PHYLOGENETICS ANALYSIS OF NORTH AMERICAN NATIVE CYNTHIANA/NORTON GRAPE VARIETY USING DNA MICROSATELLITE MARKERS

Proc. Fla. State Hort. Soc. 119:61-65. 2006. PHYLOGENETICS ANALYSIS OF NORTH AMERICAN NATIVE CYNTHIANA/NORTON GRAPE VARIETY USING DNA MICROSATELLITE MARKERS LELAN PARKER, PATRICIA BORDALLO AND VIOLETA COLOVA* Florida A & M University Center for Viticulture and Small Fruit Research 6505 Mahan Drive Tallahassee, FL 32317 Additional index words. Vitis aestivalis, Michaux, SSR markers, ampelography, grape genetics Abstract. Cynthiana / Norton is considered the best American native grape variety for fine wine with suspected tolerance to PD (Pierce s disease) and proven low susceptibility to foliar and fruit disease. This variety has been successfully grown for commercial wine production for more then a century in the Eastern United States, and over a decade in Louisiana under the PD area. Given that this grape variety also produces color stable wines, it has great possibility in Florida but needs to be evaluated before it can be recommended. DNA microsatellites are proven to be the markers of choice for this purpose because they are diffused in a codominant Mendelian manner. Using the methods of molecular analysis the parent cultivars and the offspring can be recognized and the pedigree describing the genetic history of the grape variety can be reconstructed. By examining the microsatellite allele composition of an individual and its two presumptive parents, it is possible to confirm or reject the proposed parentage. We are using an already published set of 13 pairs of SSR primers designed for other Vitis sp. For purpose to develop more specific markers, Norton publicly available expressed sequence tags (ESTs) were used to screen for SSR. Two hundred two SSRs out of 2,101 ESTs were located, and the promising SSR primers were synthesized for specific amplification in the targeted grape accessions. It is assumed that Cynthiana / Norton is originated from Vitis aestivalis Michaux. We are investigating the phylogenetics of Cynthiana / Norton grape via data mining in the existing North American grape germplasm collections, ampelographic analysis and specifically expressed in the variety microsatellite markers. This study is expected to identify the phylogenetics and genetic make up of Cynthiana / Norton grape and to verify to the growers that this particular grape variety carry tolerance to Pierce s disease and is appropriate for high quality red wine production under Florida environmental conditions. Cynthiana / Norton is reported to be of predominantly Vitis aestivalis Michaux ancestry, developed during the mid part of the 19th century (Reisch et al., 1993). Vitis aestivalis can be found almost anywhere in the eastern and central USA, from New England to Florida and from Wisconsin to Texas (Galet, 1998). Vitis aestivalis has several closely related species and many variants have created numerous confusions and contradictions between the taxonomists trying to classify these native American grape species. Cynthiana / Norton has excellent wine characteristics and is particularly well suited to humid regions with comparatively long growing seasons (Reisch et al., 1993). The general resistance that Cynthiana / This research is funded by FDACS, VAC Grant # 009083. *Corresponding author; e-mail: Violetka.Colova@famu.edu Norton exhibits makes it attractive to wine growers, especially in this area of environmental protection and pesticide avoidance. Ampelography shows a close connection between Cynthiana / Norton and the native Vitis aestivalis, but the rest of the parentage remains controversial. It is believed that Cynthiana / Norton is a seedling of Bland and