Analysis of nutritional composition in opaque2- and crtRB1-based single- and double-biofortified super sweet corn.

Sweet corn has emerged as a favorite food item worldwide owing to its kernel sweetness. However, traditional sweet corn cultivars are poor in provitamin-A (proA) and essential amino acids, viz., lysine and tryptophan. So far, no sweet corn hybrid with high nutritional qualities has been commercialized elsewhere. Here, we analyzed accumulation of provitamin-A (proA), lysine, and tryptophan in a set of mutant versions of (i) crtRB1-, (ii) o2-, and (iii) crtRB1 + o2-based sweet corn inbreds and hybrids with (iv) traditional sweet corn (wild-type: O2 + CrtRB1). The crtRB1- and crtRB1 + o2-based genotypes possessed significantly higher proA (17.31 ppm) over traditional sweet corn (2.83 ppm), while o2- and crtRB1 + o2-based genotypes possessed significantly higher lysine (0.345%) and tryptophan (0.080%) over traditional sweet corn (lysine 0.169%, tryptophan 0.036%). Late sowing favored high kernel lysine, proA, and green cob yield among hybrids. Sweetness (17.87%) among the improved inbreds and hybrids was comparable to the original sweetcorn genotypes (17.84%). Among the four genotypic classes, crtRB1 + o2-based improved genotypes showed stronger association among traits over genotypes with o2 and crtRB1 genes alone. Significant association was observed among (i) proA and BC (r = 0.99), (ii) proA and BCX (r = 0.93), (iii) lysine and tryptophan (r = 0.99), and (iv) green cob yield with fodder yield (r = 0.73) in sweet corn hybrids. The study demonstrated that combining crtRB1 and o2 genes did not pose any negative impact on nutritional, yield, and agronomic performance. Sweet corn with crtRB1 + o2 assumes significance for alleviating malnutrition through sustainable and cost-effective approach.

Prediction of matrilineal specific patatin-like protein governing in-vivo maternal haploid induction in maize using support vector machine and di-peptide composition.

The mutant matrilineal (mtl) gene encoding patatin-like phospholipase activity is involved in in-vivo maternal haploid induction in maize. Doubling of chromosomes in haploids by colchicine treatment leads to complete fixation of inbreds in just one generation compared to 6-7 generations of selfing. Thus, knowledge of patatin-like proteins in other crops assumes great significance for in-vivo haploid induction. So far, no online tool is available that can classify unknown proteins into patatin-like proteins. Here, we aimed to optimize a machine learning-based algorithm to predict the patatin-like phospholipase activity of unknown proteins. Four different kernels [radial basis function (RBF), sigmoid, polynomial, and linear] were used for building support vector machine (SVM) classifiers using six different sequence-based compositional features (AAC, DPC, GDPC, CTDC, CTDT, and GAAC). A total of 1170 protein sequences including both patatin-like (585 sequences) from various monocots, dicots, and microbes; and non-patatin-like proteins (585 sequences) from different subspecies of Zea mays were analyzed. RBF and polynomial kernels were quite promising in the prediction of patatin-like proteins. Among six sequence-based compositional features, di-peptide composition attained > 90% prediction accuracies using RBF and polynomial kernels. Using mutual information, most explaining dipeptides that contributed the highest to the prediction process were identified. The knowledge generated in this study can be utilized in other crops prior to the initiation of any experiment. The developed SVM model opened a new paradigm for scientists working in in-vivo haploid induction in commercial crops. This is the first report of machine learning of the identification of proteins with patatin-like activity.

Mutant crtRB1 gene negates the unfavourable effects of opaque2 gene on germination and seed vigour among shrunken2-based biofortified sweet corn genotypes.

