Enhancing kernel oil and tailoring fatty acid composition by genomics-assisted selection for dgat1-2 and fatb genes in multi-nutrient-rich maize: new avenue for food, feed and bioenergy.

Enhancing maize kernel oil is vital for improving the bioavailability of fat-soluble vitamins. Here, we combined favourable alleles of dgat1-2 and fatb into parental lines of four multi-nutrient-rich maize hybrids (APTQH1, APTQH4, APTQH5 and APTQH7) using marker-assisted selection (MAS). Parental lines possessed favourable alleles of crtRB1, lcyE, vte4 and opaque2 genes. Gene-specific markers enabled successful foreground selection in BC1F1, BC2F1 and BC2F2, while background selection using genome-wide microsatellite markers (127-132) achieved 93% recurrent parent genome recovery. Resulting inbreds exhibited significantly higher oil (6.93%) and oleic acid (OA, 40.49%) and lower palmitic acid (PA, 14.23%) compared to original inbreds with elevated provitamin A (11.77 ppm), vitamin E (16.01 ppm), lysine (0.331%) and tryptophan (0.085%). Oil content significantly increased from 4.80% in original hybrids to 6.73% in reconstituted hybrids, making them high-oil maize hybrids. These hybrids displayed 35.70% increment in oil content and 51.56% increase in OA with 36.32% reduction in PA compared to original hybrids, while maintaining higher provitamin A (two-fold), vitamin E (nine-fold), lysine (two-fold) and tryptophan (two-fold) compared to normal hybrids. Lipid health indices showed improved atherogenicity, thrombogenicity, cholesterolaemic, oxidability, peroxidizability and nutritive values in MAS-derived genotypes over original versions. Besides, the MAS-derived inbreds and hybrids exhibited comparable grain yield and phenotypic characteristics to the original versions. The maize hybrids developed in the study possessed high-yielding ability with high kernel oil and OA, low PA, better fatty acid health and nutritional properties, higher multi-vitamins and balanced amino acids, which hold immense significance to address malnutrition and rising demand for oil sustainably in a fast-track manner.

Low expression of carotenoids cleavage dioxygenase 1 (ccd1) gene improves the retention of provitamin-A in maize grains during storage.

Provitamin-A (proA) is essentially required for vision in humans but its deficiency affects children and pregnant women especially in the developing world. Biofortified maize rich in proA provides new opportunity for sustainable and cost-effective solution to alleviate malnutrition, however, significant loss of carotenoids during storage reduces its efficacy. Here, we studied the role of carotenoid cleavage dioxygenase 1 (ccd1) gene on degradation of carotenoids in maize. A set of 24 maize inbreds was analyzed for retention of proA during storage. At harvest, crtRB1-based maize inbreds possessed significantly high proA (ß-carotene: 12.30 µg/g, ß-cryptoxanthin: 4.36 µg/g) than the traditional inbreds (ß-carotene: 1.74 µg/g, ß-cryptoxanthin: 1.28 µg/g). However, crtRB1-based inbreds experienced significant degradation of proA carotenoids (ß-carotene: 20%, ß-cryptoxanthin: 32% retention) following 5 months. Among the crtRB1-based genotypes, V335PV had the lowest retention of proA (ß-carotene: 1.63 µg/g, ß-cryptoxanthin: 0.82 µg/g), while HKI161PV had the highest retention of proA (ß-carotene: 4.17 µg/g, ß-cryptoxanthin: 2.32 µg/g). Periodical analysis revealed that ~ 60-70% of proA degraded during the first three months. Expression analysis revealed that high expression of ccd1 led to low retention of proA carotenoids in V335PV, whereas proA retention in HKI161PV was higher due to lower expression. Highest expression of ccd1 was observed during first 3 months of storage. Copy number of ccd1 gene varied among yellow maize (1-6 copies) and white maize (7-35 copies) while wild relatives contained 1-4 copies of ccd1 gene per genome. However, copy number of ccd1 gene did not exhibit any correlation with proA carotenoids. We concluded that lower expression of ccd1 gene increased the retention of proA during storage in maize. Favourable allele of ccd1 can be introgressed into elite maize inbreds for higher retention of proA during storage.

