University of Florida Thesis Or Dissertation Formatting

INTROGRESSION OF IMPORTANT ECONOMICAL FEATURES OF Vaccinium elliottii INTO SOUTHERN HIGHBUSH BLUEBERRY

DIEGO CABEZAS

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

2019

To my wife and children

ACKNOWLEDGMENTS

I thank my parents, brother and sisters for their continued support and love throughout my life, for always being there when I need them, for their example and great influence in my life. I thank my wife Constanza for her love, constant care, and patience during this path. I thank my mentor Dr. Paul Genho for turning the course of my career in a positive direction. I thank him for giving me the tools to become a better person and for showing me the beauty of plant breeding. I thank Dr. Patricio Munoz for his constant advice and patience. I will be always grateful for his example and care. I thank Dr. Paul Lyrene for his support, time and efforts to make this project possible, for dedicating the time to answer my questions. I thank Elliot Norden for preparing the materials, so I was able to undertake this study. I thank Dr. Ivone de Bem Oliveira for her friendship, constant support and advice from the beginning. I thank Ayron Utreras for his influence throughout my life and his cheerfulness. I thank the blueberry breeding family for their support and care through my master. Specially Lauren Scott and her team for the long hours they dedicated to this project. I express gratitude to my committee members, Dr. Jose Chaparro, Dr. Esteban Rios and Dr. Pam Soltis.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 6

LIST OF FIGURES ...... 7

ABSTRACT ...... 8

CHAPTER

1 INTRODUCTION ...... 10

2 EVALUATING CROSS DIRECTION FOR INTERSPECIFIC HYBRIDIZATION OF VACCINIUM ELLIOTTII AND SOUTHERN HIGHBUSH BLUEBERRY ...... 15

Background Information ...... 15 Materials and Methods...... 16 Results ...... 20 Discussion ...... 22

3 PHENOTYPIC DIFFERENCES BETWEEN SOUTHERN HIGHBUSH, VACCINIUM ELLIOTTII AND THEIR HYBRIDS ...... 32

Background Information ...... 32 Materials and Methods...... 33 Results ...... 37 Discussion ...... 40

4 SUMMARY AND CONCLUSIONS ...... 53

APPENDIX

CROSS DIRECTION EVALUATION FOR INTERSPECIFIC HYBRIDIZATION OF VACCINIUM ELLIOTTII AND SOUTHERN HIGHBUSH BLUEBERRY ...... 56

LIST OF REFERENCES ...... 57

BIOGRAPHICAL SKETCH ...... 64

LIST OF TABLES

Table page

2-1 Number of flowers pollinated…...... 27

2-2 Interspecific hybridization between SHB and V. elliottii ...... 28

3-1 Planting year, and years of evaluation for different hybrid family types...... 45

A-1 Crossability in interspecific hybridization between V. elliottii and SHB ...... 56

A-2 Seed germination in interspecific hybridization between V. elliottii and SHB ...... 56

LIST OF FIGURES

Figure page

2-1 Seedlings placed in a greenhouse...... 29

2-2 Fruit set for 10 reciprocal crosses between V. elliottii and V. corymbosum...... 30

2-3 Average seeds per berry...... 30

2-4 Number of seeds obtained per pollinated flower...... 31

2-5 Germination percentage...... 31

3-1 pH of families...... 46

3-2 Total Soluble Solids...... 46

3-3 Soluble solids by Total titratable acids...... 47

3-4 Fruit firmness...... 47

3-5 Fruit firmness by size...... 48

3-6 Fruit diameter...... 48

3-7 Total Titratable Acids...... 49

3-8 Total soluble solids by size...... 49

3-9 Fruit size and color...... 50

3-10 Plant height...... 50

3-11 Plant vigor...... 51

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

INTROGRESSION OF IMPORTANT ECONOMICAL FEATURES OF Vaccinium elliottii INTO SOUTHERN HIGHBUSH BLUEBERRY

Diego Cabezas

December 2019 Chair: Patricio Munoz Major: Horticultural Sciences

Vaccinium elliottii is a wild diploid blueberry species relative to the tetraploid cultivated, Vaccinium corymbosum. This wild species possesses important horticultural traits that can contribute to enhance fruit quality and adaptation to the Florida’s environment. The ultimate goal of this study was to generate information towards introgressing favorable traits from V. elliottii into southern highbush blueberry (SHB).

Two studies were performed, the first one consisting of evaluating the effect of cross direction on interspecific hybridizations between V. elliottii and SHB. For this, ten synthetic autotetraploid genotypes of V. elliottii derived from colchicine treatment were used as both male and female parents in distinct crosses with SHB. The results showed that when SHB was used as the female parent, significantly more berries were produced per pollinated flower compared to when V. elliottii was the female parent.

However, a significant decrease in the average number of seeds per berry was detected when SHB as used as female parent. Overall, it was found that V. elliottii can be successfully hybridized with cultivated SHB in both directions, and that direction of the cross had a small effect on number of seeds per pollinated flower or on germination percent. The second project aimed at evaluating different stages of breeding using V.

elliottii. For this, the performance of genotype crossing combinations was evaluated in terms of fruit quality traits as fruit size, firmness, acidity, brix⁰, weight, and color, considering different breeding generations (i.e., F1, F2, and BC1). The results showed an increase in berry size on F1, F2, and BC1 berries when compared with V. elliottii, while F1 and F2 showed a decrease in size when compared to SHB berries. The highest levels of soluble solids, an indicator of sugar content, were detected in V. elliottii when compared to SHB, F1, F2, and BC1. SHB and BC1 families planted in the 2017 nursery had significantly lower acidity compared with V. elliottii. No significant differences in firmness were found between hybrids, SHB, and V. elliottii. Overall, fruit quality in BC1 hybrids increased when V. elliottii was introgressed into SHB. Overall results confirm the potential of exploiting interspecific hybridization between SHB and synthetic tetraploid

V. elliottii in blueberry breeding. Genetic gains can be achieved independent of the direction of crossing. The evaluation of hybrid populations showed that fruit quality increased when V. elliottii is introgressed into SHB. In addition, a large amount of variability was obtained, generating material to be exploited by the University of Florida

Blueberry Breeding Program. Therefore, these results provide essential information towards the introgression of important traits from V. elliottii into SHB.

CHAPTER 1 INTRODUCTION

Blueberry is a perennial shrub grown mainly for its high-value edible fruit. The major producers in 2017 were United States, Chile and Canada, which combined account for 79.5% of global blueberry production. Europe, Asia, and Oceania also produced blueberries, comprising 18.7%, 0.3% and 1.4% of total production, respectively (FAO, 2018).

Several Vaccinium species in North America are grouped under the common name “blueberry” in the commercial market: V. corymbosum (highbush blueberry), V. angustifolium (lowbush blueberry), V. virgatum (rabbiteye blueberry). Differing taxonomic classifications of blueberries have been proposed by different taxonomists

(Vander Kloet, 1980; Poster et al., 2017). Classifying blueberries is a challenging task, given the existence of multiple ploidy series in the genus (e.g., 2n= 2x, 4x, 6x) and the occurrence of natural interspecific hybrids (Lyrene and Vorsa, 2003). It has been assessed that there are between 150-450 species of Vaccinium around the world, classified within 30 sections. Cyanococcus is the largest section, comprising 20 species.

Important species in this section include the three cultivated species mentioned earlier

V. elliottii (Elliott’s blueberry) and V. darrowii (Darrow’s or Evergreen blueberry)

(Hancock et al., 2008).

Wild species of section Cyanococcus are considered a valuable source of variability and novel traits for blueberry breeding programs (Ballington, 1980, 2001;

Darrow and Camp, 1945; Lyrene, 2011; Rousi, 1963). A successful example of the role of wild species in blueberry breeding was the development of southern highbush blueberry (SHB) cultivars (Lyrene, 2007).

Florida blueberry cultivation started in 1893 and expanded during the 1920s, being composed mostly of rabbiteye blueberries and restricted to north Florida (Vander

Kloet, 1988). However, factors such as lack of handling and marketing expertise contributed to the collapse of the first Florida blueberry industry. Although rabbiteye blueberries usually grow well in Florida soils and environment, they have large variation in fruit quality, time of ripening, and productivity (Lyrene and Sherman, 1984; Coville,

1937; Vander Kloet, 1988). Rabbiteye blueberries ripened mainly from June to August, when prices were low and harvest weather is hot and rainy. Professor Ralph Sharpe saw the potential of earlier-ripening blueberries in Florida and anticipated a market benefit of elevated prices for early season blueberries. He started the University of

Florida Breeding program in 1949 with the goal of developing early ripening tetraploid highbush cultivars that were adapted to the Florida environment (Lyrene, 1997).

Southern highbush blueberry cultivars are the result of breeding efforts to introgress traits from wild species to suit the low number of chilling hours of Florida.

