that were used as female parents were emasculated before anthesis by removing the corolla and anthers with forceps. Open flowers were collected from the pollen parents to provide pollen for the crosses. Pollen from the selected pollen parent was shed onto the fingernail and applied to the stigma of the emasculated flower. The goal was to pollinate a minimum of 50 flowers per plant, but on some plants chosen as females, there were not enough flowers to pollinate 50.

Backcrosses

Examination of flowers from several plants that were dug and potted from the highbush x V. elliottii field nursery indicated that they were not the intended hybrid. They were probably highbush selfs or highbush seedlings from seed splash. These plants were used in pseudo-backcrosses to highbush x V. elliottii hybrids. F1 hybrids that produced less than 15% fully formed tetrads were considered possible triploids, and these were backcrossed to diploid V. elliottii (Table 2-2).

Berry studies

Berries began to ripen in late March of 2017. The first 10 berries that were harvested from each plant in the crossing study were evaluated for phenotypic characteristics. Berries were processed within 48 hours of harvesting. Berries that were not processed the same day were stored in a cooler at 4°C, until processing. Fruit firmness and diameter were measured using the Firmtech 2 (BioWorks, Inc., Wamego,

KS), which was calibrated before each use. Berries were cut in half using a scalpel, squeezed onto an American Optical Company hand refractometer, and Brix was measured. Seeds were harvested by smashing individual berries onto a paper towel.

22 Number of plump seeds per berry was recorded as the mean per-berry seed content of the 10 berries.

Statistical analysis

Flower morphology, leaf morphology, percent fully formed tetrads, pollen size, 2n chromosome number, and flow cytometry RFI (relative fluorescence intensity) were used for distinguishing and confirming hybrids. Fruit set percentage, number of plump seeds per pollinated flower, and berry characteristics were used to measure the results of pollination treatments. Data from flower morphology, leaf morphology, and berry characteristics were analyzed by ANOVA with a Tukey’s HSD (honest significant difference) test to determine whether means differed significantly at the 5% level. All residual plots and qqplots (quantile quantile plots) were observed to ensure normality and homogeneity of variances (Ghasemi and Zahediasl, 2012). In addition, means from flower and leaf morphology measurements were used to generate a principle component analysis. Means from berry characteristics were used to generate a principle component analysis with k-means clustering (Ding and He, 2004). All data analysis was conducted using R and Rstudio (Version 1.0.136 – © 2009-2016 RStudio, Inc.).

Results and Discussion

Studies with Vaccinium elliottii

Interspecific hybridization data

In comparative crosses with V. corymbosum (4x) and V. elliottii (2x) made in

2014, V. corymbosum x V. corymbosum crosses fruit set averaged 98%, and V. elliottii x V. elliottii crosses fruit set averaged 73%. Variation in fruit set was attributed to pollen fertility and female-male interaction (Table 2-7).

23 In V. corymbosum x V. elliottii crosses made in 2014, fruit set percentage ranged from 1-100% (Table 2-1). Variation in fruit set was attributed to fertility, ploidy differences and to varying levels of parthenocarpy among the highbush clones used as female parents. The crosses FL05-603 x V. elliottii, ‘Raven’ x V. elliottii, and FL 06-161 x V. elliottii all yielded a fruit set percentage above 90, but most of the berries had no seed. These crosses gave few viable seeds per 100 flowers pollinated (Lyrene, 2015).

Therefore, the high fruit set percentage was attributed to parthenocarpy. Fruit with plump seeds were most likely due to diploid V. elliottii producing unreduced gametes

(Ortiz et al., 1992). Unreduced gamete formation via first division restitution has been described in other Vaccinium species (Vorsa, 1986). Unreduced gametes formed from first division restitution preserve the heterozygosity of the 2x parent due to the non- segregation of homologous chromosomes. This gives the 2x parent the opportunity to transfer its exact genotype to the 4x offspring (Ortiz et al., 1992). This is a great benefit when attempting to transfer traits across ploidy levels.

Putative F1 hybrid selection

In December 2015, putative F1 hybrids were selected from the high-density nursery in Citra, Florida. 38 vigorous putative hybrids were selected with serrate leaves, stem and leaf petiole pubescence (all V. elliottii traits). Plants not selected were either too weak, dead, or clearly lacked V. elliottii characteristics. There was continuous variation in most phenotypic characteristics among plants. From this initial selection, 3 genotypes stood out among the rest, N-4, N-10 and N-32. All three were pubescent and highly vigorous, with leaf size intermediate between the parents. They also were evergreen, a trait inherited from the highbush parent in this cross.

24 Morphological Studies of Putative F1 Hybrids (Southern Highbush Cultivars x V. elliottii)

Flower characteristics

This study was conducted in a bee-proof greenhouse under natural day-length and temperatures that averaged 15°C night/25°C day. Temperatures fluctuated according to the seasons (Gainesville, FL). In the wild the flowering period for V. elliottii extended from November 2015 through June 2016. In the field, the flowering season for

V. corymbosum extended from December 2015 to April 2016. The putative F1 hybrids began to flower in late January 2016, and flowering on some plants continued through

June 2016. Differences in flowering times and numbers between the taxa are controlled by many factors such as plant maturity, plant size, plant architecture and genotype

(Spann, et al., 2003).

Flower morphology data clustered most of the putative F1’s together when a multivariate analysis (Principal Component Analysis) was run (Figure 2-1). The Principal

Component Analysis (PCA) found two components that explained 76.5% of the variation in flower morphology. Most variation was explained by traits that were strongly correlated. When the loadings for each component were viewed, the variation explained by component one was heavily weighted on the size traits corolla length, corolla diameter, and corolla aperture size. The variation explained by component two was weighted heavily on the other traits not used in the first component that were not as strongly correlated. This PCA method used eigenvalue decomposition with a correlation matrix to partition out the variance into two main components. This is the simplest of true eigenvector multivariate analysis methods (Abdi and Williams, 2010).

25 Results from the analysis of variance and Tukey’s HSD for the flower morphology data revealed that the two parental types (V. elliottii and highbush) significantly differed from one another in all floral features measured (Table 2-3). Furthermore, the putative

F1 hybrids significantly differed from both parental types in all features measured, except for pollen size and the distance between the distal ends of the corolla to the distal end of the style. In most hybrids and interspecific hybrids within Vaccinium spp., the F1 progeny are intermediate between the parental types for quantitative traits

(Darrow and Camp, 1945) (Figure 2-4).

Leaf characteristics

The putative F1 hybrids derived from the original interspecific crosses made in

2014 were used for this study. Results from the ANOVA and Tukey’s HSD for the leaf morphology data revealed that the two parental types (V. elliottii and highbush) significantly differed from one another in both length and width (P-value<0.01) (Table 2-

3). Furthermore, the putative F1 hybrids also significantly differed from both parental types in both length and width. They were intermediate between the two parental types.

The principal component analysis clustered the two parental types and the F1 hybrids separately based on the correlation between length and width of each leaf (Figure 2-2).

The PCA used two components that explained 100% of the variation in leaf size among the genotypes. Both components were comprised of size vectors, which explained all the variation of the length and width of these leaves.

Desired leaf traits from V. elliottii showed some segregation in the F1 population.

Most of the F1 hybrids showed increased leaf retention through the winter, compared to highbush cultivars. Although, V. elliottii is a deciduous species in the northern ranges of its growing regions (Lyrene and Sherman, 1984), in north Florida’s climate, it can

26 remain fairly evergreen during mild winters. By allowing photosynthesis through the winter months, this trait can be useful for producing blueberries under tunnels, and increased fruit quality early in the year. V. elliottii also has a very desirable growth habit described by Lyrene and Sherman (1984). Some clones are fastigiate in nature with low suckering. This trait could be selected for in later generations for aiding in mechanical harvesting. There also appeared to be less leaf spot diseases within this population based on observations of plants grown outside in an environment conducive for infection.

