Low expression of lipoxygenase 3 (LOX3) enhances the retention of kernel tocopherols in maize during storage.

BACKGROUND: Deficiency of vitamin E results in several neurological and age-related disorders in humans. Utilization of maize mutants with favourable vte4-allele led to the development of several α-tocopherol (vitamin E) rich (16-19 µg/g) maize hybrids worldwide. However, the degradation of tocopherols during post-harvest storage substantially affects the efficacy of these genotypes. METHODS AND RESULTS: We studied the role of lipoxygenase enzyme and Lipoxygenase 3 (LOX3) gene on the degradation of tocopherols at monthly intervals under traditional storage up to six months in two vte4-based contrasting-tocopherol retention maize inbreds viz. HKI323-PVE and HKI193-1-PVE. The analysis revealed significant degradation of tocopherols across storage intervals in both the inbreds. Lower retention of α-tocopherol was noticed in HKI193-1-PVE. HKI323-PVE with the higher retention of α-tocopherol showed lower lipoxygenase activity throughout the storage intervals. LOX3 gene expression was higher (~ 1.5-fold) in HKI193-1-PVE compared to HKI323-PVE across the storage intervals. Both lipoxygenase activity and LOX3 expression peaked at 120 days after storage (DAS) in both genotypes. Further, a similar trend was observed for LOX3 expression and lipoxygenase activity. The α-tocopherol exhibited a significantly negative correlation with lipoxygenase enzyme and expression of LOX3 across the storage intervals. CONCLUSIONS: HKI323-PVE with high tocopherol retention, low -lipoxygenase activity, and -LOX3 gene expression can act as a potential donor in the vitamin E biofortification program. Protein-protein association network analysis also indicated the independent effect of vte4 and LOX genes. This is the first comprehensive report analyzing the expression of the LOX3 gene and deciphering its vital role in the retention of α-tocopherol in biofortified maize varieties under traditional storage.

Allelic variation in sugary1 gene affecting kernel sweetness among diverse-mutant and -wild-type maize inbreds.

Sweet corn is popular worldwide as vegetable. Though large numbers of sugary1 (su1)-based sweet corn germplasm are available, allelic diversity in su1 gene encoding SU1 isoamylase among diverse maize inbreds has not been analyzed. Here, we characterized the su1 gene in maize and compared with allied species. The entire su1 gene (11,720 bp) was sequenced among six mutant (su1) and five wild (Su1) maize inbreds. Fifteen InDels of 2-45 bp were selected to develop markers for studying allelic diversity in su1 gene among 19 mutant- (su1) and 29 wild-type (Su1) inbreds. PIC ranged from 0.15 (SU-InDel7) to 0.37 (SU-InDel13). Major allele frequency varied from 0.52 to 0.90, while gene diversity ranged from 0.16 to 0.49. Phylogenetic tree categorized 48 maize inbreds in two clusters each for wild- type (Su1) and mutant (su1) types. 44 haplotypes of su1 were observed, with three haplotypes (Hap6, Hap22 and Hap29) sharing more than one genotype. Further, comparisons were made with 23 orthologues of su1 from 16 grasses and Arabidopsis. Maize possessed 15-19 exons in su1, while it was 11-24 exons among orthologues. Introns among the orthologues were longer (77-2206 bp) than maize (859-1718 bp). SU1 protein of maize and orthologues had conserved α-amylase and CBM_48 domains. The study also provided physicochemical properties and secondary structure of SU1 protein in maize and its orthologues. Phylogenetic analysis showed closer relationship of maize SU1 protein with P. hallii, S. bicolor and E. tef than Triticum sp. and Oryza sp. The study showed that presence of high allelic diversity in su1 gene which can be utilized in the sweet corn breeding program. This is the first report of comprehensive characterization of su1 gene and its allelic forms in diverse maize and related orthologues.

On-farm crop diversity, conservation, importance and value: a case study of landraces from Western Ghats of Karnataka, India.