Sweet corn is one of the most popular vegetables worldwide. However, traditional shrunken2 (sh2 )-based sweet corn varieties are poor in nutritional quality. Here, we analysed the effect of (1) ß-carotene hydroxylase1 (crtRB1 ), (2) opaque2 (o2 ) and (3) o2+crtRB1 genes on nutritional quality, germination, seed vigour and physico-biochemical traits in a set of 27 biofortified sh2 -based sweet corn inbreds. The biofortified sweet corn inbreds recorded significantly higher concentrations of proA (16.47µg g-1 ), lysine (0.36%) and tryptophan (0.09%) over original inbreds (proA: 3.14µg g-1 , lysine: 0.18%, tryptophan: 0.04%). The crtRB1 -based inbreds had the lowest electrical conductivity (EC), whereas o2 -based inbreds possessed the highest EC. The o2 +crtRB1 -based inbreds showed similar EC to the original inbreds. Interestingly, o2 -based inbreds also had the lowest germination and seed vigour compared to original inbreds, whereas crtRB1 and o2 +crtRB1 introgressed sweet corn inbreds showed similar germination and seed vigour traits to their original versions. This suggested that the negative effect of o2 on germination, seed vigour and EC is nullified by crtRB1 in the double mutant sweet corn. Overall, o2 +crtRB1 -based sweet corn inbreds were found the most desirable over crtRB1 - and o2 -based inbreds alone.

Assessing sequence variation, haplotype analysis and molecular characterisation of aspartate kinase2 (ask2) gene regulating methionine biosynthesis in diverse maize inbreds.

Traditional maize grain is deficient in methionine, an essential amino acid required for proper growth and development in humans and poultry birds. Thus, development of high methionine maize (HMM) assumes great significance in alleviating malnutrition through sustainable and cost-effective approach. Of various genetic loci, aspartate kinase2 (ask2) gene plays a pivotal role in regulating methionine accumulation in maize. Here, we sequenced the entire ask2 gene of 5394 bp with 13 exons in five wild and five mutant maize inbreds to understand variation at nucleotide level. Sequence analysis revealed that an SNP in exon-13 caused thymine to adenine transversion giving rise to a favourable mutant allele associated with leucine to glutamine substitution in mutant ASK2 protein. Gene-based diversity analysis with 11 InDel markers grouped 48 diverse inbreds into three major clusters with an average genetic dissimilarity of 0.570 (range, 0.0-0.9). The average major allele frequency, gene diversity and PIC are 0.693, 0.408 and 0.341, respectively. A total of 45 haplotypes of the ask2 gene were identified among the maize inbreds. Evolutionary relationship analysis performed among 22 orthologues grouped them into five major clusters. The number of exons varied from 7 to 17, with length varying from 12 to 495 bp among orthologues. ASK2 protein with 565 amino acids was predicted to be in homo-dimeric state with lysine and tartaric acid as binding ligands. Amino acid kinase and ACT domains were found to be conserved in maize and orthologues. The study depicted the presence of enough genetic diversity in ask2 gene in maize, and development of HMM can be accelerated through introgression of favourable allele of ask2 into the parental lines of elite hybrids using molecular breeding.

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.

Enhancement of nutritional quality in maize kernel through marker-assisted breeding for vte4, crtRB1, and opaque2 genes.

Traditional maize is poor in vitamin-E [α-tocopherol (α-T): 6-8 ppm], vitamin-A [provitamin-A (proA): 1-2ppm], lysine (0.150-0.2-50%), and tryptophan (0.030-0.040%). Here, we combined favourable alleles of vte4, crtRB1, and opaque2 (o2) genes in the parents of maize hybrids, viz., APQH-10 (PMI-PV-9 × PMI-PV-14) and APQH-11 (PMI-PV-9 × PMI-PV-15) using molecular breeding. Gene-specific markers were successfully used to select vte4, crtRB1, and o2 in BC1F1, BC2F1, and BC2F2 generations. Simple sequence repeats (104-109) were used for background selection, leading to an average recovery of 94% recurrent parent genome. The introgressed inbreds possessed significantly higher α-T: 18.38 ppm, α-/γ-tocopherol (α-/γ-T: 52%), and α-/total tocopherol (α-/TT: 32%) compared to original inbreds (α-T: 8.17 ppm, α-/γ-T: 25%, α-/TT: 18%). These newly derived inbreds also possessed higher ß-carotene (BC: 8.91 ppm), ß-cryptoxanthin (BCX: 1.27 ppm), proA (9.54 ppm), lysine (0.348%), and tryptophan (0.082%) compared to traditional maize inbreds. The reconstituted hybrids recorded higher α-T (2.1-fold), α-/γ-T (1.9-fold), and α-/TT (1.6-fold) over the original hybrids. These reconstituted hybrids were also rich in BC (5.7-fold), BCX (3.3-fold), proA (5.3-fold), lysine (1.9-fold), and tryptophan (2.0-fold) over the traditional hybrids. The reconstituted hybrids had similar grain yield and phenotypic characteristics to original versions. These multinutrient-rich maize hybrids hold great potential to alleviate malnutrition in sustainable and cost-effective manner.