Vitis aestivalis, unfortunately Bland is no longer in existence and its parentage is indistinct (Ambers and Ambers, 2004). It is hypothesized that Bland was a hybrid of a labrusca vinifera cross (Hedrick, 1908), the vinifera possibly being that of the white grape variety European Chasselas. Therefore, a clarification of the pedigree of the variety is of great importance not only to the Florida grape and wine industry, but also nationwide. In our studies we are aiming to reconstruct the parentage of Cynthiana / Norton by combining the use of DNA fingerprinting via microsatellite markers, and Simple Sequence Repeats (SSR) with data mining in the existing germplasm collections and comparative morphological description and ampelographic analysis of the variety itself and close wild grape relatives. This paper presents some preliminary results accumulated in the course of study and our findings for the variety via employed comparative morphological descriptors, ampelographic analysis and DNA fingerprinting approach. Materials and Methods Plant material. Our experimental plot, 0.7 acres, was planted with Cynthiana grape plants in 2003 at FAMU/CESTA, Center for Viticulture & Small Fruit Research located in Leon, Co., Fla. For proven authenticity, the planting material was taken from Post Winery and Vineyard, Altus, Ark., the chief recognized nursery-distributor for Cynthiana in the U.S. Three year old, well established vines at anthesis and fruit set were marked in the vineyard to serve as a donor material for morphological similarity and also for DNA isolation as an accession of Cynthiana. One accession of Southern Vitis aestivalis, vine growing at the site of Carriage Factory Restaurant, Quincy, Fla., was identified and included in this study. From the Grape National Germplasm Repositories at Geneva, N.Y. and Davis, Calif., 7 more accessions (5 Vitis aestivalis; 2 Vitis aestivalis var. aestivalis) from wild aestivalis were identified for inclusion in the study. Noble vines from the experimental vineyard (Henscratch Nursery, Lake Placid, Fla.), Chardonnay vines (Vintage Nursery, Indio, Calif.) and Concord vines (Ison s Nursery, Brooks, Ga.) were used as individual accessions for V. muscadinia, V. vinifera, V. labrusca, and Vitis riparia grape species, respectively. Ampelographic analysis. Morphological characterizations were performed upon Cynthiana, Southern aestivalis, and Vitis labrusca following the descriptor list for the distinction of genus and varieties of the Office International de la Vigne et du Vin (OIV) and Union for Protection of New Varieties of Plants (UPOV). Ampelographic data was recorded for the three plants young shoot, and shoot, young leaf, and mature leaf. Proc. Fla. State Hort. Soc. 119: 2006. 61

DNA Isolation and Molecular Analysis. DNA was extracted using the Qiagen Protocol for Isolation of DNA from Plant Tissue (DNAeasy Plant Mini Kit, 2003). DNA was extracted and kept in a freezer at -20 C. Isolated DNA from each of the 7 samples was quantified using a Hoefer DyNA Quant 200 Flourometer. Ten Simple Sequence Repeat (SSR) markers were developed for Norton, based upon their Expressed Sequence Tags (ESTs), by Dr. Patricia Bordallo. Retrieving information from the National Center for Biotechnology Information (NCBI), the DNA sequence information was able to be found for Norton. The primers were then tested on Norton, Cynthiana, Southern aestivalis, Northern aestivalis, Vitis labrusca, Vitis vinifera, and Vitis riparia. The primers were given codes of SSR 1, SSR 2, SSR 3, SSR 4, SSR 5, SSR 6, SSR 7, SSR 8, SSR 9, and SSR 10. A set of 12 SSR markers already published for Vitis riparia (Sefc et al., 1999), which give amplification within the