Landraces are important genetic resources that have a significant role in maintaining the long-term sustainability of traditional agro-ecosystems, food, nutrition, and livelihood security. In an effort to document landraces in the on-farm conservation context, Central Western Ghat region in India was surveyed. A total of 671 landraces belonging to 60 crops were recorded from 24 sites. The custodian farmers were found to conserve a variety of crops including vegetables, cereals and pulses, perennial fruits, spices, tuber and plantation crops. The survey indicated a difference in the prevalence of landraces across the sites. A significant difference with respect to the Shannon-diversity index, Gini-Simpson index, evenness, species richness, and abundance was observed among the different survey sites. Computation of a prevalence index indicated the need for immediate intervention in the form of collecting and ex situ conservation of landraces of some crops as a back-up to on-farm conservation. The study also identified the critical determinants of on-farm conservation, including (i) suitability to regional conditions, (ii) relevance in regional cuisine and local medicinal practices, (iii) cultural and traditional significance, and (iv) economic advantage. The information documented in this study is expected to promote the collection and conservation of landraces ex situ. The National Genebank housed at ICAR-NBPGR, New Delhi conserves around 550 accessions of landraces collected from the Central Western Ghats region surveyed in this report. Information collected from custodian farmers on specific uses will be helpful to enhance the utilization of these accessions.

Multilocus functional characterization of indigenous and exotic inbreds for dgat1-2, fatb, ge2 and wri1a genes affecting kernel oil and fatty acid profile in maize.

Demand for maize oil is progressively increasing due to its diverse industrial applications, aside from its primary role in human nutrition and animal feed. Oil content and composition are two crucial determinants of maize oil in the international market. As kernel oil in maize is a complex quantitative trait, improving this trait presents a challenge for plant breeders and biotechnologists. Here, we characterized a set of 292 diverse maize inbreds of both indigenous and exotic origin by exploiting functional polymorphism of the dgat1-2, fatb, ge2, and wri1a genes governing kernel oil in maize. Genotyping using gene-based functional markers revealed a lower frequencies of dgat1-2 (0.15) and fatb (0.12) mutant alleles and a higher frequencies of wild-type alleles (Dgat1-2: 0.85; fatB: 0.88). The favorable wri1a allele was conserved across genotypes, while its wild-type allele (WRI1a) was not detected. In contrast, none of the genotypes possessed the ge2 favorable allele. The frequency of favorable alleles of both dgat1-2 and fatb decreased to 0.03 when considered together. Furthermore, pairwise protein-protein interactions among target gene products were conducted to understand the effect of one protein on another and their responses to kernel oil through functional enrichments. Thus, the identified maize genotypes with dgat1-2, fatb, and wri1a favourable alleles, along with insights gained through the protein-protein association network, serve as prominent and unique genetic resources for high-oil maize breeding programs. This is the first comprehensive report on the functional characterization of diverse genotypes at the molecular and protein levels.

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.

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.

Nutritional Potential of Adzuki Bean Germplasm and Mining Nutri-Dense Accessions through Multivariate Analysis.

The adzuki bean (Vigna angularis), known for its rich nutritional composition, holds significant promise in addressing food and nutritional security, particularly for low socioeconomic classes and the predominantly vegetarian and vegan populations worldwide. In this study, we assessed a total of 100 diverse adzuki bean accessions, analyzing essential nutritional compounds using AOAC's official analysis procedures and other widely accepted standard techniques. Our analysis of variance revealed significant genotype variations for all the traits studied. The variability range among different traits was as follows: moisture: 7.5-13.3 g/100 g, ash: 1.8-4.2 g/100 g, protein: 18.0-23.9 g/100 g, starch: 31.0-43.9 g/100 g, total soluble sugar: 3.0-8.2 g/100 g, phytic acid: 0.65-1.43 g/100 g, phenol: 0.01-0.59 g/100 g, antioxidant: 11.4-19.7 mg/100 g GAE. Noteworthy accessions included IC341955 and EC15256, exhibiting very high protein content, while IC341957 and IC341955 showed increased antioxidant activity. To understand intertrait relationships, we computed correlation coefficients between the traits. Principal Component Analysis (PCA) revealed that the first four principal components contributed to 63.6% of the variation. Further, hierarchical cluster analysis (HCA) identified nutri-dense accessions, such as IC360533, characterized by high ash (>4.2 g/100 g) and protein (>23.4 g/100 g) content and low phytic acid (0.652 g/100 g). These promising compositions provide practical support for the development of high-value food and feed varieties using effective breeding strategies, ultimately contributing to improved global food security.

