Genetic and Biotechnological Approaches to Improve Fruit Bioactive Content: A Focus on Eggplant and Tomato Anthocyanins.

Anthocyanins are a large group of water-soluble flavonoid pigments. These specialized metabolites are ubiquitous in the plant kingdom and play an essential role not only in plant reproduction and dispersal but also in responses to biotic and abiotic stresses. Anthocyanins are recognized as important health-promoting and chronic-disease-preventing components in the human diet. Therefore, interest in developing food crops with improved levels and compositions of these important nutraceuticals is growing. This review focuses on work conducted to elucidate the genetic control of the anthocyanin pathway and modulate anthocyanin content in eggplant (Solanum melongena L.) and tomato (Solanum lycopersicum L.), two solanaceous fruit vegetables of worldwide relevance. While anthocyanin levels in eggplant fruit have always been an important quality trait, anthocyanin-based, purple-fruited tomato cultivars are currently a novelty. As detailed in this review, this difference in the anthocyanin content of the cultivated germplasm has largely influenced genetic studies as well as breeding and transgenic approaches to improve the anthocyanin content/profile of these two important solanaceous crops. The information provided should be of help to researchers and breeders in devising strategies to address the increasing consumer demand for nutraceutical foods.

Mapping of quantitative trait loci for the nutritional value of fresh market tomato.

The incidence of many diseases, such as cancer, cardiovascular diseases, and diabetes, is associated with malnutrition and an unbalanced daily diet. Vegetables are an important source of vitamins and essential compounds for human health. As a result, such metabolites have increasingly become the focus of breeding programs. Tomato is one of the most popular components of our daily diet. Therefore, the improvement of tomato's nutritional quality is an important goal. In the present study, we performed targeted metabolic profiling of an interspecific Solanum pimpinellifolium × S. lycopersicum inbred backcross line (IBL) population and identified quantitative trait loci responsible for the nutritional value of tomato. Transgressive segregation was apparent for many of the nutritional compounds such that some IBLs had extremely high levels of various amino acids and vitamins compared to their parents. A total of 117 QTLs for nutritional traits including 62 QTLs for amino acids, 18 QTLs for fatty acids, 12 QTLs for water-soluble vitamins, and 25 QTLs for fat-soluble vitamins were identified. Moreover, almost 24% of identified QTLs were confirmed in previous studies, and 40 possible gene candidates were found for 18 identified QTLs. These findings can help breeders to improve the nutritional value of tomato.

GRAS-Di SNP-based molecular characterization and fingerprinting of a Turkish Corylus avellana core set provide insights into the cultivation and breeding of hazelnut in Turkey.

Hazelnut (Corylus avellana L.) is an economically and socially important product for Turkey, the country that leads global production of this crop. The preservation of Turkish hazelnut genetic diversity and informed breeding of new cultivars are crucial for maintaining quality and crop yield stability. In this study, genotyping by random amplicon sequencing (GRAS-Di) was used to identify single-nucleotide polymorphisms (SNPs) in a panel of 96 individuals representing the Turkish national hazelnut collection. The resulting 7609 high-quality SNPs were physically mapped to the Tombul cultivar reference genome and used for population structure and diversity analyses. These analyses revealed that cultivars are not less diverse than wild accessions and that 44% of the panel had admixed ancestry. The results also indicated that recently released Turkish cultivars are highly similar to each other, suggesting that diversity analysis is an important tool that should be employed to prevent future genetic bottlenecks in this crop. A minimal marker algorithm was used to select a set of seven SNP markers that were capable of differentiating the panel accessions. These fingerprinting markers should be useful for the propagation of true-to-type elite cultivars that can be used to renew Turkey's aging hazelnut orchards.

Interaction With Fungi Promotes the Accumulation of Specific Defense Molecules in Orchid Tubers and May Increase the Value of Tubers for Biotechnological and Medicinal Applications: The Case Study of Interaction Between Dactylorhiza sp. and Tulasnella calospora.

