Molecular-assisted breeding for soybean with high oleic/low linolenic acid and elevated vitamin E in the seed oil

The uses of vegetable oils are determined by functional properties arising from their chemical composition. Soybean oil was previously used in margarines and baked foods after partial hydrogenation to achieve heat and oxidative stability. This process, however, generates trans fats that are now excluded from food use because of cardiovascular health risks. Also present in soybean oil are the anti-oxidant tocopherols, with α- tocopherol (vitamin E) typically present as a minor component compared to γ-tocopherol. Genetic improvement of the fatty acid profile and tocopherol profile is an attractive solution to increase the functional and health qualities of soybean oil. The objective of this research was to develop resources to directly select with molecular markers for the elevated vitamin E trait in soybean oil and to use a molecular breeding approach to combine elevated vitamin E with the high oleic/low linolenic acid seed oil trait that improves oil functionality and nutrition. New soybean germplasm was developed from the molecular breeding strategy that selected for alleles of six targeted genes. Seed oil from the novel soybean germplasm was confirmed to contain increased vitamin E α-tocopherol along with a high oleic acid/low linolenic acid profile

Validation of QTL mapping and transcriptome profiling for identification of candidate genes associated with nitrogen stress tolerance in sorghum

Background: Quantitative trait loci (QTLs) detected in one mapping population may not be detected in other mapping populations at all the time. Therefore, before being used for marker assisted breeding, QTLs need to be validated in different environments and/or genetic backgrounds to rule out statistical anomalies. In this regard, we mapped the QTLs controlling various agronomic traits in a recombinant inbred line (RIL) population in response to Nitrogen (N) stress and validated these with the reported QTLs in our earlier study to find the stable and consistent QTLs across populations. Also, with Illumina RNA-sequencing we checked the differential expression of gene (DEG) transcripts between parents and pools of RILs with high and low nitrogen use efficiency (NUE) and overlaid these DEGs on to the common validated QTLs to find candidate genes associated with N-stress tolerance in sorghum. Results: An F7 RIL population derived from a cross between CK60 (N-stress sensitive) and San Chi San (N-stress tolerant) inbred sorghum lines was used to map QTLs for 11 agronomic traits tested under different N-levels. Composite interval mapping analysis detected a total of 32 QTLs for 11 agronomic traits. Validation of these QTLs revealed that of the detected, nine QTLs from this population were consistent with the reported QTLs in earlier study using CK60/China17 RIL population. The validated QTLs were located on chromosomes 1, 6, 7, 8, and 9. In addition, root transcriptomic profiling detected 55 and 20 differentially expressed gene (DEG) transcripts between parents and pools of RILs with high and low NUE respectively. Also, overlay of these DEG transcripts on to the validated QTLs found candidate genes transcripts for NUE and also showed the expected differential expression. For example, DEG transcripts encoding Lysine histidine transporter 1 (LHT1) had abundant expression in San Chi San and the tolerant RIL pool, whereas DEG transcripts encoding seed storage albumin, transcription factor IIIC (TFIIIC) and dwarfing gene (DW2) encoding multidrug resistance-associated protein-9 homolog showed abundant expression in CK60 parent, similar to earlier study. Conclusions: The validated QTLs among different mapping populations would be the most reliable and stable QTLs across germplasm. The DEG transcripts found in the validated QTL regions will serve as future candidate genes for enhancing NUE in sorghum using molecular approaches

Production of tocotrienols in seeds of cotton (Gossypium hirsutum L.) enhances oxidative stability and offers nutraceutical potential

Upland cotton (Gossypium hirsutum L.) is an economically important multi-purpose crop cultivated globally for fibre, seed oil and protein. Cottonseed oil also is naturally rich in vitamin E components (collectively known as tocochromanols), with a- and c-tocopherols comprising nearly all of the vitamin E components. By contrast, cottonseeds have little or no tocotrienols, tocochromanols with a wide range of health benefits. Here, we generated transgenic cotton lines expressing the barley (Hordeum vulgare) homogentisate geranylgeranyl transferase coding sequence under the control of the Brassica napus seed-specific promoter, napin. Transgenic cottonseeds had ~twofold to threefold increases in the accumulation of total vitamin E (tocopherols + tocotrienols), with more than 60% c-tocotrienol. Matrix assisted laser desorption ionization-mass spectrometry imaging showed that c-tocotrienol was localized throughout the transgenic embryos. In contrast, the native tocopherols were distributed unequally in both transgenic and non-transgenic embryos. a- Tocopherol was restricted mostly to cotyledon tissues and c-tocopherol was more enriched in the embryonic axis tissues. Production of tocotrienols in cotton embryos had no negative impact on plant performance or yield of other important seed constituents including fibre, oil and protein. Advanced generations of two transgenic events were field grown, and extracts of transgenic seeds showed increased antioxidant activity relative to extracts from non-transgenic seeds. Furthermore, refined cottonseed oil from the two transgenic events showed 30% improvement in oxidative stability relative to the non-transgenic cottonseed oil. Taken together, these materials may provide new opportunities for cottonseed co-products with enhanced vitamin E profile for improved shelf life and nutrition

