Evaluation Of Seed Oil,Fatty Acids And Association Mapping Of The Lipid Biosynthesis Genes In Cucumis Sativus L. Cultivars And Related Wild Species

Cucumber(Cucumis sativus L.)belongs to the cucurbitaceae family and it is widely cultivated and consumed in many countries for its edible fruit either fresh or pickled.It is an important part of the traditional Mediterranean diet since ancient times due to its nutritional and medicinal properties.Young shoots are consumed as a leafy vegetable;kernels of the seeds are extracted and used in confectionary.The seeds yield edible oil used in salad dressing and the fruits are used during summer as a cooling food.The literature outlines the traditional uses of cucumber as well as other cucurbits and also gives scientific evidence that validates the traditional uses.Cucurbitaceae seeds are well endowed with fatty acids that confer them medicinal properties which enhance human health.Cucumber seed oil has high economic potential so we sought to extract the seed oil and measure the fatty acids content.The objectives of this study were to,(?)Investigation of the high seed oil producing cultivars of cucumber,by assessing number of mature seeds per fruit and amount of oil produced.(?)Assessing the fatty acids in seed oil,as well as their variation in different cucumber cultivars and Cucumis wild relatives(?)Assessing the seasonal variation of seed oil and fatty acids in cucumber cultivars as well as their wild relatives and(?)undertake association mapping of lipid biosynthesis genes in Cucumis sativus and some related wild species.The experimental design used was a completely randomized block design where mature seeds of 80 cucumber cultivars and 11 wild species of cucumber were selected from the laboratory germplasm bank and planted in the Jiangpu experimental site.Seed oil extraction was determined according to the AO AC(1990)method with an automated Soxhlet extractor(Buchi Universal Extraction System B-811/B-811 LSV BUCHI Labortechnik AG,Flawil,Switzerland).Fatty acids were determined by the AO AC(1990)method and methyl esters of the fatty acids were analyzed using the GC-MS analysis with an Agilent 7890 Series Gas Chromatograph machine.Genomic DNA was extracted using the CTAB method(Murray and Thompson,1980)with minor modification.Associationmapping was carried out using the STRUCTURE 2.3.3 software to get population stratification while trait association mapping was carried out using TASSEL 2.1 software.Phenotypic data analysis was carried out using GenStat and Minitab statistical packages.The key results are summarized below:1.Cultivar and seasonal effects on seed oil content and fatty acid composition ofcucumber as a potential industrial cropCucumber(Cucumis sativus)seed oil has potential for use as edible oil,pharmaceutical,cosmetic,insecticidal and as an industrial product.In this paper,we investigated,for the first time,the effect of cultivar and season on seed number,oil quantity and fatty acid profiles as well as their proportions in different cucumber cultivars.We examined the effects of spring and autumn seasons on seed oil quantity and fatty acid composition in 46 cucumber cultivars and one wild species of cucumber grown in greenhouse experiments in 2013 and 2014.Seed oil was determined using the Soxhlet method and fatty acids using the Gas Chromatograph-Mass Spectrometer(GC-MS)method.Seed oil quantity in the cucumber seeds ranged from 41.07%in 'Hazerd' to 29.24%in 'Lubao' while Cucumis anguria the wild species had 23.3%.Fatty acids detected were linoleic(C18:2),palmitic(C16:0),oleic(C18:1),stearic(C18:0),linolenic(C18:3),behenic(C22:0),arachidic C20:0),lignoceric(C24:0),eicosenoic(C20:1),palmitoleic(C16:1),myristic(C14:0)among other unidentified fatty acids.The results showed significant effects of cultivar genotype,growing season and interactions on the variables examined.The quantities of seed oil and fatty acids differed significantly among the cultivar genotypes.Spring grown cucumbers had higher quantities of oil than the autumn grown cucumbers.The quantities of fatty acids,mainly;palmitic(C16:0),palmitoleic(C16:1),stearic(C18:0),oleic(C18:1),eicosenoic(C20:1)and