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Interspecific hybrid identification of Vitis aestivalis-derived ‘Norton’-based populations using microsatellite markers
Scientia Horticulturae 179 (2014) 363–366
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Interspecific hybrid identification of Vitis aestivalis-derived ‘Norton’-based populations using microsatellite markers Pragya Adhikari a , Li-Ling Chen a , Xu Chen a,b , Surya D. Sapkota a , Chin-Feng Hwang a,∗ a b
State Fruit Experiment Station at Mountain Grove Campus, Darr School of Agriculture, Missouri State University, Springfield, MO 65897, USA Pennington Seed, Inc., Greenfield, MO 65661, USA
a r t i c l e
i n f o
Article history: Received 11 July 2014 Received in revised form 25 September 2014 Accepted 27 September 2014 Available online 21 October 2014 Keywords: Vitis Interspecific hybrids Molecular marker Marker-assisted selection
a b s t r a c t The Vitis aestivalis-derived ‘Norton’ is one of the very few commercial red grape varieties that can be grown in regions with high disease pressure and cold temperatures in winter and spring where V. vinifera is difficult to grow. This study began with the objectives of generating interspecific hybrids of Norton and V. vinifera (Cabernet Sauvignon, Pinot Noir, Syrah, and Merlot) in order to combine the disease resistance and cold hardiness characters of V. aestivalis with the excellent wine quality of V. vinifera. Norton grapes were also crossed with the Vitis interspecific hybrid ‘Vignoles’ for the same reason. The true hybrids obtained from all of the crosses were identified using microsatellite markers. A high percentage of hybrids (70–92%) were obtained in all crosses except Norton × Merlot (17%). Results demonstrate the reliability of SSR markers for Norton-based interspecific hybrid identification. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Norton is a unique grape with good wine characteristics and thought to originate from an accidental cross of a lost grapevine ‘Bland’ (a hybrid of V. labrusca and Chasselas) with pollen from V. aestivalis (Ambers, 2013). It produces a dark, full-bodied, red wine without the intense “foxy” flavors and aromas that taint the wines of most American hybrid grapes (Herald and Herald, 1988). The Norton grape is mainly grown in many U.S. wine growing regions with high humidity and comparatively long growing seasons where V. vinifera cultivars are difficult to grow and require extensive pesticide use (Reisch et al., 1993). Two prominent characteristics of Norton grape are: (1) high levels of resistance to powdery mildew, downy mildew, black rot, bitter rot and Botrytis bunch rot, which are collectively the most destructive fungal diseases of grape worldwide; (2) high levels of anthocyanins that are associated with the health benefits of consuming grapes and wine (Hogan et al., 2009). Norton was selected as the official grape variety of the State of Missouri on 11 July 2003 (Missouri Code of State Regulation, 2010). The disease resistance and cold hardiness of Norton have enhanced its attractiveness to wine growers due to increasing concerns regarding environmental protection and pesticide avoidance. However, only limited breeding efforts have been made using this variety. Research in the genetics and genomics of Norton traits
∗ Corresponding author. Tel.: +1 4175477538; fax: +1 4175477540. E-mail address: [email protected] (C.-F. Hwang). http://dx.doi.org/10.1016/j.scienta.2014.09.048 0304-4238/© 2014 Elsevier B.V. All rights reserved.