Berry characteristics

For the berry characteristics that were measured, the F1 hybrids were intermediate between the parental types (P-value<0.01) (Table 2-3) (Figure 2-3). Mean berry weight of the F1 hybrids was 1.4 g, a little more than half of the southern highbush cultivars (mean berry weight 2.3 g), and more than 10x larger than V. elliottii clones

(mean berry weight 0.12 g). With such a large increase in berry size in the first generation, it can be expected that with successive backcrossing to highbush cultivars the berry size can be quickly recovered. The mean berry weight of the F1 hybrids was greatly reduced by triploid hybrids that had a high number of parthenocarpic fruit, which contained no seeds and were small in size. After the first hybridization event with highbush cultivars, segregation of negative V. elliottii attributes (dark berries, small berry size, and powdery mildew susceptibility) was evident. Multiple generation of intercrossing, backcrossing, and selection will allow for the elimination of undesired characteristics from the V. elliottii germplasm (Sharpe and Sherman, 1971). Wax retention on the berries, which gives the berries a powdery-blue appearance, is an

27 important commercial trait desired by consumers, and has been reported as being polymorphic within the species V. elliotti (Albrigo, Lyrene and Freeman, 1980).

V. elliottii berries had a significantly higher Brix (18.7) than both highbush (12.7) and F1 hybrids (12.2) (Table 2-3). Although, the Brix of the F1 hybrids was significantly lower than both parental types, it was closer to highbush cultivars and could be increased by backcrossing to V. elliottii or highbush clones with recorded higher Brix.

However, because of the increased level of acidity within the V. elliottii fruit, the increase in sugars is not always perceived by the consumer (Lyrene, 1997).

Data from fruit firmness revealed that V. elliottii had the firmest fruit (314.7 g/mm) and highbush cultivars were significantly lower (253.3 g/mm). The F1 hybrids showed a significantly lower firmness than both the parental types (220.1 g/mm) (Table 2-3). It would have been expected that the firmness of the F1 hybrids would be intermediate.

Fertility Studies of Putative F1 Hybrids (Southern Highbush Cultivars x V. elliottii)

Pollen data. The F1 hybrids (V. corymbosum x V. elliottii) used for this study were generated from crosses in 2014. Controls consisted of highbush cultivars and V. elliottii clones. Highbush cultivars percent fully formed tetrads ranged from 90-98%. V. elliottii percent fully formed tetrads ranged from 80-90%. Percent fully formed tetrads ranged from 0-90% in the F1 hybrids (Table 2-5). The differences between percent fully formed tetrads in the putative F1 hybrids were attributed to ploidy level differences from the initial hybridization event., but other factors may have contributed to the variation.

Heteroploid crosses (V. corymbosum (4x) x V. elliottii (2x)) are likely to give rise to some highly sterile triploid progeny, both in nature and in synthetic breeding (Ramanna and

Jacobsen, 2003). Therefore, plants that contained high percentages of aborted pollen grains were classified as triploids for crossing purposes. Plants that contained percent

28 fully formed tetrads above 15% with high pollen shed were classified as fertile, presumably tetraploid F1 hybrids for crossing purposes. From these criteria, there were

12 plants selected as fertile tetraploids. Ten of these tetraploids were used for backcrossing and intercrossing. A few plants that had high percentages of fully formed tetrads and lacking phenotypic features originating from V. elliottii were discarded from the experiment at this point.

When polyploidization occurs within plants, there is a strongly correlated increase in cell size, which leads to increased pollen size and other enlarged morphological features (Ramanna and Jacobsen, 2003). Diameter of fully formed pollen tetrads was measured in an attempt to separate triploid F1 hybrids from tetraploid F1 hybrids.

Highbush pollen size (4x) ranged from 50.7-60 μm and V. elliottii (2x) pollen size ranged from 48.2-53.9 μm. Triploid pollen size was expected to fall between the two parental types. Triploid pollen size ranged from 49.3-52.4 μm. This was intermediate and overlapping between the two parental types. Distinguishing the tetraploids (pollen size range 50.7-60 μm) was more difficult due to the overlapping size range with triploids, but pollen size, percent fully formed tetrads, and pollen shed were combined to make an estimation of ploidy. In total, 12 plant were selected as tetraploids from these methods, and none of the F1 hybrids (pollen size range 54.8-59.7) fell within the range for triploids.

Cytogenetic Studies

Flow cytometry data. In previous protocols for DNA content determination by flow cytometry, using DAPI as a fluorescent stain in Vaccinium species, it was necessary to etiolate tissue to produce tissue that was pigment free and would reduce noise in the flow cytometer. However, comparative results in my tests showed no

29 significant difference between etiolated tissue and non-etiolated tissue when using a propidium iodide stain (Table 2-4). This was also found to be true in blueberries by

Costich et al. (1993). However, Costich et al. (1993) also found no genome size differences between diploid, tetraploid, and hexaploid Vaccinium species. Our study found the tetraploids to be approximately twice the size of the diploids and the triploids to be intermediate between the two.

In total, DNA content of 29 putative F1 hybrids was estimated using flow cytometry (Table 2-5). The 29 plants clearly segregated into two distinct ploidy levels

(3x and 4x) based on the internal standard Vigna radiata, which has a known genome size of 1.06 pg (Bennett and Leitch, 1995). The diploid control (V. elliottii) DNA 2C-value ranged from 0.58-0.66 pg, and the tetraploid control (V. corymbosum) DNA 2C-value ranged from 1.11-1.27 pg. Tetraploid F1 hybrids DNA 2C-value ranged from 1.12-1.44 pg. Variation in DNA content among the tetraploid F1 hybrids was attributed to genome rearrangements that may have occurred during the interspecific hybridization event.

Triploid F1 hybrids were intermediate between the parental types (DNA 2C-value range 0.89-1.07 pg). Few triploids were expected in this population due to the strong triploid block that exists in the genus Vaccinium (Lyrene and Sherman, 1983). Genetic imprinting regulates the triploid block. In nature, this form of postzygotic reproductive isolation is a major path for sympatric speciation (Kradolfer et al., 2009). The genetic imprinting genes regulate the development of the endosperm, an essential structure for embryo development. Imprinted genes are dosage sensitive and can lead to seed abortion.

30 Chromosome Counting

N-37

Chromosome counting confirmed the findings from flow cytometry that N-37was a tetraploid. The meiotic analysis of N-37 revealed normal pairing of chromosomes during metaphase I, 24 bivalents (Figure 2-5), to be a rare event. Most of the PMCs

(pollen mother cells) observed during metaphase formed a combination of univalents, bivalents and multivalents. This could be a reason why the fertility of N-37 was lower than expected.

N-32

Chromosome counting confirmed the findings from flow cytometry that N-32 was a triploid. The meiotic analysis of N-32 revealed 12 bivalents and 12 univalents (Figure

2-5). This explains the sterility of N-32 when used as a parent in crosses.

Crossing Data 2016

Intercrosses

The F1 hybrid plants used for the following studies were generated from crosses made in 2014. In 2016, based on plant morphology, pollen fertility, and flow cytometry data, the plants were segregated into three distinct groups: highbush selfs, tetraploid F1 hybrids and triploid F1 hybrids. The valid F1 hybrids were intercrossed to produce a segregating F2 generation.