Hybridization of the Northern highbush blueberry (V. corymbosum L.) with several native species found in the southern regions of the U.S. (Lyrene, 1990) made it possible to obtain cultivars with fewer chilling hour requirements, early ripening berries, high fruit quality and productivity (Galletta, 1975). The three main species that were used to create the SHB germplasm were Northern highbush blueberry, Darrow's blueberry, and rabbiteye blueberry (Sharpe, 1954). Several inter-ploidy Vaccinium hybrids have been produced by overcoming the ploidy barrier using different techniques (Chavez, 2009;

Ballington, 2001). One of these techniques, the use of colchicine, allowed breeders to circumvent the triploid block and generate tetraploid hybrids using diploid species.

Colchicine works by suppressing the formation of spindle fibers during mitosis.

Colchicine interrupts microtubule formation and interferes with chromosome movement during anaphase (Hadlaczky et al., 1983; Blakeslee, 1937). It is expected that the used of colchicine to double the ploidy of V. elliottii would generate morphological variation among natural diploid genotypes. This effect is commonly known as the “gigas effect”.

This effect explains larger organs that polyploid plants present when compared to their diploid relatives (Stebbins, 1971). This phenomenon has been previously identified in polyploidization of V. elliottii by Dweikat (1991).

The development of SHB cultivars, allied with the increase in blueberry consumption worldwide, allowed Florida growers to increase acreage, productivity and profitability (Brazelton, 2013). Between 2007 and 2012, Florida blueberry acreage increased by 73% and production increased by 132% (Williamson et al., 2014). Due to the early ripeness of SHB (March, April, and May), Florida growers were also able to target a very lucrative marketing window when no other U.S. region was producing blueberries.

Considering the benefits that interspecific hybridization can bring to blueberry breeding, it would be useful to assess the potential contribution that other species could make (Lyrene, 1997). In the southern U.S., there are ten native Vaccinium species of section Cyanococcus (Retamales and Hancock, 2012). One of these, V. elliottii, is a diploid deciduous wild blueberry that can be found from Northern Florida to southeast

Virginia, and as far west as eastern Texas. V. elliottii flowers from February to March, and produces numerous small, dark, non-glaucous berries with a distinctive flavor. The berries ripen from late April to early May in north Florida. V. elliottii also has a desirable

growth habit (upright without excessive suckering) and it is adapted to a more upland type of soil compared to SHB cultivars (Lyrene and Sherman, 1980).

The interspecific hybridization of SHB with V. elliottii could bring important traits into blueberry breeding programs, such as short bloom to ripe period, distinctive aroma, and better flavor (Lyrene and Sherman, 1984). Moreover, adaptation to sandy soils could improve blueberry plant growth in Florida and decrease the need for pine bark inputs during planting (Lyrene, 1997), which is a significant cost for producers.

V. elliottii is a diploid species (2n=2x=24) but doubling the chromosome number by colchicine treatment was successful on different V. elliottii seedlings. This facilitated hybridization with tetraploid SHB, and different hybrid populations have been generated

(Dweikat and Lyrene, 1991; Norden, 2017). However, the effect of cross direction during the hybridizations remains unexplored. Comparing the phenotypic differences among reciprocal crosses can guide breeding decisions and provide a better understanding of maternal effects on hybrids. Maternal effects are the causal effect of the genetic contribution of the female parent on the offspring phenotype (Wolf and Wade, 2009).

The genetic contribution of the female parent goes beyond the one made by the paternal parent. This can be due to mitochondria and chloroplast inheritance, retention of epigenetic markers, genomic imprinting, delivery of messenger RNA or proteins into the egg (Lynch and Walsh, 1998). The potential contribution that maternal effects could have to the phenotype of an individual is the main rationale to evaluate the effects and efficiency of cross direction, which is one of the objectives of this project.

Hybridization between two different species has advantageous and disadvantageous consequences, therefore it is important to analyze the progenies and

select materials to advance to the next breeding cycles. Crossing divergent parental lines, such as different species, is expected to result in extremely heterozygous progenies compared with their parents, which may result in heterosis (hybrid vigor).

Heterosis is a phenomenon whereby hybrid offspring show increased vigor relative to their parents probably due to heterozygote advantage and/or by concealing recessive mutations with detrimental effects (Hegarty and Hiscock 2005; Whitney et al. 2010). On the other hand, hybridization could result in the interruption of favorable allele and gene interactions causing a deleterious effect on the offspring hybrids (Arnold, 1997; Hegarty et al., 2008). In the additive genetic model, both parents are expected to contribute equally to the genetic component of a trait. In the dominance genetic model, dominant alleles determine the phenotype expression, thereby offspring are equivalent to one of the parents (Omholt et al., 2000). The phenotypic results of hybridization between V. elliottii and SHB were expressed on its offspring, which made it plausible to study such effects to guide breeding decisions for the future of the Blueberry Breeding Program.

In this sense, the main goal of the present study is to generate information towards introgressing favorable traits from V. elliottii into southern highbush blueberry

(SHB) through interspecific hybridization. For this two projects were developed, the first had the objective of evaluating the effects of cross direction for interspecific hybridization of V. elliottii and SHB blueberry and the second aimed to study the phenotypic differences between SHB, V. elliottii and their hybrids. Each one is detailed in the chapters of this thesis.

CHAPTER 2 EVALUATING CROSS DIRECTION FOR INTERSPECIFIC HYBRIDIZATION OF Vaccinium elliottii AND SOUTHERN HIGHBUSH BLUEBERRY

Background Information

The genus Vaccinium comprises approximately 130 known species, many of which have commercial importance in the blueberry industry. Twenty-four of these species are native to eastern North America (Camp, 1945), and some have been eaten and harvested by man for thousands of years (Moerman, 1998). A large density of species can be found in several regions of the world, such as the Himalayas, New

Guinea, and the Andean regions of South America. Section Cyanococcus is one of the most important economically species groups, comprising several species used for cultivar development and commercial production (Lyrene, 1997).

Wide hybridization has been fundamental to the development of “southern” highbush blueberry cultivars. Efforts made by the blueberry breeding program at the

University of Florida starting in 1949 resulted in the successful adaptation of V. corymbosum (highbush blueberry) to low chilling environments (Sharpe, 1953).

Southern highbush blueberry cultivars are interspecific hybrids derived by crossing northern highbush cultivars with wild species native to the southeastern United States

(Sharpe and Darrow, 1960; Lyrene and Ballington, 1986; Ballington, 1990; Draper,

1997). The introgression of wild germplasm into highbush blueberries gave SHB blueberries traits such as heat tolerance, drought, and mineral soils with little humus or organic matter, as well as reduced chilling requirements (less than 600 h below 7⁰ C), and introduced disease resistance, among other traits (Draper, 1997). To incorporate novel traits from wild germplasm found in the primary gene pool, homoploid and

heteroploid interspecific hybridizations were used to introgress valuable traits throughout the period of SHB blueberry development.

A wild species that has been used successfully for interspecific hybridization with

SHB is V. elliottii. It is a wild diploid blueberry that belongs to section Cyanococcus.

Vaccinium elliottii produces small black, with some exceptions of glaucous, fruit. High sugar content, pleasant flavor and aroma, and short bloom to ripe periods are traits that make this species valuable for breeding (Lyrene, 1997). This diploid species presents a strong triploid block when crossed with SHB, which are tetraploid, making hybridization challenging (Dweikat and Lyrene, 1991). Several synthetic fertile autotetraploid V. elliottii genotypes were successfully created by chromosome doubling (Lyrene, 2014) and later evaluated and selected by Norden (2017). The objective of this study was to investigate the effect of cross direction of interspecific hybridization between tetraploid

V. elliottii and SHB.

Materials and Methods

Plant Material

The tetraploid V. elliottii plants used in this study were generated from two sets of

V. elliottii seedlings collected by Lyrene (2014) and Norden (2017). In summary, the first set was collected from berries of V. elliottii population, growing in the wild near the

Blackwater River in Santa Rosa County (30°34'24.2"N 87°00'36.4"W), which is the far western panhandle of Florida. The second set was collected along Perone Creek in

Baldwin County (30°34'48.0"N 87°45'18.7"W ), in southern Alabama. Both locations are fewer than 100 meters above sea level and are near 30 ⁰ north latitude. In November

2014, open pollinated seeds extracted from these berries were germinated in pots filled with Canadian peat. Seedlings 1 cm tall were removed from the peat, washed, and

submerged in flasks of 0.2% aqueous colchicine (Colchicine, 97%, ACROS Organics™) for 72 hours at room temperature. After being treated, seedlings were washed and replanted in trays with Canadian peat. In June 2015, surviving seedlings were transplanted to a high-density nursery in Citra, FL (29°24'24.18"N, 82° 8'29.53"W ). The second set was generated in 2016, where four hundred seedlings were soaked for approximately 48 hours in flasks of 0.2% aqueous colchicine (Colchicine, 97%, ACROS

Organics™) at 22⁰ C. After rinsing, seedlings were grown in the greenhouse for six months, and then transplanted into a high-density nursery in June 2016 at Citra, FL. For both sets, high-density nurseries were planted at a spacing of 15 cm between plants and 46 cm between rows.

