Progress 04/01/13 to 03/31/18
Outputs
Progress Report Objectives (from AD-416): 1: Identify and characterize the molecular, genetic, functional genomic, and biological factors controlling grape resistance to fungal and oomycete pathogens, with primary emphasis on powdery mildew. 1.A. Determine the genetic basis, pathogen race-specificity, and cellular mechanisms of powdery mildew and downy mildew resistance in grape breeding populations. 1.B. Determine the genetic basis by which abiotic stresses transiently alter foliar gene expression and induce resistance to grape powdery mildew and to extreme weather events. 1.C. Determine the genetic basis of grapevine ontogenic resistance to powdery mildew through quantitative trait locus analysis. 2: Characterize the genetic, genomic, and physiological aspects of grapevine abiotic stress tolerance and environmental adaptation. 2.A. Evaluate physiological variation in candidate thermal and dehydration stress traits from wild grapevine germplasm and mapping populations. 2.B. Identify and characterize candidate loci, linked genetic markers, or expression level changes for improved deep supercooling and environmental adaptation in grapevine. 3: Elucidate the key genetic and genomic factors controlling grape quality, including grape flavor and aroma, natural drying-on-vine, and berry size. 3.A. Determine genetic control of grape berry secondary metabolite production utilizing rapid-cycling grape germplasm populations. 3.B. Identify candidate genes or linked genetic markers associated with natural Dry-on-Vine raisins. 3.C. Determine the genetic basis of berry quality traits of muscadine grapes through quantitative trait locus analysis. 4: Develop and utilize novel or improved methods and genetic systems for grapevine breeding and genetic studies including, but not limited to, marker-assisted selection, germplasm for rapid trait evaluation, and transgenic solutions for introducing novel variation. 4.A. Develop Scout as a rapid-cycling system for trait evaluation and integration. 4.B. Develop and utilize transgenic and mutation technologies to introduce novel genetic variation for trait improvement. 4.C. Develop, evaluate, and apply computational methods for linking high quality large volume grapevine genotypic data with economically important grapevine traits and phenotypes. Approach (from AD-416): Grape production and product quality are substantially harmed by diseases and adverse environmental conditions. This research project provides genetic solutions to biotic and abiotic stresses, while maintaining high fruit quality. Our general strategies are: A) Utilization of existing or created populations and germplasm for characterizing traits of interest and their underlying molecular mechanisms through trait, marker, and functional analyses; B) Integration of desirable alleles into prebreeding lines via molecular markers and rapid-cycling breeding systems; and C) Development of alternative solutions to complex problems through transgenic technologies. The following knowledge and products will be delivered at the end of this project: 1) Molecular markers useful in grape scion breeding that predict multiple sources of disease resistance, abiotic stress tolerance, fruit quality, and precocious flowering; 2) Enhanced understanding of critical genetic processes underlying these traits; 3) Development of novel germplasm resources, including rapidly flowering genetic lines, for the genetic dissection and stacking of traits of interest; and 4) Improved capacity of grape breeders to develop superior lines through marker-assisted breeding and other technology- driven approaches. Successful accomplishment of this project will accelerate development of improved grape cultivars to maintain the competiveness of the US grape industry, by reducing fungicide applications in new plantings, expanding the options for grape growers faced with climate change or in new production regions, and opening new market niches for novel grape products. This is the final report for the project 8060-21220-006-00D which was concluded on March 31, 2018. The new project 8060-21220-007-00D, built upon the success of 8060-21220-006-00D, is a continuation of the broad objectives and efforts of 8060-21220-006-00D for improving grape fruit quality, yield and stress tolerance. During this period USDA-ARS researchers in Geneva, New York successfully achieved each of the objectives and made substantial contribution of the knowledge and methods for genetically improving grape fruit quality, yield and tolerance to biotic and abiotic stress. Our Objective 1 was to identify and characterize the molecular, genetic, functional genomic, and biological factors controlling grape resistance to fungal and oomycete pathogens, with primary emphasis on powdery mildew. The most significant accomplishments and impacts under Objective 1 are: 1) Resistance mechanisms, strength, and breadth were characterized for 12 loci conferring powdery mildew resistance. Genetic analysis of several phenotyping approaches in the vineyard and laboratory led to improved methods for measuring heritable components of powdery mildew resistance for the identification of QTL. Strategies for powdery mildew resistance gene stacking were developed for improved durability of resistance. 2) DNA markers linked with resistance genes were identified for gene stacking of RUN1, REN1, REN2, REN3, REN4, REN6, REN7, REN9, and REN10. These markers were implemented in U.S. grape breeding programs for marker- assisted selection along with other key traits. 3) We collaborated with scientists at Cornell University and Rensselaer Polytechnic Institute and developed tools for automated image capture and computer vision quantification of powdery mildew disease. From the results researchers can learn which treatments or varieties control disease best. The computer vision methods are of broad interest to basic and applied scientists studying various aspects of disease biology and management. 4) Finally, genetic tools were developed and biological knowledge gained for the study of fungicide resistance and other pathogen traits. Knowledge, germplasm, and research tools developed in the first three topics forms the foundation of marker discovery and application in our new project plan Sub-Objective 1.A., which is focused on the genetic basis of host resistance. Progress in the fourth topic forms the foundation of Sub- Objective 1.B. in our new project plan; this sub-objective focuses on pathogen genes required for infection of grapevine Our Objective 2 was to characterize the genetic, genomic, and physiological aspects of grapevine abiotic stress tolerance and environmental adaptation. ARS researchers in Geneva, New York made significant research progress in the studies of abiotic (cold) stress. Several multi-year studies were performed evaluating cold hardiness and dormancy in multiple wild and cultivated grapes. Major advances in the knowledge of the physiology and genetics of abiotic stress were made, including comprehensive descriptions of supercooling ability and dormancy requirement in wild and hybrid Vitis genotypes. Major strides were made in understanding the chilling requirement of wild grapevines and identifying specific species with enhanced dormancy, a trait necessary for avoiding elevated frost in a changing climate. Comprehensive analysis of the pattern of dormant bud cold hardiness across several years resulted in new phenotyping methods. Specifically, early, mid, and late winter cold hardiness responses have been isolated and predictive models are nearly complete. Substantial progress has been achieved in the increase in phenotyping throughput, though proposed future studies will build on and increase the rigor of cold hardiness phenotypes. These phenotypes are being used to develop a new model to improve current predictive methods for evaluating cold hardiness. The future release of this model will enhance local stakeholders ability to assess the potential freeze risk in their vineyards and adjust viticultural management to compensate, should killing freezes occur. As a result of these studies, cold hardy wild germplasm with high midwinter hardiness and increased dormancy response is now being used in breeding programs. Additional progress has been made in identifying candidate genes, pathways, and processes that impact cold hardiness. Gene expression studies have been successful in identifying several candidate pathways for future genetic manipulation including the starch/sucrose pathway, ABA regulation, and stilbene synthase pathways. The genetic regulation of cold response has been greatly resolved through examination of gene expression datasets and through the detailed analysis of genetic variation in the key transcription factors controlling cold response through the CBF gene regulon. Additionally, the Grape Genetics Research Unit has successfully created multi-tissue gene expression references for seven wild grape species. These references are being used in conjunction with genome sequencing projects in order to increase accuracy of the Vitis gene annotation and to describe aspects of non-coding RNA regulation. Research in our new project plan will leverage the phenotyping and genotyping that has been successful thus far. The overall impacts of the accomplishments for abiotic stress in grapevine from ARS researchers in Geneva, New York, have been moderate to date, but significant impact has been realized in the areas of identification of specific wild germplasm with cold hardiness and dormancy traits and development of the predictive model of cold hardiness in the next few years as the new project plan�s objectives are achieved. Our Objective 3 was to elucidate the key genetic and genomic factors controlling grape quality, including grape flavor and aroma, natural drying-on-vine, and berry size. Most significant accomplishments and impacts in this research area were: 1. Characterized berry and seed content of anthocyanins and other polyphenolic compounds in the USDA-ARS Vitis germplasm, providing much needed knowledge for future improvement of grapevine cultivars for these health-beneficial phytochemicals; 2. Developed molecular markers for the 5-O glucosyltransferase gene which coverts monoglucoside to di-glucoside anthocyanins. Di-glucoside anthocyanins are only present in the North American grape and the markers developed for the 5-O glucosyltransferase gene will help keep track of North American Vitis germplasm in a breeding program for fruit and wine quality improvement. The markers have been used in the U.S. grape breeding