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.

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.

Allelic variation and haplotype diversity of Matrilineal (MTL) gene governing in vivo maternal haploid induction in maize.

Diverse haploid inducer lines with > 6% of haploid induction rate are now routinely used to develop doubled haploid lines. Though MTL gene regulates haploid induction, its molecular characterization and haplotype analysis in maize and its related species have not been undertaken so far. In the present study, the entire 1812 bp long MTL gene was sequenced among two mutant and eight wild-type inbreds. A 4 bp insertion differentiated the mutant from the wild-type allele. Sequence analysis further revealed 103 polymorphic sites including 38 InDels and 65 SNPs. A total of 15 conserved regions were detected, of which exon-4 was the most conserved. Ten gene-based markers specific to MTL revealed the presence of 40 haplotypes among diverse 48 inbreds of exotic and indigenous origin. It generated 20 alleles with an average of two alleles per locus. The mean polymorphic information content was 0.3247 with mean gene diversity of 0.4135. A total of 15 paralogous sequences of MTL were detected in maize genome with 3-7 exons. Maize MTL proteins of both wild-type and mutant were non-polar in nature, and they possessed four domains. R1-nj-based haploid inducer (HI) lines viz., Pusa-HI-101 and Pusa-HI-102 had an average haploid induction rate of 8.45 ± 0.96% and 10.46 ± 1.15%, respectively. Lines wild-type MTL gene did not generate any haploid. In comparison with 27 orthologues of 21 grass species, maize MTL gene had the closest ancestry with Saccharum spontaneum and Sorghum. The information generated here assumes great significance in understanding the diversity of MTL gene and presence of paralogues and orthologues. This is the first report on haplotype analysis and molecular characterization of MTL gene in maize and related grass species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01456-3.

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.

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.

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.

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.].

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.

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.

Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985.

Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.

High amylopectin in waxy maize synergistically affects seed germination and seedling vigour over traditional maize genotypes.

Waxy maize grains rich in amylopectin have emerged as a popular food and industrial raw materials. Here, a set of waxy inbreds having recessive waxy1 (wx1) gene derived through marker-assisted selection (MAS), and their original versions were evaluated for germination, seed vigour index-I and vigour index-II, electrical conductivity (EC) and enzymatic activities viz., dehydrogenase (DH), esterase (EST), peroxidase (POX), superoxide dismutase (SOD) and α-amylase (AMY). Waxy inbreds under study possessed average 97.8% amylopectin compared to 72.4% in original inbreds. Waxy versions showed 15.2% more test weight, 4.3% increase in germination, 22.7% higher seed vigour index-I and 28.3% higher seed vigour index-II, respectively, over the original inbreds. Further, activity of DH, EST, POX, SOD and AMY of MAS-derived waxy inbreds was more than that of original inbreds, whereas EC was less in improved inbreds compared to originals. Amylopectin exhibited strong positive correlations (r = 0.69 to 0.97**) with seed germination, vigour index-I and -II, DH, SOD, POX, EST and AMY activity. However, amylopectin showed negative correlation of - 0.82** with EC. Seed germination and seed vigour indices were also positively correlated with all enzymatic activities (r = 0.58 to 0.92**). The analysis revealed that waxy inbreds possess better seed vigour and enzymatic activities over traditional inbreds. This is the first report of synergistic effects of wx1 gene on seed germination, vigour and enzymatic activities in maize endosperm.

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.

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.

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.

Development and validation of rapid and cost-effective protocol for estimation of amylose and amylopectin in maize kernels.

Maize possesses wide variation in amylose and amylopectin which assumes significance as a part of both food-chain and different industrial applications. Estimation of amylose and amylopectin in maize kernels is important for developing suitable hybrids. The existing protocols for estimation of amylose and amylopectin in maize are elaborate and lengthy, and involve high cost. Here, we developed a rapid and cost-effective method for estimation of amylose and amylopectin in maize kernels. 10% toluene and 80% ethanol were used for removal of proteins (~ 10%) and lipids (~ 4%) from maize flour. The over-estimation of amylose was minimized using NaOH with KI to stop free KI to bind with amylopectin. Standards were improved by mixing amylose and amylopectin in different concentrations (0-100%), rather than using amylose or amylopectin alone. Standard curve generated regression equation of y = 90.436x + 0.8535 with R 2 = 0.9989. Two types of samples viz., (1) protein, amylose and amylopectin (2) amylose and amylopectin, showed that starch fractions were highly comparable to expected values with correlation coefficient (r) of 0.9998 and mean standard deviation of 0.54. The protocol successfully estimated wide range of amylose (2.79-50.04%) and amylopectin (59.96-97.21%) among diverse maize inbreds including amylose extender1 (ae1) and waxy1 (wx1) mutants. Present protocol required 75% less time and 92.5% less cost compared to existing protocols. The newly developed method would be highly useful in developing maize hybrids high in amylose or amylopectin. This is the first report of rapid and cost-effective protocol for estimation of starch fractions in maize kernels.