Material Selection

Flow cytometry, pollen, and leaf stomate size analysis were used to identify tetraploid plants and tetraploid chimeras (Norden, 2017). Then, fifteen individual V. elliottii genotypes identified as tetraploid were dug from the high-density nursery, potted, and placed in a cooler at 5⁰ C for three weeks to accumulate chilling hours. To confirm the previous results obtained by Norden (2017) regarding ploidy level, the pollen from all 15 V. elliottii main branches was harvested to be analyzed under microscope (Leitz

Wetzlar, model 664012, Wetzlar, Germany) using 250x magnification. The average pollen tetrad size for tetraploid V. elliottii varied from 48 to 56 µm. Branches producing pollen tetrads with diameter smaller than 48 µm were removed from the plant, and plants identified as non-tetraploid were removed from the experiment. After the analysis, a total of 11 V. elliottii genotypes were identified as tetraploid and used in the downstream analyses; for these, genotypes were placed in a bee-inaccessible greenhouse. The temperature in the greenhouse ranged between 5⁰ C and 27⁰ C.

Crosses and Data Collection

Based on the flowering time of the V. elliottii genotypes, 11 SHB genotypes were selected and paired to make reciprocal crosses (Table 2-1). Flowers from each plant were manually emasculated and cross-pollinated. This process consisted of, removing corollas and anthers of unopened flowers forceps from the plants that were designated to be female parents for each cross. Pollen was collected from male plants and manually applied to the stigma of the emasculated flower. Three people performed crosses at different time of the days every day.

The total number of pollinated flowers on each plant was recorded by date.

Approximately 300 flowers were targeted to be pollinated in each plant, however, this number varied depending on the availability of flowers in each genotype (Table 2-1).

During May 2018, ripe fruits were harvested from each bush. The number of harvested berries per plant and date of harvest were recorded. Fruit weight (g) of ten berries was estimated placing each individual berry in a scale (CP2245, Sartorious Corp., Bohemia,

NY). Seeds from each of the ten berries were extracted, hand cleaned, and counted.

Remaining unopened berries were stored in a cooler at 4⁰ C and were processed for seed extraction by the end of the season in June 2018.

Seeds were extracted using the Blueberry Breeding Program’s protocol. In summary, berries from each cross were placed in a food blender with an equal volume of water. After blending, the seeds were decantated with purified water to separate pulp and debris from seeds. Dried and cleaned plump seeds were counted and stored in coin envelopes in at 4⁰ C for a period of six months.

Seed Germination Percentage

In November of 2018, three cross combinations were selected as they had enough seeds out of the 10 reciprocal crosses. F1 seeds from three reciprocal crosses

(three SHB x V. elliottii and three V. elliottii x SHB) were separated in two groups. One group was composed of three families where SHB genotypes were used as female flowers. The second group was composed of the same three families where V. elliottii genotypes were used as female flowers. Each family present in both groups had six replicates. Each replicate consisted of one-liter container with 30 seeds. A total of 180 seeds were used for each cross in a treatment. The one-liter pots were arranged on a complete randomized design in a greenhouse. Thirty seeds were sown on each single pot placed on the surface of Canadian peat moss. Pots were under a mist running for five seconds every five minutes for nine hours during the day (Figure 2-1). Seeds were sprayed with Germination percentage was recorded after 60 days.

In February 2019, germinated seedlings were transplanted to flat trays of

Canadian peat. Seedlings were grown in a greenhouse for five months. They were watered daily as needed and fertilized with a water-soluble fertilizer once a month to provide the following (in mg/L): 20 N, 20 P2O5, 20 K2O (Base formulation; Peters

Professional). In June 2019, seedlings were transplanted to a high-density nursery at the University of Florida research station in Citra, FL. (Sharpe and Sherman, 1973).

Seed germination percentage was calculated by dividing the number of planted seeds by the number of germinated seedlings.

Statistical Analysis

Of the 11 reciprocal crosses performed in this experiment, one was found to be an outlier and was not included in the statistical analysis. The SHB genotype used for

this cross bloomed outside the greenhouse, and it is possible that contamination occurred due to natural cross pollination facilitated by bees. Fruit set percentage, number of seeds per berry, and seeds per pollinated flower showed a Poisson-like distribution. Consequently, the data were analyzed as a generalized linear model

ANOVA with a Tukey’s HSD test to determine differences among the means. A 5% significance level was used. All data analyses were conducted using R statistical software (R Core Team 2019).

Results

Crossability

Ten different autotetraploid V. elliottii genotypes were reciprocally crossed with ten tetraploid SHB genotypes. A total of 17,137 seeds were obtained from all of the reciprocal crosses between colchicine-induced autotetraploid V. elliottii and SHB genotypes (Table 2-1). The total consisted of 7874 and 9263 seeds from female V. elliottii and female SHB genotype, respectively. The great majority of the seeds were viable with an average of 85% seed germination in both directions (Table A-2). After the seedlings were transplanted into a high-density nursery in June 2019, the great majority of the them grew into vigorous plants.

Average fruit set, average number of seeds per berry, and total number of seeds per pollinated flower were considered to generate an estimation in crossability of interspecific hybrids between SHB and V. elliottii. On average, we observed that SHB female plants yielded a significantly larger number of berries per pollinated flower than

V. elliottii female genotypes (Figure 2-2). Also, on average SHB female genotypes presented more than double the amount of fruit set in comparison to V. elliottii female genotypes (Table 2-2). However, large differences in fruit set were observed among

crosses. For example, SHB female parent in cross number four presented more than

80% higher fruit set compared to cross 10 of the same direction (Figure 2-2). In contrast to the fruit set results, the average number of seeds per berry was higher in crosses where V. elliottii served as a female parent (Figure 2-3). On average, V. elliottii female parents produced double the amount of seeds when compared to SHB female parents.

The average number of seeds per pollinated flower were not significantly different between cross direction. Female V. elliottii genotypes yielded almost equal amounts of seeds per pollinated flower in comparison to SHB female genotypes (Figure 2-4).

Cross combination presented large differences on fruit set, average number of seeds per berry, and number of seeds per pollinated flower. It was observed that crosses number four and five yielded the largest number of seeds per pollinated flower among the rest of the crosses (i.e., 16.8 seeds and 21.6 seeds, respectively), (Table A-

1). On the other hand, crosses seven and ten showed the lowest number of seeds per pollinated flower among the rest of the combinations with 0.1 seed per pollinated flower in both cases, (Table A-1).

Seed Germination Percentage

Three different V. elliottii genotypes were selected after being reciprocally crossed with three different SHB genotypes. The three reciprocal crosses were selected as they had enough seeds to carry a replicated germination experiment. Seeds from female V. elliottii genotypes did not present significant differences in seed germination percentage when compared to female SHB seeds (Figure 2-5). On the other hand, genotype cross combinations between female V. elliottii germination ratio showed significant differences between crosses one and two (Figure 2-5).

Discussion

Wild relative species of commercialized crops are a valuable pool of genetic variability for distinct economic and agronomic characteristics (Knott and Dvorak, 1976;

Stalker, 1980; Sears, 1981). However, several barriers such as sterility, low vigor, and developmental abnormalities could be encountered when wild germplasm is used for introgression into a cultivated species. Blueberry species are not an exception to this condition. The generation of hybrids between SHB and the wild diploid V. elliottii have a high level of difficulty. Munoz (1984) reported that V. elliottii possesses a strong post- fertilization barrier that hinders the development of hybrid seeds due to embryo abortion. On top of this, V. ellilottii possesses a strong triploid block which is a barrier that plants encounter when they differ in ploidy level during hybridization (Dweikat and

Lyrene, 1988). Therefore, the creation of hybrids between SHB and the wild species V. elliottii are very difficult, hindering the introgression of important economic traits from V. elliottii into SHB.

Crosses between colchicine-treated synthetic autotetraploid V. elliottii and tetraploid SHB yielded a large number of seeds compared to crosses between diploid V. elliottii and tetraploid SHB previously made by Norden (2017) and Lyrene (2014).

Originally, Dweikat and Lyrene (1988) obtained a rate of 0.001 seedling per pollinated flower when diploid V. elliottii was crossed as a pollen parent. We demonstrated that by using a tetraploid parent, we observed a substantial increase in the number of seedlings per pollinated flower (Table 2-1). Our results agree with Dweikat and Lyrene (1991), where they obtained 3.86 seedlings per pollinated flower. Based on this data, we suggest that the crossing barrier between these species is not due to genome incompatibility but, mainly to the difference in ploidy levels (Lyrene and Brooks, 1996).

Differences in reciprocal interspecific crosses have been reported in several species. This differences are more evident when the ploidies of the parents differ

(Dweikat and Lyrene, 1988). One reason that could cause reciprocal differences on interspecific hybrid progeny is due to the cytoplasmic differences between the two species or different embryo: endosperm ratio (Kalloo and Chowdhury, 2012; Thompson,

1930; Myashita, 2012). Cross direction between interspecific crosses of Vaccinium species of different ploidy levels had been investigated for effects in crossability.

Dweikat and Lyrene (1988) and Myashita (2012) found significant differences in crossability between tetraploid V. corymbosum and hexaploid V. virgatum. It was shown that V. corymbosum female genotypes (4x) had a higher fruit set than V. virgatum female genotypes. The present study showed similar results, on average, SHB female genotypes presented a significantly higher fruit set than V. elliottii female genotypes.