programs. The work under this objective has provided some potential genome-editing targets for improving fruit quality traits as proposed in Objective 3.A. in our new project plan. Our Objective 4 was to develop and utilize novel or improved methods and genetic systems for grapevine breeding and genetic studies including, but not limited to, marker-assisted selection, germplasm for rapid trait evaluation, and transgenic solutions for introducing novel variation. Most significant accomplishments and impacts from this research area were: 1. identified several promising breeding lines for development of new-generation of rootstocks for RKN resistance. Through collaboration with scientists from the University of California, Davis, these rootstock breeding lines are being further evaluated for potential commercial releases; 2. demonstrated genome-scale mRNA exchanges between grapevine rootstocks and scions, providing new genetic clues for how rootstocks and scions interact with each other to produce superior grape grafts; 3. demonstrated the feasibility of using an RNAi approach for controlling root-knot nematodes in grapevine, opening an alternative avenue for breeding new rootstock resistant to root-knot nematodes; and 4. identified several promising clonal lines for the development of a new Vignoles which has reduced cluster compactness and is resistant to Botrytis bunch rot. In addition, we released one tetraploid rootstock for reducing scion vigor in grafted vines and three dwarf and continuous-flowering genetic stocks for genetic research. During this period, all subordinate projects made significant progress and met their research goals and objectives. Accomplishments 01 Rootstock variety, named as �A4�, released for controlling scion vigor in grafted vines. VR O39-16 is a grapevine rootstock which provides protection against both the dagger nematode Xiphinema index and fanleaf degeneration disease. However, the VR O39-16 rootstock is not favored by growers because it is very vigorous and causes the scions grafted on it to grow vigorously, producing large vines with high pruning weights. These large vines can be subject to the problems associated with excessively vigorous plants, such as dense canopies and poor fruit quality. A lower vigor rootstock that provides protection against both the dagger nematode Xiphinema index and fanleaf degeneration disease would be useful for grape growers because such a rootstock would allow growers to grow vines with appropriate vigor and canopy size. USDA-ARS researchers in Geneva, New York, developed a grapevine rootstock variety, named as �A4�, through doubling the chromosome numbers of a VR O39-16 rootstock vine. A4 is a low vigor grapevine rootstock variety and can reduce vigor in the scions grafted to it when compared to the diploid rootstock from which they are created. This new autotetraploid rootstock variety provides a valuable rootstock choice for grapevine growers to control scion vigor as well as damage from the dagger nematode Xiphinema index and fanleaf degeneration disease. 02 Three dwarf, continuous-flowering genetic stocks for grapevine genetic research and breeding released. �Pixie�, a dwarf and continuous- flowering variety, serves as a useful genetic stock for grapevine researchers due to its small size and continuous flowering. However, the Pixie vine is not vigorous and requires emasculation when it is used in cross hybridization. USDA-ARS researchers in Geneva, New York, crossed Pixie with other varieties and developed three new dwarf, continuous-flowering varieties which have only female flowers and do not self-fertilize. These varieties are useful germplasm for genetics, genomics, breeding, pathology, physiology, and other grapevine research, as the space required per experimental grapevine is greatly reduced; they can be grown in the greenhouse to maturity without the need to plant them in a vineyard; no emasculation is needed for making a cross; and the continuous flowering habit provides researchers with the opportunity to study flowers and berries at all stages of development throughout the year. 03 DNA markers discovered for 7 new disease resistance genes. Planting resistant varieties is a natural, low-cost and sustainable method of managing diseases. To track resistance genes in a traditional breeding program requires the identification of DNA markers close to each resistance gene. USDA-ARS researchers in Geneva, New York, published DNA markers for grape breeders to track 7 new disease resistance genes: RPV17, RPV18, RPV19, RPV20, and RPV21 for downy mildew resistance; and RDA1 and RDA2 for resistance to phomopsis canker. Already these DNA markers have been adopted for marker-assisted selection in two U.S. grape breeding programs. If commercialized, varieties carrying these resistance genes would enable fewer fungicide applications, less disease, and increased yield and fruit quality. 04 Cold hardiness response deconstructed into high-throughput phenotypes. Grapevine dormant buds change their level of cold hardiness throughout winter as temperatures change. Winter damage results when cultivars of grapevine are unable to gain sufficient cold hardiness or lose their level of cold hardiness during winter. Specific wild species with enhanced temperature response were identified and will contribute to future breeding efforts for rapid responding grapevines. Sources of germplasm for extended dormancy were identified and will allow breeders to select for this trait, decreasing the risk of midwinter loss of cold hardiness in new varieties. Finally, temperature dependent responses during late winter were determined to be genotype specific and contribute strongly to frost risk. By measuring the rate of cold hardiness loss at multiple temperatures, this response can be modeled and used as a quantifiable trait in genetic mapping analysis. Combining these new parameters for cold hardiness has enabled the preliminary construction of a predicative cold hardiness model with high accuracy for grapevine. 05 Key differences in gene expression between chill and frost damage identified. Understanding how gene expression changes in cold sensitive and resistant grapevines is key to identifying candidate genes for breeding efforts or future functional analysis. Leaf tissue from sensitive and resistant genotypes is often used due to easy access, with results extrapolated to the tissues which actually survive winter (e.g. buds and canes). ARS researchers in Geneva, New York, examined cold sensitivity of leaf tissue from 5 cultivars of grapevine and compared patterns of gene expression to identify �resistance� genes. Phenotypic screens for leaf damage revealed that the cultivar �Sangiovese� has enhanced leaf freeze resistance. Gene expression analysis revealed that chill and frost exposure produce very different, and in some cases opposite, patterns. These results indicate the need to utilize freezing stress to identify cold hardiness candidate genes. Results demonstrated the enhanced freeze resistance of �Sangiovese� as well as identified the stilbene synthase pathway as a potential freeze resistance candidate pathway in this genotype. This result can be leveraged in breeding programs for enhanced frost resistance.
Impacts
(N/A)
Publications
- Harris, Z., Kovacs, L., Londo, J.P. 2017. RNASeq-based genome annotation and identification of long-noncoding RNAs in the grapevine cultivar 'Riesling'. Biomed Central (BMC) Genomics. 18:937.
- Bai, Y., Dougherty, L., Cheng, L., Zhong, G., Xu, K. 2015. Uncovering co- expression gene network regulating fruit acidity in diverse apples. Biomed Central (BMC) Genomics. 16(1):612. DOI:10.1186/s12864-015-1816-6.
- Sawler, J., Reisch, B., Aradhya, M.K., Prins, B.H., Zhong, G., Schwaninger, H.R., Simon, C.J., Buckler, E., Myles, S. 2013. Genomics assisted ancestry deconvolution in grape. PLoS One. 8(11):1-8.
- Wan, Y., Schwaninger, H.R., Baldo, A.M., Labate, J.A., Zhong, G., Simon, C. 2013. A phylogenetic analysis of the grape genus (Vitis L.) reveals broad reticulation and concurrent diversification during neogene and quaternary climate change. BMC Evolutionary Biology. 13:141.
- Money, D., Gardner, K., Migicovsky, Z., Schwaninger, H.R., Zhong, G., Myles, S. 2015. LinkImpute: fast and accurate genotype imputation for non- model organisms. G3, Genes/Genomes/Genetics. doi: 10.1534/G3.115.021667.
- Wan, Y., Schwaninger, H.R., Baldo, A.M., Labate, J.A., Simon, C., Zhong, G. 2013. A phylogenomic analysis of the grape genus (Vitis) reveals broad reticulation and concurrent diversification during quaternary climate change. BMC Evlutionary Biology. 13:141.
- Yang, Y., Jittayasothorn, Y., Chronis, D.N., Wang, X., Cousins, P., Zhong, G. 2013. Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots. PLoS One. 8(7):e69463. doi: 10.1371/journal.pone.0069463.
- Miller, A., Matasci, N., Schwaninger, H.R., Aradhya, M.K., Prins, B.H., Zhong, G., Simon, C., Buckler IV, E.S., Myles, S. 2013. Vitis phylogenomics: hybridization intensities from a SNP array outperform genotype calls. PLoS One. doi: 10.1371/journal.pone.0078680.
- Volk, G.M., Chao, C.T., Norelli, J.L., Brown, S.K., Fazio, G., Peace, C., McFerson, J., Zhong, G., Bretting, P.K. 2015. The vulnerability of US apple (Malus) genetic resources. Genetic Resources and Crop Evolution. 62(5):765-794. doi:10.1007/s10722-014-0194-2.
- Migicovsky, Z., Sawler, J., Gardner, K.M., Aradhya, M.K., Prins, B.H., Schwaninger, H.R., Bustamante, C.D., Buckler, E.S., Zhong, G., Brown, P.J., Myles, S. 2017. Patterns of genomic and phenomic diversity in wine and table grapes. Horticulture Research. 4(17035):1-11.
- Migicovsky, Z., Gardner, K., Money, D., Sawler, J., Bloom, J., Moffett, P., Chao, C.T., Schwaninger, H.R., Fazio, G., Zhong, G., Myles, S. 2016. Genome to phenome mapping in apple using historical data. The Plant Genome. 9(2). doi: 10.3835/plantgenome2015.11.0113.
- Duan, N., Bai, Y., Sun, H., Wang, N., Ma, Y., Jiao, C., Legal, N., Mao, L., Wan, S., Wang, K., He, T., Feng, S., Zhang, Z., Mao, Z., Shen, X., Chen, X., Jiang, Y., Wu, S., Yin, C., Ge, S., Yang, L., Jiang, S., Xu, H., Jiang, Y., Wu, S., Yin, C., Liu, J., Wang, D., Qu, C., Wang, Y., Zuo, W., Xiang, L., Liu, C., Zhang, D., Gao, Y., Xu, Y., Li, M., Xu, K., Chao, C.T., Shu, H., Zhong, G., Cheng, L., Fei, Z., Chen, X. 2017. Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement. Nature Communications. 8:A249.
- Londo, J.P., Kovaleski, A.P., Lillis, J.A. 2018. Divergence in the transcriptional landscape between low temperature and freeze shock in cultivated grapevine (Vitis vinifera). Horticulture Research. 5(10).