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.

Expression analysis of ß-carotene hydroxylase1 and opaque2 genes governing accumulation of provitamin-A, lysine and tryptophan during kernel development in biofortified sweet corn.

Traditional sweet corn possesses low levels of provitamin-A (proA), lysine and tryptophan. Mutant version of ß-carotene hydroxylase1 (crtRB1) gene affecting the accumulation of ß-carotene (BC), ß-cryptoxanthin (BCX) and proA, and opaque2 (o2) gene governing the enhancement of lysine and tryptophan were introgressed together into elite sweet corn inbreds through marker-assisted selection. Here, we analyzed the expression pattern of crtRB1 and o2 genes among introgressed and traditional sweet corn inbreds at 20-, 24- and 28-days after pollination (DAP). The introgressed inbreds possessed two- to sevenfolds higher BC, BCX, proA, lysine and tryptophan compared to their original inbreds. However, all the nutrients attained the peak at 20-DAP (BC: 9.95 µg/g, BCX: 8.21 µg/g, proA: 14.05 µg/g, lysine: 0.301%, tryptophan: 0.074%), which gradually reduced through 24-DAP (BC: 8.24 µg/g, BCX: 7.53 µg/g, proA: 12.01 µg/g, lysine: 0.273%, tryptophan: 0.057%) and 28-DAP (BC: 5.84 µg/g, BCX: 5.82 µg/g, proA: 8.75 µg/g, lysine: 0.202%, tryptophan: 0.037%). Biofortified sweet corn inbreds possessed significantly lower expression levels of crtRB1 (4.1-fold) and o2 (2.2-fold) compared to their wild type alleles in traditional sweet corn inbreds across DAPs. The expression of crtRB1 and o2 increased from 20-DAP to attain the highest peak at 24-DAP, and further decreased by 28-DAP. The transcript levels of crtRB1 were negatively correlated with BC (r = - 0.83), BCX (r = - 0.79) and proA (r = - 0.83) across dates of harvest. Lysine (r = - 0.83) and tryptophan (r = - 0.73) were also inversely associated with o2 transcript levels. This is the first report on expression of crtRB1 and o2 genes during kernel development in biofortified sweet corn. This information holds immense promise in understanding the dynamics of gene-regulation during kernel development in sweet corn.

Development of multinutrient-rich biofortified sweet corn hybrids through genomics-assisted selection of shrunken2, opaque2, lcyE and crtRB1 genes.

Sweet corn has gained worldwide popularity. Traditional sweet corn possesses low concentration of essential nutrients such as lysine (0.15-0.25%), tryptophan (0.03-0.04%) and provitamin-A (proA 3-4 ppm), and deficiency leads to serious health problems in humans. Here, stacking of shrunken2 (sh2), opaque2 (o2), lycopene epsilon cyclase (lcyE) and ß-carotene hydroxylase (crtRB1) genes  were undertaken in the parents of four hybrids viz., APQH1, APHQ4, APHQ5 and APHQ7 using marker-assisted backcross breeding (MABB). Gene-linked markers (umc2276 and umc1320) for sh2, while gene-based markers for o2 (umc1066 and phi057), lcyE (5'TE-InDel) and crtRB1 (3'TE-InDel), were used for genotyping in BC1F1, BC2F1 and BC2F2. Selected backcross progenies showed high recovery of recurrent parent genome (92.4-97.7%). The reconstituted sweet corn hybrids possessed significantly high lysine (0.390%), tryptophan (0.082%) and proA (21.14 ppm), coupled with high kernel sweetness (brix 18.96%). The improved sweet corn hybrids had high cob yield (12.22-15.33 t/ha) across three environments. These newly developed biofortified sweet corn hybrids possess great significance in providing balanced nutrition. This is the first report of combining sh2, o2, lcyE and crtRB1 genes for enrichment of sweet corn hybrids with multiple essential nutrients.