Dweikat and Lyrene (1988) and Myashita (2012), also found a significant higher number of seeds per berry. However, our results do not match their findings, because in our case, the wild species (i.e. V. elliottii) used as a female presented a significantly higher average number of seeds per berry. This difference between the results of these studies could be due to the difference in ploidy that SHB (4x) and V. virgatum (6x) have.

A study involving a tetraploid V. elliottii genotype was performed by Dweikat and

Lyrene (1991), were a reciprocal cross between Fla. 519 a tetraploid V. elliottii and

O’Neal a SHB cultivar correlates with our study. In their case colchicine treated autotetraploid V. elliottii female parent yielded larger amounts of seeds per berry in comparison to the SHB female parent. On the other hand, their result on fruit set differs from the one obtained in this study. Fruit set resulted to be 10% lower on the SHB

female parent whereas our results showed that in average SHB female parents yielded a significantly higher fruit set percentage in comparison to the reciprocal cross. The difference in the results could be due to differences in compatibility between Fla. 519 and O’Neal.

The large contrast in fruit set and number of seeds per berry between cross direction in the case of V. elliottii and SHB hybridization in this study could be due to the differences in degree of self-infertility between the two species. Embryo abortion after fertilization is a barrier for self-infertility. This happens during the early stages of sporophyte development, yielding a low seed set to avoid inbreeding (Levin, 1984). It could be the case that differences in self-infertility and embryo abortion between the two species could cause the differences in fruit set and average number of seeds per berry.

In addition, it has been found that some SHB genotypes have the capacity of being highly parthenocarpic, raising the possibility that some SHB genotypes presented larger fruit set than V. elliottii (even with a higher rate of seed abortion) due parthenocarpy

(Coville, 1921; Merrill and Johnston, 1940; Morrow, 1943; White and Clark, 1939).

Interspecific crosses between autotetraploid V. elliottii and SHB evaluated in this study did not show significant differences in number of seeds per pollinated flower. For a breeder, the number of seeds per pollinated flower is a trait of interest. This trait has a direct relationship with cross efficiency. If the number of seeds per pollinated flowers is high the breeder could decrease the time and effort used to create large F1 progenies.

Large F1 progenies are important for the breeding program in order to have sufficient genetic variability to evaluate and select elite genotypes for crosses in the future. On the other hand, a high variability among parental combinations was observed. Specific

female and male parents of both species yielded larger amount of seeds per pollinated flower in comparison to the rest of the parental combinations. Out of the ten parental combinations, crosses number four and five yielded the largest number of seeds per pollinated flower. This indicates that cross success and efficiency could be greatly affected by parental combinations. This being the scenario, it is believed that, the low number of seeds per pollinated flower from different parental combinations may be due to failure of the synthetic conversion from diploid to autotetraploid in V. elliottii genotypes. Although, the V. elliottii genotypes were subject to pollen size, stomata size, and flowcytometry studies to differentiate ploidy level, genotypes with low number of seeds per pollinated flower could be mericlinal or sectorial chimeras in which only a portion of the plant was autotetraploid. Norden (2017) and Chavez (2009) identified several chimeras in colchicine treated populations of V. elliottii and V. darrowii.

Therefore, we can conclude that cross direction does not have an influence in the number of seeds obtained per pollinated flower, but parental combination involving the

V. elliottii genotype could increase or decrease the number of seeds obtained per cross.

Seed germination percentage studies are fundamental to comprehend the viability of the seeds produced by the reciprocal interspecific crosses. The average of seed germination from this study did not show significant differences between seeds produced by female SHB genotypes and female V. elliottii genotypes (Figure 2-5).

These results differ from interspecific reciprocal crosses made at different ploidy levels.

Miyashita (2012) found that seeds from female V. ashei (6x) plants yielded 1.7 times higher average germination percentage than seeds from female V. corymbosum (4x) genotypes. We suggest that, the doubling of chromosome number in V. elliottii, it was

possible to overcome the genetic barriers that affect interspecific reciprocal crosses at different ploidy levels. Seeds from cross one where V. elliottii served as the female parent presented a significantly higher germination percentage than cross two of the same direction. Therefore, germination ability can also be expected to change among different parental combinations. On the other hand, the rest of the crosses of the same direction did not present significative differences on germination percentage.

Interspecific hybridization between SHB and V. elliottii can be achieved after generating synthetic autotetraploid V. elliottii genotypes. The ploidy difference between

V. elliottii and SHB and a strong triploid block from V. elliottii are large obstacles in generating large numbers of F1 populations. It was hypothesized that cross direction would have an effect on progeny number. Nevertheless, no significant difference in seed per pollinated flower and seed germination ratio was found for cross direction. On the other hand, we observed a large variability in parental cross combination. Therefore, it is suggested that the generation of synthetic autotetraploid V. elliottii genotypes has the potential to breed a large amount of F1 seeds when crossed with SHB in either direction. Nonetheless, the results will depend on the success of synthetic chromosome doubling in V. elliottii. The generation of large F1 progeny has the potential to produce a larger pool of germplasm from which breeders can select elite genotypes with important economical traits.

Table 2-1. Number of flowers pollinated in each interspecific reciprocal cross between V. elliottii and SHB Parents Cross Direction* Number of Crosses Female Male HB x ell 11-051 17-662 237 ell x HB 17-662 11-051 57 HB x ell 06-19 17-724 346 ell x HB 17-724 06-19 401 HB x ell 13-168 17-728 260 ell x HB 17-728 13-168 152 HB x ell 12-213 17-726 292 ell x HB 17-726 12-213 164 HB x ell 12-113 16-799 247 ell x HB 16-799 12-113 48 HB x ell 05-603 16-793 416 ell x HB 16-793 05-603 360 HB x ell 11-155 17-730 232 ell x HB 17-730 11-155 360 HB x ell 11-184 16-800A 488 ell x HB 16-800A 11-184 255 HB x ell 02-178 17-725 97 ell x HB 17-725 02-178 80 HB x ell 09-279 17-729 204 ell x HB 17-729 09-279 47 HB x ell 13-161 16-800B 183 ell x HB 16-800B 13-161 119 HB x ell 16-144 16-801B 184 ell x HB 16-801B 16-144 313 * Cross direction where the first species was the female parent and the second is the pollen source (male) parent (ell = V. elliottii; HB = V. corymbosum (southern highbush))

Table 2-2. Interspecific hybridization between SHB and V. elliottii and the average effect on the success of reciprocal crosses. Total Seeds per crosses Seeds per berry Germination Type of cross seeds Fruit set (%)* pollinated (no.) (no.) (%) (no.) flower (no.) SHB x V. elliottii 2982 9263 28.66 ± 8.20 8.29 ± 6.05 86.48 ± 1.23 3.37 ± 1.28 V. elliottii x SHB 2309 7874 13.52 ± 4.01*** 16.82 ± 37.05*** 83.88 ± 2.14 2.67 ± 1.19 ***significant at p < 0.01. Means ± standard error. *significant results were calculated with a T-test comparison of the mean

A. . .B

C. .D

Figure 2-1. Blueberry seedlings placed in a greenhouse as part of the seed germination percentage study A) Mist hydration of seedlings and substrate, B) Seedlings placed on a bench inside the greenhouse, C) Seedlings from cross between SHB and V. elliottii and D) Seedlings from cross between V. elliottii and SHB.

Individual cross number 1 2 3 4 5 6 7 8 9 10 mean 90%

80%

70%

60%

50%

40% Fruit Set

30%

20%

10%

06-19 x 17-724 17-724 x 06-19 SHB x V. elliottii V. elliottii x SHB 13-168 x 17-728 17-728 x 13-168 12-213 x 17-726 17-726 x 12-213 12-113 x 16-799 16-799 x 12-113 05-603 x 16-793 16-793 x 05-603 11-155 x 17-730 17-730 x 11-155 02-178 x 17-725 17-725 x 02-178

11-184 x 16-800A 16-800A x 11-148 13-161 x 16-800B 16-800B x 13-161 16-144 x 16-801B 16-801B x 16-144 Genotype ID Cross Direction SHB ♀ V. elliottii ♀ Figure 2-2. Fruit set for 10 reciprocal crosses and their average in crosses between V. elliottii and tetraploid V. corymbosum (SHB). Bars correspond to standard errors.

Individual cross number 1 2 3 4 5 6 7 8 9 10 mean 28 26 24 22 20 18 16 14 12 10

8 Average seeds per berry 6 4 2 0

11-184 x 16-800A 16-800A x 11-148 13-161 x 16-800B 16-800B x 13-161 16-144 x 16-801B 16-801B x 16-144 Genotype ID Cross Direction SHB ♀ V. elliottii ♀ Figure 2-3. Average number of seeds per berry among 10 reciprocal crosses and their average in crosses between tetraploid V. elliottii and Southern highbush blueberry (SHB). Bars correspond to standard errors.