Development of novel gene-based markers for waxy1 gene and their validation for exploitation in molecular breeding for enhancement of amylopectin in maize.

Waxy corn possessing high amylopectin is widely employed as an industrial product. Traditional corn contains ~ 70-75% amylopectin, whereas waxy corn with the mutant waxy1 (wx1) gene possesses ~ 95-100% amylopectin. Marker-assisted breeding can greatly hasten the transfer of the wx1 allele into normal corn. However, the available gene-based marker(s) for wx1 are not always polymorphic between recipient and donor parents, thereby causing a considerable delay in the molecular breeding program. Here, a 4800 bp sequence of the wx1 gene was analyzed among seven wild-type and seven mutant inbreds employing 16 overlapping primers. Three polymorphisms viz., 4 bp InDel (at position 2406 bp) in intron-7 and two SNPs (C to A at position 3325 bp in exon-10 and G to T at position 4310 bp in exon-13) differentiated the dominant (Wx1) and recessive (wx1) allele. Three breeder-friendly PCR markers (WxDel4, SNP3325_CT1, and SNP4310_GT2) specific to InDel and SNPs were developed. WxDel4 amplified 94 bp among mutant-type inbreds, while 90 bp was amplified among wild-type inbreds. SNP3325_CT1 and SNP4310_GT2 revealed the presence-absence polymorphisms with an amplification of 185 bp and 189 bp of amplicon, respectively. These newly developed markers showed 1:1 segregation in both BC1F1 and BC2F1 generations, while 1:2:1 segregation was observed in BC2F2. The recessive homozygotes (wx1wx1) of BC2F2 identified by the markers possessed significantly higher amylopectin (97.7%) compared to the original inbreds (Wx1Wx1: 72.7% amylopectin). This is the first report of novel wx1 gene-based markers. The information generated here would help in accelerating the development of waxy maize hybrids.

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.

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.

Provitamin A, lysine and tryptophan enrichment in shrunken2-based sweet corn genotypes through genomics-assisted breeding for crtRB1 and opaque2 genes.

BACKGROUND: Malnutrition affects large section of population worldwide. Vitamin A and protein deficiencies have emerged as the major global health-issue. Traditional shrunken2 (sh2)-based sweet corn is deficient in provitamin A (proA), lysine and tryptophan. Natural variant of ß-carotene hydroxylase1 (crtRB1) and opaque2 (o2) enhances proA, lysine and tryptophan in maize. So far, no sweet corn hybrid rich in these nutrients has been released elsewhere. Development of biofortified sweet corn hybrids would help in providing the balanced nutrition. METHODS AND RESULTS: We targeted three sh2-based sweet corn inbreds (SWT-19, SWT-20 and SWT-21) for introgression of mutant crtRB1 and o2 genes using molecular breeding. The gene-based 3'TE-InDel and simple sequence repeat (SSR) (umc1066) markers specific to crtRB1 and o2, respectively were utilized in foreground selection in BC1F1, BC2F1 and BC2F2. Segregation distortion was observed for crtRB1 and o2 genes in majority of populations. Background selection using 91-100 SSRs revealed recovery of recurrent parent genome (RPG) up to 96%. The introgressed progenies possessed significantly higher proA (13.56 µg/g) as compared to the original versions (proA: 2.70 µg/g). Further, the introgressed progenies had accumulated moderately higher level of lysine (0.336%) and tryptophan (0.082%) over original versions (lysine: 0.154% and tryptophan: 0.038%). Kernel sweetness among introgressed progenies (17.3%) was comparable to original sweet corn (17.4%). The introgressed inbreds exhibited higher resemblance with their recurrent parents for yield and morphological characters. CONCLUSION: These newly developed biofortified sweet corn genotypes hold immense promise to alleviate malnutrition.

