Progress in Blueberry Research in New Zealand

Abstract

Blueberries were first introduced to New Zealand in the 1950s from North America and by 1970 around 30 cultivars had been imported. Selection for new material better suited to the New Zealand climate then began using F₁ seedlings and open pollinated crosses. The Plant & Food Research Ltd blueberry breeding program (led by Dr. Jessica Scalzo) is currently run at three sites: at Hamilton and Motueka on the North and South Island, respectively, of New Zealand, and a smaller program at Dierking's Nursery in Germany. Plant & Food Research Ltd's breeding targets are to improve yield, ripening season, and disease resistance, as well as fruit quality traits, such as fruit size, firmness, flavor, color, and grittiness. Areas of research include molecular genetics, functional genomics, biochemical composition, and health, and in 2008, we joined the Speciality Crop Research Initiative project to generate genomic tools for blueberry improvement, led by Dr. Lisa Rowland. We present a summary of the microsatellite and single nucleotide polymorphism-based markers we have developed as part of the Speciality Crop Research Initiative program, as well as an update on other blueberry research undertaken at Plant & Food Research Ltd. This will include anthocyanin composition studies, research into polyphenolics and skeletal muscle damage, as well as work on our expressed sequence tag library and candidate genes within the anthocyanin pathway. We will also discuss the impact of this research on the future of blueberry breeding at Plant & Food Research Ltd.

Keywords:

• Vaccinium
• breeding
• genomics
• DNA markers
• health
• fruit composition

INTRODUCTION

In the 1950’s, the first blueberry cultivars were introduced to New Zealand (NZ), as a potential crop for the wetland peat areas of the Waikato. A breeding research program was then initiated at what is now The New Zealand Institute for Plant & Food Research Limited (PFR). The program primarily focused on developing blueberries better suited to the NZ climate, but as the industry expanded, PFR began to look into other aspects of blueberry research. Two large projects have recently been undertaken, funded by the New Zealand Foundation for Research Science and Technology (now known as the Ministry of Science and Innovation [MSI], New Zealand) and the key NZ industry groups including blueberry. From 2004 until 2008, the Fresh and Processed Berryfruit Products to Enhance Gut Health (previously Health Enhancing Food Products from Berryfruit, C06X0407), aimed to examine fruit composition, crop genetics, maintain germplasm and support the breeding programs in blackcurrant, blueberry and raspberry (and other Rubus species and hybrids) in the development of new cultivars. In 2008, this work continued into a current, second project, New Healthy and Flavorsome Berries and Products (C06X0807), which focuses more on the various health-attributes and their related benefits. In 2008, PFR was also invited to join the USDA Speciality Crop Research Initiative (USDA-SCRI) project in the USA to generate genomic tools for blueberry, led by Dr Lisa Rowland. Herein, we give an overview of the NZ blueberry industry, as well as the strategies used within the PFR blueberry breeding program and report progress on our current research on blueberry fruit composition, health effects, blueberry genomics and marker development.

THE NEW ZEALAND BLUEBERRY INDUSTRY

The NZ blueberry industry is primarily based on northern highbush blueberry types; however, both southern highbush and rabbiteye types are grown commercially and are attracting growers’ interest for their potential cultivation in lower winter chilling areas of the country. Commercial plantings have traditionally been in the North Island of NZ with recent interest in extending the cultivation into warmer areas; however, parts of the South Island with cold winters have also recently been planted for northern highbush production with a range of medium- to high-chill blueberry varieties. The industry product group, Blueberry New Zealand Inc. (BBNZ), represents industry interests and is run by an elected executive consisting of growers, exporters, and representatives of the fresh and processed blueberry industries. The NZ blueberry industry contributes levies that are used to support research to benefit its members and leverage further government investment. The BBNZ also has input into the blueberry breeding program and other PFR research programs.

Because of increasing consumer appeal of blueberries, the NZ industry has grown from 1,000 tonnes in 1999 to over 2,700 tonnes in 2008 (Fig. 1). This has been mainly due to a doubling in blueberry plantings within NZ, from 263 to 522 hectares with an increase in the number of growers, 78 to 95, over the same period, as well as improvements in crop management and harvest technologies. Although the revenue gained from blueberry exports has expanded two fold over this time (Fig. 1), the domestic market has increased from NZ$3.3 million in 1999 to NZ$24.2 million in 2008 (HortResearch, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007; Plant & Food Research, 2008, 2009).

