One of the first steps is an evaluation of germplasm for their nutrient content and health-promoting compounds. Other concurrent efforts will need to be made to ensure success, both in commercialization of the bioenriched product and impact on consumers. Second, a compendium of the studies performed on the genomic association of metabolic data in crops is also presented here.
Finally, an overview is also provided of the current regulations and the future prospects for the development of nutritionally enriched crops. Their predominance is also a result of the development of next-generation sequencing (NGS), including second- and third-generation sequencing (SGS and TGS), mainly SGS, which emerged from the sequencing of short DNA fragments (first-generation sequencing, FGS) to high-throughput technologies (SGS) and finally single-molecule sequencing (TGS). Their predominance is also a consequence of the development of next generation sequencing (NGS), including second and third generation sequencing (SGS and TGS), mainly SGS, which arose from the sequencing of short DNA fragments (sequencing of the first generation, FGS) to high-throughput technologies (SGS) and finally single-molecule sequencing (TGS).
Comparison of the most commonly used SNP genotyping techniques in tumors grouped according to the platforms in the throughput level. Regardless of the allele at the specific locus, both probes hybridize to the DNA sample although the signal becomes fainter in the case of a mismatch. The mass of the allele-specific product is determined using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry.
Among all the methods developed, including restriction site-associated DNA sequencing (RAD-Seq), sequencing diversity array technology (DArT-Seq), restriction fragment sequencing (REST-Seq), multiple gun genotyping (MSG), sequence-based genotyping (SBG), site-specific amplified fragment sequencing (SAF-Seq), etc., one of the most widely used in agricultural crops is sequence genotyping ( GBS).
Introducing Allelic Variants to Biofortify Crops
Instead of phenotyping at every stage of population building (as . in the MAS strategy), it is performed only in what is known as training population (DHs, F2, marker-assisted recurrent selections, etc.). Overexpression of the bacterial genes crtB (for phytoene synthase) and crtW and bkt1 (ketolase genes) under the control of seed specific. In biofortification, transgenic approaches consist of the transfer of one or more alleles of genes responsible for increasing the nutritional value of one or more organisms to the crop of interest.
The identification and characterization of the genes is necessary to eventually introduce them into the crop. One of the most notable examples is Golden Rice, obtained to alleviate vitamin A deficiency [17]. As in the case of conventional breeding, many different strategies have been applied to almost every type of crop, including cereals, legumes, vegetables, fruits and oilseeds, where the targets for biofortification are fatty acids, essential amino acids and antioxidants. among others (Table 3).
The identification and characterization of the genes are necessary to eventually introduce them into the crop. One of the most notable examples is Golden Rice, obtained to alleviate vitamin A deficiency [17]. As with conventional breeding, many different strategies have been applied to almost every kind of crop, including grains, legumes, vegetables, fruits, and oilseeds, with the targets of biofortification being fatty acids, essential amino acids, and antioxidants, among others (Table 3).
In cisgenesis, the transgene is a complete DNA copy of the gene as found in the donor plant (with promoter, introns, and terminator) in the normal orientation (Figure 3B). In both cases, when Agrobacterium-mediated transformation is used, T-DNA boundaries (flanking DNA sequences to be transferred) can also be inserted into the plant genome. Second, sequences similar to the T-DNA boundary found in plant genomes, known as P-DNA boundaries, can be used upstream and downstream of the gene to be transferred [216,221].
A rearrangement of the original gene is thus required, as it was in the donor plant, which is why this option should only be chosen if it is an intragenic plant. The introduction of the complete natural gene (cisgenesis) and changes in promoters and terminators (intragenesis) could increase expression levels, whereas the use of silencing constructs (intragenesis) could reduce them. These barley plants with a single copy of the gene showed a 2.8-fold increase in phytase activity and an improved bioavailability of phosphate.
In the case of intragenesis, potatoes are the most recurrent crop for gene suppression strategies. In the case of tomato, the content of carotenoid and flavonoid was simultaneously increased through the repression of the DE-ETIOLATED1 (DET1) gene by using RNAi technology and fruit-specific promoters [28].
Regulation of Plant Breeding Methods
In fact, the first intragenic application was the increase of amylopectin content in potatoes by silencing the granule-bound starch synthase (GBSS) gene responsible for the synthesis of amylose in potatoes [204]. Nevertheless, this potato was released into the field in the EU in 2007 (B/NL/07/04) with the potato GBSS terminator, i.e. a fully inbred potato plant. Another intragenic potato was engineered to reduce acrylamide content in processed potatoes (without yield penalty or affecting tuber shape) by silencing one asparagine synthase gene (StAs1) [205].