Metabolic Engineering of Soybean (Glycine max) and Camelina (Camelina sativa) for Improved Oil Functionality

Vegetable oils are important sources of food, feed and fuel. The value and usage can be enhanced by increased production of vitamin E and wax esters in the oil. To this end, this thesis explored the vitamin E and wax ester biosynthetic pathways in oil seed crops like soybean and camelina. Vitamin E consists of tocopherols, tocotrienols and provides oxidative stability to vegetable oils. Due to high antioxidant nature and limited occurence, production of tocotrienols in dicotyledonous oil seeds is expexted to improve oil functionality. Also, high alpha tocochromanols in vegetable oils enhances nutritional value. We generated soybean seeds with 6.5- to 7.1- fold increases in vitamin E, mainly through tocotrienols production by transgenic expression of barley homogentisate geranylgeranyl transferase (HGGT). Also, generated seeds with 5.4- and 16-fold increase in total vitamin E and alpha tocochromanols respectively by co-expressing soybean γ-tocopherol methyltransferase (γ-TMT/VTE4) with barley HGGT. Further, introgression of high vitamin E trait into stearidonic rich soybean had improved oxidative stability of oil. We also, addressed the limitation of homogentisate production for increased vitamin E, using metabolic engineering strategies in camelina. Seed specific expression of down regulated homogentisate oxygenase ( HGO) and de-regulated synthesis genes, Arabidopsis hydroxyphenylpyruvate dioxigenase (HPPD) and Eschericia coli bi-functional chorismate mutase/prephenate dehydrogenase (TyrA) alone or in combination resulted drastic increase in homogentisate with limited increase in vitamin E content. Where as, in presence of HGGT expression, tremendous increase in homogentisate and total vitamin E content suggests that homogentisate is not a limiting factor for vitamin E synthesis, except in lines expressing HGGT and HGGT/HGO RNAi where free HGA was not accumulated. Similarly, by exploring wax biosynthetic pathway we generated camelina lines producing novel wax esters with reduced chain length, mainly C42 and C44 consisting of C20 and C22 fatty acids and fatty alcohols, by co-expressing fatty acid reductase (FAR), wax synthase (WS) from jojoba with fatty acid elongase (FAE1) from carmbe. In summary, we conclude that enhanced vitamin E increaed the oxidative stability of polyunsaturated fatty acids and increased alpha forms improves nutritional value of soybean oil. Also, wax esters with reduced chain length improve stability and functionality

Metabolic Engineering of Soybean (Glycine max) and Camelina (Camelina sativa) for Improved Oil Functionality

Vegetable oils are important sources of food, feed and fuel. The value and usage can be enhanced by increased production of vitamin E and wax esters in the oil. To this end, this thesis explored the vitamin E and wax ester biosynthetic pathways in oil seed crops like soybean and camelina. Vitamin E consists of tocopherols, tocotrienols and provides oxidative stability to vegetable oils. Due to high antioxidant nature and limited occurence, production of tocotrienols in dicotyledonous oil seeds is expexted to improve oil functionality. Also, high alpha tocochromanols in vegetable oils enhances nutritional value. We generated soybean seeds with 6.5- to 7.1- fold increases in vitamin E, mainly through tocotrienols production by transgenic expression of barley homogentisate geranylgeranyl transferase (HGGT). Also, generated seeds with 5.4- and 16-fold increase in total vitamin E and alpha tocochromanols respectively by co-expressing soybean γ-tocopherol methyltransferase (γ-TMT/VTE4) with barley HGGT. Further, introgression of high vitamin E trait into stearidonic rich soybean had improved oxidative stability of oil. We also, addressed the limitation of homogentisate production for increased vitamin E, using metabolic engineering strategies in camelina. Seed specific expression of down regulated homogentisate oxygenase ( HGO) and de-regulated synthesis genes, Arabidopsis hydroxyphenylpyruvate dioxigenase (HPPD) and Eschericia coli bi-functional chorismate mutase/prephenate dehydrogenase (TyrA) alone or in combination resulted drastic increase in homogentisate with limited increase in vitamin E content. Where as, in presence of HGGT expression, tremendous increase in homogentisate and total vitamin E content suggests that homogentisate is not a limiting factor for vitamin E synthesis, except in lines expressing HGGT and HGGT/HGO RNAi where free HGA was not accumulated. Similarly, by exploring wax biosynthetic pathway we generated camelina lines producing novel wax esters with reduced chain length, mainly C42 and C44 consisting of C20 and C22 fatty acids and fatty alcohols, by co-expressing fatty acid reductase (FAR), wax synthase (WS) from jojoba with fatty acid elongase (FAE1) from carmbe. In summary, we conclude that enhanced vitamin E increaed the oxidative stability of polyunsaturated fatty acids and increased alpha forms improves nutritional value of soybean oil. Also, wax esters with reduced chain length improve stability and functionality