N-1, N-10, N-4, N-32, and N-28 were the five F1 hybrids used for intercrossing in

2016. N-4 was the primary pollen donor due to its prolific flower production. N-1, used as a female, when pollinated by N-4, had a 64.4% fruit set with an average of 24 plump seeds per pollinated flower (PPF) (Table 2-2). N-10 was the best female, pollinated by

N-4, producing 78.6% fruit set with an average of 29.8 PPF. N-4 used as female with N-

31 32 pollen produced a 19.7% fruit set with 0.1 PPF. This low fruit and seed set was attributed to N-32 being identified as triploid after flow cytometry was performed later that year. N-28 was crossed with N-4 and had a 36.2% fruit set with 15.3 PPF. The variability in fruit set was attributed to female and male interaction and variation in fertility among the female parents.

Backcrosses

Plants were chosen for backcrossing based on hybrid validity, expected ploidy, and fertility measurements. Plants that were determined to be triploid and low in fertility were backcrossed to V. elliottii, in an attempt to regain some favorable alleles from the diploid species. Every cross made where a triploid was backcrossed to V. elliottii failed

(Table 2-2). This failure was probably due to low viability of gametes from the triploid parent resulting from meiotic irregularities in the Vaccinium triploids.

Plants that were determined to be tetraploid, but did not appear to be the intended interspecific hybrids (most likely highbush selfs or seed splashes), were backcrossed to highbush clones to see if the berries showed any signs of V. elliottii introgression and to compare the berries with those of the valid F1 hybrids. The backcrosses fruit set percentage ranged from 0%-97.3%, with a PPF range from 0-29.2.

The differences in fruit set and seeds were attributed to some of the putative highbush selfs actually being triploid hybrids. N-2, N-7, and N-24 had less than 30% for fruit set, and were below 3 PPF. These figures are similar to numbers reported by Vorsa and

Ballington (1991) in 3x x 4x Vaccinium crosses. Berries that developed on these three plants were small and dark in color, much like diploid V. elliottii berries. N-36 had a fruit set of 40.7% and a PPF of 14.8. Chavez and Lyrene (2009) reported similar numbers in backcrossing experiments using F1 hybrids of V. darrowi x highbush and crossing them

32 back to highbush clones. The berries produced were also similar in size to the true F1 hybrids that were being intercrossed. Therefore, it seems likely that N-36 was a true hybrid. N-38 produced a fruit set of 97.3% and a PPF of 29.2, almost identical to highbush x highbush crosses reported in Table 2-7. Berries from this cross also highly resembled highbush berries in size and color.

Crossing Data 2017

Intercrosses

The F1 hybrid plants used for the following studies were generated from crosses made in 2014. In 2017, flow cytometry and cytological studies had been performed on this population. Data from these studies allowed selection of two types of plants for use in the 2017 crosses: tetraploid F1 hybrids and triploid hybrids that produced high rates of unreduced gametes. Intercrosses consisted of 12 different genotypes crossed among each other to produce segregating F2 populations. Fruit set ranged from 38.2%-99.2% with a PPF ranging from 0.9 to 63 among the 12 genotypes (Table 2-6). The most productive cross was N-2 x N-28, which resulted in 99.2% fruit set and an average of 63 plump seeds per pollinated flower (PPF). The least productive cross was N-37 x N-4, which resulted in 38.2% fruit set and an average of 0.9 PPF.

Backcrosses

Triploid F1 hybrids were backcrossed to various highbush clones. Microscopic examination of the pollen indicated that these triploids produced unreduced gametes at a higher rate than normal diploid V. elliottii. Pentaploid hybrids would be the expected offspring from this cross. A triploid plant could also conceivably produce a viable 2x or

2x-1 gamete, resulting in tetraploid or near-tetraploid hybrids in crosses with tetraploid plants. Four plants were selected for these backcrosses. The putative triploid hybrids

33 were used as the female parents. Fruit set ranged from 11.2%-99% and an average

PPF ranging from 0-0.1. There was a high level of parthenocarpy in these crosses, which is why such high fruit set was observed with low fully developed seeds. A backcross was also made with a high confidence tetraploid F1 hybrid, N-64. This backcross was made both ways, and more seeds were obtained when N-64 was the pollen parent (Table 2-6).

Conclusions

Intra-specific crosses (tetraploid V. corymbosum x tetraploid V. corymbosum and diploid V. elliottii x diploid V. elliottii) had high fruit set and produced copious amounts of plump seeds per pollinated flower. Inter-specific hybridization (tetraploid V. corymbosum x diploid V. elliottii) produced low fruit set and few plump seed per pollinated flower compared to intra-specific, homoploid crosses. All tetraploid F1 hybrids produced from the 4x-2x crosses were attributed to V. elliottii clones producing unreduced gametes, which fused with the haploid gamete of the tetraploid parent. Triploid progeny were the result of the fusion of two haploid gametes from the 4x-2x crosses. Variation in frequency of unreduced gametes both in microspores and megaspores was observed within plants and across ploidy levels. Triploid plants produced from the 4x-2x crosses generated a higher rate of unreduced gametes than any of the 2x V. elliottii plants.

Differences between 2016 and 2017 seasons in the rate of success from intercrossing plants from the F1 population were attributed to lack of ploidy and cytological information in 2016. In 2016, the plants were selected for intercrossing based solely on their phenotypes, which explains the low rate of fruit set and PPF. In

2017, the plants had been screened to identify tetraploids using flow cytometry, chromosome counting, and the previous year’s crossing information. All crosses in 2017

34 had fruit set and plump seeds. The failed backcrosses in 2016 were attributed to low fertility of the triploid plants and to an imbalance in the endosperm balance number.

Triploid plants did not produce enough viable gametes to fertilize the egg and endosperm cells, it is likely that the endosperm aborts and embryo death occurs.

Flow cytometry clearly distinguished diploids, triploids, and tetraploids within the population of F1 hybrids. No diploids were found. However, there were many triploids

(DNA 2C-value range 0.89-1.07 pg), and the use of these as parents explains the low productivity of crosses made in 2016. Tetraploids that were identified by flow cytometry and by high fertility in 2016 crosses were used in intercrossing the following year

(2017). All crosses succeeded after triploid plants were identified using flow cytometry and eliminated from the crossing plan. Flow cytometry was an efficient and cost- effective way to determine ploidy level, if the appropriate tissue is used for the analysis.

Identification of F1 hybrids in the field based on phenotypic characteristics (leaf, flower and berry characteristics) was very efficient and accurate. Most F1 hybrids’ features (leaf, flower, and berry) were intermediate between highbush and V. elliottii.

However, it was not possible to differentiate between triploid and tetraploid F1’s using vegetative traits.

Pollen fertility (% fully formed tetrads) for the F1 hybrids was lower than for the parental types. This was true for the entire F1 population, including the tetraploid hybrids. Plants with high pollen fertility were classified as tetraploids with normal meiotic paring. Plants with low pollen fertility, which produced mostly aborted tetrads, were classified as triploids.

35 Crossing data from 2017 were the most relevant data for comparing fruit set and

PPF (Table 2-6). Fruit set and PPF were highly variable for the tetraploid F1 hybrids.

Approximately 75% of the F1 hybrid crosses were not significantly different from parental crosses with regards to fruit set and PPF. The remaining 25% were significantly lower in both fruit set and PPF from the parental types. This variability was attributed to variation in pollen fertility resulting from multivalent formation during meiosis and male-female interactions.