Vitis aestivalis species, were tested in the 7 samples. These twelve primers are coded as VrSSR 7, VrSSR 12, VrSSR 14, VrSSR 15, VrSSR 21, VrSSR 25, VrSSR 26, VrSSR 47, VrSSR 62, VrSSR 64, VrSSR 67, and VrSSR 79. Six Simple Sequence Repeat (SSR) markers from Vitis vinifera have been tested in Cynthiana out of a set of 25 already published (Lamboy and Alpha, 1998, Thomas and Scott, 1993) for Vitis SSR markers which have been acquired through Sigma-Genosys. These 6 primers are coded as VVMD5, VVMD6, VVMD7, VVMD8, VVS2, and VVS4. PCR (Polymerase Chain Reaction) were carried out in 20 µl volume for SSR primers, containing various samples of 28-182 ng (nanogram) of genomic DNA. For each of the primers, 1 µm of each primer pair, 0.2 mm of 10 mm dntp, 1 of 10 PCR buffer, 1 mm of 50 mm MgCl 2, and 0.5 units of 5U/ µl of Taq DNA Polymerase were used. PCR reactions were carried out using a Eppendorff Thermocycler with the following profile for primers SSR 1, SSR 2, SSR 3, SSR 4, SSR 5, SSR 6, SSR 7, SSR 8, SSR 9, and SSR 10: (i) 94 C for 2 min; (ii) 94 C for 15 s, 52 C for 15 s, 72 C for 20 s per 30 cycles; and (iii) 72 C for 1 min. For primers VrSSR 7, VrSSR 12, VrSSR 14, VrSSR 15, VrSSR 21, VrSSR 25, VrSSR 26, VrSSR 47, VrSSR 62, VrSSR 64, VrSSR 67, and VrSSR 79: (i) 95 C for 5 min; (ii) 94 C for 15 s, 50 C for 10 s per 23 cycles; (iii) 89 C for 15 s, 50 C for 15 s per 23 cycles. For SSR primers VVMD5, VVMD6, VVMD7, and VVMD8 with the following profile for primer VVMD5: (i) 94 C for 2 min; (ii) 92 C for 30 s, 56 C for 30 s, 72 C for 2 min per 40 cycles; (iii) 72 C for 7 min; for primers VVMD6 and VVMD7: (i) 94 C for 2 min; (ii) 92 C for 30 s, 52 C for 30 s, 72 C for 2 min per 40 cycles; (iii) 72 C for 7 min; for primer VVMD8: (i) 94 C for 2 min, (ii) 92 C for 30 s, 54 C for 30 s, 72 C for 1 min per 5 cycles; (iii) 80 C for 30 s, 54 C for 30 s and 72 C for 1 min per 35 cycles; (iv) 72 C for 7 min; for primers VVS2 and VVS4: (i) 94 C for 6 min, (ii) 94 C for 1 min, 55 C for 1 min, 72 C for 100 s per 45 cycles, (iii) 72 C for 7 min. A 100bp DNA ladder and 1kb was used. Amplification was confirmed after running PCR product in 2% Metaphor agarose, 2% regular agarose gel and 1% regular Agarose + 1% NuSieve Agarose and staining with ethidium bromide and observed under UV light. More experiments are underway testing 7 accessions of European Chasselas acquired from the National Germplasm Repository in Davis, Calif. with each of the primers designed for Norton, Vitis riparia, and Vitis vinifera. Also, fragment analysis will be performed using a DNA fragment analysis software program. Results and Discussions The systematics of the Vitis aestivalis species are a specifically complicated area of taxonomy. The definition of a species may be based on comparative morphology with support from environmental characteristics and geographical location (Subden et al., 1987). Evidence for natural hybridization may be based on the phenotypic resemblance of the supposed hybrid with two or more other species (Meredith et al., 1999). Given the extreme morphological variation among and within Vitis aestivalis species, and the genetic variability of hybrid populations, it is not surprising that there has been a lack of identifying ancestral species. For example Galet (1998), described and accepted Southern Aestivalis, V. Bourquina (or Bourquiniana), as a natural hybrid of aestivalis represented by the cultivars Black Spanish and Herbemont. Bailey (1934), established special section Aestivales in the genus Vitis and in Florida he named 5 separate species under this section. Rogers and Mortensen (1979), tried to put some order in the description of the native grape species for Florida, but they