FIGURE 1 Trends in crop volume, planted area, and export revenue from New Zealand-grown blueberries from 1999 to 2008 (data obtained from the annual HortResearch, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007; Plant & Food Research, 2008, 2009).

THE PFR BLUEBERRY BREEDING PROGRAM

The blueberry breeding program was established at the Ruakura Research Centre in 1969. Improving yield, fruit quality and adaptation to the NZ climate were the initial targets, as well as expanding the fruit season to meet the Northern hemisphere off-season fresh market (Patel and George, 1997). Selection for new material began using F₁ seedlings and open pollinated crosses obtained from the USA. Some of the early releases included the northern highbush cultivars Puru, Nui, and Reka in 1987.

Dr. Jessica Scalzo now leads the breeding program, which has expanded onto two sites, at Ruakura (37°48′S 175°17′E) and Motueka (41°6′S 172°58′E). Currently the program involves many of the major commercial blueberry types: northern highbush, southern highbush, and rabbiteye, utilizing many of the standard varieties grown around the world as well as those developed specifically for the NZ climate. The core germplasm collection consists primarily of Vaccinium corymbosum, V. corymbosum hybrids, and V. virgatum; however, a small collection of V. myrtilloides, V. simulatum, and V. angustifolium is also maintained as pre-breeding material. A number of the varieties released from the program are widely cultivated around the world. PFR's latest releases include low chill varieties, for example, ‘Island Blue’ and ‘Blue Bayou’, which are well adapted to warmer climates, and late season rabbiteye blueberries, such as ‘Centra Blue’ and ‘Sky Blue’. We have also recently released ‘Blue Moon’ and ‘Sunset Blue’, which have larger, firmer fruit more suited to the fresh market, and ‘Hortblue Poppins’, ‘Hortblue Petite’, and ‘Hortblue Onyx’, which are home garden and ornamental types. The key objectives for our breeding program include improving adaptation to the NZ climate, increasing disease resistance, and enhancing amounts of antioxidants and other health-related compounds, while simultaneously improving quality, taste, and suitability for mechanical harvesting.

BREEDING FOR ADAPTATION TO NEW ZEALAND ENVIRONMENTS

The highbush varieties initially introduced to NZ varied from mid to high winter-chilling requirements. NZ growers wish to extend the cultivation area to include mid to low chill regions; thus, there is a need to improve the adaptability to a lower winter chill requirement. The variation of winter chill requirement in the current PFR germplasm includes those adapted to lower-chill areas, and such individuals are being used as parents within the breeding program to develop new varieties with low winter chilling requirements.

Currently, we are extending the production window by selecting for very early and very late cropping periods. Phenotyping our germplasm demonstrated that low winter chill blueberries have the potential for producing a second late-summer crop that could be of interest in niche production areas with specific environments. We are evaluating the best strategy for managing this second crop in a double cropping production system. We are also examining new genotypes with late flowering in order to avoid spring frosts combined with short fruit development time, so that the late flowering will not conflict with the requirement for early-season fruit. We will also explore adaptability to various soil conditions, including higher pH, in order to extend the cultivation area.

DISEASE RESISTANCE BREEDING

As there is a general desire by growers to minimize fungicide and pesticide use, and ideally to achieve a fully sustainable, nil-residue and high value export crop, breeders are under pressure to develop new disease-resistant varieties. This is of particular importance in a crop where their “health-promoting” properties are a key market driver. In the last 5 years we have carried out preliminary characterization of disease resistance in our germplasm collection. Field observations strongly suggest that there is a range of resistance/tolerance present for the two main diseases affecting NZ blueberries, foliar rust and anthracnose fruit rot, and we are currently advancing key lines that have shown resistance to rust, for further evaluation for fruit quality and yield traits.