36 Table 2-1. Results from 14 tetraploid highbush x Vaccinium elliottii crosses done in the spring of 2014. All highbush flowers were pollinated with pollen bulked from diploid Vaccinium elliottii clonesz. Highbush parent Flowers Fruit set % Viable seeds Plump seeds pollinated per 100 flowers 05-603 682 100 16 2.34 Raven 115 100 1 0.87 O6-161 99 92 5 5.05 02-22 545 70 10 1.83 06-340 262 60 14 5.34 12-64 60 55 1 1.67 12-275 144 15 5 3.47 Primadonna 203 13 0 0.00 04-245 259 12 1 0.39 12-228 204 10 10 4.90 01-234 373 9 3 0.80 12-215 206 8 4 1.94 12-252 217 4 8 3.69 06-399 181 1 0 0.00 z Data from Lyrene, 2014.

37 Table 2-2. Results from the 2016 crosses including: intercrossing of F1 hybrids and b backcrossing of F1 hybrids to diploid VE (Vaccinium elliottii) and tetraploid VCc (Vaccinium corymbosum) varieties. Female Male Flowers (No.) Berries (No.) Fruit set (%) PPFa N-9d x 131163(VE) 190 0 0 0 N-6 x 131163(VE) 97 0 0 0 N-12 x 131163(VE) 223 0 0 0 N-22 x 131163(VE) 43 0 0 0 N-23 x 131163(VE) 121 0 0 0 N-8 x 131163(VE) 169 0 0 0 N-5 x 131163(VE) 20 0 0 0 N-11 x 131163(VE) 42 0 0 0 N-29 x 131163(VE) 29 0 0 0 N-37 x 131163(VE) 22 0 0 0 N-3 x 131163(VE) 28 0 0 0 N-39 x 131163(VE) 15 0 0 0 N-15 x 131163(VE) 31 0 0 0 N-30 x 131163(VE) 55 0 0 0 N-13 x 131163(VE) 217 0 0 0 N-21 x 131163(VE) 79 0 0 0 N-35 x 131163(VE) 146 0 0 0 N-33 x 131163(VE) 68 0 0 0 N-38e x 11-67(VC) 150 146 97.3 29.2 N-7 x 14-28(VC) 81 20 24.7 1.9 N-36 x 14-15(VC) 54 22 40.7 14.8 N-2 x 15-155(VC) 67 19 28.4 2.14 N-24 x 15-108(VC) 100 0 0 0 N-1f x N-4 59 38 64.4 24 N-10 x N-4 42 33 78.6 29.8 N-4 x N-32 223 44 19.7 0.1 N-28 x N-4 185 67 36.2 15.3 a PPF = number of plump seeds per pollinated flower. b VE- 2n=2x=24, diploid Vaccinium elliottii clone. c VC- 2n=4x=48, tetraploid Vaccinium corymbosum selection clones. d Plants with % fully-formed tetrads < 15% were backcrossed to diploid V. elliottii. e Plants with high % fully-formed tetrads and had features similar to the highbush parent were backcrossed to V. corymbosum selections. f Plants with high % fully-formed tetrads and features indicative of being a hybrid were intercrossed among each other.

38 Table 2-3. Leaf, flower, and berry characteristics of three taxa: V. elliottii (VE), southern highbush (HB) and highbush cultivars x V. elliottii F1 hybrids (HB x VE). z Taxa VE HB F1(HBxVE) No. leaves PGx 1 1 2 No. genotypes 10 10 10 Leaf Length 2.6y (0.35) 5.9 (1.88) 4.4 (0.36) characteristics a* b c (cm) Width 1.1 (0.35) 3.6 (0.47) 2.3 (0.38) a b c No. flowers PG 10 10 10 No. genotypes 2 2 30 Corolla length 6.2 (0.57) 10.1 (0.82) 7.8 (0.84) a b c Flower Corolla diameter 3.3 (0.47) 6.8 (0.46) 5.7 (1.10) characteristics a b c (mm) Distal end of style to distal end of corollay 1.7 (0.54) 1.3 (.33) 0.97 (0.71) a b b Style length 4.9 (0.68) 8.3 (0.72) 7.50 (0.69) a b c No. berries PG 5 5 10 No. genotypes 10 10 38 Weight (g) 0.12 (0.04) 2.3 (0.43) 1.40 (0.72) a b c Berry Diameter (mm) 11.1 (1.40) 17.8 (1.53) 14.2 (3.20) Characteristics a b c Firmness (g/mm) 314.7 (29.9) 253.3 (55.5) 220.1 (58.1) a b c Brix 18.7 (1.10) 12.7 (1.30) 12.2 (2.40) a b c z F1 (HB x VE). HB= Highbush cultivars (Vaccinium corymbosum). VE= Vaccinium elliottii. y Means and (standard deviations). x PG= per genotype. *Similar letters within a row indicates means not significantly different, Tukey’s HSD test, α=0.05. y Positive values indicate the corolla is longer than the style.

39 Table 2-4. Comparative analysis of DNA content of etiolated tissue versus Non-etiolated tissue from V. elliottii (VE), highbush (HB) and highbush x V. elliottii F1 hybrids using flow cytometry. Selection number Etiolated tissue DNA content Non-etiolated tissue DNA (pg) content (pg) VE 0.59 0.62 HB 1.13 1.14 N-1 1.24 1.22 N-2 1.35 1.32 N-3 0.93 1.00 N-4 1.34 1.35 N-9 0.91 0.89 N-10 1.23 1.24 N-15 1.04 1.07 N-24 1.00 1.06 N-26 1.38 1.42 N-29 0.98 0.90

40 Table 2-5. Genome size estimation by flow cytometry of VC (Vaccinium corymbosum) x VE (Vaccinium elliottii) F1 hybrids utilizing Vigna radiata as a diploid standard with a genome size of 1.06 pg. Pollen size of VE selections was also used as a predictor of ploidy level. Pollen shed and % fully formed tetrads were used as indicators of fertility. Selection Flow cytometry Pollen sizea Fertility measurements Ploidy determination number (µm) technique Diploid 2C- Triploid 2C- Tetraploid 2C- Pollen % Fully- Flow cytometry Pollen value mean value mean value mean shed (0- formed size (pg) (pg) (pg) 9)b tetrads Diploid 0.61c - - - - - 2x - standard V. elliottii range 0.58-0.66 - - 48.2-53.9 8-9 80-90 2x 2x (2x) Triploid range - 0.89-1.07 - 49.3-52.4 0-9 0-40 3x NA V. corym. range - - 1.11-1.27 50.7-60 8-9 5-90 4x 4x (4x) N-3 - 0.93 - 51.1 9 0.1 3x NA N-5 - 1.01 - 49.8 5 5.0 3x 3x N-9 - 0.90 - 52.0 0 0.1 3x 3x N-11 - 1.06 - NA 1 0 3x NA N-12 - 0.92 - NA 1 0 3x NA N-13 - 1.02 - NA 5 0 3x NA N-14 - 1.03 - NA 0 0 3x NA N-15 - 1.04 - 52.4 1 1 3x 3x N-21 - 1.04 - 51.7 9 1 3x 3x N-22 - 0.95 - 49.3 8 10 3x 3x N-23 - 1.01 - 52.2 1 0.1 3x 3x N-24 - 1.06 - 50.9 2 0.01 3x 3x N-30 - 0.98 - NA 9 0 3x NA N-32 - 0.99 - 50.3 8 40 3x 3x N-33 - 1.04 - NA 1 0 3x NA N-39 - 0.96 - NA 1 0 3x NA N-1 - - 1.24 56.4 9 15 4x 4x N-2 - - 1.32 57.6 9 80 4x 4x N-4 - - 1.34 59.7 9 60 4x 4x N-10 - - 1.24 56.0 9 25 4x 4x N-19 - - 1.30 58.1 9 60 4x 4x N-25 - - 1.22 55.6 9 75 4x 4x N-28 - - 1.31 54.8 9 90 4x 4x

41 Table 2-5. Continued Selection Flow cytometry Pollen sizea Fertility measurements Ploidy determination number (µm) technique Diploid 2C- Triploid 2C- Tetraploid 2C- Pollen % Fully- Flow cytometry Pollen value mean value mean value mean shed (0- formed size (pg) (pg) (pg) 9)b tetrads N-36 - - 1.44 56.8 9 5 4x 4x N-37 - - 1.12 56.6 8 10 4x 4x N-40 - - 1.30 57.3 9 80 4x 4x N-60 - - 1.23 58.1 9 90 4x 4x N-63 - - 1.19 57.8 9 90 4x 4x N-64 - - 1.18 59.0 9 90 4x 4x a Fully formed tetrads were measured at longest point. b A scale ranging from 0-9, with 0= none 1= very low, 2= low, 3= high low, 4= low medium, 5= medium, 6= high medium, 7= low high, 8= high, 9= very high. c Mean DNA content(pg) of 10 Vigna radiata diploid standards.

