Final Report Summary - HIPPOHEALTH (Evaluation of fruit quality and disease resistance in sea buckthorn germplasm, based on molecular markers and metabolomics)
• 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. SSR 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.
• a description of the work performed since the beginning of the project
From 10/06/2013 to 09/06/2014, to perform the tasks towards the objectives of the project, we have i) developed simple sequence repeat (SSR) markers of sea buckthorn (Hippophae L.) using amplified fragments of ISSR markers and RNA-sequences of sea buckthorn from NCBI; ii) identified ISSR and SRAP markers associated with sea buckthorn DSD-resistance; iii) measured the fungal infection-induced metabolites in sea buckthorn using 1H NMR and fourier transform infrared (FT-IR) spectroscopy; vi) tested the changes of oil contents and components of fatty acids in sea buckthorn berries of maternal half-sib families and cultivars during ripening using targeted GC-MS spectroscopy and principal component analysis (PCA); and v) investigated the changes of metabolites of pathogenic fungi infected leaves of sea buckthorn.
• a description of the main results achieved so far
Eight SSR markers were developed by a simple method of using amplified fragments of ISSR. Stepwise multiple regression analysis were used to identified four ISSR markers (887190, 835700, 809290, and 811280) associated with DSD-resistance with highly negative correlation (p < 0.001) and eleven SRAP markers (Me1-Em3600, Me1-Em1680, Me2-Em1650, Me2-Em1950, Me3-Em61300, Me2-Em6320, Me2-Em6400, Me1-Em2600, Me1-Em11200, Me1-Em11700, Me2-Em2250) were significantly correlated with DSD-resistance (P < 0.001).
There are significant differences in oil contents of dry-pulp among different individuals of maternal half-sib family. Significant differences in oil contents of dry-pulp did not found in different berry ripe time of two cultivars of BHi72715 and BHi7278.
The metabolic interactions between three cultivars of sea buckthorn and three pathogenic fungi such as Fusarium oxysporum, Fusarium acuminatum and Fusarium camptoceras were investigated using 1H NMR spectroscopy. Our results showed that the metabolites in infected sea buckthorn leaves such as flavonoids, glucosinolates and phenylpropanoids were highly associated with the fungal infection.
A co-culture approach of sea buckthorn suspension cell and Fusarium oxysporum was used to assess the intracellular metabolomes (metabolic fingerprints) of both host and pathogen and their extruded (extracellular) metabolites (metabolic footprints) under conditions relevant to disease and resistance, using FT-IR spectroscopy.
• the expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
i) SSR markers of sea buckthorn developed in our study will provide co-dominant markers for identification of sea buckthorn genotypes and relationships between cultivars, and for differentiation of different cultivars based on SSR fingerprinting.
ii) ISSR and SRAP markers associated with DSD-resistance will be applied for selection and identification of sea buckthorn germplasm with DSD-resistance. This can be used for screen of seedling with DSD-resistance for sea buckthorn plantation.
iii) The data on oil contents for maternal half-sib families and cultivars first presents the changes of oil contents in berry pulp of sea buckthorn half-sib family, providing scientific basis for sea buckthorn breeding. And, the data for different ripen time may provide index for harvest and processing of sea buckthorn berries.
iv) NMR-based metabolomics used in this study revealed that fungal infection altered a manifold of secondary metabolites, though specific patterns were observed for each fungus and each sea buckthorn cultivar. The co-culture system between suspension cell and pathogenic fungi will enable the metabolomic profiling of the separated host and pathogen.
v) There is a strong need for developing improved sea buckthorn cultivars with DSD-resistance. 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.
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. SSR 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.
• a description of the work performed since the beginning of the project
From 10/06/2013 to 09/06/2014, to perform the tasks towards the objectives of the project, we have i) developed simple sequence repeat (SSR) markers of sea buckthorn (Hippophae L.) using amplified fragments of ISSR markers and RNA-sequences of sea buckthorn from NCBI; ii) identified ISSR and SRAP markers associated with sea buckthorn DSD-resistance; iii) measured the fungal infection-induced metabolites in sea buckthorn using 1H NMR and fourier transform infrared (FT-IR) spectroscopy; vi) tested the changes of oil contents and components of fatty acids in sea buckthorn berries of maternal half-sib families and cultivars during ripening using targeted GC-MS spectroscopy and principal component analysis (PCA); and v) investigated the changes of metabolites of pathogenic fungi infected leaves of sea buckthorn.
• a description of the main results achieved so far
Eight SSR markers were developed by a simple method of using amplified fragments of ISSR. Stepwise multiple regression analysis were used to identified four ISSR markers (887190, 835700, 809290, and 811280) associated with DSD-resistance with highly negative correlation (p < 0.001) and eleven SRAP markers (Me1-Em3600, Me1-Em1680, Me2-Em1650, Me2-Em1950, Me3-Em61300, Me2-Em6320, Me2-Em6400, Me1-Em2600, Me1-Em11200, Me1-Em11700, Me2-Em2250) were significantly correlated with DSD-resistance (P < 0.001).
There are significant differences in oil contents of dry-pulp among different individuals of maternal half-sib family. Significant differences in oil contents of dry-pulp did not found in different berry ripe time of two cultivars of BHi72715 and BHi7278.
The metabolic interactions between three cultivars of sea buckthorn and three pathogenic fungi such as Fusarium oxysporum, Fusarium acuminatum and Fusarium camptoceras were investigated using 1H NMR spectroscopy. Our results showed that the metabolites in infected sea buckthorn leaves such as flavonoids, glucosinolates and phenylpropanoids were highly associated with the fungal infection.
A co-culture approach of sea buckthorn suspension cell and Fusarium oxysporum was used to assess the intracellular metabolomes (metabolic fingerprints) of both host and pathogen and their extruded (extracellular) metabolites (metabolic footprints) under conditions relevant to disease and resistance, using FT-IR spectroscopy.
• the expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
i) SSR markers of sea buckthorn developed in our study will provide co-dominant markers for identification of sea buckthorn genotypes and relationships between cultivars, and for differentiation of different cultivars based on SSR fingerprinting.
ii) ISSR and SRAP markers associated with DSD-resistance will be applied for selection and identification of sea buckthorn germplasm with DSD-resistance. This can be used for screen of seedling with DSD-resistance for sea buckthorn plantation.
iii) The data on oil contents for maternal half-sib families and cultivars first presents the changes of oil contents in berry pulp of sea buckthorn half-sib family, providing scientific basis for sea buckthorn breeding. And, the data for different ripen time may provide index for harvest and processing of sea buckthorn berries.
iv) NMR-based metabolomics used in this study revealed that fungal infection altered a manifold of secondary metabolites, though specific patterns were observed for each fungus and each sea buckthorn cultivar. The co-culture system between suspension cell and pathogenic fungi will enable the metabolomic profiling of the separated host and pathogen.
v) There is a strong need for developing improved sea buckthorn cultivars with DSD-resistance. 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.