BREEDING FOR INCREASED FRUIT ANTIOXIDANT CONTENT

Consumers now desire fruits and vegetables that have high health benefits and nutritional values. Although increasing fruit and vegetable consumption has been associated with improvements in general health, the impacts of these health benefits can be increased still further if the intrinsic amounts of these “healthy compounds” are improved. For this reason, increasing the amounts of specific health-related compounds is now one of the main objectives of the PFR breeding program. We have characterized our Vaccinium germplasm for their fruit antioxidant activity and anthocyanin composition (see the following section). Two newly released PFR varieties, ‘Hortblue Petite’ and ‘Hortblue Onyx’, have fruit with exceptionally high phytochemical composition and we are trialing the use of these varieties as breeding parents. In order to increase the content of health-related phytochemicals in fruit while maintaining other important traits, we are developing molecular markers that are co-inherited with high amounts of particular phytochemicals or phytochemical classes.

BLUEBERRY FRUIT COMPOSITIONAL RESEARCH

The polyphenolics present in the fruit are some of the key chemicals credited for blueberries’ putative health benefits. The anthocyanins are part of this group and provide blueberries with their characteristic color. They can be present in several different forms, including galactosides, glucosides, and the arabinosides of the aglycones delphinidin, cyanidin, petunidin, peonidin, and malvidin. In addition, these glycosides can be acetylated. In a recent study by Scalzo et al. (2008), around 500 individuals from our germplasm and breeding line collection were analyzed for anthocyanin content. The most representative individual anthocyanin within the northern and southern highbush, the rabbiteye and rabbiteye x northern highbush hybrids, studied was malvidin 3-galactoside, with the highest average amounts observed in the rabbiteye group. While the ornamental blueberries group not only contained the highest total anthocyanin amount, at 3,090 μg/g, followed by rabbiteye V. virgatum (previously V. ashei), at 2,102 μg/g, they also contained the highest sources of delphinidin 3-galactoside, malvidin 3-glucoside, malvindin 3-arabinoside, and petunidin anthocyanin. These ornamental blueberries were also particularly abundant in the acylated anthocyanins. This research will be used to select parents for use in the breeding program, to develop new cultivars with elevated total anthocyanins, as well as specific anthocyanin contents.

BLUEBERRIES AND HEALTH

Although blueberries are particularly known for their high antioxidant content (Harborne and Williams, 2000; Prior et al., 1998), and overall high nutritional value, the mechanism by which berry-fruit polyphenolics confer the putative health benefits is still unclear.

As part of the current MSI project, we are examining the mechanism for health benefits associated with blueberry consumption. A recent study led by Dr. Roger Hurst used differentiated skeletal muscle myotubes to assess the protective capacity of blueberry extracts against oxidative stress (Hurst et al., 2010). In this study, muscle myotubes were simultaneously exposed to the oxidative stress-inducing compound, calcium ionophore A23187, and extracts obtained from blueberry. The protective capacity of the fruit extracts was assessed by measuring the release of lactate dehydrogenase a cytosolic enzyme, upon membrane damage to the cells. Dr. Hurst and co-workers found that some blueberry extracts displayed a significant protective capacity in these experiments. The highest extract concentration (50 μg/ml) showed a 54% (P < 0.05) protection against oxidative stress. Using functional and chemical analysis of further sub-extracts, followed by investigation using purified compounds, they concluded that malvidin galactoside and glucoside were the likely active compounds. These preliminary in vitro findings indicate that blueberry polyphenolics may be beneficial in alleviating oxidative stress-induced damage in muscle tissue, offering potential for functional food outcomes following more detailed investigation.

BLUEBERRY GENOMICS AT PFR

PFR has a well-established and world-respected fruit genomics team. Our blueberry genomics work began with the development of four expressed sequence tag (EST) libraries created from floral and fruiting tissues from three different V. corymbosum genotypes (‘Duke’, ‘Puru’, and E118 BC4–41). These EST libraries encompass over 9,000 unigenes, approximately 2,500 of which are singletons and 6,500 are contigs. The library is accessed using the in-house PFR BioView platform using a report card system for individual EST sequences, where information on individual ESTs can be stored and relationships between EST sequences visualized. These ESTs have subsequently been used to investigate several genes in the anthocyanin and related pathways. These include full gene sequences of the P450 gene responsible for generating blue anthocyanins, VcF3′5′H and partial sequences of nine biosynthetic genes, and six regulatory genes. Included in the regulatory genes are transcription factors, which activate transcription of the biosynthetic genes (VcMYB10, bHLHs, TTG1-like) and or repress the transcription of the same genes (VcMYB17). The full list of which, with their closest BLAST match hits in Arabidopsis, raspberry, apple, and bilberry, are shown in Table 1.

