Existing public and unpublished databases of grapevine SSR profiles .
Grapevine cultivation is increasing worldwide as people realize the benefits of grape and wine consumption. To improve yield and enhance the quality of grapes, biotechnology research plays an ever-increasing role. In recent years, the sequencing of multiple grape genomes has led to increased vibrant research initiatives on grape improvement. These novel approaches include those related to the application of transgenic technology toward the improvement of grape varieties. These advancements include the development of molecular markers for valuable traits, improved plant transformation systems, genetic engineering to enhance disease tolerance in grape cultivars, and the identification of flavor and aroma components to improve the enological quality of grapes. Some of the results obtained by various researchers have direct application, whereas others are yet to gain direct application in grape quality improvement, although such techniques possess potential qualities, which can be exploited for genetic breeding of Vitis species. This chapter highlights selected advancements in grape biotechnology from recently reported research activities.
- Vitis spp.
Worldwide, grapes are one of the most widely cultivated fruit crops, encompassing 6.9 million hectares of arable land from which 74.3 million metric tons were produced in 2017 . From the 2017 data, grapes ranked third among crops such as bananas, apples, and oranges that produced 113.9, 83.1, and 73.3 million metric tons, respectively. Since most of the harvested grapes are usually fermented into wine, it is suggested that its economic potential is greater than those of other comparative commodity crops. For example, wine sales from California alone in 2018 generated approximately $40 billion in sales . According to 2015 statistics, the California wine industry contributed $57.6 and $114 billion to both the state and the US economies, respectively. The three major uses for grapes are winemaking, fresh fruit (table grapes), and dried fruit (raisins) production. The products derived from grapes or winemaking include grape juice, jelly products, ethanol, vinegar, grape seed oil, tartaric acid, and fertilizer.
Potential health benefits of certain grape-derived antioxidant compounds (polyphenols, resveratrol) have also contributed to increased research to investigate its compounds for their nutraceutical value. Grape extracts are used food additive, cosmetic, and pharmaceutical industries. Statistics from winemaking is steeped in history and tradition—perhaps more than any other food or beverage industry. From the soil, climate, and harvesting of grapes to the crushing and aging processes, painstaking attention to detail dictates the flavor, bouquet, and the overall sensory experience of the final product. Hence, it may not be a surprise to learn that the grape industry is increasingly looking toward biotechnology for new opportunities to improve strategies for combating crop diseases and lower production costs for producing healthier and more flavorful products.
1.1 Historical development of grape biotechnology research
Grape breeding started very early, first for wine grapes and, by the end of the nineteenth century, for table grapes. Breeding for rootstocks started toward the end of the nineteenth century after the era of
Globally, table grape production represents 27% of the 750,000 hectares planted with this species. Although table grape production in North and South America mainly represents c.a. 18% of the total world production, North America accounts for almost 50% of the global exports. Main exporters are Chile and Italy, followed by the USA, South Africa, and Mexico. Of the thousands of existing cultivars, only about 20 are grown for fresh consumption, with “Sultanina” (“Sultani,” “Sultana,” “Kishmish,” or “Thompson Seedless”) representing about 40% of the grapes grown for fresh consumption. This cultivar has been used extensively as a parental line for the development of new cultivars, such as “Flame Seedless” and “Crimson Seedless.” These varieties, together with “Red Globe” (an important seeded cultivar due to its excellent postharvest life, high productivity, and public acceptance), are some of the most cultivated worldwide. During the last decade, new biological and genetic information are available to plant breeders, particularly in the area of biotechnology.
Biotechnological tools have been incorporated into breeding programs focused on the improvement of genetic diversity [3, 4]; fingerprinting applications based on codominant markers; quantitative trait loci (QTL) mapping and identification of candidate genes linked to QTLs for quality traits; development of cDNA libraries designed for the identification of genes involved in plant and berry development and host-pathogen interactions; and finally, the establishment of a genetic transformation platform available for the introduction of genes of interest as well as for the evaluation of gene function(s) using the grapevine as a model for woody plant species. The grape genome project was started in 2005 with collaborators in France and Italy within the framework of the International Grape Genome Project (IGGP).