that are involved in disease resistance and berry quality have not yet been fully explored or understood. Genetic mapping of Norton × V. vinifera populations may elucidate the underlying genetic and molecular mechanisms of berry disease resistance and berry quality. Simple Sequence Repeats (SSR) or microsatellite markers allow for the selection of true hybrids at the seedling stage; therefore, offtypes in a cross from both selfings and outcrossing can be discarded in the F1 generation (Schuck et al., 2011). DNA microsatellites are abundant in the grape genome, follow Mendelian inheritance, are co-dominant, highly polymorphic, and have proved their usefulness for the genetic analysis of a heterozygous species like grape (Powell et al., 1996; Riaz et al., 2004). The SSR markers are also highly valuable because of their interspecies transferability, allowing direct comparison of linkage groups with previously published maps of Vitis species (Doligez et al., 2006). Six highly polymorphic SSR markers (VVMD5, VVMD7, VVMD27, VVS2, VrZAG62 and VrZAG79) have been proposed as standard markers for the analysis of grapevine cultivars following the analysis of 46 grape cultivars at these six loci in 10 different laboratories in seven countries (This et al., 2004). However, microsatellites have not been implemented for the analysis of Norton-based hybrid populations. Once true hybrids are identified, these hybrid populations can subsequently be used to construct a linkage map of Norton and to identify markers associated with desirable traits. In this study, 448 progenies resulting from the crosses between V. aestivalis-derived ‘Norton’ and each of five different cultivars (Cabernet Sauvignon, Pinot Noir, Syrah, Merlot, and Vignoles)
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were screened to determine interspecific hybrids using the six internationally-adopted standard SSR markers listed above. Additional markers were used when no polymorphism was found. This work emphasizes the efficiency of microsatellite markers that can be used to greatly accelerate the breeding process and allow a much more accurate selection of progeny. 2. Materials and methods 2.1. Plant materials Crosses using V. aestivalis-derived ‘Norton’ × V. vinifera ‘Cabernet Sauvignon’ were made in 2005 and resulted in 98 individuals of which 24 were from the reciprocal cross. This F1 population was planted in a Missouri State Fruit Experiment Station (MSFES) vineyard in 2007 and has yielded fruit for the past 3 years. This population was expanded to 310 genotypes in 2011. Additional crosses between Norton and other V. vinifera species including Pinot Noir, Syrah, Merlot and the French–American hybrid ‘Vignoles’ were also made and generated 30, 34, 42 and 32 progenies, respectively. For crosses, female parents were emasculated before anthesis and covered with paper bags to avoid contamination from unwanted pollen. The pollen of desired paternal parents was collected, placed under a 60 W lamp, dried overnight and stored at room temperature for further use. Pollination was done with a brush the morning after emasculation, and the inflorescences were tagged and re-covered with the paper bags. After harvest, the seeds were extracted and washed with water to remove the pulp. The seeds were then placed in sterilized sand and stratified at 4 ◦ C for 3 months. Seeds were planted in seedling trays and germinated within 3 weeks in a greenhouse. 2.2. DNA extraction One hundred mg of young leaf tissue was ground to fine powder in liquid nitrogen. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Concentration and purity of DNA were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The DNA was normalized to 15 ng/L and stored at −20 ◦ C. 2.3. PCR amplification PCR amplification of microsatellite marker alleles used three primers per reaction: an M13-tailed, 5 -TGTAAAACGACGGCCAGT3 , forward primer (Oetting et al., 1995), a reverse primer and a WellRED (Sigma–Aldrich, St. Louis, MO) labeled M13 sequence. A PCR reaction was performed in a total volume of 10 L that consisted of 15 ng of template DNA, 0.1 M of forward and 2 M of reverse primer, 2 M WellRed M13 primer, 5 mM MgCl2 , and 5 L AmpliTaq GoldR 360 Master Mix buffer (Life Technologies, Grand Island, NY). Amplification was performed using touchdown PCR consisting of 10 min of denaturation at 95 ◦ C, 10 cycles of denaturation at 94 ◦ C for 30 s, primer annealing at 62 ◦ C for 30 s and extension at 72 ◦ C for 1 min where annealing temperature was decreased by 1 ◦ C at each cycle. This was followed by 24 cycles of denaturation at 94 ◦ C for 30 s, annealing at 56 ◦ C for 30 s and extension at 72 ◦ C for 1 min. The reaction was completed with a post extension cycle at 72 ◦ C for 7 min. Four L of reaction product was visualized on 1.5% agarose gel to confirm the amplifications as well as estimate the length of amplified products (Bio-Rad, Hercules, CA). 2.4. SSR genotyping To expedite the true hybrid screen, the F1 progenies from several Norton-based populations were genotyped at six microsatellite
loci as recommended in This et al. (2004). An additional 16 markers were randomly selected and further tested due to the non-polymorphic nature of the six standard markers on specific populations. The expressed sequence tag (EST)-SSR FAM primer sequences were described in Huang et al. (2011). Other primer sequences are available in the NCBI database uni-STS (www.ncbi.nlm.nih.gov). PCR products were run on a GenomeLab GeXP capillary sequencer (Beckman Coulter, Brea, CA), and alleles were sized with the GenomeLab GeXP Genetic Analysis software, Fragment Analysis Module. The 18 bp M13 sequence was then subtracted from each recorded allele to calculate the actual allele size. 3. Results In 2005 and 2011, a total of 416 genotypes were produced from crosses between V. aestivalis-derived ‘Norton’ and V. vinifera cultivars including Cabernet Sauvignon, Pinot Noir, Syrah and Merlot. DNA fingerprinting of these F1 progenies with the six standard SSR loci indicated that a high percentage of hybrids were acquired in all crosses (70–92%) except for Norton × Merlot (Table 1). Each sample was first analyzed by using six standard SSR markers. All six markers showed polymorphisms between Norton and Cabernet Sauvignon; five markers showed polymorphism for Norton and Pinot Noir (VVMD27 did not); four markers showed polymorphism between Norton and Syrah (VVS2 and VrZAG79 did not); and four markers showed polymorphism between Norton and Merlot (VVMD7 and VVMD5 did not) (Table 2). Therefore, interspecific hybrids were identified using markers showing polymorphism for all crosses. In the case of the Norton and Vignoles crosses, none of the six standard markers could be used. Although VVMD5 and VRZAG62 (Table 2) showed polymorphism when screened through the parents, random amplification patterns were seen in the progenies (data not shown). PCR and capillary electrophoresis were repeated for these progenies and data were re-recorded, but the same result was obtained. Sixteen additional SSR markers (VVMC4GC, CTG0393, UDV-041, VMC3F3, VVIP28, VVIP77, CF6881, FAM12, FAM 24, FAM26, FAM46, FAM56, FAM71, FAM79, FAM115 and FAM132) were randomly selected and screened for polymorphisms. UDV-041, VMC3F3, VVIP28, VVIP77, CF6881 and FAM26 showed the random amplification pattern in the progenies generating peaks different from the parents. These markers also had one identical allele between the parents except CF6881 (2 identical alleles), thus making these markers unsuitable for the identification of hybrids. VVMC4GC, CTG0393, FAM24, FAM46 and FAM132 showed the normal amplification patterns generating the peaks similar to that of parents, but they also had one identical allele between the parents. Therefore, these markers were discarded. FAM56 showed non-polymorphisms generating two identical peaks in both parents. The markers of FAM12, FAM 71, FAM79, and FAM115 showed polymorphisms and normal amplification patterns (Table 2), and hence were used for identification of hybrids. Twenty-five out of 32 progenies (78%) were identified as hybrids between the cross of Norton and Vignoles using these four markers (Table 1). 4. Discussion The ability to produce novel grapevine cultivars by conventional breeding is frequently hampered by the long juvenile period before fruit is produced. Thus, assessment of progeny purity is one of the most important quality control components in interspecific hybrid production. The morphological differences between the true hybrids and off-types are not always easily recognized. The sensitivity of morphological traits to the environment
P. Adhikari et al. / Scientia Horticulturae 179 (2014) 363–366
365
Table 1 Crosses tested for true hybrid production. Crosses
# Plants evaluated
# True hybrids
% True hybrids
Norton × Cabernet Sauvignon Norton × Pinot Noir Norton × Syrah Norton × Merlot Norton × Vignoles
310 30 34 42 32
273 21 31 7 25
88.1 70.0 91.7 16.7 78.1
Table 2 Genetic profiles (allele sizes in bp) of six grape varieties at various microsatellite loci. Primers
Norton
Cabernet Sauvignon
Pinot Noir
Syrah
Merlot
Vignoles
VVMD5 VVMD7 VVMD27 VVS2 VrZAG62 VrZAG79 FAM12 FAM71 FAM79 FAM115
233/247 237/246 184/186 135/137 181/205 250/254 368/377 181/184 147/149 316/336
231/240 239/239 173/187 141/154 189/195 246/246
228/238 239/243 184/188 139/154 189/195 239/244
225/231 239/239 188/190 135/135 189/195 244/250
225/233 239/246 188/190 141/154 195/195 258/258
232/246 239/246 175/186 137/154 189/195 246/254 356/365 188/190 150/156 318/318
further limits its application. This work presented the usefulness of SSR markers for the identification of V. aestivalis-derived ‘Norton’based hybrid populations. Although the allelic profiles of the six standard markers described in this study have already been published (Moreno-Sanz et al., 2008; Stover et al., 2009), this research is the first to use them for the study of V. aestivalis-derived ‘Norton’based populations. As described in This et al. (2004), due to different laboratory equipment and individually adapted protocols, allele size discrepancy of these six standard markers often existed. This may explain the few base pair differences between our results and the previously published data. Norton has not been widely used in grape breeding programs. In this study, SSR markers enabled accurate and quick identification of hybrids in the F1 generation, allowed the early disposal of nonhybrids in their seedling stage thus delivering substantial savings in time and resources. This procedure ensures that only true hybrid plants are used for mapping population development. Our research has highlighted the importance and potential of Norton grapes in grape breeding programs to enhance viticultural performance and enological quality. The low percentage of hybrids obtained from the cross between Norton and Merlot, where all the non-hybrids showed the Norton (maternal) alleles, indicating a likely emasculation or pollination error that led to self-pollination. Each and every flower in the cluster should be emasculated – a single missed stamen is sufficient to cause self-pollination in a whole cluster. Sometimes, if pollination is conducted in the presence of wind, pollen from other Norton clusters may cause self-fertilization. This suggests that hand emasculation has been a technical challenge and the importance of proper emasculation and pollination in the hybrid production. The interspecific hybrids of Norton × V. vinifera were easily identified by the six standard SSR markers. Confirmation of a subset of these results was subsequently provided using the 182 Norton × Cabernet Sauvignon true hybrids identified from this study. Following the SSR analysis, this subset was processed using genotyping-by-sequencing (GBS) technology with single nucleotide polymorphism (SNP) markers as part of the VitisGen project (www.vitisgen.org). VitisGen is a Cornell University-based collaborative project with the long-term goal of accelerating grape cultivar improvement using advanced molecular marker technologies. The GBS analysis confirmed that these 182 genotypes were true hybrids between Norton and Cabernet Sauvignon (data not shown).