Individual cross number 1 2 3 4 5 6 7 8 9 10 mean 13 12 11 10 9 8 7 6 5 4 3

2 Number of seeds per polinated flower 1 0

11-184 x 16-800A 16-800A x 11-148 13-161 x 16-800B 16-800B x 13-161 16-144 x 16-801B 16-801B x 16-144 Genotype ID Cross Direction SHB ♀ V. elliottii ♀

Figure 2-4. Number of seeds obtained per pollinated flower for 10 reciprocal crosses and their average of V. elliottii and V. corymbosum (SHB). Bars correspond to standard errors.

Individual Cross Number 1 2 3 100%

90%

80%

70%

60%

50%

40% Germination rate 30%

20%

10%

06-19 x 17-724 17-724 x 06-19 12-113 x 16-799 16-799 x 12-113 05-603 x 16-793 16-793 x 05-603 Genotype ID Cross Direction SHB x V. elliottii V. elliottii x SHB

Figure 2-5. Germination percentage of three different reciprocal interspecific crosses between V. elliottii and V. corymbosum (SHB).

CHAPTER 3 PHENOTYPIC DIFFERENCES BETWEEN SOUTHERN HIGHBUSH, Vaccinium elliottii AND THEIR HYBRIDS

Background Information

Annual harvest for cultivated blueberry has more than tripled since the 1970s.

Increasing at a rate of 10–20% annually since 2000, Americans’ desire for a healthy and beneficial diet has propelled this growth (USDA Economic Research Service, 2003;

USDA National Agricultural Statistics Service, 2006; Sinelli et al., 2008). This increase in demand has forced the industry to develop new growing systems, increase yields, and produce in areas with challenging environmental conditions for blueberry plants

(Strik 2005, Strik and Yarborough 2005). Florida’s southern highbush blueberries have the capacity to ripen earlier than blueberries in the rest of the U.S. This early ripening fruit allows growers to access a high price window (Williamson and Lyrene, 2004). With other countries starting to produce and exporting in the same window it is necessary for

Florida producers to obtain earlier ripening and high-quality varieties, in order to compete in the market.

Fruit quality and storage life are directly impacted by fruit soluble solids content, acidity, fruit firmness, fruit size, aroma, and the ratio between soluble solids and acidity

(Retamales and Hancock, 2012). Therefore, these traits will have a direct influence on determining consumer acceptability and preference. The University of Florida blueberry breeding program has successfully bred and introgressed economically important fruit quality traits from interspecific hybridization, as improved fruit quality, and abiotic and biotic stress adaptability (Ballington, 2008; Brevis et all.,2008; Moore, 1965). The goal of the program today is to develop early ripening cultivars with better disease resistance, late flowering dates, and high fruit quality, so that consumers will prefer those over other

varieties for their outstanding fruit quality. Fruit quality in the blueberry breeding program considers firm berries (150> g/mm), with high sugar concentration (10> Brix⁰), intermediate acidity (1< %), and large fruit size (14> mm). V. elliottii can be used as an important genetic resource to develop new cultivars with outstanding characteristics by performing interspecific hybridization, as was previously done within Vaccinium section

Cyanococcus (Ballington 1990). Several attempts to cross V. elliottii wild genotypes into

SHB had been made in the past (Lyrene and Sherman, 1983; Dweikat and Lyrene,

1991). Their success led to the creation of cultivars, such as ‘Snowchaser’ and ‘Kestrel’ which are known by their highly aromatic flavor components and excellent flavor.

Previous work in generating hybrids was performed by Lyrene (2014) and Norden

(2017) creating, selecting, and planting large F1, F2, and BC1 populations involving tetraploid V. elliottii germplasm that is useful to perform a study that could help breeders acknowledge the potential of V. elliottii in blueberry breeding.

This study aimed to facilitate the development of cultivars with favorable traits from V. elliottii by phenotyping hybrids populations generated by Lyrene (2014) and

Norden (2017). The information generated here will help guide genotype selection towards the development of new improved varieties for the University of Florida blueberry breeding program.

Materials and Methods

Plant Material - 2017 Nursery

The population used in this experiment comprised hybrid seedlings from two high-density nurseries planted in two different years at the University of Florida

Research Station in Citra Florida (29°24'24.18"N, 82° 8'29.53"W). The first hybrid population, hereafter called 2017 nursery, was composed of F1, F2, and BC1 hybrid

families, generated by Norden (2017), where F1 families are the product of interspecific hybridization between SHB and a diploid V. elliottii, F2 families are the product of crossing between different F1 hybrid genotypes, and BC1 families are the product of backcrossing of F1 hybrids to a non-related SHB genotype. In summary, seeds from two

F2, one BC1, and one V. elliottii family were germinated in 2016. The seeds were germinated in 2 L pots in November 2016. Plants were transplanted into a high-density blueberry breeding nursery at the University of Florida research station (Citra, FL) in

June 2017. A V. elliottii population was used as a control, which was generated from open-pollinated crosses with wild V. elliottii genotypes. For more details, see Norden

(2017).

Plant Material - 2018 Nursery

The second population, hereafter called 2018 nursery was generated by Lyrene

(2014) from a set of seeds from nine F1, four F2, two BC1, and two V. elliottii families. A wide number of seeds were germinated in 2 L pots in November 2017 and later transplanted into a high-density blueberry breeding nursery at the University of Florida research station (Citra, FL) in June 2018. Each family consisted approximately of 100 seedlings, from which a variable number of genotypes were evaluated by family (Table

3-1).

Both nurseries received standard cultivation procedures, including irrigation three times a week for 1.5 hours with an overhead sprinkler irrigation system at a flow of 10 to

17 liters per minute. Both nurseries were tilled, and the soil amended with pine bark before planting. Slow release fertilizer was applied approximately monthly during the season (in mg/L): 15 N, 5 P2O5, 10 K2O (Blueberry Mix, Growers Fertilizer Corporation,

Lake Alfred, FL). Weed control was performed manually. At the beginning of the second

year, coarse pine bark was applied as a mulch, in order to retain humidity and control weeds. The experimental unit consisted of hybrid generation type sampled inside the nursery. Plants were planted in an area of 153 square meters with not replications each genotype being unique.

Phenotypic Evaluations

In May 2018, the first phenotypic evaluations were performed on the 2017 nursery. Plant vigor and plant height were visually evaluated in the field. Evaluations were performed during fruit ripening in April and early May. Vigor was evaluated using a

1-5 rating scale, where 1 represented an individual with very low vegetative growth and

5 represented high vegetative growth, when comparable to standard SHB cultivars. In the spring of 2019 these same traits were evaluated for the second time in the 2017

Nursery and for the first time for the 2018 Nursery. Fruit color, wax coating, scar, and stem detachment were visually evaluated in the field and compared with SHB genotypes from the same nursery. Individual genotypes that reached the quality standards of the blueberry breeding program were flagged and left in the nurseries for future evaluation.

Lab Analysis

Six fruit-related traits (i.e., soluble solids, total titratable acids, fruit diameter, fruit weight, fruit firmness, and pH) were measured at the University of Florida blueberry breeding laboratory in Gainesville, FL. In May 2018, twenty individual plants were sampled for each family. Each plant was flagged and tagged with a sample ID number.

Fifteen fully mature berries were picked from each individual plant. Fruit weight (g) of the 15-berry sample was determined using an analytical balance (CP2245, Sartorious

Corp., Bohemia, NY). The same 15 berries were then used to measure firmness (g.mm-

1 of compression force) and fruit size diameter (mm) using FirmTech II equipment

(BioWorks Inc., Wamego, KS). Firmness measurements were performed by equatorially orienting the berries with the scar facing to the side in a consistent direction to ensure accuracy. To compare sensory quality traits, soluble solids (⁰Brix), total titratable acids

(TTA) and pH were measured after extracting juice from the same 15 berries. Juice extracted from these pooled 15 berries yielded roughly 14 ml. Next, 1 mL of juice was pipetted onto a digital pocket refractometer (Atago, U.S.A., Inc., Bellevue, WA) to determine soluble solid content. A glass electrode was used to measure the pH of the remaining juice (Mettler-Toldeo, Inc., Schwerzenbach, Switzerland). Lastly, TTA was measured by diluting three mL of juice in 50 mL of nanopure water. More details of the methodology of these techniques are provided by Amadeu et al. (2016) and Cellon et al.

(2018). The same evaluations were performed in April 2019 for the second time on fruits from the 2017 nursery and for the first time on fruit from the 2018 nursery. Among the hybrid families, plants that did not yield enough fruit or had poor vegetative growth characteristics were removed from the nursery and were not evaluated. Random samples consisting on ~25 berries each sample from 30 individual SHB genotypes corresponding to different families were collected to be used as reference.

Statistical Analysis

The statistical analyses were performed using the packages agricolae

(Mendiburu and Mendiburu, 2019) and lsmeans (Length, 2016), implemented on the R platform (R Core Team, 2019). One-way ANOVA analyses were performed. Factors presenting significant differences (p≤ 0.05) were submitted to a post hoc test, using

Tukey’s correction for multiple comparisons (alpha= 0.05).

Results

Fruit pH

All families from 2017 and 2018 nurseries had their fruit quality traits measured and compared. For the 2017 nursery in the first year of evaluation, pH of the F2 families did not differ significantly from average pH of the SHB families (Figure 3-1). In the second season, this nursery showed that pH concentration of SHB and BC1 families was significantly higher than pH of V. elliottii and F2 families (Figure 3-1). However, a different pattern was observed for the 2018 nursery, where only the F2 families presented significantly higher pH than all other families.