The iron content in wheat flour is increased more than 2-fold after the expression of a vacuolar iron transporter gene (TaVIT2) under the control of a wheat endosperm-specific promoter [12]. Finally, Dupont-Pioneer and Monsanto have developed two high oleic soybean oils, Plenish® and Vistive®Gold, respectively, which are currently available in the US market. In many African countries, there is no or very restrictive regulatory framework, despite being considered the part of the world with the greatest potential to benefit from the adoption of GM crops due to high levels of hunger and malnutrition.
Nevertheless, the number of countries embracing GM crops on this continent has doubled from three in 2018 to six in 2019 [232]. Despite the huge number of crops developed with improved traits through genetic engineering, only four different biotech crops cover more than 95% of the cultivated area (soybean, maize, cotton and canola) and in most cases the altered traits are related to herbicide tolerance and insect resistance [232]. Therefore, additional efforts are needed to approve genetically modified crops with increased nutritional value to contribute to the end of world hunger.
Today, transgenesis, cisgenesis and intragenesis are subject to the same regulation in the vast majority of countries. However, cisgenic and intragenic crops are generally more accepted by the public and are expected to be less strictly regulated in the coming years in some countries [236]. In fact, the regulatory system in Canada is based on the final product rather than on the process of obtaining it, which has relaxed the control of this kind of crops compared to the transgenic crops [237].
In the case of intragenesis, EFSA stated that the risks are less predictable due to the recombination of different genetic elements, despite belonging to the same gene pool [240]. Moreover, many studies have confirmed a higher acceptance by consumers and farmers of cisgenic and intragenic crops than transgenic ones, because they are considered to be more natural [227-229]. This, along with favorable opinions on cisgenesis and intragenesis from public organizations, should pave the way for less stringent regulations for these types of cultures.
Future Perspectives
However, until then, genetically modified plants will still be considered GMOs and their commercial cultivation will have to be allowed under current legislation. In Australia, cisgenic plants are not considered GMOs as defined in the Genetic Technology Regulations, with non-GMO organisms including "a mutated organism in which the mutational event did not involve the introduction of any foreign nucleic acid" [238]. Furthermore, a recent global study showed that consumers are willing to pay up to 23.9% more for GM-fortified crops [242].
At the same time, an effort to highlight the safety of genetically engineered crops in a clear and understandable way is essential to increase their acceptance among the general public and political organizations. It would also be interesting to improve research and development of bioenriched crops in developing countries where malnutrition is a real burden.
Conclusions
A powerful tool for genome analysis in maize: Development and evaluation of the high-density 600 k SNP genotyping array. Genome-wide association analyzes provide genetic and biochemical insights into natural variation in rice metabolism. Genome-wide comparative diversity reveals multiple targets of selection for improvement in hexaploid wheat landraces and cultivars.
Development of a large SNP genotypic array and generation of high-density genetic maps in tomato. Development and evaluation of a 6K SNP genomic array for diplomatic sweet cherry and tetraploid cherry. Exploring genome-wide diversity in the national peach (Prunus persica) germplasm collection at CITA (Zaragoza, Spain).
Identification of genetic loci and candidate genes associated with β-glucan content in barley grain by genome-wide association study in International Barley Core Selected Collection. Genome-wide association studies in apple reveal loci for volatile aromas, sugar composition and harvest date. Genome-wide SNP identification, linkage map construction and QTL mapping for seed mineral concentrations and contents in pea (Pisum sativum L.).
Genome-wide association study reveals genetic complexity of fructan accumulation patterns in barley grain. Metabolome-Based Genome Association Study Provides Genetic Insights into Natural Variation in Foxtail Millet. A basis for provitamin A biofortification of maize: Genome-wide association and genomic prediction models of carotenoid levels.
Metabolome genome-wide association study unravels the genetic architecture for generating natural variation in rice secondary metabolism. Identification of a candidate gene associated with isoflavone content in soybean seeds using genome-wide linkage and linkage mapping. Genomic predictions and genome-wide association studies based on RAD-seq of quality-related metabolites for genomics-assisted breeding of tea plants.
Metabolite-based genome-wide association study enables dissection of the flavonoid decoration pathway of wheat grains. Promotion of provitamin A biofortification in sorghum: genome-wide association studies of cereal carotenoids in global germplasm.