The grape genome is attractive to genomic research due to its diploid chromosome with a small genome size of 475–500 Mb. The economic importance of the
2. Grapevine breeding
Most breeding programs initially were publicly funded, but nowadays many of them are privately owned. Mostly new cultivars are protected by intellectual property rights, and, hence, growers need to pay royalties for their use or they may not gain access to some of the cultivars stored in closed commercialized “entities.” Due to this new scenario, many countries and companies started their own private breeding programs. In 1988, the Chilean Institute for Agricultural Research started a breeding program to develop new table grape cultivars with emphasis on seedless grapes, disease resistance, and postharvest life . Since the production of seedless cultivars, crosses were made among the seedless cultivars followed by in vitro embryo rescue. Early in the program, researchers have realized that certain cultivars were more efficient for embryo rescue. For example, in “Ruby Seedless” and “Red Seedless,” 68% and 40% of the embryos, respectively, could be rescued, but with “Superior Seedless” or “Black Seedless,” less than 30% of the embryos could be rescued .
As with other crops, plant breeders faced difficult task to develop high-vigor cultivars that would combine high yield with good quality traits. Quality in table grapes is associated with genetic factors, but also with environmental factors, most of which can be managed by different agricultural practices which can influence yield. Quality traits in table grapes are also influenced by consumer preferences, an important factor to be considered by grape breeders. Good berry quality characteristics include seedlessness, berry size, skin thickness, uniformity, aroma, firmness, flavor, texture, etc. present during harvest and after prolonged storage [7, 8]. More recently, characters such as the presence of nutritional components and nutraceutical determinants have gained increased traction. Postharvest traits of importance include resistance to prolonged storage and transport, rachis tolerance to oxidation and dehydration, low susceptibility of the berries to browning and spotting, as well as resistance to decay.
2.1 Application of biotechnology research to grapevine breeding and genetics
Research in grapevine genetics is restrained by the lack of genetic stocks, high heterozygosity, inbreeding depression, large space requirements, and the relatively long juvenile period. In 1957, De Lattin  summarized his work on 53 genes identified in
Genetic resources possessing genes for resistance to many fungal diseases were found within
2.2 Marker-assisted selection
Marker-assisted selection can be used for pyramiding genes for resistance. Genetic pyramiding is a process used for the development of new breeding lines with homozygous resistance loci and consequently selecting new parental lines with the desired traits. To understand the potential value of molecular markers, it is imperative to identify the major markers. Isozymes have different electrophoretic mobility and, hence, can be visualized following gel electrophoresis. Over 20 polymorphic isozymes have been identified in grapes. Restriction fragment length polymorphisms (RFLPs) can be used for their rapid detection using restriction enzymes and involves cutting genomic DNA molecules at unique nucleotide sequences (restriction sites) yielding DNA fragments with varied sizes. However, identification of RFLPs requires a high concentration of DNA and could be relatively expensive to assay.
Polymerase chain reaction (PCR)-based assays are generally much less expensive and can reveal higher levels of polymorphism [11, 13]. The selection process of a DNA fragment for amplification involves “primer annealing” in which two primer pairs (5–30 bases long) complementarily bind onto genomic DNA strands in a reaction process. The primer-DNA complex is a critical step for the replication of adjacent DNA sequences by a thermostable polymerase supplied in the reaction mixture.
A commonly used PCR analysis is based on random amplified polymorphic DNA (RAPDs). These markers are based on the occurrence of an inverted pair of 9–11 base repeats (occasionally longer or shorter, as well) as within between 200 and 2000 base pairs. This is a single primer reaction that amplifies one-to-many segments of DNA through PCR. Amplified fragment length polymorphisms (AFLPs) are based on the selective amplification of restriction enzyme-digested DNA fragments. Multiple bands (50–100) are generated during each amplification reaction resulting in random DNA markers. Neither RAPDs nor AFLPs are “anchored,” i.e., their primary use is within and not between crosses. On the other hand, several sequence-tagged site (STS) markers are useful as anchoring loci between crosses. The most important of these is a
2.2.1 RAPD markers
Genetic analyses have progressed rapidly since the discovery of polymorphic regions or loci with two or more alleles in genomic DNA . Variation in location, copy number, length, and base pair sequence of these highly repetitive DNA regions provide a rich source of markers for unique identification. Random amplified polymorphic DNA analysis has been applied to several aspects of the winemaking process [15, 16]. Several investigators have attempted to discriminate between grape plant clones utilizing a variety of genetic typing techniques [17, 18, 19, 20, 21, 22, 23]. However, Regner et al.  utilized SSR, RAPD, and AFLP markers and were successful in detecting differences within clones of the Grüner, Veltliner, Pinot Blanc, Morillion, and Chardonnay varieties. Using RAPD markers, Moreno et al.  discriminated between clones of
Microsatellite genotyping requires the determination of the number of repeat units at a given locus in a given cultivar. This is achieved by electrophoretic sizing of the fragment containing the repeat region (the microsatellite allele), which was amplified by PCR with primers situated upstream and downstream of the microsatellite DNA. The initial grapevine microsatellite study conducted by Thomas and Scott  at CSIRO Plant Industry, Australia, reportedly identified DNA isolated from 26
Since then researchers have accumulated microsatellite profiles of hundreds of grapevine cultivars from many different regions. The data is available in public databases (Table 1). Among the 19 chromosomes of grape genome from a homozygous line, PN40024, about 10,948 contained trinucleotide repeats, 4386 had tetranucleotide repeats, and 3347 had penta-nucleotide repeats .