Soluble Solids

Considering the trait soluble solids, no significant difference was found between

F2 and SHB families in the both years on the 2017 nursery. In addition, total soluble solids of V. elliottii families which was evaluated only the second year presented significantly higher Brix⁰ content when compared to the rest of the families on the data collected throughout the second year. Furthermore, BC1 families showed significantly higher total soluble solids concentration in comparison to F2 families (Figure 3-2). Total soluble solids for 2018 nursery presented different results, where BC1 presented significantly lower total soluble solid values when compared to F2.

The average of the total soluble solids were divided by the diameter (mm) of the berry to obtain the concentration of Brix⁰ per unit of area. The 2017 nursery results from the first year were not consistent with previous analysis performed for soluble solids.

SHB presented significantly smaller ratios than F2 families. In the second year of collected data, V. elliottii presented a significantly higher concentration of sugars per area in comparison to the rest of the family types (Figure 3-3). F2, BC1, and SHB did not

present significant differences in sugar concentration per area. For 2018 nursery results from ratio between total soluble solids and size showed that F1 and F2 families presented significantly higher values than BC1 and SHB.

Fruit Firmness and Diameter

Fruit firmness data from all nurseries did not present significant differences between families (figure 3-5). Fruit firmness was divided by fruit diameter to obtain firmness per area of the berry. Fruit firmness per area was significantly higher in the F2 families when compared to SHB for all the scenarios. For both nurseries V. elliottii presented a significantly higher firmness per area among the other families (Figure 3-5).

Fruit diameter for V. elliottii families presented significantly smaller fruit diameter in all the scenarios in comparison to the rest of the family types (Figure 3-6 ). BC1 families presented no significant difference in fruit diameter when compared to SHB families in 2017 nursery for the second year of evaluations. However, BC1 was significantly smaller than SHB in 2018 nursery. Fruit diameter was significantly higher in

SHB when compared to the F2 families for the two consecutive years of data (Figure 3-

6).

Total Titratable Acids

For 2017 nursery, total titratable acidity of the fruit was significantly higher in F2 families when compared with SHB for the two consecutive years (Figure 3-7). In the second year for the same nursery V. elliottii and F2 families did not present significant differences for total titratable acids, presenting significantly higher values when compared to BC1 and SHB families. For 2018 nursery, total titratable acidity was significantly higher for F1 and BC1 families in comparison to F2 families (Figure 3-7).

Considering the ratio between soluble solids (⁰Brix) and TTA, for 2017 nursery

SHB families presented a significative higher ratio for the two consecutive years with respect to the rest of the families evaluated. On the other hand, in 2018 nursery, F2 families presented the largest soluble solids: TTA ratio in comparison to the rest of the families (Figure 3-8).

Fruit Color

The visual assessments of fruit color, wax persistence, scar, and stem detachment was performed during the analysis. Fruit color was highly variable across genotypes. For example, most of the F2 genotypes present in 2017 nursery showed to have low wax coating and BC1 genotypes evaluated for wax coating always presented wax. However, the visual assessment of the persistence of the wax on the fruit showed that it varied within families and between genotypes (Figure 3-9). Considering stem detachment and scar, F1 and F2 families had a low rate of stem detachment in comparison to SHB and BC1 genotypes and most of the time fruit scar from F2 genotypes tore when it was harvested.

Plant Height and Vigor

Plant height and vigor were measured in the first year in families planted on 2018 nursery. No significant differences among families were found for plant height (Figure 3-

10). F2 families mean presented significantly less plant vigor in comparison to the rest of the families evaluated that year (Figure 3-11).

Fruit Weight

Average fruit weight was estimated throughout the second season in both nurseries. In the 2017 nursery, BC1 families yielded significantly higher berry weight in comparison to F2 and V. elliottii families. SHB Families planted in 2018 nursery

presented significantly higher berry weight when compared to F2, F1, and V. elliottii families. However, BC1 families did not showed significant differences when compared with SHB families for both nurseries (Figure 3-11). Average yield per bush was estimated by calculating the weight of all the berries harvested in season 2018. In the

2017 nursery, SHB and BC1 families presented significantly higher yields when compared to V. elliottii and F2 families (Figure 3-12).

Discussion

Fruit diameter and size presented significant higher values for hybrid families in comparison to V. elliottii families. Lyrene (1997) and Lyrene and Sherman (1984) reported that berry size of BC1 genotypes have the tendency to average below the normal commercial standards. Therefore, fruit size is a difficult trait to recover when

SHB is interbreed with V. elliottii. However, the BC1 families evaluated in the second- year of 2017 nursery presented an average diameter of 16.64 millimeters (mm) and an average berry weight of 2.37 g (Figure 3-12). This values are above the minimum commercial standard for blueberry cultivars of the University of Florida blueberry breeding program. This results show that is feasible to recover fruit size and weight when interbreeding V. elliottii and SHB. Environmental components have the capacity to produce significant variability on phenotype (Aalders et al., 1975). The differences observed between nurseries for fruit diameter and also for the average berry weight of

BC1 could be a result of this factor. The average fruit weight of BC1 families in 2018 nursery did not reach the minimum standard of 2 g per berry. However, this smaller fruit weight was not significantly different with respect to the values obtained for SHB families in the same nursery. This indicates that variations among experimental units could be due to different environments effects specific of each nursery location and

management. In addition, genotypes were not the same across the nurseries, therefore genetic variability can also be playing and important role in the differences observed.

Hybrid genotypes that had been selected on different generations families could be clonally propagated and tested at different locations to understand how they would perform at different environments.

Fruit production, evaluated as yield per bush, showed to be significantly higher in BC1 families when compared to V. elliottii and F2 values (Figure 3-13). Therefore, promising results were obtained for BC1 genotypes, which is an evidence that it could produce enough yield allowing their progress through the breeding program, in order to become future cultivars.

Sugars, expressed in total soluble solids, is one of the principal contributors to consumer preference, since sugars and acids in fruit crops have a direct impact on taste

(Retamales and Hancock, 2012; Yarmolinsky, 2009). Vaccinium species greatly differ in their chemical composition, such as organic acids and sugars (Gunduz et al., 2014;

Perkins-Veazie et al., 1995; Bremer et al., 2008; Hancock et al., 2008; Saftner et al.,

2008). We observed these significant differences between V. elliottii families and SHB, where the presence of total soluble solids (TSS) were significantly higher in V. elliottii in comparison to SHB (Figure 3-2). We had hypothesized that V. elliottii high sugar content could produce a significant increase on TSS for hybrid families. However, the amount of TSS on the hybrids was more affected by the SHB parents. For example, F2 families in 2017 nursery showed a significative smaller TSS in comparison to V. elliottii families, and for the second year of evaluation, F2 families presented smaller TSS values than all other families. A different result was observed for the 2018 nursery,

where TSS of F1 and F2 families was not significantly different than the values observed for SHB families. Therefore, the combination of the genotypes used in the crosses might have a bigger effect on the accumulation of TSS than expected.

Another factor that has a big role in consumer desirability is fruit acidity (TTA), which was significantly higher in V. elliottii and F2 families when analyzing the 2017 nursery (Figure 3-7). However, in 2018 nursery F2 hybrids presented significantly lower

TTA values than F1 and BC1, not differing from SHB values. Once more, the effects observed might be attributed to the genotypes chosen for crossing, however further investigation is necessary in order to validate this hypothesis. However not all the hybrids presented good values of TTA. High TTA negatively affects the perception of flavor and consumer preference (Gilbert et al., 2015). F2 families in their majority tasted more sour and less sweet than SHB families grown in the same environment (i.e., 2017 nursery). These results are in accordance with Lyrene’s (1997), where the fruit of intersection hybrids between V. elliottii and SHB remains acidic after numerous days of being ripe. This could negatively affect consumption preference in future SHB cultivars.

Gilbert et al., (2015) found that overall liking of blueberry fruit was positively correlated with sweetness (R2 = 0.70), texture (R2 = 0.68), and flavor (R2 = 0.63). They also found that sourness had a negative correlation with overall liking (R2 = 0.55). Further improvement on TTA present on V. elliottii could improve the results obtained for these traits, which could generate better results in hybridization.

The introgression of V. elliottii germplasm into SHB at the BC1 level could also significantly help in the improvement of other fruit quality traits, such as TSS. BC1

Families presented similar TSS in comparison to SHB, positively affecting TSS/TTA

ratio (Figure 3-8). The expected TSS/TTA ratio in SHB cultivars for the blueberry breeding program is above 14, the results obtained for BC1 in 2017 nursery year 2 were almost double in comparation with this value. However, SHB genotypes also presented high values for this ratio, which shows that the choice of the genotypes used during crosses can be improved in order to obtain better results. In contrast, BC1 families in

2018 nursery presented a much lower TSS/TTA ratio with an average of 14.5 units. This demonstrates once again that there is a high variability in fruit quality traits due to the environment and also associated with the genotype effect. It has been suggested by

Beaudry (1992) that high quality fruit in blueberries should present soluble solids >10% with pH values with a range of 2.25 to 4.25, a TTA ranging from 0.3 to 1.3%, and

TSS/TTA ratio between 10 and 33. Values of BC1 families from both nurseries, in average, presented values that are inside the established margins from Beaudry (1992).