- Adam-Blondon, A., Alaux, M., Pommier, C., Cantu, D., Cheng, Z., Cramer, G. R., Davies, C., Delrot, S., Deluc, L., Di Gaspero, G., Grimplet, J., Fennell, A., Londo, J.P., Kersey, P., Mattivi, F., Naithani, S., Neveu, P., Nikolski, M., Pezzotti, M., Reisch, B., Topfer, R., Vivier, M., Ware, D., Quesneville, H. 2016. Towards an open grapevine information system. Horticulture Research. doi: 10.1038/hortres.2016.56.
- Li, M., An, H., Angelovici, R., Bagaza, C., Albert, B., Clark, L., Coneva, V., Donoghue, M., Edwards, E., Fajardo, D., Fang, H., Frank, M.H., Gallaher, T., Gebken, S., Hill, T., Jansky, S.H., Kaur, B., Klahs, P.C., Klein, L., Kuraparthy, V., Londo, J.P., Migicovsky, Z., Miller, A., Mohn, R., Myles, S., Otoni, W.C., Pires, J.C., Rieffer, E., Schmerler, S., Spriggs, E., Topp, C.N., Van Deynze, A., Zhang, K., Zhu, L., Zink, B.M., Chitwood, D.H. 2018. Topological data analysis as a morphometric method: using persistent homology to demarcate a leaf morphospace. Frontiers in Plant Science. 9:553.
- Shellie, K., Kovaleski, A., Londo, J.P. 2018. Water deficit severity during berry development alters timing of dormancy transitions in wine grape cultivar Malbec. Scientia Horticulturae. 232:226-230.
- Klein, L., Miller, A.J., Ciotir, C., Hyma, K., Migicovsky, Z., Uribe- Convers, S., Londo, J.P. 2018. High-throughput sequencing data clarify evolutionary relationships among North American Vitis species and improve identification in USDA Vitis germplasm collections. American Journal of Botany. 105(2):215-226.
- Fresnedo, J., Yang, S., Sun, Q., Cote, L., Schweitzer, P., Reisch, B., Ledbetter, C.A., Luby, J., Clark, M., Londo, J.P., Gadoury, D., Kozma, P., Cadle Davidson, L.E. 2017. An integrative AmpSeq platform for highly multiplexed marker-assisted pyramiding of grapevine powdery mildew resistance loci. Molecular Breeding. 37(145).
- Norelli, J.L., Wisniewski, M.E., Fazio, G., Burchard, E.A., Gutierrez, B.L. , Levin, E., Droby, S. 2017. Genotyping-by-sequencing markers facilitate the identification of quantitative trait loci controlling resistance to Penicillium expansum in Malus sieversii. PLoS One. doi: 10.1371/journal. pone.0172949.
- Gutierrez, B.L., Zhong, G., Brown, S. 2018. Genetic diversity of dihydrochalcone content in Malus germplasm. Genetic Resources and Crop Evolution. 65(5):1485-1502.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 1: Identify and characterize the molecular, genetic, functional genomic, and biological factors controlling grape resistance to fungal and oomycete pathogens, with primary emphasis on powdery mildew. 1.A. Determine the genetic basis, pathogen race-specificity, and cellular mechanisms of powdery mildew and downy mildew resistance in grape breeding populations. 1.B. Determine the genetic basis by which abiotic stresses transiently alter foliar gene expression and induce resistance to grape powdery mildew and to extreme weather events. 1.C. Determine the genetic basis of grapevine ontogenic resistance to powdery mildew through quantitative trait locus analysis. 2: Characterize the genetic, genomic, and physiological aspects of grapevine abiotic stress tolerance and environmental adaptation. 2.A. Evaluate physiological variation in candidate thermal and dehydration stress traits from wild grapevine germplasm and mapping populations. 2.B. Identify and characterize candidate loci, linked genetic markers, or expression level changes for improved deep supercooling and environmental adaptation in grapevine. 3: Elucidate the key genetic and genomic factors controlling grape quality, including grape flavor and aroma, natural drying-on-vine, and berry size. 3.A. Determine genetic control of grape berry secondary metabolite production utilizing rapid-cycling grape germplasm populations. 3.B. Identify candidate genes or linked genetic markers associated with natural Dry-on-Vine raisins. 3.C. Determine the genetic basis of berry quality traits of muscadine grapes through quantitative trait locus analysis. 4: Develop and utilize novel or improved methods and genetic systems for grapevine breeding and genetic studies including, but not limited to, marker-assisted selection, germplasm for rapid trait evaluation, and transgenic solutions for introducing novel variation. 4.A. Develop Scout as a rapid-cycling system for trait evaluation and integration. 4.B. Develop and utilize transgenic and mutation technologies to introduce novel genetic variation for trait improvement. 4.C. Develop, evaluate, and apply computational methods for linking high quality large volume grapevine genotypic data with economically important grapevine traits and phenotypes. Approach (from AD-416): Grape production and product quality are substantially harmed by diseases and adverse environmental conditions. This research project provides genetic solutions to biotic and abiotic stresses, while maintaining high fruit quality. Our general strategies are: A) Utilization of existing or created populations and germplasm for characterizing traits of interest and their underlying molecular mechanisms through trait, marker, and functional analyses; B) Integration of desirable alleles into prebreeding lines via molecular markers and rapid-cycling breeding systems; and C) Development of alternative solutions to complex problems through transgenic technologies. The following knowledge and products will be delivered at the end of this project: 1) Molecular markers useful in grape scion breeding that predict multiple sources of disease resistance, abiotic stress tolerance, fruit quality, and precocious flowering; 2) Enhanced understanding of critical genetic processes underlying these traits; 3) Development of novel germplasm resources, including rapidly flowering genetic lines, for the genetic dissection and stacking of traits of interest; and 4) Improved capacity of grape breeders to develop superior lines through marker-assisted breeding and other technology- driven approaches. Successful accomplishment of this project will accelerate development of improved grape cultivars to maintain the competiveness of the US grape industry, by reducing fungicide applications in new plantings, expanding the options for grape growers faced with climate change or in new production regions, and opening new market niches for novel grape products. Novel methods for semi-automated, computer vision quantification of disease. Objective and quantitative analysis of diseases such as powdery mildew and downy mildew is an important challenge for the development of new disease management tools, such as resistant varieties for reduced chemical input. To address this problem, ARS researchers in Geneva, New York partnered with scientists at Cornell University and Rensselaer Polytechnic Institute to develop tools for semi-automated, computer vision quantification of disease. An imaging robot scans an array of infected leaf discs and captures microscopy images of each disc. Then a series of image analysis software processes the disc images to generate a quantitative assessment of disease. From the results researchers can learn which treatments or cultivars control disease best. The computer vision methods are of broad interest to basic and applied scientists studying various aspects of disease biology and management. Stress Tolerance: Grapevine production that occurs outside of Mediterranean climate regions is limited due to environmental stresses such as cold temperatures. ARS researchers in Geneva, New York completed field based phenotyping of chilling requirement, dormancy, budburst rate and dormant bud freeze resistance for eight different wild grapevine species frequently used in breeding programs for scion and rootstock germplasm and an in-depth analysis on 43 V. riparia genotypes. V. riparia is used frequently for cold hardy hybrid grape breeding due to its superior bud freeze resistance. New germplasm accessions with increased dormancy and delayed deacclimation have been identified and these accessions are being used in local breeding efforts. High resolution studies have uncovered a new mechanism driving cold hardiness tied to the initiation of fall acclimation. Additionally, studies examining the impact of different daily temperature cycling treatments have revealed aspects of temperature variation that contribute to increased dormant bud hardiness. This data is being used to create a predictive model of cold hardiness for the Northeast grapevine production regions with hope for wider applicability in other regions. Putative grapevine cryo-proteins are being evaluated in the model plant Arabidopsis thaliana via gene overexpression to determine the potential for these cryo-proteins to enhance cold hardiness. Early phenotypes suggest role of these cryoproteins in modulating germination and freezing tests will be conducted when sufficient plant material is available. Reference transcriptome atlases have been produced for 24 different grapevine genotypes, representing eight species. The ninth species, V. acerifolia, has been collected and will be sequenced in FY2017. These reference transcriptomes are currently being used in several collaborator projects including de novo genome annotation of V. cinerea and V. riparia as well as in tool development for non-coding RNA identification. Genome-SNP data and genetic maps have been constructed for a number of candidate mapping populations for cold hardiness and winter survival studies. Vine Architecture: Vine architecture plays a critical role in enhancing yield and fruit quality of grapes as well as reducing viticulture management cost of grape production. Among many genes discovered for controlling plant architecture, Gibberellic Acid Insensitive (GAI) genes are best characterized and they are the genetic basis of �green revolution� in cereal crops in 1960s-1980s. ARS researchers in Geneva, New York have introduced several different versions of the grape GAI gene into a Vitis vinifera cultivar and observed dramatic changes of vine architecture including internode length, leaf size, overall vine height, and other traits. In the fiscal year 2017, we collected more evaluation data on these transgenic lines. The work will be completed and summarized in the fiscal year 2018. ARS researchers in Geneva, New York are also conducting a research project for modifying cluster architecture of a well-known wine grape cultivar, �Vignoles�. Vignoles is a valuable cultivar in the eastern United States due to its distinctive apricot, peach, and citrus notes in wines and its winter hardiness. However, Vignoles has compact clusters prone to secondary spread of Botrytis bunch rot late in the season and losses up to one third of the crop are possible in rainy years. ARS Researchers in Geneva, New York have used gamma irradiation to induce and identify 20 loose-clustered clones of Vignoles. These selections were grafted to rootstock and