also count 4 subspecies for V. aestivalis in Florida and their description suffered lack of systematical approach and ampelographic knowledge about the internationally recognized morphological descriptors for grape species and varieties. Nevertheless, use of these standard methods alone would have enabled some longstanding errors to be corrected. Morphological characterizations performed upon Cynthinana, Southern aestivalis, and Vitis labrusca young shoot and shoot, young leaf, and mature leaf using the OIV and UPOV descriptor list demonstrated similarities and differences within the varieties (Table 1). Morphological characterizations for the three varieties woody shoot, bunch and berry size are under way. Simple Sequence Repeats (SSRs), also known as microsatellites are tandem repeated DNA sequences. These repeated sequences are common in genomic DNA. Primers designed to test these repeated sequences at different loci have been used to distinguish genetically different grapevines. Genetic markers produced with SSR primers have several advantages over other DNA-based markers. SSRs are detected at specific loci; they are highly reproducible; and because SSRs use the Polymerase Chain Reaction (PCR) process, the amount of sample tissue DNA is very low (Lin and Walker, 1998). SSR markers are ubiquitously distributed throughout genomes making these markers particularly useful in many applications such as parentage studies (Meredith et al., 1999), genetic mapping studies (Blondon-Adam et al., 2004), and fingerprinting plant varieties (Bowers et al., 1993). Primers VVMD 6 and VVMD 7 (Fig. 1) gave specific amplification for Southern aestivalis. SSR primers labeled SSR 1 (Fig. 3), SSR 2 (Fig. 3), SSR 5 (Fig. 4), and SSR 10 (Fig. 5) amplified only within Cynthiana, Norton, Vitis riparia, and Vitis labrusca. These five primers are good candidates for, and may be used as, specific markers within the Vitis aestivalis species. Primer labeled VrSSR 7 (Fig. 6) amplified within Cynthiana, Northern aestivalis, Vitis vinifera, and Vitis riparia. Primer VrSSR 79 (Fig. 7) amplified within Cynthiana, Norton, Northern aestivalis, Vitis labrusca, Vitis vinifera, and Vitis riparia. The DNA fragments that were visible within the primers will also be further analyzed using fragment analysis software that is yet determined. Conclusion A minimal amount of other crops claim as many varieties as does the grape (Bowers et al., 1993). Estimates place the 62 Proc. Fla. State Hort. Soc. 119: 2006.

Table 1. Ampelographic characters. Ampelographic descriptors Cynthiana Southern aestivalis Vitis labrusca 051 Young leaf: color of upper surface 1 1 1 053 Young leaf: density of prostrate hairs between veins 9 5 9 054 Young leaf: density of erect hairs between veins 3 9 5 055 Young leaf: density of prostrate hairs on main veins 9 9 9 056 Young leaf: density of erect hairs on main veins 3 3 0 001 Young shoot: form of tip 5 3 3 003 Young shoot: anthocyanin coloration of tip 9 9 3 004 Young shoot: density of prostrate hairs on tip 1 0 0 005 Young shoot: density of erect hairs on tip 5 3 0 006 Shoot: attitude (habit) 5 5 9 007 Shoot: color of dorsal side of internode 2 2 1 008 Shoot: color of ventral side of internode 2 2 1 009 Shoot: color of dorsal side of node 1 2 1 010 Shoot: color of ventral side of node 1 1 1 011 Shoot: density of erect hairs on node 3 1 0 012 Shoot: erect hairs on internode 1 1 1 013 Shoot: density of prostrate hairs on node 9 9 0 014 Shoot: density of prostrate hairs on internode 9 9 9 016 Shoot: number of consecutive tendrils 2 2 2 017 Shoot: length of tendril 3 3 3 065 Mature leaf: size of blade 7 7 7 067 Mature leaf: shape of blade 4 4 3 068 Mature leaf: number of lobes 2 