Maize genotypes with favourable dgat1-2 and fatb alleles possess stable high kernel oil and better fatty acid health and nutritive indices.

Diverse uses of maize oil attracted various stakeholders, including food, feed, and bioenergy, highlighting the increased demand for sustainable production. Here, 48 diverse sub-tropical maize genotypes varying for dgat1-2 and fatb genes governing oil attributes, were evaluated in three diverse locations to assess trends of oil content, fatty acid (FA) profile, the effect of environment on oil attributes, the impact of different gene combinations and determine FA health and nutritional properties. The genotypes revealed wide variation in oil content (OC: 3.4-6.8 %) and FA compositional traits, namely palmitic (PA, 11.3-24.1 %), oleic (OA, 21.5-42.7 %), linoleic (LA, 36.6-61.7 %), and linolenic (ALA, 0.7-2.3 %) acids. Double-mutants with both favourable alleles (dd/ff) exhibited 51.6 % higher oil, 33.2 % higher OA, and 30.2 % reduced PA compared to wild-types (d+d+/f+f+) across locations. These double-mutants had lower saturated FA (12.2 %), and higher unsaturated FA (87.0 %), indicating reduced susceptibility to autooxidation, with lower atherogenicity (0.14), thrombogenicity (0.27) and peroxidisability (48.15), higher cholesterolemic index (7.16), optimum oxidability (5.27) and higher nutritive-value-index (3.35) compared to d+d+/f+f+, making them promising for significant health and nutritional benefits. Locally adapted stable novel double-mutants with high-oil and better FA properties identified here can expedite the maize breeding programs, meeting production demands and addressing long-standing challenges for breeders.

Genetic dissection of embryo size and weight related traits for enhancement of kernel oil in maize.

Embryo is a key determinant of kernel-oil in maize. Higher calorific value of maize kernel is attributed to increment in kernel-oil and it stores in specialised structure called embryo. Understanding the genetic behaviour of embryo size and weight related-traits is inevitable task for genetic improvement of kernel-oil. Here, the six-basic generations (P1, P2, F1, F2, BC1P1 and BC1P2) of three crosses (CRPBIO-962 × EC932601, CRPBIO-973 × CRPBIO-966 and CRPBIO-966 × CRPBIO-979) between contrasting embryo-sized maize inbreds were field evaluated at three locations to decipher the genetics of twenty embryo, kernel and embryo-to-kernel related-traits through generation-mean-analysis (GMA). Combined ANOVA revealed the significance of all the traits among generations; however, location and generation × location were found to be non-significant (P > 0.05) for most of the traits. Significance (P < 0.05) of scaling and joint-scaling tests revealed the presence of non-allelic interactions. Elucidation of six-parameters disclosed the predominance of dominance main-effect (h) and dominance × dominance interaction-effect (l) for most of traits. The signs of (h) and (l) indicated the prevalence of duplicate-epistasis type across crosses and locations. Thus, the population improvement approaches along with heterosis breeding method could be effective for improvement of these traits. Quantitative inheritance pattern was observed for all the traits with high broad-sense heritability and better-stability across locations. The study also predicted one to three major-gene blocks/QTLs for embryo-traits and up to 11 major-gene blocks/QTLs for embryo-to-kernel traits. These findings could provide deep insights to strategize extensive breeding methods to improve embryo traits for enhancing kernel-oil in sustainable manner.

Heat-Stress-Induced Changes in Physio-Biochemical Parameters of Mustard Cultivars and Their Role in Heat Stress Tolerance at the Seedling Stage.