initially evaluated in field trials in the prior year. Second field trial data will be collected later this season. This work will help develop improved Vignoles with loose clusters and better resistance to Botrytis bunch rot. Resistance to Root-Knot Nematodes: Root-knot nematodes are a major pest of vineyards across California and the United States. Nematode management through fumigation can be useful in mitigating the problem, but use of chemicals is costly and environmentally undesirable. Resistant rootstocks are a sustainable method for managing nematodes in vineyards, but development of a successful resistant rootstock is a long process involving many years of effort. To overcome this challenge, ARS researchers in Geneva, New York has been developing a molecular-marker- based tool which can be used to accelerate the selection process of resistance to root-knot nematodes in a breeding program. Several candidate molecular markers have been identified for this purpose from a population segregating for resistance to root knot nematodes. These markers are being validated and, once confirmed, will be provided to rootstock breeders for use. In parallel, ARS researchers in Geneva, New York have been exploring a gene silencing technology which may offer a complementary approach for improving root-knot nematode resistance of grapevine rootstocks. In the previous years, we developed and evaluated transgenic vines containing small double strand RNAs (dsRNAs) of a nematode gene required for parasitism in grapevine roots. In the fiscal year 2017, we continued the evaluation to collect more data to make a robust conclusion. Data collection is in progress and will be summarized in the fiscal year 2018. All subordinate projects are making good progress toward meeting their research goals and objectives. Accomplishments 01 The molecular basis of the arrangement of leaves on grapevine revealed. The arrangement of leaves on grapevine (Tendril phyllotaxy) is an important vine architecture trait and has significant implications in the improvement of flowering and patterns of flower inflorescence. However, the underlying molecular and genetic mechanisms for leave distribution and flowering arrangements are largely unknown. ARS researchers in Geneva, New York analyzed gene expression patterns of wild and mutant types of vine phyllotaxy and discovered the involvement of several key genes in controlling vine phyllotaxy. The findings have important theoretical and practical implications in improving inflorescence and cluster architecture in grapevines. 02 Novel technology applied to powdery mildew resistance. Low-cost genetic tools are needed for analysis of multiple traits simultaneously. ARS researchers in Geneva, New York developed a $5 assay that can determine up to 400 DNA markers simultaneously in up to 4600 samples in one experiment. This technique was validated and applied in U.S. grape breeding programs for seven powdery mildew resistance genes, one downy mildew resistance gene, seedlessness, and flower sex, saving years of research time. As a result, breeders were able to select vines having multiple resistance genes for improved durability, and discard undesirable vines. Validated markers are being made publicly available for public and private grape breeders to use. 03 Phenotypic characterization of mid-winter dormant bud supercooling potential. Cold hardiness and winter survival is a complex interplay between genetic background and environmental variation. Wild grapevines have much greater supercooling ability than cultivated grape and this trait would be beneficial in hybrid varieties. Seven wild species were evaluated for midwinter cold hardiness using low temperature exotherms to determine lethal temperatures. Results demonstrated three different classes of wild grapevines with different abilities to perceive and respond to temperature fluctuations; high, medium and low responders. These findings have thus identified novel germplasm pools for breeding hybrid grapes with reduced temperature response, preventing mid-winter loss of hardiness and subsequent freeze damage.
Impacts
(N/A)
Publications
- Cadle Davidson, L.E., Gadoury, D., Fresnedo, J., Yang, S., Barba, P., Sun, Q., Seem, R., Schaub, M.L., Nowogrodzki, A., Kasinathan, H., Ledbetter, C. A., Reisch, B. 2016. Lessons from a phenotyping center revealed by the genome-guided mapping of powdery mildew resistance loci. Phytopathology. 106:1159-1169.
- Arro, J., Cuenca, J., Yang, Y., Liang, Z., Cousins, P., Zhong, G. 2017. A transcriptome analysis of two grapevine populations segregating for tendril phyllotaxy. Horticulture Research. 4:11. doi:10.1038/hortres.2017. 32.
- Centinari, M., Smith, M., Londo, J.P. 2016. Assessment of freeze injury of grapevine green tissues in response to cultivars and a cryoprotectant product. HortScience. 51(7):856-860.
- Migicovsky, Z., Sawler, J., Gardner, K., Aradhya, M.K., Prins, B.H., Schwaninger, H.R., Bustamente, C., Buckler IV, E.S., Zhong, G., Brown, P., Myles, S. 2017. Patterns of genomic and phenomic diversity in wine and table grapes. Horticulture Research. 4:17035. DOI: 10.1038/hortres.2017.35.
- Adam-Blondon, A., Alaux, M., Pommier, C., Cantu, D., Cheng, Z., Cramer, G., Davies, C., Delrot, S., Deluc, L., Di Gaspero, G., Grimplet, J., Fennell, A., Londo, J.P., Kersey, P., Mattivi, F., Naithani, S., Neveu, P., Nikolski, M., Pezzotti, M., Reisch, B., Topfer, R., Vivier, M., Ware, D., Quesneville, H. 2016. Towards an open grapevine information system. Horticulture Research. 3:16056. doi:10.1038/hortres.2016.56.
- Fresnedo-Ramirez, J., Sun, Q., Hwang, C., Ledbetter, C.A., Ramming, D.W., Fennell, A., Walker, A., Luby, J., Clark, M., Londo, J.P., Cadle Davidson, L.E., Zhong, G., Reisch, B. 2016. Towards the elucidation of the cytoplasmic diversity of North American Grape Breeding Programs. Molecular Breeding. 36:116.
- Londo, J.P., Kovaleski, A.P. 2017. Characterization of wild north american grapevine cold hardiness using differential thermal analysis. American Journal of Enology and Viticulture. ajev.2016.16090. DOI: 10.5344/ajev. 2016.16090.
- Chitwood, D., Klein, L., O'Hanlon, R., Chacko, S., Greg, M., Kitchen, C., Miller, A., Londo, J.P. 2016. Latent developmental and evolutionary shapes embedded within the grapevine leaf. New Phytologist. 210(1):343-355.
- Teh, S., Fresnedo, J., Clark, M., Sun, Q., Cadle Davidson, L.E., Luby, J. 2016. Genetic dissection of powdery mildew resistance in interspecific half-sib grapevine families using SNP-based maps. Molecular Breeding. 37:1.
- Wang, Y., Liu, X., Ren, C., Zhong, G., Yang, L., Li, S., Liang, Z. 2016. Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome. Biomed Central (BMC) Plant Biology. DOI: 10. 1186/x12870-016-0787-3.
- Guo, D., Xi, F., Yu, Y., Zhang, X., Zhang, G., Zhong, G. 2016. Comparative RNA-Seq profiling of berry development between table grape �Kyoho� and its early-ripening mutant �Fengzao�. BMC Genomics. 17(1):795. doi:10/1186/ s12864-016-3051-1.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 1: Identify and characterize the molecular, genetic, functional genomic, and biological factors controlling grape resistance to fungal and oomycete pathogens, with primary emphasis on powdery mildew. 1.A. Determine the genetic basis, pathogen race-specificity, and cellular mechanisms of powdery mildew and downy mildew resistance in grape breeding populations. 1.B. Determine the genetic basis by which abiotic stresses transiently alter foliar gene expression and induce resistance to grape powdery mildew and to extreme weather events. 1.C. Determine the genetic basis of grapevine ontogenic resistance to powdery mildew through quantitative trait locus analysis. 2: Characterize the genetic, genomic, and physiological aspects of grapevine abiotic stress tolerance and environmental adaptation. 2.A. Evaluate physiological variation in candidate thermal and dehydration stress traits from wild grapevine germplasm and mapping populations. 2.B. Identify and characterize candidate loci, linked genetic markers, or expression level changes for improved deep supercooling and environmental adaptation in grapevine. 3: Elucidate the key genetic and genomic factors controlling grape quality, including grape flavor and aroma, natural drying-on-vine, and berry size. 3.A. Determine genetic control of grape berry secondary metabolite production utilizing rapid-cycling grape germplasm populations. 3.B. Identify candidate genes or linked genetic markers associated with natural Dry-on-Vine raisins. 3.C. Determine the genetic basis of berry quality traits of muscadine grapes through quantitative trait locus analysis. 4: Develop and utilize novel or improved methods and genetic systems for grapevine breeding and genetic studies including, but not limited to, marker-assisted selection, germplasm for rapid trait evaluation, and transgenic solutions for introducing novel variation. 4.A. Develop Scout as a rapid-cycling system for trait evaluation and integration. 4.B. Develop and utilize transgenic and mutation technologies to introduce novel genetic variation for trait improvement. 4.C. Develop, evaluate, and apply computational methods for linking high quality large volume grapevine genotypic data with economically important grapevine traits and phenotypes. Approach (from AD-416): Grape production and product quality are substantially harmed by diseases and adverse environmental conditions. This research project provides genetic solutions to biotic and abiotic stresses, while maintaining high fruit quality. Our general strategies are: A) Utilization of existing or created populations and germplasm for characterizing traits of interest and their underlying molecular mechanisms through trait, marker, and functional analyses; B) Integration of desirable alleles into prebreeding lines via molecular markers and rapid-cycling breeding systems; and C) Development of alternative solutions to complex problems through transgenic technologies. The following knowledge and products will be delivered at the end of this project: 1) Molecular markers useful in grape scion breeding that predict multiple sources of disease resistance, abiotic stress tolerance, fruit quality, and precocious flowering; 2) Enhanced understanding of critical genetic processes underlying these traits; 3) Development of novel germplasm resources, including rapidly flowering genetic lines, for the genetic dissection and stacking of traits of interest; and 4) Improved capacity of grape breeders to develop superior lines through marker-assisted breeding and other technology- driven approaches. Successful accomplishment of this project will accelerate development of improved grape cultivars to maintain the competiveness of the US grape industry, by reducing fungicide applications in new plantings, expanding the options for grape growers faced with climate