TABLE 1 Anthocyanin-related genes of blueberry and other fruit species (actin is included as a useful house-keeping gene)

	Blueberry	EST number	Bilberry	Arabidopsis	Raspberry	Apple
PAL	VcPAL	28265	AY123770	At2g37040	AF304366	EB147511
CHS	VcCHS2	122717	AY123765	AF1122086	AF292367	MdCHS4 CN944824
	VcCHS1	123399
CHI	VcCHI1	96484	None	AT5G05270	None	MdCHI CN946541
F3H	VcF3H	287684	AY123766	U33932	None	MdF3H CN491664
F3′H	VcF3′H	123063	None	TT7 AT5G07990		MdF3’H1 CX024459
F3‘5’H	VcF3′5′H	124528	None	None	None	none
DFR	VcDFR1	122903	AY123767	AJ251982	None	MdDFR1 AF117268
LDOX	VcLDOX1	94183	AY123768	U70478	None	MdLDOX1 AF117269
	VcLDOX2	50247
UFGT	VcUFGT	122878	None	AT5G17050	None	MdUFGT AF117267
ANL2	VcANL2	96822	None	NM116298	None	MdANL2 DR992895
MYB10-activators	VcMYB10	None	None	AtMYB75	RiMYB10	MdMYB10 DQ267897
					EU155165
MYB-repressors	VcMYB17	51894	None	AT4G09460 AtMYB6	None	MdMYB17 CO867070
TT8-bHLH	VcbHLH-TT8	83379	None	AT4G09820	None	MdbHLH3 CN934367
DELILA-like	VcbHLH-GL3	97108	None	AT1G63650	None	MdbHLH33 DQ266451
TTG1	VcTTG1	124942	None	AT5G24520	None	MdTTG1 AF220203
difF	VcdifF	82931	None	AT2G32720	None	MddifF EB124543
Actin	VcActin2	48821	None	AT5G09810	None	MdActin CN893577

DNA MARKER DEVELOPMENT AND GENETICS

The EST library is the key resource for the development of blueberry molecular markers at PFR. Markers have been developed for key genes in the anthocyanin pathway (Buck et al., unpublished), but the majority of the markers developed so far have been microsatellite, or simple sequence repeat (SSR)-based markers. The EST BioView platform indicates the presence of SSR sequences within the ESTs, and a preliminary mining of the blueberry database indicated that there are over 500 SSR-containing ESTs.

In 2006, a set of 28 EST-SSR markers were developed and eight of them, in combination with VCC-K4 (Boches et al., 2005), were used to screen a set of 55 individuals from the blueberry germplasm collection (Wiedow et al., 2007). These samples consisted of 23 V. corymbosum, 10 southern highbush (V. corymbosum hybrid), 9 V. simulatum, 10 V. virgatum, 1 V. myrtilloides hybrid, 1 V. ovatum, and 1 V. consanguineum individual. In total, 152 alleles were observed from the nine SSR markers within these 55 samples. In addition, six of the SSRs amplified products in all the species screened. The southern highbush samples appeared to be genetically indistinct among the samples analyzed, a reflection of their mixed parentage. However, the V. simulatum samples were genetically distinct, generally clustering apart from the other samples. These genetic relationships will be used to aid parental selection within the breeding program. These samples include the potential parents of our own mapping population, and this initial screening will be used to identify markers for future map construction. Following this initial work, we were invited to join the USDA Speciality Crop Research Initiative and this has enabled us to develop more SSR markers from the EST database.

SPECIALITY CROP RESEARCH INITIATIVE—DEVELOPING BLUEBERRY MOLECULAR TOOLS

Within the USDA-SCRI program, we concentrated initially on developing EST-SSR markers. Of an additional 186 markers developed, 150 and 115 have so far been screened, respectively, across two subsets of the two mapping populations utilized within the program: a diploid mapping population (Rowland et al., 1999) and a tetraploid mapping population (Brevis et al., 2007). So far, we have identified 50 markers as polymorphic in the tetraploid mapping population and these markers were provided to the USDA-SCRI group for screening on the whole tetraploid mapping population.