Terrestrial orchids can form tubers, organs modified to store energy reserves. Tubers are an attractive source of nutrients, and salep, a flour made from dried orchid tubers, is the source of traditional beverages. Tubers also contain valuable secondary metabolites and are used in traditional medicine. The extensive harvest of wild orchids is endangering their populations in nature; however, orchids can be cultivated and tubers mass-produced. This work illustrates the importance of plant-fungus interaction in shaping the content of orchid tubers in vitro. Orchid plants of Dactylorhiza sp. grown in asymbiotic culture were inoculated with a fungal isolate from Tulasnella calospora group and, after 3 months of co-cultivation, tubers were analyzed. The fungus adopted the saprotrophic mode of life, but no visible differences in the morphology and biomass of the tubers were detected compared to the mock-treated plants. To elucidate the mechanisms protecting the tubers against fungal infestation, proteome, metabolome, and lipidome of tubers were analyzed. In total, 1,526, 174, and 108 proteins, metabolites, and lipids were quantified, respectively, providing a detailed snapshot of the molecular process underlying plant-microbe interaction. The observed changes at the molecular level showed that the tubers of inoculated plants accumulated significantly higher amounts of antifungal compounds, including phenolics, alkaloid Calystegine B2, and dihydrophenanthrenes. The promoted antimicrobial effects were validated by observing transient inhibition of Phytophthora cactorum growth. The integration of omics data highlighted the promotion of flavonoid biosynthesis, the increase in the formation of lipid droplets and associated production of oxylipins, and the accumulation of auxin in response to T. calospora. Taken together, these results provide the first insights into the molecular mechanisms of defense priming in orchid tubers and highlight the possible use of fungal interactors in biotechnology for the production of orchid secondary metabolites.

Association analysis of germination level cold stress tolerance and candidate gene identification in Upland cotton (Gossypium hirsutum L.).

Cotton originated from ancestors in the Gossypium genus that grew in semi-desert habitats. As a result, it is adversely affected by low temperatures especially during germination and the first weeks of growth. Despite this, there are relatively few molecular studies on cold stress in cotton. This limitation may present a future breeding handicap, as recent years have witnessed increased low temperature damage to cotton production. Cold tolerance is a sustainable approach to obtain good production in case of extreme cold. In the present study, 110 Upland cotton (Gossypium hirsutum) genotypes were evaluated for cold tolerance at the germination stage. We identified vigorous genotypes with cold-related parameters that outperformed the panel's average performance ( x ¯ = 76.9% CG, 83.9% CSI, 167.5 CWVI). Molecular genetic diversity analysis with 101 simple sequence repeat (SSR) markers yielding 416 loci was used to select tolerant genotypes that could be important materials for breeding this trait. A total of 16 marker-cold tolerance trait associations (p < 0.005) were identified with 10 of them having major effects (PVE > 10%). Based on the positions of these markers, candidate genes for cold tolerance in the G. hirsutum genome were identified. Three of these markers (BNL0569, CIR081 and CIR202) are important candidates for use in marker-assisted breeding for cold tolerance because they mapped to genes previously associated with cold tolerance in other plant species such as Arabidopsis thaliana, rice and tomato. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01184-6.

Chromatographic Analysis for Targeted Metabolomics of Antioxidant and Flavor-Related Metabolites in Tomato.