change or in new production regions, and opening new market niches for novel grape products. Novel Technology Applied to Powdery Mildew Resistance: Genetic tools are needed for low-cost analysis of multiple traits simultaneously. To address this ARS researchers in Geneva, New York developed AmpSeq, which is a $5 assay to determine up to 200 DNA markers simultaneously. This technique was validated for five powdery mildew resistance genes and applied in U.S. grape breeding programs. As a result, breeders were able to select vines having multiple resistance genes for improved durability, and discard undesirable vines. AmpSeq markers are now being validated for the prediction of flower sex and malic acid concentration in fruit. Technology Development: Development of an effective functional genomics platform for evaluating functions of genes and creating new genetic variation is critical to grape genetic research and trait development. ARS researchers in Geneva, New York are making significant progress in developing embryogenic callus from a rapid cycling Vitis vinifera variety, �Pixie� and using �Pixie� as a potential functional genomics platform. �Pixie� can flower and produce berries continuously and will be excellent genetic material for studying gene functions through transformation. Being able to produce embryogenic callus from �Pixie� is the first important step in developing the platform. Stress-Induced Disease Resistance (SIDR) to Powdery Mildew: Each Spring, grapevine powdery mildew epidemics proceed more slowly than predicted by epidemiological models. Working with colleagues at Cornell University, we have shown that these delays are due to the induction of host resistance by cool evening temperatures (below 50F), which commonly occur everywhere grapes are grown. These findings have now been extended to other crops, including hops, brassicas, and strawberries, showing that SIDR drastically reduces powdery mildew severity even in the most susceptible varieties. The work has resulted in decreased application of fungicides early in the growing season, and molecular genetic studies may suggest novel approaches for the development of disease resistant varieties. Stress Tolerance: Grapevine production that occurs outside of Mediterranean climate regions is limited due to environmental stresses such as cold temperatures. ARS researchers in Geneva, New York are pursuing a strategy that combines field based vine phenotyping and molecular biology-based laboratory analysis to identify elite cold hardy germplasm and elucidate the genetic attributes of the trait of cold hardiness. Field based assessments of chilling requirement, dormancy, budburst rate and supercooling ability have been completed for eight different wild grapevine species frequently used in breeding programs for scion and rootstock germplasm. In addition to the germplasm screen, an in- depth analysis has been performed on 43 V. riparia genotypes. Several new germplasm accessions with enhanced dormancy and deacclimation resistance have been identified for use in cold hardy grape breeding programs (manuscript in preparation). Studies examining polyamine concentrations in dormant bud tissue has revealed a correlation between cold hardiness and putricine levels as well as the ability to modify putricine metabolic pools throughout the winter season. Second year replicates are in progress with a publication to follow. In tandem with phenotyping, candidate gene evaluation of cold responsive regulatory factors and downstream effector genes (KIN-like genes) are being evaluated through overexpression in the model plant species Arabidopsis thaliana. Several independent transgenic lines have been generated and lines are being brought forward to advanced T4 generations. Analysis of gene expression data (RNASeq) is being used to identify and categorize differences in cold response between these wild species and with cultivated grape including several high resolution collections of dormant tissue throughout the winter and during loss of dormancy. Results of this study will be used to identify candidate genes for cold hardiness and identify the gene expression regulatory networks which are active during periods of winter where temperatures do not reach above the freezing point, yet changes in bud hardiness are observed. Reference transcriptome atlases have been produced for 24 different grapevine genotypes, representing eight species. Studies designed to identify novel gene transcripts, annotate allelic variation, and determine effects of SNP mutations are underway. Cluster Architecture: Vine architecture plays a critical role in enhancing yield and fruit quality of grapes as well as reducing viticulture management cost of grape production. Among many genes discovered for controlling plant architecture, GAI genes are best characterized and they are the genetic basis of �green revolution� in cereal crops in 1960s-1980s. ARS researchers in Geneva, New York have introduced several different versions of the grape Gibberellic Acid Insensitive (GAI) gene into a Vitis vinifera cultivar and observed dramatic changes of vine architecture including internode length, leaf size, overall vine height, and other traits. More evaluation and characterization of these transgenic lines are in progress. ARS researchers in Geneva, New York are also conducting a research project for modifying cluster architecture of a well-known wine grape cultivar, �Vignoles�. Vignoles is a valuable cultivar in the eastern United States due to its distinctive apricot, peach, and citrus notes in wines and its winter hardiness. However, Vignoles has compact clusters prone to secondary spread of Botrytis bunch rot late in the season and losses up to one third of the crop are possible in rainy years. ARS Researchers in Geneva, New York have used gamma irradiation to induce and identify 20 loose-clustered clones of Vignoles. These selections were grafted to rootstock and are being evaluated in field trials. First field trial data will be collected later this season. This work will help develop improved Vignoles with loose clusters and better resistance to Botrytis bunch rot. Resistance to Root-Knot Nematodes: Root-knot nematodes are a major pest of vineyards across California and the United States. Nematode management through fumigation can be useful in mitigating the problem, but use of chemicals is costly and environmentally undesirable. Resistant rootstocks are a sustainable method for managing nematodes in vineyards, but development of a successful resistant rootstock is a long process involving many years of effort. The challenge is further superimposed by the rapid evolving of aggressive nematodes and the desire of stacking other rootstock traits with nematode resistance. ARS researchers in Geneva, New York have been exploring a gene silencing technology which may offer a complementary approach for improving root-knot nematode resistance of grapevine rootstocks. In fiscal year 2016, we continued the evaluation of transgenic vines containing small double strand RNAs (dsRNAs) of a nematode gene required for parasitism in grapevine roots. The evaluation has been carried out on more than 10 independent transgenic lines in both greenhouse and in vitro conditions. While the results are promising, more tests and developmental work are required to prove the concept and then translate the concept into actual products. All subordinate projects are making good progress toward meeting their research goals and objectives. Accomplishments 01 Demonstrated genomic-scale mRNA exchanges between scions and rootstocks in grafted grapevines. Grafting has been widely practiced for centuries in the propagation and production of many vegetable and fruit species. However, the underlying molecular and genetic mechanisms for how the graft partners interact with each other to produce a successful graft are largely unknown. ARS researchers in Geneva, New York identified more than three thousand genes transporting mRNAs across graft junctions in grafted grapevines grown in the in vitro and field conditions. While many biological processes and mechanisms involved in such mRNA movement are yet to be elucidated, one obvious benefit from such exchange of mRNAs between two genetically distinct graft partners would increase diversity of the mRNA pool accessible to both scion and rootstock in a graft, which in turn can make the grafted plants more productive and adaptive to various biotic and abiotic conditions through complementation and synergistic interactions of the two genetic systems from scions and rootstocks. These findings have important theoretical and practical implications in enhancing efficiency of graft breeding. 02 Novel methods for highly controlled assessment of powdery mildew resistance. Across many crops, powdery mildew resistance is desired by farmers for reduced chemical input and for maintaining yield and quality. However, technical challenges in reproducibly measuring powdery mildew resistance in research fields have slowed the development of new varieties with resistance. These challenges include among other factors inability to control pathogen genetics, spatial variability of disease, and subjectivity. To address this problem, ARS researchers in Geneva, New York developed highly controlled laboratory evaluation methods that enabled 4-fold increase in statistical power to detect powdery mildew resistance genes. These methods enabled detection, localization, and DNA marker development for five powdery mildew resistance genes that are being used in U.S. grape breeding programs. These methods are of broad interest to basic and applied scientists studying various aspects of powdery mildew biology and management.
Impacts
(N/A)
Publications
- Summers, C., Gulliford, C., Lillis, J.A., Carlson, M., Cadle Davidson, L.E. , Gent, D.H., Smart, C. 2015. Identification of genetic variation between obligate plant pathogens Psuedoperonospora cubensis and P. humuli using RNA sequencing and genotyping-by-sequencing. PLoS One. doi: 10.1371/ journal.pone.0143665.
- Yang, S., Fresnedo, J., Sun, Q., Manns, D., Sacks, G., Awale, M., Mansfield, A., Luby, J., Londo, J.P., Reisch, B., Cadle Davidson, L.E., Fennell, A. 2016. Next generation mapping of enological traits in an F2 interspecific grapevine hybrid family. PLoS One. 11(3):e0149560. doi:10. 1371/journal.pone.0149560.
- Wen, Y., Zhong, G., Gao, Y., Lan, Y., Duan, C., Pan, Q. 2015. Using the combined analysis of transcripts and metabolites to propose key genes for differential terpene accumulation across two regions. Biomed Central (BMC) Plant Biology. 15:240. DOI: 10.1186/s12870-015-0631-1.
- Yang, Y., Mao, L., Jittayasothorn, Y., Kang, Y., Jiao, C., Fei, Z., Zhong, G. 2015. Messenger RNA exchange between scions and rootstocks in grafted grapevines. Biomed Central (BMC) Plant Biology. 15:251.