2 2 070 Mature leaf: anthocyanin colouration of main veins on upper side of blade 5 3 1 074 Mature leaf: profile 2 2 2 075 Mature leaf: blistering of blade upper surface 5 1 9 076 Mature leaf: shape of teeth 3 1 3 077 Mature leaf: length of teeth 1 1 1 078 Mature leaf: ratio length/width of teeth 1 1 1 079 Mature leaf: general shape of petiole sinus 3 2 3 081 Mature leaf: tooth at petiole sinus 0 0 0 081.2 Mature leaf: petiole sinus limited by veins 0 0 0 082 Mature leaf: shape of upper lateral sinus 1 1 1 605 Mature leaf: depth of upper lateral sinus 3 3 3 084 Mature leaf: density of prostrate hairs between veins 7 5 9 085 Mature leaf: density of erect hairs between veins 9 7 1 086 Mature leaf: density of prostrate hairs 9 7 9 087 Mature leaf: density of erect hairs on main veins 3 1 0 088 Mature leaf: density of prostrate hairs on main veins 1 1 1 093 Mature leaf: density of petiole compared to middle vein 2 2 1 Fig. 1. SSR genotypes of 5 grape species at locus VVMD6 and VVMD7. Amplification products were detected by Metaphor Agarose 3%. Lane 1: Muscadinia (Noble); Lane 2: V. labrusca; Lane 3: Cynthiana; Lane 4: Cynthiana; Lane 5, Southern aestivalis; Lane 6: V. vinifera (Chardonnay); Lane 7: CK-; L, 100bp DNA ladder. Fig. 2. SSR genotypes of 6 grape species at locus VVS2 and VVS4. Amplification products were detected by Metaphor Agarose 3%. Lane 1: Muscadinia (Noble); Lane 2: Vitis. labrusca; Lane 3: Cynthiana; Lane 4: Cynthiana; Lane 5: Southern aestivalis; Lane 6: Vitis vinifera (Chardonnay); Lane 7: CK-; L, 100 bp DNA ladder. Proc. Fla. State Hort. Soc. 119: 2006. 63

Fig. 3. Amplification products were ran on Metaphor Agarose 2%. Lane Lane 5: Southern aestivalis; Lane 6: Vitis vinifera; Lane 7: Northern aestivalis; Lane 8: Vitis riparia; Lane 9: CK-. Fig. 6. Amplification products were ran on Metaphor Agarose 2%. Lane 1: DNA ladder 50 bp; Lane 2: Cynthiana; Lane 3: Northern aestivalis; Lane 4: Southern aestivalis; Lane 5: Vitis labrusca; Lane 6: Vitis vinifera; Lane 7: Vitis riparia; Lane 8: CK-; Lane 9: DNA ladder 50 bp. Fig. 4. Amplification products were ran on Metaphor Agarose 2%. Lane Lane 5: Southern aestivalis; Lane 6: Vitis vinifera; Lane 7: Northern aestivalis; Lane 8: Vitis riparia; Lane 9: CK-; Lane 10: DNA ladder 50 bp. Fig. 7. Amplification products were ran on Metaphor Agarose 2%. Lane 1: DNA ladder 50 bp; Lane 2: Cynthiana; Lane 3: Norton; Lane 4: Northern aestivalis; Lane 5: Southern aestivalis; Lane 6: Vitis labrusca; Lane 7: Vitis vinifera; Lane 8: Vitis riparia; Lane 9: CK-; Lane 10: DNA ladder 50 bp. Fig. 5. Amplification products were ran on Metaphor Agarose 2%. Lane Lane 5: Southern aestivalis; Lane 6: Vitis vinifera; Lane 7: Northern aestivalis; Lane 8: Vitis riparia; Lane 9: CK-; Lane 10: DNA ladder 50 bp. number of cultivars in V. Vinifera species alone between 5,000 and 15,000 (Galet, 1979). When viticulture was a traditional pastime centered on native cultivars, differentiating the parentage of varieties was less important. As viticulture continues to grow, a great necessity has been placed upon the genetic background of numerous grape varieties. Today, the demands of international wine trade, the need to protect patented cultivars, and improved communication among researchers in various countries have made accurate identification of grape pedigrees essential. By discovering and analyzing the phylogenetics and parentage of Cynthiana / Norton, a new and suitable for growth in Florida grape variety, yielding quality red, color-stable wine when grown in Florida may be scientifically recommended for commercial production. The use of 64 Proc. Fla. State Hort. Soc. 119: 2006.

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