Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models. The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content. Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.

/pdf/mutagenesis-of-fad2-genes-in-peanut-with-crispr-cas9-based-1k5x6x4im3.pdf

Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing

Background The nutritional quality, flavor, and shelf-life of both peanut products and its seeds are dependent on relative quantity of various fatty acids (FAs) like saturated, mono unsaturated fatty acid (MUFA) and poly unsaturated fatty acid (PUFA) present in its oil. High oleic (HO) peanut oils are extremely valued due to its superior nutritional composition for human health and augmented thermo-oxidative stability for industrial purposes. Scope and approach From the research perspective, noteworthy progress has been made during last three decades for the development of peanut lines having HO trait in its oil. In this review, the research achievements, perspectives, and prospects of peanut genetic improvement for HO trait is thoroughly discussed. Key findings and conclusions The research has helped not only understanding the genetics of HO traits and its genotype (G) by environment (E) interaction but also produced an enormous number of HO line throughout the world. Although, as of now, most of the high O/L cultivars developed are the outcome of traditional breeding efforts. But, with the advent of novel molecular techniques like CAPS and AS-PCR assay for HO peanut breeding program, it is extremely easy to achieve the traits through marker assisted selection (MAS) rather than through either conventional or genetic engineering approaches. The availability of peanut genome sequence and identification of different ahFAD2 gene families is also expediting the research for the breeding of HO peanut genotypes.

High oleic peanut breeding: Achievements, perspectives, and prospects

In peanut (Arachis hypogaea L.), the customization of fatty acid profile is an evolving area to fulfil the nutritional needs in the modern market. A total of 174 peanut genotypes, including 167 Indian cultivars, 6 advanced breeding lines and ‘SunOleic’‒ a double mutant line, were investigated using AS-PCRs, CAPS and gene sequencing for the ahFAD2 allele polymorphism, along with its fatty acid compositions. Of these, 80 genotypes were found having substitution (448G>A) mutation only in ahFAD2A gene, while none recorded 1-bp insertion (441_442insA) mutation in ahFAD2B gene. Moreover, 22 wild peanut accessions found lacking both the mutations. Among botanical types, the ahFAD2A mutation was more frequent in ssp. hypogaea (89%) than in ssp. fastigiata (17%). This single allele mutation, found affecting not only oleic to linoleic acid fluxes, but also the composition of other fatty acids in the genotypes studied. Repeated use of a few selected genotypes in the Indian varietal development programs were also eminently reflected in its ahFAD2 allele polymorphism. Absence of known mutations in the wild-relatives indicated the possible origin of these mutations, after the allotetraploidization of cultivated peanut. The SNP analysis of both ahFAD2A and ahFAD2B genes, revealed haplotype diversity of 1.05% and 0.95%, while Ka/Ks ratio of 0.36 and 0.39 respectively, indicating strong purifying selection pressure on these genes. Cluster analysis, using ahFAD2 gene SNPs, showed presence of both mutant and non-mutant genotypes in the same cluster, which might be due the presence of ahFAD2 gene families. This investigation provided insights into the large number of Indian peanut genotypes, covering various aspects related to O/L flux regulation and ahFAD2 gene polymorphism.

/pdf/insights-into-the-indian-peanut-genotypes-for-ahfad2-gene-2frsdfgc9f.pdf

Insights into the Indian Peanut Genotypes for ahFAD2 Gene Polymorphism Regulating Its Oleic and Linoleic Acid Fluxes.

This research aimed to compare the effects of monosodium glutamate (MSG) and its alternatives on sensory characteristics of chicken soup. High-performance liquid chromatography analysis was carried out to quantify umami substances in potential MSG alternatives. Two mushroom extracts (CE and MC), one tomato extract (TC), and one yeast extract (YE) powders were selected due to their high equivalent umami concentration (EUC). These extracts together with MSG were then applied individually at four different levels (CE, MC, TC, MSG: 0.05%, 0.1%, 0.2%, 0.4%; YE: 0.0125%, 0.025%, 0.05%, 0.1%) in chicken soup in order to compare their impact on major sensory attributes using the degree of difference from control (DODC) test. Our results showed that all four extracts at all the usage levels exhibited an enhancement effect on the overall flavor, meaty flavor, saltiness, and umami taste. The extent of enhancement depended on the type of the alternative and its usage level. Higher levels of MSG alternatives (except YE) suppressed the chicken flavor. YE had similar enhancement effects as MSG on umami and salty tastes already at lower usage levels. At the lowest concentration, TC showed a stronger enhancement effect than MSG, but its effect on most attributes decreased as the usage dose increased. Compared to CE, the other mushroom extract MC resembled MSG at most levels. Overall, the closest synergistic effect in chicken soup was noted with 0.1% MSG, 0.1% MC, and 0.025% YE. PRACTICAL APPLICATION: This study compared the enhancement effects of MSG and selected alternatives in chicken soup. Results will help food manufacturers who would like to replace MSG with natural umami substances in soup products to enhance flavor and reduce sodium chloride.

Investigation of Monosodium Glutamate Alternatives for Content of Umami Substances and Their Enhancement Effects in Chicken Soup Compared to Monosodium Glutamate.

Identification of potential QTLs and genes associated with seed composition traits in peanut (Arachis hypogaea L.) using GWAS and RNA-Seq analysis.