|Database name||Physical address||Internet address of public databases||Number of genotypes|
|European ||IRZ, Siebeldingen, Germany||http://www.genres.de/eccdb/vitis/||In preparation|
|Grape Microsatellite Collection (GMC)||IASMA, San Michele, Italy||Not public|
|Grape SSR database||Australian Wine Research Institute (AWRI)||Not public|
|International ||IRZ, Siebeldingen, Germany||http://www.vivc.bafz.de/index.php||46|
|SSR profiles (not searchable)||BOKU, Vienna, Austria||http://www.boku.ac.at/zag/forsch/grapeSSR2.htm||162|
|The Bulgarian Plant Genomics Database||Agrobioinstitute, Sofia, Bulgaria||http://bulgenom.abi.bg/AgroBioInstitute%20Selected.htm||76|
|The Greek ||University of Crete, Heraklion, Greece||http://gvd.biology.uoc.gr/gvd/index.htm||298|
|The Swiss ||University of Neuchâtel, Switzerland||http://hydra.unine.ch/svmd/||170|
|Ukrainian, Moldovan and Russian ||Magarach Institute, Yalta, Ukraine||Not public||104|
|University of California, Davis, USA||Not public|
|INRA Montpellier, France||Not public|
2.2.3 Single-nucleotide polymorphism (SNP)
Single-nucleotide polymorphism (SNP)-based genetic markers have attracted significant attention when researchers are creating dense genetic linkage maps. SNPs are the most abundant class of polymorphisms, and they provide gene-based markers that may prove useful when identifying candidate genes of interest to be associated with quantitative trait loci.
In grapes, polymorphic DNA loci are relatively frequent. Salmaso et al.  found a single SNP in every 116 bp in the coding regions of 25 genes using EST-derived primers in the analysis of seven
3. Grape genome sequence and its applications
The genome project was informed by the realization that the
The first high-quality reference grape genome sequence was obtained from a Pinot Noir clone ENTAV 115, a variety grown in wide range of soil types to produce red and sparkling wines. The reference genome sequence information has been useful toward understanding its overall genetic organization, including the content of genes and the structural components of the DNA of the 19 linkage groups (LGs) of
The estimated genome size of
The NCBI taxonomy web portal for
Genetic maps have been produced [33, 61, 62, 63, 64], and physical maps are being produced in several laboratories  with a consensus map in progress. A grape BAC library is available from the French National Resources Center for Plant Genomics (CNRGV). Affymetrix (http://www.affymetrix.com/index.affx) released a grape array that represents 14,000
Qiagen (http://www1.qiagen.com) also released a new grape (
4. Genetic transformation in grapevines
Genetic transformation offers new perspectives for introducing important traits like that of disease resistance into traditional
Leaf disc derived embryogenic callus for grapevine cv. Koshusanjaku by Hoshino et al. , who subsequently established an
4.1 Genomics and transgenic research
In the past, various studies have been conducted on the origin and regulation of sugar and acid concentrations. Of these two processes, the regulation of acid levels is probably well-understood. It is clear, for example, that the two most important acids, namely, tartaric acid and malic acid, have different origins. Tartaric acid is produced directly out of the sugar pool, while malic acid is probably formed by reactions of the Krebs cycle and phosphoenolpyruvate carboxylase (PEPcase) . During ripening, malic acid is used for the synthesis of sugar and as a respiration substrate . Less is known about the control of tartaric acid concentration, which is also far more slowly metabolized than malic acid. Recently researchers have identified two important wine quality genes in grapevine related to tannin synthesis. By looking at when and wherein the plant tannins are produced throughout berry development and comparing similar genes in tobacco and the model plant
In grape, flavonoids are the major portion of soluble phenolics and represent the most concentrated natural antioxidants in the berry . The predominant flavonoids occurring in grape berries and seeds belong to varied classes such as tannins, anthocyanins, flavan-3-ols, and flavonols . These compounds in addition to phenolic acids (mainly benzoic and hydroxycinnamic acids) contribute in different ways and/or manner to organoleptic features of the wine and other by-products . Flavonoids are synthesized along the general phenylpropanoid pathway by the activity of a cytosolic multienzyme complex loosely associated at the cytoplasmic surface of the endoplasmic reticulum. This pathway has largely been characterized in different plant species  but also in
In berry pulp their expression is low, and phenylalanine ammonia-lyase (
Anthocyanins are responsible for the red and white color in grapes. Grapes are primarily distinguished based on the level of anthocyanin in berry skin. Geneticists discovered that the grape skin color is controlled by two
Until now, the models of flavonoid transport have been mainly based on genetic approaches where this process has been correlated to the expression of several specific genes in reproductive organs during development or in response to environmental factors. Limited information is available for direct identification and characterization of proteins involved in the uptake and accumulation of these metabolites. Therefore, it is crucial that future research should be more focused on the understanding of the biochemical mechanisms responsible for flavonoid transport and regulation .