This suggest that it can be feasible for the breeding program to select elite genotypes with outstanding fruit quality from interspecific hybridization between V. elliottii and SHB.

It has been shown in different fruit quality perception studies, that fruit texture in blueberry, such as mealiness and toughness had a strong impact in decreasing consumer liking. And that fruit firmness and crispness were generally preferred by consumers (Gilbert et all, 2015). All the families presented similar results in fruit firmness in both nurseries (Figure 3-4). The minimum standard for cultivars in the

Florida blueberry breeding program for firmness is above 150 g/mm. BC1 families presented a firmness above 220 g/mm in both nurseries (Figure 3-4). This values are close to the values presented by the cultivar ‘Optimus’, a machine harvestable cultivar released in 2017 for its excellent fruit quality and firmness allowing growers to machine

harvest. Taking in consideration that post-harvest longevity, berry quality, and harvest mechanization have a direct correlation with fruit firmness (Gallardo et al., 2018), we can suggest that hybrids between V. elliottii and SHB are capable of producing fruit with the standard firmness values that are required by the market.

Consumers prefer blue-colored fruit with good wax presence since it increases shelf-life of the fruit (Chu, et al., 2018; Saftner, et al., 2008). Wax presence presented a large variation among genotypes. Lyrene (1997) reported that it is difficult to find blue colored fruit in BC genotypes. We found that most of the F2 genotypes presented a black color lacking the presence of wax in their fruit (Figure 3-9). However, all harvested

BC1 genotypes presented medium to high amounts of wax content. Lyrene (1997) mentioned that although hybrids present wax content in the berries it is abraded easily by handling and packing. It would be important to perform further research on the wax persistence in hybrids.

The work developed here reinforces the feasibility of interspecific crosses between V. elliottii and SHB and demonstrate the possibilities that these crosses have in generating improvement on fruit quality, and consequently, in consumer preference.

The results presented here can help guiding decisions towards improving genotype selection in order to generate cultivars for the blueberry breeding program at the

University of Florida. Promising results are expected, given the results obtained.

However, further research is necessary to improve accuracy in selection and, consequently, the results obtained with it.

Table 3-1. Planting year, and years of evaluation for different hybrid family types. Parental information and number of genotypes evaluated. # Genotypes Nursery Planting date Year of evaluation Parents Family type Cross evaluated 2017 2018 - 2019 N1 x N4 F2 F1 x F1 19 2017 2018 - 2019 N10 x N4 F2 F1 x F1 17 2017 2019 N-38 x 11-67 BC1 F1 x SHB* 11 2017 2018 unknown SHB SHB x SHB 30 2017 2019 open pollinated V. elliottii V. elliottii x V. elliottii 9 2018 2019 Indigo x C217 F1 SHB x V. elliottii 5 2018 2019 12-8 x 16-791 F1 SHB x V. elliottii 20 2018 2019 05-603 x 16-791 F1 SHB x V. elliottii 4 2018 2019 Indigo x 16-791 F1 SHB x V. elliottii 14 2018 2019 12-179 x 16-791 F1 SHB x V. elliottii 9 2018 2019 12-8 x 16-793 F1 SHB x V. elliottii 1 2018 2019 12-213 x 16-791 F1 SHB x V. elliottii 7 2018 2019 11-184 x 16-800 F1 SHB x V. elliottii 3 2018 2019 N-4 x N-60 F2 F1 x F1 2 2018 2019 N-1 x N-10 F2 F1 x F1 5 2018 2019 N-10 x N-63 F2 F1 x F1 2 2018 2019 N-64 x 05-603 BC1 F1 x SHB 1 2018 2019 05-603 x N-64 BC1 SHB x F1 5 2018 2019 open pollinated V. elliottii V. elliottii x V. elliottii 4 2018 2019 05-603 x 04-202 SHB SHB x SHB 2 2018 2019 04-202 x 05-603 SHB SHB x SHB 5 2018 2019 05-603 x 05-603 SHB SHB x SHB 2 2018 2019 12-179 x 13-168 SHB SHB x SHB 37 *SHB = southern highbush (Vaccinium corymbosum)

Nursery 2017 - Year 1 Nursery 2017 - Year 2 Nursery 2018 . a a a 4.0 3.5 3.5 b b b 3.5 b b 3.0 3.0 3.0 2.5 2.5 2.5 2.0 2.0

pH 2.0 Frut pH 1.5 Fruit pH 1.5 1.5

1.0 1.0 1.0 0.5 0.5 0.5

0.0 0.0 0.0 F2 Highbush V. elliottii F2 BC1 Highbush F1 F2 BC1 Highbush Type Figure 3-1. pH of family types planted in the 2017 nursery (evaluated for two consecutive seasons) And 2018 nursery (evaluated for a single season).

Nursery 2017 - Year 1 Nursery 2017 - Year 2 Nursery 2018

17 . 17 a 13 a 16 16 12 15 15 ab 11 ab 14 14 b 13 13 10 12 12 b bc 9 11 11 c 8 10 10 9 9 7

8 8 6 °Brix (%) 7 °Brix (%) 7 °Brix (%) 5 6 6 5 5 4 4 4 3 3 3 2 2 2 1 1 1 0 0 0 F2 Highbush V. elliottii F2 BC1 Highbush F1 F2 BC1 Highbush Family Type

Figure 3-2. Total soluble solids (⁰Brix) of family types planted in the 2017 nursery (evaluated for two consecutive seasons) and 2018 nursery (evaluated for a single season).

Nursery 2017 - Year 1 Nursery 2017 - Year 2 Nursery 2018 1.08 . a a

2.5 2.5 0.96 a 0.84 2.0 2.0 0.72 b b 0.60 1.5 1.5

0.48

°Brix/Size °Brix/Size 1.0 °Brix/Size 1.0 b a 0.36 b b b 0.24 0.5 0.5 0.12

0.0 0.0 0.00 F2 Highbush V. elliottii F2 BC1 Highbush F1 F2 BC1 Highbush

Figure 3-3. Soluble solids by total titratable acids of family types planted in the 2017 nursery (evaluated for two consecutive seasons) and 2018 nursery (evaluated for a single season).

Nursery 2017 - Year 1 Nursery 2017 - Year 2 Nursery 2018 240 240 260 . 220 220 240 200 200 220

180 180 200

160 160 180 160 140 140 140 120 120 120 100 100 100 80 80

Firmness (g/mm)

Firmness (g/mm) Firmness (g/mm) 60 60 60 40 40 40 20 20 20 0 0 0 F2 Highbush V. elliottii F2 BC1 Highbush V. elliottii F1 F2 BC1 Highbush Family Type Figure 3-4. Fruit firmness of family types planted in the 2017 nursery (evaluated for two consecutive seasons) and 2018 nursery (evaluated for a single season).

Nursery 2017 - Year 1 Nursery 2017 - Year 2 Nursery 2018 36 36 38

. 34 34 a 36 a 32 32 34 30 30 32 28 28 30 26 26 28 26 24 24 24 22 22 22 20 20 b b 20 18 18 b bc a 18 16 16 c c 16 c

14 b 14 14

Firmness/Size Firmness/Size 12 Firmness/Size 12 12 10 10 10 8 8 8 6 6 6 4 4 4 2 2 2 0 0 0 F2 Highbush V. elliottii F2 BC1 Highbush V. elliottii F1 F2 BC1 Highbush

Figure 3-5. Fruit firmness by size of berry for family types planted in the 2017 nursery (evaluated for two consecutive seasons) and 2018 nursery (evaluated for a single season).

Nursery 2017 - Year 1 Nursery 2017 - Year 2 Nursery 2018

a . a 18 18 a 18 a 16 16 16 b b 14 14 b b 14 b 12 12 12

10 10 10

8 8 c 8 c

6 6 6

Fruit Diameter (mm) Fruit Diameter (mm) Fruit Diameter (mm) 4 4 4

2 2 2

0 0 0 F2 Highbush V. elliottii F2 BC1 Highbush V. elliottii F1 F2 BC1 Highbush Family Type Figure 3-6. Fruit diameter of family types planted in the 2017 nursery (evaluated for two consecutive seasons) and 2018 nursery (evaluated for a single season).

Nursery 2017 - Year 1 Nursery 2017 - Year 2 Nursery 2018

1.0 . 1.0 1.0 a a a a 0.8 0.8 0.8 a ab 0.6 0.6 0.6

b b b

0.4 0.4 b 0.4 Total Titratable Acids (%)

0.2 Total Titratable Acids (%) 0.2 0.2 Total Titratable Acids (%)

0.0 0.0 0.0 F2 Highbush V. elliottii F2 BC1 Highbush F1 F2 BC1 Highbush Family Type Figure 3-7. Total titratable acids of family types planted in the 2017 nursery (evaluated for two consecutive seasons) and 2018 nursery (evaluated for a single season).