42 Table 2-6. Results from the 2017 crosses, including: Intercrosses of F1 hybrids to obtain F2 populations and backcrosses to VCb (Vaccinium corymbosum) varieties. Female Male Flowers (No.) Berries Fruit set (%) PPFa (No.) N-1 x N-10 132 65 49.2 6 N-2 x N-28 234 232 99.2 63 N-4 x N-60 331 233 70.4 10 N-10 x N-63 250 229 91.6 12 N-19 x N-2 75 33 44.0 1 N-28 x N-2 254 244 96.1 21 N-36 x N-1 34 22 64.7 2 N-37 x N-4 173 66 38.2 0.9 N-40 x N-36 41 18 43.9 12.9 N-60 x N-4 11 7 63.6 20 N-63 x N-10 99 64 64.6 25.6 N-64 x N-4 10 4 40.0 25.4 N-30 x 13-168(VC) 100 99 99.0 0.1 N-32 x 11-199(VC) 337 55 16.3 0 N-21 x 13-121(VC) 115 92 80.0 0 N-22 x 13-161(VC) 258 29 11.2 0 N-64 x 05-603(VC) 27 12 44.4 6.17 05-603(VC) x N-64 143 117 81.8 16.4 a PPF = number of plump seeds per pollinated flower. b VC = 2n=4x=48, Vaccinium corymbosum selections.

Table 2-7. Comparative success of Highbush x Highbush, Vaccinium elliottii x Vaccinium elliottii, and Highbush x Vaccinium elliottii crosses 2014z. Type of Number of Flowers Berries Number of Fruit PPFa Cross Pollinated Harvested Seeds set % HB x HB 349 342 6720 98 19.6 VE x VE 380 277 8000 73 28.9 HB x VE 4301 1677 78 39 0.05 a PPF = number of plump seeds per pollinated flower. z Data from Lyrene, 2014.

43 A

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Figure 2-2. Principal component analysis that explains 100% of the variation in leaf length and width from V. elliottii, F1 hybrids and highbush leaves.

44

Figure 2-3. Principal component analysis and K-means clustering that explains 60% of the variation, with K=3, for fruit traits from V. elliottii, F1 hybrids and highbush blueberries.

45 VE F1 HB VE F1 HB

VE F1 HB

Figure 2-4. Typical flowers of diploid V. elliottii, F1 hybrids and a tetraploid highbush cultivar. Photo courtesy of author.

A) B)

Figure 2-5. Meiotic analysis at metaphase I of F1 (Highbush x V. elliottii) hybrids. A) Tetraploid N-37, metaphase I, 24II. B) Triploid N-32, Metaphase I, 12II+12I. 1000x. Photo courtesy of author.

46 CHAPTER 3 PLOIDY LEVEL DETERMINATION IN COLCHICINE-TREATED VACCINIUM ELLIOTII SEEDLINGS AND SUBSEQUENT INTERSPECIFIC HYBRIDIZATION BETWEEN HIGHBUSH BLUEBERRY CULTIVARS AND AUTOTETRAPLOID V. ELLIOTTII

Vaccinium section Cyanococcus contains multiple species at three ploidy levels.

There are nine diploid (2n=2x=24), twelve tetraploid (2n=4x=48), and three hexaploid

(2n=6x=72) species (Camp, 1944; Darrow et al., 1944). Useful traits exist at all three ploidy levels, and certain traits are confined within different ploidy levels (Moore, 1965).

Transferring genes across ploidy levels is difficult. Currently, cultivated blueberries only exist at the tetraploid and hexaploid levels. Diploid species have not been domesticated.

Vaccinium elliottii is a wild diploid species native to the southeast United States.

Its potentially useful traits include low-chilling requirement, adaptation to upland sandy soils, disease resistance, an upright growth habit, and early ripening. Its berries are very small but have small seeds and good flavor. There have been major efforts in the past to transfer desirable traits from V. elliottii into southern highbush cultivars

(Ballington, 1990, 2001; Lyrene, 1997). A few seedlings were obtained from crosses between tetraploid highbush cultivars and diploid V. elliottii (Lyrene and Sherman,

1980). Several early ripening backcross selections were made from this population, one of which became the cultivar ‘Snowchaser’. However, V. elliottii was largely abandoned as a parent due to lack of fruit quality and size in backcross generations and disease issues (Ballington 2001; Lyrene 1997).

Chromosome doubling of diploid plant species has been used to overcome ploidy barriers. Blueberry autotetraploids showed changes in growth habit and fertility (Munoz and Lyrene, 1985; Dweikat and Lyrene, 1991). Production of autotetraploid V. elliottii plants has been previously accomplished by colchicine treatment of seed

47 (Chandler, 1980) and by in vitro colchicine treatments (Dweikat and Lyrene,

1991; Lyrene and Perry, 1992). Only a very few tetraploid V. elliottii genotypes have previously been produced and these are no longer available. Tetraploid V. elliottii plants produced using colchicine have been crossed with highbush cultivars, producing fertile offspring.

The purpose of this study was to develop a protocol for creating and identifying tetraploid V. elliottii plants using colchicine treatments, to produce a larger number of interspecific hybrids between V. elliottii and tetraploid highbush cultivars, and to study the characteristics of these hybrids.

Materials and Methods

Materials

Two sets of V. elliottii seedlings were used in this study. Open-pollinated seeds were collected by Lyrene (2014), from a large population of V. elliottii plants growing in forests near the Blackwater River in Santa Rosa County in the western Florida panhandle and along Perone Creek in Baldwin County in southwestern Alabama. The seeds were planted on the surface of peat moss in a greenhouse in November 2014.

When the seedlings were about 1cm tall, they were dug and the peat was washed from the roots. The seedlings were then submerged in a flask of 0.2% aqueous colchicine

(Colchicine, 97%, ACROS Organics™) for approximately 72 hours. The flask was kept on a lightly-shaded bench in the greenhouse during treatment. Treated seedlings were rinsed in water and transplanted to a tray of Canadian peat in a greenhouse where they were fertilized and watered for 6 months. In June 2015, seedlings that survived were transplanted to a high-density nursery at the University of Florida’s Research and

48 Education Center in Citra, FL. This population grew in the field for 1 year before being analyzed. Between January and May 2016, these plants were examined for morphological features that might indicate an increase in ploidy level, such as larger leaves, flowers, pollen and stomata (Figure 3-1). Forty-three plants were selected for flow cytometry study because they had one or more features that indicated possible tetraploidy. Plants that showed no morphological changes were discarded.