In the era of global warming, heat stress, particularly at the seedling stage, is a major problem that affects the production and productivity of crops such as mustard that are grown in cooler climates. Nineteen mustard cultivars were exposed to contrasting temperature regimes-20 °C, 30 °C, 40 °C and a variable range of 25-40 °C-and evaluated for changes in physiological and biochemical parameters at the seedling stage to study their role in heat-stress tolerance. Exposure to heat stress showed detrimental effects on seedling growth as revealed by reduced vigor indices, survival percentages, antioxidant activity and proline content. The cultivars were grouped into tolerant, moderately tolerant and susceptible based on the survival percentage and biochemical parameters. All the conventional and three single-zero cultivars were found to be tolerant and moderately tolerant, respectively, while double-zero cultivars were reckoned to be susceptible except for two cultivars. Significant increases in proline content and catalase and peroxidase activities were found associated with thermo-tolerant cultivars. More efficient antioxidant system activity and proline accumulation were noticed in conventional along with three single-zero (PM-21, PM-22, PM-30) and two double-zero (JC-21, JC-33) cultivars that might have provided better protection to them under heat stress than the remaining one single- and nine double-zero cultivars. Tolerant cultivars also resulted in significantly higher values of most of the yield attributing traits. Heat-stress-tolerant cultivars could easily be selected based on the survival percentage, proline and antioxidants at the seedling stage and included as efficient cultivars in breeding programs.

Expression Dynamics of lpa1 Gene and Accumulation Pattern of Phytate in Maize Genotypes Possessing opaque2 and crtRB1 Genes at Different Stages of Kernel Development.

Phytic acid (PA) acts as a storehouse for the majority of the mineral phosphorous (P) in maize; ~80% of the total P stored as phytate P is not available to monogastric animals and thereby causes eutrophication. In addition, phytic acid chelates positively charged minerals making them unavailable in the diet. The mutant lpa1-1 allele reduces PA more than the wild-type LPA1 allele. Further, mutant gene opaque2 (o2) enhances lysine and tryptophan and crtRB1 enhances provitamin-A (proA) more than wild-type O2 and CRTRB1 alleles, respectively. So far, the expression pattern of the mutant lpa1-1 allele has not been analysed in maize genotypes rich in lysine, tryptophan and proA. Here, we analysed the expression pattern of wild and mutant alleles of LPA1, O2 and CRTRB1 genes in inbreds with (i) mutant lpa1-1, o2 and crtRB1 alleles, (ii) wild-type LPA1 allele and mutant o2 and crtRB1 alleles and (iii) wild-type LPA1, O2 and CRTRB1 alleles at 15, 30 and 45 days after pollination (DAP). The average reduction of PA/total phosphorous (TP) in lpa1-1 mutant inbreds was 29.30% over wild-type LPA1 allele. The o2 and crtRB1-based inbreds possessed ~two-fold higher amounts of lysine and tryptophan, and four-fold higher amounts of proA compared to wild-type alleles. The transcript levels of lpa1-1, o2 and crtRB1 genes in lpa1-1-based inbreds were significantly lower than their wild-type versions across kernel development. The lpa1-1, o2 and crtRB1 genes reached their highest peak at 15 DAP. The correlation of transcript levels of lpa1-1 was positive for PA/TP (r = 0.980), whereas it was negative with inorganic phosphorous (iP) (r = -0.950). The o2 and crtRB1 transcripts showed negative correlations with lysine (r = -0.887) and tryptophan (r = -0.893), and proA (r = -0.940), respectively. This is the first comprehensive study on lpa1-1 expression in the maize inbreds during different kernel development stages. The information generated here offers great potential for comprehending the dynamics of phytic acid regulation in maize.

Allelic Variation in Zmfatb Gene Defines Variability for Fatty Acids Composition Among Diverse Maize Genotypes.