Nursery 2017 - Year 1 Nursery 2017 - Year 2 Nursery 2018 50 . a a 50 50 45 45 45 40 40 40 a b 35 35 35 30 30 30 b c 25 b 25 25 b 20 20 b 20 c 15

15 15

Soluble Solids/TTA

Soluble Solids/TTA Soluble Solids/TTA 10 10 10

5 5 5

0 0 0 F2 Highbush V. elliottii F2 BC1 Highbush F1 F2 BC1 Highbush Type Type Family Type Figure 3-8. Total soluble solids by size of berry for family types planted in the 2017 nursery (evaluated for two consecutive seasons) and 2018 nursery (evaluated for a single season)

Figure 3-9. Fruit size and color of different generation of intersectional hybrids between V. elliottii and SHB.

Plant Height Nursery 2018 105

Plant Height (cm) 30

0 V. ellottii F1 F2 BC1 SHB

Family Type V. ellottii F1 F2 BC1 SHB

Figure 3-10. Plant height of different family types planted in the 2018 nursery, evaluated in one-year old plants.

Nursery 2018

5 .

3 a a a b

2 Plant Vigor

0 V. elliottii F1 F2 SHB Family Type

Figure 3-11. Plant vigor (scale 1 to 5) of family types planted in the 2018 nursery evaluated in one-year old plants. (5 is the highest vigor observed)

Nursery 2017 - Year 2 Nursery 2018

a . 2.5 2.5 a ab a 2.0 2.0

b b 1.5 b 1.5

1.0 1.0 Average Weight (g) c c

0.5 0.5 Average Berry Weight (g)

0.0 0.0 V. elliottii F2 BC1 Highbush V. elliottii F1 F2 BC1 Highbush Family Type Family Type . Figure 3-12. Average berry weight for family types planted in the 2018 nursery, evaluated in one-year old plants.

Nursery 2017 - Year 2 800 a 750 700 650 600 550 a 500 450 400 350 300 b 250 b 200 150 Average Yield per bush 100(g) 50 0 V. elliottii F2 BC1 Highbush Family Type

Figure 3-13. Average yield per bush of family types planted in the 2017 nursery, evaluated in two-year-old plants

CHAPTER 4 CONCLUSIONS

Vaccinium elliottii has been characterized as an important diploid wild species for blueberry improvement. Its adaptability to the Florida environment and capacity to produce pleasant highly aromatic berries with a short bloom to ripe period make it a strong candidate for introgression to SHB. A strong triploid block was observed previously, and several unsuccessful attempts to create large F1 populations were made in the past. This study was undertaken to generate further information related to the efficiency and feasibility of introgression of wild V. elliottii germplasm into the SHB blueberry breeding program at the University of Florida.

This study investigated the effects that cross direction could have in the generation of new hybrid progeny for interspecific crosses between V. elliottii and SHB.

In addition, hybrid populations in different stages of introgression (i.e., F1, F2, and BC1) were evaluated to generate information towards genotype selection from material containing economically important traits from V. elliottii.

In this study, the feasibility of intercrossing synthetic autotetraploid V. elliottii and

SHB genotypes was demonstrated. Large numbers of F1 hybrids were recovered from all crosses performed, independently of the direction of the cross. Cross direction did not have an effect on number of seeds per pollinated flower, or on germination success.

Therefore, breeders could perform crosses in either direction considering the objective of obtaining high numbers of individuals from interspecific crosses, and consequently generating highly genetically variable hybrid populations. However, the specific combination between V. elliottii and SHB genotypes could affect the results of

hybridization. Further studies could explore the importance of genotype combination on the generation of hybrids.

Consumer preference in the first purchase is driven by fruit appearance and texture. Subsequently, future purchases by consumers depends on the satisfaction they will have with fruit flavor, texture, and aroma. Continued increasing blueberry consumption will be possible if fruit quality traits continue to improve. Vaccinium elliottii presents highly aromatic and pleasantly tasting fruit. Fruit quality was rapidly increased by introgressing wild germplasm into the SHB blueberry breeding program. Several BC1 families presented promising characteristics as high fruit firmness, high sugars, and low acidity, which are characteristics known to be preferred by consumers.

The results obtained here suggest that interspecific crosses between V. elliottii and SHB have high potential in generating genetic variability for later phases of breeding. It was found that the success of obtaining hybrids increased when using synthetic autotetraploid V. elliottii. Also, the direction of crosses did not affect the number of viable seeds obtained. By evaluating different stages of hybridization, it was found that hybrids presented fruit-quality values superior than the minimum thresholds acceptable for commercialization, and this was obtained with only one generation of backcrossing. Therefore, promising results are expected for introgressing V. elliottii into an SHB background, and thus to maintain the needed variability to keep making genetic gains in important commercial traits.

Further investigation of adaptability, disease resistance, and volatile components can generate information about the success of introgression of these traits from V. elliottii into SHB material. It is imperative to mention that this research generated a high

amount of genetic variability to be used in further stages of the University of Florida blueberry breeding program, and that potential genotypes were identified that can be used for crosses.

APPENDIX CROSS DIRECTION EVALUATION FOR INTERSPECIFIC HYBRIDIZATION OF VACCINIUM ELLIOTTII AND SOUTHERN HIGHBUSH BLUEBERRY

Table A-1. Crossability in interspecific hybridization between V. elliottii and SHB Parents Flowers- Seeds per Cross Fruit set Seeds per Cross direction pollinated pollinated ID (no.) (%) berry (no.) Female Male (no.) flower(no.)

06-19 17-724 1 SHB x V. elliottii 346 41.0 10.6 5.5 17-724 06-19 1 V. elliottii x SHB 401 9.2 19.2 1.7 13-168 17-728 2 SHB x V. elliottii 260 6.2 5.8 0.1 17-728 13-168 2 V. elliottii x SHB 152 28.3 27.3 5.5 12-213 17-726 3 SHB x V. elliottii 292 8.2 12.1 0.4 17-726 12-213 3 V. elliottii x SHB 164 1.2 11.0 0.1 12-113 16-799 4 SHB x V. elliottii 247 87.0 14.8 12.1 16-799 12-113 4 V. elliottii x SHB 48 22.9 24.8 4.7 05-603 16-793 5 SHB x V. elliottii 416 63.7 15.0 8.7 16-793 05-603 5 V. elliottii x SHB 360 42.5 26.6 12.9 11-155 17-730 6 SHB x V. elliottii 232 37.5 1.0 0.1 17-730 11-155 6 V. elliottii x SHB 360 15.6 28.0 3.8 11-184 16-800A 7 SHB x V. elliottii 488 13.9 2.3 0.1 16-800A 11-184 7 V. elliottii x SHB 255 0.4 12.0 0.0 02-178 17-725 8 SHB x V. elliottii 97 8.2 11.7 0.8 17-725 02-178 8 V. elliottii x SHB 80 16.3 10.6 0.3 13-161 16-800B 9 SHB x V. elliottii 183 33.9 7.6 2.1 16-800B 13-161 9 V. elliottii x SHB 119 2.5 9.7 0.2 16-144 16-801B 10 SHB x V. elliottii 184 1.6 2.0 0.0 16-801B 16-144 10 V. elliottii x SHB 313 2.9 6.5 0.1

Table A-2. Seed germination in interspecific hybridization between V. elliottii and SHB

Parents Cross ID Sown seeds Germinated seedlings Cross direction Germination (%) Female Male (no.) (no.) (no.) 06-19 17-724 2 SHB x V. elliottii 180 155 86.1 17-724 06-19 2 V. elliottii x SHB 180 169 93.9* 12-113 16-799 5 SHB x V. elliottii 180 165 91.7 16-799 12-113 5 V. elliottii x SHB 180 140 77.8* 05-603 16-793 6 SHB x V. elliottii 180 147 81.7 16-793 05-603 6 V. elliottii x SHB 180 144 80.0 *significant at p < 0.05

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BIOGRAPHICAL SKETCH

Diego Cabezas, born in Santiago, Chile, in 1989, is the second child of Alejandro

Cabezas and Marisol Catalan. He graduated with an aeronautic mechanic’s degree from high school. Later in life he experienced the beauty of working with plants, which helped him on making the decision to study agriculture in a community college in

Santiago, Chile. After two years of graduating with an associate degree in agronomical technician he moved to Idaho and entered Brigham Young University-Idaho to obtain a bachelor’s degree on agronomy.

Many people has contributed to the life of Diego his father and mother raised him with love and care and have always believed on his potential. Diego’s agricultural career has had a strong influence by his mentor Paul Genho who gave him the opportunity to study and find new horizons on his life. Paul showed Diego the beauty of plant breeding. His curiosity and persistence drove him to move to Florida where he had the opportunity to work and learn from David Norden as a field assistant for the blueberry breeding program at the University of Florida.

Dr. Patricio Munoz took him as a student in January 2017. Many people played a very important role in his life here at the University of Florida. Dr. Munoz and Dr. Paul

Lyrene supported him continuously trough his learning path. Their patience and knowledge helped him through every step. Diego’s goal is to become a positive influence and support to his family, friends, and country.