The second set of plants was generated in 2016 by soaking 400 seedlings in flasks of 0.2% aqueous colchicine (Colchicine, 97%, ACROS Organics™) for approximately 48 hours at 22°C. Treated seedlings were rinsed twice with water and transplanted to trays of Canadian peat in a greenhouse, where they were fertilized and watered for 6 months. In June 2016, seedlings that survived were transplanted into a high-density nursery at the University of Florida’s Research and Education Center in

Citra, FL. This population grew in the field for 2 months before being analyzed. In

August 2016, these plants were examined for morphological features that might indicate an increase in ploidy level such as larger leaves and stomata. Thirty-seven plants were selected for flow cytometry study because they had one or more features that indicated possible tetraploidy. Plants that showed no morphological changes were left in the nursery.

Flow Cytometry Analyses

Tissue for flow cytometry analysis from both populations of colchicine-treated plants consisted of tender new growth harvested from plants in the field and placed between wet napkins until it was processed. Flow cytometry analysis was done using the BD Accuri™ C6 machine at the University of Florida’s ICBR (Interdisciplinary Center for Biotechnology Research), Gainesville, FL. Tissue was prepared using the Sysmex

49 CyStain® PI Absolute P kit. Prior to tissue preparation, RNAseA stock solution and staining solution were prepared. RNAseA stock solution was prepared by adding 1.5 ml

H2O to 1 tube of RNAse A. The stock solution was stored at -20°C. The staining solution was prepared by combining 20 ml staining buffer, 120 μL propidium iodide, and 60 μL

RNAseA stock solution. The staining solution was stored at 4°C in bottles wrapped in aluminum foil to exclude light until it was used. Five hundred mg of tissue was harvested from each plant. Within 8 hours, the tissue was chopped for 60 seconds in

500 μL of nuclei-extraction buffer on a 7.5 cm petri dish using a microtome blade. The solution was filtered through 50μm mesh into a 5ml glass tube. 2 ml of staining solution was added to each 5 ml tube, and the tube was immediately placed on ice in the dark to incubate until being analyzed.

The equipment settings were held constant for all samples. Each sample ran

10,000 events at a medium flow rate of 35 μl/min and a threshold of 1,000 on FL2-H.

The Gate was set according to each sample. Samples were vortexed before being analyzed. To determine the ploidy of each sample, a V. elliottii nuclei solution was prepared and used as an internal diploid standard. The diploid standard was run first and then the unknown samples were run individually. Since, a tetraploid was expected to have double the DNA content of a diploid, and the diploid RFI peak was known, any peak from an unknown sample that had a an RFI peak twice as big as the diploid standard was considered a potential tetraploid. Cytochimeras within this population showed 3 peaks, one at the diploid G1 phase, one that combined the diploid G2 phase and the tetraploid G1 phase, and one at the tetraploid G2 phase of the cell cycle (Table

3-1; Table 3-2).

50 Stomata Analyses

Stomata size was measured by harvesting a single mature leaf per plant. The leaf was removed and the abaxial surface was immediately painted with clear nail polish. The leaves were left at 22°C until the nail polish was completely dry. The nail polish was then removed, placed in a drop of water on a microscope slide and covered with a cover slip. To view the stomata, a microscope (Leitz Wetzlar Model 664012,

Wetzlar, Germany) was used. Samples were observed at 400x. Stomata were measured with an internal scale built into the occular. Measurements from the internal scale were converted to metric size using a 2 mm external scale. Only open stomata were measured. Measuring only the length of the aperture hole between the guard cells standardized the measurements across samples (Table 3-1; Table 3-2). Ten randomly selected stomata were measured from each leaf, avoiding stomata around the main leaf veins.

Pollen Analyses

Plants with indications of both 2x and 4x peaks from flow were further examined to identify and delineate the 4x sectors of the plant. Chimeric plants were dug and moved into a bee-proof greenhouse. When the chimeras flowered, the first flower from the tetraploid sector was used for pollen size measurements. In a sectorial chimera, the various limbs could differ in ploidy, so limbs that had the largest flowers were tagged and flowers were taken from those limbs for pollen studies. Multiple flowers were examined per plant. Open flowers were harvested and the pollen was shed into a drop of 45% acetic acid on a microscope slide for evaluation. Pollen was observed at 250x magnification using a microscope (Leitz Wetzlar Model 664012, Wetzlar, Germany).

Pollen tetrad size was recorded (Table 3-1; Table 3-2). The branches on sectors of the

51 plant that produced diploid-size pollen were removed to enhance growth on branches that produced large pollen and were presumably tetraploid.

Interspecific Hybridization of Colchicine-Derived Tetraploid Vaccinium elliottii with Tetraploid Highbush Blueberry Cultivars

Nine plants whose flow cytometry results identified them to be cytochimeras (2x and 4x) or as complete tetraploids (4x only) were dug from the field nursery, potted into pots of Canadian peat, and placed in a bee-proof greenhouse for flowering. Tetraploid highbush selections that had been accumulating chilling in a cooler were taken out and placed in the same greenhouse. When the highbush clones and the colchicine-treated

V. elliottii selections began to flower, cross-pollinations were begun. Pollinations were done by removing the corolla and emasculating the flower, then shedding pollen from the selected pollen parent onto the fingernail and applying it to the stigma of the emasculated flower. The highbush selections were used as female parents and were pollinated by pollen from 4x V. elliottii (Table 3-3).

Results and Discussion

Colchicine Treatment

Morphological abnormalities were observed in seedlings treated with colchicine.

Visual identification of morphological changes allowed for early selection and prescreening of putative tetraploid V. elliottii plants. These characteristics included an increase in leaf size, broadening of leaves and rounding of leaf apices, abnormal shoots, unusual growth habit, and reduction or loss of apical dominance (Chavez and

Lyrene, 2009). However, visual identification of stable tetraploid V. elliottii plants was difficult. Vegetative characteristics of young blueberry seedlings are quite variable, and differences in diploid and tetraploid seedlings were not clear-cut, especially when the

52 seedlings are transitioning from juvenile to adult morphology. Furthermore, ploidy increases are often confined to certain tissue layers and sections within the same plant, resulting in cytochimeras. Cytochimeras have been previously described following colchicine treatment of blueberries (Chavez and Lyrene, 2009). A cytochimera may or may not be useful in breeding depending on which type of chimera is present. Sectorial chimeras, in plants, are unstable and are distinguished by complete limbs, or sectors of a plant that differ from the rest. Periclinal chimeras are more stable and characterized by all branches in the periclinal sector of the plant being affected (Satina et al., 1940).

Periclinal chimeras in which the L2 layer is tetraploid are the only ones that are expected to cross readily with tetraploid cultivars, because this is the tissue layer that gives rise to the reproductive organs. Eight of the nine tetraploid plants found by flow appeared to be chimeric. These plants required pruning to remove the diploid sectiors and to promote the growth of the tetraploid sectors.

Flow Cytometry Data

Plants that were phenotypically different from the rest of the population were selected as candidates for flow cytometry. Of the original 400 treated seedlings, 37 were visually selected for flow cytometry (Table 3-1; Table 3-2). The diploid range for

DNA content was 0.58-0.66 pg and the flow cytometry results indicated that 28 plants were diploids, indicating no colchicine effect. The tetraploid range for DNA content was

1.11-1.27 pg, and 9 plants had genome size estimations in or near this range. Variation in genome size estimates in this population was attributed to the effects of colchicine, and to variation in genome size before colchicine treatment among the diploid plants in the original population. Costich et al. (1993) using flow cytometry described the variation in the genome size of different blueberry species. However, his results showed that all

53 Vaccinium species, even different ploidy levels, had the same size genomes. In reality, diploid species are expected to have half the genome size of a tetraploid and hexaploids three times the genome size of diploids. Other sources of variation in the flow cytometry process include laser drift during analysis and sample processing over multiple days (Dolezel et al., 1998). The presence of three peaks in the flow graphs are indicative of multiple ploidy levels within the tissue layers of the plant, and therefore cytochimeras. Samples with only two peaks indicated nonchimeral diploid or tetraploid shoots. Solid tetraploid plants and periclinal cytochimeras with tetraploid LII tissue layers would be the most desirable, because the gametes are produced from this layer, and periclinal chimeras are more stable. However, most plants screened appeared to be cytochimeras, with sections of the plant being diploid and other sections being chimeric diploid-tetraploid.

