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.

Pollen staining is a rapid and cost-effective alternative to marker-assisted selection for recessive waxy1 gene governing high amylopectin in maize.

Waxy maize is popular for food-, feed- and industrial usage. It possesses a recessive waxy1 (wx1) gene that enhances amylopectin to ~ 95-100%, compared to ~ 70-75% in traditional maize. Marker-assisted selection (MAS) is a preferred approach to converting normal maize into a waxy version. However, it requires specialized expertise, a well-equipped laboratory, and high cost. Here, pollen staining was used as an alternative approach to MAS. BC1F1, BC1F2 and BC2F2 populations in seven genetic backgrounds segregating for the wx1 gene were used. Pollens treated with iodine-potassium iodide showed that wild types (Wx1Wx1) were dark purple, while waxy pollens (wx1wx1) exhibited red colour. Heterozygotes (Wx1wx1) showed a mix of both dark purple and red colour. Staining of endosperm flour also confirmed the same findings. Wx1-based genotyping using phi022 and wx2507F/RG confirmed the same genotypic status. The average amylopectin among genotypes having red coloured pollens was 97.6%, while it was 72.5% among dark purple. Heterozygotes with both dark purple and red pollens had 85.2% amylopectin. Pollen staining showed complete agreement with the genotyping as well as amylopectin contents. Pollen staining saved 81% cost, and 54% time compared to MAS. This is the first report on the utilization of pollen staining for selecting the wx1 allele in segregating populations used for the development of waxy maize hybrids. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01240-1.

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.

Protective effect of alpha-lipoic acid and omega-3 fatty acids against cyclophosphamide-induced ovarian toxicity in rats.

BACKGROUND AND AIM: Cyclophosphamide therapy is known to be associated with the risk of female infertility as a result of ovarian toxicity. Alpha-lipoic acid (LA) and omega-3 fatty acids are known for their antioxidant and anti-inflammatory activities. The present study investigated the potential protective effect of alpha-LA, omega-3 fatty acids, and its combination against cyclophosphamide-induced ovarian toxicity in rats. MATERIALS AND METHODS: Thirty rats were equally divided into Groups I, II, III, IV, and V. Group I was normal control, wherein the rats were fed with normal feed and water ad libitum. Group II served as cyclophosphamide-induced group, wherein the rats were injected with cyclophosphamide at 75 mg/kg through intraperitoneal route once a week to induce ovarian toxicity. Groups III and IV were treated with alpha-LA at the rate of 25 mg/kg and omega-3 fatty acids at the rate of 400 mg/kg, respectively, in parallel to cyclophosphamide induction as in Group II. Group V animals were coadministered with alpha-LA (25 mg/kg) and omega-3 fatty acids (400 mg/kg) along with cyclophosphamide induction as in Group II. The respective treatments were administered daily through oral route for a period of 30 days. Regularity of estrous cycle was evaluated by vaginal cytology. Post-treatment period, the animals were humanely sacrificed, and the blood samples were subjected to the estimation of follicle-stimulating hormone (FSH) and estrogen. The ovarian tissue was weighed and subjected to histopathology, transmission electron microscopy, estimation of decreased glutathione (GSH), and tumor necrosis factor (TNF)-alpha. RESULTS: Rats treated with cyclophosphamide alone manifested irregularity in estrous cycle, increased FSH, and reduced estrogen levels. The ovaries showed decreased GSH and increased TNF-alpha concentrations. Histopathological and transmission electron microscopic analysis of the ovarian follicles revealed degenerative changes. Administration of alpha-LA and omega-3 fatty acids as well as the combination of both the treatments demonstrated significant normalization of the estrous cycle and antioxidant defense mechanism as well as ameliorated the hormonal profile and histological architecture of the ovarian follicles. However, appreciable synergistic efficacy of the combination therapy (alpha-LA+omega-3 fatty acids) with respect to the monotherapies was not observed in the present study. CONCLUSION: The efficacy of alpha-LA and omega-3 fatty acids against cyclophosphamide-induced ovarian toxicity could be attributed to its antioxidant and anti-inflammatory activities that prevented the oxidative damage to the ovaries caused by cyclophosphamide. Hence, our findings suggest that dietary supplementation of alpha-LA and omega-3 fatty acids in women receiving cyclophosphamide therapy could carry potential benefits in preventing cyclophosphamide-induced infertility in childbearing women.

Effect of feeding higher concentrations of limiting amino acids on performance, slaughter variables and nitrogen retention in broiler chicken fed graded levels of toasted guar (Cyamopsis tetragonoloba) meal.

1. An experiment was conducted to study the effect of supplementing higher concentrations (100% vs. 110%) of critical amino acids (CAA) on performance (body weight gain - BWG, feed efficiency - FE), slaughter variables and nitrogen retention in broiler chicken (1-6 weeks of age) fed graded levels of toasted guar meal (TGM) as a protein source in diets. 2. The TGM was included at five graded concentrations (0, 50, 100, 150 and 200 g/kg) in iso-caloric and iso-protein diets with either the recommended concentration (100%) of CAA (lysine, total sulphur amino acids, threonine, tryptophan and valine) or at 10% higher (110%) concentration. A metabolism trial of 3-day duration was conducted during 6th week of age to study nitrogen retention. 3. The TGM levels and CAA concentration at 21 or 42 d of age did not influence BWG, FI and FE. BWG was not affected with inclusion of TGM up to 100 g/kg in starter and overall production (1-42 d of age) phases. The FE improved with TGM supplementation during starter phase, while at the end of experiment (42 d), FE was depressed by inclusion of TGM in dose dependant manner. All performance variables improved with increase in concentration of CAA from 100% to 110%. 4. Breast meat weight improved and abdominal fat weight reduced with higher levels of CAA in diet. Retention of nitrogen reduced with increase in level of TGM in broiler diet. Increasing concentrations of CAA in diet improved nitrogen retention. 5. It was concluded that TGM could be incorporated up to 100 g/kg with 100% CAA and up to 150 g/kg with 110% CAA without affecting performance. Increasing CAA concentration (110%) in diets significantly improved BWG and FE (21 and 42 d), breast meat weight and nitrogen retention in broiler chicken.

