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"Evaluation of fruit quality and disease resistance in sea buckthorn germplasm, based on molecular markers and metabolomics"

Final Report Summary - HIPPOHEALTH (Evaluation of fruit quality and disease resistance in sea buckthorn germplasm, based on molecular markers and metabolomics)

1. The work carried out to achieve the project's objectives

1.1 A summary description of the project objectives
Sea buckthorn (Hippophae L.) is a woody, dioecious plant that can be grown in areas characterized by severe drought, high salinity and poor soil conditions. It has recently received considerable attention in Europe and China as a new berry crop with a very high nutritional value and unique medicinal properties, as well as a means of combating soil erosion. Methods must, however, be developed that enable fast and efficient identification of genotypes carrying genes for desirable plant architecture and fruit quality as well as resistance to the very detrimental dried-shrink disease (DSD) in order to achieve a sustainable commercial production of this crop. The objectives of this proposal are to investigate fruit quality parameters and DSD-resistance in already collected germplasm presently conserved in China and in Sweden, respectively, and then develop molecular techniques that will allow fast and accurate development of elite cultivars destined both for direct usage and as a parental material in plant breeding programs. Fruit quality traits and disease resistance will be screened in European and Chinese plant materials. Molcecular markers associated with these traits will be identified using the germplasm-regression-combined marker-trait association analysis. Relationships between genotypic and environmental effects on desirable traits will be investigated using metabolomic techniques based on GC/LC-MS and 1H-NMR. Carefully selected genetic resource collections (gene banks) will be established both at Balsgard in Sweden and at Fuxin in China. A germplasm evaluation system, based on markers associated with desirable traits and results from the metabolomics analysis, will be constructed that provides fast and efficient identification and selection of elite genotypes with high fruit quality in combination with DSD-resistance. These tools will provide a platform for marker- and metabolomics-assisted breeding to develop further improved cultivars.

1.2 The work carried out to achieve the project's objectives
From 06/07/2011 to 06/06/2013, to perform the tasks towards the objectives of the project, we have i) tested bioactive compounds of sea buckthorn berry from four maternal half-sib families in 2011 and 2012, including vitamin C, malic and quinic acids, sugars mainly including ethyl-glucose, fructose and glucose, ursolic and oleanic acids and polyphenols including 18 compoounds; ii) measured variation of oil content in berry pulp during berry ripening; iii) measured dynamic changes of metabolites during berry ripening with untargeted (LC-MS) and targeted methods; iv) identified the differences in metabolites between berry pulp and seeds with untargeted (NMR and GC-MS) methods; v) tested genetic variation of maternal half-sib families using ISSR and SRAP markers; and vi) investigated agronomic traits of maternal half-sib families and symptoms of infection by the dried-shrink disease in a germplasm collection of sea buckthorn (Hippophae L.) growing at Balsgard, Sweden.

2 The main results, conclusions and their potential impact and use and any socio-economic impact of the project

2.1 The main results and conclusions
Based on samples collected from sea buckthorn germplasm collection at Balsgard in Sweden and different regions in China, we first studied genetic variation of bioactive compounds in berry of sea buckthorn maternal half-sib families using HPLC and GC-MS methods, and also a metabolic profiling of sea buckthorn berry based on untargeted NMR and GC/LC-MS. The main achievements based on these studies are shown as follows: i) genetic variation of bioactive compounds of sea buckthorn berry in maternal half-sib families: there were significant differences for contents of vitamin C, ethyl-glucose, fructose and glucose, quinic and malic acids, ursolic and oleanic acids, and 18 polyphenols in fresh berries; significant differences of these compounds were also found between maternal half-sib families; ii) Sourness and astringency are negatively correlated with pleasantness and thus consumer acceptance, whereas sweetness has a strong positive effect on the perceived pleasantness of sea buckthorn berry. We found that the palatability is correlated with the sugar/acid ratios; in fresh berries a ratio of about 1.8–2.0 is perceived as pleasant; iii) metabolic profiling during berry ripening reveals key metabolic networks: metabolic networks in sea buckthorn berry development, especially for compounds (e.g. vitamin C, qualic and malic acids) that showed a difference in patterns of change (stability vs. variation). Presently, we are performing these analyses; and iv) There was a strong separation between pulp and seeds in the NMR signal intensity of bioactive compounds and GC-MS analyses, such as amino acids (GABA, aspartate, glutamate, theanine and proline), organic acids (citrate, succinate, malate, acetate, quinate and heriguard), carbohydrates (sucrose, fructose, glucose and melibiose), aldehydes, phenol and some fatty acids. Sea buckthorn cultivars could be separated into two well-defined groups using PCA based on the NMR spectroscopy and GC-MS data of bioactive compounds in the pulp and seeds. Several metabolic compounds like sugar, organic acids, amino acids, like 2,4-dimethyl benzaldehyde, 2,6-ditert-butyl-Phenol and special fatty acid could serve as biomarkers for prediction of berry quality and for classification of germplasm collections. This dataset provides potential information concerning the mechanisms of berry quality formation and contributes to the breeding of high-quality sea buckthorn.

2.2 Potential impact and use and any socio-economic impact of the project
i) Significant differences within maternal half-sib families will provide a valuable basis for pre-breeding and breeding of sea buckthorn cultivars with desirable traits, such as high vitamin C content in fresh berry, optimal ratio of sugar to acid for fresh-berry for consumer acceptance, high contents in berry-oil, ursolic acid and polyphenols.
ii) Metabolic-based biomarkers snf ISSR snf SRAP markers associated with desirable traits identified in this project will be applied for selection and identification of sea buckthorn germplasm with desirable traits.
iii) There is a strong need for developing improved sea buckthorn cultivars with desirable berry traits (e.g. optimal ratio of sugar and acid) due to the world-wide market demand for bioactive sea buckthorn berries. The germplasm evaluation system based on bio- and DNA-markers will be perfected through this project, thus resulting in cost-efficient preservation and use of sea buckthorn collections. Access to different markers will support the ongoing sea buckthorn breeding and research achieves in Sweden and China, ultimately resulting in increased efficient commercial and sustainable utilization of sea buckthorn for alleviating poverty and improving the living standard and health situation.