Stomata and Pollen Size Data

Stomata size was in almost perfect agreement with flow cytometry data across the two populations (Table 3-1; Table 3-2). The range for diploid stomata size for population one was 11.9-15.8 μm. No chimeric or full tetraploid plants were found in population one; all screened plants were diploid. The range for diploid stomata size in population two was 12.1-15.6 μm. The tetraploid range for stomata size in population two was 17.2-22.4 μm. Eight plants were identified as tetraploid in population two form stomata data. A few colchicine-treated plants fell outside the upper limit of the range for the non-treated diploid V. elliottii stomata size (12.2-15.5 μm), but these were interpreted as diploid since they were closer to the diploid size than the tetraploid size

(18.7-22.1 μm). The small amount of variation observed in the stomata size of colchicine-treated plants was attributed to plant-to-plant variation. In previous studies of

54 Vaccinium and Alocasia species, there was also strong correlation between flow data and stomata data as a predictor of ploidy in colchicine treated plants (Tsuda et al. 2013;

Thao et al., 2003; Chandler, 1980).

Pollen size was not as indicative of ploidy level in these two populations when compared with flow and stomata data. There was significant variation in pollen tetrad size among plants in the second population (diploid range: 46.5-56.6 μm), but not so much in the first population (diploid range: 47.1-55.2 μm). There was a lot of overlap between the range for diploid V. elliottii (48.2-53.9 μm) and tetraploid V. corymbosum

(50.7-60 μm) for pollen size, thus confounding some of the ploidy predictions based solely on pollen size. Although, pollen size was in agreement with tetraploids confirmed by flow cytometry (tetraploid range: 52.8-65.4 μm), the variation prevented the use of pollen size as the sole character for accurate prediction of ploidy.

In this study, diploid V. elliottii showed much variation in pollen size. Previous studies with V. elliottii did not find such variation (Dweikat and Lyrene, 1991). However, the previous study cited involved only one clone of V. elliottii. Our study compared pollen size of 10 different V. elliottii genotypes, and the colchicine-treated populations were seedlings from similar genotypes.

Interspecific Hybridization of Colchicine-Derived Autotetraploid Vaccinium elliottii with Tetraploid Highbush Cultivars

The eight confirmed V. elliottii tetraploids were subsequently crossed as males with multiple southern highbush cultivars. All but one cross produced plump seeds.

Among the tetraploid V. elliottii clones, FL 16-791 and FL 16-802 performed best, producing a PPF (number of plump seeds per pollinated flower) above 1 for every cross. FL 16-791 was crossed to five different southern highbush cultivars, with a

55 highest PPF of 5.71. FL 16-802 was crossed to only one southern highbush cultivar but produced a PPF of 8.60 (Table 3-3). Some V. elliottii plants were crossed more than once based on availability of female V. corymbosum plants for use as female parents.

Tetraploid V. elliottii plants that were identified later in the crossing season were used in fewer crosses, because few highbush plants were still available when they were identified. In general, the number of plump seeds per pollinated flower was much lower than would be expected after pollinating highbush cultivars from pollen of a tetraploid species from section Cyanococcus. Of the 8 putative tetraploid V. elliottii plants that were used as pollen parents, only 3 gave an average of 1.0 or more good seeds per pollinated flower, and three averaged fewer than 0.2 seeds per pollinated flower. By contrast, crosses between two highbush cultivars normally give 10 to 30 good seeds per pollinated flower. It is not known why seed numbers were so low in these crosses.

Crossing barriers may exist between V. elliottii and highbush cultivars additional to the ploidy-level difference, but this seems unlikely based on previous experience. It may be that the autotetraploid V. elliottii is not very fertile, even when pollen looks good under the microscope. It is also likely that some highbush flowers were pollinated using pollen from V. elliottii branches that contained diploid LII layers rather than tetraploid. Several of the V. elliottii plants used in crosses appeared to have both diploid and tetraploid branches. Because the V. elliottii plants were young and most of them did not produce many flowers, flowers from ambiguous branches were sometimes used along with pollen from known tetraploid branches. Whatever the explanation for the low yield of hybrid seed, the amount of F1 hybrid seed obtained from these crosses was sufficient to allow further breeding. Since valuable traits exist at all ploidy levels within the genus

56 Vaccinium, overcoming the ploidy barrier with colchiploids provides a convenient way to transfer traits across ploidy level with minimal difficulty (Lyrene and Sherman, 1983).

Conclusions

Combining multiple methods (flow cytometry, stomata size, and pollen size), nine colchicine-treated V. elliottii clones were selected as tetraploids or diploid/tetraploid chimeras, from an original population of 400 treated seedlings. The tetraploid V. elliottii genome size was highly variable, with some clones having genome sizes larger than tetraploid V. corymbosum, others smaller. Variation was attributed to variation in the genome size of diploid V. elliottii genotypes.

Eight of the nine V. elliottii clones found to be tetraploid by flow cytometry were cytochimeras. Using a leaf for stomata size measurements from the same stem that provided the tissue for flow cytometry allowed determination that these eight plants were chimeras with tetraploid and diploid sectors. Using these two methods together was more informative than relying solely on pollen size data, which were highly variable.

57 Table 3-1. Population one, genome size estimation by flow cytometry of colchicine-treated V. elliottii clones utilizing Vigna radiata as a diploid standard with a genome size of 1.06 pg. Stomata size and pollen size were also used as predictors of ploidy level. Selection number Flow Cytometry Stomata Size (μm) Pollen Tetrad Size Ploidy Estimation (μm) Diploid 2C-value Tetraploid 2C-value mean (pg) mean (pg) Diploid standard 0.61a - - - 2x V. elliottii range (2x)b 0.58-0.66 - 12.2-15.5 48.2-53.9 2x V. corymbosum range - 1.11-1.27 18.7-22.1 50.7-60 4x (4x)c C-1 0.61 - 14.1 50.2 2x C-2 0.66 - 12.0 53.1 2x C-3 0.56 - 15.5 54.2 2x C-4 0.53 - 13.6 53.7 2x C-5 0.62 - 17.8 49.5 2x C-6 0.61 - 16.3 51.3 2x C-7 0.67 - 14.3 53.2 2x C-8 0.58 - 13.2 50.1 2x C-9 0.63 - 15.0 50.4 2x C-10 0.57 - 11.9 49.3 2x C-11 0.67 - 14.0 48.1 2x C-12 0.68 - 13.5 53.2 2x C-13 0.48 - 12.5 54.1 2x C-14 0.46 - 15.2 50.8 2x C-15 0.53 - 15.1 51.6 2x C-16 0.48 - 13.4 53.3 2x C-17 0.67 - 14.1 49.0 2x C-18 0.63 - 14.6 51.5 2x C-19 0.49 - 12.8 47.1 2x C-20 0.68 - 13.5 48.9 2x C-21 0.50 - 15.0 49.2 2x C-22 0.48 - 14.2 50.7 2x C-23 0.66 - 14.8 55.2 2x C-24 0.55 - 13.3 53.1 2x C-25 0.57 - 13.9 50.9 2x