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.

Allelic variation in shrunken2 gene affecting kernel sweetness in exotic-and indigenous-maize inbreds.

Sweet corn has become a popular food worldwide. It possesses six-times more sugar than field corn due to the presence of recessive shrunken2 (sh2) gene. Despite availability of diverse sweet corn germplasm, comprehensive characterization of sh2 has not been undertaken so far. Here, entire Sh2 gene (7320 bp) among five field corn-(Sh2Sh2) and six sweet corn-(sh2sh2) inbreds was sequenced. A total of 686 SNPs and 372 InDels were identified, of which three SNPs differentiated the wild-(Sh2) and mutant-(sh2) allele. Ten InDel markers were developed to assess sh2 gene-based diversity among 23 sweet corn and 25 field corn lines. Twenty-five alleles and 47 haplotypes of sh2 were identified among 48 inbreds. Among markers, MGU-InDel-2, MGU-InDel-3, MGU-InDel-5 and MGU-InDel-8 had PIC>0.5. Major allele frequency varied from 0.458-0.958. The gene sequence of these maize inbreds was compared with 25 orthologues of monocots. Sh2 gene possessed 15-18 exons with 6-225bp among maize, while it was 6-21 exons with 30-441bp among orthologues. While intron length across maize genotypes varied between 67-2069bp, the same among orthologues was 57-2713 bp. Sh2-encoded AGPase domain was more conserved than NTP transferase domain. Nucleotide and protein sequences of sh2 in maize and orthologues revealed that rice orthologue was closer to maize than other monocots. The study also provided details of motifs and domains present in sh2 gene, physicochemical properties and secondary structure of SH2 protein in maize inbreds and orthologues. This study reports detailed characterization and diversity analysis in sh2 gene of maize and related orthologues in various monocots.

Combining higher accumulation of amylopectin, lysine and tryptophan in maize hybrids through genomics-assisted stacking of waxy1 and opaque2 genes.

Waxy maize rich in amylopectin has emerged as a preferred food. However, waxy maize is poor in lysine and tryptophan, deficiency of which cause severe health problems. So far, no waxy hybrid with high lysine and tryptophan has been developed and commercialized. Here, we combined recessive waxy1 (wx1) and opaque2 (o2) genes in the parental lines of four popular hybrids (HQPM1, HQPM4, HQPM5, and HQPM7) using genomics-assisted breeding. The gene-based markers, wx-2507F/RG and phi057 specific for wx1 and o2, respectively were successfully used to genotype BC1F1, BC2F1 and BC2F2 populations. Background selection with > 100 SSRs resulted in recovering > 94% of the recurrent parent genome. The reconstituted hybrids showed 1.4-fold increase in amylopectin (mean: 98.84%) compared to the original hybrids (mean: 72.45%). The reconstituted hybrids also showed 14.3% and 14.6% increase in lysine (mean: 0.384%) and tryptophan (mean: 0.102%), respectively over the original hybrids (lysine: 0.336%, tryptophan: 0.089%). Reconstituted hybrids also possessed similar grain yield (mean: 6248 kg/ha) with their original versions (mean: 6111 kg/ha). The waxy hybrids with high lysine and tryptophan assume great significance in alleviating malnutrition through sustainable and cost-effective means. This is the first report of development of lysine and tryptophan rich waxy hybrids using genomics-assisted selection.