It is noted that these six standard markers were unable to confirm the parentage of Norton × Vignoles hybrids. The random amplification and non-parental banding patterns seen in the progenies of Norton × Vignoles implies that these markers are not specific for Vignoles. The ancestry of Vignoles is still under debate (Bautista et al., 2009) and its genome sequence is not available. Further characterization of Vignoles would be necessary to understand our observed nonspecific amplification. Nevertheless, this study provided four additional EST-SSR markers (FAM12, FAM 71, FAM 79 and FAM 115) for future Vignoles hybrid populations. 5. Conclusion It can be extremely difficult to identify interspecific grape hybrids through the sole use of morphological features. This study demonstrated that the use of six standard SSR markers was sufficient to efficiently identify hybrids developed from V. aestivalis-derived ‘Norton,’ an under-utilized genetic resource with large potential for enhanced disease and stress resistance. In one cross using Vignoles, a French–American hybrid of uncertain ancestry, these six markers proved ineffective for hybrid identification. In this case, additional SSR markers were generated that successfully identified interspecific hybrids and are described. Acknowledgments The first two authors contributed equally to this work. The authors thank Marilyn Odneal and Kevin Fort for valuable discussions and constructive comments on the manuscript. The authors also thank Dr. Wenping Qiu for providing the plant materials from crosses he made in 2005. This work is supported by Agriculture and Food Research Initiative Competitive Grant no. 2013-67014-21360 and Capacity Building Grants for Non-Land Grant College of Agriculture no. 2013-70001-21268 from the USDA National Institute of Food and Agriculture. References Ambers, C.P., 2013. A historical hypothesis on the origin of the Norton grape. J. Wine Res. 24, 85–95. Bautista, J., Dangl, G.S., Yang, J., Reisch, B., Stover, E., 2009. Use of genetic markers to assess pedigrees of grape cultivars and breeding program selection. Am. J. Enol. Vitic. 59, 248–254.
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Doligez, A., Adam-Blondon, A., Cipriani, G., Di Gaspero, G., Laucou, V., Merdinoglu, D., Meredith, C., Riaz, S., Roux, C., This, P., 2006. An integrated SSR map of grapevine based on five mapping populations. Theor. Appl. Genet. 113, 369–382. Herald, R., Herald, E., 1988. Cynthiana/Norton: the reliable red. Practical Winery Vineyard 18, 56. Hogan, S., Zhang, L., Li, J., Zoecklein, B., Zhou, K., 2009. Antioxidant properties and bioactive components of Norton (Vitis aestivalis) and Cabernet Franc (Vitis vinifera) wine grapes. Food Sci. Technol. 42, 1269–1274. Huang, H., Lu, J., Ren, Z., Hunter, W., Dowd, S.E., Dang, P., 2011. Mining and validating grape (Vitis L.) ESTs to develop EST-SSR markers for genotyping and mapping. Mol. Breed. 28, 241–254. Missouri Code of State Regulations, 2010. Revised Statutes of Missouri 10.160. Secretary of State, Jefferson city, Missouri. Moreno-Sanz, P., Suarez, B., Loureiro, M.D., 2008. Identification of synonyms and homonyms in grapevine cultivars (Vitis vinifera L.) from Asturias (Spain). J. Horti. Sci. Biotechnol. 83, 683–688. Oetting, W.S., Lee, H.K., Flanders, D.J., Wiesner, G.L., Sellers, T.A., King, R.A., 1995. Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers. Genomics 30, 450–458.