Edible oil with lower saturated fatty acids is desired for perceived quality and health benefits to humans and livestock. fatb gene encoding acyl-ACP thioesterase is a key player in the conversion of palmitic acid to oleic acid, thereby modifying the ratio of saturated to unsaturated fatty acids in maize kernels. The present investigation characterised the full-length sequence of the Zmfatb gene (4.63 kb) in two mutants (Zmfatb) and eight wild-types (ZmfatB) inbreds to study allelic variation, gene-based diversity, phylogenetic-relationship, protein-modelling, and molecular-docking to identify novel candidates for modification of fatty acid profile. Sequence alignment revealed wide genomic variability for Zmfatb among the inbreds; identified five novel SNPs and two InDels that clearly differentiated the wild-type and mutant genotypes. Gene-based diversity using 11-InDel markers categorised 48-diverse maize-inbreds into two-clusters. The majority of mutant and wild-type inbreds were grouped in separate clusters and led to the generation of 41 haplotypes. Genetic relationship of maize fatb gene with orthologues among 40 accessions of 12 oilseed-crops using both nucleotide and protein sequence clustered maize, soybean, sunflower, opium-poppy, Citrulus lanata, quinoa, and prunus species into one cluster; and brassica, camelina, and arabidopsis into the different cluster. The clustering pattern revealed that the plant oil with higher unsaturated fatty acids, particularly oleic, linoleic, and linolenic acids grouped together in one cluster and higher proportions of other fractions like arachidic, eicosenoic, and erucic acids grouped in another cluster. Physico-chemical properties highlighted more similarity between maize and 29 orthologue proteins, but orthologues were found to have better thermostability. Homology models have been developed for maize mutant and wild-type inbreds using Umbellularia californica (PDB ID: 5x04) as a template. Predicted protein models possessed optimum confidence-score and RMSD values and validated stability via., Ramachandran plots. Molecular docking indicated most of the interactions of protein-ligand were having similar binding-affinity due to the broader specificity of fatty acyl-ACP thioesterases and the presence of conserved-domains across crops. This is the first report on the comprehensive molecular characterisation of the fatb gene in maize and various orthologues. The information generated here provided new insights into the genetic diversity of fatb gene which can be utilised for the enhanced nutritive value of oil in the breeding programme.

Experimental rice seed aging under elevated oxygen pressure: Methodology and mechanism.

Seed aging during storage results in loss of vigor and germination ability due to the accumulation of damage by oxidation reactions. Experimental aging tests, for instance to study genetic variation, aim to mimic natural aging in a shorter timeframe. As the oxidation rate is increased by elevating the temperature, moisture, and oxygen levels, this study aimed to (1) investigate the effect of experimental rice seed aging by an elevated partial pressure of oxygen (EPPO), (2) elucidate the mechanism of dry-EPPO aging and (3) compare aging under dry-EPPO conditions to aging under traditional moist-controlled deterioration (CD) conditions and to long-term ambient storage. Dry seeds from 20 diverse rice accessions were experimentally aged under EPPO (200 times higher oxygen levels), at 50% relative humidity (RH), along with storage under high-pressure nitrogen gas and ambient conditions as controls. While no decline in germination was observed with ambient storage, there was significant aging of the rice seeds under EPPO storage, with considerable variation in the aging rate among the accessions, with an average decline toward 50% survival obtained after around 21 days in EPPO storage and total loss of germination after 56 days. Storage under high-pressure nitrogen gas resulted in a small but significant decline, by an average of 5% germination after 56 days. In a second experiment, seven rice seed lots were stored under EPPO as compared to a moist-CD test and two different long-term ambient storage conditions, i.e., conditioned warehouse seed storage (CWSS) and traditional rice seed storage (TRSS). Untargeted metabolomics (with identification of lipid and volatile compounds profiles) showed a relatively high increase in levels of oxidized lipids and related volatiles under all four storage conditions. These compounds had a high negative correlation with seed viability, indicating oxidation as a main deteriorating process during seed aging. Correlation analysis indicated that EPPO storage at 50% RH is more related to aging under TRSS at 60% and CD-aging at 75% ERH rather than CWSS at 40% ERH. In conclusion, aging rice seeds under EPPO conditions is a suitable experimental aging method for analyzing variation among seed lots or genotypes for longevity under storage.

Ex-foliar application of glycine betaine and its impact on protein, carbohydrates and induction of ROS scavenging system during drought stress in flax (Linum usitatissimum).