We then focused on the diploid population. Two different methods were used to screen the SSR markers across the diploid mapping population. The first method enables size variations in PCR products to be detected, using an ABI377 sequencer using a fluorescently labeled primer (Schuelke, 2000). Table 2 shows the numbers of markers designed around the various types of SSRs (di-nucleotide, tri-nucleotide, tetra-nucleotide, and complex) and the polymorphism observed using the ABI377 from each type. The majority of the polymorphic markers (80) were from 124 di-nucleotide repeat-based SSR markers, followed by 7 polymorphic markers from the 14 tri-nucleotide repeat-based SSRs. Of the 150 markers screened, 96 were polymorphic. The second method used enables sequence variations to be detected, including single nucleotide polymorphism (SNP) but also small size variations, both within and among samples. This was achieved using the high resolution melting (HRM) analysis in a Roche Light Cycler 480 (Roche Diagnostics Corporation, Indianapolis, IN, USA). With this technique, the 49 markers previously identified as monomorphic with ABI377 were re-assessed, and 39 of them were found to contain sequence variations. Some of these markers produced complex profiles, so only the 10 with distinct profiles were screened over the whole population. Similarly, 39 SSR markers previously identified as monomorphic in size in the diploid population (Bassil et al., unpublished) were re-screened at PFR using the HRM technique. This revealed an additional 23 polymorphic markers, 14 of which were screened across the whole population. So far, data from 24 markers have been provided for mapping within the diploid population; 4 from ABI377, 6 from HRM screening of the PFR-developed EST-SSRs, and 14 from HRM screening of the SSR markers developed in the U.S. Further markers will be screened across the diploid population to increase marker density on this map and it will then be used to dissect the genetics underlying important plant and fruit traits, to improve our breeding efficiencies by enabling the development and application of marker assisted selection.

TABLE 2 Numbers of Microsatellite Markers That Are Polymorphic, Monomorphic, or Failed to Amplify a Product When Screened Over the Parents and a Subset of Seedlings from the Diploid Blueberry Mapping Population (Rowland et al., 1999) Using an ABI377 and Fluorescent Primer Method ^z

Microsatellite marker type	Polymorphic	Monomorphic	Failed	Total
Di-nucleotide	80	40	4	124
Tri-nucleotide	7	7	1	15
Tetra-nucleotide	4	0	0	4
Complex	5	2	0	7
Total	96	49	5	150
^z“Complex” markers amplify products containing multiple microsatellites consisting of different types.

DISCUSSION AND CONCLUSION

PFR has a well-established blueberry breeding program, which has developed key cultivars important to the NZ industry. This is now well supported by several research projects on compositional chemistry, health, genomics, and genetic marker development. These different research areas have enhanced our understanding and opened new directions for the breeding program. In particular, compositional analysis and health projects have revealed that the ornamental blueberries within the germplasm contain high amounts of important anthocyanins, such as malvidin glucoside, that have been found to be potent antioxidants capable of protecting skeletal muscle under oxidative stress, and as such, are a potential new breeding goal. In addition, the marker team has developed an important resource of EST-SSR markers for use within the USDA-SCRI project and other research programs internationally. We have also successfully used HRM technologies to reveal sequence difference in previous un-mappable SSR markers. We plan to expand this technology into the development of SNP markers for key candidate genes involved in the polyphenolic pathway and the control of chilling requirements, to aid the USDA-SCRI target of increasing our understanding of this key environmental relationship to enhance blueberry production. This information will be used to develop “designer” blueberry cultivars that contain very high amounts of health-related compounds and have been tailored to grow in a range of environments.

ACKNOWLEDGMENTS

We would like to thank the various research teams based at PFR who collected and analyzed the data included in the publications used in this review. In addition, we would like to thank the New Zealand Ministry of Science and Innovation (C06X0407, C06X0807); Blueberry New Zealand Limited, the USDA Speciality Crop Research Initiative and Plant & Food Research for internal funding to support the various research projects covered in this review.