Powell, W., Morgante, M., Andre, C., Hanafey, M., Vogel, J., Tingey, S., Rafalski, A., 1996. The comparison of RFLP, RAPD AFLP and SSR (microsatellite) markers for germplasm analysis. Mol. Breed. 2, 225–238. Reisch, B.I., Goodman, R.N., Martens, M.H., Weeden, N.F., 1993. The relationship between Norton and Cynthiana, red wine cultivars derived from Vitis aestivalis. Am. J. Enol. Vitic. 44, 441–444. Riaz, S., Dangl, G.S., Edwards, K.J., Meredith, C.P., 2004. A microsatellite marker based framework linkage map of Vitis vinifera L. Theor. Appl. Genet. 108, 864–872. Schuck, M.R., Biasi, L.A., Mariano, A.M., Lipski, B., Riaz, S., Walker, M.A., 2011. Obtaining interspecific hybrids, and molecular analysis by microsatellite markers in grapevine. Pesq. Agropec. Bras. 46, 1480–1488. Stover, E., Aradhya, M., Yang, J., Bautista, J., Dangl, G.S., 2009. Investigations into the origin of ‘Norton’ grape using SSR markers. Proc. Fla. State Hort. Soc. 122, 19–24. This, P., Jung, A., Boccacci, P., Borrego, J., Bolta, R., Costantini, L., Crespan, M., Dangl, G.S., Eisenheld, C., Ferreira, F., 2004. Development of a standard set of microsatellite reference alleles for the identification of grape cultivars. Theor. Appl. Genet. 109, 1448–1458.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Interspecific hybrid identification of Vitis aestivalis-derived ‘Norton’-based populations using microsatellite markers Pragya Adhikari a , Li-Ling Chen a , Xu Chen a,b , Surya D. Sapkota a , Chin-Feng Hwang a,∗ a b
State Fruit Experiment Station at Mountain Grove Campus, Darr School of Agriculture, Missouri State University, Springfield, MO 65897, USA Pennington Seed, Inc., Greenfield, MO 65661, USA
a r t i c l e
i n f o
Article history: Received 11 July 2014 Received in revised form 25 September 2014 Accepted 27 September 2014 Available online 21 October 2014 Keywords: Vitis Interspecific hybrids Molecular marker Marker-assisted selection
a b s t r a c t The Vitis aestivalis-derived ‘Norton’ is one of the very few commercial red grape varieties that can be grown in regions with high disease pressure and cold temperatures in winter and spring where V. vinifera is difficult to grow. This study began with the objectives of generating interspecific hybrids of Norton and V. vinifera (Cabernet Sauvignon, Pinot Noir, Syrah, and Merlot) in order to combine the disease resistance and cold hardiness characters of V. aestivalis with the excellent wine quality of V. vinifera. Norton grapes were also crossed with the Vitis interspecific hybrid ‘Vignoles’ for the same reason. The true hybrids obtained from all of the crosses were identified using microsatellite markers. A high percentage of hybrids (70–92%) were obtained in all crosses except Norton × Merlot (17%). Results demonstrate the reliability of SSR markers for Norton-based interspecific hybrid identification. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Norton is a unique grape with good wine characteristics and thought to originate from an accidental cross of a lost grapevine ‘Bland’ (a hybrid of V. labrusca and Chasselas) with pollen from V. aestivalis (Ambers, 2013). It produces a dark, full-bodied, red wine without the intense “foxy” flavors and aromas that taint the wines of most American hybrid grapes (Herald and Herald, 1988). The Norton grape is mainly grown in many U.S. wine growing regions with high humidity and comparatively long growing seasons where V. vinifera cultivars are difficult to grow and require extensive pesticide use (Reisch et al., 1993). Two prominent characteristics of Norton grape are: (1) high levels of resistance to powdery mildew, downy mildew, black rot, bitter rot and Botrytis bunch rot, which are collectively the most destructive fungal diseases of grape worldwide; (2) high levels of anthocyanins that are associated with the health benefits of consuming grapes and wine (Hogan et al., 2009). Norton was selected as the official grape variety of the State of Missouri on 11 July 2003 (Missouri Code of State Regulation, 2010). The disease resistance and cold hardiness of Norton have enhanced its attractiveness to wine growers due to increasing concerns regarding environmental protection and pesticide avoidance. However, only limited breeding efforts have been made using this variety. Research in the genetics and genomics of Norton traits
∗ Corresponding author. Tel.: +1 4175477538; fax: +1 4175477540. E-mail address: [email protected] (C.-F. Hwang). http://dx.doi.org/10.1016/j.scienta.2014.09.048 0304-4238/© 2014 Elsevier B.V. All rights reserved.