Crop plants have an innate capacity to acclimatize and survive myriad stresses in field conditions. This acclimatization to stress enhances crop stand in field and productivity of plant. Inter alia field crops withstand drought stress (hydropenia) by inducing synthesis or accumulation of osmolytes such as (i) proline and other amino acids, (ii) glycine betaine (GB), (iii) soluble carbohydrates, and (iv) reactive oxygen species (ROS) scavenging system as intrinsic drought antagonizing molecules. Precise in vivo induction of osmolytes and their effect on ROS scavenging system in flax/linseed has not been elucidated. The investigation was carried out to identify a tolerant and susceptible cultivar of flax from a core collection of 53 core accessions and evaluate the role of compatible osmolytes in Linum usitatissimum under hydropenia. We screened eight morphometrically diverse flax genotypes in field under irrigated and un-irrigated condition and classified them as tolerant and susceptible genotypes. Further, we examined the effect of ex-foliar glycine betaine application - a signature molecule involved in drought tolerance, on selected tolerant and susceptible varieties. Our results showed stimulatory impact of glycine betaine on accumulation of ROS scavenging antioxidants, total soluble protein and on its own accumulation. While the ex-foliar application had no inhibitory effect on the growth of plants; accumulation of free proline, amino acids and carbohydrates are inhibited par se in flax. Our findings reveal, flax is a non-accumulator of glycine betaine and exogenous application of glycine betaine enhances its own levels during drought stress.

Unraveling the Relationship Between Seed Yield and Yield-Related Traits in a Diversity Panel of Brassica juncea Using Multi-Traits Mixed Model.

The response to selection in any crop improvement program depends on the degree of variance and heritability. The objective of the current study was to explain variance and heritability components in Indian mustard Brassica juncea (L). Czern & Coss to recognize promising genotypes for effective breeding. Two hundred and eighty-nine diverse accessions of Indian mustard belonging to four continents were analyzed for yield and yield-related traits (20 traits) over two seasons (2017-2018 and 2018-2019) using an alpha lattice design. The genetic variance was found to be significant (P ≤ 0.01) for the individual and under pooled analysis for all of the evaluated traits, demonstrating the presence of significant genetic variability in the diversity panel, which bids greater opportunities for utilizing these traits in future breeding programs. High heritability combined with high genetic advance as percent of mean and genotypic coefficient of variation was observed for flowering traits, plant height traits, seed size, and seed yield/plant; hence, a better genetic gain is expected upon the selection of these traits over subsequent generations. Both correlation and stepwise regression analysis indicated that the main shoot length, biological yield, total seed yield, plant height up to the first primary branch, seed size, total siliqua count, days to flowering initiation, plant height at maturity, siliquae on the main shoot, main shoot length, and siliqua length were the most significant contributory traits for seed yield/plant. Also, promising genotypes were identified among the diversity panel, which can be utilized as a donor to improve Indian mustard further. These results indicated a greater scope for improving seed yield per plant directly through a selection of genotypes having the parsimonious combination of these nine traits.

Microsatellites in Brassica unigenes: relative abundance, marker design, and use in comparative physical mapping and genome analysis.