Metabolic and Gene Expression Changes Triggered by Nitrogen Deprivation in the Photoautotrophically Grown Microalgae \u3ci\u3eChlamydomonas reinhardtii\u3c/i\u3e and \u3ci\u3eCoccomyxa\u3c/i\u3e sp. C-169

Microalgae are emerging as suitable feedstocks for renewable biofuel production. Characterizing the metabolic pathways involved in the biosynthesis of energy-rich compounds, such as lipids and carbohydrates, and the environmental factors influencing their accumulation is necessary to realize the full potential of these organisms as energy resources. The model green alga Chlamydomonas reinhardtii accumulates significant amounts of triacylglycerols (TAGs) under nitrogen starvation or salt stress in medium containing acetate. However, since cultivation of microalgae for biofuel production may need to rely on sunlight as the main source of energy for biomass synthesis, metabolic and gene expression changes occurring in Chlamydomonas and Coccomyxa subjected to nitrogen deprivation were examined under strictly photoautotrophic conditions. Interestingly, nutrient depletion triggered a similar pattern of early synthesis of starch followed by substantial TAG accumulation in both of these fairly divergent green microalgae. A marked decrease in chlorophyll and protein contents was also observed, including reduction in ribosomal polypeptides and some key enzymes for CO2 assimilation like ribulose-1,5-bisphosphate carboxylase/oxygenase. These results suggest that turnover of nitrogen-rich compounds such as proteins may provide carbon/energy for TAG biosynthesis in the nutrient deprived cells. In Chlamydomonas, several genes coding for diacylglycerol:acyl-CoA acyltransferases, catalyzing the acylation of diacylglycerol to TAG, displayed increased transcript abundance under nitrogen depletion but, counterintuitively, genes encoding enzymes for de novo fatty acid synthesis, such as 3-ketoacyl-ACP synthase I, were down-regulated. Understanding the interdependence of these anabolic and catabolic processes and their regulation may allow the engineering of algal strains with improved capacity to convert their biomass into useful biofuel precursors

Identification of differentially expressed genes between sorghum genotypes with contrasting nitrogen stress tolerance by genome-wide transcriptional profiling

Background: Sorghum is an important cereal crop, which requires large quantities of nitrogen fertilizer for achieving commercial yields. Identification of the genes responsible for low-N tolerance in sorghum will facilitate understanding of the molecular mechanisms of low-N tolerance, and also facilitate the genetic improvement of sorghum through marker-assisted selection or gene transformation. In this study we compared the transcriptomes of root tissues from seven sorghum genotypes having differential response to low-N stress. Results: Illumina RNA-sequencing detected several common differentially expressed genes (DEGs) between four low-N tolerant sorghum genotypes (San Chi San, China17, KS78 and high-NUE bulk) and three sensitive genotypes (CK60, BTx623 and low-NUE bulk). In sensitive genotypes, N-stress increased the abundance of DEG transcripts associated with stress responses including oxidative stress and stimuli were abundant. The tolerant genotypes adapt to N deficiency by producing greater root mass for efficient uptake of nutrients. In tolerant genotypes, higher abundance of transcripts related to high affinity nitrate transporters (NRT2.2, NRT2.3, NRT2.5, and NRT2.6) and lysine histidine transporter 1 (LHT1), may suggest an improved uptake efficiency of inorganic and organic forms of nitrogen. Higher abundance of SEC14 cytosolic factor family protein transcript in tolerant genotypes could lead to increased membrane stability and tolerance to N-stress. Conclusions: Comparison of transcriptomes between N-stress tolerant and sensitive genotypes revealed several common DEG transcripts. Some of these DEGs were evaluated further by comparing the transcriptomes of genotypes grown under full N. The DEG transcripts showed higher expression in tolerant genotypes could be used for transgenic over-expression in sensitive genotypes of sorghum and related crops for increased tolerance to N-stress, which results in increased nitrogen use efficiency for sustainable agriculture