Natural genetic variation exists in animals and plants. Mining and utilizing this variation may provide benefits for new breed/cultivar development. From screening over 4000 cultivated peanut germplasm accessions, we identified two natural mutant lines (PI 342664 and PI 342666) with 80 % oleic acid by gas chromatography analysis. It is known that FAD2A and FAD2B are the two major genes involved in the conversion of oleic to linoleic acid in peanuts by fatty acid desaturase. Functional mutations in one or both genes can alter the oleate level. By sequencing the coding region of these two genes, we identified a substitution of G448A in FAD2A and a substitution of C301G in FAD2B for both mutant lines. The substitution in FAD2A is the same as a previously identified one, resulting in a missense amino acid substitution of D150N, but the substitution in FAD2B is a new one, resulting in H101D. The new amino acid substitution on FAD2B is located in the first histidine box (one of the active sites) of the fatty acid desaturase and may significantly decrease its activity. Our mutants did not have flowers on the main stem (subspecies hypogaea), but F435 (a previously identified natural high-oleate mutant) had flowers on the main stem (subspecies fastigiata). Therefore, we identified a class of natural mutants from the subspecies hypogaea and provided new additional genetic resources for breeders to use. Our results also demonstrate a good example of the importance and usefulness for preserving natural genetic diversity and utilizing plant germplasm collections.

Newly identified natural high-oleate mutant from Arachis hypogaea L. subsp. hypogaea

Bamboo shoots and leaves are nutritious, providing food for human and animal consumption. However their nutrient value depends on a number of factors including the bamboo species (e.g. taxon), harvesting season and growing location. Leaf crude protein content, amino acid composition and mineral element concentration were quantified from leaf samples collected from 222 accessions representing two bamboo types (temperate/tropical), two growing seasons (dry/wet) and three locations (PGRCU-Byron, GA; TARSMayaguez, PR; and Govardhan Gardens-Mayaguez, PR). Significant variability in leaf nutrient value was identified among accessions, types, growing locations and harvesting seasons. On average, bamboo leaf tissue contained 12.92% protein with relatively high percentages of leucine (Leu, 8.97%) and proline (Pro, 7.98%) and low percentages of tryptophan (Trp, 1.69%) and histidine (His, 2.01%). Bamboo leaves also contained relatively high concentrations of the macro-mineral elements potassium (K, 12.17mg/g) and calcium (Ca, 5.37mg/g), high concentrations of the micro-mineral elements manganese (Mn, 388.76 μg/g) and iron (Fe, 123.19 μg/g), and low concentrations of boron (B, 7.8 μg/g) and zinc (Zn, 28.56 μg/g). PI 647932 and TARS 182857 contained the highest and lowest protein content (21.69% and 5.78%), respectively. Temperate bamboos contained a significantly higher percentage of protein (13.02%) than tropical bamboos (12.71%). Leaf samples harvested from the dry season contained a significantly higher percentage of protein (13.12%) than leaf samples harvested from the wet season (12.70%). The leaf samples harvested from Govardhan Garden location contained a higher percentage of protein (13.40%) than from Byron (12.65%) and TARS (12.89%) locations. The leaf samples harvested from Govardhan Garden contained a significantly higher iron concentration (151.22 μg/g) than from the other two locations (118.19 μg/g, 111.52 μg/g), whereas the leaf samples collected from Byron contained a significantly higher zinc concentration (36.03 μg/g) than what was observed from the other two locations (27.83 μg/g, 22.11 μg/g). There was no significant difference in iron and zinc concentrations between leaf samples collected from dry or wet seasons. Tropical bamboo leaves contained a higher iron concentration (131.74 μg/g) than temperate bamboo leaves (118.95 μg/g), whereas temperate bamboo leaves contained a significantly higher zinc concentration (29.82 μg/g) than tropical bamboo leaves (26.01 μg/g). The information on the bamboo leaf nutrient value related to bamboo accessions, types, growing location and harvesting season will be very useful for bamboo growers, processors and consumers.

Exploring Bamboo Leaf Nutrient Value in the USNPGS Germplasm Collection

High oleate is an important seed quality trait frequently incorporated in peanut varieties. Crop wild relatives (CWR) are potentially useful genetic resources for cultivar improvement through genetic introgression; but for wild peanut species, many chemical or nutritional traits are not well characterized. A mutant from Arachis veigae S. H. Santana & Valls (2n = 2x = 20), with increased oleic and very long chain (C ≥ 22) fatty acid content was identified from screening 209 accessions of 45 species using gas chromatography (GC). The A. veigae (formerly A. sylvestris) accession, VVeSv 8373 (PI 688970) contained 55.5% oleic acid in seeds, significantly higher than the average (18.3%) of other accessions within the same species and also significantly higher than the average (37.0%) of all wild peanut accessions evaluated. A C37T substitution was identified by sequencing the coding region of FAD2H, resulting in the nonsense mutation of Q13* (a premature stop codon). This functional mutation may significantly reduce the fatty acid desaturase (FAD) activity and result in the enhanced oleate level. Arachis veigae also contained a high percentage of very long-chain (C ≥ 22) fatty acids, and their variation identified in this study is also discussed and compared with other species. The mutant with such an altered fatty acid composition may be useful for potentially improving seed or food nutrition quality.

/pdf/identification-of-a-mutant-from-arachis-veigae-with-enhanced-3jl04zfqdm.pdf

Identification of a mutant from Arachis veigae with enhanced seed oleic and very long-chain fatty acid content

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Newly identified natural high-oleate mutant from Arachis hypogaea L. subsp. hypogaea

Exploring Bamboo Leaf Nutrient Value in the USNPGS Germplasm Collection

Identification of a mutant from Arachis veigae with enhanced seed oleic and very long-chain fatty acid content