Variation in anthocyanin pigmentation by R1-navajo gene, development and validation of breeder-friendly markers specific to C1-Inhibitor locus for in-vivo haploid production in maize.

BACKGROUND: In-vivo maternal haploids serve as the basis of doubled haploid (DH) breeding in maize. R1-navajo (R1-nj) gene governing anthocyanin colouration in the endosperm and embryo is widely used to identify haploid seeds. However, the expression of R1-nj depends on genetic-background of source-germplasm used for deriving DH-lines. Further, presence of C1-Inhibitor (C1-I) gene suppresses the expression of R1-nj, thus makes the selection of haploids difficult. METHODS: In the present study, 178 subtropically-adapted maize inbreds were crossed with two R1-nj donors 'that do not have haploid induction genes'. Of these, 76.4% inbreds developed purple colour in endosperm, while 23.6% did not show any colouration. In case of scutellum, 62.9% inbreds possessed colour and 37.1% were colourless. The anthocyanin intensity varied greatly, with 19.66% and 42.98% inbreds displayed the least intensity, while 16.85% and 0.84% inbreds showed the highest intensity in endosperm and scutellum, respectively. Two C1-I specific breeder-friendly markers (MGU-CI-InDel8 and MGU-C1-SNP1) covering (i) 8 bp InDel and (ii) A to G SNP, respectively, were developed. MGU-CI-InDel8 and MGU-C1-SNP1 markers predicted presence of C1-I allele with 92.9% and 84.7% effectiveness, respectively. However, when both markers were considered together, they provided 100% effectiveness. CONCLUSIONS: These markers of C1-I gene would help in saving valuable resources and time during haploid induction in maize. The information generated here assume great significance in DH breeding of maize.

Correction to: Pollen staining is a rapid and cost­effective alternative to marker­assisted selection for recessive waxy1 gene governing high amylopectin in maize.

[This corrects the article DOI: 10.1007/s12298-022-01240-1.].

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 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.

Characterization of crtRB1- and vte4-based biofortified sweet corn inbreds for seed vigour and physico-biochemical traits.

Sweet corn possessing recessive shrunken2 (sh2) gene is popular worldwide. Traditional sweet corn is poor in vitamin A and vitamin E. Plant breeders during the selection of sweet corn genotypes mainly emphasize on plant architecture and yield. Seed germination and seedling vigour play important role for early establishment in field, thereby increasing yield and income. Here, we analysed a set of 15 sh2-based biofortified sweet corn inbreds with crtRB1 (ß-carotene hydroxylase1) and vte4 (γ-tocopherol methyltransferase) genes and three traditional sh2-based sweet corn inbreds for nutritional quality, seed vigour and various physico-biochemical traits. The newly developed inbreds possessed significantly higher provitamin A (proA: 15.60 µg/g) and vitamin E [α-tocopherol (α-T): 20.42 µg/g] than the traditional sweet corn inbreds (proA: 2.51 µg/g, α-T: 11.16 µg/g). The biofortified sweet corn inbreds showed wide variation for germination (80.67-87.33%), vigour index-I (2097.17-2925.28 cm), vigour index-II (134.27-216.19 mg) and electrical conductivity (10.19-13.21 µS cm-1 g-1). Wide variation was also observed for dehydrogenase (1.29-1.59 OD g-1 ml-1), super oxide dismutase (4.01-9.82 g-1), peroxidase (11.66-16.47 µM min-1 g-1), esterase (22.98-34.76 nM min-1 g-1) and α-amylase (5.91-8.15 OD g-1 ml-1). Enrichment of proA and vitamin E in sweet corn did not affect seed vigour and physico-biochemical traits. Correlation analysis revealed that electrical conductivity and α-amylase activity was the reliable indicator for assessing seed germination and vigour. The study identified superior biofortified sweet corn genotypes that would contribute to better vigour and establishment in field. This is the first report of analysis of biofortified sweet corn genotypes for seed vigour and physico-biochemical traits.

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.

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.