58 Table 3-1. Continued Selection number Flow Cytometry Stomata Size (μm) Pollen Tetrad Size Ploidy Estimation (μm) Diploid 2C-value Tetraploid 2C-value mean (pg) mean (pg) C-26 0.62 - 14.9 48.3 2x C-27 0.61 - 12.8 49.6 2x C-28 0.58 - 15.1 50.8 2x C-29 0.59 - 14.1 49.0 2x C-30 0.63 - 13.2 52.5 2x C-31 0.54 - 15.4 50.5 2x C-32 0.64 - 14.5 48.6 2x C-33 0.67 - 14.8 49.9 2x C-34 0.58 - 13.5 54.5 2x C-35 0.61 - 13.8 51.2 2x C-36 0.66 - 12.7 51.6 2x C-37 0.65 - 13.0 53.4 2x C-38 0.59 - 15.8 49.8 2x C-39 0.64 - 12.2 50.1 2x C-40 0.61 - 15.3 50.3 2x C-41 0.56 - 14.5 48.9 2x C-42 0.59 - 13.6 52.2 2x C-43 0.63 - 12.3 48.3 2x a Mean DNA content(pg) of 10 Vigna radiata diploid standards. b Range for 10 randomly sampled diploid V. elliottii plants that were not colchicine-treated. c Range for 10 randomly sampled tetraploid highbush cultivars.

59 Table 3-2. Population two, genome size estimation by flow cytometry of colchicine-treated V. elliottii clones utilizing Vigna radiata as a diploid standard with a genome size of 1.06 pg. Stomata size and pollen size were also used as predictors of ploidy level. Selection number Flow Cytometry Stomata Size (μm) Pollen Tetrad Size Ploidy Estimation (μm) Diploid 2C-value Tetraploid 2C-value mean (pg) mean (pg) Diploid standard 0.61a - - - 2x V. elliottii range (2x)b 0.58-0.66 - 12.2-15.5 48.2-53.9 2x V. corymbosum range - 1.11-1.27 18.7-22.1 50.7-60 4x (4x)c C-200 0.58 - 14.3 49.1 2x C-201 0.68 - 15.4 51.7 2x C-202 0.64 - 14.7 56.6 2x C-203 0.66 - 14.5 54.3 2x C-204 0.62 - 12.8 50.1 2x C-205 0.64 - 14.2 53.0 2x C-206 0.56 - 13.9 46.5 2x C-207 0.40 - 15.1 47.8 2x C-208 0.59 - 12.1 50.5 2x C-209 0.60 - 12.6 48.0 2x C-210 0.64 - 14.4 54.1 2x C-211 0.61 - 15.3 52.2 2x C-212 0.47 - 15.2 49.7 2x C-213 0.64 - 14.8 47.3 2x C-214 0.65 - 13.1 51.6 2x C-215 0.52 - 14.5 50.5 2x C-216 0.44b 0.90 17.2 59.2 2x, 4x C-217 0.65 1.31 20.1 62.5 2x, 4x C-218 0.62 - 15.6 51.0 2x C-219 0.64 - 13.0 47.9 2x C-220 0.57 - 14.8 48.2 2x C-221 0.60 1.20 19.5 60.1 2x, 4x C-222 0.68 1.37 20.7 52.8 2x, 4x C-223 0.67 - 15.4 50.6 2x C-224 0.63 1.24 21.3 59.7 2x, 4x

60 Table 3-2. Continued Selection number Flow Cytometry Stomata Size (μm) Pollen Tetrad Size Ploidy Estimation (μm) Diploid 2C-value Tetraploid 2C-value mean (pg) mean (pg) C-225 0.66 1.34 22.4 58.3 2x, 4x C-226 0.50 - 13.7 51.1 2x C-227 - 1.16 20.6 53.6 4x C-228 0.63 - 14.1 53.2 2x C-229 0.62 - 14.9 48.1 2x C-230 0.67 - 15.2 51.6 2x C-231 0.63 - 12.5 51.4 2x C-232 0.57 - 13.6 50.3 2x C-233 0.63 - 13.2 48.9 2x C-234 0.37 - 14.5 54.4 2x C-235 0.53 - 15.1 50.2 2x C-236 0.57 1.15 20.8 65.4 2x, 4x C-237 0.48 0.97 17.9 61.0 2x, 4x a Mean DNA content(pg) of 10 Vigna radiata diploid standards. b Range for 10 randomly sampled diploid V. elliottii plants that were not colchicine-treated. c Range for 10 randomly sampled tetraploid highbush cultivars.

61 Table 3-3. Results of crosses between southern highbush cultivars and tetraploid colchicine-treated V. elliottii during winter and spring 2017. Female Male Flowers (No.) Berries (No.) Fruit set (%) PPFa Highbush V. elliottii 12-213 16-791b 117 66 56.41 3.79 (C-217) 05-603 16-791 209 217 103.82 1.28 Indigocrisp 16-791 113 70 61.95 5.71 12-179 16-791 46 47 102.17 1.74 12-8 16-791 68 67 98.53 2.00 12-8 16-793 22 26 118.18 0.96 (C-221) 13-123 16-793 18 45 250.00 0.22 07-186 16-799 107 126 117.76 0.21 (C-222) 12-76 16-799 131 40 30.53 1.20 Indigocrisp 16-799 14 0 0 0 11-184 16-800 231 370 160.17 0.008 (C-224) Indigocrisp 16-800 155 37 23.87 0.14 12-76 16-801 18 6 33.33 1.67 (C-225) 12-213 16-801 10 2 20.00 2.50 12-179 16-801 38 9 23.68 0.22 Indigocrisp 16-802 162 38 23.46 8.60 (C-227) 05-603 16-803 52 55 100.58 0.21 (C-236) Indigocrisp 16-804 26 20 76.92 0.05 (C-237) a PPF = number of plump seeds per pollinated flower. b Plants were renumbered for record keeping.

4x 2x 4x 2x

Figure 3-1. Pictures showing flower size variation between autotetraploid and diploid V. elliottii. Photo courtesy of author.

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65 BIOGRAPHICAL SKETCH

Elliot Norden, born in Gainesville, Florida, in 1992, is the middle child of David and Claudia Norden. His father taught him the importance of the little things in life, and to immerse himself in nature whenever possible. Even though he was surrounded by agriculture and the outdoors his whole adolescent life, he was still unsure of his studies after graduating high school.

Although Elliot never had the chance to get to know his grandfather, Alan

Norden’s great success in breeding peanuts inspired Elliot to study plant breeding and genetics. Elliot studied agriculture at Santa Fe community college before being accepted to the University of Florida’s College of Agricultural and Life Sciences (CALS).

It was here Elliot learned the fundamentals of plant physiology, biology, genetics and plant breeding, and in 2 years earned his Bachelor of Science in horticultural sciences.

During his time at the University of Florida, Elliot worked as a research assistant for the blueberry-breeding program and learned how much work goes into operating and maintaining a breeding program. He also had the chance to work closely with his father, David Norden, and his mentor, Dr. Paul Lyrene who together opened his eyes to the beauty of plant breeding. Elliot’s hard work and perseverance led him to being offered an assistantship with the blueberry-breeding program from Dr. Jim Olmstead, which he accepted.

Throughout his life, Elliot, met many people who changed the way he viewed things, but none as much as his father, mother, and mentors Dr. Paul Lyrene and Dr.

Jose Chaparro. Their continuous support and knowledge helped him through every step. Elliot’s new goal is to be a successful plant breeder as he enters into his new job.

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