Marker-assisted pyramiding of opaque2 and novel opaque16 genes for further enrichment of lysine and tryptophan in sub-tropical maize.

The improvement of protein quality in maize so far has been based on recessive opaque2 (o2) mutant that along with endosperm-modifiers led to development of quality protein maize (QPM). Recent discovery of nutritional benefits of recessive opaque16 (o16) mutant was of immense significance for further improvement of protein quality. In the present study, o16 was introgressed into o2-based parental inbreds (HKI161, HKI193-1, HKI193-2 and HKI163) of four commercial QPM hybrids (HQPM-1, HQPM-4, HQPM-5 and HQPM-7) released in India, using marker-assisted backcross breeding. Background selection led to high recovery of recurrent parent genome (RPG) to maximum of 95%, and introgressed progenies showed considerable phenotypic resemblance for plant-, ear- and grain- characteristics to their respective recurrent parents. Selection of markers for o2 and o16 led to development of pyramided lines (o2o2/o16o16) that possessed as high as 76% and 91% more lysine and tryptophan over the recurrent parents, respectively. Reconstituted hybrids showed an average enhancement of 49% and 60% in lysine and tryptophan over the original hybrids, with highest enhancement amounting 64% and 86%, respectively. This is first report of enhancement of both lysine and tryptophan by o16 in maize genotypes adaptable to sub-tropics. Moderate variation in lysine and tryptophan was also observed in pyramided lines. Multi-location evaluation of reconstituted hybrids revealed similar grain yield and attributing traits to their original versions. This study signified the role of o16 as supplementary to o2 for nutritional quality enhancement in maize, and improved elite inbreds and hybrids developed here hold great significance in maize biofortification programme.

Effect of supplementing germinated sprouts of pulses on performance, carcass variables, immune and oxidative stress indicators in broiler chickens reared during tropical summer season.

An experiment was conducted to study the effects of supplementing sprouts of pulses on performance, carcass variables, immune responses, and anti-oxidant variables in broiler chicken (day 1 to 6 weeks of age) reared during summer season in tropical region. Sprouts of black gram (BG, Vigna mungo), green gram (GG, Vigna radiata), and wild gram (WG, Vigna trilobata) were produced by soaking the pulses in water for 16 h and incubating at 37 °C for 24 h. Total phenolic content in sprouts of WG, BG, and GG was 102, 96.1, and 79.2 mg GAE/g, respectively, while the anti-radical activity in the sprouts was 61, 58, and 52%, respectively. A total of 200-day-old broiler male chicks were equally and randomly distributed in to 4 groups, each having 10 replicates of 5 chicks and housed in battery brooders in open-sided poultry house. Each of these groups was fed sprouts of BG, GG, or WG at 5% of feed intake, while the control group without feeding sprouts was kept for comparison. The trial was conducted during mid summer season (April and May, 2017). Feed conversion ratio (FCR) was reduced (P < 0.05) in broilers fed sprouted pulses compared to the control group at day 21. However, the body weight gain and FCR at 42 days of age, slaughter variables, and immune responses were not affected due to feeding of sprouted pulses. Feeding of sprouts significantly (P < 0.05) reduced lipid peroxidation and increased (P < 0.05) the activities of glutathione peroxidase, glutathione reductase, and superoxide dismutase in liver and spleen of broilers compared to the control group. Based on the results, it is concluded that oxidative stress in broiler chicken reared in tropical summer could be reduced by supplementing sprouted pulses without affecting performance, carcass variables, and immune responses.

Effect of Supplemental Trace Minerals on Hsp-70 mRNA Expression in Commercial Broiler Chicken.

The effects of supplementing the organic forms of selenium (Se), chromium (Cr), and zinc (Zn) on Hsp-70 mRNA expression and body weight in broiler chickens were evaluated. 200 chicks were equally distributed into stainless steel battery brooders at the rate of 5 birds per pen and reared under heat stress condition up to 42nd day. The chicks were fed with three experimental diets supplemented with organic forms of Se (0.30 mg/kg), Cr (2 mg/kg), and Zn (40 mg/kg) during the starter and finisher phases and a control diet without any supplementation. On the 21st and 42nd day, 20 birds from each period were sacrificed and samples were collected for analysis. Organic Se, Cr, and Zn supplementation significantly (P < 0.05) reduced the expression of Hsp-70 mRNA levels. The Hsp-70 mRNA expression levels were significantly (P < 0.05) different between the tissues studied with spleen having the lowest expression level. Hsp-70 mRNA expression level was not affected by age of the birds. The study concluded that organic trace mineral (oTM) supplementation resulted in low Hsp-70 mRNA expression, indicating reduced heat stress in broilers.