4.1.3 Nutraceutical value
Previous  study conducted at the Center for Viticulture and Small Fruit Research, Tallahassee, Florida, which involved the analysis of metabolites in local grape varieties with high-performance liquid chromatography (HPLC), determined a high phenolic content in muscadines as compared to bunch and Florida hybrid bunch grapes . Grape seed extracts from some muscadine grape cultivars showed high anticancer activity. Characterization of these compounds confirmed the presence of resveratrol. One other advantage of resveratrol is its
4.1.4 Disease control
|Causal agent||Properties of pathogen||Disease||Specific characters of disease|
|Uncinula necator||Obligate biotrophic fungus||Powdery mildew||The most economically important disease of |
|Plasmopara viticola||Obligate biotrophic oomycete||Downy mildew||Affects |
|Botrytis cinerea||Necrotrophic fungus||Grey mold rot||One of the most common and widely distributed grapevine diseases|
|Elsinoe ampelina||Non-obligate fungus||Anthracnose||Affects |
|Phomopsis viticola||Non-obligate fungus||Phomopsis cane blight and leaf spot||A wood disease|
|Non-obligate fungus||Excoriosis||A wood disease|
|Ascomycete fungus||Eutypa dieback||A major grapevine disease in many countries that infects the vine stock; a wood disease|
Knowledge and experience in the field of genetically engineered grapevines have increased enormously. Several ongoing projects are aimed at the improvement of transformation efficiency, allowing its use as a standard strategy for various purposes. The great interest in the transgenic approach is due to its capability to establish disease tolerance or resistance in both elite grapevine varieties and rootstocks without changing their genotype-specific traits. Progress made in grapevine genomics along with the availability of reference genome sequence obtained from Pinot Noir  has made the transgenic approach attractive for both basic research and functional genomics. Included herein is a list of major transgenic disease-tolerant plants, which are currently in field trials in the last 5 years for improved bacterial and fungal resistance (Table 3).
|Cornell University||07/21/09||Issued||Coat protein—donor: Grapevine fan leaf virus resistant||Grapevine Fan leaf Nepovirus Resistant||CA, USA||4|
|University of Florida||09/27/07||Acknowledged||FL, USA||1.1|
|University of Florida||09/27/07||Acknowledged||Lytic peptide gene for bacterial resistance ||Powdery Mildew Resistant BR—||FL, USA||1.1|
|University of Florida||09/27/07||Acknowledged||Endogenous gene for fungal resistance—grape lytic peptide gene for bacterial resistance||Powdery Mildew Resistant BR—||FL, USA||1.1|
|University of Florida||09/27/07||Acknowledged||Lytic peptide gene for bacterial resistance||FL, USA||1.1|
|University of Florida||09/27/07||Acknowledged||Lytic peptide gene for bacterial resistance||FL, USA||1.1|
|University of Florida||09/13/06||Acknowledged||Synthetic lytic peptide gene|
Grape thaumatin-like protein gene
|Fungal Resistant, Bacteria Resistant||FL, USA|
|University of Florida||09/13/06||Acknowledged||Neomycin phosphotransferase (NPTII)* Synthetic lytic peptide gene cercopin of Silkworm|
Grape thaumatin-like protein gene
|Fungal Resistant, FR—Fungal Resistant, —Bacteria Resistant||FL, USA|
|University of Florida||09/13/06||Acknowledged||Synthetic lytic peptide gene cercopin of Silkworm|
Grape thaumatin-like protein gene
|Bacteria Resistant, Fungal Resistant||FL, USA|
|State University of New York||08/02/06||Acknowledged||Lignan biosynthesis protein from peas||Powdery Mildew Resistant||NY, USA||1|
|Cornell University||03/03/06||Acknowledged||Antimicrobial peptide from |
|Pathogen resistant||TX, USA||0.1|
|State University of New York||04/11/05||Acknowledged||Lignan biosynthesis protein||Powdery Mildew Resistant||NY, USA||1|
22.214.171.124 Downy mildew
Different strategies and genes have been used in genetic engineering to enhance resistance to major plant pathogens . Expression of a fungal endochitinase gene in cv. “Chardonnay” led to reduced symptoms of powdery mildew and
The expression of synthetic magainins, such as Myp30  and MSI99 in transgenic plants via either the chloroplast genome  or in the nuclear genome , led to enhanced resistance against bacterial and fungal pathogens. The studies in “Chardonnay” (
126.96.36.199 Pierce’s disease
The most devastating diseases in the southeastern United States include Pierce’s disease commonly present on bunch grapes and anthracnose that infects Florida hybrid bunch grapes (Figure 1) . Pierce’s disease, caused by the bacterium