- Warschefsky, E., Bishop-Von Wettberg, E., Chitwood, D., Frank, M., Klein, L., Londo, J.P., Miller, A. 2015. Rootstocks: diversity, domestication and impacts on shoot phenotypes. Trends in Plant Science. DOI: http//dx.doi. org/10.106/j.tplants.2015.11.008.
- Yang, S., Fresnedo, J., Wang, M., Cote, L., Bogdanowicz, S., Schweitzer, P. , Barba, P., Takacs, E., Clark, M., Luby, J., Manns, D., Sacks, G., Mansfield, A., Londo, J.P., Fennel, A., Gadoury, D., Reisch, B., Cadle Davidson, L.E., Sun, Q. 2016. A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker assisted selection in grapevine. Horticulture Research. 3:16002.doi:10.1038/hortres.2016.2.
- Londo, J.P., Garris, A. 2014. Genetic and protein sequence variation of CBF1-4 in cold hardy wild grapevine germplasm. Vitis. 53(4):201-206.
- Londo, J.P., Mckinney, J., Schwartz, M., Bollman, M., Sagers, C., Watrud, L. 2014. Sub-lethal glyphosate exposure increases outcrossing potential in Brassica spp. by altering flowering phenology and causing transient male- sterility. Biomed Central (BMC) Plant Biology. doi:10.1186/1471-2229-14-70.
- Londo, J.P., Martinson, T. 2015. Geographic trend of bud hardiness response in Vitis riparia. Acta Horticulturae. (ISHS) 1082:299-304.
- Sagers, C., Londo, J.P., Bautista, N., Lee, E.H., King, G., Watrud, L. 2015. Benefits of transgenic insect resistance in Brassica hybrids under selection. Journal of Agronomy. 5(1):21-34.
- Moyer, M., Londo, J.P., Gadoury, D., Cadle Davidson, L.E. 2015. Cold Stress-Induced Disease Resistance(SIDR): Indirect effects of low temperatures on host-pathogen interactions and disease progress in the grapevine powdery mildew pathosystem. European Journal of Plant Pathology. 2:5-8. doi: 10.1007/s10658-015-0745-1.
- Pap, D., Miller, A., Londo, J.P., Kovacs, L. 2015. Population structure of Vitis rupestris, an important resource for viticulture. American Journal of Enology and Viticulture. 66:403-410.
- Hyma, K., Barba, P., Wang, M., Londo, J.P., Acharya, C., Mitchell, S., Sun, Q., Reisch, B., Cadle Davidson, L.E. 2015. Heterozygous mapping strategy (HetMapps) for high resolution genotyping-by-sequencing markers: a case study in grapevine. PLoS One. 10(8):e0134880. doi: 10.1371/journal/pone. 0134880.
- Chitwood, D., Klein, L., Miller, A., Londo, J.P. 2016. Leaves as composites of latent developmental and evolutionary shapes. New Phytologist. 210(1):343-355.
- Chitwood, D.H., Rundell, S.M., Li, D.Y., Woodford, Q.L., Yu, T.T., Lopez, J.R., Kang, J., Londo, J.P. 2016. Climate and developmental plasticity: interannual variability in grapevine leaf morphology. Plant Physiology. doi: 10.1101/030957.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 1: Identify and characterize the molecular, genetic, functional genomic, and biological factors controlling grape resistance to fungal and oomycete pathogens, with primary emphasis on powdery mildew. 1.A. Determine the genetic basis, pathogen race-specificity, and cellular mechanisms of powdery mildew and downy mildew resistance in grape breeding populations. 1.B. Determine the genetic basis by which abiotic stresses transiently alter foliar gene expression and induce resistance to grape powdery mildew and to extreme weather events. 1.C. Determine the genetic basis of grapevine ontogenic resistance to powdery mildew through quantitative trait locus analysis. 2: Characterize the genetic, genomic, and physiological aspects of grapevine abiotic stress tolerance and environmental adaptation. 2.A. Evaluate physiological variation in candidate thermal and dehydration stress traits from wild grapevine germplasm and mapping populations. 2.B. Identify and characterize candidate loci, linked genetic markers, or expression level changes for improved deep supercooling and environmental adaptation in grapevine. 3: Elucidate the key genetic and genomic factors controlling grape quality, including grape flavor and aroma, natural drying-on-vine, and berry size. 3.A. Determine genetic control of grape berry secondary metabolite production utilizing rapid-cycling grape germplasm populations. 3.B. Identify candidate genes or linked genetic markers associated with natural Dry-on-Vine raisins. 3.C. Determine the genetic basis of berry quality traits of muscadine grapes through quantitative trait locus analysis. 4: Develop and utilize novel or improved methods and genetic systems for grapevine breeding and genetic studies including, but not limited to, marker-assisted selection, germplasm for rapid trait evaluation, and transgenic solutions for introducing novel variation. 4.A. Develop Scout as a rapid-cycling system for trait evaluation and integration. 4.B. Develop and utilize transgenic and mutation technologies to introduce novel genetic variation for trait improvement. 4.C. Develop, evaluate, and apply computational methods for linking high quality large volume grapevine genotypic data with economically important grapevine traits and phenotypes. Approach (from AD-416): Grape production and product quality are substantially harmed by diseases and adverse environmental conditions. This research project provides genetic solutions to biotic and abiotic stresses, while maintaining high fruit quality. Our general strategies are: A) Utilization of existing or created populations and germplasm for characterizing traits of interest and their underlying molecular mechanisms through trait, marker, and functional analyses; B) Integration of desirable alleles into prebreeding lines via molecular markers and rapid-cycling breeding systems; and C) Development of alternative solutions to complex problems through transgenic technologies. The following knowledge and products will be delivered at the end of this project: 1) Molecular markers useful in grape scion breeding that predict multiple sources of disease resistance, abiotic stress tolerance, fruit quality, and precocious flowering; 2) Enhanced understanding of critical genetic processes underlying these traits; 3) Development of novel germplasm resources, including rapidly flowering genetic lines, for the genetic dissection and stacking of traits of interest; and 4) Improved capacity of grape breeders to develop superior lines through marker-assisted breeding and other technology- driven approaches. Successful accomplishment of this project will accelerate development of improved grape cultivars to maintain the competiveness of the US grape industry, by reducing fungicide applications in new plantings, expanding the options for grape growers faced with climate change or in new production regions, and opening new market niches for novel grape products. Pathogen Resistance: Genotyping strategies for high-diversity species. Like many specialty crops, grapevines have high genetic diversity, providing opportunity for genetic improvement but scientific challenges in genetic analysis. ARS scientists in Geneva, New York, and Ithaca, New York, developed a genotyping microarray for grapevines, and we developed analytical approaches that are relevant to high diversity species. We demonstrated these approaches in the USDA-ARS germplasm repositories and in breeding populations, including the genetic analysis of three key traits: fruit color, flower sex, and powdery mildew resistance. While we have since identified superior genotyping strategies for high diversity species, these analytical approaches should be of broad interest to scientists using genotyping microarrays. Many economically important oomycete plant pathogens, such as downy mildews, cannot be cultured and thus have poor genetic resources available for research. To discover new genetic markers for characterizing oomycete biology, we applied genome-wide DNA and RNA sequencing to three downy mildews � on grapevine, hops, and cucurbits. We showed that DNA and RNA approaches are robust and provide an excess of markers (>100,000) for population genetic analyses. Tolerance to Abiotic Stress: Grapevine production that occurs outside of Mediterranean climate regions is limited due to environmental stresses such as cold temperatures. We are pursuing a strategy that combines field based vine phenotyping and molecular biology-based laboratory analysis to identify elite cold hardy germplasm and elucidate the genetic attributes of the trait of cold hardiness. Field based assessments have been completed for 8 different wild grapevine species frequently used in breeding programs for scion and rootstock germplasm. The traits of chilling hour requirement, endo- and eco- dormancy, budburst rate, supercooling ability, and deacclimation resistance have been determined for these species. Initial results have identified new sources of cold hardy, deacclimation resistant wild grapevine that can be used to enhance breeding programs. Assessments of the wild V. riparia germplasm have identified specific elite accessions for cold hardiness breeding. Preliminary studies examining polyamine concentrations in dormant bud tissue has revealed a correlation between cold hardiness and putricine levels as well as the ability to modify putricine metabolic pools throughout the winter season. In tandem with phenotyping, candidate gene evaluation of cold responsive regulatory factors and downstream effector genes (KIN-like genes) are being evaluated through overexpression in the model plant species Arabidopsis thaliana. Finally, next generation analysis of gene expression data (RNASeq) is being used to identify and categorize differences in cold response between these wild species and with cultivated grape. Reference transcriptome atlases have been produced from 6-8 different plant organs, for 24 different grapevine genotypes, representing 8 species. Studies designed to identify novel gene transcripts, annotate allelic variation, and determine effects of SNP mutations are underway. Fruit Quality Improvement: Traditional grapevine cultivars grown for raisin production, such as Thompson Seedless, are heavily dependent on manual labor and favorable weather conditions to ensure adequate drying of the raisins post-harvest by sun-drying fruit on trays in the vineyard, i.e. tray drying. An improvement over tray drying has come through the adoption of Dry-on-Vine (DOV) in which the grapes dry into raisins while still in the trellis and are mechanically harvested. To elucidate the genetic mechanisms controlling this important trait, a large population of 480 seedlings derived from a cross of two numbered selections from the USDA-ARS grape breeding program in Parlier, California, was screened for variation in drying traits. Additionally, DNA was isolated from the seedlings, and genotyping-by-sequencing (GBS) was conducted to generate a large number of molecular markers for constructing a linkage map from this cross so that important regions of the genome controlling these traits can be identified. Muscadine grapes (V. rotundifolia) are an important fruit crop within the Southeastern U.S. but very little genetic information is known, particularly compared to the European bunch grape (V. vinifera). In order to develop molecular markers useful for assisting selection of several important fruit quality traits in muscadine grape breeding programs, two experimental populations that segregate for many fruit quality characteristics were evaluated. 