Targeted metabolomics is a useful approach to evaluate crop breeding studies. Antioxidant and flavor-related traits are of increasing interest and are considered quality traits in tomato breeding. The present study presents chromatographic methods to study antioxidants (carotenoids, vitamin C, vitamin E, phenolic compounds, and glutathione) and flavor-related characters (sugars and organic acids) in tomato. Two different extraction methods (for polar and apolar entities) were applied to isolate the targeted compounds. The extraction methods developed in this work were time and cost-effective since no further purification was needed. Carotenoids, vitamin C, glutathione, and phenolic acids were analyzed by HPLC-PDA using a RP C18 column at an appropriate wavelength for each compound. Vitamin E and sugars were analyzed by HPLC with RP C18 and NH2 columns and detected by FLD and RI detectors, respectively. In addition, organic acids were analyzed with GC-FID using a Rtx 5DA column after derivatization with MSTFA. As a result, sensitive analytical methods to quantify important plant metabolites were developed and are described herein. These methods are not only applicable in tomato but are also useful to characterize other species for flavor-related and antioxidant compounds. Thus, these protocols can be used to guide selection in crop breeding.

Exploring wild alleles from Solanum pimpinellifolium with the potential to improve tomato flavor compounds.

Most consumers complain about the flavor of current tomato cultivars and many pay a premium for alternatives such as heirloom varieties. Breeding for fruit flavor is difficult because it is a quantitatively inherited trait influenced by taste, aroma and environmental factors. A lack of genetic diversity in modern tomato cultivars also necessitates exploration of new sources for flavor alleles. Wild tomato S. pimpinellifolium and inbred backcross lines were assessed for individual sugars and organic acids which are two of the main components of tomato flavor. S. pimpinellifolium was found to harbor alleles that could be used to increase glucose and fructose content and adjust acidity by altering malic and citric acid levels. Single nucleotide polymorphism markers were used to detect 14 quantitative trait loci (QTLs) for sugars and 71 for organic acids. Confirmation was provided by comparing map locations with previously identified loci. Thus, seven (50 %) of the sugar QTLs and 22 (31 %) of the organic acids loci were supported by analyses in other tomato populations. Examination of the genomic sequence containing the QTLs allowed identification of potential candidate genes for several flavor components.

Mapping of quantitative trait loci for antioxidant molecules in tomato fruit: Carotenoids, vitamins C and E, glutathione and phenolic acids.

The nutritional value of a crop lies not only in its protein, lipid, and sugar content but also involves compounds such as the antioxidants lycopene, ß-carotene and vitamin C. In the present study, wild tomato Solanum pimpinellifolium LA 1589 was assessed for its potential to improve antioxidant content. This wild species was found to be a good source of alleles for increasing ß-carotene, lycopene, vitamin C and vitamin E contents in cultivated tomato. Characterization of an LA 1589 interspecific inbred backcross line (IBL) mapping population revealed many individuals with transgressive segregation for the antioxidants confirming the usefulness of this wild species for breeding of these traits. Molecular markers were used to identify QTLs for the metabolites in the IBL population. In total, 64 QTLs were identified for the antioxidants and their locations were compared to the map positions of previously identified QTLs for confirmation. Four (57 %) of the carotenoid QTLs, four (36 %) of the vitamin QTLs, and 11 (25 %) of the phenolic acid QTLs were supported by previous studies. Furthermore, several potential candidate genes were identified for vitamins C and E and phenolic acids loci. These candidate genes might be used as markers in breeding programs to increase tomato's antioxidant content.

Transcriptomic analysis of selenium accumulation in Puccinellia distans (Jacq.) Parl., a boron hyperaccumulator.

Selenium (Se) is present in a wide variety of natural and man-made materials on Earth. Plants are able to tolerate and (hyper)accumulate Se to different extents. In fact, some species can tolerate and accumulate multiple elements. Puccinellia distans (P. distans), weeping alkali grass, is known to hyperaccumulate extreme concentrations of boron and tolerate high levels of salinity, therefore, we investigated the Se accumulation and tolerance capacities of this species. In addition, P. distans' Se tolerance mechanism was studied using a transcriptomic approach. The results of this study indicated that, when grown in a hydroponic system containing 80 or 120 µM Se, P. distans shoots accumulated from 1500 to 2500-fold more Se than plants grown without the element. Thus, P. distans was discovered to be a novel Se accumulator plant. RNA sequencing results and biochemical analyses helped to shed light on the Se tolerance and accumulation mechanism of P. distans. Here, we suggest that upregulation of Se assimilation and stress response genes may be due to induction of jasmonic acid signaling. In addition, we propose that the cell wall may play an important role in restriction of Se movement to the cytoplasm. Also, we hypothesize that Se accumulates in cells by sequestration of selenate in the vacuole.