Microsatellites present in the transcribed regions of the genome have the potential to reveal functional diversity. Unigene sequence databases are the sources of such genic microsatellites with unique flanking sequences and genomic locations even in complex polyploids. The present study was designed to assay the unigenes of Brassica napus and B. rapa for various microsatellite repeats, and to design markers and use them in comparative genome analysis and study of evolution. The average frequency of microsatellites in Brassica unigenes was one in every 7.25 kb of sequence, as compared with one in every 8.57 kb of sequence in Arabidopsis thaliana. Trinucleotide motifs coding for serine and the dinucleotide motif GA were most abundant. We designed 2374 and 347 unigene-based microsatellite (UGMS) markers including 541 and 58 class I types in B. napus and B. rapa, respectively, and evaluated their use across diverse species and genera. Most of these markers (93.3%) gave successful amplification of target microsatellite motifs, which was confirmed by sequencing. Interspecific polymorphism between B. napus and B. rapa detected in silico for the UGMS markers was 4.16 times higher in 5' untranslated regions than in coding sequences. Physical anchoring of Brassica UGMS markers on the A. thaliana genome indicated their significance in studying the evolutionary history of A. thaliana genomic duplications in relation to speciation. Comparative physical mapping identified 85% of Brassica unigenes as single copy and gave clues for the presence of conserved primordial gene order. Complex chromosomal rearrangements such as inversions, tandem and segmental duplications, and insertions/deletions were evident between A. thaliana and B. rapa genomes. The results obtained have encouraging implications for the use of UGMS markers in comparative genome analysis and for understanding evolutionary complexities in the family Brassicaceae.

Development and validation of breeder-friendly gene-based markers for lpa1-1 and lpa2-1 genes conferring low phytic acid in maize kernel.

Based on C (wild) to T (mutant) transition at amino acid position 1432 bp of lpa1-1 gene, two dominant markers each specific to wild type (LPA1) and mutant (lpa1-1) allele were developed and validated across seven F2 populations. Joint segregation of these markers behaved in co-dominant fashion, clearly distinguishing heterozygote from two other homozygote genotypes. Full length sequence alignment between wild type (LPA2) and mutant (lpa2-1) allele revealed one transition mutation (A to G) and a co-dominant CAPS marker was developed which differentiated all three types of segregants across seven F2 populations. Across populations, segregants with lpa1-1/lpa1-1 (1.77 mg/g) and lpa2-1/lpa2-1 (1.85 mg/g) possessed significantly lower phytic acid compared to LPA1/LPA1 (2.58 mg/g) and LPA2/LPA2 (2.53 mg/g). Inorganic phosphorus was however higher in recessive homozygotes (lpa1-1/lpa1-1: 0.77 mg/g, lpa2-1/lpa2-1: 0.53 mg/g) than the dominant homozygotes (LPA1/LPA1: 0.33 mg/g, LPA2/LPA2: 0.19 mg/g). Overall, homozygous segregants of lpa1-1 and lpa2-1 showed 31% and 27% reduction of phytic acid, respectively. Analysis of phytate and inorganic phosphorous in the maize kernel in these segregating populations confirmed co-segregation of trait and markers specific to lpa1-1 and lpa2-1. This is the first report of the development of breeder-friendly gene-based markers for lpa1-1 and lpa2-1; and it holds great significance for maize biofortification.

Genome-wide analysis of drought induced gene expression changes in flax (Linum usitatissimum).

A robust phenotypic plasticity to ward off adverse environmental conditions determines performance and productivity in crop plants. Flax (linseed), is an important cash crop produced for natural textile fiber (linen) or oilseed with many health promoting products. This crop is prone to drought stress and yield losses in many parts of the world. Despite recent advances in drought research in a number of important crops, related progress in flax is very limited. Since, response of this plant to drought stress has not been addressed at the molecular level; we conducted microarray analysis to capture transcriptome associated with induced drought in flax. This study identified 183 differentially expressed genes (DEGs) associated with diverse cellular, biophysical and metabolic programs in flax. The analysis also revealed especially the altered regulation of cellular and metabolic pathways governing photosynthesis. Additionally, comparative transcriptome analysis identified a plethora of genes that displayed differential regulation both spatially and temporally. These results revealed co-regulated expression of 26 genes in both shoot and root tissues with implications for drought stress response. Furthermore, the data also showed that more genes are upregulated in roots compared to shoots, suggesting that roots may play important and additional roles in response to drought in flax. With prolonged drought treatment, the number of DEGs increased in both tissue types. Differential expression of selected genes was confirmed by qRT-PCR, thus supporting the suggested functional association of these intrinsic genes in maintaining growth and homeostasis in response to imminent drought stress in flax. Together the present study has developed foundational and new transcriptome data sets for drought stress in flax.