lignoceric(C24:0)were also higher in spring.In autumn there were more seeds,and higher linoleic(C18:2),linolenic(C18:3)and other unspecified fatty acids.The higher the oleic acid(C18:1)content the lower was the linoleic acid(C18:2)indicating a strong negative relationship in these two fatty acids.The higher the seed oil content the higher was linoleic(C18:2)and oleic(C18:1)indicating a positive relationship between the seed oil and the two fatty acids.Results of this study provide important information applicable in improving management and production of cucumber seed oil,considering its versatility in uses.Furthermore,the wide range of fatty acids found in thestudied cucumber cultivars,could be used in the production of novel industrial oils through genetic engineering.2.Seasonal profiles of the oil content and fatty acid in unexploited wild CucumisspeciesCucurbitaceae wild species are natural resources that have been used since antiquity as food,human and animal medicine,cosmetics and insecticides.They can be conserved by utilizing their seed oil.We examined the oil content and fatty acid profiles of 9 wild species and a cucurbita pepo in two seasons in the open field and in the greenhouse.We report for the first time the seed oil and fatty acid content of C.melo dudaim,C.ficifolius,C.myriocarpus and Zehneria scabra.The highest oil content was observed in C.metuliferus at 32.29%and the lowest was 22.45%in C.ficifolius.The major fatty acids detected were linoleic(C18:2),oleic(C18:1),palmitic(C16:0),stearic(C18:0)while the minor ones were linolenic(C18:3),lignoceric(C24:0),eicosenoic(C20:1),behenic(C22:0),arachidic(C20:0),palmitoleic(C16:1),myristic(C14:0)and other undetected fatty acids.The highest linoleic(C18:3)content was 74.62%in C.myriocarpus while Cucurbita pepo had the highest oleic(C18:1)at 17.26%.Palmitic(C16:0)was highest in C.melo dudaim at 13.56%while C.metuliferus had the most stearic(C18:0)at 8.31%.The species may also have been affected by seasons since in spring they had increased oil,oleic(C18:1),stearic(C18:0),lignoceric(C24:0),eicosenoic(C20:1),behenic(C22:0),arachidic(C20:0),palmitoleic(C16:1),myristic(C14:0)and OF As whereas in autumn linoleic(C18:2),linolenic(C18:3)and palmitic(C16:0)were increased.Utilizing and commercializing wild species seed oil will conserve them as well as give economic empowerment to rural communities where they grow freely.These wild species are an important gene pool for improving cultivated species and utilizing them will avoid their extinction by weeding and rapid development in their natural habitats.3.Association mapping of seed oil and fatty acid traits in cucumber cultivars andrelated Cucumis speciesWe carried out genetic analyses and association mapping on 80 cucumber cultivars and 11 related wild species from different geographic regions.We evaluated 14 traits of seed oil,seed number,linoleic(C18:2),palmitic(C16:0),oleic(C18:1),stearic(C18:0),linolenic(C18:3),behenic(C22:0),arachidic C20:0),lignoceric(C24:0),eicosenoic(C20:1),palmitoleic(C16:1),myristic(C14:0)and other fatty acids.Phenotypic data was collectedfor two seasons in 2013 autumn and 2014 spring.The phenotypic variables were highly variable within cultivars as well as in the different seasons.The germplasm accessions were structured into seven subpopulations in which wild relatives and Xishuangbanna cultivar had distinct groups.The association of 172 SSR candidate gene markers with the seed oil and fatty acids were analyzed using the general linear model,where structure subgroups were used to control for spurious marker-trait associations.Out of the 172 markers used 13 candidate gene markers were associated with the seed oil and fatty acid traits at P?0.05.The significant associations were detected in:Palmitic(2),Stearic(2),Oleic(3),Linoleic(4),Linolenic(1),Arachidic(3),Behenic(1),Eicosenoic(3),Lignoceric(4),Myristic(2)and Other Fatty Acids(3)making a total of 28 marker trait associations.However,these marker trait associations were not consistent across the seasons due to the nature of seed oil and fatty acids variations in different environmental conditions in the different seasons.