Analysis of European hazelnut (Corylus avellana) reveals loci for cultivar improvement and the effects of domestication and selection on nut and kernel traits.

Turkey is a rich source of European hazelnut (Corylus avellana) germplasm with nearly 400 accessions in the national collection. This genetic material encompasses cultivars, landraces and wild genotypes which were characterized for 12 nut and 13 kernel traits over 2 years in the 1990s. Analysis of these attributes revealed both the positive and negative impacts that human selection and breeding have had on hazelnut. Thus, while selection has resulted in larger nuts and kernels, cultivars have fewer nuts per cluster and kernels with larger internal cavities. Breeding has also resulted in a propensity for cultivars to have higher proportions of double kernels and empty nuts, two traits which reduce quality and yield. In addition, it is clear that while selection has successfully increased hazelnut fat content it has not impacted overall flavor, a much more complex trait. The nut and kernel phenotypic data were combined with genotypic data from 406 simple sequence repeat marker alleles for association mapping of the quantitative trait loci (QTL) for the traits. A total of 78 loci were detected in the population with the highest proportions for nut (24%) and kernel (26%) appearance parameters followed by quality (19%), shell thickness (16%) and yield-related (15%) traits. It is hoped that some of the identified QTL will be useful for future breeding of hazelnut for improved nut and kernel yield and quality.

Health benefits and bioactive compounds of eggplant.

Eggplant is a vegetable crop that is grown around the world and can provide significant nutritive benefits thanks to its abundance of vitamins, phenolics and antioxidants. In addition, eggplant has potential pharmaceutical uses that are just now becoming recognized. As compared to other crops in the Solanaceae, few studies have investigated eggplant's metabolic profile. Metabolomics and metabolic profiling are important platforms for assessing the chemical composition of plants and breeders are increasingly concerned about the nutritional and health benefits of crops. In this review, the historical background and classification of eggplant are shortly explained; then the beneficial phytochemicals, antioxidant activity and health effects of eggplant are discussed in detail.

Transcriptomic analysis of boron hyperaccumulation mechanisms in Puccinellia distans.

Puccinellia distans, common alkali grass, is found throughout the world and can survive in soils with boron concentrations that are lethal for other plant species. Indeed, P. distans accumulates very high levels of this element. Despite these interesting features, very little research has been performed to elucidate the boron tolerance mechanism in this species. In this study, P. distans samples were treated for three weeks with normal (0.5 mg L-1) and elevated (500 mg L-1) boron levels in hydroponic solution. Expressed sequence tags (ESTs) derived from shoot tissue were analyzed by RNA sequencing to identify genes up and down-regulated under boron stress. In this way, 3312 differentially expressed transcripts were detected, 67.7% of which were up-regulated and 32.3% of which were down-regulated in boron-treated plants. To partially confirm the RNA sequencing results, 32 randomly selected transcripts were analyzed for their expression levels in boron-treated plants. The results agreed with the expected direction of change (up or down-regulation). A total of 1652 transcripts had homologs in A. thaliana and/or O. sativa and mapped to 1107 different proteins. Functional annotation of these proteins indicated that the boron tolerance and hyperaccumulation mechanisms of P. distans involve many transcriptomic changes including: alterations in the malate pathway, changes in cell wall components that may allow sequestration of excess boron without toxic effects, and increased expression of at least one putative boron transporter and two putative aquaporins. Elucidation of the boron accumulation mechanism is important in developing approaches for bioremediation of boron contaminated soils.

Relationship between geographical origin, seed size and genetic diversity in faba bean (Vicia faba L.) as revealed by SSR markers.