Development of Maize Hybrids With Enhanced Vitamin-E, Vitamin-A, Lysine, and Tryptophan Through Molecular Breeding.

Malnutrition is a widespread problem that affects human health, society, and the economy. Traditional maize that serves as an important source of human nutrition is deficient in vitamin-E, vitamin-A, lysine, and tryptophan. Here, favorable alleles of vte4 (α-tocopherol methyl transferase), crtRB1 (ß-carotene hydroxylase), lcyE (lycopene ε-cyclase), and o2 (opaque2) genes were combined in parental lines of four popular hybrids using marker-assisted selection (MAS). BC1F1, BC2F1, and BC2F2 populations were genotyped using gene-based markers of vte4, crtRB1, lcyE, and o2. Background selection using 81-103 simple sequence repeats (SSRs) markers led to the recovery of recurrent parent genome (RPG) up to 95.45%. Alpha (α)-tocopherol was significantly enhanced among introgressed progenies (16.13 µg/g) as compared to original inbreds (7.90 µg/g). Provitamin-A (proA) (10.42 µg/g), lysine (0.352%), and tryptophan (0.086%) were also high in the introgressed progenies. The reconstituted hybrids showed a 2-fold enhancement in α-tocopherol (16.83 µg/g) over original hybrids (8.06 µg/g). Improved hybrids also possessed high proA (11.48 µg/g), lysine (0.367%), and tryptophan (0.084%) when compared with traditional hybrids. The reconstituted hybrids recorded the mean grain yield of 8,066 kg/ha, which was at par with original hybrids (mean: 7,846 kg/ha). The MAS-derived genotypes resembled their corresponding original hybrids for the majority of agronomic and yield-related traits, besides characteristics related to distinctness, uniformity, and stability (DUS). This is the first report for the development of maize with enhanced vitamin-E, vitamin-A, lysine, and tryptophan.

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.

Development of sub-tropically adapted diverse provitamin-A rich maize inbreds through marker-assisted pedigree selection, their characterization and utilization in hybrid breeding.

Malnutrition has emerged as one of the major health problems worldwide. Traditional yellow maize has low provitamin-A (proA) content and its genetic base in proA biofortification breeding program of subtropics is extremely narrow. To diversify the proA rich germplasm, 10 elite low proA inbreds were crossed with a proA rich donor (HP702-22) having mutant crtRB1 gene. The F2 populations derived from these crosses were genotyped using InDel marker specific to crtRB1. Severe marker segregation distortion was observed. Seventeen crtRB1 inbreds developed through marker-assisted pedigree breeding and seven inbreds generated using marker-assisted backcross breeding were characterized using 77 SSRs. Wide variation in gene diversity (0.08 to 0.79) and dissimilarity coefficient (0.28 to 0.84) was observed. The inbreds were grouped into three major clusters depicting the existing genetic diversity. The crtRB1-based inbreds possessed high ß-carotene (BC: 8.72µg/g), ß-cryptoxanthin (BCX: 4.58µg/g) and proA (11.01µg/g), while it was 2.35µg/g, 1.24µg/g and 2.97µg/g in checks, respectively. Based on their genetic relationships, 15 newly developed crtRB1-based inbreds were crossed with five testers (having crtRB1 gene) using line × tester mating design. 75 experimental hybrids with crtRB1 gene were evaluated over three locations. These experimental hybrids possessed higher BC (8.02µg/g), BCX (4.69µg/g), proA (10.37µg/g) compared to traditional hybrids used as check (BC: 2.36 µg/g, BCX: 1.53µg/g, proA: 3.13µg/g). Environment and genotypes × environment interaction had minor effects on proA content. Both additive and dominance gene action were significant for proA. The mean proportion of proA to total carotenoids (TC) was 44% among crtRB1-based hybrids, while 11% in traditional hybrids. BC was found to be positively correlated with BCX (r = 0.68) and proA (r = 0.98). However, no correlation was observed between proA and grain yield. Several hybrids with >10.0 t/ha grain yield with proA content >10.0 µg/g were identified. This is the first comprehensive study on development of diverse proA rich maize hybrids through marker-assisted pedigree breeding approach. The findings provides sustainable and cost-effective solution to alleviate vitamin-A deficiency.