A study was conducted at the Center for Viticulture and Small Fruit Research, Tallahassee, Florida, USA, to understand the molecular basis of Pierce’s disease tolerance by employing subtractive hybridization (SH) and real-time PCR for the detection and characterization of transcripts, which are differentially expressed in the xylem tissue challenged by PD bacterium. Results obtained from the SH analysis of 300 partial cDNAs indicated high to moderate expression patterns in PD-tolerant
Transgenic Pierce’s disease-resistant plants have been developed using polygalacturonase-inhibiting proteins (PGIPs) and antimicrobial peptides. PGIPs are plant cell wall proteins that specifically inhibit fungal endo-polygalacturonases (PGs) that contribute to an aggressive decomposition of susceptible plant tissues. The inhibition of fungal PGs by PGIPs suggested that PGIPs have a role in plant tolerance to fungal infections, and this has been confirmed in transgenic plants expressing PGIPs. The bacterium,
AMPs are particularly effective against bacteria since they disrupt cell membranes. To date, transgenic plants with antimicrobial peptides have been generated. The plants included 76 “Chardonnay” lines and transformed with two magainin-type genes, mag2 and MSI99, as well as a PGL class gene. The primary objective of the research was to study the potential resistance to Pierce’s disease of magainin- and PGL-producing vines. A newly designed antimicrobial peptide was developed based on natural antibacterial toxins. For example, shiva-1 peptide was designed with a significantly different sequence from natural cecropin B (46% homology) [85, 86]. A more advanced generation of lytic peptides was based on the synthesis of newly designed antimicrobial peptides instead of its natural antibacterial toxin . The design of synthetic antimicrobial peptides with predetermined structures and properties led to improved stability of these gene products and enhanced their protection property against proteases in the transformed plants.
In another research with the objective to control Pierce’s disease, Aguero et al.  studied transgenic plants of grape cvs. “Chardonnay” and “Thompson Seedless” by expressing the pear polygalacturonase protein (pGIP). They reported a delayed development of Pierce’s disease in some of the transgenic lines. The lines had reduced leaf scorching, lower titers of
Genetic resources possessing genes for resistance to many fungal diseases have been found in
Recently, researchers at the Center for Viticulture and Small Fruit Research, Tallahassee, Florida, USA, successfully identified genes/gene products from Florida hybrid grape that are uniquely expressed in response to anthracnose infestation postinoculation with pure cultures of
The grape industry must maintain and expand grape production despite increasing constraints caused by pests, diseases, and abiotic stressors. Biotechnology represents one of the most promising approaches that can bridge the knowledge gap that exists since it is crucial for the introduction of single gene determinants with defined phenotypic traits. In addition to aiding the production of grapevine varieties (disease-resistant and stress-tolerant), biotechnology has also contributed to the modification of numerous quality traits, such as color, flavor, ripening characteristics, and modulation of specific metabolites with potential health benefits. Multiple genes for disease resistance and/or modification of quality traits should be inserted simultaneously into grape cultivars. However, researchers are still concerned that the product of a single gene could readily be overcome by virulent pathogens. New genes are being sought from grapevines and other close relatives with an attempt to create a comprehensive genetic gene pool for the improvement of grapevines. Genetically altered vines should be subjected to stringent field testing to assure the public that ultimately the technology will be safe and will not alter essential traits of both the vine and the fruit. These advantages can only be realized if concrete strategies are put in place to overcome potential technical hurdles. Strategies should be put in place that involve a comprehensive regulatory framework to improve the general public acceptance of such biotechnology-derived foods.
The corresponding author Kambiranda D would like to acknowledge the funds received from USDA-Evans-Allen project 621660.