Faba bean (Vicia faba L.) is an important legume species because of its high protein and starch content. Broad bean can be grown in different climatic conditions and is an ideal rotation crop because of the nitrogen fixing bacteria in its roots. In this work, 255 faba bean germplasm accessions were characterized using 32 SSR primers which yielded 302 polymorphic fragments. According to the results, faba bean individuals were divided into two main groups based on the neighbor-joining algorithm (r = 0.91) with some clustering based on geographical origin as well as seed size. Population structure was also determined and agreed with the dendrogram analysis in splitting the accessions into two subpopulations. Analysis of molecular variance (AMOVA) revealed high levels of within population genetic variation. Genetic similarity and geographical proximity were related with separation of European accessions from African and Asian ones. Interestingly, there was no significant difference between landrace (38%) and cultivar (40%) diversity indicating that genetic variability has not yet been lost due to breeding. A total of 44 genetically well-characterized faba bean individuals were selected for a core collection to be further examined for yield and nutritional traits.

Newly developed SSR markers reveal genetic diversity and geographical clustering in spinach (Spinacia oleracea).

Spinach is a popular leafy green vegetable due to its nutritional composition. It contains high concentrations of vitamins A, E, C, and K, and folic acid. Development of genetic markers for spinach is important for diversity and breeding studies. In this work, Next Generation Sequencing (NGS) technology was used to develop genomic simple sequence repeat (SSR) markers. After cleaning and contig assembly, the sequence encompassed 2.5% of the 980 Mb spinach genome. The contigs were mined for SSRs. A total of 3852 SSRs were detected. Of these, 100 primer pairs were tested and 85% were found to yield clear, reproducible amplicons. These 85 markers were then applied to 48 spinach accessions from worldwide origins, resulting in 389 alleles with 89% polymorphism. The average gene diversity (GD) value of the markers (based on a GD calculation that ranges from 0 to 0.5) was 0.25. Our results demonstrated that the newly developed SSR markers are suitable for assessing genetic diversity and population structure of spinach germplasm. The markers also revealed clustering of the accessions based on geographical origin with clear separation of Far Eastern accessions which had the overall highest genetic diversity when compared with accessions from Persia, Turkey, Europe, and the USA. Thus, the SSR markers have good potential to provide valuable information for spinach breeding and germplasm management. Also they will be helpful for genome mapping and core collection establishment.

Genome-wide SNP discovery and QTL mapping for fruit quality traits in inbred backcross lines (IBLs) of solanum pimpinellifolium using genotyping by sequencing.

BACKGROUND: Solanum pimpinellifolium has high breeding potential for fruit quality traits and has been used as a donor in tomato breeding programs. Unlocking the genetic potential of S. pimpinellifolium requires high-throughput polymorphism identification protocols for QTL mapping and introgression of favourable alleles into cultivated tomato by both positive and background selection. RESULTS: In this study we identified SNP loci using a genotyping by sequencing (GBS) approach in an IBL mapping population derived from the cross between a high yielding fresh market tomato and S. pimpinellifolium (LA1589) as the recurrent and donor parents, respectively. A total of 120,983,088 reads were generated by the Illumina HiSeq next-generation sequencing platform. From these reads 448,539 sequence tags were generated. A majority of the sequence tags (84.4%) were uniquely aligned to the tomato genome. A total of 3.125 unique SNP loci were identified as a result of tag alignment to the genome assembly and were used in QTL analysis of 11 fruit quality traits. As a result, 37 QTLs were identified. S. pimpinellifolium contributed favourable alleles for 16 QTLs (43.2%), thus confirming the high breeding potential of this wild species. CONCLUSIONS: The present work introduced a set of SNPs at sufficiently high density for QTL mapping in populations derived from S. pimpinellifolium (LA1589). Moreover, this study demonstrated the high efficiency of the GBS approach for SNP identification, genotyping and QTL mapping in an interspecific tomato population.

Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil.

The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil.

GDdom: An Online Tool for Calculation of Dominant Marker Gene Diversity.

Gene diversity (GD), also called polymorphism information content, is a commonly used measure of molecular marker polymorphism. Calculation of GD for dominant markers such as AFLP, RAPD, and multilocus SSRs is valuable for researchers. To meet this need, we developed a free online computer program, GDdom, which provides easy, quick, and accurate calculation of dominant marker GD with a commonly used formula. Results are presented in tabular form for quick interpretation.

Authentication of Botanical Origin in Herbal Teas by Plastid Noncoding DNA Length Polymorphisms.

The aim of this study was to develop a DNA barcode assay to authenticate the botanical origin of herbal teas. To reach this aim, we tested the efficiency of a PCR-capillary electrophoresis (PCR-CE) approach on commercial herbal tea samples using two noncoding plastid barcodes, the trnL intron and the intergenic spacer between trnL and trnF. Barcode DNA length polymorphisms proved successful in authenticating the species origin of herbal teas. We verified the validity of our approach by sequencing species-specific barcode amplicons from herbal tea samples. Moreover, we displayed the utility of PCR-CE assays coupled with sequencing to identify the origin of undeclared plant material in herbal tea samples. The PCR-CE assays proposed in this work can be applied as routine tests for the verification of botanical origin in herbal teas and can be extended to authenticate all types of herbal foodstuffs.

Cultivar origin and admixture detection in Turkish olive oils by SNP-based CAPS assays.

The aim of this study was to establish a DNA-based identification key to ascertain the cultivar origin of Turkish monovarietal olive oils. To reach this aim, we sequenced short fragments from five olive genes for SNP (single nucleotide polymorphism) identification and developed CAPS (cleaved amplified polymorphic DNA) assays for SNPs that alter restriction enzyme recognition motifs. When applied on the oils of 17 olive cultivars, a maximum of five CAPS assays were necessary to discriminate the varietal origin of the samples. We also tested the efficiency and limit of our approach for detecting olive oil admixtures. As a result of the analysis, we were able to detect admixing down to a limit of 20%. The SNP-based CAPS assays developed in this work can be used for testing and verification of the authenticity of Turkish monovarietal olive oils, for olive tree certification, and in germplasm characterization and preservation studies.

Genomic Simple Sequence Repeat Markers Reveal Patterns of Genetic Relatedness and Diversity in Sesame.

Sesame (Sesamum indicum L. syn. Sesamum orientale L.) is an orphan crop species with most molecular genetic research work done in the last decade. In this study, we used a pyrosequencing approach for the development of genomic simple-sequence repeat (SSR) markers in sesame. Our approach proved successful in identifying 19,816 nonredundant SSRs, 5727 of which were identified in a contig assembly that covers 19.29% of the sesame genome. Mononucleotide repeats were the most abundant SSR type identified in the sesame genome (48.5% of all SSRs), followed by dinucleotide SSRs (45.0%). Adenine-thymine-rich motifs were predominant, representing 81.7, 51.7, 66.5, and 22.1% of the mononucleotide, dinucleotide, trinucleotide, and tetranucleotide SSRs, respectively. As a result of this work, we introduce 933 experimentally validated sesame specific markers, 849 of which are also applicable in Sesamum mulayanum (syn. Sesamum orientale var. malabaricum Nar.), the wild progenitor of cultivated sesame. Using a subset of the newly identified SSR markers, we analyzed molecular genetic diversity and population structure of a collection of world accessions. Results of the two analyses almost overlapped and suggested correlation between genetic similarity and geographical proximity. Indeed, a pattern of gene flow among sesame diversity centers was apparent, with levels of variability in some regions similar to that seen in the domestication origin of the crop. Taken together with the high rate of genomic marker transferability detected between S. indicum and S. mulayanum, our results represent additional molecular genetic evidence for designating the two taxa as cultivated and wild forms of the same species.