172 seedlings of a cross of Black Beauty X Nesbitt and 174 seedlings of a cross of Supreme x Nesbitt were evaluated. Specific traits that have been characterized in each of the two populations are: berry weight, berry juice pH, soluble solids at harvest, berry color, percent dry picking scar, petiole color, flower sex, date of bloom, and date of harvest. DNA was isolated from the seedlings, and GBS was conducted to generate a large number of molecular markers for constructing a linkage map from these crosses so that important regions of the genome controlling these traits can be identified. Grape aroma is a primary determinant of table grape, unfermented juice, and wine quality. One key class of aromatic compounds that are important in several widely grown cultivars are the 3-alkyl-2-methoxypyrazines (MPs) . Previous work had shown two major unlinked QTL that controlled a significant amount of the phenotypic variation within a cross of a cultivar producing a large amount of MPs (Cabernet Franc) and a variety that does not produce MPs (St. Pepin). Individual seedlings showing extreme phenotypic values within the segregating population and recombination near the identifying QTL were chosen for generating additional populations. These parents were crossed with a rapid-cycling grape genotype so that fruit from the resulting seedlings can be generating at a younger age. Cluster Architecture: Vignoles is a valuable cultivar in the eastern United States due to its distinctive apricot, peach, and citrus notes in wines and its winter hardiness. However, Vignoles has compact clusters prone to secondary spread of Botrytis bunch rot late in the season and losses up to one third of the crop are possible in rainy years. We have used gamma irradiation to induce and identify 20 loose-clustered clones of Vignoles. These selections have now been grafted to rootstock for field trials. Field trials may take several years, but generation and identification of these mutant selections are critical steps toward the development of improved Vignoles with loose clusters and better resistance to Botrytis bunch rot. Resistance to root-knot nematodes: Root-knot nematodes are a major pest of vineyards across California and the United States. Nematode management through fumigation can be useful in mitigating the problem, but use of chemicals is costly and environmentally undesirable. Resistant rootstocks are a sustainable method for managing nematodes in vineyards. USDA-ARS Grape Genetics Research Unit released three nematode resistant rootstocks developed through hybridization. A gene silencing technology may offer a complementary approach for improving root-knot nematode resistance of grapevine rootstocks. In FY15, we continued the evaluation of transgenic vines containing small double strand RNAs (dsRNAs) of a nematode gene required for parasitism in grapevine roots. Accomplishments 01 Developed strategies to stack grape genes for durable powdery mildew resistance. Powdery mildew is the most costly grape disease, in part because nearly all grape varieties lack powdery mildew resistance. Grape breeders across the U.S. and around the world are developing powdery mildew resistant varieties. ARS researchers in Geneva, New York, showed that individual resistance genes were not effective against some powdery mildew clones, and that some resistance gene combinations were no more effective than a single gene. Then they identified resistance gene combinations that would be most effective and durable in long-term grape plantings. With this approach they have developed research-based strategies and methods for breeders to incorporate the best combinations of resistance genes in their grape breeding programs for durable management of powdery mildew. 02 Molecular markers for an important grape quality gene developed. Anthocyanins in red grapes are important components of wine and beneficial to human health, but some of them are undesirable for wine- making if they are in a chemical state called �diglucoside�. This chemical state is under genetic control of a gene and development of molecular markers for detecting this gene in a breeding program is important. ARS researchers in Geneva, New York, surveyed different forms of the gene in more than 100 different types of cultivated and wild grapes and developed molecular markers for the gene. This accomplishment advances our understanding of the genetic diversity of the gene and provides a molecular basis for improving this important wine quality trait.
Impacts
(N/A)
Publications
- Londo, J.P., Garris, A. 2014. Genetic and protein sequence variation of CBF1-4 in cold hardy wild grapevine germplasm. Vitis. 53(4):201-206.
- Londo, J.P., Johnson, L.M. 2014. Variation in the chilling requirement and bud burst rate of wild Vitis species. Environmental and Experimental Botany. 160: 138-147.
- Kono, A., Sato, A., Reisch, B., Cadle Davidson, L.E. 2015. Effect of detergent on the quantification of grapevine downy mildew Sporangia from leaf discs. HortScience. 50(5):656-660.
- Frenkel, O., Cadle Davidson, L.E., Wilcox, W., Milgroom, M. 2014. Mechanisms of resistance to an azole fungicide in the grapevine powdery mildew fungus, Erysiphe necator. Phytopathology. 105(3):370-377.
- Yang, Y., Labate, J.A., Liang, Z., Cousins, P., Prins, B.H., Preece, J.E., Aradhya, M.K., Zhong, G. 2014. Multiple loss-of-function 5-O- Glucosyltransferase alleles revealed in Vitis vinifera, but not in other Vitis species. Theoretical and Applied Genetics. 127(11):2433-2451.
- Liang, Z., Cheng, L., Zhong, G., Liu, R. 2014. Total antioxidant and antiproliferative activities of twenty-four Vitis vinifera grapes. PLoS One. 9(8): e105146. DOI:10.1371/journal.pone.0105146.
- Owens, C.L. 2015. Grape Pigments in Pigments in Fruits and Vegetables: Genomics and Dietetics. Springer. Chunxian, C. (ed.), pp. 189-204.
- Feechan, A., Kocsis, M., Riaz, S., Zhang, W., Walker, A., Dry, I., Reisch, B., Cadle Davidson, L.E. 2015. Strategies for RUN1 deployment using RUN2 and REN2 to manage grapevine powdery mildew informed by studies of race- specificity. Phytopathology. DOI: 10.1094/Phyto-09-14-0244-R.
- Wang, L., Zhu, W., Fang, L., Sun, X., Su, L., Wang, N., Liang, Z., Londo, J.P., Li, S., Xin, H. 2014. Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera. Biomed Central (BMC) Plant Biology. 14(1):103. DOI:10.1186/1471-2229-14-103.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): 1: Identify and characterize the molecular, genetic, functional genomic, and biological factors controlling grape resistance to fungal and oomycete pathogens, with primary emphasis on powdery mildew. 1.A. Determine the genetic basis, pathogen race-specificity, and cellular mechanisms of powdery mildew and downy mildew resistance in grape breeding populations. 1.B. Determine the genetic basis by which abiotic stresses transiently alter foliar gene expression and induce resistance to grape powdery mildew and to extreme weather events. 1.C. Determine the genetic basis of grapevine ontogenic resistance to powdery mildew through quantitative trait locus analysis. 2: Characterize the genetic, genomic, and physiological aspects of grapevine abiotic stress tolerance and environmental adaptation. 2.A. Evaluate physiological variation in candidate thermal and dehydration stress traits from wild grapevine germplasm and mapping populations. 2.B. Identify and characterize candidate loci, linked genetic markers, or expression level changes for improved deep supercooling and environmental adaptation in grapevine. 3: Elucidate the key genetic and genomic factors controlling grape quality, including grape flavor and aroma, natural drying-on-vine, and berry size. 3.A. Determine genetic control of grape berry secondary metabolite production utilizing rapid-cycling grape germplasm populations. 3.B. Identify candidate genes or linked genetic markers associated with natural Dry-on-Vine raisins. 3.C. Determine the genetic basis of berry quality traits of muscadine grapes through quantitative trait locus analysis. 4: Develop and utilize novel or improved methods and genetic systems for grapevine breeding and genetic studies including, but not limited to, marker-assisted selection, germplasm for rapid trait evaluation, and transgenic solutions for introducing novel variation. 4.A. Develop Scout as a rapid-cycling system for trait evaluation and integration. 4.B. Develop and utilize transgenic and mutation technologies to introduce novel genetic variation for trait improvement. 4.C. Develop, evaluate, and apply computational methods for linking high quality large volume grapevine genotypic data with economically important grapevine traits and phenotypes. Approach (from AD-416): Grape production and product quality are substantially harmed by diseases and adverse environmental conditions. This research project provides genetic solutions to biotic and abiotic stresses, while maintaining high fruit quality. Our general strategies are: A) Utilization of existing or created populations and germplasm for characterizing traits of interest and their underlying molecular mechanisms through trait, marker, and functional analyses; B) Integration of desirable alleles into prebreeding lines via molecular markers and rapid-cycling breeding systems; and C) Development of alternative solutions to complex problems through transgenic technologies. The following knowledge and products will be delivered at the end of this project: 1) Molecular markers useful in grape scion breeding that predict multiple sources of disease resistance, abiotic stress tolerance, fruit quality, and precocious flowering; 2) Enhanced understanding of critical genetic processes underlying these traits; 3) Development of novel germplasm resources, including rapidly flowering genetic lines, for the genetic dissection and stacking of traits of interest; and 4) Improved capacity of grape breeders to develop superior lines through marker-assisted breeding and other technology- driven approaches. Successful accomplishment of this project will accelerate development of improved grape cultivars to maintain the competiveness of the US grape industry, by reducing fungicide applications in new plantings, expanding the options for grape growers faced with climate change or in new production regions, and opening new market niches for novel grape products. Pathogen Resistance: In protecting grapes from fungal and oomycete pathogens, we pursued a strategy to know the enemy then identify tools effective against that enemy. Ren4 is a resistance locus that provides broad-spectrum resistance against diverse clones of powdery mildew. We identified and