that are involved in disease resistance and berry quality have not yet been fully explored or understood. Genetic mapping of Norton × V. vinifera populations may elucidate the underlying genetic and molecular mechanisms of berry disease resistance and berry quality. Simple Sequence Repeats (SSR) or microsatellite markers allow for the selection of true hybrids at the seedling stage; therefore, offtypes in a cross from both selfings and outcrossing can be discarded in the F1 generation (Schuck et al., 2011). DNA microsatellites are abundant in the grape genome, follow Mendelian inheritance, are co-dominant, highly polymorphic, and have proved their usefulness for the genetic analysis of a heterozygous species like grape (Powell et al., 1996; Riaz et al., 2004). The SSR markers are also highly valuable because of their interspecies transferability, allowing direct comparison of linkage groups with previously published maps of Vitis species (Doligez et al., 2006). Six highly polymorphic SSR markers (VVMD5, VVMD7, VVMD27, VVS2, VrZAG62 and VrZAG79) have been proposed as standard markers for the analysis of grapevine cultivars following the analysis of 46 grape cultivars at these six loci in 10 different laboratories in seven countries (This et al., 2004). However, microsatellites have not been implemented for the analysis of Norton-based hybrid populations. Once true hybrids are identified, these hybrid populations can subsequently be used to construct a linkage map of Norton and to identify markers associated with desirable traits. In this study, 448 progenies resulting from the crosses between V. aestivalis-derived ‘Norton’ and each of five different cultivars (Cabernet Sauvignon, Pinot Noir, Syrah, Merlot, and Vignoles)
364
P. Adhikari et al. / Scientia Horticulturae 179 (2014) 363–366
were screened to determine interspecific hybrids using the six internationally-adopted standard SSR markers listed above. Additional markers were used when no polymorphism was found. This work emphasizes the efficiency of microsatellite markers that can be used to greatly accelerate the breeding process and allow a much more accurate selection of progeny. 2. Materials and methods 2.1. Plant materials Crosses using V. aestivalis-derived ‘Norton’ × V. vinifera ‘Cabernet Sauvignon’ were made in 2005 and resulted in 98 individuals of which 24 were from the reciprocal cross. This F1 population was planted in a Missouri State Fruit Experiment Station (MSFES) vineyard in 2007 and has yielded fruit for the past 3 years. This population was expanded to 310 genotypes in 2011. Additional crosses between Norton and other V. vinifera species including Pinot Noir, Syrah, Merlot and the French–American hybrid ‘Vignoles’ were also made and generated 30, 34, 42 and 32 progenies, respectively. For crosses, female parents were emasculated before anthesis and covered with paper bags to avoid contamination from unwanted pollen. The pollen of desired paternal parents was collected, placed under a 60 W lamp, dried overnight and stored at room temperature for further use. Pollination was done with a brush the morning after emasculation, and the inflorescences were tagged and re-covered with the paper bags. After harvest, the seeds were extracted and washed with water to remove the pulp. The seeds were then placed in sterilized sand and stratified at 4 ◦ C for 3 months. Seeds were planted in seedling trays and germinated within 3 weeks in a greenhouse. 2.2. DNA extraction One hundred mg of young leaf tissue was ground to fine powder in liquid nitrogen. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Concentration and purity of DNA were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The DNA was normalized to 15 ng/L and stored at −20 ◦ C. 2.3. PCR amplification PCR amplification of microsatellite marker alleles used three primers per reaction: an M13-tailed, 5 -TGTAAAACGACGGCCAGT3 , forward primer (Oetting et al., 1995), a reverse primer and a WellRED (Sigma–Aldrich, St. Louis, MO) labeled M13 sequence. A PCR reaction was performed in a total volume of 10 L that consisted of 15 ng of template DNA, 0.1 M of forward and 2 M of reverse primer, 2 M WellRed M13 primer, 5 mM MgCl2 , and 5 L AmpliTaq GoldR 360 Master Mix buffer (Life Technologies, Grand Island, NY). Amplification was performed using touchdown PCR consisting of 10 min of denaturation at 95 ◦ C, 10 cycles of denaturation at 94 ◦ C for 30 s, primer annealing at 62 ◦ C for 30 s and extension at 72 ◦ C for 1 min where annealing temperature was decreased by 1 ◦ C at each cycle. This was followed by 24 cycles of denaturation at 94 ◦ C for 30 s, annealing at 56 ◦ C for 30 s and extension at 72 ◦ C for 1 min. The reaction was completed with a post extension cycle at 72 ◦ C for 7 min. Four L of reaction product was visualized on 1.5% agarose gel to confirm the amplifications as well as estimate the length of amplified products (Bio-Rad, Hercules, CA). 2.4. SSR genotyping To expedite the true hybrid screen, the F1 progenies from several Norton-based populations were genotyped at six microsatellite