sequenced a region of the grape genome linked to Ren4. In that region, we checked the expression of genes in susceptible and resistant lines and identified 20 Ren4 candidate genes. We annotated and analyzed these 20 candidate genes across a panel of diverse grape lines, and selected ten genes that we are isolating for functional characterization. We previously showed that when susceptible grapevines are subjected to abiotic stress, such as cool spring nights below 50F, they become temporarily resistant to powdery mildew. This phenomenon is called Stress-Induced Disease Resistance (SIDR). We identified several wild Vitis that lack SIDR and developed an F1 family of 131 vines that should vary in the expression of SIDR. In FY14, we completed two screens of this family for cold SIDR. In addition, we germinated 600 F2 seedlings, in case the F1 vines had no variation in SIDR. Cultivated grape berries only remain susceptible to powdery mildew for 2 to 3 weeks post-bloom before they develop ontogenic resistance. We identified one wild Vitis accession that remains susceptible throughout development and developed an F1 family that should vary in the expression of ontogenic resistance. In FY14, we collected microscopy data on ontogenic resistance for a small subset of this population, and are preparing for a field screen in July 2014. Tolerance to Abiotic Stress: Grapevine production that occurs outside of Mediterranean climate regions is limited due to environmental stresses such as cold temperatures. We are pursuing a strategy that combines field based vine phenotyping and molecular biology based laboratory analysis to identify elite cold hardy germplasm and elucidate the genetic attributes of the trait of cold hardiness. Field based assessments have been performed to measure chilling hour requirement, endo- and eco- dormancy, budburst rate, supercooling ability, and deacclimation resistance of eight different wild grapevine species that are frequently utilized in breeding programs for scion and rootstock germplasm. In tandem with phenotyping, candidate gene evaluation of cold responsive regulatory factors and downstream effector genes are used to identify underlying genetic variation that may contribute to phenotypic differences. Finally, next generation analysis of gene expression data (RNASeq) is being used to identify and categorize differences in cold response between these wild species and with cultivated grape. Initial results have identified new sources of cold hardy, deacclimation resistant wild grapevine that can be used to enhance breeding programs. Genetic variation has been quantified for several regulatory genes responsible for cold response. Finally, reference transcriptome atlases have been produced from 6-8 different plant organs, for 24 different grapevine genotypes, representing 8 species. Fruit Quality Improvement: Traditional grapevine cultivars grown for raisin production, such as Thompson Seedless, are heavily dependent on manual labor and favorable weather conditions to ensure adequate drying of the raisins post-harvest by sun-drying fruit on trays in the vineyard, i.e. tray drying. An improvement over tray drying has come through the adoption of Dry-on-Vine (DOV) in which the grapes dry into raisins while still in the trellis and are mechanically harvested. To elucidate the genetic mechanisms controlling this important trait, a large population of 480 seedlings derived from a cross of two numbered selections from the USDA-ARS grape breeding program in Parlier, CA, was screened for variation in drying traits. Additionally, DNA was isolated from the seedlings, and genotyping-by-sequencing (GBS) was conducted to generate a large number of molecular markers for constructing a linkage map from this cross so that important regions of the genome controlling these traits can be identified. Muscadine grapes (V. rotundifolia) are an important fruit crop within the Southeastern U.S. but very little genetic information is known, particularly compared to the European bunch grape (V. vinifera). In order to develop molecular markers useful for assisting selection of several important fruit quality traits in muscadine grape breeding programs, two experimental populations that segregate for many fruit quality characteristics were evaluated. 172 seedlings of a cross of Black Beauty X Nesbitt and 174 seedlings of a cross of Supreme x Nesbitt were evaluated. Specific traits that have been characterized in each of the two populations are: berry weight, berry juice pH, soluble solids at harvest, berry color, percent dry picking scar, petiole color, flower sex, date of bloom, and date of harvest. DNA was isolated from the seedlings, and genotyping-by-sequencing (GBS) was conducted to generate a large number of molecular markers for constructing a linkage map from these crosses so that important regions of the genome controlling these traits can be identified. Grape aroma is a primary determinant of table grape, unfermented juice, and wine quality. One key class of aromatic compounds that are important in several widely grown cultivars are the 3-alkyl-2-methoxypyrazines (MPs) . Previous work had shown two major unlinked QTL that controlled a significant amount of the phenotypic variation within a cross of a cultivar producing a large amount of MPs (Cabernet Franc) and a variety that does not produce MPs (St. Pepin). Individual seedlings showing extreme phenotypic values within the segregating population and recombination near the identifying QTL were chosen for generating additional populations. These parents were crossed with a rapid-cycling grape genotype so that fruit from the resulting seedlings can be generated at a younger age. Vine Development and Cluster Architecture: In 2010 USDA-ARS GGRU released Scout, a grapevine cultivar that flowers freely on prompt laterals below the 25th node. This precocious flowering property of Scout makes it a useful genetic system for rapid trait evaluation. Elucidation of the genetic control of precocious flowering in Scout will enhance its use as a genetic system for accelerating grapevine breeding and genetic studies. In FY14, we have phenotyped and genotyped a segregation population segregating for the precocious flowering trait, constructed a genetic map and identified a potential major QTL for the trait. Resistance to root-knot nematodes: Root-knot nematodes are a major pest of vineyards across California and the United States. Nematode management through fumigation can be useful in mitigating the problem, but use of chemicals is costly and environmentally undesirable. Resistant rootstocks are a sustainable method for managing nematodes in vineyards. USDA-ARS GGRU released three nematode resistant rootstocks developed through hybridization. A gene silencing technology may offer a complementary approach for improving root-knot nematode resistance of grapevine rootstocks. In FY14, we generated transgenic vines containing small double strand RNAs (dsRNAs) of a nematode gene required for parasitism in grapevine roots. Evaluation of these transgenic vines against root-knot nematodes is in progress. Significant Activities that Support Special Target Populations: For an undergraduate summer scholar with vision and hearing impairment, we provided research training on a project that introduced her to plant pathology, molecular biology, genetics, and statistics, accommodating her specific needs to complete the project. At the end of the project, she presented a poster on her research at a Cornell University poster session. Accomplishments 01 DNA-informed breeding for durable powdery mildew resistance. Powdery mildew is the most costly grape disease, in part because nearly all grape varieties lack powdery mildew resistance. Grape breeders across the U.S. and around the world are developing powdery mildew resistant varieties. In FY14, ARS researchers at Geneva, New York facilitated DNA marker screening of over 5000 grape breeding lines to track powdery mildew resistance genes and to combine multiple resistance genes in a single vine for more durable resistance. They showed that individual resistance genes were not effective against some powdery mildew clones, and that some resistance gene combinations were no more effective than a single gene. Then they identified resistance gene combinations that would be most effective and durable in long-term grape plantings. With this approach they have developed research-based strategies and methods for breeders to incorporate the best combinations of resistance genes in their grape breeding programs for durable management of powdery mildew. 02 Identification of cold hardy, deacclimation resistant grapevine germplasm. Grapevine breeders selecting for cold hardiness traits have traditionally utilized species which are characterized by an ability to deep supercool and survive temperatures of -35 degrees C. However, this same germplasm is prone to rapid fulfillment of dormancy, rapid deacclimation in response to warming temperatures, and rapid budburst. These traits expose vines to acute cold snaps or early spring frosts, which can damage or destroy the fruiting tissues. ARS researchers at Geneva, New York have used field based studies examining the maximal bud hardiness and deacclimation rate traits of wild grapevine to identify germplasm accessions where the trait of midwinter hardiness has been decoupled from low chilling requirement and rapid budburst. Identification of this germplasm provides a breeding tool for grapevine breeders who want to retain the deep midwinter hardiness phenotype but with greater control over chilling requirement and budburst rate. 03 Identification of Vignoles mutant selections with reduced cluster compactness. Vignoles is a valuable cultivar in the eastern United States due to its distinctive apricot, peach, and citrus notes in wines and its winter hardiness. However, Vignoles has compact clusters prone to secondary spread of Botrytis bunch rot late in the season and losses up to one third of the crop are possible in rainy years. ARS researchers in Geneva, NY have used gamma irradiation to induce several thousands of Vignoles mutants and identified 20 loose-clustered clones of Vignoles through 3 years of field evaluation. Generation and identification of these mutant selections are critical steps toward the development of improved Vignoles with loose clusters and better resistance to Botrytis bunch rot.
Impacts
(N/A)
Publications
- Saito, S., Cadle Davidson, L.E., Wilcox, W. 2014. Selection, fitness, and control of grape isolates of Botrytis cinerea variably sensitive to fenhexamid. Plant Disease. 98:233-240.
- Barba, P., Cadle Davidson, L.E., Harriman, J., Glaubitz, J., Mahanil, S., Hyma, K., Reisch, B. 2014. Grapevine powdery mildew resistance and susceptibility loci identified on a high-resolution SNP map. Journal of Theoretical and Applied Genetics. 127:73-84.
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