loci as recommended in This et al. (2004). An additional 16 markers were randomly selected and further tested due to the non-polymorphic nature of the six standard markers on specific populations. The expressed sequence tag (EST)-SSR FAM primer sequences were described in Huang et al. (2011). Other primer sequences are available in the NCBI database uni-STS (www.ncbi.nlm.nih.gov). PCR products were run on a GenomeLab GeXP capillary sequencer (Beckman Coulter, Brea, CA), and alleles were sized with the GenomeLab GeXP Genetic Analysis software, Fragment Analysis Module. The 18 bp M13 sequence was then subtracted from each recorded allele to calculate the actual allele size. 3. Results In 2005 and 2011, a total of 416 genotypes were produced from crosses between V. aestivalis-derived ‘Norton’ and V. vinifera cultivars including Cabernet Sauvignon, Pinot Noir, Syrah and Merlot. DNA fingerprinting of these F1 progenies with the six standard SSR loci indicated that a high percentage of hybrids were acquired in all crosses (70–92%) except for Norton × Merlot (Table 1). Each sample was first analyzed by using six standard SSR markers. All six markers showed polymorphisms between Norton and Cabernet Sauvignon; five markers showed polymorphism for Norton and Pinot Noir (VVMD27 did not); four markers showed polymorphism between Norton and Syrah (VVS2 and VrZAG79 did not); and four markers showed polymorphism between Norton and Merlot (VVMD7 and VVMD5 did not) (Table 2). Therefore, interspecific hybrids were identified using markers showing polymorphism for all crosses. In the case of the Norton and Vignoles crosses, none of the six standard markers could be used. Although VVMD5 and VRZAG62 (Table 2) showed polymorphism when screened through the parents, random amplification patterns were seen in the progenies (data not shown). PCR and capillary electrophoresis were repeated for these progenies and data were re-recorded, but the same result was obtained. Sixteen additional SSR markers (VVMC4GC, CTG0393, UDV-041, VMC3F3, VVIP28, VVIP77, CF6881, FAM12, FAM 24, FAM26, FAM46, FAM56, FAM71, FAM79, FAM115 and FAM132) were randomly selected and screened for polymorphisms. UDV-041, VMC3F3, VVIP28, VVIP77, CF6881 and FAM26 showed the random amplification pattern in the progenies generating peaks different from the parents. These markers also had one identical allele between the parents except CF6881 (2 identical alleles), thus making these markers unsuitable for the identification of hybrids. VVMC4GC, CTG0393, FAM24, FAM46 and FAM132 showed the normal amplification patterns generating the peaks similar to that of parents, but they also had one identical allele between the parents. Therefore, these markers were discarded. FAM56 showed non-polymorphisms generating two identical peaks in both parents. The markers of FAM12, FAM 71, FAM79, and FAM115 showed polymorphisms and normal amplification patterns (Table 2), and hence were used for identification of hybrids. Twenty-five out of 32 progenies (78%) were identified as hybrids between the cross of Norton and Vignoles using these four markers (Table 1). 4. Discussion The ability to produce novel grapevine cultivars by conventional breeding is frequently hampered by the long juvenile period before fruit is produced. Thus, assessment of progeny purity is one of the most important quality control components in interspecific hybrid production. The morphological differences between the true hybrids and off-types are not always easily recognized. The sensitivity of morphological traits to the environment
P. Adhikari et al. / Scientia Horticulturae 179 (2014) 363–366
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Table 1 Crosses tested for true hybrid production. Crosses
# Plants evaluated
# True hybrids
% True hybrids
Norton × Cabernet Sauvignon Norton × Pinot Noir Norton × Syrah Norton × Merlot Norton × Vignoles
310 30 34 42 32
273 21 31 7 25
88.1 70.0 91.7 16.7 78.1
Table 2 Genetic profiles (allele sizes in bp) of six grape varieties at various microsatellite loci. Primers
Norton
Cabernet Sauvignon
Pinot Noir
Syrah
Merlot
Vignoles
VVMD5 VVMD7 VVMD27 VVS2 VrZAG62 VrZAG79 FAM12 FAM71 FAM79 FAM115
233/247 237/246 184/186 135/137 181/205 250/254 368/377 181/184 147/149 316/336
231/240 239/239 173/187 141/154 189/195 246/246
228/238 239/243 184/188 139/154 189/195 239/244
225/231 239/239 188/190 135/135 189/195 244/250
225/233 239/246 188/190 141/154 195/195 258/258
232/246 239/246 175/186 137/154 189/195 246/254 356/365 188/190 150/156 318/318
further limits its application. This work presented the usefulness of SSR markers for the identification of V. aestivalis-derived ‘Norton’based hybrid populations. Although the allelic profiles of the six standard markers described in this study have already been published (Moreno-Sanz et al., 2008; Stover et al., 2009), this research is the first to use them for the study of V. aestivalis-derived ‘Norton’based populations. As described in This et al. (2004), due to different laboratory equipment and individually adapted protocols, allele size discrepancy of these six standard markers often existed. This may explain the few base pair differences between our results and the previously published data. Norton has not been widely used in grape breeding programs. In this study, SSR markers enabled accurate and quick identification of hybrids in the F1 generation, allowed the early disposal of nonhybrids in their seedling stage thus delivering substantial savings in time and resources. This procedure ensures that only true hybrid plants are used for mapping population development. Our research has highlighted the importance and potential of Norton grapes in grape breeding programs to enhance viticultural performance and enological quality. The low percentage of hybrids obtained from the cross between Norton and Merlot, where all the non-hybrids showed the Norton (maternal) alleles, indicating a likely emasculation or pollination error that led to self-pollination. Each and every flower in the cluster should be emasculated – a single missed stamen is sufficient to cause self-pollination in a whole cluster. Sometimes, if pollination is conducted in the presence of wind, pollen from other Norton clusters may cause self-fertilization. This suggests that hand emasculation has been a technical challenge and the importance of proper emasculation and pollination in the hybrid production. The interspecific hybrids of Norton × V. vinifera were easily identified by the six standard SSR markers. Confirmation of a subset of these results was subsequently provided using the 182 Norton × Cabernet Sauvignon true hybrids identified from this study. Following the SSR analysis, this subset was processed using genotyping-by-sequencing (GBS) technology with single nucleotide polymorphism (SNP) markers as part of the VitisGen project (www.vitisgen.org). VitisGen is a Cornell University-based collaborative project with the long-term goal of accelerating grape cultivar improvement using advanced molecular marker technologies. The GBS analysis confirmed that these 182 genotypes were true hybrids between Norton and Cabernet Sauvignon (data not shown).
It is noted that these six standard markers were unable to confirm the parentage of Norton × Vignoles hybrids. The random amplification and non-parental banding patterns seen in the progenies of Norton × Vignoles implies that these markers are not specific for Vignoles. The ancestry of Vignoles is still under debate (Bautista et al., 2009) and its genome sequence is not available. Further characterization of Vignoles would be necessary to understand our observed nonspecific amplification. Nevertheless, this study provided four additional EST-SSR markers (FAM12, FAM 71, FAM 79 and FAM 115) for future Vignoles hybrid populations. 5. Conclusion It can be extremely difficult to identify interspecific grape hybrids through the sole use of morphological features. This study demonstrated that the use of six standard SSR markers was sufficient to efficiently identify hybrids developed from V. aestivalis-derived ‘Norton,’ an under-utilized genetic resource with large potential for enhanced disease and stress resistance. In one cross using Vignoles, a French–American hybrid of uncertain ancestry, these six markers proved ineffective for hybrid identification. In this case, additional SSR markers were generated that successfully identified interspecific hybrids and are described. Acknowledgments The first two authors contributed equally to this work. The authors thank Marilyn Odneal and Kevin Fort for valuable discussions and constructive comments on the manuscript. The authors also thank Dr. Wenping Qiu for providing the plant materials from crosses he made in 2005. This work is supported by Agriculture and Food Research Initiative Competitive Grant no. 2013-67014-21360 and Capacity Building Grants for Non-Land Grant College of Agriculture no. 2013-70001-21268 from the USDA National Institute of Food and Agriculture. References Ambers, C.P., 2013. A historical hypothesis on the origin of the Norton grape. J. Wine Res. 24, 85–95. Bautista, J., Dangl, G.S., Yang, J., Reisch, B., Stover, E., 2009. Use of genetic markers to assess pedigrees of grape cultivars and breeding program selection. Am. J. Enol. Vitic. 59, 248–254.
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